U.S. patent application number 15/872581 was filed with the patent office on 2019-01-10 for biological methods for preparing a fatty dicarboxylic acid.
The applicant listed for this patent is Verdezyne, Inc.. Invention is credited to Tom BEARDSLEE, Dudley EIRICH, Jose LAPLAZA, Stephen PICATAGGIO.
Application Number | 20190010524 15/872581 |
Document ID | / |
Family ID | 50001251 |
Filed Date | 2019-01-10 |
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United States Patent
Application |
20190010524 |
Kind Code |
A1 |
LAPLAZA; Jose ; et
al. |
January 10, 2019 |
BIOLOGICAL METHODS FOR PREPARING A FATTY DICARBOXYLIC ACID
Abstract
Provided are engineered microorganisms capable of producing
fatty dicarboxylic acids and products expressed by such
microorganisms. Also provided are biological methods for producing
fatty dicarboxylic acids.
Inventors: |
LAPLAZA; Jose; (Carlsbad,
CA) ; BEARDSLEE; Tom; (Carlsbad, CA) ; EIRICH;
Dudley; (Carlsbad, CA) ; PICATAGGIO; Stephen;
(Carlsbad, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Verdezyne, Inc. |
Carlsbad |
CA |
US |
|
|
Family ID: |
50001251 |
Appl. No.: |
15/872581 |
Filed: |
January 16, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14654442 |
Jun 19, 2015 |
9909151 |
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PCT/US13/76664 |
Dec 19, 2013 |
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15872581 |
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61739656 |
Dec 19, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/52 20130101;
C12P 7/44 20130101; C12N 15/81 20130101 |
International
Class: |
C12P 7/44 20060101
C12P007/44; C12N 15/81 20060101 C12N015/81; C12N 15/52 20060101
C12N015/52 |
Claims
1.-98. (canceled)
99. A method for producing a desired diacid by a yeast from a
feedstock toxic to the yeast, comprising: (a) contacting a
genetically modified yeast in culture with a feedstock not
substantially toxic to the yeast, thereby performing an induction;
and (b) contacting the yeast after the induction in (a) with a
feedstock toxic to the yeast, whereby the desired diacid is
produced by the yeast from the feedstock toxic to the yeast in an
amount greater than the amount of the diacid produced from the
feedstock toxic to the yeast when the induction is not
performed.
100. The method of claim 99, wherein the feedstock not
substantially toxic to the yeast has the same number of carbons as
the feedstock toxic to the yeast.
101. The method of claim 99, wherein the feedstock not
substantially toxic to the yeast has a different number of carbons
compared to the feedstock toxic to the yeast.
102. The method of claim 99, wherein the feedstock not
substantially toxic to the yeast comprises a fatty acid methyl
ester.
103. The method of claim 99, wherein the feedstock not
substantially toxic to the yeast comprises a free fatty acid.
104. The method of claim 99, wherein the feedstock not
substantially toxic to the yeast comprises more than twelve
carbons.
105. A diacid produced by the method of claim 99.
106.-176. (canceled)
177. The method of claim 102, wherein the fatty acid ethyl ester is
methyl myristate.
178. The method of claim 99, wherein the feedstock toxic to the
yeast is a fatty acid methyl ester.
179. The method of claim 103, wherein the free fatty acid is
decane.
180. The method of claim 178, wherein the fatty acid methyl ester
is methyl decanoate.
181. The method of claim 99, wherein the feedstock toxic to the
yeast is a free fatty acid.
182. The method of claim 181, wherein the free fatty acid is lauric
acid.
183. The method of claim 99, wherein the genetically modified yeast
comprises at least one genetic modification that substantially
blocks beta oxidation activity.
184. The method of claim 99, wherein the genetically modified yeast
comprises at least one genetic modification at least one genetic
modification that increase at least one activity selected from the
group consisting of: monooxygenase activity, monooxygenase
reductase activity, thioesterase activity, acyltransferase
activity, isocitrate dehydrogenase activity,
glyceraldehyde-3-phosphate dehydrogenase activity,
glucose-6-phosphate dehydrogenase activity, acyl-coA oxidase
activity, fatty alcohol oxidase activity, acyl-CoA hydrolase
activity, alcohol dehydrogenase activity, peroxisomal biogenesis
factor activity, fatty aldehyde dehydrogenase activity, CTF, UTR,
FAT1.
185. The method of claim 99, wherein the genetically modified yeast
comprises at least one genetic modification that decreases MIG1
activity, as compared to corresponding yeast not comprising said
genetic modification.
186. The method of claim 184, wherein the genetically modified
yeast comprises at least one genetic modification that increases
monooxygase activity selected from the group consisting of: a
CYP52A12 monooxygenase activity, CYP52A13 monooxygenase activity,
CYP52A14 monooxygenase activity, CYP52A15 monooxygenase activity,
CYP52A16 monooxygenase activity, CYP52A17 monooxygenase activity,
CYP52A18 monooxygenase activity, CYP52A19 monooxygenase activity,
CYP52A20 monooxygenase activity, CYP52D2 monooxygenase activity and
BM3 monooxygenase activity.
187. The method of claim 184, wherein the genetically modified
yeast comprises at least one genetic modification that increases
glucose-6-phosphate dehydrogenase activity selected from the group
consisting of: a ZWF1 glucose-6-phosphate dehydrogenase activity
and ZWF2 glucose-6-phosphate dehydrogenase activity.
188. The method of claim 184, wherein the genetically modified
yeast comprises at least one genetic modification that increases
fatty alcohol oxidase activity selected from the group consisting
of: FAO1 fatty alcohol oxidase activity, FA02A fatty alcohol
oxidase activity, FA02B fatty alcohol oxidase activity, FAO13 fatty
alcohol oxidase activity, FAO17 fatty alcohol oxidase activity,
FA018 fatty alcohol oxidase activity and FA020 fatty alcohol
oxidase activity.
189. The method of claim 184, wherein the genetically modified
yeast comprises at least one genetic modification that increases
alcohol dehydrogenase activity selected from the group consisting
of: ADH1 alcohol dehydrogenase activity, ADH2 alcohol dehydrogenase
activity, ADH3 alcohol dehydrogenase activity, ADH4 alcohol
dehydrogenase activity, ADH5 alcohol dehydrogenase activity, ADH7
alcohol dehydrogenase activity, ADH8 alcohol dehydrogenase activity
and SFA alcohol dehydrogenase activity.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. .sctn.
119(e) of U.S. Provisional Patent Application No. 61/739,656, filed
Dec. 19, 2012 which is herein incorporated by reference in its
entirety.
FIELD
[0002] The technology relates in part to biological methods for
producing a fatty dicarboxylic acid and engineered microorganisms
capable of such production.
BACKGROUND
[0003] Microorganisms employ various enzyme-driven biological
pathways to support their own metabolism and growth. A cell
synthesizes native proteins, including enzymes, in vivo from
deoxyribonucleic acid (DNA). DNA first is transcribed into a
complementary ribonucleic acid (RNA) that comprises a
ribonucleotide sequence encoding the protein. RNA then directs
translation of the encoded protein by interaction with various
cellular components, such as ribosomes. The resulting enzymes
participate as biological catalysts in pathways involved in
production of molecules by the organism.
[0004] These pathways can be exploited for the harvesting of the
naturally produced products. The pathways also can be altered to
increase production or to produce different products that may be
commercially valuable. Advances in recombinant molecular biology
methodology allow researchers to isolate DNA from one organism and
insert it into another organism, thus altering the cellular
synthesis of enzymes or other proteins. Advances in recombinant
molecular biology methodology also allow endogenous genes, carried
in the genomic DNA of a microorganism, to be increased in copy
number, thus altering the cellular synthesis of enzymes or other
proteins. Such genetic engineering can change the biological
pathways within the host organism, causing it to produce a desired
product. Microorganic industrial production can minimize the use of
caustic chemicals and the production of toxic byproducts, thus
providing a "clean" source for certain compounds. The use of
appropriate plant derived feedstocks allows production of "green"
compounds while further minimizing the need for and use of
petroleum derived compounds.
SUMMARY
[0005] Provided in certain aspects is a genetically modified yeast,
comprising: one or more genetic modifications that substantially
block beta oxidation activity; and one or more genetic
modifications that increase one or more activities chosen from
monooxygenase activity; monooxygenase reductase activity,
thioesterase activity, acyltransferase activity, isocitrate
dehydrogenase activity, glyceraldehyde-3-phosphate dehydrogenase
activity, glucose-6-phosphate dehydrogenase activity, acyl-coA
oxidase-activity, fatty alcohol oxidase activity, acyl-CoA
hydrolase activity, alcohol dehydrogenase activity, peroxisomal
biogenesis factor activity, and fatty aldehyde dehydrogenase
activity.
[0006] The one or more genetic modifications sometimes increase one
or more of: (a) one or more monooxygase activities chosen from
monooxygenase activity chosen from CYP52A12 monooxygenase activity,
CYP52A13 monooxygenase activity, CYP52A14 monooxygenase activity,
CYP52A15 monooxygenase activity, CYP52A16 monooxygenase activity,
CYP52A17 monooxygenase activity, CYP52A18 monooxygenase activity,
CYP52A19 monooxygenase activity, CYP52A20 monooxygenase activity,
CYP52D2 monooxygenase activity and BM3 monooxygenase activity; (b)
one or more monooxygenase reductase activities chosen from CPRA
monooxygenase reductase activity, CPRB monooxygenase reductase
activity and CPR750 monooxygenase reductase activity; (c) an IDP2
isocitrate dehydrogenase activity; (d) a GDP1
glyceraldehyde-3-phosphate dehydrogenase activity; (e) one or more
glucose-6-phosphate dehydrogenase activities chosen from a ZWF1
glucose-6-phosphate dehydrogenase activity and ZWF2
glucose-6-phosphate dehydrogenase activity; (f) one or more fatty
alcohol oxidase activities chosen from FAO1 fatty alcohol oxidase
activity, FAO2A fatty alcohol oxidase activity, FAO2B fatty alcohol
oxidase activity, FAO13 fatty alcohol oxidase activity, FAO17 fatty
alcohol oxidase activity, FAO18 fatty alcohol oxidase activity and
FAO20 fatty alcohol oxidase activity; (g) one or more alcohol
dehydrogenase activities chosen from ADH1 alcohol dehydrogenase
activity, ADH2 alcohol dehydrogenase activity, ADH3 alcohol
dehydrogenase activity, ADH4 alcohol dehydrogenase activity, ADH5
alcohol dehydrogenase activity, ADH7 alcohol dehydrogenase
activity, ADH8 alcohol dehydrogenase activity and SFA alcohol
dehydrogenase activity; (h) one or more acyl-CoA hydrolase
activities chosen from ACH-A acyl-CoA hydrolase activity and ACH-B
acyl-CoA hydrolase activity; (i) one or more acyltransferase
activities chosen from acyl-CoA sterol acyl transferase activity,
diacylglycerol, acyltransferase activity and
phospholipid:diacylglycerol acyltransferase activity; (j) one or
more acyl transferase activities chosen from ARE1 acyl-CoA sterol
acyltransferase activity, ARE2 acyl-CoA sterol acyltransferase
activity, DGA1 diacylglycerol acyl transferase activity, and LRO1
phospholipid:diacylglycerol acyltransferase activity; (k) an
acyl-coA thioesterase activity (e.g., a TESA acyl:coA thioesterase
activity); (l) a PEX11 peroxisomal biogenesis factor activity; (m)
one or more fatty aldehyde dehydrogenase activities chosen from
HFD1 fatty aldehyde dehydrogenase activity and HFD2 fatty aldehyde
dehydrogenase activity; and (n) a POX5 acyl-coA oxidase
activity.
[0007] In certain aspects, a genetically modified yeast is fully
beta oxidation blocked. In some cases all alleles of
polynucleotides encoding a polypeptide having acyl-coA oxidase
activity are disrupted in a genetically modified yeast. In certain
cases where a genetically modified yeast is a Candida spp. yeast,
all alleles of POX4 and POX5 are disrupted.
[0008] In some aspects, a genetic modification that increases an
activity in a genetically modified yeast comprises incorporating in
the yeast multiple copies of a polynucleotide that encodes a
polypeptide having the activity. Sometimes a genetic modification
that increases an activity in a genetically modified yeast
comprises incorporating in the yeast a promoter in operable linkage
with a polynucleotide that encodes a polypeptide having the
activity. In some cases the promoter is chosen from a POX4
promoter, PEX11 promoter, TEF1 promoter, PGK promoter and FAO1
promoter.
[0009] In certain aspects, a genetically modified yeast comprises
one or more genetic modifications that decrease an acyl-coA
synthetase activity. In some cases the one or more genetic
modifications decrease one or more acyl-coA synthetase activities
chosen from an ACS1 acyl-coA synthetase activity and a FAT1
long-chain acyl-CoA synthetase activity. In some aspects, a
genetically modified yeast is chosen from a Candida spp. yeast
(e.g., C. tropicalis, C. viswanathii, genetically modified
ATCC20336 yeast), Yarrowia spp. yeast, Pichia spp. yeast,
Saccharomyces spp. yeast and Kluyveromyces spp. yeast.
[0010] Any suitable combination of genetic modifications described
herein can be incorporated into a genetically modified yeast for
production of a diacid target product. In some cases, a genetically
modified yeast includes one or more of (a) a genetic modification
that increases an activity, (b) a genetic modification that
decreases an activity, and (c) a promoter insertion, as described
herein, in any suitable combination.
[0011] In some aspects, provided is a method for producing a
diacid, comprising: contacting a genetically modified yeast
described herein with a feedstock capable of being converted by the
yeast to a diacid; and culturing the yeast under conditions in
which the diacid is produced from the feedstock. In some cases the
feedstock comprises one or more components from a vegetable oil,
and sometimes the diacid is a C4 to C24 diacid.
[0012] In certain aspects, provided is a method for producing a
diacid by a yeast from a feedstock toxic to the yeast, comprising:
(a) contacting a genetically modified yeast in culture with a
feedstock not substantially toxic to the yeast, thereby performing
an induction; and (b) contacting the yeast after the induction in
(a) with a feedstock toxic to the yeast, whereby a diacid is
produced by the yeast from the feedstock toxic to the yeast in an
amount greater than the amount of the diacid produced from the
feedstock toxic to the yeast when the induction is not
performed.
[0013] Provided also herein in some aspects are particular isolated
nucleic acids.
[0014] Certain embodiments are described further in the following
description, examples, claims and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The drawings illustrate embodiments of the technology and
are not limiting. For clarity and ease of illustration, the
drawings are not made to scale and, in some instances, various
aspects may be shown exaggerated or enlarged to facilitate an
understanding of particular embodiments.
[0016] FIG. 1 is a schematic representation of the conversion of
decane to sebacic acid in a beta-oxidation blocked microorganism.
Capric acid is formed as an intermediate during omega
oxidation.
[0017] FIG. 2 is a schematic representation of the conversion of
dodecane to dodecanedioic acid in a beta-oxidation blocked
microorganism. Lauric acid is formed as an intermediate during
omega oxidation.
[0018] FIG. 3 is a schematic representation of the conversion of a
feedstock containing mixed chain-length alkanes to mixed diacids
products, including sebacic acid in a beta-oxidation blocked
microorganism. Mixed chain-length fatty acids are formed as
intermediates during omega oxidation. Sebacic acid can be separated
from other diacid products by the use of appropriate separation
techniques.
[0019] FIG. 4 is a schematic representation of the conversion of a
feedstock containing mixed chain-length alkanes to mixed diacids
products, including dodecanedioic acid in a beta-oxidation blocked
microorganism. Mixed chain-length fatty acids are formed as
intermediates during omega oxidation. Dodecanedioic acid can be
separated from other diacid products by the use of appropriate
separation techniques.
[0020] FIG. 5 is a schematic representation of the conversion of a
long-chain alkane into sebacic acid in a partially beta-oxidation
blocked microorganism. The long-chain alkane is first converted
into a long-chain fatty acid and then into a long-chain diacid by
activities in the omega-oxidation pathway. The long-chain diacid
can be converted to sebacic acid by activities in the
beta-oxidation pathway, with the simultaneous generation of
acetyl-CoA.
[0021] FIG. 6 is a schematic representation of the conversion of a
long-chain alkane into dodecanedioic acid in a partially
beta-oxidation blocked microorganism. The long-chain alkane is
first converted into a long-chain fatty acid and then into a
long-chain diacid by activities in the omega-oxidation pathway. The
long-chain diacid can be converted to dodecanedioic acid by
activities in the beta-oxidation pathway, with the simultaneous
generation of acetyl-CoA.
[0022] FIG. 7 is a schematic representation of the conversion of a
feedstock containing mixed chain-length alkanes into sebacic acid
in a partially beta-oxidation blocked microorganism. The mixed
chain-length alkanes are first converted into mixed chain-length
fatty acids and then mixed diacids by activities in the
omega-oxidation pathway. Mixed diacids can be converted to sebacic
acid by activities in the beta-oxidation pathway, with the
simultaneous generation of acetyl-CoA.
[0023] FIG. 8 is a schematic representation of the conversion of a
feedstock containing mixed chain-length alkanes into dodecanedioic
acid in a partially beta-oxidation blocked microorganism. The mixed
chain-length alkanes are first converted into mixed chain-length
fatty acids and then mixed diacids by activities in the
omega-oxidation pathway. Mixed diacids can be converted to
dodecanedioic acid by activities in the beta-oxidation pathway,
with the simultaneous generation of acetyl-CoA.
[0024] FIG. 9 graphically illustrates the conversion of decane to
sebacic acid in a fully beta-oxidation blocked C. tropicalis yeast
strain. After incubation for the times shown in the graph, the
media was subjected to gas chromatography. The results indicate
that greater than 99% of the decane was converted into sebacic
acid, with a minimal amount of capric acid also detected by gas
chromatography. No significant accumulation of any other monoacid
or diacid was detected by gas chromatography. Experimental details
and results are given in Example 1.
[0025] FIG. 10 graphically illustrates the conversion of capric
acid to sebacic acid in a C. tropicalis yeast strain. GC analysis
was performed after a predetermined period of growth. Nearly all
the capric acid added was converted to sebacic acid using a
starting concentration of capric acid. Experimental details and
results are given in Example 2.
[0026] FIG. 11 graphically illustrates the distribution of diacids
produced during the conversion of long-chain fatty acids to mixed
diacids under fermentation conditions using a partially
beta-oxidation blocked Candida tropicalis strain (e.g., sAA106).
Experimental details and results are given in Example 5.
[0027] FIG. 12 graphically illustrates the conversion of decane to
sebacic acid in a fully beta-oxidation blocked C. tropicalis yeast
strain having additional genetic modifications. Strain sAA003 is
the fully beta-oxidation blocked control strain. +CPR indicates the
fully beta-oxidation blocked strain also includes an increased
number of copies of cytochrome P450 reductase. +CPR+A12 indicates
starting strain sAA003 includes the addition genetic modifications
of an increased number of copies of cytochrome P450 reductase and
also includes an increased number of copies of cytochrome P450 A12
(e.g., CYP52A12). +CPR+A18 indicates starting strain sAA003
includes the addition genetic modifications of an increased number
of copies of cytochrome P450 reductase and also includes an
increased number of copies of cytochrome P450 A18 (e.g., CYP52A18).
+CPR+A19 indicates starting strain sAA003 includes the addition
genetic modifications of an increased number of copies of
cytochrome P450 reductase and also includes an increased number of
copies of cytochrome P450 A19 (e.g., CYP52A19). +CPR+A20 indicates
starting strain sAA003 includes the addition genetic modifications
of an increased number of copies of cytochrome P450 reductase and
also includes an increased number of copies of cytochrome P450 A20
(e.g., CYP52A20). Experimental details and results are given in
Example 7.
[0028] FIG. 13 graphically illustrates the results of conversion of
methyl laurate to dodecanedioic acid in a fully beta-oxidation
blocked C. tropicalis yeast strain also contain genetic alterations
to a monooxygenase reductase activity, a monooxygenase activity, or
a monooxygenase reductase activity and a monooxygenase activity.
After 48 hours of incubation the media was subjected to gas
chromatography. The results indicate that Candida strains
containing an increased number of copies of a CYP52A18
monooxygenase activity and an increased number of copies of a
monooxygenase reductase activity (e.g., CPR750) gave the highest
yield of dodecanedioic acid (e.g., DDDA), in shake flask
fermentation experiments. Experimental details and results are
given in Example 8.
[0029] FIG. 14 and FIG. 15 schematically illustrate a screening
and/or selection method for identifying acyl-CoA oxidase activities
with specific substrate specificities. The method can be utilized
in conjunction with generating and/or identifying acyl-CoA oxidase
activities with altered chain-length substrate specificities.
Screening/selection method details are given in Example 9.
[0030] FIG. 16 graphically illustrates the results of engineered
microorganisms described herein converting decane to sebacic acid
under fermentation conditions using different amounts of decane as
the feedstock. Experimental details and results are given in
Example 3.
[0031] FIG. 17 graphically illustrates the results of engineered
microorganisms described herein converting a mixed fatty acid
feedstock (e.g., mixed chain-length fatty acids) to sebacic acid
under fermentation conditions. Experimental details and results are
given in Example 4.
[0032] FIG. 18 shows a diagram of a plasmid designated pAA073
containing a POX4 promoter and a POX4 terminator.
[0033] FIG. 19 shows a diagram of a plasmid designated pAA298.
[0034] FIG. 20 shows the production of either dodecanedioic acid
from methyl laurate (ML) or tetradecanedioic acid from methyl
myristate (MM) utilizing strains sAA1306 and sAA003.
[0035] FIG. 21 shows the production of either dodecanedioic acid
from methyl laurate (ML) or tetradecanedioic acid from methyl
myristate (MM) using strains sAA1082 and sAA003.
[0036] FIG. 22 shows the production cis-9-octadecenedioic acid
(C18:1 diacid) from oleic acid for four fully beta-oxidation
blocked strains. The data points are derived from the averages of
three identical fermentations.
[0037] FIG. 23 shows the concentrations of HFAs produced during the
omega oxidation of oleic acid by strains sAA003, sAA1233, sAA1306
and sAA1485.
[0038] FIG. 24 shows the production of decanedioic acid (sebacic
acid) and compares the productivity of the two strains under the
two different induction conditions.
[0039] FIG. 25 shows the amount of decanoic acid produced under the
different fermentation conditions.
[0040] FIG. 26 shows the production of DDDA and
12-hydroxy-dodecanoic acid (HFA) from methyl laurate.
[0041] FIG. 27 shows the production of DDDA from methyl
laurate.
[0042] FIG. 28 shows the production of DDDA.
[0043] FIG. 29 shows the production of HFAs from methyl
laurate.
[0044] FIG. 30 shows a prompter replacement strategy.
[0045] FIG. 31 shows an example of the production of HFAs during
the first oxidation step in the omega-oxidation pathway.
[0046] FIG. 32A shows the production of omega hydroxyl-oleic acid
and cis-9 C18 diacid from strains sAA003 and sAA2047.
[0047] FIG. 32B shows the production of 12-HDDA and DDDA from
strains sAA003 and sAA2047.
DETAILED DESCRIPTION
[0048] Certain fatty dicarboxylic acids (i.e., diacids, e.g.,
dodecanedioic acid or sebacic acid) are chemical intermediates in
manufacturing processes used to make certain polyamides,
polyurethanes and plasticizers, all of which have wide applications
in producing items such as antiseptics, top-grade coatings,
hot-melt coating and adhesives, painting materials, corrosion
inhibitor, surfactant, engineering plastics and can also be used as
a starting material in the manufacture of fragrances, for example.
For example dodecanedioic acid, also known as 1,12 dodecanedioic
acid, and DDDA, is a 12 carbon organic molecule that is a fatty
dicarboxylic acid. In another example, sebacic acid, also known as
1,10 decanedioic acid, and 1,8 octanedicarboxylic acid, is a 10
carbon organic molecule that is a fatty dicarboxylic acid.
[0049] Provided herein are methods for producing a fatty
dicarboxylic acid (also referred to herein as a diacid). Any
suitable diacid can be produced, and a diacid produced often
includes acid moieties at each terminus of the molecule (e.g.,
alpha omega diacids). A diacid sometimes is a C4 to a C24 diacid
(i.e., a diacid containing 4 carbons to 24 carbons) and sometimes
is a C8, C10, C12, C14, C16, C18, or C20 diacid. Yeast and
processes herein are capable of producing a diacid containing an
odd number of carbons, and sometimes a product contains one or more
diacids chosen from a C5, C7, C9, C11, C13, C15, C17, C19, C21 and
C23 diacid. A hydrocarbon portion of a diacid sometimes is fully
saturated and sometimes a diacid includes one or more unsaturations
(e.g., double bonds).
[0050] Non-limiting examples of diacids include octanedioic acid,
decanedioic acid, dodecanedioic acid, tetradecanedioic acid,
hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid) and
other organic intermediates using biological systems. Non-limiting
examples of fatty dicarboxylic acids include suberic acid (i.e.,
octanedioic acid, 1,8-octanedioic acid, octanedioic acid,
octane-1,8-dioic acid, 1,6-hexanedicarboxylic acid, capryllic
diacids), sebacic acid (i.e., 1,10-decanedioic acid, decanedioic
acid, decane-1,10-dioic acid, 1,8-octanedicarboxylic acid, capric
diacid), dodecanedioic acid (i.e., DDDA, 1,12-dodecanedioic acid,
dodecanedioic acid, dodecane-1,12-dioic acid,
1,10-decanedicarboxylic acid, decamethylenedicaboxylic acid,
1,10-dicarboxydecane, lauric diacid), tetradecanedioic acid (i.e.,
TDDA, 1,14-tetradecanedioic acid, tetradecancedioic acid,
tetradecane-1,14-dioic acid, 1,12-dodecanedicarboxylic acid,
myristic diacid), thapsic acid (i.e., hexadecanedioic acid,
1,16-hexadecanedioic acid, hexadecanedioic acid,
hexadecane-1,16-dioic acid, 1,14-tetradecanedicarboxylic acid,
palmitic diacid), cis-9-hexadecenedioic acid (i.e., palmitoleic
diacids), octadecanedioic acid (i.e., 1,18-octadecanedioic acid,
octadecanedioic acid, octadecane-1,18-dioic acid,
1,16-hexadecanedicarboxylic acid, stearic diacid),
cis-9-octadecenedioic acid (i.e., oleic diacids),
cis-9,12-octadecenedioic acid (i.e., linoleic diacids),
cis-9,12,15-octadecenedioic acid (i.e., linolenic diacids),
arachidic diacid (i.e., eicosanoic diacid, icosanoic diacid),
11-eicosenoic diacid (i.e., cis-11-eicosenedioic acid),
13-eicosenoic diacids (i.e., cis-13-eicosenedioic acid),
arachidonic diacid (i.e., cis-5,8,11,14-eicosatetraenedioic
acid).
[0051] A genetically modified yeast can be provided with a
feedstock to produce a diacid, and the feedstock sometimes includes
a substantially pure aliphatic molecule from which the diacid is
produced. In certain embodiments, the feedstock contains a mixed
set of aliphatic molecules from which diacids may be produced. In
some embodiments, an aliphatic molecule in the feedstock is the
predominant aliphatic species and sometimes a particular diacid
produced from that aliphatic molecule is the predominant diacid
species produced. A predominant species generally is 51% or more by
weight of aliphatic molecule species in a feedstock or 51% or more
by weight of diacid species in a product (e.g., about 55% or more,
60% or more, 65% or more, 70% or more, 75% or more, 80% or more,
85% or more, 90% or more or 95% or more).
[0052] Such production systems may have significantly less
environmental impact and could be economically competitive with
current manufacturing systems. Thus, provided in part herein are
methods for manufacturing a fatty dicarboxylic acid (e.g.,
octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) by engineered microorganisms. In some
embodiments microorganisms are engineered to contain at least one
heterologous gene encoding an enzyme, where the enzyme is a member
of a novel and/or altered pathway engineered into the
microorganism. In certain embodiments, an organism may be selected
for elevated activity of a native enzyme.
Microorganisms
[0053] A microorganism selected often is suitable for genetic
manipulation and often can be cultured at cell densities useful for
industrial production of a target fatty dicarboxylic acid product.
A microorganism selected often can be maintained in a fermentation
device.
[0054] The term "engineered microorganism" as used herein refers to
a modified microorganism that includes one or more activities
distinct from an activity present in a microorganism utilized as a
starting point (hereafter a "host microorganism"). An engineered
microorganism includes a heterologous polynucleotide in some
embodiments, and in certain embodiments, an engineered organism has
been subjected to selective conditions that alter an activity, or
introduce an activity, relative to the host microorganism. Thus, an
engineered microorganism has been altered directly or indirectly by
a human being. A host microorganism sometimes is a native
microorganism, and at times is a microorganism that has been
engineered to a certain point.
[0055] In some embodiments an engineered microorganism is a single
cell organism, often capable of dividing and proliferating. A
microorganism can include one or more of the following features:
aerobe, anaerobe, filamentous, non-filamentous, monoploid, dipoid,
auxotrophic and/or non-auxotrophic. In certain embodiments, an
engineered microorganism is a prokaryotic microorganism (e.g.,
bacterium), and in certain embodiments, an engineered microorganism
is a non-prokaryotic microorganism. In some embodiments, an
engineered microorganism is a eukaryotic microorganism (e.g.,
yeast, fungi, amoeba). In some embodiments, an engineered
microorganism is a fungus. In some embodiments, an engineered
organism is a yeast.
[0056] Any suitable yeast may be selected as a host microorganism,
engineered microorganism, genetically modified organism or source
for a heterologous or modified polynucleotide. Yeast include, but
are not limited to, Yarrowia yeast (e.g., Y. lipolytica (formerly
classified as: Candida lipolytia)), Candida yeast (e.g., C.
revkaufi, C. viswanathii, C. pulcherrima, C. tropicalis, C.
utilis), Rhodotorula yeast (e.g., R. glutinus, R. graminis),
Rhodosporidium yeast (e.g., R. toruloides), Saccharomyces yeast
(e.g., S. cerevisiae, S. bayanus, S. pastorianus, S.
carlsbergensis), Cryptococcus yeast, Trichosporon yeast (e.g., T.
pullans, T. cutancum), Pichia yeast (e.g., P. pastoris) and
Lipomyces yeast (e.g., L. starkeyii, L. lipoferus). In some
embodiments, a suitable yeast is of the genus Arachniotus,
Aspergillus, Aureobasidium, Auxarthron, Blastomyces, Candida,
Chrysosporuim, Chrysosporuim Debaryomyces, Coccidiodes,
Cryptococcus, Gymnoascus, Hansenula, Histoplasma, Issatchenkia,
Kluyveromyces, Lipomyces, Lssatchenkia, Microsporum, Myxotrichum,
Myxozyma, Oidiodendron, Pacysolen, Penicillium, Pichia,
Rhodosporidium, Rhodotorula, Rhodoturala, Saccharomyces,
Schizosaccharomyces, Scopulariopsis, Sepedonium, Trichosporon, or
Yarrowia. In some embodiments, a suitable yeast is of the species
Arachniotus flavolutcus, Aspergillus flavus, Aspergillus
furnigatus, Aspergillus niger, Aurcobasidium pullulans, Auxarthron
thaxteri, Blastomyces dermatitidis, Candida albicans, Candida
dubliniensis, Candida famata, Candida glabrata, Candida
guilliermondii, Candida kefyr, Candida krusei, Candida lambica,
Candida lipolytica, Candida lustitaniae, Candida parapsilosis,
Candida pulcherrima, Candida revkaufi, Candida rugosa, Candida
tropicalis, Candida utilis, Candida viswanathii, Candida xestobii,
Chrysosporuim keratinophilum, Coccidiodes immitis, Cryptococcus
albidus var. diffluens, Cryptococcus laurentii, Cryptococcus
neoformans, Debaryomyces hansenii, Gymnoscus dugwayensis, Hansenula
anomala, Histoplasma capsulatum, Issatchenkia occidentalis,
Isstachenkia orientalis, Kluyveromyces lactis, Kluyveromyces
marxianus, Kluyveromyces thermotolerans, Kluyveromyces waltii,
Lipomyces lipoferus, Lipomyces starkeyii, Microsporum gypseum,
Myxotrichum deflexum, Oidiodendron echinulatum, Pachysolen
tannophilis, Penicillium notatum, Pichia anomala, Pichia pastoris,
Pichia stipitis, Rhodosporidium toruloides, Rhodotorula glutinus,
Rhodotorula graminis, Saccharomyces cerevisiae, Saccharomyces
kluyveri, Schizosaccharomyces pombe, Scopulariopsis acremonium,
Sepedonium chrysospermum, Trichosporon cutancum, Trichosporon
pullas, Yarrowia lipolytica, or Yarrowia lipolytica (formerly
classified as Candida lipolytica). In some embodiments, a yeast is
a Y. lipolytica strain that includes, but is not limited,
ATCC20362, ATTC 8862, ATTC18944, ATCC20228, ATCC76982 and LGAM
S(7)1 strains (Papanikoalaou S., and Agellis G., Bioresour.
Technol. 82(1):43-9(2002)). In certain embodiments, a yeast is a
Candida species (i.e., Candida spp.) yeast. Any suitable Candida
species can be used and/or genetically modified for production of a
fatty dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid). In some embodiments,
suitable Candida species include, but are not limited to Candida
albicans, Candida dubliniensis, Candida famata, Candida glabrata,
Candida guilliermondii, Candida kefyr, Candida krusei, Candida
lambica, Candida lipolytica, Candida lustitaniae, Candida
parapsilosis, Candida pulcherrima, Candida revkaufi, Candida
rugosa, Candida tropicalis, Candida utilis, Candida viswanathii,
Candida xestobii and any other Candida spp. yeast described herein.
Non-limiting examples of Candida spp. strains include, but are not
limited to sAA001 (ATCC20336), sAA002 (ATCC20913, sAA003
(ATCC20962), sAA496(US2012/077252), sAA106 (US2012/0077252, SU-2
(ura3-/ura3-), H5343 (beta oxidation blocked, U.S. Pat. No.
5,648,247), strains. Any suitable strains from Candida spp. yeast
may be utilized as parental strains for genetic modification.
[0057] Yeast genera, species and strains are often so closely
related in genetic content that they can be difficult to
distinguish, classify and/or name. In some cases strains of C.
lipolytica and Y. lipolytica can be difficult to distinguish,
classify and/or name and can be, in some cases, considered the same
organism. In some cases, various strains of C. tropicalis and C.
viswanathii can be difficult to distinguish, classify and/or name
(for example see Arie et. al., J. Gen. Appl. Microbiol., 46,
257-262 (2000). Some C. tropicalis and C. viswanathii strains
obtained from ATCC as well as from other commercial or academic
sources can be considered equivalent and equally suitable for the
embodiments described herein. In some embodiments, some parental
stains of C. tropicalis and C. viswanathii are considered to differ
in name only.
[0058] Any suitable fungus may be selected as a host microorganism,
engineered microorganism or source for a heterologous
polynucleotide. Non-limiting examples of fungi include, but are not
limited to, Aspergillus fungi (e.g., A. parasiticus, A. nidulans),
Thraustochytrium fungi, Schizochytrium fungi and Rhizopus fungi
(e.g., R. arrhizus, R. oryzae, R. nigricans). In some embodiments,
a fungus is an A. parasiticus strain that includes, but is not
limited to, strain ATCC24690, and in certain embodiments, a fungus
is an A. nidulans strain that includes, but is not limited to,
strain ATCC38163.
[0059] Any suitable prokaryote may be selected as a host
microorganism, engineered microorganism or source for a
heterologous polynucleotide. A Gram negative or Gram positive
bacteria may be selected. Examples of bacteria include, but are not
limited to, Bacillus bacteria (e.g., B. subtilis, B. megaterium),
Acinetobacter bacteria, Norcardia baceteria, Xanthobacter bacteria,
Escherichia bacteria (e.g., E. coli (e.g., strains DH10B, Stb12,
DH5-alpha, DB3, DB3.1), DB4, DB5, JDP682 and ccdA-over (e.g., U.S.
application Ser. No. 09/518,188)), Streptomyces bacteria, Erwinia
bacteria, Klebsiella bacteria; Serratia bacteria (e.g., S.
marcessans), Pseudomonas bacteria (e.g., P. acruginosa), Salmonella
bacteria (e.g., S. typhimurium, S. typhi), Megasphaera bacteria
(e.g., Megasphaera elsdenii). Bacteria also include, but are not
limited to, photosynthetic bacteria (e.g., green non-sulfur
bacteria, Choroflexus bacteria (e.g., C. aurantiacus), Chloronema
bacteria (e.g., C. gigateum)), green sulfur bacteria (e.g.,
Chlorobium bacteria (e.g., C. limicola)), Pelodictyon bacteria
(e.g., P. luteolum), purple sulfur bacteria (e.g., Chromatium
bacteria (e.g., C. okenii)), and purple non-sulfur bacteria (e.g.,
Rhodospirillum bacteria (e.g., R. rubrum), Rhodobacter bacteria
(e.g., R. Sphaeroides, R. capsulatus), and Rhodomicrobium bacteria
(e.g., R. vanellii).
[0060] Cells from non-microbial organisms can be utilized as a host
microorganism, engineered microorganism or source for a
heterologous polynucleotide. Examples of such cells, include, but
are not limited to, insect cells (e.g., Drosophila (e.g., D.
melanogaster), Spodoptera (e.g., S. frugiperda Sf9 or Sf21 cells)
and Trichoplusa (e.g., High-Five cells); nematode cells (e.g., C.
elegans cells); avian cells; amphibian cells (e.g., Xenopus laevis
cells); reptilian cells; mammalian cells (e.g., NIH3T3, 293, CHO,
COS, VERO, C127, BHK, Per-C6, Bowes melanoma and HeLa cells); and
plant cells (e.g., Arabidopsis thaliana, Nicotania tabacum, Cuphea
acinifolia, Cuphea acquipetala, Cuphea angustifolia, Cuphea
appendiculata, Cuphea avigera, Cuphea avigera var. pulcherrima,
Cuphea axillifora, Cuphea bahiensis, Cuphea baillonis, Cuphea
brachypoda, Cuphea bustamanta, Cuphea calcarata, Cuphea calophylla,
Cuphea calophylla subsp. mesostemon, Cuphea carthagenensis, Cuphea
circaeoides, Cuphea confertiflora, Cuphea cordata, Cuphea
crassiflora, Cuphea cyanea, Cuphea decandra, Cuphea denticulata,
Cuphea disperma, Cuphea epilobiifolia, Cuphea ericoides, Cuphea
Cuphea flava, Cuphea flavisetula, Cuphea fuchsiifolia, Cuphea
gaurneri, Cuphea glutinosa, Cuphea heterophylla, Cuphea hookeriana,
Cuphea hyssopifolia (Mexican-heather), Cuphea hyssopoides, Cuphea
ignca, Cuphea ingrata, Cuphea jorullensis, Cuphea lanccolata,
Cuphea linarioides, Cuphea llavca, Cuphea lophostoma, Cuphea lutca,
Cuphea lutescens, Cuphea melanium, Cuphea melvilla, Cuphea
micrantha, Cuphea micropetala, Cuphea mimuloides, Cuphea nitidula,
Cuphea palustris, Cuphea parsonia, Cuphea pascuorum, Cuphea
paucipetala, Cuphea procumbens, Cuphea pseudosilene, Cuphea
pseudovaccinium, Cuphea pulchra, Cuphea racemosa, Cuphea repens,
Cuphea salicifolia, Cuphea salvadorensis, Cuphea schumannii, Cuphea
sessiflifora, Cuphea sessilifolia, Cuphea setosa, Cuphea
spectabilis, Cuphea spermacoce, Cuphea splendida, Cuphea splendida
var. viridiflava, Cuphea strigulosa, Cuphea subuligera, Cuphea
teleandra, Cuphea thymoides, Cuphea tolucana, Cuphea urens, Cuphea
utriculosa, Cuphea viscosissima, Cuphea watsoniana, Cuphea
wrightii, Cuphea lanccolata)).
[0061] Microorganisms or cells used as host organisms or source for
a heterologous polynucleotide are commercially available.
Microorganisms and cells described herein, and other suitable
microorganisms and cells are available, for example, from
Invitrogen Corporation (Carlsbad, Calif.), American Type Culture
Collection (Manassas, Va.), and Agricultural Research Culture
Collection (NRRL, Peoria, Ill.).
[0062] Host microorganisms and engineered microorganisms may be
provided in any suitable form. For example, such microorganisms may
be provided in liquid culture or solid culture (e.g., agar-based
medium), which may be a primary culture or may have been passaged
(e.g., diluted and cultured) one or more times. Microorganisms also
may be provided in frozen form or dry form (e.g., lyophilized).
Microorganisms may be provided at any suitable concentration.
[0063] Carbon Processing Pathways and Activities
[0064] FIGS. 1-8 schematically illustrate non-limiting embodiments
of engineered pathways that can be used to produce a fatty
dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid) from various starting
carbon sources or feedstocks. FIG. 1 depicts an embodiment of a
non-limiting, engineered biological pathway for producing sebacic
acid in microorganisms having a fully blocked beta-oxidation
pathway, using decane as the carbon source starting material. FIG.
2 depicts an embodiment of a non-limiting engineered biological
pathway for producing dodecanedioic acid in microorganisms having a
fully blocked beta-oxidation pathway, using dodecane as the carbon
source starting material. FIG. 3 and FIG. 4 depict an embodiment of
a non-limiting engineered biological pathway for producing mixed
chain-length diacids in a microorganism having a fully blocked
beta-oxidation pathway, using mixed chain-length alkanes as the
carbon source starting material. Sebacic acid (FIG. 3) and
dodecanedioic acid (FIG. 4) can be separated and/or purified away
from other diacid products using a suitable combination of
centrifugation, organic solvent extraction, chromatography, and/or
other purification/separation techniques. FIG. 5 and FIG. 6 depict
an embodiment of a non-limiting engineered biological pathway for
producing sebacic acid (FIG. 5) and dodecanedioic acid (FIG. 6) in
microorganisms having a partially blocked beta oxidation pathway,
using long-chain alkanes as the carbon source starting material.
FIG. 7 and FIG. 8 depict an embodiment of a non-limiting engineered
biological pathway for producing sebacic acid (FIG. 7) and
dodecanedioic acid (FIG. 8) in microorganisms having a partially
blocked beta oxidation pathway, using mixed-chain length alkanes as
the carbon source starting material.
[0065] The alkane carbon source starting materials are initially
metabolized using naturally occurring and/or engineered activities
in naturally occurring and/or engineered pathways to yield an
intermediate alcohol which can then be converted to a carboxylic
acid (e.g., fatty acid) by the action of other naturally occurring
and/or engineered activities in the omega-oxidation pathway
depicted in FIGS. 1-8.
[0066] Alkanes are omega-hydroxylated by the activity of cytochrome
P450 enzymes, thereby generating the equivalent chain-length
alcohol derivative of the starting alkane carbon-source material.
In certain embodiments, a cytochrome P450 activity can be increased
by increasing the number of copies of a cytochrome P450 gene (e.g.,
2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more copies of the
genes), by increasing the activity of a promoter that regulates
transcription of a cytochrome P450 gene, or by increasing the
number of copies of a cytochrome P450 gene and increasing the
activity of a promoter that regulates transcription of a cytochrome
P450 gene, thereby increasing the production of target product
(e.g., sebacic or dodecanedioic acid) via increased activity of one
or more cytochrome P450 enzymes. In some embodiments, a cytochrome
P450 enzyme is endogenous to the host microorganism. One or more
cytochrome P450 activities can be added and/or increased dependent
on the carbon source starting material, in certain embodiments.
Cytochrome P450's sometimes exhibit increased activities in
response to stimulation by certain feedstocks or carbon source
starting materials. In some embodiments, an engineered
microorganism includes an increased number of copies of one or more
cytochrome P450s that are stimulated by a chosen carbon source
starting material or feedstock. Cytochrome P450 responsiveness to a
chosen starting carbon source or feedstock can be determined using
any suitable assay. Non-limiting examples of assays suitable for
identification of cytochrome P450 responsiveness to a starting
carbon source or feedstock include RT-PCR or qRT-PCR after the host
microorganism has been exposed to the chosen carbon source or
feedstock for varying amounts of time.
[0067] Cytochrome P450 is reduced by the activity of cytochrome
P450 reductase (CPR), thereby recycling cytochrome P450 to allow
further enzymatic activity. In certain embodiments, the CPR enzyme
is endogenous to the host microorganism. In some embodiments, host
CPR activity can be increased by increasing the number of copies of
a CPR gene (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more
copies of the gene), by increasing the activity of a promoter that
regulates transcription of a CPR gene, or by increasing the number
of copies of a CPR gene and increasing the activity of a promoter
that regulates transcription of a CPR gene, thereby increasing the
production of target product (e.g., sebacic or dodecanedioic acid)
via increased recycling of cytochrome P450. In certain embodiments,
the promoter can be a heterologous promoter (e.g., endogenous or
exogenous promoter). In some embodiments, the CPR gene is
heterologous and exogenous and can be isolated from any suitable
organism. Non-limiting examples of organisms from which a CPR gene
can be isolated include C. tropicalis, S. cerevisiae and Bacillus
megaterium.
[0068] Oxidation of the alcohol to an aldehyde may be performed by
an enzyme in the fatty alcohol oxidase family (e.g., long-chain
fatty alcohol oxidase EC 1.1.3.20), or an enzyme in the alcohol
dehydrogenase family (e.g., fatty alcohol dehydrogenase; EC
1.1.1.1). The aldehyde may be oxidized to a carboxylic acid (e.g.,
sebacic or dodecanedioic acid) by the activity of the enzyme
aldehyde dehydrogenase (e.g., long-chain-aldehyde dehydrogenase or
fatty aldehyde dehydrogenase; EC 1.2.1.48). In some embodiments,
the long chain fatty alcohol oxidase, fatty alcohol dehydrogenase
and/or the long-chain-aldehyde dehydrogenase exist in a host
organism. Flux through these two steps may sometimes be augmented
by increasing the copy number of the enzymes, or by increasing the
activity of the promoter transcribing the genes. In some
embodiments alcohol and aldehyde dehydrogenases specific for 10, 12
or 14 carbon substrates may be isolated from another organism, and
inserted into the host organism.
[0069] FIG. 1 depicts a non-limiting embodiment of an engineered
biological pathway for making sebacic acid using decane (e.g., a
C10 alkane) as the carbon source starting material. Due to the
carbon chain length of decane, no chain shortening is necessary to
arrive at the 10 carbon diacid, sebacic acid. Thus a fully beta
oxidation blocked microorganism can be utilized to minimize
conversion of the desired 10 carbon diacid into diacids having
shorter chain lengths.
[0070] FIG. 2 depicts a non-limiting embodiment of an engineered
biological pathway for making dodecanedioic acid using dodecane
(e.g., a C12 alkane) as the carbon source starting material. Due to
the carbon chain length of dodecane, no chain shortening is
necessary to arrive at the 12 carbon diacid, dodecanedioic acid.
Thus a fully beta oxidation blocked microorganism can be utilized
to minimize conversion of the desired 12 carbon diacid into diacids
having shorter chain lengths.
[0071] FIGS. 3 and 4 depict a non-limiting embodiment of an
engineered biological pathway for generating a mixed population of
diacid (fatty dicarboxylic acid) products, including sebacid acid
(FIG. 3) and dodecanedioic acid (FIG. 4), using a carbon source or
feedstock that contains mixed-chain-length alkanes as the carbon
source starting material. Any suitable mixed-chain-length alkane,
fatty alcohol, mixed chain length fatty alcohol feedstock, fatty
acid, mixed fatty acid feedstock, paraffin, fat or oil can be used.
In some embodiments, the distribution of carbon chain lengths in
the starting material is substantially similar to the desired
carbon chain length distribution in the mixed diacid product. In
certain embodiments, the feedstock is enriched for a desired chain
length. In some embodiments, the enriched fraction is enriched for
carbon chain lengths of about 10 carbons. In some embodiments, the
enriched fraction is enriched for carbon chain lengths of about 12
carbons. Because, in some embodiments, the diacids generated have
substantially the same chain lengths as the chain lengths found in
the carbon source starting material, a fully beta-oxidation blocked
microorganism can be utilized to minimize conversion of the diacids
of desired chain length into diacids of shorter chain lengths. The
lower part of the pathways in FIG. 3 and FIG. 4 show the separation
of sebacic acid and dodecanedioic acid, respectively, away from the
mixed diacid products by the use of separation techniques described
herein, or those known in the art.
[0072] In certain embodiments involving genetically modified
organisms having partially blocked beta-oxidation pathways (see
FIGS. 5-8), feedstocks suitable for use include, but are not
limited to, fatty acid distillates or soapstocks of renewable oils
(palm oil fatty acid distillate, soybean oil soapstock, coconut oil
soapstock), renewable oils (coconut oil, palm oil, palm kernel oil,
soybean oil, corn oil, etc.), fatty acids of chain length equal to
or greater than C10 (in substantially single form (e.g., in
substantially pure form) or in mixture form, alkanes of chain
length equal to or greater than C10 in substantially single form
(e.g., substantially pure form) or in mixture form.
[0073] Carbon sources with longer chain lengths (e.g., 12 carbons
or greater in length) can be metabolized using naturally occurring
and/or engineered pathways to yield molecules that can be further
metabolized using the beta oxidation pathway shown in the lower
portion of FIGS. 5-8. In some embodiments, beta-oxidation
activities in the pathways shown in FIGS. 5-8 also can be
engineered (e.g., as described herein) to enhance metabolism and
target product formation. In some embodiments, one acyl-CoA oxidase
activity of the beta-oxidation pathway is engineered to be
enhanced, and in certain embodiments, the other acyl-CoA oxidase
activity in the beta-oxidation pathway is altered to reduce or
eliminate the activity, thereby optimizing the production of a
diacid of a desired chain-length or diacids with a distribution of
desired chain lengths. In some embodiments, an acyl-CoA oxidase is
selected and/or engineered to alter the substrate specificity of
the enzyme. In certain embodiments, the substrate specificity of a
heterologous and/or engineered acyl-CoA oxidase is for carbon chain
lengths of between about 12 carbons and about 18 carbons, and in
some embodiments a heterologous and/or engineered acyl-CoA oxidase
exhibits no activity on substrates below 12 carbons in length. In
certain embodiments, a heterologous acyl-CoA oxidase with a desired
chain length specificity can be isolated from any suitable
organism. Non-limiting examples of organisms that include, or can
be used as donors for acyl-CoA oxidase enzymes include yeast (e.g.,
Candida, Saccharomyces, Debaryomyces, Meyerozyma, Lodderomyces,
Scheffersomyces, Clavispora, Yarrowia, Pichia, Kluyveromyces,
Eremothecium, Zygosaccharomyces, Lachancea, Nakaseomyces), animals
(e.g., Homo, Rattus), bacteria (e.g., Escherichia, Pseudomonas,
Bacillus), or plants (e.g., Arabidopsis, Nictotania, Cuphea).
[0074] In certain embodiments, a carbon source starting material
(e.g., alkane, fatty acid, fatty alcohol, dicarboxylic acid) of
intermediate or long chain length (e.g., between about 10 carbons
and 22 carbons) is converted into an acyl-CoA derivative for entry
into the beta-oxidation pathway. The acyl-CoA derivative can be
generated by the activity of an acyl-CoA ligase enzyme, in some
embodiments. The acyl-CoA-derivative is subsequently oxidized by
the activity of an acyl-CoA oxidase-enzyme (e.g., also known as
acyl-CoA oxidoreductase and fatty acyl-coenzyme A oxidase) of
natural or altered substrate specificity, in certain embodiments.
The trans-2,3-dehydroacyl-CoA derivative long chain fatty alcohol,
fatty acid or dicarboxylic acid may be further converted to
3-hydroxyacyl-CoA by the activity of enoyl-CoA hydratase.
3-hydroxyacyl-CoA can be converted to 3-oxoacyl-CoA by the activity
of 3-hydroxyacyl-CoA dehydrogenase. 3-oxoacyl-CoA may be converted
to an acyl-CoA molecule, shortened by 2 carbons and an acetyl-CoA,
by the activity of Acetyl-CoA C-acyltransferase (e.g., also known
as beta-ketothiolase and beta-ketothiolase). In some embodiments,
acyl-CoA molecules may be repeatedly shortened by beta oxidation
until a desired carbon chain length is generated (e.g., 10 or 12
carbons, sebacic acid or dodecanedioic acid, respectively). A
shortened fatty acid can be further processed using omega,
oxidation to yield a dicarboxylic acid (e.g., dodecanedioic
acid).
[0075] Beta-Oxidation Activities
[0076] The term "beta oxidation pathway" as used herein, refers to
a series of enzymatic activities utilized to metabolize fatty
alcohols, fatty acids, or dicarboxylic acids. The activities
utilized to metabolize fatty alcohols, fatty acids, or dicarboxylic
acids include, but are not limited to, acyl-CoA ligase activity,
acyl-CoA oxidase activity, acyl-CoA hydrolase activity, acyl-CoA
thioesterase activity, enoyl-CoA hydratase activity,
3-hydroxyacyl-CoA dehydrogenase activity and acetyl-CoA
C-acyltransferase activity. The term "beta oxidation activity"
refers to any of the activities in the beta oxidation pathway
utilized to metabolize fatty alcohols, fatty acids or dicarboxylic
acids.
[0077] Beta-Oxidation--Acyl-CoA Ligase
[0078] An acyl-CoA ligase enzyme sometimes is encoded by the host
organism and can be added to generate an engineered organism. In
some embodiments, host acyl-CoA ligase activity can be increased by
increasing the number of copies of an acyl-CoA ligase gene, by
increasing the activity of a promoter that regulates transcription
of an acyl-CoA ligase gene, or by increasing the number copies of
the gene and by increasing the activity of a promoter that
regulates transcription of the gene, thereby increasing production
of target product (e.g., sebacic or dodecanedioic acid) due to
increased carbon flux through the pathway. In certain embodiments,
the acyl-CoA ligase gene can be isolated from any suitable
organism. Non-limiting examples of organisms that include, or can
be used as donors for, acyl-CoA ligase enzymes include Candida,
Saccharomyces, or Yarrowia.
[0079] Beta-Oxidation--Enoyl-CoA Hydratase
[0080] An enoyl-CoA hydratase enzyme catalyzes the addition of a
hydroxyl group and a proton to the unsaturated .beta.-carbon on a
fatty-acyl CoA and sometimes is encoded by the host organism and
sometimes can be added to generate an engineered organism. In
certain embodiments, the enoyl-CoA hydratase activity is unchanged
in a host or engineered organism. In some embodiments, the host
enoyl-CoA hydratase activity can be increased by increasing the
number of copies of an enoyl-CoA hydratase gene, by increasing the
activity of a prompter that regulates transcription of an enoyl-CoA
hydratase gene, or by increasing the number copies of the gene and
by increasing the activity of a promoter that regulates
transcription of the gene, thereby increasing the production of
target product (e.g., sebacic or dodecanedioic acid) due to
increased carbon flux through the pathway. In certain embodiments,
the enoyl-CoA hydratase gene can be isolated from any suitable
organism. Non-limiting examples of organisms that include, or can
be used as donors for, enoyl-CoA hydratase enzymes include Candida,
Saccharomyces, or Yarrowia.
[0081] Beta-Oxidation--3-Hydroxyacyl-CoA Dehydrogenase
[0082] 3-hydroxyacyl-CoA dehydrogenase enzyme catalyzes the
formation of a 3-ketoacyl-CoA by removal of a hydrogen from the
newly formed hydroxyl group created by the activity of enoyl-CoA
hydratase. In some embodiments, the activity is encoded by the host
organism and sometimes can be added or increased to generate an
engineered organism. In certain embodiments, the 3-hydroxyacyl-CoA
activity is unchanged in a host or engineered organism. In some
embodiments, the host 3-hydroxyacyl-CoA dehydrogenase activity can
be increased by increasing the number of copies of a
3-hydroxyacyl-CoA dehydrogenase gene, by increasing the activity of
a promoter that regulates transciption of a 3-hydroxyacyl-CoA
dehydrogenase gene, or by increasing the number copies of the gene
and by increasing the activity of a promoter that regulates
transciption of the gene, thereby increasing production of target
product (e.g., sebacic or dodecanedioic acid) due to increased
carbon flux through the pathway. In certain embodiments, the
3-hydroxyacyl-CoA dehydrogenase gene can be isolated from any
suitable organism. Non-limiting examples of organisms that include,
or can be used as donors for, 3-hydroxyacyl-CoA dehydrogenase
enzymes include Candida, Saccharomyces, or Yarrowia.
[0083] Beta-Oxidation--Acetyl-CoA C-Acyltransferase
[0084] An Acetyl-CoA C-acyltransferase (e.g.,
beta-ketothiolase)enzyme catalyzes the formation of a fatty
acyl-CoA shortened by 2 carbons by cleavage of the 3-ketoacyl-CoA
by the thiol group of another molecule of CoA. The thiol is
inserted between C-2 and C-3, which yields an acetyl CoA molecule
and an acyl CoA molecule that is two carbons shorter. An Acetyl-CoA
C-acyltransferase sometimes is encoded by the host organism and
sometimes can be added to generate an engineered organism. In
certain embodiments, the acetyl-CoA C-acyltransferase activity is
unchanged in a host or engineered organism. In some embodiments,
the host acetyl-CoA C-acyltransferase activity can be increased by
increasing the number of copies of an acetyl-CoA C-acyltransferase
gene, or by increasing the activity of a promoter that regulates
transcription of an acetyl-CoA C-acyltransferase gene, thereby
increasing the production of target product (e.g., sebacic or
dodecanedioic acid) due to increased carbon flux through the
pathway. In certain embodiments, the acetyl-CoA C-acyltransferase
gene can be isolated from any suitable organism. Non-limiting
examples of organisms that include, or can be used as donors, for
acetyl-CoA C-acyltransferase enzymes include Candida,
Saccharomyces, or Yarrowia.
[0085] Omega Oxidation Activities
[0086] Targets for improving the productivity of diacid product
formation from fatty acid feedstocks in .beta.-oxidation blocked
strains are often those which can improve carbon flux through the
.omega.-oxidation pathway. In some embodiments, these targets are:
1) enzymes performing the rate-limiting step in the
.omega.-oxidation pathway (e.g., CPR and CYP450), 2) enzymes
performing fatty acid, transport into the cell (e.g., Acyl Co A
Synthetases), and 3) enzymes that provide the cofactors required
for the .omega.-oxidation pathway (e.g., G6PDH).
[0087] The term "omega oxidation activity" refers to any of the
activities in the omega oxidation pathway utilized to metabolize
alkanes, fatty alcohols, fatty acids, dicarboxylic acids, or
sugars. The activities utilized to metabolize fatty alcohols, fatty
acids, or dicarboxylic acids include, but are not limited to,
monooxygenase activity (e.g., cytochrome P450 activity),
monooxygenase reductase activity (e.g., cytochrome P450 reductase
activity), alcohol dehydrogenase activity (e.g., fatty alcohol
dehydrogenase activity, or long-chain alcohol dehydrogenase
activity), fatty alcohol oxidase activity; fatty aldehyde
dehydrogenase activity, and thioesterase activity.
[0088] Omega Oxidation--Monooxygenases
[0089] A cytochrome P450 enzyme (e.g., monooxygenase activity)
often catalyzes the insertion of one atom of oxygen into an organic
substrate (RH) while the other oxygen atom is reduced to water.
Insertion of the oxygen atom near the omega carbon of a substrate
yields an alcohol derivative of the original starting substrate
(e.g., yields a fatty alcohol). A cytochrome P450 sometimes is
encoded by the host organism and sometimes can be added to generate
an engineered organism.
[0090] In certain embodiments, the monooxygenase activity is
unchanged in a host or engineered organism. In some embodiments,
the host monooxygenase activity can be increased by increasing the
number of copies of a cytochrome P450 gene, or by increasing the
activity of a promoter that regulates transcription of a cytochrome
P450 gene, thereby increasing the production of target product
(e.g., sebacic or dodecanedioic acid) due to increased carbon flux
through the pathway. In certain embodiments, the cytochrome P450
gene can be isolated from any suitable organism. Non-limiting
examples of organisms that include, or can be used as donors for,
cytochrome P450 enzymes include yeast (e.g., Candida,
Saccharomyces, Debaryomyces, Meyerozyma, Lodderomyces,
Scheffersomyces, Clavispora, Yarrowia, Pichia, Kluyveromyces,
Eremothecium, Zygosaccharomyces, Lachancea, Nakaseomyces), animals
(e.g., Homo, Rattus), bacteria (e.g., Escherichia, Pseudomonas,
Bacillus), or plants (e.g., Arabidopsis, Nictotania, Cuphea).
[0091] The rate limiting step of .omega.-oxidation is the
hydroxylation of the .omega.-carbon of a fatty acid which is
carried out by an enzyme system composed of two enzymes, NADPH
cytochrome, P450 reductase (CPR) and cytochrome P450 monooxygenase
(e.g., CYP52, EC 1.14.14.1). The P450's are a gene family that
produces isozymes with different substrate specificities. In
Candida the gene family is typically composed of CYP52A12,
CYP52A13, CYP52A14, CYP52A15, CYP52A16, CYP52A17, CYP52A18,
CYP52A19, CYP52A20, and CYP52D2. The P450 enzyme is encoded by a
gene family of CTP genes designated A12-A20, and D2 in Candida spp.
Each member of the P450 gene family displays unique substrate
chain-length specificity. Using engineered Candida strains we have
identified the P450 isozymes that improve performance upon
different chain-length fatty acid feedstocks. For short- or
medium-chain fatty acid feedstocks (C6-C14) CYP52A19 amplification
improved performance more than the other isozymes. For long-chain
fatty acid feedstocks (>C16) CYP52A14 amplification improved
performance more than the other isozymes. In some embodiments, to
increase the carbon flux through the .omega.-oxidation pathway the
enzyme activity for one or both of the CPR and the P450 enzyme
families is amplified. In some embodiments, care is taken to select
the P450 family member with substrate specificity that matches the
chain length of the exogenously supplied fatty acid feedstock. In
some embodiments, to increase the carbon flux through the
.omega.-oxidation pathway the enzyme activity of a CYP52A19 is
amplified. In some embodiments, to increase the carbon flux through
the .omega.-oxidation pathway the enzyme activity of a CYP52A14 is
amplified.
[0092] The term "monooxygenase activity" as used herein refers to
inserting one atom of oxygen from O.sub.2 into an organic substrate
(RH) and reducing the other oxygen atom to water. In some
embodiments, monooxygenase activity refers to incorporation of an
oxygen atom onto a six-carbon organic substrate. In certain
embodiments, monooxygenase activity refers to conversion of
hexanoate to 6-hydroxyhexanoic acid. Monooxygenase activity can be
provided by any suitable polypeptide, such as a cytochrome P450
polypeptide (hereafter "CYP450") in certain embodiments. Nucleic
acid sequences conferring CYP450 activity can be obtained from a
number of sources, including Bacillus megaterium and may be induced
in organisms including but not limited to Candida tropicalis,
Yarrowia lipolytica, Aspergillus nidulans, and Aspergillus
parasiticus. Examples of oligonucleotide sequences utilized to
isolate a polynucleotide sequence encoding a polypeptide, having
CYP450 activity (e.g., CYP52A12 polynucleotide, a CYP52A13
polynucleotide, a CYP52A14 polynucleotide, a CYP52A15
polynucleotide, a CYP52A16 polynucleotide, a CYP52A17
polynucleotide, a CYP52A18 polynucleotide, a CYP52A19
polynucleotide, a CYP52A20 polynucleotide, a CYP52D2
polynucleotide, and/or a BM3 polynucleotide) are presented herein.
In some embodiments, monooxygenase activity is not altered in a
host micro organism, and in certain embodiments, the activity is
added or increased in the engineered microorganism relative to the
host microorganism. In some embodiments, the altered monooxygenase
activity is an endogenous activity, and in certain embodiments, the
altered monooxygenase activity is an exogenous activity. In some
embodiments, the exogenous activity is a single polypeptide with
both monooxygenase and monooxygenase reductase activities (e.g., B.
megaterium cytochrome P450:NADPH P450 reductase).
[0093] Presence, absence or amount of cytochrome P450 activity can
be detected by any suitable method known in the art. For example,
detection can be performed by assaying a reaction containing
cytochrome P450 (CYP52A family) and NADPH-cytochrome P450 reductase
(see Appl Environ Microbiol 69: 5983 and 5992). Briefly, cells are
grown under standard conditions and harvested for production of
microsomes, which are used to detect CYP activity. Microsomes are
prepared by lysing cells in Tris-buffered sucrose (10 mM Tris-HCl
pH 7.5, 1 mM EDTA, 0.25M sucrose). Differential centrifugation is
performed first at 25,000.times.g then at 100,000.times.g to pellet
cell debris then microsomes, respectively. The microsome pellet is
resuspended in 0.1M phosphate buffer (pH 7.5), 1 mM EDTA to a final
concentration of approximately 10 mg protein/mL. A reaction mixture
containing approximately 0-3 mg microsomes, 0.1 mM sodium
hexanoate, 0.7 mM NADPH, 50 mM Tris-HCl pH 7.5 in 1 mL is initiated
by the addition of NADPH and incubated at 37.degree. C. for 10
minutes. The reaction is terminated by addition of 0.25 mL 5M HCl
and 0.25 mL 2.5 ug/mL 10-hydroxydecanoic acid is added as an
internal standard (3.3 nmol). The mixture is extracted with 4.5 mL
diethyl ether under NaCl-saturated conditions. The organic phase is
transferred to a new tube and evaporated to dryness. The residue is
dissolved in acetonitrile containing 10 mM
3-bromomethyl-7-methoxy-1,4-benzoxazin-2-one (BrMB) and 0.1 mL of
15 mg/mL 18-crown-6 in acetonitrile saturated with K.sub.2CO.sub.3.
The solution is incubated at 40.degree. C. for 30 minutes before
addition of 0.05 m L2% acetic acid. The fluorescently labeled
omega-hydroxy fatty acids are resolved via HPLC with detection at
430 nm and excitation at 355 nm (Yamada et al., 1991, Anal Biochem
199: 132-136). Optionally, specifically induced CYP gene(s) may be
detected by Northern blotting and/or quantitative RT-PCR, (Craft et
al., 2003, App Environ Micro 69: 5983-5991).
[0094] Omega Oxidation--Monooxygenase Reductases
[0095] A cytochrome P450 reductase (e.g., monooxygenase reductase
activity) catalyzes the reduction of the heme-thiolate moiety in
cytochrome P450 by transferring an electron to the cytochrome P450.
A cytochrome P450 reductase sometimes is encoded by the host
organism and sometimes can be added to generate an engineered
organism. In certain embodiments, the monooxygenase reductase
activity is unchanged in a host or engineered organism. In some
embodiments, the host monooxygenase reductase activity can be
increased by increasing the number of copies of a cytochrome P450
reductase gene, or by increasing the activity of a promoter that
regulates transcription of a cytochrome P450 reductase gene,
thereby increasing the production of target product (e.g., sebacic
or dodecanedioic acid) due to increased carbon flux through the
pathway. In certain embodiments, the cytochrome P450 reductase gene
can be isolated from any suitable organism. Non-limiting examples
of organisms that include, or can be used as donors for, cytochrome
P450 reductase enzymes include yeast (e.g., Candida, Saccharomyces,
Debaryomyces, Meyerozyma, Lodderomyces, Scheffersomyces,
Clavispora, Yarrowia, Pichia, Kluyveromyces, Eremothecium,
Zygosaccharomyces, Lachancea, Nakaseomyces), animals (e.g., Homo,
Rattus), bacteria (e.g., Escherichia, Pseudomonas, Bacillus), or
plants, (e.g., Arabidopsis, Nictotania, Cuphea).
[0096] The reductase (CPR) enzyme (EC 1.6.2:4) is able to work with
any of the P450 isozymes. The reductase is encoded by the genes
CPRA and CPRB in Candida sp. In some embodiments, to increase the
carbon flux through the .omega.-oxidation pathway the enzyme
activity of a CPR is amplified. In some embodiments a CPRA gene is
amplified. In some embodiments a CPRB gene is amplified.
[0097] The term "monooxygenase reductase activity" as used herein
refers to the transfer of an electron from NAD(P)H, FMN, or FAD by
way of an electron transfer chain, reducing the ferric heme iron of
cytochrome P450 to the ferrous state. The term "monooxygenase
reductase activity" as used herein also can refer to the transfer
of a second electron via the electron transport system, reducing a
dioxygen adduct to a negatively charged peroxo group. In some
embodiments, a monooxygenase activity can donate electrons from the
two-electron donor NAD(P)H to the heme of cytochrome P450 (e.g.,
monooxygenase activity) in a coupled two-step reaction in which
NAD(P)H can bind to the NAD(P)H-binding domain of the polypeptide
having the monooxygenase reductase activity and electrons are
shuttled from NAD(P)H through FAD and FMN to the heme of the
monooxygenase activity, thereby regenerating an active
monooxygenase-activity (e.g., cytochrome P450). Monooxygenase
reductase activity can be provided by any suitable polypeptide,
such as a cytochrome P450 reductase polypeptide (hereafter "CPR")
in certain embodiments. Nucleic acid sequences conferring CPR
activity can be obtained from and/or induced in a number of
sources, including but not limited to Bacillus megaterium, Candida
tropicalis, Yarrowia lipolytica, Aspergillus nidulans, and
Aspergillus parasiticus. Examples of oligonucleotide sequences
utilized to isolate a polynucleotide sequence encoding a
polypeptide having CPR activity are presented herein. In some
embodiments, monooxygenase reductase activity is not altered in a
host microorganism, and in certain embodiments, the activity is
added or increased in the engineered microorganism relative to the
host microorganism. In some embodiments, the altered monooxygenase
reductase activity is an endogenous activity, and in certain
embodiments, the altered monooxygenase reductase activity is an
exogenous activity. In some embodiments, the exogenous activity is
a single polypeptide with both monooxygenase and monooxygenase
reductase activities (e.g., B. megaterium cytochrome P450:NADPH
P450 reductase).
[0098] Presence, absence or amount of CPR activity can be detected
by any suitable method known in the art. For example, an engineered
microorganism having an increased number of genes encoding a CPR
activity, relative to the host microorganism, could be detected
using quantitative nucleic acid detection methods (e.g., southern
blotting, PCR, primer extension, the like and combinations
thereof). An engineered microorganism having increased expression
of genes encoding a CPR activity, relative to the host
microorganism, could be detected using quantitative expression
based analysis (e.g., RT-PCR, western blot analysis, northern blot
analysis, the like and combinations thereof). Alternately, an
enzymatic assay can be used to detect Cytochrome P450 reductase
activity, where the enzyme activity alters the optical absorbance
at 550 nanometers of a substrate solution (Masters, B. S. S.,
Williams, C. H., Kamin, H. (1967) Methods in Enzymology, X,
565-573).
[0099] Omega Oxidation--Hydroxy Fatty Acids Omega-hydroxy fatty
acids (HFAs) are intermediates in oxidation of the terminal methyl
group of fatty acids (FIG. 31). HFAs can be produced during the
first oxidation step in the omega-oxidation pathway, which is
catalyzed by cyctochrome P450 using molecular oxygen and electrons
supplied by NADPH. Electron transfer from NADPH can be performed
using the enzyme, cytochrome P450 reductase (CPR). HFAs can be
further oxidized to form the omega-oxo-fatty acid. This oxidation
of HFAs can occur through three different enzymatic mechanisms: 1)
Over-oxidation by cytochrome P450 which requires molecular oxygen,
NADPH, and CPR; 2) Alcohol dehydrogenase (ADH), which requires
either NAD+ or NADP+, depending upon the specificity of the ADH; or
3) Fatty alcohol oxidase (FAO), which requires molecular oxygen and
produces hydrogen peroxide as a byproduct in the reaction. FAO
enzymes are membrane-bound and associated with peroxisomes in
Candida. Omega-oxo-fatty acids can be oxidized to the dicarboxylic
acid either through the over-oxidation reaction by cytochrome P450s
or through the enzyme aldehyde dehydrogenase (ALD). HFAs are
frequently found in small, but economically significant amounts in
dicarboxylic acid fermentations in which
beta-oxidation-blocked-strains of Candida using fatty acids or
fatty acid methyl esters as feedstock. Although HFAs only
constitute approximately 5-10% of the final oxidation product, the
presence of HFAs can result in decreased yields and purity of a
final fatty dicarboxylic acid product and can be undesirable.
[0100] Omega Oxidation--Alcohol Dehydrogenases
[0101] An alcohol dehydrogenase (e.g., fatty alcohol dehydrogenase,
long-chain alcohol dehydrogenase) catalyzes the removal of a
hydrogen from an alcohol to yield an aldehyde or ketone and a
hydrogen atom and NADH, in the endoplasmic reticulum of a cell. In
the case of longer chain alcohols (e.g., hexadecanol), water is
utilized in the dehydrogenation to yield a long chain carboxylate,
2 NADH and H.sub.2. An alcohol dehydrogenase sometimes is encoded
by the host organism and sometimes can be added to generate an
engineered organism. In certain embodiments, the alcohol
dehydrogenase activity is unchanged in a host or engineered
organism. In some embodiments, the host alcohol dehydrogenase
activity can be increased by increasing the number of copies of an
alcohol dehydrogenase gene, or by increasing the activity of a
promoter that regulates transcription of an alcohol dehydrogenase
gene, thereby increasing the production of target product (e.g.,
sebacic or dodecanedioic acid) due to increased carbon flux through
the pathway. In certain embodiments, the alcohol dehydrogenase gene
can be isolated from any suitable organism. Non-limiting examples
of organisms that include, or can be used as donors for, alcohol
dehydrogenase enzymes include yeast (e.g., Candida, Saccharomyces,
Debaryomyces, Meyerozyma, Lodderomyces, Scheffersomyces,
Clavispora, Yarrowia, Pichia, Kluyveromyces, Eremothecium,
Zygosaccharomyces, Lachancea, Nakaseomyces), animals (e.g., Homo,
Rattus), bacteria (e.g., Escherichia, Pseudomonas, Bacillus), or
plants, (e.g., Arabidopsis, Nictotania, Cuphea). Non-limiting
examples of fatty alcohol dehydrogenases are ADH1, ADH2a, ADH2b,
ADH3, ADH4, ADH6, ADH7, ADH8, SFA1, FAO1, EC 1.1.1.66, EC 1.1.1.164
and/or EC 1.1.1.192. In some embodiments, the expression of ADH1,
ADH2a, ADH2b, ADH3, ADH4, ADH6, ADH7, ADH8, SFA1, FAO1, EC
1.1.1.66, EC 1.1.1.164 and/or EC 1.1.1.192 is increased in a fatty
dicarboxylic acid producing organism.
[0102] Omega Oxidation--Fatty Alcohol Oxidases
[0103] A fatty alcohol oxidase (e.g., long-chain alcohol oxidase,
EC 1.1.3.20) enzyme catalyzes the addition of oxygen to two
molecules of a long-chain alcohol to yield 2 long chain aldehydes
and 2 molecules of water, in the peroxisome of a cell. A fatty
alcohol oxidase sometimes is encoded by the host organism and
sometimes can be added to generate an engineered organism. In
certain embodiments, the fatty alcohol oxidase activity is
unchanged in a host or engineered organism. In some embodiments,
the host fatty alcohol oxidase activity can be increased by
increasing the number of copies of a fatty alcohol oxidase gene, or
by increasing the activity of a promoter that regulates
transcription of a fatty alcohol oxidase gene, thereby increasing
the production of target product (e.g., sebacic or dodecanedioic
acid) due to increased carbon flux through the pathway. In certain
embodiments, the fatty alcohol oxidase gene can be isolated from
any suitable organism. Non-limiting examples of fatty alcohol
oxidases include FAO1; FAO2a, FAO2b, FAO13, FAO17, FAO18, FAO20 and
FAO.DELTA.PTS1. Non-limiting examples of organisms that include, or
can be used as donors for, fatty alcohol oxidase enzymes include
yeast (e.g., Candida, Saccharomyces, Debaryomyces, Meyerozyma,
Lodderomyces, Scheffersomyces, Clavispora, Yarrowia, Pichia,
Kluyveromyces, Eremothecium, Zygosaccharomyces, Lachancea,
Nakaseomyces), animals (e.g., Homo, Rattus), bacteria (e.g.,
Escherichia, Pseudomonas, Bacillus), or plants, (e.g., Arabidopsis,
Nictotania, Cuphea).
[0104] Omega Oxidation--Aldehyde Dehydrogenases
[0105] A fatty aldehyde dehydrogenase (e.g., long chain aldehyde
dehydrogenase) enzyme catalyzes the oxidation of long chain
aldehydes to a long chain dicarboxylic acid, NADH and H.sub.2. A
fatty aldehyde dehydrogenase sometimes is encoded by the host
organism and sometimes can be added to generate an engineered
organism. In certain embodiments, the fatty aldehyde dehydrogenase
activity is unchanged in a host or engineered organism. In some
embodiments, the host fatty aldehyde dehydrogenase activity can be
increased by increasing the number of copies of a fatty aldehyde
dehydrogenase gene; or by increasing the activity of a promoter
that regulates transcription of a fatty aldehyde dehydrogenase
gene, thereby increasing the production of target product (e.g.,
sebacic or dodecanedioic acid) due to increased carbon flux through
the pathway. In certain embodiments, the fatty aldehyde
dehydrogenase gene can be isolated from any suitable organism.
Non-limiting examples of organisms that include, or can be used as
donors for, fatty aldehyde dehydrogenase enzymes include yeast
(e.g., Candida, Saccharomyces, Debaryomyces, Meyerozyma,
Lodderomyces, Scheffersomyces, Clavispora, Yarrowia, Pichia,
Kluyveromyces, Eremothecium, Zygosaccharomyces, Lachancea,
Nakaseomyces), animals (e.g., Homo, Rattus), bacteria (e.g.,
Escherichia, Pseudomonas, Bacillus), or plants, (e.g., Arabidopsis,
Nictotania, Cuphea). Non-limiting examples of aldehyde
dehydrogenases are ALD1, ALD5, HFD1, HFD1a, EC 1.2.1.3, EC 1.2.1.48
and/or HFD2. In some embodiments, the expression of ALD1, ALD5,
HFD1 and/or HFD2 is increased in a fatty dicarboxylic acid
producing organism.
[0106] Omega Oxidation--Thioesterases
[0107] A thioesterase enzyme (e.g., acyl-Co A thioesterase
activity, acyl-ACP thioesterase activity) catalyzes the removal of
Coenzyme A or acyl carrier protein (e.g., ACP) from a fatty acid
including acyl-CoA or acyl carrier protein (e.g., esterified fatty
acid) to yield a fatty acid and an alcohol. The reaction occurs in
the presence of water and Coenzyme A or acyl carrier protein is
specifically removed at a thiol group. A thioesterase sometimes is
encoded by the host organism and sometimes can be added to generate
an engineered organism. In certain embodiments, the thioesterase
activity is unchanged in a host or engineered organism. In some
embodiments, the host thioesterase activity can be increased by
increasing the number of copies of a thioesterase gene, or by
increasing the activity of a promoter that regulates transcription
of a thioesterase gene, thereby increasing the production of target
product (e.g., sebacic or dodecanedioic acid) due to increased
carbon flux through the pathway. In certain embodiments, a
thioesterase gene can be isolated from any suitable organism.
Non-limiting examples of organisms that include, or can be used as
donors for, thioesterase enzymes include yeast (e.g., Candida,
Saccharomyces, Debaryomyces, Meyerozyma, Lodderomyces,
Scheffersomyces, Clavispora, Yarrowia, Pichia, Kluyveromyces,
Eremothecium, Zygosaccharomyces, Lachancea, Nakaseomyces), animals
(e.g., Homo, Rattus), bacteria (e.g., Escherichia, Pseudomonas,
Bacillus), or plants, (e.g., Arabidopsis, Nictotania, Cuphea).
[0108] Transcription Factors
[0109] MIG1 ("Multicopy inhibitor of GAL Gene Expression") is a
transcription factor that primarily functions to repress the
transcription of genes whose expression is turned off when glucose
is present. Examples of such genes are enzymes involved in the
utilization of sugars. When cells are glucose limited, MIG1 has
been shown to be phosphorylated and removed from the nucleus such
that it cannot repress transcription of its targeted genes. Without
being limited by mechanism, it is believed that deletion of the MIG
gene may increase activity in those genes involved in omega
oxidation and transport required for the production of diacids. In
some instances, deletion of one or both MIG1 alleles in
microorganisms engineered to produce diacids may serve to decrease
the amount of omega-hydroxy fatty acids produced by the
microorganism.
[0110] CTF1 is a putative zinc-finger transcriptional factor and is
apparently similar to the Aspergillus nidulans FarA and FarB
transcription factors. CTF1 is believed to activate genes required
for fatty acid degradation that are induced by the presence of
oleic acid. Overexpression of CTF1 is expected to increase
expression of genes involved in omega and beta oxidation thereby
increasing productivity of the engineered microorganisms.
[0111] UTR is a NADH kinase that phosphorylates both NAD and NADH
into NADP and NADPH. The EC numbers for UTR 2.7.1.23 and 2.7.1.86,
respectively. In the situation that there is an excess of NAD or
NADH or a deficiency of NADP or NADPH, it can convert one into the
other. During omega oxidation there maybe an increase NADH but a
decrease of NADPH. In some embodiments, overexpression of a NASH
kinase increases production of a diacid.
[0112] Engineered Pathways
[0113] FIGS. 1-8 depict embodiments of biological pathways for
making sebacic acid and dodecanedioic acid, using various alkanes,
fatty acids, fatty alcohols or combinations thereof. Any suitable
alkane, fatty acid, fatty alcohol, plant based oil, seed based oil,
non-petroleum derived soap stock or the like can be used as the
feedstock for the organism (e.g., dodecane, methyl laurate, lauric
acid, carbon sources having 10 or greater carbons, (e.g. for
sebacic acid production) or carbon sources having 12 or greater
carbons (e.g. for dodecanedioic acid production). In some
embodiments, carbon sources with greater than 12 carbons can be
metabolized using naturally occurring and/or engineered pathways to
yield molecules that can be further metabolized using the beta
oxidation pathway shown in the lower portion of FIGS. 5-8. In some
embodiments, the activities in the pathways depicted in FIGS. 1-8
can be engineered, as described herein, to enhance metabolism and
target product formation.
[0114] In certain embodiments, one or more activities in one or
more metabolic pathways can be engineered to increase carbon flux
through the engineered pathways to produce a desired product (e.g.,
sebacic or dodecanedioic acid). The engineered activities can be
chosen to allow increased production of metabolic intermediates
that can be utilized in one or more other engineered pathways to
achieve increased production of a desired product with respect to
the unmodified host organism. The engineered activities also can be
chosen to allow decreased activity of enzymes that reduce
production of a desired intermediate or end product (e.g., reverse
activities). This "carbon flux management" can be optimized for any
chosen feedstock, by engineering the appropriate activities in the
appropriate pathways. Non-limiting examples are given herein using
pure alkanes (e.g., single chain length alkanes, dodecane or
example), mixed chain-length alkanes, long-chain alkanes, pure
fatty acids (e.g., single chain length fatty acids, capric acid for
example) and mixed chain length fatty acids (see FIGS. 1-8). The
process of "carbon flux management" through engineered pathways
produces a dicarboxylic acid (e.g. sebacic acid or dodecanedioic
acid) at a level and rate closer to the calculated maximum
theoretical yield for any given feedstock, in certain embodiments.
The terms "theoretical yield" or "maximum theoretical yield" as
used herein refer to the yield of product of a chemical or
biological reaction that can be formed if the reaction went to
completion. Theoretical yield is based on the stoichiometry of the
reaction and ideal conditions in which starting material is
completely consumed, undesired side reactions do not occur, the
reverse reaction does not occur, and there no losses in the work-up
procedure.
[0115] A microorganism may be modified and engineered to include or
regulate one or more activities in a fatty dicarboxylic acid (e.g.,
octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) pathway. The term "activity" as used herein
refers to the functioning of a microorganism's natural or
engineered biological pathways to yield various products including
a fatty dicarboxylic acid (e.g., octanedioic acid, decanedioic
acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic
acid, octadecanedioic acid, eicosanedioic acid) and its precursors.
A fatty dicarboxylic acid (e.g., octanedioic acid, decanedioic
acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic
acid, octadecanedioic acid, eicosanedioic acid) producing activity
can be provided by any non-mammalian source in certain embodiments.
Such sources include, without limitation, eukaryotes such as yeast
and fungi and prokaryotes such as bacteria. In some embodiments, a
reverse activity in a pathway described herein can be altered
(e.g., disrupted, reduced) to increase carbon flux through a beta
oxidation pathway, an omega oxidation pathway, or a beta oxidation
and omega oxidation pathway, towards the production of target
product (e.g., sebacic or dodecanedioic acid). In some embodiments,
a genetic modification disrupts an activity in the beta oxidation
pathway, or disrupts polynucleotide that encodes a polypeptide that
carries out a forward reaction in the beta oxidation pathway, which
renders beta oxidation activity undetectable. The term
"undetectable" as used herein refers to an amount of an analyte
that is below the limits of detection, using detection methods or
assays known (e.g., described herein). In certain embodiments, the
genetic modification partially reduces beta oxidation activity. The
term "partially reduces beta oxidation activity" as used here
refers to a level of activity in an engineered organism that is
lower than the level of activity found in the host or starting
organism.
[0116] In some embodiments, a beta-oxidation activity can be
modified to alter the catalytic specificity of the chosen activity.
In certain embodiments, an acyl-CoA oxidase activity can be altered
by modifying a catalytic domain associated with carbon chain length
preference and/or specificity. In some embodiments, the altered
catalytic specificity can be found by screening naturally occurring
variant or mutant populations of a host organism. In certain
embodiments, the altered catalytic can be generated by various
mutagenesis techniques in conjunction with selection and/or
screening for the desired activity. In some embodiments, the
altered catalytic activity can be generated by generating chimeric
acyl-CoA oxidases using a mix and match approach, followed by
selection and/or screening for the desired catalytic activity.
Examples of experiments performed to generate acyl-CoA oxidases
with altered catalytic activity are described herein.
[0117] An activity within an engineered microorganism provided
herein can include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14 or all) of the following activities:
6-oxohexanoic acid dehydrogenase activity; 6-hydroxyhexanoic acid
dehydrogenase activity; cytochrome P450 activity; cytochrome P450
reductase activity; fatty alcohol oxidase activity; acyl-CoA ligase
activity, acyl-CoA oxidase activity; enoyl-CoA hydratase activity,
3-hydroxyacyl-CoA dehydrogenase activity, fatty acid synthase
activity, lipase activity, acetyl-CoA carboxylase activity,
acyltransferase activity (diacylglycerol acyl transferase,
lecithin-cholesterol acyltransferase, phospholipids acyl glycerol
acyltransferase) and thioesterase activity (e.g., acyl-CoA
hydrolase, acyl-CoA thioesterase, acyl-ACP thioesterase, acetyl-CoA
C-acyltransferase, beta-ketothiolase, and the like). In certain
embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or
all) of the foregoing activities is altered by way of a genetic
modification. In some embodiments, one or more (e.g., 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or all) of the foregoing
activities is altered by way of (i) adding a heterologous
polynucleotide that encodes a polypeptide having the activity,
and/or (ii) altering or adding a regulatory sequence that regulates
the expression of a polypeptide having the activity. In certain
embodiments, one or more of the foregoing activities is altered by
way of (i) disrupting an endogenous polynucleotide that encodes a
polypeptide having the activity (e.g., insertional mutagenesis),
(ii) deleting a regulatory sequence that regulates the expression
of a polypeptide having the activity, and/or (iii) deleting the
coding sequence that encodes a polypeptide having the activity
(e.g., knock out mutagenesis).
[0118] The term "omega hydroxyl fatty acid dehydrogenase activity"
as used herein refers to conversion of an omega hydroxyl fatty acid
to an omega oxo fatty acid. The omega hydroxyl fatty acid
dehydrogenase activity can be provided by a polypeptide. In some
embodiments, the polypeptide is encoded by a heterologous
nucleotide sequence introduced to a host microorganism. In certain
embodiments, an endogenous polypeptide having the omega hydroxyl
fatty acid dehydrogenase activity is identified in the host
microorganism, and the host microorganism is genetically altered to
increase the amount of the polypeptide produced (e.g., a
heterologous promoter is introduced in operable linkage with a
polynucleotide that encodes the polypeptide; the copy number of a
polynucleotide that encodes the polypeptide is increased (e.g., by
introducing a plasmid that includes the polynucleotide)). Nucleic
acid sequences conferring omega hydroxyl fatty acid dehydrogenase
activity can be obtained from a number of sources, including
Actinobacter, Norcardia, Pseudomonas and Xanthobacter bacteria.
Examples of an amino acid sequence of a polypeptide having omega
hydroxyl fatty acid dehydrogenase activity and a nucleotide
sequence of a polynucleotide that encodes the polypeptide, are
presented herein. Presence, absence or amount of omega hydroxyl
fatty acid dehydrogenase activity can be detected by any suitable
method known in the art. In some embodiments, omega hydroxyl fatty
acid dehydrogenase activity is not altered in a host microorganism,
and in certain embodiments, the activity is added or increased in
the engineered microorganism relative to the host
microorganism.
[0119] Increasing NADPH Production in Yeast Producing a Fatty
Dicarboxylic Acid
[0120] The .omega.-oxidation pathway requires the cofactors NADPH
in the first step and NAD.sup.1 in the second and third steps.
Since the first step in .omega.-oxidation is the rate-limiting
step, amplification of the enzyme activity performing this step in
the cell would also require a sufficient supply of the NADPH
cofactor for the reaction. There are a number of cellular reactions
that produce NADPH that may be used by the first step in
.omega.-oxidation. Some of the enzymes performing NADPH-producing
reactions in the cell are glucose-6-phosphate dehydrogenase,
isocitrate dehydrogenase, and glycerol-3-phosphate dehydrogenase.
Amplification of the activity levels of any of these genes can
increase cellular levels of NADPH to provide enough cofactor for an
amplified .omega.-oxidation activity.
[0121] NADPH is required for both .omega.-oxidation and fatty acid
synthesis. Genetic changes that increase the amount of NADPH in the
cell can result in a production boost for the production of diacid
from either single fatty acids and/or fatty acids mixtures. In
addition, if the number of NADPH obtained per glucose is increased,
the amount of glucose required as co-feed can be reduced.
[0122] Increasing NADPH Production: by Increasing
Glucoses-6-Phosphate Dehydrogenase Activity through Overexpression
of ZWF1 or ZWF2 genes
[0123] The ZWF1 and ZWF2 genes encode two isozymes of
glucose-6-phosphate dehydrogenase (G6PDH, e.g., EC 1.1.1.49). In S.
cerevisiae increasing glucose-6-phosphate dehydrogenase (G6PDH)
activity results in an increase in cytosolic NADPH. This technique
has been used to create strains with increased xylitol production
and increased furfurals resistance. In some embodiments the ZWF1
open reading frame will be amplified frorn either Candida strain
ATCC20336 or Scheffersomyces stipitis and placed under the control
of the ZWF1 promoter, TEF1 promoter, POX4 promoter, or another
strong constitutive or inducible promoter. These cassettes can be
transformed into suitable yeast strains for either specific or
random integration using the URA3 auxotrophic marker for selection.
Ura+ strains can be analyzed by PCR and qPCR for proper integration
or copy number. Increased glucose-6-phosphate dehydrogenase
activity can be confirmed by activity assays. Strains can then be
tested for production of the desired fatty dicarboxylic acid. In
some embodiments strains can then be tested for production of a
octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid/octadecanedioic acid,
eicosanedioic acid, suberic or adipic acid depending on the strain
and feedstock used. The fermentation performance of a yeast strain
engineered for increased NADPH production can be compared to the
parental strain.
[0124] Two examples of the amino acid sequences for G6PDH are shown
below: [0125] >Scheffersomyces_stipitis_ZWF1--SEQ ID NO: 157
[0126] Candida strain ATCC20336_ZWF1--SEQ ID NO: 74 [0127]
Increasing NADPH Production by Decreasing Glycolysis and Increasing
Pentose Pathway through Disruption of the PGI1 Gene [0128] In some
embodiments a disruption cassette for PGI1 is constructed. For
example 300 to 700 bp of the 5' and 3' untranslated region or open
reading frame of the PGI1 gene can be amplified. The two pieces can
be ligated together leaving a unique restriction site between them
where an URA3 can be cloned into. This URA3 cassette can have
either the terminator or promoter duplicated in either the
beginning of the end of the URA3 cassette, respectively. The direct
repeat can allow loop-out of the URA3. The disruption cassette can
then be transformed into a suitable yeast strain and select by
growing in uracil deficient plates. Disruption of the first copy of
PGI1 can be verified by PCR. URA3 loopout events can be selected by
growth in 5-Fluorootic acid containing plates. The loop-out event
can be verified by PCR using primers outside the region
encompassing the transformation cassette. This strain can be
transformed with the PGI1 disruption cassette previously used or a
new disruption cassette that targets regions not present in the
first disruption. Ura+ strains can be screened for the complete
loss of the PGI1 gene. Strains can then be tested for production of
the desired fatty dicarboxylic acid. In some embodiments strains
can then be tested for production of an octanedioic acid,
decanedioic acid, dodecanedioic acid, tetradecanedioic acid,
hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid,
suberic or adipic acid depending on the strain and feedstock used.
The fermentation performance of a yeast strain engineered for
increased NADPH production can be compared to the parental strain.
[0129] An example of the amino acid sequences for PGI1 is shown
below: [0130] Candida strain ATCC20336_PGI1--SEQ ID NO: 78
[0131] Increasing NADPH Production by Overexpression of K1GDP1
[0132] GDP1 (e.g., GDP1 of Kluyveromyces lactis, i.e., K1GDP1)
encodes an NADP+ depending glyceraldehyde dehydrogenase (EC
1.2.1.9) that converts glyceraldehyde 3-phosphate into 1,3
biphosphoglycerate producing NADPH instead of NADH. This activity
can increase the production of NADPH from glucose. K1GDP1 open
reading can be mutagenized to change the CTG codon to another
leucine encoding codon. The open reading frame can be placed under
the control of the TEF1 promoter, POX4 promoter or another strong
constitutive or inducible promoter. These cassettes can be
integrated into any suitable yeast strain by targeted or random
integration using the URA3 auxotrophic marker to select for
transformation events. Ura+ strains can be analyzed by PCR and qPCR
for proper integration or copy number determination. In addition,
increased NADP+ dependent glyceraldehyde 3-phosphate dehydrogenase
activity can be confirmed by activity assay. Strains can then be
tested for production of the desired fatty dicarboxylic acid. In
some embodiments strains can then be tested for production of an
octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosancdioic acid, suberic or adipic acid depending on the strain
and feedstock used. The fermentation performance of a yeast strain
engineered for increased NADPH production can be compared to the
parental strain.
[0133] An example of the amino acid sequence for GDP1 is shown
below: [0134] >GDP1, K1--SEQ ID NO: 72 [0135] Increasing NADPH
Production by Overexpression of IDPs [0136] IDP1 and IDP2 (e.g.,
from Candida strain 20336) encode proteins with an isocitrate
dehydrogenase activity that converts isocitrate to
.alpha.-ketoglutarate producing NADPH instead of NADH (e.g., EC
1.1.1.42). The IDP1 protein is targeted to the mitochondria while
the IDP2 protein is targeted to the peroxisome and it can be
present in the ER where .omega.-oxidation happens. IDP2 expression
has been shown to be induced by the presence of alkanes and
overexpression may increase NADPH availability.
[0137] The open reading frame can be placed under the TEF1
promoter, POX4 promoter, or another strong constitutive or
inducible promoter. These cassettes can be integrated into any
suitable yeast strains either by targeted integration or random
integration using the URA3 auxotrophic marker to select for
transformation events. Ura+ strains can be verified by PCR and qPCR
for proper integration or copy number. In addition, increased NADP+
dependent isocitrate dehydrogenase activity can be confirmed by
activity assay. Strains can then be tested for production of the
desired fatty dicarboxylic acid. In some embodiments strains can
then be tested for production of an octanedioic acid, decanedioic
acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic
acid, octadecanedioic acid, eicosanedioic acid, suberic or adipic
acid depending on the strain and feedstock used. The fermentation
performance of a yeast strain engineered for increased NADPH
production can be compared to the parental strain.
[0138] Another IDP to be tested can be IDP3 (e.g. from
Saccharomyces cerevisiae, i.e., ScIdp3). This protein is targeted
to the peroxisome and may also be present in the ER. A similar
approach can be taken for IDP2 except that the open reading frame
may need to be mutagenized if there are any CTG codons.
[0139] An example of the amino acid sequences for an IDP2 and IDP3
are shown below: [0140] Candida strain ATCC20336_IDP2--SEQ ID NO:
67 [0141] >Saccharomyces_cerevisiae_IDP3--SEQ ID NO: 69
[0142] Increasing NADPH Production by Overexpression of ScMAE1 and
ScPYC2
[0143] MAE encodes a malic enzyme (e.g., 1.1.1.40) converting malic
acid to pyruvate producing NADPH (as shown below). [0144]
(S)-malate+NADP+.fwdarw.pyruvate+CO2+NADPH+H+
[0145] When overexpressed in the cytosol in the presence of PYC2
(i.e., pyruvate carboxylase, e.g., 6.4.1.1) that converts pyruvate
to oxaloacetate) a shunt is formed that produces one NADPH at the
expense of one ATP and NADH. MAE expression can be directed to the
cytosol by expressing a truncated version that prevents its
translocation into the mitochondria.
[0146] MAE1 (e.g., from a Candida strain or Saccharomyces
cerevisiae, i.e., ScMAE1) and PYC2 (e.g., from Saccharomyces
cerevisiae, i.e., ScPYC2) open reading frames can be amplified and
mutagenized to replace any CTG codons for other leucine encoding
codons. The genes can be placed under the control of the TEF1
promoter, POX4 promoter, or another strong constitutive or
inducible promoter. These cassettes can be integrated into any
suitable yeast strain by targeting integration or random
integration using the URA3 auxotrophic marker to select for
transformation events. Ura+ strains can be verified by PCR and qPCR
for proper integration or copy number. Strains can then be tested
for production of the desired fatty dicarboxylic acid. In some
embodiments strains can then be tested for production of an
octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid, suberic or adipic acid depending on the strain
and feedstock used. The fermentation performance of a yeast strain
engineered for increased NADPH production can be compared to the
parental strain.
[0147] An example of the amino acid sequences for a ScMAE1 and
ScPYC2 are shown below: [0148] >ScMAE1--SEQ ID NO: 191 [0149]
>ScPYC2--SEQ ID NO: 107 [0150] >Candida strain, truncated
cytosolic MAE1--SEQ: ID 143
[0151] Increasing NADPH Production when Using Glycerol as a
Co-Feed
[0152] Archacoglobus fulgidus gpsA encodes a glycerol 3-phosphate
dehydrogenase using NADP+ as a co-factor. The gene encoding this
enzyme can be mutagenized to change any CTG codons to other leucine
encoding codons. This gene can be placed under either a
constitutive or glycerol inducible promoter with a loop-out capable
URA3 auxotrophic marker in a disruption cassette for GUT2. This
cassette can be transformed into any suitable yeast strain
disrupting the first copy of GUT2. The URA3 marker can be recycled
and the resulting strain can be retransformed with the integration
cassette. Strains that have both copies of GUT2 disrupted can be
selected. This strain should produce NADPH instead of FADH in the
conversion of glycerol-3-phosphate to dihydroxyacetone. Strains can
then be tested for production of the desired fatty dicarboxylic
acid. In some embodiments strains can then be tested for production
of an octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid, suberic or adipic acid depending on the strain
and feedstock used. The fermentation performance of a yeast strain
engineered for increased NADPH production can be compared to the
parental strain.
[0153] An example of the amino acid sequences for a GUT2 and
Archacoglobus fulgidus gpsA are shown below: [0154] >Candida
strain ATCC20336 GUT2--SEQ ID NO: 109 [0155] >AfgpsA--SEQ ID NO:
111
[0156] Acyl-CoA Oxidases
[0157] The term "acyl-CoA oxidase activity" as used herein refers
to the oxidation of a long chain fatty-acyl-CoA to a
trans-2,3-dehydroacyl-CoA fatty alcohol. In some embodiments, the
acyl-CoA activity is from a peroxisome. In certain embodiments, the
acyl-CoA oxidase activity is a peroxisomal acyl-CoA oxidase (POX)
activity, carried out by a POX polypeptide. In some embodiments the
acyl-CoA oxidase activity is encoded by the host organism and
sometimes can be altered to generate an engineered organism.
Acyl-CoA oxidase activity is encoded by the POX4 and POX5 genes of
C. tropicalis. In certain embodiments, endogenous acyl-CoA oxidase
activity can be increased. In some embodiments, acyl-CoA oxidase
activity of the POX4 polypeptide or the POX5 polypeptide can be
altered independently of each other (e.g., increase activity of
POX4 alone, POX5 alone, increase one and disrupt the other, and the
like). Increasing the activity of one POX activity, while
disrupting the activity of another POX activity, may alter the
specific activity of acyl-CoA oxidase with respect to carbon chain
length, while maintaining or increasing overall flux through the
beta oxidation pathway, in certain embodiments.
[0158] In certain embodiments, host acyl-CoA oxidase activity of
one of the POX genes can be increased by genetically altering
(e.g., increasing) the amount of the polypeptide produced (e.g., a
strongly transcribed or constitutively expressed heterologous
promoter is introduced in operable linkage with a polynucleotide
that encodes the polypeptide, the copy number of a polynucleotide
that encodes the polypeptide is increased (e.g., by introducing a
plasmid that includes the polynucleotide, integration of additional
copies in the host genome)). In some embodiments, the host acyl-CoA
oxidase activity can be decreased by disruption (e.g., knockout,
insertion mutagenesis, the like and combinations thereof) of an
acyl-CoA oxidase gene, or by decreasing the activity of the
promoter (e.g., addition of repressor sequences to the promoter or
5'UTR) which transcribes an acyl-CoA oxidase gene.
[0159] As noted above, disruption of nucleotide sequences encoding
POX4, POX5, or POX4 and POX5 sometimes can alter pathway
efficiency, specificity and/or specific activity with respect to
metabolism of carbon chains of different lengths (e.g., carbon
chains including fatty alcohols, fatty acids, paraffins,
dicarboxylic acids of between about 1 and about 60 carbons in
length). In some embodiments, the nucleotide sequence of POX4,
POX5, or POX4 and POX5is disrupted with a URA3 nucleotide sequence
encoding a selectable marker, and introduced to a host
microorganism, thereby generating an engineered organism deficient
in POX4, POX5 or POX4 and POX5 activity. Nucleic acid sequences
encoding POX4 and POX5 can be obtained from a number of sources,
including Candida tropicalis, for example. Examples of POX4 and
POX5 amino acid sequences and nucleotide sequences of
polynucleotides that encode the polypeptides, are presented herein.
Described in the examples are experiments conducted to amplify the
activity encoded by the POX5 gene.
[0160] Also as noted above, catalytic specificity of acyl-CoA
oxidases (e.g., POX4, POX5) can be altered by a variety of methods.
Altering the binding and/or catalytic specificity of acyl-CoA
oxidases may prove advantageous for generating novel acyl-CoA
oxidases with altered chain length recognition, altered chain
length catalytic activity, and/or generation of an acyl-CoA oxidase
activity with a narrow or specific chain length specificity,
thereby allowing further increases in pathway efficiency,
specificity and/or specific activity with respect to metabolism of
carbon chains of different lengths or metabolism of carbon chain
distributions found in a particular chosen feedstock. In some
embodiments the altered acyl-CoA oxidase sequences are identified
and/or generated by; (i) screening naturally occurring variant
populations; (ii) mutagenesis of endogenous sequences; (iii)
introduction of heterologous sequences having a desired
specificity; (iv) generation of chimeric sequences having a portion
of the coding sequence from one polynucleotide source (e.g., gene,
organism) and a portion of the coding sequence from another source
and/or (v) intelligent design using nucleotide sequences an three
dimensional structure analysis from an acyl-CoA oxidase having a
desired specificity to remodel an endogenous acyl-Co A oxidase,
thereby generating a novel specificity enzyme. In some embodiments
a chimeric acyl-CoA oxidase sequence can have polynucleotide
sequence contributions from two or more sources. In some
embodiments, a chimeric acyl-CoA oxidase sequence comprises a
portion of the coding sequences from an endogenous polynucleotide
and a portion of the coding sequence from a heterologous
polynucleotide. Described in the examples are methods utilized to
identify and/or generate acyl-CoA oxidases with novel catalytic and
binding specificities.
[0161] Presence, absence or amount of POX4 and/or POX5 activity can
be detected by any suitable method known in the art. For example,
using enzymatic assays as described in Shimizu et al, 1979, and as
described herein in the Examples. Alternatively, nucleic acid
sequences representing native and/or disrupted POX4 and POX5
sequences also can be detected using nucleic acid detection methods
(e.g., PCR, primer extension, nucleic acid hybridization, the like
and combinations thereof), or quantitative expression based
analysis (e.g., RT-PCR, western blot analysis, northern blot
analysis, the like and combinations thereof), where the engineered
organism exhibits decreased RNA and/or polypeptide levels as
compared to the host organism.
[0162] Thioesterase
[0163] The term "thioesterase activity" as used herein refers to
removal of Coenzyme A from hexanoate. The term "thioesterase
activity" as used herein also refers to the removal of Coenzyme A
from an activated fatty acid (e.g., fatty-acyl-CoA). A Non-limiting
example of an enzyme with thioesterase activity includes acyl-CoA
hydrolase (e.g., EC 3.1.2.20; also referred to as acyl coenzyme A
thioesterase, acyl-CoA thioesterase, acyl coenzyme A hydrolase,
thioesterase B, thioesterase II, lecithinase B, lysophopholipase
L1, acyl-CoA thioesterase 1, and acyl-CoA thioesterase).
Thioesterases that remove Coenzyme A from fatty-acyl-CoA molecules
catalyze the reaction. [0164] acyl-CoA+H2O--.fwdarw.CoA+a
carboxylate, where the carboxylate often is a fatty acid. The
released Coenzyme A can then be reused for other cellular
activities.
[0165] The thioesterase activity can be provided by a polypeptide.
In certain embodiments, the polypeptide is an endogenous nucleotide
sequence that is increased in copy number, operably linked to a
heterologous and/or endogenous promoter, or increased in copy
number and operably linked to a heterologous and/or endogenous
promoter. In some embodiments, the polypeptide is encoded by a
heterologous nucleotide sequence introduced to a host
microorganism. Nucleic acid sequences conferring thioesterase
activity can be obtained from a number of sources, including Cuphea
lanceolata, C. tropicalis (e.g., see SEQ ID NOS: 33 and 35), and E.
coli (e.g., see SEQ ID NO: 37). Additional organisms that can be
used as thioesterase polynucleotide sequence donors are given
herein. Examples of such polypeptides include, without limitation,
acyl-(ACP) thioesterase type B from Cuphea lanecolata (see SEQ ID
NO: 1), acyl-CoA hydrolase (e.g., ACHA and ACHB, see SEQ ID NOS: 34
and 36)) from C. tropicalis, acyl-CoA thioesterase (e.g., TESA, sec
SEQ ID NO: 38) from E. coli. A non-limiting example of a
thioesterase polynucleotide sequences is referenced by accession
number CAB60830 at the World Wide Web Uniform Resource Locator
(URL) ncbi.nlm.nih.gov of the National Center for Biotechnology
Information NCBI).
[0166] Presence, absence or amount of thioesterase activity can be
detected by any suitable method known in the art. An example of
such a method is described Chemistry and Biology 9: 981-988. In
some embodiments, thioesterase activity is not altered in a host
microorganism, and in certain embodiments, the activity is added or
increased in the engineered microorganism relative to the host
microorganism. In some embodiments, a polypeptide having
thioesterase activity is linked to another polypeptide (e.g., a
hexanoate synthase A or hexanoate synthase B polypeptide).
Non-limiting examples of polynucleotide sequences encoding
thioesterase activities and polypeptides having thioesterase
activity are provided in Example 33.
[0167] Reducing Omega Fatty Acid Conversion--General
[0168] The term "a genetic modification that reduces omega hydroxyl
fatty acid conversion" as used herein refer to genetic alterations
of a host microorganism that reduce an endogenous activity that
converts an omega hydroxyl fatty acid to another product. In some
embodiments, an endogenous omega hydroxyl fatty acid dehydrogenase
activity is reduced. Such alterations can advantageously increase
the amount of a dicarboxylic acid, which can be purified and
further processed.
[0169] Reducing Beta Oxidation--General
[0170] The term "a genetic modification that reduces beta-oxidation
activity" as used herein refers to a genetic alteration of a host
microorganism that reduces an endogenous activity that oxidizes a
beta carbon of carboxylic acid containing organic molecules. In
certain embodiments, the organic molecule is a ten or twelve carbon
molecule, and sometimes contains one or two carboxylic acid
moieties located at a terminus of the molecule (e.g., sebacic or
dodecanedioic acid). Such alterations can advantageously increase
yields of end products, such as a fatty dicarboxylic acid (e.g.,
octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid).
[0171] The term "a genetic modification that results in increased
fatty acid synthesis" as used herein also refers to a genetic
alteration of a host microorganism that reduces an endogenous
activity that converts fatty acids into fatty-acyl-CoA
intermediates. In some embodiments, an endogenous activity that
converts fatty acids into fatty-acyl-CoA intermediates is reduced.
In certain embodiments, an acyl-CoA synthetase activity is reduced.
Such alterations can advantageously increase yields of end
products, such as a fatty-dicarboxylic acid (e.g., octanedioic
acid, decanedioic acid, dodecanedioic acid, tetradecanedioic acid,
hexadecanedioic acid, octadecanedioic acid, eicosanedioic
acid).
[0172] Acyl-CoA Synthetase
[0173] Organisms that have a complete block of the .beta.-oxidation
pathway cannot utilize fatty acids or diacids for energy. In these
.beta.-oxidation blocked organisms, the chain length of the diacid
produced mimics the chain length of the fatty acid feedstock.
Blocking the .beta.-oxidation pathway removes the primary route for
diacid product yield loss. In some embodiments, genetic
modifications that alter the cell's ability to utilize fatty acids
in other biochemical pathways results in increased diacid
production. In some embodiments, blocking a fatty acid activation
pathway by knocking out or modifying an acyl CoA synthetase results
in increased diacid production.
[0174] The activation of fatty acids to fatty acyl-CoA thioesters
is performed by an enzyme called acyl-CoA synthetase (ACS).
Acyl-CoA synthetases are a member of the ligase class of enzymes
and catalyzes the reaction,
ATP+Fatty
Acid+CoA.revreaction.AMP+Pyrophosphate+Fatty-Acyl-CoA.
[0175] Fatty acids can be converted into fatty-acyl-CoA
intermediates by the activity of an acyl-CoA synthetase (e.g.,
ACS1, ACS2; EC 6.2.1.3; also referred to as acyl-CoA synthetase,
acyl-CoA ligase), in many organisms. Yeast cells contain multiple
genes for ACS enzymes that are targeted to different cellular
locations and may have different substrate chain-length
specificities. S. cerevisiae has six genes with ACS activity named
FAA1, FAA2, FAA3, FAA4, FAT1, and FAT2. The corresponding proteins
produced by these genes are often called Faa1p, Faa2p, Faa3p,
Faa4p, Fat1p and Fat2p respectively. The Faa1p isozyme can exhibit
broad substrate chain-length specificity, represents 90% of the
cellular ACS activity, and is localized in the cytosolic and
microsomal fractions. The Faa2p isozyme is targeted to the
peroxisome and has broad chain-length specificity. The Faa3p
isozyme has a substrate specificity for long-chain or very
long-chain fatty acids and its cellular localization is unknown.
Faa4p has broad chain-length specificity and has been shown to be
important in protein myristoylation. Fat1p is a dual function
protein localized to the cellular membrane that has activity for
both fatty acid transport and fatty acid activation. Fat2p is
targeted to the peroxisomal membrane for medium chain fatty acid
transport and activation.
[0176] Acyl-CoA synthetase has six isoforms encoded by ACS1, FAT1,
AC-S2A, ACS2B, ACS2C, and ACS2D, respectively, in some Candida spp.
(e.g., homologous to FAA1, FAT1, and FAA2 in S. cerevisiae).
[0177] Disruption of the genes encoding ACS isozymes with activity
targeted to the cellular membrane and to the cytosolic fraction can
leave the exogenously supplied fatty acids in the free fatty acid
form which is a substrate for entry into the
.omega.-oxidation-pathway. This essentially redirects exogenously
supplied fatty acids from normal cellular utilization (energy,
triacylglycerides, phospholipids) to the production of the desired
diacid product. In some embodiments, in Candida strain ATCC20336
these gene targets are ACS1 and FAT1. [0178] Candida strain
ATCC20336_ACS1--SEQ ID NO: 40 [0179] Candida strain
ATCC20336_FAT1--SEQ ID NO: 148
[0180] Disruption of the genes encoding ACS isozymes with activity
targeted to the peroxisome can prevent the activation of any
exogenously supplied fatty acids that are transported to the
interior of the peroxisomal compartment. In a .beta.-oxidation
blocked organism fatty acyl-CoA molecules cannot enter
.beta.-oxidation, but they can be substrates for the synthesis of
phospholipids. Knocking out the genes encoding these ACS isozymes
can increase the yield of a diacid product by redirecting the free
fatty acids to .omega.-oxidation instead of the phospholipid
synthesis pathway. Candida strain ATCC20336 homologs to the
peroxisomal S. cerevisiae FAA2 are named ACS2A, ACS2B, ACS2C, and
ACS2D and the protein sequences of ACS2A, ACS2B and ACS2C are shown
below. [0181] Candida strain ATCC20336_ACS2A--SEQ ID NO: 80 [0182]
Candida strain ATCC20336_ACS2B--SEQ ID NO: 158 [0183] Candida
strain ATCC20336_ACS2C--SEQ ID NO: 159
[0184] Fatty acids and Coenzyme A often are utilized in the
activation of fatty acids to fatty-acyl-CoA intermediates for entry
into various cellular processes. Without being limited by theory,
it is believed that reduction in the amount of fatty-acyl-CoA
available for various cellular processes can increase the amount of
fatty acids available for conversion into a fatty dicarboxylic acid
(e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) by other engineered pathways in the same host
organism (e.g., omega oxidation pathway, beta oxidation pathway,
omega oxidation pathway and beta oxidation pathway). Acyl-CoA
synthetase can be inactivated by any suitable means. Described
herein are gene knockout methods suitable for use to disrupt the
nucleotide sequence that encodes a polypeptide having ACS1
activity. A nucleotide sequence of ACS1 is provided in Example 33,
SEQ ID NO: 39. An example of an integration/disruption construct,
configured to generate a deletion mutant for ACS1 is also provided
in the Examples.
[0185] The presence, absence or amount of acyl-CoA synthetase
activity can be detected by any suitable method known in the art.
Non-limiting examples of suitable detection methods include
enzymatic assays (e.g., Lageweg et al "A Fluorimetric Assay for
Acyl-CoA Synthetase Activity", Analytical Biochemistry,
197(2):384-388 (1991)), PCR based assays (e.g., qPCR, RT-PCR),
immunological detection methods (e.g., antibodies specific for
acyl-CoA synthetase), the like and combinations thereof.
[0186] The term "a genetic modification that results in increased
fatty acid synthesis" as used herein also refers to a genetic
alteration of a host microorganism that reduces an endogenous
activity that converts long chain and very long chain fatty acids
into activated fatty-acyl-CoA intermediates. In some embodiments,
an endogenous activity that converts long chain and very long chain
fatty acids into activated fatty-acyl-CoA intermediates is reduced.
In certain embodiments, a long chain acyl-CoA synthetase activity
is reduced. Such alterations can advantageously increase yields of
end products, such as a fatty dicarboxylic acid (e.g., octanedioic
acid, decanedioic acid, dodecanedioic acid, tetradecanedioic acid,
hexadecanedioic acid, octadecanedioic acid, eicosanedioic
acid).
[0187] Long chain fatty acids (e.g., C12-C18 chain lengths) and
very long chain fatty acids (e.g., C20-C26) often are activated
and/or transported by the thioesterification activity of a
long-chain acyl-CoA synthetase (e.g., FAT1; EC 6.2.1.3;
also-referred to as long-chain fatty acid-CoA ligase, acyl-CoA
synthetase; fatty acid thiokinase (long chain); acyl-activating
enzyme; palmitoyl-CoA synthase; lignoceroyl-CoA synthase;
arachidonyl-CoA synthetase; acyl coenzyme A synthetase; acyl-CoA
ligase; palmitoyl coenzyme A synthetase; thiokinase; palmitoyl-CoA
ligase; acyl-coenzyme A ligase; fatty acid CoA ligase; long-chain
fatty acyl coenzyme A synthetase; oleoyl-COA synthetase;
stearoyl-CoA synthetase; long chain fatty acyl-CoA synthetase;
long-chain acyl CoA synthetase; fatty acid elongase (ELO);
LCFA-synthetase; pristanoyl-CoA synthetase; ACS3; long-chain
acyl-CoA synthetase I; long-chain acyl-CoA synthetase II; fatty
acyl-coenzyme A synthetase; long-chain acyl-coenzyme A synthetase;
and acid:CoA ligase (AMP-forming)), in some organisms. Fatty acids
also can be transported into the host organism from feedstocks by
the activity of long chain acyl-CoA synthetase.
[0188] Long-chain acyl-CoA synthetase catalyzes the reaction,
ATP+a long-chain carboxylic acid+CoA=AMP+diphosphate+an
acyl-CoA,
where "an acyl-CoA" refers to a fatty-acyl-CoA molecule. As noted
herein, activation of fatty acids is often necessary for entry of
fatty acids into various cellular processes (e.g., as an energy
source, as a component for membrane formation and/or remodeling, as
carbon storage molecules). Deletion mutants of FAT1 have been shown
to accumulate very long chain fatty acids and exhibit decreased
activation of these fatty acids. Without being limited by theory,
it is believed that reduction in the activity of long-chain
acyl-CoA synthetase may reduce the amount of long chain fatty acids
converted into fatty-acyl-CoA intermediates, thereby increasing the
amount of fatty acids available for conversion into a fatty
dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid) by other engineered
pathways in the same host organism (e.g., omega oxidation pathway,
beta oxidation pathway, omega oxidation pathway and beta oxidation
pathway). Long-chain-acyl-CoA synthetase activity can be reduced or
inactivated by any suitable means. Described herein are gene
knockout methods suitable for disrupting the nucleotide sequence
that encodes the polypeptide having FAT1 activity. The nucleotide
sequence of FAT1 is provided to Example 33, SEQ ID NO: 41. DNA
vectors suitable for use in constructing "knockout" constructs are
described herein.
[0189] The presence, absence or amount of long-chain-acyl-CoA
synthetase activity can be detected by any suitable method known in
the art. Non-limiting examples of suitable detection methods
include enzymatic assays, binding assays (e.g., Erland et al,
Analytical Biochemistry 295(1):38-44 (2001)), PCR based assays
(e.g., qPCR, RTPCR), immunological detection methods (e.g.,
antibodies specific for long-chain-acyl-CoA synthetase), the like
and combinations thereof.
[0190] Selective Modification of Fat1p to Retain Transport
Activity
[0191] Transport of free fatty acids across a cellular membrane can
occur by passive diffusion or by protein-mediated active transport.
The mechanism of passive diffusion can be manipulated (increased
rate or decreased rate) to some extent, by the choice of fatty acid
feedstock or by changing the extracellular environment. The rate of
active transport of free fatty acids into the cell may be increased
by amplifying transport proteins involved in fatty acid import. One
such enzyme is Fat1p (e.g., Fat1p of S. cerevisiae) which is a dual
function protein with both fatty acid transport and acyl-CoA
synthetase activities.
[0192] Discussed above were the benefits of knocking out enzymes
with acyl-CoA synthetase activity. In order to increase fatty acid
transport into the cell without also increasing ACS activity,
mutants of the Fat1p can be constructed that are transport
competent but ACS incompetent.
[0193] Fat1p can transport a free fatty acid across a cellular
membrane and "activate" the fatty acid to an acyl-CoA thio ester on
the inner side of the cellular membrane. Once converted to an
acyl-CoA thioester a fatty acid can enter the following biochemical
pathways: 1) peroxisomal beta-oxidation, 2) triacylglyceride
synthesis, 3) cholesteryl ester synthesis or 4) phospholipid
synthesis. All of these possible fates for fatty acyl-CoA can
prevent the metabolism of an imported fatty acid into a
dicarboxylic acid and result in a low yield production of
dicarboxylic acids from fatty acid feedstocks. Therefore, in some
embodiments, strains are being developed with a mutant Fat1p enzyme
(i.e., Fat1p-mut), that retains fatty acid transport activity
(e.g., the ability to transport fatty acids across the cellular
membrane) but lacks a thioesterase activity (e.g., the ability to
activate a fatty acid to an acyl-CoA thioester). Fat1p mutants have
been described in the literature for S. cerevisiae. Some such
mutants are known in the literature and correspond to the mutants
S244A or D495A in the Candida strain ATCC20336 Fat1p enzyme.
[0194] Knocking Out Transport into the Peroxisome
[0195] The mechanism of transport of fatty acids into the
peroxisome differs upon the chain length of the fatty acid. Long
chain fatty acids, C16-C18, are not able to diffuse across the
peroxisomal membrane in free acid form but are instead transported
across as fatty acyl-CoA esters in an ATP-dependent process
catalyzed by the Pxa1p/Pxa2p heterodimer (e.g. EC 3.6.3.47). Short
and medium chain fatty acids, C6-C14, are thought to be able to
diffuse across the peroxisomal membrane in the free acid form,
however may also by aided in transport into the peroxisomal matrix
by Pex11p or by other as yet unknown transporters. In some
embodiments, knocking put the genes encoding these transport
proteins would again improve diacid yields by redirecting
exogenously supplied fatty acids from biochemical use in the
peroxisome to the .omega.-oxidation pathway.
[0196] Examples of the sequences of Pxa1p, Pxa2p and Pex11p from
Candida strain ATCC20336 are shown below. [0197] Candida strain
ATCC20336_PXA1--SEQ ID NO: 92 [0198] Candida strain
ATCC20336_PXA2--SEQ ID NO: 94 [0199] Candida strain
ATCC20336_PEX11--SEQ ID NO: 96
[0200] Acyl-CoA Sterol Acyltransferase
[0201] The term "a genetic modification that results in increased
fatty acid synthesis" as used herein also refers to a genetic
alteration of a host microorganism that reduces an endogenous
activity that converts fatty acids into cholesterol esters. In some
embodiments, an endogenous activity that converts fatty acids into
cholesterol esters is reduced. In certain embodiments, an acyl-CoA
sterol acyltransferase activity is reduced. Such alterations can
advantageously increase yields of end products, such as a fatty
dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid).
[0202] Fatty acids can be converted into a cholesterol-ester by the
activity of acyl-CoA sterol acyltransferase (e.g., ARE1, ARE2, EC
2.3.1.26; also referred to as sterol O-acyltransferase; cholesterol
acyltransferase; sterol-ester synthase; sterol-ester-synthetase;
sterol-ester synthase; acyl coenzyme
A-cholesterol-O-acyltransferase; acyl-CoA:cholesterol acyl
transferase; ACAT; acylcoenzyme A:cholesterol O-acyltransferase;
cholesterol ester synthase; cholesterol ester synthetase; and
cholesterol ester synthetase), in many organisms. Without being
limited by any theory, cholesterol esterification may be involved
in directing fatty acids away from incorporation into cell
membranes and towards storage forms of lipids. Acyl-CoA sterol
acyltransferase catalyzes the reaction,
acyl-CoA+cholesterol=CoA+cholesterol ester.
[0203] The esterificaiton of cholesterol is believed to limit its
solubility in cell membrane lipids and thus promotes accumulation
of cholesterol ester in the fat droplets (e.g., a form of carbon
storage molecule) within cytoplasm. Therefore, without being
limited by any theory esterification of cholesterol may cause the
accumulation of lipid storage molecules, and disruption of the
activity of acyl-CoA sterol acyltransferase may cause an increase
in acyl-CoA levels that can be converted into a fatty dicarboxylic
acid (e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) by other engineered pathways in the same host
organism (e.g., omega oxidation pathway, beta oxidation pathway,
omega oxidation pathway and beta oxidation pathway). Acyl-CoA
sterol acyltransferase can be inactivated by any suitable means.
Described herein are gene knockout methods suitable for disrupting
nucleotide sequences that encode polypeptides having ARE1 activity,
ARE2 activity or ARE1 activity and ARE2 activity. The nucleotide
sequences of ARE1 and ARE2 are provided in Example 33, SEQ ID NOS:
43 and 45. DNA vectors suitable for use in constructing "knockout"
constructs are described herein.
[0204] The presence absence or amount of acyl-CoA sterol
acyltransferase activity can be detected by any suitable method
known in the art. Non-limiting examples of suitable detection
methods include enzymatic assays (e.g., Chen et al, Plant
Physiology 145:974-984 (2007)), binding assays, PCR based assays
(e.g., qPCR, RT-PCR), immunological detection methods (e.g.,
antibodies specific for long-chain-acyl-CoA synthetase), the like
and combinations thereof.
[0205] Diacylglycerol Acyltransferase & Acyltransferases
[0206] The term "a genetic modification that results in increased
fatty acid synthesis" as used herein also refers to a genetic
alteration of a host microorganism that reduces an endogenous
activity that catalyzes diacylglycerol esterification (e.g.,
addition of acyl group to a diacylglycerol to form a
triacylglycerol). In some embodiments, an endogenous activity that
converts diacylglycerol into triacylglycerol is reduced. In certain
embodiments, an acyltransferase activity is reduced. In some
embodiments a diacylglycerol acyltransferase activity is reduced.
In some embodiments a diacylglycerol acyltransferase (e.g., DGA1,
EC 2.3.1.20) activity and an acyltransferase (e.g., LRO1) activity
are reduced. Such alterations can advantageously increase yields of
end products, such as a fatty dicarboxylic acid (e.g., octanedioic
acid, decanedioic acid, dodecanedioic acid, tetradecanedioic acid,
hexadecanedioic acid, octadecanedioic acid, eicosanedioic
acid).
[0207] Diacylglycerol can be converted into triacylglycerol by the
activity of diacylglycerol acyltransferase (e.g., DGA1; EC
2.3.1.20; also referred to as diglyceride-acyltransferase;
1,2-diacylglycerol acyltransferase; diacylglycerol acyltransferase;
diglyceride O-acyltransferase; palmitoyl-CoA-sn-1,2-diacylglycerol
acyltransferase; acyl-CoA: 1,2-diacyl glycerol O-acyltransferase
and acyl-CoA:1,2-diacyl-sn-glycerol O-acyltransferase), in many
organisms. Diacylglycerol acyltransferase catalyzes the
reaction,
Acyl-CoA+1,2-diacyl-sn-glycerol=CoA+triacylglycerol,
and is generally considered the terminal and only committed step in
triglyceride synthesis. The product of THE DGA1 gene in yeast
normally is localized to lipid particles.
[0208] In addition to the diacylglycerol esterification activity
described for DGA1, many organisms also can generate triglycerides
by the activity of other acyltransferase activities, non-limiting
examples of which include lecithin-cholesterol acyl transferase
activity (e.g., LRO1; EC 2.3.1.43; also referred to as
phosphatidylcholine-sterol O-acyltransferase activity;
lecithin-cholesterol acyltransferase activity;
phospholipid-cholesterol acyltransferase activity; LCAT
(lecithin-cholesterol acyltransferase) activity;
lecithin:cholesterol acyltransferase activity; and lysolecithin
acyltransferase activity) and phospholipid:diacylglycerol
acyltransferase (e.g., EC 2.3.1.158; also referred to as PDAT
activity and phospholipid:1,2-diacyl-sn-glycerol O-acyltransferase
activity). Acyltransferases of the families EC 2.3.1.43 and EC-
2.3.1.58 catalyze the general reaction,
phospholipid+1,2-diacylglycerol=lysophospholipid+triacylglycerol.
Triacylglycerides often are utilized as carbon (e.g., fatty acid or
lipid) storage molecules. Without being limited by any theory, it
is believe that reducing the activity of acyltransferase may reduce
the conversion of diacylglycerol to triacylglycerol, which may
cause increased accumulation of fatty acid, in conjunction with
additional genetic modifications (e.g., lipase to further remove
fatty acids from the glycerol backbone) that can be converted into
a fatty dicarboxylic acid (e.g., octanedioic acid, decanedioic
acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic
acid, octadecanedioic acid, eicosanedioic acid) by other engineered
pathways in the same host organism (e.g., omega oxidation pathway,
beta oxidation pathway, omega oxidation pathway and beta oxidation
pathway). Acyltransferases can be inactivated by any suitable
means. Described herein are gene knockout methods suitable for
disrupting nucleotide sequences that encode polypeptides having
DGA1 activity, LRO1 activity or DGA1 activity and LRO1 activity.
The nucleotide sequence of DGA1 is provided in Example 33, SEQ ID
NO: 47 The nucleotide sequence of LRO1 is provided in Example 33,
SEQ ID NO: 49. DNA vectors suitable for use in constructing
"knockout" constructs are described herein.
[0209] The genes ARE1 and ARE2 in S. cerevisiae are also involved
in triacylglyceride synthesis. Knocking out genes encoding these
enzymes can redirect exogenously supplied fatty acids to
.omega.-oxidation.
[0210] The presence, absence or amount of acyltransferase activity
can be detected by any suitable method known in the art.
Non-limiting examples of suitable detection methods include
enzymatic assays (e.g., Geelen, Analytical Biochemistry
322(2):264-268 (2003), Dahlqvist et al, PNAS 97(12):6487-6492
(2000)), binding assays, PCR based assays (e.g., qPCR, RTPCR),
immunological detection methods (e.g., antibodies specific for a
DGA1 or LRO1 acyltransferase), the like and combinations
thereof.
[0211] Carnitine Acetyltransferase
[0212] Carnitine acetyltransferase (i.e., Cat2, Cat2p) is an enzyme
targeted to both the peroxisomal and mitochondrial compartments. It
catalyzes the transfer of an acetyl group from a CoA group to a
carnitine (or vice versa depending on location) as shown below.
[0213] Acetyl-CoA+carnitine.fwdarw.acetylcarnitine+CoA
[0214] An acetyl-carnitine molecule is transported across the
peroxisomal and mitochondrial membranes whereas an acetyl-CoA
molecule is not. Therefore the action of Cat2 (e.g., 2.3.1.7)
provides one of three possible routes for acetyl groups to leave
the peroxisome. Acetyl-CoA produced by beta-oxidation can be
converted to acetyl-carnitine and transported to the mitochondria
for entry into the TCA cycle. In some embodiments, the activity of
Cat2 is decreased or eliminated in order to slow the exit of
acetyl-CoA from the peroxisome. In some embodiments, providing a
bottle-neck downstream of the adipic acid intermediate that is
derived from beta-oxidation is a strategy for improving the yield
of adipic acid.
[0215] Carnitine O-acyltransferase
[0216] Carnitine O-acyltransferase (i.e., CROT, e.g., 2.3.1.137) is
a peroxisomal enzyme that can transfer an acyl chain from a CoA
group to a carnitine group as shown below. [0217]
Acyl-CoA+carnitine.fwdarw.acylcarnitine+CoA
[0218] The acyl-carnitine produced may then be transported out of
the peroxisome for use elsewhere in the cell. The enzyme may act on
acyl chains of different chain lengths but is most active on short
chains (C6-C8). Diacids that are prematurely pulled out of
beta-oxidation and sent to other cellular compartments can
represent a yield loss.
[0219] UDP-glucosyltransferase
[0220] The UDP-glucosyltransferase enzyme (i.e., UGTA1, UgtA1p,
e.g., 2.4.1.-) performs the first reaction in the synthesis of
sophorolipids. Sophorolipids are a class of biosurfactant molecules
produced by some yeast when exposed to hydrophobic environments.
They are made up of sophorose
(2-O-.beta.-D-glucopyranosyl-D-glucopyranose) attached through its
anomeric carbon to an .omega.- or (.omega.-1)-hydroxylated fatty
acid of 16 or 18 carbons. The most well-known yeast for producing
sophorolipids is Candida bombicola. The pathway for sophorolipid
production in this yeast proceeds via a step-wise transfer of two
glucose molecules to a hydroxy-fatty acid. The first step is
carried out by UgtA1p and the second step by UgtB1p (Saerens K M J,
Roelants S L K W, VanBogaert I N A, Soetaert W (2011) FEMS Yeast
Res 11: 123-132; Saerens K M J, Zhang J, Saey L, VanBogaert I N A,
Soetaert W (2011) Yeast 28: 279-292). The stepwise transfer of
glucose from UDP-glucose to the .omega.-end of the hydroxyl-fatty
acid could represent a yield loss if .omega.-hydroxy fatty acids
produced in the first step of .omega.-oxidation are pulled into
sophorolipid production rather than diacid production.
[0221] Elongase(s)
[0222] "Elongase(s)" means those enzyme(s) in an organism that have
ability to (i) extend the chain length of fatty acyl-CoA molecules,
as for example converting C-12 to C-16 fatty acyl-CoA molecules to
C16-C18 fatty acids; (ii) elongate palmitoyl-CoA and stearoyl-CoA
up to about 22 carbon fatty acids; or (iii) synthesize longer chain
carbon fatty acids from shorter chain CoA primers such as C-18-CoA.
In some embodiments, the expression of an elongase is decreased or
knocked out in a fatty dicarboxylic acid producing yeast.
[0223] Polynucleotides and Polypeptides
[0224] A nucleic acid (e.g., also referred to herein as nucleic
acid reagent, target nucleic acid, target nucleotide sequence,
nucleic acid sequence of interest or nucleic acid region of
interest) can be from any source or composition, such as DNA, cDNA,
gDNA (genomic DNA), RNA, siRNA (short inhibitory RNA), RNAi, tRNA
or mRNA, for example, and can be in any form (e.g., linear,
circular, supercoiled, single-stranded, double-stranded, and the
like). A nucleic acid can also comprise DNA or RNA analogs (e.g.,
containing base analogs, sugar analogs and/or a non-native backbone
and the like). It is understood that the term "nucleic acid" does
not refer to or infer a specific length of the polynucleotide
chain, thus polynucleotides and oligonucleotides are also included
in the definition. Deoxyribonucleotides include deoxyadenosine,
deoxycytidine, deoxyguanosine and deoxythymidine. For RNA, the
uracil base is uridine.
[0225] A nucleic acid sometimes is a plasmid, phage, autonomously
replicating sequence (ARS), centromere, artificial chromosome,
yeast artificial chromosome (e.g., YAC) or other nucleic acid able
to replicate or be replicated in a host cell. In certain
embodiments a nucleic acid can be from a library or can be obtained
from enzymatically digested, sheared or sonicated genomic DNA
(e.g., fragmented) from an organism of interest. In some
embodiments, nucleic acid subjected to fragmentation or cleavage
may have a nominal, average or mean length about 5 to about 10,000
base pairs, about 100 to about 1,000 base pairs, about 100 to about
500 base pairs, or about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700,
800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or
10000 base pairs. Fragments can be generated by any suitable method
in the art, and the average, mean or nominal length of nucleic acid
fragments can be controlled by selecting an appropriate
fragment-generating procedure by the person of ordinary skill. In
some embodiments, the fragmented DNA can be size selected to obtain
nucleic acid fragments of a particular size range.
[0226] Nucleic acid can be fragmented by various methods known to
the person of ordinary skill, which include without limitation,
physical, chemical and enzymic processes. Examples of such
processes are described in U.S. Patent Application Publication No.
20050112590 (published on May 26, 2005, entitled
"Fragmentation-based methods and systems for sequence variation
detection and discovery," naming Van Den Boom et al.). Certain
processes can be selected by the person of ordinary skill to
generate non-specifically cleaved fragments or specifically cleaved
fragments. Examples of processes that can generate non-specifically
cleaved fragment sample nucleic acid include, without limitation,
contacting sample nucleic acid with apparatus that expose nucleic
acid to shearing force (e.g., passing nucleic acid through a
syringe needle; use of a French press); exposing sample nucleic
acid to irradiation (e.g., gamma, x-ray, UV irradiation; fragment
sizes can be controlled by irradiation intensity); boiling nucleic
acid in water (e.g., yields about 500 base pair fragments) and
exposing nucleic acid to an acid and base hydrolysis process.
[0227] Nucleic acid may be specifically cleaved by contacting the
nucleic acid with one or more specific cleavage agents. The term
"specific cleavage agent" as used herein refers to an agent,
sometimes a chemical or an enzyme that can cleave a nucleic acid at
one or more specific sites. Specific cleavage agents often will
cleave specifically according to a particular nucleotide sequence
at a particular site. Examples of enzymic specific cleavage agents
include without limitation endonucleases (e.g., DNase (e.g., DNase
I, II); RNase (e.g., RNase E, F, H, P); Cleavase.TM. enzyme; Taq
DNA polymerase E. coli DNA polymerase I and eukaryotic
structure-specific endonucleases; murine FEN-1 endonucleases;
type-I, II or III restriction endonucleases such as Acc I, Afl III,
Alu I, Alw44 I, Apa I, Asn I, Ava I, Ava II, BamH I, Ban II, Bcl I,
Bgl I, Bgl II, Bln I, Bsm I, BssH II, BstE II, Cfo I, CIa I, Dde I,
Dpn I, Dra I, EcIX I, EcoR I, EcoR I, EcoR II, EcoR V, Hac II, Hac
II; Hind II, Hind III, Hpa I, Hpa II, Kpn I, Ksp I, Mlu I, MluN I,
Msp I, Nci I, Nco I, Ndc I, Ndc II, Nhc I, Not I, Nru I, Nsi I, Pst
I, Pvu I, Pvu II, Rsa I, Sac I, Sal I, Sau3A I, Sca I, ScrF I, Sfi
I; Sma I, Spc I, Sph I, Ssp I, Stu I, Sty I, Swa I, Taq I, Xba I,
Xho I); glycosylases (e.g., uracil-DNA glycolsylase (UDG),
3-methyladenine DNA glycosylase, 3-methyladenine DNA, glycosylase
II, pyrimidine hydrate-DNA glycosylase, FaPy-DNA glycosylase,
thymine mismatch-DNA glycosylase, hypoxanthine-DNA glycosylase,
5-Hydroxymethyluracil DNA-glycosylase (HmUDG),
5-Hydroxymethylcytosine DNA glycosylase, or 1,N6-etheno-adenine DNA
glycosylase); exonucleases (e.g.; exonuclease III); ribozymes, and
DNAzymes. Sample nucleic acid may be treated with a chemical agent,
or synthesized using modified nucleotides, and the modified nucleic
acid may be cleaved. In non-limiting examples, sample nucleic acid
may be treated with (i) alkylating agents such as methylnitrosourea
that generate several alkylated bases, including N3-methyladenine
and N3-methylguanine, which are recognized and cleaved by alkyl
purine DNA-glycosylase; (ii) sodium bisulfite, which causes
deamination of cytosine residues in DNA to form uracil residues
that can be cleaved by uracil N-glycosylase; and (iii) a chemical
agent that converts guanine to its oxidized form, 8-hydroxyguanine,
which can be cleaved by formamidopyrimidine DNA N-glycosylase.
Examples of chemical cleavage processes include without limitation
alkylation, (e.g., alkylation of phosphorothioate-modified nucleic
acid); cleavage of acid lability of
P3'-N5'-phosphoroamidate-containing nucleic acid; and osmium
tetroxide and piperidine treatment of nucleic acid.
[0228] As used herein, the term "complementary cleavage reactions"
refers to cleavage reactions that are carried out on the same
nucleic acid using different cleavage reagents or by altering the
cleavage specificity of the same cleavage reagent such that
alternate cleavage patterns of the same target or reference nucleic
acid or protein are generated. In certain embodiments, nucleic
acids of interest may be treated with one or more specific cleavage
agents (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more specific
cleavage agents) in one or more reaction vessels (e.g., nucleic
acid of interest is treated with each specific cleavage agent in a
separate vessel).
[0229] A nucleic acid suitable for use in the embodiments described
herein sometimes is amplified by any amplification process known in
the art (e.g., PCR, RT-PCR and the like). Nucleic acid
amplification may be particularly beneficial when using organisms
that are typically difficult to culture (e.g., slow growing,
require specialize culture conditions and the like). The terms
"amplify", "amplification", "amplification reaction", or
"amplifying" as used herein refer to any in vitro processes, for
multiplying the copies of a target sequence of nucleic acid.
Amplification sometimes refers to an "exponential" increase in
target nucleic acid. However, "amplifying" as used herein can also
refer to linear increases in the numbers of a select target
sequence of nucleic acid, but is different than a one-time, single
primer extension step. In some embodiments, a limited amplification
reaction, also known as pre-amplification, can be performed.
Pre-amplification is a method in which a limited amount of
amplification occurs due to a small number of cycles, for example
10 cycles, being performed. Pre-amplification can allow some
amplification, but stops amplification prior to the exponential
phase, and typically produces about 500 copies of the desired
nucleotide sequence(s). Use of pre-amplification may also limit
inaccuracies associated with depleted reactants in standard PCR
reactions.
[0230] In some embodiments, a nucleic acid reagent sometimes is
stably integrated into the chromosome of the host organism, or a
nucleic acid reagent can be a deletion of a portion of the host
chromosome, in certain embodiments (e.g., genetically modified
organisms, where alteration of the host genome confers the ability
to selectively or preferentially maintain the desired organism
carrying the genetic modification). Such nucleic acid reagents
(e.g., nucleic acids or genetically modified organisms whose
altered genome confers a selectable trait to the organism) can be
selected for their ability to guide production of a desired protein
or nucleic acid molecule. When desired, the nucleic acid reagent
can be altered such that codons encode for (i) the same amino acid,
using a different tRNA than that specified in the native sequence;
or (ii) a different amino acid than is normal, including
unconventional or unnatural amino acids (including detectably
labeled amino acids). As described herein, the term "native
sequence" refers to an unmodified nucleotide sequence as found in
its natural setting (e.g., a nucleotide sequence as found in an
organism).
[0231] A nucleic acid or nucleic acid reagent can comprise certain
elements often selected according to the intended use of the
nucleic acid. Any of the following elements can be included in or
excluded from a nucleic acid reagent. A nucleic acid reagent, for
example, may include one or more or all of the following nucleotide
elements: one or more promoter elements, one or more 5'
untranslated regions (5'UTRs), one or more regions into which a
target nucleotide sequence may be inserted (an "insertion
element"), one or more target nucleotide sequences, one or more 3'
untranslated regions (3'UTRs), and one or more selection elements.
A nucleic acid reagent can be provided with one or more of such
elements and other elements may be inserted into the nucleic acid
before the nucleic acid is introduced into the desired organism. In
some embodiments, a provided nucleic acid reagent comprises a
promoter, 5'UTR, optional 3'UTR and insertion element(s) by which a
target nucleotide sequence is inserted (i.e., cloned) into the
nucleotide acid reagent. In certain embodiments, a provided nucleic
acid reagent comprises a promoter, insertion element(s) and
optional 3'UTR, and a 5' UTR/target nucleotide sequence is inserted
with an optional 3'UTR. The elements can be arranged in any order
suitable for expression in the chosen expression system (e.g.,
expression in a chosen organism, or expression in a cell free
system, for example), and in some embodiments a nucleic acid
reagent comprises the following elements in the 5' to 3' direction:
(1) promoter element, 5'UTR, and insertion element(s); (2) promoter
element, 5'UTR, and target nucleotide sequence; (3) promoter
element, 5'UTR, insertion element(s) and 3'UTR; and (4) promoter
element, 5'UTR, target nucleotide sequence and 3'UTR.
[0232] Promoters
[0233] A promoter element typically is required for DNA synthesis
and/or RNA synthesis. A promoter element often comprises a region
of DNA that can facilitate the transcription of a particular gene,
by providing a start site for the synthesis of RNA corresponding to
a gene.
[0234] Promoters generally are located near the genes they
regulate, are located upstream of the gene (e.g., 5' of the gene),
and are on the same strand of DNA as the sense strand of the gene,
in some embodiments. In some embodiments, a promotor element can be
isolated from a gene or organism and inserted in functional
connection with a polynucleotide sequence to allow altered and/or
regulated expression. A non-native promoter (e.g., promoter not
normally associated with a given nucleic acid sequence) used for
expression of a nucleic acid often is referred to as a heterologous
promoter. In certain embodiments, a heterologous promoter and/or a
5'UTR can be inserted in functional connection with a
polynucleotide that encodes a polypeptide having a desired activity
as described herein. The terms "operably linked" and "in functional
connection with" as used herein with respect to promoters, refer to
a relationship between a coding sequence and a promoter element.
The promoter is operably linked or in functional connection with
the coding sequence when expression from the coding sequence via
transcription is regulated, or controlled by, the promoter element.
The terms "operably linked" and "in functional connection with" are
utilized interchangeably herein with respect to promoter
elements.
[0235] A promoter often interacts with a RNA polymerase. A
polymerase is an enzyme that catalyses synthesis of nucleic acids
using a preexisting nucleic acid reagent. When the template is a
DNA template, an RNA molecule is transcribed before protein is
synthesized. Enzymes having polymerase activity suitable for use in
the present methods include any polymerase that is active in the
chosen system with the chosen template to synthesize protein. In
some embodiments, a promoter (e.g., a heterologous promoter) also
referred to herein as a promoter element, can be operably linked to
a nucleotide sequence or an open reading frame (ORF). Transcription
from the promoter element can catalyze the synthesis of an RNA
corresponding to the nucleotide sequence or ORF sequence operably
linked to the promoter, which in turn leads to synthesis of a
desired peptide, polypeptide or protein.
[0236] Promoter elements sometimes exhibit responsiveness to
regulatory control. Promoter elements also sometimes can be
regulated by a selective agent. That is, transcription from
promoter elements sometimes can be turned on, turned off,
up-regulated or down-regulated, in response to a change in
environmental, nutritional or internal conditions or signals (e.g.,
heat inducible promoters, light regulated promoters, feedback
regulated promoters, hormone influenced promoters, tissue specific
promoters, oxygen and pH influenced promoters, promoters that are
responsive to selective agents (e.g., kanamycin) and the like, for
example). Promoters influenced by environmental, nutritional or
internal signals frequently are influenced by a signal (direct or
indirect) that binds at or near the promoter and increases or
decreases expression of the target sequence under certain
conditions.
[0237] Non-limiting examples of selective or regulatory agents that
can influence transcription from a promoter element used in
embodiments described herein include, without limitation, (1)
nucleic acid segments that encode products that provide resistance
against otherwise toxic compounds (e.g., antibiotics); (2) nucleic
acid segments that encode products that are otherwise lacking in
the recipient cell (e.g., essential products, tRNA genes,
auxotrophic markers); (3) nucleic acid segments that encode
products that suppress the activity of a gene product; (4) nucleic
acid segments that encode products that can be readily identified
(e.g., phenotypic markers such as antibiotics (e.g.,
.beta.-lactamase), .beta.-galactosidase, green fluorescent protein
(GFP), yellow fluorescent protein (YFP), red fluorescent protein
(RFP), cyan fluorescent protein (CFP), and cell surface proteins);
(5) nucleic acid segments that bind products that are otherwise
detrimental to cell survival and/or function; (6) nucleic acid
segments that otherwise inhibit the activity of any of the nucleic
acid segments described in Nos. 1-5 above (e.g., antisense
oligonucleotides); (7) nucleic acid segments that bind products
that modify a substrate (e.g., restriction endonucleases); (8)
nucleic acid segments that can be used to isolate or identify a
desired molecule (e.g., specific protein binding sites); (9)
nucleic acid segments that encode a specific nucleotide sequence
that can be otherwise non-functional (e.g., for PCR amplification
of subpopulations of molecules); (10) nucleic acid segments that,
when absent, directly or indirectly confer resistance or
sensitivity to particular compounds; (11) nucleic acid segments
that encode products that either are toxic or convert a relatively
non-toxic compound to a toxic compound (e.g., Herpes simplex
thymidine kinase, cytosine deaminase) in recipient cells; (12)
nucleic acid segments that inhibit replication, partition or
heritability of nucleic acid molecules that contain them; and/or
(13) nucleic acid segments that encode conditional replication
functions, e.g., replication in certain hosts or host cell strains
or under certain environmental conditions (e.g., temperature,
nutritional conditions, and the like). In some embodiments, the
regulatory or selective agent can be added to change the existing
growth conditions to which the organism is subjected (e.g., growth
in liquid culture, growth in a fermenter, growth on solid nutrient
plates and the like for example).
[0238] In some embodiments, regulation of a promoter element can be
used to alter (e.g., increase, add, decrease or substantially
eliminate) the activity of a peptide, polypeptide or protein (e.g.,
enzyme activity for example). For example, a microorganism can be
engineered by genetic modification to express a nucleic acid
reagent that can add a novel activity (e.g., an activity not
normally found in the host organism) or increase the expression of
an existing activity by increasing transcription from a homologous
or heterologous promoter operably linked to a nucleotide sequence
of interest (e.g., homologous or heterologous nucleotide sequence
of interest), in certain embodiments. In some embodiments, a
microorganism can be engineered by genetic modification to express
a nucleic acid reagent that can decrease expression of an activity
by decreasing or substantially eliminating transcription from a
homologous or heterologous promoter operably linked to a nucleotide
sequence of interest, in certain embodiments.
[0239] In some embodiments the activity can be altered using
recombinant DNA and genetic techniques known to the artisan.
Methods for engineering microorganisms are further described
herein. Tables herein provide non-limiting lists of yeast promoters
that are up-regulated by oxygen, yeast promoters that are
down-regulated by oxygen, yeast transcriptional repressors and
their associated genes, DNA binding motifs as determined using the
MEME sequence analysis software. Potential regulator binding motifs
can be identified using the program MEME to search intergenic
regions bound by regulators for overrepresented sequences. For each
regulator, the sequences of intergenic regions bound with p-values
less than 0.001 were extracted to use as input for motif discovery.
The MEME software was run using the following settings: a motif
width ranging from 16 to 18 bases, the "zoops" distribution model,
a 6.sup.th order Markov background model and a discovery limit of
20 motifs. The discovered sequence motifs were scored for
significance by two criteria: an E-value calculated by MEME and a
specificity score. The motif with the best score using each metric
is shown for each regulator. All motifs presented are derived from
datasets generated in rich growth conditions with the exception of
a previously published dataset for eptiope-tagged Ga14 grown in
galactose.
[0240] In some embodiments, the altered activity can be found by
screening the organism under conditions that select for the desired
change in activity. For example, certain microorganisms can be
adapted to increase or decrease an activity by selecting or
screening the organism in question on a media containing substances
that are poorly metabolized or even toxic. An increase in the
ability of an organism to grow a substance that is normally poorly
metabolized may result in an increase in the growth rate on that
substance, for example. A decrease in the sensitivity to a toxic
substance might be manifested by growth on higher concentrations of
the toxic substance, for example. Genetic modifications that are
identified in this manner sometimes are referred to as naturally
occurring mutations or the organisms that carry them can sometimes
be referred to as naturally occurring mutants. Modifications
obtained in this manner are not limited to alterations in promoter
sequences. That is, screening microorganisms by selective pressure,
as described above, can yield genetic alterations that can occur in
non-promoter sequences, and sometimes also can occur in sequences
that are not in the nucleotide sequence of interest, but in a
related nucleotide sequences (e.g., a gene involved in a different
step of the same pathway, a transport gene, and the like).
Naturally occurring mutants sometimes can be found by isolating
naturally occurring variants from unique environments, in some
embodiments.
[0241] Homology and Identity
[0242] In addition to the regulated promoter sequences, regulatory
sequences, and coding polynucleotides provided herein, a nucleic
acid reagent may include a polynucleotide sequence 80% or more
identical to the foregoing (or to the complementary sequences).
That is, a nucleotide sequence that is at least 80% or more, 81% or
more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or
more, 87% or more, 88% or more, 89% or more; 90% or more, 91% or
more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or
more, 97% or more, 98% or more, or 99% or more identical to a
nucleotide sequence described herein can be utilized. The term
"identical" as used herein refers to two or more nucleotide
sequences having substantially the same nucleotide sequence when
compared to each other. One test for determining whether two
nucleotide sequences or amino acids sequences are substantially
identical is to determine the percent of identical nucleotide
sequences or amino acid sequences shared.
[0243] Calculations of sequence identity can be performed as
follows. Sequences are aligned for optimal comparison purposes
(e.g., gaps can be introduced in one or both of a first and a
second amino acid or nucleic acid sequence for optimal alignment
and non-homologous sequences can be disregarded for comparison
purposes). The length of a reference sequence aligned for
comparison purposes is sometimes 30% or more, 40% or more, 50% or
more, often 60% or more, and more often 70% or more, 80%or more,
90% or more, or 100% of the length of the reference sequence. The
nucleotides or amino acids at corresponding nucleotide or
polypeptide positions, respectively, are then compared among the
two sequences. When a position in the first sequence is occupied by
the same nucleotide or amino acid as the corresponding position in
the second sequence, the nucleotides or amino acids are deemed to
be identical at that position. The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences, taking into account the number of gaps, and the
length of each gap, introduced for optimal alignment of the two
sequences.
[0244] Comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. Percent identity between two amino acid or
nucleotide sequences can be determined using the algorithm of
Meyers & Miller, CABIOS 4:11-17 (1989), which has been
incorporated into the ALIGN program (version 2.0), using a PAM 120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4. Also, percent identity between two amino acid sequences can
be determined using the Needleman & Wunsch, J. Mol. Biol.
48:444-453 (1970) algorithm which has been incorporated into the
GAP program in the GCG software package (available at the http
address www.gcg.com), using either a Blossum 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6. Percent identity between two
nucleotide sequences can be determined using the GAP program in the
GCG software package (available at http address www:gcg.com), using
a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80
and a length weight of 1, 2, 3, 4, 5, or 6. A set of parameters
often used is a Blossum 62 scoring matrix with a gap open penalty
of 12, a gap extend penalty of 4, and a frameshift gap penalty of
5.
[0245] Sequence identity can also be determined by hybridization
assays conducted under stringent conditions. As use herein, the
term "stringent conditions" refers to conditions for hybridization
and washing. Stringent conditions are known to those skilled in the
art and can be found in Current Protocols in Molecular Biology,
John Wiley & Sons, N.Y., 6.3.1-6.3.6 (1989). Aqueous and
non-aqueous methods are described in that reference and either can
be used. An example of stringent hybridization conditions is
hybridization in 6.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C.; followed by one or more washes in
0.2.times.SSC, 0.1% SDS at 50.degree. C. Another example of
stringent hybridization conditions are hybridization in
6.times.sodium chloride/sodium citrate (SSC) at about 45.degree.
C., followed by one or more washes in 0.2.times. SSC, 0.1% SDS at
55.degree. C. A further example of stringent hybridization
conditions is hybridization in 6.times. sodium chloride/sodium
citrate (SSC) at about 45.degree. C., followed by one or more
washes in 0.2.times.SSC, 0.1% SDS at 60.degree. C. Often, stringent
hybridization conditions are hybridization in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by
one or more washes in 0.2.times.SSC, 0.1% SDS at 65.degree. C. More
often, stringency conditions are 0.5M sodium phosphate, 7% SDS at
65.degree. C., followed by one or more washes at 0.2.times.SSC, 1%
SDS at 65.degree. C.
[0246] UTRs
[0247] As noted above, nucleic acid reagents may also comprise one
or more 5' UTR's, and one or more 3'UTR's. A 5' UTR may comprise
one or more elements endogenous to the nucleotide sequence from
which it originates, and sometimes includes one or more exogenous
elements. A 5' UTR can originate from any suitable nucleic acid,
such as genomic DNA, plasmid DNA, RNA or mRNA, for example, from
any suitable organism (e.g., virus, bacterium, yeast, fungi, plant,
insect or mammal). The artisan may select appropriate elements for
the 5' UTR based upon the chosen expression system (e.g.,
expression in a chosen organism, or expression in a cell free
system, for example). A 5' UTR sometimes comprises one or more of
the following elements known to the artisan: enhancer sequences
(e.g., transcriptional or translational), transcription initiation
site, transcription factor binding site, translation regulation
site, translation initiation site, translation factor binding site,
accessory protein binding site, feedback regulation agent binding
sites, Pribnow box, TATA box, -35 clement, E-box (helix-loop-helix
binding element), ribosome binding site, replicon, internal
ribosome entry site (IRES), silencer element and the like. In some
embodiments, a promoter element may be isolated such that all 5'
UTR elements necessary for proper conditional regulation are
contained in the promoter element fragment, or within a functional
subsequence of a promoter element fragment.
[0248] A 5' UTR in the nucleic acid reagent can comprise a
translational enhancer nucleotide sequence. A translational
enhancer nucleotide sequence often is located between the promoter
and the target nucleotide sequence in a nucleic acid reagent. A
translational enhancer sequence often binds to a ribosome,
sometimes is an 18S rRNA-binding ribonucleotide sequence (i.e., a
40S ribosome binding sequence) and sometimes is an internal
ribosome entry sequence (IRES). An IRES generally forms an RNA
scaffold with precisely placed RNA tertiary structures that contact
a 40S ribosomal subunit via a number of specific intermolecular
interactions. Examples of ribosomal enhancer sequences are known
and can be identified by the artisan (e.g., Mignone et al., Nucleic
Acids Research 33: D141-D146 (2005); Paulous et al., Nucleic Acids
Research 31: 722-733 (2003); Akbergenov et al., Nucleic Acids
Research 32:239-247 (2004); Mignone et al., Genome Biology 3 (3):
reviews 0004.1-0001.10 (2002); Gallie, Nucleic Acids Research 30:
3401 -3411 (2002); Shaloiko et al., http address
www.interscience.wiley.com, DOI; 10,1002/bit 20267; and Gallie et
al., Nucleic Acids Research 15: 3257-3273 (1987)).
[0249] A translational enhancer sequence sometimes is a eukaryotic
sequence, such as a Kozak consensus sequence or other sequence
(e.g., hydroid polyp sequence, GenBank accession no. U07128). A
translational enhancer sequence sometimes is a prokaryotic
sequence, such as a Shine-Dalgarno consensus sequence. In certain
embodiments, the translational enhancer sequence is a viral
nucleotide sequence. A translational enhancer sequence sometimes is
from a 5' UTR of a plant virus, such as Tobacco Mosaic Virus (TMV),
Alfalfa Mosaic Virus (AMV); Tobacco Etch Virus (ETV); Potato Virus
Y (PVY); Turnip Mosaic (poty) Virus and Pea Seed Borne Mosaic
Virus; for example. In certain embodiments, an omega sequence about
67 bases in length from TMV is included in the nucleic acid reagent
as a translational enhancer sequence (e.g., devoid of guanosine
nucleotides and includes a 25 nucleotide long poly (CAA) central
region).
[0250] A 3' UTR may comprise one or more elements endogenous to the
nucleotide sequence from which it originates and sometimes includes
one or more exogenous elements. A 3' UTR may originate from any
suitable nucleic acid, such as genomic DNA, plasmid DNA, RNA or
mRNA, for example, from any suitable organism (e.g., a virus,
bacterium, yeast, fungi, plant, insect or mammal). The artisan can
select appropriate elements for the 3' UTR based upon the chosen
expression system (e.g., expression in a chosen organism, for
example). A 3' UTR sometimes comprises one or more of the following
elements known to the artisan: transcription regulation site,
transcription initiation site, transcription termination site,
transcription factor binding site, translation regulation site,
translation termination site, translation initiation site,
translation factor binding site, ribosome binding site, replicon,
enhancer element, silencer element and polyadenosine tail. A 3' UTR
often includes a polyadenosine tail and sometimes does not, and if
a polyadenosine tail is present, one or more adenosine moieties may
be added or deleted from it (e.g., about 5, about 10, about 15,
about 20, about 25, about 30, about 35, about 40, about 45 or about
50 adenosine moieties may be added or subtracted).
[0251] In some embodiments, modification of a 5' UTR and/or a 3'
UTR can be used to alter (e.g., increase, add, decrease or
substantially eliminate) the activity of a promoter. Alteration of
the promoter activity can in turn alter the activity of a peptide,
polypeptide or protein (e.g., enzyme activity for example), by a
change in transcription of the nucleotide sequence(s) of interest
from an operably linked promoter element comprising the modified 5'
or 3' UTR. For example, a microorganism can be engineered by
genetic modification to express a nucleic acid reagent comprising a
modified 5' or 3' UTR that can add a novel activity (e.g., an
activity not normally found in the host organism) or increase the
expression of an existing activity by increasing transcription from
a homologous or heterologous promoter operably linked to a
nucleotide sequence of interest (e.g., homologous or heterologous
nucleotide sequence of interest), in certain embodiments. In some
embodiments, a microorganism can be engineered by genetic
modification to express a nucleic acid reagent comprising a
modified 5' or 3' UTR that can decrease the expression of an
activity by decreasing or substantially eliminating transcription
from a homologous or heterologous promoter operably linked to a
nucleotide sequence of interest, in certain embodiments.
[0252] Target Nucleotide Sequence
[0253] A nucleotide reagent sometimes can comprise a target
nucleotide sequence. A "target nucleotide sequence" as used herein
encodes a nucleic acid, peptide, polypeptide of protein of
interest, and may be a ribonucleotide sequence or a
deoxyribonucleotide sequence. A target nucleic acid sometimes is an
untranslated ribonucleic acid and sometimes is a translated
ribonucleic acid. An untranslated ribonucleic acid may include, but
is not limited to, a small interfering ribonucleic acid (siRNA), a
short hairpin ribonucleic acid (shRNA), other ribonucleic acid
capable of RNA interference (RNAi), an antisense ribonucleic acid,
or a ribozyme. A translatable target nucleotide sequence (e.g., a
target ribonucleotide sequence) sometimes encodes a peptide,
polypeptide or protein, which are sometimes referred to herein as
"target peptides," "target polypeptides" or "target proteins".
[0254] Any peptides, polypeptides or proteins, or an activity
catalyzed by one of more peptides, polypeptides or proteins may be
encoded by a target nucleotide sequence and may be selected by a
user. Representative proteins include enzymes (e.g., acetyl-CoA
carboxylase, acyl-CoA oxidase, thioesterase, monooxygenase,
monooxygenase reductase, fatty alcohol oxidase, acyltransferase and
the like, for example), antibodies, serum proteins (e.g., albumin),
membrane bound proteins, hormones (e.g., growth hormone,
erythropoietin, insulin, etc.), cytokines, etc., and include both
naturally occurring and exogenously expressed polypeptides.
Representative activities (e.g., enzymes or combinations of enzymes
which are functionally associated to provide an activity) include
thioesterase activity, monooxygenase activity, monooxygenase
reductase activity, acyltransferase activity, omega hydroxyl fatty
acid dehydrogenase activity, beta-oxidation activity,
omega-oxidation activity and the like, for example. The term
"enzyme" as used herein refers to a protein which can act as a
catalyst to induce a chemical change in other compounds, thereby
producing one or more products from one or more substrates.
[0255] Specific polypeptides (e.g., enzymes) useful for embodiments
described herein are listed herein. The term "protein" as used
herein refers to a molecule having a sequence of amino acids linked
by peptide bonds. This term includes fusion proteins,
oligopeptides, peptides, cyclic peptides, polypeptides and
polypeptide derivatives, whether native or recombinant, and also
includes fragments, derivatives, homologs, and variants thereof. A
protein or polypeptide sometimes is of intracellular origin (e.g.,
located in the nucleus, cytosol, or interstitial space of host
cells in vivo) and sometimes is a cell membrane protein in vivo. In
some embodiments (described above, and in further detail hereafter
in Engineering and Alteration Methods), a genetic modification can
result in a modification (e.g., increase, substantially increase,
decrease or substantially decrease) of a target activity.
[0256] A translatable nucleotide sequence generally is located
between a start codon (AUG in ribonucleic acids and ATG in
deoxyribonucleic acids) and a stop codon (e.g., UAA (ochre), UAG
(amber) or UGA (opal) in ribonucleic acids and TAA, TAG or TGA in
deoxyribonucleic acids), and sometimes is referred to herein as an
"open reading frame" (ORF). A translatable nucleotide sequence
(e.g., ORF) sometimes is encoded differently in one organism (e.g.,
most organisms encode CTG as leucine) than in another organism
(e.g., C. tropicalis encodes CTG as serine). In some embodiments, a
translatable nucleotide sequence is altered to correct alternate
genetic code (e.g., codon usage) differences between a nucleotide
donor organism and an nucleotide recipient organism (e.g.,
engineered organism). In certain embodiments, a translatable
nucleotide sequence is altered to improve; (i) codon usage, (ii)
transcriptional efficiency, (iii) transitional efficiency, (iv) the
like, and combinations thereof.
[0257] Nucleic Acid Reagents & Tools
[0258] A nucleic acid reagent sometimes comprises one or more ORFs.
An ORF may be from any suitable source, sometimes from genomic DNA,
mRNA, reverse transcribed RNA or complementary DNA (cDNA) or a
nucleic acid library comprising one or more of the foregoing, and
is from any organism species that contains a nucleic acid sequence
of interest, protein of interest, or activity of interest.
Non-limiting examples of organisms from which an ORF can be
obtained include bacteria, yeast, fungi, human, insect, nematode,
bovine, equine, canine, feline, rat or mouse, for example.
[0259] A nucleic acid reagent sometimes comprises a nucleotide
sequence adjacent to an ORF that is translated in conjunction with
the ORF and encodes an amino acid tag. The tag-encoding nucleotide
sequence is located 3' and/or 5' of an ORF in the nucleic acid
reagent, thereby encoding a tag at the C-terminus or N-terminus of
the protein or peptide encoded by the ORF. Any tag that does not
abrogate in vitro transcription and/or translation may be utilized
and may be appropriately selected by the artisan. Tags may
facilitate isolation and/or purification of the desired ORF product
from culture or fermentation media.
[0260] A tag sometimes specifically binds a molecule or moiety of a
solid phase or a detectable label, for example, thereby having
utility for isolating, purifying and/or detecting a protein or
peptide encoded by the ORF. In some embodiments, a tag comprises
one or more of the following elements: FLAG (e.g., DYKDDDDKG); V5
(e.g., GKPIPNPLLGLDST), c-MYC (e.g., EQKLISEEDL), HSV (e.g.,
QPELAPEDPED), influenza hemaglutinin, HA (e.g., YPYDVPDYA), VSV-G
(e.g., YTDIEMNRLGK), bacterial glutathione-S-transferase, maltose
binding protein, a streptavidin- or avidin-binding tag (e.g.,
pcDNA.TM.6 BioEase.TM. Gateway.RTM. Biotinylation System
(Invitrogen)), thioredoxin, .beta.-galactosidase, VSV-glycoprotein,
a fluorescent protein (e.g., green fluorescent protein or one of
its many color variants (e.g., yellow, red, blue)), a polylysine or
polyarginine sequence, a polyhistidine sequence (e.g., His6) or
other sequence that chelates a metal (e.g., cobalt, zinc, copper),
and/or a cysteine-rich sequence that binds to an arsenic-containing
molecule. In certain embodiments, a cysteine-rich tag comprises the
amino acid sequence CC-Xn-CC, wherein X is any amino acid and n is
1 to 3, and the cysteine-rich sequence sometimes is CCPGCC. In
certain embodiments, the tag comprises a cysteine-rich element and
a polyhistidine element (e.g., CCPGCC and His6).
[0261] A tag often conveniently binds to a binding partner. For
example, some tags bind to an antibody (e.g., FLAG) and sometimes
specifically bind to a small molecule. For example, a polyhistidine
tag specifically chelates a bivalent metal, such as copper, zinc
and cobalt; a polylysine or polyarginine tag specifically binds to
a zinc finger; a glutathione S-transferase tag binds to
glutathione; and a cysteine-rich tag specifically binds to an
arsenic-containing molecule. Arsenic-containing molecules include
LUMIO.TM. agents (Invitrogen, California), such as FlAsH.TM.
(EDT2[4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethan-
edithio)2]) and RcAsH reagents (e.g., U.S. Pat. No. 5,932,474 to
Tsien et al., entitled "Target Sequences for Synthetic Molecules;"
U.S. Pat. No. 6,054,271 to Tsien et al., entitled "Methods of Using
Synthetic Molecules and Target Sequences;" U.S. Pat. Nos. 6,451,569
and 6,008,378; published U.S. Patent Application 2003/0083373, and
published PCT Patent Application WO 99/21013, all to Tsien et al.
and all entitled "Synthetic Molecules that Specifically React with
Target Sequences"). Such antibodies and small molecules sometimes
are linked to a solid phase for convenient isolation of the target
protein or target peptide.
[0262] A tag sometimes comprises a sequence that localizes a
translated protein or peptide to a component in a system, which is
referred to as a "signal sequence" or "localization signal
sequence" herein. A signal sequence often is incorporated at the
N-terminus of a target protein or target peptide, and sometimes is
incorporated at the C-terminus. Examples of signal sequences are
known to the artisan, are readily incorporated into a nucleic acid
reagent, and often are selected according to the organism in which
expression of the nucleic acid reagent is performed. A signal
sequence in some embodiments localizes a translated protein or
peptide to a cell membrane. Examples of signal sequences include,
but are not limited to, a nucleus targeting signal (e.g., steroid
receptor sequence and N-terminal sequence of SV40 virus large T
antigen); mitochondrial targeting signal (e.g., amino acid sequence
that forms an amphipathic helix); peroxisome targeting signal
(e.g., C-terminal sequence in YFG from S. cerevisiae); and a
secretion signal (e.g., N-terminal sequences from invertase, mating
factor alpha, PHO5 and SUC2 in S. cerevisiae; multiple N-terminal
sequences of B. subtilis proteins (e.g., Tjalsma et al., Microbiol.
Molec. Biol. Rev. 64: 515-547 (2000)); alpha amylase signal
sequence (e.g., U.S. Pat. No. 6,288,302); pectate lyase signal
sequence (e.g., U.S. Pat. No. 5,846,818); precollagen signal
sequence (e.g., U.S. Pat. No. 5,712,114); OmpA signal sequence
(e.g., U.S. Pat. No. No. 5,470,719); lam beta signal sequence
(e.g., U.S. Pat. No. 5,389,529); B. brevis signal sequence (e.g.,
U.S. Pat. No. 5,232,841); and P. pastoris signal sequence (e.g.,
U.S. Pat. No. 5,268,273)).
[0263] A tag sometimes is directly adjacent to the amino acid
sequence encoded by an ORF (i.e., there is no intervening sequence)
and sometimes a tag is substantially adjacent to an ORF encoded
amino acid sequence (e.g., an intervening sequence is present). An
intervening sequence sometimes includes a recognition site for a
protease, which is useful for cleaving a tag from a target protein
or peptide. In some embodiments, the intervening sequence is
cleaved by Factor Xa (e.g., recognition site I (E/D)GR), thrombin
(e.g., recognition site LVPRGS), enterokinase (e.g., recognition
site DDDDK), TEV protease (e.g., recognition site ENLYFQG) or
PreScission.TM. protease (e.g., recognition, site LEVLFQGP), for
example.
[0264] An intervening sequence sometimes is referred to herein as a
"linker sequence," and may be of any suitable length selected by
the artisan. A linker sequence sometimes is about 1 to about 20
amino acids in length, and sometimes about 5 to about 10 amino
acids in length. The artisan may select the linker length to
substantially preserve target protein or peptide function (e.g., a
tag may reduce target protein or peptide function unless separated
by a linker), to enhance disassociation of a tag from a target
protein or peptide when a protease cleavage site is present (e.g.,
cleavage may be enhanced when a linker is present), and to enhance
interaction of a tag/target protein product with a solid phase. A
linker can be of any suitable amino acid content, and often
comprises a higher proportion of amino acids having relatively
short side chains (e.g., glycine, alanine, serine and
threonine).
[0265] A nucleic acid reagent sometimes includes a stop codon
between a tag element and an insertion element or ORF, which can be
useful for translating an ORF with or without the tag. Mutant tRNA
molecules that recognize stop codons (described above) suppress
translation termination and thereby are designated "suppressor
tRNAs." Suppressor tRNAs can result in the insertion of amino acids
and continuation of translation past stop codons (e.g., U.S. Patent
Application No. 60/587,583, filed Jul. 14, 2004, entitled
"Production of Fusion Proteins by Cell-Free Protein Synthesis,";
Eggertsson, et al., (1988) Microbiological Review 52(3):354-374,
and Engleerg-Kukla, et al. (1996) in Escherichia coli and
Salmonella Cellular and Molecular Biology, Chapter 60, pps 909-921,
Neidhardt, et al. eds., ASM Press, Washington, D.C.). A number of
suppressor tRNAs are known, including but not limited to, supE,
supP, supD, supF and supZ suppressors, which suppress the
termination of translation of the-amber stop codon; supB; glT, sup
L, supN, supC and supM suppressors, which suppress the function of
the ochre stop codon and glyT, trpT and Su-9 suppressors, which
suppress the function of the opal stop codon. In general,
suppressor tRNAs contain one or more mutations in the anti-codon
loop of the tRNA that allows the tRNA to base pair with a codon
that ordinarily functions as a stop codon. The mutant tRNA is
charged with its cognate amino acid residue and the cognate amino
acid residue is inserted into the translating polypeptide when the
stop codon is encountered. Mutations that enhance the efficiency of
termination suppressors (i.e., increase stop codon read-through)
have been identified. These include, but are not limited to,
mutations in the uar gene (also known as the prfA gene), mutations
in the ups gene, mutations in the sucA, sucB and sucC genes,
mutations in the rpsD (ramA) and rpsE (spcA) genes and mutations in
rplL gene.
[0266] Thus, a nucleic acid reagent comprising a stop codon located
between an ORF and a tag can yield a translated ORF alone when no
suppressor tRNA is present in the translation system, and can yield
a translated ORF-tag fusion when a suppressor tRNA is present in
the system. Suppressor tRNA can be generated in cells transfected
with a nucleic acid encoding the tRNA (e.g., a replication
incompetent adenovirus containing the human tRNA-Ser suppressor
gene can be transfected into cells, or a YAC containing a yeast or
bacterial tRNA suppressor gene can be transfected into yeast cells,
for example). Vectors for synthesizing suppressor tRNA and for
translating ORFs with or without a tag are available to the artisan
(e.g., Tag-On-Demand.TM. kit (Invitrogen Corporation, California);
Tag-On-Demand.TM. Suppressor Supernatant Instruction Manual,
Version B, 6 Jun. 2003, at http address
www.invitrogen.com/content/sfs/manuals/tagondemand_supernatant_man.pdf;
Tag-On-Demand.TM. Gateway.RTM. Vector Instruction Manual, Version
B, 20 Jun. 2003 at http address
www.invitrogen.com/content/sfs/manuals/tagondemand_vectors_man.pdf;
and Capone et al., Amber, ochre and opal suppressor tRNA genes
derived from a human serine tRNA gene. EMBO J, 4:213, 1985).
[0267] Any convenient cloning strategy known in the art may be
utilized to incorporate an element such as an ORF, into a nucleic
acid reagent. Known methods can be utilized to insert an element
into the template independent of an insertion element, such as (1)
cleaving the template, at one or more existing restriction enzyme
sites and ligating an element of interest and (2) adding
restriction enzyme sites to the template by hybridizing
oligonucleotide primers that include one or more suitable
restriction enzyme sites and amplifying by polymerase chain
reaction (described in greater detail herein). Other cloning
strategies take advantage of one or more insertion sites present or
inserted into the nucleic acid reagent, such as an oligonucleotide
primer hybridization site for PCR, for example, and others
described herein. In some embodiments, a cloning strategy can be
combined with genetic manipulation such as recombination (e.g.,
recombination of a nucleic acid reagent with a nucleic acid
sequence of interest into the genome of the organism to be
modified, as described further herein). In some embodiments, the
cloned ORF(s) can produce (directly or indirectly) a fatty
dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid), by engineering a
microorganism with one or more ORFs of interest, which
microorganism comprises one or more altered activities selected
from the group consisting of omega-hydroxyl fatty acid
dehydrogenase activity, acyl-CoA oxidase activity, acyl transferase
activity, thioesterase activity, monooxygenase activity and
monooxygenase reductase activity.
[0268] In some embodiments, the nucleic acid reagent includes one
or more recombinase insertion sites. A recombinase insertion site
is a recognition sequence on a nucleic acid molecule that
participates in an integration/recombination reaction by
recombination proteins. For example, the recombination site for Cre
recombinase loxP, which is a 34 base pair sequence comprised of two
13 base pair inverted repeats (serving as the recombinase binding
sites) flanking an 8 base pair core sequence (e.g., Figure 1 of
Sauer, B., Curr. Opin. Biotech. 5:521-527 (1994)), Other examples
of recombination sites include attB, attP, attL, and attR
sequences, and mutants, fragments, variants and derivatives
thereof, which are recognized by the recombination protein
.lamda.Int and by the auxiliary proteins integration host factor
(IHF), FIS and excisionase (Xis) (e.g., U.S. Pat. Nos. 5,888,732;
6,143,557; 6,171,861; 6,270,969; 6,277,608; and 6,720,140; U.S.
patent application Ser. No. 09/517,466, filed Mar. 2, 2000, and
Ser. No. 09/732,914, filed Aug. 14, 2003, and in U.S. patent
publication no. 2002-0007051-A1; Landy, Curr. Opin. Biotech.
3:699-707 (1993)).
[0269] Examples of recombinase cloning nucleic acids are in
Gateway.RTM. systems (Invitrogen, California), which include at
least one recombination site for cloning a desired nucleic acid
molecules in vivo or in vitro. In some embodiments, the system
utilizes vectors that contain at least two different site-specific
recombination sites, often based on the bacteriophage lambda system
(e.g., att1 and att2), and are mutated from the wild-type (att0)
sites. Each mutated site has a unique specificity for its cognate
partner att site (i.e., its binding partner recombination site) of
the same type (for example attB1 with attP1, or attL1 with attR1)
and will not cross-react with recombination sites of the other
mutant type or with the wild-type att0 site. Different site
specificities allow directional cloning or linkage of desired
molecules thus providing desired orientation of the cloned
molecules. Nucleic acid fragments flanked by recombination sites
are cloned and subcloned using the Gateway.RTM. system by replacing
a selectable marker (for example, ccdB) flanked by att sites on the
recipient plasmid molecule, sometimes termed the Destination
Vector. Desired clones are then selected by transformation of a
ccdB sensitive host strain and positive selection for a marker on
the recipient molecule. Similar strategies for negative selection
(e.g., use of toxic genes) can be used in other organisms such as
thymidine kinase (TK) in mammals and insects.
[0270] A recombination system useful for engineering yeast is
outlined briefly. The system makes use of the URA3 gene (e.g., for
S. cerevisieae and C. albicans, for example) or URA4 and URA5 genes
(e.g., for S. pombe, for example) and toxicity of the nucleotide
analogue 5-Fluoroorotic acid (5-FOA). The URA3 or URA4 and URA5
genes encode orotine-5'-monophosphate (OMP) dicarboxylase. Yeast
with an active URA3 or URA4 and URA5 gene (phenotypically Ura+)
convert 5-FOA to fluorodeoxyuridine, which is toxic to yeast cells.
Yeast carrying a mutation in the appropriate gene(s) or having a
knock out of the appropriate gene(s) can grow in the presence of
5-FOA, if the media is also supplemented with uracil.
[0271] A nucleic acid engineering construct can be made which may
comprise the URA3 gene or cassette (for S. cerevisieae), flanked on
either side by the same nucleotide sequence in the same
orientation. The URA3 cassette comprises a promoter, the URA3 gene
and a functional transcription terminator. Target sequences which
direct the construct to a particular nucleic acid region of
interest in the organism to be engineered are added such that the
target sequences are adjacent to and abut the flanking sequences on
either side of the URA3 cassette. Yeast can be transformed with the
engineering construct and plated on minimal media without uracil.
Colonies can be screened by PCR to determine those transformants
that have the engineering construct inserted in the proper location
in the genome. Checking insertion location prior to selecting for
recombination of the ura3 cassette may reduce the number of
incorrect clones carried through to later stages of the procedure.
Correctly inserted transformants can then be replica plated on
minimal media containing 5-FOA to select for recombination of the
URA3 cassette out of the construct, leaving a disrupted gene and an
identifiable footprint (e.g., nucleic acid sequence) that can be
use to verify the presence of the disrupted gene. The technique
described is useful for disrupting or "knocking out" gene function,
but also can be used to insert genes or constructs into a host
organisms genome in a targeted, sequence specific manner.
[0272] In certain embodiments, a nucleic acid reagent includes one
or more topoisomerase insertion sites. A topoisomerase insertion
site is a defined nucleotide sequence recognized and bound by a
site-specific topoisomerase. For example, the nucleotide sequence
5'-(C/T)CCTT-3' is a topoisomerase recognition site bound
specifically by most pox virus topoisomerases, including vaccinia
virus DNA topoisomerase I. After binding to the recognition
sequence, the topoisomerase cleaves the strand at the 3'-most
thymidine of the recognition site to produce a nucleotide sequence
comprising 5'-(C/T)CCTT-PO4-TOPO, a complex of the topoisomerase
covalently bound to the 3' phosphate via a tyrosine in the
topoisomerase (e.g., Shuman, J. Biol. Chem. 266:11372-11379, 1991;
Sekiguchi and Shuman, Nucl. Acids Res. 22:5360-5365, 1994; U.S.
Pat. No. 5,766,891; PCT/US95/16099; and PCT/US98/12372). In
comparison, the nucleotide sequence 5'-GCAACTT-3' is a
topoisomerase recognition site for type IA E. coli topoisomerase
III. An element to be inserted often is combined with topo
isomerase-reacted template and thereby incorporated into the
nucleic acid reagent (e.g., World-Wide Web URL
invitrogen.com/downloads/F-13512_Topo_Flyer.pdf; World Wide Web URL
invitrogen.com/content/sfs/brochures/710_021849%20_B_TOPOCloning_bro.pdf;
TOPO TA Cloning.RTM. Kit and Zero Blunt.RTM. TOPO.RTM. Cloning Kit
product information).
[0273] A nucleic acid reagent sometimes contains one or more origin
of replication (ORI) elements. In some embodiments, a template
comprises two or more ORIs, where one functions efficiently in one
organism (e.g., a bacterium) and another functions efficiently in
another organism (e.g., a eukaryote, like yeast for example). In
some embodiments, an ORI may function efficiently in one species
(e.g., S. cerevisieae, for example) and another ORF may function
efficiently in a different species (e.g., S. pombe, for example). A
nucleic acid reagent also sometimes includes one or more
transcription regulation sites.
[0274] A nucleic acid reagent can include one or more selection
elements (e.g., elements for selection of the presence of the
nucleic acid reagent, and not for activation of a promoter element
which can be selectively regulated). Selection elements often are
utilized using known processes to determine whether a nucleic acid
reagent is included in a cell. In some embodiments, a nucleic acid
reagent includes two or more selection elements, where one
functions efficiently in one organism and another functions
efficiently in another organism. Examples of selection elements
include, but are not limited to, (1) nucleic acid segments that
encode products that provide resistance against otherwise toxic
compounds (e.g., antibiotics); (2) nucleic acid segments that
encode products that are otherwise lacking in the recipient cell
(e.g., essential products, tRNA genes, auxotrophic markers); (3)
nucleic acid segments that encode products that suppress the
activity of a gene product; (4) nucleic acid segments that encode
products that can be readily identified (e.g., phenotypic markers
such as antibiotics (e.g., .beta.-lactamase), .beta.-galactosidase,
green fluorescent protein (GFP), yellow fluorescent protein (YFP),
red fluorescent protein (RFP), cyan fluorescent protein (CFP), and
cell surface proteins); (5) nucleic acid segments that bind
products that are otherwise detrimental to-cell survival and/or
function; (6) nucleic acid segments that otherwise inhibit the
activity of any of the nucleic acid segments described in Nos. 1-5
above (e.g., antisense oligonucleotides); (7) nucleic acid segments
that bind products that modify a substrate (e.g., restriction,
endonucleases); (8) nucleic acid segments that can be used to
isolate or identify a desired molecule (e.g., specific protein
binding sites); (9) nucleic acid segments that encode a specific
nucleotide sequence that can be otherwise non-functional (e.g., for
PCR amplification of subpopulations of molecules); (10) nucleic
acid segments that, when absent, directly or indirectly confer
resistance or sensitivity to particular compounds; (11) nucleic
acid segments that encode products that either are toxic or convert
a relatively non-toxic compound to a toxic compound (e.g., Herpes
simplex thymidine kinase, cytosine deaminase) in recipient cells;
(12) nucleic acid segments that inhibit replication, partition or
heritability of nucleic acid molecules that contain them; and/or
(13) nucleic acid segments that encode conditional replication
functions, e.g., replication in certain hosts or host cell strains
or under certain environmental conditions (e.g., temperature,
nutritional conditions, and the like).
[0275] A nucleic acid reagent is of any form useful for in vivo
transcription and/or translation. A nucleic acid sometimes is a
plasmid, such as a supercoiled plasmid, sometimes is a yeast
artificial chromosome (e.g., YAC), sometimes is a linear nucleic
acid (e.g., a linear nucleic acid produced by PCR or by restriction
digest), sometimes is single-stranded and sometimes is
double-stranded. A nucleic acid reagent sometimes is prepared by an
amplification process, such as a polymerase chain reaction (PCR)
process or transcription-mediated amplification process (TMA). In
TMA, two enzymes are used in an isothermal reaction to produce
amplification products detected by light emission (see, e.g.,
Biochemistry 1996 Jun. 25; 35(25):8429-38 and http address
www.devicelink.com/ivdt/archive/00/11/007.html). Standard PCR
processes are known (e.g., U.S. Pat. Nos. 4,683,202; 4,683,195;
4,965,188; and 5,656,493), and generally are performed in cycles.
Each cycle includes heat denaturation, in which hybrid nucleic
acids dissociate; cooling, in which primer oligonucleotides
hybridize; and extension of the oligonucleotides by a polymerase
(i.e., Taq polymerase). An example of a PCR cyclical process is
treating the sample at 95.degree. C. for 5 minutes; repeating
forty-five cycles of 95.degree. C. for 1 minute, 59.degree. C. for
1 minute, 10 seconds, and 72.degree. C. for minute 30 seconds; and
then treating the sample at 72.degree. C. for 5 minutes. Multiple
cycles frequently are performed using a commercially available
thermal cycler. PCR amplification products sometimes are stored for
a time at a lower temperature (e.g., at 4.degree. C.) and sometimes
are frozen (e.g., at -20.degree. C.) before analysis.
[0276] In some embodiment, a nucleic acid reagent, protein reagent,
protein fragment reagent or other reagent described herein is
isolated or purified. The term "isolated" as used herein refers to
material removed from its original environment (e.g., the natural
environment if it is naturally occurring, or a host cell if
expressed exogenously), and thus is altered "by the hand of man"
from its original environment. The term "purified" as used herein
with reference to molecules does not refer to absolute purity.
Rather, "purified" refers to a substance in a composition that
contains fewer substance species in the same class (e.g., nucleic
acid or protein species) other than the substance of interest in
comparison to the sample from which it originated. "Purified," if a
nucleic acid or protein for example, refers to a substance in a
composition that contains fewer nucleic acid species or protein
species other than the nucleic acid or protein of interest in
comparison to the sample from which it originated. Sometimes, a
protein or nucleic acid is "substantially pure," indicating that
the protein of nucleic acid represents at least 50% of protein or
nucleic acid on a mass basis of the composition. Often, a
substantially pure protein or nucleic acid is at least 75% on a
mass basis of the composition, and sometimes at least 95% on a mass
basis of the composition.
[0277] Engineering and Alteration Methods
[0278] Methods and compositions (e.g., nucleic acid reagents)
described herein can be used to generate engineered microorganisms.
As noted above, the term "engineered microorganism" as used herein
refers to a modified organism that includes one or more activities
distinct from an activity present in a microorganism utilized as a
starting point for modification (e.g., host microorganism or
unmodified organism). Engineered microorganisms typically arise as
a result of a genetic modification, usually introduced or selected
for, by one of skill in the art using readily available techniques.
Non-limiting examples of methods useful for generating an altered
activity include, introducing a heterologous polynucleotide (e.g.,
nucleic acid or gene integration, also referred to as "knock in"),
removing an endogenous polynucleotide, altering the sequence of an
existing endogenous nucleic acid sequence (e.g., site-directed
mutagenesis), disruption of an existing endogenous nucleic acid
sequence (e.g., knock-outs and transposon or insertion element
mediated mutagenesis), selection for an altered activity where the
selection causes a change in a naturally occurring activity that
can be stably inherited (e.g., causes a change in a nucleic acid
sequence in the genome of the organism or in an epigenetic nucleic
acid that is replicated and passed on to daughter cells), PCR-based
mutagenesis, and the like. The term "mutagenesis" as used herein
refers to any modification to a nucleic acid (e.g., nucleic acid
reagent, or host chromosome, for example) that is subsequently used
to generate a product in a host or modified organism. Non-limiting
examples of mutagenesis include deletion, insertion, substitution,
rearrangement, point mutations, suppressor mutations, and the like.
Mutagenesis methods are known in the art and are readily available
to the artisan. Non-limiting examples of mutagenesis methods are
described herein and can also be found in Maniatis, T., E. F.
Fritsch and J. Sambrook (1982) Molecular Cloning: a Laboratory
Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Another non-limiting example of mutagenesis can be conducted using
a Stratagene (San Diego, Calif.) "QuickChange" kit according to the
manufacturer's instructions.
[0279] The term "genetic modification" as used herein refers to any
suitable nucleic acid addition, removal or alteration that
facilitates production of a target fatty dicarboxylic acid product
(e.g., sebacic or dodecanedioic acid) in an engineered
microorganism. Genetic modifications include, without limitation,
insertion of one or more nucleotides in a native nucleic acid of a
host organism in one or more locations, deletion of one or more
nucleotides in a native nucleic acid of a host organism in one or
more locations, modification or substitution of one or more
nucleotides in a native nucleic acid of a host organism in one or
more locations, insertion of a non-native nucleic acid into a host
organism (e.g., insertion of an autonomously replicating vector),
and removal of a non-native nucleic acid in a host organism (e.g.,
removal of a vector).
[0280] The term "heterologous polynucleotide" as used herein refers
to a nucleotide sequence not present in a host microorganism in
some embodiments. In certain embodiments, a heterologous
polynucleotide is present in a different amount (e.g., different
copy number) than in a host microorganism, which can be
accomplished; for example, by introducing more copies of a
particular nucleotide sequence to a host microorganism (e.g., the
particular nucleotide sequence may be in a nucleic acid autonomous
of the host chromosome or may be inserted into a chromosome). A
heterologous polynucleotide is from a different organism in some
embodiments, and in certain embodiments, is from the same type of
organism but from an outside source (e.g., a recombinant
source).
[0281] In some embodiments, an organism engineered using the
methods and nucleic acid reagents described herein can produce a
fatty dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid). In certain embodiments,
an engineered microorganism described herein that produces a fatty
dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid) may comprise one or more
altered activities selected from the group consisting of omega oxo
fatty acid dehydrogenase activity, omega hydroxyl fatty acid
dehydrogenase activity, fatty acid synthase activity, acetyl CoA
carboxylase activity, acyl-CoA oxidase activity, monooxygenase
activity and monooxygenase reductase activity. In some embodiments,
an engineered microorganism as described herein may comprise a
genetic modification that adds or increases the omega oxo fatty
acid dehydrogenase activity, omega hydroxyl fatty acid
dehydrogenase activity, fatty acid synthase activity, acetyl CoA
carboxylase activity, acyl-CoA oxidase activity, monooxygenase
activity and monooxygenase reductase activity.
[0282] In certain embodiments, an engineered microorganism
described herein can comprise an altered thioesterase activity. In
some embodiments, the engineered microorganism may comprise a
genetic alteration that adds or increases a thioesterase activity.
In some embodiments, the engineered microorganism comprising a
genetic alteration that adds or increases a thioesterase activity,
may further comprise a heterologous polynucleotide encoding a
polypeptide having thioesterase activity.
[0283] The term "altered activity" as used herein refers to an
activity in an engineered microorganism that is added or modified
relative to the host microorganism (e.g., added, increased,
reduced, inhibited or removed activity). An activity can be altered
by introducing a genetic modification to a host microorganism that
yields an engineered microorganism having added, increased,
reduced, inhibited or removed activity.
[0284] An added activity often is an activity not detectable in a
host microorganism. An increased activity generally is an activity
detectable in a host microorganism that has been increased in an
engineered microorganism. An activity can be increased to any
suitable level for production of a target fatty dicarboxylic acid
product (e.g., sebacic or dodecanedioic acid), including but not
limited to less than 2-fold (e.g., about 10% increase to about 99%
increase; about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% increase),
2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, of
10-fold increase, or greater than about 10-fold increase. A reduced
or inhibited activity generally is an activity detectable in a host
microorganism that has been reduced or inhibited in an engineered
microorganism. An activity can be reduced to undetectable levels in
some embodiments, or detectable levels in certain embodiments. An
activity can be decreased to any suitable level for production of a
target fatty dicarboxylic acid product (e.g., sebacic or
dodecanedioic acid), including but not limited to less than 2-fold
(e.g., about 10% decrease to about 99% decrease; about 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90% decrease), 2-fold, 3-fold, 4-fold,
5-fold, 6-fold, 7-fold, 8-fold, 9-fold, of 10-fold decrease, or
greater than about 10-fold decrease.
[0285] An altered activity sometimes is an activity not detectable
in a host organism and is added to an engineered organism. An
altered activity also may be an activity detectable in a host
organism and is increased in an engineered organism. An activity
may be added or increased by increasing the number of copies of a
polynucleotide that encodes a polypeptide having a target activity,
in some embodiments. In some embodiments, the activity of a native
polypeptide can be increased by increasing in the modified organism
the number copies of a polynucleotide that encodes the polypeptide
(e.g., introducing 1 to about 100 additional copies of the
polynucleotide (e.g., introducing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30 or more
additional copies of the polynucleotide). In certain embodiments an
activity can be added or increased by inserting into a host
microorganism a polynucleotide that encodes a heterologous
polypeptide having the added activity or encodes a modified
endogenous polypeptide. In such embodiments, 1 to about 100 copies
of the polynucleotide can be introduced (e.g., introducing 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22,
24, 26, 28, 30 copies). A "modified endogenous polypeptide" often
has an activity different than an activity of a native polypeptide
counterpart (e.g., different catalytic activity and/or different
substrate specificity), and often is active (e.g., an activity
(e.g., substrate turnover) is detectable). In certain embodiments,
an activity can be added or increased by inserting into a host
microorganism a heterologous polynucleotide that is (i) operably
linked to another polynucleotide that encodes a polypeptide having
the added activity, and (ii) up regulates production of the
polynucleotide. Thus, an activity can be added or increased by
inserting or modifying a regulatory polynucleotide operably linked
to another polynucleotide that encodes a polypeptide having the
target activity. In certain embodiments, an activity can be added
or increased by subjecting a host microorganism to a selective
environment and screening for microorganisms that have a detectable
level of the target activity. Examples of a selective environment
include, without limitation, a medium containing a substrate that a
host organism can process and a medium lacking a substrate that a
host organism can process.
[0286] An altered activity sometimes is an activity detectable in a
host organism and is reduced, inhibited or removed (i.e., not
detectable) in an engineered organism. An activity may be reduced
or removed by decreasing the number of copies of a polynucleotide
that encodes a polypeptide having a target activity, in some
embodiments. In some embodiments, an activity can be reduced or
removed by (i) inserting a polynucleotide within a polynucleotide
that encodes a polypeptide having the target activity (disruptive
insertion), and/or (ii) removing a portion of or all of a
polynucleotide that encodes a polypeptide having the target
activity (deletion or knockout, respectively). In certain
embodiments, an activity can be reduced or removed by inserting
into a host microorganism a heterologous polynucleotide that is (i)
operably linked to another polynucleotide that encodes a
polypeptide having the target activity, and (ii) down regulates
production of the polynucleotide. Thus, an activity can be reduced
or removed by inserting or modifying a regulatory polynucleotide
operably linked to another polynucleotide that encodes a
polypeptide having the target activity.
[0287] An activity also can be reduced or removed by (i) inhibiting
a polynucleotide that encodes a polypeptide having the activity or
(ii) inhibiting a polynucleotide operably linked to another
polynucleotide that encodes a polypeptide having the activity. A
polynucleotide can be inhibited by a suitable technique known in
the art, such as by contacting an RNA encoded by the polynucleotide
with a specific inhibitory RNA (e.g., RNAi, siRNA, ribozyme). An
activity also can be reduced or removed by contacting a polypeptide
having the activity with a molecule that specifically inhibits the
activity (e.g., enzyme inhibitor, antibody). In certain
embodiments, an activity can be reduced or removed by subjecting a
host microorganism to a selective environment and screening for
microorganisms that have a reduced level or removal of the target
activity.
[0288] In some embodiments, an untranslated ribonucleic acid, or a
cDNA can be used to reduce the expression of a particular activity
or enzyme. For example, a microorganism can be engineered by
genetic modification to express a nucleic acid reagent that reduces
the expression of an activity by producing an RNA molecule that is
partially or substantially homologous to a nucleic acid sequence of
interest which encodes the activity of interest. The RNA molecule
can bind to the nucleic acid sequence of interest and inhibit the
nucleic acid sequence from performing its natural function, in
certain embodiments. In some embodiments, the RNA may alter the
nucleic acid sequence of interest which encodes the activity of
interest in a manner that the nucleic acid sequence of interest is
no longer capable of performing its natural function (e.g., the
action of a ribozyme for example).
[0289] In certain embodiments, nucleotide sequences sometimes are
added to, modified or removed from one or more of the nucleic acid
reagent elements, such as the promoter, 5'UTR, target sequence, or
3'UTR elements, to enhance, potentially enhance, reduce, or
potentially reduce transcription and/or translation before or after
such elements are incorporated in a nucleic acid reagent. In some
embodiments, one or more of the following sequences may be modified
or removed if they are present in a 5'UTR: a sequence that forms a
stable secondary structure (e.g., quadruplex structure or stem loop
stem structure (e.g., EMBL sequences X12949, AF274954, AF139980,
AF152961, S95936, U194144, AF116649 or substantially identical
sequences that form such stem loop stem structures)); a translation
initiation codon upstream of the target nucleotide sequence start
codon; a stop codon upstream of the target nucleotide sequence
translation initiation codon; an ORF upstream of the target
nucleotide sequence translation initiation codon; an iron
responsive element (IRE) or like sequence; and a 5' terminal
oligopyrimidine tract (TOP, e.g., consisting of 5-15 pyrimidines
adjacent to the cap). A translational enhancer sequence and/or an
internal ribosome entry site (IRES) sometimes is inserted into a
5'UTR (e.g., EMBL, nucleotide sequences J04513, X87949, M95825,
M12783, AF025841, AF013263, AF006822, M17169, M13440, M22427,
D14838 and M17446 and substantially identical nucleotide
sequences).
[0290] An AU-rich element (ARE, e.g., AUUUA repeats) and/or
splicing junction that follows a non-sense codon sometimes is
removed from or modified in a 3'UTR. A polyadenosine tail sometimes
is inserted into a 3'UTR if none is present, sometimes is removed
if it is present, and adenosine moieties sometimes are added to or
removed from a polyadenosine tail present in a 3'UTR. Thus, some
embodiments are directed to a process comprising: determining
whether any nucleotide sequences that increase, potentially
increase, reduce or potential reduce translation efficiency are
present in the elements, and adding, removing or modifying one or
more of such sequences if they are identified. Certain embodiments
are directed to a process comprising: determining whether any
nucleotide sequences that increase or potentially increase
translation efficiency are not present in the elements, and
incorporating such sequences into the nucleic acid reagent.
[0291] In some embodiments, an activity can be altered by modifying
the nucleotide sequence of an ORF. An ORF sometimes is mutated or
modified (for example, by point mutation, deletion mutation,
insertion mutation, PCR based mutagenesis and the like) to alter,
enhance or increase, reduce, substantially reduce or eliminate the
activity of the encoded protein or peptide. The protein or peptide
encoded, by a modified ORF sometimes is produced in a lower amount
or may not be produced at detectable levels, and in other
embodiments, the product or protein encoded by the modified ORF is
produced at a higher level (e.g., codons sometimes are modified so
they are compatible with tRNA's preferentially used in the host
organism or engineered organism). To determine the relative
activity, the activity from the product of the mutated ORF (or cell
containing it) can be compared to the activity of the product or
protein encoded by the unmodified ORF (or cell containing it).
[0292] In some embodiments, an ORF nucleotide sequence sometimes is
mutated or modified to alter the triplet nucleotide sequences used
to encode amino acids (e.g., amino acid codon triplets, for
example). Modification of the nucleotide sequence of an ORF to
alter codon triplets sometimes is used to change the codon found in
the original sequence to better match the preferred codon usage of
the organism in which the ORF or nucleic acid reagent will be
expressed. The codon usage and therefore the codon triplets encoded
by a nucleic acid sequence, in bacteria may be different from the
preferred codon usage in eukaryotes, like yeast or plants for
example. Preferred codon usage also may be different between
bacterial species. In certain embodiments an ORF nucleotide
sequences sometimes is modified to eliminate codon pairs and/or
eliminate mRNA secondary structures that can cause pauses during
translation of the mRNA encoded by the ORF nucleotide sequence.
Translational pausing sometimes occurs when nucleic acid secondary
structures exist in an mRNA, and sometimes occurs due to the
presence of codon pairs that slow the rate of translation by
causing ribosomes to pause. In some embodiments, the use of lower
abundance codon triplets can reduce translational pausing due to a
decrease in the pause time needed to load a charged tRNA into the
ribosome translation machinery. Therefore, to increase
transcriptional and translational efficiency in bacteria (e.g.,
where transcription and translation are concurrent, for example) or
to increase translational efficiency in eukaryotes (e.g., where
transcription and translation are functionally separated), the
nucleotide sequence of a nucleotide sequence of interest can be
altered to better suit the transcription and/or translational
machinery of the host and/or genetically modified microorganism. In
certain embodiments, slowing the rate of translation by the use of
lower abundance codons, which slow or pause the ribosome, can lead
to higher yields of the desired product due to an increase in
correctly folded proteins and a reduction in the formation of
inclusion bodies.
[0293] Codons can be altered and optimized according to the
preferred usage by a given organism by determining the codon
distribution of the nucleotide sequence donor organism and
comparing the distribution of codons to the distribution of codons
in the recipient or host organism. Techniques described herein
(e.g., site directed mutagenesis and the like) can then be used to
alter the codons accordingly. Comparisons of codon usage can be
done by hand or using nucleic acid analysis software commercially
available to the artisan.
[0294] Modification of the nucleotide sequence of an ORF also can
be used to correct codon triplet sequences that have diverged in
different organisms. For example, certain yeast (e.g., C.
tropicalis and C. maltose) use the amino acid triplet CUG (e.g.,
CTG in the DNA sequence) to encode serine. CUG typically encodes
leucine in most organisms. In order to maintain the correct amino
acid in the resultant polypeptide or protein, the CUG codon must be
altered to reflect the organism in which the nucleic acid reagent
will be expressed. Thus, if an ORF from a bacterial donor is to be
expressed in either Candida yeast strain mentioned above, the
heterologous nucleotide sequence must first be altered or modified
to the appropriate leucine codon. Therefore, in some embodiments,
the nucleotide sequence of an ORF sometimes is altered or modified
to correct for differences that have occurred in the evolution of
the amino acid codon triplets between different organisms. In some
embodiments, the nucleotide sequence can be left unchanged at a
particular amino acid codon, if the amino acid encoded is a
conservative or neutral change in amino acid when compared to the
originally encoded amino acid.
[0295] In some embodiments, an activity can be altered by modifying
translational regulation signals, like a stop codon for example. A
stop codon at the end of an ORF sometimes is modified to another
stop codon, such as an amber stop codon described above. In some
embodiments, a stop codon is introduced within an ORF, sometimes by
insertion or mutation of an existing codon. An ORF comprising a
modified terminal stop codon and/or internal stop codon often is
translated in a system comprising a suppressor tRNA that recognizes
the stop codon. An ORF comprising a stop codon sometimes is
translated in a system comprising a suppressor tRNA that
incorporates an unnatural amino acid during translation of the
target protein or target peptide. Methods for incorporating
unnatural amino acids into a target protein or peptide are known,
which include, for example, processes utilizing a heterologous
tRNA/synthetase pair, where the tRNA recognizes an amber stop codon
and is loaded with an unnatural amino acid (e.g., World Wide Web
URL iupac.org/news/prize/2003/wang.pdf).
[0296] Depending on the portion of a nucleic acid reagent (e.g.,
Promoter, 5' or 3'UTR, ORI, ORF, and the like) chosen for
alteration (e.g., by mutagenesis, introduction or deletion, for
example) the modifications described above can alter a given
activity by (i) increasing or decreasing feedback inhibition
mechanisms, (ii) increasing or decreasing promoter initiation,
(iii) increasing or decreasing translation initiation, (iv)
increasing or decreasing translational efficiency, (v) modifying
localization of peptides or products expressed from nucleic acid
reagents described herein, or (vi) increasing or decreasing the
copy number of a nucleotide sequence of interest, (vii) expression
of an anti-sense RNA, RNAi, siRNA, ribozyme and the like. In some
embodiments, alteration of a nucleic acid reagent or nucleotide
sequence can alter a region involved in feedback inhibition (e.g.,
5' UTR; promoter and the like). A modification sometimes is made
that can add or enhance binding of a feedback regulator and
sometimes a modification is made that can reduce, inhibit or
eliminate binding of a feedback regulator.
[0297] In certain embodiments, alteration of a nucleic acid reagent
or nucleotide sequence can alter sequences involved in
transcription initiation (e.g., promoters, 5' UTR, and the like). A
modification sometimes can be made that can enhance or increase
initiation from an endogenous or heterologous promoter element. A
modification sometimes can be made that removes or disrupts
sequences that increase or enhance transcription initiation,
resulting in a decrease or elimination of transcription from an
endogenous or heterologous promoter element.
[0298] In some embodiments, alteration of a nucleic acid reagent or
nucleotide sequence can alter sequences involved in translational
initiation or translational efficiency (e.g., 5' UTR, 3' UTR, codon
triplets of higher or lower abundance, translational terminator
sequences and the like, for example). A modification sometimes can
be made that can increase or decrease translational initiation,
modifying a ribosome binding site for example. A modification
sometimes can be made that can increase or decrease translation
efficiency. Removing or adding sequences that form hairpins and
changing codon triplets to a more or less preferred codon are
non-limiting examples of genetic modifications that can be made to
alter translation initiation and translation efficiency.
[0299] In certain embodiments, alteration of a nucleic acid reagent
or nucleotide sequence can alter sequences involved in localization
of peptides, proteins or other desired products (e.g., a sebacic
acid or dodecanedioic acid, for example). A modification sometimes
can be made that can alter, add or remove sequences responsible for
targeting a polypeptide, protein or product to an intracellular
organelle, the periplasm, cellular membranes, or extracellularly.
Transport of a heterologous product to a different intracellular
space or extracellularly sometimes can reduce or eliminate the
formation of inclusion bodies (e.g., insoluble aggregates of the
desired product).
[0300] In some embodiments, alteration of a nucleic acid reagent or
nucleotide sequence can alter sequences involved in increasing or
decreasing the copy number of a nucleotide sequence of interest. A
modification sometimes can be made that increases or decreases the
number of copies of an ORF stably integrated into the genome of an
organism or on an epigenetic nucleic acid reagent. Non-limiting
examples of alterations that can increase the number of copies of a
sequence of interest include, adding copies of the sequence of
interest by duplication of regions in the genome (e.g., adding
additional copies by recombination or by causing gene amplification
of the host genome, for example), cloning additional copies of a
sequence onto a nucleic acid reagent, or altering an ORI to
increase the number of copies of an epigenetic nucleic acid
reagent. Non-limiting examples of alteration that can decrease the
number of copies of a sequence of interest include, removing copies
of the sequence of interest by deletion or disruption of regions in
the genome, removing additional copies of the sequence from
epigenetic nucleic acid reagents, or altering an ORI to decrease
the number of copies of an epigenetic nucleic acid reagent.
[0301] In certain embodiments, increasing or decreasing the
expression of a nucleotide sequence of interest can also be
accomplished by altering, adding or removing sequences involved in
the expression of an anti-sense RNA, RNAi, siRNA, ribozmye and the
like. The methods described above can be used to modify expression
of anti-sense RNA, RNAi, siRNA, ribozyme and the like.
[0302] The methods and nucleic acid reagents described herein can
be used to generate genetically modified microorganisms with
altered activities in cellular processes involved in a fatty
dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid) synthesis. In some
embodiments, an engineered microorganism described herein may
comprise an increased number of copies of an endogenous
polynucleotide encoding a polypeptide having omega oxo fatty acid
dehydrogenase activity. In certain embodiments, an engineered
microorganism described herein may comprise an increased number of
copies of an endogenous polynucleotide encoding a polypeptide
having omega hydroxyl fatty acid dehydrogenase activity. In some
embodiments, an engineered microorganism described herein may
comprise a heterologous polynucleotide encoding a polypeptide
having omega oxo fatty acid dehydrogenase activity. In some
embodiments, an engineered microorganism described herein may
comprise a heterologous polynucleotide encoding a polypeptide
having omega hydroxyl fatty acid dehydrogenase activity. In some
embodiments, the heterologous polynucleotide can be from a
bacterium. In some embodiments, the bacterium can be Acinetobacter,
Nocardia, Pseudomonas or Xanthobacter bacterium.
[0303] In some embodiments, an engineered microorganism described
herein may comprise a heterologous polynucleotide encoding a
polypeptide having monooxygenase activity. In certain embodiments,
the heterologous polynucleotide can be from a bacterium. In some
embodiments, the bacterium can be a Bacillus bacterium. In certain
embodiments, the Bacillus bacterium is B. megaterium.
[0304] In certain embodiments, an engineered microorganism
described herein may comprise a genetic modification that reduces
omega hydroxyl fatty acid conversion. In some embodiments, the
genetic modification can reduce omega hydroxyl fatty acid,
dehydrogenase activity. In certain embodiments, an engineered
microorganism described herein may comprise a genetic modification
that reduces beta-oxidation activity. In some embodiments, the
genetic modification can reduce a target activity described
herein.
[0305] Engineered microorganisms that produce a fatty dicarboxylic
acid (e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid), as described herein, can comprise an altered
monooxygenase activity, in certain embodiments. In some
embodiments, the engineered microorganism described herein may
comprise a genetic modification that alters the monooxygenase
activity. In certain embodiments, the engineered microorganism
described herein can comprise an increase number of copies of an
endogenous polynucleotide encoding a polypeptide having
monooxygenase activity. In some embodiments, the engineered
microorganism described herein can comprise a heterologous
polynucleotide encoding a polypeptide having monooxygenase
activity. In certain embodiments, the heterologous polynucleotide
can be from a bacterium. In some embodiments, the bacterium can be
a Bacillus bacterium. In certain embodiments, the Bacillus
bacterium is B. megaterium. In some embodiments, the genetic
modification can reduce a polyketide synthase activity.
[0306] Engineered microorganisms that produce a fatty dicarboxylic
acid (e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid), as described herein, can comprise an altered
thioesterase activity, in certain embodiments. In some embodiments,
the engineered microorganism may comprise a genetic modification
that adds or increases the thioesterase activity. In certain
embodiments, the engineered microorganism may comprise a
heterologous polynucleotide encoding a polypeptide having
thioesterase activity.
[0307] In some embodiments, the engineered microorganism with an
altered thioesterase activity may comprise an altered omega oxo
fatty acid dehydrogenase activity. In certain embodiments, the
engineered microorganism with an altered thioesterase activity may
comprise a genetic modification that adds or increases omega oxo
fatty acid dehydrogenase activity. In some embodiments, the
engineered microorganism may comprise a heterologous polynucleotide
encoding a polypeptide having altered omega oxo fatty acid
dehydrogenase activity. In certain embodiments, the heterologous
polynucleotide can be from a bacterium. In some embodiments, the
bacterium can be an Acinetobacter, Nocardia, Pseudomonas or
Xanthobacter bacterium.
[0308] Engineered microorganisms that produce a fatty dicarboxylic
acid (e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid), as described herein, can comprise an altered
omega hydroxyl fatty acid dehydrogenase activity. In certain
embodiments, the engineered microorganism may comprise a genetic
modification that adds or increases the omega hydroxyl fatty acid
dehydrogenase activity. In certain embodiments, the engineered
microorganism may comprise a heterologous polynucleotide encoding a
polypeptide having altered omega hydroxyl fatty acid dehydrogenase
activity. In some embodiments, the heterologous polynucleotide is
from a bacterium. In certain embodiments, the bacterium can be an
Acinetobacter, Nocardia, Pseudomonas or Xanthobacter bacterium. In
some embodiments, the engineered microorganism can be a eukaryote.
In certain embodiments, the eukaryote can be a yeast. In some
embodiments, the eukaryote may be a fungus. In certain embodiments,
the yeast can be a Candida yeast. In some embodiments, the Candida
yeast maybe C. tropicalis. In certain embodiments, the fungus can
be a Yarrowia fungus. In some embodiments the Yarrowia fungus may
be Y. lipolytica. In certain embodiments, the fungus can be an
Aspergillus fungus. In some embodiments, the Aspergillus fungus,
may be A. parasiticus or A. nidulans. In some embodiments, an
engineered microorganism as described above may comprise a genetic
modification that reduces omega hydroxyl fatty acid conversion. In
certain embodiments, the genetic modification can reduce omega
hydroxyl fatty acid dehydrogenase activity. In some embodiments the
genetic may reduce beta-oxidation activity. In certain embodiments,
the genetic modification may reduce a target activity described
herein.
[0309] Engineered microorganisms can be prepared by altering,
introducing or removing nucleotide sequences in the host genome or
in stably maintained epigenetic nucleic acid reagents, as noted
above. The nucleic acid reagents use to alter, introduce or remove
nucleotide sequences in the host genome or epigenetic nucleic acids
can be prepared using the method described herein or available to
the artisan.
[0310] Nucleic acid sequences having a desired activity can be
isolated from cells of a suitable organism using lysis and nucleic
acid purification procedures described in a known reference manual
(e.g., Maniatis, T., E. F. Fritsch and J. Sambrook (1982) Molecular
Cloning: a Laboratory Manual; Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y.) or using commercially available cell lysis and
DNA purification reagents and kits. In some embodiments, nucleic
acids used to engineer microorganisms can be provided for
conducting methods described herein after processing of the
organism containing the nucleic acid. For example, the nucleic acid
of interest may be extracted, isolated, purified or amplified from
a sample (e.g., from an organism of interest or culture containing
a plurality of organisms of interest, like yeast or bacteria for
example). The term "isolated" as used herein refers to nucleic acid
removed from its original environment (e.g., the natural
environment if it is naturally occurring, or a host cell if
expressed exogenously), and thus is altered "by the hand of man"
from its original environment. An isolated nucleic acid generally
is provided with fewer non-nucleic acid components (e.g., protein,
lipid) than the amount of components present in a source sample. A
composition comprising isolated sample nucleic acid can be
substantially isolated (e.g., about 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or greater than 99% free of non-nucleic acid
components). The term "purified" as used herein refers to sample
nucleic acid provided that contains fewer nucleic acid species than
in the sample source from which the sample nucleic acid is derived.
A composition comprising sample nucleic acid may be substantially
purified (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or greater than 99% free of other nucleic acid species). The
term "amplified" as used herein refers to subjecting nucleic acid
of a cell, organism or sample to a process that linearly or
exponentially generates amplicon nucleic acids having the same or
substantially the same nucleotide sequence as the nucleotide
sequence of the nucleic acid in the sample, or portion thereof. As
noted above, the nucleic acids used to prepare nucleic acid
reagents as described herein can be subjected to fragmentation or
cleavage.
[0311] Amplification of nucleic acids is sometimes necessary when
dealing with organisms that are difficult to culture. Where
amplification may be desired, any suitable amplification technique
can be utilized. Non-limiting examples of methods for amplification
of polynucleotides include, polymerase chain reaction (PCR);
ligation amplification (or ligase chain reaction (LCR));
amplification methods based on the use Of Q-beta replicase or
template-dependent polymerase (see US Patent Publication Number
US20050287592); helicase-dependent isothermal amplification
(Vincent et al., "helicase-dependent isothermal DNA amplification".
EMBO reports 5 (8): 795-800 (2004)); strand displacement
amplification (SDA); thermophilic SDA nucleic acid sequence based
amplification (3SR or NASBA) and transcription-associated
amplification (TAA). Non-limiting examples of PCR amplification
methods include standard PCR, AFLP-PCR, Allele-specific PCR,
Alu-PCR, Asymmetric PCR, Colony PCR, Hot start PCR, Inverse PCR
(IPCR), In situ-PCR (ISH), Intersequence-specific PCR (ISSR-PCR),
Long PCR, Multiplex PCR, Nested PCR, Quantitative PCR, Reverse
Transcriptase PCR (RT-PCR), Real Time PCR, Single cell PCR, Solid
phase PCR, combinations thereof, and the like. Reagents and
hardware for conducting PCR are commercially available.
[0312] Protocols for conducting the various type of PCR listed
above are readily available to the artisan. PCR conditions can be
dependent upon primer sequences, target abundance, and the desired
amount of amplification, and therefore, one of skill in the art may
choose from a number of PCR protocols available (see, e.g., U.S.
Pat. Nos. 4,683,195 and 4,683,202; and PCR Protocols: A Guide to
Methods and Applications, Innis et al., eds, 1990. PCR often is
carried out as an automated process with a thermostable enzyme. In
this process, the temperature of the reaction mixture is cycled
through a denaturing region, a primer-annealing region, and an
extension reaction region automatically. Machines specifically
adapted for this purpose are commercially available. A non-limiting
example of a PCR protocol that may be suitable for embodiments
described herein is, treating the sample at 95.degree. C. for 5
minutes; repeating forty-five cycles of 95.degree. C. for 1 minute,
59.degree. C. for 1 minute, 10 seconds, and 72.degree. C. for 1
minute 30 seconds; and then treating the sample at 72.degree. C.
for 5 minutes. Additional PCR protocols are described in the
example section. Multiple cycles frequently are performed using a
commercially available thermal cycler. Suitable isothermal
amplification processes known and selected by the person of
ordinary skill in the art also may be applied, in certain
embodiments. In some embodiments, nucleic acids encoding
polypeptides with a desired activity can be isolated by amplifying
the desired sequence from an organism having the desired activity
using oligonucleotides or primers designed based on sequences
described herein.
[0313] Amplified, isolated and/or purified nucleic acids can be
cloned into the recombinant DNA vectors described in Figures herein
or into suitable commercially available recombinant DNA vectors.
Cloning of nucleic acid sequences of interest into recombinant DNA
vectors can facilitate further manipulations of the nucleic acids
for preparation of nucleic acid reagents, (e.g., alteration of
nucleotide sequences by mutagenesis, homologous recombination,
amplification and the like, for example). Standard cloning
procedures (e.g., enzymic digestion, ligation, and the like) are
known (e.g., described in Maniatis, T., E. F. Fritsch and J.
Sambrook (1982) Molecular Cloning: a Laboratory Manual: Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y.).
[0314] In some embodiments, nucleic acid sequences prepared by
isolation or amplification can be used, without any further
modification, to add an activity to a microorganism and thereby
create a genetically modified or engineered microorganism. In
certain embodiments, nucleic acid sequences prepared by isolation
or amplification can be genetically modified to alter (e.g.,
increase or decrease, for example) a desired activity. In some
embodiments, nucleic acids, used to add an activity to an organism,
sometimes are genetically modified to optimize the heterologous
polynucleotide sequence encoding the desired activity (e.g.,
polypeptide or protein, for example). The term "optimize" as used
herein can refer to alteration to increase or enhance expression by
preferred codon usage. The term optimize can also refer to
modifications to the amino acid sequence to increase the activity
of a polypeptide or protein, such that the activity exhibits a
higher catalytic activity as compared to the "natural" version of
the polypeptide or protein.
[0315] Nucleic acid sequences of interest can be genetically
modified using methods known in the art. Mutagenesis techniques are
particularly useful for small scale (e.g., 1, 2, 5, 10 or more
nucleotides) or large scale (e.g., 50, 100, 150, 200, 500, or more
nucleotides) genetic modification. Mutagenesis allows the artisan
to alter the genetic information of an organism in a stable manner,
either naturally (e.g., isolation using selection and screening) or
experimentally by the use of chemicals, radiation or inaccurate DNA
replication (e.g., PCR mutagenesis). In some embodiments, genetic
modification can be performed by whole scale synthetic synthesis of
nucleic acids, using a native nucleotide sequence as the reference
sequence, and modifying nucleotides that can result in the desired
alteration of activity. Mutagenesis methods sometimes are specific
or targeted to specific regions or nucleotides (e.g., site-directed
mutagenesis, PCR-based site-directed mutagenesis, and in vitro
mutagenesis techniques such as transplacement and in vivo
oligonucleotide site-directed mutagenesis, for example).
Mutagenesis methods sometimes are non-specific or random with
respect to the placement of genetic modifications (e.g., chemical
mutagenesis, insertion element (e.g., insertion or transposon
elements) and inaccurate PCR based methods, for example).
[0316] Site directed mutagenesis is a procedure in which a specific
nucleotide or specific nucleotides in a DNA molecule are mutated or
altered. Site directed mutagenesis typically is performed using a
nucleic acid sequence of interest cloned into a circular plasmid
vector. Site-directed mutagenesis requires that the wild type
sequence be known and used a platform for the genetic alteration.
Site-directed mutagenesis sometimes is referred to as
oligonucleotide-directed mutagenesis because the technique can be
performed using oligonucleotides which have the desired genetic
modification incorporated into the complement a nucleotide sequence
of interest. The wild type sequence and the altered nucleotide are
allowed to hybridize and the hybridized nucleic acids are extended
and replicated using a DNA polymerase. The double stranded nucleic
acids are introduced into a host (e.g., E. coli, for example) and
further rounds of replication are carried out in vivo. The
transformed cells carrying the mutated nucleic acid sequence are
then selected and/or screened for those cells carrying the
correctly mutagenized sequence. Cassette mutagenesis and PCR-based
site-directed mutagenesis are further modifications of the
site-directed mutagenesis technique. Site-directed mutagenesis can
also be performed in vivo (e.g., transplacement "pop-in pop-out",
In vivo site-directed mutagenesis with synthetic oligonucleotides
and the like, for example). PCR-based mutagenesis can be performed
using PCR with oligonucleotide primers that contain the desired
mutation or mutations. The technique functions in a manner similar
to standard site-directed mutagenesis, with the exception that a
thermocycler and PCR conditions are used to replace replication and
selection of the clones in a microorganism host. As PCR-based
mutagenesis also uses a circular plasmid vector, the amplified
fragment (e.g., linear nucleic acid molecule) containing the
incorporated genetic modifications can be separated from the
plasmid containing the template sequence after a sufficient number
of rounds of thermocycler amplification, using standard
electrophorectic procedures. A modification of this method uses
linear amplification methods and a pair of mutagenic primers that
amplify the entire plasmid. The procedure takes advantage of the E.
coli. Dam methylase system which causes DNA replicated in vivo to
be sensitive to the restriction endonucleases DpnI. PCR synthesized
DNA is not methylated and is therefore resistant to DpnI. This
approach allows the template plasmid to be digested, leaving the
genetically modified, PCR synthesized plasmids to be isolated and
transformed into a host bacteria for DNA repair and replication,
thereby facilitating subsequent cloning and identification steps. A
certain amount of randomness can be added to PCR-based sited
directed mutagenesis by using partially degenerate primers.
[0317] Recombination sometimes can be used as a tool for
mutagenesis. Homologous recombination allows the artisan to
specifically target regions of known sequence for insertion of
heterologous nucleotide sequences using the host organisms natural
DNA replication and repair enzymes. Homologous recombination
methods sometimes are referred to as "pop in pop out" mutagenesis,
transplacement, knock out mutagenesis or knock in mutagenesis.
Integration of a nucleic acid sequence into a host genome is a
single cross over event, which inserts the entire nucleic acid
reagent (e.g., pop in). A second cross over event excises all but a
portion of the nucleic acid reagent, leaving behind a heterologous
sequence, often referred to as a "footprint" (e.g., pop out).
Mutagenesis by insertion (e.g., knock in) or by double
recombination leaving behind a disrupting heterologous nucleic acid
(e.g., knock out) both server to disrupt or "knock out" the
function of the gene or nucleic acid sequence in which insertion
occurs. By combining selectable markers and/or auxotrophic markers
with nucleic acid reagents designed to provide the appropriate
nucleic acid target sequences, the artisan can target a selectable
nucleic acid reagent to a specific region, and then select for
recombination events that "pop out" a portion of the inserted
(e.g., "pop in") nucleic acid reagent.
[0318] Such methods take advantage of nucleic acid reagents that
have been specifically designed with known target nucleic acid
sequences at or near a nucleic acid or genomic region of interest.
Popping out typically leaves a "foot print" of left over sequences
that remain after the recombination event. The left over sequence
can disrupt a gene and thereby reduce or eliminate expression of
that gene. In some embodiments, the method can be used to insert
sequences, upstream or downstream of genes that can result in an
enhancement or reduction in expression of the gene. In certain
embodiments, new genes can be introduced into the genome of a host
organism using similar recombination or "pop in" methods. An
example of a yeast recombination system using the ura3 gene and
5-FOA were described briefly above and further detail is presented
herein.
[0319] A method for modification is described in Alani et al., "A
method for gene disruption that allows repeated use of URA3
selection in the construction of multiply disrupted yeast strains",
Genetics 116(4):541-545 August 1987. The original method uses a
Ura3 cassette with 1000 base pairs (bp) of the same nucleotide
sequence cloned in the same orientation on either side of the URA3
cassette. Targeting sequences of about 50 bp are added to each side
of the construct. The double stranded targeting sequences are
complementary to sequences in the genome of the host organism. The
targeting sequences allow site-specific recombination in a region
of interest. The modification of the original technique replaces
the two 1000 bp sequence direct repeats with two 200 bp direct
repeats. The modified method also uses 50 bp targeting sequences.
The modification reduces or eliminates recombination of a second
knock out into the 1000 bp repeat left behind in a first
mutagenesis, therefore allowing multiply knocked out yeast.
Additionally, the 200 bp sequences used herein are uniquely
designed self-assembling sequences that leave behind identifiable
footprints. The technique used to design the sequences incorporate
design features such as low identity to the yeast genome, and low
identity to each other. Therefore a library of the self-assembling
sequences can be generated to allow multiple knockouts in the same
organism, while reducing or eliminating the potential for
integration into a previous knockout.
[0320] As noted above, the URA3 cassette makes use of the toxicity
of 5-FOA in yeast carrying a functional URA3 gene. Uracil synthesis
deficient yeast are transformed with the modified URA3 cassette,
using standard yeast transformation protocols, and the transformed
cells are plated on minimal media minus uracil. In some
embodiments, PCR can be used to verify correct insertion into the
region of interest in the host genome, and certain embodiments the
PCR step can be omitted. Inclusion of the PCR step can reduce the
number of transformants that need to be counter selected to "pop
out" the URA3 cassette. The transformants (e.g., all or the ones
determined to be correct by PCR, for example) can then be
counter-selected on media containing 5-FOA, which will select for
recombination out (e.g., popping out) of the URA3 cassette, thus
rendering the yeast ura3 deficient again, and resistant to 5-FOA
toxicity. Targeting sequences used to direct recombination events
to specific regions are presented herein. A modification of the
method described above can be used to integrate genes in to the
chromosome, where after recombination a functional gene is left in
the chromosome next to the 200 bp footprint.
[0321] In some embodiments, other auxotrophic or dominant selection
markers can be used in place of URA3 (e.g., an auxotrophic
selectable marker), with the appropriate change in selection media
and selection agents. Auxotrophic selectable markers are used in
strains deficient for synthesis of a required biological molecule
(e.g., amino acid or nucleoside, for example). Non-limiting
examples of additional auxotrophic markers include; HIS3, TRP-1,
LEU2, LEU2-d, and LYS2. Certain auxotrophic markers (e.g., URA3 and
LYS2) allow counter selection to select for the second
recombination event that pops out all but one of the direct repeats
of the recombination construct. HIS3 encodes an activity involved
in histidine synthesis. TRP1 encodes an activity involved in
tryptophan synthesis. LEU2 encodes an activity involved in leucine
synthesis. LEU2-d is a low expression version of LEU2 that selects
for increased copy number (e.g., gene or plasmid copy number, for
example) to allow survival on minimal media without leucine. LYS2
encodes an activity involved in lysine synthesis, and allows
counter selection for recombination out of the LYS2 gene using
alpha-amino adipate (.alpha.-amino adipate).
[0322] Dominant selectable markers are useful because they also
allow industrial and/or prototrophic strains to be used for genetic
manipulations. Additionally, dominant selectable markers provide
the advantage that rich medium can be used for plating and culture
growth, and thus growth rates are markedly increased. Non-limiting
examples of dominant selectable markers include: Tn903kan.sup.r,
Cm.sup.4, Hyg.sup.r, CUP1, and DHFR. Tn903 kan.sup.r encodes an
activity involved in kanamycin antibiotic resistance (e.g.,
typically neomycin phosphotransferase II or NPTII, for example).
Cm.sup.r encodes an activity involved in chloramphenicol antibiotic
resistance (e.g., typically chloramphenicol acetyl transferase or
CAT, for example). Hyg.sup.T encodes an activity involved in
hydgromycin resistance by phosphorylation of hygromycin B (e.g.,
hygromycin phosphotransferase, or HPT). CUP1 encodes an activity
involved in resistance to heavy metal (e.g., copper, for example)
toxicity. DHFR encodes a dihydrofolate reductase activity which
confers resistance to methotrexate and sulfanilamide compounds.
[0323] In contrast to site-directed or specific mutagenesis, random
mutagenesis does not require any sequence information and can be
accomplished by a number of widely different methods. Random
mutagenesis often is used to create mutant libraries that can be
used to screen for the desired genotype or phenotype. Non-limiting
examples of random mutagenesis include; chemical mutagenesis,
UV-induced mutagenesis, insertion element or transposon-mediated
mutagenesis, DNA shuffling, error-prone PCR mutagenesis, and the
like.
[0324] Chemical mutagenesis often involves chemicals like ethyl
methanesulfonate (EMS), nitrous acid, mitomycin C,
N-methyl-N-nitrosourea (MNU), diepoxybutane (DEB),
1,2,7,8-diepoxyoctane (DEO), methyl methane sulfonate (MMS),
N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline
1-oxide (4-NQO),
2-methyloxy-6-chloro-9(3-[ethyl-2-chloroethyl]-aminopropylamino)-acridine-
dihydrochloride (ICR-170), 2-amino purine (2AP), and hydroxylamine
(HA), provided herein as non-limiting examples. These chemicals can
cause base-pair substitutions, frameshift mutations, deletions,
transversion mutations, transition mutations, incorrect
replication, and the like. In some embodiments, the mutagenesis can
be carried out in vivo. Sometimes the mutagenic process involves
the use of the host organisms DNA replication and repair mechanisms
to incorporate and replicate the mutagenized base or bases.
[0325] Another type of chemical mutagenesis involves the use of
base-analogs. The use of base-analogs cause incorrect base pairing
which in the following round of replication is corrected to a
mismatched nucleotide when compared to the starting sequence. Base
analog mutagenesis introduces a small amount of non-randomness to
random mutagenesis, because specific base analogs can be chose
which can be incorporated at certain nucleotides in the starting
sequence. Correction of the mispairing typically yields a known
substitution. For example, Bromo-deoxyuridine (BrdU) can be
incorporated into DNA and replaces T in the sequence. The host DNA
repair and replication machinery can sometime correct the defect,
but sometimes will mispair the BrdU with a G. The next round of
replication then causes a G-C transversion from the original A-T in
the native sequence. Ultra violet (UV) induced mutagenesis is
caused by the formation of thymidine dimers when UV light
irradiates chemical bonds between two adjacent thymine residues.
Excision repair mechanism of the host organism correct the lesion
in the DNA, but occasionally the lesion is incorrectly repaired
typically resulting in a C to T transition.
[0326] Insertion element or transposon-mediated mutagenesis makes
use of naturally occurring or modified naturally occurring mobile
genetic elements. Transposons often encode accessory activities in
addition to the activities necessary for transposition (e.g.,
movement using a transposase activity, for example). In many
examples, transposon accessory activities are antibiotic resistance
markers (e.g., see Tn903 kan.sup.r described above, for example).
Insertion elements typically only encode the activities necessary
for movement of the nucleic acid sequence. Insertion element and
transposon mediated mutagenesis often can occur randomly, however
specific target sequences are known for some transposons. Mobile
genetic elements like IS elements or Transposons (Tn) often have
inverted repeats, direct repeats or both inverted and direct
repeats flanking the region coding for the transposition genes.
Recombination events catalyzed by the transposase cause the element
to remove itself from the genome and move to a new location,
leaving behind a portion of an inverted or direct repeat. Classic
examples of transposons are the "mobile genetic elements"
discovered in maize. Transposon mutagenesis kits are commercially
available which are designed to leave behind a 5 codon insert
(e.g., Mutation Generation System kit, Finnzymes, World Wide Web
URL finnzymes.us, for example). This allows the artisan to identify
the insertion site, without fully disrupting the function of most
genes.
[0327] DNA shuffling is a method which uses DNA fragments from
members of a mutant library and reshuffles the fragments randomly
to generate new mutant sequence combinations. The fragments are
typically generated using DNaseI, followed by random annealing and
re-joining using self priming PCR. The DNA overhanging ends, from
annealing of random fragments, provide "primer" sequences for the
PCR process. Shuffling can be applied to libraries generated by any
of the above mutagenes is methods.
[0328] Error prone PCR and its derivative rolling circle error
prone PCR uses increased magnesium and manganese concentrations in
conjunction with limiting amounts of one or two nucleotides to
reduce the fidelity of the Taq polymerase. The error rate can be as
high as 2% under appropriate conditions, when the resultant mutant
sequence is compared to the wild type starting sequence. After
amplification, the library of mutant coding sequences must be
cloned into a suitable plasmid. Although point mutations are the
most common types of mutation in error prone PCR, deletions and
frameshift mutations are also possible. There are a number of
commercial error-prone PCR kits available, including those from
Stratagene and Clontech (e.g., World Wide Web URL strategene.com
and World Wide Web URL clontech.com, respectively, for example).
Rolling circle error-prone PCR is a variant of error-prone PCR in
which wild-type sequence is first cloned into a plasmid, then the
whole plasmid is amplified under error-prone conditions.
[0329] As noted above, organisms with altered activities can also
be isolated using genetic selection and screening of organisms
challenged on selective media or by identifying naturally occurring
variants from unique environments. For example, 2-Deoxy-D-glucose
is a toxic glucose analog. Growth of yeast on this substance yields
mutants that are glucose-deregulated. A number of mutants have been
isolated using 2-Deoxy-D-glucose including transport mutants, and
mutants that ferment glucose and galactose simultaneously instead
of glucose first then galactose when glucose is depleted. Similar
techniques have been used to isolate mutant microorganisms that can
metabolize plastics (e.g., from landfills), petrochemicals (e.g.,
from oil spills), and the like, either in a laboratory setting or
from unique environments.
[0330] Similar methods can be used to isolate naturally occurring
mutations in a desired activity when the activity exists at a
relatively low or nearly undetectable level in the organism of
choice, in some embodiments. The method generally consists of
growing the organism to a specific density in liquid culture,
concentrating the cells, and plating the cells on various
concentrations of the substance to which an increase in metabolic
activity is desired. The cells are incubated at a moderate growth
temperature, for 5 to 10 days. To enhance the selection process,
the plates can be stored for another 5 to 10 days at a low
temperature. The low temperature sometimes can allow strains that
have gained or increased an activity to continue growing while
other strains are inhibited for growth at the low temperature.
Following the initial selection and secondary growth at low
temperature, the plates can be replica plated on higher or lower
concentrations of the selection substance to further select for the
desired activity.
[0331] A native, heterologous or mutagenized polynucleotide can be
introduced into a nucleic acid reagent for introduction into a host
organism; thereby generating an engineered microorganism. Standard
recombinant DNA techniques (restriction enzyme digests, ligation,
and the like) can be used by the artisan to combine the mutagenized
nucleic acid of interest into a suitable nucleic acid reagent
capable of (i) being stably maintained by selection in the host
organism, or (ii) being integrating into the genome of the host
organism. As noted above, sometimes nucleic acid reagents comprise
two replication origins to allow the same nucleic acid reagent to
be manipulated in bacterial before final introduction of the final
product into the host organism (e.g., yeast or fungus for example).
Standard molecular biology and recombinant DNA methods are known
(e.g., described in Maniatis, T., E. F. Fritsch and J. Sambrook
(1982) Molecular Cloning: a Laboratory Manual; Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.).
[0332] Nucleic acid reagents can be introduced into microorganisms
using various techniques. Non-limiting examples of methods used to
introduce heterologous nucleic acids into various organisms
include; transformation, transaction, transduction,
electroporation, ultrasound-mediated transformation, particle
bombardment and the like. In some instances the addition of carrier
molecules (e.g., bis-benzimdazolyl compounds, for example, see U.S.
Pat. No. 5,595,899) can increase the uptake of DNA in cells
typically thought to be difficult to transform by conventional
methods. Conventional methods of transformation are known (e.g.,
described in Maniatis, T., E. F. Fritsch and J. Sambrook (1982).
Molecular Cloning: a Laboratory Manual; Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.).
[0333] Feedstocks, Media, Supplements & Additives
[0334] Engineered microorganisms often are cultured under
conditions that optimize yield of a fatty dicarboxylic acid (e.g.,
an eight to eighteen-carbon fatty dicarboxylic acid). Non-limiting
examples of fatty dicarboxylic acids include suberic acid (i.e.,
octanedioic acid, 1,8-octanedioic acid, octanedioic acid,
octane-1,8-dioic acid, 1,6-hexanedicarboxylic acid, capryllic
diacids), sebacic acid (i.e., 1,10-decanedioic acid, decanedioic
acid, decane-1,10-dioic acid, 1,8-octanedicarboxylic acid, capric
diacid), dodecanedioic acid,(i.e., DDDA, 1,12-dodecanedioic acid,
dodecanedioic acid, dodecane-1,12-dioic acid,
1,10-decanedicarboxylic acid, decamethylenedicaboxylic acid,
1,10-dicarboxydecane, lauric diacid), tetradecanedioic acid (i.e.,
TDDA, 1,14-tetradecanedioic acid, tetradecanedioic acid,
tetradecane-1,14-dioic acid, 1,12-dodecanedicarboxylic acid,
myristic diacid), thapsic acid (i.e., hexadecanedioic acid,
1,16-hexadecanedioic acid, hexadecanedioic acid,
hexadecane-1,16-dioic acid, 1,14-tetradecanedicarboxylic acid,
palmitic diacid), cis-9-hexadocenedioic acid (i.e., palmitoleic
diacids), octanedioic acid (i.e., 1,18-octadecanedioic acid,
octadecanedioic acid, octadecane-1,18-dioic acid,
1,16-hexadecanedicarboxylic acid, stearic diacid),
cis-9-octadecenedioic acid (i.e., oleic diacids),
cis-9,12-octadocenedioic acid (i.e., linoleic diacids),
cis-9,12,15-octadocenedioic acid (i.e., linolenic diacids),
arachidic diacid (i.e., eicosanoic diacid, icosanoic diacid),
11-eicosenoic diacid (i.e., cis-11-eicosenedioic acid),
13-eicosenoic diacids (i.e., cis-13-eicosenedioic acid),
arachidonic diacid (i.e., cis-5,8,11,14-eicosatetraenedioic acid).
Culture conditions often optimize activity of one or more of the
following activities: omega oxo fatty acid dehydrogenase activity,
omega hydroxyl fatty acid dehydrogenase activity, acetyl CoA
carboxylase activity, monooxygenase activity, monooxygenase
reductase activity, fatty alcohol oxidase, acyl-CoA ligase,
acyl-CoA oxidase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA
dehydrogenase, and/or acyltransferase (e.g., acetyl-CoA
C-acyltransferase) activities. In general, non-limiting examples of
conditions that may be optimized include the type and amount of
carbon source, the type and amount of nitrogen source, the
carbon-to-nitrogen ratio, the oxygen level, growth temperature, pH,
length of the biomass production phase, length of target product
accumulation phase, and time of cell harvest.
[0335] Culture media generally contain a suitable carbon source.
Carbon sources useful for culturing microorganisms and/or
fermentation processes sometimes are referred to as feedstocks. The
term "feedstock" as used herein refers to a composition containing
a carbon source that is provided to an organism, which is used by
the organism to produce energy and metabolic products useful for
growth. A feedstock may be a natural substance, a "man-made
substance," a purified or isolated substance, a mixture of purified
substances, a mixture of unpurified substances or combinations
thereof. A feedstock often is prepared by and/or provided to an
organism by a person, and a feedstock often is formulated prior to
administration to the organism. A carbon source may comprise, but
is not limited to including, one or more of the following
substances: alkanes; alkenes, mono-carboxylic acids, di-carboxylic
acids, monosaccharides (e.g., also referred to as "saccharides,"
which include 6-carbon sugars (e.g., glucose, fructose), 5-carbon
sugars (e.g., xylose and other pentoses) and the like),
disaccharides (e.g., lactose, sucrose), oligosaccharides (e.g.,
glycans, homopolymers of a monosaccharide); polysaccharides (e.g.,
starch, cellulose, heteropolymers of monosaccharides or mixtures
thereof), sugar alcohols (e.g., glycerol), and renewable feedstocks
(e.g., cheese whey permeate, cornsteep liquor, sugar beet molasses,
barley malt).
[0336] Carbon sources also can be selected from one or more of the
following non-limiting examples: paraffin (e.g.,
saturated-paraffin, unsaturated paraffin, substituted paraffin,
linear paraffin, branched paraffin, or combinations thereof);
alkanes (e.g., dodecane), alkenes or alkynes, each of which may be
linear, branched, saturated, unsaturated, substituted or
combinations thereof (described in greater detail below); linear or
branched alcohols (e.g., dodecanol); fatty acids (e.g., about 1
carbon to about 60 carbons, including free fatty acids such as,
without limitation, caproic acid, capryllic acid, capric acid,
lauric acid, myristic acid, palmitic acid, palmitoleic acid,
stearic acid, oleic acid, linoleic acid, linolenic acid), or soap
stock, for example; esters (such as methyl esters, ethyl esters,
butyl estes, and the like) of fatty acids including, without
limitation, esters such as methyl caprate, ethyl caprate, methyl
laurate, ethyl laurate, methyl myristate, ethyl myristate, methyl
caprolate, ethyl caprolate, ethyl caprillic, methyl caprillic,
methyl palmitate, or ethyl palmitate; monoglycerides; diglycerides;
triglycerides, phospholipids. Non-limiting commercial sources of
products for preparing feedstocks include plants, plant oils or
plant products (e.g., vegetable oils (e.g., almond oil, canola oil,
cocoa butter, coconut oil, corn oil, cottonseed oil, flaxseed oil,
grape seed oil, illipe, olive oil, palm oil, palm olein, palm
kernel oil, safflower oil, peanut oil, soybean oil, sesame oil,
shea nut oil, sunflower oil walnut oil, the like and combinations
thereof) and animal fats (e.g., beef tallow, butterfat, lard, cod
liver oil). A carbon source may included petroleum product and/or a
petroleum distillate (e.g., diesel, fuel oils, gasoline, kerosene,
paraffin wax, paraffin oil, petrochemicals). In some embodiments, a
feedstock comprises petroleum distillate. A carbon source can be a
fatty acid distillate (e.g., a palm oil distillate or corn oil
distillate). Fatty acid distillates can be by-products from the
refining of crude plant oils. In some embodiments, a feedstock
comprises a fatty acid distillate.
[0337] In some embodiments, a feedstock comprises a soapstock (i.e.
soap stock). A widely practiced method for purifying crude
vegetable oils for edible use is the alkali or caustic refining
method. This process employs a dilute aqueous solution of caustic
soda to react with the free fatty acids present which results in
the formation of soaps. The soaps together with hydrated
phosphatides, gums and prooxidant metals are typically separated
from the refined oil as the heavy phase discharge from the refining
centrifuge and are typically known as soapstock.
[0338] A carbon source also may include a metabolic product that
can be used directly as a metabolic substrate in an engineered
pathway described herein, or indirectly via conversion to a
different molecule using engineered or native biosynthetic pathways
in an engineered microorganism. In certain embodiments, metabolic
pathways can be preferentially biased towards production of a
desired product by increasing the levels of one or more activities
in one or more metabolic pathways having and/or generating at least
one common metabolic and/or synthetic substrate. In some
embodiments, a metabolic byproduct (e.g., fatty acid) of an
engineered activity (e.g., omega oxidation activity) can be used in
one or more metabolic pathways selected from gluconeogenesis,
pentose phosphate pathway, glycolysis, fatty acid synthesis beta
oxidation, and omega oxidation, to generate a carbon source that
can be converted to a fatty dicarboxylic acid (e.g., octanedioic
acid, decanedioic acid, dodecanedioic acid, tetradecanedioic acid,
hexadecanedioic acid, octadecanedioic acid, eicosanedioic
acid).
[0339] The term "paraffin" as used herein refers to the common name
for alkane hydrocarbons, independent of the source (e.g., plant
derived, petroleum derived, chemically synthesized, fermented by a
microorganism), or carbon chain length. A carbon source sometimes
comprises a paraffin, and in some embodiments, a paraffin is
predominant in a carbon source (e.g., about 75%, 80%, 85%, 90% or
95% paraffin). A paraffin sometimes is saturated (e.g., fully
saturated), sometimes includes one or more unsaturations (e.g.,
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 unsaturations) and sometimes is
substituted with one or more non-hydrogen substituents.
Non-limiting examples of non-hydrogen substituents include halo;
acetyl, .dbd.O, .dbd.N--CN, .dbd.N--OR, .dbd.NR, OR, NR.sub.2, SR,
SO.sub.2R, SO.sub.2NR.sub.2, NRCONR.sub.2, NRCOOR, NRCOR, CN, COOR,
CONR.sub.2, OOCR, COR, and NO.sub.2, and where each R is
independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8
heteroacyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8alkynyl, C2-C8
heteroalkynyl, C6-C10 aryl, or C5-C10 heteroaryl, and each R is
optionally substituted with halo, .dbd.O, .dbd.N--CN, .dbd.N--OR',
.dbd.NR', OR.dbd., NR'.sub.2, SR', SO.sub.2R', SO.sub.2NR'.sub.2,
NR'SO.sub.2R', NR'CONR'.sub.2, NR'COOR', NR'COR', CN, COOR',
CONR'.sub.2, OOCR', COR', and NO.sub.2, where each R' is
independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8
heteroacyl, C6-C10 aryl or C5-C10 heteroaryl.
[0340] In some embodiments a feedstock is selected according to the
genotype and/or phenotype of the engineered microorganism to be
cultured. For example, a feedstock rich in 12-carbon fatty acids,
12-carbon dicarboxylic acids or 12-carbon paraffins, or a mixture
of 10, 12 and 14-carbon compounds can be useful for culturing yeast
strains harboring an alteration that partially blocks beta
oxidation by disrupting POX4 activity, as described herein.
Non-limiting examples of carbon sources having 10 to 14 carbons
include fats (e.g., coconut oil, palm kernel oil), paraffins (e.g.,
alkanes, alkenes, or alkynes) having 10 to 14 carbons, (e.g.,
dodecane (also referred to as adakane12, bihexyl, dihexyl and
duodecane); tetradecane), alkene and alkyne derivatives), fatty
acids (dodecanoic acid, tetradecanoic acid), fatty alcohols
(dodecanol, tetradecanol), the like, non-toxic substituted
derivatives or combinations thereof.
[0341] A carbon source sometimes comprises an alkyl, alkenyl or
alkynyl compound or molecule (e.g., a compound that includes an
alkyl, alkenyl or alkynyl moiety (e.g., alkane, alkene, alkyne)).
In certain embodiments, an alkyl, alkenyl or alkynyl molecule, or
combination thereof, is predominant in a carbon source (e.g., about
75%, 80%, 85%, 90% or 95% of such molecules). As used herein, the
terms "alkyl," "alkenyl" and "alkynyl" include straight-chain
(referred to herein as "linear"), branched-chain (referred to
herein as "non-linear"), cyclic monovalent hydrocarbyl radicals,
and combinations of these, which contain only C and H atoms when
they are unsubstituted. Non-limiting examples of alkyl moieties
include methyl, ethyl, isobutyl, cyclohexyl, cyclopentylethyl,
2-propenyl, 3-butynyl, and the like. An alkyl that contains only C
and H atoms and is unsubstituted sometimes is referred to as
"saturated." An alkenyl or alkynyl generally is "unsaturated" as it
contains one or more double bonds or triple bonds, respectively. An
alkenyl can include any number of double bonds, such as 1, 2, 3, 4
or 5 double bonds, for example. An alkynyl can include any number
of triple bonds, such as 1, 2, 3, 4 or 5 triple bonds, for example.
Alkyl, alkenyl and alkynyl molecules sometimes contain between
about 2 to about 60 carbon atoms (C). For example, an alkyl,
alkenyl and alkynyl molecule can include about 1 carbon atom, about
2 carbon atoms, about 3 carbon atoms, about 4 carbon atoms, about 5
carbon atoms, about 6 carbon atoms, about 7 carbon atoms, about 8
carbon atoms, about 9 carbon atoms, about 10 carbon atoms, about 12
carbon atoms, about 14 carbon atoms, about 16 carbon atoms, about
18 carbon atoms, about 20 carbon atoms, about 22 carbon atoms,
about 24 carbon atoms, about 26 carbon atoms, about 28 carbon
atoms, about 30 carbon atoms, about 32 carbon atoms, about 34
carbon atoms, about 36 carbon atoms, about 38 carbon atoms, about
40 carbon atoms, about 42 carbon atoms, about 44 carbon atoms,
about 46 carbon atoms, about 48 carbon atoms, about 50 carbon
atoms, about 52 carbon atoms, about 54 carbon atoms, about 56
carbon atoms, about 58 carbon atoms or about 60 carbon atoms. In
some embodiments, paraffins can have a mean number of carbon atoms
of between about 8 to about 18 carbon atoms (e.g., about 8 carbon
atoms, about 9 carbon atoms, about 10 carbon atoms, about 11 carbon
atoms, about 12 carbon atoms, about 13 carbon atoms, about 14
carbon atoms, about 15 carbon atoms, about 16 carbon atoms, about
17 carbon atoms and about 18 carbon atoms). A single group can
include more than one type of multiple bond, or more than one
multiple bond. Such groups are included within the definition of
the term "alkenyl" when they contain at least one carbon-carbon
double bond, and are included within the term "alkynyl" when they
contain at least one carbon-carbon triple bond. Alkyl, alkenyl and
alkynyl molecules include molecules that comprise an alkyl, alkenyl
and/or alkynyl moiety, and include molecules that consist of an
alkyl, alkenyl or alkynyl moiety (i.e., alkane, alkene and alkyne
molecules). Non-limiting examples of unsaturated fatty acid
feedstocks useful for practicing certain embodiments herein include
oleic acid, linoleic acid, linolenic acid, eicosenoic acid,
palmitoleic acid and arachidonic acid.
[0342] Alkyl, alkenyl and alkynyl substituents sometimes contain
1-20C (alkyl) or 2-20C (alkenyl or alkynyl). They can contain about
8-20C or about 10-20C in some embodiments. A single group can
include more than one type of multiple bond, or more than one
multiple bond. Such groups are included within the definition of
the term "alkenyl" when they contain at least one carbon-carbon
double bond; and are included within the term "alkynyl" when they
contain at least one carbon-carbon triple bond.
[0343] Alkyl, alkenyl and alkynyl groups or compounds sometimes are
substituted to the extent that such substitution can be synthesized
and can exist. Typical substituents include, but are not limited
to, halo, acetyl, .dbd.O, .dbd.N--CN, .dbd.N--OR, .dbd.NR, OR,
NR.sub.2, SR, SO.sub.2R, SO.sub.2NR.sub.2, NRSO.sub.2R,
NRCONR.sub.2, NRCOOR, NRCOR, CN, COOR, CONR.sub.2, OOCR, COR, and
NO.sub.2, where each R is independently H, C1-C8 alkyl, C2-C8
heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C2-C8 alkenyl, C2-C8
heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C11 aryl, or
C5-C11 heteroaryl, and each R is optionally substituted with halo,
.dbd.O, .dbd.N--CN, .dbd.N--OR', .dbd.NR', OR', NR'.sub.2, SR',
SO.sub.2R', SO.sub.2NR'.sub.2, NR'SO.sub.2R', NR'CONR'.sub.2,
NR'COOR', NR'COR', CN, COOR', CONR'.sub.2, OOCR', COR', and
NO.sub.2, where each R' is independently H, C1-C8 alkyl, C2-C8
heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl or C5-C10
heteroaryl. Alkyl, alkenyl and alkynyl groups can also be
substituted by C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl or C5-C10
heteroaryl, each of which can be substituted by the substituents
that are appropriate for the particular group.
[0344] "Acetylene" or "acetyl" substituents are 2-10C alkynyl
groups that are optionally substituted, and are of the formula
--C.ident.C--Ri, where Ri is H or C1-C8 alkyl, C2-C8 heteroalkyl,
C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8
heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl, C5-C10
heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl, and each
Ri group is optionally substituted with one or more substituents
selected from halo, .dbd.O, .dbd.N--CN, .dbd.N--OR', .dbd.NR', OR',
NR'2, SR', SO.sub.2R', SO.sub.2NR'.sub.2, NR'SO.sub.2R,
NR'CONR'.sub.2, NR'COOR', NR'COR', CN, COOR', CONR'.sub.2, OOCR',
COR', and NO.sub.2, each R' is independently H, C1-C6 alkyl, C2-C6
heteroalkyl, C1-C6 acyl, C2-C6 heterooacyl, C6-C10 aryl, C5-C10
heteroaryl, C7-12 arylalkyl, or C6-12 heteroarylalkyl, each of
which is optionally substituted with one or more groups selected
from halo, C1-C4 alkyl, C1-C4 heteroalkyl, C1-C6 acyl, C1-C6
heteroacyl, hydroxy, amino, and .dbd.O; and where two R' can be
linked to form a 3-7 membered ring optionally containing up to
three heteroatoms selected from N, O and S. In some embodiments, Ri
of --C.ident.C--Ri is H or Me.
[0345] A carbon source sometimes comprises a heteroalkyl,
heteroalkenyl and/or heteroalkynyl molecule or compound (e.g.,
comprises heteroalkyl, heteroalkenyl and/or heteroalkynyl moiety
(e.g., heteroalkane, heteroalkene or heteroalkyne)). "Heteroalkyl",
"heteroalkenyl", and "heteroalkynyl" and the like are defined
similarly to the corresponding hydrocarbyl (alkyl, alkenyl and
alkynyl) groups, but the `hetero` terms refer to groups that
contain one to three O, S or N heteroatoms or combinations thereof
within the backbone; thus at least one carbon atom of a
corresponding alkyl, alkenyl, or alkynyl group is replaced by one
of the specified heteroatoms to form a heteroalkyl, heteroalkenyl,
or heteroalkynyl group. The typical and sizes for heteroforms of
alkyl, alkenyl and alkynyl groups are generally the same as for the
corresponding hydrocarbyl groups, and the substituents that may be
present on the heteroforms are the same as those described above
for the hydrocarbyl groups. For reasons of chemical stability, it
is also understood that, unless otherwise specified, such groups do
not include more than two contiguous heteroatoms except where an
oxo group is present on N or S as in a nitro or sulfonyl group.
[0346] The term "alkyl" as used herein includes cycloalkyl and
cycloalkylalkyl groups and compounds, the term "cycloalkyl" may be
used herein to describe a carbocyclic non-aromatic compound or
group that is connected via a ring carbon atom, and
"cycloalkylalkyl" may be used to describe a carbocyclic
non-aromatic compound or group that is connected to a molecule
through an alkyl linker. Similarly, "heterocyclyl" may be used to
describe a non-aromatic cyclic group that contains at least one
heteroatom as a ring member that is connected to the molecule via a
ring atom, which may be C or N; and "heterocyclylalkyl" may be used
to describe such a group that is connected to another molecule
through a linker. The sizes and substituents that are suitable for
the cycloalkyl, cycloalkylalkyl, heterocyclyl, and
heterocyclylalkyl groups are the same as those described above for
alkyl groups. As used herein, these terms also include rings that
contain a double bond or two, as long as the ring is not
aromatic.
[0347] A carbon source sometimes comprises an acyl compound or
moiety (e.g., compound comprising an acyl moiety). As used herein,
"acyl" encompasses groups comprising an alkyl, alkenyl, alkynyl,
aryl or arylalkyl radical attached at one of the two available
valence positions of a carbonyl carbon atom, and heteroacyl refers
to the corresponding groups where at least one carbon other than
the carbonyl carbon has been replaced by a heteroatom chosen from
N, O and S. Thus heteroacyl includes, for example, --C(.dbd.O)OR
and --C(.dbd.O)NR.sub.2 as well as --C(.dbd.O)-- heteroaryl.
[0348] Acyl and heteroacyl groups are bonded to any group or
molecule to which they are attached through the open valence of the
carbonyl carbon atom. Typically, they are C1-C8 acyl groups, which
include formyl, acetyl, pivaloyl, and benzoyl, and C2-C8 heteroacyl
groups, which include methoxyacetyl, ethoxycarbonyl, and
4-pyridinoyl. The hydrocarbyl groups, aryl groups, and heteroforms
of such groups that comprise an acyl or heteroacyl group can be
substituted with the substituents described herein as generally
suitable substituents for each of the corresponding component of
the acyl or heteroacyl group.
[0349] A carbon source sometimes comprises one or more aromatic
moieties and/or heteroaromatic moieties. "Aromatic" moiety or
"aryl" moiety refers to a monocyclic or fused bicyclic moiety
having the well-known characteristics of aromaticity; examples
include phenyl and naphthyl. Similarly, "heteroaromatic" and
"heteroaryl" refer to such monocyclic or fused bicyclic ring
systems which contain as ring members one or more heteroatoms
selected from O, S and N. The inclusion of a heteroatom permits
aromaticity in 5 membered rings as well as 6 membered rings.
Typical heteroaromatic systems include monocyclic C5-C6 aromatic
groups such as pyridyl, pyrimidyl, pyrazinyl, thienyl, furanyl,
pyrrolyl, pyrazolyl, thiazolyl, oxazolyl, and imidazolyl and the
fused bicyclic moieties formed by fusing one of these monocyclic
groups with a phenyl ring or with any of the heteroaromatic
monocyclic groups to form a C8-C10 bicyclic group such as indolyl,
benzimidazolyl, indazolyl, benzotriazolyl, isoquinolyl, quinolyl,
benzothiazolyl, benzofuranyl, pyrazolopyridyl, quinazolinyl,
quinoxalinyl, cinnolinyl, and the like. Any monocyclic or fused
ring bicyclic system which has the characteristics of aromaticity
in terms of electron distribution throughout the ring system is
included in this definition. It also includes bicyclic groups where
at least the ring which is directly attached to the remainder of
the molecule has the characteristics of aromaticity. Typically, the
ring systems contain 5-12 ring member atoms. The monocyclic
heteroaryls sometimes contain 5-6 ring members, and the bicyclic
heteroaryls sometimes contain 8-10 ring members.
[0350] Aryl and heteroaryl moieties may be substituted with a
variety of substituents including C1-C8 alkyl, C2-C8 alkenyl, C2-C8
alkynyl, C5-C12 aryl, C1-C8 acyl, and heteroforms of these, each of
which can itself be further substituted; other substituents for
aryl and heteroaryl moieties include halo, OR, NR.sub.2, SR,
SO.sub.2R, SO.sub.2NR.sub.2, NRSO.sub.2R, NRCONR.sub.2, NRCOOR,
NRCOR, CN, COOR, CONR.sub.2, OOCR, COR, and NO.sub.2, where each R
is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl,
C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C10
aryl, C5-C10 heteroaryl, C7-C12 arylalkyl, or C6-C12
heteroarylalkyl, and each R is optionally substituted as described
above for alkyl groups. The substituent groups on an aryl or
heteroaryl group may be further substituted with the groups
described herein as suitable for each type of such substituents or
for each component of the substituent. Thus, for example, an
arylalkyl substituent may be substituted on the aryl portion with
substituents typical for aryl groups, and it may be further
substituted on the alkyl portion with substituents as typical or
suitable for alkyl groups.
[0351] Similarly, "arylalkyl" and "heteroarylalkyl" refer to
aromatic and heteroaromatic ring systems, which are stand-alone
molecules (e.g., benzene or substituted benzene, pyridine or
substituted pyridine), or which are bonded to an attachment point
through a linking group such as an alkylene, including substituted
or unsubstituted, saturated or unsaturated, cyclic or acyclic
linkers. A linker often is C1-C8 alkyl or a hetero form thereof.
These linkers also may include a carbonyl group, thus making them
able to provide substituents as an acyl or heteroacyl moiety. An
aryl or heteroaryl ring in an arylalkyl or heteroarylalkyl group
may be substituted with the same substituents described above for
aryl groups. An arylalkyl group sometimes includes a phenyl ring
optionally substituted with the groups defined above for aryl
groups and a C1-C4 alkylene that is unsubstituted or is substituted
with one or two C1-C4 alkyl groups or heteroalkyl groups, where the
alkyl or heteroalkyl groups can optionally cyclize to form a ring
such as cyclopropane, dioxolane, or oxacyclopentane. Similarly, a
heteroarylalkyl group often includes a C5-C6 monocyclic heteroaryl
group optionally substituted with one or more of the groups
described above as substituents typical on aryl groups and a C1-C4
alkylene that is unsubstituted. A heteroarylalkyl group sometimes
is substituted with one or two C1-C4 alkyl groups or heteroalkyl
groups, or includes an optionally substituted phenyl ring or C5-C6
monocyclic heteroaryl and a C1-C4 heteroalkylene that is
unsubstituted or is substituted with one or two C1-C4 alkyl or
heteroalkyl groups, where the alkyl or heteroalkyl groups can
optionally cyclize to form a ring such as cyclopropane, dioxolane,
or oxacyclopentane.
[0352] Where an arylalkyl or heteroarylalkyl group is described as
optionally substituted, the substituents may be on the alkyl or
heteroalkyl portion or on the aryl or heteroaryl portion of the
group. The substituents optionally present on the alkyl or
heteroalkyl portion sometimes are the same as those described above
for alkyl groups, and the substituents optionally present on the
aryl or heteroaryl portion often are the same as those described
above for aryl groups generally.
[0353] "Arylalkyl" groups as used herein are hydrocarbyl groups if
they are unsubstituted, and are described by the total number of
carbon atoms in the ring and alkylene or similar linker. Thus a
benzyl group is a C7-arylalkyl group, and phenylethyl is a
C8-arylalkyl.
[0354] "Heteroarylalkyl" as described above refers to a moiety
comprising an aryl group that is attached through a linking group,
and differs from "arylalkyl" in that at least one ring atom of the
aryl moiety or one atom in the linking group is a heteroatom
selected from N, O and S. The heteroarylalkyl groups are described
herein according to the total number of atoms in the ring and
linker combined, and they include aryl groups linked through a
heteroalkyl linker; heteroaryl groups linked through a hydrocarbyl
linker such as an alkylene; and heteroaryl groups linked through a
heteroalkyl linker. Thus, for example, C7-heteroarylalkyl includes
pyridylmethyl, phenoxy, and N-pyrrolylmethoxy.
[0355] "Alkylene" as used herein refers to a divalent hydrocarbyl
group. Because an alkylene is divalent, it can link two other
groups together. An alkylene often is referred to as
--(CH.sub.2).sub.n-- wherein can be 1-20, 1-10, 1-8, or 1-4, though
where specified, an alkylene can also be substituted by other
groups, and can be of other lengths, and the open valences need not
be at opposite ends of a chain. Thus --CH(Me)-- and --C(Me).sub.2--
may also be referred to as alkylenes, as can a cyclic group such as
cyclopropan-1,1-diyl. Where an alkylene group is substituted, the
substituents include those typically present on alkyl groups as
described herein.
[0356] In some embodiments, a feedstock includes a mixture of
carbon sources, where each carbon source in the feedstock is
selected based on the genotype of the engineered microorganism. In
certain embodiments, a mixed carbon source feedstock includes one
or more carbon sources selected from sugars, cellulose, alkanes,
fatty acids, triacylglycerides, paraffins, the like and
combinations thereof.
[0357] Nitrogen may be supplied from an inorganic (e.g.;
(NH.sub.4).sub.2SO.sub.4) or organic source (e.g., urea or
glutamate). In addition to appropriate carbon and nitrogen sources;
culture media also can contain suitable minerals, salts, cofactors,
buffers, vitamins, metal ions (e.g., Mn.su.p.+2, Co.sup.+2,
Zn.sup.+2, Mg.sup.+2) and other components suitable for culture of
microorganisms.
[0358] Engineered microorganisms sometimes are cultured in complex
media (e.g., yeast extract-peptone-dextrose broth (YPD)). In some
embodiments, engineered microorganisms are cultured in a defined
minimal media that lacks a component necessary for growth and
thereby forces selection of a desired expression cassette (e.g.,
Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.)).
[0359] Culture media in some embodiments are common commercially
prepared media, such as Yeast Nitrogen Base (DIFCO Laboratories,
Detroit, Mich.). Other defined or synthetic growth media may also
be used and the appropriate medium for growth of the particular
microorganism are known. A variety of host organisms can be
selected for the production of engineered microorganisms.
Non-limiting examples include yeast (e.g., Candida tropicalis
(e.g., ATCC20336; ATCC20913, ATCC20962), Yarrowia lipolytica (e.g.,
ATCC20228)) and filamentous fungi (e.g., Aspergillus nidulans
(e.g., ATCC38164) and Aspergillus parasiticus (e.g., ATCC 24690)).
In specific embodiments, yeast are cultured in YPD media (10 g/L
Bacto Yeast Extract, 20 g/L Bacto Peptone, and 20 g/L Dextrose).
Filamentous fungi, in particular embodiments, are grown in CM
(Complete Medium) containing 10 g/L Dextrose, 2 g/L Bacto Peptone,
1 g/L Bacto Yeast Extract, 1 g/L Casamino acids, 50 mL/L20X Nitrate
Salts (120 g/L NaNO.sub.3, 10.4 g/L KCl, 10.4 g/L MgSO.sub.4.7
H.sub.2O), 1 mL/L 1000.times. Trace Elements (22 g/L ZnSO.sub.4.7
H.sub.2O, 11 g/L H.sub.3BO.sub.3, 5 g/L MnCl.sub.2.7 H.sub.2O, 5
g/L FeSO.sub.4.7 H.sub.2O, 1.7 g/L CoCl.sub.2.6 H.sub.2O, 1.6 g/L
CuSO.sub.4.5 H.sub.2O, 1.5 g/L Na.sub.2MoO.sub.4.2 H.sub.2O, and 50
g/L Na.sub.4EDTA), and 1 mL/L Vitamin Solution (100 mg each of
Biotin, pyridoxine, thiamine, riboflavin, p-aminobenzoic acid, and
nicotinic acid in 100 mL water).
[0360] In those embodiments in which a feedstock comprising an
unsaturated fatty acid or ester thereof is employed, the resulting
diacid may be unsaturated as well. If a saturated diacid is desired
as the final product, the unsaturated diacid may be hydrogenated to
remove one or all carbon-carbon double bonds. Hydrogenation may be
accomplished using methods known in the art. The addition of
hydrogen across the double bond can be accomplished with metallic
chemical catalysts, non-metallic chemical catalysts, or enzymatic
catalysts. The source of hydrogen may be molecular hydrogen in the
case of chemical catalysis or enzymatic cofactors (ie. NADH, NADPH,
FADH.sub.2) in the case of enzymatic catalysis.
[0361] Catalytic hydrogenation with metallic catalysts may take
advantage of many different types of catalysts. The metal may be
platinum, palladium, rhodium, ruthenium, nickel, or other metals.
The catalysts may be homogenous or heterogeneous catalysts.
Elevated temperatures and pressures may be employed to increase the
reaction rate. Catalytic hydrogenation may also occur with
nonmetallic catalysts such as frustrated Lewis pair compounds.
[0362] Enzymatic hydrogenation may occur in vivo or in vitro with
native or engineered enzymes that catalyze redox reactions that use
unsaturated-diacids or fatty acids as a substrate or a product.
Examples of such enzymes are acyl-CoA dehydrogenase (EC# 1.3.1.8),
trans-2-enoyl-CoA reductase (EC# 1.3.1.44), or stearoyl-CoA
9-desaturase (EC# 1.14.19.1). In some instances, the desired
reaction producing a saturated diacid may actually require the
enzyme to operate in the reverse direction from its normal in vivo
reaction; such reversal can be accomplished via genetic
manipulation of the enzyme.
[0363] Growth Conditions & Fermentation
[0364] A suitable pH range for the fermentation often is between
about pH 4.0 to about pH 8.0, where a pH in the range of about pH
5.5 to about pH 7.0 sometimes is utilized for initial culture
conditions. Depending on the host organism, culturing may be
conducted under aerobic or anaerobic conditions, where microaerobic
conditions sometimes are maintained. A two-stage process may be
utilized, where one stage promotes microorganism proliferation and
another state promotes production of target molecule. In a
two-stage process, the first stage may be conducted under aerobic
conditions (e.g., introduction of air and/or oxygen) and the second
stage may be conducted under anaerobic conditions (e.g., air or
oxygen are not introduced to the culture conditions). In some
embodiments, the first stage may be conducted under anaerobic
conditions and the second stage may be conducted under aerobic
conditions. In certain embodiments, a two-stage process may include
two more organisms, where one organism generates an intermediate
product in one stage and another organism processes the
intermediate product into a target fatty dicarboxylic acid product
(e.g., sebacic or dodecanedioic acid) in another stage, for
example.
[0365] A variety of fermentation processes may be applied for
commercial biological production of a target fatty dicarboxylic
acid product. In some embodiments, commercial production of a
target fatty dicarboxylic acid product from a recombinant microbial
host is conducted using a batch, fed-batch or continuous
fermentation process, for example.
[0366] A batch fermentation process often is a closed system where
the media composition is fixed at the beginning of the process and
not subject to further additions beyond those required for
maintenance of pH and oxygen level during the process. At the
beginning of the culturing process the media is inoculated with the
desired organism and growth or metabolic activity is permitted to
occur without adding additional sources (i.e., carbon and nitrogen
sources) to the medium. In batch processes the metabolite and
biomass compositions of the system change constantly up to the time
the culture is terminated. In a typical batch process, cells
proceed through a static lag phase to a high-growth log phase and
finally to a stationary phase, wherein the growth rate is
diminished or halted. Left untreated, cells in the stationary phase
will eventually die.
[0367] A variation of the standard batch process is the fed-batch
process, where the carbon source is continually added to the
fermentor over the course of the fermentation process. Fed-batch
processes are useful when catabolite repression is apt to inhibit
the metabolism of the cells or where it is desirable to have
limited amounts of carbon source in the media at any one time.
Measurement of the carbon source concentration in fed-batch systems
may be estimated on the basis of the changes of measurable factors
such as pH, dissolved oxygen and the partial pressure of waste
gases (e.g., CO.sub.2).
[0368] Batch and fed-batch culturing methods are known in the art.
Examples of such methods may be found in Thomas D. Brock in
Biotechnology: A Textbook of Industrial Microbiology, 2.sup.nd ed.,
(1989) Sinauer Associates Sunderland, Mass. and Deshpande, Mukund
V., Appl. Biochem. Biotechnol., 36:227 (1992).
[0369] In continuous fermentation process a defined media often is
continuously added to a bioreactor while an equal amount of culture
volumes is removed simultaneously for product recovery. Continuous
cultures generally maintain cells in the log phase of growth at a
constant cell density. Continuous or semi-continuous culture
methods permit the modulation of one factor or any number of
factors that affect cell growth or end product concentration. For
example, an approach may limit the carbon source and allow all
other parameters to moderate metabolism. In some systems, a number
of factors affecting growth may be altered continuously while the
cell concentration, measured by media turbidity, is kept constant.
Continuous systems often maintain steady state growth and thus the
cell growth rate often is balanced against cell loss due to media
being drawn off the culture. Methods of modulating nutrients and
growth factors for continuous culture processes, as well as
techniques for maximizing the rate of product formation, are known
and a variety of methods are detailed by Brock, supra.
[0370] In some embodiments involving fermentation, the fermentation
can be carried out using two or more microorganisms (e.g., host
microorganism, engineered microorganism, isolated naturally
occurring microorganism, the like and combinations thereof), where
a feedstock is partially or completely utilized by one or more
organisms in the fermentation (e.g., mixed fermentation), and the
products of cellular respiration or metabolism of one or more
organisms can be further metabolized by one or more other organisms
to produce a desired target product (e.g., sebacic acid,
dodecanedioic acid, hexanoic acid). In certain embodiments, each
organism can be fermented independently and the products of
cellular respiration or metabolism purified and contacted with
another organism to produce a desired target products. In some
embodiments, one or more organisms are partially or completely
blocked in a metabolic pathway (e.g., beta oxidation, omega
oxidation, the like or combinations thereof), thereby producing a
desired product that can be used as a feedstock for one or more
other organisms. Any suitable combination of microorganisms can be
utilized to carry out mixed fermentation or sequential
fermentation.
[0371] Enhanced Fermentation Processes
[0372] It has been determined that certain feedstock components are
toxic to, or produce a by-product (e.g., metabolite) that is toxic
to, yeast utilized in a fermentation process for the purpose of
producing a target product (e.g., a C4 to C24 diacid). A toxic
component or metabolite from a feedstock sometimes is utilized by
the yeast to produce a target product (e.g., target molecule).
[0373] In some instances, a fatty acid component having 12 carbons
(i.e., C12), or fewer carbons can be toxic to yeast. Components
that are not free fatty acids, but are processed by yeast to a
fatty acid having twelve or fewer carbons, also can have a toxic
effect. Non-limiting examples of such components are esters of
fatty acids (e.g., methyl esters) that are processed by yeast into
a fatty acid having twelve or fewer carbons. Feedstocks containing
molecules that are directly toxic, or indirectly toxic by
conversion of a nontoxic component to a toxic metabolite, are
collectively referred to as "toxic feedstocks" and "toxic
components." Providing yeast with a feedstock that comprises or
delivers one or more toxic components can reduce the viability of
the yeast and/or reduce the amount of target product produced by
the yeast.
[0374] In some embodiments, a process for overcoming the toxic
effect of certain components in a feedstock includes first inducing
yeast with a feedstock not containing a substantially toxic
component and then providing the yeast with a feedstock that
comprises a toxic component. Thus, in some embodiments, provided is
a method for producing a diacid by a yeast from a feedstock toxic
to the yeast, comprising: (a) contacting a genetically modified
yeast in culture with a first feedstock comprising a component not
substantially toxic to the yeast, thereby performing an induction;
and (b) contacting the yeast after the induction in (a) with a
second feedstock that comprises or delivers a component toxic to
the yeast ("toxic component"), whereby a diacid is produced by the
yeast in an amount greater than the amount of the diacid produced
when the induction is not performed.
[0375] A toxic component provided by the second feedstock sometimes
is processed by the yeast into a target product (e.g., diacid).
Sometimes a component not substantially toxic to the yeast in the
first feedstock (e.g., an inducer) is processed by the yeast into a
target product or byproduct (e.g., diacid containing a different
number of carbons than the target product). The first feedstock
sometimes comprises a component not substantially toxic to the
yeast having the same number of carbons as the component in the
second feedstock, or a metabolite processed by the yeast from a
component in the second feedstock, that is substantially toxic to
the yeast. In some embodiments, the first feedstock comprises a
component not substantially toxic to the yeast having a different
number of carbons as the component in the second feedstock, or a
metabolite processed by yeast from a component in the second
feedstock, that is substantially toxic to the yeast. In certain
embodiments, the first feedstock comprises a component that is not
substantially toxic to the yeast (e.g., an inducer) that has the
same number of carbons as the target product. Sometimes the first
feedstock comprises component not substantially toxic to the yeast
(e.g., an inducer) that has a different number of carbons as the
target product (e.g., diacid).
[0376] In some embodiments, the first feedstock comprises an ester
of a fatty acid that is not substantially toxic to the yeast (e.g.,
methyl ester), and sometimes the fatty acid has more than 12
carbons. The first feedstock sometimes comprises a fatty acid that
is not substantially toxic to the yeast, and in some cases the
fatty acid has more than 12 carbons. The first feedstock sometimes
comprises a triglyceride, which triglyceride often contains various
chain-length fatty acids, that is not substantially toxic to the
yeast. In certain cases the first feedstock comprises an aliphatic
chain, which aliphatic chain often contains more than 6 carbons,
that is not substantially toxic to the yeast. In some embodiments,
the first feedstock comprises one or more alkanes (e.g., linear
alkanes, branched alkanes, substituted alkanes) with chain lengths
greater than 6 carbons. In some embodiments a target product is a
C12 diacid, the first feedstock comprises an alkane (e.g., alkane
inducer) and the second feedstock comprises a C12 fatty acid or an
ester of a C12 fatty acid, where the alkane sometimes is a C12
alkane. In some embodiments a target product is a C10 diacid, the
first feedstock comprises an alkane (e.g., alkane inducer) and the
second feedstock comprises a C10 fatty acid or an ester of a C10
fatty acid, where the alkane sometimes is a C10 alkane. In some
embodiments a target product is a C18 diacid, the first feedstock
comprises an alkane (e.g., alkane inducer) and the second feedstock
comprises a C18 fatty acid or an ester of a C18 fatty acid, where
the alkane sometimes is a C18 alkane. In certain embodiments, one
or more of the (i) components in the first feedstock and/or the
second feedstock and (ii) products (e.g., target product) are
saturated. In some embodiments, one or more of the (i) components
in the first feedstock and/or the second feedstock and (ii)
products (e.g., target product) include one or more unsaturations
(e.g., one or more double bonds).
[0377] In some embodiments, the second feedstock is provided to the
yeast a certain amount of time after the first feedstock is
provided to the yeast. The amount of time sometimes is about 1 hour
to about 48 hours, sometimes is about 1 hour to about 12 hours
(e.g., about 2 hours, 3, hours, 4, hours, 5, hours, 6 hours, 7
hours, 8, hours, 9 hours, 10 hours or 11 hours), and sometimes is
about 3 hours to about 9 hours. In some embodiments, the yeast is a
Candida spp. yeast, or another yeast described herein.
[0378] Target Product Production, Isolation and Yield
[0379] In various embodiments a fatty dicarboxylic acid (e.g.,
octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) is isolated or purified from the culture media
or extracted from the engineered microorganisms. In some
embodiments, fermentation of feedstocks by methods described herein
can produce a target fatty dicarboxylic acid product (e.g., sebacic
or dodecanedioic acid) at a level of about 10% to about 100% of
theoretical yield (e.g., about 15%, about 20%, about 25% or more of
theoretical yield (e.g., 25% or more, 26% or more, 27% or more, 28%
or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or
more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or
more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or
more, 44% or more, 45% or more, 46% or more, 47% or more, 48% or
more; 49% or more, 50% or more, 51% or more, 52% or more, 53% or
more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or
more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or
more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or
more, 69% or more, 70% or more, 71 % or more, 72% or more, 73% or
more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or
more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or
more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or
more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or
more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or
more; or 99% or more of theoretical yield). The term "theoretical
yield" as used herein refers to the amount of product that could be
made from a starting material if the reaction is 100% complete.
Theoretical yield is based on the stoichiometry of a reaction and
ideal conditions in which starting material is completely consumed,
undesired side reactions do not occur, the reverse reaction does
not occur, and there are no losses in the workup procedure. Culture
media may be tested for target product (e.g., sebacic or
dodecanedioic acid) concentration and drawn off when the
concentration reaches a predetermined level. Detection methods are
known in the art, including but not limited to chromatographic
methods (e.g., gas chromatography) or combined chromatographic/mass
spectrometry (e.g., GC-MS) methods. Target product (e.g., sebacic
or dodecanedioic acid) may be present at arrange of levels as
described herein.
[0380] A target fatty dicarboxylic acid product sometimes is
retained within an engineered microorganism after a culture process
is completed, and in certain embodiments, the target product is
secreted out of the microorganism into the culture medium. For the
latter embodiments, (i) culture media may be drawn from the culture
system and fresh medium may be supplemented, and/or (ii) target
product may be extracted from the culture media during or after the
culture process is completed. Engineered microorganisms may be
cultured on or in solid, semi-solid or liquid media. In some
embodiments media, is drained from cells adhering to a plate. In
certain embodiments, a liquid-cell mixture is centrifuged at a
speed sufficient to pellet the cells but not disrupt the cells and
allow extraction of the media, as known in the art. The cells may
then be resuspended in fresh media. Target product may be purified
from culture media according to known methods know in the art.
[0381] In some embodiments, a target diacid is present in a product
containing other diacids and/or byproducts. The target diacid can
be purified from the other diacids and/or byproducts using a
suitable purification procedure. A partially purified or
substantially purified target diacid may be produced using a
purification process.
[0382] Provided herein are non-limiting examples of methods useful
for recovering target product from fermentation broth, and/or
isolating/partially purifying a target fatty dicarboxylic acid
product from non-target products when utilizing mixed chain length
feedstocks. Recovery of a fatty dicarboxylic acid (e.g.,
octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) from fermentation broth can be accomplished
using a variety of methods. Optionally, one can first employ a
centrifugation step to separate cell mass and a fatty dicarboxylic
acid (e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) from the aqueous phase. A fatty dicarboxylic
acid (e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid/octadecanedioic acid,
eicosanedioic acid) has limited solubility in water under
fermentation conditions, and has a density similar to that of
water. Upon centrifugation, the majority of fatty dicarboxylic acid
(e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) will be pulled away from the water stream, and
be concentrated in the cell mass stream. The concentrated fatty
dicarboxylic acid stream will then be further concentrated via
filtration steps (e.g., solid dodecanedioic acid will be retained
on a filter, allowing water to pass through, concentrating the
product). Once the fatty dicarboxylic acid (e.g., octanedioic acid,
decanedioic acid, dodecanedioic acid, tetradecanedioic acid,
hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid) is
concentrated to the desired level, the temperature will be
increased to above its melting point of 130.degree. C. After the
fatty dicarboxylic acid is melted, the remaining impurities are
removed via filtration; the final product is recovered by
decreasing the temperature, allowing the fatty dicarboxylic acid to
solidify, and collecting the solid product.
[0383] Alternatively, a fatty dicarboxylic acid (e.g., octanedioic
acid, decanedioic acid, dodecanedioic acid, tetradecanedioic acid,
hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid) can
be recovered from fermentation broth by first extracting the broth
with an organic solvent in which a fatty dicarboxylic acid (e.g.,
octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) is soluble (e.g., ethanol). The organic solvent
phase can then be filtered through various membranes to further
purify the fatty dicarboxylic acid. Subsequent extractions with the
same or a different organic solvent can then be performed and each
round of extraction can be followed by membrane filtration to
further concentrate the fatty dicarboxylic acid. The organic
solvent can be evaporated, leaving the fatty dicarboxylic acid
behind as a residue and the residue can be dried to provide the
fatty dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid) in solid form.
[0384] In certain embodiments, target product is extracted from the
cultured engineered microorganisms. The microorganism cells may be
concentrated through centrifugation at a speed sufficient to shear
the cell membranes. In some embodiments, the cells may be
physically disrupted (e.g., shear force, sonication) or chemically
disrupted (e.g., contacted with detergent or other lysing agent).
The phases may be separated by centrifugation or other method known
in the art and target product may be isolated according to known
methods.
[0385] Commercial grade target product sometimes is provided in
substantially pure form (e.g., 90% pure or greater, 95% pure or
greater, 99% pure or greater or 99.5% pure or greater). In some
embodiments, target product may be modified into any one of a
number of downstream products. For example, a fatty dicarboxylic
acid (e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) may be polycondensed with hexamethylenediamine
to produce nylon. Nylon may be further processed into fibers for
applications in carpeting, automobile tire cord and clothing. A
fatty dicarboxylic acid (e.g., octanedioic, acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid) is also used for
manufacturing plasticizers, lubricant components and polyester
polyols for polyurethane systems. Various esters of food grade
fatty dicarboxylic acids (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid) are used as components in
fragrance manufacture, gelling aids, flavorings, acidulant,
leavening and buffering agent. A fatty dicarboxylic acid (e.g.,
octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) has two carboxylic acid (--COOH) groups, which
can yield two kinds of salts. Its derivatives, acyl halides,
anhydrides, esters, amides and nitriles, are used in making a
variety of downstream products through further reactions of
substitution, catalytic reduction, metal hydride reduction,
diborane reduction, keto formation with organometallic reagents,
electrophile bonding at oxygen, and condensation.
[0386] Target product may be provided within cultured microbes
containing target product, and cultured microbes may be supplied
fresh or frozen in a liquid media or dried. Fresh or frozen
microbes may be contained in appropriate moisture-proof containers
that may also be temperature controlled as necessary. Target
product sometimes is provided in culture medium that is
substantially cell-free. In some embodiments target product or
modified target product purified from microbes is provided, and
target product sometimes is provided in substantially pure form. In
certain embodiments crystallized or powdered target product is
provided. Dodecanedioic acid (1,12 dodecanedioic acid; DDDA) is a
white powder or crystal with a melting point of between 260.degree.
F. and 266.degree. F. Sebacic acid (1,8 ocatanedicarboxylic acid)
is also a white powder or crystal with a melting point of between
268.degree. F. and 274.degree. F. A crystallized or powdered fatty
dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid) may be transported in a
variety of containers including one ton cartons, drums, 50 pound
bags and the like.
[0387] In certain embodiments, a fatty dicarboxylic acid target
product (e.g., dodecanedioic acid or sebacic acid) is produced with
a yield of about 0.50 grams of target product per gram of feedstock
added, or greater; 0.51 grams of target product per gram of
feedstock added, or greater; 0.52 grams of target product per gram
of feedstock added, or greater; 0.53 grams of target product per
gram of feedstock added, or greater; 0.54 grams of target product
per gram of feedstock added, or greater; 0.55 grams of target
product per gram of feedstock added, or greater; 0.56 grams of
target product per gram of feedstock added, or greater; 0.57 grams,
of target product per gram of feedstock added, or greater; 0.58
grams of target product per gram of feedstock added, or greater;
0.59 grams of target product per gram of feedstock added, or
greater; 0.60 grams of target product per gram of feedstock added,
or greater; 0.61 grams of target product per gram of feedstock
added, or greater; 0.62 grams of target product per gram of
feedstock added, or greater; 0.63 grams of target product per gram
of feedstock added, or greater; 0.64 grams of target product per
gram of feedstock added, or greater; 0.65 grams of target product
per gram of feedstock added, or greater; 0:66 grams of target
product per gram of feedstock added, or greater; 0.67 grams of
target product per gram of feedstock added, or greater; 0.68 grams
of target product per gram of feedstock added, or greater; 0.69
grams of target product per gram of feedstock added, or greater;
0.70 grams of target product per gram of feedstock added or
greater; 0.71 grams of target product per gram of feedstock added,
or greater; 0.72 grams of target product per gram of feedstock
added, or greater; 0.73 grams of target product per gram of
feedstock added, of greater; 0.74: grams of target product per gram
of feedstock added, or greater; 0.75 grams of target product per
gram of feedstock added, or greater; 0.76 grams of target product
per gram of feedstock added; or greater; 0.77 grams of target
product per gram of feedstock added, or greater; 0.78 grams of
target product per gram of feedstock added, or greater; 0.79 grams
of target product per gram of feedstock added, or greater; 0.80
grams of target product per gram of feedstock added, or greater;
0.81 grams of target product per gram of feedstock added, or
greater; 0.82 grams of target product per gram of feedstock added,
or greater; 0.83 grams of target product per gram of feedstock
added, or greater; 0.84 grams of target product per gram of
feedstock added, or greater; 0.85 grams of target product per gram
of feedstock added, or greater; 0.86 grams of target product per
gram of feedstock added; or greater; 0.87 grams of target product
per gram of feedstock added, or greater; 0.88 grams of target
product per gram of feedstock added, or greater; 0.89 grams of
target product per gram of feedstock added, or greater; 0:90 grams
of target product per gram of feedstock added, or greater; 0.91
grams of target product per gram of feedstock added, or greater;
0.92 grams of target product per gram of feedstock added, or
greater; 0.93 grams of target product per gram of feedstock added,
or greater; 0.94 grams of target product per gram of feedstock
added, or greater; 0.95 grams of target product per gram of
feedstock added, or greater; 0.96 grams of target product per gram
of feedstock added, or greater; 0.97 grams of target product per
gram of feedstock added, or greater; 0.98 grams of target product
per gram of feedstock added, or greater; 0.99 grams of target
product per gram of feedstock added, or greater; 1.0 grams of
target product per gram of feedstock added, or greater; 1.1 grams
of target product per gram of feedstock added, or greater; 1.2
grams of target product per gram of feedstock added, or greater;
1.3 grams of target product per gram of feedstock added, or
greater; 1.4 grams of target product per gram of feedstock added,
or greater; or about 1.5 grams of target product per gram of
feedstock added, or greater.
[0388] In certain embodiments, a fatty dicarboxylic acid (e.g.,
octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) is produced with a yield of greater than about
0.15 grams per gram of the feedstock (e.g., dodecane, mixed chain
length alkanes, lauric acid, mixed chain length fatty acids, oil,
the like or combinations of the foregoing). In some embodiments, a
fatty dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,
dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,
octadecanedioic acid, eicosanedioic acid) is produced at between
about 10% and about 100% of maximum theoretical yield of any
introduced feedstock ((e.g., about 15%, about 20%, about 25% or
more of theoretical yield (e.g., 25% or more, 26% or more, 27% or
more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or
more, 33% or more, 34% or more, 35% of more, 36% or more, 37% or
more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or
more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or
more, 48% or more, 49% or more, 50% or more, 51% or more, 52% or
more, 53 % or more, 54% or more, 55% or more, 56% or more, 57% or
more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or
more, 63% or more, 64% or more, 65% or more, 66% of more, 67% or
more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or
more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or
more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or
more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or
more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or
more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or
more, 98% or more, or 99% or more of theoretical maximum yield). In
certain embodiments, a fatty dicarboxylic acid (e.g., octanedioic
acid, decanedioic acid, dodecanedioic acid, tetradecanedioic acid,
hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid) is
produced in a concentration range of between about 50 g/L to about
1000 g/L of culture media (e.g., about 50 g/L, about 55 g/L, about
60 g/L, about 65 g/L, about 70 g/L, about 75 g/L, about 80 g/L,
about 85 g/L, about 90 g/L, about 95 g/L, about 100 g/L, about 110
g/L, about 120 g/L, about 130 g/L, about 140 g/L, about 150 g/L,
about 160 g/L, about 170 g/L, about 180 g/L, about 190 g/L, about
200 g/L, about 225 g/L, about 250 g/L, about 275 g/L, about 300
g/L, about 325 g/L, about 350 g/L, about 375 g L, about 400 g/L,
about 425 g/L, about 450 g/L, about 475 g/L, about 500 g/L, about
550 g/L, about 600 g/L, about 650 g/L, about 700 g/L, about 750
g/L, about 800 g/L, about 850 g/L, about 900 g/L, about 950 g/L, of
about 1000 g/L).
[0389] In some embodiments, a fatty dicarboxylic acid (e.g.,
octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) is produced at a rate of between about 0.5
g/L/hour to about 5 g/L/hour (e.g., about 0.5 g/L/hour, about 0.6
g/L/hour, about 0.7 g/L/hour, about 0.8 g/L/hour, about 0.9
g/L/hour, about 1.0 g/L/hour, about 1.1 g/L/hour, about 1.2
g/L/hour, about 1.3 g/L/hour, about 1.4 g/L/hour, about 1.5
g/L/hour, about 1.6 g/L/hour, about 1.7 g/L/hour, about 1.8
g/L/hour, about 1.9 g/L/hour, about 2.0 g/L/hour, about 2.25
g/L/hour, about 2.5 g/L/hour, about 2.75 g/L/hour, about 3.0
g/L/hour, about 3.25 g L/hour, about 3.5 g/L/hour, about
3.75'g/L/hour, about 4.0 g/L/hour, about 4.25 g/L/hour, about 4.5
.g/L/hour, about 4.75 g/L/hour, or about 5.0 g/L/hour.) In certain,
embodiments, the engineered organism comprises between about a
5-fold to about a 500-fold increase in a fatty-dicarboxylic acid
(e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,
tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,
eicosanedioic acid) production when compared to wild-type or
partially engineered organisms of the same strain, under identical
fermentation conditions (e.g., about a 5-fold increase, about a
10-fold increase, about a 15-fold increase, about a 20-fold
increase, about a 25-fold increase, about a 30-fold increase, about
a 35-fold increase, about a 40-fold increase, about a 45-fold
increase, about a 50-fold increase, about a 55-fold increase, about
a 60-fold increase, about a 65-fold increase, about a 70-fold
increase, about a 75-fold increase, about a 80-fold increase, about
a 85-fold increase, about a 90-fold increase, about a 95-fold
increase, about a 100-fold increase, about a 125-fold increase,
about a 150-fold increase, about a 175-fold increase, about a
200-fold increase, about a 250-fold increase, about 300-fold
increase, about a 350-fold increase, about a 400-fold increase,
about a 450-fold increase, or about a 500-fold increase).
[0390] In certain embodiments, the maximum theoretical yield
(Y.sub.max) of dodecanedioic and in a fully beta-oxidation blocked
engineered microorganism is about 1.15 grams of dodecanedioic acid
produced per gram of lauric acid added. In some embodiments, the
maximum theoretical yield (Y.sub.max) of dodecanedioic acid in a
fully beta-oxidation blocked engineered microorganism is about 1.07
grams of dodecanedioic acid produced per gram of methyl laurate
added. In certain embodiments, the maximum theoretical yield
(Y.sub.max) of dodecanedioic acid in a partially beta-oxidation
blocked engineered microorganism is about 0.82 grams of
dodecanedioic acid produced per gram of oleic acid added. In some
embodiments, the maximum theoretical yield (Y.sub.max) of
dodecanedioic acid in a partial beta-oxidation blocked engineered
microorganism is about 0.95 grams of dodecanedioic acid produced
per gram of coconut oil added. The percentage of Y.sub.max for the
engineered microorganism under conditions in which dodecanedioic
acid is produced is calculated as (%
Y.sub.max)=Y.sub.p/s/Y.sub.max*100, where
(Y.sub.p/s)=[dodecanedioic acid (g/L)]*final volume of culture in
flask (L)]/[feedstock added to flask (g)]. In some embodiments, the
engineered microorganism produces dodecanedioic acid at about 10%
to about 100% of maximum theoretical yield.
[0391] In certain embodiments, the maximum theoretical yield
(Y.sub.max) of sebacic acid in a fully beta-oxidation blocked
engineered microorganism is about 1.42 grams of sebacic-acid
produced per gram of decane added. In some embodiments, the maximum
theoretical yield (Y.sub.max) of sebacic acid in a fully
beta-oxidation blocked engineered microorganism is about 1.17 grams
of sebacic acid produced per gram of capric acid added. In certain
embodiments, the maximum theoretical yield (Y.sub.max) of sebacic
acid in a partially beta-oxidation blocked engineered microorganism
is about 0.83 grams of sebacic acid produced per gram of coconut
oil added. In some embodiments, the maximum theoretical yield
(Y.sub.max) of sebacic acid in a partially beta-oxidation blocked
engineered microorganism is about 0.72 grams of sebacic acid
produced per gram of oleic acid added. The percentage of Y.sub.max
for the engineered microorganism under conditions in which sebacic
acid is produced is calculated as (%
Y.sub.max)=Y.sub.p/s/Y.sub.max*100, where (Y.sub.p/s)=[sebacic acid
(g/L)]*final volume of culture in flask (L)]/[feedstock added to
flask (g)]. In some embodiments, the engineered microorganism
produces sebacic acid at about 10% to about 100% of maximum
theoretical yield.
EXAMPLES
[0392] The examples set forth below illustrate certain embodiments
and do not limit the technology. Certain examples set forth below
utilize standard recombinant DNA and other biotechnology protocols
known in the art. Many such techniques are described in detail in
Maniatis, T., E. F. Fritsch and J. Sambrook (1982) Molecular
Cloning a Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. DNA mutagenesis can be accomplished using the
Stratagene (San Diego, Calif.) "QuickChange" kit according to the
manufacturer's instructions.
[0393] Non-limiting examples of recombinant DNA techniques and
genetic manipulation of microorganisms are described herein. In
some embodiments, strains of engineered organisms described herein
are mated to combine genetic backgrounds to further enhance carbon
flux management through native and/or engineered pathways described
herein, for the production of a desired target product (e.g.,
sebacic or dodecanedioic acid).
Example 1
Conversion of Decane to Sebacic Acid in Shake Flask
Fermentation
[0394] 50 mL of SP92 medium (6.7 g/L yeast nitrogen base, 3.0 g/L
yeast extract, 3.0 g/L (NH.sub.4).sub.2SO.sub.4, 1.0 g/L
K.sub.2HPO.sub.4, 1.0 g/L KH.sub.2PO.sub.4, 75 g/L dextrose) was
inoculated with a single colony of a completely beta-oxidation
blocked strain of Candida tropicalis (ATCC20962) and the culture
was grown overnight at 30.degree. C., with shaking at about 300
rpm. Cells were pelleted by centrifugation for 10 minutes at
4.degree. C. and 1,050.times.g and the supernatant discarded. Cells
were resuspended in 20 mL TB-low nitrogen (low-N) media (1.7 g/L
yeast nitrogen base without ammonium sulfate, 3.0 g/L yeast
extract, 1.0 g/L K.sub.2HPO.sub.4, 1.0 g/L KH.sub.2PO.sub.4) and
transferred to a new sterile 250 mL glass baffled flask and
incubated at 30.degree. C., with shaking at about 250 rpm,
utilizing the following feeding schedule: dextrose fed to 0.1% at
0, 1, 2, 3, 4, and 5 hours, dextrose fed to 5% at 30 hours, decane
fed to 0.7% at 0, 5, 30, and 48 hours. Samples were removed for gas
chromatographic (GC) analysis at 0, 4, 30, and 72 hours. The GC
profile showed that the culture accumulated the C10 dicarboxylic
acid (sebacic acid) with very little accumulation of the C10
monocarboxylic acid (capric acid), as shown in FIG. 9. After 72
hours of incubation the concentration of sebacic acid was 0.94 g/L
and the capric acid concentration was 0.01 g/L. There was no
significant accumulation of any other monoacid or diacid.
Example 2
Conversion of Capric Acid to Sebacic Acid in Shake Flask
Fermentation
[0395] 5 mL of SP92-glycerol medium (6.7 g/L yeast nitrogen base,
3.0 g/L yeast extract, 3.0 g/L (NH4)2SO4, 1.0 g/L K2HPO4, 1.0 g/L
KH2PO4, 75 g/L glycerol) was inoculated with a single colony of
Candida tropicalis (ATCC20962) and the starter culture was grown
overnight at 30.degree. C., with shaking at about 250 rpm.
Variations of SP92 media recipes are known, non-limiting examples
of which include the addition of dextrose and/or glycerol, the like
or combinations thereof. SP92 media, as referred to herein, can
include dextrose and/or glycerol. The starter culture was then used
to inoculate 25 mL cultures in the same medium to an initial
OD.sub.600nm of 0.4 and grown overnight at 30.degree. C., with
shaking at about 300 rpm. Cells were pelleted by centrifugation for
10 minutes at 4.degree. C. and 1,050.times.g and the supernatant
discarded. Cells were resuspended in 12.5 mL TB-lowN media+glycerol
(1.7 g/L yeast nitrogen base without ammonium sulfate, 3.0 g/L
yeast extract; 1.0 g/L K2HPO4, 1.0 g/L KH2PO4, 75 g/L glycerol) and
transferred to a new sterile 250 mL glass baffled flask. Cultures
were fed 0.05% or 0.1% capric acid and incubated at 30.degree. C.,
with shaking at about 300 rpm. After 24 hours incubation cultures
were fed glycerol to 75 g/L and incubation continued before
sampling for GC at 48 hours. GC analysis showed that nearly all
capric acid was converted to sebacic acid under both starting
concentrations of capric acid, as shown in FIG. 10.
Example 3
Fermentation Procedure for Conversion of Decline to Sebacic
Acid
[0396] Filter sterilized modified SP92-glycerol fermentation medium
(6.7 g/L yeast nitrogen base, 3.0 g/L yeast extract, 3.0 g/L
(NH4)2SO4, 1.0 g/L K2HPO4, 1.0 g/L KH2PO4, 20 g/L glycerol) is
transferred to a sterile fermentation vessel. Growth of Candida
tropicalis (ATCC20962) is inoculated to an initial OD.sub.600nm of
about 1.0 with a 5% inoculum and growth carried out under the
following conditions: 30.degree. C. with shaking at about 1000 rpm,
1 volume per volume per minute aeration (vvm), pH 5.8 and initial
volume of 0.3 L. Growth proceeds for approximately 8 hours and the
conversion phase is initiated by the addition of decane to 2 g/L.
Continuous feeds for decane (1 g/L-h) and glucose (1.5 g/L-h) are
initiated at the same time as the addition of the decane bolus.
Fermentation conditions are maintained at 30.degree. C., 1000 rpm,
1 vvm, and pH 5.8 for 44 hours.
[0397] Samples were collected for GC analysis at 44 hours after
initiating the conversion phase. The data, presented in FIG. 16,
shows that the decane was converted exclusively to the C10
dicarboxylic acid, sebacic acid. Significant evaporative losses
from the decane feed bottles prevented an accurate determination of
product yield.
Example 4
Conversion of Mixed Fatty Acid Feedstock to Mixed Diacid Products
Containing Sebacic Acid in Shake Flask Fermentation
[0398] 5 mL of SP92-glycerol medium (6.7 g/L yeast nitrogen base,
3.0 g/L yeast extract, 3.0 g/L (NH4)2SO4, 1.0 g/L K2HPO4, 1.0 g/L
KH2PO4, 75 g/L glycerol) is inoculated with a single colony of
Candida tropicalis (ATCC20962) and grown as described in Example 2.
25 mL of the same media is inoculated using overnight cultures to
an initial OD.sub.600nm of 0.4 and grown overnight at 30.degree.
C., with shaking at about 300 rpm. Cells are pelleted by
centrifugation for 10 minutes at 4.degree. C. and 1,050.times.g and
the supernatant discarded. Cells are suspended in 12.5 mL TB-lowN
media without carbon source (1.7 g/L yeast nitrogen base-without
ammonium sulfate; 3.0 g/L yeast extract, 1.0 g/L K2HPO4, 1.0 g/L
KH2PO4) and transferred to a new sterile 250 mL glass baffled
flask. Cultures are fed 0.05% capric acid, 0.05% methyl laurate,
and 30 g/L glycerol and incubated at 30.degree. C., 300 rpm. After
24 hours of incubation cultures are sampled for GC analysis.
[0399] The results, presented in FIG. 17, show that the C12 and C10
fatty acids were converted to dicarboxylic acids of the same chain
length (e.g., C12 and C10 dicarboxylic acids), with no evidence of
chain shortening of the diacids (e.g., no significant levels of
monocarboxylic acids were detected).
Example 5
Conversion of Long Chain Fatty Acids to Mixed Diacids
[0400] SP92 fermentation medium was filter sterilized and
transferred to a sterile fermentation vessel. Growth of a partially
beta-oxidation blocked strain of Candida tropicalis (sAA106) was
initiated with a 10% inoculum (initial OD.sub.600nm=3.0) and grown
under the following conditions: of 30.degree. C. with shaking at
about 1200 rpm, 1 vvm, pH 6.1, and initial volume of 0.3 L. Growth
continued until the glucose concentration dropped to less than 2
g/L at which time the conversion phase was initiated by increasing
the pH to 8.0 by the addition of 6N KOH and by the addition of
methyl myristate to 30 g/L. Immediately following the methyl
myristate bolus a continuous feed of glucose was initiated at a
rate of 1.5 g/L-h. Fermentation conditions were maintained at
30.degree. C., 1200 rpm, 1 vvm, and pH 8.0 for 90 hours with
boluses of 30 g/L methyl myristate at 24, 48, and 72 hours after
initiation of conversion. Samples for GC were collected at 24, 48,
72, and 90 hours. The diacid profile graphically illustrated in
FIG. 11 shows an accumulation of dicarboxylic acids ranging in
chain-length from 6 to 14 carbons long, including sebacic acid. The
methyl myristate substrate (methyl ester of myristic acid) is first
converted to the C14 dicarboxylic acid via the .beta.-oxidation
pathway before being shortened by two carbon increments via the
cyclic .beta.-oxidation pathway. The glucose co-feed employed
during the fermentation represses the .beta.-oxidation pathway such
that all chain-lengths of diacid accumulate. Manipulation of diacid
chain-length distribution is being investigated by altering the
glucose co-feed, rate in the fermentation medium, thereby allowing
growth under varying glucose concentrations.
Example 6
Fermentation Procedure for Conversion of Mixed Long-Chain Fatty
Acids to Mixed Diacids of Shorter Chain Length
[0401] SP92 fermentation medium without glycerol was filter
sterilized and transferred to a sterile fermentation vessel.
Autoclaved virgin coconut oil was added to the vessel to a final
concentration of 80 g/L. A partially beta-oxidation blocked Candida
tropicalis strain (sAA496) was inoculated to an initial
OD.sub.600nm of 1.0 with a 5% inoculum and grown under the
following conditions: 30.degree. C. with shaking at about 1200 rpm,
1 vvm, initial pH 6.5 and initial volume of 1.0 L. The effect of pH
on the distribution of fatty acid chain lengths was determined by
manipulating the pH of the fermentation media. The pH of the
fermentation was either 1) increased to pH 7.5 and controlled at
that pH for the entire run, 2) allowed to drop-naturally due to the
growth of the culture before controlling at pH 6.0 for the rest of
the run; or 3) allowed to drop naturally due to the growth of the
culture before controlling at pH 4.5 for the rest of the run.
Samples were collected for GC analysis after 140 hours of
fermentation time. The product diacid composition was shown to
shift to longer chain diacids with increasing pH, as shown in the
TABLE 1 below.
TABLE-US-00001 TABLE 1 Diacid composition (fraction of total
diacids) C12 Diacid Sebacic Acid Suberic Acid Adipic Acid pH 4.5
0.00 0.00 0.68 0.32 pH 6.0 0.03 0.10 0.75 0.12 pH 7.5 0.16 0.17
0.62 0.05
Example 7
Conversion of Capric Acid to Sebacic Acid in Shake Flask
Fermentations using Fully Beta-Oxidation Blocked Strains Having
Additional Genetic Modifications in the Omega Oxidation Pathway
[0402] Various genetically modified strains of Candida tropicalis
were inoculated into 5 mL of SP92 medium (6.7 g/L yeast nitrogen
base, 3.0 g/L yeast extract, 3.0 g/L (NH.sub.4).sub.2SO.sub.4, 1.0
g/L K.sub.2HPO.sub.4, 1.0 g/L KH.sub.2PO.sub.4, 75 g/L glycerol).
The strains included a completely beta-oxidation blocked strain of
Candida tropicalis (sAA003), as well as derivatives of sAA003 with
amplified components of the omega-oxidation pathway (e.g., various
cytochrome P450s, cytochrome P450 reductase or combinations
thereof) and the cultures grown overnight at 30.degree. C., with
shaking at about 250 rpm. These starter cultures were then used to
inoculate 25 mL cultures in the same medium and grown overnight at
30.degree. C., with shaking at about 250 rpm. Cells were pelleted
by centrifugation for 10 minutes at 4.degree. C. and 1,050.times.g
and the supernatant discarded. Cells were resuspended in 12.5 mL
TB-lowN media+glycerol (1.7 g/L yeast nitrogen base without
ammonium sulfate, 3.0 g/L yeast extract, 1.0 g/L K.sub.2HPO.sub.4,
1.0 g/L KH.sub.2PO.sub.4, 75 g/L glycerol) and transferred to a new
sterile 250 mL glass baffled flask. Cultures were fed 0.05% from a
5% capric acid solution in ethanol and incubated at 30.degree. C.,
with shaking at about 300 rpm. After 24 hours incubation cultures
were fed glycerol to 30 g/L and an additional bolus of 0.05% capric
acid. Incubation continued before sampling for GC at 24, 48, and 72
hours. The results are shown in FIG. 12. GC analysis showed that a
greater proportion of capric acid was converted to sebacic acid
when particular elements of the omega-oxidation pathway are
amplified. The data are presented as % of theoretical maximum
yield. Strains which include genetic modifications to CYPA18 and
CYPA19 achieve approximately 80% of theoretical maximum yield in
conversion of capric acid to sebacic acid. The strain designated
+CPR+A18 has about 30 copies of CYPA18, whereas the strain
designated +CPR+A19 has about 7 copies of CYPA19.
Example 8
Conversion of Methyl-Laurate to Dodecanedioic Acid in Shake Flask
Fermentation
[0403] 5 mL of SP92 glycerol medium (6.7 g/L yeast nitrogen base,
3.0 g/L yeast extract, 3.0 g/L (NH4)2SO4, 1.0 g/L K2HPO4, 1.0 g/L
KH2PO4, 75 g/L glycerol) was inoculated with a single colony of a
completely beta-oxidation blocked strain of Candida tropicalis
(ATCC20962), as well as, modified derivatives of this strain with
amplified components of the omega-oxidation pathway, and the
cultures grown overnight at 30.degree. C., with shaking at about
250 rpm. The starter cultures were then used to inoculate 25 mL
cultures of the same medium and grown overnight at 30.degree. C.,
with shaking at about 250 rpm. Cells were pelleted by
centrifugation for 10 minutes at 4.degree. C. and 1,050.times.g and
the supernatant discarded. Cells were resuspended in 12.5 mL SP-92
glycerol medium and transferred to a sterile 250 mL glass baffled
flask. Cultures were fed 2% (v/v) methyl laurate and incubated at
30.degree. C., with shaking at about 300 rpm. After 24 hours
incubation, cultures were fed glycerol to 60 g/L and incubation
continued before sampling for GC at 48 hours. GC analysis showed
that amplification of certain components of the omega oxidation
pathway allow for increased conversion to dodecanedioic acid (FIG.
13).
Example 9
Alteration of Acyl CoA Oxidase Substrate Specificity
[0404] The substrate specificity of the peroxisomal-acyl-CoA
oxidase enzymes POX4 and POX5 have been shown to be involved in the
control of the diacid product chain-length in fermentations of
Candida tropicalis fed a mixed chain-length fatty acid feedstock.
Reduction or elimination of POX4 activity, POX5 activity or POX4
activity and POX5 activity, effects the carbon chain-length
distribution of dicarboxylic acids produced in C. tropicalis.
Acyl-CoA oxidase is the first enzyme in the cyclic beta-oxidation
pathway that shortens a substrate by two carbons each cycle. Thus
the acyl-CoA oxidase activity serves as the pathway entry point for
substrates entering into the beta-oxidation pathway. Altering the
substrate specificity an acyl-CoA oxidase activity such that it is
not active on substrate carbon chains shorter than a desired carbon
chain length (e.g., C8, C10, C12, C14 and the like), can inhibit
shortening of carbon chains below a chosen threshold, allowing
accumulation of a desired target chain length and product (e.g.,
C12, dodecanedioic acid).
[0405] The native acyl-CoA oxidase isozymes in C. tropicalis, Pox4p
and Pox5p have different substrate specificities. The Pox4p isozyme
has a broad substrate specificity while the Pox5p isozyme has a
narrow substrate specificity. In strains that are Pox4, Pox5 the
chain length of the diacid product is determined by the substrate
specificity of the Pox5p isozyme and the main product as adipic
acid.
[0406] To maximize production of desired diacid products of longer
chain lengths (e.g., C12) in fermentations, genetically modified
organisms containing an acyl-CoA oxidase activity with a substrate
chain-length, specificity appropriate for the chain-length of the
desired diacid product can be engineered, in some embodiments. The
source of the acyl-CoA oxidase activity or the method of
engineering the acyl-CoA oxidase activity may vary. Non-limiting
examples of organisms which can be used to provide polynucleotide
sequences suitable for use in engineering altered substrate
specificity acyl-CoA oxidase activities include: plants (e.g.,
Arabidopsis, Cucurbita (e.g., pumpkin, squash), Oryza (e.g.,
rice)); animals (e.g., Bos (e.g., bovine), Cavia (e.g., guinea
pig), Mus (e.g., mouse), Rattus (e.g., rat), Phascolarctos (e.g.,
Koala), primates (e.g., orangutans)); molds (e.g., Dictyostelium
(e.g., slime molds)); insects (e.g., Drosophila); Yeast (e.g.,
Yarrowia lipolytica, Candida maltosa, Candida glabrata, Ashbya
gossypii, Debaryomyces hansenii, Kluyveromyces lactis, Pichia
pastoris, Saccharomyces cerevisiae); bacteria (e.g., Eschericia
coli); cyanobacteria; nematodes (e.g., Caenorhabditis); and
humans.
[0407] Acyl-CoA oxidase activities with different substrate
chain-length specificities can be identified by: [0408] 1)
Selecting acyl-CoA oxidase genes from heterologous organisms that
contain different substrate chain-length specificities. The
identified genes can be transferred into a Candida strain deleted
for all acyl-CoA oxidase activity. The only acyl-CoA oxidase
activity detectable in such a genetically modified organism may be
that imparted by the heterologous gene. [0409] 2) Engineering an
acyl-CoA oxidase gene library by domain swapping from multiple
acyl-CoA oxidase genes to produce a library of non-native chimeric
acyl-CoA oxidase genes. The library of chimeric genes can be
transferred into a strain of C. tropicalis deleted for all acyl-CoA
oxidase activity. The only detectable acyl-CoA oxidase activity may
be that imparted by an engineered gene from the library of
non-native chimeric acyl-CoA oxidase genes. [0410] 3) Engineering
an acyl-CoA oxidase gene library by random mutagenesis. A naturally
occurring or engineered acyl-CoA oxidase activity with a substrate
chain-length specificity close to that desired can be used as the
basis for random mutagenesis, followed by screening and/or
selection in an effort to generate and identify an altered activity
with the desired substrate chain-length specificity. The library of
genes can be transferred into Candida strain deleted for all
acyl-CoA oxidase activity. The only detectable acyl-CoA oxidase
activity maybe that imparted by the gene from the randomly
mutagenized library. [0411] 4) Engineering an acyl-CoA oxidase gene
by intelligent design and directed mutation using protein
structural information to guide the position and identity of the
amino acid(s) to be replaced. The engineered gene(s) can be
transferred into a Candida strain deleted for all acyl-CoA oxidase
activity. The only detectable acyl-CoA oxidase activity may be that
imparted by the engineered gene(s).
[0412] A non-limiting example of a post-engineering method for
selecting genes that impart the desired substrate chain-length
specificity is provided herein. Selection is performed by growth on
substrates of different chain lengths that are provided as the only
carbon source. Growth of the cells on certain substrates but not
others often reflects the substrate chain-length specificity of the
acyl-CoA oxidase enzyme present in the strain. Candida tropicalis
can utilize alkanes provided in the gas phase as its sole carbon
source for growth. Alkanes of different chain lengths are provided
by soaking a filter paper in the appropriate alkane, and inverting
a solid growth media without a carbon source over the filter paper,
with each specific carbon source (e.g., specific chain length
alkane) provided in a different petri dish. Serially diluted C.
tropicalis carrying the altered specificity acyl-CoA oxidase genes
are spotted on the solid growth media as a growth selection for the
chain-length specificity of the acyl-CoA oxidase enzyme in each
strain. Shown in FIGS. 14 and 15 are a schematic representation of
the selection process, which provides an alkane as a gas phase
carbon source, as described herein The solid growth media is an
agar medium containing yeast nitrogen base without amino acids or
any other carbon source. The plated cells are inverted over a lid
containing a filter paper soaked with an alkane of appropriate
chain length that evaporates and provides the carbon source through
the gas phase, as shown in FIG. 14.
[0413] Candida strains containing altered acyl-CoA oxidase
activities generated as described herein are selected and/or
screened using the method described herein. Strains carrying
different altered acyl-CoA oxidase activities (e.g., strain 1 (S1),
strain 2 (S2), strain 3 (S3), strain 4 (S4)) are grown overnight in
a rich medium (e.g., YPD). Overnight cultures are centrifuged and
washed to remove any traces of residual rich medium and serial
dilutions of the cells are prepared in a phosphate buffered
solution. The serial dilutions of each strain are spotted onto
multiple YNB agar plates (growth medium having no amino acids or
other carbon sources), the individual plates inverted over filter
papers soaked in the appropriate chain length alkane, and the plate
incubated at 30.degree. C. The growth of the strains is dependent
upon the chain-length specificity of the acyl-CoA oxidase. In order
to utilize the particular alkane for growth the provided
chain-length must be able to enter the beta-oxidation pathway. The
shortest chain-length at which a certain strain is able to grow
indicates the shortest chain-length of the acyl-CoA oxidase
isozymes substrate specificity. An example is provided in FIG. 15.
FIG. 15 illustrates that strain S4 can grow on decane, but is
unable to grow on octane. Therefore the modified acyl-CoA oxidase
activity of strain S4 has a substrate chain-length specificity that
inhibits the utilization of 8 carbon molecules and the diacid
product from fermentations with this strain typically result in an
8 carbon diacid. Acyl-CoA oxidase activities with any desired
specificity can be selected and/or screened using the method
described herein.
[0414] It will be understood that the example presented herein is a
generalized method used to describe the selection/screening
process. The feedstocks used for the selection and screening
process are altered to suit the acyl-CoA oxidase activity being
sought. For example, for acyl-CoA oxidases having specificity for
longer chain substrates, feedstocks having longer carbon chain
lengths could be substituted to allow selection and or screening
for acyl-CoA oxidase activities with specificities for longer
carbon chain lengths.
Example 10
Transformation of C. tropicalis Procedure
[0415] 5 mL YPD start cultures were inoculated with a single colony
of C. tropicalis and incubated overnight at 30.degree. C., with
shaking at about 200 rpm. The following day, fresh 25 mL YPD
cultures, containing 0.05% Antifoam B, were inoculated to an
initial OD.sub.600nm of 0.4 and the culture incubated at 30.degree.
C., with shaking at about 200 rpm until an OD.sub.600nm of 1.0-2.0
was reached. Cells were pelleted by centrifugation at
1,000.times.g, 4.degree. C. for 10 minutes. Cells were washed by
resuspending in 10 mL sterile water, pelleted, resuspended in 1 mL
sterile water and transferred to a 1.5 mL microcentrifuge tube. The
cells were then washed in 1 mL sterile TE/LiOAC solution, pH 7.5,
pelleted, resuspended in 0.25 mL TE/LiOAC solution and incubated
with shaking at 30.degree. C. for 30 minutes.
[0416] The cell solution was divided into 50 uL aliquots in 1.5 mL
tubes to which was added 5.8 ug of linearized DNA and 5 uL of
carrier DNA (boiled and cooled salmon sperm DNA, 10 mg/mL). 300 uL
of sterile PEG solution (40% PEG 3500, 1.times.TE, 1.times.LiOAC)
was added, mixed thoroughly and incubated at 30.degree. C. for 60
minutes with gentle mixing every 15 minutes. 40 uL of DMSO was
added, mixed thoroughly and the cell solution was incubated at
42.degree. C. for 15 minutes. Cells were then pelleted by
centrifugation at 1,000.times.g 30 seconds, resuspended in 500 uL
of YPD media and incubated at 30.degree. C. with shaking at about
200 rpm for 2 hours. Cells were then pelleted by centrifugation and
resuspended in 1 mL 1.times. TE, cells were pelleted again,
resuspended in 0.2 mL 1.times. TE and plated on selective media.
Plates were incubated at 30.degree. C. for growth of
transformants.
Example 11
Procedure for Recycling of the URA3 Marker
[0417] The URA3 gene was obtained from genomic DNA of Candida yeast
culture ATCC20336. C. tropicalis has a limited number of selectable
marker, as compared to S. cerevisiae, therefore, the URA3 marker is
"recycled" to allow multiple rounds of selection using URA3. To
reutilize the URA3 marker for subsequent engineering of C.
tropicalis, a single colony having the Ura' phenotype was
inoculated into 3 mL YPD and grown overnight at 30.degree. C. with
shaking. The overnight culture was then harvested by centrifugation
and resuspended in mL YNB+YE (6.7 g/L Yeast Nitrogen Broth, 3 g/L
Yeast Extract). The resuspended cells were then serially diluted in
YNB+YE and 100 uL aliquots plated on YPD plates (incubation
overnight at 30.degree. C.) to determine titer of the original
suspension. Additionally, triplicate 100 uL aliquots of the
undiluted suspension were plated on SC Dextrose (Bacto Agar 20 g/L,
Uracil 0.3 g/L, Dextrose 20 g/L, Yeast Nitrogen Broth 6.7 g/L,
Amino Acid Dropout Mix 2.14 g/L) and 5-FOA at 3 different
concentrations (0.5, 0.75, 1 mg/mL).
[0418] Plates were incubated for at least 5 days at 30.degree. C.
Colonies arising on the SC Dextrose+5-FOA plates were resuspended
in 50 uL sterile, distilled water and 5 uL utilized to streak on to
YPD and SC-URA (SC Dextrose medium without Uracil) plates. Colonies
growing only on YPD and hot on SC-URA plates were then inoculated
into 3 mL YPD and grown overnight at 30.degree. C. with shaking.
Overnight cultures were harvested by centrifugation and resuspended
in 1.5 mL YNB (6.7 g/L Yeast Nitrogen Broth). The resuspended cells
were serially diluted in YNB and 100 uL aliquots plated on YPD
plates and incubation overnight at 30.degree. C. to determine
initial titer. 1 mL of each undiluted cell suspension also was
plated on SC-URA and incubated for up to 7 days at 30.degree. C.
Colonies on the SC-URA plates are revertants and the isolate with
the lowest reversion frequency (<10.sup.-7) was used for
subsequent strain engineering.
Example 12
Cloning and Analysis of C. tropicalis Fatty Alcohol Oxidase (FAO)
Alleles
[0419] Isolation of Fatty Alcohol Oxidase Genes from C.
tropicalis
[0420] C. tropicalis (ATCC20336) fatty alcohol oxidase genes were
isolated by PCR amplification using primers generated to amplify
the sequence region covering promoter, fatty alcohol oxidase gene
(FAO) and terminator of the FAO1 sequence (GenBank accession number
of FAO1 AY538780). The primers used to amplify the fatty alcohol
oxidase nucleotide sequences from Candida strain ATCC20336, are
showing in the TABLE 2 below.
TABLE-US-00002 TABLE 2 Oligonucleotides for cloning FAO alleles
Oligo Sequence oAA01 AACGACAAGATTAGATTGGTTGAGA 44 oAA01
GTCGAGTTTGAAGTGTGTGTCTAAG 45 oAA02
AGATCTCATATGGCTCCATTTTTGCCCGACCAGGTCGA 68 CTACAAACACGTC oAA02
ATCTGGATCCTCATTACTACAACTTGGCTTTGGTCTTC 69 AAGGAGTCTGCCAAACCTAAC
oAA02 ACATCTGGATCCTCATTACTACAACTTGGCCTTGGTCT 82 oAA04
CACACAGCTCTTCTAGAATGGCTCCATTTTTGCCCGAC 21 CAGGTCGAC oAA04
CACACAGCTCTTCCTTTCTACAACTTGGCTTTGGTCTT 22 CAAGGAGTCTGC oAA04
GTCTACTGATTCCCCTTTGTC 29 oAA02 TTCTCGTTGTACCCGTCGCA 81
[0421] PCR reactions contained 25 .mu.L 2.times. master mix, 1.5 uL
of oAA0144 and oAA0145 (10 uM), 3.0 uL genomic DNA, and 19 uL
sterile H.sub.2O. Thermocycling parameters used were 98.degree. C.
for 2 minutes, 35 cycles of 98.degree. C. 20 seconds, 52.degree. C.
20 seconds, 72.degree. C. 1 minute, followed by 72.degree. C. 5
minutes and a 4.degree. C. hold. PCR products of the correct size
were gel purified, ligated into pCR-Blunt II-TOPO (Invitrogen) and
transformed into competent TOP 10 E. coli cells (Invitrogen).
Clones containing PCR inserts were sequenced to confirm correct DNA
sequence. Four FAO alleles were identified from sequence analysis
and designated as FAO-13, FAO-17, FAO-18 and FAO-20. The sequence
of the clone designated FAO-18 had a sequence that was
substantially identical to the sequence of FAO1 from GenBank. The
resulting plasmids of the four alleles were designated pAA083,
pAA084, pAA059 and pAA085, respectively. Sequence identity
comparisons of FAO genes isolated as described herein are shown in
the TABLE 3-5 below.
TABLE-US-00003 TABLE 3 DNA sequence identity FAO- FAO- FAO- FAO-
FAO1 18 17 13 20 FAO2a FAO2b FAO1 100 100 98 96 95 83 82 FAO-18 100
98 96 95 83 82 FAO-17 100 98 98 83 82 FAO-13 100 99 83 83 FAO-20
100 83 83 FAO2a 100 96 FAO2b 100
TABLE-US-00004 TABLE 4 Protein sequence identity FAO- FAO- FAO-
FAO- FAO1 18 17 13 20 FAO2a FAO2b FAO1 100 100 99 98 98 81 80 FAO-
100 99 98 98 81 80 18 FAO- 100 99 99 82 81 17 FAO- 100 99 82 81 13
FAO- 100 82 81 20 FAO2a 100 97 FAO2b 100
TABLE-US-00005 TABLE 5 Amino acid differences in FAO alleles 32 75
89 179 185 213 226 352 544 590 FAO1 E M G L Y T R H S P FAO- Q T A
L Y A K Q A A 13 FAO- Q T A M D A K Q A A 20
[0422] Expression of FAO Alleles in E. coli
[0423] To determine the levels of FAO enzyme activity with respect
to various carbon sources, the four isolated FAO alleles were
further cloned and over-expressed in E. coli. The FAOs were
amplified using the plasmids mentioned above as DNA template by PCR
with primers oAA0268 and oAA0269 for FAO-13 and FAO-20 and oAA0268
and oAA0282 for FAO-17 and FAO-18, using conditions as described
herein. PCR products of the correct size were gel purified and
ligated into pET11a vector between NdcI and BamHI sites and
transformed into BL21 (DE3) E. coli cells. The colonies containing
corresponding FAOs were confirmed by DNA sequencing. Unmodified
pET11a vector also was transformed into BL21 (DE3) cells, as a
control. The resulting strains and plasmids were designated sAA153
(pET11a), sAA154 (pAA079 containing FAO-13), sAA155 (pAA080
containing FAO-17), sAAA156 (pAA081 containing FAO-18) and sAA157
(pAA082 containing FAO-20), respectively. The strains and plasmids
were used for FAO over-expression in E. coli. One colony of each
strain was transferred into 5 mL of LB medium containing 100 g/mL
ampicillin and grown overnight at 37.degree. C., 200 rpm. The
overnight culture was used to inoculate a new culture to
OD.sub.600nm 0.2 in 25 ml LB containing 100 .quadrature.g/ml
ampicillin. Cells were induced at OD.sub.600nm 0.8 with 0.3 mM IPTG
for 3 hours and harvested by centrifugation at 4.degree. C.
1,050.times.g for 10 minutes. The cell pellet was stored at
-20.degree. C..
[0424] Expression of FAOs in C. tropicalis
[0425] Two alleles, FAO-13 and FAO-20, were chosen for
amplification in C. tropicalis based on their substrate specificity
profile, as determined from enzyme assays of soluble cell extracts
of E. coli with over expressed FAOs. DNA fragments containing
FAO-13 and FAO-20 were amplified using plasmids pAA079 and pAA082
as DNA templates, respectively, by PCR with primers oAA0421 and
oAA0422. PCR products of the correct sizes were gel purified and
ligated into pCR-Blunt II-TOPO (Invitrogen), transformed into
competent TOP10 E. coli cells (Invitrogen) and clones containing
FAO inserts were sequenced to confirm correct DNA sequence.
Plasmids containing FAO-13 and FAO-20 were digested with SapI and
ligated into vector pAA105, which includes the C. tropicalis PGK
promoter and terminator. The resulting plasmids were confirmed by
restriction digestion and DNA sequencing and designated as pAA115
(FAO-13) and pAA116 (FAO-20), respectively. Plasmids pAA115 and
pAAT16 were linearized with SpeI, transformed into competent C.
tropicalis Ura.sup.- strains sAA002 (SU-2, ATCC20913) and sAA103.
The integration of FAO-13 and FAO-20 was confirmed by colony PCR
using primers oAA0429 and oAA0281. The resulting strains were
designated as sAA278 (pAA115 integrated in strain sAA002), sAA280
(pAA116 integrated in sAA002), sAA282(pAA115 integrated in sAA103),
and sAA284 (pAA116 integrated in sAA103), and were used for fatty
alcohol oxidase over-expression in C. tropicalis.
[0426] One colony of each strain was inoculated into 5 ml YPD and
grown overnight as described herein. The overnight culture was used
to inoculate a new 25 mL YPD culture to about OD.sub.600nm 0.5. FAO
over-expression was regulated by the PGK promoter/terminator,
induced with glucose in the medium and expressed constitutively.
Strains sAA002 and sAA103 (e.g., untransformed starting strains)
were included as negative controls for FAO over-expression. Cells
were harvested at early log phase (OD.sub.600nm=in the range of
between about 3 to about 5) by centrifugation at 4.degree. C. for
10 minutes at 1,050.times.g. Cell pellets were stored at
-20.degree. C.
[0427] Cell Extract Preparation from E. coli
[0428] Cell pellets from 25 mL of FAO expressing E. coli cultures
were resuspended in 10 mL phosphate-glycerol buffer containing 50
mM potassium phosphate buffer (pH 7.6), 20% glycerol, 1 mM
Phenylmethylsulfonyl fluoride (PMSF), 2 uL Benzonase 25 U/uL, 20 uL
10 mg/mL lysozyme. The cells were then lysed by incubation at room
temperature for 50 minutes on a rotating shaker, and the cell
suspension centrifuged for 30 minutes at 4.degree. C. using
15,000.times.g for. The supernatant was aliquoted in 1.5 ml
microcentrifuge tubes and stored at -20.degree. C. for FAO enzyme
activity assays.
[0429] Cell Extract Preparation from C. tropicalis
[0430] Frozen C. tropicalis cell pellets were resuspended in 1.2 ml
of phosphate-glycerol buffer containing 50 mM potassium phosphate
buffer (pH 7.6), 20% glycerol, 1 mM Phenylmethylsulfonyl fluoride
(PMSF). Resuspended cells were transferred to 1.5 mL screw-cap
tubes containing about 500 uL of zirconia beads on ice. The cells
were lysed with a Bead Beater (Biospec) using 2 minute pulses and 1
minute rest intervals on ice. The process was repeated 3 times. The
whole cell extract was then transferred to a new 1.5 ml tube and
centrifuged at 16,000.times.g for 15 minutes at 4.degree. C. The
supernatant was transferred into a new tube and used for FAO enzyme
activity assays.
[0431] Protein Concentration Determination
[0432] Protein concentration of the cell extracts was determined
using the Bradford Reagent following manufacturers' recommendations
(Cat# 23238, Thermo Scientific).
[0433] FAO Enzyme Activity Assay
[0434] FAO enzyme activity assays were performed using a
modification of Eirich et al., 2004). The assay utilizes a
two-enzyme coupled reaction (e.g., FAO and horseradish peroxidase
(HRP)) and can be monitored by spectrophotometry. 1-Dodecanol was
used as a standard substrate for fatty alcohol oxidase enzymatic
activity assays. FAO oxidizes the dodecanol to dodecanal while
reducing molecular oxygen to hydrogen peroxide simultaneously. HRP
reduces (2,2'-azino-bis 3-ethylbenzthiazoline-6-sulfonic acid;
ABTS) in the two-enzyme coupled reaction, where the electron
obtained from oxidizing hydrogen peroxide to ABTS, which can be
measured by spectrometry at 405 nm. The assay was modified using
aminotriazole (AT) to prevent the destruction of H.sub.2O.sub.2 by
endogenous catalase, thus eliminating the need for microsomal
fractionation. The final reaction mixture (1.0 mL) for FAO enzyme
assay consisted of 500 .mu.L of 200 mM HEPES buffer, pH 7.6; 50
.mu.L of a 10 mg/mL ABTS solution in deionized water; 10 .mu.L of 5
mM solution of dodecanol in acetone; 40 .mu.L of 1 M AT and 5 .mu.L
of a 2 mg/mL horseradish peroxidase solution in 5.0 mM potassium
phosphate buffer, pH 7.6. Reaction activity was measured by
measuring light absorbance at 405 nm for 10 minutes at room
temperature after adding the extract. The amount of extract added
to the reaction mixture was varied so that the activity fell within
the range of 0.2 to 1.0 .DELTA.A.sub.405nm/min. The actual amounts
of extract used were about 1.69 U/mg for E. coli expressed FAO-13,
0.018 U/mg for E. coli expressed FAO-17, 0.35 U/mg for E. coli
expressed FAO-18 (e.g., FAO1), 0.47 U/mg E. coli expressed FAO-20,
0.036 U/mg C. tropicalis (strain sAA278) expressed FAO-13, 0.016
U/mg C. tropicalis (strain sAA282) expressed FAO-13, 0.032 U/mg C.
tropicalis (strain sAA280) expressed FAO-20 and 0.029 U/mg C.
tropicalis (strain sAA284) expressed FAO-20. FAO activity was
reported as activity units/mg of total protein (1 unit=1
.quadrature.mole substrate oxidized/min). An extinction coefficient
at 405 nm of 18.4 was used for ABTS and was equivalent to 0.5 mM
oxidized substrate. The results of the activity assays are shown in
TABLES 6-7 below.
TABLE-US-00006 TABLE 6 FAO activity (units/mg total protein) on
primary alcohols 1- 1- 1- 1- 1- 1- 1- Butanol Pentanol Hexanol
Octanol Decanol Dodecanol Tetradecanol Hexadecanol FAO- 0.01 0.09
1.17 82.67 70.94 100 79.35 58.88 13 FAO- 0.72 0.26 1.06 66.23 22.00
100 47.86 60.98 17 FAO- 0.07 0.11 0.26 60.56 54.56 100 114.47 50.65
18 FAO- 0.07 0.11 0.91 55.96 74.57 100 89.52 42.59 20
TABLE-US-00007 TABLE 7 FAO activity (units/mg total protein) on
omega hydroxy fatty acids 1- 10-OH- 12-OH- 16-OH- Dodecanol 6-OH-HA
DA DDA HDA FAO-13 100 4.18 4.14 6.87 8.57 FAO-17 100 1.18 0.00 0.59
0.94 FAO-18 100 0.00 0.00 4.87 2.94 FAO-20 100 0.03 0.04 2.25
7.46
Example 13
Construction of C. tropicalis Shuttle Vector pAA061
[0435] Vector pAA061 was constructed from a pUC19 backbone to
harbor the selectable marker URA3 from Candida strain ATCC20336 as
well as modifications to allow insertion of C. tropicalis promoters
and terminators. A 1,507 bp DNA fragment containing the promoter,
ORF, and terminator of URA3 from C. tropicalis ATCC20336 was
amplified using primers oAA0124 and oAA0125, shown in the TABLE 8
below. The URA3 PCR product was digested with NdeI/MluI and ligated
into the 2,505 bp fragment of pUC19 digested with NdeI/BsmBI (an
MluI compatible overhang was produced by BsmBI). In order to
replace the lac promoter with a short 21 bp linker sequence, the
resulting plasmid was digested with SphI/SapI and filled in with a
linker produced by annealing oligos oAA0173 and oAA0174. The
resulting plasmid was designated pAA061.
TABLE-US-00008 TABLE 8 Oligonucleotides for construction of pAA061
PCR product Oligos Sequence (bp) oAA012
cacacacatatgCGACGGGTACAACGAGAATT 1507 4 oAA012
cacacaacgcgtAGACGAAGCCGTTCTTCAAG 5 oAA017 ATGATCTGCCATGCCGAACTC 21
3 (linker) oAA017 AGCGAGTTCGGCATGGCAGATCATCATG 4
Example 14
Cloning of C. tropicalis PGK Promoter and Terminator
[0436] Vector pAA105 was constructed from base vector pAA061 to
include the phosphoglycerate kinase (PGK) promoter and terminator
regions from C. tropicalis ATCC20336 with an intervening multiple
cloning site (MCS) for insertion of open reading frames (ORF's).
The PGK promoter region was amplified by PCR using primers oAA0347
and oAA0348, shown in the TABLE 9 below. The 1,029 bp DNA fragment
containing the PGK promoter was digested with restriction enzymes
PstI/XmaI. The PGK terminator region was amplified by PCR using
primers oAA0351 and oAA0352, also shown in the TABLE 9 below. The
396 bp DNA fragment containing the PGK terminator was digested with
restriction enzymes XmaI/EcoRI. The 3,728 bp PstI/EcoRI DNA
fragment from pAA061 was used in a three piece ligation reaction
with the PGK promoter and terminator regions to produce pAA105. The
sequence between the PGK promoter and terminator contains
restriction sites for incorporating ORF's to be controlled by the
functionally linked constitutive PGK promoter.
TABLE-US-00009 TABLE 9 Oligonucleotides for cloning C. tropicalis
PGK promoter and terminator PCR product Oligos Sequence (bp)
oAA0347 CACACACTGCAGTTGTCCAATGTAATAATTTT 1028 oAA0348
CACACATCTAGACCCGGGCTCTTCTTCTGAAT AGGCAATTGATAAACTTACTTATC oAA0351
GAGCCCGGGTCTAGATGTGTGCTCTTCCAAAG 396 TACGGTGTTGTTGACA oAA0352
CACACACATATGAATTCTGTACTGGTAGAGCT AAATT
Example 15
Cloning of the POX4 Locus
[0437] Primers oAA0138 and oAA0141 (TABLE 10) were generated to
amplify the entire sequence of NCBI accession number M12160 for the
YSAPOX4 locus from genomic DNA prepared from Candida strain
ATCC20336. The 2,845 bp PCR product was cloned into the vector,
pCR-BluntII-TOPO (Invitrogen), sequenced and designated pAA052.
TABLE-US-00010 TABLE 10 Oligonucleotides for cloning of POX4 PCR
product Oligos Sequence (bp) oAA013 GAGCTCCAATTGTAATATTTCGGG 2845 8
oAA014 GTCGACCTAAATTCGCAACTATCAA 1
Example 16
Cloning of the POX5 Locus
[0438] Primers oAA0179 and oAA0182 (TABLE 11) were generated to
amplify the entire sequence of NCBI accession number M12161 for the
YSAPOX5 locus from genomic DNA prepared from Candida strain
ATCC20336. The 2,624 bp PCR product was cloned into the vector
pCR-BluntII-TOPO (Invitrogen), sequenced and designated pAA049.
TABLE-US-00011 TABLE 11 Oligonucleot1des for cloning of POX5 PCR
product Oligos Sequence (bp) oAA017
GAATTCACATGGCTAATTTGGCCTCGGTTCCACAA 2624 9 CGCACTCAGCATTAAAAA
oAA018 GAGCTCCCCTGCAAACAGGGAAACACTTGTCATCT 2 GATTT
Example 17
Construction of Strains with Amplified CPR and CYP52 Genes
[0439] Strains having an increased number of copies of cytochrome
P450 reductase (CPR) and/or for cytochrome P450 monooxygenase
(CYP52) genes were constructed to determine how over expression of
CPR and CYP52 affected diacid production.
[0440] Cloning and Integration of the CPR Gene
[0441] A 3,019 bp DNA fragment encoding the CPR promoter, ORF, and
terminator from C. tropicalis ATCC750 was amplified by PCR using
primers oAA0171 and oAA0172 (TABLE 12), incorporating unique SapI
and SphI sites. The amplified DNA fragment was cut with the
indicated restriction enzymes and ligated into plasmid pAA061,
(described in Example 13) to produce plasmid pAA067. Plasmid pAA067
was linearized with ClaI and transformed into C. tropicalis
Ura.sup.- strain sAA103 (ura3/ura3, pox4::ura3/pox4::ura3,
pox5::ura3/pox5::ura3). Transformations were performed with plasmid
pAA067 alone and in combination with plasmids harboring the
CYP52A15 or CYP52A16 genes, described below.
[0442] Cloning and Integration of CYP52A15 Gene
[0443] A 2,842 bp DNA fragment encoding the CYP52A15 promoter, ORF,
and terminator from C. tropicalis ATCC20336 was amplified by PCR
using primers oAA0175 and oAA0178 (TABLE 12) and cloned into
pCR-BluntII-TOPO for DNA sequence verification. The cloned CYP52A15
DNA fragment was isolated by restriction digest with XbaI/BamHI
(2,742 bp) and ligated into plasmid pAA061, (described in Example
13), to produce plasmid pAA077. Plasmid pAA077 was linearized with
PmlI and transformed into C. tropicalis Ura.sup.- strain sAA103
(ura3/ura3; pox4::ura3/pox4::ura3, pox5::ura3/pox5::ura3). pAA077
was cotransformed with plasmid pAA067 harboring the CPR gene.
[0444] Cloning and Integration of CYP52A16 Gene
[0445] A 2,728 bp DNA fragment encoding the CYP52A16 promoter, ORF,
and terminator from C. tropicalis ATCC20336 was amplified by PCR
using primers oAA0177 and oAA0178 (TABLE 12) and cloned into
pCR-BluntII-TOPO for DNA sequence verification. The cloned CYP52A16
DNA fragment was amplified with primers oAA0260 and oAA0261 (TABLE
12) which incorporated unique SacI/XbaI restriction sites. The
amplified DNA fragment was digested with SacI and XbaI restriction
enzymes and ligated into plasmid pAA061 to produce plasmid pAA078.
Plasmid pAA078 was linearized with ClaI and transformed into C.
tropicalis Ura.sup.- strain sAA103 (ura3/ura3,
pox4::ura3/pox4::ura3, pox5::ura3/pox5::ura3). pAA078 was
cotransformed with plasmid pAA067 harboring the CPR gene.
TABLE-US-00012 TABLE 12 Oligonucleotides for cloning of CPR,
CYP52A15 and CYP52A16 PCR product Oligos Sequence (bp) oAA017
cacctcgctcttccAGCTGTCATGTCTATTCAATG 3019 1 CTTCGA oAA017
cacacagcatgcTAATGTTTATATCGTTGACGGTG 2 AAA oAA017
cacaaagcggaagagcAAATTTTGTATTCTCAGTA 2842 5 GGATTTCATC oAA017
cacacagcatgCAAACTTAAGGGTGTTGTAGATAT 8 CCC oAA017
cacacacccgggATCGACAGTCGATTACGTAATCC 2772 7 ATATTATTT oAA017
cacacagcatgCAAACTTAAGGGTGTTGTAGATAT 8 CCC oAA026
cacacagagctcACAGTCGATTACGTAATCCAT 2772 0 oAA026
cacatctagaGCATGCAAACTTAAGGGTGTTGTA 1
[0446] Preparation of Genomic DNA
[0447] Genomic DNA was prepared from transformants for PCR
verification and for Southern blot analysis. Isolated colonies were
inoculated into 3 mL YPD and grown overnight at 30.degree. C. with
shaking. Cells were pelleted by centrifugation. To each pellet, 200
uL Breaking Buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM
Tris pH 8 and, 1 mM EDTA) was added, and the pellet resuspended and
transferred to a fresh tube containing 200 uL 0.5 mm
Zirconia/Silica Beads. 200 uL Phenol:Chloroform:Isoamyl Alcohol
(25:24:1) was added to each tube, followed by vortexing for 1
minute. Sterile distilled water was added (200 uL) to each tube and
the tubes were centrifuged at 13000 rpm for 10 minutes. The aqueous
layer was ethanol precipitated and washed with 70% ethanol. The
pellet was resuspended in 100-200 uL 10 mM Tris, after drying.
Genomic DNA preparation for Southern blot analysis was performed
using the same procedure on 25 mL cultures for each colony
tested.
[0448] Characterization of Strains with Amplified CPR and CYP52
Genes
[0449] Verification of gene integration was performed by PCR using
primers oAA0252 and oAA0256 (CPR), oAA0231 and oAA0281 (CYP52A15),
and oAA242 and oAA0257 (CYP52A16). The primers used for
verification are shown in the TABLE 13.
TABLE-US-00013 TABLE 13 Oligonucleotides for PCR verification of
CPR, CYP52A15 and CYP52A16 PCR product Oligos Sequence (bp) oAA025
TTAATGCCTTCTCAAGACAA 743 2 oAA025 GGTTTTCCCAGTCACGACGT 6 oAA023
CCTTGCTAATTTTCTTCTGTATAGC 584 1 oAA028 TTCTCGTTGTACCCGTCGCA 1
oAA024 CACACAACTTCAGAGTTGCC 974 2 oAA025 TCGCCACCTCTGACTTGAGC 7
[0450] Southern blot analysis was used to determine the copy number
of the CPR, CYP52A15 and CYP52A16 genes. Biotinylated DNA probes
were prepared with gene specific oligonucleotides using the NEBlot
Phototope Kit from New England BioLabs (Catalog #N7550S) on PCR
products generated from each gene target as specified in TABLE 14.
Southern Hybridizations were performed using standard methods
(e.g., Sambrook, J. and Russell, D. W., (2001) Molecular Cloning: A
Laboratory Manual, (3rd ed.), pp. 6.33-6.64. Cold Spring Harbor
Laboratory Press). Detection of hybridized probe was performed
using the Phototope-Star Detection Kit from New England BioLabs
(Catalog #N7020S). Copy number was determined by densitometry of
the resulting bands.
TABLE-US-00014 TABLE 14 Oligonucleotides for Probe Template PCR of
CPR, CYP52A15 and CYP52A16 PCR Tem- product Oligos Sequence Gene
plate (bp) oAA0250 AATTGAACATCAGAACAGGA CPR pAA067 1313 oAA0254
CCTGAAATTTCCAAATGGTG TCTAA oAA0227 TTTTTTGTGCGCAAGTACAC CYP52A
pAA077 905 oAA0235 CAACTTGACGTGAGAAACCT 15 oAA0239
AGATGCTCGTTTTACACCCT CYP52A pAA078 672 oAA0247 ACACAGCTTTGATGTTCTCT
16
Example 18
Addition and/or Amplification of Monooxygenase and Monooxygenase
Reductase Activities
[0451] Cytochrome P450's often catalyze a monooxygenase reaction,
e.g., insertion of one atom of oxygen into an organic substrate
(RH) while the other oxygen atom is reduced to water:
RH+O2+2H++2e-.fwdarw.ROH+H.sub.2O
[0452] The substrates sometimes are of a homogeneous carbon chain
length. Enzymes with monooxygenase activity sometimes recognize
substrates of specific carbon chain lengths, or a subgroup of
carbon chain lengths with respect to organic substrates of
homogenous carbon chain length. Addition of novel cytochrome
activities (e.g., B. megaterium BM3) and/or amplification of
certain or all endogenous or heterologous monooxygenase activities
(e.g., CYP52A12 polynucleotide, CYP52A13 polynucleotide, CYP52A14
polynucleotide, CYP52A15 polynucleotide, CYP52A16 polynucleotide,
CYP52A17 polynucleotide, CYP52A18 polynucleotide, CYP52A19
polynucleotide, CYP52A20 polynucleotide, CYP52D2 polynucleotide,
BM3 polynucleotide) can contribute to an overall increase in carbon
flux through native and/or engineered metabolic pathways, in some
embodiments. In certain embodiments, adding a novel monooxygenase
or increasing certain or all endogenous or heterologous
monooxygenase activities can increase the flux of substrates of
specific carbon chain length or subgroups of substrates with
mixtures of specific carbon chain lengths. In some embodiments, the
selection of a monooxygenase activity for amplification in an
engineered strain is related to the feedstock utilized for growth
of the engineered strain, pathways for metabolism of the chosen
feedstock and the desire end product (e.g., dodecanedioic
acid).
[0453] Strains engineered to utilize plant-based oils for
conversion to dodecanedioic acid can benefit by having one or more
monooxygenase activities with substrate specificity that matches
the fatty acid chain-length distribution of the oil. For example,
the most prevalent fatty acid in coconut oil is lauric acid (12
carbons long), therefore, the monooxygenase activity chosen for a
coconut oil-utilizing strain, can have a substrate preference for
C12 fatty acids. For strains engineered to utilize other plant
based oils with different fatty acid chain-length distributions it
may be desirable to amplify a monooxygenase activity that has a
matching substrate preference. In some embodiments, a genetic
modification that alters monooxygenase activity increases the
activity of one or more monooxygenase activities with a substrate
preference for feedstocks having carbon chain lengths of between
about 12 and about 24 carbons (e.g., mixed chain length alkanes,
mixed chain length fatty acids, soapstocks, the like and
combinations thereof). In certain embodiments, the genetic
modification increases the activity of a monooxygenase activity
with a preference for fatty acids having a carbon chain-length
distribution of between about 10 carbons and about 16 carbons.
[0454] As mentioned previously, the enzymes that carry out the
monooxygenase activity are reduced by the activity of monooxygenase
reductase, thereby regenerating the enzyme. Selection of a CPR for
amplification in an engineered strain depends upon which P450 is
amplified, in some embodiments. A particular CPR may interact
preferentially with one or more monooxygenase activities, in some
embodiments, but not well with other monooxygenases. A
monooxygenase reductase from Candida strain ATCC750, two
monooxygenase reductase activities from Candida strain ATCC20336
and a monooxygenase reductase activity from Bacillus megaterium are
being evaluated for activity with the added and/or amplified
monooxygenases described herein. Provided in the tables below are
nucleotide sequences used to add or amplify monooxygenase and
monooxygenase reductase activities.
Example 19
Amplification of Selected Beta Oxidation Activities
[0455] Described herein are methods of amplifying a POX5 beta
oxidation activity. Substantially similar methods can be utilized
to amplify different beta oxidation activities including non-POX
(e.g., acyl-CoA oxidase) activities and or acyl-CoA oxidase
activities with altered substrate specificities, as described
herein.
[0456] Construction of POX5 Amplified Strains
[0457] Plasmid pAA166 (P.sub.POX4POX5T.sub.POX4)
[0458] A PCR product containing the nucleotide sequence of POX5 was
amplified from C. tropicalis 20336 genomic DNA using primers oAA540
and oAA541. The PCR product was gel purified and ligated into
pCR-BluntII-TOPO (Invitrogen), transformed into competent TOP10 E.
coli cells (Invitrogen) and clones containing PCR inserts were
sequenced to confirm correct DNA sequence. One such plasmid was
designated, pAA165. Plasmid pAA165 was digested with BspQI and a
2-kb fragment was isolated. Plasmid pAA073 which contained a POX4
promoter and POX4 terminator was also digested with BspQI and gel
purified. The isolated fragments were ligated together to generate
plasmid pAA166. Plasmid pAA166 contains a
P.sub.POX4POX5T.sub.POX4fragment.
[0459] Plasmid pAA204 (Thiolase Deletion Construct)
[0460] A PCR product containing the nucleotide sequence of a
short-chain thiolase (e.g., acetyl-coA acetyltransferase) was
amplified from C. tropicalis 20336 genomic DNA using primers oAA640
and oAA641. The PCR product was gel purified and ligated into
pCR-Blunt II-TOPO (Invitrogen), transformed into competent TOP10 E.
coli cells (Invitrogen) and clones containing PCR inserts were
sequenced to confirm correct DNA sequence. One such plasmid was
designated, pAA184. A URA3 PCR product was amplified from pAA061
using primers oAA660 and oAA661. The PCR product was gel purified
and ligated into pCR-Blunt II-TOPO (Invitrogen), transformed as
described and clones containing PCR inserts were sequenced to
confirm the correct DNA sequence. One such plasmid was designated
pAA192. Plasmid pAA184 was digested with BglII/SalI and gel
purified. Plasmid pAA192 was digested with BglII/SalI and a 1.5 kb
fragment was gel purified. The isolate fragments were ligated
together to create pAA199. An alternative P.sub.URA3 PCR product
was amplified from plasmid pAA061 using primers oAA684 and oAA685.
The PCR product was gel purified and ligated into pCR-Blunt II-TOPO
(Invitrogen), transformed as described and clones containing PCR
inserts were sequenced. One such plasmid was designated, pAA201.
Plasmid pAA199 was digested with SalI and gel purified. Plasmid
pAA201 was digested with SalI and a 0.43 kb P.sub.URA3 was gel
purified. The isolated fragments were ligated to create plasmid
pAA204 that contains a direct repeat of P.sub.URA3.
[0461] Plasmid pAA221 (P.sub.POX4POX5T.sub.POX4 in Thiolase
Deletion Construct)
[0462] A PCR product containing the nucleotide sequence of
P.sub.POX4POX5T.sub.POX4 was amplified from plasmid pAA166 DNA
using primers oAA728 an oAA729. The PCR product was gel purified
and ligated into pCR-Blunt II-TOPO, transformed as described and
clones containing PCR inserts were sequenced to confirm the
sequence of the insert. One such plasmid was designated, pAA220.
Plasmid pAA204 was digested with BglII, treated with shrimp
alkaline phosphatase (SAP), and a 6.5 kb fragment was gel purified.
Plasmid pAA220 was digested with BglII and a 2.7 kb fragment
containing P.sub.POX4POX5T.sub.POX4 was gel purified. The isolated
fragments were ligated to create plasmid pAA221.
[0463] Strain sAA617 (P.sub.POX4POX5T.sub.POX4 in sAA451)
[0464] Strain sAA451 is a ura-, partially .beta.-oxidation blocked
Candida strain (ura3/ura3 pox4a::ura3/pox4::ura3 POX5/POX5).
Plasmid pAA221 was digested with EcoRI to release a DNA fragment
containing P.sub.POX4POX5T.sub.POX4 in a thiolase deletion
construct. The DNA was column purified and transformed to strain
sAA451 to plate on SCD-ura plate. After two days, colonies were
streaked out on YPD plates, single colonies selected and again
streaked out on YPD plates. Single colonies were selected from the
second YPD plates and characterized by colony PCR. The insertion of
P.sub.POX4POX5T.sub.POX4 in strain sAA451, disrupting the
short-chain thiolase gene, was confirmed by PCR and one such strain
was designated sAA617.
[0465] Strain sAA620
[0466] Strain sAA617 was grown overnight on YPD medium and plated
on SCD+URA+5-FOA, to select for loop-out of URA3. Colonies were
streaked out onto YPD plates twice as described for strain sAA617,
and single colonies characterized by colony PCR. The loop-out of
URA3 by direct repeats of PURA3 was confirmed by PCR. One such
strain was designated sAA620. Strain sAA620 has one additional copy
of POX5 under control of the POX4 promoter.
[0467] Plasmid pAA156
[0468] A PCR product containing the nucleotide sequence of CYP52A19
was amplified from Candida strain 20336 genomic DNA, using primers
oAA525 and oAA526. The PCR product was gel purified and ligated
into pCR-Blunt II-TOPO, transformed as described, and clones
containing PCR inserts were sequenced to confirm correct
DNA-sequence. One such plasmid was designated, pAA144. Plasmid
pAA144 was digested with BspQI and a 1.7-kb fragment was isolated.
Plasmid pAA073, which includes a POX4 promoter and POX4 terminator,
also was digested with BspQI and gel purified. The isolated
fragments were ligated together to generate plasmid, pAA156.
Plasmid pAA156 included P.sub.POX4CYP52A19T.sub.POX4 fragment and
URA3.
[0469] Strain sAA496
[0470] Plasmid pAA156 was digested with ClaI and column purified.
Strain sAA451 was transformed with this linearized DNA and plated
on SCD-ura plate. Colonies were checked for CYP52A19 integration.
Colonies positive for plasmid integration were further analyzed by
qPCR to determine the number of copies of CYP52A19 integrated. One
such strain, designated contained about 13 copies of the
monooxygenase activity encoded by CYP52A19.
[0471] Strains sAA632 and sAA635
[0472] Strain sAA620 was transformed with linearized pAA156 DNA and
plated on SCD-ura plates. Several colonies were checked for
CYP52A19 integration. Colonies positive for plasmid integration
were further analyzed by qPCR to determine the number of copies of
CYP52A19 integrated. One such strain, designated sAA632 contained
about 27 copies of the monooxygenase activity encoded by CYP52A19.
Another strain, designated sAA635, contained about 12 copies of the
monooxygenase activity encoded by CYP52A19.
Example 20
Cloning of C. tropicalis ACH Genes
[0473] ACH PCR product was amplified from C. tropicalis 20336
genomic DNA using primers oAA1095 and oAA1096, shown in TABLE 15.
The PCR product was gel purified and ligated into pCR-Blunt II-TOPO
(Invitrogen), transformed into competent TOP10 E. coli cells
(Invitrogen) and clones containing PCR inserts were sequenced to
confirm correct DNA sequence.
[0474] Sequence analysis of multiple transformants revealed the
presence of allelic sequences for the ACH gene, which were
designated ACHA and ACHB. A vector containing the DNA sequence for
the ACHA allele was generated and designated pAA310 (see FIG. 51).
A vector containing the DNA sequence for the ACHB allele was
generated and designated pAA311.
TABLE-US-00015 TABLE 15 Primer sequence oAA1095
CACACACCCGGGATGATCAGAACCGTCCGTTATCAAT oAA1096
CACACATCTAGACTCTCTTCTATTCTTAATTGCCGCT TCCACTAAACGGCAAAGTCTCCACG
Example 21
Cloning of C. tropicalis FAT1 Gene
[0475] FAT1 PCR product was amplified from C. tropicalis 20336
genomic DNA using primers oAA1023 and oAA1024, shown in TABLE 16.
The PCR product was gel purified and ligated into pCR-Blunt II-TOPO
(Invitrogen), transformed into competent TOP10 E. coli cells
(Invitrogen) and clones containing PCR inserts were sequenced to
confirm correct DNA sequence. A vector containing the DNA sequence
for the FAT1 gene was designated pAA296.
TABLE-US-00016 TABLE 16 Primer sequence oAA1023
GATATTATTCCACCTTCCCTTCATT oAA1024 CCGTTAAACAAAAATCAGTCTGTAAA
Example 22
Cloning of C. tropicalis ARE1 and ARE2 genes
[0476] ARE1 and ARE2 PCR products were amplified from C. tropicalis
20336 genomic DNA using primers oAA2006/oAA2007 and
oAA1012/oAA1018; respectively, shown in TABLE 17. The PCR products
were gel purified and ligated into pCR-Blunt II-TOPO (Invitrogen),
transformed into competent TOP10 E. coli cells (Invitrogen) and
clones containing PCR inserts were sequenced to confirm correct DNA
sequence. A vector containing the DNA sequence for the ARE1 gene
was designated pAA318. A vector containing the DNA sequence for the
ARE2 gene was designated pAA301.
TABLE-US-00017 TABLE 17 Primer sequence oAA1012
ATGTCCGACGACGAGATAGCAGGAATAGTCAT oAA1018
TCAGAAGAGTAAATACAACGCACTAACCAAGCT oAA2006
ATGCTGAAGAGAAAGAGACAACTCGACAAG oAA2007
GTGGTTATCGGACTCTACATAATGTCAACG
Example 23
Construction of an Optimized TESA Gene for Expression in C.
tropicalis
[0477] The gene sequence for the E. coli TESA gene was optimized
for expression in C. tropicalis by codon replacement. A new TESA
gene sequence was constructed using codons from C. tropicalis with
similar usage frequency for each of the codons in the native E.
coli TESA gene, (avoiding the use of the CTG codon due to the
alternative yeast nuclear genetic code utilized by C. tropicalis).
The optimized TESA gene was synthesized with flanking BspQI
restriction sites and provided in vector pIDTSMART-Kan (Integrated
DNA Technologies). The vector was designated as pAA287. Plasmid
pAA287 was cut with BspQI and the 555 bp DNA fragment was gel
purified. Plasmid pAA073 also was cut with BspQI and the linear DNA
fragment was gel purified. The two DNA fragments were ligated
together to place the optimized TESA gene under the control of the
C. tropicalis POX4 promoter. The resulting plasmid was designated
pAA294.
Example 24
Cloning of C. tropicalis DGA1 Gene
[0478] DGA1 PCR product was amplified from C tropicalis 20336
genomic DNA using primers oAA996 and oAA997, shown in TABLE 18. The
PCR product was gel purified and ligated into pCR-Blunt II-TOPO
(Invitrogen), transformed into competent TOP10 E. coli cells
(Invitrogen) and clones containing PCR inserts were sequenced to
confirm correct DNA sequence. A vector containing the DNA sequence
of the DGA1 gene was designated pAA299.
TABLE-US-00018 TABLE 18 Primer Sequence oAA996
ATGACTCAGGACTATAAAGACGATAGTCCTACGT CCACTGAGTTG oAA997
CTATTCTACAATGTTTAATTCAACATCACCGTAG CCAAACCT
Example 25
Cloning of C. tropicalis LRO1 Gene
[0479] LRO1 PCR product was amplified from C. tropicalis 20336
genomic DNA using primers oAA998 and oAA999, shown in TABLE 19. The
PCR product was gel purified and ligated into pCR-Blunt II-TOPO
(Invitrogen), transformed into competent TOP10 E. coli cells
(Invitrogen) and clones containing PCR inserts were sequenced to
confirm correct DNA sequence. A vector containing the DNA sequence
of the LRO1 gene was designated pAA300.
TABLE-US-00019 TABLE 19 Primer sequence oAA998
ATGTCGTCTTTAAAGAACAGAAAATC oAA999 TTATAAATTTATGGCCTCTACTATTTCT
Example 26
Cloning of C. tropicalis ACS1 Gene and Construction of Deletion
Cassette
[0480] ACS1 PCR product was amplified from C. tropicalis 20336
genomic DNA using primers oAA951 and oAA952, shown in TABLE 20. The
PCR product was gel purified and ligated into pGR-Blunt II-TOPO
(Invitrogen), transformed into competent TOP 10 E. coli cells
(Invitrogen) and clones containing PCR inserts were sequenced to
confirm the DNA sequence. One such plasmid was designated pAA275.
Plasmid pAA280 was digested with BamHI to release a 2.0 kb
P.sub.URA 3URA3T.sub.URA3P.sub.URA3 cassette. Plasmid pAA275 was
digested with BgIII and gel purified. The two pieces were ligated
together to generate plasmid pAA26 and pAA282. Plasmid pAA276 and
pAA282 have the P.sub.URA3URA3T.sub.URA3P.sub.URA3 cassette
inserted into the ACS gene in opposite orientations.
TABLE-US-00020 TABLE 20 Primer sequence oAA951
CCTACTTCCACAGCTTTAATCTACTATCAT oAA952
TTTAAGAAAACAACTAAGAGAAGCCAC
Example 27
Construction of Strain sAA722 (pox4a::ura3/pox4b::ura3 POX5/POX5
ACS1/acs1::P.sub.URA3URA3T.sub.URA3P.sub.URA3)
[0481] Plasmid pAA276 was digested with BamHI/XhoI and column
purified. Strain sAA329 (ura3/ura3 pox4a::ura3/pox4b::ura3
POX5/POX5) was transformed with the linearized DNA and plated on
SCD-ura plate. Several colonies were checked for ACS1 disruption.
One such strain was designated sAA722.
Example 28
Construction of Strain sAA741 (pox4a::ura3/pox4b::ura3 POX5/POX5
ACS1/acs1::P.sub.URA3)
[0482] Strain sAA722 was grown in YPD media overnight and plated on
5-FOA plate. Colonies that grew in the presence of 5-FOA were PCR
screened for the looping out of the URA3 gene leaving behind only
the URA3 promoter (P.sub.URA3) in the ACS1 site. Out of 30 colonies
analyzed, only one strain showed the correct genetic modification.
The strain was designated sAA741.
Example 29
Construction of Strain sAA776 (pox4a::ura3/pox4b::ura3 POX5/POX5
acs1::P.sub.URA3URA3T.sub.URA3P.sub.URA3/acs1::P.sub.URA3)
[0483] Plasmid pAA282 was digested with BamHI/XhoI and column
purified. Strain sAA741 (see Example 28) was transformed with the
linearized DNA and plated on SCD-ura plate. Several colonies were
checked for double ACS1 knockout by insertional inactivation. One
such strain was designated sAA776.
Example 30
Construction of Strain sAA779 (pox4a::ura3/pox4b::ura3 POX5/POX5
acs1::P.sub.URA3/acs1::P.sub.URA)
[0484] Strain sAA776 (see Example 29) was grown in YPD media
overnight and plated on 5-FOA plates. Colonies that grew in the
presence of 5-FOA were PCR screened for the looping out of the URA3
gene leaving behind only the URA3 promoter (P.sub.URA3) in both
ACS1 copies. One such strain was designated sAA779.
Example 31
Construction of Strain sAA811 (pox4a::ura3/pox4b::ura3 POX5/POX5
acs1::P.sub.URA3/acs1::P.sub.URA3 ura3::3xP.sub.POX4P450A19)
[0485] Plasmid pAA156 containing a P450A19 integration cassette was
digested with ClaI and column purified. Strain sAA779 (sec Example
30) was transformed with the linearized DNA and plated on SCD-ura
plate. Several colonies were checked for P450A19 integration. From
those colonies, qPCR was performed to check the copy number of
P450A19 integration. One strain, designated sAA811, contained 3
copies of P450A19.
Example 32
Construction of Strain sAA810 (pox4a::ura3/pox4b::ura3 POX5/POX5
acs1::P.sub.URA3/acs1::P.sub.URA3ura3::5xP.sub.POX4P450A19
ura3::8xP.sub.POX4TESA)
[0486] Plasmid pAA156 containing a P450-A19 integration cassette
was digested with ClaI and column purified. Plasmid pAA294
containing a TESA integration cassette also was digested with ClaI
and column purified. Strain sAA779 was cotransformed with both
linearized DNAs and plated on SCD-ura plate. Several colonies were
checked for both P450A19 integration and TESA integration. Colonies
that were positive for both TESA and P450A19 were further analyzed
by qPCR. qPCR was performed to check the copy number of the P450A19
and TESA integration events. One strain, designated sAA810,
contained 5 copies of P450A19 and 8 copies of TESA.
Example 33
General Techniques & Methods (Used for Examples 34-55)
Growth Media, Reagents and Conditions
[0487] YPD, ScD-ura media and plates, and 5-FOA containing plates
were made as described in Methods in Yeast Genetics: a Cold Spring
Harbor Laboratory Manual/David C. Amberg, Daniel J. Burke, Jeffrey
Strathern,--2005 ed.).
[0488] SP92+glycerol was made by adding 6.7 g of Bacto yeast
nitrogen base without amino acids (BD, Franklin Lakes, N.J., USA),
3.0 g of Bacto yeast extract (BD, Franklin Lakes, N.J., USA), 3.0 g
of ammonium sulfate, 1.0 g of potassium phosphate monobasic, 1.0 g
of potassium phosphate dibasic, and 75 g of glycerol to water to a
final volume of one liter. The media was then filtered
sterilized.
[0489] TB-low N Media was made by adding 1.7 g Bacto yeast nitrogen
base without ammonium sulfate, 3 g of Bacto yeast extract, 1 g of
potassium phosphate monobasic and 1 g potassium phosphate dibasic
per liter of water. The media was filtered sterilized.
[0490] Overnight cultures were typically grown in 2 to 5 ml of
either ScD-ura media or YPD media in standard culture tubes at
3.degree. C. on a shaker at about 250 rpm.
Molecular Methods
[0491] Gel purifications of DNA fragments were done as recommended
by the manufacturer using either the GeneJET Gel Extraction Kit
(Fermentas Inc, Glen Burnie, Md., USA) or the Zymoclean Gel DNA
Recovery Kit (ZymoResearch, Irvine, Calif., USA).
[0492] PCR was performed using either PFU Ultra II DNA Polymerase
(Agilent Technologies, Santa Clara, Calif., USA), Taq DNA
polymerase (New England Biolabs, Ipswich, Mass., USA), DreamTaq PCR
Master Mix (Fermentas Inc, Glen Burnie, Md., USA) or Quick Load
Midas Mix (Monserate, San Diego, Calif., USA). Each enzyme was used
according to the manufacturer's instructions.
[0493] Restriction enzyme digestions were conducted as recommended
by each manufacturer (New England Biolabs, Ipswich, Mass., USA or
Fermentas lnc., Glen Burnie, Md., USA). DNA ligations were
conducted using either the Rapid Ligation Kit (Fermentas Inc., Glen
Burnie, Md., USA) or using T4 DNA Ligase (New England Biolabs,
Ipswich, Mass., USA) according to the manufacturer's
instructions.
[0494] Yeast transformations were performed as described in Example
10.
[0495] Genomic DNA Preparation
[0496] The URA3 gene was obtained from genomic DNA of Candida yeast
culture ATCC20336. Genomic DNA from Candida strain ATCC20336 was
prepared as follows: About 1.5 ml of an overnight culture of cells
was and the pellet was resuspended in about 200 .mu.l of a solution
containing 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 MM Tris pH 8.0,
and 1 mM EDTA. About 200 .mu.l of acid washed glass beads were
added with about 200 .mu.l of phenol:chloroform:isoamyl alcohol
(25:24:1) at a pH of about 8:0. The sample was vortexed for about 2
minutes after which about 200 .mu.l of water was added. The sample
was then centrifuged at 13000 rpm for about 10 minutes. The aqueous
layer was transferred to a new microcentrifuge tube and an equal
volume of chloroform:isoamyl alcohol (24:1) solution was added.
This sample was vortexed for 10 seconds and then centrifuged at
13000 rpm for about 2 minutes. The aqueous layer was transferred to
a new microfuge tube and 1 ml of ethanol was added. The tube was
then placed at -80.degree. C. for about 15 minutes and then spun at
13000 rpm for 15 minutes to pellet the DNA The DNA was washed with
70% ethanol and air-dried. The DNA was then resuspended in about
500 .mu.l of water.
[0497] Genomic DNA for Klyveromyces lactis (ATCC8585) was purchased
from the American Type Culture Collection (Manassas, Va., USA).
[0498] To calculate gene copy number, a qPCR method was used as
described by Jin et al (Appl. Environ. Microbiol. January 2003 vol.
69, no. 1, 495-503). qPCR was performed according to the
manufacturer's instructions using either the Brilliant III
Ultra-Fast SYBR.RTM. Green QPCR Master Mix (Agilent Technologies,
Englewood, Colo., USA) or the QuantiTect Multiplex PCR NoROX Kit
(Qiagen). Genomic DNA from Candida strain ATCC20336 or plasmid DNA
containing the actin gene from ATCC20336 and the gene of interest
were used as standards.
[0499] Primers and probes used throughout these Examples were made
via standard DNA synthesis techniques by Integrated DNA
Technologies (Coralville, Iowa, USA).
Example 34
Construction of Cloning Plasmid AA073
[0500] The plasmid pAA073 was designed to contain the POX4 promoter
and terminator from Candida strain ATCC20336 (this strain is also
referred to herein as strain sAA001). This plasmid was derived from
the publicly available plasmid pUC19 which contains an ampicillin
resistance marker. pAA073 was designed to have two SapI restriction
enzyme sites located between the POX4 promoter and terminator which
allows unidirectional cloning of any gene of interest in tandem
with the POX4 promoter. The Candida strain ATCC20336 URA3 gene
including the open reading frame and the endogenous regulatory
regions was also placed into pAA073 as a selection marker for
transformants. Plasmid pAA073 allows the direct integration of
multiple copies of any gene of interest by digesting the plasmid
with a unique restriction enzyme such as SpeI, ClaI or BstZ171.
These multiple cloning sites for are contained in the URA3
auxotrophic marker region and can be selectively be used to avoid
cutting the gene of interest (i.e., the DNA sequence for the gene
of interest can be searched for particular restriction enzyme cut
sites and those enzymes can be avoided). In addition, this plasmid
can serve as a template to create an antibiotic free-DNA cassette
containing the gene of interest and the POX 4 regulatory regions
inserted between the 3' and 5' regions of the URA3 gene; this
cassette can be PCR amplified using the plasmid as a template, and
the isolated PCR product can be inserted into any microorganism
strain.
[0501] A diagram of pAA073 is set forth in FIG. 18 and the sequence
of pAA073 is set forth as SEQ ID NO: 160.
Example 35
Generic Procedure for Creating Yeast Transformation Plasmids and
Integration Cassettes and Creation of a ZWF1 Gene Transformation
Plasmid
[0502] One of two procedures was used to generate DNA constructs
useful to make transformed Candida yeast strains that contained
either amplified levels of endogenous genes or exogenous genes
inserted into the genomic DNA of the Candida yeast host. The
following endogenous genes were amplified from genomic Candida
ATCC20336 genomic DNA: fatty alcohol dehydrogenase ("ADH")--ADH1,
2, 3, 4, 5, 7 and 8; ZWF1 (glucose-6-phosphate dehydrogenase); FAT1
(fatty acyl transporter 1); PEX11 (peroxisomal biogenesis factor
11); HFD1 and HFD2 (human fatty aldehyde dehydrogenase 1 and 2),
CPRB (cytochrome p450 reductase B), P450A12-A20 and P450D2
(cytochrome p450 oxidases 12-20 and D2); FAT1 (fatty acyl
transporter 1); and IDP2 (cytoplasmic isocitrate dehydrogenase
NADP+). The gene GDP1 (glyceraldehyde 3 phosphate dehydrogenase)
was obtained from Klyveromyces lactis genomic DNA and is sometimes
referred to as "K1GDP1". In the case of the ADH1 gene, the alleles
were separately cloned; these alleles are referred to as "ADH1-1
and ADH-1-2. In addition, the ADH1 allele 1 was cloned as the
"short" version and thus is referred to as "ADH1-1short"; the ADH1
allele 2 was cloned as both short and regular versions and these
genes are referred to as "ADH1-2-short" and "ADH1-2". For ADH2, two
separate genes have been identified; each of them was cloned and
amplified herein and they are referred to as "ADH2a" and "ADH2b".
The first procedure ("Procedure 1") resulted in generating a
plasmid that was directly transformed into yeast; this plasmid
contained the antibiotic resistance gene kanamycin.
[0503] The second procedure ("Procedure 2") included all of the
steps of the first procedure, but added an additional final step to
remove the antibiotic resistance gene such that the transformed
Candida strain did not contain any exogenous antibiotic resistance
genes.
[0504] The first step in Procedure 1 was to amplify the gene of
interest from Candida strain ATCC20336 genomic DNA using
appropriately designed primers and standard PCR techniques. as set
forth above. The sequence of each primer is set forth in TABLE 25,
26 and 27. The amplified gene of interest was then inserted into
plasmid pCR-Blunt II-Topo (Life Technologies, Carlsbad, Calif.,
USA) using standard techniques recommended by the manufacturer. The
sequence of the gene or interest was then verified using standard
sequencing techniques. The name of the resulting plasmid for each
gene of interest is set forth in TABLE 21 under the column labeled
"Plasmid 1". Next, Plasmid 1 was digested with appropriate
restriction enzymes to isolate the gene of interest insert. This
gene of interest was then inserted into pAA073 (described in
Example 35) to create "Plasmid 3" for each gene of interest. The
name of each Plasmid 3 for each gene of interest is set forth in
TABLE 21 in the column labeled "Plasmid 3". It is possible to clone
the PCR fragment directly into Plasmid 3 thereby avoiding
construction of Plasmid 1. Each resulting Plasmid 3 contained the
gene of interest under the control of the POX 4 promoter and
terminator, the URA3 gene and regulatory regions, and the
ampicillin resistance marker gene. For some constructs, this
Plasmid 3 was cut in the URA3 gene and the entire linearized
plasmid was transformed into Candida strain ATCC20336. Such
transformed Candida strains contained the ampicillin resistance
gene.
[0505] In the second procedure, the entire first procedure was
followed. After creation of Plasmid 3 however, two PCR reactions
were conducted. The first reaction was designed to amplify only the
3' region of the URA3 gene; the amplified fragment was then gel
purified. A second PCR reaction amplified, as a single fragment,
the POX4 promoter, the gene of interest, the POX4 terminator, and
the 5' region of the URA3 gene. This fragment was also gel
purified. The two fragments were fused together by PCR and this PCR
product was inserted into plasmid pCR-Blunt II-Topo, this plasmid
was transformed into E coli cells and colonies were then selected
for sequence verification of the plasmid insert. The plasmid
containing the correct sequence was named and is referred to as
"Plasmid 4" in TABLE 21. Plasmid 4 was then used for PCR
amplification of the entire URA3'-POX4 promoter-gene of
interest-POX4 terminator-URA5 construct and this construct was then
used to transform Candida cells. The resulting transformed cells
contained the gene of interest but no antibiotic resistance genes
were introduced into the strain.
[0506] Preparation of a ZWF1 Transformation Plasmid
[0507] Procedure 1 described immediately above was used to create
this plasmid. The ZWF1 gene was PCR amplified from Candida strain
ATCC20336 genomic DNA using primers oAA831 and oAA832. The PCR
fragment was gel purified, cloned into the plasmid pCR-Blunt
II-Topo (Life Technologies, Carlsbad, Calif., USA) using standard
techniques recommended by the manufacturer and the sequences were
verified. The plasmid pCR-Blunt II-Topo contains a kanamycin
resistance gene. The resulting plasmid containing the gene encoding
the ZWF1 polypeptide was named pAA246 ("Plasmid 1"). The open
reading frame of ZWF1 was then cloned as a Sap1 fragment into
pAA073. The resulting plasmid was named pAA253 ("Plasmid 3").
Example 36
Creation of an Antibiotic-Free Yeast Integration Cassette for the
ADH2a Gene
[0508] Procedure 2 described in the previous Example was used to
create an integration cassette to introduce the gene encoding ADH2a
into Candida yeast cells. The ADH2a gene was PCR amplified using
standard procedures from Candida strain genomic DNA using primers
oAA3018 and oAA3019. The PCR fragment was gel purified, cloned into
pCR-Blunt II-Topo (Invitrogen, Carlsbad, Calif., USA) using
standard cloning techniques and the sequence was verified. Plasmid
1 containing the correct sequence was named pAA571. The ADH2A
fragment from pAA671 was then subcloned into pAA073 using SapI
restriction enzyme sites to form Plasmid 3, referred to as pAA683,
which places the ADH2a open reading frame under the control of the
POX4 promoter and POX4 terminator. An antibiotic-free cassette was
then created by assembly PCR. The 3' region of URA3 had a separate
fragment containing the POX4 promoter, ADH2a open reading frame,
POX4 terminator, and 5' region URA3 were each amplified using PCR
with either primers oAA2206 and oAA2207, or with primers oAA2208
and oAA2209, respectively. The PCR products were gel-purified,
combined and re-amplified using primers oAA2206 and oAA2209. The
resulting PCR fragment was cloned into pCR-Blunt II Topo (Life
Technologies, Carlsbad, Calif., USA) and sequence verified. A
plasmid with the correct sequence ("Plasmid") was named pAA711.
Example 37
Creation of an Antibiotic-Free Yeast Integration Cassette for the
K. lactis GDP1 Gene
[0509] K1GDP1 was cloned from genomic DNA at the same time that it
was mutagenized to replace an internal CUG codon to another leucine
encoding codon by replacing guanosine at position 774 with an
adenosine. The 5' region or 3' region of K1GDP1 was PCR amplified
from K. lactis genomic DNA using either oAA2457 and oAA2459 or
oAA2458 and oJHR4, respectively.
[0510] The PCR fragments were gel purified and combined to be used
as template for a PCR amplification with oAA2457 and oJHR4. The PCR
fragment was gel purified and cloned into pCR-Blunt II-TOPO as
recommended by the manufacturer. Plasmids were sequenced and a
plasmid with the right sequence named pAA541. This plasmid was the
template for the PCR with primers oAA2854 and oAA2855 to create
plasmid pAA578. All other procedures for preparing this cassette
were as described for the ADH2a using appropriate primers for
cloning and gene amplification.
[0511] <GDP1, K1--SEQ ID NO: 71
Example 38
Other Gene Amplification Cassette Constructs
[0512] In addition to ZWF1 and ADH2a, several other genes were
placed into either transformation plasmids or amplification
cassettes using either Procedure 1 (transformation plasmids) or
Procedure 2 (amplification cassettes) above. The genes included in
these plasmids or cassettes are set forth in TABLE 21. The genes
that were inserted into antibiotic-free amplification cassettes
have a Plasmid 4 on the TABLE 21; those genes that were put into
transformation plasmids do not have Plasmid 4. Tables 25-28 list
some oligonucleotides and oligonucleotide sequences that were used
to subclone and clone some of the genes described in the Examples
herein.
Example 39
Creation of a Candida Strain Overexpressing ZWF1
[0513] Plasmid pAA253 was digested with the restriction enzyme
ClaI. The linearized plasmid was transformed into Candida strain
sAA103 using standard transformation procedures. Transformants were
selected by growth in ScD-ura plates using standard procedures.
Plates were streaked to generate single colonies and transformants
were verified by PCR and sequence analysis. ZWF1 copy number was
determined using qPCR. A strain with approximately six copies of
ZWF1 was designated as sAA1233.
Example 40
Creation of a Candida Strain Overexpressing ADH2a
[0514] A 3'URA3-P.sub.POX4-ADH2A-T.sub.POX4-5'URA3 fragment was
constructed by using plasmid pAA711 as a template and PCR
amplifying the desired region of the plasmid with of primers
oAA2206and oAA2209. The PCR fragment was gel-purified and
transformed into Candida strain sAA103. Transformants were selected
by growth in ScD-ura plates. Colonies were streaked for single
isolates and transformant isolates were verified by PCR. Gene copy
number was then determined by qPCR. A strain was identified with
approximately seven copies of P.sub.POX4-ADH2A-T.sub.POX4-4 and was
named sAA1803.
Example 41
Creation of Additional Candida Strains
[0515] Several other transformation plasmids or amplification
cassettes were generated and were transformed in to Candida strain
sAA103 using Procedure 1 or Procedure 2 described above to create
novel plasmids and Candida strains. The genes, plasmid names and
strain names are set forth in TABLE 21.
TABLE-US-00021 TABLE 21 Plasmid Plasmid Plasmid Plasmid 4 Gene 1 2
3 Plasmid 2 Strain ADH1-1- pAA698 short ADH1-2 pAA670 pAA682 pAA716
sAA1817 ADH1-2- pAA697 pAA700 pAA728 sAA1848 short ADH2A pAA671
pAA683 pAA711 sAA1803 ADH2B pAA672 pAA691 pAA717 sAA1805 ADH7
pAA673 pAA692 pAA714 sAA1841 ADH5 pAA674 pAA693 pAA718 sAA1844 ADH3
pAA675 pAA715 pAA730 pAA739 sAA1901 ADH4 pAA676 pAA694 pAA719
sAA1839 SFA1 pAA680 pAA699 pAA727 sAA1808 ADH8 pAA729 pAA738 pAA741
sAA1904 ZWF1 pAA246 pAA253 sAA1233 FAT1 pAA635 PEX11 N/A pAA336
HFD1 pAA677 HFD2 pAA678 pAA695 pAA712 sAA1819 CPRB N/A pAA218
pAA391 P450 A12 pAA139 pAA151 P450 A13 pAA140 pAA152 P450 A14
pAA141 pAA153 pAA367 P450 A15 pAA160 P450 A16 pAA161 P450 A17
pAA142 pAA154 P450 A18 pAA143 pAA155 P450 A19 pAA144 pAA156 pAA392
P450 A20 pAA145 pAA157 P450 D2 pAA146 pAA158 FAT1 S244A PAA637 FAT1
pAA639 D495A IDP2 pAA462 sAA1306 KIGDP1 pAA578 pAA581 pAA592
sAA1485 Note: "Plasmid 1", "Plasmid 3", and "Plasmid 4" are as
described in Example-35; "Plasmid 2" was generated only for the
gene alcohol dehydrogenase 3 in which the guanosine at position 600
was mutated to an adenosine by site directed mutagenesis. To
prepare this plasmid, 30 to 50 ng of pAA675 was used as template in
a 50 .mu.l PCR reaction using primers oAA3073 and oAA3074 and PFU
Ultra II DNA Polymerase (Agilent Technologies, Santa Glara,
California, USA) as recommended by manufacture. After the PGR was
completed, 20 units of DpnI (New England Biolabs, Ipswich,
Massachusetts, USA) was added to the PCR reaction and incubated for
2 hours-at 37.degree. C. 5 .mu.l of the reaction was used to
transform DH5.alpha. cells (Monserate Biotechnology, San Diego CA
USA) as recommended by manufacture. The resulting plasmids were
sequence verified, and a plasmid with the right sequence was named
pAA715.
Example 42
Creation of Two FAT1 Mutant Genes
[0516] Two mutants of the FAT1 gene were created in an attempt to
reduce the acyl CoA synthetase activity of the enzyme while
maintaining its fatty acid transport activity. The first mutant
substituted an alanine at position 244 for the native serine; the
second mutant substituted an alanine at position 495 for the native
aspartic acid.
[0517] To prepare a gene containing the S244A mutation of FAT1,
oligonucleotides oAA2839 and oAA2805 were used to amplify the 5'
end of the native FAT1 gene from Candida ATCC20336 genomic DNA,
while oligonucleotides oAA2804 and oAA2875 were used to amplify the
3' end of the gene. Both products were gel purified and used as
templates for a second round of PCR using oligonucleotides oAA2839
and oAA2875. The resultant PCR product was digested along with
pAA073 using the restriction enzyme BspQ1 (New England Biolabs) and
the gel purified products were ligated with T4 DNA ligase
(Fermentas). The ligations were transformed into E. coli DH5a
(Montserrat) and plated on LB ampicillin. Minipreps (Qiagen) were
completed oil several colonies and sequence confirmed.
[0518] The above process was repeated fop the FAT1 D495A mutant
gene using oAA2839 and oAA2842 for the 5' end of the gene and
oAA2841 and oAA2875 for the 3' end. The two ends of the gene were
used as described with oAA2839 and oAA2875 to make the full
product, digested, cloned and verified as above.
[0519] Each mutant gene was inserted into plasmid pAA073.
Example 43
Preparation of Candida Strains Containing Multiple Amplified
Genes
[0520] In addition to creating novel Candida strains in which a
single gene was amplified, several strains were created with more
than one gene amplified. These strains were generated by
co-transforming strain sAA103 with the individual transformation
plasmids or amplification cassettes for each of the genes of
interest. TABLE 22 below sets forth the name of each such Candida
strain created and the genes transformed into the strain.
TABLE-US-00022 TABLE 22 Strain Gene 1 Source of Gene Gene 2 Source
of Gene Gene 3 Final Plasmid sAA1082 CPRB pAA391 P450 A19 pAA392
sAA1569 CPRB pAA391 p450 A14 pAA367 sAA1633 CPRB pAA391 p450 A19
pAA392 ZWF1 pAA246 sAA1644 CPRB pAA391 p450 A19 pAA391 IDP2 pAA462
sAA1304 CPRB pAA391 p450 A19 pAA392
Example 44
Creation of a FAT1 Knockout Strain
[0521] To create a Candida strain with decreased FAT1 gene
expression, knock out cassettes for each FAT1 allele were
generated. For the first allele, the 5' homology region
(nucleotides 27 to 488 of the open reading frame of FAT1) was
amplified using primers oAA2055 and oAA2056 with Candida strain
ATCC20336 genomic DNA as a template. The 3' homology region
(consisting of nucleotides 1483 to 1891 of the FAT1 open reading
frame) was amplified using primers oAA2069 and oAA2060 from the
same genomic DNA. A cassette containing the URA3 marker with the
promoter repeated at the 3' end was amplified from pAA298 to
contain overlaps with both homology regions with oAA2057 and
oAA2068. These three DNA fragments pieces were then used in a
subsequent PCR reaction to generate the deletion cassette using
oligos oAA2055 and oAA2060. The PCR purified cassette was then
transformed into strain sAA103 and transformants verified by PCR to
obtain sAA919. This strain was plated on 5FOA to cure the URA3
marker ad was verified by PCR. This strain without URA3 was
designated as sAA986.
[0522] The second FAT1 allele disruption cassette was generated as
follows: A 5' homology region (nucleotides 487 to 951 of the open
reading frame) was amplified using primers oAA2070 and oAA2071. A
3' homology region (nucleotides 987 to 1439 of the open reading
frame) was amplified using primers oAA2074 and oAA2075 and Candida
ATCC20336 genomic DNA as a template. A cassette containing the URA3
marker with the promoter repeated at the 3' end was constructed to
have overlaps with homology to primers oAA2072 and oAA2073. The
three fragments were then used in a subsequent PCR reaction to
generate the deletion cassette using oligos oAA2070 and oAA2075.
This purified product was then used to transform sAA986, and
transformants were verified by PCR as having the second allele
disrupted. A strain with the correct genotype was named sAA1000.
This strain was plated on 5FOA and was verified for removal of the
URA3 marker using PCR. This strain was designated as sAA1182.
Example 45
Creation of a FAT1/ACS1 Double Deletion Strain
[0523] Functional POX5 alleles were restored in Candida strain
sAA003 by transformation of sAA003 with POX5 linear DNA to replace
the URA3-disrupted loci with/a functional allele. A 2,584 bp DNA
fragment was amplified by PCR using primers oAA0179 and oAA0182
that contained the POX5 ORF as well as 456 bp upstream and 179 bp
downstream of the ORF using plasmid pAA049 as template. The
purified PCR product was used to transform competent sAA003 cells
which were plated on YNB-agar plates supplemented with dodecane
vapor as the carbon source (e.g., by placing a filter paper soaked
with dodecane in the lid of the inverted petri dish) and incubated
at 30.degree. C. for 4-5 days. Colonies growing on dodecane as the
sole carbon source were re-streaked onto YPD-agar and incubated at
30.degree. C. Single colonies were grown in YPD cultures and used
for the preparation of genomic DNA. PCR analysis of the genomic DNA
prepared from the transformants was performed with oligos oAA0179
and oAA0182. An ura3-disrupted POX5 would produce a PCR product of
4,784 bp while a functional POX5 would produce a PCR product of
2,584 bp. In the resulting strain, sAA235, a PCR product of 2,584
bp was amplified indicating that both POX5 alleles had been
functionally restored. An unintended consequence of the
selection/strategy (YNB-agar with dodecane) was that the cells
reverted back to an Ura' phenotype. Without being limited by any
theory, it is believed the absence of uracil in the solid media and
the replacement of the only functional URA3 forced the cells to
mutate one of the other ura3 loci back to a functional allele.
Plasmid pAA276 was digested with BamHI/XhoI and column purified.
Strain sAA329 (ura3/ura3 pox4a::ura3/pox4b::ura3 POX5/POX5) was
transformed with the linearized DNA and plated on SCD-ura plate.
Several colonies were checked for ACS1 disruption. One-such strain
was designated sAA722.
[0524] Strain sAA722 was grown in YPD media overnight and plated on
5-FOA plate. Colonies that grew in the presence of 5-FOA were PCR
screened for the looping out of the URA3 gene leaving behind only
the URA3 promoter (P.sub.URA3) in the ACS1 site. Out of 30 colonies
analyzed, only one strain showed the correct genetic modification.
The strain was designated sAA741.
[0525] Plasmid pAA282 was digested with BamHI/XhoI and column
purified. Strain sAA741 was transformed with the linearized DNA and
plated on SCD-ura plate. Several colonies were checked for doubled
ACS1 knockout by insertional inactivation. One such strain was
designated sAA776.
[0526] Strain sAA776 was grown in YPD media overnight and plated on
5-FOA plate. Colonies that grew in the presence of 5-FOA were PCR
screened for the looping out of the URA3 gene leaving behind only
the URA3 promoter (PURA3) in both ACS1 gene alleles. One such
strain was named sAA779. The full-length coding sequence of the
Fat1 gene was amplified from Candid strain ATCC20336 genomic DNA
using primers oAA1023 and oAA1024. The 2,086 bp PCR product was gel
purified and ligated into pCR-Blunt II-TOPO (Invitrogen),
transformed into competent TOP10 E. coli cells (Invitrogen) and
clones containing PCR inserts were sequenced to confirm correct DNA
sequence. One such plasmid was named pAA296.
[0527] Deletion of each FAT1 allele was achieved by transforming
cells with linear DNA cassettes constructed by overlap extension
PCR (OE-PCR). The deletion-cassette for the first FAT1 allele in
sAA779 was created from three DNA fragments. The first DNA fragment
(FAT1 5' homology) was amplified from plasmid pAA296 using primers
oAA2055 and oAA2056. The second DNA fragment (PURA3URA3TURA3PURA3)
was amplified from plasmid pAA298 using primers oAA2057 and
oAA2068. A diagram of plasmid pAA298 is set forth in FIG. 19 and
the sequence of this plasmid is set forth as SEQ ID NO: 161.
>PAA298--SEQ ID NO: 161
[0528] The third DNA fragment (FAT1 3' homology) was amplified from
plasmid pAA296 using primers oAA2069 and oAA2060. The location of
primer annealing sites in pAA296 that amplify FAT1 DNA fragments
are shown in FIG. 59. All three DNA fragments were combined in the
same reaction to generate the full-length deletion cassette by
OE-PCR using primers oAA2055 and oAA2060. Strain sAA779 was
transformed with the full-length deletion cassette and plated on
SCD-Ura-plate. Several colonies were screened by PCR for
integration of the deletion cassette at the first FAT1 allele. One
such strain was named sAA865.
[0529] Strain sAA865 was grown in YPD media overnight and plated on
5-FOA plate. Colonies that grew in the presence of 5-FOA were PCR
screened for the looping out of the URA3 gene leaving behind only
the URA3 promoter (PURA3) in the first FAT1 allele. One such strain
was named sAA869.
[0530] The deletion of the second FAT1 allele in sAA869 was
performed by transformation with a deletion cassette created by
OE-PCR. The deletion cassette for the second FAT1 allele was
constructed from three DNA fragments. The first DNA fragment (FAT1
5' homology) was amplified from plasmid pAA296 using primers
oAA2070 and oAA2075. The second DNA fragment (PURA3URA3TURA3PURA3)
was amplified from plasmid pAA298 using primers oAA2072 and
oAA2073. The third DNA fragment (FAT1 3' homology) was amplified
from plasmid pAA296 using primers oAA2074 and oAA2075. All three
DNA fragments were combined in the same reaction to create a
full-length deletion cassette by OE-PCR using primers oAA2070 and
oAA2071. Strain sAA869 was transformed with the full-length
deletion cassette and plated on SCD-Ura-plate. Several colonies
were screened by PCR for integration of the deletion cassette at
the second FAT1 allele. One such strain was named sAA875. Candida
strain sAA875 was grown overnight in YPD media and then streaked on
to in5-fluorotic acid containing plates. Single colonies were
tested for URA3 reversion frequency, and the isolate with least
reversion frequency was named sAA886.
[0531] A disruption cassette for the first allele of the POX5 gene
was constructed by overlapping PCR. A 5' POX5 (+34 to +488 of the
ORF) or 3' POX5 (+1487 to +1960 of the OrF) fragment was PCR
amplified using genomic DNA from ATCC20336 as the template and
primers oAA2173 and oAA2174 (for the 5' fragment) or oAA2177 and
oAA2178 (for the 3' fragment). A Candida URA3 gene fragment with
direct repeat was PCR amplified using oAA2175 and oAA2176 as
primers. The three gene fragments were then gel purified, combined,
ligated and used as template for to male the full length construct
via PCR using oAA2173 and oAA2178 as primers. This approximately
2.9 Kb fragment was gel purified and used to transform sAA886.
Transformants were selected by growth in ScD-ura plates. Colonies
were re-streaked to isolate individual transformants. Disruption of
the first allele of POX5 was verified by PCR. A strain with the
right genotype was named sAA940.
[0532] Strain sAA940 was grown overnight in YPD and then streaked
in 5-fluorotic acid containing plates. Strains were screened by PCR
for the present of the POX5 deletion. A strain with the right
genotype was renamed sAA969.
[0533] A disruption cassette for the second allele of the POX5 gene
was constructed by overlapping PCR. A 5' POX5 (+489 to +960 of the
ORF) or 3' POX5 (+1014 to +1479 of the ORF) fragment was PCR
amplified using genomic DNA from ATCC20336 and primers oAA2188 and
oAA2189 or oAA2192 and oAA2193, respectively. A Candida URA3 gene
fragment with the terminator as a direct repeat was PCR amplified
using oAA2190 and pAA2191 as primers and pAA298 as template. These
three DNA fragments were gel purified, combined, ligated and used
as template for PCR using oAA2188 and oAA2193 as primers. This
approximately 2.9 Kb fragment was gel purified and used to
transform strain sAA969. Transformants were selected by growth in
ScD-ura plates. Colonies were re-streaked to isolate individual
transformants. Strains were screened for disruption of both POX5
alleles by PCR. A strain with the right genotype was named
sAA988.
Example 46
Construction of a POX4, POX5, ACS1 Deletion Strain
[0534] A disruption cassette for the first ACS1 gene allele was
constructed by overlapping PCR. A 5' ACS1 (+101 to +601 of the ORF)
or 3' ACS1 fragment (+1546 to +1960 of the ORF) was PCR amplified
using genomic DNA from ATCC20962 and primers oAA2406 and oAA2407 or
oAA2408 and oAA2409, respectively. A Candida URA3 gene fragment was
PCR amplified using oAA2410 and oAA2411 as primers and pAA244
(described in Example 58) as template. The three gene fragments
were gel purified, combined, ligated and used as template for PCR
using oAA2406 and oAA2409 as primers. This PCR fragment was gel
purified and used to transform sAA103. Transformants were selected
by growth in ScD-ura plates. Colonies were re-streaked to isolate
individual transformants. Disruption of the first allele of ACS1
was verified by PCR. A strain with the right genotype was named
sAA1185.
[0535] sAA1185 was grown overnight in YPD and streaked in streaked
in 5-fluoorotic acid containing plates. Strains were screened by
PCR for the present of the ACS1 deletion. A strain with the right
genotype was renamed sAA1313.
[0536] A nested disruption cassette was constructed by overlapping
PCR. A 5' ACS1 (+626 to +1021 of the ORF) or a 3' ACS1 (+1151 to
+1518 of the ORF) fragment was PCR amplified using genomic DNA from
ATCC20336 and primers oAA2412 and oAA2413 or oAA2414 and oAA2415,
respectively. A Candida URA3 fragment was PCR amplified using
oAA2416 and oAA2417 as primers for amplification of the URA3 gene.
The three fragments were gel purified, combined and used as
template for PCR with oAA2412 and oAA2415 as primers for this PCR
reaction. The correct PCR fragment was gel purified and used to
transform sAA1184. Transformants were selected by growth in ScD-ura
plates. Colonies were re-streaked to isolate individual
transformants. These transformants were screened for disruption of
both ACS1 alleles by PCR. A strain with the correct genotype was
named sAA1371.
Example 47
Construction and Evaluation of Certain CPR750-CYP450 Strains
[0537] Plasmids comprising a combination the CPR750 gene and one or
more CYP450 genes were created ligating either the CPR750 gene
containing the endogenous CPR750 promoter (see plasmid pAA067 in
Example 16) into each of pAA151-158, pAA160 or pAA161 as follows
(TABLE 23).
[0538] Plasmid pAA151 was digested with SbfI/SpeI restriction
enzymes and the 2584 bp fragment encoding CPR750 was isolated and
ligated into the 6198 bp fragment of pAA067 when digested with SbfI
and SpeI. The ligation mixture was transformed into E. coli cells
(DH5alpha). Plasmids were verified by restriction enzyme analysis
and sequencing. A plasmid with the correct sequence was named
pAA223.
TABLE-US-00023 TABLE 23 P450 CPR750 Frag- Frag- Final En- ment En-
ment Plasmid P450 Plasmid zymes size Plasmid zymes size pAA223 A12
pAA151 Sbf1/ 2584 pAA067 Sbf1/ 6198 SpeI SpeI pAA224 A13 pAA152
Sbf1/ 2609 pAA067 Sbf1/ 6198 SpcI Spcl pAA225 A14 pAA153 Sbf1/ 2581
pAA067 Sbf1/ 6198 SpcI Spcl pAA226 A15 pAA160 SbfI/ 5712 pAA067
SbfI/PciI/Ap 3121 PciI aL1 pAA227 A16 pAA161 SbfI/ 5712 pAA067
SbfI/ 3121 PciI PciI pAA228 A17 pAA154 Sbf1/ 2594 pAA067 Sbf1/ 6198
Spcl Spcl pAA229 A18 pAA155 Sbf1/ 2566 pAA067 Sbf1/ 6198 SpcI Spcl
pAA230 A19 AA156 Sbf1/ 2551 pAA067 Sbf1/ 6198 SpeI SpeI pAA231 A20
pAA157 Sbf1/ 2551 pAA067 Sbf1/ 6198 SpeI SpeI pAA232 D2 pAA148
Sbf1/ 3512 pAA067 Sbf1/ 6198 SpcI Spcl
[0539] Plasmids pAA223 and pAA233 were linearized with SpeI (New
England Biolabs) while the remaining plasmids were linearized with
ClaI (New England Biolabs). sAA103 was transformed with the
linearized plasmids. Transformants were selected by growth in
ScD-ura plates. Colonies were streaked for single isolates and
transformants in each isolate were selected and verified by
PCR.
[0540] The strains prepared above were then tested for production
of di-acids using coconut oil as a substrate ("feedstock").
[0541] Strains were grown overnight in SP92+glycerol (5 mL), then
transferred to 50 mL SP92+glycerol (50 mL) at a starting OD=0.4.
Each strain was centrifuged and the pellet resuspended in TB lowN
medium (12.5 mL). To each flask 2% coconut oil was added. Flasks
were incubated at 300 RPMs 30.degree. C. Samples (1 mL) were taken
at 30 and 96 hrs. for GC analysis.
[0542] As can be seen in TABLE 24, P450 A19 showed the biggest
improvement in diacid formation on C10, C12 and C14 fatty
acids.
TABLE-US-00024 TABLE 24 Gene Diacid formed from total acid at 30
hrs. Strain P450 C6 C8 C10 C12 C14 sAA003 N/A 0.24 0.03 0.61 0.40
0.16 sAA0797 P450 A12 0.21 0.04 0.31 0.11 0.04 sAA0798 P450 A13
0.15 0.03 0.71 0.71 0.35 sAA0799 P450 A14 0.18 0.03 0.29 0.08 0.03
sAA0800 P450 A15 0.13 0.04 0.60 0.35 0.14 sAA0801 P450 A15 0.16
0.06 0.75 0.65 0.33 sAA0802 P450 A15 0.20 0.08 0.75 0.67 0.38
sAA0803 P450 A16 0.20 0.03 0.67 0.46 0.19 sAA0804 P450 A17 0.26
0.07 0.74 0.64 0.41 sAA0805 P450 A18 0.19 0.08 0.81 0.81 0.55
sAA0806 P450 A19 0.24 0.56 0.95 0.92 0.73 sAA0807 P450 A20 0.22
0.38 0.83 0.64 0.32
Example 48
Conversion of Methyl Laurate and Methyl Myristate to the
Corresponding Diacid--Comparison of Strain sAA1304 to sAA003
[0543] A pre-culture of 80 mL SP92 (6.7 g/L yeast nitrogen base,
3.0 g/L yeast extract, 3.0 g/L (NH.sub.4).sub.2SO.sub.4, 1.0 g/L
K.sub.2HPO.sub.4, 1.0 g/L KH.sub.2PO.sub.4, 75 g L dextrose) in a
500 mL baffled flask with foam plugs was inoculated with 1.0 mL
from a frozen glycerol stock of strain sAA003 (beta-oxidation
blocked strain) or strain sAA1304 (beta-oxidation blocked strain
plus amplified CPRB and CYP52A19) and incubated for 24 h at
30.degree. C. and 250 RPM. Fermentation medium (MM1) of composition
27 g/L dextrose, 7.0 g/L ammonium sulfate, 5.1 g/L potassium
phosphate monobasic, 1.024 g/L magnesium sulfate heptahydrate,
0.155 g/L calcium sulfate dihydrate, 0.06 g/L citric acid
anhydrous, 0.04 g/L ferrous sulfate heptahydrate, 0.0002 mg/L
biotin, 1.0 mL trace minerals solution (0.9 g/L boric acid, 0.11
g/L cupric sulfate pentahydrate, 0.18 g/L potassium iodide, 0.806
g/L manganese sulfate monohydrate, 0.360 g/L sodium molybdate,
0.720 g/L zinc sulfate), pH 5.8 was filter sterilized and
transferred to a sterile fermentation vessel. Growth was initiated
with an inoculum of pre-culture to an initial OD.sub.600nm=1.0 and
growth conditions of 35.degree. C., 1000 rpm, 1 vvm, pH 5.8. Growth
continued for approximately 10-12 h at which point the conversion
phase was initiated by the addition of a bolus of 5 g/L of
feedstock (methyl myristate only), followed immediately by a
continuous feed of feedstock. Because of the toxicity of lauric
acid, which is formed during the conversion process by
demethylation of methyl laurate at high concentrations, no initial
bolus was given. Feedstock feed rates varied as follows: methyl
myristate (Sigma-Aldrich #W272205), 1.0 g/L-h for the first 24 h;
1.5 g/L-h from 24 h to termination; methyl laurate (Sigma-Aldrich
#W271500), 0.5 g/L-h for the first 24 h; 1.2 g/L-h from 24 h to
termination. In addition, a co-feed of glucose was fed at a rate of
1.25 g/L-h when using methyl myristate as substrate or at a rate of
1.0 g/L-h when using methyl laurate as substrate. At induction, the
temperature was changed to 30.degree. C. and the pH was maintained
at 6.0 by addition of 6N KOH. The data in FIG. 20 shows the
production of either dodecanedioic acid from methyl laurate or
tetradecanedioic acid from methyl myristate and demonstrates the
improved productivity of strain sAA1306 over sAA003 on both
feedstocks. When methyl myristate was used as feedstock, sAA1306
showed an approximately 25% improvement in productivity over
sAA003.
Example 49
Conversion of Methyl Laurate and Methyl Myristate to the
Corresponding Diacid--Comparison of Strain sAA1082 to sAA003
[0544] A pre-culture of 80 mL SP92 in a 500 mL baffled flask with
foam plugs was inoculated with 1.0 mL from a frozen glycerol stock
of strain sAA003 (beta-oxidation blocked strain) or strain sAA1082
(beta-oxidation blocked strain plus amplified CPRB and CYP52A19)
and incubated for 24 h at 30.degree. C. and 250 RPM. Fermentation
medium (MM1) at pH 5.8 was filter sterilized and transferred to a
sterile fermentation vessel. Growth was initiated with an inoculum
of pre-culture to an initial OD.sub.600nm=1.0 and growth conditions
of 35.degree. C., 1000 rpm, 1 vvm, pH 5.8. Growth continued for
approximately 10-12 h at which point the conversion phase was
initiated by the addition of a bolus of 5 g/L of feedstock (methyl
myristate only), followed immediately by a continuous feed of
feedstock. Because of the toxicity of lauric acid which is formed
during the conversion process by demethylation of methyl laurate at
high concentrations, no initial bolus was given. Feedstock feed
rates varied as follows: methyl myristate, 1.0 g/L-h for the first
24 h; 1.5 g/L-h from 24 h to termination; methyl laurate, 0.75
g/L-h for the first 24 h; 1.4 g/L-h from 24 h to termination. In
addition, a co-feed of glucose was fed at a rate of 1.25 g/L-h for
all fermentations. At induction, the temperature was changed to
30.degree. C. and the pH was maintained at 6.0 by addition of 6N
KOH. The data in FIG. 21 show the production of either
dodecanedioic acid from methyl laurate or tetradecanedioic acid
from methyl myristate and demonstrate improved productivity of
strain sAA1082 over sAA003 on both feedstocks. When methyl laurate
was used as feedstock, sAA1082 demonstrated about 23% productivity
improvement over sAA003. With methyl myristate as feedstock,
sAA1082 showed an approximately 37% improvement over sAA003.
Example 50
Conversion of Oleic to cis-9-octadecenedioic Acid--Comparison of
Strains sAA1233, sAA1306 and sAA1485 to sAA003
[0545] A pre-culture of 80 mL SP92 in a 500 mL baffled flask with
foam plugs was inoculated with 1.0 mL from a frozen glycerol stock
of strain sAA003 (beta-oxidation blocked strain), strain sAA1233
(beta-oxidation blocked strain plus amplified ZWF1), strain sAA1306
(beta-oxidation blocked strain plus amplified IDP2), or strain
sAA1485 (beta-oxidation blocked strain plus amplified K1GDP1) and
incubated for 24 h at 30.degree. C. and 250 RPM. Fermentation
medium (MM1) at pH 5.8 was filter sterilized and transferred to a
sterile fermentation vessel. Growth was initiated with an inoculum
of pre-culture to an initial OD.sub.600nm=1.0 and growth conditions
of 35.degree. C., 1000 rpm, 1 vvm, pH 5.8. Growth continued for
approximately 10-12 h at which point the conversion phase was
initiated by the addition of a bolus of 5 g/L of oleic acid
(Sigma-Aldrich #W281506), followed immediately by a continuous feed
of feedstock at a rate of 2.0 g/L-h throughout the conversion
phase. In addition, a co-feed of glucose was fed at a rate of 1.25
g/L-h for all fermentations. At induction, the temperature was
changed to 30.degree. C. and the pH was maintained at 6.0 by
addition of 6N KOH. The data in FIG. 22 are averages of three
identical fermentations and show the production
cis-9-octadecenedioic acid (C18:1 diacid) from oleic acid.
[0546] All three amplified genes (ZWF1, IDP2 and K1GDP1) code for
enzymes that produce NADPH during the biochemical reaction and,
because of that, increased expression of those enzymes should
result in increased intracellular levels of NADPH. Omega-hydroxy
fatty acids (HFAs) are observed to be produced as a result of
incomplete oxidation of the fatty acid feedstock to the
corresponding diacid. One reason for this incomplete oxidation may
be reduced levels of NADPH, which is required for the
over-oxidation reaction of HFAs by cytochrome P450 Thus, increasing
the intracellular pool of NADPH should result in decreased levels
of HFA in the fermentation broth. The concentrations of HFAs
produced during the omega oxidation of oleic acid by strains
sAA003, sAA1233, sAA1306 and sAA1485 are shown in FIG. 23. The
results (averages of three fermentations) demonstrate that all
three test strains (sAA1233, sAA1306 and sAA1485) produced lower
levels of HFAs than the base strain, sAA003. Production of HFAs in
a commercial diacid fermentation process is undesirable, since it
results in lower molar yields and has to be removed during
purification of the diacid. These results indicate that
amplification of either ZWF1, IDP2 or K1GDP1 should result in an
improved diacid fermentation having lower levels of HFAs.
Example 51
Conversion of Methyl Decanoate to Sebacic Acid--Comparison of
Strain sAA1082 to sAA003
[0547] Omega-oxidation of decanoic acid to produce sebacic acid by
a Candida strain can be impractical due to the high degree of
toxicity of this potential feedstock (ref). An alternative is to
use methyl decanoate, which has very low toxicity. Methyl esters of
fatty acids can be converted to the corresponding diacid by
beta-oxidation-blocked strains of Candida since Candida produces an
esterase that demethylates the fatty acid ester during the
omega-oxidation process, allowing conversion of methyl decanoate
into the non-toxic diacid, sebacic acid. Unfortunately, having an
excess of methyl decanoate in the fermentation broth prior to
induction of the enzymes involved in omega-oxidation process
results in sufficient demethylation of methyl decanoate to produce
toxic levels of decanoic acid, resulting in rapid cell death and a
failed fermentation. The standard fermentation procedure would
utilize the feedstock (methyl decanoate) as inducer. However, an
alternative would be to induce with a non-toxic inducer, such as
decane, which has the same carbon chain-length as decanoic acid,
but which does not produce decanoic acid during bioconversion to
sebacic acid. A set of four fermentations was performed to compare
decane to methyl decanoate as inducer as well as to compare the
beta-oxidation blocked base strain, sAA003 to strain sAA1082, which
has amplified CPRB and CYP52A19 genes. In a previous example,
sAA1082 demonstrated increased productivity over sAA003 with both
methyl laurate and methyl myristate.
[0548] A pre-culture of 80 mL SP92 in a 500 mL baffled flask with
foam plugs was inoculated with 1.0 mL from a frozen glycerol stock
of strain sAA003 (beta-oxidation blocked strain) or strain sAA1082
(beta-oxidation blocked strain plus amplified CPRB and CYP52A19)
and incubated for 24 h at 30.degree. C. and 250 RPM. Fermentation
medium (MM1) at pH 5.8 was filter sterilized and transferred to a
sterile fermentation vessel. Growth was initiated with an inoculum
of pre-culture to an initial OD.sub.600nm=1.0 and growth conditions
of 35.degree. C., 1000 rpm, 1 vvm, pH 5.8. Growth continued for
approximately 10-12 h at which point the conversion phase was
induced by the addition of either: 1) a bolus of 10 g/L of decane
(Sigma-Aldrich #457116) for 6 h after which a continuous feed of
methyl decanoate (TCI America #D0023) at 0.25 g/L-h was initiated
or 2) no addition of decane. Induction was performed by initiating
a continuous feed of methyl decanoate at 0.25 g/L-h. Because of the
volatility of decane, the aeration rate was reduced to 0.3 vvm
during the 6-h induction phase with decane as inducer. In addition,
a co-feed of glucose was fed at a rate of 1.25 g/L-h for all
fermentations. At induction, the temperature was changed to
30.degree. C. and the pH was maintained at 6.0 by addition of 6N
KOH. The data in FIG. 24 show the production of decanedioic acid
(sebacic acid) and compare the productivity of the two strains
under the two different induction conditions. When induced only
with methyl decanoate, neither strain sAA003 nor sAA1082 produced
significant quantities of sebacic acid over the course of the
fermentation. However, both strains produced sebacic acid when
induced with decane prior to beginning a slow feed of methyl
decanoate. Strain sAA1082, however, yielded an over four times
higher titer of sebacic acid at 84 h fermentation time than strain
sAA003, indicating that sAA1082 is a superior strain for diacid
production on methyl decanoate as well as methyl laurate and methyl
myristate as feedstock. One of the reasons why productivity was
better with strain sAA1082 induced with decane is illustrated in
FIG. 25, which shows the amount of decanoic acid produced under the
different fermentation conditions. The only fermentation that did
not produce a detectable quantity of the toxic by-product, decanoic
acid, was the fermentation with strain sAA1082 induced with decane.
The other three fermentations produced between 1 and 4 g/L decanoic
acid. Viable cell count data demonstrates the toxicity of decanoic
acid. The only fermentation where viable cell counts remained high
throughout the fermentation was the fermentation with strain
sAA1082 induced with decane. The other three fermentations lost
between 10.sup.3-10.sup.5 viable cells/mL of culture broth. That
significant reduction of biologically-active cells resulted in a
large accumulation of both methyl decanoate and dextrose in all
fermentations except the fermentation with sAA1082 induced with
decane. That fermentation showed little to no accumulation of
either methyl decanoate or dextrose. These data indicate that it
would probably have been possible to use a higher methyl decanoate
feed rate than 0.25 g/L-h. These results demonstrate that under the
right induction conditions and with an improved production strain;
it is possible to produce significant quantities of sebacic acid
from the methyl ester of a toxic fatty acid.
[0549] Other non-toxic inducers, such as alkanes with chain lengths
greater than C6, fatty acids with chain-lengths greater than C12,
various esters of fatty acids greater than C12, triglycerides
containing various chain-length fatty acids, or other non-toxic
chemicals containing a long aliphatic chain greater than C6 could
be used as a non-toxic inducer. However, as in this example, using
an inducer that would hot produce sebacic acid during the
omega-oxidation process, would likely result in an oxidation
product that would need to be purified from the desired product,
sebacic acid.
[0550] The method described in this example--for employing a
non-toxic feedstock to induce diacid production from the methyl
ester of a toxic fatty acid--could be used with fermentations
utilizing methyl laurate as feedstock. Lauric acid is not as toxic
to Candida as is decanoic acid, but care must be exercised in the
induction process to feed methyl laurate at a rate sufficient to
allow good induction without overfeeding, which would result in the
production and accumulation of toxic levels of lauric acid due to
demethylation by esterases.
Example 52
Conversion of Methyl Laurate to DDDA--Comparison of Strain sAA1569
to sAA003
[0551] A pre-culture of 80 mL SP92 in a 500 mL baffled flask with
foam plugs was inoculated with 1.0 mL from a frozen glycerol stock
of strain sAA003 (beta-oxidation blocked strain); or strain sAA1569
(beta-oxidation blocked strain plus amplified CPRB and CYP52A14),
and incubated, for 24 h at 30.degree. C. and 250 RPM. Fermentation
medium (MM1) at pH 5.8 was filter sterilized and transferred to a
sterile fermentation vessel. Growth was initiated with an inoculum
of pre-culture to an initial OD.sub.600nm=1.0 and growth conditions
of 35.degree. C., 1000 rpm, 1 vvm, pH 5.8. Growth continued for
approximately 10-12 h at which point the conversion phase was
initiated by a continuous feed of methyl laurate at a rate of 0.75
g/L-h for the first 24 h; 1.5 g/L-h from 24 h to termination. In
addition, a co-feed of glucose was fed at a rate of 1.25 g/L-h for
all fermentations. At induction, the temperature-was changed to
30.degree. C. and the pH was maintained at 6.0 by addition of 6N
KOH. The data in FIG. 26 are averages of two identical
fermentations and show the production of DDDA and
12-hydroxy-dodecanoic acid (HFA) from methyl laurate. Although
strain sAA1569 did not exhibit increased productivity over sAA003,
it did produce less than half the amount of HFA as sAA003. This
result is likely due to CYP52A14 exhibiting a greater rate of
over-oxidation of omega-hydroxy dodecanoic acid than the native
P450s.
Example 53
Conversion of Methyl Laurate to DDDA--Comparison of Strains sAA1082
and sAA1633 to sAA003
[0552] A pre-culture of 80 mL SP92 in a 500 mL baffled flask with
foam plugs was inoculated with 1.0 mL from a frozen glycerol stock
of strain sAA003 (beta-oxidation blocked strain), strain sAA1082
(beta-oxidation blocked strain plus amplified CPRB and CYP52A19) or
sAA1633 (beta-oxidation blocked strain plus amplified CPRB,
CYP52A19 and ZWF1), and incubated for 24 h at 30.degree. C. and 250
RPM. Fermentation medium (MM1) at pH 5.8 was filter sterilized and
transferred to a sterile fermentation vessel. Growth was initiated
with an inoculum of pre-culture to an initial OD.sub.600nm=1.0 and
growth conditions of 35.degree. C., 1000 rpm, 1 vvm, pH 5.8. Growth
continued for approximately 10-12 h at which point the conversion
phase was initiated by a continuous feed of methyl laurate at a
rate of 0.75 g/L-h for the first 24 h; 1.5 g/L-h from 24 h to
termination. In addition, a co-feed of glucose was fed at a rate of
1.25 g/L-h for all fermentations. At induction, the temperature was
changed to 30.degree. C. and the pH was maintained at 6.0 by
addition of 6N KOH. The data in FIG. 27 are averages of two
identical fermentations and show the production of DDDA from methyl
laurate. Strain sAA1082 again demonstrated about 23% increase in
productivity over sAA003. Strain sAA1633 exhibited an even greater
productivity increase of about 30% over sAA003. This additional
productivity increase was probably due to the amplification of
ZWF1, leading to increased production of NADPH, which provides
electrons for the omega-oxidation pathway.
Example 54
Conversion of Methyl Laurate to DDDA--Comparison of Strains
sAA1901, sAA1904, sAA1803 and sAA1805 to sAA003
[0553] A pre-culture of 80 mL SP92 in a 500 mL baffled flask with
foam plugs was inoculated with 1.0 mL from a frozen glycerol stock
of strain sAA003 (beta-oxidation blocked strain), sAA1901
(beta-oxidation blocked strain plus amplified ADH3), sAA1904
(beta-oxidation blocked strain plus amplified ADH8), strain sAA1803
(beta-oxidation blocked strain plus amplified ADH2a) or sAA1805
(beta-oxidation blocked strain plus amplified ADH2b), and incubated
for 24 h at 30.degree. C. and 250 RPM. Fermentation medium (MMI) at
pH 5.8 was filter sterilized and transferred to a
TABLE-US-00025 TABLE 25 Supplemental List 1 of Oligonucleotides
used in Examples 1-56 Oligo Designation Nucleotide sequence
(optional) oAA0179
GAATTCACATGGCTAATTTGGCCTCGGTTCCACAACGCACTCAGCATTAAAAA oAA0182
GAGCTCCCCTGCAAACAGGGAAACACTTGTCATCTGATTT oAA0509
CACACAGCTCTTCCATAATGTCGTCTTCTCCATCGT oAA0510
CACACAGCTCTTCCCTCTCTTCTATTCTTAGTACATTCTAACATC oAA0511
CACACAGCTCTTCCATAATGTCGTCTTCTCCATCGT oAA0512
CACACAGCTCTTCCCTCTCTTCTATTCTTAGAACATTCTAACGTC oAA0515
CACACAGCTCTTCCATAatggccacacaagaaatcatcg oAA0516
CACACAGCTCTTCCctctcttctattcttacatcttgacaaagacaccatcg oAA0517
CACACACCCGGGatgactgtacacgatattatcgccac oAA0518
CACACACCCGGGctaatacatctcaatattggcaccg oAA0519
CACACAGCTCTTCCATAatgactgcacaggatattatcgcc oAA0520
CACACAGCTCTTCCctctcttctattcctaatacatctcaatgttggcaccg oAA0521
CACACACCCGGGatgattgaacaactcctagaatattgg oAA0522
CACACACCCGGGctagtcaaacttgacaatagcacc oAA0523
CACACAGCTCTTCCATAatgattgaacaaatcctagaatattgg oAA0524
CACACAGCTCTTCCctctcttctattcctagtcaaacttgacaatagcacc oAA0525
CACACAGCTCTTCCATAatgctcgatcagatcttacattactg oAA0526
CACACAGCTCTTCCctctcttctattcctatgacatcttgacgtgtgcaccg oAA0527
CACACAGCTCTTCCATAatgctcgaccagatcttccattactg oAA0528
CACACAGCTCTTCCctctcttctattcctattgcatcttgacgtatgccccg oAA0529
CACACAGCTCTTCCATAatggctatatctagtttgctatcgtg oAA0530
CACACAGCTCTTCCctctcttctattctcaagttctagttcggatgtacaccc oAA0694
CACACAGCTCTTCCATAATGGCTTTAGACAAGTTAGA oAA0695
CACACAGCTCTTCCctctcttctattcCTACCAAACATCTTCTTG oAA0831
CACACAGCTCTTCCATAatgtcttatgattcattcggtgactacgtc oAA0832
CACACAGCTCTTCCctctcttctattcttagatcttacctttgacatcggtgtttg oAA1023
GATATTATTCCACCTTCCCTTCATT oAA1024 CCGTTAAACAAAAATCAGTCTGTAAA
oAA2053 CACACAGCTCTTCCATAATGGGCGAAATTCAGAAAA oAA2054
CACACAGCTCTTCCCTCTCTTCTATTCCTAGTAGCCCAAGTTTTT oAA2055
TGCCATCCTTGGTAGTCAGTTATT oAA2056
CCGAAACAACCGTAGATACCTTTAATGGCTTGTCCTTGGTGTTGA oAA2057
TCAACACCAAGGACAAGCCATTAAAGGTATCTACGGTTGTTTCGG oAA2060
TGTCGCCATTCAACCAGTAGAT
TABLE-US-00026 TABLE 26 Supplemental List II of Oligonucleotides
used in Examples 1-56 Oligo Designation Nucleotide sequence
(optional) oAA2068 TCCTCGTCCATCTTCAACAAGTCGGTACCGAGCTCTGCGAATT
oAA2069 AATTCGCAGAGCTCGGTACCGACTTGTTGAAGATGGACGAGGA oAA2070
TTGATCCACTGTCTTAAGATTGTCAA oAA2071
CCGAAACAACCGTAGATACCTTTAACCAGAACGAAGTAGCGGAGAAT oAA2072
ATTCTCCGCTACTTCGTTCTGGTTAAAGGTATCTACGGTTGTTTCGG oAA2073
CGACAGACCTCACCGACGTATGGTACCGAGCTCTGCGAATT oAA2074
AATTCGCAGAGCTCGGTACCATACGTCGGTGAGGTCTGTCG oAA2075
AGGATTTTGCTGTTGGTGGC oAA2127 CACACAGCTCTTCCATAATGGTCGCCGATTCTTTAGT
oAA2128 CACACAGCTCTTCCCTCTCTTCTATTCTTAAGTGGCCTTCCACAAGT oAA2173
ACCAAGTTCAACCCAAAGGAGT oAA2174
CCGAAACAACCGTAGATACCTTTAATCTTCGTCAAAAGTGGCGGT oAA2175
ACCGCCACTTTTGACGAAGATTAAAGGTATCTACGGTTGTTTCGG oAA2176
AATGTCGAAACCCTTGTCTTCAGGGTACCGAGCTCTGCGAATT oAA2177
AATTCGCAGAGCTCGGTACCCTGAAGACAAGGGTTTCGACATT oAA2178
CGGACTTTTCACCTCTTTCTCTG oAA2188 CACTGACGAGTTTGTCATCAACAC oAA2189
CCGAAACAACCGTAGATACCTTTAAGGTATCGGTGTCCTTCTTCTTGA oAA2190
TCAAGAAGAAGGACACCGATACCTTAAAGGTATCTACGGTTGTTTCGG oAA2191
ACAAGTAAGCGGCAGCCAAGGGTACCGAGCTCTGCGAATT oAA2192
AATTCGCAGAGCTCGGTACCCTTGGCTGCCGCTTACTTGT oAA2193
ACCAATGTCTCTGGCCAAGC oAA2206 TTCCGCTTAATGGAGTCCAAA oAA2209
TAAACGTTGGGCAACCTTGG oAA2406 CAGACTCAAAGGCAACCACTT oAA2407
tttattggagctccaattgtaatatttcggGATGACATACTTGACGGAGGTG oAA2408
aaacaaccataaagctgcttgacaaAGAACGAAGAAGAAACCAAGGC oAA2409
GCAACAATTCAATACCTTTCAAACC oAA2410
CACCTCCGTCAAGTATGTCATCccgaaatattacaattggagctccaataaa
TABLE-US-00027 TABLE 27 Supplemental List III of Oligonucleotides
used in Examples 1-56 Oligo Designation Nucleotide sequence
(optional) oAA2411 GCCTTGGTTTCTTCTTCGTTCTttgtcaagcagctttatggttgttt
oAA2412 ACAAGAGACAGGGCGGCAAA oAA2413
tttattggagctccaattgtaatatttcggCGGTCAAAGTCTTGACATTGG oAA2414
aaacaaccataaagctgcttgacaaACAAAAGATCTTCTGGGCTGC oAA2415
TTTCAACCAGATTTCACCCTG oA42416
CCAATGTCAAGACTTTGACCGccgaaatattacaattggagctccaataaa oAA2417
GCAGCCCAGAAGATCTTTTGTttgtcaagcagctttatggttgttt oAA2804
gatttacaccgcgggtaccaccggtttgcc oAA2805
GGCAAACCGGTGGTACCCGCGGTGTAAATC oAA2839
CACACAGCTCTTCCATAATGTCAGGATTAGAAATAGCCGCTG oAA2854
CACACAGCTCTTCCATAATGCCCGATATGACAAACGAAT oAA2855
CACACAGCTCTTCCCTCTCTTCTATTCAACACCAGCTTCGAAGTCCTTT oAA2875
CACACAGCTCTTCCCTCTCTTCTATTCCTACAATTTGGCTTTACCGGTACAAA oAA3016
CACACAGCTCTTCCATAATGCATGCATTATTCTCAAAATC oAA3017
CACACAGCTCTTCCCTCTCTTCTATTCTCATTTGGAGGTATCCAAGA oAA3018
CACACAGCTCTTCCATAATGTCAATTCCAACTACTCA oAA3019
CACACAGCTCTTCCCTCTCTTCTATTCTTACTTAGAGTTGTCCAAGA oAA3020
CACACAGCTCTTCCATAATGTCAATTCCAACTACCCA oAA3021
CACACAGCTCTTCCCTCTCTTCTATTCCTACTTGGCAGTGTCAACAA oAA3022
CACACAGCTCTTCCATAATGACTGTTGACGCTTCTTC oAA3023
CACACAGCTCTTCCCTCTCTTCTATTCCTAATTGCCAAAAGCTTTGT oAA3024
CACACAGCTCTTCCATAATGTCACTTGTCCTCAAGCG oAA3025
CACACAGCTCTTCCCTCTCTTCTATTCTTATGGGTGGAAGACAACTC oAA3026
CACACAGCTCTTCCATAATGTCAACTCAATCAGGTTA oAA3027
CACACAGCTCTTCCCTCTCTTCTATTCCTACAACTTACTTGGTCTAA oAA3028
CACACAGCTCTTCCATAATGTCATTATCAGGAAAGAC oAA3029
CACACAGCTCTTCCCTCTCTTCTATTCTTAACGAGCAGTGAAACCAC oAA3030
CACACAGCTCTTCCATAATGAGTAAGTCATACAAGTT oAA3031
CACACAGCTCTTCCCTCTCTTCTATTCCTACAAAGAGGCACCAATAAA oAA3032
CACACAGCTCTTCCATAATGTCCCCACCATCTAAATT oAA3033
CACACAGCTCTTCCCTCTCTTCTATTCTCTATTGCTTATTAGTGATG oAA3035
CACACAGCTCTTCCCTCTCTTCTATTCTCACCACATGTTGACAACAG oAA3036
CACACAGCTCTTCCATAatgtctgaatcaaccgttggaacaccaatcacctgtaaagccg
oAA3054 CACACAGCTCTTCCATAATGTCTGCTAATATCCCAAAAACTCAAAAAG oAA3073
gttaggcttcaacgctattcaaatattgaaaagctacaattgttacattg oAA3074
caatgtaacaattgtagcttttcaatatttgaatagcgttgaagcctaac oAA3120
CACACAGCTCTTCCATAATGTCCGTTCCAACTACTCA oAA3121
CACACAGCTCTTCCCTCTCTTCTATTCCTACTTTGACGTATCAACGA oAA1023
GATATTATTCCACCTTCCCTTCATT oAA1024 CCGTTAAACAAAAATCAGTCTGTAAA oBS1
GGTTTCATAAGCCTTTTCACGGTCTTC oBS2 GAGTTGACAAAGTTCAAGTTTGCTGTC oJRH4
AGTCAGTACTCGAGTTAAACACCAGCTTCGAAGTCC
TABLE-US-00028 TABLE 28 Supplemental list of genes and the names of
the oligonucleotides used to clone or subclone them. Gene Primer 1
Primer 2 ADH1-1-short oAA3054 oAA3017 ADH1-2 oAA3016 oAA3017
ADH1-2-short oAA3054 oAA3017 ADH2A oAA3018 oAA3019 ADH2B oAA3020
oAA3021 ADH7 oAA3022 oAA3023 ADH5 oAA3024 oAA3025 ADH3 oAA3026
oAA3027 ADH4 oAA3028 oAA3029 SFA1 oAA3036 oAA3035 ADH8 oAA3120
oAA3121 ZWF1 oAA831 oAA832 FAT1 oAA2839 oAA2875 FAO1.DELTA.PTS1
oAA3068 oAA3069 PEX11 oAA2127 oAA2128 HFD1 oAA3030 oAA3031 HFD2
oAA3032 oAA3033 CPRB oAA694 oAA695 P450 A12 oAA515 oAA516 P450 A13
oAA517 oAA518 P450 A14 oAA519 oAA520 P450 A15 oAA509 oAA510 P450
A16 oAA511 oAA512 P450 A17 oAA521 oAA522 P450 A18 oAA523 oAA524
P450 A19 oAA525 oAA526 P450 A20 oAA527 oAA528 P450 D2 oAA529 oAA530
IDP2 oAA2053 oAA2054 KIGDP1* oAA2854 oAA2855
Example 55
Replacement of the FAO1 Promoter with a Stronger or Constitutive
Promoter
[0554] The following Promoter Replacement DNA I molecule is
constructed by either overlapping PCR, DNA synthesis or a
combination of both from five different DNA fragments (Pieces A to
E) as illustrated in FIG. 30.
[0555] Piece A (e.g.; SEQ ID NO: 162)about 250 bp piece of the 5'
untranslated region of Candida strain ATCC20336 FAO1 gene (from
about position -500 to about -250).
[0556] Piece B (e.g., SEQ ID NO: 163)URA3 marker.
[0557] Piece C (e.g., SEQ ID NO: 164)about 50 bp of untranslated
region of Candida strain ATCC20336 FAO1 (from position -300 to
-250).
[0558] Piece D (e.g., SEQ ID NOS: 165, 166, 167)500 bp to 1 kb
piece of the promoter of POX4, PEX11 or TEF1 gene, each obtained
from Candida strain ATCC20336.
[0559] Piece E (SEQ ID NO: 168)First 250 bp of the coding sequence
of FAO1 from Candida strain ATCC20336.
[0560] This Promoter Replacement DNA I integrates into at least one
of the chromosomes but it may also integrate in both chromosomes
depending on the nucleotide sequence/divergence between the two
chromosomes. The region of -1500 to +500 of the FAO1 gene is
sequenced for both chromosomes. The -1500 to +500 area is PCR
amplified with primers oBS1 and oBS2 using genomic DNA from
ATCC20336. The PCR fragment is cloned into pCR-Blunt II Topo
Multiple cones are sequenced and the sequence of the second allele
is determined. Pieces A, C and E and are changed to match the
sequence of the second allele. A second promoter replacement
cassette is constructed, sequence verified and named Promoter
Replacement DNA II.
[0561] A Candida strain-such as sAA103 is transformed with Promoter
Replacement DNA cassette I. Transformants are selected by growth in
ScD-ura plates. Colonies are streaked for single isolates. Correct
insertion of the integrated piece is verified by PCR. A correct
strain is grown in YPD overnight and plated in 5-FOA containing
plates to select for the loop-out of the URA 3 marker. Ura-strains
are streaked for single isolates and loop out of URA3 is verified
by PCR. This strain now has one FAO1 allele under the control of
the POX4, PEX11 or TEF1 promoter.
[0562] The ura-strain is then transformed with the Promoter Replace
DNA II molecule. Transformants are selected by growth in ScD-ura
plates. Colonies are streaked for single isolates. Correct
insertion of the integrated piece in the second sister chromosome
is verified by PCR. A correct strain is grown in YPD overnight and
plated in 5-FOA containing plates to select for the loop-out of the
URA3 marker. Ura-strains are streaked for single isolates and the
loop out of URA3 is verified by PCR. This strain now has both
alleles under the control of the POX4, PEX11 or TEF1 promoter.
[0563] This strain is then tested in fermentation for improved
performance as compared with a strain not containing this genetic
modification. DNA sequences for each fragment used in the
constructs are set forth below: [0564] SEQ ID NO: 162--Piece A (5'
untranslated region of FAO1 (from position -500 to -250). [0565]
SEQ ID NO: 163--Piece B--URA3 marker. [0566] SEQ ID NO: 164--Piece
C. [0567] SEQ ID NO : 165--Piece D--Promoter ROX4. [0568] SEQ ID:
NO: 166--Piece D--Promoter PEX11. [0569] SEQ ID NO: 167--Piece
D--Promoter TEF1. [0570] SEQ ID NO: 168--Piece E--First 250 bp of
the coding sequence of FAO1.
Example 56
Increasing NADPH Production by Overexpression of Cytosolic MAE1 and
PYC2
[0571] The open reading frame of ScMAE1 (non-mitochondrial) and
ScPYC2 are mutagenized to replace any CTG codon with other
leucine-encoding codons. Two plasmids are constructed that replaces
the Candida HFD2 of pAA712 open reading frame with either the
ScMAE1 or the ScPYC2 open reading frame. A
3'URA3-P.sub.POX4-ScMAE1*-T.sub.POX4-5'URA3 or
3'URA3-P.sub.POX4-ScPYC2-T.sub.POX4-5'URA3 fragment are amplified
with PCR using primers oAA2206 and oAA2209 and the corresponding
plasmid, as template. The two PCR fragments are gel-purified,
combined, and transformed into sAA103. Transformants are selected
by growth in ScD-ura plates. Colonies are streaked for single
colonies and transformants verified by PCR and copy number
determined by qPCR. A strain is identified with approximately 5-10
copies of P.sub.POX4-ScMAE1*-T.sub.POX4 and 5-10 copies of
P.sub.POX4-ScPYC2-T.sub.POX4 strain.
[0572] A parallel approach is taken by replacing ScMAE1
(non-mitochondrial) with the Candida MAE I (non-mitochondrial). A
strain is identified with approximately 5-10 copies of
P.sub.POX4-MAE1*-T.sub.POX4 and 5-10 copies of
P.sub.POX4-ScPYC2-T.sub.POX.
[0573] The oligonucleotides listed in TABLE 29 below were
selectively used in some of the following Examples.
TABLE-US-00029 TABLE 29 Oligo Designation Nucleotide sequence
oAA0108 CGACGGGTACAACGAGAATT oAA0109 AGACGAAGCCGTTCTTCAAG oAA0634
CACACACTGCAGTTTTCTTTCGTTCTCGTTCCGTCCTTC oAA0635
CACACATCTAGACCCGGGCTCTTCTCCTAGGGGTTATTTTATGT
GATGATTATTATGATATAGTAGTC oAA0722
CCCGAAATATTACAATTGGAGCTCCAATAAATATTGTAATAAA
TAGGTCTATATACATACACTAAGCTTC oAA0723
CAATACAATTCTCGTTGTACCCGTCGAGACGAAGCCGTTCTTC AAGGTG oAA0724
CGACGGGTACAACGAGAATTGTATTG oAA0727
TTGTCAAGCAGCTTTATGGTTGTTTAGACGAAGCCGTTCTTCAA GGTG oAA0730
TTGTCAAGCAGCTTTATGGTTGTTT oAA0731 CCCGAAATATTACAATTGGAGCTCC oAA2107
CACACACTGCAGCTAGCAAAGGCTTGATCAGAGAAAGCAACA oAA2108
CACACATCTAGACCCGGGCTCTTCTGATTGTAGGGCGTTGGTG AGTAAGAACATT oAA2109
GAGCCCGGGTCTAGATGTGTGCTCTTCCTGGAAATCGAACTCG ACGGTCACAA oAA2110
CACACACATATGAATTCGCCAACATGTTGGTTACTCTATGCAT
CGTACTTGGTATATTGATGTTGTTAATAGACTATA oAA2135
GAGCCCGGGTCTAGATGTGTGCTCTTCCGCTCCAGGCTTGTTAT GACTCTAGAGAGAAGTGTG
oAA2136 CACACACATATGAATTCGGTCGGGTTTTGACCTTGGATATGAA
ACTCAAAAATCATCAAATT oAA2138
CACACATCTAGACCCGGGCTCTTCTGGCTGCGTTGTGTATGGG TT oAA2164
CACACACTGCAGGAGGATGAAGAAGACGAAGA oAA2206 TTCCGCTTAATGGAGTCCAAA
oAA2207 ATGATCTGCCATGCCGAACTAGACGAAGCCGTTCTTCAAG oAA2208
CTTGAAGAACGGCTTCGTCTAGTTCGGCATGGCAGATCAT oAA2209
TAAACGTTGGGCAACCTTGG oAA2388 AAGCTTTTAATTAACGTTGGGGTAAAACAACAGAGAG
oAA2389 GGATCCGCATGCGGCCGGCCGTCGTGAAGATTTGAACAATGTT AGTG oAA2390
GGATCCGAGCTCGCGGCCGCGAATAGAAGAGAGTGACTCTTTT GATAAGAGTC oAA2391
GAATTCTTAATTAACATTGTTTGGAGAAAGAAGAAGAAGAAG oAA2392
GGATCCGAGCTCGCGGCCGCCCGAAACAACCATAAAGCTGC oAA2394
aacaGCGGCCGCtaaatattgtaataaataggtctatatacatacactaagcttctag oAA2395
aacaGCGGCCGCagacgaagccgttcttcaaggt oAA2396
aggaatacagatttatacaataaattgccatACaAGTcacgtgagatatctcatccattc
oAA2397
gaatggatgagatatctcacgtgACTtGTatggcaatttattgtataaatctgtattcct
oAA2398 acaGGATCCtagaggttgttctagcaaataaagtgtttca oAA2399
acaGCGGCCGCcgacgacgtgagtcagaacttg oAA2400
acaGGCCGGCCcgacgacgtgagtcagaacttg oAA2401
acaGGATCCcatcaagatcatctatggggataattacg oAA2403
acaGGCCGGCCcgacgacgtgagccagaact oAA2656
AAGCTTTTAATTAAAGATAATCACAGGGGTAGAGACCTTG oAA2657
GGATCCGCATGCGGCCGGCCGATAGCGTGGTATGAATGAATA AGTGTG oAA2658
GGATCCGAGCTCGCGGCCGCGAGCACTAGGTTTTGATAATTTG GTTCTTAC oAA2659
GAATTCTTAATTAACGGCGAAGAACATAGTGTGATG oAA2888
CACACAGCATGCGAGCTCCAATTGTAATATTTCG oAA2888
CACACAGCATGCGAGCTCCAATTGTAATATTTCG oAA2889
CACACACCCGGGGTCGACCTAAATTCGCAACTATCAACTAAGG oAA2889
CACACACCCGGGGTCGACCTAAATTCGCAACTATCAACTAAGG oAA2890
CACACACCCGGGGAGCTCCAATTGTAATATTTCGGG oAA2890
CACACACCCGGGGAGCTCCAATTGTAATATTTCGGG oAA2891
CACACAGCGGCCGCGTCGACCTAAATTCGCAACTATCAAC oAA2891
CACACAGCGGCCGCGTCGACCTAAATTCGCAACTATCAAC oAA2902
GTGGCAGCGTACAACTTACCG oAA2903 CTCCAACGTCAGAATCCCAAG oAA2904
cttgggattctgacgttggagcgacgggtacaacgagaattg oAA2905
CGAAGCCGTTCTTCAAGGTG oAA2906
caccttgaagaacggcttcgccgttatcgataccatctctaccc oAA2907
CGTTAGTGTATCACAAGGTCCTGACC oAA3329 ACAAGTGCACGTACTGTGACAAGGC
oAA3330 ggagctccaattgtaatatttcggcgttacggtcgatagcaaaggggat oAA3331
aaacaaccataaagctgcttgacaaaataccgtctcagccatcatctacatcc oAA3332
TCAACACCGATCTAATTGGCGGCAACTGTGTTCCTG oAA3333
atcccctttgctatcgaccgtaacgccgaaatattacaattggagctcc oAA3334
ggatgtagatgatggctgagacggtattttgtcaagcagctttatggttgttt oAA3335
TGTACCCTCAACCATACCCTGTGTTT oAA3336
ggagctccaattgtaatatttcggtgatttggacggtttgggacatttt oAA3337
aaacaaccataaagctgcttgacaacaaacagggtgtttagccaaccaaa oAA3338
CGGGTGTCGAGTTTGTAGATGTCTG oAA3339
aaaatgtcccaaaccgtccaaatcaccgaaatattacaattggagctcc oAA3340
tttggttggctaaacaccctgtttgttgtcaagcagctttatggttgttt oAA3358
CACACAGCTCTTCAGCCATGCTCGATCAGATCTTACATTACTG GTAC oAA3359
CACACAGCTCTTCGAGCCTATGACATCTTGACGTGTGCACC oAA3378
ATGCCTACCGAACTTCAAAAAGAA OAA3379 AACTCGTCAGTGTCTTCGTCAAAA oAA3380
TTTTGACGAAGACACTGACGAGTTCGACGGGTACAACGAGAA CTG oAA3381
caccttgaagaacggcttcgGACAAGGGTTTCGACATTACCG oAA3382
TATTAACTGGACAAGATTTCAGCAGC oAA3573 GGCCTGACTGGCCTAATCAGGCGGCTCCTTCC
oAA3574 GGCCTGGAAGGCCACGGCGGGTTGTTTGAGTTG oAA3640
GGCCTTCCTGGCCGAGGATGAAGAAGACGAAGACGAATTG oAA3641
GGCCTGAGAGGCCGGTCGGGTTTTGACCTTGG oAA3643
GGCCTCTCTGGCCTGAATTTTCTCAGGGCCGTG oAA3644
GGCCTTGCAGGCCTCTGTCTTGTTTGAGTTGATCGACTC oAA3645
GAGCTCGGCCGOCCATGGCTATGCTCAGTCAACCAAAC oAA3648
AAGCTTGGCCGGCCGCAGATGGTAAGGGTTCTACTTGG oAA3692
GAATTCGGCCGGCCACAAGTGCACGTACTGTGACAAGG oAA3693
GGCCCGAATGCGGATCCCCGGTCTGGCCCGTTACGGTCGATAG CAAAGGG oAA3694
GGATCCGCATTCGGGCCAGCACGGCCGCGGCCGCAATACCGTC TCAGCCATCATCTACATC
oAA3695 TAGCGCATGCGGCCGGCCTCAACACCGATCTAATTGGCG oAA3696
GGCCCGAATGCGGATCCCCGGTCTGGCCCCTCCTTCTTGTTGG ACCAAAAG oAA3697
GGATCCGCATTCGGGCCAGCACGGCCGCGGCCGCCCATTGGTG CTCAAAGAGTCATC oAA3711
Gggccagaccggccggatccgcattc oAA3712 Gaatgcggatccggccggtctggccc
oAA3789 CACACAGCTCTTCCATAATGTATGCGACCAACGAAAAAAAAAT
TGAAATCTCCGACCTA oAA3790
CACACAGCTCTTCCCTCTCTTCTATTCTCAGCTTTTGCCGCCAC TCAAG oAA3804
CACACACCTAGGATGTCAAGCTCAGATGAAGGAGATCACACT CCTGAGTTACAAC oAA3805
CACACATCTAGACTATTGGTTCATCATGTTAAACAAAGAATGA ATGTCCTCGTCC oAA3806
CACACAGCTCTTCAATCATGTCAAGCTCAGATGAAGGAGATCA CACTCCTGAGTTACAAC
oAA3807 CACACAGCTCTTCTCCACTATTGGTTCATCATGTTAAACAAAG
AATGAATGTCCTCGTC oAA3930 TTCCGCTTAATGGAGTCCAAAAAGA oAA3931
TCCCGAAATATTACAATTGGAGCTCTAGACGAAGCCGTTCTTC AAGGTGT oAA3932
ACACCTTGAAGAACGGCTTCGTCTAGAGCTCCAATTGTAATAT TTCGGGA oAA3933
ATTCTCGTTGTACCCGTCGCATATGGTCGACCTAAATTCGCAA CTATCAA oAA3934
TTGATAGTTGCGAATTTAGGTCGACCATATGCGACGGGTACAA CGAGAAT oAA3935
TAAACGTTGGGCAACCTTGGAGGGGTGTGCTGATGCC oAA4083
GGCCTCTCTGGCCggtcgggttttgaccttg oAA4084
GGCCTACGAGGCCGAGGATGAAGAAGACGAAGACGAATTG oAA4085
GGCCTCGTTGGCCCAATTGTAATATTTCGGGAGAAATATCG oAA4086
GGCCTTGCAGGCCGTCGACCTAAATTCGCAACTATCAAC
[0574] TABLE 30 sets forth the genetic modifications of selected
Candida yeast strains described in the following Examples. The
symbol ".DELTA." refers to a deletion of the gene preceding the
symbol; "P450A" refers to CYP450A; "mig1.DELTA./MIG1" refers to
deletion of one allele of the MIG1 gene.
TABLE-US-00030 TABLE 30 Strain Genetic Modifications sAA2014
pox4.DELTA. pox5.DELTA. mig1.DELTA./MIG1 ura3 sAA2047 pox4.DELTA.
pox5.DELTA. mig1.DELTA. sAA2174 pox4.DELTA. pox5.DELTA. CPRB
P450A19 ADH2 ADH8 HFD2 sAA2178 pox4.DELTA. pox5.DELTA. CPRB P450A19
ADH2 ADH8 HFD2 sAA1400 pox4.DELTA. pox5.DELTA. CPRB ura3 sAA1544
pox4.DELTA. pox5.DELTA. CPRB ura3 sAA2115 pox4.DELTA. pox5.DELTA.
CPRB P450A19 ura3 sAA1598 pox4.DELTA. pox5.DELTA. CPRA CPRB ura3
sAA1656 pox4.DELTA. pox5.DELTA. CPRA ura3 sAA2187 pox4.DELTA.
pox5.DELTA. CPRA CPRB P450A19 ura3 sAA2313 pox4.DELTA. pox5.DELTA.
CPRA CPRB P450A19 sAA2314 pox4.DELTA. pox5.DELTA. CPRA CPRB P450A19
sAA2355 pox4.DELTA. pox5.DELTA. CPRA CPRB P450A19 ura3 sAA2390
pox4.DELTA. pox5.DELTA. mig1.DELTA./MIG1 CPRA CPRB P450A19 ADH8
PEX11 ZWF1 sAA2433 pox4.DELTA. pox5.DELTA. mig1.DELTA. CPRA CPRB
P450A19 ADH8 PEX11 ZWF1 sAA2593 pox4.DELTA. pox5.DELTA. mig1.DELTA.
CPRA CPRB P450A19 ADH8 PEX11 ZWF1 sAA2466 pox4.DELTA. pox5.DELTA.
mig1.DELTA. CPRA CPRB P450A19 ADH8 PEX11 ZWF1 ura3 sAA2274
pox4.DELTA. pox5.DELTA. CPRB P450A19 sAA2687 pox4.DELTA.
pox5.DELTA. mig1.DELTA. CPRA CPRB P450A19 ADH8 CcFAO1 PEX11 ZWF1
sAA2693 pox4.DELTA. pox5.DELTA. mig1.DELTA. CPRA CPRB P450A19 ADH8
CCFAO1 PEX11 ZWF1 sAA2356 pox4.DELTA. pox5.DELTA. CPRA CPRB ura3
sAA2552 pox4.DELTA. pox5.DELTA. CPRA CPRB P450A19 UTR1 sAA2671
pox4.DELTA. pox5.DELTA. CPRA CPRB P450A19 ADH8 PEX11 ZWF1 FAT1
sAA2672 pox4.DELTA. pox5.DELTA. CPRA CPRB P450A19 ADH8 PEX11 ZWF1
FAT1 S244A sAA2479 pox4.DELTA. pox5.DELTA. CPRB P450A19 CTF1
sAA1082 pox4.DELTA. pox5.DELTA. CPRB P450A19
Example 57
Construction of Strain sAA2047 with Disrupted MIG1 Alleles
[0575] To create a Candida strain with decreased MIG1 expression,
knockout cassettes for each MIG1 allele were generated. For the
first allele, the 5'-homology region of MIG1 was amplified using
primers oAA3329 and oAA3330 and ATCC20962 genomic DNA as template.
The 3' homology region was amplified using the same genomic DNA and
primers oAA3331 and oAA3332. A cassette containing the URA3 marker
with a terminator repeated at the 5' end was amplified from plasmid
pAA244 (described in Example 58) using oligos oAA3333 and oAA3334;
this plasmid contains overlaps with both MIG1 homology regions.
After PCR amplification, the DNA fragments from each PCR reaction
were gel purified. All resulting DNA fragments were then used in a
subsequent PCR reaction using oligos oAA3329 and oAA3332. The PCR
purified cassette was then transformed into strain sAA103
(described above) and transformants were verified by PCR. One
correctly transformed strain was named sAA1979. This strain was
plated on 5FOA (procedure described above) to cure the URA3 marker
and was verified by PCR. The strain without URA3 was designated as
sAA2014.
[0576] The second MIG1 allele disruption cassette was generated as
follows. A 5' homology region was amplified using primers oAA3335
and oAA3336. A 3' homology region was amplified using primers
oAA3337 and oAA3338 and Candida ATCC20962 genomic DNA as a
template. A cassette containing the URA3 marker with the terminator
repeated was amplified using primers oAA3339 and oAA3340. The three
fragments were then used in a subsequent PCR reaction to generate
the deletion cassette using oligos oAA3335 and oAA3338. The
purified cassette product was then transformed into strain sAA2014,
and transformants were verified by PCR as having the second allele
disrupted. A strain with the correct genotype was named sAA2047.
MIG1 ORF and flanking regions are provided in SEQ ID NO: 198.
Example 58
Construction of Strains sAA2174 and sAA2178
[0577] A 5'POX4-CPRB-T.sub.URA 3-URA3-3'POX4 ("T.sub.URA 3" means
the terminator region of the URA3 gene) cassette was constructed as
follows. The 5'flanking region of POX4 was amplified using primers
oAA2388 and oAA2389 with genomic DNA obtained from ATCC strain
20336 as a template. The resulting fragment was gel purified and
cloned into pCRTOPO Blunt II, sequence verified and the resulting
plasmid was named pAA395. Separately, the 3' flanking region of
POX4 was amplified from genomic DNA of ATCC strain 20336 using the
primers oAA2390 and oAA2391. This amplified DNA fragment was cloned
into pCR Topo-Blunt II and sequence verified and the plasmid was
named pAA396. Separately, the POX4 coding region from pAA395 was
cut with HindIII and BamHI and ligated with the BamHI and EcoRI to
the POX4 coding sequence of pAA396; the ligation product was cloned
into the HindIII and EcoRI sites of pUC 9. The resulting plasmid
was named pAA406.
[0578] A CPRB gene was amplified from genomic DNA of ATCC20336
using primers oAA2398 and oAA2399 or oAA2398 and oAA2400. The two
resulting PCR fragments were separately cloned into pCR Topo Blunt
II and each resulting CPRB insert was sequence verified to create
plasmids pAA398 and pAA399, respectively. The CPRB coding region
from pAA398 was cut with BamHI and NotI and ligated into the BamHI
and NotI sites of pAA406 to form plasmid pAA418. Due to the
orientation of CPRB in pAA399, the CPRB fragment was released by
cutting the plasmid with BamHI. This BamHI piece was ligated into
the BamHI site of pAA418 to form plasmid pAA415.
[0579] Plasmid pAA244 was prepared as follows: The URA3 terminator
was amplified with primers oAA0722 and oAA0723 using pAA061 as a
template. The URA3 gene cassette (promoter, open reading frame and
terminator) was also amplified from pAA061 using primers oAA0724
and oAA0727. Both PCR products were combined and fused by PCR using
primers oAA0730 and oAA0731. The T.sub.uRA3-URA3 PCR fragment was
then cloned into pCR Topo Blunt II and a plasmid with the correct
sequence was named pAA244.
[0580] Plasmid pAA244 was used as a template to PCR amplify a
T.sub.uRA3-URA3 cassette using oligos oAA2394 and oAA2395. The
amplified fragment was cloned into pCR Topo Blunt II, sequence
verified and then designated as plasmid pAA402. Plasmid pAA402
served as a template for site directed mutagenesis with primers
pAA2396 and pAA2397 to remove an internal SpeI site. The resulting
plasmid was named pAA408. This URA3 cassette was cut with NotI and
ligated into the NotI site of pAA415 to create plasmid pAA450.
[0581] Strain sAA103 was transformed with a gel purified PacI
fragment of pAA450 and transformants were selected by growth on
ScD-ura plates. Correct integration of the 5'POX4.sup.-
CPRB.sup.-CPRB T.sub.URA3-URA3-3'POX4 was verified by PCR. A strain
with the correct genotype was named sAA1396. This strain was plated
on 5FOA to cure the URA3 marker and was verified by PCR. The strain
without URA3 was designated as sAA1400.
[0582] Strain sAA1400 was transformed with a PCR fragment
containing URA3 flanked by POX4 flanking regions. The URA3 cassette
was made by amplifying the 5' or 3' flanking region of POX4 from
genomic DNA of ATCC20336 with primers oAA2902 and oAA2903 and
oAA2906 and oAA2907. The URA3 of pAA450 was amplified using primers
oAA2904 and oAA2905. The final cassette fragment was assembled by
PCR using primers oAA2902 and oAA2907. Correct integration of the
URA3 gene was verified by PCR. A strain with the correct genotype
was named sAA1486. This strain was plated on 5FOA to cure the URA3
gene. This results in insertion of the CPRB gene The correct strain
was named sAA1544.
[0583] Separately, a 5'POX502xP450A19-T.sub.URA3-URA3-3'POX5 was
constructed as follows. The 5'flanking region of POX5 was amplified
using primers oAA2656 and oAA2657. The 3' flanking region of POX5
was amplified from genomic DNA of ATCC20336 using primers oAA2658
and oAA2659. Each PCR fragment was separately cloned into pCR Topo
Blunt II and the resulting POX5 insert of plasmid was sequence
verified. The plasmids were named pAA494 and pAA495, respectively.
The POX5 piece from pAA495 was cut with HindIII and BamHI and
ligated with the BamHI and EcoRI POX5 piece of pAA494 and cloned
into the HindIII and EcoRI sites of pUC19. The resulting plasmid
was named pAA620.
[0584] The gene encoding CYP450A19 from Candida strain ATCC 20336
was amplified from plasmid pAA156 using primers oAA2888 and oAA288
or oAA2890 and oAA2891. The PCR fragments were separately cloned
into pCR Topo Blunt II and sequence verified. The P450A19 pieces
were digested with either SphI and XmaI or XmaI and NotI,
respectively, and ligated into the SphI and NotI restriction sites
of pAA620 to form pAA 625. The NotI URA3 piece of pAA448 was
ligated into the NotI restriction site of pAA625 to form
pAA505.
[0585] Strain sAA1544 was transformed with a gel purified PAC1
fragment of pAA505 and transformants were selected by growth on
ScD-ura plates. Correct integration of the
5'POX5-2xP450A19-T.sub.URA3-UKA3-3'POX4 was confirmed. A strain
with the correct genotype was named sAA1816. This strain was plated
on 5FOA to cure the URA3 marker and was verified by PCR. The strain
without URA3 was designated as sAA1957.
[0586] Strain sAA1957 was transformed with a PCR fragment
containing the URA3 gene flanked by POX5 flanking regions. This
URA3 cassette was made by amplifying the 5' or 3' flanking region
of POX5 from genomic DNA of ATCC20336 with primers oAA3378 and
oAA3379 and oAA338T and oAA3382. The URA3 gee from pAA450 was
amplified using primers oA3380 and oAA2905. The complete fragment
was assembled by PCR using primers oAA3378 and oAA3382. Correct
integration was verified. A strain with the correct genotype was
named sAA2042. This strain was plated on 5FOA to cure the URA3
marker. The correct strain was named sAA2115 (TABLE 30).
[0587] To create strains sAA2178 and sAA2174, strain sAA2115 was
transformed with gel purified PCR amplification product for each of
the following ATCC20962 genes: ADH2a, ADH8, HFD2 and CYP450A19
(TABLE 31). The sequence of each gene is set forth in Table 40. Two
strains containing all of these genes were selected and named
sAA2178 and sAA2174 (see TABLE 31).
TABLE-US-00031 TABLE 31 Strain Gene 1 Source Gene 2 Source Gene 3
Source Gene 4 Source sAA2174 ADH2a pAA711 ADH8 pAA- HFD2 pAA- P450-
pAA392 741 712 A19 sAA2178 ADH2a pAA711 ADH8 pAA- HFD2 pAA- P450A
pAA392 741 712 19
Example 59
Construction of Strain sAA2433
[0588] A 5'POX4-CPRA-CPRB-T.sub.URA3-URA3-3'POX 4 cassette was
constructed as follows: A CPRA gene was amplified from genomic DNA
of Candida strain ATCC 20336 using primers oAA2401 and oAA2403. The
resulting PCR fragment was cloned into pCR TOPO Blunt II and
sequence verified and the resulting plasmid was named pAA401. A
CPRB gene fragment was obtained by cutting plasmid p AA398 with
BamHI and Not I; this fragment was ligated to the CPRA fragment
amplified from pAA401 and the ligation product was cloned into the
FscI and NotI piece of pAA406 to create plasmid pAA417. The NotI
URA3 cassette from pAA408 was ligated into the NotI site of paA417
to create plasmid pAA452.
[0589] Strain sAA103 was transformed with a gel purified PacI
fragment of pAA452 and transformants were selected by growth on
ScD-ura plates. Correct integration of the
5'POX4-CPRA-CPRB-T.sub.URA3-URA3-3'POX4 cassette into the first
allele of POX 4 was verified. A strain with the correct genotype
was named sAA1596. This strain was plated on 5FOA to cure the URA3
gene. The strain without URA3 was designated as sAA1598.
[0590] Strain sAA1598 was transformed using standard transformation
procedures with a PCR fragment prepared as follows: A URA3 cassette
was created by amplifying the 5' or 3' flanking region of POX4 from
genomic DNA of ATCC20336 with primers oAA2904 and oAA2905 (5') or
primers oAA2906 and oAA2907 (3'). Separately, a URA3 gene from
plasmid pAA450 was amplified using primers oAA2904 and oAA2905. A
cassette fragment containing all 3 of the fragments described
immediately above was assembled by PCR using primers oAA2902 and
oAA2907. After transformation, the correct integration of the URA3
cassette was verified by PCR. A strain with the correct genotype
was named sAA1653. This strain was plated on 5FOA to cure the URA3
gene, and the final strain (minus URA3) was named sAA1656.
[0591] Next, a cassette was generated that places the gene
CYP450A19 under the control of either the POX4 or PEX11 promoter at
both of the POX5 loci. To accomplish this, a
5'-POX5-2xP450A19-TURA3-URA3-3'-POX5 cassette was constructed as
follows. A fragment containing the POX4 promoter ("PPOX4") and the
CYP450A14 gene was created by amplifying two regions of pAA153
using primers oAA2888 and oAA2889 or oAA2890 and oAA2891. The two
resulting PCR fragments were cloned into separate pCR Topo-Blunt II
plasmids and each was sequence verified. The PPOX4 and CYP450A19
pieces were digested with either SphI and XmaI, or XmaI and NotI
respectively and ligated into the SphI and NotI restriction sites
of pAA620 to form pAA634. The NotI URA3 piece of pAA448 was ligated
into the NotI restriction site of pAA634 to form pAA500.
[0592] The promoter and terminator of PEX11 were PCR amplified from
genomic DNA obtained from Candida strain ATCC20336 using oligos
oAA2164 and oAA2138 or oAA2135 and oAA2136, respectively. The two
pieces were digested with either PstI and SmaI or NdcI and SmaI and
ligated into the PstI and NdcI sites of pAA61 to form pAA335.
[0593] The open reading frame of P450A19 was amplified using oligos
oAA3358 and oAA3359 and the PCR product was cloned as a SapI piece
into the SapI restriction sites of pAA335. The resulting plasmid
was sequenced and named pAA798. The PPEX11-P450A19-TPEX11 was moved
as a XmaI-SphI fragment into the SmaI-SphI1 sites of pAA500. The
resulting plasmid was named pAA800. The XmaI-ClaI restriction
fragment of pAA505 (3137 bp in length) was ligated into the
XmaI-ClaI fragment (6988 bp) of pAA800 to form pAA866.
[0594] Strain sAA1656 was transformed with a gel purified Pad
fragment of pAA866 and transformants selected in ScD-ura plates.
Correct integration of the 5'POX5-2xP450A19-TURA3-URA3-3'POX 5
cassette was verified by PCR. A strain with the correct genotype
was named sAA2123. This strain was plated on 5FOA to cure the URA3
marker and was verified by PCR. The strain without URA3 was
designated as sAA2187. Strain sAA2187 was transformed with a with a
gel purified PacI fragment of pAA866 and transformants selected in
ScD-ura plates. Genotype was verified by PCR and two correct
strains were named sAA2313 and sAA2314. sAA2313 was plated on 5FOA
to cure the URA3 marker and was verified by PCR. The strain without
URA3 was designated as sAA2355.
[0595] A 5'MIG1-ADH8-PEX11-ZWF1-3'MIG1 deletion cassette was
constructed. The 5' flanking region or 3' flanking region of MIG1
was amplified with primers oAA3692 and oAA3693 or oAA3694 and
oAA3695, respectively. The two PCR fragments were combined by PCR
using primers oAA3693 and oAA3695. Each fragment was purified and
cloned separately into pCR Topo Blunt II. The insertion fragment in
each plasmid was sequenced verified and cloned as a EcoRI and SphI
piece into pUC19. This plasmid was then modified by site-directed
mutagenesis to generate a SfaI restriction site using oligos
oAA3711 and oAA3712. The fragment was sequence and a clone having
the sequence set forth in SEQ ID NO: 196 was selected. This plasmid
was named pAA917.
[0596] The ADH8, PEX11, and ZWF1 genes were amplified from strain
ATCC 20336 genomic DNA using primers oAA3573 and oAA3574, oAA3640
and oAAoAA3641, or oAA3643 and oAA3644, respectively. Each PCR
fragment was separately cloned into pCR Topo Blunt II and the
insert of each plasmid was sequenced. Plasmids containing the
correct inserts were named pAA906, pAA881 and pAA914, respectively.
All three gene inserts were cut out of their respective plasmids as
SfiI fragments and ligated into the SfiI sites of pAA917. The
resulting plasmid was named pAA944. The NotI URA3 fragment of
pAA408 was cloned into the NotI site of pAA944 to form pAA946.
[0597] Strain sAA2355 was transformed with a gel purified PacI
fragment of pAA946 and transformants selected in ScD-ura plates.
Correct integration of the 5'MIG1-ADH8-PEX11-ZWF1-3'MIG1 was
verified. A strain with the correct genotype was named sAA2390.
This strain was plated on 5FOA to cure the URA3 marker and was
verified. The strain without URA3 was designated as sAA2415. Strain
sAA2415 was transformed with a gel-purified PacI fragment of pAA946
and transformants were selected by growth on ScD-ura plates. A
strain with the correct genotype was named sAA2433.
Example 60
Construction of Strains 2593 and 2687
[0598] A synthetic gene encoding Candida cloacae FAO1 linked on the
5' end to the ATCC20962 POX4 promoter and on the 3' end to the
ATCC20962 terminator was created by standard DNA synthesis
procedures. This synthetic construct was cloned into pCR Topo Blunt
II. The plasmid was sequenced and named pAA1009. The sequence of
this promoter-FAO1-terminator insert in pAA1009 is set forth as SEQ
ID NO: 197.
[0599] Two URA3 fragments were amplified from pAA0073 using primers
oAA3930 and oAA3931 or oAA3934 and oAA3935. These two pieces were
combined with the PCR product of oAA3932 and oAA3935 from pAA1009.
The piece was fused by PCR using primers oAA3930 and oAA3935. The
PCR product was then cloned into pCR Topo Blunt II, sequence
verified and named pAA1015.
[0600] Separately, a PEX11 expression cassette was prepared as
follows: Two fragments were amplified from pAA336 using primers
oAA2206 and oAA2207 or oAA2208 and oAA2209. These fragments were
fused by PCR using primers oAA2206 and oAA2209 and the PCR product
was cloned in pCR Topo Blunt II and sequence verified. A plasmid
with the correct sequence was named pAA996.
[0601] Strain sAA2433 was plated on 5FOA to cure the URA3 marker.
The genotype was verified and the strain was named sAA2466.
[0602] To create strains sAA2687 and sAA2693, strain sAA2466 was
transformed with amplification cassettes for ATCC20962 ADH8, PEX11,
Candida cloacae FAO1 and ATCC20962 CYP450A19. Each cassette was
prepared using standard PCR techniques together with a template for
each gene; the sequence of each gene used as 4 template is set
forth in Example 75. Two strains containing all of these genes were
named sAA2687 and sAA2693 (TABLE 32).
TABLE-US-00032 TABLE 32 Strain Gene 1 Source Gene 2 Source Gene 3
Source Gene 4 Source sAA2687 ADH8 pAA741 P450A19 AA392 PEX11 pAA996
CcFAO1 pAA1015 sAA2693 ADH8 pAA741 P450A19 AA392 PEX11 pAA996
CcFAO1 pAA1015
Example 61
Production of a CTF+ Strain
[0603] The TEF1 promoter and terminator were PCR amplified from
ATCC strain 20336 genomic DNA using oligos oAA2107 and oAA2108 or
oAA2109 and oAA2110, respectively. The two PCR products were
digested with either Pst1 and SmaI or Pst1 and NdeI and ligated
into the Pst1 and NdeI sites of plasmid pAA61 to form pAA332.
[0604] The promoter of SSA3 was amplified from ATCC strain 20336
genomic DNA using primers oAA0634 and o0AA635. The PCR product was
cloned into pCR Topo BluntII and the sequence was verified. The
promoter piece was moved as a Pst1 and XbaI fragment into plasmid
pAA73 to form plasmid pAA181.
[0605] To place CTF1 under the SSA1 promoter, CTF1 was PCR
amplified from ATCC strain 20962 genomic DNA using primers oAA3804
and oAA3805. The PCR fragment was digested with AvrII and XbaI and
ligated into the AvrII and XbaI sites of pAA181. This plasmid was
verified by sequencing and was named pAA990.
[0606] To place CTF1 under the TEF1 promoter, CTF1 was PCR
amplified as using primers oAA3806 and oAA3807. The PCR fragment
was digested with SapI and the two fragments were ligated into the
SapI sites of plasmid pAA332. Two plasmids were selected and the
insert in each was verified by sequencing. These identical plasmids
were named pAA1017 and pAA1018.
[0607] Plasmids pAA990, pAA1017, and pAA1018 were linearized within
the URA3 gene at the ClaI site by restriction digestion and the
linear plasmid was purified. The linearized products were then
transformed into strains sAA2115 and sAA2356. Resultant
transformants were verified by PCR and the following strains were
created (TABLE 33).
TABLE-US-00033 TABLE 33 Parental Strain Plasmid New Strain sAA2115
pAA990 sAA2479 sAA2356 pAA990 sAA2481 sAA2115 pAA1017 sAA2574
sAA2356 pAA1018 sAA2577
[0608] Strain sAA2115 was transformed with the URA3 PCR fragment
amplified from pAA061 using oligos oAA0108 and oAA0109.
Transformants were selected in ScD-ura. A Ura+ strain was named
sAA2274.
Example 62
Creation of a UTR1 Overexpression Strain
[0609] A gene encoding UTR1 (SEQ ID NO: 200) was amplified from
strain ATCC20336 genomic DNA using oligos oAA3789 and oAA3790. The
PCR fragment was cloned as a SapI fragment. into the SapI sites of
pAA73 to form pAA975. The gene was verified by sequencing. This
plasmid was digested with SpeI and transformed into strain sAA2466.
Transformants were selected in ScD-ura. A Ura+ transformant with
integrated URT1 was named sAA2552.
Example 63
Creation of FAT1 and FAT1 S244A Mutant Overexpression Strains
[0610] Plasmids pAA635 and pAA637 were digested with ClaI and
transformed into sAA2466. Transformants were selected in ScD-ura. A
Ura+ with multiple copies of FAT1 integrated was named-sAA2671 and
a Ura+ strain with multiple copies of FAT1 S244A was named
sAA2672
Example 64
Di-Acid Production by Selected CTF+ and CTF- Strains
[0611] Shake flask fermentations were conducted to determine the
effect of CTF1 on production of di-acids from a methyl laurate
feedstock as follows: Strains sAA2274, sAA2479, sAA2574, sAA2314,
sAA2481 and sAA2577 were inoculated into separate shake flasks
containing 50 mL of SP92 medium containing 50 g/L glucose and grown
overnight. The cultures for each were pelleted by centrifugation
and the cells resuspended in 25 mL of DCA3 medium prepared as
follows: About 6.7 g of yeast nitrogen and 50 g of dextrose were
dissolved in water to a final volume of 500 mL. Separately, a
phosphate solution was prepared by adding 7.7 g of monobasic
potassium phosphate and 42.4 g of dibasic potassium phosphate to
water to a final volume of 500 ml. The solutions were combined and
filter sterilized.
[0612] Bottom baffled flasks were used for this fermentation. To
duplicate flasks for each strain 12.5 mL of culture was added. To
each flask 250 uL of methyl-laurate was added and the flasks
incubated at 30.degree. C. for 40 hrs. with shaking at 300 rpm.
Flasks were sampled by removal of 1 mL at 18 and 40 hrs. for gas
chromatograph analysis. Results (in grams of DDDA per liter of
fermentation broth) are shown in TABLE 34.
TABLE-US-00034 TABLE 34 HDDA (g/L) DDDA (g/L) Strain 18 hr. 40 hr.
18 hr. 40 hr. CTF1 2274-1 0.14 0.41 0.51 0.62 4 .times. CPRB - 4
.times. P450A19 2274-2 0.65 0.97 1.06 1.27 4 .times. CPRB - 4
.times. P450A19 2479-1 0.47 0.64 2.27 2.67 4 .times. CPRB + 4
.times. P450A19 2479-2 0.47 0.67 2.31 2.86 4 .times. CPRB + 4
.times. P450A19 2574-1 0.67 0.79 1.59 1.78 4 .times. CPRB + 4
.times. P450A19 2574-2 0.62 0.8 1.37 1.58 4 .times. CPRB + 4
.times. P450A19 2314-1 0.23 0.38 0.72 0.73 2 .times. CPRA 2 .times.
- CPRB 4 .times. P450A19 2314-2 0.26 0.49 0.92 0.99 2 .times. CPRA
2 .times. - CPRB 4 .times. P450A19 2481-1 0.11 0.31 1.77 2.07 2
.times. CPRA 2 .times. + CPRB 4 .times. P450A19 2481-2 0.08 0 1.47
3.06 2 .times. CPRA 2 .times. + CPRB 4 .times. P450A19 2577-1 0.29
0.41 0.69 0.75 2 .times. CPRA 2 .times. + CPRB 4 .times. P450A19
2577-2 0.25 0.38 0.64 0.69 2 .times. CPRA 2 .times. + CPRB 4
.times. P450A19
[0613] The results indicate that the promoter for SSA3 expressing
CTF1b boosts conversion of methyl-laurate to dodecanedioic acid in
both host backgrounds, with a reduction in omega-hydroxy fatty acid
(HDDA) seen in the sAA2356 host background. The strain containing
the TEF promoter expressing CTF1b also displayed increased
dodecanedioic acid (DDDA) production compared to the control strain
for the sAA2115 background. However, this was not observed for the
sAA2356 background and may be due to lower copy number of the CTF1b
gene.
Example 65
Conversion of Methyl Laurate to DDDA--Comparison of Strain sAA2047
to Strain ATCC 20962
[0614] A pre-culture of 80 mL of SP92 medium (67 g/L yeast nitrogen
base, 3.0 g/L yeast extract, 3.0 g/L (NH.sub.4).sub.2SO.sub.4, 1.0
g/L K.sub.2HPO.sub.4, 1.0 g/L K.sub.2HPO.sub.4, 75 g/L dextrose) in
a 500 mL baffled flask with foam plugs was inoculated with 1.0 mL
from a frozen glycerol stock of strain ATCC 20962 or strain sAA2047
and incubated for 24 h at 30.degree. C. and 250 RPM. Fermentation
medium (MM1), pH 5.8 was filter sterilized and transferred to a
sterile fermentation vessel. Growth was initiated with an inoculum
of pre-culture to an initial OD.sub.600nm=1.0 and growth conditions
of 35.degree. C.; 1000 rpm, 1 vvm, pH 5.8. Growth continued for
approximately 10-12 h at which point the conversion phase was
initiated by a continuous feed of methyl laurate. Feedstock feed
rates varied as follows: methyl laurate (Sigma-Aldrich #W271500);
0.7 g/L-h for the first 24 h; 1.0 g/L-h from 24 h to termination.
In addition, a co-feed of dextrose was fed at a rate of 1.25 g/L-h.
At induction, the temperature was changed to 30.degree. C. and the
pH was maintained at 6.0 by addition of 6N KOH.
[0615] DDDA productivity (average of triplicate fermentations) for
strain sAA2047 was 0.585 g/L-h (grams of DDDA per liter of
fermentation broth per hour) while strain sAA0003 showed a
productivity of 0.543 g/L-h, which is a difference of about 8
percent. Strain sAA2047 also produced lower levels of both lauric
acid and 12-hydroxy lauric acid than strain sAA0003. Lauric acid
levels were reduced almost 60% and 12-hydroxy lauric acid levels
were reduced by 25%.
Example 66
Conversion of Methyl Laurate to DDDA--Comparison of Strains sAA2174
and sAA2178 to sAA1082
[0616] A pre-culture of 80 mL of SP92 medium in a 500 mL baffled
flask with foam plugs was inoculated with 1.0 mL from a frozen
glycerol stock either of strain sAA1082, sAA2074, or sAA2078 and
incubated for 24 h at 30.degree. C. and 250 RPM. Fermentation
medium (MM1) at pH 5.8 was filter sterilized and transferred to a
sterile fermentation vessel. Growth was initiated with an inoculum
of pre-culture to an initial OD.sub.600nm=1.0 and growth conditions
of 35.degree. C., 1000 rpm, 1 vvm, pH 5.8. Growth continued for
approximately 10-12 h at which point the conversion phase was
initiated by a continuous feed of feedstock. Methyl laurate feed
rates were 0.75 g/L-h for the first 24 h and 1.4 g/L-h from 24 h to
termination. In addition, a co-feed of dextrose was fed at a rate
of 1.25 g/L-h. At induction, the temperature was changed to
30.degree. C. and the pH was maintained at 6.0. by addition of 6N
KOH.
[0617] DDDA productivity (average of triplicate fermentations) for
strain sAA2178 was 0.896 g/L-h and strain sAA2174 was 0.879 g/L-h
while sAA1082 showed a productivity of 0.820 g/L-h, an improvement
of about 8% for sAA2178 and 7.2% for sAA2174 over strain sAA1082.
Both sAA2178 and sAA2174 produced lower levels of lauric acid than
strain sAA1082. Lauric acid levels were reduced by 50% for sAA2174
and by 26% for sAA2178 relative to sAA1082. Lowering the amount of
lauric acid improves the overall yield, of DDDA from methyl
laurate.
Example 66.1
Conversion of Ethyl Laurate to DDDA--Comparison of Strain sAA2433
to sAA2671
[0618] A pre-culture of 80 mL SP92 (6.7 g/L yeast nitrogen base,
3.0 g/L yeast extract, 3.0 g/L. (NH4)2S04, 1.0 g/L K2HPO4, 1.0 g/L
KH2PO4, 75 g/L dextrose) in a 500 mL baffled flask with foam plugs
was inoculated with 1.0 mL from a frozen glycerol stock of strain,
sAA2433 or strain sAA2047 and incubated for 24 h at 30.degree. C.
and 250 RPM. Fermentation medium (MM1), pH 5.8 was filter
sterilized and transferred to a sterile fermentation vessel. Growth
was initiated with an inoculum of pre-culture to an initial
OD.sub.600nm=1.0 and growth conditions of 35.degree. C. 1000 rpm, 1
vvm, pH 5.8. Growth continued for approximately 10-12 h at which
point the conversion phase was initiated by a continuous feed of
ethyl laurate, which was fed at a rate of 0.75 g/L-h for the first
24 h; 1.28 g/L-h from 24 h to termination. In addition, a co-feed
of dextrose was fed at a rate of 0.9 g/L-h. At induction, the
temperature was changed to 30.degree. C. and the pH was maintained
at 6.0 by addition of 6N KOH. The main difference between these two
strains is the fact that strain sAA2671 has a gene for fatty acid
transport (FAT1) amplified, which is not the case for sAA2433. DDDA
productivity for strains was similar at about 0.8 g/L-h. However,
strain 2433 produced 4.0 g/L of lauric acid and strain sAA2671
produced only 0.3 g/L. This difference can be attributed to the
fact that, if lauric acid is produced by de-esterification of ethyl
laurate and is subsequently released into the broth, it will be
more readily transported back into the cell for bioconversion to
DDDA in a strain that has been amplified for FAT1 i.e. sAA2671. A
25% reduction in the accumulation of 12-hydroxy lauric acid in the
broth was also observed with strain sAA2671.
Example 66.2
Conversion of Ethyl Laurate to DDDA--Comparison of Strains sAA2593,
sAA2687 and sAA2693 to sAA2178
[0619] A pre-culture of 80 mL SP92 in a 500 mL baffled flask with
foam plugs was inoculated with 1.0 mL from a frozen glycerol stock
either of strain sAA2593 sAA2687 or sAA2693, or sAA2178 and was
incubated for 24 h at 30.degree. C. and 250 RPM. Fermentation
medium (MM1) at pH 5.8 was filter sterilized and transferred to a
sterile fermentation vessel. Growth was initiated with an inoculum
of pre-culture to an initial OD.sub.600nm=1.0 and growth conditions
of 35.degree. C., 1000 rpm, 1 vvm, pH 5.8. Growth continued for
approximately 10-12 h at which point the conversion phase was
initiated by a continuous feed of feedstock. Ethyl laurate feed
rates were 0.75 g/L-h for the first 24 h and 1.49 g/L-h from 24 h
to termination. In addition, a co-feed of dextrose was fed at a
rate of 0.9 g/L-h. At induction, the temperature was changed to
30.degree. C. and the pH was maintained at 6.0 by addition of 6N
KOH. DDDA productivity (average of duplicate fermentations) for all
strains was similar at about 0.83 g/L-h. However, strains sAA2687
and 2693 (both with Candida cloacae FAO1 genes amplified),
accumulated 24% and 47% lower levels of hydroxyl fatty acids,
respectively, than either sAA2178 or sAA2593, neither of which have
an amplified FAO gene. In addition, strain sAA2687 accumulated at
least 55% less lauric acid than any of the other three strains.
Example 67
Production of DDDA from Ethyl Laurate or Methyl Laurate
Feedstock
[0620] Fermentations were carried out with strain sAA1082 grown in
MM1 medium with dextrose as growth substrate but using either
methyl laurate or ethyl laurate (Sigma-Aldrich #W244112) as
feedstock. A pre-culture of 80 mL SP92 in a 500 mL baffled flask
with foam plugs was inoculated with 1.0 mL from a frozen glycerol
stock of strain sAA1082 (beta-oxidation blocked strain plus
amplified CPRB and CYP52A19 and incubated for 24 h at 30.degree. C.
and 250 RPM. Fermentation medium (MM1) at pH 5.8 was filter
sterilized and transferred to a sterile fermentation vessel. Growth
was initiated with an inoculum of pre-culture to an initial
OD.sub.600nm=1.0 and growth conditions of 35.degree. C., 1000 rpm,
1 vvm, pH 5.8. Growth continued for approximately 10-12 h at which
point the conversion phase was initiated by a continuous feed of
feedstock. Methyl laurate feed rates varied as follows: 0.75 g/L-h
for the first 24 h; 1.2 g/L-h from 24 h to termination. Ethyl
laurate was fed at a rate of 0.75 gL-h for the first 24 h; 1.28
g/L-h from 24 h to termination. At induction, the temperature was
changed to 30.degree. C. and the pH was maintained at 6.0 by
addition of 6N KOH. Also at induction a co-feed of dextrose was fed
at a rate of either 1.25 g/L-h or 0.9 g/L-h. In TABLE 35, data from
these two ethyl laurate fermentations were compared to similar data
obtained from methyl laurate fermentations run under the same
conditions.
[0621] The results are the averages of duplicate fermentations.
These data indicate that ethyl laurate substitutes quite well for
methyl laurate. Both ethyl laurate fermentations with dextrose feed
rates of either 1.25 g/L-h or 0.9 g/L-h showed good DDDA
productivity. The higher dextrose feed had an improvement in
productivity of about 6% over the methyl laurate fermentation with
similar co-feed rate. Both ethyl laurate fermentations showed a
significant decrease in the 12-hydroxy lauric acid (HFA)
concentration compared to either of the methyl laurate
fermentations. The ethyl laurate fermentations also had the lowest
lauric acid accumulation, which is an advantage, since lauric acid
can accumulate to toxic levels and eventually inhibit productivity.
An additional benefit for using ethyl laurate is that no
significant quantity of ethanol was observed to accumulate in the
broth. Ethanol in the off-gas was not determined, but it should be
quite low, since the ethanol would be slowly released from ethyl
laurate and would be consumed quickly before it would evaporate to
the off-gas. The final beneficial result was that the dextrose:DDDA
ratio decreased significantly, since ethanol is a very good energy
source and substitutes well for dextrose. When the ethyl laurate
fermentation results are compared to methyl laurate fermentations
having the same co-feed rates (1.25 g/L-h and 0.9 g/L-h) a very
large difference is seen. Decreasing the co-feed to 0.9 g/L-h with
methyl laurate caused a decrease in productivity (relative to the
1.25 g/L-h co-feed rate) and resulted in a significant increase in
lauric acid accumulation and also resulting in a significant
decrease in DDDA purity. In contrast, with the ethyl laurate
fermentations the lower dextrose feed resulted in only a slight
decrease in productivity and both the lauric acid concentration and
the HFA concentration showed a small increase in concentration,
resulting in a small decrease in purity. The lower dextrose:DDDA
ratio could result in a significant decrease in the cost of
producing DDDA from ethyl laurate compared to methyl laurate. In
TABLE 35, "Purity" refers to the amount of DDDA in the fermentation
broth relative to all other chain lengths of fatty acids and
diacids in the broth.
TABLE-US-00035 TABLE 35 Co-Feed Lauric Dextrose: Rate Productivity
HFA Acid DDDA Purity Feed (g/L-h) (g/L-h) (g/L) (g/L) (g/g) (%)
Methyl Laurate 1.25 0.832 3.83 3.88 1.60 84.8 Methyl Laurate 0.9
0.638 3.31 8.36 1.90 73.0 Ethyl Laurate 1.25 0.879 1.64 0.71 1.40
92.5 Ethyl Laurate 0.9 0.832 2.48 2.00 1.07 90.0
Example 68
Production of DDDA Using Sucrose or Dextrose as a Co-Feed
[0622] A pre-culture of 80 mL SP92 in a 500 mL baffled flask with
foam plugs was inoculated with 1.0 mL from a frozen glycerol stock
of strain sAA2178 (beta-oxidation blocked strains plus amplified
CPRB, CYP52A19, ADH2a, ADH8, and HFD2) and incubated for 24 h at
30.degree. C. and 250 RPM. Fermentation medium (MM1) at pH 5.8 was
filter sterilized and transferred to a sterile fermentation vessel.
For the control, dextrose was used for both growth and co-feed. For
the experimental fermentations, sucrose was substituted on a g/g
basis for dextrose in both the growth medium (MM1) and in the
co-feed. Growth was initiated an inoculum of pre-culture to an
initial OD.sub.600nm=1.0 and growth conditions of 35.degree. C.,
1000 rpm, 1 vvm, pH 5.8. Growth continued for approximately 10-12 h
at which point the conversion phase was initiated by a continuous
feed of ethyl laurate. Ethyl laurate was fed at a rate of 0.75
g/L-h for the first 24 h; 1.28 g/L-h from 24 h to termination. In
addition, a co-feed of either dextrose or sucrose was fed at a rate
of 0.9 g/L-h for all fermentations. At induction, the temperature,
was changed to 30.degree. C. and the pH was maintained at 6.0 by
addition of 6N KOH. The data from three fermentations were averaged
for the comparison.
[0623] With dextrose as co-feed, the production of DDDA was 0.872
g/L-h and with sucrose the production of DDDA was only about 3%
less at 0.847 g/L-h. The fermentations with sucrose as co-feed
accumulated a bit more lauric acid (1.56 g/L) and HFA (1.78 g/L)
than the fermentations with dextrose as co-feed (lauric acid=0.52
g/L and HFA=1:44 g/L). Because of this the fermentations with
sucrose as co-feed had slightly lower purity (92.2%) than the
fermentations with glucose as co-feed (94.3%). Overall, sucrose
substituted well for dextrose.
Example 69
Alternative-Co-Feed--Substitution of Glycerol or Xylose for
Dextrose in DDDA Production
[0624] A pre-culture of 80 mL SP92 in a 500 mL baffled flask with
foam plugs was inoculated with 1.0 mL from a frozen glycerol stock
of strain sAA2178 (beta-oxidation blocked strains plus amplified
CPRB, CYP52A19, ADH2a, ADH8, and HFD2) and incubated for 24 h at
30.degree. C. and 250 RPM. Fermentation medium (MM1) at pH 5.8 was
filter sterilized and transferred to a sterile fermentation vessel.
For the control, dextrose was used for both growth and co-feed. For
the experimental fermentations, glycerol or xylose was substituted
on a g/g basis for dextrose in both the growth medium (MM1) and in
the co-feed. Growth was initiated with an inoculum of pre-culture
to an/initial OD.sub.600nm=1.0 and growth conditions of 35.degree.
C., 1000 rpm, 1 vvm; pH 5.8. Growth continued for approximately
10-12 h at which point the conversion phase was initiated by a
continuous feed of ethyl laurate. Ethyl laurate was fed at a rate
of 0.75 g/L-h for the first 24 h; 1.28 g/L-h from 24 h to
termination. In addition, a co-feed of either dextrose, glycerol or
xylose was fed at a rate of 1.25 g/L-h for all fermentations. At
induction, the temperature was changed to 30.degree. C. and the pH
was maintained at 6.0 by addition of 6N KOH.
[0625] With dextrose as co-feed, the production of DDDA was 0.825
g/L-h; with glycerol the production of DDDA was about 60% less at
0.327 g/L-h and with xylose the production of DDDA was about 75%
less at 0.211 g/L-h. The fermentations with glycerol and xylose as
co-feed accumulated a lot more lauric acid (12.28 g/L and 19.04
g/L, respectively) than the fermentation with dextrose as co-feed
(0.37 g/L). HFA levels were very low in the fermentations with
glycerol (0.33 g/L) or xylose (0.20 g/L) relative to dextrose (1.24
g/L) in part because they produced significantly less DDDA. Because
of the large accumulation of lauric acid, the DDDA purity in the
fermentations with glycerol or xylose (39.6% and 28.9% purity,
respectively) as co-feed was significantly worse than the DDDA
purity in the dextrose fermentation (93.4%). On a g/g basis,
substituting either glycerol or xylose for dextrose as co-feed
results in fermentations with lower productivity, greater
accumulation of lauric acid, and lower overall DDDA purity.
Example 70
Production of DDDA: Use of Ethanol or Dextrose as a Co-Feed
[0626] A pre-culture of 80 mL SP92 in a 500 ml baffled flask with
foam plugs was inoculated with 1.0 mL from a frozen glycerol stock
of Strain sAA1082 (beta-oxidation blocked strains plus amplified
CPRB and CYP52A19) and was incubated for 24 h at 30.degree. C. and
250 RPM. Fermentation medium (MM1) at pH 5.8 was filter sterilized
and transferred to a sterile fermentation vessel. For the control,
dextrose was used for both growth and co-feed. For the experimental
fermentations, glycerol or xylose was substituted oh a g/g basis
for dextrose in both the growth medium (MM1) and in the co-feed.
Growth was initiated with an inoculum of pre-culture to an initial
OD.sub.600nm=1.0 and growth conditions of 35.degree. C., 1000 rpm,
1 vvm, pH 5.8. Growth continued for approximately 10-12 h at which
point the conversion phase was initiated by a continuous feed of
methyl laurate. Methyl laurate was fed at a rate of 0.7 g/L-h for
the first 24 h; 1.2 g/L-h from 24 h to termination. In addition, a
co-feed of ethanol was fed at a rate of 0.7 g/L-h. The ethanol
feed-rate was reduced relative to what would normally be used for
glucose (1.2 g/l-h), since ethanol is theoretically a more
energy-rich compound. At induction, the temperature was changed to
30.degree. C. and the pH was maintained at 6.0 by addition of 6N
KOH.
[0627] Substituting ethanol for dextrose as co-feed at the feed
rate selected resulted in a fermentation with lower DDDA
productivity (0.446 gL) than that seen for dextrose, which resulted
in a greater accumulation of lauric acid (8.5 g/L) HFA (5.3 g/L),
and methyl laurate (38 g/L) and a lower overall DDDA purity
(33%).
Example 71
Conversion of Methyl Decanoate to Sebacic Acid (Decanedioic
Acid)--Comparison of Strain sAA1082 to sAA2178
[0628] A pre-culture of 80 mL SP92 in a 500 mL baffled flask with
foam plugs was inoculated with 1.0 mL from a frozen glycerol stock
of strain sAA2178 or strain sAA1082 and incubated for 24 h at
30.degree. C. and 250 RPM. Fermentation medium (MM1) at pH 5.8 was
filter sterilized and transferred to a sterile fermentation vessel.
Growth was initiated with an inoculum of pre-culture to an initial
OD.sub.600nm=1.0 and growth conditions of 35.degree. C., 1000 rpm,
1 vvm, pH 5.8. Growth continued for approximately 10-12 h at which
point the conversion phase was induced by the a bolus of 10 g/L of
decane (Sigma-Aldrich #457116) for 6 h after which a continuous
feed of methyl decanoate (TCI America #D0023) at 0.25 or 0.50 g/L-h
was initiated. Because of the volatility of decane, the aeration
rate was reduced to 0.3 vvm during the 6-h induction phase with
decane as inducer. In addition, a co-feed of dextrose was fed at a
rate of 1.25 g/L-h for all fermentations. At induction, the
temperature was changed to 30.degree. C. and the pH was maintained
at 6.0 by addition of 6N KOH.
[0629] The results in TABLE 36 below show the production of
decanedioic acid (sebacic acid) reported as "Productivity" (grams
of sebacic acid produced per liter of fermentation broth per hour)
and compare the productivity of the two strains under the different
feed rates. "Methyl Decanoate" refers to the amount of methyl
decanoate, the feedstock, present at the end of each fermentation.
Both strains sAA1082 and sAA2178 were able to produce sebacic acid
well when the feed rate was at 0.25 g/L-h, but, when the feed rate
was increased to 0.5 g/L-h, strain sAA 1082 produced significant
amounts of decanoic acid and 10-hydroxy decanoic acid, and
accumulated methyl decanoate; the sebacic acid purity was greatly
reduced as a result. Because of the toxicity of accumulated
decanoic acid, the productivity was significantly reduced. Strain
sAA2178, however, showed a strong increase in productivity when the
feed rate of methyl decanoate was increased to 0.5 g/L-h and had
little to no accumulation of either decanoic acid, 10-hydroxy
decanoic acid or methyl decanoate.
TABLE-US-00036 TABLE 36 Methyl De- 10-OH Methyl Decanoate Pro-
canoic Decanoic De- Feed Rate ductivity Acid Acid canoate Purity
Strain (g/L-h) (g/L-h) (g/L) (g/L) (g/L) (%) sAA1082 0.25 0.143 0
0.03 0.07 92.6 sAA1082 0.5 0.092 8.8 3 10.2 28.2 sAA2178 0.25 0.194
0 0.06 0 93.3 sAA2178 0.5 0.361 0 0.184 0 95.6
Example 72
Diacid Production from Feedstocks of Varying Carbon Chain
Lengths
[0630] The bioconversion tests in this Example were undertaken
following a shake flask protocol. On day 1, 5 ml of YPD was
inoculated with a fresh colony of Candida strain ATTC 20962,
sAA1082, or sAA2178. The YPD contained 10 g/L yeast extract, 20 g/L
peptone, and 20 g/L dextrose. These cultures were then placed in a
30.degree. C. shaking incubator at 250 rpm for 18 to 20 hours.
[0631] After this growth phase, these cultures were used to
inoculate 350 ml of SP92-glycerol media dispensed into 10-250 ml
wide mouth flasks in 35 ml aliquots for each strain. The
SP92-glycerol media contained 6.7 g/L yeast nitrogen base, 3 g/L
yeast extract, 3 g/L ammonium sulfate, 1 g/L potassium phosphate
monobasic; 1 g/L potassium phosphate, dibasic, and 75 g/L glycerol
(Picataggio S., et al., Biotechnology (NY) 1992 August; 10
(8):894-8). The cultures were then placed in a 30.degree. C.
shaking incubator at 300 rpm for 20 to 24 hours.
[0632] The cells were then centrifuged for 5 minutes at 3000 rpm
and the supernatant discarded. The cells were resuspended in DCA3
medium supplemented with 1.0% triton-X100. DCA3 is a 0.3 M
potassium phosphate buffer, pH 7.5 containing 6.7 g/L yeast
nitrogen base and 50 g/L glycerol. Variations of DCA3 media recipes
are known, non-limiting examples of which include the addition of
dextrose and/or glycerol, the like or combinations thereof. DCA3
media, as referred to herein, can include dextrose and/or glycerol.
After re-suspension, the OD600 nm for each culture was adjusted to
a common OD (based on the available OD for the strain with the
least growth) and 10 ml was transferred to pre-weighed 250 ml
bottom baffled shake flasks. The flasks were then reweighed to
determine the starting weight of the culture. The substrate to be
converted was then added at 1% v/v for ethyl esters and 3% v/v for
alkanes. The increased amount of alkanes added was due to their
volatility to ensure that sufficient substrate remained in solution
for bioconversion. The flasks were weighed once more to determine
the exact amount of substrate added to each flask and were then
placed, in a 30.degree. C. shaking incubator at 300 rpm for 4
hours. Bioconversion tests for ethyl-stearate and ethyl palmitate
were performed at 35.degree. C. due to melting point limitations
for these substrates.
[0633] After 48 hours, the flasks were weighed once more to
determine the weight of the final culture. The cultures were mixed
well by swirling and 1 ml was transferred to a pre-weighed 15 ml
falcon tube containing 0.8 ml of 6N HCl. The tube was weighed to
determine the density of the final culture and this number used to
determine the culture volume present at the final time point.
Samples were submitted to analytical for analysis by GC.
TABLE-US-00037 TABLE 37 Feedstock Yield (g diacid/g % improvement
Carbon added feedstock) over ATTC 20962 Chain Temp Starting ATTC-
sAA- sAA- sAA- sAA- Feedstock length (.degree. C.) OD 20962 1082
2178 1082 2178 Ethyl 14 30 125 0.347 0.404 0.444 16.5 28.1
tetradecanoate Ethyl palmitate 16 35 75 0.059 0.084 0.090 41.8 51.2
Ethyl stearate 18 35 75 0.074 0.085 0.143 14.5 92.7 n-Undecane 11
30 125 0.312 0.386 0.403 23.7 29.3 n-Tridecane 13 30 75 0.680 0.941
0.868 38.3 27.7 n-pentadecane 15 30 75 0.505 0.673 0.590 33.1 16.8
n-heptadecane 17 30 75 0.473 0.555 0.514 17.3 8.6
Example 73
Conversion of Ethyl Decanoate to Decanedoic Acid (Sebacic Acid)
[0634] By conversion tests for ethyl decanoate were performed in
0.5 L Infors HT Multifors bioreactors (Infors AG, Switzerland) with
working volumes of 0.4 L each. A pre-culture of 50 mL SP92 (6.7 g/L
yeast nitrogen base, 3.0 g/L yeast extract, 3.0 g/L
(NH.sub.4).sub.2SO.sub.4, 1.0 g/L K.sub.2HPO.sub.4, 1.0 g/L
K.sub.2HPO.sub.4, 75 g/L dextrose) in a 250 mL baffled flask with
foam plugs was inoculated with a single colony from a fresh plate
of Candida strain ATCC 20962, strain sAA1082, or strain sAA2178 and
incubated for 24 h at 30.degree. C. and 250 RPM. Fermentation
medium. (MM1), pH 5.8 was filter sterilized and transferred to a
sterile fermentation vessel. Bioreactors were equipped with two
rushton impellers rotating at 1000 rpm. Bioreactors were inoculated
to an OD of approximately 0.2 and growth continued for
approximately 12-14 hours, at which point the conversion phase was
initiated by evenly space boluses of ethyl decanoate. Culture pH
was kept constant at 5.8 during the growth phase by automatic
addition of 6 N KOH and temperature and airflow were controlled at
30.degree. C. and 1 vvm respectively.
[0635] At induction, the pH was changed to 6.2 and maintained by
addition of 6N KOH. Bioreactors were induced with an initial 4.0 ml
bolus of decane for 6 hours with an airflow rate of 0.25 vvm to
minimize evaporation. Minimal decanedioic acid (sebacic acid) was
observed following this initial induction period, indicating that
the majority of the decane was lost to evaporation.
[0636] Following this initial 6 hour induction, airflow was
increased to 1 vvm and 58 .mu.l of ethyl decanoate was added every
30 minutes for the next 6 hours. Ethyl decanoate boluses were then
increased to 116 .mu.l at a frequency of once per hour for the next
36 hours. In addition, a co-feed of dextrose was fed at a rate of
1.25 g/L-h. Final samples were taken 12 hours after the final ethyl
decanoate bolus. Yield calculations were made for all cultures
taking only added ethyl decanoate into account due to the
volatility of decane and the lack of decanedioic acid formation
following the initial induction period. The results are shown In
TABLE 38 below. As can be seen, strains sAA1082 and sAA2178
produced significantly more sebacic acid than did strain ATCC
20962.
TABLE-US-00038 TABLE 38 Feedstock Yield (g sebacic acid/g
feedstock) % improvement Chain Temp Starting ATCC over ATTC 20962
Feedstock length (.degree. C.) OD 20962 sAA1082 sAA2178 sAA1082
sAA2178 Ethyl 10 30 0.2 0.685 1.088 1.032 58.9 50.7 decanoate
Example 74
Shake Flask Evaluation of a MIG1 Mutant Stain
[0637] Shake flask fermentations were conducted to determine the
effect of deletion of MIG1 on production of di-acids from a methyl
laurate or oleic acid feedstock as follows: Strains ATCC ATCC20962
(sAA003) and sAA2047 were inoculated into separate shake flasks
containing 50 mL of SP92 medium containing 50 g/L glucose and grown
overnight. The cultures for each were pelleted by centrifugation
and the cells resuspended in 25 mL of DCA3 medium prepared as
follows: About 6.7 g of yeast nitrogen and 50 g of dextrose were
dissolved in water to a final volume of 500 mL. Separately
phosphate solution was prepared by adding 7.7 g of monobasic
potassium phosphate and 42.4 g of dibasic potassium phosphate to
water to a final volume of 500 ml. The solutions were combined and
filter sterilized.
[0638] Bottom baffled flasks were used for this fermentation. To
duplicate flasks for each strain 12.5 mL of culture was added. To
each flask 250 uL of methyl-laurate or oleic acid was added and the
flasks incubated at 30.degree. C. for 40 hrs. with shaking at 300
rpm. Flasks were sampled by removal of 1 mL 24 hrs. for gas
chromatograph analysis.
[0639] The results are shown in TABLE 39A & 39B below. In the
tables, "HDDA" refers to hydroxy dodecane acid. As is apparent,
strain sAA2047 containing a MIG1 deletion showed higher production
of diacids from both oleic acid and methyl-laurate fermentation as
compared with strain ATCC 20962. Significantly, there was a
concomitant reduction of hydoxy fatty acids in the fermentation
broth of strain sAA2047 as compared with strain ATCC 20962.
TABLE-US-00039 TABLE 39A Oleic Acid Shake Flask Fermentation Omega
hydroxy-Oleic cis-9 C18 Acid (g/L) diacid (g/L) ATCC20962 0.503
2.933 sAA2047 0.260 3.057
TABLE-US-00040 TABLE 39B Methyl Laurate Shake Flask Fermentation
12-HDDA (g/L) DDDA (g/L) ATCC20962 0.960 1.493 sAA2047 0.300
1.637
Example 75
Certain Nucleotide and Amino Acid Sequences for Genetic
Modification
TABLE-US-00041 [0640] TABLE 40 SEQ ID NO: Description Sequence SEQ
Thioesterase MVAAAATSAFFPVPAPGTSPKPGKSGNWPSSLSPTFKPKSIPN ID
activity AGFQVKANASAHPKANGSAVNLKSGSLNTQEDTSSSPPPRAF NO: Cuphca
LNQLPDWSMLLTAITTVFVAAEKQWTMLDRKSKRPDMLVD 1 lanccolata Amino
SVGLKSIVRDGLVSRQSFLIRSYEIGADRTASIETLMNHLQET acid (A.A. Seq)
SINHCKSLGLLNDGFGRTPGMCKNDLIWVLTKMQIMVNRYP
TWGDTVEINTWFSQSGKIGMASDWLISDCNTGEILIRATSVW
AMMNQKTRRFSRLPYEVRQELTPHFVDSPHVIEDNDQKLHK
FDVKTGDSIRKGLTPRWNDLDVNQHVSNVKYIGWILESMPIE
VLETQELCSLTVEYRRECGMDSVLESVTAVDPSENGGRSQY
KHLLRLEDGTDIVKSRTEWRPKNAGTNGAISTSTAKTSNGNS AS SEQ FAO-13 (fatty
atggctccatttttgcccgaccaggtcgactacaaacacgtcgacacccttatgttattatgtgacg
ID alcohol oxidase
ggatcatccacgaaaccaccgtcgaccaaatcaaagacgttattgctcctgacttccctgctgac
NO: activity)
aagtacgaagagtacgtcaggacattcaccaaaccctccgaaaccccagggttcagggaaacc 2
C. Tropicalis
gtctacaacacagtcaacgcaaacaccacggacgcaatccaccagttcattatcttgaccaatgtt
Nucleotide (Nuc.
ttggcatccagggtcttggctccagctttgaccaactcgttgacgcctatcaaggacatgagcttg
Seq)
gaagaccgtgaaaaattgttggcctcgtggcgcgactccccaatcgctgccaaaaggaagttgtt
caggttggtttctacgcttaccttggtcacgttcacgagattggccaatgagttgcatttgaaagcc
attcattatccaggaagagaagaccgtgaaaaggcttatgaaacccaggagattgacccttttaa
gtaccagtttttggaaaaaccgaagttttacggcgctgagttgtacttgccagatattgatgtgatca
ttattggatctggtgccggtgctggtgttgtggcccacactttggccaacgatggcttcaagagttt
ggttttggaaaagggcaaatactttagcaactccgagttgaactttgatgacaaggacggcgttca
agaattataccaaagtggaggtactttgactacagtcaaccaacagttgtttgttcttgctggttcca
cttttggtggcggtaccactgtcaattggtcagcctgtcttaagacgccattcaaggtgcgtaagg
aatggtatgatgagtttggtgttgactttgctgctgatgaagcatacgataaagcgcaggattatgtt
tggcagcaaatgggagcttctaccgaaggcatcacccactctttggctaacgagattattattgaa
ggtggtaagaaattaggttacaaggccaaggtattagaccaaaacagcggtggtcatcctcagc
acagatgcggtttctgttatttgggctgtaagcacggtatcaagcagggttctgttaataactggttt
agagacgcagctgcccacggttcccagttcatgcaacaggttagagttttgcaaatacttaacaa
gaaggggatcgcttacggtatcttgtgtgaggatgttgtaaccggcgccaagttcaccattactgg
ccccaaaaagtttgttgttgctgccggtgctttgaacactccatctgtgttggtcaactccggcttca
agaacaagaacatcggtaagaacttaactttgcacccagtttctgtcgtgtttggtgattttggcaaa
gacgttcaagcagaccacttccacaactccatcatgactgccctttgttcagaagccgctgattta
gacggcaagggccatggatgcagaattgaaaccatcttgaacgctccattcatccaggcttcatt
cttaccatggagaggtagtaacgaggctagacgagacttgttgcgttacaacaacatggtggcg
atgttgctccttagtcgtgacaccaccagtggttccgtttctgctcatccaaccaaacctgaagcttt
ggttgtcgagtacgacgtgaacaagtttgacagaaactcgatcttgcaggcattgttggtcactgc
tccgacaagccaaaggataagagatcaatcaaggacgaggactatgtcgaatggagagccaa
ggttgccaagattcctttcgacacctacggctcaccttatggttcggcacatcaaatgtcttcttgcc
gtatgtcaggtaagggtcctaaatacggtgctgttgacaccgatggtagattgtttgaatgttcgaa
tgtttatgttgccgatgcaagtcttttgccaactgcaagcggtgccaaccctatggtcaccaccatg
actcttgccagacatgttgcgttaggtttggcagactccttgaagaccaaagccaagttgtag SEQ
FAO-13 (fatty MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDQIKDVIAPDFP ID alcohol
oxidase ADKYEEYVRTFTKPSETPGFRETVYNTVNANTTDAIHQFIKT NO: activity)
NVLASRVLAPALTNSLTPIKDMSLEDREKLLASWRDSPIAAK 3 C. Tropicalis
RKLFRLVSTLTLVTFTRLANELHLKAIHYPGREDREKAYETQ A.A. Seq
EIDPFKYQFLEKPKFYGAELYLPDIDVIIIGSGAGAGVVAHTL
ANDGFKSLVLEKGKYFSNSELNFDDKDGVQELYQSGGTLTT
VNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFG
VDFAADEAYDKAQDYVWQQMGASTEGITHSLANEIIIEGGK
KLGYKAKVLDQNSGGHPQHRCGFCYLGCKHGIKQGSVNNW
FRDAAAHGSQFMQQVRVLQILNKKGIAYGILCEDVVTGAKF
TITGPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSV
VFGDFGKDVQADHFHNSIMTALCSEAADLDGKGHGCRIETIL
NAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSG
SVSAHPTKPEALVVEYDVNKFDRNSILQALLVTADLLYIQGA
KRILSPQAWVPIFESDKPKDKRSIKDEDYVEWRAKVAKIPFD
TYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGRLFECSNVY
VADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKL SEQ FAO-17(fatty
atggctccatttttgcccgaccaggtcgacacaaacacgtcgacacccttatgttattatgtgacg
ID alcohol oxidase
ggatcatccacgaaaccaccgtggacgaaatcaaagacgtcattgcccctgacttccccgccga
NO: activity)
caaatacgaggagtacgtcaggacattcaccaaaccctccgaaaccccagggttcagggaaac 4
C. Tropicalis
cgtctataacaccgtcaacgcaaacaccatggatgcaatccaccagttcattatcttgaccaatgt
Nuc. Seq
tttgggatcaagggtcttggcaccagctttgaccaactcgttgactcctatcaaggacatgagc-
ttg
gaagaccgtgaaaagttgttagcctcgtggcgtgactcccctattgctgctaaaaggaagttgttc
aggttggtttctacgcttaccttggtcacgttcacgagattggccaatgagttgcatttgaaagccat
tcattatccaggaagagaagaccgtgaaaaggcttatgaaacccaggagattgacccttttaagt
accagtttttggaaaaaccgaagttttacggcgctgagttgtacttgccagatattgatgtgatcatt
attggatctggtgccggtgctggtgttgtggcccacactttggccaacgatggcttcaagagtttg
gttttggaaaagggcaaatactttagcaactccgagttgaactttgatgacaaggacggcgttcaa
gaattataccaaagtggaggtactttgactacagtcaaccaacagttgtttgttcttgctggttccac
ttttggtggcggtaccactgtcaattggtcagcctgtcttaagacgccattcaaggtgcgtaagga
atggtatgatgagtttggtgttgactttgctgctgatgaagcatacgataaagcgcaggattatgttt
ggcagcaaatgggagcttctaccgaaggcatcacccactctttggctaacgagattattattgaag
gtggtaagaaattaggttacaaggccaaggtattagaccaaaacagcggtggtcatcctcagca
cagatgcggtttctgttatttgggttgtaagcacggtatcaagcagggctctgttaataactggttta
gagacgcagctgcccacggttctcagttcatgcaacaggttagagttttgcaaatccttaacaaga
agggcatcgcttatggtatcttgtgtgaggatgttgtaaccggtgccaagttcaccattactggccc
caaaaagtttgttgttgccgccggcgccttaaacactccatctgtgttggtcaactccggattcaag
aacaagaacatcggtaagaacttaactttgcatccagtttctgtcgtgtttggtgattttggcaaaga
cgttcaagcagaccacttccacaactccatcatgactgccctttgttcagaagccgctgatttagac
ggcaagggccatggatgcagaattgaaaccatcttgaacgctccattcatccaggcttcattctta
ccatggagaggtagtaacgaggctagacgagacttgttgcgttacaacaacatggtggcgatgtt
gctccttagtcgtgacaccaccagtggttccgtttctgctcatccaaccaaacctgaagctttggtt
gtcgagtacgacgtgaacaagtttgacagaaactcgatcttgcaggcattgttggtcactgctgac
ttgttgtatatccaaggtgccaagagaatccttagtccacaggcatgggtgccaatttttgaatccg
acaagccaaaggataagagatcaatcaaggacgaggactatgtcgaatggagagccaaggttg
ccaagattcctttcgacacctacggctcaccttatggttcggcacatcaaatgtcttcttgccgtatg
tcaggtaagggtcctaaatacggtgctgttgacaccgatggtagattgtttgaatgttcgaatgttta
tgttgccgatgcaagtcttttgccaactgcaagcggtgccaaccctatggtcaccaccatgactctt
gcaagacatgttgcgttaggtttggcagactccttgaagaccaaggccaagttgtag SEQ
FAO-17(fatty MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDEIKDVIAPDFP ID alcohol
oxidase ADKYEEYVRTFTKPSETPGFRETVYNTVNANTMDAIHQFIIL NO: activity)
TNVLGSRVLAPALTNSLTPIKDMSLEDREKLLASWRDSPIAA 5 C. Tropicalis
KRKLFRLVSTLTLVTFTRLANELHLKAIHYPGREDREKAYET A.A. Seq
QEIDPFKYQFLEKPKFYGAELYLPDIDVIIIGSGAGAGVVAHT
LANDGFKSLVLEKGKYFSNSELNFDDKDGQELYQSGGTLT
TVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEF
GVDFAADEAYDKAQDYVWQQMGASTEGITHSLANEIIIEGG
KKLGYKAKVLDQNSGGHPQHRCGFCYLGCKHGIKQGSVNN
WFRDAAAHGSQFMQQVRVLQILNKKGIAYGILCEDVVTGA
KFTITQPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPV
SVVFGDFGKDVQADHFHNSIMTALCSEAADLDGKGHGCRIE
TILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTT
SGSVSAHPTKPEALVVEYDVNKFDRNSILQALLVTADLLYIQ
GAKRILSPQAWVPIFESDKPKDKRSIKDEDYVEWRAKVAKIP
FDTYGSPYGSAHQMSSCRMSCKGPKYGAVDTDGRLFECSN
VYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKA KI SEQ FAO-20(fatty
atggctccatttttgcccgaccaggtcgactacaaacacgtcgacacccttatgttattatgtgacg
ID alcohol oxidase
ggatcatccacgaaaccaccgtcgaccaaatcaaagacgttattgctcctgacttccctgctgac
NO: activity)
aagtacgaagagtacgtcaggacattcaccaaaccctccgaaaccccagggttcagggaaacc 6
C. Tropicalis
gtctacaacacagtcaacgcaaacaccacggacgcaatccaccagttcattatcttgaccaatgtt
Nuc. Seq
ttggcatccagggtcttggctccagctttgaccaactcgttgacgcctatcaaggacatgagct-
tg
gaagaccgtgaaaaattgttggcctcgtggcgcgactccccaatcgctgccaaaaggaaattgtt
caggttggtttccacgcttaacttggttactttcacgagattggccaatgagttgcatttgaaagcca
ttcactatccaggaagagaagaccgtgaaaaggcttatgaaacccaggagattgaccctttcaag
taccagtttatggaaaagccaaagtttgacggcgctgagttgtacttgccagatattgatgttatcat
tattggatctggtgccggtgctggtgttgtggcccacactttggccaacgatggcttcaagagtttg
gttttggaaaagggcaaatactttagcaactccgagttgaactttgatgaaaaggacggcgttcaa
gaattataccaaagtggaggtactttgactacagtcaaccaacagttgtttgttcttgctggttccac
ttttggtggcggtaccactgtcaattggtcagcctgtcttaagacgccattcaaggtgcgtaagga
atggtatgatgagtttggtgttgactttgctgctgatgaagcatacgataaagcgcaggattatgttt
ggcagcaaatgggagcttctaccgaaggcatcacccactctttggctaacgagattattattgaag
gtggtaagaaattaggttacaaggccaaggtattagaccaaaacagcggtggtcatcctcagca
cagatgcggtttctgttatttgggctgtaagcacggtatcaagcagggttctgttaataactggttta
gagacgcagctgcccacggttcccagttcatgcaacaggttagagttttgcaaatacttaacaag
aaggggatcgcttacggtatcttgtgtgaggatgttgtaaccggcgccaagttcaccattactggc
cccaaaaagtttgttgttgctgccggtgctttgaacactccatctgtgttggtcaactccggcttcaa
gaacaagaacatcggtaagaacttaactttgcacccagtttctgtcgtgtttggtgattttggcaaa
gacgttcaagcagaccacttccacaactccatcatgactgccctttgttcagaagccgctgattta
gacggcaagggccatggatgcagaattgaaaccatcttgaacgctccattcatccaggcttcatt
cttaccatggagaggtagtaacgaggctagacgagacttgttgcgttacaacaacatggtggcg
atgttgctccttagtcgtgacaccaccagtggttccgtttctgctcatccaaccaaacctgaagcttt
ggttgtcgagtacgacgtgaacaagtttgacagaaactcgatcttgcaggcattgttggtcactgc
tgacttgttgtatatccaaggtgccaagagaatccttagtccacaggcatgggtgccaatttttgaa
tccgacaagccaaaggataagagatcaatcaaggacgaggactatgtcgaatggagagccaa
ggttgccaagattcctttcgacacctacggctcaccttatggttcggcacatcaaatgtcttcttgcc
gtatgtcaggtaagggtcctaaatacggtgctgttgacaccgatggtagattgtttgaatgttcgaa
tgtttatgttgccgatgcaagtcttttgccaactgcaagcggtgccaaccctatggtcaccaccatg
actcttgccagacatgttgcgttaggtttggcagactccttgaagaccaaagccaagttgtag SEQ
FAO-20(fatty MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDQIKDVIAPDFP ID alcohol
oxidase ADKYEEYVRTFTKPSETPGFRETVYNTVNANTTDAIHQFIILT NO: activity)
NVLASRVLAPALTNSLTPIKDMSLEDREKLLASWRDSPIAAK 7 C. Tropicalis
RKLFRLVSTLTLVTFTRLANELHLKAIHYPGREDKEKAYETQ A.A. Seq
EIDPFKYQFMEKPKFDGAELYLPDIDVIIIGSGAGAGVVAHTL
ANDGFKSLVLEKGKYFSNSELNFDDKDGVQELYQSGGTLTT
VNQQLFVLAGSTEGGGTTVNWSACLKTPFKVRKEWYDEFG
VDFAADEAYDKAQDYVWQQMGASTEGITHSLANEIIIEGGK
KLGYKAKVLDQNSGGHPQHRCGFCYLGCKHGIKQGSVNNW
FRDAAAHGSQFMQQVRVLQILNKKGIAYGILCEDVVTGAKF
TITGPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSV
VFGDFGKDVQADHFHNSIMTALCSEAADLDGKGHGCRIETIL
NAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSG
SVSAHPTKPEALVVEYDVNKFDRNSILQALLVTADLLYIQGA
KRILSPQAWVPIFESDKPKDKRSIKDEDYVEWRAKVAKIPFD
TYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGRLFECSNVY
VADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKL SEQ FAO-2a(fatty
atgaataccttcttgccagacgtgctcgaatacaaacacgtcgacacccttttgttattgtgtgacg
ID alcohol oxidase
ggatcatccacgaaaccacagtcgatcagatcaaggacgccattgctcccgacttccctgagga
NO: activity)
ccagtacgaggagtatctcaagaccttcaccaagccatctgagacccctgggttcagagaagcc 8
C. Tropicalis
gtctacgacacgatcaacgccaccccaaccgatgccgtgcacatgtgtattgtcttgaccaccgc
Nuc: Seq
attggactccagaatcttggcccccacgttgaccaactcgttgacgcctatcaaggatatgacc-
tt
gaaggagcgtgaacaattgttggcctcttggcgtgattccccgattgcggcaaagagaagattgt
tcagattgatttcctcgcttaccttgacgacgtttacgagattggccagcgaattgcacttgaaagc
catccactaccctggcagagacttgcgtgaaaggcgtatgaaacccaggtggttgaccctttca
ggtacctgtttatggagaaaccaaagtttgacggcgccgaattgtacttgccagatatcgacgtca
tcatcattggatcaggcgccggtgctggtgtcatggcccacactctcgccaacgacgggttcaa
gaccttggttttggaaaagggaaagtatttcagcaactccgagttgaactttaatgacgctgatgg
cgtgaaagagttgtaccaaggtaaaggtgctttggccaccaccaatcagcagatgtttattcttgc
cggttccactttgggcggtggtaccactgtcaactggtctgcttgccttaaaacaccatttaaagtg
cgtaaggagtggtacgacgagtttggtcttgaatttgctgccgatgaagcctacgacaaagcgca
ggattatgtttggaaacaaatgggtgcttcaacagatggaataactctatccttggccaacgaagt
tgtggttgaaggaggtaagaagttgggctacaagagcaaggaaattgagcagaacaacggtgg
ccaccctgaccacccatgtggtttctgttacttgggctgtaagtacggtattaaacagggttctgtg
aataactggtttagagacgcagctgcccacgggtccaagttcatgcaacaagtcagagttgtgca
aatcctcaacaagaatggcgtcgcttatggtatcttgtgtgaggatgtcgaaaccggagtcaggtt
cactattagtggccccaaaaagtttgttgtttctgctggttctttgaacacgccaactgtgttgacca
actccggattcaagaacaagcacattggtaagaacttgacgttgcacccagtttccaccgtgtttg
gtgactttggcagagacgtgcaagccgaccatttccacaaatctattatgacttcgctttgttacga
ggttgctgacttggacggcaagggccacggatgcagaatcgaaaccatcttgaacgctccattc
atccaagcttctttgttgccatggagaggaagtgacgaggtcagaagagacttgttgcgttacaac
aacatggtggccatgttgcttatcacgcgtgataccaccagtggttcagcttctgctgacccaaag
aagcccgacgctttgattgtcgactatgagattaacaagtttgacaagaatgccatcttgcaagctt
tcttgatcacttccgacatgttgtacattgaaggtgccaagagaatcctcagtccacagccatggg
tgccaatctttgagtcgaacaagccaaaggagcaaagaacgatcaaggacaaggactatgttga
gtggagagccaaggctgctaagatacctttcgacacctacggttctgcatatgggtccgcacatc
aaatgtccacctgtcgtatgtccggaaagggtcctaaatacggtgctgttgatactgatggtagatt
gtttgaatgttcgaatgtctatgttgctgatgctagtgttttgcctactgccagcggtgccaacccaa
tgatatccaccatgacctttgctagacagattgcgttaggtttggctgactccttgaagaccaaacc
caagttgtag SEQ FAO-2a(fatty
MNTFLPDVLEYKHVDTLLLLCDGIIHETTVDQIKDAIAPDFPE ID alcohol oxidase
DQYEEYLKTFTKPSETPGFREAVYDTINATPTDAVHMCIVLT NO: activity)
TALDSRILAPTLTNSLTPIKDMTLKEREQLLASWRDSPIAAKR 9 C. Tropicalis
RLFRLISSLTLTTFTRLASELHLKAIHYPGRDLREKAYETQVV A.A. Seq.
DPFRYSFMEKPKFDGAELYLPDIDVIIIGSGAGAGVMAHTLA
NDGFKTLVLEKGKYFSNSELNFNDADGVKELYQGKGALATT
NQQMFILAGSTLGGGTTVNWSACLKTPFKVRKEWYDEFGLE
FAADEAYDKAQDYVWKQMGASTDGITHSLANEVVVEGGK
KLGYKSKEIEQNNGGHPDHPCGFCYLGCKYGIKQGSVNNWF
RDAAAHGSKFMQQVRVVQILNKNGVAYGILCEDVETGVRF
TISGPKKFVVSAGSLNTPTVLTNSGFKNKHIGKNLTLHPVSTV
FGDFGRDVQADHFHKSIMTSLCYEVADLDGKGHGCRIETILN
APFIQASLLTWRGSDEVRRDLLRYNNMVAMLLITRDTTSGSV
SADPKKPDALIVDYEINKFDKNAILQAFLITSDMLYIEGAKRI
LSPQPWVPIFESNKPKEQRTIKDKDYVEWRAKAAKIPFDTYG
SAYGSAHQMSTCRMSGKGPKYGAVDTDGRLFECSNVYVAD
ASVLPTASGANPMISTMTFARQIALGLADSLKTKPKL SEQ FAO-2b(fatty
atgaataccttcttgccagacgtgctcgaatacaaacacgtcgatacccttttgttattatgtgacgg
ID alcohol oxidase
gatcatccacgaaaccacagtcgaccagatcagggacgccattgctcccgacttccctgaagac
NO: activity)
cagtacgaggagtatctcaagaccttcaccaagccatctgagacccctgggttcagagaagccg 10
C. Tropicalis
tctacgacacgatcaacagcaccccaaccgaggctgtgcacatgtgtattgtattgaccaccgca
Nuc. Seq
ttggactcgagaatcttggcccccacgttgaccaactcgttgacgcctatcaaggatatgacct-
tg
aaagagcgtgaacaattgttggctgcctggcgtgattccccgatcgcggccaagagaagattgtt
cagattgatttcctcacttaccttgacgacctttaggagattggccagcgacttgcacttgagagcc
atccactaccctggcagagacttgcgtgaaaaggcatatgaaacccaggtggttgaccctttcag
gtacctgtttatggaaaaaccaaagtttgacggcaccgagttgtacttgccagatatcgacgtcat
catcattggatccggtgccggtgctggtgtcatggcccacactttagccaacgacgggtacaag
accttggttttggaaaagggaaagtatttcagcaactccgagttgaactttaatgatgccgatggta
tgaaagagttgtaccaaggtaaatgtgcgttgaccaccacgaaccagcagatgtttattcttgccg
gttccactttgggcggtggtaccactgttaactggtctgcttgtcttaaaacaccatttaaagtgcgt
aaggagtggtacgacgagtttggtcttgaatttgctgccgacgaagcctacgacaaagcacaag
actatgtttggaaacaaatgggcgcttctaccgaaggaatcactcactctttggcgaacgcggttg
tggttgaaggaggtaagaagttgggttacaagagcaaggaaatcgagcagaacaatggtggcc
atcctgaccacccctgtggtttctgttacttgggctgtaagtacggtattaagcagggttctgtgaat
aactggtttagagacgcagctgcccacgggtccaagttcatgcaacaagtcagagttgtgcaaat
cctccacaataaaggcgtcgcttatggcatcttgtgtgaggatgtcgagaccggagtcaaattca
ctatcagtggccccaaaaagtttgttgtttctgcaggttctttgaacacgccaacggtgttgaccaa
ctccggattcaagaacaaacacatcggtaagaacttgacgttgcacccagtttcgaccgtgtttgg
tgactttggcagagacgtgcaagccgaccatttccacaaatctattatgacttcgctctgttacgaa
gtcgctgacttggacggcaagggccacggatgcagaatcgagaccatcttgaacgctccattca
tccaagcttctttgttgccatggagaggaagcgacgaggtcagaagagacttgttgcgttacaac
aacatggtggccatgttgcttatcacccgtgacaccaccagtggttcagtttctgctgacccaaag
aagcccgacgctttgattgtcgactatgacatcaacaagtttgacaagaatgccatcttgcaagctt
tcttgatcacctccgacatgttgtacatcgaaggtgccaagagaatcctcagtccacaggcatgg
gtgccaatctttgagtcgaacaagccaaaggagcaaagaacaatcaaggacaaggactatgtc
gaatggagagccaaggctgccaagatacctttcgacacctacggttctgcctatgggtccgcac
atcaaatgtccacctgtcgtatgtccggaaagggtcctaaatacggcgccgttgataccgatggt
agattgtttgaatgttcgaatgtctatgttgctgatgctagtgttttgcctactgccagcggtgccaac
ccaatgatctcccaccatgacgtttgctagacagattgcgttaggtttggctgactctttgaagacca
aacccaagttgtag SEQ FAO-2b(fatty
MNTFLPDVLEYKHVDTLLLLCDGIIHETTVDQIRDAIAPDFPE ID alcohol oxidase
DQYEEYLKTFTKPSETPGFREAVYDTINSTPTEAVHMCIVLTT NO: activity)
ALDSRILAPTLTNSLTPIKDMTLKEREQLLAAWRDSPIAAKRR 11 C. Tropicalis
LFRLISSLTLTTFTRLASDLHLRAIHYPGRDLREEKAYETQVVD A.A. Seq
PFRYSFMEKPKFDGTELYLPDIDVIIGSGAGAGVMAHTLAN
DGYKTLVLEKGKYFSNSELNFNDADGMKELYQGKCALTTT
NQQMFILAGSTLGGGTTVNWSACLKTPFKVRKEWYDEFGLE
FAADEAYDKAQDYVWKQMGASTEGITHSLANAVVVEGGK
KLGYKSKEIEQNNGGHPDHPCGFCYLGCKYGIKQGSVNNWF
RDAAAHGSKFMQQVRVVQILHNKGVAYGILCEDVETGVKF
TISGPKKFVVSAGSLNTPTVLTNSGFKNKHIGKNLTLHPVSTV
FGDFGRDVQADHFHKSIMTSLCYEVADLDGKGHGCRIETILN
APFIQASLLPWRGSDEVRRDLLRYNNMVAMLLITRDTTSGSV
SADPKKPDALIVDYDINKFDKNAILQAFLITSDMLYIEGAKRI
LSPQAWVPIFESNKPKEQRTIKDKDYVEWRAKAAKIPFDTYG SAYGSAHQMST SEQ
FAO-18(fatty
atggctccctatttttgcccgaccaggtcgactacaaacacgtcgacacccttatgttattatgtgacg
ID alcohol oxidase
ggatcatccacgaaaccaccgtggacgaaatcaaagacgtcattgcccctgacttccccgccga
NO: activity)
caaatacgaggagtacgtcaggacattcaccaaaccctccgaaaccccagggttcagggaaac 12
C. Tropicalis
cgtctacaacaccgtcaacgcaaacaccatggatgcaatccaccagttcattatcttgaccaatgt
Nuc. Seq
tttgggatcaagggtcttggcaccagctttgaccaactcgttgactcctatcaaggacatgagc-
ttg
gaagaccgtgaaaagttgttagcctcgtggcgtgactcccctattgctgctaaaaggaagttgttc
aggttggtttctacgcttaccttggtcacgttcacgagattggccaatgagttgcatttgaaagccat
tcattatccaggaagagaagaccgtgaaaaggcttatgaaacccaggagattgacccttttaagt
accagtttttggaaaaaccgaagttttacggcgctgagttgtacttgccagatattgatgtgatcatt
attggatctggggccggtgctggtgtcgtggcccacactttgaccaacgacggcttcaagagttt
ggttttggaaaagggcagatactttagcaactccgagttgaactttgatgacaaggacggggttc
aagaattataccaaagtggaggtactttgaccaccgtcaaccagcagttgtttgttcttgctggttcc
acttttggtggtggtaccactgtcaattggtcggcctgtcttaaaacgccattcaaggtgcgtaagg
aatggtatgatgagtttggcgttgactttgctgccgatgaagcctacgacaaagcacaggattatg
tttggcagcaaatgggagcttctaccgaaggcatcacccactctttggctaacgagattattattga
aggtggcaagaaattaggttacaaggccaaggtattagaccaaaacagcggtggtcatcctcat
cacagatgcggtttctgttatttgggttgtaagcacggtatcaagcagggctctgttaataactggtt
tagagacgcagctgcccacggttctcagttcatgcaacaggttagagttttgcaaatccttaacaa
gaagggcatcgcttatggtatcttgtgtgaggatgttgtaaccggtgccaagttcaccattactggc
cccaaaaagtttgttgttgccgccggcgccttaaacactccatctgtgttggtcaactccggattca
agaacaagaacatcggtaagaacttaactttgcatccagtttctgtcgtgtttggtgattttggcaaa
gacgttcaagcagatcacttccacaactccatcatgactgctctttgttcagaagccgctgatttag
acggcaagggtcatggatgcagaattgaaaccatcttgaacgctccattcatccaggcttcattctt
accatggagaggtagtaacgaggctagacgagacttgttgcgttacaacaacatggtggccatg
ttacttcttagtcgtgataccaccagtggttccgtttcgtcccatccaactaaacctgaagcattagtt
gtcgagtacgacgtgaacaagtttgacagaaactccatcttgcaggcattgttggtcactgctgac
ttgttgtacattcaaggtgccaagagaatccttagtccccaaccatgggtgccaattttgaatccg
acaagccaaaggataagagatcaatcaaggacgaggactatgtcgaatggagagccaaggttg
ccaagattccttttgacacctacggctcgccttatggttcggcgcatcaaatgtcttcttgtcgtatgt
caggtaagggtcctaaatacggtgctgttgataccgatggtagattgtttgaatgttcgaatgtttat
gttgctgacgctagtcttttgccaactgctagcggtgctaatcctatggtcaccaccatgactcttgc
aagacatgttgcgttaggtttggcagactccttgaagaccaaggccaagttgtag SEQ
FAO-1(fatty MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDEIKDVIAPDFP ID alcohol
oxidase ADKYEEYVRTFTKPSETPGFRETVYNTVNANTMDAIHQFIIL NO: activity)
TNVLGSRVLAPALTNSLTPIKDMSLEDREKLLASWRDSPIAA 13 C. Tropicalis
KRKLFRLVSTLTLVTFTRLANELHLKAIHYPGREDREKAYET A.A. Seq
QEIDPFKYQFLEKPKFYGAELYLPDIDVIIIGSGAGAGVVAHT
LTNDGFKSLVLEKGRYFSNSELNFDDKDGVQELYQSGGTLT
TVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEF
GVDFAADEAYDKAQDYVWQQMGASTEGITHSLANEIIIEGG
KKLGYKAKVLDQNSGGHPHHRCGFCYLGCKHGIKQGSVNN
WFRDAAAHGSQFMQQVRVLQILNKKGIAYGILCEDVVTGA
KFTITGPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPV
SVVFGDFGKDVQADHFHNSIMTALCSEAADLDGKGHGCRIE
TILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTT
SGSVSSHPTKPEALVVEYDVNKFDRNSILQALLVTADLLYIQ
GAKRILSPQPWVPIFESDKPKDKRSIKDEDYVEWRAKVAKIP
FDTYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGRLFECSN
VYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKA KL SEQ cytochrome P450
atggccacacaagaaatcatcgattctgtacttccgtacttgaccaaatggtacactgtgattactg
ID A12 (CYP52A12)
cagcagtattagtcttccttatctccacaaacatcaagaactacgtcaaggcaaagaaattgaaat
NO: Nuc. Seq
gtgtcgatccaccatacttgaaggatgccggtctcactggtattctgtctttgatcgccgccatcaa
14 ggccaagaacgacggtagattggctaactttgccgatgaagttttcgacgagtacccaaaccac
accttctacttgtctgttgccggtgctttgaagattgtcatgactgttgacccagaaaacatcaaggc
tgtcttggccacccaattcactgacttctccttgggtaccagacacgcccactttgctcctttgttgg
gtgacggtatcttcaccttggacggagaaggttggaagcactccagagctatgttgagaccaca
gtttgctagagaccagattggacacgttaaagccttggaaccacacatccaaatcatggctaagc
agatcaagttgaaccagggaaagactttcgatatccaagaattgttctttagatttaccgtcgacac
cgctactgagttcttgtttggtgaatccgttcactccttgtacgatgaaaaattgggcatcccaactc
caaacgaaatcccaggaagagaaaactttgccgctgctttcaacgtttcccaacactacttggcc
accagaagttactcccagactttttactttttgaccaaccctaaggaattcagagactgtaacgcca
aggtccaccacttggccaagtactttgtcaacaaggccttgaactttactcctgaagaactcgaag
agaaatccaagtccggttacgttttcttgtacgaattggttaagcaaaccagagatccaaaggtctt
gcaagatcaattgttgaacattatggttgccggaagagacaccactgccggtttgttgtcctttgctt
tgtttgaattggctagacacccagagatgtggtccaagttgagagaagaaatcgaagttaactttg
gtgttggtgaagactcccgcgttgaagaaattaccttcgaagccttgaagagatgtgaatacttga
aggctatccttaacgaaaccttgcgtatgtacccatctgttcctgtcaactttagaaccgccaccag
agacaccactttgccaagaggtggtggtgctaacggtaccgacccaatctacattcctaaaggct
ccactgttgcttacgttgtctacaagacccaccgtttggaagaatactacggtaaggacgctaacg
acttcagaccagaaagatggtttgaaccatctactaagaagttgggctgggcttatgttccattcaa
cggtggtccaagagtctgcttgggtcaacaattcgccttgactgaagcttcttatgtgatcactaga
ttggcccagatgtttgaaactgtctcatctgatccaggtctcgaataccctccaccaaagtgtattca
cttgaccatgagtcacaacgatggtgtctttgtcaagatgtaa SEQ cytochrome P450
atgactgtacacgatattatcgccacatacttcaccaaatggtacgtgatagtaccactcgctttgat
ID A13 (CYP52A13)
tgcttatagagtcctcgactacttctatggcagatacttgatgtacaagcttggtgctaaaccattttt
NO: Nuc. Seq
ccagaaacagacagacggctgtttcggattcaaagctccgcttgaattgttgaagaagaagagc 15
gacggtaccctcatagacttcacactccagcgtatccacgatctcgatcgtcccgatatcccaact
ttcacattcccggtcttttccatcaaccttgtcaatacccttgagccggagaacatcaaggccatctt
ggccactcagttcaacgatttctccttgggtaccagacactcgcactttgctcctttgttgggtgatg
gtatctttacgttggatggcgccggctggaagcacagcagatctatgttgagaccacagtttgcca
gagaacagatttcccacgtcaagttgttggagccacacgttcaggtgttcttcaaacacgtcagaa
aggcacagggcaagacttttgacatccaggaattgtttttcagattgaccgtcgactccgccaccg
agtttttgtttggtgaatccgttgagtccttgagagatgaatctatcggcatgtccatcaatgcgcttg
actttgacggcaaggctggctttgctgatgcttttaactattcgcagaattatttggcttcgagagcg
gttatgcaacaattgtactgggtgttgaacgggaaaaagtttaaggagtgcaacgctaaagtgca
caagtttgctgactactacgtcaacaaggctttggacttgacgcctgaacaattggaaaagcagg
atggttatgtgtttttgtacgaattggtcaagcaaaccagagacaagcaagtgttgagagaccaatt
gttgaacatcatggttgctggtagagacaccaccgccggtttgttgtcgtttgttttctttgaattggc
cagaaacccagaagttaccaacaagttgagagaagaaattgaggacaagtttggactcggtga
gaatgctagtgttgaagacatttcctttgagtcgttgaagtcctgtgaatacttgaaggctgttctca
acgaaaccttgagattgtacccatccgtgccacagaatttcagagttgccaccaagaacactacc
ctcccaagaggtggtggtaaggacgggttgtctcctgttttggtgagaaagggtcagaccgttatt
tacggtgtctacgcagcccacagaaacccagctgtttacggtaaggacgctcttgagtttagacc
agagagatggtttgagccagagacaaagaagcttggctgggccttcctcccattcaacggtggt
ccaagaatctgtttgggacagcagtttgccttgacagaagcttcgtatgtcactgtcaggttgctcc
aggagtttgcacacttgtctatggacccagacaccgaatatccacctaagaaaatgtcgcatttga
ccatgtcgcttttcgacggtgccaatattgagatgtattag SEQ cytochrome P450
atgactgcacaggatattatcgccacatacatcaccaaatggtacgtgatagtaccactcgctttg
ID A14 (CYP52A14)
attgcttatagggtcctcgactacttttacggcagatacttgatgtacaagcttggtgctaaaccgttt
No: Nuc. Seq
ttccagaaacaaacagacggttatttcggattcaaagctccacttgaattgttaaaaaagaagagt
16
gacggtaccctcatagacttcactctcgagcgtatccaagcgctcaatcgtccagatatcccaact
tttacattcccaatcttttccatcaaccttatcagcacccttgagccggagaacatcaaggctatctt
ggccacccagttcaacgatttctccttgggcaccagacactcgcactttgctcctttgttgggcgat
ggtatctttaccttggacggtgccggctggaagcacagcagatctatgttgagaccacagtttgcc
agagaacagatttcccacgtcaagttgttggagccacacatgcaggtgttcttcaagcacgtcag
aaaggcacagggcaagacttttgacatccaagaattgtttttcagattgaccgtcgactccgccac
tgagtttttgtttggtgaatccgttgagtccttgagagatgaatctattgggatgtccatcaatgcact
tgactttgacggcaaggctggctttgctgatgcttttaactactcgcagaactatttggcttcgagag
cggttatgcaacaattgtactgggtgttgaacgggaaaaagtttaaggagtgcaacgctaaagtg
cacaagtttgctgactattacgtcagcaaggctttggacttgacacctgaacaattggaaaagcag
gatggttatgtgttcttgtacgagttggtcaagcaaaccagagacaggcaagtgttgagagacca
gttgttgaacatcatggttgccggtagagacaccaccgccggtttgttgtcgtttgttttctttgaatt
ggccagaaacccagaggtgaccaacaagttgagagaagaaatcgaggacaagtttggtcttgg
tgagaatgctcgtgttgaagacatttcctttgagtcgttgaagtcatgtgaatacttgaaggctgttct
caacgaaactttgagattgtacccatccgtgccacagaatttcagagttgccaccaaaaacactac
ccttccaaggggaggtggtaaggacgggttatctcctgttttggtcagaaagggtcaaaccgttat
gtacggtgtctacgctgcccacagaaacccagctgtctacggtaaggacgcccttgagtttagac
cagagaggtggtttgagccagagacaaagaagcttggctgggccttccttccattcaacggtggt
ccaagaatttgcttgggacagcagtttgccttgacagaagcttcgtatgtcactgtcagattgctcc
aagagtttggacacttgtctatggaccccaacaccgaatatccacctaggaaaatgtcgcatttga
ccatgtcccttttcgacggtgccaacattgagatgtattag SEQ cytochrome P450
atgtcgtcttctccatcgtttgcccaagaggttctcgctaccactagtccttacatcgagtactttctt
ID A15 (CYP52A15)
gacaactacaccagatggtactacttcatacctttggtgcttctttcgttgaactttataagtttgctcc
NO: Nuc. Seq
acacaaggtacttggaacgcaggttccacgccaagccactcggtaactttgtcagggaccctac 17
gtttggtatcgctactccgttgcttttgatctacttgaagtcgaaaggtacggtcatgaagtttgcttg
gggcctctggaacaacaagtacatcgtcagagacccaaagtacaagacaactgggctcaggat
tgttggcctcccattgattgaaaccatggacccagagaacatcaaggctgttttggctactcagttc
aatgatttctctttgggaaccagacacgatttcttgtactccttgttgggtgacggtattttcaccttgg
acggtgctggctggaaacatagtagaactatgttgagaccacagtttgctagagaacaggtttctc
acgtcaagttgttggagccacacgttcaggtgttcttcaagcacgttagaaagcaccgcggtcaa
acgttcgacatccaagaattgttcttcaggttgaccgtcgactccgccaccgagttcttgtttggtg
agtctgctgaatccttgagggacgaatctattggattgaccccaaccaccaaggatttcgatggca
gaagagatttcgctgacgctttcaactattcgcagacttaccaggcctacagatttttgttgcaaca
aatgtactggatcttgaatggctcggaattcagaaagtcgattgctgtcgtgcacaagtttgctgac
cactatgtgcaaaaggctttggagttgaccgacgatgacttgcagaaacaagacggctatgtgtt
cttgtacgagttggctaagcaaaccagagacccaaaggtcttgagagaccagttattgaacatttt
ggttgccggtagagacacgaccgccggtttgttgtcatttgttttctacgagttgtcaagaaaccct
gaggtgtttgctaagttgagagaggaggtggaaaacagatttggactcggtgaagaagctcgtg
ttgaagagatctcgtttgagtccttgaagtcttgtgagtacttgaaggctgtcatcaatgaaaccttg
agattgtacccatcggttccacacaactttagagttgctaccagaaacactaccctcccaagaggt
ggtggtgaagatggatactcgccaattgtcgtcaagaagggtcaagttgtcatgtacactgttattg
ctacccacagagacccaagtatctacggtgccgacgctgacgtcttcagaccagaaagatggttt
gaaccagaaactagaaagttgggctgggcatacgttccattcaatggtggtccaagaatctgtttg
ggtcaacagtttgccttgaccgaagcttcatacgtcactgtcagattgctccaggagtttgcacact
tgtctatggacccagacaccgaatatccaccaaaattgcagaacaccttgaccttgtcgctctttga
tggtgctgatgttagaatgtactaa SEQ cytochrome P450
atgtcgtcttctccatcgtttgctcaggaggttctcgctaccactagtccttacatcgagtactttcttg
ID A16
acaactacaccagatggtactacttcatccctttggtgcttctttcgttgaacttcatcagcttgct-
cc NO: (CYP52A16)
acacaaagtacttggaacgcaggttccacgccaagccgctcggtaacgtcgtgttggatcctac 18
Nuc. Seq
gtttggtatcgctactccgttgatcttgatctacttaaagtcgaaaggtacagtcatgaagtttgcctg
gagcttctggaacaacaagtacattgtcaaagacccaaagtacaagaccactggccttagaattg
tcggcctcccattgattgaaaccatagacccagagaacatcaaagctgtgttggctactcagttca
acgatttctccttgggaactagacacgatttcttgtactccttgttgggcgatggtatttttaccttgga
cggtgctggctggaaacacagtagaactatgttgagaccacagtttgctagagaacaggtttccc
acgtcaagttgttggaaccacacgttcaggtgttcttcaagcacgttagaaaacaccgcggtcag
acttttgacatccaagaattgttcttcagattgaccgtcgactccgccaccgagttcttgtttggtga
gtctgctgaatccttgagagacgactctgttggtttgaccccaaccaccaaggatttcgaaggca
gaggagatttcgctgacgctttcaactactcgcagacttaccaggcctacagatttttgttgcaaca
aatgtactggattttgaatggcgcggaattcagaaagtcgattgccatcgtgcacaagtttgctga
ccactatgtgcaaaaggctttggagttgaccgacgatgacttgcagaaacaagacggctatgtgt
tcttgtacgagttggctaagcaaactagagacccaaaggtcttgagagaccagttgttgaacatttt
ggttgccggtagagacacgaccgccggtttgttgtcgtttgtgttctacgagttgtcgagaaaccc
tgaagtgtttgccaagttgagagaggaggtggaaaacagatttggactcggcgaagaggctcgt
gttgaagagatctcttttgagtccttgaagtcctgtgagtacttgaaggctgtcatcaatgaagcctt
gagattgtacccatctgttccacacaacttcagagttgccaccagaaacactacccttccaagag
gcggtggtaaagacggatgctcgccaattgttgtcaagaagggtcaagttgtcatgtacactgtc
attggtacccacagagacccaagtatctacggtgccgacgccgacgtcttcagaccagaaagat
ggttcgagccagaaactagaaagttgggctgggcatatgttccattcaatggtggtccaagaatct
gtttgggtcagcagtttgccttgactgaagcttcatacgtcactgtcagattgctccaagagtttgga
aacttgtccctggatccaaacgctgagtacccaccaaaattgcagaacaccttgaccttgtcactc
tttgatggtgctgacgttagaatgttctaa SEQ cytochrome P450
atgattgaacaactcctagaatattggtatgtcgttgtgccagtgttgtacatcatcaaacaactcctt
ID A17 (CYP52A17)
gcatacacaaagactcgcgtcttgatgaaaaagttgggtgctgctccagtcacaaacaagttgta
NO: Nuc. Seq
cgacaacgctttcggtatcgtcaatggatggaaggctctccagttcaagaaagagggcagggct 19
caagagtacaacgattacaagtttgaccactccaagaacccaagcgtgggcacctacgtcagta
ttcttttcggcaccaggatcgtcgtgaccaaagatccagagaatatcaaagctattttggcaaccc
agtttggtgatttttctttgggcaagaggcacactctttttaagcctttgttaggtgatgggatcttcac
attggacggcgaaggctggaagcacagcagagccatgttgagaccacagtttgccagagaac
aagttgctcatgtgacgtcgttggaaccacacttccagttgttgaagaagcatattcttaagcacaa
gggtgaatactttgatatccaggaattgttctttagatttaccgttgattcggccacggagttcttattt
ggtgagtccgtgcactccttaaaggacgaatctattggtatcaaccaagacgatatagattttgctg
gtagaaaggactttgctgagtcgttcaacaaagcccaggaatacttggctattagaaccttggtgc
agacgttctactggttggtcaacaacaaggagtttagagactgtaccaagctggtgcacaagttc
accaactactatgttcagaaagctttggatgctagcccagaagagcttgaaaagcaaagtgggta
tgtgttcttgtacgagcttgtcaagcagacaagagaccccaatgtgttgcgtgaccagtctttgaac
atcttgttggccggaagagacaccactgctgggttgttgtcgtttgctgtctttgagttggccagac
acccagagatctgggccaagttgagagaggaaattgaacaacagtttggtcttggagaagactc
tcgtgttgaagagattacctttgagagcttgaagagatgtgagtacttgaaagcgttccttaatgaa
accttgcgtatttacccaagtgtcccaagaaacttcagaatcgccaccaagaacacgacattgcc
aaggggcggtggttcagacggtacctcgccaatcttgatccaaaagggagaagctgtgtcgtat
ggtatcaactctactcatttggaccctgtctattacggccctgatgctgctgagttcagaccagaga
gatggtttgagccatcaaccaaaaagctcggctgggcttacttgccattcaacgtggtccaaga
atctgtttgggtcagcagtttgccttgacggaagctggctatgtgttggttagattggtgcaagagtt
ctcccacgttaggctggacccagacgaggtgtacccgccaaagaggttgaccaacttgaccatg
tgtttgcaggatggtgctattgtcaagtttgactag SEQ cytochrome P450
atgattgaacaaatcctagaatattggtatattgttgtgcctgtgttgtacatcatcaaacaactcatt
ID A18 (CYP52A18)
gcctacagcaagactcgcgtcttgatgaaacagttgggtgctgctccaatcacaaaccagttgta
NO: Nuc. Seq
cgacaacgttttcggtatcgtcaacggatggaaggctctccagttcaagaaagagggcagagct 20
caagagtacaacgatcacaagtttgacagctccaagaacccaagcgtcggcacctatgtcagta
ttctttttggcaccaagattgtcgtgaccaaggatccagagaatatcaaagctattttggcaaccca
gtttggcgatttttctttgggcaagagacacgctctttttaaacctttgttaggtgatgggatcttcacc
ttggacggcgaaggctggaagcatagcagatccatgttaagaccacagtttgccagagaacaa
gttgctcatgtgacgtcgttggaaccacacttccagttgttgaagaagcatatccttaaacacaag
ggtgagtactttgatatccaggaattgttctttagatttactgtcgactcggccacggagttcttatttg
gtgagtccgtgcactccttaaaggacgaaactatcggtatcaaccaagacgatatagattttgctg
gtagaaaggactttgctgagtcgttcaacaaagcccaggagtatttgtctattagaattttggtgca
gaccttctactggttgatcaacaacaaggagtttagagactgtaccaagctggtgcacaagtttac
caactactatgttcagaaagctttggatgctaccccagaggaacttgaaaagcaaggcgggtatg
tgttcttgtatgagcttgtcaagcagacgagagaccccaaggtgttgcgtgaccagtctttgaaca
tcttgttggcaggaagagacaccactgctgggttgttgtcctttgctgtgtttgagttggccagaaa
cccacacatctgggccaagttgagagaggaaattgaacagcagtttggtcttggagaagactctc
gtgttgaagagattacctttgagagcttgaagagatgtgagtacttgaaagcgttccttaacgaaa
ccttgcgtgtttacccaagtgtcccaagaaacttcagaatcgccaccaagaatacaacattgccaa
ggggtggtggtccagacggtacccagccaatcttgatccaaaagggagaaggtgtgtcgtatg
gtatcaactctacccacttagatcctgtctattatggccctgatgctgctgagttcagaccagagag
atggtttgagccatcaaccagaaagctcggctgggcttacttgccattcaacggtgggccacgaa
tctgtttgggtcagcagtttgccttgaccgaagctggttacgttttggtcagattggtgcaagagttc
tcccacattaggctggacccagatgaagtgtatccaccaaagaggttgaccaacttgaccatgtg
tttgcaggatggtgctattgtcaagtttgactag SEQ cytochrome P450
atgctcgatcagatcttacattactggtacattgtcttgccattgttggccattatcaaccagatcgtg
ID A19 (CYP52A19)
gctcatgtcaggaccaattatttgatgaagaaattgggtgctaagccattcacacacgtccaacgt
NO: Nuc. Seq
gacgggtggttgggcttcaaattcggccgtgaattcctcaaagcaaaaagtgctgggagactgg 21
ttgatttaatcatctcccgtttccacgataatgaggacactttctccagctatgcttttggcaaccatgt
ggtgttcaccagggaccccgagaatatcaaggcgcttttggcaacccagtttggtgatttttcattg
ggcagcagggtcaagttcttcaaaccattattggggtacggtatcttcacattggacgccgaagg
ctggaagcacagcagagccatgttgagaccacagtttgccagagaacaagttgctcatgtgacg
tcgttggaaccacacttccagttgttgaagaagcatatccttaaacacaagggtgagtactttgata
tccaggaattgttctttagatttactgtcgactcggccacggagttcttatttggtgagtccgtgcact
ccttaaaggacgaggaaattggctacgacacgaaagacatgtctgaagaaagacgcagatttgc
cgacgcgttcaacaagtcgcaagtctacgtggccaccagagttgctttacagaacttgtactggtt
ggtcaacaacaaagagttcaaggagtgcaatgacattgtccacaagtttaccaactactatgttca
gaaagccttggatgctaccccagaggaacttgaaaagcaaggcgggtatgtgttcttgtatgagc
ttgtcaagcagacgagagaccccaaggtgttgcgtgaccagtctttgaacatcttgttggcagga
agagacaccactgctgggttgttgtcctttgctgtgtttgagttggccagaaacccacacatctgg
gccaagttgagagaggaaattgaacagcagtttggtcttggagaagactctcgtgttgaagagat
tacctttgagagcttgaagagatgtgagtacttgaaggccgtgttgaacgaaactttgagattaca
cccaagtgtcccaagaaacgcaagatttgcgattaaagacacgactttaccaagaggcggtggc
cccaacggcaaggatcctatcttgatcaggaaggatgaggtggtgcagtactccatctcggcaa
ctcagacaaatcctgcttattatggcgccgatgctgctgattttagaccggaaagatggtttgaacc
atcaactagaaacttgggatgggctttcttgccattcaacggtggtccaagaatctgtttgggacaa
cagtttgctttgactgaagccggttacgttttggttagacttgttcaggagtttccaaacttgtcacaa
gaccccgaaaccaagtacccaccacctagattggcacacttgacgatgtgcttgtttgacggtgc
acacgtcaagatgtcatag SEQ cytochrome P450
atgctcgaccagatcttccattactggtacattgtcttgccattgttggtcattatcaagcagatcgtg
ID A20 (CYP52A20)
gctcatgccaggaccaattatttgatgaagaagttgggcgctaagccattcacatgtccaacta
NO: Nuc. Seq
gacgggtggtttggcttcaaatttggccgtgaattcctcaaagctaaaagtgctgggaggcaggtt
22
gatttaatcatctcccgtttccacgataatgaggacactttctccagctatgcttttggcaaccatgtg
gtgttcaccagggaccccgagaatatcaaggcgcttttggcaacccagtttggtgatttttcattgg
gaagcagggtcaaattcttcaaaccattgttggggtacggtatcttcaccttggacggcgaaggct
ggaagcacagcagagccatgttgagaccacagtttgccagagagcaagttgctcatgtgacgtc
gttggaaccacatttccagttgttgaagaagcatattcttaagcacaagggtgaatactttgatatcc
aggaattgttctttagatttaccgttgattcagcgacggagttcttatttggtgagtccgtgcactcctt
aagggacgaggaaattggctacgatacgaaggacatggctgaagaaagacgcaaatttgccg
acgcgttcaacaagtcgcaagtctatttgtccaccagagttgctttacagacattgtactggttggtc
aacaacaaagagttcaaggagtgcaacgacattgtccacaagttcaccaactactatgttcagaa
agccttggatgctaccccagaggaacttgaaaaacaaggcgggtatgtgttcttgtacgagcttg
ccaagcagacgaaagaccccaatgtgttgcgtgaccagtctttgaacatcttgttggctggaagg
gacaccactgctgggttgttgtcctttgctgtgtttgagttggccaggaacccacacatctgggcc
aagttgagagaggaaattgaatcacactttgggctgggtgaggactctcgtgttgaagagattac
ctttgagagcttgaagagatgtgagtacttgaaagccgtgttgaacgaaacgttgagattacaccc
aagtgtcccaagaaacgcaagatttgcgattaaagacacgactttaccaagaggcggtggcccc
aacggcaaggatcctatcttgatcagaaagaatgaggtggtgcaatactccatctcggcaactca
gacaaatcctgcttattatggcgccgatgctgctgattttagaccggaaagatggtttgagccatca
actagaaacttgggatgggcttacttgccattcaacggtggtccaagaatctgcttgggacaaca
gtttgctttgaccgaagccggttacgttttggttagacttgttcaggaattccctagcttgtcacagg
accccgaaactgagtacccaccacctagattggcacacttgacgatgtgcttgtttgacggggca
tacgtcaagatgcaatag SEQ cytochrome P450
atggctatatctagtttgctatcgtgggatgtgatctgtgtcgtcttcatttgcgtttgtgtttatttcgg
ID D2 (CYP52D2)
gtatgaatattgttatactaaatacttgatgcacaaacatggcgctcgagaaatcgagaatgtgatc
NO: Nuc. Seq.
aacgatgggttctttgggttccgcttacctttgctactcatgcgagccagcaatgagggccgactt
23 atcgagttcagtgtcaagagattcgagtcggcgccacatccacagaacaagacattggtcaacc
gggcattgagcgttcctgtgatactcaccaaggacccagtgaatatcaaagcgatgctatcgacc
cagtttgatgacttttcccttgggttgagactacaccagtttgcgccgttgttggggaaaggcatctt
tactttggacggcccagagtggaagcagagccgatctatgttgcgtccgcaatttgccaaagatc
gggtttctcatatcctggatctagaaccgcattttgtgttgcttcggaagcacattgatggccacaat
ggagactacttcgacatccaggagctctacttccggttctcgatggatgtggcgacggggtttttg
tttggcgagtctgtggggtcgttgaaagacgaagatgcgaggttcctggaagcattcaatgagtc
gcagaagtatttggcaactagggcaacgttgcacgagttgtactttctttgtgacgggtttaggtttc
gccagtacaacaaggttgtgcgaaagttctgcagccagtgtgtccacaaggcgttagatgttgca
ccggaagacaccagcgagtacgtgtttctccgcgagttggtcaaacacactcgagatcccgttgt
tttacaagaccaagcgttgaacgtcttgcttgctggacgcgacaccaccgcgtcgttattatcgttt
gcaacatttgagctagcccggaatgaccacatgtggaggaagctacgagaggaggttatcctga
cgatgggaccgtccagtgatgaaataaccgtggccgggttgaagagttgccgttacctcaaagc
aatcctaaacgaaactcttcgactatacccaagtgtgcctaggaacgcgagatttgctacgagga
atacgacgcttcctcgtggcggaggtccagatggatcgtttccgattttgataagaaagggccag
ccagtggggtatttcatttgtgctacacacttgaatgagaaggtatatgggaatgatagccatgtgt
ttcgaccggagagatgggctgcgttagagggcaagagtttgggctggtcgtatcttccattcaac
ggcggcccgagaagctgccttggtcagcagtttgcaatccttgaagcttcgtatgttttggctcgat
tgacacagtgctacacgacgatacagcttagaactaccgagtacccaccaaagaaactcgttcat
ctcacgatgagtcttctcaacggggtgtacatccgaactagaacttga SEQ cytochrome
P450:
atgacaattaaagaaatgcctcagccaaaaacgtttggagagcttaaaaatttaccgttattaaaca
ID NADPH P450
cagataaaccggttcaagctttgatgaaaattgcggatgaattaggagaaatctttaaattcgagg
NO: reductase
cgcctggtcgtgtaacgcgctacttatcaagtcagcgtctaattaaagaagcatgcgatgaatcac
24 (Bacillus
gctttgataaaaacttaagtcaagcgcttaaatttgtacgtgattttgcaggagacgggttatttaca
megaterium)
agctggacgcatgaaaaaaattggaaaaaagcgcataatatcttacttccaagcttcagtcagca
nucleotide
ggcaatgaaaggctatcatgcgatgatggtcgatatcgccgtgcagcttgttcaaaagtgggagc
Nuc. Seq
gtctaaatgcagatgagcatattgaagtaccggaagacatgacacgtttaacgcttgatacaat-
tg
gtctttgcggctttaactatcgctttaacagcttttaccgagatcagcctcatccatttattacaagtat
ggtccgtgcactggatgaagcaatgaacaagctgcagcgagcaaatccagacgacccagctta
tgatgaaaacaagcgccagtttcaagaagatatcaaggtgatgaacgacctagtagataaaatta
ttgcagatcgcaaagcaagcggtgaacaaagcgatgatttattaacgcatatgctaaacggaaaa
gatccagaaacgggtgagccgcttgatgacgagaacattcgctatcaaattattacattcttaattg
cgggacacgaaacaacaagtggtcttttatcatttgcgctgtatttcttagtgaaaaatccacatgta
ttacaaaaagcagcagaagaagcagcacgagttctagtagatcctgttccaagctacaaacaag
tcaaacagcttaaatatgtcggcatggtcttaaacgaagcgctgcgcttatggccaactgctcctg
cgttttccctatatgcaaaagaagatacggtgcttggaggagaatatcctttagaaaaaggcgac
gaactaatggttctgattcctcagcttcaccgtgataaaacaatttggggagacgatgtggaagag
ttccgtccagagcgttttgaaaatccaagtgcgattccgcagcatgcgtttaaaccgtttggaaac
ggtcagcgtgcgtgtatcggtcagcagttcgctcttcatgaagcaacgctggtacttggtatgatg
ctaaaacactttgactttgaagatcatacaaactacgagctggatattaaagaaactttaacgttaaa
acctgaaggctttgtggtaaaagcaaaatcgaaaaaaattccgcttggcggtattccttcacctag
cactgaacagtctgctaaaaaagtacgcaaaaaggcagaaaacgctcataatacgccgctgctt
gtgctatacggttcaaatatgggaacagctgaaggaacggcgcgtgatttagcagatattgcaat
gagcaaaggatttgcaccgcaggtcgcaacgcttgattcacacgccggaaatcttccgcgcgaa
ggagctgtattaattgtaacggcgtcttataacggtcatccgcctgataacgcaaagcaatttgtcg
actggttagaccaagcgtctgctgatgaagtaaaaggcgttcgctactccgtatttggatgcggc
gataaaaactgggctactacgtatcaaaaagtgcctgcttttatcgatgaaacgcttgccgctaaa
ggggcagaaaacatcgctgaccgcggtgaagcagatgcaagcgacgactttgaaggcacata
tgaagaatggcgtgaacatatgtggagtgacgtagcagcctactttaacctcgacattgaaaaca
gtgaagataataaatctactctttcacttcaatttgtcgacagcgccgcggatatgccgcttgcgaa
aatgcacggtgcgttttcaacgaacgtcgtagcaagcaaagaacttcaacagccaggcagtgca
cgaagcacgcgacatcttgaaattgaacttccaaaagaagcttcttatcaagaaggagatcattta
ggtgttattcctcgcaactatgaaggaatagtaaaccgtgtaacagcaaggttcggcctagatgc
atcacagcaaatccgtctggaagcagaagaagaaaaattagctcatttgccactcgctaaaacag
tatccgtagaagagcttctgcaatacgtggagcttcaagatcctgttacgcgcacgcagcttcgc
gcaatggctgctaaaacggtctgcccgccgcataaagtagagcttgaagccttgcttgaaaagc
aagcctacaaagaacaagtgctggcaaaacgtttaacaatgcttgaactgcttgaaaaatacccg
gcgtgtgaaatgaaattcagcgaatttatcgcccttctgccaagcatacgcccgcgctattactcg
atttcttcatcacctcgtgtcgatgaaaaacaagcaagcatcacggtcagcgttgtctcaggagaa
gcgtggagcggatatggagaatataaaggaattgcgtcgaactatcttgccgagctgcaagaag
gagatacgattacgtgctttatttccacaccgcagtcagaatttacgctgccaaaagaccctgaaa
cgccgcttatcatggtcggaccgggaacaggcgtcgcgccgtttagaggctttgtgcaggcgc
gcaaacagctaaaagaacaaggacagtcacttggagaagcacatttatacttcggctgccgttca
cctcatgaagactatctgtatcaagaagagcttgaaaacgcccaaagcgaaggcatcattacgct
tcataccgctttttctcgcatgccaaatcagccgaaaacatacgttcagcacgtaatggaacaaga
cggcaagaaattgattgaacttcttgatcaaggagcgcacttctatatttgcggagacggaagcc
aaatggcacctgccgttgaagcaacgcttatgaaaagctatgctgacgttcaccaagtgagtgaa
gcagacgctcgcttatggctgcagcagctagaagaaaaaggccgatacgcaaaagacgtgtg
ggctgggtaa SEQ NADPH
atggcattagataagttagatttatatgttattataacattggtggttgcaattgcagcttatt-
ttgcaaa ID cytochrome P450
gaatcagtttcttgaccaacaacaagataccgggttccttaatactgatagtggagatggtaattca
NO: reductase, CPR
agagatatcttacaagctttgaagaagaacaataaaaatacgttattattatttggatcccaaacag
25 (Candida strain
gtacagcagaagattatgccaacaaattgtcaagagaattgcattcaagatttggtttgaaaaccat
ATCC750)
ggttgctgatttcgctgattatgatttcgaaaacttcggagatattactgaagatatcttggtt-
ttcttta Nuc. Seq
ttgttgctacttatggtgaaggtgaaccaaccgataatgctgacgaatttcacacttggttgac-
tga
agaagctgacaccttgagtactttgaaatatactgtttttggttgggtaattcaacttatgaattcttc
aatgctattggtagaaaatttgacagattgttgggagaaaaaggtggtgacagatttgctgaatac
ggtgaaggtgacgatggtactggtactttagatgaagatttcttggaaggataacgtgtttg
attccttaaagaatgatttgaattttgaagaaaaagagttgaaatacgaaccaaatgttaaattgact
gaaagagatgatttatctggcaatgatccagatgtctccttgggtgaaccaaatgtcaaatacatta
aatctgaagtgttgacttaactaaaggtccatttgatcatactcatccatttttggctagaattgttaa
aactaaagaattgtttacttctgaagacagacattgtgttcatgttgaatttgatatttctgaatcaaac
ttgaaatataccaccggtgatcatcttgcaatctggccatctaactctgatgaaaacattaagcaatt
tgccaaatgttttggtttagaagacaaacttgatactgttattgaattgaaagctttggattccacttat
tccatcccattccctaatccaatcacttatggagctgttattagacaccatttggaaatttcaggtcct
gtttctagacaatttttcttatctattgctggatttgcccctgatgaagaaactaaaaagtcatttacta
gaattggtggtgataagcaagaatttgctagtaaagtcacccgtagaaaattcaacattgccgatg
ctttattatttgcttccaacaacagaccatggtccgatgttccattcgaattccttattgaaaatgtcca
acacttaactcctcgttattactccatttcttcttcctcattaagtgaaaagcaaaccattaatgttactg
ctgttgttgaagccgaagaagaagctgatggaagaccagttactggtgttgtcaccaacttgttga
agaatattgaaattgaacaaaacaaaactggtgaaaccccaatggttcattatgatttgaatggtcc
aagaggcaaatttagcaagttcagattgccagttcacgttagaagatctaatttcaaattaccaaag
aatagcactaccccagttattttgattggtccaggtaccggtgttgcaccattgagaggttttgttag
agaaagagttcaacaagttaaaaatggtgttaatgttggtaagactgtattgttttatggatgtagaa
attccgaacaagatttcttgtacaaacaagaatggagtgaatatgcctcagtattgggagaaaattt
cgaaatgtttaatgccttctcaagacaagatccaactaagaaagtttatgttcaagataagattttag
aaaatagtgctcttgttgatgagttattatctagtggagcaattatttatgtttgtggtgatgccagtag
aatggctagagatgttcaagctgcaattgccaagattgttgccaaaagtagagatatccacgaag
ataaagctgctgaattggttaaatcttggaaagttcaaaatagataccaagaagatgtctggtaa
SEQ NADPH
atggctttagacaagttagatttgtatgtcatcataacattggtggtcgctgtagccgcctatt-
ttgct ID cytochrome P450
aagaaccagttccttgatcagccccaggacaccgggttcctcaacacggacagcggaagcaac NO:
reductase A,
tccagagacgtcttgctgacattgaagaagaataataaaaacacgttgttgttgtttgggtcccaga
26 CPRA (Candida
cgggtacggcagaagattacgccaacaaattgtccagagaattgcactccagatttggcttgaaa
strain
acgatggttgcagatttcgctgattacgattgggataacttcggagatatcaccgaagacatcttg
ATCC20336)
gtgtttttcattgttgccacctatggtgagggtgaacctaccgataatgccgacgagttccacacct
Nuc. Seq
ggttgactgaagaagctgacactttgagtaccttgaaatacaccgtgttcgggttgggtaactc-
ca
cgtacgagttcttcaatgccattggtagaaagtttgacagattgttgagcgagaaaggtggtgaca
ggtttgctgaatacgctgaaggtgatgacggtactggcaccttggacgaagatttcatggcctgg
aaggacaatgtctttgacgccttgaagaatgatttgaactttgaagaaaaggaattgaagtacgaa
ccaaacgtgaaattgactgagagagacgacttgtctgctgctgactcccaagtttccttgggtgag
ccaaacaagaagtacatcaactccgagggcatcgacttgaccaagggtccattcgaccacacc
cacccatacttggccagaatcaccgagacgagagagttgttcagctccaaggacagacactgta
tccacgttgaatttgacatttctgaatcgaacttgaaatacaccaccggtgaccatctagctatctgg
ccatccaactccgacgaaaacattaagcaatttgccaagtgtttcggattggaagataaactcgac
actgttattgaattgaaggcgttggactccacttacaccatcccattcccaaccccaattacctacg
gtgctgtcattagacaccatttagaaatctccggtccagtctcgagacaattctttttgtcaattgctg
ggtttgctcctgatgaagaaacaaagaaggcttttaccagacttggtggtgacaagcaagaattc
gccgccaaggtcacccgcagaaagttcaacattgccgatgccttgttatattcctccaacaacgct
ccatggtccgatgttccttttgaattccttattgaaaacgttccacacttgactccacgttactactcc
atttcgtcttcgtcattgagtgaaaagcaactcatcaacgttactgcagttgttgaagccgaagaag
aagctgatggcagaccagtcactggtgttgtcaccaacttgttgaagaacgttgaaattgtgcaaa
acaagactggcgaaaagccacttgtccactacgatttgagcggcccaagaggcaagttcaaca
agttcaagttgccagtgcatgtgagaagatccaactttaagttgccaaagaactccaccacccca
gttatcttgattggtccaggtactggtgttgccccattgagaggttttgtcagagaaagagttcaac
aagtcaagaatggtgtcaatgttggcaagactttgttgttttatggttgcagaaactccaacgagga
ctttttgtacaagcaagaatgggccgagtacgcttctgttttgggtgaaaactttgagatgttcaatg
ccttctccagacaagacccatccaagaaggtttacgtccaggataagattttagaaaacagccaa
cttgtgcacgagttgttgactgaaggtgccattatctacgtctgtggtgatgccagtagaatggcta
gagacgtgcagaccacaatttccaagattgttgctaaaagcagagaaattagtgaagacaaggc
tgctgaattggtcaagtcctggaaggtccaaaatagataccaagaagatgtttggtag SEQ
NADPH
atggctttagacaagttagatttgtatgtcatcataacattggtggtcgctgtggccgcctatt-
ttgct ID cytochrome P450
aagaaccagttccttgatcagccccaggacaccgggttcctcaacacggacagcggaagcaac NO:
reductase B,
tccagagacgtcttgctgacattgaagaagaataataaaaacacgttgttgttgtttgggtcccaga
27 CPRB (Candida
ccggtacggcagaagattacgccaacaaattgtcaagagaattgcactccagatttggcttgaaa
strain
accatggttgcagatttcgctgattacgattgggataacttcggagatatcaccgaagatatcttg-
g ATCC20336)
tgtttttcatcgttgccacctacggtgagggtgaacctaccgacaatgccgacgagttccacacct
Nuc. Seq
ggttgactgaagaagctgacactttgagtactttgagatataccgtgttcgggttgggtaactc-
cac
ctacgagttcttcaatgctattggtagaaagtttgacagattgttgagtgagaaaggtggtgacaga
tttgctgaatatgctgaaggtgacgacggcactggcaccttggacgaagatttcatggcctggaa
ggataatgtctttgacgccttgaagaatgacttgaactttgaagaaaaggaattgaagtacgaacc
aaacgtgaaattgactgagagagatgacttgtctgctgccgactcccaagtttccttgggtgagcc
aaacaagaagtacatcaactccgagggcatcgacttgaccaagggtccattcgaccacaccca
cccatacttggccaggatcaccgagaccagagagttgttcagctccaaggaaagacactgtattc
acgttgaatttgacatttctgaatcgaacttgaaatacaccaccggtgaccatctagccatctggcc
atccaactccgacgaaaacatcaagcaatttgccaagtgtttcggattggaagataaactcgaca
ctgttattgaattgaaggcattggactccacttacaccattccattcccaactccaattacttacggtg
ctgtcattagacaccatttagaaatctccggtccagtctcgagacaattctttttgtcgattgctgggt
ttgctcctgatgaagaaacaaagaagactttcaccagacttggtggtgacaaacaagaattcgcc
accaaggttacccgcagaaagttcaacattgccgatgccttgttatattcctccaacaacactccat
ggtccgatgttccttttgagttccttattgaaaacatccaacacttgactccacgttactactccatttc
ttcttcgtcgttgagtgaaaaacaactcatcaatgttactgcagtcgttgaggccgaagaagaagc
cgatggcagaccagtcactggtgttgttaccaacttgttgaagaacattgaaattgcgcaaaacaa
gactggcgaaaagccacttgttcactacgatttgagcggcccaagaggcaagttcaacaagttc
aagttgccagtgcacgtgagaagatccaactttaagttgccaaagaactccaccaccccagttat
cttgattggtccaggtactggtgttgccccattgagaggtttcgttagagaaagagttcaacaagtc
aagaatggtgtcaatgttggcaagactttgttgttttatggttgcagaaactccaacgaggacttttt
gtacaagcaagaatgggccgagtacgcttctgttttgggtgaaaactttgagatgttcaatgccttc
tctagacaagacccatccaagaaggtttacgtccaggataagattttagaaaacagccaacttgtg
cacgaattgttgaccgaaggtgccattatctacgtctgtggtgacgccagtagaatggccagaga
cgtccagaccacgatctccaagattgttgccaaaagcagagaaatcagtgaagacaaggccgc
tgaattggtcaagtcctggaaagtccaaaatagataccaagaagatgtttggtag SEQ
cytochrome P450:
mtikempqpktfgelknlpllntdlpvqalmkiadelgeifkfeapgrvtrylssqrlikeacde
ID NADPH P450
srfdknlsqalkfvrdfagdglftswthenwkkahnillpsfsqqamkgyhammvdiavql NO:
reductase
vqkwerlnadehievpedmtrltldtiglcgfnyrfnsfyrdqphpfitsmvrasdeamnksq 28
(Bacillus
ranpddpaydenkrqfqedikvmndlvdkiiadrkasgeqsddllthmlngkdpetgepld
megaterium)
deniryqiitfliaghettsgllsfasyflvknphvlqkaaeeaarvlvdpvpsykqvkqlkyvg
amino acid [P450
mvlneasrlwptapafslyakedtvlggeyplekgdelmvsipqlhrdktiwgddveefrper
activity shown in
fenpsaipqhafkpfgngqracigqqfalheatsvlgmmlkhfdfedhtnyesdiketltlkpe
italics, P450
gfvvkakskkiplggipspsteqsakkvrkkaenahntpslvlygsnmgtaegtardladiam
reductase
skgfapqvatldshagnlpregavlivtasynghppdnakqfvdwldqasadevlgvrysv
activity
fgcgdknwattyqkvpafidetlaakgaeniadrgeadasddfegtyeewrehmwsdvaa shown
in yfnldiensednkstlslqfvdsaadmplakmhgafstnvvaskelqqpgsarstrhleielpk
normal font]
casyqcgdhlgviprnycgivnrvtarfgldasqqirscacccklahlplaktvsvcclsqyvcl
A.A. Seq
qdpvtrtqlramaaktvcpphkvclcallckqaykcqvsakrltmlcslckypaccmkfscfi
alspsirpryysisssprvdckqasitvsvvsgcawsgygcykgiasnylacsqcgdtitcfist
pqscftspkdpctplimvgpgtgvapfrgfvqarkqlkcqgqslgcahlyfgcrsphcdysy
qeelenaqsegiitlhtafsrmpnqpktyvqhvmeqdgkklielldqgahfyicgdgsqma
paveatlmksyadvhqvseadarlwsqqleekgryakdvwag* SEQ acyl CoA
ATGACTTTTACAAAGAAAAACGTTAGTGTATCACAAGGTC ID oxidase, POX4
CTGACCCTAGATCATCCATCCAAAAGGAAAGAGACAGCTC NO: (Candida strain
CAAATGGAACCCTCAACAAATGAACTACTTCTTGGAAGGC 29 ATCC20336)
TCCGTCGAAAGAAGTGAGTTGATGAAGGCTTTGGCCCAAC nucleotide
AAATGGAAAGAGACCCAATCTTGTTCACAGACGGCTCCTA
CTACGACTTGACCAAGGACCAACAAAGAGAATTGACCGC
CGTCAAGATCAACAGAATCGCCAGATACAGAGAACAAGA
ATCCATCGACACTTTCAACAAGAGATTGTCCTTGATTGGT
ATCTTTGACCCACAGGTCGGTACCAGAATTGGTGTCAACC
TCGGTTTGTTCCTTTCTTGTATCAGAGGTAACGGTACCACT
TCCCAATTGAACTACTGGGCTAACGAAAAGGAAACCGCTG
ACGTTAAAGGTATCTACGGTTGTTTCGGTATGACCGAATT
GGCCCACGGTTCCAACGTTGCTGGTTTGGAAACCACCGCC
ACATTTGACAAGGAATCTGACGAGTTTGTCATCAACACCC
CACACATTGGTGCCACCAAGTGGTGGATTGGTGGTGCTGC
TCACTCCGCCACCCACTGTTCTGTCTACGCCAGATTGATTG
TTGACGGTCAAGATTACGGTGTCAAGACTTTTGTTGTCCC
ATTGAGAGACTCCAACCACGACCTCATGCCAGGTGTCACT
GTTGGTGACATTGGTGCCAAGATGGGTAGAGATGGTATCG
ATAACGGTTGGATCCAATTCTCCAACGTCAGAATCCCAAG
ATTCTTTATGTTGCAAAAGTTCTGTAAGGTTTCTGCTGAAG
GTGAAGTCACCTTGCCACCTTTGGAACAATTGTCTTACTCC
GCCTTGTTGGGTGGTAGAGTCATGATGGTTTTGGACTCCT
ACAGAATGTTGGCTAGAATGTCCACCATTGCCTTGAGATA
CGCCATTGGTAGAAGACAATTCAAGGGTGACAATGTCGAT
CCAAAAGATCCAAACGCTTTGGAAACCCAATTGATAGATT
ACCCATTGCACCAAAAGAGATTGTTCCCATACTTGGCTGC
TGCCTACGTCATCTCCGCTGGTGCCCTCAAGGTTGAAGAC
ACCATCCATAACACCTTGGCTGAATTGGACGCTGCCGTTG
AAAAGAACGACACCAAGGCTATCTTTAAGTCTATTGACGA
CATGAAGTCATTGTTTGTTGACTCTGGTTCCTTGAAGTCCA
CTGCCACTTGGTTGGGTGCTGAAGCCATTGACCAATGTAG
ACAAGCCTGTGGTGGTCACGGTTACTCGTCCTACAACGGC
TTCGGTAAAGCCTACAACGATTGGGTTGTCCAATGTACTT
GGGAAGGTGACAACAATGTCTTGGCCATGAGTGTTGGTAA
GCCAATTGTCAAGCAAGTTATCAGCATTGAAGATGCCGGC
AAGACCGTCAGAGGTTCCACCGCTTTCTTGAACCAATTGA
AGGACTACACTGGTTCCAACAGCTCCAAGGTTGTTTTGAA
CACTGTTGCTGACTTGGACGACATCAAGACTGTCATCAAG
GCTATTGAAGTTGCCATCATCAGATTGTCCCAAGAAGCTG
CTTCTATTGTCAAGAAGGAATCTTTCGACTATGTCGGCGCT
GAATTGGTTCAACTCTCCAAGTTGAAGGCTCACCACTACT
TGTTGACTGAATACATCAGAAGAATTGACACCTTTGACCA
AAAGGACTTGGTTCCATACTTGATCACCCTCGGTAAGTTG
TACGCTGCCACTATTGTCTTGGACAGATTTGCCGGTGTCTT
CTTGACTTTCAACGTTGCCTCCACCGAAGCCATCACTGCTT
TGGCCTCTGTGCAAATTCCAAAGTTGTGTGCTGAAGTCAG
ACCAAACGTTGTTGCTTACACCGACTCCTTCCAACAATCC
GACATGATTGTCAATTCTCCTATTGGTACATACGATGGTG
ACATCTATGAGAACTACTTTGACTTGGTCAAGTTGCAGAA
CCCACCATCCAAGACCAAGGCTCCTTACTCTGATGCTTTG
GAAGCCATGTTGAACAGACCAACCTTGGACGAAAGAGAA
AGATTTGAAAAGTCTGATGAAACCGCTGCTATCTTGTCCA AGTAA SEQ acyl CoA
MTFTKKNVSVSQGPDPRSSIQKERDSSKWNPQQMNYFLEGS ID oxidase, POX4
VERSELMKALAQQMERDPILFTDGSYYDLTKDQQRELTAVK NO: (Candida strain
INRIARYREQESIDTFNKRLSLIGIFDPQVGTRIGVNLGLFLSCI 30 ATCC20336)
RGNGTTSQLNYWANEKETADVKGIYGCFGMTELAHGSNVA amino acid
GLETTATFDKESDEFVINTPHIGATKWWIGGAAHSATHCSVY
ARLIVDGQDYGVKTFVVPLRDSNHDLMPGVTVGDIGAKMG
RDGIDNGWIQFSNVRIPRFFMLQKFCKVSAEGEVTLPPLEQLS
YSALLGGRVMMVLDSYRMLARMSTIALRYAIGRRQFKGDN
VDPKDPNALETQLIDYPLHQKRLFPYLAAAYVISAGALKVED
TIHNTLAELDAAVEKNDTKAIFKSIDDMKSLFVDSGSLKSTA
TWLGAEAIDQCRQACGGHGYSSYNGFGKAYNDWVVQCTW
EGDNNVLAMSVGKPIVKQVISIEDAGKTVRGSTAFLNQLKD
YTGSNSSKVVLNTVADLDDIKTVIKAIEVAIIRLSQEAASIVK
KESFDYVGAELVQLSKLKAHHYLLTEYIRRIDTFDQKDLVPY
LITLGKLYAATIVLDRFAGVFLTFNVASTEAITALASVQIPKL
CAEVRPNVVAYTDSFQQSDMIVNSAIGRYDGDIYENYFDLV
KLQNPPSKTKAPYSDALEAMLNRPTLDERERFEKSDETAAIL SK* SEQ acyl CoA
ATGCCTACCGAACTTCAAAAAGAAAGAGAACTCACCAAG ID oxidase, POX5
TTCAACCCAAAGGAGTTGAACTACTTCTTGGAAGGTTCCC NO: (Candida strain
AAGAAAGATCCGAGATCATCAGCAACATGGTCGAACAAA 31 ATCC20336)
TGCAAAAAGACCCTATCTTGAAGGTCGACGCTTCATACTA nucleotide
CAACTTGACCAAAGACCAACAAAGAGAAGTCACCGCCAA
GAAGATTGCCAGACTCTCCAGATACTTTGAGCACGAGTAC
CCAGACCAACAGGCCCAGAGATTGTCGATCCTCGGTGTCT
TTGACCCACAAGTCTTCACCAGAATCGGTGTCAACTTGGG
TTTGTTTGTTTCCTGTGTCCGTGGTAACGGTACCAACTCCC
AGTTCTTCTACTGGACCATAAATAAGGGTATCGACAAGTT
GAGAGGTATCTATGGTTGTTTTGGTATGACTGAGTTGGCC
CACGGTTCCAACGTCCAAGGTATTGAAACCACCGCCACTT
TTGACGAAGACACTGACGAGTTTGTCATCAACACCCCACA
CATTGGTGCCACCAAGTGGTCGATCGGTGGTGCTGCGCAC
TCCGCCACCCACTCCTCCGTCTACGCCAGATTGAAGGTCA
AAGGAAAGGACTACGGTGTCAAGACCTTTGTTGTCCCATT
GAGAGACTCCAACCACGACCTCGAGCCAGOTGTGACTGTT
GGTGACATTGGTGCCAAGATGGGTAGAGACGGTATCGAT
AACGGTTGGATCCAGTTCTCCAACGTCAGAATCCCAAGAT
TCTTTATGTTGCAAAAGTACTGTAAGGTTTCCCGTCTGGGT
GAAGTCACCATGCCACCATCTGAACAATTGTCTTACTCGG
CTTTCATTGGTGGTAGAGTCACCATGATGATGGACTCCTA
CAGAATGACCAGTAGATTCATCACCATTGCCTTGAGATAC
GCCATCCACAGAAGACAATTCAAGAAGAAGGACACCGAT
ACCATTGAAACCAAGTTGATTGACTACCCATTGCATCAAA
AGAGATTGTTCCCATTCTTGGCTGCCGCTTACTTGTTCTCC
CAAGGTGCCTTGTACTTAGAACAAACCATGAACGCAACCA
ACGACAAGTTGGACGAAGCTGTCAGTGCTGGTGAAAAGG
AAGCCATTGACGCTGCCATTGTCGAATCCAAGAAATTGTT
CGTCGCTTCCGGTTGTTTGAAGTCCACCTGTACCTGGTTGA
CTGCTGAAGCCATTGACGAAGCTCGTCAAGCTTGTGGTGG
TCACGGTTACTCGTCTTACAACGGTTTCGGTAAAGCCTACT
CCGACTGGGTTGTCCAATGTACCTGGGAAGGTGACAACAA
CATCTTGGCCATGAACGTTGCCAAGCCAATGGTTAGAGAC
TTGTTGAAGGAGCCAGAACAAAAGGGATTGGTTCTCTCCA
GCGTTGCCGACTTGGACGACCCAGCCAAGTTGCTTAAGGC
TTTCGACCACGCCCTTTCCGGCTTGGCCAGAGACATTGGT
GCTGTTGCTGAAGACAAGGGTTTCGACATTACCGGTCCAA
GTTTGGTTTTGGTTTCCAAGTGAACGCTCACAGATTCTTG
ATTGACGGTTTCTTCAAGCGTATCACCCCAGAATGGTCTG
AAGTCTTGAGACCTTTGGGTTTCTTGTATGCCGACTGGATC
TTGACCAACTTTGGTGCCACCTTCTTGCAGTACGGTATCAT
TACCCCAGATGTCAGCAGAAAGATTTCCTCCGAGCACTTC
CCAGCCTTGTGTGCCAAGGTTAGACCAAACGTTGTTGGTT
TGACTGATGGTTTCAACTTGACTGACATGATGACCAATGC
TGCTATTGGTAGATATGATGGTAACGTCTACGAACACTAC
TTCGAAACTGTCAAGGCTTTGAACCCACCAGAAAACACCA
AGGCTCCATACTCCAAGGCTTTGGAAGACATGTTGAACCG
TCCAGACCTTGAAGTCAGAGAAAGAGGTGAAAAGTCCGA
AGAAGCTGCTGAAATCTTGTCCAGTTAA SEQ acyl CoA
MPTELQKERELTKFNPKELNYFLEGSQERSEIISNMVEQMQK ID oxidase, POX5
DPILKVDASYYNLTKDQQREVTAKKIARLSRVFEHEYPDQQ NO: (Candida strain
AQRLSILGVFDPQVFTRIGVNLGLFVSCVRGNGTNSQFFYWT 32 ATCC20336)
INKGIDKLRGIYGCFGMTELAHGSNVQGIETTATFDEDTDEF amino acid
VINTPHIGATKWWIGGAAHSATHCSVYARLKVKGKDYGVK
TFVVPLRDSNHDLEPGVTVGDIGAKMGRDGIDNGWIQFSNV
RIPRFFMLQKYCKVSRSGEVTMPPSEQLSYSALIGGRVTMM
MDSYRMTSRFITIALRYAIHRRQFKKDTDTIETKLIDYPLHQ
KRLFPFLAAAYLFSQGALYLEQTMNATNDKLDEAVSAGEKE
AIDAAIVESKKLFVASGCLKSTCTWLTAEAIDEARQACGGHG
YSSYNGFGKAYSDWVVQCTWEGDNNILAMNVAKPMVRDL
LKEPEQKGLVLSSVADLDDPAKLVKAFDHALSGLARDIGAV
AEDKGFDITGPSLVLVSKLNAHRFLIDGFFKRITPEWSEVLRP
LGFLYADWILTNFGATFLQYGIITPDVSRKISSEHFPALCAKV
RPNVVGLTDGFNLTDMMTNAAIGRYDGNVYEHYFETVKAL
NPPENTKAPYSKALEDMLNRPDLEVRERGEKSEEAAEILSS* SEQ Acyl-CoA
atgatcagaaccgtccgttatcaatccctcaagaggttcagacctctggctttgtctcctgtttttcgt
ID Hydrolase
ccacgctacaactcccagaaggccaatttccaccgtccagaccaccctgggtccgacgagcca NO:
(ACHA)
gctgaagccgccgacgccgccgccacgatcctcgccgagttgcgagacaagcagacgaacc 33
Nucleotide Seq
cgaacaaggccacctggctcgatgcgttaacggagcgggagaagttgcgtgccgagggcaag
acgattgacagtttcagctacgttgaccccaagacgaccgtcgtgggggagaagacacgcagt
gactcgttctcgttcttgttgttgccgttcaaggacgacaagtggttgtgtgacgcgtacatcaatg
cgtttggccggttgcgtgtagcgcagttgttccaggacttggacgccttggcggggcgcatcgc
gtacaggcactgttccccagcggagcccgtgaatgtcacggcgagcgtggatagggtgtacat
ggtgaagaaagtggacgagattaacaattacaatttcgtgttggcggggtccgtgacgtggacc
gggagatcgtcgatggagatcacggtgaaagggtatgcttttgaagacgccgtgccggatataa
cgaacgaggagtccttgccggcagagaatgtgtttttggctgctaatttcaccttcgtggcacgga
acccacttacacacaagtcctttgctattaacagattgttgcccgtgactgagaaggactgggtcg
actatcgccgtgctgagtcccacaacgccaagaagaagttgatggcaaagaacaagaagatctt
ggagcctaccgcggaagagtccaagttgatctacgacatgtggagatcgtccaagtccttacag
aacatcgagagggccaacgatgggatcgcgttcatgaaggacacgaccatgaagtccaccttg
ttcatgcagccccagtaccgtaacagacactcatacatgattttcggagggtacttgttaagacaa
actttcgaattggcctactgtaccgcggcaacgttttccctggccgggccccgtttcgtcagcttg
gactccaccacgttcaagaaccccgtgcccgtggggtcggtgctcaccatggactcgtcgatct
cgtacacggagcacgtgcacgagggagtggaggagattgacgcggactcaccgttcaacttca
gcttgcctgccacgaacaagatctcgaagaaccccgaggcgttcttgtcggaacccggcacgtt
gattcaagtcaaggtcgacacatacatccaggagttagagcagagtgtgaagaagcccgcggg
tacgttcatctactcgttctatgttgataaagaaagcgttactgttgatggaaaggcgtcgttttgttc
agttatcccgcagacgtactccgagatgatgacttatgtgggcgggagaagaagagcccagga
tactgctaactacgtggagactttgccgtttagtggaagcggcaattaa SEQ Acyl-CoA
MIRTVRYQSLKRFRPSALSPVFRPRYNSQKANFHRPDHPGSD ID Hydrolase
EPAEAADAAATILAELRDKQTNPNKATWLDALTEREKLRAE NO: (ACHA) Amino
GKTIDSFSYVDPKTTVVGEKTRSDSFSFLLLPFKDDKWLCDA 34 Acid Seq
YINAFGRLRVAQLFQDLDALAGRIAYRHCSPAEPVNVTASV
DRVYMVKKVDEINNYNFVLAGSVTWTGRSSMEITVKGYAF
EDAVPDITNEESLPAENVFLAANFTFVARNPLTHKSFAINRLL
PVTEKDWVDYRRAESHNAKKKLMAKNKKILEPTAEESKLIY
DNWRSSKSLQNIERANDGIAFMKDTTMKSTLFMQPQYRNR
HSYMIFGGYLLRQTFELAYCTAATFSSAGPRFVSLDSTTFKNP
VPVGSVLTMDSSISYTEHVHEGVEEIDADSPFNFSLPATNKIS
KNPEAFLSEPGTLIQVKVDTYIQELEQSVKKPAGTFIYSFYVD
KESVTVDGKASFCSVIPQTYSEMMTYVGGRRRAQDTANYVE TLPFSGSGN SEQ Acyl-CoA
atgatcagaaccgtccgttatcaatccttcaagaggttcaaacctctgactttatcccccgttttccgt
ID Hydrolase
ccacgctacaactcccagaaggccaatttccaccgtccagaccacgctgggtccgacgagcca NO:
(ACHB)
gccgaagccgccgacgccgctgccacgatcctcgccgagttgcgagacaagcagacgaacc 35
Nucleotide Seq
cgaacaaggccacctggctcgatgcgttaacggagcgggagaagttgcgtgccgagggcaag
acaatcgacagcttcagctacgttgaccccaagacaaccgtcgtgggggagaagacacgcag
cgactcgttctcgttcttgttgttgccgttcaaggacgacaagtggttgtgtgacgcgtacatcaat
gcgtttggccggttgcgtgtagcgcagttgttccaggacttggacgccttggcgggccgcatcg
cgtacaggcactgttcccccgctgagcccgtgaatgtcacggcgagcgtggatagagtgtatat
ggtgaagaaagtggacgagattaataattacaatttcgtgttggcggggtccgtgacgtggaccg
ggagatcgtcgatggagatcacggtcaaagggtatgcttttgaagacgccgtgccggagataac
taacgaggagtccttgccggcagagaatgtgttcttggctgttaatttcaccttcgtggcacgtaac
ccactcacacacaagtccttcgctattaacagattgttgcccgtgactgagaaggactgggtcgat
tatcgccgtgctgagtcccacaacgccaagaagaagttgatggcaaagaacaagaagatcttgg
agcctaccccggaagagtccaagttgatctacgacatgtggagatcgtccaagtccttacagaac
atcgagaaggccaacgacgggatcgcgttcatgaaggacacgataatgaagtccaccttgttca
tgcagccccagtaccgtaacagacactcatacatgattttcggtgggtatttgttaagacaaacttt
cgaattggcctattgtaccgcagcaacgttttccctggcgggaccccgtttcgtcagcttggactc
caccacgttcaagaaccccgtgcccgtggggtcggtgctcaccatggactcgtcgatctcgtac
acggagcacgtccacgatggcgttgaggagattgacgccgactccccgttcaacttcagcttgc
ctgccacgaacaagatctcgaagaaccccgaggcgttcttgtcggagcccggcacgttgatcca
agtcaaggtcgacacgtacatccaggagttagagcaaagtgtgaagaagcctgcgggaacgtt
catctactcgttctatgttgataaagagagcgttactgtggatggaaaggcgtcgttttgttcagttat
cccgcagacgtactccgagatgatgacttatgtgggcgggagaagaagagcccaggatactgc
taattacgtggagactttgccgtttagtggaagcggcaattaa SEQ ACyl-CoA
MIRTVRYQSFKRFKPLTLSPVFRPRYNSQKANFHRPDHAGSD ID Hydrolase
EPAEAADAAATILAELRDKQTNPNKATWLDALTEREKLRAE NO: (ACHB)
GKTIDSFSYVDPKTTVVGEKTRSDSFSFLLLPFKDDKWLCDA 36
YINAFGRLRVAQLFQDLDALAGRIAYRHCSPAEPVNVTASV
DRVYMVKKVDEINNYNFVLAGSVTWTGRSSMEITVKGYAF
EDAVPEITNEESLPAENVFLAVNFTFVARNPLTHKSFAINRLL
PVTEKDWVDYRRAESHNAKKKLMAKNKKILEPTPEESKLIY
DMWRSSKSLQNIEKANDGIAFMKDTIMKSTLFMQPQYRNRH
SYMIFGGYLLRQTFELAYCTAATFSLAGPRFVSLDSTTFKNP
VPVGSVLTMDSSISYTEHVHDGVEEIDADSPFNFSLPATNKIS
KNPEAFLSEPGTLIQVKVDTYIQELEQSVKKPAGTFIYSFYVD
KESVTVDGKASFCSVIPQTYSEMMTYVGGRRRAQDTANYVE TLPFSGSGN SEQ E. coli
Acyl-CoA
atggccgatacattgctcatcttgggtgactctttgtctgcagggtatcggatgtccgcatctgccg
ID Thioesterase
catggcctgcactcctcaatgacaaatggcaaagcaagacatcggtcgtgaatgcatctatctct
NO: (TESA) gene
ggcgatacctcgcagcaggggttggcccgtctcccagccttgttgaagcaacatcaaccacgtt 37
without signal
gggtcttggtcgaattgggcggcaatgatggtctcagaggttttcaacctcaacagaccgagcag
peptide sequence
acattgcgtcaaatcctccaagacgtgaaggcagcaaacgccgaacctctcttgatgcagataa
optimized for C.
gattgcctgccaactatggtcgtagatacaatgaagccttttctgcaatctacccgaagcttgcaaa
tropicalis
ggagtttgacgtcccattgttgccgtttttgatggaagaggtgtaccttaagcctcagtggatgcaa
Nucleotide Seq
gacgatggtatccatccgaaccgtgatgcacaaccattcatcgcagattggatggccaaacaact
ccaacctttggtcaatcatgatagctaa SEQ E. coli Acyl-CoA
MADTLLILGDSLSAGYRMSASAAWPALLNDKWQSKTSVVN ID Thioesterase
ASISGDTSQQGLARLPALLKQHQPRWVLVELGGNDGLRGFQ NO: (TESA) without
PQQTEQTLRQILQDVKAANAEPLLMQIRLPANYGRRYNEAFS 38 signal peptide
AIYPKLAKEFDVPLLPFLMEEVYLKPQWMQDDGIHPNRDAQ Amino Acid Seq
PFIADWMAKQLQPLVNHDS SEQ Acyl-CoA
atgggtgcccctttaacagtcgccgttggcgaagcaaaaccaggcgaaaccgctccaagaaga ID
Synthetase
aaagccgctcaaaaaatggcctctgtcgaacgcccaacagactcaaaggcaaccactttgcca NO:
(ACS1)Nuc. Seq
gacttcattgaagagtgttttgccagaaacggcaccagagatgccatggcctggagagacttggt
39 cgaaatccacgtcgaaaccaaacaggttaccaaaatcattgacggcgaacagaaaaaggtcga
taaggactggatctactacgaaatgggtccttacaactacatatcctaccccaagttgttgacgttg
gtcaagaactactccaagggtttgttggagttgggcttggccccagatcaagaatccaagttgatg
atctttgccagtacctcccacaagtggatgcagaccttcttagcctccagtttccaaggtatccccg
ttgtcaccgcctacgacaccttgggtgagtcgggcttgacccactccttggtgcaaaccgaatcc
gatgccgtgttcaccgacaaccaattgttgtcctccttgattcgtcctttggagaaggccacctccg
tcaagtatgtcatccacggggaaaagattgaccctaacgacaagagacagggcggcaaaatct
accaggatgcggaaaaggccaaggagaagattttacaaattagaccagatattaaatttatttcttt
cgacgaggttgttgcattgggtgaacaatcgtccaaagaattgcatttcccaaaaccagaagacc
caatctgtatcatgtacacctcgggttccaccggtgctccaaagggtgtggttatcaccaatgcca
acattgttgccgccgtgggtggtatctccaccaatgctactagagacttggttagaactgtcgaca
gagtgattgcatttttgccattggcccacattttcgagttggcctttgagttggttaccttctggtggg
gggctccattgggttacgccaatgtcaagactttgaccgaagcctcctgcagaaactgtcagcca
gacttgattgaattcaaaccaaccatcatggttggtgttgctgccgtttgggaatcggtcagaaag
ggtgtcttgtctaaattgaaacaggcttctccaatccaacaaaagatcttctgggctgcattcaatg
ccaagtctactttgaaccgttatggcttgccaggcggtgggttgtttgacgctgtcttcaagaaggt
taaagccgccactggtggccaattgcgttatgtgttgaatggtgggtccccaatctctgttgatgcc
caagtgtttatctccaccttgcttgcgccaatgttgttgggttacggtttgactgaaacctgtgccaat
accaccattgtcgaacacacgcgcttccagattggtactttgggtaccttggttggatctgtcactg
ccaagttggttgatgttgctgatgctggatactacgccaagaacaaccagggtgaaatctggttga
aaggcggtccagttgtcaaggaatactacaagaacgaagaagaaaccaaggctgcattcaccg
aagatggctggttcaagactggtgatattggtgaatggaccgccgacggtggtttgaacatcattg
accgtaagaagaacttggtcaagactttgaatggtgaatacattgctttggagaaattggaaagtat
ttacagatccaaccacttgattttgaacttgtgtgtttacgctgaccaaaccaaggtcaagccaattg
ctattgtcttgccaattgaagccaacttgaagtctatgttgaaggacgaaaagattatcccagatgc
tgattcacaagaattgagcagcttggttcacaacaagaaggttgcccaagctgtcttgagacactt
gctccaaaccggtaaacaacaaggtttgaaaggtattgaattgttgcagaatgttgtcttgttggat
gacgagtggaccccacagaatggttttgttacttctgcccaaaagttgcagagaaagaagatttta
gaaagttgtaaaaaagaagttgaagaggcatacaagtcgtcttag SEQ Acyl-CoA
MGAPLTVAVGEAKPGETAPRRKAAQKMASVERPTDSKATT ID Synthetase
LPDFIEECFARNGTRDAMAWRDLVEIHVETKQVTKIIDGEQK NO: (ACS1)
KVDKDWIYYEMGPYNYISYPKLLTLVKNYSKGLLELGLAPD 40 A.A. Seq
QESKLMIFASTSHKWMQTFLASSFQGIPVVTAYDTLGESGLT
HSLVQTESDAVFTDNQLLSSLIRPLEKATSVKYVIHGEKIDPN
DKRQGGKIYQDAEKAKEKILQIRPDIKFISFDEVVALGEQSSK
ELHFPKPEDPICIMYTSGSTGAPKGVVITNANIVAAVGGISTN
ATRDLVRTVDRVIAFLPLAHIFELAFELVTFWWGAPLGYAN
VKTLTEASCRNCQPDLIEFKPTIMVGVAAVWESVRKGVLSK
LKQASPIQQKIFWAAFNAKSTENRYGLPGGGLFDAVFKKVK
AATGGQLRYVLNGGSPISVDAQVFISTLLAPMLLGYGLTETC
ANTTIVEHTRFQIGTLGTLVCSVTAKLVDVADAGYYAKNNQ
GEIWLKGGPVVKEYYKNEEETKAAFTEDGWFKTGDIGEWT
ADGGLNIIDRKKNLVKTLNGEYIALEKLESIYRSNHLILNLCV
YADQTKVKPIAIVLPIEANLKSMLKDEKIIPDADSQELSSLVH
NKKVAQAVLRHLLQTGKQQGLKGIELLQNVVLLDDEWTPQ
NGFVTSAQKLQRKKILESCKKEVEEAYKSS SEQ Long-chain Acyl-
atgtcaggattagaaatagccgctgctgccatccttggtagtcagttattggaagccaaatatttaat
ID CoA Synthetase
tgccgacgacgtgctgttagccaagacagtcgctgtcaatccctcccatacttgtggaaagcca
NO: (FAT1)
gcagaggtaaggcatcatactggtactttttcgagcagtccgtgttcaagaacccaaacaaca- aa
41 Nuc. Seq
gcgttggcgttcccaagaccaagaaagaatgcccccacccccaagaccgacgccgagggatt
ccagatctacgacgatcagtttgacctagaagaatacacctacaaggaattgtacgacatggtttt
gaagtactcatacatcttgaagaacgagtacggcgtcactgccaacgacaccatcggtgtttcttg
tatgaacaagccgcttttcattgtcttgtggttggcattgtggaacattggtgccttgcctgcgttctt
gttgacccggactgtgattccccaatcagagataccgaggctcagatcagagaggaattgccac
atgtgcaaataaactacattgacgagtttgccttgtttgacagattgagactcaagtcgactccaaa
acacagagccgaggacaagaccagaagaccaaccgatactgactcctccgcttgtgcattgatt
tacacctcgggtaccaccggtttgccaaaagccggtatcatgtcctggagaaaagccttcatggc
ctcggttttctttggccacatcatgaagattgactcgaaatcgaacgtcttgaccgccatgcccttgt
accactccaccgcggccatgttggggttgtgtcctactttgattgtcggtggctgtgtctccgtgtc
ccagaaattctccgctacttcgttctggacccaggccagattatgtggtgccacccacgtgcaata
cgtcggtgaggtctgtcgttacttgttgaactccaagcctcatccagaccaagacagacacaatgt
cagaattgcctacggtaacgggttgcgtccagatatatggtctgagttcaagcgcagattccacat
tgaaggtatcggtgagttctacgccgccaccgagtcccctatcgccaccaccaacttgcagtacg
gtgagtacggtgtcggcgcctgtcgtaagtacgggtccctcatcagcttgttattgtctacccagc
agaaattggccaagatggacccagaagacgagagtgaaatctacaaggaccccaagaccggg
ttctgtaccgaggccgcttacaacgagccaggtgagttgttgatgagaatcttgaaccctaacga
cgtgcagaaatccttccagggttattatggtaacaagtccgccaccaacagcaaaatcctcacca
atgttttcaaaaaaggtgacgcgtggtacagatccggtgacttgttgaagatggacgaggacaaa
ttgttgtactttgtcgacagattaggtgacactttccgttggaagtccgaaaacgtctccgccaccg
aggtcgagaacgaattgatgggctccaaggccttgaagcagtccgtcgttgtcggtgtcaaggt
gccaaaccacgaaggtagagcctgttttgccgtctgtgaagccaaggacgagttgagccatgaa
gaaatcttgaaattgattcactctcacgtgaccaagtctttgcctgtgtatgctcaacctgcgttcatc
aagattggcaccattgaggcttcgcacaaccacaaggttcctaagaaccaattcaagaaccaaa
agttgccaaagggtgaagacggcaaggatttgatctactggttgaatggcgacaagtaccagga
gttgactgaagacgattggtctttgatttgtaccggtaaagccaaattg SEQ Long-chain
Acyl- MSGLEIAAAAILGSQLLEAKKYLIADDVSLAKTVAVNALPYL ID CoA Synthetase
WKASRGKASYWYFFEQSVFKNPNNKALAFPRPRKNAPTPKT NO: (FAT1)
DAEGFQIYDDQFDLEEYTYKELYDMVLKYSYILKNEYGVTA 42 A.A. Seq
NDTIGVSCMNKPLFIVLWLALWNIGALPAFLNFNTKDKPLIH
CLKIVNASQVFVDPDCDSPIRDTEAQIREELPHVQINYIDEFAL
FDRLRLKSTPKHRAEDKTRRPTDTDSSACALIYTSGTTGLPK
AGIMSWRKAFMASVFFGHIMKIDSKSNVLTAMPLYHSTAAM
LGLCPTLIVGGCVSVSQKFSATSFWTQARLCGATHVQYVGE
VCRYLLNSKPHPDQDRHNVRIAYGNGLRPDIWSEFKRRFHIE
GIGEFYAATESPIATINLQYGEYGVGACRKYGSLISLLLSTQQ
KLAKMDPEDESEIYKDPKTGFCTEAAYNEPGELLMRILNPND
VQRSFQGYYGNKSATNSKILTNVFKKGDAWYRSGDLLKMD
EDKLLYFVDRLGDTFRWKSENVSATEVENELMGSKALKQSV
VVGVKVPNHEGRACFAVCEAKDELSHEEILKLIHSHVTKSLP
VYAQPAFIKIGTIEASHNHKVPKNQFKNQKLPKGEDGKDLIY
WLNGDKYQELTEDDWSLICTGKAKL SEQ Acyl-CoA Sterol
atgtccgacgacgagatagcaggaatagtcattgaaatcgacgatgacgtgaaatccacgtcttc
ID acyl transferase
gttccaggaagaactagtcgaggttgaaatgtccaactcgtccattaacgaatcccagaccgatg
NO: (ARE1)
agtcgtaccgtcctgaagaaacctcattgcattacaggaggaagtcccacaggaccccgtcag- a
43 Nuc. Seq
ggagtcgttcctagagatcaccaagaacgtgaatgatccggatctagtttccaagattgagaacct
aaggggcaaagtaagccaacgggaagacaggttgaggaagcactaccttcacacctcccagg
acgtcaagttcttgtcccggttcaacgacatcaagttcaagctgaactccgcgacgattctagattc
ggatgcgttttacaagagtgaatactttggagtcttgaccatcttctgggtggttatcgcactctaca
tattgtcaacgttgtcagatgtttactttggcatggccaagcccttactggactggatcatcatagga
atgttcaagcaggacttggtgaaagttgcactcgttgatcttgccatgtacctatcctcgtattttcctt
atttcttgcaggttgcatgcaaacggggtgatgtatcttggcatggtcttggatgggcaatacagg
gggtttacagcttggtgtttttgacgttctggacggtagttccgcaggagttggccatggatcttcct
tggattgcacgaattttcttgatcttgcattgcttggtgtttattatgaagatgcagtcgtatgggcatt
acaatggatacctttgggatgtgtatcaggaaggattggcctctgaggctgatctcagggaccttt
ctgagtatgatgaagatttccccctggatcacgtggaggttctagaacagagcttgtggtttgcca
aacacgagttggagtttcaatcgaatggaactgctgagaggaaggaccaccatcacctgtattc
gacgaaaaggatgtcaacaaaccaatacgtgtcttgcaagaagagggaattatcaagtttccgg
caaacatcaacttcaaggattatttcgagtacagtatgttcccaacgctagtctacacgttgagcttc
ccccgaactcgacagattagatggacgtatgtgttgcagaaggttttgggaacatttgccttagtgt
ttgccatgattatcgtcgccgaagagagtttctgccccttgatgcaagaagttgatcagtacacaa
aattgccaaccaaccaaaggttcccaaaatacttcgtcgttctttcccacttgatattaccgctcggc
aagcagtacttgctctcattcatcctcatctggaatgaaattctcaacggcatagcggagttaagca
ggtttggcgaccggcatttctacggcgcttggtggtcgagcgtcgattacatggactattcaagaa
aatggaacaccatcgtgcaccgattcctccgtcggcacgtttacaattcgagcattcacatcctcg
gtatttccaggacgcaagccgcgatagttacacttttgctttctgccacaatccacgaactcgttat
gtacgtcctatttggcaaattacgagggtacctattccttacgatgcttgtccagatccccatgacc
gtcacctccaagttcaacaaccgtgtttggggcaacatcatgttctggttgacgtatttatctggccc
cagcttggttagtgcgttgtatttactcttctag SEQ Acyl-CoA Sterol
MSDDEIAGIVIEIDDDVKSTSSFQEELVEVEMSNSSINESQTDE ID acyl transferase
SYRPEETSLHYRRKSHRTPSEESFLEITKNVNDPDLVSKIENL NO: (ARE1)
RGKVSQREDRLRKHYLHTSQDVKFLSRFNDIKFKLNSATILD 44 A.A. Seq
SDAFYKSEYFGVLTIFWVVIALYILSTLSDVYFGMAKPLLDW
IIIGMFKQDLVKVALVDLAMYLSSYFPYFLQVACKRGDVSW
HGLGWAIQGVYSLVFLTFWTVVPQELAMDLPWIARIFLILHC
LVFIMKMQSYGHYNGYLWDVYQEGLASEADLRDLSEYDED
FPLDHVEVLEQSLWFAKHELEFQSNGTAERKDHHHHVFDEK
DVNKPIRVLQEEGIIKFPANINFKDYFEYSMFPTLVYTLSFPRT
RQIRWTYVLQKVLGTFALVFAMIIVAEESFCPLMQEVDQYT
KLPTNQRFPKYFVVLSHLILPLGKQYLLSFILIWNEILNGIAEL
SRFGDRHFYGAWWSSVDYMDYSRKWNTIVHRFLRRHVYNS
SIHILGISRTQAAIVTLLLSATIHELVMYVLFGKLRGYLFLTML
VQIPMTVISKFNNRVWGNIMFWLTYLSGPSLVSALYLLF SEQ Acyl-coA Sterol
atgtccgacgacgagatagcaggaatagtcattgaaatcgacgatgacgtgaaatctacgtcttc
ID acyl transferase
gttccaggaagacctagtcgaggttgagatgtccaactcgtccattaacgaatcccagacggatg
NO: (ARE2)
agttgtcgtaccgtcctgaagaaatctcattgcattcgagaaggaagtcccacaagaccccgt- ca
45 Nuc. Seq
gatgagtcgttcctagagatcaccaagaacgtgaatgatccggatctagtctccaagattgagaa
cttaaggggcaaagtaagccaacgggaagacaggttgaggaaacactacctccacacatccca
ggacgtcaagttcttgtctcggttcaacgacatcaagttcaagctgaactccgcgacgattctaga
ttcggatgcgttttacaagagcgagcactttggagtcttgactatcttctgggtggttatcggactct
acataatgtcaacgttgtcagacatgtattttggcatggccaagcccttactggactggataatcat
aggaatgttcaagaaggatttgatgcaagttgcactcgttgatcttgtcatgtacttatcctcgtatttt
ccttatttcctacaggttgcatgcaagaccggagctatatcttggcatggtcttggatgggccatac
agggggtttacagcttggtgtttttaactttctgggcggtacttccgctggagctggccatggatctt
ccttggattgcacgagttttcttgatcttgcattgcttggtgtttattatgaagatgcaatcatatggac
attacaatggatacctttgggatgtatatcaggaaggattggtctcggaagctgatctcacggctgt
ttctgagtatgatgatgatttccccctggatcacggggaggttctagaacagagcttgtggttcgcc
aaacacgagttggagtttcaatctaatggaactacggagaggaaggatcaccatcatcatgtattc
gacgaaaaggatgtcaacaaaccaatgcgtgtcttgcaagaagagggaattatcaaatttccgg
caaacatcaatttcaaggattatttcgagtacagtatgttccccacgctagtctacacattgaacttc
cccagaattcgacatattagatgggcgtatgtgttgcagaaagttttgggaacatttgccttagtgtt
tgccatgattatcgtcgccgaagagagtttctgtcccttgatgcaagaagttgaacagtacacaag
attgccaaccaaccaaaggttctcaaagtacttcgtcgttctttcccacttgatattgcccctcggca
aacagtacttgctctcgtttatcctcatttggaacgaaattctcaacgggatagcggagttaagcag
gtttggggatcgccatttctacggcgcctggtggtcaagcgtcgactacatggactattcaagaaa
atggaacacgatcgtgcaccgattcctccgccggcacgtttacaattcgaccattcgcatcctcg
gtatttccaggacccaagccgcgataattacacttttgctttcagccacaatccacgaactcgttat
gtacatcctatttggaaaattacgagggtacctattccttacgatgcttgtccagatccccatgaca
gtcaccgccaagttcaacaaccgtttgtggggcaacatcatgttctggttgacgtatttatctggcc
ccagcttggttagtgcgttgtatttactcttctga SEQ Acyl-CoA Sterol
MSDDEIAGIVIEIDDDVKSTSSFQEDLVEVEMSNSSINESQTDE ID acyl transferase
LSYRPEEISLHSRRKSHKTPSDESFLEITKNVNDPDLVSKIENL NO: (ARE2)
RGKVSQREDRLRKHYLHTSQDVKFLSRFNDIKFKSNSATILD 46 A.A. Seq
SDAFYKSEHFGVLTIFWVVIGLYIMSTLSDMYFGMAKPLSD
WIIIGMFKKDLMQVALVDLVMYLSSYFPYFLQVACKTGAIS
WHGLGWAIQGVYSLVFLTFWAVLPSESAMDLPWIARVFLIL
HCLVFIMKMQSYGHYNGYLWDVYQEGLVSEADLTAVSEYD
DDFPSDHGEVLEQSLWFAKHELEFQSNGTTERKDHHHHVFD
EKDVNKPMRVLQEEGIIKFPANINFKDYFEYSMFPTLVYTLN
FPRIRHIRWAYVLQKVLGTFALVFAMIIVAEESECPLMQEVE
QYTRLPTNQRFSKYFVVLSHLILPLGKQYLLSFILIWNEILNGI
AELSRFGDRHFYGAWWSSVDYMDYSRKWNTIVHRFLRRHV
YNSTIRILGISRTQAAIITLLLSATIHELVMYILFGKLRGYLFLT
MLVQIPMTVTAKFNNRLWGNIMFWLTYLSGPSLVSALYLLF SEQ Diacylglycerol
atgactcaggactataaagacgatagtcctacgtccactgagttggacactaacatagaagaggt
ID acyltransferase
ggaaagcactgcaaccctagagtcggaactcagacagagaaaacagaccacggaaactccag NO:
(DGA1)
catcaaccccaccaccacctccacaacaacagcaggcgcataagaaagccctgaagaatggc 47
Nuc. Seq
aagaggaagagaccatttataaacgtggcgccgctcaacaccccgttggctcacaggctcgag
actttggctgttgtttggcactgtgtcagttcccgttctttatgtttttgttcttgcttacggtctccatg
gggttgcttgggtggttctttatcattttgccatatttcatttggtggtacggtttcgacttgcacactcc
atcgaatggtaaagttgtctatcgtgtgcgcaactcgttcaagaatttcatcatttgggactggtttgt
caagtatttcccgattgaagtgcacaagacggtcgagttggatcctacttttagcgaattgcctgtg
gaagagagcggcgacagttcggacgacgacgaacaagacttggtgtctgagcacagcagaac
tttggttgatcaaatcttcaagtttttcgggttgaagaaacgcttgaatgacacctccctgggcaaac
cagagacattcaagaatgtgcctacgggtccaaggtatatttttgggtaccacccacacggagtg
atttctatgggggcagtggggttgtttgccaacaacgccttgaggaacgaaccatatacgccaatt
tccaaatggttaaaaccattcttccacgacagctccaagggcgagagattgttccctggtattggc
aatatcttcccattgacgcttaccacacagtttgcgctcccattttaccgtgactacttgatggctttg
gggatcactagtgcatcggctaaaaacattagaagcttgatcaacaatggagacaactctgtgtgt
ctcgtcgttggcggtgcacaagaatcgttgttgaacaatatgattgccaagcacgccagagtcgg
gtacggttacaaagagagcctagatattcatggcgaccagtccgaagaagaagaagaagaaga
ggatgataccaagcagctagagaacccaagtcctaaacgtgaagtgcaattggtcttgaacaaa
cgtaaaggttttgtgaagttggctatcgaactaggaaatgtttccttggtgcctatttttgcattcgga
gaagctgatgtttacagattggcccagccagcaccaggctcgttcttgtacaagttccagcaatg
gatgaaggcaacttttcaattcaccatcccattgtttagtgctcgaggcgtgttcatctatgatttcgg
attgttgccattcagaaacccaataaacatttgcgtcggtagacccgtctacattccgcacaacgtc
ttgcaagaatacaagcaaaagcacccagaggagtttgccgaagaggaacctgccagtaccccg
atgaagaagtctggatctttcaccgatatgttcaaagctggtgaaaagaagcccaagacttcaagt
atcaagactaaaatcccacctgcattactagacaagtaccacaagctatacgtcgacgagttgaa
gaaggtctatgaagagaacaaggaaaggtttggctacggtgatgttgaattaaacattgtagaata
g SEQ Diacylglycerol MTQDYKDDSPTSTELDTNIEEVESTATLESELRQRKQTTETP ID
acyltransferase ASTPPPPPQQQQAHKKASKNGKRKRPFINVAPLNTPLAHRLE NO:
(DGA1) TLAVVWHCVSIPFFMFLFLLTVSMGLLGWFFIILPYFIWWYG 48 A.A. Seq
FDLHTPSNGKVVYRVRNSFKNFIIWDWFVKYFPIEVHKTVEL
DPTFSELPVEESCDSSDDDEQDLVSEHSRTLVDQIFKFFGLKK
RLNDTSSGKPETFKNVPTGPRYIFGYHPHGVISMGAVGLFAN
NALRNEPYTPISKWLKPFFHDSSKGERLFPGIGNIFPLTLTTQF
ALPFYRDYLMALGITSASAKNIRSLINNGDNSVCLVVGGAQE
SLLNNMIAKHARVGYGYKESLDIHGDQSEEEEEEEDDTKQL
ENPSPKREVQLVLNKRKGFVKLAIELGNVSLVPIFAFGEADV
YRLAQPAPGSFLYKFQQWMKATFQFTIPLFSARGVFIYDFGL
LPFRNPINICVGRPVYIPHNVLQEYKQKHPEEFAEEEPASTPM
KKSGSFTDMFKAGEKKPKTSSIKTKIPPALLDKYHKLYVDEL KKVYEENKERFGYGDVELNIVE
SEQ Diacylglycerol
atgtcgtctttaaagaacagaaaatccgcaagcgtcgccacaagcgatacagaagactcagaaa ID
acyltransferase
cagaggcagtatcctcctcaattgatcccaacggcaccatattgcgaccagtcctacatgacgaa
NO: (LRO1)
ccccaccacagccatcaccaccacaacataactagaccagtattggaggacgatggcagcatc 49
Nuc. Seq
ctggtgtccagaagatcgtcgatctccaaatccgacgacctgcaggcaaagcaaaagaagaag
aaacccaagaagqaagatcttggagtctcgtcgggtcatgtttatctttggtaccctcattgggttaat
ctttgcgtgggcgtttaccacagacacgcatcctttcaatggcgacttggagaagtttatcaacttt
gaccagctcaacgggatctttgacgactggaagaactggaaggatatcttgcccaacagcatcc
agacgtacttgcaggaatcgggcaagggcgaagataacgacgggttgcatggtctggccgatt
ccttctccgtcgggctccgcttgaaagcccagaagaacttcactgacaaccacaatgtcgtgttg
gttcctggtgtggtgagcacggggttggaatcgtggggaacaaccaccaccggtgattgtccat
ctatcggatacttcaggaagagattgtggggatcattttatatgttaaggacaatgattttggagaaa
acgtgctggttgaagcatatccagttggacgagaagacggggttggatcctcccaatattaaggt
ccgtgcggcgcagggtttcgaagcggcagatttctttatggctgggtactggatctggaacaaga
tcttgcagaacttggcggttattgggtacggaccaaataacatggtgagtgctagttatgactgga
gattggcttacattgacttggagagaagagatggatatttttcgaaacttaaagcgcagattgagtt
gaataacaagttgaacaacaagaagactgtgttgattggccactcgatggggacccagattatttt
ctactttttgaaatgggtcgaagccaccgggaaaccatactatggcaatggcggaccaaactgg
gtgaatgatcatattgagtcgattattgacatcagtgggtcgactttgggtacccccaagagtattc
ctgtgttgatctctggggaaatgaaagacaccgttcaattgaacgcgttggcggtttacgggttgg
agcaatttttcagcaggcgtgaaagagtcgatatgttgcgtacatttggtggcgttgccagtatgtt
acccaaggggggagacaagatatggggcaacttgacgcatgcgccagatgatccaatttccac
attcagtgatgacgaagttacggacagccacgaacctaaagatcgttcttttggtacgtttatccaa
ttcaagaaccaaactagcgacgctaagccatacagggagatcaccatggctgaaggtatcgatg
aattgttggacaaatcaccagactggtattccaagagagtccgtgagaactactcttacggcatta
cagacagcaaggcgcaattagagaagaacaacaatgaccacctgaagtggtcgaacccattag
aagctgccttgcctaaagcacccgacatgaagatctattgtttctacggagttggaaatcctaccg
aaagggcatacaagtatgtgactgccgataaaaaagccacgaaattggactacataatagacgc
cgacgatgccaatggagtcatattaggagacggagacggcactgtttcgttattaacccactcga
tgtgccatgagtgggccaagggagacaagtcgagatacaacccagccaactcgaaggttacca
ttgttgaaatcaagcacgagccagacagatttgatttacgaggcggcgccaagactgcggaaca
tgttgatattttggggagtgccgagttgaacgagttgattttgactgtggttagcgggaacgggga
cgagattgagaatagatatgtcagcaacttaaaagaaatagtagaggccataaatttataa SEQ
Diacylglycerol MSSLKNRKSASVATSDTEDSETEAVSSSIDPNGTILRPVLHDE ID
acyltransferase PHHSHHHHNITRPVLEDDGSISVSRRSSISKSDDSQAKQKKKK NO:
(LRO1) PKKKILESRRVMFIFGTLIGLIFAWAFTTDTHPFNGDLEKFINF 50 A.A. Seq
DQLNGIFDDWKNWKDILPNSIQTYLQESGKGEDNDGLHGSA
DSFSVGLRLKAQKNFTDNHNVVLVPGVVSTGLESWGTTTTG
DCPSIGYFRKRLWGSFYMLRTMILEKTCWLKHIQLDEKTGL
DPPNIKVRAAQGFEAADFFMAGYWIWNKILQNLAVIGYGPN
NMVSASYDWRLAYIDLERRDGYFSKLKAQIELNNKLNNKKT
VLIGHSMGTQIIFYFLKWVEATGKPYYGNGGPNWVNDHIESI
IDISGSTLGTPKSIPVLISGEMKDTVQLNALAVYGLEQFFSRRE
RVDMLRTFGGVASMLPKGGDKIWGNLTHAPDDPISTFSDDE
VTDSHEPKDRSFGTFIQFKNQTSDAKPYREITMAEGIDELLDK
SPDWYSKRVRENYSYGITDSKAQLEKNNNDHSKWSNPLEAA
LPKAPDMKIYCFYGVGNPTERAYKYVTADKKATKLDYIIDA
DDANGVILGDGDGTVSLLTHSMCHEWAKGDKSRYNPANSK
VTIVEIKHEPDRFDLRGGAKTAEHVDILGSAELNELILTVVSG NGDEIENRYVSNLKEIVEAINL
SEQ Thioesterase
atggtggctgctgcagcaacttctgcattcttccccgttccagccccgggaacctcccctaaacccggg
ID activity,
aagtccggcaactggccatcgagcttgagccctaccttcaagcccaagtcaatccccaatgctggattt
NO: Cuphea
caggttaaggcaaatgccagtgcccatcctaaggctaacggttctgcagtaaatctaaagtct-
ggcagc 51 lanceolata
ctcaacactcaggaggacacttcgtcgtcccctcctccccgggctttccttaaccagttgcctgattgga
(Nucleic Acid
gtatgcttctgactgcaatcacgaccgtcttcgtggcggcagagaagcagtggactatgcttgatagga
Seq)
aatctaagaggcctgacatgctcgtggactcggttgggttgaagagtattgttcgggatgggctcgtg-
t
ccagacagagttttttgattagatcttatgaaataggcgctgatcgaacagcctctatagagacgctgat
gaaccacttgcaggaaacatctatcaatcattgtaagagtttgggtcttctcaatgacggctttggtcgta
ctcctgggatgtgtaaaaacgacctcatttgggtgcttacaaaaatgcagatcatggtgaatcgctaccc
aacttggggcgatactgttgagatcaatacctggttctctcagtcggggaaaatcggtatggctagcgat
tggctaataagtgattgcaacacaggagaaattcttataagagcaacgagcgtgtgggctatgatgaat
caaaagacgagaagattctcaagacttccatacgaggttcgccaggagttaacacctcattttgtggact
ctcctcatgtcattgaagacaatgatcagaaattgcataagtttgatgtgaagactggtgattccattcgc
aagggtctaactccgaggtggaatgacttggatgtgaatcagcacgtaagcaacgtgaagtacattgg
gtggattctcgagagtatgccaatagaagttttggagacccaggagctatgctctctcaccgttgaatat
aggcgggaatgcggaatggacagtgtgctggagtccgtgactgctgtggatccctcagaaaatggag
gccggtctcagtacaagcaccttttgcggcttgaggatgggactgatatcgtgaagagcagaactgag
tggcgaccgaagaatgcaggaactaacggggcgatatcaacatcaacagcaaagacttcaaatgga
aactcggcctcttag SEQ CYP52A12,
MATQEIIDSVLPYLTKWYTVITAAVLVFLISTNIKNYVKAKKLKC ID ATCC20336
andidaDPPYLKDAGLTGISSLIAAIKAKNDGRLANFADEVFDEYP NO: (Amino Acid
NHTFYLSVAGALKIVMTVDPENIKAVLATQFTDFSLGTRHAHFA 52 Seq)
PLLGDGIFTLDGEGWKHSRAMLRPQFARDQIGHVKALEPHIQIM
AKQIKLNQGKTFDIQELFFRFTVDTATEFLFGESVHSLYDEKLGIP
TPNEIPGRENFAAAFNVSQHYLATRSYSQTFYFLTNPKEFRDCNA
KVHHLAKYFVNKALNFTPEELEEKSKSGYVFLYELVKQTRDPK
VLQDQLLNIMVAGRDTTAGLLSFALFELARHPEMWSKLREEIEV
NFGVGEDSRVEEITFEALKRCEYLKAILNETLRMYPSVPVNFRTA
TRDTTLPRGGGANGTDPIYIPKGSTVAYVVYKTHRLEEYYGKDA
NDFRPERWFEPSTKKLGWAYVPFNGGPRVCLGQQFALTEASYVI
TRLAQMFETVSSDPGLEYPPPKCIHLTMSHNDGVFVKM* SEQ CYP52A13,
MTVHDIIATYFTKWYVIVPLALIAYRVLDYFYGRYLMYKLGAK ID ATCC20336
PFFQKQTDGCFGFKAPLELLKKKSDGTLIDFTLQRIHDLDRPDIPT NO: (Amino Acid
FTFPVFSINLVNTLEPENIKAILATQFNDFSLGTRHSHFAPLLGDGI 53 Seq.)
FTLDGAGWKHSRSMLRPQFAREQISHVKLLEPHVQVFFKHVRK
AQGKTFDIQELFFRLTVDSATEFLFGESVESLRDESIGMSINALDF
DGKAGFADAFNYSQNYLASRAVMQQLYWVLNGKKFKECNAK
VHKFADYYVNKALDLTPEQLEKQDGYVFLYELVKQTRDKQVL
RDQLLNIMVAGRDTTAGLLSFVFFELARNPEVTNKLREEIEDKF
GLGENASVEDISFESLKSCEYLKAVLNETLRLYPSVPQNFRVATK
NTTLPRGGGKDGLSPVLVRKGQTVIYGVYAAHRNPAVYGKDAL
EFRPERWFEPETKKLGWAFLPFNGGPRICLGQQFALTEASYVTV
RLLQEFAHLSMDPDTEYPPKKMSHLTMSLFDGANIEMY* SEQ CYP52A14,
MTAQIIATYITKWYVIVPLALIAYRYLDYFYGRYLMYKLGAKP ID ATCC20336
FFQKQTDGYFGFKAPLELLKKKSDGTLIDFTLERIQALNRPDIPTF NO: (Amino Acid
TFPIFSINLISTLEPENIKAILATQFNDFSLGTRHSHFAPLLGDGIFT 54 Seq.)
LDGAGWKHSRSMLRPQFAREQISHVKLLEPHMQVFEKHVRKAQ
GKTFDIQELFFRLTVDSATEFLFGESVESLRDESIGMSINALDFDG
KAGFADAFNYSQNYLASRAVMQQLYWVLNGKKFKECNAKVH
KFADYYVSKALDLTPEQLEKQDGYVFLYELVKQTRDRQVLRDQ
LLNIMVAGRDTTAGLLSFVFFELARNPEVTNKLREEIEDKFGLGE
NARVEDISFESLKSCEYLKAVLNETLRLYPSVPQNFRVATKNTTL
PRGGGKDGLSPYLVRKGQTVMYGVYAAHRNPAVYGKDALEFR
PERWFEPETKKLGWAFLPFNGGPRICLGQQFALTEASYVTVRLL
QEFGHLSMDPNTEYPPRKMSHLTMSLFDGANIEMY* SEQ CYP52A15,
MSSSPSFAQEVLATTSPYIEYFLDNYTRWYYFIPLVLLSLNFISLL ID ATCC20336
HTRYLERRFHAKPLGNFVRDPTFGIATPLLLIYLKSKGTVMKFA NO: (Amino Acid
WGLWNNKYIVDPKYKTTGLRIVGLPLIETMDPENIKAVLATQF 55 Seq.)
NDFSLGTRHDFLYSLLGDGIFTLDGAGWKHSRTMLRPQFAREQ
VSHVKLLEPHVQVFFKHVRKHRGQTFDIQELFFRLTVDSNTEFLF
GESAESLRDESIGLTPTTKDFDGRRDFADAFNYSQTYQAYRFLL
QQMYWILNGSEFRKSIAVVHKFADHYVQKALELTDDDLQKQD
GYVFLYELAKQTRDPKVLRDQLLNILVAGRDTTAGLLSFVFYEL
SRNPEVFAKLREEVENRFGLGEEARVEEISFESLKSCEYLKAVIN
ETLRLYPSVPHNFRVATRNTTLPRGGGEDGYSPIVVKKGQVVM
YTVIATHRDPSIYGADADVFRPERWFEPETRKLGWAYVPFNGGP
RICLGQQFALTEASYVTVRLLQEFAHLSMDPDTEYPPKLQNTLT LSLFDGADVRMY* SEQ
CYP52A16, MSSSPSFAQEVLATTSPYIEYFLDNYTRWYYFIPLVLLSLNFISLL ID
ATCC20336 HTKYLERRFHAKPLGNVVLDPTFGIATPLILIYLKSKGTVMKFA NO: (Amino
Acid WSFWNNKYIVKDPKYKTTGLRIVGLPLIETIDPENIKAVLATQFN 56 Seq.)
DFSLGTRHDFLYSLLGDGIFTLDGAGWKHSRTMLRPQFAREQVS
HVKLLEPHVQVFEKHVRKHRGQTFDIQELFFRLTVDSATEFLFG
ESAESLRDDSVGLTPTTKDFEGkGDFADAFNYSQTYQAYRFLLQ
QMYWILNGAEFRKSIAIVHKFADHYVQKALELTDDDLQKQDGY
VFLYELAKQTRDPKVLRDQLLNILVAGDTTAGLLSFVFYELSR
NPEVTAKLREEVENRFGLGEEARVEEISFESLKSCEYLKAVINEA
LRLYPSVPHNFRVATRNTTLPRGGGKDGCSPIVVKKGQVVMYT
VIGTHRDPSIYGADADVFRPERWFEPETRKLGWAYVPFNGGPRI
CLGQQFALTEASYVTYRLLQEFGNLSSDPNAEYPPKLQNTLTLS LFDGADVRMF* SEQ
CYP52A17, MIEQLLEYWYVVVPVLYIIKQLLAYTKTRVLMKKLGAAPVTNK ID ATCC20336
LYDNAFGIVNGWKALQFKKEGRAQEYNDYKFDHSKNPSVGTY NO: (Amino Acid
VSILFGTRIVVTKDPENIKAILATQFGDFSLGKRHTLFKPLLGDGI 57 Seq.)
FTLDGEGWKHSRAMLRPQFAREQVAHVTSLEPHFQLLKKHILK
HKGEYFDIQELFFRFTVDSATEFLFGESVHSLKDESIGINQDDIDF
AGRKDFAESFNKAQEYLAIRTLVQTFYWLVNNKEFRDCTKSVH
KFTNYYVQKALDASPEELEKQSGYVFLYELVKQTRDPNVLRDQ
SLNILLAGRDTTAGLLSFAVFELARHPEIWAKLREEIEQQFGLGE
DSRVEEITFESLKRCEYLKAFLNETLRIYPSVPRNFRIATKNTTLP
RGGGSDGTSPILIQKGEAVSYGINSTHLDPVYYGPDAAEFRPER
WFEPSTKKLGWAYLPFNGGPRICLGQQFALTEAGYVLVRLVQE
FSHVRSDPDEVYPPKRLTNLTMCLQDGAIVKFD* SEQ CYP52A18,
MIEQILEYWYIVVPVLYIIKQLIAYSKTRYLMKQLGAAPITNQLY ID ATCC20336
DNVFGIVNGWKALQFKKEGRAQEYNDHKFDSSKNPSVGTYVSI NO: (Amino Acid
LFGTKIVVTKDPENIKAILATQFGDFSLGKRHALFKPLLGDGIFTL 58 Seq.)
DGEGWKHSRSMLRPQFATEQVAHYTSLEPHFQLLKKHILKHKG
EYFDIQELFFRFTVDSATEFLFGESVHSLKDETIGINQDDIDFAGR
KDFAESFNKAQEYLSIRILVQTFYWLINNKEFRDCTKSVHKFTNY
YVQKALDATPEELEKQGGYVFLYELVKQTRDPKVLRDQSLNILL
AGRDTTAGIISFAVFELARNPHIWAKLREEIEQQFGLGEDSRVE
EITFESLKRCEYLKAFLNETLRVYPSVPRNFRIATKNTTLPRGGGP
DGTQPILIQKGEGVSYGINSTHLDPVYYGPDAAEFRPERWFEPST
RKLGWAYLPFNGGPRICLGQQFALTEAGYVLVRLYQEFSHIRSD
PDEVYPPKRLTNLTMCLQDGAIVKFD* SEQ CYP52A19,
MLDQILHYWYIVLPLLAIINQIVAHVRTNYLMKKLGAKPFTHVQ ID ATCC20336
RDGWLGFKFGREFLKAKSAGRSVDLIISRFHDNEDTFSSYAFGN NO: (Amino Acid
HVVFTRDPENIKALLATQFGDFSLGSRVKFFKPLLGYGIFTLDAE 59 Seq.)
GWKHSRAMLRPQFAREQVAHVTSLEPHFQLLKKHILKHKGEYF
DIQELFFRFTVDSATEFLFGESVHSLKDEEIGYDTKDMSEERRRF
ADAFNKSQVYVATRVALQNLYWLYNNKEFKECNDIVHKFTNY
YVQKALDATPEELEKQGGYVFLYELVKQTRDPKVLRDQSLNILL
AGRDTTAGLLSFAVFELARNPHIWAKLREEIEQQFGLGEDSRVE
EITFESLKRCEYLKAVLNETLRLHPSVPRNARFAIKDTTLPRGGG
PNGKDPILIRKDEVVQYSISATQTNPAYYGADAADFRERWFEPS
TRNLGWAFLPFNGGPRICLGQQFALTEAGYVLVRLVQEFPNLSQ
DPETKYPPPRLAHLTMCLFDGAHVKMS* SEQ CYP52A20,
MLDQIFHYWYIVLPLLVIIKQIVAHARTNYLMKKLGAKPFTHVQ ID ATCC20336
LDGWFGFKFGREFLKAKSAGRQVDLIISRFHDNEDTFSSYAFGN NO: (Amino Acid
HVVFTRDPENIKALLATQFGDFSLGSRVKFFKPLLGYGIFTLDGE 60 Seq.)
GWKHSRAMLRPQFAREQVAHVTSLEPHFQLLKKHILKHKGEYF
DIQELFFRFTVDSATEFLFGESVHSLRDEEIGYDTKDMAEERRKF
ADAFNKSQVYLSTRVALQTLYWLVNNKEFKECNDIVHKFTNYY
VQKALDATPEELEKQGGYVFLYELAKQTKDPNVLRDQSLNILL
AGRDTTAGLLSFAVFELARNPHIWAKLREEIESHFGSGEDSRVEE
ITFESLKRCEYLKAVLNETLRLHPSYPRNARFAIKDTTLPRGGGP
NGKDPILIRKNEVVQYSISATQTNPAYYGADAADFRPERWFEPS
TRNLGWAYLPFNGGPRICLGQQFALTEAGYVLYRLVQEFPSLSQ
DPETEYPPPRLAHLTMCLFDGAYVKMQ* SEQ CYP52D2,
MAISSLLSWDVICandidaVFICandidaCandidaYFGYEYCYTKYLMH ID ATCC20336
KHGAREIENVINDGFFGFRLPLLLMRASNEGRLIEFSVKRFESAP NO: (Amino Acid
HPQNKTLVNRALSVPVILTKDMINIKAMISTQFDDFSLGLRLHQ 61 Seq.)
FAPLLGKGIFTLDGPEWKQSRSMLRPQFAKDRVSHISDLEPHFVL
LRKHIDGHNGDYFDIQELYFRFSMDVATGFLFGESVGSLKDEDA
RFSEAFNESQKYLATRATLHELYFLCDGFRFRQYNKVVRKFCSQ
CandidaHKALDVAPEDTSEYVFLRELVKHTRDPVVLQDQALNVL
LAGRDTTASLLSFATFELARNDHMWRKLREEVISTMGPSSDEIT
VAGLKSCRYLKAILNETLRLYPSVPRNARFATRNTTLPRGGGPD
GSFPILIRKGQPVGYFICATHLNEKVYGNDSHVFRPERWAALEG
KSLGWSYLPFNGGPRSCLGQQFAILEASYVLARLTQCYTTIQLRT
TEYPPKKLVHLTMSLLNGVYIRTRT* SEQ ADH1-1,
MHALFSKSVFLKYVSSPTTSAIPHSSEFIVPRSFYLRRSISPYLPHS ID Candida
SLFPSFSYSSSSVYTKKSFHTMSANIPKTQKAVVFEKNGGELEYK NO: (Amino Acid
DIPVPTPKANELLINVKYSGVCHTDLHAWKGDWPLATKLPLVG 62 Seq.)
GHEGAGVVVGMGENVKGWKIGDFAGIKWLNGSCMSCEFCQQG
AEPNCGEADLSGYTHDGSFEQYATADAVQAARIPAGTDLAEVA
PILCAGVTVYKALKTADLAAGQWVAISGAGGGLGSLAVQYAV
AMGLRVVAIDGGDEKGAFVKSLGAEAYIDFLKEKDIVSAVKKA
TDGGPHGAINVSVSEKAIDQSVEYVRPLGKVVLVGLPAGSKVTA
GVFEAVVKSIEIKGSYVGNRKDTAEAVDFFSRGLIKCPIKIVGLSE
LPQVFKLMEEGKILGRYVLDTSK SEQ NADPH
MALDKLDLYVIITLVVAIAAYFAKNQFLDQQQDTGFLNTDSGD ID cytochrome
GNSRDILQALKKNNKNTLLLFGSQTGTAEDYANKLSRELHSRFG NO: P450
LKTMVADFADYDFENFGDITEDILVFFIVATYGEGEPTDNADEF 63 reductase,
HTWLTEEADTLSTLKYTVFGLGNSTYEFFNAIGRKFDRLLGEKG CPR (Candida
GDRFAEYGEGDDGTGTLDEDFLAWKDNVFDSLKNDLNFEEKEL strain
KYEPNVKLTERDDLSGNDPDVSLGEPNVKYIKSEGVDLTKGPFD ATCC750)
HTHPFLARIVKTKELFTSEDRHCandidaHVEFDISESNLKYTTGDH (Amino Acid
LAIWPSNSDENIKQFAKCFGLEDKLDTVIELKAEDSTYSIPFPNPI Seq.)
TYGAVIRHHLEISGPVSRQFFLSIAGFAPDEETKKSFTRIGGDKQE
FASKVTRRKFNIADALLFASNNRPWSDVPFEFLIENVQHLTPRYY
SISSSSLSEKQTINVTAVVEAEEEADGRPVTGVVTNLLKNIEIEQN
KTGETPMVHYDLNGPRGKFSKFRLPVHVRRSNFKLPKNSTTPVI
LIGPGTGVAPLRGFVRERVQQVKNGVNVGKTVLFYGCRNSEQD
FLYKQEWSEYASVLGENFEMFNAFSRQDPTKKVYVQDKILENS
ALVDELLSSGAIIYVCGDASRMARDVQAAIAKIVAKSRDIHEDK AAELVKSWKVQNRYQEDVW
SEQ NADPH MALDKLDLYVIITLVVAVAAYFAKNQFLDQPQDTGFLNTDSGS ID cytochrome
NSRDVLSTLKKNNKNTLLLFGSQTGTAEDYANKLSRELHSRFGL NO: P450
KTMVADFADYDWDNFGDITEDILVFFIVATYGEGEPTDNADEFH 64 reductase A,
TWLTEEADTLSTLKYTVFGLGNSTYEFFNAIGRKFDRLLSEKGG CPRA
DRFAEYAEGDDGTGTLDEDFMAWKDNVFDALKNDLNFEEKEL (Candida
KYEPNVKLTERDDLSAADSQVSLGEPNKKYINSEGIDLTKGPFD strain
HTHPYLARITETRELFSSKDRHCIHVEFDISESNLKYTTGDHLAIW ATCC20336)
PSNSDENIKQFAKCFGLEDKLDTVIELKALDSTYTIPFPTPITYGA (Amino Acid
VIRHHLEISGPVSRQFFLSIAGFAPDEETKKAFTRLGGDKQEFAA Seq.)
KVTRRKFNIADALLYSSNNAPWSDVPFEFLIENVPHLTPRYYSISS
SSLSEKQLINVTAVVEAEEEADGRPVTGVVTNLLKNVEIVQNKT
GEKPLVHYDLSGPRGKFNKFKLPVHVRRSNFKLPKNSTTPVILIG
PGTGVAPLRGFVRERVQQVKNGVNVGKTLLFYGCRNSNEDFLY
KQEWAEYASVLGENFEMFNAFSRQDPSKKVYVQDKILENSQLV
HELLTEGAIIYVCGDASRMARDVQTTISKIVAKSREISEDKAAEL VKSWKVQNRYQEDVW SEQ
NADPH MALDKLDLYVIITLYVAVAAYFAKNQFLDQPQDTGFLNTDSGS ID cytochrome
NSRDVLSTLKKNNKNTLLLFGSQTGTAEDYANKLSRELHSRFGL NO: P450
KTMVADFADYDWDNFGDITEDILVFFIVATYGEGEPTDNADEFH 65 reductase B,
TWLTEEADTLSTLRYTVFGLGNSTYEFFNAIGRKFDRLLSEKGG CPRB
DRFAEYAEGDDGTGTLDEDFMAWKDNVFDALKNDLNFEEKEL (Candida
KYEPNVKLTERDDLSAADSQVSLGEPNKKYINSEGIDLTKGPFD strain
HTHPYLARITETRELFSSKERHCIHVEFDISESNLKYTTGDHLAIW ATCC20336)
PSNSDENIKQFAKCFGLEDKLDTVIELKALDSTYTIPFPTPITYGA (Amino Acid
VIRHHLEISGPVSRQFFLSIAGFAPDEETKKTFTRLGGDKQEFATK Seq.)
VTRRKFNIADALLYSSNNTPWSDVPFEFLIENIQHLTPRYYSISSS
SLSEKQLINVTAVVEAEEEADGRPVTGVVTNLLKNIEIAQNKTG
EKPLVHYDLSGPRGKFNKFKLPVHVRRSNFKLPKNSTTPVILIGP
GTGVAPLRGFVRERVQQVKNGVNVGKTLLFYGCRNSNEDFLYK
QEWAEYASVLGENFEMFNAFSRQDPSKKVYVQDKILENSQLVH
ELLTEGAIIYVCGDASRMARDVQTTISKIVAKSREISEDKAAELV KSWKVQNRYQEDVW SEQ
NADPH P450
atgacaattaaagaaatgcctcagccaaaaacgtttggagcttaaaaatttaccgttattaaacacag
ID cytochrome
ataaaccggttcaagctttgatgaaaattgcggatgaattaggagaaatctttaaattcgaggcgcctgg
NO: reductase
tcgtgtaacgcgctacttatcaagtcagcgtctaattaaagaagcatgcgatgaatcacgctttgataaa
66 (Bacillus
aacttaagtcaagcgcttaaatttgtacgtgattttgcaggagacgggttatttacaagctggacgcatga
megaterium)
aaaaaattggaaaaaagcgcataatatcttacttccaagcttcagtcagcaggcaatgaaaggctatcat
(Nucleic Acid
gcgatgatggtcgatatcgccgtgcagcttgttcaaaagtgggagcgtctaaatgcagatgagcatatt
Seq.)
gaagtaccggaagacatgacacgtttaacgcttgatacaattggtctttgcggctttaactatcgct-
ttaa
cagcttttaccgagatcagcctcatccatttattacaagtatggtccgtgcactggatgaagcaatgaac
aagctgcagcgagcaaatccagacgacccagcttatgatgaaaacaagcgccagtttcaagaagata
tcaaggtgatgaacgacctagtagataaaattattgcagatcgcaaagcaagcggtgaacaaagcgat
gatttattaacgcatatgctaaacgaaaagatccagaaacgggtgagccgcttgatgacgagaacatt
cgctatcaaattattacattcttaattgcgggacacgaaacaacaagtggtcttttatcatttgcgctgtat-
t
cttagtgaaaaatccacatgtattacaaaaagcagcagaagaagcagcacgagttctagtagatcctgt
tccaagctacaaacaagtcaaacagcttaaatatgtcggcatggtcttaaacgaagcgctgcgcttatg
gccaactgctcctgcgttttccctatatgcaaaagaagatacggtgcttggaggagaatatcctttagaa
aaaggcgacgaactaatggttctgattcctcagcttcaccgtgataaaacaatttggggagacgatgtg
gaagagttccgtccagagcgttttgaaaatccaagtgcgattccgcagcatgcgtttaaaccgtttggaa
acggtcagcgtgcgtgtatcggtcagcagttcgctcttcatgaagcaacgctggtacttggtatgatgct
aaaacactttgactttgaagatcatacaaactacgagctggatattaaagaaactttaacgttaaaacctg
aaggctttgtggtaaaagcaaaatcgaaaaaaattccgcttggcggtattccttcacctagcactgaaca
gtctgctaaaaaagtacgcaaaaaggcagaaaacgctcataatacgccgctgcttgtgctatacggttc
aaatatgggaacagctgaaggaacggcgcgtgatttagcagatattgcaatgagcaaaggatttgcac
cgcaggtcgcaacgcttgattcacacgccggaaatcttccgcgcgaaggagctgtattaattgtaacg
gcgtcttataacggtcatccgcctgataacgcaaagcaatttgtcgactggttagaccaagcgtctgctg
atgaagtaaaaggcgttcgctactccgtatttggatgcggcgataaaaactgggctactacgtatcaaa
aagtgcctgcttttatcgatgaaacgcttgccgctaaaggggcagaaaacatcgctgaccgcggtgaa
gcagatgcaagcgacgactttgaaggcacatatgaagaatggcgtgaacatatgtggagtgacgtag
cagcctactttaacctcgacattgaaaacagtgaagataataaatctactctttcacttcaatttgtcgaca
gcgccgcggatatgccgcttgcgaaaatgcacggtgcgttttcaacgaacgtcgtagcaagcaaaga
acttcaacagccaggcagtgcacgaagcacgcgacatcttgaaattgaacttccaaaagaagcttctta
tcaagaaggagatcatttaggtgttattcctcgcaactatgaaggaatagtaaaccgtgtaacagcaag
gttcggcctagatgcatcacagcaaatccgtctggaagcagaagaagaaaaattagctcatttgccact
cgctaaaacagtatccgtagaagagcttctgcaatacgtggagcttcaagatcctgttacgcgcacgca
gcttcgcgcaatggctgctaaaacggtctgcccgccgcataaagtagagcttgaagccttgcttgaaa
agcaagcctacaaagaacaagtgctggcaaaacgtttaacaatgcttgaactgcttgaaaaatacccg
gcgtgtgaaatgaaattcagcgaatttatcgcccttctgccaagcatacgcccgcgctattactcgatttc
ttcatcacctcgtgtcgatgaaaaacaagcaagcatcacggtcagcgttgtctcaggagaagcgtgga
gcggatatggagaatataaaggaattgcgtcgaactatcttgccgagctgcaagaaggagatacgatt
acgtgctttatttccacaccgcagtcagaatttacgctgccaaaagaccctgaaacgccgcttatcatgg
tcggaccgggaacaggcgtcgcgccgtttagaggctttgtgcaggcgcgcaaacagctaaaagaac
aaggacagtcacttggagaagcacatttatacttcggctgccgttcacctcatgaagactatctgtatca
agaagagcttgaaaacgcccaaagcgaaggcatcattacgcttcataccgctttttctcgcatgccaaa
tcagccgaaaacatacgttcagcacgtaatggaacaagacggcaagaaattgattgaacttcttgatca
aggagcgcacttctatatttgcggagacggaagccaaatggcacctgccgttgaagcaacgcttatga
aaagctatgctgacgttcaccaagtgagtgaagcagacgctcgcttatggctgcagcagctagaagaa
aaaggccgatacgcaaaagacgtgtgggctggg SEQ IDP2, Candida
MGEIQKITVKNPIVEMDGDEMTRIIWQFIKDKLILPYLNVDLKYY ID (Amino Acid
DLGIEYRDQTDDKVTTDAAEAILKYGVGVKCATITPDEARVKEF NO: Seq.)
NLKKMWLSPNGTLRNVLGGTVFREPIVIDNIPRIVPSWEKPIIIGR 67
HAFGDQYKATDVVIPAAGDLKLVFKPKDGGEVQEYPVYQFDGR
GVALSMYNTDASITDFAESSFQLAIERKLNLFSSTKNTILKKYDG
KFKDIFEGLYASKYKTKMDELGIWYEHRLIDDMVAQMLKSKGG
YIIAMKNYDGDVQSDIVAQGFGSLGLMTSVLVTPDGKAFESEAA
HGTVTRHYRQHQQGKETSTNSIASIYAWTRGLVQRGKLDDTPE
VVKFAEELEKAVIETVSKDNIMTKDLALTQGKTDRSSYVTTEEFI DGVANRLNKNLGY SEQ
IDP2, Candida
atgggcgaaattcagaaaataacagtcaagaacccaatcgtcgaaatggacggtgacgaaatgaccc
ID (Nucleic Acid
gtatcatctggcaattcatcaaagacaagttgatcttgccttatttgaacgttgacttgaaatactacgactt
NO: Seq.)
gggcatcgagtacagagaccagaccgacgacaaggtcaccaccgatgctgctgaagccatcttg-
aa 68
atacggtgtcggtgtcaagtgtgccaccatcaccccagacgaagccagagttaaagaattcaacttga
agaagatgtggctctctccaaacggtactttgagaaacgtccttggtggtactgtcttcagagaaccaat
tgtcattgacaacatcccaagaatcgtgcctagctgggaaaaaccaatcatcattggtagacacgctttc
ggcgaccaatacaaggccaccgacgtggttatcccagctgccggtgacttgaagttggtgttcaagcc
aaaagacggcggtgaagtgcaagaatacccagtctaccagttcgacggtcgaggtgtcgccttgagc
atgtacaacaccgacgcttcaatcactgatttcgctgaaagttccttccaattggccattgagcgtaaatt
gaacttgttttcttccaccaaaaacaccatcttgaagaaatacgacgggaaattcaaagacatattcgaa
ggcttgtacgccagcaaatacaagaccaagatggacgaattgggcatctggtacgagcacagattgat
tgacgatatggttgcacagatgttgaagtctaaaggtggttacatcatcgccatgaaaaactacgatggt
gatgtccaatccgacattgtcgcacaaggtttcggttccttgggtttgatgacctctgttttggttacccca
gacggcaaggcttttgaatccgaggctgcccacggtactgtcactagacattatagacaacaccaaca
aggtaaagagacctcgaccaactccattgcctccatctacgcctggaccagagggttggtccaaaga
ggtaagttggatgacactccggaagttgtcaagtttgctgaagagttggaaaaggctgtcattgagact
gtctccaaggacaacatcatgaccaaagatttggccttgacccaaggtaagaccgacagatcttcgtat
gtcacgactgaagaattcatcgatggtgttgctaatagattgaacaaaaacttgggctac SEQ
IDP3, Sc MSKIKVVHPIVEMDGDEQTRVIWKLIKEKLILPYLDVDLKYYDL ID (Amino Add
SIQERDRTNDQVTKDSSYATLKYGVAVKCATITPDEARMKEFNL NO: Seq.)
KEMWKSPNGTIRNILGGTVFREPIIIPKIPRLVPHWEKPIIIGRHAF 69
GDQYRATDIKIKKAGKLRLQFSSDDGKENIDLKVYEFPKSGGIA
MAMFNTNDSIKGFAKASFELALKRKLPLFFTTKNTILKNYDNQF
KQIFDNLFDKEYKEKFQALKITYEHRLIDDMVAQMLKSKGGFII
AMKNYDGDVQSDIVAQGFGSLGLMTSILITPDGKTFESEAAHGT
VTRHFRKHQRGEETSTNSIASIFAWTRAIIQRGKLDNTDDVIKFG
NLLEKATLDTVQVGGKMTKDLALMLGKTNRSSYVTTEEFIDEV AKRLQNMNLSSNEDKKGMCKL*
SEQ IDP3, Sc
atgagtaaaattaaagttgttcatcccatcgtggaaatggacggtgatgagcagacaagagttatttgga
ID (Nucleic Acid
aacttatcaaagaaaaattgatattgccatatttagatgtggatttaaaatactatgacctttcaatccaaga
NO: Seq.)
gcgtgataggactaatgatcaagtaacaaaggattcttcttatgctaccctaaaatatggggtt-
gctgtca 70
aatgtgccactataacacccgatgaggcaagaatgaaagaatttaaccttaaagaaatgtggaaatctc
caaatggaacaatcagaaacatcctaggtggaactgtatttagagaacccatcattattccaaaaatacc
tcgtctagtccctcactgggagaaacctataattataggccgtcatgcttttggtgaccaatatagggcta
ctgacatcaagattaaaaaagcaggcaaactaaggttacagtttagctcagatgacggtaaagaaaac
atcgatttaaaggtttatgaatttcctaaaagtggtgggatcgcaatggcaatgtttaatacaaatgattcc
attaaagggttcgcaaaggcatccttcgaattagctctcaaaagaaaactaccgttattctttacaaccaa
aaacactattctgaaaaattatgataatcagttcaaacaaattttcgataatttgttcgataaagaatataa-
g
gaaaagttcaggctttaaaaataacgtacgagcatcgtttgattgatgatatggtagcacagatgctaaa
atcaaagggcgggtttataatcgccatgaagaattatgatggcgatgtccagtctgacattgtggcaca
aggatttgggtctcttggtttaatgacgtccatattgattacacctgatggtaaaacgtttgaaagcgagg
ctgcccatggtacggtgaccagacattttagaaaacatcaaagaggcgaagaaacatcaacaaattca
atagcctcaatatttgcctggacaagggcaattatacaaagaggaaaattagacaatacagatgatgtta
taaaatttggaaacttactagaaaaggctactttggacacagttcaagtgggcggaaaaatgaccaagg
atttagcattgatgcttggaaagactaatagatcatcatatgtaaccacagaagagtttattgatgaagttg
ccaagaggcttcaaaacatgatgctcagctccaatgaagacaagaaaggtatgtgcaaactataa
SEQ GDP1, K1
atgcccgatatgacaaacgaatcttcttctaagccagctcaaattaacattggtatcaatggttttggtaga
ID (Nucleic Acid
atcggtagattggttctacgtgctgctttgacgcacccagaagttaaggtcagattaatcaataatccatc
NO: Seq.)
cacaacaccagaatacgctgcttatttgttcaaatacgattctactcacggcaagtatcgtggt-
gaagttg 71
aattcgacgatgaacgtatcatcattcaaaatgaccatgtttcggctcatatccctctatctcattttag-
gga
accagagcgtatcccatgggcttcctacaacgtcgattatgtaattgactcaaccggtgtcttcaaggaa
gtcgatacagcctctagacataaaggtgtcaaaaaagttatcattactgctccatcaaagaccgcgcca
atgtacgtctatggtgttaaccacgttaaatacaacccattgacggatcacgtggtctctaatgcctcctgt
actaccaactgtttggctccgttggttaaggctttggacgatgagttcggtatcgaagaagccttgatga
caactattcatgcaactactgcttctcaaaagactgtcgatggtaccagttctggtggtaaggactggag
aggcggtagatcttgccagggaaatatcattccttcatctactggtgcagctaaggctgtagggaaaat
cttgcctgaacttaatggtaagatcaccggtatgtctataagagtcccaacaattaatatttccctggttga
cttgacattccgtacagcaaagaaaacttcttacgatgacattatgaaggccctagaacaaagatctcgc
agcgatatgaagggtgttttgggtgttaccaaagacgccgttgtgtcctctgacttcacatccgattcacg
ttcatctattgttgatgccaaggccggtattgaattgaacgaccattttttcaaggtcctttcttggtatga-
ta
atgaatatggttactcttcaagagtggttgatttatccattttcatggctcaaaaggacttcgaagctggtg-
t ttaa SEQ GDP1, K1 MPDMTNESSSKPAQINIGINGFGRIGRLVLRAALTHPEVKVRLIN
ID (Amino Acid NPSTTPEYAAYLFKYDSTHGKYRGEVEFDDERIIIQNDHVSAHIP NO:
Seq.) LSHFREPERIPWASYNVDYVIDSTGVFKEVDTASRHKGVKKVIIT 72
APSKTAPMYVGVNHVKYNPLTDHVVSNASCTTNCLAPLVKAL
DDEFGIEEALMTTIHATTASQKTVDGTSSGGKDWRGGRSCQGNI
IPSSTGAAKAVGKILPELNGKITGMSIRVPTINISLVDLTFRTAKK
TSYDDIMKALEQRSRSDMKGVLGVTKDAVVSSDFTSDSRSSIVD
AKAGIELNDHFFKVLSWYDNEYGYSSRVVDLSIFMAQKDFEAG V SEQ ZWF1,
atgtcttatgattcattcggtgactacgtcactatcgtcgttttcggtgcttccggtgacttgg-
ccagcaaa ID Candida
aaaaccttccctgccttgtttggcttgtttagagaaaagcaattgccccaaccgtccagatca-
ttggcta NO: (Nucleic Acid
tgccagatcccatttgtccgacaaggacttcaaaaccaagatctcctcccacttcaagggcggcgacg
73 Seq.)
aaaaaaccaagcaagacttcttgaacttgtgtacttatatcagcgacccatacgacactgacgat-
ggtta
caagagattggaagccgccgctcaagaatacgaatccaagcacaacgtcaaggtccctgaaagattg
ttttacttggccttgcctccttctgtcttccacaccgtctgtgagcaagtcaagaagatcgtctaccctaag
gacggtaagctcagaatcatcattgaaaagccgttcggacgtgatttggccacctaccgtgaattgcaa
aagcaaatctccccattgttcaccgaagacgaactctacagaattgaccactacttgggtaaagaaatg
gtcaagaacttgttggttttgagattcggtaacgaattgttcagtgggatctggaacaacaagcacatca
cctcggtgcaaatctccttcaaggaacccttcggtaccgaaggtagaggtggctactttgacaacattg
gtatcatcagagatgtcatgcaaaaccacttgttgcaagtcttgaccttgttgaccatggaaagaccagt
ctcttttgacccagaagctgtcagagacgaaaaggtcaaggttttgaaagcttttgacaagattgacgtc
aacgacgttcttttgggacaatacgccaagtctgaggatggctccaagccaggttacttggatgactcc
accgtcaagccaaactccaaggctgtcacctacgccgctttcagagtcaacatccacaacgaaagatg
ggacggtgttccaattgttttgagagccggtaaggctttagacgaaggtaaagttgaaattagaatccaa
ttcaagccagttgccaaaggtatgtttaaggagatccaaagaaacgaattggttattagaatccaaccag
acgaagccatctacttgaagatcaactccaagatcccaggtatctccaccgaaacttccttgaccgactt
ggacttgacttactccaagcgttactccaaggacttctggatcccagaagcatacgaagccttgatcag
agactgttacttgggcaaccactccaactttgtcagagacgatgaattggaagttgcttggaagctcttc
accccattgttggaagccgttgaaaaagaagacgaagtcagcttgggaacctacccatacggatccaa
gggtcctaaagaattgagaaagtacttggtcgaccacggttacgtcttcaacgacccaggtacttacca
atggccattgaccaacaccgatgtcaaaggtaagatctaa SEQ ZWF1,
MSYDSFGDYVTIVVFGASGDEASKKTFPALFGLFREKQLPPTVQI ID Candida
IGYARSHLSDKDFKTKISSHFKGGDEKTKQDFLNLCTYISDPYDT NO (Amino Acid
DDGYKRLEAAAQEYESKHNVKVPERLFYLALPPSVFHTVCEQV 74 Seq.)
KKIVYPKDGKLRIIIEKPFGRDLATYRELQKQISPLFTEDELYRID
HYLGKEMVKNLLVLRFGNELFSGIWNNKHITSVQISFKEPFGTE
GRGGYFDNIGIIRDVMQNHLLQVLTLLTMERPVSFDPEAVRDEK
VKVLKAFDKIDVNDVLLGQYAKSEDGSKPGYLDDSTVKPNSKA
VTYAAFRVNIHNERWDGVPIVLRAGKALDEGKVEIRIQFKPVAK
GMFKEIQRNELVIRIQPDEAIYLKINSKIPGISTETSLTDLDLTYSK
RYSKDFWIPEAYEALIRDCYLGNHSNFVRDDELEVAWKLFTPLL
EAVEKEDEVSLGTYPYGSKGPKELRKYLVDHGYVFNDPGTYQ WPLTNTDVKGKI SEQ ZWF2,
atgtcttatgattcattcggtgactacgtcactatcgtcgtttcggtgcttccggtgacttggc-
cagaaaa ID Candida
aaaaccttccctgccttgtttggcttgtttagagaaaagcaattgcccccaaccgtccagatc-
attggcta NO: Nucleic Acid
tgccagatcccatttgtccgacaaggacttcaaaaccaagatctcctcccacttcaagggcggcgacg
75
aaaaaaccaagcaagacttcttgaacttgtgctcttatatgagtgacccatacgacaccgacgacggtt
acaagaaattggaagccaccgctcaagaatacgaatccaagcacaacgtaaaggtcccagaaagatt
gttctacttggccttgcctccttccgtcttccacaccgtctgtgagcaagtcaagaagctcgtctacccta
aggacggtaagctcagaatcatcattgaaaagccgtttggccgtgacttggccacctaccgtgaattgc
aaaagcaaatctccccattgttcaccgaagacgaagtctacagaatcgaccactacttgggtaaagaa
atggtcaagaacttgttggttttgagattcggtaatgaattgttcagtgggatctggaacaacaagcacat
cacctcggtgcaaatttccttcaaggaacccttcggtaccgaaggcagaggtggctactttgacaacat
tggtatcatcagagatgtcatgcaaaaccacttgttgcaagtcttgaccttgttgaccatggaaagacca
gtctcttttgacccagaagcagtcagagacgaaaaggtcaaagttttgaaagcttttgacaacattgacg
tcaatgacgttcttttgggacaatacgccaagtccgaggatggctccaagccaggttacttggatgactc
caccgtcaagccaaactccaaggctgtcacctacgccgctttcagagtcaacatccacaacgaaagat
gggacggtgttccaattgttttgagagccggtaaggctttagacgaaggtaaagttgaaattagaatcca
attcaagccagtcgctaaaggtatgtttaaggaaatccaaagaaacgaattggttattagaatccaacca
gacgaagccatctacttgaagatcaactccaagatcccaggtatctccaccgaaacctccttgaccgac
ttggacttgacttactccaagcgttactccaaagacttctggatcccagaagcatacgaagccttgatca
gagactgttacttgggcaaccactccaactttgtcagagacgatgaattggaagttgcttggaagctctt
caccccattgttggaagccgttgaaaaagaagacgaagtcagcttgggaacctacccatacggatcca
agggtcctaaagaattgagaaagtacttggtcgaccacggttacgtcttcaacgacccaggtacttacc
aatggccattgaccaacaccgatgtcaaaggtaagatctaa SEQ ZWF2,
MSYDSFGDYVTIVVFGASGDLARKKTFPALFGLFREKQLPPTVQI ID Candida
IGYARSHLSDKDFKTKISSHFKGGDEKTKQDFLNLCSYMSDPYD NO: (Amino Acid
TDDGYKKLEATAQEYESKHNVKVPERLFYLALPPSVFHTVCEQ 76 Seq.)
VKKLVYPKDGKLRIIEKPFGRDLATYRELQKQISPLFTEDEVYRI
DHYLGKEMVKNLLVLRFGNELFSGIWNNKHITSVQISFKEPFGT
EGRGGYFDNIGIIRDVMQNHLLQVLTLLTMERPVSFDPEAVRDE
KVKVLKAFDNIDVNDVLLGQYAKSEDGSKPGYLDDSTVKPNSK
AVTYAAFRVNIHNERWDGVPIVLRAGKALDEGKVEIRIQFKPVA
KGMFKEIQRNELVIRIQPDEAIYLKINSKIPGISTETSLTDLDLTYS
KRYSKDFWIPEAYEALIRDCYLGNHSNFVRDDELEVAWKLFTPL
LEAVEKEDEVSLGTYPYGSKGPKELRKYLVDHGYVFNDPGTYQ WPLTNTDVKGKI SEQ PGI1,
Candida
atgtccactttcaagttagccaccgaattgccagaatggaaaaaattggaacaaacctacaagtccgtg
ID Nucleic Acid
ggtgaaaagttcagcgtcagagatgccttcgctaacgacaagaacagattcgaagagttctcctggat
NO:
ctaccaaaactacgacgactccaagatcttgtttgatttctccaagaacttggtcaacaaggagatctt-
gg 77
accaattgatcaccttggccaaagaagctggtgtcgagaaattgagagacgctatgtttgctggtgatc
acatcaacaccaccgaagacagagccgtgtaccacgttgccttgagaaaccgtgccttgagaaaaat
gccagtcgacggtaaggacaccgccaaggaagttgacgacgtcttgcaacacatgaaggaattctcc
gactccatcagagacggctcttggaccggttacactggtaaggccatcaccgatgttgtcaacattggt
attggtggttccgacttgggtccagtcatggttactgaagccttgaaggcctacagcaagccaggcttg
aacgtccactttatctccaacattgacggtacccacacccacgaaaccttgaagaacttgaacccagaa
accaccttgttcttggttgcttccaagaccttcaccaccgctgaaaccatcaccaacgccacctccgcca
aaaactggttcttagctgccgctaaggatccaaagcacattgccaagcatttcgctgctttgtccaccaa
cgaagctgaggttgaaaaattcggtatcgacgtcaagaacatgtttggtttcgaaagttgggtcggtggt
cgttactctgtctggtccgccattggtttgtctgtcgccatctacattggtttcgaaaacttcaacgacttc-
tt
gaagggtggtgaagccatggacaacactttttgaccactcctttggaaaacaacatcccagtcattgg
tggtttgttgtctgtctggtacaacaacttctttggtgctcaaacccacttggttgttccattcgaccaata-
ct
tgcacagattcccagcttgacttgcaacaattgtctatggaatccaacggtaagtctgtcactagagccaa
cgtcttcaccaactaccaaaccggtaccatcttgtttggtgagccagccaccaacgcccaacactcctt
cttccaattggtgcaccaaggtaccaagttgatcccagctgacttcatcttggctgctcaatcccacaac
ccaattgaaaacaacttgcaccaaaagatgttggcctccaacttctttgctcaatccgaagctttgatggt
tggtaaggacgaagccaaggttaaagccgaaggtgccactggtggtttggttccacacaaggaattct
ctggtaacagaccaaccacctccatcttggctcaaaagatcaccccagccgctttgggttctttgattgc
ctactacgaacacgttactttcaccgaaggtgctatctggaacatcaactctttcgaccaatggggtgttg
aattgggtaaggttttggctaaggtcattggtaaggaattggatgacaagtccgctgttgttacccacgat
gcctccaccaacggtttgatcaaccaattcaagaaatgggaagcttga SEQ PGI1, Candida
MSTFKLATELPEWKKLEQTYKSVGEKFSVRDAFANDKNRFEEFS ID (Amino Acid
WIYQNYDDSKILFDFSKNLVNKEILDQLITLAKEAGVEKLRDAM NO: Seq.)
FAGDHINTTEDRAVYHVALRNRALRKMPVDGKDTAKEVDDVL 78
QHMKEFSDSIRDGSWTGYTGKAITDVVNIGIGGSDLGPVMVTEA
LKAYSKPGLNVHFISNIDGTHTHETLKNLNPETTLFLVASKTFTT
AETITNATSAKNWFLAAAKDPKHIAKHFAALSTNEAEVEKFGID
VKNMFGFESWVGGRYSVWSAIGLSVAIYIGFENFNDFLKGGEA
MDQHFLTTPLENNIPVIGGLLSVWYNNFFGAQTHLVVPFDQYLH
RFPAYLQQLSMESNGKSVTRANVFTNYQTGTILFGEPATNAQHS
FFQLVHQGTKLIPADFILAAQSHNPIENNLHQKMLASNFFAQSEA
LMVGKDEAKVKAEGATGGLVPHKEFSGNRPTTSILAQKITPAAL
GSLIAYYEHVTFTEGAIWNINSFDQWGVELGKVLAKVIGKELDD
KSAVVTHDASTNGLINQFKKWEA SEQ ACS2A,
atgccagcattattcaaagattctgcccaacacatacttgacaccatcaagtctgaactccca-
cttgatcc ID ATCC20336
cctcaaaaccgcatatgctgtgccgcttgaaaattcagccgaaccaggctactctgccatctacagaaa
NO: (Nucleic Acid
caaatactccatcgataagttaattgataccccataccccggcttggacaccttgtacaagttgtttgagg
79 Seq.)
ttgccactgaagcatacggtgataaaccatgtcttggtgccagagtcaagaacgccgatggcacc-
tttg
gagaatacaagttccaagactacaacaccattcaccaaagaagaaacaacttcgggtcaggtattttctt
tgtcttacagaacaacccatacaagaccgattctgaagcccactccaagttgaagtacgacccaacaa
gcaaggattccttcatcttgacaatcttcagtcacaaccgtcctgaatgggccttgtgtgatttgaccagt
attgcctattccatcaccaacaccgctttgtacgacactttgggtcccgacaccagtaagtacattttggg
tttgactgagtcgccaattgtcatctgttccaaggataagattagaggtcttattgacttgaagaagaaca
acccagacgaattgtccaacttgattgttttagtgtccatggatgacttgaccaccgctgatgcctctttga
agaactacggtagtgaacacaacgtcactgtttttgacatcaagcaggttgagaaattgggtgagatca
acccattggacccaatcgagccaaccccagacaccaatttcaccattactttcacctctggtaccaccg
gtgctaacccaaagggtgtcttgttgaaccacagaaacgctgtcgctggtgtcactttgtgctcagtag
atacgatggccatttcaacccaacagcttattctttccttcccttggcccacatttacgaaagagctagcat
ccagtttgcattgactatcggttccgccattggattcccacaaggtccatctccattgactttgattgaaga
tgccaaggtgttgcaaccagacggtttggctttggttcctagagtcttgaccaagttggaagctgccatc
agggcacaaactgtcaacaatgacgaaaaaccattggttaaatcgtctttggagctgctatcaacgcc
aagatggaagcacaaatgaaagaagaaaacgaaaacttcaacccaagtttcattgtctatgatcgtttgt
tgaacttgttgagaaagaaggttggtttgcaaaaagttacccaaatcagtaccggaagtgctccaatctc
tccatcaactattcaattcttgaaagcttccttgaatgtcggtatcttgcaaggttacggtttgagtgaatc-
a
tttgctggatgtatggcttcttccaaattcgaaccagcggcagccacttgtggtcctactggtgtcaccac
tgaagtcaagttgaaggatcttgaagaaatgggttacacttccaaggatgaaggtggtccaagaggtg
agttattattgagaggtccacaaatcttcaagggatatttcaagaaccctgaggaaactgccaaggccat
tgacgaagatggttggttccatactggtgatgttgccaagatcaacgacaaaggcagaatttccatcatt
gatagagcaaagaatttcttcaaattggctcaaggtgaatacgttaccccagagaaaatcgaaggtttgt
acttgtccaagttcccatacattgcccaattatttgtccatggtgactctaaggaatcgtacttggttggtg-
t
tgtcggattagacccagttgctggtaagcagtacatggagtcgagattccacgacaagatcatcaagg
aagaggacgttgttgagttcttcaagtccccaagaaacagaaagatcttagtgcaagacatgaacaag
ctgattgctgaccaattgcaaggttttgagaagttgcacaacatctacgttgactttgacccattgacggt
cgaaagaggcgtcattactccaaccatgaagatcagaagaccacttgctgccaagttcttccaggatca
aatcgatgctatgtacagcgaaggatcattggttagaaatggttctttgtag SEQ ACS2A,
MPALFKDSAQHILDTIKSELPLDPLKTAYAVPLENSAEPGYSAIY ID ATCC20336
RNKYSIDKLIDTPYPGLDTLYKLFEVATEAYGDKPCLGARVKNA NO: (Amino Acid
DGTFGEYKFQDYNTIHQRRNNFGSGIFFVLQNNPYKTDSEAHSK 80 Seq.)
LKYDPTSKDSFILTIFSFNRPEWALCDLTSIAYSITNTALYDTLGP
DTSKYILGLTESPIVICSKDKIRGLIDLKKNNPDELSNLIVLVSMD
DLTTADASLKNYGSEHNVTVFDIKQVEKLGEINPLDPIEPTPDTN
FTITFTSGTTGANPKGVLLNHRNAVAGVTFVLSRYDGHFNPTAY
SFLPLAHIYERASIQFALTIGSAIGFPQGPSPLTLIEDAKVLQPDGL
ALVPRVLTKLEAAIRAQTVNNDEKPLVKSVFGAAINAKMEAQM
KEENENFNPSFIVYDRLLNLLRKKVGLQKVTQISTGSAPISPSTIQ
FLKASLNVGILQGYGLSESFAGCMASSKFEPAAATCGPTGVTTE
VKLKDLEEMGYTSKDEGGPRGELLLRGPQIFKGYFKNPEETAKA
IDEDGWFHTGDVAKINDKGRISIIDRAKNFFKLAQGEYVTPEKIE
GLYLSKFPYIAQLFVHGDSKESYLVGVVGLDPVAGKQYMESRF
HDKIIKEEDVVEFFKSPRNRKILVQDMNKSIADQLQGFEKLHNIY
VDFDPLTVERGVITPTMKIRRPLAAKFFQDQIDAMYSEGSLVRN GSL* SEQ ACS2B,
atgacatcgacacagagtatcttctccggcgagaagtacactaaggaagaagcgttagcacaa-
ttacc ID ATCC20336
tttcgggagtgctgttgagaatgctgtcgctataaacgagccagttacaaaccccaagtattctgcaatct
NO: (Nucleic Acid
tcagaaatgcagcccatctcgaccgcatggttcagaacgtgcaccctgatttaaacacccactacaag
81 Seq.)
ctcttcaacaatgctgctgagatgtaccgtgaccgtccatgtcttggtaagcgtccatacaacta-
cacca
cacaccaactggatgattacttcctgcactggacgtatggtgaggtctttacgaagaagaataacattgg
tgctgggtttattcgcgcgttattggagaaccctttccttgacgtgacgttggagtcgcataggaagattg
tcaatcatttacgtgactggcccactttcggcatcaacaaatcaccaagggagaacttgaactatgagat
tgagaagaattgttcgttcattttgactatttttgccgtcaatcgtgctgaatggatcttgactgatttggc-
at
gcagttcgtatgcaatcacaaacaccgcgttgtatgatacattaggtcccgacgtgtctcagtatatcttg
aacttgactgaatcaccaattgtcgtttgcacccatgacaagatccaggttttgttgaacttgaaaagaaa
gtaccctgagcagaccaagaacttgatttctatcgtgtcaatggacccaattgatttagtcacgcaggga
acaatcgaacaagcttatgagttgggggtcacaattcaaggtttgaatcagattgagaaaattggtgttct
gaacccaattcaacaattggaaactggtacagaagctttgtttaccatttcattcacttcaggaactactg
gtagtaaacccaaaggagtgatgatttctcaaggtggtgctgctgcctacgtcacgtgggggttgagct
gttgtccacaggctaaacctggtgataaggcgtatattttcttgccgttgactcatttgtatgaaagagaaa
cttgtgcgtttgcctatagttctgggtactatttggggttcccgcagattaacctaggcaaaaagaaggtg
aatccttttgagaacatgcttaacgatttgagaatcttcaaaccaacatatatgtccatggttcctagatta-
tt
gaccaggttggaagcgttgatcaagagtaagatcaaggagttgccacaagctgaccaggacagagt
gaatggtataattgagatcaagataagggagcaaagcaaggctgacggcgccaaagggtttgacgc
aaccttggacaacgaccctacctacaaatcattagctaagtttgttgggtatgaaaacatgagatgggtt
cagactgcaagtgcaccaattgcccccaccacacttgtctacttgaaagcgtctttgaacatcggtgcc
agacaacaatacgggttgactgaaagtggggccgccatcacaagtaccggggagtacgaagcatcc
ccaggaggttgtggtgttgtattaccaactgggcagtgccgactctactccgtttccgagatggggtact
ccttggacaaattagaaggcgaggtgttgctccagggcccacagatgttcaaagggtactactacaac
tacgaggaaaccgagaatgcagttactgaagacggatggttccattcaggagacattgctcgggttga
ccccgcgacaggtcgcctcgacataattgaccgagtcaagcatttcttcaaattggctcagggagagta
catctccccagagcgtatcgagaacaggtacttgtcgtcgaacccagacatctgccagctctgggtgc
acggggactctaaggagcactacttgattggcatagtgggggtggagtacgaaaaaggattgaagttt
atcaatgaggagtttggatataacaagattgacatgcagccggatgatttgttggacattttgaactcggc
ggaggtgaaggcacggttcttgagcaagctaaatagactggttaaagataagttgaacgggtttgagat
cttgcataatatctttattgagtttgagccgttgacggtccagagagaagttgttactccgacattcaaaat-
t
agaagaccaatctgtcgtaagttcttcaaggcccagcttgatgcgatgtacgccgaggggtccttaatc
agcgctdccaagttgtag SEQ ACS2B,
MTSTQSIFSGEKYTKEEALAQLPFGSAVENAVAINEPVTNPKYSA ID ATCC20336
IFRNAAHLDRMVQNVHPDLNTHYKLFNNAAEMYRDRPCLGKR NO: (Amino Acid
PYNYTTHQSDDYFSHWTYGEVFTKKNNIGAGFIRALLENPFLDV 82 Seq.)
TLESHRKIVNHLRDWPTFGINKSPRENLNYEIEKNCSFILTIFAVN
RAEWILTDLACSSYAITNTALYDTLGPDVSQYILNLTESPIVVCT
HDKIQVLLNLKRKYPEQTKNLISIVSMDPIDLVTQGTIEQAYELG
VTIQGLNQIEKIGVSNPIQQLETGTEALFTISFTSGTTGSKPKGVMI
SQGGAAAYVTWGLSCCPQAKPGDKAYIFLPLTHLYERETCAFA
YSSGYYLGFPQINLGKKKVNPFENMLNDLRIFKPTYMSMVPRLL
TRLEALIKSKIKELPQADQDRVNGIIEIKIREQSKADGAKGFDATL
DNDPTYKSLAKFVGYENMRWVQTASASPIAPTTLVYLKASLNIG
ARQQYGLTESGAAITSTGEYEASPGGCGVVLPTGQCRLYSVSEM
GYSLDKLEGEVLLQGPQMFKGYYYNYEETENAVTEDGWFHSG
DIARVDPATGRLDIIDRVKHFFKLAQGEYISPERIENRYLSSNPDI
CQLWVHGDSKEHYLIGIVGVEYEKGLKFINEEFGYNKIDMQPDD
LLDILNSAEVKARFLSKLNRSVKDKLNGFEILHNIFIEFEPLTVQR
EVVTPTFKIRRPICRKFFKAQLDAMYAEGSLISAAKL* SEQ ACS2C,
atgtccaccttattcaacgagccacctgaacagatctaccagagtctcttgactcagtacaac-
aacccac ID ATCC20336
tcgattacgcatccagtgttgcactcccaaatacgcaagaaccgggatattcatctatctacaggaatgt
NO: (Nucleic Acid
gtttgatcccagtaagcttgttacctgtccacatcccgagttagacacgttgtacaaaatttttgagttcagt
83 Seq.)
gttattgtttacggagacaagccgttccttggccaccggatcaaaaacccagatggtacctttgg-
cgagt
acacgtttgaaacttacaagcaggtctatgaaagaagaaacaatcttggttctggtatctactacgtcttg
gaaaacagtccataccggacctcatccgaagctcatgcgaaattaaagtatgaccctaccaacgacaa
cccattcattttggcgcttttcagtcataatcgtccagaatgggcattgtgtgatgtcacaaccagtgctta-
t
ggtttcatcaacactgcattgtacagcaccttgggtccagataccagcagatacatcttgtctgtcaccg
attgtccaattgtagttgccacgaaggacaagattgaagggttgatcaacttgaagaagaaaaatccca
aagacttggtcaacttgattgttcttgtttcattagatgaacttaccgttgaagacgataagttgagatcat-
t
gggtcgtgaaaacaacattgtggtttatgctttgaaggaagttgaaagactcggtgcagctaacccattg
gcaccaattgctccaaccccagacactgtcttcacgatttcgtttacttcgggtaccagcggagcagca
cctaaaggtgttgtgttgacaaatagaatcttggcttgcggaatagcatcccattgttctctcgttgggttt
ggtcctgaccgtgtcgagtacagtttcttgcctttggctcatatctacgagagaatggtgcttcagtttgga
ataatagctggagtgaagatcgggtatccacagggtccttctccaacaaccttgtttgaagacatcaag
gttttgcaaccaacgatgttgtgtttggttcccagagtattcacgaagatagaagctgccatcaaggccc
agacagttgaaaatgaatcagatccagaactcagagctaagtttatcgaaataatcaacaagaaagtgg
agttgcaacaacaacaggactttacaaatccaagtctcccagaaggtgacaagctcttacaacaattgc
gtgagacccttggaatgggtaaacttcaattcatgaacactggatcagctccactgtcagaggagtctta
tcgttttatccaagctgtgatgaacatgcctaatgggttccgttgtggttacggtttgacagaaagcgctg
ctggtcttgcaatctccccaccatacgccaatgaattctcatgtgggcccatttcccagaccaccgagttt
agattgaaagatcttgttgatatgggttacacttcgaaagataaagagggagtaaggggtgaattgttatt
gagaggtcctcaaatcttctcttactactacaagaacccagaggagacggccaaggctatagacaagg
acggatggttccatactggtgatgtggcttctctcaccgaagcacatggaaacaggtttcagattatcga
cagagccaagaacttcttcaagttgtcccaaggagagtatgtgtctccagagaagattgaaaatgtgta
catggctcagtttccgttcatctcgcagttgtttgttcatggagattcattggagtcatatttggttggtgt-
cg
ttggaatcgacaaagcattggttgatccttatttgaagaaacgcttcaacgtggagttggacacaccagc
tgagatcttcaagttttttgagaacccacggaacagaaagacattgttgcaagacatgaacaaggcggt
tggtagtgagttgcaaggattcgaaaaattgcataatgtttttgttgattttgaaccattgacccttgagag-
a
ggagttattacgccaactgtcaagatcagaagagctaactgtgtcaacttctttaagcagcacatccaaa
gtatgtatggcgaaggatctttgctcaagaatagtaacttatag SEQ ACS2C,
MSTLFNEPPEQIYQSLLTQYNNPLDYASSVALPNTQEPGYSSIYR ID ATCC20336
NVFDPSKLVTCPHPELDTLYKIFEFSVIVYGDKPFLGHRIKNPDG NO: (Amino Acid
TFGEYTFETYKQVYERRNNLGSGIYYVLENSPYRTSSEAHAKLK 84 Seq.)
YDPTNDNPFILALFSHNRPEWALCDVTTSAYGFINTALYSTLGPD
TSRYILSVTDCPIVVATKDKIEGLINLKKKNPKDLVNLIVLVSLDE
LTVEDDKLRSLGRENNIVVYALKEVERLGAANPLAPIAPTPDTV
FTISFTSGTSGAAPKGVVLTNRILACGIASHCSLVGFGPDRVEYSF
LPLAHIYERMVLQFGIIAGVKIGYPQGPSPTTLFEDIKVLQPTMLC
LVPRVFTKIEAAIKAQTVENESDPELRAKFIEIINKKVELQQQQDF
TNPSLPEGDKLLQQLRETLGMGKLQFMNTGSAPSSPPSYRFIQA
VMNMPNGFRCGYGLTESAAGLAISPPYANEFSCGPISQTTEFRLK
DLVDMGYTSKDKEGVRGELLLRGPQIFSYYYKNPEETAKAIDK
DGWFHTGDVASLTEAHGNRFQIIDRAKNFFKLSQGEYVSPEKIE
NVYMAQFPFISQLFVHGDSLESYLVGVVGIDKALVDPYLKKRFN
VELDTPAEIFKFFENPRNRKTLLQDMNKAVGSELQGFEKLHNVF
VDFEPLTLERGVITPTVKIRRANCandidaNFFKQHIQSMYGEGSLL KNSNL* SEQ CAT2,
atgtttaactttaagttgtcgcaacaagtattaaagaattccaccaaatccattatgccaattt-
tgaaaaaac ID ATCC20336
cattctccaccagccacgcaaagggtgacttgttcaaataccagtcacaattacccaagttgcctgttcc
NO: (Nucleic Acid
tactttggaagaaaccgcatccaagtacctcaagaccgttgagccattcttgaaccaagagcaattgga
85 Seq.)
atccaccaaggccaaagtcgctgagtttgttagaccaggtggtgccggtgaagccttgcaagcca-
gat
tgaacaactttgccgccgacaaggacaactggttggctgaattttgggacgactatgcatacatgtctta
tagagatcctgttgttccatatgtttcttactttttcagtcacaaggatgtcaagaacatcattggccaaga-
c
caattgttgaaggccactttgattgcttactacactattgagttccaagaaaaggttttggacgaaagtttg
gacccagaagtcatcaagggtaacccattctgtatgaacgccttcaagtacatgttcaacaactcgaga
gttccagctgaaggctccgacatcacccaacactacaacggtgaagaaaaccaatttttcgttgtcatct
acaagaacaacttctacaaggttccaacccacaagaacggccaaagattgaccaagggtgaaatcta
cagctacttgcaagaaatcaagaacgatgccactccaaagggtctcggtttgggtgctttgacctcattg
aacagagacgaatggttgagtgcctacaacaacttgttgaagtccccaatcaacgaagcttccttggga
tccatctttgcttccagctttgtcattgccttggactccaacaacccagtcaccattgaagaaaaatccaa
gaactgctggcacggggacggtcaaaacagattctttgacaagcctttggaattcttcgtcagtgctaac
ggtaactctggtttccttggtgaacactccagaatggacgctaccccaaccgtgcaattgaacaacacc
atctacaagcaaatcttggaaaccaatccaaacgacttgattgttgaaattggttcttctgctccaagattc
ggcaatgctgaaatcttgcctttcgacatcaacccaaccaccagagccaacatcaaagacgctattgcc
aagtttgacgccaccattgctgcccacgacgaagaaatcttccaacactacgttacgtaagggattg
atcaagaagttcaaggtctccccagatgcctacgtgcaattgttgatgcaattggcatacttcaagtaca
ccggcaagatcagaccaacttatgaatccgccgccaccagaaagttcttgaagggtagaaccgaaac
cggtagaactgtctccaacgaatccaagaagtttgttgagacctggtccgatccaaaggccagcagcg
ccgacaaggttgccactttccaagctgctgctaagcaacacgttgcctatttgtctgctgctgccgatgg
taagggtgttgaccgtcacttgtttggtttgaagcaaatgattcaaccaggcgaaccaatccctgaaatct
tcactgacccaatcttcagctattctcaaacctggtacatttcttcttcccaagttccatctgaattcttcc-
aa
tcttggggttggtcgcaagtcattgatgacggtttcggtttggcttacttgatcaacaacgactggatcca
cgttcacatttcttgtaagagaggcaacggcttgcaatctgaccacttgaaatggtacttggttgaaagtg
ctaatgaaatgaaggatgttttgactaagggattattgactgatgctaagcctaagttgtaa SEQ
CAT2, MFNFKLSQQVLKNSTKSIMPILKKPFSTSHAKGDLFKYQSQLPKL ID ATCC20336
PVPTLEETASKYLKTVEPFLNQEQLESTKAKVAEFVRPGGAGEA NO: (Amino Acid
LQARLNNFAADKDNWLAEFWDDYAYMSYRDPVVPYVSYFFSH 86 Seq.)
KDVKNIIGQDQLLKATLIAYYTIEFQEKVLDESLDPEVIKGNPFC
MNAFKYMFNNSRVPAEGSDITQHYNGEENQFFVVIYKNNFYKV
PTHKNGQRLTKGEIYSYLQEIKNDATPKGLGLGALTSLNRDEWL
SAYNNLLKSPINEASLGSIFASSFVIALDSNNPVTIEEKSKNCWHG
DGQNRFFDKPLEFFVSANGNSGFLGEHSRMDATPTVQLNNTIYK
QILETNPNDLIVEIGSSAPRFGNAEILPFDINPTTRANIKDAIAKFD
ATIAAHDEEIFQHYGYGKGLIKKFKVSPDAYVQLLMQLAYFKYT
GKIRPTYESAATRKFLKGRTETGRTVSNESKKFVETWSDPKASS
ADKVATFQAAAKQHVAYLSAAADGKGVDRHLFGLKQMIQPGE
PIPEIFTDPIFSYSQTWYISSSQVPSEFFQSWGWSQVIDDGFGLAY
LINNDWIHVHISCKRGNGLQSDHLKWYLVESANEMKDVLTKGL LTDAKPKL* SEQ CROT
MPQEDYSDIVSEATEFVSSSLINLIQRHLEAVAEREEFSNYLNVV ID (Amino Acid
NNDMTPSIYGEIRGDILPRNPYLILEEDPYSKTINPPNQAQRAANL NO: Seq.)
INSSLKFIITLRNETLKPDVTPKHGNPLTMKCYRNLFGTTRIPEFE 87
EHNHDIKMRKYEHINDSRHILIIANNQFYTLEVITEYTEEEYQETK
SKHKIWFNDHELSLILQQIIDESKKVDAVKSINNSIGSITTQTLKH
WKLARLELEQSNLENIKKIDDALFVVILDSNAPETDGEKTAVISH
GTSVLSADNVQVGTCTSRWYDKLQLIVTQNSVAGVVWESMSM
DSTAILRFISDIYTDSVLKLAKNINGSEYTLFDSNIVFASSSISKPE
AVMIYFNKTKELQNIIHLSETRLADLINQHEYMTHRIKLDSYLTS
KFNLSVDSIMQVCFQIAYYSLYGRVVNTLEPITTRKFKDARTELI
PVQNEVLGNLVKLYITSASALEKFEAFKKCCELHTHQYHDAMIG
KGFERHLMTIIQVIKKPKAVVRLNELNSHLPPIPDLTKEPVTIPLL
LNPAIDKLLSPELLISNCGNPALRLFGIPPAIDQGFGIGYIIHRDKV
LITVCSKHRQTERFLDTFHRVVRDLKVNLRQKSNFLXXXXSVTR NKENTSCRGCESSMS SEQ
CROT
atgccgcaggaggactactcagacatcgtgagcgaggccaccgagtttgtctctagcagcttgat-
caa ID (Nucleic Acid
cttgatccagcggcacttggaagctgttgctgagagagaggagttctcaaactacttgaacgtcgtcaa
NO: Seq.)
caatgacatgactccgtccatctacggtgagattaggggtgacatattgcccagaaacccgtac-
cttatc 88
ttggaagaagacccgtacagcaagacgatcaacccaccgaaccaggcccagagagcagccaactt
gatcaactcgagtttgaagttcattattacgttgaggaacgagacgttgaagccagatgtcactccaaag
cacggaaacccgttgacgatgaagtgctacaggaacttgtttggcacgacgaggatcccggaatttga
agagcacaaccacgacatcaagatgagaaagtacgaacacatcaacgactcacgacacattttgatta
tcgccaacaaccagttctacacattggaagtgatcacggagtacacagaggaggagtaccaggagac
caagtccaagcacaagatctggttcaatgaccatgagctttcgttgatcttgcagcaaatcatagacgag
tccaagaaggttgacgccgtcaagtccatcaacaactccattgggtcgttgaccacgcagacgttgaa
gcattggaagttggctaggttggagttggagcagtcgaatctggaaaacatcaagaagattgacgatg
cgttgtttgtcgtgattttggactctaacgcaccagaaactgacggagagaaaacggcggtgatttccc
acggtacgtccgtgttgtcagcggacaacgtgcaggttggtqcctgtacctcgcgttggtacgataagt
tgcagttgattgtcacccagaactccgttgctggtgtggtgtgggaatccatgtcgatggacagtactgc
tattttgagattcatcagtgatatctacaccgactcggtgttgaagttggccaagaacatcaacgggtcc
gagtacactttgtttgactccaatattgtgtttgcgtcttcttcaatcagcaagccggaagccgtcatgatc-
t
atttcaacaaaacaaaggagttgcagaacattatccatctttcggaaaccagattggctgacttgatcaa
ccaacacgagtacatgacgcaccgtatcaagttggattcgtacttgacgagcaagtttaacctttccgtg
gactccatcatgcaggtgtgtttccagattgcttattactcgttgtacggtagagttgtcaacacgttggag
ccaatcaccaccagaaaattcaaggacgccagaaccgagttgatcccggttcagaatgaagttcttgg
caacttggtcaagctctacatcaccagcgccagtgcactggagaagtttgaagcattcaagaaatgttg
tgagttgcacacccaccagtaccacgatgccatgattggtaaaggtttcgaaagacacttgatgacgat
catccaagtgatcaagaaaccgaaagctgtggtcagattaaacgaacttaacagccacttgccgccaa
ttcctgacttgaccaaggaaccagtgaccattccgttattgctaaacccagccattgacaagttactgag
ccccgagttgttgatttccaactgcggtaatcctgcattgagattgtttggtatcccgccagccatcgacc
aagggtttggtattgggtacattatccaccgcgacaaggtgttgatcactgtttgttccaagcacagaca
aacggaaaggttcttggacactttccaccgtgttgttcgtgatttgaggtcaacttgagacaaaagagc
aactttttgnnnnnnnnnngatcagtgactcggaacaaagaaaacacgagttgcagaggttgcgaat
cgagcatgagt SEQ FAT1,
atgtcaggattagaaatagccgctgctgccatccttggtagtcagttattggaagccaaatatt-
taattgc ID ATCC20336
cgacgacgtgctgttagccaagacagtcgctgtcaatgccctcccatacttgtggaaagccagcagag
NO: (Nucleic Acid
gtaaggcatcatactggtactttttcgagcagtccgtgttcaagaacccaaacaacaaagcgttggcgtt
89 Seq.)
cccaagaccaagaaagaatgccccacccccaagaccgacgccgagggattccagatctacgacg
atcagtttgacctagaagaatacacctacaaggaattgtacgacatggttttgaagtactcatacatcttg
aagaacgagtacggcgtcactgccaacgacaccatcggtgtttcttgtatgaacaagccgcttttcattg
tcttgtggttggcattgtggaacattggtgccttgcctgcgttcttgaacttcaacaccaaggacaagcca
ttgatccactgtcttaagattgtcaacgcttcgcaagttttcgttgacccggactgtgattccccaatcaga
gataccgaggctcagatcagagaggaattgccacatgtgcaaataaactacattgacgagtttgccttg
tttgacagattgagactcaagtcgactccaaaacacagagccgaggacaagaccagaagaccaacc
gatactgactcctccgcttgtgcattgatttacacctcgggtaccaccggtttgccaaaagccggtatcat
gtcctggagaaaagccttcatggcctcggttttctttggccacatcatgaagattgactcgaaatcgaac
gtcttgaccgccatgcccttgtaccactccaccgcggccatgttggggttgtgtcctactttgattgtcgg
tggctgtgtctccgtgtcccagaaattctccgctacttcgttctggacccaggccagattatgtggtgcca
cccacgtgcaatacgtcggtgaggtctgtcgttacttgttgaactccaagcctcatccagaccaagaca
gacacaatgtcagaattgcctacggtaacgggttgcgtccagatatatggtctgagttcaagcgcagat
tccacattgaaggtatcggtgagttctacgccgccaccgagtcccctatcgccaccaccaacttgcagt
acggtgagtacggtgtcggcgcctgtcgtaagtacgggtccctcatcagcttgttattgtctacccagca
gaaattggccaagatggacccagaagacgagagtgaaatctacaaggaccccaagaccgggttctg
taccgaggccgcttacaacgagccaggtgagttgttgatgagaatcttgaaccctaacgacgtgcaga
aatccttccagggttattatggtaacaagtccgccaccaacagcaaaatcctcaccaatgttttcaaaaa
aggtgacgcgtggtacagatccggtgacttgttgaagatggacgaggacaaattgttgtactttgtcga
cagattaggtgacactttccgttggaagtccgaaaacgtctccgccaccgaggtcgagaacgaattga
tgggctccaaggccttgaagcagtccgtcgttgtcggtgtcaaggtgccaaaccacgaaggtagagc
ctgttttgccgtctgtgaagccaaggacgagttgagccatgaagaaatcttgaaattgattcactctcac
gtgaccaagtctttgcctgtgtatgctcaacctgcgttcatcaagattggcaccattgaggcttcgcacaa
ccacaaggttcctaagaaccaattcaagaaccaaaagttgccaaagggtgaagacggcaaggatttg
atctactggttgaatggcgacaagtaccaggagttgactgaagacgattggtctttgatttgtaccggta
aagccaaattgtag SEQ FAT1,
MSGLEIAAAAILGSQLLEAKYLIADDVSLAKTVAVNALPYLWK ID ATCC20336
ASRGKASYWYFFEQSVFKNPNNKALAFPRPRKNAPTPKTDAEG NO: (Amino Acid
FQIYDDQFDLEEYTYKELYDMVLKYSYILKNEYGVTANDTIGVS 90 Seq.)
CMNKPLFIVLWLALWNIGALPAFLNFNTKDKPLIHCLKIVNASQ
VFVDPDCDSPIRDTEAQIREELPHVQINYIDEFALFDRLRLKSTPK
HRAEDKTRRPTDTDSSACALIYTSGTTGLPKAGIMSWRKAFMAS
VFFGHIMKIDSKSNVLTAMPLYHSTAAMLGLCPTLIVGGCandida
SVSQKFSATSFWTQARLCGATHVQYVGEVCRYLLNSKPHPDQD
RHNVRIAYGNGLRPDIWSEFKRRFHIEGIGEFYAATESPIATTNLQ
YGEYGVGACRKYGSLISLLLSTQQKLAKMDPEDESEIYKDPKTG
FCTEAAYNEPGELLMRILNPNDVQKSFQGYYGNKSATNSKILTN
VFKKGDAWYRSGDLLKMDEDKLLYFVDRLGDTFRWKSENVSA
TEVENELMGSKALKQSVVVGVKVPNHEGRACFAVCEAKDELS
HEEILKLIHSHVTKSLPVYAQPAFIKIGTIEASHNHKVPKNQFKNQ
KLPKGEDGKDLIYWLNGDKYQELTEDDWSLICTGKAKL* SEQ PXA1,
atggtcaacatatcgaaattgacgggttataacaagcaggacatcaggaatgtggtgctattgc-
tacag ID ATCC20336
gagtttgtcaagacctacaaagacaacaagatcaaactcaactacctgagtagacctgtcatcttgttctt
NO: (Nucleic Acid
gagtaccttggttgcaactgccggtattggggtgtttttcaccttgagaagcatcgtcactaagtacaacg
91 Seq.)
agtacctactcaacaagagattgagacgcccaagtcttatcagacaatcctccaatatcttgaag-
aacg
gatcccgtgagatctttatccagaagggcaacggcaaagtaacaagaatcatcatcccaaaagcaaac
aacgaccagtatgccgccgacaagtatttgtataaagattttgcccgcaacgagcaaatattgcaacag
caaaagggaaggctcttcaattccagattcttgaaccagttgaccattatctggaagatcttgattccaaa
gttctactgccaaaacacttccttgttgttatcgcagtgcttctttttgattttcagaacatggttgtcctt-
gttg
attgccaagctagatggtcagattgtcaagaacttgattgctgcagacggtaggaagtttgcccgtgact
tgatttactttttgttgattgccttccctgcttcgtacaccaacgccgctatcaaatacttggaattgagat-
tg
gcgttaggattcagaactaatcttaccagatacatccatgacatgtacttggacaaaaccatgtcgtacta
caaagtgggattgaacggcgccgatatccaaaacatagaccagtacatcaccgaagatgtcaccaaa
ttctgtatgtcgttgtgttcgttgttttcctccatgggtaagccattcattgacttgatctttttcagtgtt-
tatttg
agagacaatttgggtactggtgccattattggcatttttgccaactattttgctaccgccatcatgttgaaa-
a
aggcaacaccaagattcggtaagttggctgccaaaagaacccacttggaaggtgtttatttcaaccaac
agttgaacataatgaccaacagtgaagagattgggttctacaaaggatcgaagattgagaagtccaag
cttgcggagaactttgacaagttgatgggtcacgtatcgagagaaatcaatttatcgtccagctatgccg
ctctagaagactacgtgcttaaatacacgtggctggcctggggttacatcttttctggtctacctgtgtttt-
t
ggatgtgcttttccctaaagaagacccaagtagtggccatattgctgatatagatgacgatgaccatgcc
catggacacgggcacaccggggaagagaccagctcaacaactgaaaacatgaagaccttcgtcacc
aacaagcgattattgttgagtcttgccgatgctggttccagattgatggttagtttgaaagaggtcaccac
gttgacaggtataacgaatagagtcttcaacatgttgactcagctccaccgtgtccatgatcctaaatttg
actacggtgacaagtatggtttgcctgatattcacgggacttatcaattgaactacgatggtttgagattg
gaacatgttccaattactgtgccaactgccgagggttcttactccacaccattgatcccagacctcactttt
gacatcaagggcaagaatttgttatttgttggtccaaacggttcgggcaaaacttctgttgccagggttct
tgcaggtctttggcccttgtatgccgggttagtgctgaaaccactggatttgttctttaacccacaaaaga
gttatttcaccaccggaagtttgcgtgaccaagttgtttaccctaatagatccgaaaacaccaccaacga
tcaaattttccacatcttacactgtgtacacttagaccatattgttaaacggtacggattgaaccagaactt
ggatttcgctaaaacattgagtggaggtgagaagcaaagattgagtttcgccagagtgttgtttaacaga
ccaagtattgtcattcttgatgattcgacgtcggcgttgtccccagatatggaagagttgatgtaccaggt
gttgcaagatcacaagatcaattacgtcacactttcaaatcgtccctctctcagtaagttccatgataaagt
atttgaaatataa SEQ PXA1,
MVNISKLTGYNKQDIRNVVLLLQEFVKTYKDNKIKLNYSSRPVI ID ATCC20336
LFLSTLVATAGIGVFFTLRSIVTKYNEYLLNKRLRRPSLIRQSSNIL NO: (Amino Acid
KNGSREIFIQKGNGKVTRIIIPKANNDQYAADKYLYKDFARNEQI 92 Seq.)
LQQQKGRLFNSRFLNQLTIIWKILIPKFYCQNTSLLLSQCFFLIFRT
WLSLLIAKLDGQIVKNLIAADGRKFARDLIYFLLIAFPASYTNAAI
KYLELRLALGFRTNLTRYIHDMYLDKTMSYYKVGLNGADIQNI
DQYITEDVTKFCMSLCSLFSSMGKPEIDLIFFSVYLRDNLGTGAII
GIFANYFATAIMLKKATPRFGKLAAKRTHLEGVYFNQQLNIMTN
SEEIGFYKGSKIEKSKLAENFDKLMGHVSREINLSSSYAALEDYV
LKYTWSAWGYIFSGLPVFLDVLFPKEDPSSGHIADIDDDDHAHG
HGHTGEETSSTTENMKTFVTNKRLLLSLADAGSRLMVSLKEVTT
LTGITNRVFNMLTQLHRVHDPKFDYGDKYGLPDIHGTYQLNYD
GLRLEHVPITVPTAEGSYSTPLIPDLTFDIKGKNLLFVGPNGSGKT
SVARVLAGLWPLYAGLVSKPSDLFFNPQKSYFTTGSLRDQVVYP
NRSENTTNDQIFHILHCandidaHLDHIVKRYGLNQNLDFAKTLSG
GEKQRLSFARVLFNRPSIVILDDSTSALSPDMEELMYQVLQDHKI
NYVTLSNRPSLSKFHDKVFEI* SEQ PXA2,
atgacagtggagaatgcaaaactacagaagaactcgttggcggttctgctcttgaaggtgtaca-
aatcc ID ATCC20336
aacagatcattattgttaaacacctcatacatcatattaatcattgctgccttcactggcgcaacgaatacc
NO: (Nucleic Acid
gggcgaggcacctcctccagatcatcggcaaaagtagagaccgatgaagaacaatcggttaaaaag
93 Seq.)
aaacaccccaagctctctagagagtccttccatagactaagaaaagcaatcttgccaactttctt-
tgatag
aactatagtttactttttcgccaacttgactttgttggtggtcagagcattattgacacttagagttgctac-
cc
ttgacggtcagcttgtgggggcattggtttcaagaagaataagggtgtttgccaagtacttgttgtactgg
atgcttcttggtatccccgctgctttgacaaatgccttgttgaactggaccaaactgaacttgagcaagag
cattagaatgaacttgaataataacatcatggaggaatacttgccagataacttggacccaaactattatt
cattgatccatttgactgataacaagattagagacccaaatcagagaataaccactgatactagtcgttg
agcgatgccttggcaagcttgcccggtcacatattgaagccaacgttggatatcatattgtgtgcgcaac
agttaagcaagagcggtgttggtaatggggaaggtacgttggcattaggtatattggcacacttctcaa
ccatgatcatccgtttcttctccccgccatttgccaagttggcggctgagagagctaaccttgaaggtca
gttgcgttccgcgcattccaagattgttgccaacagtgaagaaattgctttcttgggtggtcatgaccgtg
agttggatcacatcgaccactgctactatactttggagagattctcgaaaggcgaatattggaagcgag
ccatacacgaaatcacacaaacgtttattgtgaagtacttttggggtgttgcaggtttagtgttgtgttctg-
c
accggttttcattgccaaatacttgggtgagccggaagataagaatgttgctggtaatttcatcaccaaca
gaagattgttgatgagtgcctcggattccttggatcgtttaatctattctagaagatacttgttgcaagttg-
t
cggtcatgctaccagagtgtctgacttcttggacactttacatgaagtggaggagaagaagaagagaat
cacatcgaatgtgcagtttaacaacgacgagattactttcgatcatgttagattgatgactccaacggaa
gtgaccttgatcccagacttgaacttttccattaaaccaggtgaccatttgttgattgtggggccaaacgg
ttcaggtaagtcgtcgttgttcagaatgttgggtgggttgtggcccgttaggtttggtactattagaattcc
aaacacagagaacatgttctacttgccgcaaaaggcttaccttgttgaaggatcattcagagagcaaat
catttatccacacaacgtgactcaacagaagaagactgatcaacaattgaaagagatcttgaaggttttg
aaattggaagattactcagggcaattggatgaggttaagaaatggagcgaagaattgtccattggtgct
caacaaagattggctatggctagattgtactaccacgaacctaagtttgctgtcttggacgaatgtacttc
agctgtgtcaccagacatggaacaactcatgtaccaacacgcacaaggtttgggtatcacgcttttgtcc
gttgcccatagacctgcattgtggcacttccacaaatacttgttggaattcgacgggaagggtagttact
actttggtacgttggatgaaaagcacaaaatgaagttagaagaagaagaacgactcaagaaggagaa
tgaaaagaagagtgtcgccaagaagtag SEQ PXA2,
MTVENAKLQKNSLAVSLLKVYKSNRSLLLNTSYILLIIAAFTGAT ID ATCC20336
NTGRGTSSRSSAKVETDEEQSVKKKHPKLSRESFHRLRKAILPTF NO: (Amino Acid
FDRTIVYFFANLTLLVVRALLTERVATLDGQLVGALVSRRIRVF 94 Seq.)
AKYLLYWMLLGIPAALTNALLNWTKSNLSKSIRMNLNNNIMEE
YLPDNLDPNYYSLIHLTDNKIRDPNQRITTDTSRLSDALASLPGHI
LKPTLDIILCAQQLSKSGVGNGEGTLALGILAHFSTMIIRFFSPPFA
KLAAERANLEGQLRSAHSKIVANSEEIAFLGGHDRELDHIDHCY
YTLERFSKGEYWKRAIHEITQTFIVKYFWGVAGLVLCSAPVFIAK
YLGEPEDKNVAGNFITNTRRLLMSASDSLDRLIYSRRYLLQVVGH
ATRVSDFLDTLHEVEEKKKRITSNVQFNNDEITFDHVRLMTPTE
VTLIPDLNFSIKPGDHLLIVGPNGSGKSSLFRMLGGLWPVRFGTIR
IPNTENMFYLPQKAYLVEGSFREQIIYPHNVTQQKKTDQQLKEIL
KVLKLEDYSGQLDEVKKWSEELSIGAQQRLAMARLYYHEPKFA
VLDECTSAVSPDMEQLMYQHAQGLGITLLSVAHRPALWHFHKY
LLEFDGKGSYYFGTLDEKHKMKLEEEERLKKENEKKSVAKK* SEQ PEX11,
atggtcgccgattctttagtctaccacccaaccgtctccaaattagtcaagttcttggacaca-
accccaaa ID ATCC20336
gagggaaaaggtcttcagattattgtcctacttgtccagattcttgggctactacgcctacagaaagggc
NO: (Nucleic Acid
tactccaaggaaaccatcgcccttttcgccaacttgaaaggaaacttcacattcatcagaaaggccatg
95 Seq.)
agattcttgaagccaataaatcacttgcaattggcctccaaggcatacgacaacaagttgttgga-
ccca
gtcttgcagatcaccaccatcatcagaaacttggcctacgccggctacttgaccatcgacggtgtcatat
tcttcaagttgttgggtctcattgacgccaagaagttccctaacttggctacatacgcctccagattctggt
tgatcgggttgattgccggtttgatcaactccttgagaatcatctactccttgaaggactacgagcacca
ggagggcgacaaggagaaggagaccgacgctaaggctatccacactaagttgtacgccgctaaga
gaaaattggtctgggacttgttggatacttttattgctttgaactccttggacatcttgcatttcaccgagg-
g
tgacgtcgggttcgctggtactatcacctccctcttgggattggaagacttgtggaaggccacttaa
SEQ PEX11, MVADSLVYHPTVSKLVKFLDTTPKREKVFRLLSYLSRFLGYYAY ID
ATCC20336 RKGYSKETIALFANLKGNFTFIRKAMRFLKPINHLQLASKAYDN NO: (Amino
Acid KLLDPVLQITTIIRNLAYAGYLTIDGVIFFKLLGLIDAKKFPNLAT 96 Seq.)
YASRFWLIGLIAGLINSLRIIYSLKDYEHQEGDKEKETDAKAIHT
KLYAAKRKLVWDLLDTFIALNSLDILHFTEGDVGFAGTITSLLGL EDLWKAT* SEQ UGTA1
MFEDRLMAASGLDVPIILEDSPFFKTEMKPSTSYNITLLTIGSRGD ID (Amino Acid
VQPYMALGKGLVKEGHNVTIATHGEFGDWIKKSGLNFKEIAGN NO: Seq.)
PAELMSFMVTHNTMSVGFLKDAQKKFKSWIATLLTTSWKACQ 97
GSDILIESPSAMAGIHIAEALGIPYFRAFTMPWTRTRAYPHAFFVP
DQKKGGSYNYLTHVLFENIFWKGISGQVNKWRVQELDLPKTNL
YRLQQTRVPFLYNVSLTVLPPAVDFPDWIKVTGYWFLDEGSGD
YKPPEELVKFMSDAAADGKKIVYIGFGSIVVKDAKSLTKAVVEA
VKRADVRCILNKGWSDRLDKKQKDDIEVELPPEVYNSGAIPHD
WLFPRVDAAVHHGGSGTTGASLRAGTPTIIKPFFGDQFFYATRV
EDLGAGLGLKKLTAKTLANALVTVTEDLKIIEKAKRVSEQIKHE
HGVLSAIEAIYSELEYSRNLILVKDIYNQNYKRHHPDFRSQSGIQS
PVEPSDDEDEEDEEEEDSNEDDDEDNEDYEDESSDQRSKAS SEQ UGTA1
atgtttgaagacagattgatggctgcgtctggtttggatgttcctattatcttggaagactcgc-
cattcttta ID (Nucleic Acid
agacagaaatgaagccatcaacctcttacaatatcacgttgttgactattgggtcgcgaggtgatgtgca
NO: Seq.)
accatacatggctttaggtaaaggcttagttaaagagggccacaatgttactatcgctactcac-
ggaga 98
gttcggagactggatcaagaagagtggattgaactttaaagaaattgctggtaatcctgccgagttgatg
tcgtttatggttacccacaacaccatgtctgttggtttccttaaggatgcccgaagaagttcaaatcctg
gattgccacgttgttgactaccagttggaaggcgtgtcaaggttctgatatcttgattgaaagtccttcag
ccatggccggtatccacattgccgaagccttaggcattccttatttcagagcattcacgatgccatggac
tagaactagagcgtacccgcatgcattctttgtgcctgatcaaaagaagggtggttcatacaattatttga
cacatgtcttgtttgaaaatatcttctggaaagtatttctggacaggttaacaagtggagagtgcaagag
ttggatttgcccaagaccaacttgtacagattgcagcaaacgagagtgccgttcttgtacaatgtctcgct
cactgttttgccaccggccgttgacttccccgattggattaaagtcactggttactggtttttggatgaagg
ttctggtgattacaaacctcctgaagagcttgtcaagtttatgagtgatgccgctgctgatggcaaaaag
attgtctacattggatttggttccattgttgttaaggacgccaagtcgttgacgaaagctgttgttgaagct
gttaagcgtgccgacgtccgttgtatcttgaacaaaggttggtccgacaggctagacaagaagggtaa
ggacgatatcgaagttgagttaccgccagaagtgtacaactcgggtgctatccctcatgattggttgttc
ccacgtgtcgacgcagctgtacaccacggtggttctggtaccactggtgctagtttgcgtgctggtaca
cctactattatcaaaccattctttggagaccaatttttctacgccacgcgagttgaagatttaggcgccgg
gttggggttgaaaaaattgactgcaaaaacgttggcaaacgcgcttgttaccgttaccgaagacttgaa
gattattgaaaaggcaaagagagtcagcgagcaaatcaaacacgagcatggtgttctcagtgccatcg
caagcgtcaccacccagatttcagatcacaatctggtattcaatcgccagttgagcctagcgatgacga
ggacgaagaagacgaagaagaagaagacagcaatgaagacgacgatgaggacaatgaagattatg
aagacgagtcatcagaccagcggtcaaaggcttcatag SEQ IDP2, S.c
atgacaaagattaaggtagctaaccccattgtggaaatggacggcgatgagcaaacaagaataatctg
ID (Nucleic Acid
gcatttaatcagggacaagttagtcttgccctatcttgacgttgatttgaagtactacgatctttccgtgga
NO: Seq.)
gtatcgtgaccagactaatgatcaagtaactgtggattctgccaccgcgactttaaagtatgga-
gtagct 99
gtcaaatgcgcgactattacacccgatgaggcaagggtcgaggaatttcatttgaaaaagatgtggaa
atctccaaatggtactattagaaacattttgggtggtacagtgttcagagaacctattattatccctagaat-
t
ccaaggctagttcctcaatgggagaagcccatcatcattgggagacacgcattcggcgatcagtacaa
agctaccgatgtaatagtccctgaagaaggcgagttgaggcttgtttataaatccaagagcggaactca
tgatgtagatctgaaggtatttgactacccagaacatggtggggttgccatgatgatgtacaacactaca
gattcgatcgaagggtttgcgaaggcctcctttgaattggccattgaaaggaagttaccattatattccac
tactaagaatactattttgaagaagtatgatggtaaattcaaagatgttttcgaagccatgtatgctagaag
ttataaagagaagtttgaatcccttggcatctggtacgagcaccgtttaattgatgatatggtggcccaaa
tgttgaaatctaaaggtggatacataattgccatgaaaaattacgacggtgacgtagaatcagatattgtt
gcacaaggatttggctccttggggttaatgacatctgtgttgattaccccggacggtaaaacctttgaaa
gcgaagccgcccacggtacagtaacaagacattttagacagcatcagcaaggaaaggagacgtcaa
caaattccattgcatcaattttcgcgtggactagaggtattattcaaaggggtaaacttgataatactccag
atgtagttaagttcggccaaatattggaaagcgctacggtaaatacagtgcaagaagatggaatcatga
ctaaagatttggcgctcattctcggtaagtctgaaagatccgcttatgtcactaccgaggagttcattgac
gcggtggaatctagattgaaaaaagagttcgaggcagctgcattgtaa SEQ IDP2, S. c
MTKIKVANPIVEMDGDEQTRIIWHLIRDKLVLPYLDVDLKYYDL ID (Amino Acid
SVEYRDQTNDQVTVDSATATLKYGVAVKCATITPDEARVEEFH NO: Seq.)
LKKMWKSPNGTIRNILGGTVFREPIIIPRIPRLVPQWEKPIIIGRHA 100
FGDQYKATDVIVPEEGELRLVYKSKSGTHDVDLKVFDYPEHGG
VAMMMYNTTDSIEGFAKASFELAIERKLPLYSTTKNTILKKYDG
KFKDVFEAMYARSYKEKFESLGIWYEHRLIDDMVAQMLKSKG
GYIIAMKNYDGDVESDIVAQGFGSLGLMTSVLITPDGKTFESEA
AHGTVTRHFRQHQQGKETSTNSIASIFAWTRGIIQRGKLDNTPDV
VKFGQILESATVNTVQEDGIMTKDLALILGKSERSAYVTTEEFID AVESRLKKEFEAAAL SEQ
MAE1 (non- MWPIQQSRLYSSNTRSHKATTTRENTFQKPYSDEEVTKTPVGSR ID
mitochondrial), ARKIFEAPHPHATRLTVEGAIECPLESFQLLNSPLFNKGSAFTQEE NO:
Sc.(Amino REAFNLEALLPPQVNTLDEQLERSYKQLCYLKTPLAKNDFMTSL 101 Acid
Seq.) RVQNKVLYFALIRRHIKELVPIIYTPTEGDAIAAYSHRFRKPEGVF
LDITEPDSIECRLATYGGDKDVDYIVVSDSEGILGIGDQGIGGVRI
AISKLALMTLCGGIHPGRVLPVCLDVGTNNKKLARDELYMGNK
FSRIRGKQYDDFLEKFIKAVKKVYPSAVLHFEDFGVKNARRLLE
KYRYELPSFNDDIQGTGAVVMASLIAALKHTNRDLKDTRVLIYG
AGSAGLGIADQIVNHMVTHGVDKEEARKKIFLMDRRGLILQSYE
ANSTPAQHVYAKSDAEWAGINTRSLHDVVENVKPTCLVGCSTQ
AGAFTQDVVEEMHKHNPRPIIFPLSNPTRLHEAVPADLMKWTN
NNALVATGSPFPPVDGYRISENNNCYSFPGIGLGAVLSRATTITD
KMISAAVDQLAELSPLREGDSRPGLLPGLDTITNTSARLATAVIL
QALEEGTARIEQEQVPGGAPGETVKVPRDFDECLQWVKAQMW EPVYRPMKVQHDPSVHTNQL SEQ
MAE1 (non- ATGTGGCCTATTCAGCAATCGCGTTTTATATTCTTCTAACACTA ID
mitochondrial), GATCGCATAAAGCTACCACAACAAGAGAAAATACTTTCCAAA NO: Sc
(Nucleic AGCCATACAGCGACGAGGAGGTCACTAAAACACCCGTCGGTT 102 Acid Seq.)
CTCGCGCCAGAAAGATCTTCGAAGCTCCTCACCCACATGCCA
CTCGTTTGACTGTAGAAGGTGCCATAGAATGTCCCTTGGAGA
GCTTTCAACTTTTAAACTCTCCTTTATTTAACAAGGGTTCTGC
ATTTACACAAGAAGAAAGGGAAGCGTTTAATTTAGAAGCATT
GCTACCACCACAAGTGAACACTTTGGACGAACAACTGGAAAG
AAGCTACAAGCAGTTATGCTATTTGAAGACGCCCTTGGCCAA
AAACGACTTCATGACGTCTTTGAGAGTACAGAACAAAGTCCT
ATATTTTGCATTAATAAGGAGACATATCAAGGAATTAGTTCC
TATCATTTACACCCCAACCGAAGGTGATGCTATIGCTGCCTAT
TCCCACAGGTTCAGAAAGCCAGAAGGTGTGTTTTTAGACATT
ACCGAACCTGATTCCATCGAATGTAGATTGGCTACATACGGT
GGAGACAAAGATGTAGACTACATCGTTGTGTCGGATTCGGAA
GGTATTCTGGGAATTGGTGACCAAGGTATCGGTGGTGTACGT
ATTGCTATCTCCAAATTGGCATTGATGACGCTGTGCGGTGGTA
TTCATCCCGGCCGTGTGCTACCTGTGTGTTTGGACGTCGGTAC
TAACAACAAGAAACTAGCCCGTGACGAATTGTACATGGGTAA
CAAGTTCTCCAGAATCAGGGGTAAGCAATATGACGACTTCTT
GGAAAAATTCATCAAGGCCGTTAAGAAAGTGTATCCAAGCGC
CGTTCTGCATTTCGAAGATTTCGGTGTTAAGAACGCTAGAAG
ATTACTAGAAAAGTACAGGTACGAATTGCCATCATTCAACGA
TGACATTCAGGGCACCGGTGCCGTCGTGATGGCCTCGTTGAT
TGCTGCTTTGAAACATACCAACAGAGACTTGAAAGACACCAG
AGTGCTTATTTACGGTGCCGGGTCTGCGGGCCTCGGTATCGC
AGATCAAATTGTGAATCATATGGTCACGACGGCGTTGACAA
GGAAGAAGCGCGCAAGAAAATCTTCTTGATGGACAGACGTG
GGTTAATTCTACAATCTTACGAGGCTAACTCCACTCCCGCCCA
ACACGTATACGCTAAGAGTGATGCGGAATGGGCTGGTATCAA
CACCCGCTCTTTACATGATGTGGTGGAGAACGTCAAACCAAC
GTGTTTGGTTGGCTGCTCCACACAAGCAGGCGCATTCACTCA
AGATGTCGTAGAAGAAATGCACAAGCACAATCCTAGACCGAT
CATTTTCCCATTATOCAACCCTACTAGACTACACGAAGCCGTT
CCTGCCGATTIAATGAAGTGGACCAACAAGAACGCTCTTGTA
GCTACCGGATCTCCTTTCCCACCTGTTGATGGTTACCGTATCT
CGGAGAACAACAATTGTTACTCTTTCCCAGGTATCGGTTTAG
GTGCCGTACTATCGCGTGCCACCACCATCACAGACAAGATGA
TCTCCGCTGCAGTGGACCAACTAGCCGAATTGTCGCCACTAA
GAGAGGGCGACTCGAGACCTGGCTTGCTACCCGGCCTGGACA
CCATCACCAACACTTCTGCGCGTCTAGCTACCGCTGTGATCTT
GCAAGCACTCGAGGAOGGAACCGCCCGTATCGAGCAAGAAC
AAGTACCGGGAGGAGCTCCCGGCGAAACTGTCAAGGTTCCTC
GTGACTTTGACGAATGTTTACAGTGGGTCAAAGCCCAAATGT
GGGAGCCTGTGTACAQACCTATGATCAAGGTCCAACATGACC
CATCGGTGCACACCAACCAATTGTAG SEQ MAE1,
MLKFNKISARFVSSTATASATSGEMRTVKTPVGIKAAIESLKPKA ID Candida
TRVSMDGPVECPLTDFALLNSPQFNKGSAFSLEERKSFKLTGLLP NO: (Amino Acid
SQVNTLDEQVERAYRQFTYLKTPLAKNDFCTSMRLQNKVLYVE 103 Seq.)
LVRRNIREMLPIIYTPTEGDAIASYSDRFRKPEGCFLDINDPDNID
ERLAAYGENKDIDYIVMSDGEGIXXXSDRFRKPEGCFLDINDPD
NIDERLAAYGENKDIDYIVMSDGEGILGIGDQGVGGIRTAIAKLG
LMTLCGGIHPARVLPITLDVGTNNDRLLNDDLYMGNKFPRVRG
ERYWDFVDKVIHAITKRFPSAVMHYEDFGVTTGRDMLHKYRTA
LPSFNDDIQGTGAVVMASITAALKFSNRSLKDIEVLIYGAGSAGL
GIADQITNHLVSHGATPEQARSRIHCMDRYGLITTESNNASPAQ
MNYADKASDWEGVDTSSLLACandidaEKVKPTVLVGCSTQAGA
FTEEVVKTMYKYNPQPIIFPLSNPTRLHEAVPADLMKWTDNNAL
IATGSPFEPVDGYYISENNNCFTFPGIGLGAVLSRCSTISDTMISA
AVDRLASMSPKMENPKNGLLPRLEEIDEVSAHVATAVILQSLKE
GTARVESEKKPDGGYVEVPRDYDDCLKWVQSQMWKPVYRPYI KVEYVSNIHTYQY SEQ MAE1,
atgctcaaattcaataaaatactggccagattcgtctcctccacggccaccgcatccgccacgt-
caggg ID Candida
gaaatgcgtaccgtcaagaccccagtggggatcaaggcggccatcgaatcattaaaaccaaaa-
gcta NO: (Nucleic Acid
ctagagtctccatggacggacctgtcgaatgcccattgaccgatttcgccttgttgaactcccctcaattc
104 Seq.)
aacaaaggttcggcattttctttggaagaaaggaaaagtttcaagttgaccgggctcctccctt-
ctcaagt
caacactttggtgaacaggttgaaagagcctatagacaattcacatacttgaagaccccattggccaa
gaacgatttctgcacgtctatgagattgcagaacaaagtgctttactacgagttggttagaagaaatatcc
gtgagatgttgcccatcatctacaccccaaccgaaggggacgccatcgccagttattccgacaggttc
agaaaaccagagggctgtttcttggatatcaacgaccccgacaacatcgatgagagattagctgcctat
ggggagaacaaagacatagattacattgtcatgagtgacggagaaggtatcnnnnnnnnctccgac
aggttcagaaaaccagagggctgcttcttggacatcaatgacccagacaacatcgacgagagattgg
ctgcctatggggagaacaaagacatagattacattgtcatgagtgacggagaaggtatcctcggtattg
gagaccaaggcgtcggtggtatcagaattgccattgctaaattggggttgatgaccctttgtggtggtat
tcacccggccagagttttgcccatcactttggatgttggtacaaataacgacaggttgttgaatgatgattt
gtacatgggcaacaagttccctagagtcagaggagaaagatactgggactttgtcgataaggtcatac
acgcaattacgaaacggttcccaagtgccgtgatgcattacgaagatttcggagtcacaactggtagg
gacatgttgcacaagtaccgtacggctcttccttctttcaacgacgacatccaaggtaccggtgcagttg
tcatggcatcgatcacagctgccttgaagttctccaaccgtagcctaaaggacatcgaggttttgatttac
ggtgccggctcagctggtttaggtattgctgaccagatcaccaaccacttggtcagccacggcgctact
ccagaacaagccagatctaggatccattgtatggaccgttatgggttgatcacaactgaatccaacaac
gccagtcctgctcaaatgaactacgccgacaaggcatctgattgggaaggtgtcgatacctcgagtct
acttgcctgtgttgagaaagtcaaaccaactgtcttggttgggtgttccactcaggcaggtgcattcacc
gaagaggttgtcaaaaccatgtacaagtacaacccacagccaattattttcccattgtccaaccctacca
gattgcatgaagccgtgccggctgatttgatgaaatggaccgacaacaacgcgttgattgccaccggtt
ctccatttgaacctgtcgatggctactacatttccgaaaacaacaactgtttcaccttcccaggtattgggt
tgggtgctgtcttgtccagatgtagcaccatttcggataccatgatttctgccgccgttgatagattggctt
cgatgtcgccaaagatggagagaacccaaagaacggattgttgcctagattggaagaaatcgacgaagt
cagtgcccatgttgccacggctgttatcttgcaatctttgaaggaaggcaccgctagagtcgaaagcga
gaagaagccagacggtggttacgttgaagttccaagagactatgatgattgtcttaagtgggtgcaatc
acaaatgtggaagccagtgtacagaccatacatcaaggttgagtacgtttcgaatattcacacctatcaa
tat SEQ: MAE1, Sc MLRTRLSVSVAARSQLTRSLTASRTAPLRRWPIQQSRLYSSNTRS ID
(Amino Acid HKATTTRENTFQKPYSDEEVTKTPVGSRARKIFEAPHPHATRLT NO: Seq.)
VEGAIECPLESFQLLNSPLFNKGSAFTQEEREAFNLEALLPPQVN 105
TLDEQLERSYKQLCYLKTPLAKNDFMTSLRVQNKVLYFALIRRH
IKELVPIIYTPTEGDAIAAYSHRFRKPEGVFLDITEPDSIECRLATY
GGDKDVDYIVVSDSEGILGIGDQGIGGVRIAISKLALMTLCGGIH
PGRVLPVCLDVGTNNKKLARDELYMGNKFSRIRGKQYDDFLEK
FIKAVKKVYPSAVLHFEDFGVKNARRLLEKYRYELPSFNDDIQG
TGAVVMASLIAALKHTNRDLKDTRVLIYGAGSAGLGIADQIVNH
MVTHGVDKEEARKKIFLMDRRGLILQSYEANSTPAQHVYAKSD
AEWAGINTRSLHDVVENVKPTCLVGCSTQAGAFTQDVVEEMH
KHNPRPIIFPLSNPTRLHEAVPADLMKWTNNNALVATGSPFPPV
DGYRISENNNCYSFPGIGLGAVLSRATTITDKMISAAVDQLAELS
PLREGDSRPGLLPGLDTITNTSARLATAVILQALEEGTARIEQEQ
VPGGAPGETVKVPRDFDECLQWVKAQMWEPVYRPMIKVQHDP SVHTNQL SEQ MAE1, Sc
ATGCTTAGAACCAGACTATCCGTTTCCGTTGCTGCTAGATCGC ID (Nucleic Acid
AACTAACCAGATCCTTGACAGCATCAAGGACAGCACCATTAA NO: Seq.)
GAAGATGGCCTATTCAGCAATCGCGTTTATATTCTTCTAACAC 106
TAGATCGCATAAAGCTACCACAACAAGAGAAAATACTTTCCA
AAAGCCATACAGCGACGAGGAGGTCACTAAAACACCCGTCG
GTTCTCGCGCCAGAAAGATCTTCGAAGCTCCTCACCCACATG
CCACTCGTTTGACTGTAGAAGGTGCCATAGAATGTCCCTTGG
AGAGCTTTCAACTTTTAAACTCTCCTTTATTTAACAAGGGTTC
TGCATTTACACAAGAAGAAAGGGAAGCGTTTAATTTAGAAGC
ATTGCTACCACCACAAGTGAACACTTTGGACGAACAACTGGA
AAGAAGCTACAACCAGTTATGCTATTTGAAGACGCCCTTGGC
CAAAAACGACTTCATGACGTCTTTGAGAGTACAGAACAAAGT
CCTATATTTTGCATTAATAAGGAGACATATCAAGGAATTAGT
TCCTATCATTTACACCCCAACCGAAGGTGATGCTATTGCTGCC
TATTCCCACAGGTTCAGAAAGCCAGAAGGTGTGTTTTTAGAC
ATTACCGAACCTGATTCCATCGAATGTAGATTGGCTACATAC
GGTGGAGACAAAGATGTAGACTACATCGTTGTGTCGGATTCG
GAAGGTATTCTGGGAATTGGTGACCAAGGTATCGGTGGTGTA
CGTATTGCTATCTCCAAATTGGCATTGATGACGCTGTGCGGTG
GTATTCATCCCGGCCGTGTGCTACCTGGOTGTTTGGACCTCGG
TACTAACAACAAGAAACTAGCCCGTGACGAATTGTACATGGG
TAACAAGTTCTCCAGAATCAGGGGTAAGCAATATGACGACTT
CTTGGAAAAATTCATCAAGGCCGTTAAGAAAGTGTATCCAAG
CGCCGTTCTGCATTTCGAAGATTTCGGTGTTAAGAACGCTAG
AAGATTACTAGAAAAGTACAGGTACGAATTGCCATCATTCAA
CGATGACATTCAGGGCACCGGTGCCGTCGTGATGGCCTCGTT
GATTGCTGCTTTGAAACATACCCACAGACTTGAAAGACAC
CAGAGTGCTTATTTACGGTGCCGGGTCTGCGGOCCTCGGTAT
CGCAGATCAAATTGTGAATCATATGGTCACGCACGGCGTTGA
CAAGGAAGAAGCGCGCAAGAAAATCTTCTTGATGGACAGAC
GTGGGTTAATTCTACAATCTTACGAGGCTAACTCCACTCCCGG
CCAACACGTATACGCTAAGAGTGATGCGGAATGGGCTGGTAT
CAACACCCGCTCTTTACATGATGTGGTGGAGAACGTCAAACC
AACGTGTTTGGTTGGCTGCTCCACACAAGCCAGGCGCTTCAC
TCAAGATGTCGTAGAAGAAATGCACAAGCACAATCCTAGACC
GATCATTTTCCCATTATCCAACCCTACTAGACTACACGAAGCC
GTTCCTGCCGATTTAATGAAGTGGACCAACAACAACGCTCTT
GTAGCTACCGGATCTCCTTTCCCACCTGTTGATGGTTACCGTA
TCTCGGAGAACAACAATTGTTACTCTTTCCCAGGTATCGGTTT
AGGTGCCGTACTATCGCGTGCCACCACCATCACAGACAAGAT
GATCTCCGCTGCAGTGGACCAACTAGCCGAATTGTCGCCACT
AAGAGAGGGCGACTCGAGACCTGGGTTGCTACCCGGCCTGGA
CACCATCACCAACACTTCTGCGCGTCTAGCTACCGCTGTGATC
TTGCAAGCACTCGAGGAGGGAACCGCCCGTATCGAGCAAGA
ACAAGTACCOGGAGGAGCTCCCGGCGAAACTGTCAAGGTTCC
TCGTGACTTTGACGAATGTTTACAGTGGGTCAAAGCCCAAAT
GTGGGAGCCTGTGTACAGACCTATGATCAAGGTCCAACATGA
CCCATCGGTGCACACCAACCAATTGTAG SEQ PYC2, Sc
MSSSKKLAGLRDNFSLLGEKNKILVANRGELPIRIFRSAHELSMR ID (Amino Acid
TIAIYSHEDRLSMHRLKADEAYVIGEEGQYTPVGAYLAMDEIIEI NO: Seq.)
AKKHKVDFIHPGYGFLSENSEFADKVVKAGITWIGPPAEVIDSV 107
GDKVSARHLAARANVPTVPGTPGPIETVQEALDFVNEYGYPVII
KAAFGGGGRGMRVVREGDDVADAFQRATSEARTAFGNGTCFV
ERFLDKPKHIEVQLLADNHGNVVHLFERDCSVQRRHQKVVEVA
PAKTLPREVRDAILTDAVKLAKVCGYRNAGTAEFLVDNQNRHY
FIEINPRIQVEHTTTEEITGIDIVSAQIQIAAGATLTQLGLLQPKITT
RGFSIQCRITTEDPSKNFQPDTGRLEVYRSAGGNGVRLDGGNAY
AGATISPHYDSMLVKCSCSGSTYEIVRRKMIRALIEFRIRGVKTNI
PFLLTLLTNPVFIEGTYWTTFIDDTPQLFQMVSSQNRAQKLLHYL
ADLAVNGSSIKGQIGLPKLKSNPSVPHLHDAQGNVINVTKSAPPS
GWRQVLLEKGPSEFAKQVRQFNGTLLMDTTWRDAHQSLLATR
VRTHDLATIAPTTAHALAGAFALECWGGATFDVAMRFLHEDPW
ERLRKLRSLVPNIPFQMLLRGANGVAYSSLPDNAIDHFVKQAKD
NGVDIFRVFDALNDLEQLKVGVNAVKKAGGVVEATVCYSGDM
LQPGKKYNLDYYLEVVEKIVQMGTHILGIKDMAGTMKPAAAKL
LIGSLRTRYPDLPIHVHSHDSAGTAVASMTACALAGADVVDVAI
NSMSGLTSQPSINALLASLEGNIDTGINVEHVRELDAYWAEMRL
LYSCFEADLKGPDPEVYQHEIPGGQLTNLLFQAQQLGLGEQWA
ETKRAYREANYLLGDIVKVTPTSKVVGDLAQFMVSNKLTSDDIR
RLANSLDFPDSVMDFFEGLIGQPYGGFPEPLRSDVLRNKRRKLT
CRPGLELEPFDLEKIREDLQNRFQDIDECDVASYNMYPRVYEDF
QKIRETYGDLSVLPTKNFLAPAEPDEEIEVTIEQGKTLIIKLQAVG
DLNKKTGQREVYFELNGELRKIRVADKSQNIQSVAKPKADVHD
THQIGAPMAGVIIEVKVHKGSLVKKGESIAVLSAMKMEMVVSSP
ADGQVKDVFIKDGESVDASDLLVVLEEETLPPSQKK* SEQ PYC2, Sc
ATGAGCAGTAGCAAGAAATTGGCCGGTCTTAGGGACAATTTG ID (Nucleic Acid
AGTTTGCTCGGCGAAAAGAATAAGATCTTGGTCGCCAATAGA NO: Seq.)
GGTGAAATTCCGATTAGAATTTTTAGATCTGCTCATGAGCTGT 108
CTATGAGAACCATCGCCATATACTCCCATGAGGACCGTCTTTC
AATGCACAGGTTGAAGGCGGACCAAGCGTATGTTATCGGGG
AGGAGGGCCAGTATACACCTGTGGGTGCTTACTTGGCAATGG
ACGAGATCATCGAAATTGCAAAGAAGCATAAGGTGGATTTCA
TCCATCCAGGTTATGGGTTCTTGTCTGAAAATTCGGAATTTGC
CGACAAAGTAGTGAAGGCCGGTATCACTTGGATCGGCCCTCC
AGCTGAAGTTATTGACTCTGTGGGTGACAAAGTCTCTGCCAG
ACACTTGGCAGCAAGAGCTAACGTTCCTACCGTTCCCGGTAC
TCCAGGAGGTATCGAAACTGTGCAAGAGGCACTTGACTTCGT
TAATGAATACGGCTACCCGGTGATCATTAAGGCCGCCTTTGG
TGGTGGTGGTAGAGGTATGAGAGTCGTTAGAGAAGGTGACG
ACGTGGCAGATGCCTTTCAACGTGCTACCTCCGAAGCCCGTA
CTGCCTTCGGTAATGGTACCTGCTTTGTGGAAAGATTCTTGGA
CAAGCCAAAGCATATTGAAGTTCAATTGTTGGCTGATAACCA
CGGAAACGTGGTTCATCTTTTCGAAAGAGACTGTTCTGTGCA
AAGAAGACACCAAAAAGTTGTCGAAGTCGCTCCAGCAAAGA
CTTTGCCCCGTGAAGTTCGTGACGCTATTTTGACAGATGCTGT
TAAATTAGCTAAGGTATGTGGTTACAGAAACGCAGGTACCGC
CGAATTCTTGGTTGACAACCAAAACAGACACTATTTCATTGA
AATTAATCCAAGAATTCAAGTGGAGCATACCATCACTGAAGA
AATCACCGGTATTGACATTGTTTCTGCCCAAATCCAGATTGCC
GCAGGTGCCACTTTGACTCAACTAGGTCTATTACAGGATAAA
ATCACCACCCGTGGGTTTTCCATCCAATGTCGTATTACCACTG
AAGATCCCTCTAAGAATTTCCAACGGGATACCGGTCGCCTGG
AGGTCTATCGTTCTGCCGGTGGTAATGGTGTGAGATTGGACG
GTGGTAACGCTTATGCAGGTGCTACTATCTCGCCTCACTACGA
CTCAATGCTGGTCAAATGTTCATGCTaGGTTCTACTTATGAA
ATCGTCCGTAGGAAGATGATTCGTGCCCTGATCGAATTCAGA
ATCAGAGGTGTTAAGACCAACATTCCCTTCCTATTGACTCTTT
TGACCAATCCAGTTTTTATTGACGGTACATACTGGACGACTTT
TATTGACGACACCCCACAACTGTTCCAAATGGTATCGTCACA
AAACAGAGCGCAAAAACTGTTACACTATTTGGCAGACTTGGC
AGTTAACGGTTCTTCTATTAAGGGTCAAATTGGCTTGCCAAA
ACTAAAATCAAATCCAAGTCTCCCCCATTTGCACGATGCTCA
GGGCAATGTCATCAACGTTACAAAGTCTGCACCACCATCCGG
ATGGAGACAAGTGCTACTGGAAAAGGGACCATCTGAATTTGC
CAAGCAAGTCAGACAGTTCAATGGTACTCTACTGATGGACAC
CACCTGGAGAGACGCTCATCAATCTCTACTTGCAACAAGAGT
CAGAACCCACGATTTGGCTACAATCGCTCCAACAACCGCACA
TGCCCTTCTCAGGTGCTTTCGCTTTAGAATGTTGGGGTGGTGCT
ACATTCGACdTTGCAATGAGATTCTTGCATGAGGATCCATGG
GAACGTGTGAGAAAATTAAGATCTCTGGTGCCTAATATTCCA
TTCCAAATGTTATTACGTGGTGCCAACCGTGTGGCTTACTCTT
CATTACCTGACAATGCTATTGACCATTTTGTCAAGCAAGCCA
AGGATAATGGTGTTGATATATTTAGAGTTTTTGATGCCTTGAA
TGATTTAGAACAATTAAAAGTTGGTGTGAATGCTGTCAAGAA
GGCCGGTGGTGTTGTCGAAGCTACTGTTTGTTACTCTGGTGAC
ATGCTTCAGCCAGGTAAGAAATACAACTTAGACTACTACCTA
GAAGTTGTTGAAAAAATAGTTCAAATGGGTACACATATCTTG
GGTATTAAGGATATGGCAGGTACTATGAAACCGGCCGCTGCC
AAATTATTAATTGGCTCCCTAAGAACCAGATATCCGGATTTA
CCAATTCATGTTCACAGTCATGACTCCGCAGGTACTGCTGTTG
CGTCTATGACTGCATGTGCCCTAGCAGGTGCTGATGTTGTCGA
TGTAGCTATCAATTCAATGTCGGGCTTAACTTCCCAACCATCA
ATTAATGCACTGTTGGCTTCATTAGAAGGTAACATTGATACTG
GGATTAACGTTGAGCATGTTCGTGAATTAGATGCATACTGGG
CCGAAATGAGACTGTTGTATTCTTGTTTCGAGGCCGACTTGAA
GGGACCAGATCCAGAAGTTTACCAACATGAAATCCCAGGTGG
TCAATTGACTAACTTGTTATTCCAAGCTCAACAACTGGGTCTT
GGTGAACAATGGGCTGAAACTAAAAGAGCTTACAGAGAAGC
CAATTACCTACTGGGAGATATTGTTAAAGTTACCCCAACTTCT
AAGGTTGTCGGTGATTTAGCTCAATTCATGGTTTCTAACAAAG
TGACTTCCGACGATATTAGACGTTTAGCTAATTCTTTGGACTT
TCCTGACTCTGTTATGGACTTTTTTGAAGGTTTAATTGGTCAA
CCATACGGTGGGTTCCCAGAACCATTAAGATCTGATGTATTG
AGAAACAAGAGAAGAAAGTTGACGTGCCGTCCAGGTTTAGA
ATTAGAACCATTTGATCTCGAAAAAATTAGAGAAGACTTGCA
GAACAGATTCGGTGATATTGATGAATGCGATGTTGCTTCTTAC
AATATGTATGCAAGGGTCTATGAAGATTTCCAAAAGATCAGA
GAAACATACGGTGATTTATCAGTTCTACCAACCAAAAATTTC
CTAGGACCAGCAGAACCTGATGAAGAAATCGAAGTCACCATC
GAACAAGGTAAGACTTTGATTATCAAATTGCAAGCTGTTGGT
GACTTAAATAAGAAAACTGGGCAAAGAGAAGTGTATTTTGAA
TTGAACGGTGAATTAAGAAAGATCAGAGTTGCAGACAAGTCA
CAAAACATACAATCTGTTGCTAAACCAAAGGCTGATGTCCAC
GATACTCACCAAATCGGTGCACCAATGGCTGGTGTTATCATA
GAAGTTAAAGTACATAAAGGGTCTTTGGTGAAAAAGGGCGA
ATCGATTGCTGTTTTGAGTGCCATGAAAATGGAAATGGTTGT
CTCTTCACCAGCAGATGGTCAAGTTAAAGACGTTTTCATTAA
GGATGGTGAAAGTGTTGACGCATCAGATTTGTTGGTTGTCCT
AGAAGAAGAAACCCTACCCCCATCCCAAAAAAAGTAA SEQ GUT2,
MSRFLKSTFAKSALAAGAAVGGTIVYLDFIKPKNETHLATTYPA ID Candida
FNKNIPAPPPRESLIENLKKTPQFDVLVIGGGAVGTGTALDAATR NO: (Amino Acid
GLNVCLLEKTDFGAGTSSKSTKMAHGGVRYLEKAIFQLSRAQL 109 Seq.)
DLVIEALNERGNMLRTAPHLCSVLPIMIPVYNWWQVPYFFAGC
KMYDWFAGKQNLRSSTIFTTEQAAAIAPMMDTSNLKAACandida
YHDGSFNDTRYNVSLAVTAIKNGATVLNYFEVEQLLKDDKGKL
YGVKAKDLETNETYEIKATSVVNATGPFADKILEMDEDPKGLPP
KVEQPPRMVVPSSGVHIVLPEYYCPTNYGLLDPSTSDGRVMFFL
PWQGKVLAGTTDTPLKTVPANPVPTEEEIQDIIKEMQKYLVFPID
RNDVLSAWSGIRPLVRDPSTIPKGQEGSGKTEGLVRSHLLVQSPT
GLVTISGGKWTTYREMAQETVDYLVDHFSYGDKKLLPCQTNKL
LLVGGENYTKNYSARLIHEYKIPLKLAKHLSHNYGSRAPLVLDL
YAESDFNKLPVTLAATKEFEPSEKKANEDNQLSYQSFDEPFTVA
ELKYSLKYEYPRTPLDFLARRTKLAFLNAREALNAVDGVVEIMS
QEYGWDKETEDRLRKEARQYIGNMGISPKKFDVEKIVIQ
SEQ GUT2,
atgtcaagattcttaaagtcaacctttgcaaagtcagccttagctgctggtgctgcagtcggtg-
gtaccat ID Candida
cgtgtacttggatttcatcaagccaaagaacgaaacccacttggccactacctacccagcctt-
caacaa NO: (Nucleic Acid
gaacataccagctcctcccccacgtgagtccttgatcgaaaacttgaagaagactcctcaattcgacgt
110 Seq.)
cttggtcattggtggtggtgcagttggtaccggtactgctctcgacgctgccacgcgtgggtta-
aatgtg
tgtcttttggaaaaaaccgacttcggtgcaggaacttcgtcgaagtccaccaaaatggcccacggtggt
gtccgttacttggagaaggccatttttcaattgtccagagcccaattggacttggtcattgaagccttgaa
cgaaagaggtaacatgttgagaactgctcctcacttgtgctctgttttgccaatcatgattccagtctacaa
ctggtggcaggttccttactttttcgctggttgtaagatgtacgattggtttgccggtaagcaaaacttgcg
ttcctccactatcttcaccactgaacaggctgccgctattgccccaatgatggatacttctaacttgaaag
ccgcctgtgtctaccacgatggtagtttcaacgataccagatacaacgtctccttggctgtcaccgccat
caagaacggcgccactgtcttgaactatttcgaagttgagcaattgttgaaagatgacaagggtaagtt
gtacggtgtcaaggccaaggatttggaaaccaacgagacctacgagatcaaagccactagtgttgtca
acgctaccggtcctttcgctgataagatcttggagatggacgaagatccaaagggtttgcctccaaagg
tcgagcaaccaccaagaatggttgttccatcttccggtgtccacattgttttgcctgaatactactgccca
accaactacggtttgttagacccatccacctccgacggtagagtcattgttctttttgccatggcaaggtaa
ggtcttggctggtaccactgatactccattgaagactgttcctgccaaccctgttccaactgaggaagaa
atccaagacatcatcaaagaaatgcaaaagtaccttgttttcccaatcgacagaaacgacgttttgtctg
cttggtccggtatcagaccattggttagagacccatctactattccaaagggccaagaaggctctggta
agactgaaggtttagttcgttcccacttgttagttcaatccccaactggtttggtcaccatctccggtggta
aatggaccacctacagagaaatggcccaagaaactgttgactacctcgttgatcactttagctacggcg
acaagaagctcttgccatgccagaccaacaaattgctcttggttggtggtgaaaactacaccaagaact
actctgccagattgatccacgaatacaagatcccattgaagttggccaagcacttgtcccacaactacg
gttccagagctccattggtcttggacttgtacgctgaaagtgatttcaacaagttgccagtcaccttggct
gccaccaaggagtttgagccatctgaaaagaaggccaacgaagacaaccagttgagttaccagagtt
tcgacgagccattcaccgttgctgagttgaagtactccttgaagtacgagtacccaagaaccccactcg
atttcttggccagaagaacgagattggctttcttgaacgccagagaagccctcaatgcggttgatgggg
ttgttgaaatcatgagtcaagagtacggctgggacaaggagaccgaagatagattgagaaaagaagc
cacacaatacattggtaatatgggtatttctccaaagaaatttgacgttgaaaagattgttattcaa
SEQ gpsA, Af MIVSILGAGAMGSALSVPLVDNGNEVRIWCTEFDTEILKSISAGR ID
(Amino Acid EHPRLGVKLNGVEIFWPEQLEKCLENAEVVLLGVSTDGVLPVM NO: Seq.)
SRILPYLKDQYIVLISKGLIDFDNSVLTVPEAVWRLKHDLRERTV 111
AITGPAIAREVAKRMPTTVVFSSPSESSANKMKEIFETEYFGVEV
TTDIIGTEITSALKNVYSIAIAWIRGYESRKNVEMSNAKGVIATRA
INEMAELIEILGGDRETAFGLSGFGDLIATFRGGRNGMLGELLGK
GLSIDEAMEELERRGVGVVEGYKTAEKAYRLSSKINADTKLLDS IYRVLYEGLKVEEVLEELATFK
SEQ gpsA, Af
atgattgtttcgatactgggagcgggtgcaatgggctcagccctctccgtcccgctcgtagataacggc
ID (Nucleic Acid
aacgaagtgagaatctgggggaccgagttcgatacggagattttaaaatcaatctcagccggcagag
NO: Seq.)
agcatccaaggcttggtgtaaagctcaatggcgtggaaattttctggccagagcagcttgaaaa-
atgttt 112
ggagaatgcagaggttgtacttctgggtgttagcacggatggcgtgctgcccgtaatgagcagaattct
cccgtatctcaaggaccagtacatcgtactcatctctaaagggctgattgattttgataacagtgttctgac
ggttcccgaagctgtatggaggttaaagcacgatttgagggaaaggactgtggcgataaccgggccc
gctattgcaagagaggtggcgaaacgcatgcccacaaccgttgttttcagcagcccatccgaaagctc
ggccaataaaatgaaagaaatctttgagacagagtactttggcgttgaagtaacaacagacataattgg
cacggaaataacctccgccctcaaaaacgtttattccatagccattgcatggataaggggctacgaga
gcagaaaaaacgttgagatgagcaatgcaaagggagtgattgcaacgagagccataaacgagatgg
cagagctgatagagattctcggaggggatagagagaccgcctttggcctttccggatttggagacctc
atcgcaaccttcaggggaggaaggaacgggatgctgggagagctgcttggaaaggggcttagcatc
gatgaggcgatggaggagcttgagaggagaggagttggtgtggttgagggctacaaaacggcaga
gaaagcatacaggctgtccagcaaaataaatgcagacacaaagctgctcgacagcatctacagagtc
ctttatgaaggactgaaggttgaggaagtgctgtttgaactcgctacatttaaataa SEQ
ADH1-2, MHALFSKSVFLKYVSSPTTSAIPHSLEFIVSRSSYLRRRIPPYLPRC ID Candida
SHEPSFYYSSSSVYTKKSFHTMSANIPKTQKAVVFEKNGGELKY NO: (Amino Acid
KDIPVPTPKANELLINVKYSGVCHTDLHAWKGDWPLDTKLPLV 113 Seq.)
GGHEGAGVVVGMGENVKGWKIGDFAGIKWLNGSCMSCEFCQQ
GAEPNCGEADLSGYTHDGSFEQYATADAVQAARIPAGTDLAEV
APILCAGVTVYKALKTADLAAGQWVAISGAGGGLGSLAVQYA
VAMGLRVVAIDGGDEKGDFVKSLGAEAYIDFLKEKGIVAAVKK
ATDGGPHGAINVSYSEKAIDQSVEYVRPLGKVVLVGLPAGSKVT
AGVFEAVVKSIEIKGSYVGNRKDTAEAVDFFSRGLIKCPIKIVGL
SELPQVFKLMEEGKILGRYVLDTSK SEQ ADH1-2,
atgtctgctaatatcccaaaaactcaaaaagctgtcgtcttcgagaagaacggtggtgaattaaaataca
ID Candida
aagacatcccagtgccaaccccaaaggccaacgaattgctcatcaacgtcaagtactcgggtg-
tctgt NO: (Nucleic Acid
cacactgatttgcacgcctggaagggtgactggccattggacaccaaattgccattggttggtggtcac
114 Seq.)
gaaggtgctggtgttgttgtcggcatgggtgaaaacgtcaagggctggaaaatcggtgatttcg-
ccgg
tatcaaatggttgaacggttcttgtatgtcctgtgagttctgtcagcaaggtgctgaaccaaactgtggtg
aagctgacttgtctggttacaccacgatggttctttcgaacaatacgccactgctgatgctgtgcaagc
cgccagaatcccagctggcactgatttggccgaagttgccccaatcttgtgtgctggtgtcaccgtctac
aaagccttgaagactgccgacttggctgctggtcaatgggtcgctatctccggtgctggtggtggtttg
ggctccttggctgtccaatacgccgtcgccatgggtttgagagtcgttgccattgacggtggtgacgaa
aagggtgactttgtcaagtccttgggtgctgaagcctacattgatttcctcaaggaaaagggcattgttg
ctgctgtcaagaaggccactgatggcggtccacacggtgctatcaatgtttccgtttccgaaaaagcca
ttgaccaatctgtcgagtacgttagaccattgggtaaggttgttttggttggtttgccagctggctccaag
gtcactgctggtgttttcgaagccgttgtcaagtccattgaaatcaagggttcttacgtcggtaacagaaa
ggatactgccgaagccgttgactttttctccagaggcttgatcaagtgtccaatcaagattgtgggcttga
gtgaattgccacaggtcttcaagttgatggaagaaggtaagatcttgggtagatacgtcttggatacctc
caaa SEQ ADH2a, MSIPTTQKAIIFETNGGKLEYKDIPVPKPKPNELLINVKYSGVCHT ID
Candida DLHAWKGDWPLDTKLPLVGGHEGAGVVVAIGDNVKGWKVGD NO: (Amino Acid
LAGVKWLNGSCMNCEYCQQGAEPNCPQADLSGYTHDGSFQQY 115 Seq.)
ATADAVQAARIPAGTDLANVAPILCAGVTVYKALKTADLQPGQ
WVAISGAAGGLGSLAVQYAKAMGYRVVAIDGGADKGEFVKSL
GAEVFVDELKEKDIVGAVKKATDGGPHGAVNVSISEKAINQSVD
YVRTLGKVVLVGLPAGSKVSAPVFDSVVKSIQIKGSYVGNRKDT
AEAVDFFSRGLIKCPIKVVGLSELPEVYKLMEEGKILGRYVLDNS K SEQ ADH2a,
atgtcaattccaactactcaaaaagctatcattttcgaaaccaacggtggaaaattagaatac-
aaggaca ID Candida
tcccagttccaaagccaaagccaaacgaattgctcatcaacgtcaagtactccggtgtctgcc-
acactg NO: (Nucleic Acid
atttacacgcctggaagggtgactggccattggacaccaagttgccattggtgggtggtcacgaaggt
116 Seq.)
gctggtgttgttgttgccattggtgacaatgtcaagggatggaaggtcggtgatttggccggtg-
tcaagt
ggttgaacggttcctgtatgaactgtgagtactgtcaacagggtgccgaaccaaactgtccacaggctg
acttgtctggttacacccacgacggttctttccagcaatacgccactgcagatgccgtgcaagccgcta
gaattccagctggtactgatttagccaacgttgcccccatcttgtgtgctggtgtcactgtttacaaggcct
tgaagaccgccgacttgcagccaggtcaatgggtcgccatttccggtgccgctggtggtttgggttcttt
ggccgttcaatacgccaaggccatgggctacagagttgtcgccatcgatggtggtgccgacaagggt
gagttcgtcaagtctttgggcgctgaggtctttgttgatttcctcaaggaaaaggacattgttggtgctgtc
aagaaggcaaccgatggtggcccacacggtgccgttaacgtttccatctccgaaaaggccatcaacc
aatctgtcgactacgttagaaccttgggtaaggttgtcttggtcggtttgccagctggctccaaggtttct
gctccagtctttgactccgtcgtcaagtccatccaaatcaagggttcctatgtcggtaacagaaaggaca
ctgccgaagctgttgactttttctccagaggcttgatcaagtgtccaatcaaggttgtcggtttgagtgaat
tgccagaagtctacaagttgatggaagaaggtaagatcttgggtagatacgtcttggacaactctaag
SEQ ADH2b, MSIPTTQKAVIYEANSAPLQYTDIPVPVPKPNELLVHVKYSQVCH ID Candida
SDIHVWKDWFPASKLPVVGGHEGAGVVVAIGENVQGWKVGD NO: (Aminio Acid
LAGIKMLNGSCMNCEYCQQGAEPNCPHADVSGYSHDGTFQQY 117 Seq.)
ATADAVQAAKFPAGSDLASIAPISCAGVTVYKALKTAGLQPGQ
WVAISGAAGGLGSLAVQYAKAMGLRVVAIDGGDERGVFVKSL
GAEVFVDFTKEANVSEAIIKATDGGAHGVINVSISEKAINQSVEY
VRTLGTVVLVGLPAGAKLEAPIFNAVAKSIQIKGSYVGNRRDTA
EAVDFFARGLVKCPIKVVGLSELPEIFKLLEEGKILGRYVVDTAK SEQ ADH2b,
atgtcaattccaactacccaaaaagctgttatctacgaagccaactctgctccattgcaatac-
accgatat ID Candida
cccagttccagtccctaagccaaacgaattgctcgtccacgtcaaatactccggtgtttgtca-
ctcagat NO: (Nucleic Acid
atacacgtctggaagggtgactggttcccagcatcgaaattgcccgttgttggtggtcacgaaggtgcc
118 Seq.)
ggtgttgtcgttgccattggtgaaaacgtccaaggctggaaagtaggtgacttggcaggtataa-
agatg
ttgaatggttcctgtatgaactgtgaatactgtcaacaaggtgctgaaccaaactgtccccacgctgatgt
ctcgggttactcccacgacggtactttccaacagtacgctaccgccgatgctgttcaagctgctaaattc
ccagctggttctgatttagctagcatcgcacctatatcctgcgccggtgttactgtttacaaagcattgaa
aactgcaggcttgcagccaggtcaatgggttgccatctctggtgcagctggtggtttgggttctttggct
gtgcaatacgccaaggccatgggtttgagagtcgtggccattgacggtggtgacgaaagaggagtgt
ttgtcaaatcgttgggtgctgaagttttcgttgatttcaccaaagaggccaatgtctctgaggctatcatca
aggctaccgacggtggtgcccatggcgtcatcaacgtttccatttctgaaaaagccatcaaccagtctg
ttgaatatgttagaactttgggaactgttgtcttggttggtttgccagctggtgcaaagctcgaagctccta
tcttcaatgccgttgccaaatccatccaaatcaaaggttcttacgtgggaaacagaagagacactgctg
aggctgttgatttcttcgctagaggtttggtcaaatgtccaattaaggttgttgggttgagtgaattgccag
agattttcaaattgttggaagagggtaagatcttgggtagatacgttgttgacactgccaag SEQ
ADH3 MSTQSGYGYVKGQKTIQKYTDIPIPTPGPNEVLLKVEAAGLCLS ID (Amino Acid
DPHTLIGGPIESKPPLPNATKFIMGHEIAGSISQVGANLANDPYYK NO: Seq.)
KGGRFALTIAQACGICENCRDGYDAKCESTTQAYGLNEDGGFQ 119
QYLLIKNLRTMLPIPEGVSYEEAAVSTDSVLTPFHAIQKVAHLLH
PTTKVLVQGCGGLGFNAIQILKSYNCYIVATDVKPELEKLALEY
GANEYHTDLTKSKHEPMSFDLIFDLVGIQPTFDLSDRYIKARGKI
LMIGLGRSKLFIPNYKLGIREVEIIFNFGGTSAEQIECMKWVAKG
LIKNIHVADFASLPEYLEDLAKGKLTGRIVFRPSKL SEQ ADH3
Atgtcaactcaatcaggttacggatacgtgaaaggacaaaagaccattcagaaatacaccgacat-
cc ID (Nucleic Acid
cgatccctacgccgggccccaacgaagtcttgttgaaagtcgaagctgccggcttgtgtctctcggatc
NO: Seq.)
cacacacgttgatcgggggtcccattgagagcaagccgccgttgccgaacgccacgaagttcat-
cat 120
gggtcacgaaatcgcggggctgattagccaagtaggcgccaacttggccaacgatccatactataaa
aagggaggtaggttcgccttgactatcgcgcaggcttgtgggatttgtgagaattgtcgtgatgggtatg
atgcaaagtgtgagtctacgacgcaggcttatgggttgaacgaggacggtggattccagcaatacttgt
tgattaagaacttgcgtacgatgttgcctatccctgagggtgtgagttacgaagaagccgctgtgtctac
tgactctgtgttgactccattccatgcgattcagaaggtcgctcatttgttgcacccaactactaaggtgtt
ggttcagggttgtggtgggttaggcttcaacgctattcaaatattgaagagctacaattgttacattgttgc
cactgatgtcaaaccagagcttgaaaaattagctttggagtatggtgccaacgaataccacactgatctc
accaagtccaagcatgagccaatgtcgttcgatttgattttcgaccttgtgggaatccaacctacttttgat
ttgtccgacaggtacatcaaagcaaggggtaagattcttatgattggcttaggcagatccaagttgtttat
tccaaattataaattgggtatccgtgaagtcgagatcattttcaattttggtggtacttcggccgagcaaat
tgagtgcatgaaatgggttgcaaaaggcttgatcaaacctaatattcacgtggctgattttgcttccttgcc
tgagtacctcgaggacttggccaagggtaaactcactggtagaattgtatttagaccaagtaagttg
SEQ ADH4 MSLSGKTSLIAAGTKNLGGASAKELAKAGSNLFLHYRSNPDEAE ID (Amino
Acid KFKQEILKEFPNVKVETYQSKLDRAADLTNLFAAAKKAFPSGID NO: Seq.)
VAVNFVGKVIKGPITEVTEEQFDEMDVANNKIAFFFIKEAAINLN 121
KNGSIISIVTSLLPAYTDSYGLYQGTKGAVEYYSKSISKELIPKGIT
SNCIGPGPASTSFLFNSETKESVEFFKTVAIDQRLTEDSDIAPIVLF
LATGGRWATGQTTYASGGFTAR SEQ ADH4
Atgtcattatcaggaaagacctcattaattgctgctggtaccaagaacttgggtggtgcaagtgc-
caaa ID (Nucleic Acid
gaattggccaaagccggctccaacctcttcttgcactacagatccaacccagacgaggctgaaaagtt
NO: Seq.)
caagcaagagatwcaaggagttccctaacgtcaaggtcgaaacctaccaatccaaatttggacc-
gtg 122
ccgccgacctcaccaacttgtttgctgctgccaagaaggcattccctagtggtattgacgtcgctgtca-
a
ctttgtcggtaaggtcatcaagggcccaatcactgaggtcactgaagaacagtttgacgagatggatgt
tgccaacaacaagattgcctttttcttcatcaaggaggccgctatcaacttgaacaagaacggtagtatc
atttccatcgttactagtttgctcccagcttacaccgattcttacggtttgtaccagggtactaaaggagct
gttgaatactattcgaaatctatcctgaaggagttgattccaaagggtatcaccagtaactgtattggtcct
ggtcctgcttctacttcctttttgtttaattccgaaaccaaggagagtgttgagttcttcaagaccgttgct-
at
tgaccaacgtttgactgaagacagcgacattgccccaattgtgttgttcctcgccactggaggtcgttgg
gcaactggtcaaactatttacgctagtggtggtttcactgctcgt SEQ ADH5
MSLVLKRLLPIRSPTLLNSKFIQLQSQIRTMAIPATQTGFRFKQE ID (Amino Acid
GLNYRTDIPVRKPQAGQLLLKVNAVGLGHSDLHVIDKELECGD NO Seq.)
NYVMGHEIAGTVAEVGPEVEGYKVGDRVACandidaGPNGCGVC 123
KHCLTGNDNVCKTAFLDWFGLGSDGGYEEYLLVRRPRNLVKVP
DNVSIEEAAAITDAVLTPYHAVKTAKVKPTSNVLVIGAGGLGGN
GIQIVKAFGGKVTVVDKKDKARDQAKALGADEVYSEIPASIEPG
TFDVCLDFVSVQATYDLCQKYCEPKGIIIPVGLGATKLTIDLADL
DLREITVTGTFWGTANDLREAFDLVSQGKIKPIVSHAPLKELPNY MEKLKQGAYEGRVVFHP SEQ
ADH5
Atgtcacttgtcctcaagcgattacttccaatcagatctcctactttactcaattcgaagttcat-
acagttacA ID (Nucleic Acid
aatctcaaattcgcacaatggctatccccgctactcaaactggattcttcttcaccaaacaagaaggttta
NO: Seq.)
aactacagaaccgacattcctgtccgcaagccacaagccggtcagttgttgttgaaggtcaatg-
ccgtt 124
ggtctctgccactcggacrtgcacgtgattgacaaggagcttgaatgtggtgacaactatgtcat
cacgaaattgccggteccgttgctgaagttggtcccgaagtgaaggctacaaggttggcgaccgtgt
cgcttgtgttggtcctaacgggtgcggtgtctgtaagcactgcttgactggtaacgacaatgtctgtaag
actgctttcctcgactggncgggttgggctccgatggtgggtacgaagagtacttgttggtgagaaga
ccaagaaacttggttaaggtcccggacaacgtctcgattgaggaggctgctgctatcactgatgctgtg
ttgactccttaccatgctgtcaagactgccaaggtcaagccaaccagtaacgttttggttattggtgctgg
tggattaggtggtaacggtatccagattgtcaaggcttttggcggtaaggttactgttgtcgataagaag
gataaggcacgtgaccaagctaaggctttgggtgctgatgaagtctacagtgaaatcccagcaagtatt
gaaccgggtacttttgatgtctgtcagattttgtttccgtgcaagccacctatgatctctgccaaaagtact
gtgagccaaagggtatcattatcccagttgggttggggtgctaccaagctcaccattgatttggcagattt
ggatctccgtgaaatcacggttactggtaccttctggggaactgccaatgacttgagagaggcgtttgat
ttggttagtcaaggtaagatcaagccgattgtttcacatgccccartgaaggagttgccaaactatatgg
agaagttgaagcagggagcatatgaaggattgagttgtcttccaccca SEQ ADH7
MTVDASSVPDKFQGFASDKRENWEHRKLISYDRKQLNDHDVVL ID (Amino Acid
KNETCGLCYSDIHTLRSTWGPYGTNELVVGHEICGTVIAVGPKV NO: Seq.)
TEFKVGDRAGIGAASSSCRHCSRCTHDNEQYCKEQYSTYNSVDP 125
KAAGYVTIKGGYSSHSIADELFVFKVPDDLPFEYASPLFCAGITTF
SPLYRNLVGSDKDATGKTVGIIGVGGLGHLAIQFASKALNAKVV
AFSRSSSKKEEALELOAAEFVATNEDKNWTSRYEDQFDLILNCA
SGIDGLKSDYLSVLKVDKKFVSYGLPPIDDEFNVSPFTFLKQGA
SFGSSLLGSKAEVNIMLELAAKHNIRPWIEKVPISEENVAKALKR CFEGDVRYRFVFTEFDKAFGN
SEQ ADH7
atgactgttgacgcttcttctgttccagacaagttccaagggtttgcctccgacaagagagaaaa-
ctggg ID (Nucleic Acid
aacacccaaagttgatctcctacgacagaaagcttactcaatgaccacgacgttgtcttgaagaacgag
NO: Seq.)
acctgtggtttgtgttactcggacatccacaccttgcgttccacgtggggaccatacggcacca-
atgag 126
cttgtcgttggccacgaaatctgtggtaccgtcattgctgtcggtccaaaggtcactgagttcaaggtc-
g
gtgacagagccggtattggtgctgcctcttcgtcttgtcgtcactgttccagatgtacccacgataacga
gcaatactgtaaggaacaagtctccacttacaattctgttgatccaaaggccgctggttacgtcaccaag
ggtggttactcctcccactccatcgctgacgaattgtttgtcttcaaggttccagatgacttgccattcgag
tacgcttccccattattctgtgctggtatcacaactttctccccattgtaccgtaacttggttgggtccgat-
a
aagacgccactggtaagaccgttggtatcattggtgttggtggtcttggtcaccttgccatccagtttgcg
tctaaagctttgaacgctaaggtcgttgctttctccagatcctcctccaagaaggaagaagctctcgaatt
gggtgctgctgagtttgtcgccaccaacgaagacaagaactggaccagcagatacgaggaccaattc
gacctcatcttgaactgtgcgagcggtatcgatggcttgaacttgtctgactacttgagtgtcttgaaagt
cgacaagaagtttgtcttttgttggtttgccaccaatcgacgacgagttcaacgtctctcctttcactttct-
tg
aagcaaggtgccagtttcggtagttccttgttgggatccaaggctgaagtcaacatcatgttggaattgg
ctgccaagcacaacatcagaccatggattgaaaaggtcccaatcagtgaggaaaacgtcgccaagg
ctttgaagagatgttttgaaggtgatgtcagatacagattcgtcttcactgagtttgacaaagcttttggca
at SEQ ADH8 MSVPTTQKAVIFETNGGKLEYKDVPVPVPKPNELINNVKYSGV ID (Amino
Acid CHSDLHVWKODWPIPAKLPINGGHEGAGVVVGMGDNVKGWK NO: Seq.)
VGDLAGIKWLNGSCIVINCEFCQQGAEPNCSRADMSGYTHDGTE 127
QQYATADAVQAAKIPEGADIVIASIAPMCAGVTVYKALKNADLL
AGQWVAISGAGGGLGSLGVQYAKAMGYRVLAIDGGDERGEFV
KSLGAEVYIDFLKEQDIVSAIRKATGGGPHGYINVSVSEKAINQS
VEYVRTLGKVVINSLPAQGKILTAPLFESVARSIQIRTTCandidaGN
RKDTTEAIDFFSVRGLIDCPIKVAGLSEVPEIFDLMEQGKILGRYV VDTSK SEQ ADH8
Atgtccgttccaactactcagaaagctgttatctttgaaaccaatggtggcaagttagaatacaa-
agac ID (Nucleic Acid
gtgccggtccctgtccctaaacccaacgaattgcttgtcaacgtcaagtactcgggtgtgtgtcattctg
NO: Seq.)
acttgcatgtctggaaaggcgactggcccattcctgccaagttgcccttggtgggaggtcacga-
aggt 128
gctggtgtcgttgtcggcatgggtgacaacgtcaagggctggaaggtgggggacttggctggtatca
agtggttgaatggttcgtgtatgaactgtgagttttgccaacagggcgcagaacctaactgttcaagagc
cgacatgtctgggtatacccacgatggaactttccaacaatacgccactgctgatgctgtccaagctgc
caagatcccagaaggcgccgacatggctagtatcgccccgatcttgtgcgctggtgtgaccgtgtaca
aggctttgaagaacgccgacttgttggctggccaatgggaggctatctctggtgctggtggtggtttgg
gctccttgggtgtgcagtacgctaaagccatgggttacagagtgttggctatcgacggtggtgacgag
agaggagagtttgtcaagtccttgggcgccgaagtgtacattgacttccttaaggaacaggacatcgtt
agtgctatcagaaaggcaactggtggtggtccacacggtgttattaacgtctcagtgtccgaaaaggca
atcaaccagtcggtggagtacgtcagaactttggggaaagtggttttagttagcttgccggcaggtggt
aaactcactgctcctcttttcgagtctgttgctagatcaatccagattagaactacgtgtgttggcaacaga
aaggatactactgaagctattgatttctltgttagagggttgatcgattgcccaattaaagtcgctggttta-
a
gtgaagtgccagagatttttgacttgatggagcagggaaagatcttgggtagatatgtcgttgatacgtc
aaag SEQ SFA1 (Amino MSESTVGKPITCKAAVAWEACKPLTIEDVTVAPPKAHEVRIKIL
ID Acid. Seq.) YTGVCHTDAYILSGVDPEGAPPVILGHEGAGIVESVGEGVITVK NO:
PGDHVIALYTPECGECKECKSGKTNLCQKIRATQGKGVMPDGTP 129
RFTCKGKELIHFMGCSTFSQYTVVTDISVVAINDKAELDKACLL
GCGITTGYGAATITANVQKGDNVAVFGGGAVGLSVLQGCKERE
AQIILVDVNNKKKEWGEKFGATAFINFILELPKGVTIVDKLIEMT
DGGCDETFACTQNVINIMMRDALEACITIKGWGtSVAGVAAAGKEI
STRPEQLVTGRVWKGAAFGGKGRSQLPGVEDYMVGKLKVEE
FITHRKPLEQINEAFEDMHAGDCIRAVVNMW SEQ SFA1
atgtctgaatcaaccgttggaaaaccaatcacctgtaaagccgctgttgcctgggaagcaggcaa-
gcc ID (Nucleic Acid
tttgaccatcgaagacgtcactgttgctccaccaaaggcccacgaagtgcgtatcaagatcttgtacact
NO: Seq.) no
ggtgtctgtcacactgatgcctacaccttgagtggtgttgatccagagggtgccttcccagtcatcttgg
130 introns
gacacgaaggtgccggtattgttgaaagtgttggtgaaggtgtcaccactgtcaagccaggcgqaccac
gtcattgcattatacactccagaatgtggtgagtggtgagtgtaagttctgtaaatcgggtaagaccaactt-
gtgtgg
taaaatcagagctacccaaggcaaaggtgtgatgccagacggaactccaagattcacttgcaagggc
aaagaattgattcactttatgggtatgctccaccttctcccaatacaccgttgtcactgacatttccgtcgt-
g
gccatcaacgacaaagccgaacttgaaaggcttgtttgttgggatgtggtatcactactggttatggtg
ctgccaccatcactgccaatgttcaaaagggtgacaatgtcgcagttttcggtggtggtgctgtcggatt
gtccgtcctccaaggatgtaaagaaagagaagctgcccaaatcattttggttgatgtcaacaacaagaa
gaaggaatggggtgaaaagttcggtgccactgcttttatcaacccattagaattaccaaaaggcgtcac
tattgttgacaagttgattgaaatgactgacggtggttgtgactttacttttgactgtaccggtaatgtcaa-
t
gttatgagagatgccttaaagcttgtcataagggttggggtacttcagtcatcatcggtgttgccgctg
ccggtaaggaaatctccaccagaccattccaattggtcactggtagagtctggaagggtgctgctttcg
gtggcgtcaagggtagatcccaattgccaggaatcgttgaagactacatggttggtaagttgaaggttg
aagagtttatcacccacagaaaaccattggaacaaatcaatgaagcatttgaagacatgcatgctggtg
attgtattagagctgttgtcaacatgtgg SEQ FAO1,
atggctccatttttgcccgaccaggtcgactacaaacacgtcgacacccttatgttattatgtg-
acgggat ID Candida
catccacgaaaccaccgtcgaccaaatcaaagacgttatttgctcctgacttccctgctgaca-
agtacga NO: (Nucleic Acid
agagtacgtcaggacattcaccaaaccctccgaaaccccagggttcagggaaaccgtctacaacaca
131 Seq.)
gtcaacgcaaacaccacggacgcaatccaccagttcattatcttgaccaagtttttggcatcca-
gggtct
tggctccagctttgaccaactcgttgacgcctatcaaggacatgagcnggaagaccgtgaaaaattgtt
ggcctcgtggcgcgactccccaatcgctgccaaaaggaagttgttcaggttggtttctacgcttaccttg
gtcacgucacgagattggccaatgagttgcatttgaaagccattcattatccaggaagagaagaccgt
gaaaaggcttatgaaacccaggagattgacccttttaagtaccagtttttggaaaaaccgaagtttacg
gcgctgagttgtaottgccagatattgatgtgatcattattggatctggtgccggtgctggtgttgtggcc
cacactttggccaacgatggcttcaagagtttggttttggaaaagggcaaatactttagcaactccgagt
tgaactttgatgacaaggacggcgttcaagaattataccaaagtggaggtactttgactacagtcaacc
aacagttgtttgttcttgctggttccacttttggtggcggtaccactgtcaattggtcagcctgtcttaaga-
c
gccattcaaggtgcgtaaggaatggtatgatgagtttggtgrtgactttgctgctgatgaagcatacgata
aagcgcaggattatgtttggcagcaaatgggagcttctaccgaaggcatcacccactctttggctaacg
agattattattgaaggtggtaagaaattaggttacaaggccaaggtattagaccaaaacagcggtggtc
atcctcagcacagatgcggtttctgttatttgggctgtaagcacggtatcaagcagggttctgttaataac
tggtttagagacgcagctgcccacggttcccagttcatgcaacaggttagagttttgcaaatacttaaca
agaaggggatcgcttacggtatcttgtgtgaggatgttgtaaccggcgccaagtcaccattactggcc
ccaaaaagtttgttgttgctgccggtgctttgaacactccatctgtgttggtcaactccggcttcaagaac
aagaacatcggtaagaacttaactttgcacccagtttctgtcgtgtttgagattttggaaagacgttcaa
gcagaccacttccacaactccatcatgactgccctttgttcagaagccgctgatttagacggcaagggc
catggatgcagaattgaaaccatcttgaacgctccattcatccaggcttcattcttaccatggagaggta
gtaacgaggctagacgagacttgttgcgttacaacaacatggtggcgatgttgctccttagtcgtgaca
ccaccagtggttccgtttctgctcatccaaccaaacctgaagctttggttgtcgagtacgacgtgaacaa
gtttgacagaaactcgatcttgcaggcattgttggtcactgctgacttgttgtatatccaaggrgccaaga
gaatccttagtccacaggcatgggtgccaatttttgaatccgacaagccaaaggataagagatcaatca
aggacgaggactttgtcgaatggagagccaaggttgccaagattcctttcgacacctacggctcacct
tatggttcggcacatcaaatgtcttcttgccgtatgtcaggtaagggtcctaaatacggtgctgttgacac
cgatggtagattgtttgaatgttcgaatgtttatgttgccgatgcaagtcttttgccaactgcaagcggtgc
caaccctatggtcaccaccatgactcttgccagacatgttgcgttaggtttggcagactccttgaagacc
aaagccaagttgtag SEQ FAO1,
MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDQIKDVIAPDFPAD ID Candida
KYEEYVRTFTKPSETPGFRETVYNTVNANTTDAIHQFIILTNVLA NO: (Amino Acid
SRVLAPALTNSLTPIKDMSLEDREKLLASWRDSPIAAKRKLFRLV 132 Seq.)
STLTLVTFTRLANELHLKAIHYPGREDREKAYETQEIDPFKYQFL
EKPKFYGAELVLPDIDVIIIGSGAGAGVVAHTLANDGFKSLVLEK
GKYFSNSEILNFDDKDGWEINQSGGITTTVNQQLFVLAGSTFG
GGTTVNWSACLKTPFKVRKEYVYDEFGVDFAADEAYDKAQDYV
WQQMGASTEGITHSLANEMEGGKKLGYIEAKVLDQNSGGHPQH
RCGFCYLGCKHGIKQGSVNNWFRDAAAHCSOFMOOVIWLQIL
NKKGIAYGILCWDVVTGAKFTITGPKKFVVAAGALNTPSVLVNS
FFKNKNIGKNLTLHPVSVVFCDFGKDVQADHHNSIMTALCSE
AADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNN
MVANILLLSRDTTSGSVSAIIPTKPEALVVEYDVNKFDRNSILQAL
INTADLLYIQOAKRILSPQAWVPIFESDKPKDKRSIKDEDYVEW
RAKVAKIPFDTYGSPYGSAHQMSSCGISGKGPKYGAVDTDGRL
FECSNVYVADASLLPTASGANMVTVTTMTLARHVALGLADSLKT KAKL SEQ
FAO1.DELTA.pts1,
atggctccatttttgcccgaccaggtcgactacaaacacgtcgacacccttatgttattatgtgacgggat
ID Candida
catccacgaaaccaccgtcgaccaaatcaaagacgttattgctcctgacttccctgctgacaa-
gtacga NO (Nucleic Acid
agagtacgtcaggacattcaccaaaccctccgaaaccccagggttcagggaaaccgtctacaacaca
133 Seq.)
gtcaacgcaaacaccacggacgcaatccaccagttcattatcttgaccaatgttttggcatcca-
gggtct
tggctccagctttgaccaactcgttgacgcctatcaaggacatgagcttggaagaccgtgaaaaattgtt
ggcctcgtggcgcgactcccaatcgctgccaaaaggaagttgttcaggttggtttctacgcttaccttg
gtcacgttcacgagattggccaatgagttgcatttgaaagccattcattatccaggaagagaagaccgt
gaaaaggcttatgaaacccaggagattgacccttttaagtaccagtttttggaaaaaccgaagttttacg
gcgctgagttgtacttgccagatattgatgtgatcattattggatctggtgccggtgctggtgttgtggc
cacactttggccaacgatggcttcaagagtttggttttggaaaagggcaaatactttagcaactccgagt
tgaactttgatgacaaggacggcgttcaagaattataccaaagtggaggtactttgactacagtcaacc
aacagttgtttgttcttgctggtccacttttggtggcggtaccactgtcaattggtcagcctgtcttaagac
gccattcaaggtgcgtaaggaatggtatgatgagtttggtgttgactttgctgctgatgaagcatacgata
aagcgagganatgtttggcagcaaatgggagcttctaccgaaggcatcacccactctttggctaacg
agattattattgaaggtggtaagaaattaggttacaaggccaaggtattagaccaaaacagcggtggtc
atcctcagcacagatgcggtttctgttatttgggctgtaagcacggtatcaagcagggttctgttaataac
tggtttagagacgcagctgcccacggttcccagttcatgcaacaggttagagttttgcaaatacttaaca
agaaggggatcgcttacggtatcttgtgtgaggatgttgtaaccggcgccaagttcaccattactggcc
ccaaaaaagtttgttgatgctgccggtgctttgaacactccatctgtgttggtcaactccggcttcaagaac
aagaacatcggtaagaacttaactttgcacccagtttctgtcgtgtttggtgattttggcaaagacgttcaa
gcagaccacttccacaactccatcatgactgccctttgttcagaagccgctgatnagacggcaagggc
catggatgcagaattgaaaccatcttgaacgctccattcatccaggcttcattcttaccatggagaggta
gtaacgaggctagacgagacttgttgcgttacaacaacatggtggcgatgttgctccttagtcgtgaca
ccaccagtggttccgtttctgctcatccaaccaaacctgaagctttggttgtcgagtacgacgtgaacaa
gtttgacagaaactcgatcttgcaggcattgttggtcactgctgacttgttgtatatccaaggtgccaaga
aggacgaggactatgtcgaatggagagccaaggttgccaagattcctttcgacacctacggctcacct
tatggttcggcatatcaaatgtcttcttgccgtatgtcaggtaagggtcctaaatacggtgctgttgacac
cgatggtagattgtttgaatgttcgaatgtttatgttgccgatgcaagtcttttgccaactgcaagcggtgc
caaccctggtcaccaccatgactcttgccagacatgttgcgttaggtttggcagactccttgaagacc
aaatag SEQ FAO1.DELTA.pts1,
MIAPFLPDQVDYKHVDTLMLLCDGIIHETTVDQIKDVIAPDFPAD ID Candida
KYEEYVRTFTKPSETPGFRETVYNTVNANTTDAIHQFIILTNVLA NO: (Amino Acid
SRVLAPALTNSLTPIKDMSLEDREKLLASVVRDSPIAAKRKLFRLV 134 Seq.)
STLTLVTFTRLANELHLKAIHYPGREDREKAYETQEIDPFKYQFL
EKPKFYGAELYLPDIDVIIIGSGAGAGWAHTLANDGEKSLVLEK
GKYFSNSELNFDDKDGyQELYQSGGTLITVNQGLFVLAGSTFG
GGTTVNWSACLKTPFKYRKEWYDEFGVDFAADEAYDKAQDYV
WQQMGASTEGITHSLANEITIEGGKKLGYKAKVLDQNSGGHPQR
RCGFCYLGCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVRVQIL
NKKGIAYGILCEDVVTQAKFTITGPKKFVVAAGALNTPSVLVNS
GFKNKNIGKNLTLHPVSVVFGDFGKDVQADFIFHNSIMTALCSE
AADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNN
MVAMLLLSRDTTSGSVSAHPTKPEALVVEYDVNKFDRNSILQAL
LVTADLLYIQGAKRILSPQAWVPIFESDKPKDKRSIKDEDYVEW
RAKVAKIPFDTYGSPYGSAHQMSSCRMSGKEPKYGAVDTDGRL
FECSNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKT K SEQ ALD1,
MLSRVLFKTKPRVPTKSITAMAIRNKSPVTLSSTIISTYPTDHTTPS ID Candida
TEPYITPSFVNNEFIKSDSNTWFDVHDPATNYVVSKVPQSTPEEL NO: (Amino Acid
EEAIASAHAAFPKWIDTSIIKRQGIAFISFVQLLRENMDKIASVIV 135 Seq.)
LEQGKTFVDAQGDVTRGLQVAEAACNITNDLKGESLEVSTDME
TKMIREPLGVVGSICPFNFPAMVPLWSLPLVLVTGNTAVIKPSER
VPGASMIKELAAKAGVPPGYLNIVHGKHDTVNKLIEDPRIKALT
FVGGDKAGKYIYEKGSSLGKRVQANLGAKNIHLVVLPDAHKQSF
VNAVNGAAFGAAGQRCMAISVLVTVGKTKEWVQDVIKDAKLL
NTGSGFDPKSDLGPVINPESLTRAEEHADSVANGAVLELDGRGY
RPEDARFAKGNFLGPTILTNVKPGLRAYDEEIFAPVLSVVNVDTI
DEAIELINNNKYGNGVSLFTSSGGSAQYFTKRIDVGQVGINVPIP
VPLPMFSFTGSRGSFLGDLNFYGKAGITFLTKPKTITSAWKINLI DDEILKPSTSMPVQQ SEQ
ALD1,
atgttatccagagttcttttcaagactaaaccaagagttcctactaaatcaatcaccgccatgg-
ccatcag ID Candida
aaacaaatccatcgtgactttatcctccaccacctccacatacccaaccgaccacacgacccc-
gtccac NO: (Nucleic Acid
ggagccatacatcacgccatccttcgtgaacaacgagttcatcaagtcggactccaacacctggttcga
136 Seq.)
cgtgcacgacccggccacgaactacgtcgtgtccaaggtgccacagtcgacgccggaggagttg-
ga
agaagcaatcgcgtcggcccacgccgcgttccccaaatggcgcgacaccagcatcatcaagcgtca
agggatcgcgttcaagtttgtgcagttgttgcgcgagaacatggacagaatcgcaagcgtcattgtctt
ggaacaaggtaagacgtttgtcgatgcccagggtgacgtgactagaggattacaggttgctgaggctg
cgtgcaacatcactaacgacttgaaaggtgagtcgttggaagtgtctactgatatggagaccaagatga
ttagagaacctttgggtgttgtgggatccatctgtccttttaacttcccagctatggtcccattgtggtctt-
tg
cctttggttttggtcacgggtaacaccgctgtgattaagccttccgagagagtcccgggcgcaagtatg
attatttgtgaattggccgccaaggcaggtgttccacctggtgtgttgattcattgtccacggtaagcacg
acaccgtcaacaagttgatcgaggacccaagaatcaaggcattgacttttgtcggtggtgacaaggcc
ggtaagtacatttacgaaaagggttccagtttgggcaagagagtgcaggccaacttgggtgctaagaa
ccacttggttgtgttgccagacgcacacaagcagagttttgtcaatgccgtcaagggtgccgctttcggt
gctgctggacagagatgtatggctattttctgtcttggtcaccgtgggtaagaccaaggaatgggtgcag
gatgtcatcaaggacgccaagttgttgaacaccggaagtggatttgacccaaagagtgacttgggtcc
agtcatcaacccagagtccttgactcgtgctgaagaaatcattgctgattccgtggccaacggtgc
gttggaattggacggaagaggatacagaccagaagacgccagattcgccaagggtaacttcttgggt
ccaaccatcttgaccaacgtcaagccgggcttgagagcatacgacgaggagattttcgctcctgttttgt
cggtggttaacgtcgacaccattgacgaagccattgagttgatcaacaataacaagtacggtaacggt
gtttcattatttacttcctccggtggctcagcccagtatttcactaagagaatcgatgtcggccaagtcggt
atcaatgtcccaatccctgttccattgcctatgttctcttcactggttccagaggctccttcttgggtgact-
t
gaacttctacggtaaggccggtatcaccttcttgaccaagccaaagaccatcactagtgcctggaaga
ccaacttgattgatgacgagatcttgaaaccatctacctcgatgcctgtccaacag SEQ ALD5,
MSLPVVTKLTTPKGLSYNQPLGLFINNEFVVPKSKQTFEWSPST ID Candida
EEKITDVYEALAEDVDVAAEAAYAAYEINDWALGAPEQRAKILL NO: (Amino Acid
KLADLVEEHAETLAQIETWDNGKSLQNARGDIGFTAAYFISCG 137 Seq.)
GWADKNTGDNINTGGTHLTYTQRVPLVCGQIIPWNASTLMASW
KLGPVIATGGTTVLKSAEATPLAVLYLAQLLVEAGLPKGVVNIV
SGFGTTAGSAIASHFKIDKVAFTGSTNTGKLIMKLAAESNLKKVT
LELGGKSPHIVFNDADLDRAVSYLVAAIFSNSGETCAAGSRVLV
QSGVYDEVVAKFKKGAEAVKVGDPFDEETFMGSQVNEVQLSRI
LQYIESGKEQGATVVTGGGRAGDKGYFVKPTIFADVHKDMTIV
KEEIFGPVVSVVKFDTIEEAIALANDSEYGLAAGIHTTNISTGVTV
ANRIKSGTVWVNTYNDLHPMVPFGGFGASGIGREMGAEVMKE YTEVKAVRIKLT SEQ ALD5,
atgtctttgccagtcgtcaccaaactcactactcctaagggtctctcctacaaccaaccattag-
gtttgttc ID Candida
atcaacaacgagttcgttgttccaaaatccaagcaaaccttcgaagtcttctccccttccacc-
gaagaga NO: (Nucleic Acid
agatcaccgatgtctacgaagctttagccgaagatgtcgacgttgctgccgaagcagcttacgccgcc
138 Seq.)
taccacaacgactgggcccttggtgctccagaacaaagagccaagatcttgctcaagttggccg-
actt
ggtcgaagaacacgccgagaccttggcccagatcgaaacctgggacaacggtaagtccttgcagaa
cgccagaggcgatatcggattcactgccgcttactttagatcctgtggtggatgggccgacaagaaca
ccggtgacaacatcaacaccggtggcacccaccttacttacacccagagagtcccattggtgtgtggt
caaatcatcccttggaacgcaagtaccttgatggccagttggaagcttggtcccgttatcgctaccggtg
gtaccactgtgcttaaatcagctgaagctaccccattagctgtcttgtacctcgcccaattgttagttgaag
ccggtcttccaaagggtgtcgttaacattgtttccggtttcggtaccactgccggttccgctatcgctagc
catccaaagatcgacaaggtcgcctttactggttccaccaacaccggtaagatcatcatgaagttggct
gcggagtccaacttgaagaaggtcactttggaattgggtggtaagtccccacacattgttttcaacgac
gctgacttggaccgcgccgtcagctacttggttgctgccattttcagtaactccggcgagacctgtgctg
ccggatcccgtgtcttggtgcaatccggtgtctacgacgaagttgttgctaagttcaagaagggcgccg
aggccgttaaagttggtgacccattcgacgaagaaaccttcatgggttcccaagtcaacgaagtccaat
tgtctagaatcttgtaatacatcgagctgggtaaggaacattggtgccactgttgtcaccggtggtggta
gagccggggacaagggttacttcgtcaagccaactattttcgccgacgttcacaaggacatgactatc
gtcaaggaagaaatctttggtcctgttgtctccgtcgtcaaattcgataccattgaagaagctatcgctttg
gctaacgactccgaatacggtttggccgctggtatccacaccactaacatcagcaccggtgtcaccgt
cgctaacagaatcaagtccggtactgtctgggtcaaacacttcaatgacttgcacccccatggttccattc
ggtggtttcggcgcttctggtatcggcagagaaatgggtgcagaagtcatgaaggaatacaccgaagt
taaggctgttagaattaagctcact SEQ HFD1,
MSKSYKLPKSSKISPIVKGKTSAKSKSSSKTPSPPSGSPPTSARIAA ID Candida
PELEPVEQTSDNELPATKVAVRRSSSASSKSTNGSAAATSAAAA NO: (Amino Acid
NAAAPQKTPVEAKPAPKPEPVQSKGNDNDSDDSKLDTAESYVD 139 Seq.)
VKKETEALVESKSVASTVDDTSVLQYTPLSEIPGGVKRVVDGFH
TGKTHPLEFRLKQLRNLYFAVRDNQEAICDALSKDFHRVSSETR
NYELVTGLNELLYTMSQLHKWSKPLPVDELPLNLMINPTYVERI
PVGTVLVIAAFNYPLFVSISPIAGAIAAGNTVVFKPSELTPHFSKL
FTDLMAKALDPDVDYAVNGSVPETTWLLNQKFDKIIYTGSETVG
KIIAKKAAETLTPVILELGGKSPAFVLDDVADKDLPIVARRIAWG
RYANAGQTCIGVDYVLVAKSKHDKFIKALRDVIEKEFFPNVDAN
SNFTHLIHDRAFHKMKNIIDKTTGKIIIGGQMDSASRYVSPTVIDD
ATWDDSSMQEEIFGPILPVLTYTDLTDACRDVVSHHDTPLAEYIF
TSGSTSRKYNSQINTIATIIRSGGLVINDVLMHIALHNAPFGGIGK
SGHGAYHGEFSYRAFTHERTVLEQNLWNDWIIKSRYPPYSNKK
DRLVASSQGKYGGRVWFGKQGDVKIDGPSTFFSAWTNVLGVA GVVCDFIGASL SEQ HFD1,
atgagtaagtcatacaagttgccaaaatcatctaagatctcgcctatcgtaaagggtaagacct-
cggca ID Candida
aaatcaaaaagtagctcaaaaacaccatcaccaccactgggatccccaccaacatcagccagg-
atcg NO: (Nucleic Acid
ctgcgcctgaattggaaccggtcgaacaaacatccgacaacgagctcccagctactaaagtggccgtt
140 Seq.)
cgcagaagcagcagcgcttcatcgaagtcaaccaacgggtccgcggctgccacttctgccgctg-
ctg
ccaatgctgctgccccacaaaaaactccagttgaagcaaagccagcccctaagccagagccagttca
gtccaagggtaacgacaacgactccgatgactccaaguagacaccgctgaatcatatgtcgacgtga
agaaagaaaccgaagctctcgttgagtcaaagtcggttgcttcaacagtcgatgatacttcttgca
gtacaccccgttatctgagatccctggcggcgtcaagagagttgtcgatgggttccacaccggcaaga
cccacccattggagtttagattgaagcaattgagaaacttgtacttcgccgtgagggataaccaagaag
ccatctgtgacgccttgagcaaggatttccaccgtgtctcatccgaaactagaaactatgaattggttac
cgggttgaatgaattgttgtacacgatgtcgcaattgcacaagtggagcaagccattgccagtggatga
gttgccattgaacttgatgatcaaccctacttatgttgaaagaatcccagttggtacggttttggtcatcgc
cgctttcaactatccattgtttgtttcgatttcccctattgccggtgccattgctgctggtaacactgttgt-
gtt
caagccatctgaattgactccgcacttttccaagttgttcactgatttgatggctaaagcgttggaccag
atgtcttttatgccgtgaacggttccgtgcctgaaactaccgagttgttgaaccagaagttcgacaagat
catttacaccggtagtgaaactgttggtaagatcattgccaagaaggcggccgagacgttgacgccag
tgattttggaacttggaggcaaatcgccagcctttgttttggacgacgttgccgacaaagacttgccaatt
gttgcgcgtcgtattgcttggggaagatatgctaatgctggacagacctgtattggtgttgattatgtgttg
gttgcaaagtccaaacacgacaagttcatcaaggctttgagggatgttattgagaaagagttcttcccca
atgttgacgctaacagcaactttacccatttgatccacgacagggcattccacaaaatgaagaacatcat
tgacaaaaccacagggaagataatcatcggtggtcaaatggatagtgcttccaggtatgtctcaccaac
tgtgattgacgatgctacctgggacgactcgtccatgcaggaggaaatatttggccctatcttgccggtt
ctcacttatactgaccttactgatgcatgtcgtgacgttgtttctcaccatgataaccgttggctgaataca
tcttcaccagcgggtccacttcaagaaagtacaactcgcagatcaacaccattgctactatcatcagatc
cggtgggttagtcataaatgatgtgttgatgcacattgccttgcacaacgctccgttcggtggtattggta
aatctggacatggtgcctaccacggagaattctcctacagggcttttacccacgagagaaccgttcttg
aacaaaacttgtggaatgactggatcattaagtcaagatacccaccatattccaataagaaagacaggtt
ggttgctagttcacaaggcaagtatggtggaagagtttggttcggacgtcaaggtgatgtcaagattga
cggaccatccacgttcttctcggcatggaccaatgtccnggtgtagctggtgtagtgtgtgattttattgg
tgcctctttg SEQ HFD2, MSPPSKLEDSSSATTAADTLGDSWYTKVSDIAPGVQRLTESFHR
ID Candida DQKTHDIQFRLNQLRNLYFAVQDNADALCAALDKDFYRPPSET NO: (Amino
Acid KNLELVGGLNELVHTISSLHEWMKPEKVTDLPLTLRSNPIYIERIP 141 Seq.)
LGVVLIISPFNYPFFLSFSAVVGAIAGGNAVVLKGSELTPNFSSLF
SKILTKALDPDIFFAVDGAIPETTELLEQKFDKIMYTGNNTVGKII
AKKAAETLTPVILELGGKSPAFILDDVKDKNLEVIARRIAWGRFT
NAGQTCandidaAVDYVLVPTKLHKKFIAALTKVLSQEFYPNLTK
DTKGYTHVIHDRAFNNLSKIISTTKGDIVFGGDTDAATRFIAPTVI
DNATWEDSSMKGEIFGPILPVLTYDKLTTAIRQVVSTHDTPLAQ
YIFTSGSTSRKYNRQLDQILTGVRSGGVIVNDVLMHVALINAPFG
GVGDSGYGSYHGKFSFRSFTHERTTMEQKLWNDGMVKVRYPP
YNSNKDKLIQVSQQNYNGKVWFDRNGDVPVNGPGALFSAWTT FTGVFHLLGEFITNKQ SEQ
HFD2,
atgtccccaccatctaaattagaagactcctcctccgcaaccaccgctgccgatacccttggcg-
actcc ID Candida
tggtacaccaaagtgtccgacattgcgcctggcgtgcagagattgaccgagtcattccacagg-
gatca NO: (Nucleic Acid
aaagacgcacgacattcagttccgcttgaaccaattgcgtaacctttactttgcggtccaggacaatgcc
142 Seq.)
gacgcgctctgtgctgccttggacaaggacttctaccgtccccccagtgaaaccaagaacttgg-
aact
cgtgggtggcttgaatgagttggtgcacaccatttcgagcttgcatgagtggatgaagccggaaaaag
tcacggatttgccacttactttgaggtcaaacccgatttatattgaaagaatcccattgggggtcgtgttga
tcatctcgcctttcaactaccctttcttcttgtcgttttcggccgtcgtgggtgcgattgctggtggtaacg-
c
ggttgttttgaagggctctgagttgacgccaaagttctccagtttgttctcaaagatcttgactaaggcttt-
g
gaccctgatattttctttgcagtcgatggtgctatccctgagacgaccgagttgttggaacaaaagtttga
caagatcatgtatactggtaacaacaccgtgggtaagattattgccaagaaggctgctgagaccttgac
gccagttatcttggaattgggtggtaagtcgccagctttcatcttggacgacgtcaaggataaaaacttg
gaagtcatcgccagaagaatcgcatggggtagattcaccaacgccggtcaaacctgtgttgctgtcga
ctacgtcttggttccaaccaaactccacaagaagttcattgctgcgttgaccaaggtcttgagtcaagaa
ttctaccctaacttgaccaaagacaccaagggctacacccacgtcatccacgaccgtgcattcaacaat
ttgtccaagatcatcagcaccaccaagggtgacattgtctttggcggcgacaccgatgccgccacccg
cttcatcgcccccaccgtcatcgacaacgccacctgggaggattcttccatgaagggcgaaatctttgg
tcccatcttgcccgtcttgacctacgacaagctcaccaccgccatcaggcaagttgtgtccacgcacga
cacgccattagcgcagtacatcttcaccagcgggtccacatcccgcaagtacaaccgccagctcgac
cagatcttgactggtgtccggtccgggggtgtgattgtcaacgatgtcttgatgcacgttgcgttgatca
atgcgccatttggcggcgttggtgactccgggtacggctcgtaccacggcaagttctcgttccgcagct
tcacgcacgaacgtaccaccatggagcagaagttgtggaacgacgggatggtcaaggtcagatacc
ctccttataactccaacaaggacaagttgatccaggtctcccagcagaactacaacggcaaggtctggt
tcgatagaaacggcgacgtgcctgtgaatggaccaggtgcgttgtttagcgcttggactacgttcactg
gtgtcttccatttgcttggtgagttcatcactaataagcaataga SEQ MAE1 (non-
MVSSTATASATSGEMRTVKTPVGIKAAIESLKPKATRVSMDGPV ID mitochondrial),
ECPLTDFALLNSPQFNKGSAFSLEERKSFKLTGLLPSQVNTLDEQ NO: Candida
VERAYRQFTYLKTPLAKNDFVTSMTLQNKVLYYELVRRNIREM 143 (Amino Acid
LPIIYTPTEGDAIASYSDRFRKPEGCFLDINDPDNIDERLAAYGEN Seq.)
KDIDYIVMSDGEGIXXXSDRFRKPEGCFLDINDPDNIDERLAAYG
ENKDIDYIVMSDGEGILGIGDQGVGGIRIAIAKLGLMTLCGGIHP
ARVLPITLDVGTNNDRLLNDDLYMGNKFPRVRGERYWDFVDK
VIHAITKRFPSAVMHYEDFGVTTGRDMLHKYRTALPSFNDDIQG
TGAVVMASITAALKFSNRSLKDIEVLIYGAGSAGLGIADQITNHL
VSHGATPEQARSRIHCMDRYGLITTESNNASPAQMNYADKASD
WEGVDTSSLLACandidaEKVKPTVLVGCSTQAGAFTEEVVKTMY
KYNPQPIIFPLSNPTRLHEAVPADLMKWTDNNALIATGSPFEPVD
GYYISENNNCFTFPGIGLGAVLSRCSTISDTMISAAVDRLASMSP
KMENPKNGLLPRLEEIDEVSAHVATAVILQSLKEGTARVESEKK
PDGGYVEVPRDYDDCLKWVQSQMWKIWYRPYIKVEYVSNIHT YQY SEQ MAE1 (non-
atggtctcctccacggccaccgcatccgccacgtcaggggaaatgcgtaccgtcaagaccccagtgg
ID mitochondrial),
ggatcaaggcggccatcgaatcattaaaaccaaaagctactagagtctccatggacggacctgtcgaa
NO: Candida
tgcccattgaccgatttcgccttgttgaactcccacaattcaacaaaggttcggcattttctttggaagaa
144 (Nucleic Acid
aggaaaagtttcaagttgaccgggctcctcccttctcaagtcaacactttggatgaacaggttgaaaga
Seq.)
gcctatagacaattcacatacttgaagaccccattggccaagaacgatttctgcacgtctatgagat-
tgc
agaacaaagtgctttactacgagttggttagaagaaatatccgtgagatgttgcccatcatctacacccc
aaccgaaggggacgccatcgccagttattccgacaggttcagaaaaccagagggctgtttcttggata
tcaacgaccccgacaacatcgatgagagattagctgcctatggggagaacaaagacatagattacatt
gtcatgagtgacggagaaggtatnnnnnnnnctccgacaggttcagaaaaccagagggctgcttc
ttggacatcaatgacccagacaacatcgacgagagattggctgcctatggggagaacaaagacatag
attacattgtcatgagtgacggagaaggtatccticggtattggagaccaaggcgtcggtggtatcagaa
ttgccattgctaaattggggttgatgaccctttgtggtggtattcacccggccagagtttttcccatcactt-
t
ggatgttggtacaaataacgacaggttgttgaatgatgatttgtacatgggcaacaagttccctagagtc
agaggagaaagatactgggactttgtcgataaggtcatacacgcaattacgaaacggttcccaagtgc
cgtgatgcattacgaagatttcggagtcacaactggtagggacatgttgcacaagtaccgtacggctct
tccttctttcaacgacgacatccaaggtaccggtgcagttgtcatggcatcgatcacagctgccttgaag
ttctccaaccgtagcctaaaggacatcgaggttttgatttacggtgccggctcagctggtttaggtattgc
tgaccagatcaccaaccacttggtcagccacggcgctactccagaacaagccagatctaggatccatt
gtatggaccgttatgggttgatcacaactgaatccaacaacgccagtcctgctcaaatgaactacgccg
acaaggcatctgattgggaaggtgtcgatacctcgagtctacttgcctgtgttgagaaagtcaaaccaa
ctgtcttggttgggtgttccactcaggcaggtgcattcaccgaagaggttgtcaaaaccatgtacaagta
caacccacagccaattattttcccattgtccaaccctaccagattgcatgaagccgtgccggctgatttg
atgaaatggaccgacaacaacgcgttgattgccaccggttctccatttgaacctgtcgatggctactaca
tttccgaaaacaacaactgtttcaccttcccaggtattgggttgggtgctgccttgtccagatgtagcacc
atttcggataccatgatttctgccgccgttgatagattggcttcgatgtcgccaaagatggagaacccaa
agaacggattgttgcctagattggaagaaatcgacgaagtcagtgcccatgttgccacggctgttatctt
gcaatctttgaaggaaggcaccgctagagtcgaaagcgagaagaagccagacggtggttacgttga
agttccaagagactatgatgattgtcttaagtgggtgcaatcacaaatgtggaagccagtgtacagacc
atacatcaaggttgagtacgtttcgaatattcacacctatcaatat SEQ PYC2, Sc
MPESRLQRLANLKIGTPQQLRRTSIIGTIGPKTNSCEAITALRKAG ID (Amino Acid
LNIIRLNFSHGSYEEHQSVIENAVKSEQQFPGRPLAIALDTKGPEI NO: Seq.)
RTGRTLNDQDLYIPVDHQMIFTTDASFANTSNDKIMYIDYANLT 145
KVIVPGRFIYVDDGILSFKVLQIIDESNLRVQAVNSGYIASHKGV
NLPNTDVDLPPLSAKDMKDLQFGVRNGIHIVFASFIRTSEDVLSI
RKALGSEGQDIKIISKIENQQGLDNFDEILEVTDGVMIARGDLGIE
ILAPEVLAIQKKLIAKCNLAGKPVICATQMLDSMTHNPRPTRAE
VSDVGNAVLDGADCandidaMLSGETAKGDYPVNAVNIMAATAL
IAESTIAHLALYDDLRDATPKPTSTTETVAAAATAAILEQDGKAI
VVILSTTGNTARLLSKYRPSCPIILVTRHARTARIAHLYRGVFPFL
YEPKRLDDWGEDVHRRLKFGVEMARSFGMVDNGDTVVSIQGF KGGVGHSNTLRISTVGQEF* SEQ
PYC2, Sc ATGCCAGAGTCCAGATIGCAGAGACTAGCTAATTTGAAAATA ID (Nucleic
Acid GGAACTCCGCAGCAGCTCAGACGCACGTCCATAATAGGTACC NO: Seq.)
ATTGGGCCCAAGACAAATAGCTGCGAGGCCATTACTGCTCTG 146
AGAAAAGCTGGTTTGAACATCATTCGATTCAACTTTTCCCATG
GCTCCTACGAATTCCATCAATCAGTAATCGAAAATGCTGTGA
AATCGGAACAGCAATTCCCTGGCAGGCCGCTCGCCATTGCCG
TGGATACCAAGGGTCCCGAGATCAGAACAGGTCGCACGTTAA
ATGACCAAGATCTTTATATCCCCGTAGACCACCAAATGATCTT
TACCACTGACGCAAGTTTTGCAAACACCTCCAATGATAAAAT
CATGTATATAGACTATGCTAACCTGACAAAAGTTATCGTTCC
GGGGAGATTTATATACGTGGACGACGGGATTCTCTCTTTTAA
AGTGCTCCAAATCATTGACGAATCTAATTTAAGGGTGCAAGC
GGTAAACTCGGGTTATATCGCATCTCATAAAGGTGTTAATCT
GCCTAATACCGACGTTGATTTGCCCCCGTTGTCCGCCAAAGAC
ATGAAGGACTTGCAATTCGGAGTCCGCAATGGCATTCACATC
GTATTTGCCTCTTTCATAAGAACTTCAGAAGATGTGTTGTCTA
TCAGAAAAGCGTTGGGTTCTGAAGGGCAAGATATCAAGATTA
TATCCAAGATAGAAAACCAGCAAGGGTTGGATAATTTTGACG
AAATCCTGGAAGTCACGGATGGTGTTATGATAGCGAGAGGCG
ATTTAGGAATTGAAATCCTGGCACCTGAAGTATTAGCCATTC
AAAAAAAGCTGATTGGAAAATGTAATTTGGCGGGCAAACCTG
TCATTTGCGCGACTCAGATGCTGGATTCAATGACACACAATC
CGAGACCGACAAGGGCTGAAGTATCGGATGTGGGTAACGCT
GTGTTGGATGGTGCTGATTGTGTTATGCTTTCTGGAGAAACGG
CGAAGGGTGATTATCCGGTGAATGCAGTTAATATTATGGCGG
CGACCGCTCTGATTGCTGAAAGTACTATCGCTCATTTGGCTCT
TTATGACGATCTCAGAGACGCCACTCCCAAACCTACTTCCACT
ACGGAAACTGTAGCAGCGCAGCTACCGCAGCAATCTTGGAG
CAAGATGGTAAGGCCATCGTTGTATTATCTACTACAGGGAAC
ACGGCAAGGCTACTGTCGAAGTATAGACCAAGCTGCCCTATC
ATATTAGTAACAAGACACGCAAGAACGGCAAGAATTGCGCA
TTTGTATAGAGGTGTTTTCCCATTTCTGTATGAACCGAAACGC
CTAGACGACTGGGGTGAGGATGTTCATAGGCGCCTAAAGTTT
GGTGTTGAAATGGCGAGGTCTTTCGGAATGGTGGACAACGGT
GATACTGTTGTTTCCATTCAAGGATTCAAAGGAGGAGTCGGC
CATTCCAATACCTTACGCATTTCTACTGTTGGTCAAGAATTCT AG SEQ FAT1 S244A,
atgtcaggattagaaatagccgctgcigccatccttggtagtcagttattggaagccaaatatttaattgc
ID ATCC20336
cgacgacgtgctgttagccaagacagtcgctgtcaatgccctcccatacttgtggaaagccagcagag
NO: (Nucleic Acid
gtaaggcatcatactggtactttttcgagcagtccgtgttcaagaacccaaacaacaaagcgttggcgtt
147 seq.)
cccaagaccaaagaaagaatgcccccacccccaagaccgacgccgagggattccagatctacga-
cg
atcagtttgacctagaagaatacacctacaaggaattgtacgacatggttttgaagtactcatacatctt
aagaacgagtacggcgtcactgccaacgacaccatcggtgtttcttgtatgaacaagccgcttttcattg
tcttgtggttggcattgtggaacattggtgccttgcctgcgttcttgaacttcaacaccaaggacaagcca
ttgatccactgtcttaagattgtcaacgcttcgcaagttttcgttgacccggactgtgattccccaatcaga
gataccgaggctcagatcagagaggaattgccacatgtgcaaataaactacattgacgagtttgccttg
tttgacagattgagactcaagtcgactccaaaacacagagccgaggacaagaccagaagaccaacc
gatactgactcctccgcttgtgcattgatttacaccgcgggtaccaccggtttgccaaaagccggtatca
tgtcctggagaaaagccttcatggcctcggttttctttggccacatcatgaagattgactcgaaatcgaac
gtcttgaccgccatgcccttgtaccactccaccgcggccatgttggggttgtgtcctactttgattgtcgg
tggctgtgtctccgtgtcccagaaattctccgctacttcgttctggacccaggccagattatgtggtgcca
cccacgtgcaatacgtcggtgaggtctgtcgttacttgttgaactccaagcctcatccagaccaagaca
gacacaatgtcagaattgcctacggtaacgggttgcgtccagatatatggtctgagttcaagcgcagat
tccacattgaaggtatcggtgagttctacgccgccaccgagtcccctatcgccaccaccaacttgcagt
acggtgagtacggtgtcggcgcctgtcgtaagtacgggtccctcatcagcttgttattgtctacccagca
gaaattggccaagatggacccagaagacgagagtgaaatctacaaggaccccaagaccgggttctg
taccgaggccgcttacaacgagccaggtgagttgttgatgagaatcttgaaccctaacgacgtgcaga
aatccttccagggttattatggtaacaagtccgccaccaacagcaaaatcctcaccaatgttttcaaaaa
aggtgacgcgtggtacagatccggtgacttgttgaagatggacgaggacaaattgttgtactttgtcga
cagattaggtgacactttccgttggaagtccgaaaacgtctccgccaccgaggtcgagaacgaattga
tgggctccaaggccttgaagcagtccgtcgttgtcggtgtcaaggtgccaaaccacgaaggtagagc
ctgttttgccgtctgtgaagccaaggacgagttgagccatgaagaaatcttgaaattgattcactctcac
gtgaccaagtctttgcctgtgtatgctcaacctgcgttcatcaagattggcaccattgaggcttcgcacaa
ccacaaggttcctaagaaccaattcaagaaccaaaagttgccaaagggtgaagacggcaaggatttg
atctactggttgaatggcgacaagtaccaggagttgactgaagacgattggtctttgatttgtaccggta
aagccaaattgtag SEQ FAT1 S244A,
msgleiaaaailgsqlteakyliaddvslaktvavnalpylwkasrgkasywyffeqsvfknpnnk
ID ATCC20336
alafprprknaptpktdacgfqiyddqfdlccytykclydmylkysyilkncygvtandtigvscm
NO: (Amino Acid
nkplfivlwlalwnigalpaflnfntkdkplihclkivnasqvfvdpdcdspirdtcaqireelphvq
148 seq.)
rnyidcfalfdrlrlkstpkhracdktrrptdtdssacaliytagttglpkagimswrkafmas-
vffghi
mkidsksnvltamplyhstaamlglcptlivggCandidasvsqkfsatsfwtqarlcgathvqyv
gevcryllnskphpdqdrhnvriaygnglrpdiwsefkrrfhiegigefyaaespiattnlqygey
gvgacrkygslislllstqqklakmdpedeseiykdpktgfeteaaynepgellmrilnpndvqksf
qgyygnksatnskiltnvfkkgdawyrsgdllkmdedkllyfvdrlgdtfrwksenvsatevenel
mgskalkqsvvvgvkvpnhegracfavceakdelsheeilkihshvtkslpvyaqpafikigtie
ashnhkvpknqfknqklpkgedgkdliywlngdkyqelteddwslictgkakl SEQ FAT1
D495A,
atgtcaggattagaaatagccgctgctgccatccttggtagtcagttattggaagccaaatatttaattgc
ID ATCC20336
cgacgacgtgctgttagccaagacagtcgctgtcaatgccctcccatacttgtggaaagccagcagag
NO: (Nucleic Acid
gtaaggcatcatactggtactttttcgagcagtccgtgttcaagaacccaaacaacaaagcgttggcgtt
149 Seq.)
cccaagaccaagaaagaatgcccccacccccaagaccgacgccgagggattccagatctacgac- g
atcagtttgacctagaagaatacacctacaaggaattgtacgacatggtmgaagtactcatacatcttg
aagaacgagtacggcgtcactgccaacgacaccatcggtgtttcttgtatgaacaagccgcttttcattg
tcttgtggttggcattgtggaacattggtgccttgcctgcgttcttgaacttcaacaccaaggacaagcca
ttgatccactgtcttaagattgtcaacgcttcgcaagttttcgttgacccggactgtgattccccaatcaga
gataccgaggctcagatcagagaggaattgccacatgtgcaaataaactacattgacgagtttgccttg
tttgacagattgagactcaagtcgactccaaaacacagagccgaggacaagaccagaagaccaacc
gatactgactcctccgcttgtgcattgatttacacctcgggtaccaccggtttgccaaaagccggtatcat
gtcctggagaaaagccttcatggcctcggttttctttggccacatcatgaagattgactcgaaatcgaac
gtcttgaccgccatgcccttgtaccactccaccgcggccatgttggggttgtgtcctactttgattgtcgg
tggctgtgtctccgtgtcccagaaattctccgctacttcgttctggacccaggccagattatgtggtgcca
cccacgtgcaatacgtcggtgaggtctgtcgttacttgttgaactccaagcctcatccagaccaagaca
gacacaatgtcagaattgcctacggtaacgggttgcgtccagatatatggtctgagttcaagcgcagat
tccacattgaaggtatcggtgagttctacgccgccaccgagtcccctatcgccaccaccaacttgcagt
acggtgagtacggtgtcggcgcctgtcgtaagtacgggtccctcatcagcttgttattgtctacccagca
gaaattggccaagatggacccagaagacgagagtgaaatctacaaggaccccaagaccgggttctg
taccgaggccgcttacaacgagccaggtgagttgttgatgagaatcttgaaccctaacgacgtgcaga
aatccttccagggttattatggtaacaagtccgccaccaacagcaaaatcctcaccaatgttttcaaaaa
aggtgacgcgtggtacagatccggtgccttgttgaagatggacgaggacaaattgttgtactttgtcga
cagattaggtgacactttccgttggaagtccgaaaacgtctccgccaccgaggtcgagaacgaattga
tgggctccaaggccttgaagcagtccgtcgttgtcggtgtcaaggtgccaaaccacgaaggtagagc
ctgttttgccgtctgtgaagccaaggacgagttgagccatgaagaaatcttgaaattgattcactctcac
gtgaccaagtctttgcctgtgtatgctcaacctgcgttcatcaagattggcaccattgaggcttcgcacaa
ccacaaggttcctaagaaccaattcaagaaccaaaagttgccaaagggtgaagacggcaaggatttg
atctactggttgaatggcgacaagtaccaggagttgactgaagacgattggtctttgatttgtaccggta
aagccaaattgtag SEQ FAT1 D495A,
Msgleiaaaailgsqlleakyliaddvslaktvaavnalpylwkasrgkasywyffeqsvfknpnnk
ID ATCC20336
alafprprknaptpktdaegfqiyddqfdleeytykelydmvlkysyilkneygvtandtigvscm
NO: (Amino Acid
nkplfivlwlalwnigalpaflnfntkdkplihclkivnasqvfvdpdspirdtcaqireelphvq
150 seq.)
inyidcfalfdrlrlkstpkhracdktrrptdssacaliytsgttglpkagimswrkafmasvf-
fghi
mkidsksnvltamplyhslaamlglcptlivggcvsvsqkfsatsfwlqarlcgathvqyvgcvcr
yllnskphpdqdrhnvriaygnglrpdiwscfkrrfhicgigcfyaatcspiattnlqygcygvgac
rkygslislllstqqklakmdpedeseiykdpktgfcteaaynepgellmrilnpndvqksfqgyy
gnksatnskiltnvfkkgdawyrsgalkmdedlkkyvdrlgdtfrwksenvsatevenelmgs
kalkqsvvvgvkvpnhegracfavceakdelshceilklihshvtkslpvyaqpafikigtieashn
hkvpknqfknqklpkgedgkdliywlngdkyqelteddwslictgkakl SEQ SFA 1
atgtctgaatcaaccgttggaaagtatgtcaccttaacaattgagtctcgtaattgctcgccat-
tgggaa ID (Nucleic Acid
atcatcggctgtatttataatccagatctttcctaccctagtagttaccacagaacaattgcaacagaataa
NO: Seq.) genomic
atactaacctttttgtcttagccaatcacctgtaaagccgctgttgcctgggaagcaggcaagcctt
151
catcgaagacgtcactgttgctccaccaaaggcccacgaagtgcgtatcaagatcttgtacactggtgt
ctgtcacactgatgcctacaccttgagtggtgttgatccagagggtgccttcccagtcatcttgggacac
gaaggtgccggtattgttgaaagtgttggtgaaggtgtcaccactgtcaagccaggcgaccacgtcatt
gcattatacactccagaatgtggtgagtgtaagttctgtaaatcgggtaagaccaacttgtgtggtaaaat
cagagctacccaaggcaaaggtgtgatgccagacggaactccaagattcacttgcaagggcaaaga
attgattcactttatgggatgctccaccttctcccaatacaccgttgtcactgacatttccgtcgtggccat-
c
aacgacaaagccgaacttgacaaggcttgtttgttgggatgtggtatcactactggttatggtgctgcca
ccatcactgccaatgttcaaaagggtgacaatgtcgcagttttcggtggtggtgctgtcggattgtccgt
cctccaaggatgtaaagaaagagaagctgcccaaatcattttggttgatgtcaacaacaagaagaagg
aatggggtgaaaagttcggtgccactgcttttatcaacccattagaattaccaaaaggcgtcactattgtt
gacaagttgattgaaatgattgacggtggttgtgactttacttttgactgtaccggtaatgtcaatgttatg
agagatgccttggaagcttgtcataagggttggggtacttcagtcatcatcggtgttgccgctgccggta
aggaaatctccaccagaccattccaattggtcactggtagagtctggaagggtgctgctttcggtggcg
tcaagggtagatcccaattgccaggaatcgttgaagactacatggttggcaagttgaaggttgaagagtt
tatcacccacagaaaaccattggaacaaatcaatgaagcatttgaagacatgcatgctggtgattgtatt
agagctgttgtcaacatgtgg SEQ ADH1-1short,
MSANIPKTQKAVVFEKNGGELEYKDIPVPTPKANELLINVKYSG ID Candida
VCHTDLHAWKGDWPLATKLPLVGGHEGAGVVVGMGENVKG NO: (Amino Acid
WKIGDFAGIKWLNGSCMSCEFCQQGAEPNCGEADLSGYTHDGS 152 Seq.)
FEQYATADAVQAAAIPAGTDLAEVAPILCAGVTVYKALKTADL
AAGQWVAISGAGGGLGSLAVQYAVAMGLRVVAIDGGDEKGAF
VKSLGAEAYIDFLKEKDIVSAVKKATDGGPHGAINVSVSEKAID
QSVEYVRPLGKVVLVGLPAGSKVTAGVFEAVVKSIEIKGSYVGN
RKDTAEAVDFFSRGLIKCPIKIVGLSELPQVFKLMEEGKILGRYV LDTSK SEQ
ADH1-1short,
atgtctgctaatatcccaaaaactcaaaaagctgtcgtctttgagaagaacggtggtgaattagaataca
ID Candida
aagatatcccagtgccaaccccaaaggccaacgaattgctcatcaacgtcaaatactcgggtg-
tctgc NO: (Nucleic Acid
cacactgatttgcacgcctggaagggtgactggccattggccaccaagttgccattggttggtggtcac
153 Seq.)
gaaggtgctggtgtcgttgtcggcatgggtgaaaacgtcaagggctggaagattggtgacttcg-
ccgg
tatcaaatggttgaacggttcctgtatgtcctgtgagttctgtcaacaaggtgctgaaccaaactgtggtg
aggccgacttgtctggttacacccacgatggttctttcgaacaatacgccactgctgatgctgttcaagc
cgccagaatcccagctggtactgatttggccgaagttgccccaatcttgtgtgcgggtgtcaccgtcta
caaagccttgaagaagccgacttggccgctggtcaatgggtcgctatctccggtgctggtggtggttt
gggttccttggctgtccaatacgccgtcgccatgggcttgagagtcgttgccattgacggtggtgacga
aaagggtgcctttgtcaagtccttgggtgctgaagcctacattgatttcctcaaggaaaaggacattgtct
ctgctgtcaagaaggccaccgatggaggtccacacggtgctatcaatgtttccgtttccgaaaaagcca
ttgaccaatccgtcgagtacgttagaccattgggtaaggttgttttggttggtttgccagctggctccaag
gtcactgctggtgttttcgaagccgttgtcaagtccattgaaatcaagggttcctatgtcggtaacagaaa
ggataccgccgaagccgttgactttttctccagaggcttgatcaagtgtccaatcaagattgttggcttga
gtgaattgccacaggtcttcaagttgatggaaggtaggttaagatcttgggtagatacgtcttggatacctc
caaa SEQ ADH1-2 MSANIPKTQKAVVFEKNGCELKYKDIPVPTPKANELLINVKYSG ID
short, Candida VCHTQLHAWKGDWPLDTKLPLVGGHEGAGVVVGMGENVKG NO: (Amino
Acid WKIGDFAGIKWLNGSCMSCEFCQQGAEPNCGEADLSGYTHDGS 154 Seq.)
FEQYATADAVQAARIPAGTDLAEVAPILCAGVTVYKALKTADL
AAGQWVAISGAGGGLGSLAVQYAVAMGLRVVAIDGGDEKGDF
VKSLGAEAYIDFLKEKQIVAAVKKATDGGPHGAINVSVSEKAID
QSVEYVRPLGKVVLVGLPAGSKVTAGVFEAVVKSIEIKGSYVGN
RKDTAEAVDFFSRGLIKCPIKIVGLSELPQVFKLMEEGKILGRYV LDTSK SEQ ADH1-2
atgcatgcattattctcaaaatcagtttttctcaagtatgtgagtctgcccactacctctgct-
atcccccattc ID short, Candida
cctagaattcattgtctcccgaagctcctatttaaggagacgaattcccccatatcttccacgttgctccca
NO: (Nucleic Acid
ctttccttccttctattattcttcttcttcagtctacaccaagaaatcatttcacacaatgtctgctaatatc-
cca 155 Seq.)
aaaactcaaaaagctgtcgtcttcgagaagaacggtggtgaattaaaatacaaagacatcccag-
tgcc
aaccccaaaggccaacgaattgctcatcaacgtcaagtactcgggtgtctgtcacactgatttgcacgc
ctggaagggtgactggccattggacaccaaattgccattggttggtggtcacgaaggtgctggtgttgt
tgtcggcatgggtgaaaacgtcaagggctggaaaatcggtgatttcgccggtatcaaatggttgaacg
gttcttgtatgtcctgtgagttctgtcagcaaggtgctgaaccaaactgtggtgaagctgacttgtctggtt
acacccacgatggttctttcgaacaatacgccactgctgatgctgtgcaagccgccagaatcccagct
ggcactgatttggccgaagttgccccaatcttgtgtgctggtgtcaccgtctacaaagccttgaagactg
ccgacttggctgctggtcaatgggtcgctatctccggtgctggtggtggtttgggctccttggctgtcca
atacgccgtcgccatgggtttgagagtcgttgccattgacggtggtgacgaaaagggtgactttgtcaa
gtccttgggtgctgaagcctacattgatttcctcaaggaaaagggcattgttgctgctgtcaagaaggcc
actgatggcggtccacacggtgctatcaatgtttccgtttccgaaaaagccattgaccaatctgtcgagt
acgttagaccattgggtaaggttgttttggttggtttgccagctggctccaaggtcactgctggtgttttcg
aagccgttgtcaagtccattgaaatcaagggttcttacgtcggtaacagaaaggatactgccgaagcc
gttgactttttctccagaggcttgatcaagtgtccaatcaagattgtgggcttgagtgaattgccacaggt
cttcaagttgatggaagaaggtaagatcttgggtagatacgtcttggatacctccaaa SEQ
MAE1, Sc MWPIQQSRLYSSNTRSHKATTTRENTFQKPYSDEEVTKTPVGSR ID (Amino
Acid ARKIFEAPHPHATILTVEGAIECPLESFQLLNSPLENKGSAFTQEE NO: Seq.) in
Spec. REAFNLEALLPPQVNTLDEQLERSYKQLCYLKTPLAKNDFMTSL 156
RVQNKVLYFALIRRHIKELVPIIYTPTEGDAIAAYSHRFRKPEGVF
LDITEPDSIECRLATYGGDKDVDYIVVSDSEGILGIGDQGIGGVRI
AISKLALMTLCGGLEIKRVLFVCLPVCTNNKKLARDELYMGNK
FSRIRGKQYDDFLEKFIKAVKKVYPSAVIEFEDFGVKNARRLLE
KYRYELPSENDDIQGICAVVMASLIAALKHTNRDLKDTRVLIYG
AGSAGLGIADQIVNHMVIHGVDKEEARKKIFLIVIDRRGLILQSVE
ANSTPAQHVYAKSDAEWAQINTRSIHDVVENVKPTCLVGCSTQ
AGAFTQDVVEEMHKHNPRPIIFPLSNPTRLHEAVPADLMKWTN
NNALVATGSPFPPVDGYRISENNNCYSFPGIGLGAVLSRATTITD
KMISAAVDQLAELSPLREGDSRPGLLPGLDITTNTSARLATAVIL
QALEEPTARIEQEQVPGGAPGETVKVPRDFDECLQWVKAQMW EPVYKPMIKVQHDPSVHTNQL*
SEQ ZWF1, MSFDPEGSTATIVVFGASGDLAKKKTFPALFGLFREGHLSSDVKI ID
Scheffersomy IGYARSHLEEDDFKKRISANFKGGNPETVEQFLKLTSYISGPYDT NO: ccs
stipitis DEGYQTLLKSIEDYEAANNVSTPERLFYLALPPSVFTTVASQLKK 157 (Amino
Acid NVYSPTGKTRIIVEKPFGHDLESSRQLQKDLSPLFTEEELYRIDHY Seq.)
LGKEMVKNLLVLRFGNELFNGVWNKNHIKSIQISFKEAFGTDGR
GGYFDSIGIVRDVMQNHLLQVLTLLTMDRPVSFDPEAVRDEKV
KILKAFDALDPEDILLGQYGKSEDGSKPGYLDDSTVPKDSKCandi
daTYAALGIKIHNERWEGVPIVMRAGKALDESKVEIRIQFKPVAR
GMFKEIQRNELVIRVQPNESIYLKINSKIPGTSTETSLTDLDLTYST
RYSKDFWIPEAYEALIRDCYLGNHSNFVRDDELDVSWKLFTPLL
QYIESDKSPQPEAYAYGSKGPKGLREFLNKHDYIFADEGTYQWP LTTPKVKGKI* SEQ ACS2B,
MPALFKDSAQHILDTIKSELPLDPLKTAYAVPLENSAEPGYSAIY ID Candida
RNKYSTDKLIDTPYPGLDTLYKLFEVSTEANGDKPCLGGRVKNA NO: (Amino Acid
DGTFGEYKFQDYNTIHQRRNNLGSGIFFVLQNNPYKTNSEAHSK 158 Seq.) (from
LKYDPTSKDSFILTIFSHNRPEWALCDLTSIAYSITNTALYDTLGP spec)
DTSKYILGLTESPIVVCSKDKIRGLIDLKKNNPDELSNLIVLVSMD
DLTTADASLKNYGSEHNVTVYDIKQVEKLGEINPLDPIEPTPDTN
FTITFTSGTTGANPKGVLLNHRNAVAGVTFVLSRYDGQFNPTAY
SFLPLAHIYERASIQFALTIGSAIGFPQGPSPLTLIEDAKVLQPDGL
ALVPRVLTKLEAAIRAQTVNNDEKPLVKSVFGAAINAKMEAQM
KEENENFNPSFIVYDRLLNLLRKKVGLQKVSQISTGSAPISPSTIQ
FLKASLNVGILQGYGLSESFAGCMASSKFEPAAVTCGPPGITTEV
KLKDLEEMGYTSKDEGGPRGELLLRGPQIFKEYFKNPEETAEAI
DEDGWFHTGDVAKINNKGRISIIDRAKNFFKLAQGEYVTPEKIEG
LYLSKFPYIAQIFVHGDSKESYLVGVVGLDPVAGKQYMESRFHD
KIIKEEDVVEFFKSPRNRKILLQDMNKSIADQLQGFEKLHNIYVD
FDPLTVERGVITPTMKIRRPLAAKFFQDQIDAMYSEGSLVRNGSL
* SEQ ACS2C, MPALFKDSAKHIFDTIKSELPLDPLKTAYAVPLENSAEPGYSAIY ID
Candida RNKYSIDKLIDTPYPGLDTLYKLFEVATEAYGDKPCLGARVKNA NO: (Amino
Acid DGTFGEYKFQDYNTIHQRRNNFGSGIFFVLQNNPYKTDSEAHSK 159 Seq.) (from
LKYDPTSKDSFILTIFSHNRPEWALCDLTSIAYSITNTALYDTLGP spec)
DTSKYILGLTESPIVKSKDKIRGLIDLKKNNPDELSNLIVLVSMD
DLTTADASLKNYGSEHNVTVFDIKQVEKLGEINPLDPIEPTPDTN
FTITFTSGTTGANPKGVLLNHRNAVAGVTFVLSRYDGHFNPTAY
SFLPLAHIYERASIQFALTIGSAIGFPQGPSPLTLIEDAKVLQPDGL
ALVPRVLTKLEAAIRAQTVNNDEKPLVKSVFGAAINAKMEAQM
KEENENFNPSFIVYDRLLNLLRKKVGLQKVTQISTGSAPISPSTIQ
FLKASLNVGILQGYGLSESFAGCMASSKFEPAAATCGPTGVTTE
VKLKDLEEMGYTSKDEGGPRGELLLRGPQIFKGYFKNPEETAKA
IDEDGWFHTGDVAKINDKGRISIIDRAKNFFKLAQGEYVTPEKIE
GLYLSKFPYIAQLFVHGDSKESYLVGVVGLDPVAGKQYMESRF
HDKIIKEEDVVEFFKSPRNRKILVQDMNKSIADQLQGFEKLHNIY
VDFDPLTVERGVITPTMKIRRPLAAKFFQDQIDAMYSEGSLVRN GSL* SEQ PAA073
aaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgtt-
ctttcctgc ID (Nucleic Acid
gttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccga
NO: Seq.)
acgaccgagcgcagcgagtcagtgagcgaggaagcgagttcggcatggcagatcatcatgcctg-
ca 160
ggagctccaattgtaatatttcgggagaaatatcgttggggtaaaacaacagagagagagagggaga
gatggttctggtagaattataatctggttgttgcaaatgctactgatcgactctggcaatgtctgtagctcg
ctagttgtatgcaacttaggtgttatgcatacacacggttattcggttgaattgtggagtaaaaattgtctg-
a
gttgtgtcttagctactggctggccccccgcgaaagataatcaaaattacacttgtgaatttttgcacaca
caccgattaacatttcccttttttgtccaccgatacacgcttgcctcttcttttttttctctgtgcttcccc-
ctcct
gtgactttttccuccattgatataaaatcaactccatttccctaaaatctccccagattctaaaaacaactt-
ct
tctcttctgcttttcctttttttttgttatatttatttaccatcccttttttttgaatagttattccccact-
aacattgttc
aaatcttcacgacataagaagagcccgggtctagatgtgtgctcttccgagtgactcttttgataagagt
cgcaaatttgatttcataagtatatattcattatgtaaagtagtaaatggaaaattcattaaaaaaaaagca-
a
atttccgttgtatgcatactccgaacacaaaactagccccggaaaaacccttagttgatagttgcgaattt
aggtcgaccatatgcgacgggtacaacgagaattgtattgaattgatcaagaacatgatcttggtgttac
agaacatcaagttcttggaccagactgagaatgcacagatatacaaggcgtcatgtgataaaatggatg
agatttatccacaattgaagaaagagtttatggaaagtggtcaaccagaagctaaacaggaagaagca
aacgaagaggtgaaacaagaagaagaaggtaaataagtattttgtattatataacaaacaaagtaagg
aatacagatttatacaataaattgccatactagtcacgtgagatatctcatccattccccaatcccaaga
aaaaaaaaaagtgaaaaaaaaaatcaaacccaaagatcaacctccccatcatcatcgtcatcaaaccc
ccagctcaattcgcaatggttagcacaaaaacatacacagaaagggcatcagcacacccctccaagg
ttgcccaacgtttattccgcttaatggagtccaaaaagaccaacctctgcgcctcgatcgacgtgacca
caaccgccgagttcctttcgctcatcgacaagctcggtccccacatctgtctcgtgaagacgcacatcg
atatcatctcagacttcagctacgagggcacgattgagccgttgcttgtgcttgcagagcgccacgggt
tcttgatattcgaggacaggaagtttgctgatatcggaaacaccgtgatgttgcagtacacctcgggggt
ataccggatcgcggcgtggagtgacatcacgaacgcgcacggagtgactgggaagggcgtcgttga
agggttgaaacgcggtgcggagggggtagaaaaggaaaggggcgtgttgatgttggcggagttgtc
gagtaaaggctcgttggcgcatggtgaatatacccgtgagacgatcgagattgcgaagagtgatcgg
gagttcgtgattgggttcatcgcgcagcgggacatggggggtagagaagaagggtttgattggatcat
catgacgcctggtgtggggttggatgataaaggcgatgcgttgggccagcagtataggactgttgatg
aggtggttctgactggtaccgatgtgattattgtcgggagagggttgtttggaaaaggaagagaccctg
aggtggagggaaagagatacagggatgctggatggaaggcatacttgaagagaactggtcagttag
aataaatattgtaataaataggtctatatacatacactaagcttctaggacgtcattgtaatttcgaagttg
tctgctagtttctcatgatttcgaaaaccaataacgcaatggatgtagcagggatggtggttagtgc
gttcctgacaaacccagagtacgccgcctcaaaccacgtcacattcgccctttgcttcatccgcatcact
tgcttgaaggtatccacgtacgagttgtaatacaccttgaagaacggcttcgtctacgcgcgagacgaa
agggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggca
cttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctca-
tg
agacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgt
cgcccttattccctttttgcggcaattgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaa
gatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatcctt
gagagttttcgccgcgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtatt
atcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttga
gtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccat
aaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaacc
gcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagcc
ataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaa
ctggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcag
gaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtg
ggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacga
cggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaa
gcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaa-
aag
gatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagc
gtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgca
aacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaa
ggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccacca
cttcaagaactctgtagcaccgcctacatacctcgctgctaatcctgttaccagtggctgctgccagt
ggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgg
gctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacc
tacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaa
gcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttata
gtcctgtcgggtttcgccacctctgatttgagtgtcgatttttgtgatgctcgtcaggggggcggagcct
atggaa SEQ pAA298
gatctggaatccctcggcgtcggtcttgggggtgggggcattctttcttggtcttgggaacgc-
caacgc ID (Nucleic Acid
tttgttgtttgggttatgaacagggactgctcgaaaaagtaccagtatgatccttacctctgctggctttc
NO: Seq.)
cacaagtatgggagggcattgacagcgactgtcttggcgtacagcacgtcgtcggcaattaaat-
atttg 161
gcttccaataactgactaccaaggatggcagcagcggctatttctaatcctgacatgtttctcgtacgt-
ag
tagtgaatgaagggaaggtggaatttatatcaagggcgaattctgtcagatatccatcacaatggcggcc
gctcgagcatgcatctagagggcccaattcgccctatagtgagtcgtattacaattcactggccgtcgtt
ttacaacgtcgtggqtgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcg
ccagaggcgtaatagcgaagaggcccgcaccgatcgccatcccaacagttgcgcagcctatacgt
acggcagtttaaggtttacacctataaaagagagagcagttatcgtctgtttgtggatgtacagagtgata
ttattgacacgccggggcgacggatggtgatccccctggccagtgoadgtctgctgtcagattaagtc
tcccgtgaactttacccggtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggc
cagtgtgccggtctccgttatcggggaagaagtggctgatacagctaccgcgaaaatgacatcaaaa
acgccattaacctgatgttctggggaatataatttgtcaggcatgagattatcaaaaaggatcttcaccta
gatccttttcacgtagaaagccagtccgcagaaacggtgagttccccggatgaatgtcagctactggg
ctatctggacaagggaaaacgcaagcgcaaagagaaagcaggtagcttgcagtgggcttacatagc
gatagctagactgggcggttttatggacagccagcgaaccggaattgccagctggggcgctctctgg
taaggttgggaagccctgcaaagtaaactggatggctttctcgccgccttaggatctgatggcgcagg
ggatcaagctctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgc
aggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgct
ctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccg
gtgccctgaatgaactgcaagacgaggcagcgcggctatcgtactggccacgatgggcgttccttg
cgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggg
gcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcg
gctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagca
cgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgc
cagccgaactgttcgccaggctcaaggcgagcatgcccgacggagaggatctcgtcgtgacccatg
gcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggct
gggtgtgggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggc
gaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatc
gccttcttgacgagttctrctgaattattaacgcttacaatttcctgatgcggtattttctccttacgcatc-
tgt
gcggtatttcacaccgcatacaggtggcacttttcggggaaatgtgcgcggaacccctatttgttattttt
ctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatagcacgtga
ggagggccaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccgga
gcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtg
tggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccc
tggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacga
acttccgggacgcctccgggccaccatgaccgagatcggcgagcagccgtgggggcgggagttc
gccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtgcta
aaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaa-
cg
tgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttct-
g
cgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagag
ctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgta
gccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgtta
ccagtggctgctgccagaggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggat
aaggcgcagcggtcgggctgaacgggggttcgtgcacacagcccagcttggagcgaacgaccta
caccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggc
ggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccaggggga
aacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcg-
t
caggggggcggagcctatggaaaaacgccagcaacgcggccttttttacggttcctgggcttttgctgg
ccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtga-
gc tgatacpgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagag
cgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtt
tcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcacccc
aggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacagga
aacagctatgaccatgattacgccaagctatttaggtgacactatagaatactcaagctatgcatcaagc
ttggtaccgagctcggatccactagtaacggccgccagtgtgctggaattcgcccttccgttaaacaaa
aatcagtctgtaaaaaaggttctaaataaatattctgtctagtgtacacattctcccaaaatagtgaaatcc
agctctacaatttggctttaccggtacaaatcaaagaccaatcgtcttcagtcaactcctggtacttgcg
ccattcaaccagtagatcaaatccttgccgtcttcaccctttggcaacttttggttcttgaattggttctta-
gg
aaccttgtggttgtgcgaagcctcaatggtgccaatcttgatgaacgcaggttgagcatacacaggcaa
agacttggtcacgtgagagtgaatcaatttcaagatttcttcatggctcaactcgtccttggcttcacaga
cggcaaaacaggctctaccttcgtggtttggcaccttgacaccgacaacgacggactgcttcaaggcc
ttggagcccatcaattcgttctcgacctcggtggcggagacgttttcggacttccaacggaaagtgtca
cctaatctgtcgacaaagtacaacaatttgtcctcgtccatcttcaacaagtcaccggatctgtaccacgc
gtcaccttttttgaaaacattggtgaggattttgctgttggtggcggacttgttaccataataaccctggaa
ggatttctgcacgtcgttagggttcaagattctcatcaacaactcacctggctcgttgtaagcggcctcg
gtacagaacccggtcttggggtccttgtagatttcactctcgtcttctggtccatcttggccaatttctgct
gggtagacaataacaagctgatgagggacccgtacttacgacaggcgccgacaccgtactcaccgta
ctgcaagttggtggtggcgataggggactcggtggcggcgtagaactcaccgataccttcaatgtgga
atctgcgcttgaactcaaccatatatctggacgcaacccgttaccgtaggcaattctgacattgtgtctg
tcttggtctggatgaggcttggagttcaacaagtaacgacagacctcaccgacgtattgcacgtgggtg
gcaccacataatctggcctgggtccagaacgaagtagcggagaatttctgggacacggagacacag
ccaccgacaatcaaagtaggacacataccccaacatggccgcggtggagtggtacaagggcatggc
ggtcaagacgttcgatttcgagtcaatcttcatgatgtggccaaagaaaacgaggccatgaaggctttt
ctccaggacatgataccggcttttggcaaaccggtggtacccgaggtgtaaatcaatgcacaagcgga
ggagtcagtatcggttggtcttctggtcttgtcctcggctctctgttttggagtcgacttgagtctcaatct-
g
tcaaacaaggcaaactcgtcaatgtagtttatttgcacatgtggcaattcctctctgatctgagcctcggta
tctctgattggggaatcacagtccgggtcaacgaaaacttgcgaagcgttgacaatcttaagacagtgg
atcaatggcttgtccttggtgttgaagttcaagaacgcaggcaaggcaccaatgttccacaatgccaac
cacaagacaatgaaaagcggcttgttcatacaagaaacaccgatggtgtcgttggcagtgacgccgta
ctcgttcttcaagatgtatgaatacttcaaaaccatatcgtacaattccttgtaggtgtattcttctaggtc-
aa
actgatcgtcgtagatccactagtaacggccgccagtgtgctatggaattcgcccttgggctaacgaaaa
ggaaaccgctgacgttaaagatatctatggttgtttcggtatgacccccggggatctgacgggtacaa
cgagaattgtattgaattgatcaagaacatgatcttggtgttacagaacatcaagttcttggaccagactg
agaatgcacagatatacaaggcgtcatgtgataaaatggatgagatttatccacaattgaagaaagagt
ttatggaaagtggtcaaccagaagctaaacaggaagaagcaaacgaagaggtgaaacaagaagaa
gaaggtaaataagtattttgtattatataacaaacaaagtaaggaatacagatttatacaataaattgccat
actagtcacgtgagatatctcatccattcccaactcccaagaaaaaaaaaagtgaaaaaaaaaaaaatca
aacccaaagatcaacctccccatcatcatcgtcatcaaacccccagctcaattcgcaatggttagcaca
aaaacatacacagaaagggcattcagcacacccctccnaggttgcccaacgtttattccgcttaatgga
gtccaaaaagaccaacctctgcgcctcgatcgacgtgaccacaaccgccgagttcctttcgctcatcg
acaagctcggtccccacatctgtctcgtgaagacgcacatcgattttcatctcagacttcagctacgagg
gcacgattgagccgttgcttgtgcttgcagagcgccacgggttcttgatattcgaggacaggaagtttg
ctgatatcggaaacaccgtgatgttgcagtacacctcgggggtataccggatcgcggcgtggagtga
catcacgaacgcgcacggagtgactgggaaggcgtcgttgaagggttgaaacgcggtgcggagg
gggtagaaaaggaaaggggcgtgttgatgtnggcggagttgtcgagtaaaggctcgttggcgcatg
gtgaatatacccgtgagacgatcgagattgcgaagagtgatcgggagttcgtgattgggttcatcgcg
cagcgggacatgggggtagagaagggtttgattggatcatcatgacgcctggtgtggggttgg
atgataaaggcgatgcgttgggccagcagtataggactgttgatgaggtggttctgactggtaccgatg
tgattattgtcgggagagggttgtttggaaaaggaagagaccctgaggtggagggaaagagatacag
ggatgctggatggaaggcatacttgaagagaactggtcagttagaataaatattgtaataaataggtcta
tatacatacactaagcttctaggacgtcattgtagtcttcgaagttgtctgctagtttagttctcatgattt-
cg
aaaaccaataacgcaatggatgtagcagggatggtggttagtgcgttcctgacaaacccagagtacgc
cgcctcaaaccacgtcattcgccctttgcttcatccgcatcacttgcttgaaggtatccacgtacgagt
tgtaatacaccttgaagaacggcttcgtctacggtcgacgacgggtacaacgagaattgtattgaattga
tcaagaacatgatcttggtgttacagaacatcaagttcttggaccagactgagaatgcacagatatcaa
ggcgtcatgtgataaaatggatgagatttatccacaattgaagaaagagtttatggaaagtggtcaacca
gaagctaaacaggaagaagcaaacgaagaggtgaaacaagaagaagaaggtaaataagtattttgta
ttatataacaaacaaagtaaggaatacagatttatacaataaattgccatactagtcacgtgagatatctc
atccattccccaactcccaagaaaaaaaaaagtgaaaaaaaaaatcaaacccaaagatcaacctccc
catcatcatcgtcatcaaacccccagctcaattcgcagagctcggtacccggg SEQ >Piece
A (5'
gtttgtctatgttttccgtttgccttttctttctagtacgagacgttattgaacgaagtttttatatatctag-
atc ID untranslated
aatacatattccatgtctgttcatttttgacggagtttcataaggtggcagtttctaatcaaaggtccgtcat-
t NO: region of
ggcgtcgtggcattggcggctcgcatcaactcgtatgtcaatattttctgttaactccgccagacatacg
162 FAO1 (from atcaaaacctacaageaaaaaaattccae position -500 to -250)
(Nucleic Acid Seq.) SEQ >Piece B-
cgacgggtacaacgagaattgtattgaattgatcaagaacatgatcttggtgttacagaacatcaagttct
ID URA3 marker
tggaccagactgagaatgcacagatatacaaggcgtcatgtgataaaatggatgagatttatccacaat
NO: (Nucleic Acid
tgaagaaagagtttatggaaagtggtcaaccagaagctaaacaggaagaagcaaacgaagaggtga
163 Seq.)
aacaagaagaagaaggtaaataagtattttgtattatataacaaacaaagtaaggaatacagat-
ttatac
aataaattgccatactagtcacgtgagatatctcatccattccccaactcccaagaaaaaaaaaaagtga
aaaaaaaaatcaaacccaaagatcaaactccccatcatcatcgtcatcaaacccccagctcaattcgca
atggttagcacaaaaacatacacagaaagggcatcagcacacccctccaaggttgcccaacgtttatt
ccgcttaatggagtccaaaaagaccaacctctgcgcctcgatcgacgtgaccacaaccgccgttgttc
ctttcgctcatcgacaagctcggtccccacatctgtctcgtgaagacgcacatcgatatcatctcagactt
cagctacgagggcacgattgagccgttgcttgtgcttgcagagcgccacgggttcttgatattcgagga
caggaagtttgctgatatcggaaacaccgtgatgttgcagtacacctcgggggtataccggatcgcgg
ctttggagtgacatcacgaacgcgcacggagtgactgggaagggcgtcgttgaagggttgaaacgc
ggtgcggagggggtagaaaaggaaaggggcgtgttgatgttggcggagttgtcgagtaaaggctcg
ttggcgcatggtgaatatacccgtgagacgatcgagattgcgaagagtgatcgggagttcgtgattgg
gttcatcgcgcagcgggacatggggggtagagaagaagggtttgattggatcatcatgacgcctggt
gtggggttggatgataaaggcgatgcgttgggccagcagtataggactgttgatgaggtggttctgact
ggtaccgatagattattgtcgggagacggttaggaaaaggaagagaccctgaggtggagggaa
agagatacagggatgclggatggaaggcatacttgaagagaactggtcagttagaataaatattgtaat
aaataggtctatatacatacactaagcttctaggacgtcattgtagtcttcgaagttgtctgctagtttagt-
t
ctcatgatttcgaaaaccaataacgcaatggatgtagcagggatggtggttagtgcgttcctgacaaac
ccagagtacgccgcctcaaaccacgtcacattcgccctttgcttcatccgcatcacttgcttgaaggtat
ccacgtacgagttgtaatacaccttgaagaacggcttcgtct SEQ >Piece C
Gttaactccgccagacatacgatcaaaacctacaagcaaaaaaattccac ID (Nucleic Acid
NO: Seq.) 164 SEQ >Piece D-
Gagctccaattgtaatatttcgggagaaaatatcgttggggtaaaacaacagagagagagagggagag
ID Promoter
atggttctggtagaattataatctggttgttgcaaatgctactgatcgactctggcaatgtctgtagctcgct
NO: POX4
agttgtatgcaacttaggtgatgcatacacacggttattcggttgaattgtggagtaaaaattgt-
ctgag 165 (Nucleic Acid
ttgtgtcttagctactggctggccccccgcgaaagataatcaaaattacacttgtgaatttttgcacacac
Seq.)
accgattaacatttcccttttttgtccaccgatacacgcttgcctcttcttttttttctctgtgctt-
ccccctcct
gtgactttttccaccattgatataaaatcaactccatttccctaaaatctccccagattctaaaaaacaact-
tct
tctcttctgcttttcctttttttttgttatatttatttaccatcccttttttttgaatagttattccccact-
aacattgttc aaatcttcacgacata SEQ >Piece D-
Gaagatgaagcgtatgagtattatgagtactgtcggacgttggaaggtggcagagttaagcccgaga
ID Promoter
aagcaaggaaggagtgggagatgatgagtgatgcggccaaagaggatgtgaaggctgcgtatctgtt
NO: POX11
tttgatagctggtggtagccgaatagaggaaggcaagcttgttcatattggatgatgatggtag-
atggtg 166 (Nucleic Acid
gctgccattagtggttgtaaatagaaaaaagtgggtttgggtctgttgatttgttagtggtggcggctgtct
Seq.)
gtgattacgtcagcaagtagcacctcggcagttaaaacagcagcaacagaaaaaaaatgtgtgaaag-
t
ttgattcccccacagtctaccacacccagagttccatttatccataatatcacaagcaatagaaaaataaa
aaattatcaacaaatcacaacgaaaagattctgcaaaattattttcacttcttcttttgacttcctcttctt-
g ttaggttctttccatattttccccttaaacccatacacaacgcagcc SEQ >Piece D-
Ctagcaaaggcttgatcagagaaagcaacaaaaaaaaaaactctaatactccagaatacactccttta
ID Promoter
gaaacacacaacaaacaagcctagactaccatggacctacgatgaagacgatttagattacatttctcaa
NO: TEF1
ggagaagaggaagagtttgacgaaaacaagttgaacaacgaagagtacgacttgttgcatgacat-
gc 167 (Nucleic Acid
ttccggagttgaagacaaaattgaaagattacaatgatgagatcccagattacgatttaaaggaagcgtt
Seq.)
atactacaactatttcgagatagaccctaccattgaagaattgaagacgaaattcaaaaagagtacg-
tat
atacaactaacatcaacgcctttctagtttctgttctgtctccaatgcttctcctggtttcttcatggttct-
ctct
gtaccaacaaggaaaaaaaaaaaaatctggcaaaaaaaaccaaaccaaccaatgttcttactcaccaa
cgccctacaatc SEQ >Piece E-
atggctccatttttgcccgaccaggtcgactacaaacacgtcgacacccttatgttattatgtgacgggat
ID First 250 bp of
catccacgaaaccaccgtggacgaaatcaaagacgtcattgcccctgacttcccgccgacaaatac
NO: the coding
gaggagtacgtcaggacattcaccaaaccctccgaaaccccagggttcagggaaaccgtctacaaca
168 sequence of ccgtcaacgcaaacaccatggatgcaatccaccagttcattatct FAO1.
(Nucleic Acid Seq.) SEQ >PGKpromoter
ttgtccaatgtaataatttttccatgactaaaaagtgtgtgttggtgtaaagaagaaagtggaagggacgt
ID _Candida_A
tggtgatggtgagttcgtctatcccttttttatagttgcttgtatagtaggaactcttctagggactcgatgg
NO: TCC20336
gggaaggttcttgatatttgcttagttcgagaaggttccagatgagcgagacatttttggtacgacattgg
169
gtggatgatctgcacgacattttgtgattcttgcgacacgctgcactaccaagtgtgagtctggctgaa-
cg
gatcacaagataaacctctgaaaaattatctcagggcatgcaacaacaattatacatagaagagggagt
cacgatatacacctgtgaaggaatcatgtggtcggctctccttgaactttgaattcatgcaattattaagaa
gaagcacaggtgagcaacccaccatacgttcatttgcaccacctgatgattaaaagccaaagaaagaa
aaaaaaaaaagaaacaggcggtgggaattgttacaacccacgcgaacccgaaaatggagcaatcttc
cccggggcctccaaataccaactcacccgagagagagaaagagacaccacccaccacgagacgg
agtatatccaccaaggtaagtaactcagggttaatgatacaggtgtacacagctccttccctagccattg
agtgggtatcacatgacactggcaggttacaaccacgtttagtagttattttgtgcaattccatggggatc
aggaagtttggtttggtgggtgcgtctaagattcccctttgtctctgaaaatcttttccctagtggaacact
ttggctgaatgatataaattcaccttgattcccaccctcccttctttctctctctctctgttacacccaatt-
gaa
ttttcttttttttttttttactttccctccttctttatcatcaaagataagtaagtttatcaattgcctatt-
caga SEQ pAA335
ggtttgattggatcatcatgacgcctggtgtggggttggatgataaaggcgatgcgttgggcc-
agcagt ID
ataggactgttgatgaggtggttctgactggtaccgatgtgattattgtcgggagagggttgtttggaaa
NO:
aggaagagaccctgaggtggagggaaagagatacagggatgctggatggaaggcatacttgaaga
170
gaactggtcagttagaataaatattgtaataaataggtctatatacatacactaagcttctaggacgtc-
att
gtagtcttcgaagttgtctgctagtttagttctcatgatttcgaaaaccaataacgcaatggatgtagcagg
gatggtggttagtgcgttcctgacaaacccagagtacgccgcctcaaaccacgtcacattcgccctttg
cttcatccgcatcacttgcttgaaggtatccacgtacgagttgtaatacaccttgaagaacggcttcgtct
acgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttctta
gacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaa
tatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagta
ttcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaa-
cgc
tggtcaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaac
agcggtaatccttgagagttttcgccccgaagaacgaagaacgtttccaatgatgagcacttttaaagttct-
gct
atgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctca
gaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcaigacagtaagagaatt
atgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggac
cgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccgg
agctgaatgaagccataccaaacgacgagcgtgarcaccacgatgcctgtagcaatggcaacaacgtt
gcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggc
ggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctgga
gccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgt
agttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggt
gcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaactt-
ca
tttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagtt-
ttc
gttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttcrgcgcgtaa
tctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaa
ctctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtag
ttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtgg
ctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagtttccggataaggcg
cagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccga
actgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggaca
ggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgc
ctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcagg-
g
gggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttg
ctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgata-
c cgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcgagttcggcatgg
cagatcatcatgcctgcaggaagatgaagcgtatgagtattatgagtactgtcggacgttggaaggtgg
cagagttaagcccgagaaagcaaggaaggagtgggagatgatgagtgatgcggccaaagaggatg
tgaaggctgcgtatctgtttttgatagctggtggtagccgaatagaggaaggcaagcttgttcatattgg
atgatgatggtagatggtggctgccaaagtggttgtaaatagaaaaaagtgggtttgggtctgttgatag
ttagtggtggcggctgtctgtgattacgtcagcaagtagcacctcggcagttaaaacagcagcaacag
aaaaaaaatgtgtgaaagtttgattcccccacagtctaccacacccagagttccatttatccataatatca
caagcaatagaaaaataaaaaattatcaacaaatcacaacgaaaagattctgcaaaattattttcacttctt
cttttgacttcctcttcttcttgttaggttctttccatattttccttaaacccatacacaacgcagccagaa-
g
agcccgggtctagatgtgtgctcttccgctccaggcttgttatgactctagagagaagtgtgtgtgtgtgt
gtgcgtttgttttactatacattcaacatgttctttttcttttttgatatttattccaactataattataca-
cagattc
gtatatactttactttaccttctttcgtagttttttaatttgatgatttttgagtttcatatccaaggtcaa-
aaccc
gaccgaattcatatgcgacgggtacaacgagaattgtattgaattgatcaagaacatgatcttggtgtta
cagaacatcaagttcttggaccagactgagaatgcacagatatacaaggcgtcatgtgataaaatggat
gagatttatccacaattgaagaaagagtttatggaaagtggtcaaccagaagctaaacaggaagaagc
aaacgaagaggtgaaacaagaagaagaaggtaaataagtattttgtattatataacaaacaaagtaag
gaatacagatttatacaataaattgccatactagtcacgtgagatatctcatccattccccaactcccaag
aaaaaaaaaaagtgaaaaaaaaaatcaaacccaaagatcaacctccccatcatcatcgtcatcaaacc
cccagctcaattcgcaatgttagcacaaaaacatacacagaaagggcatcagcacacccctccaag
gttgcccaacgtttattccgcttaatggagtccaaaaagaccaacctctgcgcctcgatcgacgtgacc
acaaccgccgagttcctttcgctcatcgacaagctcggtccccacatctgtctcgtgaagacgcacatc
gatatcatctcagacttcagctacgagggcacgattgagccgttgcttgtgcttgcagagcgccacgg
gttcttgatattcgaggacaggaagtttgctgatatcggaaacaccgtgatgttgcagtacacctcgggg
gtataccggatcgcggcgtggagtgacatcacgaacgcgcacggagtgactgggaagggcgtcgtt
gaagggttgaaacgcggtgcggagggggtagaaaaggaaaggggcgtgttgatgttggcggagtt
gtcgagtaaaggctcgttggcgcatggtgaatatacccgtgagacgatcgagattgcgaagagtgatc
gggagttcgtgattgggttcatcgcgcagcgggacatggggggtagagaagaag SEQ CvMIG1
MLTPKPKKNKEDRPYKCTYCDKAFHRLEHQTRHIRTHTGEKPH ID
ACTFPGCVKRFSRSDELTRHLRIHTNPSSRKRRNKSQDLMGPTP NO:
MSINGQNLPPGSYAVTNTGIPFAIDRNGNHVYPQPYPVFFVPQPN 172
GYMQPVVQAPGLSIVPPPPQQQQQQGQAPQQQQQQLHSQQQQ
QRMGSPHSNMTTPTHLQQEGSAVFSIPSSPTNSYQNVPNRPNQQ
PLEPPQPVTMSTPMSTRSMSSDAIRLPPLTSNNSFQQQQQQQPRP
PPTILKSESTTSKDSNRVFSQPNSNLHSLGTSPDTSPSAMPPPTVV
PTPSFSNLNEYFQQKSNNPRIFNASSSSLSSLSGKIRSTSSTNLAGL
QRLTPLVQTSTNSTPGKSIIPSQPSSTSLNLEFYNQGNASHASKKS
RPNSPCQSAMNISSIMSSPSETPLQTPSQSPRLHNGPGNSSVIEGA
QAKLESIATTGTQLPPIRSVLSFTNLSDYPPPPSNR SEQ ScMIG1-
MQSPYPMTQVSNVDDGSLLKESKSKSKVAAKSEAPRPHACPICH ID Amino Acid
RAFHRLEHQTRHMRIHTGEKPHACDFPGCVKRESRSDELTRHRR NO:
IHTNSHPRGKRGRKKKVVGSPINSASSSATSIPDLNTANFSPPLPQ 173
QHLSPLIPIAIAPKENSSRSSTRKGRKTKFEIGESGGNDPYMVSSP
KTMAKIPYSVKPPPSLALNNMNYQTSSASTALSSLSNSHSGSRLK
LNALSSLQMMTPIASSAPRTVFIDGPEQKQLQQQQNSLSPRYSNT
VILPRPRSLTDFQGLNNANPNNNGSLRAQTQSSVQLKRPSSVLSL
NDLLVGQRNTNESDSDFTTGGEDEEDGLKDPSNSSIDNLEQDYL
QEQSRKKSKTSTPTTMLSRSTSGTNLHHTLGYVMNQNHLHFSSSS
PDFQKELNNRLLNVQQQQQEQHTLLQSQNTSNQSQNQNQNQM
MASSSSLSTTPLLLSPRVNMINTAISTQQTPISQSDSQYQELETLPP IRSLPLPFPHMD SEQ
SvMIG2- ATGCAAAGCCCATATCCAATGACACAAGTGTCTAACGTTGAT ID Nucleotide
GATGGGTCACTATTGAAGGAGAGTAAAAGCAAGTCGAAAGT NO:
AGCTGCGAAGTCAGAGGCGCCAAGACCACATGCTTGTCCTAT 174
CTGTCATAGAGGTTTTCACAGACTGGAACATCAGACGAGACA
CATGAGAATTCATAGAGGTGAGAAGCCTCACGCGTGTGACTT
CCCCGGATGTGTGAAAAGGTTCAGTAGAAGCGATGAACTGAC
GAGACACAGAAGAATTCATACAAACTCCCAGCCTCGAGGTAA
AAGAGGCAGAAAGAAGAAGGTTGTGGGCTCTCCAATAAATA
GTGCTAGTTCTAGTGCTAGCAGTATAGCAGATTTAAATAGGG
CAAATTTTTCACCGCCATTACCACAGCAACACCTATCGCCTTT
AATTCCTATTGCTATTGCTCGGAAAGAAAATTCAAGTCGATCT
TCTACAAGAAAAGGTAGAAAAACCAAATTCGAAATCGGCGA
AAGTGGTGGGAATGACCCATATATGGTTTCTTCTCCCAAAAC
GATGGCTAAGATTCCCGTCTCGGTGAAGCCTCCACCTTCTTTA
GCACTGAATAATATGAACTACCAAACTTCATCCGCTTCCACT
GCTTTGTCTTCGTTGAGCAATAGCCATAGTGGCAGTAGACTG
AAACTGAACGCGTTATCGTCCCTACAAATGATGACGCCCATT
GCTAGCAGTGCGCCAAGGACTGTTTTCATAGACGGTCCTGAA
CAGAAACAACTACAACAACAACAAAATTCTCTTTCACCACGT
TATTCCAACACTGTTATATTACCAAGGCCGCGATCTTTAACGG
ATTTTCAAGGATTGAACAATGCAAATCCAAACAACAATGGAA
GTCTCAGAGCACAAACTCAGAGTTCCGTACAGTTGAAGAGAC
CAAGTTCAGTTTTAAGTTTGAACGACTTGTTGGTTGGCCAAAG
AAATACCAACGAATCTGACTCTGATTTTACTACTGGTGGTGA
GGATGAAGAAGACGGACTAAAGGACCCGTCTAACTCTAGTAT
CGATAACCTTGAGCAAGACTATTTGCAAGAGCAATCAAGAAA
GAAATCTAAGACTTCCACGCCCACGACAATGCTAAGTAGATC
CACTAGTGGTACGAATTTGCACACTTTGGGGTATGTAATGAA
CCAAAATCACTTGCATTTCTCCTCATCATCTCCTGATTTCCAA
AAGGAGTTGAACAACAGATTACTGAACGTTCAACAACAGCA
GCAAGAGCAACATACCCTACTGCAATCACAAAATACGTCAAA
CCAAAGTCAAAATCAAAATCAAAATCAAATGATGGCTTCCAG
TAGTTCGTTAAGTACAACCCCGTTATTATTGTCACCAAGGGTG
AATATGATTAATACTGCTATATCCACCCAACAAACCCCCATTT
CTCAGTCGGATTCACAAGTTCAAGAACTGGAAACATTACCAC
CCATAAGAAGTTTACCGTTGCCCTTCCCACACATGGACTGA SEQ KIMIG1
MTEAIIEKKNHKKSINDHDKDGPRPYVCPICQRGFHRLEHQTRHI ID
RTHTGERPHACDFPGCSKRFSRSDELTRHRRIHDSDKPKGKRGR NO:
KKKSETIAREKELELQRQKQRNANDSAAVDSAGGTSANVIEPNH 175
KLLKSTNSIKQDGSTFTEPLKSLRSKPMFDLGSDESDECGIYSVPP
IRSQNNSGNIDLLLNAAKFESDKASSSFKIDKLPLTSSSSSPSLSF
TSHSINNSSSGLLLPRPASRAKLSALSSLQRMTPLSQNSESYNHSQ
QNLVHLHHPAPNRPLTEFVDNEYISNGLPRTRSWTNLSEQQSPS
GFSSSALNSRFSSSNSLNQLIDQHSRNSSTVSISTLLKQETVISQDE
DMSTEDAYGRPLKKSKAIMPIMRPSTMPPSSGSATEGEFYDEL
HSRLRSMDQLPVRNSKDEKDYYFQSHFSSLLCTPTHSPPPEGLLP
SLNQNKPVQLPSLRSLDLLPPK SEQ KIMIG1
ATGACAGAGGCGATTATAGAGAAAAAAAATCATAAGAAGTC ID (XP_454446)
TATCAATGATCATGACAAGGATGGACCAAGGCCTTAGGTCTG NO:
TCCCATATGTGAAAGGGGATTCCATCGACTGGAACATCAGAG 176
TAGGCAGATCAGAAGAGACACTGGGGAAAGAGCGCATGCAT
GTGATTTCCCGGGATGTTGAAAACGCTTTTAGTAGAAGCGATG
AACTAACAAGACATAGAAGGATACATGAGAAGCATAACACCA
AAGGGGAAAAGAGGAAGGAAAAAGAAGAGTGAGACGATAG
CTCGTGAAAGGAATTAGAATTGCAGCGGCAAAAACAACGA
AACGCAAACGACTCTGCGGGGTTGATTCTGCTGGTGGAACG
AGCGCTAATGTCATAGAACGAAACCACAAACTTCTGAAATCC
ACTAATTGGATTAAACAAGATGGTTCAACATTTAGTGAACCT
CTGAAATCGTTGAGGTCGAACCCAATGTTTGATCTCGGTAGC
GATGAATCGGATGAATCCGGTATATATAGTGTCCCACCTATT
AGATCTCAGAATAATAGTCGTAACATAGACCTTCTCCTGAAT
GCTGCAAAATTTGAGTCTGACAAAGCCTCATCCTCTTTCAAAT
TTATTGATAAACTACCGTTGACTTCATCTTCATCCTCTCCGTC
ACTTTCGTTTACATCTCATTCAGTCAGCAACAGCAGTACCGG
ACTATTGTTACCAAGACCAGCTTCACGTGCTAACCTTTCTGCT
TTATCATCATTACAAAGAATGACACCCTTGTCTCAAAATTCAG
AGTCATATAATCATTCGCAACAGAATCTAGTACATCTTCACC
ATCCCGCACCCAACCGACCATTGACCGACTTTGTTGATAACG
AGTATATAACTAACGTTCTGCCTAGAACCAGATCGTGGACAA
ATCTGTCGGAACAGCAATCACCATCGGGCTTCAGCTCCTCTG
CACTTAACTCCAGATTCTCGTCATCCAATAATCTCAACCAACT
GATAGATCAACATTCAAGAAATTCAACTACTGTAAGCATATC
TACTCTACTGAAGCAAGAAACCGTAATCTCACAACATGAGGA
TATGAGTACAGAAGATCCATATGGCCGGCCACTTAAGAAATC
AAAAGCCATAATGCCCATCATGAGACCTAGTTCTACAATGCC
ACCAAGTTCCGGCTCAGCTACAGAAGGACAATTTTATGATCA
ACTTCATTCAAGGCTTAGATCGATGGATCAACTGCCGGTAAG
GAACAGTAAGGACGAAAAAGATTACTATTTCCAAAGTCATTT
TTCAAGTTTACTGTGCACTCCAACGCACAGTCCTCCACCGGA
AGGATTGTTACCCAGTTTGAATCAGAATAAGCCAGTGCAGTT
GCCATCACTTCGAAGCTTAGACCTTTTACCACCAAAATAA SEQ CtMIG1
MNNQKMLSSKEKKNKEDRPYKCTYCDKAFHRLEHQTRHIRTHT ID
GEKPHACTFPGCVKRFSRSDELTRHLRIHTNPSSRKRKNKNQDL NO:
VEPTPMNVPPGSYAVPNTAIPFSIDRNGNHVYHQPYPVFFVPQPN 177
GYMQPVVQAPGLSIVPPPPHAHAQQAPQGPVPIQIQPPQPQRIAS
PHSNMSTPTHLQQEGSAVFSIPSSPTNSYQNAPNRQNLPQQPQPQ
PQITVQAVPMTTRSTSSDAIRLPPLTPNTTAQQPQPQRPQSAIFKS
ESNTSLYSDTSKVFSQPNSTLHSVGTSPDTSPSAMPPPIVVPAPSF
SNLNEYFQQKSNNPRIFNASSSSLSSLSGKIRSTSSTNLAGLQRLT
PLVPTTSTNTSNTTKSNIIPKQPSSTSLNLEFFNGNGTVGHANKKS
RPNSPCQSAMNIMSSPNETPLQTPSQSPRLNASNGPQSNIIEAA
QAKLESIATTGTQLPPIRSVLSFTNLSDYPQTSN SEQ CtMIG1
ATGAATAATCAAAAAATGTTATCTTGTAAGGAGAAAAAGAAT ID (XP_0025465
AAGGAGGATAGACCTTACAAGTGTACTTATTGTGATAAAGCA NO: 01)
TTCCACAGATTGGAACATCAAACAAGACATATTGGAACCCAT 178
ACTGGTGAAAAACCTCATGCGTGTACTTTTCCTGGATGTGTTA
AAAGATTTAGTAGATCAGATGAACTAACAAGACATTTAAGAA
TTCATACTAATCCAAGTTCAAGAAAGAGAAAGAATAAGAATC
AAGATTTGGTTGAACCTACTCCAATGAATGTTCCTCCAGGTTC
ATATGCTGTTCCAAACACTGCAATTCCATTTTCTATTGATCGT
AATGGTAATCATGTCTACCATCAACCATATCCAGTATTTTTTG
TTCCTCAACCAAATGGTTATATGCAACCTGTTGTTCAAGCTCC
AGGCCTTTCTATTGTTCCACCACCACCTCATGCACATGCACAA
CAAGCACCACAAGGACCGGTTCCTATTCAAATTCAACCACCT
CAACCACAACGCATAGCAAGTCCACATAGTAATATGTCTACT
CCTACACATTTGCAACAAGAAGGTTCTGCTGTTTTCTCAATTC
CTTCATCTCCTACAAATTCATACCAAAATGCTCCAAATCGTCA
AAACCTACCACAACAACCACAACCACAGCCACAAATTACTGT
CCAAGCTGTTCCAATGACTACAAGATCAACATCATCAGATGC
TATTAGATTACCACCACTAACACCAAACACCACTGCCCAACA
ACCACAACCTCAACGTCCACAATCTGCCATTTTCAAATCAGA
ATCTAATACTAGCCTATACTCTGATACTAGTAAAGTATTTAGT
CAACCAAATTCTACTTTACATTCAGTAGGTACATCACCAGAT
ACAAGTCCTTCAACAATGCCACCACCAATTGTTGTTCCTGCTC
CTTCTTTTAGTAATTTGAATGAATATTTTCAACAAAAGAGTAA
TAATCCAAGAATTTCAATGCTAGTTCTTCATCTTTAAGTTCA
TTAAGTGGTAAGATTAGATCTACATCATCTACAAATCTTGCA
GGTTTACAAAGATTGACTCCATTAGTTCCAACAACTAGTACA
AATACTTCTAACACTACAAAATCTAATATTAATACCCAAACAG
CCTTCATCAACTTCATTAAATTTGGAATTTTTCAATGGTAATG
GAACTGTTGGACATCCAAATAAGAAATCAAGACCAAATTCAC
CATGTCAATCAGCAATGAATATTTCTTCAATAATGAGTTCACC
AAATGAAACACCATTACAAACACCTTCACAATCACCACGTTT
GAATGCTAGTAATGGTCCTCAAAGTAACATCATTGAAGCTC
TCAAGCTAAATTGGAAAGTATAGCTACTACAGGTACTCAATT
ACCACCAATTAGATCGGTTTTAAGTTTTACTAATTTGTCAGAT
TATCCACAACCCACGTCAAATTAG SEQ YIMIG1
MEFTATNSDREMHPPPVAVPSHLQQSTTHRTVPKTNPKTGKSE ID (XP_503678)
MPRPYKCPICDKAFHRLEHQTRHIRTHTGEKPHECTFPGCTKRFS NO:
RSDELTRHSRIHLNPNTRRAKNMNSAAAAAHNAAAQQKGPPSN 179
QQPKDDRTDRLVAISNLVDHHDAHPPIPGGSGIKSEYSSAYSTPY
SSVPSSPTMGQASLYRPYGAGPGGPPPPGGPGGLGGPAPPGPPG
HHGGPPTHIGLPPLHHPPPPGGPHSAAPSAPNSTPGSPMVRVPH
STYPRSTFDMNMLATAASQQLERENAPPSGLSSGAPSAAPSGTSS
PFNHSPSSSPLQSTQSSPALASYFSRPPAPSSNPGTPGSSHGPPGF
SSGTSLSSHGNTHSNHFQHPGHLHPPHHHHHGLQGHIFAGLQRMT
PMSGRDDSEPWHRSKKSRPNSPSSTAPSSPTFSNSESPTPDHTPLA
TPAHSPRIHPRDLEGVQLPSIRSLSIGRHVPPTLPPMEIAPRSSGGT
HTGTHTPGGGSASHTPFGTSPNSGVPPPHPNAAQGAASSLSML
AMAGLNHAAGGAAPGGSGAAATSSTRDEPSNDTSPASGSGPS APSTASSSTRMAVSDLIDR SEQ
YLMIG1 ATGGAATTCGCAGCCACCAATTCAGACCGAGAAATGCACCCT ID
CCCCCGGTGGCAGTGCCGTCGCATCTGCAGCAGTCCACGACC NO:
CACCGCACGGTGCCCAAAACCAACCCCAAGACGGGCAAGTC 180
CGAAATGCCCCGACCCTACAAGTGCCCCATCTGCGACAAGGC
CTTCCACCGGCTGGAGCATCAGACCCGCCACATCCGGACACA
GACGGGCAAGAAGCCCCACGAGTGCACCTTCCCAGGTTGCAC
CAAGCGGTTCAGCCGTAGCGACGAGCTCACCCGACACTCGCG
CATACATTTAAATCCCAACACTAGACGGGCCAAGAACATGAA
CAGCGCCGCCGCCGCCGCTCACAATGCCGCCGCTCAGCAGAA
GGGCCCTCCTTCTAACCAGCAGCCCAAGGACGACCGAACAGA
CCGCCTTGTGGCCATCAGTAATCTCGTCGACCACCACGACGC
CCACCCTCCTATTCCCGGAGGCAGTGGTATCAAGAGCGAGTA
CTCGTCCGCATACTCGACTCCATATTCTTCGCTGCCGTCGTCG
CCAACCATGGGACAGGCCTCGTTGTACAGACCTTATGGTGCT
GGTCCAGGCGGTCCTCCTCCTCCTGGTGGACCTGGTGGACTG
GGTGGACCTGCTCCTCCTGGCCCTCCCGGCCACCATGGAGGA
CCCCCCACTCACATAGGTCTACCACCCCTACACCATCCTCCTC
CTCCAGGAGGCCCTCATTCGGCCGCGCCCAGTGCTCCCAACA
GTACGCCCGGGTCGCCCATGTTTGTGCGAGTGCCACATTCAA
CCTACCCAAGATCGACTTTCGACATGAACATGCTCGCGACTG
CGGCCTCTCAGGAGCTCGAGCGCGAGAACGCGCCTCCATCGG
GTCTCTCTTCTGGTGCTCCGTCGGCTGCGGCTTCCGGCACCTG
CAGCCCCTTCAACCATTCACCTCTCGTCGTCGCCACTGCAGTCC
ACGCAGTCGTCCGCCGCTGTGGCGTCGTATTTCAGTCGGCCTC
CTGCTCCGTCTTCCAACCCCGGAACCCCCGGTAGCTCGCACG
CTCCTCCCTACGGCTTCTCGTCGGGCACGTCGCTGTCCAAGCA
TGGAAACACGCACTCCAATCACTTCCAGCACCCACAACACTT
GCATCCAACGCATCATCACCATGGTCTGCAGGGACACATCTTT
GCGGGTCTGCAGCGCATGACGCCCATGTCTGGAAGAGACGAC
TCGGAGCCATGGCACCCGTCTAAGAAGTCGCGCCCCAACTCA
AGAGCCCCACGCCGGACCACACGCCCCTGGCGACCCCCCCAC
ATTCGCCTCGAATCCACCCTCGAGATCTCGAGGGAGTGCAGC
TGCCTTCCATTCGCTCGCTGTCGATTGGCCGCCACGTGCCCCC
CACGCTACCGCCTATGGAGATTGCGCCTCGGTCAAGTGGAGG
CACCCATACCGGCACCCATACGCCGTACGGCGGCGCATCTGC
GTCGCACACGCCGTTTGGCACATCACCCAACTCGGGAGTTCC
TCCTCCGGATCCAAACGCGCGCCAGGGCGCTGCTCTTCGCTT
AGCACTGCTAGCCATGGCTGGCCTGAACCACGCTGCTGGGGGA
GCTGGTCCTGGAGGCAGTGGGGCGGCAGCTAGGTCGTCCACG
CGCGACGAGCCCAGCAACGATACATCTGGGCCTGCGTCAGGC
AGCGGGCCTTCTGCTCCGTCCACCGCGTCGTCCAGTACTCGA
ATGGCCGTCAGTGACTTGATTGATAGATGA SEQ >CvCTF1
MSSSDEGDHTPELQQEKVTQNTHSDAPSATPQTTPAQTTTTTT ID
TTSSSQTTTTTKKPKSDKEDKPKAFTIKYKRPGSRACTVCRSRKV NO:
RCDAEIHIPCINCITFGCECILPEAKKRGNQSGESKAKRQKTQPK 181
DKAATTAASTKKKATTTTTTENGDEDGSPLEQTSSSRSAKSPDL
VTPTKESETSSHTSSTAEPIINISGVSVPPSILTSTYKNRPSMHKKEL
LDSKAKTALTFLGSSSIGVVEQRVGENHVELTIDVEDTSDIKLDS
VELEILKIVIRGAFLLPSKELSLELINAYFEHVHPLMPVINRISLFMK
KFNDPNDNPSLMVGAVLLTCCAASKNPLLEDSNGINDLASTIT
FRRAKALYETNYESDPVSIIQTLILIGSYWDGPEDVTKNSFYWTR
VAVGLAQGEGFQRDYSKSHQGINSEKKINATGRIWWCLFEKDRNV
AIAFGRGVVIDGNDCDVPMETVDDFDETDPELGITDPYPVNETG
ALYFIHLVKLAEITGIIIKHQYSVKSETMKRRNAFSIIEHCDMLMG
IWFTNLPSNLVFSLADSSTHNFFACLLNAGYYNRLYLIHRSNLIR
MARSSSTNPNNYKYPSWGISFQSARMISIISKILMDKDLIQFVPV
MYVYIAFSALVMLIYDVDSANSVIAATASDSLFVSRAVLRELQK
AWIEWAAVLLKLFPKYANDKIKRTKITIENGNMIVEYKENAAKE
KMKKSINEYGTNTSPTAMPIPNRSPISQPQQQPQQQQQHRQVVQ
QQLPLPQPAKPRDRTLFSPSSVTSGVSPPGNVYNNYSEQTPVKQ
EFSSVSPVAANPPQQQVQGSNPTPQIEELVKQYKLPMKRAAKAN
NGNGDSSDKQTTDTSPASSTKSFPDISMVTENLQNKQNFFENFEP
TQLFPTFSIPPTRAQSPTPNFDDGAIGSNVYNTMNTTMEIKEERTP
QPQQSGENQQEQPHNGNAAASLAAPAQTLKDEHDILDLHTPGF
QTNFIDSSFNYLNLQSGMNMDEDIHSLFNMMNQ SEQ CtCTF1
MSDQKDGVAKLDQQKETENSHPETQTATTTITTTATQTTSTISN ID (XP_0025469
KKPKSDAKDKPKAFTIKYKRPRGSRACTVCRSRKVRCDAETHIP NO: 74)
CTNCITFGCECILPEAKKRGNQSGESKAKRQKVNQSKDKPLSAT 182
ALKKKSSSNSTINTPINENGNSLEKSTSPVNNKSSDMATTIVISSN
NDTSENSNTTSTAEPIINISQVSMPPSILTSTYKNRPSIVIHKKELLDS
KAKTALTFLGSSSIGVVPQRAGENHVELTTDVFDTSDTKLDSVE
LEILKMRGAFLITSKELSLELINAYFEHVHPIIMFVINRSLFMKKF
NDPNDNFSLIVIVLHAVLLLGCRASKNPLLLDSKGTNDLASITFFR
RAKALYETNYESDPVSIIQTVILIGSYWDGPEDVTKNSFYWTIW
AVGLAQGFGFQRDVSKSHQLTISEKKIWRRIWWCLFEKDRNVAI
AFGRPVVIDLNDCDVPIVILTVEDFDETDPELGIVDPYPINETQALY
FIFILVKLAEITGIIKEIQYSVICSETMKRRNAFSIICHCDMLMGIWF
TNLPSKLVFSLADTSTHNFYACLLNAQYYNRLYLIHNSNLIRMA
RSSSSTNPNNYKYPSVVGISFQSARMISISKILLDRNLIQYVPVMYV
YIAESALITMLIYHVDSDNSVIAATAADSLYVSRAVLKELSKYWP
VAGVLLKLFDKYANDKLKRTRLIENCNIVIIVEYKENAAKEKIVIK
QSIGEYDSHSSSATMSIPNRQQQQQQASPLPQNRQSQQPLPLPQT
AKSKDHTMFSPTSATSGISFQGGWNGYPIKQTPIKPFSSASSHE
HPQQQQQQQSGQLNQTQNNNQTFQIEELIRQYKLPMKRAAKGT
NGNNNVASDSSDKQTTETSPASSTKSFPDISMVTENLQNKQNFP
ENFEPTQLFPTFSIPPTRAQSPTPHFEDDGIGSNSVYNSMNSTMML
KSDKQQKSQPSTEQQQQSQQLQQQQQPTHNGNSNQLNAGESHK
AEHDILDPNTPAFQTNFIDSSFNYLNLQSGMTMDDDIHSLFNIAM NQ SEQ CtCTF1
ATGTCGGATCAAAAAGACGGGGTAGGTAAACTAGACCAACA ID
GAAGGAGACTGAAAATTCACACCCTGAAACTCAAACTGCAAC NO:
AACAACGACAACAACAACAGCCACCCAGACAACATCAACAA 183
CTTCAAATAACAAACCAAAATCAGATGCAAAGGATAAACCA
AAAGCTTTCACTATAAAATATAAACGTCCTAGAGGTTCACCT
GCTTGTACCGTTTGTCGTTCAAGAAAAGTACGTTGTGATGCA
GAAATCCATATTCCTTGTACAAATTGCATTACTTTTGGTTGTG
AATGTATTTTACCAGAAGCAAAGAAAAGAGGCAATCAATCCG
GTGAATCAAAGGCAAAACGACAGAAAGTAAATCAATCAAAG
GATAAACCACTTTCGGCAACTGCCTTAAAAAAAGAAATCCTCA
TCTAATTCTACAATAAACACACCAATAAATGAAAATGGCAAT
TCACTAGAGAAATCAACCTCTCCTGTATCGAACAACAAATCT
TCAGACATGGCGACAACAATGTCATCAAATAATGATACATCT
GAAAATTCAAATACTACGTCCACGGCGGAACCAATAATTAAT
ATATCACAAGTTAGTGTACCCCCTTCATTGACATCGACATATA
AAAACAGACCTTCTATGCATAAAAAGGAACTACTTGATTCAA
AAGCCAAAACTGCATTAACATTTTTGGGTCTGTCATCAATTGG
TGTTGTTCCTCAACGTGCAGGTGAAAATCATGTTGAATTAACT
ACCGATGTATTTGATACAAGTGATACTAAATTGGATTCTGTTG
AATTAGAAATTTTAAAAATGAGAGGTGCATTCTTATTACCAA
GCAAAGAATTATCCCTAGAATTGATTAATGCTTATTTTGAGCA
TGTTCATCCATGATGCCAGTTATTAACCGATCGTTATTTATG
AAGAAATTTAATGATCCCAATGATAATCCAACTTTAATGGTT
CTTCATGCCGTGTTGTTGTTGGGATGTAGGGCATCTAAAAATC
CATTGTTGTTGGATTCCAAAGGTACCAACGATTTAGCCAGTAT
AACATTTTTCCGAAGAGCAAAAGCGTTATATGAAACAAATTA
TGAAAGTGATCCGGTATCAATTATTCAGACTGTAATTTTAATT
GGTTCCTATTGGGATGGTCCTGAGGATGTCACCAAGAATTCTT
TTTACTGGACTAGAGTTGCTGTGGGATTAGGTCAAGGGTTTG
GTTTTCAACGTGATGTTAGTAAATCACATCAATTGACTATTTC
AGAAAAGAAAATATGGAGAAGAATTTGGTGGTGTTTATTTGA
AAAGGATCGTAATGTAGCTATAGCATTTGGAAGACCAGTTGT
TATTGATTTAAATGATTGTGATGTACCAGTGTTAACCGTGGAA
GATTTTGATGAAACTGATCCGGAATTGGGAATAGTTGACCCA
TATCCTATTAATGAAACCCAAGCCTTGTATTTTATTCATTTAG
TTAAATTGGCAGAAATAACTGGTATTATTATCAAACATCAAT
ATAGCGTAAAATCTGAAACCATGAAGAGAAGAAATGCATTTT
CCATTATTGAACATTGTGATATGTTGATGGGTATTTGGTTTAC
AAACTTACCCTCAAAATTGGTATTTTCATTGGCAGATACCCTG
ACTCATAATTTTTATGCCTGCTTATTAAATGCACAATATTATA
ATCGTTTATATTTGATTCATAGATCAAATTTGATTAGAATGGC
TAGATCATCATCAACAAACCCAAACAATTATAAATATCCAAG
TTGGGGTATTTCTTTCCAATCAGCAAGAATGATTTCTATAATT
TCAAAAATATTATTGGATAGAAATTTGATTGAATATGTTCCAG
TCATGTACGTTTACATTGCATTTAGTGCATTGGTTATGTTGAT
TTATCATGTGGACTCGGATAATTCAGTTATTGCTGCCACTGCA
GCTGATTCGTTATATGTTTCAAGAGCAGTATTGAAAGAGCTTC
TGAAATATTGGCCAGTTGCAGGCGTGTTGTTGAAATTGTTTGA
TAAATATGCTAACGATAAATTGAAGAGAACAAGATTAATTGA
AAATGGTAATATGATTGTTGAATACAAGGAGAATGCAGCTAA
AGAAAAGATGAAACAATCTATTGGTGAATACGATAGTCATAG
CAGTTCCGCTACCATGTCGATTGCAAATAGACAACAACAACA
ACAACAGGCATCACCATTGCTCACAGAATCGTCAGTCACAACA
ACCTTTACCGTTACCACAGACCGCAAAGAGTAAAGATCATAC
AATGTTTTCGCCAACGTCAGCAACTTCTGGAATATCGCCAGG
TGGAGGTGTTTACAATGGTTATCCAGATCAAACACCAATTAA
GAGAGATTTTTCACTGGCTTCATCACACCCACACCCACAACA
ACAACAACAACAGCAATCAGGTCAATTAAATCAAACTCAAA
ATAATAACCAAACACCACAGATTGAAGAATTAATAAGACAAT
ATAAATTACCAATGAAGCGTGCTGCAAAAGGTACTAATGGGA
ATAACAATGTTGCTAGTGATAGTAGTGATAAACAAACTACTG
AAACATCTCCTGCTTCATCTACAAAATCATTTCCAGATATATC
CATGGTTACTGAAAATTTACAAAATAAACAGAATTTCTTTGA
AAATTTCGAACCAACACAATTATTTCCTACATTCAGTATTCCA
CCAACAAGAGCACAATCCCCAACACCACATTTTGAAGATGAT
GGTATTGGTAGTAATGTTTATAATTCAATGAATTCAACCATGA
TGCTTAAATCAGACAAACAACAGAAAAGTCAACCATCCACAG
AACAGCAACAGCAACTGCAGCAACTTCAGCAGCAACAACAA
CCAACTCATAATGGGAATTCTAATCAACTTGTTGCAGGGGAA
TCACATAAAGCCGAACATGACATTCTTGATCCAAATACACCA
GCATTTCAAACTAATTTCATTGATAGTTCATTTAATTATTTGA
ATTTACAATCAGGAATGACTATGGATGATGATATTCATTCTTT ATTTAATATGATGAATCAATAG
SEQ YICTF1 MSSKYKEEEGATPGGSGNAKKYRRAKPRASRACENTCHARKVR ID
(XP_502753) CDVTERMPCTNCQAFGCECKIPEVKRKKNDKKAAAEKVADKG NO:
TKDRKRRKTDGGEESDEGSVPPQAPSNASSSTASSPNTAPTAQQ 184
RLMHFQQETKQQQYQQHEGETKPDANPNLSDSSNTWLKMLDS
KVVKQSGRVAFLGSSSNLNLIDDANPDNEAYHYPLPAEITGGNP
VFHELDPEEIEILKIRGAFLITPRELCDDIWSYFEFAXPVIPIVNRT
QFMRRYNDPVNTPSLLLLQAVLLAGSRVCRNPALLDANGSSDQ
ASLTFYKKAKALYDSNYENDRISIVQSLALIVIGWWWEGPEDVTK
NVEYWSRVGKVAQOPGIERSVENSSISVAEKRIVIWKRVWWVI
FFRDKAIAVSLGRPVITNLGASPVPMTTEDDRIEDERDVPSGVPV
NRLFISINEIHAVKLSEIMGLVLICQQFSVGAEFISHRLNFGNSHC
DMAMQSWIVINNLPPELKYSVKDMGSHNFYETALLHSQYYTILCIL
VHRSNILQRFGTSEANESAYPSWGIAFQAAHMIAKIMENMLAYNE
LRDTPAFMVYTLFSAMINILHVYQTESICSPSVVESANRSLGCMK
ALLECGKTWVVARMVIKLFKHIYINESQPIRNHMAKTIRRHARG
VSKPDAAKQQQMPQAQHQASQPPHGAQQHHAQQQRHAQQQQ
QQQQHRQQQQQQQQQQQQHTQQQQQTHGPQPERKTNNHAF
DQQQQSQQEGEEYLGDRQPPTNPGTPFNGTLPSKPGTPHPDFYE
VTNTPPASNTFFESFQPMQLFPDVTGDMSNLQSQLQEPATSALPP
DMFAHADGASHTEGGTYKSSPEDEPTAASGFGYAPQSLNIGDW
YQYLMMNGEGGAAAGAAAGTAAGAPGSVPPGAPAEAHPEAAT SNSWNESH SEQ YICTF1
ATGTCTTCCAAGGTCAAAGAGGAGGAGGGCGCCACCCCGGG ID
CGGCTCAGGCAACGCCAAATTTGGCTATCGAAGGGCCAAGCC NO:
GCGAGCTTCCAGGGCTTGTGAGGTGTGAGTGAGAGAGGAGG 185
GAAGATTGTCAGACGGACGGATAGGTGGAGCTGATCGCGAC
GAACCACTCAGTACTAACACAGATGCCATGCCCGAAAAGTCC
GATGTGACGTTACAGAACGAATGCCGTGTACCGTGAGTATGG
TGGGGTGGAAGAAACAAACACTCCAGGAACAAAAGCGTTGT
CGACGTGTGAGTGACATGAGGCCATATCAACTAACATAGAAT
TGTCAAGCATTTGGATGCGAGTGCAAGATACCCGAAGTGAAG
CGGAAAAAGAACGACAAAAAGGCGGCGGCAGAAAAGGTGG
CGGACAAGGGAACCAAGGACCGCAAACGAGGCAAGACGGAC
GGCGGCGAGGAGTCCGACGAGGGGTCCGTGCCCGCCCAGGC
GCCCTCCAACGCGTCTTCCTCCAGAGCGTCGTCGCCCAACAC
GGCCCCCACTGCCCAGCAGCOGCTGATGGAGTTTCAGCAGGA
GACCAAACAGCAACAGTACCAGCAACACGAGGGCGAGACGA
AACCCGGAGGCCAACCCCAACCTCAGCCACTCGTCCAACACGT
GGCTCAAAATGCTCGACTGCAAGGTGGTTAAACAGAGTGGCC
GGGTGGCGTACTGGGCTCGTCGTCCAACCTCAATCTATTGTT
GGACGCTAACCO9GATAATGAAGCCTACCACTACCCCTTGCC
AGCCGAAATCACAGGTGGAAACCCCGAATTGCATGAATTGGA
TCCCGAAGAGATTGAGATTCTCAAACTGAGAGGAGCCTTCCT
GTTACCTCCCCGAGAACTGTGTGACGACATTGTGGAGAGCTA
TTTTGAAAAGATCCACCCGGTCATCCGAATTGTCAACAGAAC
ACAGTTCATGCGCCGCTACAACGACCCCGTCAACACGCCGTC
GCTGCTGTTGGTTCAGGCCGTTCTGGTGGCTGCTTCTCGAGTG
TGCAGAAACCAAGCTCTACTGGACGCCAATGGGTCGTCAGAC
CAGGCGTCGCTCACCTTCTACAAGCGAGCCTTAGGCACTGTAC
GACTCCAACTACGAAAATGATCGTATTTCCAATTGTGCAGTCG
CTGGCTCCTCATGGGCTGGTGGTGGGAGGGTCCCCAAGACGTG
ACCAAAAACGTGTTGTACTGGTCCCGTGTGGGTCTGTGTGTG
GCGCAAGGATTCGGTGTGCATGGATCTGTGGAAAACTGGTCG
CTGTCGGTGGCCGAAAAGCGAATGTGGAAGCGGGTGTGGGTG
GGTCATCTTTTTCCGAGACCGAGCCATCGCCGTATCTTTGGGG
CGACCTGTTATGATCAACCTGGAAGACTCGGATGTGCCAATG
CTCACCGAAGACGATTTCATTGAGGACGAGCCTGACTACCCG
TCTCCCTACCCCGTCAACAGACTCCACTCGGTCTATTTTATTC
ACGCCGTCAAGCTGTCTGAAATCATGGGGCTTGTTCTGCGAC
AACAATTCAGTGTGGGTGCCGAGCACTCGCACCGACTAAACC
GAATTCCCGTCGTCTCGCACTCTGACATGGGCATGGGCTCGT
GGATGAACAACCTGCCGCCGGAGCTCAAGTACTCCGTCAAGG
ATATGGGCTCTCACAACTTCTACAAGGCCCTCCTGCACTCACA
ATACTACACGATTCTGTGTCTTGTGCACCGAAGCAACATTCTG
CAACGGCGAACGTCAGAGGCTAACGAGAGCGCATACCCTTCG
TGGGGTATCGCGTTCCAGGCGGTCACATGATTGCCAAAATC
ATGGAGAACATGTTAGCGTATAACGAGCTGCGTGACACCCCG
GCGTTCATGGTGTACACGCTCTTTTCGGCATGATCATGCTTG
TGTACCAGACCGAGTCCAAGTCGCGTCGGTGGTTGAGTCTG
CCAACCGGTCGCTGGACGTGTGTATGAAGGCCCTGGAGGAAT
GCGGCAAGACGTGGGTGGTGGCACGAATGGTGCTCAAGCTTT
TCAAGCACATGAACGAGTCGCAGCCCATTCGAAATCACATGG
CCAAGACCATCCGACCTCATGCTAGAGGCGTCAGCAAACGCG
ACGCAGCAAAGCAGCAGCAAATGCCTCAAGCTCAGCACCAG
GCTTCACAACCACCACATGGCGCCCAACAGCACATGCCCAA
CAGCAACGACATGCCCAGCAACAACAACAACAGCAGGCAGCA
TGCTCGACAGCAACAGCAGCAGCAGCAGCAACAACAACAGC
AACACACACAACAGCAACAACAGACGCATCAGCCACAGCCA
GAACGCAAGTTCAACAATCATGCGTTTGACCAGCAGCAGCAA
TCGCAGCAGGAAGGAGAGGAGTACCTGGGCGATAGACAGCC
TCCTACAAATCCCGGCACCCCTTTCAACGGCACGCTGCCATCC
AAGCCCGGCACCCCGCATCCGGACTTTTACTTTGTCACTAACA
CCCCGCCGGCTTCGAACACCTTCTTTGAGTCGTTCCAGCCCAT
GCAGCTTTTCCCCGACGTGACTGGCGATATGTGTAAGCTGGA
GTCGCAGCTCCAGGAGCCCGCCACCAGCGCGCTTCCTCCGGA
CATGTTTGCCCACGCCGACGGAGCCAGCCACACCGAAGGCGG
CACCTACAAGTCGTCGCCCGAGGACGAGCCCACCGCGGCCAG
TGGATTTGGATACGCCCCTCAATCGCTCAATATTGGCGACTG
GTACCAATATCTGATGATGAACGGTGAAGGGGGTGCTGCTGC
TGGCGCTGCTGCTGGCACTGCTGCAGGCGCCCGTGGTTCTGTT
CCTCCAGGTGCTCCTGCTGAGGCTCATCCTGAAGCTGCCACTT SEQ KIUTR1
MVEGHPLEKVLSASALTSSSNSSSRSSIPLTFEVTHQHKTQIKRFQ ID (XP_455838)
NVLTSDSATQDDGNDDPSRNQGNEWSEQFHLLQYPEQHQHQHQ NO:
NKHQQHQQQHEKGDLDEVLCTQRMIRKLSTGSDDVKKVYSH 186
AQLSSTAHGVRLLSKNILSNTKVALEVKKLMIVTKRQDDSLIYLT
RELVEWILVNYPTIDVYVEYGFERNESENAKELCIGSKCGSHKI
QYWSPEFVKEHEDEFDLAITLGGDGTVLYVSSIFQKNVPPVMSFA
LQSLGFLTNFQFEDFKHALSKILQNKIKIKMRMRLCCQLFRKRIK
KVDEEARISTHIKYTMEGEYHVLNELITIDRGPSPFISMLEINGDG
SLLTVAQADGLIIASPTGSTAYSLSAGGSLVYPSVNAIAVTPKPH
TLSF2PITLPDSMTLKVICYPKASRSTAWAAFDGKNRVENIKRGDY
IVINASPYSEPTLEARSTEFIDSISETTLNWNVKESQKSFTHMLSRK
NQQKYELEITVRTRQDSEEEEELEDDQSDDYSTDSDSELNE SEQ KIUTR1
ATGGTTGAAGGACACCCTTTGGAGAAGGTTCTGAGTGCTAGT ID
GCACTAACTTCAAGCAGTAACAGCTCATCAAGAAGTAGTATT NO:
CCCTTGACATTTGAAGTCACTCATCAACACAAGACTCAGATC 187
AAGCGGTTCCAGAATGTGTTAACTAGTGATAGTGCCACCCAA
GATGACGGCAACGATGACCCTAGTAGAAATCAAGGGAATGA
GGTGAGTGAACAATTTCATTTGCTGCAATATCCTGAGCAGCA
TCAACATCAACATCAAAATAAACATCAACATCAACATCAGCA
GCAGCACGAAAAAGGAGATCTGGACGAGGTGCTCTGTACCC
AACGGATGTTCAGGAAACTATCTACTGGGAACGATGATGTGA
AAAAGGTTTATTCGCATGCACAGCTATCGTCCACTGCACATG
GAGTGAGATTATTATCCAAAAATCTATCCAACACCAAAGTGG
CATTAGAAGTTAAGAAATTAATGATAGTAACTAAAAGGCAAG
ATGATTAATGATCTACTTGACAAGGGAGTTGGTAGAGTGGA
TCCTTGTCAATTATCCAACAAGATGTTTATGTGGAATACGG
TTTTGAACGAAACGAATCTTTCAACGCAAAAGAGTTGTGTAA
AGAGAGTAAATGTGGTTCGCATAAGATTCAATATTGGTCTCC
AGAATTTGTGAAAGAACACGAAGATTTCTTTGACCTAATCAT
AACACTTGGTGGAGATGGCACAGTATTATACGTATCATCAAT
TTTCCAAAAGAATGTGCCACCGGTCATGTCATTTGCCTTGGGG
TCTCTAGGATTCTTGACGAACTTCCAATTTGAAGACTTCAAAC
ATGCTTTGTGCAAGATTTTAGAAAACAAGATTAAAACTAAGA
TGAGAATGCGACTATGTTGTCAACTTTCAGGAAAAGGATCA
AGAAAGTGGACGAGGAAGCACGCAAGACGCATATTAAGTAC
ACAATGGAGGGAGAGTACCATGTTTTGAATGAACTTACGATT
GACAGAGGTCCTAGTCCGTTGATTTCAATGTTAGAATTATATG
GAGATGGATGTTTATTAACAGTGGCACAAGCAGACGGTCTGA
TCATAGGCTGGCCCACTGGATCCACAGGGTATTCCTTAAGTGC
TGGTGGTTCCTTGGTATACCCCAGCGTCAAGGGTATTGCAGTT
ACACCAATCTGTCCTCATACTCTAAGTTTCAGACCCATCATCT
TGCCGGATAGTATGACGTTAAAAGTGAAAGTGCCAAAGGCTA
GCAGAAGCACCGCATGGGCAGCATTTGACGGTAAGAACAGA
GTGGAAATGAAGAGGGQCGACTACATAGTGATAAACGCAAG
TCCGTATTCATTCCCTACACTCGAAGCCGGCAGCACAGAATIT
ATTGACAGTATCAGTAGAACATTGAATTGGAACGTCAGGGAA
TCCCAAAAGTGGTTCACACATATGCTCTGAAGAAAGAATCAA
CAGAAGTACGAAATAACACCGTGAGAAGCAGGCAGGATTC
TGAAGAAGAGGAAGAACTCGAAGACGACCAAAGTGATGATT
ATTCTACTGACTCTGACAGTGAACTGAACGAGTAA SEQ YIUTR1
MSTPVSESPYVHQRFDAALSAASALQELSAEDIQSSRLSHAFIEV ID (XP_504486)
QTATGVRRIAKHLGKASVHIDVTKVMLITKARDNSLVYLTRDM NO:
ARWLMGGVVVYVDAKLEICSGILFDAPTLTANTPAIMERYWT 188
AEMATQKPELFDLVITLGGDGTVLWASWLFQGTAPPVIPFALGS
LGFLTNFEYHDFGKHLTKAMTQGVHVHLIGMRFKTVFKREMN
PETGKRDKHHSKIGRHEVLNEIVVDRGPSPFISMLELYGDDNLLT
IVQADGLILSTPTGSTAYSLSAGGSLVIEIPAIGVTPIGPHTLSFR
PMLLPDSMTLKVVVPRKNSRTSAWVSEDGRSRVELKSGDYITV
RASKFPFPTVIRSDMDYIESVSGTLKWNTRELQKPLTSLSRPAST
VSVNRSASLEASSSVPSGAGYSRLRSITSLYIKGTPGVIVITPISQLTA
ALPTIQQSTPSVPNTTNSNTPTSTGTANSNINNNYNPTTNPTTNIN
TNPTITTTQKPYNIRDIVQPSRSGSIWNGTASLPQSFSTSYIVIPNT
NIPSGMTSINSATPQQYSATASTTCLNSPPVTRGFTPIQGQSPAQIQ
AFESMIDFDIDDEGTSTFADCSRRGGLVMSPTSVFSPTGSNDGML
TLGDLGKQVSDSDSDSYHSSEYEEEEYDIDIDLAHKTENLHVLD KDEDRNNEQEDTK SEQ
YIUTR1 ATGAGCACTCCGGTCAGCGAGTCTCCGTACGTGCACCACCGG ID
TTCGACGCTGCTGTCAGCGCCGCCTCGGCCCTCCAGGAGCTCT NO:
CAGCTGAAGAGATTCAGAGCTCACGACTTAGCCATGCTCACT 189
TCGTCCAAACTGCGACAGGAGTTCGACGAATTGCCAAACATC
TCGTCCAAGGCCTCCGTCCACATCGATGTGACTAAGGTCATGA
TCATCAAAAGGCCCGAGACAACTCGCTTGTCTACCTGACCC
GAGATATGGCCCGGTGGTTGATGGACCGAGGAGTGGTGGTCT
ATGTGGATGCCAAACTCGAGAAGAGTGGCCGCTTCGAGGCTC
CTACACTGACTGCAAACACCCCAGGACGTATGCTCAGATACT
GGACTGCCGAAATGGCGATCCAGAAGCCCGAGCTCITTGATC
TAGTCATTACCCTGGGAGGAGACGGGACGGTTCMTGGGCCT
CGTGGCTTTTTCAAGGCACCGCTCCCCCCGTATTCCCTTTGC
GCTGGGTTGGCTGGGTTCGTACCAATTTTGAATACCATGAC
TTTGGCAAGCATCTTACAAAGGCCATGACTCAGGGAGTACAT
GTGCATCTGAGAATGCGATAACATGCACCGTGTTCAAGCGG
GAAATGAACCCGGAAACCGGAAAGAGAGATAAGCATCACTC
CAAGATTGCGGACACGAAGTTCTCAACGAAATTGTCGTCGA
TAGAGGTCCTTCACCTTCATcTCAATGCTGAGCTCTATGGG
GATGATAACCTGTTGACCATCGTACAGGCTGATGGCCTCATT
CTATCGACTCCCACAGGATCCACCGCATACTCTTTGTCACA
GGAGGCTCTCGGTTCACCGGAGATTCGAAACATCTGTGTGA
CTCCAATTTGTACCCATACCTGTCCTTCAGACGATGCTTCT
GCCTGATTCAATGACACTTAAGGTGGTTGTTCCTCGAAAGAA
CTCACGGACTTCAGCATGGGTCTCATTTGATGGCAGATCACG
TGTGGAGCTCAAGTCGGGCGACTACATCACAGTGCGAGCTTC
AAAGTTTCCATTCCCCACCGTGATCCGATCAGACATGGACTA
CATTGAGTCTGTGAGTCGAACACTCAAGTGGAACACCCGAAA
ACTCCAGAAGCCTCTGACGTCACTTTCCAGACCTGCATCGAC
CGTTTCTGTCAAcAGAAGCGCAATCTCCATGCCTCTTCGTCA
GTGCCAAGTGGAGCAGGGTACAGCCGTCTGAGAAGTAGATC
GATGAAGGGCACCCCAGGAGTCATGACTCCCATCAGTCAGCT
TACTGCAGCACTGCCAACCATCCAGCAGAGCACTCCTTCCGT
TCCCAACACAACTAATAGTAACACACCTACAAGCACGGGTAC
TGCCAACTCCAACATCAACAACAATTACAACCCAACCACAAA
CCCAACCACAAACACGAACACAAACCCGACGACGACCACTA
CACAGAAACCCTACAATCTCAGAGACAACCAGCCCAGCAGA
ACCAACAGCAGAGATAACGGAACTGCTTCTCTTCCTCAAAGT
TTCAGCACAAGTTATATGCCCAACACAATGCCCAGCGGAATG
ACTAGCACTAACTCTGCCACTCCCCAGCAGTACTCGGCAGCC
TCTACTACATGTCTCAACTCACCCCCTGTTACCCAAGGCTTCA
CACCCATTCAGCAGCAGTCGCGGGCTCAGATCCAGGCGTTTG
AGAGTATGATTGACTTTGATATCGACGACGAAGGCACCTCCA
CCTTTGCCGACTGCTCTCGAAGAGGAGGACTGGTCATGTCCC
CGACATCTGTCTTCTCTCCCACCGGCTCCAACGACGGCATGCT
GACCTTGGACGATCTGGGTAAGCAGGTGTCGGACTCGGACTC
GGACTCGTACCACTCTTCGGAGTACGAGGAAGACTGAGTATGA
TATTGACATTGACCTTGCCCACAAGACGGAGAATTTGCATGT
GCTGGACAAGGACGAAGATAGGAACAACGAACAGGAGGACA CGAAGTAA SEQ CtUTR1
MYATEEKKIEISDLRFLLQQATEYSTAMNNNNSSSSNSNYH ID (XP_0025479
RAQFTTGSSTTTNSSTSSLSELTTSSSFQRKNNFVPHSPKIESKLK NO: 52)
DDVKAASGGNTPRSIKSHIELAETANGVRLLAKNLARATIQLDV 190
KAIMVITKARDNSLITLTKQLVEWLLESHPHIVVFVDSKLQQSKR
FGVAPCNSLKFWTKRLVKKQPELFDLVVTLGGDGTVLYASTLF
QHIAPPVLPFSLGSLGFLTNFQFQDFKRILNRCIESGVKANLRMRF
TCRVHSSDGKLIGTMTLNELVVDRGPSPYVTQLELYGDGSLLT
VAQADGLIIATPTGSTAYSLSAGGSLVHPGVSAISVTPKPEITLSF
RPVLLPDGMFLKVKVPDGSRATAWCSFDGKARTELKKGDYVTI
QASSFPFPTVIASPTEYFDSVSRNLHWNVREQQKPLGNQTKAIDC
DMDNLHISSEQDEESEPDITEDDEEDDEFAINFTDTERSSYSSTPS
SDDIHYLSTNGAETPQSMSYLNNVDERCCFAHPNARVHLSGGKS SEQ CtUTR1
ATGTATGCAACTGAAGAAAAAAAAATTGAAATCTCCGACCTA ID
CGGTTTCTTTTACAACAAGCTATTGAATACTCCACAGCAATGA NO:
ATAACAACAACAACAATAATAGTAGTAGTAGTAATAGTAATT 191
ATCATAGAGCACAGTTTTACTACTGGTTCTAGTACTACTACAA
ATTCATCAACTTCATCATTATCTGAACTTACAACTTCATCTTC
TTTTAACGCAAGAATAATTTTGTCCCCCACTCTCCAAAAATC
CATTCCAAGTTGATTTGTGATGATGTTAAAGCTGCTCTGGGTG
GGAATACACGTGGTTGTATTAAATCACATACTGAATTGGCAG
AAACAGCTAATGGTGTTAGATTACTTGCCAAAAACTTAGCTA
GAGCTACTATTCAACTTGATGTCAAAGCAATCATGGTTATTAC
TAAAGCAAGAGATAATAGTTTAATCAGATTAACCAAACAATT
AGTTGAATGGCTTTTGGAAAGTCATCCACATATTGTTGTCTTT
GTTGATTCAAAGTTGCAACAATCAAAAAGATTTGGTGTTGCA
CCATGTAACTCATTAAAATTTTGGACTAAAAGATTGGTTAAA
AAACAACCTGAATTATTTGATTTGCTTGTTACATTAGGTGGTG
ATGGTACTGTTTTGTATGCTTCTACATTATTCCAACATATTGC
TCCTCCAGTTTTACCATTTAGTCTTGGGTCTCTTGGTTTTTGA
CAAATTTCCAATTCCAAGATTTCAAACGTATTTTGAATCGTTG
TATTGAATCAGGTGTCAAAGCAAATCTTCGTATGCGTTTCACT
TGTAGAGTTCATTCCAGTGATGGCAAATTAATTGGGCAATAT
CAAACATTGAATGAGTTGGTTGTCGATAGAGGTCCTTCTCCTT
ATGTTACTCAATTAGAATTGTATGGTGATGGTTCTTTGTTGAC
TGTTGCTCAAGCTGATGGGTTAATTATTGCTACTCCTACTGGT
TCAAGTGCTTATTCATTATCAGCTGGTGGTTCATTGGTTCATC
CAGGTGTTAGTGCTATCAGTGTTACTGCAATTTGTCCACATAC
TTTATCATTTAGACCAGTTTTGTTACCTGATGGTATGTTTTTAA
AAGTCAAAGTTCCAGATGGTAGTAGAGCAACTGCTTGGTGTT
CATTTGATGGTAAAGATAGAACTGAGTTGAAAAAAGGTGATT
ATGTCACCATTCAAGGTTCATCAGTCCCATTCCCAACAGTGAT
AGCATCACCAACTGAATATAAGACTCTGTTAGTAGAAACTTG
CACTGGAATGTCAGAGAACAACAGAAACCATTAGGAAATCA
AACTAAAGATATTGATGAAAATATGGATAACTTGCATATTTC
AAGTGAACAGGATGAAGAAAGTGAACCGGACATTACTGAAG
ATGATGAAAAAGATGATGAATTTGATATTAATTTCACAGACA
CAGAACGTTCTTCATATAGTTCAACTCCATCTAGTGATGATAT
TCATTATCTTTCTACAAATGGGGCAGAAACCCCACAATCCAT
GTCTTATCTTAACAATGTTGATGAAAGATGTTGTTTTGGTCAT
CCTAATGCTAGAGTGCATTTAAGTGGAGGTAAGAGCTGA SEQ Promoter of
ttttcttrcgttctcgttccgtcctrcaacccatatggatcgtgcgggaaaaatctgttttgcagcatgggcc
ID SSA1 in
agtcgcatggaatccgatagaaagttagcaatcttgagattttatttctttagctgtttcggc-
cccttttgtgt NO: pAA181
acccacgattttttttttttcaactttcttggttccatctcgtttgtcaacctcaattgaatg-
gtcaattgaagctt 192
gttcatgtctgtagaagctcaattgggtacaccatgtagtcacgatacctcgtcgcccagttgttctgc-
cc
cttgcgcgaatgttccagcaggttctagcaagctctcattggggcattccagcaccttcgatggtgttcta
tgagcttcgatcgaaaaacacaaacctatatgcaggacttttgcagctcctccccataaaactaaaatca
acctcccccttaagtttgctgtttcttcatgcctataaatatacgccaacctagcccgacttccagactttt-
t
tttctaatccctttctttttgttttccttctaaatcaatcaattagatttattagactactatatcataaca-
tca cataaaataacc SEQ Promoter of
ctagcaaaggcttgatcagagaaagcaacaaaaaaaaaaactctaatactccagaatacactcctttag
ID TEF1 in
aaacacacaacaaacaagcctagactaccatggactacgatgaagacgatttagattacattt-
ctcaag NO: pAA332
gagaagaggattgagtttgacgaaaacaagttgaacaacgaagagtagcacttgttgcatgaa-
atgctt 193
ccggagttgaagacaaaattgaaagattacaatgatgagatcccagattacgatttaaaggaagcgtta
tactacaactatttcgagatagaccctaccattgaagaattgaagacgaaattcaaaaagagtacgtata
tacaactaacatcaacgcctttctagtttctgttctactccaatgcttctcctgatcttcatggttctctct-
g
taccaacaaggaaaaaaaaaaaaataggcaaaaaaaaccaaaccaaccaatgttcttactcaccaac
gccctacaatc SEQ Terminator of
tggaaatcgaactcgacggtcacaacacgctcccattaaccaagtacgaagacaacaagatactcgg
ID TEF1 in
aaggattgtcatcagaagagaaggggtgactatcggggctggtacggttgtcgattattcaga-
agattg NO: pAA332
atatatagtctattaacaacatcaatataccaagtacgatgcatagagtaaccaacatgttgg- c
194 SEQ CTF1 open
atgtcaagctcagatgaaggagatcactcctgagttacaacaagagaaggttacccaaaataccca
ID reading frame
ctcggacgcaccttctgcaacaccacaaacaaccccggctcagacaacaaccacaaccacaaccac
NO:
cacttcctcctcccagacaaccactactaagaaaccaaaatcagatacaaaagataaaccaaaagcttt
195
caccattaaatataaacgtcctagaggttcaagagcttgtaccgtctgtcgctcaagaaaagtccgttg-
t
gacgcagaaatccacattccttgcacaaactgtataacatttggctgtgaatgcatcttgcccgaagcaa
aaaagagaggcaatcaatcaggcgaatccaaagccaaacgacaaaagactcaaccaaaagacaaa
gcagcaacaacagctgcttctaccaaaaagaaagcaacaactacaactacaaccgaaaacggcgat
gaggatggctccccactagaacagacatcgtcgtctcgcagtgctaaatccccggacttggtaacccc
taccaaagaatcagaaacatccagtcacacatgtctcgcagtgctaaatccccggacttggtaacccc
gagtccctccgtctttaacctcaacttataaaaacagactgatatcaagttggattctgtcgaattgga
cgaaagccaaaacagcattgacattcttggggctgtcgtcaataggtgttgttcctcaacgtgtcgggg
agaaccacgtcgagttaaccactgacgtttttgacaccagtgatatcaagttggattctgtcgaattgga
gattttgaagatgagaggcgccttcttgttaccaagtaaagaactatcattggaattgatcaatgcgtattt
tgaacacgtccacccattgatgcccgttataaatagatccttgtttatgaagaaattcaacgatccaaacg
acaacccaagtttaatggttctccacgccgtgctacttttgggctgtcgtgcctccaagaatccgttgttgt
tggactcgaatggaucaaacgatttagcaagcattacatcttttcagaagagccaaagcgttgtacgaga
caaaacacgaaagtgaccccagtcaattatccaaaccttgattttgattggctcgtattgggatggtcat
gaggatgttaccaagaactccttctactggacaagagtggctgtggggttggcccaaggttttgggttc
caacgtgatggcagtaaatctcaccaattgacaaaccttgattttgattggctcgtattgggatggtcat
gtctatttgagaaagatcgtaatgtggctattgcattcggtagaccagttgtgattgatttaaatgattgtg-
a
tgtccccatgttgaccgtggatgattttgacgaaactgacacgagacgggcatcaccgatccatacgta
gtaaacgaaactcaagcattatatttcatacatttggtgaagttggcagaaatcacaggtatcattatcaa
acatcaatacagcgtcaagtctgaaaccatgaagagaaggaacgcgttctccattatcgaacattgtga
tatgttgatgggtatttggtttactaacttgccgtccaacatggtgttctcattggcggatagtctgacaca-
c
aacttctttgcttgtttgctaaatgcacaatattacaaccgtttgtacttgatccacaggtccaatttgatc-
ag
aatggctagatcgtcatcaaccaacccaaacaactacaagtacccaagttggggtatttctttccaatcg
gccagaatgatttccatcatttccaaaatcttgatggacaaggatttgatacaattcgttcctgtcatgtat-
g
tttacatcgcgttcagtgcattggttatgttaatctacaagtacccaagttggggtatttctttccaatcg
tgttttttcagactcatgtttgtgtcaagagccgtcttgagggaacttcaaaaagcaggccagttgctgcg
gtgctattgaaattgtttgacaagtatgctaacgacaagttgaagagaaccaagcttattgagaacggta
acatgatcgtggagtacaaagagaatgctgctaaagaaaagatgaagaagtctattaatgaatatggca
ccaacaccagtccaacagcaatgccaattccaaatagactgccattatcacaaccacagcaacaacca
cagcaacaacaacaacaccgacaagttgtttcaacagcaattgccgcttccgcagccggcaaattccaa
gagaccgtacaatctcgccaagttctgtcacatcaggggtttctccgggtggtaatgtttacaacaac
tactcagagcagacaccggtcaaacaagagttttcgcgcaattgccgcttccgcagccggcaaattccaa
agcaagtacaaggtagcaaccctactccacagattgaagaattggttaaacaatacaagttgccaatga
aacgtgctgctaaggcgaacaatggaaacggtgatagcagtgataaacaaaccaccgatacttctcca
gcatcctccaccaagtcgttcccagatatctccatggttactgagaacttgcagaacaagcagaacttttt
tgaaaactttgaaccaacacagttgttccctactttcagcatcccaccaactagagcgcagtctccaatcg
ccaaatttcgatgatggagctattggcagtaacgtgtacaataccatgaacaccaccatggagatcaaa
gaagaaaggacaccacagccacaacaatcgggggaaaatcagcaggaacaacctcacaatggtaa
tgctgctgcaagtcttgctgctccagcacaaacactcaaggacgaacacgacattcttgatctacatact
ccaggattccaaaccaactttattgatagctccttcaactacttgaacttgcatcaggaatgaacatgga
cgaggacattcattctttgtttaacatgatgaaccaatag SEQ Amplicon
gaattcggccggccacaagtgcacgtactgtgacaaggcattccacagattggagcaccagacaaga
ID Fragment
cacatacgaacccacacgggcgaaaaaccccatgcctgcacctttcctgggtgtgtcaagcgcttcag
NO:
caggtcagacgaactcacaagacacttgagaatccacaccaacccatcgtcaagaaagagaaagaa
196
caagagtcaggacttgatgggaccaactcctatgagcatcaatggccagaacttgcctccaggctcgt
atgccgtgacaaacacgggtatcccctttgctatcgaccgtaacgggccagaccggccggatccgca
ttcgggccagaccggccggatccgcattcgggccagaaggccggatccgcattcgggccagacc
ggccggatccgcattcgggccagaccggccggatccgcattcgggccagcacggccgcggccgc
aataagtctcagccatcatctacatccttaaacctagagttctacaaccagggtaacgcttcccacgca
agcaaaaaatcgcgaccaaactccccatgccagtccgccatgaacatctcgtccatcatgagctcacc
aagcgaaaccccgctacagacgccatcacagtccccacgcttgcacgcaagcaacgggcctggtaa
tagcagtgtcattgagggtgcacaggcaaagttggagagtatagcgacgacaggaacacagttgccg
ccaattagatcggtgttgaggccggccgcatgc SEQ Sequence of
gagctccaattgtaatatttcgggagaaatatcgttggggtaaaacaacagagagagagagggagag
ID the fragment
atggttctgaagaattataatctggttgttgcaaatgctactgatcgactaggcaatgtctgtagctcgct
NO: in pAA1099
agttgtatgcaacttaggtgttatgcatacacacggttattcggttgaattgtggagtaaaaattgtctgag
197
ttgtgtcttagctactggctggcccaagcgaaagataatcaaaattacacttgtgaatttttgcacaca-
c
accgattaacatttcccttttttgtccaccgatacacgcttgcctcttcttttttttctctgtgcttccccc-
tcct
gtgactttttccaccattgatataaaatcaactccatttccctaaaatctccccagattctaaaaacaactt-
ct
tctcttctgcttttcctttttttttgttatattaccatccttttttttggatagcccccactaacattgttc
aaatcttcacgacataatgtcccatcaagttgaagaccacgacttagacgtgttctgtttattggccgatg
ccgtgaccatgaaancctcccagcgaaatcgtggagtaccttcatcctgacttcccaaaagataagat
cgaagagtatttgacaggcttttcccgtccgtctgctgttcctcagtttagacaatgtgccaagaagcttat
caacagaggctccgagctgtcgatcaagttgtttttgtacttgaccactgcgttggactcaagaatccttg
cacctgccttgaccaattcgttgactttgatcagggatatggatctttcccaaagagaggagttgttgaga
tcatggagagactctcctttaactgcaaaaagaagattatttagagtgtatgcctcttttaccttggctact-
tt
taacaagttgggaactgacttgcactttaaggcgttgggctacccaggtagagagctcagaacgcaaa
ttcaagactacgaagtcgacccttttagatattcgtttatggagaaacttaaacacgagggccacgaatt
gttccttcctgatattgacgttttaatcatcgggtcgggatcaggagcaggtgtggttgcacaaactctta
ctgaaagtggcctcaaatcattggttttggaaaagggcaaatactttgccagtgaagaattgtgcatgac
ggacttggacggtaacgaggcattattcgaaagtggaggaacaattccttccaccaaccaacaacaattgtt
catgattgcaggttcgacttttggtggtggttctacagttaattggtctgcatgtttgaagaccccattcaa-
a
gtaagaaaggaatggtatgacgatltcggacttgattttgtcgctactcaacaatacgacgattgtatgga
ttacgtgtggaagaaaatgggtgcttcgaccgaacatatcgaacattctgctgcaaatgccgtpatcatg
gacggggcagcaaaacttggctacgcacacagagcacttgagcagaataccgggggccatgttcac
gactgtgggatgtgccacttgggatgtagattcggtatcaaacaaggtggtgtaaattgctggttccgtg
aacctagtgaaaagggttctaagttcatggaacaagttgttgttgaaaagattttgcagcacaagggtaa
agctactggagattttgtaagagataagaaagtgggattaaattcaaaatcactggaccaaagaaatac
gttgtttccggtggttctttgcaaaccccagttttgttacaaaaatctggtttcaagaataaacatattgga-
g
ctaacttaaaacttcacccagtctcggttgcccttggggactttggtaatgaagtggactttgaagcntac
aagagaccacttatgaccgccgtttgtaatgccgtcgatgatttagatggcaaggcccatggaacaag
aattgaagccattttgcatgctccatacgtcactgccccattttacccatggcaatcaggtgctcaagcaa
gaaagaacctcttgaaatataaacaaactgtgccgttattacttctttctagagatacatcatcaggtaccg
ttacatatgataaacaaaagcctgacgtattggtagttgactacactgttaacaagtttgacagaaattcg
attttacagatttttggttgcttccgacatcttgtatattgaaggtgctaaagagattttgtcaccacaag
cttgggtaccaaccttcaagagcaacaaaccaaaacatgctagatcgatcaaagacgaagattacgtc
aaatggagagaaaccgtggccaagatcccatttgactcctacggttcgccatacggttctgctcatcaa
atgagttcgtgtagaatgtctggtaagggaccaggatacggtgcttgtgacactaaaggaagattatttg
aatgtaacaacgtttacgttgctgatgcttcggttatgcctactgcatcgggagtcaatcctatgatcacta
caatggcttttgcaagacatgtggccttatgtcttgctaaagacttgcaaccacaaactaaactttaggaa
tagaagagagtgactcttttgataagagtcgcaaatttgatttcataagtatatattcattatgtaaagtag-
t
aaatggaaaattcattaaaaaaaaagcaaatttccgttgtatgcatactccgaacacaaaactagccccg
gaaaaacccttagttgatagttgcgaatttaggtcgac SEQ MIG1
cggattatcttctgttttttttctttttttttttttttatagaaaaagcagtgagaaaatttttc-
ctccagtatgtgg ID sequence ORF
gcaccaacaccaccacctgcacaaaagatgttcctccccccacttgatctttgtgggattctttgcttttttt-
tc NO: and flanking
ggggagcagatatgtcagctggtggtgatccataggagctctcttgctcatgcttattaaaaaaaaaga
198 region
cagcaaagccttcgacgaggtccatggttcctaatcggagtgtcaagcacgacgaacattgcc-
cacca
gtaatattcctccaactgggagttaaaaaaaggccagtttttgttccctatccggagttgcacaaacggttg
tacacatcgccaaactggataccacaggggcactacaatcttgtttcattgtttttgttgacccacgggtg
gtacttcgttgtatcgaatattttctggcacccatttttcttggggcgagatggagagagagacagagag
agaccaccatgaggctgatgcaggtgcccaggttgaacaagatgaaattcgctcggactggggtttcc
gccccttccagaccgcttttctcagcacgatgatgtcgactgttgaaacgggggcgggcagggaaga
cagaaaacagaaattttcattaaattactgattgggttaattggcgggttagattctggagcttgaggagg
aagaggaggagaaggagaaggaggaagcctaaaaagtttttccgaaaattggaccagttraaggcat
cagacatatattacacaaagcaagagagtgcatgggaatataatatgcgtggttttgtgtagtttgttc
aagaaaagcgaacgagtgggcagaaattgcgggttacggtcgtttctttttcctgggtgtgcttttgtcttt
ctggctgtaagaattgtcatataaatttaactccagatctctctttctctcccccggtcccgtaagacaaaa
aaaaattgccctgtttccttctgcatcaccaaattcaccaccaccaccatgttaacccctaaggacaaga
agaacaaggaggacagaccatacaagtgcacgtactgtgacaaggcattccacagattggagcacc
agccaagacacatacgaacccacacgggcgaaaaaccccatgcctgcacctttcctgggtgtgtcaa
gcgcttcagcaggtcagacgaactcacaagacacttgagaatccacaccaacccatcgtcaagaaag
agaaagaacaagaggcaggacttgatgggatcaactcttgatgggaccaactcctatgaaacttgcctcc
aggctcgtatgccgtgacaaacacgggtatcccctttgctatcgaccgtaacggcaatcacgtgtaccc
tcaaccataccctgtgtttttcgtcccacagccaaacggatacatgcagcctgttgttcaagcaccaggg
ctctccattgttccaccaccaccaccacaacaacaacaacaacaaggacaagcaccacaacagcagc
aacaacaactacattcacaacagcaacaacagcgtatgggtagcccacacagcaatatgacaacacc
tacacatttgcaacaggaaggatcagcagttttctccattccttcgtcccctacaaactcataccaaaatg
tcccaaaccgtccaaatcaacaaccattaccacctccacaaccagttaccatgtccacaccaatgtcta
cgagatcaatgtcgtcagatgctattagattgccaccactaacgtcaaacaactccttccaacaacaaca
acaacagcagccacgcccaccaccaaccattctcaaatcggagtctacaactagcctatactcggatt
caaacagggtgtttagccaaccaaactcaaatttgcactctttgggtacgtcgccagacacaagcccat
ccgccatgccaccaccaacagttgtcccaactccgtctttcagcaacttgaatgaatacttccagcaaaa
gagcaacaacccaaggattttcaacgccagttcatcatccttgagctcgttgagtggcaagatcagatc
cacgtcatccacaaaccttgcaggattgcaaagattgactccattggttcagacatctacaaactcgaca
cccggaaaatccataataccgtctcagccatcatctacatccttaaacctagagttctacaaccagggta
acgcttcccacgcaagcaaaaaatcgcgaccactccccatgccagtccgccatgaacatctcgtcc
atcatgagctcaccaagcgaaaccccgctacagacgccatcacagtccccacgcttgcacgcaagca
acgggcctggtaatagcagtgtcattgagggtgcacaacaaagttggagagtatagcgacgacag
gaacacagttgccgccaattagatcggtgttgagctttacaaacttgtcggattatcctccgcctccttcta
atagatagatatacataattattagtaagagttgataaaaatacatacaaaaaaaccaactaagggatac
cgttatacatattacttcagtttcgtaacaatcagacgagtgtatatctggctggctatgtaattaacgcgg
gatgggagcattgtttctctcccccgtgggcgaagtctgcttaacccccaggatctgcagcctgcaaac
ccagtgcaactccattgaacagagcctgcgaagcaaattttctcgaaacattttccagaccttggacttat
tccaccgagaaatgcaaccaacgaacaacgcaagtttcagtgtttcctttttgtattttgcaggtgcatac
gcaagtgtgagtgaatgagtagtgtgatgggtttctgcttttgtggatttccaggttacgtcctggtattaa-
t
tgttctttgggggttggtctcttttgggtagcttgttggtacaaggcgggttttttcattgcagcttccttt-
gg
attacaacagctttctccccacggacagttgggcatggacggaaatataataacagccgggagaata
atacggaaggtgacgaagataattatttcttcggattaaatttcccctggaggttcaagagtgaattattat
catccatcgtcatcgccatcacttcctcttcctcccccaacttcatctccagatcaatccccccagggattt
ttcacttctgattattttttaccacaaaaaataaccaggcttttctttcttttttttccatttttgaggagc-
tgcc
cacaatggacccagtttacggccgcagaatcattcagtttcaaatacctggggaagacaagacaaagg
gaaggtgattagaacacatttgtgtgccgtacttgttcgccctacggggttgctcgcaacgactttctctc
cgccctgcgaaaaactcccgtgctccctccaggagaatcttactccgatgtttagactttttctcaacaact
ataaagggatcacgaatcctcagacattcttgtcattgctttcctagaccatttatcaacttaccaaacaac
aaacatgtctactgctactgcttcccc SEQ CvUTR1
atgtatgcgaccaacgaaaaaaaaattgaaatctccgacctacgatttcttttacaacaagca-
atcgaat ID
actccacagccatgaataacaacaataatcatagctacaacaccaataacaactcccaccgagctgtat
NO:
tcacctctagcagctcaaccaccaactcatcgacctcctccttgtctgagctcaccacgtcatcgtctt-
tc 199
caccgcaagaacaatatgatcccgcattctcccaaggtccactccaagttggtctgtgacgaaatctcc
gccgctctgactagtggaacaagcacgccgcgctccatcaagtctcacaccgagttggccgaaactg
ccaacggggtcagattgctcgccaagaacttgtcccgggccacgatccagctcgacgtgagagcaat
catggttatcaccaaggctagagacaacagcttgatcacattgaccaaacaactagtagagtggttgtt
ggaaactcaccctcacatcaccatctttgtcgacgccaaattgcagcaatctaagcgtttcggcgtagct
acttgcaactcgttgaagttctggaacaaaagattggttaaaaaacagccagagttgtttgacttggtcgt
cacgttgggcggtgatggtaccgtgttgtatgcgtctactttgttccagagcattgcaccaccggtcttgc
cattcagtcttgggtccctaggtrtcttgacaaatttccagttccaggatttcaagcgcattttgaaccgct-
g
tatcgagtcaggtgttaaggcaaacctccgtatgcgtttcacctgtagggttcatgccaacgacggcaa
gttgattggacagtaccagactttgaatgagcttgttgttgatagaggtccttctccttacgtcaccctgtt-
g
gaattgtacggtgacggctcgttgacggttgctcaagcagacgggttgattatcgctactccaactg
ggttgactgcttattcgttgtctgctggaggttctttagtgcatcctggtgttagtgctattagtgtcaccc-
c
aatctgtccgcacaccctatcttttaggccaattttgttgcccgatggaatgttttgaaagtcaaagttcca
gacggtagtagagctactgcctggtgttcgatggtaaggacagaaccgaattgcacaagggtga
ttacgttaccatccaagcttcgccattcccattccctaccgtgatagcttctccaaacgaatactttgattc-
t
gtcagtagaaacttgcattggaacgtcagagaacaacagaagccattgagtgataactccaaggatgtt
gatggggccatggacaacttgcacatttcgagtgaacaagacgaagaggacgaggaacctgagatta
ctgaagaggaggatgattttgatatcaatttcaccgatactgagcgttcttcctacagttctactccttcaa
gtgacgacatgaactttcttgcgaaatggcacagctaccccacagaacatgtcttatcttaacaatgtt
gatgagagatgttgttttgcccaccctaatgctagagttcacttgagtggcggcaaaagc SEQ
UTR1
atgtatgcgaccaacgaaaaaaaaattgaaatctccgacctacgatttctttacaacaagcaatc-
gaat ID (ATCC20962)
actccacagccatgaataacaacaataatcatagctacaacaccaataacaactcccaccgagctgtat
NO: sequence
tcacctctagcagctcaaccaccaactcatcgacctcctccttgtctgagctcaccacgtcatcgtctttc
200
caccgcaagaacaatatgatcccgcattctcccaaggtccactccaagttggtctgtgacgaaatctcc
gccgctctgactagtggaacaagcacgccgcgctccatcaagtctcacaccgagttggccgaaactg
ccaacggggtcagattgctcgccaagaacngtcccgggccacgatccagctcgacgtgagagcaat
catggttatcaccaaggctagagacaacagcttgatcacattgaccaaacaactagtagagtggttgtt
ggaaactcaccctcacatcaccatctttgtcgacgccaaattgcagcaatctaagcgtttcggcgtagct
acttgcaactcgttgaagttctggaacaaaagattggttaaaaaacagccagagttgtttgacttggtcgt
cacgttgggcggtgatggtaccgtgttgtatgcgtctactttgttccagagcattgcaccaccggtcttgc
cattcagtcttgggtccctaggtttcttgacaaatttccagttccaggatttcaagcgcattttgaaccgct-
g
tatcgagtcttggtgttaaggcaaacctccgtatgcgtttcacctgtagggttcatgccaacgacggcaa
gttgattggaaagtaccagactttgaatgagcttgttgttgatagaggtccttctccttacgtcaccctgtt-
g
gaattgtacggtgacggctcgttgttgacggttgctcaagcagacgggttgattatcgctactccaactg
ggtcgactgctcattcgttgtctgctggaggttctttagtgcatcctggtgttagtgctattagtgtcaccc-
c
aatctgtccgcacaccctatcttttaggccaattttgttgcccgatggaatgtttttgaaagtcaaagttcc-
a
gacggtagtagagctactgcctggtgttcgttcgatggtaaggacagaaccgaattgcacaagggtga
ttacgttaccatccaagcttcgccattcccattccctaccgtgatagcttctccaaacgaatactttgattc-
t
gtcagtagaaacttgcattggaacgtcagagaacaacagaagccattgagrgataactccaaggatgtt
gatggggccatggacaacttgcacatttcgagtgaacaagacgaagaggacgaggaacctgagatta
ctgaagaggaggatgattttgatatcaatttcaccgatactgagcgttcttcctacagttctaactccttca-
a
gtgacgacatgaactttcttgcgggtaatggcacagctaccccacagaacatgtcttatcttaacaatgtt
gatgagagatgttgttttgcccaccctaatgctagagttcacttgagtggcggcaaaagc SEQ
Promoter of
ttttctttcgttctcgttccgtctttcaacccatatggatcgtgcgggaaaaatctgttttgcagcatgggcc
ID SSA1 in
agtcgcatggaatccgatagaaagttagcaatcttgagattttatttctttagctgtttcggc-
cccttttgtgt NO: pAA181
acccacgattttttttttttcaactttcttggttccatctcgtttgtcaacctcaattgaatg-
gtcaattgaagctt 201
gttcatgtctgtagaagctcaattgggtacaccatgtagtcacgatacctcgtcgcccagttgttctgc-
cc
cttgcgcgaatgttccagcaggttctagcaagctctcattggggcattccagcaccttcgatggtgttcta
tgagcttcgatcgaaaaacacaaacctatatgcaggacttttgcagctcctccccataaaactaaaaatca
acctcccccttaagtttgctgtttcctcatgcctataaatatacgccaacctagcccgacttccagactttt
tttctaatccctttctttttgttttccttctaaatcaatcaattagatttattagactactatatcataata-
atcatca cataaaataacc SEQ Promoter of
ctagcaaaggcttgatcagagaaagcaacaaaaaaaaaaactctaatactccagaatacactcctttag
ID TEF1 in
aaacacacaacaaacaagcctagactaccatggactacgatgaagacgatttagattacattt-
ctcaag NO: pAA332
gagaagaggaagagtttgacgaaaacaagttgaacaacgaagagtacgacttgttgcatgaca-
tgctt 202
ccggagttgaagacaaaattgaaagattacaatgatgagatcccagattacgatttaaaggaagcgtta
tactacaactatttcgagatagaccctaccattgaagaattgaagacgaaattcaaaaagagtacgtata
tacaactaacatcaacgcctttctagtttctgttctgtctccaatgcttctcctggtttcttcatggttctc-
tctg
taccaacaaggaaaaaaaaaaaaatctggcaaaaaaaaccaaaccaaccaatgttcttactcaccaac
gccctacaatc SEQ Terminator of
tggaaatcgaactcgacggtcacaacacgctcccattaaccaagtacgaagacaacaagatactcgg
ID TEF2 in
aaggattgtcatcagaagagaaggggtgactatcggggctggtcggttgtcgattattcagaa-
gattg NO: pAA332
atatatagtctattaacaacatcaatataccaagtacgatgcatagagtaaccaacatgttgg- c
203 SEQ CIT1 open
atgtcaagctcagatgaaggagatcacactcctgagttacaacaagagaaggttacccaaaataccca
ID reading frame
ctcggacgcaccttctgcaacaccacaaacaaccccggctcagacaacaaccacaaccacaaccac
NO:
cacttcctcctcccagacaaccactactaagaaaccaaaatcagatacaaaagataaaccaaaagcttt
204
caccattaaatataaacgtcctagaggttcaagagcttgtaccgtctgtcgctcaagaaaagtccgttg-
t
gacgcagaaatccacattccttgcacaaactgtataacatttggctgtgaatgcatcttgcccgaagcaa
aaaagagaggcaatcaatcaggcgaatccaaagccaaacgacaaaagactcaaccaaaagacaaa
gcagcaacaacagctgcttctaccaaaaagaaagcaacaactacaactacaaccgaaaacggcgat
gaggatggctccccactagaacagacatcgtcgtctcgcagtgctaaatccccggacttggtaacccc
taccaaagaatcagaaacatccagtcacacatcatccacagcggagccaataataaatatatcacaagt
gagtgttcctccgtctttaacctcaacttataaaaacagaccatcaatgcacaaaaaggaacttctcgact
cgaaagccaaaacagcattgacattcttggggctgtcgtcaataggtgttgttcctcaacgtgtcgggg
agaaccacgtcgagttaaccactgacgtttttgacaccagtgatatcaaatggattctgtcgaattgga
gattttgaagatgagaggcgccttcttgttaccaagtaaagaactatcattggaattgatcaatgcgtattt
tgaacacgtccacccattgatgcccgttataaatagatccttgtttatgaagaaattcaacgatccaacg
acaacccaagtttaatggttctccacgccgtgctacttttgggctgtcgtgcctccaagaatccgttgttgt
tggactcgaatggaacaaacgatttagcaagcattacatttttcagaagagccaaagcgttgtacgaga
caaactacgaaagtgaccccgtgtcaattatccaaaccttgattttgattggctcgtattgggatggtccg
gaggatgttaccaagaactccttctactggacaagagtggctgtggggttggcccaaggttttgggttc
caacgtgatgtcagtaaatctcaccaattgacggtttctgaaaagaagatctggagaagaatctggtggt
gtctatttgagaaagatcgtaatgtggctattgcattcggtagaccagttgtgattgatttaaatgattgtg-
a
tgccccatgttgaccgtggatgattttgacgaaactgacccagagttgggcatcaccgatccatacct
gtaaacgaaactcaagcattatatttcatacatttggtgaagttggcagaaatcacaggtatcattatcaa
acatcaatacagcgtcaagtctgaaaccatgaagagaaggaacgcgttctccattatcgaacattgtga
tatgttgatgggtatttggtttactaacttgccgtccaacttggtgttctcattggcggatagtctgacaca-
c
aacttctttgcttgtttgctaaatgcacaatattacaaccgtttgtacttgatccacaggtccaatttgatc-
ag
aatggctagatcgtcatcaaccaacccaaacaactacaagtacccaagttggggtatttctttccaatcg
gccagaatgatttccatcatttccaaaatcttgatggacaaggatttgatacaattcgttcctgtcatgtat-
g
tttacatcgcgttcagtgcattggttatgttaatctaccatgtggactcggccaactcagtcattgctgcta-
c
tgcttcagactccttgtttgtgtcaagagccgtcttgagggaacttcaaaaagcctggccagttgctgcg
gtgctattgaaattgtttgacaagtatgctaacgacaagttgaagagaaccaagcttattgagaacggta
acatgatccgtggagtacaaagagaatgctgctaaagaaaagatgaagaagtctattaatgaatatggca
ccaacaccagtccaacagcaatgccaattccaaatagactgccattatcacaaccacagcaacaacca
cagcaacaacaacaacaccgacaagttgttcaacagcaattgccgcttccgcagccggcaaaaccaa
gagaccgtacgttgttctcgccaagttctgtcacatcaggggtttctccgggtggtaatgtttacaacaac
tactcagagcagacaccggtcaaacaagagttttcgctggtttctcctgttgctgcaaacccacctcaac
agcaagtacaaggtagcaaccctactccacagattgaagaattggttaaacaatacaagttgccaatga
aacgtgctgctaaggcgaacaatggaaacgaagatagcagtgataaacaaaccaccgatacttctcca
gcatcctccaccaagtcgttcccagatatctccatggttactgagaacttgcagaacaagcagaacttttt
tgaaaactttgaaccaacacagttgttccctactttcagcatcccaccaactagagcgcagtctccaacg
ccaaatttcgatgatggagctattggcagtaacgtgtacaataccatgaacaccaccatggagatcaaa
gaagaaaggacaccacagccacaacaatcgggggaaaatcagcaggaacaacctcacaatggtaa
tgctgctgcaagtcttgctgctccagcacaaacactcaaggacgaacacgacattcttgatctacatact
ccaggattccaaacaactttattgatagctccttcaactacttgaacttgcaatcaggaatgaacatgga
cgaggacattcattctttgtttaacatgatgaaccaatag
Example 76
Description of Some Strains Referenced Herein
TABLE-US-00042 [0641] TABLE 41 Description of some strains
referenced herein. Strain Genetic Modifications sAA496 pox4.DELTA.,
CPR750, P450A19 sAA617 pox4.DELTA., CPR750, acoata.DELTA./ACOATB,
POX5 sAA620 pox4.DELTA., CPR750, acoata.DELTA./ACOATB, POX5, ura3
sAA632 pox4.DELTA., CPR750, acoata.DELTA./ACOATB, P450A19 sAA635
pox4.DELTA., CPR750, acoata.DELTA./ACOATB, P450A19 sAA722
pox4.DELTA., acs1.DELTA./ACS1 sAA741 pox4.DELTA., acs1.DELTA./ACS1,
ura3 sAA776 pox4.DELTA., acs1.DELTA. sAA779 pox4.DELTA.,
acs1.DELTA., ura3 sAA811 pox4.DELTA., acs1.DELTA., P450A19 sAA810
pox4.DELTA., acs1.DELTA., P450A19, EcTESA sAA865 pox4.DELTA.,
acs1.DELTA., fat1.DELTA./FAT1 sAA869 pox4.DELTA., acs1.DELTA.,
fat1.DELTA./FAT1 ura3 sAA875 pox4.DELTA., acs1.DELTA., fat1.DELTA.
s4A886 pox4.DELTA., acs1.DELTA., fat1.DELTA., ura3 sAA1764
pox4.DELTA., acs1.DELTA., fat1.DELTA., eci1.DELTA./ECI1 sAA1860
pox4.DELTA., acs1.DELTA., fat1.DELTA., eci1.DELTA./ECI1, ura3
sAA2058 pox4.DELTA., pox5.DELTA., acs1.DELTA., fat1.DELTA.,
POX5(T287A) sAA2109 pox4.DELTA., pox5.DELTA., acs1.DELTA.,
fat1.DELTA., POX4(D96A) sAA2220 pox4.DELTA., acs1.DELTA.,
fat1.DELTA., eci1A sAA2291 pox4.DELTA., pox5.DELTA./POX5,
acs1.DELTA., fat1.DELTA. sAA2310 pox4.DELTA., pox5.DELTA./POX5,
acs1.DELTA., fat1.DELTA., ura3 sAA2399 pox4.DELTA., pox5.DELTA.,
acs1.DELTA., fat1.DELTA. sAA2428 pox4.DELTA., pox5.DELTA.,
acs1.DELTA., fat1.DELTA., ura3 sAA2570 pox4.DELTA., pox5.DELTA.,
acs1.DELTA., fat1.DELTA., POX5(F98G) sAA2645 pox4.DELTA.,
pox5.DELTA., acs1.DELTA., fat1.DELTA., POX5(S88A) sAA2646
pox4.DELTA., pox5.DELTA., acs1.DELTA., fat1.DELTA., POX5(G284E)
sAA2648 pox4.DELTA., pox5.DELTA., acs1.DELTA., fat1.DELTA.,
POX5(S292A) sAA2651 pox4.DELTA., pox5.DELTA., acs1.DELTA.,
fat1.DELTA., POX5(p95A,Q96A) **sAA2780 pox4.DELTA., pox5.DELTA.,
acs1.DELTA., fat1.DELTA., POX4 Note: Genes in lower case and/or
with a .DELTA. symbol indicate a deleted gene. Strains sAA6171,
sAA620 and sAA632 comprise a deletion of one allele of "acoat" and
are heterozygous for the acoat gene knock out (e.g., acetoacetyl
CoA thiolase .sup.-/+). **Strain AA2780 comprises a deletion of the
endogenous POX4 gene (i.e. pox4.DELTA.) and re-introduction of the
wild type POX4 gene (i.e., POX4) under the control of a PEX11
promoter.
Example 77
Examples of Certain Non-Limiting Embodiments
[0642] A1. A genetically modified yeast, comprising: [0643] one or
more genetic modifications that substantially block beta oxidation
activity; [0644] one or more genetic modifications that increase
one or more activities chosen from monooxygenase activity,
monooxygenase reductase activity, thioesterase activity,
acyltransferase activity, isocitrate dehydrogenase activity,
glyceraldehyde-3-phosphate dehydrogenase activity,
glucose-6-phosphate dehydrogenase activity, acyl-coA oxidase
activity, fatty alcohol oxidase activity, acyl-CoA hydrolase
activity, alcohol dehydrogenase activity, peroxisomal biogenesis
factor activity, fatty aldehyde dehydrogenase activity, CTF, UTR,
FAT1; and a genetic modification that decreases MIG1 activity.
[0645] A2. The genetically modified yeast of embodiment A1, wherein
the one or more genetic modifications increase one or more
monooxygase activities chosen from a CYP52A12 monooxygenase
activity, CYP52A13 monooxygenase activity, YCP52A14 monooxygenase
activity, CYP52A15 monooxygenase activity; CYP52A16 monooxygenase
activity, CYP52A17 monooxygenase activity, CYP52A18 monooxygenase
activity, CYP52A19 monooxygenase activity, CYP52A20 monooxygenase
activity, CYP52D2 monooxygenase activity and BM3 monooxygenase
activity.
[0646] A3. The genetically modified yeast of embodiment A1 or A2,
wherein the one or more genetic modifications increase one or more
monooxygenase reductase activities chosen from CPRA monooxygenase
reductase activity, CPRB monooxygenase reductase activity and
CPR750 monooxygenase reductase activity.
[0647] A4. The genetically modified yeast of anyone of embodiments
A1 to A3, wherein the one or more genetic modifications increase a
IDP2 isocitrate dehydrogenase activity.
[0648] A5. The genetically modified yeast of any one of embodiments
A1 to A4, wherein the one or more genetic modifications increase a
GDP1 glyceraldehyde-3-phosphate dehydrogenase activity.
[0649] A6. The genetically modified yeast of any one of embodiments
A1 to A5; wherein the one or more genetic modifications increase
one or more glucose-6-phosphate dehydrogenase activities chosen
from a ZWF1 glucose-6-phosphate dehydrogenase activity and ZWF2
glucose-6-phosphate dehydrogenase-activity.
[0650] A7. The genetically modified yeast of any one of embodiments
A1 to A6, wherein the one or more genetic modifications increase
one or more fatty alcohol oxidase activities chosen from FAO1 fatty
alcohol oxidase activity, FAO2A fatty alcohol oxidase activity,
FAO2B fatty alcohol oxidase activity, FAO13 fatty alcohol oxidase
activity, FAO17 fatty alcohol oxidase activity, FAO18 fatty alcohol
oxidase activity and FAO20 fatty alcohol oxidase activity.
[0651] A8. The genetically modified yeast of any one of embodiments
A1 to A7, wherein the one or more genetic modifications increase
one or more alcohol dehydrogenase activities chosen from ADH1
alcohol dehydrogenase activity, ADH2 alcohol dehydrogenase
activity, ADH3 alcohol dehydrogenase activity, ADH4 alcohol
dehydrogenase activity, ADH5 alcohol dehydrogenase activity, ADH7
alcohol dehydrogenase activity, ADH8 alcohol dehydrogenase activity
and SFA alcohol dehydrogenase activity.
[0652] A9. The genetically modified yeast of any one of embodiments
A1 to A8 wherein the one or more genetic modifications increase one
or more acyl-CoA hydrolase activities chosen from ACH-A acyl-CoA
hydrolase activity and ACH-B acyl-CoA hydrolase activity.
[0653] A10. The genetically modified yeast of any one of
embodiments A1 to A9, wherein the one or more genetic modifications
increase one or more acyltransferase activities chosen from
acyl-CoA sterol acyltransferase activity, diacylglycerol
acyltransferase activity and phospholipid:diacylglycerol
acyltransferase activity.
[0654] A11. The genetically modified yeast of embodiment A10,
wherein the one or more acyltransferase activities are chosen from
ARE1 acyl-CoA sterol acyltransferase activity, ARE2 acyl-CoA sterol
acyltransferase activity, DGA1 diacylglycerol acyltransferase
activity, and LRO1 phospholipid:diacylglycerol acyltransferase
activity.
[0655] A12. The genetically modified yeast of any one of
embodiments A1 to A11, wherein the one or more genetic
modifications increase an acyl-coA thioesterase activity.
[0656] A13. The genetically modified yeast of embodiment A12,
wherein the acyl-coA thioesterase activity is a TESA acyl-coA
thioesterase activity.
[0657] A14. The genetically modified yeast of any one of
embodiments A1 to A13, wherein the one or more genetic
modifications increase a PEX11 peroxisomal biogenesis factor
activity;
[0658] A15. The genetically modified yeast of any one of
embodiments A1 to A14, wherein the one or more genetic
modifications increase one or more fatty aldehyde dehydrogenase
activities chosen from HFD1 fatty aldehyde dehydrogenase activity
and HFD2 fatty aldehyde dehydrogenase activity.
[0659] A16. The genetically modified yeast of any one of
embodiments A1 to A15, wherein the one or more genetic
modifications increase a POX5 acyl-coA oxidase activity.
[0660] A17. The genetically modified yeast of any one of
embodiments A1 to A16, wherein the one or more genetic
modifications increase a monooxygenase activity and a monooxygenase
reductase activity.
[0661] A18. The genetically modified yeast of embodiment A17,
wherein the one or more genetic modifications increase a CYP52A19
monooxygenase activity and a CPRB monooxygenase reductase
activity.
[0662] A19. The genetically modified yeast of embodiment A17,
wherein the one or more genetic modifications increase a CYP52A14
monooxygenase activity and a CPRB monooxygenase reductase
activity.
[0663] A20. The genetically modified yeast of any one of
embodiments A1 to A19, wherein the one or more genetic
modifications increase a monooxygenase activity, a monooxygenase
reductase activity, and a isocitrate dehydrogenase activity.
[0664] A21. The genetically modified yeast of embodiment A20,
wherein the one or more genetic modifications increase a CYP52A19
monooxygenase activity, a CPRB monooxygenase reductase activity,
and a IDP2 isocitrate dehydrogenase activity.
[0665] A22. The genetically modified yeast of any one of
embodiments A1 to A21, wherein the one or more genetic
modifications increase a monooxygenase activity, a monooxygenase
reductase activity, and a glucose-6-phosphate dehydrogenase
activity.
[0666] A23. The genetically modified yeast of embodiment A22,
wherein the one or more genetic modifications increase a CYP52A19
monooxygenase activity, a CPRB monooxygenase reductase-activity,
and a ZWF1 glucose-6-phosphate dehydrogenase activity.
[0667] A24. The genetically modified yeast of any one of
embodiments A1 to A23, wherein a monooxygenase activity is by a
polypeptide comprising an amino acid sequence chosen from SEQ ID
NOs: 52, 53, 54, 55, 56, 57, 58, 59, 60 and 61.
[0668] A25. The genetically modified yeast of embodiment A24,
wherein the polypeptide is encoded by a polynucleotide chosen from
SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24.
[0669] A26. The genetically modified yeast of any one of
embodiments A1 to A25, wherein a monooxygenase reductase activity
is by a polypeptide comprising an amino acid sequence chosen from
SEQ ID NOs: 28, 63, 64 and 65.
[0670] A27. The genetically modified yeast of embodiment A26,
wherein the polypeptide is encoded by a polynucleotide chosen from
SEQ ID NOs: 24, 25, 26 and 27.
[0671] A28. The genetically modified yeast of any one of
embodiments A1 to A27, wherein a thioesterase activity is by a
polypeptide comprising an amino acid sequence of SEQ ID NO: 38.
[0672] A29. The genetically modified yeast of embodiment A28,
wherein the polypeptide is encoded by a polynucleotide of SEQ ID
NO: 37.
[0673] A30. The genetically modified yeast of any one of
embodiments A1 to A29, wherein an acyltransferase activity is by a
polypeptide comprising an amino acid sequence chosen from SEQ ID
NOs: 44, 46, 48 and 50.
[0674] A31. The genetically modified yeast of embodiment A30,
wherein the polypeptide is encoded by a polynucleotide chosen from
SEQ ID NOs: 43, 45, 47 and 49.
[0675] A32. The genetically modified yeast of any one of
embodiments A1 to A31, wherein an isocitrate dehydrogenase activity
is by a polypeptide comprising an amino acid sequence of SEQ ID NO:
67, 69 or 100.
[0676] A33. The genetically modified yeast of embodiment A32,
wherein the polypeptide is encoded by a polynucleotide of SEQ ID
NO: 68, 70 or 99.
[0677] A34. The genetically modified yeast of any one of
embodiments A1 to A33, wherein a glyceraldehyde-3-phosphate
dehydrogenase activity is by a polypeptide comprising an amino acid
sequence of SEQ ID NO: 72.
[0678] A35. The genetically modified yeast of embodiment A34,
wherein the polypeptide is encoded by a polynucleotide of SEQ. ID
NO: 71.
[0679] A36. The genetically modified yeast of any one of
embodiments A1 to A35, wherein a glucose-6-phosphate dehydrogenase
activity is by a polypeptide comprising an amino acid sequence of
SEQ ID NO: 74, 76 or 157.
[0680] A37. The genetically modified yeast of embodiment A36,
wherein the polypeptide is encoded by a polynucleotide of SEQ ID
NO: 73 or 75.
[0681] A38. The genetically modified yeast of any one of
embodiments A1 to A37, wherein an acyl-coA oxidase activity is by a
polypeptide comprising an amino acid sequence of SEQ ID NO: 32.
[0682] A39. The genetically modified yeast of embodiment A38,
wherein the polypeptide is encoded by a polynucleotide of SEQ ID
NO: 31.
[0683] A40. The genetically modified yeast of any one of
embodiments A1 to A39, wherein a fatty alcohol oxidase activity is
by a polypeptide comprising an amino acid sequence chosen from SEQ
ID NOs: 3, 5, 7, 9, 11, 13, 132 and 134.
[0684] A41. The genetically modified yeast of embodiment A40,
wherein the polypeptide is encoded by a polynucleotide chosen from
SEQ ID NOs: 2, 4, 6, 8, 10, 12, 131 and 133.
[0685] A42. The genetically modified yeast of any one of
embodiments A1 to A41, wherein an acyl-CoA hydrolase activity is by
a polypeptide comprising an amino acid sequence chosen from SEQ ID
NOs: 34 and 36.
[0686] A43. The genetically modified yeast of embodiment A42,
wherein the polypeptide is encoded by a polynucleotide chosen from
SEQ ID NOs: 33 and 35.
[0687] A44. The genetically modified yeast of any one of
embodiments A1 to A43, wherein an alcohol dehydrogenase activity is
by a polypeptide comprising an amino acid sequence chosen from SEQ
ID NOs: 129, 113, 115, 117, 119, 121, 123, 125, 127, 152 and
154.
[0688] A45. The genetically modified yeast of embodiment A44,
wherein the polypeptide is encoded by a polynucleotide chosen from
SEQ ID NOs: 130, 114, 116, 118, 120, 122, 124, 126, 128, 153 and
155.
[0689] A46. The genetically modified yeast of any one of
embodiments A1 to A45, wherein a peroxisomal biogenesis factor
activity is by a polypeptide comprising an amino acid sequence of
SEQ ID NO: 96.
[0690] A47. The genetically modified yeast of embodiment A46,
wherein the polypeptide is encoded by a polynucleotide of SEQ ID
NO: 95.
[0691] A48. The genetically modified yeast of any one of
embodiments A1 to A47, wherein a fatty aldehyde dehydrogenase
activity is by a polypeptide comprising an amino acid sequence
chosen from SEQ ID NOs: 139 and 141.
[0692] A49. The genetically modified yeast of embodiment A48,
wherein the polypeptide is encoded by a polynucleotide chosen from
SEQ ID NOs: 140 and 142.
[0693] A50. The genetically modified yeast of any one of
embodiments A1 to A49, comprising one or more genetic modifications
that decrease an acyl-coA synthetase activity.
[0694] A51. The genetically modified yeast of embodiment A50,
wherein the one or more genetic modifications decrease one or more
acyl-coA synthetase activities chosen from ACS1 acyl-coA synthetase
activity and FAT1 long-chain acyl-CoA synthetase activity.
[0695] A52. The genetically modified yeast of embodiment A50 or
A51, wherein the one or more genetic modifications disrupt a
nucleic acid that encodes a polypeptide having the acyl-coA
synthetase activity.
[0696] A53. The genetically modified yeast of any one of
embodiments A50 to A52, wherein the acyl-coA synthetase activity is
by a polypeptide comprising an amino acid sequence chosen from SEQ
ID NOs: 40, 42, 80, 82, 84, 90, 158 and 159.
[0697] A54. The genetically modified yeast of embodiment A53,
wherein the polypeptide is encoded by a polynucleotide chosen from
SEQ ID NOs: 39, 41, 79, 81, 83 and 89.
[0698] A55. The genetically modified yeast of any one of
embodiments, A1 to A54, which is a Candida spp. yeast.
[0699] A56. The genetically modified yeast of embodiment A55,
wherein the Candida spp. yeast is chosen from C. tropicalis and C.
viswanathii.
[0700] A57. The genetically modified yeast of embodiment A56,
wherein the Candida spp. yeast is a genetically modified ATCC20336
yeast.
[0701] A58. The genetically modified yeast of any one of
embodiments A1 to A57, which is chosen from a Yarrowia spp. yeast,
Pichia spp. yeast, Saccharomyces spp. yeast and Kluyveromyces spp.
yeast.
[0702] A59. The genetically modified yeast of embodiment A58, which
is chosen from Y. lipolytica, P. pastoris, P. membranifaciens, P.
kluyveri, P. guilliermondii, P. heedii, P. subpelliculosa, S.
cerevisiae, S. bayanus, S. pastorianus, S. carlsbergensis, K.
lactis and K. marxianus.
[0703] A60. The genetically modified yeast of any one of
embodiments A1 to A59, which is capable of producing a diacid from
a feedstock comprising one or more components from a vegetable
oil.
[0704] A61. The genetically modified yeast of embodiment A60,
wherein the diacid is a C4 to C24 diacid.
[0705] A62. The genetically modified yeast of embodiment A61,
wherein the diacid is a C10, C12, C14, C16, C18 or C20 diacid.
[0706] A62.1. The genetically modified yeast of embodiment A62,
wherein the diacid is a C10 diacid.
[0707] A63. The genetically modified yeast of embodiment A62,
wherein the diacid is a C12 diacid.
[0708] A64. The genetically modified yeast of embodiment A62,
wherein the diacid is a C18 diacid.
[0709] A65. The genetically modified yeast of any one of
embodiments A60 to A64, wherein the diacid contains no
unsaturation.
[0710] A66. The genetically modified yeast of any one of
embodiments A60 to A64, wherein the diacid contains one or more
unsaturations.
[0711] A67. The genetically modified yeast of any one of
embodiments A60 to A66, wherein the diacid is the predominant
diacid in a mixture of diacids.
[0712] A68. The genetically modified yeast of any one of
embodiments A60 to A67, wherein the feedstock comprises a
substantially pure oil.
[0713] A69. The genetically modified yeast of any one of
embodiments A60 to A68, wherein the feedstock comprises a plurality
of fatty acids.
[0714] A70. The genetically modified yeast of embodiment A69,
wherein the feedstock comprises a soapstock.
[0715] A71. The genetically modified yeast of embodiment A69,
wherein the feedstock comprises a fatty acid distillate.
[0716] A72. The genetically modified yeast of any one of
embodiments A60 to A71, wherein the vegetable oil is from a plant
chosen from palm, palm kernel, coconut, soy, safflower, canola or
combination thereof.
[0717] A73. The genetically modified yeast of any one of
embodiments A1 to A73, wherein a genetic modification that
increases an activity comprises incorporating in the yeast multiple
copies of a polynucleotide that encodes a polypeptide having the
activity.
[0718] A74. The genetically modified yeast of any one of
embodiments A1 to A73, wherein a genetic modification that
increases an activity comprises incorporating in the yeast a
promoter in operable linkage with a polynucleotide that encodes a
polypeptide having the activity.
[0719] A75. The genetically modified yeast of embodiment A74,
wherein the promoter is native to the yeast.
[0720] A76. The genetically modified yeast of embodiment A74 or
A75, wherein the promoter is chosen from a POX4 promoter; PEX11
promoter, TEF1 promoter, PGK promoter and FAO1 promoter.
[0721] A77. The genetically modified yeast of embodiment A76,
wherein the promoter comprises a polynucleotide chosen from SEQ ID
NOs: 162, 165, 166, 167 and 169.
[0722] B1. A method for producing a diacid, comprising: [0723]
contacting a genetically modified yeast of any one of embodiments
A1 to A77 with a feedstock capable of being converted by the yeast
to a diacid; and [0724] culturing the yeast under conditions in
which the diacid is produced from the feedstock.
[0725] B2. The method of embodiment B1, wherein the feedstock
comprises one or more components from a vegetable oil.
[0726] B3. The method of embodiment B1 or B2, wherein the diacid is
a C4 to C24 diacid.
[0727] B4. The method of embodiment B3, wherein the diacid is a
C10, C12, C14, C16, C18 or C20 diacid.
[0728] B5. The method of embodiment B4, wherein the diacid is a C10
diacid.
[0729] B6. The method of embodiment B4, wherein the diacid is a C12
diacid.
[0730] B7. The method of embodiment B4, wherein the diacid is a C18
diacid.
[0731] B8. The method of any one of embodiments B1 to B7, wherein
the diacid contains no unsaturation.
[0732] B9. The method of any one of embodiments B1 to B7, wherein
the diacid contains one or more unsaturations.
[0733] B10. The method of any one of embodiments B1 to B9, wherein
the diacid is the predominant diacid in a mixture of diacids.
[0734] B11. The method of any one of embodiments B1 to B10, wherein
the feedstock comprises a substantially pure oil.
[0735] B12. The method of any one of embodiments B1 to B10, wherein
the feedstock comprises a plurality of fatty acids.
[0736] B13. The method of embodiment B12, wherein the feedstock
comprises a soapstock.
[0737] B14. The method of embodiment B12, wherein the feedstock
comprises a fatty acid distillate.
[0738] B15. The method of any one of embodiments B1 to B14, wherein
the vegetable oil is from a plant chosen from palm, palm kernel,
coconut, soy, safflower, canola or combination thereof.
[0739] B16. The method of any one of embodiments B1 to B8 wherein
the feedstock comprises a fatty acid methyl ester.
[0740] B17. The method of embodiment B16 wherein the fatty acid
methyl ester is methyl laurate and the diacid comprises
dodecanedioic acid.
[0741] B18. The method of any one of embodiments B1 to B8 wherein
the feedstock comprises a fatty acid ethyl ester and the diacid
comprises dodecanedioic acid.
[0742] B19. The method of any of embodiments B1 to B8 wherein the
feedstock comprises lauric acid and the diacid comprises
dodecanedioic acid.
[0743] B20. The method of any of embodiments B1 to B8 wherein the
feedstock comprises ethyl caprate and the diacid comprises sebacic
acid.
[0744] C1. A method for producing a diacid by a yeast from a
feedstock toxic to the yeast, comprising: [0745] (a) contacting a
genetically modified yeast in culture with a feedstock not
substantially toxic to the yeast, thereby performing an induction;
and [0746] (b) contacting the yeast after the induction in (a) with
a feedstock toxic to the yeast, [0747] whereby a diacid is produced
by the yeast from the feedstock toxic to the yeast in an amount
greater than the amount of the diacid produced from the feedstock
toxic to the yeast when the induction is not performed.
[0748] C2. The method of embodiment C1, wherein the feedstock not
substantially toxic to the yeast has the same number of carbons as
the feedstock toxic to the yeast.
[0749] C3. The method of embodiment C1, wherein the feedstock not
substantially toxic to the yeast has a different number of carbons
compared to the feedstock toxic to the yeast.
[0750] C4. The method of any one of embodiments C1 to C3, wherein
the feedstock not substantially toxic to the yeast comprises a
fatty acid methyl ester.
[0751] C5. The method of any one of embodiments C1 to C3, wherein
the feedstock not substantially toxic to the yeast comprises a free
fatty acid.
[0752] C6. The method of any one of embodiments C1 to C5, wherein
the feedstock not substantially toxic to the yeast comprises more
than twelve carbons.
[0753] D1. An isolated nucleic acid comprising a polynucleotide
that encodes a polypeptide of SEQ ID NO: 148 or 150.
[0754] D2. The isolated nucleic acid of embodiment D1, wherein the
polynucleotide comprises the nucleotide sequence of SEQ ID NO: 147
or 149.
[0755] D3. An isolated nucleic acid, comprising a polynucleotide
that comprises: the nucleotide sequence of SEQ ID NO: 37 or a
nucleotide sequence having greater than 75% identity to SEQ ID NO:
37.
[0756] D4. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 44; a polypeptide
comprising an amino acid sequence having greater than 71% identity
to SEQ ID NO: 44; or a polypeptide of SEQ ID NO: 44 having 1 to 5
amino acid substitutions.
[0757] D5. The isolated nucleic acid of embodiment D4, wherein the
polynucleotide comprises the nucleotide sequence of SEQ ID NO: 43
or a nucleotide sequence having greater than 69% identity to SEQ ID
NO: 43.
[0758] D6. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 46; a polypeptide
comprising an amino acid sequence having greater than 71% identity
to SEQ ID NO: 46; or a polypeptide of SEQ ID NO: 46 having 1 to 5
amino acid substitutions.
[0759] D7. The isolated nucleic acid of embodiment D6, wherein the
polynucleotide comprises the nucleotide sequence of SEQ ID NO: 45
or a nucleotide sequence having greater than 70% identity to SEQ ID
NO: 45.
[0760] D8. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 48; a polypeptide
comprising an amino acid sequence having greater than 87% identity
to SEQ ID NO: 48; or a polypeptide of SEQ ID NO: 48 having 1 to 5
amino acid substitutions.
[0761] D9: The isolated nucleic acid of embodiment D8, wherein the
polynucleotide comprises the nucleotide sequence of SEQ ID NO: 47
or a nucleotide sequence having greater than 78% identity to SEQ ID
NO: 47.
[0762] D10. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 50; a polypeptide
comprising an amino acid sequence having greater than 80% identity
to SEQ ID NO: 50; or a polypeptide of SEQ ID NO: 50 having 1 to 5
amino acid substitutions.
[0763] D11. The isolated nucleic acid of embodiment D10, wherein
the polynucleotide comprises the nucleotide sequence of SEQ ID NO:
49 or a nucleotide sequence having greater than 75% identity to SEQ
ID NO: 49.
[0764] D12. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 67; a polypeptide
comprising an amino acid sequence having greater than 99% identity
to SEQ ID NO: 67; or a polypeptide of SEQ ID NO: 67 having 1 to 5
amino acid substitutions.
[0765] D13. The isolated nucleic acid of embodiment D12, wherein
the polynucleotide comprises the nucleotide sequence of SEQ ID NO:
68 or a nucleotide sequence having greater than 97% identity to SEQ
ID NO: 68.
[0766] D14. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 74; a polypeptide
comprising an amino acid sequence having greater than 99% identity
to SEQ ID NO: 74; or a polypeptide of SEQ ID NO: 74 having 1 to 5
amino acid substitutions.
[0767] D15. The isolated nucleic acid of embodiment D14, wherein
the polynucleotide comprises the nucleotide sequence of SEQ ID NO:
73 or a nucleotide sequence having greater than 97% identity to SEQ
ID NO: 73.
[0768] D16. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 76; a polypeptide
comprising an amino acid sequence having greater than 99% identity
to SEQ ID NO: 76; or a polypeptide of SEQ ID NO: 76 having 1 to 5
amino acid substitutions.
[0769] D17. The isolated nucleic acid of embodiment D16, wherein
the polynucleotide comprises the nucleotide sequence of SEQ ID NO:
75 or a nucleotide sequence having greater than 99% identity to SEQ
ID NO: 75.
[0770] D18. An isolated-nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 3; a polypeptide
comprising an amino acid sequence having greater than 99% identity
to SEQ ID NO: 3; or a polypeptide of SEQ ID NO: 3 having 1 to 5
amino acid substitutions.
[0771] D19. The isolated nucleic acid of embodiment D18, wherein
the polynucleotide comprises the nucleotide sequence of SEQ ID NO:
2 or a nucleotide sequence having greater than 99% identity to SEQ
ID NO: 2.
[0772] D20. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 5; a polypeptide
comprising an amino acid sequence having greater than 99% identity
to SEQ ID NO: 5; or a polypeptide of SEQ ID NO: 5 having 1 to 5
amino acid substitutions.
[0773] D21. The isolated nucleic acid of embodiment D20, wherein
the polynucleotide comprises the nucleotide sequence of SEQ ID NO:
4 or a nucleotide sequence having greater than 98% identity to SEQ
ID NO: 4.
[0774] D22. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 7; a polypeptide
comprising an amino acid sequence having greater than 99% identity
to SEQ ID NO: 7; or a polypeptide of SEQ ID NO: 7 having 1 to 5
amino acid substitutions.
[0775] D23. The isolated nucleic acid of embodiment D22, wherein
the polynucleotide comprises the nucleotide sequence of SEQ ID NO:
6 or a nucleotide sequence having greater than 99% identity to SEQ
ID NO: 6.
[0776] D24. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 34; a polypeptide
comprising an amino acid sequence having greater than 95% identity,
to SEQ ID NO: 34; or a polypeptide of SEQ ID NO: 34 having 1 to 5
amino acid substitutions.
[0777] D25. The isolated nucleic acid of embodiment D24, wherein
the polynucleotide comprises the nucleotide sequence of SEQ. ID NO:
33 or a nucleotide sequence having greater than 73% identity to SEQ
ID NO: 33.
[0778] D26. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 36; a polypeptide
comprising an amino acid sequence having greater than 94% identity
to SEQ ID NO: 36; or a polypeptide of SEQ ID NO: 36 having 1 to 5
amino acid substitutions.
[0779] D27. The isolated nucleic acid of embodiment D3, wherein the
polynucleotide comprises the nucleotide sequence of SEQ ID NO: 35
or a nucleotide sequence having greater than 73% identity to SEQ ID
NO: 35.
[0780] D28. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 129; a polypeptide
comprising an amino acid sequence having greater than 89% identity
to SEQ ID NO: 129; or a polypeptide of SEQ ID NO: 129 having 1 to 5
amino acid substitutions.
[0781] D29. The isolated nucleic acid of embodiment D28, wherein
the polynucleotide comprises the nucleotide sequence of SEQ ID NO:
130 or a nucleotide sequence having greater than 84% identity to
SEQ ID NO: 130.
[0782] D30. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 113; a polypeptide
comprising an amino acid sequence having greater than 85% identity
to SEQ ID NO: 113; or a polypeptide of SEQ ID NO: 113 having 1 to 5
amino acid substitutions.
[0783] D31. The isolated nucleic acid of embodiment D30, wherein
the polynucleotide comprises the nucleotide sequence of SEQ ID NO:
114 or a nucleotide sequence having greater than 84% identity to
SEQ ID NO: 114.
[0784] D32. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 115; a polypeptide
comprising an amino acid sequence having greater than 97% identity
to SEQ ID NO: 115; or a polypeptide of SEQ ID NO: 115 having 1 to 5
amino acid substitutions.
[0785] D33. The isolated nucleic acid of embodiment D32, wherein
the polynucleotide comprises the nucleotide sequence of SEQ ID NO:
116 or a nucleotide sequence having greater than 86% identity to
SEQ ID NO: 116
[0786] D34. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 117; a polypeptide
comprising an amino acid sequence having greater than 80% identity
to SEQ ID NQ: 117; or a polypeptide of SEQ ID NO: 117 having 1 to 5
amino acid substitutions.
[0787] D35. The isolated nucleic acid of embodiment D34, wherein
the polynucleotide comprises the nucleotide sequence of SEQ ID NO:
118 or a nucleotide sequence having greater than 80% identity to
SEQ ID NO: 118.
[0788] D36. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 119; a polypeptide
comprising an amino acid sequence having greater than 84% identity
to SEQ ID NO: 119; or a polypeptide of SEQ ID NO: 119 having 1 to 5
amino acid substitutions. D37. The isolated nucleic acid of
embodiment D36, wherein the polynucleotide comprises the nucleotide
sequence of SEQ ID NO: 120 or a nucleotide sequence having greater
than 76% identity to SEQ ID NO: 120.
[0789] D38. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 121; a polypeptide
comprising an amino acid sequence having greater than 81% identity
to SEQ ID NO: 121; or a polypeptide of SEQ ID NO: 121 having 1 to 5
amino acid substitutions.
[0790] D39. The isolated nucleic acid of embodiment D38, wherein
the polynucleotide comprises the nucleotide sequence of SEQ ID NO:
122 or a nucleotide sequence having greater than 74%, identity to
SEQ ID NO: 122.
[0791] D40. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 123; a polypeptide
comprising an amino acid sequence having greater than 90% identity
to SEQ ID NO: 123; or a polypeptide of SEQ ID NO: 123 having 1 to 5
amino acid substitutions.
[0792] D41. The isolated nucleic acid of embodiment D40, wherein
the polynucleotide comprises the nucleotide sequence of SEQ ID NO:
124 or a nucleotide sequence having greater than 82% identity to
SEQ ID NO: 124.
[0793] D42. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 125; a polypeptide
comprising an amino acid sequence having greater than 80% identity
to SEQ ID NO: 125; or a polypeptide of SEQ ID NO: 125 having 1 to 5
amino acid substitutions.
[0794] D43. The isolated nucleic acid of embodiment D42, wherein
the polynucleotide comprises the nucleotide sequence of SEQ ID NO:
126 or a nucleotide sequence having greater than 77% identity to
SEQ ID NO: 126.
[0795] D44. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 127; a polypeptide
comprising an amino acid sequence having greater than 81% identity
to SEQ ID NO: 127; or a polypeptide of SEQ ID NO: 127 having 1 to 5
amino acid substitutions.
[0796] D45. The isolated nucleic acid of embodiment D44, wherein
the polynucleotide comprises the nucleotide sequence of SEQ ID NO:
128 or a nucleotide sequence having greater than 78% identity to
SEQ ID NO: 128.
[0797] D46. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 96; a polypeptide
comprising an amino acid sequence having greater than 85% identity
to SEQ ID NO: 96; or a polypeptide of SEQ ID NO: 96 having 1 to 5
amino acid substitutions.
[0798] D47. The isolated nucleic acid of embodiment D46, wherein
the polynucleotide comprises the nucleotide sequence of SEQ ID NO:
95 or a nucleotide sequence having greater than 72% identity to SEQ
ID NO:95
[0799] D48. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 139; a polypeptide
comprising an amino acid sequence having greater than 76% identity
to SEQ ID NO: 139; or a polypeptide of SEQ ID NO: 139 having 1 to 5
amino acid substitutions.
[0800] D49. The isolated nucleic acid of embodiment D48, wherein
the polynucleotide comprises the nucleotide sequence of SEQ ID NO:
140 or a nucleotide sequence having greater than 77% identity to
SEQ ID NO: 140.
[0801] D50. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 141; a polypeptide
comprising an amino acid sequence having greater than 83% identity
to SEQ ID NO: 141; or a polypeptide of SEQ ID NO: 141 having 1 to 5
amino acid substitutions.
[0802] D51. The isolated nucleic acid of embodiment D50, wherein
the polynucleotide comprises the nucleotide sequence of SEQ ID NO:
142 or a nucleotide sequence having greater than 73% identity to
SEQ ID NO: 142.
[0803] D52. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 90; a polypeptide
comprising an amino acid sequence having greater than 95% identity
to SEQ ID NO: 90; or a polypeptide of SEQ ID NO:90 having 1 to 5
amino acid substitutions.
[0804] D53. The isolated nucleic acid of embodiment D52, wherein
the polynucleotide comprises the nucleotide sequence of SEQ ID NO:
89 or a nucleotide sequence having greater than 81% identity to SEQ
ID NO: 89.
[0805] D54. An isolated nucleic acid comprising a polynucleotide
that encodes: the polypeptide of SEQ ID NO: 40; a polypeptide
comprising an amino acid sequence having greater than 92% identity
to SEQ ID NO: 40; or a polypeptide of SEQ ID NO: 40 having 1 to 5
amino acid substitutions.
[0806] D55. The isolated nucleic acid of embodiment D54, wherein
the polynucleotide comprises the nucleotide sequence of SEQ ID NO:
39 or a nucleotide sequence having greater than 84% identity to SEQ
ID NO: 39.
[0807] D56. An isolated nucleic acid, comprising a polynucleotide
that comprises: the nucleotide sequence of SEQ ID NO: 166 or a
nucleotide sequence having greater than 84% identity to SEQ ID NO:
166.
[0808] D57. An isolated nucleic acid, comprising a polynucleotide
that comprises: the nucleotide sequence of SEQ ID NO: 167 or a
nucleotide sequence having greater than 85% identity to SEQ ID NO:
167.
[0809] D58. An isolated nucleic acid, comprising a polynucleotide
that comprises: the nucleotide sequence of SEQ ID NO: 164 or a
nucleotide sequence having greater than 92% identity to SEQ ID NO:
169.
[0810] D59. The isolated nucleic acid of any one of embodiments D3
to D58, wherein at least one of the 1 to 5 amino acid substitutions
is conservative.
[0811] D60. The isolated nucleic acid of any one of embodiments D3
to D58, wherein at least one of the 1 to 5 amino acid substitutions
is non-conservative.
[0812] E1. The isolated nucleic acid of any one of embodiments D1
to D60, which is an expression vector.
[0813] E2. A cell comprising a nucleic acid of any one of
embodiments D1 to E1.
[0814] E3. The cell of embodiment E2, which is a bacterium.
[0815] E4. The cell of embodiment E1, which is a yeast.
[0816] E5. The cell of embodiment E4, which is a Candida spp.
yeast.
[0817] E6. The cell of embodiment E5, wherein the Candida spp.
yeast is chosen from C. tropicalis and C. viswanathii.
[0818] E7. The cell of embodiment E6, wherein the Candida spp yeast
is a genetically modified ATCC20336 yeast.
[0819] E8. The cell of embodiment E4, which is chosen from a
Yarrowia spp. yeast, Pichia spp. yeast, Saccharomyces spp. yeast
and Kluyveromyces spp. yeast.
[0820] E9. The cell of embodiment E8, which is chosen from Y.
lipolytica, P. pastoris, P. membranifaciens, P. kluyveri, P.
guilliermondii, P. heedii, P. subpelliculosa, S. cerevisiae, S.
bayanus, S. pastorianus, S. carlsbergensis, K. lactis and K.
marxianus.
[0821] The entirety of each patent, patent application, publication
and document referenced herein hereby is incorporated by reference.
Citation of the above patents, patent applications, publications
and documents is not an admission that any of the foregoing is
pertinent prior art, nor does it constitute any admission as to the
contents or date of these publications or documents.
[0822] Modifications may be made to the foregoing without departing
from the basic aspects of the technology. Although the technology
has been described in substantial detail with reference to one or
more specific embodiments, those of ordinary skill in the art will
recognize that changes may be made to the embodiments specifically
disclosed in this application, yet these modifications and
improvements are within the scope and spirit of the technology.
[0823] The technology illustratively described herein suitably may
be practiced in the absence of any element(s) not specifically
disclosed herein. Thus, for example, in each instance herein any of
the terms "comprising," "consisting essentially of," and
"consisting of" may be replaced with either of the other two terms.
The terms and expressions which have been employed are used as
terms of description and not of limitation, and use of such terms
and expressions do not exclude any equivalents of the features
shown and described or portions thereof, and various modifications
are possible within the scope of the technology claimed. The term
"a" or "an" can refer to one of or a plurality of the elements it
modifies (e.g., "a reagent" can mean one or more reagents) unless
it is contextually clear either one of the elements or more than
one of the elements is described. The term "about" as used herein
refers to a value within 10% of the underlying parameter (i.e.,
plus or minus 10%), and use of the term "about" at the beginning of
a string of values modifies each of the values (i.e., "about 1, 2
and 3" refers to about 1, about 2 and about 3). For example, a
weight of "about 100 grams" can include weights between 90 grams
and 110 grams. Further, when a listing of values is described
herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%) the listing
includes all intermediate and fractional values thereof (e.g., 54%,
85.4%). Thus, it should be understood that although the present
technology has been specifically disclosed by representative
embodiments and optional features, modification and variation of
the concepts herein disclosed may be resorted to by those skilled
in the art, and such modifications and variations are considered
within the scope of this technology.
[0824] Certain embodiments of the technology are set forth in the
claim(s) that follow(s).
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20190010524A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20190010524A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References