U.S. patent application number 13/382902 was filed with the patent office on 2012-07-19 for engineered microorganisms with enhanced fermentation activity.
This patent application is currently assigned to Verdezyne, Inc.. Invention is credited to Jose Miguel LaPlaza, Stephen Picataggio, Kirsty Anne Lily Salmon.
Application Number | 20120184020 13/382902 |
Document ID | / |
Family ID | 43429863 |
Filed Date | 2012-07-19 |
United States Patent
Application |
20120184020 |
Kind Code |
A1 |
Picataggio; Stephen ; et
al. |
July 19, 2012 |
ENGINEERED MICROORGANISMS WITH ENHANCED FERMENTATION ACTIVITY
Abstract
Provided herein are genetically modified microorganisms that
have enhanced fermentation activity, and methods for making and
using such microorganisms.
Inventors: |
Picataggio; Stephen;
(Carlsbad, CA) ; Salmon; Kirsty Anne Lily;
(Carlsbad, CA) ; LaPlaza; Jose Miguel; (Carlsbad,
CA) |
Assignee: |
Verdezyne, Inc.
Carlsbad
CA
|
Family ID: |
43429863 |
Appl. No.: |
13/382902 |
Filed: |
July 9, 2010 |
PCT Filed: |
July 9, 2010 |
PCT NO: |
PCT/US2010/041618 |
371 Date: |
March 26, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61224430 |
Jul 9, 2009 |
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61316780 |
Mar 23, 2010 |
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61334097 |
May 12, 2010 |
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Current U.S.
Class: |
435/254.21 ;
435/254.2 |
Current CPC
Class: |
Y02E 50/16 20130101;
Y02E 50/10 20130101; C12P 7/10 20130101; C12N 9/88 20130101; C12N
9/92 20130101; Y02E 50/17 20130101 |
Class at
Publication: |
435/254.21 ;
435/254.2 |
International
Class: |
C12N 1/19 20060101
C12N001/19 |
Claims
1-52. (canceled)
53. A eukaryotic cell comprising a polypeptide having xylose
isomerase activity, which polypeptide comprises an amino acid
sequence greater than that 65.8% identical to SEQ ID NO: 31.
54. The eukaryotic cell of claim 53, wherein the polypeptide
comprises an amino acid sequence that is 75% or more identical to
SEQ ID NO: 31.
55. The eukaryotic cell of claim 54, wherein the polypeptide
comprises an amino acid sequence that is 80% or more identical to
SEQ ID NO: 31.
56. The eukaryotic cell of claim 55, wherein the polypeptide
comprises an amino acid sequence that is 85% or more identical to
SEQ ID NO: 31.
57. The eukaryotic cell of claim 56, wherein the polypeptide
comprises an amino acid sequence that is 90% or more identical to
SEQ ID NO: 31.
58. The eukaryotic cell of claim 57, wherein the polypeptide
comprises an amino acid sequence that is 95% or more identical to
SEQ ID NO: 31.
59. The eukaryotic cell of claim 58, wherein the polypeptide
comprises an amino acid sequence of SEQ ID NO: 31.
60. The eukaryotic cell of claim 59, wherein the polypeptide
consists of an amino acid sequence of SEQ ID NO: 31.
61. The eukaryotic cell of claim 53, which is a yeast cell.
62. The eukaryotic cell of claim 61, which is a Saccharomyces spp.
cell.
63. The eukaryotic cell of claim 62, which is a Saccharomyces
cerevisiae cell.
64. An engineered yeast comprising a chimeric enzyme having xylose
isomerase activity, wherein: the C-terminal region of the chimeric
enzyme comprises at least 5 contiguous amino acids from a bacterial
xylose isomerase enzyme; and the N-terminal region of the chimeric
enzyme comprises at least 5 contiguous amino acids from a fungal
xylose isomerase enzyme.
65. The engineered yeast of claim 64, wherein the bacterial xylose
isomerase enzyme is a Ruminococcus spp. xylose isomerase
enzyme.
66. The engineered yeast of claim 65, wherein the Ruminococcus spp.
xylose isomerase enzyme is from a Ruminococcus flavefaciens
strain.
67. The engineered yeast of claim 66, wherein the Ruminococcus
flavefaciens strain is selected from the group consisting of
Ruminococcus flavefaciens strain 17, Ruminococcus flavefaciens
strain Siijpesteijn 1948, and Ruminococcus flavefaciens strain
FD1.
68. The engineered yeast of claim 66, wherein the at least 5
contiguous amino acids from the bacterial xylose isomerase enzyme
are from SEQ ID NO: 31.
69. The engineered yeast of claim 64, wherein the fungal xylose
isomerase enzyme is from a fungus selected from the group
consisting of an Aspergillus spp. fungus, Thraustochytrium spp.
fungus, Schizochytrium spp. fungus, Rhizopus spp. fungus,
Orpinomyces spp. fungus and Piromyces spp. fungus.
70. The engineered yeast of claim 69, wherein the fungal xylose
isomerase enzyme is from a Piromyces spp. fungus.
71. The engineered yeast of claim 70, wherein the Piromyces spp.
fungus is a Piromyces strain E2 fungus.
72. The engineered yeast of claim 71, wherein the at least 5
contiguous amino acids from the fungal xylose isomerase enzyme are
from SEQ ID NO: 35.
73. The engineered yeast of claim 64, which is an engineered
Saccharomyces spp. yeast.
74. The engineered yeast of claim 73, which is an engineered
Saccharomyces cerevisiae yeast.
Description
RELATED PATENT APPLICATION(S)
[0001] This patent application is a national stage of international
patent application no. PCT/2010/041618 filed Jul. 9, 2010, entitled
ENGINEERED MICROORGANISMS WITH ENHANCED FERMENTATION ACTIVITY,
naming Stephen Picataggio, Kirsty Anne Lily Salmon, and Jose Miguel
LaPlaza as inventors, and designated by Attorney Docket No.
VRD-1002-PC, which claims the benefit of U.S. provisional patent
application No. 61/224,430 filed on Jul. 9, 2009, entitled USE OF
ENGINEERED MICROORGANISMS WITH ENHANCED FERMENTATION ACTIVITY,
naming Stephen Picataggio as inventor and designated by Attorney
Docket No. VRD-1002-PV; claims the benefit of U.S. provisional
patent application No. 61/316,780 filed on Mar. 23, 2010, entitled
USE OF ENGINEERED MICROORGANISMS WITH ENHANCED FERMENTATION
ACTIVITY, naming Stephen Picataggio as inventor and designated by
Attorney Docket No. VRD-1002-PV2; and claims the benefit of U.S.
provisional patent application No. 61/334,097 filed on May 12,
2010, entitled ENGINEERED MICROORGANISMS WITH ENHANCED FERMENTATION
ACTIVITY, naming Stephen Picataggio as inventor and designated by
Attorney Docket No. VRD-1002-PV3. The entire contents of the
foregoing patent applications are incorporated herein by reference,
including, without limitation, all text, tables and drawings.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Feb. 10, 2012, is named VRD102US.txt and is 489,276 bytes in
size.
FIELD
[0003] The technology relates in part to genetically modified
microorganisms that have enhanced fermentation activity, and
methods for making and using such microorganisms.
BACKGROUND
[0004] Microorganisms employ various enzyme-driven biological
pathways to support their own metabolism and growth. A cell
synthesizes native proteins, including enzymes, in vivo from
deoxyribonucleic acid (DNA). DNA first is transcribed into a
complementary ribonucleic acid (RNA) that comprises a
ribonucleotide sequence encoding the protein. RNA then directs
translation of the encoded protein by interaction with various
cellular components, such as ribosomes. The resulting enzymes
participate as biological catalysts in pathways involved in
production of molecules utilized or secreted by the organism.
[0005] These pathways can be exploited for the harvesting of the
naturally produced products. The pathways also can be altered to
increase production or to produce different products that may be
commercially valuable. Advances in recombinant molecular biology
methodology allow researchers to isolate DNA from one organism and
insert it into another organism, thus altering the cellular
synthesis of enzymes or other proteins. Such genetic engineering
can change the biological pathways within the host organism,
causing it to produce a desired product. Microorganic industrial
production can minimize the use of caustic chemicals and production
of toxic byproducts, thus providing a "clean" source for certain
products.
SUMMARY
[0006] Provided herein are engineered microorganisms having
enhanced fermentation activity. In certain non-limiting
embodiments, such microorganisms are capable of generating a target
product with enhanced fermentation efficiency by, for example, (i)
preferentially utilizing a particular glycolysis pathway, which
increases yield of a target product, upon a change in fermentation
conditions; (ii) reducing cell division rates upon a change in
fermentation conditions, thereby diverting nutrients towards
production of a target product; (iii) having the ability to readily
metabolize five-carbon sugars; and/or (iv) having the ability to
readily metabolize carbon dioxide; and combinations of the
foregoing. In some embodiments, a target product is ethanol or
succinic acid.
[0007] Thus, provided in certain embodiments are engineered
microorganisms that comprise: (a) a functional Embden-Meyerhoff
glycolysis pathway that metabolizes six-carbon sugars under aerobic
fermentation conditions, and (b) a genetic modification that
reduces an Embden-Meyerhoff glycolysis pathway member activity upon
exposure of the engineered microorganism to anaerobic fermentation
conditions, whereby the engineered microorganisms preferentially
metabolize six-carbon sugars by the Enter-Doudoroff pathway under
the anaerobic fermentation conditions. In some embodiments, the
genetic modification is insertion of a promoter into genomic DNA in
operable linkage with a polynucleotide that encodes the
Embden-Meyerhoff glycolysis pathway member activity. In certain
embodiments, the genetic modification is provision of a
heterologous promoter polynucleotide in operable linkage with a
polynucleotide that encodes the Embden-Meyerhoff glycolysis pathway
member activity. In some embodiments, the genetic modification is a
deletion or disruption of a polynucleotide that encodes, or
regulates production of, the Embden-Meyerhoff glycolysis pathway
member, and the microorganism comprises a heterologous nucleic acid
that includes a polynucleotide encoding the Embden-Meyerhoff
glycolysis pathway member operably linked to a polynucleotide that
down-regulates production of the member under anaerobic
fermentation conditions. In certain embodiments, the
Embden-Meyerhoff glycolysis pathway member activity is a
phosphofructokinase activity, a phosphoglucose isomerase activity,
or a phosphofructokinase activity and a phosphoglucose isomerase
activity. In some embodiments, the activity of one or more (e.g.,
2, 3, 4, 5 or more) pathway members in an EM pathway is reduced or
removed to undetectable levels. In certain embodiments, one or more
activities in an EM pathway, not mentioned herein, also can be
modified to further enhance production of a desired product (e.g.,
alcohol).
[0008] Also provided in some embodiments are engineered
microorganisms that comprise a genetic modification that inhibits
cell division upon exposure to a change in fermentation conditions,
where: the genetic modification comprises introduction of a
heterologous promoter operably linked to a polynucleotide encoding
a polypeptide that regulates the cell cycle of the microorganism;
and the promoter activity is altered by the change in fermentation
conditions. Provided also in certain embodiments are engineered
microorganisms that comprise a genetic modification that inhibits
cell division and/or cell proliferation upon exposure of the
microorganisms to a change in fermentation conditions. In certain
embodiments, the genetic modification inhibits cell division,
inhibits cell proliferation, inhibits the cell cycle and/or induces
cell cycle arrest. In some embodiments, the change in fermentation
conditions is a switch to anaerobic fermentation conditions, and in
certain embodiments, the change in fermentation conditions is a
switch to an elevated temperature. In some embodiments, the
polypeptide that regulates the cell cycle has thymidylate synthase
activity. In certain embodiments, the promoter activity is reduced
by the change in fermentation conditions. In some embodiments, the
genetic modification is a temperature sensitive mutation.
[0009] Provided also in some embodiments are methods for
manufacturing a target product produced by an engineered
microorganism, which comprise: (a) culturing an engineered
microorganism described herein under aerobic conditions; and (b)
culturing the engineered microorganism after (a) under anaerobic
conditions, whereby the engineered microorganism produces the
target product. Also provided in some embodiments are methods for
producing a target product by an engineered microorganism, which
comprise: (a) culturing an engineered microorganism described
herein under a first set of fermentation conditions; and (b)
culturing the engineered microorganism after (a) under a second set
of fermentation conditions different than the first set of
fermentation conditions, whereby the second set of fermentation
conditions inhibits cell division and/or cell proliferation of the
engineered microorganism. In certain embodiments, the target
product is ethanol or succinic acid. In some embodiments, the host
microorganism from which the engineered microorganism is produced
does not produce a detectable amount of the target product. In
certain embodiments, the culture conditions comprise fermentation
conditions, comprise introduction of biomass, comprise introduction
of a six-carbon sugar (e.g., glucose), and/or comprise introduction
of a five-carbon sugar (e.g., xylulose, xylose); or combinations of
the foregoing. In some embodiments, the target product is produced
with a yield of greater than about 0.3 grams per gram of glucose
added, and in certain embodiments, a method comprises purifying the
target product from the cultured microorganisms. In some
embodiments, a method comprises modifying the target product,
thereby producing modified target product. In certain embodiments,
a method comprises placing the cultured microorganisms, the target
product or the modified target product in a container, and in
certain embodiments, a method comprises shipping the container. In
some embodiments, the second set of fermentation conditions
comprises an elevated temperature as compared to the temperature in
the first set of fermentation conditions. In certain embodiments,
the genetic modification inhibits the cell cycle of the engineered
microorganism upon exposure to the second set of fermentation
conditions. In some embodiments, the genetic modification inhibits
cell proliferation, inhibits cell division, inhibits the cell cycle
and/or induces cell cycle arrest upon exposure to the second set of
fermentation conditions. In certain embodiments, the genetic
modification inhibits thymidylate synthase activity upon exposure
to the change in fermentation conditions, and sometimes the genetic
modification comprises a temperature sensitive mutation.
[0010] Also provided in certain embodiments are methods for
manufacturing an engineered microorganism, which comprise: (a)
introducing a genetic modification to a host microorganism that
reduces an Embden-Meyerhoff glycolysis pathway member activity upon
exposure of the engineered microorganism to anaerobic conditions;
and (b) selecting for engineered microorganisms that (i) metabolize
six-carbon sugars by the Embden-Meyerhoff glycolysis pathway under
aerobic fermentation conditions, and (ii) preferentially metabolize
six-carbon sugars by the Entner-Doudoroff pathway under the
anaerobic fermentation conditions. In some embodiments, the genetic
modification is insertion of a promoter into genomic DNA in
operable linkage with a polynucleotide that encodes the
Embden-Meyerhoff glycolysis pathway member activity. The genetic
modification sometimes is provision of a heterologous promoter
polynucleotide in operable linkage with a polynucleotide that
encodes the Embden-Meyerhoff glycolysis pathway member activity. In
certain embodiments, the genetic modification is a deletion or
disruption of a polynucleotide that encodes, or regulates
production of, the Embden-Meyerhoff glycolysis pathway member, and
the microorganism comprises a heterologous nucleic acid that
includes a polynucleotide encoding the Embden-Meyerhoff glycolysis
pathway member operably linked to a polynucleotide that
down-regulates production of the member under anaerobic
fermentation conditions. In some embodiments, the Embden-Meyerhoff
glycolysis pathway member activity is a phosphofructokinase
activity, and in certain embodiments, the Embden-Meyerhoff
glycolysis pathway member activity is a phosphoglucose isomerase
activity. In some embodiments, the activity of one or more (e.g.,
2, 3, 4, 5 or more) pathway members in an EM pathway is reduced or
removed to undetectable levels.
[0011] Provided also in some embodiments are methods for
manufacturing an engineered microorganism, which comprise: (a)
introducing a genetic modification to a host microorganism that
inhibits cell division upon exposure to a change in fermentation
conditions, thereby producing engineered microorganisms; and (b)
selecting for engineered microorganisms with inhibited cell
division upon exposure of the engineered microorganisms to the
change in fermentation conditions. In certain embodiments, the
change in fermentation conditions comprises a change to anaerobic
fermentation conditions. The change in fermentation conditions
sometimes comprises a change to an elevated temperature. In some
embodiments, the genetic modification inhibits the cell cycle of
the engineered microorganism upon exposure to the change in
fermentation conditions. The genetic modification sometimes
inhibits cell division, inhibits the cell cycle, inhibits cell
proliferation and/or induces cell cycle arrest upon exposure to the
change in fermentation conditions. In some embodiments, the genetic
modification inhibits thymidylate synthase activity upon exposure
to the change in fermentation conditions, and in certain
embodiments, the genetic modification comprises a temperature
sensitive mutation.
[0012] In certain embodiments pertaining to engineered
microorganisms, and methods of making or using such microorganisms,
the microorganism comprises a genetic modification that adds or
alters a five-carbon sugar metabolic activity. In some embodiments,
the microorganism comprises a genetic alteration that adds or
alters xylose isomerase activity. In certain embodiments, the
microorganism comprises a genetic alteration that adds or alters a
xylose reductase (XR) activity and a xylitol dehydrogenase (XD)
activity. In some embodiments, the microorganism comprises a
xylulokinase (XK) activity. In certain embodiments, the
microorganism comprises a genetic alteration that adds or alters
five-carbon sugar transporter activity, and sometimes the
transporter activity is a transporter facilitator activity or an
active transporter activity. In some embodiments, the microorganism
comprises a genetic alteration that adds or alters carbon dioxide
fixation activity, and sometimes the genetic alteration that adds
or alters phosphoenolpyruvate (PEP) carboxylase activity. In
certain embodiments, the microorganism comprises a genetic
modification that reduces or removes an alcohol dehydrogenase 2
activity. In certain embodiments, the microorganism comprises a
genetic alteration that adds or alters a 6-phosphogluconate
dehydrogenase (decarboxylating) activity. In some embodiments the
microorganism is an engineered yeast, such as a Saccharomyces yeast
(e.g., S. cerevisiae), for example.
[0013] In some embodiments, provided are nucleic acids, comprising
a polynucleotide that encodes a polypeptide from Ruminococcus
possessing a xylose to xylulose xylose isomerase activity, or a
polypeptide possessing xylose reductase activity and xylitol
dehydrogenase activity. In certain embodiments, provided are
nucleic acids, comprising a polynucleotide that encodes a
polypeptide possessing xylulokinase activity. Also provided in
certain embodiments are expression vectors comprising a
polynucleotide that encodes a polypeptide from Ruminococcus
possessing a xylose isomerase activity. Also provided in some
embodiments are expression vectors comprising a polynucleotide that
encodes a polypeptide possessing a xylose reductase activity and
xylitol dehydrogenase activity. Also provided in some embodiments
are expression vectors comprising a polynucleotide that encodes a
polypeptide possessing a xylulokinase activity. The polynucleotide
sometimes includes one or more substituted codons, and in some
embodiments, the one or more substituted codons are yeast codons
(e.g., some or all codons are optimized with yeast codons (e.g., S.
cerevisiae codons).
[0014] The polynucleotide sometimes includes a nucleotide sequence
of SEQ ID NO: 29, 30, 32 or 33, fragment thereof, or sequence
having 50% identity or greater (e.g., about 55, 60, 65, 70, 75, 80,
85, 90, 95% identity or greater) to the foregoing, and in certain
embodiments the polypeptide includes an amino acid sequence of SEQ
ID NO: 31, fragment thereof, or sequence having 75% identity or
greater (e.g., about 80, 85, 90, 95% identity or greater) to the
foregoing. In certain embodiments, a stretch of contiguous
nucleotides of the polynucleotide is from another organism, and
sometimes the stretch of contiguous nucleotides from the other
organism is from a nucleotide sequence that encodes a polypeptide
possessing a xylose isomerase activity. The other organism
sometimes is a fungus, such as a Piromyces fungus (e.g., Piromyces
strain E2 or another Piromyces strain) for example, and at times
the stretch of contiguous nucleotides from the other organism is
from SEQ ID NO: 34, or sequence having 50% identity or greater
(e.g., about 55, 60, 65, 70, 75, 80, 85, 90, 95% identity or
greater) to the foregoing. In some embodiments, the stretch of
contiguous nucleotides from the other organism encodes an amino
acid sequence from SEQ ID NO: 35, or sequence having 75% identity
or greater (e.g., about 80, 85, 90, 95% identity or greater) to the
foregoing. The stretch of contiguous nucleotides from the other
organism sometimes is about 1% to about 30% (e.g., about 2, 3, 4,
5, 6, 7, 8, 9, 10, 15, 20, 25%) of the total number of nucleotides
in the polynucleotide that encodes the polypeptide possessing
xylose isomerase activity. In some embodiments, about 30 contiguous
nucleotides from the polynucleotide from Ruminococcus are replaced
by about 10 to about 20 nucleotides from the other organism.
Sometimes, the contiguous stretch of polynucleotides from the other
organism is at the 5' end of the polynucleotide. In some
embodiments, the polynucleotide includes a nucleotide sequence of
SEQ ID NO: 55, 56, 57, 59 or 61, fragment thereof, or sequence
having 50% identity or greater (e.g., about 55, 60, 65, 70, 75, 80,
85, 90, 95% identity or greater) to the foregoing. The
polynucleotide sometimes encodes a polypeptide that includes an
amino acid sequence of SEQ ID NO: 58, 60 or 62, fragment thereof,
or sequence having 75% identity or greater (e.g., about 80, 85, 90,
95% identity or greater) to the foregoing. In some embodiments, the
polynucleotide comprises one or more point mutations, and sometimes
the point mutation is at a position corresponding to position 179
of the R. flavefaciens strain Siijpesteijn 1948 polypeptide having
xylose isomerase activity (e.g., the point mutation is a glycine
179 to alanine point mutation). In certain embodiments, an
expression vector includes a regulatory nucleotide sequence in
operable linkage with the polynucleotide. A regulatory nucleotide
sequence sometimes includes a promoter sequence (e.g., an inducible
promoter sequence, constitutively active promoter sequence. In
certain embodiments, provided are methods for preparing an
expression vector of any one of embodiments H1 to H24, comprising:
(i) providing a nucleic acid that contains a regulatory sequence,
and (ii) inserting the polynucleotide into the nucleic acid in
operable linkage with the regulatory sequence.
[0015] Thus, in some embodiments, provided are chimeric xylose
isomerase enzymes, and polynucleotides that encode them, which
include subsequences from two or more xylose isomerase donor
sequences. The xylose isomerase donor sequences sometimes are
naturally occurring native sequences from an organism, and
sometimes are modified sequences. In certain embodiments, a
subsequence from one donor may represent a majority of the chimeric
xylose isomerase sequence (e.g., about 55% to about 99% of the
chimeric xylose isomerase nucleotide or amino acid sequence (e.g.,
about 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98%) or all but 30 or fewer nucleotides
or amino acids of the chimeric sequence (e.g., all but about 25,
24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8,
7, 6, 5, 4, 3, 2 or 1 nucleotides or amino acids of the chimeric
molecule). In some embodiments, a subsequence from one donor may
represent a minority of the chimeric xylose isomerase sequence
(e.g., about 1% to about 45% of the chimeric xylose isomerase
nucleotide or amino acid sequence (e.g., about 40, 35, 30, 25, 24,
23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,
5, 4, 3, 2%) or about 1 to about 30 nucleotides or amino acids of
the chimeric sequence (e.g., about 25, 24, 23, 22, 21, 20, 19, 18,
17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
nucleotides or amino acids of the chimeric molecule). In some
embodiments, one or more donor sequences for a chimeric xylose
isomerase molecule are from a xylose isomerase described in the
following Table:
TABLE-US-00001 SEQ ID NO (nucleotide sequence/ Accession Number
amino (nucleotide sequence/ Organism Name (XI Donor) acid sequence)
amino acid sequence) Clostridiales_genomosp. 104 and YP_003474614.1
BVAB3 str UPII9-5 109/112 Ruminococcus flavefaciens 22 and 30/31
AJ132472/CAB51938.1 Ruminococcus_FD1 102 and ZP_06143883.1 107/110
Ruminococcus_18P13 103 and CBL17278.1 108/111 Thermus thermophilus
106 P26997.1 Bacillus stercoris 105 ZP_02435145.1 Clostridium
cellulolyticum 101 YP_002507697.1 Bacillus uniformis 100
ZP_02069286.1 Bacillus stearothermophilus 99 ABI49954.1 Bacteroides
thetaiotaomicron 98 NP_809706.1 Clostridium 97 P22842.1
thermohydrosulfuricum Orpinomyces sp. ukk1 96 ACA65427.1
Clostridium phytofermentans 95 YP_001558336.1 Escherichia coli 94
Piromyces strain E2 24 and 34 AJ249909/CAB76571.1 and 93/35
or a nucleotide sequence or amino acid sequence that is (a) about
80% or more identical to one of the foregoing sequences referenced
in the Table (e.g., about 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99% identical), and/or (b) has
about 1 to about 20 nucleotide or amino acid modifications (e.g.,
substitutions, deletions or insertions) relative to one of the
foregoing sequences referenced in the Table (e.g., about 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 nucleotide or
amino acid modifications). In certain embodiments, (a) the majority
of a chimeric xylose isomerase molecule is from a Ruminococcus
xylose isomerase described in the foregoing Table (e.g., about 80%
or more of the nucleotides or amino acids of the chimeric molecule
(e.g., about 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99% of the nucleotides or amino acids) or all
but about 30 of the nucleotides or amino acids in the chimeric
molecule (e.g., all but about 25, 24, 23, 22, 21, 20, 19, 18, 17,
16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides
or amino acids of the chimeric molecule)), and (b) a minority of
the chimeric xylose isomerase is from a xylose isomerase of another
organism (e.g., about 20% or fewer of the nucleotides or amino
acids of the chimeric molecule (e.g., about 19, 18, 17, 16, 15, 14,
13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of the nucleotides or
amino acids) or about 30 of the nucleotides or amino acids in the
chimeric molecule (e.g., about 25, 24, 23, 22, 21, 20, 19, 18, 17,
16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides
or amino acids of the chimeric molecule)). In the latter
embodiments, the minority of the chimeric xylose isomerase
sometimes is from a xylose isomerase referenced in the foregoing
Table, such as a xylose isomerase from the Piromyces strain, for
example. In some embodiments, a donor sequence includes one or more
nucleotide or amino acid mutations, examples of which are described
herein.
[0016] In some embodiments, provided are nucleic acids, including a
polynucleotide that includes a first stretch of contiguous nucleic
acids from a first organism and a second stretch of contiguous
nucleic acids from a second organism, where the polynucleotide
encodes a polypeptide possessing a xylose to xylulose xylose
isomerase activity. In certain embodiments, an expression vector,
comprising a polynucleotide that includes a first stretch of
contiguous expression vectors from a first organism and a second
stretch of contiguous expression vectors from a second organism,
where the polynucleotide encodes a polypeptide possessing a xylose
to xylulose, xylose isomerase activity. In some embodiments, the
first organism and the second organism are the same, and in certain
embodiments, the first organism and the second organism are
different. In some embodiments, the first stretch of contiguous
nucleotides and the second stretch of contiguous nucleotides
independently are selected from nucleotide sequence that encodes a
polypeptide having xylose isomerase activity. In certain
embodiments, the first organism is a bacterium. In some
embodiments, the bacterium is a Ruminococcus bacterium, and in
certain embodiments, the bacterium is a Ruminococcus flavefaciens
bacterium (e.g., Ruminococcus flavefaciens strain 17, Ruminococcus
flavefaciens strain Siijpesteijn 1948, Ruminococcus flavefaciens
strain FD1, Ruminococcus flavefaciens strain 18P13). In some
embodiments, the stretch of contiguous nucleotides is from SEQ ID
NO: 29, 30, 32, 33, or a sequence having 50% identity or greater
(e.g., about 55, 60, 65, 70, 75, 80, 85, 90, 95% identity or
greater) to the foregoing. In certain embodiments, the stretch of
contiguous nucleotides from the other organism encodes an amino
acid sequence from SEQ ID NO: 31, or a sequence having 75% identity
or greater (e.g., about 80, 85, 90, 95% identity or greater) to the
foregoing. In certain embodiments, the second organism is a fungus.
In some embodiments, the fungus is a Piromyces fungus, and in some
embodiments, the fungus is a Piromyces strain E2 fungus. In certain
embodiments, the stretch of contiguous nucleotides is from SEQ ID
NO: 34, or a sequence having 50% identity or greater (e.g., about
55, 60, 65, 70, 75, 80, 85, 90, 95% identity or greater) to the
foregoing. In some embodiments, the stretch of contiguous
nucleotides from the other organism encodes an amino acid sequence
from SEQ ID NO: 35, or a sequence having 75% identity or greater
(e.g., about 80, 85, 90, 95% identity or greater) to the foregoing.
In certain embodiments, the polynucleotide includes one or more
substituted codons. In some embodiments, the one or more
substituted codons are yeast codons. In certain embodiments, the
stretch of contiguous nucleotides from the first organism or second
organism is about 1% to about 30% (e.g., about 2, 3, 4, 5, 6, 7, 8,
9, 10, 15, 20, 25%) of the total number of nucleotides in the
polynucleotide that encodes the polypeptide possessing xylose
isomerase activity. In some embodiments, the stretch of contiguous
nucleotides from the second organism is about 1% to about 30%
(e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25) of the total
number of nucleotides in the polynucleotide, the polynucleotide
includes a nucleotide sequence of SEQ ID NO: 55, 56, 57, 59 or 61,
fragment thereof, or sequence having 50% identity or greater (e.g.,
about 55, 60, 65, 70, 75, 80, 85, 90, 95% identity or greater) to
the foregoing. In certain embodiments, the polynucleotide encodes a
polypeptide that includes an amino acid sequence of SEQ ID NO: 58,
60 or 62, fragment thereof, or sequence having 75% identity or
greater (e.g., about 80, 85, 90, 95% identity or greater) to the
foregoing. In some embodiments, the expression vector can include
one or more point mutations. In certain embodiments, the point
mutation is at a position corresponding to position 179 of R.
flavefaciens polypeptide having xylose isomerase activity. In some
embodiments, the point mutation is a glycine 179 to alanine point
mutation.
[0017] In certain embodiments, the microbes described herein can be
used in fermentation methods. In some embodiments, a method
includes, contacting a microbe described herein with a feedstock
comprising a five carbon molecule under conditions for generating
ethanol. In certain embodiments, the five carbon molecule includes
xylose. In some embodiments, about 15 grams per liter of ethanol,
or more, is generated within about 372 hours. In certain
embodiments, about 2.0 grams per liter dry cell weight, or more, is
generated within about 372 hours.
[0018] In some embodiments, provided are nucleic acids, including a
polynucleotide that includes a first stretch of contiguous nucleic
acids from a first organism and a second stretch of contiguous
nucleic acids from a second organism, where the polynucleotide
encodes a polypeptide possessing a phosphogluconate dehydratase
activity. In certain embodiments, an expression vector, comprising
a polynucleotide that includes a first stretch of contiguous
expression vectors from a first organism and a second stretch of
contiguous expression vectors from a second organism, where the
polynucleotide encodes a polypeptide possessing a phosphogluconate
dehydratase activity. In some embodiments, the first organism and
the second organism are the same, and in certain embodiments, the
first organism and the second organism are different. In some
embodiments, the first stretch of contiguous nucleotides and the
second stretch of contiguous nucleotides independently are selected
from nucleotide sequence that encodes a polypeptide having a
phosphogluconate dehydratase activity.
[0019] In some embodiments, provided are nucleic acids, including a
polynucleotide that includes a first stretch of contiguous nucleic
acids from a first organism and a second stretch of contiguous
nucleic acids from a second organism, where the polynucleotide
encodes a polypeptide possessing a
2-keto-3-deoxygluconate-6-phosphate aldolase activity. In certain
embodiments, an expression vector, comprising a polynucleotide that
includes a first stretch of contiguous expression vectors from a
first organism and a second stretch of contiguous expression
vectors from a second organism, where the polynucleotide encodes a
polypeptide possessing a 2-keto-3-deoxygluconate-6-phosphate
aldolase activity. In some embodiments, the first organism and the
second organism are the same, and in certain embodiments, the first
organism and the second organism are different. In some
embodiments, the first stretch of contiguous nucleotides and the
second stretch of contiguous nucleotides independently are selected
from nucleotide sequence that encodes a polypeptide having a
2-keto-3-deoxygluconate-6-phosphate aldolase activity.
[0020] In certain embodiments, the expression vector includes a
regulatory nucleotide sequence in operable linkage with the
polynucleotide. In some embodiments, the regulatory nucleotide
sequence comprises a promoter sequence. In certain embodiments, the
promoter sequence is an inducible promoter sequence. In some
embodiments, the promoter sequence is a constitutively active
promoter sequence. In certain embodiments, a method for preparing
an expression vector as described herein, includes (i) providing a
nucleic acid that contains a regulatory sequence, and (ii)
inserting the polynucleotide into the nucleic acid in operable
linkage with the regulatory sequence. In some embodiments, a
microbe as described herein includes the nucleic acid of anyone of
the foregoing embodiments. In certain embodiments, a microbe
includes an expression vector of any one of the foregoing
embodiments. In some embodiments, the microbe is a yeast. In
certain embodiments, the microbe is a Saccharomyces yeast, and in
some embodiments, the microbe is a Saccharomyces cerevisiae
yeast.
[0021] In various embodiments, provided herein is a nucleic acid
comprising polynucleotide subsequences that encode a
phosphogluconate dehydratase enzyme (e.g., EDD), a
2-keto-3-deoxygluconate-6-phosphate aldolase enzyme (e.g., EDA), a
transaldolase enzyme (e.g., TAD), a transketolase enzyme (e.g.,
TKL1, TKL2, or TKL1 and TKL2), a glucose-6-phosphate dehydrogenase
enzyme (e.g., ZWF1), a 6-phosphogluconolactonase enzyme (e.g.,
SOL3, SOL4, or SOL3 and SOL4) and a xylose isomerase enzyme or a
xylose reductase (XR) enzyme and a xylitol dehydrogenase (XD)
enzyme, and a xylulokinase (XK) enzyme. In some embodiments,
polynucleotide subsequences encoding the phosphogluconate
dehydratase enzyme and the 3-deoxygluconate-6-phosphate aldolase
enzyme independently are from an Escherichia spp. (e.g.,
Escherichia coli) or Pseudomonas spp. (e.g., Pseudomonas
aeruginosa), and in certain embodiments, the polynucleotide
encoding the phosphogluconate dehydratase enzyme and/or the
3-deoxygluconate-6-phosphate aldolase enzyme is a chimeric
polynucleotide that includes part of such a sequence and part of
another phosphogluconate dehydratase enzyme and the
3-deoxygluconate-6-phosphate aldolase enzyme sequence (e.g., from a
different organism). In certain embodiments, the polynucleotide
subsequence that encodes the xylose isomerase enzyme is from a
Ruminococcus spp. (e.g., Ruminococcus flavefaciens), and in some
embodiments, is a chimeric polynucleotide that includes part of
such a sequence and part of another xylose isomerase sequence
(e.g., from a Piromyces spp.). Non-limiting examples of xylose
isomerase chimeric sequences are described herein. In some
embodiments, a nucleic acid includes a polynucleotide subsequence
that encodes a glucose-6-phosphate dehydrogenase enzyme (e.g.,
ZWF1) and/or a polynucleotide subsequence that encodes a
6-phosphogluconolactonase enzyme (e.g., SOL3/SOL4). In certain
embodiments, the polynucleotide subsequences that encode the
glucose-6-phosphate dehydrogenase enzyme and the
6-phosphogluconolactonase enzyme are from a yeast, non-limiting
examples of which are Saccharomyces spp. (e.g., Saccharomyces
cerevisiae). In some embodiments, a nucleic acid includes a
polynucleotide subsequence that encodes a glucose transporter
(e.g., GAL2, GXS1, GXF1, HXT7). In certain embodiments, the
polynucleotide subsequence that encodes the glucose transporter is
from a yeast, non-limiting examples of which are Saccharomyces spp.
(e.g., Saccharomyces cerevisiae). In some embodiments, a nucleic
acid includes a polynucleotide subsequence that alters the activity
of 6-phosphogluconate dehydrogenase (decarboxylating) enzyme (e.g.,
GND1, GND2). In certain embodiments, the polynucleotide
subsequences that alter the activity of 6-phosphogluconate
dehydrogenase (decarboxylating) enzyme are from a yeast. In some
embodiments, a nucleic acid includes a polynucleotide subsequence
that decreases expression of, or disrupts, a 6-phosphogluconate
dehydrogenase (decarboxylating) enzyme. In some embodiments, a
nucleic acid includes a polynucleotide subsequence that disrupts a
phosphoglucose isomerase enzyme (e.g., PGI1). In some embodiments,
a nucleic acid includes a polynucleotide subsequence that decreases
expression of, or disrupts, a phosphoglucose isomerase enzyme. In
some embodiments, a nucleic acid includes a polynucleotide
subsequence that encodes a transaldolase enzyme (e.g., TAD). In
certain embodiments, the polynucleotide subsequences that encode
the transaldolase enzyme are from a yeast, non-limiting examples of
which are Kluyveromyces, Pichia, Escherichia, Bacillus,
Ruminococcus, Schizosaccharomyces, and Candida. In some
embodiments, a nucleic acid includes a polynucleotide subsequence
that decreases expression of, or disrupts, transaldolase enzyme. In
some embodiments, a nucleic acid includes a polynucleotide
subsequence that encodes a transketolase enzyme (e.g., TKL1, TKL2,
or TKL1 and TKL2). In certain embodiments, the polynucleotide
subsequences that encode the transketolase enzyme are from a yeast,
non-limiting examples of which are Kluyveromyces, Pichia,
Escherichia, Bacillus, Ruminococcus, Schizosaccharomyces, and
Candida. In some embodiments, a nucleic acid includes a
polynucleotide subsequence that decreases expression of, or
disrupts, transketolase enzyme.
[0022] In some embodiments, a nucleic acid includes one or more
promoters operable in a yeast (e.g., Saccharomyces spp. (e.g.,
Saccharomyces cerevisiae), and in operable connection with one or
more polynucleotide subsequences described above. Such promoters
often are constitutively active and sometimes are operable under
anaerobic and aerobic conditions. Non-limiting examples of
promoters include those that control glucose phosphate
dehydrogenase (GPD), translation elongation factor (TEF-1),
phosphoglucokinase (PGK-1) and triose phosphate dehydrogenase
(TDH-1). A nucleic acid can be one or two nucleic acids in some
embodiments, and each nucleic acid can include one or two or more
of the polynucleotide subsequences and or promoters described
above. A nucleic acid can be in circular (e.g., plasmid) or linear
form, in some embodiments, and sometimes functions as an expression
vector. In some embodiments, a nucleic acid functions as a tool for
integrating the polynucleotide subsequences, and optionally
promoter sequences, included in the nucleic acid, into genomic DNA
of a host organism.
[0023] In some embodiments, provided herein is an engineered
microbe comprising heterologous polynucleotide subsequences that
encode a phosphogluconate dehydratase enzyme (e.g., EDD), a
2-keto-3-deoxygluconate-6-phosphate aldolase enzyme (e.g., EDA), a
xylose isomerase enzyme or a xylose reductase (XR) enzyme and a
xylitol dehydrogenase (XD) enzyme, and a xylulokinase (XK) enzyme.
In certain embodiments, the microbe is a yeast, non-limiting
examples of which are Saccharomyces spp. (e.g., Saccharomyces
cerevisiae). In some embodiments, polynucleotide subsequences
encoding the phosphogluconate dehydratase enzyme and the
3-deoxygluconate-6-phosphate aldolase enzyme independently are from
an Escherichia spp. (e.g., Escherichia coli) or Pseudomonas spp.
(e.g., Pseudomonas aeruginosa), and in certain embodiments, the
polynucleotide encoding the phosphogluconate dehydratase enzyme
and/or the 3-deoxygluconate-6-phosphate aldolase enzyme is a
chimeric polynucleotide that includes part of such a sequence and
part of another phosphogluconate dehydratase enzyme and the
3-deoxygluconate-6-phosphate aldolase enzyme sequence (e.g., from a
different organism). In certain embodiments, the polynucleotide
subsequence that encodes the xylose isomerase enzyme is from a
Ruminococcus spp. (e.g., Ruminococcus flavefaciens), and in some
embodiments, is a chimeric polynucleotide that includes part of
such a sequence and part of another xylose isomerase sequence
(e.g., from a Piromyces spp.). Non-limiting examples of xylose
isomerase chimeric sequences are described herein. In some
embodiments, the engineered microbe expresses a glucose-6-phosphate
dehydrogenase enzyme (e.g., ZWF1) and/or a
6-phosphogluconolactonase enzyme (e.g., SOL3/SOL4). In certain
embodiments, the polynucleotide subsequences that encode the
glucose-6-phosphate dehydrogenase enzyme and the
6-phosphogluconolactonase enzyme are from a yeast, non-limiting
examples of which are Saccharomyces spp. (e.g., Saccharomyces
cerevisiae). In certain embodiments, the polynucleotide
subsequences that disrupt the 6-phosphogluconate dehydrogenase
(decarboxylating) enzyme are from a yeast. In some embodiments, a
nucleic acid includes a polynucleotide subsequence that decreases
expression of, or disrupts, a 6-phosphogluconate dehydrogenase
(decarboxylating) enzyme.
[0024] Thus, an engineered microbe sometimes expresses
higher-than-normal levels (e.g., over-express) of an endogenous
glucose-6-ph6-phosphogluconolactonase enzyme glucose-6-phosphate
dehydrogenase enzyme (e.g., under control of a constitutive
promoter, or multiple copies of the nucleotide subsequences that
encode such enzymes are inserted in the microbe). In some
embodiments, the engineered microbe includes a polynucleotide
subsequence that encodes a glucose transporter (e.g., GAL2, GSX1,
GXF1, HXT7). In certain embodiments, the polynucleotide subsequence
that encodes the glucose transporter is from a yeast, non-limiting
examples of which are Saccharomyces spp. (e.g., Saccharomyces
cerevisiae). Thus, an engineered microbe sometimes expresses
higher-than-normal levels (e.g., over-express) of one or more
endogenous glucose transport enzymes (e.g., under control of a
constitutive promoter, or multiple copies of the nucleotide
subsequences that encode such enzymes are inserted in the microbe).
In some embodiments, the engineered microbe includes a genetic
alteration that reduces the activity of an endogenous
phosphofructokinase enzyme activity. In certain embodiments, a
polynucleotide subsequence that encodes such an enzyme is altered
such that enzyme activity is significantly reduced or not
detectable in the engineered microbe. In some embodiments, a
nucleic acid includes a polynucleotide subsequence that alters the
activity of 6-phosphogluconate dehydrogenase (decarboxylating)
enzyme (e.g., GND1, GND2). In certain embodiments, the
polynucleotide subsequences that alter the activity of
6-phosphogluconate dehydrogenase (decarboxylating) enzyme are from
a yeast. In some embodiments, a nucleic acid includes a
polynucleotide subsequence that decreases expression of, or
disrupts, a 6-phosphogluconate dehydrogenase (decarboxylating)
enzyme. In some embodiments, a nucleic acid includes a
polynucleotide subsequence that alters a phosphoglucose isomerase
enzyme (e.g., PGI1) activity. In certain embodiments, the
polynucleotide subsequences that alter the phosphoglucose isomerase
enzyme are from a yeast. In some embodiments, a nucleic acid
includes a polynucleotide subsequence that decreases expression of,
or disrupts, a phosphoglucose isomerase enzyme. In some
embodiments, a nucleic acid includes a polynucleotide subsequence
that alters a transaldolase enzyme (e.g., TAL1). In certain
embodiments, the polynucleotide subsequences that alter the
transaldolase enzyme activity, increase the transaldolase activity,
and in some embodiments the polynucleotide sequences are from a
yeast, non-limiting examples of which are Kluyveromyces, Pichia,
Escherichia, Bacillus, Ruminococcus, Schizosaccharomyces, and
Candida. In certain embodiments, the polynucleotide subsequences
that alter transaldolase enzyme activity, decrease the
transaldolase activity. In some embodiments, a nucleic acid
includes a polynucleotide subsequence that decreases expression of,
or disrupts, transaldolase enzyme. In some embodiments, a nucleic
acid includes a polynucleotide subsequence that alters a
transketolase enzyme (e.g., TKL1, TKL2, or TKL1 and TKL2). In
certain embodiments, the polynucleotide subsequences that alter the
transketolase enzyme increase transketolase activity and in some
embodiments, the polynucleotide sequences are from a yeast,
non-limiting examples of which are Kluyveromyces, Pichia,
Escherichia, Bacillus, Ruminococcus, Schizosaccharomyces, and
Candida. In certain embodiments, the polynucleotide subsequences
that alter transketolase enzyme activity, decrease the
transketolase activity. In some embodiments, a nucleic acid
includes a polynucleotide subsequence that decreases expression of,
or disrupts, transketolase enzyme.
[0025] In some embodiments, the engineered microbe includes one or
more promoters operable in a yeast (e.g., Saccharomyces spp. (e.g.,
Saccharomyces cerevisiae), and in operable connection with one or
more polynucleotide subsequences described above. Such promoters
often are constitutively active and sometimes are operable under
anaerobic and aerobic conditions. Non-limiting examples of
promoters include those that control glucose phosphate
dehydrogenase (GPD), translation elongation factor (TEF-1),
phosphoglucokinase (PGK-1) and triose phosphate dehydrogenase
(TDH-1). The polynucleotide sequences and promoters described above
sometimes are non-stably associated with the microbe (e.g., they
are in a non-integrated nucleic acid (e.g., a plasmid), and in some
embodiments, are integrated in genomic DNA of the microbe. In some
embodiments, the polynucleotide sequences are integrated in a
transposition integration event, a homologous recombination
integration event or a transposition integration event and a
homologous recombination integration event. In some embodiments, a
transposition integration event includes transposition of an operon
comprising two or more of the polynucleotide subsequences and/or
promoters described above. In certain embodiments, a homologous
recombination integration event includes homologous recombination
of an operon comprising two or more of the polynucleotide
subsequences and or promoters described above. In certain
embodiments, provided are methods for producing xylulose and/or
ethanol using an engineered microbe described above, which comprise
contacting the engineered microbe with a medium (e.g., feedstock)
under conditions in which the microbe synthesizes xylulose and/or
ethanol. In some embodiments, the engineered microbe synthesizes
xylulose and/or ethanol to about 85% to about 99% of theoretical
yield (e.g., about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% of theoretical xylulose and/or ethanol
yield). In some embodiments, the medium (e.g., feedstock) contains
a six-carbon sugar (e.g., hexose, glucose) and/or a five-carbon
sugar (e.g., pentose, xylose). In certain embodiments, the ethanol
is separated and/or recovered from the engineered
microorganism.
[0026] Additional embodiments can be found in Example 42: Examples
of the embodiments. Certain embodiments are described further in
the following description, examples, claims and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The drawings illustrate embodiments of the technology and
are not limiting. For clarity and ease of illustration, the
drawings are not made to scale and, in some instances, various
aspects may be shown exaggerated or enlarged to facilitate an
understanding of particular embodiments.
[0028] FIG. 1 depicts a metabolic pathway that produces ethanol as
by product of cellular respiration. The solid lines represent
activities present in the Embden-Meyerhoff pathway (e.g., aerobic
respiration). Dashed lines represent activities associated with the
Entner-Doudoroff pathway (e.g., anaerobic respiration). One or both
pathways often can be operational in a microorganism. The level of
activity of each pathway can vary from organism to organism. The
arrow from FBP (e.g., Fructose-1,6-bisphosphate, also referred to
as F-1,6-BP) to G3P (e.g., glcyeraldehyde-3-phosphate), illustrates
wild type levels of conversion of FBP to two molecules of G3P. In
the embodiments shown in FIGS. 2, 3 and 5 a smaller arrow from FBP
to G3P is illustrated, indicating reduced or no conversion of FBP
to G3P. The reduction in conversion of FBP to G3P illustrated in
FIGS. 2, 3 and 5 is a result of the reduction or elimination of the
previous activity that converts fructose-6-phosphate (F6P) to FBP
(e.g., the activity of PFK).
[0029] FIG. 2 depicts an engineered metabolic pathway that can be
used to produce ethanol more efficiently in a host microorganism in
which the pathway has been engineered. The solid lines in FIGS. 2-5
represent the metabolic pathway naturally found in a host organism
(e.g., Saccharomyces cerevisiae, for example). The dashed lines in
FIGS. 2-5 represent a novel activity or pathway engineered into a
microorganism to allow increased ethanol production efficiency. In
FIG. 2 the activity of an enzyme in the Embden-Meyerhoff pathway,
phosphofructokinase (e.g., PFK) is permanently or temporarily
reduced or eliminated. The inactivation is shown as the "X" in FIG.
2. Disruption of the activity of PFK serves to inactivate the
Embden-Meyerhoff pathway (EM pathway). To allow cells to survive
with a non-functional PFK, two activities from the Entner-Doudoroff
pathway (ED pathway) have been introduced into a host organism
engineered with the reduced or non-functional EM pathway. The
introduced activities allow survival with an inactivated EM pathway
in addition to increased efficiency of ethanol production.
[0030] FIG. 3 depicts an engineered metabolic pathway that can be
used to produce ethanol using xylose as a carbon source by
introducing the activity into a microorganism. The engineered
microorganism can convert xylose to xylulose in a single reaction
using the introduced xylose isomerase activity. Xylose also can be
metabolized by the combined activities of xylose reductase, and
xylitol dehydrogenase, as depicted in FIG. 20. Xylulose then can be
fermented to ethanol by entering the EM pathway. Engineered
microorganisms also can use the increased efficiency of ethanol
production associated with inactivation of the EM pathway and
introduction of activities of the ED pathway, shown in FIG. 2 and
discussed below. The ability to utilize xylose efficiently (e.g.,
concurrently with six-carbon sugars or prior to the depletion of
six-carbon sugars) can be provided by the introduction of the novel
activities, xylose isomerase, or xylose reductase and xylitol
dehydrogenase.
[0031] FIG. 4 depicts an engineered metabolic pathway that can be
used to increase the efficiency of ethanol production (and other
products) by introducing the ability to fix atmospheric carbon
dioxide into a microorganism. The engineered microorganism can
incorporate or fix atmospheric carbon dioxide into organic
molecules using the introduced phosphoenolpyruvate carboxylase
activity. Carbon dioxide incorporated in this manner can be used as
an additional carbon source that can increase production of many
organic molecules, including ethanol. Non-limiting examples of
other products whose production can benefit from carbon fixation
include; pyruvate, oxaloacetate, glyceraldehyde-3-phosphate and the
like. The pathway depicted in FIG. 4 illustrates the introduction
of the novel carbon dioxide fixation activity in the background of
a fully functional EM pathway, and an introduced ED pathway. It is
understood the introduction of the carbon fixation activity can
benefit microorganisms that have no other modifications to any
metabolic pathways. It also is understood that microorganism
modified in one, or multiple, other metabolic pathways can benefit
from the introduction of a carbon fixation activity.
[0032] FIG. 5 shows a combination of some engineered metabolic
pathways described herein. The combination of engineered metabolic
pathways shown in FIG. 5 can provide significant increases in the
production of ethanol (or other products) when compared to the wild
type organism or organisms lacking one, two, three or more of the
modifications. Other combinations of engineered metabolic pathways
not shown in FIG. 5 are possible, including but not limited to,
combinations including increased alcohol tolerance, modified
alcohol dehydrogenase 2 activity and/or modified thymidylate
synthase activity, as described herein. Therefore, FIG. 5 also
illustrates an embodiment of a method for generating an engineered
microorganism with the ability to produce a greater amount of
target product comprising expressing one or more genetically
modified activities, described herein, in a host organism that
produces the desired target (e.g., ethanol, pyruvate, oxaloacetate
and the like, for example) via one or more metabolic pathways. In
some embodiments, the combination of metabolic pathways includes
those depicted in FIG. 5 in addition to combinations including one,
two or three of the following activities; increased alcohol
tolerance, modified alcohol dehydrogenase 2 activity and modified
thymidylate synthase activity.
[0033] FIG. 6 shows DNA and amino acid sequence alignments for the
nucleotide sequences of EDA (FIG. 6A, 6B) (SEQ ID NOS 670-677,
respectively, in order of appearance) and EDD (FIG. 6C, 6D) (SEQ ID
NOS 678-685, respectively, in order of appearance) genes from
Zymomonas mobilis (native and optimized) and Escherichia coli. FIG.
7 shows a representative western blot used to detect the presence
of an enzyme associated with an activity described herein.
[0034] FIG. 8 illustrates schematic representations of native,
modified and chimeric xylose isomerase genes.
[0035] FIG. 9 shows a representative Western blot used to detect
gene products.
[0036] FIG. 10 graphically illustrates a comparison of specific
activities of engineered mutant xylose isomerase enzymes. Results
are presented as percent activity over wild type (WT) activity.
Experimental details and results of the kinetic assays are present
in Example 12.
[0037] FIG. 11 illustrates comparative growth analysis results of
yeast strains carrying vector only or a vector containing native
Ruminococcus xylose isomerase, grown on media containing xylose.
Experimental details and results of the growth assays are described
in Example 13.
[0038] FIG. 12 illustrates comparative growth analysis results and
measurement of ethanol production in yeast strains carrying vector
only or a vector containing native Ruminococcus xylose isomerase.
Growth of cells is shown by the lines connected by "diamonds"
(vector with xylose isomerase) or "squares" (vector only). Ethanol
production is shown by the lines connected by "x's" (vector with
xylose isomerase) or "circles" (vector only). Experimental
conditions and results are described in Example 13.
[0039] FIGS. 13A and 13B show representative Western blots used to
detect levels of various exogenous EDD and EDA gene combinations
expressed in a host organism. Experimental conditions and results
are described in Example 17. FIG. 14 graphically displays the
relative activities of the various EDD/EDA combinations generated
as described in Example 18.
[0040] FIG. 15 graphically represents the fermentation efficiency
of engineered yeast strains carrying exogenous EDD/EDA gene
combinations. Vector=p426GPD/p425GPD; EE=EDD-E. coli/EDA-E. coli,
EP=EDD-E. coli/EDA-PAO1; PE=EDD=PAO1/EDA-E. coli,
PP=EDD=PAO1/EDA-PAO1. Experimental conditions and results are
described in Example 19. FIGS. 16A and 16B graphically illustrate
fermentation data (e.g., cell growth, glucose usage and ethanol
production) for engineered yeast strains generated as described
herein. FIG. 16A illustrates the fermentation data for engineered
strain BF428 (BY4742 with vector controls), and FIG. 16B
illustrates the fermentation data for engineered strain BF591
(BY4742 with EDD-PAO1/EDA-PAO1). Experimental conditions and
results are described in Example 20.
[0041] FIGS. 17A and 17B graphically illustrate fermentation data
for engineered yeast strains described herein. FIG. 17A illustrates
the fermentation data for engineered strain BF738 (BY4742 tal1 with
vector controls p426GPD and p425GPD). FIG. 17B illustrates the
fermentation data for engineered strain BF741 (BY4742 tal1 with
plasmids pBF290 (EDD-PAO1) and pBF292 (EDA-PAO1). Experimental
conditions and results are described in Example 21.
[0042] FIGS. 18A and 18B graphically illustrate fermentation data
for engineered yeast strains as described herein. FIG. 18A
illustrates the fermentation data for BF740 grown on 2% dextrose,
and FIG. 18B illustrates the fermentation data for BF743 grown on
2% dextrose. Strain descriptions, experimental conditions and
results are described in Example 22. FIG. 19 graphically
illustrates the results of coupled assay kinetics for single
plasmid and two plasmid edd/eda expression vector systems. Vector
construction and experimental conditions are described in Example
24.
[0043] FIG. 20 depicts an engineered metabolic pathway that can be
used to produce ethanol using xylose as a carbon source by
introducing the activity into a microorganism. The engineered
microorganism can convert xylose to xylulose by the activities of
xylose reductase and xylitol dehydrogenase. Xylose also can be
metabolized by the combined activities of xylose reductase, and
xylitol dehydrogenase, as depicted in FIG. 20. Xylulose then can be
fermented to ethanol by entering the EM pathway. Engineered
microorganisms also can use the increased efficiency of ethanol
production associated with inactivation of the EM pathway and
introduction of activities of the ED pathway, shown in FIG. 2 and
discussed below. The ability to utilize xylose efficiently (e.g.,
concurrently with six-carbon sugars or prior to the depletion of
six-carbon sugars) can be provided by the introduction of the novel
activities, xylose isomerase, or xylose reductase and xylitol
dehydrogenase.
[0044] FIG. 21 graphically illustrates the results of xylose
isomerase chimera generated with various 5' edge sequences.
Experimental methods and results are described in Example 28. FIG.
22 shows the results of western blots performed on xylose isomerase
chimera generated with various 5' edge sequences. Experimental
methods and results are described in Example 28.
[0045] FIG. 23 shows a western blot of E. coli crude extract
illustrated the presence of the EDD protein at the expected size.
Lane 1 is a standard size ladder (Novex Sharp standard), Lane 2 is
1 .mu.g BF1055 cell lysate, Lane 3 is 10 .mu.g BF1055 cell lysate,
Lane 4 is 1.5 .mu.g BF1706 cell lysate, Lane 5 is 15 .mu.g BF1706
cell lysate. Experimental methods and results are described in
Example 34. FIG. 24 graphically illustrates the results of activity
evaluations of EDA genes expressed in yeast. Experimental methods
and results are described in Example 34. FIG. 25 graphically
illustrates the specific activity of various xylose isomerase
candidate activities. Experimental methods and results are
described in Example 41.
DETAILED DESCRIPTION
[0046] Ethanol is a two carbon, straight chain, primary alcohol
that can be produced from fermentation (e.g., cellular respiration
processes) or as a by-product of petroleum refining. Ethanol has
widespread use in medicine, consumables, and in industrial
processes where it often is used as an essential solvent and a
precursor, or feedstock, for the synthesis of other products (e.g.,
ethyl halides, ethyl esters, diethyl ether, acetic acid, ethyl
amines and to a lesser extent butadiene, for example). The largest
use of ethanol, worldwide, is as a motor fuel and fuel additive.
Greater than 90% of the cars produced world wide can run
efficiently on hydrous ethanol (e.g., 95% ethanol and 5% water).
Ethanol also is commonly used for production of heat and light.
[0047] World production of ethanol exceeds 50 gigaliters (e.g.,
1.3.times.10.sup.10 US gallons), with 69% of the world supply
coming from Brazil and the United States. The United States fuel
ethanol industry is based largely on corn biomass. The use of corn
biomass for ethanol production may not yield a positive net energy
gain, and further has the potential of diverting land that could be
used for food production into ethanol production. It is possible
that cellulosic crops may displace corn as the main fuel crop for
producing bio-ethanol. Non-limiting examples of cellulosic crops
and waste materials include switchgrass and wood pulp waste from
paper production and wood milling industries.
[0048] Biomass produced in the paper pulping and wood milling
industries contains both 5 and six-carbon sugars. Use of this
wasted biomass could allow production of significant amounts of
bio-fuels and products, while reducing the use of land that could
be used for food production. Predominant forms of sugars in the
biomass produced in wood and paper pulping and wood milling
industries are glucose and xylose.
[0049] Provided herein are methods for producing ethanol, ethanol
derivatives and/or conjugates and other organic chemical
intermediates (e.g., pyruvate, acetaldehyde,
glyceraldehyde-3-phospate, and the like) using biological systems.
Such production systems may have significantly less environmental
impact and could be economically competitive with current
manufacturing systems. Thus, provided herein are methods for
manufacturing ethanol and other organic chemical intermediates by
engineered microorganisms. In some embodiments microorganisms are
engineered to contain at least one heterologous gene encoding an
enzyme, where the enzyme is a member of a novel pathway engineered
into the microorganism. In certain embodiments, an organism may be
selected for elevated activity of a native enzyme.
[0050] Genetically engineered microorganisms described herein
produce organic molecules for industrial uses. The organisms are
designed to be "feedstock flexible" in that they can use
five-carbon sugars (e.g., pentose sugars such as xylose, for
example), six-carbon sugars (e.g., hexose sugars such as glucose or
fructose, for example) or both as carbon sources. Further, the
organisms described herein have been designed to be highly
efficient in their use of hexose sugars to produce desired organic
molecules. To that end, the microorganisms described herein are
"pathway flexible" such that the microorganisms are able to direct
hexose sugars primarily to either (i) the traditional glycolysis
pathway (the Embden-Meyerhoff pathway) thereby generating ATP
energy for cell growth and division at certain times, or (ii) a
separate glycolytic pathway (the Entner-Doudoroff pathway) thereby
producing significant levels of pyruvic acid, a key 3-carbon
intermediate for producing many desired industrial organic
molecules.
[0051] Pathway selection in the microorganism can be directed via
one or more environmental switches such as a temperature change,
oxygen level change, addition or subtraction of a component of the
culture medium, or combinations thereof. The metabolic pathway
flexibility of microorganisms described herein allow the
microorganisms to efficiently use hexose sugars, which ultimately
can lead to microorganisms capable of producing a greater amount of
industrial chemical product per gram of feedstock as compared with
conventional microorganisms (e.g., the organism from which the
engineered organism was generated, for example). In some
embodiments, the metabolic pathway flexibility of the engineered
microorganisms described herein is generated by adding or
increasing metabolic activities associated with the
Entner-Doudoroff pathway. In certain embodiments the metabolic
activities added are phosphogluconate dehydratase (e.g., EDD gene),
2-keto-3-deoxygluconate-6-phosphate aldolase (e.g., EDA gene) or
both.
[0052] A number of industrially useful microorganisms (e.g.,
microorganisms used in fermentation processes, yeast for example),
metabolize xylose inefficiently or are incapable of metabolizing
xylose. Many organisms that can metabolize xylose do so only after
all glucose and/or other six-carbon sugars have been depleted. The
microorganisms described herein have been engineered to efficiently
utilize five-carbon sugars (e.g., xylose, for example) as an
alternative or additional source of carbon, concurrently with
and/or prior to six-carbon sugar usage, by the incorporation of a
heterologous nucleic acid (e.g., gene) encoding a xylose isomerase,
in some embodiments, and in certain embodiments, by the
incorporation of a heterologous nucleic acid encoding a xylose
reductase and a xylitol dehydrogenase. Xylose isomerase converts
the five-carbon sugar xylose to xylulose, in some embodiments. In
certain embodiments, xylose reductase and xylitol dehydrogenase
convert xylose to xylulose. Xylulose can ultimately be converted to
pyruvic acid or to ethanol through metabolism via the
Embden-Meyerhoff or Entner-Doudoroff pathways.
[0053] Many non-photosynthetic organisms are not capable of
incorporating inorganic atmospheric carbon into organic carbon
compounds, via carbon fixation pathways, to any appreciable degree,
or at all. Often, microorganisms used in industrial fermentation
process also are incapable of significant carbon fixation. The
ability to incorporate atmospheric carbon dioxide, or carbon
dioxide waste from respiration in fermentation processes, can
increase the amount of industrial chemical product produced per
gram of feedstock, in certain embodiments. Thus, the microorganisms
described herein also can be modified to add or increase the
ability to incorporate carbon from carbon dioxide into industrial
chemical products, in some embodiments. In certain embodiments, the
microorganisms described herein are engineered to express enzymes
such as phosphoenolpyruvate carboxylase ("PEP" carboxylase) and/or
ribulose 1,5-bis-phosphate carboxylase ("Rubisco"), thus allowing
the use of carbon dioxide as an additional source of carbon.
[0054] A particularly useful industrial chemical product produced
by fermentation is ethanol. Ethanol is an end product of cellular
respiration and is produced from acetaldehyde by an alcohol
dehydrogenase activity (e.g., by an enzyme like alcohol
dehydrogenase 1 or ADH1, for example). However, ethanol can readily
be converted back to acetaldehyde by the action of the enzyme
alcohol dehydrogenase 2 (e.g., ADH2), thus lowering the yield of
ethanol produced. In some embodiments, microorganisms described
herein are modified to reduce or eliminate the activity of ADH2, to
allow increased yields of ethanol. In certain embodiments, the
engineered microorganisms described herein also are modified to
have a higher tolerance to alcohol, thus enabling even higher
yields of alcohol as a fermentation product without inhibition of
cellular processes due to increased levels of alcohol in the growth
medium.
Microorganisms
[0055] A microorganism selected often is suitable for genetic
manipulation and often can be cultured at cell densities useful for
industrial production of a target product. A microorganism selected
often can be maintained in a fermentation device.
[0056] The term "engineered microorganism" as used herein refers to
a modified microorganism that includes one or more activities
distinct from an activity present in a microorganism utilized as a
starting point (hereafter a "host microorganism"). An engineered
microorganism includes a heterologous polynucleotide in some
embodiments, and in certain embodiments, an engineered organism has
been subjected to selective conditions that alter an activity, or
introduce an activity, relative to the host microorganism. Thus, an
engineered microorganism has been altered directly or indirectly by
a human being. A host microorganism sometimes is a native
microorganism, and at times is a microorganism that has been
engineered to a certain point.
[0057] In some embodiments an engineered microorganism is a single
cell organism, often capable of dividing and proliferating. A
microorganism can include one or more of the following features:
aerobe, anaerobe, filamentous, non-filamentous, monoploid, dipoid,
auxotrophic and/or non-auxotrophic. In certain embodiments, an
engineered microorganism is a prokaryotic microorganism (e.g.,
bacterium), and in certain embodiments, an engineered microorganism
is a non-prokaryotic microorganism. In some embodiments, an
engineered microorganism is a eukaryotic microorganism (e.g.,
yeast, fungi, amoeba).
[0058] Any suitable yeast may be selected as a host microorganism,
engineered microorganism or source for a heterologous
polynucleotide. Yeast include, but are not limited to, Yarrowia
yeast (e.g., Y. lipolytica (formerly classified as Candida
lipolytica)), Candida yeast (e.g., C. revkaufi, C. pulcherrima, C.
tropicalis, C. utilis), Rhodotorula yeast (e.g., R. glutinus, R.
graminis), Rhodosporidium yeast (e.g., R. toruloides),
Saccharomyces yeast (e.g., S. cerevisiae, S. bayanus, S.
pastorianus, S. carlsbergensis), Cryptococcus yeast, Trichosporon
yeast (e.g., T. pullans, T. cutaneum), Pichia yeast (e.g., P.
pastoris) and Lipomyces yeast (e.g., L. starkeyii, L. lipoferus).
In some embodiments, a yeast is a S. cerevisiae strain including,
but not limited to, YGR240CBY4742 (ATCC accession number 4015893)
and BY4742 (ATCC accession number 201389). In some embodiments, a
yeast is a Y. lipolytica strain that includes, but is not limited
to, ATCC20362, ATCC8862, ATCC18944, ATCC20228, ATCC76982 and LGAM
S(7)1 strains (Papanikolaou S., and Aggelis G., Bioresour. Technol.
82(1):43-9 (2002)). In certain embodiments, a yeast is a C.
tropicalis strain that includes, but is not limited to, ATCC20336,
ATCC20913, SU-2 (ura3-/ura3-), ATCC20962, H5343 (beta oxidation
blocked; U.S. Pat. No. 5,648,247) strains.
[0059] Any suitable fungus may be selected as a host microorganism,
engineered microorganism or source for a heterologous
polynucleotide. Non-limiting examples of fungi include, but are not
limited to, Aspergillus fungi (e.g., A. parasiticus, A. nidulans),
Thraustochytrium fungi, Schizochytrium fungi and Rhizopus fungi
(e.g., R. arrhizus, R. oryzae, R. nigricans), Orpinomyces or
Piromyces. In some embodiments, a fungus is an A. parasiticus
strain that includes, but is not limited to, strain ATCC24690, and
in certain embodiments, a fungus is an A. nidulans strain that
includes, but is not limited to, strain ATCC38163.
[0060] Any suitable prokaryote may be selected as a host
microorganism, engineered microorganism or source for a
heterologous polynucleotide. A Gram negative or Gram positive
bacteria may be selected. Examples of bacteria include, but are not
limited to, Bacillus bacteria (e.g., B. subtilis, B. megaterium, B.
stearothermophilus), Bacteroides bacteria (e.g., Bacteroides
uniformis, Bacteroides thetaiotaomicron), Clostridium bacteria
(e.g., C. phytofermentans, C. thermohydrosulfuricum, C.
cellulyticum (H10)), Acinetobacter bacteria, Norcardia baceteria,
Lactobacillus bacterial (e.g., Lactobacillus pentosus),
Xanthobacter bacteria, Escherichia bacteria (e.g., E. coli (e.g.,
strains DH10B, Stb12, DH5-alpha, DB3, DB3.1), DB4, DB5, JDP682 and
ccdA-over (e.g., U.S. application Ser. No. 09/518,188))),
Streptomyces bacteria (e.g., Streptomyces rubiginosus, Streptomyces
murinus), Erwinia bacteria, Klebsiella bacteria, Serratia bacteria
(e.g., S. marcessans), Pseudomonas bacteria (e.g., P. aeruginosa),
Salmonella bacteria (e.g., S. typhimurium, S. typhi), Thermus
bacteria (e.g., Thermus thermophilus), and Thermotoga bacteria
(e.g., Thermotoga maritiima, Thermotoga neopolitana) and
Ruminococcus (e.g., Ruminococcus environmental samples,
Ruminococcus albus, Ruminococcus bromii, Ruminococcus callidus,
Ruminococcus flavefaciens, Ruminococcus gauvreauii, Ruminococcus
gnavus, Ruminococcus lactaris, Ruminococcus obeum, Ruminococcus
sp., Ruminococcus sp. 14531, Ruminococcus sp. 15975, Ruminococcus
sp. 16442, Ruminococcus sp. 18P13, Ruminococcus sp. 25F6,
Ruminococcus sp. 25F7, Ruminococcus sp. 25F8, Ruminococcus sp.
4.sub.--1.sub.--47FAA, Ruminococcus sp. 5, Ruminococcus sp.
5.sub.--1.sub.--39BFAA, Ruminococcus sp. 7L75, Ruminococcus sp.
8.sub.--1.sub.--37FAA, Ruminococcus sp. 9SE51, Ruminococcus sp.
C36, Ruminococcus sp. CB10, Ruminococcus sp. CB3, Ruminococcus sp.
CCUG 37327 A, Ruminococcus sp. CE2, Ruminococcus sp. CJ60,
Ruminococcus sp. CJ63, Ruminococcus sp. CO1, Ruminococcus sp. CO12,
Ruminococcus sp. CO22, Ruminococcus sp. CO27, Ruminococcus sp.
CO28, Ruminococcus sp. CO34, Ruminococcus sp. CO41, Ruminococcus
sp. CO47, Ruminococcus sp. CO7, Ruminococcus sp. CS1, Ruminococcus
sp. CS6, Ruminococcus sp. DJF_VR52, Ruminococcus sp. DJF_VR66,
Ruminococcus sp. DJF_VR67, Ruminococcus sp. DJF_VR70k1,
Ruminococcus sp. DJF_VR87, Ruminococcus sp. Eg2, Ruminococcus sp.
Egf, Ruminococcus sp. END-1, Ruminococcus sp. FD1, Ruminococcus sp.
GM2/1, Ruminococcus sp. ID1, Ruminococcus sp. ID8, Ruminococcus sp.
K-1, Ruminococcus sp. KKA Seq234, Ruminococcus sp. M-1,
Ruminococcus sp. M10, Ruminococcus sp. M22, Ruminococcus sp. M23,
Ruminococcus sp. M6, Ruminococcus sp. M73, Ruminococcus sp. M76,
Ruminococcus sp. MLG080-3, Ruminococcus sp. NML 00-0124,
Ruminococcus sp. Pei041, Ruminococcus sp. SC101, Ruminococcus sp.
SC103, Ruminococcus sp. Siijpesteijn 1948, Ruminococcus sp. WAL
17306, Ruminococcus sp. YE281, Ruminococcus sp. YE58, Ruminococcus
sp. YE71, Ruminococcus sp. ZS2-15, Ruminococcus torques). Bacteria
also include, but are not limited to, photosynthetic bacteria
(e.g., green non-sulfur bacteria (e.g., Choroflexus bacteria (e.g.,
C. aurantiacus), Chloronema bacteria (e.g., C. gigateum)), green
sulfur bacteria (e.g., Chlorobium bacteria (e.g., C. limicola),
Pelodictyon bacteria (e.g., P. luteolum), purple sulfur bacteria
(e.g., Chromatium bacteria (e.g., C. okenii)), and purple
non-sulfur bacteria (e.g., Rhodospirillum bacteria (e.g., R.
rubrum), Rhodobacter bacteria (e.g., R. sphaeroides, R.
capsulatus), and Rhodomicrobium bacteria (e.g., R. vanellii)).
[0061] Cells from non-microbial organisms can be utilized as a host
microorganism, engineered microorganism or source for a
heterologous polynucleotide. Examples of such cells, include, but
are not limited to, insect cells (e.g., Drosophila (e.g., D.
melanogaster), Spodoptera (e.g., S. frugiperda Sf9 or Sf21 cells)
and Trichoplusa (e.g., High-Five cells); nematode cells (e.g., C.
elegans cells); avian cells; amphibian cells (e.g., Xenopus laevis
cells); reptilian cells; and mammalian cells (e.g., NIH3T3, 293,
CHO, COS, VERO, C127, BHK, Per-C6, Bowes melanoma and HeLa
cells).
[0062] Microorganisms or cells used as host organisms or source for
a heterologous polynucleotide are commercially available.
Microorganisms and cells described herein, and other suitable
microorganisms and cells are available, for example, from
Invitrogen Corporation, (Carlsbad, Calif.), American Type Culture
Collection (Manassas, Va.), and Agricultural Research Culture
Collection (NRRL; Peoria, Ill.).
[0063] Host microorganisms and engineered microorganisms may be
provided in any suitable form. For example, such microorganisms may
be provided in liquid culture or solid culture (e.g., agar-based
medium), which may be a primary culture or may have been passaged
(e.g., diluted and cultured) one or more times. Microorganisms also
may be provided in frozen form or dry form (e.g., lyophilized).
Microorganisms may be provided at any suitable concentration.
Six-Carbon Sugar Metabolism and Activities
[0064] Six-carbon or hexose sugars can be metabolized using one of
two pathways in many organisms. One pathway, the Embden-Meyerhoff
pathway (EM pathway), operates primarily under aerobic (e.g.,
oxygen rich) conditions. The other pathway, the Entner-Doudoroff
pathway (ED pathway), operates primarily under anaerobic (e.g.,
oxygen poor) conditions, producing pyruvate that can be converted
to lactic acid. Lactic acid can be further metabolized upon a
return to appropriate conditions. The EM pathway produces two ATP
for each six-carbon sugar metabolized, as compared to one ATP
produced for each six-carbon sugar metabolized in the ED pathway.
Thus the ED pathway yields ethanol more efficiently than the EM
pathway with respect to a given amount of input carbon, as seen by
the lower net energy yield. However, yeast preferentially use the
EM pathway for metabolism of six-carbon sugars, thereby
preferentially using the pathway that yields more energy and less
desired product.
[0065] The following steps and enzymatic activities metabolize
six-carbon sugars via the EM pathway. Six-carbon sugars (glucose,
sucrose, fructose, hexose and the like) are converted to
glucose-6-phosphate by hexokinase or glucokinase (e.g., HXK or GLK,
respectively). Glucose-6-phosphate can be converted to
fructose-6-phosphate by phosphoglucoisomerase (e.g., PGI).
Fructose-6-phosphate can be converted to fructose-1,6-bisphosphate
by phosphofructokinase (e.g., PFK). Fructose-1,6-bisphosphate
(F1,6BP) represents a key intermediate in the metabolism of
six-carbon sugars, as the next enzymatic reaction converts the
six-carbon sugar into two 3 carbon sugars. The reaction is
catalyzed by fructose bisphosphate aldolase and yields a mixture of
dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate
(G-3-P). The mixture of the two 3 carbon sugars is preferentially
converted to glyceraldehyde-3-phosphate by the action of
triosephosphate isomerase. G-3-P is converted is converted to
1,3-diphosphoglycerate (1,3-DPG) by glyceraldehyde-3-phosphate
dehydrogenase (GLD). 1,3-DPG is converted to 3-phosphoglycerate
(3-P-G by phosphoglycerate kinase (PGK). 3-P-G is converted to
2-phosphoglycerate (2-P-G) by phophoglycero mutase (GPM). 2-P-G is
converted to phosphoenolpyruvate (PEP) by enolase (ENO). PEP is
converted to pyruvate (PYR) by pyruvate kinase (PYK). PYR is
converted to acetaldehyde by pyruvate dicarboxylase (PDC).
Acetaldehyde is converted to ethanol by alcohol dehydrogenase 1
(ADH1).
[0066] Many enzymes in the EM pathway are reversible. The enzymes
in the EM pathway that are not reversible, and provide a useful
activity with which to control six-carbon sugar metabolism, via the
EM pathway, include, but are not limited to phosphofructokinase and
alcohol dehydrogenase. In some embodiments, reducing or eliminating
the activity of phosphofructokinase may inactivate the EM pathway.
Engineering microorganisms with modified activities in PFK and/or
ADH may yield increased product output as compared to organisms
with the wild type activities, in certain embodiments. In some
embodiments, modifying a reverse activity (e.g., the enzyme
responsible for catalyzing the reverse activity of ADH, for
example) may also yield an increase in product yield by reducing or
eliminating the back conversion of products by the backwards
reaction. The activity which catalyzes the conversion of ethanol to
acetaldehyde is alcohol dehydrogenase 2 (ADH2). Reducing or
eliminating the activity of ADH2 can increase the yield of ethanol
per unit of carbon input due to the inactivation of the conversion
of ethanol to acetaldehyde, in certain embodiments. In addition to
enzyme activities that are not reversible, certain reversible
activities also can be used to control six-carbon sugar metabolism
via the EM pathway, in some embodiments. A non-limiting example of
a reversible enzymatic activity that can be utilized to control
six-carbon sugar metabolism includes phosphoglucose isomerase
(PGI).
[0067] A microorganism may be engineered to include or regulate one
or more activities in the Embden-Meyerhoff pathway, for example. In
some embodiments, one or more of these activities may be altered
such that the activity or activities can be increased or decreased
according to a change in environmental conditions. In certain
embodiments, one or more of the activities (e.g., PGI, PFK or ADH2)
can be altered to allow regulated control and an alternative
pathway for more efficient carbon metabolism can be provided (e.g.,
one or more activities from the ED pathway, for example). An
engineered organism with the EM pathway under regulatable control
and a novel or enhanced ED pathway would be useful for producing
significantly more ethanol or other end product from a given amount
of input feedstock. The term "activity" as used herein refers to
the functioning of a microorganism's natural or engineered
biological pathways to yield various products including ethanol and
its precursors. Ethanol (or other product) producing activity can
be provided by any non-mammalian source in certain embodiments.
Such sources include, without limitation, eukaryotes such as yeast
and fungi and prokaryotes such as bacteria. In some embodiments,
the activity of one or more (e.g., 2, 3, 4, 5 or more) pathway
members in an EM pathway is reduced or removed to undetectable
levels.
[0068] An engineered microorganism may, in some embodiments,
preferentially metabolize six-carbon sugars via the ED pathway as
opposed to the EM pathway under certain conditions. Such engineered
microorganisms may metabolize about 60% or more of the available
six-carbon sugars via the ED pathway (e.g., about 62%, 64%, 66%,
68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or greater than any one
of the foregoing), and such fraction of the available six-carbon
sugars are not metabolized by the EM pathway, under certain
conditions. A microorganism may metabolize six-carbon sugars
substantially via the ED pathway, and not the EM pathway, in
certain embodiments (e.g., 99% or greater, or 100%, of the
available six-carbon sugars are metabolized via the ED pathway). A
six-carbon sugar is deemed as being metabolized via a particular
pathway when the sugar is converted to end metabolites of the
pathway, and not intermediate metabolites only, of the particular
pathway. A microorganism may preferentially metabolize certain
sugars under the ED pathway after a certain time after the
microorganism is exposed to a certain set of conditions (e.g.,
there may be a time delay after a microorganism is exposed to a
certain set of conditions before the microorganism preferentially
metabolizes sugars by the ED pathway). Certain novel activities
involved in the metabolism of six-carbon sugars by the ED pathway
can be engineered into a desired yeast strain to increase the
efficiency of ethanol (or other products) production. Yeast do not
have an activity that converts 6-phophogluconate to
2-keto-3-deoxy-6-p-gluconate or an activity that converts
2-keto-3-deoxy-6-p-gluconate to pyruvate. Addition of these
activities to engineered yeast can allow the engineered
microorganisms to increase fermentation efficiency by allowing
yeast to ferment ethanol under anaerobic condition without having
to use the EM pathway and expend additional energy. Therefore, by
providing novel activities associated with converting
6-phophogluconate to 2-keto-3-deoxy-6-p-gluconate and
2-keto-3-deoxy-6-p-gluconate to pyruvate, the engineered
microorganism can benefit by producing ethanol more efficiently,
with respect to a given amount of input carbon, than by using the
native EM pathway.
[0069] Bacteria often have enzymatic activities that confer the
ability to anaerobically metabolize six-carbon sugars to ethanol.
These activities are associated with the ED pathway and include,
but are not limited to, phosphogluconate dehydratase (e.g., the EDD
gene, for example), and 2-keto-3-deoxygluconate-6-phosphate
aldolase (e.g., the EDA gene, for example). Phosphogluconate
dehydratase converts 6-phophogluconate to
2-keto-3-deoxy-6-p-gluconate. 2-keto-3-deoxygluconate-6-phosphate
aldolase converts 2-keto-3-deoxy-6-p-gluconate to pyruvate. In some
embodiments, these activities can be introduced into a host
organism to generate an engineered microorganism which gains the
ability to use the ED pathway to produce ethanol more efficiently
than the non-engineered starting organism, by virtue of the lower
net energy yield by the ED pathway. A microorganism may be
engineered to include or regulate one or more activities in the
Entner-Doudoroff pathway. In some embodiments, one or more of these
activities may be altered such that the activity or activities can
be increased or decreased according to a change in environmental
conditions. Nucleic acid sequences encoding Embden-Meyerhoff
pathway and Entner-Doudoroff pathway activities can be obtained
from any suitable organism (e.g., plants, bacteria, and other
microorganisms, for example) and any of these activities can be
used herein with the proviso that the nucleic acid sequence is
naturally active in the chosen microorganism when expressed, or can
be altered or modified to be active.
[0070] Yeast also can have endogenous or heterologous enzymatic
activities that enable the organism to anaerobically metabolize six
carbon sugars. Saccharomyces cerevisiae used in fermentation often
convert glucose-6-phospate (G-6-P) to fructose-6-phosphate (F-6-P)
via phosphoglucose isomerase (EC 5.3.1.9), up to 95% of G-6-P is
converted to F-6-P in this manner for example. Only a minor
proportion of G-6-P is converted to 6-phophoglucono-lactone (6-PGL)
by an alternative enzyme, glucose-6-phosphate dehydrogenase (EC
1.1.1.49). Yeast engineered to carry both Entner-Doudoroff (ED) and
Embden-Meyerhoff (EM) pathways often covert sugars to ethanol using
the EM pathway preferentially. Inactivation of one or more
activities in the EM pathway can result in conversion of sugars to
ethanol using the ED pathway preferentially, in some
embodiments.
[0071] Phosphoglucose isomerase (EC 5.3.1.9) catalyzes the
reversible interconversion of glucose-6-phosphate and
fructose-6-phosphate. Phosphoglucose isomerase is encoded by the
PGI1 gene in S. cerevisiae. The proposed mechanism for sugar
isomerization involves several steps and is thought to occur via
general acid/base catalysis. Since glucose 6-phosphate and fructose
6-phosphate exist predominantly in their cyclic forms, PGI is
believed to catalyze first the opening of the hexose ring to yield
the straight chain form of the substrates. Glucose 6-phosphate and
fructose 6-phosphate then undergo isomerization via formation of a
cis-enediol intermediate with the double bond located between C-1
and C-2. Phosphoglucose isomerase sometimes also is referred to as
glucose-6-phosphate isomerase or phosphohexose isomerase.
[0072] PGI is involved in different pathways in different
organisms. In some higher organisms PGI is involved in glycolysis,
and in mammals PGI also is involved in gluconeogenesis. In plants
PGI is involved in carbohydrate biosynthesis, and in some bacteria
PGI provides a gateway for fructose into the Entner-Doudoroff
pathway. PGI also is known as neuroleukin (a neurotrophic factor
that mediates the differentiation of neurons), autocrine motility
factor (a tumor-secreted cytokine that regulates cell motility),
differentiation and maturation mediator and myofibril-bound serine
proteinase inhibitor, and has different roles inside and outside
the cell. In the cytoplasm, PGI catalyses the second step in
glycolysis, while outside the cell it serves as a nerve growth
factor and cytokine. PGI activity is involved in cell cycle
progression and completion of the gluconeogenic events of
sporulation in S. cerevisiae.
[0073] In certain embodiments, phosphoglucose isomerase activity is
altered in an engineered microorganism. In some embodiments
phosphoglucose isomerase activity is decreased or disrupted in an
engineered microorganism. In certain embodiments, decreasing or
disrupting phosphoglucose isomerase activity may be desirable to
decrease or eliminate the isomerization of glucose-6-phosphate to
fructose-6-phosphate, thereby increasing the proportion of
glucose-6-phosphate converted to gluconolactone-6-phosphate by the
activity encoded by ZWF1 (e.g., glucose-6-phosphate dehydrogenase).
Increased levels of gluconolactone-6-phosphate can be further
metabolized and thereby improve fermentation of sugar to ethanol
via activities in the Entner-Doudoroff pathway, even in the
presence of the enzymes comprising the Embden-Meyerhoff pathway.
Decreased or disrupted phosphoglucose isomerase (EC 5.3.1.9)
activity in yeast may be achieved by any suitable method, or as
described herein. Non-limiting examples of methods suitable for
decreasing or disrupting the activity of phosphoglucose isomerase
include use of a regulated promoter, use of a weak constitutive
promoter, disruption of one of the two copies of the gene in a
diploid yeast, disruption of both copies of the gene in a diploid
yeast, expression of an anti-sense nucleic acid, expression of an
siRNA, over expression of a negative regulator of the endogenous
promoter, alteration of the activity of an endogenous or
heterologous gene, use of a heterologus gene with lower specific
activity, the like or combinations thereof. In some embodiments, a
gene used to knockout one activity can also introduce or increase
another activity. PGI1 genes may be native to S. cerevisiae, or may
be obtained from a heterologous source.
[0074] Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) catalyzes
the first step of the pentose phosphate pathway, and is encoded by
the S. cerevisiae gene, zwf1. The reaction for the first step in
the PPP pathway is;
D-glucose 6-phosphate+NADP.sup.+=D-glucono-1,5-lactone
6-phosphate+NADPH+H.sup.+
[0075] This reaction is irreversible and rate-limiting for
efficient fermentation of sugar via the Entner-Doudoroff pathway.
The enzyme regenerates NADPH from NADP+ and is important both for
maintaining cytosolic levels of NADPH and protecting yeast against
oxidative stress. Zwf1p expression in yeast is constitutive, and
the activity is inhibited by NADPH such that processes that
decrease the cytosolic levels of NADPH stimulate the oxidative
branch of the pentose phosphate pathway. Amplification of
glucose-6-phosphate dehydrogenase activity in yeast may be
desirable to increase the proportion of glucose-6-phosphate
converted to 6-phosphoglucono-lactone and thereby improve
fermentation of sugar to ethanol via the Entner-Doudoroff pathway,
even in the presence of the enzymes comprising the Embden-Meyerhoff
pathway.
[0076] Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity in
yeast may be amplified by over-expression of the zwf1 gene by any
suitable method. Non-limiting examples of methods suitable to
amplify or over express zwf1 include amplifying the number of ZWF1
genes in yeast following transformation with a high-copy number
plasmid (e.g., such as one containing a 2 uM origin of
replication), integration of multiple copies of ZWF1 into the yeast
genome, over-expression of the ZWF1 gene directed by a strong
promoter, the like or combinations thereof. The ZWF1 gene may be
native to S. cerevisiae, or it may be obtained from a heterologous
source. 6-phosphogluconolactonase (EC 3.1.1.31) catalyzes the
second step of the ED (e.g., pentose phosphate pathway), and is
encoded by S. cerevisiae genes SOL3 and SOL4. The reaction for the
second step of the pentose phosphate pathway is;
6-phospho-D-glucono-1,5-lactone+H2O=6-phospho-D-gluconate
[0077] Amplification of 6-phosphogluconolactonase activity in yeast
may be desirable to increase the proportion of
6-phospho-D-glucono-1,5-lactone converted to 6-phospho-D-gluconate
and thereby improve fermentation of sugar to ethanol via the
Entner-Doudoroff pathway, even in the presence of the enzymes
comprising the Embden-Meyerhoff pathway. For example, over
expression of SOL3 is known to increase the rate of carbon source
utilization to result in faster growth on xylose than wild
type.
[0078] The Saccharomyces cerevisiae SOL protein family includes
Sol3p and Sol4p. Both localize predominantly in the cytosol,
exhibit 6-phosphogluconolactonase activity and function in the
pentose phosphate pathway. 6-phosphogluconolactonase (EC 3.1.1.31)
activity in yeast may be amplified by over-expression of the SOL3
and/or SOL4 gene(s) by any suitable method. Non-limiting examples
of methods to amplify or over express SOL3 and SOL4 include
increasing the number of SOL3 and/or SOL4 genes in yeast by
transformation with a high-copy number plasmid, integration of
multiple copies of SOL3 and/or SOL4 gene(s) into the yeast genome,
over-expression of the SOL3 and/or SOL4 gene(s) directed by a
strong promoter, the like or combinations thereof. The SOL3 and/or
SOL4 gene(s) may be native to S. cerevisiae, or may be obtained
from a heterologous source. For example, Sol3p and Sol4p have
similarity to each other, and to Candida albicans Sol1p,
Schizosaccharomyces pombe Sol1p, human PGLS which is associated
with 6-phosphogluconolactonase deficiency, and human H6PD which is
associated with cortisone reductase deficiency. Sol3p and Sol4p are
also similar to the 6-phosphogluconolactonases in bacteria
(Pseudomonas aeruginosa) and eukaryotes (Drosophila melanogaster,
Arabidopsis thaliana, and Trypanosoma brucei), to the
glucose-6-phosphate dehydrogenase enzymes from bacteria
(Mycobacterium leprae) and eukaryotes (Plasmodium falciparum and
rabbit liver microsomes), and have regions of similarity to
proteins of the Nag family, including human GNPI and Escherichia
coli NagB.
[0079] Phosphogluconate dehydrogenase (EC:1.1.1.44) catalyzes the
second oxidative reduction of NADP+ to NADPH in the cytosolic
oxidative branch of the pentose phosphate pathway, and is encoded
by the S. cerevisiae genes GND1 and GND2. GND1 encodes the major
isoform of the enzyme accounting for up to 80% of phosphogluconate
dehydrogenase activity, while GND2 encodes the minor isoform of the
enzyme. Phosphogluconate dehydrogenase sometimes also is referred
to as phosphogluconic acid dehydrogenase, 6-phosphogluconic
dehydrogenase, 6-phosphogluconic carboxylase, 6-phosphogluconate
dehydrogenase (decarboxylating), and 6-phospho-D-gluconate
dehydrogenase. Phosphogluconate dehydrogenase belongs to the family
of oxidoreductases, specifically those acting on the CH--OH group
of donor with NAD or NADP as the acceptor. The reaction for the
second oxidative reduction of NADP+ to NADPH in the cytosolic
oxidative branch of the pentose phosphate pathway is;
6-phospho-D-gluconate+NADP.sup.+ribulose
5-phosphate+CO.sub.2+NADPH
[0080] Decreasing the level of 6-phosphogluconolactonase activity
in yeast may be desirable to decrease the proportion of
6-phospho-D-gluconate converted to D-ribulose 5-phosphate thereby
increasing the levels of the intermediate gluconate-6-phosphate
available for conversion to
6-dehydro-3-deoxy-gluconate-6-phosphate, in some embodiments
involving engineered microorganisms including increased EDA and EDD
activities, thereby improving fermentation of sugar to ethanol via
the Entner-Doudoroff pathway, even in the presence of the enzymes
comprising the Embden-Meyerhoff pathway.
[0081] Decreasing or disrupting 6-phosphogluconolactonase activity
in yeast may be achieved by any suitable method, or as described
herein. Non-limiting examples of methods suitable for decreasing
the activity of 6-phosphogluconate dehydrogenase include use of a
regulated promoter, use of a weak constitutive promoter, disruption
of one of the two copies of the gene in a diploid yeast (e.g.,
partial gene knockout), disrupting both copies of the gene in a
diploid yeast (e.g., complete gene knockout) expression of an
anti-sense nucleic acid, expression of an siRNA, over expression of
a negative regulator of the endogenous promoter, alteration of the
activity of an endogenous or heterologous gene, use of a
heterologus gene with lower specific activity, the like or
combinations thereof. In some embodiments, a gene used to knockout
one activity can also introduce or increase another activity. GND1
and/or GND2 gene(s) may be native to S. cerevisiae, or may be
obtained from a heterologous source. For example, S. cerevisiae
GND1 and GND2 have similarity to each other, and to the
phosphogluconate dehydrogenase nucleotide sequences of Candida
parapsilosis, Cryptococcus neoformans and humans.
Five-Carbon Sugar Metabolism and Activities
[0082] As noted above, five-carbon sugars are the second most
predominant form of sugars in lignocelluosic waste biomass produced
in wood pulp and wood milling industries. Furthermore, xylose is
the second most abundant carbohydrate in nature. However, the
conversion of biomass to energy (e.g., ethanol, for example) has
not proven economically attractive because many organisms cannot
metabolize hemicellulose. Biomass and waste biomass contain both
cellulose and hemicellulose. Many industrially applicable organisms
can metabolize five-carbon sugars (e.g., xylose, pentose and the
like), but may do so at low efficiency, or may not begin
metabolizing five-carbon sugars until all six-carbon sugars have
been depleted from the growth medium. Many yeast and fungus grow
slowly on xylose and other five-carbon sugars. Some yeast, such as
S. cerevisiae do not naturally use xylose, or do so only if there
are no other carbon sources. An engineered microorganism (e.g.,
yeast, for example) that could grow rapidly on xylose and provide
ethanol and/or other products as a result of fermentation of xylose
can be useful due to the ability to use a feedstock source that is
currently underutilized while also reducing the need for
petrochemicals.
[0083] The pentose phosphate pathway (PPP), which is a biochemical
route for xylose metabolism, is found in virtually all cellular
organisms where it provides D-ribose for nucleic acid biosynthesis,
D-erythrose 4-phosphate for the synthesis of aromatic amino acids
and NADPH for anabolic reactions. The PPP is thought of as having
two phases. The oxidative phase converts the hexose, D-glucose 6P,
into the pentose, D-ribulose 5P, plus CO2 and NADPH. The
non-oxidative phase converts D-ribulose 5P into D-ribose 5P,
D-xylulose 5P, D-sedoheptulose 7P, D-erythrose 4P, D-fructose 6P
and D-glyceraldehyde 3P. D-Xylose and L-arabinose enter the PPP
through D-xylulose.
[0084] Certain organisms (e.g., yeast, filamentous fungus and other
eukaryotes, for example) require two or more activities to convert
xylose to a usable from that can be metabolized in the pentose
phosphate pathway. The activities are a reduction and an oxidation
carried out by xylose reductase (XR; XYL1) and xylitol
dehydrogenase (XD; XYL2), respectively. Xylose reductase converts
D-xylose to xylitol. Xylitol dehydrogenase converts xylitol to
D-xylulose. The use of these activities sometimes can inhibit
cellular function due to cofactor and metabolite imbalances.
[0085] In some embodiments, the xylose reductase activity and/or
xylitol dehydrogenase activity selected for inclusion in an
engineered organism can be chosen from an organism whose XR and/or
XD activities utilize NADPH or NADH (e.g., co-factor flexible
activities), thereby reducing or eliminating inhibition of cellular
function due to cofactor and metabolite imbalances. Non-limiting
examples of yeast whose xylose reductase enzyme and/or xylitol
dehydrogenase enzyme can use NADP.sup.+/NADPH and/or NAD.sup.+/NADH
include C. shehatae, C. parapsilosis, P. segobiensis, P. stipitis,
and Pachysolen tannophilus. In certain embodiments, xylose
reductase and/or xylitol dehydrogenase activities can be engineered
to alter cofactor preference and/or specificity. Some organisms
(e.g., certain bacteria, for example) require only one activity,
xylose isomerase (xylA). Xylose isomerase converts xylose directly
to xylulose.
[0086] Xylulose is converted to xylulose-5-phophate by the activity
of a xylulokinase enzyme (EC 2.7.1.17). Xylulose kinase (e.g.,
XYK3, XYL3) catalyzes the chemical reaction,
ATP+D-xyluloseADP+D-xylulose 5-phosphate
[0087] Xylulokinase sometimes also is referred to as ATP:D-xylulose
5-phosphotransferase, xylulokinase (phosphorylating), and
D-xylulokinase. Increasing the activity of xylose isomerase or
xylose reductase and xylitol dehydrogenase may cause an increase of
xylulose in an engineered microorganism. Therefore, increasing
xylulokinase activity levels in embodiments involving increased
levels of XI or XR and XD may be desirable to allow increased flux
through the respective metabolic pathways. Xylulokinase activity
levels can be increased using any suitable method. Non-limiting
examples of methods suitable for increasing xylulokinase activity
include increasing the number of xylulokinase genes in yeast by
transformation with a high-copy number plasmid, integration of
multiple copies of xylulokinase genes into the yeast genome,
over-expression of the xylulokinase gene directed by a strong
promoter, the like or combinations thereof. The xylulokinase
genemay be native to S. cerevisiae, or may be obtained from a
heterologous source.
[0088] Phosphorylation of xylulose by xylulokinase allows the
five-carbon sugar to be further converted by transketolase (e.g.,
TKL1/TKL2) to enter the EM pathway for further metabolism at either
fructose-6-phosphate or glyceraldehyde-3-phosphate. In some
embodiments, where the EM pathway is inactivated, five-carbon
sugars enter the EM pathway and are further converted for use by
the ED pathway. Therefore, engineering a microorganism with xylose
isomerase activity or co-factor flexible xylose reductase activity
and xylitol dehydrogenase activity, along with increased
xylulokinase activity may allow rapid growth on xylose when
compared to the non-engineered microorganism, while avoiding
cofactor and metabolite imbalances, in some embodiments. In certain
embodiments, engineering a microorganism with co-factor flexible
xylose reductase activity and xylitol dehydrogenase activity, may
allow rapid growth on xylose when compared to the non-engineered
microorganism, while avoiding cofactor and metabolite imbalances.
The term "co-factor flexible" as used herein with respect to xylose
reductase activity and xylose isomerase activity refers to the
ability to use NADP.sup.+/NADPH and/or NAD.sup.+/NADH as a cofactor
for electron transport.
[0089] A microorganism may be engineered to include or regulate one
or more activities in a five-carbon sugar metabolism pathway (e.g.,
pentose phosphate pathway, for example). In some embodiments, an
engineered microorganism can comprise a xylose isomerase activity.
In some embodiments, the xylose isomerase activity may be altered
such that the activity can be increased or decreased according to a
change in environmental conditions. Nucleic acid sequences encoding
xylose isomerase activities can be obtained from any suitable
bacteria (e.g., Piromyces, Orpinomyces, Bacteroides
thetaiotaomicron, Clostridium phytofermentans, Thermus thermophilus
and Ruminococcus (e.g., R. flavefaciens, R. flavefaciens strain
FD1, R. Flavefaciens strain 18P13) are non-limiting examples) and
any of these activities can be used herein with the proviso that
the nucleic acid sequence is naturally active in the chosen
microorganism when expressed, or can be altered or modified to be
active. In some embodiments, an engineered microorganism can
comprise a xylose reductase activity and a xylitol dehydrogenase
activity. In certain embodiments, an engineered microorganism can
comprise a xylulokinase activity. In some embodiments, the xylose
reductase activity, xylitol dehydrogenase activity and/or
xylulokinase activity may be altered such that the activity can be
increased or decreased according to a change in environmental
conditions. Nucleic acid sequences encoding xylose reductase
activity, xylitol dehydrogenase activity and/or xylulokinase
activities can be obtained from any suitable organism, and any of
these activities can be used herein with the proviso that the
nucleic acid sequence is naturally active in the chosen
microorganism when expressed, or can be altered or modified to be
active.
Activities Linking 5-Carbon and 6-Carbon Sugar Metabolic
Pathways
[0090] In some embodiments, an engineered microorganism includes
one or more altered activities that function to link 5-carbon sugar
and 6-carbon sugar metabolic pathways (e.g., provide intermediates
that enter and/or are metabolized by the pentose phosphate pathway,
the glycolytic pathway, or the pentose phosphate and glycolytic
pathways). In certain embodiments, the altered linking activity is
added, increased or amplified, with respect to a host or starting
organism. In some embodiments, the altered activity is decreased or
disrupted, with respect to a host or starting organism.
Non-limiting examples of activities that function to reversibly
link 5-carbon sugar and 6-carbon sugar metabolic pathways include
transaldolase, transketolase, the like, or combinations thereof.
Transketolase and transaldolase catalyze transfer of 2 carbon and 3
carbon molecular fragments respectively, in each case from a ketose
donor to an aldose acceptor.
[0091] Transaldolase (EC:2.2.1.2) catalyses the reversible transfer
of a three-carbon ketol unit from sedoheptulose 7-phosphate to
glyceraldehyde 3-phosphate to form erythrose 4-phosphate and
fructose 6-phosphate. The cofactor-less enzyme acts through a
Schiff base intermediate (e.g., bound dihydroxyacetone).
Transaldolase is encoded by the gene TAL1 in S. cerevisiae, and is
an enzyme in the non-oxidative pentose phosphate pathway that
provides a link between the pentose phosphate and the glycolytic
pathways.
[0092] Transaldolase activity is thought to be found in
substantially all organisms, and include 5 subfamilies. Three
transaldolase subfamilies have demonstrated transaldolase activity,
one subfamily comprises an activity of undetermined function and
the remaining subfamily includes a fructose 6-phosphate aldolase
activity. Transaldolase deficiency is well tolerated in many
microorganisms, and without being limited by any theory, is thought
to be involved in oxidative stress responses and apoptosis.
Transaldolase sometimes also is referred to as dihydroxyacetone
transferase, glycerone transferase, or dihydroxyacetonetransferase,
sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphate
glyceronetransferase, and catalyzes the reaction:
sedoheptulose 7-phosphate+glyceraldehyde 3-phosphateerythrose
4-phosphate+fructose 6-phosphate
[0093] In some embodiments, increasing or amplifying transaldolase
activity in yeast may be desirable to increase the proportion of
sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate converted
to fructose-6-phosphate and erythrose-4-phosphate, thereby
increasing levels of fructose-6-phosphate. Increased levels of
fructose-6-phosphate can be further metabolized and thereby improve
fermentation of sugar to ethanol via activities in the
Entner-Doudoroff pathway, even in the presence of the enzymes
comprising the Embden-Meyerhoff pathway. Transaldolase (EC:2.2.1.2)
activity in yeast may be amplified by over-expression of the TAL1
gene by any suitable method. Non-limiting examples of methods to
amplify or over express TAL1 include increasing the number of TAL1
genes in yeast by transformation with a high-copy number plasmid,
integration of multiple copies of TAL1 genes into the yeast genome,
over-expression of TAL1 genes directed by a strong promoter, the
like or combinations thereof. The TAL1 genes may be native to S.
cerevisiae, or may be obtained from a heterologous source.
[0094] In certain embodiments, decreasing or disrupting
transaldolase activity may be desirable to decrease the proportion
of sedoheptulose-7-phosphate and glyceraldehyde-3-phosphate
converted to fructose-6-phosphate and erythrose-4-phosphate,
thereby increasing levels of glyceraldehyde-3-phosphate in the
engineered microorganism. Increased levels of
glyceraldehyde-3-phosphate can be further metabolized and thereby
improve fermentation of sugar to ethanol via activities in the
Entner-Doudoroff pathway, even in the presence of the enzymes
comprising the Embden-Meyerhoff pathway. Decreased or disrupted
transaldolase (EC:2.2.1.2) activity in yeast may be achieved by any
suitable method, or as described herein. Non-limiting examples of
methods suitable for decreasing or disrupting the activity of
transaldolase include use of a regulated promoter, use of a weak
constitutive promoter, disruption of one of the two copies of the
gene in a diploid yeast, disruption of both copies of the gene in a
diploid yeast, expression of an anti-sense nucleic acid, expression
of an siRNA, over expression of a negative regulator of the
endogenous promoter, alteration of the activity of an endogenous or
heterologous gene, use of a heterologus gene with lower specific
activity, the like or combinations thereof. In some embodiments, a
gene used to knockout one activity can also introduce or increase
another activity.
[0095] Transketolase (EC:2.2.1.1) catalyzes the reversible transfer
of a two-carbon ketol unit from a ketose (e.g., xylulose
5-phosphate, fructose 6-phosphate, sedoheptulose 7-phosphate) to an
aldose receptor (e.g., ribose 5-phosphate, erythrose 4-phosphate,
glyceraldehyde 3-phosphate). Transketolase is encoded by the TKL1
and TKL2 genes in S. cerevisiae. TKL1 encodes the major isoform of
the enzyme and TKL2 encodes a minor isoform. Transketolase
sometimes also is referred to as glycoaldehyde transferase,
glycolaldehydetransferase,
sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphate
glycolaldehydetransferase, or fructose
6-phosphate:D-glyceraldehyde-3-phosphate
glycolaldehydetransferase.
[0096] Transketolase double null mutants (e.g., tkl1/tkl2) are
viable but are auxotrophic for aromatic amino acids, indicating the
genes are involved in the synthesis of aromatic amino acids.
Transketolase activity also is thought to be involved in the
efficient use of fermentable carbon sources, and has been shown to
catalyze a one-substrate reaction utilizing only xylulose
5-phosphate to produce glyceraldehyde 3-phosphate and erythrulose.
Transketolase activity requires thiamine pyrophosphate as a
cofactor, and has been purified as a homodimer of approximately 70
kilodalton subunits, from S. cerevisiae. Sequences from a variety
of eukaryotic and prokaryotic sources indicate transketolase
enzymes have been evolutionarily conserved. Tkl1p has similarity to
S. cerevisiae Tkl2p, Escherichia coli transketolase, Rhodobacter
sphaeroides transketolase, Streptococcus pneumoniae recP, Hansenula
polymorpha dihydroxyacetone synthase, Kluyveromyces lactis TKL1,
Pichia stipitis TKT, rabbit liver transketolase, rat TKT, mouse
TKT, and human TKT. Tkl1p is also related to E. coli pyruvate
dehydrogenase E1 subunit, which is another vitamin B1-dependent
enzyme.
[0097] In some embodiments, increasing or amplifying transketolase
activity in yeast may be desirable to increase the proportion of
xylulose 5-phosphate converted to glyceraldehyde 3-phosphate,
thereby increasing levels of glyceraldehyde 3-phosphate available
for entry into a 6-carbon sugar metabolic pathway directly and/or
conversion to fructose-6-phosphate. Increased levels of
fructose-6-phosphate and/or glyceraldehyde 3-phosphate can be
further metabolized and thereby improve fermentation of sugar to
ethanol via activities in the Entner-Doudoroff pathway, even in the
presence of the enzymes comprising the Embden-Meyerhoff pathway.
Transketolase (EC 2.2.1.1) activity in yeast may be increased or
amplified by over-expression of the TKL1 and/or TKL2 gene(s) by any
suitable method. Non-limiting examples of methods to amplify or
over express TKL1 and TKL2 include increasing the number of TKL1
and/or TKL2 gene(s) in yeast by transformation with a high-copy
number plasmid, integration of multiple copies of TKL1 and/or TKL2
gene(s) into the yeast genome, over-expression of TKL1 and/or TKL2
gene(s) directed by a strong promoter, the like or combinations
thereof.
[0098] In certain embodiments, decreasing or disrupting
transketolase activity may be desirable to decrease the proportion
of xylulose 5-phosphate converted to glyceraldehyde 3-phosphate,
thereby increasing levels of xylulose 5-phosphate in the engineered
microorganism. Increased levels of xylulose 5-phosphate can be
further metabolized and thereby improve fermentation of sugar to
ethanol via activities in the Entner-Doudoroff pathway, even in the
presence of the enzymes comprising the Embden-Meyerhoff pathway.
Decreased or disrupted transketolase (EC 2.2.1.1) activity in yeast
may be achieved by any suitable method, or as described herein.
Non-limiting examples of methods suitable for decreasing or
disrupting the activity of transketolase include use of a regulated
promoter, use of a weak constitutive promoter, disruption of one of
the two copies of the gene in a diploid yeast, disruption of both
copies of the gene in a diploid yeast, expression of an anti-sense
nucleic acid, expression of an siRNA, over expression of a negative
regulator of the endogenous promoter, alteration of the activity of
an endogenous or heterologous gene, use of a heterologus gene with
lower specific activity, the like or combinations thereof. In some
embodiments, a gene used to knockout one activity can also
introduce or increase another activity. TKL1 and/or TKL2 gene(s)
may be native to S. cerevisiae, or may be obtained from a
heterologous source.
Sugar Transport Activities
[0099] Sugar metabolized as a carbon source by organisms typically
is transported from outside a cell into the cell for use as an
energy source and/or a raw material for synthesis of cellular
products. Sugar can be transported into the cell using active or
passive transport mechanisms. Active transport systems frequently
utilize energy to transport the sugar across the cell membrane.
Sugars often are modified by phosphorylation, once transported
inside the cell or organism, to prevent diffusion out of the cell.
Sugar transport activities are thought also to act as sugar sensors
and have high affinity and low affinity transporters. The rate of
glucose utilization in yeast often is dictated by the activity and
concentration of glucose transporters in the plasma membrane.
[0100] In yeast, sugar transporters have been found to be part of a
multi-gene family. Some sugar transport systems transport certain
sugars preferentially and other non-preferred sugars at a lower
rate. Certain sugar transport systems transport one or more
structurally similar sugars at substantially similar rates.
Non-limiting examples of sugar transporters include high affinity
glucose transporters (e.g., HXT (e.g., HXT1, HXT7}), glucose-xylose
transporters (e.g., GXF1, GXS1), and high affinity galactose
transporters (e.g., GAL2), the like and combinations thereof.
[0101] Galactose permease is a high affinity galactose transport
enzyme activity that also can transport glucose. Galactose permease
is encoded by the GAL2 gene, and sometimes also is referred to as a
galactose/glucose (methylgalactoside) porter. Gal2p is an integral
plasma membrane protein belonging to a super family of sugar
transporters that are predicted to contain 12 transmembrane domains
separated by charged residues. Structurally and functionally
similar sugar transporters have been identified in bacteria, rat,
and humans.
[0102] Glucose often is transported by high affinity glucose
transporters. High affinity glucose transporters (e.g., HXT) are
members of the major facilitator gene super family, and include the
genes HXT6 (Hxt6p) and HXT7 (Hxt7p). HXT6 and HXT7 are
substantially similar activities, and are expressed at high basal
levels relative to other high affinity glucose transporters.
[0103] Approximately 20 HXT genes have been identified. High
affinity glucose transporters sometimes also are referred to as
hexose transporters.
[0104] Certain sugar transport systems include high and low
affinity transport activities that act on more than one sugar. A
non-limiting example of such a sugar transport system includes the
glucose/xylose transport system from Candida yeast. Glucose and
xylose are transported into certain Candida by a high affinity
xylose-proton symporter (e.g., GXS1) and a low affinity diffusion
facilitator (e.g., GXF1). S. cerevisiae normally lacks an efficient
transport system for xylose, although xylose can enter the cell at
low efficiency via non-specific transport systems sometimes
involving HXT activities. Addition of the Candida GSX1, GXF1 or
GXS1 and GXF1 activities to S. cerevisiae engineered to metabolize
xylose can further enhance the ability to ferment xylose to alcohol
or other desired products.
[0105] In some embodiments, an engineered microorganism includes
one or more sugar transport activities that has been genetically
added or altered. In certain embodiments, the sugar transport
activity is amplified or increased. Sugar transport activities can
be added, amplified by over expression or increased by any suitable
method. Non-limiting methods of adding, amplifying or increasing
the activity of sugar transport systems include increasing the
number of genes of a sugar transport activity (e.g., GAL2, GXF1,
GXS1, HXT7) gene(s) in yeast by transformation with a high-copy
number plasmid, integration of multiple copies of sugar transport
activity (e.g., GAL2, GXF1, GXS1, HXT7) gene(s) into the yeast
genome, over-expression of sugar transport activity gene(s)
directed by a strong promoter, the like or combinations thereof.
The sugar transport activity (e.g., GAL2, GXF1, GXS1, HXT7) gene(s)
may be native to S. cerevisiae, or may be obtained from a
heterologous source.
Carbon Dioxide Metabolism and Activities
[0106] Microorganisms grown in fermentors often are grown under
anaerobic conditions, with limited or no gas exchange. Therefore
the atmosphere inside fermentors sometimes is carbon dioxide rich.
Unlike photosynthetic organisms, many microorganisms suitable for
use in industrial fermentation processes do not incorporate
atmospheric carbon (e.g., CO.sub.2) to any significant degree, or
at all. Thus, to ensure that increasing levels of carbon dioxide do
not inhibit cell growth and the fermentation process, methods to
remove carbon dioxide from the interior of fermentors can be
useful.
[0107] Photosynthetic organisms make use of atmospheric carbon by
incorporating the carbon available in carbon dioxide into organic
carbon compounds by a process known as carbon fixation. The
activities responsible for a photosynthetic organism's ability to
fix carbon dioxide include phosphoenolpyruvate carboxylase (e.g.,
PEP carboxylase) or ribulose 1,5-bis-phosphate carboxylase (e.g.,
Rubisco). PEP carboxylase catalyzes the addition of carbon dioxide
to phosphoenolpyruvate to generate the four-carbon compound
oxaloacetate. Oxaloacetate can be used in other cellular processes
or be further converted to yield several industrially useful
products (e.g., malate, succinate, citrate and the like). Rubisco
catalyzes the addition of carbon dioxide and
ribulose-1,5-bisphosphate to generate 2 molecules of
3-phosphoglycerate. 3-phosphoglycerate can be further converted to
ethanol via cellular fermentation or used to produce other
commercially useful products. Nucleic acid sequences encoding PEP
carboxylase and Rubisco activities can be obtained from any
suitable organism (e.g., plants, bacteria, and other
microorganisms, for example) and any of these activities can be
used herein with the proviso that the nucleic acid sequence is
either naturally active in the chosen microorganism when expressed,
or can be altered or modified to be active.
Examples of Altered Activities
[0108] In some embodiments, engineered microorganisms can include
modifications to one or more (e.g., 1, 2, 3, 4, 5, 6 or all) of the
following activities: phosphofructokinase activity (PFK1 A subunit,
PFK2 B subunit), phosphogluconate dehydratase activity (EDD),
2-keto-3-deoxygluconate-6-phosphate aldolase activity (EDA), xylose
isomerase activity (xylA), xylose reductase activity (XYL1),
xylitol dehydrogenase activity (XYL2), xylulokinase activity (XKS1,
XYL3), phosphoenolpyruvate carboxylase activity (PEP carboxylase),
alcohol dehydrogenase 2 activity (ADH2), thymidylate synthase
activity, phosphoglucose isomerase activity (PGI1), transaldolase
activity (TAD), transketolase activity (TKL1, TKL2),
6-phosphogluconolactonase activity (SOL3, SOL4),
Glucose-6-phosphate dehydrogenase activity (ZWF1),
6-phosphogluconate dehydrogenase (decarboxylating) activity (GND1,
GND2), galactose permease activity (GAL2), high affinity glucose
transport activity (HXT7), glucose/xylose transport activity (GXS1,
GXF1) and combinations of the foregoing.
[0109] The term "phosphofructokinase activity" as used herein
refers to conversion of fructose-6-phosphate to
fructose-1,6-bisphosphate. Phosphofructokinase activity may be
provided by an enzyme that includes one or two subunits (referred
to hereafter as "subunit A" and/or "subunit B"). The term
"inactivating the Embden-Meyerhoff pathway" as used herein refers
to reducing or eliminating the activity of one or more activities
in the Embden-Meyerhoff pathway, including but not limited to
phosphofructokinase activity. In some embodiments, the
phosphofructokinase activity can be reduced or eliminated by
introduction of an untranslated RNA molecule (e.g., antisense RNA,
RNAi, and the like, for example). In certain embodiments, the
untranslated RNA is encoded by a heterologous nucleotide sequence
introduced to a host microorganism.
[0110] In some embodiments, the phosphofructokinase activity can be
temporarily or permanently reduced or eliminated by genetic
modification, as described below. In certain embodiments, the
genetic modification renders the activity responsive to changes in
the environment. In some embodiments, the genetic modification
disrupts synthesis of a functional nucleic acid encoding the
activity or produces a nonfunctional polypeptide or protein.
Nucleic acid sequences that can be used to reduce or eliminate the
activity of phosphofructokinase activity can have sequences
partially or substantially complementary to sequences described
herein. Presence or absence of the amount of phosphofructokinase
activity can be detected by any suitable method known in the art,
including requiring a five-carbon sugar carbon source or a
functional Entner-Doudoroff pathway for growth. Inactivation of the
Embden-Meyerhoff pathway is described in further detail below. As
referred to herein, "substantially complementary" with respect to
sequences refers to nucleotide sequences that will hybridize with
each other. The stringency of the hybridization conditions can be
altered to tolerate varying amounts of sequence mismatch. Included
are regions of counterpart, target and capture nucleotide sequences
55% or more, 56% or more, 57% or more, 58% or more, 59% or more,
60% or more, 61% or more, 62% or more, 63% or more, 64% or more,
65% or more, 66% or more, 67% or more, 68% or more, 69% or more,
70% or more, 71% or more, 72% or more, 73% or more, 74% or more,
75% or more, 76% or more, 77% or more, 78% or more, 79% or more,
80% or more, 81% or more, 82% or more, 83% or more, 84% or more,
85% or more, 86% or more, 87% or more, 88% or more, 89% or more,
90% or more, 91% or more, 92% or more, 93% or more, 94% or more,
95% or more, 96% or more, 97% or more, 98% or more or 99% or more
complementary to each other.
[0111] The term "phosphogluconate dehydratase activity" as used
herein refers to conversion of 6-phophogluconate to
2-keto-3-deoxy-6-p-gluconate. The phosphogluconate dehydratase
activity can be provided by a polypeptide. In some embodiments, the
polypeptide is encoded by a heterologous nucleotide sequence
introduced to a host microorganism. Nucleic acid sequences
conferring phosphogluconate dehydratase activity can be obtained
from a number of sources, including Zymomonas mobilis and
Escherichia coli. Examples of an amino acid sequence of a
polypeptide having phosphogluconate dehydratase activity, and a
nucleotide sequence of a polynucleotide that encodes the
polypeptide, are presented below in tables. Presence, absence or
amount of phosphogluconate dehydratase activity can be detected by
any suitable method known in the art, including western blot
analysis.
[0112] The term "2-keto-3-deoxygluconate-6-phosphate aldolase
activity" as used herein refers to conversion of
2-keto-3-deoxy-6-p-gluconate to pyruvate. The
2-keto-3-deoxygluconate-6-phosphate aldolase activity can be
provided by a polypeptide. In some embodiments, the polypeptide is
encoded by a heterologous nucleotide sequence introduced to a host
microorganism. Nucleic acid sequences conferring
2-keto-3-deoxygluconate-6-phosphate aldolase activity can be
obtained from a number of sources, including Zymomonas mobilis and
Escherichia coli. Examples of an amino acid sequence of a
polypeptide having 2-keto-3-deoxygluconate-6-phosphate aldolase
activity, and a nucleotide sequence of a polynucleotide that
encodes the polypeptide, are presented below in tables. Presence,
absence or amount of 2-keto-3-deoxygluconate-6-phosphate aldolase
activity can be detected by any suitable method known in the art,
including western blot analysis.
[0113] The term "xylose isomerase activity" as used herein refers
to conversion of xylose to xylulose. The xylose isomerase activity
can be provided by a polypeptide. In some embodiments, the
polypeptide is encoded by a heterologous nucleotide sequence
introduced to a host microorganism. Nucleic acid sequences
conferring xylose isomerase activity can be obtained from a number
of sources, including Piromyces, Orpinomyces, Bacteroides (e.g., B.
thetaiotaomicron, B. uniformis, B. stercoris), Clostrialies (e.g.,
Clostrialies BVAB3), Clostridium (e.g., C. phytofermentans, C.
thermohydrosulfuricum, C. cellulyticum), Thermus thermophilus,
Eschericia coli, Streptomyces (e.g., S. rubiginosus, S. murinus),
Bacillus stearothermophilus, Lactobacillus pentosus, Thermotoga
(e.g., T. maritime, T. neopolitana) and Ruminococcus (e.g.,
Ruminococcus environmental samples, Ruminococcus albus,
Ruminococcus bromii, Ruminococcus callidus, Ruminococcus
flavefaciens, Ruminococcus gauvreauii, Ruminococcus gnavus,
Ruminococcus lactaris, Ruminococcus obeum, Ruminococcus sp.,
Ruminococcus sp. 14531, Ruminococcus sp. 15975, Ruminococcus sp.
16442, Ruminococcus sp. 18P13, Ruminococcus sp. 25F6, Ruminococcus
sp. 25F7, Ruminococcus sp. 25F8, Ruminococcus sp.
4.sub.--1.sub.--47FAA, Ruminococcus sp. 5, Ruminococcus sp.
5.sub.--1.sub.--39BFAA, Ruminococcus sp. 7L75, Ruminococcus sp.
8.sub.--1.sub.--37FAA, Ruminococcus sp. 9SE51, Ruminococcus sp.
C36, Ruminococcus sp. CB10, Ruminococcus sp. CB3, Ruminococcus sp.
CCUG 37327 A, Ruminococcus sp. CE2, Ruminococcus sp. CJ60,
Ruminococcus sp. CJ63, Ruminococcus sp. CO1, Ruminococcus sp. CO12,
Ruminococcus sp. 0022, Ruminococcus sp. CO27, Ruminococcus sp.
CO28, Ruminococcus sp. CO34, Ruminococcus sp. CO41, Ruminococcus
sp. CO47, Ruminococcus sp. CO7, Ruminococcus sp. CS1, Ruminococcus
sp. CS6, Ruminococcus sp. DJF_VR52, Ruminococcus sp. DJF_VR66,
Ruminococcus sp. DJF_VR67, Ruminococcus sp. DJF_VR70k1,
Ruminococcus sp. DJF_VR87, Ruminococcus sp. Eg2, Ruminococcus sp.
Egf, Ruminococcus sp. END-1, Ruminococcus sp. FD1, Ruminococcus sp.
GM2/1, Ruminococcus sp. ID1, Ruminococcus sp. ID8, Ruminococcus sp.
K-1, Ruminococcus sp. KKA Seq234, Ruminococcus sp. M-1,
Ruminococcus sp. M10, Ruminococcus sp. M22, Ruminococcus sp. M23,
Ruminococcus sp. M6, Ruminococcus sp. M73, Ruminococcus sp. M76,
Ruminococcus sp. MLG080-3, Ruminococcus sp. NML 00-0124,
Ruminococcus sp. Pei041, Ruminococcus sp. SC101, Ruminococcus sp.
SC103, Ruminococcus sp. Siijpesteijn 1948, Ruminococcus sp. WAL
17306, Ruminococcus sp. YE281, Ruminococcus sp. YE58, Ruminococcus
sp. YE71, Ruminococcus sp. ZS2-15, Ruminococcus torques). Examples
of an amino acid sequence of a polypeptide having xylose isomerase
activity, and a nucleotide sequence of a polynucleotide that
encodes the polypeptide, are presented below in tables. Presence,
absence or amount of xylose isomerase activity can be detected by
any suitable method known in the art, including western blot
analysis.
[0114] The term "phosphoenolpyruvate carboxylase activity" as used
herein refers to the addition of carbon dioxide to
phosphoenolpyruvate to generate the four-carbon compound
oxaloacetate. The phosphoenolpyruvate carboxylase activity can be
provided by a polypeptide. In some embodiments, the polypeptide is
encoded by a heterologous nucleotide sequence introduced to a host
microorganism. Nucleic acid sequences conferring
phosphoenolpyruvate carboxylase activity can be obtained from a
number of sources, including Zymomonas mobilis. Examples of an
amino acid sequence of a polypeptide having phosphoenolpyruvate
carboxylase activity, and a nucleotide sequence of a polynucleotide
that encodes the polypeptide, are presented below in tables.
Presence, absence or amount of xylose isomerase activity can be
detected by any suitable method known in the art.
[0115] The term "alcohol dehydrogenase 2 activity" as used herein
refers to conversion of ethanol to acetaldehyde, which is the
reverse of the forward action catalyzed by alcohol dehydrogenase 1.
The term "inactivation of the conversion of ethanol to
acetaldehyde" refers to a reduction or elimination in the activity
of alcohol dehydrogenase 2. Reducing or eliminating the activity of
alcohol dehydrogenase 2 activity can lead to an increase in ethanol
production. In some embodiments, the alcohol dehydrogenase 2
activity can be reduced or eliminated by introduction of an
untranslated RNA molecule (e.g., antisense RNA, RNAi, and the like,
for example). In certain embodiments, the untranslated RNA is
encoded by a heterologous nucleotide sequence introduced to a host
microorganism.
[0116] In some embodiments, the alcohol dehydrogenase 2 activity
can be temporarily or permanently reduced or eliminated by genetic
modification, as described below. In certain embodiments, the
genetic modification renders the activity responsive to changes in
the environment. In some embodiments, the genetic modification
disrupts synthesis of a functional nucleic acid encoding the
activity or produces a nonfunctional polypeptide or protein.
Nucleic acid sequences that can be used to reduce or eliminate the
activity of alcohol dehydrogenase 2 can have sequences partially or
substantially complementary to nucleic acid sequences that encode
alcohol dehydrogenase 2 activity. Presence or absence of the amount
of alcohol dehydrogenase 2 activity can be detected by any suitable
method known in the art, including inability to grown in media with
ethanol as the sole carbon source.
[0117] The term "thymidylate synthase activity" as used herein
refers to a reductive methylation, where deoxyuridine monophosphate
(dUMP) and N5,N10-methylene tetrahydrofolate are together used to
generate thymidine monophosphate (dTMP), yielding dihydrofolate as
a secondary product. The term "temporarily inactivate thymidylate
synthase activity" refers to a temporary reduction or elimination
in the activity of thymidylate synthase when the modified organism
is shifted to a non-permissive temperature. The activity can return
to normal upon return to a permissive temperature. Temporarily
inactivating thymidylate synthase uncouples cell growth from cell
division while under the non permissive temperature. This
inactivation in turn allows the cells to continue fermentation
without producing biomass and dividing, thus increasing the yield
of product produced during fermentation.
[0118] In some embodiments, the thymidylate synthase activity can
be temporarily reduced or eliminated by genetic modification, as
described below. In certain embodiments, the genetic modification
renders the activity responsive to changes in the environment.
Nucleic acid sequences conferring temperature sensitive thymidylate
synthase activity can be obtained from S. cerevisiae strain 172066
(accession number 208583). The cdc21 mutation in S. cerevisiae
strain 172066 has a point mutation at position G139S relative to
the initiating methionine. Examples of nucleotide sequences used to
PCR amplify the polynucleotide encoding the temperature sensitive
polypeptide, are presented below in tables. Presence, absence or
amount of thymidylate synthase activity can be detected by any
suitable method known in the art, including growth arrest at the
non-permissive temperature.
[0119] Thymidylate synthase is one of many polypeptides that
regulate the cell cycle. The cell cycle may be inhibited in
engineered microorganisms under certain conditions (e.g.,
temperature shift, dissolved oxygen shift), which can result in
inhibited or reduced cell proliferation, inhibited or reduced cell
division, and sometimes cell cycle arrest (collectively "cell cycle
inhibition"). Upon exposure to triggering conditions, a
microorganism may display cell cycle inhibition after a certain
time after the microorganism is exposed to the triggering
conditions (e.g., there may be a time delay after a microorganism
is exposed to a certain set of conditions before the microorganism
displays cell cycle inhibition). Where cell cycle inhibition
results in reduced cell proliferation, cell proliferation rates may
be reduced by about 50% or greater, for example (e.g., reduced by
about 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%,
76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99%, or greater than any one of the foregoing).
Where cell cycle inhibition results a reduced number of cells
undergoing cell division, the rate of cell division may be reduced
by about 50% or greater, for example (e.g., the number of cells
undergoing division is reduced by about 52%, 54%, 56%, 58%, 60%,
62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%,
88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or greater
than any one of the foregoing). Where cell cycle inhibition results
in cell cycle arrest, cells may be arrested at any stage of the
cell cycle (e.g., resting G.sub.0 phase, interphase (e.g., G.sub.1,
S, G.sub.2 phases), mitosis (e.g., prophase, prometaphase,
metaphase, anaphase, telophase)) and different percentages of cells
in a population can be arrested at different stages of the cell
cycle.
[0120] The term "phosphoglucose isomerase activity" as used herein
refers to the conversion of glucose-6-phosphate to
fructose-6-phosphate. The term "inactivation of the conversion of
glucose-6-phosphate to fructose-6-phosphate" refers to a reduction
or elimination in the activity of phosphoglucose isomerase.
Reducing or eliminating the activity of phosphoglucose isomerase
activity can lead to an increase in ethanol production. In some
embodiments, the phosphoglucose isomerase activity can be reduced
or eliminated by introduction of an untranslated RNA molecule
(e.g., antisense RNA, RNAi, and the like, for example). In certain
embodiments, the untranslated RNA is encoded by a heterologous
nucleotide sequence introduced to a host microorganism.
[0121] In some embodiments, the phosphoglucose isomerase activity
can be temporarily or permanently reduced or eliminated by genetic
modification, as described below. In certain embodiments, the
genetic modification renders the activity responsive to changes in
the environment. In some embodiments, the genetic modification
disrupts synthesis of a functional nucleic acid encoding the
activity or produces a nonfunctional polypeptide or protein.
Nucleic acid sequences that can be used to reduce or eliminate the
activity of phosphoglucose isomerase can have sequences partially
or substantially complementary to nucleic acid sequences that
encode phosphoglucose isomerase activity. Presence or absence of
the amount of phosphoglucose isomerase activity can be detected by
any suitable method known in the art, including nucleic acid based
analysis and western blot analysis.
[0122] The term "glucose-6-phosphate dehydrogenase activity" as
used herein refers to conversion of glucose-6-phosphate to
gluconolactone-6-phosphate coupled with the generation of NADPH.
The glucose-6-phosphate dehydrogenase aldolase activity can be
provided by a polypeptide. In some embodiments, the polypeptide is
encoded by a heterologous nucleotide sequence introduced to a host
microorganism. Nucleic acid sequences conferring
glucose-6-phosphate dehydrogenase activity can be obtained from a
number of sources, including, but not limited to S. cerevisiae
Examples of a nucleotide sequence of a polynucleotide that encodes
the polypeptide, are presented below in tables. Presence, absence
or amount of glucose-6-phosphate dehydrogenase activity can be
detected by any suitable method known in the art, including western
blot analysis.
[0123] The term "6-phosphogluconolactonase activity" as used herein
refers to conversion of gluconolactone-6-phosphate to
gluconate-6-phosphate. The 6-phosphogluconolactonase activity can
be provided by a polypeptide. In some embodiments, the polypeptide
is encoded by a heterologous nucleotide sequence introduced to a
host microorganism. Nucleic acid sequences conferring
6-phosphogluconolactonase activity can be obtained from a number of
sources, including, but not limited to S. cerevisiae. Examples of
an amino acid sequence of a polypeptide having
6-phosphogluconolactonase activity, and a nucleotide sequence of a
polynucleotide that encodes the polypeptide, are presented below in
tables. Presence, absence or amount of 6-phosphogluconolactonase
activity can be detected by any suitable method known in the art,
including nucleic acid based analysis and western blot
analysis.
[0124] The term "6-phosphogluconate dehydrogenase (decarboxylating)
activity" as used herein refers to the conversion of
gluconate-6-phosphate to ribulose-5-phosphate. The term
"inactivation of the conversion of gluconate-6-phosphate to
ribulose-5-phosphate" refers to a reduction or elimination in the
activity of 6-phosphogluconate dehydrogenase. Reducing or
eliminating the activity of 6-phosphogluconate dehydrogenase
(decarboxylating) activity can lead to an increase in ethanol
production. In some embodiments, the 6-phosphogluconate
dehydrogenase (decarboxylating) activity can be reduced or
eliminated by introduction of an untranslated RNA molecule (e.g.,
antisense RNA, RNAi, and the like, for example). In certain
embodiments, the untranslated RNA is encoded by a heterologous
nucleotide sequence introduced to a host microorganism.
[0125] In some embodiments, the 6-phosphogluconate dehydrogenase
(decarboxylating) activity can be temporarily or permanently
reduced or eliminated by genetic modification, as described below.
In certain embodiments, the genetic modification renders the
activity responsive to changes in the environment. In some
embodiments, the genetic modification disrupts synthesis of a
functional nucleic acid encoding the activity or produces a
nonfunctional polypeptide or protein. Nucleic acid sequences that
can be used to reduce or eliminate the activity of
6-phosphogluconate dehydrogenase (decarboxylating) can have
sequences partially or substantially complementary to nucleic acid
sequences that encode 6-phosphogluconate dehydrogenase
(decarboxylating) activity. Presence or absence of the amount of
6-phosphogluconate dehydrogenase (decarboxylating) activity can be
detected by any suitable method known in the art, including nucleic
acid based analysis and western blot analysis.
[0126] The term "transketolase activity" as used herein refers to
conversion of xylulose-5-phosphate and ribose-5-phosphate to
sedoheptulose-7-phosphate and glyceraldehyde-3-phosphate. The
transketolase activity can be provided by a polypeptide. In some
embodiments, the polypeptide is encoded by a heterologous
nucleotide sequence introduced to a host microorganism. Nucleic
acid sequences conferring transketolase activity can be obtained
from a number of sources, including, but not limited to S.
cerevisiae, Kluyveromyces, Pichia, Escherichia, Bacillus,
Ruminococcus, Schizosaccharomyces, and Candida. Examples of an
amino acid sequence of a polypeptide having transketolase activity,
and a nucleotide sequence of a polynucleotide that encodes the
polypeptide, are presented below in the examples. The term
"inactivation of the conversion of xylulose-5-phosphate and
ribose-5-phosphate to sedoheptulose-7-phosphate and
glyceraldehyde-3-phosphate" refers to a reduction or elimination in
the activity of transketolase. Reducing or eliminating the activity
of transketolase activity can lead to an increase in ethanol
production. In some embodiments, the transketolase activity can be
reduced or eliminated by introduction of an untranslated RNA
molecule (e.g., antisense RNA, RNAi, and the like, for example). In
certain embodiments, the untranslated RNA is encoded by a
heterologous nucleotide sequence introduced to a host
microorganism.
[0127] In some embodiments, the transketolase activity can be
temporarily or permanently reduced or eliminated by genetic
modification, as described below. In certain embodiments, the
genetic modification renders the activity responsive to changes in
the environment. In some embodiments, the genetic modification
disrupts synthesis of a functional nucleic acid encoding the
activity or produces a nonfunctional polypeptide or protein.
Nucleic acid sequences that can be used to reduce or eliminate the
activity of transketolase can have sequences partially or
substantially complementary to nucleic acid sequences that encode
transketolase activity. Presence, absence or amount of
transketolase activity can be detected by any suitable method known
in the art, including nucleic acid based analysis and western blot
analysis.
[0128] The term "transaldolase activity" as used herein refers to
conversion of sedoheptulose 7-phosphate and glyceraldehyde
3-phosphate to erythrose 4-phosphate and fructose 6-phosphate. The
transaldolase activity can be provided by a polypeptide. In some
embodiments, the polypeptide is encoded by a heterologous
nucleotide sequence introduced to a host microorganism. Nucleic
acid sequences conferring transaldolase activity can be obtained
from a number of sources, including, but not limited to S.
cerevisiae, Kluyveromyces, Pichia, Escherichia, Bacillus,
Ruminococcus, Schizosaccharomyces, and Candida. Examples of an
amino acid sequence of a polypeptide having transaldolase activity,
and a nucleotide sequence of a polynucleotide that encodes the
polypeptide, are presented below in the examples. The term
"inactivation of the conversion of sedoheptulose 7-phosphate and
glyceraldehyde 3-phosphate to erythrose 4-phosphate and fructose
6-phosphate" refers to a reduction or elimination in the activity
of transaldolase. Reducing or eliminating the activity of
transaldolase activity can lead to an increase in ethanol
production. In some embodiments, the transaldolase activity can be
reduced or eliminated by introduction of an untranslated RNA
molecule (e.g., antisense RNA, RNAi, and the like, for example). In
certain embodiments, the untranslated RNA is encoded by a
heterologous nucleotide sequence introduced to a host
microorganism.
[0129] In some embodiments, the transaldolase activity can be
temporarily or permanently reduced or eliminated by genetic
modification, as described below. In certain embodiments, the
genetic modification renders the activity responsive to changes in
the environment. In some embodiments, the genetic modification
disrupts synthesis of a functional nucleic acid encoding the
activity or produces a nonfunctional polypeptide or protein.
Nucleic acid sequences that can be used to reduce or eliminate the
activity of transaldolase can have sequences partially or
substantially complementary to nucleic acid sequences that encode
transaldolase activity. Presence, absence or amount of
transaldolase activity can be detected by any suitable method known
in the art, including nucleic acid based analysis and western blot
analysis.
[0130] The term "galactose permease activity" as used herein refers
to the import of galactose into a cell or organism by an activity
that transports galactose across cell membranes. The galactose
permease activity can be provided by a polypeptide. In some
embodiments, the polypeptide is encoded by a heterologous
nucleotide sequence introduced to a host microorganism. Nucleic
acid sequences conferring galactose permease activity can be
obtained from a number of sources, including, but not limited to S.
cerevisiae, Candida albicans, Debaryomyces hansenii,
Schizosaccharomyces pombe, Arabidopsis thaliana, and Colweffia
psychrerythraea. Examples of an amino acid sequence of a
polypeptide having galactose permease activity, and a nucleotide
sequence of a polynucleotide that encodes the polypeptide, are
presented below in the Examples. Presence, absence or amount of
galactose permease activity can be detected by any suitable method
known in the art, including nucleic acid based analysis and western
blot analysis.
[0131] The term "glucose/xylose transport activity" as used herein
refers to the import of glucose and/or xylose into a cell or
organism by an activity that transports glucose and/or xylose
across cell membranes. The glucose/xylose transport activity can be
provided by a polypeptide. In some embodiments, the polypeptide is
encoded by a heterologous nucleotide sequence introduced to a host
microorganism. Nucleic acid sequences conferring glucose/xylose
transport activity can be obtained from a number of sources,
including, but not limited to Pichia yeast, S. cerevisiae, Candida
albicans, Debaryomyces hansenii, Schizosaccharomyces pombe,
Arabidopsis thaliana, and Colweffia psychrerythraea. Examples of an
amino acid sequence of a polypeptide having glucose/xylose
transport activity, and a nucleotide sequence of a polynucleotide
that encodes the polypeptide, are presented below in the Examples.
Presence, absence or amount of glucose/xylose transport activity
can be detected by any suitable method known in the art, including
nucleic acid based analysis and western blot analysis.
[0132] The terms "high affinity glucose transport activity" and
"hexose transport activity" as used herein refer to the import of
glucose and other hexose sugars into a cell or organism by an
activity that transports glucose and other hexose sugars across
cell membranes. The high affinity glucose transport activity or
hexose transport activity can be provided by a polypeptide. In some
embodiments, the polypeptide is encoded by a heterologous
nucleotide sequence introduced to a host microorganism. Nucleic
acid sequences conferring high affinity glucose transport activity
or hexose transport activity can be obtained from a number of
sources, including, but not limited to S. cerevisiae, Candida
albicans, Debaryomyces hansenii, Schizosaccharomyces pombe,
Arabidopsis thaliana, and Colweffia psychrerythraea. Presence,
absence or amount of glucose/xylose transport activity can be
detected by any suitable method known in the art, including nucleic
acid based analysis and western blot analysis.
[0133] The term "xylose reductase activity" as used herein refers
to the conversion of xylose to xylitol. In some embodiments, the
polypeptide is encoded by a heterologous nucleotide sequence
introduced to a host microorganism. Nucleic acid sequences
conferring xylose reductase activity can be obtained from a number
of sources. Presence, absence or amount of xylose reductase
activity can be detected by any suitable method known in the art,
including activity assays, nucleic acid based analysis and western
blot analysis.
[0134] The term "xylitol dehydrogenase activity" as used herein
refers to the conversion of xylitol to xylulose. In some
embodiments, the polypeptide is encoded by a heterologous
nucleotide sequence introduced to a host microorganism. Nucleic
acid sequences conferring xylitol dehydrogenase activity can be
obtained from a number of sources. Presence, absence or amount of
xylitol dehydrogenase activity can be detected by any suitable
method known in the art, including activity assays, nucleic acid
based analysis and western blot analysis.
[0135] The term "xylulokinase activity" as used herein refers to
the conversion of xylulose to xylulose-5-phosphate. In some
embodiments, the polypeptide is encoded by a heterologous
nucleotide sequence introduced to a host microorganism. Nucleic
acid sequences conferring xylulokinase activity can be obtained
from a number of sources. Presence, absence or amount of
xylulokinase activity can be detected by any suitable method known
in the art, including activity assays, nucleic acid based analysis
and western blot analysis.
[0136] Activities described herein can be modified to generate
microorganisms engineered to allow a method of independently
regulating or controlling (e.g., ability to independently turn on
or off, or increase or decrease, for example) six-carbon sugar
metabolism, five-carbon sugar metabolism, atmospheric carbon
metabolism (e.g., carbon dioxide fixation) or combinations thereof.
In some embodiments, regulated control of a desired activity can be
the result of a genetic modification. In certain embodiments, the
genetic modification can be modification of a promoter sequence. In
some embodiments the modification can increase of decrease an
activity encoded by a gene operably linked to the promoter element.
In certain embodiments, the modification to the promoter element
can add or remove a regulatory sequence. In some embodiments the
regulatory sequence can respond to a change in environmental or
culture conditions. Non-limiting examples of culture conditions
that could be used to regulate an activity in this manner include,
temperature, light, oxygen, salt, metals and the like. Additional
methods for altering an activity by modification of a promoter
element are given below.
[0137] In some embodiments, the genetic modification can be to an
ORF. In certain embodiments, the modification of the ORF can
increase or decrease expression of the ORF. In some embodiments
modification of the ORF can alter the efficiency of translation of
the ORF. In certain embodiments, modification of the ORF can alter
the activity of the polypeptide or protein encoded by the ORF.
Additional methods for altering an activity by modification of an
ORF are given below.
[0138] In some embodiments, the genetic modification can be to an
activity associated with cell division (e.g., cell division cycle
or CDC activity, for example). In certain embodiments the cell
division cycle activity can be thymidylate synthase activity. In
certain embodiments, regulated control of cell division can be the
result of a genetic modification. In some embodiments, the genetic
modification can be to a nucleic acid sequence that encodes
thymidylate synthase. In certain embodiments, the genetic
modification can temporarily inactivate thymidylate synthase
activity by rendering the activity temperature sensitive (e.g.,
heat resistant, heat sensitive, cold resistant, cold sensitive and
the like).
[0139] In some embodiments, the genetic modification can modify a
promoter sequence operably linked to a gene encoding an activity
involved in control of cell division. In some embodiments the
modification can increase of decrease an activity encoded by a gene
operably linked to the promoter element. In certain embodiments,
the modification to the promoter element can add or remove a
regulatory sequence. In some embodiments the regulatory sequence
can respond to a change in environmental or culture conditions.
Non-limiting examples of culture conditions that could be used to
regulate an activity in this manner include, temperature, light,
oxygen, salt, metals and the like. In some embodiments, an
engineered microorganism comprising one or more activities
described above or below can be used in to produce ethanol by
inhibiting cell growth and cell division by use of a temperature
sensitive cell division control activity while allowing cellular
fermentation to proceed, thereby producing a significant increase
in ethanol yield when compared to the native organism.
Polynucleotides and Polypeptides
[0140] A nucleic acid (e.g., also referred to herein as nucleic
acid reagent, target nucleic acid, target nucleotide sequence,
nucleic acid sequence of interest or nucleic acid region of
interest) can be from any source or composition, such as DNA, cDNA,
gDNA (genomic DNA), RNA, siRNA (short inhibitory RNA), RNAi, tRNA
or mRNA, for example, and can be in any form (e.g., linear,
circular, supercoiled, single-stranded, double-stranded, and the
like). A nucleic acid can also comprise DNA or RNA analogs (e.g.,
containing base analogs, sugar analogs and/or a non-native backbone
and the like). It is understood that the term "nucleic acid" does
not refer to or infer a specific length of the polynucleotide
chain, thus polynucleotides and oligonucleotides are also included
in the definition. Deoxyribonucleotides include deoxyadenosine,
deoxycytidine, deoxyguanosine and deoxythymidine. For RNA, the
uracil base is uridine.
[0141] A nucleic acid sometimes is a plasmid, phage, autonomously
replicating sequence (ARS), centromere, artificial chromosome,
yeast artificial chromosome (e.g., YAC) or other nucleic acid able
to replicate or be replicated in a host cell. In certain
embodiments a nucleic acid can be from a library or can be obtained
from enzymatically digested, sheared or sonicated genomic DNA
(e.g., fragmented) from an organism of interest. In some
embodiments, nucleic acid subjected to fragmentation or cleavage
may have a nominal, average or mean length of about 5 to about
10,000 base pairs, about 100 to about 1,000 base pairs, about 100
to about 500 base pairs, or about 10, 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500,
600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,
9000 or 10000 base pairs. Fragments can be generated by any
suitable method in the art, and the average, mean or nominal length
of nucleic acid fragments can be controlled by selecting an
appropriate fragment-generating procedure by the person of ordinary
skill. In some embodiments, the fragmented DNA can be size selected
to obtain nucleic acid fragments of a particular size range.
[0142] Nucleic acid can be fragmented by various methods known to
the person of ordinary skill, which include without limitation,
physical, chemical and enzymic processes. Examples of such
processes are described in U.S. Patent Application Publication No.
20050112590 (published on May 26, 2005, entitled
"Fragmentation-based methods and systems for sequence variation
detection and discovery," naming Van Den Boom et al.). Certain
processes can be selected by the person of ordinary skill to
generate non-specifically cleaved fragments or specifically cleaved
fragments. Examples of processes that can generate non-specifically
cleaved fragment sample nucleic acid include, without limitation,
contacting sample nucleic acid with apparatus that expose nucleic
acid to shearing force (e.g., passing nucleic acid through a
syringe needle; use of a French press); exposing sample nucleic
acid to irradiation (e.g., gamma, x-ray, UV irradiation; fragment
sizes can be controlled by irradiation intensity); boiling nucleic
acid in water (e.g., yields about 500 base pair fragments) and
exposing nucleic acid to an acid and base hydrolysis process.
[0143] Nucleic acid may be specifically cleaved by contacting the
nucleic acid with one or more specific cleavage agents. The term
"specific cleavage agent" as used herein refers to an agent,
sometimes a chemical or an enzyme that can cleave a nucleic acid at
one or more specific sites. Specific cleavage agents often will
cleave specifically according to a particular nucleotide sequence
at a particular site. Examples of enzymic specific cleavage agents
include without limitation endonucleases (e.g., DNase (e.g., DNase
I, II); RNase (e.g., RNase E, F, H, P); Cleavase.TM. enzyme; Taq
DNA polymerase; E. coli DNA polymerase I and eukaryotic
structure-specific endonucleases; murine FEN-1 endonucleases; type
I, II or III restriction endonucleases such as Acc I, Afl III, Alu
I, Alw44 I, Apa I, Asn I, Ava I, Ava II, BamH I, Ban II, Bcl I, Bgl
I. Bgl II, Bln I, Bsm I, BssH II, BstE II, Cfo I, Cla I, Dde I, Dpn
I, Dra I, EcIX I, EcoR I, EcoR I, EcoR II, EcoR V, Hae II, Hae II,
Hind III, Hind III, Hpa I, Hpa II, Kpn I, Ksp I, Mlu I, MIuN I, Msp
I, Nci I, Nco I, Nde I, Nde II, Nhe I, Not I, Nru I, Nsi I, Pst I,
Pvu I, Pvu II, Rsa I, Sac I, Sal I, Sau3A I, Sca I, ScrF I, Sfi I,
Sma I, Spe I, Sph I, Ssp I, Stu I, Sty I, Swa I, Taq I, Xba I, Xho
I); glycosylases (e.g., uracil-DNA glycolsylase (UDG),
3-methyladenine DNA glycosylase, 3-methyladenine DNA glycosylase
II, pyrimidine hydrate-DNA glycosylase, FaPy-DNA glycosylase,
thymine mismatch-DNA glycosylase, hypoxanthine-DNA glycosylase,
5-Hydroxymethyluracil DNA glycosylase (HmUDG),
5-Hydroxymethylcytosine DNA glycosylase, or 1,N6-etheno-adenine DNA
glycosylase); exonucleases (e.g., exonuclease III); ribozymes, and
DNAzymes. Sample nucleic acid may be treated with a chemical agent,
or synthesized using modified nucleotides, and the modified nucleic
acid may be cleaved. In non-limiting examples, sample nucleic acid
may be treated with (i) alkylating agents such as methylnitrosourea
that generate several alkylated bases, including N3-methyladenine
and N3-methylguanine, which are recognized and cleaved by alkyl
purine DNA-glycosylase; (ii) sodium bisulfite, which causes
deamination of cytosine residues in DNA to form uracil residues
that can be cleaved by uracil N-glycosylase; and (iii) a chemical
agent that converts guanine to its oxidized form, 8-hydroxyguanine,
which can be cleaved by formamidopyrimidine DNA N-glycosylase.
Examples of chemical cleavage processes include without limitation
alkylation, (e.g., alkylation of phosphorothioate-modified nucleic
acid); cleavage of acid lability of
P3'-N5'-phosphoroamidate-containing nucleic acid; and osmium
tetroxide and piperidine treatment of nucleic acid.
[0144] As used herein, the term "complementary cleavage reactions"
refers to cleavage reactions that are carried out on the same
nucleic acid using different cleavage reagents or by altering the
cleavage specificity of the same cleavage reagent such that
alternate cleavage patterns of the same target or reference nucleic
acid or protein are generated. In certain embodiments, nucleic
acids of interest may be treated with one or more specific cleavage
agents (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more specific
cleavage agents) in one or more reaction vessels (e.g., nucleic
acid of interest is treated with each specific cleavage agent in a
separate vessel).
[0145] A nucleic acid suitable for use in the embodiments described
herein sometimes is amplified by any amplification process known in
the art (e.g., PCR, RT-PCR and the like). Nucleic acid
amplification may be particularly beneficial when using organisms
that are typically difficult to culture (e.g., slow growing,
require specialize culture conditions and the like). The terms
"amplify", "amplification", "amplification reaction", or
"amplifying" as used herein refer to any in vitro processes for
multiplying the copies of a target sequence of nucleic acid.
Amplification sometimes refers to an "exponential" increase in
target nucleic acid. However, "amplifying" as used herein can also
refer to linear increases in the numbers of a select target
sequence of nucleic acid, but is different than a one-time, single
primer extension step. In some embodiments, a limited amplification
reaction, also known as pre-amplification, can be performed.
Pre-amplification is a method in which a limited amount of
amplification occurs due to a small number of cycles, for example
10 cycles, being performed. Pre-amplification can allow some
amplification, but stops amplification prior to the exponential
phase, and typically produces about 500 copies of the desired
nucleotide sequence(s). Use of pre-amplification may also limit
inaccuracies associated with depleted reactants in standard PCR
reactions.
[0146] In some embodiments, a nucleic acid reagent sometimes is
stably integrated into the chromosome of the host organism, or a
nucleic acid reagent can be a deletion of a portion of the host
chromosome, in certain embodiments (e.g., genetically modified
organisms, where alteration of the host genome confers the ability
to selectively or preferentially maintain the desired organism
carrying the genetic modification). Such nucleic acid reagents
(e.g., nucleic acids or genetically modified organisms whose
altered genome confers a selectable trait to the organism) can be
selected for their ability to guide production of a desired protein
or nucleic acid molecule. When desired, the nucleic acid reagent
can be altered such that codons encode for (i) the same amino acid,
using a different tRNA than that specified in the native sequence,
or (ii) a different amino acid than is normal, including
unconventional or unnatural amino acids (including detectably
labeled amino acids). As described herein, the term "native
sequence" refers to an unmodified nucleotide sequence as found in
its natural setting (e.g., a nucleotide sequence as found in an
organism).
[0147] A nucleic acid or nucleic acid reagent can comprise certain
elements often selected according to the intended use of the
nucleic acid. Any of the following elements can be included in or
excluded from a nucleic acid reagent. A nucleic acid reagent, for
example, may include one or more or all of the following nucleotide
elements: one or more promoter elements, one or more 5'
untranslated regions (5'UTRs), one or more regions into which a
target nucleotide sequence may be inserted (an "insertion
element"), one or more target nucleotide sequences, one or more 3'
untranslated regions (3'UTRs), and one or more selection elements.
A nucleic acid reagent can be provided with one or more of such
elements and other elements may be inserted into the nucleic acid
before the nucleic acid is introduced into the desired organism. In
some embodiments, a provided nucleic acid reagent comprises a
promoter, 5'UTR, optional 3'UTR and insertion element(s) by which a
target nucleotide sequence is inserted (i.e., cloned) into the
nucleotide acid reagent. In certain embodiments, a provided nucleic
acid reagent comprises a promoter, insertion element(s) and
optional 3'UTR, and a 5' UTR/target nucleotide sequence is inserted
with an optional 3'UTR. The elements can be arranged in any order
suitable for expression in the chosen expression system (e.g.,
expression in a chosen organism, or expression in a cell free
system, for example), and in some embodiments a nucleic acid
reagent comprises the following elements in the 5' to 3' direction:
(1) promoter element, 5'UTR, and insertion element(s); (2) promoter
element, 5'UTR, and target nucleotide sequence; (3) promoter
element, 5'UTR, insertion element(s) and 3'UTR; and (4) promoter
element, 5'UTR, target nucleotide sequence and 3'UTR.
[0148] A promoter element typically is required for DNA synthesis
and/or RNA synthesis. A promoter element often comprises a region
of DNA that can facilitate the transcription of a particular gene,
by providing a start site for the synthesis of RNA corresponding to
a gene. Promoters generally are located near the genes they
regulate, are located upstream of the gene (e.g., 5' of the gene),
and are on the same strand of DNA as the sense strand of the gene,
in some embodiments. A promoter often interacts with a RNA
polymerase. A polymerase is an enzyme that catalyses synthesis of
nucleic acids using a preexisting nucleic acid reagent. When the
template is a DNA template, an RNA molecule is transcribed before
protein is synthesized. Enzymes having polymerase activity suitable
for use in the present methods include any polymerase that is
active in the chosen system with the chosen template to synthesize
protein. In some embodiments, a promoter (e.g., a heterologous
promoter) also referred to herein as a promoter element, can be
operably linked to a nucleotide sequence or an open reading frame
(ORF). Transcription from the promoter element can catalyze the
synthesis of an RNA corresponding to the nucleotide sequence or ORF
sequence operably linked to the promoter, which in turn leads to
synthesis of a desired peptide, polypeptide or protein. The term
"operably linked" as used herein with respect to promoters refers
to a nucleic acid sequence (e.g., a coding sequence) present on the
same nucleic acid molecule as a promoter element and whose
expression is under the control of said promoter element.
[0149] Promoter elements sometimes exhibit responsiveness to
regulatory control. Promoter elements also sometimes can be
regulated by a selective agent. That is, transcription from
promoter elements sometimes can be turned on, turned off,
up-regulated or down-regulated, in response to a change in
environmental, nutritional or internal conditions or signals (e.g.,
heat inducible promoters, light regulated promoters, feedback
regulated promoters, hormone influenced promoters, tissue specific
promoters, oxygen and pH influenced promoters, promoters that are
responsive to selective agents (e.g., kanamycin) and the like, for
example). Promoters influenced by environmental, nutritional or
internal signals frequently are influenced by a signal (direct or
indirect) that binds at or near the promoter and increases or
decreases expression of the target sequence under certain
conditions.
[0150] Non-limiting examples of selective or regulatory agents that
can influence transcription from a promoter element used in
embodiments described herein include, without limitation, (1)
nucleic acid segments that encode products that provide resistance
against otherwise toxic compounds (e.g., antibiotics); (2) nucleic
acid segments that encode products that are otherwise lacking in
the recipient cell (e.g., essential products, tRNA genes,
auxotrophic markers); (3) nucleic acid segments that encode
products that suppress the activity of a gene product; (4) nucleic
acid segments that encode products that can be readily identified
(e.g., phenotypic markers such as antibiotics (e.g.,
.beta.-lactamase), .beta.-galactosidase, green fluorescent protein
(GFP), yellow fluorescent protein (YFP), red fluorescent protein
(RFP), cyan fluorescent protein (CFP), and cell surface proteins);
(5) nucleic acid segments that bind products that are otherwise
detrimental to cell survival and/or function; (6) nucleic acid
segments that otherwise inhibit the activity of any of the nucleic
acid segments described in Nos. 1-5 above (e.g., antisense
oligonucleotides); (7) nucleic acid segments that bind products
that modify a substrate (e.g., restriction endonucleases); (8)
nucleic acid segments that can be used to isolate or identify a
desired molecule (e.g., specific protein binding sites); (9)
nucleic acid segments that encode a specific nucleotide sequence
that can be otherwise non-functional (e.g., for PCR amplification
of subpopulations of molecules); (10) nucleic acid segments that,
when absent, directly or indirectly confer resistance or
sensitivity to particular compounds; (11) nucleic acid segments
that encode products that either are toxic or convert a relatively
non-toxic compound to a toxic compound (e.g., Herpes simplex
thymidine kinase, cytosine deaminase) in recipient cells; (12)
nucleic acid segments that inhibit replication, partition or
heritability of nucleic acid molecules that contain them; and/or
(13) nucleic acid segments that encode conditional replication
functions, e.g., replication in certain hosts or host cell strains
or under certain environmental conditions (e.g., temperature,
nutritional conditions, and the like). In some embodiments, the
regulatory or selective agent can be added to change the existing
growth conditions to which the organism is subjected (e.g., growth
in liquid culture, growth in a fermentor, growth on solid nutrient
plates and the like for example).
[0151] In some embodiments, regulation of a promoter element can be
used to alter (e.g., increase, add, decrease or substantially
eliminate) the activity of a peptide, polypeptide or protein (e.g.,
enzyme activity for example). For example, a microorganism can be
engineered by genetic modification to express a nucleic acid
reagent that can add a novel activity (e.g., an activity not
normally found in the host organism) or increase the expression of
an existing activity by increasing transcription from a homologous
or heterologous promoter operably linked to a nucleotide sequence
of interest (e.g., homologous or heterologous nucleotide sequence
of interest), in certain embodiments. In some embodiments, a
microorganism can be engineered by genetic modification to express
a nucleic acid reagent that can decrease expression of an activity
by decreasing or substantially eliminating transcription from a
homologous or heterologous promoter operably linked to a nucleotide
sequence of interest, in certain embodiments.
[0152] In some embodiments the activity can be altered using
recombinant DNA and genetic techniques known to the artisan.
Methods for engineering microorganisms are further described
herein. Tables herein provide non-limiting lists of yeast promoters
that are up-regulated by oxygen, yeast promoters that are
down-regulated by oxygen, yeast transcriptional repressors and
their associated genes, DNA binding motifs as determined using the
MEME sequence analysis software. Potential regulator binding motifs
can be identified using the program MEME to search intergenic
regions bound by regulators for overrepresented sequences. For each
regulator, the sequences of intergenic regions bound with p-values
less than 0.001 were extracted to use as input for motif discovery.
The MEME software was run using the following settings: a motif
width ranging from 6 to 18 bases, the "zoops" distribution model, a
6th order Markov background model and a discovery limit of 20
motifs. The discovered sequence motifs were scored for significance
by two criteria: an E-value calculated by MEME and a specificity
score. The motif with the best score using each metric is shown for
each regulator. All motifs presented are derived from datasets
generated in rich growth conditions with the exception of a
previously published dataset for epitope-tagged Gal4 grown in
galactose
[0153] In some embodiments, the altered activity can be found by
screening the organism under conditions that select for the desired
change in activity. For example, certain microorganisms can be
adapted to increase or decrease an activity by selecting or
screening the organism in question on a media containing substances
that are poorly metabolized or even toxic. An increase in the
ability of an organism to grow a substance that is normally poorly
metabolized would result in an increase in the growth rate on that
substance, for example. A decrease in the sensitivity to a toxic
substance might be manifested by growth on higher concentrations of
the toxic substance, for example. Genetic modifications that are
identified in this manner sometimes are referred to as naturally
occurring mutations or the organisms that carry them can sometimes
be referred to as naturally occurring mutants. Modifications
obtained in this manner are not limited to alterations in promoter
sequences. That is, screening microorganisms by selective pressure,
as described above, can yield genetic alterations that can occur in
non-promoter sequences, and sometimes also can occur in sequences
that are not in the nucleotide sequence of interest, but in a
related nucleotide sequences (e.g., a gene involved in a different
step of the same pathway, a transport gene, and the like).
Naturally occurring mutants sometimes can be found by isolating
naturally occurring variants from unique environments, in some
embodiments.
[0154] In addition to the regulated promoter sequences, regulatory
sequences, and coding polynucleotides provided herein, a nucleic
acid reagent may include a polynucleotide sequence 70% or more
identical to the foregoing (or to the complementary sequences).
That is, a nucleotide sequence that is at least 70% or more, 71% or
more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or
more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or
more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or
more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or
more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or
more, 97% or more, 98% or more, or 99% or more identical to a
nucleotide sequence described herein can be utilized. The term
"identical" as used herein refers to two or more nucleotide
sequences having substantially the same nucleotide sequence when
compared to each other. One test for determining whether two
nucleotide sequences or amino acids sequences are substantially
identical is to determine the percent of identical nucleotide
sequences or amino acid sequences shared.
[0155] Calculations of sequence identity can be performed as
follows. Sequences are aligned for optimal comparison purposes
(e.g., gaps can be introduced in one or both of a first and a
second amino acid or nucleic acid sequence for optimal alignment
and non-homologous sequences can be disregarded for comparison
purposes). The length of a reference sequence aligned for
comparison purposes is sometimes 30% or more, 40% or more, 50% or
more, often 60% or more, and more often 70% or more, 80% or more,
90% or more, or 100% of the length of the reference sequence. The
nucleotides or amino acids at corresponding nucleotide or
polypeptide positions, respectively, are then compared among the
two sequences. When a position in the first sequence is occupied by
the same nucleotide or amino acid as the corresponding position in
the second sequence, the nucleotides or amino acids are deemed to
be identical at that position. The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences, taking into account the number of gaps, and the
length of each gap, introduced for optimal alignment of the two
sequences.
[0156] Comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. Percent identity between two amino acid or
nucleotide sequences can be determined using the algorithm of
Meyers & Miller, CABIOS 4: 11-17 (1989), which has been
incorporated into the ALIGN program (version 2.0), using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4. Also, percent identity between two amino acid sequences can
be determined using the Needleman & Wunsch, J. Mol. Biol. 48:
444-453 (1970) algorithm which has been incorporated into the GAP
program in the GCG software package (available at the http address
www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix,
and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight
of 1, 2, 3, 4, 5, or 6. Percent identity between two nucleotide
sequences can be determined using the GAP program in the GCG
software package (available at http address www.gcg.com), using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and
a length weight of 1, 2, 3, 4, 5, or 6. A set of parameters often
used is a Blossum 62 scoring matrix with a gap open penalty of 12,
a gap extend penalty of 4, and a frameshift gap penalty of 5.
[0157] Sequence identity can also be determined by hybridization
assays conducted under stringent conditions. As use herein, the
term "stringent conditions" refers to conditions for hybridization
and washing. Stringent conditions are known to those skilled in the
art and can be found in Current Protocols in Molecular Biology,
John Wiley & Sons, N.Y., 6.3.1-6.3.6 (1989). Aqueous and
non-aqueous methods are described in that reference and either can
be used. An example of stringent hybridization conditions is
hybridization in 6.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by one or more washes in
0.2.times.SSC, 0.1% SDS at 50.degree. C. Another example of
stringent hybridization conditions are hybridization in 6.times.
sodium chloride/sodium citrate (SSC) at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
55.degree. C. A further example of stringent hybridization
conditions is hybridization in 6.times. sodium chloride/sodium
citrate (SSC) at about 45.degree. C., followed by one or more
washes in 0.2.times.SSC, 0.1% SDS at 60.degree. C. Often, stringent
hybridization conditions are hybridization in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by
one or more washes in 0.2.times.SSC, 0.1% SDS at 65.degree. C. More
often, stringency conditions are 0.5M sodium phosphate, 7% SDS at
65.degree. C., followed by one or more washes at 0.2.times.SSC, 1%
SDS at 65.degree. C.
[0158] As noted above, nucleic acid reagents may also comprise one
or more 5' UTR's, and one or more 3'UTR's. A 5' UTR may comprise
one or more elements endogenous to the nucleotide sequence from
which it originates, and sometimes includes one or more exogenous
elements. A 5' UTR can originate from any suitable nucleic acid,
such as genomic DNA, plasmid DNA, RNA or mRNA, for example, from
any suitable organism (e.g., virus, bacterium, yeast, fungi, plant,
insect or mammal). The artisan may select appropriate elements for
the 5' UTR based upon the chosen expression system (e.g.,
expression in a chosen organism, or expression in a cell free
system, for example). A 5' UTR sometimes comprises one or more of
the following elements known to the artisan: enhancer sequences
(e.g., transcriptional or translational), transcription initiation
site, transcription factor binding site, translation regulation
site, translation initiation site, translation factor binding site,
accessory protein binding site, feedback regulation agent binding
sites, Pribnow box, TATA box, -35 element, E-box (helix-loop-helix
binding element), ribosome binding site, replicon, internal
ribosome entry site (IRES), silencer element and the like. In some
embodiments, a promoter element may be isolated such that all 5'
UTR elements necessary for proper conditional regulation are
contained in the promoter element fragment, or within a functional
subsequence of a promoter element fragment.
[0159] A 5'UTR in the nucleic acid reagent can comprise a
translational enhancer nucleotide sequence. A translational
enhancer nucleotide sequence often is located between the promoter
and the target nucleotide sequence in a nucleic acid reagent. A
translational enhancer sequence often binds to a ribosome,
sometimes is an 18S rRNA-binding ribonucleotide sequence (i.e., a
40S ribosome binding sequence) and sometimes is an internal
ribosome entry sequence (IRES). An IRES generally forms an RNA
scaffold with precisely placed RNA tertiary structures that contact
a 40S ribosomal subunit via a number of specific intermolecular
interactions. Examples of ribosomal enhancer sequences are known
and can be identified by the artisan (e.g., Mignone et al., Nucleic
Acids Research 33: D141-D146 (2005); Paulous et al., Nucleic Acids
Research 31: 722-733 (2003); Akbergenov et al., Nucleic Acids
Research 32: 239-247 (2004); Mignone et al., Genome Biology 3(3):
reviews0004.1-0001.10 (2002); Gallie, Nucleic Acids Research 30:
3401-3411 (2002); Shaloiko et al., http address
www.interscience.wiley.com, DOI: 10.1002/bit.20267; and Gallie et
al., Nucleic Acids Research 15: 3257-3273 (1987)).
[0160] A translational enhancer sequence sometimes is a eukaryotic
sequence, such as a Kozak consensus sequence or other sequence
(e.g., hydroid polyp sequence, GenBank accession no. U07128). A
translational enhancer sequence sometimes is a prokaryotic
sequence, such as a Shine-Dalgarno consensus sequence. In certain
embodiments, the translational enhancer sequence is a viral
nucleotide sequence. A translational enhancer sequence sometimes is
from a 5'UTR of a plant virus, such as Tobacco Mosaic Virus (TMV),
Alfalfa Mosaic Virus (AMV); Tobacco Etch Virus (ETV); Potato Virus
Y (PVY); Turnip Mosaic (poty) Virus and Pea Seed Borne Mosaic
Virus, for example. In certain embodiments, an omega sequence about
67 bases in length from TMV is included in the nucleic acid reagent
as a translational enhancer sequence (e.g., devoid of guanosine
nucleotides and includes a 25 nucleotide long poly (CAA) central
region).
[0161] A 3' UTR may comprise one or more elements endogenous to the
nucleotide sequence from which it originates and sometimes includes
one or more exogenous elements. A 3' UTR may originate from any
suitable nucleic acid, such as genomic DNA, plasmid DNA, RNA or
mRNA, for example, from any suitable organism (e.g., a virus,
bacterium, yeast, fungi, plant, insect or mammal). The artisan can
select appropriate elements for the 3' UTR based upon the chosen
expression system (e.g., expression in a chosen organism, for
example). A 3' UTR sometimes comprises one or more of the following
elements known to the artisan: transcription regulation site,
transcription initiation site, transcription termination site,
transcription factor binding site, translation regulation site,
translation termination site, translation initiation site,
translation factor binding site, ribosome binding site, replicon,
enhancer element, silencer element and polyadenosine tail. A 3' UTR
often includes a polyadenosine tail and sometimes does not, and if
a polyadenosine tail is present, one or more adenosine moieties may
be added or deleted from it (e.g., about 5, about 10, about 15,
about 20, about 25, about 30, about 35, about 40, about 45 or about
50 adenosine moieties may be added or subtracted).
[0162] In some embodiments, modification of a 5' UTR and/or a 3'
UTR can be used to alter (e.g., increase, add, decrease or
substantially eliminate) the activity of a promoter. Alteration of
the promoter activity can in turn alter the activity of a peptide,
polypeptide or protein (e.g., enzyme activity for example), by a
change in transcription of the nucleotide sequence(s) of interest
from an operably linked promoter element comprising the modified 5'
or 3' UTR. For example, a microorganism can be engineered by
genetic modification to express a nucleic acid reagent comprising a
modified 5' or 3' UTR that can add a novel activity (e.g., an
activity not normally found in the host organism) or increase the
expression of an existing activity by increasing transcription from
a homologous or heterologous promoter operably linked to a
nucleotide sequence of interest (e.g., homologous or heterologous
nucleotide sequence of interest), in certain embodiments. In some
embodiments, a microorganism can be engineered by genetic
modification to express a nucleic acid reagent comprising a
modified 5' or 3' UTR that can decrease the expression of an
activity by decreasing or substantially eliminating transcription
from a homologous or heterologous promoter operably linked to a
nucleotide sequence of interest, in certain embodiments.
[0163] A nucleotide reagent sometimes can comprise a target
nucleotide sequence. A "target nucleotide sequence" as used herein
encodes a nucleic acid, peptide, polypeptide or protein of
interest, and may be a ribonucleotide sequence or a
deoxyribonucleotide sequence.
[0164] A target nucleic acid sometimes can comprise a chimeric
nucleic acid (or chimeric nucleotide sequence), which can encode a
chimeric protein (or chimeric amino acid sequence). The term
"chimeric" as used herein refers to a nucleic acid or nucleotide
sequence, or encoded product thereof, containing sequences from two
or more different sources. Any suitable source can be selected,
including, but not limited to, a sequence from a nucleic acid,
nucleotide sequence, ribosomal nucleic acid, RNA, DNA, regulatory
nucleotide sequence (e.g., promoter, URL, enhancer, repressor and
the like), coding nucleic acid, gene, nucleic acid linker, nucleic
acid tag, amino acid sequence, peptide, polypeptide, protein,
chromosome, and organism. A chimeric molecule can include a
sequence of contiguous nucleotides or amino acids from a source
including, but not limited to, a virus, prokaryote, eukaryote,
genus, species, homolog, ortholog, paralog and isozyme, nucleic
acid linkers, nucleic acid tags, the like and combinations
thereof). A chimeric molecule can be generated by placing in
juxtaposition fragments of related or unrelated nucleic acids,
nucleotide sequences or DNA segments, in some embodiments. In
certain embodiments the nucleic acids, nucleotide sequences or DNA
segments can be native or wild type sequences, mutant sequences or
engineered sequences (completely engineered or engineered to a
point, for example).
[0165] In some embodiments, a chimera includes about 1, 2, 3, 4 or
5 sequences (e.g., contiguous nucleotides, contiguous amino acids)
from one organism and 1, 2, 3, 4 or 5 sequences (e.g., contiguous
nucleotides, contiguous amino acids) from another organism. The
organisms sometimes are a microbe, such as a bacterium (e.g., gram
positive, gram negative), yeast or fungus (e.g., aerobic fungus,
anaerobic fungus), for example. In some embodiments, the organisms
are bacteria, the organisms are yeast or the organisms are fungi
(e.g., different species), and sometimes one organism is a
bacterium or yeast and another is a fungus. A chimeric molecule may
contain up to about 99% of sequences from one organism (e.g., about
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99%) and the balance
percentage from one or more other organisms. In certain
embodiments, a chimeric molecule includes altered codons (in the
case of a chimeric nucleic acid) and one or more mutations (e.g.,
point mutations, nucleotide substitutions, amino acid
substitutions). In some embodiments, the chimera comprises a
portion of a xylose isomerase from one bacteria species and a
portion of a xylose isomerase from another bacteria species. In
still other embodiments, the chimera comprises a portion of a
xylose isomerase from one species of fungus and another portion of
a xylose isomerase from another species of fungus. In still other
embodiments, the chimera comprises one portion of a xylose
isomerase from a plant, and another portion of a xylose isomerase
from a non-plant (such as a bacteria or fungus).
[0166] In other embodiments, the chimera comprises one portion of a
xylose isomerase from a plant, another portion of a xylose
isomerase from a bacteria, and yet another portion of a xylose
isomerase from a fungus.
[0167] In specific embodiments, a gene encoding a xylose isomerase
protein is chimeric, and includes a portion of a xylose isomerase
encoding sequence from one organism (e.g. a fungus (e.g.,
Piromyces, Orpinomyces, Neocallimastix, Caecomyces, Ruminomyces,
and the like)) and a portion of a xylose isomerase encoding
sequence from another organism (e.g., bacterium (e.g.,
Ruminococcus, Thermotoga, Clostridium)). Sometimes a fungal
sequence is located at the N-terminal portion of the encoded xylose
isomerase polypeptide and the bacterial sequence is located at the
C-terminal portion of the polypeptide. In some embodiments one
contiguous fungal xylose isomerase sequence is about 1% to about
30% of overall sequence (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29%) and the remaining sequence is a contiguous bacterial
xylose isomerase sequence. In certain embodiments, a chimeric
xylose isomerase includes one or more point mutations.
[0168] A chimera sometimes is the result of recombination between
two or more nucleic acids, nucleotide sequences or genes, and
sometimes is the result of genetic manipulation (e.g., designed
and/or generated by the hand of a human being). Any suitable
nucleic acid or nucleotide sequence and method for combining
nucleic acids or nucleotide sequences can be used to generate a
chimeric nucleic acid or nucleotide sequence. Non-limiting examples
of nucleic acid and nucleotide sequence sources and methods for
generating chimeric nucleic acids and nucleotide sequences are
presented herein.
[0169] In some embodiments, fragments used to generate a chimera
can be juxtaposed as units (e.g., nucleic acid from the sources are
combined end to end and not interspersed. In embodiments where a
chimera includes one stretch of contiguous nucleotides for each
organism, nucleotide sequence combinations can be noted as DNA
source 1DNA source 2 or DNA source 1/DNA source 2/DNA source 3, the
like and combinations thereof, for example. In certain embodiments,
fragments used to generate a chimera can be juxtaposed such that
one or more fragments from one or more sources can be interspersed
with other fragments used to generate the chimera (e.g., DNA source
1/DNA source 2/DNA source 1/DNA source 3/DNA source 2/DNA source
1). In some embodiments, the nucleotide sequence length of the
fragments used to generate a chimera can be in the range from about
5 base pairs to about 5,000 base pairs (e.g., about 5 base pairs
(bp), about 10 bp, about 15 bp, about 20 bp, about 25 bp, about 30
bp, about 35 bp, about 40 bp, about 45 bp, about 50 bp, about 55
bp, about 60 bp, about bp, about 65 bp, about 70 bp, about 75 bp,
about 80 bp, about 85 bp, about 90 bp, about 95 bp, about 100 bp,
about 125 bp, about 150 bp, about 175 bp, about 200 bp, about 250
bp, about 300 bp, about 350 bp, about 400 bp, about 450 bp, about
500 bp, about 550 bp, about 600 bp, about 650 bp, about 700 bp,
about 750 bp, about 800 bp, about 850 bp, about 900 bp, about 950
bp, about 1000 bp, about 1500 bp, about 2000 bp, about 2500 bp,
about 3000 bp, about 3500 bp, about 4000 bp, about 4500 bp, or
about 5000 bp).
[0170] In certain embodiments, a chimeric nucleic acid or
nucleotide sequence encodes the same activity as the activity
encoded by the source nucleic acids or nucleotide sequences. In
some embodiments, a chimeric nucleic acid or nucleotide sequence
has a similar or the same activity, but the amount of the activity,
or kinetics of the activity, are altered (e.g., increased,
decreased). In certain embodiments, a chimeric nucleic acid or
nucleotide sequence encodes a different activity, and in some
embodiments a chimeric nucleic acid or nucleotide sequences encodes
a chimeric activity (e.g., a combination of two or more
activities).
[0171] A target nucleic acid sometimes is an untranslated
ribonucleic acid and sometimes is a translated ribonucleic acid. An
untranslated ribonucleic acid may include, but is not limited to, a
small interfering ribonucleic acid (siRNA), a short hairpin
ribonucleic acid (shRNA), other ribonucleic acid capable of RNA
interference (RNAi), an antisense ribonucleic acid, or a ribozyme.
A translatable target nucleotide sequence (e.g., a target
ribonucleotide sequence) sometimes encodes a peptide, polypeptide
or protein, which are sometimes referred to herein as "target
peptides," "target polypeptides" or "target proteins."
[0172] Any peptides, polypeptides or proteins, or an activity
catalyzed by one or more peptides, polypeptides or proteins may be
encoded by a target nucleotide sequence and may be selected by a
person of ordinary skill in the art. Representative proteins
include enzymes (e.g., phosphofructokinase activity,
phosphogluconate dehydratase activity,
2-keto-3-deoxygluconate-6-phosphate aldolase activity, xylose
isomerase activity, phosphoenolpyruvate carboxylase activity,
alcohol dehydrogenase 2 activity and thymidylate synthase activity
and the like, for example), antibodies, serum proteins (e.g.,
albumin), membrane bound proteins, hormones (e.g., growth hormone,
erythropoietin, insulin, etc.), cytokines, etc., and include both
naturally occurring and exogenously expressed polypeptides.
Representative activities (e.g., enzymes or combinations of enzymes
which are functionally associated to provide an activity) include
phosphofructokinase activity, phosphogluconate dehydratase
activity, 2-keto-3-deoxygluconate-6-phosphate aldolase activity,
xylose isomerase activity, phosphoenolpyruvate carboxylase
activity, alcohol dehydrogenase 2 activity and thymidylate synthase
activity and the like for example. The term "enzyme" as used herein
refers to a protein which can act as a catalyst to induce a
chemical change in other compounds, thereby producing one or more
products from one or more substrates.
[0173] Specific polypeptides (e.g., enzymes) useful for embodiments
described herein are listed hereafter. The term "protein" as used
herein refers to a molecule having a sequence of amino acids linked
by peptide bonds. This term includes fusion proteins,
oligopeptides, peptides, cyclic peptides, polypeptides and
polypeptide derivatives, whether native or recombinant, and also
includes fragments, derivatives, homologs, and variants thereof. A
protein or polypeptide sometimes is of intracellular origin (e.g.,
located in the nucleus, cytosol, or interstitial space of host
cells in vivo) and sometimes is a cell membrane protein in vivo. In
some embodiments (described above, and in further detail below in
Engineering and Alteration Methods), a genetic modification can
result in a modification (e.g., increase, substantially increase,
decrease or substantially decrease) of a target activity.
[0174] A translatable nucleotide sequence generally is located
between a start codon (AUG in ribonucleic acids and ATG in
deoxyribonucleic acids) and a stop codon (e.g., UAA (ochre), UAG
(amber) or UGA (opal) in ribonucleic acids and TAA, TAG or TGA in
deoxyribonucleic acids), and sometimes is referred to herein as an
"open reading frame" (ORF). A nucleic acid reagent sometimes
comprises one or more ORFs. An ORF may be from any suitable source,
sometimes from genomic DNA, mRNA, reverse transcribed RNA or
complementary DNA (cDNA) or a nucleic acid library comprising one
or more of the foregoing, and is from any organism species that
contains a nucleic acid sequence of interest, protein of interest,
or activity of interest. Non-limiting examples of organisms from
which an ORF can be obtained include bacteria, yeast, fungi, human,
insect, nematode, bovine, equine, canine, feline, rat or mouse, for
example.
[0175] A nucleic acid reagent sometimes comprises a nucleotide
sequence adjacent to an ORF that is translated in conjunction with
the ORF and encodes an amino acid tag. The tag-encoding nucleotide
sequence is located 3' and/or 5' of an ORF in the nucleic acid
reagent, thereby encoding a tag at the C-terminus or N-terminus of
the protein or peptide encoded by the ORF. Any tag that does not
abrogate in vitro transcription and/or translation may be utilized
and may be appropriately selected by the artisan. Tags may
facilitate isolation and/or purification of the desired ORF product
from culture or fermentation media.
[0176] A tag sometimes specifically binds a molecule or moiety of a
solid phase or a detectable label, for example, thereby having
utility for isolating, purifying and/or detecting a protein or
peptide encoded by the ORF. In some embodiments, a tag comprises
one or more of the following elements: FLAG (e.g., DYKDDDDKG) (SEQ
ID NO: 132), V5 (e.g., GKPIPNPLLGLDST) (SEQ ID NO: 133), c-MYC
(e.g., EQKLISEEDL) (SEQ ID NO: 134), HSV (e.g., QPELAPEDPED) (SEQ
ID NO: 135), influenza hemaglutinin, HA (e.g., YPYDVPDYA) (SEQ ID
NO: 136), VSV-G (e.g., YTDIEMNRLGK) (SEQ ID NO: 137), bacterial
glutathione-5-transferase, maltose binding protein, a streptavidin-
or avidin-binding tag (e.g., pcDNA.TM. 6 BioEase.TM. Gateway.RTM.
Biotinylation System (Invitrogen)), thioredoxin,
.beta.-galactosidase, VSV-glycoprotein, a fluorescent protein
(e.g., green fluorescent protein or one of its many color variants
(e.g., yellow, red, blue)), a polylysine or polyarginine sequence,
a polyhistidine sequence (e.g., His6) (SEQ ID NO: 138) or other
sequence that chelates a metal (e.g., cobalt, zinc, copper), and/or
a cysteine-rich sequence that binds to an arsenic-containing
molecule. In certain embodiments, a cysteine-rich tag comprises the
amino acid sequence CC-Xn-CC (SEQ ID NO: 139), wherein X is any
amino acid and n is 1 to 3, and the cysteine-rich sequence
sometimes is CCPGCC (SEQ ID NO: 140). In certain embodiments, the
tag comprises a cysteine-rich element and a polyhistidine element
(e.g., CCPGCC (SEQ ID NO: 140) and His6 (SEQ ID NO: 138)).
[0177] A tag often conveniently binds to a binding partner. For
example, some tags bind to an antibody (e.g., FLAG) and sometimes
specifically bind to a small molecule. For example, a polyhistidine
tag specifically chelates a bivalent metal, such as copper, zinc
and cobalt; a polylysine or polyarginine tag specifically binds to
a zinc finger; a glutathione S-transferase tag binds to
glutathione; and a cysteine-rich tag specifically binds to an
arsenic-containing molecule. Arsenic-containing molecules include
LUMIO.TM. agents (Invitrogen, California), such as FIAsH.TM.
(EDT2[4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethan-
edithiol)2]) and ReAsH reagents (e.g., U.S. Pat. No. 5,932,474 to
Tsien et al., entitled "Target Sequences for Synthetic Molecules;"
U.S. Pat. No. 6,054,271 to Tsien et al., entitled "Methods of Using
Synthetic Molecules and Target Sequences;" U.S. Pat. Nos. 6,451,569
and 6,008,378; published U.S. Patent Application 2003/0083373, and
published PCT Patent Application WO 99/21013, all to Tsien et al.
and all entitled "Synthetic Molecules that Specifically React with
Target Sequences"). Such antibodies and small molecules sometimes
are linked to a solid phase for convenient isolation of the target
protein or target peptide.
[0178] A tag sometimes comprises a sequence that localizes a
translated protein or peptide to a component in a system, which is
referred to as a "signal sequence" or "localization signal
sequence" herein. A signal sequence often is incorporated at the
N-terminus of a target protein or target peptide, and sometimes is
incorporated at the C-terminus. Examples of signal sequences are
known to the artisan, are readily incorporated into a nucleic acid
reagent, and often are selected according to the organism in which
expression of the nucleic acid reagent is performed. A signal
sequence in some embodiments localizes a translated protein or
peptide to a cell membrane. Examples of signal sequences include,
but are not limited to, a nucleus targeting signal (e.g., steroid
receptor sequence and N-terminal sequence of SV40 virus large T
antigen); mitochondrial targeting signal (e.g., amino acid sequence
that forms an amphipathic helix); peroxisome targeting signal
(e.g., C-terminal sequence in YFG from S. cerevisiae); and a
secretion signal (e.g., N-terminal sequences from invertase, mating
factor alpha, PHO5 and SUC2 in S. cerevisiae; multiple N-terminal
sequences of B. subtilis proteins (e.g., Tjalsma et al., Microbiol.
Molec. Biol. Rev. 64: 515-547 (2000)); alpha amylase signal
sequence (e.g., U.S. Pat. No. 6,288,302); pectate lyase signal
sequence (e.g., U.S. Pat. No. 5,846,818); precollagen signal
sequence (e.g., U.S. Pat. No. 5,712,114); OmpA signal sequence
(e.g., U.S. Pat. No. 5,470,719); lam beta signal sequence (e.g.,
U.S. Pat. No. 5,389,529); B. brevis signal sequence (e.g., U.S.
Pat. No. 5,232,841); and P. pastoris signal sequence (e.g., U.S.
Pat. No. 5,268,273)).
[0179] A tag sometimes is directly adjacent to the amino acid
sequence encoded by an ORF (i.e., there is no intervening sequence)
and sometimes a tag is substantially adjacent to an ORF encoded
amino acid sequence (e.g., an intervening sequence is present). An
intervening sequence sometimes includes a recognition site for a
protease, which is useful for cleaving a tag from a target protein
or peptide. In some embodiments, the intervening sequence is
cleaved by Factor Xa (e.g., recognition site I (E/D)GR), thrombin
(e.g., recognition site LVPRGS) (SEQ ID NO: 141), enterokinase
(e.g., recognition site DDDDK) (SEQ ID NO: 142), TEV protease
(e.g., recognition site ENLYFQG) (SEQ ID NO: 143) or
PreScission.TM. protease (e.g., recognition site LEVLFQGP) (SEQ ID
NO: 144), for example.
[0180] An intervening sequence sometimes is referred to herein as a
"linker sequence," and may be of any suitable length selected by
the artisan. A linker sequence sometimes is about 1 to about 20
amino acids in length, and sometimes about 5 to about 10 amino
acids in length. The artisan may select the linker length to
substantially preserve target protein or peptide function (e.g., a
tag may reduce target protein or peptide function unless separated
by a linker), to enhance disassociation of a tag from a target
protein or peptide when a protease cleavage site is present (e.g.,
cleavage may be enhanced when a linker is present), and to enhance
interaction of a tag/target protein product with a solid phase. A
linker can be of any suitable amino acid content, and often
comprises a higher proportion of amino acids having relatively
short side chains (e.g., glycine, alanine, serine and
threonine).
[0181] A nucleic acid reagent sometimes includes a stop codon
between a tag element and an insertion element or ORF, which can be
useful for translating an ORF with or without the tag. Mutant tRNA
molecules that recognize stop codons (described above) suppress
translation termination and thereby are designated "suppressor
tRNAs." Suppressor tRNAs can result in the insertion of amino acids
and continuation of translation past stop codons (e.g., U.S. Patent
Application No. 60/587,583, filed Jul. 14, 2004, entitled
"Production of Fusion Proteins by Cell-Free Protein Synthesis,";
Eggertsson, et al., (1988) Microbiological Review 52(3):354-374,
and Engleerg-Kukla, et al. (1996) in Escherichia coli and
Salmonella Cellular and Molecular Biology, Chapter 60, pps 909-921,
Neidhardt, et al. eds., ASM Press, Washington, D.C.). A number of
suppressor tRNAs are known, including but not limited to, supE,
supP, supD, supF and supZ suppressors, which suppress the
termination of translation of the amber stop codon; supB, gIT,
supL, supN, supC and supM suppressors, which suppress the function
of the ochre stop codon and glyT, trpT and Su-9 suppressors, which
suppress the function of the opal stop codon. In general,
suppressor tRNAs contain one or more mutations in the anti-codon
loop of the tRNA that allows the tRNA to base pair with a codon
that ordinarily functions as a stop codon. The mutant tRNA is
charged with its cognate amino acid residue and the cognate amino
acid residue is inserted into the translating polypeptide when the
stop codon is encountered. Mutations that enhance the efficiency of
termination suppressors (i.e., increase stop codon read-through)
have been identified. These include, but are not limited to,
mutations in the uar gene (also known as the prfA gene), mutations
in the ups gene, mutations in the sueA, sueB and sueC genes,
mutations in the rpsD (ramA) and rpsE (spcA) genes and mutations in
the rpIL gene.
[0182] Thus, a nucleic acid reagent comprising a stop codon located
between an ORF and a tag can yield a translated ORF alone when no
suppressor tRNA is present in the translation system, and can yield
a translated ORF-tag fusion when a suppressor tRNA is present in
the system. Suppressor tRNA can be generated in cells transfected
with a nucleic acid encoding the tRNA (e.g., a replication
incompetent adenovirus containing the human tRNA-Ser suppressor
gene can be transfected into cells, or a YAC containing a yeast or
bacterial tRNA suppressor gene can be transfected into yeast cells,
for example). Vectors for synthesizing suppressor tRNA and for
translating ORFs with or without a tag are available to the artisan
(e.g., Tag-On-Demand.TM. kit (Invitrogen Corporation, California);
Tag-On-Demand.TM. Suppressor Supernatant Instruction Manual,
Version B, 6 Jun. 2003, at http address
www.invitrogen.com/content/sfs/manuals/tagondemand_supernatant_man.pdf;
Tag-On-Demand.TM. Gateway.RTM. Vector Instruction Manual, Version
B, 20 Jun., 2003 at http address
www.invitrogen.com/content/sfs/manuals/tagondemand_vectors_man.pdf;
and Capone et al., Amber, ochre and opal suppressor tRNA genes
derived from a human serine tRNA gene. EMBO J. 4:213, 1985).
[0183] Any convenient cloning strategy known in the art may be
utilized to incorporate an element, such as an ORF, into a nucleic
acid reagent. Known methods can be utilized to insert an element
into the template independent of an insertion element, such as (1)
cleaving the template at one or more existing restriction enzyme
sites and ligating an element of interest and (2) adding
restriction enzyme sites to the template by hybridizing
oligonucleotide primers that include one or more suitable
restriction enzyme sites and amplifying by polymerase chain
reaction (described in greater detail herein). Other cloning
strategies take advantage of one or more insertion sites present or
inserted into the nucleic acid reagent, such as an oligonucleotide
primer hybridization site for PCR, for example, and others
described hereafter. In some embodiments, a cloning strategy can be
combined with genetic manipulation such as recombination (e.g.,
recombination of a nucleic acid reagent with a nucleic acid
sequence of interest into the genome of the organism to be
modified, as described further below). In some embodiments, the
cloned ORF(s) can produce (directly or indirectly) a desire
product, by engineering a microorganism with one or more ORFs of
interest, which microorganism comprises one or more altered
activities selected from the group consisting of
phosphofructokinase activity, phosphogluconate dehydratase
activity, 2-keto-3-deoxygluconate-6-phosphate aldolase activity,
xylose isomerase activity, phosphoenolpyruvate carboxylase
activity, alcohol dehydrogenase 2 activity, sugar transport
activity, phosphoglucoisomerase activity, transaldolase activity,
transketolase activity, glucose-6-phosphate dehydrogenase activity,
6-phosphogluconolactonase activity, 6-phosphogluconate
dehydrogenase (decarboxylating) activity, xylose reductase
activity, xylitol dehydrogenase activity, xylulokinase activity and
thymidylate synthase activity.
[0184] In some embodiments, the nucleic acid reagent includes one
or more recombinase insertion sites. A recombinase insertion site
is a recognition sequence on a nucleic acid molecule that
participates in an integration/recombination reaction by
recombination proteins. For example, the recombination site for Cre
recombinase is loxP, which is a 34 base pair sequence comprised of
two 13 base pair inverted repeats (serving as the recombinase
binding sites) flanking an 8 base pair core sequence (e.g., FIG. 1
of Sauer, B., Curr. Opin. Biotech. 5:521-527 (1994)). Other
examples of recombination sites include attB, attP, attL, and attR
sequences, and mutants, fragments, variants and derivatives
thereof, which are recognized by the recombination protein A Int
and by the auxiliary proteins integration host factor (IHF), FIS
and excisionase (Xis) (e.g., U.S. Pat. Nos. 5,888,732; 6,143,557;
6,171,861; 6,270,969; 6,277,608; and 6,720,140; U.S. patent
application Ser. No. 09/517,466, filed Mar. 2, 2000, and
09/732,914, filed Aug. 14, 2003, and in U.S. patent publication no.
2002-0007051-A1; Landy, Curr. Opin. Biotech. 3:699-707 (1993)).
[0185] Examples of recombinase cloning nucleic acids are in
Gateway.RTM. systems (Invitrogen, California), which include at
least one recombination site for cloning a desired nucleic acid
molecules in vivo or in vitro. In some embodiments, the system
utilizes vectors that contain at least two different site-specific
recombination sites, often based on the bacteriophage lambda system
(e.g., att1 and att2), and are mutated from the wild-type (att0)
sites. Each mutated site has a unique specificity for its cognate
partner att site (i.e., its binding partner recombination site) of
the same type (for example attB1 with attP1, or attL1 with attR1)
and will not cross-react with recombination sites of the other
mutant type or with the wild-type att0 site. Different site
specificities allow directional cloning or linkage of desired
molecules thus providing desired orientation of the cloned
molecules. Nucleic acid fragments flanked by recombination sites
are cloned and subcloned using the Gateway.RTM. system by replacing
a selectable marker (for example, ccdB) flanked by att sites on the
recipient plasmid molecule, sometimes termed the Destination
Vector. Desired clones are then selected by transformation of a
ccdB sensitive host strain and positive selection for a marker on
the recipient molecule. Similar strategies for negative selection
(e.g., use of toxic genes) can be used in other organisms such as
thymidine kinase (TK) in mammals and insects.
[0186] A recombination system useful for engineering yeast is
outlined briefly. The system makes use of the ura3 gene (e.g., for
S. cerevisiae and C. albicans, for example) or ura4 and ura5 genes
(e.g., for S. pombe, for example) and toxicity of the nucleotide
analogue 5-Fluoroorotic acid (5-FOA). The ura3 or ura4 and ura5
genes encode orotine-5'-monophosphate (OMP) dicarboxylase. Yeast
with an active ura3 or ura4 and ura5 gene (phenotypically Ura+)
convert 5-FOA to fluorodeoxyuridine, which is toxic to yeast cells.
Yeast carrying a mutation in the appropriate gene(s) or having a
knock out of the appropriate gene(s) can grow in the presence of
5-FOA, if the media is also supplemented with uracil.
[0187] A nucleic acid engineering construct can be made which may
comprise the URA3 gene or cassette (for S. cerevisiae), flanked on
either side by the same nucleotide sequence in the same
orientation. The ura3 cassette comprises a promoter, the ura3 gene
and a functional transcription terminator. Target sequences which
direct the construct to a particular nucleic acid region of
interest in the organism to be engineered are added such that the
target sequences are adjacent to and abut the flanking sequences on
either side of the ura3 cassette. Yeast can be transformed with the
engineering construct and plated on minimal media without uracil.
Colonies can be screened by PCR to determine those transformants
that have the engineering construct inserted in the proper location
in the genome. Checking insertion location prior to selecting for
recombination of the ura3 cassette may reduce the number of
incorrect clones carried through to later stages of the procedure.
Correctly inserted transformants can then be replica plated on
minimal media containing 5-FOA to select for recombination of the
ura3 cassette out of the construct, leaving a disrupted gene and an
identifiable footprint (e.g., nucleic acid sequence) that can be
use to verify the presence of the disrupted gene. The technique
described is useful for disrupting or "knocking out" gene function,
but also can be used to insert genes or constructs into a host
organisms genome in a targeted, sequence specific manner. Further
detail will be described below in the engineering section and in
the example section.
[0188] In certain embodiments, a nucleic acid reagent includes one
or more topoisomerase insertion sites. A topoisomerase insertion
site is a defined nucleotide sequence recognized and bound by a
site-specific topoisomerase. For example, the nucleotide sequence
5'-(C/T)CCTT-3' is a topoisomerase recognition site bound
specifically by most poxvirus topoisomerases, including vaccinia
virus DNA topoisomerase I. After binding to the recognition
sequence, the topoisomerase cleaves the strand at the 3'-most
thymidine of the recognition site to produce a nucleotide sequence
comprising 5'-(C/T)CCTT-PO4-TOPO, a complex of the topoisomerase
covalently bound to the 3' phosphate via a tyrosine in the
topoisomerase (e.g., Shuman, J. Biol. Chem. 266:11372-11379, 1991;
Sekiguchi and Shuman, Nucl. Acids Res. 22:5360-5365, 1994; U.S.
Pat. No. 5,766,891; PCT/US95/16099; and PCT/US98/12372). In
comparison, the nucleotide sequence 5'-GCAACTT-3' is a
topoisomerase recognition site for type IA E. coli topoisomerase
III. An element to be inserted often is combined with
topoisomerase-reacted template and thereby incorporated into the
nucleic acid reagent (e.g., http address
www.invitrogen.com/downloads/F-13512_Topo_Flyer.pdf; http address
at world wide web uniform resource locator
invitrogen.com/content/sfs/brochures/710.sub.--021849%20_B_TOPOCloning_br-
o.pdf; TOPO TA Cloning.RTM. Kit and Zero Blunt.RTM. TOPO.RTM.
Cloning Kit product information).
[0189] A nucleic acid reagent sometimes contains one or more origin
of replication (ORI) elements. In some embodiments, a template
comprises two or more ORIs, where one functions efficiently in one
organism (e.g., a bacterium) and another functions efficiently in
another organism (e.g., a eukaryote, like yeast for example). In
some embodiments, an ORI may function efficiently in one species
(e.g., S. cerevisiae, for example) and another ORI may function
efficiently in a different species (e.g., S. pombe, for example). A
nucleic acid reagent also sometimes includes one or more
transcription regulation sites.
[0190] A nucleic acid reagent can include one or more selection
elements (e.g., elements for selection of the presence of the
nucleic acid reagent, and not for activation of a promoter element
which can be selectively regulated). Selection elements often are
utilized using known processes to determine whether a nucleic acid
reagent is included in a cell. In some embodiments, a nucleic acid
reagent includes two or more selection elements, where one
functions efficiently in one organism and another functions
efficiently in another organism. Examples of selection elements
include, but are not limited to, (1) nucleic acid segments that
encode products that provide resistance against otherwise toxic
compounds (e.g., antibiotics); (2) nucleic acid segments that
encode products that are otherwise lacking in the recipient cell
(e.g., essential products, tRNA genes, auxotrophic markers); (3)
nucleic acid segments that encode products that suppress the
activity of a gene product; (4) nucleic acid segments that encode
products that can be readily identified (e.g., phenotypic markers
such as antibiotics (e.g., .beta.-lactamase), .beta.-galactosidase,
green fluorescent protein (GFP), yellow fluorescent protein (YFP),
red fluorescent protein (RFP), cyan fluorescent protein (CFP), and
cell surface proteins); (5) nucleic acid segments that bind
products that are otherwise detrimental to cell survival and/or
function; (6) nucleic acid segments that otherwise inhibit the
activity of any of the nucleic acid segments described in Nos. 1-5
above (e.g., antisense oligonucleotides); (7) nucleic acid segments
that bind products that modify a substrate (e.g., restriction
endonucleases); (8) nucleic acid segments that can be used to
isolate or identify a desired molecule (e.g., specific protein
binding sites); (9) nucleic acid segments that encode a specific
nucleotide sequence that can be otherwise non-functional (e.g., for
PCR amplification of subpopulations of molecules); (10) nucleic
acid segments that, when absent, directly or indirectly confer
resistance or sensitivity to particular compounds; (11) nucleic
acid segments that encode products that either are toxic or convert
a relatively non-toxic compound to a toxic compound (e.g., Herpes
simplex thymidine kinase, cytosine deaminase) in recipient cells;
(12) nucleic acid segments that inhibit replication, partition or
heritability of nucleic acid molecules that contain them; and/or
(13) nucleic acid segments that encode conditional replication
functions, e.g., replication in certain hosts or host cell strains
or under certain environmental conditions (e.g., temperature,
nutritional conditions, and the like).
[0191] A nucleic acid reagent is of any form useful for in vivo
transcription and/or translation. A nucleic acid sometimes is a
plasmid, such as a supercoiled plasmid, sometimes is a yeast
artificial chromosome (e.g., YAC), sometimes is a linear nucleic
acid (e.g., a linear nucleic acid produced by PCR or by restriction
digest), sometimes is single-stranded and sometimes is
double-stranded. A nucleic acid reagent sometimes is prepared by an
amplification process, such as a polymerase chain reaction (PCR)
process or transcription-mediated amplification process (TMA). In
TMA, two enzymes are used in an isothermal reaction to produce
amplification products detected by light emission (see, e.g.,
Biochemistry 1996 Jun. 25; 35(25):8429-38 and http address world
wide web uniform resource locator
devicelink.com/ivdt/archive/00/11/007.html). Standard PCR processes
are known (e.g., U.S. Pat. Nos. 4,683,202; 4,683,195; 4,965,188;
and 5,656,493), and generally are performed in cycles. Each cycle
includes heat denaturation, in which hybrid nucleic acids
dissociate; cooling, in which primer oligonucleotides hybridize;
and extension of the oligonucleotides by a polymerase (i.e., Taq
polymerase). An example of a PCR cyclical process is treating the
sample at 95.degree. C. for 5 minutes; repeating forty-five cycles
of 95.degree. C. for 1 minute, 59.degree. C. for 1 minute, 10
seconds, and 72.degree. C. for 1 minute 30 seconds; and then
treating the sample at 72.degree. C. for 5 minutes. Multiple cycles
frequently are performed using a commercially available thermal
cycler. PCR amplification products sometimes are stored for a time
at a lower temperature (e.g., at 4.degree. C.) and sometimes are
frozen (e.g., at -20.degree. C.) before analysis.
[0192] In some embodiments, a nucleic acid reagent, protein
reagent, protein fragment reagent or other reagent described herein
is isolated or purified. The term "isolated" as used herein refers
to material removed from its original environment (e.g., the
natural environment if it is naturally occurring, or a host cell if
expressed exogenously), and thus is altered "by the hand of man"
from its original environment. The term "purified" as used herein
with reference to molecules does not refer to absolute purity.
Rather, "purified" refers to a substance in a composition that
contains fewer substance species in the same class (e.g., nucleic
acid or protein species) other than the substance of interest in
comparison to the sample from which it originated. "Purified," if a
nucleic acid or protein for example, refers to a substance in a
composition that contains fewer nucleic acid species or protein
species other than the nucleic acid or protein of interest in
comparison to the sample from which it originated. Sometimes, a
protein or nucleic acid is "substantially pure," indicating that
the protein or nucleic acid represents at least 50% of protein or
nucleic acid on a mass basis of the composition. Often, a
substantially pure protein or nucleic acid is at least 75% on a
mass basis of the composition, and sometimes at least 95% on a mass
basis of the composition.
Engineering and Alteration Methods
[0193] Methods and compositions (e.g., nucleic acid reagents)
described herein can be used to generate engineered microorganisms.
As noted above, the term "engineered microorganism" as used herein
refers to a modified organism that includes one or more activities
distinct from an activity present in a microorganism utilized as a
starting point for modification (e.g., host microorganism or
unmodified organism). Engineered microorganisms typically arise as
a result of a genetic modification, usually introduced or selected
for, by one of skill in the art using readily available techniques.
Non-limiting examples of methods useful for generating an altered
activity include, introducing a heterologous polynucleotide (e.g.,
nucleic acid or gene integration, also referred to as "knock in"),
removing an endogenous polynucleotide, altering the sequence of an
existing endogenous nucleic acid sequence (e.g., site-directed
mutagenesis), disruption of an existing endogenous nucleic acid
sequence (e.g., knock outs and transposon or insertion element
mediated mutagenesis), selection for an altered activity where the
selection causes a change in a naturally occurring activity that
can be stably inherited (e.g., causes a change in a nucleic acid
sequence in the genome of the organism or in an epigenetic nucleic
acid that is replicated and passed on to daughter cells), PCR-based
mutagenesis, and the like. The term "mutagenesis" as used herein
refers to any modification to a nucleic acid (e.g., nucleic acid
reagent, or host chromosome, for example) that is subsequently used
to generate a product in a host or modified organism. Non-limiting
examples of mutagenesis include, deletion, insertion, substitution,
rearrangement, point mutations, suppressor mutations and the like.
Mutagenesis methods are known in the art and are readily available
to the artisan. Non-limiting examples of mutagenesis methods are
described herein and can also be found in Maniatis, T., E. F.
Fritsch and J. Sambrook (1982) Molecular Cloning: a Laboratory
Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
[0194] The term "genetic modification" as used herein refers to any
suitable nucleic acid addition, removal or alteration that
facilitates production of a target product (e.g., phosphogluconate
dehydratase activity, 2-keto-3-deoxygluconate-6-phosphate aldolase
activity, xylose isomerase activity, or phosphoenolpyruvate
carboxylase activity, for example). in an engineered microorganism.
Genetic modifications include, without limitation, insertion of one
or more nucleotides in a native nucleic acid of a host organism in
one or more locations, deletion of one or more nucleotides in a
native nucleic acid of a host organism in one or more locations,
modification or substitution of one or more nucleotides in a native
nucleic acid of a host organism in one or more locations, insertion
of a non-native nucleic acid into a host organism (e.g., insertion
of an autonomously replicating vector), and removal of a non-native
nucleic acid in a host organism (e.g., removal of a vector).
[0195] The term "heterologous polynucleotide" as used herein refers
to a nucleotide sequence not present in a host microorganism in
some embodiments. In certain embodiments, a heterologous
polynucleotide is present in a different amount (e.g., different
copy number) than in a host microorganism, which can be
accomplished, for example, by introducing more copies of a
particular nucleotide sequence to a host microorganism (e.g., the
particular nucleotide sequence may be in a nucleic acid autonomous
of the host chromosome or may be inserted into a chromosome). A
heterologous polynucleotide is from a different organism in some
embodiments, and in certain embodiments, is from the same type of
organism but from an outside source (e.g., a recombinant
source).
[0196] The term "altered activity" as used herein refers to an
activity in an engineered microorganism that is added or modified
relative to the host microorganism (e.g., added, increased,
reduced, inhibited or removed activity). An activity can be altered
by introducing a genetic modification to a host microorganism that
yields an engineered microorganism having added, increased,
reduced, inhibited or removed activity.
[0197] An added activity often is an activity not detectable in a
host microorganism. An increased activity generally is an activity
detectable in a host microorganism that has been increased in an
engineered microorganism. An activity can be increased to any
suitable level for production of a target product (e.g., adipic
acid, 6-hydroxyhexanoic acid), including but not limited to less
than 2-fold (e.g., about 10% increase to about 99% increase; about
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% increase), 2-fold, 3-fold,
4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, of 10-fold
increase, or greater than about 10-fold increase. A reduced or
inhibited activity generally is an activity detectable in a host
microorganism that has been reduced or inhibited in an engineered
microorganism. An activity can be reduced to undetectable levels in
some embodiments, or detectable levels in certain embodiments. An
activity can be decreased to any suitable level for production of a
target product (e.g., adipic acid, 6-hydroxyhexanoic acid),
including but not limited to less than 2-fold (e.g., about 10%
decrease to about 99% decrease; about 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90% decrease), 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,
8-fold, 9-fold, of 10-fold decrease, or greater than about 10-fold
decrease.
[0198] An altered activity sometimes is an activity not detectable
in a host organism and is added to an engineered organism. An
altered activity also may be an activity detectable in a host
organism and is increased in an engineered organism. An activity
may be added or increased by increasing the number of copies of a
polynucleotide that encodes a polypeptide having a target activity,
in some embodiments. In certain embodiments an activity can be
added or increased by inserting into a host microorganism a
heterologous polynucleotide that encodes a polypeptide having the
added activity. In certain embodiments, an activity can be added or
increased by inserting into a host microorganism a heterologous
polynucleotide that is (i) operably linked to another
polynucleotide that encodes a polypeptide having the added
activity, and (ii) up regulates production of the polynucleotide.
Thus, an activity can be added or increased by inserting or
modifying a regulatory polynucleotide operably linked to another
polynucleotide that encodes a polypeptide having the target
activity. In certain embodiments, an activity can be added or
increased by subjecting a host microorganism to a selective
environment and screening for microorganisms that have a detectable
level of the target activity. Examples of a selective environment
include, without limitation, a medium containing a substrate that a
host organism can process and a medium lacking a substrate that a
host organism can process.
[0199] An altered activity sometimes is an activity detectable in a
host organism and is reduced, inhibited or removed (i.e., not
detectable) in an engineered organism. An activity may be reduced
or removed by decreasing the number of copies of a polynucleotide
that encodes a polypeptide having a target activity, in some
embodiments. In some embodiments, an activity can be reduced or
removed by (i) inserting a polynucleotide within a polynucleotide
that encodes a polypeptide having the target activity (disruptive
insertion), and/or (ii) removing a portion of or all of a
polynucleotide that encodes a polypeptide having the target
activity (deletion or knock out, respectively). In certain
embodiments, an activity can be reduced or removed by inserting
into a host microorganism a heterologous polynucleotide that is (i)
operably linked to another polynucleotide that encodes a
polypeptide having the target activity, and (ii) down regulates
production of the polynucleotide. Thus, an activity can be reduced
or removed by inserting or modifying a regulatory polynucleotide
operably linked to another polynucleotide that encodes a
polypeptide having the target activity.
[0200] An activity also can be reduced or removed by (i) inhibiting
a polynucleotide that encodes a polypeptide having the activity or
(ii) inhibiting a polynucleotide operably linked to another
polynucleotide that encodes a polypeptide having the activity. A
polynucleotide can be inhibited by a suitable technique known in
the art, such as by contacting an RNA encoded by the polynucleotide
with a specific inhibitory RNA (e.g., RNAi, siRNA, ribozyme). An
activity also can be reduced or removed by contacting a polypeptide
having the activity with a molecule that specifically inhibits the
activity (e.g., enzyme inhibitor, antibody). In certain
embodiments, an activity can be reduced or removed by subjecting a
host microorganism to a selective environment and screening for
microorganisms that have a reduced level or removal of the target
activity.
[0201] In some embodiments, an untranslated ribonucleic acid, or a
cDNA can be used to reduce the expression of a particular activity
or enzyme. For example, a microorganism can be engineered by
genetic modification to express a nucleic acid reagent that reduces
the expression of an activity by producing an RNA molecule that is
partially or substantially homologous to a nucleic acid sequence of
interest which encodes the activity of interest. The RNA molecule
can bind to the nucleic acid sequence of interest and inhibit the
nucleic acid sequence from performing its natural function, in
certain embodiments. In some embodiments, the RNA may alter the
nucleic acid sequence of interest which encodes the activity of
interest in a manner that the nucleic acid sequence of interest is
no longer capable of performing its natural function (e.g., the
action of a ribozyme for example).
[0202] In certain embodiments, nucleotide sequences sometimes are
added to, modified or removed from one or more of the nucleic acid
reagent elements, such as the promoter, 5'UTR, target sequence, or
3'UTR elements, to enhance, potentially enhance, reduce, or
potentially reduce transcription and/or translation before or after
such elements are incorporated in a nucleic acid reagent. In some
embodiments, one or more of the following sequences may be modified
or removed if they are present in a 5'UTR: a sequence that forms a
stable secondary structure (e.g., quadruplex structure or stem loop
stem structure (e.g., EMBL sequences X12949, AF274954, AF139980,
AF152961, S95936, U194144, AF116649 or substantially identical
sequences that form such stem loop stem structures)); a translation
initiation codon upstream of the target nucleotide sequence start
codon; a stop codon upstream of the target nucleotide sequence
translation initiation codon; an ORF upstream of the target
nucleotide sequence translation initiation codon; an iron
responsive element (IRE) or like sequence; and a 5' terminal
oligopyrimidine tract (TOP, e.g., consisting of 5-15 pyrimidines
adjacent to the cap). A translational enhancer sequence and/or an
internal ribosome entry site (IRES) sometimes is inserted into a
5'UTR (e.g., EMBL nucleotide sequences J04513, X87949, M95825,
M12783, AF025841, AF013263, AF006822, M17169, M13440, M22427,
D14838 and M17446 and substantially identical nucleotide
sequences).
[0203] An AU-rich element (ARE, e.g., AUUUA repeats) and/or
splicing junction that follows a non-sense codon sometimes is
removed from or modified in a 3'UTR. A polyadenosine tail sometimes
is inserted into a 3'UTR if none is present, sometimes is removed
if it is present, and adenosine moieties sometimes are added to or
removed from a polyadenosine tail present in a 3'UTR. Thus, some
embodiments are directed to a process comprising: determining
whether any nucleotide sequences that increase, potentially
increase, reduce or potentially reduce translation efficiency are
present in the elements, and adding, removing or modifying one or
more of such sequences if they are identified. Certain embodiments
are directed to a process comprising: determining whether any
nucleotide sequences that increase or potentially increase
translation efficiency are not present in the elements, and
incorporating such sequences into the nucleic acid reagent.
[0204] In some embodiments, an activity can be altered by modifying
the nucleotide sequence of an ORF. An ORF sometimes is mutated or
modified (for example, by point mutation, deletion mutation,
insertion mutation, PCR based mutagenesis and the like) to alter,
enhance or increase, reduce, substantially reduce or eliminate the
activity of the encoded protein or peptide. The protein or peptide
encoded by a modified ORF sometimes is produced in a lower amount
or may not be produced at detectable levels, and in other
embodiments, the product or protein encoded by the modified ORF is
produced at a higher level (e.g., codons sometimes are modified so
they are compatible with tRNA's preferentially used in the host
organism or engineered organism). To determine the relative
activity, the activity from the product of the mutated ORF (or cell
containing it) can be compared to the activity of the product or
protein encoded by the unmodified ORF (or cell containing it).
[0205] In some embodiments, an ORF nucleotide sequence sometimes is
mutated or modified to alter the triplet nucleotide sequences used
to encode amino acids (e.g., amino acid codon triplets, for
example). Modification of the nucleotide sequence of an ORF to
alter codon triplets sometimes is used to change the codon found in
the original sequence to better match the preferred codon usage of
the organism in which the ORF or nucleic acid reagent will be
expressed. For example, the codon usage, and therefore the codon
triplets encoded by a nucleic acid sequence from bacteria may be
different from the preferred codon usage in eukaryotes like yeast
or plants. Preferred codon usage also may be different between
bacterial species. In certain embodiments an ORF nucleotide
sequences sometimes is modified to eliminate codon pairs and/or
eliminate mRNA secondary structures that can cause pauses during
translation of the mRNA encoded by the ORF nucleotide sequence.
Translational pausing sometimes occurs when nucleic acid secondary
structures exist in an mRNA, and sometimes occurs due to the
presence of codon pairs that slow the rate of translation by
causing ribosomes to pause. In some embodiments, the use of lower
abundance codon triplets can reduce translational pausing due to a
decrease in the pause time needed to load a charged tRNA into the
ribosome translation machinery. Therefore, to increase
transcriptional and translational efficiency in bacteria (e.g.,
where transcription and translation are concurrent, for example) or
to increase translational efficiency in eukaryotes (e.g., where
transcription and translation are functionally separated), the
nucleotide sequence of a nucleotide sequence of interest can be
altered to better suit the transcription and/or translational
machinery of the host and/or genetically modified microorganism. In
certain embodiment, slowing the rate of translation by the use of
lower abundance codons, which slow or pause the ribosome, can lead
to higher yields of the desired product due to an increase in
correctly folded proteins and a reduction in the formation of
inclusion bodies.
[0206] Codons can be altered and optimized according to the
preferred usage by a given organism by determining the codon
distribution of the nucleotide sequence donor organism and
comparing the distribution of codons to the distribution of codons
in the recipient or host organism. Techniques described herein
(e.g., site directed mutagenesis and the like) can then be used to
alter the codons accordingly. Comparisons of codon usage can be
done by hand, or using nucleic acid analysis software commercially
available to the artisan.
[0207] Modification of the nucleotide sequence of an ORF also can
be used to correct codon triplet sequences that have diverged in
different organisms. For example, certain yeast (e.g., C.
tropicalis and C. maltosa) use the amino acid triplet CUG (e.g.,
CTG in the DNA sequence) to encode serine. CUG typically encodes
leucine in most organisms. In order to maintain the correct amino
acid in the resultant polypeptide or protein, the CUG codon must be
altered to reflect the organism in which the nucleic acid reagent
will be expressed. Thus, if an ORF from a bacterial donor is to be
expressed in either Candida yeast strain mentioned above, the
heterologous nucleotide sequence must first be altered or modified
to the appropriate leucine codon. Therefore, in some embodiments,
the nucleotide sequence of an ORF sometimes is altered or modified
to correct for differences that have occurred in the evolution of
the amino acid codon triplets between different organisms. In some
embodiments, the nucleotide sequence can be left unchanged at a
particular amino acid codon, if the amino acid encoded is a
conservative or neutral change in amino acid when compared to the
originally encoded amino acid.
[0208] In some embodiments, an activity can be altered by modifying
translational regulation signals, like a stop codon for example. A
stop codon at the end of an ORF sometimes is modified to another
stop codon, such as an amber stop codon described above. In some
embodiments, a stop codon is introduced within an ORF, sometimes by
insertion or mutation of an existing codon. An ORF comprising a
modified terminal stop codon and/or internal stop codon often is
translated in a system comprising a suppressor tRNA that recognizes
the stop codon. An ORF comprising a stop codon sometimes is
translated in a system comprising a suppressor tRNA that
incorporates an unnatural amino acid during translation of the
target protein or target peptide. Methods for incorporating
unnatural amino acids into a target protein or peptide are known,
which include, for example, processes utilizing a heterologous
tRNA/synthetase pair, where the tRNA recognizes an amber stop codon
and is loaded with an unnatural amino acid (e.g., World Wide Web
URL iupac.org/news/prize/2003/wang.pdf).
[0209] Depending on the portion of a nucleic acid reagent (e.g.,
Promoter, 5' or 3' UTR, ORI, ORF, and the like) chosen for
alteration (e.g., by mutagenesis, introduction or deletion, for
example) the modifications described above can alter a given
activity by (i) increasing or decreasing feedback inhibition
mechanisms, (ii) increasing or decreasing promoter initiation,
(iii) increasing or decreasing translation initiation, (iv)
increasing or decreasing translational efficiency, (v) modifying
localization of peptides or products expressed from nucleic acid
reagents described herein, or (vi) increasing or decreasing the
copy number of a nucleotide sequence of interest, (vii) expression
of an anti-sense RNA, RNAi, siRNA, ribozyme and the like. In some
embodiments, alteration of a nucleic acid reagent or nucleotide
sequence can alter a region involved in feedback inhibition (e.g.,
5' UTR, promoter and the like). A modification sometimes is made
that can add or enhance binding of a feedback regulator and
sometimes a modification is made that can reduce, inhibit or
eliminate binding of a feedback regulator.
[0210] In certain embodiments, alteration of a nucleic acid reagent
or nucleotide sequence can alter sequences involved in
transcription initiation (e.g., promoters, 5' UTR, and the like). A
modification sometimes can be made that can enhance or increase
initiation from an endogenous or heterologous promoter element. A
modification sometimes can be made that removes or disrupts
sequences that increase or enhance transcription initiation,
resulting in a decrease or elimination of transcription from an
endogenous or heterologous promoter element.
[0211] In some embodiments, alteration of a nucleic acid reagent or
nucleotide sequence can alter sequences involved in translational
initiation or translational efficiency (e.g., 5' UTR, 3' UTR, codon
triplets of higher or lower abundance, translational terminator
sequences and the like, for example). A modification sometimes can
be made that can increase or decrease translational initiation,
modifying a ribosome binding site for example. A modification
sometimes can be made that can increase or decrease translational
efficiency. Removing or adding sequences that form hairpins and
changing codon triplets to a more or less preferred codon are
non-limiting examples of genetic modifications that can be made to
alter translation initiation and translation efficiency.
[0212] In certain embodiments, alteration of a nucleic acid reagent
or nucleotide sequence can alter sequences involved in localization
of peptides, proteins or other desired products (e.g., adipic acid,
for example). A modification sometimes can be made that can alter,
add or remove sequences responsible for targeting a polypeptide,
protein or product to an intracellular organelle, the periplasm,
cellular membranes, or extracellularly. Transport of a heterologous
product to a different intracellular space or extracellularly
sometimes can reduce or eliminate the formation of inclusion bodies
(e.g., insoluble aggregates of the desired product).
[0213] In some embodiments, alteration of a nucleic acid reagent or
nucleotide sequence can alter sequences involved in increasing or
decreasing the copy number of a nucleotide sequence of interest. A
modification sometimes can be made that increases or decreases the
number of copies of an ORF stably integrated into the genome of an
organism or on an epigenetic nucleic acid reagent. Non-limiting
examples of alterations that can increase the number of copies of a
sequence of interest include, adding copies of the sequence of
interest by duplication of regions in the genome (e.g., adding
additional copies by recombination or by causing gene amplification
of the host genome, for example), cloning additional copies of a
sequence onto a nucleic acid reagent, or altering an ORI to
increase the number of copies of an epigenetic nucleic acid
reagent. Non-limiting examples of alterations that can decrease the
number of copies of a sequence of interest include, removing copies
of the sequence of interest by deletion or disruption of regions in
the genome, removing additional copies of the sequence from
epigenetic nucleic acid reagents, or altering an ORI to decrease
the number of copies of an epigenetic nucleic acid reagent.
[0214] In certain embodiments, increasing or decreasing the
expression of a nucleotide sequence of interest can also be
accomplished by altering, adding or removing sequences involved in
the expression of an anti-sense RNA, RNAi, siRNA, ribozyme and the
like. The methods described above can be used to modify expression
of anti-sense RNA, RNAi, siRNA, ribozyme and the like.
[0215] Engineered microorganisms can be prepared by altering,
introducing or removing nucleotide sequences in the host genome or
in stably maintained epigenetic nucleic acid reagents, as noted
above. The nucleic acid reagents use to alter, introduce or remove
nucleotide sequences in the host genome or epigenetic nucleic acids
can be prepared using the methods described herein or available to
the artisan.
[0216] Nucleic acid sequences having a desired activity can be
isolated from cells of a suitable organism using lysis and nucleic
acid purification procedures available in Maniatis, T., E. F.
Fritsch and J. Sambrook (1982) Molecular Cloning: a Laboratory
Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. or
with commercially available cell lysis and DNA purification
reagents and kits. In some embodiments, nucleic acids used to
engineer microorganisms can be provided for conducting methods
described herein after processing of the organism containing the
nucleic acid. For example, the nucleic acid of interest may be
extracted, isolated, purified or amplified from a sample (e.g.,
from an organism of interest or culture containing a plurality of
organisms of interest, like yeast or bacteria for example). The
term "isolated" as used herein refers to nucleic acid removed from
its original environment (e.g., the natural environment if it is
naturally occurring, or a host cell if expressed exogenously), and
thus is altered "by the hand of man" from its original environment.
An isolated nucleic acid generally is provided with fewer
non-nucleic acid components (e.g., protein, lipid) than the amount
of components present in a source sample. A composition comprising
isolated sample nucleic acid can be substantially isolated (e.g.,
about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater
than 99% free of non-nucleic acid components). The term "purified"
as used herein refers to sample nucleic acid provided that contains
fewer nucleic acid species than in the sample source from which the
sample nucleic acid is derived. A composition comprising sample
nucleic acid may be substantially purified (e.g., about 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% free of
other nucleic acid species). The term "amplified" as used herein
refers to subjecting nucleic acid of a cell, organism or sample to
a process that linearly or exponentially generates amplicon nucleic
acids having the same or substantially the same nucleotide sequence
as the nucleotide sequence of the nucleic acid in the sample, or
portion thereof. As noted above, the nucleic acids used to prepare
nucleic acid reagents as described herein can be subjected to
fragmentation or cleavage.
[0217] Amplification of nucleic acids is sometimes necessary when
dealing with organisms that are difficult to culture. Where
amplification may be desired, any suitable amplification technique
can be utilized. Non-limiting examples of methods for amplification
of polynucleotides include, polymerase chain reaction (PCR);
ligation amplification (or ligase chain reaction (LCR));
amplification methods based on the use of Q-beta replicase or
template-dependent polymerase (see US Patent Publication Number
US20050287592); helicase-dependant isothermal amplification
(Vincent et al., "Helicase-dependent isothermal DNA amplification".
EMBO reports 5 (8): 795-800 (2004)); strand displacement
amplification (SDA); thermophilic SDA nucleic acid sequence based
amplification (3SR or NASBA) and transcription-associated
amplification (TAA). Non-limiting examples of PCR amplification
methods include standard PCR, AFLP-PCR, Allele-specific PCR,
Alu-PCR, Asymmetric PCR, Colony PCR, Hot start PCR, Inverse PCR
(IPCR), In situ PCR (ISH), Intersequence-specific PCR (ISSR-PCR),
Long PCR, Multiplex PCR, Nested PCR, Quantitative PCR, Reverse
Transcriptase PCR(RT-PCR), Real Time PCR, Single cell PCR, Solid
phase PCR, combinations thereof, and the like. Reagents and
hardware for conducting PCR are commercially available.
[0218] Protocols for conducting the various type of PCR listed
above are readily available to the artisan. PCR conditions can be
dependent upon primer sequences, target abundance, and the desired
amount of amplification, and therefore, one of skill in the art may
choose from a number of PCR protocols available (see, e.g., U.S.
Pat. Nos. 4,683,195 and 4,683,202; and PCR Protocols: A Guide to
Methods and Applications, Innis et al., eds, 1990. PCR often is
carried out as an automated process with a thermostable enzyme. In
this process, the temperature of the reaction mixture is cycled
through a denaturing region, a primer-annealing region, and an
extension reaction region automatically. Machines specifically
adapted for this purpose are commercially available. A non-limiting
example of a PCR protocol that may be suitable for embodiments
described herein is, treating the sample at 95.degree. C. for 5
minutes; repeating forty-five cycles of 95.degree. C. for 1 minute,
59.degree. C. for 1 minute, 10 seconds, and 72.sup.2C for 1 minute
30 seconds; and then treating the sample at 72.degree. C. for 5
minutes. Additional PCR protocols are described in the example
section. Multiple cycles frequently are performed using a
commercially available thermal cycler. Suitable isothermal
amplification processes known and selected by the person of
ordinary skill in the art also may be applied, in certain
embodiments. In some embodiments, nucleic acids encoding
polypeptides with a desired activity can be isolated by amplifying
the desired sequence from an organism having the desired activity
using oligonucleotides or primers designed based on sequences
described herein
[0219] Amplified, isolated and/or purified nucleic acids can be
cloned into the recombinant DNA vectors described in Figures herein
or into suitable commercially available recombinant DNA vectors.
Cloning of nucleic acid sequences of interest into recombinant DNA
vectors can facilitate further manipulations of the nucleic acids
for preparation of nucleic acid reagents, (e.g., alteration of
nucleotide sequences by mutagenesis, homologous recombination,
amplification and the like, for example). Standard cloning
procedures (e.g., enzymic digestion, ligation, and the like) are
readily available to the artisan and can be found in Maniatis, T.,
E. F. Fritsch and J. Sambrook (1982) Molecular Cloning: a
Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y.
[0220] In some embodiments, nucleic acid sequences prepared by
isolation or amplification can be used, without any further
modification, to add an activity to a microorganism and thereby
generate a genetically modified or engineered microorganism. In
certain embodiments, nucleic acid sequences prepared by isolation
or amplification can be genetically modified to alter (e.g.,
increase or decrease, for example) a desired activity. In some
embodiments, nucleic acids, used to add an activity to an organism,
sometimes are genetically modified to optimize the heterologous
polynucleotide sequence encoding the desired activity (e.g.,
polypeptide or protein, for example). The term "optimize" as used
herein can refer to alteration to increase or enhance expression by
preferred codon usage. The term optimize can also refer to
modifications to the amino acid sequence to increase the activity
of a polypeptide or protein, such that the activity exhibits a
higher catalytic activity as compared to the "natural" version of
the polypeptide or protein.
[0221] Nucleic acid sequences of interest can be genetically
modified using methods known in the art. Mutagenesis techniques are
particularly useful for small scale (e.g., 1, 2, 5, 10 or more
nucleotides) or large scale (e.g., 50, 100, 150, 200, 500, or more
nucleotides) genetic modification. Mutagenesis allows the artisan
to alter the genetic information of an organism in a stable manner,
either naturally (e.g., isolation using selection and screening) or
experimentally by the use of chemicals, radiation or inaccurate DNA
replication (e.g., PCR mutagenesis). In some embodiments, genetic
modification can be performed by whole scale synthetic synthesis of
nucleic acids, using a native nucleotide sequence as the reference
sequence, and modifying nucleotides that can result in the desired
alteration of activity. Mutagenesis methods sometimes are specific
or targeted to specific regions or nucleotides (e.g., site-directed
mutagenesis, PCR-based site-directed mutagenesis, and in vitro
mutagenesis techniques such as transplacement and in vivo
oligonucleotide site-directed mutagenesis, for example).
Mutagenesis methods sometimes are non-specific or random with
respect to the placement of genetic modifications (e.g., chemical
mutagenesis, insertion element (e.g., insertion or transposon
elements) and inaccurate PCR based methods, for example).
[0222] Site directed mutagenesis is a procedure in which a specific
nucleotide or specific nucleotides in a DNA molecule are mutated or
altered. Site directed mutagenesis typically is performed using a
nucleic acid sequence of interest cloned into a circular plasmid
vector. Site-directed mutagenesis requires that the wild type
sequence be known and used a platform for the genetic alteration.
Site-directed mutagenesis sometimes is referred to as
oligonucleotide-directed mutagenesis because the technique can be
performed using oligonucleotides which have the desired genetic
modification incorporated into the complement a nucleotide sequence
of interest. The wild type sequence and the altered nucleotide are
allowed to hybridize and the hybridized nucleic acids are extended
and replicated using a DNA polymerase. The double stranded nucleic
acids are introduced into a host (e.g., E. coli, for example) and
further rounds of replication are carried out in vivo. The
transformed cells carrying the mutated nucleic acid sequence are
then selected and/or screened for those cells carrying the
correctly mutagenized sequence. Cassette mutagenesis and PCR-based
site-directed mutagenesis are further modifications of the
site-directed mutagenesis technique. Site-directed mutagenesis can
also be performed in vivo (e.g., transplacement "pop-in pop-out",
In vivo site-directed mutagenesis with synthetic oligonucleotides
and the like, for example).
[0223] PCR-based mutagenesis can be performed using PCR with
oligonucleotide primers that contain the desired mutation or
mutations. The technique functions in a manner similar to standard
site-directed mutagenesis, with the exception that a thermocycler
and PCR conditions are used to replace replication and selection of
the clones in a microorganism host. As PCR-based mutagenesis also
uses a circular plasmid vector, the amplified fragment (e.g.,
linear nucleic acid molecule) containing the incorporated genetic
modifications can be separated from the plasmid containing the
template sequence after a sufficient number of rounds of
thermocycler amplification, using standard electrophorectic
procedures. A modification of this method uses linear amplification
methods and a pair of mutagenic primers that amplify the entire
plasmid. The procedure takes advantage of the E. coli Dam methylase
system which causes DNA replicated in vivo to be sensitive to the
restriction endonucleases DpnI. PCR synthesized DNA is not
methylated and is therefore resistant to DpnI. This approach allows
the template plasmid to be digested, leaving the genetically
modified, PCR synthesized plasmids to be isolated and transformed
into a host bacteria for DNA repair and replication, thereby
facilitating subsequent cloning and identification steps. A certain
amount of randomness can be added to PCR-based sited directed
mutagenesis by using partially degenerate primers.
[0224] Recombination sometimes can be used as a tool for
mutagenesis. Homologous recombination allows the artisan to
specifically target regions of known sequence for insertion of
heterologous nucleotide sequences using the host organisms natural
DNA replication and repair enzymes. Homologous recombination
methods sometimes are referred to as "pop in pop out" mutagenesis,
transplacement, knock out mutagenesis or knock in mutagenesis.
Integration of a nucleic acid sequence into a host genome is a
single cross over event, which inserts the entire nucleic acid
reagent (e.g., pop in). A second cross over event excises all but a
portion of the nucleic acid reagent, leaving behind a heterologous
sequence, often referred to as a "footprint" (e.g., pop out).
Mutagenesis by insertion (e.g., knock in) or by double
recombination leaving behind a disrupting heterologous nucleic acid
(e.g., knock out) both server to disrupt or "knock out" the
function of the gene or nucleic acid sequence in which insertion
occurs. By combining selectable markers and/or auxotrophic markers
with nucleic acid reagents designed to provide the appropriate
nucleic acid target sequences, the artisan can target a selectable
nucleic acid reagent to a specific region, and then select for
recombination events that "pop out" a portion of the inserted
(e.g., "pop in") nucleic acid reagent.
[0225] Such methods take advantage of nucleic acid reagents that
have been specifically designed with known target nucleic acid
sequences at or near a nucleic acid or genomic region of interest.
Popping out typically leaves a "foot print" of left over sequences
that remain after the recombination event. The left over sequence
can disrupt a gene and thereby reduce or eliminate expression of
that gene. In some embodiments, the method can be used to insert
sequences, upstream or downstream of genes that can result in an
enhancement or reduction in expression of the gene. In certain
embodiments, new genes can be introduced into the genome of a host
organism using similar recombination or "pop in" methods. An
example of a yeast recombination system using the ura3 gene and
5-FOA were described briefly above and further detail is presented
herein.
[0226] A method for modification is described in Alani et al., "A
method for gene disruption that allows repeated use of URA3
selection in the construction of multiply disrupted yeast strains",
Genetics 116(4):541-545 August 1987. The original method uses a
Ura3 cassette with 1000 base pairs (bp) of the same nucleotide
sequence cloned in the same orientation on either side of the URA3
cassette. Targeting sequences of about 50 bp are added to each side
of the construct. The double stranded targeting sequences are
complementary to sequences in the genome of the host organism. The
targeting sequences allow site-specific recombination in a region
of interest. The modification of the original technique replaces
the two 1000 bp sequence direct repeats with two 200 bp direct
repeats. The modified method also uses 50 bp targeting sequences.
The modification reduces or eliminates recombination of a second
knock out into the 1000 bp repeat left behind in a first
mutagenesis, therefore allowing multiply knocked out yeast.
Additionally, the 200 bp sequences used herein are uniquely
designed, self-assembling sequences that leave behind identifiable
footprints. The technique used to design the sequences incorporate
design features such as low identity to the yeast genome, and low
identity to each other. Therefore a library of the self-assembling
sequences can be generated to allow multiple knockouts in the same
organism, while reducing or eliminating the potential for
integration into a previous knockout.
[0227] As noted above, the URA3 cassette makes use of the toxicity
of 5-FOA in yeast carrying a functional URA3 gene. Uracil synthesis
deficient yeast are transformed with the modified URA3 cassette,
using standard yeast transformation protocols, and the transformed
cells are plated on minimal media minus uracil. In some
embodiments, PCR can be used to verify correct insertion into the
region of interest in the host genome, and certain embodiments the
PCR step can be omitted. Inclusion of the PCR step can reduce the
number of transformants that need to be counter selected to "pop
out" the URA3 cassette. The transformants (e.g., all or the ones
determined to be correct by PCR, for example) can then be
counter-selected on media containing 5-FOA, which will select for
recombination out (e.g., popping out) of the URA3 cassette, thus
rendering the yeast ura3 deficient again, and resistant to 5-FOA
toxicity. Targeting sequences used to direct recombination events
to specific regions are presented herein. A modification of the
method described above can be used to integrate genes in to the
chromosome, where after recombination a functional gene is left in
the chromosome next to the 200 bp footprint.
[0228] In some embodiments, other auxotrophic or dominant selection
markers can be used in place of URA3 (e.g., an auxotrophic
selectable marker), with the appropriate change in selection media
and selection agents. Auxotrophic selectable markers are used in
strains deficient for synthesis of a required biological molecule
(e.g., amino acid or nucleoside, for example). Non-limiting
examples of additional auxotrophic markers include; HIS3, TRP1,
LEU2, LEU2-d, and LYS2. Certain auxotrophic markers (e.g., URA3 and
LYS2) allow counter selection to select for the second
recombination event that pops out all but one of the direct repeats
of the recombination construct. HIS3 encodes an activity involved
in histidine synthesis. TRP1 encodes an activity involved in
tryptophan synthesis. LEU2 encodes an activity involved in leucine
synthesis. LEU2-d is a low expression version of LEU2 that selects
for increased copy number (e.g., gene or plasmid copy number, for
example) to allow survival on minimal media without leucine. LYS2
encodes an activity involved in lysine synthesis, and allows
counter selection for recombination out of the LYS2 gene using
alpha-amino adipate (.alpha.-amino adipate).
[0229] Dominant selectable markers are useful because they also
allow industrial and/or prototrophic strains to be used for genetic
manipulations. Additionally, dominant selectable markers provide
the advantage that rich medium can be used for plating and culture
growth, and thus growth rates are markedly increased. Non-limiting
examples of dominant selectable markers include; Tn903 kan.sup.r,
Cm.sup.r, Hyg.sup.r, CUP1, and DHFR. Tn903 kan.sup.r encodes an
activity involved in kanamycin antibiotic resistance (e.g.,
typically neomycin phosphotransferase II or NPTII, for example).
Cmr encodes an activity involved in chloramphenicol antibiotic
resistance (e.g., typically chloramphenicol acetyl transferase or
CAT, for example). Hyg.sup.r encodes an activity involved in
hygromycin resistance by phosphorylation of hygromycin B (e.g.,
hygromycin phosphotransferase, or HPT). CUP1 encodes an activity
involved in resistance to heavy metal (e.g., copper, for example)
toxicity. DHFR encodes a dihydrofolate reductase activity which
confers resistance to methotrexate and sulfanilamde compounds.
[0230] In contrast to site-directed or specific mutagenesis, random
mutagenesis does not require any sequence information and can be
accomplished by a number of widely different methods. Random
mutagenesis often is used to generate mutant libraries that can be
used to screen for the desired genotype or phenotype. Non-limiting
examples of random mutagenesis include; chemical mutagenesis,
UV-induced mutagenesis, insertion element or transposon-mediated
mutagenesis, DNA shuffling, error-prone PCR mutagenesis, and the
like.
[0231] Chemical mutagenesis often involves chemicals like ethyl
methanesulfonate (EMS), nitrous acid, mitomycin C,
N-methyl-N-nitrosourea (MNU), diepoxybutane (DEB), 1, 2, 7,
8-diepoxyoctane (DEO), methyl methane sulfonate (MMS),
N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline
1-oxide (4-NQO),
2-methyloxy-6-chloro-9(3-[ethyl-2-chloroethyl]-aminopropylamino)-acridine-
dihydrochloride (ICR-170), 2-amino purine (2AP), and hydroxylamine
(HA), provided herein as non-limiting examples. These chemicals can
cause base-pair substitutions, frameshift mutations, deletions,
transversion mutations, transition mutations, incorrect
replication, and the like. In some embodiments, the mutagenesis can
be carried out in vivo. Sometimes the mutagenic process involves
the use of the host organisms DNA replication and repair mechanisms
to incorporate and replicate the mutagenized base or bases. Another
type of chemical mutagenesis involves the use of base-analogs. The
use of base-analogs cause incorrect base pairing which in the
following round of replication is corrected to a mismatched
nucleotide when compared to the starting sequence. Base analog
mutagenesis introduces a small amount of non-randomness to random
mutagenesis, because specific base analogs can be chose which can
be incorporated at certain nucleotides in the starting sequence.
Correction of the mispairing typically yields a known substitution.
For example, Bromo-deoxyuridine (BrdU) can be incorporated into DNA
and replaces T in the sequence. The host DNA repair and replication
machinery can sometime correct the defect, but sometimes will
mispair the BrdU with a G. The next round of replication then
causes a G-C transversion from the original A-T in the native
sequence.
[0232] Ultra violet (UV) induced mutagenesis is caused by the
formation of thymidine dimers when UV light irradiates chemical
bonds between two adjacent thymine residues. Excision repair
mechanism of the host organism correct the lesion in the DNA, but
occasionally the lesion is incorrectly repaired typically resulting
in a C to T transition.
[0233] Insertion element or transposon-mediated mutagenesis makes
use of naturally occurring or modified naturally occurring mobile
genetic elements. Transposons often encode accessory activities in
addition to the activities necessary for transposition (e.g.,
movement using a transposase activity, for example). In many
examples, transposon accessory activities are antibiotic resistance
markers (e.g., see Tn903 kan.sup.r described above, for example).
Insertion elements typically only encode the activities necessary
for movement of the nucleic acid sequence. Insertion element and
transposon mediated mutagenesis often can occur randomly, however
specific target sequences are known for some transposons. Mobile
genetic elements like IS elements or Transposons (Tn) often have
inverted repeats, direct repeats or both inverted and direct
repeats flanking the region coding for the transposition genes.
Recombination events catalyzed by the transposase cause the element
to remove itself from the genome and move to a new location,
leaving behind a portion of an inverted or direct repeat. Classic
examples of transposons are the "mobile genetic elements"
discovered in maize. Transposon mutagenesis kits are commercially
available which are designed to leave behind a 5 codon insert
(e.g., Mutation Generation System kit, Finnzymes, World Wide Web
URL finnzymes.us, for example). This allows the artisan to identify
the insertion site, without fully disrupting the function of most
genes.
[0234] DNA shuffling is a method which uses DNA fragments from
members of a mutant library and reshuffles the fragments randomly
to generate new mutant sequence combinations. The fragments are
typically generated using DNaseI, followed by random annealing and
re-joining using self priming PCR. The DNA overhanging ends, from
annealing of random fragments, provide "primer" sequences for the
PCR process. Shuffling can be applied to libraries generated by any
of the above mutagenesis methods.
[0235] Error prone PCR and its derivative rolling circle error
prone PCR uses increased magnesium and manganese concentrations in
conjunction with limiting amounts of one or two nucleotides to
reduce the fidelity of the Taq polymerase. The error rate can be as
high as 2% under appropriate conditions, when the resultant mutant
sequence is compared to the wild type starting sequence. After
amplification, the library of mutant coding sequences must be
cloned into a suitable plasmid. Although point mutations are the
most common types of mutation in error prone PCR, deletions and
frameshift mutations are also possible. There are a number of
commercial error-prone PCR kits available, including those from
Stratagene and Clontech (e.g., World Wide Web URL strategene.com
and World Wide Web URL clontech.com, respectively, for example).
Rolling circle error-prone PCR is a variant of error-prone PCR in
which wild-type sequence is first cloned into a plasmid, the whole
plasmid is then amplified under error-prone conditions.
[0236] As noted above, organisms with altered activities can also
be isolated using genetic selection and screening of organisms
challenged on selective media or by identifying naturally occurring
variants from unique environments. For example, 2-Deoxy-D-glucose
is a toxic glucose analog. Growth of yeast on this substance yields
mutants that are glucose-deregulated. A number of mutants have been
isolated using 2-Deoxy-D-glucose including transport mutants, and
mutants that ferment glucose and galactose simultaneously instead
of glucose first then galactose when glucose is depleted. Similar
techniques have been used to isolate mutant microorganisms that can
metabolize plastics (e.g., from landfills), petrochemicals (e.g.,
from oil spills), and the like, either in a laboratory setting or
from unique environments.
[0237] Similar methods can be used to isolate naturally occurring
mutations in a desired activity when the activity exists at a
relatively low or nearly undetectable level in the organism of
choice, in some embodiments. The method generally consists of
growing the organism to a specific density in liquid culture,
concentrating the cells, and plating the cells on various
concentrations of the substance to which an increase in metabolic
activity is desired. The cells are incubated at a moderate growth
temperature, for 5 to 10 days. To enhance the selection process,
the plates can be stored for another 5 to 10 days at a low
temperature. The low temperature sometimes can allow strains that
have gained or increased an activity to continue growing while
other strains are inhibited for growth at the low temperature.
Following the initial selection and secondary growth at low
temperature, the plates can be replica plated on higher or lower
concentrations of the selection substance to further select for the
desired activity.
[0238] A native, heterologous or mutagenized polynucleotide can be
introduced into a nucleic acid reagent for introduction into a host
organism, thereby generating an engineered microorganism. Standard
recombinant DNA techniques (restriction enzyme digests, ligation,
and the like) can be used by the artisan to combine the mutagenized
nucleic acid of interest into a suitable nucleic acid reagent
capable of (i) being stably maintained by selection in the host
organism, or (ii) being integrating into the genome of the host
organism. As noted above, sometimes nucleic acid reagents comprise
two replication origins to allow the same nucleic acid reagent to
be manipulated in bacterial before final introduction of the final
product into the host organism (e.g., yeast or fungus for example).
Standard molecular biology and recombinant DNA methods available to
one of skill in the art can be found in Maniatis, T., E. F. Fritsch
and J. Sambrook (1982) Molecular Cloning: a Laboratory Manual; Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
[0239] Nucleic acid reagents can be introduced into microorganisms
using various techniques. Non-limiting examples of methods used to
introduce heterologous nucleic acids into various organisms
include; transformation, transfection, transduction,
electroporation, ultrasound-mediated transformation, particle
bombardment and the like. In some instances the addition of carrier
molecules (e.g., bis-benzimdazolyl compounds, for example, see U.S.
Pat. No. 5,595,899) can increase the uptake of DNA in cells
typically though to be difficult to transform by conventional
methods. Conventional methods of transformation are readily
available to the artisan and can be found in Maniatis, T., E. F.
Fritsch and J. Sambrook (1982) Molecular Cloning: a Laboratory
Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Culture, Production and Process Methods
[0240] Engineered microorganisms often are cultured under
conditions that optimize yield of a target molecule. A non-limiting
example of such a target molecule is ethanol. Culture conditions
often can alter (e.g., add, optimize, reduce or eliminate, for
example) activity of one or more of the following activities:
phosphofructokinase activity, phosphogluconate dehydratase
activity, 2-keto-3-deoxygluconate-6-phosphate aldolase activity,
xylose isomerase activity, phosphoenolpyruvate carboxylase
activity, alcohol dehydrogenase 2 activity and thymidylate synthase
activities. In general, conditions that may be optimized include
the type and amount of carbon source, the type and amount of
nitrogen source, the carbon-to-nitrogen ratio, the oxygen level,
growth temperature, pH, length of the biomass production phase,
length of target product accumulation phase, and time of cell
harvest.
[0241] The term "fermentation conditions" as used herein refers to
any culture conditions suitable for maintaining a microorganism
(e.g., in a static or proliferative state). Fermentation conditions
can include several parameters, including without limitation,
temperature, oxygen content, nutrient content (e.g., glucose
content), pH, agitation level (e.g., revolutions per minute), gas
flow rate (e.g., air, oxygen, nitrogen gas), redox potential, cell
density (e.g., optical density), cell viability and the like. A
change in fermentation conditions (e.g., switching fermentation
conditions) is an alteration, modification or shift of one or more
fermentation parameters. For example, one can change fermentation
conditions by increasing or decreasing temperature, increasing or
decreasing pH (e.g., adding or removing an acid, a base or carbon
dioxide), increasing or decreasing oxygen content (e.g.,
introducing air, oxygen, carbon dioxide, nitrogen) and/or adding or
removing a nutrient (e.g., one or more sugars or sources of sugar,
biomass, vitamin and the like), or combinations of the foregoing.
Examples of fermentation conditions are described herein. Aerobic
conditions often comprise greater than about 50% dissolved oxygen
(e.g., about 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%,
74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99%, or greater than any one of the
foregoing). Anaerobic conditions often comprise less than about 50%
dissolved oxygen (e.g., about 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%,
16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%,
42%, 44%, 46%, 48%, or less than any one of the foregoing).
[0242] Culture media generally contain a suitable carbon source.
Carbon sources may include, but are not limited to, monosaccharides
(e.g., glucose, fructose, xylose), disaccharides (e.g., lactose,
sucrose), oligosaccharides, polysaccharides (e.g., starch,
cellulose, hemicellulose, other lignocellulosic materials or
mixtures thereof), sugar alcohols (e.g., glycerol), and renewable
feedstocks (e.g., cheese whey permeate, cornsteep liquor, sugar
beet molasses, barley malt). Carbon sources also can be selected
from one or more of the following non-limiting examples: linear or
branched alkanes (e.g., hexane), linear or branched alcohols (e.g.,
hexanol), fatty acids (e.g., about 10 carbons to about 22 carbons),
esters of fatty acids, monoglycerides, diglycerides, triglycerides,
phospholipids and various commercial sources of fatty acids
including vegetable oils (e.g., soybean oil) and animal fats. A
carbon source may include one-carbon sources (e.g., carbon dioxide,
methanol, formaldehyde, formate and carbon-containing amines) from
which metabolic conversion into key biochemical intermediates can
occur. It is expected that the source of carbon utilized may
encompass a wide variety of carbon-containing sources and will only
be limited by the choice of the engineered microorganism(s).
[0243] Nitrogen may be supplied from an inorganic (e.g.,
(NH.sub.4).sub.2SO.sub.4) or organic source (e.g., urea or
glutamate). In addition to appropriate carbon and nitrogen sources,
culture media also can contain suitable minerals, salts, cofactors,
buffers, vitamins, metal ions (e.g., Mn.sup.+2, Co.sup.+2,
Zn.sup.+2, Mg.sup.+2) and other components suitable for culture of
microorganisms. Engineered microorganisms sometimes are cultured in
complex media (e.g., yeast extract-peptone-dextrose broth (YPD)).
In some embodiments, engineered microorganisms are cultured in a
defined minimal media that lacks a component necessary for growth
and thereby forces selection of a desired expression cassette
(e.g., Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.)).
Culture media in some embodiments are common commercially prepared
media, such as Yeast Nitrogen Base (DIFCO Laboratories, Detroit,
Mich.). Other defined or synthetic growth media may also be used
and the appropriate medium for growth of the particular
microorganism are known.
[0244] A variety of host organisms can be selected for the
production of engineered microorganisms. Non-limiting examples
include yeast and fungi. In specific embodiments, yeast are
cultured in YPD media (10 g/L Bacto Yeast Extract, 20 g/L Bacto
Peptone, and 20 g/L Dextrose). Filamentous fungi, in particular
embodiments, are grown in CM (Complete Medium) containing 10 g/L
Dextrose, 2 g/L Bacto Peptone, 1 g/L Bacto Yeast Extract, 1 g/L
Casamino acids, 50 mL /L 20.times. Nitrate Salts (120 g/L
NaNO.sub.3, 10.4 g/L KCl, 10.4 g/L MgSO.sub.4.7 H.sub.2O), 1 mL/L
1000.times. Trace Elements (22 g/L ZnSO.sub.4.7 H.sub.2O, 11 g/L
H.sub.3BO.sub.3, 5 g/L MnCl.sub.2.7 H.sub.2O, 5 g/L FeSO.sub.4.7
H.sub.2O, 1.7 g/L CoCl.sub.2.6 H.sub.2O, 1.6 g/L CuSO.sub.4.5
H.sub.2O, 1.5 g/L Na.sub.2MoO.sub.4.2 H.sub.2O, and 50 g/L
Na.sub.4EDTA), and 1 mL/L Vitamin Solution (100 mg each of Biotin,
pyridoxine, thiamine, riboflavin, p-aminobenzoic acid, and
nicotinic acid in 100 mL water).
[0245] A suitable pH range for the fermentation often is between
about pH 4.0 to about pH 8.0, where a pH in the range of about pH
5.5 to about pH 7.0 sometimes is utilized for initial culture
conditions. Culturing may be conducted under aerobic or anaerobic
conditions, where microaerobic conditions sometimes are maintained.
A two-stage process may be utilized, where one stage promotes
microorganism proliferation and another state promotes production
of target molecule. In a two-stage process, the first stage may be
conducted under aerobic conditions (e.g., introduction of air
and/or oxygen) and the second stage may be conducted under
anaerobic conditions (e.g., air or oxygen are not introduced to the
culture conditions).
[0246] A variety of fermentation processes may be applied for
commercial biological production of a target product. In some
embodiments, commercial production of a target product from a
recombinant microbial host is conducted using a batch, fed-batch or
continuous fermentation process, for example.
[0247] A batch fermentation process often is a closed system where
the media composition is fixed at the beginning of the process and
not subject to further additions beyond those required for
maintenance of pH and oxygen level during the process. At the
beginning of the culturing process the media is inoculated with the
desired organism and growth or metabolic activity is permitted to
occur without adding additional sources (i.e., carbon and nitrogen
sources) to the medium. In batch processes the metabolite and
biomass compositions of the system change constantly up to the time
the culture is terminated. In a typical batch process, cells
proceed through a static lag phase to a high-growth log phase and
finally to a stationary phase, wherein the growth rate is
diminished or halted. Left untreated, cells in the stationary phase
will eventually die.
[0248] A variation of the standard batch process is the fed-batch
process, where the carbon source is continually added to the
fermentor over the course of the fermentation process. Fed-batch
processes are useful when catabolite repression is apt to inhibit
the metabolism of the cells or where it is desirable to have
limited amounts of carbon source in the media at any one time.
Measurement of the carbon source concentration in fed-batch systems
may be estimated on the basis of the changes of measurable factors
such as pH, dissolved oxygen and the partial pressure of waste
gases (e.g., CO.sub.2).
[0249] Batch and fed-batch culturing methods are known in the art.
Examples of such methods may be found in Thomas D. Brock in
Biotechnology: A Textbook of Industrial Microbiology, 2.sup.nd ed.,
(1989) Sinauer Associates Sunderland, Mass. and Deshpande, Mukund
V., Appl. Biochem. Biotechnol., 36:227 (1992).
[0250] In continuous fermentation process a defined media often is
continuously added to a bioreactor while an equal amount of culture
volume is removed simultaneously for product recovery. Continuous
cultures generally maintain cells in the log phase of growth at a
constant cell density. Continuous or semi-continuous culture
methods permit the modulation of one factor or any number of
factors that affect cell growth or end product concentration. For
example, an approach may limit the carbon source and allow all
other parameters to moderate metabolism. In some systems, a number
of factors affecting growth may be altered continuously while the
cell concentration, measured by media turbidity, is kept constant.
Continuous systems often maintain steady state growth and thus the
cell growth rate often is balanced against cell loss due to media
being drawn off the culture. Methods of modulating nutrients and
growth factors for continuous culture processes, as well as
techniques for maximizing the rate of product formation, are known
and a variety of methods are detailed by Brock, supra.
[0251] In various embodiments ethanol may be purified from the
culture media or extracted from the engineered microorganisms.
Culture media may be tested for ethanol concentration and drawn off
when the concentration reaches a predetermined level. Detection
methods are known in the art, including but not limited to the use
of a hydrometer and infrared measurement of vibrational frequency
of dissolved ethanol using the CH band at 2900 cm.sup.-1. Ethanol
may be present at a range of levels as described herein.
[0252] A target product sometimes is retained within an engineered
microorganism after a culture process is completed, and in certain
embodiments, the target product is secreted out of the
microorganism into the culture medium. For the latter embodiments,
(i) culture media may be drawn from the culture system and fresh
medium may be supplemented, and/or (ii) target product may be
extracted from the culture media during or after the culture
process is completed. Engineered microorganisms may be cultured on
or in solid, semi-solid or liquid media. In some embodiments media
is drained from cells adhering to a plate. In certain embodiments,
a liquid-cell mixture is centrifuged at a speed sufficient to
pellet the cells but not disrupt the cells and allow extraction of
the media, as known in the art. The cells may then be resuspended
in fresh media. Target product may be purified from culture media
according to methods known in the art.
[0253] In certain embodiments, target product is extracted from the
cultured engineered microorganisms. The microorganism cells may be
concentrated through centrifugation at speed sufficient to shear
the cell membranes. In some embodiments, the cells may be
physically disrupted (e.g., shear force, sonication) or chemically
disrupted (e.g., contacted with detergent or other lysing agent).
The phases may be separated by centrifugation or other method known
in the art and target product may be isolated according to known
methods.
[0254] Commercial grade target product sometimes is provided in
substantially pure form (e.g., 90% pure or greater, 95% pure or
greater, 99% pure or greater or 99.5% pure or greater). In some
embodiments, target product may be modified into any one of a
number of downstream products. For example, ethanol may be
derivatized or further processed to produce ethyl halides, ethyl
esters, diethyl ether, acetic acid, ethyl amines, butadiene,
solvents, food flavorings, distilled spirits and the like.
[0255] Target product may be provided within cultured microbes
containing target product, and cultured microbes may be supplied
fresh or frozen in a liquid media or dried. Fresh or frozen
microbes may be contained in appropriate moisture-proof containers
that may also be temperature controlled as necessary. Target
product sometimes is provided in culture medium that is
substantially cell-free. In some embodiments target product or
modified target product purified from microbes is provided, and
target product sometimes is provided in substantially pure form. In
certain embodiments, ethanol can be provided in anhydrous or
hydrous forms. Ethanol may be transported in a variety of
containers including pints, quarts, liters, gallons, drums (e.g.,
10 gallon or 55 gallon, for example) and the like.
[0256] In certain embodiments, a target product (e.g., ethanol,
succinic acid) is produced with a yield of about 0.30 grams of
target product, or greater, per gram of glucose added during a
fermentation process (e.g., about 0.31 grams of target product per
gram of glucose added, or greater; about 0.32 grams of target
product per gram of glucose added, or greater; about 0.33 grams of
target product per gram of glucose added, or greater; about 0.34
grams of target product per gram of glucose added, or greater;
about 0.35 grams of target product per gram of glucose added, or
greater; about 0.36 grams of target product per gram of glucose
added, or greater; about 0.37 grams of target product per gram of
glucose added, or greater; about 0.38 grams of target product per
gram of glucose added, or greater; about 0.39 grams of target
product per gram of glucose added, or greater; about 0.40 grams of
target product per gram of glucose added, or greater; about 0.41
grams of target product per gram of glucose added, or greater; 0.42
grams of target product per gram of glucose added, or greater; 0.43
grams of target product per gram of glucose added, or greater; 0.44
grams of target product per gram of glucose added, or greater; 0.45
grams of target product per gram of glucose added, or greater; 0.46
grams of target product per gram of glucose added, or greater; 0.47
grams of target product per gram of glucose added, or greater; 0.48
grams of target product per gram of glucose added, or greater; 0.49
grams of target product per gram of glucose added, or greater; 0.50
grams of target product per gram of glucose added, or greater; 0.51
grams of target product per gram of glucose added, or greater; 0.52
grams of target product per gram of glucose added, or greater; 0.53
grams of target product per gram of glucose added, or greater; 0.54
grams of target product per gram of glucose added, or greater; 0.55
grams of target product per gram of glucose added, or greater; 0.56
grams of target product per gram of glucose added, or greater; 0.57
grams of target product per gram of glucose added, or greater; 0.58
grams of target product per gram of glucose added, or greater; 0.59
grams of target product per gram of glucose added, or greater; 0.60
grams of target product per gram of glucose added, or greater; 0.61
grams of target product per gram of glucose added, or greater; 0.62
grams of target product per gram of glucose added, or greater; 0.63
grams of target product per gram of glucose added, or greater; 0.64
grams of target product per gram of glucose added, or greater; 0.65
grams of target product per gram of glucose added, or greater; 0.66
grams of target product per gram of glucose added, or greater; 0.67
grams of target product per gram of glucose added, or greater; 0.68
grams of target product per gram of glucose added, or greater; 0.69
or O.70 grams of target product per gram of glucose added or
greater). In some embodiments, 0.45 grams of target product per
gram of glucose added, or greater, is produced during the
fermentation process.
EXAMPLES
[0257] The examples set forth below illustrate certain embodiments
and do not limit the technology. Certain examples set forth below
utilize standard recombinant DNA and other biotechnology protocols
known in the art. Many such techniques are described in detail in
Maniatis, T., E. F. Fritsch and J. Sambrook (1982) Molecular
Cloning: a Laboratory Manual; Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. DNA mutagenesis can be accomplished using the
Stratagene (San Diego, Calif.) "QuickChange" kit according to the
manufacturer's instructions, or by one of the other types of
mutagenesis described above.
Example 1
Activation of the Entner-Doudoroff Pathway in Yeast Cells
[0258] Genomic DNA from Zymomonas mobilis (ZM4) was obtained from
the American Type Culture Collection (ATCC accession number 31821
D-5). The genes encoding phosphogluconate dehydratase EC 4.2.1.12
(referred to as "edd") and 2-keto-3-deoxygluconate-6-phosphate
aldolase EC 4.2.1.14 (referred to as "eda") were isolated from the
ZM4 genomic DNA using the following oligonucleotides:
TABLE-US-00002 The ZM4 eda gene: (SEQ ID No: 1)
5'-aactgactagtaaaaaaatgcgtgatatcgattcc-3' (SEQ ID No: 2)
5'-agtaactcgagctactaggcaacagcagcgcgcttg-3' The ZM4 edd gene: (SEQ
ID NO: 3) 5'-aactgactagtaaaaaaatgactgatctgcattcaacg-3' (SEQ ID NO:
4) 5'-agtaactcgagctactagataccggcacctgcatatattgc-3'
[0259] E. coli genomic DNA was prepared using Qiagen DNeasy blood
and tissue kit according to the manufacture's protocol. The E. coli
edd and eda constructs were isolated from E. coli genomic DNA using
the following oligonucleotides:
TABLE-US-00003 The E. coli eda gene: (SEQ ID NO: 5)
5'-aactgactagtaaaaaaatgaaaaactggaaaacaagtgcagaatc- 3' (SEQ ID NO:
6) 5'-agtaactcgagctactacagcttagcgccttctacagcttcacg-3' The E. coli
edd gene: (SEQ ID NO: 7)
5'-aactgactagtaaaaaaatgaatccacaattgttacgcgtaacaaat cg-3' (SEQ ID
NO: 8) 5'agtaactcgagctactaaaaagtgatacaggttgcgccctgttcggca c-3'
[0260] All oligonucleotides set forth above were purchased from
Integrated DNA technologies ("IDT", Coralville, Iowa). These
oligonucleotides were designed to incorporate a SpeI restriction
endonuclease cleavage site upstream and a XhoI restriction
endonuclease cleavage site downstream of the edd and eda gene
constructs such that these sites could be used to clone these genes
into yeast expression vectors p426GPD (ATCC accession number 87361)
and p425GPD (ATCC accession number 87359). In addition to
incorporating restriction endonuclease cleavage sites, the forward
oligonucleotides were designed to incorporate six consecutive
AAAAAA nucleotides immediately upstream of the ATG initiation
codon. This ensured that there was a conserved kozak sequence
important for efficient translation initiation in yeast.
[0261] Cloning the edd and eda genes from ZM4 and E. coli genomic
DNA was accomplished using the following procedure: About 100 ng of
ZM4 or E. coli genomic DNA, 1 .mu.M of the oligonucleotide primer
set listed above, 2.5 U of PfuUltra High-Fidelity DNA polymerase
(Stratagene), 300 .mu.M dNTPs (Roche), and 1.times.PfuUltra
reaction buffer was mixed in a final reaction volume of 50 .mu.l. A
BIORAD DNA Engine Tetrad 2 Peltier thermal cycler was used for the
PCR reactions and the following cycle conditions were used: 5 min
denaturation step at 95.degree. C., followed by 30 cycles of 20 sec
at 95.degree. C., 20 sec at 55.degree. C., and 1 min at 72.degree.
C., and a final step of 5 min at 72.degree. C.
[0262] In an attempt to maximize expression of the ZM4 edd and eda
genes in yeast, two different approaches were undertaken to
optimize the ZM4 edd and eda genes. The first approach was to
remove translational pauses from the polynucleotide sequence by
designing the gene to incorporate only codons that are preferred in
yeast. This optimization is referred to as the "hot rod"
optimization. In the second approach, translational pauses which
are present in the native organism gene sequence are matched in the
heterologous expression host organism by substituting the codon
usage pattern of that host organism. This optimization is referred
to as the "matched" optimization. The final gene and protein
sequences for edd and eda from the ZM4 native, hot rod (HR) and
matched versions, as well as the E. coli native are shown in FIG.
6. Certain sequences in FIG. 6 are presented at the end of this
Example 1. The matched version of ZM4 edd and ZM4 eda genes were
synthesized by IDT, and the hot rod version was constructed using
methods described in Larsen et al. (Int. J. Bioinform. Res. Appl;
2008:4[3]; 324-336).
[0263] Each version of each edd and eda gene was inserted into the
yeast expression vector p426GPD (GPD promoter, 2 micron, URA3)
(ATCC accession number 87361) between the SpeI and XhoI cloning
sites. Each version of the eda gene was also inserted into the SpeI
and XhoI sites of the yeast expression vector p425GPD (GPD
promoter, 2 micron, LEU3) (ATCC accession number 87359). For each
edd and eda version, 3' His tagged and non tagged p426 GPD
constructs were made. Please refer to table 1 for all
oligonucleotides used for PCR amplification of edd and eda
constructs for cloning into p425 and p426 GPD vectors. All cloning
procedures were conducted according to standard cloning procedures
described by Maniatis et al.
[0264] Each edd and eda p426GPD construct was transformed into
Saccharomyces cerevisiae strain BY4742 (MATalpha his3delta1
leu2delta0 lys2delta0 ura3delta0) (ATCC accession number 201389).
This strain has a deletion of the his3 gene, an
imidazoleglycerol-phosphate dehydratase which catalyzes the sixth
step in histidine biosynthesis; a deletion of leu2 gene, a
beta-isopropylmalate dehydrogenase which catalyzes the third step
in the leucine biosynthesis pathway; a deletion of the lys2 gene,
an alpha aminoadipate reductase which catalyzes the fifth step in
biosynthesis of lysine; and a deletion of the ura3 gene, an
orotidine-5'-phosphate decarboxylase which catalyzes the sixth
enzymatic step in the de novo biosynthesis of pyrimidines. The
genotype of BY4742 makes it an auxotroph for histidine, leucine,
lysine and uracil.
[0265] Transformation of the p426GPD plasmids containing an edd or
an eda variant gene into yeast strain BY4742 was accomplished using
the Zymo Research frozen-EZ yeast transformation II kit according
to the manufacturer's protocol. The transformed BY4742 cells were
selected by growth on a synthetic dextrose medium (SD) (0.67% yeast
nitrogen base-2% dextrose) containing complete amino acids minus
uracil (Krackeler Scientific Inc). Plates were incubated at about
30.degree. C. for about 48 hours. Transformant colonies for each
edd and eda variant were inoculated onto 5 ml of SD minus uracil
medium and cells were grown at about 30.degree. C. and shaken at
about 250 rpm for about 24 hours. Cells were harvested by
centrifugation at 1000.times.g for about 5 minutes, after which
protein crude extract was prepared with Y-PER Plus (Thermo
Scientific) according to the manufacturer's instructions. Whole
cell extract protein concentrations were determined using the
Coomassie Plus Protein Assay (Thermo Scientific) according to the
manufacturer's directions. For each edd and eda variant His-tagged
construct, about 10 .mu.g of soluble and insoluble fractions were
loaded on 4-12% NuPAGE Novex Bis-Tris protein gels (Invitrogen) and
proteins were analyzed by western using anti-(His).sub.6 (SEQ ID
NO: 138) mouse monoclonal antibody (Abcam) and HRP-conjugated
secondary antibody (Abcam). Supersignal West Pico Chemiluminescent
substrate (Thermo Scientific) was used for western detection
according to manufacturer's instructions. All edd variants showed
expression in both soluble and insoluble fractions whereas only the
E. coli eda variant showed expression in the soluble fraction.
[0266] In order to confirm that edd and eda variants were
functional in yeast, the combined edd and eda activities were
assayed by the formation of pyruvate, coupled to the NADH-dependent
activity of lactate dehydrogenase. Transformation of combined edd
(in p426GPD) and edd (in p425GPD) constructs was accomplished with
the Zymo Research frozen-EZ yeast transformation II kit based on
manufacturer's protocol. As a negative control, p425GPD and p426GPD
vectors were also transformed into BY4742. Transformants (16
different combinations total including the variant edd and eda
combinations plus vector controls) were selected on synthetic
dextrose medium (SD) (0.67% yeast nitrogen base-2% dextrose)
containing complete amino acids minus uracil and leucine.
Transformants of edd and eda variant combinations were inoculated
onto 5 ml of SD minus uracil and leucine and cells were grown at
about 30.degree. C. in shaker flasks at about 250 rpm for about 24
hours. Fresh overnight culture was used to inoculate about 100 ml
of (SD media minus uracil and leucine containing about 0.01 g
ergosterol /L and about 400 .mu.l of Tween80) to an initial
inoculum OD.sub.600nm of about 0.1 and grown anaerobically at about
30.degree. C. for approximately 14 hours until cells reached an
OD.sub.600nm of 3-4. The cells were centrifuged at about 3000 g for
about 10 minutes. The cells were then washed with 25 ml deionized
H.sub.2O and centrifuged at 3000 g for 10 min. the cells were
resuspended at about 2 ml/g of cell pellet) in lysis buffer (50 mM
TrisCl pH7, 10 mM MgCl.sub.2 1.times. Calbiochem protease inhibitor
cocktail set III). Approximately 900 .mu.l of glass beads were
added and cells were lysed by vortexing at maximum speed for
4.times.30 seconds. Cell lysate was removed from the glass beads,
placed into fresh tubes and spun at about 10,000 g for about 10
minutes at about 4.degree. C. The supernatant containing whole cell
extract (WCE) was transferred to a fresh tube. WCE protein
concentrations were measured using the Coomassie Plus Protein Assay
(Thermo Scientific) according to the manufacturer's directions. A
total of about 750 .mu.g of WCE was used for the edd and eda
coupled assay. For this assay, about 750 .mu.g of WCE was mixed
with about 2 mM 6-phosphogluconate and about 4.5 U lactate
dehydrogenase in a final volume of about 400 .mu.l. A total of
about 100 .mu.l of NADH was added to this reaction to a final
molarity of about 0.3 mM, and NADH oxidation was monitored for
about 10 minutes at about 340 nM using a DU800
spectrophotometer.
TABLE-US-00004 ZM4 HR EDA GENE (SEQ ID NO: 145)
ATGAGAGACATTGATTCTGTTATGAGATTGGCTCCAGTTATGCCAGTCTT
GGTTATAGAAGATATAGCTGATGCTAAGCCAATTGCTGAGGCTTTGGTTG
CTGGTGGTTTAAATGTTTTGGAAGTTACATTGAGAACTCCATGTGCTTTG
GAAGCTATTAAAATTATGAAGGAAGTTCCAGGTGCTGTTGTTGGTGCTGG
TACTGTTTTAAACGCTAAAATGTTGGATCAAGCTCAAGAAGCTGGTTGTG
AGTTCTTTGTATCACCAGGTTTGACTGCTGATTTGGGAAAACATGCTGTT
GCTCAAAAAGCGGCTCTTCTACCAGGGGTTGCTAATGCTGCTGATGTTAT
GTTGGGATTGGATTTGGGTTTGGATAGATTTAAATTCTTCCCAGCTGAAA
ATATAGGTGGTTTGCCAGCTTTAAAATCTATGGCTTCTGTTTTTAGACAA
GTTAGATTTTGTCCAACTGGAGGAATTACTCCGACTTCTGCTCCAAAATA
TTTGGAAAATCCATCTATTTTGTGTGTTGGTGGTTCTTGGGTTGTTCCAG
CGGGTAAACCAGATGTTGCGAAAATTACTGCTTTGGCTAAAGAGGCTTCA
GCTTTTAAAAGAGCTGCTGTGGCGTAG ZM4 HR EDD GENE (SEQ ID NO: 146)
ATGACGGATTTGCATTCAACTGTTGAGAAAGTAACTGCTAGAGTAATTGA
AAGATCAAGGGAAACTAGAAAGGCTTATTTGGATTTGATACAATATGAGA
GGGAAAAAGGTGTTGATAGACCAAATTTGTCTTGTTCTAATTTGGCTCAT
GGTTTTGCTGCTATGAATGGTGATAAACCAGCTTTGAGAGATTTTAATAG
AATGAATATAGGTGTAGTTACTTCTTATAATGATATGTTGTCTGCTCATG
AACCATATTATAGATATCCAGAACAAATGAAGGTTTTTGCTCGTGAAGTT
GGTGCTACAGTTCAAGTTGCTGGTGGTGTTCCTGCAATGTGTGATGGTGT
TACTCAAGGTCAACCAGGTATGGAAGAATCTTTGTTTTCCAGAGATGTAA
TTGCTTTGGCTACATCTGTTTCATTGTCTCACGGAATGTTTGAAGGTGCT
GCATTGTTGGGAATTTGTGATAAAATTGTTCCAGGTTTGTTGATGGGTGC
TTTGAGGTTCGGTCATTTGCCAACTATTTTGGTTCCATCTGGTCCAATGA
CTACTGGAATCCCAAATAAAGAAAAGATTAGAATTAGACAATTGTATGCT
CAAGGAAAAATTGGTCAAAAGGAATTGTTGGATATGGAAGCTGCCTGTTA
TCATGCTGAAGGTACTTGTACTTTTTATGGTACTGCTAACACTAATCAGA
TGGTTATGGAAGTTTTGGGTTTGCACATGCCAGGTAGTGCATTCGTTACT
CCAGGTACTCCACTGAGACAGGCTTTGACTAGAGCTGCTGTTCATAGAGT
TGCAGAGTTGGGTTGGAAAGGTGATGATTATAGACCTTTGGGTAAAATTA
TTGATGAGAAATCTATTGTTAATGCTATTGTTGGTTTGTTAGCTACAGGT
GGTTCTACAAATCATACAATGCATATTCCGGCCATAGCTAGAGCAGCAGG
GGTTATAGTTAATTGGAATGATTTTCATGATTTGTCTGAAGTTGTTCCAT
TGATTGCTAGAATTTATCCAAATGGTCCTAGAGATATAAATGAATTTCAA
AATGCAGGAGGAATGGCTTATGTAATTAAAGAATTGTTGAGTGCGAATTT
GTTAAATAGAGATGTTACTACTATTGCTAAAGGAGGGATAGAAGAATATG
CTAAAGCTCCAGCTCTGAACGATGCGGGTGAATTGGTGTGGAAACCGGCT
GGCGAACCTGGGGACGACACAATTTTGAGACCAGTATCTAATCCATTTGC
TAAAGATGGTGGTTTGCGTCTCTTGGAAGGTAATTTGGGTAGAGCAATGT
ATAAGGCTTCTGCTGTAGATCCAAAATTCTGGACTATTGAAGCTCCCGTT
AGAGTTTTCTCTGATCAAGATGATGTTCAAAAGGCTTTTAAAGCAGGCGA
GTTAAATAAAGATGTTATAGTTGTTGTTAGATTTCAAGGTCCTCGTGCTA
ATGGTATGCCTGAATTGCATAAGTTGACTCCTGCGCTAGGCGTATTGCAA
GATAATGGTTATAAGGTTGCTTTAGTTACTGATGGTAGAATGTCTGGTGC
AACTGGTAAAGTACCGGTGGCTCTGCATGTTTCACCAGAGGCTTTAGGAG
GTGGGGCGATTGGCAAGTTGAGAGATGGCGATATAGTTAGAATTTCTGTT
GAAGAAGGTAAATTAGAGGCTCTTGTCCCCGCCGACGAGTGGAATGCTAG
ACCACATGCTGAGAAGCCCGCTTTTAGACCTGGTACTGGGAGAGAATTGT
TTGACATTTTTAGACAAAACGCTGCTAAGGCTGAGGATGGTGCAGTTGCA
ATTTATGCTGGGGCAGGGATCTAG ZM4 MATCHED EDA GENE (SEQ ID NO: 147)
ATGAGGGATATTGATAGTGTGATGAGGTTAGCCCCTGTTATGCCTGTTCT
CGTTATTGAAGATATTGCAGATGCCAAACCTATTGCCGAAGCACTCGTTG
CAGGTGGTCTAAACGTTCTAGAAGTGACACTAAGGACTCCTTGTGCACTA
GAAGCTATTAAGATTATGAAGGAAGTTCCTGGTGCTGTTGTTGGTGCTGG
TACAGTTCTAAACGCCAAAATGCTCGACCAGGCACAAGAAGCAGGTTGCG
AATTTTTCGTTTCACCTGGTCTAACTGCCGACCTCGGAAAGCACGCAGTT
GCTCAAAAAGCCGCATTACTACCCGGTGTTGCAAATGCAGCAGATGTGAT
GCTAGGTCTAGACCTAGGTCTAGATAGGTTCAAGTTCTTCCCTGCCGAAA
ACATTGGTGGTCTACCTGCTCTAAAGAGTATGGCATCAGTTTTCAGGCAA
GTTAGGTTCTGCCCTACTGGAGGTATAACTCCTACAAGTGCACCTAAATA
TCTAGAAAACCCTAGTATTCTATGCGTTGGTGGTTCATGGGTTGTTCCTG
CCGGAAAACCCGATGTTGCCAAAATTACAGCCCTCGCAAAAGAAGCAAGT
GCATTCAAGAGGGCAGCAGTTGCTTAG ZM4 MATCHED EDD GENE (SEQ ID NO: 148)
ATGACGGATCTACATAGTACAGTGGAGAAGGTTACTGCCAGGGTTATTGA
AAGGAGTAGGGAAACTAGGAAGGCATATCTAGATTTAATTCAATATGAGA
GGGAAAAAGGAGTGGACAGGCCCAACCTAAGTTGTAGCAACCTAGCACAT
GGATTCGCCGCAATGAATGGTGACAAGCCCGCATTAAGGGACTTCAACAG
GATGAATATTGGAGTTGTGACGAGTTACAACGATATGTTAAGTGCACATG
AACCCTATTATAGGTATCCTGAGCAAATGAAGGTGTTTGCAAGGGAAGTT
GGAGCCACAGTTCAAGTTGCTGGTGGAGTGCCTGCAATGTGCGATGGTGT
GACTCAGGGTCAACCTGGAATGGAAGAATCCCTATTTTCAAGGGATGTTA
TTGCATTAGCAACTTCAGTTTCATTATCACATGGTATGTTTGAAGGGGCA
GCTCTACTCGGTATATGTGACAAGATTGTTCCTGGTCTACTAATGGGAGC
ACTAAGGTTTGGTCACCTACCTACTATTCTAGTTCCCAGTGGACCTATGA
CAACGGGTATACCTAACAAAGAAAAAATTAGGATTAGGCAACTCTATGCA
CAAGGTAAAATTGGACAAAAAGAACTACTAGATATGGAAGCCGCATGCTA
CCATGCAGAAGGTACTTGCACTTTCTATGGTACAGCCAACACTAACCAGA
TGGTTATGGAAGTTCTCGGTCTACATATGCCCGGTAGTGCCTTTGTTACT
CCTGGTACTCCTCTCAGGCAAGCACTAACTAGGGCAGCAGTGCATAGGGT
TGCAGAATTAGGTTGGAAGGGAGACGATTATAGGCCTCTAGGTAAAATTA
TTGACGAAAAAAGTATTGTTAATGCAATTGTTGGTCTATTAGCCACTGGT
GGTAGTACTAACCATACGATGCATATTCCTGCTATTGCAAGGGCAGCAGG
TGTTATTGTTAACTGGAATGACTTCCATGATCTATCAGAAGTTGTTCCTT
TAATTGCTAGGATTTACCCTAATGGACCTAGGGACATTAACGAATTTCAA
AATGCCGGAGGAATGGCATATGTTATTAAGGAACTACTATCAGCAAATCT
ACTAAACAGGGATGTTACAACTATTGCTAAGGGAGGTATAGAAGAATACG
CTAAGGCACCTGCCCTAAATGATGCAGGAGAATTAGTTTGGAAGCCCGCA
GGAGAACCTGGTGATGACACTATTCTAAGGCCTGTTTCAAATCCTTTCGC
CAAAGATGGAGGTCTAAGGCTCTTAGAAGGTAACCTAGGAAGGGCCATGT
ACAAGGCTAGCGCCGTTGATCCTAAATTCTGGACTATTGAAGCCCCTGTT
AGGGTTTTCTCAGACCAGGACGATGTTCAAAAAGCCTTCAAGGCAGGAGA
ACTAAACAAAGACGTTATTGTTGTTGTTAGGTTCCAAGGACCTAGGGCCA
ACGGTATGCCTGAATTACATAAGCTAACTCCTGCATTAGGTGTTCTACAA
GATAATGGATACAAAGTTGCATTAGTGACGGATGGTAGGATGAGTGGTGC
AACTGGTAAAGTTCCTGTTGCATTACATGTTTCACCCGAAGCACTAGGAG
GTGGTGCTATTGGTAAACTTAGGGATGGAGATATTGTTAGGATTAGTGTT
GAAGAAGGAAAACTTGAAGCACTCGTTCCCGCAGATGAGTGGAATGCAAG
GCCTCATGCAGAAAAACCTGCATTCAGGCCTGGGACTGGGAGGGAATTAT
TTGATATTTTCAGGCAAAATGCAGCAAAAGCAGAAGACGGTGCCGTTGCC
ATCTATGCCGGTGCTGGTATATAG
Example 2
Inactivation of the Embden-Meyerhof Pathway in Yeast
[0267] Saccharomyces cerevisiae strain YGR240CBY4742 was obtained
from the ATCC (accession number 4015893). This strain is
genetically identical to S. cerevisiae strain BY4742, except that
YGR420C, the gene encoding the PFK1 enzyme, which is the alpha
subunit of heterooctameric phosphofructokinase, has been deleted. A
DNA construct designed to delete the gene encoding the PFK2 enzyme
via homologous recombination was prepared. This construct
substituted the gene encoding HIS3 (imidazoleglycerol-phosphate
dehydratase, an enzyme required for synthesis of histidine) for the
PFK2 gene. The DNA construct comprised, in the 5' to 3' direction,
100 bases of the 5' end of the open reading frame of PFK2, followed
by the HIS3 promoter, HIS3 open reading frame, HIS3 terminator, and
100 bp of the 3' end of the PFK2 open reading frame.
[0268] This construct was prepared by two rounds of PCR. In the
first round, about 100 ng of BY4742 genomic DNA was used as a
template. The genomic DNA was prepared from cells using the Zymo
Research Yeastar kit according to the manufacturer's instructions.
PCR was performed using the following primers:
TABLE-US-00005 (SEQ ID NO: 9)
5'-tgcatattccgttcaatcttataaagctgccatagatttttacacca
agtcgttttaagagcttggtgagcgcta-3' (SEQ ID NO: 10)
5'-cttgccagtgaatgacctttggcattctcatggaaacttcagtttca
tagtcgagttcaagagaaaaaaaaagaa-3'
[0269] The PCR reaction conditions were the same as those set forth
in Example 1 for preparing the edd and eda genes.
[0270] For the second round of PCR, approximately 1 .mu.l of the
first PCR product was used as a template. The second round of PCR
reaction was performed with the following primer set:
TABLE-US-00006 (SEQ ID NO: 11)
5'-atgactgttactactccttttgtgaatggtacttcttattgtaccgt
cactgcatattccgttcaatcttataaa-3' (SEQ ID NO: 12)
5'-ttaatcaactctctttcttccaaccaaatggtcagcaatgagtctgg
tagcttgccagtgaatgacctttggcat-3'
[0271] PCR conditions for this reaction were the same as for the
first reaction immediately above. The final PCR product was
separated by agarose gel electrophoresis, excised, and purified
using MP Biomedicals Geneclean II kit according to the
manufacturer's instructions.
[0272] Approximately 2 .mu.g of the purified DNA was used for
transformation of the yeast strain YGR240CBY4742 by lithium acetate
procedure as described by Shiestl and Gietz with an additional
recovery step added after the heat shock step. Essentially after
heat shock, cells were centrifuged at 500.times.g for 2 min and
resuspended in 1 ml of YP-Ethanol (1% yeast extract-2% peptone-2%
ethanol) and incubated at 30.degree. C. for 2 hours prior to
plating on selective media containing SC-Ethanol (0.67% yeast
nitrogen base-2% ethanol) containing complete amino acids minus
histidine. The engineered transformant strain referred to as
YGR420CBY4742APFK2 has PFK1 and PFK2 genes deleted and is an
auxotroph for leucine, uracil and lysine.
[0273] The YGR420CBY4742.DELTA.PFK2 strain was used for
transformation of the combination of edd-p426 GPD (edd variants in
p426 GPD) and eda-p425 GPD (eda variants in p425 GPD) variant
constructs. A total of 16 combinations of edd-p426 GPD and eda-p425
GPD variant constructs were tested. Each combination was
transformed into YGR420CBY4742.DELTA.PFK2. For all transformation,
1 .mu.g of edd-p426 GPD and 1 .mu.g of eda-p425 GPD was used. All
transformants from each edd-p426 GPD and eda-p425 GPD construct
combination were selected on SC-Ethanol (0.67% yeast nitrogen
base-2% ethanol) containing complete amino acids minus uracil and
leucine.
[0274] To confirm that the edd and eda variants are functional in
yeast, a complementation test for growth of
YGR420CBY4742.DELTA.PFK2 strain on YPD (1% yeast extract-2%
peptone-2% dextrose) and YPGluconate (1% yeast extract-2%
peptone-2% gluconate) was performed. Viable colonies of edd-p426
GPD and eda-p425 GPD variant construct combinations grown on
SC-Ethanol minus uracil and leucine were patched to plates
containing SC-ethanol minus uracil and leucine and incubated at
30.degree. C. for 48 hrs. These patches were used to inoculate 5 ml
of YPD media to an initial inoculum OD.sub.600nm of 0.1 and the
cells were grown anaerobically at 30.degree. C. for 3 to 7
days.
Example 3
Preparation of Carbon Dioxide Fixing Yeast Cells
[0275] Total genomic DNA from Zymomonas mobilis was obtained from
ATCC (ATCC Number 31821). The Z. mobilis gene encoding the enzyme
phosphoenolpyruvate carboxylase ("PEP carboxylase") was isolated
from this genomic DNA and cloned using PCR amplification. PCR was
performed in a total volume of about 50 micro-liters in the
presence of about 20 nanograms of Z. mobilis genomic DNA, about 0.2
mM of 5' forward primer, about 0.2 mM of 3' reverse primer, about
0.2 mM of dNTP, about 1 micro-liter of pfu UltraII DNA polymerase
(Stratagene, La Jolla, Calif.), and 1.times.PCR buffer (Stratagene,
La Jolla, Calif.). PCR was carried out in a thermocycler using the
following program: Step One "95.degree. C. for 10 minutes" for 1
cycle, followed by Step Two "95.degree. C. for 20 seconds,
65.degree. C. for 30 seconds, and 72.degree. C. for 45 seconds" for
35 cycles, followed by Step Three "72.degree. C. for 5 minutes" for
1 cycle, and then Step Four "4.degree. C. Hold" to stop the
reaction. The primers for the PCR reaction were:
TABLE-US-00007 (SEQ ID NO: 13)
5'GACTAACTGAACTAGTAAAAAAATGACCAAGCCGCGCACAATTAATCA G-3' (SEQ ID NO:
14) 5'AAGTGAGTAACTCGAGTTATTAACCGCTGTTGCGAAGTGCCGTCGC- 3'
[0276] The DNA sequence of native Z. Mobilis PEP carboxylase is set
forth as SEQ ID NO:20.
[0277] The cloned gene was inserted into the vector pGPD426 (ATCC
Number: 87361) in between the SpeI and XhoI sites. The final
plasmid containing the PEP carboxylase gene was named pGPD426
PEPC.
[0278] Separately, a similar plasmid, referred to as pGPD426 N-his
PEPC was constructed to insert a six-histidine tag (SEQ ID NO: 138)
at the N-terminus of the PEPC sequence for protein expression
verification in yeast. This plasmid was constructed using two
rounds of PCR to extend the 5' end of the PEPC gene to incorporate
a six-histidine tag (SEQ ID NO: 138) at the N-terminus of the PEPC
protein. The two 5' forward primers used sequentially were:
TABLE-US-00008 (SEQ ID NO: 15)
5'ATGTCTCATCATCATCATCATCATACCAAGCCGCGCACAATTAATCAG AAC-3' and (SEQ
ID NO: 16) 5'GACTAACTGAACTAGTAAAAAAATGTCTCATCATCATCATCATCATAC
CAAG-3'
[0279] The same 3' primer was used as described above. The PCR was
performed in a total volume of about 50 micro-liters in the
presence of about 20 nanograms of Z. Mobilis PEP carboxylase
polynucleotide, about 0.2 mM of 5' forward primer, about 0.2 mM of
3' reverse primer, about 0.2 mM of dNTP, about 1 micro-liter of pfu
UltraII DNA polymerase (Stratagene, La Jolla, Calif.), and
1.times.PCR buffer (Stratagene, La Jolla, Calif.). The PCR was
carried out in a thermocycler using the following program: Step One
"95.degree. C. for 10 minutes" for 1 cycle, followed by Step Two
"95.degree. C. for 20 seconds, 65.degree. C. for 30 seconds, and
72.degree. C. for 45 seconds" for 35 cycles, followed Step Three
"72.degree. C. for 5 minutes" for 1 cycle, and then Step Four
"4.degree. C. Hold" to stop the reaction.
[0280] To increase protein expression level of Z. Mobilis PEP
carboxylase in yeast, the PEPC coding sequence was optimized to
incorporate frequently used codons obtained from yeast glycolytic
genes. The resulting PEP carboxylase amino acid sequence remains
identical to the wild type.
[0281] The codon optimized PEP carboxylase DNA sequence was ordered
from IDT and was inserted into the vector pGPD426 at the SpeI and
XhoI site. The final plasmid containing the codon optimized PEP
carboxylase gene was named pGPD426 PEPC_opti. A similar plasmid,
named pGPD426 N-his PEPC_opti was constructed to insert a
six-histidine tag (SEQ ID NO: 138) at the N-terminus of the
optimized PEPC gene for protein expression verification in
yeast.
[0282] To construct pGPD426 N-his PEPC_opti, two rounds of PCR were
performed to extend the 5' end of the codon optimized PEPC gene to
incorporate the six-histidine tag (SEQ ID NO: 138) at the
N-terminus of the PEPC protein. Two 5' forward primers used in
sequential order were:
TABLE-US-00009 (SEQ ID NO: 17)
5'ATGTCTCATCATCATCATCATCATATGACCAAGCCAAGAACTATTAAC CAAAACCC-3' and
(SEQ ID NO: 18) 5'GACTAACTGAACTAGTAAAAAAATGTCTCATCATCATCATCATCATAT
GACCAAGCCAAG 3'
[0283] The 3' reverse primer sequence used for both PCR reactions
was:
TABLE-US-00010 (SEQ ID NO: 19)
5'AAGTGAGTAACTCGAGTTATTAACCGGAGTTTCTCAAAGCAGTAGCGA TAG3'
[0284] Both PCR reactions were performed in a total volume of about
50 micro-liters in the presence of about 20 nanograms of the codon
optimized PEP carboxylase polynucleotide, about 0.2 mM of 5'
forward primer, about 0.2 mM of 3' reverse primer, about 0.2 mM of
dNTP, about 1 micro-liter of pfu UltraII DNA polymerase
(Stratagene, La Jolla, Calif.), and 1.times.PCR buffer (Stratagene,
La Jolla, Calif.). PCR reactions were carried out in a thermocycler
using the following program: Step One "95.degree. C. for 10
minutes" for 1 cycle, followed by Step Two "95.degree. C. for 20
seconds, 65.degree. C. for 30 seconds, and 72.degree. C. for 45
seconds" for 35 cycles, followed Step Three "72.degree. C. for 5
minutes" for 1 cycle, and then Step Four "4.degree. C. Hold" to
stop the reaction.
[0285] Saccharomyces cerevisiae strain BY4742 was cultured in YPD
medium to an OD of about 1.0, and then prepared for transformation
using the Frozen-EZ Yeast Transformation II kit (Zymo Research,
Orange, Calif.) and following the manufacturer's instructions.
Approximately 500 micrograms of each plasmid was added to the
cells, and transformation was accomplished by addition of PEG
solution ("Solution 3" in the Frozen-EZ Yeast Transformation II
kit) and incubation at about 30.degree. C. for an hour. After
transformation, the cells were plated on synthetic complete medium
(described in Example IV below) minus uracil (sc-ura) medium, grown
for about 48 hours at about 30.degree. C., and transformants were
selected based on auxotrophic complementation.
[0286] Following a similar procedure, the same plasmids were
individually transformed using the procedure described above into
the following yeast mutant strains: YKR097W (ATCC Number 4016013,
.DELTA.PCK, in the phosphoenolpyruvate carboxykinase gene is
deleted), YGL062W (ATCC Number 4014429, .DELTA.PYC1, in which the
pyruvate carboxylase 1 gene is deleted), and YBR218C (ATCC Number
4013358, .DELTA.PYC2, in which the pyruvate carboxylase 2 gene is
deleted).
[0287] The transformed yeast cells were grown aerobically in a
shake flask in synthetic complete medium minus uracil (see Example
IV) containing 1% glucose to mid-log phase (an OD of 2.0). The
mid-log phase cultures were then used to inoculate a fresh culture
(in sc-ura medium with 1% glucose) to an initial OD of 0.1 at which
time the cultures were then grown anaerobically in a serum bottle.
Culture samples were drawn periodically to monitor the level of
glucose consumption and ethanol production.
TABLE-US-00011 DNA sequence of the native Z. mobilis PEP
carboxylase gene (SEQ ID NO: 20):
ACTAGTAAAAAAATGACCAAGCCGCGCACAATTAATCAGAACCCAGACCTTCGCTATTTTGGT
AACCTGCTCGGTCAGGTTATTAAGGAACAAGGCGGAGAGTCTTTATTCAACCAGATCGAGCAA
ATTCGCTCTGCCGCGATTAGACGCCATCGGGGTATTGTTGACAGCACCGAGCTAAGTTCTCG
CTTAGCCGATCTCGACCTTAATGACATGTTCTCTTTTGCACATGCCTTTTTGCTGTTTTCAATG
CTGGCCAATTTGGCTGATGATCGTCAGGGAGATGCCCTTGATCCTGATGCCAATATGGCAAGT
GCCCTTAAGGACATAAAAGCCAAAGGCGTCAGTCAGCAGGCGATCATTGATATGATCGACAAA
GCCTGCATTGTGCCTGTTCTGACAGCACATCCGACCGAAGTCCGTCGGAAAAGTATGCTTGA
CCATTATAATCGCATTGCAGGTTTAATGCGGTTAAAAGATGCTGGACAAACGGTGACCGAAGA
TGGTCTTCCGATCGAAGATGCGTTAATCCAGCAAATCACGATATTATGGCAGACTCGTCCGCT
CATGCTGCAAAAGCTGACCGTGGCTGATGAAATCGAAACTGCCCTGTCTTTCTTAAGAGAAAC
TTTTCTGCCTGTTCTGCCCCAGATTTATGCAGAATGGGAAAAATTGCTTGGTAGTTCTATTCCA
AGCTTTATCAGACCTGGTAATTGGATTGGTGGTGACCGTGACGGTAACCCCAATGTCAATGCC
GATACGATCATGCTGTCTTTGAAGCGCAGCTCGGAAACGGTATTGACGGATTATCTCAACCGT
CTTGATAAACTGCTTTCCAACCTTTCGGTCTCAACCGATATGGTTTCGGTATCCGATGATATTC
TACGTCTAGCCGATAAAAGTGGTGACGATGCTGCGATCCGTGCGGATGAACCTTATCGTCGT
GCCTTAAATGGTATTTATGACCGTTTAGCCGCTACCTATCGTCAGATCGCCGGTCGCAACCCT
TCGCGCCCAGCCTTGCGTTCTGCAGAAGCCTATAAACGGCCTCAAGAATTGCTGGCTGATTT
GAAGACCTTGGCCGAAGGCTTGGGTAAATTGGCAGAAGGTAGTTTTAAGGCATTGATCCGTTC
GGTTGAAACCTTTGGTTTCCATTTGGCCACCCTCGATCTGCGTCAGAATTCGCAGGTTCATGA
AAGAGTTGTCAATGAACTGCTACGGACAGCCACCGTTGAAGCCGATTATTTATCTCTATCGGA
AGAAGATCGCGTTAAGCTGTTAAGACGGGAATTGTCGCAGCCGCGGACTCTATTCGTTCCGC
GCGCCGATTATTCCGAAGAAACGCGTTCTGAACTTGATATTATTCAGGCAGCAGCCCGCGCC
CATGAAATTTTTGGCCCTGAATCCATTACGACTTATTTGATTTCGAATGGCGAAAGCATTTCCG
ATATTCTGGAAGTCTATTTGCTTTTGAAAGAAGCAGGGCTGTATCAAGGGGGTGCTAAGCCAA
AAGCGGCGATTGAAGCTGCGCCTTTATTCGAGACGGTGGCCGATCTTGAAAATGCGCCAAAG
GTCATGGAGGAATGGTTCAAGCTGCCTGAAGCGCAAGCCATTGCAAAGGCACATGGCGTTCA
GGAAGTGATGGTTGGCTATTCTGACTCCAATAAGGACGGCGGATATCTGACCTCGGTTTGGG
GTCTTTATAAGGCTTGCCTCGCTTTGGTGCCGATTTTTGAGAAAGCCGGTGTACCGATCCAGT
TTTTCCATGGACGGGGTGGTTCCGTTGGTCGCGGTGGTGGTTCCAACTTTAATGCCATTCTGT
CGCAGCCAGCCGGAGCCGTCAAAGGGCGTATCCGTTATACAGAACAGGGTGAAGTCGTGGC
GGCCAAATATGGCACCCATGAAAGCGCTATTGCCCATCTGGATGAGGCCGTAGCGGCGACTT
TGATTACGTCTTTGGAAGCACCGACCATTGTCGAGCCAGAGTTTAGTCGTTACCGTAAGGCCT
TGGATCAGATCTCAGATTCAGCTTTCCAGGCCTATCGCCAATTGGTCTATGGAACGAAGGGCT
TCCGTAAATTCTTTAGTGAATTTACGCCTTTGCCGGAAATTGCCCTGTTAAAGATCGGGTCACG
CCCACCTAGCCGCAAAAAATCCGACCGGATTGAAGATCTACGCGCTATTCCTTGGGTGTTTAG
CTGGTCTCAAGTTCGAGTCATGTTACCCGGTTGGTTCGGTTTCGGTCAGGCTTTATATGACTT
TGAAGATACCGAGCTGTTACAGGAAATGGCAAGCCGTTGGCCGTTTTTCCGCACGACTATTCG
GAATATGGAACAGGTGATGGCACGTTCCGATATGACGATCGCCAAGCATTATCTGGCCTTGGT
TGAGGATCAGACAAATGGTGAGGCTATCTATGATTCTATCGCGGATGGCTGGAATAAAGGTTG
TGAAGGTCTGTTAAAGGCAACCCAGCAGAATTGGCTGTTGGAACGCTTTCCGGCGGTTGATA
ATTCGGTGCAGATGCGTCGGCCTTATCTGGAACCGCTTAATTACTTACAGGTCGAATTGCTGA
AGAAATGGCGGGGAGGTGATACCAACCCGCATATCCTCGAATCTATTCAGCTGACAATCAATG
CCATTGCGACGGCACTTCGCAACAGCGGTTAATAACTCGAG DNA sequence of the codon
optimized PEP carboxylase gene (SEQ ID NO: 21):
ACTAGTAAAAAAATGACCAAGCCAAGAACTATTAACCAAAACCCAGACTTGAGATACTTCGGTA
ACTTGTTGGGTCAAGTTATCAAGGAACAAGGTGGTGAATCTTTGTTCAACCAAATTGAACAAAT
CAGATCCGCTGCTATTAGAAGACACAGAGGTATCGTCGACTCTACCGAATTGTCCTCTAGATT
GGCTGACTTGGACTTGAACGACATGTTCTCCTTCGCTCACGCTTTCTTGTTGTTCTCTATGTTG
GCTAACTTGGCTGACGACAGACAAGGTGACGCTTTGGACCCAGACGCTAACATGGCTTCCGC
TTTGAAGGACATTAAGGCTAAGGGTGTTTCTCAACAAGCTATCATTGACATGATCGACAAGGC
TTGTATTGTCCCAGTTTTGACTGCTCACCCAACCGAAGTCAGAAGAAAGTCCATGTTGGACCA
CTACAACAGAATCGCTGGTTTGATGAGATTGAAGGACGCTGGTCAAACTGTTACCGAAGACG
GTTTGCCAATTGAAGACGCTTTGATCCAACAAATTACTATCTTGTGGCAAACCAGACCATTGAT
GTTGCAAAAGTTGACTGTCGCTGACGAAATTGAAACCGCTTTGTCTTTCTTGAGAGAAACTTTC
TTGCCAGTTTTGCCACAAATCTACGCTGAATGGGAAAAGTTGTTGGGTTCCTCTATTCCATCCT
TCATCAGACCAGGTAACTGGATTGGTGGTGACAGAGACGGTAACCCAAACGTCAACGCTGAC
ACCATCATGTTGTCTTTGAAGAGATCCTCTGAAACTGTTTTGACCGACTACTTGAACAGATTGG
ACAAGTTGTTGTCCAACTTGTCTGTCTCCACTGACATGGTTTCTGTCTCCGACGACATTTTGAG
ATTGGCTGACAAGTCTGGTGACGACGCTGCTATCAGAGCTGACGAACCATACAGAAGAGCTT
TGAACGGTATTTACGACAGATTGGCTGCTACCTACAGACAAATCGCTGGTAGAAACCCATCCA
GACCAGCTTTGAGATCTGCTGAAGCTTACAAGAGACCACAAGAATTGTTGGCTGACTTGAAGA
CTTTGGCTGAAGGTTTGGGTAAGTTGGCTGAAGGTTCCTTCAAGGCTTTGATTAGATCTGTTG
AAACCTTCGGTTTCCACTTGGCTACTTTGGACTTGAGACAAAACTCCCAAGTCCACGAAAGAG
TTGTCAACGAATTGTTGAGAACCGCTACTGTTGAAGCTGACTACTTGTCTTTGTCCGAAGAAG
ACAGAGTCAAGTTGTTGAGAAGAGAATTGTCTCAACCAAGAACCTTGTTCGTTCCAAGAGCTG
ACTACTCCGAAGAAACTAGATCTGAATTGGACATCATTCAAGCTGCTGCTAGAGCTCACGAAA
TCTTCGGTCCAGAATCCATTACCACTTACTTGATCTCTAACGGTGAATCCATTTCTGACATCTT
GGAAGTCTACTTGTTGTTGAAGGAAGCTGGTTTGTACCAAGGTGGTGCTAAGCCAAAGGCTG
CTATTGAAGCTGCTCCATTGTTCGAAACCGTTGCTGACTTGGAAAACGCTCCAAAGGTCATGG
AAGAATGGTTCAAGTTGCCAGAAGCTCAAGCTATCGCTAAGGCTCACGGTGTTCAAGAAGTCA
TGGTTGGTTACTCCGACTCTAACAAGGACGGTGGTTACTTGACTTCCGTCTGGGGTTTGTACA
AGGCTTGTTTGGCTTTGGTTCCAATTTTCGAAAAGGCTGGTGTCCCAATCCAATTCTTCCACG
GTAGAGGTGGTTCTGTTGGTAGAGGTGGTGGTTCCAACTTCAACGCTATTTTGTCTCAACCAG
CTGGTGCTGTCAAGGGTAGAATCAGATACACCGAACAAGGTGAAGTTGTCGCTGCTAAGTAC
GGTACTCACGAATCCGCTATTGCTCACTTGGACGAAGCTGTTGCTGCTACCTTGATCACTTCT
TTGGAAGCTCCAACCATTGTCGAACCAGAATTCTCCAGATACAGAAAGGCTTTGGACCAAATC
TCTGACTCCGCTTTCCAAGCTTACAGACAATTGGTTTACGGTACTAAGGGTTTCAGAAAGTTCT
TCTCTGAATTCACCCCATTGCCAGAAATTGCTTTGTTGAAGATCGGTTCCAGACCACCATCTAG
AAAGAAGTCCGACAGAATTGAAGACTTGAGAGCTATCCCATGGGTCTTCTCTTGGTCCCAAGT
TAGAGTCATGTTGCCAGGTTGGTTCGGTTTCGGTCAAGCTTTGTACGACTTCGAAGACACTGA
ATTGTTGCAAGAAATGGCTTCTAGATGGCCATTCTTCAGAACCACTATTAGAAACATGGAACAA
GTTATGGCTAGATCCGACATGACCATCGCTAAGCACTACTTGGCTTTGGTCGAAGACCAAACT
AACGGTGAAGCTATTTACGACTCTATCGCTGACGGTTGGAACAAGGGTTGTGAAGGTTTGTTG
AAGGCTACCCAACAAAACTGGTTGTTGGAAAGATTCCCAGCTGTTGACAACTCCGTCCAAATG
AGAAGACCATACTTGGAACCATTGAACTACTTGCAAGTTGAATTGTTGAAGAAGTGGAGAGGT
GGTGACACTAACCCACACATTTTGGAATCTATCCAATTGACCATTAACGCTATCGCTACTGCTT
TGAGAAACTCCGGTTAATAACTCGAG
Example 4
Production of Pentose Sugar Utilizing Yeast Cells
[0288] The full length gene encoding the enzyme xylose isomerase
from Ruminococcus flavefaciens strain 17 (also known as
Ruminococcus flavefaciens strain Siijpesteijn 1948) with a
substitution at position 513 (in which cytidine was replaced by
guanidine) was synthesized by Integrated DNA Technologies, Inc.
("IDT", Coralville, Iowa; www.idtdna.com). The sequence of this
gene is set forth below as SEQ ID NO:22.
TABLE-US-00012 SEQ ID NO: 22
atggaatttttcagcaatatcggtaaaattcagtatcagggaccaaaaagtactgatcctctctcatttaagta-
ctataaccctgaagaagtca
tcaacggaaagacaatgcgcgagcatctgaagttcgctctttcatggtggcacacaatgggcggcgacggaaca-
gatatgttcggctgc
ggcacaacagacaagacctggggacagtccgatcccgctgcaagagcaaaggctaaggttgacgcagcattcga-
gatcatggataa
gctctccattgactactattgtttccacgatcgcgatctttctcccgagtatggcagcctcaaggctaccaacg-
atcagcttgacatagttacag
actatatcaaggagaagcagggcgacaagttcaagtgcctctggggtacagcaaagtgcttcgatcatccaaga-
ttcatgcacggtgca
ggtacatctccttctgctgatgtattcgctttctcagctgctcagatcaagaaggctctGgagtcaacagtaaa-
gctcggcggtaacggttac
gttttctggggcggacgtgaaggctatgagacacttcttaatacaaatatgggactcgaactcgacaatatggc-
tcgtcttatgaagatggct
gttgagtatggacgttcgatcggcttcaagggcgacttctatatcgagcccaagcccaaggagcccacaaagca-
tcagtacgatttcgata
cagctactgttctgggattcctcagaaagtacggtctcgataaggatttcaagatgaatatcgaagctaaccac-
gctacacttgctcagcata
cattccagcatgagctccgtgttgcaagagacaatggtgtgttcggttctatcgacgcaaaccagggcgacgtt-
cttcttggatgggataca
gaccagttccccacaaatatctacgatacaacaatgtgtatgtatgaagttatcaaggcaggcggcttcacaaa-
cggcggtctcaacttcg
acgctaaggcacgcagagggagcttcactcccgaggatatcttctacagctatatcgcaggtatggatgcattt-
gctctgggcttcagagct
gctctcaagcttatcgaagacggacgtatcgacaagttcgttgctgacagatacgcttcatggaataccggtat-
cggtgcagacataatcgc
aggtaaggcagatttcgcatctcttgaaaagtatgctcttgaaaagggcgaggttacagcttcactctcaagcg-
gcagacaggaaatgctg gagtctatcgtaaataacgttcttttcagtctgtaa
[0289] Separately, PCR was conducted to add a DNA sequence encoding
6 histidines (SEQ ID NO: 138) to the 3' terminus of this gene.
[0290] Two variants designed to remove the translational pauses in
the gene were prepared using the DNA self-assembly method of Larsen
et al., supra. One variant contained DNA sequence encoding a
6-hisitidine tag (SEQ ID NO: 138) at the 5' terminus, and the other
version did not. The annealing temperature for the self assembly
reactions was about 48 degrees Celsius. This gene variant is
referred to as a "Hot Rod" or "HR" gene variant. The sequence of
this HR gene is set forth below as SEQ ID NO: 23:
TABLE-US-00013 SEQ ID NO: 23
ATGGAGTTCTTTTCTAATATAGGTAAAATTCAGTATCAAGGTCCAAAATC
TACAGATCCATTGTCTTTTAAATATTATAATCCAGAAGAAGTTATAAATG
GTAAAACTATGAGAGAACATTTAAAATTTGCTTTGTCTTGGTGGCATACT
ATGGGTGGTGATGGTACTGATATGTTCGGTTGTGGTACTACTGATAAAAC
TTGGGGTCAATCTGATCCAGCTGCTAGAGCAAAAGCCAAAGTAGATGCAG
CCTTTGAAATTATGGATAAATTGTCTATTGATTATTATTGTTTTCATGAT
AGAGATTTGTCTCCTGAATATGGTTCTTTAAAAGCAACTAATGATCAATT
GGACATTGTTACGGATTATATTAAAGAAAAACAAGGTGATAAATTTAAAT
GTTTGTGGGGCACTGCGAAATGTTTTGATCATCCACGTTTTATGCATGGT
GCGGGGACGAGTCCTTCTGCTGATGTTTTTGCTTTTTCTGCCGCTCAAAT
TAAGAAGGCATTGGAATCAACTGTTAAATTAGGTGGGAACGGGTATGTAT
TCTGGGGAGGAAGGGAAGGTTATGAAACATTATTAAACACTAATATGGGT
TTGGAATTGGATAATATGGCTAGATTGATGAAAATGGCTGTAGAATACGG
AAGGTCTATTGGTTTTAAGGGTGACTTTTATATTGAACCAAAACCTAAAG
AGCCTACTAAACATCAATATGATTTTGATACTGCTACAGTTTTGGGATTC
TTGAGAAAATATGGTCTGGATAAAGATTTTAAAATGAATATAGAAGCTAA
TCATGCAACACTCGCACAACATACTTTTCAACATGAATTGAGAGTTGCCA
GAGATAACGGAGTTTTTGGATCTATCGATGCAAACCAGGGAGACGTTTTG
CTAGGATGGGATACTGATCAATTTCCAACTAACATTTATGATACTACTAT
GTGTATGTATGAAGTAATTAAGGCAGGAGGCTTTACTAATGGCGGATTAA
ACTTTGATGCGAAGGCTAGGCGTGGTAGTTTCACTCCAGAGGATATATTC
TATTCTTATATTGCTGGAATGGATGCTTTCGCGTTAGGTTTCAGGGCAGC
ACTAAAATTGATTGAAGATGGTAGAATTGATAAGTTTGTAGCTGATAGAT
ATGCTTCTTGGAATACTGGAATAGGAGCAGATATAATCGCTGGGAAAGCC
GACTTCGCCAGTCTGGAAAAATATGCGCTTGAAAAAGGAGAAGTTACTGC
CAGCTTAAGTTCCGGTCGTCAAGAAATGTTGGAATCTATTGTAAACAATG
TTTTATTTTCTCTG
[0291] For cloning purposes, PCR was used to engineer a unique SpeI
restriction site into the 5' end of each of the xylose isomerase
genes, and to engineer a unique XhoI restriction site at the 3'
end. In addition, a version of each gene was created that contained
a 6-HIS tag (SEQ ID NO: 138) at the 3' end of each gene to enable
detection of the proteins using Western analysis.
[0292] PCR amplifications were performed in about 50 .mu.l
reactions containing 1.times.PfuII Ultra reaction buffer
(Stratagene, San Diego, Calif.), 0.2 mM dNTPs, 0.2 .mu.M specific
5' and 3' primers, and 1 U PfuUltra II polymerase (Stratagene, San
Diego, Calif.). The reactions were cycled at 95.degree. C. for 10
minutes, followed by 30 rounds of amplification (95.degree. C. for
30 seconds, 62.degree. C. for 30 seconds, 72.degree. C. for 30
seconds) and a final extension incubation at 72.degree. C. for 5
minutes. Amplified PCR products were cloned into pCR Blunt II TOPO
(Life Sciences, Carlsbad, Calif.) and confirmed by sequencing
(GeneWiz, La Jolla, Calif.). The PCR primers for these reactions
were:
TABLE-US-00014 (SEQ ID NO: 26)
5'ACTTGACTACTAGTATGGAGTTCTTTTCTAATATAGGTAAAATT 3' (without the His
tag): (SEQ ID NO: 27) AGTCAAGTCTCGAGCAGAGAAAATAAAACATTGTTTACAATAGA
3' (with the His tag): (SEQ ID NO: 28)
AGTCAAGTCTCGAGCTAATGATGATGATGATGATGCAGAGAAAATAAAAC ATTGTTTAC
[0293] Separately, the xylose isomerase gene from Piromyces, strain
E2 (Harhangi et al., Arch. Microbiol., 180(2): 134-141 (2003)) was
synthesized by IDT. The sequence of this gene is set forth below as
SEQ ID NO: 24.
TABLE-US-00015 1 atggctaagg aatatttccc acaaattcaa aagattaagt
tcgaaggtaa ggattctaag 61 aatccattag ccttccacta ctacgatgct
gaaaaggaag tcatgggtaa gaaaatgaag 121 gattggttac gtttcgccat
ggcctggtgg cacactcttt gcgccgaagg tgctgaccaa 181 ttcggtggag
gtacaaagtc tttcccatgg aacgaaggta ctgatgctat tgaaattgcc 241
aagcaaaagg ttgatgctgg tttcgaaatc atgcaaaagc ttggtattcc atactactgt
301 ttccacgatg ttgatcttgt ttccgaaggt aactctattg aagaatacga
atccaacctt 361 aaggctgtcg ttgcttacct caaggaaaag caaaaggaaa
ccggtattaa gcttctctgg 421 agtactgcta acgtcttcgg tcacaagcgt
tacatgaacg gtgcctccac taacccagac 481 tttgatgttg tcgcccgtgc
tattgttcaa attaagaacg ccatagacgc cggtattgaa 541 cttggtgctg
aaaactacgt cttctggggt ggtcgtgaag gttacatgag tctccttaac 601
actgaccaaa agcgtgaaaa ggaacacatg gccactatgc ttaccatggc tcgtgactac
661 gctcgttcca agggattcaa gggtactttc ctcattgaac caaagccaat
ggaaccaacc 721 aagcaccaat acgatgttga cactgaaacc gctattggtt
tccttaaggc ccacaactta 781 gacaaggact tcaaggtcaa cattgaagtt
aaccacgcta ctcttgctgg tcacactttc 841 gaacacgaac ttgcctgtgc
tgttgatgct ggtatgctcg gttccattga tgctaaccgt 901 ggtgactacc
aaaacggttg ggatactgat caattcccaa ttgatcaata cgaactcgtc 961
caagcttgga tggaaatcat ccgtggtggt ggtttcgtta ctggtggtac caacttcgat
1021 gccaagactc gtcgtaactc tactgacctc gaagacatca tcattgccca
cgtttctggt 1081 atggatgcta tggctcgtgc tcttgaaaac gctgccaagc
tcctccaaga atctccatac 1141 accaagatga agaaggaacg ttacgcttcc
ttcgacagtg gtattggtaa ggactttgaa 1201 gatggtaagc tcaccctcga
acaagtttac gaatacggta agaagaacgg tgaaccaaag 1261 caaacttctg
gtaagcaaga actctacgaa gctattgttg ccatgtacca ataa
[0294] Two hot rod ("HR") versions of the Piromyces xylose
isomerase gene were prepared using the method of Larsen et al.,
supra. One version contained DNA sequence encoding a 6-histidine
tag (SEQ ID NO: 138) at the 5' terminus and the other did not. The
annealing temperature for the self-assembling oligonucleotides was
about 48 degrees Celsius. The sequence of this gene is set forth
below as
TABLE-US-00016 SEQ ID NO: 25
ATGGCTAAAGAATATTTTCCACAAATTCAGAAAATTAAATTTGAAGGTAAAGATTCTAAAAATCCATTGGCTTT-
CCATTA
TTATGATGCTGAAAAAGAAGTTATGGGTAAAAAGATGAAAGATTGGTTGAGATTCGCTATGGCTTGGTGGCATA-
CTCTAT
GTGCTGAAGGAGCTGATCAATTTGGAGGAGGTACTAAATCTTTTCCTTGGAATGAAGGTACTGACGCTATTGAA-
ATTGCT
AAGCAGAAAGTAGACGCGGGTTTTGAAATTATGCAAAAATTGGGAATACCATATTATTGTTTTCATGATGTTGA-
TTTGGT
ATCTGAGGGTAATTCTATTGAAGAATATGAATCTAATTTAAAAGCTGTTGTTGCTTACTTAAAAGAAAAACAAA-
AAGAAA
CTGGAATTAAATTGTTGTGGTCTACAGCTAATGTTTTCGGTCATAAAAGATATATGAATGGTGCTTCTACAAAT-
CCAGAT
TTTGATGTTGTAGCTAGAGCTATTGTTCAAATTAAAAATGCTATAGATGCAGGAATTGAATTAGGTGCCGAAAA-
TTATGT
TTTCTGGGGAGGTAGAGAAGGTTATATGTCTTTGTTAAATACTGATCAAAAACGTGAAAAGGAACACATGGCAA-
CTATGT
TGACAATGGCTAGGGATTATGCTAGATCTAAAGGTTTTAAAGGTACTTTCTTGATTGAGCCAAAACCTATGGAA-
CCAACT
AAACATCAATATGACGTTGACACTGAAACTGCTATTGGTTTCTTAAAAGCTCATAATTTGGATAAAGATTTTAA-
GGTTAA
TATAGAAGTTAATCATGCTACACTAGCTGGTCATACTTTTGAACATGAATTAGCTTGTGCAGTTGATGCCGGTA-
TGTTAG
GTTCTATCGACGCAAATAGAGGTGATTATCAAAATGGTTGGGACACAGATCAATTTCCAATAGATCAATATGAA-
TTGGTT
CAAGCATGGATGGAAATTATTAGGGGTGGAGGCTTCGTTACAGGTGGAACTAATTTTGATGCTAAAACTAGGAG-
AAATTC
TACAGATCTTGAAGATATAATTATTGCTCATGTATCTGGTATGGATGCGATGGCCCGTGCTTTGGAAAATGCAG-
CTAAAT
TACTTCAAGAATCTCCTTATACTAAAATGAAAAAGGAAAGATATGCTTCTTTTGATTCTGGAATAGGTAAGGAT-
TTTGAA
GATGGTAAATTGACATTGGAACAAGTTTATGAATATGGTAAGAAGAATGGAGAACCAAAACAAACTTCTGGTAA-
ACAAGA ATTATATGAGGCTATAGTAGCTATGTATCAAtaa.
[0295] For cloning purposes, a unique SpeI restriction site was
engineered at the 5' end of each of the XI genes, and a unique XhoI
restriction site was engineered at the 3' end. When needed, a 6-HIS
tag (SEQ ID NO: 138) was engineered at the 3' end of each gene
sequence to enable detection of the proteins using Western
analysis. The primers are listed in Table X. PCR amplifications
were performed in 50 .mu.l reactions containing 1.times.PfuII Ultra
reaction buffer (Stratagene, San Diego, Calif.), 0.2 mM dNTPs, 0.2
.mu.M specific 5' and 3' primers, and 1 U PfuUltra II polymerase
(Stratagene, San Diego, Calif.). The reactions were cycled at
95.degree. C. for 10 minutes, followed by 30 rounds of
amplification (95.degree. C. for 30 seconds, 62.degree. C. for 30
seconds, 72.degree. C. for 30 seconds) and a final extension
incubation at 72.degree. C. for 5 minutes. Amplified PCR products
were cloned into pCR Blunt II TOPO (Life Sciences, Carlsbad,
Calif.) and confirmed by sequencing (GeneWiz, La Jolla,
Calif.).
[0296] The primers used for PCR were:
TABLE-US-00017 5' (native gene) (SEQ ID NO: 149)
ACTAGTATGGCTAAGGAATATTTCCCACAAATTCAAAAG 3' (native gene) (SEQ ID
NO: 150) CTCGAGCTACTATTGGTACATGGCAACAATAGC 3' (native gene plus His
tag) (SEQ ID NO: 151)
CTCGAGCTACTAATGATGATGATGATGATGTTGGTACATGGCAACAATAG CTTCG 5' (hot
rod gene) (SEQ ID NO: 152) ACTAGTATGGCTAAAGAATATTTTCCACAAATTCAG 3'
(hot rod gene) (SEQ ID NO: 153) CTCGAGTTATTGATACATAGCTACTATAGCCTC
3' (hot rod gene plus His tag) (SEQ ID NO: 154)
CTCGAGTTAATGATGATGATGATGATGTTGATACATAGCTACTATAGCCT CATTGTTTAC
[0297] The genes encoding the native and HR versions of xylose
isomerase were separately inserted into the vector p426GDP (ATCC
catalog number 87361).
[0298] Saccharomyces cerevisiae strain BY4742 cells (ATCC catalog
number 201389) were cultured in YPD media (10 g Yeast Extract, 20 g
Bacto-Peptone, 20 g Glucose, 1 L total) at about 30.degree. C.
Separate aliquots of the cells were transformed with the plasmid
constructs containing the various xylose isomerase constructs or
with the vector alone. Transformation was accomplished using the
Zymo kit (Catalog number T2001; Zymo Research Corp., Orange, Calif.
92867) using about 1 .mu.g plasmid DNA and cultured on SC media
(set forth below) containing glucose but no uracil (20 g glucose;
2.21 g SC dry mix, 6.7 g Yeast Nitrogen Base, 1 L total) for 2-3
days at about 30.degree. C.
[0299] Synthetic Complete Medium mix (minus uracil) contained:
TABLE-US-00018 0.4 g Adenine hemisulfate 3.5 g Arginine 1 g
Glutamic Acid 0.433 g Histidine 0.4 g Myo-Inositol 5.2 g Isoleucine
2.63 g Leucine 0.9 g Lysine 1.5 g Methionine 0.8 g Phenylalanine
1.1 g Serine 1.2 g Threonine 0.8 g Tryptophan 0.2 g Tyrosine 1.2 g
Valine
[0300] For expression and activity analysis, transformed cells
containing the various xylose isomerase constructs were selected
from the cultures and grown in about 100 ml of SC-Dextrose (minus
uracil) to an OD.sub.600 of about 4.0. The S. cerevisiae cultures
that were transformed with the various xylose isomerase-histidine
constructs were then lysed using YPER-Plus reagent (Thermo
Scientific, catalog number 78999) according to the manufacturer's
directions. Protein quantitation of the lysates was performed using
the Coomassie-Plus kit (Thermo Scientific, catalog number 23236) as
directed by the manufacturer. Denaturing and native Western blot
analyses were then conducted. To detect his-tagged xylose isomerase
polypeptides Western analysis was employed. Gels were transferred
onto a nitrocellulose membrane (0.45 micron, Thermo Scientific, San
Diego, Calif.) using Western blotting filter paper (Thermo
Scientific) using a Bio-Rad Mini Trans-Blot Cell (BioRad, Hercules,
Calif.) system for approximately 90 minutes at 40V. Following
transfer, the membrane was washed in 1.times.PBS (EMD, San Diego,
Calif.), 0.05% Tween-20 (Fisher Scientific, Fairlawn, N.J.) for 2-5
minutes with gentle shaking. The membrane was blocked in 3% BSA
dissolved in 1.times.PBS and 0.05% Tween-20 at room temperature for
about 2 hours with gentle shaking. The membrane was washed once in
1.times.PBS and 0.05% Tween-20 for about 5 minutes with gentle
shaking. The membrane was then incubated at room temperature with
the 1:5000 dilution of primary antibody (Ms mAB to 6.times.His Tag
(SEQ ID NO: 138), AbCam, Cambridge, Mass.) in 0.3% BSA (Fraction V,
EMD, San Diego, Calif.) dissolved in 1.times.PBS and 0.05% Tween-20
with gentle shaking. Incubation was allowed to proceed for about 1
hour with gentle shaking. The membrane was then washed three times
for 5 minutes each with 1.times.PBS and 0.05% Tween-20 with gentle
shaking. The secondary antibody [Dnk pAb to Ms IgG (HRP), AbCam,
Cambridge, Mass.] was used at 1:15000 dilution in 0.3% BSA and
allowed to incubate for about 90 minutes at room temperature with
gentle shaking. The membrane was washed three times for about 5
minutes using 1.times.PBS and 0.05% Tween-20 with gentle shaking.
The membrane was then incubated with 5 ml of Supersignal West Pico
Chemiluminescent substrate (Thermo Scientific, San Diego, Calif.)
for 1 minute and then was exposed to a phosphorimager (Bio-Rad
Universal Hood II, Bio-Rad, Hercules, Calif.) for about 10-100
seconds. The results are shown in FIG. 7. As can be seen, both
Piromyces ("P" in FIG. 7) and Ruminococcus ("R" in FIG. 7) xylose
isomerases are expressed in both the soluble and insoluble
fractions of the yeast cells.
[0301] To measure activity of the various xylose isomerase
constructs, assays were performed according to Kuyper et al. (FEMS
Yeast Res., 4:69 [2003]). About 20 .mu.g of soluble whole cell
extract was incubated in the presence of 100 mM Tris, pH 7.5, 10 mM
MgCl.sub.2, 0.15 mM NADH (Sigma, St. Louis, Mo.), and about 2 U
sorbitol dehydrogenase (Roche) at about 30.degree. C. To start the
reaction, about 100 .mu.l of xylose was added at various final
concentrations of 40-500 mM. A Beckman DU-800 was utilized with an
Enzyme Mechanism software package (Beckman Coulter, Inc.), and the
change in the A.sub.340 was monitored for 2-3 minutes.
Example 5
Preparation of Selective Growth Yeast
[0302] The yeast gene cdc21 encodes thymidylate synthase, which is
required for de novo synthesis of pyrimidine deoxyribonucleotides.
A cdc 21 mutant, strain 17206, (ATCC accession number 208583) has a
point mutation G139S relative to the initiating methionine. The
restrictive temperature of this temperature sensitive mutant is
37.degree. C., which arrests cell division at S phase, so that
little or no cell growth and division occurs at or above this
temperature.
[0303] Saccharomyces cerevisiae strain YGR420CBY4742.DELTA.PFK2 was
used as the starting cell line to create the cdc21 growth sensitive
mutant. A construct for homologous recombination was prepared to
replace the wild type thymidylate synthase YGR420CBY4742.DELTA.PFK2
for the cdc21 mutant. This construct was made in various steps.
First, the cdc21 mutant region from Saccharomyces cerevisiae strain
17206 was PCR amplified using the following primers:
TABLE-US-00019 CDC21_fwd: (SEQ ID NO: 155)
5'-aatcgatcaaagcttctaaatacaagacgtgcgatgacgactatact ggac-3'
CDC21_rev: (SEQ ID NO: 156)
5'-taccgtactacccgggtatatagtctttttgccctggtgttccttaa taatttc-3'
[0304] For this PCR amplification reaction Saccharomyces cerevisiae
17206 genomic DNA was used. The genomic DNA was extracted using
Zymo research YeaStar Genomic DNA kit according to instructions. In
the PCR amplification reaction 100 ng of 17206 genomic DNA, 1 .mu.M
of the oligonucleotide primer set listed above, 2.5 U of PfuUltra
High-Fidelity DNA polymerase (Stratagene), 300 .mu.M dNTPs (Roche),
and 1.times.PfuUltra reaction buffer was mixed in a final reaction
volume of 50 .mu.l. Using a BIORAD DNA Engine Tetrad 2 Peltier
thermal cycler the following cycle conditions were used: 5 min
denaturation step at 95.degree. C., followed by 30 cycles of 20 sec
at 95.degree. C., 20 sec at 50.degree. C., and 1 min at 72.degree.
C., and a final step of 5 min at 72.degree. C. This PCR product was
digested with HindIII and XmaI restriction endonucleases and cloned
in the HindIII and XmaI sites of PUC19 (NEB) according to standard
cloning procedures described by Maniatis in Molecular Cloning.
[0305] The genomic DNA of BR214-4a (ATTC accession number 208600)
was extracted using Zymo research YeaStar Genomic DNA kit according
to instructions. The lys2 gene with promoter and terminator regions
was PCR amplified from BR214-4a genomic DNA using the following
primers:
TABLE-US-00020 Lys2Fwd: (SEQ ID NO: 157)
5'-tgctaatgacccgggaattccacttgcaattacataaaaaattccgg cgg-3' Lys2Rev:
(SEQ ID NO: 158) 5'-atgatcattgagctcagcttcgcaagtattcattttagacccatggt
gg-3'.
[0306] The PCR cycle was identical to that just described above but
with genomic DNA of BR214-4a instead. XmaI and SacI restriction
sites were designed to flank this DNA construct to clone it into
the XmaI and SacI sites of the PUC19-cdc21 vector according to
standard cloning procedures described by Maniatis in Molecular
Cloning. The new construct with the cdc21 mutation with a lys2
directly downstream of that will be referred to as
PUC19-cdc2'-lys2.
[0307] The final step involved the cloning of the downstream region
of thymidylate synthase into the PUC19-cdc2'-lys2 vector
immediately downstream of the lys2 gene. The downstream region of
the thymidylate synthase was amplified from BY4742 genomic DNA
(ATCC accession number 201389D-5 using the following primers:
TABLE-US-00021 ThymidylateSynthase_DownFwd: (SEQ ID NO: 159)
5'-tgctaatgagagctctcattttttggtgcgatatgtttttggttgat g-3' and
ThymidylateSynthatse_DownRev: (SEQ ID NO: 160)
5'-aatgatcatgagctcgtcaacaagaactaaaaaattgttcaaaaatg c-3'.
[0308] This final construct is referred as
PUC19-cdc2'-lys2-ThymidylateSynthase_down. The sequence is set
forth in the tables. A final PCR amplification reaction of this
construct was performed using the following PCR primers:
TABLE-US-00022 ThymidylateSynthase::cdc21 fwd: (SEQ ID NO: 161)
5'-ctaaatacaagacgtgcgatgacgactatactgg-3' and
ThymidylateSynthase::cdc21 rev: (SEQ ID NO: 162)
5'-gtcaacaagaactaaaaaattgttcaaaaatgcaattgtc-3'.
[0309] The PCR reaction was identical to that described above but
using 100 ng of the PUC19-cdc2'-lys2-ThymidylateSynthase_down
construct as a template.
[0310] The final PCR product was separated by agarose gel
electrophoresis, excised, and purified using MP Biomedicals
Geneclean II kit as recommended. Homologous recombination of
YGR420CBY4742.DELTA.PFK2 to replace the wt thymidylate synthase for
the cdc21 mutant was accomplished using 10 .mu.g of the purified
PCR product to transform YGR420CBY4742.DELTA.PFK2 strain using same
transformation protocol described above. Transformants were
selected by culturing the cells on selective media containing
SC-Ethanol (0.67% yeast nitrogen base-2% ethanol) containing
complete amino acids minus lysine.
[0311] The genome of this final engineered strain contains the
mutated cdc21 gene, and has both the PFK1 and PFK2 genes deleted.
This final engineered strain will be transformed with the best
combination of edd-p426 GPD and eda-p425 GPD variant constructs.
Ethanol and glucose measurements will be monitored during aerobic
and anaerobic growth conditions using Roche ethanol and glucose
kits according to instructions.
Example 6
Examples of Polynucleotide Regulators
[0312] Provided in the tables hereafter are non-limiting examples
of regulator polynucleotides that can be utilized in embodiments
herein. Such polynucleotides may be utilized in native form or may
be modified for use herein. Examples of regulatory polynucleotides
include those that are regulated by oxygen levels in a system
(e.g., up-regulated or down-regulated by relatively high oxygen
levels or relatively low oxygen levels)
Regulated Yeast Promoters--Up-Regulated by Oxygen
TABLE-US-00023 [0313] Relative Relative Gene mRNA level mRNA level
ORF name name (Aerobic) (Anaerobic) Ratio YPL275W 4389 30 219.5
YPL276W 2368 30 118.4 YDR256C CTA1 2076 30 103.8 YHR096C HXT5 1846
30 72.4 YDL218W 1189 30 59.4 YCR010C 1489 30 48.8 YOR161C 599 30
29.9 YPL200W 589 30 29.5 YGR110W 1497 30 27 YNL237W YTP1 505 30
25.2 YBR116C 458 30 22.9 YOR348C PUT4 451 30 22.6 YBR117C TKL2 418
30 20.9 YLL052C 635 30 20 YNL195C 1578 30 19.4 YPR193C 697 30 15.7
YDL222C 301 30 15 YNL335W 294 30 14.6 YPL036W PMA2 487 30 12.8
YML122C 206 30 10.3 YGR067C 236 30 10.2 YPR192W 204 30 10.2 YNL014W
828 30 9.8 YFL061W 256 30 9.1 YNR056C 163 30 8.1 YOR186W 153 30 7.6
YDR222W 196 30 6.5 YOR338W 240 30 6.3 YPR200C 113 30 5.7 YMR018W
778 30 5.2 YOR364W 123 30 5.1 YNL234W 93 30 4.7 YNR064C 85 30 4.2
YGR213C RTA1 104 30 4 YCL064C CHA1 80 30 4 YOL154W 302 30 3.9
YPR150W 79 30 3.9 YPR196W MAL63 30 30 3.6 YDR420W HKR1 221 30 3.5
YJL216C 115 30 3.5 YNL270C ALP1 67 30 3.3 YHL016C DUR3 224 30 3.2
YOL131W 230 30 3 YOR077W RTS2 210 30 3 YDR536W STL1 55 30 2.7
YNL150W 78 30 2.6 YHR212C 149 30 2.4 YJL108C 106 30 2.4 YGR069W 49
30 2.4 YDR106W 60 30 2.3 YNR034W SOL1 197 30 2.2 YEL073C 104 30 2.1
YOL141W 81 30 1.8
Regulated Yeast Promoters--Down-Regulated by Oxygen
TABLE-US-00024 [0314] Relative Relative Gene mRNA level mRNA level
ORF name name (Aerobic) (Anaerobic) Ratio YJR047C ANB1 30 4901
231.1 YMR319C FET4 30 1159 58 YPR194C 30 982 49.1 YIR019C STA1 30
981 22.8 YHL042W 30 608 12 YHR210C 30 552 27.6 YHR079B SAE3 30 401
2.7 YGL162W STO1 30 371 9.6 YHL044W 30 334 16.7 YOL015W 30 320 6.1
YCLX07W 30 292 4.2 YIL013C PDR11 30 266 10.6 YDR046C 30 263 13.2
YBR040W FIG1 30 257 12.8 YLR040C 30 234 2.9 YOR255W 30 231 11.6
YOL014W 30 229 11.4 YAR028W 30 212 7.5 YER089C 30 201 6.2 YFL012W
30 193 9.7 YDR539W 30 187 3.4 YHL043W 30 179 8.9 YJR162C 30 173 6
YMR165C SMP2 30 147 3.5 YER106W 30 145 7.3 YDR541C 30 140 7 YCRX07W
30 138 3.3 YHR048W 30 137 6.9 YCL021W 30 136 6.8 YOL160W 30 136 6.8
YCRX08W 30 132 6.6 YMR057C 30 109 5.5 YDR540C 30 83 4.2 YOR378W 30
78 3.9 YBR085W AAC3 45 1281 28.3 YER188W 47 746 15.8 YLL065W GIN11
50 175 3.5 YDL241W 58 645 11.1 YBR238C 59 274 4.6 YCR048W ARE1 60
527 8.7 YOL165C 60 306 5.1 YNR075W 60 251 4.2 YJL213W 60 250 4.2
YPL265W DIP5 61 772 12.7 YDL093W PMT5 62 353 5.7 YKR034W DAL80 63
345 5.4 YKR053C 66 1268 19.3 YJR147W 68 281 4.1
TABLE-US-00025 Known and putative DNA binding motifs Regulator
Known Consensus Motif Abf1 TCRNNNNNNACG (SEQ ID NO: 532) Cbf1
RTCACRTG Gal4 CGGNNNNNNNNNNNCCG (SEQ ID NO: 533) Gcn4 TGACTCA Gcr1
CTTCC Hap2 CCAATNA Hap3 CCAATNA Hap4 CCAATNA Hsf1 GAANNTTCNNGAA
(SEQ ID NO: 534) Ino2 ATGTGAAA Mata(A1) TGATGTANNT (SEQ ID NO: 535)
Mcm1 CCNNNWWRGG (SEQ ID NO: 536) Mig1 WWWWSYGGGG (SEQ ID NO: 537)
Pho4 CACGTG Rap1 RMACCCANNCAYY (SEQ ID NO: 538) Reb1 CGGGTRR Ste12
TGAAACA Swi4 CACGAAA Swi6 CACGAAA Yap1 TTACTAA Putative DNA Binding
Motifs Best Motif (scored by E- Best Motif (scored by Regulator
value) Hypergeometric) Abf1 TYCGT--R-ARTGAYA (SEQ TYCGT--R-ARTGAYA
(SEQ ID ID NO: 539) NO: 539) Ace2 RRRAARARAA-A-RARAA
GTGTGTGTGTGTGTG (SEQ (SEQ ID NO: 540) ID NO: 541) Adr1
A-AG-GAGAGAG-GGCAG YTSTYSTT-TTGYTWTT (SEQ (SEQ ID NO: 542) ID NO:
543) Arg80 T--CCW-TTTKTTTC (SEQ ID GCATGACCATCCACG (SEQ NO: 544) ID
NO: 545) Arg81 AAAAARARAAAARMA (SEQ GSGAYARMGGAMAAAAA ID NO: 546)
(SEQ ID NO: 547) Aro80 YKYTYTTYTT----KY (SEQ ID TRCCGAGRYW-SSSGCGS
NO: 548) (SEQ ID NO: 549) Ash1 CGTCCGGCGC (SEQ ID NO: CGTCCGGCGC
(SEQ ID NO: 550) 550) Azf1 GAAAAAGMAAAAAAA (SEQ AARWTSGARG-A--CSAA
ID NO: 551) (SEQ ID NO: 552) Bas1 TTTTYYTTYTTKY-TY-T CS-CCAATGK--CS
(SEQ ID (SEQ ID NO: 553) NO: 554) Cad1 CATKYTTTTTTKYTY (SEQ
GCT-ACTAAT (SEQ ID NO: ID NO: 555) 556) Cbf1 CACGTGACYA (SEQ ID NO:
CACGTGACYA (SEQ ID NO: 557) 557) Cha4 CA---ACACASA-A (SEQ ID
CAYAMRTGY-C (SEQ ID NO: NO: 558) 559) Cin5 none none Crz1
GG-A-A--AR-ARGGC-(SEQ TSGYGRGASA (SEQ ID NO: ID NO: 560) 561) Cup9
TTTKYTKTTY-YTTTKTY K-C-C---SCGCTACKGC (SEQ (SEQ ID NO: 562) ID NO:
563) Dal81 WTTKTTTTTYTTTTT-T (SEQ SR-GGCMCGGC-SSG (SEQ ID NO: 564)
ID NO: 565) Dal82 TTKTTTTYTTC (SEQ ID NO: TACYACA-CACAWGA (SEQ 566)
ID NO: 567) Dig1 AAA--RAA-GARRAA-AR CCYTG-AYTTCW-CTTC (SEQ (SEQ ID
NO: 568) ID NO: 569) Dot6 GTGMAK-MGRA-G-G (SEQ GTGMAK-MGRA-G-G (SEQ
ID ID NO: 570) NO: 570) Fhl1 -TTWACAYCCRTACAY-Y -TTWACAYCCRTACAY-Y
(SEQ ID NO: 571) (SEQ ID NO: 571) Fkh1 TTT-CTTTKYTT-YTTTT
AAW-RTAAAYARG (SEQ ID (SEQ ID NO: 572) NO: 573) Fkh2
AAARA-RAAA-AAAR-AA GG-AAWA-GTAAACAA (SEQ (SEQ ID NO: 574) ID NO:
575) Fzf1 CACACACACACACACAC SASTKCWCTCKTCGT (SEQ (SEQ ID NO: 576)
ID NO: 577) Gal4 TTGCTTGAACGSATGCCA TTGCTTGAACGSATGCCA (SEQ ID NO:
578) (SEQ ID NO: 578) Gal4 (Gal) YCTTTTTTTTYTTYYKG
CGGM---CW-Y--CCCG (SEQ (SEQ ID NO: 579) ID NO: 580) Gat1 none none
Gat3 RRSCCGMCGMGRCGCGCS RGARGTSACGCAKRTTCT (SEQ ID NO: 581) (SEQ ID
NO: 582) Gcn4 AAA-ARAR-RAAAARRAR TGAGTCAY (SEQ ID NO: 583) Gcr1
GGAAGCTGAAACGYMWRR GGAAGCTGAAACGYMWRR (SEQ ID NO: 584) (SEQ ID NO:
584) Gcr2 GGAGAGGCATGATGGGGG AGGTGATGGAGTGCTCAG (SEQ ID NO: 585)
(SEQ ID NO: 586) Gln3 CT-CCTTTCT (SEQ ID NO: GKCTRR-RGGAGA-GM (SEQ
587) ID NO: 588) Grf10 GAAARRAAAAAAMRMARA -GGGSG-T-SYGT-CGA (SEQ
(SEQ ID NO: 589) ID NO: 590) Gts1 G-GCCRS--TM (SEQ ID NO:
AG-AWGTTTTTGWCAAMA 591) (SEQ ID NO: 592) Haa1 none none Hal9
TTTTTTYTTTTY-KTTTT KCKSGCAGGCWTTKYTCT (SEQ ID NO: 593) (SEQ ID NO:
594) Hap2 YTTCTTTTYT-Y-C-KT-(SEQ G-CCSART-GC (SEQ ID NO: ID NO:
595) 596) Hap3 T-SYKCTTTTCYTTY (SEQ ID SGCGMGGG--CC-GACCG NO: 597)
(SEQ ID NO: 598) Hap4 STT-YTTTY-TTYTYYYY YCT-ATTSG-C-GS (SEQ ID
(SEQ ID NO: 599) NO: 600) Hap5 YK-TTTWYYTC (SEQ ID NO:
T-TTSMTT-YTTTCCK-C (SEQ 601) ID NO: 602) Hir1 AAAA-A-AARAR-AG (SEQ
ID CCACKTKSGSCCT-S (SEQ ID NO: 603) NO: 604) Hir2
WAAAAAAGAAAA-AAAAR CRSGCYWGKGC (SEQ ID (SEQ ID NO: 605) NO: 606)
Hms1 AAA-GG-ARAM (SEQ ID NO: -AARAAGC-GGGCAC-C (SEQ 607) ID NO:
608) Hsf1 TYTTCYAGAA--TTCY (SEQ TYTTCYAGAA--TTCY (SEQ ID ID NO:
609) NO: 609) Ime4 CACACACACACACACACA CACACACACACACACACA (SEQ ID
NO: 610) (SEQ ID NO: 610) Ino2 TTTYCACATGC (SEQ ID
SCKKCGCKSTSSTTYAA NO: 611) (SEQ ID NO: 612) Ino4 G--GCATGTGAAAA
(SEQ ID G--GCATGTGAAAA (SEQ ID NO: 613) NO: 613) Ixr1
GAAAA-AAAAAAAARA-A CTTTTTTTYYTSGCC (SEQ ID (SEQ ID NO: 614) NO:
615) Leu3 GAAAAARAARAA-AA (SEQ GCCGGTMMCGSYC--(SEQ ID NO: 616) ID
NO: 617) Mac1 YTTKT--TTTTTYTYTTT (SEQ A--TTTTTYTTKYGC (SEQ ID ID
NO: 618) NO: 619) Mal13 GCAG-GCAGG (SEQ ID NO: AAAC-TTTATA-ATACA
(SEQ 620) ID NO: 621) Mal33 none none Mata1 GCCC-C CAAT-TCT-CK (SEQ
ID NO: 622) Mbp1 TTTYTYKTTT-YYTTTTT G-RR-A-ACGCGT-R (SEQ ID (SEQ ID
NO: 623) NO: 624) Mcm1 TTTCC-AAW-RGGAAA (SEQ TTTCC-AAW-RGGAAA (SEQ
ID NO: 625) ID NO: 625) Met31 YTTYYTTYTTTTYTYTTC (SEQ ID NO: 626)
Met4 MTTTTTYTYTYTTC (SEQ ID NO: 627) Mig1 TATACA-AGMKRTATATG (SEQ
ID NO: 628) Mot3 TMTTT-TY-CTT-TTTWK (SEQ ID NO: 629) Msn1
KT--TTWTTATTCC-C (SEQ ID NO: 630) Msn2 ACCACC Msn4
R--AAAA-RA-AARAAAT (SEQ ID NO: 631) Mss11 TTTTTTTTCWCTTTKYC (SEQ ID
NO: 632) Ndd1 TTTY-YTKTTTY-YTTYT (SEQ ID NO: 633) Nrg1
TTY--TTYTT-YTTTYYY (SEQ ID NO: 634) Pdr1 T-YGTGKRYGT-YG (SEQ ID NO:
635) Phd1 TTYYYTTTTTYTTTTYTT (SEQ ID NO: 636) Pho4 GAMAAAAAARAAAAR
(SEQ ID NO: 637)
Put3 CYCGGGAAGCSAMM-CCG (SEQ ID NO: 638) Rap1 GRTGYAYGGRTGY (SEQ ID
NO: 639) Rcs1 KMAARAAAAARAAR (SEQ ID NO: 640) Reb1 RTTACCCGS Rfx1
AYGRAAAARARAAAARAA (SEQ ID NO: 641) Rgm1 GGAKSCC-TTTY-GMRTA (SEQ ID
NO: 642) Rgt1 CCCTCC Rim101 GCGCCGC Rlm1 TTTTC-KTTTYTTTTTC (SEQ ID
NO: 643) Rme1 ARAAGMAGAAARRAA (SEQ ID NO: 644) Rox1
YTTTTCTTTTY-TTTTT (SEQ ID NO: 645) Rph1 ARRARAAAGG-(SEQ ID NO: 646)
Rtg1 YST-YK-TYTT-CTCCCM (SEQ ID NO: 647) Rtg3 GARA-AAAAR-RAARAAA
(SEQ ID NO: 648) Sfl1 CY--GGSSA-C (SEQ ID NO: 649) Sfp1
CACACACACACACAYA (SEQ ID NO: 650) Sip4 CTTYTWTTKTTKTSA (SEQ ID NO:
651) Skn7 YTTYYYTYTTTYTYYTTT (SEQ ID NO: 652) Sko1 none Smp1
AMAAAAARAARWARA-AA (SEQ ID NO: 653) Sok2 ARAAAARRAAAAAG-RAA (SEQ ID
NO: 654) Stb1 RAARAAAAARCMRSRAAA (SEQ ID NO: 655) Ste12
TTYTKTYTY-TYYKTTTY (SEQ ID NO: 656) Stp1 GAAAAMAA-AAAAA-AAA (SEQ ID
NO: 657) Stp2 YAA-ARAARAAAAA-AAM (SEQ ID NO: 658) Sum1
TY-TTTTTTYTTTTT-TK (SEQ ID NO: 659) Swi4 RAARAARAAA-AA-R-AA (SEQ ID
NO: 660) Swi5 CACACACACACACACACA (SEQ ID NO: 610) Swi6
RAARRRAAAAA-AAAMAA (SEQ ID NO: 661) Thi2 GCCAGACCTAC (SEQ ID NO:
662) Uga3 GG-GGCT Yap1 TTYTTYTTYTTTY-YTYT (SEQ ID NO: 663) Yap3
none Yap5 YKSGCGCGYCKCGKCGGS (SEQ ID NO: 664) Yap6
TTTTYYTTTTYYYYKTT (SEQ ID NO: 665) Yap7 none Yfl044c TTCTTKTYYTTTT
(SEQ ID NO: 666) Yjl206c TTYTTTTYTYYTTTYTTT (SEQ ID NO: 667) Zap1
TTGCTTGAACGGATGCCA (SEQ ID NO: 668) Zms1 MG-MCAAAAATAAAAS (SEQ ID
NO: 669)
Transcriptional Repressors
TABLE-US-00026 [0315] Associated Gene(s) Description(s) WHI5
Repressor of G1 transcription that binds to SCB binding factor
(SBF) at SCB target promoters in early G1; phosphorylation of Whi5p
by the CDK, Cln3p/Cdc28p relieves repression and promoter binding
by Whi5; periodically expressed in G1 TUP1 General repressor of
transcription, forms complex with Cyc8p, involved in the
establishment of repressive chromatin structure through
interactions with histones H3 and H4, appears to enhance expression
of some genes ROX1 Heme-dependent repressor of hypoxic genes;
contains an HMG domain that is responsible for DNA bending activity
SFL1 Transcriptional repressor and activator; involved in
repression of flocculation-related genes, and activation of stress
responsive genes; negatively regulated by cAMP-dependent protein
kinase A subunit Tpk2p RIM101 Transcriptional repressor involved in
response to pH and in cell wall construction; required for alkaline
pH-stimulated haploid invasive growth and sporulation; activated by
proteolytic processing; similar to A. nidulans PacC RDR1
Transcriptional repressor involved in the control of multidrug
resistance; negatively regulates expression of the PDR5 gene;
member of the Gal4p family of zinc cluster proteins SUM1
Transcriptional repressor required for mitotic repression of middle
sporulation-specific genes; also acts as general replication
initiation factor; involved in telomere maintenance, chromatin
silencing; regulated by pachytene checkpoint XBP1 Transcriptional
repressor that binds to promoter sequences of the cyclin genes,
CYS3, and SMF2; expression is induced by stress or starvation
during mitosis, and late in meiosis; member of the Swi4p/Mbp1p
family; potential Cdc28p substrate NRG2 Transcriptional repressor
that mediates glucose repression and negatively regulates
filamentous growth; has similarity to Nrg1p NRG1 Transcriptional
repressor that recruits the Cyc8p-Tup1p complex to promoters;
mediates glucose repression and negatively regulates a variety of
processes including filamentous growth and alkaline pH response
CUP9 Homeodomain-containing transcriptional repressor of PTR2,
which encodes a major peptide transporter; imported peptides
activate ubiquitin-dependent proteolysis, resulting in degradation
of Cup9p and de-repression of PTR2 transcription YOX1
Homeodomain-containing transcriptional repressor, binds to Mcm1p
and to early cell cycle boxes (ECBs) in the promoters of cell
cycle- regulated genes expressed in M/G1 phase; expression is cell
cycle- regulated; potential Cdc28p substrate RFX1 Major
transcriptional repressor of DNA-damage-regulated genes, recruits
repressors Tup1p and Cyc8p to their promoters; involved in DNA
damage and replication checkpoint pathway; similar to a family of
mammalian DNA binding RFX1-4 proteins MIG3 Probable transcriptional
repressor involved in response to toxic agents such as hydroxyurea
that inhibit ribonucleotide reductase; phosphorylation by Snf1p or
the Mec1p pathway inactivates Mig3p, allowing induction of damage
response genes RGM1 Putative transcriptional repressor with
proline-rich zinc fingers; overproduction impairs cell growth YHP1
One of two homeobox transcriptional repressors (see also Yox1p),
that bind to Mcm1p and to early cell cycle box (ECB) elements of
cell cycle regulated genes, thereby restricting ECB-mediated
transcription to the M/G1 interval HOS4 Subunit of the Set3
complex, which is a meiotic-specific repressor of sporulation
specific genes that contains deacetylase activity; potential Cdc28p
substrate CAF20 Phosphoprotein of the mRNA cap-binding complex
involved in translational control, repressor of cap-dependent
translation initiation, competes with eIF4G for binding to eIF4E
SAP1 Putative ATPase of the AAA family, interacts with the Sin1p
transcriptional repressor in the two-hybrid system SET3 Defining
member of the SET3 histone deacetylase complex which is a
meiosis-specific repressor of sporulation genes; necessary for
efficient transcription by RNAPII; one of two yeast proteins that
contains both SET and PHD domains RPH1 JmjC domain-containing
histone demethylase which can specifically demethylate H3K36 tri-
and dimethyl modification states; transcriptional repressor of
PHR1; Rph1p phosphorylation during DNA damage is under control of
the MEC1-RAD53 pathway YMR181C Protein of unknown function; mRNA
transcribed as part of a bicistronic transcript with a predicted
transcriptional repressor RGM1/YMR182C; mRNA is destroyed by
nonsense-mediated decay (NMD); YMR181C is not an essential gene
YLR345W Similar to
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase enzymes
responsible for the metabolism of fructoso-2,6- bisphosphate; mRNA
expression is repressed by the Rfx1p-Tup1p- Ssn6p repressor
complex; YLR345W is not an essential gene MCM1 Transcription factor
involved in cell-type-specific transcription and pheromone
response; plays a central role in the formation of both repressor
and activator complexes PHR1 DNA photolyase involved in
photoreactivation, repairs pyrimidine dimers in the presence of
visible light; induced by DNA damage; regulated by transcriptional
repressor Rph1p HOS2 Histone deacetylase required for gene
activation via specific deacetylation of lysines in H3 and H4
histone tails; subunit of the Set3 complex, a meiotic-specific
repressor of sporulation specific genes that contains deacetylase
activity RGT1 Glucose-responsive transcription factor that
regulates expression of several glucose transporter (HXT) genes in
response to glucose; binds to promoters and acts both as a
transcriptional activator and repressor SRB7 Subunit of the RNA
polymerase II mediator complex; associates with core polymerase
subunits to form the RNA polymerase II holoenzyme; essential for
transcriptional regulation; target of the global repressor Tup1p
GAL11 Subunit of the RNA polymerase II mediator complex; associates
with core polymerase subunits to form the RNA polymerase II
holoenzyme; affects transcription by acting as target of activators
and repressors
Transcriptional Activators
TABLE-US-00027 [0316] Associated Gene(s) Description(s) SKT5
Activator of Chs3p (chitin synthase III), recruits Chs3p to the bud
neck via interaction with Bni4p; has similarity to Shc1p, which
activates Chs3p during sporulation MSA1 Activator of G1-specific
transcription factors, MBF and SBF, that regulates both the timing
of G1-specific gene transcription, and cell cycle initiation;
potential Cdc28p substrate AMA1 Activator of meiotic anaphase
promoting complex (APC/C); Cdc20p family member; required for
initiation of spore wall assembly; required for Clb1p degradation
during meiosis STB5 Activator of multidrug resistance genes, forms
a heterodimer with Pdr1p; contains a Zn(II)2Cys6 zinc finger domain
that interacts with a PDRE (pleotropic drug resistance element) in
vitro; binds Sin3p in a two-hybrid assay RRD2 Activator of the
phosphotyrosyl phosphatase activity of PP2A, peptidyl- prolyl
cis/trans-isomerase; regulates G1 phase progression, the
osmoresponse, microtubule dynamics; subunit of the Tap42p-Pph21p-
Rrd2p complex BLM10 Proteasome activator subunit; found in
association with core particles, with and without the 19S
regulatory particle; required for resistance to bleomycin, may be
involved in protecting against oxidative damage; similar to
mammalian PA200 SHC1 Sporulation-specific activator of Chs3p
(chitin synthase III), required for the synthesis of the chitosan
layer of ascospores; has similarity to Skt5p, which activates Chs3p
during vegetative growth; transcriptionally induced at alkaline pH
NDD1 Transcriptional activator essential for nuclear division;
localized to the nucleus; essential component of the mechanism that
activates the expression of a set of late-S-phase-specific genes
IMP2' Transcriptional activator involved in maintenance of ion
homeostasis and protection against DNA damage caused by bleomycin
and other oxidants, contains a C-terminal leucine-rich repeat LYS14
Transcriptional activator involved in regulation of genes of the
lysine biosynthesis pathway; requires 2-aminoadipate semialdehyde
as co- inducer MSN1 Transcriptional activator involved in
regulation of invertase and glucoamylase expression, invasive
growth and pseudohyphal differentiation, iron uptake, chromium
accumulation, and response to osmotic stress; localizes to the
nucleus HAA1 Transcriptional activator involved in the
transcription of TPO2, YRO2, and other genes putatively encoding
membrane stress proteins; involved in adaptation to weak acid
stress UGA3 Transcriptional activator necessary for
gamma-aminobutyrate (GABA)- dependent induction of GABA genes (such
as UGA1, UGA2, UGA4); zinc-finger transcription factor of the
Zn(2)-Cys(6) binuclear cluster domain type; localized to the
nucleus GCR1 Transcriptional activator of genes involved in
glycolysis; DNA-binding protein that interacts and functions with
the transcriptional activator Gcr2p GCR2 Transcriptional activator
of genes involved in glycolysis; interacts and functions with the
DNA-binding protein Gcr1p GAT1 Transcriptional activator of genes
involved in nitrogen catabolite repression; contains a GATA-1-type
zinc finger DNA-binding motif; activity and localization regulated
by nitrogen limitation and Ure2p GLN3 Transcriptional activator of
genes regulated by nitrogen catabolite repression (NCR),
localization and activity regulated by quality of nitrogen source
PUT3 Transcriptional activator of proline utilization genes,
constitutively binds PUT1 and PUT2 promoter sequences and undergoes
a conformational change to form the active state; has a
Zn(2)-Cys(6) binuclear cluster domain ARR1 Transcriptional
activator of the basic leucine zipper (bZIP) family, required for
transcription of genes involved in resistance to arsenic compounds
PDR3 Transcriptional activator of the pleiotropic drug resistance
network, regulates expression of ATP-binding cassette (ABC)
transporters through binding to cis-acting sites known as PDREs
(PDR responsive elements) MSN4 Transcriptional activator related to
Msn2p; activated in stress conditions, which results in
translocation from the cytoplasm to the nucleus; binds DNA at
stress response elements of responsive genes, inducing gene
expression MSN2 Transcriptional activator related to Msn4p;
activated in stress conditions, which results in translocation from
the cytoplasm to the nucleus; binds DNA at stress response elements
of responsive genes, inducing gene expression PHD1 Transcriptional
activator that enhances pseudohyphal growth; regulates expression
of FLO11, an adhesin required for pseudohyphal filament formation;
similar to StuA, an A. nidulans developmental regulator; potential
Cdc28p substrate FHL1 Transcriptional activator with similarity to
DNA-binding domain of Drosophila forkhead but unable to bind DNA in
vitro; required for rRNA processing; isolated as a suppressor of
splicing factor prp4 VHR1 Transcriptional activator, required for
the vitamin H-responsive element (VHRE) mediated induction of VHT1
(Vitamin H transporter) and BIO5 (biotin biosynthesis intermediate
transporter) in response to low biotin concentrations CDC20
Cell-cycle regulated activator of anaphase-promoting
complex/cyclosome (APC/C), which is required for metaphase/anaphase
transition; directs ubiquitination of mitotic cyclins, Pds1p, and
other anaphase inhibitors; potential Cdc28p substrate CDH1
Cell-cycle regulated activator of the anaphase-promoting
complex/cyclosome (APC/C), which directs ubiquitination of cyclins
resulting in mitotic exit; targets the APC/C to specific substrates
including Cdc20p, Ase1p, Cin8p and Fin1p AFT2 Iron-regulated
transcriptional activator; activates genes involved in
intracellular iron use and required for iron homeostasis and
resistance to oxidative stress; similar to Aft1p MET4
Leucine-zipper transcriptional activator, responsible for the
regulation of the sulfur amino acid pathway, requires different
combinations of the auxiliary factors Cbf1p, Met28p, Met31p and
Met32p CBS2 Mitochondrial translational activator of the COB mRNA;
interacts with translating ribosomes, acts on the COB mRNA
5'-untranslated leader CBS1 Mitochondrial translational activator
of the COB mRNA; membrane protein that interacts with translating
ribosomes, acts on the COB mRNA 5'-untranslated leader CBP6
Mitochondrial translational activator of the COB mRNA;
phosphorylated PET111 Mitochondrial translational activator
specific for the COX2 mRNA; located in the mitochondrial inner
membrane PET494 Mitochondrial translational activator specific for
the COX3 mRNA, acts together with Pet54p and Pet122p; located in
the mitochondrial inner membrane PET122 Mitochondrial translational
activator specific for the COX3 mRNA, acts together with Pet54p and
Pet494p; located in the mitochondrial inner membrane RRD1
Peptidyl-prolyl cis/trans-isomerase, activator of the
phosphotyrosyl phosphatase activity of PP2A; involved in G1 phase
progression, microtubule dynamics, bud morphogenesis and DNA
repair; subunit of the Tap42p-Sit4p-Rrd1p complex YPR196W Putative
maltose activator POG1 Putative transcriptional activator that
promotes recovery from pheromone induced arrest; inhibits both
alpha-factor induced G1 arrest and repression of CLN1 and CLN2 via
SCB/MCB promoter elements; potential Cdc28p substrate; SBF
regulated MSA2 Putative transcriptional activator, that interacts
with G1-specific transcription factor, MBF and G1-specific
promoters; ortholog of Msa2p, an MBF and SBF activator that
regulates G1-specific transcription and cell cycle initiation
PET309 Specific translational activator for the COX1 mRNA, also
influences stability of intron-containing COX1 primary transcripts;
localizes to the mitochondrial inner membrane; contains seven
pentatricopeptide repeats (PPRs) TEA1 Ty1 enhancer activator
required for full levels of Ty enhancer-mediated transcription; C6
zinc cluster DNA-binding protein PIP2 Autoregulatory
oleate-specific transcriptional activator of peroxisome
proliferation, contains Zn(2)-Cys(6) cluster domain, forms
heterodimer with Oaf1p, binds oleate response elements (OREs),
activates beta- oxidation genes CHA4 DNA binding transcriptional
activator, mediates serine/threonine activation of the catabolic
L-serine (L-threonine) deaminase (CHA1); Zinc-finger protein with
Zn[2]-Cys[6] fungal-type binuclear cluster domain SFL1
Transcriptional repressor and activator; involved in repression of
flocculation-related genes, and activation of stress responsive
genes; negatively regulated by cAMP-dependent protein kinase A
subunit Tpk2p RDS2 Zinc cluster transcriptional activator involved
in conferring resistance to ketoconazole CAT8 Zinc cluster
transcriptional activator necessary for derepression of a variety
of genes under non-fermentative growth conditions, active after
diauxic shift, binds carbon source responsive elements ARO80 Zinc
finger transcriptional activator of the Zn2Cys6 family; activates
transcription of aromatic amino acid catabolic genes in the
presence of aromatic amino acids SIP4 C6 zinc cluster
transcriptional activator that binds to the carbon source-
responsive element (CSRE) of gluconeogenic genes; involved in the
positive regulation of gluconeogenesis; regulated by Snf1p protein
kinase; localized to the nucleus SPT10 Putative histone acetylase,
sequence-specific activator of histone genes, binds specifically
and highly cooperatively to pairs of UAS elements in core histone
promoters, functions at or near the TATA box MET28 Basic leucine
zipper (bZIP) transcriptional activator in the Cbf1p- Met4p-Met28p
complex, participates in the regulation of sulfur metabolism GCN4
Basic leucine zipper (bZIP) transcriptional activator of amino acid
biosynthetic genes in response to amino acid starvation; expression
is tightly regulated at both the transcriptional and translational
levels CAD1 AP-1-like basic leucine zipper (bZIP) transcriptional
activator involved in stress responses, iron metabolism, and
pleiotropic drug resistance; controls a set of genes involved in
stabilizing proteins; binds consensus sequence TTACTAA INO2
Component of the heteromeric Ino2p/Ino4p basic helix-loop-helix
transcription activator that binds inositol/choline-responsive
elements (ICREs), required for derepression of phospholipid
biosynthetic genes in response to inositol depletion THI2 Zinc
finger protein of the Zn(II)2Cys6 type, probable transcriptional
activator of thiamine biosynthetic genes SWI4 DNA binding component
of the SBF complex (Swi4p-Swi6p), a transcriptional activator that
in concert with MBF (Mbp1-Swi6p) regulates late G1-specific
transcription of targets including cyclins and genes required for
DNA synthesis and repair HAP5 Subunit of the heme-activated,
glucose-repressed Hap2/3/4/5 CCAAT- binding complex, a
transcriptional activator and global regulator of respiratory gene
expression; required for assembly and DNA binding activity of the
complex HAP3 Subunit of the heme-activated, glucose-repressed
Hap2p/3p/4p/5p CCAAT-binding complex, a transcriptional activator
and global regulator of respiratory gene expression; contains
sequences contributing to both complex assembly and DNA binding
HAP2 Subunit of the heme-activated, glucose-repressed
Hap2p/3p/4p/5p CCAAT-binding complex, a transcriptional activator
and global regulator of respiratory gene expression; contains
sequences sufficient for both complex assembly and DNA binding HAP4
Subunit of the heme-activated, glucose-repressed Hap2p/3p/4p/5p
CCAAT-binding complex, a transcriptional activator and global
regulator of respiratory gene expression; provides the principal
activation function of the complex YML037C Putative protein of
unknown function with some characteristics of a transcriptional
activator; may be a target of Dbf2p-Mob1p kinase; GFP- fusion
protein co-localizes with clathrin-coated vesicles; YML037C is not
an essential gene TRA1 Subunit of SAGA and NuA4 histone
acetyltransferase complexes; interacts with acidic activators
(e.g., Gal4p) which leads to transcription activation; similar to
human TRRAP, which is a cofactor for c-Myc mediated oncogenic
transformation YLL054C Putative protein of unknown function with
similarity to Pip2p, an oleate- specific transcriptional activator
of peroxisome proliferation; YLL054C is not an essential gene RTG2
Sensor of mitochondrial dysfunction; regulates the subcellular
location of Rtg1p and Rtg3p, transcriptional activators of the
retrograde (RTG) and TOR pathways; Rtg2p is inhibited by the
phosphorylated form of Mks1p
YBR012C Dubious open reading frame, unlikely to encode a functional
protein; expression induced by iron-regulated transcriptional
activator Aft2p JEN1 Lactate transporter, required for uptake of
lactate and pyruvate; phosphorylated; expression is derepressed by
transcriptional activator Cat8p during respiratory growth, and
repressed in the presence of glucose, fructose, and mannose MRP1
Mitochondrial ribosomal protein of the small subunit; MRP1 exhibits
genetic interactions with PET122, encoding a COX3-specific
translational activator, and with PET123, encoding a small subunit
mitochondrial ribosomal protein MRP17 Mitochondrial ribosomal
protein of the small subunit; MRP17 exhibits genetic interactions
with PET122, encoding a COX3-specific translational activator TPI1
Triose phosphate isomerase, abundant glycolytic enzyme; mRNA half-
life is regulated by iron availability; transcription is controlled
by activators Reb1p, Gcr1p, and Rap1p through binding sites in the
5' non-coding region PKH3 Protein kinase with similarity to
mammalian phosphoinositide- dependent kinase 1 (PDK1) and yeast
Pkh1p and Pkh2p, two redundant upstream activators of Pkc1p;
identified as a multicopy suppressor of a pkh1 pkh2 double mutant
YGL079W Putative protein of unknown function; green fluorescent
protein (GFP)- fusion protein localizes to the endosome; identified
as a transcriptional activator in a high-throughput yeast
one-hybrid assay TFB1 Subunit of TFIIH and nucleotide excision
repair factor 3 complexes, required for nucleotide excision repair,
target for transcriptional activators PET123 Mitochondrial
ribosomal protein of the small subunit; PET123 exhibits genetic
interactions with PET122, which encodes a COX3 mRNA- specific
translational activator MHR1 Protein involved in homologous
recombination in mitochondria and in transcription regulation in
nucleus; binds to activation domains of acidic activators; required
for recombination-dependent mtDNA partitioning MCM1 Transcription
factor involved in cell-type-specific transcription and pheromone
response; plays a central role in the formation of both repressor
and activator complexes EGD1 Subunit beta1 of the nascent
polypeptide-associated complex (NAC) involved in protein targeting,
associated with cytoplasmic ribosomes; enhances DNA binding of the
Gal4p activator; homolog of human BTF3b STE5 Pheromone-response
scaffold protein; binds Ste11p, Ste7p, and Fus3p kinases, forming a
MAPK cascade complex that interacts with the plasma membrane and
Ste4p-Ste18p; allosteric activator of Fus3p that facilitates
Ste7p-mediated activation RGT1 Glucose-responsive transcription
factor that regulates expression of several glucose transporter
(HXT) genes in response to glucose; binds to promoters and acts
both as a transcriptional activator and repressor TYE7 Serine-rich
protein that contains a basic-helix-loop-helix (bHLH) DNA binding
motif; binds E-boxes of glycolytic genes and contributes to their
activation; may function as a transcriptional activator in
Ty1-mediated gene expression VMA13 Subunit H of the eight-subunit
V1 peripheral membrane domain of the vacuolar H+-ATPase (V-ATPase),
an electrogenic proton pump found throughout the endomembrane
system; serves as an activator or a structural stabilizer of the
V-ATPase GAL11 Subunit of the RNA polymerase II mediator complex;
associates with core polymerase subunits to form the RNA polymerase
II holoenzyme; affects transcription by acting as target of
activators and repressors VAC14 Protein involved in regulated
synthesis of PtdIns(3,5)P(2), in control of trafficking of some
proteins to the vacuole lumen via the MVB, and in maintenance of
vacuole size and acidity; interacts with Fig4p; activator of
Fab1p
Example 7
Heterologous Xylose Isomerase Expression in Yeast
[0317] Provided hereafter are non-limiting examples of certain
organisms from which nucleic acids that encode a polypeptide having
xylose isomerase activity can be obtained. Certain nucleic acid
encoded polypeptides having active xylose isomerase activity can be
expressed in an engineered yeast (S. cerevisiae).
TABLE-US-00028 Active? Xylose isomerase type Donor Organism
(yes/no) (Type 1/Type 2) Piromyces Yes Type 2 Orpinomyces Yes
Bacteroides thetaiotaomicron Yes Clostridium phytofermentans Yes
Thermus thermophilus Yes Type 1 Ruminococcus flavefaciens Yes
Escherichia coli No Bacillus subtilis No Lactobacillus pentoses No
Leifsoria xyli subsp. Cynodontis No Clostridium thermosulfurogenes
No Bacillus licheniformis No Burkholderia xenovorans No Psudomonas
savastanoi No Robiginitalea biformata No Saccharophagus degradans
No Staphylococcus xylosus No Streptomyces diastaticus subsp No
diastaticus Xanthomonas campestris No Salmonella enterica serovar
No Typhimurium Agrobacterium tumefaciens No Arabidopsis thaliana No
Pseudomonas syringae No Actinoplanes missouriensis No Streptomyces
rubiginosus No Epilopiscium No
Example 8
Examples of Nucleic Acid and Amino Acid Sequences
[0318] Provided hereafter and non-limiting examples of certain
nucleic acid sequences.
TABLE-US-00029 Nucleic acid Organism/ Accession No. or Gene Name
ATCC identifier other identifier Nucleotide Sequence Xylose
Ruminococcus AJ132472 atggaatttt tcagcaatat cggtaaaatt cagtatcagg
gaccaaaaag tactgatcct Isomerase flavefaciens ctctcattta agtactataa
ccctgaagaa gtcatcaacg gaaagacaat gcgcgagcat (XI-RF strain 17
ctgaagttcg ctctttcatg gtggcacaca atgggcggcg acggaacaga tatgttcggc
Native) tgcggcacaa cagacaagac ctggggacag tccgatcccg ctgcaagagc
aaaggctaag gttgacgcag cattcgagat catggataag ctctccattg actactattg
tttccacgat cgcgatcttt ctcccgagta tggcagcctc aaggctacca acgatcagct
tgacatagtt acagactata tcaaggagaa gcagggcgac aagttcaagt gcctctgggg
tacagcaaag tgcttcgatc atccaagatt catgcacggt gcaggtacat ctccttctgc
tgatgtattc gctttctcag ctgctcagat caagaaggct ctcgagtcaa cagtaaagct
cggcggtaac ggttacgttt tctggggcgg acgtgaaggc tatgagacac ttcttaatac
aaatatggga ctcgaactcg acaatatggc tcgtcttatg aagatggctg ttgagtatgg
acgttcgatc ggcttcaagg gcgacttcta tatcgagccc aagcccaagg agcccacaaa
gcatcagtac gatttcgata cagctactgt tctgggattc ctcagaaagt acggtctcga
taaggatttc aagatgaata tcgaagctaa ccacgctaca cttgctcagc atacattcca
gcatgagctc cgtgttgcaa gagacaatgg tgtgttcggt tctatcgacg caaaccaggg
cgacgttctt cttggatggg atacagacca gttccccaca aatatctacg atacaacaat
gtgtatgtat gaagttatca aggcaggcgg cttcacaaac ggcggtctca acttcgacgc
taaggcacgc agagggagct tcactcccga ggatatcttc tacagctata tcgcaggtat
ggatgcattt gctctgggct tcagagctgc tctcaagctt atcgaagacg gacgtatcga
caagttcgtt gctgacagat acgcttcatg gaataccggt atcggtgcag acataatcgc
aggtaaggca gatttcgcat ctcttgaaaa gtatgctctt gaaaagggcg aggttacagc
ttcactctca agcggcagac aggaaatgct ggagtctatc gtaaataacg ttcttttcag
tctgtaa (SEQ ID NO: 30) Xylose Based on Based on AJ132472
atggaatttttcagcaatatcggtaaaattcagtatcagggaccaaaaagtactgatcctctctcatttaagt-
actataacc isomerase Ruminococcus
ctgaagaagtcatcaacggaaagacaatgcgcgagcatctgaagttcgctctttcatggtggcacacaatggg-
cggc (point flavefaciens
gacggaacagatatgttcggctgcggcacaacagacaagacctggggacagtccgatcccgctgcaagagcaa-
a mutation) strain 17
ggctaaggttgacgcagcattcgagatcatggataagctctccattgactactattgtttccacgatcgcgat-
ctttctccc
gagtatggcagcctcaaggctaccaacgatcagcttgacatagttacagactatatcaaggagaagcaggg-
cgaca
agttcaagtgcctctggggtacagcaaagtgcttcgatcatccaagattcatgcacggtgcaggtacatct-
ccttctgctg
atgtattcgctttctcagctgctcagatcaagaaggctctGgagtcaacagtaaagctcggcggtaacggt-
tacgttttct
ggggcggacgtgaaggctatgagacacttcttaatacaaatatgggactcgaactcgacaatatggctcgt-
cttatga
agatggctgttgagtatggacgttcgatcggcttcaagggcgacttctatatcgagcccaagcccaaggag-
cccacaa
agcatcagtacgatttcgatacagctactgttctgggattcctcagaaagtacggtctcgataaggatttc-
aagatgaat
atcgaagctaaccacgctacacttgctcagcatacattccagcatgagctccgtgttgcaagagacaatgg-
tgtgttcg
gttctatcgacgcaaaccagggcgacgttcttcttggatgggatacagaccagttccccacaaatatctac-
gatacaac
aatgtgtatgtatgaagttatcaaggcaggcggcttcacaaacggcggtctcaacttcgacgctaaggcac-
gcagag
ggagcttcactcccgaggatatcttctacagctatatcgcaggtatggatgcatttgctctgggcttcaga-
gctgctctcaa
gcttatcgaagacggacgtatcgacaagttcgttgctgacagatacgcttcatggaataccggtatcggtg-
cagacata
atcgcaggtaaggcagatttcgcatctcttgaaaagtatgctcttgaaaagggcgaggttacagcttcact-
ctcaagcg gcagacaggaaatgctggagtctatcgtaaataacgttcttttcagtctgtaa (SEQ
ID NO: 29) Xylose
atggagttcttttctaatataggtaaaattcagtatcaaggtccaaaatc isomerase
tacagatccattgtcttttaaatattataatccagaagaagttataaatg (XI-RF_HR)
gtaaaactatgagagaacatttaaaatttgctttgtcttggtggcatact
atgggtggtgatggtactgatatgttcggttgtggtactactgataaaac
ttggggtcaatctgatccagctgctagagcaaaagccaaagtagatgcag
cctttgaaattatggataaattgtctattgattattattgttttcatgat
agagatttgtctcctgaatatggttctttaaaagcaactaatgatcaatt
ggacattgttacggattatattaaagaaaaacaaggtgataaatttaaat
gtttgtggggcactgcgaaatgttttgatcatccacgttttatgcatggt
gcggggacgagtccttctgctgatgtttttgctttttctgccgctcaaat
taagaaggcattggaatcaactgttaaattaggtgggaacgggtatgtat
tctggggaggaagggaaggttatgaaacattattaaacactaatatgggt
ttggaattggataatatggctagattgatgaaaatggctgtagaatacgg
aaggtctattggttttaagggtgacttttatattgaaccaaaacctaaag
agcctactaaacatcaatatgattttgatactgctacagttttgggattc
ttgagaaaatatggtctggataaagattttaaaatgaatatagaagctaa
tcatgcaacactcgcacaacatacttttcaacatgaattgagagttgcca
gagataacggagtttttggatctatcgatgcaaaccagggagacgttttg
ctaggatgggatactgatcaatttccaactaacatttatgatactactat
gtgtatgtatgaagtaattaaggcaggaggctttactaatggcggattaa
actttgatgcgaaggctaggcgtggtagtttcactccagaggatatattc
tattcttatattgctggaatggatgctttcgcgttaggtttcagggcagc
actaaaattgattgaagatggtagaattgataagtttgtagctgatagat
atgcttcttggaatactggaataggagcagatataatcgctgggaaagcc
gacttcgccagtctggaaaaatatgcgcttgaaaaaggagaagttactgc
cagcttaagttccggtcgtcaagaaatgttggaatctattgtaaacaatg ttttattttctctg
(SEQ ID NO: 23) Xylose Piromyces sp. E2 AJ249909 atggctaagg
aatatttccc acaaattcaa aagattaagt tcgaaggtaa ggattctaag isomerase
aatccattag ccttccacta ctacgatgct gaaaaggaag tcatgggtaa gaaaatgaag
(XI-P Native) gattggttac gtttcgccat ggcctggtgg cacactcttt
gcgccgaagg tgctgaccaa ttcggtggag gtacaaagtc tttcccatgg aacgaaggta
ctgatgctat tgaaattgcc aagcaaaagg ttgatgctgg tttcgaaatc atgcaaaagc
ttggtattcc atactactgt ttccacgatg ttgatcttgt ttccgaaggt aactctattg
aagaatacga atccaacctt aaggctgtcg ttgcttacct caaggaaaag caaaaggaaa
ccggtattaa gcttctctgg agtactgcta acgtcttcgg tcacaagcgt tacatgaacg
gtgcctccac taacccagac tttgatgttg tcgcccgtgc tattgttcaa attaagaacg
ccatagacgc cggtattgaa cttggtgctg aaaactacgt cttctggggt ggtcgtgaag
gttacatgag tctccttaac actgaccaaa agcgtgaaaa ggaacacatg gccactatgc
ttaccatggc tcgtgactac gctcgttcca agggattcaa gggtactttc ctcattgaac
caaagccaat ggaaccaacc aagcaccaat acgatgttga cactgaaacc gctattggtt
tccttaaggc ccacaactta gacaaggact tcaaggtcaa cattgaagtt aaccacgcta
ctcttgctgg tcacactttc gaacacgaac ttgcctgtgc tgttgatgct ggtatgctcg
gttccattga tgctaaccgt ggtgactacc aaaacggttg ggatactgat caattcccaa
ttgatcaata cgaactcgtc caagcttgga tggaaatcat ccgtggtggt ggtttcgtta
ctggtggtac caacttcgat gccaagactc gtcgtaactc tactgacctc gaagacatca
tcattgccca cgtttctggt atggatgcta tggctcgtgc tcttgaaaac gctgccaagc
tcctccaaga atctccatac accaagatga agaaggaacg ttacgcttcc ttcgacagtg
gtattggtaa ggactttgaa gatggtaagc tcaccctcga acaagtttac gaatacggta
agaagaacgg tgaaccaaag caaacttctg gtaagcaaga actctacgaa gctattgttg
ccatgtacca ataa (SEQ ID NO: 24) Xylose Based on
ATGGCTAAAGAATATTTTCCACAAATTCAGAAAATTAAATTTGAAGGTAAAGATTC Isomerase
Piromyces sp. E2
TAAAAATCCATTGGCTTTCCATTATTATGATGCTGAAAAAGAAGTTATGGGTAAAA (XI-P-HR1)
AGATGAAAGATTGGTTGAGATTCGCTATGGCTTGGTGGCATACTCTATGTGCTG
AAGGAGCTGATCAATTTGGAGGAGGTACTAAATCTTTTCCTTGGAATGAAGGTA
CTGACGCTATTGAAATTGCTAAGCAGAAAGTAGACGCGGGTTTTGAAATTATGC
AAAAATTGGGAATACCATATTATTGTTTTCATGATGTTGATTTGGTATCTGAGGGT
AATTCTATTGAAGAATATGAATCTAATTTAAAAGCTGTTGTTGCTTACTTAAAAGA
AAAACAAAAAGAAACTGGAATTAAATTGTTGTGGTCTACAGCTAATGTTTTCGGT
CATAAAAGATATATGAATGGTGCTTCTACAAATCCAGATTTTGATGTTGTAGCTA
GAGCTATTGTTCAAATTAAAAATGCTATAGATGCAGGAATTGAATTAGGTGCCGA
AAATTATGTTTTCTGGGGAGGTAGAGAAGGTTATATGTCTTTGTTAAATACTGAT
CAAAAACGTGAAAAGGAACACATGGCAACTATGTTGACAATGGCTAGGGATTAT
GCTAGATCTAAAGGTTTTAAAGGTACTTTCTTGATTGAGCCAAAACCTATGGAAC
CAACTAAACATCAATATGACGTTGACACTGAAACTGCTATTGGTTTCTTAAAAGC
TCATAATTTGGATAAAGATTTTAAGGTTAATATAGAAGTTAATCATGCTACACTAG
CTGGTCATACTTTTGAACATGAATTAGCTTGTGCAGTTGATGCCGGTATGTTAGG
TTCTATCGACGCAAATAGAGGTGATTATCAAAATGGTTGGGACACAGATCAATTT
CCAATAGATCAATATGAATTGGTTCAAGCATGGATGGAAATTATTAGGGGTGGA
GGCTTCGTTACAGGTGGAACTAATTTTGATGCTAAAACTAGGAGAAATTCTACAG
ATCTTGAAGATATAATTATTGCTCATGTATCTGGTATGGATGCGATGGCCCGTGC
TTTGGAAAATGCAGCTAAATTACTTCAAGAATCTCCTTATACTAAAATGAAAAAGG
AAAGATATGCTTCTTTTGATTCTGGAATAGGTAAGGATTTTGAAGATGGTAAATT
GACATTGGAACAAGTTTATGAATATGGTAAGAAGAATGGAGAACCAAAACAAACT
TCTGGTAAACAAGAATTATATGAGGCTATAGTAGCTATGTATCAAtaa (SEQ ID NO: 25)
PEP Zymomonas ATCC 31821
ACTAGTAAAAAAATGACCAAGCCGCGCACAATTAATCAGAACCCAGACCTTCGC Carboxylase
mobilis TATTTTGGTAACCTGCTCGGTCAGGTTATTAAGGAACAAGGCGGAGAGTCTTTAT
(PEPC- TCAACCAGATCGAGCAAATTCGCTCTGCCGCGATTAGACGCCATCGGGGTATTG
Native) TTGACAGCACCGAGCTAAGTTCTCGCTTAGCCGATCTCGACCTTAATGACATGT
TCTCTTTTGCACATGCCTTTTTGCTGTTTTCAATGCTGGCCAATTTGGCTGATGA
TCGTCAGGGAGATGCCCTTGATCCTGATGCCAATATGGCAAGTGCCCTTAAGGA
CATAAAAGCCAAAGGCGTCAGTCAGCAGGCGATCATTGATATGATCGACAAAGC
CTGCATTGTGCCTGTTCTGACAGCACATCCGACCGAAGTCCGTCGGAAAAGTAT
GCTTGACCATTATAATCGCATTGCAGGTTTAATGCGGTTAAAAGATGCTGGACAA
ACGGTGACCGAAGATGGTCTTCCGATCGAAGATGCGTTAATCCAGCAAATCACG
ATATTATGGCAGACTCGTCCGCTCATGCTGCAAAAGCTGACCGTGGCTGATGAA
ATCGAAACTGCCCTGTCTTTCTTAAGAGAAACTTTTCTGCCTGTTCTGCCCCAGA
TTTATGCAGAATGGGAAAAATTGCTTGGTAGTTCTATTCCAAGCTTTATCAGACC
TGGTAATTGGATTGGTGGTGACCGTGACGGTAACCCCAATGTCAATGCCGATAC
GATCATGCTGTCTTTGAAGCGCAGCTCGGAAACGGTATTGACGGATTATCTCAA
CCGTCTTGATAAACTGCTTTCCAACCTTTCGGTCTCAACCGATATGGTTTCGGTA
TCCGATGATATTCTACGTCTAGCCGATAAAAGTGGTGACGATGCTGCGATCCGT
GCGGATGAACCTTATCGTCGTGCCTTAAATGGTATTTATGACCGTTTAGCCGCTA
CCTATCGTCAGATCGCCGGTCGCAACCCTTCGCGCCCAGCCTTGCGTTCTGCA
GAAGCCTATAAACGGCCTCAAGAATTGCTGGCTGATTTGAAGACCTTGGCCGAA
GGCTTGGGTAAATTGGCAGAAGGTAGTTTTAAGGCATTGATCCGTTCGGTTGAA
ACCTTTGGTTTCCATTTGGCCACCCTCGATCTGCGTCAGAATTCGCAGGTTCAT
GAAAGAGTTGTCAATGAACTGCTACGGACAGCCACCGTTGAAGCCGATTATTTA
TCTCTATCGGAAGAAGATCGCGTTAAGCTGTTAAGACGGGAATTGTCGCAGCCG
CGGACTCTATTCGTTCCGCGCGCCGATTATTCCGAAGAAACGCGTTCTGAACTT
GATATTATTCAGGCAGCAGCCCGCGCCCATGAAATTTTTGGCCCTGAATCCATT
ACGACTTATTTGATTTCGAATGGCGAAAGCATTTCCGATATTCTGGAAGTCTATT
TGCTTTTGAAAGAAGCAGGGCTGTATCAAGGGGGTGCTAAGCCAAAAGCGGCG
ATTGAAGCTGCGCCTTTATTCGAGACGGTGGCCGATCTTGAAAATGCGCCAAAG
GTCATGGAGGAATGGTTCAAGCTGCCTGAAGCGCAAGCCATTGCAAAGGCACA
TGGCGTTCAGGAAGTGATGGTTGGCTATTCTGACTCCAATAAGGACGGCGGATA
TCTGACCTCGGTTTGGGGTCTTTATAAGGCTTGCCTCGCTTTGGTGCCGATTTTT
GAGAAAGCCGGTGTACCGATCCAGTTTTTCCATGGACGGGGTGGTTCCGTTGG
TCGCGGTGGTGGTTCCAACTTTAATGCCATTCTGTCGCAGCCAGCCGGAGCCG
TCAAAGGGCGTATCCGTTATACAGAACAGGGTGAAGTCGTGGCGGCCAAATAT
GGCACCCATGAAAGCGCTATTGCCCATCTGGATGAGGCCGTAGCGGCGACTTT
GATTACGTCTTTGGAAGCACCGACCATTGTCGAGCCAGAGTTTAGTCGTTACCG
TAAGGCCTTGGATCAGATCTCAGATTCAGCTTTCCAGGCCTATCGCCAATTGGT
CTATGGAACGAAGGGCTTCCGTAAATTCTTTAGTGAATTTACGCCTTTGCCGGAA
ATTGCCCTGTTAAAGATCGGGTCACGCCCACCTAGCCGCAAAAAATCCGACCG
GATTGAAGATCTACGCGCTATTCCTTGGGTGTTTAGCTGGTCTCAAGTTCGAGT
CATGTTACCCGGTTGGTTCGGTTTCGGTCAGGCTTTATATGACTTTGAAGATACC
GAGCTGTTACAGGAAATGGCAAGCCGTTGGCCGTTTTTCCGCACGACTATTCGG
AATATGGAACAGGTGATGGCACGTTCCGATATGACGATCGCCAAGCATTATCTG
GCCTTGGTTGAGGATCAGACAAATGGTGAGGCTATCTATGATTCTATCGCGGAT
GGCTGGAATAAAGGTTGTGAAGGTCTGTTAAAGGCAACCCAGCAGAATTGGCTG
TTGGAACGCTTTCCGGCGGTTGATAATTCGGTGCAGATGCGTCGGCCTTATCTG
GAACCGCTTAATTACTTACAGGTCGAATTGCTGAAGAAATGGCGGGGAGGTGAT
ACCAACCCGCATATCCTCGAATCTATTCAGCTGACAATCAATGCCATTGCGACG
GCACTTCGCAACAGCGGTTAATAACTCGAG (SEQ ID NO: 20) PEP Based on
ACTAGTAAAAAAATGACCAAGCCAAGAACTATTAACCAAAACCCAGACTTGAGAT Carboxylase
Zymomonas ACTTCGGTAACTTGTTGGGTCAAGTTATCAAGGAACAAGGTGGTGAATCTTTGTT
(PEPC-HR) mobilis
CAACCAAATTGAACAAATCAGATCCGCTGCTATTAGAAGACACAGAGGTATCGT
CGACTCTACCGAATTGTCCTCTAGATTGGCTGACTTGGACTTGAACGACATGTT
CTCCTTCGCTCACGCTTTCTTGTTGTTCTCTATGTTGGCTAACTTGGCTGACGAC
AGACAAGGTGACGCTTTGGACCCAGACGCTAACATGGCTTCCGCTTTGAAGGA
CATTAAGGCTAAGGGTGTTTCTCAACAAGCTATCATTGACATGATCGACAAGGCT
TGTATTGTCCCAGTTTTGACTGCTCACCCAACCGAAGTCAGAAGAAAGTCCATG
TTGGACCACTACAACAGAATCGCTGGTTTGATGAGATTGAAGGACGCTGGTCAA
ACTGTTACCGAAGACGGTTTGCCAATTGAAGACGCTTTGATCCAACAAATTACTA
TCTTGTGGCAAACCAGACCATTGATGTTGCAAAAGTTGACTGTCGCTGACGAAA
TTGAAACCGCTTTGTCTTTCTTGAGAGAAACTTTCTTGCCAGTTTTGCCACAAAT
CTACGCTGAATGGGAAAAGTTGTTGGGTTCCTCTATTCCATCCTTCATCAGACCA
GGTAACTGGATTGGTGGTGACAGAGACGGTAACCCAAACGTCAACGCTGACAC
CATCATGTTGTCTTTGAAGAGATCCTCTGAAACTGTTTTGACCGACTACTTGAAC
AGATTGGACAAGTTGTTGTCCAACTTGTCTGTCTCCACTGACATGGTTTCTGTCT
CCGACGACATTTTGAGATTGGCTGACAAGTCTGGTGACGACGCTGCTATCAGAG
CTGACGAACCATACAGAAGAGCTTTGAACGGTATTTACGACAGATTGGCTGCTA
CCTACAGACAAATCGCTGGTAGAAACCCATCCAGACCAGCTTTGAGATCTGCTG
AAGCTTACAAGAGACCACAAGAATTGTTGGCTGACTTGAAGACTTTGGCTGAAG
GTTTGGGTAAGTTGGCTGAAGGTTCCTTCAAGGCTTTGATTAGATCTGTTGAAAC
CTTCGGTTTCCACTTGGCTACTTTGGACTTGAGACAAAACTCCCAAGTCCACGA
AAGAGTTGTCAACGAATTGTTGAGAACCGCTACTGTTGAAGCTGACTACTTGTCT
TTGTCCGAAGAAGACAGAGTCAAGTTGTTGAGAAGAGAATTGTCTCAACCAAGA
ACCTTGTTCGTTCCAAGAGCTGACTACTCCGAAGAAACTAGATCTGAATTGGAC
ATCATTCAAGCTGCTGCTAGAGCTCACGAAATCTTCGGTCCAGAATCCATTACCA
CTTACTTGATCTCTAACGGTGAATCCATTTCTGACATCTTGGAAGTCTACTTGTT
GTTGAAGGAAGCTGGTTTGTACCAAGGTGGTGCTAAGCCAAAGGCTGCTATTGA
AGCTGCTCCATTGTTCGAAACCGTTGCTGACTTGGAAAACGCTCCAAAGGTCAT
GGAAGAATGGTTCAAGTTGCCAGAAGCTCAAGCTATCGCTAAGGCTCACGGTGT
TCAAGAAGTCATGGTTGGTTACTCCGACTCTAACAAGGACGGTGGTTACTTGAC
TTCCGTCTGGGGTTTGTACAAGGCTTGTTTGGCTTTGGTTCCAATTTTCGAAAAG
GCTGGTGTCCCAATCCAATTCTTCCACGGTAGAGGTGGTTCTGTTGGTAGAGGT
GGTGGTTCCAACTTCAACGCTATTTTGTCTCAACCAGCTGGTGCTGTCAAGGGT
AGAATCAGATACACCGAACAAGGTGAAGTTGTCGCTGCTAAGTACGGTACTCAC
GAATCCGCTATTGCTCACTTGGACGAAGCTGTTGCTGCTACCTTGATCACTTCTT
TGGAAGCTCCAACCATTGTCGAACCAGAATTCTCCAGATACAGAAAGGCTTTGG
ACCAAATCTCTGACTCCGCTTTCCAAGCTTACAGACAATTGGTTTACGGTACTAA
GGGTTTCAGAAAGTTCTTCTCTGAATTCACCCCATTGCCAGAAATTGCTTTGTTG
AAGATCGGTTCCAGACCACCATCTAGAAAGAAGTCCGACAGAATTGAAGACTTG
AGAGCTATCCCATGGGTCTTCTCTTGGTCCCAAGTTAGAGTCATGTTGCCAGGT
TGGTTCGGTTTCGGTCAAGCTTTGTACGACTTCGAAGACACTGAATTGTTGCAA
GAAATGGCTTCTAGATGGCCATTCTTCAGAACCACTATTAGAAACATGGAACAAG
TTATGGCTAGATCCGACATGACCATCGCTAAGCACTACTTGGCTTTGGTCGAAG
ACCAAACTAACGGTGAAGCTATTTACGACTCTATCGCTGACGGTTGGAACAAGG
GTTGTGAAGGTTTGTTGAAGGCTACCCAACAAAACTGGTTGTTGGAAAGATTCC
CAGCTGTTGACAACTCCGTCCAAATGAGAAGACCATACTTGGAACCATTGAACT
ACTTGCAAGTTGAATTGTTGAAGAAGTGGAGAGGTGGTGACACTAACCCACACA
TTTTGGAATCTATCCAATTGACCATTAACGCTATCGCTACTGCTTTGAGAAACTC
CGGTTAATAACTCGAG (SEQ ID NO: 21) EDA Zymomonas 31821D-5
5'-aactgactagtaaaaaaatgcgtgatatcgattcc-3' (SEQ ID No: 1) Primers
mobilis (ZM4) 5'-agtaactcgagctactaggcaacagcagcgcgcttg-3' (SEQ ID
No: 2) EDD Zymomonas 31821D-5
5'-aactgactagtaaaaaaatgactgatctgcattcaacg-3' (SEQ ID NO: 3) Primers
mobilis (ZM4) 5'-agtaactcgagctactagataccggcacctgcatatattgc-3' (SEQ
ID NO: 4) EDA Escherichia coli
5'-aactgactagtaaaaaaatgaaaaactggaaaacaagtgcagaatc-3' (SEQ ID NO: 5)
Primers 5'-agtaactcgagctactacagcttagcgccttctacagcttcacg-3' (SEQ ID
NO: 6) EDD Escherichia coli
5'-aactgactagtaaaaaaatgaatccacaattgttacgcgtaacaaatcg-3'(SEQ ID NO:
7) Primers 5'agtaactcgagctactaaaaagtgatacaggttgcgccctgttcggcac-3'
(SEQ ID NO: 8) PFK primers Saccharomyces 4015893
5'-tgcatattccgttcaatcttataaagctgccatagatttttacaccaagtcgttttaagagcttggtgag-
cgcta-3' cerevisiae (SEQ ID NO: 9) YGR240CBY4742
5'-cttgccagtgaatgacctttggcattctcatggaaacttcagtttcatagtcgagttcaagagaaaaaaa-
aagaa- 3' (SEQ ID NO: 10)
5'-atgactgttactactccttttgtgaatggtacttcttattgtaccgtcactgcatattccgttcaatc-
ttataaa-3' (SEQ ID NO: 11)
5'-ttaatcaactctctttcttccaaccaaatggtcagcaatgagtctggtagcttgccagtgaatgacct-
ttggcat- 3'(SEQ ID NO: 12) Thymidilate Saccharomyces 208583
CDC21_fwd:
5'-aatcgatcaaagcttctaaatacaagacgtgcgatgacgactatactggac-3' (SEQ ID
synthase cerevisiae strain NO: 155) Primers 17206 CDC21_rev:
5'-taccgtactacccgggtatatagtctttttgccctggtgttccttaataatttc-3' (SEQ
ID NO: (cdc21) 156) ThymidylateSynthase::cdc21 fwd:
5'-ctaaatacaagacgtgcgatgacgactatactgg-3' (SEQ ID NO: 161)
ThymidylateSynthase::cdc21 rev:
5'-gtcaacaagaactaaaaaattgttcaaaaatgcaattgtc-3' (SEQ ID NO: 162).
LYS2 BR214-4a 208600 Lys2Fwd:
5'-tgctaatgacccgggaattccacttgcaattacataaaaaattccggcgg-3' (SEQ ID
NO: 157) Lys2Rev:
5'-atgatcattgagctcagcttcgcaagtattcattttagacccatggtgg-3' (SEQ ID NO:
158). PEPC Zymomonas 5' forward (5'- Primers mobilis
GACTAACTGAACTAGTAAAAAAATGACCAAGCCGCGCACAATTAATCAG-3') (SEQ ID NO:
13) 3' reverse (5'-
AAGTGAGTAACTCGAGTTATTAACCGCTGTTGCGAAGTGCCGTCGC-3') (SEQ ID NO:
14).
[0319] Provided hereafter are non-limiting examples of certain
amino acid sequences.
TABLE-US-00030 Amino acid Accession No. Organism/ATCC or Gene Name
identifier other identifier Amino Acid Sequence Xylose Ruminococcus
CAB51938.1
MEFFSNIGKIQYQGPKSTDPLSFKYYNPEEVINGKTMREHLKFALSWWHTMGGDGTDM
Isomerase flavefaciens
FGCGTTDKTWGQSDPAARAKAKVDAAFEIMDKLSIDYYCFHDRDLSPEYGSLKATNDQL (XI-RF
strain 17
DIVTDYIKEKQGDKFKCLWGTAKCFDHPRFMHGAGTSPSADVFAFSAAQIKKALESTVKL
Native) GGNGYVFWGGREGYETLLNTNMGLELDNMARLMKMAVEYGRSIGFKGDFYIEPKPKEP
TKHQYDFDTATVLGFLRKYGLDKDFKMNIEANHATLAQHTFQHELRVARDNGVFGSIDA
NQGDVLLGWDTDQFPTNIYDTTMCMYEVIKAGGFTNGGLNFDAKARRGSFTPEDIFYSY
IAGMDAFALGFRAALKLIEDGRIDKFVADRYASWNTGIGADIIAGKADFASLEKYALEKGE
VTASLSSGRQEMLESIVNNVLFSL (SEQ ID NO: 31) Xylose Piromyces sp.
CAB76571.1 MAKEYFPQIQKIKFEGKDSKNPLAFHYYDAEKEVMGKKMKDWLR isomerase
E2 FAMAWWHTLCAEGADQFGGGTKSFPWNEGTDAIEIAKQKVDAGFEIMQKLGIPYYCFH (XI-P
DVDLVSEGNSIEEYESNLKAVVAYLKEKQKETGIKLLWSTANVFGHKRYMNGASTNPD Native)
FDVVARAIVQIKNAIDAGIELGAENYVFWGGREGYMSLLNTDQKREKEHMATMLTMAR
DYARSKGFKGTFLIEPKPMEPTKHQYDVDTETAIGFLKAHNLDKDFKVNIEVNHATLA
GHTFEHELACAVDAGMLGSIDANRGDYQNGWDTDQFPIDQYELVQAWMEIIRGGGFVT
GGTNFDAKTRRNSTDLEDIIIAHVSGMDAMARALENAAKLLQESPYTKMKKERYASFDS
GIGKDFEDGKLTLEQVYEYGKKNGEPKQTSGKQELYEAIVAMYQ (SEQ ID NO: 35)
Example 9
Preparation and Expression of Xylose Isomerase Genes
[0320] A full length native gene encoding a xylose isomerase from
Ruminococcus flavefaciens was synthesized by IDT DNA, Inc.
(Coralville, Iowa), with a single silent point mutation (a "C" to a
"G") at position 513. The sequence of this gene is set forth as SEQ
ID NO: 29 the point mutation is indicated as the larger bold
capital letter "G".
TABLE-US-00031 SEQ ID NO: 29
ATGGAATTTTTCAGCAATATCGGTAAAATTCAGTATCAGGGACCAAAAAGTACTGATCCTCTC
TCATTTAAGTACTATAACCCTGAAGAAGTCATCAACGGAAAGACAATGCGCGAGCATCTGAA
GTTCGCTCTTTCATGGTGGCACACAATGGGCGGCGACGGAACAGATATGTTCGGCTGCGGC
ACAACAGACAAGACCTGGGGACAGTCCGATCCCGCTGCAAGAGCAAAGGCTAAGGTTGACG
CAGCATTCGAGATCATGGATAAGCTCTCCATTGACTACTATTGTTTCCACGATCGCGATCTTT
CTCCCGAGTATGGCAGCCTCAAGGCTACCAACGATCAGCTTGACATAGTTACAGACTATATC
AAGGAGAAGCAGGGCGACAAGTTCAAGTGCCTCTGGGGTACAGCAAAGTGCTTCGATCATC
CAAGATTCATGCACGGTGCAGGTACATCTCCTTCTGCTGATGTATTCGCTTTCTCAGCTGCT
CAGATCAAGAAGGCTCTGGAGTCAACAGTAAAGCTCGGCGGTAACGGTTACGTTTTCTGGG
GCGGACGTGAAGGCTATGAGACACTTCTTAATACAAATATGGGACTCGAACTCGACAATATG
GCTCGTCTTATGAAGATGGCTGTTGAGTATGGACGTTCGATCGGCTTCAAGGGCGACTTCTA
TATCGAGCCCAAGCCCAAGGAGCCCACAAAGCATCAGTACGATTTCGATACAGCTACTGTTC
TGGGATTCCTCAGAAAGTACGGTCTCGATAAGGATTTCAAGATGAATATCGAAGCTAACCAC
GCTACACTTGCTCAGCATACATTCCAGCATGAGCTCCGTGTTGCAAGAGACAATGGTGTGTT
CGGTTCTATCGACGCAAACCAGGGCGACGTTCTTCTTGGATGGGATACAGACCAGTTCCCC
ACAAATATCTACGATACAACAATGTGTATGTATGAAGTTATCAAGGCAGGCGGCTTCACAAAC
GGCGGTCTCAACTTCGACGCTAAGGCACGCAGAGGGAGCTTCACTCCCGAGGATATCTTCT
ACAGCTATATCGCAGGTATGGATGCATTTGCTCTGGGCTTCAGAGCTGCTCTCAAGCTTATC
GAAGACGGACGTATCGACAAGTTCGTTGCTGACAGATACGCTTCATGGAATACCGGTATCG
GTGCAGACATAATCGCAGGTAAGGCAGATTTCGCATCTCTTGAAAAGTATGCTCTTGAAAAG
GGCGAGGTTACAGCTTCACTCTCAAGCGGCAGACAGGAAATGCTGGAGTCTATCGTAAATA
ACGTTCTTTTCAGTCTGTAA
[0321] The nucleotide sequence of the native gene is set forth as
SEQ ID NO. 30 and in GenBank as accession number AJ132472
(CAB51938.1).
TABLE-US-00032 SEQ ID NO. 30
ATGGAATTTTTCAGCAATATCGGTAAAATTCAGTATCAGGGACCAAAAAGTACTGATCCTCTC
TCATTTAAGTACTATAACCCTGAAGAAGTCATCAACGGAAAGACAATGCGCGAGCATCTGAA
GTTCGCTCTTTCATGGTGGCACACAATGGGCGGCGACGGAACAGATATGTTCGGCTGCGGC
ACAACAGACAAGACCTGGGGACAGTCCGATCCCGCTGCAAGAGCAAAGGCTAAGGTTGACG
CAGCATTCGAGATCATGGATAAGCTCTCCATTGACTACTATTGTTTCCACGATCGCGATCTTT
CTCCCGAGTATGGCAGCCTCAAGGCTACCAACGATCAGCTTGACATAGTTACAGACTATATC
AAGGAGAAGCAGGGCGACAAGTTCAAGTGCCTCTGGGGTACAGCAAAGTGCTTCGATCATC
CAAGATTCATGCACGGTGCAGGTACATCTCCTTCTGCTGATGTATTCGCTTTCTCAGCTGCT
CAGATCAAGAAGGCTCTCGAGTCAACAGTAAAGCTCGGCGGTAACGGTTACGTTTTCTGGG
GCGGACGTGAAGGCTATGAGACACTTCTTAATACAAATATGGGACTCGAACTCGACAATATG
GCTCGTCTTATGAAGATGGCTGTTGAGTATGGACGTTCGATCGGCTTCAAGGGCGACTTCTA
TATCGAGCCCAAGCCCAAGGAGCCCACAAAGCATCAGTACGATTTCGATACAGCTACTGTTC
TGGGATTCCTCAGAAAGTACGGTCTCGATAAGGATTTCAAGATGAATATCGAAGCTAACCAC
GCTACACTTGCTCAGCATACATTCCAGCATGAGCTCCGTGTTGCAAGAGACAATGGTGTGTT
CGGTTCTATCGACGCAAACCAGGGCGACGTTCTTCTTGGATGGGATACAGACCAGTTCCCC
ACAAATATCTACGATACAACAATGTGTATGTATGAAGTTATCAAGGCAGGCGGCTTCACAAAC
GGCGGTCTCAACTTCGACGCTAAGGCACGCAGAGGGAGCTTCACTCCCGAGGATATCTTCT
ACAGCTATATCGCAGGTATGGATGCATTTGCTCTGGGCTTCAGAGCTGCTCTCAAGCTTATC
GAAGACGGACGTATCGACAAGTTCGTTGCTGACAGATACGCTTCATGGAATACCGGTATCG
GTGCAGACATAATCGCAGGTAAGGCAGATTTCGCATCTCTTGAAAAGTATGCTCTTGAAAAG
GGCGAGGTTACAGCTTCACTCTCAAGCGGCAGACAGGAAATGCTGGAGTCTATCGTAAATA
ACGTTCTTTTCAGTCTGTAA
[0322] The corresponding amino acid sequence of the native
Ruminococcus flavefaciens is set forth in SEQ ID NO: 31.
TABLE-US-00033 SEQ ID NO: 31
MEFFSNIGKIQYQGPKSTDPLSFKYYNPEEVINGKTMREHLKFALSWWHT
MGGDGTDMFGCGTTDKTWGQSDPAARAKAKVDAAFEIMDKLSIDYYCFHD
RDLSPEYGSLKATNDQLDIVTDYIKEKQGDKFKCLWGTAKCFDHPRFMHG
AGTSPSADVFAFSAAQIKKALESTVKLGGNGYVFWGGREGYETLLNTNMG
LELDNMARLMKMAVEYGRSIGFKGDFYIEPKPKEPTKHQYDFDTATVLGF
LRKYGLDKDFKMNIEANHATLAQHTFQHELRVARDNGVFGSIDANQGDVL
LGWDTDQFPTNIYDTTMCMYEVIKAGGFTNGGLNFDAKARRGSFTPEDIF
YSYIAGMDAFALGFRAALKLIEDGRIDKFVADRYASWNTGIGADIIAGKA
DFASLEKYALEKGEVTASLSSGRQEMLESIVNNVLFSL
[0323] An additional nucleic acid variant of the native
Ruminococcus xylose isomerase gene was designed to eliminate
over-represented codon pairs, improve codon preferences, and reduce
mRNA secondary structures. The amino acid sequence of the hot rod
xylose isomerase gene is substantially identical to the wild type.
This sequence variant, referred to as the "hot rod" variant, is set
forth in SEQ ID NO: 32.
TABLE-US-00034 SEQ ID NO: 32
ATGGAGTTCTTTTCTAATATAGGTAAAATTCAGTATCAAGGTCCAAAATC
TACAGATCCATTGTCTTTTAAATATTATAATCCAGAAGAAGTTATAAATG
GTAAAACTATGAGAGAACATTTAAAATTTGCTTTGTCTTGGTGGCATACT
ATGGGTGGTGATGGTACTGATATGTTCGGTTGTGGTACTACTGATAAAAC
TTGGGGTCAATCTGATCCAGCTGCTAGAGCAAAAGCCAAAGTAGATGCAG
CCTTTGAAATTATGGATAAATTGTCTATTGATTATTATTGTTTTCATGAT
AGAGATTTGTCTCCTGAATATGGTTCTTTAAAAGCAACTAATGATCAATT
GGACATTGTTACGGATTATATTAAAGAAAAACAAGGTGATAAATTTAAAT
GTTTGTGGGGCACTGCGAAATGTTTTGATCATCCACGTTTTATGCATGGT
GCGGGGACGAGTCCTTCTGCTGATGTTTTTGCTTTTTCTGCCGCTCAAAT
TAAGAAGGCATTGGAATCAACTGTTAAATTAGGTGGGAACGGGTATGTAT
TCTGGGGAGGAAGGGAAGGTTATGAAACATTATTAAACACTAATATGGGT
TTGGAATTGGATAATATGGCTAGATTGATGAAAATGGCTGTAGAATACGG
AAGGTCTATTGGTTTTAAGGGTGACTTTTATATTGAACCAAAACCTAAAG
AGCCTACTAAACATCAATATGATTTTGATACTGCTACAGTTTTGGGATTC
TTGAGAAAATATGGTCTGGATAAAGATTTTAAAATGAATATAGAAGCTAA
TCATGCAACACTCGCACAACATACTTTTCAACATGAATTGAGAGTTGCCA
GAGATAACGGAGTTTTTGGATCTATCGATGCAAACCAGGGAGACGTTTTG
CTAGGATGGGATACTGATCAATTTCCAACTAACATTTATGATACTACTAT
GTGTATGTATGAAGTAATTAAGGCAGGAGGCTTTACTAATGGCGGATTAA
ACTTTGATGCGAAGGCTAGGCGTGGTAGTTTCACTCCAGAGGATATATTC
TATTCTTATATTGCTGGAATGGATGCTTTCGCGTTAGGTTTCAGGGCAGC
ACTAAAATTGATTGAAGATGGTAGAATTGATAAGTTTGTAGCTGATAGAT
ATGCTTCTTGGAATACTGGAATAGGAGCAGATATAATCGCTGGGAAAGCC
GACTTCGCCAGTCTGGAAAAATATGCGCTTGAAAAAGGAGAAGTTACTGC
CAGCTTAAGTTCCGGTCGTCAAGAAATGTTGGAATCTATTGTAAACAATG
TTTTATTTTCTCTGTAA
[0324] This gene was synthesized by assembling the oligonucleotides
set forth below first into seven separate "primary fragments" (also
referred to as "PFs"). The PFs were then assembled into three
"secondary fragments" ("SFs") which in turn were assembled into the
full length gene. All oligonucleotides were obtained from IDT. All
of the oligonucleotides used for gene construction are set forth in
the table below.
TABLE-US-00035 Oligonucleotide Sequence (SEQ ID NOS 163-228, Name
respectively, in order of appearance) 4329 On1
ATGGAGTTCTTTTCTAATATAGGTAAAATTCAGTATCAAGGTC 43-mer fwd 4329 On2
AATGGATCTGTAGATTTTGGACCTTGATACTGAATTTTA 39-mer rev 4329 On3
CAAAATCTACAGATCCATTGTCTTTTAAATATTATAATCCAGA 43-mer fwd 4329 On4
GTTTTACCATTTATAACTTCTTCTGGATTATAATATTTAAAAGAC 45-mer rev 4329 On5
AGAAGTTATAAATGGTAAAACTATGAGAGAACATTTAAAATTT 43-mer fwd 4329 On6
ATAGTATGCCACCAAGACAAAGCAAATTTTAAATGTTCTCTCATA 45-mer rev 4329 On7
GCTTTGTCTTGGTGGCATACTATGGGTGGTGATGGTACTGATATG 45-mer fwd 4329 On8
TTATCAGTAGTACCACAACCGAACATATCAGTACCATCACCACCC 45-mer rev 4329 On9
TTCGGTTGTGGTACTACTGATAAAACTTGGGGTCAATCTGATC 43-mer fwd 4329
GGCTTTTGCTCTAGCAGCTGGATCAGATTGACCCCAAGTT 40-mer On10 rev 4329
CAGCTGCTAGAGCAAAAGCCAAAGTAGATGCAGCCTTTGAAAT 43-mer On11 fwd 4329
ATCAATAGACAATTTATCCATAATTTCAAAGGCTGCATCTACTTT 45-mer On12 rev 4329
TATGGATAAATTGTCTATTGATTATTATTGTTTTCATGATAGAGA 45-mer On13 fwd 4329
AGAACCATATTCAGGAGACAAATCTCTATCATGAAAACAATAATA 45-mer On14 rev 4329
TTTGTCTCCTGAATATGGTTCTTTAAAAGCAACTAATGATCAA 43-mer On15 fwd 4329
AATATAATCCGTAACAATGTCCAATTGATCATTAGTTGCTTTTAA 45-mer On16 rev 4329
TTGGACATTGTTACGGATTATATTAAAGAAAAACAAGGTGATAAA 45-mer On17 fwd 4329
CGCAGTGCCCCACAAACATTTAAATTTATCACCTTGTTTTTCTTT 45-mer On18 rev 4329
TTTAAATGTTTGTGGGGCACTGCGAAATGTTTTGATCATCCACGT 45-mer On19 fwd 4329
ACTCGTCCCCGCACCATGCATAAAACGTGGATGATCAAAACATTT 45-mer On20 rev 4329
TTTATGCATGGTGCGGGGACGAGTCCTTCTGCTGATGTTTTTGCT 45-mer On21 fwd 4329
CTTCTTAATTTGAGCGGCAGAAAAAGCAAAAACATCAGCAGAAGG 45-mer On22 rev 4329
TTTTCTGCCGCTCAAATTAAGAAGGCATTGGAATCAACTGTTAAA 45-mer On23 fwd 4329
GAATACATACCCGTTCCCACCTAATTTAACAGTTGATTCCAATGC 45-mer On24 rev 4329
TTAGGTGGGAACGGGTATGTATTCTGGGGAGGAAGGGAAGGTTAT 45-mer On25 fwd 4329
CATATTAGTGTTTAATAATGTTTCATAACCTTCCCTTCCTCCCCA 45-mer On26 rev 4329
GAAACATTATTAAACACTAATATGGGTTTGGAATTGGATAATATG 45-mer On27 fwd 4329
TACAGCCATTTTCATCAATCTAGCCATATTATCCAATTCCAAACC 45-mer On28 rev 4329
GCTAGATTGATGAAAATGGCTGTAGAATACGGAAGGTCTATTGGT 45-mer On29 fwd 4329
TTCAATATAAAAGTCACCCTTAAAACCAATAGACCTTCCGTATTC 45-mer On30 rev 4329
TTTAAGGGTGACTTTTATATTGAACCAAAACCTAAAGAGCCTACT 45-mer On31 fwd 4329
AGTATCAAAATCATATTGATGTTTAGTAGGCTCTTTAGGTTTTGG 45-mer On32 rev 4329
AAACATCAATATGATTTTGATACTGCTACAGTTTTGGGATTCTTG 45-mer On33 fwd 4329
ATCTTTATCCAGACCATATTTTCTCAAGAATCCCAAAACTGTAGC 45-mer On34 rev 4329
AGAAAATATGGTCTGGATAAAGATTTTAAAATGAATATAGAAGCT 45-mer On35 fwd 4329
ATGTTGTGCGAGTGTTGCATGATTAGCTTCTATATTCATTTTAAA 45-mer On36 rev 4329
AATCATGCAACACTCGCACAACATACTTTTCAACATGAATTGAGA 45-mer On37 fwd 4329
AAAAACTCCGTTATCTCTGGCAACTCTCAATTCATGTTGAAAAGT 45-mer On38 rev 4329
GTTGCCAGAGATAACGGAGTTTTTGGATCTATCGATGCAAACCAG 45-mer On39 fwd 4329
ATCCCATCCTAGCAAAACGTCTCCCTGGTTTGCATCGATAGATCC 45-mer On40 rev 4329
GGAGACGTTTTGCTAGGATGGGATACTGATCAATTTCCAACTAAC 45-mer On41 fwd 4329
CATACACATAGTAGTATCATAAATGTTAGTTGGAAATTGATCAGT 45-mer On42 rev 4329
ATTTATGATACTACTATGTGTATGTATGAAGTAATTAAGGCAGGA 45-mer On43 fwd 4329
GTTTAATCCGCCATTAGTAAAGCCTCCTGCCTTAATTACTTCATA 45-mer On44 rev 4329
GGCTTTACTAATGGCGGATTAAACTTTGATGCGAAGGCTAGGCGT 45-mer On45 fwd 4329
TATATCCTCTGGAGTGAAACTACCACGCCTAGCCTTCGCATCAAA 45-mer On46 rev 4329
GGTAGTTTCACTCCAGAGGATATATTCTATTCTTATATTGCTGGA 45-mer On47 fwd 4329
GAAACCTAACGCGAAAGCATCCATTCCAGCAATATAAGAATAGAA 45-mer On48 rev 4329
ATGGATGCTTTCGCGTTAGGTTTCAGGGCAGCACTAAAATTGATT 45-mer On49 fwd 4329
CTTATCAATTCTACCATCTTCAATCAATTTTAGTGCTGCCCT 42-mer On50 rev 4329
GAAGATGGTAGAATTGATAAGTTTGTAGCTGATAGATATGCTTCT 45-mer On51 fwd 4329
TGCTCCTATTCCAGTATTCCAAGAAGCATATCTATCAGCTACAAA 45-mer On52 rev 4329
TGGAATACTGGAATAGGAGCAGATATAATCGCTGGGAAAGCCGAC 45-mer On53 fwd 4329
ATATTTTTCCAGACTGGCGAAGTCGGCTTTCCCAGCGATTATATC 45-mer On54 rev 4329
TTCGCCAGTCTGGAAAAATATGCGCTTGAAAAAGGAGAAGTTACT 45-mer On55 fwd 4329
ACGACCGGAACTTAAGCTGGCAGTAACTTCTCCTTTTTCAAGCGC 45-mer On56 rev 4329
GCCAGCTTAAGTTCCGGTCGTCAAGAAATGTTGGAATCTAT 41-mer On57 fwd 4329
CAGAGAAAATAAAACATTGTTTACAATAGATTCCAACATTTCTTG 45-mer On58 rev 4329
ACTTGACTAACTGAAGCTTCATATGATGGAGTTCTTTTCTAATATAG 55-mer On59 fwd
GTAAAATT 4329 ACTTGACTACTAGTATGGAGTTCTTTTCTAATATAGGTAAAATT 44-mer
On60 fwd 4329 ACTTGACTAACTGAAGCTTCATATGTTGGACATTGTTACGGATTAT 55-mer
On61 fwd ATTAAAGAA 4329
ACTTGACTAACTGAAGCTTCATATGAAACATCAATATGATTTTGATA 55-mer On62 fwd
CTGCTACA 4329 AGTTAAGTGAGTAAACTAGTGAATTCCAGAGAAAATAAAACATTGT 56-mer
On63 for TTACAATAGA 4329
AGTCAAGTCTCGAGCTACAGAGAAAATAAAACATTGTTTACAATAGA 44-mer On64 rev
4329 AGTTAAGTGAGTAAACTAGTGAATTCCATATTAGTGTTTAATAATGT 56-mer On65
rev TTCATAACC 4329 AGTTAAGTGAGTAAACTAGTGAATTCCATACACATAGTAGTATCAT
56-mer On66 rev AAATGTTAGT
[0325] The 7 primary fragments ("PFs") were first separately
assembled using polymerase chain reaction (PCR) mixture containing
about 1.times.Pfu Ultra II reaction buffer (Agilent, La Jolla,
Calif.), about 0.2 mM about , 0.04 .mu.mol of assembly primers (see
table below), about 0.21 .mu.mol of end primers (see table below),
and about 1 U Pfu Ultra II polymerase (Agilent, La Jolla, Calif.).
The reaction conditions were 95.degree. C. for 10 minutes, 30
cycles of 95.degree. C. for 20 seconds, 44.degree. C. for 30
seconds, and 72.degree. C. for 15 seconds, and a final extension of
5 minutes at 72.degree. C.
TABLE-US-00036 Primary Fragment Assembly Primers 5' and 3' End
Primers PF1 4329 On1 fwd 4329 On1 fwd 4329 On2 rev 4329 On10 rev
4329 On3 fwd 4329 On4 rev 4329 On5 fwd 4329 On6 rev 4329 On7 fwd
4329 On8 rev 4329 On9 fwd 4329 On10 rev PF2 4329 On9 fwd 4329 On9
fwd 4329 On10 rev 4329 On18 rev 4329 On11 fwd 4329 On12 rev 4329
On13 fwd 4329 On14 rev 4329 On15 fwd 4329 On16 rev 4329 On17 fwd
4329 On18 rev PF3 4329 On17 fwd 4329 On17 fwd 4329 On18 rev 4329
On26 rev 4329 On19 fwd 4329 On20 rev 4329 On21 fwd 4329 On22 rev
4329 On23 fwd 4329 On24 rev 4329 On25 fwd 4329 On26 rev PF4 4329
On25 fwd 4329 On25 fwd 4329 On26 rev 4329 On34 rev 4329 On27 fwd
4329 On28 rev 4329 On29 fwd 4329 On30 rev 4329 On31 fwd 4329 On32
rev 4329 On33 fwd 4329 On34 rev PF5 4329 On33 fwd 4329 On33 fwd
4329 On34 rev 4329 On42 rev 4329 On35 fwd 4329 On36 rev 4329 On37
fwd 4329 On38 rev 4329 On39 fwd 4329 On40 rev 4329 On41 fwd 4329
On42 rev PF6 4329 On41 fwd 4329 On41 fwd 4329 On42 rev 4329 On50
rev 4329 On43 fwd 4329 On44 rev 4329 On45 fwd 4329 On46 rev 4329
On47 fwd 4329 On48 rev 4329 On49 fwd 4329 On50 rev PF7 4329 On49
fwd 4329 On49 fwd 4329 On50 rev 4329 On58 rev 4329 On51 fwd 4329
On52 rev 4329 On53 fwd 4329 On54 rev 4329 On55 fwd 4329 On56 rev
4329 On57 fwd 4329 On58 rev
[0326] Each assembled primary fragment was separately PCR purified
using a Qiagen PCR purification kit (Qiagen, Valencia, Calif.)
according to the manufacturer's directions and then reassembled
into 3 secondary fragments ("SFs") in a PCR reaction containing
about 1.times.Pfu Ultra II reaction buffer (Agilent, La Jolla,
Calif.), about 0.2 mM dNTPs, about 0.1 .mu.mol of each primary
fragment (SF1=PF1+PF2+PF3; SF2=PF3+PF4+PF5; SF3=PF5+PF6+PF7), about
0.2 .mu.mol of end primers (see table below), and about 1 U Pfu
Ultra II polymerase (Agilent, La Jolla, Calif.). The reaction
conditions were 95.degree. C. for 10 minutes, 30 cycles of
95.degree. C. for 20 seconds, 62.degree. C. for 30 seconds, and
72.degree. C. for 15 seconds, and a final extension of 5 minutes at
72.degree. C.
TABLE-US-00037 Secondary Fragment Primary Fragments 5' and 3' End
Primers SF1 PF1 4329 On59 fwd (Tf1-5P1 Sf1-5P1) PF2 4329 On65 rev
(Sf1-3P1) PF3 SF2 PF3 4329 On61 fwd (Sf2-5P1) PF4 4329 On66 rev
(Sf2-3P1) PF5 SF3 PF5 4329 On62 fwd (Sf3-5P1) PF6 4329 On63 rev
(Tf1-3P1 Sf3 3P1) PF7
[0327] Each secondary fragment was PCR purified using a Qiagen PCR
purification kit (Qiagen, Valencia, Calif.) according to the
manufacturer's directions, and the final, full length gene was
assembled in a PCR reaction containing 1.times.Pfu Ultra II
reaction buffer (Agilent, La Jolla, Calif.), 0.2 mM dNTPs, 0.1
.mu.mol of each secondary fragment (SF1+SF2+SF3), 0.2 .mu.mol of
end primers (43290n60 fwd and 43290n64 rev), and 1 U Pfu Ultra II
polymerase (Agilent, La Jolla, Calif.). The reaction conditions
were 95.degree. C. for 10 minutes, 30 cycles of 95.degree. C. for
20 seconds, 62.degree. C. for 30 seconds, and 72.degree. C. for 30
seconds, and a final extension of 5 minutes at 72.degree. C. The
final product was PCR purified using a Qiagen PCR purification kit
(Qiagen, Valencia, Calif.) according to the manufacturer's
directions and then cloned into pCR Blunt II-TOPO (Invitrogen,
Carlsbad, Calif.) according to the manufacturer's directions.
Sequence confirmation of the final construct was performed at
GeneWiz (La Jolla, Calif.).
[0328] An additional variant of the native R. flavefaciens xylose
isomerase gene (XI-R-COOP) was prepared in which all of the codons
were optimized for expression in Saccharomyces cerevisiae. This
variant gene was synthesized by IDT DNA Inc. and the sequence is
set forth below as SEQ ID NO: 33.
TABLE-US-00038 SEQ ID NO: 33
ATGGAATTCTTCTCTAACATTGGTAAGATCCAATACCAAGGTCCAAAGTC
CACCGACCCATTGTCTTTCAAGTACTACAACCCAGAAGAAGTTATTAACG
GTAAGACTATGAGAGAACACTTGAAGTTCGCTTTGTCCTGGTGGCACACC
ATGGGTGGTGACGGTACTGACATGTTCGGTTGTGGTACCACTGACAAGAC
CTGGGGTCAATCTGACCCAGCTGCTAGAGCTAAGGCTAAGGTCGACGCTG
CTTTCGAAATCATGGACAAGTTGTCCATTGACTACTACTGTTTCCACGAC
AGAGACTTGTCTCCAGAATACGGTTCCTTGAAGGCTACTAACGACCAATT
GGACATCGTTACCGACTACATTAAGGAAAAGCAAGGTGACAAGTTCAAGT
GTTTGTGGGGTACTGCTAAGTGTTTCGACCACCCAAGATTCATGCACGGT
GCTGGTACCTCTCCATCCGCTGACGTCTTCGCTTTCTCTGCTGCTCAAAT
CAAGAAGGCTTTGGAATCCACTGTTAAGTTGGGTGGTAACGGTTACGTCT
TCTGGGGTGGTAGAGAAGGTTACGAAACCTTGTTGAACACTAACATGGGT
TTGGAATTGGACAACATGGCTAGATTGATGAAGATGGCTGTTGAATACGG
TAGATCTATTGGTTTCAAGGGTGACTTCTACATCGAACCAAAGCCAAAGG
AACCAACCAAGCACCAATACGACTTCGACACTGCTACCGTCTTGGGTTTC
TTGAGAAAGTACGGTTTGGACAAGGACTTCAAGATGAACATTGAAGCTAA
CCACGCTACTTTGGCTCAACACACCTTCCAACACGAATTGAGAGTTGCTA
GAGACAACGGTGTCTTCGGTTCCATCGACGCTAACCAAGGTGACGTTTTG
TTGGGTTGGGACACTGACCAATTCCCAACCAACATTTACGACACTACCAT
GTGTATGTACGAAGTCATCAAGGCTGGTGGTTTCACTAACGGTGGTTTGA
ACTTCGACGCTAAGGCTAGAAGAGGTTCTTTCACCCCAGAAGACATTTTC
TACTCCTACATCGCTGGTATGGACGCTTTCGCTTTGGGTTTCAGAGCTGC
TTTGAAGTTGATTGAAGACGGTAGAATCGACAAGTTCGTTGCTGACAGAT
ACGCTTCTTGGAACACTGGTATTGGTGCTGACATCATTGCTGGTAAGGCT
GACTTCGCTTCCTTGGAAAAGTACGCTTTGGAAAAGGGTGAAGTCACCGC
TTCTTTGTCCTCTGGTAGACAAGAAATGTTGGAATCCATCGTTAACAACG
TCTTGTTCTCTTTGTAA
[0329] Separately, the gene encoding xylose isomerase from
Piromyces strain E2 was synthesized by IDT DNA, Inc. The sequence
of this gene is set forth as SEQ ID NO: 34.
TABLE-US-00039 SEQ ID NO: 34
ACTAGTAAAAAAATGGCTAAGGAATATTTCCCACAAATTCAAAAGATTAA
GTTCGAAGGTAAGGATTCTAAGAATCCATTAGCCTTCCACTACTACGATG
CTGAAAAGGAAGTCATGGGTAAGAAAATGAAGGATTGGTTACGTTTCGCC
ATGGCCTGGTGGCACACTCTTTGCGCCGAAGGTGCTGACCAATTCGGTGG
AGGTACAAAGTCTTTCCCATGGAACGAAGGTACTGATGCTATTGAAATTG
CCAAGCAAAAGGTTGATGCTGGTTTCGAAATCATGCAAAAGCTTGGTATT
CCATACTACTGTTTCCACGATGTTGATCTTGTTTCCGAAGGTAACTCTAT
TGAAGAATACGAATCCAACCTTAAGGCTGTCGTTGCTTACCTCAAGGAAA
AGCAAAAGGAAACCGGTATTAAGCTTCTCTGGAGTACTGCTAACGTCTTC
GGTCACAAGCGTTACATGAACGGTGCCTCCACTAACCCAGACTTTGATGT
TGTCGCCCGTGCTATTGTTCAAATTAAGAACGCCATAGACGCCGGTATTG
AACTTGGTGCTGAAAACTACGTCTTCTGGGGTGGTCGTGAAGGTTACATG
AGTCTCCTTAACACTGACCAAAAGCGTGAAAAGGAACACATGGCCACTAT
GCTTACCATGGCTCGTGACTACGCTCGTTCCAAGGGATTCAAGGGTACTT
TCCTCATTGAACCAAAGCCAATGGAACCAACCAAGCACCAATACGATGTT
GACACTGAAACCGCTATTGGTTTCCTTAAGGCCCACAACTTAGACAAGGA
CTTCAAGGTCAACATTGAAGTTAACCACGCTACTCTTGCTGGTCACACTT
TCGAACACGAACTTGCCTGTGCTGTTGATGCTGGTATGCTCGGTTCCATT
GATGCTAACCGTGGTGACTACCAAAACGGTTGGGATACTGATCAATTCCC
AATTGATCAATACGAACTCGTCCAAGCTTGGATGGAAATCATCCGTGGTG
GTGGTTTCGTTACTGGTGGTACCAACTTCGATGCCAAGACTCGTCGTAAC
TCTACTGACCTCGAAGACATCATCATTGCCCACGTTTCTGGTATGGATGC
TATGGCTCGTGCTCTTGAAAACGCTGCCAAGCTCCTCCAAGAATCTCCAT
ACACCAAGATGAAGAAGGAACGTTACGCTTCCTTCGACAGTGGTATTGGT
AAGGACTTTGAAGATGGTAAGCTCACCCTCGAACAAGTTTACGAATACGG
TAAGAAGAACGGTGAACCAAAGCAAACTTCTGGTAAGCAAGAACTCTACG
AAGCTATTGTTGCCATGTACCAATAGTAGCTCGAG
[0330] The amino acid sequence of the xylose isomerase from
Piromyces strain E2 is set for in SEQ ID NO. 35
TABLE-US-00040 SEQ ID NO. 35
MAKEYFPQIQKIKFEGKDSKNPLAFHYYDAEKEVMGKKMKDWLRFAMAWW
HTLCAEGADQFGGGTKSFPWNEGTDAIEIAKQKVDAGFEIMQKLGIPYYC
FHDVDLVSEGNSIEEYESNLKAVVAYLKEKQKETGIKLLWSTANVFGHKR
YMNGASTNPDFDVVARAIVQIKNAIDAGIELGAENYVFWGGREGYMSLLN
TDQKREKEHMATMLTMARDYARSKGFKGTFLIEPKPMEPTKHQYDVDTET
AIGFLKAHNLDKDFKVNIEVNHATLAGHTFEHELACAVDAGMLGSIDANR
GDYQNGWDTDQFPIDQYELVQAWMEIIRGGGFVTGGTNFDAKTRRNSTDL
EDIIIAHVSGMDAMARALENAAKLLQESPYTKMKKERYASFDSGIGKDFE
DGKLTLEQVYEYGKKNGEPKQTSGKQELYEAIVAMYQ
[0331] For detection purposes, each gene was PCR amplified using a
3' oligonucleotide that added a 6-HIS tag (SEQ ID NO: 138) onto the
c-terminal end of each xylose isomerase gene. The oligonucleotides
used for this purpose are set forth below.
TABLE-US-00041 Gene Primer Name Primer Sequence XI-R-hotrod 4329
On63 for agttaagtgagtaaactagtgaattccagagaaaataaaacattgtttacaataga
(SEQ ID NO. 36) 4329-HIS REV
agtcaagtctcgagtcaatggtgatggtggtgatgcagagaaaataaaacattgtttac (SEQ ID
NO. 37) XI-R-native KAS/5-XI-RF-NATIVE
actagtatggaatttttcagcaatatcggtaaaattc (SEQ ID NO. 38)
KAS/3-XI-RF-NATIVE-HIS ctcgagttacagactgaaaagaacgttatttacg (SEQ ID
NO. 39) XI-R-coop KAS/5-XI-RF-COOP actagtatggaattcttctctaacattgg
(SEQ ID NO. 40) KAS/3-XI-RF-COOP-HIS
ctcgagttacaaagagaacaagacgttgttaacgatgg (SEQ ID NO. 41) XI-P
XI-P_Native FL 5' actagtaaaaaaatggctaaggaatatttcccacaaattcaaaag
(SEQ ID NO. 42) XI-P_Native FL 3'His-tag
atgactcgagctactaatgatgatgatgatgatgttggtacatggcaacaatagcttcg (SEQ ID
NO. 43)
[0332] Each xylose isomerase gene described above (plus or minus
the HIS tag) was cloned into the yeast expression vector p426GPD
(Mumberg et al., 1995, Gene 156: 119-122; obtained from ATCC
#87361; PubMed: 7737504) using the SpeI and XhoI sites located at
the 5' and 3' ends of each gene. Each of the bacterial vectors
containing a xylose isomerase gene (with or without the 6-HIS
c-terminal tag (SEQ ID NO: 138)) and the p426GPD yeast expression
vector were digested with SpeI and XhoI. The generated fragments
were gel extracted using a Qiagen gel purification kit (Qiagen,
Valencia, Calif.), the p426GPD vector reaction was cleaned up using
a Qiagen PCR purification kit. About 30 ng of each fragment was
ligated to 50 ng of the p426GPD vector using T4 DNA ligase
(Fermentas, Glen Burnie, Md.) in a 10 .mu.l volume reaction
overnight at 16.degree. C. and transformed into NEB-5a competent
cells (NEB, Ipswich, Mass.) and plated onto LB media with
ampicillin (100 .mu.g/ml). Constructs were confirmed by sequence
analysis (GeneWiz, La Jolla, Calif.).
[0333] A haploid Saccharomyces cerevisiae strain (BY4742; ATCC
catalog number 201389) was cultured in YPD media (10 g Yeast
Extract, 20 g Bacto-Peptone, 20 g Glucose, 1 L total) at about
30.degree. C. Separate aliquots of these cultured cells were
transformed with a plasmid construct containing a xylose isomerase
gene or with vector alone. Transformation was accomplished using
the Zymo frozen yeast transformation kit (Catalog number T2001;
Zymo Research Corp., Orange, Calif. 92867). To about 50 .mu.l of
cells was added approximately 0.5-1 .mu.g plasmid DNA and the cells
were cultured on SC drop out media with glucose minus uracil (about
20 g glucose; about 2.21 g SC drop-out mix [described below], about
6.7 g yeast nitrogen base, all in about 1 L of water); this mixture
was cultured for 2-3 days at about 30.degree. C.
[0334] SC drop-out mix contained the following ingredients (Sigma);
all indicated weights are approximate:
TABLE-US-00042 0.4 g Adenine hemisulfate 3.5 g Arginine 1 g
Glutamic Acid 0.433 g Histidine 0.4 g Myo-Inositol 5.2 g Isoleucine
2.63 g Leucine 0.9 g Lysine 1.5 g Methionine 0.8 g Phenylalanine
1.1 g Serine 1.2 g Threonine 0.8 g Tryptophan 0.2 g Tyrosine 1.2 g
Valine
[0335] For expression and activity analysis, cultures expressing
the various xylose isomerase wild type and variant gene constructs
were grown in about 100ml SC-Dextrose (2%) at about 30.degree. C.
to an OD600 of about 4.0.
[0336] S. cerevisiae cultures were lysed using YPER-Plus reagent
(Thermo Scientific, San Diego, Calif.; catalog number 78999)
according to the manufacturer's instructions. Quantification of the
lysates was performed using the Coomassie-Plus kit (Thermo
Scientific, San Diego, Calif.; Catalog number 23236) as directed by
the manufacturer. About 5-10 .mu.g of total cell extract was used
for SDS-gel [NuPage 4-12% Bis-Tris gels (Life Technologies,
Carlsbad, Calif.)] and native gel electrophoresis and for native
Western blot analyses.
[0337] SDS-PAGE gels were run according to the manufacturer's
recommendation using NuPage MES-SDS Running Buffer at 1.times.
concentration with the addition of NuPage antioxidant into the
cathode chamber at a 1.times. concentration. Novex Sharp Protein
Standards (Life Technologies, Carlsbad, Calif.) were used as
standards. For Western analysis, gels were transferred onto a
nitrocellulose membrane (0.45 micron, Thermo Scientific, San Diego,
Calif.) using Western blotting filter paper (Thermo Scientific)
using a Bio-Rad Mini Trans-Blot Cell (BioRad, Hercules, Calif.)
system for approximately 90 minutes at 40V. Following transfer, the
membrane was washed in 1.times.PBS (EMD, San Diego, Calif.), 0.05%
Tween-20 (Fisher Scientific, Fairlawn, N.J.) for 2-5 minutes with
gentle shaking. The membrane was blocked in 3% BSA dissolved in
1.times.PBS and 0.05% Tween-20 at room temperature for about 2
hours with gentle shaking. The membrane was washed once in
1.times.PBS and 0.05% Tween-20 for about 5 minutes with gentle
shaking. The membrane was then incubated at room temperature with
the 1:5000 dilution of primary antibody (Ms mAB to 6.times.His Tag
(SEQ ID NO: 138), AbCam, Cambridge, Mass.) in 0.3% BSA (Fraction V,
EMD, San Diego, Calif.) dissolved in 1.times.PBS and 0.05% Tween-20
with gentle shaking. Incubation was allowed to proceed for about 1
hour with gentle shaking. The membrane was then washed three times
for 5 minutes each with 1.times.PBS and 0.05% Tween-20 with gentle
shaking. The secondary antibody [Dnk pAb to Ms IgG (HRP), AbCam,
Cambridge, Mass.] was used at 1:15000 dilution in 0.3% BSA and
allowed to incubate for about 90 minutes at room temperature with
gentle shaking. The membrane was washed three times for about 5
minutes using 1.times.PBS and 0.05% Tween-20 with gentle shaking.
The membrane was then incubated with 5 ml of Supersignal West Pico
Chemiluminescent substrate (Thermo Scientific, San Diego, Calif.)
for 1 minute and then was exposed to a phosphorimager (Bio-Rad
Universal Hood II, Bio-Rad, Hercules, Calif.) for about 10-100
seconds.
[0338] The results are shown in FIG. 7. As can be seen, the wild
type R. flavefaciens xylose isomerase gene protein and the wild
type Piromyces xylose isomerase gene are both expressed in the
soluble fraction of the cells. The expected size of the xylose
isomerase R. flavefaciens polypeptide is approximately 49.8
kDa.
Example 10
In Vitro Xylose Isomerase Activity Assays
[0339] Enzyme assays of the various xylose isomerase variants were
performed according to Kuyper et al. (FEMS Yeast Res., 4:69 [2003])
with a few modifications. Approximately 20 .mu.g of soluble whole
cell extract from each transformed cell line, prepared using Y-PER
plus reagent as described above, was incubated in a solution
containing about 100 mM Tris, pH 7.5, 10 mM MgCl-2, 0.15 mM NADH
(Sigma, St. Louis, Mo.), and 2 U Sorbitol Dehydrogenase (SDH)
(Roche, Indianapolis, Ind.) at about 30.degree. C. To start the
reaction, about 100 .mu.l of xylose was added at various final
concentrations of about 40 to about 500 mM. A Beckman DU-800
spectrophotometer was utilized with an Enzyme Mechanism software
package (Beckman Coulter, Inc, Brea, Calif.), and the change in the
A340 was monitored for 2-3 minutes. Assays were repeated as
described above in the absence and in the presence of about 0 to
about 50 mM xylitol, an inhibitor of xylose isomerase, in order to
determine the K.sub.i. Regular assays (no xylitol) were done
independently about 5 to 10 times over the entire range of xylose
concentrations and 2 times in the presence of the entire range of
xylitol concentrations. The results are set forth in the table
below.
TABLE-US-00043 Specific Activity Enzyme K.sub.m (mM) (.mu.mol min-1
mg-1) K.sub.i (mM xylitol) Piromyces xylose 49 2.02 4.79 isomerase
Ruminococcus 82.12 1.718 43.64 wild type xylose isomerase
Ruminococcus 103 1.31 ND "hot rod" xylose isomerase Ruminococcus
100 1.61 ND "codon optimized" xylose isomerase
Example 11
Construction of Ruminococcus Xylose Isomerase Chimeric Variants
[0340] Several xylose isomerase gene variants were designed and
constructed in which 6 adenosine bases were added to each variant
directly 5' of the ATG "start" codon. Additionally, 15, 20, 45 or
60 base pairs of the 5' end of the Ruminococcus xylose isomerase
gene were replaced with various portions of the 5' end of the
Piromyces xylose isomerase gene to create "chimeric" or "hybrid"
xylose isomerase genes. Diagrams representative of non-limiting
xylose isomerase chimeric variant gene embodiments is shown in FIG.
8.
[0341] PCR amplification was used to generate novel chimeric
constructs. For all PCR reactions, approximately 0.2 .mu.mol of
each oligonucleotide was added to 25-30 ng of the appropriate
purified DNA template with 0.2 mM dNTPs (5' and 3'), 1.times.Pfu
Ultra II buffer and 1 unit (U) Pfu Ultra II polymerase (Agilent).
The PCR reactions were thermocycled as follows; 95.degree. C. for
10 minutes, followed by 30 cycles of 95.degree. C. for 10 sec,
58.degree. C. for 30 sec, and 72.degree. C. for 30 seconds. A 5
minute 72.degree. C. extension reaction completed the amplification
rounds.
TABLE-US-00044 Oligo Name Sequence 5' Oligonucleotides: KAS/XI-R-
actagtaaaaaaATGGAATTTTTCAGCAATATCGGTA 6A AAATTC (SEQ ID NO. 44)
KAS/XI-R- actagtaaaaaaatggctaaggaatatTTCAGCAATA P1-5 TCGGTAAAATTCAG
(SEQ ID NO. 45) KAS/XI-R- ttcccacaaattcaaAAAATTCAGTATCAGGGACCAA
P6-10 AAAG (SEQ ID NO. 46) KAS/XI-R-
ACTAGTaaaaaaatggctaaggaatatttcccacaaa P1-10
ttcaaAAAATTCAGTATCAGGGACCAAAAAG (SEQ ID NO. 47) KAS/XI-R-
aagattaagttcgaaGGACCAAAAAGTACTGATCCTC P11-15 TCTC (SEQ ID NO. 48)
KAS/XI-R- ttcccacaaattcaaaagattaagttcgaaGGACCAA P6-15
AAAGTACTGATCCTCTCTC (SEQ ID NO. 49) KAS/XI-R-
ACTAGTaaaaaaatggctaaggaatatttcccacaaa P1-15 ttcaaaagattaagttc (SEQ
ID NO. 50) KAS/XI-R- ggtaaggattctaagGATCCTCTCTCATTTAAGTACT P16-20
ATAACCCTG (SEQ ID NO. 51) KAS/XI-R-
caaaagattaagttcgaaggtaaggattctaagGATC P10-20 CTCTCTCATTTAAGTAC (SEQ
ID NO. 52) 3'-Oligonucleotides: KAS/3-XI-RF- (SEQ ID NO. 53)
ctcgagttacagactgaaaag NATIVE aacgttatttacg KAS/3-XI-RF- (SEQ ID NO.
54) ctcgagttacagactgaaaag NATIVE-HIS aacgttatttacg
[0342] The novel XI-R constructs were generated using PCR with the
relevant primers and template gene. The 5' primer KAS/XI-R-6A and
either 3' primer KAS/3-XI-RF-NATIVE or 3' primer
KAS/3-XI-RF-NATIVE-HIS were used in combination with the full
length native Ruminococcus xylose isomerase (XI-R) gene to generate
the constructs referred to as "XI-Rf-6A" and "XI-Rf-6AHis".
[0343] To generate the chimeric XI-Rp5 gene, the 5' primer
KAS/XI-R-P1-5 and either 3' primer KAS/3-XI-RF-NATIVE or 3' primer
KAS/3-XI-RF-NATIVE-HIS were used in combination with the full
length native xylose isomerase Ruminococcus gene. The chimeric
XI-Rp5 gene includes the first 5 amino acids of the Piromyces
xylose isomerase (XI-P) polypeptide followed by amino acids 6 to
1323 of the native Ruminococcus xylose isomerase.
[0344] To generate the chimeric XI-Rp10 gene, the 5' primer
KAS/XI-R-P6-10 and 3' primer KAS/3-XI-RF-NATIVE were first used to
add nucleotides 16-30 from the XI-P gene to the 5' end of the XI-R
gene keeping the remainder of the XI-R gene in-frame. The chimeric
XI-Rp10 gene includes the first 10 amino acids of the Piromyces
xylose isomerase followed by amino acids 11 to 438 of the
Ruminococcus xylose isomerase. Following PCR purification of the
resulting XI-Rp6-10 amplified product, 5' primer KAS/XI-R-P1-10 and
either the 3' primer KAS/3-XI-RF-NATIVE or the 3' primer
KAS/3-XI-RF-NATIVE-HIS oligonucleotides were used to add additional
sequences.
[0345] To generate the chimeric XI-Rp15 gene, the 5' primer
KAS/XI-R-P11-15 and 3' primer KAS/3-XI-RF-NATIVE were first used on
the XI-Rp10 construct to add nucleotides 16 to 45 from the XI-P
gene to the 5' end of the XI-R native gene. The chimeric XI-Rp15
gene includes the first 15 amino acid of the Piromyces xylose
isomerase followed by amino acids 16 to 438 of the Ruminococcus
xylose isomerase. Following PCR purification of the resulting
XI-Rp6-15 amplified product, 5' primer KAS/XI-R-P1-15 either 3'
primer KAS/3-XI-RF-NATIVE or 3' primer KAS/3-XI-RF-NATIVE-HIS
oligonucleotides were used to add additional sequences.
[0346] To generate the chimeric XI-Rp20 gene, the 5' primer
KAS/XI-R-P10-20 and 3' primer KAS/3-XI-RF-NATIVE were used on the
XI-Rp15 construct to add nucleotides 30-60 to the 5' end of the
XI-R native gene. The chimeric XI-Rp20 gene includes the first 20
amino acids of the Piromyces xylose isomerase followed by amino
acids 21 to 438 of the Ruminococcus xylose isomerase. Following PCR
purification of the resulting XI-Rp10-20 amplified product, 5'
primer KAS/XI-R-P1-15 and either 3' primer KAS/3-XI-RF-NATIVE or 3'
primer KAS/3-XI-RF-NATIVE-HIS oligonucleotides were used to add
additional sequences.
[0347] Each of the novel chimeric xylose isomerase genes (with and
without the c-terminal 6-HIS tag (SEQ ID NO: 138)) were cloned into
the bacterial cloning vector pCR Blunt II TOPO (Life Technologies,
Carlsbad, Calif.) according to the manufacturer's recommendations.
Following sequence verification (GeneWiz, La Jolla, Calif.), the
approximate 1330 bp SpeI-XhoI fragment from each construct was
subcloned into the yeast expression vector p426GPD by first
extracting each fragment from a gel slice using a gel purification
kit (Qiagen, Valencia, Calif.), and then preparing the p426GPD
vector for ligation by purifying it using a PCR purification kit
(Qiagen, Valencia, Calif.), according to the manufacturer's
instructions. About 30 ng of each of the chimeric genes was
separately ligated to about 50 ng of the p426GPD vector using T4
DNA ligase (Fermentas, Glen Burnie, Md.) in a 10 .mu.l volume
reaction overnight at about 16.degree. C., followed by
transformation using standard protocols into NEB-5.alpha. competent
cells (NEB, Ipswich, Mass.). The transformed cell culture was
plated onto LB media with ampicillin (100 .mu.g/ml). The constructs
containing the chimeric genes were confirmed by sequence analysis
(GeneWiz, La Jolla, Calif.).
[0348] A haploid Saccharomyces cerevisiae strain (BY4742; ATCC
catalog number 201389) was cultured in YPD media (10 g Yeast
Extract, 20 g Bacto-Peptone, 20 g Glucose, 1 L total) at about
30.degree. C. Separate aliquots of these cultured cells were
transformed with plasmid constructs containing the novel xylose
isomerase chimeric genes as well as with the Piromyces and
Ruminococcus native gene constructs described herein.
Transformation was performed using the Zymo frozen yeast
transformation kit (Catalog number T2001; Zymo Research Corp.,
Orange, Calif. 92867). Approximately about 0.5 .mu.g to about 1
.mu.g plasmid DNA was added to about 50 .mu.l of cells, and the
transformed cells were cultured on SC drop out media with glucose
minus uracil (e.g., about 20 g glucose; about 2.21 g SC drop-out
mix], about 6.7 g yeast nitrogen base, per 1 L of water) for 2-3
days at about 30.degree. C.
[0349] S. cerevisiae cultures were lysed using YPER-Plus reagent
(Thermo Scientific, San Diego, Calif.; catalog number 78999)
according to the manufacturer's instructions. Quantification of the
lysates was performed using the Coomassie-Plus kit (Thermo
Scientific, San Diego, Calif., catalog number 23236) as directed by
the manufacturer. About 5 to 10 .mu.g of total cell extract was
used for SDS-gel (NuPage 4-12% Bis-Tris gels, Life Technologies,
Carlsbad, Calif.) and native gel electrophoresis and Western blot
analyses. SDS-PAGE gels were run according to the manufacturer's
recommendation using NuPage MES-SDS Running Buffer (Life
Technologies, Carlsbad, Calif.) at 1.times. concentration with the
addition of NuPage antioxidant to the cathode chamber at a 1.times.
concentration. Novex Sharp Protein Standards (Life Technologies,
Carlsbad, Calif.) were used as standards.
[0350] For Western analysis, gels were transferred onto a
nitrocellulose membrane (0.45 micron, Thermo Scientific, San Diego,
Calif.) using Western blotting filter paper (Thermo Scientific)
using a Bio-Rad Mini Trans-Blot Cell (BioRad, Hercules, Calif.)
system for approximately 90 minutes at 40V. Following transfer, the
membrane was washed in 1.times.PBS (EMD, San Diego, Calif.), 0.05%
Tween-20 (Fisher Scientific, Fairlawn, N.J.) for 2-5 minutes with
gentle shaking. The membrane was blocked in about 3% BSA dissolved
in 1.times.PBS and 0.05% Tween-20 at room temperature for about 2
hours with gentle shaking. The membrane was washed once in
1.times.PBS and 0.05% Tween-20 for about 5 minutes with gentle
shaking. The membrane was then incubated at room temperature with
an approximately 1:5000 dilution of primary antibody (anti-His
mouse monoclonal antibody , AbCam, Cambridge, Mass.) in about 0.3%
BSA (Fraction V, EMD, San Diego, Calif.) dissolved in 1.times.PBS
and 0.05% Tween-20 with gentle shaking. Incubation was allowed to
proceed for about 1 hour with gentle shaking. The membrane was then
washed three times for about 5 minutes each with 1.times.PBS and
0.05% Tween-20 with gentle shaking. The secondary antibody (donkey
anti mouse IgG polyclonal antibody linked to horse radish
peroxidase, AbCam, Cambridge, Mass.) was used at about a 1:15000
dilution in 0.3% BSA and allowed to incubate for about 90 minutes
at room temperature with gentle shaking. The membrane was washed
three times for about 5 minutes each time using 1.times.PBS and
0.05% Tween-20 with gentle shaking. The membrane was then incubated
with 5 ml of Supersignal West Pico Chemiluminescent substrate
(Thermo Scientific, San Diego, Calif.) for 1 minute and then was
exposed to a phosphorimager (Bio-Rad Universal Hood II, Bio-Rad,
Hercules, Calif.) for about 10-100 seconds. The expected size of
the chimeric xylose isomerase constructs is approximately 49.8 kDa.
The expected size of the xylose isomerase protein is approximately
50.2 kDa. The results of Western blot analysis are shown in FIG. 9.
For each protein 2 lanes were run: T=total protein from whole cell
extract, S=soluble portion of the whole cell extract.
[0351] As can be seen in FIG. 9, adding 6 adenosine bases directly
upstream of the 5' end of the xylose isomerase Ruminococcus wild
type gene did not improve expression of the polypeptide. However,
replacing the 5' end of the Ruminococcus wild type xylose isomerase
gene with 15, 30 or 45 of the 5' base pairs of the Piromyces wild
type xylose isomerase gene improved expression of the enzyme.
[0352] Enzyme assays of the various novel xylose isomerase chimeric
polypeptides were performed according to Kuyper et al. (FEMS Yeast
Res., 4:69 [2003]) with a few modifications as described above.
Approximately 20 .mu.g of soluble whole cell extract from each
transformed cell line was prepared using Y-PER plus reagent as
described above was incubated in a solution containing about 100 mM
Tris, pH 7.5, 10 mM MgCl.sub.2, 0.15 mM NADH (Sigma, St. Louis,
Mo.), and 2 U sorbitol dehydrogenase (SDH) (Roche, Indianapolis,
Ind.) at about 30.degree. C. To start the reaction, about 100 .mu.l
of xylose was added at various final concentrations of 40-500 mM. A
Beckman DU-800 spectrophotometer was utilized with an Enzyme
Mechanism software package (Beckman Coulter, Inc, Brea, Calif.),
and the change in the A340 was monitored for 2-3 minutes. The
results of the assays are set forth in the table below.
TABLE-US-00045 K.sub.m Specific Activity (.mu.mol K.sub.i (mM
Enzyme (mM) min-1 mg-1) xylitol) XI-P 49 2.02 4.79 XI-Rnative 82.12
1.72 43.64 XI-Rp5 66.5 1.45 ND XI-Rp10 75.8 2.10 ND XI-Rp15 49.3
1.53 ND
[0353] The results indicate that substituting 30 base pairs of DNA
from the 5' end of the Ruminococcus xylose isomerase gene with the
first 15 base pairs of the Piromyces wild type xylose isomerase
gene increased both the specific activity and the expression level
to a level comparable to that of the wild type Piromyces xylose
isomerase. The DNA and amino acid sequence for each chimeric gene
is set forth below as SEQ ID NOs. 55 to 62. Small, bold "a"
nucleotides indicated the 6 added adenosines. Large capital bold
"A, T, G or C" nucleotides indicate the portion of the chimeric
sequences donated by Piromyces and combined with the Ruminococcus
sequence (e.g., small non-bold nucleotides).
TABLE-US-00046 SEQ ID NO. 55: XI-Rp5 DNA
aaaaaaATGGCTAAGGAATATTTCAGCAATATCGGTAAAATTCAGTATCAGGGACCAAAAA
GTACTGATCCTCTCTCATTTAAGTACTATAACCCTGAAGAAGTCATCAACGGAAAGACAATGC
GCGAGCATCTGAAGTTCGCTCTTTCATGGTGGCACACAATGGGCGGCGACGGAACAGATAT
GTTCGGCTGCGGCACAACAGACAAGACCTGGGGACAGTCCGATCCCGCTGCAAGAGCAAA
GGCTAAGGTTGACGCAGCATTCGAGATCATGGATAAGCTCTCCATTGACTACTATTGTTTCC
ACGATCGCGATCTTTCTCCCGAGTATGGCAGCCTCAAGGCTACCAACGATCAGCTTGACATA
GTTACAGACTATATCAAGGAGAAGCAGGGCGACAAGTTCAAGTGCCTCTGGGGTACAGCAA
AGTGCTTCGATCATCCAAGATTCATGCACGGTGCAGGTACATCTCCTTCTGCTGATGTATTC
GCTTTCTCAGCTGCTCAGATCAAGAAGGCTCTGGAGTCAACAGTAAAGCTCGGCGGTAACG
GTTACGTTTTCTGGGGCGGACGTGAAGGCTATGAGACACTTCTTAATACAAATATGGGACTC
GAACTCGACAATATGGCTCGTCTTATGAAGATGGCTGTTGAGTATGGACGTTCGATCGGCTT
CAAGGGCGACTTCTATATCGAGCCCAAGCCCAAGGAGCCCACAAAGCATCAGTACGATTTC
GATACAGCTACTGTTCTGGGATTCCTCAGAAAGTACGGTCTCGATAAGGATTTCAAGATGAA
TATCGAAGCTAACCACGCTACACTTGCTCAGCATACATTCCAGCATGAGCTCCGTGTTGCAA
GAGACAATGGTGTGTTCGGTTCTATCGACGCAAACCAGGGCGACGTTCTTCTTGGATGGGA
TACAGACCAGTTCCCCACAAATATCTACGATACAACAATGTGTATGTATGAAGTTATCAAGGC
AGGCGGCTTCACAAACGGCGGTCTCAACTTCGACGCTAAGGCACGCAGAGGGAGCTTCACT
CCCGAGGATATCTTCTACAGCTATATCGCAGGTATGGATGCATTTGCTCTGGGCTTCAGAGC
TGCTCTCAAGCTTATCGAAGACGGACGTATCGACAAGTTCGTTGCTGACAGATACGCTTCAT
GGAATACCGGTATCGGTGCAGACATAATCGCAGGTAAGGCAGATTTCGCATCTCTTGAAAAG
TATGCTCTTGAAAAGGGCGAGGTTACAGCTTCACTCTCAAGCGGCAGACAGGAAATGCTGG
AGTCTATCGTAAATAACGTTCTTTTCAGTCTGTAA SEQ ID NO. 56: XI-Rp5
Polypeptide
MAKEYFSNIGKIQYQGPKSTDPLSFKYYNPEEVINGKTMREHLKFALSWWHTMGGDGT
DMFGCGTTDKTWGQSDPAARAKAKVDAAFEIMDKLSIDYYCFHDRDLSPEYGSLKATN
DQLDIVTDYIKEKQGDKFKCLWGTAKCFDHPRFMHGAGTSPSADVFAFSAAQIKKALES
TVKLGGNGYVFWGGREGYETLLNTNMGLELDNMARLMKMAVEYGRSIGFKGDFYIEP
KPKEPTKHQYDFDTATVLGFLRKYGLDKDFKMNIEANHATLAQHTFQHELRVARDNGV
FGSIDANQGDVLLGWDTDQFPTNIYDTTMCMYEVIKAGGFTNGGLNFDAKARRGSFTP
EDIFYSYIAGMDAFALGFRAALKLIEDGRIDKFVADRYASWNTGIGADIIAGKADFASLEK
YALEKGEVTASLSSGRQEMLESIVNNVLFSL SEQ ID NO. 57: XI-Rp10 DNA
aaaaaaaTGGCTAAGGAATATTTCCCACAAATTCAACAGTATCAGGGACCAAAAAGTACT
GATCCTCTCTCATTTAAGTACTATAACCCTGAAGAAGTCATCAACGGAAAGACAATGCGCGA
GCATCTGAAGTTCGCTCTTTCATGGTGGCACACAATGGGCGGCGACGGAACAGATATGTTC
GGCTGCGGCACAACAGACAAGACCTGGGGACAGTCCGATCCCGCTGCAAGAGCAAAGGCT
AAGGTTGACGCAGCATTCGAGATCATGGATAAGCTCTCCATTGACTACTATTGTTTCCACGAT
CGCGATCTTTCTCCCGAGTATGGCAGCCTCAAGGCTACCAACGATCAGCTTGACATAGTTAC
AGACTATATCAAGGAGAAGCAGGGCGACAAGTTCAAGTGCCTCTGGGGTACAGCAAAGTGC
TTCGATCATCCAAGATTCATGCACGGTGCAGGTACATCTCCTTCTGCTGATGTATTCGCTTTC
TCAGCTGCTCAGATCAAGAAGGCTCTGGAGTCAACAGTAAAGCTCGGCGGTAACGGTTACG
TTTTCTGGGGCGGACGTGAAGGCTATGAGACACTTCTTAATACAAATATGGGACTCGAACTC
GACAATATGGCTCGTCTTATGAAGATGGCTGTTGAGTATGGACGTTCGATCGGCTTCAAGGG
CGACTTCTATATCGAGCCCAAGCCCAAGGAGCCCACAAAGCATCAGTACGATTTCGATACAG
CTACTGTTCTGGGATTCCTCAGAAAGTACGGTCTCGATAAGGATTTCAAGATGAATATCGAA
GCTAACCACGCTACACTTGCTCAGCATACATTCCAGCATGAGCTCCGTGTTGCAAGAGACAA
TGGTGTGTTCGGTTCTATCGACGCAAACCAGGGCGACGTTCTTCTTGGATGGGATACAGAC
CAGTTCCCCACAAATATCTACGATACAACAATGTGTATGTATGAAGTTATCAAGGCAGGCGG
CTTCACAAACGGCGGTCTCAACTTCGACGCTAAGGCACGCAGAGGGAGCTTCACTCCCGAG
GATATCTTCTACAGCTATATCGCAGGTATGGATGCATTTGCTCTGGGCTTCAGAGCTGCTCT
CAAGCTTATCGAAGACGGACGTATCGACAAGTTCGTTGCTGACAGATACGCTTCATGGAATA
CCGGTATCGGTGCAGACATAATCGCAGGTAAGGCAGATTTCGCATCTCTTGAAAAGTATGCT
CTTGAAAAGGGCGAGGTTACAGCTTCACTCTCAAGCGGCAGACAGGAAATGCTGGAGTCTA
TCGTAAATAACGTTCTTTTCAGTCTGTAA SEQ ID NO. 58: XI-Rp10 Polypeptide
MAKEYFPQIQQYQGPKSTDPLSFKYYNPEEVINGKTMREHLKFALSWWHTMGGDGTD
MFGCGTTDKTWGQSDPAARAKAKVDAAFEIMDKLSIDYYCFHDRDLSPEYGSLKATND
QLDIVTDYIKEKQGDKFKCLWGTAKCFDHPRFMHGAGTSPSADVFAFSAAQIKKALEST
VKLGGNGYVFWGGREGYETLLNTNMGLELDNMARLMKMAVEYGRSIGFKGDFYIEPK
PKEPTKHQYDFDTATVLGFLRKYGLDKDFKMNIEANHATLAQHTFQHELRVARDNGVF
GSIDANQGDVLLGWDTDQFPTNIYDTTMCMYEVIKAGGFTNGGLNFDAKARRGSFTPE
DIFYSYIAGMDAFALGFRAALKLIEDGRIDKFVADRYASWNTGIGADIIAGKADFASLEKY
ALEKGEVTASLSSGRQEMLESIVNNVLFSL SEQ ID NO. 59: XI-Rp15 DNA
aaaaaaaTGGCTAAGGAATATTTCCCACAAATTCAAAAGATTAAGTTCGAAAAAAGTA
CTGATCCTCTCTCATTTAAGTACTATAACCCTGAAGAAGTCATCAACGGAAAGACAATGCGC
GAGCATCTGAAGTTCGCTCTTTCATGGTGGCACACAATGGGCGGCGACGGAACAGATATGT
TCGGCTGCGGCACAACAGACAAGACCTGGGGACAGTCCGATCCCGCTGCAAGAGCAAAGG
CTAAGGTTGACGCAGCATTCGAGATCATGGATAAGCTCTCCATTGACTACTATTGTTTCCAC
GATCGCGATCTTTCTCCCGAGTATGGCAGCCTCAAGGCTACCAACGATCAGCTTGACATAGT
TACAGACTATATCAAGGAGAAGCAGGGCGACAAGTTCAAGTGCCTCTGGGGTACAGCAAAG
TGCTTCGATCATCCAAGATTCATGCACGGTGCAGGTACATCTCCTTCTGCTGATGTATTCGC
TTTCTCAGCTGCTCAGATCAAGAAGGCTCTGGAGTCAACAGTAAAGCTCGGCGGTAACGGTT
ACGTTTTCTGGGGCGGACGTGAAGGCTATGAGACACTTCTTAATACAAATATGGGACTCGAA
CTCGACAATATGGCTCGTCTTATGAAGATGGCTGTTGAGTATGGACGTTCGATCGGCTTCAA
GGGCGACTTCTATATCGAGCCCAAGCCCAAGGAGCCCACAAAGCATCAGTACGATTTCGAT
ACAGCTACTGTTCTGGGATTCCTCAGAAAGTACGGTCTCGATAAGGATTTCAAGATGAATAT
CGAAGCTAACCACGCTACACTTGCTCAGCATACATTCCAGCATGAGCTCCGTGTTGCAAGAG
ACAATGGTGTGTTCGGTTCTATCGACGCAAACCAGGGCGACGTTCTTCTTGGATGGGATACA
GACCAGTTCCCCACAAATATCTACGATACAACAATGTGTATGTATGAAGTTATCAAGGCAGG
CGGCTTCACAAACGGCGGTCTCAACTTCGACGCTAAGGCACGCAGAGGGAGCTTCACTCCC
GAGGATATCTTCTACAGCTATATCGCAGGTATGGATGCATTTGCTCTGGGCTTCAGAGCTGC
TCTCAAGCTTATCGAAGACGGACGTATCGACAAGTTCGTTGCTGACAGATACGCTTCATGGA
ATACCGGTATCGGTGCAGACATAATCGCAGGTAAGGCAGATTTCGCATCTCTTGAAAAGTAT
GCTCTTGAAAAGGGCGAGGTTACAGCTTCACTCTCAAGCGGCAGACAGGAAATGCTGGAGT
CTATCGTAAATAACGTTCTTTTCAGTCTGTAA SEQ ID NO. 60: XI-Rp15 Polypeptide
MAKEYFPQIQKIKFEKSTDPLSFKYYNPEEVINGKTMREHLKFALSWWHTMGGDGTDM
FGCGTTDKTWGQSDPAARAKAKVDAAFEIMDKLSIDYYCFHDRDLSPEYGSLKATNDQ
LDIVTDYIKEKQGDKFKCLWGTAKCFDHPRFMHGAGTSPSADVFAFSAAQIKKALESTV
KLGGNGYVFWGGREGYETLLNTNMGLELDNMARLMKMAVEYGRSIGFKGDFYIEPKP
KEPTKHQYDFDTATVLGFLRKYGLDKDFKMNIEANHATLAQHTFQHELRVARDNGVFG
SIDANQGDVLLGWDTDQFPTNIYDTTMCMYEVIKAGGFTNGGLNFDAKARRGSFTPED
IFYSYIAGMDAFALGFRAALKLIEDGRIDKFVADRYASWNTGIGADIIAGKADFASLEKYA
LEKGEVTASLSSGRQEMLESIVNNVLFSL SEQ ID NO. 61: XI-Rp20 DNA
aaaaaaTGGCTAAGGAATATTTCCCACAAATTCAAAAGATTAAGTTCGAAGGTAAG
GATTCTAAGCTCTCATTTAAGTACTATAACCCTGAAGAAGTCATCAACGGAAAGACAATGC
GCGAGCATCTGAAGTTCGCTCTTTCATGGTGGCACACAATGGGCGGCGACGGAACAGATAT
GTTCGGCTGCGGCACAACAGACAAGACCTGGGGACAGTCCGATCCCGCTGCAAGAGCAAA
GGCTAAGGTTGACGCAGCATTCGAGATCATGGATAAGCTCTCCATTGACTACTATTGTTTCC
ACGATCGCGATCTTTCTCCCGAGTATGGCAGCCTCAAGGCTACCAACGATCAGCTTGACATA
GTTACAGACTATATCAAGGAGAAGCAGGGCGACAAGTTCAAGTGCCTCTGGGGTACAGCAA
AGTGCTTCGATCATCCAAGATTCATGCACGGTGCAGGTACATCTCCTTCTGCTGATGTATTC
GCTTTCTCAGCTGCTCAGATCAAGAAGGCTCTGGAGTCAACAGTAAAGCTCGGCGGTAACG
GTTACGTTTTCTGGGGCGGACGTGAAGGCTATGAGACACTTCTTAATACAAATATGGGACTC
GAACTCGACAATATGGCTCGTCTTATGAAGATGGCTGTTGAGTATGGACGTTCGATCGGCTT
CAAGGGCGACTTCTATATCGAGCCCAAGCCCAAGGAGCCCACAAAGCATCAGTACGATTTC
GATACAGCTACTGTTCTGGGATTCCTCAGAAAGTACGGTCTCGATAAGGATTTCAAGATGAA
TATCGAAGCTAACCACGCTACACTTGCTCAGCATACATTCCAGCATGAGCTCCGTGTTGCAA
GAGACAATGGTGTGTTCGGTTCTATCGACGCAAACCAGGGCGACGTTCTTCTTGGATGGGA
TACAGACCAGTTCCCCACAAATATCTACGATACAACAATGTGTATGTATGAAGTTATCAAGGC
AGGCGGCTTCACAAACGGCGGTCTCAACTTCGACGCTAAGGCACGCAGAGGGAGCTTCACT
CCCGAGGATATCTTCTACAGCTATATCGCAGGTATGGATGCATTTGCTCTGGGCTTCAGAGC
TGCTCTCAAGCTTATCGAAGACGGACGTATCGACAAGTTCGTTGCTGACAGATACGCTTCAT
GGAATACCGGTATCGGTGCAGACATAATCGCAGGTAAGGCAGATTTCGCATCTCTTGAAAAG
TATGCTCTTGAAAAGGGCGAGGTTACAGCTTCACTCTCAAGCGGCAGACAGGAAATGCTGG
AGTCTATCGTAAATAACGTTCTTTTCAGTCTGTAA SEQ ID NO. 62: XI-Rp20
Polypeptide
MAKEYFPQIQKIKFEGKDSKLSFKYYNPEEVINGKTMREHLKFALSWWHTMGGDGTDM
FGCGTTDKTWGQSDPAARAKAKVDAAFEIMDKLSIDYYCFHDRDLSPEYGSLKATNDQ
LDIVTDYIKEKQGDKFKCLWGTAKCFDHPRFMHGAGTSPSADVFAFSAAQIKKALESTV
KLGGNGYVFWGGREGYETLLNTNMGLELDNMARLMKMAVEYGRSIGFKGDFYIEPKP
KEPTKHQYDFDTATVLGFLRKYGLDKDFKMNIEANHATLAQHTFQHELRVARDNGVFG
SIDANQGDVLLGWDTDQFPTNIYDTTMCMYEVIKAGGFTNGGLNFDAKARRGSFTPED
IFYSYIAGMDAFALGFRAALKLIEDGRIDKFVADRYASWNTGIGADIIAGKADFASLEKYA
LEKGEVTASLSSGRQEMLESIVNNVLFSL
Example 12
Production of Additional Xylose Isomerase Variants
[0354] A series of specific point mutations were made to the "hot
rod" Ruminococcus xylose isomerase gene using site-directed
mutagenesis. The particular point mutations that were generated are
set forth in the table below.
W136F
F184S
G179A
G179A F184S
W136F G179A F184S
W1361 G179A F184S
W136S G179A F184S
F87L W136F G179A F184S
F87M W136F G179A F184S
F87L W136F G179A F184S V214G
G179S F184A
F85S W136F G179A F184A V214G Q273T
F85S W136F G179A F184A V214 D257A
W136F G179A F184A
W136F G179A F184S D257E
[0355] Site directed mutagenesis was performed as follows: About 50
ng of template DNA was added to 1.times.Pfu Ultra II buffer, 0.3 mM
dNTPs, 0.3 .mu.mol of the relevant mutagenesis primers depending on
the mutant being constructed and 1 U Pfu Ultra II polymerase
(Agilent, La Jolla, Calif.) in a 50 .mu.l reaction mix. The
sequence of the mutagenesis primers used is set forth in the table
below. The "hot rod" Ruminococcus xylose isomerase gene was used as
the template DNA for constructing single point mutation variants.
Previously engineered mutants sometimes were used as "template" DNA
to generate other mutants. The sequence of the oligonucleotides
used to prepare each mutant is indicated in the table below. Each
reaction was PCR cycled as follows: 95.degree. C. 10 minutes
followed by 30 rounds of 95.degree. C. for 20 seconds, 55.degree.
C. for 30 seconds, and 72.degree. C. for 5 minutes. A final 5
minute extension reaction at 72.degree. C. was also included.
Following PCR, 1.5 .mu.l of DpnI (NEB, Ipswich, Mass.) was added
and allowed to digest the reaction mixture for 1 to 1.5 hours at
37.degree. C. 5 .mu.l of this mixture was then used to transform
NEB-5.alpha. cells (NEB, Ipswich, Mass.) and plated onto LB media
with ampicillin (100 .mu.g/ml). (Table below discloses SEQ ID NOS
229-256, respectively, in order of appearance)
TABLE-US-00047 GGCGACAAGTTCAAGTGCCTCTTCGGTACAGCAAAG W136F_Forward
CTTTGCTGTACCGAAGAGGCACTTGAACTTGTCGCC W136F_Reverse
TCGGCGGTAACGGTTACGTTAGCTGGGGCGGAC F184S_Forward
GTCCGCCCCAGCTAACGTAACCGTTACCGCCGA F184S_Reverse
AACAGTAAAGCTCGGCGCTAACGGTTACGTTTTCT G179A_Forward
AGAAAACGTAACCGTTAGCGCCGAGCTTTACTGTT G179A_Reverse
AACAGTAAAGCTCGGCGCTAACGGTTACGTTAGCTGGGGCGGAC G179A-F184S_Forward
GTCCGCCCCAGCTAACGTAACCGTTAGCGCCGA G179A-F184S_Reverse
GCTAAGGTTGACGCAGCAATGGAGATCATGGATAAGCTC F85M_Forward
GAGCTTATCCATGATCTCCATTGCTGCGTCAACCTTAGC F85M_Reverse
CTAAGGTTGACGCAGCATTAGAGATCATGGATAAGCTC F85L_Forward
GAGCTTATCCATGATCTCTAATGCTGCGTCAACCTTAG F85L_Reverse
AGCTAACCACGCTACACTTGCTACGCATACATTCCAGCATG Q273T_Forward
CATGCTGGAATGTATGCGTAGCAAGTGTAGCGTGGTTAGCT Q273T_Reverse
GCGACAAGTTCAAGTGCCTCATAGGTACAGCAAAGTGCTTCGA W136I_Forward
TCGAAGCACTTTGCTGTACCTATGAGGCACTTGAACTTGTCGC W136I_Reverse
GCGACAAGTTCAAGTGCCTCTCGGGTACAGCAA W136S_Forward
TTGCTGTACCCGAGAGGCACTTGAACTTGTCGC W136S_Reverse
GCTAACCACGCTACACTTGCTGGTCATACATTCCAGCAT Q273G_Forward
ATGCTGGAATGTATGACCAGCAAGTGTAGCGTGGTTAGC Q273G_Reverse
CCTCAGAAAGTACGGTCTCGCTAAGGATTTCAAGATGAATA D257A_Forward
TATTCATCTTGAAATCCTTAGCGAGACCGTACTTTCTGAGG D257A_Reverse
CCTCAGAAAGTACGGTCTCGAGAAGGATTTCAAGATGAATATC D257E_Forward
GATATTCATCTTGAAATCCTTCTCGAGACCGTACTTTCTGAGG D257E_Reverse
GTCTTATGAAGATGGCTGGTGAGTATGGACGTTCGAT V214G Forward
ATCGAACGTCCATACTCACCAGCCATCTTCATAAGAC V214G Reverse
GTCAACAGTAAAGCTCGGCAGTAACGGTTACGTTAGCTGG G179S_Forward
CCAGCTAACGTAACCGTTACTGCCGAGCTTTACTGTTGAC G179S_Reverse
[0356] Following sequence verification (GeneWiz, La Jolla, Calif.),
the approximate 1330 bp SpeI-XhoI fragment from each construct was
subcloned into the yeast expression vector p426GPD by first gel
extracting each fragment using a Qiagen gel purification kit
(Qiagen, Valencia, Calif.), and then preparing the p426GPD vector
for ligation by purifying it using a Qiagen PCR purification kit
according to the manufacturer's instructions. About 30 ng of each
of the chimeric genes was separately ligated to about 50 ng of the
p426GPD vector using T4 DNA ligase followed by transformation using
known protocols into NEB-5.alpha. competent cells (NEB, Ipswich,
Mass.). The transformed cells were plated onto LB media with
ampicillin (100 .mu.g/ml). Constructs containing the chimeric genes
were confirmed by sequence analysis (GeneWiz, La Jolla,
Calif.).
[0357] A haploid Saccharomyces cerevisiae strain (BY4742; ATCC
catalog number 201389) was cultured in YPD media (10 g Yeast
Extract, 20 g Bacto-Peptone, 20 g Glucose, 1 L total) at about
30.degree. C. Separate aliquots of these cultured cells were
transformed with a plasmid constructs containing the novel xylose
isomerase chimeric genes as well as with the Piromyces and
Ruminococcus native gene constructs made above. Transformation was
accomplished using the Zymo frozen yeast transformation kit
(Catalog number T2001; Zymo Research Corp., Orange, Calif. 92867).
To about 50 .mu.l of cells was added approximately 0.5-1 .mu.g
plasmid DNA and the cells were cultured on SC drop out media with
glucose minus uracil (about 20 g glucose; about 2.21 g SC drop-out
mix, about 6.7 g yeast nitrogen base, all in about 1 L of water);
this mixture was cultured for 2-3 days at about 30.degree. C.
[0358] Assays of the various novel xylose isomerase point mutation
polypeptides were performed according to Kuyper et al. (FEMS Yeast
Res., 4:69 [2003]) with a few modifications as described above.
Approximately 20 .mu.g of soluble whole cell extract from each
transformed cell line was prepared using Y-PER plus reagent as
described above was incubated in a solution containing about 100 mM
Tris, pH 7.5, 10 mM MgCl2, 0.15 mM NADH (Sigma, St. Louis, Mo.),
and 2 U Sorbitol Dehydrogenase (SDH) (Roche, Indianapolis, Ind.) at
about 30.degree. C. To start the reaction, about 1000 of xylose was
added at various final concentrations of 40-500 mM. A Beckman
DU-800 spectrophotometer was utilized with an Enzyme Mechanism
software package (Beckman Coulter, Inc, Brea, Calif.), and the
change in the A.sub.340 was monitored for 2-3 minutes. The results
of the assays are shown in FIG. 10.
[0359] Mutant G179A had the highest activity as compared to the
Ruminococcus wild type xylose isomerase, as shown in FIG. 10. The
kinetics of the G179A mutant were further analyzed using kinetic
assays described herein, adding various concentrations of xylose
ranging from about 40 to about 500 mM. The results of the kinetic
assays, shown below, for the G179A mutant illustrate that the
mutant xylose isomerase activity has a higher specific activity
than the Piromyces xylose isomerase.
TABLE-US-00048 Km Specific Activity Enzyme (mM) (.mu.mol min-1
mg-1) Piromyces xylose 49 2.02 isomerase Ruminococcus 82.12 1.718
xylose isomerase wild type Ruminococcus 83.82 2.24 xylose isomerase
G179A
Example 13
In Vivo Evaluation of Xylose Isomerase Constructs
[0360] The yeast strain BY4742 was specifically engineered to more
readily utilize xylose as a carbon source. The engineered strain
was designed to include the following genetic modifications: the
native Pho13 gene (alkaline phosphatase specific for p-nitrophenyl
phosphate) was disrupted by inserting a construct containing the
native TLK1 gene (Transketolase-1); the native aldose reductase
gene (Gre3) was disrupted by inserting a construct containing the
native high-affinity glucose transporter-7 gene (HXT7); the native
glucose-repressible alcohol dehydrogenase II gene (adh2) was
disrupted by inserting a construct containing the native
xylulokinase gene (XYLK); and the native orotidine-5' phosphate
decarboxylase gene (ura3) was disrupted by inserting a construct
containing the native transaldolase 1 gene (TAD). The resulting
strain had the following genotype: pho13::TKL1, gre3::HXT7,
adh2::XYLK, ura3::TAL1. The final strain is referred to as the "C5"
strain and was used for in vivo evaluation of the xylose isomerase
variants.
[0361] The C5 strain was transformed using standard protocols with
either p426GPD (as a control) or the chimeric variants XI-R,
XI-Rp5, XI-Rp10, or XI-Rp15. The transformed cells were grown on
SC-glucose minus uracil initially and then passaged onto SC-xylose
minus uracil. Cultures of each of the above constructs were made in
SC-xylose minus uracil--and grown for one week. The cultures were
grown aerobically at 30.degree. C. with 250 rpm agitation, 1 vvm
sparge of process air, 21% O2. The pH was controlled at about 5.0
with 1N NaOH. Ethanol, glucose and xylose concentrations in the
fermentation broth were monitored by a YSI 2700 BioAnalyzer during
aerobic fermentation. At 24 hours elapsed fermentation time the
fermentation was switched to anaerobic conditions. Before changing
to anaerobic conditions, samples were taken to measure ethanol,
glucose and xylose concentrations, and biomass was measured by
OD600 nm and dry cell weight. At the start of anaerobic
fermentation, 4 ml/L of 2.5 g/L ergosterol in EtOH, 0.4 ml/L Tween
80, and 0.01% AF-204 were added to each fermentor. Oxygen was
purged with 100% N2 sparge at 1 vvm until percent O2 was below 1%.
Aeration was then set at 0.25 vvm 100% N2.
[0362] Samples were taken every 24 to 48 hours and measured for
ethanol concentration, glucose concentration, xylose concentration,
and cell density (OD600 nm). The fermentation was harvested when
xylose concentration was below 4 g/L in the XI-R strains, at 372
hours after commencing fermentation. The final sample also measured
biomass by dry cell weight. The results are presented in the table
below.
TABLE-US-00049 Dry Cell Ethanol Glucose Xylose Strain OD.sub.600 nm
Wt (g/L) (g/L) (g/L) (g/L) XI-R 7.72 2.20 15.65 0 3.14 Vector 7.34
1.78 6.705 0 23.21 XI-R 7.32 2.03 15.5 0 3.85 Vector 7.96 1.87 9.91
0 23.11
[0363] The data presented indicate that the Ruminococcus xylose
isomerase containing yeast cells were able to utilize xylose as a
carbon source, and the cells containing vector only (e.g., vector
with no xylose isomerase gene) utilized very little xylose.
Industrial Yeast Strain Evaluation
[0364] To evaluate the activity of the various native, modified and
engineered (e.g., mutant and/or chimeric) Ruminococcus xylose
isomerases in a commercial yeast strain, the Ruminococcus wild type
gene or Ruminococcus Rp10 and Rp15 chimeric constructs were
inserted into a yeast vector containing a 2.mu. origin and a KANMX4
(G418R) cassette (cloned from vector HO-poly-KANMX4-HO; ATCC Cat.
No. 87804; Voth et al., 2001 NAR 29(12): e59, DDBJ/EMBL/GenBank
accession nos. AF324723-9). A commercially available industrial
diploid strain of Saccharomyces cerevisiae (strain BF903;
"Stillspirits" triple distilled yeast, Brewcraft, Albany, New
Zealand; available at Hydrobrew, Oceanside, Calif.) was obtained
and was made competent for transformation using known yeast cell
transformation procedures. The transformed cells containing either
vector alone or the various Ruminococcus xylose isomerase gene
constructs were passaged in YPD medium containing about 100
.mu.g/ml G418 (EMD, San Diego, Calif.), and about 2% glucose.
Transformed yeast containing each construct were grown overnight
aerobically in a 15 ml culture tube on YPD media containing 2%
glucose. After about 24 hours, about 25 ml of YP media containing
2% glucose and 100 .mu.g/ml G418 was seeded with the cells at an
initial OD.sub.600 of 0.5 in a 250 ml Erlenmeyer flask and grown
aerobically at 30.degree. C. The cultures were then passaged once
every 7 days into fresh media, also at an initial OD.sub.600 of
0.5. The fresh media contained increasing amounts of xylose and
decreasing amounts of glucose as set forth below.
TABLE-US-00050 Week Glucose Xylose 1 1% 1% 2 0.50% 1.50% 3 0.25%
1.75% 4 0.10% 1.90%
[0365] Measurements were taken of the cell optical density
(OD.sub.600) to assess cell density and plated onto YPD with 100
.mu.g/ml G418 to ensure that the plasmid was stable. Glucose,
xylose and ethanol in the culture media were assayed using YSI 2700
Bioanalyzer instruments (World Wide Web uniform resource locator
ysi.com), according to the manufacturer's recommendations. The
strains were then grown overnight in YPD (with 100 .mu.g/ml G418)
and used to inoculate about 50 ml YP-xylose (with 100 .mu.g/ml
G418) into disposable 250 ml Erlenmeyer flasks with vented caps at
an initial OD.sub.600 of about 1. The cultures were allowed to grow
aerobically at 30.degree. C. at 200 rpm for 7 days. The results are
shown in FIG. 11. The results shown in FIG. 11 indicate that the
commercial yeast strain expressing the Ruminococcus xylose
isomerase is more efficient at consuming xylose than the strain
carrying the vector control only.
[0366] To evaluate ethanol production, the transformed cells
containing either the vector control or the gene encoding native
Ruminococcus xylose isomerase were grown overnight in YP glucose
(with 100 .mu.g/ml G418) and then used to inoculate serum bottles
containing 50 ml YP plus xylose (with 100 .mu.g/ml G418) at an
initial OD.sub.600 of about 1. The serum bottles were sealed with a
butyl rubber stopper to prevent air exchange. As a result, the
cultures became anaerobic once the available oxygen in the serum
bottle was utilized. In general, anaerobiosis (e.g., the onset of
anaerobic conditions) occurred a few hours after the culture was
inoculated. Xylose utilization, ethanol production and cell growth
were measured every twenty four hours. The results are shown in
FIG. 12. The results suggest that yeast strains containing the
Ruminococcus xylose isomerase, grew to a higher cell density and
produced more ethanol using xylose as a carbon source than did
cells carrying the vector only control (e.g., see FIG. 12).
Example 14
High Diversity Library of Xylose Isomerase of Variants
[0367] To generate additional Ruminococcus xylose isomerase
variants, a high diversity library of mutants was generated using
known molecular biology procedures. The library contained the
combinations and permutations of substitutions listed in the table
below. The Ruminococcus xylose isomerase variants listed below and
highlighted in boldface type have been transformed into yeast
strains, and are evaluated for growth and ethanol production on
xylose media utilizing protocols described above. Yeast
transformation of the variants listed below not highlighted in
boldface is conducted and resulting variants are tested. In the
table below "position" refers to the amino acid position in the
Ruminococcus xylose isomerase amino acid sequence, "AA1" refers to
the first of the considered amino acid substitutions for that
position, "CODON1" refers to the nucleotide sequence selected for
the amino acid chosen in "AA1", "AA2" refers to the second of the
considered amino acid substitutions for that position, "CODON2"
refers to the nucleotide sequence selected for the amino acid
chosen in "AA2", "AA3" refers to the third of the considered amino
acid substitutions for that position, "CODON3" refers to the
nucleotide sequence selected for the amino acid chosen in "AA3",
"AA4" refers to the fourth of the considered amino acid
substitutions for that position and "CODON4" refers to the
nucleotide sequence selected for the amino acid chosen in
"AA4".
TABLE-US-00051 High Diversity Library of Xylose Isomerase of
Variants # Position AA1 CODON1 AA2 CODON2 AA3 CODON3 AA4 CODON4
substitutions LIB1-A LIB1-B 3 F TTT Y TAT 2 1 1 45 L TTG M ATG 2 1
1 46 S TCT A GCT 2 1 1 51 M ATG L TTG 2 1 1 52 G GGT C TGT 2 1 1 53
G GGT A GCT 2 1 1 58 M ATG Q CAA 2 1 1 85 F TTT M ATG L TTG 3 1 3
101 R AGA V GTT 2 1 1 107 Y TAT G GGT 2 2 2 121 T ACT V GTT 2 2 2
131 K AAA G GGT 2 2 2 136 W TGG F TTT 2 2 1 140 K AAA N AAT 2 2 2
147 F TTT Y TAT 2 2 2 179 G GGT R AGA A GCT 3 1 3 184 F TTT S TCT 2
1 1 204 D GAT E GAA 2 2 2 214 V GTT R AGA G GGT 3 1 1 257 D GAT A
GCT E GAA 3 1 1 273 Q CAA F TTT T ACT G GGT 4 1 4 292 I ATT V GTT 2
2 2 296 Q CAA R AGA 2 1 1 345 T ACT D GAT E GAA 3 3 3 373 D GAT E
GAA 2 2 2 509607936 1536 27648 Number of possible variants:
5.096e+08 Expected completeness: 0.95 Required library size:
1.527e+09
[0368] The xylose isomerase mutants listed above are generated
using oligonucleotides listed below and a 3 step PCR method,
described in further detail below.
[0369] (Table below discloses SEQ ID NOS 257-336, respectively, in
order of appearance)
TABLE-US-00052 KAS/XI-LIB-for-1
ctagaactagtaaaaaaatggctaaggaatattattctaatataggtaaaattcagtat
KAS/XI-LIB-for-2 Actatgagagaacatttaaaatttgctatgtcttggtggcatactwt
KAS/XI-LIB-for-3 Agagaacatttaaaatttgctttggcttggtggcatactwtgkgtg
KAS/XI-LIB-for-4
Actatgagagaacatttaaaatttgctatggcttggtggcatactwtgkgtg
KAS/XI-LIB-for-5 Ttgctttgtcttggtggcatactttgkgtgstgatg
KAS/XI-LIB-for-6 Cttggtggcatactatgtgtgstgatggtactgats
KAS/XI-LIB-for-7 Gtggcatactatgggtgctgatggtactgatatgt
KAS/XI-LIB-for-8 Gtggcatactwtgkgtgctgatggtactgatcaat
KAS/XI-LIB-for-9 Gcatactwtgkgtgstgatggtactgatcaattcggttgtggtact
KAS/XI-LIB-for-10
gcaaaagccaaagtagatgcagccwtggaaattatggataaattgtctattg
KAS/XI-LIB-for-11
ttatggataaattgtctattgattattattgttttcatgatgttgatttgtctcctgaatatggttctttaaa-
ag KAS/XI-LIB-for-12
ttatggataaattgtctattgattattattgttttcatgatgttgatttgtctcctgaaggtggttctttaaa-
ag KAS/XI-LIB-for-13
gttttcatgatagagatttgtctcctgaaggtggttctttaaaagcaactaatg
KAS/XI-LIB-for-14
gttctttaaaagcaactaatgatcaattggacattgttgttgattatattaaagaaaaacaaggtgataaatt
taaatg KAS/XI-LIB-for-15
gttctttaaaagcaactaatgatcaattggacattgttgttgattatattaaagaaaaacaaggtgatggttt
taaatg KAS/XI-LIB-for-16
cggattatattaaagaaaaacaaggtgatggttttaaatgtttgtkkggcactgcgaawt
KAS/XI-LIB-for-17
ttatattaaagaaaaacaaggtgataaatttaaatgtttgtttggcactgcgaawtgttttgat
KAS/XI-LIB-for-18 Gtttgtggggcactgcgaattgttttgatcatcc
KAS/XI-LIB-for-19 Ttttgatcatccacgttatatgcatggtgcgggga
KAS/XI-LIB-for-20 Ggaatcaactgttaaattaggtagaaacgggtatgtattctgggga
KAS/XI-LIB-for-21 Gaatcaactgttaaattaggtgctaacgggtatgtattctggggag
KAS/XI-LIB-for-22 Ggaatcaactgttaaattaggtagaaacgggtatgtatcttgggga
KAS/XI-LIB-for-23 Gaatcaactgttaaattaggtgctaacgggtatgtatcttggggag
KAS/XI-LIB-for-24 Aaattaggtgggaacgggtatgtatcttggggaggaaggg
KAS/XI-LIB-for-25 Cactaatatgggtttggaattggaaaatatggctagattgatgaaaatg
KAS/XI-LIB-for-26
ggataatatggctagattgatgaaaatggctagagaatacggaaggtcta
KAS/XI-LIB-for-27 Gctagattgatgaaaatggctggtgaatacggaaggtctattggtt
KAS/XI-LIB-for-28
cagttttgggattcttgagaaaatatggtttggctaaagattttaaaatgaatatagaagcta
KAS/XI-LIB-for-29
agttttgggattcttgagaaaatatggtttggaaaaagattttaaaatgaatatagaagctaa
KAS/XI-LIB-for-30
atagaagctaatcatgcaacactcgcatttcatacttttcaacatgaattgagagtt
KAS/XI-LIB-for-31
atagaagctaatcatgcaacactcgcaactcatacttttcaacatgaattgagagtt
KAS/XI-LIB-for-32
agaagctaatcatgcaacactcgcaggtcatacttttcaacatgaattgagag
KAS/XI-LIB-for-33 Taacggagtttttggatctgttgatgcaaaccagggagacg
KAS/XI-LIB-for-34 Taacggagtttttggatctgttgatgcaaacagaggagacg
KAS/XI-LIB-for-35 Ttttggatctatcgatgcaaacagaggagacgttttgctaggatggg
KAS/XI-LIB-for-36 Aaggctaggcgtggtagtttcgatccagaggatatattctattc
KAS/XI-LIB-for-37 Cgaaggctaggcgtggtagtttcgaaccagaggatatattctattctta
KAS/XI-LIB-for-38 Cagggcagcactaaaattgattgaagaaggtagaattgataagtttg
KAS/XI-LIB-for-39 Tgggaaagccgacttcgccagtttggaaaaatatg
KAS/XI-LIB-rev-1
atactgaattttacctatattagaataatattccttagccatttttttactagttctag
KAS/XI-LIB-rev-2 Awagtatgccaccaagacatagcaaattttaaatgttctctcatagt
KAS/XI-LIB-rev-3 Cacmcawagtatgccaccaagccaaagcaaattttaaatgttctct
KAS/XI-LIB-rev-4
cacmcawagtatgccaccaagccatagcaaattttaaatgttctctcatagt
KAS/XI-LIB-rev-5 Catcascacmcaaagtatgccaccaagacaaagcaa
KAS/XI-LIB-rev-6 Satcagtaccatcascacacatagtatgccaccaag
KAS/XI-LIB-rev-7 Acatatcagtaccatcagcacccatagtatgccac
KAS/XI-LIB-rev-8 Attgatcagtaccatcagcacmcawagtatgccac
KAS/XI-LIB-rev-9 Agtaccacaaccgaattgatcagtaccatcascacmcawagtatgc
KAS/XI-LIB-rev-10
Caatagacaatttatccataatttccawggctgcatctactttggcttttgc
KAS/XI-LIB-rev-11
cttttaaagaaccatattcaggagacaaatcaacatcatgaaaacaataataatcaatagacaatttatccat-
aa KAS/XI-LIB-rev-12
cttttaaagaaccaccttcaggagacaaatcaacatcatgaaaacaataataatcaatagacaatttatccat-
aa KAS/XI-LIB-rev-13
cattagttgcttttaaagaaccaccttcaggagacaaatctctatcatgaaaac
KAS/XI-LIB-rev-14
catttaaatttatcaccttgtttttctttaatataatcaacaacaatgtccaattgatcattagttgctttta
aagaac KAS/XI-LIB-rev-15
catttaaaaccatcaccttgtttttctttaatataatcaacaacaatgtccaattgatcattagttgctttta
aagaac KAS/XI-LIB-rev-16
awttcgcagtgccmmacaaacatttaaaaccatcaccttgtttttctttaatataatccg
KAS/XI-LIB-rev-17
atcaaaacawttcgcagtgccaaacaaacatttaaatttatcaccttgtttttctttaatataa
KAS/XI-LIB-rev-18 Ggatgatcaaaacaattcgcagtgccccacaaac
KAS/XI-LIB-rev-19 Tccccgcaccatgcatataacgtggatgatcaaaa
KAS/XI-LIB-rev-20 Tccccagaatacatacccgtttctacctaatttaacagttgattcc
KAS/XI-LIB-rev-21 Ctccccagaatacatacccgttagcacctaatttaacagttgattc
KAS/XI-LIB-rev-22 Tccccaagatacatacccgtttctacctaatttaacagttgattcc
KAS/XI-LIB-rev-23 Ctccccaagatacatacccgttagcacctaatttaacagttgattc
KAS/XI-LIB-rev-24 Cccttcctccccaagatacatacccgttcccacctaattt
KAS/XI-LIB-rev-25 Cattttcatcaatctagccatattttccaattccaaacccatattagtg
KAS/XI-LIB-rev-26
Tagaccttccgtattctctagccattttcatcaatctagccatattatcc
KAS/XI-LIB-rev-27 Aaccaatagaccttccgtattcaccagccattttcatcaatctagc
KAS/XI-LIB-rev-28
tagcttctatattcattttaaaatctttagccaaaccatattttctcaagaatcccaaaactg
KAS/XI-LIB-rev-29
ttagcttctatattcattttaaaatctttttccaaaccatattttctcaagaatcccaaaact
KAS/XI-LIB-rev-30
aactctcaattcatgttgaaaagtatgaaatgcgagtgttgcatgattagcttctat
KAS/XI-LIB-rev-31
aactctcaattcatgttgaaaagtatgagttgcgagtgttgcatgattagcttctat
KAS/XI-LIB-rev-32
Ctctcaattcatgttgaaaagtatgacctgcgagtgttgcatgattagcttct
KAS/XI-LIB-rev-33 Cgtctccctggtttgcatcaacagatccaaaaactccgtta
KAS/XI-LIB-rev-34 Cgtctcctctgtttgcatcaacagatccaaaaactccgtta
KAS/XI-LIB-rev-35 Cccatcctagcaaaacgtctcctctgtttgcatcgatagatccaaaa
KAS/XI-LIB-rev-36 Gaatagaatatatcctctggatcgaaactaccacgcctagcctt
KAS/XI-LIB-rev-37 Taagaatagaatatatcctctggttcgaaactaccacgcctagccttcg
KAS/XI-LIB-rev-38 Caaacttatcaattctaccttcttcaatcaattttagtgctgccctg
KAS/XI-LIB-rev-39 Catatttttccaaactggcgaagtcggctttccca
KAS/FOR-XI-LIB Cctgaaattattcccctacttgact KAS/REV-XI-LIB
Ccttctcaagcaaggttttcagtat
[0370] The nucleotide sequences of the oligonucleotides above
include IUPAC nucleotide symbol designations, in some embodiments.
The IUPAC nucleotide symbol designations used in the listing above
and the nucleotides they represent are; m (A or C nucleotides), k
(G or T nucleotides), s (C or G nucleotides), w (A or T
nucleotides).
[0371] Oligonucleotides are prepared as 100 micromolar stocks to be
diluted as needed for use as PCR and/or primer extension primers.
Step 1 of the 3 step PCR protocol included initial primer extension
reactions performed four times, each with a different concentration
of mutant oligonucleotide (e.g., about 7.5 nanomolar, about 37.5
nanomolar, about 75 nanomolar, and about 150 nanomolar). An
appropriate amount (e.g., dependent on the reaction) of each of the
desired primers is contacted with Ruminococcus xylose isomerase
nucleotide sequences, under PCR/primer extension conditions to
generate the xylose isomerase mutant variants. The forward and
reverse primers listed above are designated with "-for-" or "-rev-"
as part of the primer name. A non-limiting example of the
PCR/primer extension conditions utilized for generating the xylose
isomerase mutant variants listed above include 200 micromolar of
each deoxyribonucleotide (dNTP), 1.times.Pfu ultra II buffer, and 1
unit Pfu ultra II polymerase and a thermocycle profile of; (a) an
initial 10 minute denaturation at 94.degree. C., (b) 40 cycles of
(i) 94.degree. C. for 20 seconds, (ii) 56.degree. C. for 30
seconds, and (iii) 72.degree. C. for 45 seconds, and (c) a final
extension at 72.degree. C. for 5 minutes. The initial extension
products are analyzed by gel electrophoresis on 1.2% Tris-acetate
agarose gels. The reactions are column purified and the resultant
purified nucleic acids are used for subsequent steps in the 3 step
PCR protocol.
[0372] The second step of the 3 step protocol includes contacting
the purified nucleic acids from the first step with Ruminococcus
xylose isomerase gene primers (e.g., KAS/FOR-XI-LIB and
KAS/REV-XI-LIB, as listed in the table above, under substantially
similar PCR/primer extension conditions, with the modification of 5
units of Pfu ultra II polymerase instead of 1 unit. The PCR
reactions also are performed four times, each with a differing
amount of gene primers (e.g., about 20 nanomolar, about 100
nanomolar, about 2 micromolar and about 5 micromolar. The reaction
products are analyzed by gel electrophoresis as described above and
column purified in preparation for the final step of the 3 step
PCR/primer extension protocol.
[0373] The final step of the 3 step protocol generated full length
nucleic acids of xylose isomerase mutants. The column purified
nucleic acid of the second step was contacted with about 200
nanomolar of each gene primer under extension conditions as
described for the second step. The protocol described herein was
used to generate a wide range of mutant xylose isomerase variants,
each with between about 1 and about 9 mutations per gene.
Example 15
Construction of Mutant G179A with p10 or p15 Extensions
[0374] Site directed mutagenesis was performed as follows: 50 ng of
the vector pBF348 (pCR Blunt 11/XI-R-P10), pBF370 (pCR Blunt
II/XI-R-P10-HIS), pBF349 (pCR Blunt II/XI-R-P15), or pBF370 (pCR
Blunt II/XI-R-P10-HIS) was added to 1.times.Pfu Ultra II buffer,
0.3 mM dNTPs, 0.3 .mu.mol mutagenesis primers [JML/5
(aacagtaaagctcggcgctaacggttacgttttct) and JML/6
(agaaaacgtaaccgttagcgccgagctttactgtt)], and 1 U Pfu Ultra II
polymerase (Agilent, La Jolla, Calif.) in a 50 .mu.l reaction mix.
This reaction mixture was cycled as follows: (a) 95.degree. C. 10
minutes, followed by (b) 30 rounds of (i) 95.degree. C. for 20
seconds, (ii) 55.degree. C. for 30 seconds, and (iii) 72.degree. C.
for 5 minutes. A final 5 minute extension reaction at 72.degree. C.
was also included. Following the cycling times, 1.5 .mu.l of DpnI
(NEB, Ipswich, Mass.) was added and allowed to digest the reaction
mixture for 1 to 1.5 hours at 37.degree. C. 5 .mu.l of this mixture
was then used to transform NEB-5.alpha. cells (NEB, Ipswich, Mass.)
and plated onto LB media with kanamycin (35 .mu.g/ml). The
following plasmids were generated using the procedure described
herein; pBF613 (XI-R-P15 with G179A mutation), pBF614 (XI-R-P15-HIS
with G179A mutation), pBF615 (XI-R-P10 with G179A mutation), and
pBF616 (XI-R-P10-HIS with G179A mutation), where XI=xylose
isomerase, R=Ruminococcus, P=Piromyces, HIS=Histidine Tag, and the
numbers that follow P indicate how many amino acids of the
Piromyces xylose isomerase was fused to the 5' end of the
Ruminococcus xylose isomerase gene.
[0375] Following sequence verification, the approximately 1330 base
pair SpeI-XhoI fragment from each construct was subcloned into the
yeast expression vector p426GPD. The generated xylose isomerase
fragments were first gel extracted using a Qiagen gel purification
kit (Qiagen, Valencia, Calif.), and the p426GPD vector reaction was
cleaned up using a Qiagen PCR purification kit. 30 ng of the
XI-fragments was ligated to 50 ng of the p426GPD vector using T4
DNA ligase (Fermentas, Glen Burnie, Md.) in a 10 .mu.l volume
reaction overnight at 16.degree. C. and transformed into
NEB-5.alpha. competent cells (NEB, Ipswich, Mass.) and plated onto
LB media with ampicillin (100 .mu.g/ml). Constructs were confirmed
by sequence analysis. The following plasmids were generated using
the procedure described herein: pBF677 (p426GPD/XI-R-P15_G179A),
pBF678 (p426GPD/XI-R-P15-HIS_G179A), pBF679
(p426GPD/XI-R-P10_G179A), pBF680 (p426GPD/XI-R-P10-HIS_G179A).
Example 16
Additional Xylose Isomerase High Diversity Variants
[0376] An additional library of high diversity mutants containing
changes not listed above also is generated. The table below lists
positions in the Ruminococcus xylose isomerase gene at which one of
two further amino acid codons is substituted to generate additional
high diversity xylose isomerase variants. In the table below "XI-R
position" refers to the amino acid position in the Ruminococcus
xylose isomerase amino acid sequence, "AA1" refers to the first of
two considered amino acid substitutions for that position, "CODON1"
refers to the nucleotide sequence selected for the amino acid
chosen in "AA1", "AA2" refers to the second of two considered amino
acid substitutions for that position and "CODON2" refers to the
nucleotide sequence selected for the amino acid chosen in "AA2".
The nucleotide sequences for each codon are chosen using sequence
and codon optimization methods described herein.
TABLE-US-00053 XI-R position AA1 CODON1 AA2 CODON2 5 S TCT P CCA 6
N AAT Q CAA 42 K AAA R AGA 54 D GAT E GAA 56 T ACT A GCT 84 A GCT G
GGT 137 G GGT S TCT 141 C TGT V GTT 180 N AAT E GAA 181 G GGT N AAT
203 L TTG K AAA 205 N AAT H CAT 208 R AGA T ACT 209 L TTG M ATG 210
M ATG L TTG 211 K AAA T ACT 215 E GAA D GAT 252 R AGA K AAA 253 K
AAA A GCT 254 Y TAT H CAT 255 G GGT N AAT 277 Q CAA E GAA 299 V GTT
Y TAT 300 L TTG Q CAA 301 L TTG N AAT 344 F TTT T ACT 346 P CCA L
TTG 372 E GAA Q CAA 374 G GGT S TCT 375 R AGA P CCA
Example 17
Activation of the Entner-Doudoroff Pathway in Yeast Cells Using EDD
and EDA Genes from Pseudomonas aeruginosa Strain PAO1
[0377] Pseudomonas aeruginosa strain PAO1 DNA was prepared using
Qiagen DNeasy Blood and Tissue kit (Qiagen, Valencia, Calif.)
according to the manufacture's instructions. The P. aeruginosa edd
and eda constructs were isolated from P. aeruginosa genomic DNA
using the following oligonucleotides:
TABLE-US-00054 The P. aeruginosa edd gene: (SEQ ID NO: 63)
5'-aactgaactgactagtaaaaaaatgcaccctcgtgtgctcgaagt- 3' (SEQ ID NO:
64) 5'-agtaaagtaaaagcttctactagcgccagccgttgaggctct-3' The P.
aeruginosa edd gene with 6-HIS c-terminal tag (SEQ ID NO: 138):
(SEQ ID NO 63) 5'-aactgaactgactagtaaaaaaatgcaccctcgtgtgctcgaagt- 3'
(SEQ ID NO: 65) 5'-agtaaagtaaaagcttctactaatgatgatgatgatgatggcgccag
ccgttgaggctc-3' The P. aeruginosa eda gene: (SEQ ID NO: 66)
5'-aactgaactgactagtaaaaaaatgcacaaccttgaacagaagacc- 3' (SEQ ID NO:
67) 5'-agtaaagtaactcgagctattagtgtctgcggtgctcggcgaa-3' The P.
aeruginosa eda gene with 6-HIS c-terminal tag (SEQ ID NO: 138):
(SEQ ID NO: 66) 5'-aactgaactgactagtaaaaaaatgcacaaccttgaacagaagacc-
3' (SEQ ID NO: 68)
5'-taaagtaactcgagctactaatgatgatgatgatgatggtgtctgcg
gtgctcggcgaa-3'
[0378] All oligonucleotides set forth above were purchased from
Integrated technologies ("IDT", Coralville, Iowa). These
oligonucleotides were designed to incorporate a SpeI restriction
endonuclease cleavage site upstream of a HindIII restriction
endonuclease cleavage site or downstream of an XhoI restriction
endonuclease cleavage site, with respect to the edd and eda gene
constructs. These restriction endonuclease sites could be used to
clone the edd and eda genes into yeast expression vectors p426GPD
(ATCC accession number 87361) and p425GPD (ATCC accession number
87359). In addition to incorporating restriction endonuclease
cleavage sites, the forward oligonucleotides also incorporate six
consecutive A nucleotides (e.g., AAAAAA) immediately upstream of
the ATG initiation codon. The six consecutive A nucleotides ensured
that there was a conserved ribosome binding sequence for efficient
translation initiation in yeast.
[0379] PCR amplification of the genes were performed as follows:
about 100 ng of the genomic P. aeruginosa PAO1 DNA was added to
1.times.Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3 .mu.mol
gene-specific primers (SEQ. ID. NOS: 63-68, and combinations as
indicated), and 1 U Pfu Ultra II polymerase (Agilent, La Jolla,
Calif.) in a 50 .mu.l reaction mix. This was cycled as follows:
95.degree. C. 10 minutes followed by 30 rounds of 95.degree. C. for
20 seconds, 50.degree. C. (eda amplifications) or 53.degree. C.
(edd amplifications) for 30 seconds, and 72.degree. C. for 15
seconds (eda amplifications) or 30 seconds (edd amplifications). A
final 5 minute extension reaction at 72.degree. C. also was
included. The about 670 bp (eda) or 1830 bp product (edd) was TOPO
cloned into the pCR Blunt II TOPO vector (Life Technologies,
Carlsbad, Calif.) according to the manufacturer's
recommendations.
[0380] The nucleotide and amino acid sequences of the P. aeruginosa
edd and eda genes are given below as SEQ ID NOS. 69-72.
TABLE-US-00055 P. aeruginosa edd nucleotide sequence: SEQ ID NO: 69
ATGCACCCTCGTGTGCTCGAAGTCACCCGCCGCATCCAGGCCCGTAGCGCGGCCACTCGCC
AGCGCTACCTCGAGATGGTCCGGGCTGCGGCCAGCAAGGGGCCGCACCGCGGCACCCTGC
CGTGCGGCAACCTCGCCCACGGGGTCGCGGCCTGTGGCGAAAGCGACAAGCAGACCCTGC
GGCTGATGAACCAGGCCAACGTGGCCATCGTTTCCGCCTACAACGACATGCTCTCGGCGCAC
CAGCCGTTCGAGCGCTTTCCGGGGCTGATCAAGCAGGCGCTGCACGAGATCGGTTCGGTCG
GCCAGTTCGCCGGCGGCGTGCCGGCCATGTGCGACGGGGTGACCCAGGGCGAGCCGGGCA
TGGAACTGTCGCTGGCCAGCCGCGACGTGATCGCCATGTCCACCGCCATCGCGCTGTCTCA
CAACATGTTCGATGCAGCGCTGTGCCTGGGTGTTTGCGACAAGATCGTGCCGGGCCTGCTGA
TCGGCTCGCTGCGCTTCGGCCACCTGCCCACCGTGTTCGTCCCGGCCGGGCCGATGCCGAC
CGGCATCTCCAACAAGGAAAAGGCCGCGGTGCGCCAACTGTTCGCCGAAGGCAAGGCCACT
CGCGAAGAGCTGCTGGCCTCGGAAATGGCCTCCTACCATGCACCCGGCACCTGCACCTTCTA
TGGCACCGCCAATACCAACCAGTTGCTGGTGGAGGTGATGGGCCTGCACTTGCCCGGTGCC
TCCTTCGTCAACCCGAACACCCCCCTGCGCGACGAACTCACCCGCGAAGCGGCACGCCAGG
CCAGCCGGCTGACCCCCGAGAACGGCAACTACGTGCCGATGGCGGAGATCGTCGACGAGAA
GGCCATCGTCAACTCGGTGGTGGCGCTGCTCGCCACCGGCGGCTCGACCAACCACACCCTG
CACCTGCTGGCGATCGCCCAGGCGGCGGGCATCCAGTTGACCTGGCAGGACATGTCCGAGC
TGTCCCATGTGGTGCCGACCCTGGCGCGCATCTATCCGAACGGCCAGGCCGACATCAACCA
CTTCCAGGCGGCCGGCGGCATGTCCTTCCTGATCCGCCAACTGCTCGACGGCGGGCTGCTT
CACGAGGACGTACAGACCGTCGCCGGCCCCGGCCTGCGCCGCTACACCCGCGAGCCGTTC
CTCGAGGATGGCCGGCTGGTCTGGCGCGAAGGGCCGGAACGGAGTCTCGACGAAGCCATC
CTGCGTCCGCTGGACAAGCCGTTCTCCGCCGAAGGCGGCTTGCGCCTGATGGAGGGCAACC
TCGGTCGCGGCGTGATGAAGGTCTCGGCGGTGGCGCCGGAACACCAGGTGGTCGAGGCGC
CGGTACGGATCTTCCACGACCAGGCCAGCCTGGCCGCGGCCTTCAAGGCCGGCGAGCTGGA
GCGCGACCTGGTCGCCGTGGTGCGTTTCCAGGGCCCGCGGGCGAACGGCATGCCGGAGCT
GCACAAGCTCACGCCGTTCCTCGGGGTCCTGCAGGATCGTGGCTTCAAGGTGGCGCTGGTC
ACCGACGGGCGCATGTCCGGGGCGTCGGGCAAGGTGCCCGCGGCCATCCATGTGAGTCCG
GAAGCCATCGCCGGCGGTCCGCTGGCGCGCCTGCGCGACGGCGACCGGGTGCGGGTGGAT
GGGGTGAACGGCGAGTTGCGGGTGCTGGTCGACGACGCCGAATGGCAGGCGCGCAGCCTG
GAGCCGGCGCCGCAGGACGGCAATCTCGGTTGCGGCCGCGAGCTGTTCGCCTTCATGCGCA
ACGCCATGAGCAGCGCGGAAGAGGGCGCCTGCAGCTTTACCGAGAGCCTCAACGGCTGGCG
CTAGTAG P. aeruginosa edd amino sequence: SEQ ID NO: 70
MHPRVLEVTRRIQARSAATRQRYLEMVRAAASKGPHRGTLPCGNLAHGVAACGESDKQTLRLMN
QANVAIVSAYNDMLSAHQPFERFPGLIKQALHEIGSVGQFAGGVPAMCDGVTQGEPGMELSLASR
DVIAMSTAIALSHNMFDAALCLGVCDKIVPGLLIGSLRFGHLPTVFVPAGPMPTGISNKEKAAVRQL
FAEGKATREELLASEMASYHAPGTCTFYGTANTNQLLVEVMGLHLPGASFVNPNTPLRDELTREA
ARQASRLTPENGNYVPMAEIVDEKAIVNSVVALLATGGSTNHTLHLLAIAQAAGIQLTWQDMSELS
HVVPTLARIYPNGQADINHFQAAGGMSFLIRQLLDGGLLHEDVQTVAGPGLRRYTREPFLEDGRLV
WREGPERSLDEAILRPLDKPFSAEGGLRLMEGNLGRGVMKVSAVAPEHQVVEAPVRIFHDQASLA
AAFKAGELERDLVAVVRFQGPRANGMPELHKLTPFLGVLQDRGFKVALVTDGRMSGASGKVPAAI
HVSPEAIAGGPLARLRDGDRVRVDGVNGELRVLVDDAEWQARSLEPAPQDGNLGCGRELFAFM
RNAMSSAEEGACSFTESLNGWR P. aeruginosa eda nucleotide sequence: SEQ
ID NO: 71
ATGCACAACCTTGAACAGAAGACCGCCCGCATCGACACGCTGTGCCGGGAGGCGCGCATCC
TCCCGGTGATCACCATCGACCGCGAGGCGGACATCCTGCCGATGGCCGATGCCCTCGCCGC
CGGCGGCCTGACCGCCCTGGAGATCACCCTGCGCACGGCGCACGGGCTGACCGCCATCCG
GCGCCTCAGCGAGGAGCGCCCGCACCTGCGCATCGGCGCCGGCACCGTGCTCGACCCGCG
GACCTTCGCCGCCGCGGAAAAGGCCGGGGCGAGCTTCGTGGTCACCCCGGGTTGCACCGA
CGAGTTGCTGCGCTTCGCCCTGGACAGCGAAGTCCCGCTGTTGCCCGGCGTGGCCAGCGCT
TCCGAGATCATGCTCGCCTACCGCCATGGCTACCGCCGCTTCAAGCTGTTTCCCGCCGAAGT
CAGCGGCGGCCCGGCGGCGCTGAAGGCGTTCTCGGGACCATTCCCCGATATCCGCTTCTGC
CCCACCGGAGGCGTCAGCCTGAACAATCTCGCCGACTACCTGGCGGTACCCAACGTGATGT
GCGTCGGCGGCACCTGGATGCTGCCCAAGGCCGTGGTCGACCGCGGCGACTGGGCCCAGG
TCGAGCGCCTCAGCCGCGAAGCCCTGGAGCGCTTCGCCGAGCACCGCAGACACTAATAG P.
aeruginosa eda amino sequence: SEQ ID NO: 72
MHNLEQKTARIDTLCREARILPVITIDREADILPMADALAAGGLTALEITLRTAHGLTAIRRLSEERPH
LRIGAGTVLDPRTFAAAEKAGASFVVTPGCTDELLRFALDSEVPLLPGVASASEIMLAYRHGYRRF
KLFPAEVSGGPAALKAFSGPFPDIRFCPTGGVSLNNLADYLAVPNVMCVGGTWMLPKAVVDRGD
WAQVERLSREALERFAEHRRH
[0381] Cloning of PAO1 edd and eda Genes into Yeast Expression
Vectors
[0382] Following sequence confirmation (GeneWiz), the about 670 bp
SpeI-XhoI eda and about 1830 bp SpeI-HindIII edd fragments were
cloned into the corresponding restriction sites in plasmids p425GPD
and p426GPD vectors (Mumberg et al., 1995, Gene 156: 119-122;
obtained from ATCC #87361; PubMed: 7737504), respectively. Briefly,
about 50 ng of SpeI-XhoI-digested p425GPD vector was ligated to
about 50 ng of SpeI/XhoI-restricted eda fragment in a 100 reaction
with 1.times.T4 DNA ligase buffer and 1 U T4 DNA ligase (Fermentas)
overnight at 16.degree. C. About 30 of this reaction was used to
transform DH5.alpha. competent cells (Zymo Research) and plated
onto LB agar media containing 100 .mu.g/ml ampicillin. Similarly,
about 50 ng of SpeI-HindIII-digested p426GPD vector was ligated to
about 42 ng of SpeI/HindIII-restricted edd fragment in a 100
reaction with 1.times.T4 DNA ligase buffer and 1 U T4 DNA ligase
(Fermentas) overnight at 16.degree. C. About 30 of this reaction
was used to transform DH5.alpha. competent cells (Zymo Research)
and plated onto LB agar media containing 100
.mu.g/mlampicillin.
[0383] A haploid Saccharomyces cerevisiae strain (BY4742; ATCC
catalog number 201389) was cultured in YPD media (10 g Yeast
Extract, 20 g Bacto-Peptone, 20 g Glucose, 1 L total) at about
30.degree. C. Separate aliquots of these cultured cells were
transformed with a plasmid construct(s) containing the eda gene
alone, the eda and edd genes, or with vector alone. Transformation
was accomplished using the Zymo frozen yeast transformation kit
(Catalog number T2001; Zymo Research Corp., Orange, Calif.). To 50
.mu.l of cells was added approximately 0.5-1 .mu.g plasmid DNA and
the cells were cultured on SC drop out media with glucose minus
leucine (eda), minus uracil and minus leucine (eda and edd) (about
20 g glucose; about 2.21 g SC drop-out mix [described below], about
6.7 g yeast nitrogen base, all in about 1 L of water); this mixture
was cultured for 2-3 days at about 30.degree. C. SC drop-out mix
contained the following ingredients (Sigma); all indicated weights
are approximate:
TABLE-US-00056 0.4 g Adenine hemisulfate 3.5 g Arginine 1 g
Glutamic Acid 0.433 g Histidine 0.4 g Myo-Inositol 5.2 g Isoleucine
2.63 g Leucine 0.9 g Lysine 1.5 g Methionine 0.8 g Phenylalanine
1.1 g Serine 1.2 g Threonine 0.8 g Tryptophan 0.2 g Tyrosine 0.2 g
Uracil 1.2 g Valine
[0384] Activity and Western Analyses
[0385] Cell lysates of the various EDD and EDA expressing strains
were prepared as follows. About 50 to 100 ml of SCD-ura-leu media
containing 10 mM MnCl2 was used to culture strains containing the
desired plasmid constructs. When cultured aerobically, strains were
grown in a 250 ml baffled shaker flask. When grown anaerobically,
400 .mu.l/L Tween-80 (British Drug Houses, Ltd., West Chester, Pa.)
plus 0.01 g/L Ergosterol (Alef Aesar, Ward Hill, Mass.) were added
and the culture was grown in a 250 ml serum bottle outfitted with a
butyl rubber stopper with an aluminum crimp cap. Each strain was
inoculated at an initial OD.sub.600 of about 0.2 and grown to an
OD.sub.600 of about 3-4. Cells were grown at 30.degree. C. at 200
rpm.
[0386] Yeast cells were harvested by centrifugation at 1046.times.g
(e.g., approximately 3000 rpm) for 5 minutes at 4.degree. C. The
supernatant was discarded and the cells were resuspended in 25 mL
cold sterile water. This wash step was repeated once. Washed cell
pellets were resuspended in 1 mL sterile water, transferred to 1.5
mL screw cap tube, and centrifuged at 16,100.times.g (e.g.,
approximately 13,200 rpm) for 3 minutes at 4.degree. C.
[0387] Cell pellets were resuspended in about 800-1000 .mu.l of
freshly prepared lysis buffer (50 mM Tris-Cl pH 7.0, 10 mM MgCl2,
1.times. protease inhibitor cocktail EDTA-free (Thermo Scientific,
Waltham, Mass.) and the tube filled with zirconia beads to avoid
any headspace in the tube. The tubes were placed in a Mini
BeadBeater (Bio Spec Products, Inc., Bartlesville, Okla.) and
vortexed twice for 30 seconds at room temperature. The supernatant
was transferred to a new 1.5 mL microcentrifuge tube and
centrifuged twice to remove cell debris at 16,100.times.g (e.g.,
approximately 13,200 rpm) for 10 minutes, at 4.degree. C.
Quantification of the lysates was performed using the
Coomassie-Plus kit (Thermo Scientific, San Diego, Calif.) as
directed by the manufacturer.
TABLE-US-00057 Strain EDD EDA BF428 p426GPD (vector control)
p425GPD (vector control) BF604 E. coli native E. coli native BF460
E. coli native with 6-HIS E. coli native with 6-HIS BF591 PAO1
native PAO1 native BF568 PAO1 native with 6-HIS PAO1 native with
6-HIS BF592 PAO1 native E. coli native BF603 E. coli native PAO1
native ("6-HIS" in above table is disclosed as SEQ ID NO: 138)
About 5-10 .mu.g of total cell extract was used for SDS-gel [NuPage
4-12% Bis-Tris gels (Life Technologies, Carlsbad, CA)]
electrophoresis and Western blot analyses.
[0388] SDS-PAGE gels were performed according to the manufacturer's
recommendation using NuPage MES-SDS Running Buffer at 1.times.
concentration with the addition of NuPage antioxidant into the
cathode chamber at a 1.times. concentration. Novex Sharp Protein
Standards (Life Technologies, Carlsbad, Calif.) were used as
standards. For Western analysis, gels were transferred onto a
nitrocellulose membrane (0.45 micron, Thermo Scientific, San Diego,
Calif.) using Western blotting filter paper (Thermo Scientific)
using a Bio-Rad Mini Trans-Blot Cell (BioRad, Hercules, Calif.)
system for approximately 90 minutes at 40V. Following transfer, the
membrane was washed in 1.times.PBS (EMD, San Diego, Calif.), 0.05%
Tween-20 (Fisher Scientific, Fairlawn, N.J.) for 2-5 minutes with
gentle shaking. The membrane was blocked in 3% BSA dissolved in
1.times.PBS and 0.05% Tween-20 at room temperature for about 2
hours with gentle shaking. The membrane was washed once in
1.times.PBS and 0.05% Tween-20 for about 5 minutes with gentle
shaking. The membrane was then incubated at room temperature with
the 1:5000 dilution of primary antibody (Ms mAB to 6.times.His Tag
(SEQ ID NO: 138), AbCam, Cambridge, Mass.) in 0.3% BSA (Fraction V,
EMD, San Diego, Calif.) dissolved in 1.times.PBS and 0.05% Tween-20
with gentle shaking.
[0389] Incubation was allowed to proceed for about 1 hour with
gentle shaking. The membrane was then washed three times for 5
minutes each with 1.times.PBS and 0.05% Tween-20 with gentle
shaking. The secondary antibody [Dnk pAb to Ms IgG (HRP), AbCam,
Cambridge, Mass.] was used at 1:15000 dilution in 0.3% BSA and
allowed to incubate for about 90 minutes at room temperature with
gentle shaking. The membrane was washed three times for about 5
minutes using 1.times.PBS and 0.05% Tween-20 with gentle shaking.
The membrane incubated with 5 ml of Supersignal West Pico
Chemiluminescent substrate (Thermo Scientific, San Diego, Calif.)
for 1 minute and then was exposed to a phosphorimager (Bio-Rad
Universal Hood II, Bio-Rad, Hercules, Calif.) for about 10-100
seconds.
[0390] The results of the Western blots, shown in FIGS. 13A and
13B. Included in the expression data are engineered and/or
optimized versions of certain eda and edd genes. The genes were
modified to include a C-terminal HIS tag to facilitate
purification. The two letters refer to the EDD and EDA source,
respectively. P is from P. aeruginosa, PAO1, E is from E. coli, Z
is from Zymomonas mobilis ZM4, hot rod is the optimized version of
Zymomonas mobilis, Harmonized is the codon harmonized version of
Zymomonas mobilis, V refers to the vector(s). Both total crude
extract and the solubilized extract are shown. The results
presented in FIGS. 13A and 13B indicate that the PAO1 EDD protein
is expressed and soluble in S. cerevisiae. The results also
demonstrate that the E. coli EDA protein is expressed and soluble.
It was not clear from these experiments if the PAO1 EDA was soluble
in yeast.
Example 18
EDD and EDA Activity Assays
[0391] Cell lysates of the various EDD and EDA expressing strains
were prepared as follows. About 50 to 100 ml of SCD-ura-leu media
containing 10 mM MnCl2 was used. When cultured aerobically, strains
were grown in a 250 ml baffled shake flask. When grown
anaerobically, 400 .mu.l/L Tween-80 (British Drug Houses, Ltd.,
West Chester, Pa.) plus 0.01 g/L Ergosterol (Alef Aesar, Ward Hill,
Mass.) were added and the culture was grown in a 250 ml serum
bottle outfitted with a butyl rubber stopper with an aluminum crimp
cap. Each strain was inoculated at an initial OD.sub.600 of about
0.2 and grown to an OD.sub.600 of about 3-4. Cells were grown at
30.degree. C. at 200 rpm.
[0392] Yeast cells were harvested by centrifugation at 1046.times.g
(3000 rpm) for 5 minutes at 4.degree. C. The supernatant was
discarded and the cells were resuspended in 25 mL cold sterile
water. This wash step was repeated once. Washed cell pellets were
resuspended in 1 mL sterile water, transferred to 1.5 mL screw cap
tube, and centrifuged at 16,100.times.g (13,200 rpm) for 3 minutes
at 4.degree. C. Cell pellets were resuspended in about 800-1000
.mu.l of freshly prepared lysis buffer (50 mM Tris-Cl pH 7.0, 10 mM
MgCl2, 1.times. protease inhibitor cocktail EDTA-free (Thermo
Scientific, Waltham, Mass.) and the tube filled with zirconia beads
to avoid any headspace in the tube. The tubes were placed in a Mini
BeadBeater (Bio Spec Products, Inc., Bartlesville, Okla.) and
vortexed twice for 30 seconds at room temperature. The supernatant
was transferred to a new 1.5 mL microcentrifuge tube and
centrifuged twice to remove cell debris at 16,100.times.g (13,200
rpm) for 10 minutes, at 4.degree. C. Quantification of the lysates
was performed using the Coomassie-Plus kit (Thermo Scientific, San
Diego, Calif.) as directed by the manufacturer.
[0393] About 750 .mu.g of crude extract was assayed using 1.times.
assay buffer (50 mM Tris-Cl pH 7.0, 10 mM MgCl2), 3 U lactate
dehydrogenase (5 .mu.g/.mu.L in 50 mM Tris-Cl pH 7.0), and 10 .mu.l
mM 6-phosphogluconate dissolved in 50 mM Tris-Cl pH 7.0 were mixed
in a reaction of about 400 .mu.l. This reaction mix was transferred
to a 1 ml Quartz cuvette and allowed to incubate about 5 minutes at
30.degree. C. To this reaction, 100 .mu.l of 1.5 mM NADH (prepared
in 50 mM Tris-Cl pH 7.0) was added, and the change in Abs.sub.340nm
over the course of 5 minutes at 30.degree. C. was monitored in a
Beckman DU-800 spectrophotometer using the Enzyme Mechanism
software package (Beckman Coulter, Inc, Brea, Calif.).
[0394] The table below presents the relative specific activities
for BY4742 strains expressing EDD and EDA from either P. aeruginosa
(PAO1) or E. coli sources. The results presented in the table below
indicate that each of the listed combinations of EDD and EDA genes,
when expressed in S. cerevisiae strain BY4742, confers
activity.
TABLE-US-00058 Gene Km Vmax Specific Activity Combination
(M.sup.-1) (mmol min.sup.-1) (mmol min.sup.-1 mg.sup.-1)
EDD-P/EDA-P 1.04 .times. 10.sup.-3 0.21930 0.3451 EDD-P/EDA-E 2.06
.times. 10.sup.-3 0.27280 0.3637 EDD-E/EDA-P 1.43 .times. 10.sup.-3
0.09264 0.1235 EDD-E/EDA-E 0.839 .times. 10.sup.-3 0.16270
0.2169
[0395] The data presented above is also presented graphically in
FIG. 14. FIG. 14 graphically displays the relative activities of
the various EDD/EDA combinations presented in the table above, as
measured in assays using 750 micrograms of crude extract. From the
height of the PE bar in FIG. 14, and the data presented in the
table above, it is evident that the combinations conferring the
highest level of activity were the EDD-P/EDA-E (e.g., PE) and
EDD-P/EDA-P (e.g., PP) combinations.
Example 19
Improved Ethanol Yield from Yeast Strains Expressing EDD and EDA
Constructs
[0396] Strains BF428 (vector control), BF591 (EDD-PAO1/EDA-PAO1),
BF592 (EDD-PAO1/EDA-E. coli), BF603 (EDD-E. coli/EDA-PAO1) and
BF604 (EDD-E. coli/EDA-E. coli) were inoculated into 15 ml
SCD-ura-leu media containing 400 .mu.l/L Tween-80 (British Drug
Houses, Ltd., West Chester, Pa.) plus 0.01 g/L Ergosterol (EMD, San
Diego, Calif.) in 20 ml Hungate tubes outfitted with a butyl rubber
stopper and sealed with an aluminum crimped cap to prevent oxygen
from entering the culture at an initial OD.sub.600 of 0.5 and grown
for about 20 hours. Glucose and ethanol in the culture media were
assayed using YSI 2700 BioAnalyzer instruments (world wide web
uniform resource locator ysi.com), according to the manufacturer's
recommendations at 0 and 20 hours post inoculation. The results of
the fermentation of glucose to ethanol are showing graphically in
FIG. 15. The results presented in FIG. 15 indicate that the
presence of the EDD/EDA combinations in S. cerevisiae increase the
yield of ethanol produced, when compared to a vector-only control.
The EDD/EDA combinations that showed the greatest fermentation
efficiency in yeast were EDD-P/EDA-E (e.g., PE) and EDD-E/EDA-P
(e.g., EP).
Example 20
Improved Ethanol Yield from Yeast Strains Expressing EDD and EDA
from PAO1 in Fermentors
[0397] A fermentation test of the strain BF591 [BY4742 with
plasmids pBF290 (p426GPD-EDD_PAO1) and pBF292 (p425GPD-EDA_PAO1)]
was conducted against BF428 (BY4742 p426GPD/p425GPD) control strain
in 700 ml w.v. Multifors multiplexed fermentors. The fermentation
medium was SC-Ura-Leu with about 2% glucose. Vessels were
inoculated with about a 6.25% inoculum from overnight cultures
grown in about 50 ml SC-Ura-Leu with about 2% glucose.
[0398] The cultures were grown aerobically at about 30.degree. C.
with about 250 rpm agitation, 1 vvm sparge of process air, (21%
O.sub.2). The pH was controlled at around 5.0 with 0.25 N NaOH.
Once glucose concentrations dropped below 0.5 g/L the fermentation
was switched to anaerobic conditions. Before changing to anaerobic
conditions, samples were taken to measure glucose concentrations
and biomass by OD.sub.600 as reported in Table B. Ethanol and
glucose concentrations in the fermentation broth were monitored
using YSI 2700 BioAnalyzer instruments.
[0399] The table below presents the elapsed fermentation time
(EFT), the biomass and glucose at the start of anaerobic
fermentation in a 400 ml fermentor. The edd and eda combinations
carried by the strains are described above.
TABLE-US-00059 Glucose Strain EFT (hrs) OD.sub.600 nm (g/L) BF591
32 4.50 .047 BF428 27 4.81 .062
[0400] At the beginning of the anaerobic portion of the
fermentation, a bolus of 20 g/L glucose plus 3.35 g/L of yeast
nitrogen base without amino acids was added to the fermentors. In
addition, 4 ml/L of 2.5 g/L ergosterol in ethanol, 0.4 ml/L Tween
80, and 0.01% AF-204 were added to each fermentor. Oxygen was
purged with 100% N2 sparged at about 1 vvm until pO2 was below
1%.
[0401] Samples were taken every 2 to 7 hours and measured for
ethanol and glucose concentrations and OD.sub.600. The fermentation
was harvested when the glucose concentration was below 0.05 g/L, at
50 hours elapsed fermentation time (EFT). Ethanol and glucose
concentrations and OD.sub.600 of the final sample are reported in
the table below.
TABLE-US-00060 Ethanol Glucose Strain OD.sub.600 nm (g/L) (g/L)
BF591 5.6 17.1 .04 BF428 5.6 15.8 0
[0402] The data presented in the table above also is presented
graphically in FIGS. 16A and 16B. FIG. 16A presents the
fermentation data from strain BF428 (BY4742 with vector controls)
and FIG. 16B presents the fermentation data from strain BF591
(BY4742 with EDD-PAO1/EDA-PAO1). Fermentation profiles for strains
BF 428 and BF 591, grown on 2% dextrose, were calculated and are
presented in the table below.
TABLE-US-00061 Strain Yx/s Yp/s Yp/x Qp qp BF428 0.24 0.40 7.19
0.02 0.05 BF591 0.23 0.43 7.44 0.02 0.07 Yx/s = OD/g glucose Yp/s =
q ethanol/g glucose Yp/x = g ethanol/OD Qp = g ethanol/Lh.sup.-1 qp
= g ethanol/ODh.sup.-1
[0403] The results from the fermentation show that the BF591 has a
higher ethanol yield (triangles, compare FIG. 16A and FIG. 16B)
than the control BF428 strain. The calculated yield of ethanol was
also determined to be higher in the engineered BF591 strain (0.43 g
ethanol/g glucose) than that of the BF428 control strain (0.40 g
ethanol/g glucose).
Example 21
Improved Ethanol Yield in a Tall Strain of S. cerevisiae Expressing
EDD and EDA from PAO1
[0404] To generate BY4741 and BY4742 tal1 mutant strains, the
following procedure was used:
TABLE-US-00062 Oligonucleotides (SEQ ID NO: 337)
#350-5'-TAAAACGACGGCCAGTGAAT-3' (SEQ ID NO: 338)
#351-5'-TGCAGGTCGACTCTAGAGGAT-3' (SEQ ID NO: 339)
#352-5'-GTGTGCGTGTATGTGTACACCTGTATTTAATTTCCTTACTCG
CGGGTTTTTCTAAAACGACGGCCAGTGAAT-3' (SEQ ID NO: 340)
#353-5'-TGTACCAGTCTAGAATTCTACCAACAAATGGGGAAATCAAAG
TAACTTGGGCTGCAGGTCGACTCTAGAGGA-3'
[0405] All oligonucleotides set forth above were purchased from
Integrated Technologies ("IDT", Coralville, Iowa). PCR
amplification of the genes were performed as follows: about 50 ng
of the pBFU-719 DNA (e.g., plasmid with unique 200-mer sequence)
was added to 1.times.Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3 .mu.mol
gene-specific primers (#350/#351 in the first round), and 1 U Pfu
Ultra II polymerase (Agilent, La Jolla, Calif.) in a 50 .mu.l
reaction mix. The reaction mixture was cycled as follows:
95.degree. C. 10 minutes followed by 30 rounds of 95.degree. C. for
20 seconds, 60.degree. C. for 30 seconds, and 72.degree. C. for 45
seconds. A final 5 minute extension reaction at 72.degree. C. was
also included. A second round of PCR amplification was done using
50 ng of the first round PCR amplification with 1.times.Pfu Ultra
II buffer, 0.3 mM dNTPs, 0.3 .mu.mol gene-specific primers
(#352/#353 in the second round), and 1 U Pfu Ultra II polymerase
(Agilent, La Jolla, Calif.) in a 50 .mu.l reaction mix. The second
reaction mixture was cycled as follows: 95.degree. C. 10 minutes
followed by 30 rounds of 95.degree. C. for 20 seconds, 60.degree.
C. for 30 seconds, and 72.degree. C. for 45 seconds. A final 5
minute extension reaction at 72.degree. C. was also included. The
final PCR product was purified using the Zymo Research DNA Clean
& Concentrator-25 kit (Zymo Research, Orange, Calif.).
[0406] Transformation was accomplished by a high-efficiency
competency method. A 5 ml culture of the BY4742 or BY4741 strain
was grown overnight at about 30.degree. C. with shaking at about
200 rpm. A suitable amount of this overnight culture was added to
60 ml of YPD media to obtain an initial OD600 of about 0.2
(approximately 2.times.10.sup.6 cells/ml). The cells were allowed
to grow at 30.degree. C. with agitation (about 200 rpm) until the
OD.sub.600 was about 1. The cells were then centrifuged at 3000 rpm
for 5 min, washed with 10 ml sterile water and re-centrifuged. The
cell pellet was resuspended in 1 ml sterile water, transferred to a
1.5 ml sterile microcentrifuge tube and spun down at 4000.times.g
for about 5 minutes. This cell pellet was resuspended in 1 ml
sterile 1.times.TE/LiOAC solution (10 mM Tris-HCl, 1 mM EDTA, 100
mM LiOAc, pH7.5) and re-centrifuged at about 4000.times.g for about
5 minutes. The cell pellet was resuspended in 0.25 ml
1.times.TE/LiOAc solution. For the transformation, 50 .mu.l of
these cells were aliquoted to a 1.5 ml microcentrifuge tube and
about 1 .mu.g purified PCR product and 5 .mu.l of salmon sperm DNA
that had been previously boiled for about 5 minutes and placed on
ice. 300 .mu.l of a sterile PEG solution was then added (40% PEG
3500, 10 mM Tris-HCl, 1 mM EDTA, 100 mM LiOAc, pH7.5). This mixture
was allowed to incubate at 30.degree. C. for about one hour with
gentle mixing every 15 minutes. About 40 .mu.l DMSO (Sigma, St.
Louis, Mo.) was added to the incubating mixture, and the mixture
heat shocked at about 42.degree. C. for about 15 minutes. The cells
were pelleted in a microcentrifuge at 13000 rpm for about 30
seconds and the supernatant removed. The cells were resuspended in
1 ml 1.times.TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5), centrifuged at
13000 rpm for about 30 seconds and resuspended in 1 ml 1.times.TE.
About 100-200 .mu.l of cells were plated onto SCD-URA media, as
described above, and allowed to grow at about 30.degree. C. for
about 3 days. After 3 days, transformed colonies were streaked for
single colonies on SCD-URA plates and allowed to grow at about
30.degree. C. for about 3 days. From these plates, single colonies
were streaked onto SCD agar plates (20 g/L agar in SCD media)
containing 1 g/L 5-FOA (Research Products International Corp, Mt.
Prospect, Ill.), and also inoculated into YPD liquid broth. The
plates were allowed to grow at about 30.degree. C. for about 4 days
and the liquid culture was grown overnight at about 30.degree. C.
with agitation of about 200 rpm.
[0407] To confirm that integration of the construct was correct,
genomic DNA was prepared from the YPD overnight cultures. Briefly,
the yeast cells were pelleted by centrifugation at room temperature
for 5 minutes at approximately 3000 rpm. The cell pellet was
resuspended in 200 .mu.l of breaking buffer (2% Triton X-100, 1%
SDS, 100 mM NaCl, 10 mM Tris pH8, 1 mM EDTA) and placed into a 1.5
ml microcentrifuge tube containing about 200 .mu.l glass beads and
about 200 .mu.l of phenol:chloroform:isoamyl alcohol (Ambion,
Austin, Tex.). The mixture was vortexed for about 2 to 5 minutes at
room temperature. About 200 .mu.l of sterile water was then added
and the mixture vortexed again. The mixture was centrifuged for
about 10 minutes at about 13000 rpm and the aqueous layer
transferred to a new microcentrifuge tube. About 1/10th of the
aqueous layers volume of 3M NaOAc ((British Drug Houses, Ltd., West
Chester, Pa.) was added to the aqueous layer and 2.5.times. the
total volume of the mixture of ethanol was added and mixed well.
The genomic DNA was then precipitated by placing the tubes at
-80.degree. C. for at least one hour (or in a dry ice/ethanol bath
for about 30 minutes). The tubes were then centrifuged at about
13000 rpm for 5 minutes at about 4.degree. C. to pellet the DNA.
The DNA pellet was then washed two times or more times with about
200 .mu.l of 70% ethanol and re-centrifuged. The DNA pellet was
dried using vacuum assisted air drying and resuspended in about 50
to 200 .mu.l 1.times.TE.
[0408] The genomic DNA isolated as described above was used in a
PCR amplification reaction consisting of about 50 ng of the genomic
DNA was added to 1.times.Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3
.mu.mol gene-specific primers (#276/#277), and 1 U Pfu Ultra II
polymerase (Agilent, La Jolla, Calif.) in a 50 .mu.l reaction mix.
The reaction mix was cycled as follows: 95.degree. C. 10 minutes
followed by 30 rounds of 95.degree. C. for 20 seconds, 60.degree.
C. for 30 seconds, and 72.degree. C. for 45 seconds. A final 5
minute extension reaction at 72.degree. C. was also included. A
second round of PCR amplification was done using 50 ng of the first
round PCR amplification with 1.times.Pfu Ultra II buffer, 0.3 mM
dNTPs, 0.3 .mu.mol gene-specific primers (#352/#353 in the second
round), and 1 U Pfu Ultra II polymerase (Agilent, La Jolla, Calif.)
in a 50 .mu.l reaction mix. The second mixture was cycled as
follows: 95.degree. C. 10 minutes followed by 30 rounds of
95.degree. C. for 20 seconds, 55.degree. C. for 30 seconds, and
72.degree. C. for about 30 seconds. A final 5 minute extension
reaction at 72.degree. C. was also included.
[0409] Positive colonies from the screen in YPD that had a PCR
product of about 1600 bp indicating the insertion of the
integration construct in the TAL1 locus, and that grew on the
plates containing 5-FOA were grown overnight in YPD at about
30.degree. C. with agitation of about 200 rpm. Genomic DNA was
prepared as above and checked by PCR amplification using primers
#276 and #277 (described below). Positive clones were identified
which had a PCR product of 359 bp indicating the deletion of the
tal1 locus and the remaining portion of the 200-mer tag. The strain
carrying the correct traits was labeled as BF716. The BY4741
version was labeled as BF717.
TABLE-US-00063 Oligonucleotides (SEQ ID NO: 341)
#276-5'-GTCGACTGGAAATCTGGAAGGTTGGT-3' (SEQ ID NO: 342)
#277-5'-GTCGACGCTTTGCTGCAAGGATTCAT-3'
[0410] The BY4742 tal1 strain was then made competent using the
high efficiency competent method as described above. About 500 ng
of plasmids pBF290 and pBF292 or with plasmids p426GPD and p425GPD
were used to transform the BY4742 tal1 strain. The final
transformation mixture was plated onto SCD-ura-leu plates and grown
at about 30.degree. C. for about 3 days. Strain BF716 (BY4742 tal1)
with p426GPD/p425GPD was labeled as BF738. Strain BF716 with
pBF290/pBF292 was labeled as BF741.
[0411] A fermentation test of the BF738 was conducted against BF741
in a 400 ml multiplexed fermentor. The fermentation medium utilized
was SC-Ura -Leu with 2% glucose. Cultures were grown overnight in
50 ml SC-Ura -Leu 2% glucose and used to inoculate the fermentors
at 4 to 5% inoculum. OD.sub.600 readings of the inoculum are shown
in the table below.
TABLE-US-00064 Strain OD.sub.600 nm BF741 (tal1 PP) 3.70 BF738
(tal1 VV) 3.80
[0412] The cultures were grown aerobically at about 30.degree. C.
with about 250 rpm agitation, 0.5 vvm sparge of process air, 21%
O.sub.2. pH was controlled at 5.0 with 1N NaOH. Glucose
concentrations in the fermentation broth were monitored by YSI 2700
BioAnalyzers during aerobic fermentation. Once glucose was depleted
the fermentation was switched to anaerobic conditions. Before
changing to anaerobic conditions samples were taken to measure
glucose usage. Biomass was measured by monitoring the optical
density of the growth medium at 600 nanometers (e.g., OD.sub.600).
EFT at glucose depletion, glucose concentrations and OD.sub.600 are
shown in the table below. The table below reports the amount of
biomass in the fermentor and the amount of ethanol produced in
grams per liter, after the specified amount of time (EFT), by the
respective strains.
TABLE-US-00065 Strain EFT (hrs) OD.sub.600 nm Glucose (g/L) BF741
(tal1 PP) 43.5 2.50 0.045 BF738 (tal1 VV) 31 2.95 0.192
[0413] At the beginning of anaerobic fermentation, about 19 g/L
glucose, 3.7 g/L YNB, 4 ml/L of 2.5 g/L ergosterol (in ethanol),
0.4 ml/L Tween 80, and 0.01% AF-204 were added to each fermentor.
Oxygen was purged with 100% N.sub.2 sparged at 0.25 vvm for the
remainder of the fermentation. Samples were taken every 4 to 12
hours and analyzed for ethanol production and glucose utilization
using the YSI Bioanalyzers, and amount of biomass by OD.sub.600.
The fermentations were harvested when the glucose bolus was
depleted. Anaerobic ethanol produced, anaerobic glucose consumption
and OD.sub.600 of the final sample are shown in the table
below.
TABLE-US-00066 Ethanol Produced Glucose Consumed Strain OD.sub.600
nm (g/L) (g/L) BF741 (tal1 PP) 3.75 8.1 18.99 BF738 (tal1 VV) 3.6
6.5 18.168
[0414] The results are also presented graphically in FIGS. 17A and
17B. FIG. 17A illustrates the fermentation data for strain BF738
(BY4742 tal1 with vector controls p426GPD and p425GPD) and FIG. 17B
illustrates the fermentation data for strain BF741 (BY4742 tal1
with plasmids pBF290 (EDD-PAO1) and pBF292 (EDA-PAO1). The results
presented above and in FIGS. 17A and 17B indicate that strain
BF741, which expresses the activities encoded by the eda and edd
genes, yields more ethanol than control strain BF738. Strain BF741
produced about 0.43 g ethanol per gram of glucose consumed whereas
strain BF738 produced only 0.36 g ethanol per gram of glucose
consumed. Fermentation profiles were calculated for strains BF738
and BF741 and are presented below.
TABLE-US-00067 Strain Yx/s Yp/s Yp/x Qp qp BF738 0.198 0.358 3.76
0.371 0.103 BF741 0.203 0.439 2.16 0.439 0.131 Yx/s = OD/g glucose,
Yp/s = q ethanol/g glucose, Yp/x = g ethanol/OD Qp = g
ethanol/Lh.sup.-1, qp = g ethanol/ODh.sup.-1
Example 22
Complementation and Improved Ethanol Yield in a pfk1 Strain of S.
cerevisiae Expressing the EDA and EDD Genes from P. aeruginosa
[0415] Strain BF205 (YGR240C/BY4742, ATCC Cat. No. 4015893; PubMed:
10436161) was transformed with plasmids p426GPD and p425GPD or with
plasmids pBF290 (p426GPD/EDD-PAO1) and pBF292 (p426GPD/EDA-PAO1),
generating strains BF740 (vector controls) and BF743, respectively.
Transformation was accomplished by a high-efficiency competency
method using 500 ng of plasmids p426GPD and p425GPD or plasmids
pBF290 and pBF292. Transformants were plated onto SCD-ura-leu agar
plates and grown at about 30.degree. C. for about 3 days. The final
strains were named BF740 (BY4742 pfk1 with plasmids p426GPD and
p425GPD) and BF743 (BY4742-pfk1, pBF290/pBF292).
[0416] A fermentation test of the control strain BF740 (BY4742 pfk1
with plasmids p426GPD and p425GPD) was conducted against BF743
(BY4742-pfk1, pBF290/pBF292) in 400 ml w.v. Multifors multiplexed
fermentors. The fermentation medium was SC-Ura-Leu with 2% glucose.
Vessels were inoculated with about a 10% inoculum from overnight
cultures grown in about 50 ml SC-Ura-Leu with about 2% glucose and
normalized to 0.5 OD.sub.600. The actual inoculated ODs for the
fermentations are shown in the table below.
TABLE-US-00068 Strain OD.sub.600 nm BF740 (pfk1 VV) 0.571 BF743
(pfk1 PP) 0.535
[0417] The cultures were grown aerobically at about 30.degree. C.
with about 250 rpm agitation, 1 vvm sparge of process air, (21%
O.sub.2). The pH was controlled at around 5.0 with 0.25 N NaOH.
Once glucose concentrations dropped below 0.5 g/L the fermentation
was switched to anaerobic conditions. Before changing to anaerobic
conditions, samples were taken to measure glucose concentrations
and biomass by OD.sub.600 as shown in the table below. The table
below shows the beginning cell biomass and glucose concentration
(in grams per liter of nutrient broth). Ethanol and glucose
concentrations in the fermentation broth were monitored using a YSI
2700 BioAnalyzer.
TABLE-US-00069 Ethanol Glucose Strain OD.sub.600 nm (g/L) (g/L)
BF740 5.94 5.67 0.033 BF743 5.82 5.82 0.034
[0418] At the beginning of the anaerobic portion of the
fermentation, a bolus of about 18 g/L glucose plus about 4 ml/L of
2.5 g/L ergosterol in Ethanol, 0.4 ml/L Tween 80, and 0.01% AF-204
were added to each fermentor. Oxygen was purged with 100% N.sub.2
sparged at about 1 vvm until pO.sub.2 was below 1%. Samples were
taken every 4 to 8 hours and measured for ethanol and glucose
concentrations and biomass (OD.sub.600). The fermentation was
harvested when the glucose concentration was below 0.05 g/L, at
about 42 hours elapsed fermentation time (EFT). Ethanol and glucose
concentrations and OD.sub.600 of the final sample are shown in the
table below.
TABLE-US-00070 Ethanol Glucose Strain OD.sub.600 nm (g/L) (g/L)
BF740 6.4 5.07 14.6 BF743 5.09 13.37 0.042
[0419] The results also are present graphically in FIGS. 18A and
18B. The results presented in FIG. 18A illustrate the fermentation
data for strain BF740 grown on 2% dextrose and the results
presented in FIG. 18B illustrate the fermentation data for strain
BF743 grown on 2% dextrose. The results indicate that the BY4742
pfk1 mutant strain, BF740 cannot utilize glucose nor produce
ethanol under anaerobic conditions. However, the engineered strain
BF743 is capable of both utilizing glucose and producing ethanol
under anaerobic conditions. Strain BF743 has a yield of about 0.39
g ethanol per gram of glucose consumed versus no yield in the
control strain BF740. The fermentation profile for strains BF740
and BF743 are presented in the table below.
TABLE-US-00071 Strain Yx/s Yp/s Yp/x Qp qp BF740 2.133 -0.700
-0.328 -0.022 -0.003 BF743 0.264 0.390 1.483 0.178 0.035 Yx/s =
OD/g glucose, Yp/s = q ethanol/g glucose, Yp/x = g ethanol/OD Qp =
g ethanol/Lh.sup.-1, qp = g ethanol/ODh.sup.-1
Example 23
EDD and EDA Activities from Other Sources
[0420] The EDD and EDA genes also have been isolated from
additional sources and tested for the ability to direct
fermentation in yeast. The additional EDD and EDA genes have been
isolated from Shewanella oneidensis, Gluconobacter oxydans, and
Ruminococcus flavefaciens. Genomic DNA was purchased from ATCC for
both S. oneidensis (Cat. No. 700550D) and G. oxydans (621 HD-5). R.
flavefaciens, strain C94 (NCDO 2213) was also purchased from ATCC
(Cat. No. 19208). To prepare genomic DNA, R. flavefaciens was grown
in cooked meat media (Becton Dickinson, Franklin Lakes, N.J. USA)
overnight at 37.degree. C. and genomic DNA was isolated using a
Qiagen DNeasy Blood and Tissue kit according to the manufacture's
protocol. The eda and edd genes were PCR amplified from the
corresponding genomic DNA using the following sets of PCR
oligonucleotides. The nucleotide and amino acid sequences of eda
and edd genes PCR amplified using the following sets of PCR
oligonucleotide primers, also is given below.
TABLE-US-00072 The S. oneidensis edd gene: (SEQ. ID. NO: 73)
5'-GTTCACTGCactagtaaaaaaATGCACTCAGTCGTTCAATCTG-3' (SEQ. ID. NO: 74)
5'-CTTCGAGATCTCGAGTTAGTAAAGTTCATCGATGGC-3' The S. oneidensis eda
gene: (SEQ. ID. NO: 75)
5'-GTTCACTGCactagtaaaaaaATGCTTGAGAATAACTGGTC-3' (SEQ. ID. NO: 76)
5'-CTTCGAGATCTCGAGTTAAAGTCCGCCAATCGCCTC-3' The G. oxydans edd gene:
(SEQ. ID. NO: 77) 5'-GTTCACTGCactagtaaaaaaATGTCTCTGAATCCCGTCGTC-3'
(SEQ. ID. NO: 78) 5'-CTTCGAGATCTCGAGTTAGTGAATGTCGTCGCCAAC-3' The G.
oxydans eda gene: (SEQ. ID. NO: 79)
5'-GTTCACTGCactagtaaaaaaATGATCGATACTGCCAAACTC-3' (SEQ. ID. NO: 80)
5'-CTTCGAGATCTCGAGTCAGACCGTGAAGAGTGCCGC-3' The R. flavefaciens edd
gene: (SEQ. ID. NO: 81)
5'-GTTCACTGCactagtaaaaaaATGAGCGATAATTTTTTCTGCG-3' (SEQ. ID. NO: 82)
5'-CTTCGAGATCTCGAGCTATTTCCTGTTGATGATAGC-3' S. oneidensis
6-phosphogluconate dehydratase (edd) (SEQ. ID. NO: 83)
ATGCACTCAGTCGTTCAATCTGTTACTGACAGAATTATTGCCCGTAGCAAAGCATCTCGTGAA
GCATACCTTGCTGCGTTAAACGATGCCCGTAACCATGGTGTACACCGAAGTTCCTTAAGTTGC
GGTAACTTAGCCCACGGTTTTGCGGCTTGTAATCCCGATGACAAAAATGCATTGCGTCAATTG
ACGAAGGCCAATATTGGGATTATCACCGCATTCAACGATATGTTATCTGCACACCAACCCTAT
GAAACCTATCCTGATTTGCTGAAAAAAGCCTGTCAGGAAGTCGGTAGTGTTGCGCAGGTGGC
TGGCGGTGTTCCCGCCATGTGTGACGGCGTGACTCAAGGTCAGCCCGGTATGGAATTGAGCT
TACTGAGCCGTGAAGTGATTGCGATGGCAACCGCGGTTGGCTTATCACACAATATGTTTGATG
GAGCCTTACTCCTCGGTATTTGCGATAAAATTGTACCGGGTTTACTGATTGGTGCCTTAAGTTT
TGGCCATTTACCTATGTTGTTTGTGCCCGCAGGCCCAATGAAATCGGGTATTCCTAATAAGGA
AAAAGCTCGCATTCGTCAGCAATTTGCTCAAGGTAAGGTCGATAGAGCACAACTGCTCGAAGC
GGAAGCCCAGTCTTACCACAGTGCGGGTACTTGTACCTTCTATGGTACCGCTAACTCGAACCA
ACTGATGCTCGAAGTGATGGGGCTGCAATTGCCGGGTTCATCTTTTGTGAATCCAGACGATCC
ACTGCGCGAAGCCTTAAACAAAATGGCGGCCAAGCAGGTTTGTCGTTTAACTGAACTAGGCA
CTCAATACAGTCCGATTGGTGAAGTCGTTAACGAAAAATCGATAGTGAATGGTATTGTTGCATT
GCTCGCGACGGGTGGTTCAACAAACTTAACCATGCACATTGTGGCGGCGGCCCGTGCTGCA
GGTATTATCGTCAACTGGGATGACTTTTCGGAATTATCCGATGCGGTGCCTTTGCTGGCACGT
GTTTATCCAAACGGTCATGCGGATATTAACCATTTCCACGCTGCGGGTGGTATGGCTTTCCTT
ATCAAAGAATTACTCGATGCAGGTTTGCTGCATGAGGATGTCAATACTGTCGCGGGTTATGGT
CTGCGCCGTTACACCCAAGAGCCTAAACTGCTTGATGGCGAGCTGCGCTGGGTCGATGGCC
CAACAGTGAGTTTAGATACCGAAGTATTAACCTCTGTGGCAACACCATTCCAAAACAACGGTG
GTTTAAAGCTGCTGAAGGGTAACTTAGGCCGCGCTGTGATTAAAGTGTCTGCCGTTCAGCCAC
AGCACCGTGTGGTGGAAGCGCCCGCAGTGGTGATTGACGATCAAAACAAACTCGATGCGTTA
TTTAAATCCGGCGCATTAGACAGGGATTGTGTGGTGGTGGTGAAAGGCCAAGGGCCGAAAGC
CAACGGTATGCCAGAGCTGCATAAACTAACGCCGCTGTTAGGTTCATTGCAGGACAAAGGCTT
TAAAGTGGCACTGATGACTGATGGTCGTATGTCGGGCGCATCGGGCAAAGTACCTGCGGCGA
TTCATTTAACCCCTGAAGCGATTGATGGCGGGTTAATTGCAAAGGTACAAGACGGCGATTTAA
TCCGAGTTGATGCACTGACCGGCGAGCTGAGTTTATTAGTCTCTGACACCGAGCTTGCCACC
AGAACTGCCACTGAAATTGATTTACGCCATTCTCGTTATGGCATGGGGCGTGAGTTATTTGGA
GTACTGCGTTCAAACTTAAGCAGTCCTGAAACCGGTGCGCGTAGTACTAGCGCCATCGATGA
ACTTTACTAA S. oneidensis 6-phosphogluconate dehydratase (edd)-Amino
Acid sequence (SEQ. ID. NO: 84)
MHSVVQSVTDRIIARSKASREAYLAALNDARNHGVHRSSLSCGNLAHGFAACNPDDKNALRQLTK
ANIGIITAFNDMLSAHQPYETYPDLLKKACQEVGSVAQVAGGVPAMCDGVTQGQPGMELSLLSRE
VIAMATAVGLSHNMFDGALLLGICDKIVPGLLIGALSFGHLPMLFVPAGPMKSGIPNKEKARIRQQF
AQGKVDRAQLLEAEAQSYHSAGTCTFYGTANSNQLMLEVMGLQLPGSSFVNPDDPLREALNKMA
AKQVCRLTELGTQYSPIGEVVNEKSIVNGIVALLATGGSTNLTMHIVAAARAAGIIVNWDDFSELSD
AVPLLARVYPNGHADINHFHAAGGMAFLIKELLDAGLLHEDVNTVAGYGLRRYTQEPKLLDGELR
WVDGPTVSLDTEVLTSVATPFQNNGGLKLLKGNLGRAVIKVSAVQPQHRVVEAPAVVIDDQNKLD
ALFKSGALDRDCVVVVKGQGPKANGMPELHKLTPLLGSLQDKGFKVALMTDGRMSGASGKVPAA
IHLTPEAIDGGLIAKVQDGDLIRVDALTGELSLLVSDTELATRTATEIDLRHSRYGMGRELFGVLRSN
LSSPETGARSTSAIDELY G. oxydans 6-phosphogluconate dehydratase (edd)
(SEQ. ID. NO: 85)
ATGTCTCTGAATCCCGTCGTCGAGAGCGTGACTGCCCGTATCATCGAGCGTTCGAAAGTCTC
CCGTCGCCGGTATCTCGCCCTGATGGAGCGCAACCGCGCCAAGGGTGTGCTCCGGCCCAAG
CTGGCCTGCGGTAATCTGGCGCATGCCATCGCAGCGTCCAGCCCCGACAAGCCGGATCTGA
TGCGTCCCACCGGGACCAATATCGGCGTGATCACGACCTATAACGACATGCTCTCGGCGCAT
CAGCCGTATGGCCGCTATCCCGAGCAGATCAAGCTGTTCGCCCGTGAAGTCGGTGCGACGG
CCCAGGTTGCAGGCGGCGCACCAGCAATGTGTGATGGTGTGACGCAGGGGCAGGAGGGCAT
GGAACTCTCCCTGTTCTCCCGTGACGTGATCGCCATGTCCACGGCGGTCGGGCTGAGCCAC
GGCATGTTTGAGGGCGTGGCGCTGCTGGGCATCTGTGACAAGATTGTGCCGGGCCTTCTGAT
GGGCGCGCTGCGCTTCGGTCATCTCCCGGCCATGCTGATCCCGGCAGGGCCAATGCCGTCC
GGTCTTCCAAACAAGGAAAAGCAGCGCATCCGCCAGCTCTATGTGCAGGGCAAGGTCGGGC
AGGACGAGCTGATGGAAGCGGAAAACGCCTCCTATCACAGCCCGGGCACCTGCACGTTCTAT
GGCACGGCCAATACGAACCAGATGATGGTCGAAATCATGGGTCTGATGATGCCGGACTCGGC
TTTCATCAATCCCAACACGAAGCTGCGTCAGGCAATGACCCGCTCGGGTATTCACCGTCTGG
CCGAAATCGGCCTGAACGGCGAGGATGTGCGCCCGCTCGCTCATTGCGTAGACGAAAAGGC
CATCGTGAATGCGGCGGTCGGGTTGCTGGCGACGGGTGGTTCGACCAACCATTCGATCCATC
TTCCTGCTATCGCCCGTGCCGCTGGTATCCTGATCGACTGGGAAGACATCAGCCGCCTGTCG
TCCGCGGTTCCGCTGATCACCCGTGTTTATCCGAGCGGTTCCGAGGACGTGAACGCGTTCAA
CCGCGTGGGTGGTATGCCGACCGTGATCGCCGAACTGACGCGCGCCGGGATGCTGCACAAG
GACATTCTGACGGTCTCTCGTGGCGGTTTCTCCGATTATGCCCGTCGCGCATCGCTGGAAGG
CGATGAGATCGTCTACACCCACGCGAAGCCGTCCACGGACACCGATATCCTGCGCGATGTGG
CTACGCCTTTCCGGCCCGATGGCGGTATGCGCCTGATGACTGGTAATCTGGGCCGCGCGAT
CTACAAGAGCAGCGCTATTGCGCCCGAGCACCTGACCGTTGAAGCGCCGGCACGGGTCTTC
CAGGACCAGCATGACGTCCTCACGGCCTATCAGAATGGTGAGCTTGAGCGTGATGTTGTCGT
GGTCGTCCGGTTCCAGGGACCGGAAGCCAACGGCATGCCGGAGCTTCACAAGCTGACCCCG
ACTCTGGGCGTGCTTCAGGATCGCGGCTTCAAGGTGGCCCTGCTGACGGATGGACGCATGT
CCGGTGCGAGCGGCAAGGTGCCGGCCGCCATTCATGTCGGTCCCGAAGCGCAGGTTGGCG
GTCCGATCGCCCGCGTGCGGGACGGCGACATGATCCGTGTCTGCGCGGTGACGGGACAGAT
CGAGGCTCTGGTGGATGCCGCCGAGTGGGAGAGCCGCAAGCCGGTCCCGCCGCCGCTCCC
GGCATTGGGAACGGGCCGCGAACTGTTCGCGCTGATGCGTTCGGTGCATGATCCGGCCGAG
GCTGGCGGATCCGCGATGCTGGCCCAGATGGATCGCGTGATCGAAGCCGTTGGCGACGACA
TTCACTAA G. oxydans 6-phosphogluconate dehydratase (edd)-Amino Acid
sequence (SEQ. ID. NO: 86)
MSLNPVVESVTARIIERSKVSRRRYLALMERNRAKGVLRPKLACGNLAHAIAASSPDKPDLMRPTG
TNIGVITTYNDMLSAHQPYGRYPEQIKLFAREVGATAQVAGGAPAMCDGVTQGQEGMELSLFSRD
VIAMSTAVGLSHGMFEGVALLGICDKIVPGLLMGALRFGHLPAMLIPAGPMPSGLPNKEKQRIRQL
YVQGKVGQDELMEAENASYHSPGTCTFYGTANTNQMMVEIMGLMMPDSAFINPNTKLRQAMTR
SGIHRLAEIGLNGEDVRPLAHCVDEKAIVNAAVGLLATGGSTNHSIHLPAIARAAGILIDWEDISRLSS
AVPLITRVYPSGSEDVNAFNRVGGMPTVIAELTRAGMLHKDILTVSRGGFSDYARRASLEGDEIVY
THAKPSTDTDILRDVATPFRPDGGMRLMTGNLGRAIYKSSAIAPEHLTVEAPARVFQDQHDVLTAY
QNGELERDVVVVVRFQGPEANGMPELHKLTPTLGVLQDRGFKVALLTDGRMSGASGKVPAAIHV
GPEAQVGGPIARVRDGDMIRVCAVTGQIEALVDAAEWESRKPVPPPLPALGTGRELFALMRSVHD
PAEAGGSAMLAQMDRVIEAVGDDIH R. flavefaciens phosphogluconate
dehydratase/DHAD (SEQ. ID. NO: 87)
ATGAGCGATAATTTTTTCTGCGAGGGTGCGGATAAAGCCCCTCAGCGTTCACTTTTCAATGCA
CTGGGCATGACTAAAGAGGAAATGAAGCGTCCCCTCGTTGGTATCGTTTCTTCCTACAATGAG
ATCGTTCCCGGCCATATGAACATCGACAAGCTGGTCGAAGCCGTTAAGCTGGGTGTAGCTAT
GGGCGGCGGCACTCCTGTTGTTTTCCCTGCTATCGCTGTATGCGACGGTATCGCTATGGGTC
ACACAGGCATGAAGTACAGCCTTGTTACCCGTGACCTTATTGCCGATTCTACAGAGTGTATGG
CTCTTGCTCATCACTTCGACGCACTGGTAATGATACCTAACTGCGACAAGAACGTTCCCGGCC
TGCTTATGGCGGCTGCACGTATCAATGTTCCTACTGTATTCGTAAGCGGCGGCCCTATGCTTG
CAGGCCATGTAAAGGGTAAGAAGACCTCTCTTTCATCCATGTTCGAGGCTGTAGGCGCTTACA
CAGCAGGCAAGATAGACGAGGCTGAACTTGACGAATTCGAGAACAAGACCTGCCCTACCTGC
GGTTCATGTTCGGGTATGTATACCGCTAACTCCATGAACTGCCTCACTGAGGTACTGGGTATG
GGTCTCAGAGGCAACGGCACTATCCCTGCTGTTTACTCCGAGCGTATCAAGCTTGCAAAGCA
GGCAGGTATGCAGGTTATGGAACTCTACAGAAAGAATATCCGCCCTCTCGATATCATGACAGA
GAAGGCTTTCCAGAACGCTCTCACAGCTGATATGGCTCTTGGATGTTCCACAAACAGTATGCT
CCATCTCCCTGCTATCGCCAACGAATGCGGCATAAATATCAACCTTGACATGGCTAACGAGAT
AAGCGCCAAGACTCCTAACCTCTGCCATCTTGCACCGGCAGGCCACACCTACATGGAAGACC
TCAACGAAGCAGGCGGAGTTTATGCAGTTCTCAACGAGCTGAGCAAAAAGGGACTTATCAACA
CCGACTGCATGACTGTTACAGGCAAGACCGTAGGCGAGAATATCAAGGGCTGCATCAACCGT
GACCCTGAGACTATCCGTCCTATCGACAACCCATACAGTGAAACAGGCGGAATCGCCGTACT
CAAGGGCAATCTTGCTCCCGACAGATGTGTTGTGAAGAGAAGCGCAGTTGCTCCCGAAATGC
TGGTACACAAAGGCCCTGCAAGAGTATTCGACAGCGAGGAAGAAGCTATCAAGGTCATCTAT
GAGGGCGGTATCAAGGCAGGCGACGTTGTTGTTATCCGTTACGAAGGCCCTGCAGGCGGCC
CCGGCATGAGAGAAATGCTCTCTCCTACATCAGCTATACAGGGTGCAGGTCTCGGCTCAACT
GTTGCTCTAATCACTGACGGACGTTTCAGCGGCGCTACCCGTGGTGCGGCTATCGGACACGT
ATCCCCCGAAGCTGTAAACGGCGGTACTATCGCATATGTCAAGGACGGCGATATTATCTCCAT
CGACATACCGAATTACTCCATCACTCTTGAAGTATCCGACGAGGAGCTTGCAGAGCGCAAAAA
GGCAATGCCTATCAAGCGCAAGGAGAACATCACAGGCTATCTGAAGCGCTATGCACAGCAGG
TATCATCCGCAGACAAGGGCGCTATCATCAACAGGAAATAG R. flavefaciens
phosphogluconate dehydratase/DHAD-Amino Acid sequence (SEQ. ID. NO:
88)
MSDNFFCEGADKAPQRSLFNALGMTKEEMKRPLVGIVSSYNEIVPGHMNIDKLVEAVKLGVAMGG
GTPVVFPAIAVCDGIAMGHTGMKYSLVTRDLIADSTECMALAHHFDALVMIPNCDKNVPGLLMAAA
RINVPTVFVSGGPMLAGHVKGKKTSLSSMFEAVGAYTAGKIDEAELDEFENKTCPTCGSCSGMYT
ANSMNCLTEVLGMGLRGNGTIPAVYSERIKLAKQAGMQVMELYRKNIRPLDIMTEKAFQNALTAD
MALGCSTNSMLHLPAIANECGININLDMANEISAKTPNLCHLAPAGHTYMEDLNEAGGVYAVLNEL
SKKGLINTDCMTVTGKTVGENIKGCINRDPETIRPIDNPYSETGGIAVLKGNLAPDRCVVKRSAVAP
EMLVHKGPARVFDSEEEAIKVIYEGGIKAGDVVVIRYEGPAGGPGMREMLSPTSAIQGAGLGSTVA
LITDGRFSGATRGAAIGHVSPEAVNGGTIAYVKDGDIISIDIPNYSITLEVSDEELAERKKAMPIKRKE
NITGYLKRYAQQVSSADKGAIINRK
[0421] Pair wise homology comparisons for various edd proteins are
presented in the table below. The comparisons were made using
ClustalW software (ClustalW and ClustalX version 2; Larkin M. A.,
Blackshields G., Brown N. P., Chema R., McGettigan P. A., McWilliam
H., Valentin F., Wallace I. M., Wilm A., Lopez R., Thompson J. D.,
Gibson T. J. and Higgins D. G., Bioinformatics 2007 23(21):
2947-2948). ClustalW is a free alignment tool available at the
European Bioinformatics Institute website (e.g., world wide web
uniform resource locator ebi.ac.uk, specific ClustalW location is
ebi.ac.uk/Tools/clustalw2/index.html). PAO1=Pseudomonas aeruginosa
PAO1, E.C.=Eschericia coli, S.O.=S. oneidensis, G.O.=G. oxydans,
R.F.=Ruminococcus flavefaciens.
TABLE-US-00073 PAO1 E.C. S.O. G.O. R.F. PAO1 100 62 62 55 29 E.C.
62 100 66 56 30 S.O. 62 66 100 56 28 G.O. 55 56 56 100 28 R.F. 29
30 28 28 100
TABLE-US-00074 S. oneidensis keto-hydroxyglutarate-aldolase/keto-
deoxy-phosphogluconate aldolase (eda) (SEQ. ID. NO: 89)
ATGCTTGAGAATAACTGGTCATTACAACCACAAGATATTTTTAAACGCA
GCCCTATTGTTCCTGTTATGGTGATTAACAAGATTGAACATGCGGTGCC
CTTAGCTAAAGCGCTGGTTGCCGGAGGGATAAGCGTGTTGGAAGTGACA
TTACGCACGCCATGCGCCCTTGAAGCTATCACCAAAATCGCCAAGGAAG
TGCCTGAGGCGCTGGTTGGCGCGGGGACTATTTTAAATGAAGCCCAGCT
TGGACAGGCTATCGCCGCTGGTGCGCAATTTATTATCACTCCAGGTGCG
ACAGTTGAGCTGCTCAAAGCGGGCATGCAAGGACCGGTGCCGTTAATTC
CGGGCGTTGCCAGTATTTCCGAGGTGATGACGGGCATGGCGCTGGGCTA
CACTCACTTTAAATTCTTCCCTGCTGAAGCGTCAGGTGGCGTTGATGCG
CTTAAGGCTTTCTCTGGGCCGTTAGCAGATATCCGCTTCTGCCCAACAG
GTGGAATTACCCCGAGCAGCTATAAAGATTACTTAGCGCTGAAGAATGT
CGATTGTATTGGTGGCAGCTGGATTGCTCCTACCGATGCGATGGAGCAG
GGCGATTGGGATCGTATCACTCAGCTGTGTAAAGAGGCGATTGGCGGACT TTAA S.
oneidensis keto-hydroxyglutarate-aldolase/keto-
deoxy-phosphogluconate aldolase (eda)-Amino Acid sequence (SEQ. ID.
NO: 90) MLENNWSLQPQDIFKRSPIVPVMVINKIEHAVPLAKALVAGGISVLEVT
LRTPCALEAITKIAKEVPEALVGAGTILNEAQLGQAIAAGAQFIITPGA
TVELLKAGMQGPVPLIPGVASISEVMTGMALGYTHFKFFPAEASGGVDA
LKAFSGPLADIRFCPTGGITPSSYKDYLALKNVDCIGGSWIAPTDAMEQ GDWDRITQLCKEAIGGL
G. oxydans keto-hydroxyglutarate-aldolase/keto-
deoxy-phosphogluconate aldolase (eda) (SEQ. ID. NO: 91)
ATGATCGATACTGCCAAACTCGACGCCGTCATGAGCCGTTGTCCGGTCA
TGCCGGTGCTGGTGGTCAATGATGTGGCTCTGGCCCGCCCGATGGCCGA
GGCTCTGGTGGCGGGTGGACTGTCCACGCTGGAAGTCACGCTGCGCACG
CCCTGCGCCCTTGAAGCTATTGAGGAAATGTCGAAAGTACCAGGCGCGC
TGGTCGGTGCCGGTACGGTGCTGAATCCGTCCGACATGGACCGTGCCGT
GAAGGCGGGTGCGCGCTTCATCGTCAGCCCCGGCCTGACCGAGGCGCTG
GCAAAGGCGTCGGTTGAGCATGACGTCCCCTTCCTGCCAGGCGTTGCCA
ATGCGGGTGACATCATGCGGGGTCTGGATCTGGGTCTGTCACGCTTCAA
GTTCTTCCCGGCTGTGACGAATGGCGGCATTCCCGCGCTCAAGAGCTTG
GCCAGTGTTTTTGGCAGCAATGTCCGTTTCTGCCCCACGGGCGGCATTA
CGGAAGAGAGCGCACCGGACTGGCTGGCGCTTCCCTCCGTGGCCTGCGT
CGGCGGATCCTGGGTGACGGCCGGCACGTTCGATGCGGACAAGGTCCGT
CAGCGCGCCACGGCTGCGGCACTCTTCACGGTCTGA G. oxydans
keto-hydroxyglutarate-aldolase/keto- deoxy-phosphogluconate
aldolase (eda)-Amino Acid (SEQ. ID. NO: 92)
MIDTAKLDAVMSRCPVMPVLVVNDVALARPMAEALVAGGLSTLEVTLRT
PCALEAIEEMSKVPGALVGAGTVLNPSDMDRAVKAGARFIVSPGLTEAL
AKASVEHDVPFLPGVANAGDIMRGLDLGLSRFKFFPAVTNGGIPALKSL
ASVFGSNVRFCPTGGITEESAPDWLALPSVACVGGSWVTAGTFDADKVR QRATAAALFTV
[0422] Pair wise homology comparisons for various eda proteins are
presented in the table below. The comparisons were made using
ClustalW software (ClustalW and ClustalX version 2; Larkin M. A.,
Blackshields G., Brown N. P., Chema R., McGettigan P. A., McWilliam
H., Valentin F., Wallace I. M., Wilm A., Lopez R., Thompson J. D.,
Gibson T. J. and Higgins D. G., Bioinformatics 2007 23(21):
2947-2948). PAO1=Pseudomonas aeruginosa PAO1, E.C.=Eschericia coli,
S.O.=S. oneidensis, G.O.=G. oxydans, R.F.=Ruminococcus
flavefaciens.
TABLE-US-00075 PAO1 E.C. S.O. G.O. PAO1 100 41 44 40 E.C. 41 100 60
46 S.O. 44 60 100 45 G.O. 40 46 45 100
[0423] All oligonucleotides set forth above were purchased from
Integrated technologies ("IDT", Coralville, Iowa). These
oligonucleotides were designed to incorporate a SpeI restriction
endonuclease cleavage site upstream and an XhoI restriction
endonuclease cleavage site downstream of the edd and eda gene
constructs, such that the sites could be used to clone the genes
into yeast expression vectors p426GPD (ATCC accession number 87361)
and p425GPD (ATCC accession number 87359). In addition to
incorporating restriction endonuclease cleavage sites, the forward
oligonucleotides were designed to incorporate six consecutive A
nucleotides immediately upstream of the ATG initiation codon.
[0424] PCR amplification of the genes were performed as follows:
about 100 ng of the genomic DNA was added to 1.times.Pfu Ultra II
buffer, 0.3 mM dNTPs, 0.3 .mu.mol gene-specific primers and 1 U Pfu
Ultra II polymerase (Agilent, La Jolla, Calif.) in a 50 .mu.l
reaction mix. The reaction mixture was cycled as follows:
95.degree. C. 10 minutes followed by 30 rounds of 95.degree. C. for
20 seconds, 50.degree. C. (eda amplifications) or 53.degree. C.
(edd amplifications) for 30 seconds, and 72.degree. C. for 15
seconds (eda amplifications) or 30 seconds (edd amplifications). A
final 5 minute extension reaction at 72.degree. C. was also
included. Each amplified product was TOPO cloned into the pCR Blunt
II TOPO vector (Life Technologies, Carlsbad, Calif.) according to
the manufacturer's recommendations and the sequences verified
(GeneWiz, La Jolla, Calif.).
[0425] Cloning of New edd and eda Genes into Yeast Expression
Vectors
[0426] Each of the sequence-verified eda and edd fragments were
subcloned into the corresponding restriction sites in plasmids
p425GPD and p426GPD vectors (ATCC #87361; PubMed: 7737504).
Briefly, about SOng of SpeI-XhoI-digested p425GPD vector was
ligated to about 50 ng of SpeI/XhoI -restricted eda or edd fragment
in a 10 .mu.l reaction with 1.times.T4 DNA ligase buffer and 1 U T4
DNA ligase (Fermentas) overnight at 16.degree. C. About 3 .mu.l of
this reaction was used to transform DH5.alpha. competent cells
(Zymo Research) and plated onto LB agar media containing 100
.mu.g/ml ampicillin. Final constructs were confirmed by restriction
endonuclease digests and sequence verification (GeneWiz, La Jolla,
Calif.).
[0427] In Vivo Assay to Determine Optimal EDD/EDA Combination
[0428] To determine the optimal EDD/EDA gene combinations, a yeast
strain was developed to enable in vivo gene combination evaluation.
Growth on glucose was impaired in this strain by disrupting both
copies of phosphofructokinase (PFK), however, the strain could grow
normally on galactose due to the presence of a single plasmid copy
of the PFK2 gene under the control of a GAL1 promoter. The strain
can only grow on glucose if a functional EDD/EDA is present in the
cell. The strain was generated using strain BF205 (YGR240C/BY4742,
ATCC Cat. No. 4015893; Winzeler E A, et al. Science 285: 901-906,
1999, PubMed: 10436161) as the starting strain.
[0429] PFK2 Expressing Plasmid
[0430] The plasmid expressing the PFK2 gene under the control of
the GAL1 promoter, for use in the in vivo edd/eda gene combination
evaluations, was constructed by first isolating the PFK2 gene.
Primers JML/89 and JML/95 were used to amplify the PFK2 gene from
BY4742 in a PCR reaction containing about 100 ng of the genomic
DNA, 1.times.Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3 .mu.mol
gene-specific primers, and 1 U Pfu Ultra II polymerase (Agilent, La
Jolla, Calif.) in a 50 .mu.l reaction mix. The reactions were
cycled as follows: 95.degree. C. for 10 minutes followed by 10
rounds of 95.degree. C. for 20 seconds, 55.degree. C. for 20
seconds, and 72.degree. C. for 90 seconds and 25 rounds of
95.degree. C. for 20 seconds, 62.degree. C. for 20 seconds, and
72.degree. C. for 90 seconds. A final 5 minute extension reaction
at 72.degree. C. was also included. Each amplified product was TOPO
cloned into the pCR Blunt II TOPO vector (Life Technologies,
Carlsbad, Calif.) according to the manufacturer's recommendations
and sequence verified (GeneWiz, San Diego, Calif.). The sequences
of JML/89 and JML/95 are given below.
TABLE-US-00076 JML/89 (SEQ ID NO: 343)
ACTAGTATGACTGTTACTACTCCTTTTGTGAATGGTAC JML/95 (SEQ ID NO: 344)
CTCGAGTTAATCAACTCTCTTTCTTCCAACCAAATGGTC
[0431] The primers used were designed to include a unique SpeI
restriction site at the 5' end of the gene and a unique XhoI
restriction site at the 3' end of the gene. This SpeI-XhoI fragment
(approximately 2900 bp) was cloned into the SpeI-XhoI sites of the
yeast vector p416GAL (ATCC Cat. No. 87332; Mumberg D, et al.,
Nucleic Acids Res. 22: 5767-5768, 1994. PubMed: 7838736) in a 10
.mu.l ligation reaction containing about 50 ng of the p416GAL
plasmid and about 100 ng of the PFK2 fragment with 1.times.
ligation buffer and 1 U T4 DNA ligase (Fermentas). This ligation
reaction was allowed to incubate at room temperature for about one
hour and was transformed into competent DH5.alpha. (Zymo Research,
Orange, Calif.) and plated onto LB plates containing 100 .mu.g/ml
ampicillin. The final plasmid was verified by restriction digests
and sequence confirmed (GeneWiz, San Diego, Calif.) and was called
pBF744. Plasmid pBF744 was transformed in yeast strain BF205
(BY4742 pfk1) using the procedure outlined below. This resulting
strain was called BF1477. [0432] 1. Inoculate 5 mLs YPD with a
single yeast colony. Grow 0/N at 30.degree. C. [0433] 2. Next day:
add 50 .mu.l culture to 450 .mu.l fresh YPD, check A660. Add
suitable amount of cells to 60 mLs fresh YPD to give an A660=0.2
(2.times.10.sup.6 cells/mL). Grow to A660=1.0 (2.times.10.sup.7
cells/mL), approximately 5 hours. [0434] 3. Boil a solution of 10
mg/ml salmon sperm DNA for 5 min, then quick chill on ice. [0435]
4. Spin down 50 mL cells at 3000 rpm for 5 min, wash in 10 mL
sterile water, recentrifuge. [0436] 5. Resuspend in 1 mL sterile
water. Transfer to 1.5 mL sterile microfuge tube, spin down. [0437]
6. Resuspend in 1 mL sterile TE/LiOAC solution. Spin down,
resuspend in 0.25 mLs TE/LiOAc (4.times.10.sup.9 cells). [0438] 7.
In a 1.5 mL microfuge tube, mix 50 .mu.l yeast cells with 1-5 .mu.g
transforming DNA and 5 .mu.l single stranded carrier DNA (boiled
salmon sperm DNA). [0439] 8. Add 300 .mu.l sterile PEG solution.
Mix thoroughly. Incubate at 30.degree. C. for 60 min with gentle
mixing every 15 min. [0440] 9. Add 40 .mu.l DMSO, mix thoroughly.
Heat shock at 42.degree. C. for 15 min. [0441] 10. Microfuge cells
at 13000 rpm for 30 seconds, remove supernatant. Resuspend in 1 mL
1.times.TE, microfuge 30 sec. Resuspend in 1 mL 1.times.TE. Plate
100-200 .mu.l on selective media (SCD-ura).
[0442] pfk2 Knockout Cassette
[0443] A knockout cassette for the PFK2 gene was constructed by
first PCR amplifying about 300 bp of the 5' and 3' flanking regions
of the PFK2 gene from S. cerevisiae, strain BY4742 using primers
JML/85 and JML/87 and primers JML/86 and JML/88, respectively.
These flanking regions were designed such that the 5' flanking
region had a HindIII site at its 5' edge and a BamHI site at its 3'
end. The 3' flanking region had a BamHI site at its 5' edge and a
EcoRI site at its 3' edge. The nucleotide sequence of the PFK2 gene
and the primers used for amplification of the PFK2 gene are given
below.
TABLE-US-00077 S. cerevisiae PFK2 (from genomic sequence) SEQ. ID.
NO: 121 ATGACTGTTACTACTCCTTTTGTGAATGGTACTTCTTATTGTACCGTCA
CTGCATATTCCGTTCAATCTTATAAAGCTGCCATAGATTTTTACACCAA
GTTTTTGTCATTAGAAAACCGCTCTTCTCCAGATGAAAACTCCACTTTA
TTGTCTAACGATTCCATCTCTTTGAAGATCCTTCTACGTCCTGATGAAA
AAATCAATAAAAATGTTGAGGCTCATTTGAAGGAATTGAACAGTATTAC
CAAGACTCAAGACTGGAGATCACATGCCACCCAATCCTTGGTATTTAAC
ACTTCCGACATCTTGGCAGTCAAGGACACTCTAAATGCTATGAACGCTC
CTCTTCAAGGCTACCCAACAGAACTATTTCCAATGCAGTTGTACACTTT
GGACCCATTAGGTAACGTTGTTGGTGTTACTTCTACTAAGAACGCAGTT
TCAACCAAGCCAACTCCACCACCAGCACCAGAAGCTTCTGCTGAGTCTG
GTCTTTCCTCTAAAGTTCACTCTTACACTGATTTGGCTTACCGTATGAA
AACCACCGACACCTATCCATCTCTGCCAAAGCCATTGAACAGGCCTCAA
AAGGCAATTGCCGTCATGACTTCCGGTGGTGATGCTCCAGGTATGAACT
CTAACGTTAGAGCCATCGTGCGTTCCGCTATCTTCAAAGGTTGTCGTGC
CTTTGTTGTCATGGAAGGTTATGAAGGTTTGGTTCGTGGTGGTCCAGAA
TACATCAAGGAATTCCACTGGGAAGACGTCCGTGGTTGGTCTGCTGAAG
GTGGTACCAACATTGGTACTGCCCGTTGTATGGAATTCAAGAAGCGCGA
AGGTAGATTATTGGGTGCCCAACATTTGATTGAGGCCGGTGTCGATGCT
TTGATCGTTTGTGGTGGTGACGGTTCTTTGACTGGTGCTGATCTGTTTA
GATCAGAATGGCCTTCTTTGATCGAGGAATTGTTGAAAACAAACAGAAT
TTCCAACGAACAATACGAAAGAATGAAGCATTTGAATATTTGCGGTACT
GTCGGTTCTATTGATAACGATATGTCCACCACGGATGCTACTATTGGTG
CTTACTCTGCCTTGGACAGAATCTGTAAGGCCATCGATTACGTTGAAGC
CACTGCCAACTCTCACTCAAGAGCTTTCGTTGTTGAAGTTATGGGTAGA
AACTGTGGTTGGTTAGCTTTATTAGCTGGTATCGCCACTTCCGCTGACT
ATATCTTTATTCCAGAGAAGCCAGCCACTTCCAGCGAATGGCAAGATCA
AATGTGTGACATTGTCTCCAAGCACAGATCAAGGGGTAAGAGAACCACC
ATTGTTGTTGTTGCAGAAGGTGCTATCGCTGCTGACTTGACCCCAATTT
CTCCAAGCGACGTCCACAAAGTTCTAGTTGACAGATTAGGTTTGGATAC
AAGAATTACTACCTTAGGTCACGTTCAAAGAGGTGGTACTGCTGTTGCT
TACGACCGTATCTTGGCTACTTTACAAGGTCTTGAGGCCGTTAATGCCG
TTTTGGAATCCACTCCAGACACCCCATCACCATTGATTGCTGTTAACGA
AAACAAAATTGTTCGTAAACCATTAATGGAATCCGTCAAGTTGACCAAA
GCAGTTGCAGAAGCCATTCAAGCTAAGGATTTCAAGAGAGCTATGTCTT
TAAGAGACACTGAGTTCATTGAACATTTAAACAATTTCATGGCTATCAA
CTCTGCTGACCACAACGAACCAAAGCTACCAAAGGACAAGAGACTGAAG
ATTGCCATTGTTAATGTCGGTGCTCCAGCTGGTGGTATCAACTCTGCCG
TCTACTCGATGGCTACTTACTGTATGTCCCAAGGTCACAGACCATACGC
TATCTACAATGGTTGGTCTGGTTTGGCAAGACATGAAAGTGTTCGTTCT
TTGAACTGGAAGGATATGTTGGGTTGGCAATCCCGTGGTGGTTCTGAAA
TCGGTACTAACAGAGTCACTCCAGAAGAAGCAGATCTAGGTATGATTGC
TTACTATTTCCAAAAGTACGAATTTGATGGTTTGATCATCGTTGGTGGT
TTCGAAGCTTTTGAATCTTTACATCAATTAGAGAGAGCAAGAGAAAGTT
ATCCAGCTTTCAGAATCCCAATGGTCTTGATACCAGCTACTTTGTCTAA
CAATGTTCCAGGTACTGAATACTCTTTGGGTTCTGATACCGCTTTGAAT
GCTCTAATGGAATACTGTGATGTTGTTAAACAATCCGCTTCTTCAACCA
GAGGTAGAGCCTTCGTTGTCGATTGTCAAGGTGGTAACTCAGGCTATTT
GGCCACTTACGCTTCTTTGGCTGTTGGTGCTCAAGTCTCTTATGTCCCAG
AAGAAGGTATTTCTTTGGAGCAATTGTCCGAGGATATTGAATACTTAGC
TCAATCTTTTGAAAAGGCAGAAGGTAGAGGTAGATTTGGTAAATTGATT
TTGAAGAGTACAAACGCTTCTAAGGCTTTATCAGCCACTAAATTGGCTG
AAGTTATTACTGCTGAAGCCGATGGCAGATTTGACGCTAAGCCAGCTTA
TCCAGGTCATGTACAACAAGGTGGTTTGCCATCTCCAATTGATAGAACA
AGAGCCACTAGAATGGCCATTAAAGCTGTCGGCTTCATCAAAGACAACC
AAGCTGCCATTGCTGAAGCTCGTGCTGCCGAAGAAAACTTCAACGCTGA
TGACAAGACCATTTCTGACACTGCTGCTGTCGTTGGTGTTAAGGGTTCA
CATGTCGTTTACAACTCCATTAGACAATTGTATGACTATGAAACTGAAG
TTTCCATGAGAATGCCAAAGGTCATTCACTGGCAAGCTACCAGACTCAT
TGCTGACCATTTGGTTGGAAGAAAGAGAGTTGATTAA JML/85 (SEQ ID NO: 345)
AAGCTTTTAATTAATATAACGCTATGACGGTAGTTGAATGTTAAAAAC JML/86 (SEQ ID NO:
346) GAATTCTTAATTAAAGAGAACAAAGTATTTAACGCACATGTATAAATAT TG JML/87
(SEQ ID NO: 347) GGATCCGCATGCGGCCGGCCAGCTTTTAATCAAGGAAGTAATAAATAA
AGGAC JML/88 (SEQ ID NO: 348)
GGATCCGAGCTCGCGGCCGCAGCTTTTGAACAATGAATTTTTTGTTCCTT TC
[0444] The nucleic acid fragments were amplified using the
following conditions; about 100 ng of the BY4742 genomic DNA was
added to 1.times.Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3 .mu.mol
gene-specific primers, and 1 U Pfu Ultra II polymerase (Agilent, La
Jolla, Calif.) in a 50 .mu.l reaction mix. The reaction was cycled
at 95.degree. C. for 10 minutes, followed by 30 rounds of
95.degree. C. for 20 seconds, 58.degree. C. for 30 seconds, and
72.degree. C. for 20 seconds. A final 5 minute extension reaction
at 72.degree. C. was also included. Each amplified product was TOPO
cloned into the pCR Blunt II TOPO vector (Life Technologies,
Carlsbad, Calif.) according to the manufacturer's recommendations
and the sequence of the construct was verified (GeneWiz, San Diego,
Calif.). The resulting plasmids were named pBF648 (5' flanking
region) and pBF649 (3' flanking region). A three fragment ligation
was performed using about 100 ng of the 5' flanking region
HindIII-BamHI fragment, about 100 ng of the 3' flanking region
BamHI-EcoRI fragment and about 50 ng of pUC19 digested with HindIII
and EcoRI in a 5 .mu.l ligation reaction containing 1.times.
ligation buffer and 1 U T4 DNA ligase (Fermentas). This reaction
was incubated at room temperature for about one hour. About 2 .mu.l
of this reaction mix was used to transform competent DH5.alpha.
cells (Zymo Research, Orange, Calif.) and plated onto LB agar media
containing 100 .mu.g/ml ampicillin. The final construct was
confirmed by restriction endonuclease digests and sequence
verification (GeneWiz, San Diego, Calif.), resulting in plasmid
pBF653.
[0445] Lys 2 Gene Cloning
[0446] The Lys2 gene was isolated by PCR amplification from pRS317
(ATCC Cat. No. 77157; Sikorski R S, Boeke J D. Methods Enzymol.
194: 302-318, 1991. PubMed: 2005795) using primers JML/93 and
JML/94. PCR amplification was performed as follows: about 25 ng of
the pRS317 plasmid DNA was added to 1.times.Pfu Ultra II buffer,
0.3 mM dNTPs, 0.3 .mu.mol gene-specific primers, and 1 U Pfu Ultra
II polymerase (Agilent, La Jolla, Calif.) in a 50 .mu.l reaction
mix. The reactions were cycled at: 95.degree. C. 10 minutes
followed by 10 rounds of 95.degree. C. for 20 seconds, 55.degree.
C. for 30 seconds, and 72.degree. C. for 2 minutes, followed by 25
more rounds of 95.degree. C. for 20 seconds, 62.degree. C. for 30
seconds, and 72.degree. C. for 2 minutes. A final 5 minute
extension reaction at 72.degree. C. was also included. The
amplified product was TOPO cloned into the pCR Blunt II TOPO vector
as described herein, resulting in plasmid pBF656. The nucleotide
sequence of Lys2 gene and the primers used for amplification of the
Lys2 gene are given below.
TABLE-US-00078 JML/93 (SEQ ID NO: 349)
GCGGCCGCAGCTTCGCAAGTATTCATTTTAGACCCATG JML/94 (SEQ ID NO: 350)
GGCCGGCCGGTACCAATTCCACTTGCAATTACATAAAAAATTCC Lys 2 (from genomic
sequence database), SEQ. ID. NO: 122
ATGACTAACGAAAAGGTCTGGATAGAGAAGTTGGATAATCCAACTCTTTCAGTGTTACCACAT
GACTTTTTACGCCCACAACAAGAACCTTATACGAAACAAGCTACATATTCGTTACAGCTACCTC
AGCTCGATGTGCCTCATGATAGTTTTTCTAACAAATACGCTGTCGCTTTGAGTGTATGGGCTG
CATTGATATATAGAGTAACCGGTGACGATGATATTGTTCTTTATATTGCGAATAACAAAATCTTA
AGATTCAATATTCAACCAACGTGGTCATTTAATGAGCTGTATTCTACAATTAACAATGAGTTGAA
CAAGCTCAATTCTATTGAGGCCAATTTTTCCTTTGACGAGCTAGCTGAAAAAATTCAAAGTTGC
CAAGATCTGGAAAGGACCCCTCAGTTGTTCCGTTTGGCCTTTTTGGAAAACCAAGATTTCAAAT
TAGACGAGTTCAAGCATCATTTAGTGGACTTTGCTTTGAATTTGGATACCAGTAATAATGCGCA
TGTTTTGAACTTAATTTATAACAGCTTACTGTATTCGAATGAAAGAGTAACCATTGTTGCGGAC
CAATTTACTCAATATTTGACTGCTGCGCTAAGCGATCCATCCAATTGCATAACTAAAATCTCTC
TGATCACCGCATCATCCAAGGATAGTTTACCTGATCCAACTAAGAACTTGGGCTGGTGCGATT
TCGTGGGGTGTATTCACGACATTTTCCAGGACAATGCTGAAGCCTTCCCAGAGAGAACCTGTG
TTGTGGAGACTCCAACACTAAATTCCGACAAGTCCCGTTCTTTCACTTATCGCGACATCAACC
GCACTTCTAACATAGTTGCCCATTATTTGATTAAAACAGGTATCAAAAGAGGTGATGTAGTGAT
GATCTATTCTTCTAGGGGTGTGGATTTGATGGTATGTGTGATGGGTGTCTTGAAAGCCGGCGC
AACCTTTTCAGTTATCGACCCTGCATATCCCCCAGCCAGACAAACCATTTACTTAGGTGTTGCT
AAACCACGTGGGTTGATTGTTATTAGAGCTGCTGGACAATTGGATCAACTAGTAGAAGATTAC
ATCAATGATGAATTGGAGATTGTTTCAAGAATCAATTCCATCGCTATTCAAGAAAATGGTACCA
TTGAAGGTGGCAAATTGGACAATGGCGAGGATGTTTTGGCTCCATATGATCACTACAAAGACA
CCAGAACAGGTGTTGTAGTTGGACCAGATTCCAACCCAACCCTATCTTTCACATCTGGTTCCG
AAGGTATTCCTAAGGGTGTTCTTGGTAGACATTTTTCCTTGGCTTATTATTTCAATTGGATGTC
CAAAAGGTTCAACTTAACAGAAAATGATAAATTCACAATGCTGAGCGGTATTGCACATGATCCA
ATTCAAAGAGATATGTTTACACCATTATTTTTAGGTGCCCAATTGTATGTCCCTACTCAAGATGA
TATTGGTACACCGGGCCGTTTAGCGGAATGGATGAGTAAGTATGGTTGCACAGTTACCCATTT
AACACCTGCCATGGGTCAATTACTTACTGCCCAAGCTACTACACCATTCCCTAAGTTACATCAT
GCGTTCTTTGTGGGTGACATTTTAACAAAACGTGATTGTCTGAGGTTACAAACCTTGGCAGAA
AATTGCCGTATTGTTAATATGTACGGTACCACTGAAACACAGCGTGCAGTTTCTTATTTCGAAG
TTAAATCAAAAAATGACGATCCAAACTTTTTGAAAAAATTGAAAGATGTCATGCCTGCTGGTAA
AGGTATGTTGAACGTTCAGCTACTAGTTGTTAACAGGAACGATCGTACTCAAATATGTGGTATT
GGCGAAATAGGTGAGATTTATGTTCGTGCAGGTGGTTTGGCCGAAGGTTATAGAGGATTACCA
GAATTGAATAAAGAAAAATTTGTGAACAACTGGTTTGTTGAAAAAGATCACTGGAATTATTTGG
ATAAGGATAATGGTGAACCTTGGAGACAATTCTGGTTAGGTCCAAGAGATAGATTGTACAGAA
CGGGTGATTTAGGTCGTTATCTACCAAACGGTGACTGTGAATGTTGCGGTAGGGCTGATGATC
AAGTTAAAATTCGTGGGTTCAGAATCGAATTAGGAGAAATAGATACGCACATTTCCCAACATCC
ATTGGTAAGAGAAAACATTACTTTAGTTCGCAAAAATGCCGACAATGAGCCAACATTGATCACA
TTTATGGTCCCAAGATTTGACAAGCCAGATGACTTGTCTAAGTTCCAAAGTGATGTTCCAAAGG
AGGTTGAAACTGACCCTATAGTTAAGGGCTTAATCGGTTACCATCTTTTATCCAAGGACATCAG
GACTTTCTTAAAGAAAAGATTGGCTAGCTATGCTATGCCTTCCTTGATTGTGGTTATGGATAAA
CTACCATTGAATCCAAATGGTAAAGTTGATAAGCCTAAACTTCAATTCCCAACTCCCAAGCAAT
TAAATTTGGTAGCTGAAAATACAGTTTCTGAAACTGACGACTCTCAGTTTACCAATGTTGAGCG
CGAGGTTAGAGACTTATGGTTAAGTATATTACCTACCAAGCCAGCATCTGTATCACCAGATGAT
TCGTTTTTCGATTTAGGTGGTCATTCTATCTTGGCTACCAAAATGATTTTTACCTTAAAGAAAAA
GCTGCAAGTTGATTTACCATTGGGCACAATTTTCAAGTATCCAACGATAAAGGCCTTTGCCGC
GGAAATTGACAGAATTAAATCATCGGGTGGATCATCTCAAGGTGAGGTCGTCGAAAATGTCAC
TGCAAATTATGCGGAAGACGCCAAGAAATTGGTTGAGACGCTACCAAGTTCGTACCCCTCTCG
AGAATATTTTGTTGAACCTAATAGTGCCGAAGGAAAAACAACAATTAATGTGTTTGTTACCGGT
GTCACAGGATTTCTGGGCTCCTACATCCTTGCAGATTTGTTAGGACGTTCTCCAAAGAACTAC
AGTTTCAAAGTGTTTGCCCACGTCAGGGCCAAGGATGAAGAAGCTGCATTTGCAAGATTACAA
AAGGCAGGTATCACCTATGGTACTTGGAACGAAAAATTTGCCTCAAATATTAAAGTTGTATTAG
GCGATTTATCTAAAAGCCAATTTGGTCTTTCAGATGAGAAGTGGATGGATTTGGCAAACACAG
TTGATATAATTATCCATAATGGTGCGTTAGTTCACTGGGTTTATCCATATGCCAAATTGAGGGA
TCCAAATGTTATTTCAACTATCAATGTTATGAGCTTAGCCGCCGTCGGCAAGCCAAAGTTCTTT
GACTTTGTTTCCTCCACTTCTACTCTTGACACTGAATACTACTTTAATTTGTCAGATAAACTTGT
TAGCGAAGGGAAGCCAGGCATTTTAGAATCAGACGATTTAATGAACTCTGCAAGCGGGCTCA
CTGGTGGATATGGTCAGTCCAAATGGGCTGCTGAGTACATCATTAGACGTGCAGGTGAAAGG
GGCCTACGTGGGTGTATTGTCAGACCAGGTTACGTAACAGGTGCCTCTGCCAATGGTTCTTCA
AACACAGATGATTTCTTATTGAGATTTTTGAAAGGTTCAGTCCAATTAGGTAAGATTCCAGATAT
CGAAAATTCCGTGAATATGGTTCCAGTAGATCATGTTGCTCGTGTTGTTGTTGCTACGTCTTTG
AATCCTCCCAAAGAAAATGAATTGGCCGTTGCTCAAGTAACGGGTCACCCAAGAATATTATTC
AAAGACTACTTGTATACTTTACACGATTATGGTTACGATGTCGAAATCGAAAGCTATTCTAAAT
GGAAGAAATCATTGGAGGCGTCTGTTATTGACAGGAATGAAGAAAATGCGTTGTATCCTTTGC
TACACATGGTCTTAGACAACTTACCTGAAAGTACCAAAGCTCCGGAACTAGACGATAGGAACG
CCGTGGCATCTTTAAAGAAAGACACCGCATGGACAGGTGTTGATTGGTCTAATGGAATAGGTG
TTACTCCAGAAGAGGTTGGTATATATATTGCATTTTTAAACAAGGTTGGATTTTTACCTCCACCA
ACTCATAATGACAAACTTCCACTGCCAAGTATAGAACTAACTCAAGCGCAAATAAGTCTAGTTG
CTTCAGGTGCTGGTGCTCGTGGAAGCTCCGCAGCAGCTTAA
[0447] The knockout cassette was fully assembled by cloning the
NotI-FseI LYS2 fragment from plasmid pBF656 into the NotI-FseI
sites located between the 5' and 3' flanking PFK2 regions in
plasmid pBF653. About 50 ng of plasmid pBF653 digested with NotI
and FseI was ligated to about 100 ng of the NotI-FseI LYS2 fragment
from plasmid pBF656 in a 5 .mu.l reaction containing 1.times.
ligation buffer and 1 U T4 DNA ligase (Fermentas) for about 1 hour
at room temperature. About 2 .mu.l of this reaction was used to
transform competent DH5.alpha. (Zymo Research, Orange, Calif.) and
plated on 100 .mu.g/ml ampicillin. The structure of the final
plasmid, pBF745, was confirmed by restriction enzyme digests. The
approximately 5 kbp PacI fragment containing the LYS2 cassette and
PFK2 flanking regions was gel extracted using the Zymoclean Gel DNA
Recovery Kit (Zymo Research, Orange, Calif.) according to the
manufacturer's conditions.
[0448] Strain BF1477 was transformed with the about 5 kbp PacI
fragment using the method described above (LiOAc/PEG method)
generating strain BF1411. Strain BF1411 has the ability to grow on
galactose as a carbon source, but cannot grow on glucose. Various
combinations of the EDD and EDA constructs can be expressed in this
strain and monitored for growth on glucose. Strains which show
growth on glucose (or the highest growth rate on glucose) can be
further characterized to determine which combination of EDD and EDA
genes is present. Using the strain and method described herein,
libraries of EDD and EDA genes can be screened for improved
activities and activity combinations in a host organism.
Example 24
Single Plasmid System for Industrial Yeast
[0449] A single plasmid system expressing EDD and EDA for
industrial yeast was constructed as follows: The approximately 2800
bp fragment containing the GPD1 promoter, EDD-PAO1 gene and CYC1
terminator from plasmid pBF291 (p426GPD with EDD-PAO1) was PCR
amplified using primers KAS/5'-BamHI-Pgpd and KAS/3'-NdeI-CYCt,
described below. About 25 ng of the plasmid DNA was added to
1.times.Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3 .mu.mol
gene-specific primers, and 1 U Pfu Ultra II polymerase (Agilent, La
Jolla, Calif.) in a 50 .mu.l reaction mix. The reaction was cycled
at 95.degree. C. for 10 minutes, followed by 30 rounds of
95.degree. C. for 20 seconds, 55.degree. C. for 30 seconds, and
72.degree. C. for 45 seconds. A final 5 minute extension reaction
at 72.degree. C. was also included. The amplified product was TOPO
cloned into the pCR Blunt II TOPO vector, as described herein, and
the final plasmid was sequence verified and designated, pBF475.
TABLE-US-00079 KAS/5'-BamHI-Pgpd (SEQ ID NO: 351)
GGATCCgtttatcattatcaatactcgccatttcaaag KAS/3'-NdeI-CYCt (SEQ ID NO:
352) CATATGttgggtaccggccgcaaattaaagccttcgagcg
[0450] An approximately 1500 bp KANMX4 cassette was PCR amplified
from plasmid pBF413 HO-poly-KanMX4-HO (ATCC Cat. No. 87804) using
primers KAS/5'-Bam_NdeI-KANMX4 and KAS/3'-Sal_NheI-KANMX4,
described below.
TABLE-US-00080 KAS/5'-Bam_NdeI-KANMX4 (SEQ ID NO: 353)
GGATTCagtcagatCATATGggtacccccgggttaattaaggcgcgccag atctg
KAS/3'-Sal_NheI-KANMX4 (SEQ ID NO: 354)
GTCGACaggcctactgtacgGCTAGCgaattcgagctcgttttcgacac tggatggcggc
[0451] About 25 ng of plasmid pBF413 HO-poly-KanMX4-HO DNA was
added to 1.times.Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3 .mu.mol
gene-specific primers and 1 U Pfu Ultra II polymerase (Agilent, La
Jolla, Calif.) in a 50 .mu.l reaction mix. The reaction was cycled
at 95.degree. C. for 10 minutes, followed by 30 rounds of
95.degree. C. for 20 seconds, 55.degree. C. for 30 seconds, and
72.degree. C. for 30 seconds. A final 5 minute extension reaction
at 72.degree. C. was also included. The amplified product was TOPO
cloned into the pCR Blunt II TOPO vector, as described herein. The
resulting plasmid was sequence verified and designated, pBF465.
[0452] An approximately 225 bp ADH1 terminator was PCR amplified
from the genome of BY4742 using primers KAS/5'-Xba-XhoI-ADHt and
KAS/3'-StuI-ADHS. The sequence of primers KAS/5'-Xba-XhoI-ADHt and
KAS/3'-StuI-ADHS is given below.
TABLE-US-00081 KAS/5'-Xba-XhoI-ADHt (SEQ ID NO: 355)
tctagaCTCGAGtaataagcgaatttcttatgatttatg KAS/3'-StuI-ADH5 (SEQ ID
NO: 356) aagcttAGGCCTggagcgatttgcaggcatttgc
[0453] About 100 ng of genomic DNA from BY4742 was added to
1.times.Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3 .mu.mol
gene-specific primers and 1 U Pfu Ultra II polymerase (Agilent, La
Jolla, Calif.) in a 50 .mu.l reaction mix. The reaction was cycled
at 95.degree. C. for 10 minutes, followed by 30 rounds of
95.degree. C. for 20 seconds, 55.degree. C. for 30 seconds, and
72.degree. C. for 15 seconds. A final 5 minute extension reaction
at 72.degree. C. was also included. The amplified product was TOPO
cloned into the pCR Blunt II TOPO vector according to the
manufacturer's recommendations and sequence verified. The resulting
plasmid was designated pBF437.
[0454] The TEF2 promoter was PCR amplified from the genome of
BY4742 using primers KAS/5'-Xba-XhoI-ADHt and KAS/3'-StuI-ADHS,
described below.
TABLE-US-00082 KAS/5'-Bam-NheI-Ptef (SEQ ID NO: 357)
GGATCCgctagcACCGCGAATCCTTACATCACACCC KAS/3'-XbaI-SpeI-Ptef (SEQ ID
NO: 358) tctagaCTCGAGtaataagcgaatttcttatgatttatg
[0455] About 100 ng of genomic DNA from BY4742 was added to
1.times.Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3 .mu.mol
gene-specific primers, and 1 U Pfu Ultra II polymerase (Agilent, La
Jolla, Calif.) in a 50 .mu.l reaction mix. This was cycled at
95.degree. C. for 10 minutes, followed by 30 rounds of 95.degree.
C. for 20 seconds, 55.degree. C. for 30 seconds, and 72.degree. C.
for 15 seconds. A final 5 minute extension reaction at 72.degree.
C. was also included. The amplified product was TOPO cloned into
the pCR Blunt II TOPO vector (Life Technologies, Carlsbad, Calif.)
according to the manufacturer's recommendations and sequence
verified (GeneWiz, San Diego, Calif.). The resulting plasmid was
called pBF440.
[0456] The EDA gene cassettes were constructed as follows: First
the TEF2 promoter from the plasmid pBF440 was digested with BamHI
and XbaI and was cloned into the BamHI and XbaI sites of pUC19
creating plasmid pBF480. Plasmid pBF480 was then digested with XbaI
and HindIII and was ligated to the XbaI-HindIII fragment from
plasmid pBF437 containing the ADH1 terminator, creating plasmid
pBF521. Plasmid pBF521 was then digested with SpeI and XhoI and
then ligated to either SpeI-XhoI fragment containing either the
PAO1 eda gene from plasmid pBF292 or the E. coli eda gene from
plasmid pBF268. The 2 plasmids generated, depending on the eda gene
chosen, were designated pBF523 (e.g., containing the PAO1-eda) and
pBF568 (e.g., containing the E. coli-eda), respectively. The
approximately 1386 bp TEF-EDA-ADHt cassette from either plasmid pBF
523 or pBF568 was then gel extracted using the NheI-StuI sites.
[0457] The final vector was generated by first altering the NdeI
site in pUC19 using the mutagenesis primers described below.
TABLE-US-00083 KAS/SDM-NdeI-pUC18-5 (SEQ ID NO: 359)
gattgtactgagagtgcacaatatgcggtgtgaaatacc KAS/SDM-NdeI-pUC18-3 (SEQ
ID NO: 360) ggtatttcacaccgcatattgtgcactctcagtacaatc
[0458] About 50 ng of pUC19 plasmid DNA was added to 1.times.Pfu
Ultra II buffer, 0.3 mM dNTPs, 0.3 .mu.mol SDM-specific primers and
1 U Pfu Ultra II polymerase (Agilent, La Jolla, Calif.) in a 50
.mu.l reaction mix. The reaction was cycled at 95.degree. C. for 10
minutes, followed by 15 rounds of 95.degree. C. for 15 seconds,
55.degree. C. for 40 seconds, and 72.degree. C. for 3 minutes. A
final 10 minute extension reaction at 72.degree. C. was also
included. The PCR reaction mixture was then digested with 30 U of
DpnI for about 2 hours and 5 .mu.l of the digested PCR reaction
mixture was used to transform competent DH5.alpha. (Zymo Research,
Orange, Calif.) and plated onto LB plates containing 100 .mu.g/ml
ampicillin. The structure of the final plasmid, pBF421, was
confirmed by restriction digests.
[0459] An approximately 1359 bp EcoRI fragment containing the 2.mu.
yeast origin cassette was cloned into the EcoRI site of plasmid
pBF421 in a 10 .mu.l ligation reaction mixture containing 1.times.
ligation buffer, 50 ng of EcoRI-digested pBF421 80 ng of
EcoRI-digested 2.mu. cassette, and 1 U T4 DNA ligase (Fermentas).
The reaction was incubated at room temperature for about 2 hours
and 3 .mu.l of this was used to transform competent DH5.alpha.
(Zymo Research, Orange, Calif.). The structure of the resultant
plasmid, pBF429, was confirmed by restriction enzyme digests.
[0460] Plasmid pBF429 was then digested with BamHI and SalI and
ligated to the BamHI-SalI KANMX4 cassette described above. The
resultant plasmid, designated pBF515, was digested with BamHI and
NdeI and ligated to the BamHI-NdeI fragment containing the 2802 bp
GPD-EDD-CYCt fragment from pBF475. The resulting plasmid,
designated pBF522, was digested with NheI-StuI and was ligated to
the 1386 bp NheI-StuI TEF-EDA-ADHt fragment from plasmids pBF523 or
pBF568, creating final plasmids pBF524 and pBF612.
[0461] Expression levels of each of the single plasmid eda/edd
expression system vectors was assayed and compared against the
original eda/edd two plasmid expression system vectors. The
results, presented in FIG. 19, graphically illustrate edd/eda
coupled assay kinetics for the single and two plasmid systems. The
kinetics graphs for both expression systems show substantially
similar enzyme kinetics over the major of the time course.
Example 25
Chimeric Xylose Isomerase Activities
[0462] Chimeric Xylose Isomerase nucleotide sequences and
functional activities were generated that included an N-terminal
portion of Xylose isomerase from one donor organism and a
C-terminal portion of Xylose isomerase from a different donor
organism. In some embodiments the second donor organism was a
Ruminococcus bacteria. Given below are oligonucleotides utilized to
isolate and modify a nucleotide sequence encoding a xylose
isomerase activity. Also given below are non-limiting examples of
native and chimeric nucleotide and amino acid sequences encoding
xylose isomerase activities.
[0463] The native Ruminococcus flavefaciens nucleotide sequence
(SEQ ID NO: 22) utilized to generate chimeric xylose isomerase
activities is given below.
TABLE-US-00084 ATGGAATTTTTCAGCAATATCGGTAAAATTCAGTATCAGGGACCAAAAA
GTACTGATCCTCTCTCATTTAAGTACTATAACCCTGAAGAAGTCATCAA
CGGAAAGACAATGCGCGAGCATCTGAAGTTCGCTCTTTCATGGTGGCAC
ACAATGGGCGGCGACGGAACAGATATGTTCGGCTGCGGCACAACAGACA
AGACCTGGGGACAGTCCGATCCCGCTGCAAGAGCAAAGGCTAAGGTTGA
CGCAGCATTCGAGATCATGGATAAGCTCTCCATTGACTACTATTGTTTC
CACGATCGCGATCTTTCTCCCGAGTATGGCAGCCTCAAGGCTACCAACG
ATCAGCTTGACATAGTTACAGACTATATCAAGGAGAAGCAGGGCGACAA
GTTCAAGTGCCTCTGGGGTACAGCAAAGTGCTTCGATCATCCAAGATTC
ATGCACGGTGCAGGTACATCTCCTTCTGCTGATGTATTCGCTTTCTCAG
CTGCTCAGATCAAGAAGGCTCTGGAGTCAACAGTAAAGCTCGGCGGTAA
CGGTTACGTTTTCTGGGGCGGACGTGAAGGCTATGAGACACTTCTTAAT
ACAAATATGGGACTCGAACTCGACAATATGGCTCGTCTTATGAAGATGG
CTGTTGAGTATGGACGTTCGATCGGCTTCAAGGGCGACTTCTATATCGA
GCCCAAGCCCAAGGAGCCCACAAAGCATCAGTACGATTTCGATACAGCT
ACTGTTCTGGGATTCCTCAGAAAGTACGGTCTCGATAAGGATTTCAAGA
TGAATATCGAAGCTAACCACGCTACACTTGCTCAGCATACATTCCAGCA
TGAGCTCCGTGTTGCAAGAGACAATGGTGTGTTCGGTTCTATCGACGCA
AACCAGGGCGACGTTCTTCTTGGATGGGATACAGACCAGTTCCCCACAA
ATATCTACGATACAACAATGTGTATGTATGAAGTTATCAAGGCAGGCGG
CTTCACAAACGGCGGTCTCAACTTCGACGCTAAGGCACGCAGAGGGAGC
TTCACTCCCGAGGATATCTTCTACAGCTATATCGCAGGTATGGATGCAT
TTGCTCTGGGCTTCAGAGCTGCTCTCAAGCTTATCGAAGACGGACGTAT
CGACAAGTTCGTTGCTGACAGATACGCTTCATGGAATACCGGTATCGGT
GCAGACATAATCGCAGGTAAGGCAGATTTCGCATCTCTTGAAAAGTATG
CTCTTGAAAAGGGCGAGGTTACAGCTTCACTCTCAAGCGGCAGACAGGA
AATGCTGGAGTCTATCGTAAATAACGTTCTTTTCAGTCTGTAA
[0464] The first 10 amino acids are underlined, and amino acids
11-15 are in bold font. The native sequence was originally cloned
into a pUC57 vector, called pBF202, which was utilized as the PCR
template for the 5' chimera constructs. The oligonucleotides used
to generate the 5' replacement nucleotide sequences (e.g.,
oligonucleotides used to replace the first 10 amino acids of the
Ruminococcus xylose isomerase protein) are given in the table
below. In some embodiments, greater or fewer than 10 amino acids
were replaced to maintain proper amino acid alignment between
xylose isomerase activities.
TABLE-US-00085 Oligonucleotide sequence (SEQ ID NOS 361--374, Name
respectively, in order of appearance) KAS/5-XR_Cp10
ACTAGTAAAAAATGAAAAATTACTTTCCAAATGTTCCAGAAGTACAGTATCAGGG ACCAAAAAG
KAS/5-XR-O10
ACTAGTAAAAAATGACTAAGGAATATTTCCCAACTATCGGCAAGATTCAGTATCA
GGGACCAAAAAG KAS/5-XR-Cth10
ACTAGTAAAAAATGGAATACTTCAAAAATGTACCACAAATAAAACAGTATCAGGG ACCAAAAAG
KAS/5-XR-Bth10
ACTAGTAAAAAATGGCAACAAAAGAATTTTTTCCGGGAATTGAAAAGATTCAGTA
TCAGGGACCAAAAAG KAS/5-XR-Bst10
ACTAGTAAAAAATGGCTTATTTTCCGAATATCGGCAAGATTCAGTATCAGGGACC AAAAAG
KAS/5-XR-Bun10
ACTAGTAAAAAATGGCTACCAAGGAATACTTCCCAGGTATTGGTAAGATCCAGT
ATCAGGGACCAAAAAG KAS/5-XR-Cce10
ACTAGTAAAAAATGTCAGAAGTATTTAGCGGTATTTCAAACATTCAGTATCAGGG ACCAAAAAG
KAS/5-XR-RF10
ACTAGTAAAAAATGGAATTTTTCAAGAACATAAGCAAGATCCAGTATCAGGGACC AAAAAG
KAS/5-XR-18P10
ACTAGTAAAAAATGAGCGAATTTTTTACAGGCATTTCAAAGATCCAGTATCAGGG ACCAAAAAG
KAS/5-XR-BV10
ACTAGTAAAAAATGAAATTTTTTGAAAATGTCCCTAAGGTACAGTATCAGGGACC AAAAAG
KAS/XI-Re6-10
CCTATTTTGACCAGCTCGATCGCGTTCAGTATCAGGGACCAAAAAGTACTGATC CTCTC
KAS/XI-Re1-10
actagtaaaaaaATGCAAGCCTATTTTGACCAGCTCGATCGCGTTCAGTATCAGG
KAS/3-XI-RF- ctcgagttacagactgaaaagaacgttatttacg NATIVE KAS/3-XI-RF-
ctcgagttagtgatggtggtggtgatgcagactgaaaagaacgttatttacg
Native-HISb
[0465] In the table above the following abbreviations are used:
Cp--Clostridium phytofermentans; O--Orpinomyces; Cth--Clostridium
thermohydrosulfuricum, Bth--Bacteroides thetaiotaomicron,
Bst--Bacillus stearothermophilus; Bun--Bacillus uniformis;
Cce--Clostridium cellulolyticum; RF--Ruminococcus flavefaciens FD1,
18P10--Ruminococcus 18P13; BV10--Clostridials genomosp BVAB3 str
UPII9-5; Re--E. coli.
[0466] All oligonucleotides set forth above were purchased from
Integrated Technologies ("IDT", Coralville, Iowa). The
oligonucleotides were designed to incorporate a SpeI restriction
endonuclease cleavage site upstream and an XhoI restriction
endonuclease cleavage site downstream of the new XI gene
constructs, to allow cloning into the yeast expression vector
p426GPD (ATCC accession number 87361), as described herein. In
addition to incorporating restriction endonuclease cleavage sites,
the forward oligonucleotides were designed to incorporate six
consecutive A nucleotides immediately upstream of the ATG
initiation codon.
[0467] PCR reactions to amplify the xylose isomerase genes were
performed using about 40 ng of the pBF202 plasmid (containing the
native XI-R gene in pUC57) DNA. The reactions were performed as
described previously herein, using the oligonucleotide primers
shown in the table above. Gene specific and for first and second
rounds of PCR amplification were added at a final concentration of
0.3 mmol. The about 1350 bp products were TOPO cloned into the pCR
Blunt II TOPO vector (Life Technologies, Carlsbad, Calif.)
according to the manufacturer's recommendations and sequenced
confirmed (GeneWiz, La Jolla, Calif.). For the 5' E. coli 10 amino
acid extension, the PCR reactions also were performed in two steps
with the following exceptions; the first reaction the nucleotides
corresponding to amino acids 6-10 from the E. coli XI were added
first using the 5' oligonucleotide KAS/XI-Re6-10 (see table above)
using the 3' oligonucleotide KAS/3-XI-RF-NATIVE (see table above).
Once the PCR product was confirmed by agarose gel electrophoresis,
nucleic acid was purified using the Zymo Research DNA Clean &
Concentrator-25 kit (Zymo Research, Orange, Calif.). In a second
PCR reaction, about 40 ng of this cleaned PCR product was used in a
second PCR reaction as outlined above but this time using the 5'
oligonucleotide KAS/XI-Re1-10 and either KAS/3-XI-RF-NATIVE or
KAS/3-XI-RF-Native-HISb, which generated the XI-R with 5' XI-E.
coli extensions. These products were also TOPO cloned as detailed
above and the sequence confirmed by sequence analysis. Following
sequence confirmation, the approximately 1350 bp SpeI-XhoI
fragments were cloned into the corresponding restriction sites in
the p425GPD vectors, as described above.
[0468] Chimeric Xylose Isomerase Activities with a 5' 150 Amino
Acid Replacement
[0469] Chimeric xylose isomerase proteins were also generated that
included greater that 10 or 15 5' amino acid replacements, in some
embodiments. Described herein are non-limiting examples of chimeric
xylose isomerase activities with a replacement of approximately 150
5' amino acids from a different donor organism. The first 450
nucleotides of the native xylose isomerase sequence given above can
be replaced with any of the sequences given in the table below to
create chimeric xylose isomerase activities with approximately the
150 5' amino acids donated by a different organism than
Ruminococcus flavefaciens.
TABLE-US-00086 5' Extension Source Nucleotides Piromyces
ATGGCTAAGGAATATTTCCCACAAATTCAAAAGATTAAGTTCGA (SEQ ID NO: 93)
AGGTAAGGATTCTAAGAATCCATTAGCCTTCCACTACTACGAT
GCTGAAAAGGAAGTCATGGGTAAGAAAATGAAGGATTGGTTAC
GTTTCGCCATGGCCTGGTGGCACACTCTTTGCGCCGAAGGTG
CTGACCAATTCGGTGGAGGTACAAAGTCTTTCCCATGGAACGA
AGGTACTGATGCTATTGAAATTGCCAAGCAAAAGGTTGATGCT
GGTTTCGAAATCATGCAAAAGCTTGGTATTCCATACTACTGTTT
CCACGATGTTGATCTTGTTTCCGAAGGTAACTCTATTGAAGAAT
ACGAATCCAACCTTAAGGCTGTCGTTGCTTACCTCAAGGAAAA
GCAAAAGGAAACCGGTATTAAGCTTCTCTGGAGTACTGCTAAC
GTCTTCGGTCACAAGCGTTACATGAAC Escherichia coli (SEQ
ATGCAAGCCTATTTTGACCAGCTCGATCGCGTTCGTTATGAAG ID NO: 94)
GCTCAAAATCCTCAAACCCGTTAGCATTCCGTCACTACAATCC
CGACGAACTGGTGTTGGGTAAGCGTATGGAAGAGCACTTGCG
TTTTGCCGCCTGCTACTGGCACACCTTCTGCTGGAACGGGGC
GGATATGTTTGGTGTGGGGGCGTTTAATCGTCCGTGGCAGCA
GCCTGGTGAGGCACTGGCGTTGGCGAAGCGTAAAGCAGATGT
CGCATTTGAGTTTTTCCACAAGTTACATGTGCCATTTTATTGCT
TCCACGATGTGGATGTTTCCCCTGAGGGCGCGTCGTTAAAAGA
GTACATCAATAATTTTGCGCAAATGGTTGATGTCCTGGCAGGC
AAGCAAGAAGAGAGCGGCGTGAAGCTGCTGTGGGGAACGGC
CAACTGCTTTACAAACCCTCGCTACGGCGCG Clostridium
ATGAAAAATTACTTTCCAAATGTTCCAGAAGTAAAATACGAAGG phytofermentans
CCCAAATTCAACGAATCCATTTGCTTTTAAATATTATGACGCAA (SEQ ID NO: 95)
ATAAAGTTGTAGCGGGTAAAACAATGAAAGAGCACTGTCGTTT
TGCATTATCTTGGTGGCATACTCTTTGTGCAGGTGGTGCTGAT
CCATTCGGTGTAACAACTATGGATAGAACCTACGGAAATATCA
CAGATCCAATGGAACTTGCTAAGGCAAAAGTTGACGCTGGTTT
CGAATTAATGACTAAATTAGGAATTGAATTCTTCTGTTTCCATG
ACGCAGATATTGCTCCAGAAGGTGATACTTTTGAAGAGTCAAA
GAAGAATCTTTTTGAAATCGTTGATTACATCAAAGAGAAGATGG
ATCAGACTGGTATCAAGTTATTATGGGGTACTGCTAATAACTTT AGTCATCCAAGATTTATGCAT
Orpinomyces ATGACTAAGGAATATTTCCCAACTATCGGCAAGATTAGATTCGA (SEQ ID
NO: 96) AGGTAAGGATTCTAAGAATCCAATGGCCTTCCACTACTATGAT
GCTGAAAAGGAAGTCATGGGTAAGAAAATGAAGGATTGGTTAC
GTTTCGCCATGGCCTGGTGGCACACTCTTTGCGCCGATGGTG
CTGACCAATTCGGTGTTGGTACTAAGTCTTTCCCATGGAATGA
AGGTACTGACCCAATTGCTATTGCCAAGCAAAAGGTTGATGCT
GGTTTTGAAATCATGACCAAGCTTGGTATTGAACACTACTGTTT
CCACGATGTTGATCTTGTTTCTGAAGGTAACTCTATTGAAGAAT
ACGAATCCAACCTCAAGCAAGTTGTTGCTTACCTTAAGCAAAA
GCAACAAGAAACTGGTATTAAGCTTCTCTGGAGTACTGCCAAT
GTTTTCGGTAACCCACGTTACATGAAC Clostridium
ATGGAATACTTCAAAAATGTACCACAAATAAAATATGAAGGACC thermohydrosulfuricum
AAAATCAAACAATCCATATGCATTTAAATTTTACAATCCAGATGA (SEQ ID NO: 97)
AATAATAGACGGAAAACCTTTAAAAGAACACTTGCGTTTTTCAG
TAGCGTACTGGCACACATTTACAGCCAATGGGACAGATCCATT
TGGAGCACCCACAATGCAAAGGCCATGGGACCATTTTACTGAC
CCTATGGATATTGCCAAAGCGAGAGTAGAAGCAGCCTTTGAAC
TATTTGAAAAACTCGACGTACCATTTTTCTGTTTCCATGACAGA
GATATAGCTCCGGAAGGAGAGACATTAAGGGAGACGAACAAA
AATTTAGATACAATAGTTGCAATGATAAAAGACTACTTAAAGAC
GAGCAAAACAAAAGTATTATGGGGCACAGCGAACCTTTTTTCA AATCCGAGATTTGTACAT
Bacteroides ATGGCAACAAAAGAATTTTTTCCGGGAATTGAAAAGATTAAATT
thetaiotaomicron (SEQ TGAAGGTAAAGATAGTAAGAACCCGATGGCATTCCGTTATTAC
ID NO: 98) GATGCAGAGAAGGTGATTAATGGTAAAAAGATGAAGGATTGGC
TGAGATTCGCTATGGCATGGTGGCACACATTGTGCGCTGAAG
GTGGTGATCAGTTCGGTGGCGGAACAAAGCAATTCCCATGGA
ATGGTAATGCAGATGCTATACAGGCAGCAAAAGATAAGATGGA
TGCAGGATTTGAATTCATGCAGAAGATGGGTATCGAATACTATT
GCTTCCATGACGTAGACTTGGTTTCGGAAGGTGCCAGTGTAGA
AGAATACGAAGCTAACCTGAAAGAAATCGTAGCTTATGCAAAA
CAGAAACAGGCAGAAACCGGTATCAAACTACTGTGGGGTACT
GCTAATGTATTCGGTCACGCCCGCTATATGAAC Bacillus
ATGGCTTATTTTCCGAATATCGGCAAGATTGCGTATGAAGGGC stearothermophilus
CGGAGTCGCGCAATCCGTTGGCGTTTAAGTTTTATAATCCAGA (SEQ ID NO: 99)
AGAAAAAGTCGGCGACAAAACAATGGAGGAGCATTTGCGCTTT
TCAGTGGCCTATTGGCATACGTTTACGGGGGATGGGTCGGAT
CCGTTTGGCGTCGGCAATATGATTCGTCCATGGAATAAGTACA
GCGGCATGGATCTGGCGAAGGCGCGCGTCGAGGCGGCGTTT
GAGCTGTTTGAAAAGCTGAACGTTCCGTTTTTCTGCTTCCATGA
CGTCGACATCGCGCCGGAAGGGGAAACGCTCAGCGAGACGT
ACAAAAATTTGGATGAAATTGTCGATATGATTGAAGAATACATG
AAAACAAGCAAAACGAAGCTGCTTTGGAATACGGCGAACTTGT TCAGCCATCCGCGCTTCGTTCAC
Bacillus uniformis ATGGCTACCAAGGAATACTTCCCAGGTATTGGTAAGATCAAAT (SEQ
ID NO: 100) TCGAAGGTAAGGAATCCAAGAACCCAATGGCCTTCAGATACTA
CGATGCTGACAAGGTTATCATGGGTAAGAAGATGTCTGAATGG
TTAAAGTTCGCTATGGCTTGGTGGCATACCTTGTGTGCTGAAG
GTGGTGACCAATTCGGTGGTGGTACCAAGAAATTCCCATGGAA
CGGTGAAGCTGACAAGGTCCAAGCTGCTAAGAACAAGATGGA
CGCTGGTTTCGAATTTATGCAAAAGATGGGTATTGAATACTACT
GTTTCCACGATGTTGACTTGTGTGAAGAAGCTGAAACCATCGA
AGAATACGAAGCTAACTTGAAGGAAATTGTTGCTTACGCTAAG
CAAAAGCAAGCTGAAACTGGTATCAAGCTATTATGGGGTACTG
CTAACGTCTTTGGTCATGCCAGATACATGAAC Clostridium
ATGTCAGAAGTATTTAGCGGTATTTCAAACATTAAATTTGAAGG cellulolyticum
AAGCGGGTCAGATAATCCATTAGCTTTTAAGTACTATGACCCTA (SEQ ID NO: 101)
AGGCAGTTATCGGCGGAAAGACAATGGAAGAACATCTGAGATT
CGCAGTTGCCTACTGGCATACTTTTGCAGCACCAGGTGCTGAC
ATGTTCGGTGCAGGATCATATGTAAGACCTTGGAATACAATGT
CCGATCCTCTGGAAATTGCAAAATACAAAGTTGAAGCAAACTTT
GAATTCATTGAAAAGCTGGGAGCACCTTTCTTCGCTTTCCATG
ACAGGGATATTGCTCCTGAAGGCGACACACTCGCTGAAACAAA
TAAAAACCTTGATACAATAGTTTCAGTAATTAAAGATAGAATGA
AATCCAGTCCGGTAAAGTTATTATGGGGAACTACAAATGCTTTC GGAAACCCAAGATTTATGCAT
Ruminococcus ATGGAATTTTTCAAGAACATAAGCAAGATCCCTTACGAGGGCA
flavefaciens FD1 AGGACAGCACAAATCCTCTCGCATTCAAGTACTACAATCCTGA (SEQ
ID NO: 102) TGAGGTAATTGACGGCAAGAAGATGCGTGACATTATGAAGTTT
GCTCTCTCATGGTGGCATACAATGGGCGGCGACGGAACAGAT
ATGTTCGGCTGCGGTACAGCTGACAAGACATGGGGCGAAAAT
GATCCTGCTGCAAGAGCTAAGGCTAAGGTTGACGCAGCTTTC
GAGATCATGCAGAAGCTCTCTATCGATTACTTCTGTTTCCACGA
CCGTGATCTTTCTCCTGAGTACGGCTCACTGAAGGACACAAAC
GCTCAGCTGGACATCGTTACAGATTACATCAAGGCTAAGCAGG
CTGAGACAGGTCTCAAGTGCCTCTGGGGTACAGCTAAGTGCTT CGATCACCCAAGATTCATGCAC
Ruminococcus 18P13 ATGAGCGAATTTTTTACAGGCATTTCAAAGATCCCCTTTGAGG (SEQ
ID NO: 103) GAAAGGCATCCAACAATCCCATGGCGTTCAAGTACTACAACCC
GGATGAGGTCGTAGGCGGCAAGACCATGCGGGAGCAGCTGA
AGTTTGCGCTGTCCTGGTGGCATACTATGGGGGGAGACGGTA
CGGACATGTTTGGTGTGGGTACCACCAACAAGAAGTTCGGCG
GAACCGATCCCATGGACATTGCTAAGAGAAAGGTAAACGCTGC
GTTTGAGCTGATGGACAAGCTGTCCATCGATTATTTCTGTTTCC
ACGACCGGGATCTGGCGCCGGAGGCTGATAATCTGAAGGAAA
CCAACCAGCGTCTGGATGAAATCACCGAGTATATTGCACAGAT
GATGCAGCTGAACCCGGACAAGAAGGTTCTGTGGGGTACTGC
AAATTGCTTCGGCAATCCCCGGTATATGCAT Clostriales genomosp
ATGAAATTTTTTGAAAATGTCCCTAAGGTAAAATATGAGGGAAG BVAB3 str UPII9-5
CAAGTCTACCAACCCGTTTGCATTTAAGTATTACAATCCTGAAG (SEQ ID NO: 104)
CGGTGATTGCCGGTAAAAAAATGAAGGATCACCTGAAATTCGC
GATGTCCTGGTGGCACACCATGACGGCGACCGGGCAAGACCA
GTTCGGTTCGGGGACGATGAGCCGAATATATGACGGGCAAAC
TGAACCGCTGGCCTTGGCCAAAGCCCGAGTGGATGCGGCTTT
CGATTTCATGGAAAAATTAAATATCGAATATTTTTGTTTTCATGA
TGCCGACTTGGCTCCAGAAGGTAACAGTTTGCAGGAACGCAA
CGAAAATTTGCAGGAAATGGTGTCTTACCTGAAACAAAAGATG
GCCGGAACTTCGATTAAGCTTTTATGGGGAACCTCGAATTGTT TCAGCAACCCTCGTTTTATGCAC
Bacillus stercoris ATGGCAACAAAAGAGTATTTTCCCGGAATAGGAAAAATCAAATT
(SEQ ID NO: 105) CGAAGGCAAAGAAAGTAAGAATCCTATGGCATTCCGCTACTAC
GATGCGGAAAAAGTAATCATGGGCAAGAAGATGAAAGATTGGT
TGAAGTTCTCTATGGCATGGTGGCATACACTCTGTGCAGAGGG
TGGTGACCAGTTCGGCGGCGGAACGAAACATTTCCCCTGGAA
CGGTGATGCCGATAAACTGCAGGCTGCCAAGAACAAAATGGA
TGCTGGTTTCGAGTTCATGCAGAAAATGGGCATCGAATATTAC
TGCTTCCACGATGTTGACCTTTGCGACGAGGCCGATACAATCG
AAGAGTACGAAGCAAACCTGAAAGCCATCGTTGCATACGCCAA
GCAAAAGCAGGAGGAAACAGGTATCAAACTGTTGTGGGGTAC
TGCCAACGTATTCGGTCATGCACGTTACATGAACG Thermus thermophilus
ATGTACGAGCCCAAACCGGAGCACAGGTTTACCTTTGGCCTTT (SEQ ID NO: 106)
GGACTGTGGGCAATGTGGGCCGTGATCCCTTCGGGGACGCG
GTTCGGGAGAGGCTGGACCCGGTTTACGTGGTTCATAAGCTG
GCGGAGCTTGGGGCCTACGGGGTAAACCTTCACGACGAGGAC
CTGATCCCGCGGGGCACGCCTCCTCAGGAGCGGGACCAGAT
CGTGAGGCGCTTCAAGAAGGCTCTCGATGAAACCGGCCTCAA
GGTCCCCATGGTCACCGCCAACCTCTTCTCCGACCCTGCTTTC AAGGAC
[0470] Xylose isomerase genes from additional bacteria were also
utilized as the C-terminal portion of chimeric xylose isomerase
activities. In some embodiments, the bacteria used as xylose
isomerase nucleotide sequence donors were additional Ruminococcus
bacteria. In certain embodiments, the bacteria used as xylose
isomerase nucleotide sequences donors were Clostridiales bacteria.
The native nucleotide and amino acid sequences of the additional
xylose isomerase genes utilized to create chimeric xylose isomerase
activities are given below. The 5' approximately 150 amino acids of
the sequences below can be replaced as described above, using the
sequences above, to create novel chimeric xylose isomerase
activities.
Nucleotide Sequences:
TABLE-US-00087 [0471] Ruminococcus_FD1 Xylose Isomerase
(ZP_06143883.1, SEQ ID NO: 107)
ATGGAATTTTTCAAGAACATAAGCAAGATCCCTTACGAGGGCAAGGACAGCACAAATCCTCTC
GCATTCAAGTACTACAATCCTGATGAGGTAATTGACGGCAAGAAGATGCGTGACATTATGAAG
TTTGCTCTCTCATGGTGGCATACAATGGGCGGCGACGGAACAGATATGTTCGGCTGCGGTAC
AGCTGACAAGACATGGGGCGAAAATGATCCTGCTGCAAGAGCTAAGGCTAAGGTTGACGCAG
CTTTCGAGATCATGCAGAAGCTCTCTATCGATTACTTCTGTTTCCACGACCGTGATCTTTCTCC
TGAGTACGGCTCACTGAAGGACACAAACGCTCAGCTGGACATCGTTACAGATTACATCAAGG
CTAAGCAGGCTGAGACAGGTCTCAAGTGCCTCTGGGGTACAGCTAAGTGCTTCGATCACCCA
AGATTCATGCACGGTGCAGGTACTTCACCATCCGCAGACGTATTCGCTTTCTCAGCTGCACAG
ATCAAGAAGGCTCTCGAGTCTACTGTAAAGCTCGGCGGTACAGGCTACGTATTCTGGGGCGG
ACGTGAGGGTTATGAGACTCTCCTCAACACAAACATGGGCCTTGAGCTTGACAACATGGCTC
GTCTCATGAAGATGGCTGTTGAGTACGGACGTTCTATCGGCTTCAAGGGCGATTTCTACATCG
AGCCTAAGCCAAAGGAGCCAACAAAGCACCAGTACGATTTCGATACTGCTACTGTTCTCGGCT
TCCTCAGAAAGTACGGTCTCGACAAGGATTTCAAGATGAACATCGAAGCTAACCACGCTACAC
TGGCTCAGCACACATTCCAGCACGAGCTCTGCGTAGCAAGAACAAACGGTGCTTTCGGTTCA
ATCGACGCAAACCAGGGCGATCCTCTCCTCGGATGGGATACAGACCAGTTCCCGACAAATAT
CTATGACACAACAATGTGTATGTACGAAGTTATCAAGGCTGGCGGCTTCACAAACGGCGGTCT
CAACTTCGATGCAAAGGCAAGACGTGGAAGCTTCACACCTGAGGATATCTTCTACAGCTACAT
TGCAGGTATGGATGCATTCGCTCTCGGCTACAAGGCTGCAAGCAAGCTCATCGCTGACGGAC
GTATCGACAGCTTCATTTCCGACCGCTACGCTTCATGGAGCGAGGGAATCGGTCTCGACATC
ATCTCAGGCAAGGCTGATATGGCTGCTCTTGAGAAGTATGCTCTCGAAAAGGGCGAGGTTAC
AGACTCTATTTCCAGCGGCAGACAGGAACTCCTCGAGTCTATCGTAAACAACGTTATATTCAAT
CTTTGA Ruminococcus_18P13 Xylose Isomerase (CBL17278.1, SEQ ID NO:
108)
ATGAGCGAATTTTTTACAGGCATTTCAAAGATCCCCTTTGAGGGAAAGGCATCCAACAATCCC
ATGGCGTTCAAGTACTACAACCCGGATGAGGTCGTAGGCGGCAAGACCATGCGGGAGCAGC
TGAAGTTTGCGCTGTCCTGGTGGCATACTATGGGGGGAGACGGTACGGACATGTTTGGTGTG
GGTACCACCAACAAGAAGTTCGGCGGAACCGATCCCATGGACATTGCTAAGAGAAAGGTAAA
CGCTGCGTTTGAGCTGATGGACAAGCTGTCCATCGATTATTTCTGTTTCCACGACCGGGATCT
GGCGCCGGAGGCTGATAATCTGAAGGAAACCAACCAGCGTCTGGATGAAATCACCGAGTATA
TTGCACAGATGATGCAGCTGAACCCGGACAAGAAGGTTCTGTGGGGTACTGCAAATTGCTTC
GGCAATCCCCGGTATATGCATGGTGCCGGCACTGCGCCCAATGCGGACGTGTTTGCATTTGC
AGCTGCGCAGATCAAAAAGGCAATTGAGATCACCGTAAAGCTGGGTGGCAAGGGCTATGTAT
TCTGGGGCGGCAGAGAGGGCTACGAAACGCTGCTGAACACCAATATGGGTCTGGAACTGGA
TAATATGGCACGGCTGCTGCATATGGCAGTGGACTATGCAAGAAGCATCGGCTTTACCGGCG
ACTTCTACATCGAGCCCAAGCCCAAGGAGCCTACCAAGCATCAGTATGATTTTGATACCGCAA
CCGTGATCGGCTTCCTGCGCAAGTATAATCTGGACAAGGACTTCAAGATGAACATCGAAGCCA
ACCACGCAACCCTTGCACAGCACACCTTCCAGCATGAACTGCGGGTAGCACGGGAGAACGG
CTTCTTTGGCTCCATCGATGCTAACCAGGGTGACACCCTGCTGGGCTGGGATACGGATCAGT
TCCCCACTAATACCTATGACGCAGCACTGTGTATGTACGAGGTACTCAAGGCTGGCGGTTTTA
CCAATGGCGGTCTGAACTTTGACTCCAAGGCACGGCGTGGATCCTTTGAGATGGAGGATATC
TTCCACAGCTACATTGCCGGTATGGACACCTTTGCACTGGGTCTGAAGATTGCGCAGAAGATG
ATCGATGACGGACGGATCGACCAGTTCGTGGCTGATCGGTATGCAAGCTGGAACACCGGCAT
CGGTGCGGATATCATTTCCGGCAAGGCAACCATGGCAGATTTGGAGGCTTACGCACTGAGCA
AGGGCGATGTGACCGCATCCCTCAAGAGCGGTCGTCAGGAATTGCTGGAAAGCATCCTGAAC
AATATTATGTTCAATCTTTAA Clostridiales_genomosp_BVAB3_UPII9-5 Xylose
Isomerase (YP_003474614.1, SEQ ID NO: 109)
ATGAAATTTTTTGAAAATGTCCCTAAGGTAAAATATGAGGGAAGCAAGTCTACCAACCCGTTTG
CATTTAAGTATTACAATCCTGAAGCGGTGATTGCCGGTAAAAAAATGAAGGATCACCTGAAATT
CGCGATGTCCTGGTGGCACACCATGACGGCGACCGGGCAAGACCAGTTCGGTTCGGGGACG
ATGAGCCGAATATATGACGGGCAAACTGAACCGCTGGCCTTGGCCAAAGCCCGAGTGGATGC
GGCTTTCGATTTCATGGAAAAATTAAATATCGAATATTTTTGTTTTCATGATGCCGACTTGGCTC
CAGAAGGTAACAGTTTGCAGGAACGCAACGAAAATTTGCAGGAAATGGTGTCTTACCTGAAAC
AAAAGATGGCCGGAACTTCGATTAAGCTTTTATGGGGAACCTCGAATTGTTTCAGCAACCCTC
GTTTTATGCACGGGGCAGCCACATCTTGCGAAGCGGATGTGTTTGCTTGGACCGCCACTCAG
TTGAAAAATGCCATCGATGCTACCATCGCGCTTGGCGGTAAAGGCTATGTTTTCTGGGGCGG
CCGGGAAGGCTATGAAACCTTGCTGAACACTGATGTCGGCCTGGAGATGGATAATTATGCAA
GAATGCTGAAAATGGCGGTTGCATATGCGCATTCTAAAGGTTATACGGGTGACTTTTATATTGA
ACCTAAGCCAAAAGAACCCACTAAACATCAATATGATTTCGATGTCGCCACTTGCGTTGCTTTC
CTTGAAAAATACGATTTGATGCGTGATTTTAAAGTAAACATTGAGGCTAATCACGCTACTTTGG
CCGGTCATACTTTCCAACATGAGTTACGCATGGCGCGTACCTTCGGGGTATTCGGCTCGGTT
GATGCCAATCAGGGCGACAGCAATCTGGGCTGGGATACCGATCAGTTCCCGGGCAATATTTA
TGATACGACTTTGGCCATGTATGAGATTTTGAAGGCCGGTGGATTTACCAACGGAGGCTTGAA
CTTTGATGCTAAAGTGCGTCGTCCGTCATTTACCCCGGAAGATATTGCTTATGCTTATATTTTG
GGCATGGATACGTTTGCCTTAGGCTTGATTAAGGCGCAACAGCTGATTGAGGATGGCAGAATT
GATCGTTTCGTAGCGGAAAAATATGCTAGTTATAAGTCGGGCATCGGTGCTGAAATCTTGAGT
GGTAAAACCGGTTTGCCGGAATTGGAGGCTTACGCATTGAAGAAAGGCGAGCCTAAGTTGTA
TAGTGGGCGGCAGGAATATCTTGAAAGTGTCGTTAATAACGTAATTTTCAACGGAAATCTTTGA
Amino Acid Sequences:
TABLE-US-00088 [0472] Ruminococcus_FD1 Xylose Isomerase (SEQ ID NO:
110)
MEFFKNISKIPYEGKDSTNPLAFKYYNPDEVIDGKKMRDIMKFALSWWHTMGGDGTDMFGCGTA
DKTWGENDPAARAKAKVDAAFEIMQKLSIDYFCFHDRDLSPEYGSLKDTNAQLDIVTDYIKAKQAE
TGLKCLWGTAKCFDHPRFMHGAGTSPSADVFAFSAAQIKKALESTVKLGGTGYVFWGGREGYET
LLNTNMGLELDNMARLMKMAVEYGRSIGFKGDFYIEPKPKEPTKHQYDFDTATVLGFLRKYGLDK
DFKMNIEANHATLAQHTFQHELCVARTNGAFGSIDANQGDPLLGWDTDQFPTNIYDTTMCMYEVI
KAGGFTNGGLNFDAKARRGSFTPEDIFYSYIAGMDAFALGYKAASKLIADGRIDSFISDRYASWSE
GIGLDIISGKADMAALEKYALEKGEVTDSISSGRQELLESIVNNVIFNL
Ruminococcus_18P13 Xylose Isomerase (SEQ ID NO: 111)
MSEFFTGISKIPFEGKASNNPMAFKYYNPDEVVGGKTMREQLKFALSWWHTMGGDGTDMFGVG
TTNKKFGGTDPMDIAKRKVNAAFELMDKLSIDYFCFHDRDLAPEADNLKETNQRLDEITEYIAQMM
QLNPDKKVLWGTANCFGNPRYMHGAGTAPNADVFAFAAAQIKKAIEITVKLGGKGYVFWGGREG
YETLLNTNMGLELDNMARLLHMAVDYARSIGFTGDFYIEPKPKEPTKHQYDFDTATVIGFLRKYNL
DKDFKMNIEANHATLAQHTFQHELRVARENGFFGSIDANQGDTLLGWDTDQFPTNTYDAALCMY
EVLKAGGFTNGGLNFDSKARRGSFEMEDIFHSYIAGMDTFALGLKIAQKMIDDGRIDQFVADRYAS
WNTGIGADIISGKATMADLEAYALSKGDVTASLKSGRQELLESILNNIMFN
Clostridiales_genomosp. BVAB3 str UPII9-5 Xylose Isomerase (SEQ ID
NO: 112)
MKFFENVPKVKYEGSKSTNPFAFKYYNPEAVIAGKKMKDHLKFAMSWWHTMTATGQDQFGSGT
MSRIYDGQTEPLALAKARVDAAFDFMEKLNIEYFCFHDADLAPEGNSLQERNENLQEMVSYLKQK
MAGTSIKLLWGTSNCFSNPRFMHGAATSCEADVFAWTATQLKNAIDATIALGGKGYVFWGGREG
YETLLNTDVGLEMDNYARMLKMAVAYAHSKGYTGDFYIEPKPKEPTKHQYDFDVATCVAFLEKYD
LMRDFKVNIEANHATLAGHTFQHELRMARTFGVFGSVDANQGDSNLGWDTDQFPGNIYDTTLAM
YEILKAGGFTNGGLNFDAKVRRPSFTPEDIAYAYILGMDTFALGLIKAQQLIEDGRIDRFVAEKYASY
KSGIGAEILSGKTGLPELEAYALKKGEPKLYSGRQEYLESVVNNVIFNGNL
[0473] Amino acid similarity comparisons were performed on the
various xylose isomerase proteins whose sequences were analyzed to
generate the chimeric xylose isomerase activity nucleotide
sequences. The results of the amino acid similarity comparison are
presented in the table below.
TABLE-US-00089 Ruminococcus_FD1 Ruminococcus_18P13
Clostriales_BVAB3 XI-R 88 77 65.8 Piromyces 50.9 50 51.6
Clostridium phytofermentans 64.9 64.4 68.3 Thermus thermophilus
25.1 22.9 26.6 Orpinomyces 51.6 51.8 52.5 Bacteroides
thetaiotaomicron 52.6 53 53 Escherichia coli 50.3 50.5 51.4
Clostridium 60.7 61.3 59.8 thermohydrosulfuricum Streptomyces
rubiginosus 22.7 23.8 24.7 Thermotoga maritima 61 60 61.3
Thermotoga neopolitana 60.5 59.7 60.8 Streptomyces murinus 24.2 23
25.1 Lactobacillus pentosus 49.7 50.7 47.7 Bacillus
stearothermophilus 55 57 56.5 Bacteroides uniformis 52.6 54.2 52.8
Clostridium cellulyticum 58.8 64.1 58.7 Ruminococcus_FD1 100 77.1
65.6 Ruminococcus_18P13 77.1 100 64 Clostriales_BVAB3 65.6 64 100
Bacteroides stercoris 51.9 52.4 53
Example 26
Nucleotide and Amino Acid Sequences of Over Expressed Activities
Useful for Increasing Sugar Transport and/or Sugar Metabolism
[0474] As noted herein, increased or over expression of certain
activities can result in increased ethanol production due to an
increase in the utilization of the fermentation substrate,
sometimes due to an increase in transport and/or metabolism of a
desired sugar. Non-limiting examples of activities that can be over
expressed to increase ethanol production by increasing sugar
transport and/or metabolism include activities encoded by the genes
gxf1, gxs1, hxt7, zwf1, gal2, soI3, sol4, the like, homologs
thereof (e.g., Candida albicans Sol1p, Schizosaccharomyces pombe
Sol1p, human PGLS and human H6PD), that can be expressed in a
desired host organism, and combinations thereof. Nucleotide and
amino acid sequences for some of these additional activities are
given below. In some embodiments, 1, 2, 3, 4, 5, 6 or more of the
non-limiting additional activities can be increased in expression
or over expressed in an engineered host, thereby increasing
transport and/or metabolism of a desired carbon source, wherein
increased transport and/or metabolism of a desired carbon source
results in increased ethanol production.
Nucleotide Sequences
TABLE-US-00090 [0475] Debaryomyces hansenii gxf1 (SEQ ID NO: 113)
ATGTCTCAAGAAGAATATAGTTCTGGGGTACAAACCCCAGTTTCTAACCATTCTGGTTTAGAGA
AAGAAGAGCAACACAAGTTAGACGGTTTAGATGAGGATGAAATTGTCGATCAATTACCTTCTTT
ACCAGAAAAATCAGCTAAGGATTATTTATTAATTTCTTTCTTCTGTGTATTAGTTGCATTTGGTG
GTTTTGTTTTCGGTTTCGATACTGGTACTATCTCAGGTTTCGTTAACATGAGTGATTACTTGGA
AAGATTCGGTGAGCTTAATGCAGATGGTGAATATTTCTTATCTAATGTTAGAACTGGTTTGATT
GTTGCTATTTTTAATGTTGGTTGTGCTGTCGGTGGTATTTTCTTATCTAAGATTGCTGATGTTTA
TGGTAGAAGAATTGGTCTTATGTTTTCCATGATTATTTATGTGATTGGTATAATTGTTCAAATCT
CAGCTTCTGACAAGTGGTATCAAATCGTTGTTGGTAGAGCTATTGCAGGTTTAGCTGTTGGTA
CCGTTTCTGTCTTATCCCCATTATTCATTGGTGAATCAGCACCTAAAACCTTAAGAGGTACTTT
AGTGTGTTGTTTCCAATTATGTATTACCTTAGGTATCTTCTTAGGTTACTGTACTACATATGGTA
CTAAAACCTACACCGACTCTAGACAATGGAGAATTCCATTAGGTTTATGTTTTGTTTGGGCTAT
CATGTTGGTTATTGGTATGGTTTGCATGCCAGAATCACCAAGATACTTAGTTGTCAAGAACAAG
ATTGAAGAAGCTAAGAAATCGATTGGTAGATCCAACAAGGTTTCACCAGAAGATCCTGCTGTT
TACACCGAAGTCCAATTGATTCAAGCAGGTATTGAAAGAGAAAGTTTAGCTGGTTCTGCCTCTT
GGACCGAATTGGTTACTGGTAAGCCAAGAATCTTTCGTAGAGTCATTATGGGTATTATGTTACA
ATCTTTACAACAATTGACTGGTGACAACTATTTCTTCTACTATGGTACTACTATTTTCCAAGCTG
TCGGTATGACTGATTCCTTCCAAACATCTATTGTTTTAGGTGTTGTTAACTTTGCATCTACATTT
CTCGGTATCTACACAATTGAAAGATTCGGTAGAAGATTATGTTTGTTAACTGGTTCTGTCTGTA
TGTTCGTTTGTTTCATCATTTACTCCATTTTGGGTGTTACAAACTTATATATTGATGGCTACGAT
GGTCCAACTTCGGTTCCAACCGGTGATGCGATGATTTTCATTACTACCTTATACATTTTCTTCT
TCGCATCCACCTGGGCTGGTGGTGTCTACTGTATCGTTTCCGAAACATACCCATTGAGAATTA
GATCTAAGGCCATGTCCGTTGCCACCGCTGCTAACTGGATTTGGGGTTTCTTGATCTCTTTCT
TCACTCCATTCATCACCTCGGCTATCCACTTCTACTACGGTTTCGTTTTCACAGGATGTTTGTT
ATTCTCGTTCTTTTACGTTTACTTCTTTGTTGTTGAAACTAAGGGATTAACTTTAGAAGAAGTTG
ATGAATTGTATGCCCAAGGTGTTGCCCCATGGAAGTCATCGAAATGGGTTCCACCAACCAAGG
AAGAAATGGCCCATTCTTCAGGATATGCTGCTGAAGCCAAACCTCACGATCAACAAGTATAA
Saccharomyces cerevisiae gal2 (SEQ ID NO: 114)
ATGGCAGTTGAGGAGAACAATATGCCTGTTGTTTCACAGCAACCCCAAGCTGGTGAAGAC
GTGATCTCTTCACTCAGTAAAGATTCCCATTTAAGCGCACAATCTCAAAAGTATTCTAAT
GATGAATTGAAAGCCGGTGAGTCAGGGTCTGAAGGCTCCCAAAGTGTTCCTATAGAGATA
CCCAAGAAGCCCATGTCTGAATATGTTACCGTTTCCTTGCTTTGTTTGTGTGTTGCCTTC
GGCGGCTTCATGTTTGGCTGGGATACCGGTACTATTTCTGGGTTTGTTGTCCAAACAGAC
TTTTTGAGAAGGTTTGGTATGAAACATAAGGATGGTACCCACTATTTGTCAAACGTCAGA
ACAGGTTTAATCGTCGCCATTTTCAATATTGGCTGTGCCTTTGGTGGTATTATACTTTCC
AAAGGTGGAGATATGTATGGCCGTAAAAAGGGTCTTTCGATTGTCGTCTCGGTTTATATA
GTTGGTATTATCATTCAAATTGCCTCTATCAACAAGTGGTACCAATATTTCATTGGTAGA
ATCATATCTGGTTTGGGTGTCGGCGGCATCGCCGTCTTATGTCCTATGTTGATCTCTGAA
ATTGCTCCAAAGCACTTGAGAGGCACACTAGTTTCTTGTTATCAGCTGATGATTACTGCA
GGTATCTTTTTGGGCTACTGTACTAATTACGGTACAAAGAGCTATTCGAACTCAGTTCAA
TGGAGAGTTCCATTAGGGCTATGTTTCGCTTGGTCATTATTTATGATTGGCGCTTTGACG
TTAGTTCCTGAATCCCCACGTTATTTATGTGAGGTGAATAAGGTAGAAGACGCCAAGCGT
TCCATTGCTAAGTCTAACAAGGTGTCACCAGAGGATCCTGCCGTCCAGGCAGAGTTAGAT
CTGATCATGGCCGGTATAGAAGCTGAAAAACTGGCTGGCAATGCGTCCTGGGGGGAATTA
TTTTCCACCAAGACCAAAGTATTTCAACGTTTGTTGATGGGTGTGTTTGTTCAAATGTTC
CAACAATTAACCGGTAACAATTATTTTTTCTACTACGGTACCGTTATTTTCAAGTCAGTT
GGCCTGGATGATTCCTTTGAAACATCCATTGTCATTGGTGTAGTCAACTTTGCCTCCACT
TTCTTTAGTTTGTGGACTGTCGAAAACTTGGGACATCGTAAATGTTTACTTTTGGGCGCT
GCCACTATGATGGCTTGTATGGTCATCTACGCCTCTGTTGGTGTTACTAGATTATATCCT
CACGGTAAAAGCCAGCCATCTTCTAAAGGTGCCGGTAACTGTATGATTGTCTTTACCTGT
TTTTATATTTTCTGTTATGCCACAACCTGGGCGCCAGTTGCCTGGGTCATCACAGCAGAA
TCATTCCCACTGAGAGTCAAGTCGAAATGTATGGCGTTGGCCTCTGCTTCCAATTGGGTA
TGGGGGTTCTTGATTGCATTTTTCACCCCATTCATCACATCTGCCATTAACTTCTACTAC
GGTTATGTCTTCATGGGCTGTTTGGTTGCCATGTTTTTTTATGTCTTTTTCTTTGTTCCA
GAAACTAAAGGCCTATCGTTAGAAGAAATTCAAGAATTATGGGAAGAAGGTGTTTTACCT
TGGAAATCTGAAGGCTGGATTCCTTCATCCAGAAGAGGTAATAATTACGATTTAGAGGAT
TTACAACATGACGACAAACCGTGGTACAAGGCCATGCTAGAATAA Saccharomyces
cerevisiae sol3 (SEQ ID NO: 115)
ATGGTGACAGTCGGTGTGTTTTCTGAGAGGGCTAGTTTGACCCATCAATTGGGGGAATTC
ATCGTCAAGAAACAAGATGAGGCGCTGCAAAAGAAGTCAGACTTTAAAGTTTCCGTTAGC
GGTGGCTCTTTGATCGATGCTCTGTATGAAAGTTTAGTAGCGGACGAATCACTATCTTCT
CGAGTGCAATGGTCTAAATGGCAAATCTACTTCTCTGATGAAAGAATTGTGCCACTGACG
GACGCTGACAGCAATTATGGTGCCTTCAAGAGAGCTGTTCTAGATAAATTACCCTCGACT
AGTCAGCCAAACGTTTATCCCATGGACGAGTCCTTGATTGGCAGCGATGCTGAATCTAAC
AACAAAATTGCTGCAGAGTACGAGCGTATCGTACCTCAAGTGCTTGATTTGGTACTGTTG
GGCTGTGGTCCTGATGGACACACTTGTTCCTTATTCCCTGGAGAAACACATAGGTACTTG
CTGAACGAAACAACCAAAAGAGTTGCTTGGTGCCACGATTCTCCCAAGCCTCCAAGTGAC
AGAATCACCTTCACTCTGCCTGTGTTGAAAGACGCCAAAGCCCTGTGTTTTGTGGCTGAG
GGCAGTTCCAAACAAAATATAATGCATGAGATCTTTGACTTGAAAAACGATCAATTGCCA
ACCGCATTGGTTAACAAATTATTTGGTGAAAAAACATCCTGGTTCGTTAATGAGGAAGCT
TTTGGAAAAGTTCAAACGAAAACTTTTTAG Saccharomyces cerevisiae zwf1 (SEQ
ID NO: 116)
ATGAGTGAAGGCCCCGTCAAATTCGAAAAAAATACCGTCATATCTGTCTTTGGTGCGTCA
GGTGATCTGGCAAAGAAGAAGACTTTTCCCGCCTTATTTGGGCTTTTCAGAGAAGGTTAC
CTTGATCCATCTACCAAGATCTTCGGTTATGCCCGGTCCAAATTGTCCATGGAGGAGGAC
CTGAAGTCCCGTGTCCTACCCCACTTGAAAAAACCTCACGGTGAAGCCGATGACTCTAAG
GTCGAACAGTTCTTCAAGATGGTCAGCTACATTTCGGGAAATTACGACACAGATGAAGGC
TTCGACGAATTAAGAACGCAGATCGAGAAATTCGAGAAAAGTGCCAACGTCGATGTCCCA
CACCGTCTCTTCTATCTGGCCTTGCCGCCAAGCGTTTTTTTGACGGTGGCCAAGCAGATC
AAGAGTCGTGTGTACGCAGAGAATGGCATCACCCGTGTAATCGTAGAGAAACCTTTCGGC
CACGACCTGGCCTCTGCCAGGGAGCTGCAAAAAAACCTGGGGCCCCTCTTTAAAGAAGAA
GAGTTGTACAGAATTGACCATTACTTGGGTAAAGAGTTGGTCAAGAATCTTTTAGTCTTG
AGGTTCGGTAACCAGTTTTTGAATGCCTCGTGGAATAGAGACAACATTCAAAGCGTTCAG
ATTTCGTTTAAAGAGAGGTTCGGCACCGAAGGCCGTGGCGGCTATTTCGACTCTATAGGC
ATAATCAGAGACGTGATGCAGAACCATCTGTTACAAATCATGACTCTCTTGACTATGGAA
AGACCGGTGTCTTTTGACCCGGAATCTATTCGTGACGAAAAGGTTAAGGTTCTAAAGGCC
GTGGCCCCCATCGACACGGACGACGTCCTCTTGGGCCAGTACGGTAAATCTGAGGACGGG
TCTAAGCCCGCCTACGTGGATGATGACACTGTAGACAAGGACTCTAAATGTGTCACTTTT
GCAGCAATGACTTTCAACATCGAAAACGAGCGTTGGGAGGGCGTCCCCATCATGATGCGT
GCCGGTAAGGCTTTGAATGAGTCCAAGGTGGAGATCAGACTGCAGTACAAAGCGGTCGCA
TCGGGTGTCTTCAAAGACATTCCAAATAACGAACTGGTCATCAGAGTGCAGCCCGATGCC
GCTGTGTACCTAAAGTTTAATGCTAAGACCCCTGGTCTGTCAAATGCTACCCAAGTCACA
GATCTGAATCTAACTTACGCAAGCAGGTACCAAGACTTTTGGATTCCAGAGGCTTACGAG
GTGTTGATAAGAGACGCCCTACTGGGTGACCATTCCAACTTTGTCAGAGATGACGAATTG
GATATCAGTTGGGGCATATTCACCCCATTACTGAAGCACATAGAGCGTCCGGACGGTCCA
ACACCGGAAATTTACCCCTACGGATCAAGAGGTCCAAAGGGATTGAAGGAATATATGCAA
AAACACAAGTATGTTATGCCCGAAAAGCACCCTTACGCTTGGCCCGTGACTAAGCCAGAA
GATACGAAGGATAATTAG
Amino Acid Sequences
TABLE-US-00091 [0476] Debaryomyces hansenii gxf1 (SEQ ID NO: 117) 1
MSQEEYSSGV QTPVSNHSGL EKEEQHKLDG LDEDEIVDQL PSLPEKSAKD YLLISFFCVL
61 VAFGGFVFGF DTGTISGFVN MSDYLERFGE LNADGEYFLS NVRTGLIVAI
FNVGCAVGGI 121 FLSKIADVYG RRIGLMFSMI IYVIGIIVQI SASDKWYQIV
VGRAIAGLAV GTVSVLSPLF 181 IGESAPKTLR GTLVCCFQLC ITLGIFLGYC
TTYGTKTYTD SRQWRIPLGL CFVWAIMLVI 241 GMVCMPESPR YLVVKNKIEE
AKKSIGRSNK VSPEDPAVYT EVQLIQAGIE RESLAGSASW 301 TELVTGKPRI
FRRVIMGIML QSLQQLTGDN YFFYYGTTIF QAVGMTDSFQ TSIVLGVVNF 361
ASTFLGIYTI ERFGRRLCLL TGSVCMFVCF IIYSILGVTN LYIDGYDGPT SVPTGDAMIF
421 ITTLYIFFFA STWAGGVYCI VSETYPLRIR SKAMSVATAA NWIWGFLISF
FTPFITSAIH 481 FYYGFVFTGC LLFSFFYVYF FVVETKGLTL EEVDELYAQG
VAPWKSSKWV PPTKEEMAHS 541 SGYAAEAKPH DQQV Saccharomyces cerevisiae
gal2 (SEQ ID NO: 118) 1 MAVEENNMPV VSQQPQAGED VISSLSKDSH LSAQSQKYSN
DELKAGESGS 51 EGSQSVPIEI PKKPMSEYVT VSLLCLCVAF GGFMFGWDTG
TISGFVVQTD 101 FLRRFGMKHK DGTHYLSNVR TGLIVAIFNI GCAFGGIILS
KGGDMYGRKK 151 GLSIVVSVYI VGIIIQIASI NKWYQYFIGR IISGLGVGGI
AVLCPMLISE 201 IAPKHLRGTL VSCYQLMITA GIFLGYCTNY GTKSYSNSVQ
WRVPLGLCFA 251 WSLFMIGALT LVPESPRYLC EVNKVEDAKR SIAKSNKVSP
EDPAVQAELD 301 LIMAGIEAEK LAGNASWGEL FSTKTKVFQR LLMGVFVQMF
QQLTGNNYFF 351 YYGTVIFKSV GLDDSFETSI VIGVVNFAST FFSLWTVENL
GHRKCLLLGA 401 ATMMACMVIY ASVGVTRLYP HGKSQPSSKG AGNCMIVFTC
FYIFCYATTW 451 APVAWVITAE SFPLRVKSKC MALASASNWV WGFLIAFFTP
FITSAINFYY 501 GYVFMGCLVA MFFYVFFFVP ETKGLSLEEI QELWEEGVLP
WKSEGWIPSS 551 RRGNNYDLED LQHDDKPWYK AMLE Saccharomyces cerevisiae
zwf1 (SEQ ID NO: 119) 1 MSEGPVKFEK NTVISVFGAS GDLAKKKTFP ALFGLFREGY
LDPSTKIFGY 51 ARSKLSMEED LKSRVLPHLK KPHGEADDSK VEQFFKMVSY
ISGNYDTDEG 101 FDELRTQIEK FEKSANVDVP HRLFYLALPP SVFLTVAKQI
KSRVYAENGI 151 TRVIVEKPFG HDLASARELQ KNLGPLFKEE ELYRIDHYLG
KELVKNLLVL 201 RFGNQFLNAS WNRDNIQSVQ ISFKERFGTE GRGGYFDSIG
IIRDVMQNHL 251 LQIMTLLTME RPVSFDPESI RDEKVKVLKA VAPIDTDDVL
LGQYGKSEDG 301 SKPAYVDDDT VDKDSKCVTF AAMTFNIENE RWEGVPIMMR
AGKALNESKV 351 EIRLQYKAVA SGVFKDIPNN ELVIRVQPDA AVYLKFNAKT
PGLSNATQVT 401 DLNLTYASRY QDFWIPEAYE VLIRDALLGD HSNFVRDDEL
DISWGIFTPL 451 LKHIERPDGP TPEIYPYGSR GPKGLKEYMQ KHKYVMPEKH
PYAWPVTKPE 501 DTKDN Saccharomyces cerevisiae sol3 (SEQ ID NO: 120)
1 MVTVGVFSER ASLTHQLGEF IVKKQDEALQ KKSDFKVSVS GGSLIDALYE 51
SLVADESLSS RVQWSKWQIY FSDERIVPLT DADSNYGAFK RAVLDKLPST 101
SQPNVYPMDE SLIGSDAESN NKIAAEYERI VPQVLDLVLL GCGPDGHTCS 151
LFPGETHRYL LNETTKRVAW CHDSPKPPSD RITFTLPVLK DAKALCFVAE 201
GSSKQNIMHE IFDLKNDQLP TALVNKLFGE KTSWFVNEEA FGKVQTKTF
Example 27
Cloning of Additional ZWF1 Candidate Genes
[0477] A variety of ZWF1 genes were cloned from S. cerevisiae,
Zymomonas mobilis, Pseudomonas fluorescens (zwf1 and zwf2), and P.
aeruginosa strain PAO1. The sequences of these additional ZWF1
genes are given below.
TABLE-US-00092 zwf1 from P. fluorescens Amino Acid Sequence (SEQ.
ID. NO: 123)
MTTTRKKSKALPAPPTTLFLFGARGDLVKRLLMPALYNLSRDGLLDEGLRIVGVDHNAVSDAEFAT
LLEDFLRDEVLNKQGQGAAVDAAVWARLTRGINYVQGDFLDDSTYAELAARIAASGTGNAVFYLA
TAPRFFSEVVRRLGSAGLLEEGPQAFRRVVIEKPFGSDLQTAEALNGCLLKVMSEKQIYRIDHYLG
KETVQNILVSRFSNSLFEAFWNNHYIDHVQITAAETVGVETRGSFYEHTGALRDMVPNHLFQLLAM
VAMEPPAAFGADAVRGEKAKVVGAIRPWSVEEARANSVRGQYSAGEVAGKALAGYREEANVAP
DSSTETYVALKVMIDNWRWVGVPFYLRTGKRMSVRDTEIVICFKPAPYAQFRDTEVERLLPTYLRI
QIQPNEGMWFDLLAKKPGPSLDMANIELGFAYRDFFEMQPSTGYETLIYDCLIGDQTLFQRADNIE
NGWRAVQPFLDAWQQDASLQNYPAGVDGPAAGDELLARDGRVWRPLG Nucleotide Sequence
(SEQ. ID. NO: 124)
ATGACCACCACGCGAAAGAAGTCCAAGGCGTTGCCGGCGCCGCCGACCACGCTGTTCCTGT
TCGGCGCCCGCGGTGATCTGGTCAAGCGCCTGCTGATGCCGGCGCTGTACAACCTCAGCCG
CGACGGTTTGCTGGATGAGGGGCTGCGGATTGTCGGCGTCGACCACAACGCGGTGAGCGAC
GCCGAGTTCGCCACGCTGCTGGAAGACTTCCTTCGCGATGAAGTGCTCAACAAGCAAGGCCA
GGGGGCGGCGGTGGATGCCGCCGTCTGGGCCCGCCTGACCCGGGGCATCAACTATGTCCA
GGGCGATTTTCTCGACGACTCCACCTATGCCGAACTGGCGGCGCGGATTGCCGCCAGCGGC
ACCGGCAACGCGGTGTTCTACCTGGCCACCGCACCGCGCTTCTTCAGTGAAGTGGTGCGCC
GCCTGGGCAGCGCCGGGTTGCTGGAGGAGGGGCCGCAGGCTTTTCGCCGGGTGGTGATCG
AAAAACCCTTCGGCTCCGACCTGCAGACCGCCGAAGCCCTCAACGGCTGCCTGCTCAAGGTC
ATGAGCGAGAAGCAGATCTATCGCATCGACCATTACCTGGGCAAGGAAACGGTCCAGAACAT
CCTGGTCAGCCGTTTTTCCAACAGCCTGTTCGAGGCATTCTGGAACAACCATTACATCGACCA
CGTGCAGATCACCGCGGCGGAAACCGTCGGCGTGGAAACCCGTGGCAGCTTTTATGAACAC
ACCGGTGCCCTGCGGGACATGGTGCCCAACCACCTGTTCCAGTTGCTGGCGATGGTGGCCA
TGGAGCCGCCCGCTGCCTTTGGCGCCGATGCGGTACGTGGCGAAAAGGCCAAGGTGGTGG
GGGCTATCCGCCCCTGGTCCGTGGAAGAGGCCCGGGCCAACTCGGTGCGCGGCCAGTACA
GCGCCGGTGAAGTGGCCGGCAAGGCCCTGGCGGGCTACCGCGAGGAAGCCAACGTGGCGC
CGGACAGCAGCACCGAAACCTACGTTGCGCTGAAGGTGATGATCGACAACTGGCGCTGGGT
CGGGGTGCCGTTCTACCTGCGCACCGGCAAGCGCATGAGTGTGCGCGACACCGAGATCGTC
ATCTGCTTCAAGCCGGCGCCCTATGCACAGTTCCGCGATACCGAGGTCGAGCGCCTGTTGCC
GACCTACCTGCGGATCCAGATCCAGCCCAACGAAGGCATGTGGTTCGACCTGCTGGCGAAAA
AGCCCGGGCCGAGCCTGGACATGGCCAACATCGAACTGGGTTTTGCCTACCGCGACTTTTTC
GAGATGCAGCCCTCCACCGGCTACGAAACCCTGATCTACGACTGCCTGATCGGCGACCAGAC
CCTGTTCCAGCGCGCCGACAACATCGAGAACGGCTGGCGCGCGGTGCAACCCTTCCTCGAT
GCCTGGCAACAGGACGCCAGCTTGCAGAACTACCCGGCGGGCGTGGATGGCCCGGCAGCC
GGGGATGAACTGCTGGCCCGGGATGGCCGCGTATGGCGACCCCTGGGGTGA zwf2 from P.
fluorescens Amino Acid Sequence (SEQ. ID. NO: 125)
MPSITVEPCTFALFGALGDLALRKLFPALYQLDAAGLLHDDTRILALAREPGSEQEHLANIETELHKY
VGDKDIDSQVLQRFLVRLSYLHVDFLKAEDYVALAERVGSEQRLIAYFATPAAVYGAICENLSRVGL
NQHTRVVLEKPIGSDLDSSRKVNDAVAQFFPETRIYRIDHYLGKETVQNLIALRFANSLFETQWNQ
NYISHVEITVAEKVGIEGRWGYFDKAGQLRDMIQNHLLQLLCLIAMDPPADLSADSIRDEKVKVLKA
LAPISPEGLTTQVVRGQYIAGHSEGQSVPGYLEEENSNTQSDTETFVALRADIRNWRWAGVPFYL
RTGKRMPQKLSQIVIHFKEPSHYIFAPEQRLQISNKLIIRLQPDEGISLRVMTKEQGLDKGMQLRSG
PLQLNFSDTYRSARIPDAYERLLLEVMRGNQNLFVRKDEIEAAWKWCDQLIAGWKKSGDAPKPYA
AGSWGPMSSIALITRDGRSWYGDI Nucleotide Sequence (SEQ. ID. NO: 126)
ATGCCTTCGATAACGGTTGAACCCTGCACCTTTGCCTTGTTTGGCGCGCTGGGCGATCTGGC
GCTGCGTAAGCTGTTTCCTGCCCTGTACCAACTCGATGCCGCCGGTTTGCTGCATGACGACA
CGCGCATCCTGGCCCTGGCCCGCGAGCCTGGCAGCGAGCAGGAACACCTGGCGAATATCGA
AACCGAGCTGCACAAGTATGTCGGCGACAAGGATATCGATAGCCAGGTCCTGCAGCGTTTTC
TCGTCCGCCTGAGCTACCTGCATGTGGACTTCCTCAAGGCCGAGGACTACGTCGCCCTGGCC
GAACGTGTCGGCAGCGAGCAGCGCCTGATTGCCTACTTCGCCACGCCGGCGGCGGTGTATG
GCGCGATCTGCGAAAACCTCTCCCGGGTCGGGCTCAACCAGCACACCCGTGTGGTCCTGGA
AAAACCCATCGGCTCGGACCTGGATTCATCACGCAAGGTCAACGACGCGGTGGCGCAGTTCT
TCCCGGAAACCCGCATCTACCGGATCGACCACTACCTGGGCAAGGAAACGGTGCAGAACCTG
ATTGCCCTGCGTTTCGCCAACAGCCTGTTCGAAACCCAGTGGAACCAGAACTACATCTCCCAC
GTGGAAATCACCGTGGCCGAGAAGGTCGGCATCGAAGGTCGCTGGGGCTATTTCGACAAGG
CCGGCCAACTGCGGGACATGATCCAGAACCACTTGCTGCAACTGCTCTGCCTGATCGCGATG
GACCCGCCGGCCGACCTTTCGGCCGACAGCATCCGCGACGAGAAGGTCAAGGTGCTCAAGG
CCCTGGCGCCCATCAGCCCGGAAGGCCTGACCACCCAGGTGGTGCGCGGCCAGTACATCGC
CGGCCACAGCGAAGGCCAGTCGGTGCCGGGCTACCTGGAGGAAGAAAACTCCAACACCCAG
AGCGACACCGAGACCTTCGTCGCCCTGCGCGCCGATATCCGCAACTGGCGCTGGGCCGGTG
TGCCTTTCTACCTGCGCACCGGCAAGCGCATGCCACAGAAGCTGTCGCAGATCGTCATCCAC
TTCAAGGAACCCTCGCACTACATCTTCGCCCCCGAGCAGCGCCTGCAGATCAGCAACAAGCT
GATCATCCGCCTGCAGCCGGACGAAGGTATCTCGTTGCGGGTGATGACCAAGGAGCAGGGC
CTGGACAAGGGCATGCAACTGCGCAGCGGTCCGTTGCAGCTGAATTTTTCCGATACCTATCG
CAGTGCACGGATCCCCGATGCCTACGAGCGGTTGTTGCTGGAAGTGATGCGCGGCAATCAG
AACCTGTTTGTGCGCAAAGATGAAATCGAAGCCGCGTGGAAGTGGTGTGACCAGTTGATTGC
CGGGTGGAAGAAATCCGGCGATGCGCCCAAGCCGTACGCGGCCGGGTCCTGGGGGCCGAT
GAGCTCCATTGCACTGATCACGCGGGATGGGAGGTCTTGGTATGGCGATATCTaA zwf1 from
P. aeruginosa, PAO1 Amino Acid Sequence (SEQ. ID. NO: 127)
MPDVRVLPCTLALFGALGDLALRKLFPALYQLDRENLLHRDTRVLALARDEGAPAEHLATLEQRLR
LAVPAKEWDDVVWQRFRERLDYLSMDFLDPQAYVGLREAVDDELPLVAYFATPASVFGGICENLA
AAGLAERTRVVLEKPIGHDLESSREVNEAVARFFPESRIYRIDHYLGKETVQNLIALRFANSLFETQ
WNQNHISHVEITVAEKVGIEGRWGYFDQAGQLRDMVQNHLLQLLCLIAMDPPSDLSADSIRDEKV
KVLRALEPIPAEQLASRVVRGQYTAGFSDGKAVPGYLEEEHANRDSDAETFVALRVDIRNWRWS
GVPFYLRTGKRMPQKLSQIVIHFKEPPHYIFAPEQRSLISNRLIIRLQPDEGISLQVMTKDQGLGKG
MQLRTGPLQLSFSETYHAARIPDAYERLLLEVTQGNQYLFVRKDEVEFAWKWCDQLIAGWERLSE
APKPYPAGSWGPVASVALVARDGRSWYGDF Nucleotide Sequence (SEQ. ID. NO:
128) ATGCCTGATGTCCGCGTTCTGCCTTGCACGTTAGCGCTGTTCGGTGCGCTGGGCGATCTCGC
CTTGCGCAAGCTGTTCCCGGCGCTCTACCAACTCGATCGTGAGAACCTGCTGCACCGCGATA
CCCGCGTCCTGGCCCTGGCCCGTGACGAAGGCGCTCCCGCCGAACACCTGGCGACGCTGG
AGCAGCGCCTGCGCCTGGCAGTGCCGGCGAAGGAGTGGGACGACGTGGTCTGGCAGCGTT
TCCGCGAACGCCTCGACTACCTGAGCATGGACTTCCTCGACCCGCAGGCCTATGTCGGCTTG
CGCGAGGCGGTGGATGACGAACTGCCGCTGGTCGCCTACTTCGCCACGCCGGCCTCGGTGT
TCGGCGGCATCTGCGAGAACCTCGCCGCCGCCGGTCTCGCCGAGCGCACCCGGGTGGTGC
TGGAGAAGCCCATCGGTCATGACCTGGAGTCGTCCCGCGAGGTCAACGAGGCAGTCGCCCG
GTTCTTCCCGGAAAGCCGCATCTACCGGATCGACCATTACCTGGGCAAGGAGACGGTGCAGA
ACCTGATCGCCCTGCGCTTCGCCAACAGCCTCTTCGAGACCCAGTGGAACCAGAACCACATC
TCCCACGTGGAGATCACCGTGGCCGAGAAGGTCGGCATCGAAGGCCGCTGGGGCTACTTCG
ACCAGGCCGGGCAACTGCGCGACATGGTGCAGAACCACCTGCTGCAACTGCTCTGCCTGAT
CGCCATGGATCCGCCCAGCGACCTTTCGGCGGACAGCATTCGCGACGAGAAGGTCAAGGTC
CTCCGCGCCCTCGAGCCGATTCCCGCAGAACAACTGGCTTCGCGCGTGGTGCGTGGGCAGT
ACACCGCCGGTTTCAGCGACGGCAAGGCAGTGCCGGGCTACCTGGAGGAGGAACATGCGAA
TCGCGACAGCGACGCGGAAACCTTCGTCGCCCTGCGCGTGGACATCCGCAACTGGCGCTGG
TCGGGCGTGCCGTTCTACCTGCGCACCGGCAAGCGCATGCCGCAGAAGCTGTCGCAGATCG
TCATCCACTTCAAGGAGCCGCCGCACTACATCTTCGCTCCCGAGCAGCGTTCGCTGATCAGC
AACCGGCTGATCATCCGCCTGCAGCCGGACGAAGGTATCTCCCTGCAAGTGATGACCAAGGA
CCAGGGCCTGGGCAAGGGCATGCAATTGCGTACCGGCCCGCTGCAACTGAGTTTTTCCGAG
ACCTACCACGCGGCGCGGATTCCCGATGCCTACGAGCGTCTGCTGCTGGAGGTCACCCAGG
GCAACCAGTACCTGTTCGTGCGCAAGGACGAGGTGGAGTTCGCCTGGAAGTGGTGCGACCA
GCTGATCGCTGGCTGGGAACGCCTGAGCGAAGCGCCCAAGCCGTATCCGGCGGGGAGTTG
GGGGCCGGTGGCCTCGGTGGCCCTGGTGGCCCGCGATGGGAGGAGTTGGTATGGCGATTT CTGA
zwf1 from Z. mobilis Amino Acid Sequence (SEQ. ID. NO: 129)
MTNTVSTMILFGSTGDLSQRMLLPSLYGLDADGLLADDLRIVCTSRSEYDTDGFRDFAEKALDRFV
ASDRLNDDAKAKFLNKLFYATVDITDPTQFGKLADLCGPVEKGIAIYLSTAPSLFEGAIAGLKQAGLA
GPTSRLALEKPLGQDLASSDHINDAVLKVFSEKQVYRIDHYLGKETVQNLLTLRFGNALFEPLWNS
KGIDHVQISVAETVGLEGRIGYFDGSGSLRDMVQSHILQLVALVAMEPPAHMEANAVRDEKVKVF
RALRPINNDTVFTHTVTGQYGAGVSGGKEVAGYIDELGQPSDTETFVAIKAHVDNWRWQGVPFYI
RTGKRLPARRSEIVVQFKPVPHSIFSSSGGILQPNKLRIVLQPDETIQISMMVKEPGLDRNGAHMRE
VWLDLSLTDVFKDRKRRIAYERLMLDLIEGDATLFVRRDEVEAQWVWIDGIREGWKANSMKPKTY
VSGTWGPSTAIALAERDGVTWYD Nucleotide Sequence (SEQ. ID. NO: 130)
ATGACAAATACCGTTTCGACGATGATATTGTTTGGCTCGACTGGCGACCTTTCACAGCGTATG
CTGTTGCCGTCGCTTTATGGTCTTGATGCCGATGGTTTGCTTGCAGATGATCTGCGTATCGTC
TGCACCTCTCGTAGCGAATACGACACAGATGGTTTCCGTGATTTTGCAGAAAAAGCTTTAGAT
CGCTTTGTCGCTTCTGACCGGTTAAATGATGACGCTAAAGCTAAATTCCTTAACAAGCTTTTCT
ACGCGACGGTCGATATTACGGATCCGACCCAATTCGGAAAATTAGCTGACCTTTGTGGCCCG
GTCGAAAAAGGTATCGCCATTTATCTTTCGACTGCGCCTTCTTTGTTTGAAGGGGCAATCGCT
GGCCTGAAACAGGCTGGTCTGGCTGGTCCAACTTCTCGCCTGGCGCTTGAAAAACCTTTAGG
TCAAGATCTTGCTTCTTCCGATCATATTAATGATGCGGTTTTGAAAGTTTTCTCTGAAAAGCAA
GTTTATCGTATTGACCATTATCTGGGTAAAGAAACGGTTCAGAATCTTCTGACCCTGCGTTTTG
GTAATGCTTTGTTTGAACCGCTTTGGAATTCAAAAGGCATTGACCACGTTCAGATCAGCGTTG
CTGAAACGGTTGGTCTTGAAGGTCGTATCGGTTATTTCGACGGTTCTGGCAGCTTGCGCGATA
TGGTTCAAAGCCATATCCTTCAGTTGGTCGCTTTGGTTGCAATGGAACCACCGGCTCATATGG
AAGCCAACGCTGTTCGTGACGAAAAGGTAAAAGTTTTCCGCGCTCTGCGTCCGATCAATAACG
ACACCGTCTTTACGCATACCGTTACCGGTCAATATGGTGCCGGTGTTTCTGGTGGTAAAGAAG
TTGCCGGTTACATTGACGAACTGGGTCAGCCTTCCGATACCGAAACCTTTGTTGCTATCAAAG
CGCATGTTGATAACTGGCGTTGGCAGGGTGTTCCGTTCTATATCCGCACTGGTAAGCGTTTAC
CTGCACGTCGTTCTGAAATCGTGGTTCAGTTTAAACCTGTTCCGCATTCGATTTTCTCTTCTTC
AGGTGGTATCTTGCAGCCGAACAAGCTGCGTATTGTCTTACAGCCTGATGAAACCATCCAGAT
TTCTATGATGGTGAAAGAACCGGGTCTTGACCGTAACGGTGCGCATATGCGTGAAGTTTGGCT
GGATCTTTCCCTCACGGATGTGTTTAAAGACCGTAAACGTCGTATCGCTTATGAACGCCTGAT
GCTTGATCTTATCGAAGGCGATGCTACTTTATTTGTGCGTCGTGACGAAGTTGAGGCGCAGTG
GGTTTGGATTGACGGAATTCGTGAAGGCTGGAAAGCCAACAGTATGAAGCCAAAAACCTATGT
CTCTGGTACATGGGGGCCTTCAACTGCTATAGCTCTGGCCGAACGTGATGGAGTAACTTGGT
ATGACTGA
[0478] All the above genes were PCR amplified from their genomic
DNA sources with and without c-terminal 6-HIS tags (SEQ ID NO: 138)
and cloned into the yeast expression vector p426GPD for
testing.
[0479] Assays of Candidate ZWF1 Genes
[0480] Strain BY4742 zwf1 (ATCC Cat. No. 4011971; Winzeler E A, et
al. Science 285: 901-906, 1999. PubMed: 10436161) was used as the
base strain for all ZWF1 assays. The assays were performed as
follows: A 5 ml overnight of the strain expressing the ZWF1 gene
was grown in SCD-ura. A 50 ml culture of the strain was then grown
for about 18 hours from an initial OD.sub.600 of about 0.2 until it
had reached about OD.sub.600 of about 4. The cells were centrifuged
at 1046.times.g washed twice with 25 ml cold sterile water, and
resuspended in 2 ml/g Yper Plus (Thermo Scientific) plus
1.times.protease inhibitors (EDTA-free). The cells were allowed to
lyse at room temperature for about 30 minutes with constant
rotation of the tubes. The lysate was centrifuged at 16,100.times.g
for 10 minutes at 4.degree. C. and the supernatants were
transferred to a new 1.5 ml microcentrifuge tube. Quantification of
the lysates was performed using the Coomassie-Plus kit (Thermo
Scientific, San Diego, Calif.) as directed by the manufacturer.
[0481] Each kinetic assay was done using approximately 50 to 60
.mu.g of crude extract in a reaction mixture containing 50 mM
Tris-HCl, pH 8.9, and 1 mM NADP+ or NAD+. The reaction was started
with 20 mM glucose-6-phosphate and the reaction was monitored at
A340. The specific activity was measured as the .mu.mol
substrate/min/mg protein. The results of the assays are presented
in the table below.
TABLE-US-00093 Specific Vmax Km Activity (.mu.mol Zwf1 Cofactors
(.mu.mol min.sup.-1) (M.sup.-1) min.sup.-1 mg.sup.-1) S. cerevisiae
NAD+ NA NA NA NADP+ 0.9523 0.4546 224.07 S. cerevisiae + His NAD+
NA NA NA NADP+ 0.7267 0.4109 164.79 ZM4 NAD+ NA NA NA NADP+ NA NA
NA ZM4 + His NAD+ 0.0213 0.0156 0.1267 NADP+ 0.0027 0.0140 0.0160
P. fluorescens 1 NAD+ 0.0158 0.6201 0.3132 NADP+ 0.0213 0.8171
0.4208 P. fluorescens 1 + NAD+ 0.0126 4.9630 0.2473 His NADP+
0.0139 0.9653 0.2739 P. fluorescens 2 NAD+ ND ND ND NADP+ NA NA NA
P. fluorescens 2 + NAD+ NA NA NA His NADP+ ND ND ND PAO1 NAD+ NA NA
NA NADP+ 0.0104 0.6466 0.1564 PAO1 + His NAD+ 0.0074 0.0071 0.1098
NADP+ 0.0123 3.9050 0.1823 NA = cannot be calculated (substrate not
used by enzyme) ND = was not determined (either not enough crude
available or cells did not grow)
[0482] Altering Cofactor Preference of S. cerevisiae ZWF1
[0483] ZWF1 from S. cerevisiae is an NADP.sup.+-only utilizing
enzyme. Site-directed mutagenesis was used to alter of ZWF1 so that
the altered ZWF1 could also utilize NAD+, thereby improving the
REDOX balance within the cell. Site directed mutagenesis reactions
were performed in the same manner for all mutations, and for
mutants which include more than one mutation, each mutation was
performed sequentially. About 50 ng of plasmid DNA was added to
1.times.Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3 .mu.mol site
directed mutagenesis specific primers, and 1 U Pfu Ultra II
polymerase (Agilent, La Jolla, Calif.) in a 50 .mu.l reaction mix.
The reaction was cycled at 95.degree. C. for 10 minutes, followed
by 15 rounds of 95.degree. C. for 15 seconds, 55.degree. C. for 40
seconds, and 72.degree. C. for 3 minutes. A final 10 minute
extension reaction at 72.degree. C. was also included. The PCR
reaction mixture was then digested with 30 U of DpnI for about 2
hours and 5 .mu.l of the digested PCR reaction mixture was used to
transform competent DH5.alpha. (Zymo Research, Orange, Calif.) and
plated onto LB plates containing the appropriate antibiotics. The
table below lists mutants generated in a first round of
mutagenesis.
TABLE-US-00094 Mutant # zwf1_sc Codon changes 1 A24G GCA -> GGT
2 A24G/D28G GCA -> GGT, ACT -> GGT 3 A51N GCC -> AAT 4
A51D GCC -> GAT 5 D28F ACT -> TTT 6 K46R AAG -> AGA 7 Y40L
TAC -> TTG 8 F33Y TTT -> TAC 9 D28L ACT -> TTG 10 V16L GTC
-> TTG 11 V13T GTC -> ACT 12 L66E CTA -> GAA 13 A24G/A51D
GCA -> GGT, GCC -> GAT 14 A24G/D28G/A51D GCA -> GGT, ACT
-> GGT, GCC -> GAT 15 R52D CGG -> GAT 16 A51D/R52A GCC
-> GAT, CGG -> GCT 17 A24G/A51D/R52A GCA -> GGT, GCC ->
GAT, CGG -> GCT 18 A24G/D28G/A51D/R52A GCA -> GGT, ACT ->
GGT, GCC -> GAT, CGG -> GCT 19 A51D/R52H GCC -> GAT, CGG
-> CAT 20 R52H CGG -> CAT 21 D22R GAT -> AGA
[0484] The oligonucleotides, utilized to generate the mutants
listed in the table above, are listed in the table below. All
oligonucleotides were purchased from Integrated DNA Technologies
(IDT).
TABLE-US-00095 Base Nucleotide sequence (SEQ ID NOS 375-412,
Mutation plasmid Oligo Name respectively, in order of appearance) 1
pBF300 ka/zwf1sc_A24Gfor gtgcgtcaggtgatctgggtaagaagaagacttttccc 1
pBF300 ka/zwf1sc_A24Grev gggaaaagtcttcttcttacccagatcacctgacgcac 2
pBF300 ka/zwf1sc_D28Gfor gtgatctgggtaagaagaagggttttcccgccttatttgg 2
pBF300 ka/zwf1sc_D28Grev CCAAATAAGGCGGGAAAACCCTTCTTCTTAC CCAGATCAC
3 pBF300 ka/zwf1sc_A51Nfor
ccttgatccatctaccaagatcttcggttataatcggtccaaattgtc cat 3 pBF300
ka/zwf1sc_A51Nrev atggacaatttggaccgattataaccgaagatcttggtagatggat
caagg 4 pBF300 ka/zwf1sc_A51Dfor
atctaccaagatcttcggttatgatcggtccaaattgtccatg 4 pBF300
ka/zwf1sc_A51Drev catggacaatttggaccgatcataaccgaagatcttggtagat 5
pBF300 ka/zwf1sc_D28Ffor ggtgatctggcaaagaagaagttttttcccgccttatttggg
5 pBF300 ka/zwf1sc_D28Frev
cccaaataaggcgggaaaaaacttcttctttgccagatcacc 6 pBF300
ka/zwf1sc_K46Rfor taccttgatccatctaccagaatcttcggttatgcccggt 6 pBF300
ka/zwf1sc_K46Rrev accgggcataaccgaagattctggtagatggatcaaggta 7 pBF300
ka/zwf1sc_Y39Lfor gggcttttcagagaaggtttgcttgatccatctaccaaga 7 pBF300
ka/zwf1sc_Y39Lrev tcttggtagatggatcaagcaaaccttctctgaaaagccc 8 pBF300
ka/zwf1sc_F33Yfor gaagaagacttttcccgccttatacgggcttttcagagaag 8
pBF300 ka/zwf1sc_F33Yrev cttctctgaaaagcccgtataaggcgggaaaagtcttcttc
9 pBF300 ka/zwf1sc_D28Lfor
gtcaggtgatctggcaaagaagaagttgtttcccgccttatttgg 9 pBF300
ka/zwf1sc_D28Lrev ccaaataaggcgggaaacaacttcttctttgccagatcacctgac 10
pBF300 ka/zwf1sc_V16Lfor
cgaaaaaaataccgtcatatctttgtttggtgcgtcaggtgatctg 10 pBF300
ka/zwf1sc_V16rev cagatcacctgacgcaccaaacaaagatatgacggtatttttttcg 12
pBF300 ka/zwf1sc_L66Efor gacctgaagtcccgtgtcgaaccccacttgaaaaaacc 12
pBF300 ka/zwf1sc_L66Erev ggttttttcaagtggggttcgacacgggacttcaggtc 13
pBF374 ka/zwf1sc_A24Gfor gtgcgtcaggtgatctgggtaagaagaagacttttccc 13
pBF374 ka/zwf1sc_A24Grev gggaaaagtcttcttcttacccagatcacctgacgcac 14
pBF374 ka/zwf1sc_A24Gfor gtgcgtcaggtgatctgggtaagaagaagacttttccc 14
pBF374 ka/zwf1sc_A24Grev gggaaaagtcttcttcttacccagatcacctgacgcac 15
pBF300 KA/zwf1mut15for
accaagatcttcggttatgccgattccaaattgtccatggaggag 15 pBF300
KA/zwf1mut15rev ctcctccatggacaatttggaatcggcataaccgaagatcttggt 16
pBF374 KA/zwf1mut16for
tccatctaccaagatcttcggttatgatgcttccaaattgtccatgga ggaggac 16 pBF374
KA/zwf1mut16rev gtcctcctccatggacaatttggaagcatcataaccgaagatcttg
gtagatgga 17 pBF441 KA/zwf1mut16for
tccatctaccaagatcttcggttatgatgcttccaaattgtccatgga ggaggac 17 pBF441
KA/zwf1mut16rev gtcctcctccatggacaatttggaagcatcataaccgaagatcttg
gtagatgga 18 pBF442 KA/zwf1mut16for
tccatctaccaagatcttcggttatgatgcttccaaattgtccatgga ggaggac 18 pBF442
KA/zwf1mut16rev gtcctcctccatggacaatttggaagcatcataaccgaagatcttg
gtagatgga 19 pBF374 KA/zwf1sc_mut19for
aagatcttcggttatgatcattccaaattgtccatggagg 19 pBF374
KA/zwf1sc_mut19rev cctccatggacaatttggaatgatcataaccgaagatctt 20
pBF300 KA/zwf1sc_mut20for aagatcttcggttatgcccattccaaattgtccatggagg
20 pBF300 KA/zwf1sc_mut20rev
cctccatggacaatttggaatgggcataaccgaagatctt
[0485] Initial kinetic screening of the ZWF1 mutants generated as
described above, identified the following altered ZWF1 genes and
preliminary cofactor phenotype.
TABLE-US-00096 NAD+ NADP+ Mutant # zwf1_sc usage usage 1 A24G No
Yes 2 A24G/T28G No No 3 A51N No Yes 4 A51D Yes No 5 T28F No Yes 6
K46R No Yes 7 Y40L No Yes 8 F33Y No Yes 9 T28L No Yes 10 V16L No
Yes 11 V13T ND ND 12 L66E No Yes 13 A24G/A51D Yes No 14
A24G/T28G/A51D No No 15 R52D No No 16 A51D/R52A No No 17
A24G/A51D/R52A No No 18 A24G/T28G/A51D/R52A ND ND 19 A51D/R52H ND
ND 20 R52H ND ND 21 D22R ND ND ND = not determined
[0486] Mutants 4 (A51 D) and 13 (A24G/A51 D) were identified as
mutants which enabled NAD+ utilization with concomitant loss of
NADP+ utilization.
[0487] Cloning of SOL3
[0488] The SOL3 gene from S. cerevisiae was cloned as follows. The
approximately 750 bp SOL3 gene was PCR amplified from the BY4742
genome using primers KAS/5-SOL3-NheI and KAS/3'-SOL3-SalI, shown
below.
TABLE-US-00097 KAS/5-SOL3-NheI (SEQ ID NO: 413)
gctagcatggtgacagtcggtgtgttttctgag KAS/3'-SOL3-SalI (SEQ ID NO: 414)
gtcgacctaaaaagttttcgtttgaacttttcc
[0489] About 100 ng of genomic DNA from S. cerevisiae strain BY4742
was added to 1.times.Pfu Ultra II buffer, 0.3 mM dNTPs, 0.3 .mu.mol
gene-specific primers, and 1 U Pfu Ultra II polymerase (Agilent, La
Jolla, Calif.) in a 50 .mu.l reaction mix. The reaction was cycled
at 95.degree. C. for 10 minutes, followed by 30 rounds of
95.degree. C. for 20 seconds, 55.degree. C. for 30 seconds, and
72.degree. C. for 15 seconds. A final 5 minute extension reaction
at 72.degree. C. was also included. The amplified product was TOPO
cloned into the pCR Blunt II TOPO vector (Life Technologies,
Carlsbad, Calif.) according to the manufacturer's recommendations
and sequence verified (GeneWiz, San Diego, Calif.). The resultant
plasmid was designated pBF301. The sequence of the S. cerevisiae
SOL3 gene is given below.
TABLE-US-00098 S. cerevisiae SOL3 (SEQ. ID. NO: 131)
ATGGTGACAGTCGGTGTGTTTTCTGAGAGGGCTAGTTTGACCCATCAATT
GGGGGAATTCATCGTCAAGAAACAAGATGAGGCGCTGCAAAAGAAGTCAG
ACTTTAAAGTTTCCGTTAGCGGTGGCTCTTTGATCGATGCTCTGTATGAA
AGTTTAGTAGCGGACGAATCACTATCTTCTCGAGTGCAATGGTCTAAATG
GCAAATCTACTTCTCTGATGAAAGAATTGTGCCACTGACGGACGCTGACA
GCAATTATGGTGCCTTCAAGAGAGCTGTTCTAGATAAATTACCCTCGACT
AGTCAGCCAAACGTTTATCCCATGGACGAGTCCTTGATTGGCAGCGATGC
TGAATCTAACAACAAAATTGCTGCAGAGTACGAGCGTATCGTACCTCAAG
TGCTTGATTTGGTACTGTTGGGCTGTGGTCCTGATGGACACACTTGTTCC
TTATTCCCTGGAGAAACACATAGGTACTTGCTGAACGAAACAACCAAAAG
AGTTGCTTGGTGCCACGATTCTCCCAAGCCTCCAAGTGACAGAATCACCT
TCACTCTGCCTGTGTTGAAAGACGCCAAAGCCCTGTGTTTTGTGGCTGAG
GGCAGTTCCAAACAAAATATAATGCATGAGATCTTTGACTTGAAAAACGA
TCAATTGCCAACCGCATTGGTTAACAAATTATTTGGTGAAAAAACATCCT
GGTTCGTTAATGAGGAAGCTTTTGGAAAAGTTCAAACGAAAACTTTTTAG
[0490] The NheI-SalI SOL3 gene fragment from plasmid pBF301 will be
cloned into the SpeI-XhoI site in plasmids p413GPD and p423GPD
(HIS3 marker-based plasmids; ATCC 87354 and ATCC 87355).
[0491] Testing of ZWF1/SOL3 Combinations in BY4742
[0492] A URA blaster cassette was digested with NotI and ligated
into the MET17 integration cassette plasmid pBF691 to generate the
Met17 knockout plasmid pBF772. Plasmid pBF772 was digested with
PacI and linear fragments were purified by Zymo PCR purification
kit (Zymo Research, Orange, Calif.) and concentrated in 10 .mu.A
ddH2O. LiCl2 high efficiency transformation was performed as shown
described. About 1 .mu.g linear MET17 knockout fragment was
transformed into 50 .mu.l fresh made BY4742 competent cells and
cells were plated onto SCD-Ura plates at 30.degree. C. for about
2-3 days. A single URA+ colony was streaked out on a SCD-Ura plate
and grown at 30.degree. C. for about 2-3 days. A single colony was
inoculated overnight in YPD medium at 30.degree. C. 50 .mu.l of the
overnight culture was then plated onto SCD complete -5FOA plates
and incubated at 30.degree. C. for about 3 days.
[0493] A single colony which grew on SCD complete-5FOA plates was
then picked and inoculated in YPD medium and grown at 30.degree. C.
overnight. Yeast genomic DNA was extracted by YeaStar genomic
extraction kit (Zymo Research, Orange, Calif.) and confirmation of
the strain was confirmed by PCR using primers JML/237 and JML/238,
shown below.
TABLE-US-00099 JML/237: (SEQ ID NO: 415)
CCAACACTAAGAAATAATTTCGCCATTTCTTG JML/238: (SEQ ID NO: 416)
GCCAACAATTAAATCCAAGTTCACCTATTCTG
[0494] The PCR amplification was performed as follows: 10 ng of
yeast genomic DNA with 0.1 .mu.mol gene specific primers,
1.times.Pfu Ultra II buffer, 0.2 mmol dNTPs, and 0.2 U Taq DNA
polymerase. The PCR mixture was cycled at 95.degree. C. for 2
minutes, followed by 30 cycles of 95.degree. C. for 20 seconds,
55.degree. C. for 30 seconds and 72.degree. C. for 45 seconds. A
final step of 72.degree. C. for 5 minutes was also included. The
resultant strain was designated BF1618.
[0495] Strain BF1618 is undergoing transformation with the
following plasmid combinations. Additionally, the affect of the
ZWF1 mutant constructs will also be evaluated with and without SOL3
constructs. The table below shows the plasmid combinations being
transformed into strain BF1618.
TABLE-US-00100 Test Strain EDD EDA ZWF1 SOL3 1 2.mu. 2.mu. cen/ars
NONE 2 2.mu. 2.mu. 2.mu. NONE 3 2.mu. 2.mu. cen/ars cen/ars 4 2.mu.
2.mu. 2.mu. 2.mu. 5 2.mu. 2.mu. NONE cen/ars 6 2.mu. 2.mu. NONE
2.mu.
[0496] Strains with improved ethanol production may benefit from
two or more copies of the ZWF1 gene due to increased flux of the
carbon towards the alternative pathway. A strain embodiment
currently under construction has the phenotype; pfk1, ZWF1, SOL3,
tal1, EDD-PAO1*, EDA-E. coli*, where the "*" represents additional
copies of the gene. It is believed that multiple copies of the EDD
and EDA genes may provide additional increases in ethanol
production.
Example 28
Identification of Additional Xylose Isomerase 5' Ends that can
Increase Expression Levels of Ruminococcus Xylose Isomerase in
Yeast
[0497] To determine if the 5' end of other xylose isomerase genes
could also be used to increase the expression of the Ruminococcus
xylose isomerase in yeast, as demonstrated herein for the 5' end of
Piromyces xylose isomerase, additional chimeric molecules were
generated as described herein, using approximately 10 amino acids
from the xylose isomerase genes described in Example 25. The
alternate xylose isomerase gene 5' ends were selected from xylose
isomerase sequences previously shown to be expressed and active inn
yeast. The xylose isomerase gene donors and the 5' end of the
nucleotide sequence from each are presented in the table below.
TABLE-US-00101 Clostridium
ATGAAAAATTACTTTCCAAATGTTCCAGAAGTAAAATACGAAGGCCCAAATTCAACG
phytofermentans
AATCCATTTGCTTTTAAATATTATGACGCAAATAAAGTTGTAGCGGGTAAAACAATG
AAAGAGCACTGTCGTTTTGCATTATCTTGGTGGCATACTCTTTGTGCAGGTGGTGC
TGATCCATTCGGTGTAACAACTATGGATAGAACCTACGGAAATATCACAGATCCAA
TGGAACTTGCTAAGGCAAAAGTTGACGCTGGTTTCGAATTAATGACTAAATTAGGA
ATTGAATTCTTCTGTTTCCATGACGCAGATATTGCTCCAGAAGGTGATACTTTTGAA
GAGTCAAAGAAGAATCTTTTTGAAATCGTTGATTACATCAAAGAGAAGATGGATCA
GACTGGTATCAAGTTATTATGGGGTACTGCTAATAACTTTAGTCATCCAAGATTTAT GCAT (SEQ
ID NO: 95) Orpinomyces
ATGACTAAGGAATATTTCCCAACTATCGGCAAGATTAGATTCGAAGGTAAGGATTC
TAAGAATCCAATGGCCTTCCACTACTATGATGCTGAAAAGGAAGTCATGGGTAAGA
AAATGAAGGATTGGTTACGTTTCGCCATGGCCTGGTGGCACACTCTTTGCGCCGA
TGGTGCTGACCAATTCGGTGTTGGTACTAAGTCTTTCCCATGGAATGAAGGTACTG
ACCCAATTGCTATTGCCAAGCAAAAGGTTGATGCTGGTTTTGAAATCATGACCAAG
CTTGGTATTGAACACTACTGTTTCCACGATGTTGATCTTGTTTCTGAAGGTAACTCT
ATTGAAGAATACGAATCCAACCTCAAGCAAGTTGTTGCTTACCTTAAGCAAAAGCA
ACAAGAAACTGGTATTAAGCTTCTCTGGAGTACTGCCAATGTTTTCGGTAACCCAC
GTTACATGAAC (SEQ ID NO: 96) Clostridium
ATGGAATACTTCAAAAATGTACCACAAATAAAATATGAAGGACCAAAATCAAACAAT
thermohydrosulfuricum
CCATATGCATTTAAATTTTACAATCCAGATGAAATAATAGACGGAAAACCTTTAAAA
GAACACTTGCGTTTTTCAGTAGCGTACTGGCACACATTTACAGCCAATGGGACAGA
TCCATTTGGAGCACCCACAATGCAAAGGCCATGGGACCATTTTACTGACCCTATG
GATATTGCCAAAGCGAGAGTAGAAGCAGCCTTTGAACTATTTGAAAAACTCGACGT
ACCATTTTTCTGTTTCCATGACAGAGATATAGCTCCGGAAGGAGAGACATTAAGGG
AGACGAACAAAAATTTAGATACAATAGTTGCAATGATAAAAGACTACTTAAAGACGA
GCAAAACAAAAGTATTATGGGGCACAGCGAACCTTTTTTCAAATCCGAGATTTGTA CAT (SEQ
ID NO: 97) Bacteroides
ATGGCAACAAAAGAATTTTTTCCGGGAATTGAAAAGATTAAATTTGAAGGTAAAGAT
thetaiotaomicron
AGTAAGAACCCGATGGCATTCCGTTATTACGATGCAGAGAAGGTGATTAATGGTAA
AAAGATGAAGGATTGGCTGAGATTCGCTATGGCATGGTGGCACACATTGTGCGCT
GAAGGTGGTGATCAGTTCGGTGGCGGAACAAAGCAATTCCCATGGAATGGTAATG
CAGATGCTATACAGGCAGCAAAAGATAAGATGGATGCAGGATTTGAATTCATGCAG
AAGATGGGTATCGAATACTATTGCTTCCATGACGTAGACTTGGTTTCGGAAGGTGC
CAGTGTAGAAGAATACGAAGCTAACCTGAAAGAAATCGTAGCTTATGCAAAACAGA
AACAGGCAGAAACCGGTATCAAACTACTGTGGGGTACTGCTAATGTATTCGGTCA
CGCCCGCTATATGAAC (SEQ ID NO: 98) Bacillus
ATGGCTTATTTTCCGAATATCGGCAAGATTGCGTATGAAGGGCCGGAGTCGCGCA
stearothermophilus
ATCCGTTGGCGTTTAAGTTTTATAATCCAGAAGAAAAAGTCGGCGACAAAACAATG
GAGGAGCATTTGCGCTTTTCAGTGGCCTATTGGCATACGTTTACGGGGGATGGGT
CGGATCCGTTTGGCGTCGGCAATATGATTCGTCCATGGAATAAGTACAGCGGCAT
GGATCTGGCGAAGGCGCGCGTCGAGGCGGCGTTTGAGCTGTTTGAAAAGCTGAA
CGTTCCGTTTTTCTGCTTCCATGACGTCGACATCGCGCCGGAAGGGGAAACGCTC
AGCGAGACGTACAAAAATTTGGATGAAATTGTCGATATGATTGAAGAATACATGAA
AACAAGCAAAACGAAGCTGCTTTGGAATACGGCGAACTTGTTCAGCCATCCGCGC TTCGTTCAC
(SEQ ID NO: 99) Bacillus uniformis
ATGGCTACCAAGGAATACTTCCCAGGTATTGGTAAGATCAAATTCGAAGGTAAGGA
ATCCAAGAACCCAATGGCCTTCAGATACTACGATGCTGACAAGGTTATCATGGGTA
AGAAGATGTCTGAATGGTTAAAGTTCGCTATGGCTTGGTGGCATACCTTGTGTGCT
GAAGGTGGTGACCAATTCGGTGGTGGTACCAAGAAATTCCCATGGAACGGTGAAG
CTGACAAGGTCCAAGCTGCTAAGAACAAGATGGACGCTGGTTTCGAATTTATGCAA
AAGATGGGTATTGAATACTACTGTTTCCACGATGTTGACTTGTGTGAAGAAGCTGA
AACCATCGAAGAATACGAAGCTAACTTGAAGGAAATTGTTGCTTACGCTAAGCAAA
AGCAAGCTGAAACTGGTATCAAGCTATTATGGGGTACTGCTAACGTCTTTGGTCAT
GCCAGATACATGAAC (SEQ ID NO: 100) Clostridium cellulolyticum
ATGTCAGAAGTATTTAGCGGTATTTCAAACATTAAATTTGAAGGAAGCGGGTCAGA
TAATCCATTAGCTTTTAAGTACTATGACCCTAAGGCAGTTATCGGCGGAAAGACAA
TGGAAGAACATCTGAGATTCGCAGTTGCCTACTGGCATACTTTTGCAGCACCAGGT
GCTGACATGTTCGGTGCAGGATCATATGTAAGACCTTGGAATACAATGTCCGATCC
TCTGGAAATTGCAAAATACAAAGTTGAAGCAAACTTTGAATTCATTGAAAAGCTGG
GAGCACCTTTCTTCGCTTTCCATGACAGGGATATTGCTCCTGAAGGCGACACACTC
GCTGAAACAAATAAAAACCTTGATACAATAGTTTCAGTAATTAAAGATAGAATGAAA
TCCAGTCCGGTAAAGTTATTATGGGGAACTACAAATGCTTTCGGAAACCCAAGATT TATGCAT
(SEQ ID NO: 101) Ruminococcus
ATGGAATTTTTCAAGAACATAAGCAAGATCCCTTACGAGGGCAAGGACAGCACAAA
flavefaciens FD1
TCCTCTCGCATTCAAGTACTACAATCCTGATGAGGTAATTGACGGCAAGAAGATGC
GTGACATTATGAAGTTTGCTCTCTCATGGTGGCATACAATGGGCGGCGACGGAAC
AGATATGTTCGGCTGCGGTACAGCTGACAAGACATGGGGCGAAAATGATCCTGCT
GCAAGAGCTAAGGCTAAGGTTGACGCAGCTTTCGAGATCATGCAGAAGCTCTCTA
TCGATTACTTCTGTTTCCACGACCGTGATCTTTCTCCTGAGTACGGCTCACTGAAG
GACACAAACGCTCAGCTGGACATCGTTACAGATTACATCAAGGCTAAGCAGGCTG
AGACAGGTCTCAAGTGCCTCTGGGGTACAGCTAAGTGCTTCGATCACCCAAGATT CATGCAC
(SEQ ID NO: 102) Ruminococcus 18P13
ATGAGCGAATTTTTTACAGGCATTTCAAAGATCCCCTTTGAGGGAAAGGCATCCAA
CAATCCCATGGCGTTCAAGTACTACAACCCGGATGAGGTCGTAGGCGGCAAGACC
ATGCGGGAGCAGCTGAAGTTTGCGCTGTCCTGGTGGCATACTATGGGGGGAGAC
GGTACGGACATGTTTGGTGTGGGTACCACCAACAAGAAGTTCGGCGGAACCGATC
CCATGGACATTGCTAAGAGAAAGGTAAACGCTGCGTTTGAGCTGATGGACAAGCT
GTCCATCGATTATTTCTGTTTCCACGACCGGGATCTGGCGCCGGAGGCTGATAAT
CTGAAGGAAACCAACCAGCGTCTGGATGAAATCACCGAGTATATTGCACAGATGA
TGCAGCTGAACCCGGACAAGAAGGTTCTGTGGGGTACTGCAAATTGCTTCGGCAA
TCCCCGGTATATGCAT (SEQ ID NO: 103) Clostriales genomosp
ATGAAATTTTTTGAAAATGTCCCTAAGGTAAAATATGAGGGAAGCAAGTCTACCAA BVAB3 str
UPII9-5 CCCGTTTGCATTTAAGTATTACAATCCTGAAGCGGTGATTGCCGGTAAAAAAATGA
AGGATCACCTGAAATTCGCGATGTCCTGGTGGCACACCATGACGGCGACCGGGC
AAGACCAGTTCGGTTCGGGGACGATGAGCCGAATATATGACGGGCAAACTGAACC
GCTGGCCTTGGCCAAAGCCCGAGTGGATGCGGCTTTCGATTTCATGGAAAAATTA
AATATCGAATATTTTTGTTTTCATGATGCCGACTTGGCTCCAGAAGGTAACAGTTTG
CAGGAACGCAACGAAAATTTGCAGGAAATGGTGTCTTACCTGAAACAAAAGATGG
CCGGAACTTCGATTAAGCTTTTATGGGGAACCTCGAATTGTTTCAGCAACCCTCGT TTTATGCAC
(SEQ ID NO: 104)
[0498] The first 10 amino acids (30 bp) of XI-R was replaced with
the 5' edge from the xylose isomerase genes presented in the table
above using a single oligonucleotide in a PCR reaction, described
herein. The oligonucleotides used for the PCR reactions are shown
in the table below. The last 2 oligonucleotides were used as 3'
oligonucleotides to amplify each resulting chimeric molecule with
or without a c-terminal 6-HIS tag (SEQ ID NO: 138).
TABLE-US-00102 Oligonucleotide sequence (SEQ ID NOS 361-370 and
373-374, Name respectively, in order of appearance) KAS/5-XR_Cp10
ACTAGTAAAAAATGAAAAATTACTTTCCAAATGTTCCAGAAGTACAGTATCAGGGACCAAAAAG
KAS/5-XR-O10
ACTAGTAAAAAATGACTAAGGAATATTTCCCAACTATCGGCAAGATTCAGTATCAGGGACCAAA
AAG KAS/5-XR-Cth10
ACTAGTAAAAAATGGAATACTTCAAAAATGTACCACAAATAAAACAGTATCAGGGACCAAAAAG
KAS/5-XR-Bth10
ACTAGTAAAAAATGGCAACAAAAGAATTTTTTCCGGGAATTGAAAAGATTCAGTATCAGGGACC
AAAAAG KAS/5-XR-Bst10
ACTAGTAAAAAATGGCTTATTTTCCGAATATCGGCAAGATTCAGTATCAGGGACCAAAAAG
KAS/5-XR-
ACTAGTAAAAAATGGCTACCAAGGAATACTTCCCAGGTATTGGTAAGATCCAGTATCAGGGAC
Bun10 CAAAAAG KAS/5-XR-
ACTAGTAAAAAATGTCAGAAGTATTTAGCGGTATTTCAAACATTCAGTATCAGGGACCAAAAAG
Cce10 KAS/5-XR-RF10
ACTAGTAAAAAATGGAATTTTTCAAGAACATAAGCAAGATCCAGTATCAGGGACCAAAAAG
KAS/5-XR-
ACTAGTAAAAAATGAGCGAATTTTTTACAGGCATTTCAAAGATCCAGTATCAGGGACCAAAAAG
18P10 KAS/5-XR-BV10
ACTAGTAAAAAATGAAATTTTTTGAAAATGTCCCTAAGGTACAGTATCAGGGACCAAAAAG
KAS/3-XI-RF- CTCGAGTTACAGACTGAAAAGAACGTTATTTACG NATIVE KAS/3-XI-RF-
CTCGAGTTAGTGATGGTGGTGGTGATGCAGACTGAAAAGAACGTTATTTACG
NATIVE-HISb
[0499] Each new PCR product was TOPO cloned using a pCR Blunt II
vector (Invitrogen), verified by sequencing and subcloned into
p426GPD, also as described herein. The resulting plasmids were
transformed into BY4742 (S. cerevisiae) and selected on SCD-ura
medium. Assays to detect levels of expressed xylose isomerase were
performed as described herein.
[0500] Results
[0501] Each of the new chimeric genes was evaluated for expression
against the native Ruminococcus xylose isomerase gene. Each
chimeric variant (e.g., 5' end of an alternate XI donor gene
attached to the Ruminococcus acceptor gene) was evaluated under
saturating xylose conditions (e.g., 500 mM), using 20 .mu.g crude
extract. The assays were repeated several times and the results are
presented graphically in FIG. 21. As shown in FIG. 21, the 5' edge
of 4 of the 10 genes tested showed increased expression in yeast,
with respect to the native Ruminococcus xylose isomerase control.
The 5' edges (e.g., 5' ends) that showed increased expression or
improved he activity of the Ruminococcus xylose isomerase gene were
from Orpinomyces, Bacteroides thetaiotaomicron, Bacillus
stearothermophilus, and B. uniformis. These results suggest that
exchanging the 5' edge of a xylose isomerase gene with low
expression and/or activity for the 5' edge of a different xylose
isomerase gene can be used as a method to improve the activity
and/or expression of xylose isomerase genes when expressed in
eukaryotes such as yeast. 5' edge nucleic acid sequences that can
improve activity or expression are not necessarily associated with
native xylose isomerase genes that themselves show high levels of
expressions. Therefore, these results also suggest that an "ideal"
chimera can be created for organism specific expression of xylose
isomerase, using the method described herein with routine levels of
experimentation to determine the best 5' edges and best acceptor
gene combinations for a specific organism.
[0502] The top 4 chimeric variants (e.g., new 5' edges combined
with the Ruminococcus xylose isomerase acceptor gene) were further
analyzed using a full kinetic assay using varying xylose
concentrations ranging from about 40 mM to about 500 mM. The
results are presented in the table below.
TABLE-US-00103 Km Specific Activity Samples (M - 1) mmol min-1 mg-1
XI-R native 75.03 2.171 XI-R O10 (BF 1754) 75.04 4.817 XI-R Bth10
(BF 1755) 66.70 4.185 XI-R Bst10 (BF 1756) 70.86 4.383 XI-R Bun10
(BF 1757) 90.14 5.000
[0503] These results demonstrate that each of these 5' edge
replacements confers increased activity to the native XI-R enzyme,
with the XI-R-Bun10 enzyme being the most active. The results of
western blots are presented in FIG. 22. The western blot analysis
presented in FIG. 22 shows the levels of expression of each
chimeric construct in total crude extract and the soluble portion
of the crude extract. The results of the western blot analysis are
in good agreement with the results of the kinetic assays. G179A
mutations, similar to those generated in the Ruminococcus native
and chimeric genes described herein, are being generated for the 4
alternate 5' edge chimeras described in this example.
Example 29
Increased Expression of Ribulose-5-phosphate ketol-isomerase and
Ribulose-5-phosphate-3-epimerase
[0504] Ribulose-5-phosphate ketol-isomerase (RKI1) and
ribulose-5-phosphate-3-epimerase (RPE1) catalyze reactions in the
non-oxidative portion of the Pentose Phosphate pathway.
Ribulose-5-phosphate ketol-isomerase catalyzes the interconversion
of ribulose-5-phosphate and ribose-5-phosphate.
Ribulose-5-phosphate-3-epimerase catalyzes the interconversion of
ribulose-5-phosphate to xylulose-5-phosphate. Increasing the
activity of one or both of these enzymes can lead to increased
ethanol production. Ribulose-5-phosphate ketol-isomerase activity
and ribulose-5-phosphate-3-epimerase activity each can be
independently provided by a peptide. In some embodiments, the
polypeptide is encoded by a heterologous nucleotide sequence
introduced to a host microorganism. Nucleic acid sequences
conferring Ribulose-5-phosphate ketol-isomerase activity and
ribulose-5-phosphate-3-epimerase activity can be obtained from a
number of sources, including, but not limited to S. cerevisiae,
including but not limited to Kluyveromyces, Pichia, Escherichia,
Bacillus, Ruminococcus, Schizosaccharomyces, and Candida.
[0505] Examples of an amino acid sequence of a polypeptide having
ribulose-5-phosphate ketol-isomerase activity or
ribulose-5-phosphate-3-epimerase activity, and a nucleotide
sequence of a polynucleotide that encodes the respective
polypeptide, are presented below. Increased activity of
Ribulose-5-phosphate ketol-isomerase and
Ribulose-5-phosphate-3-epimerase can be achieved using any suitable
method. Non-limiting examples of methods suitable for adding,
amplifying or over expressing ribulose-5-phosphate ketol-isomerase
activity, ribulose-5-phosphate-3-epimerase activity, or
ribulose-5-phosphate ketol-isomerase activity and
ribulose-5-phosphate-3-epimerase activity include amplifying the
number of RKI1 and/or RPE1 gene(s) in yeast following
transformation with a high-copy number plasmid (e.g., such as one
containing a 2 uM origin of replication), integration of multiple
copies of RKI1 and/or RPE1 gene(s) into the yeast genome,
over-expression of the RKI1 and/or RPE1 gene(s) directed by a
strong promoter, the like or combinations thereof. Presence,
absence or amount of 6-phosphogluconolactonase activity can be
detected by any suitable method known in the art, including nucleic
acid based analysis and western blot analysis.
TABLE-US-00104 RKI1 nucleotide sequence (SEQ ID NO: 417)
ATGGCTGCCGGTGTCCCAAAAATTGATGCGTTAGAATCTTTGGGCAATCC
TTTGGAGGATGCCAAGAGAGCTGCAGCATACAGAGCAGTTGATGAAAATT
TAAAATTTGATGATCACAAAATTATTGGAATTGGTAGTGGTAGCACAGTG
GTTTATGTTGCCGAAAGAATTGGACAATATTTGCATGACCCTAAATTTTA
TGAAGTAGCGTCTAAATTCATTTGCATTCCAACAGGATTCCAATCAAGAA
ACTTGATTTTGGATAACAAGTTGCAATTAGGCTCCATTGAACAGTATCCT
CGCATTGATATAGCGTTTGACGGTGCTGATGAAGTGGATGAGAATTTACA
ATTAATTAAAGGTGGTGGTGCTTGTCTATTTCAAGAAAAATTGGTTAGTA
CTAGTGCTAAAACCTTCATTGTCGTTGCTGATTCAAGAAAAAAGTCACCA
AAACATTTAGGTAAGAACTGGAGGCAAGGTGTTCCCATTGAAATTGTACC
TTCCTCATACGTGAGGGTCAAGAATGATCTATTAGAACAATTGCATGCTG
AAAAAGTTGACATCAGACAAGGAGGTTCTGCTAAAGCAGGTCCTGTTGTA
ACTGACAATAATAACTTCATTATCGATGCGGATTTCGGTGAAATTTCCGA
TCCAAGAAAATTGCATAGAGAAATCAAACTGTTAGTGGGCGTGGTGGAAA
CAGGTTTATTCATCGACAACGCTTCAAAAGCCTACTTCGGTAATTCTGAC
GGTAGTGTTGAAGTTACCGAAAAGTGA RKI1 amino acid sequence (SEQ ID NO:
418) MAAGVPKIDALESLGNPLEDAKRAAAYRAVDENLKFDDHKIIGIGSGSTV
VYVAERIGQYLHDPKFYEVASKFICIPTGFQSRNLILDNKLQLGSIEQYP
RIDIAFDGADEVDENLQLIKGGGACLFQEKLVSTSAKTFIVVADSRKKSP
KHLGKNWRQGVPIEIVPSSYVRVKNDLLEQLHAEKVDIRQGGSAKAGPVV
TDNNNFIIDADFGEISDPRKLHREIKLLVGVVETGLFIDNASKAYFGNSD GSVEVTEK RPE1
nucleotide sequence (SEQ ID NO: 419)
ATGGTCAAACCAATTATAGCTCCCAGTATCCTTGCTTCTGACTTCGCCAA
CTTGGGTTGCGAATGTCATAAGGTCATCAACGCCGGCGCAGATTGGTTAC
ATATCGATGTCATGGACGGCCATTTTGTTCCAAACATTACTCTGGGCCAA
CCAATTGTTACCTCCCTACGTCGTTCTGTGCCACGCCCTGGCGATGCTAG
CAACACAGAAAAGAAGCCCACTGCGTTCTTCGATTGTCACATGATGGTTG
AAAATCCTGAAAAATGGGTCGACGATTTTGCTAAATGTGGTGCTGACCAA
TTTACGTTCCACTACGAGGCCACACAAGACCCTTTGCATTTAGTTAAGTT
GATTAAGTCTAAGGGCATCAAAGCTGCATGCGCCATCAAACCTGGTACTT
CTGTTGACGTTTTATTTGAACTAGCTCCTCATTTGGATATGGCTCTTGTT
ATGACTGTGGAACCTGGGTTTGGAGGCCAAAAATTCATGGAAGACATGAT
GCCAAAAGTGGAAACTTTGAGAGCCAAGTTCCCCCATTTGAATATCCAAG
TCGATGGTGGTTTGGGCAAGGAGACCATCCCGAAAGCCGCCAAAGCCGGT
GCCAACGTTATTGTCGCTGGTACCAGTGTTTTCACTGCAGCTGACCCGCA
CGATGTTATCTCCTTCATGAAAGAAGAAGTCTCGAAGGAATTGCGTTCTA
GAGATTTGCTAGATTAG RPE1 amino acid sequence (SEQ ID NO: 420)
MVKPIIAPSILASDFANLGCECHKVINAGADWLHIDVMDGHFVPNITLGQ
PIVTSLRRSVPRPGDASNTEKKPTAFFDCHMMVENPEKWVDDFAKCGADQ
FTFHYEATQDPLHLVKLIKSKGIKAACAIKPGTSVDVLFELAPHLDMALV
MTVEPGFGGQKFMEDMMPKVETLRAKFPHLNIQVDGGLGKETIPKAAKAG
ANVIVAGTSVFTAADPHDVISFMKEEVSKELRSRDLLD
Example 30
Xylulokinase Over Expression
[0506] As described herein, metabolism of xylose as a carbon
source, either by xylose isomerase or the combination of xylose
reductase and xylitol dehydrogenase, produces xylulose, which must
be phosphorylated to enter the pentose phosphate pathway. Increased
ethanol fermentation via the over expression of xylose isomerase or
xylose reductase and xylitol dehydrogenase also may be further
enhanced by the over expression of xylulokinase, in some
embodiments. Presented herein are the nucleotide and amino acid
sequence of the S. cerevisiae xylulokinase (XKS1) gene. The
activity of xylulokinase was increased using methods described
herein (e.g., strong promoter, multiple copies, the like and
combinations thereof). The XKS1 gene of S. cerevisiae is
functionally similar to the XYL3 gene of Pichia stipitis.
TABLE-US-00105 XKS1 (SEQ ID NO: 421)
ATGTTGTGTTCAGTAATTCAGAGACAGACAAGAGAGGTTTCCAACACAAT
GTCTTTAGACTCATACTATCTTGGGTTTGATCTTTCGACCCAACAACTGA
AATGTCTCGCCATTAACCAGGACCTAAAAATTGTCCATTCAGAAACAGTG
GAATTTGAAAAGGATCTTCCGCATTATCACACAAAGAAGGGTGTCTATAT
ACACGGCGACACTATCGAATGTCCCGTAGCCATGTGGTTAGAGGCTCTAG
ATCTGGTTCTCTCGAAATATCGCGAGGCTAAATTTCCATTGAACAAAGTT
ATGGCCGTCTCAGGGTCCTGCCAGCAGCACGGGTCTGTCTACTGGTCCTC
CCAAGCCGAATCTCTGTTAGAGCAATTGAATAAGAAACCGGAAAAAGATT
TATTGCACTACGTGAGCTCTGTAGCATTTGCAAGGCAAACCGCCCCCAAT
TGGCAAGACCACAGTACTGCAAAGCAATGTCAAGAGTTTGAAGAGTGCAT
AGGTGGGCCTGAAAAAATGGCTCAATTAACAGGGTCCAGAGCCCATTTTA
GATTTACTGGTCCTCAAATTCTGAAAATTGCACAATTAGAACCAGAAGCT
TACGAAAAAACAAAGACCATTTCTTTAGTGTCTAATTTTTTGACTTCTAT
CTTAGTGGGCCATCTTGTTGAATTAGAGGAGGCAGATGCCTGTGGTATGA
ACCTTTATGATATACGTGAAAGAAAATTCAGTGATGAGCTACTACATCTA
ATTGATAGTTCTTCTAAGGATAAAACTATCAGACAAAAATTAATGAGAGC
ACCCATGAAAAATTTGATAGCGGGTACCATCTGTAAATATTTTATTGAGA
AGTACGGTTTCAATACAAACTGCAAGGTCTCTCCCATGACTGGGGATAAT
TTAGCCACTATATGTTCTTTACCCCTGCGGAAGAATGACGTTCTCGTTTC
CCTAGGAACAAGTACTACAGTTCTTCTGGTCACCGATAAGTATCACCCCT
CTCCGAACTATCATCTTTTCATTCATCCAACTCTGCCAAACCATTATATG
GGTATGATTTGTTATTGTAATGGTTCTTTGGCAAGGGAGAGGATAAGAGA
CGAGTTAAACAAAGAACGGGAAAATAATTATGAGAAGACTAACGATTGGA
CTCTTTTTAATCAAGCTGTGCTAGATGACTCAGAAAGTAGTGAAAATGAA
TTAGGTGTATATTTTCCTCTGGGGGAGATCGTTCCTAGCGTAAAAGCCAT
AAACAAAAGGGTTATCTTCAATCCAAAAACGGGTATGATTGAAAGAGAGG
TGGCCAAGTTCAAAGACAAGAGGCACGATGCCAAAAATATTGTAGAATCA
CAGGCTTTAAGTTGCAGGGTAAGAATATCTCCCCTGCTTTCGGATTCAAA
CGCAAGCTCACAACAGAGACTGAACGAAGATACAATCGTGAAGTTTGATT
ACGATGAATCTCCGCTGCGGGACTACCTAAATAAAAGGCCAGAAAGGACT
TTTTTTGTAGGTGGGGCTTCTAAAAACGATGCTATTGTGAAGAAGTTTGC
TCAAGTCATTGGTGCTACAAAGGGTAATTTTAGGCTAGAAACACCAAACT
CATGTGCCCTTGGTGGTTGTTATAAGGCCATGTGGTCATTGTTATATGAC
TCTAATAAAATTGCAGTTCCTTTTGATAAATTTCTGAATGACAATTTTCC
ATGGCATGTAATGGAAAGCATATCCGATGTGGATAATGAAAATTGGGATC
GCTATAATTCCAAGATTGTCCCCTTAAGCGAACTGGAAAAGACTCTCATC TAA XKS1 amino
acid sequence (SEQ ID NO: 422)
MLCSVIQRQTREVSNTMSLDSYYLGFDLSTQQLKCLAINQDLKIVHSETV
EFEKDLPHYHTKKGVYIHGDTIECPVAMWLEALDLVLSKYREAKFPLNKV
MAVSGSCQQHGSVYWSSQAESLLEQLNKKPEKDLLHYVSSVAFARQTAPN
WQDHSTAKQCQEFEECIGGPEKMAQLTGSRAHFRFTGPQILKIAQLEPEA
YEKTKTISLVSNFLTSILVGHLVELEEADACGMNLYDIRERKFSDELLHL
IDSSSKDKTIRQKLMRAPMKNLIAGTICKYFIEKYGFNTNCKVSPMTGDN
LATICSLPLRKNDVLVSLGTSTTVLLVTDKYHPSPNYHLFIHPTLPNHYM
GMICYCNGSLARERIRDELNKERENNYEKTNDWTLFNQAVLDDSESSENE
LGVYFPLGEIVPSVKAINKRVIFNPKTGMIEREVAKFKDKRHDAKNIVES
QALSCRVRISPLLSDSNASSQQRLNEDTIVKFDYDESPLRDYLNKRPERT
FFVGGASKNDAIVKKFAQVIGATKGNFRLETPNSCALGGCYKAMWSLLYD
SNKIAVPFDKFLNDNFPWHVMESISDVDNENWDRYNSKIVPLSELEKTLI
Example 31
Construction of the KanMX-ATO1-L750 Cassette
[0507] A unique disruption cassette suitable for use when
auxotrophic markers are unavailable, such as in diploid industrial
strains or haploids derived from such strains, was constructed to
allow homologous recombination or integration of sequences in the
absence of traditional auxotrophic marker selection. The primers
used for amplification of nucleic acids utilized to generate the
disruption cassette are described in the table below.
[0508] (Table discloses SEQ ID NOS 423-441, respectively, in order
of appearance)
TABLE-US-00106 JML/ ACTAGTATGTCTGACAAGGAACAAACGAGC 5'ScAto1SpeI 51
JML/ CTCGAGTTAAAAGATTACCCTTTCAGTAGATGGTAATG 3'ScAto1XhoI 52 JML/
caagcctttggtggtacccagaatccagggttagctcc ScATO(L75Q)_For 55 JML/
ggagctaaccctggattctgggtaccaccaaaggcttg ScATO(L75Q)_Rev 56 JML/
ggtacaacgcatatgcagatgttgctacaaagcagaa ScATO1G259D_For 57 JML/
ttctgctttgtagcaacatctgcatatgcgttgtacc ScATO1G259D_Rev 58 JML/
GACGACGTCTAGAAAAGAATACTGGAGAAATGAAAAGAAAAC ReplacesJML/30 59 JML/
GCATGCTTAATTAATGCGAGGCATATTTATGGTGAAGG F'of5'FlankingRegionof 63
ScURA3 JML/ GGCCGGCCAGATCTGCGGCCGCGGCCAGCAAAACTAAAAAAC
F'of3'FlankingRegionof 64 TGTATTATAAG ScURA3 JML/
GCGGCCGCAGATCTGGCCGGCCGATTTATCTTCGTTTCCTGC R'of5'FlankingRegionof
65 AGGTTTTTG ScURA3 JML/ GAATTCTTAATTAACTTTTGTTCCACTACTTTTTGGAACTCT
R'of3FlankingRegionofSc 66 TG URA3 JML/
GCATGCGCGGCCGCACGTCGGCAGGCCCG F'200mer-R 67 JML/
CGAAGGACGCGCGACCAAGTTTATCATTATCAATACTCGCCA F'200mer-R-pGPD-ATO1- 68
TTTC CYC JML/ GAAATGGCGAGTATTGATAATGATAAACTTGGTCGCGCGTCC
R'pGPD-ATO1-CYC- 69 TTCG 200mer-R JML/ GTCGACCCGCAAATTAAAGCCTTCGAGC
R-pGPD-ATO1-CYC 70 JML/ GTCGACGTACCCCCGGGTTAATTAAGGCG F-KanMX 71
JML/ GTCGAAAACGAGCTCGAATTCGACGTCGGCAGGCCCG F-KanMX-200mer-R 72 JML/
CGGGCCTGCCGACGTCGAATTCGAGCTCGTTTTCGAC R-200mer-R-KanMX 73 JML/
GGATCCGCGGCCGCTGGTCGCGCGTCCTTCG R-200mer-R 74
[0509] ScATO1 was amplified from genomic DNA (gDNA) isolated from
BY4742 with primers oJML51 and oJML52 and cloned into pCR Blunt
II-TOPO (Invitrogen, Carlsbad, Calif.). Site Directed Mutagenesis
(SDM) was performed on that plasmid with oJML55 and oJML56, as
described herein. The mutagenized clone was re-amplified with
primers oJML51 and oJML52 and cloned into pCR Blunt II-TOPO
(Invitrogen, Carlsbad, Calif.), and designated ATO1-L75Q. ATO1-L75Q
was subcloned into p416GPD using SpeI/XhoI restriction enzyme
sites. The resulting plasmid was designated pJLVO48.
[0510] The 5' and 3' flanking regions of URA3 were amplified via
PCR of the 5' regions with primers oJML63 and oJML65, the 3' region
with primers oJML64 and oJML66. The amplified nucleic acids were
annealed and re-amplified with oligonucleotides oJML63 and oJML66.
The template used was TURBO gDNA. The PCR product was Topo cloned
into pCR-Blunt II. The desired sequence was moved as an EcoRI-SphI
fragment into vector pUC19 and designated pJLV63.
[0511] The R-KanMX fragment was made as follows: The KANMX fragment
was first amplified from pBF524 with primers oJML71 and oJML73. The
R-200-mer from plasmid pBF32 was then amplified using primers
oJML72 and oJML74. The two fragments were annealed together and PCR
amplified using primers oJML67 and oJML70 and topo cloned using
pCR-Blunt II. The final plasmid construct was designated pJLV062.
The R-P.sub.TDH3-ATO1-L75Q construct was generated by amplifying a
mixture of PCR oJML67-oJM L69 (pBF32)+PCR oJML68-oJML70 (pJLVO48).
The resulting plasmid was designated pJLV065. The R-PT.sub.DH3-ATO1
L75Q (SalI/SphI) fragment from pJLV065 was ligated in a 3 piece
ligation to the SalI/BamHI (R-KanMX) fragment from pJLV063 into the
BamHI/SphI site of pUC19. The entire R-KanMX-P.sub.TDH3-ATO1-L75Q-R
fragment was ligated as a NotI piece into the NotI site of pJLV63
and designated pJLV74. The letter "R" with reference to nucleic
acid fragments, primers, plasmids and unique 200-mer sequence tags,
refers to a unique 200-mer tag identification number. The unique
sequence tags are described in Example 40. A table describing the
intermediate and final plasmids is presented below.
TABLE-US-00107 pJLV0035 pBF493 pCR-Topo BluntII-ScATO1 PCR oJML51,
oJML52 (SDM L75Q oJML55, oJML56 (Clone of ScATO1 Not Kept) pJLV0048
pBF506 pRS416-ProGPD-ScATO1 XhoI-SpeI (pRAS416-GPD) + XhoI- L75Q
SpeI(pJLV035) pJLV0061 pBF604 pCR-Topo BluntII-5' + 3' PCR oJML63,
oJML66 (PCR oJML63, oJML65 ScURA3 gDNA ScTURBO + PCR oJML64, oJML66
gDNA ScTURBO) pJLV0062 pBF605 pCR-Topo BluntII-KanMX- PCR
oJML71-oJML74 (PCR oJML71, oJML73 200m-448 pBF524 + PCR oJML72,
oJML74 pBF32) pJLV0063 pBF606 pUC19-5 + 3' ScURA3
EcoR1-SphI(pJLV0061) + EcoR1- SphI(pUC19) pJLV0065 pBF608 pCR-Topo
BluntII-200m448- PCR oJML67-oJML70 (PCR oJML67-oJML59 ProGDP-ScATO1
L75Q (pBF32) + PCR oJML68-oJML70 (pJLV048)) pJLV0070 pBF650
pUC19-200m448-ProGDP- SalI/SphI (pJLV0065) + BamHI/SalI ScATO1
L75Q-KanMX- (pJLV0062) + SphI/BamHI (pUC19) 200m448 pJLV0074 pBF654
pUC19-5' URA3-200m448- NotI(pJLV070) + NotI(pJLV063) ProGDP-ScATO1
L75Q- KanMX-200m448-3' URA3
Example 32
Construction of the ura3 Disruptions in Each Haploid
[0512] Haploid yeast strains were transformed with 2 to 3 .mu.g of
a PvuII, SphI digested ura3::R-KanMX-ATO1-L75Q-R disruption
cassette using the high-efficiency L1-PEG procedure with a heat
shock time of 8 minutes. Transformants were plated on YPD plus G418
(200 .mu.g/ml) plates. Colonies were re-streaked onto ScD FOA
plates. Single colonies were replica plated on ScD-ura, ScD+FOA,
YPD, and YPD G418 200 .mu.g/ml plates. Ura-FOA.sup.R G418.sup.R
colonies were grown overnight in YPD. Genomic DNA was extracted and
the presence of the KanMX-ATO1-L75Q gene in the URA3 loci was
verified by PCR. 50 .mu.l of each overnight culture was plated on
ScD Acetate (2 g/L), pH 4.0, plates. Colonies were restreaked on
ScD Acetate plates and single colonies grown overnight in YPD.
Disruptions of the URA3 loci were verified by PCR with primers
complementary to a region outside of the flanking region used for
the disruption. The presence of the unique 200-mer sequence was
verified by PCR with primers complementary to the 200-mer in
combination with primers complementary to a region outside of the
flanking region used for the disruption. The absence of the URA3
loci was verified by PCR that amplifies a 500 bp region of the
Actin gene open reading frame and a 300 bp region of the URA3 open
reading frame. The primers utilized for amplification and
verification are presented, respectively, in the tables below.
[0513] Primers used for amplification of URA and Actin (SEQ ID NOS
442-445, respectively, in order of appearance)
TABLE-US-00108 JML/211 GAGGGCACAGTTAAGCCGCTAAAGG URA3 JML/212
GTCAACAGTACCCTTAGTATATTCTCCAGTAGCTAGG URA3 GAG JML/213
CGTTACCCAATTGAACACGGTATTGTCAC ACT1 JML/214
GAAGATTGAGCAGCGGTTTGCATTTC ACT1
[0514] Primers used to verify the presence or absence of URA3 (SEQ
ID NOS 434, 441 and 446-447, respectively, in order of
appearance)
TABLE-US-00109 JML/ GCATGCgcggccgcACGT F'200mer-R 67 CGGCAGGCCCG
JML/ GGATCCgcggccgcTGGT R-200mer-R 74 CGCGCGTCCTTCG JML/
gagtcaaacgacgttgaa PCRtoverifydisruptionofURA3 102 attgaggctactgc
JML/ GATTACTGCTGCTGTTC PCRtoverifydisruptionofURA3 103
CAGCCCATATCCAAC
Example 33
EDA Gene Integration Method and Constructs
[0515] Plasmid DNA was digested with PacI using manufacturers
suggestions. The digestions were purified using the GeneJET.TM. Gel
Extraction Kit I (Fermentas). Each column was eluted with 20 .mu.l
of Elution buffer and multiple digests were combined. S. cerevisiae
was transformed using the high-efficiency L1-PEG procedure with 2
to 3 .mu.g of DNA and transformants were selected on ScD-ura solid
media. Correct integrations were confirmed by PCR analysis with
primers outside the flanking regions used as the disruption
cassette and primers complementary to either the open reading frame
of EDA or the 200-mer repeat. Oligonucleotide primers utilized for
verification are described in the tables below.
[0516] Primers--Outside (SEQ ID NOS 448-451, respectively, in order
of appearance)
TABLE-US-00110 YBR110.5 5' GGCAATCAAATTGGGAACGAACAATG JML/187 3'
CTCAAGGTATCCTCATGGCCAAGCAATAC JML/188 YDL075.5 5'
GGGTCTACAAACTGTTGTTGTCGAAGAAGA JML/189 TG 3'
CATTCAGTTCCAATGATTTATTGACAGTGC JML/190 AC
[0517] Primers--Repeat and EDA going out (SEQ ID NOS 452-454,
respectively, in order of appearance)
TABLE-US-00111 JML/276 CCTACCCGCCTCGGATCCCAGCTACC R-repeat JML/277
GGTAGCTGGGATCCGAGGCGGGTAGG R-repeat JML/278
CCTCCCGGCACAGCGTGTCGATGC R at the 5'EDA
[0518] PaEDA going out and similar primers for EcEDA (SEQ ID NOS
455-457, respectively, in order of appearance)
TABLE-US-00112 JML/297 CGAAGCCCTGGAGCGCTTCGC PCR for PaEDA going
out at the 3' of the ORF JML/298 GTGGTCAGGATTGATTCTGCA PCR for
EcEDA Reverse CTTGTTTTCCAG at the 5' end JML/299
CGCGTGAAGCTGTAGAAGGCG PCR for EcEDA Forward CTAAG at the 3' end
[0519] The PCR reactions were performed in a final reaction volume
of 25 .mu.l using the following amplification profile; 1 cycle at
94 degrees C. for 2 minutes, followed by 35 cycles of 94 degrees C.
for 30 seconds, 52 degrees C. for 30 second and 72 degrees C. for 2
minutes.
[0520] Construction of EDA Disruption Cassettes
[0521] P.sub.TDH3-PaEDA was amplified from pBF292 using primers
oJML225 and oJML226, shown in the table below and Topo cloned in
pCR Blunt II to make pJLV95. (SEQ ID NOS 458-459, respectively, in
order of appearance)
TABLE-US-00113 JML/225 GAGCTCGGCCGCAAATTAAAGCCTTC 3'cyCTERMINATOR
GAG JML/226 GGCCGGCCGTTTATCATTATCAATAC 5'PROMOTERgpd
TCGCCATTTCAAAGAATACG
[0522] The desired fragment was moved as a FseI-SacI piece into
pBF730 or pBF731 (the integration cassette of either YBR110.5 or
YDL075.5, respectively) to make plasmids pJLV114 and pJLV115,
respectively. YBR110.5 is located inbetween loci YBR110 and YBR111,
and YDL075.5 is located in between loci YDL075 and YDL076. The
R-URA3-R sequence was moved into these plasmids as a NotI fragment
to make pJLV119 and pJLV120. The resultant plasmids are described
in the table below.
TABLE-US-00114 pJLV0095 pBF777 pCR-Topo BluntII-PaEDA PCR
oJML225-oJML226 (pBF292) pJLV0114 pBF862
pUC19-5'-YBR110.5-PGDP1-PaEDA- FseI-SacI(pBF730) + FseI-
TCYC-3'YBR110.5 SacI(pJLV95) pJLV0115 pBF863
pUC19-5'-YDL075.5-PGDP1-PaEDA- FseI-SacI(pBF731) + FseI-
TCYC-3'YDL075.5 SacI(pJLV95) pJLV0119 pBF867
pUC19-5'-YBR110.5-PGDP1-PaEDA- NotI(pBF742) + NotI(pJLV114)
TCYC-R-URA3-R-3'YBR110.5 pJLV0120 pBF868
pUC19-5'-YDL075.5-PGDP1-PaEDA- NotI(pBF742) + NotI(pJLV115)
TCYC-R-URA3-R-3'YDL075.5
Example 34
Isolation and Evaluation of Additional EDA Genes
[0523] EDA genes isolated from a variety of sources were expressed
in yeast and evaluated independently of EDA activity, to identify
EDA activities suitable of inclusion in an engineered yeast strain.
The EDA activities were was independently assessed by adding
saturating amounts of over expressed E. coli EDD extracts to S.
cerevisiae EDA extracts lacking EDD (Chemyan et al., Protein
Science 16:2368-2377, 2007). The relative activities of EDAs,
expressed in S. cerevisiae, were compared and ranked in this way.
The activity of integrated EDAs in Thermosacc-Gold haploids, were
also evaluated in this manner. The table below describes
oligonucleotide primers used to isolate the various EDA genes.
TABLE-US-00115 Sequence (SEQ ID NOS 75, 76, 79-80 and 460-473,
respectively, in order Name Description of appearance) KA/EDA-
Cloning primer for Shewanella GTTCACTGCACTAGTAAAAAAATGCTTGAGAATAACT
SoFor oneidensis EDA GGTC KA/EDA- Cloning primer for Shewanella
CTTCGAGATCTCGAGTTAAAGTCCGCCAATCGCCTC SoRev oneidensis EDA KA/EDA-
Cloning primer for GTTCACTGCACTAGTAAAAAAATGATCGATACTGCCA GoFor
Gluconobacter oxydans EDA AACTC KA/EDA- Cloning primer for
CTTCGAGATCTCGAGTCAGACCGTGAAGAGTGCCGC GoRev Gluconobacter oxydans
EDA KA/EDA- Cloning primer for Bacilluis
GTTCACTGCACTAGTAAAAAAATGGTATTGTCACACA BLFor licheniformis EDA
TCGAAG KA/EDA- Cloning primer for Bacilluis
CTTCGAGATCTCGAGTTACTGTTTTGCTGCTTCAACA BLRev licheniformis EDA AATTG
KA/EDA- Cloning primer for Bacillus
GTTCACTGCACTAGTAAAAAAATGGAGTCCAAAGTCG BsFor subtilis EDA TTGAAAACC
KA/EDA- Cloning primer for Bacillus
CTTCGAGATCTCGAGTTACACTTGGAAAACAGCCTGC BsRev subtilis EDA AAATCC
KA/EDA- Cloning primer for GTTCACTGCACTAGTAAAAAAATGACAAACCTCGCCC
PfFor Pseudomonas fluorescens EDA CGACC KA/EDA- Cloning primer for
CTTCGAGATCTCGAGTCAGTCCAGCAGGGCCAGG PfRev Pseudomonas fluorescens
EDA KA/EDA- Cloning primer for
GTTCACTGCACTAGTAAAAAAATGACACAGAACGAAA PsFor Pseudomonas syringae
ATAATCAGCCGC EDA KA/EDA- Cloning primer for
CTTCGAGATCTCGAGTCAGTCAAACAGCGCCAGCGC PsRev Pseudomonas syringae EDA
KA/EDA- Cloning primer for GTTCACTGCACTAGTAAAAAAATGGCTATTACAAAAG
SdFor Saccharaophagus AATTTTTAGCTCCAG degradans EDA KA/EDA- Cloning
primer for CTTCGAGATCTCGAGTTAGCTAGAAATTTTAGCGGTA SdRev
Saccharaophagus GTTGCC degradans EDA KA/EDA- Cloning primer for
GTTCACTGCACTAGTAAAAAAATGACGATTGCCCAGA XaFor Xanthamonas axonopodis
CCCAG EDA KA/EDA- Cloning primer for
CTTCGAGATCTCGAGTCAGCCCGCCCGCACC XaRev Xanthamonas axonopodis EDA
KA/Nde Cloning primer for E. coli
GTTCACTGCCATATGAATCCACAATTGTTACGCGTAA IEDDfor EDD CAAATCGAATCATTG
KA/Xho Cloning primer for E. coli
CTTCGAGATCTCGAGTTAAAAAGTGATACAGGTTGCG IEDDrev EDD CCCTGTTCGGC
[0524] Listed below are the amino acid sequences, nucleotide
sequences and accession numbers of the EDA genes evaluated as
described in this Example.
TABLE-US-00116 Accession Strain Amino Acid Number Species Number
Nucleotide Sequence Sequence YP_526856.1 Saccharophagus 2-40
ATGGCTATTACAAAAGAATTTTTAGCTCCAGTTGGCGTAATGCCTGT MAITKEFLAPVGVMPVV
degradans TGTGGTTGTGGATCGTGTAGAAGATGCGGTGCCTATTACAAACGCAT
VVDRVEDAVPITNALKA TAAAAGCCGGCGGTATTAAAGCAGTTGAGATTACTTTACGTACTCCT
GGIKAVEITLRTPAALD GCGGCACTGGATGCTATTCGCGCTATTAAAGCTGAGTGTGAAGACAT
AIRAIKAECEDILVGVG CCTGGTGGGGGTAGGTACGGTTATTAACCATCAAAACCTTAAAGATA
TVINHQNLKDIAAIGVD TTGCTGCAATTGGTGTTGATTTCGCCGTATCTCCTGGTTACACCCCA
FAVSPGYTPTLLKQAQD ACATTGCTGAAGCAAGCGCAAGATTTGGGCGTAGAAATGTTGCCTGG
LGVEMLPGVTSPSEVML TGTAACTTCGCCTTCTGAAGTTATGCTTGGTATGGAGCTAGGTTTGT
GMELGLSCFKLFPAVAV CTTGCTTCAAGCTATTCCCTGCGGTTGCAGTAGGTGGTTTGCCATTA
GGLPLLKSIGGPLPQVS CTTAAGTCTATTGGTGGCCCATTACCACAGGTTTCCTTCTGTCCAAC
FCPTGGLTIDTFTDFLA AGGCGGTTTGACTATCGATACTTTCACCGACTTCTTGGCATTGCCTA
LPNVACVGGTWLVPADA ACGTTGCTTGTGTGGGTGGTACTTGGTTGGTGCCTGCAGATGCTGTT
VAAKNWQAITDIAAATT GCAGCTAAAAACTGGCAAGCTATTACTGATATTGCGGCGGCAACTAC
AKISS (SEQ ID NO: CGCTAAAATTTCTAGCTAA (SEQ ID NO: 474) 475)
Xanthomonas ATCC ATGACGATTGCCCAGACCCAGAACACCGCCGAACAGTTGCTGCGCGA
MTIAQTQNTAEQLLRDA axonopodis 13902
TGCCGGCATCTTGCCCGTGGTCACCGTGGACACGCTGGATCAGGCGC GILPVVTVDTLDQARRV
pv. GCCGCGTCGCCGATGCGTTGCTCGAAGGCGGCCTGCCCGCGATCGAG
ADALLEGGLPAIELTLR Vasculorum
CTGACCCTTCGCACGCCAGTGGCGATCGACGCGCTGGCGATGCTCAA TPVAIDALAMLKRELPN
GCGCGAGCTTCCTAACATCTTGATCGGTGCCGGCACCGTGCTGAGCG ILIGAGTVLSELQLRQS
AATTGCAGCTGCGTCAGTCGGTGGATGCCGGTGCAGACTTCCTGGTG VDAGADFLVTPGTPAPL
ACCCCGGGCACGCCGGCGCCGCTGGCGCGCCTGCTGGCGGATGCGCC ARLLADAPIPAVPGAAT
GATCCCGGCCGTTCCCGGCGCGGCCACTCCGACCGAGCTGCTGACCT PTELLTLMGLGFRVCKL
TGATGGGTCTTGGCTTTCGCGTCTGCAAGCTGTTCCCGGCCACCGCC FPATAVGGLQMLRGLAG
GTGGGCGGTCTGCAGATGCTCAGGGGCCTGGCCGGCCCGCTGTCCGA PLSELKLCPTGGISEAN
GCTCAAGCTGTGCCCCACCGGCGGCATCAGCGAGGCCAACGCCGCCG AAEFLSQPNVLCIGGSW
AGTTCCTGTCGCAGCCGAACGTGCTGTGCATCGGCGGTTCGTGGATG MVPKDWLAHGQWDKVKE
GTCCCCAAGGATTGGCTGGCGCACGGCCAATGGGACAAGGTCAAGGA SSAKAAAIVRQVRAG
AAGCTCGGCCAAGGCGGCGGCGATCGTGCGGCAGGTGCGGGCGGGCT (SEQ ID NO: 477) GA
(SEQ ID NO: 476) AAO55695.1 Pseudomonas Pv.
ATGACACAGAACGAAAATAATCAGCCGCTCACCAGCATGGCGAACAA MTQNENNQPLTSMANKI
syringiae Tomato GATTGCCCGGATCGACGAACTCTGCGCCAAGGCAAAGATTCTGCCGG
ARIDELCAKAKILPVIT str
TCATCACCATTGCCCGTGATCAGGACGTATTGCCACTGGCCGACGCG IARDQDVLPLADALAAG
DC3000 CTGGCCGCTGGTGGCATGACGGCTCTGGAAATCACCCTGCGCTCGGC
GMTALEITLRSAFGLSA GTTCGGACTGAGTGCGATCCGCATTTTGCGCGAGCAGCGCCCAGAGC
IRILREQRPELCTGAGT TGTGCACTGGCGCCGGGACCATTCTGGACCGCAAGATGCTGGCCGAC
ILDRKMLADAEAAGSQF GCCGAGGCGGCGGGCTCGCAATTCATTGTGACCCCCGGCAGCACGCA
IVTPGSTQELLQAALDS GGAACTGTTGCAGGCGGCGCTCGACAGCCCGTTGCCCCTGTTGCCAG
PLPLLPGVSSASEIMIG GCGTCAGCAGCGCGTCGGAAATCATGATCGGCTATGCCTTGGGTTAT
YALGYRRFKLFPAEISG CGCCGCTTCAAGCTGTTCCCGGCAGAAATCAGCGGCGGTGTGGCAGC
GVAAIKALGGPFNEVRF GATCAAGGCCTTGGGCGGGCCTTTCAACGAGGTGCGTTTCTGCCCGA
CPTGGVNEQNLKNYMAL CGGGCGGCGTCAACGAGCAGAACCTCAAGAACTACATGGCCTTGCCC
PNVMCVGGTWMIDNAWV AACGTCATGTGCGTCGGCGGGACATGGATGATTGATAACGCCTGGGT
KNGDWGRIQEATAQALA CAAGAATGGCGACTGGGGCCGCATTCAGGAAGCCACGGCACAGGCGC
LFD (SEQ ID NO: TGGCGCTGTTTGACTGA (SEQ ID NO: 478) 479) NP_718073.1
Shewanella MR-1 ATGCTTGAGAATAACTGGTCATTACAACCACAAGATATTTTTAAACG
MLENNWSLQPQDIFKRS oneidensis
CAGCCCTATTGTTCCTGTTATGGTGATTAACAAGATTGAACATGCGG PIVPVMVINKIEHAVPL
TGCCCTTAGCTAAAGCGCTGGTTGCCGGAGGGATAAGCGTGTTGGAA AKALVAGGISVLEVTLR
GTGACATTACGCACGCCATGCGCCCTTGAAGCTATCACCAAAATCGC TPCALEAITKIAKEVPE
CAAGGAAGTGCCTGAGGCGCTGGTTGGCGCGGGGACTATTTTAAATG ALVGAGTILNEAQLGQA
AAGCCCAGCTTGGACAGGCTATCGCCGCTGGTGCGCAATTTATTATC IAAGAQFIITPGATVEL
ACTCCAGGTGCGACAGTTGAGCTGCTCAAAGCGGGCATGCAAGGACC LKAGMQGPVPLIPGVAS
GGTGCCGTTAATTCCGGGCGTTGCCAGTATTTCCGAGGTGATGACGG ISEVMTGMALGYTHFKF
GCATGGCGCTGGGCTACACTCACTTTAAATTCTTCCCTGCTGAAGCG FPAEASGGVDALKAFSG
TCAGGTGGCGTTGATGCGCTTAAGGCTTTCTCTGGGCCGTTAGCAGA PLADIRFCPTGGITPSS
TATCCGCTTCTGCCCAACAGGTGGAATTACCCCGAGCAGCTATAAAG YKDYLALKNVDCIGGSW
ATTACTTAGCGCTGAAGAATGTCGATTGTATTGGTGGCAGCTGGATT IAPTDAMEQGDWDRITQ
GCTCCTACCGATGCGATGGAGCAGGGCGATTGGGATCGTATCACTCA LCKEAIGGL (SEQ ID
GCTGTGTAAAGAGGCGATTGGCGGACTTTAA (SEQ ID NO: 89) NO: 90) YP_261692
Pseudomonas Pf-5 ATGACAAACCTCGCCCCGACCGTTTCCATGGCGGACAAAGTTGCCCT
MTNLAPTVSMADKVALI fluorescens
GATCGACAGCCTCTGCGCCAAGGCGCGGATCCTGCCGGTGATCACCA DSLCAKARILPVITIAR
TTGCCCGCGAGCAGGATGTCCTGCCGCTGGCCGATGCCCTGGCGGCC EQDVLPLADALAAGGLT
GGCGGCCTGACCGCCCTGGAAGTGACCCTGCGTTCGCAGTTCGGCCT ALEVTLRSQFGLKAIQI
CAAGGCGATCCAGATCCTGCGCGAACAGCGCCCGGAGCTGGTGACCG LREQRPELVTGAGTVLD
GTGCCGGCACCGTGCTCGACCCGCAGATGCTGGTGGCGGCGGAAGCG PQMLVAAEAAGSQFIVT
GCAGGTTCGCAGTTCATCGTCACCCCGGGCATCACCCGCGACCTGCT PGITRDLLQASVASPIP
GCAAGCCAGCGTGGCCAGCCCGATTCCCCTGCTGCCGGGGATCAGCA LLPGISNASGIMEGYAL
ATGCCTCCGGGATCATGGAGGGTTATGCCCTGGGCTACCGCCGCTTC GYRRFKLFPAEVSGGVA
AAGCTGTTCCCGGCGGAAGTCAGTGGTGGCGTGGCGGCGATCAAGGC AIKALGGPFGEVKFCPT
CCTGGGCGGGCCGTTCGGCGAGGTCAAGTTCTGCCCTACCGGCGGCG GGVGPANIKSYMALKNV
TCGGCCCGGCCAATATCAAGAGCTACATGGCGCTCAAGAATGTGATG MCVGGSWMLDPEWIKNG
TGTGTCGGCGGTAGCTGGATGCTCGATCCCGAGTGGATCAAGAACGG DWARIQECTAEALALLD
CGACTGGGCACGGATCCAGGAGTGCACGGCCGAGGCCCTGGCCCTGC (SEQ ID NO: 481)
TGGACTGA (SEQ ID NO: 480) ZP_03591973.1 Bacillus subtilis
ATGGAGTCCAAAGTCGTTGAAAACCGTCTGAAAGAAGCAAAGCTGAT MESKVVENRLKEAKLIA
subtilis str. TGCAGTCATTCGTTCAAAGGATAAGCAGGAGGCCTGTCAGCAGATTG
VIRSKDKQEACQQIESL 168
AGAGTTTATTAGATAAAGGGATTCGTGCAGTTGAAGTGACGTATACG LDKGIRAVEVTYTTPGA
ACCCCCGGGGCATCAGATATTATCGAATCCTTCCGTAATAGGGAAGA SDIIESFRNREDILIGA
TATTTTAATTGGCGCGGGTACGGTCATCAGCGCGCAGCAAGCTGGGG GTVISAQQAGEAAKAGA
AAGCTGCTAAGGCTGGCGCGCAGTTTATTGTCAGTCCGGGTTTTTCA QFIVSPGFSADLAEHLS
GCTGATCTTGCTGAACATCTATCTTTTGTAAAGACACATTATATCCC FVKTHYIPGVLTPSEIM
CGGCGTCTTGACTCCGAGCGAAATTATGGAAGCGCTGACATTCGGTT EALTFGFTTLKLFPSGV
TTACGACATTAAAGCTGTTCCCAAGCGGTGTGTTTGGCATTCCGTTT FGIPFMKNLAGPFPQVT
ATGAAAAATTTAGCGGGTCCTTTCCCGCAGGTGACCTTTATTCCGAC FIPTGGIHPSEVPDWLR
AGGCGGGATACATCCGTCTGAAGTGCCTGATTGGCTTAGAGCCGGAG AGAGAVGVGSQLGSCSK
CTGGCGCCGTCGGAGTCGGCAGCCAGTTGGGCAGCTGTTCAAAAGAG EDLQAVFQV (SEQ ID
GATTTGCAGGCTGTTTTCCAAGTGTAA (SEQ ID NO: 482) NO: 483) YP_081150.2
Bacillus ATCC ATGGTATTGTCACACATCGAAGAACAAAAACTGATTGCGATCATCCG
MVLSHIEEQKLIAIIRG licheniformis 14580
CGGATACAATCCGGAGGAGGCAGTGAGCATTGCCGGCGCCTTAAAAG YNPEEAVSIAGALKAGG
CGGGCGGCATCAGGCTTGTGGAGATTACGCTTAATTCCCCTCAAGCG IRLVEITLNSPQAIKAI
ATCAAAGCGATTGAAGCGGTTTCAGAGCATTTTGGGGACGAAATGCT EAVSEHFGDEMLVGAGT
TGTCGGAGCGGGAACCGTACTTGATCCCGAATCTGCGAGAGCGGCGC VLDPESARAALLAGARF
TTTTAGCCGGCGCGCGGTTTATCCTGTCTCCGACCGTCAATGAAGAG ILSPTVNEETIKLTKRY
ACGATCAAGCTGACAAAACGGTATGGAGCGGTCAGCATTCCAGGCGC GAVSIPGAFTPTEILTA
TTTTACCCCGACTGAAATATTGACGGCGTATGAAAGCGGGGGAGACA YESGGDIIKVFPGTMGP
TCATCAAGGTATTTCCCGGAACAATGGGGCCTGGCTATATCAAGGAT GYIKDIHGPLPHIPLLP
ATCCACGGACCGCTTCCGCATATTCCGCTGCTTCCGACTGGAGGAGT TGGVGLENLHEFLQAGA
CGGATTGGAAAACCTTCACGAGTTTCTGCAGGCCGGTGCGGTCGGAG VGAGIGGSLVRANKDVN
CGGGAATCGGCGGTTCGCTTGTTCGGGCTAATAAAGATGTTAATGAC DAFLEELSKKAKQFVEA
GCGTTTTTAGAAGAGCTGTCCAAAAAAGCAAAGCAATTTGTTGAAGC AKQ (SEQ ID NO:
AGCAAAACAGTAA (SEQ ID NO: 484) 485) YP_190869.1 Gluconobacter 62IH
ATGATCGATACTGCCAAACTCGACGCCGTCATGAGCCGTTGTCCGGT MIDTAKLDAVMSRCPVM
oxydans CATGCCGGTGCTGGTGGTCAATGATGTGGCTCTGGCCCGCCCGATGG
PVLVVNDVALARPMAEA CCGAGGCTCTGGTGGCGGGTGGACTGTCCACGCTGGAAGTCACGCTG
LVAGGLSTLEVTLRTPC CGCACGCCCTGCGCCCTTGAAGCTATTGAGGAAATGTCGAAAGTACC
ALEAIEEMSKVPGALVG AGGCGCGCTGGTCGGTGCCGGTACGGTGCTGAATCCGTCCGACATGG
AGTVLNPSDMDRAVKAG ACCGTGCCGTGAAGGCGGGTGCGCGCTTCATCGTCAGCCCCGGCCTG
ARFIVSPGLTEALAKAS ACCGAGGCGCTGGCAAAGGCGTCGGTTGAGCATGACGTCCCCTTCCT
VEHDVPFLPGVANAGDI GCCAGGCGTTGCCAATGCGGGTGACATCATGCGGGGTCTGGATCTGG
MRGLDLGLSRFKFFPAV GTCTGTCACGCTTCAAGTTCTTCCCGGCTGTGACGAATGGCGGCATT
TNGGIPALKSLASVFGS CCCGCGCTCAAGAGCTTGGCCAGTGTTTTTGGCAGCAATGTCCGTTT
NVRFCPTGGITEESAPD CTGCCCCACGGGCGGCATTACGGAAGAGAGCGCACCGGACTGGCTGG
WLALPSVACVGGSWVTA CGCTTCCCTCCGTGGCCTGCGTCGGCGGATCCTGGGTGACGGCCGGC
GTFDADKVRQRATAAAL ACGTTCGATGCGGACAAGGTCCGTCAGCGCGCCACGGCTGCGGCACT
FTV (SEQ ID NO: CTTCACGGTCTGA (SEQ ID NO: 91) 92) NP_251871.1 P.
aeruginosa PAO1 ATGAAAAACTGGAAAACAAGTGCAGAATCAATCCTGACCACCGGCCC
MKNWKTSAESILTTGPV Codon
GGTTGTACCGGTTATCGTGGTAAAAAAACTGGAACACGCGGTGCCGA VPVIVVKKLEHAVPMAK
Optimized TGGCAAAAGCGTTGGTTGCTGGTGGGGTGCGCGTTCTGGAAGTGACT
ALVAGGVRVLEVTLRTE CTGCGTACCGAGTGTGCAGTTGACGCTATCCGTGCTATCGCCAAAGA
CAVDAIRAIAKEVPEAI AGTGCCTGAAGCGATTGTGGGTGCCGGTACGGTGCTGAATCCACAGC
VGAGTVLNPQQLAEVTE AGCTGGCAGAAGTCACTGAAGCGGGTGCACAGTTCGCAATTAGCCCG
AGAQFAISPGLTEPLLK GGTCTGACCGAGCCGCTGCTGAAAGCTGCTACCGAAGGGACTATTCC
AATEGTIPLIPGISTVS TCTGATTCCGGGGATCAGCACTGTTTCCGAACTGATGCTGGGTATGG
ELMLGMDYGLKEFKFFP ACTACGGTTTGAAAGAGTTCAAATTCTTCCCGGCTGAAGCTAACGGC
AEANGGVKALQAIAGPF GGCGTGAAAGCCCTGCAGGCGATCGCGGGTCCGTTCTCCCAGGTCCG
SQVRFCPTGGISPANYR TTTCTGCCCGACGGGTGGTATTTCTCCGGCTAACTACCGTGACTACC
DYLALKSVLCIGGSWLV TGGCGCTGAAAAGCGTGCTGTGCATCGGTGGTTCCTGGCTGGTTCCG
PADALEAGDYDRITKLA GCAGATGCGCTGGAAGCGGGCGATTACGACCGCATTACTAAGCTGGC
REAVEGAKL (SEQ ID GCGTGAAGCTGTAGAAGGCGCTAAGCTGTAA (SEQ ID NO: NO:
487) 486) PAO1-Ec5 ATGAAAAACTGGAAACAGAAGACCGCCCGCATCGACACGCTGTGCCG
MKNWKQKTARIDTLCRE GGAGGCGCGCATCCTCCCGGTGATCACCATCGACCGCGAGGCGGACA
ARILPVITIDREADILP TCCTGCCGATGGCCGATGCCCTCGCCGCCGGCGGCCTGACCGCCCTG
MADALAAGGLTALEITL GAGATCACCCTGCGCACGGCGCACGGGCTGACCGCCATCCGGCGCCT
RTAHGLTAIRRLSEERP CAGCGAGGAGCGCCCGCACCTGCGCATCGGCGCCGGCACCGTGCTCG
HLRIGAGTVLDPRTFAA ACCCGCGGACCTTCGCCGCCGCGGAAAAGGCCGGGGCGAGCTTCGTG
AEKAGASFVVTPGCTDE GTCACCCCGGGTTGCACCGACGAGTTGCTGCGCTTCGCCCTGGACAG
LLRFALDSEVPLLPGVA CGAAGTCCCGCTGTTGCCCGGCGTGGCCAGCGCTTCCGAGATCATGC
SASEIMLAYRHGYRRFK TCGCCTACCGCCATGGCTACCGCCGCTTCAAGCTGTTTCCCGCCGAA
LFPAEVSGGPAALKAFS GTCAGCGGCGGCCCGGCGGCGCTGAAGGCGTTCTCGGGACCATTCCC
GPFPDIRFCPTGGVSLN CGATATCCGCTTCTGCCCCACCGGAGGCGTCAGCCTGAACAATCTCG
NLADYLAVPNVMCVGGT CCGACTACCTGGCGGTACCCAACGTGATGTGCGTCGGCGGCACCTGG
WMLPKAVVDRGDWAQVE ATGCTGCCCAAGGCCGTGGTCGACCGCGGCGACTGGGCCCAGGTCGA
RLSREALERFAEHRRH GCGCCTCAGCCGCGAAGCCCTGGAGCGCTTCGCCGAGCACCGCAGAC
(SEQ ID NO: 489) ACTAATAGCTCGAGTTACTTTACT (SEQ ID NO: 488)
PAO1-Ec10 ATGAAAAACTGGAAAACAAGTGCAGAATCAATCGACACGCTGTGCCG
MKNWKTSAESIDTLCRE GGAGGCGCGCATCCTCCCGGTGATCACCATCGACCGCGAGGCGGACA
ARILPVITIDREADILP TCCTGCCGATGGCCGATGCCCTCGCCGCCGGCGGCCTGACCGCCCTG
MADALAAGGLTALEITL GAGATCACCCTGCGCACGGCGCACGGGCTGACCGCCATCCGGCGCCT
RTAHGLTAIRRLSEERP CAGCGAGGAGCGCCCGCACCTGCGCATCGGCGCCGGCACCGTGCTCG
HLRIGAGTVLDPRTFAA ACCCGCGGACCTTCGCCGCCGCGGAAAAGGCCGGGGCGAGCTTCGTG
AEKAGASFVVTPGCTDE GTCACCCCGGGTTGCACCGACGAGTTGCTGCGCTTCGCCCTGGACAG
LLRFALDSEVPLLPGVA CGAAGTCCCGCTGTTGCCCGGCGTGGCCAGCGCTTCCGAGATCATGC
SASEIMLAYRHGYRRFK TCGCCTACCGCCATGGCTACCGCCGCTTCAAGCTGTTTCCCGCCGAA
LFPAEVSGGPAALKAFS GTCAGCGGCGGCCCGGCGGCGCTGAAGGCGTTCTCGGGACCATTCCC
GPFPDIRFCPTGGVSLN CGATATCCGCTTCTGCCCCACCGGAGGCGTCAGCCTGAACAATCTCG
NLADYLAVPNVMCVGGT CCGACTACCTGGCGGTACCCAACGTGATGTGCGTCGGCGGCACCTGG
WMLPKAVVDRGDWAQVE ATGCTGCCCAAGGCCGTGGTCGACCGCGGCGACTGGGCCCAGGTCGA
RLSREALERFAEHRRH GCGCCTCAGCCGCGAAGCCCTGGAGCGCTTCGCCGAGCACCGCAGAC
(SEQ ID NO: 491) ACTAATAGCTCGAGTTACTTTACT (SEQ ID NO: 490)
PAO1-Ec15 ATGAAAAACTGGAAAACAAGTGCAGAATCAATCCTGACCACCGGCCG
MKNWKTSAESILTTGRE GGAGGCGCGCATCCTCCCGGTGATCACCATCGACCGCGAGGCGGACA
ARILPVITIDREADILP TCCTGCCGATGGCCGATGCCCTCGCCGCCGGCGGCCTGACCGCCCTG
MADALAAGGLTALEITL GAGATCACCCTGCGCACGGCGCACGGGCTGACCGCCATCCGGCGCCT
RTAHGLTAIRRLSEERP CAGCGAGGAGCGCCCGCACCTGCGCATCGGCGCCGGCACCGTGCTCG
HLRIGAGTVLDPRTFAA ACCCGCGGACCTTCGCCGCCGCGGAAAAGGCCGGGGCGAGCTTCGTG
AEKAGASFVVTPGCTDE GTCACCCCGGGTTGCACCGACGAGTTGCTGCGCTTCGCCCTGGACAG
LLRFALDSEVPLLPGVA CGAAGTCCCGCTGTTGCCCGGCGTGGCCAGCGCTTCCGAGATCATGC
SASEIMLAYRHGYRRFK TCGCCTACCGCCATGGCTACCGCCGCTTCAAGCTGTTTCCCGCCGAA
LFPAEVSGGPAALKAFS GTCAGCGGCGGCCCGGCGGCGCTGAAGGCGTTCTCGGGACCATTCCC
GPFPDIRFCPTGGVSLN CGATATCCGCTTCTGCCCCACCGGAGGCGTCAGCCTGAACAATCTCG
NLADYLAVPNVMCVGGT CCGACTACCTGGCGGTACCCAACGTGATGTGCGTCGGCGGCACCTGG
WMLPKAVVDRGDWAQVE ATGCTGCCCAAGGCCGTGGTCGACCGCGGCGACTGGGCCCAGGTCGA
RLSREALERFAEHRRH GCGCCTCAGCCGCGAAGCCCTGGAGCGCTTCGCCGAGCACCGCAGAC
(SEQ ID NO: 493) ACTAATAGCTCGAGTTACTTTACT (SEQ ID NO: 492)
[0525] EDA extracts were prepared using the following protocol.
[0526] Day 1
[0527] Grow 5 ml LB-Kan preps of BF1055 (BL21/DE3 with pET26b empty
vector) and BF1706 (BL21DE3 with pET26b+E. coli EDD).
[0528] Grow 5 ml preps of each EDA construct expressed in S.
cerevisiae in appropriate selective media (e.g. ScD-leu).
[0529] Day 2
[0530] Grow 50 ml LB-Kan prep of BF1055, 2% (v/v) inoculate.
[0531] Grow 50 ml prep of BF1706 using Novagen's Overnight Express
(46.45 ml LB-Kan, 1 ml solution 1, 2.5 ml solution 2, 50 .mu.l
solution 3, 5 .mu.l of 1M MnCl.sub.2, 50 .mu.l of 0.5 M
FeCl.sub.2), 2% (v/v) inoculate.
[0532] Grow 50 ml prep of each EDA construct expressed in S.
cerevisiae in appropriate selective media
[0533] +10 mM MnCl.sub.2. Inoculate to OD.sub.600 of 0.2.
[0534] Day 3
[0535] EDD extractions (adapted from Chemyan et al, Protein Science
16:2368-2377, 2007): [0536] 1) Pellet cells in 50 ml conical tubes,
4.degree. C., 3,000 rpm, 10 mins, discard supernatant. [0537] 2)
Resuspend in 2 ml degassed PDGH buffer (20 mM MES pH 6.5, 30 mM
NaCl, 5 mM MnCl.sub.2, 0.5 mM FeCl.sub.2, 10 mM 2-mercaptoethanol,
10 mM cysteine, sparged with nitrogen gas). Move to hungate tube.
[0538] 3) Add 0.1% Triton X-100, 10 ng/ml DNase, 10 .mu.g/ml PMSF,
10 .mu.g/ml TAME (Na-(p-toluene sulfonyl)-L-arginine methyl ester),
100 .mu.g/ml lysozyme. [0539] 4) Sparge hungate tube with nitrogen
gas, cap and seal. Incubate 2 hours at 37.degree. C., swirl
occasionally. [0540] 5) Clarify by centrifugation in 2-ml tube,
4.degree. C., 10 mins, 14,000 rpm. Keep supernatant. [0541] 6)
Treat with 150 mM pyruvate and 10 mM sodium cyanoborohydride (work
in hood) to inactivate aldolase activity. Incubate 30 mins at room
temperature. [0542] 7) During incubation, pre-equilibrate PD-10
column from GE [0543] a. Remove top cap, pour off storage buffer.
[0544] b. Cut off bottom tip, fit in 50 ml conical with adapter.
[0545] c. Pour 5 ml of 20 mM MES buffer, pH 6.5 (total of 5 times).
Discard flow-through. [0546] 8) Run sample through column, then add
MES buffer to a total of 2.5 ml volume added. Discard flow-through.
[0547] 9) Run 3.5 ml 20 mM MES pH 6.5 buffer to elute protein.
Discard column in appropriate waste receptacle. [0548] 10) Perform
Bradford assay (1:10 or 1:20 dilution).
EDA Extractions:
[0548] [0549] 1) Spin down in 50 ml conicals, 4.degree. C., 3,400
rpm, 5 mins. Wash 2.times. with 25 ml water. [0550] 2) Resuspend in
1 ml lysis buffer (50 mM Tris-HCl, pH 7, 10 mM MgCl.sub.2, 1.times.
protease inhibitor. [0551] 3) Add 1 cap of zirconia beads, vortex
4-6 times, 15 sec bursts, ice in between. [0552] 4) Spin down cell
debris, 4.degree. C., 14,000 rpm, 10 mins. Save supernatant. [0553]
5) Perform Bradford assay (1:2 dilution).
Activity Assays:
[0554] Each reaction contains 50 mM Tris-HCl, pH 7, 10 mM
MgCl.sub.2, 0.15 mM NADH, 15 .mu.g LDH, saturating amounts of EDD
determined empirically (usually -100 .mu.g), 1-50 .mu.g EDA
(depending on level of activity), and 1 mM 6-phosphogluconate.
Reactions are started by the addition of 6-phosphogluconate and
monitored for 5 mins at 30.degree. C.
[0555] Results
[0556] The S. cerevisiae strains tested for EDA activity are
described in the table below. yCH strains are Thermosacc-based
(Lallemand). BF strains are based on BY4742.
TABLE-US-00117 Strain Vector Construct BF542 pBF150 Zymomonas
mobilis EDA BF1689 pBF892 PAO1 + 5aa E. coli EDA BF1691 pBF894 PAO1
+ 10aa E. coli EDA BF1693 pBF896 PAO1 + 15aa E. coli EDA BF1721
pBF909 Bacilluis licheniformis EDA BF1722 pBF910 Bacillus subtilis
EDA BF1723 pBF911 Pseudomonas fluorescens EDA BF1724 pBF912
Pseudomonas syringae EDA BF1725 pBF913 Saccharaophagus degradans
EDA BF1726 pBF914 Xanthamonas axonopodis EDA BF1727 pBF766
Escherichia coli EDA BF1728 pBF764 Pseudomonas aeruginosa EDA
BF1729 pBF729 Gluconobacter oxydans EDA BF1730 pBF727 Shewanella
oneidensis EDA BF1775 pBF87 p425GPD (empty vector) BF1776 pBF928
PAO1 EDA codon optimized for S. cerevisiae
[0557] E. coli expressed EDD was prepared and confirmed by western
blot analysis as shown in FIG. 23. The expected size of EDD is
approximately 66 kilodaltons (kDa). A band of approximately that
size (e.g., as determined by the nearest sized protein standard of
approximately 60 kDa) was identified by western blot. The E. coli
expressed EDD was used with S. cerevisiae expressed EDA's to
evaluate the EDA activities. The results of EDA kinetic assays are
presented in the table below.
TABLE-US-00118 EDD/EDA slope % max EC/EC 0.3467 100.00 EC/SO 0.1907
55.00 EC/BS 0.0897 25.87 EC/GO 0.0848 24.46 EC/PCO 0.084 24.23
EC/PA 0.0533 15.37 EC/PE5 0.0223 6.43 EC/PE10 0.0218 6.29 EC/SD
0.015 4.33 EC/PS 0.0135 3.89 EC/BL 0.0112 3.23 EC/ZM 0.0109 3.14
EC/PF 0.0082 2.37 EC/V 0.0074 2.13 EC/XA 0.0065 1.87 EC/PE15 0.005
1.44
[0558] In the results presented above, the slope of the E. coli
(EC) EDA is outside the linear range for accurate detection, and is
therefore underestimated. For the other EDA's, when compared to the
E. coli EDA, the calculated percentage of maximum activity (e.g., %
max) is overestimated, however the slopes are accurate. The results
of this experiment indicate that the E. coli EDA has higher
activity as compared to the other EDA activities evaluated herein,
and is approximately 16-fold more active than the EDA from P.
aeruginosa. EDA's from X. anoxopodis and a chimera between E. coli
EDA and P. aeruginosa (e.g., PE15) show less activity than the
vector control. Codon-optimized EDA from P. aeruginosa showed a
slight improvement over the native sequence, however chimeric
versions (e.g., PE5, PE10, PE15) showed less activity than native.
The experiments were repeated using 100 .mu.g of EDD and 25 .mu.g
of EDA cell lysates in each reaction (unless otherwise noted, such
as 5 .mu.g of E. coli EDA). The reactions in the repeated
experiment all were in the linear range of detection and the
results of these additional kinetic assays are shown graphically in
FIG. 24, and in the table below. E. coli EDA was again found to be
the most active of those EDA's tested.
TABLE-US-00119 EDA slope % max EC 0.462 100.00 SO 0.128 27.71 GO
0.0544 11.77 PCO 0.0539 11.67 BS 0.0505 10.93 PA 0.0273 5.91 V
0.0006 0.13
Example 35
Nucleotide and Amino Acid Sequence of S. cerevisiae Phosphoglucose
Isomerase
[0559] Phosphoglucose isomerase (PGI1) activity was decreased or
disrupted, in some embodiments, to favor the conversion of
glucose-6-phosphate to gluconolactone-6-phosphate by the activity
of ZWF1 (e.g., glucose-6-phosphate dehydrogenase). The nucleotide
sequence of the S. cerevisiae PGI1 gene altered to decrease or
disrupt phosphoglucose isomerase activity is shown below.
TABLE-US-00120 PGI1 nucleotide sequence (SEQ ID NO: 494)
ATGTCCAATAACTCATTCACTAACTTCAAACTGGCCACTGAATTGCCAGC
CTGGTCTAAGTTGCAAAAAATTTATGAATCTCAAGGTAAGACTTTGTCTG
TCAAGCAAGAATTCCAAAAAGATGCCAAGCGTTTTGAAAAATTGAACAAG
ACTTTCACCAACTATGATGGTTCCAAAATCTTGTTCGACTACTCAAAGAA
CTTGGTCAACGATGAAATCATTGCTGCATTGATTGAACTGGCCAAGGAGG
CTAACGTCACCGGTTTGAGAGATGCTATGTTCAAAGGTGAACACATCAAC
TCCACTGAAGATCGTGCTGTCTACCACGTCGCATTGAGAAACAGAGCTAA
CAAGCCAATGTACGTTGATGGTGTCAACGTTGCTCCAGAAGTCGACTCTG
TCTTGAAGCACATGAAGGAGTTCTCTGAACAAGTTCGTTCTGGTGAATGG
AAGGGTTATACCGGTAAGAAGATCACCGATGTTGTTAACATCGGTATTGG
TGGTTCCGATTTGGGTCCAGTCATGGTCACTGAGGCTTTGAAGCACTACG
CTGGTGTCTTGGATGTCCACTTCGTTTCCAACATTGACGGTACTCACATT
GCTGAAACCTTGAAGGTTGTTGACCCAGAAACTACTTTGTTTTTGATTGC
TTCCAAGACTTTCACTACCGCTGAAACTATCACTAACGCTAACACTGCCA
AGAACTGGTTCTTGTCGAAGACAGGTAATGATCCATCTCACATTGCTAAG
CATTTCGCTGCTTTGTCCACTAACGAAACCGAAGTTGCCAAGTTCGGTAT
TGACACCAAAAACATGTTTGGTTTCGAAAGTTGGGTCGGTGGTCGTTACT
CTGTCTGGTCGGCTATTGGTTTGTCTGTTGCCTTGTACATTGGCTATGAC
AACTTTGAGGCTTTCTTGAAGGGTGCTGAAGCCGTCGACAACCACTTCAC
CCAAACCCCATTGGAAGACAACATTCCATTGTTGGGTGGTTTGTTGTCTG
TCTGGTACAACAACTTCTTTGGTGCTCAAACCCATTTGGTTGCTCCATTC
GACCAATACTTGCACAGATTCCCAGCCTACTTGCAACAATTGTCAATGGA
ATCTAACGGTAAGTCTGTTACCAGAGGTAACGTGTTTACTGACTACTCTA
CTGGTTCTATCTTGTTTGGTGAACCAGCTACCAACGCTCAACACTCTTTC
TTCCAATTGGTTCACCAAGGTACCAAGTTGATTCCATCTGATTTCATCTT
AGCTGCTCAATCTCATAACCCAATTGAGAACAAATTACATCAAAAGATGT
TGGCTTCAAACTTCTTTGCTCAAGCTGAAGCTTTAATGGTTGGTAAGGAT
GAAGAACAAGTTAAGGCTGAAGGTGCCACTGGTGGTTTGGTCCCACACAA
GGTCTTCTCAGGTAACAGACCAACTACCTCTATCTTGGCTCAAAAGATTA
CTCCAGCTACTTTGGGTGCTTTGATTGCCTACTACGAACATGTTACTTTC
ACTGAAGGTGCCATTTGGAATATCAACTCTTTCGACCAATGGGGTGTTGA
ATTGGGTAAAGTCTTGGCTAAAGTCATCGGCAAGGAATTGGACAACTCCT
CCACCATTTCTACCCACGATGCTTCTACCAACGGTTTAATCAATCAATTC
AAGGAATGGATGTGA
Example 36
Nucleotide and Amino Acid Sequence of S. cerevisiae
6-Phosphogluconate Dehydrogenase (Decarboxylating)
[0560] 6-phosphogluconate dehydrogenase (decarboxylating) (GND1)
activity was decreased or disrupted, in some embodiments, to
minimize or eliminate the conversion of gluconate-6-phophate to
ribulose-5-phosphate. The nucleotide sequence of the S. cerevisiae
GND1 and GND2 genes altered to decrease or disrupt
6-phosphogluconate dehydrogenase (decarboxylating) activity is
shown below.
TABLE-US-00121 GND1/YHR183W (SEQ ID NO: 495)
ATGTCTGCTGATTTCGGTTTGATTGGTTTGGCCGTCATGGGTCAAAATTT
GATCTTGAACGCTGCTGACCACGGTTTCACTGTTTGTGCTTACAACAGAA
CTCAATCCAAGGTCGACCATTTCTTGGCCAATGAAGCTAAGGGCAAATCT
ATCATCGGTGCTACTTCCATTGAAGATTTCATCTCCAAATTGAAGAGACC
TAGAAAGGTCATGCTTTTGGTTAAAGCTGGTGCTCCAGTTGACGCTTTGA
TCAACCAAATCGTCCCACTTTTGGAAAAGGGTGATATTATCATCGATGGT
GGTAACTCTCACTTCCCAGATTCTAATAGACGTTACGAAGAATTGAAGAA
GAAGGGTATTCTTTTCGTTGGTTCTGGTGTCTCCGGTGGTGAGGAAGGTG
CCCGTTACGGTCCATCTTTGATGCCAGGTGGTTCTGAAGAAGCTTGGCCA
CATATTAAGAACATCTTCCAATCCATCTCTGCTAAATCCGACGGTGAACC
ATGTTGCGAATGGGTTGGCCCAGCCGGTGCTGGTCACTACGTCAAGATGG
TTCACAACGGTATTGAATACGGTGATATGCAATTGATTTGTGAAGCTTAT
GACATCATGAAGAGATTGGGTGGGTTTACCGATAAGGAAATCAGTGACGT
TTTTGCCAAATGGAACAATGGTGTCTTGGATTCCTTCTTGGTCGAAATTA
CCAGAGATATTTTGAAATTCGACGACGTCGACGGTAAGCCATTAGTTGAA
AAAATCATGGATACTGCTGGTCAAAAGGGTACTGGTAAGTGGACTGCCAT
CAACGCCTTGGATTTGGGTATGCCAGTTACTTTGATTGGTGAAGCTGTCT
TTGCCCGTTGTCTATCTGCTTTGAAGAACGAGAGAATTAGAGCCTCCAAG
GTCTTACCAGGCCCAGAAGTTCCAAAAGACGCCGTCAAGGACAGAGAACA
ATTTGTCGATGATTTGGAACAAGCTTTGTATGCTTCCAAGATTATTTCTT
ACGCTCAAGGTTTCATGTTGATCCGTGAAGCTGCTGCTACTTATGGCTGG
AAACTAAACAACCCTGCCATCGCTTTGATGTGGAGAGGTGGTTGTATCAT
TAGATCTGTTTTCTTGGGTCAAATCACAAAGGCCTACAGAGAAGAACCAG
ATTTGGAAAACTTGTTGTTCAACAAGTTCTTCGCTGATGCCGTCACCAAG
GCTCAATCTGGTTGGAGAAAGTCAATTGCGTTGGCTACCACCTACGGTAT
CCCAACACCAGCCTTTTCCACCGCTTTGTCTTTCTACGATGGGTACAGAT
CTGAAAGATTGCCAGCCAACTTACTACAAGCTCAACGTGACTACTTTGGT
GCTCACACTTTCAGAGTGTTGCCAGAATGTGCTTCTGACAACTTGCCAGT
AGACAAGGATATCCATATCAACTGGACTGGCCACGGTGGTAATGTTTCTT
CCTCTACATACCAAGCTTAA GND2/YGR256W (SEQ ID NO: 496)
ATGTCAAAGGCAGTAGGTGATTTAGGCTTAGTTGGTTTAGCCGTGATGGG
TCAAAATTTGATCTTAAACGCAGCGGATCACGGATTTACCGTGGTTGCTT
ATAATAGGACGCAATCAAAGGTAGATAGGTTTCTAGCTAATGAGGCAAAA
GGAAAATCAATAATTGGTGCAACTTCAATTGAGGACTTGGTTGCGAAACT
AAAGAAACCTAGAAAGATTATGCTTTTAATCAAAGCCGGTGCTCCGGTCG
ACACTTTAATAAAGGAACTTGTACCACATCTTGATAAAGGCGACATTATT
ATCGACGGTGGTAACTCACATTTCCCGGACACTAACAGACGCTACGAAGA
GCTAACAAAGCAAGGAATTCTTTTTGTGGGCTCTGGTGTCTCAGGCGGTG
AAGATGGTGCACGTTTTGGTCCATCTTTAATGCCTGGTGGGTCAGCAGAA
GCATGGCCGCACATCAAGAACATCTTTCAATCTATTGCCGCCAAATCAAA
CGGTGAGCCATGCTGCGAATGGGTGGGGCCTGCCGGTTCTGGTCACTATG
TGAAGATGGTACACAACGGTATCGAGTACGGTGATATGCAGTTGATTTGC
GAGGCTTACGATATCATGAAACGAATTGGCCGGTTTACGGATAAAGAGAT
CAGTGAAGTATTTGACAAGTGGAACACTGGAGTTTTGGATTCTTTCTTGA
TTGAAATCACGAGGGACATTTTAAAATTCGATGACGTCGACGGTAAGCCA
TTGGTGGAAAAAATTATGGATACTGCCGGTCAAAAGGGTACTGGTAAATG
GACTGCAATCAACGCCTTGGATTTAGGAATGCCAGTCACTTTAATTGGGG
AGGCTGTTTTCGCTCGTTGTTTGTCAGCCATAAAGGACGAACGTAAAAGA
GCTTCGAAACTTCTGGCAGGACCAACAGTACCAAAGGATGCAATACATGA
TAGAGAACAATTTGTGTATGATTTGGAACAAGCATTATACGCTTCAAAGA
TTATTTCATATGCTCAAGGTTTCATGCTGATCCGCGAAGCTGCCAGATCA
TACGGCTGGAAATTAAACAACCCAGCTATTGCTCTAATGTGGAGAGGTGG
CTGTATAATCAGATCTGTGTTCTTAGCTGAGATTACGAAGGCTTATAGGG
ACGATCCAGATTTGGAAAATTTATTATTCAACGAGTTCTTCGCTTCTGCA
GTTACTAAGGCCCAATCCGGTTGGAGAAGAACTATTGCCCTTGCTGCTAC
TTACGGTATTCCAACTCCAGCTTTCTCTACTGCTTTAGCGTTTTACGACG
GCTATAGATCTGAGAGGCTACCAGCAAACTTGTTACAAGCGCAACGTGAT
TATTTTGGCGCTCATACATTTAGAATTTTACCTGAATGTGCTTCTGCCCA
TTTGCCAGTAGACAAGGATATTCATATCAATTGGACTGGGCACGGAGGTA
ATATATCTTCCTCAACCTACCAAGCTTAA
Example 37
Nucleotide and Amino Acid Sequence of S. cerevisiae
Transaldolase
[0561] Transaldolase (TAL1) activity was increased in some
embodiments, and in certain embodiments transaldolase activity was
decreased or disrupted. Transaldolase converts sedoheptulose
7-phosphate and glyceraldehyde 3-phosphate to erythrose 4-phosphate
and fructose 6-phosphate. The rationale for increasing or
decreasing transaldolase activity is described herein with respect
to various embodiments. The nucleotide sequence of the S.
cerevisiae TAL1 gene altered to increase or decrease transaldolase
activity, and the encoded amino acid sequence are shown below.
TABLE-US-00122 TAL1 nucleotide sequence (SEQ ID NO: 497)
ATGTCTGAACCAGCTCAAAAGAAACAAAAGGTTGCTAACAACTCTCTAGA
ACAATTGAAAGCCTCCGGCACTGTCGTTGTTGCCGACACTGGTGATTTCG
GCTCTATTGCCAAGTTTCAACCTCAAGACTCCACAACTAACCCATCATTG
ATCTTGGCTGCTGCCAAGCAACCAACTTACGCCAAGTTGATCGATGTTGC
CGTGGAATACGGTAAGAAGCATGGTAAGACCACCGAAGAACAAGTCGAAA
ATGCTGTGGACAGATTGTTAGTCGAATTCGGTAAGGAGATCTTAAAGATT
GTTCCAGGCAGAGTCTCCACCGAAGTTGATGCTAGATTGTCTTTTGACAC
TCAAGCTACCATTGAAAAGGCTAGACATATCATTAAATTGTTTGAACAAG
AAGGTGTCTCCAAGGAAAGAGTCCTTATTAAAATTGCTTCCACTTGGGAA
GGTATTCAAGCTGCCAAAGAATTGGAAGAAAAGGACGGTATCCACTGTAA
TTTGACTCTATTATTCTCCTTCGTTCAAGCAGTTGCCTGTGCCGAGGCCC
AAGTTACTTTGATTTCCCCATTTGTTGGTAGAATTCTAGACTGGTACAAA
TCCAGCACTGGTAAAGATTACAAGGGTGAAGCCGACCCAGGTGTTATTTC
CGTCAAGAAAATCTACAACTACTACAAGAAGTACGGTTACAAGACTATTG
TTATGGGTGCTTCTTTCAGAAGCACTGACGAAATCAAAAACTTGGCTGGT
GTTGACTATCTAACAATTTCTCCAGCTTTATTGGACAAGTTGATGAACAG
TACTGAACCTTTCCCAAGAGTTTTGGACCCTGTCTCCGCTAAGAAGGAAG
CCGGCGACAAGATTTCTTACATCAGCGACGAATCTAAATTCAGATTCGAC
TTGAATGAAGACGCTATGGCCACTGAAAAATTGTCCGAAGGTATCAGAAA
ATTCTCTGCCGATATTGTTACTCTATTCGACTTGATTGAAAAGAAAGTTA CCGCTTAA TAL1
amino acid sequence (SEQ ID NO: 498)
MSEPAQKKQKVANNSLEQLKASGTVVVADTGDFGSIAKFQPQDSTTNPSL
ILAAAKQPTYAKLIDVAVEYGKKHGKTTEEQVENAVDRLLVEFGKEILKI
VPGRVSTEVDARLSFDTQATIEKARHIIKLFEQEGVSKERVLIKIASTWE
GIQAAKELEEKDGIHCNLTLLFSFVQAVACAEAQVTLISPFVGRILDWYK
SSTGKDYKGEADPGVISVKKIYNYYKKYGYKTIVMGASFRSTDEIKNLAG
VDYLTISPALLDKLMNSTEPFPRVLDPVSAKKEAGDKISYISDESKFRFD
LNEDAMATEKLSEGIRKFSADIVTLFDLIEKKVTA
Example 38
Nucleotide and Amino Acid Sequence of S. cerevisiae
Transketolase
[0562] Transketolase (TKL1 and TKL2) activity was increased in some
embodiments, and in certain embodiments transaldolase activity was
decreased or disrupted. Transketolase converts xylulose-5-phosphate
and ribose-5-phosphate to sedoheptulose-7-phosphate and
glyceraldehyde-3-phosphate. The rationale for increasing or
decreasing transketolase activity is described herein with respect
to various embodiments. The nucleotide sequence of the S.
cerevisiae TKL1 gene altered to increase or decrease transketolase
activity, and the encoded amino acid sequence are shown below.
TABLE-US-00123 TKL1 nucleotide sequence (SEQ ID NO: 499)
ATGACTCAATTCACTGACATTGATAAGCTAGCCGTCTCCACCATAAGAAT
TTTGGCTGTGGACACCGTATCCAAGGCCAACTCAGGTCACCCAGGTGCTC
CATTGGGTATGGCACCAGCTGCACACGTTCTATGGAGTCAAATGCGCATG
AACCCAACCAACCCAGACTGGATCAACAGAGATAGATTTGTCTTGTCTAA
CGGTCACGCGGTCGCTTTGTTGTATTCTATGCTACATTTGACTGGTTACG
ATCTGTCTATTGAAGACTTGAAACAGTTCAGACAGTTGGGTTCCAGAACA
CCAGGTCATCCTGAATTTGAGTTGCCAGGTGTTGAAGTTACTACCGGTCC
ATTAGGTCAAGGTATCTCCAACGCTGTTGGTATGGCCATGGCTCAAGCTA
ACCTGGCTGCCACTTACAACAAGCCGGGCTTTACCTTGTCTGACAACTAC
ACCTATGTTTTCTTGGGTGACGGTTGTTTGCAAGAAGGTATTTCTTCAGA
AGCTTCCTCCTTGGCTGGTCATTTGAAATTGGGTAACTTGATTGCCATCT
ACGATGACAACAAGATCACTATCGATGGTGCTACCAGTATCTCATTCGAT
GAAGATGTTGCTAAGAGATACGAAGCCTACGGTTGGGAAGTTTTGTACGT
AGAAAATGGTAACGAAGATCTAGCCGGTATTGCCAAGGCTATTGCTCAAG
CTAAGTTATCCAAGGACAAACCAACTTTGATCAAAATGACCACAACCATT
GGTTACGGTTCCTTGCATGCCGGCTCTCACTCTGTGCACGGTGCCCCATT
GAAAGCAGATGATGTTAAACAACTAAAGAGCAAATTCGGTTTCAACCCAG
ACAAGTCCTTTGTTGTTCCACAAGAAGTTTACGACCACTACCAAAAGACA
ATTTTAAAGCCAGGTGTCGAAGCCAACAACAAGTGGAACAAGTTGTTCAG
CGAATACCAAAAGAAATTCCCAGAATTAGGTGCTGAATTGGCTAGAAGAT
TGAGCGGCCAACTACCCGCAAATTGGGAATCTAAGTTGCCAACTTACACC
GCCAAGGACTCTGCCGTGGCCACTAGAAAATTATCAGAAACTGTTCTTGA
GGATGTTTACAATCAATTGCCAGAGTTGATTGGTGGTTCTGCCGATTTAA
CACCTTCTAACTTGACCAGATGGAAGGAAGCCCTTGACTTCCAACCTCCT
TCTTCCGGTTCAGGTAACTACTCTGGTAGATACATTAGGTACGGTATTAG
AGAACACGCTATGGGTGCCATAATGAACGGTATTTCAGCTTTCGGTGCCA
ACTACAAACCATACGGTGGTACTTTCTTGAACTTCGTTTCTTATGCTGCT
GGTGCCGTTAGATTGTCCGCTTTGTCTGGCCACCCAGTTATTTGGGTTGC
TACACATGACTCTATCGGTGTCGGTGAAGATGGTCCAACACATCAACCTA
TTGAAACTTTAGCACACTTCAGATCCCTACCAAACATTCAAGTTTGGAGA
CCAGCTGATGGTAACGAAGTTTCTGCCGCCTACAAGAACTCTTTAGAATC
CAAGCATACTCCAAGTATCATTGCTTTGTCCAGACAAAACTTGCCACAAT
TGGAAGGTAGCTCTATTGAAAGCGCTTCTAAGGGTGGTTACGTACTACAA
GATGTTGCTAACCCAGATATTATTTTAGTGGCTACTGGTTCCGAAGTGTC
TTTGAGTGTTGAAGCTGCTAAGACTTTGGCCGCAAAGAACATCAAGGCTC
GTGTTGTTTCTCTACCAGATTTCTTCACTTTTGACAAACAACCCCTAGAA
TACAGACTATCAGTCTTACCAGACAACGTTCCAATCATGTCTGTTGAAGT
TTTGGCTACCACATGTTGGGGCAAATACGCTCATCAATCCTTCGGTATTG
ACAGATTTGGTGCCTCCGGTAAGGCACCAGAAGTCTTCAAGTTCTTCGGT
TTCACCCCAGAAGGTGTTGCTGAAAGAGCTCAAAAGACCATTGCATTCTA
TAAGGGTGACAAGCTAATTTCTCCTTTGAAAAAAGCTTTCTAA TKL1 amino acid
sequence (SEQ ID NO: 500)
MTQFTDIDKLAVSTIRILAVDTVSKANSGHPGAPLGMAPAAHVLWSQMRM
NPTNPDWINRDRFVLSNGHAVALLYSMLHLTGYDLSIEDLKQFRQLGSRT
PGHPEFELPGVEVTTGPLGQGISNAVGMAMAQANLAATYNKPGFTLSDNY
TYVFLGDGCLQEGISSEASSLAGHLKLGNLIAIYDDNKITIDGATSISFD
EDVAKRYEAYGWEVLYVENGNEDLAGIAKAIAQAKLSKDKPTLIKMTTTI
GYGSLHAGSHSVHGAPLKADDVKQLKSKFGFNPDKSFVVPQEVYDHYQKT
ILKPGVEANNKWNKLFSEYQKKFPELGAELARRLSGQLPANWESKLPTYT
AKDSAVATRKLSETVLEDVYNQLPELIGGSADLTPSNLTRWKEALDFQPP
SSGSGNYSGRYIRYGIREHAMGAIMNGISAFGANYKPYGGTFLNFVSYAA
GAVRLSALSGHPVIWVATHDSIGVGEDGPTHQPIETLAHFRSLPNIQVWR
PADGNEVSAAYKNSLESKHTPSIIALSRQNLPQLEGSSIESASKGGYVLQ
DVANPDIILVATGSEVSLSVEAAKTLAAKNIKARVVSLPDFFTFDKQPLE
YRLSVLPDNVPIMSVEVLATTCWGKYAHQSFGIDRFGASGKAPEVFKFFG
FTPEGVAERAQKTIAFYKGDKLISPLKKAF
Example 39
Nucleotide and Amino Acid Sequences of Additional EDD Genes
Evaluated for Activity
TABLE-US-00124 [0563] Accession Strain Amino Acid Number Species
Number NUCLEOTIDE SEQUENCE Sequence YP_526855.1 Saccharophagus 2-40
ATGAATAGCGTAATCGAAGCTGTAACTCAGCGAATTATTGAGCGCAGT MNSVIEAVTQRIIERSR
degradans CGACATTCTCGTCAGGCGTATTTGAATTTAATGCGCAACACCATGGAG
HSRQAYLNLMRNTME CAGCATCCTCCTAAAAAGCGTCTATCTTGCGGCAATTTGGCTCATGCCT
QHPPKKRLSCGNLAHA ATGCAGCATGTGGTCAATCCGATAAGCAAACAATTCGTTTAATGCAAA
YAACGQSDKQTIRLMQ GTGCAAACATAAGTATTACTACGGCATTTAACGATATGCTTTCGGCGC
SANISITTAFNDMLSAH ATCAGCCTTTAGAAACATACCCTCAAATAATCAAAGAAACTGCGCGTG
QPLETYPQIIKETARAM CAATGGGTTCAACTGCTCAAGTTGCAGGCGGCGTGCCGGCAATGTGTG
GSTAQVAGGVPAMCD ATGGTGTAACTCAAGGCCAGCCCGGTATGGAGCTGAGTTTGTTTAGCC
GVTQGQPGMELSLFSR GCGAAGTTGTAGCAATGGCTACAGCAGTAGGCCTTTCGCACAATATGT
EVVAMATAVGLSHNM TTGATGGCAATATGTTTTTGGGTGTATGCGATAAAATTGTTCCTGGCAT
FDGNMFLGVCDKIVPG GCTAATTGGCGCGTTGCAGTTTGGTCATATTCCTGGGGTGTTTGTGCCT
MLIGALQFGHIPGVFVP GCCGGACCAATGCCTTCTGGTATTCCCAACAAAGAAAAAGCAAAAGTT
AGPMPSGIPNKEKAKV CGTCAGCAATATGCGGCGGGCATTGTGGGGGAAGATAAGCTTTTAGAA
RQQYAAGIVGEDKLLE ACCGAGTCGGCTTCCTATCACAGTGCAGGCACGTGTACTTTTTACGGTA
TESASYHSAGTCTFYGT CAGCGAATACAAACCAAATGATGGTTGAAATGTTGGGTGTTCAGTTGC
ANTNQMMVEMLGVQL CTGGCTCGTCGTTTGTTTACCCCGGTACTGAGTTGCGTGATGCCTTAAC
PGSSFVYPGTELRDALT GAGAGCTGCTGTTGAAAAGTTGGTAAAAATCACAGATTCAGCCGGTAA
RAAVEKLVKITDSAGN CTACCGTCCGCTCTACGAAGTCATTACGGAAAAATCCATCGTCAATTC
YRPLYEVITEKSIVNSII
AATAATTGGTTTGTTGGCTACCGGCGGTTCTACTAACCACACGCTACAC GLLATGGSTNHTLHIVA
ATTGTTGCTGTGGCTCGCGCTGCGGGTATAGAGGTTACGTGGGCAGAT VARAAGIEVTWADMD
ATGGACGAGCTTTCGCGTGCTGTGCCATTACTTGCACGTGTTTACCCTA ELSRAVPLLARVYPNGE
ACGGCGAAGCTGATGTTAACCAATTCCAGCAGGCTGGCGGCATGGCTT ADVNQFQQAGGMAYL
ATTTAGTAAGAGAGCTGCGCAGCGGCGGTTTGCTAAATGAAGATGTGG VRELRSGGLLNEDVVTI
TTACTATTATGGGTGAGGGCCTCGAGGCCTACGAAAAAGAGCCCATGC MGEGLEAYEKEPMLND
TTAACGATAAGGGGCAGGCTGAATGGGTAAATGATGTACCTGTTAGCC KGQAEWVNDVPVSRD
GCGACGATACCGTTGTGCGTCCAGTTACCTCGCCTTTCGATAAAGAGG DTVVRPVTSPFDKEGGL
GTGGGTTGCGTCTACTCAAGGGTAACTTAGGGCAGGGCGTAATCAAAA RLLKGNLGQGVIKISAV
TTTCTGCGGTAGCGCCAGAAAATCGCGTTGTTGAGGCCCCATGTATTGT APENRVVEAPCIVFEAQ
ATTCGAGGCCCAAGAAGAGCTAATAGCTGCGTTTAAGCGTGGTGAGCT EELIAAFKRGELEKDFV
CGAAAAAGACTTTGTTGCGGTAGTGCGCTTCCAAGGGCCTTCTGCCAA AVVRFQGPSANGMPEL
TGGCATGCCAGAACTTCATAAAATGACCCCGCCTTTAGGTGTGCTTCA HKMTPPLGVLQDKGFK
AGATAAGGGTTTCAAGGTAGCGTTAGTTACCGATGGCAGAATGTCTGG VALVTDGRMSGASGKV
TGCATCTGGTAAAGTGCCGGCCGGTATACACTTGTCGCCAGAAGCGAG PAGIHLSPEASKGGLLN
TAAGGGTGGCCTGTTGAATAAGCTGCGCACGGGTGATGTGATTCGCTT KLRTGDVIRFDAEAGVI
CGATGCCGAAGCGGGCGTTATTCAAGCGCTTGTTAGTGATGAAGAGTT QALVSDEELAAREPAV
AGCTGCGCGTGAGCCAGCTGTGCAACCGGTCGTGGAGCAGAACCTCGG QPVVEQNLGRSLFGGL
ACGCTCTCTGTTTGGTGGTTTGCGCGATTTGGCTGGTGTATCGCTACAA RDLAGVSLQGGTVFDF
GGCGGAACAGTTTTCGATTTTGAAAGAGAGTTTGGCGAAAAATAG EREFGEK (SEQ ID (SEQ
ID NO: 501) NO: 502) NP_642389.1 Xanthomonas Pv.
ATGAGCCTGCATCCGAATATCCAAGCCGTCACCGACCGTATCCGCAAG MSLHPNIQAVTDRIRKR
axonopodis citri CGCAGTGCTCCCTCGCGCGCGGCGTATCTGGCCGGCCTCGATGCCGCC
SAPSRAAYLAGIDAALR str. 306
CTGCGTGAGGGCCCGTTCCGTAGCCGGTTGAGCTGCGGCAATCTCGCG EGPFRSRLSCGNLAHGF
CATGGCTTCGCTGCGTCCGAGCCGGGCGACAAATCGCGCCTGCGCGGT AASEPTDKSRLRGAATP
GCGGCCACGCCGAACCTGGGCATCATCACTGCCTATAACGACATGTTG NLGIITAYNDMLSAHQP
TCGGCACATCAGCCGTTCGAGCACTACCCGCAGCTGATCCGCGAAACC FEHYPQLIRETARSLGA
GCGCGCTCACTTGGCGCCACTGCGCAGGTGGCCGGCGGCGTGCCGGCG TAQVAGGVPAMCDGV
ATGTGTGACGGCGTGACCCAGGGCCGCGCCGGCATGGAGCTGTCGCTG TQGRAGMELSLFSRDNI
TTCTCGCGCGACAACATCGCTCAGGCTGCGGCCATTGGCCTGAGCCAT AQAAAIGLSHDMFDSV
GACATGTTCGACAGCGTGGTGTACCTGGGGGTGTGCGACAAGATCGTG VYLGVCDKIVPGLLIGA
CCGGGTCTGCTGATCGGTGCGCTGGCGTTTGGCCATTTGCCGGCGATCT LAFGHLPAIFMPAGPMT
TCATGCCGGCTGGTCCGATGACCCCGGGCATCCCGAACAAGCAGAAAG PGIPNKQKAEVRERYA
CCGAAGTCCGCGAACGCTACGCCGCTGGCGAAGCCACCCGCGCCGAAT AGEATRAELLEAESSSY
TGCTGGAGGCCGAATCCTCGTCTTATCACTCGCCCGGCACCTGCACCTT HSPGTCTFYGTANSNQ
TTACGGCACGGCGAACTCCAACCAGGTGTTGCTCGAAGCGATGGGCGT VLLEAMGVQLPGASFV
GCAGTTGCCCGGCGCCTCGTTCGTCAATCCGGAGCTGCCGCTGCGCGA NPELPLRDALTREGTAR
TGCACTGACCCGCGAAGGCACCGCACGCGCATTGGCGATCTCCGCGCT ALAISALGDDFRPFGRLI
GGGCGATGACTTCCGCCCGTTCGGTCGTTTGATCGACGAACGGGCCAT DERAIVNAVVALMATG
CGTCAATGCCGTGGTCGCGCTGATGGCGACCGGCGGTTCGACCAACCA GSTNHTIHWIAVARAA
CACCATCCACTGGATCGCAGTGGCGCGTGCGGCCGGCATCGTGTTGAC GIVLTWDDMDLISQTVP
CTGGGACGACATGGATCTGATCTCGCAGACCGTGCCGCTGTTGACACG LLTRIYPNGEADVNRFQ
CATCTACCCGAACGGCGAAGCCGACGTGAACCGCTTCCAGGCCGCAGG AAGGTAFVFRELMDAG
CGGCACGGCGTTCGTGTTCCGCGAATTGATGGACGCCGGCTACATGCA YMHDDLPTIVEGGMRA
CGACGACCTGCCGACCATCGTCGAAGGCGGCATGCGCGCGTACGTCAA YVNEPRLQDGKVTYVP
CGAACCGCGCCTGCAGGACGGCAAGGTGACCTACGTGCCCGGCACCG GTATTADDSVARPVSD
CGACCACTGCCGACGACAGCGTCGCGCGTCCGGTCAGCGATGCATTCG AFESQGGLRLLRGNLG
AATCACAAGGCGGCCTGCGCCTGCTGCGCGGCAACCTCGGCCGCTCGT RSLIKLSAVKPQHRSIQ
TGATCAAGCTGTCGGCGGTCAAGCCGCAGCACCGCAGCATCCAAGCGC APAVVIDTPQVLNKLH
CAGCGGTGGTGATCGACACCCCGCAAGTGCTCAACAAACTGCATGCGG AAGVLPHDFVVVLRYQ
CGGGCGTACTGCCGCACGATTTCGTGGTGGTACTGCGCTATCAGGGCC GPRANGMPELHSMAPL
CACGCGCAAACGGCATGCCGGAGCTGCATTCGATGGCGCCGCTACTGG LGLLQNQGRRVALVTD
GCCTGCTGCAGAACCAGGGCCGGCGCGTGGCGTTGGTCACCGACGGCC GRLSGASGKFPAAIHMT
GTCTGTCCGGCGCCTCGGGCAAGTTCCCGGCGGCGATCCACATGACCC PEAARGGPIGRVREGDI
CGGAAGCCGCACGCGGCGGCCCGATCGGGCGCGTACGCGAAGGCGAC VRLDGEAGTLEVLVSA
ATCGTGCGACTGGACGGCGAAGCCGGCACCTTGGAAGTGCTGGTTTCG EEWASREVAPNTALAG
GCCGAAGAATGGGCATCGCGCGAGGTCGCACCGAACACTGCGTTGGC NDLGRNLFAINRQVVG
CGGCAACGACCTGGGCCGCAACCTGTTCGCCATCAACCGCCAGGTGGT PADQGAISISCGPTHPD
TGGCCCGGCCGACCAGGGCGCGATTTCCATTTCCTGCGGCCCGACCCA GALWSYDAEYELGAD
TCCGGACGGTGCGCTGTGGAGCTACGACGCCGAGTACGAACTCGGTGC AAAAAAPHESKDA
CGATGCAGCTGCAGCCGCCGCGCCGCACGAGTCCAAGGACGCCTGA (SEQ ID NO: 504)
(SEQ ID NO: 503) NP_791117.1 Pseudomonas Pv.
ATGCATCCCCGCGTCCTTGAAGTAACCGAGCGGCTCATTGCTCGCAGT MHPRVLEVTERLIARSR
syringae tomato CGCGATACCCGTCAGCGCTACCTTCAATTGATTCGAGGCGCAGCGAGC
DTRQRYLQLIRGAASD str.
GATGGCCCGATGCGCGGCAAGCTTCAATGTGCCAACTTTGCTCACGGC GPMRGKLQCANFAHG
DC3000 GTCGCCGCCTGCGGACCGGAGGACAAGCAAAGCCTGCGTTTGATGAAC
VAACGPEDKQSLRLMN GCCGCCAACGTGGCAATCGTCTCTTCCTACAATGAAATGCTCTCGGCG
AANVAIVSSYNEMLSA CATCAGCCCTACGAGCACTTTCCTGCACAGATCAAACAGGCGTTACGT
HQPYEHFPAQIKQALRD GACATTGGTTCGGTCGGTCAGTTTGCCGGCGGCGTGCCTGCCATGTGC
IGSVGQFAGGVPAMCD GATGGCGTGACTCAGGGTGAGCCGGGCATGGAACTGGCCATTGCCAGC
GVTQGEPGMELAIASRE CGCGAAGTGATTGCCATGTCCACGGCAATTGCCTTGTCACACAATATG
VIAMSTAIALSHNMFDA TTCGACGCCGCCATGATGCTGGGTATCTGCGACAAGATCGTCCCCGGC
AMMLGICDKIVPGLMM CTGATGATGGGGGCGTTGCGTTTCGGTCATCTGCCGACCATCTTCGTGC
GALRFGHLPTIFVPGGP CGGGCGGGCCGATGGTGTCAGGTATCTCCAACAAGGAAAAAGCCGAC
MVSGISNKEKADVRQR GTACGGCAGCGTTACGCTGAAGGCAAGGCCAGCCGTGAAGAGCTGCT
YAEGKASREELLDSEM GGACTCGGAAATGAAGTCCTATCACGGCCCGGGAACCTGCACGTTCTA
KSYHGPGTCTFYGTAN CGGCACCGCCAACACCAATCAGTTGGTGATGGAAGTCATGGGCATGCA
TNQLVMEVMGMHLPG CCTTCCCGGTGCCTCGTTCGTCAATCCCTACACACCACTGCGTGATGCG
ASFVNPYTPLRDALTAE CTGACAGCTGAAGCGGCTCGTCAGGTCACGCGTCTGACCATGCAAAGC
AARQVTRLTMQSGSFM GGCAGTTTCATGCCGATTGGTGAAATCGTCGACGAGCGCTCGCTGGTC
PIGEIVDERSLVNSIVAL AATTCCATCGTTGCGCTGCACGCCACCGGCGGCTCGACCAACCACACG
HATGGSTNHTLHMPAI CTGCACATGCCGGCGATTGCTCAGGCTGCGGGTATTCAGCTGACCTGG
AQAAGIQLTWQDMAD CAGGACATGGCCGACCTCTCCGAAGTGGTGCCGACCCTCAGTCACGTC
LSEVVPTLSHVYPNGK TACCCCAACGGCAAGGCCGACATCAACCATTTCCAGGCCGCAGGCGGC
ADINHFQAAGGMSFLIR ATGTCGTTCCTGATTCGCGAGCTGCTGGCAGCCGGTCTGCTGCACGAA
ELLAAGLLHENVNTVA AACGTTAACACCGTGGCCGGTTATGGCCTGAGCCGCTACACCAAAGAG
GYGLSRYTKEPFLEDG CCATTCCTGGAGGATGGCAAACTGGTCTGGCGTGAAGGCCCGCTGGAC
KLVWREGPLDSLDENIL AGCCTGGATGAAAACATCCTGCGCCCGGTGGCGCGTCCGTTCTCCCCT
RPVARPFSPEGGLRVME GAAGGCGGTTTGCGGGTCATGGAAGGCAACCTGGGTCGCGGTGTCATG
GNLGRGVMKVSAVAL AAAGTATCGGCCGTTGCGCTGGAGCATCAGATTGTCGAAGCGCCAGCC
EHQIVEAPARVFQDQK CGAGTGTTTCAGGATCAGAAGGAGCTGGCCGATGCGTTCAAGGCCGGC
ELADAFKAGELECDFV GAGCTGGAATGTGATTTCGTCGCCGTCATGCGTTTTCAGGGCCCGCGCT
AVMRFQGPRCNGMPEL GCAACGGCATGCCCGAACTGCACAAGATGACCCCGTTTCTGGGCGTGC
HKMTPFLGVLQDRGFK TGCAGGATCGTGGTTTCAAAGTGGCGCTGGTCACCGATGGACGGATGT
VALVTDGRMSGASGKI CGGGCGCCTCAGGCAAGATTCCGGCGGCGATTCACGTCTGCCCGGAAG
PAAIHVCPEAFDGGPLA CGTTCGATGGTGGCCCGTTGGCACTGGTACGCGACGGCGATGTGATCC
LVRDGDVIRVDGVKGT GCGTGGATGGCGTAAAAGGCACGTTACAAGTGCTGGTCGAAGCGTCA
LQVLVEASELAAREPAI GAATTGGCCGCCCGAGAACCGGCCATCAACCAGATCGACAACAGTGTC
NQIDNSVGCGRELFGF GGCTGCGGTCGCGAGCTTTTTGGATTCATGCGCATGGCCTTCAGCTCCG
MRMAFSSAEQGASAFT CAGAGCAAGGCGCCAGCGCCTTTACCTCTAGTCTGGAGACGCTCAAGT
SSLETLK (SEQ ID GA (SEQ ID NO: 505) NO: 506) YP_261706.1
Pseudomonas Pf-5 ATGCATCCCCGCGTTCTTGAGGTCACCGAACGGCTTATCGCCCGTAGTC
MHPRVLEVTERLIARSR fluorescens
GCGCCACTCGCCAGGCCTATCTCGCGCTGATCCGCGATGCCGCCAGCG ATRQAYLALIRDAASD
ACGGCCCGCAGCGGGGCAAGCTGCAATGTGCGAACTTCGCCCACGGC GPQRGKLQCANFAHGV
GTGGCCGGTTGCGGCACCGACGACAAGCACAACCTGCGGATGATGAA AGCGTDDKHNLRMMN
TGCGGCCAACGTGGCAATTGTTTCGTCATATAACGACATGTTGTCGGC AANVAIVSSYNDMLSA
GCACCAGCCTTACGAGGTGTTCCCCGAGCAGATCAAGCGCGCCCTGCG HQPYEVFPEQIKRALRE
CGAGATCGGCTCGGTGGGCCAGTTCGCCGGCGGCACCCCGGCCATGTG IGSVGQFAGGTPAMCD
CGATGGCGTGACCCAGGGCGAGGCCGGTATGGAACTGAGCCTGCCGA GVTQGEAGMELSLPSR
GCCGTGAAGTGATCGCCCTGTCTACGGCGGTGGCCCTCTCTCACAACA EVIALSTAVALSHNMFD
TGTTCGATGCCGCGCTGATGCTGGGGATCTGCGACAAGATTGTCCCGG AALMLGICDKIVPGLM
GGTTGATGATGGGCGCTCTGCGCTTCGGTCACCTGCCGACCATCTTCGT MGALRFGHLPTIFVPGG
TCCGGGCGGGCCCATGGTCTCGGGCATTTCCAACAAGCAGAAAGCCGA PMVSGISNKQKADVRQ
CGTGCGCCAGCGTTACGCCGAAGGCAAGGCCAGCCGCGAGGAACTGC RYAEGKASREELLESE
TGGAGTCGGAAATGAAGTCCTACCACAGCCCCGGCACCTGCACTTTCT MKSYHSPGTCTFYGTA
ACGGCACCGCCAACACCAACCAGTTGCTGATGGAAGTGATGGGCCTGC NTNQLLMEVMGLHLPG
ACCTGCCGGGCGCCTCTTTCGTCAACCCCAATACGCCGCTGCGCGACG ASFVNPNTPLRDALTHE
CCCTGACCCATGAGGCGGCGCAGCAGGTCACGCGCCTGACCAAGCAG AAQQVTRLTKQSGAFM
AGCGGGGCCTTCATGCCGATTGGCGAGATCGTCGACGAGCGCGTGCTG PIGEIVDERVLVNSIVAL
GTCAACTCCATCGTTGCCCTGCACGCCACGGGCGGCTCCACCAACCAC HATGGSTNHTLHMPAI
ACCCTGCACATGCCGGCCATCGCCCAGGCGGCGGGCATCCAGCTGACC AQAAGIQLTWQDMAD
TGGCAGGACATGGCCGACCTCTCCGAGGTGGTGCCGACCCTGTCCCAC LSEVVPTLSHVYPNGK
GTCTATCCAAACGGCAAGGCCGATATCAACCACTTCCAGGCGGCGGGC ADINHFQAAGGMSFLIR
GGCATGTCTTTCCTGATCCGCGAGCTGCTGGAAGCCGGCCTGCTCCAC ELLEAGLLHEDVNTVA
GAAGACGTCAATACCGTGGCCGGCCGCGGCCTGAGCCGCTATACCCAG GRGLSRYTQEPFLDNG
GAACCCTTCCTGGACAACGGCAAGCTGGTGTGGCGCGACGGCCCGATT KLVWRDGPIESLDENIL
GAAAGCCTGGACGAAAACATCCTGCGCCCGGTGGCCCGGGCGTTCTCT RPVARAFSAEGGLRVM
GCGGAGGGCGGCTTGCGGGTCATGGAAGGCAACCTCGGTCGCGGCGT EGNLGRGVMKVSAVAP
GATGAAGGTTTCCGCCGTGGCCCCGGAGCACCAGATCGTCGAGGCCCC EHQIVEAPAVVFQDQQ
GGCCGTGGTGTTCCAGGACCAGCAGGACCTGGCCGATGCCTTCAAGGC DLADAFKAGLLEKDFV
CGGCCTGCTGGAGAAGGACTTCGTCGCGGTGATGCGCTTCCAGGGCCC AVMRFQGPRSNGMPEL
GCGCTCCAACGGCATGCCCGAGCTGCACAAGATGACCCCCTTCCTCGG HKMTPFLGVLQDRGFK
GGTGCTGCAGGACCGCGGCTTCAAGGTGGCGCTGGTCACCGACGGGCG VALVTDGRMSGASGKI
CATGTCCGGCGCTTCGGGCAAGATTCCGGCAGCGATCCATGTCAGCCC PAAIHVSPEAQVGGAL
CGAAGCCCAGGTGGGTGGCGCGCTGGCCCGGGTGCTGGACGGCGATA ARVLDGDIIRVDGVKG
TCATCCGAGTGGATGGCGTCAAGGGCACCCTGGAGCTTAAGGTAGACG TLELKVDAAEFAAREP
CCGCAGAATTCGCCGCCCGGGAGCCGGCCAAGGGCCTGCTGGGCAAC AKGLLGNNVGTGRELF
AACGTTGGCACCGGCCGCGAACTCTTCGCCTTCATGCGCATGGCCTTC AFMRMAFSSAEQGASA
AGCTCGGCAGAGCAGGGCGCCAGCGCCTTTACCTCTGCCCTGGAGACG FTSALETLK (SEQ ID
CTCAAGTGA (SEQ ID NO: 507) NO: 508) ZP_0359148.1 Bacillus subtilis
ATGGCAGAATTACGCAGTAATATGATCACACAAGGAATCGATAGAGCT MAELRSNMITQGIDRAP
subtilis str. 168 CCGCACCGCAGTTTGCTTCGTGCAGCAGGGGTAAAAGAAGAGGATTTC
HRSLLRAAGVKEEDFG GGCAAGCCGTTTATTGCGGTGTGTAATTCATACATTGATATCGTTCCCG
KPFIAVCNSYIDIVPGHV GTCATGTTCACTTGCAGGAGTTTGGGAAAATCGTAAAAGAAGCAATCA
HLQEFGKIVKEAIREAG GAGAAGCAGGGGGCGTTCCGTTTGAATTTAATACCATTGGGGTAGATG
GVPFEFNTIGVDDGIAM ATGGCATCGCAATGGGGCATATCGGTATGAGATATTCGCTGCCAAGCC
GHIGMRYSLPSREIIADS GTGAAATTATCGCAGACTCTGTGGAAACGGTTGTATCCGCACACTGGT
VETVVSAHWFDGMVCI TTGACGGAATGGTCTGTATTCCGAACTGCGACAAAATCACACCGGGAA
PNCDKITPGMLMAAMR TGCTTATGGCGGCAATGCGCATCAACATTCCGACGATTTTTGTCAGCG
INIPTIFVSGGPMAAGRT GCGGACCGATGGCGGCAGGAAGAACAAGTTACGGGCGAAAAATCTCC
SYGRKISLSSVFEGVGA CTTTCCTCAGTATTCGAAGGGGTAGGCGCCTACCAAGCAGGGAAAATC
YQAGKINENELQELEQF AACGAAAACGAGCTTCAAGAACTAGAGCAGTTCGGATGCCCAACGTG
GCPTCGSCSGMFTANS CGGGTCTTGCTCAGGCATGTTTACGGCGAACTCAATGAACTGTCTGTC
MNCLSEALGLALPGNG AGAAGCACTTGGTCTTGCTTTGCCGGGTAATGGAACCATTCTGGCAAC
TILATSPERKEFVRKSA ATCTCCGGAACGCAAAGAGTTTGTGAGAAAATCGGCTGCGCAATTAAT
AQLMETIRKDIKPRDIV GGAAACGATTCGCAAAGATATCAAACCGCGTGATATTGTTACAGTAAA
TVKAIDNAFALDMALG AGCGATTGATAACGCGTTTGCACTCGATATGGCGCTCGGAGGTTCTAC
GSTNTVLHTLALANEA AAATACCGTTCTTCATACCCTTGCCCTTGCAAACGAAGCCGGCGTTGA
GVEYSLERINEVAERVP ATACTCTTTAGAACGCATTAACGAAGTCGCTGAGCGCGTGCCGCACTT
HLAKLAPASDVFIEDLH GGCTAAGCTGGCGCCTGCATCGGATGTGTTTATTGAAGATCTTCACGA
EAGGVSAALNELSKKE AGCGGGCGGCGTTTCAGCGGCTCTGAATGAGCTTTCGAAGAAAGAAG
GALHLDALTVTGKTLG GAGCGCTTCATTTAGATGCGCTGACTGTTACAGGAAAAACTCTTGGAG
ETIAGHEVKDYDVIHPL AAACCATTGCCGGACATGAAGTAAAGGATTATGACGTCATTCACCCGC
DQPFTEKGGLAVLFGN TGGATCAACCATTCACTGAAAAGGGAGGCCTTGCTGTTTTATTCGGTA
LAPDGAIIKTGGVQNGI ATCTAGCTCCGGACGGCGCTATCATTAAAACAGGCGGCGTACAGAATG
TRHEGPAVVFDSQDEA GGATTACAAGACACGAAGGGCCGGCTGTCGTATTCGATTCTCAGGACG
LDGIINRKVKEGDVVIIR AGGCGCTTGACGGCATTATCAACCGAAAAGTAAAAGAAGGCGACGTT
YEGPKGGPGMPEMLAP GTCATCATCAGATACGAAGGGCCAAAAGGCGGACCTGGCATGCCGGA
TSQIVGMGLGPKVALIT AATGCTGGCGCCAACATCCCAAATCGTTGGAATGGGACTCGGGCCAAA
DGRFSGASRGLSIGHVS AGTGGCATTGATTACGGACGGACGTTTTTCCGGAGCCTCCCGTGGCCT
PEAAEGGPLAFVENGD CTCAATCGGCCACGTATCACCTGAGGCCGCTGAGGGCGGGCCGCTTGC
HIIVDIEKRILDVQVPEE CTTTGTTGAAAACGGAGACCATATTATCGTTGATATTGAAAAACGCAT
EWEKRKANWKGFEPK CTTGGATGTACAAGTGCCAGAAGAAGAGTGGGAAAAACGAAAAGCGA
VKTGYLARYSKLVTSA ACTGGAAAGGTTTTGAACCGAAAGTGAAAACCGGCTACCTGGCACGTT
NTGGIMKI (SEQ ATTCTAAACTTGTGACAAGTGCCAACACCGGCGGTATTATGAAAATCT ID
NO: 510) AG (SEQ ID NO: 509) YP_091897.1 Bacillus ATCC
ATGACAGGTTTACGCAGTGACATGATTACAAAAGGGATCGACAGAGC MTGLRSDMITKGIDRAP
licheniformis 14580
GCCGCACCGCAGTTTGCTGCGCGCGGCTGGGGTAAAAGAAGAGGACTT HRSLLRAAGVKEEDFG
CGGCAAACCGTTTATTGCCGTTTGCAACTCATACATCGATATCGTACCG
KPFIAVCNSYIDIVPGHV GGTCATGTCCATTTGCAGGAGTTTGGAAAAATCGTCAAAGAGGCGATC
HLQEFGKIVKEAIREAG AGAGAGGCCGGCGGTGTTCCGTTTGAATTTAATACAATCGGGGTCGAC
GVPFEFNTIGVDDGIAM GACGGAATTGCGATGGGGCACATCGGAATGAGGTATTCTCTCCCGAGC
GHIGMRYSLPSREIIADS CGCGAAATCATCGCAGATTCAGTGGAAACGGTTGTATCGGCGCACTGG
VETVVSAHWFDGMVCI TTTGACGGAATGGTATGTATTCCAAACTGTGATAAAATCACACCGGGC
PNCDKITPGMIMAAMRI ATGATCATGGCGGCAATGCGGATCAACATTCCGACCGTGTTTGTCAGC
NIPTVFVSGGPMEAGRT GGGGGGCCGATGGAAGCGGGAAGAACGAGCGACGGACGAAAAATCTC
SDGRKISLSSVFEGVGA GCTTTCCTCTGTATTTGAAGGCGTTGGCGCTTATCAATCAGGCAAAATC
YQSGKIDEKGLEELEQF GATGAGAAAGGACTCGAGGAGCTTGAACAGTTCGGCTGTCCGACTTGC
GCPTCGSCSGMFTANS GGATCATGCTCGGGCATGTTTACGGCGAACTCGATGAACTGTCTTTCTG
MNCLSEALGIAMPGNG AAGCTCTTGGCATCGCCATGCCGGGCAACGGCACCATTTTGGCGACAT
TILATSPDRREFAKQSA CGCCCGACCGCAGGGAATTTGCCAAACAGTCGGCCCGCCAGCTGATGG
RQLMELIKSDIKPRDIVT AGCTGATCAAGTCGGATATCAAACCGCGCGACATCGTGACCGAAAAA
EKAIDNAFALDMALGG GCGATCGACAACGCGTTCGCTTTAGACATGGCGCTCGGCGGATCAACG
STNTILHTLAIANEAGV AATACGATCCTTCATACGCTTGCGATCGCCAATGAAGCGGGTGTAGAC
DYSLERINEVAARVPHL TATTCGCTTGAACGGATCAATGAGGTAGCGGCAAGGGTTCCGCATTTA
SKLAPASDVFIEDLHEA TCGAAGCTTGCACCGGCTTCCGATGTGTTTATTGAAGATTTGCATGAAG
GGVSAVLNELSKKEGA CAGGAGGCGTATCGGCAGTCTTAAACGAGCTGTCGAAAAAAGAAGGC
LHLDTLTVTGKTLGENI GCGCTTCACTTGGATACGCTGACTGTAACGGGGAAAACGCTTGGCGAA
AGREVKDYEVIHPIDQP AATATTGCCGGACGCGAAGTGAAAGATTACGAGGTCATTCATCCGATC
FSEQGGLAVLFGNLAP GATCAGCCGTTTTCAGAGCAAGGCGGACTCGCCGTCCTGTTCGGCAAC
DGAIIKTGGVQDGITRH CTGGCTCCTGACGGTGCGATCATTAAAACGGGCGGCGTCCAAGACGGG
EGPAVVFDSQEEALDGI ATTACCCGCCATGAAGGACCTGCGGTTGTCTTTGATTCACAGGAAGAA
INRKVKAGDVVIIRYEG GCGCTTGACGGCATCATCAACCGTAAAGTAAAAGCGGGAGATGTCGTC
PKGGPGMPEMLAPTSQI ATCATCCGCTATGAAGGCCCTAAAGGCGGACCGGGAATGCCTGAAATG
VGMGLGPKVALITDGR CTTGCGCCGACTTCACAGATCGTCGGAATGGGCCTCGGCCCGAAAGTC
FSGASRGLSIGHVSPEA GCCTTGATTACCGACGGCCGCTTTTCAGGAGCCTCCCGCGGTCTTTCGA
AEGGPLAFVENGDHIV TCGGCCACGTTTCACCGGAAGCAGCCGAAGGCGGCCCGCTTGCTTTCG
VDIEKRILNIEISDEEWE TAGAAAACGGCGACCATATCGTTGTCGATATCGAAAAGCGGATTTTAA
KRKANWPGFEPKVKTG ACATCGAAATCTCCGATGAGGAATGGGAAAAAAGAAAAGCAAACTGG
YLARYSKLVTSANTGGI
CCCGGCTTTGAACCGAAAGTGAAAACGGGCTATCTCGCCAGGTATTCA MKI (SEQ ID
AAGCTTGTGACATCTGCCAATACCGGCGGCATTATGAAAATCTAG NO: 512) (SEQ ID NO:
511) NP_0718074.1 Sewanella MR-1
ATGCACTCAGTCGTTCAATCTGTTACTGACAGAATTATTGCCCGTAGCA MHSVVQSVTDRIIARSK
oneidensis AAGCATCTCGTGAAGCATACCTTGCTGCGTTAAACGATGCCCGTAACC
ASREAYLAALNDARNH ATGGTGTACACCGAAGTTCCTTAAGTTGCGGTAACTTAGCCCACGGTTT
GVHRSSLSCGNLAHGF TGCGGCTTGTAATCCCGATGACAAAAATGCATTGCGTCAATTGACGAA
AACNPDDKNALRQLTK GGCCAATATTGGGATTATCACCGCATTCAACGATATGTTATCTGCACA
ANIGIITAFNDMLSAHQ CCAACCCTATGAAACCTATCCTGATTTGCTGAAAAAAGCCTGTCAGGA
PYETYPDLLKKACQEV AGTCGGTAGTGTTGCGCAGGTGGCTGGCGGTGTTCCCGCCATGTGTGA
GSVAQVAGGVPAMCD CGGCGTGACTCAAGGTCAGCCCGGTATGGAATTGAGCTTACTGAGCCG
GVTQGQPGMELSLLSR TGAAGTGATTGCGATGGCAACCGCGGTTGGCTTATCACACAATATGTT
EVIAMATAVGLSHNMF TGATGGAGCCTTACTCCTCGGTATTTGCGATAAAATTGTACCGGGTTTA
DGALLLGICDKIVPGLLI
CTGATTGGTGCCTTAAGTTTTGGCCATTTACCTATGTTGTTTGTGCCCG GALSFGHLPMLFVPAGP
CAGGCCCAATGAAATCGGGTATTCCTAATAAGGAAAAAGCTCGCATTC MKSGIPNKEKARIRQQF
GTCAGCAATTTGCTCAAGGTAAGGTCGATAGAGCACAACTGCTCGAAG AQGKVDRAQLLEAEAQ
CGGAAGCCCAGTCTTACCACAGTGCGGGTACTTGTACCTTCTATGGTA SYHSAGTCTFYGTANS
CCGCTAACTCGAACCAACTGATGCTCGAAGTGATGGGGCTGCAATTGC NQLMLEVMGLQLPGSS
CGGGTTCATCTTTTGTGAATCCAGACGATCCACTGCGCGAAGCCTTAA FVNPDDPLREALNKMA
ACAAAATGGCGGCCAAGCAGGTTTGTCGTTTAACTGAACTAGGCACTC AKQVCRLTELGTQYSPI
AATACAGTCCGATTGGTGAAGTCGTTAACGAAAAATCGATAGTGAATG GEVVNEKSIVNGIVALL
GTATTGTTGCATTGCTCGCGACGGGTGGTTCAACAAACTTAACCATGC ATGGSTNLTMHIVAAA
ACATTGTGGCGGCGGCCCGTGCTGCAGGTATTATCGTCAACTGGGATG RAAGIIVNWDDFSELSD
ACTTTTCGGAATTATCCGATGCGGTGCCTTTGCTGGCACGTGTTTATCC AVPLLARVYPNGHADI
AAACGGTCATGCGGATATTAACCATTTCCACGCTGCGGGTGGTATGGC NHFHAAGGMAFLIKEL
TTTCCTTATCAAAGAATTACTCGATGCAGGTTTGCTGCATGAGGATGTC LDAGLLHEDVNTVAGY
AATACTGTCGCGGGTTATGGTCTGCGCCGTTACACCCAAGAGCCTAAA GLRRYTQEPKLLDGEL
CTGCTTGATGGCGAGCTGCGCTGGGTCGATGGCCCAACAGTGAGTTTA RWVDGPTVSLDTEVLT
GATACCGAAGTATTAACCTCTGTGGCAACACCATTCCAAAACAACGGT SVATPFQNNGGLKLLK
GGTTTAAAGCTGCTGAAGGGTAACTTAGGCCGCGCTGTGATTAAAGTG GNLGRAVIKVSAVQPQ
TCTGCCGTTCAGCCACAGCACCGTGTGGTGGAAGCGCCCGCAGTGGTG HRVVEAPAVVIDDQNK
ATTGACGATCAAAACAAACTCGATGCGTTATTTAAATCCGGCGCATTA LDALFKSGALDRDCVV
GACAGGGATTGTGTGGTGGTGGTGAAAGGCCAAGGGCCGAAAGCCAA VVKGQGPKANGMPEL
CGGTATGCCAGAGCTGCATAAACTAACGCCGCTGTTAGGTTCATTGCA HKLTPLLGSLQDKGFK
GGACAAAGGCTTTAAAGTGGCACTGATGACTGATGGTCGTATGTCGGG VALMTDGRMSGASGK
CGCATCGGGCAAAGTACCTGCGGCGATTCATTTAACCCCTGAAGCGAT VPAAIHLTPEAIDGGLIA
TGATGGCGGGTTAATTGCAAAGGTACAAGACGGCGATTTAATCCGAGT KVQDGDLIRVDALTGE
TGATGCACTGACCGGCGAGCTGAGTTTATTAGTCTCTGACACCGAGCT LSLLVSDTELATRTATEI
TGCCACCAGAACTGCCACTGAAATTGATTTACGCCATTCTCGTTATGGC DLRHSRYGMGRELFGV
ATGGGGCGTGAGTTATTTGGAGTACTGCGTTCAAACTTAAGCAGTCCT LRSNLSSPETGARSTSAI
GAAACCGGTGCGCGTAGTACTAGCGCCATCGATGAACTTTACTAA DELY (SEQ ID (SEQ ID
NO: 83) NO: 84) YP_190870.1 Gluconobacter 621H
ATGTCTCTGAATCCCGTCGTCGAGAGCGTGACTGCCCGTATCATCGAG MSLNPVVESVTARIIER
oxydans CGTTCGAAAGTCTCCCGTCGCCGGTATCTCGCCCTGATGGAGCGCAAC
SKVSRRRYLALMERNR CGCGCCAAGGGTGTGCTCCGGCCCAAGCTGGCCTGCGGTAATCTGGCG
AKGVLRPKLACGNLAH CATGCCATCGCAGCGTCCAGCCCCGACAAGCCGGATCTGATGCGTCCC
AIAASSPDKPDLMRPTG ACCGGGACCAATATCGGCGTGATCACGACCTATAACGACATGCTCTCG
TNIGVITTYNDMLSAHQ GCGCATCAGCCGTATGGCCGCTATCCCGAGCAGATCAAGCTGTTCGCC
PYGRYPEQIKLFAREVG CGTGAAGTCGGTGCGACGGCCCAGGTTGCAGGCGGCGCACCAGCAAT
ATAQVAGGAPAMCDG GTGTGATGGTGTGACGCAGGGGCAGGAGGGCATGGAACTCTCCCTGTT
VTQGQEGMELSLFSRD CTCCCGTGACGTGATCGCCATGTCCACGGCGGTCGGGCTGAGCCACGG
VIAMSTAVGLSHGMFE CATGTTTGAGGGCGTGGCGCTGCTGGGCATCTGTGACAAGATTGTGCC
GVALLGICDKIVPGLLM GGGCCTTCTGATGGGCGCGCTGCGCTTCGGTCATCTCCCGGCCATGCTG
GALRFGHLPAMLIPAGP ATCCCGGCAGGGCCAATGCCGTCCGGTCTTCCAAACAAGGAAAAGCA
MPSGLPNKEKQRIRQLY GCGCATCCGCCAGCTCTATGTGCAGGGCAAGGTCGGGCAGGACGAGCT
VQGKVGQDELMEAEN GATGGAAGCGGAAAACGCCTCCTATCACAGCCCGGGCACCTGCACGTT
ASYHSPGTCTFYGTANT CTATGGCACGGCCAATACGAACCAGATGATGGTCGAAATCATGGGTCT
NQMMVEIMGLMMPDS GATGATGCCGGACTCGGCTTTCATCAATCCCAACACGAAGCTGCGTCA
AFINPNTKLRQAMTRSG GGCAATGACCCGCTCGGGTATTCACCGTCTGGCCGAAATCGGCCTGAA
IHRLAEIGLNGEDVRPL CGGCGAGGATGTGCGCCCGCTCGCTCATTGCGTAGACGAAAAGGCCAT
AHCVDEKAIVNAAVGL CGTGAATGCGGCGGTCGGGTTGCTGGCGACGGGTGGTTCGACCAACCA
LATGGSTNHSIHLPAIA TTCGATCCATCTTCCTGCTATCGCCCGTGCCGCTGGTATCCTGATCGAC
RAAGILIDWEDISRLSSA TGGGAAGACATCAGCCGCCTGTCGTCCGCGGTTCCGCTGATCACCCGT
VPLITRVYPSGSEDVNA GTTTATCCGAGCGGTTCCGAGGACGTGAACGCGTTCAACCGCGTGGGT
FNRVGGMPTVIAELTR GGTATGCCGACCGTGATCGCCGAACTGACGCGCGCCGGGATGCTGCAC
AGMLHKDILTVSRGGF AAGGACATTCTGACGGTCTCTCGTGGCGGTTTCTCCGATTATGCCCGTC
SDYARRASLEGDEIVYT GCGCATCGCTGGAAGGCGATGAGATCGTCTACACCCACGCGAAGCCGT
HAKPSTDTDILRDVATP CCACGGACACCGATATCCTGCGCGATGTGGCTACGCCTTTCCGGCCCG
FRPDGGMRLMTGNLGR ATGGCGGTATGCGCCTGATGACTGGTAATCTGGGCCGCGCGATCTACA
AIYKSSAIAPEHLTVEAP AGAGCAGCGCTATTGCGCCCGAGCACCTGACCGTTGAAGCGCCGGCAC
ARVFQDQHDVLTAYQN GGGTCTTCCAGGACCAGCATGACGTCCTCACGGCCTATCAGAATGGTG
GELERDVVVVVRFQGP AGCTTGAGCGTGATGTTGTCGTGGTCGTCCGGTTCCAGGGACCGGAAG
EANGMPELHKLTPTLG CCAACGGCATGCCGGAGCTTCACAAGCTGACCCCGACTCTGGGCGTGC
VLQDRGFKVALLTDGR TTCAGGATCGCGGCTTCAAGGTGGCCCTGCTGACGGATGGACGCATGT
MSGASGKVPAAIHVGP CCGGTGCGAGCGGCAAGGTGCCGGCCGCCATTCATGTCGGTCCCGAAG
EAQVGGPIARVRDGDM CGCAGGTTGGCGGTCCGATCGCCCGCGTGCGGGACGGCGACATGATCC
IRVCAVTGQIEALVDAA GTGTCTGCGCGGTGACGGGACAGATCGAGGCTCTGGTGGATGCCGCCG
EWESRKPVPPPLPALGT AGTGGGAGAGCCGCAAGCCGGTCCCGCCGCCGCTCCCGGCATTGGGA
GRELFALMRSVHDPAE ACGGGCCGCGAACTGTTCGCGCTGATGCGTTCGGTGCATGATCCGGCC
AGGSAMLAQMDRVIEA GAGGCTGGCGGATCCGCGATGCTGGCCCAGATGGATCGCGTGATCGAA
VGDDIH (SEQ ID NO: GCCGTTGGCGACGACATTCACTAA (SEQ ID NO: 85) 86)
ZP_06145432.1 Ruminococcus FD-1
ATGAGCGATAATTTTTTCTGCGAGGGTGCGGATAAAGCCCCTCAGCGT MSDNFFCEGADKAPQR
flavefaciens TCACTTTTCAATGCACTGGGCATGACTAAAGAGGAAATGAAGCGTCCC
SLFNALGMTKEEMKRP CTCGTTGGTATCGTTTCTTCCTACAATGAGATCGTTCCCGGCCATATGA
LVGIVSSYNEIVPGHMN ACATCGACAAGCTGGTCGAAGCCGTTAAGCTGGGTGTAGCTATGGGCG
IDKLVEAVKLGVAMGG GCGGCACTCCTGTTGTTTTCCCTGCTATCGCTGTATGCGACGGTATCGC
GTPVVFPAIAVCDGIAM TATGGGTCACACAGGCATGAAGTACAGCCTTGTTACCCGTGACCTTAT
GHTGMKYSLVTRDLIA TGCCGATTCTACAGAGTGTATGGCTCTTGCTCATCACTTCGACGCACTG
DSTECMALAHHFDALV GTAATGATACCTAACTGCGACAAGAACGTTCCCGGCCTGCTTATGGCG
MIPNCDKNVPGLLMAA GCTGCACGTATCAATGTTCCTACTGTATTCGTAAGCGGCGGCCCTATGC
ARINVPTVFVSGGPMLA TTGCAGGCCATGTAAAGGGTAAGAAGACCTCTCTTTCATCCATGTTCG
GHVKGKKTSLSSMFEA AGGCTGTAGGCGCTTACACAGCAGGCAAGATAGACGAGGCTGAACTT
VGAYTAGKIDEAELDE GACGAATTCGAGAACAAGACCTGCCCTACCTGCGGTTCATGTTCGGGT
FENKTCPTCGSCSGMY ATGTATACCGCTAACTCCATGAACTGCCTCACTGAGGTACTGGGTATG
TANSMNCLTEVLGMGL GGTCTCAGAGGCAACGGCACTATCCCTGCTGTTTACTCCGAGCGTATC
RGNGTIPAVYSERIKLA AAGCTTGCAAAGCAGGCAGGTATGCAGGTTATGGAACTCTACAGAAA
KQAGMQVMELYRKNI GAATATCCGCCCTCTCGATATCATGACAGAGAAGGCTTTCCAGAACGC
RPLDIMTEKAFQNALTA TCTCACAGCTGATATGGCTCTTGGATGTTCCACAAACAGTATGCTCCAT
DMALGCSTNSMLHLPA CTCCCTGCTATCGCCAACGAATGCGGCATAAATATCAACCTTGACATG
IANECGININLDMANEIS GCTAACGAGATAAGCGCCAAGACTCCTAACCTCTGCCATCTTGCACCG
AKTPNLCHLAPAGHTY GCAGGCCACACCTACATGGAAGACCTCAACGAAGCAGGCGGAGTTTA
MEDLNEAGGVYAVLN TGCAGTTCTCAACGAGCTGAGCAAAAAGGGACTTATCAACACCGACTG
ELSKKGLINTDCMTVT CATGACTGTTACAGGCAAGACCGTAGGCGAGAATATCAAGGGCTGCAT
GKTVGENIKGCINRDPE CAACCGTGACCCTGAGACTATCCGTCCTATCGACAACCCATACAGTGA
TIRPIDNPYSETGGIAVL AACAGGCGGAATCGCCGTACTCAAGGGCAATCTTGCTCCCGACAGATG
KGNLAPDRCVVKRSAV TGTTGTGAAGAGAAGCGCAGTTGCTCCCGAAATGCTGGTACACAAAGG
APEMLVHKGPARVFDS CCCTGCAAGAGTATTCGACAGCGAGGAAGAAGCTATCAAGGTCATCTA
EEEAIKVIYEGGIKAGD TGAGGGCGGTATCAAGGCAGGCGACGTTGTTGTTATCCGTTACGAAGG
VVVIRYEGPAGGPGMR CCCTGCAGGCGGCCCCGGCATGAGAGAAATGCTCTCTCCTACATCAGC
EMLSPTSAIQGAGLGST TATACAGGGTGCAGGTCTCGGCTCAACTGTTGCTCTAATCACTGACGG
VALITDGRFSGATRGAA ACGTTTCAGCGGCGCTACCCGTGGTGCGGCTATCGGACACGTATCCCC
IGHVSPEAVNGGTIAYV CGAAGCTGTAAACGGCGGTACTATCGCATATGTCAAGGACGGCGATAT
KDGDIISIDIPNYSITLE
TATCTCCATCGACATACCGAATTACTCCATCACTCTTGAAGTATCCGAC VSDEELAERKKAMPIKR
GAGGAGCTTGCAGAGCGCAAAAAGGCAATGCCTATCAAGCGCAAGGA KENITGYLKRYAQQVS
GAACATCACAGGCTATCTGAAGCGCTATGCACAGCAGGTATCATCCGC SADKGAIINRK (SEQ
AGACAAGGGCGCTATCATCAACAGGAAATAG (SEQ ID NO: 87) ID NO: 88)
Example 40
Unique 200-Mer Nucleotide Sequences Used for Integration
Constructs
TABLE-US-00125 [0564] 200-mer Sequence (SEQ ID NOS 513-531, Number
respectively, in order of appearance) 30
CACGCACGGACCGACCGTCACCGGACCGTTTCGCGCGACGTG
CGCGAGGCTCCGACACGAAAGACGGGCCCCCTATTGCGCTC
ATGTCGGCCGCACCCCTGCGTAAAGTCAGATACGTGCGCCAC
CCGAGCCGGGACCGCCCTGAGCGCATGGTCCGGGCGGCGTG
GCAAGCGCAGGAGGGCGTGCCCCGTTCGCTAGGCA 44
ACGTATGTCGGCTGATCGTACACGCCGACCAGCGCAGTCGG
CGTACTCAGGCGTTCCGAGTAGCTCACATCTGTGGGCCCCGG
CGTACCTTCGGCAGGGTTATGCGACGGGGCGGCAGGCTTGC
GCTGGCGTCGGGAATCACCGCGAACTTGACCCGCGCCGGTT
CCGTATCGGTCCGCTGCGGCCGTGCTCCGCAGTCGA 45
TGCAGTCCGCCCAGCCGGCCGTGTAGCACGGCCGACTGCAG
GTGCGACGTGCTAGGGGCCAGCACGCGAGCGGCCCTACCAC
GGGTCGTGTGGGGCGCATGACCGCCGGCCGGGTCTCGGCAC
GGGGCGACGCGGTGCTCCTAGGCTAGCAGGGCCTCACCGGG
TGATCCCCCGTGTAGCGCCGCACAACACCCCCTGCGA 49
TGCCCGCATACCGCCCGCCCACTGGGGATCCTCCGGCGCTGT
CGCGCTATGCGCGTCCATCCTGGTCGGACGGGCTCGGGCCCC
GGACCAAACCGCAGCGGCCCCTGGCAGCGACTAAGGGCGC
CGTCTCACCCTAGACTTCTTAATCGGGGTGTCCCGGTAGGCC
GGGAGTAGCCTCGGCGGGCTAGCCGCGTGACTATA 78
GCGGGTTAGTCCCCGTCGGACGTCATGCATACAGTCGGGGCT
GGCGAGACAGGAGGCTACAGGGGGCGCCCGGAGGAACACACG
TGGGACTAAGACGTCGGTCCGTGTGCCCCCGAACCGGCGTGC
TCATCGTAGGACTGGGAAGTCCGTACCGCGTGGCTCGTACCT
CGCGGTCTGAGTCCGACACCCGCTGACGCCGGA 13
CTGAGACGACTCCCGCACTACGGATCGCGAGCGTAGACTCAG
CCCGGACTCTCACGCGACCTCGGACGCGGCCTAATGTCTCGA
CTCGCGGTCCGCTGAAGGTCTCGGGGCACGCGAGACGCGGG
GTCAGGCCGGGGGGATCCCCGCACACACTCAGTCGCGGCGA
ACGGAGTCCCGTGGCCTGGCTAGGGATCGTGGGTA 60
GGGGCGTCCACTCTGGCTCGGTAGAGCGCTGGGCTCCGCGC
GACTGCGCGCACCCATCGGTTTGGCGCGACGCACCGTGGA
CTCCTGGGCTAGAGGGCGGGTCCCCGCCATACCCCGTTCT
CGTGCCGGCTGGGTAGGACCGGAGTGACGGCTGTGGCCGG
CGACTCGGGCGCGCACTGTAGTCGGATCTGGGCGGGCAGA 92
GTCGGGCGCGCGTCAGTCCACGCGTTAAACACTGGCCGACGA
CACGACGGGATCCGGGCACGCCCCGAGAGCGCGTGTTCGCG
CGAGTCGATCGGGAGGCCGCAGCGTGTCGAGCCCAGACCCC
GCTCTAGCGTGGCCATCGCGGTGCTAAGTGGGGCGGCCGGG
TCCTATACACGCTTACCGATAGTCAAGTTTGCGTGA 127
GTCTTAGGGCCCAGGGACCGCACGGGTCGACCGCGCGACTGG
TCGGAGCTTGCGCGTCTACGCCACTCGGCGGCCCCGACGGGG
GATGCCGCGGAATGTCCGCCGGCGTATGCGGCTCAAGCCGGA
CCGTCGGACTGCGAAGCGCCGTGAGCACCCCTCGACCTGAC
CGGACGCGGCGCACCCGTCCGAGTATCGTCGCGA 153
TCGGGTCTCGCCCGGCGCTAGTCCAGCCGTAGCGCTCTCCG
GCGATCACCCCGGAGCACTCTGGAGCCGAGCGGTCGGGTCT
GTTGGGCGCGCCGCGGCTACGGACGGCTCGACTCACTGGCG
CTCGACCCCGTATCCCCCGTCTCGGACGACGCACCGTTGCG
CGGGAACGATCGGCGGCGCTCACACGCACGATCGGAA 299
CTTAAGGCTGGCGCACCATGAGGGCCGCGCCACGTCCGAC
CCGCAGCCCGCGCGTAGTAGCCTAGCCGGGCGGGGTTCCTC
CCGTGCGTCACCTAGCACGGGGCCTGGCACCGAACGCGAGC
CCGTCCGGTCACCGCGGCGGGTCTGCGGACGTCCCCGGTC
GCTCGGCTCGGAGTCCCCGCTGGGGATCGCGTCGGGACA 317
CGACGGCGTAGCACTCGCGGACCTAGGGCGCGCGAGTCGGG
GGAGCCCGCGGTGCGACGCTCGGGGAGGAGCTCGCATGCC
CAAGGCACGATCTAGGGGGGGGTACGGGGGGCGTCCGTCC
GAGCGCCGGGACTGCGATCCGGGGCCACATGCTAACCGGC
GGAAGGGGGGACCTAACCGGTGTGGACTCCGGGTAATCCA 319
CGGGGGGCTGACACGTCTCGGATCGCCCCGTCAGTCAGCCC
CCTAGTCCCGGACAGGACGTCGGAGGTCGAGTCCGCACTG
TCGGGCCTGCTCGTGGGCACGGCAGGACGCGTCCCCATGG
TCAGCCGCCGTGCGATACCTCGCCACGACTCTGAGCCGGG
CGCGAGCGTGAGAGCCCGAGCCGCGGTACACGGGGCGTCA 529
GCGAGCTCGCTCTCGACTCCGGGCTCCCGTGCTGACACGGG
GTGCGACCCCGCGGCGATTGTCCGCACGCCTGTCGGACGAC
GTCGGCCCGTCGTAGTGCCGGTCAGAGGCAGGGGGGCTGCT
CGCGCTGGCCGCCTCGTCGCGCGTGGACCCTATGGGGGATC
ACGCGTGGGGTCGGGATCGGGGACCGCGCGACTTGGA 651
CGCGCCCCGTAACGGACGCGGTGAGTCGAGCTTACGCGGCT
AGGGCCGAGTCGTGTTAGCGTCTCGCGTAAGCGAATGCCAC
GTCCCCCGCCGCCCGTCGCGCAGCTGGCTACGCAACGCC
TCCGCGGCCTCCGTAGCGAGTGCGTGGGACGCTGGCCGTC
CGCGTGTTCCGGGACCTGGATGCGGGAGGGACCTAAGGCA 677
AGAACGTGCGGTCGTCCCCACGCACGGGATGACGGACGGGG
TAGACGGGCGTCGTGCGCGCGGGTAGCGTAACCGGTTACA
GTCCCCGCAACGCTCTAGCTCCGGCCCTCGCTTAGGAGTT
CGCGGCCGAGACATGAGGTGGTCCGGACGGCAGGGGGTCG
CGGAGACCGTGGAGCCGATTCTGCCGGACGCCACGTCCCA 708
CGGGACGCCCCGTACCGTGTACGAAGCCCCGGTCGGTCGGCG
GATCGTAGATCCCGGAGCCGACGCCTTGAACCCGGCTTTCCC
AGCGACTCGCGCCCCCACTGGGTCCCTCGGGACCCCGCTCC
CCCCAGACGCATACAGCCCGCAAGCGGGGGCAGTCTCGGAC
CGCCCGGACACTGGCCTTAGGCACCGTGGGCTCGA 717
GTGTCCGGGGCGCATCGGAGCTGTCCGACCGAGTTCCGGGG
ACGGCGCACGTTGTGCCGGCCTCAGACGGAGCCTGTAGCCCC
CGGACAGTGTGTGCCCGCCCACTACGGGTTAGGCACGGGGTT
GGTCGGCACGCGTCCTCCGCGTGTCACGGACCGATGCAGAC
CGCTGGCCGGGAGGTCGCCCCCCCAGGGGTGCACA 719
CGCGCAGCACGCACGTCCGGGGCACGCGCGGCTCGGAGGGTC
CGGGCTGGGACGGGAGGTTTGGAGTCGCGTGCGCGTAGCAGC
GCACCCGCCTGGTCGCCGGGTCTAGTAGGGCTGGGTTACGGA
GGACGTGCAGGCGACCCCAACCGTTGACGACGGGTCCGACC
ACGCCTTTAGCCGTGGCGTGTCCGTCGCGAGCCA
Example 41
Evaluation of Additional Xylose Isomerase Genes
[0565] As noted above, additional xylose isomerase genes were
identified and isolated and chimeric versions generated in certain
embodiments. Presented below are the results of activity assays of
three candidate xylose isomerase genes from R. flavefaciens, FD-1,
Ruminococcus 18P13, and Clostridiales genomosp., when expressed in
S. cerevisiae.
[0566] The candidate xylose isomerase enzymes (XI's) were assayed
as total soluble crude extracts (prepared as described herein in
YPER-PLUS reagent and quantified with the Coomasie-Plus kit). 100
.mu.g of each extract was compared for the candidate XI's alongside
the original XI-R (e.g., Ruminococcus xylose isomerase) native
construct. The Clostridiales enzyme was further characterized at
1974 .mu.g to confirm the presence of activity. The results in this
experiment are presented as the slope of the activity at saturating
xylose concentrations (500 mM). The results are show in FIG. 25.
The R. flavefaciens, FD-1 XI activity shows the highest activity of
the candidates tested, and shows higher activity than the XI-R
native construct used as a control. The two candidate genes from
Ruminococcus strains were further characterized in an enzyme assay
using xylose concentrations between 40 and 500 nM. Michaelis Mentin
plots were generated for each candidate in order to determine
K.sub.m and specific activity levels as compared to the native XI-R
enzyme. The results are presented in the table below. The R.
flavefaciens, FD-1 XI activity shows a greater than 2 fold specific
activity than the native XI control activity.
TABLE-US-00126 Enzyme Source Km Specific Activity (umol min.sup.-1
mg.sup.-1) XI-R native 42.57 nM 0.9605 R. flavefaciens FD-1 71.91
nM 2.3045 Ruminococcus 18P13 65.11 Nm 0.20448
Example 42
Examples of Embodiments
[0567] Provided hereafter are certain non-limiting embodiments of
the technology.
[0568] A1. An engineered microorganism that comprises: [0569] (a) a
functional Embden-Meyerhoff glycolysis pathway that metabolizes
six-carbon sugars under aerobic fermentation conditions, and [0570]
(b) a genetic modification that reduces an Embden-Meyerhoff
glycolysis pathway member activity upon exposure of the engineered
microorganism to anaerobic fermentation conditions, [0571] whereby
the engineered microorganism preferentially metabolizes six-carbon
sugars by the Enter-Doudoroff pathway under the anaerobic
fermentation conditions.
[0572] A2. The engineered microorganism of embodiment A1, wherein
the genetic modification is insertion of a promoter into genomic
DNA in operable linkage with a polynucleotide that encodes the
Embden-Meyerhoff glycolysis pathway member activity.
[0573] A3. The engineered microorganism of embodiment A1, wherein
the genetic modification is provision of a heterologous promoter
polynucleotide in operable linkage with a polynucleotide that
encodes the Embden-Meyerhoff glycolysis pathway member
activity.
[0574] A4. The engineered microorganism of embodiment A1, wherein
the genetic modification is a deletion or disruption of a
polynucleotide that encodes, or regulates production of, the
Embden-Meyerhoff glycolysis pathway member, and the microorganism
comprises a heterologus nucleic acid that includes a polynucleotide
encoding the Embden-Meyerhoff glycolysis pathway member operably
linked to a polynucleotide that down-regulates production of the
member under anaerobic fermentation conditions.
[0575] A5. The engineered microorganism of any one of embodiments
A1-A4, wherein the Embden-Meyerhoff glycolysis pathway member
activity is a phosphofructokinase activity.
[0576] A6. The engineered microorganism of any one of embodiments
A1-A5, which microorganism comprises an added or altered
five-carbon sugar metabolic activity.
[0577] A7. The engineered microorganism of embodiment A6, wherein
the microorganism comprises an added or altered xylose isomerase
activity.
[0578] A8. The engineered microorganism of any one of embodiments
A1-A7, wherein the microorganism comprises an added or altered
five-carbon sugar transporter activity.
[0579] A9. The engineered microorganism of embodiment A8, wherein
the transporter activity is a transporter facilitator activity.
[0580] A10. The engineered microorganism of embodiment A8, wherein
the transporter activity is an active transporter activity.
[0581] A11. The engineered microorganism of any one of embodiments
A1-A10, wherein the microorganism comprises an added or altered
carbon dioxide fixation activity.
[0582] A12. The engineered microorganism of embodiment A11, wherein
the microorganism comprises an added or altered phosphoenolpyruvate
(PEP) carboxylase activity.
[0583] A13. The engineered microorganism of any one of embodiments
A1-A12, wherein the microorganism comprises a genetic modification
that reduces or removes an alcohol dehydrogenase 2 activity.
[0584] A14. The engineered microorganism of any one of embodiments
A1-A13, wherein the microorganism comprises a genetic modification
described in any one of embodiments B1-B208.
[0585] B1. An engineered microorganism that comprises a genetic
modification that inhibits cell division upon exposure to a change
in fermentation conditions, wherein:
[0586] the genetic modification comprises introduction of a
heterologous promoter operably linked to a polynucleotide encoding
a polypeptide that regulates the cell cycle of the microorganism;
and [0587] the promoter activity is altered by the change in
fermentation conditions.
[0588] B2. The engineered microorganism of embodiment B1, wherein
the genetic modification induces cell cycle arrest.
[0589] B3. The engineered microorganism of embodiment B1 or B2,
wherein the change in fermentation conditions is a switch to
anaerobic fermentation conditions.
[0590] B4. The engineered microorganism of embodiment B1 or B2,
wherein the change in fermentation conditions is a switch to an
elevated temperature.
[0591] B5. The engineered microorganism of any one of embodiments
B1-B4, wherein the polypeptide that regulates the cell cycle has
thymidylate synthase activity.
[0592] B6. The engineered microorganism of any one of embodiments
B1-B5, wherein the promoter activity is reduced by the change in
fermentation conditions.
[0593] B100. An engineered microorganism that comprises a genetic
modification that inhibits cell division and/or cell proliferation
upon exposure of the microorganism to a change in fermentation
conditions.
[0594] B101. The engineered microorganism of embodiment B100,
wherein the change in fermentation conditions is a switch to
anaerobic fermentation conditions.
[0595] B102. The engineered microorganism of embodiment B100,
wherein the change in fermentation conditions is a switch to an
elevated temperature.
[0596] B103. The engineered microorganism of any one of embodiments
B100-B102, wherein the genetic modification induces cell cycle
arrest upon exposure to the change in fermentation conditions.
[0597] B104. The engineered microorganism of any one of embodiments
B100-B103, wherein the genetic modification reduces thymidylate
synthase activity upon exposure to the change in fermentation
conditions.
[0598] B200. The engineered microorganism of any one of embodiments
B1-B104, wherein the genetic modification is a temperature
sensitive mutation.
[0599] B201. The engineered microorganism of any one of embodiments
B1-B200, wherein the microorganism comprises an added or altered
five-carbon sugar metabolic activity.
[0600] B202. The engineered microorganism of embodiment B201,
wherein the microorganism comprises an added or altered xylose
isomerase activity.
[0601] B203. The engineered microorganism of any one of embodiments
B1-B202, wherein the microorganism comprises an added or altered
five-carbon sugar transporter activity.
[0602] B204. The engineered microorganism of embodiment B203,
wherein the transporter activity is a transporter facilitator
activity.
[0603] B205. The engineered microorganism of embodiment B203,
wherein the transporter activity is an active transporter
activity.
[0604] B206. The engineered microorganism of any one of embodiments
B1-B205, wherein the microorganism comprises an added or altered
carbon dioxide fixation activity.
[0605] B207. The engineered microorganism of embodiment B206,
wherein the microorganism comprises an added or altered
phosphoenolpyruvate (PEP) carboxylase activity.
[0606] B208. The engineered microorganism of any one of embodiments
B1-B207, wherein the microorganism comprises a genetic modification
that reduces or removes an alcohol dehydrogenase 2 activity.
[0607] B300. The engineered microorganism of any one of embodiments
A1-B208, wherein the microorganism is an engineered yeast.
[0608] B301. The engineered microorganism of embodiment B300,
wherein the yeast is a Saccharomyces yeast.
[0609] B302. The engineered microorganism of embodiment B301,
wherein the Saccharomyces yeast is S. cerevisiae.
[0610] C1. A method for manufacturing a target product produced by
an engineered microorganism, which comprises: [0611] (a) culturing
an engineered microorganism of any one of embodiments A1-B302 under
aerobic conditions; and [0612] (b) culturing the engineered
microorganism after (a) under anaerobic conditions, whereby the
engineered microorganism produces the target product.
[0613] C2. The method of embodiment C1, wherein the target product
is ethanol.
[0614] C3. The method of embodiment C1, wherein the target product
is succinic acid.
[0615] C4. The method of any one of embodiments C1-C3, wherein the
host microorganism from which the engineered microorganism is
produced does not produce a detectable amount of the target
product.
[0616] C5. The method of any one of embodiments C1-C4, wherein the
culture conditions comprise fermentation conditions.
[0617] C6. The method of any one of embodiments C1-C5, wherein the
culture conditions comprise introduction of biomass.
[0618] C7. The method of any one of embodiments C1-C6, wherein the
culture conditions comprise introduction of a six-carbon sugar.
[0619] C8. The method of embodiment C7, wherein the sugar is
glucose.
[0620] C9. The method of any one of embodiments C1-C8, wherein the
culture conditions comprise introduction of a five-carbon
sugar.
[0621] C10. The method of embodiment C9, wherein the sugar is
xylose.
[0622] C11. The method of any one of embodiments C1-C10, wherein
the target product is produced with a yield of greater than about
0.3 grams per gram of glucose added.
[0623] C12. The method of any one of embodiments C1-C11, which
comprises purifying the target product from the cultured
microorganisms.
[0624] C13. The method of embodiment C12, which comprises modifying
the target product, thereby producing modified target product.
[0625] C14. The method of any one of embodiments C1-C13, which
comprises placing the cultured microorganisms, the target product
or the modified target product in a container.
[0626] C15. The method of embodiment C14, which comprises shipping
the container.
[0627] D1. A method for producing a target product by an engineered
microorganism, which comprises: [0628] (a) culturing an engineered
microorganism of any one of embodiments A1-B302 under a first set
of fermentation conditions; and [0629] (b) culturing the engineered
microorganism after (a) under a second set of fermentation
conditions different than the first set of fermentation conditions,
whereby the second set of fermentation conditions inhibits cell
division and/or cell proliferation of the engineered
microorganism.
[0630] D2. The method of embodiment D1, wherein the second set of
fermentation conditions comprises anaerobic fermentation conditions
and the first set of fermentation conditions comprises aerobic
fermentation conditions.
[0631] D3. The method of embodiment D1, wherein the second set of
fermentation conditions comprises an elevated temperature as
compared to the temperature in the first set of fermentation
conditions.
[0632] D4. The method of any one of embodiments D1-D3, wherein the
genetic modification inhibits the cell cycle of the engineered
microorganism upon exposure to the second set of fermentation
conditions.
[0633] D5. The method of any one of embodiments D1-D4, wherein the
genetic modification induces cell cycle arrest upon exposure to the
second set of fermentation conditions.
[0634] D6. The method of any one of embodiments D1-D5, wherein the
genetic modification inhibits thymidylate synthase activity upon
exposure to the change in fermentation conditions.
[0635] D7. The method of embodiment D6, wherein the genetic
modification comprises a temperature sensitive mutation.
[0636] D8. The method of any one of embodiments D1-D7, wherein the
microorganism comprises an added or altered five-carbon sugar
metabolic activity.
[0637] D9. The method of embodiment D8, wherein the microorganism
comprises an added or altered xylose isomerase activity.
[0638] D10. The method of any one of embodiments D1-D9, wherein the
microorganism comprises an added or altered five-carbon sugar
transporter activity.
[0639] D11. The method of embodiment D10, wherein the transporter
activity is a transporter facilitator activity.
[0640] D12. The method of embodiment D10, wherein the transporter
activity is an active transporter activity.
[0641] D13. The method of any one of embodiments D1-D12, wherein
the microorganism comprises an added or altered carbon dioxide
fixation activity.
[0642] D14. The method of embodiment D13, wherein the microorganism
comprises an added or altered phosphoenolpyruvate (PEP) carboxylase
activity.
[0643] D15. The method of any one of embodiments D1-D14, wherein
the microorganism comprises a genetic modification that reduces or
removes an alcohol dehydrogenase 2 activity.
[0644] D16. The method of any one of embodiments D1-D15, wherein
the microorganism is an engineered yeast.
[0645] D17. The method of embodiment D16, wherein the yeast is a
Saccharomyces yeast.
[0646] D18. The method of embodiment D17, wherein the Saccharomyces
yeast is S. cerevisiae.
[0647] D19. The method of any one of embodiments D1-D18, wherein
the target product is ethanol.
[0648] D20. The method of any one of embodiments D1-D18, wherein
the target product is succinic acid.
[0649] E1. A method for manufacturing an engineered microorganism,
which comprises: [0650] (a) introducing a genetic modification to a
host microorganism that reduces an Embden-Meyerhoff glycolysis
pathway member activity upon exposure of the engineered
microorganism to anaerobic conditions; and [0651] (b) selecting for
engineered microorganisms that (i) metabolize six-carbon sugars by
the Embden-Meyerhoff glycolysis pathway under aerobic fermentation
conditions, and (ii) preferentially metabolize six-carbon sugars by
the Enter-Doudoroff pathway under the anaerobic fermentation
conditions.
[0652] E2. The method of embodiment E1, wherein the genetic
modification is insertion of a promoter into genomic DNA in
operable linkage with a polynucleotide that encodes the
Embden-Meyerhoff glycolysis pathway member activity.
[0653] E3. The method of embodiment E1, wherein the genetic
modification is provision of a heterologous promoter polynucleotide
in operable linkage with a polynucleotide that encodes the
Embden-Meyerhoff glycolysis pathway member activity.
[0654] E4. The method of embodiment E1, wherein the genetic
modification is a deletion or disruption of a polynucleotide that
encodes, or regulates production of, the Embden-Meyerhoff
glycolysis pathway member, and the microorganism comprises a
heterologous nucleic acid that includes a polynucleotide encoding
the Embden-Meyerhoff glycolysis pathway member operably linked to a
polynucleotide that down-regulates production of the member under
anaerobic fermentation conditions.
[0655] E5. The method of any one of embodiments E1-E4, wherein the
Embden-Meyerhoff glycolysis pathway member activity is a
phosphofructokinase activity.
[0656] E6. The method of any one of embodiments E1-E5, which
comprises introducing a genetic alteration that adds or alters a
five-carbon sugar metabolic activity.
[0657] E7. The method of embodiment E6, wherein the genetic
alteration adds or alters a xylose isomerase activity.
[0658] E8. The method of any one of embodiments E1-E7, which
comprises introducing a genetic modification that adds or alters a
five-carbon sugar transporter activity.
[0659] E9. The method of embodiment E8, wherein the transporter
activity is a transporter facilitator activity.
[0660] E10. The method of embodiment E8, wherein the transporter
activity is an active transporter activity.
[0661] E11. The method of any one of embodiments E1-E10, which
comprises introducing a genetic modification that adds or alters a
carbon dioxide fixation activity.
[0662] E12. The method of embodiment E11, which comprises
introducing a genetic modification that adds or alters a
phosphoenolpyruvate (PEP) carboxylase activity.
[0663] E13. The method of any one of embodiments E1-E12, which
comprises introducing a genetic modification that reduces or
removes an alcohol dehydrogenase 2 activity.
[0664] E14. The method of any one of embodiments E1-E13, which
comprises introducing a genetic modification described in any one
of embodiments B1-B208.
[0665] F1. A method for manufacturing an engineered microorganism,
which comprises: [0666] (a) introducing a genetic modification to a
host microorganism that inhibits cell division upon exposure to a
change in fermentation conditions, thereby producing engineered
microorganisms; and [0667] (b) selecting for engineered
microorganisms with inhibited cell division upon exposure of the
engineered microorganisms to the change in fermentation
conditions.
[0668] F2. The method of embodiment F2, wherein the change in
fermentation conditions comprises a change to anaerobic
fermentation conditions.
[0669] F3. The method of embodiment F1, wherein the change in
fermentation conditions comprises a change to an elevated
temperature.
[0670] F4. The method of any one of embodiments F1-F3, wherein the
genetic modification inhibits the cell cycle of the engineered
microorganism upon exposure to the change in fermentation
conditions.
[0671] F5. The method of any one of embodiments F1-F4, wherein the
genetic modification induces cell cycle arrest upon exposure to the
second set of fermentation conditions.
[0672] F6. The method of any one of embodiments F1-F5, wherein the
genetic modification inhibits thymidylate synthase activity upon
exposure to the change in fermentation conditions.
[0673] F7. The method of embodiment F6, wherein the genetic
modification comprises a temperature sensitive mutation.
[0674] F8. The method of any one of embodiments F1-F7, wherein the
genetic modification adds or alters a five-carbon sugar metabolic
activity.
[0675] F9. The method of embodiment F8, wherein the genetic
modification adds or alters a xylose isomerase activity.
[0676] F10. The method of any one of embodiments F1-F9, wherein the
genetic modification adds or alters a five-carbon sugar transporter
activity.
[0677] F11. The method of embodiment F10, wherein the transporter
activity is a transporter facilitator activity.
[0678] F12. The method of embodiment F10, wherein the transporter
activity is an active transporter activity.
[0679] F13. The method of any one of embodiments F1-F12, wherein
the genetic modification adds or alters a carbon dioxide fixation
activity.
[0680] F14. The method of embodiment F13, wherein the genetic
modification adds or alters a phosphoenolpyruvate (PEP) carboxylase
activity.
[0681] F15. The method of any one of embodiments F1-F14, wherein
the genetic modification reduces or removes an alcohol
dehydrogenase 2 activity.
[0682] F16. The method of any one of embodiments E1-E14 and F1-F15,
wherein the microorganism is an engineered yeast.
[0683] F17. The method of embodiment F16, wherein the yeast is a
Saccharomyces yeast.
[0684] F18. The method of embodiment F17, wherein the Saccharomyces
yeast is S. cerevisiae.
[0685] G1. A nucleic acid, comprising a polynucleotide that encodes
a polypeptide from Ruminococcus flavefaciens possessing a xylose to
xylulose xylose isomerase activity.
[0686] G2. The nucleic acid of embodiment G1, wherein the
polynucleotide includes one or more substituted codons.
[0687] G3. The nucleic acid of embodiment G2, wherein the one or
more substituted codons are yeast codons.
[0688] G4. The nucleic acid of any one of embodiments G1 to G3,
wherein the polynucleotide includes a nucleotide sequence of SEQ ID
NO: 29, 30, 32 or 33, fragment thereof, or sequence having 50%
identity or greater to the foregoing.
[0689] G5. The nucleic acid of any one of embodiments G1 to G4,
wherein the polypeptide includes an amino acid sequence of SEQ ID
NO: 31, fragment thereof, or sequence having 75% identity or
greater to the foregoing.
[0690] G6. The nucleic acid of any one of embodiments G1 to G5,
wherein a stretch of contiguous nucleotides of the polynucleotide
is from another organism.
[0691] G7. The nucleic acid of embodiment G6, wherein the stretch
of contiguous nucleotides from the other organism is from a
nucleotide sequence that encodes a polypeptide possessing a xylose
isomerase activity.
[0692] G8. The nucleic acid of embodiment G5 or G6, wherein the
other organism is a fungus.
[0693] G9. The nucleic acid of embodiment G8, wherein the fungus is
a Piromyces fungus.
[0694] G10. The nucleic acid of embodiment G9, wherein the fungus
is a Piromyces strain E2.
[0695] G11. The nucleic acid of embodiment G10, wherein the stretch
of contiguous nucleotides from the other organism is from SEQ ID
NO: 34, or sequence having 50% identity or greater to the
foregoing.
[0696] G12. The nucleic acid of embodiment G10, wherein the stretch
of contiguous nucleotides from the other organism encodes an amino
acid sequence from SEQ ID NO: 35, or sequence having 75% identity
or greater to the foregoing.
[0697] G13. The nucleic acid of any one of embodiments G6 to G12,
wherein the stretch of contiguous nucleotides from the other
organism is about 1% to about 30% of the total number of
nucleotides in the polynucleotide that encodes the polypeptide
possessing xylose isomerase activity.
[0698] G14. The nucleic acid of embodiment G13, wherein about 30
contiguous nucleotides from the polynucleotide from R. flavefaciens
are replaced by about 10 to about 20 nucleotides from the other
organism.
[0699] G15. The nucleic acid of embodiment G13 or G14, wherein the
contiguous stretch of polynucleotides from the other organism are
at the 5' end of the polynucleotide.
[0700] G16. The nucleic acid of any one of embodiments G6 to G15,
wherein the polynucleotide includes a nucleotide sequence of SEQ ID
NO: 55, 56, 57, 59 or 61, fragment thereof, or sequence having 50%
identity or greater to the foregoing.
[0701] G17. The nucleic acid of any one of embodiments G6 to G15,
wherein the polynucleotide encodes a polypeptide that includes an
amino acid sequence of SEQ ID NO: 58, 60 or 62, fragment thereof,
or sequence having 75% identity or greater to the foregoing.
[0702] G18. The nucleic acid of any one of embodiments G1 to G17,
which comprises one or more point mutations.
[0703] G19. The nucleic acid of embodiment G18, wherein the point
mutation is at a position corresponding to position 179 of the R.
flavefaciens polypeptide having xylose isomerase activity.
[0704] G20. The nucleic acid of embodiment G19, wherein the point
mutation is a glycine 179 to alanine point mutation.
[0705] H1. An expression vector comprising a polynucleotide that
encodes a polypeptide from Ruminococcus flavefaciens possessing a
xylose to xylulose xylose isomerase activity.
[0706] H2. The expression vector of embodiment H1, wherein the
polynucleotide includes one or more substituted codons.
[0707] H3. The expression vector of embodiment H2, wherein the one
or more substituted codons are yeast codons.
[0708] H4. The expression vector of any one of embodiments H1 to
H3, wherein the polynucleotide includes a nucleotide sequence of
SEQ ID NO: 29, 30, 32 or 33, fragment thereof, or sequence having
50% identity or greater to the foregoing.
[0709] H5. The expression vector of any one of embodiments H1 to
H4, wherein the polypeptide includes an amino acid sequence of SEQ
ID NO: 31, fragment thereof, or sequence having 75% identity or
greater to the foregoing.
[0710] H6. The expression vector of any one of embodiments H1 to
H5, wherein a stretch of contiguous nucleotides of the
polynucleotide is from another organism.
[0711] H7. The expression vector of embodiment H6, wherein the
stretch of contiguous nucleotides from the other organism is from a
nucleotide sequence that encodes a polypeptide possessing a xylose
isomerase activity.
[0712] H8. The expression vector of embodiment H5 or H6, wherein
the other organism is a fungus.
[0713] H9. The expression vector of embodiment H8, wherein the
fungus is a Piromyces fungus.
[0714] H10. The expression vector of embodiment H9, wherein the
fungus is a Piromyces strain E2.
[0715] H11. The expression vector of embodiment H10, wherein the
stretch of contiguous nucleotides from the other organism is from
SEQ ID NO: 34, or sequence having 50% identity or greater to the
foregoing.
[0716] H12. The expression vector of embodiment H10, wherein the
stretch of contiguous nucleotides from the other organism encodes
an amino acid sequence from SEQ ID NO: 35, or sequence having 75%
identity or greater to the foregoing.
[0717] H13. The expression vector of any one of embodiments H6 to
H12, wherein the stretch of contiguous nucleotides from the other
organism is about 1% to about 30% of the total number of
nucleotides in the polynucleotide that encodes the polypeptide
possessing xylose isomerase activity.
[0718] H14. The expression vector of embodiment H13, wherein about
30 contiguous nucleotides from the polynucleotide from R.
flavefaciens are replaced by about 10 to about 20 nucleotides from
the other organism.
[0719] H15. The expression vector of embodiment H13 or H14, wherein
the contiguous stretch of polynucleotides from the other organism
are at the 5' end of the polynucleotide.
[0720] H16. The expression vector of any one of embodiments H6 to
H15, wherein the polynucleotide includes a nucleotide sequence of
SEQ ID NO: 55, 56, 57, 59 or 61, fragment thereof, or sequence
having 50% identity or greater to the foregoing.
[0721] H17. The expression vector of any one of embodiments H6 to
H15, wherein the polynucleotide encodes a polypeptide that includes
an amino acid sequence of SEQ ID NO: 58, 60 or 62, fragment
thereof, or sequence having 75% identity or greater to the
foregoing.
[0722] H18. The expression vector of any one of embodiments H1 to
H17, which comprises one or more point mutations.
[0723] H19. The expression vector of embodiment H18, wherein the
point mutation is at a position corresponding to position 179 of
the R. flavefaciens polypeptide having xylose isomerase
activity.
[0724] H20. The expression vector of embodiment H19, wherein the
point mutation is a glycine 179 to alanine point mutation.
[0725] H21. The expression vector of any one of embodiments, H1 to
H20, comprising a regulatory nucleotide sequence in operable
linkage with the polynucleotide.
[0726] H22. The expression vector of embodiment J25, wherein the
regulatory nucleotide sequence comprises a promoter sequence.
[0727] H23. The expression vector of embodiment J26, wherein the
promoter sequence is an inducible promoter sequence.
[0728] H24. The expression vector of embodiment J26, wherein the
promoter sequence is a constitutively active promoter sequence.
[0729] H25. A method for preparing an expression vector of any one
of embodiments H1 to H24, comprising: (i) providing a nucleic acid
that contains a regulatory sequence, and (ii) inserting the
polynucleotide into the nucleic acid in operable linkage with the
regulatory sequence.
[0730] I1. A nucleic acid , comprising a polynucleotide that
includes a first stretch of contiguous nucleic acids from a first
organism and a second stretch of contiguous nucleic acids from a
second organism, wherein the polynucleotide encodes a polypeptide
possessing a xylose to xylulose xylose isomerase activity.
[0731] I2. The nucleic acid of embodiment I1, wherein the first
organism and the second organism are the same species.
[0732] I3. The nucleic acid of embodiment I1, wherein the first
organism and the second organism are different species.
[0733] I4. The nucleic acid of any one of embodiments I1 to I3,
wherein the first stretch of contiguous nucleotides and the second
stretch of contiguous nucleotides independently are selected from
nucleotide sequence that encodes a polypeptide having xylose
isomerase activity.
[0734] I5. The nucleic acid of any one of embodiments I1 to I4,
wherein the first organism is a bacterium.
[0735] I6. The nucleic acid of embodiment I5, wherein the bacterium
is a Ruminococcus bacterium.
[0736] I7. The nucleic acid of embodiment I6, wherein the bacterium
is a Ruminococcus flavefaciens bacterium.
[0737] I8. The nucleic acid of any one of embodiments I5 to I7,
wherein the stretch of contiguous nucleotides is from SEQ ID NO:
29, 30, 32, 33, or a sequence having 50% identity or greater to the
foregoing.
[0738] I9. The nucleic acid of embodiment I8, wherein the stretch
of contiguous nucleotides from the other organism encodes an amino
acid sequence from SEQ ID NO: 31, or a sequence having 75% identity
or greater to the foregoing.
[0739] I10. The nucleic acid of any one of embodiments I1 to I9,
wherein the second organism is a fungus.
[0740] I11. The nucleic acid of embodiment I10, wherein the fungus
is a Piromyces fungus.
[0741] I12. The nucleic acid of embodiment I11, wherein the fungus
is a Piromyces strain E2 fungus.
[0742] I13. The nucleic acid of any one of embodiments I10 to I12,
wherein the stretch of contiguous nucleotides is from SEQ ID NO:
34, or a sequence having 50% identity or greater to the
foregoing.
[0743] I14. The nucleic acid of embodiment I13, wherein the stretch
of contiguous nucleotides from the other organism encodes an amino
acid sequence from SEQ ID NO: 35, or a sequence having 75% identity
or greater to the foregoing.
[0744] I15. The nucleic acid of any one of embodiments I1 to I14,
wherein the polynucleotide includes one or more substituted
codons.
[0745] I16. The nucleic acid of embodiment I15, wherein the one or
more substituted codons are yeast codons.
[0746] I17. The nucleic acid of any one of embodiments I1 to I16,
wherein the stretch of contiguous nucleotides from the first
organism or second organism is about 1% to about 30% of the total
number of nucleotides in the polynucleotide that encodes the
polypeptide possessing xylose isomerase activity.
[0747] I18. The nucleic acid of embodiment I17, wherein the stretch
of contiguous nucleotides from the second organism is about 1% to
about 30% of the total number of nucleotides in the
polynucleotide.
[0748] I19. The nucleic acid of embodiment I18, wherein the
contiguous stretch of polynucleotides from the second organism are
at the 5' end of the polynucleotide.
[0749] I20. The nucleic acid of any one of embodiments I1 to I19,
wherein the polynucleotide includes a nucleotide sequence of SEQ ID
NO: 55, 56, 57, 59 or 61, fragment thereof, or sequence having 50%
identity or greater to the foregoing.
[0750] I21. The nucleic acid of any one of embodiments I1 to I20,
wherein the polynucleotide encodes a polypeptide that includes an
amino acid sequence of SEQ ID NO: 58, 60 or 62, fragment thereof,
or sequence having 75% identity or greater to the foregoing.
[0751] I22. The nucleic acid of any one of embodiments I1 to I21,
which comprises one or more point mutations.
[0752] I23. The nucleic acid of embodiment I22, wherein the point
mutation is at a position corresponding to position 179 of the R.
flavefaciens polypeptide having xylose isomerase activity.
[0753] I24. The nucleic acid of embodiment I23, wherein the point
mutation is a glycine 179 to alanine point mutation.
[0754] J1. An expression vector , comprising a polynucleotide that
includes a first stretch of contiguous nucleotides from a first
organism and a second stretch of contiguous nucleotides from a
second organism, wherein the polynucleotide encodes a polypeptide
possessing a xylose to xylulose xylose isomerase activity.
[0755] J2. The expression vector of embodiment J1, wherein the
first organism and the second organism are the same.
[0756] J3. The expression vector of embodiment J1, wherein the
first organism and the second organism are different.
[0757] J4. The expression vector of any one of embodiments J1 to
J3, wherein the first stretch of contiguous nucleotides and the
second stretch of contiguous nucleotides independently are selected
from nucleotide sequence that encodes a polypeptide having xylose
isomerase activity.
[0758] J5. The expression vector of any one of embodiments J1 to
J4, wherein the first organism is a bacterium.
[0759] J6. The expression vector of embodiment J5, wherein the
bacterium is a Ruminococcus bacterium.
[0760] J7. The expression vector of embodiment J6, wherein the
bacterium is a Ruminococcus flavefaciens bacterium.
[0761] J8. The expression vector of any one of embodiments J5 to
J7, wherein the stretch of contiguous nucleotides is from SEQ ID
NO: 29, 30, 32, 33, or a sequence having 50% identity or greater to
the foregoing.
[0762] J9. The expression vector of embodiment J8, wherein the
stretch of contiguous nucleotides from the other organism encodes
an amino acid sequence from SEQ ID NO: 31, or a sequence having 75%
identity or greater to the foregoing.
[0763] J10. The expression vector of any one of embodiments J1 to
J9, wherein the second organism is a fungus.
[0764] J11. The expression vector of embodiment J10, wherein the
fungus is a Piromyces fungus.
[0765] J12. The expression vector of embodiment J11, wherein the
fungus is a Piromyces strain E2 fungus.
[0766] J13. The expression vector of any one of embodiments J10 to
J12, wherein the stretch of contiguous nucleotides is from SEQ ID
NO: 34, or a sequence having 50% identity or greater to the
foregoing.
[0767] J14. The expression vector of embodiment J13, wherein the
stretch of contiguous nucleotides from the other organism encodes
an amino acid sequence from SEQ ID NO: 35, or a sequence having 75%
identity or greater to the foregoing.
[0768] J15. The expression vector of any one of embodiments J1 to
J14, wherein the polynucleotide includes one or more substituted
codons.
[0769] J16. The expression vector of embodiment J15, wherein the
one or more substituted codons are yeast codons.
[0770] J17. The expression vector of any one of embodiments J1 to
J16, wherein the stretch of contiguous nucleotides from the first
organism or second organism is about 1% to about 30% of the total
number of nucleotides in the polynucleotide that encodes the
polypeptide possessing xylose isomerase activity.
[0771] J18. The expression vector of embodiment J17, wherein the
stretch of contiguous nucleotides from the second organism is about
1% to about 30% of the total number of nucleotides in the
polynucleotide.
[0772] J19. The expression vector of embodiment J18, wherein the
contiguous stretch of polynucleotides from the second organism are
at the 5' end of the polynucleotide.
[0773] J20. The expression vector of any one of embodiments J1 to
J19, wherein the polynucleotide includes a nucleotide sequence of
SEQ ID NO: 55, 56, 57, 59 or 61, fragment thereof, or sequence
having 50% identity or greater to the foregoing.
[0774] J21. The expression vector of any one of embodiments J1 to
J20, wherein the polynucleotide encodes a polypeptide that includes
an amino acid sequence of SEQ ID NO: 58, 60 or 62, fragment
thereof, or sequence having 75% identity or greater to the
foregoing.
[0775] J22. The expression vector of any one of embodiments J1 to
J21, which comprises one or more point mutations.
[0776] J23. The expression vector of embodiment J22, wherein the
point mutation is at a position corresponding to position 179 of
the R. flavefaciens polypeptide having xylose isomerase
activity.
[0777] J24. The expression vector of embodiment J23, wherein the
point mutation is a glycine 179 to alanine point mutation.
[0778] J25. The expression vector of any one of embodiments, J1 to
J24, comprising a regulatory nucleotide sequence in operable
linkage with the polynucleotide.
[0779] J26. The expression vector of embodiment J25, wherein the
regulatory nucleotide sequence comprises a promoter sequence.
[0780] J27. The expression vector of embodiment J26, wherein the
promoter sequence is an inducible promoter sequence.
[0781] J28. The expression vector of embodiment J26, wherein the
promoter sequence is a constitutively active promoter sequence.
[0782] J29. A method for preparing an expression vector of any one
of embodiments J1 to J28, comprising: (i) providing a nucleic acid
that contains a regulatory sequence, and (ii) inserting the
polynucleotide into the nucleic acid in operable linkage with the
regulatory sequence.
[0783] K1. A microbe comprising a polynucleotide of the nucleic
acid of any one of embodiments G1 to G20 or any one of embodiments
I1 to I24.
[0784] K2. A microbe comprising an expression vector of any one of
embodiments H1 to H20 or any one of embodiments J1 to J24.
[0785] K3. The microbe of embodiment K1 or K2, which is a
yeast.
[0786] K4. The microbe of embodiment K3, which is a Saccharomyces
yeast.
[0787] K5. The microbe of embodiment K4, which is a Saccharomyces
cerevisiae yeast.
[0788] L1. A method, comprising contacting a microbe of any one of
embodiments K1 to K5 with a feedstock comprising a five carbon
molecule under conditions for generating ethanol.
[0789] L2. The method of embodiment L1, wherein the five carbon
molecule comprises xylose.
[0790] L3. The method of embodiment L1 or L2, wherein about 15
grams per liter of ethanol or more is generated within about 372
hours.
[0791] L4. The method of any one of embodiments L1 to L3, wherein
about 2.0 grams per liter dry cell weight is generated within about
372 hours.
[0792] M1. A composition comprising an engineered yeast comprising
heterologous polynucleotide subsequences that encode a
phosphogluconate dehydratase enzyme, a
2-keto-3-deoxygluconate-6-phosphate aldolase enzyme and a xylose
isomerase enzyme.
[0793] M2. The composition of embodiment M1, wherein the yeast is a
Saccharomyces spp. yeast.
[0794] M3. The composition of embodiment M2, wherein the yeast is a
Saccharomyces cerevisiae yeast strain.
[0795] M3.1. The composition of any one of embodiments M1 to M3,
wherein the polynucleotide subsequences encoding the
phosphogluconate dehydratase enzyme and the
3-deoxygluconate-6-phosphate aldolase enzyme independently are from
an Escherichia spp. microbe or Pseudomonas spp. microbe.
[0796] M4. The composition of embodiment M3.1, wherein the
Escherichia spp. microbe is an Escherichia coli strain.
[0797] M5. The composition of embodiment M3.1 or M4, wherein the
Pseudomonas spp. microbe is a Pseudomonas aeruginosa strain.
[0798] M6. The composition of any one of embodiments M1 to M5,
wherein the polynucleotide subsequence that encodes the
phosphogluconate dehydratase enzyme is an EDD gene.
[0799] M7. The composition of any one of embodiments M1 to M5,
wherein the polynucleotide subsequence that encodes the
2-keto-3-deoxygluconate-6-phosphate aldolase enzyme is an EDA
gene.
[0800] M8. The composition of any one of embodiments M1 to M7,
wherein the xylose isomerase enzyme is a chimeric enzyme.
[0801] M8.1. The composition of embodiment M8, wherein a first
portion of the polynucleotide subsequence that encodes the chimeric
xylose isomerase enzyme is from a first microbe and a second
portion of the polynucleotide subsequence that encodes the chimeric
xylose isomerase enzyme is from a second microbe.
[0802] M8.2. The composition of embodiment M8 or M8.1, wherein the
first microbe, the second microbe, or the first microbe and the
second microbe independently are selected from one or more of the
group consisting of Clostridiales spp., Ruminococcus spp., Thermus
spp., Bacillus spp., Clostridium spp., Orpinomyces spp.,
Escherichia spp. and Piromyces spp. microbes.
[0803] M8.3. The composition of embodiment M8.2, wherein the first
microbe, the second microbe, or the first microbe and the second
microbe independently are selected from one or more of the group
consisting of Clostridiales genomosp. BVAB3 str UPI19-5,
Ruminococcus flavefaciens, Ruminococcus FD1, Ruminococcus 18P13,
Thermus thermophilus, Bacillus stercoris, Clostridium
cellulolyticum, Bacillus uniformis, Bacillus stearothermophilus,
Bacteroides thetaiotaomicron, Clostridium thermohydrosulfuricum,
Orpinomyces, Clostridium phytofermentans, Escherichia coli and
Piromyces strain E2.
[0804] M8.4. The composition of any one of embodiments M1 to M7,
wherein 80% or more of the polynucleotide subsequence that encodes
the xylose isomerase enzyme is from a Ruminococcus spp. microbe
xylose isomerase-encoding sequence.
[0805] M9. The composition of embodiment M8.4, wherein all of the
polynucleotide subsequence that encodes the xylose isomerase enzyme
is from a Ruminococcus spp. microbe xylose isomerase-encoding
sequence.
[0806] M10. The composition of any one of embodiments M8.4 to M9,
wherein the Ruminococcus spp. microbe is a Ruminococcus
flavefaciens strain.
[0807] M11. The composition of any one of embodiments M8.4 to M10,
wherein the polynucleotide subsequence that encodes the xylose
isomerase enzyme is chimeric and includes a sequence that encodes a
xylose isomerase from another microbe.
[0808] M12. The composition of embodiment M11, wherein the other
microbe is a fungus.
[0809] M13. The composition of embodiment M12, wherein the fungus
is an anaerobic fungus.
[0810] M14. The composition of embodiment M12, wherein the fungus
is a Piromyces spp. fungus.
[0811] M15. The composition of embodiment M14, wherein the
Piromyces spp. fungus is a Piromyces strain E2.
[0812] M16. The composition of any one of embodiments M1 to M15,
wherein the yeast expresses a glucose-6-phosphate dehydrogenase
enzyme, a glucose-6-phosphate dehydrogenase enzyme, or a
glucose-6-phosphate dehydrogenase enzyme and a glucose-6-phosphate
dehydrogenase enzyme.
[0813] M17. The composition of embodiment M16, wherein the
polynucleotide subsequences that encode the glucose-6-phosphate
dehydrogenase enzyme, the glucose-6-phosphate dehydrogenase enzyme,
or the glucose-6-phosphate dehydrogenase enzyme and the
glucose-6-phosphate dehydrogenase enzyme are from a yeast.
[0814] M18. The composition of embodiment M17, wherein the yeast
from which the polynucleotide subsequence or subsequences are
derived is a Saccharomyces spp. yeast.
[0815] M19. The composition of embodiment 18, wherein the yeast is
a Saccharomyces cerevisiae strain.
[0816] M20. The composition of any one of embodiments M16 to M19,
wherein the yeast over-expresses an endogenous glucose-6-phosphate
dehydrogenase enzyme, an endogenous glucose-6-phosphate
dehydrogenase enzyme, or an endogenous glucose-6-phosphate
dehydrogenase enzyme and an endogenous glucose-6-phosphate
dehydrogenase enzyme.
[0817] M21. The composition of any one of embodiments M16 to M20,
wherein the glucose-6-phosphate dehydrogenase enzyme is expressed
from a ZWF gene.
[0818] M22. The composition of embodiment M21, wherein the ZWF gene
is a ZWF1 gene.
[0819] M23. The composition of any one of embodiments M16 to M22,
wherein the glucose-6-phosphate dehydrogenase enzyme is expressed
from a SOL gene.
[0820] M24. The composition of embodiment M23, wherein the SOL gene
is a SOL3 gene.
[0821] M25. The composition of any one of embodiments M1 to M25,
wherein the yeast includes a polynucleotide subsequence that
encodes a glucose transporter.
[0822] M26. The composition of embodiment M25, wherein the
polynucleotide subsequence that encodes the glucose transporter is
from a yeast.
[0823] M27. The composition of embodiment M25 or M26, wherein the
yeast over-expresses one or more endogenous glucose transport
enzymes.
[0824] M28. The composition of any one of embodiments M25 to M27,
wherein the glucose transporter is encoded by a one or more of a
GAL2, GSX1 and GXF1 gene.
[0825] M29. The composition of any one of embodiments M1 to M28,
wherein the yeast includes a genetic alteration that reduces the
activity of an endogenous phosphofructokinase (PFK) enzyme
activity.
[0826] M29.1. The composition of embodiment M29, wherein the PFK
enzyme is a PFK-2 enzyme.
[0827] M30. The composition of any one of embodiments M1 to M29.1,
wherein the yeast includes one or more extra copies of an
endogenous promoter, or a heterologous promoter operable in a
yeast, wherein the promoter is in operable connection with one or
more of the polynucleotide subsequences.
[0828] M31. The composition of embodiment M30, wherein the promoter
is selected from promoters that regulate glucose phosphate
dehydrogenase (GPD), translation elongation factor (TEF-1),
phosphoglucokinase (PGK-1) and triose phosphate dehydrogenase
(TDH-1).
[0829] M32. The composition of any one of embodiments M1 to M31,
wherein the polynucleotide subsequences, the promoters, or the
polynucleotide subsequences and the promoters are not integrated in
the yeast nucleic acid.
[0830] M33. The composition of embodiment M32, wherein the
polynucleotide subsequences, the promoters, or the polynucleotide
subsequences and the promoters are in one or more plasmids.
[0831] M34. The composition of any one of embodiments M1 to M31,
wherein the polynucleotide subsequences, the promoters, or the
polynucleotide subsequences and the promoters are integrated in
genomic DNA of the yeast.
[0832] M35. The composition of embodiment M34, wherein the
polynucleotide subsequences, the promoters, or the polynucleotide
subsequences and the promoters are integrated in a transposition
integration event, in a homologous recombination integration event,
or in a transposition integration event and a homologous
recombination integration event.
[0833] M36. The composition of embodiment M35, wherein the
transposition integration event includes transposition of an operon
comprising two or more of the polynucleotide subsequences, the
promoters, or the polynucleotide subsequences and the
promoters.
[0834] M37. The composition of embodiment 34, wherein the
homologous recombination integration event includes homologous
recombination of an operon comprising two or more of the
polynucleotide subsequences, the promoters, or the polynucleotide
subsequences and the promoters.
[0835] N1. A method, comprising contacting an engineered yeast of
any one of embodiments M1 to M37 with a feedstock that contains one
or more hexose sugars and one or more pentose sugars under
conditions in which the microbe synthesizes ethanol.
[0836] N2. The method of embodiment N1, wherein the engineered
yeast synthesizes ethanol to about 85% to about 99% of theoretical
yield.
[0837] N3. The method of embodiment N1 or N2, comprising recovering
ethanol synthesized by the engineered yeast.
[0838] N4. The method of any one of embodiments, N1 to N3, wherein
the conditions are fermentation conditions.
[0839] O1. A composition comprising a nucleic acid comprising
heterologous polynucleotides that encode a phosphogluconate
dehydratase enzyme, a 2-keto-3-deoxygluconate-6-phosphate aldolase
enzyme and a xylose isomerase enzyme.
[0840] O2. The composition of embodiment O1, wherein the yeast is a
Saccharomyces spp. yeast.
[0841] O3. The composition of embodiment O2, wherein the yeast is a
Saccharomyces cerevisiae yeast strain.
[0842] O3.1. The composition of any one of embodiments O1 to O3,
wherein the polynucleotides encoding the phosphogluconate
dehydratase enzyme and the 3-deoxygluconate-6-phosphate aldolase
enzyme independently are from an Escherichia spp. microbe or
Pseudomonas spp. microbe.
[0843] O4. The composition of embodiment O3, wherein the
Escherichia spp. microbe is an Escherichia coli strain.
[0844] O5. The composition of embodiment O3 or O4, wherein the
Pseudomonas spp. microbe is a Pseudomonas aeruginosa strain.
[0845] O6. The composition of any one of embodiments O1 to O5,
wherein the polynucleotide that encodes the phosphogluconate
dehydratase enzyme is an EDD gene.
[0846] O7. The composition of any one of embodiments O1 to O5,
wherein the polynucleotide that encodes the
2-keto-3-deoxygluconate-6-phosphate aldolase enzyme is an EDA
gene.
[0847] O8. The composition of any one of embodiments O1 to O7,
wherein the xylose isomerase enzyme is a chimeric enzyme.
[0848] O8.1. The composition of embodiment O8, wherein a first
portion of the polynucleotide that encodes the chimeric xylose
isomerase enzyme is from a first microbe and a second portion of
the polynucleotide that encodes the chimeric xylose isomerase
enzyme is from a second microbe.
[0849] O8.2. The composition of embodiment O8 or O8.1, wherein the
first microbe, the second microbe, or the first microbe and the
second microbe independently are selected from one or more of the
group consisting of Clostridiales spp., Ruminococcus spp., Thermus
spp., Bacillus spp., Clostridium spp., Orpinomyces spp.,
Escherichia spp. and Piromyces spp. microbes.
[0850] O8.3. The composition of embodiment O8.2, wherein the first
microbe, the second microbe, or the first microbe and the second
microbe independently are selected from one or more of the group
consisting of Clostridiales genomosp. BVAB3 str UPI19-5,
Ruminococcus flavefaciens, Ruminococcus FD1, Ruminococcus 18P13,
Thermus thermophilus, Bacillus stercoris, Clostridium
cellulolyticum, Bacillus uniformis, Bacillus stearothermophilus,
Bacteroides thetaiotaomicron, Clostridium thermohydrosulfuricum,
Orpinomyces, Clostridium phytofermentans, Escherichia coli and
Piromyces strain E2.
[0851] O8.4. The composition of any one of embodiments O1 to O7,
wherein 80% or more of the polynucleotide that encodes the xylose
isomerase enzyme is from a Ruminococcus spp. microbe xylose
isomerase-encoding sequence.
[0852] O9. The composition of embodiment O8.4, wherein all or a
portion of the polynucleotide that encodes the xylose isomerase
enzyme is from a Ruminococcus spp. microbe xylose
isomerase-encoding sequence.
[0853] O10. The composition of any one of embodiments O8.4 to O9,
wherein the Ruminococcus spp. microbe is a Ruminococcus
flavefaciens strain.
[0854] O11. The composition of any one of embodiments O8.4 to O10,
wherein the polynucleotide that encodes the xylose isomerase enzyme
is chimeric and includes a sequence that encodes a xylose isomerase
from another microbe.
[0855] O11.1. The composition of any one of embodiments O8.4 to
O11, wherein the portion of the polynucleotide from the
Ruminococcus spp. microbe xylose isomerase is 3' with respect to
the portion of the polynucleotide from another microbe.
[0856] O12. The composition of embodiment O11 or O11.1, wherein the
other microbe is a fungus.
[0857] O13. The composition of embodiment O12, wherein the fungus
is an anaerobic fungus.
[0858] O14. The composition of embodiment O12, wherein the fungus
is a Piromyces spp. fungus.
[0859] O15. The composition of embodiment O14, wherein the
Piromyces spp. fungus is a Piromyces strain E2.
[0860] O16. The composition of any one of embodiments O1 to O15,
wherein the nucleic acid includes one or more polynucleotides that
encode a glucose-6-phosphate dehydrogenase enzyme, a
6-phosphogluconolactonase enzyme, or a glucose-6-phosphate
dehydrogenase enzyme and a 6-phosphogluconolactonase enzyme.
[0861] O17. The composition of embodiment O16, wherein the one or
more polynucleotides that encode the glucose-6-phosphate
dehydrogenase enzyme, the 6-phosphogluconolactonase enzyme, or the
glucose-6-phosphate dehydrogenase enzyme and the
6-phosphogluconolactonase enzyme are from a yeast.
[0862] O18. The composition of embodiment O17, wherein the yeast
from which the polynucleotide or polynucleotides are derived is a
Saccharomyces spp. yeast.
[0863] O19. The composition of embodiment O18, wherein the yeast is
a Saccharomyces cerevisiae strain.
[0864] O20. The composition of any one of embodiments O16 to O19,
wherein the nucleic acid includes one or more polynucleotides that
encode an endogenous glucose-6-phosphate dehydrogenase enzyme, an
endogenous 6-phosphogluconolactonase enzyme, or an endogenous
glucose-6-phosphate dehydrogenase enzyme and an endogenous
6-phosphogluconolactonase enzyme.
[0865] O21. The composition of any one of embodiments O16 to O20,
wherein the glucose-6-phosphate dehydrogenase enzyme is expressed
from a ZWF gene.
[0866] O22. The composition of embodiment O21, wherein the ZWF gene
is a ZWF1 gene.
[0867] O23. The composition of any one of embodiments O16 to O22,
wherein the 6-phosphogluconolactonase enzyme is expressed from a
SOL gene.
[0868] O24. The composition of embodiment O23, wherein the SOL gene
is a SOL3 gene.
[0869] O25. The composition of any one of embodiments O1 to O24,
wherein the nucleic acid includes one or more polynucleotides that
encode one or more glucose transporters.
[0870] O26. The composition of embodiment O25, wherein the
polynucleotide that encodes the one or more glucose transporters is
from a yeast.
[0871] O27. The composition of embodiment O25 or O26, wherein the
one or more glucose transporters is encoded by a one or more of a
GAL2, GSX1 and GXF1 gene.
[0872] O28. The composition of any one of embodiments O1 to O27,
wherein the nucleic acid includes one or more polynucleotides that
encode a transketolase enzyme, transaldolase enzyme, or a
transketolase enzyme and transaldolase enzyme.
[0873] O29. The composition of embodiment O28, wherein the
transketolase enzyme is encoded by a TKL1 coding sequence or a TKL2
coding sequence.
[0874] O30. The composition of embodiment O28, wherein the
transaldolase is encoded by a TAL coding sequence.
[0875] O31. The composition of any one of embodiments O28 to O30,
wherein the transketolase enzyme or the transaldolase enzyme is
from a yeast.
[0876] O32. The composition of any one of embodiments O1 to O31,
wherein the nucleic acid includes one or more promoters operable in
a yeast, wherein the promoter is in operable connection with one or
more of the polynucleotides.
[0877] O33. The composition of embodiment O32, wherein the promoter
is selected from promoters that regulate glucose phosphate
dehydrogenase (GPD), translation elongation factor (TEF-1),
phosphoglucokinase (PGK-1) and triose phosphate dehydrogenase
(TDH-1).
[0878] O34. The composition of any one of embodiments O1 to O33,
wherein the nucleic acid includes one or more polynucleotides that
homologously combine in a gene of a host that encodes a
phosphofructokinase (PFK) enzyme, phosphoglucoisomerase (PGI)
enzyme or 6-phosphogluconate dehydrogenase (decarboxylating)
enzyme. 035. The composition of embodiment O34, wherein the
phosphofructokinase (PFK) enzyme is a PFK-2 enzyme or PFK-1
enzyme.
[0879] O35.1. The composition of embodiment O34, wherein the
6-phosphogluconate dehydrogenase (decarboxylating) enzyme is
encoded by a GND-1 gene or a GND-2 gene.
[0880] O36. The composition of embodiment O34, wherein the PGI is
encoded by a PGI-1 gene.
[0881] O37. The composition of any one of embodiments O1 to O36,
wherein the nucleic acid is one or two separate nucleic acid
molecules.
[0882] O38. The composition of embodiment O37, wherein each nucleic
acid molecule includes one or two or more of the polynucleotide
subsequences, one or two or more of the promoters, or one or two or
more of the polynucleotide subsequences and one or two or more of
the promoters.
[0883] O39. The composition of embodiment O37 or O38, wherein each
of the one or two nucleic acid molecules are in circular form.
[0884] O40. The composition of embodiment O37 or O38, wherein each
of the one or two nucleic acid molecules are in linear form.
[0885] O41. The composition of any one of embodiments O37 to O40,
wherein each of the one or two nucleic acid molecules functions as
an expression vector.
[0886] O42. The composition of any one of embodiments O37 to O41,
wherein each of the one or two nucleic acid molecules includes
flanking sequences for integrating the polynucleotides, the
promoter sequences, or the polynucleotides and the promoter
sequences in the nucleic acid into genomic DNA of a host
organism.
[0887] P1. The composition comprising an engineered yeast that
includes an alteration that adds or increases a phosphogluconate
dehydratase activity, a 2-keto-3-deoxygluconate-6-phosphate
aldolase activity and a xylose isomerase activity.
[0888] P2. The composition of embodiment P1, wherein the yeast is a
Saccharomyces spp. yeast.
[0889] P3. The composition of embodiment P2, wherein the yeast is a
Saccharomyces cerevisiae yeast strain.
[0890] P4. The composition of any one of embodiments P1 to P3 that
includes heterologous polynucleotides that encode a
phosphogluconate dehydratase enzyme, a
2-keto-3-deoxygluconate-6-phosphate aldolase enzyme and a xylose
isomerase enzyme.
[0891] P5. The composition of embodiment P4, wherein the
polynucleotides encoding the phosphogluconate dehydratase enzyme
and the 3-deoxygluconate-6-phosphate aldolase enzyme independently
are from an Escherichia spp. microbe or Pseudomonas spp.
microbe.
[0892] P6. The composition of embodiment P5, wherein the
Escherichia spp. microbe is an Escherichia coli strain.
[0893] P7. The composition of embodiment P5, wherein the
Pseudomonas spp. microbe is a Pseudomonas aeruginosa strain.
[0894] P8. The composition of any one of embodiments P4 to P7,
wherein the polynucleotide that encodes the phosphogluconate
dehydratase enzyme is an EDD gene.
[0895] P9. The composition of any one of embodiments P4 to P7,
wherein the polynucleotide that encodes the
2-keto-3-deoxygluconate-6-phosphate aldolase enzyme is an EDA
gene.
[0896] P10. The composition of any one of embodiments P1 to P9,
wherein the xylose isomerase enzyme is a chimeric enzyme.
[0897] P11. The composition of embodiment P10, wherein a first
portion of the polynucleotide that encodes the chimeric xylose
isomerase enzyme is from a first microbe and a second portion of
the polynucleotide that encodes the chimeric xylose isomerase
enzyme is from a second microbe.
[0898] P12. The composition of embodiment P10 or P11, wherein the
first microbe, the second microbe, or the first microbe and the
second microbe independently are selected from one or more of the
group consisting of Clostridiales spp., Ruminococcus spp., Thermus
spp., Bacillus spp., Clostridium spp., Orpinomyces spp.,
Escherichia spp. and Piromyces spp. microbes.
[0899] P13. The composition of embodiment P12, wherein the first
microbe, the second microbe, or the first microbe and the second
microbe independently are selected from one or more of the group
consisting of Clostridiales genomosp. BVAB3 str UPI19-5,
Ruminococcus flavefaciens, Ruminococcus FD1, Ruminococcus 18P13,
Thermus thermophilus, Bacillus stercoris, Clostridium
cellulolyticum, Bacillus uniformis, Bacillus stearothermophilus,
Bacteroides thetaiotaomicron, Clostridium thermohydrosulfuricum,
Orpinomyces, Clostridium phytofermentans, Escherichia coli and
Piromyces strain E2.
[0900] P14. The composition of any one of embodiments P10 to P13,
wherein 80% or more of the polynucleotide that encodes the xylose
isomerase enzyme is from a Ruminococcus spp. microbe xylose
isomerase-encoding sequence.
[0901] P15. The composition of embodiment P14, wherein all or a
portion of the polynucleotide that encodes the xylose isomerase
enzyme is from a Ruminococcus spp. microbe xylose
isomerase-encoding sequence.
[0902] P16. The composition of embodiment P15, wherein the
Ruminococcus spp. microbe is a Ruminococcus flavefaciens
strain.
[0903] P17. The composition of any one of embodiments P10 to P16,
wherein the polynucleotide that encodes the xylose isomerase enzyme
is chimeric and includes a sequence that encodes a xylose isomerase
from another microbe.
[0904] P18. The composition of any one of embodiments P10 to P17,
wherein the portion of the polynucleotide from the Ruminococcus
spp. microbe xylose isomerase is 3' with respect to the portion of
the polynucleotide from another microbe.
[0905] P19. The composition of embodiment P17 or P18, wherein the
other microbe is a fungus.
[0906] P20. The composition of embodiment P19, wherein the fungus
is an anaerobic fungus.
[0907] P21. The composition of embodiment P20, wherein the fungus
is a Piromyces spp. fungus.
[0908] P22. The composition of embodiment P21, wherein the
Piromyces spp. fungus is a Piromyces strain E2.
[0909] P23. The composition of any one of embodiments P1 to P22,
wherein one or more of the following activities are added or
increased: a glucose-6-phosphate dehydrogenase activity, a
6-phosphogluconolactonase activity, or a glucose-6-phosphate
dehydrogenase activity and a 6-phosphogluconolactonase
activity.
[0910] P24. The composition of embodiment P24, wherein the yeast
comprises one or more heterologous polynucleotides that encode one
or more of the following enzymes, or wherein the yeast comprises
multiple copies of endogenous polynucleotides that encode one or
more of the following enzymes: glucose-6-phosphate dehydrogenase
enzyme, 6-phosphogluconolactonase enzyme, or glucose-6-phosphate
dehydrogenase enzyme and 6-phosphogluconolactonase enzyme.
[0911] P25. The composition of embodiment P24, wherein the one or
more polynucleotides that encode the glucose-6-phosphate
dehydrogenase enzyme, the 6-phosphogluconolactonase enzyme, or the
glucose-6-phosphate dehydrogenase enzyme and the
6-phosphogluconolactonase enzyme are from a yeast.
[0912] P26. The composition of embodiment P25, wherein the yeast is
a Saccharomyces spp. yeast.
[0913] P27. The composition of embodiment P26, wherein the yeast is
a Saccharomyces cerevisiae strain.
[0914] P28. The composition of any one of embodiments P24 to P27,
wherein the glucose-6-phosphate dehydrogenase enzyme is expressed
from a ZWF gene.
[0915] P29. The composition of embodiment P28, wherein the ZWF gene
is a ZWF1 gene.
[0916] P30. The composition of any one of embodiments P24 to P29,
wherein the 6-phosphogluconolactonase enzyme is expressed from a
SOL gene.
[0917] P31. The composition of embodiment P31, wherein the SOL gene
is a SOL3 gene.
[0918] P32. The composition of any one of embodiments P1 to P31,
wherein the nucleic acid includes one or more polynucleotides that
encode one or more glucose transporters.
[0919] P33. The composition of embodiment P32, wherein the
polynucleotide that encodes the one or more glucose transporters is
from a yeast.
[0920] P34. The composition of embodiment P32 or P33, wherein the
one or more glucose transporters is encoded by a one or more of a
GAL2, GSX1 and GXF1 gene.
[0921] P35. The composition of any one of embodiments P1 to P34,
wherein the yeast includes one or more added activities or
increased activities selected from the group consisting of
transketolase activity, transaldolase activity, or a transketolase
activity and transaldolase activity.
[0922] P36. The composition of embodiment P35, wherein the yeast
includes one or more heterologous polynucleotides that encodes one
or more of the following enzymes, or includes multiple copies of
polynucleotides that encode one or more of the following enzymes:
transketolase enzyme, transaldolase enzyme, or a transketolase
enzyme and transaldolase enzyme
[0923] P37. The composition of embodiment P36, wherein the
transketolase enzyme is encoded by a TKL1 coding sequence or a TKL2
coding sequence.
[0924] P38. The composition of embodiment P36, wherein the
transaldolase is encoded by a TAL coding sequence.
[0925] P39. The composition of any one of embodiments P36 to P38,
wherein the transketolase enzyme or the transaldolase enzyme is
from a yeast.
[0926] P40. The composition of any one of embodiments P1 to P39,
wherein the nucleic acid includes one or more promoters operable in
a yeast, wherein the promoter is in operable connection with one or
more of the polynucleotides.
[0927] P41. The composition of embodiment P40, wherein the promoter
is selected from promoters that regulate glucose phosphate
dehydrogenase (GPD), translation elongation factor (TEF-1),
phosphoglucokinase (PGK-1) and triose phosphate dehydrogenase
(TDH-1).
[0928] P42. The composition of any one of embodiments P1 to P41,
wherein the yeast includes a reduction in one or more of the
following activities: phosphofructokinase (PFK) activity,
phosphoglucoisomerase (PGI) activity, 6-phosphogluconate
dehydrogenase (decarboxylating) activity or combination
thereof.
[0929] P43. The composition of embodiment P42, wherein the yeast
includes an alteration in one or more polynucleotides that inhibits
production of one or more enzymes selected from the group
consisting of phosphofructokinase (PFK) enzyme,
phosphoglucoisomerase (PGI) enzyme, 6-phosphogluconate
dehydrogenase (decarboxylating) enzyme or combination thereof.
[0930] P44. The composition of embodiment P43, wherein the
phosphofructokinase (PFK) enzyme is a PFK-2 enzyme or PFK-1
enzyme.
[0931] P44.1. The composition of embodiment P43, wherein the
6-phosphogluconate dehydrogenase (decarboxylating) enzyme is
encoded by a GND-1 gene or GND-2 gene.
[0932] P45. The composition of embodiment P43, wherein the PGI is
encoded by a PGI-1 gene.
[0933] P46. The composition of any one of embodiments P1 to P45,
wherein the polynucleotides, the promoters, or the polynucleotides
and the promoters are not integrated in the yeast nucleic acid.
[0934] P47. The composition of embodiment P46, wherein the
polynucleotides, the promoters, or the polynucleotides and the
promoters are in one or more plasmids.
[0935] P48. The composition of any one of embodiments P1 to P47,
wherein the polynucleotide subsequences, the promoters, or the
polynucleotide subsequences and the promoters are integrated in
genomic DNA of the yeast.
[0936] P49. The composition of embodiment P48, wherein the
polynucleotides, the promoters, or the polynucleotides and the
promoters are integrated in a transposition integration event, in a
homologous recombination integration event, or in a transposition
integration event and a homologous recombination integration
event.
[0937] P50. The composition of embodiment P49, wherein the
transposition integration event includes transposition of an operon
comprising two or more of the polynucleotide subsequences, the
promoters, or the polynucleotide subsequences and the
promoters.
[0938] P51. The composition of embodiment P49, wherein the
homologous recombination integration event includes homologous
recombination of an operon comprising two or more of the
polynucleotide subsequences, the promoters, or the polynucleotide
subsequences and the promoters.
[0939] Q1. A method, comprising contacting an engineered yeast of
any one of embodiments P1 to P51 with a feedstock that contains one
or more hexose sugars and one or more pentose sugars under
conditions in which the microbe synthesizes ethanol.
[0940] Q2. The method of embodiment Q1, wherein the engineered
yeast synthesizes ethanol to about 85% to about 99% of theoretical
yield.
[0941] Q3. The method of embodiment Q1 or Q2, comprising recovering
ethanol synthesized by the engineered yeast.
[0942] Q4. The method of any one of embodiments Q1 to Q3, wherein
the conditions are fermentation conditions.
[0943] R1. A composition comprising a nucleic acid comprising
heterologous polynucleotides that encode a phosphogluconate
dehydratase enzyme, a 2-keto-3-deoxygluconate-6-phosphate aldolase
enzyme and a 6-phosphogluconolactonase enzyme.
[0944] R2. The composition of embodiment R1, wherein the yeast is a
Saccharomyces spp. yeast.
[0945] R3. The composition of embodiment R2, wherein the yeast is a
Saccharomyces cerevisiae yeast strain.
[0946] R3.1. The composition of any one of embodiments R1 to R3,
wherein the polynucleotides encoding the phosphogluconate
dehydratase enzyme and the 3-deoxygluconate-6-phosphate aldolase
enzyme independently are from an Escherichia spp. microbe or
Pseudomonas spp. microbe.
[0947] R4. The composition of embodiment R3, wherein the
Escherichia spp. microbe is an Escherichia coli strain.
[0948] R5. The composition of embodiment R3 or R4, wherein the
Pseudomonas spp. microbe is a Pseudomonas aeruginosa strain.
[0949] R6. The composition of any one of embodiments R1 to R5,
wherein the polynucleotide that encodes the phosphogluconate
dehydratase enzyme is an EDD gene.
[0950] R7. The composition of any one of embodiments R1 to R5,
wherein the polynucleotide that encodes the
2-keto-3-deoxygluconate-6-phosphate aldolase enzyme is an EDA
gene.
[0951] R8. The composition of any one of embodiments R1 to R7,
wherein the 6-phosphogluconolactonase enzyme is expressed from a
SOL gene.
[0952] R9. The composition of embodiment R8, wherein the SOL gene
is a SOL3 gene.
[0953] R10. The composition of any one of embodiments R1 to R9,
wherein the nucleic acid includes a polynucleotide that encodes a
glucose-6-phosphate dehydrogenase enzyme.
[0954] R11. The composition of embodiment R10, wherein the
polynucleotide that encodes the glucose-6-phosphate dehydrogenase
enzyme is from a yeast.
[0955] R12. The composition of embodiment R11, wherein the yeast is
a Saccharomyces spp. yeast.
[0956] R13. The composition of embodiment R12, wherein the yeast is
a Saccharomyces cerevisiae strain.
[0957] R14. The composition of any one of embodiments R10 to R13,
wherein the nucleic acid includes a polynucleotide that encode an
endogenous glucose-6-phosphate dehydrogenase enzyme.
[0958] R15. The composition of any one of embodiments R10 to R14,
wherein the glucose-6-phosphate dehydrogenase enzyme is expressed
from a ZWF gene.
[0959] R16. The composition of embodiment R15, wherein the ZWF gene
is a ZWF1 gene.
[0960] R17. The composition of any one of embodiments R1 to R16,
wherein the nucleic acid includes one or more promoters operable in
a yeast, wherein the promoter is in operable connection with one or
more of the polynucleotides.
[0961] R18. The composition of embodiment R17, wherein the promoter
is selected from promoters that regulate glucose phosphate
dehydrogenase (GPD), translation elongation factor (TEF-1),
phosphoglucokinase (PGK-1) and triose phosphate dehydrogenase
(TDH-1).
[0962] R19. The composition of any one of embodiments R1 to R18,
wherein the nucleic acid includes one or more polynucleotides that
homologously combine in a gene of a host that encodes a
phosphofructokinase (PFK) enzyme, phosphoglucoisomerase (PGI)
enzyme, 6-phosphogluconate dehydrogenase (decarboxylating) enzyme,
transketolase enzyme, transaldolase enzyme, or combination
thereof.
[0963] R20. The composition of embodiment R19, wherein the
transketolase enzyme is encoded by a TKL-1 coding sequence or a
TKL-2 coding sequence.
[0964] R21. The composition of embodiment R19, wherein the
transaldolase is encoded by a TAL-1 coding sequence.
[0965] R22. The composition of embodiment R19, wherein the
phosphofructokinase (PFK) enzyme is a PFK-2 enzyme or PFK-1
enzyme.
[0966] R23. The composition of embodiment R19, wherein the
6-phosphogluconate dehydrogenase (decarboxylating) enzyme is
encoded by a GND-1 gene or a GND-2 gene.
[0967] R24. The composition of embodiment R19, wherein the PGI is
encoded by a PGI-1 gene.
[0968] R25. The composition of any one of embodiments R1 to R24,
wherein the nucleic acid is one or two separate nucleic acid
molecules.
[0969] R26. The composition of embodiment R25, wherein each nucleic
acid molecule includes one or two or more of the polynucleotide
subsequences, one or two or more of the promoters, or one or two or
more of the polynucleotide subsequences and one or two or more of
the promoters.
[0970] R27. The composition of embodiment R25 or R26, wherein each
of the one or two nucleic acid molecules are in circular form.
[0971] R28. The composition of embodiment R25 or R26, wherein each
of the one or two nucleic acid molecules are in linear form.
[0972] R29. The composition of any one of embodiments R25 to R28,
wherein each of the one or two nucleic acid molecules functions as
an expression vector.
[0973] R30. The composition of any one of embodiments R25 to R29,
wherein each of the one or two nucleic acid molecules includes
flanking sequences for integrating the polynucleotides, the
promoter sequences, or the polynucleotides and the promoter
sequences in the nucleic acid into genomic DNA of a host
organism.
[0974] S1. A composition comprising an engineered yeast that
includes an alteration that adds or increases a phosphogluconate
dehydratase activity, a 2-keto-3-deoxygluconate-6-phosphate
aldolase activity and a 6-phosphogluconolactonase activity.
[0975] S2. The composition of embodiment S1, wherein the yeast is a
Saccharomyces spp. yeast.
[0976] S3. The composition of embodiment S2, wherein the yeast is a
Saccharomyces cerevisiae yeast strain.
[0977] S4. The composition of any one of embodiments S1 to S3 that
includes heterologous polynucleotides that encode a
phosphogluconate dehydratase enzyme, a
2-keto-3-deoxygluconate-6-phosphate aldolase enzyme and a
6-phosphogluconolactonase enzyme.
[0978] S5. The composition of embodiment S4, wherein the
polynucleotides encoding the phosphogluconate dehydratase enzyme
and the 3-deoxygluconate-6-phosphate aldolase enzyme independently
are from an Escherichia spp. microbe or Pseudomonas spp.
microbe.
[0979] S6. The composition of embodiment S5, wherein the
Escherichia spp. microbe is an Escherichia coli strain.
[0980] S7. The composition of embodiment S5, wherein the
Pseudomonas spp. microbe is a Pseudomonas aeruginosa strain.
[0981] S8. The composition of any one of embodiments S4 to S7,
wherein the polynucleotide that encodes the phosphogluconate
dehydratase enzyme is an EDD gene.
[0982] S9. The composition of any one of embodiments S4 to S7,
wherein the polynucleotide that encodes the
2-keto-3-deoxygluconate-6-phosphate aldolase enzyme is an EDA
gene.
[0983] S10. The composition of any one of embodiments S4 to S9,
wherein the 6-phosphogluconolactonase enzyme is expressed from a
SOL gene.
[0984] S11. The composition of embodiment S10, wherein the SOL gene
is a SOL3 gene.
[0985] S12. The composition of any one of embodiments S4 to S11,
wherein a glucose-6-phosphate dehydrogenase activity is added or
increased.
[0986] S13. The composition of embodiment S12, wherein the yeast
comprises a heterologous polynucleotide that encodes a
glucose-6-phosphate dehydrogenase enzyme, or wherein the yeast
comprises multiple copies of an endogenous polynucleotide that
encodes a glucose-6-phosphate dehydrogenase enzyme.
[0987] S14. The composition of embodiment S13, wherein the
polynucleotide that encodes the glucose-6-phosphate dehydrogenase
enzyme is from a yeast.
[0988] S15. The composition of embodiment S14, wherein the yeast is
a Saccharomyces spp. yeast.
[0989] S16. The composition of embodiment S15, wherein the yeast is
a Saccharomyces cerevisiae strain.
[0990] S17. The composition of any one of embodiments S13 to S17,
wherein the glucose-6-phosphate dehydrogenase enzyme is expressed
from a ZWF gene.
[0991] S18. The composition of embodiment S17, wherein the ZWF gene
is a ZWF1 gene.
[0992] S19. The composition of any one of embodiments S1 to S18,
wherein the nucleic acid includes one or more promoters operable in
a yeast, wherein the promoter is in operable connection with one or
more of the polynucleotides.
[0993] S20. The composition of embodiment S19, wherein the promoter
is selected from promoters that regulate glucose phosphate
dehydrogenase (GSD), translation elongation factor (TEF-1),
phosphoglucokinase (SGK-1) and triose phosphate dehydrogenase
(TDH-1).
[0994] S21. The composition of any one of embodiments S1 to S20,
wherein the yeast includes a reduction in one or more of the
following activities: phosphofructokinase (PFK) activity,
phosphoglucoisomerase (PGI) activity, 6-phosphogluconate
dehydrogenase (decarboxylating) activity, transketolase activity,
transaldolase activity, or combination thereof.
[0995] S22. The composition of embodiment S21, wherein the yeast
includes an alteration in one or more polynucleotides that inhibits
production of one or more enzymes selected from the group
consisting of phosphofructokinase (PFK) enzyme,
phosphoglucoisomerase (PGI) enzyme, 6-phosphogluconate
dehydrogenase (decarboxylating) enzyme, transketolase enzyme,
transaldolase enzyme, or combination thereof.
[0996] S23. The composition of embodiment S22, wherein the
transketolase enzyme is encoded by a TKL-1 coding sequence or a
TKL-2 coding sequence.
[0997] S24. The composition of embodiment S22, wherein the
transaldolase is encoded by a TAL-1 coding sequence.
[0998] S25. The composition of embodiment S22, wherein the
phosphofructokinase (PFK) enzyme is a PFK-2 enzyme or PFK-1
enzyme.
[0999] S26. The composition of embodiment S22, wherein the
6-phosphogluconate dehydrogenase (decarboxylating) enzyme is
encoded by a GND-1 gene or GND-2 gene.
[1000] S27. The composition of embodiment S22, wherein the PGI is
encoded by a PGI-1 gene.
[1001] S28. The composition of any one of embodiments S1 to S27,
wherein the polynucleotides, the promoters, or the polynucleotides
and the promoters are not integrated in the yeast nucleic acid.
[1002] S29. The composition of embodiment S28, wherein the
polynucleotides, the promoters, or the polynucleotides and the
promoters are in one or more plasmids.
[1003] S30. The composition of any one of embodiments S1 to S29,
wherein the polynucleotide subsequences, the promoters, or the
polynucleotide subsequences and the promoters are integrated in
genomic DNA of the yeast.
[1004] S31. The composition of embodiment S30, wherein the
polynucleotides, the promoters, or the polynucleotides and the
promoters are integrated in a transposition integration event, in a
homologous recombination integration event, or in a transposition
integration event and a homologous recombination integration
event.
[1005] S32. The composition of embodiment S31, wherein the
transposition integration event includes transposition of an operon
comprising two or more of the polynucleotide subsequences, the
promoters, or the polynucleotide subsequences and the
promoters.
[1006] S33. The composition of embodiment S31, wherein the
homologous recombination integration event includes homologous
recombination of an operon comprising two or more of the
polynucleotide subsequences, the promoters, or the polynucleotide
subsequences and the promoters.
[1007] T1. A method, comprising contacting an engineered yeast of
any one of embodiments S1 to S33 with a feedstock that contains one
or more hexose sugars under conditions in which the microbe
synthesizes ethanol.
[1008] T2. The method of embodiment T1, wherein the engineered
yeast synthesizes ethanol to about 85% to about 99% of theoretical
yield.
[1009] T3. The method of embodiment T1 or T2, comprising recovering
ethanol synthesized by the engineered yeast.
[1010] T4. The method of any one of embodiments T1 to T3, wherein
the conditions are fermentation conditions.
[1011] U1. A composition comprising an engineered yeast that
includes an alteration that adds or increases a xylose isomerase
activity and a glucose transporter activity.
[1012] U2. The composition of embodiment U1, wherein the yeast is a
Saccharomyces spp. yeast.
[1013] U3. The composition of embodiment U2, wherein the yeast is a
Saccharomyces cerevisiae yeast strain.
[1014] U4. The composition of any one of embodiments U1 to U3 that
includes heterologous polynucleotides that encode a xylose
isomerase enzyme and a glucose transport enzyme.
[1015] U5. The composition of any one of embodiments U1 to U4,
wherein the xylose isomerase enzyme is a chimeric enzyme.
[1016] U6. The composition of embodiment U5, wherein a first
portion of the polynucleotide that encodes the chimeric xylose
isomerase enzyme is from a first microbe and a second portion of
the polynucleotide that encodes the chimeric xylose isomerase
enzyme is from a second microbe.
[1017] U7. The composition of embodiment U5 or U6, wherein the
first microbe, the second microbe, or the first microbe and the
second microbe independently are selected from one or more of the
group consisting of Clostridiales spp., Ruminococcus spp., Thermus
spp., Bacillus spp., Clostridium spp., Orpinomyces spp.,
Escherichia spp. and Piromyces spp. microbes.
[1018] U8. The composition of embodiment U7, wherein the first
microbe, the second microbe, or the first microbe and the second
microbe independently are selected from one or more of the group
consisting of Clostridiales_genomosp. BUAB3 str UUII9-5,
Ruminococcus flavefaciens, Ruminococcus_FD1, Ruminococcus.sub.--18
U13, Thermus thermophilus, Bacillus stercoris, Clostridium
cellulolyticum, Bacillus uniformis, Bacillus stearothermophilus,
Bacteroides thetaiotaomicron, Clostridium thermohydrosulfuricum,
Orpinomyces, Clostridium phytofermentans, Escherichia coli and
Piromyces strain E2.
[1019] U9. The composition of any one of embodiments U5 to U8,
wherein 80% or more of the polynucleotide that encodes the xylose
isomerase enzyme is from a Ruminococcus spp. microbe xylose
isomerase-encoding sequence.
[1020] U10. The composition of embodiment U9, wherein all or a
portion of the polynucleotide that encodes the xylose isomerase
enzyme is from a Ruminococcus spp. microbe xylose
isomerase-encoding sequence.
[1021] U11. The composition of embodiment U10, wherein the
Ruminococcus spp. microbe is a Ruminococcus flavefaciens
strain.
[1022] U12. The composition of any one of embodiments U5 to U12,
wherein the polynucleotide that encodes the xylose isomerase enzyme
is chimeric and includes a sequence that encodes a xylose isomerase
from another microbe.
[1023] U13. The composition of any one of embodiments U5 to U12,
wherein the portion of the polynucleotide from the Ruminococcus
spp. microbe xylose isomerase is 3' with respect to the portion of
the polynucleotide from another microbe.
[1024] U14. The composition of embodiment U12 or U13, wherein the
other microbe is a fungus.
[1025] U15. The composition of embodiment U14, wherein the fungus
is an anaerobic fungus.
[1026] U16. The composition of embodiment U15, wherein the fungus
is a Piromyces spp. fungus.
[1027] U17. The composition of embodiment U16, wherein the
Piromyces spp. fungus is a Piromyces strain E2.
[1028] U18. The composition of any one of embodiments U4 to U17,
wherein the polynucleotide that encodes the one or more glucose
transporters is from a yeast.
[1029] U19. The composition of embodiment U18, wherein the one or
more glucose transporters is encoded by a one or more of a GAL2,
GSX1 and GXF1 gene.
[1030] U20. The composition of any one of embodiments U1 to U19,
wherein the yeast includes one or more added activities or
increased activities selected from the group consisting of
transketolase activity, transaldolase activity, or a transketolase
activity and transaldolase activity.
[1031] U21. The composition of embodiment U20, wherein the yeast
includes one or more heterologous polynucleotides that encodes one
or more of the following enzymes, or includes multiple copies of
polynucleotides that encode one or more of the following enzymes:
transketolase enzyme, transaldolase enzyme, or a transketolase
enzyme and transaldolase enzyme
[1032] U22. The composition of embodiment U21, wherein the
transketolase enzyme is encoded by a TKL1 coding sequence or a TKL2
coding sequence.
[1033] U23. The composition of embodiment U21, wherein the
transaldolase is encoded by a TAL coding sequence.
[1034] U24. The composition of any one of embodiments U21 to U23,
wherein the transketolase enzyme or the transaldolase enzyme is
from a yeast.
[1035] U25. The composition of any one of embodiments U1 to U24,
wherein the nucleic acid includes one or more promoters operable in
a yeast, wherein the promoter is in operable connection with one or
more of the polynucleotides.
[1036] U26. The composition of embodiment U25, wherein the promoter
is selected from promoters that regulate glucose phosphate
dehydrogenase (GUD), translation elongation factor (TEF-1),
phosphoglucokinase (UGK-1) and triose phosphate dehydrogenase
(TDH-1).
[1037] U27. The composition of any one of embodiments U1 to U26,
wherein the polynucleotides, the promoters, or the polynucleotides
and the promoters are not integrated in the yeast nucleic acid.
[1038] U28. The composition of embodiment U27, wherein the
polynucleotides, the promoters, or the polynucleotides and the
promoters are in one or more plasmids.
[1039] U29. The composition of any one of embodiments U1 to U28,
wherein the polynucleotide subsequences, the promoters, or the
polynucleotide subsequences and the promoters are integrated in
genomic DNA of the yeast.
[1040] U30. The composition of embodiment U29, wherein the
polynucleotides, the promoters, or the polynucleotides and the
promoters are integrated in a transposition integration event, in a
homologous recombination integration event, or in a transposition
integration event and a homologous recombination integration
event.
[1041] U31. The composition of embodiment U30, wherein the
transposition integration event includes transposition of an operon
comprising two or more of the polynucleotide subsequences, the
promoters, or the polynucleotide subsequences and the
promoters.
[1042] U32. The composition of embodiment U30, wherein the
homologous recombination integration event includes homologous
recombination of an operon comprising two or more of the
polynucleotide subsequences, the promoters, or the polynucleotide
subsequences and the promoters.
[1043] V1. A method, comprising contacting an engineered yeast of
any one of embodiments U1 to U32 with a feedstock that contains one
or more pentose sugars under conditions in which the microbe
synthesizes ethanol.
[1044] V2. The method of embodiment V1, wherein the engineered
yeast synthesizes ethanol to about 85% to about 99% of theoretical
yield.
[1045] V3. The method of embodiment V1 or V2, comprising recovering
ethanol synthesized by the engineered yeast.
[1046] W1. A composition comprising a synthetic nucleic acid that
includes a polynucleotide selected from the group of twenty (20)
polynucleotides (SEQ ID NOS 513-531, respectively, in order of
appearance) consisting of:
TABLE-US-00127 CACGCACGGACCGACCGTCACCGGACCGTTTCGCGCGACGTGCGCGAGGC
TCCGACACGAAAGACGGGCCCCCTATTGCGCTCATGTCGGCCGCACCCCT
GCGTAAAGTCAGATACGTGCGCCACCCGAGCCGGGACCGCCCTGAGCGCA
TGGTCCGGGCGGCGTGGCAAGCGCAGGAGGGCGTGCCCCGTTCGCTAGGC A
ACGTATGTCGGCTGATCGTACACGCCGACCAGCGCAGTCGGCGTACTCAG
GCGTTCCGAGTAGCTCACATCTGTGGGCCCCGGCGTACCTTCGGCAGGGT
TATGCGACGGGGCGGCAGGCTTGCGCTGGCGTCGGGAATCACCGCGAACT
TGACCCGCGCCGGTTCCGTATCGGTCCGCTGCGGCCGTGCTCCGCAGTCG A
TGCAGTCCGCCCAGCCGGCCGTGTAGCACGGCCGACTGCAGGTGCGACGT
GCTAGGGGCCAGCACGCGAGCGGCCCTACCACGGGTCGTGTGGGGCGCAT
GACCGCCGGCCGGGTCTCGGCACGGGGCGACGCGGTGCTCCTAGGCTAGC
AGGGCCTCACCGGGTGATCCCCCGTGTAGCGCCGCACAACACCCCCTGCG A
TGCCCGCATACCGCCCGCCCACTGGGGATCCTCCGGCGCTGTCGCGCTAT
GCGCGTCCATCCTGGTCGGACGGGCTCGGGCCCCGGACCAAACCGCAGCG
GCCCCTGGCAGCGACTAAGGGCGCCGTCTCACCCTAGACTTCTTAATCGG
GGTGTCCCGGTAGGCCGGGAGTAGCCTCGGCGGGCTAGCCGCGTGACTAT A
GCGGGTTAGTCCCCGTCGGACGTCATGCATACAGTCGGGGCTGGCGAGAC
AGGAGGCTACAGGGGGCGCCCGGAGGAACACACGTGGGACTAAGACGTCG
GTCCGTGTGCCCCCGAACCGGCGTGCTCATCGTAGGACTGGGAAGTCCGT
ACCGCGTGGCTCGTACCTCGCGGTCTGAGTCCGACACCCGCTGACGCCGG A
CTGAGACGACTCCCGCACTACGGATCGCGAGCGTAGACTCAGCCCGGACT
CTCACGCGACCTCGGACGCGGCCTAATGTCTCGACTCGCGGTCCGCTGAA
GGTCTCGGGGCACGCGAGACGCGGGGTCAGGCCGGGGGGATCCCCGCACA
CACTCAGTCGCGGCGAACGGAGTCCCGTGGCCTGGCTAGGGATCGTGGGT A
GGGGCGTCCACTCTGGCTCGGTAGAGCGCTGGGCTCCGCGCGACTGCGCG
CACCCATCGGTTTGGCGCGACGCACCGTGGACTCCTGGGCTAGAGGGCGG
GTCCCCGCCATACCCCGTTCTCGTGCCGGCTGGGTAGGACCGGAGTGACG
GCTGTGGCCGGCGACTCGGGCGCGCACTGTAGTCGGATCTGGGCGGGCAG A
GTCGGGCGCGCGTCAGTCCACGCGTTAAACACTGGCCGACGACACGACGG
GATCCGGGCACGCCCCGAGAGCGCGTGTTCGCGCGAGTCGATCGGGAGGC
CGCAGCGTGTCGAGCCCAGACCCCGCTCTAGCGTGGCCATCGCGGTGCTA
AGTGGGGCGGCCGGGTCCTATACACGCTTACCGATAGTCAAGTTTGCGTG A
GTCTTAGGGCCCAGGGACCGCACGGGTCGACCGCGCGACTGGTCGGAGCT
TGCGCGTCTACGCCACTCGGCGGCCCCGACGGGGGATGCCGCGGAATGTC
CGCCGGCGTATGCGGCTCAAGCCGGACCGTCGGACTGCGAAGCGCCGTGA
GCACCCCTCGACCTGACCGGACGCGGCGCACCCGTCCGAGTATCGTCGCG A
TCGGGTCTCGCCCGGCGCTAGTCCAGCCGTAGCGCTCTCCGGCGATCACC
CCGGAGCACTCTGGAGCCGAGCGGTCGGGTCTGTTGGGCGCGCCGCGGCT
ACGGACGGCTCGACTCACTGGCGCTCGACCCCGTATCCCCCGTCTCGGAC
GACGCACCGTTGCGCGGGAACGATCGGCGGCGCTCACACGCACGATCGGA A
CTTAAGGCTGGCGCACCATGAGGGCCGCGCCACGTCCGACCCGCAGCCCG
CGCGTAGTAGCCTAGCCGGGCGGGGTTCCTCCCGTGCGTCACCTAGCACG
GGGCCTGGCACCGAACGCGAGCCCGTCCGGTCACCGCGGCGGGTCTGCGG
ACGTCCCCGGTCGCTCGGCTCGGAGTCCCCGCTGGGGATCGCGTCGGGAC A
CGACGGCGTAGCACTCGCGGACCTAGGGCGCGCGAGTCGGGGGAGCCCGC
GGTGCGACGCTCGGGGAGGAGCTCGCATGCCCAAGGCACGATCTAGGGGG
GGGTACGGGGGGCGTCCGTCCGAGCGCCGGGACTGCGATCCGGGGCCACA
TGCTAACCGGCGGAAGGGGGGACCTAACCGGTGTGGACTCCGGGTAATCC A
CGGGGGGCTGACACGTCTCGGATCGCCCCGTCAGTCAGCCCCCTAGTCCC
GGACAGGACGTCGGAGGTCGAGTCCGCACTGTCGGGCCTGCTCGTGGGCA
CGGCAGGACGCGTCCCCATGGTCAGCCGCCGTGCGATACCTCGCCACGAC
TCTGAGCCGGGCGCGAGCGTGAGAGCCCGAGCCGCGGTACACGGGGCGTC A
GCGAGCTCGCTCTCGACTCCGGGCTCCCGTGCTGACACGGGGTGCGACCC
CGCGGCGATTGTCCGCACGCCTGTCGGACGACGTCGGCCCGTCGTAGTGC
CGGTCAGAGGCAGGGGGGCTGCTCGCGCTGGCCGCCTCGTCGCGCGTGGA
CCCTATGGGGGATCACGCGTGGGGTCGGGATCGGGGACCGCGCGACTTGG A
CGCGCCCCGTAACGGACGCGGTGAGTCGAGCTTACGCGGCTAGGGCCGAG
TCGTGTTAGCGTCTCGCGTAAGCGAATGCCACGTCCCCCGCCGCCCGTCG
CGCAGCTGGCTACGCAACGCCTCCGCGGCCTCCGTAGCGAGTGCGTGGGA
CGCTGGCCGTCCGCGTGTTCCGGGACCTGGATGCGGGAGGGACCTAAGGC A
AGAACGTGCGGTCGTCCCCACGCACGGGATGACGGACGGGGTAGACGGGC
GTCGTGCGCGCGGGTAGCGTAACCGGTTACAGTCCCCGCAACGCTCTAGC
TCCGGCCCTCGCTTAGGAGTTCGCGGCCGAGACATGAGGTGGTCCGGACG
GCAGGGGGTCGCGGAGACCGTGGAGCCGATTCTGCCGGACGCCACGTCCC A
CGGGACGCCCCGTACCGTGTACGAAGCCCCGGTCGGTCGGCGGATCGTAG
ATCCCGGAGCCGACGCCTTGAACCCGGCTTTCCCAGCGACTCGCGCCCCC
ACTGGGTCCCTCGGGACCCCGCTCCCCCCAGACGCATACAGCCCGCAAGC
GGGGGCAGTCTCGGACCGCCCGGACACTGGCCTTAGGCACCGTGGGCTCG A
GTGTCCGGGGCGCATCGGAGCTGTCCGACCGAGTTCCGGGGACGGCGCAC
GTTGTGCCGGCCTCAGACGGAGCCTGTAGCCCCCGGACAGTGTGTGCCCG
CCCACTACGGGTTAGGCACGGGGTTGGTCGGCACGCGTCCTCCGCGTGTC
ACGGACCGATGCAGACCGCTGGCCGGGAGGTCGCCCCCCCAGGGGTGCAC A
CGCGCAGCACGCACGTCCGGGGCACGCGCGGCTCGGAGGGTCCGGGCTGG
GACGGGAGGTTTGGAGTCGCGTGCGCGTAGCAGCGCACCCGCCTGGTCGC
CGGGTCTAGTAGGGCTGGGTTACGGAGGACGTGCAGGCGACCCCAACCGT
TGACGACGGGTCCGACCACGCCTTTAGCCGTGGCGTGTCCGTCGCGAGCC A
[1047] W2. A microorganism comprising a polynucleotide that
includes a sequence selected from the group of twenty (20)
sequences (SEQ ID NOS 513-531, respectively, in order of
appearance) consisting of
TABLE-US-00128 CACGCACGGACCGACCGTCACCGGACCGTTTCGCGCGACGTGCGCGAGGC
TCCGACACGAAAGACGGGCCCCCTATTGCGCTCATGTCGGCCGCACCCCT
GCGTAAAGTCAGATACGTGCGCCACCCGAGCCGGGACCGCCCTGAGCGCA
TGGTCCGGGCGGCGTGGCAAGCGCAGGAGGGCGTGCCCCGTTCGCTAGGC A
ACGTATGTCGGCTGATCGTACACGCCGACCAGCGCAGTCGGCGTACTCAG
GCGTTCCGAGTAGCTCACATCTGTGGGCCCCGGCGTACCTTCGGCAGGGT
TATGCGACGGGGCGGCAGGCTTGCGCTGGCGTCGGGAATCACCGCGAACT
TGACCCGCGCCGGTTCCGTATCGGTCCGCTGCGGCCGTGCTCCGCAGTCG A
TGCAGTCCGCCCAGCCGGCCGTGTAGCACGGCCGACTGCAGGTGCGACGT
GCTAGGGGCCAGCACGCGAGCGGCCCTACCACGGGTCGTGTGGGGCGCAT
GACCGCCGGCCGGGTCTCGGCACGGGGCGACGCGGTGCTCCTAGGCTAGC
AGGGCCTCACCGGGTGATCCCCCGTGTAGCGCCGCACAACACCCCCTGCG A
TGCCCGCATACCGCCCGCCCACTGGGGATCCTCCGGCGCTGTCGCGCTAT
GCGCGTCCATCCTGGTCGGACGGGCTCGGGCCCCGGACCAAACCGCAGCG
GCCCCTGGCAGCGACTAAGGGCGCCGTCTCACCCTAGACTTCTTAATCGG
GGTGTCCCGGTAGGCCGGGAGTAGCCTCGGCGGGCTAGCCGCGTGACTAT A
GCGGGTTAGTCCCCGTCGGACGTCATGCATACAGTCGGGGCTGGCGAGAC
AGGAGGCTACAGGGGGCGCCCGGAGGAACACACGTGGGACTAAGACGTCG
GTCCGTGTGCCCCCGAACCGGCGTGCTCATCGTAGGACTGGGAAGTCCGT
ACCGCGTGGCTCGTACCTCGCGGTCTGAGTCCGACACCCGCTGACGCCGG A
CTGAGACGACTCCCGCACTACGGATCGCGAGCGTAGACTCAGCCCGGACT
CTCACGCGACCTCGGACGCGGCCTAATGTCTCGACTCGCGGTCCGCTGAA
GGTCTCGGGGCACGCGAGACGCGGGGTCAGGCCGGGGGGATCCCCGCACA
CACTCAGTCGCGGCGAACGGAGTCCCGTGGCCTGGCTAGGGATCGTGGGT A
GGGGCGTCCACTCTGGCTCGGTAGAGCGCTGGGCTCCGCGCGACTGCGCG
CACCCATCGGTTTGGCGCGACGCACCGTGGACTCCTGGGCTAGAGGGCGG
GTCCCCGCCATACCCCGTTCTCGTGCCGGCTGGGTAGGACCGGAGTGACG
GCTGTGGCCGGCGACTCGGGCGCGCACTGTAGTCGGATCTGGGCGGGCAG A
GTCGGGCGCGCGTCAGTCCACGCGTTAAACACTGGCCGACGACACGACGG
GATCCGGGCACGCCCCGAGAGCGCGTGTTCGCGCGAGTCGATCGGGAGGC
CGCAGCGTGTCGAGCCCAGACCCCGCTCTAGCGTGGCCATCGCGGTGCTA
AGTGGGGCGGCCGGGTCCTATACACGCTTACCGATAGTCAAGTTTGCGTG A
GTCTTAGGGCCCAGGGACCGCACGGGTCGACCGCGCGACTGGTCGGAGCT
TGCGCGTCTACGCCACTCGGCGGCCCCGACGGGGGATGCCGCGGAATGTC
CGCCGGCGTATGCGGCTCAAGCCGGACCGTCGGACTGCGAAGCGCCGTGA
GCACCCCTCGACCTGACCGGACGCGGCGCACCCGTCCGAGTATCGTCGCG A
TCGGGTCTCGCCCGGCGCTAGTCCAGCCGTAGCGCTCTCCGGCGATCACC
CCGGAGCACTCTGGAGCCGAGCGGTCGGGTCTGTTGGGCGCGCCGCGGCT
ACGGACGGCTCGACTCACTGGCGCTCGACCCCGTATCCCCCGTCTCGGAC
GACGCACCGTTGCGCGGGAACGATCGGCGGCGCTCACACGCACGATCGGA A
CTTAAGGCTGGCGCACCATGAGGGCCGCGCCACGTCCGACCCGCAGCCCG
CGCGTAGTAGCCTAGCCGGGCGGGGTTCCTCCCGTGCGTCACCTAGCACG
GGGCCTGGCACCGAACGCGAGCCCGTCCGGTCACCGCGGCGGGTCTGCGG
ACGTCCCCGGTCGCTCGGCTCGGAGTCCCCGCTGGGGATCGCGTCGGGAC A
CGACGGCGTAGCACTCGCGGACCTAGGGCGCGCGAGTCGGGGGAGCCCGC
GGTGCGACGCTCGGGGAGGAGCTCGCATGCCCAAGGCACGATCTAGGGGG
GGGTACGGGGGGCGTCCGTCCGAGCGCCGGGACTGCGATCCGGGGCCACA
TGCTAACCGGCGGAAGGGGGGACCTAACCGGTGTGGACTCCGGGTAATCC A
CGGGGGGCTGACACGTCTCGGATCGCCCCGTCAGTCAGCCCCCTAGTCCC
GGACAGGACGTCGGAGGTCGAGTCCGCACTGTCGGGCCTGCTCGTGGGCA
CGGCAGGACGCGTCCCCATGGTCAGCCGCCGTGCGATACCTCGCCACGAC
TCTGAGCCGGGCGCGAGCGTGAGAGCCCGAGCCGCGGTACACGGGGCGTC A
GCGAGCTCGCTCTCGACTCCGGGCTCCCGTGCTGACACGGGGTGCGACCC
CGCGGCGATTGTCCGCACGCCTGTCGGACGACGTCGGCCCGTCGTAGTGC
CGGTCAGAGGCAGGGGGGCTGCTCGCGCTGGCCGCCTCGTCGCGCGTGGA
CCCTATGGGGGATCACGCGTGGGGTCGGGATCGGGGACCGCGCGACTTGG A
CGCGCCCCGTAACGGACGCGGTGAGTCGAGCTTACGCGGCTAGGGCCGAG
TCGTGTTAGCGTCTCGCGTAAGCGAATGCCACGTCCCCCGCCGCCCGTCG
CGCAGCTGGCTACGCAACGCCTCCGCGGCCTCCGTAGCGAGTGCGTGGGA
CGCTGGCCGTCCGCGTGTTCCGGGACCTGGATGCGGGAGGGACCTAAGGC A
AGAACGTGCGGTCGTCCCCACGCACGGGATGACGGACGGGGTAGACGGGC
GTCGTGCGCGCGGGTAGCGTAACCGGTTACAGTCCCCGCAACGCTCTAGC
TCCGGCCCTCGCTTAGGAGTTCGCGGCCGAGACATGAGGTGGTCCGGACG
GCAGGGGGTCGCGGAGACCGTGGAGCCGATTCTGCCGGACGCCACGTCCC A
CGGGACGCCCCGTACCGTGTACGAAGCCCCGGTCGGTCGGCGGATCGTAG
ATCCCGGAGCCGACGCCTTGAACCCGGCTTTCCCAGCGACTCGCGCCCCC
ACTGGGTCCCTCGGGACCCCGCTCCCCCCAGACGCATACAGCCCGCAAGC
GGGGGCAGTCTCGGACCGCCCGGACACTGGCCTTAGGCACCGTGGGCTCG A
GTGTCCGGGGCGCATCGGAGCTGTCCGACCGAGTTCCGGGGACGGCGCAC
GTTGTGCCGGCCTCAGACGGAGCCTGTAGCCCCCGGACAGTGTGTGCCCG
CCCACTACGGGTTAGGCACGGGGTTGGTCGGCACGCGTCCTCCGCGTGTC
ACGGACCGATGCAGACCGCTGGCCGGGAGGTCGCCCCCCCAGGGGTGCAC A
CGCGCAGCACGCACGTCCGGGGCACGCGCGGCTCGGAGGGTCCGGGCTGG
GACGGGAGGTTTGGAGTCGCGTGCGCGTAGCAGCGCACCCGCCTGGTCGC
CGGGTCTAGTAGGGCTGGGTTACGGAGGACGTGCAGGCGACCCCAACCGT
TGACGACGGGTCCGACCACGCCTTTAGCCGTGGCGTGTCCGTCGCGAGCC A
[1048] W3. A method comprising detecting the presence or absence of
a nucleotide sequence identification tag in a microorganism,
wherein the nucleotide sequence is selected from the group of
twenty (20) nucleotide sequences (SEQ ID NOS 513-531, respectively,
in order of appearance) consisting of
TABLE-US-00129 CACGCACGGACCGACCGTCACCGGACCGTTTCGCGCGACGTGCGCGAGGC
TCCGACACGAAAGACGGGCCCCCTATTGCGCTCATGTCGGCCGCACCCCT
GCGTAAAGTCAGATACGTGCGCCACCCGAGCCGGGACCGCCCTGAGCGCA
TGGTCCGGGCGGCGTGGCAAGCGCAGGAGGGCGTGCCCCGTTCGCTAGGC A
ACGTATGTCGGCTGATCGTACACGCCGACCAGCGCAGTCGGCGTACTCAG
GCGTTCCGAGTAGCTCACATCTGTGGGCCCCGGCGTACCTTCGGCAGGGT
TATGCGACGGGGCGGCAGGCTTGCGCTGGCGTCGGGAATCACCGCGAACT
TGACCCGCGCCGGTTCCGTATCGGTCCGCTGCGGCCGTGCTCCGCAGTCG A
TGCAGTCCGCCCAGCCGGCCGTGTAGCACGGCCGACTGCAGGTGCGACGT
GCTAGGGGCCAGCACGCGAGCGGCCCTACCACGGGTCGTGTGGGGCGCAT
GACCGCCGGCCGGGTCTCGGCACGGGGCGACGCGGTGCTCCTAGGCTAGC
AGGGCCTCACCGGGTGATCCCCCGTGTAGCGCCGCACAACACCCCCTGCG A
TGCCCGCATACCGCCCGCCCACTGGGGATCCTCCGGCGCTGTCGCGCTAT
GCGCGTCCATCCTGGTCGGACGGGCTCGGGCCCCGGACCAAACCGCAGCG
GCCCCTGGCAGCGACTAAGGGCGCCGTCTCACCCTAGACTTCTTAATCGG
GGTGTCCCGGTAGGCCGGGAGTAGCCTCGGCGGGCTAGCCGCGTGACTAT A
GCGGGTTAGTCCCCGTCGGACGTCATGCATACAGTCGGGGCTGGCGAGAC
AGGAGGCTACAGGGGGCGCCCGGAGGAACACACGTGGGACTAAGACGTCG
GTCCGTGTGCCCCCGAACCGGCGTGCTCATCGTAGGACTGGGAAGTCCGT
ACCGCGTGGCTCGTACCTCGCGGTCTGAGTCCGACACCCGCTGACGCCGG A
CTGAGACGACTCCCGCACTACGGATCGCGAGCGTAGACTCAGCCCGGACT
CTCACGCGACCTCGGACGCGGCCTAATGTCTCGACTCGCGGTCCGCTGAA
GGTCTCGGGGCACGCGAGACGCGGGGTCAGGCCGGGGGGATCCCCGCACA
CACTCAGTCGCGGCGAACGGAGTCCCGTGGCCTGGCTAGGGATCGTGGGT A
GGGGCGTCCACTCTGGCTCGGTAGAGCGCTGGGCTCCGCGCGACTGCGCG
CACCCATCGGTTTGGCGCGACGCACCGTGGACTCCTGGGCTAGAGGGCGG
GTCCCCGCCATACCCCGTTCTCGTGCCGGCTGGGTAGGACCGGAGTGACG
GCTGTGGCCGGCGACTCGGGCGCGCACTGTAGTCGGATCTGGGCGGGCAG A
GTCGGGCGCGCGTCAGTCCACGCGTTAAACACTGGCCGACGACACGACGG
GATCCGGGCACGCCCCGAGAGCGCGTGTTCGCGCGAGTCGATCGGGAGGC
CGCAGCGTGTCGAGCCCAGACCCCGCTCTAGCGTGGCCATCGCGGTGCTA
AGTGGGGCGGCCGGGTCCTATACACGCTTACCGATAGTCAAGTTTGCGTG A
GTCTTAGGGCCCAGGGACCGCACGGGTCGACCGCGCGACTGGTCGGAGCT
TGCGCGTCTACGCCACTCGGCGGCCCCGACGGGGGATGCCGCGGAATGTC
CGCCGGCGTATGCGGCTCAAGCCGGACCGTCGGACTGCGAAGCGCCGTGA
GCACCCCTCGACCTGACCGGACGCGGCGCACCCGTCCGAGTATCGTCGCG A
TCGGGTCTCGCCCGGCGCTAGTCCAGCCGTAGCGCTCTCCGGCGATCACC
CCGGAGCACTCTGGAGCCGAGCGGTCGGGTCTGTTGGGCGCGCCGCGGCT
ACGGACGGCTCGACTCACTGGCGCTCGACCCCGTATCCCCCGTCTCGGAC
GACGCACCGTTGCGCGGGAACGATCGGCGGCGCTCACACGCACGATCGGA A
CTTAAGGCTGGCGCACCATGAGGGCCGCGCCACGTCCGACCCGCAGCCCG
CGCGTAGTAGCCTAGCCGGGCGGGGTTCCTCCCGTGCGTCACCTAGCACG
GGGCCTGGCACCGAACGCGAGCCCGTCCGGTCACCGCGGCGGGTCTGCGG
ACGTCCCCGGTCGCTCGGCTCGGAGTCCCCGCTGGGGATCGCGTCGGGAC A
CGACGGCGTAGCACTCGCGGACCTAGGGCGCGCGAGTCGGGGGAGCCCGC
GGTGCGACGCTCGGGGAGGAGCTCGCATGCCCAAGGCACGATCTAGGGGG
GGGTACGGGGGGCGTCCGTCCGAGCGCCGGGACTGCGATCCGGGGCCACA
TGCTAACCGGCGGAAGGGGGGACCTAACCGGTGTGGACTCCGGGTAATCC A
CGGGGGGCTGACACGTCTCGGATCGCCCCGTCAGTCAGCCCCCTAGTCCC
GGACAGGACGTCGGAGGTCGAGTCCGCACTGTCGGGCCTGCTCGTGGGCA
CGGCAGGACGCGTCCCCATGGTCAGCCGCCGTGCGATACCTCGCCACGAC
TCTGAGCCGGGCGCGAGCGTGAGAGCCCGAGCCGCGGTACACGGGGCGTC A
GCGAGCTCGCTCTCGACTCCGGGCTCCCGTGCTGACACGGGGTGCGACCC
CGCGGCGATTGTCCGCACGCCTGTCGGACGACGTCGGCCCGTCGTAGTGC
CGGTCAGAGGCAGGGGGGCTGCTCGCGCTGGCCGCCTCGTCGCGCGTGGA
CCCTATGGGGGATCACGCGTGGGGTCGGGATCGGGGACCGCGCGACTTGG A
CGCGCCCCGTAACGGACGCGGTGAGTCGAGCTTACGCGGCTAGGGCCGAG
TCGTGTTAGCGTCTCGCGTAAGCGAATGCCACGTCCCCCGCCGCCCGTCG
CGCAGCTGGCTACGCAACGCCTCCGCGGCCTCCGTAGCGAGTGCGTGGGA
CGCTGGCCGTCCGCGTGTTCCGGGACCTGGATGCGGGAGGGACCTAAGGC A
AGAACGTGCGGTCGTCCCCACGCACGGGATGACGGACGGGGTAGACGGGC
GTCGTGCGCGCGGGTAGCGTAACCGGTTACAGTCCCCGCAACGCTCTAGC
TCCGGCCCTCGCTTAGGAGTTCGCGGCCGAGACATGAGGTGGTCCGGACG
GCAGGGGGTCGCGGAGACCGTGGAGCCGATTCTGCCGGACGCCACGTCCC A
CGGGACGCCCCGTACCGTGTACGAAGCCCCGGTCGGTCGGCGGATCGTAG
ATCCCGGAGCCGACGCCTTGAACCCGGCTTTCCCAGCGACTCGCGCCCCC
ACTGGGTCCCTCGGGACCCCGCTCCCCCCAGACGCATACAGCCCGCAAGC
GGGGGCAGTCTCGGACCGCCCGGACACTGGCCTTAGGCACCGTGGGCTCG A
GTGTCCGGGGCGCATCGGAGCTGTCCGACCGAGTTCCGGGGACGGCGCAC
GTTGTGCCGGCCTCAGACGGAGCCTGTAGCCCCCGGACAGTGTGTGCCCG
CCCACTACGGGTTAGGCACGGGGTTGGTCGGCACGCGTCCTCCGCGTGTC
ACGGACCGATGCAGACCGCTGGCCGGGAGGTCGCCCCCCCAGGGGTGCAC A
CGCGCAGCACGCACGTCCGGGGCACGCGCGGCTCGGAGGGTCCGGGCTGG
GACGGGAGGTTTGGAGTCGCGTGCGCGTAGCAGCGCACCCGCCTGGTCGC
CGGGTCTAGTAGGGCTGGGTTACGGAGGACGTGCAGGCGACCCCAACCGT
TGACGACGGGTCCGACCACGCCTTTAGCCGTGGCGTGTCCGTCGCGAGCC A
[1049] W4. The method of embodiment W3, wherein the microorganism
includes two or more different identification tags.
[1050] W5. The method of embodiment W3, wherein the microorganism
includes multiple copies of one or more of the identification
tags.
[1051] X1. A composition comprising a nucleic acid comprising
heterologous polynucleotides that encode a phosphogluconate
dehydratase enzyme and a 2-keto-3-deoxygluconate-6-phosphate
aldolase enzyme, and one or more polynucleotides that homologously
combine in a gene of a host that encodes a 6-phosphogluconate
dehydrogenase (decarboxylating) enzyme.
[1052] X2. The composition of embodiment X1, wherein the yeast is a
Saccharomyces spp. yeast.
[1053] X3. The composition of embodiment X2, wherein the yeast is a
Saccharomyces cerevisiae yeast strain.
[1054] X3.1. The composition of any one of embodiments X1 to X3,
wherein the polynucleotides encoding the phosphogluconate
dehydratase enzyme and the 3-deoxygluconate-6-phosphate aldolase
enzyme independently are from an Escherichia spp. microbe or
Pseudomonas spp. microbe.
[1055] X4. The composition of embodiment X3, wherein the
Escherichia spp. microbe is an Escherichia coli strain.
[1056] X5. The composition of embodiment X3 or X4, wherein the
Pseudomonas spp. microbe is a Pseudomonas aeruginosa strain.
[1057] X6. The composition of any one of embodiments X1 to X5,
wherein the polynucleotide that encodes the phosphogluconate
dehydratase enzyme is an EDD gene.
[1058] X7. The composition of any one of embodiments X1 to X5,
wherein the polynucleotide that encodes the
2-keto-3-deoxygluconate-6-phosphate aldolase enzyme is an EDA
gene.
[1059] X8. The composition of any one of embodiments X1 to X7,
wherein the nucleic acid includes a polynucleotide that encodes a
6-phosphogluconolactonase enzyme.
[1060] X8.1. The composition of embodiment X8, wherein the
polynucleotide that encodes the 6-phosphogluconolactonase enzyme is
from a yeast.
[1061] X8.2. The composition of embodiment X8.1, wherein the yeast
is a Saccharomyces spp. yeast.
[1062] X8.3. The composition of embodiment X8.2, wherein the yeast
is a Saccharomyces cerevisiae strain.
[1063] X8.4. The composition of any one of embodiments X8 to X8.3,
wherein the 6-phosphogluconolactonase enzyme is expressed from a
SOL gene.
[1064] X9. The composition of embodiment X8.4, wherein the SOL gene
is a SOL3 gene.
[1065] X10. The composition of any one of embodiments X1 to X9,
wherein the nucleic acid includes a polynucleotide that encodes a
glucose-6-phosphate dehydrogenase enzyme.
[1066] X11. The composition of embodiment X10, wherein the
polynucleotide that encodes the glucose-6-phosphate dehydrogenase
enzyme is from a yeast.
[1067] X12. The composition of embodiment X11, wherein the yeast is
a Saccharomyces spp. yeast.
[1068] X13. The composition of embodiment X12, wherein the yeast is
a Saccharomyces cerevisiae strain.
[1069] X14. The composition of any one of embodiments X10 to X13,
wherein the nucleic acid includes a polynucleotide that encode an
endogenous glucose-6-phosphate dehydrogenase enzyme.
[1070] X15. The composition of any one of embodiments X10 to X14,
wherein the glucose-6-phosphate dehydrogenase enzyme is expressed
from a ZWF gene.
[1071] X16. The composition of embodiment X15, wherein the ZWF gene
is a ZWF1 gene.
[1072] X17. The composition of any one of embodiments X1 to X16,
wherein the nucleic acid includes one or more promoters operable in
a yeast, wherein the promoter is in operable connection with one or
more of the polynucleotides.
[1073] X18. The composition of embodiment X17, wherein the promoter
is selected from promoters that regulate glucose phosphate
dehydrogenase (GPD), translation elongation factor (TEF-1),
phosphoglucokinase (PGK-1) and triose phosphate dehydrogenase
(TDH-1).
[1074] X19. The composition of any one of embodiments X1 to X18,
wherein the nucleic acid includes one or more polynucleotides that
homologously combine in a gene of a host that encodes a
phosphofructokinase (PFK) enzyme, phosphoglucoisomerase (PGI)
enzyme, transketolase enzyme, transaldolase enzyme, or combination
thereof.
[1075] X20. The composition of embodiment X19, wherein the
transketolase enzyme is encoded by a TKL-1 coding sequence or a
TKL-2 coding sequence.
[1076] X21. The composition of embodiment X19, wherein the
transaldolase is encoded by a TAL-1 coding sequence.
[1077] X22. The composition of embodiment X19, wherein the
phosphofructokinase (PFK) enzyme is a PFK-2 enzyme or PFK-1
enzyme.
[1078] X23. The composition of any one of embodiments X1 to X22,
wherein the 6-phosphogluconate dehydrogenase (decarboxylating)
enzyme is encoded by a GND-1 gene or a GND-2 gene.
[1079] X24. The composition of embodiment X19, wherein the PGI is
encoded by a PGI-1 gene.
[1080] X25. The composition of any one of embodiments X1 to X24,
wherein the nucleic acid is one or two separate nucleic acid
molecules.
[1081] X26. The composition of embodiment X25, wherein each nucleic
acid molecule includes one or two or more of the polynucleotide
subsequences, one or two or more of the promoters, or one or two or
more of the polynucleotide subsequences and one or two or more of
the promoters.
[1082] X27. The composition of embodiment X25 or X26, wherein each
of the one or two nucleic acid molecules are in circular form.
[1083] X28. The composition of embodiment X25 or X26, wherein each
of the one or two nucleic acid molecules are in linear form.
[1084] X29. The composition of any one of embodiments X25 to X28,
wherein each of the one or two nucleic acid molecules functions as
an expression vector.
[1085] X30. The composition of any one of embodiments X25 to X29,
wherein each of the one or two nucleic acid molecules includes
flanking sequences for integrating the polynucleotides, the
promoter sequences, or the polynucleotides and the promoter
sequences in the nucleic acid into genomic DNA of a host
organism.
[1086] Y1. A composition comprising an engineered yeast that
includes an alteration that adds or increases a phosphogluconate
dehydratase activity and a 2-keto-3-deoxygluconate-6-phosphate
aldolase activity, and an alteration that reduces a
6-phosphogluconate dehydrogenase (decarboxylating) activity.
[1087] Y2. The composition of embodiment Y1, wherein the yeast is a
Saccharomyces spp. yeast.
[1088] Y3. The composition of embodiment Y2, wherein the yeast is a
Saccharomyces cerevisiae yeast strain.
[1089] Y4. The composition of any one of embodiments Y1 to Y3,
wherein the yeast includes an altered gene that encodes a
6-phosphogluconate dehydrogenase (decarboxylating) enzyme.
[1090] Y4.1. The composition of any one of embodiments Y1 to Y4
where the yeast includes heterologous polynucleotides, or multiple
copies of endogenous polynucleotides, that encode a
phosphogluconate dehydratase enzyme and a
2-keto-3-deoxygluconate-6-phosphate aldolase enzyme.
[1091] Y5. The composition of embodiment Y4, wherein the
polynucleotides encoding the phosphogluconate dehydratase enzyme
and the 3-deoxygluconate-6-phosphate aldolase enzyme independently
are from an Escherichia spp. microbe or Pseudomonas spp.
microbe.
[1092] Y6. The composition of embodiment Y5, wherein the
Escherichia spp. microbe is an Escherichia coli strain.
[1093] Y7. The composition of embodiment Y5, wherein the
Pseudomonas spp. microbe is a Pseudomonas aeruginosa strain.
[1094] Y8. The composition of any one of embodiments Y4 to Y7,
wherein the polynucleotide that encodes the phosphogluconate
dehydratase enzyme is an EDD gene.
[1095] Y9. The composition of any one of embodiments Y4 to Y7,
wherein the polynucleotide that encodes the
2-keto-3-deoxygluconate-6-phosphate aldolase enzyme is an EDA
gene.
[1096] Y10. The composition of any one of embodiments Y1 to Y11,
wherein a glucose-6-phosphate dehydrogenase activity is added or
increased.
[1097] Y10.1. The composition of embodiment Y10, wherein the yeast
comprises a heterologous polynucleotide that encodes a
6-phosphogluconolactonase enzyme, or wherein the yeast comprises
multiple copies of an endogenous polynucleotide that encodes a
6-phosphogluconolactonase enzyme.
[1098] Y10.2. The composition of embodiment Y10.1, wherein the
polynucleotide that encodes the 6-phosphogluconolactonase enzyme
enzyme is from a yeast.
[1099] Y10.3. The composition of embodiment Y10.2, wherein the
yeast is a Saccharomyces spp. yeast.
[1100] Y10.4. The composition of embodiment Y10.3, wherein the
yeast is a Saccharomyces cerevisiae strain.
[1101] Y10.5. The composition of any one of embodiments Y10 to
Y10.4, wherein the 6-phosphogluconolactonase enzyme is expressed
from a SOL gene.
[1102] Y11. The composition of embodiment Y10.4, wherein the SOL
gene is a SOL3 gene.
[1103] Y12. The composition of any one of embodiments Y4 to Y11,
wherein a glucose-6-phosphate dehydrogenase activity is added or
increased.
[1104] Y13. The composition of embodiment Y12, wherein the yeast
comprises a heterologous polynucleotide that encodes a
glucose-6-phosphate dehydrogenase enzyme, or wherein the yeast
comprises multiple copies of an endogenous polynucleotide that
encodes a glucose-6-phosphate dehydrogenase enzyme.
[1105] Y14. The composition of embodiment Y13, wherein the
polynucleotide that encodes the glucose-6-phosphate dehydrogenase
enzyme is from a yeast.
[1106] Y15. The composition of embodiment Y14, wherein the yeast is
a Yaccharomyces spp. yeast.
[1107] Y16. The composition of embodiment Y15, wherein the yeast is
a Yaccharomyces cerevisiae strain.
[1108] Y17. The composition of any one of embodiments Y13 to Y17,
wherein the glucose-6-phosphate dehydrogenase enzyme is expressed
from a ZWF gene.
[1109] Y18. The composition of embodiment Y17, wherein the ZWF gene
is a ZWF1 gene.
[1110] Y19. The composition of any one of embodiments Y1 to Y18,
wherein the nucleic acid includes one or more promoters operable in
a yeast, wherein the promoter is in operable connection with one or
more of the polynucleotides.
[1111] Y20. The composition of embodiment Y19, wherein the promoter
is selected from promoters that regulate glucose phosphate
dehydrogenase (GYD), translation elongation factor (TEF-1),
phosphoglucokinase (YGK-1) and triose phosphate dehydrogenase
(TDH-1).
[1112] Y21. The composition of any one of embodiments Y1 to Y20,
wherein the yeast includes a reduction in one or more of the
following activities: phosphofructokinase (PFK) activity,
phosphoglucoisomerase (PGI) activity, transketolase activity,
transaldolase activity, or combination thereof.
[1113] Y22. The composition of embodiment Y21, wherein the yeast
includes an alteration in one or more polynucleotides that inhibits
production of one or more enzymes selected from the group
consisting of phosphofructokinase (PFK) enzyme,
phosphoglucoisomerase (PGI) enzyme, 6-phosphogluconate
dehydrogenase (decarboxylating) enzyme, transketolase enzyme,
transaldolase enzyme, or combination thereof.
[1114] Y23. The composition of embodiment Y22, wherein the
transketolase enzyme is encoded by a TKL-1 coding sequence or a
TKL-2 coding sequence.
[1115] Y24. The composition of embodiment Y22, wherein the
transaldolase is encoded by a TAL-1 coding sequence.
[1116] Y25. The composition of embodiment Y22, wherein the
phosphofructokinase (PFK) enzyme is a PFK-2 enzyme or PFK-1
enzyme.
[1117] Y26. The composition of any one of embodiments Y4 to Y25,
wherein the 6-phosphogluconate dehydrogenase (decarboxylating)
enzyme is encoded by a GND-1 gene or GND-2 gene.
[1118] Y27. The composition of embodiment Y22, wherein the PGI is
encoded by a PGI-1 gene.
[1119] Y28. The composition of any one of embodiments Y1 to Y27,
wherein the polynucleotides, the promoters, or the polynucleotides
and the promoters are not integrated in the yeast nucleic acid.
[1120] Y29. The composition of embodiment Y28, wherein the
polynucleotides, the promoters, or the polynucleotides and the
promoters are in one or more plasmids.
[1121] Y30. The composition of any one of embodiments Y1 to Y29,
wherein the polynucleotide subsequences, the promoters, or the
polynucleotide subsequences and the promoters are integrated in
genomic DNA of the yeast.
[1122] Y31. The composition of embodiment Y30, wherein the
polynucleotides, the promoters, or the polynucleotides and the
promoters are integrated in a transposition integration event, in a
homologous recombination integration event, or in a transposition
integration event and a homologous recombination integration
event.
[1123] Y32. The composition of embodiment Y31, wherein the
transposition integration event includes transposition of an operon
comprising two or more of the polynucleotide subsequences, the
promoters, or the polynucleotide subsequences and the
promoters.
[1124] Y33. The composition of embodiment Y31, wherein the
homologous recombination integration event includes homologous
recombination of an operon comprising two or more of the
polynucleotide subsequences, the promoters, or the polynucleotide
subsequences and the promoters.
[1125] Z1. A method, comprising contacting an engineered yeast of
any one of embodiments Y1 to Y33 with a feedstock that contains one
or more hexose sugars under conditions in which the microbe
synthesizes ethanol.
[1126] Z2. The method of embodiment Z1, wherein the engineered
yeast synthesizes ethanol to about 85% to about 99% of theoretical
yield.
[1127] Z3. The method of embodiment Z1 or Z2, comprising recovering
ethanol synthesized by the engineered yeast.
[1128] Z4. The method of any one of embodiments Z1 to Z3, wherein
the conditions are fermentation conditions.
[1129] The entirety of each patent, patent application, publication
and document referenced herein hereby is incorporated by reference.
Citation of the above patents, patent applications, publications
and documents is not an admission that any of the foregoing is
pertinent prior art, nor does it constitute any admission as to the
contents or date of these publications or documents.
[1130] Modifications may be made to the foregoing without departing
from the basic aspects of the technology. Although the technology
has been described in substantial detail with reference to one or
more specific embodiments, those of ordinary skill in the art will
recognize that changes may be made to the embodiments specifically
disclosed in this application, yet these modifications and
improvements are within the scope and spirit of the technology.
[1131] The technology illustratively described herein suitably may
be practiced in the absence of any element(s) not specifically
disclosed herein. Thus, for example, in each instance herein any of
the terms "comprising," "consisting essentially of," and
"consisting of" may be replaced with either of the other two terms.
The terms and expressions which have been employed are used as
terms of description and not of limitation, and use of such terms
and expressions do not exclude any equivalents of the features
shown and described or portions thereof, and various modifications
are possible within the scope of the claimed technology. The term
"a" or "an" can refer to one of or a plurality of the elements it
modifies (e.g., "a reagent" can mean one or more reagents) unless
it is contextually clear either one of the elements or more than
one of the elements is described. The term "about" as used herein
refers to a value within 10% of the underlying parameter (i.e.,
plus or minus 10%), and use of the term "about" at the beginning of
a string of values modifies each of the values (i.e., "about 1, 2
and 3" refers to about 1, about 2 and about 3). For example, a
weight of "about 100 grams" can include weights between 90 grams
and 110 grams. Further, when a listing of values is described
herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%) the listing
includes all intermediate and fractional values thereof (e.g., 54%,
85.4%). Thus, it should be understood that although the present
technology has been specifically disclosed by representative
embodiments and optional features, modification and variation of
the concepts herein disclosed may be resorted to by those skilled
in the art, and such modifications and variations are considered
within the scope of this technology.
[1132] Certain embodiments of the technology are set forth in the
claim(s) that follow(s).
Sequence CWU 1
1
685135DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1aactgactag taaaaaaatg cgtgatatcg attcc
35236DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 2agtaactcga gctactaggc aacagcagcg cgcttg
36338DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 3aactgactag taaaaaaatg actgatctgc attcaacg
38441DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 4agtaactcga gctactagat accggcacct gcatatattg c
41546DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 5aactgactag taaaaaaatg aaaaactgga aaacaagtgc
agaatc 46644DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 6agtaactcga gctactacag cttagcgcct
tctacagctt cacg 44749DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 7aactgactag taaaaaaatg
aatccacaat tgttacgcgt aacaaatcg 49849DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
8agtaactcga gctactaaaa agtgatacag gttgcgccct gttcggcac
49975DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 9tgcatattcc gttcaatctt ataaagctgc catagatttt
tacaccaagt cgttttaaga 60gcttggtgag cgcta 751075DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
10cttgccagtg aatgaccttt ggcattctca tggaaacttc agtttcatag tcgagttcaa
60gagaaaaaaa aagaa 751175DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 11atgactgtta ctactccttt
tgtgaatggt acttcttatt gtaccgtcac tgcatattcc 60gttcaatctt ataaa
751275DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 12ttaatcaact ctctttcttc caaccaaatg gtcagcaatg
agtctggtag cttgccagtg 60aatgaccttt ggcat 751349DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
13gactaactga actagtaaaa aaatgaccaa gccgcgcaca attaatcag
491446DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 14aagtgagtaa ctcgagttat taaccgctgt tgcgaagtgc
cgtcgc 461551DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 15atgtctcatc atcatcatca tcataccaag
ccgcgcacaa ttaatcagaa c 511652DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 16gactaactga actagtaaaa
aaatgtctca tcatcatcat catcatacca ag 521756DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
17atgtctcatc atcatcatca tcatatgacc aagccaagaa ctattaacca aaaccc
561860DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 18gactaactga actagtaaaa aaatgtctca tcatcatcat
catcatatga ccaagccaag 601951DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 19aagtgagtaa ctcgagttat
taaccggagt ttctcaaagc agtagcgata g 51202679DNAZymomonas mobilis
20actagtaaaa aaatgaccaa gccgcgcaca attaatcaga acccagacct tcgctatttt
60ggtaacctgc tcggtcaggt tattaaggaa caaggcggag agtctttatt caaccagatc
120gagcaaattc gctctgccgc gattagacgc catcggggta ttgttgacag
caccgagcta 180agttctcgct tagccgatct cgaccttaat gacatgttct
cttttgcaca tgcctttttg 240ctgttttcaa tgctggccaa tttggctgat
gatcgtcagg gagatgccct tgatcctgat 300gccaatatgg caagtgccct
taaggacata aaagccaaag gcgtcagtca gcaggcgatc 360attgatatga
tcgacaaagc ctgcattgtg cctgttctga cagcacatcc gaccgaagtc
420cgtcggaaaa gtatgcttga ccattataat cgcattgcag gtttaatgcg
gttaaaagat 480gctggacaaa cggtgaccga agatggtctt ccgatcgaag
atgcgttaat ccagcaaatc 540acgatattat ggcagactcg tccgctcatg
ctgcaaaagc tgaccgtggc tgatgaaatc 600gaaactgccc tgtctttctt
aagagaaact tttctgcctg ttctgcccca gatttatgca 660gaatgggaaa
aattgcttgg tagttctatt ccaagcttta tcagacctgg taattggatt
720ggtggtgacc gtgacggtaa ccccaatgtc aatgccgata cgatcatgct
gtctttgaag 780cgcagctcgg aaacggtatt gacggattat ctcaaccgtc
ttgataaact gctttccaac 840ctttcggtct caaccgatat ggtttcggta
tccgatgata ttctacgtct agccgataaa 900agtggtgacg atgctgcgat
ccgtgcggat gaaccttatc gtcgtgcctt aaatggtatt 960tatgaccgtt
tagccgctac ctatcgtcag atcgccggtc gcaacccttc gcgcccagcc
1020ttgcgttctg cagaagccta taaacggcct caagaattgc tggctgattt
gaagaccttg 1080gccgaaggct tgggtaaatt ggcagaaggt agttttaagg
cattgatccg ttcggttgaa 1140acctttggtt tccatttggc caccctcgat
ctgcgtcaga attcgcaggt tcatgaaaga 1200gttgtcaatg aactgctacg
gacagccacc gttgaagccg attatttatc tctatcggaa 1260gaagatcgcg
ttaagctgtt aagacgggaa ttgtcgcagc cgcggactct attcgttccg
1320cgcgccgatt attccgaaga aacgcgttct gaacttgata ttattcaggc
agcagcccgc 1380gcccatgaaa tttttggccc tgaatccatt acgacttatt
tgatttcgaa tggcgaaagc 1440atttccgata ttctggaagt ctatttgctt
ttgaaagaag cagggctgta tcaagggggt 1500gctaagccaa aagcggcgat
tgaagctgcg cctttattcg agacggtggc cgatcttgaa 1560aatgcgccaa
aggtcatgga ggaatggttc aagctgcctg aagcgcaagc cattgcaaag
1620gcacatggcg ttcaggaagt gatggttggc tattctgact ccaataagga
cggcggatat 1680ctgacctcgg tttggggtct ttataaggct tgcctcgctt
tggtgccgat ttttgagaaa 1740gccggtgtac cgatccagtt tttccatgga
cggggtggtt ccgttggtcg cggtggtggt 1800tccaacttta atgccattct
gtcgcagcca gccggagccg tcaaagggcg tatccgttat 1860acagaacagg
gtgaagtcgt ggcggccaaa tatggcaccc atgaaagcgc tattgcccat
1920ctggatgagg ccgtagcggc gactttgatt acgtctttgg aagcaccgac
cattgtcgag 1980ccagagttta gtcgttaccg taaggccttg gatcagatct
cagattcagc tttccaggcc 2040tatcgccaat tggtctatgg aacgaagggc
ttccgtaaat tctttagtga atttacgcct 2100ttgccggaaa ttgccctgtt
aaagatcggg tcacgcccac ctagccgcaa aaaatccgac 2160cggattgaag
atctacgcgc tattccttgg gtgtttagct ggtctcaagt tcgagtcatg
2220ttacccggtt ggttcggttt cggtcaggct ttatatgact ttgaagatac
cgagctgtta 2280caggaaatgg caagccgttg gccgtttttc cgcacgacta
ttcggaatat ggaacaggtg 2340atggcacgtt ccgatatgac gatcgccaag
cattatctgg ccttggttga ggatcagaca 2400aatggtgagg ctatctatga
ttctatcgcg gatggctgga ataaaggttg tgaaggtctg 2460ttaaaggcaa
cccagcagaa ttggctgttg gaacgctttc cggcggttga taattcggtg
2520cagatgcgtc ggccttatct ggaaccgctt aattacttac aggtcgaatt
gctgaagaaa 2580tggcggggag gtgataccaa cccgcatatc ctcgaatcta
ttcagctgac aatcaatgcc 2640attgcgacgg cacttcgcaa cagcggttaa
taactcgag 2679212679DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 21actagtaaaa aaatgaccaa
gccaagaact attaaccaaa acccagactt gagatacttc 60ggtaacttgt tgggtcaagt
tatcaaggaa caaggtggtg aatctttgtt caaccaaatt 120gaacaaatca
gatccgctgc tattagaaga cacagaggta tcgtcgactc taccgaattg
180tcctctagat tggctgactt ggacttgaac gacatgttct ccttcgctca
cgctttcttg 240ttgttctcta tgttggctaa cttggctgac gacagacaag
gtgacgcttt ggacccagac 300gctaacatgg cttccgcttt gaaggacatt
aaggctaagg gtgtttctca acaagctatc 360attgacatga tcgacaaggc
ttgtattgtc ccagttttga ctgctcaccc aaccgaagtc 420agaagaaagt
ccatgttgga ccactacaac agaatcgctg gtttgatgag attgaaggac
480gctggtcaaa ctgttaccga agacggtttg ccaattgaag acgctttgat
ccaacaaatt 540actatcttgt ggcaaaccag accattgatg ttgcaaaagt
tgactgtcgc tgacgaaatt 600gaaaccgctt tgtctttctt gagagaaact
ttcttgccag ttttgccaca aatctacgct 660gaatgggaaa agttgttggg
ttcctctatt ccatccttca tcagaccagg taactggatt 720ggtggtgaca
gagacggtaa cccaaacgtc aacgctgaca ccatcatgtt gtctttgaag
780agatcctctg aaactgtttt gaccgactac ttgaacagat tggacaagtt
gttgtccaac 840ttgtctgtct ccactgacat ggtttctgtc tccgacgaca
ttttgagatt ggctgacaag 900tctggtgacg acgctgctat cagagctgac
gaaccataca gaagagcttt gaacggtatt 960tacgacagat tggctgctac
ctacagacaa atcgctggta gaaacccatc cagaccagct 1020ttgagatctg
ctgaagctta caagagacca caagaattgt tggctgactt gaagactttg
1080gctgaaggtt tgggtaagtt ggctgaaggt tccttcaagg ctttgattag
atctgttgaa 1140accttcggtt tccacttggc tactttggac ttgagacaaa
actcccaagt ccacgaaaga 1200gttgtcaacg aattgttgag aaccgctact
gttgaagctg actacttgtc tttgtccgaa 1260gaagacagag tcaagttgtt
gagaagagaa ttgtctcaac caagaacctt gttcgttcca 1320agagctgact
actccgaaga aactagatct gaattggaca tcattcaagc tgctgctaga
1380gctcacgaaa tcttcggtcc agaatccatt accacttact tgatctctaa
cggtgaatcc 1440atttctgaca tcttggaagt ctacttgttg ttgaaggaag
ctggtttgta ccaaggtggt 1500gctaagccaa aggctgctat tgaagctgct
ccattgttcg aaaccgttgc tgacttggaa 1560aacgctccaa aggtcatgga
agaatggttc aagttgccag aagctcaagc tatcgctaag 1620gctcacggtg
ttcaagaagt catggttggt tactccgact ctaacaagga cggtggttac
1680ttgacttccg tctggggttt gtacaaggct tgtttggctt tggttccaat
tttcgaaaag 1740gctggtgtcc caatccaatt cttccacggt agaggtggtt
ctgttggtag aggtggtggt 1800tccaacttca acgctatttt gtctcaacca
gctggtgctg tcaagggtag aatcagatac 1860accgaacaag gtgaagttgt
cgctgctaag tacggtactc acgaatccgc tattgctcac 1920ttggacgaag
ctgttgctgc taccttgatc acttctttgg aagctccaac cattgtcgaa
1980ccagaattct ccagatacag aaaggctttg gaccaaatct ctgactccgc
tttccaagct 2040tacagacaat tggtttacgg tactaagggt ttcagaaagt
tcttctctga attcacccca 2100ttgccagaaa ttgctttgtt gaagatcggt
tccagaccac catctagaaa gaagtccgac 2160agaattgaag acttgagagc
tatcccatgg gtcttctctt ggtcccaagt tagagtcatg 2220ttgccaggtt
ggttcggttt cggtcaagct ttgtacgact tcgaagacac tgaattgttg
2280caagaaatgg cttctagatg gccattcttc agaaccacta ttagaaacat
ggaacaagtt 2340atggctagat ccgacatgac catcgctaag cactacttgg
ctttggtcga agaccaaact 2400aacggtgaag ctatttacga ctctatcgct
gacggttgga acaagggttg tgaaggtttg 2460ttgaaggcta cccaacaaaa
ctggttgttg gaaagattcc cagctgttga caactccgtc 2520caaatgagaa
gaccatactt ggaaccattg aactacttgc aagttgaatt gttgaagaag
2580tggagaggtg gtgacactaa cccacacatt ttggaatcta tccaattgac
cattaacgct 2640atcgctactg ctttgagaaa ctccggttaa taactcgag
2679221317DNARuminococcus flavefaciens 22atggaatttt tcagcaatat
cggtaaaatt cagtatcagg gaccaaaaag tactgatcct 60ctctcattta agtactataa
ccctgaagaa gtcatcaacg gaaagacaat gcgcgagcat 120ctgaagttcg
ctctttcatg gtggcacaca atgggcggcg acggaacaga tatgttcggc
180tgcggcacaa cagacaagac ctggggacag tccgatcccg ctgcaagagc
aaaggctaag 240gttgacgcag cattcgagat catggataag ctctccattg
actactattg tttccacgat 300cgcgatcttt ctcccgagta tggcagcctc
aaggctacca acgatcagct tgacatagtt 360acagactata tcaaggagaa
gcagggcgac aagttcaagt gcctctgggg tacagcaaag 420tgcttcgatc
atccaagatt catgcacggt gcaggtacat ctccttctgc tgatgtattc
480gctttctcag ctgctcagat caagaaggct ctggagtcaa cagtaaagct
cggcggtaac 540ggttacgttt tctggggcgg acgtgaaggc tatgagacac
ttcttaatac aaatatggga 600ctcgaactcg acaatatggc tcgtcttatg
aagatggctg ttgagtatgg acgttcgatc 660ggcttcaagg gcgacttcta
tatcgagccc aagcccaagg agcccacaaa gcatcagtac 720gatttcgata
cagctactgt tctgggattc ctcagaaagt acggtctcga taaggatttc
780aagatgaata tcgaagctaa ccacgctaca cttgctcagc atacattcca
gcatgagctc 840cgtgttgcaa gagacaatgg tgtgttcggt tctatcgacg
caaaccaggg cgacgttctt 900cttggatggg atacagacca gttccccaca
aatatctacg atacaacaat gtgtatgtat 960gaagttatca aggcaggcgg
cttcacaaac ggcggtctca acttcgacgc taaggcacgc 1020agagggagct
tcactcccga ggatatcttc tacagctata tcgcaggtat ggatgcattt
1080gctctgggct tcagagctgc tctcaagctt atcgaagacg gacgtatcga
caagttcgtt 1140gctgacagat acgcttcatg gaataccggt atcggtgcag
acataatcgc aggtaaggca 1200gatttcgcat ctcttgaaaa gtatgctctt
gaaaagggcg aggttacagc ttcactctca 1260agcggcagac aggaaatgct
ggagtctatc gtaaataacg ttcttttcag tctgtaa 1317231314DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
23atggagttct tttctaatat aggtaaaatt cagtatcaag gtccaaaatc tacagatcca
60ttgtctttta aatattataa tccagaagaa gttataaatg gtaaaactat gagagaacat
120ttaaaatttg ctttgtcttg gtggcatact atgggtggtg atggtactga
tatgttcggt 180tgtggtacta ctgataaaac ttggggtcaa tctgatccag
ctgctagagc aaaagccaaa 240gtagatgcag cctttgaaat tatggataaa
ttgtctattg attattattg ttttcatgat 300agagatttgt ctcctgaata
tggttcttta aaagcaacta atgatcaatt ggacattgtt 360acggattata
ttaaagaaaa acaaggtgat aaatttaaat gtttgtgggg cactgcgaaa
420tgttttgatc atccacgttt tatgcatggt gcggggacga gtccttctgc
tgatgttttt 480gctttttctg ccgctcaaat taagaaggca ttggaatcaa
ctgttaaatt aggtgggaac 540gggtatgtat tctggggagg aagggaaggt
tatgaaacat tattaaacac taatatgggt 600ttggaattgg ataatatggc
tagattgatg aaaatggctg tagaatacgg aaggtctatt 660ggttttaagg
gtgactttta tattgaacca aaacctaaag agcctactaa acatcaatat
720gattttgata ctgctacagt tttgggattc ttgagaaaat atggtctgga
taaagatttt 780aaaatgaata tagaagctaa tcatgcaaca ctcgcacaac
atacttttca acatgaattg 840agagttgcca gagataacgg agtttttgga
tctatcgatg caaaccaggg agacgttttg 900ctaggatggg atactgatca
atttccaact aacatttatg atactactat gtgtatgtat 960gaagtaatta
aggcaggagg ctttactaat ggcggattaa actttgatgc gaaggctagg
1020cgtggtagtt tcactccaga ggatatattc tattcttata ttgctggaat
ggatgctttc 1080gcgttaggtt tcagggcagc actaaaattg attgaagatg
gtagaattga taagtttgta 1140gctgatagat atgcttcttg gaatactgga
ataggagcag atataatcgc tgggaaagcc 1200gacttcgcca gtctggaaaa
atatgcgctt gaaaaaggag aagttactgc cagcttaagt 1260tccggtcgtc
aagaaatgtt ggaatctatt gtaaacaatg ttttattttc tctg
1314241314DNAPiromyces sp. 24atggctaagg aatatttccc acaaattcaa
aagattaagt tcgaaggtaa ggattctaag 60aatccattag ccttccacta ctacgatgct
gaaaaggaag tcatgggtaa gaaaatgaag 120gattggttac gtttcgccat
ggcctggtgg cacactcttt gcgccgaagg tgctgaccaa 180ttcggtggag
gtacaaagtc tttcccatgg aacgaaggta ctgatgctat tgaaattgcc
240aagcaaaagg ttgatgctgg tttcgaaatc atgcaaaagc ttggtattcc
atactactgt 300ttccacgatg ttgatcttgt ttccgaaggt aactctattg
aagaatacga atccaacctt 360aaggctgtcg ttgcttacct caaggaaaag
caaaaggaaa ccggtattaa gcttctctgg 420agtactgcta acgtcttcgg
tcacaagcgt tacatgaacg gtgcctccac taacccagac 480tttgatgttg
tcgcccgtgc tattgttcaa attaagaacg ccatagacgc cggtattgaa
540cttggtgctg aaaactacgt cttctggggt ggtcgtgaag gttacatgag
tctccttaac 600actgaccaaa agcgtgaaaa ggaacacatg gccactatgc
ttaccatggc tcgtgactac 660gctcgttcca agggattcaa gggtactttc
ctcattgaac caaagccaat ggaaccaacc 720aagcaccaat acgatgttga
cactgaaacc gctattggtt tccttaaggc ccacaactta 780gacaaggact
tcaaggtcaa cattgaagtt aaccacgcta ctcttgctgg tcacactttc
840gaacacgaac ttgcctgtgc tgttgatgct ggtatgctcg gttccattga
tgctaaccgt 900ggtgactacc aaaacggttg ggatactgat caattcccaa
ttgatcaata cgaactcgtc 960caagcttgga tggaaatcat ccgtggtggt
ggtttcgtta ctggtggtac caacttcgat 1020gccaagactc gtcgtaactc
tactgacctc gaagacatca tcattgccca cgtttctggt 1080atggatgcta
tggctcgtgc tcttgaaaac gctgccaagc tcctccaaga atctccatac
1140accaagatga agaaggaacg ttacgcttcc ttcgacagtg gtattggtaa
ggactttgaa 1200gatggtaagc tcaccctcga acaagtttac gaatacggta
agaagaacgg tgaaccaaag 1260caaacttctg gtaagcaaga actctacgaa
gctattgttg ccatgtacca ataa 1314251314DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
25atggctaaag aatattttcc acaaattcag aaaattaaat ttgaaggtaa agattctaaa
60aatccattgg ctttccatta ttatgatgct gaaaaagaag ttatgggtaa aaagatgaaa
120gattggttga gattcgctat ggcttggtgg catactctat gtgctgaagg
agctgatcaa 180tttggaggag gtactaaatc ttttccttgg aatgaaggta
ctgacgctat tgaaattgct 240aagcagaaag tagacgcggg ttttgaaatt
atgcaaaaat tgggaatacc atattattgt 300tttcatgatg ttgatttggt
atctgagggt aattctattg aagaatatga atctaattta 360aaagctgttg
ttgcttactt aaaagaaaaa caaaaagaaa ctggaattaa attgttgtgg
420tctacagcta atgttttcgg tcataaaaga tatatgaatg gtgcttctac
aaatccagat 480tttgatgttg tagctagagc tattgttcaa attaaaaatg
ctatagatgc aggaattgaa 540ttaggtgccg aaaattatgt tttctgggga
ggtagagaag gttatatgtc tttgttaaat 600actgatcaaa aacgtgaaaa
ggaacacatg gcaactatgt tgacaatggc tagggattat 660gctagatcta
aaggttttaa aggtactttc ttgattgagc caaaacctat ggaaccaact
720aaacatcaat atgacgttga cactgaaact gctattggtt tcttaaaagc
tcataatttg 780gataaagatt ttaaggttaa tatagaagtt aatcatgcta
cactagctgg tcatactttt 840gaacatgaat tagcttgtgc agttgatgcc
ggtatgttag gttctatcga cgcaaataga 900ggtgattatc aaaatggttg
ggacacagat caatttccaa tagatcaata tgaattggtt 960caagcatgga
tggaaattat taggggtgga ggcttcgtta caggtggaac taattttgat
1020gctaaaacta ggagaaattc tacagatctt gaagatataa ttattgctca
tgtatctggt 1080atggatgcga tggcccgtgc tttggaaaat gcagctaaat
tacttcaaga atctccttat 1140actaaaatga aaaaggaaag atatgcttct
tttgattctg gaataggtaa ggattttgaa 1200gatggtaaat tgacattgga
acaagtttat gaatatggta agaagaatgg agaaccaaaa 1260caaacttctg
gtaaacaaga attatatgag gctatagtag ctatgtatca ataa
13142644DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 26acttgactac tagtatggag ttcttttcta atataggtaa aatt
442744DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 27agtcaagtct cgagcagaga aaataaaaca ttgtttacaa taga
442859DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 28agtcaagtct cgagctaatg atgatgatga tgatgcagag
aaaataaaac attgtttac 59291317DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 29atggaatttt
tcagcaatat cggtaaaatt cagtatcagg gaccaaaaag tactgatcct 60ctctcattta
agtactataa ccctgaagaa gtcatcaacg gaaagacaat gcgcgagcat
120ctgaagttcg ctctttcatg gtggcacaca atgggcggcg acggaacaga
tatgttcggc 180tgcggcacaa cagacaagac ctggggacag tccgatcccg
ctgcaagagc aaaggctaag 240gttgacgcag cattcgagat catggataag
ctctccattg actactattg tttccacgat 300cgcgatcttt ctcccgagta
tggcagcctc aaggctacca acgatcagct tgacatagtt 360acagactata
tcaaggagaa gcagggcgac aagttcaagt gcctctgggg tacagcaaag
420tgcttcgatc atccaagatt catgcacggt gcaggtacat ctccttctgc
tgatgtattc 480gctttctcag ctgctcagat caagaaggct ctggagtcaa
cagtaaagct cggcggtaac 540ggttacgttt tctggggcgg acgtgaaggc
tatgagacac ttcttaatac aaatatggga 600ctcgaactcg acaatatggc
tcgtcttatg aagatggctg ttgagtatgg acgttcgatc 660ggcttcaagg
gcgacttcta tatcgagccc aagcccaagg agcccacaaa gcatcagtac
720gatttcgata cagctactgt
tctgggattc ctcagaaagt acggtctcga taaggatttc 780aagatgaata
tcgaagctaa ccacgctaca cttgctcagc atacattcca gcatgagctc
840cgtgttgcaa gagacaatgg tgtgttcggt tctatcgacg caaaccaggg
cgacgttctt 900cttggatggg atacagacca gttccccaca aatatctacg
atacaacaat gtgtatgtat 960gaagttatca aggcaggcgg cttcacaaac
ggcggtctca acttcgacgc taaggcacgc 1020agagggagct tcactcccga
ggatatcttc tacagctata tcgcaggtat ggatgcattt 1080gctctgggct
tcagagctgc tctcaagctt atcgaagacg gacgtatcga caagttcgtt
1140gctgacagat acgcttcatg gaataccggt atcggtgcag acataatcgc
aggtaaggca 1200gatttcgcat ctcttgaaaa gtatgctctt gaaaagggcg
aggttacagc ttcactctca 1260agcggcagac aggaaatgct ggagtctatc
gtaaataacg ttcttttcag tctgtaa 1317301317DNARuminococcus
flavefaciens 30atggaatttt tcagcaatat cggtaaaatt cagtatcagg
gaccaaaaag tactgatcct 60ctctcattta agtactataa ccctgaagaa gtcatcaacg
gaaagacaat gcgcgagcat 120ctgaagttcg ctctttcatg gtggcacaca
atgggcggcg acggaacaga tatgttcggc 180tgcggcacaa cagacaagac
ctggggacag tccgatcccg ctgcaagagc aaaggctaag 240gttgacgcag
cattcgagat catggataag ctctccattg actactattg tttccacgat
300cgcgatcttt ctcccgagta tggcagcctc aaggctacca acgatcagct
tgacatagtt 360acagactata tcaaggagaa gcagggcgac aagttcaagt
gcctctgggg tacagcaaag 420tgcttcgatc atccaagatt catgcacggt
gcaggtacat ctccttctgc tgatgtattc 480gctttctcag ctgctcagat
caagaaggct ctcgagtcaa cagtaaagct cggcggtaac 540ggttacgttt
tctggggcgg acgtgaaggc tatgagacac ttcttaatac aaatatggga
600ctcgaactcg acaatatggc tcgtcttatg aagatggctg ttgagtatgg
acgttcgatc 660ggcttcaagg gcgacttcta tatcgagccc aagcccaagg
agcccacaaa gcatcagtac 720gatttcgata cagctactgt tctgggattc
ctcagaaagt acggtctcga taaggatttc 780aagatgaata tcgaagctaa
ccacgctaca cttgctcagc atacattcca gcatgagctc 840cgtgttgcaa
gagacaatgg tgtgttcggt tctatcgacg caaaccaggg cgacgttctt
900cttggatggg atacagacca gttccccaca aatatctacg atacaacaat
gtgtatgtat 960gaagttatca aggcaggcgg cttcacaaac ggcggtctca
acttcgacgc taaggcacgc 1020agagggagct tcactcccga ggatatcttc
tacagctata tcgcaggtat ggatgcattt 1080gctctgggct tcagagctgc
tctcaagctt atcgaagacg gacgtatcga caagttcgtt 1140gctgacagat
acgcttcatg gaataccggt atcggtgcag acataatcgc aggtaaggca
1200gatttcgcat ctcttgaaaa gtatgctctt gaaaagggcg aggttacagc
ttcactctca 1260agcggcagac aggaaatgct ggagtctatc gtaaataacg
ttcttttcag tctgtaa 131731438PRTRuminococcus flavefaciens 31Met Glu
Phe Phe Ser Asn Ile Gly Lys Ile Gln Tyr Gln Gly Pro Lys1 5 10 15Ser
Thr Asp Pro Leu Ser Phe Lys Tyr Tyr Asn Pro Glu Glu Val Ile 20 25
30Asn Gly Lys Thr Met Arg Glu His Leu Lys Phe Ala Leu Ser Trp Trp
35 40 45His Thr Met Gly Gly Asp Gly Thr Asp Met Phe Gly Cys Gly Thr
Thr 50 55 60Asp Lys Thr Trp Gly Gln Ser Asp Pro Ala Ala Arg Ala Lys
Ala Lys65 70 75 80Val Asp Ala Ala Phe Glu Ile Met Asp Lys Leu Ser
Ile Asp Tyr Tyr 85 90 95Cys Phe His Asp Arg Asp Leu Ser Pro Glu Tyr
Gly Ser Leu Lys Ala 100 105 110Thr Asn Asp Gln Leu Asp Ile Val Thr
Asp Tyr Ile Lys Glu Lys Gln 115 120 125Gly Asp Lys Phe Lys Cys Leu
Trp Gly Thr Ala Lys Cys Phe Asp His 130 135 140Pro Arg Phe Met His
Gly Ala Gly Thr Ser Pro Ser Ala Asp Val Phe145 150 155 160Ala Phe
Ser Ala Ala Gln Ile Lys Lys Ala Leu Glu Ser Thr Val Lys 165 170
175Leu Gly Gly Asn Gly Tyr Val Phe Trp Gly Gly Arg Glu Gly Tyr Glu
180 185 190Thr Leu Leu Asn Thr Asn Met Gly Leu Glu Leu Asp Asn Met
Ala Arg 195 200 205Leu Met Lys Met Ala Val Glu Tyr Gly Arg Ser Ile
Gly Phe Lys Gly 210 215 220Asp Phe Tyr Ile Glu Pro Lys Pro Lys Glu
Pro Thr Lys His Gln Tyr225 230 235 240Asp Phe Asp Thr Ala Thr Val
Leu Gly Phe Leu Arg Lys Tyr Gly Leu 245 250 255Asp Lys Asp Phe Lys
Met Asn Ile Glu Ala Asn His Ala Thr Leu Ala 260 265 270Gln His Thr
Phe Gln His Glu Leu Arg Val Ala Arg Asp Asn Gly Val 275 280 285Phe
Gly Ser Ile Asp Ala Asn Gln Gly Asp Val Leu Leu Gly Trp Asp 290 295
300Thr Asp Gln Phe Pro Thr Asn Ile Tyr Asp Thr Thr Met Cys Met
Tyr305 310 315 320Glu Val Ile Lys Ala Gly Gly Phe Thr Asn Gly Gly
Leu Asn Phe Asp 325 330 335Ala Lys Ala Arg Arg Gly Ser Phe Thr Pro
Glu Asp Ile Phe Tyr Ser 340 345 350Tyr Ile Ala Gly Met Asp Ala Phe
Ala Leu Gly Phe Arg Ala Ala Leu 355 360 365Lys Leu Ile Glu Asp Gly
Arg Ile Asp Lys Phe Val Ala Asp Arg Tyr 370 375 380Ala Ser Trp Asn
Thr Gly Ile Gly Ala Asp Ile Ile Ala Gly Lys Ala385 390 395 400Asp
Phe Ala Ser Leu Glu Lys Tyr Ala Leu Glu Lys Gly Glu Val Thr 405 410
415Ala Ser Leu Ser Ser Gly Arg Gln Glu Met Leu Glu Ser Ile Val Asn
420 425 430Asn Val Leu Phe Ser Leu 435321317DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
32atggagttct tttctaatat aggtaaaatt cagtatcaag gtccaaaatc tacagatcca
60ttgtctttta aatattataa tccagaagaa gttataaatg gtaaaactat gagagaacat
120ttaaaatttg ctttgtcttg gtggcatact atgggtggtg atggtactga
tatgttcggt 180tgtggtacta ctgataaaac ttggggtcaa tctgatccag
ctgctagagc aaaagccaaa 240gtagatgcag cctttgaaat tatggataaa
ttgtctattg attattattg ttttcatgat 300agagatttgt ctcctgaata
tggttcttta aaagcaacta atgatcaatt ggacattgtt 360acggattata
ttaaagaaaa acaaggtgat aaatttaaat gtttgtgggg cactgcgaaa
420tgttttgatc atccacgttt tatgcatggt gcggggacga gtccttctgc
tgatgttttt 480gctttttctg ccgctcaaat taagaaggca ttggaatcaa
ctgttaaatt aggtgggaac 540gggtatgtat tctggggagg aagggaaggt
tatgaaacat tattaaacac taatatgggt 600ttggaattgg ataatatggc
tagattgatg aaaatggctg tagaatacgg aaggtctatt 660ggttttaagg
gtgactttta tattgaacca aaacctaaag agcctactaa acatcaatat
720gattttgata ctgctacagt tttgggattc ttgagaaaat atggtctgga
taaagatttt 780aaaatgaata tagaagctaa tcatgcaaca ctcgcacaac
atacttttca acatgaattg 840agagttgcca gagataacgg agtttttgga
tctatcgatg caaaccaggg agacgttttg 900ctaggatggg atactgatca
atttccaact aacatttatg atactactat gtgtatgtat 960gaagtaatta
aggcaggagg ctttactaat ggcggattaa actttgatgc gaaggctagg
1020cgtggtagtt tcactccaga ggatatattc tattcttata ttgctggaat
ggatgctttc 1080gcgttaggtt tcagggcagc actaaaattg attgaagatg
gtagaattga taagtttgta 1140gctgatagat atgcttcttg gaatactgga
ataggagcag atataatcgc tgggaaagcc 1200gacttcgcca gtctggaaaa
atatgcgctt gaaaaaggag aagttactgc cagcttaagt 1260tccggtcgtc
aagaaatgtt ggaatctatt gtaaacaatg ttttattttc tctgtaa
1317331317DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 33atggaattct tctctaacat tggtaagatc
caataccaag gtccaaagtc caccgaccca 60ttgtctttca agtactacaa cccagaagaa
gttattaacg gtaagactat gagagaacac 120ttgaagttcg ctttgtcctg
gtggcacacc atgggtggtg acggtactga catgttcggt 180tgtggtacca
ctgacaagac ctggggtcaa tctgacccag ctgctagagc taaggctaag
240gtcgacgctg ctttcgaaat catggacaag ttgtccattg actactactg
tttccacgac 300agagacttgt ctccagaata cggttccttg aaggctacta
acgaccaatt ggacatcgtt 360accgactaca ttaaggaaaa gcaaggtgac
aagttcaagt gtttgtgggg tactgctaag 420tgtttcgacc acccaagatt
catgcacggt gctggtacct ctccatccgc tgacgtcttc 480gctttctctg
ctgctcaaat caagaaggct ttggaatcca ctgttaagtt gggtggtaac
540ggttacgtct tctggggtgg tagagaaggt tacgaaacct tgttgaacac
taacatgggt 600ttggaattgg acaacatggc tagattgatg aagatggctg
ttgaatacgg tagatctatt 660ggtttcaagg gtgacttcta catcgaacca
aagccaaagg aaccaaccaa gcaccaatac 720gacttcgaca ctgctaccgt
cttgggtttc ttgagaaagt acggtttgga caaggacttc 780aagatgaaca
ttgaagctaa ccacgctact ttggctcaac acaccttcca acacgaattg
840agagttgcta gagacaacgg tgtcttcggt tccatcgacg ctaaccaagg
tgacgttttg 900ttgggttggg acactgacca attcccaacc aacatttacg
acactaccat gtgtatgtac 960gaagtcatca aggctggtgg tttcactaac
ggtggtttga acttcgacgc taaggctaga 1020agaggttctt tcaccccaga
agacattttc tactcctaca tcgctggtat ggacgctttc 1080gctttgggtt
tcagagctgc tttgaagttg attgaagacg gtagaatcga caagttcgtt
1140gctgacagat acgcttcttg gaacactggt attggtgctg acatcattgc
tggtaaggct 1200gacttcgctt ccttggaaaa gtacgctttg gaaaagggtg
aagtcaccgc ttctttgtcc 1260tctggtagac aagaaatgtt ggaatccatc
gttaacaacg tcttgttctc tttgtaa 1317341335DNAPiromyces sp.
34actagtaaaa aaatggctaa ggaatatttc ccacaaattc aaaagattaa gttcgaaggt
60aaggattcta agaatccatt agccttccac tactacgatg ctgaaaagga agtcatgggt
120aagaaaatga aggattggtt acgtttcgcc atggcctggt ggcacactct
ttgcgccgaa 180ggtgctgacc aattcggtgg aggtacaaag tctttcccat
ggaacgaagg tactgatgct 240attgaaattg ccaagcaaaa ggttgatgct
ggtttcgaaa tcatgcaaaa gcttggtatt 300ccatactact gtttccacga
tgttgatctt gtttccgaag gtaactctat tgaagaatac 360gaatccaacc
ttaaggctgt cgttgcttac ctcaaggaaa agcaaaagga aaccggtatt
420aagcttctct ggagtactgc taacgtcttc ggtcacaagc gttacatgaa
cggtgcctcc 480actaacccag actttgatgt tgtcgcccgt gctattgttc
aaattaagaa cgccatagac 540gccggtattg aacttggtgc tgaaaactac
gtcttctggg gtggtcgtga aggttacatg 600agtctcctta acactgacca
aaagcgtgaa aaggaacaca tggccactat gcttaccatg 660gctcgtgact
acgctcgttc caagggattc aagggtactt tcctcattga accaaagcca
720atggaaccaa ccaagcacca atacgatgtt gacactgaaa ccgctattgg
tttccttaag 780gcccacaact tagacaagga cttcaaggtc aacattgaag
ttaaccacgc tactcttgct 840ggtcacactt tcgaacacga acttgcctgt
gctgttgatg ctggtatgct cggttccatt 900gatgctaacc gtggtgacta
ccaaaacggt tgggatactg atcaattccc aattgatcaa 960tacgaactcg
tccaagcttg gatggaaatc atccgtggtg gtggtttcgt tactggtggt
1020accaacttcg atgccaagac tcgtcgtaac tctactgacc tcgaagacat
catcattgcc 1080cacgtttctg gtatggatgc tatggctcgt gctcttgaaa
acgctgccaa gctcctccaa 1140gaatctccat acaccaagat gaagaaggaa
cgttacgctt ccttcgacag tggtattggt 1200aaggactttg aagatggtaa
gctcaccctc gaacaagttt acgaatacgg taagaagaac 1260ggtgaaccaa
agcaaacttc tggtaagcaa gaactctacg aagctattgt tgccatgtac
1320caatagtagc tcgag 133535437PRTPiromyces sp. 35Met Ala Lys Glu
Tyr Phe Pro Gln Ile Gln Lys Ile Lys Phe Glu Gly1 5 10 15Lys Asp Ser
Lys Asn Pro Leu Ala Phe His Tyr Tyr Asp Ala Glu Lys 20 25 30Glu Val
Met Gly Lys Lys Met Lys Asp Trp Leu Arg Phe Ala Met Ala 35 40 45Trp
Trp His Thr Leu Cys Ala Glu Gly Ala Asp Gln Phe Gly Gly Gly 50 55
60Thr Lys Ser Phe Pro Trp Asn Glu Gly Thr Asp Ala Ile Glu Ile Ala65
70 75 80Lys Gln Lys Val Asp Ala Gly Phe Glu Ile Met Gln Lys Leu Gly
Ile 85 90 95Pro Tyr Tyr Cys Phe His Asp Val Asp Leu Val Ser Glu Gly
Asn Ser 100 105 110Ile Glu Glu Tyr Glu Ser Asn Leu Lys Ala Val Val
Ala Tyr Leu Lys 115 120 125Glu Lys Gln Lys Glu Thr Gly Ile Lys Leu
Leu Trp Ser Thr Ala Asn 130 135 140Val Phe Gly His Lys Arg Tyr Met
Asn Gly Ala Ser Thr Asn Pro Asp145 150 155 160Phe Asp Val Val Ala
Arg Ala Ile Val Gln Ile Lys Asn Ala Ile Asp 165 170 175Ala Gly Ile
Glu Leu Gly Ala Glu Asn Tyr Val Phe Trp Gly Gly Arg 180 185 190Glu
Gly Tyr Met Ser Leu Leu Asn Thr Asp Gln Lys Arg Glu Lys Glu 195 200
205His Met Ala Thr Met Leu Thr Met Ala Arg Asp Tyr Ala Arg Ser Lys
210 215 220Gly Phe Lys Gly Thr Phe Leu Ile Glu Pro Lys Pro Met Glu
Pro Thr225 230 235 240Lys His Gln Tyr Asp Val Asp Thr Glu Thr Ala
Ile Gly Phe Leu Lys 245 250 255Ala His Asn Leu Asp Lys Asp Phe Lys
Val Asn Ile Glu Val Asn His 260 265 270Ala Thr Leu Ala Gly His Thr
Phe Glu His Glu Leu Ala Cys Ala Val 275 280 285Asp Ala Gly Met Leu
Gly Ser Ile Asp Ala Asn Arg Gly Asp Tyr Gln 290 295 300Asn Gly Trp
Asp Thr Asp Gln Phe Pro Ile Asp Gln Tyr Glu Leu Val305 310 315
320Gln Ala Trp Met Glu Ile Ile Arg Gly Gly Gly Phe Val Thr Gly Gly
325 330 335Thr Asn Phe Asp Ala Lys Thr Arg Arg Asn Ser Thr Asp Leu
Glu Asp 340 345 350Ile Ile Ile Ala His Val Ser Gly Met Asp Ala Met
Ala Arg Ala Leu 355 360 365Glu Asn Ala Ala Lys Leu Leu Gln Glu Ser
Pro Tyr Thr Lys Met Lys 370 375 380Lys Glu Arg Tyr Ala Ser Phe Asp
Ser Gly Ile Gly Lys Asp Phe Glu385 390 395 400Asp Gly Lys Leu Thr
Leu Glu Gln Val Tyr Glu Tyr Gly Lys Lys Asn 405 410 415Gly Glu Pro
Lys Gln Thr Ser Gly Lys Gln Glu Leu Tyr Glu Ala Ile 420 425 430Val
Ala Met Tyr Gln 4353656DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 36agttaagtga gtaaactagt
gaattccaga gaaaataaaa cattgtttac aataga 563759DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
37agtcaagtct cgagtcaatg gtgatggtgg tgatgcagag aaaataaaac attgtttac
593837DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 38actagtatgg aatttttcag caatatcggt aaaattc
373934DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 39ctcgagttac agactgaaaa gaacgttatt tacg
344029DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 40actagtatgg aattcttctc taacattgg
294138DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 41ctcgagttac aaagagaaca agacgttgtt aacgatgg
384245DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 42actagtaaaa aaatggctaa ggaatatttc ccacaaattc
aaaag 454359DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 43atgactcgag ctactaatga tgatgatgat
gatgttggta catggcaaca atagcttcg 594443DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 44actagtaaaa aaatggaatt tttcagcaat atcggtaaaa ttc
434551DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 45actagtaaaa aaatggctaa ggaatatttc
agcaatatcg gtaaaattca g 514641DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 46ttcccacaaa
ttcaaaaaat tcagtatcag ggaccaaaaa g 414768DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 47actagtaaaa aaatggctaa ggaatatttc ccacaaattc
aaaaaattca gtatcaggga 60ccaaaaag 684841DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 48aagattaagt tcgaaggacc aaaaagtact gatcctctct c
414956DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 49ttcccacaaa ttcaaaagat taagttcgaa
ggaccaaaaa gtactgatcc tctctc 565054DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 50actagtaaaa aaatggctaa ggaatatttc ccacaaattc
aaaagattaa gttc 545146DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 51ggtaaggatt
ctaaggatcc tctctcattt aagtactata accctg 465254DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 52caaaagatta agttcgaagg taaggattct aaggatcctc
tctcatttaa gtac 545334DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 53ctcgagttac
agactgaaaa gaacgttatt tacg 345434DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 54ctcgagttac
agactgaaaa gaacgttatt tacg 34551329DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
55aaaaaaatgg ctaaggaata tttcagcaat atcggtaaaa ttcagtatca gggaccaaaa
60agtactgatc ctctctcatt taagtactat aaccctgaag aagtcatcaa cggaaagaca
120atgcgcgagc atctgaagtt cgctctttca tggtggcaca caatgggcgg
cgacggaaca 180gatatgttcg gctgcggcac aacagacaag acctggggac
agtccgatcc cgctgcaaga 240gcaaaggcta aggttgacgc agcattcgag
atcatggata agctctccat tgactactat 300tgtttccacg atcgcgatct
ttctcccgag tatggcagcc tcaaggctac caacgatcag 360cttgacatag
ttacagacta tatcaaggag aagcagggcg acaagttcaa gtgcctctgg
420ggtacagcaa agtgcttcga tcatccaaga ttcatgcacg gtgcaggtac
atctccttct 480gctgatgtat tcgctttctc agctgctcag atcaagaagg
ctctggagtc aacagtaaag 540ctcggcggta acggttacgt tttctggggc
ggacgtgaag gctatgagac acttcttaat 600acaaatatgg gactcgaact
cgacaatatg gctcgtctta tgaagatggc tgttgagtat 660ggacgttcga
tcggcttcaa gggcgacttc tatatcgagc ccaagcccaa ggagcccaca
720aagcatcagt acgatttcga tacagctact
gttctgggat tcctcagaaa gtacggtctc 780gataaggatt tcaagatgaa
tatcgaagct aaccacgcta cacttgctca gcatacattc 840cagcatgagc
tccgtgttgc aagagacaat ggtgtgttcg gttctatcga cgcaaaccag
900ggcgacgttc ttcttggatg ggatacagac cagttcccca caaatatcta
cgatacaaca 960atgtgtatgt atgaagttat caaggcaggc ggcttcacaa
acggcggtct caacttcgac 1020gctaaggcac gcagagggag cttcactccc
gaggatatct tctacagcta tatcgcaggt 1080atggatgcat ttgctctggg
cttcagagct gctctcaagc ttatcgaaga cggacgtatc 1140gacaagttcg
ttgctgacag atacgcttca tggaataccg gtatcggtgc agacataatc
1200gcaggtaagg cagatttcgc atctcttgaa aagtatgctc ttgaaaaggg
cgaggttaca 1260gcttcactct caagcggcag acaggaaatg ctggagtcta
tcgtaaataa cgttcttttc 1320agtctgtaa 132956440PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
56Met Ala Lys Glu Tyr Phe Ser Asn Ile Gly Lys Ile Gln Tyr Gln Gly1
5 10 15Pro Lys Ser Thr Asp Pro Leu Ser Phe Lys Tyr Tyr Asn Pro Glu
Glu 20 25 30Val Ile Asn Gly Lys Thr Met Arg Glu His Leu Lys Phe Ala
Leu Ser 35 40 45Trp Trp His Thr Met Gly Gly Asp Gly Thr Asp Met Phe
Gly Cys Gly 50 55 60Thr Thr Asp Lys Thr Trp Gly Gln Ser Asp Pro Ala
Ala Arg Ala Lys65 70 75 80Ala Lys Val Asp Ala Ala Phe Glu Ile Met
Asp Lys Leu Ser Ile Asp 85 90 95Tyr Tyr Cys Phe His Asp Arg Asp Leu
Ser Pro Glu Tyr Gly Ser Leu 100 105 110Lys Ala Thr Asn Asp Gln Leu
Asp Ile Val Thr Asp Tyr Ile Lys Glu 115 120 125Lys Gln Gly Asp Lys
Phe Lys Cys Leu Trp Gly Thr Ala Lys Cys Phe 130 135 140Asp His Pro
Arg Phe Met His Gly Ala Gly Thr Ser Pro Ser Ala Asp145 150 155
160Val Phe Ala Phe Ser Ala Ala Gln Ile Lys Lys Ala Leu Glu Ser Thr
165 170 175Val Lys Leu Gly Gly Asn Gly Tyr Val Phe Trp Gly Gly Arg
Glu Gly 180 185 190Tyr Glu Thr Leu Leu Asn Thr Asn Met Gly Leu Glu
Leu Asp Asn Met 195 200 205Ala Arg Leu Met Lys Met Ala Val Glu Tyr
Gly Arg Ser Ile Gly Phe 210 215 220Lys Gly Asp Phe Tyr Ile Glu Pro
Lys Pro Lys Glu Pro Thr Lys His225 230 235 240Gln Tyr Asp Phe Asp
Thr Ala Thr Val Leu Gly Phe Leu Arg Lys Tyr 245 250 255Gly Leu Asp
Lys Asp Phe Lys Met Asn Ile Glu Ala Asn His Ala Thr 260 265 270Leu
Ala Gln His Thr Phe Gln His Glu Leu Arg Val Ala Arg Asp Asn 275 280
285Gly Val Phe Gly Ser Ile Asp Ala Asn Gln Gly Asp Val Leu Leu Gly
290 295 300Trp Asp Thr Asp Gln Phe Pro Thr Asn Ile Tyr Asp Thr Thr
Met Cys305 310 315 320Met Tyr Glu Val Ile Lys Ala Gly Gly Phe Thr
Asn Gly Gly Leu Asn 325 330 335Phe Asp Ala Lys Ala Arg Arg Gly Ser
Phe Thr Pro Glu Asp Ile Phe 340 345 350Tyr Ser Tyr Ile Ala Gly Met
Asp Ala Phe Ala Leu Gly Phe Arg Ala 355 360 365Ala Leu Lys Leu Ile
Glu Asp Gly Arg Ile Asp Lys Phe Val Ala Asp 370 375 380Arg Tyr Ala
Ser Trp Asn Thr Gly Ile Gly Ala Asp Ile Ile Ala Gly385 390 395
400Lys Ala Asp Phe Ala Ser Leu Glu Lys Tyr Ala Leu Glu Lys Gly Glu
405 410 415Val Thr Ala Ser Leu Ser Ser Gly Arg Gln Glu Met Leu Glu
Ser Ile 420 425 430Val Asn Asn Val Leu Phe Ser Leu 435
440571323DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 57aaaaaaatgg ctaaggaata tttcccacaa
attcaacagt atcagggacc aaaaagtact 60gatcctctct catttaagta ctataaccct
gaagaagtca tcaacggaaa gacaatgcgc 120gagcatctga agttcgctct
ttcatggtgg cacacaatgg gcggcgacgg aacagatatg 180ttcggctgcg
gcacaacaga caagacctgg ggacagtccg atcccgctgc aagagcaaag
240gctaaggttg acgcagcatt cgagatcatg gataagctct ccattgacta
ctattgtttc 300cacgatcgcg atctttctcc cgagtatggc agcctcaagg
ctaccaacga tcagcttgac 360atagttacag actatatcaa ggagaagcag
ggcgacaagt tcaagtgcct ctggggtaca 420gcaaagtgct tcgatcatcc
aagattcatg cacggtgcag gtacatctcc ttctgctgat 480gtattcgctt
tctcagctgc tcagatcaag aaggctctgg agtcaacagt aaagctcggc
540ggtaacggtt acgttttctg gggcggacgt gaaggctatg agacacttct
taatacaaat 600atgggactcg aactcgacaa tatggctcgt cttatgaaga
tggctgttga gtatggacgt 660tcgatcggct tcaagggcga cttctatatc
gagcccaagc ccaaggagcc cacaaagcat 720cagtacgatt tcgatacagc
tactgttctg ggattcctca gaaagtacgg tctcgataag 780gatttcaaga
tgaatatcga agctaaccac gctacacttg ctcagcatac attccagcat
840gagctccgtg ttgcaagaga caatggtgtg ttcggttcta tcgacgcaaa
ccagggcgac 900gttcttcttg gatgggatac agaccagttc cccacaaata
tctacgatac aacaatgtgt 960atgtatgaag ttatcaaggc aggcggcttc
acaaacggcg gtctcaactt cgacgctaag 1020gcacgcagag ggagcttcac
tcccgaggat atcttctaca gctatatcgc aggtatggat 1080gcatttgctc
tgggcttcag agctgctctc aagcttatcg aagacggacg tatcgacaag
1140ttcgttgctg acagatacgc ttcatggaat accggtatcg gtgcagacat
aatcgcaggt 1200aaggcagatt tcgcatctct tgaaaagtat gctcttgaaa
agggcgaggt tacagcttca 1260ctctcaagcg gcagacagga aatgctggag
tctatcgtaa ataacgttct tttcagtctg 1320taa 132358438PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
58Met Ala Lys Glu Tyr Phe Pro Gln Ile Gln Gln Tyr Gln Gly Pro Lys1
5 10 15Ser Thr Asp Pro Leu Ser Phe Lys Tyr Tyr Asn Pro Glu Glu Val
Ile 20 25 30Asn Gly Lys Thr Met Arg Glu His Leu Lys Phe Ala Leu Ser
Trp Trp 35 40 45His Thr Met Gly Gly Asp Gly Thr Asp Met Phe Gly Cys
Gly Thr Thr 50 55 60Asp Lys Thr Trp Gly Gln Ser Asp Pro Ala Ala Arg
Ala Lys Ala Lys65 70 75 80Val Asp Ala Ala Phe Glu Ile Met Asp Lys
Leu Ser Ile Asp Tyr Tyr 85 90 95Cys Phe His Asp Arg Asp Leu Ser Pro
Glu Tyr Gly Ser Leu Lys Ala 100 105 110Thr Asn Asp Gln Leu Asp Ile
Val Thr Asp Tyr Ile Lys Glu Lys Gln 115 120 125Gly Asp Lys Phe Lys
Cys Leu Trp Gly Thr Ala Lys Cys Phe Asp His 130 135 140Pro Arg Phe
Met His Gly Ala Gly Thr Ser Pro Ser Ala Asp Val Phe145 150 155
160Ala Phe Ser Ala Ala Gln Ile Lys Lys Ala Leu Glu Ser Thr Val Lys
165 170 175Leu Gly Gly Asn Gly Tyr Val Phe Trp Gly Gly Arg Glu Gly
Tyr Glu 180 185 190Thr Leu Leu Asn Thr Asn Met Gly Leu Glu Leu Asp
Asn Met Ala Arg 195 200 205Leu Met Lys Met Ala Val Glu Tyr Gly Arg
Ser Ile Gly Phe Lys Gly 210 215 220Asp Phe Tyr Ile Glu Pro Lys Pro
Lys Glu Pro Thr Lys His Gln Tyr225 230 235 240Asp Phe Asp Thr Ala
Thr Val Leu Gly Phe Leu Arg Lys Tyr Gly Leu 245 250 255Asp Lys Asp
Phe Lys Met Asn Ile Glu Ala Asn His Ala Thr Leu Ala 260 265 270Gln
His Thr Phe Gln His Glu Leu Arg Val Ala Arg Asp Asn Gly Val 275 280
285Phe Gly Ser Ile Asp Ala Asn Gln Gly Asp Val Leu Leu Gly Trp Asp
290 295 300Thr Asp Gln Phe Pro Thr Asn Ile Tyr Asp Thr Thr Met Cys
Met Tyr305 310 315 320Glu Val Ile Lys Ala Gly Gly Phe Thr Asn Gly
Gly Leu Asn Phe Asp 325 330 335Ala Lys Ala Arg Arg Gly Ser Phe Thr
Pro Glu Asp Ile Phe Tyr Ser 340 345 350Tyr Ile Ala Gly Met Asp Ala
Phe Ala Leu Gly Phe Arg Ala Ala Leu 355 360 365Lys Leu Ile Glu Asp
Gly Arg Ile Asp Lys Phe Val Ala Asp Arg Tyr 370 375 380Ala Ser Trp
Asn Thr Gly Ile Gly Ala Asp Ile Ile Ala Gly Lys Ala385 390 395
400Asp Phe Ala Ser Leu Glu Lys Tyr Ala Leu Glu Lys Gly Glu Val Thr
405 410 415Ala Ser Leu Ser Ser Gly Arg Gln Glu Met Leu Glu Ser Ile
Val Asn 420 425 430Asn Val Leu Phe Ser Leu 435591323DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
59aaaaaaatgg ctaaggaata tttcccacaa attcaaaaga ttaagttcga aaaaagtact
60gatcctctct catttaagta ctataaccct gaagaagtca tcaacggaaa gacaatgcgc
120gagcatctga agttcgctct ttcatggtgg cacacaatgg gcggcgacgg
aacagatatg 180ttcggctgcg gcacaacaga caagacctgg ggacagtccg
atcccgctgc aagagcaaag 240gctaaggttg acgcagcatt cgagatcatg
gataagctct ccattgacta ctattgtttc 300cacgatcgcg atctttctcc
cgagtatggc agcctcaagg ctaccaacga tcagcttgac 360atagttacag
actatatcaa ggagaagcag ggcgacaagt tcaagtgcct ctggggtaca
420gcaaagtgct tcgatcatcc aagattcatg cacggtgcag gtacatctcc
ttctgctgat 480gtattcgctt tctcagctgc tcagatcaag aaggctctgg
agtcaacagt aaagctcggc 540ggtaacggtt acgttttctg gggcggacgt
gaaggctatg agacacttct taatacaaat 600atgggactcg aactcgacaa
tatggctcgt cttatgaaga tggctgttga gtatggacgt 660tcgatcggct
tcaagggcga cttctatatc gagcccaagc ccaaggagcc cacaaagcat
720cagtacgatt tcgatacagc tactgttctg ggattcctca gaaagtacgg
tctcgataag 780gatttcaaga tgaatatcga agctaaccac gctacacttg
ctcagcatac attccagcat 840gagctccgtg ttgcaagaga caatggtgtg
ttcggttcta tcgacgcaaa ccagggcgac 900gttcttcttg gatgggatac
agaccagttc cccacaaata tctacgatac aacaatgtgt 960atgtatgaag
ttatcaaggc aggcggcttc acaaacggcg gtctcaactt cgacgctaag
1020gcacgcagag ggagcttcac tcccgaggat atcttctaca gctatatcgc
aggtatggat 1080gcatttgctc tgggcttcag agctgctctc aagcttatcg
aagacggacg tatcgacaag 1140ttcgttgctg acagatacgc ttcatggaat
accggtatcg gtgcagacat aatcgcaggt 1200aaggcagatt tcgcatctct
tgaaaagtat gctcttgaaa agggcgaggt tacagcttca 1260ctctcaagcg
gcagacagga aatgctggag tctatcgtaa ataacgttct tttcagtctg 1320taa
132360438PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 60Met Ala Lys Glu Tyr Phe Pro Gln Ile Gln Lys
Ile Lys Phe Glu Lys1 5 10 15Ser Thr Asp Pro Leu Ser Phe Lys Tyr Tyr
Asn Pro Glu Glu Val Ile 20 25 30Asn Gly Lys Thr Met Arg Glu His Leu
Lys Phe Ala Leu Ser Trp Trp 35 40 45His Thr Met Gly Gly Asp Gly Thr
Asp Met Phe Gly Cys Gly Thr Thr 50 55 60Asp Lys Thr Trp Gly Gln Ser
Asp Pro Ala Ala Arg Ala Lys Ala Lys65 70 75 80Val Asp Ala Ala Phe
Glu Ile Met Asp Lys Leu Ser Ile Asp Tyr Tyr 85 90 95Cys Phe His Asp
Arg Asp Leu Ser Pro Glu Tyr Gly Ser Leu Lys Ala 100 105 110Thr Asn
Asp Gln Leu Asp Ile Val Thr Asp Tyr Ile Lys Glu Lys Gln 115 120
125Gly Asp Lys Phe Lys Cys Leu Trp Gly Thr Ala Lys Cys Phe Asp His
130 135 140Pro Arg Phe Met His Gly Ala Gly Thr Ser Pro Ser Ala Asp
Val Phe145 150 155 160Ala Phe Ser Ala Ala Gln Ile Lys Lys Ala Leu
Glu Ser Thr Val Lys 165 170 175Leu Gly Gly Asn Gly Tyr Val Phe Trp
Gly Gly Arg Glu Gly Tyr Glu 180 185 190Thr Leu Leu Asn Thr Asn Met
Gly Leu Glu Leu Asp Asn Met Ala Arg 195 200 205Leu Met Lys Met Ala
Val Glu Tyr Gly Arg Ser Ile Gly Phe Lys Gly 210 215 220Asp Phe Tyr
Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys His Gln Tyr225 230 235
240Asp Phe Asp Thr Ala Thr Val Leu Gly Phe Leu Arg Lys Tyr Gly Leu
245 250 255Asp Lys Asp Phe Lys Met Asn Ile Glu Ala Asn His Ala Thr
Leu Ala 260 265 270Gln His Thr Phe Gln His Glu Leu Arg Val Ala Arg
Asp Asn Gly Val 275 280 285Phe Gly Ser Ile Asp Ala Asn Gln Gly Asp
Val Leu Leu Gly Trp Asp 290 295 300Thr Asp Gln Phe Pro Thr Asn Ile
Tyr Asp Thr Thr Met Cys Met Tyr305 310 315 320Glu Val Ile Lys Ala
Gly Gly Phe Thr Asn Gly Gly Leu Asn Phe Asp 325 330 335Ala Lys Ala
Arg Arg Gly Ser Phe Thr Pro Glu Asp Ile Phe Tyr Ser 340 345 350Tyr
Ile Ala Gly Met Asp Ala Phe Ala Leu Gly Phe Arg Ala Ala Leu 355 360
365Lys Leu Ile Glu Asp Gly Arg Ile Asp Lys Phe Val Ala Asp Arg Tyr
370 375 380Ala Ser Trp Asn Thr Gly Ile Gly Ala Asp Ile Ile Ala Gly
Lys Ala385 390 395 400Asp Phe Ala Ser Leu Glu Lys Tyr Ala Leu Glu
Lys Gly Glu Val Thr 405 410 415Ala Ser Leu Ser Ser Gly Arg Gln Glu
Met Leu Glu Ser Ile Val Asn 420 425 430Asn Val Leu Phe Ser Leu
435611322DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 61aaaaaatggc taaggaatat ttcccacaaa
ttcaaaagat taagttcgaa ggtaaggatt 60ctaagctctc atttaagtac tataaccctg
aagaagtcat caacggaaag acaatgcgcg 120agcatctgaa gttcgctctt
tcatggtggc acacaatggg cggcgacgga acagatatgt 180tcggctgcgg
cacaacagac aagacctggg gacagtccga tcccgctgca agagcaaagg
240ctaaggttga cgcagcattc gagatcatgg ataagctctc cattgactac
tattgtttcc 300acgatcgcga tctttctccc gagtatggca gcctcaaggc
taccaacgat cagcttgaca 360tagttacaga ctatatcaag gagaagcagg
gcgacaagtt caagtgcctc tggggtacag 420caaagtgctt cgatcatcca
agattcatgc acggtgcagg tacatctcct tctgctgatg 480tattcgcttt
ctcagctgct cagatcaaga aggctctgga gtcaacagta aagctcggcg
540gtaacggtta cgttttctgg ggcggacgtg aaggctatga gacacttctt
aatacaaata 600tgggactcga actcgacaat atggctcgtc ttatgaagat
ggctgttgag tatggacgtt 660cgatcggctt caagggcgac ttctatatcg
agcccaagcc caaggagccc acaaagcatc 720agtacgattt cgatacagct
actgttctgg gattcctcag aaagtacggt ctcgataagg 780atttcaagat
gaatatcgaa gctaaccacg ctacacttgc tcagcataca ttccagcatg
840agctccgtgt tgcaagagac aatggtgtgt tcggttctat cgacgcaaac
cagggcgacg 900ttcttcttgg atgggataca gaccagttcc ccacaaatat
ctacgataca acaatgtgta 960tgtatgaagt tatcaaggca ggcggcttca
caaacggcgg tctcaacttc gacgctaagg 1020cacgcagagg gagcttcact
cccgaggata tcttctacag ctatatcgca ggtatggatg 1080catttgctct
gggcttcaga gctgctctca agcttatcga agacggacgt atcgacaagt
1140tcgttgctga cagatacgct tcatggaata ccggtatcgg tgcagacata
atcgcaggta 1200aggcagattt cgcatctctt gaaaagtatg ctcttgaaaa
gggcgaggtt acagcttcac 1260tctcaagcgg cagacaggaa atgctggagt
ctatcgtaaa taacgttctt ttcagtctgt 1320aa 132262438PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
62Met Ala Lys Glu Tyr Phe Pro Gln Ile Gln Lys Ile Lys Phe Glu Gly1
5 10 15Lys Asp Ser Lys Leu Ser Phe Lys Tyr Tyr Asn Pro Glu Glu Val
Ile 20 25 30Asn Gly Lys Thr Met Arg Glu His Leu Lys Phe Ala Leu Ser
Trp Trp 35 40 45His Thr Met Gly Gly Asp Gly Thr Asp Met Phe Gly Cys
Gly Thr Thr 50 55 60Asp Lys Thr Trp Gly Gln Ser Asp Pro Ala Ala Arg
Ala Lys Ala Lys65 70 75 80Val Asp Ala Ala Phe Glu Ile Met Asp Lys
Leu Ser Ile Asp Tyr Tyr 85 90 95Cys Phe His Asp Arg Asp Leu Ser Pro
Glu Tyr Gly Ser Leu Lys Ala 100 105 110Thr Asn Asp Gln Leu Asp Ile
Val Thr Asp Tyr Ile Lys Glu Lys Gln 115 120 125Gly Asp Lys Phe Lys
Cys Leu Trp Gly Thr Ala Lys Cys Phe Asp His 130 135 140Pro Arg Phe
Met His Gly Ala Gly Thr Ser Pro Ser Ala Asp Val Phe145 150 155
160Ala Phe Ser Ala Ala Gln Ile Lys Lys Ala Leu Glu Ser Thr Val Lys
165 170 175Leu Gly Gly Asn Gly Tyr Val Phe Trp Gly Gly Arg Glu Gly
Tyr Glu 180 185 190Thr Leu Leu Asn Thr Asn Met Gly Leu Glu Leu Asp
Asn Met Ala Arg 195 200 205Leu Met Lys Met Ala Val Glu Tyr Gly Arg
Ser Ile Gly Phe Lys Gly 210 215 220Asp Phe Tyr Ile Glu Pro Lys Pro
Lys Glu Pro Thr Lys His Gln Tyr225 230 235 240Asp Phe Asp Thr Ala
Thr Val Leu Gly Phe Leu Arg Lys Tyr Gly Leu 245 250 255Asp Lys Asp
Phe Lys Met Asn Ile Glu Ala Asn His Ala Thr Leu Ala 260 265 270Gln
His Thr Phe Gln His Glu Leu Arg Val Ala Arg Asp Asn Gly Val 275
280 285Phe Gly Ser Ile Asp Ala Asn Gln Gly Asp Val Leu Leu Gly Trp
Asp 290 295 300Thr Asp Gln Phe Pro Thr Asn Ile Tyr Asp Thr Thr Met
Cys Met Tyr305 310 315 320Glu Val Ile Lys Ala Gly Gly Phe Thr Asn
Gly Gly Leu Asn Phe Asp 325 330 335Ala Lys Ala Arg Arg Gly Ser Phe
Thr Pro Glu Asp Ile Phe Tyr Ser 340 345 350Tyr Ile Ala Gly Met Asp
Ala Phe Ala Leu Gly Phe Arg Ala Ala Leu 355 360 365Lys Leu Ile Glu
Asp Gly Arg Ile Asp Lys Phe Val Ala Asp Arg Tyr 370 375 380Ala Ser
Trp Asn Thr Gly Ile Gly Ala Asp Ile Ile Ala Gly Lys Ala385 390 395
400Asp Phe Ala Ser Leu Glu Lys Tyr Ala Leu Glu Lys Gly Glu Val Thr
405 410 415Ala Ser Leu Ser Ser Gly Arg Gln Glu Met Leu Glu Ser Ile
Val Asn 420 425 430Asn Val Leu Phe Ser Leu 4356345DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
63aactgaactg actagtaaaa aaatgcaccc tcgtgtgctc gaagt
456442DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 64agtaaagtaa aagcttctac tagcgccagc cgttgaggct ct
426559DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 65agtaaagtaa aagcttctac taatgatgat gatgatgatg
gcgccagccg ttgaggctc 596646DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 66aactgaactg actagtaaaa
aaatgcacaa ccttgaacag aagacc 466743DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
67agtaaagtaa ctcgagctat tagtgtctgc ggtgctcggc gaa
436859DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 68taaagtaact cgagctacta atgatgatga tgatgatggt
gtctgcggtg ctcggcgaa 59691830DNAPseudomonas aeruginosa 69atgcaccctc
gtgtgctcga agtcacccgc cgcatccagg cccgtagcgc ggccactcgc 60cagcgctacc
tcgagatggt ccgggctgcg gccagcaagg ggccgcaccg cggcaccctg
120ccgtgcggca acctcgccca cggggtcgcg gcctgtggcg aaagcgacaa
gcagaccctg 180cggctgatga accaggccaa cgtggccatc gtttccgcct
acaacgacat gctctcggcg 240caccagccgt tcgagcgctt tccggggctg
atcaagcagg cgctgcacga gatcggttcg 300gtcggccagt tcgccggcgg
cgtgccggcc atgtgcgacg gggtgaccca gggcgagccg 360ggcatggaac
tgtcgctggc cagccgcgac gtgatcgcca tgtccaccgc catcgcgctg
420tctcacaaca tgttcgatgc agcgctgtgc ctgggtgttt gcgacaagat
cgtgccgggc 480ctgctgatcg gctcgctgcg cttcggccac ctgcccaccg
tgttcgtccc ggccgggccg 540atgccgaccg gcatctccaa caaggaaaag
gccgcggtgc gccaactgtt cgccgaaggc 600aaggccactc gcgaagagct
gctggcctcg gaaatggcct cctaccatgc acccggcacc 660tgcaccttct
atggcaccgc caataccaac cagttgctgg tggaggtgat gggcctgcac
720ttgcccggtg cctccttcgt caacccgaac acccccctgc gcgacgaact
cacccgcgaa 780gcggcacgcc aggccagccg gctgaccccc gagaacggca
actacgtgcc gatggcggag 840atcgtcgacg agaaggccat cgtcaactcg
gtggtggcgc tgctcgccac cggcggctcg 900accaaccaca ccctgcacct
gctggcgatc gcccaggcgg cgggcatcca gttgacctgg 960caggacatgt
ccgagctgtc ccatgtggtg ccgaccctgg cgcgcatcta tccgaacggc
1020caggccgaca tcaaccactt ccaggcggcc ggcggcatgt ccttcctgat
ccgccaactg 1080ctcgacggcg ggctgcttca cgaggacgta cagaccgtcg
ccggccccgg cctgcgccgc 1140tacacccgcg agccgttcct cgaggatggc
cggctggtct ggcgcgaagg gccggaacgg 1200agtctcgacg aagccatcct
gcgtccgctg gacaagccgt tctccgccga aggcggcttg 1260cgcctgatgg
agggcaacct cggtcgcggc gtgatgaagg tctcggcggt ggcgccggaa
1320caccaggtgg tcgaggcgcc ggtacggatc ttccacgacc aggccagcct
ggccgcggcc 1380ttcaaggccg gcgagctgga gcgcgacctg gtcgccgtgg
tgcgtttcca gggcccgcgg 1440gcgaacggca tgccggagct gcacaagctc
acgccgttcc tcggggtcct gcaggatcgt 1500ggcttcaagg tggcgctggt
caccgacggg cgcatgtccg gggcgtcggg caaggtgccc 1560gcggccatcc
atgtgagtcc ggaagccatc gccggcggtc cgctggcgcg cctgcgcgac
1620ggcgaccggg tgcgggtgga tggggtgaac ggcgagttgc gggtgctggt
cgacgacgcc 1680gaatggcagg cgcgcagcct ggagccggcg ccgcaggacg
gcaatctcgg ttgcggccgc 1740gagctgttcg ccttcatgcg caacgccatg
agcagcgcgg aagagggcgc ctgcagcttt 1800accgagagcc tcaacggctg
gcgctagtag 183070608PRTPseudomonas aeruginosa 70Met His Pro Arg Val
Leu Glu Val Thr Arg Arg Ile Gln Ala Arg Ser1 5 10 15Ala Ala Thr Arg
Gln Arg Tyr Leu Glu Met Val Arg Ala Ala Ala Ser 20 25 30Lys Gly Pro
His Arg Gly Thr Leu Pro Cys Gly Asn Leu Ala His Gly 35 40 45Val Ala
Ala Cys Gly Glu Ser Asp Lys Gln Thr Leu Arg Leu Met Asn 50 55 60Gln
Ala Asn Val Ala Ile Val Ser Ala Tyr Asn Asp Met Leu Ser Ala65 70 75
80His Gln Pro Phe Glu Arg Phe Pro Gly Leu Ile Lys Gln Ala Leu His
85 90 95Glu Ile Gly Ser Val Gly Gln Phe Ala Gly Gly Val Pro Ala Met
Cys 100 105 110Asp Gly Val Thr Gln Gly Glu Pro Gly Met Glu Leu Ser
Leu Ala Ser 115 120 125Arg Asp Val Ile Ala Met Ser Thr Ala Ile Ala
Leu Ser His Asn Met 130 135 140Phe Asp Ala Ala Leu Cys Leu Gly Val
Cys Asp Lys Ile Val Pro Gly145 150 155 160Leu Leu Ile Gly Ser Leu
Arg Phe Gly His Leu Pro Thr Val Phe Val 165 170 175Pro Ala Gly Pro
Met Pro Thr Gly Ile Ser Asn Lys Glu Lys Ala Ala 180 185 190Val Arg
Gln Leu Phe Ala Glu Gly Lys Ala Thr Arg Glu Glu Leu Leu 195 200
205Ala Ser Glu Met Ala Ser Tyr His Ala Pro Gly Thr Cys Thr Phe Tyr
210 215 220Gly Thr Ala Asn Thr Asn Gln Leu Leu Val Glu Val Met Gly
Leu His225 230 235 240Leu Pro Gly Ala Ser Phe Val Asn Pro Asn Thr
Pro Leu Arg Asp Glu 245 250 255Leu Thr Arg Glu Ala Ala Arg Gln Ala
Ser Arg Leu Thr Pro Glu Asn 260 265 270Gly Asn Tyr Val Pro Met Ala
Glu Ile Val Asp Glu Lys Ala Ile Val 275 280 285Asn Ser Val Val Ala
Leu Leu Ala Thr Gly Gly Ser Thr Asn His Thr 290 295 300Leu His Leu
Leu Ala Ile Ala Gln Ala Ala Gly Ile Gln Leu Thr Trp305 310 315
320Gln Asp Met Ser Glu Leu Ser His Val Val Pro Thr Leu Ala Arg Ile
325 330 335Tyr Pro Asn Gly Gln Ala Asp Ile Asn His Phe Gln Ala Ala
Gly Gly 340 345 350Met Ser Phe Leu Ile Arg Gln Leu Leu Asp Gly Gly
Leu Leu His Glu 355 360 365Asp Val Gln Thr Val Ala Gly Pro Gly Leu
Arg Arg Tyr Thr Arg Glu 370 375 380Pro Phe Leu Glu Asp Gly Arg Leu
Val Trp Arg Glu Gly Pro Glu Arg385 390 395 400Ser Leu Asp Glu Ala
Ile Leu Arg Pro Leu Asp Lys Pro Phe Ser Ala 405 410 415Glu Gly Gly
Leu Arg Leu Met Glu Gly Asn Leu Gly Arg Gly Val Met 420 425 430Lys
Val Ser Ala Val Ala Pro Glu His Gln Val Val Glu Ala Pro Val 435 440
445Arg Ile Phe His Asp Gln Ala Ser Leu Ala Ala Ala Phe Lys Ala Gly
450 455 460Glu Leu Glu Arg Asp Leu Val Ala Val Val Arg Phe Gln Gly
Pro Arg465 470 475 480Ala Asn Gly Met Pro Glu Leu His Lys Leu Thr
Pro Phe Leu Gly Val 485 490 495Leu Gln Asp Arg Gly Phe Lys Val Ala
Leu Val Thr Asp Gly Arg Met 500 505 510Ser Gly Ala Ser Gly Lys Val
Pro Ala Ala Ile His Val Ser Pro Glu 515 520 525Ala Ile Ala Gly Gly
Pro Leu Ala Arg Leu Arg Asp Gly Asp Arg Val 530 535 540Arg Val Asp
Gly Val Asn Gly Glu Leu Arg Val Leu Val Asp Asp Ala545 550 555
560Glu Trp Gln Ala Arg Ser Leu Glu Pro Ala Pro Gln Asp Gly Asn Leu
565 570 575Gly Cys Gly Arg Glu Leu Phe Ala Phe Met Arg Asn Ala Met
Ser Ser 580 585 590Ala Glu Glu Gly Ala Cys Ser Phe Thr Glu Ser Leu
Asn Gly Trp Arg 595 600 60571666DNAPseudomonas aeruginosa
71atgcacaacc ttgaacagaa gaccgcccgc atcgacacgc tgtgccggga ggcgcgcatc
60ctcccggtga tcaccatcga ccgcgaggcg gacatcctgc cgatggccga tgccctcgcc
120gccggcggcc tgaccgccct ggagatcacc ctgcgcacgg cgcacgggct
gaccgccatc 180cggcgcctca gcgaggagcg cccgcacctg cgcatcggcg
ccggcaccgt gctcgacccg 240cggaccttcg ccgccgcgga aaaggccggg
gcgagcttcg tggtcacccc gggttgcacc 300gacgagttgc tgcgcttcgc
cctggacagc gaagtcccgc tgttgcccgg cgtggccagc 360gcttccgaga
tcatgctcgc ctaccgccat ggctaccgcc gcttcaagct gtttcccgcc
420gaagtcagcg gcggcccggc ggcgctgaag gcgttctcgg gaccattccc
cgatatccgc 480ttctgcccca ccggaggcgt cagcctgaac aatctcgccg
actacctggc ggtacccaac 540gtgatgtgcg tcggcggcac ctggatgctg
cccaaggccg tggtcgaccg cggcgactgg 600gcccaggtcg agcgcctcag
ccgcgaagcc ctggagcgct tcgccgagca ccgcagacac 660taatag
66672220PRTPseudomonas aeruginosa 72Met His Asn Leu Glu Gln Lys Thr
Ala Arg Ile Asp Thr Leu Cys Arg1 5 10 15Glu Ala Arg Ile Leu Pro Val
Ile Thr Ile Asp Arg Glu Ala Asp Ile 20 25 30Leu Pro Met Ala Asp Ala
Leu Ala Ala Gly Gly Leu Thr Ala Leu Glu 35 40 45Ile Thr Leu Arg Thr
Ala His Gly Leu Thr Ala Ile Arg Arg Leu Ser 50 55 60Glu Glu Arg Pro
His Leu Arg Ile Gly Ala Gly Thr Val Leu Asp Pro65 70 75 80Arg Thr
Phe Ala Ala Ala Glu Lys Ala Gly Ala Ser Phe Val Val Thr 85 90 95Pro
Gly Cys Thr Asp Glu Leu Leu Arg Phe Ala Leu Asp Ser Glu Val 100 105
110Pro Leu Leu Pro Gly Val Ala Ser Ala Ser Glu Ile Met Leu Ala Tyr
115 120 125Arg His Gly Tyr Arg Arg Phe Lys Leu Phe Pro Ala Glu Val
Ser Gly 130 135 140Gly Pro Ala Ala Leu Lys Ala Phe Ser Gly Pro Phe
Pro Asp Ile Arg145 150 155 160Phe Cys Pro Thr Gly Gly Val Ser Leu
Asn Asn Leu Ala Asp Tyr Leu 165 170 175Ala Val Pro Asn Val Met Cys
Val Gly Gly Thr Trp Met Leu Pro Lys 180 185 190Ala Val Val Asp Arg
Gly Asp Trp Ala Gln Val Glu Arg Leu Ser Arg 195 200 205Glu Ala Leu
Glu Arg Phe Ala Glu His Arg Arg His 210 215 2207343DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
73gttcactgca ctagtaaaaa aatgcactca gtcgttcaat ctg
437436DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 74cttcgagatc tcgagttagt aaagttcatc gatggc
367541DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 75gttcactgca ctagtaaaaa aatgcttgag aataactggt c
417636DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 76cttcgagatc tcgagttaaa gtccgccaat cgcctc
367742DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 77gttcactgca ctagtaaaaa aatgtctctg aatcccgtcg tc
427836DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 78cttcgagatc tcgagttagt gaatgtcgtc gccaac
367942DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 79gttcactgca ctagtaaaaa aatgatcgat actgccaaac tc
428036DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 80cttcgagatc tcgagtcaga ccgtgaagag tgccgc
368143DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 81gttcactgca ctagtaaaaa aatgagcgat aattttttct gcg
438236DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 82cttcgagatc tcgagctatt tcctgttgat gatagc
36831827DNAShewanella oneidensis 83atgcactcag tcgttcaatc tgttactgac
agaattattg cccgtagcaa agcatctcgt 60gaagcatacc ttgctgcgtt aaacgatgcc
cgtaaccatg gtgtacaccg aagttcctta 120agttgcggta acttagccca
cggttttgcg gcttgtaatc ccgatgacaa aaatgcattg 180cgtcaattga
cgaaggccaa tattgggatt atcaccgcat tcaacgatat gttatctgca
240caccaaccct atgaaaccta tcctgatttg ctgaaaaaag cctgtcagga
agtcggtagt 300gttgcgcagg tggctggcgg tgttcccgcc atgtgtgacg
gcgtgactca aggtcagccc 360ggtatggaat tgagcttact gagccgtgaa
gtgattgcga tggcaaccgc ggttggctta 420tcacacaata tgtttgatgg
agccttactc ctcggtattt gcgataaaat tgtaccgggt 480ttactgattg
gtgccttaag ttttggccat ttacctatgt tgtttgtgcc cgcaggccca
540atgaaatcgg gtattcctaa taaggaaaaa gctcgcattc gtcagcaatt
tgctcaaggt 600aaggtcgata gagcacaact gctcgaagcg gaagcccagt
cttaccacag tgcgggtact 660tgtaccttct atggtaccgc taactcgaac
caactgatgc tcgaagtgat ggggctgcaa 720ttgccgggtt catcttttgt
gaatccagac gatccactgc gcgaagcctt aaacaaaatg 780gcggccaagc
aggtttgtcg tttaactgaa ctaggcactc aatacagtcc gattggtgaa
840gtcgttaacg aaaaatcgat agtgaatggt attgttgcat tgctcgcgac
gggtggttca 900acaaacttaa ccatgcacat tgtggcggcg gcccgtgctg
caggtattat cgtcaactgg 960gatgactttt cggaattatc cgatgcggtg
cctttgctgg cacgtgttta tccaaacggt 1020catgcggata ttaaccattt
ccacgctgcg ggtggtatgg ctttccttat caaagaatta 1080ctcgatgcag
gtttgctgca tgaggatgtc aatactgtcg cgggttatgg tctgcgccgt
1140tacacccaag agcctaaact gcttgatggc gagctgcgct gggtcgatgg
cccaacagtg 1200agtttagata ccgaagtatt aacctctgtg gcaacaccat
tccaaaacaa cggtggttta 1260aagctgctga agggtaactt aggccgcgct
gtgattaaag tgtctgccgt tcagccacag 1320caccgtgtgg tggaagcgcc
cgcagtggtg attgacgatc aaaacaaact cgatgcgtta 1380tttaaatccg
gcgcattaga cagggattgt gtggtggtgg tgaaaggcca agggccgaaa
1440gccaacggta tgccagagct gcataaacta acgccgctgt taggttcatt
gcaggacaaa 1500ggctttaaag tggcactgat gactgatggt cgtatgtcgg
gcgcatcggg caaagtacct 1560gcggcgattc atttaacccc tgaagcgatt
gatggcgggt taattgcaaa ggtacaagac 1620ggcgatttaa tccgagttga
tgcactgacc ggcgagctga gtttattagt ctctgacacc 1680gagcttgcca
ccagaactgc cactgaaatt gatttacgcc attctcgtta tggcatgggg
1740cgtgagttat ttggagtact gcgttcaaac ttaagcagtc ctgaaaccgg
tgcgcgtagt 1800actagcgcca tcgatgaact ttactaa 182784608PRTShewanella
oneidensis 84Met His Ser Val Val Gln Ser Val Thr Asp Arg Ile Ile
Ala Arg Ser1 5 10 15Lys Ala Ser Arg Glu Ala Tyr Leu Ala Ala Leu Asn
Asp Ala Arg Asn 20 25 30His Gly Val His Arg Ser Ser Leu Ser Cys Gly
Asn Leu Ala His Gly 35 40 45Phe Ala Ala Cys Asn Pro Asp Asp Lys Asn
Ala Leu Arg Gln Leu Thr 50 55 60Lys Ala Asn Ile Gly Ile Ile Thr Ala
Phe Asn Asp Met Leu Ser Ala65 70 75 80His Gln Pro Tyr Glu Thr Tyr
Pro Asp Leu Leu Lys Lys Ala Cys Gln 85 90 95Glu Val Gly Ser Val Ala
Gln Val Ala Gly Gly Val Pro Ala Met Cys 100 105 110Asp Gly Val Thr
Gln Gly Gln Pro Gly Met Glu Leu Ser Leu Leu Ser 115 120 125Arg Glu
Val Ile Ala Met Ala Thr Ala Val Gly Leu Ser His Asn Met 130 135
140Phe Asp Gly Ala Leu Leu Leu Gly Ile Cys Asp Lys Ile Val Pro
Gly145 150 155 160Leu Leu Ile Gly Ala Leu Ser Phe Gly His Leu Pro
Met Leu Phe Val 165 170 175Pro Ala Gly Pro Met Lys Ser Gly Ile Pro
Asn Lys Glu Lys Ala Arg 180 185 190Ile Arg Gln Gln Phe Ala Gln Gly
Lys Val Asp Arg Ala Gln Leu Leu 195 200 205Glu Ala Glu Ala Gln Ser
Tyr His Ser Ala Gly Thr Cys Thr Phe Tyr 210 215 220Gly Thr Ala Asn
Ser Asn Gln Leu Met Leu Glu Val Met Gly Leu Gln225 230 235 240Leu
Pro Gly Ser Ser Phe Val Asn Pro Asp Asp Pro Leu Arg Glu Ala 245 250
255Leu Asn Lys Met Ala Ala Lys Gln Val Cys Arg Leu Thr Glu Leu Gly
260 265 270Thr Gln Tyr Ser Pro Ile Gly Glu Val Val Asn Glu Lys Ser
Ile Val 275 280 285Asn Gly Ile Val Ala Leu Leu Ala Thr Gly Gly Ser
Thr Asn Leu Thr 290 295 300Met His Ile Val Ala Ala Ala Arg Ala Ala
Gly Ile Ile Val Asn Trp305 310 315 320Asp Asp Phe Ser Glu Leu Ser
Asp Ala Val Pro Leu Leu Ala Arg Val 325 330 335Tyr Pro Asn Gly His
Ala Asp Ile Asn His Phe His Ala Ala Gly Gly 340 345 350Met Ala Phe
Leu Ile Lys Glu Leu Leu Asp Ala Gly Leu Leu His Glu 355 360 365Asp
Val Asn Thr Val Ala Gly Tyr Gly Leu Arg Arg Tyr Thr
Gln Glu 370 375 380Pro Lys Leu Leu Asp Gly Glu Leu Arg Trp Val Asp
Gly Pro Thr Val385 390 395 400Ser Leu Asp Thr Glu Val Leu Thr Ser
Val Ala Thr Pro Phe Gln Asn 405 410 415Asn Gly Gly Leu Lys Leu Leu
Lys Gly Asn Leu Gly Arg Ala Val Ile 420 425 430Lys Val Ser Ala Val
Gln Pro Gln His Arg Val Val Glu Ala Pro Ala 435 440 445Val Val Ile
Asp Asp Gln Asn Lys Leu Asp Ala Leu Phe Lys Ser Gly 450 455 460Ala
Leu Asp Arg Asp Cys Val Val Val Val Lys Gly Gln Gly Pro Lys465 470
475 480Ala Asn Gly Met Pro Glu Leu His Lys Leu Thr Pro Leu Leu Gly
Ser 485 490 495Leu Gln Asp Lys Gly Phe Lys Val Ala Leu Met Thr Asp
Gly Arg Met 500 505 510Ser Gly Ala Ser Gly Lys Val Pro Ala Ala Ile
His Leu Thr Pro Glu 515 520 525Ala Ile Asp Gly Gly Leu Ile Ala Lys
Val Gln Asp Gly Asp Leu Ile 530 535 540Arg Val Asp Ala Leu Thr Gly
Glu Leu Ser Leu Leu Val Ser Asp Thr545 550 555 560Glu Leu Ala Thr
Arg Thr Ala Thr Glu Ile Asp Leu Arg His Ser Arg 565 570 575Tyr Gly
Met Gly Arg Glu Leu Phe Gly Val Leu Arg Ser Asn Leu Ser 580 585
590Ser Pro Glu Thr Gly Ala Arg Ser Thr Ser Ala Ile Asp Glu Leu Tyr
595 600 605851848DNAGluconobacter oxydans 85atgtctctga atcccgtcgt
cgagagcgtg actgcccgta tcatcgagcg ttcgaaagtc 60tcccgtcgcc ggtatctcgc
cctgatggag cgcaaccgcg ccaagggtgt gctccggccc 120aagctggcct
gcggtaatct ggcgcatgcc atcgcagcgt ccagccccga caagccggat
180ctgatgcgtc ccaccgggac caatatcggc gtgatcacga cctataacga
catgctctcg 240gcgcatcagc cgtatggccg ctatcccgag cagatcaagc
tgttcgcccg tgaagtcggt 300gcgacggccc aggttgcagg cggcgcacca
gcaatgtgtg atggtgtgac gcaggggcag 360gagggcatgg aactctccct
gttctcccgt gacgtgatcg ccatgtccac ggcggtcggg 420ctgagccacg
gcatgtttga gggcgtggcg ctgctgggca tctgtgacaa gattgtgccg
480ggccttctga tgggcgcgct gcgcttcggt catctcccgg ccatgctgat
cccggcaggg 540ccaatgccgt ccggtcttcc aaacaaggaa aagcagcgca
tccgccagct ctatgtgcag 600ggcaaggtcg ggcaggacga gctgatggaa
gcggaaaacg cctcctatca cagcccgggc 660acctgcacgt tctatggcac
ggccaatacg aaccagatga tggtcgaaat catgggtctg 720atgatgccgg
actcggcttt catcaatccc aacacgaagc tgcgtcaggc aatgacccgc
780tcgggtattc accgtctggc cgaaatcggc ctgaacggcg aggatgtgcg
cccgctcgct 840cattgcgtag acgaaaaggc catcgtgaat gcggcggtcg
ggttgctggc gacgggtggt 900tcgaccaacc attcgatcca tcttcctgct
atcgcccgtg ccgctggtat cctgatcgac 960tgggaagaca tcagccgcct
gtcgtccgcg gttccgctga tcacccgtgt ttatccgagc 1020ggttccgagg
acgtgaacgc gttcaaccgc gtgggtggta tgccgaccgt gatcgccgaa
1080ctgacgcgcg ccgggatgct gcacaaggac attctgacgg tctctcgtgg
cggtttctcc 1140gattatgccc gtcgcgcatc gctggaaggc gatgagatcg
tctacaccca cgcgaagccg 1200tccacggaca ccgatatcct gcgcgatgtg
gctacgcctt tccggcccga tggcggtatg 1260cgcctgatga ctggtaatct
gggccgcgcg atctacaaga gcagcgctat tgcgcccgag 1320cacctgaccg
ttgaagcgcc ggcacgggtc ttccaggacc agcatgacgt cctcacggcc
1380tatcagaatg gtgagcttga gcgtgatgtt gtcgtggtcg tccggttcca
gggaccggaa 1440gccaacggca tgccggagct tcacaagctg accccgactc
tgggcgtgct tcaggatcgc 1500ggcttcaagg tggccctgct gacggatgga
cgcatgtccg gtgcgagcgg caaggtgccg 1560gccgccattc atgtcggtcc
cgaagcgcag gttggcggtc cgatcgcccg cgtgcgggac 1620ggcgacatga
tccgtgtctg cgcggtgacg ggacagatcg aggctctggt ggatgccgcc
1680gagtgggaga gccgcaagcc ggtcccgccg ccgctcccgg cattgggaac
gggccgcgaa 1740ctgttcgcgc tgatgcgttc ggtgcatgat ccggccgagg
ctggcggatc cgcgatgctg 1800gcccagatgg atcgcgtgat cgaagccgtt
ggcgacgaca ttcactaa 184886615PRTGluconobacter oxydans 86Met Ser Leu
Asn Pro Val Val Glu Ser Val Thr Ala Arg Ile Ile Glu1 5 10 15Arg Ser
Lys Val Ser Arg Arg Arg Tyr Leu Ala Leu Met Glu Arg Asn 20 25 30Arg
Ala Lys Gly Val Leu Arg Pro Lys Leu Ala Cys Gly Asn Leu Ala 35 40
45His Ala Ile Ala Ala Ser Ser Pro Asp Lys Pro Asp Leu Met Arg Pro
50 55 60Thr Gly Thr Asn Ile Gly Val Ile Thr Thr Tyr Asn Asp Met Leu
Ser65 70 75 80Ala His Gln Pro Tyr Gly Arg Tyr Pro Glu Gln Ile Lys
Leu Phe Ala 85 90 95Arg Glu Val Gly Ala Thr Ala Gln Val Ala Gly Gly
Ala Pro Ala Met 100 105 110Cys Asp Gly Val Thr Gln Gly Gln Glu Gly
Met Glu Leu Ser Leu Phe 115 120 125Ser Arg Asp Val Ile Ala Met Ser
Thr Ala Val Gly Leu Ser His Gly 130 135 140Met Phe Glu Gly Val Ala
Leu Leu Gly Ile Cys Asp Lys Ile Val Pro145 150 155 160Gly Leu Leu
Met Gly Ala Leu Arg Phe Gly His Leu Pro Ala Met Leu 165 170 175Ile
Pro Ala Gly Pro Met Pro Ser Gly Leu Pro Asn Lys Glu Lys Gln 180 185
190Arg Ile Arg Gln Leu Tyr Val Gln Gly Lys Val Gly Gln Asp Glu Leu
195 200 205Met Glu Ala Glu Asn Ala Ser Tyr His Ser Pro Gly Thr Cys
Thr Phe 210 215 220Tyr Gly Thr Ala Asn Thr Asn Gln Met Met Val Glu
Ile Met Gly Leu225 230 235 240Met Met Pro Asp Ser Ala Phe Ile Asn
Pro Asn Thr Lys Leu Arg Gln 245 250 255Ala Met Thr Arg Ser Gly Ile
His Arg Leu Ala Glu Ile Gly Leu Asn 260 265 270Gly Glu Asp Val Arg
Pro Leu Ala His Cys Val Asp Glu Lys Ala Ile 275 280 285Val Asn Ala
Ala Val Gly Leu Leu Ala Thr Gly Gly Ser Thr Asn His 290 295 300Ser
Ile His Leu Pro Ala Ile Ala Arg Ala Ala Gly Ile Leu Ile Asp305 310
315 320Trp Glu Asp Ile Ser Arg Leu Ser Ser Ala Val Pro Leu Ile Thr
Arg 325 330 335Val Tyr Pro Ser Gly Ser Glu Asp Val Asn Ala Phe Asn
Arg Val Gly 340 345 350Gly Met Pro Thr Val Ile Ala Glu Leu Thr Arg
Ala Gly Met Leu His 355 360 365Lys Asp Ile Leu Thr Val Ser Arg Gly
Gly Phe Ser Asp Tyr Ala Arg 370 375 380Arg Ala Ser Leu Glu Gly Asp
Glu Ile Val Tyr Thr His Ala Lys Pro385 390 395 400Ser Thr Asp Thr
Asp Ile Leu Arg Asp Val Ala Thr Pro Phe Arg Pro 405 410 415Asp Gly
Gly Met Arg Leu Met Thr Gly Asn Leu Gly Arg Ala Ile Tyr 420 425
430Lys Ser Ser Ala Ile Ala Pro Glu His Leu Thr Val Glu Ala Pro Ala
435 440 445Arg Val Phe Gln Asp Gln His Asp Val Leu Thr Ala Tyr Gln
Asn Gly 450 455 460Glu Leu Glu Arg Asp Val Val Val Val Val Arg Phe
Gln Gly Pro Glu465 470 475 480Ala Asn Gly Met Pro Glu Leu His Lys
Leu Thr Pro Thr Leu Gly Val 485 490 495Leu Gln Asp Arg Gly Phe Lys
Val Ala Leu Leu Thr Asp Gly Arg Met 500 505 510Ser Gly Ala Ser Gly
Lys Val Pro Ala Ala Ile His Val Gly Pro Glu 515 520 525Ala Gln Val
Gly Gly Pro Ile Ala Arg Val Arg Asp Gly Asp Met Ile 530 535 540Arg
Val Cys Ala Val Thr Gly Gln Ile Glu Ala Leu Val Asp Ala Ala545 550
555 560Glu Trp Glu Ser Arg Lys Pro Val Pro Pro Pro Leu Pro Ala Leu
Gly 565 570 575Thr Gly Arg Glu Leu Phe Ala Leu Met Arg Ser Val His
Asp Pro Ala 580 585 590Glu Ala Gly Gly Ser Ala Met Leu Ala Gln Met
Asp Arg Val Ile Glu 595 600 605Ala Val Gly Asp Asp Ile His 610
615871665DNARuminococcus flavefaciens 87atgagcgata attttttctg
cgagggtgcg gataaagccc ctcagcgttc acttttcaat 60gcactgggca tgactaaaga
ggaaatgaag cgtcccctcg ttggtatcgt ttcttcctac 120aatgagatcg
ttcccggcca tatgaacatc gacaagctgg tcgaagccgt taagctgggt
180gtagctatgg gcggcggcac tcctgttgtt ttccctgcta tcgctgtatg
cgacggtatc 240gctatgggtc acacaggcat gaagtacagc cttgttaccc
gtgaccttat tgccgattct 300acagagtgta tggctcttgc tcatcacttc
gacgcactgg taatgatacc taactgcgac 360aagaacgttc ccggcctgct
tatggcggct gcacgtatca atgttcctac tgtattcgta 420agcggcggcc
ctatgcttgc aggccatgta aagggtaaga agacctctct ttcatccatg
480ttcgaggctg taggcgctta cacagcaggc aagatagacg aggctgaact
tgacgaattc 540gagaacaaga cctgccctac ctgcggttca tgttcgggta
tgtataccgc taactccatg 600aactgcctca ctgaggtact gggtatgggt
ctcagaggca acggcactat ccctgctgtt 660tactccgagc gtatcaagct
tgcaaagcag gcaggtatgc aggttatgga actctacaga 720aagaatatcc
gccctctcga tatcatgaca gagaaggctt tccagaacgc tctcacagct
780gatatggctc ttggatgttc cacaaacagt atgctccatc tccctgctat
cgccaacgaa 840tgcggcataa atatcaacct tgacatggct aacgagataa
gcgccaagac tcctaacctc 900tgccatcttg caccggcagg ccacacctac
atggaagacc tcaacgaagc aggcggagtt 960tatgcagttc tcaacgagct
gagcaaaaag ggacttatca acaccgactg catgactgtt 1020acaggcaaga
ccgtaggcga gaatatcaag ggctgcatca accgtgaccc tgagactatc
1080cgtcctatcg acaacccata cagtgaaaca ggcggaatcg ccgtactcaa
gggcaatctt 1140gctcccgaca gatgtgttgt gaagagaagc gcagttgctc
ccgaaatgct ggtacacaaa 1200ggccctgcaa gagtattcga cagcgaggaa
gaagctatca aggtcatcta tgagggcggt 1260atcaaggcag gcgacgttgt
tgttatccgt tacgaaggcc ctgcaggcgg ccccggcatg 1320agagaaatgc
tctctcctac atcagctata cagggtgcag gtctcggctc aactgttgct
1380ctaatcactg acggacgttt cagcggcgct acccgtggtg cggctatcgg
acacgtatcc 1440cccgaagctg taaacggcgg tactatcgca tatgtcaagg
acggcgatat tatctccatc 1500gacataccga attactccat cactcttgaa
gtatccgacg aggagcttgc agagcgcaaa 1560aaggcaatgc ctatcaagcg
caaggagaac atcacaggct atctgaagcg ctatgcacag 1620caggtatcat
ccgcagacaa gggcgctatc atcaacagga aatag 166588554PRTRuminococcus
flavefaciens 88Met Ser Asp Asn Phe Phe Cys Glu Gly Ala Asp Lys Ala
Pro Gln Arg1 5 10 15Ser Leu Phe Asn Ala Leu Gly Met Thr Lys Glu Glu
Met Lys Arg Pro 20 25 30Leu Val Gly Ile Val Ser Ser Tyr Asn Glu Ile
Val Pro Gly His Met 35 40 45Asn Ile Asp Lys Leu Val Glu Ala Val Lys
Leu Gly Val Ala Met Gly 50 55 60Gly Gly Thr Pro Val Val Phe Pro Ala
Ile Ala Val Cys Asp Gly Ile65 70 75 80Ala Met Gly His Thr Gly Met
Lys Tyr Ser Leu Val Thr Arg Asp Leu 85 90 95Ile Ala Asp Ser Thr Glu
Cys Met Ala Leu Ala His His Phe Asp Ala 100 105 110Leu Val Met Ile
Pro Asn Cys Asp Lys Asn Val Pro Gly Leu Leu Met 115 120 125Ala Ala
Ala Arg Ile Asn Val Pro Thr Val Phe Val Ser Gly Gly Pro 130 135
140Met Leu Ala Gly His Val Lys Gly Lys Lys Thr Ser Leu Ser Ser
Met145 150 155 160Phe Glu Ala Val Gly Ala Tyr Thr Ala Gly Lys Ile
Asp Glu Ala Glu 165 170 175Leu Asp Glu Phe Glu Asn Lys Thr Cys Pro
Thr Cys Gly Ser Cys Ser 180 185 190Gly Met Tyr Thr Ala Asn Ser Met
Asn Cys Leu Thr Glu Val Leu Gly 195 200 205Met Gly Leu Arg Gly Asn
Gly Thr Ile Pro Ala Val Tyr Ser Glu Arg 210 215 220Ile Lys Leu Ala
Lys Gln Ala Gly Met Gln Val Met Glu Leu Tyr Arg225 230 235 240Lys
Asn Ile Arg Pro Leu Asp Ile Met Thr Glu Lys Ala Phe Gln Asn 245 250
255Ala Leu Thr Ala Asp Met Ala Leu Gly Cys Ser Thr Asn Ser Met Leu
260 265 270His Leu Pro Ala Ile Ala Asn Glu Cys Gly Ile Asn Ile Asn
Leu Asp 275 280 285Met Ala Asn Glu Ile Ser Ala Lys Thr Pro Asn Leu
Cys His Leu Ala 290 295 300Pro Ala Gly His Thr Tyr Met Glu Asp Leu
Asn Glu Ala Gly Gly Val305 310 315 320Tyr Ala Val Leu Asn Glu Leu
Ser Lys Lys Gly Leu Ile Asn Thr Asp 325 330 335Cys Met Thr Val Thr
Gly Lys Thr Val Gly Glu Asn Ile Lys Gly Cys 340 345 350Ile Asn Arg
Asp Pro Glu Thr Ile Arg Pro Ile Asp Asn Pro Tyr Ser 355 360 365Glu
Thr Gly Gly Ile Ala Val Leu Lys Gly Asn Leu Ala Pro Asp Arg 370 375
380Cys Val Val Lys Arg Ser Ala Val Ala Pro Glu Met Leu Val His
Lys385 390 395 400Gly Pro Ala Arg Val Phe Asp Ser Glu Glu Glu Ala
Ile Lys Val Ile 405 410 415Tyr Glu Gly Gly Ile Lys Ala Gly Asp Val
Val Val Ile Arg Tyr Glu 420 425 430Gly Pro Ala Gly Gly Pro Gly Met
Arg Glu Met Leu Ser Pro Thr Ser 435 440 445Ala Ile Gln Gly Ala Gly
Leu Gly Ser Thr Val Ala Leu Ile Thr Asp 450 455 460Gly Arg Phe Ser
Gly Ala Thr Arg Gly Ala Ala Ile Gly His Val Ser465 470 475 480Pro
Glu Ala Val Asn Gly Gly Thr Ile Ala Tyr Val Lys Asp Gly Asp 485 490
495Ile Ile Ser Ile Asp Ile Pro Asn Tyr Ser Ile Thr Leu Glu Val Ser
500 505 510Asp Glu Glu Leu Ala Glu Arg Lys Lys Ala Met Pro Ile Lys
Arg Lys 515 520 525Glu Asn Ile Thr Gly Tyr Leu Lys Arg Tyr Ala Gln
Gln Val Ser Ser 530 535 540Ala Asp Lys Gly Ala Ile Ile Asn Arg
Lys545 55089642DNAShewanella oneidensis 89atgcttgaga ataactggtc
attacaacca caagatattt ttaaacgcag ccctattgtt 60cctgttatgg tgattaacaa
gattgaacat gcggtgccct tagctaaagc gctggttgcc 120ggagggataa
gcgtgttgga agtgacatta cgcacgccat gcgcccttga agctatcacc
180aaaatcgcca aggaagtgcc tgaggcgctg gttggcgcgg ggactatttt
aaatgaagcc 240cagcttggac aggctatcgc cgctggtgcg caatttatta
tcactccagg tgcgacagtt 300gagctgctca aagcgggcat gcaaggaccg
gtgccgttaa ttccgggcgt tgccagtatt 360tccgaggtga tgacgggcat
ggcgctgggc tacactcact ttaaattctt ccctgctgaa 420gcgtcaggtg
gcgttgatgc gcttaaggct ttctctgggc cgttagcaga tatccgcttc
480tgcccaacag gtggaattac cccgagcagc tataaagatt acttagcgct
gaagaatgtc 540gattgtattg gtggcagctg gattgctcct accgatgcga
tggagcaggg cgattgggat 600cgtatcactc agctgtgtaa agaggcgatt
ggcggacttt aa 64290213PRTShewanella oneidensis 90Met Leu Glu Asn
Asn Trp Ser Leu Gln Pro Gln Asp Ile Phe Lys Arg1 5 10 15Ser Pro Ile
Val Pro Val Met Val Ile Asn Lys Ile Glu His Ala Val 20 25 30Pro Leu
Ala Lys Ala Leu Val Ala Gly Gly Ile Ser Val Leu Glu Val 35 40 45Thr
Leu Arg Thr Pro Cys Ala Leu Glu Ala Ile Thr Lys Ile Ala Lys 50 55
60Glu Val Pro Glu Ala Leu Val Gly Ala Gly Thr Ile Leu Asn Glu Ala65
70 75 80Gln Leu Gly Gln Ala Ile Ala Ala Gly Ala Gln Phe Ile Ile Thr
Pro 85 90 95Gly Ala Thr Val Glu Leu Leu Lys Ala Gly Met Gln Gly Pro
Val Pro 100 105 110Leu Ile Pro Gly Val Ala Ser Ile Ser Glu Val Met
Thr Gly Met Ala 115 120 125Leu Gly Tyr Thr His Phe Lys Phe Phe Pro
Ala Glu Ala Ser Gly Gly 130 135 140Val Asp Ala Leu Lys Ala Phe Ser
Gly Pro Leu Ala Asp Ile Arg Phe145 150 155 160Cys Pro Thr Gly Gly
Ile Thr Pro Ser Ser Tyr Lys Asp Tyr Leu Ala 165 170 175Leu Lys Asn
Val Asp Cys Ile Gly Gly Ser Trp Ile Ala Pro Thr Asp 180 185 190Ala
Met Glu Gln Gly Asp Trp Asp Arg Ile Thr Gln Leu Cys Lys Glu 195 200
205Ala Ile Gly Gly Leu 21091624DNAGluconobacter oxydans
91atgatcgata ctgccaaact cgacgccgtc atgagccgtt gtccggtcat gccggtgctg
60gtggtcaatg atgtggctct ggcccgcccg atggccgagg ctctggtggc gggtggactg
120tccacgctgg aagtcacgct gcgcacgccc tgcgcccttg aagctattga
ggaaatgtcg 180aaagtaccag gcgcgctggt cggtgccggt acggtgctga
atccgtccga catggaccgt 240gccgtgaagg cgggtgcgcg cttcatcgtc
agccccggcc tgaccgaggc gctggcaaag 300gcgtcggttg agcatgacgt
ccccttcctg ccaggcgttg ccaatgcggg tgacatcatg 360cggggtctgg
atctgggtct gtcacgcttc aagttcttcc cggctgtgac gaatggcggc
420attcccgcgc tcaagagctt ggccagtgtt tttggcagca atgtccgttt
ctgccccacg 480ggcggcatta cggaagagag cgcaccggac tggctggcgc
ttccctccgt ggcctgcgtc 540ggcggatcct gggtgacggc cggcacgttc
gatgcggaca aggtccgtca gcgcgccacg 600gctgcggcac tcttcacggt ctga
62492207PRTGluconobacter oxydans 92Met Ile Asp Thr Ala Lys Leu Asp
Ala Val Met Ser Arg Cys Pro Val1 5 10 15Met Pro Val Leu Val Val Asn
Asp Val Ala Leu Ala Arg Pro Met
Ala 20 25 30Glu Ala Leu Val Ala Gly Gly Leu Ser Thr Leu Glu Val Thr
Leu Arg 35 40 45Thr Pro Cys Ala Leu Glu Ala Ile Glu Glu Met Ser Lys
Val Pro Gly 50 55 60Ala Leu Val Gly Ala Gly Thr Val Leu Asn Pro Ser
Asp Met Asp Arg65 70 75 80Ala Val Lys Ala Gly Ala Arg Phe Ile Val
Ser Pro Gly Leu Thr Glu 85 90 95Ala Leu Ala Lys Ala Ser Val Glu His
Asp Val Pro Phe Leu Pro Gly 100 105 110Val Ala Asn Ala Gly Asp Ile
Met Arg Gly Leu Asp Leu Gly Leu Ser 115 120 125Arg Phe Lys Phe Phe
Pro Ala Val Thr Asn Gly Gly Ile Pro Ala Leu 130 135 140Lys Ser Leu
Ala Ser Val Phe Gly Ser Asn Val Arg Phe Cys Pro Thr145 150 155
160Gly Gly Ile Thr Glu Glu Ser Ala Pro Asp Trp Leu Ala Leu Pro Ser
165 170 175Val Ala Cys Val Gly Gly Ser Trp Val Thr Ala Gly Thr Phe
Asp Ala 180 185 190Asp Lys Val Arg Gln Arg Ala Thr Ala Ala Ala Leu
Phe Thr Val 195 200 20593459DNAPiromyces sp. 93atggctaagg
aatatttccc acaaattcaa aagattaagt tcgaaggtaa ggattctaag 60aatccattag
ccttccacta ctacgatgct gaaaaggaag tcatgggtaa gaaaatgaag
120gattggttac gtttcgccat ggcctggtgg cacactcttt gcgccgaagg
tgctgaccaa 180ttcggtggag gtacaaagtc tttcccatgg aacgaaggta
ctgatgctat tgaaattgcc 240aagcaaaagg ttgatgctgg tttcgaaatc
atgcaaaagc ttggtattcc atactactgt 300ttccacgatg ttgatcttgt
ttccgaaggt aactctattg aagaatacga atccaacctt 360aaggctgtcg
ttgcttacct caaggaaaag caaaaggaaa ccggtattaa gcttctctgg
420agtactgcta acgtcttcgg tcacaagcgt tacatgaac
45994456DNAEscherichia coli 94atgcaagcct attttgacca gctcgatcgc
gttcgttatg aaggctcaaa atcctcaaac 60ccgttagcat tccgtcacta caatcccgac
gaactggtgt tgggtaagcg tatggaagag 120cacttgcgtt ttgccgcctg
ctactggcac accttctgct ggaacggggc ggatatgttt 180ggtgtggggg
cgtttaatcg tccgtggcag cagcctggtg aggcactggc gttggcgaag
240cgtaaagcag atgtcgcatt tgagtttttc cacaagttac atgtgccatt
ttattgcttc 300cacgatgtgg atgtttcccc tgagggcgcg tcgttaaaag
agtacatcaa taattttgcg 360caaatggttg atgtcctggc aggcaagcaa
gaagagagcg gcgtgaagct gctgtgggga 420acggccaact gctttacaaa
ccctcgctac ggcgcg 45695456DNAClostridium phytofermentans
95atgaaaaatt actttccaaa tgttccagaa gtaaaatacg aaggcccaaa ttcaacgaat
60ccatttgctt ttaaatatta tgacgcaaat aaagttgtag cgggtaaaac aatgaaagag
120cactgtcgtt ttgcattatc ttggtggcat actctttgtg caggtggtgc
tgatccattc 180ggtgtaacaa ctatggatag aacctacgga aatatcacag
atccaatgga acttgctaag 240gcaaaagttg acgctggttt cgaattaatg
actaaattag gaattgaatt cttctgtttc 300catgacgcag atattgctcc
agaaggtgat acttttgaag agtcaaagaa gaatcttttt 360gaaatcgttg
attacatcaa agagaagatg gatcagactg gtatcaagtt attatggggt
420actgctaata actttagtca tccaagattt atgcat 45696459DNAOrpinomyces
sp. 96atgactaagg aatatttccc aactatcggc aagattagat tcgaaggtaa
ggattctaag 60aatccaatgg ccttccacta ctatgatgct gaaaaggaag tcatgggtaa
gaaaatgaag 120gattggttac gtttcgccat ggcctggtgg cacactcttt
gcgccgatgg tgctgaccaa 180ttcggtgttg gtactaagtc tttcccatgg
aatgaaggta ctgacccaat tgctattgcc 240aagcaaaagg ttgatgctgg
ttttgaaatc atgaccaagc ttggtattga acactactgt 300ttccacgatg
ttgatcttgt ttctgaaggt aactctattg aagaatacga atccaacctc
360aagcaagttg ttgcttacct taagcaaaag caacaagaaa ctggtattaa
gcttctctgg 420agtactgcca atgttttcgg taacccacgt tacatgaac
45997453DNAClostridium thermohydrosulfuricum 97atggaatact
tcaaaaatgt accacaaata aaatatgaag gaccaaaatc aaacaatcca 60tatgcattta
aattttacaa tccagatgaa ataatagacg gaaaaccttt aaaagaacac
120ttgcgttttt cagtagcgta ctggcacaca tttacagcca atgggacaga
tccatttgga 180gcacccacaa tgcaaaggcc atgggaccat tttactgacc
ctatggatat tgccaaagcg 240agagtagaag cagcctttga actatttgaa
aaactcgacg taccattttt ctgtttccat 300gacagagata tagctccgga
aggagagaca ttaagggaga cgaacaaaaa tttagataca 360atagttgcaa
tgataaaaga ctacttaaag acgagcaaaa caaaagtatt atggggcaca
420gcgaaccttt tttcaaatcc gagatttgta cat 45398462DNABacteroides
thetaiotaomicron 98atggcaacaa aagaattttt tccgggaatt gaaaagatta
aatttgaagg taaagatagt 60aagaacccga tggcattccg ttattacgat gcagagaagg
tgattaatgg taaaaagatg 120aaggattggc tgagattcgc tatggcatgg
tggcacacat tgtgcgctga aggtggtgat 180cagttcggtg gcggaacaaa
gcaattccca tggaatggta atgcagatgc tatacaggca 240gcaaaagata
agatggatgc aggatttgaa ttcatgcaga agatgggtat cgaatactat
300tgcttccatg acgtagactt ggtttcggaa ggtgccagtg tagaagaata
cgaagctaac 360ctgaaagaaa tcgtagctta tgcaaaacag aaacaggcag
aaaccggtat caaactactg 420tggggtactg ctaatgtatt cggtcacgcc
cgctatatga ac 46299450DNABacillus stearothermophilus 99atggcttatt
ttccgaatat cggcaagatt gcgtatgaag ggccggagtc gcgcaatccg 60ttggcgttta
agttttataa tccagaagaa aaagtcggcg acaaaacaat ggaggagcat
120ttgcgctttt cagtggccta ttggcatacg tttacggggg atgggtcgga
tccgtttggc 180gtcggcaata tgattcgtcc atggaataag tacagcggca
tggatctggc gaaggcgcgc 240gtcgaggcgg cgtttgagct gtttgaaaag
ctgaacgttc cgtttttctg cttccatgac 300gtcgacatcg cgccggaagg
ggaaacgctc agcgagacgt acaaaaattt ggatgaaatt 360gtcgatatga
ttgaagaata catgaaaaca agcaaaacga agctgctttg gaatacggcg
420aacttgttca gccatccgcg cttcgttcac 450100462DNABacillus uniformis
100atggctacca aggaatactt cccaggtatt ggtaagatca aattcgaagg
taaggaatcc 60aagaacccaa tggccttcag atactacgat gctgacaagg ttatcatggg
taagaagatg 120tctgaatggt taaagttcgc tatggcttgg tggcatacct
tgtgtgctga aggtggtgac 180caattcggtg gtggtaccaa gaaattccca
tggaacggtg aagctgacaa ggtccaagct 240gctaagaaca agatggacgc
tggtttcgaa tttatgcaaa agatgggtat tgaatactac 300tgtttccacg
atgttgactt gtgtgaagaa gctgaaacca tcgaagaata cgaagctaac
360ttgaaggaaa ttgttgctta cgctaagcaa aagcaagctg aaactggtat
caagctatta 420tggggtactg ctaacgtctt tggtcatgcc agatacatga ac
462101456DNAClostridium cellulolyticum 101atgtcagaag tatttagcgg
tatttcaaac attaaatttg aaggaagcgg gtcagataat 60ccattagctt ttaagtacta
tgaccctaag gcagttatcg gcggaaagac aatggaagaa 120catctgagat
tcgcagttgc ctactggcat acttttgcag caccaggtgc tgacatgttc
180ggtgcaggat catatgtaag accttggaat acaatgtccg atcctctgga
aattgcaaaa 240tacaaagttg aagcaaactt tgaattcatt gaaaagctgg
gagcaccttt cttcgctttc 300catgacaggg atattgctcc tgaaggcgac
acactcgctg aaacaaataa aaaccttgat 360acaatagttt cagtaattaa
agatagaatg aaatccagtc cggtaaagtt attatgggga 420actacaaatg
ctttcggaaa cccaagattt atgcat 456102450DNARuminococcus flavefaciens
102atggaatttt tcaagaacat aagcaagatc ccttacgagg gcaaggacag
cacaaatcct 60ctcgcattca agtactacaa tcctgatgag gtaattgacg gcaagaagat
gcgtgacatt 120atgaagtttg ctctctcatg gtggcataca atgggcggcg
acggaacaga tatgttcggc 180tgcggtacag ctgacaagac atggggcgaa
aatgatcctg ctgcaagagc taaggctaag 240gttgacgcag ctttcgagat
catgcagaag ctctctatcg attacttctg tttccacgac 300cgtgatcttt
ctcctgagta cggctcactg aaggacacaa acgctcagct ggacatcgtt
360acagattaca tcaaggctaa gcaggctgag acaggtctca agtgcctctg
gggtacagct 420aagtgcttcg atcacccaag attcatgcac
450103456DNARuminococcus flavefaciens 103atgagcgaat tttttacagg
catttcaaag atcccctttg agggaaaggc atccaacaat 60cccatggcgt tcaagtacta
caacccggat gaggtcgtag gcggcaagac catgcgggag 120cagctgaagt
ttgcgctgtc ctggtggcat actatggggg gagacggtac ggacatgttt
180ggtgtgggta ccaccaacaa gaagttcggc ggaaccgatc ccatggacat
tgctaagaga 240aaggtaaacg ctgcgtttga gctgatggac aagctgtcca
tcgattattt ctgtttccac 300gaccgggatc tggcgccgga ggctgataat
ctgaaggaaa ccaaccagcg tctggatgaa 360atcaccgagt atattgcaca
gatgatgcag ctgaacccgg acaagaaggt tctgtggggt 420actgcaaatt
gcttcggcaa tccccggtat atgcat 456104453DNAClostriales sp.
104atgaaatttt ttgaaaatgt ccctaaggta aaatatgagg gaagcaagtc
taccaacccg 60tttgcattta agtattacaa tcctgaagcg gtgattgccg gtaaaaaaat
gaaggatcac 120ctgaaattcg cgatgtcctg gtggcacacc atgacggcga
ccgggcaaga ccagttcggt 180tcggggacga tgagccgaat atatgacggg
caaactgaac cgctggcctt ggccaaagcc 240cgagtggatg cggctttcga
tttcatggaa aaattaaata tcgaatattt ttgttttcat 300gatgccgact
tggctccaga aggtaacagt ttgcaggaac gcaacgaaaa tttgcaggaa
360atggtgtctt acctgaaaca aaagatggcc ggaacttcga ttaagctttt
atggggaacc 420tcgaattgtt tcagcaaccc tcgttttatg cac
453105463DNABacillus stercoris 105atggcaacaa aagagtattt tcccggaata
ggaaaaatca aattcgaagg caaagaaagt 60aagaatccta tggcattccg ctactacgat
gcggaaaaag taatcatggg caagaagatg 120aaagattggt tgaagttctc
tatggcatgg tggcatacac tctgtgcaga gggtggtgac 180cagttcggcg
gcggaacgaa acatttcccc tggaacggtg atgccgataa actgcaggct
240gccaagaaca aaatggatgc tggtttcgag ttcatgcaga aaatgggcat
cgaatattac 300tgcttccacg atgttgacct ttgcgacgag gccgatacaa
tcgaagagta cgaagcaaac 360ctgaaagcca tcgttgcata cgccaagcaa
aagcaggagg aaacaggtat caaactgttg 420tggggtactg ccaacgtatt
cggtcatgca cgttacatga acg 463106300DNAThermus thermophilus
106atgtacgagc ccaaaccgga gcacaggttt acctttggcc tttggactgt
gggcaatgtg 60ggccgtgatc ccttcgggga cgcggttcgg gagaggctgg acccggttta
cgtggttcat 120aagctggcgg agcttggggc ctacggggta aaccttcacg
acgaggacct gatcccgcgg 180ggcacgcctc ctcaggagcg ggaccagatc
gtgaggcgct tcaagaaggc tctcgatgaa 240accggcctca aggtccccat
ggtcaccgcc aacctcttct ccgaccctgc tttcaaggac
3001071320DNARuminococcus flavefaciens 107atggaatttt tcaagaacat
aagcaagatc ccttacgagg gcaaggacag cacaaatcct 60ctcgcattca agtactacaa
tcctgatgag gtaattgacg gcaagaagat gcgtgacatt 120atgaagtttg
ctctctcatg gtggcataca atgggcggcg acggaacaga tatgttcggc
180tgcggtacag ctgacaagac atggggcgaa aatgatcctg ctgcaagagc
taaggctaag 240gttgacgcag ctttcgagat catgcagaag ctctctatcg
attacttctg tttccacgac 300cgtgatcttt ctcctgagta cggctcactg
aaggacacaa acgctcagct ggacatcgtt 360acagattaca tcaaggctaa
gcaggctgag acaggtctca agtgcctctg gggtacagct 420aagtgcttcg
atcacccaag attcatgcac ggtgcaggta cttcaccatc cgcagacgta
480ttcgctttct cagctgcaca gatcaagaag gctctcgagt ctactgtaaa
gctcggcggt 540acaggctacg tattctgggg cggacgtgag ggttatgaga
ctctcctcaa cacaaacatg 600ggccttgagc ttgacaacat ggctcgtctc
atgaagatgg ctgttgagta cggacgttct 660atcggcttca agggcgattt
ctacatcgag cctaagccaa aggagccaac aaagcaccag 720tacgatttcg
atactgctac tgttctcggc ttcctcagaa agtacggtct cgacaaggat
780ttcaagatga acatcgaagc taaccacgct acactggctc agcacacatt
ccagcacgag 840ctctgcgtag caagaacaaa cggtgctttc ggttcaatcg
acgcaaacca gggcgatcct 900ctcctcggat gggatacaga ccagttcccg
acaaatatct atgacacaac aatgtgtatg 960tacgaagtta tcaaggctgg
cggcttcaca aacggcggtc tcaacttcga tgcaaaggca 1020agacgtggaa
gcttcacacc tgaggatatc ttctacagct acattgcagg tatggatgca
1080ttcgctctcg gctacaaggc tgcaagcaag ctcatcgctg acggacgtat
cgacagcttc 1140atttccgacc gctacgcttc atggagcgag ggaatcggtc
tcgacatcat ctcaggcaag 1200gctgatatgg ctgctcttga gaagtatgct
ctcgaaaagg gcgaggttac agactctatt 1260tccagcggca gacaggaact
cctcgagtct atcgtaaaca acgttatatt caatctttga
13201081326DNARuminococcus flavefaciens 108atgagcgaat tttttacagg
catttcaaag atcccctttg agggaaaggc atccaacaat 60cccatggcgt tcaagtacta
caacccggat gaggtcgtag gcggcaagac catgcgggag 120cagctgaagt
ttgcgctgtc ctggtggcat actatggggg gagacggtac ggacatgttt
180ggtgtgggta ccaccaacaa gaagttcggc ggaaccgatc ccatggacat
tgctaagaga 240aaggtaaacg ctgcgtttga gctgatggac aagctgtcca
tcgattattt ctgtttccac 300gaccgggatc tggcgccgga ggctgataat
ctgaaggaaa ccaaccagcg tctggatgaa 360atcaccgagt atattgcaca
gatgatgcag ctgaacccgg acaagaaggt tctgtggggt 420actgcaaatt
gcttcggcaa tccccggtat atgcatggtg ccggcactgc gcccaatgcg
480gacgtgtttg catttgcagc tgcgcagatc aaaaaggcaa ttgagatcac
cgtaaagctg 540ggtggcaagg gctatgtatt ctggggcggc agagagggct
acgaaacgct gctgaacacc 600aatatgggtc tggaactgga taatatggca
cggctgctgc atatggcagt ggactatgca 660agaagcatcg gctttaccgg
cgacttctac atcgagccca agcccaagga gcctaccaag 720catcagtatg
attttgatac cgcaaccgtg atcggcttcc tgcgcaagta taatctggac
780aaggacttca agatgaacat cgaagccaac cacgcaaccc ttgcacagca
caccttccag 840catgaactgc gggtagcacg ggagaacggc ttctttggct
ccatcgatgc taaccagggt 900gacaccctgc tgggctggga tacggatcag
ttccccacta atacctatga cgcagcactg 960tgtatgtacg aggtactcaa
ggctggcggt tttaccaatg gcggtctgaa ctttgactcc 1020aaggcacggc
gtggatcctt tgagatggag gatatcttcc acagctacat tgccggtatg
1080gacacctttg cactgggtct gaagattgcg cagaagatga tcgatgacgg
acggatcgac 1140cagttcgtgg ctgatcggta tgcaagctgg aacaccggca
tcggtgcgga tatcatttcc 1200ggcaaggcaa ccatggcaga tttggaggct
tacgcactga gcaagggcga tgtgaccgca 1260tccctcaaga gcggtcgtca
ggaattgctg gaaagcatcc tgaacaatat tatgttcaat 1320ctttaa
13261091323DNAClostridiales sp. 109atgaaatttt ttgaaaatgt ccctaaggta
aaatatgagg gaagcaagtc taccaacccg 60tttgcattta agtattacaa tcctgaagcg
gtgattgccg gtaaaaaaat gaaggatcac 120ctgaaattcg cgatgtcctg
gtggcacacc atgacggcga ccgggcaaga ccagttcggt 180tcggggacga
tgagccgaat atatgacggg caaactgaac cgctggcctt ggccaaagcc
240cgagtggatg cggctttcga tttcatggaa aaattaaata tcgaatattt
ttgttttcat 300gatgccgact tggctccaga aggtaacagt ttgcaggaac
gcaacgaaaa tttgcaggaa 360atggtgtctt acctgaaaca aaagatggcc
ggaacttcga ttaagctttt atggggaacc 420tcgaattgtt tcagcaaccc
tcgttttatg cacggggcag ccacatcttg cgaagcggat 480gtgtttgctt
ggaccgccac tcagttgaaa aatgccatcg atgctaccat cgcgcttggc
540ggtaaaggct atgttttctg gggcggccgg gaaggctatg aaaccttgct
gaacactgat 600gtcggcctgg agatggataa ttatgcaaga atgctgaaaa
tggcggttgc atatgcgcat 660tctaaaggtt atacgggtga cttttatatt
gaacctaagc caaaagaacc cactaaacat 720caatatgatt tcgatgtcgc
cacttgcgtt gctttccttg aaaaatacga tttgatgcgt 780gattttaaag
taaacattga ggctaatcac gctactttgg ccggtcatac tttccaacat
840gagttacgca tggcgcgtac cttcggggta ttcggctcgg ttgatgccaa
tcagggcgac 900agcaatctgg gctgggatac cgatcagttc ccgggcaata
tttatgatac gactttggcc 960atgtatgaga ttttgaaggc cggtggattt
accaacggag gcttgaactt tgatgctaaa 1020gtgcgtcgtc cgtcatttac
cccggaagat attgcttatg cttatatttt gggcatggat 1080acgtttgcct
taggcttgat taaggcgcaa cagctgattg aggatggcag aattgatcgt
1140ttcgtagcgg aaaaatatgc tagttataag tcgggcatcg gtgctgaaat
cttgagtggt 1200aaaaccggtt tgccggaatt ggaggcttac gcattgaaga
aaggcgagcc taagttgtat 1260agtgggcggc aggaatatct tgaaagtgtc
gttaataacg taattttcaa cggaaatctt 1320tga 1323110439PRTRuminococcus
flavefaciens 110Met Glu Phe Phe Lys Asn Ile Ser Lys Ile Pro Tyr Glu
Gly Lys Asp1 5 10 15Ser Thr Asn Pro Leu Ala Phe Lys Tyr Tyr Asn Pro
Asp Glu Val Ile 20 25 30Asp Gly Lys Lys Met Arg Asp Ile Met Lys Phe
Ala Leu Ser Trp Trp 35 40 45His Thr Met Gly Gly Asp Gly Thr Asp Met
Phe Gly Cys Gly Thr Ala 50 55 60Asp Lys Thr Trp Gly Glu Asn Asp Pro
Ala Ala Arg Ala Lys Ala Lys65 70 75 80Val Asp Ala Ala Phe Glu Ile
Met Gln Lys Leu Ser Ile Asp Tyr Phe 85 90 95Cys Phe His Asp Arg Asp
Leu Ser Pro Glu Tyr Gly Ser Leu Lys Asp 100 105 110Thr Asn Ala Gln
Leu Asp Ile Val Thr Asp Tyr Ile Lys Ala Lys Gln 115 120 125Ala Glu
Thr Gly Leu Lys Cys Leu Trp Gly Thr Ala Lys Cys Phe Asp 130 135
140His Pro Arg Phe Met His Gly Ala Gly Thr Ser Pro Ser Ala Asp
Val145 150 155 160Phe Ala Phe Ser Ala Ala Gln Ile Lys Lys Ala Leu
Glu Ser Thr Val 165 170 175Lys Leu Gly Gly Thr Gly Tyr Val Phe Trp
Gly Gly Arg Glu Gly Tyr 180 185 190Glu Thr Leu Leu Asn Thr Asn Met
Gly Leu Glu Leu Asp Asn Met Ala 195 200 205Arg Leu Met Lys Met Ala
Val Glu Tyr Gly Arg Ser Ile Gly Phe Lys 210 215 220Gly Asp Phe Tyr
Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys His Gln225 230 235 240Tyr
Asp Phe Asp Thr Ala Thr Val Leu Gly Phe Leu Arg Lys Tyr Gly 245 250
255Leu Asp Lys Asp Phe Lys Met Asn Ile Glu Ala Asn His Ala Thr Leu
260 265 270Ala Gln His Thr Phe Gln His Glu Leu Cys Val Ala Arg Thr
Asn Gly 275 280 285Ala Phe Gly Ser Ile Asp Ala Asn Gln Gly Asp Pro
Leu Leu Gly Trp 290 295 300Asp Thr Asp Gln Phe Pro Thr Asn Ile Tyr
Asp Thr Thr Met Cys Met305 310 315 320Tyr Glu Val Ile Lys Ala Gly
Gly Phe Thr Asn Gly Gly Leu Asn Phe 325 330 335Asp Ala Lys Ala Arg
Arg Gly Ser Phe Thr Pro Glu Asp Ile Phe Tyr 340 345 350Ser Tyr Ile
Ala Gly Met Asp Ala Phe Ala Leu Gly Tyr Lys Ala Ala 355 360 365Ser
Lys Leu Ile Ala Asp Gly Arg Ile Asp Ser Phe Ile Ser Asp Arg 370 375
380Tyr Ala Ser Trp Ser Glu Gly Ile Gly Leu Asp Ile Ile Ser Gly
Lys385 390 395 400Ala Asp Met Ala Ala Leu Glu Lys Tyr Ala Leu Glu
Lys Gly Glu Val 405 410 415Thr Asp Ser Ile Ser Ser Gly Arg Gln Glu
Leu Leu Glu Ser Ile Val 420 425 430Asn Asn Val Ile Phe Asn Leu
435111440PRTRuminococcus flavefaciens 111Met Ser Glu Phe
Phe Thr Gly Ile Ser Lys Ile Pro Phe Glu Gly Lys1 5 10 15Ala Ser Asn
Asn Pro Met Ala Phe Lys Tyr Tyr Asn Pro Asp Glu Val 20 25 30Val Gly
Gly Lys Thr Met Arg Glu Gln Leu Lys Phe Ala Leu Ser Trp 35 40 45Trp
His Thr Met Gly Gly Asp Gly Thr Asp Met Phe Gly Val Gly Thr 50 55
60Thr Asn Lys Lys Phe Gly Gly Thr Asp Pro Met Asp Ile Ala Lys Arg65
70 75 80Lys Val Asn Ala Ala Phe Glu Leu Met Asp Lys Leu Ser Ile Asp
Tyr 85 90 95Phe Cys Phe His Asp Arg Asp Leu Ala Pro Glu Ala Asp Asn
Leu Lys 100 105 110Glu Thr Asn Gln Arg Leu Asp Glu Ile Thr Glu Tyr
Ile Ala Gln Met 115 120 125Met Gln Leu Asn Pro Asp Lys Lys Val Leu
Trp Gly Thr Ala Asn Cys 130 135 140Phe Gly Asn Pro Arg Tyr Met His
Gly Ala Gly Thr Ala Pro Asn Ala145 150 155 160Asp Val Phe Ala Phe
Ala Ala Ala Gln Ile Lys Lys Ala Ile Glu Ile 165 170 175Thr Val Lys
Leu Gly Gly Lys Gly Tyr Val Phe Trp Gly Gly Arg Glu 180 185 190Gly
Tyr Glu Thr Leu Leu Asn Thr Asn Met Gly Leu Glu Leu Asp Asn 195 200
205Met Ala Arg Leu Leu His Met Ala Val Asp Tyr Ala Arg Ser Ile Gly
210 215 220Phe Thr Gly Asp Phe Tyr Ile Glu Pro Lys Pro Lys Glu Pro
Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Thr Ala Thr Val Ile
Gly Phe Leu Arg Lys 245 250 255Tyr Asn Leu Asp Lys Asp Phe Lys Met
Asn Ile Glu Ala Asn His Ala 260 265 270Thr Leu Ala Gln His Thr Phe
Gln His Glu Leu Arg Val Ala Arg Glu 275 280 285Asn Gly Phe Phe Gly
Ser Ile Asp Ala Asn Gln Gly Asp Thr Leu Leu 290 295 300Gly Trp Asp
Thr Asp Gln Phe Pro Thr Asn Thr Tyr Asp Ala Ala Leu305 310 315
320Cys Met Tyr Glu Val Leu Lys Ala Gly Gly Phe Thr Asn Gly Gly Leu
325 330 335Asn Phe Asp Ser Lys Ala Arg Arg Gly Ser Phe Glu Met Glu
Asp Ile 340 345 350Phe His Ser Tyr Ile Ala Gly Met Asp Thr Phe Ala
Leu Gly Leu Lys 355 360 365Ile Ala Gln Lys Met Ile Asp Asp Gly Arg
Ile Asp Gln Phe Val Ala 370 375 380Asp Arg Tyr Ala Ser Trp Asn Thr
Gly Ile Gly Ala Asp Ile Ile Ser385 390 395 400Gly Lys Ala Thr Met
Ala Asp Leu Glu Ala Tyr Ala Leu Ser Lys Gly 405 410 415Asp Val Thr
Ala Ser Leu Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser 420 425 430Ile
Leu Asn Asn Ile Met Phe Asn 435 440112440PRTClostridiales sp.
112Met Lys Phe Phe Glu Asn Val Pro Lys Val Lys Tyr Glu Gly Ser Lys1
5 10 15Ser Thr Asn Pro Phe Ala Phe Lys Tyr Tyr Asn Pro Glu Ala Val
Ile 20 25 30Ala Gly Lys Lys Met Lys Asp His Leu Lys Phe Ala Met Ser
Trp Trp 35 40 45His Thr Met Thr Ala Thr Gly Gln Asp Gln Phe Gly Ser
Gly Thr Met 50 55 60Ser Arg Ile Tyr Asp Gly Gln Thr Glu Pro Leu Ala
Leu Ala Lys Ala65 70 75 80Arg Val Asp Ala Ala Phe Asp Phe Met Glu
Lys Leu Asn Ile Glu Tyr 85 90 95Phe Cys Phe His Asp Ala Asp Leu Ala
Pro Glu Gly Asn Ser Leu Gln 100 105 110Glu Arg Asn Glu Asn Leu Gln
Glu Met Val Ser Tyr Leu Lys Gln Lys 115 120 125Met Ala Gly Thr Ser
Ile Lys Leu Leu Trp Gly Thr Ser Asn Cys Phe 130 135 140Ser Asn Pro
Arg Phe Met His Gly Ala Ala Thr Ser Cys Glu Ala Asp145 150 155
160Val Phe Ala Trp Thr Ala Thr Gln Leu Lys Asn Ala Ile Asp Ala Thr
165 170 175Ile Ala Leu Gly Gly Lys Gly Tyr Val Phe Trp Gly Gly Arg
Glu Gly 180 185 190Tyr Glu Thr Leu Leu Asn Thr Asp Val Gly Leu Glu
Met Asp Asn Tyr 195 200 205Ala Arg Met Leu Lys Met Ala Val Ala Tyr
Ala His Ser Lys Gly Tyr 210 215 220Thr Gly Asp Phe Tyr Ile Glu Pro
Lys Pro Lys Glu Pro Thr Lys His225 230 235 240Gln Tyr Asp Phe Asp
Val Ala Thr Cys Val Ala Phe Leu Glu Lys Tyr 245 250 255Asp Leu Met
Arg Asp Phe Lys Val Asn Ile Glu Ala Asn His Ala Thr 260 265 270Leu
Ala Gly His Thr Phe Gln His Glu Leu Arg Met Ala Arg Thr Phe 275 280
285Gly Val Phe Gly Ser Val Asp Ala Asn Gln Gly Asp Ser Asn Leu Gly
290 295 300Trp Asp Thr Asp Gln Phe Pro Gly Asn Ile Tyr Asp Thr Thr
Leu Ala305 310 315 320Met Tyr Glu Ile Leu Lys Ala Gly Gly Phe Thr
Asn Gly Gly Leu Asn 325 330 335Phe Asp Ala Lys Val Arg Arg Pro Ser
Phe Thr Pro Glu Asp Ile Ala 340 345 350Tyr Ala Tyr Ile Leu Gly Met
Asp Thr Phe Ala Leu Gly Leu Ile Lys 355 360 365Ala Gln Gln Leu Ile
Glu Asp Gly Arg Ile Asp Arg Phe Val Ala Glu 370 375 380Lys Tyr Ala
Ser Tyr Lys Ser Gly Ile Gly Ala Glu Ile Leu Ser Gly385 390 395
400Lys Thr Gly Leu Pro Glu Leu Glu Ala Tyr Ala Leu Lys Lys Gly Glu
405 410 415Pro Lys Leu Tyr Ser Gly Arg Gln Glu Tyr Leu Glu Ser Val
Val Asn 420 425 430Asn Val Ile Phe Asn Gly Asn Leu 435
4401131665DNADebaryomyces hansenii 113atgtctcaag aagaatatag
ttctggggta caaaccccag tttctaacca ttctggttta 60gagaaagaag agcaacacaa
gttagacggt ttagatgagg atgaaattgt cgatcaatta 120ccttctttac
cagaaaaatc agctaaggat tatttattaa tttctttctt ctgtgtatta
180gttgcatttg gtggttttgt tttcggtttc gatactggta ctatctcagg
tttcgttaac 240atgagtgatt acttggaaag attcggtgag cttaatgcag
atggtgaata tttcttatct 300aatgttagaa ctggtttgat tgttgctatt
tttaatgttg gttgtgctgt cggtggtatt 360ttcttatcta agattgctga
tgtttatggt agaagaattg gtcttatgtt ttccatgatt 420atttatgtga
ttggtataat tgttcaaatc tcagcttctg acaagtggta tcaaatcgtt
480gttggtagag ctattgcagg tttagctgtt ggtaccgttt ctgtcttatc
cccattattc 540attggtgaat cagcacctaa aaccttaaga ggtactttag
tgtgttgttt ccaattatgt 600attaccttag gtatcttctt aggttactgt
actacatatg gtactaaaac ctacaccgac 660tctagacaat ggagaattcc
attaggttta tgttttgttt gggctatcat gttggttatt 720ggtatggttt
gcatgccaga atcaccaaga tacttagttg tcaagaacaa gattgaagaa
780gctaagaaat cgattggtag atccaacaag gtttcaccag aagatcctgc
tgtttacacc 840gaagtccaat tgattcaagc aggtattgaa agagaaagtt
tagctggttc tgcctcttgg 900accgaattgg ttactggtaa gccaagaatc
tttcgtagag tcattatggg tattatgtta 960caatctttac aacaattgac
tggtgacaac tatttcttct actatggtac tactattttc 1020caagctgtcg
gtatgactga ttccttccaa acatctattg ttttaggtgt tgttaacttt
1080gcatctacat ttctcggtat ctacacaatt gaaagattcg gtagaagatt
atgtttgtta 1140actggttctg tctgtatgtt cgtttgtttc atcatttact
ccattttggg tgttacaaac 1200ttatatattg atggctacga tggtccaact
tcggttccaa ccggtgatgc gatgattttc 1260attactacct tatacatttt
cttcttcgca tccacctggg ctggtggtgt ctactgtatc 1320gtttccgaaa
catacccatt gagaattaga tctaaggcca tgtccgttgc caccgctgct
1380aactggattt ggggtttctt gatctctttc ttcactccat tcatcacctc
ggctatccac 1440ttctactacg gtttcgtttt cacaggatgt ttgttattct
cgttctttta cgtttacttc 1500tttgttgttg aaactaaggg attaacttta
gaagaagttg atgaattgta tgcccaaggt 1560gttgccccat ggaagtcatc
gaaatgggtt ccaccaacca aggaagaaat ggcccattct 1620tcaggatatg
ctgctgaagc caaacctcac gatcaacaag tataa 16651141725DNASaccharomyces
cerevisiae 114atggcagttg aggagaacaa tatgcctgtt gtttcacagc
aaccccaagc tggtgaagac 60gtgatctctt cactcagtaa agattcccat ttaagcgcac
aatctcaaaa gtattctaat 120gatgaattga aagccggtga gtcagggtct
gaaggctccc aaagtgttcc tatagagata 180cccaagaagc ccatgtctga
atatgttacc gtttccttgc tttgtttgtg tgttgccttc 240ggcggcttca
tgtttggctg ggataccggt actatttctg ggtttgttgt ccaaacagac
300tttttgagaa ggtttggtat gaaacataag gatggtaccc actatttgtc
aaacgtcaga 360acaggtttaa tcgtcgccat tttcaatatt ggctgtgcct
ttggtggtat tatactttcc 420aaaggtggag atatgtatgg ccgtaaaaag
ggtctttcga ttgtcgtctc ggtttatata 480gttggtatta tcattcaaat
tgcctctatc aacaagtggt accaatattt cattggtaga 540atcatatctg
gtttgggtgt cggcggcatc gccgtcttat gtcctatgtt gatctctgaa
600attgctccaa agcacttgag aggcacacta gtttcttgtt atcagctgat
gattactgca 660ggtatctttt tgggctactg tactaattac ggtacaaaga
gctattcgaa ctcagttcaa 720tggagagttc cattagggct atgtttcgct
tggtcattat ttatgattgg cgctttgacg 780ttagttcctg aatccccacg
ttatttatgt gaggtgaata aggtagaaga cgccaagcgt 840tccattgcta
agtctaacaa ggtgtcacca gaggatcctg ccgtccaggc agagttagat
900ctgatcatgg ccggtataga agctgaaaaa ctggctggca atgcgtcctg
gggggaatta 960ttttccacca agaccaaagt atttcaacgt ttgttgatgg
gtgtgtttgt tcaaatgttc 1020caacaattaa ccggtaacaa ttattttttc
tactacggta ccgttatttt caagtcagtt 1080ggcctggatg attcctttga
aacatccatt gtcattggtg tagtcaactt tgcctccact 1140ttctttagtt
tgtggactgt cgaaaacttg ggacatcgta aatgtttact tttgggcgct
1200gccactatga tggcttgtat ggtcatctac gcctctgttg gtgttactag
attatatcct 1260cacggtaaaa gccagccatc ttctaaaggt gccggtaact
gtatgattgt ctttacctgt 1320ttttatattt tctgttatgc cacaacctgg
gcgccagttg cctgggtcat cacagcagaa 1380tcattcccac tgagagtcaa
gtcgaaatgt atggcgttgg cctctgcttc caattgggta 1440tgggggttct
tgattgcatt tttcacccca ttcatcacat ctgccattaa cttctactac
1500ggttatgtct tcatgggctg tttggttgcc atgttttttt atgtcttttt
ctttgttcca 1560gaaactaaag gcctatcgtt agaagaaatt caagaattat
gggaagaagg tgttttacct 1620tggaaatctg aaggctggat tccttcatcc
agaagaggta ataattacga tttagaggat 1680ttacaacatg acgacaaacc
gtggtacaag gccatgctag aataa 1725115750DNASaccharomyces cerevisiae
115atggtgacag tcggtgtgtt ttctgagagg gctagtttga cccatcaatt
gggggaattc 60atcgtcaaga aacaagatga ggcgctgcaa aagaagtcag actttaaagt
ttccgttagc 120ggtggctctt tgatcgatgc tctgtatgaa agtttagtag
cggacgaatc actatcttct 180cgagtgcaat ggtctaaatg gcaaatctac
ttctctgatg aaagaattgt gccactgacg 240gacgctgaca gcaattatgg
tgccttcaag agagctgttc tagataaatt accctcgact 300agtcagccaa
acgtttatcc catggacgag tccttgattg gcagcgatgc tgaatctaac
360aacaaaattg ctgcagagta cgagcgtatc gtacctcaag tgcttgattt
ggtactgttg 420ggctgtggtc ctgatggaca cacttgttcc ttattccctg
gagaaacaca taggtacttg 480ctgaacgaaa caaccaaaag agttgcttgg
tgccacgatt ctcccaagcc tccaagtgac 540agaatcacct tcactctgcc
tgtgttgaaa gacgccaaag ccctgtgttt tgtggctgag 600ggcagttcca
aacaaaatat aatgcatgag atctttgact tgaaaaacga tcaattgcca
660accgcattgg ttaacaaatt atttggtgaa aaaacatcct ggttcgttaa
tgaggaagct 720tttggaaaag ttcaaacgaa aactttttag
7501161518DNASaccharomyces cerevisiae 116atgagtgaag gccccgtcaa
attcgaaaaa aataccgtca tatctgtctt tggtgcgtca 60ggtgatctgg caaagaagaa
gacttttccc gccttatttg ggcttttcag agaaggttac 120cttgatccat
ctaccaagat cttcggttat gcccggtcca aattgtccat ggaggaggac
180ctgaagtccc gtgtcctacc ccacttgaaa aaacctcacg gtgaagccga
tgactctaag 240gtcgaacagt tcttcaagat ggtcagctac atttcgggaa
attacgacac agatgaaggc 300ttcgacgaat taagaacgca gatcgagaaa
ttcgagaaaa gtgccaacgt cgatgtccca 360caccgtctct tctatctggc
cttgccgcca agcgtttttt tgacggtggc caagcagatc 420aagagtcgtg
tgtacgcaga gaatggcatc acccgtgtaa tcgtagagaa acctttcggc
480cacgacctgg cctctgccag ggagctgcaa aaaaacctgg ggcccctctt
taaagaagaa 540gagttgtaca gaattgacca ttacttgggt aaagagttgg
tcaagaatct tttagtcttg 600aggttcggta accagttttt gaatgcctcg
tggaatagag acaacattca aagcgttcag 660atttcgttta aagagaggtt
cggcaccgaa ggccgtggcg gctatttcga ctctataggc 720ataatcagag
acgtgatgca gaaccatctg ttacaaatca tgactctctt gactatggaa
780agaccggtgt cttttgaccc ggaatctatt cgtgacgaaa aggttaaggt
tctaaaggcc 840gtggccccca tcgacacgga cgacgtcctc ttgggccagt
acggtaaatc tgaggacggg 900tctaagcccg cctacgtgga tgatgacact
gtagacaagg actctaaatg tgtcactttt 960gcagcaatga ctttcaacat
cgaaaacgag cgttgggagg gcgtccccat catgatgcgt 1020gccggtaagg
ctttgaatga gtccaaggtg gagatcagac tgcagtacaa agcggtcgca
1080tcgggtgtct tcaaagacat tccaaataac gaactggtca tcagagtgca
gcccgatgcc 1140gctgtgtacc taaagtttaa tgctaagacc cctggtctgt
caaatgctac ccaagtcaca 1200gatctgaatc taacttacgc aagcaggtac
caagactttt ggattccaga ggcttacgag 1260gtgttgataa gagacgccct
actgggtgac cattccaact ttgtcagaga tgacgaattg 1320gatatcagtt
ggggcatatt caccccatta ctgaagcaca tagagcgtcc ggacggtcca
1380acaccggaaa tttaccccta cggatcaaga ggtccaaagg gattgaagga
atatatgcaa 1440aaacacaagt atgttatgcc cgaaaagcac ccttacgctt
ggcccgtgac taagccagaa 1500gatacgaagg ataattag
1518117554PRTDebaryomyces hansenii 117Met Ser Gln Glu Glu Tyr Ser
Ser Gly Val Gln Thr Pro Val Ser Asn1 5 10 15His Ser Gly Leu Glu Lys
Glu Glu Gln His Lys Leu Asp Gly Leu Asp 20 25 30Glu Asp Glu Ile Val
Asp Gln Leu Pro Ser Leu Pro Glu Lys Ser Ala 35 40 45Lys Asp Tyr Leu
Leu Ile Ser Phe Phe Cys Val Leu Val Ala Phe Gly 50 55 60Gly Phe Val
Phe Gly Phe Asp Thr Gly Thr Ile Ser Gly Phe Val Asn65 70 75 80Met
Ser Asp Tyr Leu Glu Arg Phe Gly Glu Leu Asn Ala Asp Gly Glu 85 90
95Tyr Phe Leu Ser Asn Val Arg Thr Gly Leu Ile Val Ala Ile Phe Asn
100 105 110Val Gly Cys Ala Val Gly Gly Ile Phe Leu Ser Lys Ile Ala
Asp Val 115 120 125Tyr Gly Arg Arg Ile Gly Leu Met Phe Ser Met Ile
Ile Tyr Val Ile 130 135 140Gly Ile Ile Val Gln Ile Ser Ala Ser Asp
Lys Trp Tyr Gln Ile Val145 150 155 160Val Gly Arg Ala Ile Ala Gly
Leu Ala Val Gly Thr Val Ser Val Leu 165 170 175Ser Pro Leu Phe Ile
Gly Glu Ser Ala Pro Lys Thr Leu Arg Gly Thr 180 185 190Leu Val Cys
Cys Phe Gln Leu Cys Ile Thr Leu Gly Ile Phe Leu Gly 195 200 205Tyr
Cys Thr Thr Tyr Gly Thr Lys Thr Tyr Thr Asp Ser Arg Gln Trp 210 215
220Arg Ile Pro Leu Gly Leu Cys Phe Val Trp Ala Ile Met Leu Val
Ile225 230 235 240Gly Met Val Cys Met Pro Glu Ser Pro Arg Tyr Leu
Val Val Lys Asn 245 250 255Lys Ile Glu Glu Ala Lys Lys Ser Ile Gly
Arg Ser Asn Lys Val Ser 260 265 270Pro Glu Asp Pro Ala Val Tyr Thr
Glu Val Gln Leu Ile Gln Ala Gly 275 280 285Ile Glu Arg Glu Ser Leu
Ala Gly Ser Ala Ser Trp Thr Glu Leu Val 290 295 300Thr Gly Lys Pro
Arg Ile Phe Arg Arg Val Ile Met Gly Ile Met Leu305 310 315 320Gln
Ser Leu Gln Gln Leu Thr Gly Asp Asn Tyr Phe Phe Tyr Tyr Gly 325 330
335Thr Thr Ile Phe Gln Ala Val Gly Met Thr Asp Ser Phe Gln Thr Ser
340 345 350Ile Val Leu Gly Val Val Asn Phe Ala Ser Thr Phe Leu Gly
Ile Tyr 355 360 365Thr Ile Glu Arg Phe Gly Arg Arg Leu Cys Leu Leu
Thr Gly Ser Val 370 375 380Cys Met Phe Val Cys Phe Ile Ile Tyr Ser
Ile Leu Gly Val Thr Asn385 390 395 400Leu Tyr Ile Asp Gly Tyr Asp
Gly Pro Thr Ser Val Pro Thr Gly Asp 405 410 415Ala Met Ile Phe Ile
Thr Thr Leu Tyr Ile Phe Phe Phe Ala Ser Thr 420 425 430Trp Ala Gly
Gly Val Tyr Cys Ile Val Ser Glu Thr Tyr Pro Leu Arg 435 440 445Ile
Arg Ser Lys Ala Met Ser Val Ala Thr Ala Ala Asn Trp Ile Trp 450 455
460Gly Phe Leu Ile Ser Phe Phe Thr Pro Phe Ile Thr Ser Ala Ile
His465 470 475 480Phe Tyr Tyr Gly Phe Val Phe Thr Gly Cys Leu Leu
Phe Ser Phe Phe 485 490 495Tyr Val Tyr Phe Phe Val Val Glu Thr Lys
Gly Leu Thr Leu Glu Glu 500 505 510Val Asp Glu Leu Tyr Ala Gln Gly
Val Ala Pro Trp Lys Ser Ser Lys 515 520 525Trp Val Pro Pro Thr Lys
Glu Glu Met Ala His Ser Ser Gly Tyr Ala 530 535 540Ala Glu Ala Lys
Pro His Asp Gln Gln Val545 550118574PRTSaccharomyces cerevisiae
118Met Ala Val Glu Glu Asn Asn Met Pro Val Val Ser Gln Gln Pro Gln1
5 10 15Ala Gly Glu Asp Val Ile Ser Ser Leu Ser Lys Asp Ser His Leu
Ser 20 25 30Ala Gln Ser Gln Lys Tyr Ser Asn Asp Glu Leu Lys Ala Gly
Glu Ser 35 40 45Gly Ser Glu Gly Ser Gln Ser Val Pro Ile Glu Ile Pro
Lys Lys Pro 50 55
60Met Ser Glu Tyr Val Thr Val Ser Leu Leu Cys Leu Cys Val Ala Phe65
70 75 80Gly Gly Phe Met Phe Gly Trp Asp Thr Gly Thr Ile Ser Gly Phe
Val 85 90 95Val Gln Thr Asp Phe Leu Arg Arg Phe Gly Met Lys His Lys
Asp Gly 100 105 110Thr His Tyr Leu Ser Asn Val Arg Thr Gly Leu Ile
Val Ala Ile Phe 115 120 125Asn Ile Gly Cys Ala Phe Gly Gly Ile Ile
Leu Ser Lys Gly Gly Asp 130 135 140Met Tyr Gly Arg Lys Lys Gly Leu
Ser Ile Val Val Ser Val Tyr Ile145 150 155 160Val Gly Ile Ile Ile
Gln Ile Ala Ser Ile Asn Lys Trp Tyr Gln Tyr 165 170 175Phe Ile Gly
Arg Ile Ile Ser Gly Leu Gly Val Gly Gly Ile Ala Val 180 185 190Leu
Cys Pro Met Leu Ile Ser Glu Ile Ala Pro Lys His Leu Arg Gly 195 200
205Thr Leu Val Ser Cys Tyr Gln Leu Met Ile Thr Ala Gly Ile Phe Leu
210 215 220Gly Tyr Cys Thr Asn Tyr Gly Thr Lys Ser Tyr Ser Asn Ser
Val Gln225 230 235 240Trp Arg Val Pro Leu Gly Leu Cys Phe Ala Trp
Ser Leu Phe Met Ile 245 250 255Gly Ala Leu Thr Leu Val Pro Glu Ser
Pro Arg Tyr Leu Cys Glu Val 260 265 270Asn Lys Val Glu Asp Ala Lys
Arg Ser Ile Ala Lys Ser Asn Lys Val 275 280 285Ser Pro Glu Asp Pro
Ala Val Gln Ala Glu Leu Asp Leu Ile Met Ala 290 295 300Gly Ile Glu
Ala Glu Lys Leu Ala Gly Asn Ala Ser Trp Gly Glu Leu305 310 315
320Phe Ser Thr Lys Thr Lys Val Phe Gln Arg Leu Leu Met Gly Val Phe
325 330 335Val Gln Met Phe Gln Gln Leu Thr Gly Asn Asn Tyr Phe Phe
Tyr Tyr 340 345 350Gly Thr Val Ile Phe Lys Ser Val Gly Leu Asp Asp
Ser Phe Glu Thr 355 360 365Ser Ile Val Ile Gly Val Val Asn Phe Ala
Ser Thr Phe Phe Ser Leu 370 375 380Trp Thr Val Glu Asn Leu Gly His
Arg Lys Cys Leu Leu Leu Gly Ala385 390 395 400Ala Thr Met Met Ala
Cys Met Val Ile Tyr Ala Ser Val Gly Val Thr 405 410 415Arg Leu Tyr
Pro His Gly Lys Ser Gln Pro Ser Ser Lys Gly Ala Gly 420 425 430Asn
Cys Met Ile Val Phe Thr Cys Phe Tyr Ile Phe Cys Tyr Ala Thr 435 440
445Thr Trp Ala Pro Val Ala Trp Val Ile Thr Ala Glu Ser Phe Pro Leu
450 455 460Arg Val Lys Ser Lys Cys Met Ala Leu Ala Ser Ala Ser Asn
Trp Val465 470 475 480Trp Gly Phe Leu Ile Ala Phe Phe Thr Pro Phe
Ile Thr Ser Ala Ile 485 490 495Asn Phe Tyr Tyr Gly Tyr Val Phe Met
Gly Cys Leu Val Ala Met Phe 500 505 510Phe Tyr Val Phe Phe Phe Val
Pro Glu Thr Lys Gly Leu Ser Leu Glu 515 520 525Glu Ile Gln Glu Leu
Trp Glu Glu Gly Val Leu Pro Trp Lys Ser Glu 530 535 540Gly Trp Ile
Pro Ser Ser Arg Arg Gly Asn Asn Tyr Asp Leu Glu Asp545 550 555
560Leu Gln His Asp Asp Lys Pro Trp Tyr Lys Ala Met Leu Glu 565
570119505PRTSaccharomyces cerevisiae 119Met Ser Glu Gly Pro Val Lys
Phe Glu Lys Asn Thr Val Ile Ser Val1 5 10 15Phe Gly Ala Ser Gly Asp
Leu Ala Lys Lys Lys Thr Phe Pro Ala Leu 20 25 30Phe Gly Leu Phe Arg
Glu Gly Tyr Leu Asp Pro Ser Thr Lys Ile Phe 35 40 45Gly Tyr Ala Arg
Ser Lys Leu Ser Met Glu Glu Asp Leu Lys Ser Arg 50 55 60Val Leu Pro
His Leu Lys Lys Pro His Gly Glu Ala Asp Asp Ser Lys65 70 75 80Val
Glu Gln Phe Phe Lys Met Val Ser Tyr Ile Ser Gly Asn Tyr Asp 85 90
95Thr Asp Glu Gly Phe Asp Glu Leu Arg Thr Gln Ile Glu Lys Phe Glu
100 105 110Lys Ser Ala Asn Val Asp Val Pro His Arg Leu Phe Tyr Leu
Ala Leu 115 120 125Pro Pro Ser Val Phe Leu Thr Val Ala Lys Gln Ile
Lys Ser Arg Val 130 135 140Tyr Ala Glu Asn Gly Ile Thr Arg Val Ile
Val Glu Lys Pro Phe Gly145 150 155 160His Asp Leu Ala Ser Ala Arg
Glu Leu Gln Lys Asn Leu Gly Pro Leu 165 170 175Phe Lys Glu Glu Glu
Leu Tyr Arg Ile Asp His Tyr Leu Gly Lys Glu 180 185 190Leu Val Lys
Asn Leu Leu Val Leu Arg Phe Gly Asn Gln Phe Leu Asn 195 200 205Ala
Ser Trp Asn Arg Asp Asn Ile Gln Ser Val Gln Ile Ser Phe Lys 210 215
220Glu Arg Phe Gly Thr Glu Gly Arg Gly Gly Tyr Phe Asp Ser Ile
Gly225 230 235 240Ile Ile Arg Asp Val Met Gln Asn His Leu Leu Gln
Ile Met Thr Leu 245 250 255Leu Thr Met Glu Arg Pro Val Ser Phe Asp
Pro Glu Ser Ile Arg Asp 260 265 270Glu Lys Val Lys Val Leu Lys Ala
Val Ala Pro Ile Asp Thr Asp Asp 275 280 285Val Leu Leu Gly Gln Tyr
Gly Lys Ser Glu Asp Gly Ser Lys Pro Ala 290 295 300Tyr Val Asp Asp
Asp Thr Val Asp Lys Asp Ser Lys Cys Val Thr Phe305 310 315 320Ala
Ala Met Thr Phe Asn Ile Glu Asn Glu Arg Trp Glu Gly Val Pro 325 330
335Ile Met Met Arg Ala Gly Lys Ala Leu Asn Glu Ser Lys Val Glu Ile
340 345 350Arg Leu Gln Tyr Lys Ala Val Ala Ser Gly Val Phe Lys Asp
Ile Pro 355 360 365Asn Asn Glu Leu Val Ile Arg Val Gln Pro Asp Ala
Ala Val Tyr Leu 370 375 380Lys Phe Asn Ala Lys Thr Pro Gly Leu Ser
Asn Ala Thr Gln Val Thr385 390 395 400Asp Leu Asn Leu Thr Tyr Ala
Ser Arg Tyr Gln Asp Phe Trp Ile Pro 405 410 415Glu Ala Tyr Glu Val
Leu Ile Arg Asp Ala Leu Leu Gly Asp His Ser 420 425 430Asn Phe Val
Arg Asp Asp Glu Leu Asp Ile Ser Trp Gly Ile Phe Thr 435 440 445Pro
Leu Leu Lys His Ile Glu Arg Pro Asp Gly Pro Thr Pro Glu Ile 450 455
460Tyr Pro Tyr Gly Ser Arg Gly Pro Lys Gly Leu Lys Glu Tyr Met
Gln465 470 475 480Lys His Lys Tyr Val Met Pro Glu Lys His Pro Tyr
Ala Trp Pro Val 485 490 495Thr Lys Pro Glu Asp Thr Lys Asp Asn 500
505120249PRTSaccharomyces cerevisiae 120Met Val Thr Val Gly Val Phe
Ser Glu Arg Ala Ser Leu Thr His Gln1 5 10 15Leu Gly Glu Phe Ile Val
Lys Lys Gln Asp Glu Ala Leu Gln Lys Lys 20 25 30Ser Asp Phe Lys Val
Ser Val Ser Gly Gly Ser Leu Ile Asp Ala Leu 35 40 45Tyr Glu Ser Leu
Val Ala Asp Glu Ser Leu Ser Ser Arg Val Gln Trp 50 55 60Ser Lys Trp
Gln Ile Tyr Phe Ser Asp Glu Arg Ile Val Pro Leu Thr65 70 75 80Asp
Ala Asp Ser Asn Tyr Gly Ala Phe Lys Arg Ala Val Leu Asp Lys 85 90
95Leu Pro Ser Thr Ser Gln Pro Asn Val Tyr Pro Met Asp Glu Ser Leu
100 105 110Ile Gly Ser Asp Ala Glu Ser Asn Asn Lys Ile Ala Ala Glu
Tyr Glu 115 120 125Arg Ile Val Pro Gln Val Leu Asp Leu Val Leu Leu
Gly Cys Gly Pro 130 135 140Asp Gly His Thr Cys Ser Leu Phe Pro Gly
Glu Thr His Arg Tyr Leu145 150 155 160Leu Asn Glu Thr Thr Lys Arg
Val Ala Trp Cys His Asp Ser Pro Lys 165 170 175Pro Pro Ser Asp Arg
Ile Thr Phe Thr Leu Pro Val Leu Lys Asp Ala 180 185 190Lys Ala Leu
Cys Phe Val Ala Glu Gly Ser Ser Lys Gln Asn Ile Met 195 200 205His
Glu Ile Phe Asp Leu Lys Asn Asp Gln Leu Pro Thr Ala Leu Val 210 215
220Asn Lys Leu Phe Gly Glu Lys Thr Ser Trp Phe Val Asn Glu Glu
Ala225 230 235 240Phe Gly Lys Val Gln Thr Lys Thr Phe
2451212880DNASaccharomyces cerevisiae 121atgactgtta ctactccttt
tgtgaatggt acttcttatt gtaccgtcac tgcatattcc 60gttcaatctt ataaagctgc
catagatttt tacaccaagt ttttgtcatt agaaaaccgc 120tcttctccag
atgaaaactc cactttattg tctaacgatt ccatctcttt gaagatcctt
180ctacgtcctg atgaaaaaat caataaaaat gttgaggctc atttgaagga
attgaacagt 240attaccaaga ctcaagactg gagatcacat gccacccaat
ccttggtatt taacacttcc 300gacatcttgg cagtcaagga cactctaaat
gctatgaacg ctcctcttca aggctaccca 360acagaactat ttccaatgca
gttgtacact ttggacccat taggtaacgt tgttggtgtt 420acttctacta
agaacgcagt ttcaaccaag ccaactccac caccagcacc agaagcttct
480gctgagtctg gtctttcctc taaagttcac tcttacactg atttggctta
ccgtatgaaa 540accaccgaca cctatccatc tctgccaaag ccattgaaca
ggcctcaaaa ggcaattgcc 600gtcatgactt ccggtggtga tgctccaggt
atgaactcta acgttagagc catcgtgcgt 660tccgctatct tcaaaggttg
tcgtgccttt gttgtcatgg aaggttatga aggtttggtt 720cgtggtggtc
cagaatacat caaggaattc cactgggaag acgtccgtgg ttggtctgct
780gaaggtggta ccaacattgg tactgcccgt tgtatggaat tcaagaagcg
cgaaggtaga 840ttattgggtg cccaacattt gattgaggcc ggtgtcgatg
ctttgatcgt ttgtggtggt 900gacggttctt tgactggtgc tgatctgttt
agatcagaat ggccttcttt gatcgaggaa 960ttgttgaaaa caaacagaat
ttccaacgaa caatacgaaa gaatgaagca tttgaatatt 1020tgcggtactg
tcggttctat tgataacgat atgtccacca cggatgctac tattggtgct
1080tactctgcct tggacagaat ctgtaaggcc atcgattacg ttgaagccac
tgccaactct 1140cactcaagag ctttcgttgt tgaagttatg ggtagaaact
gtggttggtt agctttatta 1200gctggtatcg ccacttccgc tgactatatc
tttattccag agaagccagc cacttccagc 1260gaatggcaag atcaaatgtg
tgacattgtc tccaagcaca gatcaagggg taagagaacc 1320accattgttg
ttgttgcaga aggtgctatc gctgctgact tgaccccaat ttctccaagc
1380gacgtccaca aagttctagt tgacagatta ggtttggata caagaattac
taccttaggt 1440cacgttcaaa gaggtggtac tgctgttgct tacgaccgta
tcttggctac tttacaaggt 1500cttgaggccg ttaatgccgt tttggaatcc
actccagaca ccccatcacc attgattgct 1560gttaacgaaa acaaaattgt
tcgtaaacca ttaatggaat ccgtcaagtt gaccaaagca 1620gttgcagaag
ccattcaagc taaggatttc aagagagcta tgtctttaag agacactgag
1680ttcattgaac atttaaacaa tttcatggct atcaactctg ctgaccacaa
cgaaccaaag 1740ctaccaaagg acaagagact gaagattgcc attgttaatg
tcggtgctcc agctggtggt 1800atcaactctg ccgtctactc gatggctact
tactgtatgt cccaaggtca cagaccatac 1860gctatctaca atggttggtc
tggtttggca agacatgaaa gtgttcgttc tttgaactgg 1920aaggatatgt
tgggttggca atcccgtggt ggttctgaaa tcggtactaa cagagtcact
1980ccagaagaag cagatctagg tatgattgct tactatttcc aaaagtacga
atttgatggt 2040ttgatcatcg ttggtggttt cgaagctttt gaatctttac
atcaattaga gagagcaaga 2100gaaagttatc cagctttcag aatcccaatg
gtcttgatac cagctacttt gtctaacaat 2160gttccaggta ctgaatactc
tttgggttct gataccgctt tgaatgctct aatggaatac 2220tgtgatgttg
ttaaacaatc cgcttcttca accagaggta gagccttcgt tgtcgattgt
2280caaggtggta actcaggcta tttggccact tacgcttctt tggctgttgg
tgctcaagtc 2340tcttatgtcc cagaagaagg tatttctttg gagcaattgt
ccgaggatat tgaatactta 2400gctcaatctt ttgaaaaggc agaaggtaga
ggtagatttg gtaaattgat tttgaagagt 2460acaaacgctt ctaaggcttt
atcagccact aaattggctg aagttattac tgctgaagcc 2520gatggcagat
ttgacgctaa gccagcttat ccaggtcatg tacaacaagg tggtttgcca
2580tctccaattg atagaacaag agccactaga atggccatta aagctgtcgg
cttcatcaaa 2640gacaaccaag ctgccattgc tgaagctcgt gctgccgaag
aaaacttcaa cgctgatgac 2700aagaccattt ctgacactgc tgctgtcgtt
ggtgttaagg gttcacatgt cgtttacaac 2760tccattagac aattgtatga
ctatgaaact gaagtttcca tgagaatgcc aaaggtcatt 2820cactggcaag
ctaccagact cattgctgac catttggttg gaagaaagag agttgattaa
28801224179DNASaccharomyces cerevisiae 122atgactaacg aaaaggtctg
gatagagaag ttggataatc caactctttc agtgttacca 60catgactttt tacgcccaca
acaagaacct tatacgaaac aagctacata ttcgttacag 120ctacctcagc
tcgatgtgcc tcatgatagt ttttctaaca aatacgctgt cgctttgagt
180gtatgggctg cattgatata tagagtaacc ggtgacgatg atattgttct
ttatattgcg 240aataacaaaa tcttaagatt caatattcaa ccaacgtggt
catttaatga gctgtattct 300acaattaaca atgagttgaa caagctcaat
tctattgagg ccaatttttc ctttgacgag 360ctagctgaaa aaattcaaag
ttgccaagat ctggaaagga cccctcagtt gttccgtttg 420gcctttttgg
aaaaccaaga tttcaaatta gacgagttca agcatcattt agtggacttt
480gctttgaatt tggataccag taataatgcg catgttttga acttaattta
taacagctta 540ctgtattcga atgaaagagt aaccattgtt gcggaccaat
ttactcaata tttgactgct 600gcgctaagcg atccatccaa ttgcataact
aaaatctctc tgatcaccgc atcatccaag 660gatagtttac ctgatccaac
taagaacttg ggctggtgcg atttcgtggg gtgtattcac 720gacattttcc
aggacaatgc tgaagccttc ccagagagaa cctgtgttgt ggagactcca
780acactaaatt ccgacaagtc ccgttctttc acttatcgcg acatcaaccg
cacttctaac 840atagttgccc attatttgat taaaacaggt atcaaaagag
gtgatgtagt gatgatctat 900tcttctaggg gtgtggattt gatggtatgt
gtgatgggtg tcttgaaagc cggcgcaacc 960ttttcagtta tcgaccctgc
atatccccca gccagacaaa ccatttactt aggtgttgct 1020aaaccacgtg
ggttgattgt tattagagct gctggacaat tggatcaact agtagaagat
1080tacatcaatg atgaattgga gattgtttca agaatcaatt ccatcgctat
tcaagaaaat 1140ggtaccattg aaggtggcaa attggacaat ggcgaggatg
ttttggctcc atatgatcac 1200tacaaagaca ccagaacagg tgttgtagtt
ggaccagatt ccaacccaac cctatctttc 1260acatctggtt ccgaaggtat
tcctaagggt gttcttggta gacatttttc cttggcttat 1320tatttcaatt
ggatgtccaa aaggttcaac ttaacagaaa atgataaatt cacaatgctg
1380agcggtattg cacatgatcc aattcaaaga gatatgttta caccattatt
tttaggtgcc 1440caattgtatg tccctactca agatgatatt ggtacaccgg
gccgtttagc ggaatggatg 1500agtaagtatg gttgcacagt tacccattta
acacctgcca tgggtcaatt acttactgcc 1560caagctacta caccattccc
taagttacat catgcgttct ttgtgggtga cattttaaca 1620aaacgtgatt
gtctgaggtt acaaaccttg gcagaaaatt gccgtattgt taatatgtac
1680ggtaccactg aaacacagcg tgcagtttct tatttcgaag ttaaatcaaa
aaatgacgat 1740ccaaactttt tgaaaaaatt gaaagatgtc atgcctgctg
gtaaaggtat gttgaacgtt 1800cagctactag ttgttaacag gaacgatcgt
actcaaatat gtggtattgg cgaaataggt 1860gagatttatg ttcgtgcagg
tggtttggcc gaaggttata gaggattacc agaattgaat 1920aaagaaaaat
ttgtgaacaa ctggtttgtt gaaaaagatc actggaatta tttggataag
1980gataatggtg aaccttggag acaattctgg ttaggtccaa gagatagatt
gtacagaacg 2040ggtgatttag gtcgttatct accaaacggt gactgtgaat
gttgcggtag ggctgatgat 2100caagttaaaa ttcgtgggtt cagaatcgaa
ttaggagaaa tagatacgca catttcccaa 2160catccattgg taagagaaaa
cattacttta gttcgcaaaa atgccgacaa tgagccaaca 2220ttgatcacat
ttatggtccc aagatttgac aagccagatg acttgtctaa gttccaaagt
2280gatgttccaa aggaggttga aactgaccct atagttaagg gcttaatcgg
ttaccatctt 2340ttatccaagg acatcaggac tttcttaaag aaaagattgg
ctagctatgc tatgccttcc 2400ttgattgtgg ttatggataa actaccattg
aatccaaatg gtaaagttga taagcctaaa 2460cttcaattcc caactcccaa
gcaattaaat ttggtagctg aaaatacagt ttctgaaact 2520gacgactctc
agtttaccaa tgttgagcgc gaggttagag acttatggtt aagtatatta
2580cctaccaagc cagcatctgt atcaccagat gattcgtttt tcgatttagg
tggtcattct 2640atcttggcta ccaaaatgat ttttacctta aagaaaaagc
tgcaagttga tttaccattg 2700ggcacaattt tcaagtatcc aacgataaag
gcctttgccg cggaaattga cagaattaaa 2760tcatcgggtg gatcatctca
aggtgaggtc gtcgaaaatg tcactgcaaa ttatgcggaa 2820gacgccaaga
aattggttga gacgctacca agttcgtacc cctctcgaga atattttgtt
2880gaacctaata gtgccgaagg aaaaacaaca attaatgtgt ttgttaccgg
tgtcacagga 2940tttctgggct cctacatcct tgcagatttg ttaggacgtt
ctccaaagaa ctacagtttc 3000aaagtgtttg cccacgtcag ggccaaggat
gaagaagctg catttgcaag attacaaaag 3060gcaggtatca cctatggtac
ttggaacgaa aaatttgcct caaatattaa agttgtatta 3120ggcgatttat
ctaaaagcca atttggtctt tcagatgaga agtggatgga tttggcaaac
3180acagttgata taattatcca taatggtgcg ttagttcact gggtttatcc
atatgccaaa 3240ttgagggatc caaatgttat ttcaactatc aatgttatga
gcttagccgc cgtcggcaag 3300ccaaagttct ttgactttgt ttcctccact
tctactcttg acactgaata ctactttaat 3360ttgtcagata aacttgttag
cgaagggaag ccaggcattt tagaatcaga cgatttaatg 3420aactctgcaa
gcgggctcac tggtggatat ggtcagtcca aatgggctgc tgagtacatc
3480attagacgtg caggtgaaag gggcctacgt gggtgtattg tcagaccagg
ttacgtaaca 3540ggtgcctctg ccaatggttc ttcaaacaca gatgatttct
tattgagatt tttgaaaggt 3600tcagtccaat taggtaagat tccagatatc
gaaaattccg tgaatatggt tccagtagat 3660catgttgctc gtgttgttgt
tgctacgtct ttgaatcctc ccaaagaaaa tgaattggcc 3720gttgctcaag
taacgggtca cccaagaata ttattcaaag actacttgta tactttacac
3780gattatggtt acgatgtcga aatcgaaagc tattctaaat ggaagaaatc
attggaggcg 3840tctgttattg acaggaatga agaaaatgcg ttgtatcctt
tgctacacat ggtcttagac 3900aacttacctg aaagtaccaa agctccggaa
ctagacgata ggaacgccgt ggcatcttta 3960aagaaagaca ccgcatggac
aggtgttgat tggtctaatg gaataggtgt tactccagaa 4020gaggttggta
tatatattgc atttttaaac aaggttggat ttttacctcc accaactcat
4080aatgacaaac ttccactgcc aagtatagaa ctaactcaag cgcaaataag
tctagttgct 4140tcaggtgctg gtgctcgtgg aagctccgca gcagcttaa
4179123505PRTPseudomonas fluorescens 123Met Thr Thr Thr Arg Lys Lys
Ser Lys Ala Leu Pro Ala Pro Pro Thr1 5 10 15Thr Leu
Phe Leu Phe Gly Ala Arg Gly Asp Leu Val Lys Arg Leu Leu 20 25 30Met
Pro Ala Leu Tyr Asn Leu Ser Arg Asp Gly Leu Leu Asp Glu Gly 35 40
45Leu Arg Ile Val Gly Val Asp His Asn Ala Val Ser Asp Ala Glu Phe
50 55 60Ala Thr Leu Leu Glu Asp Phe Leu Arg Asp Glu Val Leu Asn Lys
Gln65 70 75 80Gly Gln Gly Ala Ala Val Asp Ala Ala Val Trp Ala Arg
Leu Thr Arg 85 90 95Gly Ile Asn Tyr Val Gln Gly Asp Phe Leu Asp Asp
Ser Thr Tyr Ala 100 105 110Glu Leu Ala Ala Arg Ile Ala Ala Ser Gly
Thr Gly Asn Ala Val Phe 115 120 125Tyr Leu Ala Thr Ala Pro Arg Phe
Phe Ser Glu Val Val Arg Arg Leu 130 135 140Gly Ser Ala Gly Leu Leu
Glu Glu Gly Pro Gln Ala Phe Arg Arg Val145 150 155 160Val Ile Glu
Lys Pro Phe Gly Ser Asp Leu Gln Thr Ala Glu Ala Leu 165 170 175Asn
Gly Cys Leu Leu Lys Val Met Ser Glu Lys Gln Ile Tyr Arg Ile 180 185
190Asp His Tyr Leu Gly Lys Glu Thr Val Gln Asn Ile Leu Val Ser Arg
195 200 205Phe Ser Asn Ser Leu Phe Glu Ala Phe Trp Asn Asn His Tyr
Ile Asp 210 215 220His Val Gln Ile Thr Ala Ala Glu Thr Val Gly Val
Glu Thr Arg Gly225 230 235 240Ser Phe Tyr Glu His Thr Gly Ala Leu
Arg Asp Met Val Pro Asn His 245 250 255Leu Phe Gln Leu Leu Ala Met
Val Ala Met Glu Pro Pro Ala Ala Phe 260 265 270Gly Ala Asp Ala Val
Arg Gly Glu Lys Ala Lys Val Val Gly Ala Ile 275 280 285Arg Pro Trp
Ser Val Glu Glu Ala Arg Ala Asn Ser Val Arg Gly Gln 290 295 300Tyr
Ser Ala Gly Glu Val Ala Gly Lys Ala Leu Ala Gly Tyr Arg Glu305 310
315 320Glu Ala Asn Val Ala Pro Asp Ser Ser Thr Glu Thr Tyr Val Ala
Leu 325 330 335Lys Val Met Ile Asp Asn Trp Arg Trp Val Gly Val Pro
Phe Tyr Leu 340 345 350Arg Thr Gly Lys Arg Met Ser Val Arg Asp Thr
Glu Ile Val Ile Cys 355 360 365Phe Lys Pro Ala Pro Tyr Ala Gln Phe
Arg Asp Thr Glu Val Glu Arg 370 375 380Leu Leu Pro Thr Tyr Leu Arg
Ile Gln Ile Gln Pro Asn Glu Gly Met385 390 395 400Trp Phe Asp Leu
Leu Ala Lys Lys Pro Gly Pro Ser Leu Asp Met Ala 405 410 415Asn Ile
Glu Leu Gly Phe Ala Tyr Arg Asp Phe Phe Glu Met Gln Pro 420 425
430Ser Thr Gly Tyr Glu Thr Leu Ile Tyr Asp Cys Leu Ile Gly Asp Gln
435 440 445Thr Leu Phe Gln Arg Ala Asp Asn Ile Glu Asn Gly Trp Arg
Ala Val 450 455 460Gln Pro Phe Leu Asp Ala Trp Gln Gln Asp Ala Ser
Leu Gln Asn Tyr465 470 475 480Pro Ala Gly Val Asp Gly Pro Ala Ala
Gly Asp Glu Leu Leu Ala Arg 485 490 495Asp Gly Arg Val Trp Arg Pro
Leu Gly 500 5051241518DNAPseudomonas fluorescens 124atgaccacca
cgcgaaagaa gtccaaggcg ttgccggcgc cgccgaccac gctgttcctg 60ttcggcgccc
gcggtgatct ggtcaagcgc ctgctgatgc cggcgctgta caacctcagc
120cgcgacggtt tgctggatga ggggctgcgg attgtcggcg tcgaccacaa
cgcggtgagc 180gacgccgagt tcgccacgct gctggaagac ttccttcgcg
atgaagtgct caacaagcaa 240ggccaggggg cggcggtgga tgccgccgtc
tgggcccgcc tgacccgggg catcaactat 300gtccagggcg attttctcga
cgactccacc tatgccgaac tggcggcgcg gattgccgcc 360agcggcaccg
gcaacgcggt gttctacctg gccaccgcac cgcgcttctt cagtgaagtg
420gtgcgccgcc tgggcagcgc cgggttgctg gaggaggggc cgcaggcttt
tcgccgggtg 480gtgatcgaaa aacccttcgg ctccgacctg cagaccgccg
aagccctcaa cggctgcctg 540ctcaaggtca tgagcgagaa gcagatctat
cgcatcgacc attacctggg caaggaaacg 600gtccagaaca tcctggtcag
ccgtttttcc aacagcctgt tcgaggcatt ctggaacaac 660cattacatcg
accacgtgca gatcaccgcg gcggaaaccg tcggcgtgga aacccgtggc
720agcttttatg aacacaccgg tgccctgcgg gacatggtgc ccaaccacct
gttccagttg 780ctggcgatgg tggccatgga gccgcccgct gcctttggcg
ccgatgcggt acgtggcgaa 840aaggccaagg tggtgggggc tatccgcccc
tggtccgtgg aagaggcccg ggccaactcg 900gtgcgcggcc agtacagcgc
cggtgaagtg gccggcaagg ccctggcggg ctaccgcgag 960gaagccaacg
tggcgccgga cagcagcacc gaaacctacg ttgcgctgaa ggtgatgatc
1020gacaactggc gctgggtcgg ggtgccgttc tacctgcgca ccggcaagcg
catgagtgtg 1080cgcgacaccg agatcgtcat ctgcttcaag ccggcgccct
atgcacagtt ccgcgatacc 1140gaggtcgagc gcctgttgcc gacctacctg
cggatccaga tccagcccaa cgaaggcatg 1200tggttcgacc tgctggcgaa
aaagcccggg ccgagcctgg acatggccaa catcgaactg 1260ggttttgcct
accgcgactt tttcgagatg cagccctcca ccggctacga aaccctgatc
1320tacgactgcc tgatcggcga ccagaccctg ttccagcgcg ccgacaacat
cgagaacggc 1380tggcgcgcgg tgcaaccctt cctcgatgcc tggcaacagg
acgccagctt gcagaactac 1440ccggcgggcg tggatggccc ggcagccggg
gatgaactgc tggcccggga tggccgcgta 1500tggcgacccc tggggtga
1518125489PRTPseudomonas fluorescens 125Met Pro Ser Ile Thr Val Glu
Pro Cys Thr Phe Ala Leu Phe Gly Ala1 5 10 15Leu Gly Asp Leu Ala Leu
Arg Lys Leu Phe Pro Ala Leu Tyr Gln Leu 20 25 30Asp Ala Ala Gly Leu
Leu His Asp Asp Thr Arg Ile Leu Ala Leu Ala 35 40 45Arg Glu Pro Gly
Ser Glu Gln Glu His Leu Ala Asn Ile Glu Thr Glu 50 55 60Leu His Lys
Tyr Val Gly Asp Lys Asp Ile Asp Ser Gln Val Leu Gln65 70 75 80Arg
Phe Leu Val Arg Leu Ser Tyr Leu His Val Asp Phe Leu Lys Ala 85 90
95Glu Asp Tyr Val Ala Leu Ala Glu Arg Val Gly Ser Glu Gln Arg Leu
100 105 110Ile Ala Tyr Phe Ala Thr Pro Ala Ala Val Tyr Gly Ala Ile
Cys Glu 115 120 125Asn Leu Ser Arg Val Gly Leu Asn Gln His Thr Arg
Val Val Leu Glu 130 135 140Lys Pro Ile Gly Ser Asp Leu Asp Ser Ser
Arg Lys Val Asn Asp Ala145 150 155 160Val Ala Gln Phe Phe Pro Glu
Thr Arg Ile Tyr Arg Ile Asp His Tyr 165 170 175Leu Gly Lys Glu Thr
Val Gln Asn Leu Ile Ala Leu Arg Phe Ala Asn 180 185 190Ser Leu Phe
Glu Thr Gln Trp Asn Gln Asn Tyr Ile Ser His Val Glu 195 200 205Ile
Thr Val Ala Glu Lys Val Gly Ile Glu Gly Arg Trp Gly Tyr Phe 210 215
220Asp Lys Ala Gly Gln Leu Arg Asp Met Ile Gln Asn His Leu Leu
Gln225 230 235 240Leu Leu Cys Leu Ile Ala Met Asp Pro Pro Ala Asp
Leu Ser Ala Asp 245 250 255Ser Ile Arg Asp Glu Lys Val Lys Val Leu
Lys Ala Leu Ala Pro Ile 260 265 270Ser Pro Glu Gly Leu Thr Thr Gln
Val Val Arg Gly Gln Tyr Ile Ala 275 280 285Gly His Ser Glu Gly Gln
Ser Val Pro Gly Tyr Leu Glu Glu Glu Asn 290 295 300Ser Asn Thr Gln
Ser Asp Thr Glu Thr Phe Val Ala Leu Arg Ala Asp305 310 315 320Ile
Arg Asn Trp Arg Trp Ala Gly Val Pro Phe Tyr Leu Arg Thr Gly 325 330
335Lys Arg Met Pro Gln Lys Leu Ser Gln Ile Val Ile His Phe Lys Glu
340 345 350Pro Ser His Tyr Ile Phe Ala Pro Glu Gln Arg Leu Gln Ile
Ser Asn 355 360 365Lys Leu Ile Ile Arg Leu Gln Pro Asp Glu Gly Ile
Ser Leu Arg Val 370 375 380Met Thr Lys Glu Gln Gly Leu Asp Lys Gly
Met Gln Leu Arg Ser Gly385 390 395 400Pro Leu Gln Leu Asn Phe Ser
Asp Thr Tyr Arg Ser Ala Arg Ile Pro 405 410 415Asp Ala Tyr Glu Arg
Leu Leu Leu Glu Val Met Arg Gly Asn Gln Asn 420 425 430Leu Phe Val
Arg Lys Asp Glu Ile Glu Ala Ala Trp Lys Trp Cys Asp 435 440 445Gln
Leu Ile Ala Gly Trp Lys Lys Ser Gly Asp Ala Pro Lys Pro Tyr 450 455
460Ala Ala Gly Ser Trp Gly Pro Met Ser Ser Ile Ala Leu Ile Thr
Arg465 470 475 480Asp Gly Arg Ser Trp Tyr Gly Asp Ile
4851261470DNAPseudomonas fluorescens 126atgccttcga taacggttga
accctgcacc tttgccttgt ttggcgcgct gggcgatctg 60gcgctgcgta agctgtttcc
tgccctgtac caactcgatg ccgccggttt gctgcatgac 120gacacgcgca
tcctggccct ggcccgcgag cctggcagcg agcaggaaca cctggcgaat
180atcgaaaccg agctgcacaa gtatgtcggc gacaaggata tcgatagcca
ggtcctgcag 240cgttttctcg tccgcctgag ctacctgcat gtggacttcc
tcaaggccga ggactacgtc 300gccctggccg aacgtgtcgg cagcgagcag
cgcctgattg cctacttcgc cacgccggcg 360gcggtgtatg gcgcgatctg
cgaaaacctc tcccgggtcg ggctcaacca gcacacccgt 420gtggtcctgg
aaaaacccat cggctcggac ctggattcat cacgcaaggt caacgacgcg
480gtggcgcagt tcttcccgga aacccgcatc taccggatcg accactacct
gggcaaggaa 540acggtgcaga acctgattgc cctgcgtttc gccaacagcc
tgttcgaaac ccagtggaac 600cagaactaca tctcccacgt ggaaatcacc
gtggccgaga aggtcggcat cgaaggtcgc 660tggggctatt tcgacaaggc
cggccaactg cgggacatga tccagaacca cttgctgcaa 720ctgctctgcc
tgatcgcgat ggacccgccg gccgaccttt cggccgacag catccgcgac
780gagaaggtca aggtgctcaa ggccctggcg cccatcagcc cggaaggcct
gaccacccag 840gtggtgcgcg gccagtacat cgccggccac agcgaaggcc
agtcggtgcc gggctacctg 900gaggaagaaa actccaacac ccagagcgac
accgagacct tcgtcgccct gcgcgccgat 960atccgcaact ggcgctgggc
cggtgtgcct ttctacctgc gcaccggcaa gcgcatgcca 1020cagaagctgt
cgcagatcgt catccacttc aaggaaccct cgcactacat cttcgccccc
1080gagcagcgcc tgcagatcag caacaagctg atcatccgcc tgcagccgga
cgaaggtatc 1140tcgttgcggg tgatgaccaa ggagcagggc ctggacaagg
gcatgcaact gcgcagcggt 1200ccgttgcagc tgaatttttc cgatacctat
cgcagtgcac ggatccccga tgcctacgag 1260cggttgttgc tggaagtgat
gcgcggcaat cagaacctgt ttgtgcgcaa agatgaaatc 1320gaagccgcgt
ggaagtggtg tgaccagttg attgccgggt ggaagaaatc cggcgatgcg
1380cccaagccgt acgcggccgg gtcctggggg ccgatgagct ccattgcact
gatcacgcgg 1440gatgggaggt cttggtatgg cgatatctaa
1470127489PRTPseudomonas aeruginosa 127Met Pro Asp Val Arg Val Leu
Pro Cys Thr Leu Ala Leu Phe Gly Ala1 5 10 15Leu Gly Asp Leu Ala Leu
Arg Lys Leu Phe Pro Ala Leu Tyr Gln Leu 20 25 30Asp Arg Glu Asn Leu
Leu His Arg Asp Thr Arg Val Leu Ala Leu Ala 35 40 45Arg Asp Glu Gly
Ala Pro Ala Glu His Leu Ala Thr Leu Glu Gln Arg 50 55 60Leu Arg Leu
Ala Val Pro Ala Lys Glu Trp Asp Asp Val Val Trp Gln65 70 75 80Arg
Phe Arg Glu Arg Leu Asp Tyr Leu Ser Met Asp Phe Leu Asp Pro 85 90
95Gln Ala Tyr Val Gly Leu Arg Glu Ala Val Asp Asp Glu Leu Pro Leu
100 105 110Val Ala Tyr Phe Ala Thr Pro Ala Ser Val Phe Gly Gly Ile
Cys Glu 115 120 125Asn Leu Ala Ala Ala Gly Leu Ala Glu Arg Thr Arg
Val Val Leu Glu 130 135 140Lys Pro Ile Gly His Asp Leu Glu Ser Ser
Arg Glu Val Asn Glu Ala145 150 155 160Val Ala Arg Phe Phe Pro Glu
Ser Arg Ile Tyr Arg Ile Asp His Tyr 165 170 175Leu Gly Lys Glu Thr
Val Gln Asn Leu Ile Ala Leu Arg Phe Ala Asn 180 185 190Ser Leu Phe
Glu Thr Gln Trp Asn Gln Asn His Ile Ser His Val Glu 195 200 205Ile
Thr Val Ala Glu Lys Val Gly Ile Glu Gly Arg Trp Gly Tyr Phe 210 215
220Asp Gln Ala Gly Gln Leu Arg Asp Met Val Gln Asn His Leu Leu
Gln225 230 235 240Leu Leu Cys Leu Ile Ala Met Asp Pro Pro Ser Asp
Leu Ser Ala Asp 245 250 255Ser Ile Arg Asp Glu Lys Val Lys Val Leu
Arg Ala Leu Glu Pro Ile 260 265 270Pro Ala Glu Gln Leu Ala Ser Arg
Val Val Arg Gly Gln Tyr Thr Ala 275 280 285Gly Phe Ser Asp Gly Lys
Ala Val Pro Gly Tyr Leu Glu Glu Glu His 290 295 300Ala Asn Arg Asp
Ser Asp Ala Glu Thr Phe Val Ala Leu Arg Val Asp305 310 315 320Ile
Arg Asn Trp Arg Trp Ser Gly Val Pro Phe Tyr Leu Arg Thr Gly 325 330
335Lys Arg Met Pro Gln Lys Leu Ser Gln Ile Val Ile His Phe Lys Glu
340 345 350Pro Pro His Tyr Ile Phe Ala Pro Glu Gln Arg Ser Leu Ile
Ser Asn 355 360 365Arg Leu Ile Ile Arg Leu Gln Pro Asp Glu Gly Ile
Ser Leu Gln Val 370 375 380Met Thr Lys Asp Gln Gly Leu Gly Lys Gly
Met Gln Leu Arg Thr Gly385 390 395 400Pro Leu Gln Leu Ser Phe Ser
Glu Thr Tyr His Ala Ala Arg Ile Pro 405 410 415Asp Ala Tyr Glu Arg
Leu Leu Leu Glu Val Thr Gln Gly Asn Gln Tyr 420 425 430Leu Phe Val
Arg Lys Asp Glu Val Glu Phe Ala Trp Lys Trp Cys Asp 435 440 445Gln
Leu Ile Ala Gly Trp Glu Arg Leu Ser Glu Ala Pro Lys Pro Tyr 450 455
460Pro Ala Gly Ser Trp Gly Pro Val Ala Ser Val Ala Leu Val Ala
Arg465 470 475 480Asp Gly Arg Ser Trp Tyr Gly Asp Phe
4851281470DNAPseudomonas aeruginosa 128atgcctgatg tccgcgttct
gccttgcacg ttagcgctgt tcggtgcgct gggcgatctc 60gccttgcgca agctgttccc
ggcgctctac caactcgatc gtgagaacct gctgcaccgc 120gatacccgcg
tcctggccct ggcccgtgac gaaggcgctc ccgccgaaca cctggcgacg
180ctggagcagc gcctgcgcct ggcagtgccg gcgaaggagt gggacgacgt
ggtctggcag 240cgtttccgcg aacgcctcga ctacctgagc atggacttcc
tcgacccgca ggcctatgtc 300ggcttgcgcg aggcggtgga tgacgaactg
ccgctggtcg cctacttcgc cacgccggcc 360tcggtgttcg gcggcatctg
cgagaacctc gccgccgccg gtctcgccga gcgcacccgg 420gtggtgctgg
agaagcccat cggtcatgac ctggagtcgt cccgcgaggt caacgaggca
480gtcgcccggt tcttcccgga aagccgcatc taccggatcg accattacct
gggcaaggag 540acggtgcaga acctgatcgc cctgcgcttc gccaacagcc
tcttcgagac ccagtggaac 600cagaaccaca tctcccacgt ggagatcacc
gtggccgaga aggtcggcat cgaaggccgc 660tggggctact tcgaccaggc
cgggcaactg cgcgacatgg tgcagaacca cctgctgcaa 720ctgctctgcc
tgatcgccat ggatccgccc agcgaccttt cggcggacag cattcgcgac
780gagaaggtca aggtcctccg cgccctcgag ccgattcccg cagaacaact
ggcttcgcgc 840gtggtgcgtg ggcagtacac cgccggtttc agcgacggca
aggcagtgcc gggctacctg 900gaggaggaac atgcgaatcg cgacagcgac
gcggaaacct tcgtcgccct gcgcgtggac 960atccgcaact ggcgctggtc
gggcgtgccg ttctacctgc gcaccggcaa gcgcatgccg 1020cagaagctgt
cgcagatcgt catccacttc aaggagccgc cgcactacat cttcgctccc
1080gagcagcgtt cgctgatcag caaccggctg atcatccgcc tgcagccgga
cgaaggtatc 1140tccctgcaag tgatgaccaa ggaccagggc ctgggcaagg
gcatgcaatt gcgtaccggc 1200ccgctgcaac tgagtttttc cgagacctac
cacgcggcgc ggattcccga tgcctacgag 1260cgtctgctgc tggaggtcac
ccagggcaac cagtacctgt tcgtgcgcaa ggacgaggtg 1320gagttcgcct
ggaagtggtg cgaccagctg atcgctggct gggaacgcct gagcgaagcg
1380cccaagccgt atccggcggg gagttggggg ccggtggcct cggtggccct
ggtggcccgc 1440gatgggagga gttggtatgg cgatttctga
1470129485PRTZymomonas mobilis 129Met Thr Asn Thr Val Ser Thr Met
Ile Leu Phe Gly Ser Thr Gly Asp1 5 10 15Leu Ser Gln Arg Met Leu Leu
Pro Ser Leu Tyr Gly Leu Asp Ala Asp 20 25 30Gly Leu Leu Ala Asp Asp
Leu Arg Ile Val Cys Thr Ser Arg Ser Glu 35 40 45Tyr Asp Thr Asp Gly
Phe Arg Asp Phe Ala Glu Lys Ala Leu Asp Arg 50 55 60Phe Val Ala Ser
Asp Arg Leu Asn Asp Asp Ala Lys Ala Lys Phe Leu65 70 75 80Asn Lys
Leu Phe Tyr Ala Thr Val Asp Ile Thr Asp Pro Thr Gln Phe 85 90 95Gly
Lys Leu Ala Asp Leu Cys Gly Pro Val Glu Lys Gly Ile Ala Ile 100 105
110Tyr Leu Ser Thr Ala Pro Ser Leu Phe Glu Gly Ala Ile Ala Gly Leu
115 120 125Lys Gln Ala Gly Leu Ala Gly Pro Thr Ser Arg Leu Ala Leu
Glu Lys 130 135 140Pro Leu Gly Gln Asp Leu Ala Ser Ser Asp His Ile
Asn Asp Ala Val145 150 155 160Leu Lys Val Phe Ser Glu Lys Gln Val
Tyr Arg Ile Asp His Tyr Leu 165 170 175Gly Lys Glu Thr Val Gln Asn
Leu Leu Thr Leu Arg Phe Gly Asn Ala 180 185 190Leu Phe Glu Pro Leu
Trp Asn Ser Lys Gly Ile Asp His Val Gln Ile 195 200 205Ser Val Ala
Glu Thr Val Gly Leu Glu Gly Arg Ile Gly Tyr Phe Asp 210 215 220Gly
Ser Gly Ser Leu Arg Asp Met Val Gln Ser His Ile Leu Gln Leu225
230 235 240Val Ala Leu Val Ala Met Glu Pro Pro Ala His Met Glu Ala
Asn Ala 245 250 255Val Arg Asp Glu Lys Val Lys Val Phe Arg Ala Leu
Arg Pro Ile Asn 260 265 270Asn Asp Thr Val Phe Thr His Thr Val Thr
Gly Gln Tyr Gly Ala Gly 275 280 285Val Ser Gly Gly Lys Glu Val Ala
Gly Tyr Ile Asp Glu Leu Gly Gln 290 295 300Pro Ser Asp Thr Glu Thr
Phe Val Ala Ile Lys Ala His Val Asp Asn305 310 315 320Trp Arg Trp
Gln Gly Val Pro Phe Tyr Ile Arg Thr Gly Lys Arg Leu 325 330 335Pro
Ala Arg Arg Ser Glu Ile Val Val Gln Phe Lys Pro Val Pro His 340 345
350Ser Ile Phe Ser Ser Ser Gly Gly Ile Leu Gln Pro Asn Lys Leu Arg
355 360 365Ile Val Leu Gln Pro Asp Glu Thr Ile Gln Ile Ser Met Met
Val Lys 370 375 380Glu Pro Gly Leu Asp Arg Asn Gly Ala His Met Arg
Glu Val Trp Leu385 390 395 400Asp Leu Ser Leu Thr Asp Val Phe Lys
Asp Arg Lys Arg Arg Ile Ala 405 410 415Tyr Glu Arg Leu Met Leu Asp
Leu Ile Glu Gly Asp Ala Thr Leu Phe 420 425 430Val Arg Arg Asp Glu
Val Glu Ala Gln Trp Val Trp Ile Asp Gly Ile 435 440 445Arg Glu Gly
Trp Lys Ala Asn Ser Met Lys Pro Lys Thr Tyr Val Ser 450 455 460Gly
Thr Trp Gly Pro Ser Thr Ala Ile Ala Leu Ala Glu Arg Asp Gly465 470
475 480Val Thr Trp Tyr Asp 4851301458DNAZymomonas mobilis
130atgacaaata ccgtttcgac gatgatattg tttggctcga ctggcgacct
ttcacagcgt 60atgctgttgc cgtcgcttta tggtcttgat gccgatggtt tgcttgcaga
tgatctgcgt 120atcgtctgca cctctcgtag cgaatacgac acagatggtt
tccgtgattt tgcagaaaaa 180gctttagatc gctttgtcgc ttctgaccgg
ttaaatgatg acgctaaagc taaattcctt 240aacaagcttt tctacgcgac
ggtcgatatt acggatccga cccaattcgg aaaattagct 300gacctttgtg
gcccggtcga aaaaggtatc gccatttatc tttcgactgc gccttctttg
360tttgaagggg caatcgctgg cctgaaacag gctggtctgg ctggtccaac
ttctcgcctg 420gcgcttgaaa aacctttagg tcaagatctt gcttcttccg
atcatattaa tgatgcggtt 480ttgaaagttt tctctgaaaa gcaagtttat
cgtattgacc attatctggg taaagaaacg 540gttcagaatc ttctgaccct
gcgttttggt aatgctttgt ttgaaccgct ttggaattca 600aaaggcattg
accacgttca gatcagcgtt gctgaaacgg ttggtcttga aggtcgtatc
660ggttatttcg acggttctgg cagcttgcgc gatatggttc aaagccatat
ccttcagttg 720gtcgctttgg ttgcaatgga accaccggct catatggaag
ccaacgctgt tcgtgacgaa 780aaggtaaaag ttttccgcgc tctgcgtccg
atcaataacg acaccgtctt tacgcatacc 840gttaccggtc aatatggtgc
cggtgtttct ggtggtaaag aagttgccgg ttacattgac 900gaactgggtc
agccttccga taccgaaacc tttgttgcta tcaaagcgca tgttgataac
960tggcgttggc agggtgttcc gttctatatc cgcactggta agcgtttacc
tgcacgtcgt 1020tctgaaatcg tggttcagtt taaacctgtt ccgcattcga
ttttctcttc ttcaggtggt 1080atcttgcagc cgaacaagct gcgtattgtc
ttacagcctg atgaaaccat ccagatttct 1140atgatggtga aagaaccggg
tcttgaccgt aacggtgcgc atatgcgtga agtttggctg 1200gatctttccc
tcacggatgt gtttaaagac cgtaaacgtc gtatcgctta tgaacgcctg
1260atgcttgatc ttatcgaagg cgatgctact ttatttgtgc gtcgtgacga
agttgaggcg 1320cagtgggttt ggattgacgg aattcgtgaa ggctggaaag
ccaacagtat gaagccaaaa 1380acctatgtct ctggtacatg ggggccttca
actgctatag ctctggccga acgtgatgga 1440gtaacttggt atgactga
1458131750DNASaccharomyces cerevisiae 131atggtgacag tcggtgtgtt
ttctgagagg gctagtttga cccatcaatt gggggaattc 60atcgtcaaga aacaagatga
ggcgctgcaa aagaagtcag actttaaagt ttccgttagc 120ggtggctctt
tgatcgatgc tctgtatgaa agtttagtag cggacgaatc actatcttct
180cgagtgcaat ggtctaaatg gcaaatctac ttctctgatg aaagaattgt
gccactgacg 240gacgctgaca gcaattatgg tgccttcaag agagctgttc
tagataaatt accctcgact 300agtcagccaa acgtttatcc catggacgag
tccttgattg gcagcgatgc tgaatctaac 360aacaaaattg ctgcagagta
cgagcgtatc gtacctcaag tgcttgattt ggtactgttg 420ggctgtggtc
ctgatggaca cacttgttcc ttattccctg gagaaacaca taggtacttg
480ctgaacgaaa caaccaaaag agttgcttgg tgccacgatt ctcccaagcc
tccaagtgac 540agaatcacct tcactctgcc tgtgttgaaa gacgccaaag
ccctgtgttt tgtggctgag 600ggcagttcca aacaaaatat aatgcatgag
atctttgact tgaaaaacga tcaattgcca 660accgcattgg ttaacaaatt
atttggtgaa aaaacatcct ggttcgttaa tgaggaagct 720tttggaaaag
ttcaaacgaa aactttttag 7501329PRTArtificial SequenceDescription of
Artificial Sequence Synthetic FLAG tag 132Asp Tyr Lys Asp Asp Asp
Asp Lys Gly1 513314PRTArtificial SequenceDescription of Artificial
Sequence Synthetic V5 tag 133Gly Lys Pro Ile Pro Asn Pro Leu Leu
Gly Leu Asp Ser Thr1 5 1013410PRTArtificial SequenceDescription of
Artificial Sequence Synthetic c-MYC tag 134Glu Gln Lys Leu Ile Ser
Glu Glu Asp Leu1 5 1013511PRTArtificial SequenceDescription of
Artificial Sequence Synthetic HSV tag 135Gln Pro Glu Leu Ala Pro
Glu Asp Pro Glu Asp1 5 101369PRTArtificial SequenceDescription of
Artificial Sequence Synthetic HA tag 136Tyr Pro Tyr Asp Val Pro Asp
Tyr Ala1 513711PRTArtificial SequenceDescription of Artificial
Sequence Synthetic VSV-G tag 137Tyr Thr Asp Ile Glu Met Asn Arg Leu
Gly Lys1 5 101386PRTArtificial SequenceDescription of Artificial
Sequence Synthetic 6xHis tag 138His His His His His His1
51397PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 139Cys Cys Xaa Xaa Xaa Cys Cys1
51406PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 140Cys Cys Pro Gly Cys Cys1
51416PRTUnknownDescription of Unknown Thrombin cleavage site
peptide 141Leu Val Pro Arg Gly Ser1 51425PRTUnknownDescription of
Unknown Enterokinase cleavage site peptide 142Asp Asp Asp Asp Lys1
51437PRTUnknownDescription of Unknown TEV protease cleavage site
peptide 143Glu Asn Leu Tyr Phe Gln Gly1 51448PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 144Leu
Glu Val Leu Phe Gln Gly Pro1 5145627DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
145atgagagaca ttgattctgt tatgagattg gctccagtta tgccagtctt
ggttatagaa 60gatatagctg atgctaagcc aattgctgag gctttggttg ctggtggttt
aaatgttttg 120gaagttacat tgagaactcc atgtgctttg gaagctatta
aaattatgaa ggaagttcca 180ggtgctgttg ttggtgctgg tactgtttta
aacgctaaaa tgttggatca agctcaagaa 240gctggttgtg agttctttgt
atcaccaggt ttgactgctg atttgggaaa acatgctgtt 300gctcaaaaag
cggctcttct accaggggtt gctaatgctg ctgatgttat gttgggattg
360gatttgggtt tggatagatt taaattcttc ccagctgaaa atataggtgg
tttgccagct 420ttaaaatcta tggcttctgt ttttagacaa gttagatttt
gtccaactgg aggaattact 480ccgacttctg ctccaaaata tttggaaaat
ccatctattt tgtgtgttgg tggttcttgg 540gttgttccag cgggtaaacc
agatgttgcg aaaattactg ctttggctaa agaggcttca 600gcttttaaaa
gagctgctgt ggcgtag 6271461824DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 146atgacggatt
tgcattcaac tgttgagaaa gtaactgcta gagtaattga aagatcaagg 60gaaactagaa
aggcttattt ggatttgata caatatgaga gggaaaaagg tgttgataga
120ccaaatttgt cttgttctaa tttggctcat ggttttgctg ctatgaatgg
tgataaacca 180gctttgagag attttaatag aatgaatata ggtgtagtta
cttcttataa tgatatgttg 240tctgctcatg aaccatatta tagatatcca
gaacaaatga aggtttttgc tcgtgaagtt 300ggtgctacag ttcaagttgc
tggtggtgtt cctgcaatgt gtgatggtgt tactcaaggt 360caaccaggta
tggaagaatc tttgttttcc agagatgtaa ttgctttggc tacatctgtt
420tcattgtctc acggaatgtt tgaaggtgct gcattgttgg gaatttgtga
taaaattgtt 480ccaggtttgt tgatgggtgc tttgaggttc ggtcatttgc
caactatttt ggttccatct 540ggtccaatga ctactggaat cccaaataaa
gaaaagatta gaattagaca attgtatgct 600caaggaaaaa ttggtcaaaa
ggaattgttg gatatggaag ctgcctgtta tcatgctgaa 660ggtacttgta
ctttttatgg tactgctaac actaatcaga tggttatgga agttttgggt
720ttgcacatgc caggtagtgc attcgttact ccaggtactc cactgagaca
ggctttgact 780agagctgctg ttcatagagt tgcagagttg ggttggaaag
gtgatgatta tagacctttg 840ggtaaaatta ttgatgagaa atctattgtt
aatgctattg ttggtttgtt agctacaggt 900ggttctacaa atcatacaat
gcatattccg gccatagcta gagcagcagg ggttatagtt 960aattggaatg
attttcatga tttgtctgaa gttgttccat tgattgctag aatttatcca
1020aatggtccta gagatataaa tgaatttcaa aatgcaggag gaatggctta
tgtaattaaa 1080gaattgttga gtgcgaattt gttaaataga gatgttacta
ctattgctaa aggagggata 1140gaagaatatg ctaaagctcc agctctgaac
gatgcgggtg aattggtgtg gaaaccggct 1200ggcgaacctg gggacgacac
aattttgaga ccagtatcta atccatttgc taaagatggt 1260ggtttgcgtc
tcttggaagg taatttgggt agagcaatgt ataaggcttc tgctgtagat
1320ccaaaattct ggactattga agctcccgtt agagttttct ctgatcaaga
tgatgttcaa 1380aaggctttta aagcaggcga gttaaataaa gatgttatag
ttgttgttag atttcaaggt 1440cctcgtgcta atggtatgcc tgaattgcat
aagttgactc ctgcgctagg cgtattgcaa 1500gataatggtt ataaggttgc
tttagttact gatggtagaa tgtctggtgc aactggtaaa 1560gtaccggtgg
ctctgcatgt ttcaccagag gctttaggag gtggggcgat tggcaagttg
1620agagatggcg atatagttag aatttctgtt gaagaaggta aattagaggc
tcttgtcccc 1680gccgacgagt ggaatgctag accacatgct gagaagcccg
cttttagacc tggtactggg 1740agagaattgt ttgacatttt tagacaaaac
gctgctaagg ctgaggatgg tgcagttgca 1800atttatgctg gggcagggat ctag
1824147627DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 147atgagggata ttgatagtgt gatgaggtta
gcccctgtta tgcctgttct cgttattgaa 60gatattgcag atgccaaacc tattgccgaa
gcactcgttg caggtggtct aaacgttcta 120gaagtgacac taaggactcc
ttgtgcacta gaagctatta agattatgaa ggaagttcct 180ggtgctgttg
ttggtgctgg tacagttcta aacgccaaaa tgctcgacca ggcacaagaa
240gcaggttgcg aatttttcgt ttcacctggt ctaactgccg acctcggaaa
gcacgcagtt 300gctcaaaaag ccgcattact acccggtgtt gcaaatgcag
cagatgtgat gctaggtcta 360gacctaggtc tagataggtt caagttcttc
cctgccgaaa acattggtgg tctacctgct 420ctaaagagta tggcatcagt
tttcaggcaa gttaggttct gccctactgg aggtataact 480cctacaagtg
cacctaaata tctagaaaac cctagtattc tatgcgttgg tggttcatgg
540gttgttcctg ccggaaaacc cgatgttgcc aaaattacag ccctcgcaaa
agaagcaagt 600gcattcaaga gggcagcagt tgcttag 6271481824DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
148atgacggatc tacatagtac agtggagaag gttactgcca gggttattga
aaggagtagg 60gaaactagga aggcatatct agatttaatt caatatgaga gggaaaaagg
agtggacagg 120cccaacctaa gttgtagcaa cctagcacat ggattcgccg
caatgaatgg tgacaagccc 180gcattaaggg acttcaacag gatgaatatt
ggagttgtga cgagttacaa cgatatgtta 240agtgcacatg aaccctatta
taggtatcct gagcaaatga aggtgtttgc aagggaagtt 300ggagccacag
ttcaagttgc tggtggagtg cctgcaatgt gcgatggtgt gactcagggt
360caacctggaa tggaagaatc cctattttca agggatgtta ttgcattagc
aacttcagtt 420tcattatcac atggtatgtt tgaaggggca gctctactcg
gtatatgtga caagattgtt 480cctggtctac taatgggagc actaaggttt
ggtcacctac ctactattct agttcccagt 540ggacctatga caacgggtat
acctaacaaa gaaaaaatta ggattaggca actctatgca 600caaggtaaaa
ttggacaaaa agaactacta gatatggaag ccgcatgcta ccatgcagaa
660ggtacttgca ctttctatgg tacagccaac actaaccaga tggttatgga
agttctcggt 720ctacatatgc ccggtagtgc ctttgttact cctggtactc
ctctcaggca agcactaact 780agggcagcag tgcatagggt tgcagaatta
ggttggaagg gagacgatta taggcctcta 840ggtaaaatta ttgacgaaaa
aagtattgtt aatgcaattg ttggtctatt agccactggt 900ggtagtacta
accatacgat gcatattcct gctattgcaa gggcagcagg tgttattgtt
960aactggaatg acttccatga tctatcagaa gttgttcctt taattgctag
gatttaccct 1020aatggaccta gggacattaa cgaatttcaa aatgccggag
gaatggcata tgttattaag 1080gaactactat cagcaaatct actaaacagg
gatgttacaa ctattgctaa gggaggtata 1140gaagaatacg ctaaggcacc
tgccctaaat gatgcaggag aattagtttg gaagcccgca 1200ggagaacctg
gtgatgacac tattctaagg cctgtttcaa atcctttcgc caaagatgga
1260ggtctaaggc tcttagaagg taacctagga agggccatgt acaaggctag
cgccgttgat 1320cctaaattct ggactattga agcccctgtt agggttttct
cagaccagga cgatgttcaa 1380aaagccttca aggcaggaga actaaacaaa
gacgttattg ttgttgttag gttccaagga 1440cctagggcca acggtatgcc
tgaattacat aagctaactc ctgcattagg tgttctacaa 1500gataatggat
acaaagttgc attagtgacg gatggtagga tgagtggtgc aactggtaaa
1560gttcctgttg cattacatgt ttcacccgaa gcactaggag gtggtgctat
tggtaaactt 1620agggatggag atattgttag gattagtgtt gaagaaggaa
aacttgaagc actcgttccc 1680gcagatgagt ggaatgcaag gcctcatgca
gaaaaacctg cattcaggcc tgggactggg 1740agggaattat ttgatatttt
caggcaaaat gcagcaaaag cagaagacgg tgccgttgcc 1800atctatgccg
gtgctggtat atag 182414939DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 149actagtatgg ctaaggaata
tttcccacaa attcaaaag 3915033DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 150ctcgagctac tattggtaca
tggcaacaat agc 3315155DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 151ctcgagctac taatgatgat
gatgatgatg ttggtacatg gcaacaatag cttcg 5515236DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
152actagtatgg ctaaagaata ttttccacaa attcag 3615333DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
153ctcgagttat tgatacatag ctactatagc ctc 3315460DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
154ctcgagttaa tgatgatgat gatgatgttg atacatagct actatagcct
cattgtttac 6015551DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 155aatcgatcaa agcttctaaa tacaagacgt
gcgatgacga ctatactgga c 5115654DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 156taccgtacta cccgggtata
tagtcttttt gccctggtgt tccttaataa tttc 5415750DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
157tgctaatgac ccgggaattc cacttgcaat tacataaaaa attccggcgg
5015849DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 158atgatcattg agctcagctt cgcaagtatt cattttagac
ccatggtgg 4915948DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 159tgctaatgag agctctcatt ttttggtgcg
atatgttttt ggttgatg 4816048DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 160aatgatcatg agctcgtcaa
caagaactaa aaaattgttc aaaaatgc 4816134DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
161ctaaatacaa gacgtgcgat gacgactata ctgg 3416240DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
162gtcaacaaga actaaaaaat tgttcaaaaa tgcaattgtc 4016343DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 163atggagttct tttctaatat aggtaaaatt cagtatcaag gtc
4316439DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 164aatggatctg tagattttgg accttgatac
tgaatttta 3916543DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 165caaaatctac agatccattg
tcttttaaat attataatcc aga 4316645DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 166gttttaccat
ttataacttc ttctggatta taatatttaa aagac 4516743DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 167agaagttata aatggtaaaa ctatgagaga acatttaaaa ttt
4316845DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 168atagtatgcc accaagacaa agcaaatttt
aaatgttctc tcata 4516945DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 169gctttgtctt
ggtggcatac tatgggtggt gatggtactg atatg 4517045DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 170ttatcagtag taccacaacc gaacatatca gtaccatcac
caccc 4517143DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 171ttcggttgtg gtactactga
taaaacttgg ggtcaatctg atc 4317240DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 172ggcttttgct
ctagcagctg gatcagattg accccaagtt 4017343DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 173cagctgctag agcaaaagcc aaagtagatg cagcctttga aat
4317445DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 174atcaatagac aatttatcca taatttcaaa
ggctgcatct acttt 4517545DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 175tatggataaa
ttgtctattg attattattg ttttcatgat agaga 4517645DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 176agaaccatat tcaggagaca aatctctatc atgaaaacaa
taata 4517743DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 177tttgtctcct gaatatggtt
ctttaaaagc aactaatgat caa 4317845DNAArtificial SequenceDescription
of
Artificial Sequence Synthetic oligonucleotide 178aatataatcc
gtaacaatgt ccaattgatc attagttgct tttaa 4517945DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 179ttggacattg ttacggatta tattaaagaa aaacaaggtg
ataaa 4518045DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 180cgcagtgccc cacaaacatt
taaatttatc accttgtttt tcttt 4518145DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 181tttaaatgtt tgtggggcac tgcgaaatgt tttgatcatc
cacgt 4518245DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 182actcgtcccc gcaccatgca
taaaacgtgg atgatcaaaa cattt 4518345DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 183tttatgcatg gtgcggggac gagtccttct gctgatgttt
ttgct 4518445DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 184cttcttaatt tgagcggcag
aaaaagcaaa aacatcagca gaagg 4518545DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 185ttttctgccg ctcaaattaa gaaggcattg gaatcaactg
ttaaa 4518645DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 186gaatacatac ccgttcccac
ctaatttaac agttgattcc aatgc 4518745DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 187ttaggtggga acgggtatgt attctgggga ggaagggaag
gttat 4518845DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 188catattagtg tttaataatg
tttcataacc ttcccttcct cccca 4518945DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 189gaaacattat taaacactaa tatgggtttg gaattggata
atatg 4519045DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 190tacagccatt ttcatcaatc
tagccatatt atccaattcc aaacc 4519145DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 191gctagattga tgaaaatggc tgtagaatac ggaaggtcta
ttggt 4519245DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 192ttcaatataa aagtcaccct
taaaaccaat agaccttccg tattc 4519345DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 193tttaagggtg acttttatat tgaaccaaaa cctaaagagc
ctact 4519445DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 194agtatcaaaa tcatattgat
gtttagtagg ctctttaggt tttgg 4519545DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 195aaacatcaat atgattttga tactgctaca gttttgggat
tcttg 4519645DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 196atctttatcc agaccatatt
ttctcaagaa tcccaaaact gtagc 4519745DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 197agaaaatatg gtctggataa agattttaaa atgaatatag
aagct 4519845DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 198atgttgtgcg agtgttgcat
gattagcttc tatattcatt ttaaa 4519945DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 199aatcatgcaa cactcgcaca acatactttt caacatgaat
tgaga 4520045DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 200aaaaactccg ttatctctgg
caactctcaa ttcatgttga aaagt 4520145DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 201gttgccagag ataacggagt ttttggatct atcgatgcaa
accag 4520245DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 202atcccatcct agcaaaacgt
ctccctggtt tgcatcgata gatcc 4520345DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 203ggagacgttt tgctaggatg ggatactgat caatttccaa
ctaac 4520445DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 204catacacata gtagtatcat
aaatgttagt tggaaattga tcagt 4520545DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 205atttatgata ctactatgtg tatgtatgaa gtaattaagg
cagga 4520645DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 206gtttaatccg ccattagtaa
agcctcctgc cttaattact tcata 4520745DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 207ggctttacta atggcggatt aaactttgat gcgaaggcta
ggcgt 4520845DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 208tatatcctct ggagtgaaac
taccacgcct agccttcgca tcaaa 4520945DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 209ggtagtttca ctccagagga tatattctat tcttatattg
ctgga 4521045DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 210gaaacctaac gcgaaagcat
ccattccagc aatataagaa tagaa 4521145DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 211atggatgctt tcgcgttagg tttcagggca gcactaaaat
tgatt 4521242DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 212cttatcaatt ctaccatctt
caatcaattt tagtgctgcc ct 4221345DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 213gaagatggta
gaattgataa gtttgtagct gatagatatg cttct 4521445DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 214tgctcctatt ccagtattcc aagaagcata tctatcagct
acaaa 4521545DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 215tggaatactg gaataggagc
agatataatc gctgggaaag ccgac 4521645DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 216atatttttcc agactggcga agtcggcttt cccagcgatt
atatc 4521745DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 217ttcgccagtc tggaaaaata
tgcgcttgaa aaaggagaag ttact 4521845DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 218acgaccggaa cttaagctgg cagtaacttc tcctttttca
agcgc 4521941DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 219gccagcttaa gttccggtcg
tcaagaaatg ttggaatcta t 4122045DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 220cagagaaaat
aaaacattgt ttacaataga ttccaacatt tcttg 4522155DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 221acttgactaa ctgaagcttc atatgatgga gttcttttct
aatataggta aaatt 5522244DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 222acttgactac
tagtatggag ttcttttcta atataggtaa aatt 4422355DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 223acttgactaa ctgaagcttc atatgttgga cattgttacg
gattatatta aagaa 5522455DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 224acttgactaa
ctgaagcttc atatgaaaca tcaatatgat tttgatactg ctaca
5522556DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 225agttaagtga gtaaactagt gaattccaga
gaaaataaaa cattgtttac aataga 5622647DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 226agtcaagtct cgagctacag agaaaataaa acattgttta
caataga 4722756DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 227agttaagtga gtaaactagt
gaattccata ttagtgttta ataatgtttc ataacc 5622856DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 228agttaagtga gtaaactagt gaattccata cacatagtag
tatcataaat gttagt 5622936DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 229ggcgacaagt tcaagtgcct
cttcggtaca gcaaag 3623036DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 230ctttgctgta ccgaagaggc
acttgaactt gtcgcc 3623133DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 231tcggcggtaa cggttacgtt
agctggggcg gac 3323233DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 232gtccgcccca gctaacgtaa
ccgttaccgc cga 3323335DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 233aacagtaaag ctcggcgcta
acggttacgt tttct 3523435DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 234agaaaacgta accgttagcg
ccgagcttta ctgtt 3523544DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 235aacagtaaag ctcggcgcta
acggttacgt tagctggggc ggac 4423633DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 236gtccgcccca gctaacgtaa
ccgttagcgc cga 3323739DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 237gctaaggttg acgcagcaat
ggagatcatg gataagctc 3923839DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 238gagcttatcc atgatctcca
ttgctgcgtc aaccttagc 3923938DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 239ctaaggttga cgcagcatta
gagatcatgg ataagctc 3824038DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 240gagcttatcc atgatctcta
atgctgcgtc aaccttag 3824141DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 241agctaaccac gctacacttg
ctacgcatac attccagcat g 4124241DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 242catgctggaa tgtatgcgta
gcaagtgtag cgtggttagc t 4124343DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 243gcgacaagtt caagtgcctc
ataggtacag caaagtgctt cga 4324443DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 244tcgaagcact ttgctgtacc
tatgaggcac ttgaacttgt cgc 4324533DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 245gcgacaagtt caagtgcctc
tcgggtacag caa 3324633DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 246ttgctgtacc cgagaggcac
ttgaacttgt cgc 3324739DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 247gctaaccacg ctacacttgc
tggtcataca ttccagcat 3924839DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 248atgctggaat gtatgaccag
caagtgtagc gtggttagc 3924941DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 249cctcagaaag tacggtctcg
ctaaggattt caagatgaat a 4125041DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 250tattcatctt gaaatcctta
gcgagaccgt actttctgag g 4125143DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 251cctcagaaag tacggtctcg
agaaggattt caagatgaat atc 4325243DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 252gatattcatc ttgaaatcct
tctcgagacc gtactttctg agg 4325337DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 253gtcttatgaa gatggctggt
gagtatggac gttcgat 3725437DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 254atcgaacgtc catactcacc
agccatcttc ataagac 3725540DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 255gtcaacagta aagctcggca
gtaacggtta cgttagctgg 4025640DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 256ccagctaacg taaccgttac
tgccgagctt tactgttgac 4025759DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 257ctagaactag
taaaaaaatg gctaaggaat attattctaa tataggtaaa attcagtat
5925847DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 258actatgagag aacatttaaa atttgctatg
tcttggtggc atactwt 4725946DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 259agagaacatt
taaaatttgc tttggcttgg tggcatactw tgkgtg 4626052DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 260actatgagag aacatttaaa atttgctatg gcttggtggc
atactwtgkg tg 5226136DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 261ttgctttgtc
ttggtggcat actttgkgtg stgatg 3626236DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 262cttggtggca tactatgtgt gstgatggta ctgats
3626335DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 263gtggcatact atgggtgctg atggtactga tatgt
3526435DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 264gtggcatact wtgkgtgctg atggtactga tcaat
3526546DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 265gcatactwtg kgtgstgatg gtactgatca
attcggttgt ggtact 4626652DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 266gcaaaagcca
aagtagatgc agccwtggaa attatggata aattgtctat tg 5226775DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 267ttatggataa attgtctatt gattattatt gttttcatga
tgttgatttg tctcctgaat 60atggttcttt aaaag 7526875DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 268ttatggataa attgtctatt gattattatt gttttcatga
tgttgatttg tctcctgaag 60gtggttcttt aaaag 7526954DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 269gttttcatga tagagatttg tctcctgaag gtggttcttt
aaaagcaact aatg 5427079DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 270gttctttaaa
agcaactaat gatcaattgg acattgttgt tgattatatt aaagaaaaac 60aaggtgataa
atttaaatg 7927179DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 271gttctttaaa agcaactaat
gatcaattgg acattgttgt tgattatatt aaagaaaaac 60aaggtgatgg ttttaaatg
7927260DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 272cggattatat taaagaaaaa caaggtgatg
gttttaaatg tttgtkkggc actgcgaawt 6027364DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 273ttatattaaa gaaaaacaag gtgataaatt taaatgtttg
tttggcactg cgaawtgttt 60tgat 6427434DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 274gtttgtgggg cactgcgaat tgttttgatc atcc
3427535DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 275ttttgatcat ccacgttata tgcatggtgc gggga
3527646DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 276ggaatcaact gttaaattag gtagaaacgg
gtatgtattc tgggga 4627746DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 277gaatcaactg
ttaaattagg tgctaacggg tatgtattct ggggag 4627846DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 278ggaatcaact gttaaattag gtagaaacgg gtatgtatct
tgggga 4627946DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 279gaatcaactg ttaaattagg
tgctaacggg tatgtatctt ggggag 4628040DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 280aaattaggtg ggaacgggta tgtatcttgg ggaggaaggg
4028149DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 281cactaatatg ggtttggaat tggaaaatat
ggctagattg atgaaaatg 4928250DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 282ggataatatg
gctagattga tgaaaatggc tagagaatac ggaaggtcta 5028346DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 283gctagattga tgaaaatggc tggtgaatac ggaaggtcta
ttggtt 4628463DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 284cagttttggg attcttgaga
aaatatggtt tggctaaaga ttttaaaatg aatatagaag 60cta
6328563DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 285agttttggga ttcttgagaa aatatggttt
ggaaaaagat tttaaaatga atatagaagc 60taa 6328657DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 286atagaagcta atcatgcaac actcgcattt catacttttc
aacatgaatt gagagtt 5728757DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 287atagaagcta
atcatgcaac actcgcaact catacttttc aacatgaatt gagagtt
5728853DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 288agaagctaat catgcaacac tcgcaggtca
tacttttcaa catgaattga gag 5328941DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 289taacggagtt
tttggatctg ttgatgcaaa ccagggagac g 4129041DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 290taacggagtt tttggatctg ttgatgcaaa cagaggagac g
4129147DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 291ttttggatct atcgatgcaa acagaggaga
cgttttgcta ggatggg 4729244DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 292aaggctaggc
gtggtagttt cgatccagag gatatattct attc 4429349DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 293cgaaggctag gcgtggtagt ttcgaaccag aggatatatt
ctattctta 4929447DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 294cagggcagca ctaaaattga
ttgaagaagg tagaattgat aagtttg 4729535DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 295tgggaaagcc gacttcgcca gtttggaaaa atatg
3529659DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 296atactgaatt ttacctatat tagaataata
ttccttagcc atttttttac tagttctag 5929747DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 297awagtatgcc accaagacat agcaaatttt aaatgttctc
tcatagt 4729846DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 298cacmcawagt atgccaccaa
gccaaagcaa attttaaatg ttctct 4629952DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 299cacmcawagt atgccaccaa gccatagcaa attttaaatg
ttctctcata gt 5230036DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 300catcascacm
caaagtatgc caccaagaca aagcaa 3630136DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 301satcagtacc atcascacac atagtatgcc accaag
3630235DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 302acatatcagt accatcagca cccatagtat gccac
3530335DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 303attgatcagt accatcagca cmcawagtat gccac
3530446DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 304agtaccacaa ccgaattgat cagtaccatc
ascacmcawa gtatgc 4630552DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 305caatagacaa
tttatccata atttccawgg ctgcatctac tttggctttt gc 5230675DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 306cttttaaaga accatattca ggagacaaat caacatcatg
aaaacaataa taatcaatag 60acaatttatc cataa 7530775DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 307cttttaaaga accaccttca ggagacaaat caacatcatg
aaaacaataa taatcaatag 60acaatttatc cataa 7530854DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 308cattagttgc ttttaaagaa ccaccttcag gagacaaatc
tctatcatga aaac 5430979DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 309catttaaatt
tatcaccttg tttttcttta atataatcaa caacaatgtc caattgatca 60ttagttgctt
ttaaagaac 7931079DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 310catttaaaac catcaccttg
tttttcttta atataatcaa caacaatgtc caattgatca 60ttagttgctt ttaaagaac
7931160DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 311awttcgcagt gccmmacaaa catttaaaac
catcaccttg tttttcttta atataatccg 6031264DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 312atcaaaacaw ttcgcagtgc caaacaaaca tttaaattta
tcaccttgtt tttctttaat 60ataa 6431334DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 313ggatgatcaa aacaattcgc agtgccccac aaac
3431435DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 314tccccgcacc atgcatataa cgtggatgat caaaa
3531546DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 315tccccagaat acatacccgt ttctacctaa
tttaacagtt gattcc 4631646DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 316ctccccagaa
tacatacccg ttagcaccta atttaacagt tgattc 4631746DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 317tccccaagat acatacccgt ttctacctaa tttaacagtt
gattcc 4631846DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 318ctccccaaga tacatacccg
ttagcaccta atttaacagt tgattc 4631940DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 319cccttcctcc ccaagataca tacccgttcc cacctaattt
4032049DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 320cattttcatc aatctagcca tattttccaa
ttccaaaccc atattagtg 4932150DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 321tagaccttcc
gtattctcta gccattttca tcaatctagc catattatcc 5032246DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 322aaccaataga ccttccgtat tcaccagcca ttttcatcaa
tctagc 4632363DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 323tagcttctat attcatttta
aaatctttag ccaaaccata ttttctcaag aatcccaaaa 60ctg
6332463DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 324ttagcttcta tattcatttt aaaatctttt
tccaaaccat attttctcaa gaatcccaaa 60act 6332557DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 325aactctcaat tcatgttgaa aagtatgaaa tgcgagtgtt
gcatgattag cttctat 5732657DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 326aactctcaat
tcatgttgaa aagtatgagt tgcgagtgtt gcatgattag cttctat
5732753DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 327ctctcaattc atgttgaaaa gtatgacctg
cgagtgttgc atgattagct tct 5332841DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 328cgtctccctg
gtttgcatca acagatccaa aaactccgtt a 4132941DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 329cgtctcctct gtttgcatca acagatccaa aaactccgtt a
4133047DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 330cccatcctag caaaacgtct cctctgtttg
catcgataga tccaaaa 4733144DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 331gaatagaata
tatcctctgg atcgaaacta ccacgcctag cctt 4433249DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 332taagaataga atatatcctc tggttcgaaa ctaccacgcc
tagccttcg 4933347DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 333caaacttatc aattctacct
tcttcaatca attttagtgc tgccctg 4733435DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 334catatttttc caaactggcg aagtcggctt tccca
3533525DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 335cctgaaatta ttcccctact tgact
2533625DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 336ccttctcaag caaggttttc agtat
2533720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 337taaaacgacg gccagtgaat 2033821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
338tgcaggtcga ctctagagga t 2133972DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 339gtgtgcgtgt atgtgtacac
ctgtatttaa tttccttact cgcgggtttt tctaaaacga 60cggccagtga at
7234072DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 340tgtaccagtc tagaattcta ccaacaaatg gggaaatcaa
agtaacttgg gctgcaggtc 60gactctagag ga 7234126DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
341gtcgactgga aatctggaag gttggt 2634226DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
342gtcgacgctt tgctgcaagg attcat 2634338DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
343actagtatga ctgttactac tccttttgtg aatggtac 3834439DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
344ctcgagttaa tcaactctct ttcttccaac caaatggtc 3934548DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
345aagcttttaa ttaatataac gctatgacgg tagttgaatg ttaaaaac
4834651DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 346gaattcttaa ttaaagagaa caaagtattt aacgcacatg
tataaatatt g 5134753DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 347ggatccgcat gcggccggcc agcttttaat
caaggaagta ataaataaag gac 5334852DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 348ggatccgagc tcgcggccgc
agcttttgaa caatgaattt tttgttcctt tc 5234938DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
349gcggccgcag cttcgcaagt attcatttta gacccatg 3835044DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
350ggccggccgg taccaattcc acttgcaatt acataaaaaa ttcc
4435138DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 351ggatccgttt atcattatca atactcgcca tttcaaag
3835240DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 352catatgttgg gtaccggccg caaattaaag ccttcgagcg
4035355DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 353ggattcagtc agatcatatg ggtacccccg ggttaattaa
ggcgcgccag atctg 5535460DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 354gtcgacaggc ctactgtacg
gctagcgaat tcgagctcgt tttcgacact ggatggcggc 6035539DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
355tctagactcg agtaataagc gaatttctta tgatttatg 3935634DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
356aagcttaggc ctggagcgat ttgcaggcat ttgc 3435736DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
357ggatccgcta gcaccgcgaa tccttacatc acaccc 3635839DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
358tctagactcg agtaataagc gaatttctta tgatttatg 3935939DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
359gattgtactg agagtgcaca atatgcggtg tgaaatacc 3936039DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
360ggtatttcac accgcatatt gtgcactctc agtacaatc 3936164DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 361actagtaaaa aatgaaaaat tactttccaa atgttccaga
agtacagtat cagggaccaa 60aaag 6436267DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 362actagtaaaa aatgactaag gaatatttcc caactatcgg
caagattcag tatcagggac 60caaaaag 6736364DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 363actagtaaaa aatggaatac ttcaaaaatg taccacaaat
aaaacagtat cagggaccaa 60aaag 6436470DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 364actagtaaaa aatggcaaca aaagaatttt ttccgggaat
tgaaaagatt cagtatcagg 60gaccaaaaag 7036561DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 365actagtaaaa aatggcttat tttccgaata tcggcaagat
tcagtatcag ggaccaaaaa 60g 6136670DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 366actagtaaaa
aatggctacc aaggaatact tcccaggtat tggtaagatc cagtatcagg 60gaccaaaaag
7036764DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 367actagtaaaa aatgtcagaa gtatttagcg
gtatttcaaa cattcagtat cagggaccaa 60aaag 6436861DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 368actagtaaaa aatggaattt ttcaagaaca taagcaagat
ccagtatcag ggaccaaaaa 60g 6136964DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 369actagtaaaa
aatgagcgaa ttttttacag gcatttcaaa gatccagtat cagggaccaa 60aaag
6437061DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 370actagtaaaa aatgaaattt tttgaaaatg
tccctaaggt acagtatcag ggaccaaaaa 60g 6137159DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 371cctattttga ccagctcgat cgcgttcagt atcagggacc
aaaaagtact gatcctctc 5937255DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 372actagtaaaa
aaatgcaagc ctattttgac cagctcgatc gcgttcagta tcagg
5537334DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 373ctcgagttac agactgaaaa gaacgttatt tacg
3437452DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 374ctcgagttag tgatggtggt ggtgatgcag
actgaaaaga acgttattta cg 5237538DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 375gtgcgtcagg
tgatctgggt aagaagaaga cttttccc 3837638DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 376gggaaaagtc ttcttcttac ccagatcacc tgacgcac
3837740DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 377gtgatctggg taagaagaag ggttttcccg
ccttatttgg 4037840DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 378ccaaataagg cgggaaaacc
cttcttctta cccagatcac 4037951DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 379ccttgatcca
tctaccaaga tcttcggtta taatcggtcc aaattgtcca t 5138051DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 380atggacaatt tggaccgatt ataaccgaag atcttggtag
atggatcaag g 5138143DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 381atctaccaag atcttcggtt
atgatcggtc caaattgtcc atg 4338243DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 382catggacaat
ttggaccgat cataaccgaa gatcttggta gat 4338342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 383ggtgatctgg caaagaagaa gttttttccc gccttatttg gg
4238442DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 384cccaaataag gcgggaaaaa acttcttctt
tgccagatca cc 4238540DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 385taccttgatc
catctaccag aatcttcggt tatgcccggt 4038640DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 386accgggcata accgaagatt ctggtagatg gatcaaggta
4038740DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 387gggcttttca gagaaggttt gcttgatcca
tctaccaaga 4038840DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 388tcttggtaga tggatcaagc
aaaccttctc tgaaaagccc 4038941DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 389gaagaagact
tttcccgcct tatacgggct tttcagagaa g 4139041DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 390cttctctgaa aagcccgtat aaggcgggaa aagtcttctt c
4139145DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 391gtcaggtgat ctggcaaaga agaagttgtt
tcccgcctta tttgg 4539245DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 392ccaaataagg
cgggaaacaa cttcttcttt gccagatcac ctgac 4539346DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 393cgaaaaaaat accgtcatat ctttgtttgg tgcgtcaggt
gatctg 4639446DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 394cagatcacct gacgcaccaa
acaaagatat gacggtattt ttttcg 4639538DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 395gacctgaagt cccgtgtcga accccacttg aaaaaacc
3839638DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 396ggttttttca agtggggttc gacacgggac
ttcaggtc 3839738DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 397gtgcgtcagg tgatctgggt
aagaagaaga cttttccc 3839838DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 398gggaaaagtc
ttcttcttac ccagatcacc tgacgcac 3839938DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 399gtgcgtcagg tgatctgggt aagaagaaga cttttccc
3840038DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 400gggaaaagtc ttcttcttac ccagatcacc
tgacgcac 3840145DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 401accaagatct tcggttatgc
cgattccaaa ttgtccatgg aggag 4540245DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 402ctcctccatg gacaatttgg aatcggcata accgaagatc
ttggt 4540355DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 403tccatctacc aagatcttcg
gttatgatgc ttccaaattg tccatggagg aggac 5540455DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 404gtcctcctcc atggacaatt tggaagcatc ataaccgaag
atcttggtag atgga 5540555DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 405tccatctacc
aagatcttcg gttatgatgc ttccaaattg tccatggagg aggac
5540655DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 406gtcctcctcc atggacaatt tggaagcatc
ataaccgaag atcttggtag atgga 5540755DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 407tccatctacc aagatcttcg gttatgatgc ttccaaattg
tccatggagg aggac 5540855DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 408gtcctcctcc
atggacaatt tggaagcatc ataaccgaag atcttggtag atgga
5540940DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 409aagatcttcg gttatgatca ttccaaattg
tccatggagg 4041040DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 410cctccatgga caatttggaa
tgatcataac cgaagatctt 4041140DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 411aagatcttcg
gttatgccca ttccaaattg tccatggagg 4041240DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 412cctccatgga caatttggaa tgggcataac cgaagatctt
4041333DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 413gctagcatgg tgacagtcgg tgtgttttct gag
3341433DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 414gtcgacctaa aaagttttcg tttgaacttt tcc
3341532DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 415ccaacactaa gaaataattt cgccatttct tg
3241632DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 416gccaacaatt aaatccaagt tcacctattc tg
32417777DNASaccharomyces cerevisiae 417atggctgccg gtgtcccaaa
aattgatgcg ttagaatctt tgggcaatcc tttggaggat 60gccaagagag ctgcagcata
cagagcagtt gatgaaaatt taaaatttga tgatcacaaa 120attattggaa
ttggtagtgg tagcacagtg gtttatgttg ccgaaagaat tggacaatat
180ttgcatgacc ctaaatttta tgaagtagcg tctaaattca tttgcattcc
aacaggattc 240caatcaagaa acttgatttt ggataacaag ttgcaattag
gctccattga acagtatcct 300cgcattgata tagcgtttga cggtgctgat
gaagtggatg agaatttaca attaattaaa 360ggtggtggtg cttgtctatt
tcaagaaaaa ttggttagta ctagtgctaa aaccttcatt 420gtcgttgctg
attcaagaaa aaagtcacca aaacatttag gtaagaactg gaggcaaggt
480gttcccattg aaattgtacc ttcctcatac gtgagggtca agaatgatct
attagaacaa 540ttgcatgctg aaaaagttga catcagacaa ggaggttctg
ctaaagcagg tcctgttgta 600actgacaata ataacttcat tatcgatgcg
gatttcggtg aaatttccga tccaagaaaa 660ttgcatagag aaatcaaact
gttagtgggc gtggtggaaa caggtttatt catcgacaac 720gcttcaaaag
cctacttcgg taattctgac ggtagtgttg aagttaccga aaagtga
777418258PRTSaccharomyces cerevisiae 418Met Ala Ala Gly Val Pro Lys
Ile Asp Ala Leu Glu Ser Leu Gly Asn1 5 10 15Pro Leu Glu Asp Ala Lys
Arg Ala Ala Ala Tyr Arg Ala Val Asp Glu 20 25 30Asn Leu Lys Phe Asp
Asp His Lys Ile Ile Gly Ile Gly Ser Gly Ser 35 40 45Thr Val Val Tyr
Val Ala Glu Arg Ile Gly Gln Tyr Leu His Asp Pro 50 55 60Lys Phe Tyr
Glu Val Ala Ser Lys Phe Ile Cys Ile Pro Thr Gly Phe65 70 75 80Gln
Ser Arg Asn Leu Ile Leu Asp Asn Lys Leu Gln Leu Gly Ser Ile 85 90
95Glu Gln Tyr Pro Arg Ile Asp Ile Ala Phe Asp Gly Ala Asp Glu Val
100 105 110Asp Glu Asn Leu Gln Leu Ile Lys Gly Gly Gly Ala Cys Leu
Phe Gln 115 120 125Glu Lys Leu Val Ser Thr Ser Ala Lys Thr Phe Ile
Val Val Ala Asp 130 135 140Ser Arg Lys Lys Ser Pro Lys His Leu Gly
Lys Asn Trp Arg Gln Gly145 150 155 160Val Pro Ile Glu Ile Val Pro
Ser Ser Tyr Val Arg Val Lys Asn Asp 165 170 175Leu Leu Glu Gln Leu
His Ala Glu Lys Val Asp Ile Arg Gln Gly Gly 180 185 190Ser Ala Lys
Ala Gly Pro Val Val Thr Asp Asn Asn Asn Phe Ile Ile 195 200 205Asp
Ala Asp Phe Gly Glu Ile Ser Asp Pro Arg Lys Leu His Arg Glu 210 215
220Ile Lys Leu Leu Val Gly Val Val Glu Thr Gly Leu Phe Ile Asp
Asn225 230 235 240Ala Ser Lys Ala Tyr Phe Gly Asn Ser Asp Gly Ser
Val Glu Val Thr 245 250 255Glu Lys419717DNASaccharomyces cerevisiae
419atggtcaaac caattatagc tcccagtatc cttgcttctg acttcgccaa
cttgggttgc 60gaatgtcata aggtcatcaa cgccggcgca gattggttac atatcgatgt
catggacggc 120cattttgttc caaacattac tctgggccaa ccaattgtta
cctccctacg tcgttctgtg 180ccacgccctg gcgatgctag caacacagaa
aagaagccca ctgcgttctt cgattgtcac 240atgatggttg aaaatcctga
aaaatgggtc gacgattttg ctaaatgtgg tgctgaccaa 300tttacgttcc
actacgaggc cacacaagac cctttgcatt tagttaagtt gattaagtct
360aagggcatca aagctgcatg cgccatcaaa cctggtactt ctgttgacgt
tttatttgaa 420ctagctcctc atttggatat ggctcttgtt atgactgtgg
aacctgggtt tggaggccaa 480aaattcatgg aagacatgat gccaaaagtg
gaaactttga gagccaagtt cccccatttg 540aatatccaag tcgatggtgg
tttgggcaag gagaccatcc cgaaagccgc caaagccggt 600gccaacgtta
ttgtcgctgg taccagtgtt ttcactgcag ctgacccgca cgatgttatc
660tccttcatga aagaagaagt ctcgaaggaa ttgcgttcta gagatttgct agattag
717420238PRTSaccharomyces cerevisiae 420Met Val Lys Pro Ile Ile Ala
Pro Ser Ile Leu Ala Ser Asp Phe Ala1 5 10 15Asn Leu Gly Cys Glu Cys
His Lys Val Ile Asn Ala Gly Ala Asp Trp 20 25 30Leu His Ile Asp Val
Met Asp Gly His Phe Val Pro Asn Ile Thr Leu 35 40 45Gly Gln Pro Ile
Val Thr Ser Leu Arg Arg Ser Val Pro Arg Pro Gly 50 55 60Asp Ala Ser
Asn Thr Glu Lys Lys Pro Thr Ala Phe Phe Asp Cys His65 70 75 80Met
Met Val Glu Asn Pro Glu Lys Trp Val Asp Asp Phe Ala Lys Cys 85 90
95Gly Ala Asp Gln Phe Thr Phe His Tyr Glu Ala Thr Gln Asp Pro Leu
100 105 110His Leu Val Lys Leu Ile Lys Ser Lys Gly Ile Lys Ala Ala
Cys Ala 115 120 125Ile Lys Pro Gly Thr Ser Val Asp Val Leu Phe Glu
Leu Ala Pro His 130 135 140Leu Asp Met Ala Leu Val Met Thr Val Glu
Pro Gly Phe Gly Gly Gln145 150 155 160Lys Phe Met Glu Asp Met Met
Pro Lys Val Glu Thr Leu Arg Ala Lys 165 170 175Phe Pro His Leu Asn
Ile Gln Val Asp Gly Gly Leu Gly Lys Glu Thr 180 185 190Ile Pro Lys
Ala Ala Lys Ala Gly Ala Asn Val Ile Val Ala Gly Thr 195 200 205Ser
Val Phe Thr Ala Ala Asp Pro His Asp Val Ile Ser Phe Met Lys 210 215
220Glu Glu Val Ser Lys Glu Leu Arg Ser Arg Asp Leu Leu Asp225 230
2354211803DNASaccharomyces cerevisiae 421atgttgtgtt cagtaattca
gagacagaca agagaggttt ccaacacaat gtctttagac 60tcatactatc ttgggtttga
tctttcgacc caacaactga aatgtctcgc cattaaccag 120gacctaaaaa
ttgtccattc agaaacagtg gaatttgaaa aggatcttcc gcattatcac
180acaaagaagg gtgtctatat acacggcgac actatcgaat gtcccgtagc
catgtggtta 240gaggctctag atctggttct ctcgaaatat cgcgaggcta
aatttccatt gaacaaagtt 300atggccgtct cagggtcctg ccagcagcac
gggtctgtct actggtcctc ccaagccgaa 360tctctgttag agcaattgaa
taagaaaccg gaaaaagatt tattgcacta cgtgagctct 420gtagcatttg
caaggcaaac cgcccccaat tggcaagacc acagtactgc aaagcaatgt
480caagagtttg aagagtgcat aggtgggcct gaaaaaatgg ctcaattaac
agggtccaga 540gcccatttta gatttactgg tcctcaaatt ctgaaaattg
cacaattaga accagaagct 600tacgaaaaaa caaagaccat ttctttagtg
tctaattttt tgacttctat cttagtgggc 660catcttgttg aattagagga
ggcagatgcc tgtggtatga acctttatga tatacgtgaa 720agaaaattca
gtgatgagct actacatcta attgatagtt cttctaagga taaaactatc
780agacaaaaat taatgagagc acccatgaaa aatttgatag cgggtaccat
ctgtaaatat 840tttattgaga agtacggttt caatacaaac tgcaaggtct
ctcccatgac tggggataat 900ttagccacta tatgttcttt acccctgcgg
aagaatgacg ttctcgtttc cctaggaaca 960agtactacag ttcttctggt
caccgataag tatcacccct ctccgaacta tcatcttttc 1020attcatccaa
ctctgccaaa ccattatatg ggtatgattt gttattgtaa tggttctttg
1080gcaagggaga ggataagaga cgagttaaac aaagaacggg aaaataatta
tgagaagact 1140aacgattgga ctctttttaa tcaagctgtg ctagatgact
cagaaagtag tgaaaatgaa 1200ttaggtgtat attttcctct gggggagatc
gttcctagcg taaaagccat aaacaaaagg 1260gttatcttca atccaaaaac
gggtatgatt gaaagagagg tggccaagtt caaagacaag 1320aggcacgatg
ccaaaaatat tgtagaatca caggctttaa gttgcagggt aagaatatct
1380cccctgcttt cggattcaaa cgcaagctca caacagagac tgaacgaaga
tacaatcgtg 1440aagtttgatt acgatgaatc tccgctgcgg gactacctaa
ataaaaggcc agaaaggact 1500ttttttgtag gtggggcttc taaaaacgat
gctattgtga agaagtttgc tcaagtcatt 1560ggtgctacaa agggtaattt
taggctagaa acaccaaact catgtgccct tggtggttgt 1620tataaggcca
tgtggtcatt gttatatgac tctaataaaa ttgcagttcc ttttgataaa
1680tttctgaatg acaattttcc atggcatgta atggaaagca tatccgatgt
ggataatgaa 1740aattgggatc gctataattc caagattgtc cccttaagcg
aactggaaaa gactctcatc 1800taa 1803422600PRTSaccharomyces cerevisiae
422Met Leu Cys Ser Val Ile Gln Arg Gln Thr Arg Glu Val Ser Asn Thr1
5 10 15Met Ser Leu Asp Ser Tyr Tyr Leu Gly Phe Asp Leu Ser Thr Gln
Gln 20 25 30Leu Lys Cys Leu Ala Ile Asn Gln Asp Leu Lys Ile Val His
Ser Glu 35 40 45Thr Val Glu Phe Glu Lys Asp Leu Pro His Tyr His Thr
Lys Lys Gly 50 55 60Val Tyr Ile His Gly Asp Thr Ile Glu Cys Pro Val
Ala Met Trp Leu65 70 75 80Glu Ala Leu Asp Leu Val Leu Ser Lys Tyr
Arg Glu Ala Lys Phe Pro 85 90 95Leu Asn Lys Val Met Ala Val Ser Gly
Ser Cys Gln Gln His Gly Ser 100 105 110Val Tyr Trp Ser Ser Gln Ala
Glu Ser Leu Leu Glu Gln Leu Asn Lys 115 120 125Lys Pro Glu Lys Asp
Leu Leu His Tyr Val Ser Ser Val Ala Phe Ala 130 135 140Arg Gln Thr
Ala Pro Asn Trp Gln Asp His Ser Thr Ala Lys Gln Cys145 150 155
160Gln Glu Phe Glu Glu Cys Ile Gly Gly Pro Glu Lys Met Ala Gln Leu
165 170 175Thr Gly Ser Arg Ala His Phe Arg Phe Thr Gly Pro Gln Ile
Leu Lys 180 185 190Ile Ala Gln Leu Glu Pro Glu Ala Tyr Glu Lys Thr
Lys Thr Ile Ser 195 200 205Leu Val Ser Asn Phe Leu Thr Ser Ile Leu
Val Gly His Leu Val Glu 210 215 220Leu Glu Glu Ala Asp Ala Cys Gly
Met Asn Leu Tyr Asp Ile Arg Glu225 230 235 240Arg Lys Phe Ser Asp
Glu Leu Leu His Leu Ile Asp Ser Ser Ser Lys 245 250 255Asp Lys Thr
Ile Arg Gln Lys Leu Met Arg Ala Pro Met Lys Asn Leu 260 265 270Ile
Ala Gly Thr Ile Cys Lys Tyr Phe Ile Glu Lys Tyr Gly Phe Asn 275 280
285Thr Asn Cys Lys Val Ser Pro Met Thr Gly Asp Asn Leu Ala Thr Ile
290 295 300Cys Ser Leu Pro Leu Arg Lys Asn Asp Val Leu Val Ser Leu
Gly Thr305 310
315 320Ser Thr Thr Val Leu Leu Val Thr Asp Lys Tyr His Pro Ser Pro
Asn 325 330 335Tyr His Leu Phe Ile His Pro Thr Leu Pro Asn His Tyr
Met Gly Met 340 345 350Ile Cys Tyr Cys Asn Gly Ser Leu Ala Arg Glu
Arg Ile Arg Asp Glu 355 360 365Leu Asn Lys Glu Arg Glu Asn Asn Tyr
Glu Lys Thr Asn Asp Trp Thr 370 375 380Leu Phe Asn Gln Ala Val Leu
Asp Asp Ser Glu Ser Ser Glu Asn Glu385 390 395 400Leu Gly Val Tyr
Phe Pro Leu Gly Glu Ile Val Pro Ser Val Lys Ala 405 410 415Ile Asn
Lys Arg Val Ile Phe Asn Pro Lys Thr Gly Met Ile Glu Arg 420 425
430Glu Val Ala Lys Phe Lys Asp Lys Arg His Asp Ala Lys Asn Ile Val
435 440 445Glu Ser Gln Ala Leu Ser Cys Arg Val Arg Ile Ser Pro Leu
Leu Ser 450 455 460Asp Ser Asn Ala Ser Ser Gln Gln Arg Leu Asn Glu
Asp Thr Ile Val465 470 475 480Lys Phe Asp Tyr Asp Glu Ser Pro Leu
Arg Asp Tyr Leu Asn Lys Arg 485 490 495Pro Glu Arg Thr Phe Phe Val
Gly Gly Ala Ser Lys Asn Asp Ala Ile 500 505 510Val Lys Lys Phe Ala
Gln Val Ile Gly Ala Thr Lys Gly Asn Phe Arg 515 520 525Leu Glu Thr
Pro Asn Ser Cys Ala Leu Gly Gly Cys Tyr Lys Ala Met 530 535 540Trp
Ser Leu Leu Tyr Asp Ser Asn Lys Ile Ala Val Pro Phe Asp Lys545 550
555 560Phe Leu Asn Asp Asn Phe Pro Trp His Val Met Glu Ser Ile Ser
Asp 565 570 575Val Asp Asn Glu Asn Trp Asp Arg Tyr Asn Ser Lys Ile
Val Pro Leu 580 585 590Ser Glu Leu Glu Lys Thr Leu Ile 595
60042330DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 423actagtatgt ctgacaagga acaaacgagc
3042438DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 424ctcgagttaa aagattaccc tttcagtaga tggtaatg
3842538DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 425caagcctttg gtggtaccca gaatccaggg ttagctcc
3842638DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 426ggagctaacc ctggattctg ggtaccacca aaggcttg
3842737DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 427ggtacaacgc atatgcagat gttgctacaa agcagaa
3742837DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 428ttctgctttg tagcaacatc tgcatatgcg ttgtacc
3742942DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 429gacgacgtct agaaaagaat actggagaaa tgaaaagaaa ac
4243038DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 430gcatgcttaa ttaatgcgag gcatatttat ggtgaagg
3843153DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 431ggccggccag atctgcggcc gcggccagca aaactaaaaa
actgtattat aag 5343251DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 432gcggccgcag atctggccgg
ccgatttatc ttcgtttcct gcaggttttt g 5143344DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
433gaattcttaa ttaacttttg ttccactact ttttggaact cttg
4443429DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 434gcatgcgcgg ccgcacgtcg gcaggcccg
2943546DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 435cgaaggacgc gcgaccaagt ttatcattat caatactcgc
catttc 4643646DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 436gaaatggcga gtattgataa tgataaactt
ggtcgcgcgt ccttcg 4643728DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 437gtcgacccgc aaattaaagc
cttcgagc 2843829DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 438gtcgacgtac ccccgggtta attaaggcg
2943937DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 439gtcgaaaacg agctcgaatt cgacgtcggc aggcccg
3744037DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 440cgggcctgcc gacgtcgaat tcgagctcgt tttcgac
3744131DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 441ggatccgcgg ccgctggtcg cgcgtccttc g
3144225DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 442gagggcacag ttaagccgct aaagg
2544340DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 443gtcaacagta cccttagtat attctccagt agctagggag
4044429DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 444cgttacccaa ttgaacacgg tattgtcac
2944526DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 445gaagattgag cagcggtttg catttc
2644632DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 446gagtcaaacg acgttgaaat tgaggctact gc
3244732DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 447gattactgct gctgttccag cccatatcca ac
3244826DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 448ggcaatcaaa ttgggaacga acaatg
2644929DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 449ctcaaggtat cctcatggcc aagcaatac
2945032DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 450gggtctacaa actgttgttg tcgaagaaga tg
3245132DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 451cattcagttc caatgattta ttgacagtgc ac
3245226DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 452cctacccgcc tcggatccca gctacc
2645326DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 453ggtagctggg atccgaggcg ggtagg
2645424DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 454cctcccggca cagcgtgtcg atgc 2445521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
455cgaagccctg gagcgcttcg c 2145633DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 456gtggtcagga ttgattctgc
acttgttttc cag 3345726DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 457cgcgtgaagc tgtagaaggc
gctaag 2645829DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 458gagctcggcc gcaaattaaa gccttcgag
2945946DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 459ggccggccgt ttatcattat caatactcgc catttcaaag
aatacg 4646043DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 460gttcactgca ctagtaaaaa aatggtattg
tcacacatcg aag 4346142DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 461cttcgagatc tcgagttact
gttttgctgc ttcaacaaat tg 4246246DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 462gttcactgca ctagtaaaaa
aatggagtcc aaagtcgttg aaaacc 4646343DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
463cttcgagatc tcgagttaca cttggaaaac agcctgcaaa tcc
4346442DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 464gttcactgca ctagtaaaaa aatgacaaac ctcgccccga cc
4246534DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 465cttcgagatc tcgagtcagt ccagcagggc cagg
3446649DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 466gttcactgca ctagtaaaaa aatgacacag aacgaaaata
atcagccgc 4946736DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 467cttcgagatc tcgagtcagt caaacagcgc
cagcgc 3646852DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 468gttcactgca ctagtaaaaa aatggctatt
acaaaagaat ttttagctcc ag 5246943DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 469cttcgagatc tcgagttagc
tagaaatttt agcggtagtt gcc 4347042DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 470gttcactgca ctagtaaaaa
aatgacgatt gcccagaccc ag 4247131DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 471cttcgagatc tcgagtcagc
ccgcccgcac c 3147252DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 472gttcactgcc atatgaatcc acaattgtta
cgcgtaacaa atcgaatcat tg 5247348DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 473cttcgagatc tcgagttaaa
aagtgataca ggttgcgccc tgttcggc 48474630DNASaccharophagus degradans
474atggctatta caaaagaatt tttagctcca gttggcgtaa tgcctgttgt
ggttgtggat 60cgtgtagaag atgcggtgcc tattacaaac gcattaaaag ccggcggtat
taaagcagtt 120gagattactt tacgtactcc tgcggcactg gatgctattc
gcgctattaa agctgagtgt 180gaagacatcc tggtgggggt aggtacggtt
attaaccatc aaaaccttaa agatattgct 240gcaattggtg ttgatttcgc
cgtatctcct ggttacaccc caacattgct gaagcaagcg 300caagatttgg
gcgtagaaat gttgcctggt gtaacttcgc cttctgaagt tatgcttggt
360atggagctag gtttgtcttg cttcaagcta ttccctgcgg ttgcagtagg
tggtttgcca 420ttacttaagt ctattggtgg cccattacca caggtttcct
tctgtccaac aggcggtttg 480actatcgata ctttcaccga cttcttggca
ttgcctaacg ttgcttgtgt gggtggtact 540tggttggtgc ctgcagatgc
tgttgcagct aaaaactggc aagctattac tgatattgcg 600gcggcaacta
ccgctaaaat ttctagctaa 630475209PRTSaccharophagus degradans 475Met
Ala Ile Thr Lys Glu Phe Leu Ala Pro Val Gly Val Met Pro Val1 5 10
15Val Val Val Asp Arg Val Glu Asp Ala Val Pro Ile Thr Asn Ala Leu
20 25 30Lys Ala Gly Gly Ile Lys Ala Val Glu Ile Thr Leu Arg Thr Pro
Ala 35 40 45Ala Leu Asp Ala Ile Arg Ala Ile Lys Ala Glu Cys Glu Asp
Ile Leu 50 55 60Val Gly Val Gly Thr Val Ile Asn His Gln Asn Leu Lys
Asp Ile Ala65 70 75 80Ala Ile Gly Val Asp Phe Ala Val Ser Pro Gly
Tyr Thr Pro Thr Leu 85 90 95Leu Lys Gln Ala Gln Asp Leu Gly Val Glu
Met Leu Pro Gly Val Thr 100 105 110Ser Pro Ser Glu Val Met Leu Gly
Met Glu Leu Gly Leu Ser Cys Phe 115 120 125Lys Leu Phe Pro Ala Val
Ala Val Gly Gly Leu Pro Leu Leu Lys Ser 130 135 140Ile Gly Gly Pro
Leu Pro Gln Val Ser Phe Cys Pro Thr Gly Gly Leu145 150 155 160Thr
Ile Asp Thr Phe Thr Asp Phe Leu Ala Leu Pro Asn Val Ala Cys 165 170
175Val Gly Gly Thr Trp Leu Val Pro Ala Asp Ala Val Ala Ala Lys Asn
180 185 190Trp Gln Ala Ile Thr Asp Ile Ala Ala Ala Thr Thr Ala Lys
Ile Ser 195 200 205Ser 476660DNAXanthomonas axonopodis
476atgacgattg cccagaccca gaacaccgcc gaacagttgc tgcgcgatgc
cggcatcttg 60cccgtggtca ccgtggacac gctggatcag gcgcgccgcg tcgccgatgc
gttgctcgaa 120ggcggcctgc ccgcgatcga gctgaccctt cgcacgccag
tggcgatcga cgcgctggcg 180atgctcaagc gcgagcttcc taacatcttg
atcggtgccg gcaccgtgct gagcgaattg 240cagctgcgtc agtcggtgga
tgccggtgca gacttcctgg tgaccccggg cacgccggcg 300ccgctggcgc
gcctgctggc ggatgcgccg atcccggccg ttcccggcgc ggccactccg
360accgagctgc tgaccttgat gggtcttggc tttcgcgtct gcaagctgtt
cccggccacc 420gccgtgggcg gtctgcagat gctcaggggc ctggccggcc
cgctgtccga gctcaagctg 480tgccccaccg gcggcatcag cgaggccaac
gccgccgagt tcctgtcgca gccgaacgtg 540ctgtgcatcg gcggttcgtg
gatggtcccc aaggattggc tggcgcacgg ccaatgggac 600aaggtcaagg
aaagctcggc caaggcggcg gcgatcgtgc ggcaggtgcg ggcgggctga
660477219PRTXanthomonas axonopodis 477Met Thr Ile Ala Gln Thr Gln
Asn Thr Ala Glu Gln Leu Leu Arg Asp1 5 10 15Ala Gly Ile Leu Pro Val
Val Thr Val Asp Thr Leu Asp Gln Ala Arg 20 25 30Arg Val Ala Asp Ala
Leu Leu Glu Gly Gly Leu Pro Ala Ile Glu Leu 35 40 45Thr Leu Arg Thr
Pro Val Ala Ile Asp Ala Leu Ala Met Leu Lys Arg 50 55 60Glu Leu Pro
Asn Ile Leu Ile Gly Ala Gly Thr Val Leu Ser Glu Leu65 70 75 80Gln
Leu Arg Gln Ser Val Asp Ala Gly Ala Asp Phe Leu Val Thr Pro 85 90
95Gly Thr Pro Ala Pro Leu Ala Arg Leu Leu Ala Asp Ala Pro Ile Pro
100 105 110Ala Val Pro Gly Ala Ala Thr Pro Thr Glu Leu Leu Thr Leu
Met Gly 115 120 125Leu Gly Phe Arg Val Cys Lys Leu Phe Pro Ala Thr
Ala Val Gly Gly 130 135 140Leu Gln Met Leu Arg Gly Leu Ala Gly Pro
Leu Ser Glu Leu Lys Leu145 150 155 160Cys Pro Thr Gly Gly Ile Ser
Glu Ala Asn Ala Ala Glu Phe Leu Ser 165 170 175Gln Pro Asn Val Leu
Cys Ile Gly Gly Ser Trp Met Val Pro Lys Asp 180 185 190Trp Leu Ala
His Gly Gln Trp Asp Lys Val Lys Glu Ser Ser Ala Lys 195 200 205Ala
Ala Ala Ile Val Arg Gln Val Arg Ala Gly 210 215478675DNAPseudomonas
syringiae 478atgacacaga acgaaaataa tcagccgctc accagcatgg cgaacaagat
tgcccggatc 60gacgaactct gcgccaaggc aaagattctg ccggtcatca ccattgcccg
tgatcaggac 120gtattgccac tggccgacgc gctggccgct ggtggcatga
cggctctgga aatcaccctg 180cgctcggcgt tcggactgag tgcgatccgc
attttgcgcg agcagcgccc agagctgtgc 240actggcgccg ggaccattct
ggaccgcaag atgctggccg acgccgaggc ggcgggctcg 300caattcattg
tgacccccgg cagcacgcag gaactgttgc aggcggcgct cgacagcccg
360ttgcccctgt tgccaggcgt cagcagcgcg tcggaaatca tgatcggcta
tgccttgggt 420tatcgccgct tcaagctgtt cccggcagaa atcagcggcg
gtgtggcagc gatcaaggcc 480ttgggcgggc ctttcaacga ggtgcgtttc
tgcccgacgg gcggcgtcaa cgagcagaac 540ctcaagaact acatggcctt
gcccaacgtc atgtgcgtcg gcgggacatg gatgattgat 600aacgcctggg
tcaagaatgg cgactggggc cgcattcagg aagccacggc acaggcgctg
660gcgctgtttg actga 675479224PRTPseudomonas syringiae 479Met Thr
Gln Asn Glu Asn Asn Gln Pro Leu Thr Ser Met Ala Asn Lys1 5 10 15Ile
Ala Arg Ile Asp Glu Leu Cys Ala Lys Ala Lys Ile Leu Pro Val 20 25
30Ile Thr Ile Ala Arg Asp Gln Asp Val Leu Pro Leu Ala Asp Ala Leu
35 40 45Ala Ala Gly Gly Met Thr Ala Leu Glu Ile Thr Leu Arg Ser Ala
Phe 50 55 60Gly Leu Ser Ala Ile Arg Ile Leu Arg Glu Gln Arg Pro Glu
Leu Cys65 70 75 80Thr Gly Ala Gly Thr Ile Leu Asp Arg Lys Met Leu
Ala Asp Ala Glu 85 90 95Ala Ala Gly Ser Gln Phe Ile Val Thr Pro Gly
Ser Thr Gln Glu Leu 100 105 110Leu Gln Ala Ala Leu Asp Ser Pro Leu
Pro Leu Leu Pro Gly Val Ser 115 120 125Ser Ala Ser Glu Ile Met Ile
Gly Tyr Ala Leu Gly Tyr Arg Arg Phe 130 135 140Lys Leu Phe Pro Ala
Glu Ile Ser Gly Gly Val Ala Ala Ile Lys Ala145 150 155 160Leu Gly
Gly Pro Phe Asn Glu Val Arg Phe Cys Pro Thr Gly Gly Val 165 170
175Asn Glu Gln Asn Leu Lys Asn Tyr Met Ala Leu Pro Asn Val Met Cys
180 185 190Val Gly Gly Thr Trp Met Ile Asp Asn Ala Trp Val Lys Asn
Gly Asp 195 200 205Trp Gly Arg Ile Gln Glu Ala Thr Ala Gln Ala Leu
Ala Leu Phe Asp 210 215 220480666DNAPseudomonas fluorescens
480atgacaaacc tcgccccgac cgtttccatg gcggacaaag ttgccctgat
cgacagcctc 60tgcgccaagg cgcggatcct gccggtgatc accattgccc gcgagcagga
tgtcctgccg
120ctggccgatg ccctggcggc cggcggcctg accgccctgg aagtgaccct
gcgttcgcag 180ttcggcctca aggcgatcca gatcctgcgc gaacagcgcc
cggagctggt gaccggtgcc 240ggcaccgtgc tcgacccgca gatgctggtg
gcggcggaag cggcaggttc gcagttcatc 300gtcaccccgg gcatcacccg
cgacctgctg caagccagcg tggccagccc gattcccctg 360ctgccgggga
tcagcaatgc ctccgggatc atggagggtt atgccctggg ctaccgccgc
420ttcaagctgt tcccggcgga agtcagtggt ggcgtggcgg cgatcaaggc
cctgggcggg 480ccgttcggcg aggtcaagtt ctgccctacc ggcggcgtcg
gcccggccaa tatcaagagc 540tacatggcgc tcaagaatgt gatgtgtgtc
ggcggtagct ggatgctcga tcccgagtgg 600atcaagaacg gcgactgggc
acggatccag gagtgcacgg ccgaggccct ggccctgctg 660gactga
666481221PRTPseudomonas fluorescens 481Met Thr Asn Leu Ala Pro Thr
Val Ser Met Ala Asp Lys Val Ala Leu1 5 10 15Ile Asp Ser Leu Cys Ala
Lys Ala Arg Ile Leu Pro Val Ile Thr Ile 20 25 30Ala Arg Glu Gln Asp
Val Leu Pro Leu Ala Asp Ala Leu Ala Ala Gly 35 40 45Gly Leu Thr Ala
Leu Glu Val Thr Leu Arg Ser Gln Phe Gly Leu Lys 50 55 60Ala Ile Gln
Ile Leu Arg Glu Gln Arg Pro Glu Leu Val Thr Gly Ala65 70 75 80Gly
Thr Val Leu Asp Pro Gln Met Leu Val Ala Ala Glu Ala Ala Gly 85 90
95Ser Gln Phe Ile Val Thr Pro Gly Ile Thr Arg Asp Leu Leu Gln Ala
100 105 110Ser Val Ala Ser Pro Ile Pro Leu Leu Pro Gly Ile Ser Asn
Ala Ser 115 120 125Gly Ile Met Glu Gly Tyr Ala Leu Gly Tyr Arg Arg
Phe Lys Leu Phe 130 135 140Pro Ala Glu Val Ser Gly Gly Val Ala Ala
Ile Lys Ala Leu Gly Gly145 150 155 160Pro Phe Gly Glu Val Lys Phe
Cys Pro Thr Gly Gly Val Gly Pro Ala 165 170 175Asn Ile Lys Ser Tyr
Met Ala Leu Lys Asn Val Met Cys Val Gly Gly 180 185 190Ser Trp Met
Leu Asp Pro Glu Trp Ile Lys Asn Gly Asp Trp Ala Arg 195 200 205Ile
Gln Glu Cys Thr Ala Glu Ala Leu Ala Leu Leu Asp 210 215
220482591DNABacillus subtilis 482atggagtcca aagtcgttga aaaccgtctg
aaagaagcaa agctgattgc agtcattcgt 60tcaaaggata agcaggaggc ctgtcagcag
attgagagtt tattagataa agggattcgt 120gcagttgaag tgacgtatac
gacccccggg gcatcagata ttatcgaatc cttccgtaat 180agggaagata
ttttaattgg cgcgggtacg gtcatcagcg cgcagcaagc tggggaagct
240gctaaggctg gcgcgcagtt tattgtcagt ccgggttttt cagctgatct
tgctgaacat 300ctatcttttg taaagacaca ttatatcccc ggcgtcttga
ctccgagcga aattatggaa 360gcgctgacat tcggttttac gacattaaag
ctgttcccaa gcggtgtgtt tggcattccg 420tttatgaaaa atttagcggg
tcctttcccg caggtgacct ttattccgac aggcgggata 480catccgtctg
aagtgcctga ttggcttaga gccggagctg gcgccgtcgg agtcggcagc
540cagttgggca gctgttcaaa agaggatttg caggctgttt tccaagtgta a
591483196PRTBacillus subtilis 483Met Glu Ser Lys Val Val Glu Asn
Arg Leu Lys Glu Ala Lys Leu Ile1 5 10 15Ala Val Ile Arg Ser Lys Asp
Lys Gln Glu Ala Cys Gln Gln Ile Glu 20 25 30Ser Leu Leu Asp Lys Gly
Ile Arg Ala Val Glu Val Thr Tyr Thr Thr 35 40 45Pro Gly Ala Ser Asp
Ile Ile Glu Ser Phe Arg Asn Arg Glu Asp Ile 50 55 60Leu Ile Gly Ala
Gly Thr Val Ile Ser Ala Gln Gln Ala Gly Glu Ala65 70 75 80Ala Lys
Ala Gly Ala Gln Phe Ile Val Ser Pro Gly Phe Ser Ala Asp 85 90 95Leu
Ala Glu His Leu Ser Phe Val Lys Thr His Tyr Ile Pro Gly Val 100 105
110Leu Thr Pro Ser Glu Ile Met Glu Ala Leu Thr Phe Gly Phe Thr Thr
115 120 125Leu Lys Leu Phe Pro Ser Gly Val Phe Gly Ile Pro Phe Met
Lys Asn 130 135 140Leu Ala Gly Pro Phe Pro Gln Val Thr Phe Ile Pro
Thr Gly Gly Ile145 150 155 160His Pro Ser Glu Val Pro Asp Trp Leu
Arg Ala Gly Ala Gly Ala Val 165 170 175Gly Val Gly Ser Gln Leu Gly
Ser Cys Ser Lys Glu Asp Leu Gln Ala 180 185 190Val Phe Gln Val
195484624DNABacillus licheniformis 484atggtattgt cacacatcga
agaacaaaaa ctgattgcga tcatccgcgg atacaatccg 60gaggaggcag tgagcattgc
cggcgcctta aaagcgggcg gcatcaggct tgtggagatt 120acgcttaatt
cccctcaagc gatcaaagcg attgaagcgg tttcagagca ttttggggac
180gaaatgcttg tcggagcggg aaccgtactt gatcccgaat ctgcgagagc
ggcgctttta 240gccggcgcgc ggtttatcct gtctccgacc gtcaatgaag
agacgatcaa gctgacaaaa 300cggtatggag cggtcagcat tccaggcgct
tttaccccga ctgaaatatt gacggcgtat 360gaaagcgggg gagacatcat
caaggtattt cccggaacaa tggggcctgg ctatatcaag 420gatatccacg
gaccgcttcc gcatattccg ctgcttccga ctggaggagt cggattggaa
480aaccttcacg agtttctgca ggccggtgcg gtcggagcgg gaatcggcgg
ttcgcttgtt 540cgggctaata aagatgttaa tgacgcgttt ttagaagagc
tgtccaaaaa agcaaagcaa 600tttgttgaag cagcaaaaca gtaa
624485207PRTBacillus licheniformis 485Met Val Leu Ser His Ile Glu
Glu Gln Lys Leu Ile Ala Ile Ile Arg1 5 10 15Gly Tyr Asn Pro Glu Glu
Ala Val Ser Ile Ala Gly Ala Leu Lys Ala 20 25 30Gly Gly Ile Arg Leu
Val Glu Ile Thr Leu Asn Ser Pro Gln Ala Ile 35 40 45Lys Ala Ile Glu
Ala Val Ser Glu His Phe Gly Asp Glu Met Leu Val 50 55 60Gly Ala Gly
Thr Val Leu Asp Pro Glu Ser Ala Arg Ala Ala Leu Leu65 70 75 80Ala
Gly Ala Arg Phe Ile Leu Ser Pro Thr Val Asn Glu Glu Thr Ile 85 90
95Lys Leu Thr Lys Arg Tyr Gly Ala Val Ser Ile Pro Gly Ala Phe Thr
100 105 110Pro Thr Glu Ile Leu Thr Ala Tyr Glu Ser Gly Gly Asp Ile
Ile Lys 115 120 125Val Phe Pro Gly Thr Met Gly Pro Gly Tyr Ile Lys
Asp Ile His Gly 130 135 140Pro Leu Pro His Ile Pro Leu Leu Pro Thr
Gly Gly Val Gly Leu Glu145 150 155 160Asn Leu His Glu Phe Leu Gln
Ala Gly Ala Val Gly Ala Gly Ile Gly 165 170 175Gly Ser Leu Val Arg
Ala Asn Lys Asp Val Asn Asp Ala Phe Leu Glu 180 185 190Glu Leu Ser
Lys Lys Ala Lys Gln Phe Val Glu Ala Ala Lys Gln 195 200
205486642DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 486atgaaaaact ggaaaacaag tgcagaatca
atcctgacca ccggcccggt tgtaccggtt 60atcgtggtaa aaaaactgga acacgcggtg
ccgatggcaa aagcgttggt tgctggtggg 120gtgcgcgttc tggaagtgac
tctgcgtacc gagtgtgcag ttgacgctat ccgtgctatc 180gccaaagaag
tgcctgaagc gattgtgggt gccggtacgg tgctgaatcc acagcagctg
240gcagaagtca ctgaagcggg tgcacagttc gcaattagcc cgggtctgac
cgagccgctg 300ctgaaagctg ctaccgaagg gactattcct ctgattccgg
ggatcagcac tgtttccgaa 360ctgatgctgg gtatggacta cggtttgaaa
gagttcaaat tcttcccggc tgaagctaac 420ggcggcgtga aagccctgca
ggcgatcgcg ggtccgttct cccaggtccg tttctgcccg 480acgggtggta
tttctccggc taactaccgt gactacctgg cgctgaaaag cgtgctgtgc
540atcggtggtt cctggctggt tccggcagat gcgctggaag cgggcgatta
cgaccgcatt 600actaagctgg cgcgtgaagc tgtagaaggc gctaagctgt aa
642487213PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 487Met Lys Asn Trp Lys Thr Ser Ala Glu Ser
Ile Leu Thr Thr Gly Pro1 5 10 15Val Val Pro Val Ile Val Val Lys Lys
Leu Glu His Ala Val Pro Met 20 25 30Ala Lys Ala Leu Val Ala Gly Gly
Val Arg Val Leu Glu Val Thr Leu 35 40 45Arg Thr Glu Cys Ala Val Asp
Ala Ile Arg Ala Ile Ala Lys Glu Val 50 55 60Pro Glu Ala Ile Val Gly
Ala Gly Thr Val Leu Asn Pro Gln Gln Leu65 70 75 80Ala Glu Val Thr
Glu Ala Gly Ala Gln Phe Ala Ile Ser Pro Gly Leu 85 90 95Thr Glu Pro
Leu Leu Lys Ala Ala Thr Glu Gly Thr Ile Pro Leu Ile 100 105 110Pro
Gly Ile Ser Thr Val Ser Glu Leu Met Leu Gly Met Asp Tyr Gly 115 120
125Leu Lys Glu Phe Lys Phe Phe Pro Ala Glu Ala Asn Gly Gly Val Lys
130 135 140Ala Leu Gln Ala Ile Ala Gly Pro Phe Ser Gln Val Arg Phe
Cys Pro145 150 155 160Thr Gly Gly Ile Ser Pro Ala Asn Tyr Arg Asp
Tyr Leu Ala Leu Lys 165 170 175Ser Val Leu Cys Ile Gly Gly Ser Trp
Leu Val Pro Ala Asp Ala Leu 180 185 190Glu Ala Gly Asp Tyr Asp Arg
Ile Thr Lys Leu Ala Arg Glu Ala Val 195 200 205Glu Gly Ala Lys Leu
210488682DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 488atgaaaaact ggaaacagaa gaccgcccgc
atcgacacgc tgtgccggga ggcgcgcatc 60ctcccggtga tcaccatcga ccgcgaggcg
gacatcctgc cgatggccga tgccctcgcc 120gccggcggcc tgaccgccct
ggagatcacc ctgcgcacgg cgcacgggct gaccgccatc 180cggcgcctca
gcgaggagcg cccgcacctg cgcatcggcg ccggcaccgt gctcgacccg
240cggaccttcg ccgccgcgga aaaggccggg gcgagcttcg tggtcacccc
gggttgcacc 300gacgagttgc tgcgcttcgc cctggacagc gaagtcccgc
tgttgcccgg cgtggccagc 360gcttccgaga tcatgctcgc ctaccgccat
ggctaccgcc gcttcaagct gtttcccgcc 420gaagtcagcg gcggcccggc
ggcgctgaag gcgttctcgg gaccattccc cgatatccgc 480ttctgcccca
ccggaggcgt cagcctgaac aatctcgccg actacctggc ggtacccaac
540gtgatgtgcg tcggcggcac ctggatgctg cccaaggccg tggtcgaccg
cggcgactgg 600gcccaggtcg agcgcctcag ccgcgaagcc ctggagcgct
tcgccgagca ccgcagacac 660taatagctcg agttacttta ct
682489220PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 489Met Lys Asn Trp Lys Gln Lys Thr Ala Arg
Ile Asp Thr Leu Cys Arg1 5 10 15Glu Ala Arg Ile Leu Pro Val Ile Thr
Ile Asp Arg Glu Ala Asp Ile 20 25 30Leu Pro Met Ala Asp Ala Leu Ala
Ala Gly Gly Leu Thr Ala Leu Glu 35 40 45Ile Thr Leu Arg Thr Ala His
Gly Leu Thr Ala Ile Arg Arg Leu Ser 50 55 60Glu Glu Arg Pro His Leu
Arg Ile Gly Ala Gly Thr Val Leu Asp Pro65 70 75 80Arg Thr Phe Ala
Ala Ala Glu Lys Ala Gly Ala Ser Phe Val Val Thr 85 90 95Pro Gly Cys
Thr Asp Glu Leu Leu Arg Phe Ala Leu Asp Ser Glu Val 100 105 110Pro
Leu Leu Pro Gly Val Ala Ser Ala Ser Glu Ile Met Leu Ala Tyr 115 120
125Arg His Gly Tyr Arg Arg Phe Lys Leu Phe Pro Ala Glu Val Ser Gly
130 135 140Gly Pro Ala Ala Leu Lys Ala Phe Ser Gly Pro Phe Pro Asp
Ile Arg145 150 155 160Phe Cys Pro Thr Gly Gly Val Ser Leu Asn Asn
Leu Ala Asp Tyr Leu 165 170 175Ala Val Pro Asn Val Met Cys Val Gly
Gly Thr Trp Met Leu Pro Lys 180 185 190Ala Val Val Asp Arg Gly Asp
Trp Ala Gln Val Glu Arg Leu Ser Arg 195 200 205Glu Ala Leu Glu Arg
Phe Ala Glu His Arg Arg His 210 215 220490682DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
490atgaaaaact ggaaaacaag tgcagaatca atcgacacgc tgtgccggga
ggcgcgcatc 60ctcccggtga tcaccatcga ccgcgaggcg gacatcctgc cgatggccga
tgccctcgcc 120gccggcggcc tgaccgccct ggagatcacc ctgcgcacgg
cgcacgggct gaccgccatc 180cggcgcctca gcgaggagcg cccgcacctg
cgcatcggcg ccggcaccgt gctcgacccg 240cggaccttcg ccgccgcgga
aaaggccggg gcgagcttcg tggtcacccc gggttgcacc 300gacgagttgc
tgcgcttcgc cctggacagc gaagtcccgc tgttgcccgg cgtggccagc
360gcttccgaga tcatgctcgc ctaccgccat ggctaccgcc gcttcaagct
gtttcccgcc 420gaagtcagcg gcggcccggc ggcgctgaag gcgttctcgg
gaccattccc cgatatccgc 480ttctgcccca ccggaggcgt cagcctgaac
aatctcgccg actacctggc ggtacccaac 540gtgatgtgcg tcggcggcac
ctggatgctg cccaaggccg tggtcgaccg cggcgactgg 600gcccaggtcg
agcgcctcag ccgcgaagcc ctggagcgct tcgccgagca ccgcagacac
660taatagctcg agttacttta ct 682491220PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
491Met Lys Asn Trp Lys Thr Ser Ala Glu Ser Ile Asp Thr Leu Cys Arg1
5 10 15Glu Ala Arg Ile Leu Pro Val Ile Thr Ile Asp Arg Glu Ala Asp
Ile 20 25 30Leu Pro Met Ala Asp Ala Leu Ala Ala Gly Gly Leu Thr Ala
Leu Glu 35 40 45Ile Thr Leu Arg Thr Ala His Gly Leu Thr Ala Ile Arg
Arg Leu Ser 50 55 60Glu Glu Arg Pro His Leu Arg Ile Gly Ala Gly Thr
Val Leu Asp Pro65 70 75 80Arg Thr Phe Ala Ala Ala Glu Lys Ala Gly
Ala Ser Phe Val Val Thr 85 90 95Pro Gly Cys Thr Asp Glu Leu Leu Arg
Phe Ala Leu Asp Ser Glu Val 100 105 110Pro Leu Leu Pro Gly Val Ala
Ser Ala Ser Glu Ile Met Leu Ala Tyr 115 120 125Arg His Gly Tyr Arg
Arg Phe Lys Leu Phe Pro Ala Glu Val Ser Gly 130 135 140Gly Pro Ala
Ala Leu Lys Ala Phe Ser Gly Pro Phe Pro Asp Ile Arg145 150 155
160Phe Cys Pro Thr Gly Gly Val Ser Leu Asn Asn Leu Ala Asp Tyr Leu
165 170 175Ala Val Pro Asn Val Met Cys Val Gly Gly Thr Trp Met Leu
Pro Lys 180 185 190Ala Val Val Asp Arg Gly Asp Trp Ala Gln Val Glu
Arg Leu Ser Arg 195 200 205Glu Ala Leu Glu Arg Phe Ala Glu His Arg
Arg His 210 215 220492682DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 492atgaaaaact
ggaaaacaag tgcagaatca atcctgacca ccggccggga ggcgcgcatc 60ctcccggtga
tcaccatcga ccgcgaggcg gacatcctgc cgatggccga tgccctcgcc
120gccggcggcc tgaccgccct ggagatcacc ctgcgcacgg cgcacgggct
gaccgccatc 180cggcgcctca gcgaggagcg cccgcacctg cgcatcggcg
ccggcaccgt gctcgacccg 240cggaccttcg ccgccgcgga aaaggccggg
gcgagcttcg tggtcacccc gggttgcacc 300gacgagttgc tgcgcttcgc
cctggacagc gaagtcccgc tgttgcccgg cgtggccagc 360gcttccgaga
tcatgctcgc ctaccgccat ggctaccgcc gcttcaagct gtttcccgcc
420gaagtcagcg gcggcccggc ggcgctgaag gcgttctcgg gaccattccc
cgatatccgc 480ttctgcccca ccggaggcgt cagcctgaac aatctcgccg
actacctggc ggtacccaac 540gtgatgtgcg tcggcggcac ctggatgctg
cccaaggccg tggtcgaccg cggcgactgg 600gcccaggtcg agcgcctcag
ccgcgaagcc ctggagcgct tcgccgagca ccgcagacac 660taatagctcg
agttacttta ct 682493220PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 493Met Lys Asn Trp Lys
Thr Ser Ala Glu Ser Ile Leu Thr Thr Gly Arg1 5 10 15Glu Ala Arg Ile
Leu Pro Val Ile Thr Ile Asp Arg Glu Ala Asp Ile 20 25 30Leu Pro Met
Ala Asp Ala Leu Ala Ala Gly Gly Leu Thr Ala Leu Glu 35 40 45Ile Thr
Leu Arg Thr Ala His Gly Leu Thr Ala Ile Arg Arg Leu Ser 50 55 60Glu
Glu Arg Pro His Leu Arg Ile Gly Ala Gly Thr Val Leu Asp Pro65 70 75
80Arg Thr Phe Ala Ala Ala Glu Lys Ala Gly Ala Ser Phe Val Val Thr
85 90 95Pro Gly Cys Thr Asp Glu Leu Leu Arg Phe Ala Leu Asp Ser Glu
Val 100 105 110Pro Leu Leu Pro Gly Val Ala Ser Ala Ser Glu Ile Met
Leu Ala Tyr 115 120 125Arg His Gly Tyr Arg Arg Phe Lys Leu Phe Pro
Ala Glu Val Ser Gly 130 135 140Gly Pro Ala Ala Leu Lys Ala Phe Ser
Gly Pro Phe Pro Asp Ile Arg145 150 155 160Phe Cys Pro Thr Gly Gly
Val Ser Leu Asn Asn Leu Ala Asp Tyr Leu 165 170 175Ala Val Pro Asn
Val Met Cys Val Gly Gly Thr Trp Met Leu Pro Lys 180 185 190Ala Val
Val Asp Arg Gly Asp Trp Ala Gln Val Glu Arg Leu Ser Arg 195 200
205Glu Ala Leu Glu Arg Phe Ala Glu His Arg Arg His 210 215
2204941665DNASaccharomyces cerevisiae 494atgtccaata actcattcac
taacttcaaa ctggccactg aattgccagc ctggtctaag 60ttgcaaaaaa tttatgaatc
tcaaggtaag actttgtctg tcaagcaaga attccaaaaa 120gatgccaagc
gttttgaaaa attgaacaag actttcacca actatgatgg ttccaaaatc
180ttgttcgact actcaaagaa cttggtcaac gatgaaatca ttgctgcatt
gattgaactg 240gccaaggagg ctaacgtcac cggtttgaga gatgctatgt
tcaaaggtga acacatcaac 300tccactgaag atcgtgctgt ctaccacgtc
gcattgagaa acagagctaa caagccaatg 360tacgttgatg gtgtcaacgt
tgctccagaa gtcgactctg tcttgaagca catgaaggag 420ttctctgaac
aagttcgttc tggtgaatgg aagggttata ccggtaagaa
gatcaccgat 480gttgttaaca tcggtattgg tggttccgat ttgggtccag
tcatggtcac tgaggctttg 540aagcactacg ctggtgtctt ggatgtccac
ttcgtttcca acattgacgg tactcacatt 600gctgaaacct tgaaggttgt
tgacccagaa actactttgt ttttgattgc ttccaagact 660ttcactaccg
ctgaaactat cactaacgct aacactgcca agaactggtt cttgtcgaag
720acaggtaatg atccatctca cattgctaag catttcgctg ctttgtccac
taacgaaacc 780gaagttgcca agttcggtat tgacaccaaa aacatgtttg
gtttcgaaag ttgggtcggt 840ggtcgttact ctgtctggtc ggctattggt
ttgtctgttg ccttgtacat tggctatgac 900aactttgagg ctttcttgaa
gggtgctgaa gccgtcgaca accacttcac ccaaacccca 960ttggaagaca
acattccatt gttgggtggt ttgttgtctg tctggtacaa caacttcttt
1020ggtgctcaaa cccatttggt tgctccattc gaccaatact tgcacagatt
cccagcctac 1080ttgcaacaat tgtcaatgga atctaacggt aagtctgtta
ccagaggtaa cgtgtttact 1140gactactcta ctggttctat cttgtttggt
gaaccagcta ccaacgctca acactctttc 1200ttccaattgg ttcaccaagg
taccaagttg attccatctg atttcatctt agctgctcaa 1260tctcataacc
caattgagaa caaattacat caaaagatgt tggcttcaaa cttctttgct
1320caagctgaag ctttaatggt tggtaaggat gaagaacaag ttaaggctga
aggtgccact 1380ggtggtttgg tcccacacaa ggtcttctca ggtaacagac
caactacctc tatcttggct 1440caaaagatta ctccagctac tttgggtgct
ttgattgcct actacgaaca tgttactttc 1500actgaaggtg ccatttggaa
tatcaactct ttcgaccaat ggggtgttga attgggtaaa 1560gtcttggcta
aagtcatcgg caaggaattg gacaactcct ccaccatttc tacccacgat
1620gcttctacca acggtttaat caatcaattc aaggaatgga tgtga
16654951470DNASaccharomyces cerevisiae 495atgtctgctg atttcggttt
gattggtttg gccgtcatgg gtcaaaattt gatcttgaac 60gctgctgacc acggtttcac
tgtttgtgct tacaacagaa ctcaatccaa ggtcgaccat 120ttcttggcca
atgaagctaa gggcaaatct atcatcggtg ctacttccat tgaagatttc
180atctccaaat tgaagagacc tagaaaggtc atgcttttgg ttaaagctgg
tgctccagtt 240gacgctttga tcaaccaaat cgtcccactt ttggaaaagg
gtgatattat catcgatggt 300ggtaactctc acttcccaga ttctaataga
cgttacgaag aattgaagaa gaagggtatt 360cttttcgttg gttctggtgt
ctccggtggt gaggaaggtg cccgttacgg tccatctttg 420atgccaggtg
gttctgaaga agcttggcca catattaaga acatcttcca atccatctct
480gctaaatccg acggtgaacc atgttgcgaa tgggttggcc cagccggtgc
tggtcactac 540gtcaagatgg ttcacaacgg tattgaatac ggtgatatgc
aattgatttg tgaagcttat 600gacatcatga agagattggg tgggtttacc
gataaggaaa tcagtgacgt ttttgccaaa 660tggaacaatg gtgtcttgga
ttccttcttg gtcgaaatta ccagagatat tttgaaattc 720gacgacgtcg
acggtaagcc attagttgaa aaaatcatgg atactgctgg tcaaaagggt
780actggtaagt ggactgccat caacgccttg gatttgggta tgccagttac
tttgattggt 840gaagctgtct ttgcccgttg tctatctgct ttgaagaacg
agagaattag agcctccaag 900gtcttaccag gcccagaagt tccaaaagac
gccgtcaagg acagagaaca atttgtcgat 960gatttggaac aagctttgta
tgcttccaag attatttctt acgctcaagg tttcatgttg 1020atccgtgaag
ctgctgctac ttatggctgg aaactaaaca accctgccat cgctttgatg
1080tggagaggtg gttgtatcat tagatctgtt ttcttgggtc aaatcacaaa
ggcctacaga 1140gaagaaccag atttggaaaa cttgttgttc aacaagttct
tcgctgatgc cgtcaccaag 1200gctcaatctg gttggagaaa gtcaattgcg
ttggctacca cctacggtat cccaacacca 1260gccttttcca ccgctttgtc
tttctacgat gggtacagat ctgaaagatt gccagccaac 1320ttactacaag
ctcaacgtga ctactttggt gctcacactt tcagagtgtt gccagaatgt
1380gcttctgaca acttgccagt agacaaggat atccatatca actggactgg
ccacggtggt 1440aatgtttctt cctctacata ccaagcttaa
14704961479DNASaccharomyces cerevisiae 496atgtcaaagg cagtaggtga
tttaggctta gttggtttag ccgtgatggg tcaaaatttg 60atcttaaacg cagcggatca
cggatttacc gtggttgctt ataataggac gcaatcaaag 120gtagataggt
ttctagctaa tgaggcaaaa ggaaaatcaa taattggtgc aacttcaatt
180gaggacttgg ttgcgaaact aaagaaacct agaaagatta tgcttttaat
caaagccggt 240gctccggtcg acactttaat aaaggaactt gtaccacatc
ttgataaagg cgacattatt 300atcgacggtg gtaactcaca tttcccggac
actaacagac gctacgaaga gctaacaaag 360caaggaattc tttttgtggg
ctctggtgtc tcaggcggtg aagatggtgc acgttttggt 420ccatctttaa
tgcctggtgg gtcagcagaa gcatggccgc acatcaagaa catctttcaa
480tctattgccg ccaaatcaaa cggtgagcca tgctgcgaat gggtggggcc
tgccggttct 540ggtcactatg tgaagatggt acacaacggt atcgagtacg
gtgatatgca gttgatttgc 600gaggcttacg atatcatgaa acgaattggc
cggtttacgg ataaagagat cagtgaagta 660tttgacaagt ggaacactgg
agttttggat tctttcttga ttgaaatcac gagggacatt 720ttaaaattcg
atgacgtcga cggtaagcca ttggtggaaa aaattatgga tactgccggt
780caaaagggta ctggtaaatg gactgcaatc aacgccttgg atttaggaat
gccagtcact 840ttaattgggg aggctgtttt cgctcgttgt ttgtcagcca
taaaggacga acgtaaaaga 900gcttcgaaac ttctggcagg accaacagta
ccaaaggatg caatacatga tagagaacaa 960tttgtgtatg atttggaaca
agcattatac gcttcaaaga ttatttcata tgctcaaggt 1020ttcatgctga
tccgcgaagc tgccagatca tacggctgga aattaaacaa cccagctatt
1080gctctaatgt ggagaggtgg ctgtataatc agatctgtgt tcttagctga
gattacgaag 1140gcttataggg acgatccaga tttggaaaat ttattattca
acgagttctt cgcttctgca 1200gttactaagg cccaatccgg ttggagaaga
actattgccc ttgctgctac ttacggtatt 1260ccaactccag ctttctctac
tgctttagcg ttttacgacg gctatagatc tgagaggcta 1320ccagcaaact
tgttacaagc gcaacgtgat tattttggcg ctcatacatt tagaatttta
1380cctgaatgtg cttctgccca tttgccagta gacaaggata ttcatatcaa
ttggactggg 1440cacggaggta atatatcttc ctcaacctac caagcttaa
14794971008DNASaccharomyces cerevisiae 497atgtctgaac cagctcaaaa
gaaacaaaag gttgctaaca actctctaga acaattgaaa 60gcctccggca ctgtcgttgt
tgccgacact ggtgatttcg gctctattgc caagtttcaa 120cctcaagact
ccacaactaa cccatcattg atcttggctg ctgccaagca accaacttac
180gccaagttga tcgatgttgc cgtggaatac ggtaagaagc atggtaagac
caccgaagaa 240caagtcgaaa atgctgtgga cagattgtta gtcgaattcg
gtaaggagat cttaaagatt 300gttccaggca gagtctccac cgaagttgat
gctagattgt cttttgacac tcaagctacc 360attgaaaagg ctagacatat
cattaaattg tttgaacaag aaggtgtctc caaggaaaga 420gtccttatta
aaattgcttc cacttgggaa ggtattcaag ctgccaaaga attggaagaa
480aaggacggta tccactgtaa tttgactcta ttattctcct tcgttcaagc
agttgcctgt 540gccgaggccc aagttacttt gatttcccca tttgttggta
gaattctaga ctggtacaaa 600tccagcactg gtaaagatta caagggtgaa
gccgacccag gtgttatttc cgtcaagaaa 660atctacaact actacaagaa
gtacggttac aagactattg ttatgggtgc ttctttcaga 720agcactgacg
aaatcaaaaa cttggctggt gttgactatc taacaatttc tccagcttta
780ttggacaagt tgatgaacag tactgaacct ttcccaagag ttttggaccc
tgtctccgct 840aagaaggaag ccggcgacaa gatttcttac atcagcgacg
aatctaaatt cagattcgac 900ttgaatgaag acgctatggc cactgaaaaa
ttgtccgaag gtatcagaaa attctctgcc 960gatattgtta ctctattcga
cttgattgaa aagaaagtta ccgcttaa 1008498335PRTSaccharomyces
cerevisiae 498Met Ser Glu Pro Ala Gln Lys Lys Gln Lys Val Ala Asn
Asn Ser Leu1 5 10 15Glu Gln Leu Lys Ala Ser Gly Thr Val Val Val Ala
Asp Thr Gly Asp 20 25 30Phe Gly Ser Ile Ala Lys Phe Gln Pro Gln Asp
Ser Thr Thr Asn Pro 35 40 45Ser Leu Ile Leu Ala Ala Ala Lys Gln Pro
Thr Tyr Ala Lys Leu Ile 50 55 60Asp Val Ala Val Glu Tyr Gly Lys Lys
His Gly Lys Thr Thr Glu Glu65 70 75 80Gln Val Glu Asn Ala Val Asp
Arg Leu Leu Val Glu Phe Gly Lys Glu 85 90 95Ile Leu Lys Ile Val Pro
Gly Arg Val Ser Thr Glu Val Asp Ala Arg 100 105 110Leu Ser Phe Asp
Thr Gln Ala Thr Ile Glu Lys Ala Arg His Ile Ile 115 120 125Lys Leu
Phe Glu Gln Glu Gly Val Ser Lys Glu Arg Val Leu Ile Lys 130 135
140Ile Ala Ser Thr Trp Glu Gly Ile Gln Ala Ala Lys Glu Leu Glu
Glu145 150 155 160Lys Asp Gly Ile His Cys Asn Leu Thr Leu Leu Phe
Ser Phe Val Gln 165 170 175Ala Val Ala Cys Ala Glu Ala Gln Val Thr
Leu Ile Ser Pro Phe Val 180 185 190Gly Arg Ile Leu Asp Trp Tyr Lys
Ser Ser Thr Gly Lys Asp Tyr Lys 195 200 205Gly Glu Ala Asp Pro Gly
Val Ile Ser Val Lys Lys Ile Tyr Asn Tyr 210 215 220Tyr Lys Lys Tyr
Gly Tyr Lys Thr Ile Val Met Gly Ala Ser Phe Arg225 230 235 240Ser
Thr Asp Glu Ile Lys Asn Leu Ala Gly Val Asp Tyr Leu Thr Ile 245 250
255Ser Pro Ala Leu Leu Asp Lys Leu Met Asn Ser Thr Glu Pro Phe Pro
260 265 270Arg Val Leu Asp Pro Val Ser Ala Lys Lys Glu Ala Gly Asp
Lys Ile 275 280 285Ser Tyr Ile Ser Asp Glu Ser Lys Phe Arg Phe Asp
Leu Asn Glu Asp 290 295 300Ala Met Ala Thr Glu Lys Leu Ser Glu Gly
Ile Arg Lys Phe Ser Ala305 310 315 320Asp Ile Val Thr Leu Phe Asp
Leu Ile Glu Lys Lys Val Thr Ala 325 330 3354992043DNASaccharomyces
cerevisiae 499atgactcaat tcactgacat tgataagcta gccgtctcca
ccataagaat tttggctgtg 60gacaccgtat ccaaggccaa ctcaggtcac ccaggtgctc
cattgggtat ggcaccagct 120gcacacgttc tatggagtca aatgcgcatg
aacccaacca acccagactg gatcaacaga 180gatagatttg tcttgtctaa
cggtcacgcg gtcgctttgt tgtattctat gctacatttg 240actggttacg
atctgtctat tgaagacttg aaacagttca gacagttggg ttccagaaca
300ccaggtcatc ctgaatttga gttgccaggt gttgaagtta ctaccggtcc
attaggtcaa 360ggtatctcca acgctgttgg tatggccatg gctcaagcta
acctggctgc cacttacaac 420aagccgggct ttaccttgtc tgacaactac
acctatgttt tcttgggtga cggttgtttg 480caagaaggta tttcttcaga
agcttcctcc ttggctggtc atttgaaatt gggtaacttg 540attgccatct
acgatgacaa caagatcact atcgatggtg ctaccagtat ctcattcgat
600gaagatgttg ctaagagata cgaagcctac ggttgggaag ttttgtacgt
agaaaatggt 660aacgaagatc tagccggtat tgccaaggct attgctcaag
ctaagttatc caaggacaaa 720ccaactttga tcaaaatgac cacaaccatt
ggttacggtt ccttgcatgc cggctctcac 780tctgtgcacg gtgccccatt
gaaagcagat gatgttaaac aactaaagag caaattcggt 840ttcaacccag
acaagtcctt tgttgttcca caagaagttt acgaccacta ccaaaagaca
900attttaaagc caggtgtcga agccaacaac aagtggaaca agttgttcag
cgaataccaa 960aagaaattcc cagaattagg tgctgaattg gctagaagat
tgagcggcca actacccgca 1020aattgggaat ctaagttgcc aacttacacc
gccaaggact ctgccgtggc cactagaaaa 1080ttatcagaaa ctgttcttga
ggatgtttac aatcaattgc cagagttgat tggtggttct 1140gccgatttaa
caccttctaa cttgaccaga tggaaggaag cccttgactt ccaacctcct
1200tcttccggtt caggtaacta ctctggtaga tacattaggt acggtattag
agaacacgct 1260atgggtgcca taatgaacgg tatttcagct ttcggtgcca
actacaaacc atacggtggt 1320actttcttga acttcgtttc ttatgctgct
ggtgccgtta gattgtccgc tttgtctggc 1380cacccagtta tttgggttgc
tacacatgac tctatcggtg tcggtgaaga tggtccaaca 1440catcaaccta
ttgaaacttt agcacacttc agatccctac caaacattca agtttggaga
1500ccagctgatg gtaacgaagt ttctgccgcc tacaagaact ctttagaatc
caagcatact 1560ccaagtatca ttgctttgtc cagacaaaac ttgccacaat
tggaaggtag ctctattgaa 1620agcgcttcta agggtggtta cgtactacaa
gatgttgcta acccagatat tattttagtg 1680gctactggtt ccgaagtgtc
tttgagtgtt gaagctgcta agactttggc cgcaaagaac 1740atcaaggctc
gtgttgtttc tctaccagat ttcttcactt ttgacaaaca acccctagaa
1800tacagactat cagtcttacc agacaacgtt ccaatcatgt ctgttgaagt
tttggctacc 1860acatgttggg gcaaatacgc tcatcaatcc ttcggtattg
acagatttgg tgcctccggt 1920aaggcaccag aagtcttcaa gttcttcggt
ttcaccccag aaggtgttgc tgaaagagct 1980caaaagacca ttgcattcta
taagggtgac aagctaattt ctcctttgaa aaaagctttc 2040taa
2043500680PRTSaccharomyces cerevisiae 500Met Thr Gln Phe Thr Asp
Ile Asp Lys Leu Ala Val Ser Thr Ile Arg1 5 10 15Ile Leu Ala Val Asp
Thr Val Ser Lys Ala Asn Ser Gly His Pro Gly 20 25 30Ala Pro Leu Gly
Met Ala Pro Ala Ala His Val Leu Trp Ser Gln Met 35 40 45Arg Met Asn
Pro Thr Asn Pro Asp Trp Ile Asn Arg Asp Arg Phe Val 50 55 60Leu Ser
Asn Gly His Ala Val Ala Leu Leu Tyr Ser Met Leu His Leu65 70 75
80Thr Gly Tyr Asp Leu Ser Ile Glu Asp Leu Lys Gln Phe Arg Gln Leu
85 90 95Gly Ser Arg Thr Pro Gly His Pro Glu Phe Glu Leu Pro Gly Val
Glu 100 105 110Val Thr Thr Gly Pro Leu Gly Gln Gly Ile Ser Asn Ala
Val Gly Met 115 120 125Ala Met Ala Gln Ala Asn Leu Ala Ala Thr Tyr
Asn Lys Pro Gly Phe 130 135 140Thr Leu Ser Asp Asn Tyr Thr Tyr Val
Phe Leu Gly Asp Gly Cys Leu145 150 155 160Gln Glu Gly Ile Ser Ser
Glu Ala Ser Ser Leu Ala Gly His Leu Lys 165 170 175Leu Gly Asn Leu
Ile Ala Ile Tyr Asp Asp Asn Lys Ile Thr Ile Asp 180 185 190Gly Ala
Thr Ser Ile Ser Phe Asp Glu Asp Val Ala Lys Arg Tyr Glu 195 200
205Ala Tyr Gly Trp Glu Val Leu Tyr Val Glu Asn Gly Asn Glu Asp Leu
210 215 220Ala Gly Ile Ala Lys Ala Ile Ala Gln Ala Lys Leu Ser Lys
Asp Lys225 230 235 240Pro Thr Leu Ile Lys Met Thr Thr Thr Ile Gly
Tyr Gly Ser Leu His 245 250 255Ala Gly Ser His Ser Val His Gly Ala
Pro Leu Lys Ala Asp Asp Val 260 265 270Lys Gln Leu Lys Ser Lys Phe
Gly Phe Asn Pro Asp Lys Ser Phe Val 275 280 285Val Pro Gln Glu Val
Tyr Asp His Tyr Gln Lys Thr Ile Leu Lys Pro 290 295 300Gly Val Glu
Ala Asn Asn Lys Trp Asn Lys Leu Phe Ser Glu Tyr Gln305 310 315
320Lys Lys Phe Pro Glu Leu Gly Ala Glu Leu Ala Arg Arg Leu Ser Gly
325 330 335Gln Leu Pro Ala Asn Trp Glu Ser Lys Leu Pro Thr Tyr Thr
Ala Lys 340 345 350Asp Ser Ala Val Ala Thr Arg Lys Leu Ser Glu Thr
Val Leu Glu Asp 355 360 365Val Tyr Asn Gln Leu Pro Glu Leu Ile Gly
Gly Ser Ala Asp Leu Thr 370 375 380Pro Ser Asn Leu Thr Arg Trp Lys
Glu Ala Leu Asp Phe Gln Pro Pro385 390 395 400Ser Ser Gly Ser Gly
Asn Tyr Ser Gly Arg Tyr Ile Arg Tyr Gly Ile 405 410 415Arg Glu His
Ala Met Gly Ala Ile Met Asn Gly Ile Ser Ala Phe Gly 420 425 430Ala
Asn Tyr Lys Pro Tyr Gly Gly Thr Phe Leu Asn Phe Val Ser Tyr 435 440
445Ala Ala Gly Ala Val Arg Leu Ser Ala Leu Ser Gly His Pro Val Ile
450 455 460Trp Val Ala Thr His Asp Ser Ile Gly Val Gly Glu Asp Gly
Pro Thr465 470 475 480His Gln Pro Ile Glu Thr Leu Ala His Phe Arg
Ser Leu Pro Asn Ile 485 490 495Gln Val Trp Arg Pro Ala Asp Gly Asn
Glu Val Ser Ala Ala Tyr Lys 500 505 510Asn Ser Leu Glu Ser Lys His
Thr Pro Ser Ile Ile Ala Leu Ser Arg 515 520 525Gln Asn Leu Pro Gln
Leu Glu Gly Ser Ser Ile Glu Ser Ala Ser Lys 530 535 540Gly Gly Tyr
Val Leu Gln Asp Val Ala Asn Pro Asp Ile Ile Leu Val545 550 555
560Ala Thr Gly Ser Glu Val Ser Leu Ser Val Glu Ala Ala Lys Thr Leu
565 570 575Ala Ala Lys Asn Ile Lys Ala Arg Val Val Ser Leu Pro Asp
Phe Phe 580 585 590Thr Phe Asp Lys Gln Pro Leu Glu Tyr Arg Leu Ser
Val Leu Pro Asp 595 600 605Asn Val Pro Ile Met Ser Val Glu Val Leu
Ala Thr Thr Cys Trp Gly 610 615 620Lys Tyr Ala His Gln Ser Phe Gly
Ile Asp Arg Phe Gly Ala Ser Gly625 630 635 640Lys Ala Pro Glu Val
Phe Lys Phe Phe Gly Phe Thr Pro Glu Gly Val 645 650 655Ala Glu Arg
Ala Gln Lys Thr Ile Ala Phe Tyr Lys Gly Asp Lys Leu 660 665 670Ile
Ser Pro Leu Lys Lys Ala Phe 675 6805011830DNASaccharophagus
degradans 501atgaatagcg taatcgaagc tgtaactcag cgaattattg agcgcagtcg
acattctcgt 60caggcgtatt tgaatttaat gcgcaacacc atggagcagc atcctcctaa
aaagcgtcta 120tcttgcggca atttggctca tgcctatgca gcatgtggtc
aatccgataa gcaaacaatt 180cgtttaatgc aaagtgcaaa cataagtatt
actacggcat ttaacgatat gctttcggcg 240catcagcctt tagaaacata
ccctcaaata atcaaagaaa ctgcgcgtgc aatgggttca 300actgctcaag
ttgcaggcgg cgtgccggca atgtgtgatg gtgtaactca aggccagccc
360ggtatggagc tgagtttgtt tagccgcgaa gttgtagcaa tggctacagc
agtaggcctt 420tcgcacaata tgtttgatgg caatatgttt ttgggtgtat
gcgataaaat tgttcctggc 480atgctaattg gcgcgttgca gtttggtcat
attcctgggg tgtttgtgcc tgccggacca 540atgccttctg gtattcccaa
caaagaaaaa gcaaaagttc gtcagcaata tgcggcgggc 600attgtggggg
aagataagct tttagaaacc gagtcggctt cctatcacag tgcaggcacg
660tgtacttttt acggtacagc gaatacaaac caaatgatgg ttgaaatgtt
gggtgttcag 720ttgcctggct cgtcgtttgt ttaccccggt actgagttgc
gtgatgcctt aacgagagct 780gctgttgaaa agttggtaaa aatcacagat
tcagccggta actaccgtcc gctctacgaa 840gtcattacgg aaaaatccat
cgtcaattca ataattggtt tgttggctac cggcggttct 900actaaccaca
cgctacacat tgttgctgtg gctcgcgctg cgggtataga ggttacgtgg
960gcagatatgg acgagctttc gcgtgctgtg ccattacttg cacgtgttta
ccctaacggc 1020gaagctgatg ttaaccaatt ccagcaggct ggcggcatgg
cttatttagt aagagagctg 1080cgcagcggcg gtttgctaaa tgaagatgtg
gttactatta tgggtgaggg cctcgaggcc 1140tacgaaaaag agcccatgct
taacgataag gggcaggctg aatgggtaaa tgatgtacct 1200gttagccgcg
acgataccgt tgtgcgtcca gttacctcgc ctttcgataa agagggtggg
1260ttgcgtctac tcaagggtaa
cttagggcag ggcgtaatca aaatttctgc ggtagcgcca 1320gaaaatcgcg
ttgttgaggc cccatgtatt gtattcgagg cccaagaaga gctaatagct
1380gcgtttaagc gtggtgagct cgaaaaagac tttgttgcgg tagtgcgctt
ccaagggcct 1440tctgccaatg gcatgccaga acttcataaa atgaccccgc
ctttaggtgt gcttcaagat 1500aagggtttca aggtagcgtt agttaccgat
ggcagaatgt ctggtgcatc tggtaaagtg 1560ccggccggta tacacttgtc
gccagaagcg agtaagggtg gcctgttgaa taagctgcgc 1620acgggtgatg
tgattcgctt cgatgccgaa gcgggcgtta ttcaagcgct tgttagtgat
1680gaagagttag ctgcgcgtga gccagctgtg caaccggtcg tggagcagaa
cctcggacgc 1740tctctgtttg gtggtttgcg cgatttggct ggtgtatcgc
tacaaggcgg aacagttttc 1800gattttgaaa gagagtttgg cgaaaaatag
1830502609PRTSaccharophagus degradans 502Met Asn Ser Val Ile Glu
Ala Val Thr Gln Arg Ile Ile Glu Arg Ser1 5 10 15Arg His Ser Arg Gln
Ala Tyr Leu Asn Leu Met Arg Asn Thr Met Glu 20 25 30Gln His Pro Pro
Lys Lys Arg Leu Ser Cys Gly Asn Leu Ala His Ala 35 40 45Tyr Ala Ala
Cys Gly Gln Ser Asp Lys Gln Thr Ile Arg Leu Met Gln 50 55 60Ser Ala
Asn Ile Ser Ile Thr Thr Ala Phe Asn Asp Met Leu Ser Ala65 70 75
80His Gln Pro Leu Glu Thr Tyr Pro Gln Ile Ile Lys Glu Thr Ala Arg
85 90 95Ala Met Gly Ser Thr Ala Gln Val Ala Gly Gly Val Pro Ala Met
Cys 100 105 110Asp Gly Val Thr Gln Gly Gln Pro Gly Met Glu Leu Ser
Leu Phe Ser 115 120 125Arg Glu Val Val Ala Met Ala Thr Ala Val Gly
Leu Ser His Asn Met 130 135 140Phe Asp Gly Asn Met Phe Leu Gly Val
Cys Asp Lys Ile Val Pro Gly145 150 155 160Met Leu Ile Gly Ala Leu
Gln Phe Gly His Ile Pro Gly Val Phe Val 165 170 175Pro Ala Gly Pro
Met Pro Ser Gly Ile Pro Asn Lys Glu Lys Ala Lys 180 185 190Val Arg
Gln Gln Tyr Ala Ala Gly Ile Val Gly Glu Asp Lys Leu Leu 195 200
205Glu Thr Glu Ser Ala Ser Tyr His Ser Ala Gly Thr Cys Thr Phe Tyr
210 215 220Gly Thr Ala Asn Thr Asn Gln Met Met Val Glu Met Leu Gly
Val Gln225 230 235 240Leu Pro Gly Ser Ser Phe Val Tyr Pro Gly Thr
Glu Leu Arg Asp Ala 245 250 255Leu Thr Arg Ala Ala Val Glu Lys Leu
Val Lys Ile Thr Asp Ser Ala 260 265 270Gly Asn Tyr Arg Pro Leu Tyr
Glu Val Ile Thr Glu Lys Ser Ile Val 275 280 285Asn Ser Ile Ile Gly
Leu Leu Ala Thr Gly Gly Ser Thr Asn His Thr 290 295 300Leu His Ile
Val Ala Val Ala Arg Ala Ala Gly Ile Glu Val Thr Trp305 310 315
320Ala Asp Met Asp Glu Leu Ser Arg Ala Val Pro Leu Leu Ala Arg Val
325 330 335Tyr Pro Asn Gly Glu Ala Asp Val Asn Gln Phe Gln Gln Ala
Gly Gly 340 345 350Met Ala Tyr Leu Val Arg Glu Leu Arg Ser Gly Gly
Leu Leu Asn Glu 355 360 365Asp Val Val Thr Ile Met Gly Glu Gly Leu
Glu Ala Tyr Glu Lys Glu 370 375 380Pro Met Leu Asn Asp Lys Gly Gln
Ala Glu Trp Val Asn Asp Val Pro385 390 395 400Val Ser Arg Asp Asp
Thr Val Val Arg Pro Val Thr Ser Pro Phe Asp 405 410 415Lys Glu Gly
Gly Leu Arg Leu Leu Lys Gly Asn Leu Gly Gln Gly Val 420 425 430Ile
Lys Ile Ser Ala Val Ala Pro Glu Asn Arg Val Val Glu Ala Pro 435 440
445Cys Ile Val Phe Glu Ala Gln Glu Glu Leu Ile Ala Ala Phe Lys Arg
450 455 460Gly Glu Leu Glu Lys Asp Phe Val Ala Val Val Arg Phe Gln
Gly Pro465 470 475 480Ser Ala Asn Gly Met Pro Glu Leu His Lys Met
Thr Pro Pro Leu Gly 485 490 495Val Leu Gln Asp Lys Gly Phe Lys Val
Ala Leu Val Thr Asp Gly Arg 500 505 510Met Ser Gly Ala Ser Gly Lys
Val Pro Ala Gly Ile His Leu Ser Pro 515 520 525Glu Ala Ser Lys Gly
Gly Leu Leu Asn Lys Leu Arg Thr Gly Asp Val 530 535 540Ile Arg Phe
Asp Ala Glu Ala Gly Val Ile Gln Ala Leu Val Ser Asp545 550 555
560Glu Glu Leu Ala Ala Arg Glu Pro Ala Val Gln Pro Val Val Glu Gln
565 570 575Asn Leu Gly Arg Ser Leu Phe Gly Gly Leu Arg Asp Leu Ala
Gly Val 580 585 590Ser Leu Gln Gly Gly Thr Val Phe Asp Phe Glu Arg
Glu Phe Gly Glu 595 600 605Lys 5031917DNAXanthomonas axonopodis
503atgagcctgc atccgaatat ccaagccgtc accgaccgta tccgcaagcg
cagtgctccc 60tcgcgcgcgg cgtatctggc cggcctcgat gccgccctgc gtgagggccc
gttccgtagc 120cggttgagct gcggcaatct cgcgcatggc ttcgctgcgt
ccgagccggg cgacaaatcg 180cgcctgcgcg gtgcggccac gccgaacctg
ggcatcatca ctgcctataa cgacatgttg 240tcggcacatc agccgttcga
gcactacccg cagctgatcc gcgaaaccgc gcgctcactt 300ggcgccactg
cgcaggtggc cggcggcgtg ccggcgatgt gtgacggcgt gacccagggc
360cgcgccggca tggagctgtc gctgttctcg cgcgacaaca tcgctcaggc
tgcggccatt 420ggcctgagcc atgacatgtt cgacagcgtg gtgtacctgg
gggtgtgcga caagatcgtg 480ccgggtctgc tgatcggtgc gctggcgttt
ggccatttgc cggcgatctt catgccggct 540ggtccgatga ccccgggcat
cccgaacaag cagaaagccg aagtccgcga acgctacgcc 600gctggcgaag
ccacccgcgc cgaattgctg gaggccgaat cctcgtctta tcactcgccc
660ggcacctgca ccttttacgg cacggcgaac tccaaccagg tgttgctcga
agcgatgggc 720gtgcagttgc ccggcgcctc gttcgtcaat ccggagctgc
cgctgcgcga tgcactgacc 780cgcgaaggca ccgcacgcgc attggcgatc
tccgcgctgg gcgatgactt ccgcccgttc 840ggtcgtttga tcgacgaacg
ggccatcgtc aatgccgtgg tcgcgctgat ggcgaccggc 900ggttcgacca
accacaccat ccactggatc gcagtggcgc gtgcggccgg catcgtgttg
960acctgggacg acatggatct gatctcgcag accgtgccgc tgttgacacg
catctacccg 1020aacggcgaag ccgacgtgaa ccgcttccag gccgcaggcg
gcacggcgtt cgtgttccgc 1080gaattgatgg acgccggcta catgcacgac
gacctgccga ccatcgtcga aggcggcatg 1140cgcgcgtacg tcaacgaacc
gcgcctgcag gacggcaagg tgacctacgt gcccggcacc 1200gcgaccactg
ccgacgacag cgtcgcgcgt ccggtcagcg atgcattcga atcacaaggc
1260ggcctgcgcc tgctgcgcgg caacctcggc cgctcgttga tcaagctgtc
ggcggtcaag 1320ccgcagcacc gcagcatcca agcgccagcg gtggtgatcg
acaccccgca agtgctcaac 1380aaactgcatg cggcgggcgt actgccgcac
gatttcgtgg tggtactgcg ctatcagggc 1440ccacgcgcaa acggcatgcc
ggagctgcat tcgatggcgc cgctactggg cctgctgcag 1500aaccagggcc
ggcgcgtggc gttggtcacc gacggccgtc tgtccggcgc ctcgggcaag
1560ttcccggcgg cgatccacat gaccccggaa gccgcacgcg gcggcccgat
cgggcgcgta 1620cgcgaaggcg acatcgtgcg actggacggc gaagccggca
ccttggaagt gctggtttcg 1680gccgaagaat gggcatcgcg cgaggtcgca
ccgaacactg cgttggccgg caacgacctg 1740ggccgcaacc tgttcgccat
caaccgccag gtggttggcc cggccgacca gggcgcgatt 1800tccatttcct
gcggcccgac ccatccggac ggtgcgctgt ggagctacga cgccgagtac
1860gaactcggtg ccgatgcagc tgcagccgcc gcgccgcacg agtccaagga cgcctga
1917504638PRTXanthomonas axonopodis 504Met Ser Leu His Pro Asn Ile
Gln Ala Val Thr Asp Arg Ile Arg Lys1 5 10 15Arg Ser Ala Pro Ser Arg
Ala Ala Tyr Leu Ala Gly Ile Asp Ala Ala 20 25 30Leu Arg Glu Gly Pro
Phe Arg Ser Arg Leu Ser Cys Gly Asn Leu Ala 35 40 45His Gly Phe Ala
Ala Ser Glu Pro Thr Asp Lys Ser Arg Leu Arg Gly 50 55 60Ala Ala Thr
Pro Asn Leu Gly Ile Ile Thr Ala Tyr Asn Asp Met Leu65 70 75 80Ser
Ala His Gln Pro Phe Glu His Tyr Pro Gln Leu Ile Arg Glu Thr 85 90
95Ala Arg Ser Leu Gly Ala Thr Ala Gln Val Ala Gly Gly Val Pro Ala
100 105 110Met Cys Asp Gly Val Thr Gln Gly Arg Ala Gly Met Glu Leu
Ser Leu 115 120 125Phe Ser Arg Asp Asn Ile Ala Gln Ala Ala Ala Ile
Gly Leu Ser His 130 135 140Asp Met Phe Asp Ser Val Val Tyr Leu Gly
Val Cys Asp Lys Ile Val145 150 155 160Pro Gly Leu Leu Ile Gly Ala
Leu Ala Phe Gly His Leu Pro Ala Ile 165 170 175Phe Met Pro Ala Gly
Pro Met Thr Pro Gly Ile Pro Asn Lys Gln Lys 180 185 190Ala Glu Val
Arg Glu Arg Tyr Ala Ala Gly Glu Ala Thr Arg Ala Glu 195 200 205Leu
Leu Glu Ala Glu Ser Ser Ser Tyr His Ser Pro Gly Thr Cys Thr 210 215
220Phe Tyr Gly Thr Ala Asn Ser Asn Gln Val Leu Leu Glu Ala Met
Gly225 230 235 240Val Gln Leu Pro Gly Ala Ser Phe Val Asn Pro Glu
Leu Pro Leu Arg 245 250 255Asp Ala Leu Thr Arg Glu Gly Thr Ala Arg
Ala Leu Ala Ile Ser Ala 260 265 270Leu Gly Asp Asp Phe Arg Pro Phe
Gly Arg Leu Ile Asp Glu Arg Ala 275 280 285Ile Val Asn Ala Val Val
Ala Leu Met Ala Thr Gly Gly Ser Thr Asn 290 295 300His Thr Ile His
Trp Ile Ala Val Ala Arg Ala Ala Gly Ile Val Leu305 310 315 320Thr
Trp Asp Asp Met Asp Leu Ile Ser Gln Thr Val Pro Leu Leu Thr 325 330
335Arg Ile Tyr Pro Asn Gly Glu Ala Asp Val Asn Arg Phe Gln Ala Ala
340 345 350Gly Gly Thr Ala Phe Val Phe Arg Glu Leu Met Asp Ala Gly
Tyr Met 355 360 365His Asp Asp Leu Pro Thr Ile Val Glu Gly Gly Met
Arg Ala Tyr Val 370 375 380Asn Glu Pro Arg Leu Gln Asp Gly Lys Val
Thr Tyr Val Pro Gly Thr385 390 395 400Ala Thr Thr Ala Asp Asp Ser
Val Ala Arg Pro Val Ser Asp Ala Phe 405 410 415Glu Ser Gln Gly Gly
Leu Arg Leu Leu Arg Gly Asn Leu Gly Arg Ser 420 425 430Leu Ile Lys
Leu Ser Ala Val Lys Pro Gln His Arg Ser Ile Gln Ala 435 440 445Pro
Ala Val Val Ile Asp Thr Pro Gln Val Leu Asn Lys Leu His Ala 450 455
460Ala Gly Val Leu Pro His Asp Phe Val Val Val Leu Arg Tyr Gln
Gly465 470 475 480Pro Arg Ala Asn Gly Met Pro Glu Leu His Ser Met
Ala Pro Leu Leu 485 490 495Gly Leu Leu Gln Asn Gln Gly Arg Arg Val
Ala Leu Val Thr Asp Gly 500 505 510Arg Leu Ser Gly Ala Ser Gly Lys
Phe Pro Ala Ala Ile His Met Thr 515 520 525Pro Glu Ala Ala Arg Gly
Gly Pro Ile Gly Arg Val Arg Glu Gly Asp 530 535 540Ile Val Arg Leu
Asp Gly Glu Ala Gly Thr Leu Glu Val Leu Val Ser545 550 555 560Ala
Glu Glu Trp Ala Ser Arg Glu Val Ala Pro Asn Thr Ala Leu Ala 565 570
575Gly Asn Asp Leu Gly Arg Asn Leu Phe Ala Ile Asn Arg Gln Val Val
580 585 590Gly Pro Ala Asp Gln Gly Ala Ile Ser Ile Ser Cys Gly Pro
Thr His 595 600 605Pro Asp Gly Ala Leu Trp Ser Tyr Asp Ala Glu Tyr
Glu Leu Gly Ala 610 615 620Asp Ala Ala Ala Ala Ala Ala Pro His Glu
Ser Lys Asp Ala625 630 6355051827DNAPseudomonas syringae
505atgcatcccc gcgtccttga agtaaccgag cggctcattg ctcgcagtcg
cgatacccgt 60cagcgctacc ttcaattgat tcgaggcgca gcgagcgatg gcccgatgcg
cggcaagctt 120caatgtgcca actttgctca cggcgtcgcc gcctgcggac
cggaggacaa gcaaagcctg 180cgtttgatga acgccgccaa cgtggcaatc
gtctcttcct acaatgaaat gctctcggcg 240catcagccct acgagcactt
tcctgcacag atcaaacagg cgttacgtga cattggttcg 300gtcggtcagt
ttgccggcgg cgtgcctgcc atgtgcgatg gcgtgactca gggtgagccg
360ggcatggaac tggccattgc cagccgcgaa gtgattgcca tgtccacggc
aattgccttg 420tcacacaata tgttcgacgc cgccatgatg ctgggtatct
gcgacaagat cgtccccggc 480ctgatgatgg gggcgttgcg tttcggtcat
ctgccgacca tcttcgtgcc gggcgggccg 540atggtgtcag gtatctccaa
caaggaaaaa gccgacgtac ggcagcgtta cgctgaaggc 600aaggccagcc
gtgaagagct gctggactcg gaaatgaagt cctatcacgg cccgggaacc
660tgcacgttct acggcaccgc caacaccaat cagttggtga tggaagtcat
gggcatgcac 720cttcccggtg cctcgttcgt caatccctac acaccactgc
gtgatgcgct gacagctgaa 780gcggctcgtc aggtcacgcg tctgaccatg
caaagcggca gtttcatgcc gattggtgaa 840atcgtcgacg agcgctcgct
ggtcaattcc atcgttgcgc tgcacgccac cggcggctcg 900accaaccaca
cgctgcacat gccggcgatt gctcaggctg cgggtattca gctgacctgg
960caggacatgg ccgacctctc cgaagtggtg ccgaccctca gtcacgtcta
ccccaacggc 1020aaggccgaca tcaaccattt ccaggccgca ggcggcatgt
cgttcctgat tcgcgagctg 1080ctggcagccg gtctgctgca cgaaaacgtt
aacaccgtgg ccggttatgg cctgagccgc 1140tacaccaaag agccattcct
ggaggatggc aaactggtct ggcgtgaagg cccgctggac 1200agcctggatg
aaaacatcct gcgcccggtg gcgcgtccgt tctcccctga aggcggtttg
1260cgggtcatgg aaggcaacct gggtcgcggt gtcatgaaag tatcggccgt
tgcgctggag 1320catcagattg tcgaagcgcc agcccgagtg tttcaggatc
agaaggagct ggccgatgcg 1380ttcaaggccg gcgagctgga atgtgatttc
gtcgccgtca tgcgttttca gggcccgcgc 1440tgcaacggca tgcccgaact
gcacaagatg accccgtttc tgggcgtgct gcaggatcgt 1500ggtttcaaag
tggcgctggt caccgatgga cggatgtcgg gcgcctcagg caagattccg
1560gcggcgattc acgtctgccc ggaagcgttc gatggtggcc cgttggcact
ggtacgcgac 1620ggcgatgtga tccgcgtgga tggcgtaaaa ggcacgttac
aagtgctggt cgaagcgtca 1680gaattggccg cccgagaacc ggccatcaac
cagatcgaca acagtgtcgg ctgcggtcgc 1740gagctttttg gattcatgcg
catggccttc agctccgcag agcaaggcgc cagcgccttt 1800acctctagtc
tggagacgct caagtga 1827506608PRTPseudomonas syringae 506Met His Pro
Arg Val Leu Glu Val Thr Glu Arg Leu Ile Ala Arg Ser1 5 10 15Arg Asp
Thr Arg Gln Arg Tyr Leu Gln Leu Ile Arg Gly Ala Ala Ser 20 25 30Asp
Gly Pro Met Arg Gly Lys Leu Gln Cys Ala Asn Phe Ala His Gly 35 40
45Val Ala Ala Cys Gly Pro Glu Asp Lys Gln Ser Leu Arg Leu Met Asn
50 55 60Ala Ala Asn Val Ala Ile Val Ser Ser Tyr Asn Glu Met Leu Ser
Ala65 70 75 80His Gln Pro Tyr Glu His Phe Pro Ala Gln Ile Lys Gln
Ala Leu Arg 85 90 95Asp Ile Gly Ser Val Gly Gln Phe Ala Gly Gly Val
Pro Ala Met Cys 100 105 110Asp Gly Val Thr Gln Gly Glu Pro Gly Met
Glu Leu Ala Ile Ala Ser 115 120 125Arg Glu Val Ile Ala Met Ser Thr
Ala Ile Ala Leu Ser His Asn Met 130 135 140Phe Asp Ala Ala Met Met
Leu Gly Ile Cys Asp Lys Ile Val Pro Gly145 150 155 160Leu Met Met
Gly Ala Leu Arg Phe Gly His Leu Pro Thr Ile Phe Val 165 170 175Pro
Gly Gly Pro Met Val Ser Gly Ile Ser Asn Lys Glu Lys Ala Asp 180 185
190Val Arg Gln Arg Tyr Ala Glu Gly Lys Ala Ser Arg Glu Glu Leu Leu
195 200 205Asp Ser Glu Met Lys Ser Tyr His Gly Pro Gly Thr Cys Thr
Phe Tyr 210 215 220Gly Thr Ala Asn Thr Asn Gln Leu Val Met Glu Val
Met Gly Met His225 230 235 240Leu Pro Gly Ala Ser Phe Val Asn Pro
Tyr Thr Pro Leu Arg Asp Ala 245 250 255Leu Thr Ala Glu Ala Ala Arg
Gln Val Thr Arg Leu Thr Met Gln Ser 260 265 270Gly Ser Phe Met Pro
Ile Gly Glu Ile Val Asp Glu Arg Ser Leu Val 275 280 285Asn Ser Ile
Val Ala Leu His Ala Thr Gly Gly Ser Thr Asn His Thr 290 295 300Leu
His Met Pro Ala Ile Ala Gln Ala Ala Gly Ile Gln Leu Thr Trp305 310
315 320Gln Asp Met Ala Asp Leu Ser Glu Val Val Pro Thr Leu Ser His
Val 325 330 335Tyr Pro Asn Gly Lys Ala Asp Ile Asn His Phe Gln Ala
Ala Gly Gly 340 345 350Met Ser Phe Leu Ile Arg Glu Leu Leu Ala Ala
Gly Leu Leu His Glu 355 360 365Asn Val Asn Thr Val Ala Gly Tyr Gly
Leu Ser Arg Tyr Thr Lys Glu 370 375 380Pro Phe Leu Glu Asp Gly Lys
Leu Val Trp Arg Glu Gly Pro Leu Asp385 390 395 400Ser Leu Asp Glu
Asn Ile Leu Arg Pro Val Ala Arg Pro Phe Ser Pro 405 410 415Glu Gly
Gly Leu Arg Val Met Glu Gly Asn Leu Gly Arg Gly Val Met 420 425
430Lys Val Ser Ala Val Ala Leu Glu His Gln Ile Val Glu Ala Pro Ala
435 440 445Arg Val Phe Gln Asp Gln Lys Glu Leu Ala Asp Ala Phe Lys
Ala Gly 450 455 460Glu Leu Glu Cys Asp Phe Val Ala Val Met Arg Phe
Gln Gly Pro Arg465 470 475 480Cys Asn Gly Met Pro Glu Leu His Lys
Met Thr Pro Phe Leu Gly Val
485 490 495Leu Gln Asp Arg Gly Phe Lys Val Ala Leu Val Thr Asp Gly
Arg Met 500 505 510Ser Gly Ala Ser Gly Lys Ile Pro Ala Ala Ile His
Val Cys Pro Glu 515 520 525Ala Phe Asp Gly Gly Pro Leu Ala Leu Val
Arg Asp Gly Asp Val Ile 530 535 540Arg Val Asp Gly Val Lys Gly Thr
Leu Gln Val Leu Val Glu Ala Ser545 550 555 560Glu Leu Ala Ala Arg
Glu Pro Ala Ile Asn Gln Ile Asp Asn Ser Val 565 570 575Gly Cys Gly
Arg Glu Leu Phe Gly Phe Met Arg Met Ala Phe Ser Ser 580 585 590Ala
Glu Gln Gly Ala Ser Ala Phe Thr Ser Ser Leu Glu Thr Leu Lys 595 600
6055071827DNAPseudomonas fluorescens 507atgcatcccc gcgttcttga
ggtcaccgaa cggcttatcg cccgtagtcg cgccactcgc 60caggcctatc tcgcgctgat
ccgcgatgcc gccagcgacg gcccgcagcg gggcaagctg 120caatgtgcga
acttcgccca cggcgtggcc ggttgcggca ccgacgacaa gcacaacctg
180cggatgatga atgcggccaa cgtggcaatt gtttcgtcat ataacgacat
gttgtcggcg 240caccagcctt acgaggtgtt ccccgagcag atcaagcgcg
ccctgcgcga gatcggctcg 300gtgggccagt tcgccggcgg caccccggcc
atgtgcgatg gcgtgaccca gggcgaggcc 360ggtatggaac tgagcctgcc
gagccgtgaa gtgatcgccc tgtctacggc ggtggccctc 420tctcacaaca
tgttcgatgc cgcgctgatg ctggggatct gcgacaagat tgtcccgggg
480ttgatgatgg gcgctctgcg cttcggtcac ctgccgacca tcttcgttcc
gggcgggccc 540atggtctcgg gcatttccaa caagcagaaa gccgacgtgc
gccagcgtta cgccgaaggc 600aaggccagcc gcgaggaact gctggagtcg
gaaatgaagt cctaccacag ccccggcacc 660tgcactttct acggcaccgc
caacaccaac cagttgctga tggaagtgat gggcctgcac 720ctgccgggcg
cctctttcgt caaccccaat acgccgctgc gcgacgccct gacccatgag
780gcggcgcagc aggtcacgcg cctgaccaag cagagcgggg ccttcatgcc
gattggcgag 840atcgtcgacg agcgcgtgct ggtcaactcc atcgttgccc
tgcacgccac gggcggctcc 900accaaccaca ccctgcacat gccggccatc
gcccaggcgg cgggcatcca gctgacctgg 960caggacatgg ccgacctctc
cgaggtggtg ccgaccctgt cccacgtcta tccaaacggc 1020aaggccgata
tcaaccactt ccaggcggcg ggcggcatgt ctttcctgat ccgcgagctg
1080ctggaagccg gcctgctcca cgaagacgtc aataccgtgg ccggccgcgg
cctgagccgc 1140tatacccagg aacccttcct ggacaacggc aagctggtgt
ggcgcgacgg cccgattgaa 1200agcctggacg aaaacatcct gcgcccggtg
gcccgggcgt tctctgcgga gggcggcttg 1260cgggtcatgg aaggcaacct
cggtcgcggc gtgatgaagg tttccgccgt ggccccggag 1320caccagatcg
tcgaggcccc ggccgtggtg ttccaggacc agcaggacct ggccgatgcc
1380ttcaaggccg gcctgctgga gaaggacttc gtcgcggtga tgcgcttcca
gggcccgcgc 1440tccaacggca tgcccgagct gcacaagatg acccccttcc
tcggggtgct gcaggaccgc 1500ggcttcaagg tggcgctggt caccgacggg
cgcatgtccg gcgcttcggg caagattccg 1560gcagcgatcc atgtcagccc
cgaagcccag gtgggtggcg cgctggcccg ggtgctggac 1620ggcgatatca
tccgagtgga tggcgtcaag ggcaccctgg agcttaaggt agacgccgca
1680gaattcgccg cccgggagcc ggccaagggc ctgctgggca acaacgttgg
caccggccgc 1740gaactcttcg ccttcatgcg catggccttc agctcggcag
agcagggcgc cagcgccttt 1800acctctgccc tggagacgct caagtga
1827508608PRTPseudomonas fluorescens 508Met His Pro Arg Val Leu Glu
Val Thr Glu Arg Leu Ile Ala Arg Ser1 5 10 15Arg Ala Thr Arg Gln Ala
Tyr Leu Ala Leu Ile Arg Asp Ala Ala Ser 20 25 30Asp Gly Pro Gln Arg
Gly Lys Leu Gln Cys Ala Asn Phe Ala His Gly 35 40 45Val Ala Gly Cys
Gly Thr Asp Asp Lys His Asn Leu Arg Met Met Asn 50 55 60Ala Ala Asn
Val Ala Ile Val Ser Ser Tyr Asn Asp Met Leu Ser Ala65 70 75 80His
Gln Pro Tyr Glu Val Phe Pro Glu Gln Ile Lys Arg Ala Leu Arg 85 90
95Glu Ile Gly Ser Val Gly Gln Phe Ala Gly Gly Thr Pro Ala Met Cys
100 105 110Asp Gly Val Thr Gln Gly Glu Ala Gly Met Glu Leu Ser Leu
Pro Ser 115 120 125Arg Glu Val Ile Ala Leu Ser Thr Ala Val Ala Leu
Ser His Asn Met 130 135 140Phe Asp Ala Ala Leu Met Leu Gly Ile Cys
Asp Lys Ile Val Pro Gly145 150 155 160Leu Met Met Gly Ala Leu Arg
Phe Gly His Leu Pro Thr Ile Phe Val 165 170 175Pro Gly Gly Pro Met
Val Ser Gly Ile Ser Asn Lys Gln Lys Ala Asp 180 185 190Val Arg Gln
Arg Tyr Ala Glu Gly Lys Ala Ser Arg Glu Glu Leu Leu 195 200 205Glu
Ser Glu Met Lys Ser Tyr His Ser Pro Gly Thr Cys Thr Phe Tyr 210 215
220Gly Thr Ala Asn Thr Asn Gln Leu Leu Met Glu Val Met Gly Leu
His225 230 235 240Leu Pro Gly Ala Ser Phe Val Asn Pro Asn Thr Pro
Leu Arg Asp Ala 245 250 255Leu Thr His Glu Ala Ala Gln Gln Val Thr
Arg Leu Thr Lys Gln Ser 260 265 270Gly Ala Phe Met Pro Ile Gly Glu
Ile Val Asp Glu Arg Val Leu Val 275 280 285Asn Ser Ile Val Ala Leu
His Ala Thr Gly Gly Ser Thr Asn His Thr 290 295 300Leu His Met Pro
Ala Ile Ala Gln Ala Ala Gly Ile Gln Leu Thr Trp305 310 315 320Gln
Asp Met Ala Asp Leu Ser Glu Val Val Pro Thr Leu Ser His Val 325 330
335Tyr Pro Asn Gly Lys Ala Asp Ile Asn His Phe Gln Ala Ala Gly Gly
340 345 350Met Ser Phe Leu Ile Arg Glu Leu Leu Glu Ala Gly Leu Leu
His Glu 355 360 365Asp Val Asn Thr Val Ala Gly Arg Gly Leu Ser Arg
Tyr Thr Gln Glu 370 375 380Pro Phe Leu Asp Asn Gly Lys Leu Val Trp
Arg Asp Gly Pro Ile Glu385 390 395 400Ser Leu Asp Glu Asn Ile Leu
Arg Pro Val Ala Arg Ala Phe Ser Ala 405 410 415Glu Gly Gly Leu Arg
Val Met Glu Gly Asn Leu Gly Arg Gly Val Met 420 425 430Lys Val Ser
Ala Val Ala Pro Glu His Gln Ile Val Glu Ala Pro Ala 435 440 445Val
Val Phe Gln Asp Gln Gln Asp Leu Ala Asp Ala Phe Lys Ala Gly 450 455
460Leu Leu Glu Lys Asp Phe Val Ala Val Met Arg Phe Gln Gly Pro
Arg465 470 475 480Ser Asn Gly Met Pro Glu Leu His Lys Met Thr Pro
Phe Leu Gly Val 485 490 495Leu Gln Asp Arg Gly Phe Lys Val Ala Leu
Val Thr Asp Gly Arg Met 500 505 510Ser Gly Ala Ser Gly Lys Ile Pro
Ala Ala Ile His Val Ser Pro Glu 515 520 525Ala Gln Val Gly Gly Ala
Leu Ala Arg Val Leu Asp Gly Asp Ile Ile 530 535 540Arg Val Asp Gly
Val Lys Gly Thr Leu Glu Leu Lys Val Asp Ala Ala545 550 555 560Glu
Phe Ala Ala Arg Glu Pro Ala Lys Gly Leu Leu Gly Asn Asn Val 565 570
575Gly Thr Gly Arg Glu Leu Phe Ala Phe Met Arg Met Ala Phe Ser Ser
580 585 590Ala Glu Gln Gly Ala Ser Ala Phe Thr Ser Ala Leu Glu Thr
Leu Lys 595 600 6055091677DNABacillus subtilis 509atggcagaat
tacgcagtaa tatgatcaca caaggaatcg atagagctcc gcaccgcagt 60ttgcttcgtg
cagcaggggt aaaagaagag gatttcggca agccgtttat tgcggtgtgt
120aattcataca ttgatatcgt tcccggtcat gttcacttgc aggagtttgg
gaaaatcgta 180aaagaagcaa tcagagaagc agggggcgtt ccgtttgaat
ttaataccat tggggtagat 240gatggcatcg caatggggca tatcggtatg
agatattcgc tgccaagccg tgaaattatc 300gcagactctg tggaaacggt
tgtatccgca cactggtttg acggaatggt ctgtattccg 360aactgcgaca
aaatcacacc gggaatgctt atggcggcaa tgcgcatcaa cattccgacg
420atttttgtca gcggcggacc gatggcggca ggaagaacaa gttacgggcg
aaaaatctcc 480ctttcctcag tattcgaagg ggtaggcgcc taccaagcag
ggaaaatcaa cgaaaacgag 540cttcaagaac tagagcagtt cggatgccca
acgtgcgggt cttgctcagg catgtttacg 600gcgaactcaa tgaactgtct
gtcagaagca cttggtcttg ctttgccggg taatggaacc 660attctggcaa
catctccgga acgcaaagag tttgtgagaa aatcggctgc gcaattaatg
720gaaacgattc gcaaagatat caaaccgcgt gatattgtta cagtaaaagc
gattgataac 780gcgtttgcac tcgatatggc gctcggaggt tctacaaata
ccgttcttca tacccttgcc 840cttgcaaacg aagccggcgt tgaatactct
ttagaacgca ttaacgaagt cgctgagcgc 900gtgccgcact tggctaagct
ggcgcctgca tcggatgtgt ttattgaaga tcttcacgaa 960gcgggcggcg
tttcagcggc tctgaatgag ctttcgaaga aagaaggagc gcttcattta
1020gatgcgctga ctgttacagg aaaaactctt ggagaaacca ttgccggaca
tgaagtaaag 1080gattatgacg tcattcaccc gctggatcaa ccattcactg
aaaagggagg ccttgctgtt 1140ttattcggta atctagctcc ggacggcgct
atcattaaaa caggcggcgt acagaatggg 1200attacaagac acgaagggcc
ggctgtcgta ttcgattctc aggacgaggc gcttgacggc 1260attatcaacc
gaaaagtaaa agaaggcgac gttgtcatca tcagatacga agggccaaaa
1320ggcggacctg gcatgccgga aatgctggcg ccaacatccc aaatcgttgg
aatgggactc 1380gggccaaaag tggcattgat tacggacgga cgtttttccg
gagcctcccg tggcctctca 1440atcggccacg tatcacctga ggccgctgag
ggcgggccgc ttgcctttgt tgaaaacgga 1500gaccatatta tcgttgatat
tgaaaaacgc atcttggatg tacaagtgcc agaagaagag 1560tgggaaaaac
gaaaagcgaa ctggaaaggt tttgaaccga aagtgaaaac cggctacctg
1620gcacgttatt ctaaacttgt gacaagtgcc aacaccggcg gtattatgaa aatctag
1677510558PRTBacillus subtilis 510Met Ala Glu Leu Arg Ser Asn Met
Ile Thr Gln Gly Ile Asp Arg Ala1 5 10 15Pro His Arg Ser Leu Leu Arg
Ala Ala Gly Val Lys Glu Glu Asp Phe 20 25 30Gly Lys Pro Phe Ile Ala
Val Cys Asn Ser Tyr Ile Asp Ile Val Pro 35 40 45Gly His Val His Leu
Gln Glu Phe Gly Lys Ile Val Lys Glu Ala Ile 50 55 60Arg Glu Ala Gly
Gly Val Pro Phe Glu Phe Asn Thr Ile Gly Val Asp65 70 75 80Asp Gly
Ile Ala Met Gly His Ile Gly Met Arg Tyr Ser Leu Pro Ser 85 90 95Arg
Glu Ile Ile Ala Asp Ser Val Glu Thr Val Val Ser Ala His Trp 100 105
110Phe Asp Gly Met Val Cys Ile Pro Asn Cys Asp Lys Ile Thr Pro Gly
115 120 125Met Leu Met Ala Ala Met Arg Ile Asn Ile Pro Thr Ile Phe
Val Ser 130 135 140Gly Gly Pro Met Ala Ala Gly Arg Thr Ser Tyr Gly
Arg Lys Ile Ser145 150 155 160Leu Ser Ser Val Phe Glu Gly Val Gly
Ala Tyr Gln Ala Gly Lys Ile 165 170 175Asn Glu Asn Glu Leu Gln Glu
Leu Glu Gln Phe Gly Cys Pro Thr Cys 180 185 190Gly Ser Cys Ser Gly
Met Phe Thr Ala Asn Ser Met Asn Cys Leu Ser 195 200 205Glu Ala Leu
Gly Leu Ala Leu Pro Gly Asn Gly Thr Ile Leu Ala Thr 210 215 220Ser
Pro Glu Arg Lys Glu Phe Val Arg Lys Ser Ala Ala Gln Leu Met225 230
235 240Glu Thr Ile Arg Lys Asp Ile Lys Pro Arg Asp Ile Val Thr Val
Lys 245 250 255Ala Ile Asp Asn Ala Phe Ala Leu Asp Met Ala Leu Gly
Gly Ser Thr 260 265 270Asn Thr Val Leu His Thr Leu Ala Leu Ala Asn
Glu Ala Gly Val Glu 275 280 285Tyr Ser Leu Glu Arg Ile Asn Glu Val
Ala Glu Arg Val Pro His Leu 290 295 300Ala Lys Leu Ala Pro Ala Ser
Asp Val Phe Ile Glu Asp Leu His Glu305 310 315 320Ala Gly Gly Val
Ser Ala Ala Leu Asn Glu Leu Ser Lys Lys Glu Gly 325 330 335Ala Leu
His Leu Asp Ala Leu Thr Val Thr Gly Lys Thr Leu Gly Glu 340 345
350Thr Ile Ala Gly His Glu Val Lys Asp Tyr Asp Val Ile His Pro Leu
355 360 365Asp Gln Pro Phe Thr Glu Lys Gly Gly Leu Ala Val Leu Phe
Gly Asn 370 375 380Leu Ala Pro Asp Gly Ala Ile Ile Lys Thr Gly Gly
Val Gln Asn Gly385 390 395 400Ile Thr Arg His Glu Gly Pro Ala Val
Val Phe Asp Ser Gln Asp Glu 405 410 415Ala Leu Asp Gly Ile Ile Asn
Arg Lys Val Lys Glu Gly Asp Val Val 420 425 430Ile Ile Arg Tyr Glu
Gly Pro Lys Gly Gly Pro Gly Met Pro Glu Met 435 440 445Leu Ala Pro
Thr Ser Gln Ile Val Gly Met Gly Leu Gly Pro Lys Val 450 455 460Ala
Leu Ile Thr Asp Gly Arg Phe Ser Gly Ala Ser Arg Gly Leu Ser465 470
475 480Ile Gly His Val Ser Pro Glu Ala Ala Glu Gly Gly Pro Leu Ala
Phe 485 490 495Val Glu Asn Gly Asp His Ile Ile Val Asp Ile Glu Lys
Arg Ile Leu 500 505 510Asp Val Gln Val Pro Glu Glu Glu Trp Glu Lys
Arg Lys Ala Asn Trp 515 520 525Lys Gly Phe Glu Pro Lys Val Lys Thr
Gly Tyr Leu Ala Arg Tyr Ser 530 535 540Lys Leu Val Thr Ser Ala Asn
Thr Gly Gly Ile Met Lys Ile545 550 5555111677DNABacillus
licheniformis 511atgacaggtt tacgcagtga catgattaca aaagggatcg
acagagcgcc gcaccgcagt 60ttgctgcgcg cggctggggt aaaagaagag gacttcggca
aaccgtttat tgccgtttgc 120aactcataca tcgatatcgt accgggtcat
gtccatttgc aggagtttgg aaaaatcgtc 180aaagaggcga tcagagaggc
cggcggtgtt ccgtttgaat ttaatacaat cggggtcgac 240gacggaattg
cgatggggca catcggaatg aggtattctc tcccgagccg cgaaatcatc
300gcagattcag tggaaacggt tgtatcggcg cactggtttg acggaatggt
atgtattcca 360aactgtgata aaatcacacc gggcatgatc atggcggcaa
tgcggatcaa cattccgacc 420gtgtttgtca gcggggggcc gatggaagcg
ggaagaacga gcgacggacg aaaaatctcg 480ctttcctctg tatttgaagg
cgttggcgct tatcaatcag gcaaaatcga tgagaaagga 540ctcgaggagc
ttgaacagtt cggctgtccg acttgcggat catgctcggg catgtttacg
600gcgaactcga tgaactgtct ttctgaagct cttggcatcg ccatgccggg
caacggcacc 660attttggcga catcgcccga ccgcagggaa tttgccaaac
agtcggcccg ccagctgatg 720gagctgatca agtcggatat caaaccgcgc
gacatcgtga ccgaaaaagc gatcgacaac 780gcgttcgctt tagacatggc
gctcggcgga tcaacgaata cgatccttca tacgcttgcg 840atcgccaatg
aagcgggtgt agactattcg cttgaacgga tcaatgaggt agcggcaagg
900gttccgcatt tatcgaagct tgcaccggct tccgatgtgt ttattgaaga
tttgcatgaa 960gcaggaggcg tatcggcagt cttaaacgag ctgtcgaaaa
aagaaggcgc gcttcacttg 1020gatacgctga ctgtaacggg gaaaacgctt
ggcgaaaata ttgccggacg cgaagtgaaa 1080gattacgagg tcattcatcc
gatcgatcag ccgttttcag agcaaggcgg actcgccgtc 1140ctgttcggca
acctggctcc tgacggtgcg atcattaaaa cgggcggcgt ccaagacggg
1200attacccgcc atgaaggacc tgcggttgtc tttgattcac aggaagaagc
gcttgacggc 1260atcatcaacc gtaaagtaaa agcgggagat gtcgtcatca
tccgctatga aggccctaaa 1320ggcggaccgg gaatgcctga aatgcttgcg
ccgacttcac agatcgtcgg aatgggcctc 1380ggcccgaaag tcgccttgat
taccgacggc cgcttttcag gagcctcccg cggtctttcg 1440atcggccacg
tttcaccgga agcagccgaa ggcggcccgc ttgctttcgt agaaaacggc
1500gaccatatcg ttgtcgatat cgaaaagcgg attttaaaca tcgaaatctc
cgatgaggaa 1560tgggaaaaaa gaaaagcaaa ctggcccggc tttgaaccga
aagtgaaaac gggctatctc 1620gccaggtatt caaagcttgt gacatctgcc
aataccggcg gcattatgaa aatctag 1677512558PRTBacillus licheniformis
512Met Thr Gly Leu Arg Ser Asp Met Ile Thr Lys Gly Ile Asp Arg Ala1
5 10 15Pro His Arg Ser Leu Leu Arg Ala Ala Gly Val Lys Glu Glu Asp
Phe 20 25 30Gly Lys Pro Phe Ile Ala Val Cys Asn Ser Tyr Ile Asp Ile
Val Pro 35 40 45Gly His Val His Leu Gln Glu Phe Gly Lys Ile Val Lys
Glu Ala Ile 50 55 60Arg Glu Ala Gly Gly Val Pro Phe Glu Phe Asn Thr
Ile Gly Val Asp65 70 75 80Asp Gly Ile Ala Met Gly His Ile Gly Met
Arg Tyr Ser Leu Pro Ser 85 90 95Arg Glu Ile Ile Ala Asp Ser Val Glu
Thr Val Val Ser Ala His Trp 100 105 110Phe Asp Gly Met Val Cys Ile
Pro Asn Cys Asp Lys Ile Thr Pro Gly 115 120 125Met Ile Met Ala Ala
Met Arg Ile Asn Ile Pro Thr Val Phe Val Ser 130 135 140Gly Gly Pro
Met Glu Ala Gly Arg Thr Ser Asp Gly Arg Lys Ile Ser145 150 155
160Leu Ser Ser Val Phe Glu Gly Val Gly Ala Tyr Gln Ser Gly Lys Ile
165 170 175Asp Glu Lys Gly Leu Glu Glu Leu Glu Gln Phe Gly Cys Pro
Thr Cys 180 185 190Gly Ser Cys Ser Gly Met Phe Thr Ala Asn Ser Met
Asn Cys Leu Ser 195 200 205Glu Ala Leu Gly Ile Ala Met Pro Gly Asn
Gly Thr Ile Leu Ala Thr 210 215 220Ser Pro Asp Arg Arg Glu Phe Ala
Lys Gln Ser Ala Arg Gln Leu Met225 230 235 240Glu Leu Ile Lys Ser
Asp Ile Lys Pro Arg Asp Ile Val Thr Glu Lys 245 250 255Ala Ile Asp
Asn Ala Phe Ala Leu Asp Met Ala Leu Gly Gly Ser Thr 260 265 270Asn
Thr Ile Leu His Thr Leu Ala Ile Ala Asn Glu Ala Gly Val Asp 275 280
285Tyr Ser Leu Glu Arg Ile Asn Glu Val Ala Ala Arg Val Pro His Leu
290 295 300Ser Lys Leu Ala Pro Ala Ser Asp
Val Phe Ile Glu Asp Leu His Glu305 310 315 320Ala Gly Gly Val Ser
Ala Val Leu Asn Glu Leu Ser Lys Lys Glu Gly 325 330 335Ala Leu His
Leu Asp Thr Leu Thr Val Thr Gly Lys Thr Leu Gly Glu 340 345 350Asn
Ile Ala Gly Arg Glu Val Lys Asp Tyr Glu Val Ile His Pro Ile 355 360
365Asp Gln Pro Phe Ser Glu Gln Gly Gly Leu Ala Val Leu Phe Gly Asn
370 375 380Leu Ala Pro Asp Gly Ala Ile Ile Lys Thr Gly Gly Val Gln
Asp Gly385 390 395 400Ile Thr Arg His Glu Gly Pro Ala Val Val Phe
Asp Ser Gln Glu Glu 405 410 415Ala Leu Asp Gly Ile Ile Asn Arg Lys
Val Lys Ala Gly Asp Val Val 420 425 430Ile Ile Arg Tyr Glu Gly Pro
Lys Gly Gly Pro Gly Met Pro Glu Met 435 440 445Leu Ala Pro Thr Ser
Gln Ile Val Gly Met Gly Leu Gly Pro Lys Val 450 455 460Ala Leu Ile
Thr Asp Gly Arg Phe Ser Gly Ala Ser Arg Gly Leu Ser465 470 475
480Ile Gly His Val Ser Pro Glu Ala Ala Glu Gly Gly Pro Leu Ala Phe
485 490 495Val Glu Asn Gly Asp His Ile Val Val Asp Ile Glu Lys Arg
Ile Leu 500 505 510Asn Ile Glu Ile Ser Asp Glu Glu Trp Glu Lys Arg
Lys Ala Asn Trp 515 520 525Pro Gly Phe Glu Pro Lys Val Lys Thr Gly
Tyr Leu Ala Arg Tyr Ser 530 535 540Lys Leu Val Thr Ser Ala Asn Thr
Gly Gly Ile Met Lys Ile545 550 555513201DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
513cacgcacgga ccgaccgtca ccggaccgtt tcgcgcgacg tgcgcgaggc
tccgacacga 60aagacgggcc ccctattgcg ctcatgtcgg ccgcacccct gcgtaaagtc
agatacgtgc 120gccacccgag ccgggaccgc cctgagcgca tggtccgggc
ggcgtggcaa gcgcaggagg 180gcgtgccccg ttcgctaggc a
201514201DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 514acgtatgtcg gctgatcgta cacgccgacc
agcgcagtcg gcgtactcag gcgttccgag 60tagctcacat ctgtgggccc cggcgtacct
tcggcagggt tatgcgacgg ggcggcaggc 120ttgcgctggc gtcgggaatc
accgcgaact tgacccgcgc cggttccgta tcggtccgct 180gcggccgtgc
tccgcagtcg a 201515201DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 515tgcagtccgc
ccagccggcc gtgtagcacg gccgactgca ggtgcgacgt gctaggggcc 60agcacgcgag
cggccctacc acgggtcgtg tggggcgcat gaccgccggc cgggtctcgg
120cacggggcga cgcggtgctc ctaggctagc agggcctcac cgggtgatcc
cccgtgtagc 180gccgcacaac accccctgcg a 201516201DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
516tgcccgcata ccgcccgccc actggggatc ctccggcgct gtcgcgctat
gcgcgtccat 60cctggtcgga cgggctcggg ccccggacca aaccgcagcg gcccctggca
gcgactaagg 120gcgccgtctc accctagact tcttaatcgg ggtgtcccgg
taggccggga gtagcctcgg 180cgggctagcc gcgtgactat a
201517201DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 517gcgggttagt ccccgtcgga cgtcatgcat
acagtcgggg ctggcgagac aggaggctac 60agggggcgcc cggaggaaca cacgtgggac
taagacgtcg gtccgtgtgc ccccgaaccg 120gcgtgctcat cgtaggactg
ggaagtccgt accgcgtggc tcgtacctcg cggtctgagt 180ccgacacccg
ctgacgccgg a 201518201DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 518ctgagacgac
tcccgcacta cggatcgcga gcgtagactc agcccggact ctcacgcgac 60ctcggacgcg
gcctaatgtc tcgactcgcg gtccgctgaa ggtctcgggg cacgcgagac
120gcggggtcag gccgggggga tccccgcaca cactcagtcg cggcgaacgg
agtcccgtgg 180cctggctagg gatcgtgggt a 201519201DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
519ggggcgtcca ctctggctcg gtagagcgct gggctccgcg cgactgcgcg
cacccatcgg 60tttggcgcga cgcaccgtgg actcctgggc tagagggcgg gtccccgcca
taccccgttc 120tcgtgccggc tgggtaggac cggagtgacg gctgtggccg
gcgactcggg cgcgcactgt 180agtcggatct gggcgggcag a
201520201DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 520gtcgggcgcg cgtcagtcca cgcgttaaac
actggccgac gacacgacgg gatccgggca 60cgccccgaga gcgcgtgttc gcgcgagtcg
atcgggaggc cgcagcgtgt cgagcccaga 120ccccgctcta gcgtggccat
cgcggtgcta agtggggcgg ccgggtccta tacacgctta 180ccgatagtca
agtttgcgtg a 201521201DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 521gtcttagggc
ccagggaccg cacgggtcga ccgcgcgact ggtcggagct tgcgcgtcta 60cgccactcgg
cggccccgac gggggatgcc gcggaatgtc cgccggcgta tgcggctcaa
120gccggaccgt cggactgcga agcgccgtga gcacccctcg acctgaccgg
acgcggcgca 180cccgtccgag tatcgtcgcg a 201522201DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
522tcgggtctcg cccggcgcta gtccagccgt agcgctctcc ggcgatcacc
ccggagcact 60ctggagccga gcggtcgggt ctgttgggcg cgccgcggct acggacggct
cgactcactg 120gcgctcgacc ccgtatcccc cgtctcggac gacgcaccgt
tgcgcgggaa cgatcggcgg 180cgctcacacg cacgatcgga a
201523201DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 523cttaaggctg gcgcaccatg agggccgcgc
cacgtccgac ccgcagcccg cgcgtagtag 60cctagccggg cggggttcct cccgtgcgtc
acctagcacg gggcctggca ccgaacgcga 120gcccgtccgg tcaccgcggc
gggtctgcgg acgtccccgg tcgctcggct cggagtcccc 180gctggggatc
gcgtcgggac a 201524201DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 524cgacggcgta
gcactcgcgg acctagggcg cgcgagtcgg gggagcccgc ggtgcgacgc 60tcggggagga
gctcgcatgc ccaaggcacg atctaggggg gggtacgggg ggcgtccgtc
120cgagcgccgg gactgcgatc cggggccaca tgctaaccgg cggaaggggg
gacctaaccg 180gtgtggactc cgggtaatcc a 201525201DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
525cggggggctg acacgtctcg gatcgccccg tcagtcagcc ccctagtccc
ggacaggacg 60tcggaggtcg agtccgcact gtcgggcctg ctcgtgggca cggcaggacg
cgtccccatg 120gtcagccgcc gtgcgatacc tcgccacgac tctgagccgg
gcgcgagcgt gagagcccga 180gccgcggtac acggggcgtc a
201526201DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 526gcgagctcgc tctcgactcc gggctcccgt
gctgacacgg ggtgcgaccc cgcggcgatt 60gtccgcacgc ctgtcggacg acgtcggccc
gtcgtagtgc cggtcagagg caggggggct 120gctcgcgctg gccgcctcgt
cgcgcgtgga ccctatgggg gatcacgcgt ggggtcggga 180tcggggaccg
cgcgacttgg a 201527201DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 527cgcgccccgt
aacggacgcg gtgagtcgag cttacgcggc tagggccgag tcgtgttagc 60gtctcgcgta
agcgaatgcc acgtcccccg ccgcccgtcg cgcagctggc tacgcaacgc
120ctccgcggcc tccgtagcga gtgcgtggga cgctggccgt ccgcgtgttc
cgggacctgg 180atgcgggagg gacctaaggc a 201528201DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
528agaacgtgcg gtcgtcccca cgcacgggat gacggacggg gtagacgggc
gtcgtgcgcg 60cgggtagcgt aaccggttac agtccccgca acgctctagc tccggccctc
gcttaggagt 120tcgcggccga gacatgaggt ggtccggacg gcagggggtc
gcggagaccg tggagccgat 180tctgccggac gccacgtccc a
201529201DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 529cgggacgccc cgtaccgtgt acgaagcccc
ggtcggtcgg cggatcgtag atcccggagc 60cgacgccttg aacccggctt tcccagcgac
tcgcgccccc actgggtccc tcgggacccc 120gctcccccca gacgcataca
gcccgcaagc gggggcagtc tcggaccgcc cggacactgg 180ccttaggcac
cgtgggctcg a 201530201DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 530gtgtccgggg
cgcatcggag ctgtccgacc gagttccggg gacggcgcac gttgtgccgg 60cctcagacgg
agcctgtagc ccccggacag tgtgtgcccg cccactacgg gttaggcacg
120gggttggtcg gcacgcgtcc tccgcgtgtc acggaccgat gcagaccgct
ggccgggagg 180tcgccccccc aggggtgcac a 201531201DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
531cgcgcagcac gcacgtccgg ggcacgcgcg gctcggaggg tccgggctgg
gacgggaggt 60ttggagtcgc gtgcgcgtag cagcgcaccc gcctggtcgc cgggtctagt
agggctgggt 120tacggaggac gtgcaggcga ccccaaccgt tgacgacggg
tccgaccacg cctttagccg 180tggcgtgtcc gtcgcgagcc a
20153212DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 532tcrnnnnnna cg
1253317DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 533cggnnnnnnn nnnnccg
1753413DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 534gaannttcnn gaa
1353510DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 535tgatgtannt
1053610DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 536ccnnnwwrgg
1053710DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 537wwwwsygggg
1053813DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 538rmacccannc ayy
1353916DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 539tycgtnnrna rtgaya
1654018DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 540rrraararaa nanraraa
1854115DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 541gtgtgtgtgt gtgtg
1554218DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 542anagngagag agnggcag
1854317DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 543ytstysttnt tgytwtt
1754415DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 544tnnccwnttt ktttc
1554515DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 545gcatgaccat ccacg
1554615DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 546aaaaararaa aarma
1554717DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 547gsgayarmgg amaaaaa
1754816DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 548ykytyttytt nnnnky
1654918DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 549trccgagryw nsssgcgs
1855010DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 550cgtccggcgc
1055115DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 551gaaaaagmaa aaaaa
1555218DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 552aarwtsgarg nanncsaa
1855318DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 553ttttyyttyt tkyntynt
1855414DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 554csnccaatgk nncs
1455515DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 555catkyttttt tkyty
1555610DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 556gctnactaat
1055710DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 557cacgtgacya
1055814DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 558cannnacaca sana
1455911DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 559cayamrtgyn c
1156017DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 560ggnanannar narggcn
1756110DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 561tsgygrgasa
1056218DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 562tttkytktty nytttkty
1856318DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 563kncncnnnsc gctackgc
1856417DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 564wttkttttty tttttnt
1756515DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 565srnggcmcgg cnssg
1556611DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 566ttkttttytt c
1156715DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 567tacyacanca cawga
1556818DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 568aaannraang arraanar
1856917DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 569ccytgnaytt cwncttc
1757015DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 570gtgmaknmgr angng
1557118DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 571nttwacaycc rtacayny
1857218DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 572tttnctttky ttnytttt
1857313DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 573aawnrtaaay arg
1357418DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 574aaaranraaa naaarnaa
1857516DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 575ggnaawangt aaacaa
1657617DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 576cacacacaca cacacac
1757715DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif oligonucleotide 577sastkcwctc ktcgt
1557818DNAArtificial SequenceDescription of Artificial Sequence
Synthetic binding motif
oligonucleotide 578ttgcttgaac gsatgcca 1857917DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 579yctttttttt yttyykg 1758017DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 580cggmnnncwn ynncccg 1758118DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 581rrsccgmcgm grcgcgcs 1858218DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 582rgargtsacg cakrttct 1858318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 583aaanararnr aaaarrar 1858418DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 584ggaagctgaa acgymwrr 1858518DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 585ggagaggcat gatggggg 1858618DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 586aggtgatgga gtgctcag 1858710DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 587ctncctttct 1058816DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 588gkctrrnrgg agangm 1658918DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 589gaaarraaaa aamrmara 1859017DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 590ngggsgntns ygtncga 1759111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 591gngccrsnnt m 1159218DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 592agnawgtttt tgwcaama 1859318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 593ttttttyttt tynktttt 1859418DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 594kcksgcaggc wttkytct 1859518DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 595yttcttttyt nyncnktn 1859611DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 596gnccsartng c 1159715DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 597tnsykctttt cytty 1559818DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 598sgcgmgggnn ccngaccg 1859918DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 599sttnytttyn ttytyyyy 1860014DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 600yctnattsgn cngs 1460111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 601ykntttwyyt c 1160218DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 602tnttsmttny tttccknc 1860315DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 603aaaananaar arnag 1560415DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 604ccacktksgs cctns 1560518DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 605waaaaaagaa aanaaaar 1860611DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 606crsgcywgkg c 1160711DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 607aaanggnara m 1160817DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 608naaraagcng ggcacnc 1760916DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 609tyttcyagaa nnttcy 1661018DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 610cacacacaca cacacaca 1861111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 611tttycacatg c 1161217DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 612sckkcgckst ssttyaa 1761314DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 613gnngcatgtg aaaa 1461418DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 614gaaaanaaaa aaaarana 1861515DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 615ctttttttyy tsgcc 1561615DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 616gaaaaaraar aanaa 1561715DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 617gccggtmmcg sycnn 1561818DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 618yttktnnttt ttytyttt 1861915DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 619anntttttyt tkygc 1562010DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 620gcagngcagg 1062117DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 621aaacntttat anataca 1762211DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 622caatntctnc k 1162318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 623tttytykttt nyyttttt 1862415DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 624gnrrnanacg cgtnr 1562516DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 625tttccnaawn rggaaa 1662618DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 626yttyyttytt ttytyttc 1862714DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 627mtttttytyt yttc 1462818DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 628tatacanagm krtatatg 1862918DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 629tmtttntync ttntttwk 1863016DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 630ktnnttwtta ttccnc 1663118DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 631rnnaaaanra naaraaat 1863217DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 632ttttttttcw ctttkyc 1763318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 633tttynytktt tynyttyt 1863418DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 634ttynnttytt nytttyyy 1863514DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 635tnygtgkryg tnyg 1463618DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 636ttyyyttttt yttttytt 1863715DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 637gamaaaaaar aaaar 1563818DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 638cycgggaagc sammnccg 1863913DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 639grtgyayggr tgy 1364014DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 640kmaaraaaaa raar 1464118DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 641aygraaaara raaaaraa 1864218DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 642ggaksccntt tyngmrta 1864317DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 643ttttcnkttt ytttttc 1764415DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 644araagmagaa arraa 1564517DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 645yttttctttt ynttttt 1764611DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 646arraraaagg n 1164718DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 647ystnykntyt tnctcccm 1864818DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 648garanaaaar nraaraaa 1864911DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 649cynnggssan c 1165016DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 650cacacacaca cacaya 1665115DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 651cttytwttkt tktsa 1565218DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 652yttyyytytt tytyyttt 1865318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 653amaaaaaraa rwaranaa 1865418DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 654araaaarraa aaagnraa 1865518DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 655raaraaaaar cmrsraaa 1865618DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 656ttytktytyn tyykttty 1865718DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 657gaaaamaana aaaanaaa 1865818DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 658yaanaraara aaaanaam 1865918DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 659tyntttttty tttttntk 1866018DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 660raaraaraaa naanrnaa 1866118DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 661raarrraaaa anaaamaa 1866211DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 662gccagaccta c 1166318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 663ttyttyttyt ttynytyt 1866418DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 664yksgcgcgyc kcgkcggs 1866517DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 665ttttyytttt yyyyktt 1766613DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 666ttcttktyyt ttt 1366718DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 667ttyttttyty ytttyttt 1866818DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 668ttgcttgaac ggatgcca 1866916DNAArtificial
SequenceDescription of Artificial Sequence Synthetic binding motif
oligonucleotide 669mgnmcaaaaa taaaas 16670624DNAZymomonas mobilis
670atgcgtgata tcgattccgt aatgcgtttg gcaccggtta tgccggtcct
cgtcattgaa 60gatattgctg atgcaaaacc tatcgcagaa gctttggttg ctggtggtct
gaacgttctt 120gaagtaacgc ttcgcacccc ttgtgctctt gaagccatca
agatcatgaa agaagttccg 180ggtgccgttg ttggtgccgg tacggttctg
aacgcaaaaa tgctcgacca agctcaggaa 240gctggttgcg aatttttcgt
tagcccgggt ctgaccgctg acctcggcaa gcatgctgtt 300gcccagaaag
cagctttgct tccaggtgtt gctaatgctg ctgatgtgat gcttggtctt
360gaccttggtc ttgatcgctt caaattcttc ccggctgaaa atatcggtgg
tttacctgcc 420ctgaagtcca tggcttctgt tttccgtcag gttcgtttct
gcccgaccgg cggtatcacc 480ccgacgtcag ctcctaaata tcttgaaaac
ccgtccattc tttgcgtcgg tggtagctgg 540gttgttccgg ctggcaaacc
agatgtcgca aaaatcacgg cactcgctaa agaagcttct 600gctttcaagc
gcgctgctgt tgcc 624671624DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide
671atgagggata ttgatagtgt gatgaggtta gcccctgtta tgcctgttct
cgttattgaa 60gatattgcag atgccaaacc tattgccgaa gcactcgttg caggtggtct
aaacgttcta 120gaagtgacac taaggactcc ttgtgcacta gaagctatta
agattatgaa ggaagttcct 180ggtgctgttg ttggtgctgg tacagttcta
aacgccaaaa tgctcgacca ggcacaagaa 240gcaggttgcg aatttttcgt
ttcacctggt ctaactgccg acctcggaaa gcacgcagtt 300gctcaaaaag
ccgcattact acccggtgtt gcaaatgcag cagatgtgat gctaggtcta
360gacctaggtc tagataggtt caagttcttc cctgccgaaa acattggtgg
tctacctgct 420ctaaagagta tggcatcagt tttcaggcaa gttaggttct
gccctactgg aggtataact 480cctacaagtg cacctaaata tctagaaaac
cctagtattc tatgcgttgg tggttcatgg 540gttgttcctg ccggaaaacc
cgatgttgcc aaaattacag ccctcgcaaa agaagcaagt 600gcattcaaga
gggcagcagt tgct 624672624DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 672atgagagaca
ttgattctgt tatgagattg gctccagtta tgccagtctt ggttatagaa 60gatatagctg
atgctaagcc aattgctgag gctttggttg ctggtggttt aaatgttttg
120gaagttacat tgagaactcc atgtgctttg gaagctatta aaattatgaa
ggaagttcca 180ggtgctgttg ttggtgctgg tactgtttta aacgctaaaa
tgttggatca agctcaagaa 240gctggttgtg agttctttgt atcaccaggt
ttgactgctg atttgggaaa acatgctgtt 300gctcaaaaag cggctcttct
accaggggtt gctaatgctg ctgatgttat gttgggattg 360gatttgggtt
tggatagatt taaattcttc ccagctgaaa atataggtgg tttgccagct
420ttaaaatcta tggcttctgt ttttagacaa gttagatttt gtccaactgg
aggaattact 480ccgacttctg ctccaaaata tttggaaaat ccatctattt
tgtgtgttgg tggttcttgg 540gttgttccag cgggtaaacc agatgttgcg
aaaattactg ctttggctaa agaggcttca 600gcttttaaaa gagctgctgt ggcg
624673639DNAEscherichia coli 673atgaaaaact ggaaaacaag tgcagaatca
atcctgacca ccggcccggt tgtaccggtt 60atcgtggtaa aaaaactgga acacgcggtg
ccgatggcaa aagcgttggt tgctggtggg 120gtgcgcgttc tggaagtgac
tctgcgtacc gagtgtgcag ttgacgctat ccgtgctatc 180gccaaagaag
tgcctgaagc gattgtgggt gccggtacgg tgctgaatcc acagcagctg
240gcagaagtca ctgaagcggg tgcacagttc gcaattagcc cgggtctgac
cgagccgctg 300ctgaaagctg ctaccgaagg gactattcct ctgattccgg
ggatcagcac tgtttccgaa 360ctgatgctgg gtatggacta cggtttgaaa
gagttcaaat tcttcccggc tgaagctaac 420ggcggcgtga aagccctgca
ggcgatcgcg ggtccgttct cccaggtccg tttctgcccg 480acgggtggta
tttctccggc taactaccgt gactacctgg cgctgaaaag cgtgctgtgc
540atcggtggtt cctggctggt tccggcagat gcgctggaag cgggcgatta
cgaccgcatt 600actaagctgg cgcgtgaagc tgtagaaggc gctaagctg
639674208PRTZymomonas mobilis 674Met Arg Asp Ile Asp Ser Val Met
Arg Leu Ala Pro Val Met Pro Val1 5 10 15Leu Val Ile Glu Asp Ile Ala
Asp Ala Lys Pro Ile Ala Glu Ala Leu 20 25 30Val Ala Gly Gly Leu Asn
Val Leu Glu Val Thr Leu Arg Thr Pro Cys 35 40 45Ala Leu Glu Ala Ile
Lys Ile Met Lys Glu Val Pro Gly Ala Val Val 50 55 60Gly Ala Gly Thr
Val Leu Asn Ala Lys Met Leu Asp Gln Ala Gln Glu65 70 75 80Ala Gly
Cys Glu Phe Phe Val Ser Pro Gly Leu Thr Ala Asp Leu Gly 85 90 95Lys
His Ala Val Ala Gln Lys Ala Ala Leu Leu Pro Gly Val Ala Asn 100 105
110Ala Ala Asp Val Met Leu Gly Leu Asp Leu Gly Leu Asp Arg Phe Lys
115 120 125Phe Phe Pro Ala Glu Asn Ile Gly Gly Leu Pro Ala Leu Lys
Ser Met 130 135 140Ala Ser Val Phe Arg Gln Val Arg Phe Cys Pro Thr
Gly Gly Ile Thr145 150 155 160Pro Thr Ser Ala Pro Lys Tyr Leu Glu
Asn Pro Ser Ile Leu Cys Val 165 170 175Gly Gly Ser Trp Val Val Pro
Ala Gly Lys Pro Asp Val Ala Lys Ile 180 185 190Thr Ala Leu Ala Lys
Glu Ala Ser Ala Phe Lys Arg Ala Ala Val Ala 195 200
205675208PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 675Met Arg Asp Ile Asp Ser Val Met Arg Leu
Ala Pro Val Met Pro Val1 5 10 15Leu Val Ile Glu Asp Ile Ala Asp Ala
Lys Pro Ile Ala Glu Ala Leu 20 25 30Val Ala Gly Gly Leu Asn Val Leu
Glu Val Thr Leu Arg Thr Pro Cys 35 40 45Ala Leu Glu Ala Ile Lys Ile
Met Lys Glu Val Pro Gly Ala Val Val 50 55 60Gly Ala Gly Thr Val Leu
Asn Ala Lys Met Leu Asp Gln Ala Gln Glu65 70 75 80Ala Gly Cys Glu
Phe Phe Val Ser Pro Gly Leu Thr Ala Asp Leu Gly 85 90 95Lys His Ala
Val Ala Gln Lys Ala Ala Leu Leu Pro Gly Val Ala Asn 100 105 110Ala
Ala Asp Val Met Leu Gly Leu Asp Leu Gly Leu Asp Arg Phe Lys 115 120
125Phe Phe Pro Ala Glu Asn Ile Gly Gly Leu Pro Ala Leu Lys Ser Met
130 135 140Ala Ser Val Phe Arg Gln Val Arg Phe Cys Pro Thr Gly Gly
Ile Thr145 150 155 160Pro Thr Ser Ala Pro Lys Tyr Leu Glu Asn Pro
Ser Ile Leu Cys Val 165 170 175Gly Gly Ser Trp Val Val Pro Ala Gly
Lys Pro Asp Val Ala Lys Ile 180 185 190Thr Ala Leu Ala Lys Glu Ala
Ser Ala Phe Lys Arg Ala Ala Val Ala 195 200 205676208PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
676Met Arg Asp Ile Asp Ser Val Met Arg Leu Ala Pro Val Met Pro Val1
5 10 15Leu Val Ile Glu Asp Ile Ala Asp Ala Lys Pro Ile Ala Glu Ala
Leu 20 25 30Val Ala Gly Gly Leu Asn Val Leu Glu Val Thr Leu Arg Thr
Pro Cys 35 40 45Ala Leu Glu Ala Ile Lys Ile Met Lys Glu Val Pro Gly
Ala Val Val 50 55 60Gly Ala Gly Thr Val Leu Asn Ala Lys Met Leu Asp
Gln Ala Gln Glu65 70 75 80Ala Gly Cys Glu Phe Phe Val Ser Pro Gly
Leu Thr Ala Asp Leu Gly 85 90 95Lys His Ala Val Ala Gln Lys Ala Ala
Leu Leu Pro Gly Val Ala Asn 100 105 110Ala Ala Asp Val Met Leu Gly
Leu Asp Leu Gly Leu Asp Arg Phe Lys 115 120 125Phe Phe Pro Ala Glu
Asn Ile Gly Gly Leu Pro Ala Leu Lys Ser Met 130 135 140Ala Ser Val
Phe Arg Gln Val Arg Phe Cys Pro Thr Gly Gly Ile Thr145 150 155
160Pro Thr Ser Ala Pro Lys Tyr Leu Glu Asn Pro Ser Ile Leu Cys Val
165 170 175Gly Gly Ser Trp Val Val Pro Ala Gly Lys Pro Asp Val Ala
Lys Ile 180 185 190Thr Ala Leu Ala Lys Glu Ala Ser Ala Phe Lys Arg
Ala Ala Val Ala 195 200 205677213PRTEscherichia coli 677Met Lys Asn
Trp Lys Thr Ser Ala Glu Ser Ile Leu Thr Thr Gly Pro1 5 10 15Val Val
Pro Val Ile Val Val Lys Lys Leu Glu His Ala Val Pro Met 20 25 30Ala
Lys Ala Leu Val Ala Gly Gly Val Arg Val Leu Glu Val Thr Leu 35 40
45Arg Thr Glu Cys Ala Val Asp Ala Ile Arg Ala Ile Ala Lys Glu Val
50 55 60Pro Glu Ala Ile Val Gly Ala Gly Thr Val Leu Asn Pro Gln Gln
Leu65 70 75 80Ala Glu Val Thr Glu Ala Gly Ala Gln Phe Ala Ile Ser
Pro Gly Leu 85 90 95Thr Glu Pro Leu Leu Lys Ala Ala Thr Glu Gly Thr
Ile Pro Leu Ile 100 105 110Pro Gly Ile Ser Thr Val Ser Glu Leu Met
Leu Gly Met Asp Tyr Gly 115 120 125Leu Lys Glu Phe Lys Phe Phe Pro
Ala Glu Ala Asn Gly Gly Val Lys 130 135 140Ala Leu Gln Ala Ile Ala
Gly Pro Phe Ser Gln Val Arg Phe Cys Pro145 150 155 160Thr Gly Gly
Ile Ser Pro Ala Asn Tyr Arg Asp Tyr Leu Ala Leu Lys 165 170 175Ser
Val Leu Cys Ile Gly Gly Ser Trp Leu Val Pro Ala Asp Ala Leu 180 185
190Glu Ala Gly Asp Tyr Asp Arg Ile Thr Lys Leu Ala Arg Glu Ala Val
195 200 205Glu Gly Ala Lys Leu 2106781821DNAZymomonas mobilis
678atgactgatc tgcattcaac ggtagaaaag gttaccgcgc gcgttattga
acgctcgcgg 60gaaacccgta aggcttatct ggatttgatc cagtatgagc gggaaaaagg
cgtagaccgt 120ccaaacctgt cctgtagtaa ccttgctcat ggctttgcgg
ctatgaatgg tgacaagcca 180gctttgcgcg acttcaaccg catgaatatc
ggcgtcgtga cttcctacaa cgatatgttg 240tcggctcatg aaccatatta
tcgctatccg gagcagatga aagtatttgc tcgcgaagtt 300ggcgcaacgg
ttcaggtcgc cggtggcgtg cctgctatgt gcgatggtgt gacccaaggt
360cagccgggca tggaagaatc cctgtttagc cgcgatgtta tcgctttggc
taccagcgtt 420tctttgtctc atggtatgtt tgaaggggct gcccttctcg
gtatctgtga caagattgtc 480cctggtctgt tgatgggcgc tctgcgcttt
ggtcacctgc cgaccattct ggtcccatca 540ggcccgatga cgactggtat
cccgaacaaa gaaaaaatcc gtatccgtca gctctatgct 600cagggtaaaa
tcggccagaa agaacttctg gatatggaag cggcttgcta ccatgctgaa
660ggtacctgca ccttctatgg tacggcaaac accaaccaga tggttatgga
agtcctcggt 720cttcatatgc caggttcggc atttgttacc ccgggtaccc
cgctccgcca ggctctgacc 780cgtgctgctg tgcatcgcgt tgctgaattg
ggttggaagg gcgacgatta tcgtccgctt 840ggtaaaatca ttgacgaaaa
atcaatcgtc aatgctattg ttggtctgtt ggcaaccggt 900ggttccacca
accataccat gcatattccg gccattgctc gtgctgctgg tgttatcgtt
960aactggaatg acttccatga tctttctgaa gttgttccgt tgattgcccg
catttacccg 1020aatggcccgc gcgacatcaa tgaattccag aatgcaggcg
gcatggctta tgtcatcaaa 1080gaactgcttt ctgctaatct gttgaaccgt
gatgtcacga ccattgccaa gggcggtatc 1140gaagaatacg ccaaggctcc
ggcattaaat gatgctggcg aattggtctg gaagccagct 1200ggcgaacctg
gtgatgacac cattctgcgt ccggtttcta atcctttcgc aaaagatggc
1260ggtctgcgtc tcttggaagg taaccttggc cgtgcaatgt acaaggccag
tgcggttgat 1320cctaaattct ggaccattga agcaccggtt cgcgtcttct
ctgaccaaga cgatgttcag 1380aaagccttca aggctggcga attgaacaaa
gacgttatcg ttgttgttcg tttccagggc 1440ccgcgcgcaa acggtatgcc
tgaattgcat aagctgaccc cggctttggg tgttctgcag 1500gataatggct
acaaagttgc tttggtaact gatggtcgta tgtccggtgc taccggtaaa
1560gttccggttg ctttgcatgt cagcccagaa gctcttggcg gtggtgccat
cggtaaatta 1620cgtgatggcg atatcgtccg tatctcggtt gaagaaggca
aacttgaagc tttggttcca 1680gctgatgagt ggaatgctcg tccgcatgct
gaaaaaccgg ctttccgtcc gggaaccgga 1740cgcgaattgt ttgatatctt
ccgtcagaat gctgctaaag ctgaagacgg tgcagtcgca 1800atatatgcag
gtgccggtat c 18216791821DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 679atgacggatc
tacatagtac agtggagaag gttactgcca gggttattga aaggagtagg 60gaaactagga
aggcatatct agatttaatt caatatgaga gggaaaaagg agtggacagg
120cccaacctaa gttgtagcaa cctagcacat ggattcgccg caatgaatgg
tgacaagccc 180gcattaaggg acttcaacag gatgaatatt ggagttgtga
cgagttacaa cgatatgtta 240agtgcacatg aaccctatta taggtatcct
gagcaaatga aggtgtttgc aagggaagtt 300ggagccacag ttcaagttgc
tggtggagtg cctgcaatgt gcgatggtgt gactcagggt 360caacctggaa
tggaagaatc cctattttca agggatgtta ttgcattagc aacttcagtt
420tcattatcac atggtatgtt tgaaggggca gctctactcg gtatatgtga
caagattgtt 480cctggtctac taatgggagc actaaggttt ggtcacctac
ctactattct agttcccagt 540ggacctatga caacgggtat acctaacaaa
gaaaaaatta ggattaggca actctatgca 600caaggtaaaa ttggacaaaa
agaactacta gatatggaag ccgcatgcta ccatgcagaa 660ggtacttgca
ctttctatgg tacagccaac actaaccaga tggttatgga agttctcggt
720ctacatatgc ccggtagtgc ctttgttact cctggtactc ctctcaggca
agcactaact 780agggcagcag tgcatagggt tgcagaatta ggttggaagg
gagacgatta taggcctcta 840ggtaaaatta ttgacgaaaa aagtattgtt
aatgcaattg ttggtctatt agccactggt 900ggtagtacta accatacgat
gcatattcct gctattgcaa gggcagcagg tgttattgtt 960aactggaatg
acttccatga tctatcagaa gttgttcctt taattgctag gatttaccct
1020aatggaccta gggacattaa cgaatttcaa aatgccggag gaatggcata
tgttattaag 1080gaactactat cagcaaatct actaaacagg gatgttacaa
ctattgctaa gggaggtata 1140gaagaatacg ctaaggcacc tgccctaaat
gatgcaggag aattagtttg gaagcccgca 1200ggagaacctg gtgatgacac
tattctaagg cctgtttcaa atcctttcgc caaagatgga 1260ggtctaaggc
tcttagaagg taacctagga agggccatgt acaaggctag cgccgttgat
1320cctaaattct ggactattga agcccctgtt agggttttct cagaccagga
cgatgttcaa 1380aaagccttca aggcaggaga actaaacaaa gacgttattg
ttgttgttag gttccaagga 1440cctagggcca acggtatgcc tgaattacat
aagctaactc ctgcattagg tgttctacaa 1500gataatggat acaaagttgc
attagtgacg gatggtagga tgagtggtgc aactggtaaa 1560gttcctgttg
cattacatgt ttcacccgaa gcactaggag gtggtgctat tggtaaactt
1620agggatggag atattgttag gattagtgtt gaagaaggaa aacttgaagc
actcgttccc 1680gcagatgagt ggaatgcaag gcctcatgca gaaaaacctg
cattcaggcc tgggactggg 1740agggaattat ttgatatttt caggcaaaat
gcagcaaaag cagaagacgg tgccgttgcc 1800atctatgccg gtgctggtat a
18216801821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 680atgacggatt tgcattcaac tgttgagaaa
gtaactgcta gagtaattga aagatcaagg 60gaaactagaa aggcttattt ggatttgata
caatatgaga gggaaaaagg tgttgataga 120ccaaatttgt cttgttctaa
tttggctcat ggttttgctg ctatgaatgg tgataaacca 180gctttgagag
attttaatag aatgaatata ggtgtagtta cttcttataa tgatatgttg
240tctgctcatg aaccatatta tagatatcca gaacaaatga aggtttttgc
tcgtgaagtt 300ggtgctacag ttcaagttgc tggtggtgtt cctgcaatgt
gtgatggtgt tactcaaggt 360caaccaggta tggaagaatc tttgttttcc
agagatgtaa ttgctttggc tacatctgtt 420tcattgtctc acggaatgtt
tgaaggtgct gcattgttgg gaatttgtga taaaattgtt 480ccaggtttgt
tgatgggtgc tttgaggttc ggtcatttgc caactatttt ggttccatct
540ggtccaatga ctactggaat cccaaataaa gaaaagatta gaattagaca
attgtatgct 600caaggaaaaa ttggtcaaaa ggaattgttg gatatggaag
ctgcctgtta tcatgctgaa 660ggtacttgta ctttttatgg tactgctaac
actaatcaga tggttatgga agttttgggt 720ttgcacatgc caggtagtgc
attcgttact ccaggtactc cactgagaca ggctttgact 780agagctgctg
ttcatagagt tgcagagttg ggttggaaag gtgatgatta tagacctttg
840ggtaaaatta ttgatgagaa atctattgtt aatgctattg ttggtttgtt
agctacaggt 900ggttctacaa atcatacaat gcatattccg gccatagcta
gagcagcagg ggttatagtt 960aattggaatg attttcatga tttgtctgaa
gttgttccat tgattgctag aatttatcca 1020aatggtccta gagatataaa
tgaatttcaa aatgcaggag gaatggctta tgtaattaaa 1080gaattgttga
gtgcgaattt gttaaataga gatgttacta ctattgctaa aggagggata
1140gaagaatatg ctaaagctcc agctctgaac gatgcgggtg aattggtgtg
gaaaccggct 1200ggcgaacctg gggacgacac aattttgaga ccagtatcta
atccatttgc taaagatggt 1260ggtttgcgtc tcttggaagg taatttgggt
agagcaatgt ataaggcttc tgctgtagat 1320ccaaaattct ggactattga
agctcccgtt agagttttct ctgatcaaga tgatgttcaa 1380aaggctttta
aagcaggcga gttaaataaa gatgttatag ttgttgttag atttcaaggt
1440cctcgtgcta atggtatgcc tgaattgcat aagttgactc ctgcgctagg
cgtattgcaa 1500gataatggtt ataaggttgc tttagttact gatggtagaa
tgtctggtgc aactggtaaa 1560gtaccggtgg ctctgcatgt ttcaccagag
gctttaggag gtggggcgat tggcaagttg 1620agagatggcg atatagttag
aatttctgtt gaagaaggta aattagaggc tcttgtcccc 1680gccgacgagt
ggaatgctag accacatgct gagaagcccg cttttagacc tggtactggg
1740agagaattgt ttgacatttt tagacaaaac gctgctaagg ctgaggatgg
tgcagttgca 1800atttatgctg gggcagggat c 18216811809DNAEscherichia
coli 681atgaatccac aattgttacg cgtaacaaat cgaatcattg aacgttcgcg
cgagactcgc 60tctgcttatc tcgcccggat agaacaagcg aaaacttcga ccgttcatcg
ttcgcagttg 120gcatgcggta acctggcaca cggtttcgct gcctgccagc
cagaagacaa agcctctttg 180aaaagcatgt tgcgtaacaa tatcgccatc
atcacctcct ataacgacat gctctccgcg 240caccagcctt atgaacacta
tccagaaatc attcgtaaag ccctgcatga agcgaatgcg 300gttggtcagg
ttgcgggcgg tgttccggcg atgtgtgatg gtgtcaccca ggggcaggat
360ggaatggaat tgtcgctgct aagccgcgaa gtgatagcga tgtctgcggc
ggtggggctg 420tcccataaca tgtttgatgg tgctctgttc ctcggtgtgt
gcgacaagat tgtcccgggt 480ctgacgatgg cagccctgtc gtttggtcat
ttgcctgcgg tgtttgtgcc gtctggaccg 540atggcaagcg gtttgccaaa
taaagaaaaa gtgcgtattc gccagcttta tgccgaaggt 600aaagtggacc
gcatggcctt actggagtca gaagccgcgt cttaccatgc gccgggaaca
660tgtactttct acggtactgc caacaccaac cagatggtgg tggagtttat
ggggatgcag 720ttgccaggct cttcttttgt tcatccggat tctccgctgc
gcgatgcttt gaccgccgca 780gctgcgcgtc aggttacacg catgaccggt
aatggtaatg aatggatgcc gatcggtaag 840atgatcgatg agaaagtggt
ggtgaacggt atcgttgcac tgctggcgac cggtggttcc 900actaaccaca
ccatgcacct ggtggcgatg gcgcgcgcgg ccggtattca gattaactgg
960gatgacttct ctgacctttc tgatgttgta ccgctgatgg cacgtctcta
cccgaacggt 1020ccggccgata ttaaccactt ccaggcggca ggtggcgtac
cggttctggt gcgtgaactg 1080ctcaaagcag gcctgctgca tgaagatgtc
aatacggtgg caggttttgg tctgtctcgt 1140tatacccttg aaccatggct
gaataatggt gaactggact ggcgggaagg ggcggaaaaa 1200tcactcgaca
gcaatgtgat cgcttccttc gaacaacctt tctctcatca tggtgggaca
1260aaagtgttaa gcggtaacct gggccgtgcg gttatgaaaa cctctgccgt
gccggttgag 1320aaccaggtga ttgaagcgcc agcggttgtt tttgaaagcc
agcatgacgt tatgccggcc 1380tttgaagcgg gtttgctgga ccgcgattgt
gtcgttgttg tccgtcatca ggggccaaaa 1440gcgaacggaa tgccagaatt
acataaactc atgccgccac ttggtgtatt attggaccgg 1500tgtttcaaaa
ttgcgttagt taccgatgga cgactctccg gcgcttcagg taaagtgccg
1560tcagctatcc acgtaacacc agaagcctac gatggcgggc tgctggcaaa
agtgcgcgac 1620ggggacatca ttcgtgtgaa tggacagaca ggcgaactga
cgctgctggt agacgaagcg 1680gaactggctg ctcgcgaacc gcacattcct
gacctgagcg cgtcacgcgt gggaacagga 1740cgtgaattat tcagcgcctt
gcgtgaaaaa ctgtccggtg ccgaacaggg cgcaacctgt 1800atcactttt
1809682607PRTZymomonas mobilis 682Met Thr Asp Leu His Ser Thr Val
Glu Lys Val Thr Ala Arg Val Ile1 5 10 15Glu Arg Ser Arg Glu Thr Arg
Lys Ala Tyr Leu Asp Leu Ile Gln Tyr 20 25 30Glu Arg Glu Lys Gly Val
Asp Arg Pro Asn Leu Ser Cys Ser Asn Leu 35 40 45Ala His Gly Phe Ala
Ala Met Asn Gly Asp Lys Pro Ala Leu Arg Asp 50 55 60Phe Asn Arg Met
Asn Ile Gly Val Val Thr Ser Tyr Asn Asp Met Leu65 70 75 80Ser Ala
His Glu Pro Tyr Tyr Arg Tyr Pro Glu Gln Met Lys Val Phe 85 90 95Ala
Arg Glu Val Gly Ala Thr Val Gln Val Ala Gly Gly Val Pro Ala 100 105
110Met Cys Asp Gly Val Thr Gln Gly Gln Pro Gly Met Glu Glu Ser Leu
115 120 125Phe Ser Arg Asp Val Ile Ala Leu Ala Thr Ser Val Ser Leu
Ser His 130 135 140Gly Met Phe Glu Gly Ala Ala Leu Leu Gly Ile Cys
Asp Lys Ile Val145 150 155 160Pro Gly Leu Leu Met Gly Ala Leu Arg
Phe Gly His Leu Pro Thr Ile 165 170 175Leu Val Pro Ser Gly Pro Met
Thr Thr Gly Ile Pro Asn Lys Glu Lys 180 185 190Ile Arg Ile Arg Gln
Leu Tyr Ala Gln Gly Lys Ile Gly Gln Lys Glu 195 200 205Leu Leu Asp
Met Glu Ala Ala Cys Tyr His Ala Glu Gly Thr Cys Thr 210 215 220Phe
Tyr Gly Thr Ala Asn Thr Asn Gln Met Val Met Glu Val Leu Gly225 230
235 240Leu His Met Pro Gly Ser Ala Phe Val Thr Pro Gly Thr Pro Leu
Arg 245 250 255Gln Ala Leu Thr Arg Ala Ala Val His Arg Val Ala Glu
Leu Gly Trp 260 265 270Lys Gly Asp Asp Tyr Arg Pro Leu Gly Lys Ile
Ile Asp Glu Lys Ser 275 280 285Ile Val Asn Ala Ile Val Gly Leu Leu
Ala Thr Gly Gly Ser Thr Asn 290 295 300His Thr Met His Ile Pro Ala
Ile Ala Arg Ala Ala Gly Val Ile Val305 310 315 320Asn Trp Asn Asp
Phe His Asp Leu Ser Glu Val Val Pro Leu Ile Ala 325 330 335Arg Ile
Tyr Pro Asn Gly Pro Arg Asp Ile Asn Glu Phe Gln Asn Ala 340 345
350Gly Gly Met Ala Tyr Val Ile Lys Glu Leu Leu Ser Ala Asn Leu Leu
355 360 365Asn Arg Asp Val Thr Thr Ile Ala Lys Gly Gly Ile Glu Glu
Tyr Ala 370 375 380Lys Ala Pro Ala Leu Asn Asp Ala Gly Glu Leu Val
Trp Lys Pro Ala385 390 395 400Gly Glu Pro Gly Asp Asp Thr Ile Leu
Arg Pro Val Ser Asn Pro Phe 405 410 415Ala Lys Asp Gly Gly Leu Arg
Leu Leu Glu Gly Asn Leu Gly Arg Ala 420 425 430Met Tyr Lys Ala Ser
Ala Val Asp Pro Lys Phe Trp Thr Ile Glu Ala 435 440 445Pro Val Arg
Val Phe Ser Asp Gln Asp Asp Val Gln Lys Ala Phe Lys 450 455 460Ala
Gly Glu Leu Asn Lys Asp Val Ile Val Val Val Arg Phe Gln Gly465 470
475 480Pro Arg Ala Asn Gly Met Pro Glu Leu His Lys Leu Thr Pro Ala
Leu 485 490 495Gly Val Leu Gln Asp Asn Gly Tyr Lys Val Ala Leu Val
Thr Asp Gly 500 505 510Arg Met Ser Gly Ala Thr Gly Lys Val Pro Val
Ala Leu His Val Ser 515 520 525Pro Glu Ala Leu Gly Gly Gly Ala Ile
Gly Lys Leu Arg Asp Gly Asp 530 535 540Ile Val Arg Ile Ser Val Glu
Glu Gly Lys Leu Glu Ala Leu Val Pro545 550 555 560Ala Asp Glu Trp
Asn Ala Arg Pro His Ala Glu Lys Pro Ala Phe Arg 565 570 575Pro Gly
Thr Gly Arg Glu Leu Phe Asp Ile Phe Arg Gln Asn Ala Ala 580 585
590Lys Ala Glu Asp Gly Ala Val Ala Ile Tyr Ala Gly Ala Gly Ile 595
600 605683607PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 683Met Thr Asp Leu His Ser Thr Val
Glu Lys Val Thr Ala Arg Val Ile1 5 10 15Glu Arg Ser Arg Glu Thr Arg
Lys Ala Tyr Leu Asp Leu Ile Gln Tyr 20 25 30Glu Arg Glu Lys Gly Val
Asp Arg Pro Asn Leu Ser Cys Ser Asn Leu 35 40 45Ala His Gly Phe Ala
Ala Met Asn Gly Asp Lys Pro Ala Leu Arg Asp 50 55 60Phe Asn Arg Met
Asn Ile Gly Val Val Thr Ser Tyr Asn Asp Met Leu65 70 75 80Ser Ala
His Glu Pro Tyr Tyr Arg Tyr Pro Glu Gln Met Lys Val Phe 85 90 95Ala
Arg Glu Val Gly Ala Thr Val Gln Val Ala Gly Gly Val Pro Ala 100 105
110Met Cys Asp Gly Val Thr Gln Gly Gln Pro Gly Met Glu Glu Ser Leu
115 120 125Phe Ser Arg Asp Val Ile Ala Leu Ala Thr Ser Val Ser Leu
Ser His 130 135 140Gly Met Phe Glu Gly Ala Ala Leu Leu Gly Ile Cys
Asp Lys Ile Val145 150 155 160Pro Gly Leu Leu Met Gly Ala Leu Arg
Phe Gly His Leu Pro Thr Ile 165 170 175Leu Val Pro Ser Gly Pro Met
Thr Thr Gly Ile Pro Asn Lys Glu Lys 180 185 190Ile Arg Ile Arg Gln
Leu Tyr Ala Gln Gly Lys Ile Gly Gln Lys Glu 195 200 205Leu Leu Asp
Met Glu Ala Ala Cys Tyr His Ala Glu Gly Thr Cys Thr 210 215 220Phe
Tyr Gly Thr Ala Asn Thr Asn Gln Met Val Met Glu Val Leu Gly225 230
235 240Leu His Met Pro Gly Ser Ala Phe Val Thr Pro Gly Thr Pro Leu
Arg 245 250 255Gln Ala Leu Thr Arg Ala Ala Val His Arg Val Ala Glu
Leu Gly Trp 260 265 270Lys Gly Asp Asp Tyr Arg Pro Leu Gly Lys Ile
Ile Asp Glu Lys Ser 275 280 285Ile Val Asn Ala Ile Val Gly Leu Leu
Ala Thr Gly Gly Ser Thr Asn 290 295 300His Thr Met His Ile Pro Ala
Ile Ala Arg Ala Ala Gly Val Ile Val305 310 315 320Asn Trp Asn Asp
Phe His Asp Leu Ser Glu Val Val Pro Leu Ile Ala 325 330 335Arg Ile
Tyr Pro Asn Gly Pro Arg Asp Ile Asn Glu Phe Gln Asn Ala 340 345
350Gly Gly Met Ala Tyr Val Ile Lys Glu Leu Leu Ser Ala Asn Leu Leu
355 360 365Asn Arg Asp Val Thr Thr Ile Ala Lys Gly Gly Ile Glu Glu
Tyr Ala 370 375 380Lys Ala Pro Ala Leu Asn Asp Ala Gly Glu Leu Val
Trp Lys Pro Ala385 390 395 400Gly Glu Pro Gly Asp Asp Thr Ile Leu
Arg Pro Val Ser Asn Pro Phe 405 410 415Ala Lys Asp Gly Gly Leu Arg
Leu Leu Glu Gly Asn Leu Gly Arg Ala 420 425 430Met Tyr Lys Ala Ser
Ala Val Asp Pro Lys Phe Trp Thr Ile Glu Ala 435 440 445Pro Val Arg
Val Phe Ser Asp Gln Asp Asp Val Gln Lys Ala Phe Lys 450 455 460Ala
Gly Glu Leu Asn Lys Asp Val Ile Val Val Val Arg Phe Gln Gly465 470
475 480Pro Arg Ala Asn Gly Met Pro Glu Leu His Lys Leu Thr Pro Ala
Leu 485 490 495Gly Val Leu Gln Asp Asn Gly Tyr Lys Val Ala Leu Val
Thr Asp Gly 500 505 510Arg Met Ser Gly Ala Thr Gly Lys Val Pro Val
Ala Leu His Val Ser 515 520 525Pro Glu Ala Leu Gly Gly Gly Ala Ile
Gly Lys Leu Arg Asp Gly Asp 530 535 540Ile Val Arg Ile Ser Val Glu
Glu Gly Lys Leu Glu Ala Leu Val Pro545 550 555 560Ala Asp Glu Trp
Asn Ala Arg Pro His Ala Glu Lys Pro Ala Phe Arg 565 570 575Pro Gly
Thr Gly Arg Glu Leu Phe Asp Ile Phe Arg Gln Asn Ala Ala 580 585
590Lys Ala Glu Asp Gly Ala Val Ala Ile Tyr Ala Gly Ala Gly Ile 595
600 605684607PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 684Met Thr Asp Leu His Ser Thr Val
Glu Lys Val Thr Ala Arg Val Ile1 5 10 15Glu Arg Ser Arg Glu Thr Arg
Lys Ala Tyr Leu Asp Leu Ile Gln Tyr 20 25 30Glu Arg Glu Lys Gly Val
Asp Arg Pro Asn Leu Ser Cys Ser Asn Leu 35 40 45Ala His Gly Phe Ala
Ala Met Asn Gly Asp Lys Pro Ala Leu Arg Asp 50 55 60Phe Asn Arg Met
Asn Ile Gly Val Val Thr Ser Tyr Asn Asp Met Leu65 70 75 80Ser Ala
His Glu Pro Tyr Tyr Arg Tyr Pro Glu Gln Met Lys Val Phe 85 90 95Ala
Arg Glu Val Gly Ala Thr Val Gln Val Ala Gly Gly Val Pro Ala 100 105
110Met Cys Asp Gly Val Thr Gln Gly Gln Pro Gly Met Glu Glu Ser Leu
115 120 125Phe Ser Arg Asp Val Ile Ala Leu Ala Thr Ser Val Ser Leu
Ser His 130 135 140Gly Met Phe Glu Gly Ala Ala Leu Leu Gly Ile Cys
Asp Lys Ile Val145 150 155 160Pro Gly Leu Leu Met Gly Ala Leu Arg
Phe Gly His Leu Pro Thr Ile 165 170 175Leu Val Pro Ser Gly Pro Met
Thr Thr Gly Ile Pro Asn Lys Glu Lys 180 185 190Ile Arg Ile Arg Gln
Leu Tyr Ala Gln Gly Lys Ile Gly Gln Lys Glu 195 200 205Leu Leu Asp
Met Glu Ala Ala Cys Tyr His Ala Glu Gly Thr Cys Thr 210 215 220Phe
Tyr Gly Thr Ala Asn Thr Asn Gln Met Val Met Glu Val Leu Gly225 230
235 240Leu His Met Pro Gly Ser Ala Phe Val Thr Pro Gly Thr Pro Leu
Arg 245 250 255Gln Ala Leu Thr Arg Ala Ala Val His Arg Val Ala Glu
Leu Gly Trp 260 265 270Lys Gly Asp Asp Tyr Arg Pro Leu Gly Lys Ile
Ile Asp Glu Lys Ser 275 280 285Ile Val Asn Ala Ile Val Gly Leu Leu
Ala Thr Gly Gly Ser Thr Asn 290 295 300His Thr Met His Ile Pro Ala
Ile Ala Arg Ala Ala Gly Val Ile Val305 310 315 320Asn Trp Asn Asp
Phe His Asp Leu Ser Glu Val Val Pro Leu Ile Ala 325 330 335Arg Ile
Tyr Pro Asn Gly Pro Arg Asp Ile Asn Glu Phe Gln Asn Ala 340 345
350Gly Gly Met Ala Tyr Val Ile Lys Glu Leu Leu Ser Ala Asn Leu Leu
355 360 365Asn Arg Asp Val Thr Thr Ile Ala Lys Gly Gly Ile Glu Glu
Tyr Ala 370 375 380Lys Ala Pro Ala Leu Asn Asp Ala Gly Glu Leu Val
Trp Lys Pro Ala385 390 395 400Gly Glu Pro Gly Asp Asp Thr Ile Leu
Arg Pro Val Ser Asn Pro Phe 405 410 415Ala Lys Asp Gly Gly Leu Arg
Leu Leu Glu Gly Asn Leu Gly Arg Ala 420 425 430Met Tyr Lys Ala Ser
Ala Val Asp Pro Lys Phe Trp Thr Ile Glu Ala 435 440 445Pro Val Arg
Val Phe Ser Asp Gln Asp Asp Val Gln Lys Ala Phe Lys 450 455 460Ala
Gly Glu Leu Asn Lys Asp Val Ile Val Val Val Arg Phe Gln Gly465 470
475 480Pro Arg Ala Asn Gly Met Pro Glu Leu His Lys Leu Thr Pro Ala
Leu 485 490 495Gly Val Leu Gln Asp Asn Gly Tyr Lys Val Ala Leu Val
Thr Asp Gly 500 505 510Arg Met Ser Gly Ala Thr Gly Lys Val Pro Val
Ala Leu His Val Ser 515 520 525Pro Glu Ala Leu Gly Gly Gly Ala Ile
Gly Lys Leu Arg Asp Gly Asp 530 535 540Ile Val Arg Ile Ser Val Glu
Glu Gly Lys Leu Glu Ala Leu Val Pro545 550 555 560Ala Asp Glu Trp
Asn Ala Arg Pro His Ala Glu Lys Pro Ala Phe Arg 565 570 575Pro Gly
Thr Gly Arg Glu Leu Phe Asp Ile Phe Arg Gln Asn Ala Ala 580 585
590Lys Ala Glu Asp Gly Ala Val Ala Ile Tyr Ala Gly Ala Gly Ile 595
600 605685603PRTEscherichia coli 685Met Asn Pro Gln Leu Leu Arg Val
Thr Asn Arg Ile Ile Glu Arg Ser1 5 10 15Arg Glu Thr Arg Ser Ala Tyr
Leu Ala Arg Ile Glu Gln Ala Lys Thr 20 25 30Ser Thr Val His Arg Ser
Gln Leu Ala Cys Gly Asn Leu Ala His Gly 35 40 45Phe Ala Ala Cys Gln
Pro Glu Asp Lys Ala Ser Leu Lys Ser Met Leu 50 55 60Arg Asn Asn Ile
Ala Ile Ile Thr Ser Tyr Asn Asp Met Leu Ser Ala65 70 75 80His Gln
Pro Tyr Glu His Tyr Pro Glu Ile Ile Arg Lys Ala Leu His 85 90 95Glu
Ala Asn Ala Val Gly Gln Val Ala Gly Gly Val Pro Ala Met Cys 100 105
110Asp Gly Val Thr Gln Gly Gln Asp Gly Met Glu Leu Ser Leu Leu Ser
115 120 125Arg Glu Val Ile Ala Met Ser Ala Ala Val Gly Leu Ser His
Asn Met 130 135 140Phe Asp Gly Ala Leu Phe Leu Gly Val Cys Asp Lys
Ile Val Pro Gly145 150 155 160Leu Thr Met Ala Ala Leu Ser Phe Gly
His Leu Pro Ala Val Phe Val 165 170 175Pro Ser Gly Pro Met Ala Ser
Gly Leu Pro Asn Lys Glu Lys Val Arg 180 185 190Ile Arg Gln Leu Tyr
Ala Glu Gly Lys Val Asp Arg Met Ala Leu Leu 195 200 205Glu Ser Glu
Ala Ala Ser Tyr His Ala Pro Gly Thr Cys Thr Phe Tyr 210 215 220Gly
Thr Ala Asn Thr Asn Gln Met Val Val Glu Phe Met Gly Met Gln225 230
235 240Leu Pro Gly Ser Ser Phe Val His Pro Asp Ser Pro Leu Arg Asp
Ala 245 250 255Leu Thr Ala Ala Ala Ala Arg Gln Val Thr Arg Met Thr
Gly Asn Gly 260 265 270Asn Glu Trp Met Pro Ile Gly Lys Met Ile Asp
Glu Lys Val Val Val 275 280 285Asn Gly Ile Val Ala Leu Leu Ala Thr
Gly Gly Ser Thr Asn His Thr 290 295 300Met His Leu Val Ala Met Ala
Arg Ala Ala Gly Ile Gln Ile Asn Trp305 310 315 320Asp Asp Phe Ser
Asp Leu Ser Asp Val Val Pro Leu Met Ala Arg Leu 325 330 335Tyr Pro
Asn Gly Pro Ala Asp Ile Asn His Phe Gln Ala Ala Gly Gly 340 345
350Val Pro Val Leu Val Arg Glu Leu Leu Lys Ala Gly Leu Leu His Glu
355 360 365Asp Val Asn Thr Val Ala Gly Phe Gly Leu Ser Arg Tyr Thr
Leu Glu 370 375 380Pro Trp Leu Asn Asn Gly Glu Leu Asp Trp Arg Glu
Gly Ala Glu Lys385 390 395 400Ser Leu Asp Ser Asn Val Ile Ala Ser
Phe Glu Gln Pro Phe Ser His 405 410 415His Gly Gly Thr Lys Val Leu
Ser Gly Asn Leu Gly Arg Ala Val Met 420 425 430Lys Thr Ser Ala Val
Pro Val Glu Asn Gln Val Ile Glu Ala Pro Ala 435 440 445Val Val Phe
Glu Ser Gln His Asp Val Met Pro Ala Phe Glu Ala Gly 450 455 460Leu
Leu Asp Arg Asp Cys Val Val Val Val Arg His Gln Gly Pro Lys465 470
475 480Ala Asn Gly Met Pro Glu Leu His Lys Leu Met Pro Pro Leu Gly
Val 485 490 495Leu Leu Asp Arg Cys Phe Lys Ile Ala Leu Val Thr Asp
Gly Arg Leu 500 505 510Ser Gly Ala Ser Gly Lys Val Pro Ser Ala Ile
His Val Thr Pro Glu 515 520 525Ala Tyr Asp Gly Gly Leu Leu Ala Lys
Val Arg Asp Gly Asp Ile Ile 530 535 540Arg Val Asn Gly Gln Thr Gly
Glu Leu Thr Leu Leu Val Asp Glu Ala545 550 555 560Glu Leu Ala Ala
Arg Glu Pro His Ile Pro Asp Leu Ser Ala Ser Arg 565 570 575Val Gly
Thr Gly Arg Glu Leu Phe Ser Ala Leu Arg Glu Lys Leu Ser 580 585
590Gly Ala Glu Gln Gly Ala Thr Cys Ile Thr Phe 595 600
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References