U.S. patent application number 12/442013 was filed with the patent office on 2010-04-08 for metabolic engineering of arabinose-fermenting yeast cells.
Invention is credited to Johannes Pieter Van Dijken, Jacobus Thomas Pronk, Antonius Jeroen Adriaan Van Maris, Johannes Hendrik De Winde, Aaron Adriaan Winkler, Hendrik Wouter Wisselink.
Application Number | 20100086965 12/442013 |
Document ID | / |
Family ID | 38800716 |
Filed Date | 2010-04-08 |
United States Patent
Application |
20100086965 |
Kind Code |
A1 |
Van Maris; Antonius Jeroen Adriaan
; et al. |
April 8, 2010 |
METABOLIC ENGINEERING OF ARABINOSE-FERMENTING YEAST CELLS
Abstract
The invention relates to an eukaryotic cell expressing
nucleotide sequences encoding the ara A, ara B and ara D enzymes
whereby the expression of these nucleotide sequences confers on the
cell the ability to use L-arabinose and/or convert L-arabinose into
L-ribulose, and/or xylulose 5-phosphate and/or into a desired
fermentation product such as ethanol. Optionally, the eukaryotic
cell is also able to convert xylose into ethanol.
Inventors: |
Van Maris; Antonius Jeroen
Adriaan; (Delft, NL) ; Pronk; Jacobus Thomas;
(Schipluiden, NL) ; Wisselink; Hendrik Wouter;
(Culemborg, NL) ; Dijken; Johannes Pieter Van;
(Leidschendam, NL) ; Winkler; Aaron Adriaan; (Den
Haag, NL) ; Winde; Johannes Hendrik De; (Voorhout,
NL) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Family ID: |
38800716 |
Appl. No.: |
12/442013 |
Filed: |
October 1, 2007 |
PCT Filed: |
October 1, 2007 |
PCT NO: |
PCT/NL07/00246 |
371 Date: |
June 12, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60848357 |
Oct 2, 2006 |
|
|
|
Current U.S.
Class: |
435/47 ; 435/106;
435/136; 435/140; 435/141; 435/144; 435/145; 435/157; 435/160;
435/161; 435/167; 435/254.2; 435/254.21; 435/254.22; 435/254.23;
435/320.1; 435/74 |
Current CPC
Class: |
Y02E 50/30 20130101;
Y02E 50/17 20130101; C12N 9/1205 20130101; Y02E 50/10 20130101;
C12N 9/90 20130101; Y02E 50/343 20130101; C12P 7/08 20130101 |
Class at
Publication: |
435/47 ;
435/254.2; 435/254.21; 435/254.22; 435/254.23; 435/320.1; 435/136;
435/140; 435/141; 435/144; 435/145; 435/161; 435/157; 435/160;
435/106; 435/167; 435/74 |
International
Class: |
C12P 35/00 20060101
C12P035/00; C12N 1/19 20060101 C12N001/19; C12N 15/74 20060101
C12N015/74; C12P 7/40 20060101 C12P007/40; C12P 7/54 20060101
C12P007/54; C12P 7/52 20060101 C12P007/52; C12P 7/48 20060101
C12P007/48; C12P 7/46 20060101 C12P007/46; C12P 7/06 20060101
C12P007/06; C12P 7/04 20060101 C12P007/04; C12P 7/16 20060101
C12P007/16; C12P 13/04 20060101 C12P013/04; C12P 5/02 20060101
C12P005/02; C12P 19/44 20060101 C12P019/44 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 2, 2006 |
EP |
06121633.9 |
Claims
1. A eukaryotic cell capable of expressing the following nucleotide
sequences, wherein the expression of these nucleotide sequences
confers on the cell the ability to use L-arabinose and/or to
convert L-arabinose into L-ribulose, and/or xylulose 5-phosphate
and/or into a desired fermentation product: (a) a nucleotide
sequence encoding an arabinose isomerase (araA), wherein said
nucleotide sequence is selected from the group consisting of: i.
nucleotide sequences encoding an araA, said araA comprising an
amino acid sequence that has at least 55% sequence identity with
the amino acid sequence of SEQ ID NO:1, ii. nucleotide sequences
comprising a nucleotide sequence that has at least 60% sequence
identity with the nucleotide sequence of SEQ ID NO:2, iii.
nucleotide sequences the complementary strand of which hybridizes
to a nucleic add molecule of sequence of (i) or (ii); iv,
nucleotide sequences the sequences of which differs from the
sequence of a nucleic acid molecule of (iii) due to the degeneracy
of the genetic; code, (b) a nucleotide sequence encoding a
L-ribulokinase (araB), wherein said nucleotide sequence is selected
from the group consisting of: i. nucleotide sequences encoding an
araB, said araB comprising an amino acid sequence that has at least
20% sequence identity with the amino acid sequence of SEQ ID NO:3,
ii. nucleotide sequences comprising a nucleotide sequence that has
at least 50% sequence identity with the nucleotide sequence of SEQ
ID NO:4, iii. nucleotide sequences the complementary strand of
which hybridizes to a nucleic acid molecule of sequence of (i) or
(ii); iv. nucleotide sequences the sequences of which differs from
the sequence of a nucleic acid molecule of (iii) due to the
degeneracy of the genetic code, (c) a nucleotide sequence encoding
an L-ribulose-5-P-4-epimerase (araD), wherein said nucleotide
sequence is selected from the group consisting of: i. nucleotide
sequences encoding an araD, said araD comprising an amino acid
sequence that has at least 60% sequence identity with the amino
acid sequence of SEQ ID NO:5, ii. nucleotide sequences comprising a
nucleotide sequence that has at least 60% sequence identity with
the nucleotide sequence of SEQ ID NO:6, iii. nucleotide sequences
the complementary strand of which hybridizes to a nucleic acid
molecule of sequence of (i) or (ii); iv. nucleotide sequences the
sequences of which differs from the sequence of a nucleic acid
molecule of (iii) due to the degeneracy of the genetic code.
2. A cell according to claim 1, wherein one, two or three of the
araA, araB and araD nucleotide sequences originate from a
Lactobacillus genus, preferably a Lactobacillus plantarum
species.
3. A cell according to claim 1, wherein the cell is a yeast cell,
preferably belonging to one of the genera: Saccharomyces,
Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula,
Kloeckera, Schwanniomyces or Yarrowia.
4. A cell according to claim 3, wherein the yeast cell belongs to
one of the species: S. cerevisiae, S. bulderi, S. barnetti, S.
exiguus, S. uvarum, S. diastaticus, K. lactis, K. marxianus or K.
fragilis.
5. A cell according to claim 1, wherein the nucleotide sequences
encoding the araA, araB and/or araD are operably linked to a
promoter that causes sufficient expression of the corresponding
nucleotide sequences in the cell to confer to the cell the ability
to use L-arabinose and/or to convert L-arabinose into L-ribulose,
and/or xylulose 5-phosphate and/or into a desired fermentation
product.
6. A cell according to claim 1, wherein the cell exhibits the
ability to directly isomerise xylose into xylulose.
7. A cell according to claim 6, wherein the cell comprises a
genetic modification that increases the flux of the pentose
phosphate pathway.
8. A cell according to claim 6, wherein the genetic modification
comprises overexpression of at least one gene of the non-oxidative
part of the pentose phosphate pathway.
9. A cell according to claim 8, wherein the gene is selected from
the group consisting of the genes encoding ribulose-5-phosphate
isomerase, ribulose-5-phosphate epimerase, transketolase and
transaldolase.
10. A cell according to claim 8, wherein the genetic modification
comprises overexpression of at least the genes coding for a
transketolase and a transaldolase.
11. A cell according to claim 1, wherein the cell further comprises
a genetic modification that increases the specific xylulose kinase
activity.
12. A cell according to claim 11, wherein the genetic modification
comprises overexpression of a gene encoding a xylulose kinase.
13. A cell according to claim 8, wherein the gene that is
overexpressed is endogenous to the cell.
14. A cell according to claim 5, wherein the cell comprises a
genetic modification that reduces unspecific aldose reductase
activity in the cell.
15. A cell according to claim 14, wherein the genetic modification
reduces the expression of, or inactivates a gene encoding an
unspecific aldose reductase.
16. A cell according to claim 15, wherein the gene is inactivated
by deletion of at least part of the gene or by disruption of the
gene.
17. A cell according to claim 14, wherein the expression of each
gene in the cell that encodes an unspecific aldose reductase is
reduced or inactivated.
18. A cell according to claim 1, wherein the fermentation product
is selected from the group consisting of ethanol, lactic acid,
3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid,
citric acid, malic acid, fumaric acid, an amino acid,
1,3-propane-diol, ethylene, glycerol, butanol, a .beta.-lactam
antibiotic and a cephalosporin.
19. A nucleic acid construct comprising a nucleic acid sequence
encoding an araA, a nucleic acid sequence encoding an araB and/or a
nucleic acid sequence encoding an araD all as defined in claim
1.
20. A process for producing a fermentation product selected from
the group consisting of ethanol, lactic acid, 3-hydroxy-propionic
acid, acrylic acid, acetic acid, succinic acid, citric acid, malic
acid, fumaric acid, an amino acid, 1,3-propane-diol, ethylene,
glycerol, butanol, a .beta.-lactam, antibiotic and a cephalosporin,
whereby the process comprises: (a) fermenting a medium containing a
source of arabinose and optionally xylose with a modified cell as
defined in claim 1, whereby the cell ferments arabinose and
optionally xylose to the fermentation product; and optionally, (b)
recovering the fermentation product.
21. A process for producing a fermentation product selected from
the group consisting of ethanol, lactic acid, 3-hydroxy-propionic
acid, acrylic acid, acetic acid, succinic acid, citric acid, malic
acid, fumaric acid, an amino acid, 1,3-propane-diol, ethylene,
glycerol, butanol, a .beta.-lactam antibiotic and a cephalosporin,
wherein the process comprises: (a) fermenting a medium containing
at least a source of L-arabinose and a source of xylose with a cell
as defined in claim 1 and a cell able to use xylose and/or
exhibiting the ability to directly isomerise xylose into xylulose,
whereby each cell ferments L-arabinose and/or xylose to the
fermentation product; and optionally, (b) recovering the
fermentation product.
22. A process according to claim 20, wherein the medium also
contains a source of glucose.
23. A process according to claim 20, wherein the fermentation
product is ethanol.
24. A process according to claim 23, wherein the volumetric ethanol
productivity is at least 0.5 g ethanol per litre per hour.
25. A process according to claim 23, wherein the ethanol yield is
at least 30%.
26. A process according to claim 20, wherein the process is
anaerobic.
27. A process according to claim 20, wherein the process is
aerobic, preferably performed under oxygen limited conditions.
Description
FIELD OF THE INVENTION
[0001] The invention relates to an eukaryotic cell having the
ability to use L-arabinose and/or to convert L-arabinose into
L-ribulose, and/or xylulose 5-phosphate and/or into a desired
fermentation product and to a process for producing a fermentation
product wherein this cell is used.
BACKGROUND OF THE INVENTION
[0002] Fuel ethanol is acknowledged as a valuable alternative to
fossil fuels. Economically viable ethanol production from the
hemicellulose fraction of plant biomass requires the simultaneous
fermentative conversion of both pentoses and hexoses at comparable
rates and with high yields. Yeasts, in particular Saccharomyces
spp., are the most appropriate candidates for this process since
they can grow and ferment fast on hexoses, both aerobically and
anaerobically. Furthermore they are much more resistant to the
toxic environment of lignocellulose hydrolysates than (genetically
modified) bacteria.
[0003] EP 1 499 708 describes a process for making S. cerevisiae
strains able to produce ethanol from L-arabinose. These strains
were modified by introducing the araA (L-arabinose isomerase) gene
from Bacillus subtilis, the araB (L-ribulokinase) and araD
(L-ribulose-5-P4-epimerase) genes from Escherichia coli.
Furthermore, these strains were either carrying additional
mutations in their genome or overexpressing a TAL1 (transaldolase)
gene. However, these strains have several drawbacks. They ferment
arabinose in oxygen limited conditions. In addition, they have a
low ethanol production rate of 0.05 gg.sup.-1h.sup.-1 (Becker and
Boles, 2003). Furthermore, these strains are not able to use
L-arabinose under anaerobic conditions. Finally, these S.
cerevisiae strains have a wild type background, therefore they can
not be used to co-ferment several C5 sugars.
[0004] WO 03/062430 and WO 06/009434 disclose yeast strains able to
convert xylose into ethanol. These yeast strains are able to
directly isomerise xylose into xylulose.
[0005] Still, there is a need for alternative strains for producing
ethanol, which perform better and are more robust and resistant to
relatively harsh production conditions.
DESCRIPTION OF THE FIGURES
[0006] FIG. 1. Plasmid maps of pRW231 and pRW243.
[0007] FIG. 2. Growth pattern of shake flask cultivations of strain
RWB219 (.largecircle.) and IMS0001 ( ) in synthetic medium
containing 0.5% galactose (A) and 0.1% galactose +2% L-arabinose
(B). Cultures were grown for 72 hours in synthetic medium with
galactose (A) and then transferred to synthetic medium with
galactose and arabinose (B). Growth was determined by measuring the
OD.sub.660.
[0008] FIG. 3. Growth rate during serial transfers of S. cerevisiae
IMS0001 in shake flask cultures containing synthetic medium with 2%
(w/v) L-arabinose. Each datapoint represents the growth rate
estimated from the OD.sub.660 measured during (exponential) growth.
The closed and open circles represent duplicate serial transfer
experiments.
[0009] FIG. 4. Growth rate during an anaerobic SBR fermentation of
S. cerevisiae IMS0001 in synthetic medium with 2% (w/v)
L-arabinose. Each datapoint represents the growth rate estimated
from the CO.sub.2 profile (solid line) during exponential
growth.
[0010] FIG. 5. Sugar consumption and product formation during
anaerobic batch fermentations of strain IMS0002. The fermentations
were performed in 1 synthetic medium supplemented with: 20 g
l.sup.-1 arabinose (A); 20 g l.sup.-1 glucose and 20 g l.sup.-1
arabinose (B); 30 g l.sup.-1 glucose, 15 g l.sup.-1 xylose, and 15
g l.sup.-1 arabinose (C); Sugar consumption and product formation
during anaerobic batch fermentations with a mixture of strains
IMS0002 and RWB218. The fermentations were performed in 1 liter of
synthetic medium supplemented with 30 g l.sup.-1 glucose, 15 g
l.sup.-1 xylose, and 15 g l.sup.-1 arabinose (D). Symbols: glucose
( ); xylose (.largecircle.); arabinose (.box-solid.); ethanol
calculated from cumulative CO.sub.2 production (.quadrature.);
ethanol measured by HPLC (.tangle-solidup.); cumulative CO.sub.2
production (.DELTA.); xylitol ()
[0011] FIG. 6. Sugar consumption and product formation during an
anaerobic batch fermentation of strain IMS0002 cells selected for
anaerobic growth on xylose. The fermentation was performed in 1
liter of synthetic medium supplemented with 20 g l.sup.-1 xylose
and 20 g l.sup.-1 arabinose. Symbols: xylose (.largecircle.);
arabinose (.box-solid.); ethanol measured by HPLC
(.tangle-solidup.); cumulative CO.sub.2 production (.DELTA.);
xylitol ().
[0012] FIG. 7. Sugar consumption and product formation during an
anaerobic batch fermentation of strain IMS0003. The fermentation
was performed in 1 liter of synthetic medium supplemented with: 30
g l.sup.-1 glucose, 15 g l.sup.-1 xylose, and 15 g l.sup.-1
arabinose. Symbols: glucose ( ); xylose (.largecircle.); arabinose
(.box-solid.); ethanol calculated from cumulative CO.sub.2
production (.quadrature.); ethanol measured by HPLC
(.tangle-solidup.); cumulative CO.sub.2 production (.DELTA.);
DESCRIPTION OF THE INVENTION
Eukaryotic Cell
[0013] In a first aspect, the invention relates to a eukaryotic
cell capable of expressing the following nucleotide sequences,
whereby the expression of these nucleotide sequences confers on the
cell the ability to use L-arabinose and/or to convert L-arabinose
into L-ribulose, and/or xylulose 5-phosphate and/or into a desired
fermentation product such as ethanol: [0014] (a) a nucleotide
sequence encoding an arabinose isomerase (araA), wherein said
nucleotide sequence is selected from the group consisting of:
[0015] (i) nucleotide sequences encoding an araA, said araA
comprising an amino acid sequence that has at least 55% sequence
identity with the amino acid sequence of SEQ ID NO:1. [0016] (ii)
nucleotide sequences comprising a nucleotide sequence that has at
least 60% sequence identity with the nucleotide sequence of SEQ ID
NO:2. [0017] (iii) nucleotide sequences the complementary strand of
which hybridizes to a nucleic acid molecule of sequence of (i) or
(ii); [0018] (iv) nucleotide sequences the sequences of which
differ from the sequence of a nucleic acid molecule of (iii) due to
the degeneracy of the genetic code, [0019] (b) a nucleotide
sequence encoding a L-ribulokinase (araB), wherein said nucleotide
sequence is selected from the group consisting of: [0020] (i)
nucleotide sequences encoding an araB, said araB comprising an
amino acid sequence that has at least 20% sequence identity with
the amino acid sequence of SEQ ID NO:3. [0021] (ii) nucleotide
sequences comprising a nucleotide sequence that has at least 50%
sequence identity with the nucleotide sequence of SEQ ID NO:4.
[0022] (iii) nucleotide sequences the complementary strand of which
hybridizes to a nucleic acid molecule of sequence of (i) or (ii);
[0023] (iv) nucleotide sequences the sequences of which differ from
the sequence of a nucleic acid molecule of (iii) due to the
degeneracy of the genetic code, [0024] (c) a nucleotide sequence
encoding an L-ribulose-5-P-4-epimerase (araD), wherein said
nucleotide sequence is selected from the group consisting of:
[0025] (i) nucleotide sequences encoding an araD, said araD
comprising an amino acid sequence that has at least 60% sequence
identity with the amino acid sequence of SEQ ID NO:5. [0026] (ii)
nucleotide sequences comprising a nucleotide sequence that has at
least 60% sequence identity with the nucleotide sequence of SEQ ID
NO:6. [0027] (iii) nucleotide sequences the complementary strand of
which hybridizes to a nucleic acid molecule of sequence of (i) or
(ii); [0028] (iv) nucleotide sequences the sequences of which
differ from the sequence of a nucleic acid molecule of (iii) due to
the degeneracy of the genetic code.
[0029] A preferred embodiment relates to an eukaryotic cell capable
of expressing the following nucleotide sequences, whereby the
expression of these nucleotide sequences confers on the cell the
ability to use L-arabinose and/or to convert L-arabinose into
L-ribulose, and/or xylulose 5-phosphate and/or into a desired
fermentation product such as ethanol: [0030] (a) a nucleotide
sequence encoding an arabinose isomerase (araA), wherein said
nucleotide sequence is selected from the group consisting of:
[0031] (i) nucleotide sequences comprising a nucleotide sequence
that has at least 60% sequence identity with the nucleotide
sequence of SEQ ID NO:2, [0032] (ii) nucleotide sequences the
complementary strand of which hybridizes to a nucleic acid molecule
of sequence of (i); [0033] (iii) nucleotide sequences the sequences
of which differ from the sequence of a nucleic acid molecule of
(ii) due to the degeneracy of the genetic code, [0034] (b) a
nucleotide sequence encoding a L-ribulokinase (araB), wherein said
nucleotide sequence is selected from the group consisting of:
[0035] (i) nucleotide sequences encoding an araB, said araB
comprising an amino acid sequence that has at least 20% sequence
identity with the amino acid sequence of SEQ ID NO:3. [0036] (ii)
nucleotide sequences comprising a nucleotide sequence that has at
least 50% sequence identity with the nucleotide sequence of SEQ ID
NO:4. [0037] (iii) nucleotide sequences the complementary strand of
which hybridizes to a nucleic acid molecule of sequence of (i) or
(ii); [0038] (iv) nucleotide sequences the sequences of which
differ from the sequence of a nucleic acid molecule of (iii) due to
the degeneracy of the genetic code, [0039] (c) a nucleotide
sequence encoding an L-ribulose-5-P-4-epimerase (araD), wherein
said nucleotide sequence is selected from the group consisting of:
[0040] (i) nucleotide sequences encoding an araD, said araD
comprising an amino acid sequence that has at least 60% sequence
identity with the amino acid sequence of SEQ ID NO:5. [0041] (ii)
nucleotide sequences comprising a nucleotide sequence that has at
least 60% sequence identity with the nucleotide sequence of SEQ ID
NO:6. [0042] (iii) nucleotide sequences the complementary strand of
which hybridizes to a nucleic acid molecule of sequence of (i) or
(ii); [0043] (iv) nucleotide sequences the sequences of which
differ from the sequence of a nucleic acid molecule of (iii) due to
the degeneracy of the genetic code.
Sequence Identity and Similarity
[0044] Sequence identity is herein defined as a relationship
between two or more amino acid (polypeptide or protein) sequences
or two or more nucleic acid (polynucleotide) sequences, as
determined by comparing the sequences. Usually, sequence identities
or similarities are compared over the whole length of the sequences
compared. In the art, "identity" also means the degree of sequence
relatedness between amino acid or nucleic acid sequences, as the
case may be, as determined by the match between strings of such
sequences. "Similarity" between two amino acid sequences is
determined by comparing the amino acid sequence and its conserved
amino acid substitutes of one polypeptide to the sequence of a
second polypeptide. "Identity" and "similarity" can be readily
calculated by various methods, known to those skilled in the
art.
[0045] Preferred methods to determine identity are designed to give
the largest match between the sequences tested. Methods to
determine identity and similarity are codified in publicly
available computer programs. Preferred computer program methods to
determine identity and similarity between two sequences include
e.g. the BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et
al., J. Mol. Biol. 215:403-410 (1990), publicly available from NCBI
and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH
Bethesda, Md. 20894). A most preferred algorithm used is EMBOSS
(http://www.ebi.ac.uk/emboss/align). Preferred parameters for amino
acid sequences comparison using EMBOSS are gap open 10.0, gap
extend 0.5, Blosum 62 matrix. Preferred parameters for nucleic acid
sequences comparison using EMBOSS are gap open 10.0, gap extend
0.5, DNA full matrix (DNA identity matrix).
[0046] Optionally, in determining the degree of amino acid
similarity, the skilled person may also take into account so-called
"conservative" amino acid substitutions, as will be clear to the
skilled person. Conservative amino acid substitutions refer to the
interchangeability of residues having similar side chains. For
example, a group of amino acids having aliphatic side chains is
glycine, alanine, valine, leucine, and isoleucine; a group of amino
acids having aliphatic-hydroxyl side chains is serine and
threonine; a group of amino acids having amide-containing side
chains is asparagine and glutamine; a group of amino acids having
aromatic side chains is phenylalanine, tyrosine, and tryptophan; a
group of amino acids having basic side chains is lysine, arginine,
and histidine; and a group of amino acids having sulphur-containing
side chains is cysteine and methionine. Preferred conservative
amino acids substitution groups are: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine, and
asparagine-glutamine. Substitutional variants of the amino acid
sequence disclosed herein are those in which at least one residue
in the disclosed sequences has been removed and a different residue
inserted in its place. Preferably, the amino acid change is
conservative. Preferred conservative substitutions for each of the
naturally occurring amino acids are as follows: Ala to ser; Arg to
lys; Asn to gln or his; Asp to glu; Cys to ser or ala; Gln to asn;
Glu to asp; Gly to pro; His to asn or gln; Ile to leu or val; Leu
to ile or val; Lys to arg; gin or glu; Met to leu or ile; Phe to
met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or
phe; and, Val to ile or leu.
Hybridising Nucleic Acid Sequences
[0047] Nucleotide sequences encoding the enzymes expressed in the
cell of the invention may also be defined by their capability to
hybridise with the nucleotide sequences of SEQ ID NO.'s 2, 4, 6, 8,
16, 18, 20, 22, 24, 26, 28, 30 respectively, under moderate, or
preferably under stringent hybridisation conditions. Stringent
hybridisation conditions are herein defined as conditions that
allow a nucleic acid sequence of at least about 25, preferably
about 50 nucleotides, 75 or 100 and most preferably of about 200 or
more nucleotides, to hybridise at a temperature of about 65.degree.
C. in a solution comprising about 1 M salt, preferably 6.times.SSC
or any other solution having a comparable ionic strength, and
washing at 65.degree. C. in a solution comprising about 0.1 M salt,
or less, preferably 0.2.times.SSC or any other solution having a
comparable ionic strength. Preferably, the hybridisation is
performed overnight, i.e. at least for 10 hours and preferably
washing is performed for at least one hour with at least two
changes of the washing solution. These conditions will usually
allow the specific hybridisation of sequences having about 90% or
more sequence identity.
[0048] Moderate conditions are herein defined as conditions that
allow a nucleic acid sequences of at least 50 nucleotides,
preferably of about 200 or more nucleotides, to hybridise at a
temperature of about 45.degree. C. in a solution comprising about 1
M salt, preferably 6.times.SSC or any other solution having a
comparable ionic strength, and washing at room temperature in a
solution comprising about 1 M salt, preferably 6.times.SSC or any
other solution having a comparable ionic strength. Preferably, the
hybridisation is performed overnight, i.e. at least for 10 hours,
and preferably washing is performed for at least one hour with at
least two changes of the washing solution. These conditions will
usually allow the specific hybridisation of sequences having up to
50% sequence identity. The person skilled in the art will be able
to modify these hybridisation conditions in order to specifically
identify sequences varying in identity between 50% and 90%.
AraA
[0049] A preferred nucleotide sequence encoding a arabinose
isomerase (araA) expressed in the cell of the invention is selected
from the group consisting of: [0050] (a) nucleotide sequences
encoding an araA polypeptide said araA comprising an amino acid
sequence that has at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 97,
98, or 99% sequence identity with the amino acid sequence of SEQ ID
NO. 1; [0051] (b) nucleotide sequences comprising a nucleotide
sequence that has at least 60, 70, 80, 90, 95, 97, 98, or 99%
sequence identity with the nucleotide sequence of SEQ ID NO. 2;
[0052] (c) nucleotide sequences the complementary strand of which
hybridises to a nucleic acid molecule sequence of (a) or (b);
[0053] (d) nucleotide sequences the sequence of which differ from
the sequence of a nucleic acid molecule of (c) due to the
degeneracy of the genetic code. The nucleotide sequence encoding an
araA may encode either a prokaryotic or an eukaryotic araA, i.e. an
araA with an amino acid sequence that is identical to that of an
araA that naturally occurs in the prokaryotic or eukaryotic
organism. The present inventors have found that the ability of a
particular araA to confer to a eukaryotic host cell the ability to
use arabinose and/or to convert arabinose into L-ribulose, and/or
xylulose 5-phosphate and/or into a desired fermentation product
such as ethanol when co-expressed with araB and araD does not
depend so much on whether the araA is of prokaryotic or eukaryotic
origin. Rather this depends on the relatedness of the araA's amino
acid sequence to that of the sequence SEQ ID NO. 1.
AraB
[0054] A preferred nucleotide sequence encoding a L-ribulokinase
(AraB) expressed in the cell of the invention is selected from the
group consisting of: [0055] (a) nucleotide sequences encoding a
polypeptide comprising an amino acid sequence that has at least 20,
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 98,
or 99% sequence identity with the amino acid sequence of SEQ ID NO.
3; [0056] (b) nucleotide sequences comprising a nucleotide sequence
that has at least 50, 60, 70, 80, 90, 95, 97, 98, or 99% sequence
identity with the nucleotide sequence of SEQ ID NO.4; [0057] (c)
nucleotide sequences the complementary strand of which hybridises
to a nucleic acid molecule sequence of (a) or (b); [0058] (d)
nucleotide sequences the sequence of which differ from the sequence
of a nucleic acid molecule of (c) due to the degeneracy of the
genetic code. The nucleotide sequence encoding an araB may encode
either a prokaryotic or an eukaryotic araB, i.e. an araB with an
amino acid sequence that is identical to that of a araB that
naturally occurs in the prokaryotic or eukaryotic organism. The
present inventors have found that the ability of a particular araB
to confer to a eukaryotic host cell the ability to use arabinose
and/or to convert arabinose into L-ribulose, and/or xylulose
5-phosphate and/or into a desired fermentation product when
co-expressed with araA and araD does not depend so much on whether
the araB is of prokaryotic or eukaryotic origin. Rather this
depends on the relatedness of the araB's amino acid sequence to
that of the sequence SEQ ID NO. 3.
AraD
[0059] A preferred nucleotide sequence encoding a
L-ribulose-5-P-4-epimerase (araD) expressed in the cell of the
invention is selected from the group consisting of: [0060] (e)
nucleotide sequences encoding a polypeptide comprising an amino
acid sequence that has at least 60, 65, 70, 75, 80, 85, 90, 95, 97,
98, or 99% sequence identity with the amino acid sequence of SEQ ID
NO. 5; [0061] (f) nucleotide sequences comprising a nucleotide
sequence that has at least 60, 65, 70, 75, 80, 85, 90, 95, 97, 98,
or 99% sequence identity with the nucleotide sequence of SEQ ID
NO.6; [0062] (g) nucleotide sequences the complementary strand of
which hybridises to a nucleic acid molecule sequence of (a) or (b);
[0063] (h) nucleotide sequences the sequence of which differs from
the sequence of a nucleic acid molecule of (c) due to the
degeneracy of the genetic code. The nucleotide sequence encoding an
araD may encode either a prokaryotic or an eukaryotic araD, i.e. an
araD with an amino acid sequence that is identical to that of a
araD that naturally occurs in the prokaryotic or eukaryotic
organism. The present inventors have found that the ability of a
particular araD to confer to a eukaryotic host cell the ability to
use arabinose and/or to convert arabinose into L-ribulose, and/or
xylulose 5-phosphate and/or into a desired fermentation product
when co-expressed with araA and araB does not depend so much on
whether the araD is of prokaryotic or eukaryotic origin. Rather
this depends on the relatedness of the araD's amino acid sequence
to that of the sequence SEQ ID NO. 5.
[0064] Surprisingly, the codon bias index indicated that expression
of the Lactobacillus plantarum araA, araB and araD genes were more
favorable for expression in yeast than the prokaryolic araA, araB
and araD genes described in EP 1 499 708.
[0065] It is to be noted that L. plantarum is a Generally Regarded
As Safe (GRAS) organism, which is recognized as safe by food
registration authorities. Therefore, a preferred nucleotide
sequence encodes an araA, araB or araD respectively having an amino
acid sequence that is related to the sequences SEQ ID NO: 1, 3, or
5 respectively as defined above. A preferred nucleotide sequence
encodes a fungal araA, araB or araD respectively (e.g. from a
Basidiomycete), more preferably an araA, araB or araD respectively
from an anaerobic fungus, e.g. an anaerobic fungus that belongs to
the families Neocallimastix, Caecomyces, Pfromyces, Orpinomyces, or
Ruminomyces. Alternatively, a preferred nucleotide sequence encodes
a bacterial araA, araB or araD respectively, preferably from a
Gram-positive bacterium, more preferably from the genus
Lactobacillus, most preferably from Lactobacillus plantarum
species. Preferably, one, two or three or the araA, araB and araD
nucleotide sequences originate from a Lactobacillus genus, more
preferably a Lactobacillus plantarum species. The bacterial araA
expressed in the cell of the invention is not the Bacillus subtilis
araA disclosed in EP 1 499 708 and given as SEQ ID NO:9. SEQ ID
NO:10 represents the nucleotide acid sequence coding for SEQ ID
NO:9. The bacterial araB and araD expressed in the cell of the
invention are not the ones of Escherichia coli (E. coli) as
disclosed in EP 1 499 708 and given as SEQ ID NO: 11 and SEQ ID
NO:13. SEQ ID NO: 12 represents the nucleotide acid sequence coding
for SEQ ID NO:11. SEQ ID NO:14 represents the nucleotide acid
sequence coding for SEQ ID NO:13.
[0066] To increase the likelihood that the (bacterial) araA, araB
and araD enzymes respectively are expressed in active form in a
eukaryotic host cell of the invention such as yeast, the
corresponding encoding nucleotide sequence may be adapted to
optimise its codon usage to that of the chosen eukaryotic host
cell. The adaptiveness of a nucleotide sequence encoding the araA,
araB, and araD enzymes (or other enzymes of the invention, see
below) to the codon usage of the chosen host cell may be expressed
as codon adaptation index (CAI). The codon adaptation index is
herein defined as a measurement of the relative adaptiveness of the
codon usage of a gene towards the codon usage of highly expressed
genes. The relative adaptiveness (w) of each codon is the ratio of
the usage of each codon, to that of the most abundant codon for the
same amino acid. The CAI index is defined as the geometric mean of
these relative adaptiveness values. Non-synonymous codons and
termination codons (dependent on genetic code) are excluded. CAI
values range from 0 to 1, with higher values indicating a higher
proportion of the most abundant codons (see Sharp and Li, 1987,
Nucleic Acids Research 15: 1281-1295; also see: Jansen et al.,
2003, Nucleic Acids Res. 31(8):2242-51). An adapted nucleotide
sequence preferably has a CAI of at least 0.2, 0.3, 0.4, 0.5, 0.6
or 0.7.
[0067] In a preferred embodiment, expression of the nucleotide
sequences encoding an ara A, an ara B and an ara D as defined
earlier herein confers to the cell the ability to use L-arabinose
and/or to convert it into L-ribulose, and/or xylulose 5-phosphate.
Without wishing to be bound by any theory, L-arabinose is expected
to be first converted into L-ribulose, which is subsequently
converted into xylulose 5-phosphate which is the main molecule
entering the pentose phosphate pathway. In the context of the
invention, "using L-arabinose" preferably means that the optical
density measured at 660 nm (OD.sub.660) of transformed cells
cultured under aerobic or anaerobic conditions in the presence of
at least 0.5% L-arabinose during at least 20 days is increased from
approximately 0.5 till 1.0 or more. More preferably, the OD.sub.660
is increased from 0.5 till 1.5 or more. More preferably, the cells
are cultured in the presence of at least 1%, at least 1.5%, at
least 2% L-arabinose. Most preferably, the cells are cultured in
the presence of approximately 2% L-arabinose.
[0068] In the context of the invention, a cell is able "to convert
L-arabinose into L-ribulose" when detectable amounts of L-ribulose
are detected in cells cultured under aerobic or anaerobic
conditions in the presence of L-arabinose (same preferred
concentrations as in previous paragraph) during at least 20 days
using a suitable assay. Preferably the assay is HPLC for
L-ribulose.
[0069] In the context of the invention, a cell is able "to convert
L-arabinose into xylulose 5-phosphate" when an increase of at least
2% of xylulose 5-phosphate is detected in cells cultured under
aerobic or anaerobic conditions in the presence of L-arabinose
(same preferred concentrations as in previous paragraph) during at
least 20 days using a suitable assay. Preferably, an HPCL-based
assay for xylulose 5-phosphate has been described in Zaldivar J.,
et al ((2002), Appl. Microbiol. Biotechnol., 59:436-442). This
assay is briefly described in the experimental part. More
preferably, the increase is of at least 5%, 10%, 15%, 20%, 25% or
more.
[0070] In another preferred embodiment, expression of the
nucleotide sequences encoding an ara A, ara B and ara D as defined
earlier herein confers to the cell the ability to convert
L-arabinose into a desired fermentation product when cultured under
aerobic or anaerobic conditions in the presence of L-arabinose
(same preferred concentrations as in previous paragraph) during at
least one month till one year. More preferably, a cell is able to
convert L-arabinose into a desired fermentation product when
detectable amounts of a desired fermentation product are detected
using a suitable assay and when the cells are cultured under the
conditions given in previous sentence. Even more preferably, the
assay is HPLC. Even more preferably, the fermentation product is
ethanol.
[0071] A cell for transformation with the nucleotide sequences
encoding the araA, araB, and araD enzymes respectively as described
above, preferably is a host cell capable of active or passive
xylose transport into and xylose isomerisation within the cell. The
cell preferably is capable of active glycolysis. The cell may
further contain an endogenous pentose phosphate pathway and may
contain endogenous xylulose kinase activity so that xylulose
isomerised from xylose may be metabolised to pyruvate. The cell
further preferably contains enzymes for conversion of pyruvate to a
desired fermentation product such as ethanol, lactic acid,
3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid,
citric acid, malic acid, fumaric acid, an amino acid,
1,3-propane-diol, ethylene, glycerol, butanol, a .beta.-lactam
antibiotic or a cephalosporin. The cell may be made capable of
producing butanol by introduction of one or more genes of the
butanol pathway as disclosed in WO2007/041269.
[0072] A preferred cell is naturally capable of alcoholic
fermentation, preferably, anaerobic alcoholic fermentation. The
host cell further preferably has a high tolerance to ethanol, a
high tolerance to low pH (i.e. capable of growth at a pH lower than
5, 4, 3, or 2.5) and towards organic acids like lactic acid, acetic
acid or formic acid and sugar degradation products such as furfural
and hydroxy-methylfurfural, and a high tolerance to elevated
temperatures. Any of these characteristics or activities of the
host cell may be naturally present in the host cell or may be
introduced or modified through genetic selection or by genetic
modification. A suitable host cell is a eukaryotic microorganism
like e.g. a fungus, however, most suitable as host cell are yeasts
or filamentous fungi.
[0073] Yeasts are herein defined as eukaryotic microorganisms and
include all species of the subdivision Eumycotina (Alexopoulos, C.
J., 1962, In: Introductory Mycology, John Wiley & Sons, Inc.,
New York) that predominantly grow in unicellular form. Yeasts may
either grow by budding of a unicellular thallus or may grow by
fission of the organism. Preferred yeasts as host cells belong to
one of the genera Saccharomyces, Kluyveromyces, Candida, Pichia,
Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, or
Yarrowia. Preferably the yeast is capable of anaerobic
fermentation, more preferably anaerobic alcoholic fermentation.
[0074] Filamentous fungi are herein defined as eukaryotic
microorganisms that include all filamentous forms of the
subdivision Eumycotina. These fungi are characterized by a
vegetative mycelium composed of chitin, cellulose, and other
complex polysaccharides. The filamentous fungi of the present
invention are morphologically, physiologically, and genetically
distinct from yeasts. Vegetative growth by filamentous fungi is by
hyphal elongation and carbon catabolism of most filamentous fungi
is obligately aerobic. Preferred filamentous fungi as host cells
belong to one of the genera Aspergillus, Trichoderma, Humicola,
Acremonium, Fusarium, or Penicillium.
[0075] Over the years suggestions have been made for the
introduction of various organisms for the production of bio-ethanol
from crop sugars. In practice, however, all major bio-ethanol
production processes have continued to use the yeasts of the genus
Saccharomyces as ethanol producer. This is due to the many
attractive features of Saccharomyces species for industrial
processes, i.e., a high acid-, ethanol- and osmo-tolerance,
capability of anaerobic growth, and of course its high alcoholic
fermentative capacity. Preferred yeast species as host cells
include S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S.
uvarum, S. diastaticus, K. lactis, K. marxianus, K. fragilis.
[0076] In a preferred embodiment, the host cell of the invention is
a host cell that has been transformed with a nucleic acid construct
comprising the nucleotide sequence encoding the araA, araB, and
araD enzymes as defined above. In one more preferred embodiment,
the host cell is co-transformed with three nucleic acid constructs,
each nucleic acid construct comprising the nucleotide sequence
encoding araA, araB or araD. The nucleic acid construct comprising
the araA, araB, and/or araD coding sequence is capable of
expression of the araA, araB, and/or araD enzymes in the host cell.
To this end the nucleic acid construct may be constructed as
described in e.g. WO 03/0624430. The host cell may comprise a
single but preferably comprises multiple copies of each nucleic
acid construct. The nucleic acid construct may be maintained
episomally and thus comprise a sequence for autonomous replication,
such as an ARS sequence. Suitable episomal nucleic acid constructs
may e.g. be based on the yeast 2.mu. or pKD1 (Fleer et al., 1991,
Biotechnology 9:968-975) plasmids. Preferably, however, each
nucleic acid construct is integrated in one or more copies into the
genome of the host cell. Integration into the host cell's genome
may occur at random by illegitimate recombination but preferably
nucleic acid construct is integrated into the host cell's genome by
homologous recombination as is well known in the art of fungal
molecular genetics (see e.g. WO 90/14423, EP-A-0 481 008, EP-A-0
635 574 and U.S. Pat. No. 6,265,186). Accordingly, in a more
preferred embodiment, the cell of the invention comprises a nucleic
acid construct comprising the araA, araB, and/or araD coding
sequence and is capable of expression of the araA, araB, and/or
araD enzymes. In an even more preferred embodiment, the araA, araB,
and/or araD coding sequences are each operably linked to a promoter
that causes sufficient expression of the corresponding nucleotide
sequences in a cell to confer to the cell the ability to use
L-arabinose, and/or to convert L-arabinose into L-ribulose, and/or
xylulose 5-phosphate. Preferably the cell is a yeast cell.
Accordingly, in a further aspect, the invention also encompasses a
nucleic acid construct as earlier outlined herein. Preferably, a
nucleic acid construct comprises a nucleic acid sequence encoding
an araA, araB and/or araD. Nucleic acid sequences encoding an araA,
araB, or araD have been all earlier defined herein. Even more
preferably, the expression of the corresponding nucleotide
sequences in a cell confer to the cell the ability to convert
L-arabinose into a desired fermentation product as defined later
herein. In an even more preferred embodiment, the fermentation
product is ethanol. Even more preferably, the cell is a yeast
cell.
[0077] As used herein, the term "operably linked" refers to a
linkage of polynucleotide elements (or coding sequences or nucleic
acid sequence) in a functional relationship. A nucleic acid
sequence is "operably linked" when it is placed into a functional
relationship with another nucleic acid sequence. For instance, a
promoter or enhancer is operably linked to a coding sequence if it
affects the transcription of the coding sequence. Operably linked
means that the nucleic acid sequences being linked are typically
contiguous and, where necessary to join two protein coding regions,
contiguous and in reading frame.
[0078] As used herein, the term "promoter" refers to a nucleic acid
fragment that functions to control the transcription of one or more
genes, located upstream with respect to the direction of
transcription of the transcription initiation site of the gene, and
is structurally identified by the presence of a binding site for
DNA-dependent RNA polymerase, transcription initiation sites and
any other DNA sequences, including, but not limited to
transcription factor binding sites, repressor and activator protein
binding sites, and any other sequences of nucleotides known to one
of skill in the art to act directly or indirectly to regulate the
amount of transcription from the promoter. A "constitutive"
promoter is a promoter that is active under most environmental and
developmental conditions. An "inducible" promoter is a promoter
that is active under environmental or developmental regulation.
[0079] The promoter that could be used to achieve the expression of
the nucleotide sequences coding for araA, araB and/or araD may be
not native to the nucleotide sequence coding for the enzyme to be
expressed, i.e. a promoter that is heterologous to the nucleotide
sequence (coding sequence) to which it is operably linked. Although
the promoter preferably is heterologous to the coding sequence to
which it is operably linked, it is also preferred that the promoter
is homologous, i.e. endogenous to the host cell. Preferably the
heterologous promoter (to the nucleotide sequence) is capable of
producing a higher steady state level of the transcript comprising
the coding sequence (or is capable of producing more transcript
molecules, i.e. mRNA molecules, per unit of time) than is the
promoter that is native to the coding sequence, preferably under
conditions where arabinose, or arabinose and glucose, or xylose and
arabinose or xylose and arabinose and glucose are available as
carbon sources, more preferably as major carbon sources (i.e. more
than 50% of the available carbon source consists of arabinose, or
arabinose and glucose, or xylose and arabinose or xylose and
arabinose and glucose), most preferably as sole carbon sources.
Suitable promoters in this context include both constitutive and
inducible natural promoters as well as engineered promoters. A
preferred promoter for use in the present invention will in
addition be insensitive to catabolite (glucose) repression and/or
will preferably not require arabinose and/or xylose for
induction.
[0080] Promotors having these characteristics are widely available
and known to the skilled person. Suitable examples of such
promoters include e.g. promoters from glycolytic genes, such as the
phosphofructokinase (PPK), triose phosphate isomerase (TPI),
glyceraldehyde-3-phosphate dehydrogenase (GPD, TDH3 or GAPDH),
pyruvate kinase (PYK), phosphoglycerate kinase (PGK) promoters from
yeasts or filamentous fungi; more details about such promoters from
yeast may be found in (WO 93/03159). Other useful promoters are
ribosomal protein encoding gene promoters, the lactase gene
promoter (LAC4), alcohol dehydrogenase promoters (ADH1, ADH4, and
the like), the enolase promoter (ENO), the glucose-6-phosphate
isomerase promoter (PGI1, Hauf et al, 2000) or the hexose(glucose)
transporter promoter (HXT7) or the glyceraldehyde-3-phosphate
dehydrogenase (TDH3). The sequence of the PGI1 promoter is given in
SEQ ID NO:51. The sequence of the HXT7 promoter is given in SEQ ID
NO:52. The sequence of the TDH3 promoter is given in SEQ ID NO:49.
Other promoters, both constitutive and inducible, and enhancers or
upstream activating sequences will be known to those of skill in
the art. The promoters used in the host cells of the invention may
be modified, if desired, to affect their control characteristics. A
preferred cell of the invention is a eukaryotic cell transformed
with the araA, araB and araD genes of L. plantarum. More
preferably, the eukaryotic cell is a yeast cell, even more
preferably a S. cerevisiae strain transformed with the araA, araB
and araD genes of L. plantarum. Most preferably, the cell is either
CBS120327 or CBS120328 both deposited at the CBS Institute (The
Netherlands) on Sep. 27, 2006.
[0081] The term "homologous" when used to indicate the relation
between a given (recombinant) nucleic acid or polypeptide molecule
and a given host organism or host cell, is understood to mean that
in nature the nucleic acid or polypeptide molecule is produced by a
host cell or organisms of the same species, preferably of the same
variety or strain. If homologous to a host cell, a nucleic acid
sequence encoding a polypeptide will typically be operably linked
to another promoter sequence or, if applicable, another secretory
signal sequence and/or terminator sequence than in its natural
environment. When used to indicate the relatedness of two nucleic
acid sequences the term "homologous" means that one single-stranded
nucleic acid sequence may hybridize to a complementary
single-stranded nucleic acid sequence. The degree of hybridization
may depend on a number of factors including the amount of identity
between the sequences and the hybridization conditions such as
temperature and salt concentration as earlier presented. Preferably
the region of identity is greater than about 5 bp, more preferably
the region of identity is greater than 10 bp.
[0082] The term "heterologous" when used with respect to a nucleic
acid (DNA or RNA) or protein refers to a nucleic acid or protein
that does not occur naturally as part of the organism, cell, genome
or DNA or RNA sequence in which it is present, or that is found in
a cell or location or locations in the genome or DNA or RNA
sequence that differ from that in which it is found in nature.
Heterologous nucleic acids or proteins are not endogenous to the
cell into which it is introduced, but has been obtained from
another cell or synthetically or recombinantly produced. Generally,
though not necessarily, such nucleic acids encode proteins that are
not normally produced by the cell in which the DNA is transcribed
or expressed. Similarly exogenous RNA encodes for proteins not
normally expressed in the cell in which the exogenous RNA is
present. Heterologous nucleic acids and proteins may also be
referred to as foreign nucleic acids or proteins. Any nucleic acid
or protein that one of skill in the art would recognize as
heterologous or foreign to the cell in which it is expressed is
herein encompassed by the term heterologous nucleic acid or
protein. The term heterologous also applies to non-natural
combinations of nucleic acid or amino acid sequences, i.e.
combinations where at least two of the combined sequences are
foreign with respect to each other.
Preferred Eukaryotic Cell Able to Use and/or Convert L-Arabinose
and Xylose
[0083] In a more preferred embodiment, the cell of the invention
that expresses araA, araB and araD is able to use L-arabinose
and/or to convert it into L-ribulose, and/or xylulose 5-phosphate
and/or a desired fermentation product as earlier defined herein and
additionally exhibits the ability to use xylose and/or convert
xylose into xylulose. The conversion of xylose into xylulose is
preferably a one step isomerisation step (direct isomerisation of
xylose into xylulose). This type of cell is therefore able to use
both L-arabinose and xylose. "Using" xylose has preferably the same
meaning as "using" L-arabinose as earlier defined herein.
[0084] Enzyme definitions are as used in WO 06/009434, for xylose
isomerase (EC 5.3.1.5), xylulose kinase (EC 2.7.1.17), ribulose
5-phosphate epimerase (5.1.3.1), ribulose 5-phosphate isomerase (EC
5.3.1.6), transketolase (EC 2.2.1.1), transaldolase (EC 2.2.1.2),
and aldose reductase" (EC 1.1.1.21).
[0085] In a preferred embodiment, the eukaryotic cell of the
invention expressing araA, araB and araD as earlier defined herein
has the ability of isomerising xylose to xylulose as e.g. described
in WO 03/0624430 or in WO 06/009434. The ability of isomerising
xylose to xylulose is conferred to the host cell by transformation
of the host cell with a nucleic acid construct comprising a
nucleotide sequence encoding a xylose isomerase. The transformed
host cell's ability to isomerise xylose into xylulose is the direct
isomerisation of xylose to xylulose. This is understood to mean
that xylose isomerised into xylulose in a single reaction catalysed
by a xylose isomerase, as opposed to the two step conversion of
xylose into xylulose via a xylitol intermediate as catalysed by
xylose reductase and xylitol dehydrogenase, respectively.
[0086] The nucleotide sequence encodes a xylose isomerase that is
preferably expressed in active form in the transformed host cell of
the invention. Thus, expression of the nucleotide sequence in the
host cell produces a xylose isomerase with a specific activity of
at least 10 U xylose isomerase activity per mg protein at
30.degree. C., preferably at least 20, 25, 30, 50, 100, 200, 300 or
500 U per mg at 30.degree. C. The specific activity of the xylose
isomerase expressed in the transformed host cell is herein defined
as the amount of xylose isomerase activity units per mg protein of
cell free lysate of the host cell, e.g. a yeast cell free lysate.
Determination of the xylose isomerase activity has already been
described earlier herein.
[0087] Preferably, expression of the nucleotide sequence encoding
the xylose isomerase in the host cell produces a xylose isomerase
with a K.sub.m for xylose that is less than 50, 40, 30 or 25 mM,
more preferably, the K.sub.m for xylose is about 20 mM or less.
[0088] A preferred nucleotide sequence encoding the xylose
isomerase may be selected from the group consisting of: [0089] (e)
nucleotide sequences encoding a polypeptide comprising an amino
acid sequence that has at least 60, 65, 70, 75, 80, 85, 90, 95, 97,
98, or 99% sequence identity with the amino acid sequence of SEQ ID
NO. 7 or SEQ ID NO:15; [0090] (f) nucleotide sequences comprising a
nucleotide sequence that has at least 40, 50, 60, 70, 80, 90, 95,
97, 98, or 99% sequence identity with the nucleotide sequence of
SEQ ID NO. 8 or SEQ ID NO:16; [0091] (g) nucleotide sequences the
complementary strand of which hybridises to a nucleic acid molecule
sequence of (a) or (b); [0092] (h) nucleotide sequences the
sequence of which differs from the sequence of a nucleic acid
molecule of (c) due to the degeneracy of the genetic code.
[0093] The nucleotide sequence encoding the xylose isomerase may
encode either a prokaryotic or an eukaryotic xylose isomerase, i.e.
a xylose isomerase with an amino acid sequence that is identical to
that of a xylose isomerase that naturally occurs in the prokaryotic
or eukaryotic organism. The present inventors have found that the
ability of a particular xylose isomerase to confer to a eukaryotic
host cell the ability to isomerise xylose into xylulose does not
depend so much on whether the isomerase is of prokaryotic or
eukaryotic origin. Rather this depends on the relatedness of the
isomerase's amino acid sequence to that of the Piromyces sequence
(SEQ ID NO. 7). Surprisingly, the eukaryotic Piromyces isomerase is
more related to prokaryotic isomerases than to other known
eukaryotic isomerases. Therefore, a preferred nucleotide sequence
encodes a xylose isomerase having an amino acid sequence that is
related to the Piromyces sequence as defined above. A preferred
nucleotide sequence encodes a fungal xylose isomerase (e.g. from a
Basidiomycete), more preferably a xylose isomerase from an
anaerobic fungus, e.g. a xylose isomerase from an anaerobic fungus
that belongs to the families Neocallimastix, Caecomyces, Piromyces,
Orpinomyces, or Ruminomyces. Alternatively, a preferred nucleotide
sequence encodes a bacterial xylose isomerase, preferably a
Gram-negative bacterium, more preferably an isomerase from the
class Bacteroides, or from the genus Bacteroides, most preferably
from B. thetaiotaomicron (SEQ ID NO. 15).
[0094] To increase the likelihood that the xylose isomerase is
expressed in active form in a eukaryotic host cell such as yeast,
the nucleotide sequence encoding the xylose isomerase may be
adapted to optimise its codon usage to that of the eukaryotic host
cell as earlier defined herein.
[0095] A host cell for transformation with the nucleotide sequence
encoding the xylose isomerase as described above, preferably is a
host capable of active or passive xylose transport into the cell.
The host cell preferably contains active glycolysis. The host cell
may further contain an endogenous pentose phosphate pathway and may
contain endogenous xylulose kinase activity so that xylulose
isomerised from xylose may be metabolised to pyruvate. The host
further preferably contains enzymes for conversion of pyruvate to a
desired fermentation product such as ethanol, lactic acid,
3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid,
citric acid, malic acid, fumaric acid, an amino acid,
1,3-propane-diol, ethylene, glycerol, butanol, a .beta.-lactam
antibiotic or a cephalosporin. A preferred host cell is a host cell
that is naturally capable of alcoholic fermentation, preferably,
anaerobic alcoholic fermentation. The host cell further preferably
has a high tolerance to ethanol, a high tolerance to low pH (i.e.
capable of growth at a pH lower than 5, 4, 3, or 2.5) and towards
organic acids like lactic acid, acetic acid or formic acid and
sugar degradation products such as furfural and
hydroxy-methylfurfural, and a high tolerance to elevated
temperatures. Any of these characteristics or activities of the
host cell may be naturally present in the host cell or may be
introduced or modified by genetic modification. A suitable cell is
a eukaryotic microorganism like e.g. a fungus, however, most
suitable as host cell are yeasts or filamentous fungi. Preferred
yeasts and filamentous fungi have already been defined herein.
[0096] As used herein the wording host cell has the same meaning as
cell.
[0097] The cell of the invention is preferably transformed with a
nucleic acid construct comprising the nucleotide sequence encoding
the xylose isomerase. The nucleic acid construct that is preferably
used is the same as the one used comprising the nucleotide sequence
encoding araA, araB or araD.
[0098] In another preferred embodiment of the invention, the cell
of the invention: [0099] expressing araA, araB and araD, and
exhibiting the ability to directly isomerise xylose into xylulose,
as earlier defined herein further comprises a genetic modification
that increases the flux of the pentose phosphate pathway, as
described in WO 06/009434. In particular, the genetic modification
causes an increased flux of the non-oxidative part pentose
phosphate pathway. A genetic modification that causes an increased
flux of the non-oxidative part of the pentose phosphate pathway is
herein understood to mean a modification that increases the flux by
at least a factor 1.1, 1.2, 1.5, 2, 5, 10 or 20 as compared to the
flux in a strain which is genetically identical except for the
genetic modification causing the increased flux. The flux of the
non-oxidative part of the pentose phosphate pathway may be measured
by growing the modified host on xylose as sole carbon source,
determining the specific xylose consumption rate and substracting
the specific xylitol production rate from the specific xylose
consumption rate, if any xylitol is produced. However, the flux of
the non-oxidative part of the pentose phosphate pathway is
proportional with the growth rate on xylose as sole carbon source,
preferably with the anaerobic growth rate on xylose as sole carbon
source. There is a linear relation between the growth rate on
xylose as sole carbon source (.mu..sub.max) and the flux of the
non-oxidative part of the pentose phosphate pathway. The specific
xylose consumption rate (Q.sub.s) is equal to the growth rate
(.mu.) divided by the yield of biomass on sugar (Y.sub.xs) because
the yield of biomass on sugar is constant (under a given set of
Conditions: anaerobic, growth medium, pH, genetic background of the
strain, etc.; i.e. Q.sub.s=.mu./Y.sub.xs). Therefore the increased
flux of the non-oxidative part of the pentose phosphate pathway may
be deduced from the increase in maximum growth rate under these
conditions. In a preferred embodiment, the cell comprises a genetic
modification that increases the flux of the pentose phosphate
pathway and has a specific xylose consumption rate of at least 346
mg xylose/g biomass/h.
[0100] Genetic modifications that increase the flux of the pentose
phosphate pathway may be introduced in the host cell in various
ways. These including e.g. achieving higher steady state activity
levels of xylulose kinase and/or one or more of the enzymes of the
non-oxidative part pentose phosphate pathway and/or a reduced
steady state level of unspecific aldose reductase activity. These
changes in steady state activity levels may be effected by
selection of mutants (spontaneous or induced by chemicals or
radiation) and/or by recombinant DNA technology e.g. by
overexpression or inactivation, respectively, of genes encoding the
enzymes or factors regulating these genes.
[0101] In a more preferred host cell, the genetic modification
comprises overexpression of at least one enzyme of the
(non-oxidative part) pentose phosphate pathway. Preferably the
enzyme is selected from the group consisting of the enzymes
encoding for ribulose-5-phosphate isomerase, ribulose-5-phosphate
epimerase, transketolase and transaldolase, as described in WO
06/009434.
[0102] Various combinations of enzymes of the (non-oxidative part)
pentose phosphate pathway may be overexpressed. E.g. the enzymes
that are overexpressed may be at least the enzymes
ribulose-5-phosphate isomerase and ribulose-5-phosphate epimerase;
or at least the enzymes ribulose-5-phosphate isomerase and
transketolase; or at least the enzymes ribulose-5-phosphate
isomerase and transaldolase; or at least the enzymes
ribulose-5-phosphate epimerase and transketolase; or at least the
enzymes ribulose-5-phosphate epimerase and transaldolase; or at
least the enzymes transketolase and transaldolase; or at least the
enzymes ribulose-5-phosphate epimerase, transketolase and
transaldolase; or at least the enzymes ribulose-5-phosphate
isomerase, transketolase and transaldolase; or at least the enzymes
ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, and
transaldolase; or at least the enzymes ribulose-5-phosphate
isomerase, ribulose-5-phosphate epimerase, and transketolase. In
one embodiment of the invention each of the enzymes
ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase,
transketolase and transaldolase are overexpressed in the host cell.
More preferred is a host cell in which the genetic modification
comprises at least overexpression of both the enzymes transketolase
and transaldolase as such a host cell is already capable of
anaerobic growth on xylose. In fact, under some conditions we have
found that host cells overexpressing only the transketolase and the
transaldolase already have the same anaerobic growth rate on xylose
as do host cells that overexpress all four of the enzymes, i.e. the
ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase,
transketolase and transaldolase. Moreover, host cells
overexpressing both of the enzymes ribulose-5-phosphate isomerase
and ribulose-5-phosphate epimerase are preferred over host cells
overexpressing only the isomerase or only the epimerase as
overexpression of only one of these enzymes may produce metabolic
imbalances.
[0103] There are various means available in the art for
overexpression of enzymes in the cells of the invention. In
particular, an enzyme may be overexpressed by increasing the copy
number of the gene coding for the enzyme in the host cell, e.g. by
integrating additional copies of the gene in the host cell's
genome, by expressing the gene from an episomal multicopy
expression vector or by introducing a episomal expression vector
that comprises multiple copies of the gene.
[0104] Alternatively overexpression of enzymes in the host cells of
the invention may be achieved by using a promoter that is not
native to the sequence coding for the enzyme to be overexpressed,
i.e. a promoter that is heterologous to the coding sequence to
which it is operably linked. Suitable promoters to this end have
already been defined herein.
[0105] The coding sequence used for overexpression of the enzymes
preferably is homologous to the host cell of the invention.
However, coding sequences that are heterologous to the host cell of
the invention may likewise be applied, as mentioned in WO
06/009434.
[0106] A nucleotide sequence used for overexpression of
ribulose-5-phosphate isomerase in the host cell of the invention is
a nucleotide sequence encoding a polypeptide with
ribulose-5-phosphate isomerase activity, whereby preferably the
polypeptide has an amino acid sequence having at least 50, 60, 70,
80, 90 or 95% identity with SEQ ID NO. 17 or whereby the nucleotide
sequence is capable of hybridising with the nucleotide sequence of
SEQ ID NO. 18, under moderate conditions, preferably under
stringent conditions.
[0107] A nucleotide sequence used for overexpression of
ribulose-5-phosphate epimerase in the host cell of the invention is
a nucleotide sequence encoding a polypeptide with
ribulose-5-phosphate epimerase activity, whereby preferably the
polypeptide has an amino acid sequence having at least 50, 60, 70,
80, 90 or 95% identity with SEQ ID NO. 19 or whereby the nucleotide
sequence is capable of hybridising with the nucleotide sequence of
SEQ ID NO. 20, under moderate conditions, preferably under
stringent conditions.
[0108] A nucleotide sequence used for overexpression of
transketolase in the host cell of the invention is a nucleotide
sequence encoding a polypeptide with transketolase activity,
whereby preferably the polypeptide has an amino acid sequence
having at least 50, 60, 70, 80, 90 or 95% identity with SEQ ID NO.
21 or whereby the nucleotide sequence is capable of hybridising
with the nucleotide sequence of SEQ ID NO. 22, under moderate
conditions, preferably under stringent conditions.
[0109] A nucleotide sequence used for overexpression of
transaldolase in the host cell of the invention is a nucleotide
sequence encoding a polypeptide with transaldolase activity,
whereby preferably the polypeptide has an amino acid sequence
having at least 50, 60, 70, 80, 90 or 95% identity with SEQ ID NO.
23 or whereby the nucleotide sequence is capable of hybridising
with the nucleotide sequence of SEQ ID NO. 24, under moderate
conditions, preferably under stringent conditions.
[0110] Overexpression of an enzyme, when referring to the
production of the enzyme in a genetically modified host cell, means
that the enzyme is produced at a higher level of specific enzymatic
activity as compared to the unmodified host cell under identical
conditions. Usually this means that the enzymatically active
protein (or proteins in case of multi-subunit enzymes) is produced
in greater amounts, or rather at a higher steady state level as
compared to the unmodified host cell under identical conditions.
Similarly this usually means that the mRNA coding for the
enzymatically active protein is produced in greater amounts, or
again rather at a higher steady state level as compared to the
unmodified host cell under identical conditions. Overexpression of
an enzyme is thus preferably determined by measuring the level of
the enzyme's specific activity in the host cell using appropriate
enzyme assays as described herein. Alternatively, overexpression of
the enzyme may determined indirectly by quantifying the specific
steady state level of enzyme protein, e.g. using antibodies
specific for the enzyme, or by quantifying the specific steady
level of the mRNA coding for the enzyme. The latter may
particularly be suitable for enzymes of the pentose phosphate
pathway for which enzymatic assays are not easily feasible as
substrates for the enzymes are not commercially available.
Preferably in the host cells of the invention, an enzyme to be
overexpressed is overexpressed by at least a factor 1.1, 1.2, 1.5,
2, 5, 10 or 20 as compared to a strain which is genetically
identical except for the genetic modification causing the
overexpression. It is to be understood that these levels of
overexpression may apply to the steady state level of the enzyme's
activity, the steady state level of the enzyme's protein as well as
to the steady state level of the transcript coding for the
enzyme.
[0111] In a further preferred embodiment, the host cell of the
invention: [0112] expressing araA, araB and araD, and exhibiting
the ability to directly isomerise xylose into xylulose, and
optionally [0113] comprising a genetic modification that increase
the flux of the pentose pathway as earlier defined herein further
comprises a genetic modification that increases the specific
xylulose kinase activity. Preferably the genetic modification
causes overexpression of a xylulose kinase, e.g. by overexpression
of a nucleotide sequence encoding a xylulose kinase. The gene
encoding the xylulose kinase may be endogenous to the host cell or
may be a xylulose kinase that is heterologous to the host cell. A
nucleotide sequence used for overexpression of xylulose kinase in
the host cell of the invention is a nucleotide sequence encoding a
polypeptide with xylulose kinase activity, whereby preferably the
polypeptide has an amino acid sequence having at least 50, 60, 70,
80, 90 or 95% identity with SEQ ID NO. 25 or whereby the nucleotide
sequence is capable of hybridising with the nucleotide sequence of
SEQ ID NO. 26, under moderate conditions, preferably under
stringent conditions.
[0114] A particularly preferred xylulose kinase is a xylose kinase
that is related to the xylulose kinase xylB from Piromyces as
mentioned in WO 03/0624430. A more preferred nucleotide sequence
for use in overexpression of xylulose kinase in the host cell of
the invention is a nucleotide sequence encoding a polypeptide with
xylulose kinase activity, whereby preferably the polypeptide has an
amino acid sequence having at least 45, 50, 55, 60, 65, 70, 80, 90
or 95% identity with SEQ ID NO. 27 or whereby the nucleotide
sequence is capable of hybridising with the nucleotide sequence of
SEQ ID NO. 28, under moderate conditions, preferably under
stringent conditions.
[0115] In the host cells of the invention, genetic modification
that increases the specific xylulose kinase activity may be
combined with any of the modifications increasing the flux of the
pentose phosphate pathway as described above, but this combination
is not essential for the invention. Thus, a host cell of the
invention comprising a genetic modification that increases the
specific xylulose kinase activity in addition to the expression of
the araA, araB and araD enzymes as defined herein is specifically
included in the invention. The various means available in the art
for achieving and analysing overexpression of a xylulose kinase in
the host cells of the invention are the same as described above for
enzymes of the pentose phosphate pathway. Preferably in the host
cells of the invention, a xylulose kinase to be overexpressed is
overexpressed by at least a factor 1.1, 1.2, 1.5, 2, 5, 10 or 20 as
compared to a strain which is genetically identical except for the
genetic modification causing the overexpression. It is to be
understood that these levels of overexpression may apply to the
steady state level of the enzyme's activity, the steady state level
of the enzyme's protein as well as to the steady state level of the
transcript coding for the enzyme.
[0116] In a further preferred embodiment, the host cell of the
invention: [0117] expressing araA, araB and araD, and exhibiting
the ability to directly isomerise xylose into xylulose, and
optionally [0118] comprising a genetic modification that increase
the flux of the pentose pathway and/or [0119] further comprising a
genetic modification that increases the specific xylulose kinase
activity all as earlier defined herein further comprises a genetic
modification that reduces unspecific aldose reductase activity in
the host cell. Preferably, unspecific aldose reductase activity is
reduced in the host cell by one or more genetic modifications that
reduce the expression of or inactivate a gene encoding an
unspecific aldose reductase, as described in WO 06/009434.
Preferably, the genetic modifications reduce or inactivate the
expression of each endogenous copy of a gene encoding an unspecific
aldose reductase in the host cell. Host cells may comprise multiple
copies of genes encoding unspecific aldose reductases as a result
of di-, poly- or aneu-ploidy, and/or the host cell may contain
several different (iso)enzymes with aldose reductase activity that
differ in amino acid sequence and that are each encoded by a
different gene. Also in such instances preferably the expression of
each gene that encodes an unspecific aldose reductase is reduced or
inactivated. Preferably, the gene is inactivated by deletion of at
least part of the gene or by disruption of the gene, whereby in
this context the term gene also includes any non-coding sequence
up- or down-stream of the coding sequence, the (partial) deletion
or inactivation of which results in a reduction of expression of
unspecific aldose reductase activity in the host cell. A nucleotide
sequence encoding an aldose reductase whose activity is to be
reduced in the host cell of the invention is a nucleotide sequence
encoding a polypeptide with aldose reductase activity, whereby
preferably the polypeptide has an amino acid sequence having at
least 50, 60, 70, 80, 90 or 95% identity with SEQ ID NO. 29 or
whereby the nucleotide sequence is capable of hybridising with the
nucleotide sequence of SEQ ID NO. 30 under moderate conditions,
preferably under stringent conditions.
[0120] In the host cells of the invention, the expression of the
araA, araB and araD enzymes as defined herein is combined with
genetic modification that reduces unspecific aldose reductase
activity. The genetic modification leading to the reduction of
unspecific aldose reductase activity may be combined with any of
the modifications increasing the flux of the pentose phosphate
pathway and/or with any of the modifications increasing the
specific xylulose kinase activity in the host cells as described
above, but these combinations are not essential for the invention.
Thus, a host cell expressing araA, araB, and araD, comprising an
additional genetic modification that reduces unspecific aldose
reductase activity is specifically included in the invention.
[0121] In a preferred embodiment, the host cell is CBS120327
deposited at the CBS Institute (The Netherlands) on Sep. 27,
2006.
[0122] In a further preferred embodiment, the invention relates to
modified host cells that are further adapted to L-arabinose (use
L-arabinose and/or convert it into L-ribulose, and/or xylulose
5-phosphate and/or into a desired fermentation product and
optionally xylose utilisation by selection of mutants, either
spontaneous or induced (e.g. by radiation or chemicals), for growth
on L-arabinose and optionally xylose, preferably on L-arabinose and
optionally xylose as sole carbon source, and more preferably under
anaerobic conditions. Selection of mutants may be performed by
serial passaging of cultures as e.g. described by Kuyper et al.
(2004, FEMS Yeast Res. 4: 655-664) and/or by cultivation under
selective pressure in a chemostat culture as is described in
Example 4 of WO 06/009434. This selection process may be continued
as long as necessary. This selection process is preferably carried
out during one week till one year. However, the selection process
may be carried out for a longer period of time if necessary. During
the selection process, the cells are preferably cultured in the
presence of approximately 20 g/l L-arabinose and/or approximately
20 g/l xylose. The cell obtained at the end of this selection
process is expected to be improved as to its capacities of using
L-arabinose and/or xylose, and/or converting L-arabinose into
L-ribulose and/or xylulose 5-phosphate and/or a desired
fermentation product such as ethanol. In this context "improved
cell" may mean that the obtained cell is able to use L-arabinose
and/or xylose in a more efficient way than the cell it derives
from. For example, the obtained cell is expected to better grow:
increase of the specific growth rate of at least 2% than the cell
it derives from under the same conditions. Preferably, the increase
is of at least 4%, 6%, 8%, 10%, 15%, 20%, 25% or more. The specific
growth rate may be calculated from OD.sub.660 as known to the
skilled person. Therefore, by monitoring the OD.sub.660, one can
deduce the specific growth rate. In this context "improved cell"
may also mean that the obtained cell converts L-arabinose into
L-ribulose and/or xylulose 5-phosphate and/or a desired
fermentation product such as ethanol in a more efficient way than
the cell it derives from. For example, the obtained cell is
expected to produce higher amounts of L-ribulose and/or xylulose
5-phosphate and/or a desired fermentation product such as ethanol:
increase of at least one of these compounds of at least 2% than the
cell it derives from under the same conditions. Preferably, the
increase is of at least 4%, 6%, 8%, 10%, 15%, 20%, 25% or more. In
this context "improved cell" may also mean that the obtained cell
converts xylose into xylulose and/or a desired fermentation product
such as ethanol in a more efficient way than the cell it derives
from. For example, the obtained cell is expected to produce higher
amounts of xylulose and/or a desired fermentation product such as
ethanol: increase of at least one of these compounds of at least 2%
than the cell it derives from under the same conditions.
Preferably, the increase is of at least 4%, 6%, 8%, 10%, 15%, 20%,
25% or more.
[0123] In a preferred host cell of the invention at least one of
the genetic modifications described above, including modifications
obtained by selection of mutants, confer to the host cell the
ability to grow on L-arabinose and optionally xylose as carbon
source, preferably as sole carbon source, and preferably under
anaerobic conditions. Preferably the modified host cell produce
essentially no xylitol, e.g. the xylitol produced is below the
detection limit or e.g. less than 5, 2, 1, 0.5, or 0.3% of the
carbon consumed on a molar basis.
[0124] Preferably the modified host cell has the ability to grow on
L-arabinose and optionally xylose as sole carbon source at a rate
of at least 0.001, 0.005, 0.01, 0.03, 0.05, 0.1, 0.2, 0.25 or 0.3
h.sup.-1 under aerobic conditions, or, if applicable, at a rate of
at least 0.001, 0.005, 0.01, 0.03, 0.05, 0.07, 0.08, 0.09, 0.1,
0.12, 0.15 or 0.2 h.sup.-1 under anaerobic conditions Preferably
the modified host cell has the ability to grow on a mixture of
glucose and L-arabinose and optionally xylose (in a 1:1 weight
ratio) as sole carbon source at a rate of at least 0.001, 0.005,
0.01, 0.03, 0.05, 0.1, 0.2, 0.25 or 0.3 h.sup.-1 under aerobic
conditions, or, if applicable, at a rate of at least 0.001, 0.005,
0.01, 0.03, 0.05, 0.1, 0.12, 0.15, or 0.2 h.sup.-1 under anaerobic
conditions.
[0125] Preferably, the modified host cell has a specific
L-arabinose and optionally xylose consumption rate of at least 346,
350, 400, 500, 600, 650, 700, 750, 800, 900 or 1000 mg/g cells/h.
Preferably, the modified host cell has a yield of fermentation
product (such as ethanol) on L-arabinose and optionally xylose that
is at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 85, 90, 95
or 98% of the host cell's yield of fermentation product (such as
ethanol) on glucose. More preferably, the modified host cell's
yield of fermentation product (such as ethanol) on L-arabinose and
optionally xylose is equal to the host cell's yield of fermentation
product (such as ethanol) on glucose. Likewise, the modified host
cell's biomass yield on L-arabinose and optionally xylose is
preferably at least 55, 60, 70, 80, 85, 90, 95 or 98% of the host
cell's biomass yield on glucose. More preferably, the modified host
cell's biomass yield on L-arabinose and optionally xylose is equal
to the host cell's biomass yield on glucose. It is understood that
in the comparison of yields on glucose and L-arabinose and
optionally xylose both yields are compared under aerobic conditions
or both under anaerobic conditions.
[0126] In a more preferred embodiment, the host cell is CBS120328
deposited at the CBS Institute (The Netherlands) on Sep. 27, 2006
or CBS121879 deposited at the CBS Institute (The Netherlands) on
Sep. 20, 2007.
[0127] In a preferred embodiment, the cell expresses one or more
enzymes that confer to the cell the ability to produce at least one
fermentation product selected from the group consisting of ethanol,
lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid,
succinic acid, citric acid, malic acid, fumaric acid, an amino
acid, 1,3-propane-diol, ethylene, glycerol, butanol, a
.beta.-lactam antibiotic and a cephalosporin. In a more preferred
embodiment, the host cell of the invention is a host cell for the
production of ethanol. In another preferred embodiment, the
invention relates to a transformed host cell for the production of
fermentation products other than ethanol. Such non-ethanolic
fermentation products include in principle any bulk or fine
chemical that is producible by a eukaryotic microorganism such as a
yeast or a filamentous fungus. Such fermentation products include
e.g. lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic
acid, succinic acid, citric acid, malic acid, fumaric acid, an
amino acid, 1,3-propane-diol, ethylene, glycerol, butanol, a
.beta.-lactam antibiotic and a cephalosporin. A preferred host cell
of the invention for production of non-ethanolic fermentation
products is a host cell that contains a genetic modification that
results in decreased alcohol dehydrogenase activity.
Method
[0128] In a further aspect, the invention relates to fermentation
processes in which a host cell of the invention is used for the
fermentation of a carbon source comprising a source of L-arabinose
and optionally a source of xylose. Preferably, the source of
L-arabinose and the source of xylose are L-arabinose and xylose. In
addition, the carbon source in the fermentation medium may also
comprise a source of glucose. The source of L-arabinose, xylose or
glucose may be L-arabinose, xylose or glucose as such or may be any
carbohydrate oligo- or polymer comprising L-arabinose, xylose or
glucose units, such as e.g. lignocellulose, xylans, cellulose,
starch, arabinan and the like. For release of xylose or glucose
units from such carbohydrates, appropriate carbohydrases (such as
xylanases, glucanases, amylases and the like) may be added to the
fermentation medium or may be produced by the modified host cell.
In the latter case the modified host cell may be genetically
engineered to produce and excrete such carbohydrases. An additional
advantage of using oligo- or polymeric sources of glucose is that
it enables to maintain a low(er) concentration of free glucose
during the fermentation, e.g. by using rate-limiting amounts of the
carbohydrases. This, in turn, will prevent repression of systems
required for metabolism and transport of non-glucose sugars such as
xylose. In a preferred process the modified host cell ferments both
the L-arabinose (optionally xylose) and glucose, preferably
simultaneously in which case preferably a modified host cell is
used which is insensitive to glucose repression to prevent diauxic
growth. In addition to a source of L-arabinose, optionally xylose
(and glucose) as carbon source, the fermentation medium will
further comprise the appropriate ingredient required for growth of
the modified host cell. Compositions of fermentation media for
growth of microorganisms such as yeasts or filamentous fungi are
well known in the art.
[0129] In a preferred process, there is provided a process for
producing a fermentation product selected from the group consisting
of ethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid,
acetic acid, succinic acid, citric acid, malic acid, fumaric acid,
an amino acid, 1,3-propane-diol, ethylene, glycerol, butanol, a
.beta.-lactam antibiotic and a cephalosporin whereby the process
comprises the steps of: [0130] (a) fermenting a medium containing a
source of L-arabinose and optionally xylose with a modified host
cell as defined herein, whereby the host cell ferments L-arabinose
and optionally xylose to the fermentation product, and optionally,
[0131] (b) recovering the fermentation product.
[0132] The fermentation process is a process for the production of
a fermentation product such as e.g. ethanol, lactic acid,
3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid,
citric acid, malic acid, fumaric acid, an amino acid,
1,3-propane-diol, ethylene, glycerol, butanol, a .beta.-lactam
antibiotic, such as Penicillin G or Penicillin V and fermentative
derivatives thereof, and/or a cephalosporin. The fermentation
process may be an aerobic or an anaerobic fermentation process. An
anaerobic fermentation process is herein defined as a fermentation
process run in the absence of oxygen or in which substantially no
oxygen is consumed, preferably less than 5, 2.5 or 1 mmol/L/h, more
preferably 0 mmol/L/h is consumed (i.e. oxygen consumption is not
detectable), and wherein organic molecules serve as both electron
donor and electron acceptors. In the absence of oxygen, NADH
produced in glycolysis and biomass formation, cannot be oxidised by
oxidative phosphorylation. To solve this problem many
microorganisms use pyruvate or one of its derivatives as an
electron and hydrogen acceptor thereby regenerating NAD.sup.+.
Thus, in a preferred anaerobic fermentation process pyruvate is
used as an electron (and hydrogen acceptor) and is reduced to
fermentation products such as ethanol, lactic acid,
3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid,
citric acid, malic acid, fumaric acid, an amino acid,
1,3-propane-diol, ethylene, glycerol, butanol, a .beta.-lactam
antibiotics and a cephalosporin. In a preferred embodiment, the
fermentation process is anaerobic. An anaerobic process is
advantageous since it is cheaper than aerobic processes: less
special equipment is needed. Furthermore, anaerobic processes are
expected to give a higher product yield than aerobic processes.
Under aerobic conditions, usually the biomass yield is higher than
under anaerobic conditions. As a consequence, usually under aerobic
conditions, the expected product yield is lower than under
anaerobic conditions. According to the inventors, the process of
the invention is the first anaerobic fermentation process with a
medium comprising a source of L-arabinose that has been developed
so far.
[0133] In another preferred embodiment, the fermentation process is
under oxygen-limited conditions. More preferably, the fermentation
process is aerobic and under oxygen-limited conditions. An
oxygen-limited fermentation process is a process in which the
oxygen consumption is limited by the oxygen transfer from the gas
to the liquid. The degree of oxygen limitation is determined by the
amount and composition of the ingoing gasflow as well as the actual
mixing/mass transfer properties of the fermentation equipment used.
Preferably, in a process under oxygen-limited conditions, the rate
of oxygen consumption is at least 5.5, more preferably at least 6
and even more preferably at least 7 mmol/L/h.
[0134] The fermentation process is preferably run at a temperature
that is optimal for the modified cell. Thus, for most yeasts or
fungal cells, the fermentation process is performed at a
temperature which is less than 42.degree. C., preferably less than
38.degree. C. For yeast or filamentous fungal host cells, the
fermentation process is preferably performed at a temperature which
is lower than 35, 33, 30 or 28.degree. C. and at a temperature
which is higher than 20, 22, or 25.degree. C.
[0135] A preferred process is a process for the production of
ethanol, whereby the process comprises the steps of: (a) fermenting
a medium containing a source of L-arabinose and optionally xylose
with a modified host cell as defined herein, whereby the host cell
ferments L-arabinose and optionally xylose to ethanol; and
optionally, (b) recovery of the ethanol. The fermentation medium
may also comprise a source of glucose that is also fermented to
ethanol. In a preferred embodiment, the fermentation process for
the production of ethanol is anaerobic. Anaerobic has already been
defined earlier herein. In another preferred embodiment, the
fermentation process for the production of ethanol is aerobic. In
another preferred embodiment, the fermentation process for the
production of ethanol is under oxygen-limited conditions, more
preferably aerobic and under oxygen-limited conditions.
Oxygen-limited conditions have already been defined earlier
herein.
[0136] In the process, the volumetric ethanol productivity is
preferably at least 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 5.0 or 10.0 g
ethanol per litre per hour. The ethanol yield on L-arabinose and
optionally xylose and/or glucose in the process preferably is at
least 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 95 or 98%. The
ethanol yield is herein defined as a percentage of the theoretical
maximum yield, which, for glucose and L-arabinose and optionally
xylose is 0.51 g. ethanol per g. glucose or xylose. In another
preferred embodiment, the invention relates to a process for
producing a fermentation product selected from the group consisting
of lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic
acid, succinic acid, citric acid, malic acid, fumaric acid, an
amino acid, 1,3-propane-diol, ethylene, glycerol, butanol, a
.beta.-lactam antibiotic and a cephalosporin. The process
preferably comprises the steps of (a) fermenting a medium
containing a source of L-arabinose and optionally xylose with a
modified host cell as defined herein above, whereby the host cell
ferments L-arabinose and optionally xylose to the fermentation
product, and optionally, (b) recovery of the fermentation product.
In a preferred process, the medium also contains a source of
glucose.
[0137] In the fermentation process of the invention leading to the
production of ethanol, several advantages can be cited by
comparison to known ethanol fermentations processes: [0138]
anaerobic processes are possible. [0139] oxygen limited conditions
are also possible. [0140] higher ethanol yields and ethanol
production rates can be obtained. [0141] the strain used may be
able to use L-arabinose and optionally xylose.
[0142] Alternatively to the fermentation processes described above,
another fermentation process is provided as a further aspect of the
invention wherein, at least two distinct cells are used for the
fermentation of a carbon source comprising at least two sources of
carbon selected from the group consisting of but not limited
thereto: a source of L-arabinose, a source of xylose and a source
of glucose. In this fermentation process, "at least two distinct
cells" means this process is preferably a co-fermentation process.
In one preferred embodiment, two distinct cells are used: one being
the one of the invention as earlier defined able to use
L-arabinose, and/or to convert it into L-ribulose, and/or xylulose
5-phosphate and/or into a desired fermentation product such as
ethanol and optionally being able to use xylose, the other one
being for example a strain which is able to use xylose and/or
convert it into a desired fermentation product such as ethanol as
defined in WO 03/062430 and/or WO 06/009434. A cell which is able
to use xylose is preferably a strain which exhibits the ability of
directly isomerising xylose into xylulose (in one step) as earlier
defined herein. These two distinct strains are preferably cultived
in the presence of a source of L-arabinose, a source of xylose and
optionally a source of glucose. Three distinct cells or more may be
co-cultivated and/or three or more sources of carbon may be used,
provided at least one cell is able to use at least one source of
carbon present and/or to convert it into a desired fermentation
product such as ethanol. The expression "use at least one source of
carbon" has the same meaning as the expression "use of
L-arabinose". The expression "convert it (i.e. a source of carbon)
into a desired fermentation product has the same meaning as the
expression "convert L-arabinose into a desired fermentation
product".
[0143] In a preferred embodiment, the invention relates to a
process for producing a fermentation product selected from the
group consisting of ethanol, lactic acid, 3-hydroxy-propionic acid,
acrylic acid, acetic acid, succinic acid, citric acid, malic acid,
fumaric acid, amino acids, 1,3-propane-diol, ethylene, glycerol,
butanol, .beta.-lactam antibiotics and cephalosporins, whereby the
process comprises the steps of: [0144] (a) fermenting a medium
containing at least a source of L-arabinose and a source of xylose
with a cell of the invention as earlier defined herein and a cell
able to use xylose and/or exhibiting the ability to directly
isomerise xylose into xylulose, whereby each cell ferments
L-arabinose and/or xylose to the fermentation product, and
optionally, [0145] (b) recovering the fermentation product. All
preferred embodiments of the fermentation processes as described
above are also preferred embodiments of this further fermentation
processes: identity of the fermentation product, identity of source
of L-arabinose and source of xylose, conditions of fermentation
(aerobical or anaerobical conditions, oxygen-limited conditions,
temperature at which the process is being carried out, productivity
of ethanol, yield of ethanol).
Genetic Modifications
[0146] For overexpression of enzymes in the host cells of the
inventions as described above, as well as for additional genetic
modification of host cells, preferably yeasts, host cells are
transformed with the various nucleic acid constructs of the
invention by methods well known in the art. Such methods are e.g.
known from standard handbooks, such as Sambrook and Russel (2001)
"Molecular Cloning: A Laboratory Manual (3.sup.rd edition), Cold
Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or
F. Ausubel et al, eds., "Current protocols in molecular biology",
Green Publishing and Wiley Interscience, New York (1987). Methods
for transformation and genetic modification of fungal host cells
are known from e.g. EP-A-0 635 574, WO 98/46772, WO 99/60102 and WO
00/37671.
[0147] Promoters for use in the nucleic acid constructs for
overexpression of enzymes in the host cells of the invention have
been described above. In the nucleic acid constructs for
overexpression, the 3'-end of the nucleotide acid sequence encoding
the enzyme(s) preferably is operably linked to a transcription
terminator sequence. Preferably the terminator sequence is operable
in a host cell of choice, such as e.g. the yeast species of choice.
In any case the choice of the terminator is not critical; it may
e.g. be from any yeast gene, although terminators may sometimes
work if from a non-yeast, eukaryotic, gene. The transcription
termination sequence further preferably comprises a polyadenylation
signal. Preferred terminator sequences are the alcohol
dehydrogenase (ADH1) and the PGI1 terminators. More preferably, the
ADH1 and the PGI1 terminators are both from S. cerevisiae (SEQ ID
NO:50 and SEQ ID NO:53 respectively).
[0148] Optionally, a selectable marker may be present in the
nucleic acid construct. As used herein, the term "marker" refers to
a gene encoding a trait or a phenotype which permits the selection
of, or the screening for, a host cell containing the marker. The
marker gene may be an antibiotic resistance gene whereby the
appropriate antibiotic can be used to select for transformed cells
from among cells that are not transformed. Preferably however,
non-antibiotic resistance markers are used, such as auxotrophic
markers (URA3, TRP1, LEU). In a preferred embodiment the host cells
transformed with the nucleic acid constructs are marker gene free.
Methods for constructing recombinant marker gene free microbial
host cells are disclosed in EP-A-0 635 574 and are based on the use
of bidirectional markers. Alternatively, a screenable marker such
as Green Fluorescent Protein, lacZ, luciferase, chloramphenicol
acetyltransferase, beta-glucuronidase may be incorporated into the
nucleic acid constructs of the invention allowing to screen for
transformed cells.
[0149] Optional further elements that may be present in the nucleic
acid constructs of the invention include, but are not limited to,
one or more leader sequences, enhancers, integration factors,
and/or reporter genes, intron sequences, centromers, telomers
and/or matrix attachment (MAR) sequences. The nucleic acid
constructs of the invention may further comprise a sequence for
autonomous replication, such as an ARS sequence. Suitable episomal
nucleic acid constructs may e.g. be based on the yeast 2.mu. or
pKD1 (Fleer et al., 1991, Biotechnology 9:968-975) plasmids.
Alternatively the nucleic acid construct may comprise sequences for
integration, preferably by homologous recombination. Such sequences
may thus be sequences homologous to the target site for integration
in the host cell's genome. The nucleic acid constructs of the
invention can be provided in a manner known per se, which generally
involves techniques such as restricting and linking nucleic
acids/nucleic acid sequences, for which reference is made to the
standard handbooks, such as Sambrook and Russel (2001) "Molecular
Cloning: A Laboratory Manual (3.sup.rd edition), Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press.
[0150] Methods for inactivation and gene disruption in yeast or
fungi are well known in the art (see e.g. Fincham, 1989, Microbiol
Rev. 53(1):148-70 and EP-A-0 635 574).
[0151] In this document and in its claims, the verb "to comprise"
and its conjugations is used in its non-limiting sense to mean that
items following the word are included, but items not specifically
mentioned are not excluded. In addition, reference to an element by
the indefinite article "a" or "an" does not exclude the possibility
that more than one of the element is present, unless the context
clearly requires that there be one and only one of the elements.
The indefinite article "a" or "an" thus usually means "at least
one".
[0152] The invention is further described by the following
examples, which should not be construed as limiting the scope of
the invention.
EXAMPLES
Plasmid and Strain Construction
Strains
[0153] The L-arabinose consuming Sachharomyces cerevisiae strain
described in this work is based on strain RWB220, which is itself a
derivative of RWB217. RWB217 is a CEN.PK strain in which four genes
coding for the expression of enzymes in the pentose phosphate
pathway have been overexpressed, TAL1, TKL1, RPE1, RKI1 (Kuyper et
al., 2005a). In addition the gene coding for an aldose reductase
(GRE3), has been deleted. Strain RWB217 also contains two plasmids,
a single copy plasmid with a LEU2 marker for overexpression of the
xylulokinase (XKS1) and an episomal, multicopy plasmid with URA3 as
the marker for the expression of the xylose isomerase, XylA. RWB217
was subjected to a selection procedure for improved growth on
xylose which is described in Kuyper et al. (2005b). The procedure
resulted in two pure strains, RWB218 (Kuyper et al., 2005b) and
RWB219. The difference between RWB218 and RWB219 is that after the
selection procedure, RWB218 was obtained by plating and restreaking
on mineral medium with glucose as the carbon source, while for
RWB219 xylose was used.
[0154] Strain RWB219 was grown non-selectively on YP with glucose
(YPD) as the carbon source in order to facilitate the loss of both
plasmids. After plating on YPD single colonies were tested for
plasmid loss by looking at uracil and leucine auxotrophy. A strain
that had lost both plasmids was transformed with pSH47, containing
the cre recombinase, in order to remove a KanMX cassette (Guldener
et al., 1996), still present after integrating the RKI1
overexpression construct. Colonies with the plasmid were
resuspended in Yeast Peptone medium (YP) (10 g/l yeast extract and
20 g/l peptone both from BD Difco Belgium) with 1% galactose and
incubated for 1 hour at 30.degree. C. About 200 cells were plated
on YPD. The resulting colonies were checked for loss of the KanMX
marker (G418 resistance) and pSH47 (URA3). A strain that had lost
both the KanMX marker and the pSH47 plasmid was then named RWB220.
To obtain the strain tested in this patent, RWB220 was transformed
with pRW231 and pRW243 (table 2), resulting in strain IMS0001.
[0155] During construction strains were maintained on complex YP:
10 g l.sup.-1 yeast extract (BD Difco), 20 g l.sup.-1 peptone (BD
Difco) or synthetic medium (MY) (Verduyn et al., 1992) supplemented
with glucose (2%) as carbon source (YPD or MYD) and 1.5% agar in
the case of plates. After transformation with plasmids strains were
plated on MYD. Transformations of yeast were done according to
Gietz and Woods (2002). Plasmids were amplified in Escherichia coli
strain XL-1 blue (Stratagene, La Jolla, Calif., USA).
Transformation was performed according to Inoue et al. (1990). E.
coli was grown on LB (Luria-Bertani) plates or in liquid TB
(Terrific Broth) medium for the isolation of plasmids (Sambrook et
al, 1989).
Plasmids
[0156] In order to grow on L-arabinose, yeast needs to express
three different genes, an L-arabinose isomerase (AraA), a
L-ribulokinase (AraB), and a L-ribulose-5-P 4-epimerase (AraD)
(Becker and Boles, 2003). In this work we have chosen to express
AraA, AraB, and AraD from the lactic acid bacterium Lactobacillus
plantarum in S. cerevisiae. Because the eventual aim is to consume
L-arabinose in combination with other sugars, like D-xylose, the
genes encoding the bacterial L-arabinose pathway were combined on
the same plasmid with the genes coding for D-xylose
consumption.
[0157] In order to get a high level of expression, the L. plantarum
AraA and AraD genes were ligated into plasmid pAKX002, the 2.mu.
XylA bearing plasmid.
[0158] The AraA cassette was constructed by amplifying a truncated
version of the TDH3 promoter with SpeI5'Ptdh3 and 5'AraAPtdh3 (SEQ
ID NO: 49), the AraA gene with Ptdh5'AraA and Tadh3'AraA and the
ADH1 terminator (SEQ ID NO:50) with 3'AraATadh1 and 3'Tadh1-SpeI.
The three fragments were extracted from gel and mixed in roughly
equimolar amounts. On this mixture a PCR was performed using the
SpeI-5'Ptdh3 and 3'Tadh1SpeI oligos. The resulting
P.sub.TDH3-AraA-T.sub.ADH1 cassette was gel purified, cut at the 5'
and 3' SpeI sites and then ligated into pAKX002 cut with NheI,
resulting in plasmid pRW230.
[0159] The AraD construct was made by first amplifying a truncated
version of the HXT7 promoter (SEQ ID NO:52) with oligos SalI5'Phxt7
and 5'AraDPhxt, the AraD gene with Phxt5'AraD and Tpgi3'AraD and
the GPI1 terminator (SEQ ID NO:53) region with the 3'AraDTpgi and
3'TpgiSalI oligos. The resulting fragments were extracted from gel
and mixed in roughly equimolar amounts, after which a PCR was
performed using the SalI5'Phxt7 and 3'Tpgi1SalI oligos. The
resulting P.sub.HXT7-AraD-T.sub.PGI1 cassette was gel purified, cut
at the 5' and 3' SalI sites and then ligated into pRW230 cut with
XhoI, resulting in plasmid pRW231 (FIG. 1).
[0160] Since too high an expression of the L-ribulokinase is
detrimental to growth (Becker and Boles, 2003), the AraB gene was
combined with the XKS1 gene, coding for xylulokinase, on an
integration plasmid. For this, p415ADHXKS (Kuyper et al., 2005a)
was first changed into pRW229, by cutting both p415ADHXKS and
pRS305 with PvuI and ligating the ADHXKS-containing PvuI fragment
from p415ADHXKS to the vector backbone from pRS305, resulting in
pRW229.
[0161] A cassette, containing the L. plantarum AraB gene between
the PGI1 promoter (SEQ ID NO:51) and ADH1 terminator (SEQ ID NO:50)
was made by amplifying the PGI1 promoter with the SacI5'Ppgi1 and
5'AraBPpgi1 oligos, the AraB gene with the Ppgi5'AraB and
Tadh3'AraB oligos and the ADH1 terminator with 3'AraBTadh1 and
3'Tadh1SacI oligos. The three fragments were extracted from gel and
mixed in roughly equimolar amounts. On this mixture a PCR was
performed using the SacI-5'Ppgi 1 and 3'Tadh1SacI oligos. The
resulting P.sub.PGI1-AraB-T.sub.ADH1 cassette was gel purified, cut
at the 5' and 3' Sad sites and then ligated into pRW229 cut with
SacI, resulting in plasmid pRW243 (FIG. 1).
[0162] Strain RWB220 was transformed with pRW231 and pRW243 (table
2), resulting in strain IMS0001.
[0163] Restriction endonucleases (New England Biolabs, Beverly,
Mass., USA and Roche, Basel, Switzerland) and DNA ligase (Roche)
were used according to the manufacturers' specifications. Plasmid
isolation from E. coli was performed with the Qiaprep spin miniprep
kit (Qiagen, Hilden, Germany). DNA fragments were separated on a 1%
agarose (Sigma, St. Louis, Mo., USA) gel in 1.times.TBE (Sambrook
et al, 1989). Isolation of fragments from gel was carried out with
the Qiaquick gel extraction kit (Quiagen). Amplification of the
(elements of the) AraA, AraB and AraD cassettes was done with
Vent.sub.R DNA polymerase (New England Biolabs) according to the
manufacturer's specification. The template was chromosomal DNA of
S. cerevisiae CEN.PK113-7D for the promoters and terminators, or
Lactobacillus plantarum DSM20205 for the Ara genes. The polymerase
chain reaction (PCR) was performed in a Biometra TGradient
Thermocycler (Biometra, Gottingen, Germany) with the following
settings: 30 cycles of 1 min annealing at 55.degree. C., 60.degree.
C. or 65.degree. C., 1 to 3 min extension at 75.degree. C.,
depending on expected fragment size, and 1 min denaturing at
94.degree. C.
Cultivation and Media
[0164] Shake-flask cultivations were performed at 30.degree. C. in
a synthetic medium (Verduyn et al., 1992). The pH of the medium was
adjusted to 6.0 with 2 M KOH prior to sterilisation. For solid
synthetic medium, 1.5% of agar was added.
[0165] Pre-cultures were prepared by inoculating 100 ml medium
containing the appropriate sugar in a 500-ml shake flask with a
frozen stock culture. After incubation at 30.degree. C. in an
orbital shaker (200 rpm), this culture was used to inoculate either
shake-flask cultures or fermenter cultures. The synthetic medium
for anaerobic cultivation was supplemented with 0.01 g l.sup.-1
ergosterol and 0.42 g Tween 80 dissolved in ethanol (Andreasen and
Stier, 1953; Andreasen and Stier, 1954). Anaerobic (sequencing)
batch cultivation was carried out at 30.degree. C. in 2-1
laboratory fermenters (Applikon, Schiedam, The Netherlands) with a
working volume of 1 l. The culture pH was maintained at pH 5.0 by
automatic addition of 2 M KOH. Cultures were stirred at 800 rpm and
sparged with 0.5 l min.sup.-1 nitrogen gas (<10 ppm oxygen). To
minimise diffusion of oxygen, fermenters were equipped with
Norprene tubing (Cole Palmer Instrument company, Vernon Hills,
USA). Dissolved oxygen was monitored with an oxygen electrode
(Applisens, Schiedam, The Netherlands). Oxygen-limited conditions
were achieved in the same experimental set-up by headspace aeration
at approximately 0.05 l min.sup.-1.
Determination of Dry Weight
[0166] Culture samples (10.0 ml) were filtered over preweighed
nitrocellulose filters (pore size 0.45 .mu.m; Gelman laboratory,
Ann Arbor, USA). After removal of medium, the filters were washed
with demineralised water and dried in a microwave oven (Bosch,
Stuttgart, Germany) for 20 min at 360 W and weighed. Duplicate
determinations varied by less than 1%.
Gas Analysis
[0167] Exhaust gas was cooled in a condensor (2.degree. C.) and
dried with a Permapure dryer type MD-110-48P-4 (Permapure, Toms
River, USA). O2 and CO2 concentrations were determined with a NGA
2000 analyser (Rosemount Analytical, Orrville, USA). Exhaust
gasflow rate and specific oxygen-consumption and carbondioxide
production rates were determined as described previously (Van Urk
et al., 1988; Weusthuis et al., 1994). In calculating these
biomass-specific rates, volume changes caused by withdrawing
culture samples were taken account for.
Metabolite Analysis
[0168] Glucose, xylose, arabinose, xylitol, organic acids, glycerol
and ethanol were analysed by HPLC using a Waters Alliance 2690 HPLC
(Waters, Milford, USA) supplied with a BioRad HPX 87H column
(BioRad, Hercules, USA), a Waters 2410 refractive-index detector
and aWaters 2487 UV detector. The column was eluted at 60.degree.
C. with 0.5 g l.sup.-1 sulphuric acid at a flow rate of 0.6 ml
min.sup.-1.
Assay for Xylulose 5-Phosphate (Zaldivar J., et al, Appl.
Microbiol. Biotechnol., (2002), 59:436-442)
[0169] For the analysis of intracellular metabolites such as
xylulose 5-phosphate, 5 ml broth was harvested in duplicate from
the reactors, before glucose exhaustion (at 22 and 26 h of
cultivation) and after glucose exhaustion (42, 79 and 131 h of
cultivation). Procedures for metabolic arrest, solid-phase
extraction of metabolites and analysis have been described in
detail by Smits H. P. et al. (Anal. Biochem., 261:36-42, (1998)).
However, the analysis by high-pressure ion exchange chromatography
coupled to pulsed amperometric detection used to analyze cell
extracts, was slightly modified. Solutions used were eluent A, 75
mM NaOH, and eluent B, 500 mM NaAc. To prevent contamination of
carbonate in the eluent solutions, a 50% NaOH solution with low
carbonate concentration (Baker Analysed, Deventer, The Netherlands)
was used instead of NaOH pellets. The eluents were degassed with
Helium (He) for 30 min and then kept under a He atmosphere. The
gradient pump was programmed to generate the following gradients:
100% A and 0% B (0 min), a linear decrease of A to 70% and a linear
increase of B to 30% (0-30 min), a linear decrease of A to 30% and
a linear increase of B to 70% (30-70 min), a linear decrease of A
to 0% and a linear increase of B to 100% (70-75 min), 0% A and 100%
B (75-85 min), a linear increase of A to 100% and a linear decrease
of B to 0% (85-95 min). The mobile phase was run at a flow rate of
1 ml/min. Other conditions were according to Smits et al.
(1998).
Carbon Recovery
[0170] Carbon recoveries were calculated as carbon in products
formed, divided by the total amount of sugar carbon consumed, and
were based on a carbon content of biomass of 48%. To correct for
ethanol evaporation during the fermentations, the amount of ethanol
produced was assumed to be equal to the measured cumulative
production of CO.sub.2 minus the CO.sub.2 production that occurred
due to biomass synthesis (5.85 mmol CO.sub.2 per gram biomass
(Verduyn et al., 1990)) and the CO.sub.2 associated with acetate
formation.
Selection for Growth on L-Arabinose
[0171] Strain IMS0001 (CBS120327 deposited at the CBS on 27/09/06),
containing the genes encoding the pathways for both xylose (XylA
and XKS1) and arabinose (AraA, AraB, AraD) metabolization, was
constructed according the procedure described above. Although
capable of growing on xylose (data not shown), strain IMS0001 did
not seem to be capable of growing on solid synthetic medium
supplemented with 2% L-arabinose. Mutants of IMS0001 capable of
utilizing L-arabinose as carbon source for growth were selected by
serial transfer in shake flasks and by sequencing-batch cultivation
in fermenters (SBR).
[0172] For the serial transfer experiments, a 500-ml shake flask
containing 100 ml synthetic medium containing 0.5% galactose were
inoculated with either strain IMS0001, or the reference strain
RWB219. After 72 hours, at an optical density at 660 nm of 3.0, the
cultures were used to inoculate a new shake flask containing 0.1%
galactose and 2% arabinose. Based on HPLC determination with
D-ribulose as calibration standard, it was determined that already
in the first cultivations of strain IMS0001, on medium containing a
galactose/arabinose mixture, part of the arabinose was converted
into ribulose and subsequently excreted to the supernatant. These
HPLC analyses were performed using a Waters Alliance 2690 HPLC
(Waters, Milford, USA) supplied with a BioRad HPX 87H column
(BioRad, Hercules, USA), a Waters 2410 refractive-index detector
and a Waters 2487 UV detector. The column was eluted at 60.degree.
C. with 0.5 g sulphuric acid at a flow rate of 0.6 ml min.sup.-1.
In contrast to the reference strain RWB219, the OD.sub.660 of the
culture of strain IMS0001 increased after depletion of the
galactose. When after approximately 850 hours growth on arabinose
by strain IMS0001 was observed (FIG. 2), this culture was
transferred at an OD.sub.660 of 1.7 to a shake flask containing 2%
arabinose. Cultures were then sequentially transferred to fresh
medium containing 2% arabinose at an OD.sub.660 of 2-3. Utilization
of arabinose was confirmed by occasionally measuring arabinose
concentrations by HPLC (data not shown). The growth rate of these
cultures increased from 0 to 0.15 h.sup.-1 in approximately 3600
hours (FIG. 3).
[0173] A batch fermentation under oxygen limited conditions was
started by inoculating 1 l of synthetic medium supplemented with 2%
of arabinose with a 100 ml shake flask culture of arabinose-grown
IMS0001 cells with a maximum growth rate on 2% of L-arabinose of
approximately 0.12 h.sup.-1. When growth on arabinose was observed,
the culture was subjected to anaerobic conditions by sparging with
nitrogen gas. The sequential cycles of anaerobic batch cultivation
were started by either manual or automated replacement of 90% of
the culture with synthetic medium with 20 g l.sup.-1 arabinose. For
each cycle during the SBR fermentation, the exponential growth rate
was estimated from the CO.sub.2 profile (FIG. 4). In 13 cycles, the
exponential growth rate increased from 0.025 to 0.08 h.sup.-1.
After 20 cycles a sample was taken, and plated on solid synthetic
medium supplemented with 2% of L-arabinose and incubated at
30.degree. C. for several days. Separate colonies were re-streaked
twice on solid synthetic medium with L-arabinose. Finally, a shake
flask containing synthetic medium with 2% of L-L-arabinose was
inoculated with a single colony, and incubated for 5 days at
30.degree. C. This culture was designated as strain IMS0002
(CBS120328 deposited at the Centraal Bureau voor Schimmelculturen
(CBS) on 27/09/06). Culture samples were taken, 30% of glycerol was
added and samples were stored at -80.degree. C.
Mixed Culture Fermentation
[0174] Biomass hydrolysates, a desired feedstock for industrial
biotechnology, contain complex mixtures consisting of various
sugars amongst which glucose, xylose and arabinose are commonly
present in significant fractions. To accomplish ethanolic
fermentation of not only glucose and arabinose, but also xylose, an
anaerobic batch fermentation was performed with a mixed culture of
the arabinose-fermenting strain IMS0002, and the xylose-fermenting
strain RWB218. An anaerobic batch fermenter containing 800 ml of
synthetic medium supplied with 30 g l.sup.-1 D-glucose, 15 g
l.sup.-1 D-xylose, and 15 g l.sup.-1 L-arabinose was inoculated
with 100 ml of pre-culture of strain IMS0002. After 10 hours, a 100
ml inoculum of RWB218 was added. In contrast to the mixed sugar
fermentation with only strain IMS0002, both xylose and arabinose
were consumed after glucose depletion (FIG. 5D). The mixed culture
completely consumed all sugars, and within 80 hours 564.0.+-.6.3
mmol 1.sup.1 ethanol (calculated from the CO.sub.2 production) was
produced with a high overall yield of 0.42 g g.sup.-1 sugar.
Xylitol was produced only in small amounts, to a concentration of
4.7 mmol l.sup.-1.
Characterization of Strain IMS0002
[0175] Growth and product formation of strain IMS0002 was
determined during anaerobic batch fermentations on synthetic medium
with either L-arabinose as the sole carbon source, or a mixture of
glucose, xylose and L-arabinose. The pre-cultures for these
anaerobic batch fermentations were prepared in shake flasks
containing 100 ml of synthetic medium with 2% L-arabinose, by
inoculating with -80.degree. C. frozen stocks of strain IMS0002,
and incubating for 48 hours at 30.degree. C.
[0176] FIG. 5A shows that strain IMS0002 is capable of fermenting
20 g l.sup.-1 L-arabinose to ethanol during an anaerobic batch
fermentation of approximately 70 hours. The specific growth rate
under anaerobic conditions with L-arabinose as sole carbon source
was 0.05.+-.0.001 h.sup.-1. Taking into account the ethanol
evaporation during the batch fermentation, the ethanol yield from
20 g l.sup.-1 arabinose was 0.43.+-.0.003 g g.sup.-1. Without
evaporation correction the ethanol yield was 0.35.+-.0.01 g
g.sup.-1 of arabinose. No formation of arabinitol was observed
during anaerobic growth on arabinose. In FIG. 5B, the ethanolic
fermentation of a mixture of 20 g l.sup.-1 glucose and 20 g
l.sup.-1 L-arabinose by strain IMS0002 is shown. L-arabinose
consumption started after glucose depletion. Within 70 hours, both
the glucose and L-arabinose were completely consumed. The ethanol
yield from the total of sugars was 0.42.+-.0.003 g g.sup.-1.
[0177] In FIG. 5C, the fermentation profile of a mixture of 30 g
l.sup.-1 glucose, 15 g l.sup.-1 D-xylose, and 15 g l.sup.-1
L-arabinose by strain IMS0002 is shown. Arabinose consumption
started after glucose depletion. Within 80 hours, both the glucose
and arabinose were completely consumed. Only 20 mM from 100 mM of
xylose was consumed by strain IMS0002. In addition, the formation
of 20 mM of xylitol was observed. Apparently, the xylose was
converted into xylitol by strain IMS0002. Hence, the ethanol yield
from the total of sugars was lower than for the above described
fermentations: 0.38.+-.0.001 g g.sup.-1. The ethanol yield from the
total of glucose and arabinose was similar to the other
fermentations: 0.43.+-.0.001 g g.sup.-1.
[0178] Table 1 shows the arabinose consumption rates and the
ethanol production rates observed for the anaerobic batch
fermentation of strain IMS0002. Arabinose was consumed with a rate
of 0.23-0.75 g h.sup.-1 g.sup.-1 biomass dry weight. The rate of
ethanol produced from arabinose varied from 0.08-0.31 g h.sup.-1
g.sup.-1 biomass dry weight.
[0179] Initially, the constructed strain IMS0001 was able to
ferment xylose (data not shown). In contrast to our expectations,
the selected strain IMS0002 was not capable of fermenting xylose to
ethanol (FIG. 5C). To regain the capability of fermenting xylose, a
colony of strain IMS0002 was transferred to solid synthetic medium
with 2% of D-xylose, and incubated in an anaerobic jar at
30.degree. C. for 25 days. Subsequently, a colony was again
transferred to solid synthetic medium with 2% of arabinose. After 4
days of incubation at 30.degree. C., a colony was transferred to a
shake flask containing synthetic medium with 2% arabinose. After
incubation at 30.degree. C. for 6 days, 30% of glycerol was added,
samples were taken and stored at -80.degree. C. A shake flask
containing 100 ml of synthetic medium with 2% arabinose was
inoculated with such a frozen stock, and was used as preculture for
an anaerobic batch fermentation on synthetic medium with 20 g
l.sup.-1 xylose and 20 g l.sup.-1 arabinose. In FIG. 6, the
fermentation profile of this batch fermentation is shown. Xylose
and arabinose were consumed simultaneously. The arabinose was
completed within 70 hours, whereas the xylose was completely
consumed in 120 hours. At least 250 mM of ethanol was produced from
the total of sugars, not taking into account the evaporation of the
ethanol. Assuming an end biomass dry weight of 3.2 g l.sup.-1
(assuming a biomass yield of 0.08 g g.sup.-1 sugar), the end
ethanol concentration estimated from the cumulative CO.sub.2
production (355 mmol l.sup.-1) was approximately 330 mmol l.sup.-1,
corresponding to a ethanol yield of 0.41 g g.sup.-1 pentose sugar.
In addition to ethanol, glycerol, and organic acids, a small amount
of xylitol was produced (approximately 5 mM).
Selection of Strain IMS0003
[0180] Initially, the constructed strain IMS0001 was able to
ferment xylose (data not shown). In contrast to our expectations,
the selected strain IMS0002 was not capable of fermenting xylose to
ethanol (FIG. 5C). To regain the capability of fermenting xylose, a
colony of strain IMS0002 was transferred to solid synthetic medium
with 2% of D-xylose, and incubated in an anaerobic jar at
30.degree. C. for 25 days. Subsequently, a colony was again
transferred to solid synthetic medium with 2% of arabinose. After 4
days of incubation at 30.degree. C., a colony was transferred to a
shake flask containing synthetic medium with 2% arabinose. After
incubation at 30.degree. C. for 6 days, 30% of glycerol was added,
samples were taken and stored at -80.degree. C.
[0181] From this frozen stock, samples were spread on solid
synthetic medium with 2% of L-arabinose and incubated at 30.degree.
C. for several days. Separate colonies were re-streaked twice on
solid synthetic medium with L-arabinose. Finally, a shake flask
containing synthetic medium with 2% of L-arabinose was inoculated
with a single colony, and incubated for 4 days at 30.degree. C.
This culture was designated as strain IMS0003 (CBS 121879 deposited
at the CBS on 20/09/07). Culture samples were taken, 30% of
glycerol was added and samples were stored at -80.degree. C.
Characterization of Strain IMS0003
[0182] Growth and product formation of strain IMS0003 was
determined during an anaerobic batch fermentation on synthetic
medium with a mixture of 30 g l.sup.-1 glucose, 15 g l.sup.-1
D-xylose and 15 g l.sup.-1 L-arabinose. The pre-culture for this
anaerobic batch fermentation was prepared in a shake flasks
containing 100 ml of synthetic medium with 2% L-arabinose, by
inoculating with a -80.degree. C. frozen stock of strain IMS0003,
and incubated for 48 hours at 30.degree. C.
[0183] In FIG. 7, the fermentation profile of a mixture of 30 g
l.sup.-1 glucose, 15 g l.sup.-1 D-xylose, and 15 g l.sup.-1
L-arabinose by strain IMS0003 is shown. Arabinose consumption
started after glucose depletion. Within 70 hours, the glucose,
xylose and arabinose were completely consumed. Xylose and arabinose
were consumed simultaneously. At least 406 mM of ethanol was
produced from the total of sugars, not taking into account the
evaporation of the ethanol. The final ethanol concentration
calculated from the cumulative CO.sub.2 production was 572 mmol
l.sup.-1, corresponding to an ethanol yield of 0.46 g g.sup.-1 of
total sugar. In contrast to the fermentation of a mixture of
glucose, xylose and arabinose by strain IMS0002 (FIG. 5C) or a
mixed culture of strains IMS0002 and RWB218 (FIG. 5D), strain
IMS0003 did not produce detectable amounts of xylitol.
TABLE-US-00001 TABLE 1 S. cerevisiae strains used. Strain
Characteristics Reference RWB217 MATA ura3-52 leu2-112
loxP-P.sub.TPI::(-266, -1)TALl gre3::hphMX pUGP.sub.TPI- Kuyper et
al. 2005a TKLl pUGP.sub.TPI-RPEl KanloxP-P.sub.TPI::(-?, -1)RKIl
{p415ADHXKS, PAKX002} RWB218 MATA ura3-52 leu2-112
loxP-P.sub.TPI::(-266, -1)TALl gre3::hphMX pUGP.sub.TPI- Kuyper et
al. 2005b TKLl pUGP.sub.TPI-RPEl KanloxP-P.sub.TPI::(-?, -1)RKIl
{p415ADHXKS1, pAKX002} RWB219 MATA ura3-52 leu2-112
loxP-P.sub.TPI::(-266, -1)TALl gre3::hphMX pUGP.sub.TPI- This work
TKLl pUGP.sub.TPI-RPEl KanloxP-P.sub.TPI::(-?, -1)RKIl
{p415ADHXKS1, pAKX002} RWB220 MATA ura3-52 leu2-112
loxP-P.sub.TPI::(-266, -1)TALl gre3::hphMX pUGP.sub.TPI- This work
TKLl .sub.PUGP.sub.TPI-RPEl loxP-P.sub.TPI::(-?, -1)RKIl IMS0001
MATA ura3-52 leu2-112 loxP-P.sub.TPI::(-266, -1)TALl gre3::hphMX
pUGP.sub.TPI- This work TKLl pUGP.sub.TPI-RPEl loxP-P.sub.TPI::(-?,
-1)RKIl {pRW231, PRW243} IMS0002 MATA ura3-52 leu2-112
loxP-P.sub.TPI::(-266, -1)TALl gre3::hphMX pUGP.sub.TPI- This work
TKLl pUGP.sub.TPI-RPEl loxP-P.sub.TPI::(-?, -1)RKIl {pRW231,
PRW243} selected for anaerobic growth on L-arabinose
TABLE-US-00002 TABLE 2 Plasmids used plasmid characteristics
Reference pRS305 Integration, LEU2 Gietz and Sugino, 1988 pAKX002
2.mu., URA3, P.sub.TPIl-Piromyces xylA Kuyper et al. 2003
p415ADHXKS1 CEN, LEU2, P.sub.ADHI-S.cerXKS1 Kuyper et al., 2005a
pRW229 integration, LEU2, P.sub.ADHI-S.cerXKS1 This work pRW230
pAKX002 with P.sub.TDH3-AraA This work pRW231 pAKX002 with
P.sub.TDH3-AraA and P.sub.HXT7-AraD This work pRW243 LEU2,
integration, P.sub.ADHI-ScXKS1-T.sub.CYC, This work
P.sub.PGIl-L.plantarumAraB-T.sub.ADHI
TABLE-US-00003 TABLE 3 oligos used in this work Oligo DNA sequence
AraA expression cassette SpeI5'Ptdh3
5'GACTAGTCGAGTTTATCATTATCAATACTGC3' SEQ ID NO: 31 5'AraAPtdh
5'CTCATAATCAGGTACTGATAACATTTTGTTTGTTTATGTGTGTTTATTC3' SEQ ID NO: 32
Ptdh5'AraA 5'GAATAAACACACATAAACAAACAAAATGTTATCAGTACCTGATTATGAG3 SEQ
ID NO: 33 Tadh3'AraA
5'AATCATAAATCATAAGAAATTCGCTTACTTTAAGAATGCCTTAGTCAT3' SEQ ID NO: 34
3'AraATadh 1 5'ATGACTAAGGCATTCTTAAAGTAAGCGAATTTCTTATGATTTATGATT3'
SEQ ID NO: 35 3'Tadh 1 SpeI
5'CACTAGTCTCGAGTGTGGAAGAACGATTACAACAGG3' SEQ ID NO: 36 AraB
expression cassette SacI5'Ppgi1 5'CGAGCTCGTGGGTGTATTGGATTATAGGAAG3'
SEQ ID NO: 37 5'AraBPpgi1
5'TTGGGCTGTTTCAACTAAATTCATTTTTAGGCTGGTATCTTGATTCTA3' SEQ ID NO: 38
Ppgi5'AraB 5'TAGAATCAAGATACCAGCCTAAAAATGAATTTAGTTGAAACAGCCCAA3' SEQ
ID NO: 39 Tadh3'AraB
5'AATCATAAATCATAAGAAATTCGCTCTAATATTTGATTGCTTGCCCAG3' SEQ ID NO: 40
3'AraBTadh 1 5'CTGGGCAAGCAATCAAATATTAGAGCGAATTTCTTATGATTTATGATT3'
SEQ ID NO: 41 3'Tadh 1 SacI 5'TGAGCTCGTGTGGAAGAACGATTACAACAGG3' SEQ
ID NO: 42 AraD expression cassette SalI5'Phxt7
5'ACGCGTCGACTCGTAGGAACAATTTCGG3' SEQ ID NO: 43 5'AraDPhxt
5'CTTCTTGTTTTAATGCTTCTAGCATTTTTTGATTAAAATTAAAAAAACTT3' SEQ ID NO:
44 Phxt5'AraD
5'AAGTTTTTTTAATTTTAATCAAAAAATGCTAGAAGCATTAAAACAAGAAG3' SEQ ID NO:
45 Tpgi3'AraD 5'GGTATATATTTAAGAGCGATTTGTTTACTTGCGAACTGCATGATCC3'
SEQ ID NO: 46 3'AraDTpgi
5'GGATCATGCAGTTCGCAAGTAAACAAATCGCTCTTAAATATATACC3' SEQ ID NO: 47
3'TpgiSalI 5'CGCAGTCGACCTTTTAAACAGTTGATGAGAACC3' SEQ ID NO: 48
TABLE-US-00004 TABLE 4 Maximum observed specific glucose and
arabinose consumption rates and ethanol production rates during
anaerobic batch fermentations of S. cerevisiae IMS0002. q.sub.glu
q.sub.ara q.sub.eth, glu q.sub.eth, ara C-source g h.sup.-1
g.sup.-1 DW g h.sup.-1 g.sup.-1 DW g h.sup.-1 g.sup.-1 DW g
h.sup.-1 g.sup.-1 DW 20 g l.sup.-1 arabinose -- 0.75 .+-. 0.04 --
0.31 .+-. 0.02 20 g l.sup.-1 glucose 2.08 .+-. 0.09 0.41 .+-. 0.01
0.69 .+-. 0.00 0.19 .+-. 0.00 20 g l.sup.-1 arabinose 30 g l.sup.-1
glucose 1.84 .+-. 0.04 0.23 .+-. 0.01 0.64 .+-. 0.03 0.08 .+-. 0.01
15 g l.sup.-1 xylose 15 g l.sup.-1 arabinose q.sub.glu: specific
glucose consumption rate q.sub.ara: specific arabinose consumption
rate q.sub.eth, glu: specific ethanol production rate during growth
on glucose q.sub.eth, ara: specific ethanol production rate during
growth on arabinose
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Saccharomyces cerevisiae. I. Ergosterol requirement for growth in a
defined medium. J Cell Physiol 41:23-36 [0186] Becker J, Boles E
(2003) A modified Saccharomyces cerevisiae strain that consumes
L-Arabinose and produces ethanol, Appl Environ Microbiol
69:4144-4150 [0187] Gietz R. D., Sugino A. (1988). New
yeast-Escherichia coli shuttle vectors constructed with in vitro
mutagenized yeast genes lacking six-base pair restriction sites.
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DNA/polyethylene glycol method. Methods Enzymol. 350:87-96. [0189]
Guldener U, Heck S, Fielder T, Beinhauer J, Hegemann J H. (1996) A
new efficient gene disruption cassette for repeated use in budding
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H., H. Nojima and H. Okayama, High efficiency transformation of
Escherichia coli with plasmids. Gene 96 (1990), pp. 23-28 [0192]
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Van Dijken J P, Pronk J T (2005a) Metabolic engineering of a
xylose-isomerase-expressing Saccharomyces cerevisiae strain for
rapid anaerobic xylose fermentation. Ferns Yeast Research 5:399-409
[0193] Kuyper M, Toirkens M J, Diderich J A, Winkler A A, Van
Dijken J P, Pronk J T (2005b) Evolutionary engineering of
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K., Fritsch, E. F. and Maniatis, I. (1989) Molecular Cloning: A
Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press,
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glucose limitation to glucose excess. Yeast 4:283-291 [0196]
Verduyn C, Postma E, Scheffers W A, Van Dijken J P (1990)
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acid on metabolic fluxes in yeasts: a continuous-culture study on
the regulation of respiration and alcoholic fermentation. Yeast
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Van Dijken J P (1994) Effects of oxygen limitation on sugar
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effect. Microbiology 140:703-715
Sequence CWU 1
1
531474PRTLactobacillus plantarum 1Met Leu Ser Val Pro Asp Tyr Glu
Phe Trp Phe Val Thr Gly Ser Gln1 5 10 15His Leu Tyr Gly Glu Glu Gln
Leu Lys Ser Val Ala Lys Asp Ala Gln 20 25 30Asp Ile Ala Asp Lys Leu
Asn Ala Ser Gly Lys Leu Pro Tyr Lys Val 35 40 45Val Phe Lys Asp Val
Met Thr Thr Ala Glu Ser Ile Thr Asn Phe Met 50 55 60Lys Glu Val Asn
Tyr Asn Asp Lys Val Ala Gly Val Ile Thr Trp Met65 70 75 80His Thr
Phe Ser Pro Ala Lys Asn Trp Ile Arg Gly Thr Glu Leu Leu 85 90 95Gln
Lys Pro Leu Leu His Leu Ala Thr Gln Tyr Leu Asn Asn Ile Pro 100 105
110Tyr Ala Asp Ile Asp Phe Asp Tyr Met Asn Leu Asn Gln Ser Ala His
115 120 125Gly Asp Arg Glu Tyr Ala Tyr Ile Asn Ala Arg Leu Gln Lys
His Asn 130 135 140Lys Ile Val Tyr Gly Tyr Trp Gly Asp Glu Asp Val
Gln Glu Gln Ile145 150 155 160Ala Arg Trp Glu Asp Val Ala Val Ala
Tyr Asn Glu Ser Phe Lys Val 165 170 175Lys Val Ala Arg Phe Gly Asp
Thr Met Arg Asn Val Ala Val Thr Glu 180 185 190Gly Asp Lys Val Glu
Ala Gln Ile Lys Met Gly Trp Thr Val Asp Tyr 195 200 205Tyr Gly Ile
Gly Asp Leu Val Glu Glu Ile Asn Lys Val Ser Asp Ala 210 215 220Asp
Val Asp Lys Glu Tyr Ala Asp Leu Glu Ser Arg Tyr Glu Met Val225 230
235 240Gln Val Asp Asn Asp Ala Asp Thr Tyr Lys His Ser Val Arg Val
Gln 245 250 255Leu Ala Gln Tyr Leu Gly Ile Lys Arg Phe Leu Glu Arg
Gly Gly Tyr 260 265 270Thr Ala Phe Thr Thr Asn Phe Glu Asp Leu Trp
Gly Met Glu Gln Leu 275 280 285Pro Gly Leu Ala Ser Gln Leu Leu Ile
Arg Asp Gly Tyr Gly Phe Gly 290 295 300Ala Glu Gly Asp Trp Lys Thr
Ala Ala Leu Gly Arg Val Met Lys Ile305 310 315 320Met Ser His Asn
Lys Gln Thr Ala Phe Met Glu Asp Tyr Thr Leu Asp 325 330 335Leu Arg
His Gly His Glu Ala Ile Leu Gly Ser His Met Leu Glu Val 340 345
350Asp Pro Ser Ile Ala Ser Asp Lys Pro Arg Val Glu Val His Pro Leu
355 360 365Asp Ile Gly Gly Lys Asp Asp Pro Ala Arg Leu Val Phe Thr
Gly Ser 370 375 380Glu Gly Glu Ala Ile Asp Val Thr Val Ala Asp Phe
Arg Asp Gly Phe385 390 395 400Lys Met Ile Ser Tyr Ala Val Asp Ala
Asn Lys Pro Glu Ala Glu Thr 405 410 415Pro Asn Leu Pro Val Ala Lys
Gln Leu Trp Thr Pro Lys Met Gly Leu 420 425 430Lys Lys Gly Ala Leu
Glu Trp Met Gln Ala Gly Gly Gly His His Thr 435 440 445Met Leu Ser
Phe Ser Leu Thr Glu Glu Gln Met Glu Asp Tyr Ala Thr 450 455 460Met
Val Gly Met Thr Lys Ala Phe Leu Lys465 47021425DNALactobacillus
plantarum 2atgttatcag tacctgatta tgagttttgg tttgttaccg gttcacaaca
cctttatggt 60gaagaacaat tgaagtctgt tgctaaggat gcgcaagata ttgcggataa
attgaatgca 120agcggcaagt taccttataa agtagtcttt aaggatgtta
tgacgacggc tgaaagtatc 180accaacttta tgaaagaagt taattacaat
gataaggtag ccggtgttat tacttggatg 240cacacattct caccagctaa
gaactggatt cgtggaactg aactgttaca aaaaccatta 300ttacacttag
caacgcaata tttgaataat attccatatg cagacattga ctttgattac
360atgaacctta accaaagtgc ccatggcgac cgcgagtatg cctacattaa
cgcccggttg 420cagaaacata ataagattgt ttacggctat tggggcgatg
aagatgtgca agagcagatt 480gcacgttggg aagacgtcgc cgtagcgtac
aatgagagct ttaaagttaa ggttgctcgc 540tttggcgaca caatgcgtaa
tgtggccgtt actgaaggtg acaaggttga agctcaaatt 600aagatgggct
ggacagttga ctattatggt atcggtgact tagttgaaga gatcaataag
660gtttcggatg ctgatgttga taaggaatac gctgacttgg agtctcggta
tgaaatggtc 720caagttgata acgatgcgga cacgtataaa cattcagttc
gggttcaatt ggcacaatat 780ctgggtatta agcggttctt agaaagaggc
ggttacacag cctttaccac gaactttgaa 840gatctttggg ggatggagca
attacctggt ctagcttcac aattattaat tcgtgatggg 900tatggttttg
gtgctgaagg tgactggaag acggctgctt taggacgggt tatgaagatt
960atgtctcaca acaagcaaac cgcctttatg gaagactaca cgttagactt
gcgtcatggt 1020catgaagcga tcttaggttc acacatgttg gaagttgatc
cgtctatcgc aagtgataaa 1080ccacgggtcg aagttcatcc attggatatt
gggggtaaag atgatcctgc tcgcctagta 1140tttactggtt cagaaggtga
agcaattgat gtcaccgttg ccgatttccg tgatgggttc 1200aagatgatta
gctacgcggt agatgcgaat aagccagaag ccgaaacacc taatttacca
1260gttgctaagc aattatggac cccaaagatg ggcttgaaga agggtgcact
agaatggatg 1320caagctggtg gtggtcacca cacgatgctg tccttctcgt
taactgaaga acaaatggaa 1380gactatgcaa ccatggttgg catgactaag
gcattcttaa agtaa 14253533PRTLactobacillus plantarum 3Met Asn Leu
Val Glu Thr Ala Gln Ala Ile Lys Thr Gly Lys Val Ser1 5 10 15Leu Gly
Ile Glu Leu Gly Ser Thr Arg Ile Lys Ala Val Leu Ile Thr 20 25 30Asp
Asp Phe Asn Thr Ile Ala Ser Gly Ser Tyr Val Trp Glu Asn Gln 35 40
45Phe Val Asp Gly Thr Trp Thr Tyr Ala Leu Glu Asp Val Trp Thr Gly
50 55 60Ile Gln Gln Ser Tyr Thr Gln Leu Ala Ala Asp Val Arg Ser Lys
Tyr65 70 75 80His Met Ser Leu Lys His Ile Asn Ala Ile Gly Ile Ser
Ala Met Met 85 90 95His Gly Tyr Leu Ala Phe Asp Gln Gln Ala Lys Leu
Leu Val Pro Phe 100 105 110Arg Thr Trp Arg Asn Asn Ile Thr Gly Gln
Ala Ala Asp Glu Leu Thr 115 120 125Glu Leu Phe Asp Phe Asn Ile Pro
Gln Arg Trp Ser Ile Ala His Leu 130 135 140Tyr Gln Ala Ile Leu Asn
Asn Glu Ala His Val Lys Gln Val Asp Phe145 150 155 160Ile Thr Thr
Leu Ala Gly Tyr Val Thr Trp Lys Leu Ser Gly Glu Lys 165 170 175Val
Leu Gly Ile Gly Asp Ala Ser Gly Val Phe Pro Ile Asp Glu Thr 180 185
190Thr Asp Thr Tyr Asn Gln Thr Met Leu Thr Lys Phe Ser Gln Leu Asp
195 200 205Lys Val Lys Pro Tyr Ser Trp Asp Ile Arg His Ile Leu Pro
Arg Val 210 215 220Leu Pro Ala Gly Ala Ile Ala Gly Lys Leu Thr Ala
Ala Gly Ala Ser225 230 235 240Leu Leu Asp Gln Ser Gly Thr Leu Asp
Ala Gly Ser Val Ile Ala Pro 245 250 255Pro Glu Gly Asp Ala Gly Thr
Gly Met Val Gly Thr Asn Ser Val Arg 260 265 270Lys Arg Thr Gly Asn
Ile Ser Val Gly Thr Ser Ala Phe Ser Met Asn 275 280 285Val Leu Asp
Lys Pro Leu Ser Lys Val Tyr Arg Asp Ile Asp Ile Val 290 295 300Met
Thr Pro Asp Gly Ser Pro Val Ala Met Val His Val Asn Asn Cys305 310
315 320Ser Ser Asp Ile Asn Ala Trp Ala Thr Ile Phe Arg Glu Phe Ala
Ala 325 330 335Arg Leu Gly Met Glu Leu Lys Pro Asp Arg Leu Tyr Glu
Thr Leu Phe 340 345 350Leu Glu Ser Thr Arg Ala Asp Ala Asp Ala Gly
Gly Leu Ala Asn Tyr 355 360 365Ser Tyr Gln Ser Gly Glu Asn Ile Thr
Lys Ile Gln Ala Gly Arg Pro 370 375 380Leu Phe Val Arg Thr Pro Asn
Ser Lys Phe Ser Leu Pro Asn Phe Met385 390 395 400Leu Thr Gln Leu
Tyr Ala Ala Phe Ala Pro Leu Gln Leu Gly Met Asp 405 410 415Ile Leu
Val Asn Glu Glu His Val Gln Thr Asp Val Met Ile Ala Gln 420 425
430Gly Gly Leu Phe Arg Thr Pro Val Ile Gly Gln Gln Val Leu Ala Asn
435 440 445Ala Leu Asn Ile Pro Ile Thr Val Met Ser Thr Ala Gly Glu
Gly Gly 450 455 460Pro Trp Gly Met Ala Val Leu Ala Asn Phe Ala Cys
Arg Gln Thr Ala465 470 475 480Met Asn Leu Glu Asp Phe Leu Asp Gln
Glu Val Phe Lys Glu Pro Glu 485 490 495Ser Met Thr Leu Ser Pro Glu
Pro Glu Arg Val Ala Gly Tyr Arg Glu 500 505 510Phe Ile Gln Arg Tyr
Gln Ala Gly Leu Pro Val Glu Ala Ala Ala Gly 515 520 525Gln Ala Ile
Lys Tyr 53041602DNALactobacillus plantarum 4atgaatttag ttgaaacagc
ccaagcgatt aaaactggca aagtttcttt aggaattgag 60cttggctcaa ctcgaattaa
agccgttttg atcacggacg attttaatac gattgcttcg 120ggaagttacg
tttgggaaaa ccaatttgtt gatggtactt ggacttacgc acttgaagat
180gtctggaccg gaattcaaca aagttatacg caattagcag cagatgtccg
cagtaaatat 240cacatgagtt tgaagcatat caatgctatt ggcattagtg
ccatgatgca cggataccta 300gcatttgatc aacaagcgaa attattagtt
ccgtttcgga cttggcgtaa taacattacg 360gggcaagcag cagatgaatt
gaccgaatta tttgatttca acattccaca acggtggagt 420atcgcgcact
tataccaggc aatcttaaat aatgaagcgc acgttaaaca ggtggacttc
480ataacaacgc tggctggcta tgtaacctgg aaattgtcgg gtgagaaagt
tctaggaatc 540ggtgatgcgt ctggcgtttt cccaattgat gaaacgactg
acacatacaa tcagacgatg 600ttaaccaagt ttagccaact tgacaaagtt
aaaccgtatt catgggatat ccggcatatt 660ttaccgcggg ttttaccagc
gggagccatt gctggaaagt taacggctgc cggggcgagc 720ttacttgatc
agagcggcac gctcgacgct ggcagtgtta ttgcaccgcc agaaggggat
780gctggaacag gaatggtcgg tacgaacagc gtccgtaaac gcacgggtaa
catctcggtg 840ggaacctcag cattttcgat gaacgttcta gataaaccat
tgtctaaagt ctatcgcgat 900attgatattg ttatgacgcc agatgggtca
ccagttgcaa tggtgcatgt taataattgt 960tcatcagata ttaatgcgtg
ggcaacgatt tttcgtgagt ttgcagcccg gttgggaatg 1020gaattgaaac
cggatcgatt atatgaaacg ttattcttgg aatcaactcg cgctgatgcg
1080gatgctggag ggttggctaa ttatagttat caatccggtg agaatattac
taagattcaa 1140gctggtcggc cgctatttgt acggacacca aacagtaaat
ttagtttacc gaactttatg 1200ttgacccaat tatatgcggc gttcgcaccc
ctccaacttg gtatggatat tcttgttaac 1260gaagaacatg ttcaaacgga
cgttatgatt gcacagggtg gattgttccg aacgccggta 1320attggccaac
aagtattggc caacgcactg aacattccga ttactgtaat gagtactgct
1380ggtgaaggcg gcccatgggg gatggcagtg ttagccaact ttgcttgtcg
gcaaactgca 1440atgaacctag aagatttctt agatcaagaa gtctttaaag
agccagaaag tatgacgttg 1500agtccagaac cggaacgggt ggccggatat
cgtgaattta ttcaacgtta tcaagctggc 1560ttaccagttg aagcagcggc
tgggcaagca atcaaatatt ag 16025242PRTLactobacillus plantarum 5Met
Leu Glu Ala Leu Lys Gln Glu Val Tyr Glu Ala Asn Met Gln Leu1 5 10
15Pro Lys Leu Gly Leu Val Thr Phe Thr Trp Gly Asn Val Ser Gly Ile
20 25 30Asp Arg Glu Lys Gly Leu Phe Val Ile Lys Pro Ser Gly Val Asp
Tyr 35 40 45Gly Glu Leu Lys Pro Ser Asp Leu Val Val Val Asn Leu Gln
Gly Glu 50 55 60Val Val Glu Gly Lys Leu Asn Pro Ser Ser Asp Thr Pro
Thr His Thr65 70 75 80Val Leu Tyr Asn Ala Phe Pro Asn Ile Gly Gly
Ile Val His Thr His 85 90 95Ser Pro Trp Ala Val Ala Tyr Ala Ala Ala
Gln Met Asp Val Pro Ala 100 105 110Met Asn Thr Thr His Ala Asp Thr
Phe Tyr Gly Asp Val Pro Ala Ala 115 120 125Asp Ala Leu Thr Lys Glu
Glu Ile Glu Ala Asp Tyr Glu Gly Asn Thr 130 135 140Gly Lys Thr Ile
Val Lys Thr Phe Gln Glu Arg Gly Leu Asp Tyr Glu145 150 155 160Ala
Val Pro Ala Ser Leu Val Ser Gln His Gly Pro Phe Ala Trp Gly 165 170
175Pro Thr Pro Ala Lys Ala Val Tyr Asn Ala Lys Val Leu Glu Val Val
180 185 190Ala Glu Glu Asp Tyr His Thr Ala Gln Leu Thr Arg Ala Ser
Ser Glu 195 200 205Leu Pro Gln Tyr Leu Leu Asp Lys His Tyr Leu Arg
Lys His Gly Ala 210 215 220Ser Ala Tyr Tyr Gly Gln Asn Asn Ala His
Ser Lys Asp His Ala Val225 230 235 240Arg Lys6729DNALactobacillus
plantarum 6atgctagaag cattaaaaca agaagtttat gaggctaaca tgcagcttcc
aaagctgggc 60ctggttactt ttacctgggg caatgtctcg ggcattgacc gggaaaaagg
cctattcgtg 120atcaagccat ctggtgttga ttatggtgaa ttaaaaccaa
gcgatttagt cgttgttaac 180ttacagggtg aagtggttga aggtaaacta
aatccgtcta gtgatacgcc gactcatacg 240gtgttatata acgcttttcc
taatattggc ggaattgtcc atactcattc gccatgggca 300gttgcctatg
cagctgctca aatggatgtg ccagctatga acacgaccca tgctgatacg
360ttctatggtg acgtgccggc cgcggatgcg ctgactaagg aagaaattga
agcagattat 420gaaggcaaca cgggtaaaac cattgtgaag acgttccaag
aacggggcct cgattatgaa 480gctgtaccag cctcattagt cagccagcac
ggcccatttg cttggggacc aacgccagct 540aaagccgttt acaatgctaa
agtgttggaa gtggttgccg aagaagatta tcatactgcg 600caattgaccc
gtgcaagtag cgaattacca caatatttat tagataagca ttatttacgt
660aagcatggtg caagtgccta ttatggtcaa aataatgcgc attctaagga
tcatgcagtt 720cgcaagtaa 7297437PRTPiromyces sp. 7Met 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 43581669DNAPiromyces sp. 8gtaaatggct aaggaatatt
tcccacaaat tcaaaagatt aagttcgaag gtaaggattc 60taagaatcca ttagccttcc
actactacga tgctgaaaag gaagtcatgg gtaagaaaat 120gaaggattgg
ttacgtttcg ccatggcctg gtggcacact ctttgcgccg aaggtgctga
180ccaattcggt ggaggtacaa agtctttccc atggaacgaa ggtactgatg
ctattgaaat 240tgccaagcaa aaggttgatg ctggtttcga aatcatgcaa
aagcttggta ttccatacta 300ctgtttccac gatgttgatc ttgtttccga
aggtaactct attgaagaat acgaatccaa 360ccttaaggct gtcgttgctt
acctcaagga aaagcaaaag gaaaccggta ttaagcttct 420ctggagtact
gctaacgtct tcggtcacaa gcgttacatg aacggtgcct ccactaaccc
480agactttgat gttgtcgccc gtgctattgt tcaaattaag aacgccatag
acgccggtat 540tgaacttggt gctgaaaact acgtcttctg gggtggtcgt
gaaggttaca tgagtctcct 600taacactgac caaaagcgtg aaaaggaaca
catggccact atgcttacca tggctcgtga 660ctacgctcgt tccaagggat
tcaagggtac tttcctcatt gaaccaaagc caatggaacc 720aaccaagcac
caatacgatg ttgacactga aaccgctatt ggtttcctta aggcccacaa
780cttagacaag gacttcaagg tcaacattga agttaaccac gctactcttg
ctggtcacac 840tttcgaacac gaacttgcct gtgctgttga tgctggtatg
ctcggttcca ttgatgctaa 900ccgtggtgac taccaaaacg gttgggatac
tgatcaattc ccaattgatc aatacgaact 960cgtccaagct tggatggaaa
tcatccgtgg tggtggtttc gttactggtg gtaccaactt 1020cgatgccaag
actcgtcgta actctactga cctcgaagac atcatcattg cccacgtttc
1080tggtatggat gctatggctc gtgctcttga aaacgctgcc aagctcctcc
aagaatctcc 1140atacaccaag atgaagaagg aacgttacgc ttccttcgac
agtggtattg gtaaggactt 1200tgaagatggt aagctcaccc tcgaacaagt
ttacgaatac ggtaagaaga acggtgaacc 1260aaagcaaact tctggtaagc
aagaactcta cgaagctatt gttgccatgt accaataagt 1320taatcgtagt
taaattggta aaataattgt aaaatcaata aacttgtcaa tcctccaatc
1380aagtttaaaa gatcctatct ctgtactaat taaatatagt acaaaaaaaa
atgtataaac 1440aaaaaaaagt ctaaaagacg gaagaattta atttagggaa
aaaataaaaa taataataaa 1500caatagataa atcctttata ttaggaaaat
gtcccattgt attattttca tttctactaa 1560aaaagaaagt aaataaaaca
caagaggaaa ttttcccttt tttttttttt tgtaataaat 1620tttatgcaaa
tataaatata aataaaataa taaaaaaaaa aaaaaaaaa 16699496PRTBacillus
subtilis 9Met Leu Gln Thr Lys Asp Tyr Glu Phe Trp Phe Val Thr Gly
Ser Gln1 5 10 15His Leu Tyr Gly Glu Glu Thr Leu Glu Leu Val Asp Gln
His Ala Lys 20 25 30Ser Ile Cys Glu Gly Leu Ser Gly Ile Ser Ser Arg
Tyr Lys Ile Thr 35 40 45His Lys Pro Val Val Thr Ser Pro Glu Thr Ile
Arg Glu Leu Leu Arg 50 55 60Glu Ala Glu Tyr Ser Glu Thr Cys Ala Gly
Ile Ile Thr Trp Met His65 70 75 80Thr Phe Ser Pro Ala Lys Met Trp
Ile Glu Gly Leu Ser Ser Tyr Gln 85 90 95Lys Pro Leu Met His Leu His
Thr Gln Tyr Asn Arg Asp Ile Pro Trp 100 105 110Gly Thr Ile Asp Met
Asp Phe Met Asn Ser Asn Gln Ser Ala His Gly 115 120 125Asp Arg Glu
Tyr Gly Tyr Ile Asn Ser Arg Met Gly Leu Ser Arg Lys 130 135 140Val
Ile Ala Gly Tyr Trp Asp Asp Glu Glu Val Lys Lys Glu Met Ser145 150
155 160Gln Trp Met Asp Thr Ala Ala Ala Leu Asn Glu Ser Arg His Ile
Lys 165 170 175Val Ala Arg Phe Gly Asp Asn Met Arg His Val Ala Val
Thr Asp Gly 180 185 190Asp Lys Val Gly Ala His Ile Gln Phe Gly Trp
Gln Val Asp Gly Tyr 195 200 205Gly Ile Gly Asp Leu Val Glu Val Met
Asp Arg Ile Thr Asp Asp Glu 210 215 220Val Asp Thr Leu Tyr Ala Glu
Tyr Asp Arg Leu Tyr Val Ile Ser Glu225 230 235 240Glu Thr Lys Arg
Asp Glu Ala Lys Val Ala Ser Ile Lys Glu Gln Ala 245 250 255Lys Ile
Glu Leu Gly Leu Thr Ala Phe Leu Glu Gln Gly Gly Tyr Thr 260 265
270Ala Phe Thr Thr Ser Phe Glu Val Leu His Gly Met Lys Gln Leu Pro
275 280 285Gly Leu Ala Val Gln Arg Leu Met Glu Lys Gly Tyr Gly Phe
Ala Gly 290 295 300Glu Gly Asp Trp Lys Thr Ala Ala Leu Val Arg Met
Met Lys Ile Met305 310 315 320Ala Lys Gly Lys Arg Thr Ser Phe Met
Glu Asp Tyr Thr Tyr His Phe 325 330 335Glu Pro Gly Asn Glu Met Ile
Leu Gly Ser His Met Leu Glu Val Cys 340 345 350Pro Thr Val Ala Leu
Asp Gln Pro Lys Ile Glu Val His Ser Leu Ser 355 360 365Ile Gly Gly
Lys Glu Asp Pro Ala Arg Leu Val Phe Asn Gly Ile Ser 370 375 380Gly
Ser Ala Ile Gln Ala Ser Ile Val Asp Ile Gly Gly Arg Phe Arg385 390
395 400Leu Val Leu Asn Glu Val Asn Gly Gln Glu Ile Glu Lys Asp Met
Pro 405 410 415Asn Leu Pro Val Ala Arg Val Leu Trp Lys Pro Glu Pro
Ser Leu Lys 420 425 430Thr Ala Ala Glu Ala Trp Ile Leu Ala Gly Gly
Ala His His Thr Cys 435 440 445Leu Ser Tyr Glu Leu Thr Ala Glu Gln
Met Leu Asp Trp Ala Glu Met 450 455 460Ala Gly Ile Glu Ser Val Leu
Ile Ser Arg Asp Thr Thr Ile His Lys465 470 475 480Leu Lys His Glu
Leu Lys Trp Asn Glu Ala Leu Tyr Arg Leu Gln Lys 485 490
495101511DNABacillus subtilis 10atgagaaagg ggcagtttac atgcttcaga
caaaggatta tgaattctgg tttgtgacag 60gaagccagca cctatacggg gaagagacgc
tggaactcgt agatcagcat gctaaaagca 120tttgtgaggg gctcagcggg
atttcttcca gatataaaat cactcataag cccgtcgtca 180cttcaccgga
aaccattaga gagctgttaa gagaagcgga gtacagtgag acatgtgctg
240gcatcattac atggatgcac acattttccc ctgcaaaaat gtggatagaa
ggcctttcct 300cttatcaaaa accgcttatg catttgcata cccaatataa
tcgcgatatc ccgtggggta 360cgattgacat ggattttatg aacagcaacc
aatccgcgca tggcgatcga gagtacggtt 420acatcaactc gagaatgggg
cttagccgaa aagtcattgc cggctattgg gatgatgaag 480aagtgaaaaa
agaaatgtcc cagtggatgg atacggcggc tgcattaaat gaaagcagac
540atattaaggt tgccagattt ggagataaca tgcgtcatgt cgcggtaacg
gacggagaca 600aggtgggagc gcatattcaa tttggctggc aggttgacgg
atatggcatc ggggatctcg 660ttgaagtgat ggatcgcatt acggacgacg
aggttgacac gctttatgcc gagtatgaca 720gactatatgt gatcagtgag
gaaacaaaac gtgacgaagc aaaggtagcg tccattaaag 780aacaggcgaa
aattgaactt ggattaaccg cttttcttga gcaaggcgga tacacagcgt
840ttacgacatc gtttgaagtg ctgcacggaa tgaaacagct gccgggactt
gccgttcagc 900gcctgatgga gaaaggctat gggtttgccg gtgaaggaga
ttggaagaca gcggcccttg 960tacggatgat gaaaatcatg gctaaaggaa
aaagaacttc cttcatggaa gattacacgt 1020accattttga accgggaaat
gaaatgattc tgggctctca catgcttgaa gtgtgtccga 1080ctgtcgcttt
ggatcagccg aaaatcgagg ttcattcgct ttcgattggc ggcaaagagg
1140accctgcgcg tttggtattt aacggcatca gcggttctgc cattcaagct
agcattgttg 1200atattggcgg gcgtttccgc cttgtgctga atgaagtcaa
cggccaggaa attgaaaaag 1260acatgccgaa tttaccggtt gcccgtgttc
tctggaagcc ggagccgtca ttgaaaacag 1320cagcggaggc atggatttta
gccggcggtg cacaccatac ctgcctgtct tatgaactga 1380cagcggagca
aatgcttgat tgggcggaaa tggcgggaat cgaaagtgtt ctcatttccc
1440gtgatacgac aattcataaa ctgaaacacg agttaaaatg gaacgaggcg
ctttaccggc 1500ttcaaaagta g 151111566PRTE. coli 11Met Ala Ile Ala
Ile Gly Leu Asp Phe Gly Ser Asp Ser Val Arg Ala1 5 10 15Leu Ala Val
Asp Cys Ala Ser Gly Glu Glu Ile Ala Thr Ser Val Glu 20 25 30Trp Tyr
Pro Arg Trp Gln Lys Gly Gln Phe Cys Asp Ala Pro Asn Asn 35 40 45Gln
Phe Arg His His Pro Arg Asp Tyr Ile Glu Ser Met Glu Ala Ala 50 55
60Leu Lys Thr Val Leu Ala Glu Leu Ser Val Glu Gln Arg Ala Ala Val65
70 75 80Val Gly Ile Gly Val Asp Ser Thr Gly Ser Thr Pro Ala Pro Ile
Asp 85 90 95Ala Asp Gly Asn Val Leu Ala Leu Arg Pro Glu Phe Ala Glu
Asn Pro 100 105 110Asn Ala Met Phe Val Leu Trp Lys Asp His Thr Ala
Val Glu Arg Ser 115 120 125Glu Glu Ile Thr Arg Leu Cys His Ala Pro
Gly Asn Val Asp Tyr Ser 130 135 140Arg Tyr Ile Gly Gly Ile Tyr Ser
Ser Glu Trp Phe Trp Ala Lys Ile145 150 155 160Leu His Val Thr Arg
Gln Asp Ser Ala Val Ala Gln Ser Ala Ala Ser 165 170 175Trp Ile Glu
Leu Cys Asp Trp Val Pro Ala Leu Leu Ser Gly Thr Thr 180 185 190Arg
Pro Gln Asp Ile Arg Arg Gly Arg Cys Ser Ala Gly His Lys Ser 195 200
205Leu Trp His Glu Ser Trp Gly Gly Leu Pro Pro Ala Ser Phe Phe Asp
210 215 220Glu Leu Asp Pro Ile Leu Asn Arg His Leu Pro Ser Pro Leu
Phe Thr225 230 235 240Asp Thr Trp Thr Ala Asp Ile Pro Val Gly Thr
Leu Cys Pro Glu Trp 245 250 255Ala Gln Arg Leu Gly Leu Pro Glu Ser
Val Val Ile Ser Gly Gly Ala 260 265 270Phe Asp Cys His Met Gly Ala
Val Gly Ala Gly Ala Gln Pro Asn Ala 275 280 285Leu Val Lys Val Ile
Gly Thr Ser Thr Cys Asp Ile Leu Ile Ala Asp 290 295 300Lys Gln Ser
Val Gly Glu Arg Ala Val Lys Gly Ile Cys Gly Gln Val305 310 315
320Asp Gly Ser Val Val Pro Gly Phe Ile Gly Leu Glu Ala Gly Gln Ser
325 330 335Ala Phe Gly Asp Ile Tyr Ala Trp Phe Gly Arg Val Leu Ser
Trp Pro 340 345 350Leu Glu Gln Leu Ala Ala Gln His Pro Glu Leu Lys
Ala Gln Ile Asn 355 360 365Ala Ser Gln Lys Gln Leu Leu Pro Ala Leu
Thr Glu Ala Trp Ala Lys 370 375 380Asn Pro Ser Leu Asp His Leu Pro
Val Val Leu Asp Trp Phe Asn Gly385 390 395 400Arg Arg Ser Pro Asn
Ala Asn Gln Arg Leu Lys Gly Val Ile Thr Asp 405 410 415Leu Asn Leu
Ala Thr Asp Ala Pro Leu Leu Phe Gly Gly Leu Ile Ala 420 425 430Ala
Thr Ala Phe Gly Ala Arg Ala Ile Met Glu Cys Phe Thr Asp Gln 435 440
445Gly Ile Ala Val Asn Asn Val Met Ala Leu Gly Gly Ile Ala Arg Lys
450 455 460Asn Gln Val Ile Met Gln Ala Cys Cys Asp Val Leu Asn Arg
Pro Leu465 470 475 480Gln Ile Val Ala Ser Asp Gln Cys Cys Ala Leu
Gly Ala Ala Ile Phe 485 490 495Ala Ala Val Ala Ala Lys Val His Ala
Asp Ile Pro Ser Ala Gln Gln 500 505 510Lys Met Ala Ser Ala Val Glu
Lys Thr Leu Gln Pro Arg Ser Glu Gln 515 520 525Ala Gln Arg Phe Glu
Gln Leu Tyr Arg Arg Tyr Gln Gln Trp Ala Met 530 535 540Ser Ala Glu
Gln His Tyr Leu Pro Thr Ser Ala Pro Ala Gln Ala Ala545 550 555
560Gln Ala Val Ala Thr Leu 565121453DNAE. coli 12atggcgattg
caattggcct cgattttggc agtgattctg tgcgagcttt ggcggtggac 60tgcgccagcg
gtgaagagat cgccaccagc gtagagtggt atccccgttg gcaaaaaggg
120caattttgtg atgccccgaa taaccagttc cgtcatcatc cgcgtgacta
cattgagtca 180atggaagcgg cactgaaaac cgtgcttgca gagcttagcg
tcgaacagcg cgcagctgtg 240gtcgggattg gcgttgacag taccggctcg
acgcccgcac cgattgatgc cgacggtaac 300gtgctggcgc tgcgcccgga
gtttgccgaa aacccgaacg cgatgttcgt attgtggaaa 360gaccacactg
cggttgaaag aagcgaagag attacccgtt tgtgccacgc gccgggcaat
420gttgactact cccgctatat tggcggtatt tattccagcg aatggttctg
ggcaaaaatc 480ctgcatgtga ctcgccagga cagcgccgtg gcgcaatctg
ccgcatcgtg gattgagctg 540tgcgactggg tgccagctct gctttccggt
accacccgcc cgcaggatat tcgtcgcgga 600cgttgcagcg ccgggcataa
atctctgtgg cacgaaagct ggggcggctt gccgccagcc 660agtttctttg
atgagctgga cccgatcctc aatcgccatt tgccttcccc gctgttcact
720gacacctgga ctgccgatat tccggtgggc accttatgcc cggaatgggc
gcagcgtctc 780ggcctgcctg aaagcgtggt gatttccggc ggcgcgtttg
actgccatat gggcgcagtt 840ggcgcaggcg cacagcctaa cgcactggta
aaagttatcg gtacttccac ctgcgacatt 900ctgattgccg acaaacagag
cgttggcgag cgggcagtta aaggtatttg cggtcaggtt 960gatggcagcg
tggtgcctgg atttatcggt ctggaagcag gccaatcggc gtttggtgat
1020atctacgcct ggttcggtcg cgtactcagc tggccgctgg aacagcttgc
cgcccagcat 1080ccggaactga aagcgcaaat caacgccagc cagaaacaac
tgcttccggc gctgaccgaa 1140gcatgggcca aaaatccgtc tctggatcac
ctgccggtgg tgctcgactg gtttaacggt 1200cgtcgctcgc caaacgctaa
ccaacgcctg aaaggggtga ttaccgatct taacctcgct 1260accgacgctc
cgctgctgtt cggcggtttg attgctgcca ccgcctttgg cgcacgcgca
1320atcatggagt gctttaccga tcaggggatc gccgtcaata acgtgatggc
gctgggcggc 1380atcgcgcgga aaaaccaagt cattatgcag gcctgctgcg
acgtgctgaa tcgcccgctg 1440caaattgttg cct 145313231PRTE. coli 13Met
Leu Glu Asp Leu Lys Arg Gln Val Leu Glu Ala Asn Leu Ala Leu1 5 10
15Pro Lys His Asn Leu Val Thr Leu Thr Trp Gly Asn Val Ser Ala Val
20 25 30Asp Arg Glu Arg Gly Val Phe Val Ile Lys Pro Ser Gly Val Asp
Tyr 35 40 45Ser Ile Met Thr Ala Asp Asp Met Val Val Val Ser Ile Glu
Thr Gly 50 55 60Glu Val Val Glu Gly Ala Lys Lys Pro Ser Ser Asp Thr
Pro Thr His65 70 75 80Arg Leu Leu Tyr Gln Ala Phe Pro Ser Ile Gly
Gly Ile Val His Thr 85 90 95His Ser Arg His Ala Thr Ile Trp Ala Gln
Ala Gly Gln Ser Ile Pro 100 105 110Ala Thr Gly Thr Thr His Ala Asp
Tyr Phe Tyr Gly Thr Ile Pro Cys 115 120 125Thr Arg Lys Met Thr Asp
Ala Glu Ile Asn Gly Glu Tyr Glu Trp Glu 130 135 140Thr Gly Asn Val
Ile Val Glu Thr Phe Glu Lys Gln Gly Ile Asp Ala145 150 155 160Ala
Gln Met Pro Gly Val Leu Val His Ser His Gly Pro Phe Ala Trp 165 170
175Gly Lys Asn Ala Glu Asp Ala Val His Asn Ala Ile Val Leu Glu Glu
180 185 190Val Ala Tyr Met Gly Ile Phe Cys Arg Gln Leu Ala Pro Gln
Leu Pro 195 200 205Asp Met Gln Gln Thr Leu Leu Asn Lys His Tyr Leu
Arg Lys His Gly 210 215 220Ala Lys Ala Tyr Tyr Gly Gln225
23014696DNAE. coli 14atgttagaag atctcaaacg ccaggtatta gaggccaacc
tggcgctgcc aaaacataac 60ctggtcacgc tcacatgggg caacgtcagc gccgttgatc
gcgagcgcgg cgtctttgtg 120atcaaacctt ccggcgtcga ttacagcatc
atgaccgctg acgatatggt cgtggttagc 180atcgaaaccg gtgaagtggt
tgaaggtgcg aaaaagccct cctccgatac gccaactcac 240cgactgctct
atcaggcatt cccgtccatt ggcggcattg tgcacacaca ctcgcgccac
300gccactatct gggcgcaggc gggccagtcg attccagcaa ccggcaccac
ccacgccgac 360tatttctacg gcaccattcc ctgcacccgc aaaatgaccg
acgcagaaat caacggtgaa 420tatgagtggg aaaccggtaa cgtcatcgta
gaaaccttcg aaaaacaggg tatcgatgca 480gcgcaaatgc ccggcgtcct
ggtccattct cacggcccat ttgcatgggg caaaaatgcc 540gaagatgcgg
tgcataacgc catcgtgctg gaagaggtcg cttatatggg gatattctgc
600cgtcagttag cgccgcagtt accggatatg cagcaaacgc tgctgaataa
acactatctg 660cgtaagcatg gcgcgaaggc atattacggg cagtaa
69615438PRTBacteroides thetaiotaomicron 15Met Ala Thr Lys Glu Phe
Phe Pro Gly Ile Glu Lys Ile Lys Phe Glu1 5 10 15Gly Lys Asp Ser Lys
Asn Pro Met Ala Phe Arg Tyr Tyr Asp Ala Glu 20 25 30Lys Val Ile Asn
Gly Lys Lys Met Lys Asp Trp Leu Arg Phe Ala Met 35 40 45Ala Trp Trp
His Thr Leu Cys Ala Glu Gly Gly Asp Gln Phe Gly Gly 50 55 60Gly Thr
Lys Gln Phe Pro Trp Asn Gly Asn Ala Asp Ala Ile Gln Ala65 70 75
80Ala Lys Asp Lys Met Asp Ala Gly Phe Glu Phe Met Gln Lys Met Gly
85 90 95Ile Glu Tyr Tyr Cys Phe His Asp Val Asp Leu Val Ser Glu Gly
Ala 100 105 110Ser Val Glu Glu Tyr Glu Ala Asn Leu Lys Glu Ile Val
Ala Tyr Ala 115 120 125Lys Gln Lys Gln Ala Glu Thr Gly Ile Lys Leu
Leu Trp Gly Thr Ala 130 135 140Asn Val Phe Gly His Ala Arg Tyr Met
Asn Gly Ala Ala Thr Asn Pro145 150 155 160Asp Phe Asp Val Val Ala
Arg Ala Ala Val Gln Ile Lys Asn Ala Ile 165 170 175Asp Ala Thr Ile
Glu Leu Gly Gly Glu Asn Tyr Val Phe Trp Gly Gly 180 185 190Arg Glu
Gly Tyr Met Ser Leu Leu Asn Thr Asp Gln Lys Arg Glu Lys 195 200
205Glu His Leu Ala Gln Met Leu Thr Ile Ala Arg Asp Tyr Ala Arg Ala
210 215 220Arg Gly Phe Lys Gly Thr Phe Leu Ile Glu Pro Lys Pro Met
Glu Pro225 230 235 240Thr Lys His Gln Tyr Asp Val Asp Thr Glu Thr
Val Ile Gly Phe Leu 245 250 255Lys Ala His Gly Leu Asp Lys Asp Phe
Lys Val Asn Ile Glu Val Asn 260 265 270His Ala Thr Leu Ala Gly His
Thr Phe Glu His Glu Leu Ala Val Ala 275 280 285Val Asp Asn Gly Met
Leu Gly Ser Ile Asp Ala Asn Arg Gly Asp Tyr 290 295 300Gln Asn Gly
Trp Asp Thr Asp Gln Phe Pro Ile Asp Asn Tyr Glu Leu305 310 315
320Thr Gln Ala Met Met Gln Ile Ile Arg Asn Gly Gly Leu Gly Thr Gly
325 330 335Gly Thr Asn Phe Asp Ala Lys Thr Arg Arg Asn Ser Thr Asp
Leu Glu 340 345 350Asp Ile Phe Ile Ala His Ile Ala Gly Met Asp Ala
Met Ala Arg Ala 355 360 365Leu Glu Ser Ala Ala Ala Leu Leu Asp Glu
Ser Pro Tyr Lys Lys Met 370 375 380Leu Ala Asp Arg Tyr Ala Ser Phe
Asp Gly Gly Lys Gly Lys Glu Phe385
390 395 400Glu Asp Gly Lys Leu Thr Leu Glu Asp Val Val Ala Tyr Ala
Lys Thr 405 410 415Lys Gly Glu Pro Lys Gln Thr Ser Gly Lys Gln Glu
Leu Tyr Glu Ala 420 425 430Ile Leu Asn Met Tyr Cys
435161317DNABacteroides thetaiotaomicron 16atggcaacaa 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 acggtgcagc taccaatcct
480gacttcgatg tagtagctcg tgctgctgtt cagatcaaaa atgcgattga
tgcaacgatt 540gaacttggcg gagagaatta tgtgttttgg ggtggtcgtg
aaggctatat gtctcttctg 600aacacagatc agaaacgtga aaaagaacac
cttgcacaga tgttgacgat tgctcgtgac 660tatgcccgtg cccgtggttt
caaaggtact ttcctgatcg aaccgaaacc gatggaaccg 720actaaacatc
aatatgacgt agatacggaa actgtaatcg gcttcctgaa agctcatggt
780ctggataagg atttcaaagt aaatatcgag gtgaatcacg caactttggc
aggtcacact 840ttcgagcatg aattggctgt agctgtagac aatggtatgt
tgggctcaat tgacgccaat 900cgtggtgact atcagaatgg ctgggataca
gaccaattcc cgatcgacaa ttatgaactg 960actcaggcta tgatgcagat
tatccgtaat ggtggtctcg gtaccggtgg tacgaacttt 1020gatgctaaaa
cccgtcgtaa ttctactgat ctggaagata tctttattgc tcacatcgca
1080ggtatggacg ctatggcccg tgcactcgaa agtgcagcgg ctctgctcga
cgaatctccc 1140tataagaaga tgctggctga ccgttatgct tcatttgatg
ggggcaaagg taaagaattt 1200gaagacggca agctgactct ggaggatgtg
gttgcttatg caaaaacaaa aggcgaaccg 1260aaacagacta gcggcaagca
agaactttat gaggcaattc tgaatatgta ttgctaa 131717258PRTSaccharomyces
cerevisiae 17Met 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 Lys182467DNASaccharomyces cerevisiae 18ggatccaaga ccattattcc
atcagaatgg aaaaaagttt aaaagatcac ggagattttg 60ttcttctgag cttctgctgt
ccttgaaaac aaattattcc gctggccgcc ccaaacaaaa 120acaaccccga
tttaataaca ttgtcacagt attagaaatt ttctttttac aaattaccat
180ttccagctta ctacttccta taatcctcaa tcttcagcaa gcgacgcagg
gaatagccgc 240tgaggtgcat aactgtcact tttcaattcg gccaatgcaa
tctcaggcgg acgaataagg 300gggccctctc gagaaaaaca aaaggaggat
gagattagta ctttaatgtt gtgttcagta 360attcagagac agacaagaga
ggtttccaac acaatgtctt tagactcata ctatcttggg 420tttgatcttt
cgacccaaca actgaaatgt ctcgccatta accaggacct aaaaattgtc
480cattcagaaa cagtggaatt tgaaaaggat cttccgcatt atcacacaaa
gaagggtgtc 540tatatacacg gcgacactat cgaatgtccc gtagccatgt
ggttaggggc tctagatctg 600gttctctcga aatatcgcga ggctaaattt
ccattgaaca aagttatggc cgtctcaggg 660tcctgccagc agcacgggtc
tgtctactgg tcctcccaag ccgaatctct gttagagcaa 720ttgaataaga
aaccggaaaa agatttattg cactacgtga gctctgtagc atttgcaagg
780caaaccgccc ccaattggca agaccacagt actgcaaagc aatgtcaaga
gtttgaagag 840tgcataggtg ggcctgaaaa aatggctcaa ttaacagggt
ccagagccca ttttagattt 900actggtcctc aaattctgaa aattgcacaa
ttagaaccag aagcttacga aaaaacaaag 960accatttctt tagtgtctaa
ttttttgact tctatcttag tgggccatct tgttgaatta 1020gaggaggcag
atgcctgtgg tatgaacctt tatgatatac gtgaaagaaa attcatgtat
1080gagctactac atctaattga tagttcttct aaggataaaa ctatcagaca
aaaattaatg 1140agagcaccca tgaaaaattt gatagcgggt accatctgta
aatattttat tgagaagtac 1200ggtttcaata caaactgcaa ggtctctccc
atgactgggg ataatttagc cactatatgt 1260tctttacccc tgcggaagaa
tgacgttctc gtttccctag gaacaagtac tacagttctt 1320ctggtcaccg
ataagtatca cccctctccg aactatcatc ttttcattca tccaactctg
1380ccaaaccatt atatgggtat gatttgttat tgtaatggtt ctttggcaag
ggagaggata 1440agagacgagt taaacaaaga acgggaaaat aattatgaga
agactaacga ttggactctt 1500tttaatcaag ctgtgctaga tgactcagaa
agtagtgaaa atgaattagg tgtatatttt 1560cctctggggg agatcgttcc
tagcgtaaaa gccataaaca aaagggttat cttcaatcca 1620aaaacgggta
tgattgaaag agaggtggcc aagttcaaag acaagaggca cgatgccaaa
1680aatattgtag aatcacaggc tttaagttgc agggtaagaa tatctcccct
gctttcggat 1740tcaaacgcaa gctcacaaca gagactgaac gaagatacaa
tcgtgaagtt tgattacgat 1800gaatctccgc tgcgggacta cctaaataaa
aggccagaaa ggactttttt tgtaggtggg 1860gcttctaaaa acgatgctat
tgtgaagaag tttgctcaag tcattggtgc tacaaagggt 1920aattttaggc
tagaaacacc aaactcatgt gcccttggtg gttgttataa ggccatgtgg
1980tcattgttat atgactctaa taaaattgca gttccttttg ataaatttct
gaatgacaat 2040tttccatggc atgtaatgga aagcatatcc gatgtggata
atgaaaattg gatcgctata 2100attccaagat tgtcccctta agcgaactgg
aaaagactct catctaaaat atgtttgaat 2160aatttatcat gccctgacaa
gtacacacaa acacagacac ataatataca tacatatata 2220tatatcaccg
ttattatgcg tgcacatgac aatgcccttg tatgtttcgt atactgtagc
2280aagtagtcat cattttgttc cccgttcgga aaatgacaaa aagtaaaatc
aataaatgaa 2340gagtaaaaaa caatttatga aagggtgagc gaccagcaac
gagagagaca aatcaaatta 2400gcgctttcca gtgagaatat aagagagcat
tgaaagagct aggttattgt taaatcatct 2460cgagctc
246719238PRTSaccharomyces cerevisiae 19Met 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
235201328DNASaccharomyces cerevisiae 20gttaggcact tacgtatctt
gtatagtagg aatggctcgg tttatgtata ttaggagatc 60aaaacgagaa aaaaatacca
tatcgtatag tatagagagt ataaatataa gaaatgccgc 120atatgtacaa
ctaatctagc aaatctctag aacgcaattc cttcgagact tcttctttca
180tgaaggagat aacatcgtgc gggtcagctg cagtgaaaac actggtacca
gcgacaataa 240cgttggcacc ggctttggcg gctttcggga tggtctcctt
gcccaaacca ccatcgactt 300ggatattcaa atgggggaac ttggctctca
aagtttccac ttttggcatc atgtcttcca 360tgaatttttg gcctccaaac
ccaggttcca cagtcataac aagagccata tccaaatgag 420gagctagttc
aaataaaacg tcaacagaag taccaggttt gatggcgcat gcagctttga
480tgcccttaga cttaatcaac ttaactaaat gcaaagggtc ttgtgtggcc
tcgtagtgga 540acgtaaattg gtcagcacca catttagcaa aatcgtcgac
ccatttttca ggattttcaa 600ccatcatgtg acaatcgaag aacgcagtgg
gcttcttttc tgtgttgcta gcatcgccag 660ggcgtggcac agaacgacgt
agggaggtaa caattggttg gcccagagta atgtttggaa 720caaaatggcc
gtccatgaca tcgatatgta accaatctgc gccggcgttg atgaccttat
780gacattcgca acccaagttg gcgaagtcag aagcaaggat actgggagct
ataattggtt 840tgaccatttt ttcttgtgtg tttacctcgc tcttggaatt
agcaaatggc cttcttgcat 900gaaattgtat cgagtttgct ttatttttct
ttttacgggc ggattctttc tattctggct 960ttcctataac agagatcatg
aaagaagttc cagcttacgg atcaagaaag tacctataca 1020tatacaaaaa
tctgattact ttcccagctc gacttggata gctgttcttg ttttctcttg
1080gcgacacatt ttttgtttct gaagccacgt cctgctttat aagaggacat
ttaaagttgc 1140aggacttgaa tgcaattacc ggaagaagca accaaccggc
atggttcagc atacaataca 1200catttgatta gaaaagcaga gaataaatag
acatgatacc tctcttttta tcctctgcag 1260cgtattattg tttattccac
gcaggcatcg gtcgttggct gttgttatgt ctcagataag 1320cgcgtttg
132821680PRTSaccharomyces cerevisiae 21Met 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 680222046DNASaccharomyces cerevisiae
22atggcacagt tctccgacat tgataaactt gcggtttcca ctttaagatt actttccgtt
60gaccaggtgg aaagcgcaca atctggccac ccaggtgcac cactaggatt ggcaccagtt
120gcccatgtaa ttttcaagca actgcgctgt aaccctaaca atgaacattg
gatcaataga 180gacaggtttg ttctgtcgaa cggtcactca tgcgctcttc
tgtactcaat gctccatcta 240ttaggatacg attactctat cgaggacttg
agacaattta gacaagtaaa ctcaaggaca 300ccgggtcatc cagaattcca
ctcagcggga gtggaaatca cttccggtcc gctaggccag 360ggtatctcaa
atgctgttgg tatggcaata gcgcaggcca actttgccgc cacttataac
420gaggatggct ttcccatttc cgactcatat acgtttgcta ttgtagggga
tggttgctta 480caagagggtg tttcttcgga gacctcttcc ttagcgggac
atctgcaatt gggtaacttg 540attacgtttt atgacagtaa tagcatttcc
attgacggta aaacctcgta ctcgttcgac 600gaagatgttt tgaagcgata
cgaggcatat ggttgggaag tcatggaagt cgataaagga 660gacgacgata
tggaatccat ttctagcgct ttggaaaagg caaaactatc gaaggacaag
720ccaaccataa tcaaggtaac tactacaatt ggatttgggt ccctacaaca
gggtactgct 780ggtgttcatg ggtccgcttt gaaggcagat gatgttaaac
agttgaagaa gaggtggggg 840tttgacccaa ataaatcatt tgtagtacct
caagaggtgt acgattatta taagaagact 900gttgtggaac ccggtcaaaa
acttaatgag gaatgggata ggatgtttga agaatacaaa 960accaaatttc
ccgagaaggg taaagaattg caaagaagat tgaatggtga gttaccggaa
1020ggttgggaaa agcatttacc gaagtttact ccggacgacg atgctctggc
aacaagaaag 1080acatcccagc aggtgctgac gaacatggtc caagttttgc
ctgaattgat cggtggttct 1140gccgatttga caccttcgaa tctgacaagg
tgggaaggcg cggtagattt ccaacctccc 1200attacccaac taggtaacta
tgcaggaagg tacattagat acggtgtgag ggaacacgga 1260atgggtgcca
ttatgaacgg tatctctgcc tttggtgcaa actacaagcc ttacggtggt
1320acctttttga acttcgtctc ttatgctgca ggagccgtta ggttagccgc
cttgtctggt 1380aatccagtca tttgggttgc aacacatgac tctatcgggc
ttggtgagga tggtccaacg 1440caccaaccta ttgaaactct ggctcacttg
agggctattc caaacatgca tgtatggaga 1500cctgctgatg gtaacgaaac
ttctgctgcg tattattctg ctatcaaatc tggtcgaaca 1560ccatctgttg
tggctttatc acgacagaat cttcctcaat tggagcattc ctcttttgaa
1620aaagccttga agggtggcta tgtgatccat gacgtggaga atcctgatat
tatcctggtg 1680tcaacaggat cagaagtctc catttctata gatgcagcca
aaaaattgta cgatactaaa 1740aaaatcaaag caagagttgt ttccctgcca
gacttttata cttttgacag gcaaagtgaa 1800gaatacagat tctctgttct
accagacggt gttccgatca tgtcctttga agtattggct 1860acttcaagct
ggggtaagta tgctcatcaa tcgttcggac tcgacgaatt tggtcgttca
1920ggcaaggggc ctgaaattta caaattgttc gatttcacag cggacggtgt
tgcgtcaagg 1980gctgaaaaga caatcaatta ctacaaagga aagcagttgc
tttctcctat gggaagagct 2040ttctaa 204623335PRTSaccharomyces
cerevisiae 23Met 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 335242046DNASaccharomyces cerevisiae
24atggcacagt tctccgacat tgataaactt gcggtttcca ctttaagatt actttccgtt
60gaccaggtgg aaagcgcaca atctggccac ccaggtgcac cactaggatt ggcaccagtt
120gcccatgtaa ttttcaagca actgcgctgt aaccctaaca atgaacattg
gatcaataga 180gacaggtttg ttctgtcgaa cggtcactca tgcgctcttc
tgtactcaat gctccatcta 240ttaggatacg attactctat cgaggacttg
agacaattta gacaagtaaa ctcaaggaca 300ccgggtcatc cagaattcca
ctcagcggga gtggaaatca cttccggtcc gctaggccag 360ggtatctcaa
atgctgttgg tatggcaata gcgcaggcca actttgccgc cacttataac
420gaggatggct ttcccatttc cgactcatat acgtttgcta ttgtagggga
tggttgctta 480caagagggtg tttcttcgga gacctcttcc ttagcgggac
atctgcaatt gggtaacttg 540attacgtttt atgacagtaa tagcatttcc
attgacggta aaacctcgta ctcgttcgac 600gaagatgttt tgaagcgata
cgaggcatat ggttgggaag tcatggaagt cgataaagga 660gacgacgata
tggaatccat ttctagcgct ttggaaaagg caaaactatc gaaggacaag
720ccaaccataa tcaaggtaac tactacaatt ggatttgggt ccctacaaca
gggtactgct 780ggtgttcatg ggtccgcttt gaaggcagat gatgttaaac
agttgaagaa gaggtggggg 840tttgacccaa ataaatcatt tgtagtacct
caagaggtgt acgattatta taagaagact 900gttgtggaac ccggtcaaaa
acttaatgag gaatgggata ggatgtttga agaatacaaa 960accaaatttc
ccgagaaggg taaagaattg caaagaagat tgaatggtga gttaccggaa
1020ggttgggaaa agcatttacc gaagtttact ccggacgacg atgctctggc
aacaagaaag 1080acatcccagc aggtgctgac gaacatggtc caagttttgc
ctgaattgat cggtggttct 1140gccgatttga caccttcgaa tctgacaagg
tgggaaggcg cggtagattt ccaacctccc 1200attacccaac taggtaacta
tgcaggaagg tacattagat acggtgtgag ggaacacgga 1260atgggtgcca
ttatgaacgg tatctctgcc tttggtgcaa actacaagcc ttacggtggt
1320acctttttga acttcgtctc ttatgctgca ggagccgtta ggttagccgc
cttgtctggt 1380aatccagtca tttgggttgc aacacatgac tctatcgggc
ttggtgagga tggtccaacg 1440caccaaccta ttgaaactct ggctcacttg
agggctattc caaacatgca tgtatggaga 1500cctgctgatg gtaacgaaac
ttctgctgcg tattattctg ctatcaaatc tggtcgaaca 1560ccatctgttg
tggctttatc acgacagaat cttcctcaat tggagcattc ctcttttgaa
1620aaagccttga agggtggcta tgtgatccat gacgtggaga atcctgatat
tatcctggtg 1680tcaacaggat cagaagtctc catttctata gatgcagcca
aaaaattgta cgatactaaa 1740aaaatcaaag caagagttgt ttccctgcca
gacttttata cttttgacag gcaaagtgaa 1800gaatacagat tctctgttct
accagacggt gttccgatca tgtcctttga agtattggct 1860acttcaagct
ggggtaagta tgctcatcaa tcgttcggac tcgacgaatt tggtcgttca
1920ggcaaggggc ctgaaattta caaattgttc gatttcacag cggacggtgt
tgcgtcaagg 1980gctgaaaaga caatcaatta ctacaaagga aagcagttgc
tttctcctat gggaagagct 2040ttctaa 204625600PRTSaccharomyces
cerevisiae 25Met 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 600262467DNASaccharomyces cerevisiae
26ggatccaaga ccattattcc atcagaatgg aaaaaagttt aaaagatcac ggagattttg
60ttcttctgag cttctgctgt ccttgaaaac aaattattcc gctggccgcc ccaaacaaaa
120acaaccccga tttaataaca ttgtcacagt attagaaatt ttctttttac
aaattaccat 180ttccagctta ctacttccta taatcctcaa tcttcagcaa
gcgacgcagg gaatagccgc 240tgaggtgcat aactgtcact tttcaattcg
gccaatgcaa tctcaggcgg acgaataagg 300gggccctctc gagaaaaaca
aaaggaggat gagattagta ctttaatgtt gtgttcagta 360attcagagac
agacaagaga ggtttccaac acaatgtctt tagactcata ctatcttggg
420tttgatcttt cgacccaaca actgaaatgt ctcgccatta accaggacct
aaaaattgtc 480cattcagaaa cagtggaatt tgaaaaggat cttccgcatt
atcacacaaa gaagggtgtc 540tatatacacg gcgacactat cgaatgtccc
gtagccatgt ggttaggggc tctagatctg 600gttctctcga aatatcgcga
ggctaaattt ccattgaaca aagttatggc cgtctcaggg 660tcctgccagc
agcacgggtc tgtctactgg tcctcccaag ccgaatctct gttagagcaa
720ttgaataaga aaccggaaaa agatttattg cactacgtga gctctgtagc
atttgcaagg 780caaaccgccc ccaattggca agaccacagt actgcaaagc
aatgtcaaga gtttgaagag 840tgcataggtg ggcctgaaaa aatggctcaa
ttaacagggt ccagagccca ttttagattt 900actggtcctc aaattctgaa
aattgcacaa ttagaaccag aagcttacga aaaaacaaag 960accatttctt
tagtgtctaa ttttttgact tctatcttag tgggccatct tgttgaatta
1020gaggaggcag atgcctgtgg tatgaacctt tatgatatac gtgaaagaaa
attcatgtat 1080gagctactac atctaattga tagttcttct aaggataaaa
ctatcagaca aaaattaatg 1140agagcaccca tgaaaaattt gatagcgggt
accatctgta aatattttat tgagaagtac 1200ggtttcaata caaactgcaa
ggtctctccc atgactgggg ataatttagc cactatatgt 1260tctttacccc
tgcggaagaa tgacgttctc gtttccctag gaacaagtac tacagttctt
1320ctggtcaccg ataagtatca cccctctccg aactatcatc ttttcattca
tccaactctg 1380ccaaaccatt atatgggtat gatttgttat tgtaatggtt
ctttggcaag ggagaggata 1440agagacgagt taaacaaaga acgggaaaat
aattatgaga agactaacga ttggactctt 1500tttaatcaag ctgtgctaga
tgactcagaa agtagtgaaa atgaattagg tgtatatttt 1560cctctggggg
agatcgttcc tagcgtaaaa gccataaaca aaagggttat cttcaatcca
1620aaaacgggta tgattgaaag agaggtggcc aagttcaaag acaagaggca
cgatgccaaa 1680aatattgtag aatcacaggc tttaagttgc agggtaagaa
tatctcccct gctttcggat 1740tcaaacgcaa gctcacaaca gagactgaac
gaagatacaa tcgtgaagtt tgattacgat 1800gaatctccgc tgcgggacta
cctaaataaa aggccagaaa ggactttttt tgtaggtggg 1860gcttctaaaa
acgatgctat tgtgaagaag tttgctcaag tcattggtgc tacaaagggt
1920aattttaggc tagaaacacc aaactcatgt gcccttggtg gttgttataa
ggccatgtgg 1980tcattgttat atgactctaa taaaattgca gttccttttg
ataaatttct gaatgacaat 2040tttccatggc atgtaatgga aagcatatcc
gatgtggata atgaaaattg gatcgctata 2100attccaagat tgtcccctta
agcgaactgg aaaagactct catctaaaat atgtttgaat 2160aatttatcat
gccctgacaa gtacacacaa acacagacac ataatataca tacatatata
2220tatatcaccg ttattatgcg tgcacatgac aatgcccttg tatgtttcgt
atactgtagc 2280aagtagtcat cattttgttc cccgttcgga aaatgacaaa
aagtaaaatc aataaatgaa 2340gagtaaaaaa caatttatga aagggtgagc
gaccagcaac gagagagaca aatcaaatta 2400gcgctttcca gtgagaatat
aagagagcat tgaaagagct aggttattgt taaatcatct 2460cgagctc
246727494PRTPiromyces species 27Met Lys Thr Val Ala Gly Ile Asp Leu
Gly Thr Gln Ser Met Lys Val1 5 10 15Val Ile Tyr Asp Tyr Glu Lys Lys
Glu Ile Ile Glu Ser Ala Ser Cys 20 25 30Pro Met Glu Leu Ile Ser Glu
Ser Asp Gly Thr Arg Glu Gln Thr Thr 35 40 45Glu Trp Phe Asp Lys Gly
Leu Glu Val Cys Phe Gly Lys Leu Ser Ala 50 55 60Asp Asn Lys Lys Thr
Ile Glu Ala Ile Gly Ile Ser Gly Gln Leu His65 70 75 80Gly Phe Val
Pro Leu Asp Ala Asn Gly Lys Ala Leu Tyr Asn Ile Lys 85 90 95Leu Trp
Cys Asp Thr Ala Thr Val Glu Glu Cys Lys Ile Ile Thr Asp 100 105
110Ala Ala Gly Gly Asp Lys Ala Val Ile Asp Ala Leu Gly Asn Leu Met
115 120 125Leu Thr Gly Phe Thr Ala Pro Lys Ile Leu Trp Leu Lys Arg
Asn Lys 130 135 140Pro Glu Ala Phe Ala Asn Leu Lys Tyr Ile Met Leu
Pro His Asp Tyr145 150 155 160Leu Asn Trp Lys Leu Thr Gly Asp Tyr
Val Met Glu Tyr Gly Asp Ala 165 170 175Ser Gly Thr Ala Leu Phe Asp
Ser Lys Asn Arg Cys Trp Ser Lys Lys 180 185 190Ile Cys Asp Ile Ile
Asp Pro Lys Leu Leu Asp Leu Leu Pro Lys Leu 195 200 205Ile Glu Pro
Ser Ala Pro Ala Gly Lys Val Asn Asp Glu Ala Ala Lys 210 215 220Ala
Tyr Gly Ile Pro Ala Gly Ile Pro Val Ser Ala Gly Gly Gly Asp225 230
235 240Asn Met Met Gly Ala Val Gly Thr Gly Thr Val Ala Asp Gly Phe
Leu 245 250 255Thr Met Ser Met Gly Thr Ser Gly Thr Leu Tyr Gly Tyr
Ser Asp Lys 260 265 270Pro Ile Ser Asp Pro Ala Asn Gly Leu Ser Gly
Phe Cys Ser Ser Thr 275 280 285Gly Gly Trp Leu Pro Leu Leu Cys Thr
Met Asn Cys Thr Val Ala Thr 290 295 300Glu Phe Val Arg Asn Leu Phe
Gln Met Asp Ile Lys Glu Leu Asn Val305 310 315 320Glu Ala Ala Lys
Ser Pro Cys Gly Ser Glu Gly Val Leu Val Ile Pro 325 330 335Phe Phe
Asn Gly Glu Arg Thr Pro Asn Leu Pro Asn Gly Arg Ala Ser 340 345
350Ile Thr Gly Leu Thr Ser Ala Asn Thr Ser Arg Ala Asn Ile Ala Arg
355 360 365Ala Ser Phe Glu Ser Ala Val Phe Ala Met Arg Gly Gly Leu
Asp Ala 370 375 380Phe Arg Lys Leu Gly Phe Gln Pro Lys Glu Ile Arg
Leu Ile Gly Gly385 390 395 400Gly Ser Lys Ser Asp Leu Trp Arg Gln
Ile Ala Ala Asp Ile Met Asn 405 410 415Leu Pro Ile Arg Val Pro Leu
Leu Glu Glu Ala Ala Ala Leu Gly Gly 420 425 430Ala Val Gln Ala Leu
Trp Cys Leu Lys Asn Gln Ser Gly Lys Cys Asp 435 440 445Ile Val Glu
Leu Cys Lys Glu His Ile Lys Ile Asp Glu Ser Lys Asn 450 455 460Ala
Asn Pro Ile Ala Glu Asn Val Ala Val Tyr Asp Lys Ala Tyr Asp465 470
475 480Glu Tyr Cys Lys Val Val Asn Thr Leu Ser Pro Leu Tyr Ala 485
490282041DNAPiromyces sp. 28attatataaa ataactttaa ataaaacaat
ttttatttgt ttatttaatt attcaaaaaa 60aattaaagta aaagaaaaat aatacagtag
aacaatagta ataatatcaa aatgaagact 120gttgctggta ttgatcttgg
aactcaaagt atgaaagtcg ttatttacga ctatgaaaag 180aaagaaatta
ttgaaagtgc tagctgtcca atggaattga tttccgaaag tgacggtacc
240cgtgaacaaa ccactgaatg gtttgacaag ggtcttgaag tttgttttgg
taagcttagt 300gctgataaca aaaagactat tgaagctatt ggtatttctg
gtcaattaca cggttttgtt 360cctcttgatg ctaacggtaa ggctttatac
aacatcaaac tttggtgtga tactgctacc 420gttgaagaat gtaagattat
cactgatgct gccggtggtg acaaggctgt tattgatgcc 480cttggtaacc
ttatgctcac cggtttcacc gctccaaaga tcctctggct caagcgcaac
540aagccagaag ctttcgctaa cttaaagtac attatgcttc cacacgatta
cttaaactgg 600aagcttactg gtgattacgt tatggaatac ggtgatgcct
ctggtaccgc tctcttcgat 660tctaagaacc gttgctggtc taagaagatt
tgcgatatca ttgacccaaa acttttagat 720ttacttccaa agttaattga
accaagcgct ccagctggta aggttaatga tgaagccgct 780aaggcttacg
gtattccagc cggtattcca gtttccgctg gtggtggtga taacatgatg
840ggtgctgttg gtactggtac tgttgctgat ggtttcctta ccatgtctat
gggtacttct 900ggtactcttt acggttacag tgacaagcca attagtgacc
cagctaatgg tttaagtggt 960ttctgttctt ctactggtgg atggcttcca
ttactttgta ctatgaactg tactgttgcc 1020actgaattcg ttcgtaacct
cttccaaatg gatattaagg aacttaatgt tgaagctgcc 1080aagtctccat
gtggtagtga aggtgtttta gttattccat tcttcaatgg tgaaagaact
1140ccaaacttac caaacggtcg tgctagtatt actggtctta cttctgctaa
caccagccgt 1200gctaacattg ctcgtgctag tttcgaatcc gccgttttcg
ctatgcgtgg tggtttagat 1260gctttccgta agttaggttt ccaaccaaag
gaaattcgtc ttattggtgg tggttctaag 1320tctgatctct ggagacaaat
tgccgctgat atcatgaacc ttccaatcag agttccactt 1380ttagaagaag
ctgctgctct tggtggtgct gttcaagctt tatggtgtct taagaaccaa
1440tctggtaagt gtgatattgt tgaactttgc aaagaacaca ttaagattga
tgaatctaag 1500aatgctaacc caattgccga aaatgttgct gtttacgaca
aggcttacga tgaatactgc 1560aaggttgtaa atactctttc tccattatat
gcttaaattg ccaatgtaaa aaaaaatata 1620atgccatata attgccttgt
caatacactg ttcatgttca tataatcata ggacattgaa
1680tttacaaggt ttatacaatt aatatctatt atcatattat tatacagcat
ttcattttct 1740aagattagac gaaacaattc ttggttcctt gcaatataca
aaatttacat gaatttttag 1800aatagtctcg tatttatgcc caataatcag
gaaaattacc taatgctgga ttcttgttaa 1860taaaaacaaa ataaataaat
taaataaaca aataaaaatt ataagtaaat ataaatatat 1920aagtaatata
aaaaaaaagt aaataaataa ataaataaat aaaaattttt tgcaaatata
1980taaataaata aataaaatat aaaaataatt tagcaaataa attaaaaaaa
aaaaaaaaaa 2040a 204129327PRTSaccharomyces cerevisiae 29Met Ser Ser
Leu Val Thr Leu Asn Asn Gly Leu Lys Met Pro Leu Val1 5 10 15Gly Leu
Gly Cys Trp Lys Ile Asp Lys Lys Val Cys Ala Asn Gln Ile 20 25 30Tyr
Glu Ala Ile Lys Leu Gly Tyr Arg Leu Phe Asp Gly Ala Cys Asp 35 40
45Tyr Gly Asn Glu Lys Glu Val Gly Glu Gly Ile Arg Lys Ala Ile Ser
50 55 60Glu Gly Leu Val Ser Arg Lys Asp Ile Phe Val Val Ser Lys Leu
Trp65 70 75 80Asn Asn Phe His His Pro Asp His Val Lys Leu Ala Leu
Lys Lys Thr 85 90 95Leu Ser Asp Met Gly Leu Asp Tyr Leu Asp Leu Tyr
Tyr Ile His Phe 100 105 110Pro Ile Ala Phe Lys Tyr Val Pro Phe Glu
Glu Lys Tyr Pro Pro Gly 115 120 125Phe Tyr Thr Gly Ala Asp Asp Glu
Lys Lys Gly His Ile Thr Glu Ala 130 135 140His Val Pro Ile Ile Asp
Thr Tyr Arg Ala Leu Glu Glu Cys Val Asp145 150 155 160Glu Gly Leu
Ile Lys Ser Ile Gly Val Ser Asn Phe Gln Gly Ser Leu 165 170 175Ile
Gln Asp Leu Leu Arg Gly Cys Arg Ile Lys Pro Val Ala Leu Gln 180 185
190Ile Glu His His Pro Tyr Leu Thr Gln Glu His Leu Val Glu Phe Cys
195 200 205Lys Leu His Asp Ile Gln Val Val Ala Tyr Ser Ser Phe Gly
Pro Gln 210 215 220Ser Phe Ile Glu Met Asp Leu Gln Leu Ala Lys Thr
Thr Pro Thr Leu225 230 235 240Phe Glu Asn Asp Val Ile Lys Lys Val
Ser Gln Asn His Pro Gly Ser 245 250 255Thr Thr Ser Gln Val Leu Leu
Arg Trp Ala Thr Gln Arg Gly Ile Ala 260 265 270Val Ile Pro Lys Ser
Ser Lys Lys Glu Arg Leu Leu Gly Asn Leu Glu 275 280 285Ile Glu Lys
Lys Phe Thr Leu Thr Glu Gln Glu Leu Lys Asp Ile Ser 290 295 300Ala
Leu Asn Ala Asn Ile Arg Phe Asn Asp Pro Trp Thr Trp Leu Asp305 310
315 320Gly Lys Phe Pro Thr Phe Ala 32530984DNASaccharomyces
cerevisiae 30atgtcttcac tggttactct taataacggt ctgaaaatgc ccctagtcgg
cttagggtgc 60tggaaaattg acaaaaaagt ctgtgcgaat caaatttatg aagctatcaa
attaggctac 120cgtttattcg atggtgcttg cgactacggc aacgaaaagg
aagttggtga aggtatcagg 180aaagccatct ccgaaggtct tgtttctaga
aaggatatat ttgttgtttc aaagttatgg 240aacaattttc accatcctga
tcatgtaaaa ttagctttaa agaagacctt aagcgatatg 300ggacttgatt
atttagacct gtattatatt cacttcccaa tcgccttcaa atatgttcca
360tttgaagaga aataccctcc aggattctat acgggcgcag atgacgagaa
gaaaggtcac 420atcaccgaag cacatgtacc aatcatagat acgtaccggg
ctctggaaga atgtgttgat 480gaaggcttga ttaagtctat tggtgtttcc
aactttcagg gaagcttgat tcaagattta 540ttacgtggtt gtagaatcaa
gcccgtggct ttgcaaattg aacaccatcc ttatttgact 600caagaacacc
tagttgagtt ttgtaaatta cacgatatcc aagtagttgc ttactcctcc
660ttcggtcctc aatcattcat tgagatggac ttacagttgg caaaaaccac
gccaactctg 720ttcgagaatg atgtaatcaa gaaggtctca caaaaccatc
caggcagtac cacttcccaa 780gtattgctta gatgggcaac tcagagaggc
attgccgtca ttccaaaatc ttccaagaag 840gaaaggttac ttggcaacct
agaaatcgaa aaaaagttca ctttaacgga gcaagaattg 900aaggatattt
ctgcactaaa tgccaacatc agatttaatg atccatggac ctggttggat
960ggtaaattcc ccacttttgc ctga 9843131DNAArtificial Sequenceprimer
31gactagtcga gtttatcatt atcaatactg c 313249DNAArtificial
Sequenceprimer 32ctcataatca ggtactgata acattttgtt tgtttatgtg
tgtttattc 493349DNAArtificial Sequenceprimer 33gaataaacac
acataaacaa acaaaatgtt atcagtacct gattatgag 493448DNAArtificial
Sequenceprimer 34aatcataaat cataagaaat tcgcttactt taagaatgcc
ttagtcat 483548DNAArtificial Sequenceprimer 35atgactaagg cattcttaaa
gtaagcgaat ttcttatgat ttatgatt 483636DNAArtificial Sequenceprimer
36cactagtctc gagtgtggaa gaacgattac aacagg 363731DNAArtificial
Sequenceprimer 37cgagctcgtg ggtgtattgg attataggaa g
313848DNAArtificial Sequenceprimer 38ttgggctgtt tcaactaaat
tcatttttag gctggtatct tgattcta 483948DNAArtificial Sequenceprimer
39tagaatcaag ataccagcct aaaaatgaat ttagttgaaa cagcccaa
484048DNAArtificial Sequenceprimer 40aatcataaat cataagaaat
tcgctctaat atttgattgc ttgcccag 484148DNAArtificial Sequenceprimer
41ctgggcaagc aatcaaatat tagagcgaat ttcttatgat ttatgatt
484231DNAArtificial Sequenceprimer 42tgagctcgtg tggaagaacg
attacaacag g 314328DNAArtificial Sequenceprimer 43acgcgtcgac
tcgtaggaac aatttcgg 284450DNAArtificial Sequenceprimer 44cttcttgttt
taatgcttct agcatttttt gattaaaatt aaaaaaactt 504550DNAArtificial
Sequenceprimer 45aagttttttt aattttaatc aaaaaatgct agaagcatta
aaacaagaag 504646DNAArtificial Sequenceprimer 46ggtatatatt
taagagcgat ttgtttactt gcgaactgca tgatcc 464746DNAArtificial
Sequenceprimer 47ggatcatgca gttcgcaagt aaacaaatcg ctcttaaata tatacc
464833DNAArtificial Sequenceprimer 48cgcagtcgac cttttaaaca
gttgatgaga acc 3349676DNAArtificial Sequencepromoter 49tcgagtttat
cattatcaat actgccattt caaagaatac gtaaataatt aatagtagtg 60attttcctaa
ctttatttag tcaaaaaatt agccttttaa ttctgctgta acccgtacat
120gcccaaaata gggggcgggt tacacagaat atataacatc gtaggtgtct
gggtgaacag 180tttattcctg gcatccacta aatataatgg agcccgcttt
ttaagctggc atccagaaaa 240aaaaagaatc ccagcaccaa aatattgttt
tcttcaccaa ccatcagttc ataggtccat 300tctcttagcg caactacaga
gaacaggggc acaaacaggc aaaaaacggg cacaacctca 360atggagtgat
gcaacctgcc tggagtaaat gatgacacaa ggcaattgac ccacgcatgt
420atctatctca ttttcttaca ccttctatta ccttctgctc tctctgattt
ggaaaaagct 480gaaaaaaaag gttgaaacca gttccctgaa attattcccc
tacttgacta ataagtatat 540aaagacggta ggtattgatt gtaattctgt
aaatctattt cttaaacttc ttaaattcta 600cttttatagt tagtcttttt
tttagtttta aaacaccaag aacttagttt cgaataaaca 660cacataaaca aacaaa
67650326DNAArtificial Sequenceterminator 50gcgaatttct tatgatttat
gatttttatt attaaataag ttataaaaaa aataagtgta 60tacaaatttt aaagtgactc
ttaggtttta aaacgaaaat tcttattctt gagtaactct 120ttcctgtagg
tcaggttgct ttctcaggta tagcatgagg tcgctcttat tgaccacacc
180tctaccggca tgccgagcaa atgcctgcaa atcgctcccc atttcaccca
attgtagata 240tgctaactcc agcaatgagt tgatgaatct cggtgtgtat
tttatgtcct cagaggacaa 300cacctgttgt aatcgttctt ccacac
32651374DNAArtificial Sequencepromoter 51gtgggtgtat tggattatag
gaagccacgc gctcaacctg gaattacagg aagctggtaa 60ttttttgggt ttgcaatcat
caccatctgc acgttgttat aatgtcccgt gtctatatat 120atccattgac
ggtattctat ttttttgcta ttgaaatgag cgttttttgt tactacaatt
180ggttttacag acggaatttt ccctatttgt ttcgtcccat ttttcctttt
ctcattgttc 240tcatatctta aaaaggtcct ttcttcataa tcaatgcttt
cttttactta atattttact 300tgcattcagt gaattttaat acatattcct
ctagtcttgc aaaatcgatt tagaatcaag 360ataccagcct aaaa
37452390DNAArtificial Sequencepromoter 52ctcgtaggaa caatttcggg
cccctgcgtg ttcttctgag gttcatcttt tacatttgct 60tctgctggat aattttcaga
ggcaacaagg aaaaattaga tggcaaaaag tcgtctttca 120aggaaaaatc
cccaccatct ttcgagatcc cctgtaactt attggcaact gaaagaatga
180aaaggaggaa aatacaaaat atactagaac tgaaaaaaaa aaagtataaa
tagagacgat 240atatgccaat acttcacaat gttcgaatct attcttcatt
tgcagctatt gtaaaataat 300aaaacatcaa gaacaaacaa gctcaacttg
tcttttctaa gaacaaagaa taaacacaaa 360aacaaaaagt ttttttaatt
ttaatcaaaa 39053302DNAArtificial Sequenceterminator 53acaaatcgct
cttaaatata tacctaaaga acattaaagc tatattataa gcaaagatac 60gtaaattttg
cttatattat tatacacata tcatatttct atatttttaa gatttggtta
120tataatgtac gtaatgcaaa ggaaataaat tttatacatt attgaacagc
gtccaagtaa 180ctacattatg tgcactaata gtttagcgtc gtgaagactt
tattgtgtcg cgaaaagtaa 240aaattttaaa aattagagca ccttgaactt
gcgaaaaagg ttctcatcaa ctgtttaaaa 300gg 302
* * * * *
References