U.S. patent application number 11/773453 was filed with the patent office on 2008-09-11 for glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate mutase promoters for gene expression in oleaginous yeast.
Invention is credited to Stephen K. Picataggio, Quinn Qun Zhu.
Application Number | 20080220474 11/773453 |
Document ID | / |
Family ID | 33563842 |
Filed Date | 2008-09-11 |
United States Patent
Application |
20080220474 |
Kind Code |
A1 |
Picataggio; Stephen K. ; et
al. |
September 11, 2008 |
GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE AND PHOSPHOGLYCERATE
MUTASE PROMOTERS FOR GENE EXPRESSION IN OLEAGINOUS YEAST
Abstract
The promoter regions associated with the Yarrowia lipolytica
glyceraldehyde-3-phosphate dehydrogenase (gpd) and phosphoglycerate
mutase (gpm) genes have been found to be particularly effective for
the expression of heterologous genes in oleaginous yeast. The
promoter regions of the invention have been shown to drive
high-level expression of genes involved in the production of
.omega.-3 and .omega.-6 fatty acids.
Inventors: |
Picataggio; Stephen K.;
(Gaithersburg, MD) ; Zhu; Quinn Qun; (West
Chester, PA) |
Correspondence
Address: |
E I DU PONT DE NEMOURS AND COMPANY;LEGAL PATENT RECORDS CENTER
BARLEY MILL PLAZA 25/1122B, 4417 LANCASTER PIKE
WILMINGTON
DE
19805
US
|
Family ID: |
33563842 |
Appl. No.: |
11/773453 |
Filed: |
July 31, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10869630 |
Jun 16, 2004 |
7259255 |
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11773453 |
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60482263 |
Jun 25, 2003 |
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Current U.S.
Class: |
435/69.1 |
Current CPC
Class: |
C12N 9/90 20130101; C12N
9/0008 20130101; C12N 15/815 20130101; C12P 7/6427 20130101; C12P
7/6472 20130101 |
Class at
Publication: |
435/69.1 |
International
Class: |
C12P 21/06 20060101
C12P021/06 |
Claims
1. A method for the expression of a coding region of interest in a
transformed yeast cell comprising: a) providing a transformed yeast
cell having a chimeric gene comprising: (i) a promoter region of a
gpd Yarrowia gene and, (ii) a coding region of interest expressible
in the yeast cell; wherein the promoter region is operably linked
to the coding region of interest; and b) growing the transformed
yeast cell of step (a) under conditions whereby the chimeric gene
of step (a) is expressed.
2. A method according to claim 1 wherein the Yarrowia gene is
isolated from Yarrowia lipolytica.
3-4. (canceled)
5. A method according to claim 1 wherein the promoter region
contains at least one mutation that does not diminish its promoter
activity.
6. A method according to claim 1 wherein the promoter activity is
at least about 20% to at least about 400% of the promoter activity
of the wildtype promoter activity.
7. A method according to claim 1 wherein the transformed yeast cell
is an oleaginous yeast.
8. A method of claim 7, wherein the oleaginous yeast is a member of
a genus selected from the group consisting of Yarrowia, Candida,
Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and
Lipomyces.
9. (canceled)
10. A method according to claim 1 wherein the coding region of
interest encodes a polypeptide selected from the group consisting
of: desaturases, elongases, aminopeptidases, amylases,
carbohydrases, carboxypeptidases, catalyases, cellulases,
chitinases, cutinases, cyclodextrin glycosyltransferases,
deoxyribonucleases, esterases, .alpha.-galactosidases,
.beta.-galactosidases, glucoamylases, .alpha.-glucosidases,
.beta.-glucanases, .beta.-glucosidases, invertases, laccases,
lipases, mannosidases, mutanases, oxidases, pectinolytic enzymes,
peroxidases, phospholipases, phytases, polyphenoloxidases,
proteolytic enzymes, ribonucleases, transglutaminases and
xylanases.
11. A method for the production of an .omega.-3 or an .omega.-6
fatty acid comprising: (a) providing a transformed oleaginous yeast
comprising a chimeric gene, comprising: (i) a promoter region of a
. . . Yarrowia gene (ii) a coding region encoding at least one
enzyme of the .omega.-3/.omega.-6 fatty acid biosynthetic pathway;
wherein the promoter region and coding region are operably linked;
and (b) contacting the transformed oleaginous yeast of step (a)
under conditions whereby the at least one enzyme of the
.omega.-3.omega.-6 fatty acid biosynthetic pathway is expressed and
a .omega.-3 or .omega.-6 fatty acid is produced; and (c) optionally
recovering the .omega.-3 or .omega.-6 fatty acid.
12. A method according to claim 11 wherein the Yarrowia gene is
isolated from Yarrowia lipolytica.
13-14. (canceled)
15. A method according to claim 11 wherein the coding region of
interest encodes a polypeptide selected from the group consisting
of: desaturases and elongases.
16. A method according to claim 15 wherein the desaturase is
selected from the group consisting of: .DELTA.9 desaturase,
.DELTA.12 desaturase, .DELTA.6 desaturase, .DELTA.5 desaturase,
.DELTA.17 desaturase, .DELTA.15 desaturase and .DELTA.4
desaturase.
17. A method according to claim 11 wherein the oleaginous yeast is
a member of a genus selected from the group of consisting of:
Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus,
Trichosporon and Lipomyces.
18. A method according to claim 17 wherein the oleaginous yeast is
Yarrowia lipolytica.
19. (canceled)
20. A method according to claim 11 wherein the .omega.-3 or
.omega.-6 fatty acid is selected from the group consisting of:
linoleic acid, .alpha.-linolenic acid, .gamma.-linolenic acid,
stearidonic acid, dihomo-.gamma.-linoleic acid, eicosatetraenoic
acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic
acid and docosahexaenoic acid.
21-22. (canceled)
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/482,263, filed Jun. 25, 2003.
FIELD OF THE INVENTION
[0002] This invention is in the field of biotechnology. More
specifically, this invention pertains to promoter regions isolated
from Yarrowia lipolytica that are useful for gene expression in
oleaginous yeast.
BACKGROUND OF THE INVENTION
[0003] Oleaginous yeast are defined as those organisms that are
naturally capable of oil synthesis and accumulation, wherein oil
accumulation ranges from at least about 25% up to about 80% of the
cellular dry weight. Genera typically identified as oleaginous
yeast include, but are not limited to: Yarrowia, Candida,
Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and
Lipomyces. More specifically, illustrative oil-synthesizing yeast
include: Rhodosporidium toruloides, Lipomyces starkeyii, L.
lipoferus, Candida revkaufi, C. pulcherrima, C. tropicalis, C.
utilis, Trichosporon pullans, T. cutaneum, Rhodotorula glutinus, R.
graminis and Yarrowia lipolytica (formerly classified as Candida
lipolytica).
[0004] The technology for growing oleaginous yeast with high oil
content is well developed (for example, see EP 0 005 277B1;
Ratledge, C., Prog. Ind. Microbiol. 16:119-206 (1982)). And, these
organisms have been commercially used for a variety of purposes in
the past. For example, various strains of Yarrowa lipolytica have
historically been used for the manufacture and production of:
isocitrate lyase (DD259637); lipases (SU1454852, WO2001083773,
DD279267); polyhydroxyalkanoates (WO2001088144); citric acid
(RU2096461, RU2090611, DD285372, DD285370, DD275480, DD227448,
PL160027); erythritol (EP770683); 2-oxoglutaric acid (DD267999);
.gamma.-decalactone (U.S. Pat. No. 6,451,565, FR2734843);
.gamma.-dodecalactone (EP578388); and pyruvic acid (JP09252790).
Most recently, however, the natural abilities of oleaginous yeast
have been enhanced by advances in genetic engineering, resulting in
organisms capable of producing polyunsaturated fatty acids
("PUFAs"). Specifically, Picataggio et al. have demonstrated that
Yarrowia lipolytica can be engineered for production of .omega.-3
and .omega.-6 fatty acids, by introducing and expressing genes
encoding the .omega.-3/.omega.-6 biosynthetic pathway (co-pending
U.S. patent application Ser. No. 10/840,579).
[0005] Recombinant production of any heterologous protein is
generally accomplished by constructing an expression cassette in
which the DNA coding for the protein of interest is placed under
the control of a promoter suitable for the host cell. The
expression cassette is then introduced into the host cell (usually
by plasmid-mediated transformation or targeted integration into the
host genome) and production of the heterologous protein is achieved
by culturing the transformed host cell under conditions necessary
for the proper function of the promoter contained within the
expression cassette. Thus, the development of new host cells (e.g.,
oleaginous yeast) for recombinant production of proteins generally
requires the availability of promoters that are suitable for
controlling the expression of a protein of interest in the host
cell.
[0006] A variety of strong promoters have been isolated from
Saccharomyces cerevisiae that are useful for heterologous gene
expression in yeast. For example, a glyceraldehyde-3-phosphate
dehydrogenase (GPD) promoter was described by Bitter, G. A., and K.
M. Egan (Gene 32(3):263-274 (1984)); and, a phosphoglycerate mutase
(GPM1) promoter was investigated by Rodicio, R. et al. (Gene
125(2): 125-133 (1993)). Several promoters have also been isolated
from Yarrowia lipolytica that have been suitable for the
recombinant expression of proteins. For example, U.S. Pat. No.
4,937,189 and EP220864 (Davidow et al.) disclose the sequence of
the XPR2 gene (which encodes an inducible alkaline extracellular
protease) and upstream promoter region for use in expression of
heterologous proteins. However, this promoter is only active at a
pH above 6.0 on media lacking preferred carbon and nitrogen
sources; and full induction requires high levels of peptone in the
culture media. Subsequent analysis of the XPR2 promoter sequence by
Blanchin-Roland, S. et al. (EP832258; Mol. Cell. Biol.
14(1):327-338 (1994)) determined that hybrid promoters containing
only parts of the XPR2 promoter sequence may be used to obtain high
level expression in Yarrowia, without the limitations resulting
from use of the complete promoter sequence.
[0007] U.S. Pat. No. 6,265,185 (Muller et al.) describe yeast
promoters from Yarrowia lipolytica for the translation elongation
factor EF1-.alpha.(TEF) protein and ribosomal protein S7 that are
suitable for expression cloning in yeast and heterologous
expression of proteins. These promoters were improved relative to
the XPR2 promoter, when tested for yeast promoter activity on
growth plates (Example 9, U.S. Pat. No. 6,265,185) and based on
their activity in the pH range of 4-11.
[0008] Despite the utility of these known promoters, however, there
is a need for new improved yeast promoters for metabolic
engineering of yeast (oleaginous and non-oleaginous) and for
controlling the expression of heterologous genes in yeast.
Furthermore, possession of a suite of promoters that are
regulatable under a variety of natural growth and induction
conditions in yeast will play an important role in industrial
settings, wherein it is desirable to express heterologous
polypeptides in commercial quantities in said hosts for economical
production of those polypeptides. Thus, it is an object of the
present invention to provide such promoters that will be useful for
gene expression in a variety of yeast cultures, and preferably in
Yarrowia sp. cultures and other oleaginous yeast.
[0009] Applicants have solved the stated problem by identifying
genes encoding a glyceraldehyde-3-phosphate dehydrogenase (GPD) and
a phosphoglycerate mutase (GPM) from Yarrowia lipolytica and the
promoters responsible for driving expression of these native genes.
Both promoters are useful for expression of heterologous genes in
Yarrowia and have improved activity with respect to the TEF
promoter.
SUMMARY OF THE INVENTION
[0010] The present invention provides methods for the expression of
a coding region of interest in a transformed yeast cell, using a
promoter of the glyceraldehyde-3-phosphate dehydrogenase (gpd) or
phosphoglycerate mutase (gpm) genes. Accordingly, the present
invention provides a method for the expression of a coding region
of interest in a transformed yeast cell comprising: [0011] a)
providing a transformed yeast cell having a chimeric gene
comprising: [0012] (i) a promoter region of a Yarrowia gene
selected from the group consisting of: a gpm gene and a gpd gene;
and [0013] (ii) a coding region of interest expressible in the
yeast cell; [0014] wherein the promoter region is operably linked
to the coding region of interest; and [0015] b) growing the
transformed yeast cell of step (a) under conditions [0016] whereby
the chimeric gene of step (a) is expressed.
[0017] In a preferred embodiment the invention provides a method
for the production of an .omega.-3 or an .omega.-6 fatty acid
comprising: [0018] a) providing a transformed oleaginous yeast
comprising a chimeric gene, comprising: [0019] (i) a promoter
region of a Yarrowia gene selected from the group consisting of: a
gpm gene and a gpd gene; and [0020] (ii) a coding region encoding
at least one enzyme of the .omega.-3/.omega.-6 fatty acid
biosynthetic pathway; [0021] wherein the promoter region and coding
region are operably linked; and [0022] (b) contacting the
transformed oleaginous yeast of step (a) under conditions whereby
the at least one enzyme of the .omega.-3/.omega.-6 fatty
biosynthetic pathway is expressed and a .omega.-3 or .omega.-6
fatty acid is produced; and [0023] (c) optionally recovering the
.omega.-3 or .omega.-6 fatty acid.
[0024] Additionally the invention provides an isolated nucleic acid
molecule comprising a gpd promoter selected from the group
consisting of SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:43.
[0025] In similar fashion the invention provides an isolated
nucleic acid molecule comprising a gpm promoter selected from the
group consisting of SEQ ID NO:27, SEQ ID NO:28 and SEQ ID
NO:44.
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE
Descriptions
[0026] FIGS. 1A and 1B shows an alignment of known
glyceraldehyde-3-phosphate dehydrogenase (GPD) proteins from
Saccharomyces cerevisiae (GenBank Accession No. CAA24607),
Schizosaccharomyces pombe (GenBank Accession No. NP.sub.--595236),
Aspergillus oryzae (Gen Bank Accession No. AAK08065), Paralichthys
olivaceus (Gen Bank Accession No. BAA88638), Xenopus laevis
(GenBank Accession No. P51469) and Gallus gallus (GenBank Accession
No. DECHG3), used to identify two conserved regions within the
sequence alignment.
[0027] FIG. 2 shows an alignment of amino acids encoding portions
of the GPD protein from Yarrowia lipolytica, Schizosaccharomyces
pombe, Gallus gallus and Xenopus laevis.
[0028] FIG. 3 shows an alignment of phosphoglycerate mutase (GPM)
proteins from Yarrowia lipolytica and Saccharomyces cerevisiae.
[0029] FIG. 4 graphically represents the relationship between SEQ
ID NOs:11,12,23-26 and 43, each of which relates to
glyceraldehyde-3-phosphate dehydrogenase (GPD) in Yarrowia
lipolytica.
[0030] FIG. 5 graphically represents the relationship between SEQ
ID NOs:14-16, 27, 28 and 44, each of which relates to
phosphoglycerate mutase (GPM) in Yarrowia lipolytica.
[0031] FIG. 6 illustrates the construction of plasmid vector
pY5-4.
[0032] FIGS. 7A, 7B, 7C and 7D provide plasmid maps for pY5-10,
pY5-30, PYZGDG and PYZGMG, respectively.
[0033] FIG. 8A is an image of a cell culture comparing the promoter
activity of TEF and GPD in Yarrowia lipolytica as determined by
histochemical staining. FIG. 8B is an image of a cell culture
comparing the promoter activity of TEF and GPM in Y. lipolytica as
determined by histochemical staining.
[0034] FIG. 9A is a graph comparing the promoter activity of TEF
and GPD as determined fluorometically. FIG. 9B is a graph comparing
the promoter activity of TEF and GPM as determined
fluorometically.
[0035] FIG. 10 illustrates the .omega.-3/.omega.-6 fatty acid
biosynthetic pathway.
[0036] The invention can be more fully understood from the
following detailed description and the accompanying sequence
descriptions, which form a part of this application.
[0037] The following sequences comply with 37 C.F.R.
.sctn.1.821-1.825 ("Requirements for Patent Applications Containing
Nucleotide Sequences and/or Amino Acid Sequence Disclosures--the
Sequence Rules") and are consistent with World Intellectual
Property Organization (WIPO) Standard ST.25 (1998) and the sequence
listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis),
and Section 208 and Annex C of the Administrative Instructions).
The symbols and format used for nucleotide and amino acid sequence
data comply with the rules set forth in 37 C.F.R. .sctn.1.822.
[0038] SEQ ID NOs:1-6 correspond to the GPD amino acid sequences of
Saccharomyces cerevisiae (Gen Bank Accession No. CAA24607),
Schizosaccharomyces pombe (GenBank Accession No. NP-595236),
Aspergillus oryzae (GenBank Accession No. AAK08065), Paralichthys
olivaceus (GenBank Accession No. BAA88638), Xenopus laevis (GenBank
Accession No. P51469) and Gallus gallus (GenBank Accession No.
DECHG3), respectively.
[0039] SEQ ID NOs:7 and 8 correspond to conserved amino acid
regions of the GPD protein.
[0040] SEQ ID NOs:9 and 10 correspond to the degenerate primers
YL193 and YL194, respectively, used for isolating an internal
portion of the Yarrowia lipolytica GPD gene.
[0041] SEQ ID NO:11 encodes a 507 bp internal portion of the
Yarrowia lipolytica GPD gene, while SEQ ID NO:12 is the
corresponding amino acid sequence.
[0042] SEQ ID NO:13 corresponds to the Saccharomyces cerevisiae GPM
protein (GenBank Accession No. NP.sub.--012770).
[0043] SEQ ID NO:14 corresponds to Contig 2217, comprising the
complete nucleotide coding sequence for the Yarrowia lipolytica GPM
protein.
[0044] SEQ ID NO:15 corresponds to the deduced nucleotide sequence
of the Yarrowia lipolytica GPM coding region, while SEQ ID NO:16
corresponds to the amino acid sequence.
[0045] SEQ ID NOs:17-22 correspond to primers YL206, YL196, YL207,
YL197, YL208 and YL198, respectively, used for genome walking.
[0046] SEQ ID NO:23 corresponds to a 1848 bp fragment designated as
"GPDP", comprising 1525 bp upstream of the GPD gene and an
additional 323 bp representing a 5' portion of the GPD gene in
Yarrowia lipolytica.
[0047] SEQ ID NO:24 corresponds to an assembled 2316 bp contig of
DNA, corresponding to the -1525 to +791 region of the GPD gene,
wherein the `A` position of the `ATG` translation initiation codon
is designated as +1.
[0048] SEQ ID NO:25 corresponds to a partial cDNA sequence encoding
the Yarrowia lipolytica GPD gene, while SEQ ID NO:26 is the
corresponding amino acid sequence.
[0049] SEQ ID NO:27 corresponds to a 953 bp fragment designated as
"GPML", corresponding to the -875 to +78 region of the GPM gene,
wherein the `A` position of the `ATG` translation initiation codon
is designated as +1.
[0050] SEQ ID NO:28 corresponds to an assembled 1537 bp contig of
DNA, corresponding to the -875 to +662 region of the GPM gene,
wherein the `A` position of the `ATG` translation initiation codon
is designated as +1.
[0051] SEQ ID NOs:29 and 30 correspond to primers YL33 and YL34,
respectively, used for amplifying the reporter gene GUS.
[0052] SEQ ID NOs:31 and 32 correspond to primers TEF5' and TEF3',
respectively, used to isolate the TEF promoter.
[0053] SEQ ID NOs:33 and 34 correspond to primers XPR5' and XPR3',
respectively, used to isolate the XPR2 transcriptional
terminator.
[0054] SEQ ID NOs:35-42 correspond to primers YL1, YL2, YL3, YL4,
YL23, YL24, YL9 and YL10, respectively, used for site-directed
mutagenesis during construction of the pY5-10 plasmid.
[0055] SEQ ID NO:43 corresponds to a 971 bp fragment designated as
"GPDPro", and identified herein as the putative GPD promoter in
Yarrowia lipolytica. This fragment corresponds to the -968 to +3
region of the GPD gene, wherein the `A` position of the `ATG`
translation initiation codon is designated as +1.
[0056] SEQ ID NO:44 corresponds to a 878 bp fragment designated as
"GPMLPro", and identified herein as the putative GPM promoter in
Yarrowia lipolytica. This fragment corresponds to the -875 to +3
region of the GPM gene, wherein the `A` position of the `ATG`
translation initiation codon is designated as +1.
[0057] SEQ ID NOs:45 and 46 correspond to primers YL211 and YL212,
respectively, used to amplify the putative GPD promoter.
[0058] SEQ ID NOs:47 and 48 correspond to primers YL203 and YL204,
respectively, used to amplify the putative GPM promoter.
[0059] SEQ ID NOs:49-54 correspond to primers YL5, YL6, YL7, YL8,
YL61 and YL62, respectively, used for construction of plasmid
pY5-13.
[0060] SEQ ID NOs:55 and 56 correspond to primers GPDsense and
GPDantisense, respectively, used to amplify GPDPro.
[0061] SEQ ID NO:57 corresponds to the nucleotide sequence of the
Fusarium moniliforme strain M-8114 .DELTA.15 desaturase coding
region, while SEQ ID NO:58 corresponds to the amino acid
sequence.
[0062] SEQ ID NOs:59 and 60 correspond to primers P192 and P193,
respectively, used to amplify the F. moniliforme .DELTA.15
desaturase.
DETAILED DESCRIPTION OF THE INVENTION
[0063] In accordance with the subject invention, Applicants
describe the isolation and characterization of promoters and genes
from an oleaginous yeast, Yarrowia lipolytica. These promoter
regions, isolated upstream of the glyceraldehyde-3-phosphate
dehydrogenase (GPD) and phosphoglycerate mutase (GPM) genes, are
useful for genetic engineering in Y. lipolytica and other yeast for
the production of heterologous polypeptides.
[0064] Preferred heterologous polypeptides of the present invention
are those that are involved in the synthesis of microbial oils and
particularly polyunsaturated fatty acids (PUFAs). PUFAs, or
derivatives thereof, made by the methodology disclosed herein can
be used in many applications. For example, the PUFAs can be used as
dietary substitutes, or supplements, particularly infant formulas,
for patients undergoing intravenous feeding or for preventing or
treating malnutrition. Alternatively, the purified PUFAs (or
derivatives thereof) may be incorporated into cooking oils, fats or
margarines formulated so that in normal use the recipient would
receive the desired amount for dietary supplementation. The PUFAs
may also be incorporated into infant formulas, nutritional
supplements or other food products, and may find use as
anti-inflammatory or cholesterol lowering agents. Optionally, the
compositions may be used for pharmaceutical use (human or
veterinary). In this case, the PUFAs are generally administered
orally but can be administered by any route by which they may be
successfully absorbed, e.g., parenterally (e.g., subcutaneously,
intramuscularly or intravenously), rectally, vaginally or topically
(e.g., as a skin ointment or lotion).
[0065] Thus, the present invention advances the art by providing
methods for the expression of a coding region of interest in a
transformed yeast comprising: a) providing a transformed yeast cell
having a chimeric gene comprising (i) a promoter region of a gpd
gene or gpm gene; and (ii) a coding region of interest expressible
in the host cell, wherein the promoter region is operably linked to
the coding region of interest; and b) growing the transformed yeast
cell of step (a) in the presence of a fermentable carbon source,
wherein the chimeric gene is expressed and optionally isolated from
the cultivation medium. In preferred embodiments, the promoter
region comprises a sequence selected from the group consisting of
SEQ ID NOs: 23, 24, 27, 28, 43 and 44.
DEFINITIONS
[0066] In this disclosure, a number of terms and abbreviations are
used.
[0067] The following definitions are provided.
[0068] "Glyceraldehyde-3-phosphate dehydrogenase" is abbreviated
GPD.
[0069] "Phosphoglycerate mutase" is abbreviated GPM.
[0070] "Open reading frame" is abbreviated ORF.
[0071] "Polymerase chain reaction" is abbreviated PCR.
[0072] "Polyunsaturated fatty acid(s)" is abbreviated PUFA(s).
[0073] The term "oleaginous" refers to those organisms that tend to
store their energy source in the form of lipid (Weete, In: Fungal
Lipid Biochemistry, 2.sup.nd Ed., Plenum, 1980). Generally, the
cellular PUFA content of these microorganisms follows a sigmoid
curve, wherein the concentration of lipid increases until it
reaches a maximum at the late logarithmic or early stationary
growth phase and then gradually decreases during the late
stationary and death phases (Yongmanitchai and Ward, Appl. Environ.
Microbiol. 57:419-25 (1991)).
[0074] The term "oleaginous yeast" refers to those microorganisms
classified as yeast that can accumulate at least 25% of their dry
cell weight as oil. Examples of oleaginous yeast include (but are
no means limited to) the following genera: Yarrowia, Candida,
Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and
Lipomyces.
[0075] The term "fermentable carbon source" will refer to a carbon
source that a microorganism will metabolize to derive energy.
Typical carbon sources for use in the present invention include,
but are not limited to: monosaccharides, oligosaccharides,
polysaccharides, alkanes, fatty acids, esters of fatty acids,
monoglycerides, diglycerides, triglycerides, carbon dioxide,
methanol, formaldehyde, formate and carbon-containing amines.
[0076] The term "GPD" refers to a glyceraldehyde-3-phosphate
dehydrogenase enzyme (E.C. 1.2.1.12) encoded by the gpd gene and
which converts D-glyceraldehyde 3-phosphate to
3-phospho-D-glyceroyl phosphate during glycolysis. The partial
coding region of a respresentative gpd gene isolated from Yarrowia
lipolytica is provided as SEQ ID NOs:25 and 26; specifically, the
sequence lacks .about.115 amino acids that encode the C-terminus of
the gene (based on alignment with other known gpd sequences).
[0077] The term "GPD promoter" or "GPD promoter region" refers to
the 5' upstream untranslated region in front of the `ATG`
translation initiation codon of GPD and that is necessary for
expression. Examples of suitable GPD promoter regions are provided
as SEQ ID NOs:23 and 43, but these are not intended to be limiting
in nature.
[0078] The term "GPM" refers to a phosphoglycerate mutase enzyme
(EC 5.4.2.1) encoded by the gpm gene and which is responsible for
the interconversion of 3-phosphoglycerate and 2-phosphoglycerate
during glycolysis. A respresentative gpm gene from Saccharomyces
cerevisiae is GenBank Accession No. NP.sub.--012770 (SEQ ID NO:13);
a gpm gene isolated from Yarrowia lipolytica is provided as SEQ ID
NO:15.
[0079] The term "GPM promoter" or "GPM promoter region" refers to
the 5' upstream untranslated region in front of the `ATG`
translation initiation codon of GPM and that is necessary for
expression. Examples of suitable GPM promoter regions are provided
as SEQ ID NOs:27 and 44, but these are not intended to be limiting
in nature.
[0080] The term "promoter activity" will refer to an assessment of
the transcriptional efficiency of a promoter. This may, for
instance, be determined directly by measurement of the amount of
mRNA transcription from the promoter (e.g., by Northern blotting or
primer extension methods) or indirectly by measuring the amount of
gene product expressed from the promoter.
[0081] As used herein, an "isolated nucleic acid molecule" is a
polymer of RNA or DNA that is single- or double-stranded,
optionally containing synthetic, non-natural or altered nucleotide
bases. An isolated nucleic acid molecule in the form of a polymer
of DNA may be comprised of one or more segments of cDNA, genomic
DNA or synthetic DNA.
[0082] A nucleic acid molecule is "hybridizable" to another nucleic
acid molecule, such as a cDNA, genomic DNA, or RNA molecule, when a
single-stranded form of the nucleic acid molecule can anneal to the
other nucleic acid molecule under the appropriate conditions of
temperature and solution ionic strength. Hybridization and washing
conditions are well known and exemplified in Sambrook, J., Fritsch,
E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual,
2.sup.nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor,
N.Y. (1989), particularly Chapter 11 and Table 11.1 therein
(entirely incorporated herein by reference). The conditions of
temperature and ionic strength determine the "stringency" of the
hybridization. Stringency conditions can be adjusted to screen for
moderately similar fragments (such as homologous sequences from
distantly related organisms), to highly similar fragments (such as
genes that duplicate functional enzymes from closely related
organisms). Post-hybridization washes determine stringency
conditions. One set of preferred conditions uses a series of washes
starting with 6.times.SSC, 0.5% SDS at room temperature for 15 min,
then repeated with 2.times.SSC, 0.5% SDS at 45.degree. C. for 30
min, and then repeated twice with 0.2.times.SSC, 0.5% SDS at
50.degree. C. for 30 min. A more preferred set of stringent
conditions uses higher temperatures in which the washes are
identical to those above except for the temperature of the final
two 30 min washes in 0.2.times.SSC, 0.5% SDS was increased to
60.degree. C. Another preferred set of highly stringent conditions
uses two final washes in 0.1.times.SSC, 0.1% SDS at 65.degree. C.
An additional set of stringent conditions include hybridization at
0.1.times.SSC, 0.1% SDS, 65.degree. C. and washed with 2.times.SSC,
0.1% SDS followed by 0.1.times.SSC, 0.1% SDS, for example.
[0083] Hybridization requires that the two nucleic acids contain
complementary sequences, although depending on the stringency of
the hybridization, mismatches between bases are possible. The
appropriate stringency for hybridizing nucleic acids depends on the
length of the nucleic acids and the degree of complementation,
variables well known in the art. The greater the degree of
similarity or homology between two nucleotide sequences, the
greater the value of Tm for hybrids of nucleic acids having those
sequences. The relative stability (corresponding to higher Tm) of
nucleic acid hybridizations decreases in the following order:
RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100
nucleotides in length, equations for calculating Tm have been
derived (see Sambrook et al., supra, 9.50-9.51). For hybridizations
with shorter nucleic acids, i.e., oligonucleotides, the position of
mismatches becomes more important, and the length of the
oligonucleotide determines its specificity (see Sambrook et al.,
supra, 11.7-11.8). In one embodiment the length for a hybridizable
nucleic acid is at least about 10 nucleotides. Preferably a minimum
length for a hybridizable nucleic acid is at least about 15
nucleotides; more preferably at least about 20 nucleotides; and
most preferably the length is at least about 30 nucleotides.
Furthermore, the skilled artisan will recognize that the
temperature and wash solution salt concentration may be adjusted as
necessary according to factors such as length of the probe.
[0084] A "substantial portion" of an amino acid or nucleotide
sequence is that portion comprising enough of the amino acid
sequence of a polypeptide or the nucleotide sequence of a gene to
putatively identify that polypeptide or gene, either by manual
evaluation of the sequence by one skilled in the art, or by
computer-automated sequence comparison and identification using
algorithms such as BLAST (Basic Local Alignment Search Tool;
Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1993)). In
general, a sequence of ten or more contiguous amino acids or thirty
or more nucleotides is necessary in order to identify putatively a
polypeptide or nucleic acid sequence as homologous to a known
protein or gene. Moreover, with respect to nucleotide sequences,
gene-specific oligonucleotide probes comprising 20-30 contiguous
nucleotides may be used in sequence-dependent methods of gene
identification (e.g., Southern hybridization) and isolation (e.g.,
in situ hybridization of bacterial colonies or bacteriophage
plaques). In addition, short oligonucleotides of 12-15 bases may be
used as amplification primers in PCR in order to obtain a
particular nucleic acid molecule comprising the primers.
Accordingly, a "substantial portion" of a nucleotide sequence
comprises enough of the sequence to specifically identify and/or
isolate a nucleic acid molecule comprising the sequence.
[0085] The instant specification teaches partial or complete amino
acid and nucleotide sequences encoding one or more particular
microbial proteins and promoters. The skilled artisan, having the
benefit of the sequences as reported herein, may now use all or a
substantial portion of the disclosed sequences for purposes known
to those skilled in this art. Accordingly, the instant invention
comprises the complete sequences as reported in the accompanying
Sequence Listing, as well as substantial portions of those
sequences as defined above.
[0086] The term "oligonucleotide" refers to a nucleic acid,
generally of at least 18 nucleotides, that is hybridizable to a
genomic DNA molecule, a cDNA molecule, or an mRNA molecule. In one
embodiment, a labeled oligonucleotide can be used as a "probe" to
detect the presence of a nucleic acid according to the invention.
Thus, the term "probe" refers to a single-stranded nucleic acid
molecule that can base pair with a complementary single-stranded
target nucleic acid to form a double-stranded molecule. The term
"label" will refer to any conventional molecule which can be
readily attached to mRNA or DNA and which can produce a detectable
signal, the intensity of which indicates the relative amount of
hybridization of the labeled probe to the DNA fragment.
[0087] The term "complementary" is used to describe the
relationship between nucleotide bases that are capable of
hybridizing to one another. For example, with respect to DNA,
adenosine is complementary to thymine and cytosine is complementary
to guanine. Accordingly, the instant invention also includes
isolated nucleic acid molecules that are complementary to the
complete sequences as reported in the accompanying Sequence
Listing, as well as those substantially similar nucleic acid
sequences.
[0088] The term "percent identity", as known in the art, is a
relationship between two or more polypeptide sequences or two or
more polynucleotide sequences, as determined by comparing the
sequences. In the art, "identity" also means the degree of sequence
relatedness between polypeptide or polynucleotide sequences, as the
case may be, as determined by the match between strings of such
sequences. "Identity" and "similarity" can be readily calculated by
known methods, including but not limited to those described in: 1.)
Computational Molecular Biology (Lesk, A. M., Ed.) Oxford
University: NY (1988); 2.) Biocomputing: Informatics and Genome
Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer
Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H.
G., Eds.) Humana: NJ (1994); 4.) Sequence Analysis in Molecular
Biology (von Heinje, G., Ed.) Academic (1987); and 5.) Sequence
Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY
(1991). Preferred methods to determine identity are designed to
give the best match between the sequences tested. Methods to
determine identity and similarity are codified in publicly
available computer programs. Sequence alignments and percent
identity calculations may be performed using the Megalign program
of the LASERGENE bioinformatics computing suite (DNASTAR Inc.,
Madison, Wis.). Multiple alignment of the sequences is performed
using the Clustal method of alignment (Higgins and Sharp, CABIOS.
5:151-153 (1989)) with default parameters (GAP PENALTY=10, GAP
LENGTH PENALTY=10). Default parameters for pairwise alignments
using the Clustal method are: KTUPLE 1, GAP PENALTY=3, WINDOW=5 and
DIAGONALS SAVED=5.
[0089] Suitable nucleic acid molecules (isolated polynucleotides of
the present invention) encode polypeptides that are at least about
70% identical, preferably at least about 75% identical, and more
preferably at least about 80% identical to the amino acid sequences
reported herein. Preferred nucleic acid molecules encode amino acid
sequences that are about 85% identical to the amino acid sequences
reported herein. More preferred nucleic acid molecules encode amino
acid sequences that are at least about 90% identical to the amino
acid sequences reported herein. Most preferred are nucleic acid
molecules that encode amino acid sequences that are at least about
95% identical to the amino acid sequences reported herein. Suitable
nucleic acid molecules not only have the above homologies but
typically encode a polypeptide having at least 50 amino acids,
preferably at least 100 amino acids, more preferably at least 150
amino acids, still more preferably at least 200 amino acids, and
most preferably at least 250 amino acids.
[0090] Likewise, suitable promoter regions (isolated
polynucleotides of the present invention) encode promoter regions
that are at least about 70% identical, preferably at least about
75% identical, and more preferably at least about 80% identical to
the nucleotide sequences reported herein. Preferred nucleic acid
molecules are about 85% identical to the nucleotide sequences
reported herein, more preferred nucleic acid molecules are at least
about 90% identical, and most preferred are nucleic acid molecules
at least about 95% identical to the nucleotide sequences reported
herein. Suitable promoter regions not only have the above
homologies but typically are at least 50 nucleotides in length,
more preferably at least 100 nucleotides in length, more preferably
at least 250 nucleotides in length, and more preferably at least
500 nucleotides in length.
[0091] "Codon degeneracy" refers to the nature in the genetic code
permitting variation of the nucleotide sequence without affecting
the amino acid sequence of an encoded polypeptide. Accordingly, the
instant invention relates to any nucleic acid molecule that encodes
all or a substantial portion of the amino acid sequence encoding
the instant microbial polypeptides as set forth in SEQ ID NOs:16
and 26. The skilled artisan is well aware of the "codon-bias"
exhibited by a specific host cell in usage of nucleotide codons to
specify a given amino acid. Therefore, when synthesizing a gene for
improved expression in a host cell, it is desirable to design the
gene such that its frequency of codon usage approaches the
frequency of preferred codon usage of the host cell.
[0092] "Chemically synthesized", as related to a sequence of DNA,
means that the component nucleotides were assembled in vitro.
Manual chemical synthesis of DNA may be accomplished using
well-established procedures; or, automated chemical synthesis can
be performed using one of a number of commercially available
machines. "Synthetic genes" can be assembled from oligonucleotide
building blocks that are chemically synthesized using procedures
known to those skilled in the art. These building blocks are
ligated and annealed to form gene segments that are then
enzymatically assembled to construct the entire gene. Accordingly,
the genes can be tailored for optimal gene expression based on
optimization of nucleotide sequence to reflect the codon bias of
the host cell. The skilled artisan appreciates the likelihood of
successful gene expression if codon usage is biased towards those
codons favored by the host. Determination of preferred codons can
be based on a survey of genes derived from the host cell, where
sequence information is available.
[0093] "Gene" refers to a nucleic acid molecule that expresses a
specific protein, including regulatory sequences preceding (5'
non-coding sequences) and following (3' non-coding sequences) the
coding sequence. "Native gene" refers to a gene as found in nature
with its own regulatory sequences. "Chimeric gene" refers to any
gene that is not a native gene, comprising regulatory and coding
sequences that are not found together in nature. Accordingly, a
chimeric gene may comprise regulatory sequences and coding
sequences that are derived from different sources, or regulatory
sequences and coding sequences derived from the same source, but
arranged in a manner different than that found in nature. Chimeric
genes of the present invention will typically comprise a GPD or GPM
promoter region operably linked to a coding region of interest.
"Endogenous gene" refers to a native gene in its natural location
in the genome of an organism. A "foreign" gene refers to a gene not
normally found in the host organism, but that is introduced into
the host organism by gene transfer. Foreign genes can comprise
native genes inserted into a non-native organism, or chimeric
genes. A "transgene" is a gene that has been introduced into the
genome by a transformation procedure. A "codon-optimized gene" is a
gene having its frequency of codon usage designed to mimic the
frequency of preferred codon usage of the host cell.
[0094] "Coding sequence" refers to a DNA sequence that codes for a
specific amino acid sequence.
[0095] "Suitable regulatory sequences" refer to transcriptional and
translational nucleotide sequences located upstream (5' non-coding
sequences), within, or downstream (3' non-coding sequences) of a
coding sequence, and which influence the transcription, RNA
processing or stability, or translation of the associated coding
sequence. Regulatory sequences may include promoters, translation
leader sequences, introns, polyadenylation recognition sequences,
RNA processing sites, effector binding sites and stem-loop
structures.
[0096] "Promoter" refers to a DNA sequence capable of controlling
the expression of a coding sequence or functional RNA. In general,
a coding sequence is located 3' to a promoter sequence. Promoters
may be derived in their entirety from a native gene, or be composed
of different elements derived from different promoters found in
nature, or even comprise synthetic DNA segments. It is understood
by those skilled in the art that different promoters may direct the
expression of a gene in different tissues or cell types, or at
different stages of development, or in response to different
environmental or physiological conditions. Promoters that cause a
gene to be expressed in most cell types at most times are commonly
referred to as "constitutive promoters". It is further recognized
that since in most cases the exact boundaries of regulatory
sequences have not been completely defined, DNA fragments of
different lengths may have identical promoter activity.
[0097] The term "mutant promoter" is defined herein as a promoter
having a nucleotide sequence comprising a substitution, deletion,
and/or insertion of one or more nucleotides relative to the parent
promoter, wherein the mutant promoter has more or less promoter
activity than the corresponding parent promoter. The term "mutant
promoter" will encompass natural variants and in vitro generated
variants obtained using methods well known in the art (e.g.,
classical mutagenesis, site-directed mutagenesis and "DNA
shuffling").
[0098] The term "3' non-coding sequences" or "transcription
terminator" refers to DNA sequences located downstream of a coding
sequence. This includes polyadenylation recognition sequences and
other sequences encoding regulatory signals capable of affecting
mRNA processing or gene expression. The polyadenylation signal is
usually characterized by affecting the addition of polyadenylic
acid tracts to the 3' end of the mRNA precursor. The 3' region can
influence the transcription, RNA processing or stability, or
translation of the associated coding sequence.
[0099] "RNA transcript" refers to the product resulting from RNA
polymerase-catalyzed transcription of a DNA sequence. When the RNA
transcript is a perfect complementary copy of the DNA sequence, it
is referred to as the primary transcript or it may be a RNA
sequence derived from post-transcriptional processing of the
primary transcript and is referred to as the mature RNA. "Messenger
RNA" or "mRNA" refers to the RNA that is without introns and that
can be translated into protein by the cell. "cDNA" refers to a
double-stranded DNA that is complementary to, and derived from,
mRNA. "Sense" RNA refers to RNA transcript that includes the mRNA
and so can be translated into protein by the cell. "Antisense RNA"
refers to a RNA transcript that is complementary to all or part of
a target primary transcript or mRNA and that blocks the expression
of a target gene (U.S. Pat. No. 5,107,065; WO 99/28508). The
complementarity of an antisense RNA may be with any part of the
specific gene transcript, i.e., at the 5' non-coding sequence, 3'
non-coding sequence, or the coding sequence. "Functional RNA"
refers to antisense RNA, ribozyme RNA, or other RNA that is not
translated and yet has an effect on cellular processes.
[0100] The term "operably linked" refers to the association of
nucleic acid sequences on a single nucleic acid molecule so that
the function of one is affected by the other. For example, a
promoter is operably linked with a coding sequence when it is
capable of affecting the expression of that coding sequence (i.e.,
the coding sequence is under the transcriptional control of the
promoter). Coding sequences can be operably linked to regulatory
sequences in sense or antisense orientation.
[0101] The term "expression", as used herein, refers to the
transcription and stable accumulation of sense (mRNA) or antisense
RNA derived from a coding sequence. Expression may also refer to
translation of mRNA into a polypeptide.
[0102] "Introns" are sequences of non-coding DNA found in gene
sequences (either in the coding region, 5' non-coding region, or 3'
non-coding region) in most eukaryotes. Their full function is not
known; however, some enhancers are located in the introns
(Giacopelli F. et al., Gene Expr. 11:95-104 (2003)). These intron
sequences are transcribed, but removed from within the pre-mRNA
transcript before the mRNA is translated into a protein. This
process of intron removal occurs by self-splicing of the sequences
(exons) on either side of the intron.
[0103] The term "altered biological activity" will refer to an
activity, associated with a protein encoded by a nucleotide
sequence which can be measured by an assay method, where that
activity is either greater than or less than the activity
associated with the native sequence. "Enhanced biological activity"
refers to an altered activity that is greater than that associated
with the native sequence. "Diminished biological activity" is an
altered activity that is less than that associated with the native
sequence.
[0104] "Transformation" refers to the transfer of a nucleic acid
molecule into a host organism, resulting in genetically stable
inheritance. The nucleic acid molecule may be a plasmid that
replicates autonomously, for example; or, it may integrate into the
genome of the host organism. Host organisms containing the
transformed nucleic acid molecules are referred to as "transgenic"
or "recombinant" or "transformed" organisms.
[0105] The terms "plasmid", "vector" and "cassette" refer to an
extra chromosomal element often carrying genes that are not part of
the central metabolism of the cell, and usually in the form of
circular double-stranded DNA fragments. Such elements may be
autonomously replicating sequences, genome integrating sequences,
phage or nucleotide sequences, linear or circular, of a single- or
double-stranded DNA or RNA, derived from any source, in which a
number of nucleotide sequences have been joined or recombined into
a unique construction which is capable of introducing a promoter
fragment and DNA sequence for a selected gene product along with
appropriate 3' untranslated sequence into a cell. "Transformation
cassette" refers to a specific vector containing a foreign gene and
having elements in addition to the foreign gene that facilitates
transformation of a particular host cell. "Expression cassette"
refers to a specific vector containing a foreign gene and having
elements in addition to the foreign gene that allow for enhanced
expression of that gene in a foreign host.
[0106] The term "sequence analysis software" refers to any computer
algorithm or software program that is useful for the analysis of
nucleotide or amino acid sequences. "Sequence analysis software"
may be commercially available or independently developed. Typical
sequence analysis software will include, but is not limited to: 1.)
the GCG suite of programs (Wisconsin Package Version 9.0, Genetics
Computer Group (GCG), Madison, Wis.); 2.) BLASTP, BLASTN, BLASTX
(Altschul et al., J. Mol. Biol. 215:403-410 (1990)); 3.) DNASTAR
(DNASTAR, Inc. Madison, Wis.); and 4.) the FASTA program
incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput.
Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992,
111-20. Suhai, Sandor, Ed. Plenum: New York, N.Y.). Within the
context of this application it will be understood that where
sequence analysis software is used for analysis, that the results
of the analysis will be based on the "default values" of the
program referenced, unless otherwise specified. As used herein
"default values" will mean any set of values or parameters that
originally load with the software when first initialized.
[0107] Standard recombinant DNA and molecular cloning techniques
used herein are well known in the art and are described by
Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory: Cold
Spring Harbor, N.Y. (1989) (hereinafter "Maniatis"); by Silhavy, T.
J., Bennan, M. L. and Enquist, L. W., Experiments with Gene
Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.
(1984); and by Ausubel, F. M. et al., Current Protocols in
Molecular Biology, published by Greene Publishing Assoc. and
Wiley-Interscience (1987).
Identification of the qpd And qpm Genes In Yarrowia lipolytica
[0108] The present invention identifies the partial sequence of a
glyceraldehyde-3-phosphate dehydrogenase (gpd) gene (wherein
.about.115 amino acids of the C-terminus of the encoded protein are
not disclosed herein) and the complete sequence of the
phosphoglycerate mutase (gpm) gene contained within the Yarrowia
lipolytica genome.
[0109] Comparison of the partial gpd nucleotide base and deduced
amino acid sequences (SEQ ID NOs:25 and 26) to public databases
reveals that the most similar known sequences are about 81%
identical to the amino acid sequence of gpd reported herein over a
length of 215 amino acids using a Smith-Waterman alignment
algorithm (W. R. Pearson, supra). Preferred amino acid fragments
are at least about 70%-80% identical to the sequences herein, where
those sequences that are 85%-90% identical are particularly
suitable and those sequences that are about 95% identical are most
preferred. Similarly, preferred gpd encoding nucleic acid sequences
corresponding to the instant ORF are those encoding active proteins
and which are at least about 70%-80% identical to the nucleic acid
sequences of gpd reported herein, where those sequences that are
85%-90% identical are particularly suitable and those sequences
that are about 95% identical are most preferred.
[0110] Comparison of the gpm nucleotide base and deduced amino acid
sequences (SEQ ID NOs:15 and 16) to public databases reveals that
the most similar known sequences are about 71% identical to the
amino acid sequence of gpm reported herein over a length of 216
amino acids using a Smith-Waterman alignment algorithm (W. R.
Pearson, supra). Preferred amino acid fragments are at least about
70%-80% identical to the sequences herein, where those sequences
that are 85%-90% identical are particularly suitable and those
sequences that are about 95% identical are most preferred.
Similarly, preferred gpm encoding nucleic acid sequences
corresponding to the instant ORF are those encoding active proteins
and which are at least about 70%-80% identical to the nucleic acid
sequences of gpm reported herein, where those sequences that are
85%-90% identical are particularly suitable and those sequences
that are about 95% identical are most preferred.
Identification of Natural Promoter Regions in Yarrowia
lipolytica
[0111] The present invention also identifies putative promoter
regions that naturally regulate GPD and GPM in Yarrowia lipolytica.
These putative promoter regions have been identified as useful for
driving expression of any suitable coding region of interest in a
transformed yeast cell.
[0112] In the context of the presention invention, a promoter
useful in an oleaginous yeast should meet the following criteria:
[0113] 1.) Strength. A strong yeast promoter is a necessary premise
for a high expression level, and the low copy number of the ars18
(Fournier, P. et al. Yeast 7:25-36 (1991)) based expression vectors
or chimeric genes integrated into the genome makes this demand even
more important when Y. lipolytica is used as the host organism.
[0114] 2.) Activity in a medium suitable for expression of the
coding region of interest, and high enzymatic activity of that
coding region of interest. [0115] 3.) pH Tolerance. If the coding
region of interest is known to be produced only in e.g., an acidic
environment, then the promoter operably linked to said coding
region of interest must function at the appropriate pH. pH
tolerance is of course limited by the tolerance of the host
organism. [0116] 4.) Inducibility. A tightly regulated yeast
promoter makes it possible to separate the growth stage from the
expression stage, thereby enabling expression of products that are
known to inhibit cell growth. [0117] 5.) Activity in the stationary
phase of growth in oleaginous yeast hosts for accumulation of
PUFAs.
[0118] Additionally, it is preferable for novel yeast promoters to
possess differences in activity with respect to the known Yarrowia
lipolytica TEF and/or XPR2 promoters (U.S. Pat. No. 4,937,189;
EP220864; EP832258; U.S. Pat. No. 6,265,185). A comparative study
of the TEF promoter and the GPD and GPM promoters of the instant
invention is provided in Example 7. It is shown that the yeast
promoters of the invention have improved activity compared to the
TEF promoter. The promoter region of the instant GPD gene is
contained within several nucleic acid molecules, specifically, SEQ
ID NOs: 23, 24 and 43. In one embodiment, the GPD promoter will
comprise nucleotides -500 to +1 of SEQ ID NO:43 (wherein the `A`
position of the `ATG` translation initiation codon is designated as
+1), thereby permitting relatively strong promoter activity; in
alternate embodiments, the -100 to +1 region of SEQ ID NO:43 should
be sufficient for basal activity of the promoter.
[0119] The GPM promoter region of the instant invention is
contained in several nucleic acid molecules disclosed herein,
including SEQ ID NOs:27, 28 and 44. In one embodiment, the GPM
promoter will comprise nucleotides--500 to +1 of SEQ ID NO:44
(wherein the `A` position of the `ATG` translation initiation codon
is designated as +1), thereby permitting relatively strong promoter
activity; alternatively, the -100 to +1 region of SEQ ID NO:44
should be sufficient for basal activity of the promoter.
[0120] The promoter regions of the invention may comprise
additional nucleotides to those specified above. For example, the
promoter sequences of the invention may be constructed on the basis
of the DNA sequence presented as SEQ ID NO:23 or SEQ ID NO:27 (SEQ
ID NOs:43 and 44 are subsequences thereof, respectively). It should
be recognized that promoter fragments of various diminishing
lengths may have identical promoter activity, since the exact
boundaries of the regulatory sequences have not been completely
defined.
[0121] In alternate embodiments mutant promoters may be
constructed, wherein the DNA sequence of the promoter has one or
more nucleotide substitutions (i.e., deletions, insertions,
substitutions, or addition of one or more nucleotides in the
sequence) which do not effect (in particular impair) the yeast
promoter activity. Regions that can be modified without
significantly affecting the yeast promoter activity can be
identified by deletion studies. A mutant promoter of the present
invention has at least about 20%, preferably at least about 40%,
more preferably at least about 60%, more preferably at least about
80%, more preferably at least about 90%, more preferably at least
about 100%, more preferably at least about 200%, more preferably at
least about 300% and most preferably at least about 400% of the
promoter activity of the GPD or GPM promoter regions described
herein as SEQ ID NOs:43 and 44.
[0122] Methods for mutagenesis are well known in the art and
suitable for the generation of mutant promoters. For example, in
vitro mutagenesis and selection, PCR based random mutagenesis,
site-directed mutagenesis, or other means can be employed to obtain
mutations of the naturally occurring promoters and genes of the
instant invention. This would permit production of a putative
promoter having a more desirable level of promoter activity in the
host cell, or production of a polypeptide having more desirable
physical and kinetic parameters for function in the host cell.
[0123] If desired, the regions of a nucleotide of interest
important for promoter or enzymatic activity, respectively, can be
determined through routine mutagenesis, expression of the resulting
mutant promoters or polypeptides and determination of their
activities. Mutants may include deletions, insertions and point
mutations, or combinations thereof. A typical functional analysis
begins with deletion mutagenesis to determine either: 1.) the
minimum portion of the putative promoter necessary for activity; or
2.) the N- and C-terminal limits of the protein necessary for
function. Subsequently, internal deletions, insertions or point
mutants are made to further determine regions necessary for
function. Other techniques such as cassette mutagenesis or total
synthesis also can be used.
[0124] Deletion mutagenesis of a coding sequence is accomplished,
for example, by using exonucleases to sequentially remove the 5' or
3' coding regions. Kits are available for such techniques. After
deletion, the coding region is completed by ligating
oligonucleotides containing start or stop codons to the deleted
coding region after 5' or 3' deletion, respectively. Alternatively,
oligonucleotides encoding start or stop codons are inserted into
the coding region by a variety of methods including site-directed
mutagenesis, mutagenic PCR or by ligation onto DNA digested at
existing restriction sites.
[0125] Internal deletions in a putative promoter region or within a
coding sequence can similarly be made through a variety of methods
including the use of existing restriction sites in the DNA, by use
of mutagenic primers via site-directed mutagenesis or mutagenic
PCR. Insertions are made through methods such as linker-scanning
mutagenesis, site-directed mutagenesis or mutagenic PCR, while
point mutations are made through techniques such as site-directed
mutagenesis or mutagenic PCR.
[0126] Chemical mutagenesis also can be used for identifying
regions of a putative promoter region or polypeptide important for
activity. A mutated construct is expressed, and the ability of the
resulting altered promoter or protein, respectively, is assayed.
Such structure-function analysis can determine which regions may be
deleted, which regions tolerate insertions, and which point
mutations allow the mutant promoter or protein to function in
substantially the same way as the native promoter or protein. All
such mutant promoters and nucleotide sequences encoding
polypeptides that are derived from the instant promoters and genes
described herein are within the scope of the present invention.
Isolation Of Homologs To The qpd And qgm Genes And Putative
Promoter Regions
[0127] It will be appreciated by a person of skill in the art that
the promoter regions and genes of the present invention have
homologs in a variety of yeast species; and, the use of the
promoters and genes for heterologous gene expression are not
limited to those promoters and genes derived from Yarrowia
lipolytica, but extend to homologs in other yeast species. For
example, the invention encompasses homologs derived from oleaginous
genera including, but not limited to: Yarrowia, Candida,
Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and
Lipomyces; examples of preferred species within these genera
include: Rhodosporidium toruloides, Lipomyces starkeyii, L.
lipoferus, Candida revkaufi, C. pulcherrima, C. tropicalis, C.
utilis, Trichosporon pullans, T. cutaneum, Rhodotorula glutinus and
R. graminis.
[0128] Homology typically is measured using sequence analysis
software, wherein the term "sequence analysis software" refers to
any computer algorithm or software program (commercially available
or independently developed) that is useful for the analysis of
nucleotide or amino acid sequences. In general, such computer
software matches similar sequences by assigning degrees of homology
to various substitutions, deletions and other modifications.
[0129] As is well known in the art, isolation of homologous
promoter regions or genes using sequence-dependent protocols is
readily possible using various techniques. Examples of
sequence-dependent protocols include, but are not limited to: 1.)
methods of nucleic acid hybridization; 2.) methods of DNA and RNA
amplification, as exemplified by various uses of nucleic acid
amplification technologies [e.g., polymerase chain reaction (PCR),
Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction
(LCR), Tabor, S. et al., Proc. Acad. Sci. USA 82:1074 (1985); or
strand displacement amplification (SDA), Walker, et al., Proc.
Natl. Acad. Sci. U.S.A., 89:392 (1992)]; and 3.) methods of library
construction and screening by complementation.
[0130] For example, putative promoter regions or genes encoding
similar proteins or polypetides to those of the instant invention
could be isolated directly by using all or a portion of the instant
nucleic acid molecules as DNA hybridization probes to screen
libraries from any desired microbe using methodology well known to
those skilled in the art. Specific oligonucleotide probes based
upon the instant nucleic acid sequences can be designed and
synthesized by methods known in the art (Maniatis, supra).
Moreover, the entire sequences can be used directly to synthesize
DNA probes by methods known to the skilled artisan (e.g., random
primers DNA labeling, nick translation, or end-labeling
techniques), or RNA probes using available in vitro transcription
systems. In addition, specific primers can be designed and used to
amplify a part of (or full-length of) the instant sequences. The
resulting amplification products can be labeled directly during
amplification reactions or labeled after amplification reactions,
and used as probes to isolate full-length DNA fragments under
conditions of appropriate stringency.
[0131] Typically, in PCR-type amplification techniques, the primers
have different sequences and are not complementary to each other.
Depending on the desired test conditions, the sequences of the
primers should be designed to provide for both efficient and
faithful replication of the target nucleic acid. Methods of PCR
primer design are common and well known in the art (Thein and
Wallace, "The use of oligonucleotides as specific hybridization
probes in the Diagnosis of Genetic Disorders", in Human Genetic
Diseases: A Practical Approach, K. E. Davis (Ed.), (1986) pp 33-50
IRL: Herndon, Va.; and Rychlik, W., In Methods in Molecular
Biology, White, B. A. (Ed.), (1993) Vol. 15, pp 31-39, PCR
Protocols: Current Methods and Applications. Humania: Totowa,
N.J.).
[0132] Generally two short segments of the instant sequences may be
used in polymerase chain reaction protocols to amplify longer
nucleic acid molecules encoding homologous polynucleotides from DNA
or RNA. The polymerase chain reaction may also be performed on a
library of cloned nucleic acid molecules wherein the sequence of
one primer is derived from the instant nucleic acid molecules, and
the sequence of the other primer takes advantage of the presence of
the polyadenylic acid tracts to the 3' end of the mRNA precursor
encoding microbial genes.
[0133] Alternatively, the instant sequences may be employed as
hybridization reagents for the identification of homologs. The
basic components of a nucleic acid hybridization test include a
probe, a sample suspected of containing the nucleotide sequence of
interest, and a specific hybridization method. Probes of the
present invention are typically single-stranded nucleic acid
sequences that are complementary to the nucleic acid sequences to
be detected. Probes are "hybridizable" to the nucleic acid sequence
to be detected. The probe length can vary from 5 bases to tens of
thousands of bases, and will depend upon the specific test to be
done. Typically a probe length of about 15 bases to about 30 bases
is suitable. Only part of the probe molecule need be complementary
to the nucleic acid sequence to be detected. In addition, the
complementarity between the probe and the target sequence need not
be perfect. Hybridization does occur between imperfectly
complementary molecules with the result that a certain fraction of
the bases in the hybridized region are not paired with the proper
complementary base.
[0134] Hybridization methods are well defined. Typically the probe
and sample must be mixed under conditions that will permit nucleic
acid hybridization. This involves contacting the probe and sample
in the presence of an inorganic or organic salt under the proper
concentration and temperature conditions. The probe and sample
nucleic acids must be in contact for a long enough time that any
possible hybridization between the probe and sample nucleic acid
may occur. The concentration of probe or target in the mixture will
determine the time necessary for hybridization to occur. The higher
the probe or target concentration, the shorter the hybridization
incubation time needed. Optionally, a chaotropic agent may be
added. The chaotropic agent stabilizes nucleic acids by inhibiting
nuclease activity. Furthermore, the chaotropic agent allows
sensitive and stringent hybridization of short oligonucleotide
probes at room temperature (Van Ness and Chen, Nucl. Acids Res.
19:5143-5151 (1991)). Suitable chaotropic agents include
guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate,
lithium tetrachloroacetate, sodium perchlorate, rubidium
tetrachloroacetate, potassium iodide and cesium trifluoroacetate,
among others. Typically, the chaotropic agent will be present at a
final concentration of about 3 M. If desired, one can add formamide
to the hybridization mixture, typically 30-50% (v/v).
[0135] Various hybridization solutions can be employed. Typically,
these comprise from about 20 to 60% volume, preferably 30%, of a
polar organic solvent. A common hybridization solution employs
about 30-50% v/v formamide, about 0.15 to 1 M sodium chloride,
about 0.05 to 0.1 M buffers (e.g., sodium citrate, Tris-HCl, PIPES
or HEPES (pH range about 6-9)), about 0.05 to 0.2% detergent (e.g.,
sodium dodecylsulfate), or between 0.5-20 mM EDTA, FICOLL
(Pharmacia Inc.) (about 300-500 kdal), polyvinylpyrrolidone (about
250-500 kdal) and serum albumin. Also included in the typical
hybridization solution will be unlabeled carrier nucleic acids from
about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g., calf thymus or
salmon sperm DNA, or yeast RNA), and optionally from about 0.5 to
2% wt/vol glycine. Other additives may also be included, such as
volume exclusion agents that include a variety of polar
water-soluble or swellable agents (e.g., polyethylene glycol),
anionic polymers (e.g., polyacrylate or polymethylacrylate) and
anionic saccharidic polymers (e.g., dextran sulfate).
Recombinant Expression In Yeast
[0136] Initiation control regions or promoter regions that are
useful to drive expression of a coding gene of interest in the
desired host cell are selected from those derived from the upstream
portion of the gpd and gpm genes (SEQ ID NOs:25 and 15,
respectively). The promoter regions may be identified from the
upstream sequences of gpd and gpm genes and their homologs and
isolated according to common methods (Maniatis, supra). Once the
promoter regions are identified and isolated, they may be operably
linked to a coding region of interest to be expressed in a suitable
expression vector. These chimeric genes may then be expressed in
natural host cells and heterologous host cells, particularly in the
cells of oleaginous yeast hosts. Thus, one aspect of the present
invention provides a recombinant expression vector comprising a
yeast promoter of the invention.
[0137] In a further aspect, the invention provides a method of
expressing a coding region of interest in a transformed yeast cell,
wherein a transformed cell is provided having a chimeric gene
comprising: (i) a GPD or GPM promoter region and (ii) a coding
region of interest expressible in the host, wherein the promoter
region is operably linked to the coding region of interest; and the
transformed cell is grown under conditions wherein the chimeric
gene is expressed. The polypeptide so produced can optionally be
recovered from the culture.
[0138] Microbial expression systems and expression vectors are well
known to those skilled in the art. Any of these could be used to
construct chimeric genes comprising the promoter regions derived
from the gpm and gpd genes for production of any specific coding
region of interest suitable for expression in a desirable yeast
host cell. These chimeric genes could then be introduced into
appropriate microorganisms by integration via transformation to
provide high-level expression of the enzymes upon induction.
Alternatively, the promoters can be cloned into a plasmid that is
capable of transforming and replicating itself in the preferred
yeast host cell. The coding region of interest to be expressed can
then be cloned downstream from the promoter. Once the recombinant
host is established, gene expression can be accomplished by growing
the cells under suitable conditions (infra).
[0139] Suitable Coding Regions of Interest
[0140] Useful chimeric genes will include the promoter region of
either of the gpd and gpm genes as defined herein or a mutant
promoter thereof, operably linked to a suitable coding region of
interest to be expressed in a preferred host cell.
[0141] Coding regions of interest to be expressed in the
recombinant yeast host may be either endogenous to the host or
heterologous and must be compatible with the host organism. Genes
encoding proteins of commercial value are particularly suitable for
expression. For example, suitable coding regions of interest may
include (but are not limited to) those encoding viral, bacterial,
fungal, plant, insect, or vertebrate coding regions of interest,
including mammalian polypeptides. Further, these coding regions of
interest may be, for example, structural proteins, enzymes (e.g.,
oxidoreductases, transferases, hydrolyases, lyases, isomerases,
ligases), or peptides. A non-limiting list includes genes encoding
enzymes such as aminopeptidases, amylases, carbohydrases,
carboxypeptidases, catalyases, cellulases, chitinases, cutinases,
cyclodextrin glycosyltransferases, deoxyribonucleases, esterases,
.alpha.-galactosidases, .beta.-glucanases, .beta.-galactosidases,
glucoamylases, .alpha.-glucosidases, .beta.-glucosidases,
invertases, laccases, lipases, mannosidases, mutanases, oxidases,
pectinolytic enzymes, peroxidases, phospholipases, phytases,
polyphenoloxidases, proteolytic enzymes, ribonucleases,
transglutaminases or xylanases.
[0142] Preferred in the present invention in some embodiments are
coding regions of the enzymes involved in the production of
microbial oils, including .omega.-6 and .omega.-3 fatty acids. Many
microorganisms, including algae, bacteria, molds and yeast, can
synthesize PUFAs and omega fatty acids in the ordinary course of
cellular metabolism. Particularly well-studied are fungi including
Schizochytrium aggregatm, species of the genus Thraustochytrium and
Morteriella alpina. Additionally, many dinoflagellates
(Dinophyceaae) naturally produce high concentrations of PUFAs. As
such, a variety of genes involved in oil production have been
identified through genetic means and the DNA sequences of some of
these genes are publicly available (e.g., see GenBank Accession
No.'s AY131238, Y055118, AY055117, AF296076, AF007561, L11421,
NM.sub.--031344, AF465283, AF465282, AF465281, AF110510, AF419296,
AB052086, AJ250735, AF126799, AF126798, AF199596, AF226273,
AF320509, AB072976, AF489588, AJ510244, AF419297, AF07879,
AF067654, AB022097, AF489589.1, AY332747, AAG36933, AF110509,
AB020033, AAL13300, AF417244, AF161219, X86736, AF240777, AB007640,
AB075526, AP002063, NP.sub.--441622, BAA18302, BAA02924, AAL36934,
AF338466, AF438199, E11368, E11367, D83185, U90417, AF085500,
AY504633, NM.sub.--069854, AF230693, AX464731, NM.sub.--119617,
NM.sub.--134255, NM.sub.--134383, NM.sub.--134382, NM.sub.--068396,
NM.sub.--068392, NM.sub.--070713, NM.sub.--068746 and
NM.sub.--064685). Additionally, the patent literature provides many
additional DNA sequences of genes (and/or details concerning
several of the genes above and their methods of isolation) involved
in oil production. See, for example: U.S. Pat. No. 5,968,809
(.DELTA.6 desaturases); U.S. Pat. No. 5,972,664 and U.S. Pat. No.
6,075,183 (.DELTA.5 desaturases); WO 91/13972 and U.S. Pat. No.
5,057,419 (.DELTA.9 desaturases); WO 93/11245 (.DELTA.15
desaturases); WO 94/11516, U.S. Pat. No. 5,443,974 and WO 03/099216
(.DELTA.12 desaturases); U.S. 2003/0196217 A1 (.DELTA.17
desaturase); WO 00/12720 and U.S. 2002/0139974A1 (elongases), each
of which is herein incorporated by reference in its entirety.
[0143] Components of Vectors/DNA Cassettes
[0144] Vectors or DNA cassettes useful for the transformation of
suitable host cells are well known in the art. The specific choice
of sequences present in the construct is dependent upon the desired
expression products (supra), the nature of the host cell, and the
proposed means of separating transformed cells versus
non-transformed cells. Typically, however, the vector or cassette
contains sequences directing transcription and translation of the
relevant gene(s), a selectable marker, and sequences allowing
autonomous replication or chromosomal integration. Suitable vectors
comprise a region 5' of the gene that controls transcriptional
initiation and a region 3' of the DNA fragment that controls
transcriptional termination. It is most preferred when both control
regions are derived from genes from the transformed host cell,
although it is to be understood that such control regions need not
be derived from the genes native to the specific species chosen as
a production host.
[0145] Nucleotide sequences surrounding the translational
initiation codon `ATG` have been found to affect expression in
yeast cells. If the desired polypeptide is poorly expressed in
yeast, the nucleotide sequences of exogenous genes can be modified
to include an efficient yeast translation initiation sequence motif
to obtain optimal gene expression. For expression in yeast, this
can be done by site-directed mutagenesis of an inefficiently
expressed gene to include the favored translation initiation
motif.
[0146] The termination region can be derived from the 3' region of
the gene from which the initiation region was obtained or from a
different gene. A large number of termination regions are known and
function satisfactorily in a variety of hosts (when utilized both
in the same and different genera and species from where they were
derived). The termination region usually is selected more as a
matter of convenience rather than because of any particular
property. Preferably, the termination region is derived from a
yeast gene, particularly Saccharomyces, Schizosaccharomyces,
Candida, Yarrowia or Kluyveromyces. The 3'-regions of mammalian
genes encoding .gamma.-interferon and .alpha.-2 interferon are also
known to function in yeast. Termination control regions may also be
derived from various genes native to the preferred hosts.
Optionally, a termination site may be unnecessary; however, it is
most preferred if included.
[0147] As one of skill in the art is aware, merely inserting a
chimeric gene into a cloning vector does not ensure that it will be
successfully expressed at the level needed. In response to needs
for high expression rates, many specialized expression vectors have
been created by manipulating a number of different genetic elements
that control aspects of transcription, translation, protein
stability, oxygen limitation and secretion from the host cell. More
specifically, some of the molecular features that have been
manipulated to control gene expression include: 1.) the nature of
the relevant transcriptional promoter and terminator sequences; 2.)
the number of copies of the cloned gene and whether the gene is
plasmid-borne or integrated into the genome of the host cell; 3.)
the final cellular location of the synthesized foreign protein; 4.)
the efficiency of translation in the host organism; 5.) the
intrinsic stability of the cloned gene protein within the host
cell; and 6.) the codon usage within the cloned gene, such that its
frequency approaches the frequency of preferred codon usage of the
host cell. Each of these types of modifications are encompassed in
the present invention, as means to further optimize expression of a
chimeric gene comprising a promoter region of either of the gpd and
gpm genes as defined herein or a mutant promoter thereof, operably
linked to a suitable coding region of interest.
[0148] Transformation of Yeast Cells
[0149] Once an appropriate chimeric gene has been constructed that
is suitable for expression in a yeast cell, it is placed in a
plasmid vector capable of autonomous replication in a host cell or
it is directly integrated into the genome of the host cell.
Integration of expression cassettes can occur randomly within the
host genome or can be targeted through the use of constructs
containing regions of homology with the host genome sufficient to
target recombination with the host locus. Where constructs are
targeted to an endogenous locus, all or some of the transcriptional
and translational regulatory regions can be provided by the
endogenous locus.
[0150] Where two or more genes are expressed from separate
replicating vectors, it is desirable that each vector has a
different means of selection and should lack homology to the other
constructs to maintain stable expression and prevent reassortment
of elements among constructs. Judicious choice of regulatory
regions, selection means and method of propagation of the
introduced construct can be experimentally determined so that all
introduced genes are expressed at the necessary levels to provide
for synthesis of the desired products.
[0151] Constructs comprising a coding region of interest may be
introduced into a host cell by any standard technique. These
techniques include transformation (e.g., lithium acetate
transformation [Methods in Enzymology, 194:186-187 (1991)]),
protoplast fusion, biolistic impact, electroporation,
microinjection, or any other method that introduces the gene of
interest into the host cell. More specific teachings applicable for
oleaginous yeast (i.e., Yarrowia lipolytica) include U.S. Pat. Nos.
4,880,741 and 5,071,764 and Chen, D. C. et al. (Appl Microbiol
Biotechnol. 48(2):232-235-(1997)).
For convenience, a host cell that has been manipulated by any
method to take up a DNA sequence (e.g., an expression cassette)
will be referred to as "transformed" or "recombinant" herein. The
transformed host will have at least one copy of the expression
construct and may have two or more, depending upon whether the gene
is integrated into the genome, amplified, or is present on an
extrachromosomal element having multiple copy numbers. The
transformed host cell can be identified by selection for a marker
contained on the introduced construct. Alternatively, a separate
marker construct may be co-transformed with the desired construct,
as many transformation techniques introduce many DNA molecules into
host cells. Typically, transformed hosts are selected for their
ability to grow on selective media. Selective media may incorporate
an antibiotic or lack a factor necessary for growth of the
untransformed host, such as a nutrient or growth factor. An
introduced marker gene may confer antibiotic resistance or encode
an essential growth factor or enzyme, thereby permitting growth on
selective media when expressed in the transformed host. Selection
of a transformed host can also occur when the expressed marker
protein can be detected, either directly or indirectly. The marker
protein may be expressed alone or as a fusion to another protein.
The marker protein can be detected by: 1.) its enzymatic activity
(e.g., .beta.-galactosidase can convert the substrate X-gal
[5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside] to a
colored product; luciferase can convert luciferin to a
light-emitting product); or 2.) its light-producing or modifying
characteristics (e.g., the green fluorescent protein of Aequorea
Victoria fluoresces when illuminated with blue light).
Alternatively, antibodies can be used to detect the marker protein
or a molecular tag on, for example, a protein of interest. Cells
expressing the marker protein or tag can be selected, for example,
visually, or by techniques such as FACS or panning using
antibodies. For selection of yeast transformants, any marker that
functions in yeast may be used. Preferred for use herein are
resistance to kanamycin, hygromycin and the aminoglycoside G418, as
well as ability to grow on media lacking uracil or leucine.
[0152] Techniques to Up-Regulate Expression of a Chimeric Gene
Comprising a GPD Or GPM Promoter Operably Linked to a Coding Region
of Interest
[0153] Additional copies a particular coding region of interest
(operably linked to a promoter of the instant invention) may be
introduced into the host to increase expression. Expression of the
coding region of interest also can be increased by
removing/deleting destabilizing sequences from either the mRNA or
the encoded protein, or by adding stabilizing sequences to the mRNA
(U.S. Pat. No. 4,910,141).
[0154] Yet another approach to increase expression of a coding
region of interest is to increase the translational efficiency of
the encoded mRNAs by replacement of codons in the native gene with
those for optimal gene expression in the selected host
microorganism. As will be appreciated by one skilled in the art,
use of host preferred codons can substantially enhance the
expression of the foreign gene encoding the polypeptide. In
general, host preferred codons can be determined within a
particular host species of interest by examining codon usage in
proteins (preferably those expressed in the largest amount) and
determining which codons are used with highest frequency. Then, the
coding sequence for a polypeptide of interest can be synthesized in
whole or in part using the codons preferred in the host
species.
Preferred Hosts
[0155] Preferred host cells for expression of the instant genes and
coding regions of interest operably linked to the instant promoter
molecules herein are yeast cells (where oleaginous yeast are most
preferred where the desired use is for the production of microbial
oils, infra). Oleaginous yeast are naturally capable of oil
synthesis and accumulation, wherein the oil can comprise greater
than about 25% of the cellular dry weight, more preferably greater
than about 30% of the cellular dry weight, and most preferably
greater than about 40% of the cellular dry weight. Genera typically
identified as oleaginous yeast include, but are not limited to:
Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus,
Trichosporon and Lipomyces. More specifically, illustrative
oil-synthesizing yeast include: Rhodosporidium toruloides,
Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C.
pulcherrima, C. tropicalis, C. utilis, Trichosporon pullans, T.
cutaneum, Rhodotorula glutinus, R. graminis and Yarrowia lipolytica
(formerly classified as Candida lipolytica).
[0156] Most preferred is the oleaginous yeast Yarrowia lipolytica;
and, in a further embodiment, most preferred are the Y. lipolytica
strains designated as ATCC #20362, ATCC #8862, ATCC #18944, ATCC
#76982 and/or LGAM S(7)1 (Papanikolaou S., and Aggelis G.,
Bioresour. Technol. 82(1):43-9 (2002)). The Y. lipolytica strain
designated as ATCC #76982 was the particular strain from which the
GPD and GPM promoters and genes were isolated herein.
Industrial Production using Transformed Yeast Expressing a Suitable
Coding Region of Interest
[0157] In general, media conditions which may be optimized for
high-level expression of a particular coding region of interest
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 and
the time of cell harvest. Microorganisms of interest, such as
oleaginous yeast, are grown in complex media (e.g., yeast
extract-peptone-dextrose broth (YPD)) or a defined minimal media
that lacks a component necessary for growth and thereby forces
selection of the desired expression cassettes (e.g., Yeast Nitrogen
Base (DIFCO Laboratories, Detroit, Mich.)).
[0158] Fermentation media in the present invention must contain a
suitable carbon source. Suitable carbon sources may include, but
are not limited to: monosaccharides (e.g., glucose, fructose),
disaccharides (e.g., lactose, sucrose), oligosaccharides,
polysaccharides (e.g., starch, cellulose or mixtures thereof),
sugar alcohols (e.g., glycerol) or mixtures from renewable
feedstocks (e.g., cheese whey permeate, cornsteep liquor, sugar
beet molasses, barley malt). Additionally, carbon sources may
include alkanes, fatty acids, esters of fatty acids,
monoglycerides, diglycerides, triglycerides, phospholipids and
various commercial sources of fatty acids including vegetable oils
(e.g., soybean oil) and animal fats. Additionally, the carbon
source may include one-carbon sources (e.g., carbon dioxide,
methanol, formaldehyde, formate, carbon-containing amines) for
which metabolic conversion into key biochemical intermediates has
been demonstrated. Hence it is contemplated that the source of
carbon utilized in the present invention may encompass a wide
variety of carbon-containing sources and will only be limited by
the choice of the host organism. Although all of the above
mentioned carbon sources and mixtures thereof are expected to be
suitable in the present invention, preferred carbon sources are
sugars and/or fatty acids. Most preferred is glucose and/or fatty
acids containing between 10-22 carbons.
[0159] 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,
the fermentation media must also contain suitable minerals, salts,
cofactors, buffers, vitamins, and other components known to those
skilled in the art suitable for the growth of the
microorganism.
[0160] Preferred growth media in the present invention 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 will be known by one skilled in the art of
microbiology or fermentation science. A suitable pH range for the
fermentation is typically between about pH 4.0 to pH 8.0, wherein
pH 5.5 to pH 7.0 is preferred as the range for the initial growth
conditions. The fermentation may be conducted under aerobic or
anaerobic conditions, wherein microaerobic conditions are
preferred.
[0161] Host cells comprising a suitable coding region of interest
operably linked to the promoters of the present invention may be
cultured using methods known in the art. For example, the cell may
be cultivated by shake flask cultivation, small-scale or
large-scale fermentation in laboratory or industrial fermentors
performed in a suitable medium and under conditions allowing
expression of the coding region of interest.
[0162] Where commercial production of a product that relies on the
instant genetic chimera is desired, a variety of culture
methodologies may be applied. For example, large-scale production
of a specific gene product over-expressed from a recombinant host
may be produced by a batch, fed-batch or continuous fermentation
process.
[0163] A batch fermentation process is a closed system wherein 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. Thus, 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. A variation of the standard batch process is
the fed-batch process, wherein the source is continually added to
the fermentor over the course of the fermentation process. A
fed-batch process is also suitable in the present invention.
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 source in the media at any one time.
Measurement of the source concentration in fed-batch systems is
difficult and therefore 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). Batch and
fed-batch culturing methods are common and well known in the art
and examples may be found in Thomas D. Brock in Biotechnology: A
Textbook of Industrial Microbiology, 2.sup.nd ed., (1989) Sinauer
Associates: Sunderland, M A; or Deshpande, Mukund V., Appl.
Biochem. Biotechnol., 36:227 (1992), herein incorporated by
reference.
[0164] Commercial production may also be accomplished by a
continuous fermentation process, wherein a defined media is
continuously added to a bioreactor while an equal amount of culture
volume is removed simultaneously for product recovery. Continuous
cultures generally maintain the 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, one approach may limit the carbon source and allow all
other parameters to moderate metabolism. In other systems, a number
of factors affecting growth may be altered continuously while the
cell concentration, measured by media turbidity, is kept constant.
Continuous systems strive to maintain steady state growth and thus
the cell growth rate must be 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 well
known in the art of industrial microbiology and a variety of
methods are detailed by Brock, supra.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0165] Although the promoters of the present invention will be
suitable for expression of any suitable coding region of interest
in an oleaginous yeast, in a preferred embodiment the promoters
will be utilized in the development of an oleaginous yeast that
accumulates oils enriched in PUFAs. Toward this end, it is
necessary to introduce and express e.g., desaturases and elongases
that allow for the synthesis and accumulation of .omega.-3 and/or
.omega.-6 fatty acids.
[0166] The term "fatty acids" refers to long chain aliphatic acids
(alkanoic acids) of varying chain lengths, from about C.sub.12 to
C.sub.22 (although both longer and shorter chain-length acids are
known). The predominant chain lengths are between C.sub.16 and
C.sub.22. The structure of a fatty acid is represented by a simple
notation system of "X:Y", where X is the total number of carbon (C)
atoms and Y is the number of double bonds.
[0167] Generally, fatty acids are classified as saturated or
unsaturated. The term "saturated fatty acids" refers to those fatty
acids that have no "double bonds" between their carbon backbone. In
contrast, "unsaturated fatty acids" are cis-isomers that have
"double bonds" along their carbon backbones. "Monounsaturated fatty
acids" have only one "double bond" along the carbon backbone (e.g.,
usually between the 9.sup.th and 10.sup.th carbon atom as for
palmitoleic acid (16:1) and oleic acid (18:1)), while
"polyunsaturated fatty acids" (or "PUFAs") have at least two double
bonds along the carbon backbone (e.g., between the 9.sup.th and
10.sup.th, and 12.sup.th and 13.sup.th carbon atoms for linoleic
acid (18:2); and between the 9.sup.th and 10.sup.th, 12.sup.th and
13.sup.th, and 15.sup.th and 16.sup.th for .alpha.-linolenic acid
(18:3)).
[0168] "PUFAs" can be classified into two major families (depending
on the position (n) of the first double bond nearest the methyl end
of the fatty acid carbon chain). Thus, the ".alpha.-6 fatty acids"
(.omega.6 or .omega.-6) have the first unsaturated double bond six
carbon atoms from the omega (methyl) end of the molecule and
additionally have a total of two or more double bonds, with each
subsequent unsaturation occurring 3 additional carbon atoms toward
the carboxyl end of the molecule. In contrast, the ".alpha.-3 fatty
acids" (.omega.-3 or .omega.-3) have the first unsaturated double
bond three carbon atoms away from the omega end of the molecule and
additionally have a total of three or more double bonds, with each
subsequent unsaturation occurring 3 additional carbon atoms toward
the carboxyl end of the molecule.
[0169] For the purposes of this disclosure, the omega-reference
system will be used to indicate the number of carbons, the number
of double bonds and the position of the double bond closest to the
omega carbon, counting from the omega carbon (which is numbered 1
for this purpose). This nomenclature is shown below in Table 1, in
the column titled "Shorthand Notation". The remainder of the Table
summarizes the common names of .omega.-3 and .omega.-6 fatty acids,
the abbreviations that will be used throughout the remainder of the
specification, and each compounds' chemical name.
TABLE-US-00001 TABLE 1 Nomenclature Of Polyunsaturated Fatty Acids
Shorthand Common Name Abbreviation Chemical Name Notation Linoleic
LA cis-9,12-octadecadienoic 18:2 .omega.-6 .gamma.-Linoleic GLA
cis-6,9,12- 18:3 .omega.-6 octadecatrienoic Dihomo-.gamma.- DGLA
cis-8,11,14-eicosatrienoic 20:3 .omega.-6 Linoleic Arachidonic ARA
cis-5,8,11,14- 20:4 .omega.-6 eicosatetraenoic .alpha.-Linolenic
ALA cis-9,12,15- 18:3 .omega.-3 octadecatrienoic Stearidonic STA
cis-6,9,12,15- 18:4 .omega.-3 octadecatetraenoic Eicosatetraenoic
ETA cis-8,11,14,17- 20:4 .omega.-3 eicosatetraenoic
Eicosapentaenoic EPA cis-5,8,11,14,17- 20:5 .omega.-3
eicosapentaenoic Docosapentaenoic DPA cis-7,10,13,16,19- 22:5
.omega.-3 docosapentaenoic Docosahexaenoic DHA cis-4,7,10,13,16,19-
22:6 .omega.-3 docosahexaenoic
Microbial Biosynthesis of Fatty Acids
[0170] In general, lipid accumulation in oleaginous microorganisms
is triggered in response to the overall carbon to nitrogen ratio
present in the growth medium. When cells have exhausted available
nitrogen supplies (e.g., when the carbon to nitrogen ratio is
greater than about 40), the depletion of cellular adenosine
monophosphate (AMP) leads to the cessation of AMP-dependent
isocitrate dehydrogenase activity in the mitochondria and the
accumulation of citrate, transport of citrate into the cytosol, and
subsequent cleavage of the citrate by ATP-citrate lyase to yield
acetyl-CoA and oxaloacetate. Acetyl-CoA is the principle building
block for de novo biosynthesis of fatty acids. The first committed
step of fatty acid biosynthesis is the synthesis of malonyl-CoA,
produced via carboxylation of acetyl-CoA. Fatty acid synthesis is
catalyzed by a multi-enzyme fatty acid synthase complex and occurs
by the condensation of eight two-carbon fragments (acetyl groups
from acetyl-CoA) to form a 16-carbon saturated fatty acid,
palmitate.
[0171] Palmitate is the precursor of longer chain saturated and
unsaturated fatty acids (e.g., stearic (18:0), palmitoleic (16:1)
and oleic (18:1) acids) through the action of elongases and
desaturases present in the endoplasmic reticulum membrane.
Palmitate and stearate are converted to their unsaturated
derivatives, palmitoleic (16:1) and oleic (18:1) acids,
respectively, by the action of a .DELTA.9 desaturase.
Biosynthesis of Omega-3 and Omega-6 Fatty Acids
[0172] Simplistically, the metabolic process that converts LA to
GLA, DGLA and ARA (the .omega.-6 pathway) and ALA to STA, ETA, EPA
and DHA (the .omega.-3 pathway) involves elongation of the carbon
chain through the addition of carbon atoms and desaturation of the
molecule through the addition of double bonds. This requires a
series of special desaturation and elongation enzymes present in
the endoplasmic reticulim membrane, hereinafter referred to as
"PUFA biosynthetic pathway enzymes".
[0173] More specifically, "PUFA biosynthetic pathway enzymes" will
refer to any of the following enzymes (and genes which encode said
enzymes) associated with the biosynthesis of a PUFA, including: a
.DELTA.4 desaturase, a .DELTA.5 desaturase, a .DELTA.6 desaturase,
a .DELTA.12 desaturase, a .DELTA.15 desaturase, a .DELTA.17
desaturase, a .DELTA.9 desaturase and/or an elongase. For further
clarity within the present disclosure, the term "desaturase" refers
to a polypeptide component of a multi-enzyme complex that can
desaturate one or more fatty acids to produce a mono- or
polyunsaturated fatty acid or precursor of interest. Thus, despite
use of the omega-reference system to refer to specific fatty acids,
it is more convenient to indicate the activity of a desaturase by
counting from the carboxyl end of the source using the
delta-system. For example, a .DELTA.17 desaturase will desaturate a
fatty acid between the 17.sup.th and 18.sup.th carbon atom numbered
from the carboxyl-terminal end of the molecule and can, for
example, catalyze the conversion of ARA to EPA and/or DGLA to ETA.
In contrast, the term "elongase" refers to a polypeptide component
of a multi-enzyme complex that can elongate a fatty acid carbon
chain to produce a mono- or polyunsaturated fatty acid that is 2
carbons longer than the fatty acid source that the elongase acts
upon. This process of elongation occurs in a multi-step mechanism
in association with fatty acid synthase, whereby CoA is the acyl
carrier (Lassner et al., The Plant Cell 8:281-292 (1996)). Briefly,
malonyl-CoA is condensed with a long-chain acyl-CoA to yield
CO.sub.2 and a .beta.-ketoacyl-CoA (where the acyl moiety has been
elongated by two carbon atoms). Subsequent reactions include
reduction to .beta.-hydroxyacyl-CoA, dehydration to an enoyl-CoA
and a second reduction to yield the elongated acyl-CoA.
[0174] Synthesis of .omega.-6 fatty acids occurs in the following
fashion: oleic acid (the first of the .omega.-6 fatty acids) is
converted to LA (18:2) by the action of a .DELTA.12 desaturase
(FIG. 10). Subsequent .omega.-6 fatty acids are produced as
follows: 1.) LA is converted to GLA by the activity of a .DELTA.6
desaturase; 2.) GLA is converted to DGLA by the action of an
elongase; and 3.) DGLA is converted to ARA by the action of a
.DELTA.5 desaturase. In contrast, .omega.-3 fatty acids are all
derived from linoleic acid (LA). Specifically: 1.) LA is converted
to ALA by the action of a .DELTA.15 desaturase; 2.) ALA is
converted to STA by the activity of a .DELTA.6 desaturase; 3.) STA
is converted to ETA by the activity of an elongase; and 4.) ETA is
converted to EPA by the activity of a .DELTA.5 desaturase.
Alternatively, ETA and EPA can be produced from DGLA and ARA,
respectively, by the activity of a .DELTA.17 desaturase. EPA can be
further converted to DHA by the activity of an elongase and a
.DELTA.4 desaturase.
Production of PUFAs
[0175] As will be obvious to one skilled in the art, the particular
functionalities required to be introduced into a host organism for
production of a particular PUFA final product will depend on the
host cell (and its native PUFA profile and/or desaturase profile),
the availability of substrate and the desired end product(s). As
shown in FIG. 10, LA, GLA, DGLA, ARA, ALA, STA, ETA, EPA, DPA and
DHA may all be produced in oleaginous yeast, by introducing various
combinations of the following PUFA enzyme functionalities: a
.DELTA.4 desaturase, a .DELTA.5 desaturase, a .DELTA.6 desaturase,
a .DELTA.12 desaturase, a .DELTA.15 desaturase, a .DELTA.17
desaturase, a .DELTA.9 desaturase, and/or an elongase. One skilled
in the art will be able to identify various candidate genes
encoding each of the above enzymes, according to publicly available
literature (e.g., GenBank), the patent literature, and experimental
analysis of microorganisms having the ability to produce PUFAs.
Thus, a variety of desaturases and elongases are suitable as coding
regions of interest in the present invention. These coding regions
of interest could be operably linked to the GPD and/or GPM
promoters of the present invention or mutant promoters thereof, and
used as chimeric genes for expression of various .omega.-6 and
.omega.-3 fatty acids, using techniques well known to those skilled
in the art (see, for example co-pending U.S. patent application
Ser. No. 10/840,579, herein incorporated entirely by reference). As
such, the invention provides a method for the production of
.omega.-3 and/or .omega.-6 fatty acids comprising: [0176] a)
providing a transformed oleaginous yeast host cell comprising a
chimeric gene, comprising: [0177] 1) a promoter region of a gene
selected from the group consisting of: the promoter region of a gpm
gene and the promoter region of a gpd gene; and [0178] 2) a coding
region of interest expressible in the oleaginous yeast encoding an
enzyme of a functional .omega.-3/.omega.-6 fatty acid biosynthetic
pathway; wherein the promoter region and coding region are operably
linked; and [0179] (b) contacting the host cell of step (a) under
suitable growth conditions whereby one or more .omega.-3 or
.omega.-6 fatty acids are produced. In preferred embodiments, the
nucleic acid sequence of the promoter region is selected from the
group consisting of: SEQ ID NOs:23, 27, 43 and 44, and subsequences
and mutant promoters thereof; and the coding region of interest is
any desaturase or elongase suitable for expression in the
oleaginous yeast for the production of .omega.-3 or .omega.-6 fatty
acids.
[0180] For production of the greatest and the most economical yield
of PUFAs, the transformed oleaginous yeast host cell is grown under
conditions that optimize desaturase and elongase activities by
optimizing expression of the chimeric genes of the present
invention, wherein these chimeric genes comprise a promoter region
of a gpm or gpd gene and a coding region of interest encoding a
PUFA biosynthetic pathway enzyme.
[0181] In the fermentation media, particular attention is given to
several metal ions (e.g., Mn.sup.+2, Co+.sup.2, Zn.sup.+2,
Mg.sup.+2) that promote synthesis of lipids and PUFAs (Nakahara, T.
et al. Ind. Appl. Single Cell Oils, D. J. Kyle and R. Colin, eds.
pp 61-97 (1992)).
[0182] The preferred "fermentable carbon source" for production of
oleaginous yeast expressing various .omega.-6 and .omega.-3 fatty
acids will include, but is not limited to: monosaccharides,
oligosaccharides, polysaccharides, alkanes, fatty acids, esters of
fatty acids, monoglycerides, diglycerides, triglycerides, carbon
dioxide, methanol, formaldehyde, formate and carbon-containing
amines.
[0183] Typically, accumulation of high levels of PUFAs in
oleaginous yeast cells requires a two-stage process, since the
metabolic state must be "balanced" between growth and
synthesis/storage of fats. Thus, most preferably, a two-stage
fermentation process is necessary for the production of PUFAs in
oleaginous yeast. In this approach, the first stage of the
fermentation is dedicated to the generation and accumulation of
cell mass and is characterized by rapid cell growth and cell
division. In the second stage of the fermentation, it is preferable
to establish conditions of nitrogen deprivation in the culture to
promote high levels of lipid accumulation. The effect of this
nitrogen deprivation is to reduce the effective concentration of
AMP in the cells, thereby reducing the activity of the
NAD-dependent isocitrate dehydrogenase of mitochondria. When this
occurs, citric acid will accumulate, thus forming abundant pools of
acetyl-CoA in the cytoplasm and priming fatty acid synthesis. Thus,
this phase is characterized by the cessation of cell division
followed by the synthesis of fatty acids and accumulation of oil.
Although cells are typically grown at about 30.degree. C., some
studies have shown increased synthesis of unsaturated fatty acids
at lower temperatures (Yongmanitchai and Ward, Appl. Environ.
Microbiol. 57:419-25 (1991)). Based on process economics, this
temperature shift should likely occur after the first phase of the
two-stage fermentation, when the bulk of the organisms' growth has
occurred.
Purification of PUFAs
[0184] The PUFAs produced in a host microorganism as described
herein may be found as free fatty acids or in esterified forms such
as acylglycerols, phospholipids, sulfolipids or glycolipids, and
may be extracted from the host cell through a variety of means
well-known in the art. One review of extraction techniques, quality
analysis and acceptability standards for yeast lipids is that of Z.
Jacobs (Critical Reviews in Biotechnology 12(5/6):463-491 (1992)).
A brief review of downstream processing is also available by A.
Singh and O. Ward (Adv. Appl. Microbiol. 45:271-312 (1997)).
[0185] In general, means for the purification of PUFAs may include
extraction with organic solvents, sonication, supercritical fluid
extraction (e.g., using carbon dioxide), saponification, and
physical means such as presses, or combinations thereof. Of
particular interest is extraction with methanol and chloroform in
the presence of water (E. G. Bligh & W. J. Dyer, Can. J.
Biochem. Physiol. 37:911-917 (1959)). Where desirable, the aqueous
layer can be acidified to protonate negatively-charged moieties and
thereby increase partitioning of desired products into the organic
layer. After extraction, the organic solvents can be removed by
evaporation under a stream of nitrogen. When isolated in conjugated
forms, the products may be enzymatically or chemically cleaved to
release the free fatty acid or a less complex conjugate of
interest, and can then be subject to further manipulations to
produce a desired end product. Desirably, conjugated forms of fatty
acids are cleaved with potassium hydroxide.
[0186] If further purification is necessary, standard methods can
be employed. Such methods may include extraction, treatment with
urea, fractional crystallization, HPLC, fractional distillation,
silica gel chromatography, high-speed centrifugation or
distillation, or combinations of these techniques. Protection of
reactive groups, such as the acid or alkenyl groups, may be done at
any step through known techniques (e.g., alkylation or iodination).
Methods used include methylation of the fatty acids to produce
methyl esters. Similarly, protecting groups may be removed at any
step. Desirably, purification of fractions containing GLA, STA,
ARA, DHA and EPA may be accomplished by treatment with urea and/or
fractional distillation.
EXAMPLES
[0187] The present invention is further defined in the following
Examples. It should be understood that these Examples, while
indicating preferred embodiments of the invention, are given by way
of illustration only. From the above discussion and these Examples,
one skilled in the art can ascertain the essential characteristics
of this invention, and without departing from the spirit and scope
thereof, can make various changes and modifications of the
invention to adapt it to various usages and conditions.
General Methods
[0188] Standard recombinant DNA and molecular cloning techniques
used in the Examples are well known in the art and are described
by: 1.) Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular
Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold
Spring Harbor, N.Y. (1989) (hereinafter "Maniatis"); 2.) T. J.
Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene
Fusions; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.
(1984); and 3.) Ausubel, F. M. et al., Current Protocols in
Molecular Biology, published by Greene Publishing Assoc. and
Wiley-Interscience (1987).
[0189] Materials and methods suitable for the maintenance and
growth of microbial cultures are well known in the art. Techniques
suitable for use in the following examples may be found as set out
in Manual of Methods for General Bacteriology (Phillipp Gerhardt,
R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A.
Wood, Noel R. Krieg and G. Briggs Phillips, Eds), American Society
for Microbiology: Washington, D.C. (1994)); or by Thomas D. Brock
in Biotechnology: A Textbook of Industrial Microbiology, 2.sup.nd
ed., Sinauer Associates: Sunderland, Mass. (1989). All reagents,
restriction enzymes and materials used for the growth and
maintenance of microbial cells were obtained from Aldrich Chemicals
(Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL
(Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.),
unless otherwise specified.
[0190] A leucine autotrophic strain of Yarrowia lipolytica was
purchased from the American Type Culture Collection (Rockville,
Md.; ATCC #76982) and used for functional assays. Y. lipolytica
strains were usually grown at 28.degree. C. on YPD agar (1% yeast
extract, 2% bactopeptone, 2% glucose, 2% agar). For selection of
transformants, minimal medium (0.17% yeast nitrogen base (DIFCO
Laboratories) without ammonium sulfate or amino acids, 2% glucose,
0.1% proline, pH 6.1) was used. Supplements of adenine, leucine,
lysine and/or uracil were added to a final concentration of
0.01%.
[0191] General molecular cloning was performed according to
standard methods (Sambrook et al., supra). Oligonucleotides were
synthesized by Sigma-Genosys (Spring, Tex.). Site-directed
mutagenesis was performed using Stratagene's QuikChange.TM.
Site-Directed Mutagenesis kit (San Diego, Calif.), per the
manufacturer's instructions. When polymerase chain reaction (PCR)
or site-directed mutagenesis was involved in subcloning, the
constructs were sequenced to confirm that no errors had been
introduced to the sequence. PCR products were cloned into Promega's
pGEM-T-easy vector (Madison, Wis.).
[0192] Manipulations of genetic sequences were accomplished using
the suite of programs available from the Genetics Computer Group
Inc. (Wisconsin Package Version 9.0, Genetics Computer Group (GCG),
Madison, Wis.). The GCG program "Pileup" was used with the gap
creation default value of 12, and the gap extension default value
of 4. The GCG "Gap" or "Bestfit" programs were used with the
default gap creation penalty of 50 and the default gap extension
penalty of 3. Unless otherwise stated, in all other cases GCG
program default parameters were used.
[0193] The meaning of abbreviations is as follows: "sec" means
second(s), "min" means minute(s), "h" means hour(s), "d" means
day(s), ".mu.L" means microliter(s), "mL" means milliliter(s), "L"
means liter(s), ".mu.M" means micromolar, "mM" means millimolar,
"M" means molar, "mmol" means millimole(s), ".mu.mole" mean
micromole(s), "g" means gram(s), ".mu.g" means microgram(s), "ng"
means nanogram(s), "U" means unit(s), "bp" means base pair(s) and
"kB" means kilobase(s).
Example 1
Isolation of a Portion of the Yarrowia lipolytica GPD
[0194] The present Example describes the identification of a
portion of the Yarrowia lipolytica gene encoding GPD (SEQ ID NOs:11
and 12), by use of primers derived from conserved regions of other
GPD sequences.
[0195] A comparison of the various protein sequences encoding GPD
genes from Saccharomyces cerevisiae (GenBank Accession No.
CAA24607; SEQ ID NO:1), Schizosaccharomyces pombe (GenBank
Accession No. NP.sub.--595236; SEQ ID NO:2), Aspergillus oryzae
(GenBank Accession No. AAK08065; SEQ ID NO:3), Paralichthys
olivaceus (GenBank Accession No. BAA88638; SEQ ID NO:4), Xenopus
laevis (GenBank Accession No. P51469; SEQ ID NO:5), and Gallus
gallus (GenBank Accession No. DECHG3; SEQ ID NO:6) showed that
there were several stretches of conserved amino acid sequence
between the 6 different organisms (FIGS. 1A and 1B). Thus, two
degenerate oligonucleotides (shown below), corresponding to the
conserved `KYDSTHG` (SEQ ID NO:7) and `TGAAKAV` (SEQ ID NO:8) amino
acid sequences, respectively, were designed and used to amplify a
portion of the coding region of GPD from Y. lipolytica:
TABLE-US-00002 Degenerated oligonucleotide YL193 (SEQ ID NO:9)
AAGTACGAYTCBACYCAYGG Degenerated oligonucleotide YL194 (SEQ ID
NO:10) ACRGCCTTRGCRGCDCCRGT [Note: The nucleic acid degeneracy code
used for SEQ ID NOs:9 and 10 was as follows: R = A/G; Y = C/T; B
=C/G/T; and D = A/G/T.]
Based on the full-length sequences of the GPD sequences of FIG. 1,
it was hypothesized that the Yarrowia lipolytica GPD gene amplified
as described above would be missing .about.50 amino acids from its
N-terminus and about .about.115 amino acids from its
C-terminus.
[0196] The PCR amplification was carried out in a 50 .mu.l total
volume comprising:
[0197] PCR buffer (containing 10 mM KCl, 10 mM
(NH.sub.4).sub.2SO.sub.4, 20 mM Tris-HCl (pH 8.75), 2 mM
MgSO.sub.4, 0.1% Triton X-100), 100 .mu.g/mL BSA (final
concentration), 200 .mu.M each deoxyribonucleotide triphosphate, 10
.mu.mole of each primer, 50 ng genomic DNA of Y. lipolytica (ATCC
#76982) and 1 .mu.l of Taq DNA polymerase (Epicentre Technologies).
The thermocycler conditions were set for 35 cycles at 95.degree. C.
for 1 min, 56.degree. C. for 30 sec, and 72.degree. C. for 1 min,
followed by a final extension at 72.degree. C. for 10 min.
[0198] The PCR products were purified using a Qiagen PCR
purification kit (Valencia, Calif.), and then further purified
following gel electrophoresis in 1% (w/v) agarose. Subsequently,
the PCR products were cloned into the pGEM-T-easy vector (Promega,
Madison, Wis.). The ligated DNA was used to transform cells of E.
coli DH5.alpha. and transformants were selected on LB agar
containing ampicillin (100 .mu.g/mL). Analysis of the plasmid DNA
from one transformant confirmed the presence of a plasmid of the
expected size, and designated as "pT-GPD".
[0199] Sequence analyses showed that pT-GPD contained a 507 bp
fragment (SEQ ID NO:11). Identity of this sequence was determined
by conducting BLAST (Basic Local Alignment Search Tool; Altschul,
S. F., et al., J. Mol. Biol. 215:403-410 (1993)) searches for
similarity to sequences contained in the BLAST "nr" database
(comprising all non-redundant GenBank CDS translations, sequences
derived from the 3-dimensional structure Brookhaven Protein Data
Bank, the SWISS-PROT protein sequence database, EMBL, and DDBJ
databases). Similarity to all publicly available DNA sequences
contained in the "nr" database was determined using the BLASTN
algorithm provided by the National Center for Biotechnology
Information (NCBI). The DNA sequence was translated in all reading
frames and compared for similarity to all publicly available
protein sequences contained in the "nr" database, using the BLASTX
algorithm (Gish, W. and States, D. J. Nature Genetics 3:266-272
(1993)) provided by the NCBI. The results of the BLAST comparison
summarizing the sequence to which SEQ ID NO:11 has the most
similarity are reported according to the % identity, % similarity,
and Expectation value. "% Identity" is defined as the percentage of
amino acids that are identical between the two proteins. "%
Similarity" is defined as the percentage of amino acids that are
identical or conserved between the two proteins. "Expectation
value" estimates the statistical significance of the match,
specifying the number of matches, with a given score, that are
expected in a search of a database of this size absolutely by
chance.
[0200] The 507 bp of pT-GPD was found to encode 169 amino acids
(SEQ ID NO:12). This amino acid fragment had 77% identity and 84%
similarity (FIG. 2) with the GPD protein sequence of fission yeast
(GenBank Accession No. NP.sub.--595236), with an expectation value
of 6e-68. The Yarrowia sequence possessed the `KYDSTHG` (SEQ ID
NO:7) and `TGAAKAV` (SEQ ID NO:8) amino acid sequences
(corresponding to the degenerate primers used to amplify the
fragment) at its N- and C-termini. Further sequence comparison of
this partial GPD sequence determined that it also shared about 72%
and 74% identity with the GPD proteins from chick (GenBank
Accession No. DECHG3) and frog (GenBank Accession No. P51469),
respectively (FIG. 2).
Example 2
Identification of the Yarrowia lipolytica GPM
[0201] The present Example describes the identification of the
Yarrowia lipolytica gene encoding GPM, by use of a S. cerevisiae
GPM protein sequence as a query sequence against a Y. lipolytica
genomic database.
[0202] Specifically, the S. cerevisiae GPM protein sequence
(GenBank Accession No. NP.sub.--012770; SEQ ID NO:13) was used in
BLAST searches (as described in Example 1) against the public Y.
lipolytica database of the "Yeast project Genolevures" (Center for
Bioinformatics, LaBRI, Talence Cedex, France.
[0203] One contig ("Contig 2217"; SEQ ID NO:14) was identified that
encoded GPM in Y. lipolytica. Contig 2217 is 1049 bp in length,
although 5 nucleotide positions had ambiguous sequence (having an
"n" at nucleotide position 1020, "y" at positions 39, 62, 331; and
a "m" at position 107). The DNA sequence of Contig 2217 was
translated in all reading frames and compared for similarity to all
publicly available protein sequences contained in the "nr" database
using the BLASTX algorithm (as described in Example 1). Based on
these DNA and protein sequence analyses, it was determined that:
[0204] The GPM translation initiation codon `ATG` was at bp 388
within SEQ ID NO:14; thus, Contig 2217 possessed about 388 bp
upstream sequence relative to the `ATG` codon; and [0205] Contig
2217 was missing one base at nucleotide position 470, which
resulted in a frame shift. The deduced coding region sequence of
GPM that corresponded to Contig 2217 was 651 bp in length (SEQ ID
NO:15) and the protein sequence was encoded by SEQ ID NO:16. This
216 amino acid protein had 71% identity, 82% similarity, and an
expectation value of 3e-81 with the GPM protein sequence of S.
cerevisiae (GenBank Accession No. NP.sub.--012770; Goffeau, A., et
al., Science 274(5287): 546 (1996)) (FIG. 3).
Example 3
Isolation of the 5' Upstream Regions of the qpd and qpm Genes from
Yarrowia lipolytica
[0206] To isolate the GPD and GPM promoter regions from the genes
identified in Examples 1 and 2, a genome-walking technique
(TOPO.RTM. Walker Kit, Invitrogen, CA) was utilized.
[0207] Briefly, genomic DNA of Y. lipolytica was digested with
KpnI, SacI, SphI or PacI, and dephosphorylated with Calf Intestinal
Alkaline Phosphatase (CIP), separately. Primer extension reactions
were then carried out individually using the dephosphorylated DNA
as template and one of the following oligonucleotides as primer:
YL206 (SEQ ID NO:17) for GPD and YL196 (SEQ ID NO:18) for GPM. The
primer extended products were linked with TOPO.RTM. linker and used
as templates for the first PCR reactions using primers of LinkAmp
Primer1 and a second appropriate oligonucleotide. Specifically,
YL207 (SEQ ID NO:19) was used as the second primer targeted for the
upstream promoter region of GPD and YL197 (SEQ ID NO:20) was used
as the second primer for PCR reactions targeted to the upstream GPM
promoter region. The PCR amplifications were carried out in a 50
.mu.l total volume, comprising: PCR buffer (containing 10 mM KCl,
10 mM (NH.sub.4).sub.2SO.sub.4, 20 mM Tris-HCl (pH 8.75), 2 mM
MgSO.sub.4, 0.1% Triton X-100), 100 .mu.g/mL BSA (final
concentration), 200 .mu.M each deoxyribonucleotide triphosphate, 10
.mu.mole of each primer and 1 .mu.l of Taq DNA polymerase
(Epicentre Technologies). The thermocycler conditions were set for
35 cycles at 95.degree. C. for 1 min, 56.degree. C. for 30 sec, and
72.degree. C. for 1 min, followed by a final extension at
72.degree. C. for 10 min.
[0208] Second PCR reactions were then carried out using the first
PCR product as template and primers of LinkAmp primer 2 and the
appropriate oligonucleotide. Specifically, the first PCR product
for GPD was used as template in a reaction comprising LinkAmp
primer 2 and YL208 (SEQ ID NO:21); in contrast, the first PCR
product for GPM was used as template in a reaction comprising
LinkAmp primer 2 and YL198 (SEQ ID NO:22). The PCR amplifications
were carried out as described above.
[0209] The PCR products comprising the 5' upstream regions of the
GPD and GPM genes were each individually purified using a Qiagen
PCR purification kit, followed by gel electrophoresis in 1% (w/v)
agarose. Products were then cloned into the pGEM-T-easy vector
(Promega, Madison, Wis.). The ligated DNA was used to transform E.
coli DH5.alpha. and transformants were selected on LB agar
containing ampicillin (100 .mu.g/mL).
[0210] Analysis of the plasmid DNA from one transformant comprising
the 5' upstream region of the gpd gene confirmed the presence of
the expected plasmid, designated "pT-GPDP". Sequence analyses
showed that pT-GPDP contained a fragment of 1848 bp (SEQ ID NO:23),
which included 1525 bp of 5' upstream sequence from the nucleotide
`A` (designated as +1) of the translation initiation codon `ATG` of
the GPD gene. A complete assembly of overlapping SEQ ID NOs:23 and
11 yielded a single contig comprising 1525 bp upstream of the GPD
initiation codon and 791 bp of the gene (SEQ ID NO:24; FIG. 4).
Further analysis of the partial gene sequence (+1 to +791) revealed
the presence of an intron (base pairs +49 to +194). Thus, the
partial cDNA sequence encoding the GPD gene in Y. lipolytica is
only 645 bp in length (SEQ ID NO:25) and the corresponding protein
sequence (SEQ ID NO:26) is 215 amino acids. The protein was
compared via BLAST analysis for similarity to all publicly
available protein sequences (as described in Example 1). Based on
this analysis, it was determined that the partial GPD protein was
most similar to the GPD of Cryotococcus cyrvatus (GenBank Accession
No's Q9Y796 and AAD25080) (81% identical).
[0211] Analysis of the plasmid DNA from one transformant comprising
the 5' upstream region of the gpm gene confirmed the presence of
the expected plasmid, designated "pT-GPML". Sequence analyses
showed that pT-GPML contained a fragment of 953 bp (SEQ ID NO:27).
This clone possessed 875 bp of 5' upstream sequence from the
translation initiation codon of the GPM gene. Assembly of DNA
corresponding to overlapping SEQ ID NOs:27 and 15 yielded a single
contig of DNA represented as SEQ ID NO:28 (FIG. 5). This contig
therefore contained the -875 to +662 region of the GPM gene,
wherein the `A` position of the `ATG` translation initiation codon
was designated as +1.
Example 4
Synthesis of pY5-30
[0212] The present Example describes the synthesis of pY5-30,
comprising a TEF::GUS::XPR chimeric gene. This was required for
comparative studies investigating the promoter activity of TEF, GPD
and GPM, wherein constructs comprising each promoter and a reporter
gene were prepared and analyzed (Examples 5-7). Specifically, the
reporter was the E. coli gene encoding-glucuronidase (GUS;
Jefferson, R. A. Nature. 342(6251):837-838 (1989)).
Amplification of the GUS Coding Region
[0213] The GUS coding region was amplified using pBI101 (Jefferson,
R. A et al., EMBO J. 6:3901-3907 (1987)) as template and
oligonucleotides YL33 (SEQ ID NO:29) and YL34 (SEQ ID NO:30) as
primers. The PCR amplification was carried out in a 50 .mu.l total
volume comprising: PCR buffer (containing 10 mM KCl, 10 mM
(NH.sub.4).sub.2SO.sub.4, 20 mM Tris-HCl (pH 8.75), 2 mM
MgSO.sub.4, 0.1% Triton X-100), 100 .mu.g/mL BSA (final
concentration), 200 .mu.M each deoxyribonucleotide triphosphate, 10
pmole of each primer and 1 .mu.l of Pfu DNA polymerase (Stratagene,
San Diego, Calif.). The thermocycler conditions were set for 35
cycles at 95.degree. C. for 1 min, 56.degree. C. for 30 sec,
72.degree. C. for 1 min, followed by a final extension at
72.degree. C. for 10 min. The PCR products were digested with NcoI
and PacI.
Synthesis of Plasmid pY5-10
[0214] The plasmid pY5, a derivative of pIN.DELTA.532 (a gift from
Dr. Claude Gaillardin, Insitut National Agronomics, Centre de
biotechnologie Agro-lndustrielle, laboratoire de Genetique
Moleculaire et Cellularie INRA-CNRS, F-78850 Thiverval-Grignon,
France), was constructed for expression of heterologous genes in
Yarrowia lipolytica, as diagrammed in FIG. 6. The
partially-digested 3598 bp EcoRI fragment containing the ARS18
sequence and LEU2 gene of pIN.DELTA.532 was subcloned into the
EcoRI site of pBluescript (Strategene, San Diego, Calif.) to
generate pY2.
[0215] The TEF promoter (Muller S., et al. Yeast, 14:1267-1283
(1998)) was amplified from Y. lipolytica genomic DNA by PCR using
TEF5' (SEQ ID NO:31) and TEF3' (SEQ ID NO:32) as primers. PCR
amplification was carried out in a 50 .mu.l total volume
containing: 100 ng Yarrowia genomic DNA, PCR buffer (containing 10
mM KCl, 10 mM (NH.sub.4).sub.2SO.sub.4, 20 mM Tris-HCl (pH 8.75), 2
mM MgSO.sub.4, 0.1% Triton X-100), 100 .mu.g/mL BSA (final
concentration), 200 .mu.M each deoxyribonucleotide triphosphate, 10
pmole of each primer and 1 .mu.l of Pfu Turbo DNA polymerase
(Stratagene, San Diego, Calif.). Amplification was carried out as
follows: initial denaturation at 95.degree. C. for 3 min, followed
by 35 cycles of the following: 95.degree. C. for 1 min, 56.degree.
C. for 30 sec, 72.degree. C. for 1 min. A final extension cycle of
72.degree. C. for 10 min was carried out, followed by reaction
termination at 4.degree. C. The 418 bp PCR product was ligated into
pCR-Blunt to generate pIP-tef. The BamHI/EcoRV fragment of pIP-tef
was subcloned into the BamHI/SmaI sites of pY2 to generate pY4.
[0216] The XPR2 transcriptional terminator was amplified by PCR
using pIN.DELTA.532 as template and XPR5' (SEQ ID NO:33) and XPR3'
(SEQ ID NO:34) as primers. The PCR amplification was carried out in
a 50 .mu.l total volume, using the components and conditions
described above. The 179 bp PCR product was digested with SacII and
then ligated into the SacII site of pY4 to generate pY5. Thus, pY5
(shown in FIG. 6) contained: a Yarrowia autonomous replication
sequence (ARS18); a ColE1 plasmid origin of replication; an
ampicillin-resistance gene (AmpR) for selection in E. coli; a
Yarrowia LEU2 gene encoding isopropylmalate isomerase, for
selection in Yarrowia; the translation elongation promoter ("TEF
P"), for expression of heterologous genes in Yarrowia; and the
extracellular protease gene terminator (XPR2) for transcriptional
termination of heterologous gene expression in Yarrowia.
[0217] Plasmid pY5-10 (FIG. 7A) was constructed as a derivative of
pY5. First, pY5-4 (FIG. 6) was constructed by three rounds of
site-directed mutagenesis using pY5 as template. A NcoI site
located inside the LEU2 reporter gene was eliminated from pY5 using
oligonucleotides YL1 and YL2 (SEQ ID NOs:35 and 36) to generate
pY5-1. A NcoI site was introduced into pY5-1 between the TEF
promoter and XPR transcriptional terminator by site-directed
mutagenesis using oligonucleotides YL3 and YL4 (SEQ ID NOs:37 and
38) to generate pY5-2. A PacI site was then introduced into pY5-2
between the TEF promoter and XPR transcriptional terminator using
oligonucleotides YL23 and YL24 (SEQ ID NOs:39 and 40) to generate
pY5-4. Finally, a SalI site was introduced into pY5-4 between the
TEF promoter and the LEU2 gene by site-directed mutagenesis using
oligonucleotides YL9 (SEQ ID NO:41) and YL10 (SEQ ID NO:42) as
primers to generate pY5-10 (FIG. 7A).
Synthesis of Plasmid pY5-30
[0218] Plasmid pY5-30 (FIG. 7B), comprising a TEF::GUS::XPR
chimeric gene, was synthesized by inserting the NcoI/PacI PCR
product comprising the GUS coding region (supra) into NcoI/PacI
digested pY5-10.
Example 5
Synthesis of pYZGDG and pYZGMG
[0219] The present Example describes the synthesis of PYZGDG
(comprising a GPD::GUS::XPR chimeric gene) and PYZGMG (comprising a
GPM::GUS::XPR chimeric gene). Synthesis of these plasmids first
required identification and amplification of the putative GPD and
GPM promoter regions. Then, each putative promoter region was
cloned into a derivative of pY5-30.
Identification and Amplification of Putative Promoter Regions
[0220] After the isolation of the 5' upstream sequence of the GPD
and GPM genes by genome walking, the translation start site was
identified by looking for the consensus motif around the
translation initiation `ATG` codon and by comparison of the
translated coding region of the Yarrowia GPD and GPM genes with the
GPD and GPM genes, respectively, from other organisms. The region
upstream of the genes' `ATG` start site was used to identify
putative promoter regions.
[0221] Thus, the nucleotide region between the -968 position and
the `ATG` translation initiation site of the GPD gene (wherein the
`A` nucleotide of the `ATG` translation initiation codon was
designated as +1) was determined to contain the putative promoter
region ("GPDPro", provided as SEQ ID NO:43). In like manner, the
nucleotide region between the -875 position and the `ATG`
translation initiation site of the GPM gene was determined to
contain the putative promoter region ("GPMLPro", provided as SEQ ID
NO:44).
[0222] The putative promoter regions identified above were
amplified by PCR. Specifically, GPDPro was amplified with
oligonucleotides YL211 (SEQ ID NO:45) and YL212 (SEQ ID NO:46) as
primers and pT-GPDP (Example 3) as template. GPMLPro was amplified
with oligonucleotides YL203 (SEQ ID NO:47) and YL204 (SEQ ID NO:48)
as primers and pT-GPML (Example 3) as template. The PCR
amplifications were carried out in a 50 .mu.l total volume,
comprising: PCR buffer (containing 10 mM KCl, 10 mM
(NH.sub.4).sub.2SO.sub.4, 20 mM Tris-HCl (pH 8.75), 2 mM
MgSO.sub.4, 0.1% Triton X-100), 100 .mu.g/mL BSA (final
concentration), 200 .mu.M each deoxyribonucleotide triphosphate, 10
pmole of each primer and 1 .mu.l of Pfu DNA polymerase (Stratagene,
San Diego, Calif.). The thermocycler conditions were set for 35
cycles at 95.degree. C. for 1 min, 56.degree. C. for 30 sec,
72.degree. C. for 1 min, followed by a final extension at
72.degree. C. for 10 min.
[0223] The PCR products were then purified using a Qiagen PCR
purification kit and subjected to the following restriction
digestions and ligation reactions: [0224] GPDPro was completely
digested with SalI and then partially digested with NcoI. The
SalliNcoI fragment was purified following gel electrophoresis in 1%
(w/v) agarose and ligated to NcoI/SalI digested pY5-30 vector
(Example 4) (wherein the NcoI/SalI digestion had excised the TEF
promoter from the pY5-30 vector backbone). [0225] GPMLPro was
digested with NcoI and SalI for 1 hr at 37.degree. C. and then
purified following gel electrophoresis in 1% (w/v) agarose. The
NcoI/SalII-digested PCR product was ligated to NcoI/SalI digested
pY5-30 vector. Ligated DNA from each reaction was then used to
individually transform E. coli DH5.alpha.. Transformants were
selected on LB agar containing ampicillin (100 .mu.g/m L).
[0226] Analysis of the plasmid DNA from one transformant containing
GPDPro confirmed the presence of the expected plasmid, designated
"PYZGDG" (FIG. 7C). Thus, this plasmid contained a chimeric gene
comprising a GPD promoter, GUS reporter gene and XPR
terminator.
[0227] Analysis of the plasmid DNA from one transformant containing
GPMLPro confirmed the presence of the expected plasmid, designated
"PYZGMG", and comprising a GPM::GUS::XPR chimeric gene (FIG.
7D).
Example 6
Transformation of Y. lipolytica with pY5-30. pYZGDG and pYZGMG
[0228] The plasmids pY5-30 (Example 4; comprising a TEF::GUS::XPR
chimeric gene), PYZGDG (Example 5; comprising a GPD::GUS::XPR
chimeric gene) and PYZGMG (Example 5; comprising a GPM::GUS::XPR
chimeric gene) were transformed separately into Y. lipolytica ATCC
#76982 according to the method of Chen, D. C. et al. (Appl.
Microbiol. Biotechnol. 48(2):232-235 (1997)).
[0229] Briefly, a leucine auxotroph of Yarrowia was streaked onto a
YPD plate and grown at 30.degree. C. for approximately 18 hr.
Several large loopfuls of cells were scraped from the plate and
resuspended in 1 mL of transformation buffer containing: [0230]
2.25 mL of 50% PEG, average MW 3350; [0231] 0.125 mL of 2 M Li
acetate, pH 6.0; [0232] 0.125 mL of 2 M DTT; and [0233] 50 .mu.g
sheared salmon sperm DNA.
[0234] About 500 ng of plasmid DNA was incubated in 100 .mu.l of
resuspended cells, and maintained at 39.degree. C. for 1 hr with
vortex mixing at 15 min intervals. The cells were plated onto
minimal media plates lacking leucine and maintained at 30.degree.
C. for 2 to 3 days.
[0235] Using this technique, transformants were obtained that
contained pY5-30, PYZGDG and PYZGMG, respectively.
Example 7
Comparative Analysis of the TEF, GPD and GPM Promoter Activities in
Yarrowia lipolytica
[0236] The activity of the TEF, GPD and GPM promoters were
determined in Yarrowia lipolytica containing the pY5-30, PYZGDG and
PYZGMG constructs, each of which possessed a GUS reporter gene and
an XPR terminator. GUS activity in each expressed construct was
measured by histochemical and fluorometric assays (Jefferson, R. A.
Plant Mol. Biol. Reporter 5:387-405 (1987)).
GUS Activities, Determined by Histochemical Assay
[0237] Specifically, two Yarrowia lipolytica strains containing
plasmid pY5-30, two Yarrowia lipolytica strains containing plasmid
PYZGDG and two Yarrowia lipolytica strains containing plasmid
PYZGMG were each grown from single colonies in 3 mL minimal media
(20 g/L glucose, 1.7 g/L yeast nitrogen base without amino acids, 1
g/L L-proline, 0.1 g/L L-adenine, 0.1 g/L L-lysine, pH 6.1) at
30.degree. C. to an OD.sub.600.about.1.0. Then, 100 .mu.l of cells
were collected by centrifugation, resuspended in 100 .mu.l of
histochemical staining buffer and incubated at 30.degree. C.
[Staining buffer prepared by dissolving 5 mg of
5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) in 50 .mu.l
dimethyl formamide, followed by addition of 5 mL 50 mM NaPO.sub.4,
pH 7.0.]
[0238] The results of histochemical staining showed that the TEF
promoter in construct pY5-30, the GPD promoter in construct PYZGDG
and the GPM promoter in construct PYZGMG were all active. The GPD
promoter appeared to be much stronger than the TEF promoter (FIG.
8A), while the GPM promoter was at least as strong as the TEF
promoter (FIG. 8B).
GUS Activities, Determined by Fluorometric Assay
[0239] GUS activity was also assayed by fluorometric determination
of the production of 4-methylumbelliferone from the corresponding
substrate-glucuronide (Jefferson, R. A., supra).
[0240] Yarrowia lipolytica strains containing plasmids pY5-30,
PYZGDG and PYZGMG, respectively, were grown from single colonies in
3 mL minimal media (as described above) at 30.degree. C. to an
OD.sub.600.about.1.0. Then, the 3 mL cultures were each added to a
500 mL flask containing 50 mL minimal media and grown in a shaking
incubator at 30.degree. C. for about 24 hrs. The cells were
collected by centrifugation, resuspended in Promega Cell Lysis
Buffer and lysed using the BIO 101 Biopulverizer system (Vista,
Calif.). After centrifugation, the supernatants were removed and
kept on ice.
[0241] For each fluorometric assay, 100 .mu.l of extract was added
to 700 .mu.l of GUS assay buffer (2 mM
4-methylumbelliferyl-.beta.-D-glucuronide ("MUG") in extraction
buffer) and placed at 37.degree. C. Aliquots of 100 .mu.l were
taken at 0, 30 and 60 min time points and added to 900 .mu.l of
stop buffer (1 M Na.sub.2CO.sub.3). Each time point was read using
a Fluorimeter (CytoFluor R Series 4000, Framingham, Mass.) set to
an excitation wavelength of 360 nm and an emission wavelength of
455 nm. Total protein concentration of each sample was determined
using 10 .mu.l of extract and 200 .mu.l of BioRad Bradford reagent
(Bradford, M. M. Anal. Biochem. 72:248-254 (1976)). GUS activity
was expressed as nmoles of 4-MU per minute per mg of protein.
[0242] Results of these fluorometric assays are shown in FIG. 9.
Specifically,
[0243] FIG. 9A showed that the GPD promoter was 3 times stronger
than the bench-marker TEF promoter in Y. lipolytica; in contrast,
FIG. 9B showed that the GUS activity of the GPM promoter was about
110% as active as the bench-marker TEF promoter.
Example 8
Use of the GPD Promoter for .DELTA.15 Desaturase Expression in
Yarrowia lipolytica
[0244] The present Example describes the construction of a chimeric
gene comprising a GPD promoter, fungal .DELTA.15 desaturase and the
XPR terminator, and the expression of this gene in Y. lipolytica.
Since transformed host cells were able to produce ALA (while
wildtype Y. lipolytica do not possess any .DELTA.15 desaturase
activity), this confirms the ability of the GPD promoter to drive
expression of heterologous PUFA biosynthetic pathway enzymes in
oleaginous yeast cells such as Y. lipolytica.
Construction of Plasmid pY34, Comprising a GPD::Fml::XPR Chimeric
Gene
[0245] First, plasmid pY5-13 was constructed as a derivative of pY5
(from Example 4). Specifically, pY5-13 was constructed by 6 rounds
of site-directed mutagenesis using pY5 as template. Both SalI and
ClaI sites were eliminated from pY5 by site-directed mutagenesis
using oligonucleotides YL5 and YL6 (SEQ ID NOs:49 and 50) to
generate pY5-5. A SalI site was introduced into pY5-5 between the
LEU2 gene and the TEF promoter by site-directed mutagenesis using
oligonucleotides YL9 and YL10 (SEQ ID NOs:41 and 42) to generate
pY5-6. A PacI site was introduced into pY5-6 between the LEU2 gene
and ARS18 using oligonucleotides YL7 and YL8 (SEQ ID NOs:51 and 52)
to generate pY5-8. A NcoI site was introduced into pY5-8 around the
translation start codon of the TEF promoter using oligonucleotides
YL3 and YL4 (SEQ ID NOs:37 and 38) to generate pY5-9. The NcoI site
inside the LEU2 gene of pY5-9 was eliminated using YL1 and YL2
oligonucleotides (SEQ ID NOs:35 and 36), to generate pY5-12.
Finally, a BsiWI site was introduced into pY5-12 between the ColEI
and XPR region using oligonucleotides YL61 and YL62 (SEQ ID NOs:53
and 54) to generate pY5-13.
[0246] A purified SalliNcoI fragment comprising GPDPro (from
Example 5) was ligated to NcoI/SalI digested pY5-13 vector (wherein
the NcoI/SalI digestion had excised the TEF promoter from the
pY5-13 vector backbone) to yield "pY5-13GPD". Thus, pY5-13GPD
comprised a GPD promoter::XPR terminator expression cassette.
[0247] The Nco I site at the 3' end of the promoter fragment in
pY5-13GPD was converted to a Not I site to yield "pY5-13GPDN". For
this, the GPD promoter was re-amplified by PCR using GPDsense (SEQ
ID NO:55) and GPDantisense (SEQ ID NO:56) primers with a Not I
site. The resultant promoter fragment was digested with Sal I and
Not I and cloned into the Sal/NotI site of pY5-13 (thus removing
the TEF promoter) to produce pY5-13GPDN.
The ORF encoding the Fusarium moniliforme strain M-8114 .DELTA.15
desaturase (SEQ ID NO:57; see co-pending U.S. Provisional
Application No. 60/519,191) was PCR amplified using the cDNA clone
ffmlc.pK001.g23 (E.I. du Pont de Nemours and Co., Inc., Wilmington,
Del.) containing the full-length cDNA as the template and using
upper and lower primers P192 (SEQ ID NO:59) and P193 (SEQ ID
NO:60). The PCR was carried out in an Eppendorf Mastercycler
Gradient Cycler using Pfu polymerase, per the manufacturer's
recommendation. Amplification was carried out as follows: initial
denaturation at 95.degree. C. for 1 min, followed by 30 cycles of
denaturation at 95.degree. C. for 30 sec, annealing at 58.degree.
C. for 1 min, and elongation at 72.degree. C. for 1 min. A final
elongation cycle at 72.degree. C. for 10 min was carried out,
followed by reaction termination at 4.degree. C.
[0248] The correct-sized (ca. 1240 bp) fragment was obtained,
purified from an agarose gel using a Qiagen DNA purification kit,
digested with Not I and cloned into the Not I site between the GPD
promoter and XPR terminator of plasmid pY5-13GPDN. This resulted in
creation of plasmid "pY34", which contained a GPD::Fml::XPR
chimeric gene.
Expression of Plasmid pY34 (GPD::Fml::XPR) in Yarrowia
lipolytica
[0249] pY5 (vector alone control, from Example 4) and pY34
(GPDP::Fml::XPR) were each individually transformed into wild type
(WT) Yarrowia lipolytica ATCC #76892, using the transformation
procedure described in Example 6, and selected on Bio101
DOB/CSM-Leu plates.
[0250] Single colonies of wild type and transformant cells were
each grown in 3 mL minimal media (formulation/L: 20 g glucose, 1.7
g yeast nitrogen base, 1 g L-proline, 0.1 g L-adenine, 0.1 g
L-lysine, pH 6.1) at 30 C to an OD.sub.600.about.1.0. The cells
were harvested, washed in distilled water, speed vacuum dried and
subjected to direct trans-esterification and GC analysis.
Specifically, for fatty acid analysis cells were collected by
centrifugation and lipids were extracted as described in Bligh, E.
G. & Dyer, W. J. (Can. J. Biochem. Physiol. 37:911-917 (1959)).
Fatty acid methyl esters were prepared by transesterification of
the lipid extract with sodium methoxide (Roughan, G., and Nishida
I. Arch Biochem Biophys. 276(1):38-46 (1990)) and subsequently
analyzed with a Hewlett-Packard 6890 GC fitted with a
30-m.times.0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard) column. The
oven temperature was from 170.degree. C. (25 min hold) to 185 Cat
3.5.degree. C./min.
[0251] For direct base transesterification, Yarrowia culture (3 mL)
was harvested, washed once in distilled water, and dried under
vacuum in a Speed-Vac for 5-10 min. Sodium methoxide (100 .mu.l of
1%) was added to the sample, and then the sample was vortexed and
rocked for 20 min. After adding 3 drops of 1 M NaCl and 400 .mu.l
hexane, the sample was vortexed and spun. The upper layer was
removed and analyzed by GC as described above.
[0252] The fatty acid profile of wildtype Yarrowia and each of the
transformants are shown below in Table 2. Fatty acids are
identified as 16:0 (palmitate), 16:1 (palmitoleic acid), 18:0, 18:1
(oleic acid), 18:2 (LA) and 18:3 (ALA) and the composition of each
is presented as a % of the total fatty acids.
TABLE-US-00003 TABLE 2 Expression of Fusarium .DELTA.15 Desaturase
In Yarrowia lipolytica Y. lipolytica % % % % % % strain 16:0 16:1
18:0 18:1 18:2 ALA WT 12 9 1 34 44 0 WT + GPD:Fm1:XPR 10 10 1 37 7
31
[0253] The results above demonstrated that the GPD promoter is
suitable to drive expression of the .DELTA.15 desaturase, leading
to production of ALA in Yarrowia.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 60 <210> SEQ ID NO 1 <211> LENGTH: 332 <212>
TYPE: PRT <213> ORGANISM: Sacchromyces cerevisiae (Genbank
Accession No. CAA24607) <400> SEQUENCE: 1 Met Val Arg Val Ala
Ile Asn Gly Phe Gly Arg Ile Gly Arg Leu Val 1 5 10 15 Met Arg Ile
Ala Leu Ser Arg Pro Asn Val Glu Val Val Ala Leu Asn 20 25 30 Asp
Pro Phe Ile Thr Asn Asp Tyr Ala Ala Tyr Met Phe Lys Tyr Asp 35 40
45 Ser Thr His Gly Arg Tyr Ala Gly Glu Val Ser His Asp Asp Lys His
50 55 60 Ile Ile Val Asp Gly Lys Lys Ile Ala Thr Tyr Gln Glu Arg
Asp Pro 65 70 75 80 Ala Asn Leu Pro Trp Gly Ser Ser Asn Val Asp Ile
Ala Ile Asp Ser 85 90 95 Thr Gly Val Phe Lys Glu Leu Asp Thr Ala
Gln Lys His Ile Asp Ala 100 105 110 Gly Ala Lys Lys Val Val Ile Thr
Ala Pro Ser Ser Thr Ala Pro Met 115 120 125 Phe Val Met Gly Val Asn
Glu Val Lys Tyr Thr Ser Asp Leu Lys Ile 130 135 140 Val Ser Asn Ala
Ser Cys Thr Thr Asn Cys Leu Ala Pro Leu Ala Lys 145 150 155 160 Val
Ile Asn Asp Ala Phe Gly Ile Glu Glu Gly Leu Met Thr Thr Val 165 170
175 His Ser Leu Thr Ala Thr Gln Lys Thr Val Asp Gly Pro Ser His Lys
180 185 190 Asp Trp Arg Gly Gly Arg Thr Ala Ser Gly Asn Ile Ile Pro
Ser Ser 195 200 205 Thr Gly Ala Ala Lys Ala Val Gly Lys Val Leu Pro
Glu Leu Gln Gly 210 215 220 Lys Leu Thr Gly Met Ala Phe Arg Val Pro
Thr Val Asp Val Ser Val 225 230 235 240 Val Asp Leu Thr Val Lys Leu
Asp Lys Glu Thr Thr Tyr Asp Glu Ile 245 250 255 Lys Lys Val Val Lys
Ala Ala Ala Glu Gly Lys Leu Lys Gly Val Leu 260 265 270 Gly Tyr Thr
Glu Asp Ala Val Val Ser Ser Asp Phe Leu Gly Asp Ser 275 280 285 His
Ser Ser Ile Phe Asp Ala Ser Ala Gly Ile Gln Leu Ser Pro Lys 290 295
300 Phe Val Lys Leu Val Ser Trp Tyr Asp Asn Glu Tyr Gly Tyr Ser Thr
305 310 315 320 Arg Val Val Asp Leu Val Glu His Ile Ala Lys Ala 325
330 <210> SEQ ID NO 2 <211> LENGTH: 335 <212>
TYPE: PRT <213> ORGANISM: Schizosaccharomyces pombe (Genbank
Accession No. NP_595236) <400> SEQUENCE: 2 Met Ala Ile Pro
Lys Val Gly Ile Asn Gly Phe Gly Arg Ile Gly Arg 1 5 10 15 Ile Val
Leu Arg Asn Ala Ile Leu Thr Gly Lys Ile Gln Val Val Ala 20 25 30
Val Asn Asp Pro Phe Ile Asp Leu Asp Tyr Met Ala Tyr Met Phe Lys 35
40 45 Tyr Asp Ser Thr His Gly Arg Phe Glu Gly Ser Val Glu Thr Lys
Gly 50 55 60 Gly Lys Leu Val Ile Asp Gly His Ser Ile Asp Val His
Asn Glu Arg 65 70 75 80 Asp Pro Ala Asn Ile Lys Trp Ser Ala Ser Gly
Ala Glu Tyr Val Ile 85 90 95 Glu Ser Thr Gly Val Phe Thr Thr Lys
Glu Thr Ala Ser Ala His Leu 100 105 110 Lys Gly Gly Ala Lys Arg Val
Ile Ile Ser Ala Pro Ser Lys Asp Ala 115 120 125 Pro Met Phe Val Val
Gly Val Asn Leu Glu Lys Phe Asn Pro Ser Glu 130 135 140 Lys Val Ile
Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Leu 145 150 155 160
Ala Lys Val Ile Asn Asp Thr Phe Gly Ile Glu Glu Gly Leu Met Thr 165
170 175 Thr Val His Ala Thr Thr Ala Thr Gln Lys Thr Val Asp Gly Pro
Ser 180 185 190 Lys Lys Asp Trp Arg Gly Gly Arg Gly Ala Ser Ala Asn
Ile Ile Pro 195 200 205 Ser Ser Thr Gly Ala Ala Lys Ala Val Gly Lys
Val Ile Pro Ala Leu 210 215 220 Asn Gly Lys Leu Thr Gly Met Ala Phe
Arg Val Pro Thr Pro Asp Val 225 230 235 240 Ser Val Val Asp Leu Thr
Val Lys Leu Ala Lys Pro Thr Asn Tyr Glu 245 250 255 Asp Ile Lys Ala
Ala Ile Lys Ala Ala Ser Glu Gly Pro Met Lys Gly 260 265 270 Val Leu
Gly Tyr Thr Glu Asp Ser Val Val Ser Thr Asp Phe Cys Gly 275 280 285
Asp Asn His Ser Ser Ile Phe Asp Ala Ser Ala Gly Ile Gln Leu Ser 290
295 300 Pro Gln Phe Val Lys Leu Val Ser Trp Tyr Asp Asn Glu Trp Gly
Tyr 305 310 315 320 Ser His Arg Val Val Asp Leu Val Ala Tyr Thr Ala
Ser Lys Asp 325 330 335 <210> SEQ ID NO 3 <211> LENGTH:
338 <212> TYPE: PRT <213> ORGANISM: Aspergillus oryzae
(Genbank Accession No. AAK08065) <400> SEQUENCE: 3 Met Ala
Thr Pro Lys Val Gly Ile Asn Gly Phe Gly Arg Ile Gly Arg 1 5 10 15
Ile Val Phe Arg Asn Ala Ile Ala Ser Gly Asp Val Asp Val Val Ala 20
25 30 Val Asn Asp Pro Phe Ile Glu Thr His Tyr Ala Ala Tyr Met Leu
Lys 35 40 45 Tyr Asp Ser Thr His Gly Arg Phe Gln Gly Thr Ile Glu
Thr Tyr Asp 50 55 60 Glu Gly Leu Ile Val Asn Gly Lys Lys Ile Arg
Phe Phe Ala Glu Arg 65 70 75 80 Asp Pro Ala Ala Ile Pro Trp Gly Ser
Ala Gly Ala Ala Tyr Ile Val 85 90 95 Glu Ser Thr Gly Val Phe Thr
Thr Thr Glu Lys Ala Ser Ala His Leu 100 105 110 Lys Gly Gly Ala Lys
Lys Val Ile Ile Ser Ala Pro Ser Ala Asp Ala 115 120 125 Pro Met Phe
Val Met Gly Val Asn Asn Lys Glu Tyr Lys Thr Asp Ile 130 135 140 Asn
Val Leu Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Leu 145 150
155 160 Ala Lys Val Ile Asn Asp Asn Phe Gly Leu Val Glu Gly Leu Met
Thr 165 170 175 Thr Val His Ser Tyr Thr Ala Thr Gln Lys Thr Val Asp
Ala Pro Ser 180 185 190 Ala Lys Asp Trp Arg Gly Gly Arg Thr Ala Ala
Gln Asn Ile Ile Pro 195 200 205 Ser Ser Thr Gly Ala Ala Lys Ala Val
Gly Lys Val Ile Pro Ser Leu 210 215 220 Asn Gly Lys Leu Thr Gly Met
Ser Met Arg Val Pro Thr Ala Asn Val 225 230 235 240 Ser Val Val Asp
Leu Thr Cys Arg Thr Glu Lys Ala Val Thr Tyr Glu 245 250 255 Asp Ile
Lys Lys Thr Ile Lys Ala Ala Ser Glu Glu Gly Glu Leu Lys 260 265 270
Gly Ile Leu Gly Tyr Thr Glu Asp Asp Ile Val Ser Thr Asp Leu Ile 275
280 285 Gly Asp Ala His Ser Ser Ile Phe Asp Ala Lys Ala Gly Ile Ala
Leu 290 295 300 Asn Glu His Phe Ile Lys Leu Val Ser Trp Tyr Asp Asn
Glu Trp Gly 305 310 315 320 Tyr Ser Arg Arg Val Val Asp Leu Ile Ala
Tyr Ile Ser Lys Val Asp 325 330 335 Gly Gln <210> SEQ ID NO 4
<211> LENGTH: 333 <212> TYPE: PRT <213> ORGANISM:
Paralichthys olivaceus (Genbank Accession No. BAA88638) <400>
SEQUENCE: 4 Met Val Lys Val Gly Ile Asn Gly Phe Gly Arg Ile Gly Arg
Leu Val 1 5 10 15 Thr Arg Ala Ala Phe Thr Ser Lys Lys Val Glu Ile
Val Ala Ile Asn 20 25 30 Asp Pro Phe Ile Asp Leu Glu Tyr Met Val
Tyr Met Phe Lys Tyr Asp 35 40 45 Ser Thr His Gly Arg Phe Lys Gly
Glu Val Lys Ile Glu Gly Asp Lys 50 55 60 Leu Val Ile Asp Gly His
Lys Ile Thr Val Phe His Glu Arg Asp Pro 65 70 75 80 Thr Asn Ile Lys
Trp Gly Asp Ala Gly Ala His Tyr Val Val Glu Ser 85 90 95 Thr Gly
Val Phe Thr Thr Ile Glu Lys Ala Ser Ala His Leu Lys Gly 100 105 110
Gly Ala Lys Lys Val Ile Ile Ser Ala Pro Ser Ala Asp Ala Pro Met 115
120 125 Phe Val Met Gly Val Asn His Glu Lys Tyr Asp Lys Ser Leu Gln
Val 130 135 140 Val Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro
Leu Ala Lys 145 150 155 160 Val Ile Asn Asp Asn Phe Gly Ile Ile Glu
Gly Leu Met Ser Thr Val 165 170 175 His Ala Ile Thr Ala Thr Gln Lys
Thr Val Asp Gly Pro Ser Gly Lys 180 185 190 Leu Trp Arg Asp Gly Arg
Gly Ala Ser Gln Asn Ile Ile Pro Ala Ser 195 200 205 Thr Gly Ala Ala
Lys Ala Val Gly Lys Val Ile Pro Glu Leu Asn Gly 210 215 220 Lys Leu
Thr Gly Met Ala Phe Arg Val Pro Thr Pro Asn Val Ser Val 225 230 235
240 Val Asp Leu Thr Val Arg Leu Glu Lys Pro Ala Ser Tyr Glu Asn Ile
245 250 255 Lys Lys Val Val Lys Ala Ala Ala Glu Gly Pro Met Lys Gly
Tyr Leu 260 265 270 Ala Tyr Thr Glu His Gln Val Val Ser Thr Asp Phe
Asn Gly Asp Thr 275 280 285 His Ser Ser Ile Phe Asp Ala Gly Ala Gly
Ile Ala Leu Asn Asp His 290 295 300 Phe Val Lys Leu Val Ser Trp Tyr
Asp Asn Glu Phe Ala Tyr Ser Asn 305 310 315 320 Arg Val Cys Asp Leu
Met Ala His Met Ala Ser Lys Glu 325 330 <210> SEQ ID NO 5
<211> LENGTH: 333 <212> TYPE: PRT <213> ORGANISM:
Xenopus laevis (Genbank Accession No. P51469) <400> SEQUENCE:
5 Met Val Lys Val Gly Ile Asn Gly Phe Gly Cys Ile Gly Arg Leu Val 1
5 10 15 Thr Arg Ala Ala Phe Asp Ser Gly Lys Val Gln Val Val Ala Ile
Asn 20 25 30 Asp Pro Phe Ile Asp Leu Asp Tyr Met Val Tyr Met Phe
Lys Tyr Asp 35 40 45 Ser Thr His Gly Arg Phe Lys Gly Thr Val Lys
Ala Glu Asn Gly Lys 50 55 60 Leu Ile Ile Asn Asp Gln Val Ile Thr
Val Phe Gln Glu Arg Asp Pro 65 70 75 80 Ser Ser Ile Lys Trp Gly Asp
Ala Gly Ala Val Tyr Val Val Glu Ser 85 90 95 Thr Gly Val Phe Thr
Thr Thr Glu Lys Ala Ser Leu His Leu Lys Gly 100 105 110 Gly Ala Lys
Arg Val Val Ile Ser Ala Pro Ser Ala Asp Ala Pro Met 115 120 125 Phe
Val Val Gly Val Asn His Glu Lys Tyr Glu Asn Ser Leu Lys Val 130 135
140 Val Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Leu Ala Lys
145 150 155 160 Val Ile Asn Asp Asn Phe Gly Ile Val Glu Gly Leu Met
Thr Thr Val 165 170 175 His Ala Phe Thr Ala Thr Gln Lys Thr Val Asp
Gly Pro Ser Gly Lys 180 185 190 Leu Trp Arg Asp Gly Arg Gly Ala Gly
Gln Asn Ile Ile Pro Ala Ser 195 200 205 Thr Gly Ala Ala Lys Ala Val
Gly Lys Val Ile Pro Glu Leu Asn Gly 210 215 220 Lys Ile Thr Gly Met
Ala Phe Arg Val Pro Thr Pro Asn Val Ser Val 225 230 235 240 Val Asp
Leu Thr Cys Arg Leu Gln Lys Pro Ala Lys Tyr Asp Asp Ile 245 250 255
Lys Ala Ala Ile Lys Thr Ala Ser Glu Gly Pro Met Lys Gly Ile Leu 260
265 270 Gly Tyr Thr Gln Asp Gln Val Val Ser Thr Asp Phe Asn Gly Asp
Thr 275 280 285 His Ser Ser Ile Phe Asp Ala Asp Ala Gly Ile Ala Leu
Asn Glu Asn 290 295 300 Phe Val Lys Leu Val Ser Trp Tyr Asp Asn Glu
Cys Gly Tyr Ser Asn 305 310 315 320 Arg Val Val Asp Leu Val Cys His
Met Ala Ser Lys Glu 325 330 <210> SEQ ID NO 6 <211>
LENGTH: 333 <212> TYPE: PRT <213> ORGANISM: Gallus
gallus (Genbank Accession No. DECHG3) <400> SEQUENCE: 6 Met
Val Lys Val Gly Val Asn Gly Phe Gly Arg Ile Gly Arg Leu Val 1 5 10
15 Thr Arg Ala Ala Val Leu Ser Gly Lys Val Gln Val Val Ala Ile Asn
20 25 30 Asp Pro Phe Ile Asp Leu Asn Tyr Met Val Tyr Met Phe Lys
Tyr Asp 35 40 45 Ser Thr His Gly His Phe Lys Gly Thr Val Lys Ala
Glu Asn Gly Lys 50 55 60 Leu Val Ile Asn Gly His Ala Ile Thr Ile
Phe Gln Glu Arg Asp Pro 65 70 75 80 Ser Asn Ile Lys Trp Ala Asp Ala
Gly Ala Glu Tyr Val Val Glu Ser 85 90 95 Thr Gly Val Phe Thr Thr
Met Glu Lys Ala Gly Ala His Leu Lys Gly 100 105 110 Gly Ala Lys Arg
Val Ile Ile Ser Ala Pro Ser Ala Asp Ala Pro Met 115 120 125 Phe Val
Met Gly Val Asn His Glu Lys Tyr Asp Lys Ser Leu Lys Ile 130 135 140
Val Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Leu Ala Lys 145
150 155 160 Val Ile His Asp Asn Phe Gly Ile Val Glu Gly Leu Met Thr
Thr Val 165 170 175 His Ala Ile Thr Ala Thr Gln Lys Thr Val Asp Gly
Pro Ser Gly Lys 180 185 190 Leu Trp Arg Asp Gly Arg Gly Ala Ala Gln
Asn Ile Ile Pro Ala Ser 195 200 205 Thr Gly Ala Ala Lys Ala Val Gly
Lys Val Ile Pro Glu Leu Asn Gly 210 215 220 Lys Leu Thr Gly Met Ala
Phe Arg Val Pro Thr Pro Asn Val Ser Val 225 230 235 240 Val Asp Leu
Thr Cys Arg Leu Glu Lys Pro Ala Lys Tyr Asp Asp Ile 245 250 255 Lys
Arg Val Val Lys Ala Ala Ala Asp Gly Pro Leu Lys Gly Ile Leu 260 265
270 Gly Tyr Thr Glu Asp Gln Val Val Ser Cys Asp Phe Asn Gly Asp Ser
275 280 285 His Ser Ser Thr Phe Asp Ala Gly Ala Gly Ile Ala Leu Asn
Asp His 290 295 300 Phe Val Lys Leu Val Ser Trp Tyr Asp Asn Glu Phe
Gly Tyr Ser Asn 305 310 315 320 Arg Val Val Asp Leu Met Val His Met
Ala Ser Lys Glu 325 330 <210> SEQ ID NO 7 <211> LENGTH:
7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Conserved
protein motif in GPD <400> SEQUENCE: 7 Lys Tyr Asp Ser Thr
His Gly 1 5 <210> SEQ ID NO 8 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Conserved
protein motif in GPD <400> SEQUENCE: 8 Thr Gly Ala Ala Lys
Ala Val 1 5 <210> SEQ ID NO 9 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Degenerate
primer YL193 <400> SEQUENCE: 9 aagtacgayt cbacycaygg 20
<210> SEQ ID NO 10 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Degenerate primer YL194 <400>
SEQUENCE: 10 acrgccttrg crgcdccrgt 20 <210> SEQ ID NO 11
<211> LENGTH: 507 <212> TYPE: DNA <213> ORGANISM:
Yarrowia lipolytica <400> SEQUENCE: 11 aagtacgact ccacccacgg
ccgattcaag ggcaaggtcg aggccaagga cggcggtctg 60 atcatcgacg
gcaagcacat ccaggtcttc ggtgagcgag acccctccaa catcccctgg 120
ggtaaggccg gtgccgacta cgttgtcgag tccaccggtg tcttcaccgg caaggaggct
180 gcctccgccc acctcaaggg tggtgccaag aaggtcatca tctccgcccc
ctccggtgac 240 gcccccatgt tcgttgtcgg tgtcaacctc gacgcctaca
agcccgacat gaccgtcatc 300 tccaacgctt cttgtaccac caactgtctg
gctccccttg ccaaggttgt caacgacaag 360 tacggaatca ttgagggtct
catgaccacc gtccactcca tcaccgccac ccagaagacc 420 gttgacggtc
cttcccacaa ggactggcga ggtggccgaa ccgcctctgg taacatcatc 480
ccctcttcca ccggagccgc caaggct 507 <210> SEQ ID NO 12
<211> LENGTH: 169 <212> TYPE: PRT <213> ORGANISM:
Yarrowia lipolytica <400> SEQUENCE: 12 Lys Tyr Asp Ser Thr
His Gly Arg Phe Lys Gly Lys Val Glu Ala Lys 1 5 10 15 Asp Gly Gly
Leu Ile Ile Asp Gly Lys His Ile Gln Val Phe Gly Glu 20 25 30 Arg
Asp Pro Ser Asn Ile Pro Trp Gly Lys Ala Gly Ala Asp Tyr Val 35 40
45 Val Glu Ser Thr Gly Val Phe Thr Gly Lys Glu Ala Ala Ser Ala His
50 55 60 Leu Lys Gly Gly Ala Lys Lys Val Ile Ile Ser Ala Pro Ser
Gly Asp 65 70 75 80 Ala Pro Met Phe Val Val Gly Val Asn Leu Asp Ala
Tyr Lys Pro Asp 85 90 95 Met Thr Val Ile Ser Asn Ala Ser Cys Thr
Thr Asn Cys Leu Ala Pro 100 105 110 Leu Ala Lys Val Val Asn Asp Lys
Tyr Gly Ile Ile Glu Gly Leu Met 115 120 125 Thr Thr Val His Ser Ile
Thr Ala Thr Gln Lys Thr Val Asp Gly Pro 130 135 140 Ser His Lys Asp
Trp Arg Gly Gly Arg Thr Ala Ser Gly Asn Ile Ile 145 150 155 160 Pro
Ser Ser Thr Gly Ala Ala Lys Ala 165 <210> SEQ ID NO 13
<211> LENGTH: 245 <212> TYPE: PRT <213> ORGANISM:
Saccharomyces cerevisiae (GenBank Accesssion No. NP_012770)
<400> SEQUENCE: 13 Met Pro Lys Leu Val Leu Val Arg His Gly
Gln Ser Glu Trp Asn Glu 1 5 10 15 Lys Asn Leu Phe Thr Gly Trp Val
Asp Val Lys Leu Ser Ala Lys Gly 20 25 30 Gln Gln Glu Ala Ala Arg
Ala Gly Glu Leu Leu Lys Glu Lys Lys Val 35 40 45 Tyr Pro Asp Val
Leu Tyr Thr Ser Lys Leu Ser Arg Ala Ile Gln Thr 50 55 60 Ala Asn
Ile Ala Leu Glu Lys Ala Asp Arg Leu Trp Ile Pro Val Asn 65 70 75 80
Arg Ser Trp Arg Leu Asn Glu Arg His Tyr Gly Asp Leu Gln Gly Lys 85
90 95 Asp Lys Ala Glu Thr Leu Lys Lys Phe Gly Glu Glu Lys Phe Asn
Thr 100 105 110 Tyr Arg Arg Ser Phe Asp Val Pro Pro Pro Pro Ile Asp
Ala Ser Ser 115 120 125 Pro Phe Ser Gln Lys Gly Asp Glu Arg Tyr Lys
Tyr Val Asp Pro Asn 130 135 140 Val Leu Pro Glu Thr Glu Ser Leu Ala
Leu Val Ile Asp Arg Leu Leu 145 150 155 160 Pro Tyr Trp Gln Asp Val
Ile Ala Lys Asp Leu Leu Ser Gly Lys Thr 165 170 175 Val Met Ile Ala
Ala His Gly Asn Ser Leu Arg Gly Leu Val Lys His 180 185 190 Leu Glu
Gly Ile Ser Asp Ala Asp Ile Ala Lys Leu Asn Ile Pro Thr 195 200 205
Gly Ile Pro Leu Val Phe Glu Leu Asp Glu Asn Leu Lys Pro Ser Lys 210
215 220 Pro Ser Tyr Tyr Leu Asp Pro Glu Ala Ala Ala Ala Gly Ala Ala
Ala 225 230 235 240 Val Ala Asn Gln Gly 245 <210> SEQ ID NO
14 <211> LENGTH: 1049 <212> TYPE: DNA <213>
ORGANISM: Yarrowia lipolytica <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1020)..(1020)
<223> OTHER INFORMATION: n is a, c, g, or t <400>
SEQUENCE: 14 caattgagtg cgagcgacac aattgggtgt cacgtgccyt aattgacctc
ggatcgtgga 60 gyccccagtt atacagcaac cacgaggtgc atgagtagga
gacgtcmcca gacaataggg 120 tttttttgga ctggagaggg tagggcaaaa
gcgctcaacg ggctgtttgg ggagctatgg 180 gggaggaatt ggcgatattt
gtgaggttga cggctccgat ttgcgtgttt tgtcgcttct 240 gcatctcccc
atacccatat cttccctccc cacctctttc cacgataatt ttacggatca 300
gcaataaggt tccttctcct agtttccacg yccatatata tctatgctgc gtcgtccttt
360 tcgtgacatc accaaaacac atacaaaaat gcctaaactg attctgctgc
gacacggcca 420 gtccgactgg aacgagaaga acctgttcac cggatgggtc
gacgtcaagt ctccgagctc 480 ggccacaccg aggccaagcg agccggtact
ctgctcaagg agtccggtct caagccccag 540 attctctaca cctccgagct
ctctcgagcc atccagaccg ccaacattgc tctggatgag 600 gccgaccgac
tgtggatccc caccaagcga tcgtggcgac tcaacgagcg acactacggc 660
gctctgcagg gcaaggacaa ggccgccact ctcgccgagt acggccccga gcagttccag
720 ctctggcgac gatcttttga cgtccctcct ccccctatcg ctgacgacga
caagtggtct 780 cagtacaacg acgagcgata ccaggacatc cccaaggata
ttctgcccaa gaccgagtct 840 ctgaagctcg tgattgaccg actccttcct
tactacaact ccgacattgt ccccgacctt 900 aaggccggca agaccgtcct
cattgctgcc cacggaaact ccctccgagc tctcgtcaag 960 cacctcgacg
gtatctccga tgacgatatc gccgccctta acatccccac cggtatcccn 1020
ctcgtgctac gaccttgatg acaacctca 1049 <210> SEQ ID NO 15
<211> LENGTH: 651 <212> TYPE: DNA <213> ORGANISM:
Yarrowia lipolytica <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (633)..(633) <223> OTHER
INFORMATION: n is a, c, g, or t <400> SEQUENCE: 15 atgcctaaac
tgattctgct gcgacacggc cagtccgact ggaacgagaa gaacctgttc 60
accggatggg tcgacgtcaa gctctccgag ctcggccaca ccgaggccaa gcgagccggt
120 actctgctca aggagtccgg tctcaagccc cagattctct acacctccga
gctctctcga 180 gccatccaga ccgccaacat tgctctggat gaggccgacc
gactgtggat ccccaccaag 240 cgatcgtggc gactcaacga gcgacactac
ggcgctctgc agggcaagga caaggccgcc 300 actctcgccg agtacggccc
cgagcagttc cagctctggc gacgatcttt tgacgtccct 360 cctcccccta
tcgctgacga cgacaagtgg tctcagtaca acgacgagcg ataccaggac 420
atccccaagg atattctgcc caagaccgag tctctgaagc tcgtgattga ccgactcctt
480 ccttactaca actccgacat tgtccccgac cttaaggccg gcaagaccgt
cctcattgct 540 gcccacggaa actccctccg agctctcgtc aagcacctcg
acggtatctc cgatgacgat 600 atcgccgccc ttaacatccc caccggtatc
ccnctcgtgc tacgaccttg a 651 <210> SEQ ID NO 16 <211>
LENGTH: 216 <212> TYPE: PRT <213> ORGANISM: Yarrowia
lipolytica <400> SEQUENCE: 16 Met Pro Lys Leu Ile Leu Leu Arg
His Gly Gln Ser Asp Trp Asn Glu 1 5 10 15 Lys Asn Leu Phe Thr Gly
Trp Val Asp Val Lys Leu Ser Glu Leu Gly 20 25 30 His Thr Glu Ala
Lys Arg Ala Gly Thr Leu Leu Lys Glu Ser Gly Leu 35 40 45 Lys Pro
Gln Ile Leu Tyr Thr Ser Glu Leu Ser Arg Ala Ile Gln Thr 50 55 60
Ala Asn Ile Ala Leu Asp Glu Ala Asp Arg Leu Trp Ile Pro Thr Lys 65
70 75 80 Arg Ser Trp Arg Leu Asn Glu Arg His Tyr Gly Ala Leu Gln
Gly Lys 85 90 95 Asp Lys Ala Ala Thr Leu Ala Glu Tyr Gly Pro Glu
Gln Phe Gln Leu 100 105 110 Trp Arg Arg Ser Phe Asp Val Pro Pro Pro
Pro Ile Ala Asp Asp Asp 115 120 125 Lys Trp Ser Gln Tyr Asn Asp Glu
Arg Tyr Gln Asp Ile Pro Lys Asp 130 135 140 Ile Leu Pro Lys Thr Glu
Ser Leu Lys Leu Val Ile Asp Arg Leu Leu 145 150 155 160 Pro Tyr Tyr
Asn Ser Asp Ile Val Pro Asp Leu Lys Ala Gly Lys Thr 165 170 175 Val
Leu Ile Ala Ala His Gly Asn Ser Leu Arg Ala Leu Val Lys His 180 185
190 Leu Asp Gly Ile Ser Asp Asp Asp Ile Ala Ala Leu Asn Ile Pro Thr
195 200 205 Gly Ile Pro Leu Val Leu Arg Pro 210 215 <210> SEQ
ID NO 17 <211> LENGTH: 26 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Primer YL206 <400> SEQUENCE: 17 ccttgccggt
gaagacaccg gtggac 26 <210> SEQ ID NO 18 <211> LENGTH:
25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer YL196
<400> SEQUENCE: 18 gacgtcgacc catccggtga acagg 25 <210>
SEQ ID NO 19 <211> LENGTH: 28 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer YL207 <400> SEQUENCE:
19 gaagacctgg atgtgcttgc cgtcgatg 28 <210> SEQ ID NO 20
<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL197 <400> SEQUENCE: 20 gagcagagta
ccggctcgct tgg 23 <210> SEQ ID NO 21 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer YL208
<400> SEQUENCE: 21 gaccttgccc ttgaatcggc cgtg 24 <210>
SEQ ID NO 22 <211> LENGTH: 25 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer YL198 <400> SEQUENCE:
22 gaatctgggg cttgagaccg gactc 25 <210> SEQ ID NO 23
<211> LENGTH: 1848 <212> TYPE: DNA <213>
ORGANISM: Yarrowia lipolytica <400> SEQUENCE: 23 gtgattgcct
ctgaatactt tcaacaagtt acacccttcg cggcgacgat ctacagcccg 60
atcacatgaa ctttggccga gggatgatgt aatcgagtat cgtggtagtt caatacgtac
120 atgtacgatg ggtgcctcaa ttgtgcgata ctactacaag tgcagcacgc
tcgtgcccgt 180 accctacttt gtcggacgtc cctgctccct cgttcaacat
ctcaagctca acaatcagtg 240 ttggacactg caacgctagc agccggtacg
tggctttagc cccatgctcc atgctccatg 300 ctccatgctc tgggcctatg
agctagccgt ttggcgcaca tagcatagtg acatgtcgat 360 caagtcaaag
tcgaggtgtg gaaaacgggc tgcgggtcgc caggggcctc acaagcgcct 420
ccaccgcaga cgcccacctc gttagcgtcc attgcgatcg tctcggtaca tttggttaca
480 ttttgcgaca ggttgaaatg aatcggccga cgctcggtag tcggaaagag
ccgggaccgg 540 ccggcgagca taaaccggac gcagtaggat gtcctgcacg
ggtctttttg tggggtgtgg 600 agaaaggggt gcttggagat ggaagccggt
agaaccgggc tgcttgtgct tggagatgga 660 agccggtaga accgggctgc
ttggggggat ttggggccgc tgggctccaa agaggggtag 720 gcatttcgtt
ggggttacgt aattgcggca tttgggtcct gcgcgcatgt cccattggtc 780
agaattagtc cggataggag acttatcagc caatcacagc gccggatcca cctgtaggtt
840 gggttgggtg ggagcacccc tccacagagt agagtcaaac agcagcagca
acatgatagt 900 tgggggtgtg cgtgttaaag gaaaaaaaag aagcttgggt
tatattcccg ctctatttag 960 aggttgcggg atagacgccg acggagggca
atggcgccat ggaaccttgc ggatatcgat 1020 acgccgcggc ggactgcgtc
cgaaccagct ccagcagcgt tttttccggg ccattgagcc 1080 gactgcgacc
ccgccaacgt gtcttggccc acgcactcat gtcatgttgg tgttgggagg 1140
ccacttttta agtagcacaa ggcacctagc tcgcagcaag gtgtccgaac caaagaagcg
1200 gctgcagtgg tgcaaacggg gcggaaacgg cgggaaaaag ccacgggggc
acgaattgag 1260 gcacgccctc gaatttgaga cgagtcacgg ccccattcgc
ccgcgcaatg gctcgccaac 1320 gcccggtctt ttgcaccaca tcaggttacc
ccaagccaaa cctttgtgtt aaaaagctta 1380 acatattata ccgaacgtag
gtttgggcgg gcttgctccg tctgtccaag gcaacattta 1440 tataagggtc
tgcatcgccg gctcaattga atcttttttc ttcttctctt ctctatattc 1500
attcttgaat taaacacaca tcaacatggc catcaaagtc ggtattaacg gattcgggcg
1560 aatcggacga attgtgagta ccatagaagg tgatggaaac atgacccaac
agaaacagat 1620 gacaagtgtc atcgacccac cagagcccaa ttgagctcat
actaacagtc gacaacctgt 1680 cgaaccaatt gatgactccc cgacaatgta
ctaacacagg tcctgcgaaa cgctctcaag 1740 aaccctgagg tcgaggtcgt
cgctgtgaac gaccccttca tcgacaccga gtacgctgct 1800 tacatgttca
agtacgactc cacccacggc cgattcaagg gcaaggtc 1848 <210> SEQ ID
NO 24 <211> LENGTH: 2316 <212> TYPE: DNA <213>
ORGANISM: Yarrowia lipolytica <400> SEQUENCE: 24 gtgattgcct
ctgaatactt tcaacaagtt acacccttcg cggcgacgat ctacagcccg 60
atcacatgaa ctttggccga gggatgatgt aatcgagtat cgtggtagtt caatacgtac
120 atgtacgatg ggtgcctcaa ttgtgcgata ctactacaag tgcagcacgc
tcgtgcccgt 180 accctacttt gtcggacgtc cctgctccct cgttcaacat
ctcaagctca acaatcagtg 240 ttggacactg caacgctagc agccggtacg
tggctttagc cccatgctcc atgctccatg 300 ctccatgctc tgggcctatg
agctagccgt ttggcgcaca tagcatagtg acatgtcgat 360 caagtcaaag
tcgaggtgtg gaaaacgggc tgcgggtcgc caggggcctc acaagcgcct 420
ccaccgcaga cgcccacctc gttagcgtcc attgcgatcg tctcggtaca tttggttaca
480 ttttgcgaca ggttgaaatg aatcggccga cgctcggtag tcggaaagag
ccgggaccgg 540 ccggcgagca taaaccggac gcagtaggat gtcctgcacg
ggtctttttg tggggtgtgg 600 agaaaggggt gcttggagat ggaagccggt
agaaccgggc tgcttgtgct tggagatgga 660 agccggtaga accgggctgc
ttggggggat ttggggccgc tgggctccaa agaggggtag 720 gcatttcgtt
ggggttacgt aattgcggca tttgggtcct gcgcgcatgt cccattggtc 780
agaattagtc cggataggag acttatcagc caatcacagc gccggatcca cctgtaggtt
840 gggttgggtg ggagcacccc tccacagagt agagtcaaac agcagcagca
acatgatagt 900 tgggggtgtg cgtgttaaag gaaaaaaaag aagcttgggt
tatattcccg ctctatttag 960 aggttgcggg atagacgccg acggagggca
atggcgccat ggaaccttgc ggatatcgat 1020 acgccgcggc ggactgcgtc
cgaaccagct ccagcagcgt tttttccggg ccattgagcc 1080 gactgcgacc
ccgccaacgt gtcttggccc acgcactcat gtcatgttgg tgttgggagg 1140
ccacttttta agtagcacaa ggcacctagc tcgcagcaag gtgtccgaac caaagaagcg
1200 gctgcagtgg tgcaaacggg gcggaaacgg cgggaaaaag ccacgggggc
acgaattgag 1260 gcacgccctc gaatttgaga cgagtcacgg ccccattcgc
ccgcgcaatg gctcgccaac 1320 gcccggtctt ttgcaccaca tcaggttacc
ccaagccaaa cctttgtgtt aaaaagctta 1380 acatattata ccgaacgtag
gtttgggcgg gcttgctccg tctgtccaag gcaacattta 1440 tataagggtc
tgcatcgccg gctcaattga atcttttttc ttcttctctt ctctatattc 1500
attcttgaat taaacacaca tcaacatggc catcaaagtc ggtattaacg gattcgggcg
1560 aatcggacga attgtgagta ccatagaagg tgatggaaac atgacccaac
agaaacagat 1620 gacaagtgtc atcgacccac cagagcccaa ttgagctcat
actaacagtc gacaacctgt 1680 cgaaccaatt gatgactccc cgacaatgta
ctaacacagg tcctgcgaaa cgctctcaag 1740 aaccctgagg tcgaggtcgt
cgctgtgaac gaccccttca tcgacaccga gtacgctgct 1800 tacatgttca
agtacgactc cacccacggc cgattcaagg gcaaggtcga ggccaaggac 1860
ggcggtctga tcatcgacgg caagcacatc caggtcttcg gtgagcgaga cccctccaac
1920 atcccctggg gtaaggccgg tgccgactac gttgtcgagt ccaccggtgt
cttcaccggc 1980 aaggaggctg cctccgccca cctcaagggt ggtgccaaga
aggtcatcat ctccgccccc 2040 tccggtgacg cccccatgtt cgttgtcggt
gtcaacctcg acgcctacaa gcccgacatg 2100 accgtcatct ccaacgcttc
ttgtaccacc aactgtctgg ctccccttgc caaggttgtc 2160 aacgacaagt
acggaatcat tgagggtctc atgaccaccg tccactccat caccgccacc 2220
cagaagaccg ttgacggtcc ttcccacaag gactggcgag gtggccgaac cgcctctggt
2280 aacatcatcc cctcttccac cggagccgcc aaggct 2316 <210> SEQ
ID NO 25 <211> LENGTH: 645 <212> TYPE: DNA <213>
ORGANISM: Yarrowia lipolytica <400> SEQUENCE: 25 atggccatca
aagtcggtat taacggattc gggcgaatcg gacgaattgt cctgcgaaac 60
gctctcaaga accctgaggt cgaggtcgtc gctgtgaacg accccttcat cgacaccgag
120 tacgctgctt acatgttcaa gtacgactcc acccacggcc gattcaaggg
caaggtcgag 180 gccaaggacg gcggtctgat catcgacggc aagcacatcc
aggtcttcgg tgagcgagac 240 ccctccaaca tcccctgggg taaggccggt
gccgactacg ttgtcgagtc caccggtgtc 300 ttcaccggca aggaggctgc
ctccgcccac ctcaagggtg gtgccaagaa ggtcatcatc 360 tccgccccct
ccggtgacgc ccccatgttc gttgtcggtg tcaacctcga cgcctacaag 420
cccgacatga ccgtcatctc caacgcttct tgtaccacca actgtctggc tccccttgcc
480 aaggttgtca acgacaagta cggaatcatt gagggtctca tgaccaccgt
ccactccatc 540 accgccaccc agaagaccgt tgacggtcct tcccacaagg
actggcgagg tggccgaacc 600 gcctctggta acatcatccc ctcttccacc
ggagccgcca aggct 645 <210> SEQ ID NO 26 <211> LENGTH:
215 <212> TYPE: PRT <213> ORGANISM: Yarrowia lipolytica
<400> SEQUENCE: 26 Met Ala Ile Lys Val Gly Ile Asn Gly Phe
Gly Arg Ile Gly Arg Ile 1 5 10 15 Val Leu Arg Asn Ala Leu Lys Asn
Pro Glu Val Glu Val Val Ala Val 20 25 30 Asn Asp Pro Phe Ile Asp
Thr Glu Tyr Ala Ala Tyr Met Phe Lys Tyr 35 40 45 Asp Ser Thr His
Gly Arg Phe Lys Gly Lys Val Glu Ala Lys Asp Gly 50 55 60 Gly Leu
Ile Ile Asp Gly Lys His Ile Gln Val Phe Gly Glu Arg Asp 65 70 75 80
Pro Ser Asn Ile Pro Trp Gly Lys Ala Gly Ala Asp Tyr Val Val Glu 85
90 95 Ser Thr Gly Val Phe Thr Gly Lys Glu Ala Ala Ser Ala His Leu
Lys 100 105 110 Gly Gly Ala Lys Lys Val Ile Ile Ser Ala Pro Ser Gly
Asp Ala Pro 115 120 125 Met Phe Val Val Gly Val Asn Leu Asp Ala Tyr
Lys Pro Asp Met Thr 130 135 140 Val Ile Ser Asn Ala Ser Cys Thr Thr
Asn Cys Leu Ala Pro Leu Ala 145 150 155 160 Lys Val Val Asn Asp Lys
Tyr Gly Ile Ile Glu Gly Leu Met Thr Thr 165 170 175 Val His Ser Ile
Thr Ala Thr Gln Lys Thr Val Asp Gly Pro Ser His 180 185 190 Lys Asp
Trp Arg Gly Gly Arg Thr Ala Ser Gly Asn Ile Ile Pro Ser 195 200 205
Ser Thr Gly Ala Ala Lys Ala 210 215 <210> SEQ ID NO 27
<211> LENGTH: 953 <212> TYPE: DNA <213> ORGANISM:
Yarrowia lipolytica <400> SEQUENCE: 27 gcctctgaat actttcaaca
agttacaccc ttcattaatt ctcacgtgac acagattatt 60 aacgtctcgt
accaaccaca gattacgacc cattcgcagt cacagttcac tagggtttgg 120
gttgcatccg ttgagagcgg tttgttttta accttctcca tgtgctcact caggttttgg
180 gttcagatca aatcaaggcg tgaaccactt tgtttgagga caaatgtgac
acaaccaacc 240 agtgtcaggg gcaagtccgt gacaaagggg aagatacaat
gcaattactg acagttacag 300 actgcctcga tgccctaacc ttgccccaaa
ataagacaac tgtcctcgtt taagcgcaac 360 cctattcagc gtcacgtcat
aatagcgttt ggatagcact agtctatgag gagcgtttta 420 tgttgcggtg
agggcgattg gtgctcatat gggttcaatt gaggtggcgg aacgagctta 480
gtcttcaatt gaggtgcgag cgacacaatt gggtgtcacg tggcctaatt gacctcgggt
540 cgtggagtcc ccagttatac agcaaccacg aggtgcatgg gtaggagacg
tcaccagaca 600 atagggtttt ttttggactg gagagggttg ggcaaaagcg
ctcaacgggc tgtttgggga 660 gctgtggggg aggaattggc gatatttgtg
aggttaacgg ctccgatttg cgtgttttgt 720 cgctcctgca tctccccata
cccatatctt ccctccccac ctctttccac gataatttta 780 cggatcagca
ataaggttcc ttctcctagt ttccacgtcc atatatatct atgctgcgtc 840
gtccttttcg tgacatcacc aaaacacata caaaaatgcc taaactgatt ctgctgcgac
900 acggccagtc cgactggaac gagaagaacc tgttcaccgg atgggtcgac gtc 953
<210> SEQ ID NO 28 <211> LENGTH: 1537 <212> TYPE:
DNA <213> ORGANISM: Yarrowia lipolytica <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1507)..(1507) <223> OTHER INFORMATION: n is a, c, g, or t
<400> SEQUENCE: 28 gcctctgaat actttcaaca agttacaccc
ttcattaatt ctcacgtgac acagattatt 60 aacgtctcgt accaaccaca
gattacgacc cattcgcagt cacagttcac tagggtttgg 120 gttgcatccg
ttgagagcgg tttgttttta accttctcca tgtgctcact caggttttgg 180
gttcagatca aatcaaggcg tgaaccactt tgtttgagga caaatgtgac acaaccaacc
240 agtgtcaggg gcaagtccgt gacaaagggg aagatacaat gcaattactg
acagttacag 300 actgcctcga tgccctaacc ttgccccaaa ataagacaac
tgtcctcgtt taagcgcaac 360 cctattcagc gtcacgtcat aatagcgttt
ggatagcact agtctatgag gagcgtttta 420 tgttgcggtg agggcgattg
gtgctcatat gggttcaatt gaggtggcgg aacgagctta 480 gtcttcaatt
gaggtgcgag cgacacaatt gggtgtcacg tggcctaatt gacctcgggt 540
cgtggagtcc ccagttatac agcaaccacg aggtgcatgg gtaggagacg tcaccagaca
600 atagggtttt ttttggactg gagagggttg ggcaaaagcg ctcaacgggc
tgtttgggga 660 gctgtggggg aggaattggc gatatttgtg aggttaacgg
ctccgatttg cgtgttttgt 720 cgctcctgca tctccccata cccatatctt
ccctccccac ctctttccac gataatttta 780 cggatcagca ataaggttcc
ttctcctagt ttccacgtcc atatatatct atgctgcgtc 840 gtccttttcg
tgacatcacc aaaacacata caaaaatgcc taaactgatt ctgctgcgac 900
acggccagtc cgactggaac gagaagacct gttcaccgga tgggtcgacg tcaagctctc
960 cgagctcggc cacaccgagg ccaagcgagc cggtactctg ctcaaggagt
ccggtctcaa 1020 gccccagatt ctctacacct ccgagctctc tcgagccatc
cagaccgcca acattgctct 1080 ggatgaggcc gaccgactgt ggatccccac
caagcgatcg tggcgactca acgagcgaca 1140 ctacggcgct ctgcagggca
aggacaaggc cgccactctc gccgagtacg gccccgagca 1200 gttccagctc
tggcgacgat cttttgacgt ccctcctccc cctatcgctg acgacgacaa 1260
gtggtctcag tacaacgacg agcgatacca ggacatcccc aaggatattc tgcccaagac
1320 cgagtctctg aagctcgtga ttgaccgact ccttccttac tacaactccg
acattgtccc 1380 cgaccttaag gccggcaaga ccgtcctcat tgctgcccac
ggaaactccc tccgagctct 1440 cgtcaagcac ctcgacggta tctccgatga
cgatatcgcc gcccttaaca tccccaccgg 1500 tatcccnctc gtgctacgac
cttgatgaca acctcaa 1537 <210> SEQ ID NO 29 <211>
LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
YL33 <400> SEQUENCE: 29 tttccatggt acgtcctgta gaaaccccaa ccc
33 <210> SEQ ID NO 30 <211> LENGTH: 36 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Primer YL34 <400>
SEQUENCE: 30 cccttaatta atcattgttt gcctccctgc tgcggt 36 <210>
SEQ ID NO 31 <211> LENGTH: 19 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer TEF5' <400> SEQUENCE:
31 agagaccggg ttggcggcg 19 <210> SEQ ID NO 32 <211>
LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
TEF3' <400> SEQUENCE: 32 ttggatcctt tgaatgattc ttatactcag 30
<210> SEQ ID NO 33 <211> LENGTH: 29 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer XPR5' <400> SEQUENCE:
33 tttccgcggc ccgagattcc ggcctcttc 29 <210> SEQ ID NO 34
<211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer XPR3' <400> SEQUENCE: 34 tttccgcgga
cacaatatct ggtcaaattt c 31 <210> SEQ ID NO 35 <211>
LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
YL1 <400> SEQUENCE: 35 cagtgccaaa agccaaggca ctgagctcgt 30
<210> SEQ ID NO 36 <211> LENGTH: 31 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer YL2 <400> SEQUENCE: 36
gacgagctca gtgccttggc ttttggcact g 31 <210> SEQ ID NO 37
<211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL3 <400> SEQUENCE: 37 gtataagaat
cattcaccat ggatccacta gttcta 36 <210> SEQ ID NO 38
<211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL4 <400> SEQUENCE: 38 tagaactagt
ggatccatgg tgaatgattc ttatac 36 <210> SEQ ID NO 39
<211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL23 <400> SEQUENCE: 39 atggatccac
tagttaatta actagagcgg ccgcca 36 <210> SEQ ID NO 40
<211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL24 <400> SEQUENCE: 40 tggcggccgc
tctagttaat taactagtgg atccat 36 <210> SEQ ID NO 41
<211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL9 <400> SEQUENCE: 41 tggtaaataa
atgatgtcga ctcaggcgac gacgg 35 <210> SEQ ID NO 42 <211>
LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
YL10 <400> SEQUENCE: 42 ccgtcgtcgc ctgagtcgac atcatttatt
tacca 35 <210> SEQ ID NO 43 <211> LENGTH: 971
<212> TYPE: DNA <213> ORGANISM: Yarrowia lipolytica
<400> SEQUENCE: 43 gacgcagtag gatgtcctgc acgggtcttt
ttgtggggtg tggagaaagg ggtgcttgga 60 gatggaagcc ggtagaaccg
ggctgcttgt gcttggagat ggaagccggt agaaccgggc 120 tgcttggggg
gatttggggc cgctgggctc caaagagggg taggcatttc gttggggtta 180
cgtaattgcg gcatttgggt cctgcgcgca tgtcccattg gtcagaatta gtccggatag
240 gagacttatc agccaatcac agcgccggat ccacctgtag gttgggttgg
gtgggagcac 300 ccctccacag agtagagtca aacagcagca gcaacatgat
agttgggggt gtgcgtgtta 360 aaggaaaaaa aagaagcttg ggttatattc
ccgctctatt tagaggttgc gggatagacg 420 ccgacggagg gcaatggcgc
catggaacct tgcggatatc gatacgccgc ggcggactgc 480 gtccgaacca
gctccagcag cgttttttcc gggccattga gccgactgcg accccgccaa 540
cgtgtcttgg cccacgcact catgtcatgt tggtgttggg aggccacttt ttaagtagca
600 caaggcacct agctcgcagc aaggtgtccg aaccaaagaa gcggctgcag
tggtgcaaac 660 ggggcggaaa cggcgggaaa aagccacggg ggcacgaatt
gaggcacgcc ctcgaatttg 720 agacgagtca cggccccatt cgcccgcgca
atggctcgcc aacgcccggt cttttgcacc 780 acatcaggtt accccaagcc
aaacctttgt gttaaaaagc ttaacatatt ataccgaacg 840 taggtttggg
cgggcttgct ccgtctgtcc aaggcaacat ttatataagg gtctgcatcg 900
ccggctcaat tgaatctttt ttcttcttct cttctctata ttcattcttg aattaaacac
960 acatcaacat g 971 <210> SEQ ID NO 44 <211> LENGTH:
878 <212> TYPE: DNA <213> ORGANISM: Yarrowia lipolytica
<400> SEQUENCE: 44 gcctctgaat actttcaaca agttacaccc
ttcattaatt ctcacgtgac acagattatt 60 aacgtctcgt accaaccaca
gattacgacc cattcgcagt cacagttcac tagggtttgg 120 gttgcatccg
ttgagagcgg tttgttttta accttctcca tgtgctcact caggttttgg 180
gttcagatca aatcaaggcg tgaaccactt tgtttgagga caaatgtgac acaaccaacc
240 agtgtcaggg gcaagtccgt gacaaagggg aagatacaat gcaattactg
acagttacag 300 actgcctcga tgccctaacc ttgccccaaa ataagacaac
tgtcctcgtt taagcgcaac 360 cctattcagc gtcacgtcat aatagcgttt
ggatagcact agtctatgag gagcgtttta 420 tgttgcggtg agggcgattg
gtgctcatat gggttcaatt gaggtggcgg aacgagctta 480 gtcttcaatt
gaggtgcgag cgacacaatt gggtgtcacg tggcctaatt gacctcgggt 540
cgtggagtcc ccagttatac agcaaccacg aggtgcatgg gtaggagacg tcaccagaca
600 atagggtttt ttttggactg gagagggttg ggcaaaagcg ctcaacgggc
tgtttgggga 660 gctgtggggg aggaattggc gatatttgtg aggttaacgg
ctccgatttg cgtgttttgt 720 cgctcctgca tctccccata cccatatctt
ccctccccac ctctttccac gataatttta 780 cggatcagca ataaggttcc
ttctcctagt ttccacgtcc atatatatct atgctgcgtc 840 gtccttttcg
tgacatcacc aaaacacata caaaaatg 878 <210> SEQ ID NO 45
<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL211 <400> SEQUENCE: 45 tttgtcgacg
cagtaggatg tcctgcacgg 30 <210> SEQ ID NO 46 <211>
LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
YL212 <400> SEQUENCE: 46 tttccatggt tgatgtgtgt ttaattcaag
aatg 34 <210> SEQ ID NO 47 <211> LENGTH: 33 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Primer YL203 <400>
SEQUENCE: 47 tttccatggt tgtatgtgtt ttggtgatgt cac 33 <210>
SEQ ID NO 48 <211> LENGTH: 33 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer YL204 <400> SEQUENCE:
48 tttgtcgacc gtttaagcgc aaccctattc agc 33 <210> SEQ ID NO 49
<211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL5 <400> SEQUENCE: 49 cccccctcga
ggtcgatggt gtcgataagc ttgatatcg 39 <210> SEQ ID NO 50
<211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL6 <400> SEQUENCE: 50 cgatatcaag
cttatcgaca ccatcgacct cgagggggg 39 <210> SEQ ID NO 51
<211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL7 <400> SEQUENCE: 51 caaccgattt
cgacagttaa ttaataattt gaatcga 37 <210> SEQ ID NO 52
<211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL8 <400> SEQUENCE: 52 tcgattcaaa
ttattaatta actgtcgaaa tcggttg 37 <210> SEQ ID NO 53
<211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL61 <400> SEQUENCE: 53 acaattccac
acaacgtacg agccggaagc ata 33 <210> SEQ ID NO 54 <211>
LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
YL62 <400> SEQUENCE: 54 tatgcttccg gctcgtacgt tgtgtggaat tgt
33 <210> SEQ ID NO 55 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Primer GPDsense <400>
SEQUENCE: 55 atacgagatc gtcaaggg 18 <210> SEQ ID NO 56
<211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer GPDantisense <400> SEQUENCE: 56
gcggccgcgg attgatgtgt gtttaa 26 <210> SEQ ID NO 57
<211> LENGTH: 1209 <212> TYPE: DNA <213>
ORGANISM: Fusarium monoliforme <400> SEQUENCE: 57 atggcgactc
gacagcgaac tgccaccact gttgtggtcg aggaccttcc caaggtcact 60
cttgaggcca agtctgaacc tgtgttcccc gatatcaaga ccatcaagga tgccattccc
120 gcgcactgct tccagccctc gctcgtcacc tcattctact acgtcttccg
cgattttgcc 180 atggtctctg ccctcgtctg ggctgctctc acctacatcc
ccagcatccc cgaccagacc 240 ctccgcgtcg cagcttggat ggtctacggc
ttcgtccagg gtctgttctg caccggtgtc 300 tggattctcg gccatgagtg
cggccacggt gctttctctc tccacggaaa ggtcaacaat 360 gtgaccggct
ggttcctcca ctcgttcctc ctcgtcccct acttcagctg gaagtactct 420
caccaccgcc accaccgctt caccggccac atggatctcg acatggcttt cgtccccaag
480 actgagccca agccctccaa gtcgctcatg attgctggca ttgacgtcgc
cgagcttgtt 540 gaggacaccc ccgctgctca gatggtcaag ctcatcttcc
accagctttt cggatggcag 600 gcgtacctct tcttcaacgc tagctctggc
aagggcagca agcagtggga gcccaagact 660 ggcctctcca agtggttccg
agtcagtcac ttcgagccta ccagcgctgt cttccgcccc 720 aacgaggcca
tcttcatcct catctccgat atcggtcttg ctctaatggg aactgctctg 780
tactttgctt ccaagcaagt tggtgtttcg accattctct tcctctacct tgttccctac
840 ctgtgggttc accactggct cgttgccatt acctacctcc accaccacca
caccgagctc 900 cctcactaca ccgctgaggg ctggacctac gtcaagggag
ctctcgccac tgtcgaccgt 960 gagtttggct tcatcggaaa gcacctcttc
cacggtatca ttgagaagca cgttgttcac 1020 catctcttcc ctaagatccc
cttctacaag gctgacgagg ccaccgaggc catcaagccc 1080 gtcattggcg
accactactg ccacgacgac cgaagcttcc tgggccagct gtggaccatc 1140
ttcggcacgc tcaagtacgt cgagcacgac cctgcccgac ccggtgccat gcgatggaac
1200 aaggactag 1209 <210> SEQ ID NO 58 <211> LENGTH:
402 <212> TYPE: PRT <213> ORGANISM: Fusarium
monoliforme <400> SEQUENCE: 58 Met Ala Thr Arg Gln Arg Thr
Ala Thr Thr Val Val Val Glu Asp Leu 1 5 10 15 Pro Lys Val Thr Leu
Glu Ala Lys Ser Glu Pro Val Phe Pro Asp Ile 20 25 30 Lys Thr Ile
Lys Asp Ala Ile Pro Ala His Cys Phe Gln Pro Ser Leu 35 40 45 Val
Thr Ser Phe Tyr Tyr Val Phe Arg Asp Phe Ala Met Val Ser Ala 50 55
60 Leu Val Trp Ala Ala Leu Thr Tyr Ile Pro Ser Ile Pro Asp Gln Thr
65 70 75 80 Leu Arg Val Ala Ala Trp Met Val Tyr Gly Phe Val Gln Gly
Leu Phe 85 90 95 Cys Thr Gly Val Trp Ile Leu Gly His Glu Cys Gly
His Gly Ala Phe 100 105 110 Ser Leu His Gly Lys Val Asn Asn Val Thr
Gly Trp Phe Leu His Ser 115 120 125 Phe Leu Leu Val Pro Tyr Phe Ser
Trp Lys Tyr Ser His His Arg His 130 135 140 His Arg Phe Thr Gly His
Met Asp Leu Asp Met Ala Phe Val Pro Lys 145 150 155 160 Thr Glu Pro
Lys Pro Ser Lys Ser Leu Met Ile Ala Gly Ile Asp Val 165 170 175 Ala
Glu Leu Val Glu Asp Thr Pro Ala Ala Gln Met Val Lys Leu Ile 180 185
190 Phe His Gln Leu Phe Gly Trp Gln Ala Tyr Leu Phe Phe Asn Ala Ser
195 200 205 Ser Gly Lys Gly Ser Lys Gln Trp Glu Pro Lys Thr Gly Leu
Ser Lys 210 215 220 Trp Phe Arg Val Ser His Phe Glu Pro Thr Ser Ala
Val Phe Arg Pro 225 230 235 240 Asn Glu Ala Ile Phe Ile Leu Ile Ser
Asp Ile Gly Leu Ala Leu Met 245 250 255 Gly Thr Ala Leu Tyr Phe Ala
Ser Lys Gln Val Gly Val Ser Thr Ile 260 265 270 Leu Phe Leu Tyr Leu
Val Pro Tyr Leu Trp Val His His Trp Leu Val 275 280 285 Ala Ile Thr
Tyr Leu His His His His Thr Glu Leu Pro His Tyr Thr 290 295 300 Ala
Glu Gly Trp Thr Tyr Val Lys Gly Ala Leu Ala Thr Val Asp Arg 305 310
315 320 Glu Phe Gly Phe Ile Gly Lys His Leu Phe His Gly Ile Ile Glu
Lys 325 330 335 His Val Val His His Leu Phe Pro Lys Ile Pro Phe Tyr
Lys Ala Asp 340 345 350 Glu Ala Thr Glu Ala Ile Lys Pro Val Ile Gly
Asp His Tyr Cys His 355 360 365 Asp Asp Arg Ser Phe Leu Gly Gln Leu
Trp Thr Ile Phe Gly Thr Leu 370 375 380 Lys Tyr Val Glu His Asp Pro
Ala Arg Pro Gly Ala Met Arg Trp Asn 385 390 395 400 Lys Asp
<210> SEQ ID NO 59 <211> LENGTH: 36 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer P192 <400> SEQUENCE: 59
aaatatgcgg ccgcacaatg gcgactcgac agcgaa 36 <210> SEQ ID NO 60
<211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer P193 <400> SEQUENCE: 60 tttatagcgg
ccgcctagtc cttgttccat cgca 34
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 60 <210>
SEQ ID NO 1 <211> LENGTH: 332 <212> TYPE: PRT
<213> ORGANISM: Sacchromyces cerevisiae (Genbank Accession
No. CAA24607) <400> SEQUENCE: 1 Met Val Arg Val Ala Ile Asn
Gly Phe Gly Arg Ile Gly Arg Leu Val 1 5 10 15 Met Arg Ile Ala Leu
Ser Arg Pro Asn Val Glu Val Val Ala Leu Asn 20 25 30 Asp Pro Phe
Ile Thr Asn Asp Tyr Ala Ala Tyr Met Phe Lys Tyr Asp 35 40 45 Ser
Thr His Gly Arg Tyr Ala Gly Glu Val Ser His Asp Asp Lys His 50 55
60 Ile Ile Val Asp Gly Lys Lys Ile Ala Thr Tyr Gln Glu Arg Asp Pro
65 70 75 80 Ala Asn Leu Pro Trp Gly Ser Ser Asn Val Asp Ile Ala Ile
Asp Ser 85 90 95 Thr Gly Val Phe Lys Glu Leu Asp Thr Ala Gln Lys
His Ile Asp Ala 100 105 110 Gly Ala Lys Lys Val Val Ile Thr Ala Pro
Ser Ser Thr Ala Pro Met 115 120 125 Phe Val Met Gly Val Asn Glu Val
Lys Tyr Thr Ser Asp Leu Lys Ile 130 135 140 Val Ser Asn Ala Ser Cys
Thr Thr Asn Cys Leu Ala Pro Leu Ala Lys 145 150 155 160 Val Ile Asn
Asp Ala Phe Gly Ile Glu Glu Gly Leu Met Thr Thr Val 165 170 175 His
Ser Leu Thr Ala Thr Gln Lys Thr Val Asp Gly Pro Ser His Lys 180 185
190 Asp Trp Arg Gly Gly Arg Thr Ala Ser Gly Asn Ile Ile Pro Ser Ser
195 200 205 Thr Gly Ala Ala Lys Ala Val Gly Lys Val Leu Pro Glu Leu
Gln Gly 210 215 220 Lys Leu Thr Gly Met Ala Phe Arg Val Pro Thr Val
Asp Val Ser Val 225 230 235 240 Val Asp Leu Thr Val Lys Leu Asp Lys
Glu Thr Thr Tyr Asp Glu Ile 245 250 255 Lys Lys Val Val Lys Ala Ala
Ala Glu Gly Lys Leu Lys Gly Val Leu 260 265 270 Gly Tyr Thr Glu Asp
Ala Val Val Ser Ser Asp Phe Leu Gly Asp Ser 275 280 285 His Ser Ser
Ile Phe Asp Ala Ser Ala Gly Ile Gln Leu Ser Pro Lys 290 295 300 Phe
Val Lys Leu Val Ser Trp Tyr Asp Asn Glu Tyr Gly Tyr Ser Thr 305 310
315 320 Arg Val Val Asp Leu Val Glu His Ile Ala Lys Ala 325 330
<210> SEQ ID NO 2 <211> LENGTH: 335 <212> TYPE:
PRT <213> ORGANISM: Schizosaccharomyces pombe (Genbank
Accession No. NP_595236) <400> SEQUENCE: 2 Met Ala Ile Pro
Lys Val Gly Ile Asn Gly Phe Gly Arg Ile Gly Arg 1 5 10 15 Ile Val
Leu Arg Asn Ala Ile Leu Thr Gly Lys Ile Gln Val Val Ala 20 25 30
Val Asn Asp Pro Phe Ile Asp Leu Asp Tyr Met Ala Tyr Met Phe Lys 35
40 45 Tyr Asp Ser Thr His Gly Arg Phe Glu Gly Ser Val Glu Thr Lys
Gly 50 55 60 Gly Lys Leu Val Ile Asp Gly His Ser Ile Asp Val His
Asn Glu Arg 65 70 75 80 Asp Pro Ala Asn Ile Lys Trp Ser Ala Ser Gly
Ala Glu Tyr Val Ile 85 90 95 Glu Ser Thr Gly Val Phe Thr Thr Lys
Glu Thr Ala Ser Ala His Leu 100 105 110 Lys Gly Gly Ala Lys Arg Val
Ile Ile Ser Ala Pro Ser Lys Asp Ala 115 120 125 Pro Met Phe Val Val
Gly Val Asn Leu Glu Lys Phe Asn Pro Ser Glu 130 135 140 Lys Val Ile
Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Leu 145 150 155 160
Ala Lys Val Ile Asn Asp Thr Phe Gly Ile Glu Glu Gly Leu Met Thr 165
170 175 Thr Val His Ala Thr Thr Ala Thr Gln Lys Thr Val Asp Gly Pro
Ser 180 185 190 Lys Lys Asp Trp Arg Gly Gly Arg Gly Ala Ser Ala Asn
Ile Ile Pro 195 200 205 Ser Ser Thr Gly Ala Ala Lys Ala Val Gly Lys
Val Ile Pro Ala Leu 210 215 220 Asn Gly Lys Leu Thr Gly Met Ala Phe
Arg Val Pro Thr Pro Asp Val 225 230 235 240 Ser Val Val Asp Leu Thr
Val Lys Leu Ala Lys Pro Thr Asn Tyr Glu 245 250 255 Asp Ile Lys Ala
Ala Ile Lys Ala Ala Ser Glu Gly Pro Met Lys Gly 260 265 270 Val Leu
Gly Tyr Thr Glu Asp Ser Val Val Ser Thr Asp Phe Cys Gly 275 280 285
Asp Asn His Ser Ser Ile Phe Asp Ala Ser Ala Gly Ile Gln Leu Ser 290
295 300 Pro Gln Phe Val Lys Leu Val Ser Trp Tyr Asp Asn Glu Trp Gly
Tyr 305 310 315 320 Ser His Arg Val Val Asp Leu Val Ala Tyr Thr Ala
Ser Lys Asp 325 330 335 <210> SEQ ID NO 3 <211> LENGTH:
338 <212> TYPE: PRT <213> ORGANISM: Aspergillus oryzae
(Genbank Accession No. AAK08065) <400> SEQUENCE: 3 Met Ala
Thr Pro Lys Val Gly Ile Asn Gly Phe Gly Arg Ile Gly Arg 1 5 10 15
Ile Val Phe Arg Asn Ala Ile Ala Ser Gly Asp Val Asp Val Val Ala 20
25 30 Val Asn Asp Pro Phe Ile Glu Thr His Tyr Ala Ala Tyr Met Leu
Lys 35 40 45 Tyr Asp Ser Thr His Gly Arg Phe Gln Gly Thr Ile Glu
Thr Tyr Asp 50 55 60 Glu Gly Leu Ile Val Asn Gly Lys Lys Ile Arg
Phe Phe Ala Glu Arg 65 70 75 80 Asp Pro Ala Ala Ile Pro Trp Gly Ser
Ala Gly Ala Ala Tyr Ile Val 85 90 95 Glu Ser Thr Gly Val Phe Thr
Thr Thr Glu Lys Ala Ser Ala His Leu 100 105 110 Lys Gly Gly Ala Lys
Lys Val Ile Ile Ser Ala Pro Ser Ala Asp Ala 115 120 125 Pro Met Phe
Val Met Gly Val Asn Asn Lys Glu Tyr Lys Thr Asp Ile 130 135 140 Asn
Val Leu Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Leu 145 150
155 160 Ala Lys Val Ile Asn Asp Asn Phe Gly Leu Val Glu Gly Leu Met
Thr 165 170 175 Thr Val His Ser Tyr Thr Ala Thr Gln Lys Thr Val Asp
Ala Pro Ser 180 185 190 Ala Lys Asp Trp Arg Gly Gly Arg Thr Ala Ala
Gln Asn Ile Ile Pro 195 200 205 Ser Ser Thr Gly Ala Ala Lys Ala Val
Gly Lys Val Ile Pro Ser Leu 210 215 220 Asn Gly Lys Leu Thr Gly Met
Ser Met Arg Val Pro Thr Ala Asn Val 225 230 235 240 Ser Val Val Asp
Leu Thr Cys Arg Thr Glu Lys Ala Val Thr Tyr Glu 245 250 255 Asp Ile
Lys Lys Thr Ile Lys Ala Ala Ser Glu Glu Gly Glu Leu Lys 260 265 270
Gly Ile Leu Gly Tyr Thr Glu Asp Asp Ile Val Ser Thr Asp Leu Ile 275
280 285 Gly Asp Ala His Ser Ser Ile Phe Asp Ala Lys Ala Gly Ile Ala
Leu 290 295 300 Asn Glu His Phe Ile Lys Leu Val Ser Trp Tyr Asp Asn
Glu Trp Gly 305 310 315 320 Tyr Ser Arg Arg Val Val Asp Leu Ile Ala
Tyr Ile Ser Lys Val Asp 325 330 335 Gly Gln <210> SEQ ID NO 4
<211> LENGTH: 333 <212> TYPE: PRT <213> ORGANISM:
Paralichthys olivaceus (Genbank Accession No. BAA88638) <400>
SEQUENCE: 4 Met Val Lys Val Gly Ile Asn Gly Phe Gly Arg Ile Gly Arg
Leu Val 1 5 10 15 Thr Arg Ala Ala Phe Thr Ser Lys Lys Val Glu Ile
Val Ala Ile Asn 20 25 30 Asp Pro Phe Ile Asp Leu Glu Tyr Met Val
Tyr Met Phe Lys Tyr Asp 35 40 45 Ser Thr His Gly Arg Phe Lys Gly
Glu Val Lys Ile Glu Gly Asp Lys 50 55 60 Leu Val Ile Asp Gly His
Lys Ile Thr Val Phe His Glu Arg Asp Pro 65 70 75 80 Thr Asn Ile Lys
Trp Gly Asp Ala Gly Ala His Tyr Val Val Glu Ser 85 90 95 Thr Gly
Val Phe Thr Thr Ile Glu Lys Ala Ser Ala His Leu Lys Gly
100 105 110 Gly Ala Lys Lys Val Ile Ile Ser Ala Pro Ser Ala Asp Ala
Pro Met 115 120 125 Phe Val Met Gly Val Asn His Glu Lys Tyr Asp Lys
Ser Leu Gln Val 130 135 140 Val Ser Asn Ala Ser Cys Thr Thr Asn Cys
Leu Ala Pro Leu Ala Lys 145 150 155 160 Val Ile Asn Asp Asn Phe Gly
Ile Ile Glu Gly Leu Met Ser Thr Val 165 170 175 His Ala Ile Thr Ala
Thr Gln Lys Thr Val Asp Gly Pro Ser Gly Lys 180 185 190 Leu Trp Arg
Asp Gly Arg Gly Ala Ser Gln Asn Ile Ile Pro Ala Ser 195 200 205 Thr
Gly Ala Ala Lys Ala Val Gly Lys Val Ile Pro Glu Leu Asn Gly 210 215
220 Lys Leu Thr Gly Met Ala Phe Arg Val Pro Thr Pro Asn Val Ser Val
225 230 235 240 Val Asp Leu Thr Val Arg Leu Glu Lys Pro Ala Ser Tyr
Glu Asn Ile 245 250 255 Lys Lys Val Val Lys Ala Ala Ala Glu Gly Pro
Met Lys Gly Tyr Leu 260 265 270 Ala Tyr Thr Glu His Gln Val Val Ser
Thr Asp Phe Asn Gly Asp Thr 275 280 285 His Ser Ser Ile Phe Asp Ala
Gly Ala Gly Ile Ala Leu Asn Asp His 290 295 300 Phe Val Lys Leu Val
Ser Trp Tyr Asp Asn Glu Phe Ala Tyr Ser Asn 305 310 315 320 Arg Val
Cys Asp Leu Met Ala His Met Ala Ser Lys Glu 325 330 <210> SEQ
ID NO 5 <211> LENGTH: 333 <212> TYPE: PRT <213>
ORGANISM: Xenopus laevis (Genbank Accession No. P51469) <400>
SEQUENCE: 5 Met Val Lys Val Gly Ile Asn Gly Phe Gly Cys Ile Gly Arg
Leu Val 1 5 10 15 Thr Arg Ala Ala Phe Asp Ser Gly Lys Val Gln Val
Val Ala Ile Asn 20 25 30 Asp Pro Phe Ile Asp Leu Asp Tyr Met Val
Tyr Met Phe Lys Tyr Asp 35 40 45 Ser Thr His Gly Arg Phe Lys Gly
Thr Val Lys Ala Glu Asn Gly Lys 50 55 60 Leu Ile Ile Asn Asp Gln
Val Ile Thr Val Phe Gln Glu Arg Asp Pro 65 70 75 80 Ser Ser Ile Lys
Trp Gly Asp Ala Gly Ala Val Tyr Val Val Glu Ser 85 90 95 Thr Gly
Val Phe Thr Thr Thr Glu Lys Ala Ser Leu His Leu Lys Gly 100 105 110
Gly Ala Lys Arg Val Val Ile Ser Ala Pro Ser Ala Asp Ala Pro Met 115
120 125 Phe Val Val Gly Val Asn His Glu Lys Tyr Glu Asn Ser Leu Lys
Val 130 135 140 Val Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro
Leu Ala Lys 145 150 155 160 Val Ile Asn Asp Asn Phe Gly Ile Val Glu
Gly Leu Met Thr Thr Val 165 170 175 His Ala Phe Thr Ala Thr Gln Lys
Thr Val Asp Gly Pro Ser Gly Lys 180 185 190 Leu Trp Arg Asp Gly Arg
Gly Ala Gly Gln Asn Ile Ile Pro Ala Ser 195 200 205 Thr Gly Ala Ala
Lys Ala Val Gly Lys Val Ile Pro Glu Leu Asn Gly 210 215 220 Lys Ile
Thr Gly Met Ala Phe Arg Val Pro Thr Pro Asn Val Ser Val 225 230 235
240 Val Asp Leu Thr Cys Arg Leu Gln Lys Pro Ala Lys Tyr Asp Asp Ile
245 250 255 Lys Ala Ala Ile Lys Thr Ala Ser Glu Gly Pro Met Lys Gly
Ile Leu 260 265 270 Gly Tyr Thr Gln Asp Gln Val Val Ser Thr Asp Phe
Asn Gly Asp Thr 275 280 285 His Ser Ser Ile Phe Asp Ala Asp Ala Gly
Ile Ala Leu Asn Glu Asn 290 295 300 Phe Val Lys Leu Val Ser Trp Tyr
Asp Asn Glu Cys Gly Tyr Ser Asn 305 310 315 320 Arg Val Val Asp Leu
Val Cys His Met Ala Ser Lys Glu 325 330 <210> SEQ ID NO 6
<211> LENGTH: 333 <212> TYPE: PRT <213> ORGANISM:
Gallus gallus (Genbank Accession No. DECHG3) <400> SEQUENCE:
6 Met Val Lys Val Gly Val Asn Gly Phe Gly Arg Ile Gly Arg Leu Val 1
5 10 15 Thr Arg Ala Ala Val Leu Ser Gly Lys Val Gln Val Val Ala Ile
Asn 20 25 30 Asp Pro Phe Ile Asp Leu Asn Tyr Met Val Tyr Met Phe
Lys Tyr Asp 35 40 45 Ser Thr His Gly His Phe Lys Gly Thr Val Lys
Ala Glu Asn Gly Lys 50 55 60 Leu Val Ile Asn Gly His Ala Ile Thr
Ile Phe Gln Glu Arg Asp Pro 65 70 75 80 Ser Asn Ile Lys Trp Ala Asp
Ala Gly Ala Glu Tyr Val Val Glu Ser 85 90 95 Thr Gly Val Phe Thr
Thr Met Glu Lys Ala Gly Ala His Leu Lys Gly 100 105 110 Gly Ala Lys
Arg Val Ile Ile Ser Ala Pro Ser Ala Asp Ala Pro Met 115 120 125 Phe
Val Met Gly Val Asn His Glu Lys Tyr Asp Lys Ser Leu Lys Ile 130 135
140 Val Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Leu Ala Lys
145 150 155 160 Val Ile His Asp Asn Phe Gly Ile Val Glu Gly Leu Met
Thr Thr Val 165 170 175 His Ala Ile Thr Ala Thr Gln Lys Thr Val Asp
Gly Pro Ser Gly Lys 180 185 190 Leu Trp Arg Asp Gly Arg Gly Ala Ala
Gln Asn Ile Ile Pro Ala Ser 195 200 205 Thr Gly Ala Ala Lys Ala Val
Gly Lys Val Ile Pro Glu Leu Asn Gly 210 215 220 Lys Leu Thr Gly Met
Ala Phe Arg Val Pro Thr Pro Asn Val Ser Val 225 230 235 240 Val Asp
Leu Thr Cys Arg Leu Glu Lys Pro Ala Lys Tyr Asp Asp Ile 245 250 255
Lys Arg Val Val Lys Ala Ala Ala Asp Gly Pro Leu Lys Gly Ile Leu 260
265 270 Gly Tyr Thr Glu Asp Gln Val Val Ser Cys Asp Phe Asn Gly Asp
Ser 275 280 285 His Ser Ser Thr Phe Asp Ala Gly Ala Gly Ile Ala Leu
Asn Asp His 290 295 300 Phe Val Lys Leu Val Ser Trp Tyr Asp Asn Glu
Phe Gly Tyr Ser Asn 305 310 315 320 Arg Val Val Asp Leu Met Val His
Met Ala Ser Lys Glu 325 330 <210> SEQ ID NO 7 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Conserved protein motif in GPD <400> SEQUENCE: 7 Lys Tyr Asp
Ser Thr His Gly 1 5 <210> SEQ ID NO 8 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Conserved
protein motif in GPD <400> SEQUENCE: 8 Thr Gly Ala Ala Lys
Ala Val 1 5 <210> SEQ ID NO 9 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Degenerate
primer YL193 <400> SEQUENCE: 9 aagtacgayt cbacycaygg 20
<210> SEQ ID NO 10 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Degenerate primer YL194 <400>
SEQUENCE: 10 acrgccttrg crgcdccrgt 20 <210> SEQ ID NO 11
<211> LENGTH: 507 <212> TYPE: DNA <213> ORGANISM:
Yarrowia lipolytica <400> SEQUENCE: 11 aagtacgact ccacccacgg
ccgattcaag ggcaaggtcg aggccaagga cggcggtctg 60 atcatcgacg
gcaagcacat ccaggtcttc ggtgagcgag acccctccaa catcccctgg 120
ggtaaggccg gtgccgacta cgttgtcgag tccaccggtg tcttcaccgg caaggaggct
180 gcctccgccc acctcaaggg tggtgccaag aaggtcatca tctccgcccc
ctccggtgac 240
gcccccatgt tcgttgtcgg tgtcaacctc gacgcctaca agcccgacat gaccgtcatc
300 tccaacgctt cttgtaccac caactgtctg gctccccttg ccaaggttgt
caacgacaag 360 tacggaatca ttgagggtct catgaccacc gtccactcca
tcaccgccac ccagaagacc 420 gttgacggtc cttcccacaa ggactggcga
ggtggccgaa ccgcctctgg taacatcatc 480 ccctcttcca ccggagccgc caaggct
507 <210> SEQ ID NO 12 <211> LENGTH: 169 <212>
TYPE: PRT <213> ORGANISM: Yarrowia lipolytica <400>
SEQUENCE: 12 Lys Tyr Asp Ser Thr His Gly Arg Phe Lys Gly Lys Val
Glu Ala Lys 1 5 10 15 Asp Gly Gly Leu Ile Ile Asp Gly Lys His Ile
Gln Val Phe Gly Glu 20 25 30 Arg Asp Pro Ser Asn Ile Pro Trp Gly
Lys Ala Gly Ala Asp Tyr Val 35 40 45 Val Glu Ser Thr Gly Val Phe
Thr Gly Lys Glu Ala Ala Ser Ala His 50 55 60 Leu Lys Gly Gly Ala
Lys Lys Val Ile Ile Ser Ala Pro Ser Gly Asp 65 70 75 80 Ala Pro Met
Phe Val Val Gly Val Asn Leu Asp Ala Tyr Lys Pro Asp 85 90 95 Met
Thr Val Ile Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro 100 105
110 Leu Ala Lys Val Val Asn Asp Lys Tyr Gly Ile Ile Glu Gly Leu Met
115 120 125 Thr Thr Val His Ser Ile Thr Ala Thr Gln Lys Thr Val Asp
Gly Pro 130 135 140 Ser His Lys Asp Trp Arg Gly Gly Arg Thr Ala Ser
Gly Asn Ile Ile 145 150 155 160 Pro Ser Ser Thr Gly Ala Ala Lys Ala
165 <210> SEQ ID NO 13 <211> LENGTH: 245 <212>
TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae (GenBank
Accesssion No. NP_012770) <400> SEQUENCE: 13 Met Pro Lys Leu
Val Leu Val Arg His Gly Gln Ser Glu Trp Asn Glu 1 5 10 15 Lys Asn
Leu Phe Thr Gly Trp Val Asp Val Lys Leu Ser Ala Lys Gly 20 25 30
Gln Gln Glu Ala Ala Arg Ala Gly Glu Leu Leu Lys Glu Lys Lys Val 35
40 45 Tyr Pro Asp Val Leu Tyr Thr Ser Lys Leu Ser Arg Ala Ile Gln
Thr 50 55 60 Ala Asn Ile Ala Leu Glu Lys Ala Asp Arg Leu Trp Ile
Pro Val Asn 65 70 75 80 Arg Ser Trp Arg Leu Asn Glu Arg His Tyr Gly
Asp Leu Gln Gly Lys 85 90 95 Asp Lys Ala Glu Thr Leu Lys Lys Phe
Gly Glu Glu Lys Phe Asn Thr 100 105 110 Tyr Arg Arg Ser Phe Asp Val
Pro Pro Pro Pro Ile Asp Ala Ser Ser 115 120 125 Pro Phe Ser Gln Lys
Gly Asp Glu Arg Tyr Lys Tyr Val Asp Pro Asn 130 135 140 Val Leu Pro
Glu Thr Glu Ser Leu Ala Leu Val Ile Asp Arg Leu Leu 145 150 155 160
Pro Tyr Trp Gln Asp Val Ile Ala Lys Asp Leu Leu Ser Gly Lys Thr 165
170 175 Val Met Ile Ala Ala His Gly Asn Ser Leu Arg Gly Leu Val Lys
His 180 185 190 Leu Glu Gly Ile Ser Asp Ala Asp Ile Ala Lys Leu Asn
Ile Pro Thr 195 200 205 Gly Ile Pro Leu Val Phe Glu Leu Asp Glu Asn
Leu Lys Pro Ser Lys 210 215 220 Pro Ser Tyr Tyr Leu Asp Pro Glu Ala
Ala Ala Ala Gly Ala Ala Ala 225 230 235 240 Val Ala Asn Gln Gly 245
<210> SEQ ID NO 14 <211> LENGTH: 1049 <212> TYPE:
DNA <213> ORGANISM: Yarrowia lipolytica <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1020)..(1020) <223> OTHER INFORMATION: n is a, c, g, or t
<400> SEQUENCE: 14 caattgagtg cgagcgacac aattgggtgt
cacgtgccyt aattgacctc ggatcgtgga 60 gyccccagtt atacagcaac
cacgaggtgc atgagtagga gacgtcmcca gacaataggg 120 tttttttgga
ctggagaggg tagggcaaaa gcgctcaacg ggctgtttgg ggagctatgg 180
gggaggaatt ggcgatattt gtgaggttga cggctccgat ttgcgtgttt tgtcgcttct
240 gcatctcccc atacccatat cttccctccc cacctctttc cacgataatt
ttacggatca 300 gcaataaggt tccttctcct agtttccacg yccatatata
tctatgctgc gtcgtccttt 360 tcgtgacatc accaaaacac atacaaaaat
gcctaaactg attctgctgc gacacggcca 420 gtccgactgg aacgagaaga
acctgttcac cggatgggtc gacgtcaagt ctccgagctc 480 ggccacaccg
aggccaagcg agccggtact ctgctcaagg agtccggtct caagccccag 540
attctctaca cctccgagct ctctcgagcc atccagaccg ccaacattgc tctggatgag
600 gccgaccgac tgtggatccc caccaagcga tcgtggcgac tcaacgagcg
acactacggc 660 gctctgcagg gcaaggacaa ggccgccact ctcgccgagt
acggccccga gcagttccag 720 ctctggcgac gatcttttga cgtccctcct
ccccctatcg ctgacgacga caagtggtct 780 cagtacaacg acgagcgata
ccaggacatc cccaaggata ttctgcccaa gaccgagtct 840 ctgaagctcg
tgattgaccg actccttcct tactacaact ccgacattgt ccccgacctt 900
aaggccggca agaccgtcct cattgctgcc cacggaaact ccctccgagc tctcgtcaag
960 cacctcgacg gtatctccga tgacgatatc gccgccctta acatccccac
cggtatcccn 1020 ctcgtgctac gaccttgatg acaacctca 1049 <210>
SEQ ID NO 15 <211> LENGTH: 651 <212> TYPE: DNA
<213> ORGANISM: Yarrowia lipolytica <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(633)..(633) <223> OTHER INFORMATION: n is a, c, g, or t
<400> SEQUENCE: 15 atgcctaaac tgattctgct gcgacacggc
cagtccgact ggaacgagaa gaacctgttc 60 accggatggg tcgacgtcaa
gctctccgag ctcggccaca ccgaggccaa gcgagccggt 120 actctgctca
aggagtccgg tctcaagccc cagattctct acacctccga gctctctcga 180
gccatccaga ccgccaacat tgctctggat gaggccgacc gactgtggat ccccaccaag
240 cgatcgtggc gactcaacga gcgacactac ggcgctctgc agggcaagga
caaggccgcc 300 actctcgccg agtacggccc cgagcagttc cagctctggc
gacgatcttt tgacgtccct 360 cctcccccta tcgctgacga cgacaagtgg
tctcagtaca acgacgagcg ataccaggac 420 atccccaagg atattctgcc
caagaccgag tctctgaagc tcgtgattga ccgactcctt 480 ccttactaca
actccgacat tgtccccgac cttaaggccg gcaagaccgt cctcattgct 540
gcccacggaa actccctccg agctctcgtc aagcacctcg acggtatctc cgatgacgat
600 atcgccgccc ttaacatccc caccggtatc ccnctcgtgc tacgaccttg a 651
<210> SEQ ID NO 16 <211> LENGTH: 216 <212> TYPE:
PRT <213> ORGANISM: Yarrowia lipolytica <400> SEQUENCE:
16 Met Pro Lys Leu Ile Leu Leu Arg His Gly Gln Ser Asp Trp Asn Glu
1 5 10 15 Lys Asn Leu Phe Thr Gly Trp Val Asp Val Lys Leu Ser Glu
Leu Gly 20 25 30 His Thr Glu Ala Lys Arg Ala Gly Thr Leu Leu Lys
Glu Ser Gly Leu 35 40 45 Lys Pro Gln Ile Leu Tyr Thr Ser Glu Leu
Ser Arg Ala Ile Gln Thr 50 55 60 Ala Asn Ile Ala Leu Asp Glu Ala
Asp Arg Leu Trp Ile Pro Thr Lys 65 70 75 80 Arg Ser Trp Arg Leu Asn
Glu Arg His Tyr Gly Ala Leu Gln Gly Lys 85 90 95 Asp Lys Ala Ala
Thr Leu Ala Glu Tyr Gly Pro Glu Gln Phe Gln Leu 100 105 110 Trp Arg
Arg Ser Phe Asp Val Pro Pro Pro Pro Ile Ala Asp Asp Asp 115 120 125
Lys Trp Ser Gln Tyr Asn Asp Glu Arg Tyr Gln Asp Ile Pro Lys Asp 130
135 140 Ile Leu Pro Lys Thr Glu Ser Leu Lys Leu Val Ile Asp Arg Leu
Leu 145 150 155 160 Pro Tyr Tyr Asn Ser Asp Ile Val Pro Asp Leu Lys
Ala Gly Lys Thr 165 170 175 Val Leu Ile Ala Ala His Gly Asn Ser Leu
Arg Ala Leu Val Lys His 180 185 190 Leu Asp Gly Ile Ser Asp Asp Asp
Ile Ala Ala Leu Asn Ile Pro Thr 195 200 205 Gly Ile Pro Leu Val Leu
Arg Pro 210 215 <210> SEQ ID NO 17 <211> LENGTH: 26
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer YL206
<400> SEQUENCE: 17
ccttgccggt gaagacaccg gtggac 26 <210> SEQ ID NO 18
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL196 <400> SEQUENCE: 18 gacgtcgacc
catccggtga acagg 25 <210> SEQ ID NO 19 <211> LENGTH: 28
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer YL207
<400> SEQUENCE: 19 gaagacctgg atgtgcttgc cgtcgatg 28
<210> SEQ ID NO 20 <211> LENGTH: 23 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer YL197 <400> SEQUENCE:
20 gagcagagta ccggctcgct tgg 23 <210> SEQ ID NO 21
<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL208 <400> SEQUENCE: 21 gaccttgccc
ttgaatcggc cgtg 24 <210> SEQ ID NO 22 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer YL198
<400> SEQUENCE: 22 gaatctgggg cttgagaccg gactc 25 <210>
SEQ ID NO 23 <211> LENGTH: 1848 <212> TYPE: DNA
<213> ORGANISM: Yarrowia lipolytica <400> SEQUENCE: 23
gtgattgcct ctgaatactt tcaacaagtt acacccttcg cggcgacgat ctacagcccg
60 atcacatgaa ctttggccga gggatgatgt aatcgagtat cgtggtagtt
caatacgtac 120 atgtacgatg ggtgcctcaa ttgtgcgata ctactacaag
tgcagcacgc tcgtgcccgt 180 accctacttt gtcggacgtc cctgctccct
cgttcaacat ctcaagctca acaatcagtg 240 ttggacactg caacgctagc
agccggtacg tggctttagc cccatgctcc atgctccatg 300 ctccatgctc
tgggcctatg agctagccgt ttggcgcaca tagcatagtg acatgtcgat 360
caagtcaaag tcgaggtgtg gaaaacgggc tgcgggtcgc caggggcctc acaagcgcct
420 ccaccgcaga cgcccacctc gttagcgtcc attgcgatcg tctcggtaca
tttggttaca 480 ttttgcgaca ggttgaaatg aatcggccga cgctcggtag
tcggaaagag ccgggaccgg 540 ccggcgagca taaaccggac gcagtaggat
gtcctgcacg ggtctttttg tggggtgtgg 600 agaaaggggt gcttggagat
ggaagccggt agaaccgggc tgcttgtgct tggagatgga 660 agccggtaga
accgggctgc ttggggggat ttggggccgc tgggctccaa agaggggtag 720
gcatttcgtt ggggttacgt aattgcggca tttgggtcct gcgcgcatgt cccattggtc
780 agaattagtc cggataggag acttatcagc caatcacagc gccggatcca
cctgtaggtt 840 gggttgggtg ggagcacccc tccacagagt agagtcaaac
agcagcagca acatgatagt 900 tgggggtgtg cgtgttaaag gaaaaaaaag
aagcttgggt tatattcccg ctctatttag 960 aggttgcggg atagacgccg
acggagggca atggcgccat ggaaccttgc ggatatcgat 1020 acgccgcggc
ggactgcgtc cgaaccagct ccagcagcgt tttttccggg ccattgagcc 1080
gactgcgacc ccgccaacgt gtcttggccc acgcactcat gtcatgttgg tgttgggagg
1140 ccacttttta agtagcacaa ggcacctagc tcgcagcaag gtgtccgaac
caaagaagcg 1200 gctgcagtgg tgcaaacggg gcggaaacgg cgggaaaaag
ccacgggggc acgaattgag 1260 gcacgccctc gaatttgaga cgagtcacgg
ccccattcgc ccgcgcaatg gctcgccaac 1320 gcccggtctt ttgcaccaca
tcaggttacc ccaagccaaa cctttgtgtt aaaaagctta 1380 acatattata
ccgaacgtag gtttgggcgg gcttgctccg tctgtccaag gcaacattta 1440
tataagggtc tgcatcgccg gctcaattga atcttttttc ttcttctctt ctctatattc
1500 attcttgaat taaacacaca tcaacatggc catcaaagtc ggtattaacg
gattcgggcg 1560 aatcggacga attgtgagta ccatagaagg tgatggaaac
atgacccaac agaaacagat 1620 gacaagtgtc atcgacccac cagagcccaa
ttgagctcat actaacagtc gacaacctgt 1680 cgaaccaatt gatgactccc
cgacaatgta ctaacacagg tcctgcgaaa cgctctcaag 1740 aaccctgagg
tcgaggtcgt cgctgtgaac gaccccttca tcgacaccga gtacgctgct 1800
tacatgttca agtacgactc cacccacggc cgattcaagg gcaaggtc 1848
<210> SEQ ID NO 24 <211> LENGTH: 2316 <212> TYPE:
DNA <213> ORGANISM: Yarrowia lipolytica <400> SEQUENCE:
24 gtgattgcct ctgaatactt tcaacaagtt acacccttcg cggcgacgat
ctacagcccg 60 atcacatgaa ctttggccga gggatgatgt aatcgagtat
cgtggtagtt caatacgtac 120 atgtacgatg ggtgcctcaa ttgtgcgata
ctactacaag tgcagcacgc tcgtgcccgt 180 accctacttt gtcggacgtc
cctgctccct cgttcaacat ctcaagctca acaatcagtg 240 ttggacactg
caacgctagc agccggtacg tggctttagc cccatgctcc atgctccatg 300
ctccatgctc tgggcctatg agctagccgt ttggcgcaca tagcatagtg acatgtcgat
360 caagtcaaag tcgaggtgtg gaaaacgggc tgcgggtcgc caggggcctc
acaagcgcct 420 ccaccgcaga cgcccacctc gttagcgtcc attgcgatcg
tctcggtaca tttggttaca 480 ttttgcgaca ggttgaaatg aatcggccga
cgctcggtag tcggaaagag ccgggaccgg 540 ccggcgagca taaaccggac
gcagtaggat gtcctgcacg ggtctttttg tggggtgtgg 600 agaaaggggt
gcttggagat ggaagccggt agaaccgggc tgcttgtgct tggagatgga 660
agccggtaga accgggctgc ttggggggat ttggggccgc tgggctccaa agaggggtag
720 gcatttcgtt ggggttacgt aattgcggca tttgggtcct gcgcgcatgt
cccattggtc 780 agaattagtc cggataggag acttatcagc caatcacagc
gccggatcca cctgtaggtt 840 gggttgggtg ggagcacccc tccacagagt
agagtcaaac agcagcagca acatgatagt 900 tgggggtgtg cgtgttaaag
gaaaaaaaag aagcttgggt tatattcccg ctctatttag 960 aggttgcggg
atagacgccg acggagggca atggcgccat ggaaccttgc ggatatcgat 1020
acgccgcggc ggactgcgtc cgaaccagct ccagcagcgt tttttccggg ccattgagcc
1080 gactgcgacc ccgccaacgt gtcttggccc acgcactcat gtcatgttgg
tgttgggagg 1140 ccacttttta agtagcacaa ggcacctagc tcgcagcaag
gtgtccgaac caaagaagcg 1200 gctgcagtgg tgcaaacggg gcggaaacgg
cgggaaaaag ccacgggggc acgaattgag 1260 gcacgccctc gaatttgaga
cgagtcacgg ccccattcgc ccgcgcaatg gctcgccaac 1320 gcccggtctt
ttgcaccaca tcaggttacc ccaagccaaa cctttgtgtt aaaaagctta 1380
acatattata ccgaacgtag gtttgggcgg gcttgctccg tctgtccaag gcaacattta
1440 tataagggtc tgcatcgccg gctcaattga atcttttttc ttcttctctt
ctctatattc 1500 attcttgaat taaacacaca tcaacatggc catcaaagtc
ggtattaacg gattcgggcg 1560 aatcggacga attgtgagta ccatagaagg
tgatggaaac atgacccaac agaaacagat 1620 gacaagtgtc atcgacccac
cagagcccaa ttgagctcat actaacagtc gacaacctgt 1680 cgaaccaatt
gatgactccc cgacaatgta ctaacacagg tcctgcgaaa cgctctcaag 1740
aaccctgagg tcgaggtcgt cgctgtgaac gaccccttca tcgacaccga gtacgctgct
1800 tacatgttca agtacgactc cacccacggc cgattcaagg gcaaggtcga
ggccaaggac 1860 ggcggtctga tcatcgacgg caagcacatc caggtcttcg
gtgagcgaga cccctccaac 1920 atcccctggg gtaaggccgg tgccgactac
gttgtcgagt ccaccggtgt cttcaccggc 1980 aaggaggctg cctccgccca
cctcaagggt ggtgccaaga aggtcatcat ctccgccccc 2040 tccggtgacg
cccccatgtt cgttgtcggt gtcaacctcg acgcctacaa gcccgacatg 2100
accgtcatct ccaacgcttc ttgtaccacc aactgtctgg ctccccttgc caaggttgtc
2160 aacgacaagt acggaatcat tgagggtctc atgaccaccg tccactccat
caccgccacc 2220 cagaagaccg ttgacggtcc ttcccacaag gactggcgag
gtggccgaac cgcctctggt 2280 aacatcatcc cctcttccac cggagccgcc aaggct
2316 <210> SEQ ID NO 25 <211> LENGTH: 645 <212>
TYPE: DNA <213> ORGANISM: Yarrowia lipolytica <400>
SEQUENCE: 25 atggccatca aagtcggtat taacggattc gggcgaatcg gacgaattgt
cctgcgaaac 60 gctctcaaga accctgaggt cgaggtcgtc gctgtgaacg
accccttcat cgacaccgag 120 tacgctgctt acatgttcaa gtacgactcc
acccacggcc gattcaaggg caaggtcgag 180 gccaaggacg gcggtctgat
catcgacggc aagcacatcc aggtcttcgg tgagcgagac 240 ccctccaaca
tcccctgggg taaggccggt gccgactacg ttgtcgagtc caccggtgtc 300
ttcaccggca aggaggctgc ctccgcccac ctcaagggtg gtgccaagaa ggtcatcatc
360 tccgccccct ccggtgacgc ccccatgttc gttgtcggtg tcaacctcga
cgcctacaag 420 cccgacatga ccgtcatctc caacgcttct tgtaccacca
actgtctggc tccccttgcc 480 aaggttgtca acgacaagta cggaatcatt
gagggtctca tgaccaccgt ccactccatc 540 accgccaccc agaagaccgt
tgacggtcct tcccacaagg actggcgagg tggccgaacc 600 gcctctggta
acatcatccc ctcttccacc ggagccgcca aggct 645 <210> SEQ ID NO 26
<211> LENGTH: 215
<212> TYPE: PRT <213> ORGANISM: Yarrowia lipolytica
<400> SEQUENCE: 26 Met Ala Ile Lys Val Gly Ile Asn Gly Phe
Gly Arg Ile Gly Arg Ile 1 5 10 15 Val Leu Arg Asn Ala Leu Lys Asn
Pro Glu Val Glu Val Val Ala Val 20 25 30 Asn Asp Pro Phe Ile Asp
Thr Glu Tyr Ala Ala Tyr Met Phe Lys Tyr 35 40 45 Asp Ser Thr His
Gly Arg Phe Lys Gly Lys Val Glu Ala Lys Asp Gly 50 55 60 Gly Leu
Ile Ile Asp Gly Lys His Ile Gln Val Phe Gly Glu Arg Asp 65 70 75 80
Pro Ser Asn Ile Pro Trp Gly Lys Ala Gly Ala Asp Tyr Val Val Glu 85
90 95 Ser Thr Gly Val Phe Thr Gly Lys Glu Ala Ala Ser Ala His Leu
Lys 100 105 110 Gly Gly Ala Lys Lys Val Ile Ile Ser Ala Pro Ser Gly
Asp Ala Pro 115 120 125 Met Phe Val Val Gly Val Asn Leu Asp Ala Tyr
Lys Pro Asp Met Thr 130 135 140 Val Ile Ser Asn Ala Ser Cys Thr Thr
Asn Cys Leu Ala Pro Leu Ala 145 150 155 160 Lys Val Val Asn Asp Lys
Tyr Gly Ile Ile Glu Gly Leu Met Thr Thr 165 170 175 Val His Ser Ile
Thr Ala Thr Gln Lys Thr Val Asp Gly Pro Ser His 180 185 190 Lys Asp
Trp Arg Gly Gly Arg Thr Ala Ser Gly Asn Ile Ile Pro Ser 195 200 205
Ser Thr Gly Ala Ala Lys Ala 210 215 <210> SEQ ID NO 27
<211> LENGTH: 953 <212> TYPE: DNA <213> ORGANISM:
Yarrowia lipolytica <400> SEQUENCE: 27 gcctctgaat actttcaaca
agttacaccc ttcattaatt ctcacgtgac acagattatt 60 aacgtctcgt
accaaccaca gattacgacc cattcgcagt cacagttcac tagggtttgg 120
gttgcatccg ttgagagcgg tttgttttta accttctcca tgtgctcact caggttttgg
180 gttcagatca aatcaaggcg tgaaccactt tgtttgagga caaatgtgac
acaaccaacc 240 agtgtcaggg gcaagtccgt gacaaagggg aagatacaat
gcaattactg acagttacag 300 actgcctcga tgccctaacc ttgccccaaa
ataagacaac tgtcctcgtt taagcgcaac 360 cctattcagc gtcacgtcat
aatagcgttt ggatagcact agtctatgag gagcgtttta 420 tgttgcggtg
agggcgattg gtgctcatat gggttcaatt gaggtggcgg aacgagctta 480
gtcttcaatt gaggtgcgag cgacacaatt gggtgtcacg tggcctaatt gacctcgggt
540 cgtggagtcc ccagttatac agcaaccacg aggtgcatgg gtaggagacg
tcaccagaca 600 atagggtttt ttttggactg gagagggttg ggcaaaagcg
ctcaacgggc tgtttgggga 660 gctgtggggg aggaattggc gatatttgtg
aggttaacgg ctccgatttg cgtgttttgt 720 cgctcctgca tctccccata
cccatatctt ccctccccac ctctttccac gataatttta 780 cggatcagca
ataaggttcc ttctcctagt ttccacgtcc atatatatct atgctgcgtc 840
gtccttttcg tgacatcacc aaaacacata caaaaatgcc taaactgatt ctgctgcgac
900 acggccagtc cgactggaac gagaagaacc tgttcaccgg atgggtcgac gtc 953
<210> SEQ ID NO 28 <211> LENGTH: 1537 <212> TYPE:
DNA <213> ORGANISM: Yarrowia lipolytica <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1507)..(1507) <223> OTHER INFORMATION: n is a, c, g, or t
<400> SEQUENCE: 28 gcctctgaat actttcaaca agttacaccc
ttcattaatt ctcacgtgac acagattatt 60 aacgtctcgt accaaccaca
gattacgacc cattcgcagt cacagttcac tagggtttgg 120 gttgcatccg
ttgagagcgg tttgttttta accttctcca tgtgctcact caggttttgg 180
gttcagatca aatcaaggcg tgaaccactt tgtttgagga caaatgtgac acaaccaacc
240 agtgtcaggg gcaagtccgt gacaaagggg aagatacaat gcaattactg
acagttacag 300 actgcctcga tgccctaacc ttgccccaaa ataagacaac
tgtcctcgtt taagcgcaac 360 cctattcagc gtcacgtcat aatagcgttt
ggatagcact agtctatgag gagcgtttta 420 tgttgcggtg agggcgattg
gtgctcatat gggttcaatt gaggtggcgg aacgagctta 480 gtcttcaatt
gaggtgcgag cgacacaatt gggtgtcacg tggcctaatt gacctcgggt 540
cgtggagtcc ccagttatac agcaaccacg aggtgcatgg gtaggagacg tcaccagaca
600 atagggtttt ttttggactg gagagggttg ggcaaaagcg ctcaacgggc
tgtttgggga 660 gctgtggggg aggaattggc gatatttgtg aggttaacgg
ctccgatttg cgtgttttgt 720 cgctcctgca tctccccata cccatatctt
ccctccccac ctctttccac gataatttta 780 cggatcagca ataaggttcc
ttctcctagt ttccacgtcc atatatatct atgctgcgtc 840 gtccttttcg
tgacatcacc aaaacacata caaaaatgcc taaactgatt ctgctgcgac 900
acggccagtc cgactggaac gagaagacct gttcaccgga tgggtcgacg tcaagctctc
960 cgagctcggc cacaccgagg ccaagcgagc cggtactctg ctcaaggagt
ccggtctcaa 1020 gccccagatt ctctacacct ccgagctctc tcgagccatc
cagaccgcca acattgctct 1080 ggatgaggcc gaccgactgt ggatccccac
caagcgatcg tggcgactca acgagcgaca 1140 ctacggcgct ctgcagggca
aggacaaggc cgccactctc gccgagtacg gccccgagca 1200 gttccagctc
tggcgacgat cttttgacgt ccctcctccc cctatcgctg acgacgacaa 1260
gtggtctcag tacaacgacg agcgatacca ggacatcccc aaggatattc tgcccaagac
1320 cgagtctctg aagctcgtga ttgaccgact ccttccttac tacaactccg
acattgtccc 1380 cgaccttaag gccggcaaga ccgtcctcat tgctgcccac
ggaaactccc tccgagctct 1440 cgtcaagcac ctcgacggta tctccgatga
cgatatcgcc gcccttaaca tccccaccgg 1500 tatcccnctc gtgctacgac
cttgatgaca acctcaa 1537 <210> SEQ ID NO 29 <211>
LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
YL33 <400> SEQUENCE: 29 tttccatggt acgtcctgta gaaaccccaa ccc
33 <210> SEQ ID NO 30 <211> LENGTH: 36 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Primer YL34 <400>
SEQUENCE: 30 cccttaatta atcattgttt gcctccctgc tgcggt 36 <210>
SEQ ID NO 31 <211> LENGTH: 19 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer TEF5' <400> SEQUENCE:
31 agagaccggg ttggcggcg 19 <210> SEQ ID NO 32 <211>
LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
TEF3' <400> SEQUENCE: 32 ttggatcctt tgaatgattc ttatactcag 30
<210> SEQ ID NO 33 <211> LENGTH: 29 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer XPR5' <400> SEQUENCE:
33 tttccgcggc ccgagattcc ggcctcttc 29 <210> SEQ ID NO 34
<211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer XPR3' <400> SEQUENCE: 34 tttccgcgga
cacaatatct ggtcaaattt c 31 <210> SEQ ID NO 35 <211>
LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
YL1 <400> SEQUENCE: 35 cagtgccaaa agccaaggca ctgagctcgt 30
<210> SEQ ID NO 36 <211> LENGTH: 31 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer YL2 <400> SEQUENCE: 36
gacgagctca gtgccttggc ttttggcact g 31 <210> SEQ ID NO 37
<211> LENGTH: 36 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer YL3 <400> SEQUENCE: 37
gtataagaat cattcaccat ggatccacta gttcta 36 <210> SEQ ID NO 38
<211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL4 <400> SEQUENCE: 38 tagaactagt
ggatccatgg tgaatgattc ttatac 36 <210> SEQ ID NO 39
<211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL23 <400> SEQUENCE: 39 atggatccac
tagttaatta actagagcgg ccgcca 36 <210> SEQ ID NO 40
<211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL24 <400> SEQUENCE: 40 tggcggccgc
tctagttaat taactagtgg atccat 36 <210> SEQ ID NO 41
<211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL9 <400> SEQUENCE: 41 tggtaaataa
atgatgtcga ctcaggcgac gacgg 35 <210> SEQ ID NO 42 <211>
LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
YL10 <400> SEQUENCE: 42 ccgtcgtcgc ctgagtcgac atcatttatt
tacca 35 <210> SEQ ID NO 43 <211> LENGTH: 971
<212> TYPE: DNA <213> ORGANISM: Yarrowia lipolytica
<400> SEQUENCE: 43 gacgcagtag gatgtcctgc acgggtcttt
ttgtggggtg tggagaaagg ggtgcttgga 60 gatggaagcc ggtagaaccg
ggctgcttgt gcttggagat ggaagccggt agaaccgggc 120 tgcttggggg
gatttggggc cgctgggctc caaagagggg taggcatttc gttggggtta 180
cgtaattgcg gcatttgggt cctgcgcgca tgtcccattg gtcagaatta gtccggatag
240 gagacttatc agccaatcac agcgccggat ccacctgtag gttgggttgg
gtgggagcac 300 ccctccacag agtagagtca aacagcagca gcaacatgat
agttgggggt gtgcgtgtta 360 aaggaaaaaa aagaagcttg ggttatattc
ccgctctatt tagaggttgc gggatagacg 420 ccgacggagg gcaatggcgc
catggaacct tgcggatatc gatacgccgc ggcggactgc 480 gtccgaacca
gctccagcag cgttttttcc gggccattga gccgactgcg accccgccaa 540
cgtgtcttgg cccacgcact catgtcatgt tggtgttggg aggccacttt ttaagtagca
600 caaggcacct agctcgcagc aaggtgtccg aaccaaagaa gcggctgcag
tggtgcaaac 660 ggggcggaaa cggcgggaaa aagccacggg ggcacgaatt
gaggcacgcc ctcgaatttg 720 agacgagtca cggccccatt cgcccgcgca
atggctcgcc aacgcccggt cttttgcacc 780 acatcaggtt accccaagcc
aaacctttgt gttaaaaagc ttaacatatt ataccgaacg 840 taggtttggg
cgggcttgct ccgtctgtcc aaggcaacat ttatataagg gtctgcatcg 900
ccggctcaat tgaatctttt ttcttcttct cttctctata ttcattcttg aattaaacac
960 acatcaacat g 971 <210> SEQ ID NO 44 <211> LENGTH:
878 <212> TYPE: DNA <213> ORGANISM: Yarrowia lipolytica
<400> SEQUENCE: 44 gcctctgaat actttcaaca agttacaccc
ttcattaatt ctcacgtgac acagattatt 60 aacgtctcgt accaaccaca
gattacgacc cattcgcagt cacagttcac tagggtttgg 120 gttgcatccg
ttgagagcgg tttgttttta accttctcca tgtgctcact caggttttgg 180
gttcagatca aatcaaggcg tgaaccactt tgtttgagga caaatgtgac acaaccaacc
240 agtgtcaggg gcaagtccgt gacaaagggg aagatacaat gcaattactg
acagttacag 300 actgcctcga tgccctaacc ttgccccaaa ataagacaac
tgtcctcgtt taagcgcaac 360 cctattcagc gtcacgtcat aatagcgttt
ggatagcact agtctatgag gagcgtttta 420 tgttgcggtg agggcgattg
gtgctcatat gggttcaatt gaggtggcgg aacgagctta 480 gtcttcaatt
gaggtgcgag cgacacaatt gggtgtcacg tggcctaatt gacctcgggt 540
cgtggagtcc ccagttatac agcaaccacg aggtgcatgg gtaggagacg tcaccagaca
600 atagggtttt ttttggactg gagagggttg ggcaaaagcg ctcaacgggc
tgtttgggga 660 gctgtggggg aggaattggc gatatttgtg aggttaacgg
ctccgatttg cgtgttttgt 720 cgctcctgca tctccccata cccatatctt
ccctccccac ctctttccac gataatttta 780 cggatcagca ataaggttcc
ttctcctagt ttccacgtcc atatatatct atgctgcgtc 840 gtccttttcg
tgacatcacc aaaacacata caaaaatg 878 <210> SEQ ID NO 45
<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL211 <400> SEQUENCE: 45 tttgtcgacg
cagtaggatg tcctgcacgg 30 <210> SEQ ID NO 46 <211>
LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
YL212 <400> SEQUENCE: 46 tttccatggt tgatgtgtgt ttaattcaag
aatg 34 <210> SEQ ID NO 47 <211> LENGTH: 33 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Primer YL203 <400>
SEQUENCE: 47 tttccatggt tgtatgtgtt ttggtgatgt cac 33 <210>
SEQ ID NO 48 <211> LENGTH: 33 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer YL204 <400> SEQUENCE:
48 tttgtcgacc gtttaagcgc aaccctattc agc 33 <210> SEQ ID NO 49
<211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL5 <400> SEQUENCE: 49 cccccctcga
ggtcgatggt gtcgataagc ttgatatcg 39 <210> SEQ ID NO 50
<211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL6 <400> SEQUENCE: 50 cgatatcaag
cttatcgaca ccatcgacct cgagggggg 39 <210> SEQ ID NO 51
<211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL7 <400> SEQUENCE: 51 caaccgattt
cgacagttaa ttaataattt gaatcga 37 <210> SEQ ID NO 52
<211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL8 <400> SEQUENCE: 52 tcgattcaaa
ttattaatta actgtcgaaa tcggttg 37 <210> SEQ ID NO 53
<211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer YL61
<400> SEQUENCE: 53 acaattccac acaacgtacg agccggaagc ata 33
<210> SEQ ID NO 54 <211> LENGTH: 33 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer YL62 <400> SEQUENCE: 54
tatgcttccg gctcgtacgt tgtgtggaat tgt 33 <210> SEQ ID NO 55
<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer GPDsense <400> SEQUENCE: 55 atacgagatc
gtcaaggg 18 <210> SEQ ID NO 56 <211> LENGTH: 26
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer
GPDantisense <400> SEQUENCE: 56 gcggccgcgg attgatgtgt gtttaa
26 <210> SEQ ID NO 57 <211> LENGTH: 1209 <212>
TYPE: DNA <213> ORGANISM: Fusarium monoliforme <400>
SEQUENCE: 57 atggcgactc gacagcgaac tgccaccact gttgtggtcg aggaccttcc
caaggtcact 60 cttgaggcca agtctgaacc tgtgttcccc gatatcaaga
ccatcaagga tgccattccc 120 gcgcactgct tccagccctc gctcgtcacc
tcattctact acgtcttccg cgattttgcc 180 atggtctctg ccctcgtctg
ggctgctctc acctacatcc ccagcatccc cgaccagacc 240 ctccgcgtcg
cagcttggat ggtctacggc ttcgtccagg gtctgttctg caccggtgtc 300
tggattctcg gccatgagtg cggccacggt gctttctctc tccacggaaa ggtcaacaat
360 gtgaccggct ggttcctcca ctcgttcctc ctcgtcccct acttcagctg
gaagtactct 420 caccaccgcc accaccgctt caccggccac atggatctcg
acatggcttt cgtccccaag 480 actgagccca agccctccaa gtcgctcatg
attgctggca ttgacgtcgc cgagcttgtt 540 gaggacaccc ccgctgctca
gatggtcaag ctcatcttcc accagctttt cggatggcag 600 gcgtacctct
tcttcaacgc tagctctggc aagggcagca agcagtggga gcccaagact 660
ggcctctcca agtggttccg agtcagtcac ttcgagccta ccagcgctgt cttccgcccc
720 aacgaggcca tcttcatcct catctccgat atcggtcttg ctctaatggg
aactgctctg 780 tactttgctt ccaagcaagt tggtgtttcg accattctct
tcctctacct tgttccctac 840 ctgtgggttc accactggct cgttgccatt
acctacctcc accaccacca caccgagctc 900 cctcactaca ccgctgaggg
ctggacctac gtcaagggag ctctcgccac tgtcgaccgt 960 gagtttggct
tcatcggaaa gcacctcttc cacggtatca ttgagaagca cgttgttcac 1020
catctcttcc ctaagatccc cttctacaag gctgacgagg ccaccgaggc catcaagccc
1080 gtcattggcg accactactg ccacgacgac cgaagcttcc tgggccagct
gtggaccatc 1140 ttcggcacgc tcaagtacgt cgagcacgac cctgcccgac
ccggtgccat gcgatggaac 1200 aaggactag 1209 <210> SEQ ID NO 58
<211> LENGTH: 402 <212> TYPE: PRT <213> ORGANISM:
Fusarium monoliforme <400> SEQUENCE: 58 Met Ala Thr Arg Gln
Arg Thr Ala Thr Thr Val Val Val Glu Asp Leu 1 5 10 15 Pro Lys Val
Thr Leu Glu Ala Lys Ser Glu Pro Val Phe Pro Asp Ile 20 25 30 Lys
Thr Ile Lys Asp Ala Ile Pro Ala His Cys Phe Gln Pro Ser Leu 35 40
45 Val Thr Ser Phe Tyr Tyr Val Phe Arg Asp Phe Ala Met Val Ser Ala
50 55 60 Leu Val Trp Ala Ala Leu Thr Tyr Ile Pro Ser Ile Pro Asp
Gln Thr 65 70 75 80 Leu Arg Val Ala Ala Trp Met Val Tyr Gly Phe Val
Gln Gly Leu Phe 85 90 95 Cys Thr Gly Val Trp Ile Leu Gly His Glu
Cys Gly His Gly Ala Phe 100 105 110 Ser Leu His Gly Lys Val Asn Asn
Val Thr Gly Trp Phe Leu His Ser 115 120 125 Phe Leu Leu Val Pro Tyr
Phe Ser Trp Lys Tyr Ser His His Arg His 130 135 140 His Arg Phe Thr
Gly His Met Asp Leu Asp Met Ala Phe Val Pro Lys 145 150 155 160 Thr
Glu Pro Lys Pro Ser Lys Ser Leu Met Ile Ala Gly Ile Asp Val 165 170
175 Ala Glu Leu Val Glu Asp Thr Pro Ala Ala Gln Met Val Lys Leu Ile
180 185 190 Phe His Gln Leu Phe Gly Trp Gln Ala Tyr Leu Phe Phe Asn
Ala Ser 195 200 205 Ser Gly Lys Gly Ser Lys Gln Trp Glu Pro Lys Thr
Gly Leu Ser Lys 210 215 220 Trp Phe Arg Val Ser His Phe Glu Pro Thr
Ser Ala Val Phe Arg Pro 225 230 235 240 Asn Glu Ala Ile Phe Ile Leu
Ile Ser Asp Ile Gly Leu Ala Leu Met 245 250 255 Gly Thr Ala Leu Tyr
Phe Ala Ser Lys Gln Val Gly Val Ser Thr Ile 260 265 270 Leu Phe Leu
Tyr Leu Val Pro Tyr Leu Trp Val His His Trp Leu Val 275 280 285 Ala
Ile Thr Tyr Leu His His His His Thr Glu Leu Pro His Tyr Thr 290 295
300 Ala Glu Gly Trp Thr Tyr Val Lys Gly Ala Leu Ala Thr Val Asp Arg
305 310 315 320 Glu Phe Gly Phe Ile Gly Lys His Leu Phe His Gly Ile
Ile Glu Lys 325 330 335 His Val Val His His Leu Phe Pro Lys Ile Pro
Phe Tyr Lys Ala Asp 340 345 350 Glu Ala Thr Glu Ala Ile Lys Pro Val
Ile Gly Asp His Tyr Cys His 355 360 365 Asp Asp Arg Ser Phe Leu Gly
Gln Leu Trp Thr Ile Phe Gly Thr Leu 370 375 380 Lys Tyr Val Glu His
Asp Pro Ala Arg Pro Gly Ala Met Arg Trp Asn 385 390 395 400 Lys Asp
<210> SEQ ID NO 59 <211> LENGTH: 36 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer P192 <400> SEQUENCE: 59
aaatatgcgg ccgcacaatg gcgactcgac agcgaa 36 <210> SEQ ID NO 60
<211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer P193 <400> SEQUENCE: 60 tttatagcgg
ccgcctagtc cttgttccat cgca 34
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