U.S. patent application number 11/185301 was filed with the patent office on 2006-05-04 for ammonium transporter promoter for gene expression in oleaginous yeast.
Invention is credited to Zhixiong Xue, Quinn Qun Zhu.
Application Number | 20060094102 11/185301 |
Document ID | / |
Family ID | 36262501 |
Filed Date | 2006-05-04 |
United States Patent
Application |
20060094102 |
Kind Code |
A1 |
Xue; Zhixiong ; et
al. |
May 4, 2006 |
Ammonium transporter promoter for gene expression in oleaginous
yeast
Abstract
The promoter region associated with the Yarrowia lipolytica
ammonium transporter (yat1) gene has been found to be particularly
effective for the expression of heterologous genes in oleaginous
yeast. The promoter regions of the instant invention have been
shown to be advantageously inducible under oleaginous conditions
(i.e., nitrogen limitation) and are useful to drive expression of
genes involved in the production of .omega.-3 and .omega.-6 fatty
acids.
Inventors: |
Xue; Zhixiong; (Chadds Ford,
PA) ; Zhu; Quinn Qun; (West Chester, PA) |
Correspondence
Address: |
E I DU PONT DE NEMOURS AND COMPANY;LEGAL PATENT RECORDS CENTER
BARLEY MILL PLAZA 25/1128
4417 LANCASTER PIKE
WILMINGTON
DE
19805
US
|
Family ID: |
36262501 |
Appl. No.: |
11/185301 |
Filed: |
July 20, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60624812 |
Nov 4, 2004 |
|
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Current U.S.
Class: |
435/201 ;
435/254.2; 435/483 |
Current CPC
Class: |
C12N 15/81 20130101 |
Class at
Publication: |
435/201 ;
435/254.2; 435/483 |
International
Class: |
C12N 9/26 20060101
C12N009/26; C12N 15/74 20060101 C12N015/74; C12N 1/18 20060101
C12N001/18 |
Claims
1. A method for the expression of a coding region of interest in a
transformed yeast comprising: c) providing a transformed yeast
having a genetic construct comprising: (i) a promoter region of a
Yarrowia yat1 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, d) growing
the transformed yeast of step (a) under conditions whereby the
genetic construct of step (a) is expressed.
2. A method according to claim 1 wherein the Yarrowia yat1 gene is
isolated from Yarrowia lipolytica.
3. A method according to claim 1 wherein the promoter region of the
yat1 gene is contained within a nucleic acid molecule as set forth
in SEQ ID NO:3.
4. A method according to claim 3 wherein the promoter region
contains at least one mutation that does not diminish its promoter
activity.
5. A method according to claim 4 wherein the promoter activity is
at least about 20% to at least about 400% of the promoter activity
of the wildtype promoter activity.
6. A method according to claim 5 wherein the promoter activity is
determined by histochemical assay.
7. A method according to claim 1 wherein the transformed yeast 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. A method according to claim 8, wherein the oleaginous yeast is
selected from the group consisting of: Yarrowia lipolytica ATCC
#20362, Yarrowia lipolytica ATCC #8862, Yarrowia lipolytica ATCC
#18944, Yarrowia lipolytica ATCC #76982 and Yarrowia lipolytica
LGAM S(7)1.
10. A method according to claim 1 wherein the coding region of
interest encodes a polypeptide selected from the group consisting
of: desaturases, elongases, acyltransferases, 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 according to claim 1 wherein the conditions whereby
the genetic construct of step (a) is expressed are those of
nitrogen-limitation.
12. A method according to claim 11 wherein the conditions of
nitrogen-limitation are conditions that induce oleaginy in the
transformed yeast.
13. A method for the production of an .omega.-3 or an .omega.-6
fatty acid comprising: d) providing a transformed oleaginous yeast
comprising a genetic construct, comprising: (i) a promoter region
of a Yarrowia yat1 gene; and, (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; e) culturing 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, f)
optionally recovering the .omega.-3 or .omega.-6 fatty acid.
14. A method according to claim 13 wherein the Yarrowia yat1 gene
is isolated from Yarrowia lipolytica.
15. A method according to claim 13 wherein the promoter region of
the yat1 gene is contained within a nucleic acid molecule as set
forth in SEQ ID NO:3.
16. A method according to claim 13 wherein the coding region of
interest encodes a polypeptide selected from the group consisting
of: desaturases and elongases.
17. A method according to claim 16 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, .DELTA.8 desaturase and
.DELTA.4 desaturase.
18. A method according to claim 13 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.
19. A method according to claim 18 wherein the oleaginous yeast is
Yarrowia lipolytica.
20. A method according to claim 19, wherein the Yarrowia lipolytica
is a strain selected from the group consisting of: Yarrowia
lipolytica ATCC #20362, Yarrowia lipolytica ATCC #8862, Yarrowia
lipolytica ATCC #18944, Yarrowia lipolytica ATCC #76982 and
Yarrowia lipolytica LGAM S(7)1.
21. A method according to claim 13 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, docosahexaenoic acid, eicosadienoic acid and eicosatrienoic
acid.
22. A method according to claim 13 wherein the conditions whereby
the at least one enzyme of the .omega.-3/.omega.-6 fatty acid
biosynthetic pathway is expressed are those of nitrogen
limitation.
23. A method according to claim 22 wherein the conditions of
nitrogen-limitation are conditions that induce oleaginy in the
transformed oleaginous yeast.
Description
FIELD OF THE INVENTION
[0001] This invention is in the field of biotechnology. More
specifically, this invention pertains to a promoter region isolated
from Yarrowia lipolytica that is useful for gene expression in
oleaginous yeast.
BACKGROUND OF THE INVENTION
[0002] 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).
[0003] 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; lipases; polyhydroxy-alkanoates; citric acid;
erythritol; 2-oxoglutaric acid; .gamma.-decalactone;
.gamma.-dodecalactone; and pyruvic acid. More 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 Y. 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 (see WO 2004/101757 and
co-pending U.S. patent application Ser. No. 60/624,812).
[0004] 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 appropriate regulatory sequences (i.e., promoters)
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.
[0005] A variety of strong promoters have been isolated from
Yarrowia lipolytica that are useful for heterologous gene
expression in yeast. 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. U.S. Pat. No. 6,265,185 (Muller et al.) describes
promoters 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.
WO 2005/003310 describe regulatory sequences (e.g., promoters,
introns) of the glyceraldehyde-3-phosphate dehydrogenase (gpd) and
phosphoglycerate mutase (gpm) genes; WO 2005/049805 describes
regulatory sequences (e.g., promoters, introns) of the
fructose-bisphosphate aldolase (fba) gene; and, co-pending U.S.
patent application Ser. No. 60/610,060 describes promoters of the
glycerol-3-phosphate O-acyltransferase (gpat) gene. Similarly,
Juretzek et al. (Biotech. Bioprocess Eng., 5:320-326 (2000))
compares the glycerol-3-phosphate dehydrogenase (G3P), isocitrate
lyase (ICL1), 3-oxo-acyl-CoA thiolase (POT1) and acyl-CoA oxidase
(POX1, POX2 and POX5) promoters with respect to their regulation
and activities during growth on different carbon sources.
[0006] 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.
[0007] Applicants have solved the stated problem by identifying the
YAT1 promoter from Yarrowia lipolytica, which is responsible for
driving expression of the gene (yat1) encoding an ammonium
transporter (YAT1). Advantageously, the promoter is useful for
regulated expression of heterologous genes in Yarrowia, has
improved activity with respect to the TEF promoter and is inducible
under oleaginous conditions (i.e., nitrogen limitation).
SUMMARY OF THE INVENTION
[0008] The present invention provides methods for the regulated
expression of a coding region of interest in a transformed yeast,
using a promoter region of an ammonium transporter (yat1) gene.
Accordingly the invention provides a method for the expression of a
coding region of interest in a transformed yeast comprising: [0009]
a) providing a transformed yeast having a genetic construct
comprising: [0010] (i) a promoter region of a Yarrowia yat1 gene;
and, [0011] (ii) a coding region of interest expressible in the
yeast cell; [0012] wherein the promoter region is operably linked
to the coding region of interest; and, [0013] b) growing the
transformed yeast of step (a) under conditions whereby the genetic
construct of step (a) is expressed.
[0014] In another embodiment mutant yat1 promoters are provided
exhibiting mutations that demonstrate between 20% and 400%
increased promoter activity as compared with the wildtype
promoter.
[0015] In an alternate embodiment the invention provides a method
for the production of an .omega.-3 or an .omega.-6 fatty acid
comprising: [0016] a) providing a transformed oleaginous yeast
comprising a genetic construct, comprising: [0017] (i) a promoter
region of a Yarrowia yat1 gene; and, [0018] (ii) a coding region
encoding at least one enzyme of the .omega.-3/.omega.-6 fatty acid
biosynthetic pathway; [0019] wherein the promoter region and coding
region are operably linked; [0020] b) culturing 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, [0021] c) optionally recovering the .omega.-3 or
.omega.-6 fatty acid.
[0022] Preferred conditions suitable to induce expression of the
genetic constructs of the invention are conditions of
nitrogen-limitation; and, in a more preferred embodiment, the
nitrogen-limiting conditions are those that induce oleaginy.
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS
[0023] FIG. 1 provides plasmid maps for the following: (A) pY5-30;
(B) pDMW214; (C) pYGPAT-GUS; and (D) pYAT-GUS, respectively.
[0024] FIG. 2 graphically represents the relationship between SEQ
ID NOs:12, 15, 22, 23 and 24, each of which relates to
glycerol-3-phosphate O-acyltransferase (GPAT) in Y. lipolytica.
[0025] FIG. 3A diagrams the development of Yarrowia lipolytica
strain Y2034, producing up to 10% ARA in the total lipid fraction.
FIG. 3B provides a plasmid map for pKUNF12T6E; and FIG. 3C provides
a plasmid map for pDMW232.
[0026] FIGS. 4A, 4B and 4C illustrate the relative promoter
activities of YAT1, TEF, GPAT and FBAIN in Y. lipolytica grown in
various media as determined by histochemical staining.
[0027] FIG. 5 illustrates the .omega.-3/.omega.-6 fatty acid
biosynthetic pathway.
[0028] The invention can be more fully understood from the
following detailed description and the accompanying sequence
descriptions, which form a part of this application.
[0029] 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.
[0030] SEQ ID NOs:1-3, 10-12, 15, 23, 24, and 27-37 correspond to
ORFs (i.e., encoding genes or proteins), promoters and plasmids, as
identified in Table 1. TABLE-US-00001 TABLE 1 Summary Of Nucleotide
And Protein SEQ ID Numbers Protein Nucleotide SEQ ID Description
SEQ ID NO NO Yarrowia lipolytica ammonium transporter 1 2 (yat1)
(1461 bp) (486 AA) (GenBank Accession No. XM_504457) YAT1 promoter
3 -- (778 bp) Plasmid pY5-30 10 -- (8953 bp) FBAIN promoter 11 --
(973 bp) Yarrowia lipolytica ORF YALI-CDS1055.1 -- 12 (GenBank
Accession No. CAG81570) (727 AA) Yarrowia lipolytica
glycerol-3-phosphate O- 15 -- acyltransferase (gpat) gene (2184 bp)
Yarrowia lipolytica gpat gene: -1678 to 23 -- +2181 region (3862
bp) GPAT promoter 24 -- (1130 bp) Plasmid pKUNF12T6E 27 -- 12,649
bp) Synthetic elongase gene derived from 28 29 Mortierella alpina,
codon-optimized for (957 bp) (318 AA) expression in Yarrowia
lipolytica Synthetic .DELTA.6 desaturase, derived from 30 31
Mortierella alpina, codon-optimized for (1374 bp) (457 AA)
expression in Yarrowia lipolytica FBA promoter 32 -- (1001 bp)
Fusarium moniliforme .DELTA.12 desaturase 33 34 (1434 bp) (477 AA)
Synthetic elongase gene derived from 35 36 Thraustochytrium aureum,
codon-optimized (819 bp) (272 AA) for expression in Yarrowia
lipolytica Plasmid pDMW232 37 -- (10,945 bp)
[0031] SEQ ID NOs:4, 5 and 6 correspond to BD-Clontech's Creator
Smart.RTM. cDNA library kit primers SMART IV oligonucleotide,
CDSIII/3' PCR primer and 5'-PCR primer, respectively.
[0032] SEQ ID NO:7 corresponds to the M13 forward primer used for
sequencing of Y. lipolytica cDNA.
[0033] SEQ ID NOs:8 and 9 correspond to primers 27203-F and
27203-R, respectively, used for amplifying the 5' upstream region
of the yat1 gene.
[0034] SEQ ID NOs:13 and 14 correspond to the degenerate primers
YGPAT-F and YGPAT-R, respectively, used for amplifying Y.
lipolytica ORF YALI-CDS1055.1.
[0035] SEQ ID NOs:16 and 17 correspond to the Genome Walker adaptor
used to isolate the GPAT promoter region by genome-walking.
[0036] SEQ ID NOs:18-21 correspond to the PCR primers used in
genome-walking: Adaptor-1, YGPAT-5R-1, Nested Adaptor Primer 2 and
YGPAT-5R-2, respectively.
[0037] SEQ ID NO:22 corresponds to a 1781 bp fragment contained
within plasmid pEcoRV-G-5, the fragment containing a 1678 bp region
upstream of the translation initiation codon `ATG` of the gpat
gene.
[0038] SEQ ID NOs:25 and 26 correspond to primers GPAT-5-1 and
GPAT-5-2, respectively, used to amplify the GPAT promoter.
DETAILED DESCRIPTION OF THE INVENTION
[0039] In support of the invention described herein the following
commonly owned patents, and patent applications are incorporated
herein by reference in their entirety: 10/840,579 (filed May 6,
2004), 10/869,630 (filed Jun. 16, 2004), 10/987,548 (filed Nov. 12,
2004), U.S. Patent Provisional Application No. 60/610,060 (filed
Sep. 15, 2004), and U.S. Patent Provisional Application No.
60/624,812 (filed Nov. 4, 2004).
[0040] In accordance with the subject invention, Applicants
describe the characterization of a promoter from an oleaginous
yeast, Yarrowia lipolytica. This promoter region, isolated upstream
of the ammonium transporter (yat1) gene and inducible under
oleaginous conditions (i.e., nitrogen limitation), is useful for
genetic engineering in Y. lipolytica and other yeast for the
production of heterologous polypeptides.
[0041] 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).
[0042] Thus, the present invention advances the art by providing
methods for the regulated expression of a coding region of interest
in a transformed yeast comprising: a) providing a transformed yeast
having a chimeric gene comprising (i) a promoter region of a
Yarrowia yat1 gene; and, (ii) a coding region of interest
expressible in the yeast, wherein the promoter region is operably
linked to the coding region of interest; b) growing the transformed
yeast of step (a) under conditions whereby the chimeric gene is
expressed; and, c) optionally isolating the gene product from the
cultivation medium. In preferred embodiments, the YAT1 promoter
region comprises a sequence as set forth in SEQ ID NO:3.
Definitions
[0043] In this disclosure, a number of terms and abbreviations are
used. The following definitions are provided.
[0044] "Ammonium transporter" is abbreviated YAT.
[0045] "Open reading frame" is abbreviated ORF.
[0046] "Polymerase chain reaction" is abbreviated PCR.
[0047] "Polyunsaturated fatty acid(s)" is abbreviated PUFA(s).
[0048] 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)).
[0049] 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.
[0050] The term "ammonium transporter" refers to a family of
transporters whose physiological role within a cell is to scavenge
external ammonium for use as a nitrogen source (and, under some
circumstances, to incorporate ammonium that leaks out of cells),
based on studies of the MEP family of ammonium transporters in
Saccharomyces cerevisiae (A. M. Marini et al., EMBO J.,
13(15):3456-3463 (1994); Mol Cell Biol 17(8):4282-93 (1997)). In
general, the proteins are subject to nitrogen control and each has
different kinetic properties, specificities and regulation; for
example, the three S. cerevisiae isozymes are characterized as
follows: MEP1, a low affinity/high capacity ammonia transporter
(K.sub.m, 5-10 .mu.M); MEP2, a high affinity/low capacity ammonia
transporter (K.sub.m, 1-2 .mu.M); and MEP3, a low affinity ammonia
transporter (K.sub.m, 1.4-2.1 mM). Other known members of this
family include: the high affinity ammonium transporter (amt1), the
ammonium and methylammonium transport system, the putative ammonium
transporter amtB and nrgA.
[0051] As used herein, the term "YAT1" refers to an ammonium
transporter enzyme (TC 2.A.49) encoded by the yat1 gene and
isolated from Yarrowia lipolytica (GenBank Accession No.
XM.sub.--504457). The sequence disclosed therein
(/locus_tag="YALI0E27203g") was identified as a hypothetical
protein having similarity "to sp|P41948 Saccharomyces cerevisiae
YNL142w MEP2, hypothetical start". The Y. lipolytica yat1 gene is
presented herein as SEQ ID NO:1, while the corresponding YAT1
protein is provided herein as SEQ ID NO:2.
[0052] The term "YAT1 promoter" or "YAT1 promoter region" refers to
the 5' upstream untranslated region in front of the `ATG`
translation initiation codon of yat1 and that is necessary for
expression. An example of a suitable YAT1 promoter region is
provided as SEQ ID NO:3, but this is not intended to be limiting in
nature. One skilled in the art will recognize that since the exact
boundaries of the YAT1 promoter sequence have not been completely
defined, DNA fragments of increased or diminished length may have
identical promoter activity.
[0053] The term "GPAT" refers to a glycerol-3-phosphate
O-acyltransferase enzyme (E.C. 2.3.1.15) encoded by the gpat gene
and which converts acyl-CoA and sn-glycerol 3-phosphate to CoA and
1-acyl-sn-glycerol 3-phosphate (the first step of phospholipid
biosynthesis). The term "GPAT promoter" or "GPAT promoter region"
refers to the 5' upstream untranslated region in front of the `ATG`
translation initiation codon of gpat and that is necessary for
expression. One example of a suitable GPAT promoter region is
provided as SEQ ID NO:24, but this is not intended to be limiting
in nature (see co-pending U.S. patent application Ser. No.
60/610,060).
[0054] The term "FBA1" refers to a fructose-bisphosphate aldolase
enzyme (E.C. 4.1.2.13) encoded by the fba1 gene and which converts
D-fructose 1,6-bisphosphate into glycerone phosphate and
D-glyceraldehyde 3-phosphate. The term "FBAIN promoter" or "FBAIN
promoter region" refers to the 5' upstream untranslated region in
front of the `ATG` translation initiation codon of fba1 and that is
necessary for expression, plus a portion of 5' coding region
comprising an intron of the fba1 gene. An example of a suitable
FBAIN promoter region is provided as SEQ ID NO:11, but this is not
intended to be limiting in nature (see WO 2005/049805).
[0055] 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, as foe example by
histochemical means.
[0056] The term "conditions of nitrogen-limitation" refers to a
medium having a low concentration of nitrogen, wherein the nitrogen
may be supplied from an inorganic (e.g., (NH.sub.4).sub.2SO.sub.4)
or organic (e.g., urea or glutamate) source, or a medium having no
nitrogen source. Although one skilled in the art will be able to
determine an appropriate low concentration of nitrogen suitable to
induce the YAT1 promoter of the present invention, in one
embodiment a preferred medium would be one characterized as having
a high carbon to nitrogen (i.e., C:N) ratio and about 0.1% or less
ammonium sulfate or other suitable ammonium salts.
[0057] 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.
[0058] The term "genetic construct" is a non-limiting term meaning
any contiguous series of nucleic acids capable of being expressed
in a host organism. A genetic construct may include but is not
limited to an open reading frame, an open reading frame operably
linked to regulatory sequences, or a wildtype or mutant gene.
Genetic constructs may encode poylpeptides or be nucleic acid
fragments or molecules that are oriented for antisense
expression.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] The instant specification teaches nucleotide sequences
encoding a particular microbial promoter region. 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.
[0063] 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.
[0064] 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.
[0065] 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.) Humania: 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.
[0066] 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.
[0067] "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. 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.
[0068] "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.
[0069] "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 YAT1
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.
[0070] "Coding sequence" refers to a DNA sequence that codes for a
specific amino acid sequence.
[0071] "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.
[0072] "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.
[0073] 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").
[0074] 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.
[0075] "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.
[0076] 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.
[0077] 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.
[0078] "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.
[0079] 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. "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.
[0080] 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.
[0081] 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, 2.sup.nd 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 YAT1 and the YAT1 Promoter in Yarrowia
lipolytica
[0082] Ammonium transporters are a family of transporters whose
physiological role within a cell is to scavenge external ammonium
for use as a nitrogen source (and, under some circumstances, to
incorporate ammonium that leaks out of cells). In general, the
proteins are subject to nitrogen control and each have different
kinetic properties, specificities and regulation; for example, the
three S. cerevisiae MEP1, MEP2, and MEP3 isozymes have been
extensively characterized by A. M. Marini et al. (EMBO J., 13(15):
3456-3463 (1994); Mol Cell Biol 17(8):4282-93 (1997)) as a low
affinity/high capacity ammonia transporter, a high affinity/low
capacity ammonia transporter, and a low affinity ammonia
transporter, respectively.
[0083] A cDNA library was constructed from Yarrowia lipolytica
cells grown in an oleaginous medium containing no nitrogen source
for 4 hrs (i.e., oleaginous conditions that promoter oil
production). The relative abundance of each species of cDNA was
then examined to identify those genes that were highly expressed
under the oleaginous conditions. One of the genes that appeared
multiple times (16/9984 or 0.16%) was GenBank Accession No.
XM.sub.--504457, locus_tag="YALI0E27203g". This gene was annotated
as a hypothetical protein having similarity "to sp|P41948
Saccharomyces cerevisiae YNL142w MEP2 [the high affinity, low
capacity ammonia transporter; GenBank Accession No. X83608],
hypothetical start".
[0084] Based on sequence comparison to the MEP1, MEP2 and MEP3
genes (supra), the Applicants hypothesized that the Y. lipolytica
ORF likely encoded an ammonium transporter and designated the gene
as the "yat1" gene (SEQ ID NOs:1 and 2). The "YAT1 promoter" was
identified as the 5' upstream untranslated region in front of the
`ATG` translation initiation codon of yat1 and that is necessary
for expression. According to the invention here, this putative YAT1
promoter region will be useful for driving expression of any
suitable coding region of interest in a transformed yeast cell. In
general, a promoter useful in an oleaginous yeast should meet the
following criteria: [0085] 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. [0086] 2.) Activity in a medium suitable
for expression of the coding region of interest, and high enzymatic
activity of that coding region of interest. [0087] 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. [0088] 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. [0089] 5.) Activity in the
stationary phase of growth in oleaginous yeast hosts for
accumulation of PUFAs.
[0090] Additionally, it is preferable for novel yeast promoters to
possess differences in activity with respect to the known Yarrowia
lipolytica TEF (U.S. Pat. No. 6,265,185), XPR2 (U.S. Pat. No.
4,937,189; EP220864; EP832258), GPD, GPM (U.S. Ser. No.
10/869,630), FBA, FBAIN (U.S. Ser. No. 10/987,548), and GPAT (U.S.
Provisional Patent Application No. 60/610,060) promoters and/or the
G3P, ICL1, POT1, POX1, POX2 and POX5 promoters (Juretzek et al.,
Biotech. Bioprocess Eng., 5:320-326 (2000)).
[0091] A comparative study of the TEF, FBAIN and GPAT promoters and
the YAT1 promoter of the instant invention is provided in Examples
8 and 9. As shown in Table 7 of Example 9, the yeast promoter of
the present invention has improved activity compared to the TEF
promoter (i.e., 1.3:1 nmoles of 4-methylumbelliferone per minute
per mg of total protein) under conditions where nitrogen is not
limiting (i.e., minimal medium containing ammonium sulfate as the
nitrogen source). Furthermore, under conditions of nitrogen
limitation, the YAT1 promoter is significantly induced such that
the activity is .about.20-28 times greater than that of TEF; thus,
when grown in such medium, the activity of the YAT1 promoter is
comparable to that of the strong FBAIN promoter. Based on these
results, the Applicants characterize the YAT1 promoter as the first
promoter identified within Yarrowia that is inducible under
oleaginous conditions (i.e., nitrogen limitation).
[0092] An example of a suitable YAT1 promoter region is provided as
SEQ ID NO:3 (comprising the -778 to -1 region of the Y. lipolytica
yat1 gene (wherein the `A` position of the `ATG` translation
initiation codon is designated as +1)), but this is not intended to
be limiting in nature. One skilled in the art will recognize that
since the exact boundaries of the YAT1 promoter sequence have not
been completely defined, DNA fragments of increased or diminished
length may have identical promoter activity. For example, in an
alternate embodiment, the YAT1 promoter will comprise nucleotides
-500 to -1 of SEQ ID NO:3, thereby permitting relatively strong
promoter activity; in another embodiment, the -100 to -1 region of
SEQ ID NO:3 should be sufficient for basal activity of the
promoter. Likewise, the promoter region 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 -1000 to +1 region of the yat1 gene (e.g.,
nucleotide bases 3,222,879 to 3,223,879 of GenBank Accession No.
CR382131, comprising the complete nucleotide sequence of chromosome
E of strain CLIB99 of Y. lipolytica).
[0093] 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 YAT1 promoter region described herein as
SEQ ID NO:3.
[0094] 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 promoter of the instant
invention (wherein such mutations may include deletions, insertions
and point mutations, or combinations thereof). This would permit
production of a putative promoter having a more desirable level of
promoter activity in the host cell. Or, if desired, the regions of
a nucleotide of interest important for promoter activity can be
determined through routine mutagenesis, expression of the resulting
mutant promoters and determination of their activities. An overview
of these techniques is described in U.S. Ser. No. 10/869630. All
such mutant promoters that are derived from the instant YAT1
promoter described herein are within the scope of the present
invention.
[0095] Promoter activity is typically measured against the activity
of the wild type promoter under similar conditions. Promoter
activity is generally measured as a function of gene expression and
may be determined in a variety of ways including gene expression
profiling, measurement of the level of protein expression by SDS
gel or other means, or the measurement of reporter activity where
reporter gene fusions have been created.
Isolation of Homologs of YAT1 and the YAT1 Putative Promoter
Region
[0096] It will be appreciated by a person of skill in the art that
the promoter regions of the present invention have homologs in a
variety of yeast species; and, the use of the promoters for
regulated, heterologous gene expression are not limited to those
promoters 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.
[0097] 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.
[0098] As is well known in the art, isolation of homologous
promoter regions using sequence-dependent protocols is readily
possible using various techniques; and, these techniques can rely
on either the direct identification of a promoter having homology
to the YAT1 promoter of the invention or the indirect
identification of a promoter by initial identification of gene
having significant homology to the yat1 gene and then analysis of
the 5' upstream sequence of the homologous gene. 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.
[0099] For example, putative promoter regions to those of the
instant invention could be isolated by using all or a portion of
the YAT1 nucleic acid molecules (e.g., corresponding to the
promoter, gene or a combination thereof) 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.
[0100] 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.).
[0101] Generally two short segments of the instant sequences may be
used in PCR protocols to amplify longer nucleic acid molecules
encoding homologous polynucleotides from DNA or RNA. The PCR 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.
[0102] 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.
[0103] 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).
[0104] 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
[0105] 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 yat1 gene (SEQ ID NO:1). The promoter regions may be
identified from the upstream sequences of yat1 and its homologs and
isolated according to common methods (Maniatis, supra). Once a
promoter region is identified and isolated (e.g., SEQ ID NO:3), it
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.
[0106] In a further aspect, the invention provides a method of
expressing a coding region of interest in a transformed yeast,
wherein a transformed yeast is provided having a chimeric gene
comprising: (i) a promoter region of a Yarrowia yat1 gene; and,
(ii) a coding region of interest expressible in the yeast, wherein
the promoter region is operably linked to the coding region of
interest; and the transformed yeast is grown under conditions
wherein the chimeric gene is expressed. The polypeptide so produced
can optionally be recovered from the culture.
[0107] 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 a promoter region derived from
the yat1 gene for production of any specific coding region of
interest suitable for regulated expression in a desirable yeast
host cell. These chimeric genes could then be introduced into
appropriate microorganisms by integration via transformation to
provide 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. The
coding region of interest to be expressed can then be cloned
downstream from the promoter. Once the recombinant host is
established, regulated gene expression can be accomplished by
growing the cells under suitable conditions (infra).
[0108] Suitable Coding Regions of Interest
[0109] Useful chimeric genes will include the promoter region of
the yat1 gene 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.
[0110] 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 acyltransferases, 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.
[0111] 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.
These coding regions include desaturases and elongases (e.g., see
WO 2004/101757 for a partial review of available genes in GenBank
and/or the patent literature and considerations for choosing a
specific polypeptide having desaturase or elongase activity).
Components of Vectors/DNA Cassettes
[0112] 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.
[0113] 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.
[0114] 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.
[0115] 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.)
whether the gene is plasmid-borne or integrated into the genome of
the host cell and the number of copies of the cloned gene [e.g.,
additional copies a particular coding region of interest (operably
linked to the promoter of the instant invention) may be introduced
into the host to increase expression]; 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 [e.g., expression of
the coding region of interest 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)]; and 6.) the codon usage within the cloned gene, such
that its frequency approaches the frequency of preferred codon
usage of the host cell [e.g. translational efficiency of the
encoded mRNAs can be increased by replacement of codons in the
native gene with those for optimal gene expression in the selected
host microorganism, to thereby substantially enhance the expression
of the foreign gene encoding the polypeptide]. 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 the yat1 gene as defined herein or a mutant
promoter thereof, operably linked to a suitable coding region of
interest.
[0116] Transformation of Yeast Cells
[0117] Once an appropriate chimeric gene has been constructed that
is suitable for regulated 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.
[0118] 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.
[0119] 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. No.
4,880,741 and No. 5,071,764 and Chen, D. C. et al. (Appl Microbiol
Biotechnol. 48(2):232-235 (1997)).
[0120] 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 various selection
techniques, as described in commonly owned U.S. Ser. No.
10/869,630.
Preferred Hosts
[0121] Preferred host cells for expression of coding regions of
interest operably linked to the instant promoter fragments 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).
[0122] 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 #20362 was the particular strain from which the
YAT1 promoter was isolated therefrom.
Industrial Production using Transformed Yeast Expressing a Suitable
Coding Region of Interest
[0123] In general, media conditions that may be optimized for
regulated 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.)).
[0124] 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.
[0125] Nitrogen may be supplied from an inorganic (e.g.,
(NH.sub.4).sub.2SO.sub.4) or organic source (e.g., urea or
glutamate). Although the YAT1 promoter is active in media
containing nitrogen (e.g., up to about 0.5% ammonium sulfate), the
activity of the promoter increases when the host cell is grown in
nitrogen-limiting conditions (e.g., in medium containing very low
levels of ammonium, or lacking ammonium). Thus, a preferred medium
would be one that contains less than about 0.1% ammonium sulfate,
or other suitable ammonium salts.
[0126] In a more preferred embodiment, the YAT1 promoter is induced
when the host cell is grown in media with a high carbon to nitrogen
(i.e., C:N) ratio, such as a high glucose medium containing about
8-12% glucose, and about 0.1% or less ammonium sulfate. These
conditions are also sufficient to induce oleaginy in those yeast
that are oleaginous (e.g., Yarrowia lipolytica).
[0127] 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.
[0128] Preferred growth media in the present invention are common
commercially prepared media, such as minimal media made with 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.
[0129] 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
regulated expression of the coding region of interest. Furthermore,
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, as is well
known in the art (see, e.g., Thomas D. Brock in Biotechnoloqy: A
Textbook of Industrial Microbiology, 2.sup.nd ed., (1989) Sinauer
Associates: Sunderland, Mass.; or Deshpande, Mukund V., Appl.
Biochem. Biotechnol., 36:227 (1992), each herein incorporated by
reference).
DESCRIPTION OF PREFERRED EMBODIMENTS
[0130] Although the promoters of the present invention will be
suitable for regulated 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 high levels of 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.
[0131] 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. Additional details
concerning the differentiation between "saturated fatty acids"
versus "unsaturated fatty acids", "monounsaturated fatty acids"
versus "polyunsaturated fatty acids" (or "PUFAs"), and ".omega.-6
fatty acids" (.omega.-6 or n-6) versus ".omega.-3 fatty acids"
(.omega.-3 or n-3) are provided in commonly owned U.S. Ser. No.
10/840,579.
[0132] Nomenclature used to describe PUFAs in the present
disclosure is shown below in Table 2. In the column titled
"Shorthand Notation", the omega-reference system is 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). The
remainder of the Table summarizes the common names of .omega.-3 and
c)-6 fatty acids, the abbreviations that will be used throughout
the remainder of the specification, and each compounds' chemical
name. TABLE-US-00002 TABLE 2 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
Eicosadienoic EDA cis-11,14-eicosadienoic 20:2 .omega.-6
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 Eicosatrienoic ETrA cis-11,14,17- 20:3 .omega.-3
eicosatrienoic 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 Omega-3 and Omega-6 Fatty Acids
[0133] The process of de novo synthesis of palmitate (16:0) in
oleaginous microorganisms is described in commonly owned U.S. Ser.
No. 10/840,579. This fatty acid is the precursor of longer-chain
saturated and unsaturated fatty acid derivates, which are formed
through the action of elongases and desaturases. For example,
palmitate is converted to its unsaturated derivative [palmitoleic
acid (16:1)] by the action of a .DELTA.9 desaturase; similarly,
palmitate is elongated to form stearic acid (18:0), which can be
converted to its unsaturated derivative by a .DELTA.9 desaturase to
thereby yield oleic (18:1) acid.
[0134] 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) is well described in the literature and is
schematically depicted in FIG. 5 (see also commonly owned U.S. Ser.
No. 10/840,579 and U.S. Ser. No. 10/869,630). Simplistically, this
process involves elongation of the carbon chain through the
addition of carbon atoms and desaturation of the molecule through
the addition of double bonds, via a series of special desaturation
and elongation enzymes present in the endoplasmic reticulim
membrane (and hereinafter referred to as "PUFA biosynthetic pathway
enzymes").
[0135] More specifically, "PUFA biosynthetic pathway enzymes" or
".omega.-3/.omega.-6 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, a .DELTA.8 desaturase and/or an
elongase(s). For further clarity within the present disclosure, the
term "desaturase" refers to a polypeptide 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 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)).
[0136] As will be understood by 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/elongase profile), the availability of substrate and the
desired end product(s). As shown in FIG. 5, LA, GLA, EDA, DGLA,
ARA, ALA, STA, ETrA, 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, a .DELTA.8 desaturase and/or an elongase(s). 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 YAT1 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
(e.g., see WO 2004/101757 and co-pending U.S. patent application
Ser. No. 60/624,812). As such, the invention provides a method for
the production of .omega.-3 and/or .omega.-6 fatty acids
comprising: [0137] a) providing a transformed oleaginous yeast
comprising a genetic construct, said construct comprising: [0138]
1) a promoter region of a Yarrowia yat1 gene; and, [0139] 2) a
coding region of interest encoding at least one enzyme of the
.omega.-3/.omega.-6 fatty acid biosynthetic pathway; [0140] wherein
the promoter region and coding region are operably linked; [0141]
b) culturing 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, [0142] c)
optionally recovering the .omega.-3 or .omega.-6 fatty acid.
[0143] In preferred embodiments, the nucleic acid sequence of the
promoter region is selected from the group consisting of: SEQ ID
NO:3, and subsequences and mutant promoters thereof; the coding
region of interest is any desaturase or elongase suitable for
regulated expression in the oleaginous yeast for the production of
.omega.-3 or .omega.-6 fatty acids; and, the transformed oleaginous
yeast is cultured under conditions that induce oleaginy.
[0144] More specifically, 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 yat1 gene and a coding region of interest
encoding a PUFA biosynthetic pathway enzyme. 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.
[0145] The effect of this nitrogen deprivation is two-fold. First,
the nitrogen deprivation reduces 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. Secondly, the
nitrogen deprivation induces the YAT1 promoter of the invention,
thereby promoting expression of any chimeric genes of the invention
comprising the YAT1 promoter and a coding region of interest
encoding an enzyme of the .omega.-3/.omega.-6 fatty acid
biosynthetic pathway. Thus, this second phase of the 2-stage
fermentation is characterized by the cessation of cell division
followed by the synthesis of fatty acids and accumulation of
oil.
[0146] 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.
[0147] Additionally, 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 in the fermentation media
(Nakahara, T. et al. Ind. Appl. Single Cell Oils, D. J. Kyle and R.
Colin, eds. pp 61-97 (1992)).
Purification of PUFAs
[0148] 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)).
[0149] In general, means for the purification of fatty acids
(including 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 example of which are contained in commonly
owned U.S. Ser. No. 10/840,579.
EXAMPLES
[0150] 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
[0151] 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).
[0152] 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.
[0153] General molecular cloning was performed according to
standard methods (Sambrook et al., supra). Oligonucleotides were
synthesized by Sigma-Genosys (Spring, Tex.). When polymerase chain
reaction (PCR) 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.).
[0154] 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 "Besffit" 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.
[0155] 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).
Transformation and Cultivation of Yarrowia Lipolytica
[0156] Y. lipolytica strain ATCC #20362 was purchased from the
American Type Culture Collection (Rockville, Md.). Strains were
usually grown at 28.degree. C. on YPD agar (1% yeast extract, 2%
bactopeptone, 2% glucose, 2% agar) or in YPD liquid medium (2%
bacto-yeast extract, 3% bactopeptone, 2% glucose).
[0157] Transformation of Y. lipolytica was performed according to
the method of Chen, D. C. et al. (Appl. Microbiol Biotechnol.
48(2):232-235 (1997)), unless otherwise noted. Briefly, 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: 2.25 mL of 50% PEG, average MW 3350; 0.125 mL of 2 M Li
acetate, pH 6.0; 0.125 mL of 2 M DTT; and 50 .mu.g sheared salmon
sperm DNA. Then, approximately 500 ng of linearized 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 selection media plates and maintained at
30.degree. C. for 2 to 3 days.
[0158] For selection of transformants, minimal medium ("MM") was
generally used; the composition of MM is as follows: 0.17% yeast
nitrogen base (DIFCO Laboratories, Detroit, Mich.) without ammonium
sulfate or amino acids, 2% glucose, 0.1% proline, pH 6.1 and 20 g/L
agar). Supplements of uracil or leucine were added as appropriate
to a final concentration of 0.01% (thereby producing "MMU"
selection media or "MMLe" selection media).
[0159] Alternatively, transformants were selected on 5-fluoroorotic
acid ("FOA"; also 5-fluorouracil-6-carboxylic acid monohydrate)
selection media, comprising: 0.17% yeast nitrogen base (DIFCO
Laboratories) without ammonium sulfate or amino acids, 2% glucose,
0.1% proline, 75 mg/L uracil, 75 mg/L uridine, 900 mg/L FOA (Zymo
Research Corp., Orange, Calif.) and 20 g/L agar.
[0160] "SD" media comprises: 0.67% yeast nitrogen base with
ammonium sulfate, without amino acids and 2% glucose. And finally,
to promote conditions of oleaginy, High Glucose Media ("HGM") was
prepared as follows: 14 g/L KH.sub.2PO.sub.4, 4 g/L
K.sub.2HPO.sub.4, 2 g/L MgSO.sub.4.7H.sub.2O, 80 g/L glucose (pH
6.5).
Fatty Acid Analysis of Yarrowia Lipolytica
[0161] 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):3846 (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.degree. C. at 3.5.degree. C./min.
[0162] 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.
Example 1
Construction and Sequencing of a Yarrowia Lipolytica cDNA
Library
[0163] The present Example describes the construction of a cDNA
library of Y. lipolytica, grown under oleaginous conditions. More
specifically, Y. lipolytica cells accumulate considerable amounts
of oil when switched to a medium with a high carbon to nitrogen
ratio (>80). A cDNA library was constructed from cells grown in
an oleaginous medium containing no nitrogen source for 4 hrs, using
the BD-Clontech Creator Smart.RTM. cDNA library kit (Mississauga,
ON, Canada) according to the manufacturer's protocol.
[0164] Synthesis of the cDNA library first required growth of Y.
lipolytica strain ATCC #20362 in 30 mL YPD medium overnight at
30.degree. C. with shaking. Cells were diluted in two cultures of
100 mL fresh YPD to OD.sub.600=0.4, then grown until OD.sub.600=1.5
(Perkin-Elmer Lambda 20 UV/VIS Spectrophotometer). Cells from each
culture were pelleted twice by centrifugation at 3750 rpm in a
Beckman GH3.8 rotor for 5 min and washed with sterile water, then
resuspended in 100 mL HGM. Cells were then shaken at 30.degree. C.
for 4 hrs. Each 100 mL culture was divided into three equal
portions and pelleted by centrifugation at 3750 rpm in a Beckman
GH3.8 rotor for 5 min.
[0165] Total RNA was extracted from the pellets using the Qiagen
Rneasy Midi Kit. Cells were resusupended in 600 .mu.l buffer RLT
from the kit, with .beta.-mercaptoethanol added at a concentration
of 10 .mu.l/mL. Resuspended cells were transferred to six 2 mL
screw cap tubes each containing 0.6 mL of 0.5 mm glass beads. The
cells were homogenized at the HOMOGENIZE-setting on a Biospec
(Bartlesville, Okla.) mini bead beater for 2 min. The tubes were
briefly spun to settle the beads. Liquid was transferred to 4 fresh
2 mL microfuge tubes and 600 .mu.l of the RLT/BME mix was added to
the beads. The bead tubes were vortexed and all liquid above the
beads was transferred to the fresh 2 mL tubes. The fresh tubes were
spun for 2 min in a microfuge to pellet cells debris, and the
supernatent from each set of three tubes that came from a single
culture were pooled in a 10 mL screwcap centrifuge tube. The
procedure was then completed following the manufacturer's protocol.
The two total RNA samples were then combined and mRNA was isolated
from the combined sample using the Qiagen Oligotex Midi kit,
following the manufacturer's protocol. Purified poly(A)+RNA was
obtained with a concentration of 35.7 ng/.mu.l.
[0166] cDNA was generated, using the LD-PCR method specified by
BD-Clontech and 0.1 .mu.g of polyA(+) RNA sample. Specifically, for
1.sup.st strand cDNA synthesis, 3 .mu.l of the poly(A)+RNA sample
was mixed with 1 .mu.l of SMART IV oligo nucleotide (SEQ ID NO:4)
and 1 .mu.l of CDSIII/3' PCR primer (SEQ ID NO:5). The mixture was
heated at 72.degree. C. for 2 min and cooled on ice for 2 min. To
the tube was added the following: 2 .mu.l 1.sup.st strand buffer, 1
.mu.l 20 mM DTT, 1 .mu.l 10 mM dNTP mix and 1 .mu.l Powerscript
reverse transcriptase. The mixture was incubated at 42.degree. C.
for 1 hr and cooled on ice.
[0167] The 1.sup.st strand cDNA synthesis mixture was used as
template for the PCR reaction. Specifically, the reaction mixture
contained the following: 2 .mu.l of the 1.sup.st strand cDNA
mixture, 2 .mu.l 5'-PCR primer (SEQ ID NO:6), 2 .mu.l CDSIII/3'-PCR
primer (SEQ ID NO:5), 80 .mu.l water, 10 .mu.l 10.times. Advantage
2 PCR buffer, 2 .mu.l 50.times.dNTP mix and 2 .mu.l 50.times.
Advantage 2 polymerase mix. The thermocycler conditions were set
for 95.degree. C. for 20 sec, followed by 14 cycles of 95.degree.
C. for 5 sec and 68.degree. C. for 6 min on a GenAmp 9600
instrument. PCR product was quantitated by agarose gel
electrophoresis and ethidium bromide staining.
[0168] Seventy-five .mu.l of the above PCR products (cDNA) were
mixed with 3 .mu.l of 20 .mu.g/.mu.l proteinase K supplied with the
kit. The mixture was incubated at 45.degree. C. for 20 min, then 75
.mu.l of water was added and the mixture was extracted with 150
.mu.l phenol:chloroform:isoamyl alcohol mixture (25:24:1). The
aqueous phase was further extracted with 150 .mu.l
chloroform:isoamyl alcohol (25:1). The aqueous phase was then mixed
with 15 .mu.l of 3 M sodium acetate, 2 .mu.l of 20 .mu.g/.mu.l
glycogen and 400 .mu.l of 100% ethanol. The mixture was immediately
centrifuged at room temperature for 20 min at 14000 rpm in a
microfuge. The pellet was washed once with 150 .mu.l of 80%
ethanol, air dried and dissolved in 79 .mu.l of water.
[0169] Dissolved cDNA was subsequently digested with Sfil (79 .mu.l
of the cDNA was mixed with 10 .mu.l of 10.times. Sfil buffer, 10
.mu.l of Sfil enzyme and 1 .mu.l of 100.times.BSA and the mixture
was incubated at 50.degree. C. for 2 hrs). Xylene cyanol dye (2
.mu.l of 1%) was added. The mixture was then fractionated on the
Chroma Spin-400 column provided with the kit, following the
manufacturer's procedure exactly. Fractions collected from the
column were analyzed by agarose gel electrophoresis. The first
three fractions containing cDNA were pooled and cDNA precipitated
with ethanol. The precipitated cDNA was redissolved in 7 .mu.l of
water, and ligated into kit-supplied pDNR-LIB.
Library Sequencing
[0170] The ligation products were used to transform E. coli XL-1
Blue electroporation competent cells (Stratagene). An estimated
total of 5.4.times.10.sup.6 colonies was obtained. Sequencing of
the cDNA library was carried out by Agencourt Bioscience
Corporation (Beverly, Mass.), using a M13 forward primer (SEQ ID
NO:7)
Example 2
Identification of the Yarrowia lipolytica Gene Encoding YAT1 as a
Highly Expressed Gene Under Oleaginous Conditions
[0171] This example describes the identification of YAT1 as a
highly expressed gene under oleaginous conditions. Specifically,
the relative abundance of each species of cDNA was examined (based
on the sequencing results from Example 1) to identify those genes
that were highly expressed under oleaginous conditions. One of the
genes that appeared multiple times (16/9984 or 0.16%) was
Yali0E27203 (GenBank Accession No. XM.sub.--504457; SEQ ID NO:1
herein), annotated as a homolog of the high affinity, low capacity
ammonia transporter MEP2 of Saccharomyces cerevisiae (GenBank
Accession No. X83608; see also A. M. Marini et al., EMBO J.,
13(15):3456-3463 (1994); A. M. Marini et al., Mol Cell Biol
17(8):4282-93 (1997)).
[0172] BLAST (Basic Local Alignment Search Tool; Altschul, S. F.,
et al., J. Mol. Biol. 215:403-410 (1993)) searches were conducted
using the corresponding amino acid sequence of Yali0E27203 (SEQ ID
NO:2) as a query against the S. cerevisiae MEP1, MEP2 and
MEP3-isozymes. The results of these BLAST comparisons are shown
below in Table 3 and are reported according to the % identity, %
similarity and Expectation value. TABLE-US-00003 TABLE 3 Comparison
Of Yarrowia lipolytica Homolog To Saccharomyces cerevisiae MEP1,
MEP2 And MEP3 % % Similarity Identified Identity.sup.a
Similarity.sup.b E-value.sup.c MEP1 35 56 1.8e-79 [GenBank
Accession No. X77608] MEP2 52 66 1.9e-132 [GenBank Accession No.
X83608] MEP3 37 57 3.6e-83 [GenBank Accession No. P53390] .sup.a%
Identity is defined as percentage of amino acids that are identical
between the two proteins. .sup.b% Similarity is defined as
percentage of amino acids that are identical or conserved between
the two proteins. .sup.cExpect value. The Expect 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.
It was hypothesized that YALI0E27203 encoded a Y. lipolytica
ammonia transporter of high affinity; thus, YALI0E27203 was
tentatively named yat1.
Example 3
Isolation of the 5' Upstream Region of YAT1 from Yarrowia
Lipolytica
[0173] To isolate the YAT1 promoter region upstream of the yat1
gene identified in Example 2, primers 27203-F and 27203-R (SEQ ID
NOs:8 and 9) were designed, based on the sequence of Y. lipolytica
chromosome E between positions 3,222,879 and 3,223,875. These
primers were expected to amplify 778 bp of the 5' upstream
untranslated region in front of the `ATG` translation initiation
codon of the yat1 gene, from -778 to -1 (wherein the `A` position
of the `ATG` translation initiation codon was designated as
+1).
[0174] PCR amplification was performed in a 50 .mu.l total volume
using a 1:1 dilution of a premixed 2.times.PCR solution (TaKaRa
ExTaq, TaKaRa Bio Inc., Otsu Shiga, 520-2193, Japan). The final
composition contained 25 mM TAPS, pH 9.3, 50 mM KCl, 2 mM
MgCl.sub.2, 1 mM 2-mercaptoethanol, 200 .mu.M each
deoxyribonucleotide triphosphate, 10 pmole each primer (supra), 50
ng Y. lipolytica ATCC #20362 genomic DNA, and 1.25 units of TaKaRa
ExTaq.TM. DNA polymerase. The reaction mixture was first heated to
94.degree. C. for 150 sec. Amplification was carried out for 30
cycles at 94.degree. C. for 30 sec, 55.degree. C. for 30 sec and
72.degree. C. for 1 min. This was followed by a final extension for
7 min at 72.degree. C.
[0175] The PCR product was analyzed by agarose gel electrophoresis
and was shown to contain a single DNA fragment of .about.800 bp.
This fragment was purified using a Qiagen PCR purification kit
following the manufacturer's protocol.
Example 4
Synthesis of Plasmids pY5-30, pYGPAT-GUS and pDMW214
[0176] A series of plasmids were created comprising a chimeric gene
composed of various native Y. lipolytica promoters and the "GUS"
reporter gene, wherein "GUS" corresponds to the E. coli gene
encoding .beta.-glucuronidase (Jefferson, R. A. Nature.
342(6251):837-838 (1989)). This was required for comparative
studies investigating the promoter activity of TEF, YAT1, FBAIN and
GPAT, as described in Example 8.
Synthesis of Plasmid pY5-30 (TEF::GUS::XPR)
[0177] The synthesis of plasmid pY5-30, comprising a TEF::GUS::XPR
chimeric gene, is described in WO2005/003310. More specifically,
plasmid pY5-30 (FIG. 1A; SEQ ID NO:10) contained: a Yarrowia
autonomous replication sequence (ARS18); a ColE1 plasmid origin of
replication; an ampicillin-resistance gene (Amp.sup.R) for
selection in E. coli; a Yarrowia LEU2 gene for selection in
Yarrowia; and the chimeric TEF::GUS::XPR gene.
Synthesis of Plasmid pDMW214 (FBAIN::GUS::XPR)
[0178] The synthesis of plasmid pDMW214, comprising a
FBAIN::GUS::XPR chimeric gene, is described in WO 2005/049805.
Briefly, however, the FBAIN promoter region (SEQ ID NO:11; which
includes both an upstream DNA sequence and a downstream sequence
from the putative `ATG` translation initiation codon of the
fructose-bisphosphate aldolase (fba1) gene [wherein the downstream
region comprises an intron]) was amplified by PCR, digested with
NcoI and SaII, and then purified following gel electrophoresis. The
NcoI/SaIII-digested PCR products were ligated to NcoI/SaII digested
pY5-30 vector to produce plasmid "pDMW214" (FIG. 1B).
Synthesis of pYGPAT-GUS (GPAT::GUS::XPR)
[0179] The synthesis of plasmid pYGPAT-GUS, comprising a
GPAT::GUS::XPR chimeric gene, is described in U.S. patent
application Ser. No. 60/610,060 (filed Sep. 15, 2004) and will be
reiterated below. In general, synthesis of the plasmid required
identification and isolation of the Y. lipolytica
glycerol-3-phosphate O-acyltransferase (gpat) gene, isolation of
the promoter region upstream of the putative `ATG` translation
initiation codon via genome-walking, and then cloning of the
promoter into a derivative of pY5-30 (supra).
[0180] Isolation and Sequencing of the Putative Yarrowia Lipolytica
GPAT
[0181] Based on the gene sequences encoding two isozymes of GPAT in
Saccharomyces cerevisiae (GAT1 [GenBank Accession No. AJ311354] and
GAT2(SCT1) [GenBank Accession No. AJ314608]; see also Zheng and
Zou. J. Biol. Chem. 276(45):4171041716 (2001) and WO 02/08391 A2),
BLAST searches (supra, Example 2) were conducted against the
Genolevures genome database of Y. lipolytica ORFs (sponsored by the
Center for Bioinformatics, LaBRI, batiment A30, Universite Bordeaux
1, 351, cours de la Liberation, 33405 Talence Cedex, France) to
identify any similar sequences contained therein. The results of
the BLAST comparisons identified one homolog (Yarrowia lipolytica
ORF YAL1-CDS1055.1, a protein of 727 amino acids; SEQ ID NO:12;
also GenBank Accession No. CAG81570) annotated as a protein having
similarity to S. cerevisiae GPATs.
[0182] Following the tentative identification of ORF
YAL1-CDS1055.1, this amino acid sequence was BLASTed against the S.
cerevisiae isozymes encoding GPAT. The results of these BLAST
comparisons are shown below and are reported according to the %
identity, % similarity, and Expectation value (supra, Example 2).
TABLE-US-00004 TABLE 4 Comparison Of Yarrowia lipolytica ORF
YAL1-CDS1055.1 To Saccharomyces cerevisiae GAT1 And GAT2(SCT1) % %
Similarity Identified Identity.sup.a Similarity.sup.b E-value.sup.c
GAT1 35 54 1.2e-113 [GenBank Accession No. AJ311354] GAT2(SCT1) 43
59 3.4e-141 [GenBank Accession No. AJ314608]
Thus, it was hypothesized that ORF YALI-CDS1055.1 encoded a Y.
lipolytica GPAT.
[0183] Degenerated oligonucleotides, as shown below, were designed
to amplify the entire coding region of ORF YALI-CDS1055.1.
TABLE-US-00005 Degenerated Oligonucleotide YGPAT-F (SEQ ID NO:13)
ATGTCNGAGACYGACCAYCTNCTN Degenerated oligonucleotide YGPAT-R (SEQ
ID NO:14) YTCYTCRTCYTGYTCTCGYCGYTT [Note: The nucleic acid
degeneracy code used for SEQ ID NOs:13 and 14 was as follows: R =
A/G; Y = C/T; and N = A/C/T/G.]
[0184] The PCR amplification was carried out in a 50 .mu.l total
volume using a 1:1 dilution of a premixed 2.times.PCR solution and
1.25 units of TaKaRa Ex Taq.TM. DNA polymerase (Takara Mirus Bio,
Madison, Wis.), according to the PCR composition described in
Example 3. The thermocycler conditions were set for 30 cycles at
94.degree. C. for 2.5 min, 55.degree. C. for 30 sec and 72.degree.
C. for 2.5 min, followed by a final extension at 72.degree. C. for
6 min.
[0185] The PCR products were separated by gel electrophoresis in 1%
(w/v) agarose. A 2.2 kB DNA fragment was excised and purified using
a QiaexII gel purification kit (Qiagen, Valencia, Calif.).
Subequently, the purified 2.2 kB DNA fragment was cloned into the
pGEM-T-easy vector (Promega, Madison, Wis.). The ligated DNA was
used to transform cells of E. coli Top10 and transformants were
selected on LB (1% bacto-tryptone, 0.5% bacto-yeast extract and 1%
NaCl) 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, designated as "pGPAT-1".
[0186] Sequence analyses of pGPAT-1 showed that it contained a 2184
bp fragment of Y. lipolytica DNA encoding GPAT (SEQ ID NO:15).
Specifically, identity of this gene sequence was determined by
conducting BLAST 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). The sequence was analyzed for
similarity to all publicly available DNA sequences contained in the
"nr" database 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 BLAST
searches revealed that the translated product of SEQ ID NO:15
(comprising the putative gpat gene) had the highest BLAST hits to
annotated GPATs from: (1) S. cerevisiae (SwissPro P36148, Entrez
CAA82146): 43% identity, 59% similarity, E-146; and (2)
Schizosaccharomyces pombe SCT1(GAT2) homolog (GPAT): 48% identity,
66% similarity, E-148.
[0187] Furthermore, the translated product of SEQ ID NO:15 was 100%
identical to the amino acid sequence of ORF YAL1-CDS1055.1 (SEQ ID
NO:12; also GenBank Accession No. CAG81570).
[0188] Isolation of the 5' Upstream Region of GPAT from Y.
Lipolytica
[0189] To isolate the GPAT promoter region upstream of the gene
identified in Example 1, a genome-walking technique (Universal
GenomeWalker, ClonTech, CA) was utilized, following the
manufacturer's protocol.
[0190] Briefly, genomic DNA of Y. lipolytica was digested with
DraI, EcoRV, PvuII or StuI individually, and the digested DNA
samples were ligated with Genome Walker adaptor (SEQ ID NOs:16 [top
strand] and 17 [bottom strand]), as shown below: TABLE-US-00006
5'-GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT-3'
3'-H2N-CCCGACCA-5'
[0191] PCR reactions were then carried out using the ligation
products as templates, and the following oligonucleotides as
primers: Adaptor-1 and YGPAT-5R-1 (SEQ ID NOs:18 and 19). The PCR
amplification was carried out in a 50 .mu.l total volume using the
components and conditions previously described, with the exception
that the template used was 1 .mu.l of ligation product (versus 50
ng genomic DNA). Second PCR reactions were then carried out using 1
.mu.l of 1:50 diluted first PCR product as template, and Nested
Adaptor Primer 2 and YGPAT-5R-2 (SEQ ID NOs:20 and 21) as
primers.
[0192] A 1.7 kB DNA fragment, amplified from the EcoRV digested
sample, was purified using a Qiagen PCR purification kit and cloned
into the pGEM-T-easy vector (Promega, Madison, Wis.). The ligated
DNA was used to transform E. coli Top10 and transformants were
selected on LB agar containing ampicillin (100 .mu.g/mL).
[0193] Analysis of the plasmid DNA from one transformant confirmed
the presence of the expected plasmid, designated "pEcoRV-G-5".
Sequence analyses showed that pEcoRV-G-5 contained a fragment of
1781 bp (SEQ ID NO:22), which included 1678 bp of 5' upstream
sequence from the nucleotide `A` of the translation initiation
codon `ATG` of the GPAT gene. Assembly of DNA corresponding to
overlapping SEQ ID NOs:15 and 22 yielded a single contig of DNA
represented as SEQ ID NO:23 (FIG. 2; 3862 bp total length). This
contig therefore contained the -1678 to +2181 region of the GPAT
gene, wherein the `A` position of the `ATG` translation initiation
codon was designated as +1.
[0194] Construction of pYGPAT-GUS
[0195] The nucleotide region between the -1130 position and the
`ATG` translation initiation site of the gpat gene was considered
to represent the putative GPAT promoter region ("GPAT-Pro",
provided as SEQ ID NO:24).
[0196] GPAT-Pro was amplified by PCR using primers GPAT-5-1 and
GPAT-5-2 (SEQ ID NOs:25 and 26), and pEcoRV-G5 (supra) as template.
The PCR product was then purified using a Qiagen PCR purification
kit and was completely digested with SaII and NcoI. The digestion
product was again purified with a Qiagen PCR purification kit and
ligated to NcoI/SaII digested pY5-30 vector (supra, wherein the
NcoI/SaII digestion had excised the TEF promoter from the pY5-30
vector backbone). Ligated DNA was then used to individually
transform E. coli Top10. Transformants were selected on LB agar
containing ampicillin (100 .mu.g/mL).
[0197] Analysis of the plasmid DNA from one transformant containing
GPAT-Pro confirmed the presence of the expected plasmid, designated
"pYGPAT-GUS". Thus, this plasmid contained a chimeric gene
comprising a GPAT promoter, GUS reporter gene and XPR terminator
(FIG. 1C).
Example 5
Synthesis of pYAT-GUS
[0198] The present Example describes the synthesis of pYAT-GUS
(comprising a YAT1::GUS::XPR chimeric gene). Synthesis of this
plasmid first required amplification of the putative YAT1 promoter
region. Then, the putative promoter region was cloned into
pYGPAT-GUS (supra, Example 4).
[0199] The purified YAT1 PCR product from Example 3 was digested
with HindIII and SaII and a .about.600 bp fragment was isolated by
agarose gel electrophoresis followed by purification with Qiagen
MinElute Gel purification kit according to the manufacturer's
protocol. Furthermore, the YAT1 PCR product was also digested with
NcoI and HindIII, and a .about.200 bp fragment was isolated and
purified as above. Finally, plasmid pYGPAT-GUS was digested with
SaII and NcoI, and a .about.9.5 kB fragment was isolated and
purified. The three DNA fragments were ligated together to create
pYAT-GUS (FIG. 1D). In the resulting plasmid, the YAT1 promoter
region (SEQ ID NO:3; corresponding to 778 bp of the 5' upstream
region of YALI0E27203 (-778 to -1)) was fused to the GUS gene,
followed by the XPR terminator region.
Example 6
Generation of ARA-Producing Yarrowia lipolytica ATCC #20362 Strain
Y2034
[0200] The present Example describes the construction of strain
Y2034, derived from Y. lipolytica ATCC #20362, capable of producing
significant concentrations of ARA relative to the total lipids.
Comparison of the TEF, YAT1, GPAT and FBAIN promoters was examined
in this ARA-producing strain based on analysis of GUS expression,
as described in Example 8 (infra).
[0201] The development of strain Y2034 herein, producing 10% ARA,
required the construction of strain M4 (producing 8% DGLA) (FIG.
3A).
Construction of Strain M4 Producing 8% DGLA
[0202] Construct pKUNF12T6E (FIG. 3B; SEQ ID NO:27) was generated
to integrate four chimeric genes (comprising a .DELTA.12
desaturase, a .DELTA.6 desaturase and 2 elongases) into the Ura3
loci of wild type Yarrowia strain ATCC #20362, to thereby enable
production of DGLA. The pKUNF12T6E plasmid contained the following
components: TABLE-US-00007 TABLE 5 Description of Plasmid
pKUNF12T6E (SEQ ID NO: 27) RE Sites And Nucleotides Within SEQ ID
Description Of Fragment And NO: 27 Chimeric Gene Components
AscI/BsiWI 784 bp 5' part of Yarrowia Ura3 gene (GenBank
(9420-8629) Accession No. AJ306421) SphI/PacI 516 bp 3' part of
Yarrowia Ura3 gene (GenBank (12128-1) Accession No. AJ306421)
SwaI/BsiWI FBAIN::EL1S:Pex20, comprising: (6380-8629) FBAIN: FBAIN
promoter (SEQ ID NO: 11) EL1S: codon-optimized elongase 1 gene (SEQ
ID NO: 28), derived from Mortierella alpina (GenBank Accession No.
AX464731) Pex20: Pex20 terminator sequence from Yarrowia Pex20 gene
(GenBank Accession No. AF054613) BglII/SwaI TEF::.DELTA.6S::Lip1,
comprising: (4221-6380) TEF: TEF promoter (GenBank Accession No.
AF054508) .DELTA.6S: codon-optimized .DELTA.6 desaturase gene (SEQ
ID NO: 30), derived from Mortierella alpina (GenBank Accession No.
AF465281) Lip1: Lip1 terminator sequence from Yarrowia Lip1 gene
(GenBank Accession No. Z50020) PmeI/ClaI FBA::F..DELTA.12::Lip2,
comprising: (4207-1459) FBA: FBA promoter (SEQ ID NO: 32)
F..DELTA.12: Fusarium moniliforme .DELTA.12 desaturase gene (SEQ ID
NO: 33) Lip2: Lip2 terminator sequence from Yarrowia Lip2 gene
(GenBank Accession No. AJ012632) ClaI/PacI TEF::EL2S::XPR,
comprising: (1459-1) TEF: TEF promoter (GenBank Accession No.
AF054508) EL2S: codon-optimized elongase gene (SEQ ID NO: 35),
derived from Thraustochytrium aureum (U.S. Pat. No. 6,677,145) XPR:
XPR terminator sequence of Yarrowia Xpr gene (GenBank Accession No.
M17741)
[0203] The pKUNF12T6E plasmid was digested with AscI/SphI, and then
used for transformation of wild type Y. lipolytica ATCC #20362
according to the General Methods. The transformant cells were
plated onto FOA selection media plates and maintained at 30.degree.
C. for 2 to 3 days. The FOA resistant colonies were picked and
streaked onto MM and MMU selection plates. The colonies that could
grow on MMU plates but not on MM plates were selected as
Ura-strains. Single colonies of Ura-strains were then inoculated
into liquid MMU at 30.degree. C. and shaken at 250 rpm/min for 2
days. The cells were collected by centrifugation, lipids were
extracted, and fatty acid methyl esters were prepared by
trans-esterification, and subsequently analyzed with a
Hewlett-Packard 6890 GC.
[0204] GC analyses showed the presence of DGLA in the transformants
containing the 4 chimeric genes of pKUNF12T6E (FIG. 3B), but not in
the wild type Yarrowia control strain. Most of the selected 32
Ura.sup.- strains produced about 6% DGLA of total lipids. There
were 2 strains (i.e., strains M4 and 13-8) that produced about 8%
DGLA of total lipids.
Construction of Strain Y2034 Producing 10% ARA
[0205] Construct pDMW232 (FIG. 3C; SEQ ID NO:37) was generated to
integrate two .DELTA.5 chimeric genes into the Leu2 gene of
Yarrowia strain M4.
[0206] Plasmid pDMW232 contained the following components, as
described in Table 6: TABLE-US-00008 TABLE 6 Description of Plasmid
pDMW232 (SEQ ID NO: 37) RE Sites And Nucleotides Within SEQ ID
Description Of Fragment And NO: 37 Chimeric Gene Components
AscI/BsiWI 788 bp 5' part of Yarrowia Leu2 gene (GenBank
(5550-4755) Accession No. AF260230) SphI/PacI 703 bp 3' part of
Yarrowia Leu2 gene (GenBank (8258-8967) Accession No. AF260230)
SwaI/BsiWI FBAIN::MA.DELTA.5::Pex20, comprising: (2114-4755) FBAIN:
FBAIN Promoter (SEQ ID NO: 11) MA.DELTA.5: Mortierella alpina
.DELTA.5 desaturase gene (GenBank Accession No. AF067654) Pex20:
Pex20 terminator sequence of Yarrowia Pex20 gene (GenBank Accession
No. AF054613) SwaI/ClaI TEF::MA.DELTA.5::Lip1, comprising:
(2114-17) TEF: TEF promoter (GenBank Accession No. AF054508)
MA.DELTA.5: as described for FBAIN::MA.DELTA.5::Pex20 (supra) Lip1:
Lip1 terminator sequence of Yarrowia Lip1 gene (GenBank Accession
No. Z50020) PmeI/ClaI Yarrowia Ura3 gene (GenBank Accession No.
AJ306421) (5550-4755)
[0207] Plasmid pDMW232 was digested with AscI/SphI, and then used
to transform strain M4 (Example 4) separately according to the
General Methods. Following transformation, the cells were plated
onto MMLe plates and maintained at 30.degree. C. for 2 to 3 days.
The individual colonies grown on MMLe plates were picked and
streaked onto MM and MMLe plates. Those colonies that could grow on
MMLe plates but not on MM plates were selected as Leu2.sup.-
strains. Single colonies of Leu2.sup.- strains were then inoculated
into liquid MMLe media at 30.degree. C. and shaken at 250 rpm/min
for 2 days. The cells were collected by centrifugation, lipids were
extracted, and fatty acid methyl esters were prepared by
trans-esterification, and subsequently analyzed with a
Hewlett-Packard 6890 GC.
[0208] GC analyses showed the presence of ARA in pDMW232
transformants, but not in the parental M4 strain. Specifically,
among the 48 selected Leu2.sup.- transformants with pDMW232, there
were 34 strains that produced less than 5% ARA, 11 strains that
produced 6-8% ARA, and 3 strains that produced about 10% ARA of
total lipids in the engineered Yarrowia. One of the strains that
produced 10% ARA was named "Y2034" and was the parental strain in
Example 7.
Example 7
Transformation of Y. lipolytica with pY5-30, pYAT-GUS, pYGPAT-GUS
and pDMW214
[0209] The plasmids pY5-30 (Example 4; comprising a TEF::GUS::XPR
chimeric gene), pYAT-GUS (Example 5; comprising a YAT1::GUS::XPR
chimeric gene), pYGPAT-GUS (Example 4; comprising a GPAT::GUS::XPR
chimeric gene) and pDMW214 (Example 4; comprising a FBAIN::GUS::XPR
chimeric gene) were transformed separately into Y. lipolytica ATCC
#20362 strain Y2034, according to the General Methods. Selection
was performed on SD medium comprising 2.5% agar.
[0210] Using this technique, transformants were obtained that
contained pY5-30, pYAT-GUS, pYGPAT-GUS and pDMW214,
respectively.
Example 8
Comparative Analysis of the TEF, YAT1, GPAT and FBAIN Promoter
Activities in Yarrowia Lipolytica, as Determined by Histochemical
Assay
[0211] The activity of the TEF, YAT1, GPAT and FBAIN promoters was
determined in Y. lipolytica containing the pY5-30, pYAT-GUS,
pYGPAT-GUS and pDMW214 constructs, each of which possessed a GUS
reporter gene and an XPR terminator (from Example 7). GUS activity
in each expressed construct was measured by histochemical assays
(Jefferson, R. A. Plant Mol. Biol. Reporter 5:387-405 (1987)).
[0212] Specifically, Y. lipolytica strains containing plasmids
pY5-30, pYAT-GUS, pYGPAT-GUS and pDMW214, respectively, were grown
from single colonies in 5 mL SD media at 30.degree. C. for 24 hrs
to an OD.sub.600.about.8.0. Then, 1 mL of cells were collected by
centrifugation. The remaining cultures were centrifuged and washed
2.times. with HGM, resuspended in 5 mL each of HGM and allowed to
grow at 30.degree. C. further. After 24 and 120 hrs, .about.0.25 mL
of each culture were centrifuged to collect the cells. Cell samples
were resuspended individually in 100 .mu.l of histochemical
staining buffer [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.]. Zymolase 20T (5 .mu.l of 1 mg/mL; ICN Biomedicals, Costa
Mesa, Calif.) was added to each, and the mixture incubated at
30.degree. C.
[0213] The results of histochemical staining showed that the YAT1
promoter in construct pYAT-GUS was active. Comparatively, the YAT1
promoter appeared to be stronger than the TEF promoter (FIG. 4A)
but significantly weaker than the FBAIN promoter and GPAT promoter,
when cells were grown in SD medium for 24 hrs. More interestingly,
however, it appeared that the YAT1 promoter was stronger than the
GPAT promoter and comparable with FBAIN promoter in cells grown in
HGM for 24 hrs (FIG. 4B). This remained true after 120 hrs in HGM
(FIG. 4C). Thus, the YAT1 promoter appeared to be induced in HGM, a
medium that promotes oleaginous growth conditions due to nitrogen
limitation.
Example 9
Comparative Analysis of the TEF, YAT1, FBAIN and GPAT Promoter
Activities in Yarrowia Lipolytica, as Determined by Fluorometric
Assay
[0214] A variety of methods are available to compare the activity
of various promoters, to thereby facilitate determination of each
promoter's strength for use in future applications wherein a suite
of promoters would be necessary to construct chimeric genes. Thus,
although it may be useful to indirectly quantitate promoter
activity based on reporter gene expression using histochemical
staining (Example 8), quantification of GUS expression using more
quantitative means may be desirable. One suitable method to assay
GUS activity is by fluorometric determination of the production of
4-methylumbelliferone (4-MU) from the corresponding
.beta.-glucuronide (4-MUG; see Jefferson, R. A., Plant Mol. Biol.
Reporter 5:387-405 (1987)).
[0215] Yarrowia lipolytica strain Y2034 containing plasmids pY5-30,
pYAT-GUS, pYGPAT-GUS and pDMW214 constructs, respectively (from
Example 7), were grown from single colonies in 10 mL SD medium at
30.degree. C. for 48 hrs to an OD.sub.600 .about.5.0. Two mL of
each culture was collected for GUS activity assays, as described
below, while 5 mL of each culture was switched into HGM.
[0216] Specifically, cells from the 5 mL aliquot were collected by
centrifugation, washed once with 5 mL of HGM and resuspended in HG
medium. The cultures in HGM were then grown in a shaking incubator
at 30.degree. C. for 24 hrs. Two mL of each HGM culture were
collected for GUS activity assay, while the remaining culture was
allowed to grow for an additional 96 hrs before collecting an
additional 2 mL of each culture for the assay.
[0217] Each 2 mL culture sample in SD medium was resuspended in 1
mL of 0.5.times.cell culture lysis reagent (Promega). Resuspended
cells were mixed with 0.6 mL of glass beads (0.5 mm diameter) in a
2.0 mL screw cap tube with a rubber O-ring. The cells were then
homogenized in a Biospec mini beadbeater (Bartlesville, Okla.) at
the highest setting for 90 sec. The homogenization mixtures were
centrifuged for 2 min at 14,000 rpm in an Eppendof centrifuge to
remove cell debris and beads. The supernatant was used for GUS
assay and protein determination.
[0218] For each fluorometric assay, 200 .mu.l of extract was added
to 800 .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 (CytoFluorR 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
20 .mu.l of extract and 980 .mu.l of BioRad Bradford reagent
(Bradford, M. M. Anal. Biochem. 72:248-254 (1976)). GUS activity is
expressed as nmoles of 4-MU per minute per mg of total protein.
[0219] As shown in the Table below, the activity of the YAT1
promoter was highly induced after 24 hrs in HGM. TABLE-US-00009
TABLE 7 Comparison of TEF, FBAIN, YAT1 And GPAT Promoter Activity
Under Various Growth Conditions Culture Promoter Conditions TEF
FBAIN YAT1 GPAT 48 hr, SD 0.401 43.333 0.536 5.252 24 hr, HGM 0.942
30.694 19.154 2.969 120 hr HGM 0.466 17.200 13.400 3.050
[0220] Based on the data above wherein the activity of the YAT1
promoter was quantitated based on GUS activity of cell extracts,
the activity of the YAT1 promoter increased by .about.37 fold when
cells were switched from SD medium into HGM and grown for 24 hrs.
After 120 hrs in HGM, the activity was reduced somewhat but was
still 25.times. higher than the activity in SD medium. In contrast,
the activity of the FBAIN promoter and the GPAT promoter was
reduced by 30% and 40%, respectively, when switched from SD medium
into HGM for 24 hrs. The activity of the TEF promoter increased by
2.3 fold after 24 hrs in HGM. Thus, the YAT1 promoter is inducible
under oleaginous conditions and will thereby function as an
effective promoter for the regulated expression of heterologous
genes in Yarrowia lipolytica.
Sequence CWU 0
0
SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 37 <210>
SEQ ID NO 1 <211> LENGTH: 1461 <212> TYPE: DNA
<213> ORGANISM: Yarrowia lipolytica (GenBank Accession No.
XM_504457) <220> FEATURE: <221> NAME/KEY: CDS
<222> LOCATION: (1)..(1461) <400> SEQUENCE: 1 atg gct
gac gct acc tca acg caa gct ccc ctt ccg acc act ggc aat 48 Met Ala
Asp Ala Thr Ser Thr Gln Ala Pro Leu Pro Thr Thr Gly Asn 1 5 10 15
gga gga gac tcc ctc aca caa aac ctg aac gtc ccc ttc ctg ggt gcc 96
Gly Gly Asp Ser Leu Thr Gln Asn Leu Asn Val Pro Phe Leu Gly Ala 20
25 30 gat atg gtc tgg atc atg acc tct tcg gca ctg gtc tgg atc atg
att 144 Asp Met Val Trp Ile Met Thr Ser Ser Ala Leu Val Trp Ile Met
Ile 35 40 45 cca gga gtc ggt ctg ctc tac tct ggt atg tcg cgt aag
cac cac gcc 192 Pro Gly Val Gly Leu Leu Tyr Ser Gly Met Ser Arg Lys
His His Ala 50 55 60 ctg tct ctc ctg tgg gca tcc atc atg tgc tgt
gcc ctc gtc tct ttc 240 Leu Ser Leu Leu Trp Ala Ser Ile Met Cys Cys
Ala Leu Val Ser Phe 65 70 75 80 gag tgg ttt ttc tgg ggt tac act ctg
gca ttc tct cac aag gcc gga 288 Glu Trp Phe Phe Trp Gly Tyr Thr Leu
Ala Phe Ser His Lys Ala Gly 85 90 95 aag ttc att gga acc atg gat
aac ttc ggt ctt atg aat gtt ctc gct 336 Lys Phe Ile Gly Thr Met Asp
Asn Phe Gly Leu Met Asn Val Leu Ala 100 105 110 gct ccc tct gtt ggc
tcc tct gct gtc ccc gat atc ctg tat atg ttc 384 Ala Pro Ser Val Gly
Ser Ser Ala Val Pro Asp Ile Leu Tyr Met Phe 115 120 125 tac cag ggc
atg ttc gca tgc atc acc ggt atg ctc atg gtt ggt gga 432 Tyr Gln Gly
Met Phe Ala Cys Ile Thr Gly Met Leu Met Val Gly Gly 130 135 140 gct
cac gag cga gct cga ctc ggc ccc atg atg gta tat ctc ttc att 480 Ala
His Glu Arg Ala Arg Leu Gly Pro Met Met Val Tyr Leu Phe Ile 145 150
155 160 tgg atg act gtc gtc tac tct cct att gca tgc tgg aca tgg aac
cct 528 Trp Met Thr Val Val Tyr Ser Pro Ile Ala Cys Trp Thr Trp Asn
Pro 165 170 175 agt gga tgg ctc gcc gtc ctt gga gga ctt gat ttc gca
ggt gga gga 576 Ser Gly Trp Leu Ala Val Leu Gly Gly Leu Asp Phe Ala
Gly Gly Gly 180 185 190 cct gtt cac atg tct tcc ggt gcg ggt gcc ctt
gcc tat gct ctc tgg 624 Pro Val His Met Ser Ser Gly Ala Gly Ala Leu
Ala Tyr Ala Leu Trp 195 200 205 tgt ggt aag aga cgt gac cct gct gtt
gag aag ctg cct cac tac cgg 672 Cys Gly Lys Arg Arg Asp Pro Ala Val
Glu Lys Leu Pro His Tyr Arg 210 215 220 ccc tct tcc gtt act tcc gtt
gtt ctc ggc act gtt ttg ctt tgg ttc 720 Pro Ser Ser Val Thr Ser Val
Val Leu Gly Thr Val Leu Leu Trp Phe 225 230 235 240 gga tgg ttc gga
ttc aac ggt ggt tcc tct ggt aac gcc tcc atc cga 768 Gly Trp Phe Gly
Phe Asn Gly Gly Ser Ser Gly Asn Ala Ser Ile Arg 245 250 255 ggc ttc
tac gct gcc gct aat act aac ctt gct gct gct tgc ggt gct 816 Gly Phe
Tyr Ala Ala Ala Asn Thr Asn Leu Ala Ala Ala Cys Gly Ala 260 265 270
ctc gct tgg atg tgt gtc gac ttc ttc cga aag ggc cga aag tgg tcc 864
Leu Ala Trp Met Cys Val Asp Phe Phe Arg Lys Gly Arg Lys Trp Ser 275
280 285 act gtt ggt ctc tgt tct ggt gcc atc gca ggt ctc gtt ggc atc
acc 912 Thr Val Gly Leu Cys Ser Gly Ala Ile Ala Gly Leu Val Gly Ile
Thr 290 295 300 ccc gcc gcc ggc ttc gtc cct atc tgg tct gct gtc cct
att ggt atc 960 Pro Ala Ala Gly Phe Val Pro Ile Trp Ser Ala Val Pro
Ile Gly Ile 305 310 315 320 atc acc gcc gtc ttc gct aac att tct ggt
gac ctt aag aat ctg ctc 1008 Ile Thr Ala Val Phe Ala Asn Ile Ser
Gly Asp Leu Lys Asn Leu Leu 325 330 335 cga att gat gat ggt ctc gat
gtc ttc tct ctc cat ggt gtg gga gga 1056 Arg Ile Asp Asp Gly Leu
Asp Val Phe Ser Leu His Gly Val Gly Gly 340 345 350 ttc tgc ggc tct
gtt ctt act gcc ttc ttt gct gct gac tac att gcc 1104 Phe Cys Gly
Ser Val Leu Thr Ala Phe Phe Ala Ala Asp Tyr Ile Ala 355 360 365 cat
ctg gat ggt gcc aca gag atc aag ggt gga tgg ctc aac cac cac 1152
His Leu Asp Gly Ala Thr Glu Ile Lys Gly Gly Trp Leu Asn His His 370
375 380 tgg gct cag ctg ggt tac cag ctg gct ggt gcg ttt gct acc ctc
ggc 1200 Trp Ala Gln Leu Gly Tyr Gln Leu Ala Gly Ala Phe Ala Thr
Leu Gly 385 390 395 400 tac tcc ttt gtg gtc tct tca gtt att ctt gtc
atc atg aac aga atc 1248 Tyr Ser Phe Val Val Ser Ser Val Ile Leu
Val Ile Met Asn Arg Ile 405 410 415 ccc tac ctc aac gtc cga atg acc
gag gag gag gag atg ctt gga acc 1296 Pro Tyr Leu Asn Val Arg Met
Thr Glu Glu Glu Glu Met Leu Gly Thr 420 425 430 gac atg gcc cag atc
ggc gag ttt gcc ttc gac tgg gag gac tct gga 1344 Asp Met Ala Gln
Ile Gly Glu Phe Ala Phe Asp Trp Glu Asp Ser Gly 435 440 445 gtc ctg
gac ctg cat ggc cag aac ccc aat ggt atg ggc gtc acc ccc 1392 Val
Leu Asp Leu His Gly Gln Asn Pro Asn Gly Met Gly Val Thr Pro 450 455
460 aac gtc cag act ccc aag ccc agc agc atc aac gaa aac aag gag gct
1440 Asn Val Gln Thr Pro Lys Pro Ser Ser Ile Asn Glu Asn Lys Glu
Ala 465 470 475 480 gct gag agt gat tcg gtt taa 1461 Ala Glu Ser
Asp Ser Val 485 <210> SEQ ID NO 2 <211> LENGTH: 486
<212> TYPE: PRT <213> ORGANISM: Yarrowia lipolytica
(GenBank Accession No. XM_504457) <400> SEQUENCE: 2 Met Ala
Asp Ala Thr Ser Thr Gln Ala Pro Leu Pro Thr Thr Gly Asn 1 5 10 15
Gly Gly Asp Ser Leu Thr Gln Asn Leu Asn Val Pro Phe Leu Gly Ala 20
25 30 Asp Met Val Trp Ile Met Thr Ser Ser Ala Leu Val Trp Ile Met
Ile 35 40 45 Pro Gly Val Gly Leu Leu Tyr Ser Gly Met Ser Arg Lys
His His Ala 50 55 60 Leu Ser Leu Leu Trp Ala Ser Ile Met Cys Cys
Ala Leu Val Ser Phe 65 70 75 80 Glu Trp Phe Phe Trp Gly Tyr Thr Leu
Ala Phe Ser His Lys Ala Gly 85 90 95 Lys Phe Ile Gly Thr Met Asp
Asn Phe Gly Leu Met Asn Val Leu Ala 100 105 110 Ala Pro Ser Val Gly
Ser Ser Ala Val Pro Asp Ile Leu Tyr Met Phe 115 120 125 Tyr Gln Gly
Met Phe Ala Cys Ile Thr Gly Met Leu Met Val Gly Gly 130 135 140 Ala
His Glu Arg Ala Arg Leu Gly Pro Met Met Val Tyr Leu Phe Ile 145 150
155 160 Trp Met Thr Val Val Tyr Ser Pro Ile Ala Cys Trp Thr Trp Asn
Pro 165 170 175 Ser Gly Trp Leu Ala Val Leu Gly Gly Leu Asp Phe Ala
Gly Gly Gly 180 185 190 Pro Val His Met Ser Ser Gly Ala Gly Ala Leu
Ala Tyr Ala Leu Trp 195 200 205 Cys Gly Lys Arg Arg Asp Pro Ala Val
Glu Lys Leu Pro His Tyr Arg 210 215 220 Pro Ser Ser Val Thr Ser Val
Val Leu Gly Thr Val Leu Leu Trp Phe 225 230 235 240 Gly Trp Phe Gly
Phe Asn Gly Gly Ser Ser Gly Asn Ala Ser Ile Arg 245 250 255 Gly Phe
Tyr Ala Ala Ala Asn Thr Asn Leu Ala Ala Ala Cys Gly Ala 260 265 270
Leu Ala Trp Met Cys Val Asp Phe Phe Arg Lys Gly Arg Lys Trp Ser 275
280 285 Thr Val Gly Leu Cys Ser Gly Ala Ile Ala Gly Leu Val Gly Ile
Thr 290 295 300 Pro Ala Ala Gly Phe Val Pro Ile Trp Ser Ala Val Pro
Ile Gly Ile 305 310 315 320 Ile Thr Ala Val Phe Ala Asn Ile Ser Gly
Asp Leu Lys Asn Leu Leu 325 330 335 Arg Ile Asp Asp Gly Leu Asp Val
Phe Ser Leu His Gly Val Gly Gly 340 345 350 Phe Cys Gly Ser Val Leu
Thr Ala Phe Phe Ala Ala Asp Tyr Ile Ala 355 360 365 His Leu Asp Gly
Ala Thr Glu Ile Lys Gly Gly Trp Leu Asn His His 370 375 380 Trp Ala
Gln Leu Gly Tyr Gln Leu Ala Gly Ala Phe Ala Thr Leu Gly 385 390 395
400 Tyr Ser Phe Val Val Ser Ser Val Ile Leu Val Ile Met Asn Arg Ile
405 410 415 Pro Tyr Leu Asn Val Arg Met Thr Glu Glu Glu Glu Met Leu
Gly Thr 420 425 430 Asp Met Ala Gln Ile Gly Glu Phe Ala Phe Asp Trp
Glu Asp Ser Gly 435 440 445 Val Leu Asp Leu His Gly Gln Asn Pro Asn
Gly Met Gly Val Thr Pro 450 455 460 Asn Val Gln Thr Pro Lys Pro Ser
Ser Ile Asn Glu Asn Lys Glu Ala 465 470 475 480 Ala Glu Ser Asp Ser
Val 485 <210> SEQ ID NO 3 <211> LENGTH: 778 <212>
TYPE: DNA <213> ORGANISM: Yarrowia lipolytica
<400> SEQUENCE: 3 ataagtttgc aaaaagatcg tattatagtt ggagcaaggg
agaaatgtag agtgtgaaag 60 actcactatg gtccgggctt atctcgacca
atagccaaag tctggagttt ctgagagaaa 120 aaggcaagat acgtatgtaa
caaagcgacg catggtacaa taataccgga ggcatgtatc 180 atagagagtt
agtggttcga tgatggcact ggtgcctggt atgactttat acggctgact 240
acatatttgt cctcagacat acaattacag tcaagcactt acccttggac atctgtaggt
300 accccccggc caagacgatc tcagcgtgtc gtatgtcgga ttggcgtagc
tccctcgctc 360 gtcaattggc tcccatctac tttcttctgc ttggctacac
ccagcatgtc tgccatggct 420 cgttttcgtg ccttatctat cctcccagta
ttaccaactc taaatgacat gatgtgattg 480 ggtctacact ttcatatcag
agataaggag tagcacagtt gcataaaaag cccaactcta 540 atcagcttct
tcctttcttg taattagtac aaaggtgatt agcgaaatct ggaagcttag 600
ttggccctaa aaaaatcaaa aaaagcaaaa aacgaaaaac gaaaaaccac agttttgaga
660 acagggaggt aacgaaggat cgtatatata tatatatata tatataccca
cggatcccga 720 gaccggcctt tgattcttcc ctacaaccaa ccattctcac
caccctaatt cacaaatg 778 <210> SEQ ID NO 4 <211> LENGTH:
39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: SMART IV
oligonucleotide <400> SEQUENCE: 4 aagcagtggt atcaacgcag
agtggccatt acggccggg 39 <210> SEQ ID NO 5 <211> LENGTH:
59 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: CDSIII/3'PCR
primer <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (28)..(57) <223> OTHER INFORMATION:
thymidine (dT); see BD Biosciences Clontech's SMART cDNA technology
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (59)..(59) <223> OTHER INFORMATION: n is a, c, g,
or t <400> SEQUENCE: 5 attctagagg ccgaggcggc cgacatgttt
tttttttttt tttttttttt tttttttvn 59 <210> SEQ ID NO 6
<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: 5'-PCR primer <400> SEQUENCE: 6 aagcagtggt
atcaacgcag agt 23 <210> SEQ ID NO 7 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer M13
<400> SEQUENCE: 7 tgtaaaacga cggccagt 18 <210> SEQ ID
NO 8 <211> LENGTH: 32 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Primer 27203-F <400> SEQUENCE: 8
gatcgtcgac ataagtttgc aaaaagatcg ta 32 <210> SEQ ID NO 9
<211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer 27203-R <400> SEQUENCE: 9 gatcccatgg
ttgtgaatta gggtggtgag aatg 34 <210> SEQ ID NO 10 <211>
LENGTH: 8953 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Plasmid pY5-30 <400> SEQUENCE: 10 ggtggagctc cagcttttgt
tccctttagt gagggttaat ttcgagcttg gcgtaatcat 60 ggtcatagct
gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag 120
ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
180 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
cattaatgaa 240 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
ctcttccgct tcctcgctca 300 ctgactcgct gcgctcggtc gttcggctgc
ggcgagcggt atcagctcac tcaaaggcgg 360 taatacggtt atccacagaa
tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 420 agcaaaaggc
caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 480
cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
540 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
gttccgaccc 600 tgccgcttac cggatacctg tccgcctttc tcccttcggg
aagcgtggcg ctttctcata 660 gctcacgctg taggtatctc agttcggtgt
aggtcgttcg ctccaagctg ggctgtgtgc 720 acgaaccccc cgttcagccc
gaccgctgcg ccttatccgg taactatcgt cttgagtcca 780 acccggtaag
acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 840
cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
900 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
aaaagagttg 960 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
tggttttttt gtttgcaagc 1020 agcagattac gcgcagaaaa aaaggatctc
aagaagatcc tttgatcttt tctacggggt 1080 ctgacgctca gtggaacgaa
aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 1140 ggatcttcac
ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 1200
atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
1260 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
actacgatac 1320 gggagggctt accatctggc cccagtgctg caatgatacc
gcgagaccca cgctcaccgg 1380 ctccagattt atcagcaata aaccagccag
ccggaagggc cgagcgcaga agtggtcctg 1440 caactttatc cgcctccatc
cagtctatta attgttgccg ggaagctaga gtaagtagtt 1500 cgccagttaa
tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 1560
cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
1620 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
gtcagaagta 1680 agttggccgc agtgttatca ctcatggtta tggcagcact
gcataattct cttactgtca 1740 tgccatccgt aagatgcttt tctgtgactg
gtgagtactc aaccaagtca ttctgagaat 1800 agtgtatgcg gcgaccgagt
tgctcttgcc cggcgtcaat acgggataat accgcgccac 1860 atagcagaac
tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 1920
ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
1980 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
caaaatgccg 2040 caaaaaaggg aataagggcg acacggaaat gttgaatact
catactcttc ctttttcaat 2100 attattgaag catttatcag ggttattgtc
tcatgagcgg atacatattt gaatgtattt 2160 agaaaaataa acaaataggg
gttccgcgca catttccccg aaaagtgcca cctgacgcgc 2220 cctgtagcgg
cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 2280
ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
2340 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
tttagtgctt 2400 tacggcacct cgaccccaaa aaacttgatt agggtgatgg
ttcacgtagt gggccatcgc 2460 cctgatagac ggtttttcgc cctttgacgt
tggagtccac gttctttaat agtggactct 2520 tgttccaaac tggaacaaca
ctcaacccta tctcggtcta ttcttttgat ttataaggga 2580 ttttgccgat
ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 2640
attttaacaa aatattaacg cttacaattt ccattcgcca ttcaggctgc gcaactgttg
2700 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
ggggatgtgc 2760 tgcaaggcga ttaagttggg taacgccagg gttttcccag
tcacgacgtt gtaaaacgac 2820 ggccagtgaa ttgtaatacg actcactata
gggcgaattg ggtaccgggc cccccctcga 2880 ggtcgatggt gtcgataagc
ttgatatcga attcatgtca cacaaaccga tcttcgcctc 2940 aaggaaacct
aattctacat ccgagagact gccgagatcc agtctacact gattaatttt 3000
cgggccaata atttaaaaaa atcgtgttat ataatattat atgtattata tatatacatc
3060 atgatgatac tgacagtcat gtcccattgc taaatagaca gactccatct
gccgcctcca 3120 actgatgttc tcaatattta aggggtcatc tcgcattgtt
taataataaa cagactccat 3180 ctaccgcctc caaatgatgt tctcaaaata
tattgtatga acttattttt attacttagt 3240 attattagac aacttacttg
ctttatgaaa aacacttcct atttaggaaa caatttataa 3300 tggcagttcg
ttcatttaac aatttatgta gaataaatgt tataaatgcg tatgggaaat 3360
cttaaatatg gatagcataa atgatatctg cattgcctaa ttcgaaatca acagcaacga
3420 aaaaaatccc ttgtacaaca taaatagtca tcgagaaata tcaactatca
aagaacagct 3480 attcacacgt tactattgag attattattg gacgagaatc
acacactcaa ctgtctttct 3540 ctcttctaga aatacaggta caagtatgta
ctattctcat tgttcatact tctagtcatt 3600 tcatcccaca tattccttgg
atttctctcc aatgaatgac attctatctt gcaaattcaa 3660 caattataat
aagatatacc aaagtagcgg tatagtggca atcaaaaagc ttctctggtg 3720
tgcttctcgt atttattttt attctaatga tccattaaag gtatatattt atttcttgtt
3780 atataatcct tttgtttatt acatgggctg gatacataaa ggtattttga
tttaattttt 3840 tgcttaaatt caatcccccc tcgttcagtg tcaactgtaa
tggtaggaaa ttaccatact 3900
tttgaagaag caaaaaaaat gaaagaaaaa aaaaatcgta tttccaggtt agacgttccg
3960 cagaatctag aatgcggtat gcggtacatt gttcttcgaa cgtaaaagtt
gcgctccctg 4020 agatattgta catttttgct tttacaagta caagtacatc
gtacaactat gtactactgt 4080 tgatgcatcc acaacagttt gttttgtttt
tttttgtttt ttttttttct aatgattcat 4140 taccgctatg tatacctact
tgtacttgta gtaagccggg ttattggcgt tcaattaatc 4200 atagacttat
gaatctgcac ggtgtgcgct gcgagttact tttagcttat gcatgctact 4260
tgggtgtaat attgggatct gttcggaaat caacggatgc tcaaccgatt tcgacagtaa
4320 taatttgaat cgaatcggag cctaaaatga acccgagtat atctcataaa
attctcggtg 4380 agaggtctgt gactgtcagt acaaggtgcc ttcattatgc
cctcaacctt accatacctc 4440 actgaatgta gtgtacctct aaaaatgaaa
tacagtgcca aaagccaagg cactgagctc 4500 gtctaacgga cttgatatac
aaccaattaa aacaaatgaa aagaaataca gttctttgta 4560 tcatttgtaa
caattaccct gtacaaacta aggtattgaa atcccacaat attcccaaag 4620
tccacccctt tccaaattgt catgcctaca actcatatac caagcactaa cctaccaaac
4680 accactaaaa ccccacaaaa tatatcttac cgaatataca gtaacaagct
accaccacac 4740 tcgttgggtg cagtcgccag cttaaagata tctatccaca
tcagccacaa ctcccttcct 4800 ttaataaacc gactacaccc ttggctattg
aggttatgag tgaatatact gtagacaaga 4860 cactttcaag aagactgttt
ccaaaacgta ccactgtcct ccactacaaa cacacccaat 4920 ctgcttcttc
tagtcaaggt tgctacaccg gtaaattata aatcatcatt tcattagcag 4980
ggcagggccc tttttataga gtcttataca ctagcggacc ctgccggtag accaacccgc
5040 aggcgcgtca gtttgctcct tccatcaatg cgtcgtagaa acgacttact
ccttcttgag 5100 cagctccttg accttgttgg caacaagtct ccgacctcgg
aggtggagga agagcctccg 5160 atatcggcgg tagtgatacc agcctcgacg
gactccttga cggcagcctc aacagcgtca 5220 ccggcgggct tcatgttaag
agagaacttg agcatcatgg cggcagacag aatggtggca 5280 atggggttga
ccttctgctt gccgagatcg ggggcagatc cgtgacaggg ctcgtacaga 5340
ccgaacgcct cgttggtgtc gggcagagaa gccagagagg cggagggcag cagacccaga
5400 gaaccgggga tgacggaggc ctcgtcggag atgatatcgc caaacatgtt
ggtggtgatg 5460 atgataccat tcatcttgga gggctgcttg atgaggatca
tggcggccga gtcgatcagc 5520 tggtggttga gctcgagctg ggggaattcg
tccttgagga ctcgagtgac agtctttcgc 5580 caaagtcgag aggaggccag
cacgttggcc ttgtcaagag accacacggg aagagggggg 5640 ttgtgctgaa
gggccaggaa ggcggccatt cgggcaattc gctcaacctc aggaacggag 5700
taggtctcgg tgtcggaagc gacgccagat ccgtcatcct cctttcgctc tccaaagtag
5760 atacctccga cgagctctcg gacaatgatg aagtcggtgc cctcaacgtt
tcggatgggg 5820 gagagatcgg cgagcttggg cgacagcagc tggcagggtc
gcaggttggc gtacaggttc 5880 aggtcctttc gcagcttgag gagaccctgc
tcgggtcgca cgtcggttcg tccgtcggga 5940 gtggtccata cggtgttggc
agcgcctccg acagcaccga gcataataga gtcagccttt 6000 cggcagatgt
cgagagtagc gtcggtgatg ggctcgccct ccttctcaat ggcagctcct 6060
ccaatgagtc ggtcctcaaa cacaaactcg gtgccggagg cctcagcaac agacttgagc
6120 accttgacgg cctcggcaat cacctcgggg ccacagaagt cgccgccgag
aagaacaatc 6180 ttcttggagt cagtcttggt cttcttagtt tcgggttcca
ttgtggatgt gtgtggttgt 6240 atgtgtgatg tggtgtgtgg agtgaaaatc
tgtggctggc aaacgctctt gtatatatac 6300 gcacttttgc ccgtgctatg
tggaagacta aacctccgaa gattgtgact caggtagtgc 6360 ggtatcggct
agggacccaa accttgtcga tgccgatagc gctatcgaac gtaccccagc 6420
cggccgggag tatgtcggag gggacatacg agatcgtcaa gggtttgtgg ccaactggta
6480 aataaatgat gtcgactcag gcgacgacgg aattcctgca gcccatctgc
agaattcagg 6540 agagaccggg ttggcggcgt atttgtgtcc caaaaaacag
ccccaattgc cccaattgac 6600 cccaaattga cccagtagcg ggcccaaccc
cggcgagagc ccccttcacc ccacatatca 6660 aacctccccc ggttcccaca
cttgccgtta agggcgtagg gtactgcagt ctggaatcta 6720 cgcttgttca
gactttgtac tagtttcttt gtctggccat ccgggtaacc catgccggac 6780
gcaaaataga ctactgaaaa tttttttgct ttgtggttgg gactttagcc aagggtataa
6840 aagaccaccg tccccgaatt acctttcctc ttcttttctc tctctccttg
tcaactcaca 6900 cccgaaatcg ttaagcattt ccttctgagt ataagaatca
ttcaccatgg atggtacgtc 6960 ctgtagaaac cccaacccgt gaaatcaaaa
aactcgacgg cctgtgggca ttcagtctgg 7020 atcgcgaaaa ctgtggaatt
gatcagcgtt ggtgggaaag cgcgttacaa gaaagccggg 7080 caattgctgt
gccaggcagt tttaacgatc agttcgccga tgcagatatt cgtaattatg 7140
cgggcaacgt ctggtatcag cgcgaagtct ttataccgaa aggttgggca ggccagcgta
7200 tcgtgctgcg tttcgatgcg gtcactcatt acggcaaagt gtgggtcaat
aatcaggaag 7260 tgatggagca tcagggcggc tatacgccat ttgaagccga
tgtcacgccg tatgttattg 7320 ccgggaaaag tgtacgtatc accgtttgtg
tgaacaacga actgaactgg cagactatcc 7380 cgccgggaat ggtgattacc
gacgaaaacg gcaagaaaaa gcagtcttac ttccatgatt 7440 tctttaacta
tgccgggatc catcgcagcg taatgctcta caccacgccg aacacctggg 7500
tggacgatat caccgtggtg acgcatgtcg cgcaagactg taaccacgcg tctgttgact
7560 ggcaggtggt ggccaatggt gatgtcagcg ttgaactgcg tgatgcggat
caacaggtgg 7620 ttgcaactgg acaaggcact agcgggactt tgcaagtggt
gaatccgcac ctctggcaac 7680 cgggtgaagg ttatctctat gaactgtgcg
tcacagccaa aagccagaca gagtgtgata 7740 tctacccgct tcgcgtcggc
atccggtcag tggcagtgaa gggcgaacag ttcctgatta 7800 accacaaacc
gttctacttt actggctttg gtcgtcatga agatgcggac ttacgtggca 7860
aaggattcga taacgtgctg atggtgcacg accacgcatt aatggactgg attggggcca
7920 actcctaccg tacctcgcat tacccttacg ctgaagagat gctcgactgg
gcagatgaac 7980 atggcatcgt ggtgattgat gaaactgctg ctgtcggctt
taacctctct ttaggcattg 8040 gtttcgaagc gggcaacaag ccgaaagaac
tgtacagcga agaggcagtc aacggggaaa 8100 ctcagcaagc gcacttacag
gcgattaaag agctgatagc gcgtgacaaa aaccacccaa 8160 gcgtggtgat
gtggagtatt gccaacgaac cggatacccg tccgcaagtg cacgggaata 8220
tttcgccact ggcggaagca acgcgtaaac tcgacccgac gcgtccgatc acctgcgtca
8280 atgtaatgtt ctgcgacgct cacaccgata ccatcagcga tctctttgat
gtgctgtgcc 8340 tgaaccgtta ttacggatgg tatgtccaaa gcggcgattt
ggaaacggca gagaaggtac 8400 tggaaaaaga acttctggcc tggcaggaga
aactgcatca gccgattatc atcaccgaat 8460 acggcgtgga tacgttagcc
gggctgcact caatgtacac cgacatgtgg agtgaagagt 8520 atcagtgtgc
atggctggat atgtatcacc gcgtctttga tcgcgtcagc gccgtcgtcg 8580
gtgaacaggt atggaatttc gccgattttg cgacctcgca aggcatattg cgcgttggcg
8640 gtaacaagaa agggatcttc actcgcgacc gcaaaccgaa gtcggcggct
tttctgctgc 8700 aaaaacgctg gactggcatg aacttcggtg aaaaaccgca
gcagggaggc aaacaatgat 8760 taattaacta gagcggccgc caccgcggcc
cgagattccg gcctcttcgg ccgccaagcg 8820 acccgggtgg acgtctagag
gtacctagca attaacagat agtttgccgg tgataattct 8880 cttaacctcc
cacactcctt tgacataacg atttatgtaa cgaaactgaa atttgaccag 8940
atattgtgtc cgc 8953 <210> SEQ ID NO 11 <211> LENGTH:
973 <212> TYPE: DNA <213> ORGANISM: Yarrowia lipolytica
<220> FEATURE: <221> NAME/KEY: misc_feature <223>
OTHER INFORMATION: Promoter FBAIN <400> SEQUENCE: 11
aaattgcccc ggagaagacg gccaggccgc ctagatgaca aattcaacaa ctcacagctg
60 actttctgcc attgccacta ggggggggcc tttttatatg gccaagccaa
gctctccacg 120 tcggttgggc tgcacccaac aataaatggg tagggttgca
ccaacaaagg gatgggatgg 180 ggggtagaag atacgaggat aacggggctc
aatggcacaa ataagaacga atactgccat 240 taagactcgt gatccagcga
ctgacaccat tgcatcatct aagggcctca aaactacctc 300 ggaactgctg
cgctgatctg gacaccacag aggttccgag cactttaggt tgcaccaaat 360
gtcccaccag gtgcaggcag aaaacgctgg aacagcgtgt acagtttgtc ttaacaaaaa
420 gtgagggcgc tgaggtcgag cagggtggtg tgacttgtta tagcctttag
agctgcgaaa 480 gcgcgtatgg atttggctca tcaggccaga ttgagggtct
gtggacacat gtcatgttag 540 tgtacttcaa tcgccccctg gatatagccc
cgacaatagg ccgtggcctc atttttttgc 600 cttccgcaca tttccattgc
tcggtaccca caccttgctt ctcctgcact tgccaacctt 660 aatactggtt
tacattgacc aacatcttac aagcgggggg cttgtctagg gtatatataa 720
acagtggctc tcccaatcgg ttgccagtct cttttttcct ttctttcccc acagattcga
780 aatctaaact acacatcaca caatgcctgt tactgacgtc cttaagcgaa
agtccggtgt 840 catcgtcggc gacgatgtcc gagccgtgag tatccacgac
aagatcagtg tcgagacgac 900 gcgttttgtg taatgacaca atccgaaagt
cgctagcaac acacactctc tacacaaact 960 aacccagctc tcc 973 <210>
SEQ ID NO 12 <211> LENGTH: 727 <212> TYPE: PRT
<213> ORGANISM: Yarrowia lipolytica (ORF YALI-CDS1055.1 in
the Genolevures database of Y. lipolytica ORFs (sponsored by the
Center for Bioinformatics; http://cbi.labri.fr/Genolevures/
Y_lipolytica.php) <400> SEQUENCE: 12 Met Ser Glu Thr Asp His
Leu Leu Ala Ala Glu Pro Val Ala Glu Tyr 1 5 10 15 Pro Gln Tyr Thr
Pro Trp Pro Asn Ser Arg Lys Ser Val Asp Thr Glu 20 25 30 Phe Ser
Ala Thr Ser Trp Ile Tyr Asp Leu Val Leu Trp Ile Phe Thr 35 40 45
Ala Cys Phe Asp Ile Phe Phe Arg Glu Ile Arg Pro Arg Gly Ala Phe 50
55 60 Arg Ile Pro Arg Lys Gly Pro Val Leu Phe Val Ala Ala Pro His
Ala 65 70 75 80 Asn Gln Phe Val Asp Pro Val Ile Leu Met Asn Gln Val
Lys Gln Glu 85 90 95 Ala Gly Arg Arg Ile Ser Phe Leu Val Ala Glu
Lys Ser Met Arg Arg 100 105 110 Ala Ala Val Gly Arg Met Ala Arg Ser
Met Asn Ser Ile Pro Val Val 115 120 125 Arg Ala Gln Asp Asn Ala Lys
Lys Gly Glu Gly Lys Ile Tyr Val Asp
130 135 140 Ala Glu Asp Pro Thr Lys Ile His Gly Ile Gly Thr Gln Phe
Thr Lys 145 150 155 160 Gln Cys Glu Val Arg Gly Leu Val Val Cys Ser
Ser Ser Val Gly Ser 165 170 175 Ile Asp Val Ala Glu Ile Val Ser Asp
Thr Leu Leu Ile Ala Arg Lys 180 185 190 Glu Phe Lys Gly Pro Lys Ala
Lys Glu Ala Leu Lys Glu Ser Asn Gly 195 200 205 Gly Ile Thr Tyr Lys
Tyr Ala Asp Tyr Val Asn Gln Ala Thr Val Tyr 210 215 220 Arg Ser Val
Phe Asp Lys Leu His His Gly Gly Cys Val Gly Ile Phe 225 230 235 240
Pro Glu Gly Gly Ser His Asp Arg Thr Glu Leu Leu Pro Leu Lys Ala 245
250 255 Gly Val Ala Ile Met Ala Leu Gly Ala Leu Ala Glu Asp Pro Ser
Cys 260 265 270 Gly Val Arg Ile Val Pro Cys Gly Leu Asn Tyr Phe His
Ala Tyr Lys 275 280 285 Phe Arg Ser Arg Ala Val Val Glu Phe Gly Ser
Pro Ile Ala Ile Pro 290 295 300 Pro Asp Leu Val Glu Lys Tyr Lys Ala
Gly Gly Glu Ala Lys Arg Glu 305 310 315 320 Ala Val Lys Thr Val Leu
Asp Ile Thr Ala Ala Gly Leu Lys Ser Val 325 330 335 Thr Val Gln Val
Gln Asp Phe Asp Thr Leu Met Leu Ile Gln Ala Ile 340 345 350 Arg Arg
Leu Tyr Arg Pro Pro Gly Lys Lys Ile Pro Leu Pro Met Val 355 360 365
Val Glu Leu Asn Arg Arg Leu Val Tyr Ala Tyr Asn His Tyr Lys Asp 370
375 380 Asp Pro Arg Ile Glu Glu Met Lys Gln Glu Ile Arg Lys Tyr Asn
Lys 385 390 395 400 Phe Leu Gln Ala Met Gly Leu Lys Asp His Gln Val
Glu Lys Ala Arg 405 410 415 Ile Ser Lys Ile Glu Ile Leu Gly Arg Leu
Leu Tyr Arg Ser Ile Lys 420 425 430 Leu Val Phe Leu Ser Ile Gly Cys
Leu Pro Gly Leu Leu Leu Phe Ser 435 440 445 Pro Ile Phe Ile Ile Ser
Lys Ser Ile Ser Lys Thr Lys Ala Lys Glu 450 455 460 Ala Leu Lys Ala
Ser Ser Val Lys Ile Lys Ala Asn Asp Val Val Ala 465 470 475 480 Thr
Trp Lys Val Leu Val Ala Met Gly Leu Thr Pro Val Leu Tyr Ile 485 490
495 Leu Tyr Ser Leu Val Gly Ser Val Val Ile Arg Lys Leu Asp Leu Ile
500 505 510 Ser Trp Phe Pro Thr Ile Leu Leu Pro Gly Leu Val Leu Ser
Ile Ile 515 520 525 Ile Thr Thr Ser Tyr Ala Ala Leu Ala Met Gly Glu
Ala Gly Met Asp 530 535 540 Ile Phe Lys Ser Leu Arg Pro Leu Ala Leu
Ala Leu Asn Pro Ser Thr 545 550 555 560 Lys Asn Ser Leu Leu Lys Leu
Gln Asn Glu Arg Lys Arg Leu Val Leu 565 570 575 Lys Ser Ser Glu Leu
Val Thr Ser Leu Gly Pro Glu Leu Phe Pro Asp 580 585 590 Phe Pro Glu
Asn Ser Ile Leu Gln Gly Ser Asp Lys Phe Glu Asp Glu 595 600 605 Glu
Asn Tyr Glu Asn Glu Lys Arg Ser His Ser Arg Ser Thr Ser Ala 610 615
620 Thr Ser Leu Ser Ala Met Ser Glu Gly Asp Gly Asp Glu Leu Val Arg
625 630 635 640 Glu Val Arg Lys Gly Ala Ser Tyr Phe Pro Val Ser Thr
Thr Ser Glu 645 650 655 Asp Glu Asp Gln Ala Ile Ser Arg Val Gly Ser
Glu Ala Ser Leu Ala 660 665 670 Asp Ile Pro Leu Phe Gly Met Ser Arg
Ser Gln Ser Gly Ala Ser Leu 675 680 685 Ser Glu Ala Ser Thr His Gly
Ser Ser Thr Gly Ala Asp Ala Glu Glu 690 695 700 Ala Lys Thr Glu Val
Thr Arg Arg Ile Ala Leu Ala Met Glu Glu Lys 705 710 715 720 Arg Arg
Glu Gln Asp Glu Glu 725 <210> SEQ ID NO 13 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
YGPAT-F <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (6)..(6) <223> OTHER INFORMATION: n is
a, c, g, or t <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: n is a, c, g, or t <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (24)..(24) <223>
OTHER INFORMATION: n is a, c, g, or t <400> SEQUENCE: 13
atgtcngaga cygaccayct nctn 24 <210> SEQ ID NO 14 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
YGPAT-R <400> SEQUENCE: 14 ytcytcrtcy tgytctcgyc gytt 24
<210> SEQ ID NO 15 <211> LENGTH: 2184 <212> TYPE:
DNA <213> ORGANISM: Yarrowia lipolytica <400> SEQUENCE:
15 atgtccgaaa ccgaccatct gctggccgcc gagcccgtgg ctgagtaccc
ccagtacacg 60 ccttggccca actcccgaaa atcagtggac acggagtttt
ccgcaacctc gtggatttac 120 gacttggttc tgtggatttt cacggcttgc
tttgacattt ttttcagaga aatccggcca 180 cgtggtgcct tccgaatccc
cagaaagggc cccgtgctgt tcgtggctgc cccccacgca 240 aaccagtttg
tggaccccgt catcctcatg aaccaggtca aacaggaggc cggacgacga 300
atctccttcc ttgtggccga gaagtccatg cgacgagctg cagtcggacg aatggcccga
360 agcatgaact caattcctgt cgtgcgagct caggacaatg caaaaaaggg
agagggaaag 420 atttacgtcg acgcagagga ccccacaaag atccacggaa
tcggcaccca gttcacgaag 480 cagtgcgagg tgcgaggcct cgtggtctgc
tcgtcctctg tcggctcaat tgacgtggct 540 gagattgtgt ccgacactct
gctcattgca agaaaggaat tcaagggccc caaagccaag 600 gaggctctca
aggaatccaa cggaggaatc acatacaagt acgccgacta cgtcaaccag 660
gccacagtct accgatccgt gttcgacaaa ttgcaccatg gaggctgtgt gggtatcttc
720 ccagagggag gatctcatga ccgaaccgaa ctgctgcccc ttaaggccgg
tgttgctatc 780 atggctctgg gggctctcgc agaggacccc tcttgtggtg
tgcgaatcgt cccctgtggt 840 ctcaactact tccacgccta caagttccga
tctcgggccg tggtggagtt tggctctcct 900 attgccattc ctccggatct
cgtggagaag tacaaggcag gaggagaggc caagcgggag 960 gctgtcaaga
ccgttctaga cattactgcc gctggtctca agtctgtgac tgttcaggtg 1020
caggatttcg acaccctgat gctgatccag gccattcgac gactctaccg acctcccgga
1080 aagaagattc ctctgcccat ggttgtagag ctcaaccgtc gacttgtata
cgcctacaac 1140 cactacaagg acgatccccg tatcgaggag atgaagcagg
agattcgaaa gtacaacaag 1200 ttcctgcagg ccatgggtct caaggaccat
caggtagaga aggcccgaat ctccaagatt 1260 gagattctgg gccggcttct
gtaccggtcc atcaagcttg tgttcttgtc cattggctgt 1320 ctccccggtc
tgcttttgtt ttctcccatc ttcatcattt ctaagtccat ttccaaaacc 1380
aaggccaagg aggctctcaa ggcctccagt gtcaaaatca aggctaacga tgtggttgcc
1440 acttggaagg tgctggttgc aatgggtctg accccagttc tttacattct
ctattcactg 1500 gttggatctg tggtgattcg aaagctcgat ctcatctcct
ggttccccac aattcttctt 1560 cccggcctcg ttttaagcat catcatcaca
acctcatacg ccgccctggc tatgggagag 1620 gccggtatgg acattttcaa
gtctcttcga ccacttgcat tggctctcaa cccttccacc 1680 aaaaactctc
tgctcaagct gcaaaatgaa cgaaagcgac ttgtgctcaa gtcttccgag 1740
ctcgttacct ctttgggccc tgagctgttc cccgacttcc ccgagaactc cattctgcag
1800 ggaagcgata agtttgagga cgaggagaac tacgaaaacg agaagcgatc
gcattccaga 1860 tccacttctg ccacttctct atctgccatg agcgagggag
acggtgatga gcttgttcgg 1920 gaggtccgaa agggtgctag ctacttccct
gtgagtacca cttctgagga cgaagaccaa 1980 gccatctcgc gagtgggctc
tgaggcatct cttgctgaca ttcctctgtt tggtatgtcc 2040 cgatcacaat
ctggagcttc tctttcggaa gcctccacac acggctcttc tactggagct 2100
gatgccgagg aggctaagac ggaggtgact cgcagaattg cattggcgat ggaggaaaaa
2160 cgacgagagc aggatgagga ataa 2184 <210> SEQ ID NO 16
<211> LENGTH: 44 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Genome Walker adaptor-1 <400> SEQUENCE: 16
gtaatacgac tatagggcac gcgtggtcga cggcccgggc tggt 44 <210> SEQ
ID NO 17 <211> LENGTH: 8 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Genome Walker adaptor-2 <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: 5' end is associated with a -PO4
group <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (8)..(8) <223> OTHER INFORMATION: 3'
end is associated with a -H2N group <400> SEQUENCE: 17
accagccc 8 <210> SEQ ID NO 18 <211> LENGTH: 22
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Primer
Adaptor-1 <400> SEQUENCE: 18 gtaatacgac tcactatagg gc 22
<210> SEQ ID NO 19 <211> LENGTH: 23 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer YGPAT-5R-1 <400>
SEQUENCE: 19 gtcaaagcaa gcccgtgaaa atc 23 <210> SEQ ID NO 20
<211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Nested Adaptor Primer 2 <400> SEQUENCE: 20
actatagggc acgcgtggt 19 <210> SEQ ID NO 21 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer
YGPAT-5R-2 <400> SEQUENCE: 21 gtcgtaaatc cacgaggttg 20
<210> SEQ ID NO 22 <211> LENGTH: 1781 <212> TYPE:
DNA <213> ORGANISM: Yarrowia lipolytica <400> SEQUENCE:
22 atccagacga cggcatggga gaacggattg gttttttttt gagttcatca
tgtcctgcca 60 acccactgaa aacagggtcc gatgcggaat gatggctttt
tactgtagtc agtgtctttt 120 acagaaatac acccccagcc atcatatatc
ctctccaggc atggaccact gggtacactc 180 gtgacgtgtc ataatcactg
agccagtata cgaacaaaat acaatcctgc catcgtactc 240 actcaaacaa
ccgtgttcaa taaggaagct gggccagaca gctgaaatgt accaaccagt 300
cgaggtgaag cacagaacaa gaagacacaa accacgatct ttacaagaat gtactgtagt
360 acatacagca cgagtagtgt acgaagtttt tgtcagacac gtgacttgct
cgtaaattag 420 ccaatatgac acaccgccaa ggacctcgga tgcgtgcggt
gcgtgcggtg cgtgcggtgt 480 gtgcggtgcg tgcgtatgta ccccactctg
tgtcgatcag ccgacagtga tgttccggaa 540 gtgcggtaca acttttcttg
tcgacctgag ataccgaggt tgcgcagggg atcaactttt 600 gtgtctcaga
gggacccaag tgcgtacgga gagtacagta catactgtag ctaacggtag 660
caggcgaact actggtacat acctcccccg gaatatgtac aggcataatg cgtatctgtg
720 ggacatgtgg tcgttgcgcc attatgtaag cagcgtgtac tcctctgact
gtccatatgg 780 tttgctccat ctcaccctca tcgttttcat tgttcacagg
cggccacaaa aaaactgtct 840 tctctccttc tctcttcgcc ttagtctact
cggaccagtt ttagtttagc ttggcgccac 900 tggataaatg agacctcagg
ccttgtgatg aggaggtcac ttatgaagca tgttaggagg 960 tgcttgtatg
gatagagaag cacccaaaat aataagaata ataataaaac agggggcgtt 1020
gtcatttcat atcgtgtttt caccatcaat acacctccaa acaatgccct tcatgtggcc
1080 agccccaata ttgtcctgta gttcaactct atgcagctcg tatcttattg
agcaagtaaa 1140 actctgtcag ccgatattgc ccgacccgcg acaagggtca
acaaggtggt gtaaggcctt 1200 cgcagaagtc aaaactgtgc caaacaaaca
tctagagtct ctttggtgtt tctcgcatat 1260 atttaatcgg ctgtcttacg
tatttggcct cggtaccgga ctaatttcgg atcatcccca 1320 atacgctttt
tcttcgcagc tgtcaacagt gtccatgatc tatccaccta aatgggtcat 1380
atgaggcgta taatttcgtg gtgctgataa taattcccat atatttgaca caaaacttcc
1440 ccccctagac atacatctca caatctcact tcttgtgctt ctgtcacaca
tctcctccag 1500 ctgacttcaa ctcacacctc tgccccagtt ggtctacagc
ggtataaggt ttctccgcat 1560 agaggtgcac cactcctccc gatacttgtt
tgtgtgactt gtgggtcacg acatatatat 1620 ctacacacat tgcgccaccc
tttggttctt ccagcacaac aaaaacacga cacgctaaat 1680 gtccgaaacc
gaccatctgc tggccgccga gcccgtggct gagtaccccc agtacacgcc 1740
ttggcccaac tcccgaaaat cagtggacac ggagttttcc g 1781 <210> SEQ
ID NO 23 <211> LENGTH: 3862 <212> TYPE: DNA <213>
ORGANISM: Yarrowia lipolytica <400> SEQUENCE: 23 atccagacga
cggcatggga gaacggattg gttttttttt gagttcatca tgtcctgcca 60
acccactgaa aacagggtcc gatgcggaat gatggctttt tactgtagtc agtgtctttt
120 acagaaatac acccccagcc atcatatatc ctctccaggc atggaccact
gggtacactc 180 gtgacgtgtc ataatcactg agccagtata cgaacaaaat
acaatcctgc catcgtactc 240 actcaaacaa ccgtgttcaa taaggaagct
gggccagaca gctgaaatgt accaaccagt 300 cgaggtgaag cacagaacaa
gaagacacaa accacgatct ttacaagaat gtactgtagt 360 acatacagca
cgagtagtgt acgaagtttt tgtcagacac gtgacttgct cgtaaattag 420
ccaatatgac acaccgccaa ggacctcgga tgcgtgcggt gcgtgcggtg cgtgcggtgt
480 gtgcggtgcg tgcgtatgta ccccactctg tgtcgatcag ccgacagtga
tgttccggaa 540 gtgcggtaca acttttcttg tcgacctgag ataccgaggt
tgcgcagggg atcaactttt 600 gtgtctcaga gggacccaag tgcgtacgga
gagtacagta catactgtag ctaacggtag 660 caggcgaact actggtacat
acctcccccg gaatatgtac aggcataatg cgtatctgtg 720 ggacatgtgg
tcgttgcgcc attatgtaag cagcgtgtac tcctctgact gtccatatgg 780
tttgctccat ctcaccctca tcgttttcat tgttcacagg cggccacaaa aaaactgtct
840 tctctccttc tctcttcgcc ttagtctact cggaccagtt ttagtttagc
ttggcgccac 900 tggataaatg agacctcagg ccttgtgatg aggaggtcac
ttatgaagca tgttaggagg 960 tgcttgtatg gatagagaag cacccaaaat
aataagaata ataataaaac agggggcgtt 1020 gtcatttcat atcgtgtttt
caccatcaat acacctccaa acaatgccct tcatgtggcc 1080 agccccaata
ttgtcctgta gttcaactct atgcagctcg tatcttattg agcaagtaaa 1140
actctgtcag ccgatattgc ccgacccgcg acaagggtca acaaggtggt gtaaggcctt
1200 cgcagaagtc aaaactgtgc caaacaaaca tctagagtct ctttggtgtt
tctcgcatat 1260 atttaatcgg ctgtcttacg tatttggcct cggtaccgga
ctaatttcgg atcatcccca 1320 atacgctttt tcttcgcagc tgtcaacagt
gtccatgatc tatccaccta aatgggtcat 1380 atgaggcgta taatttcgtg
gtgctgataa taattcccat atatttgaca caaaacttcc 1440 ccccctagac
atacatctca caatctcact tcttgtgctt ctgtcacaca tctcctccag 1500
ctgacttcaa ctcacacctc tgccccagtt ggtctacagc ggtataaggt ttctccgcat
1560 agaggtgcac cactcctccc gatacttgtt tgtgtgactt gtgggtcacg
acatatatat 1620 ctacacacat tgcgccaccc tttggttctt ccagcacaac
aaaaacacga cacgctaaat 1680 gtccgaaacc gaccatctgc tggccgccga
gcccgtggct gagtaccccc agtacacgcc 1740 ttggcccaac tcccgaaaat
cagtggacac ggagttttcc gcaacctcgt ggatttacga 1800 cttggttctg
tggattttca cggcttgctt tgacattttt ttcagagaaa tccggccacg 1860
tggtgccttc cgaatcccca gaaagggccc cgtgctgttc gtggctgccc cccacgcaaa
1920 ccagtttgtg gaccccgtca tcctcatgaa ccaggtcaaa caggaggccg
gacgacgaat 1980 ctccttcctt gtggccgaga agtccatgcg acgagctgca
gtcggacgaa tggcccgaag 2040 catgaactca attcctgtcg tgcgagctca
ggacaatgca aaaaagggag agggaaagat 2100 ttacgtcgac gcagaggacc
ccacaaagat ccacggaatc ggcacccagt tcacgaagca 2160 gtgcgaggtg
cgaggcctcg tggtctgctc gtcctctgtc ggctcaattg acgtggctga 2220
gattgtgtcc gacactctgc tcattgcaag aaaggaattc aagggcccca aagccaagga
2280 ggctctcaag gaatccaacg gaggaatcac atacaagtac gccgactacg
tcaaccaggc 2340 cacagtctac cgatccgtgt tcgacaaatt gcaccatgga
ggctgtgtgg gtatcttccc 2400 agagggagga tctcatgacc gaaccgaact
gctgcccctt aaggccggtg ttgctatcat 2460 ggctctgggg gctctcgcag
aggacccctc ttgtggtgtg cgaatcgtcc cctgtggtct 2520 caactacttc
cacgcctaca agttccgatc tcgggccgtg gtggagtttg gctctcctat 2580
tgccattcct ccggatctcg tggagaagta caaggcagga ggagaggcca agcgggaggc
2640 tgtcaagacc gttctagaca ttactgccgc tggtctcaag tctgtgactg
ttcaggtgca 2700 ggatttcgac accctgatgc tgatccaggc cattcgacga
ctctaccgac ctcccggaaa 2760 gaagattcct ctgcccatgg ttgtagagct
caaccgtcga cttgtatacg cctacaacca 2820 ctacaaggac gatccccgta
tcgaggagat gaagcaggag attcgaaagt acaacaagtt 2880 cctgcaggcc
atgggtctca aggaccatca ggtagagaag gcccgaatct ccaagattga 2940
gattctgggc cggcttctgt accggtccat caagcttgtg ttcttgtcca ttggctgtct
3000 ccccggtctg cttttgtttt ctcccatctt catcatttct aagtccattt
ccaaaaccaa 3060 ggccaaggag gctctcaagg cctccagtgt caaaatcaag
gctaacgatg tggttgccac 3120 ttggaaggtg ctggttgcaa tgggtctgac
cccagttctt tacattctct attcactggt 3180 tggatctgtg gtgattcgaa
agctcgatct catctcctgg ttccccacaa ttcttcttcc 3240 cggcctcgtt
ttaagcatca tcatcacaac ctcatacgcc gccctggcta tgggagaggc 3300
cggtatggac attttcaagt ctcttcgacc acttgcattg gctctcaacc cttccaccaa
3360 aaactctctg ctcaagctgc aaaatgaacg aaagcgactt gtgctcaagt
cttccgagct 3420 cgttacctct ttgggccctg agctgttccc cgacttcccc
gagaactcca ttctgcaggg 3480
aagcgataag tttgaggacg aggagaacta cgaaaacgag aagcgatcgc attccagatc
3540 cacttctgcc acttctctat ctgccatgag cgagggagac ggtgatgagc
ttgttcggga 3600 ggtccgaaag ggtgctagct acttccctgt gagtaccact
tctgaggacg aagaccaagc 3660 catctcgcga gtgggctctg aggcatctct
tgctgacatt cctctgtttg gtatgtcccg 3720 atcacaatct ggagcttctc
tttcggaagc ctccacacac ggctcttcta ctggagctga 3780 tgccgaggag
gctaagacgg aggtgactcg cagaattgca ttggcgatgg aggaaaaacg 3840
acgagagcag gatgaggaat aa 3862 <210> SEQ ID NO 24 <211>
LENGTH: 1130 <212> TYPE: DNA <213> ORGANISM: Yarrowia
lipolytica <220> FEATURE: <221> NAME/KEY: misc_feature
<223> OTHER INFORMATION: GPAT promoter <400> SEQUENCE:
24 caacttttct tgtcgacctg agataccgag gttgcgcagg ggatcaactt
ttgtgtctca 60 gagggaccca agtgcgtacg gagagtacag tacatactgt
agctaacggt agcaggcgaa 120 ctactggtac atacctcccc cggaatatgt
acaggcataa tgcgtatctg tgggacatgt 180 ggtcgttgcg ccattatgta
agcagcgtgt actcctctga ctgtccatat ggtttgctcc 240 atctcaccct
catcgttttc attgttcaca ggcggccaca aaaaaactgt cttctctcct 300
tctctcttcg ccttagtcta ctcggaccag ttttagttta gcttggcgcc actggataaa
360 tgagacctca ggccttgtga tgaggaggtc acttatgaag catgttagga
ggtgcttgta 420 tggatagaga agcacccaaa ataataagaa taataataaa
acagggggcg ttgtcatttc 480 atatcgtgtt ttcaccatca atacacctcc
aaacaatgcc cttcatgtgg ccagccccaa 540 tattgtcctg tagttcaact
ctatgcagct cgtatcttat tgagcaagta aaactctgtc 600 agccgatatt
gcccgacccg cgacaagggt caacaaggtg gtgtaaggcc ttcgcagaag 660
tcaaaactgt gccaaacaaa catctagagt ctctttggtg tttctcgcat atatttaatc
720 ggctgtctta cgtatttggc ctcggtaccg gactaatttc ggatcatccc
caatacgctt 780 tttcttcgca gctgtcaaca gtgtccatga tctatccacc
taaatgggtc atatgaggcg 840 tataatttcg tggtgctgat aataattccc
atatatttga cacaaaactt ccccccctag 900 acatacatct cacaatctca
cttcttgtgc ttctgtcaca catctcctcc agctgacttc 960 aactcacacc
tctgccccag ttggtctaca gcggtataag gtttctccgc atagaggtgc 1020
accactcctc ccgatacttg tttgtgtgac ttgtgggtca cgacatatat atctacacac
1080 attgcgccac cctttggttc ttccagcaca acaaaaacac gacacgctaa 1130
<210> SEQ ID NO 25 <211> LENGTH: 22 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Primer GPAT-5-1 <400>
SEQUENCE: 25 caacttttct tgtcgacctg ag 22 <210> SEQ ID NO 26
<211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Primer GPAT-5-2 <400> SEQUENCE: 26 gatcccatgg
ttagcgtgtc gtgtttttgt tgtg 34 <210> SEQ ID NO 27 <211>
LENGTH: 12649 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Plasmid pKUNF12T6E <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (2507)..(2507)
<223> OTHER INFORMATION: n is a, c, g, or t <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(2512)..(2515) <223> OTHER INFORMATION: n is a, c, g, or t
<400> SEQUENCE: 27 taaccctcac taaagggaac aaaagctgga
gctccaccgc ggacacaata tctggtcaaa 60 tttcagtttc gttacataaa
tcgttatgtc aaaggagtgt gggaggttaa gagaattatc 120 accggcaaac
tatctgttaa ttgctaggta cctctagacg tccacccggg tcgcttggcg 180
gccgaagagg ccggaatctc gggccgcggt ggcggccgct tagttggtct tggacttctt
240 gggcttcttc aggtaggact ggacaaagaa gttgccgaac agagcgagca
gggtgatcat 300 gtacacgccg agcagctgga ccagagcctg agggtagtcg
caggggaaga ggtagtcgta 360 cagggactgc accagcatag ccatgaactg
ggtcatctgc agagtggtga tgtagggctt 420 gatgggcttg acgaagccga
agccctgaga ggaaaagaag tagtaggcgt acatgacggt 480 gtggacgaag
gagttgagga tgacggagaa gtaggcgtcg ccaccaggag cgtacttggc 540
aatagcccac cagatggcga agatggtggc atggtggtac acgtgcagga aggagacctg
600 gttgaacttc ttgcacagga tcatgatagc ggtgtccagg aactcgtagg
ccttggagac 660 gtagaacacg tagacgattc gggacatgcc ctgagcgtgg
gactcgttgc ccttctccat 720 gtcgttgccg aagaccttgt agccacccag
gatagcctgt cggatggtct cgacgcacat 780 gtagagggac agtccgaaga
ggaacaggtt gtggagcagc ttgatggtct tcagctcgaa 840 gggcttctcc
atctgcttca tgatgggaat gccgaagagc agcatggcca tgtagccgac 900
ctcgaaggcg agcatggtgg agacgtccat catgggcaga ccgtcggtca gagcgtaggg
960 cttagctccg tccatccact ggtcgacacc ggtctcgact cgtccgacca
cgtcgtccca 1020 gacagaggag ttggccatgg tgaatgattc ttatactcag
aaggaaatgc ttaacgattt 1080 cgggtgtgag ttgacaagga gagagagaaa
agaagaggaa aggtaattcg gggacggtgg 1140 tcttttatac ccttggctaa
agtcccaacc acaaagcaaa aaaattttca gtagtctatt 1200 ttgcgtccgg
catgggttac ccggatggcc agacaaagaa actagtacaa agtctgaaca 1260
agcgtagatt ccagactgca gtaccctacg cccttaacgg caagtgtggg aaccggggga
1320 ggtttgatat gtggggtgaa gggggctctc gccggggttg ggcccgctac
tgggtcaatt 1380 tggggtcaat tggggcaatt ggggctgttt tttgggacac
aaatacgccg ccaacccggt 1440 ctctcctgaa ttctgcatcg atcgaggaag
aggacaagcg gctgcttctt aagtttgtga 1500 catcagtatc caaggcacca
ttgcaaggat tcaaggcttt gaacccgtca tttgccattc 1560 gtaacgctgg
tagacaggtt gatcggttcc ctacggcctc cacctgtgtc aatcttctca 1620
agctgcctga ctatcaggac attgatcaac ttcggaagaa acttttgtat gccattcgat
1680 cacatgctgg tttcgatttg tcttagagga acgcatatac agtaatcata
gagaataaac 1740 gatattcatt tattaaagta gatagttgag gtagaagttg
taaagagtga taaatagcgg 1800 ccgcgcctac ttaagcaacg ggcttgataa
cagcgggggg ggtgcccacg ttgttgcggt 1860 tgcggaagaa cagaacaccc
ttaccagcac cctcggcacc agcgctgggc tcaacccact 1920 ggcacatacg
cgcactgcgg tacatggcgc ggatgaagcc acgaggacca tcctggacat 1980
cagcccggta gtgcttgccc atgatgggct taatggcctc ggtggcctcg tccgcgttgt
2040 agaaggggat gctgctgacg tagtggtgga ggacatgagt ctcgatgatg
ccgtggagaa 2100 ggtggcggcc gatgaagccc atctcacggt caatggtagc
agcggcacca cggacgaagt 2160 tccactcgtc gttggtgtag tggggaaggg
tagggtcggt gtgctggagg aaggtgatgg 2220 caacgagcca gtggttaacc
cagaggtagg gaacaaagta ccagatggcc atgttgtaga 2280 aaccgaactt
ctgaacgagg aagtacagag cagtggccat cagaccgata ccaatatcgc 2340
tgaggacgat gagcttagcg tcactgttct cgtacagagg gctgcgggga tcgaagtggt
2400 taacaccacc gccgaggccg ttatgcttgc ccttgccgcg accctcacgc
tggcgctcgt 2460 ggtagttgtg gccggtaaca ttggtgatga ggtagttggg
ccagccnacg annnnctcag 2520 taagatgagc gagctcgtgg gtcatctttc
cgagacgagt agcctgctgc tcgcgggttc 2580 ggggaacgaa gaccatgtca
cgctccatgt tgccagtggc cttgtggtgc tttcggtggg 2640 agatttgcca
gctgaagtag gggacaagga gggaagagtg aagaacccag ccagtaatgt 2700
cgttgatgat gcgagaatcg gagaaagcac cgtgaccgca ctcatgggca ataacccaga
2760 gaccagtacc gaaaagaccc tgaagaacgg tgtacacggc ccacagacca
gcgcgggcgg 2820 gggtggaggg gatatattcg ggggtcacaa agttgtacca
gatgctgaaa gtggtagtca 2880 ggaggacaat gtcgcggagg atataaccgt
atcccttgag agcggagcgc ttgaagcagt 2940 gcttagggat ggcattgtag
atgtccttga tggtaaagtc gggaacctcg aactggttgc 3000 cgtaggtgtc
gagcatgaca ccatactcgg acttgggctt ggcgatatca acctcggaca 3060
tggacgagag cgatgtggaa gaggccgagt ggcggggaga gtctgaagga gagacggcgg
3120 cagactcaga atccgtcaca gtagttgagg tgacggtgcg tctaagcgca
gggttctgct 3180 tgggcagagc cgaagtggac gccatggaga gctgggttag
tttgtgtaga gagtgtgtgt 3240 tgctagcgac tttcggattg tgtcattaca
caaaacgcgt cgtctcgaca ctgatcttgt 3300 cgtggatact cacggctcgg
acatcgtcgc cgacgatgac accggacttt cgcttaagga 3360 cgtcagtaac
aggcattgtg tgatgtgtag tttagatttc gaatctgtgg ggaaagaaag 3420
gaaaaaagag actggcaacc gattgggaga gccactgttt atatataccc tagacaagcc
3480 ccccgcttgt aagatgttgg tcaatgtaaa ccagtattaa ggttggcaag
tgcaggagaa 3540 gcaaggtgtg ggtaccgagc aatggaaatg tgcggaaggc
aaaaaaatga ggccacggcc 3600 tattgtcggg gctatatcca gggggcgatt
gaagtacact aacatgacat gtgtccacag 3660 accctcaatc tggcctgatg
agccaaatcc atacgcgctt tcgcagctct aaaggctata 3720 acaagtcaca
ccaccctgct cgacctcagc gccctcactt tttgttaaga caaactgtac 3780
acgctgttcc agcgttttct gcctgcacct ggtgggacat ttggtgcaac ctaaagtgct
3840 cggaacctct gtggtgtcca gatcagcgca gcagttccga ggtagttttg
aggcccttag 3900 atgatgcaat ggtgtcagtc gctggatcac gagtcttaat
ggcagtattc gttcttattt 3960 gtgccattga gccccgttat cctcgtatct
tctacccccc atcccatccc tttgttggtg 4020 caaccctacc catttattgt
tgggtgcagc ccaaccgacg tggagagctt ggcttggcca 4080 tataaaaagg
ccccccccta gtggcaatgg cagaaagtca gctgtgagtt gttgaatttg 4140
tcatctaggc ggcctggccg tcttctccgg ggcaattgtt cctctatagt actgcgtaca
4200 ctgtttaaac agtgtacgca gatctgcgac gacggaattc ctgcagccca
tctgcagaat 4260 tcaggagaga ccgggttggc ggcgtatttg tgtcccaaaa
aacagcccca attgccccaa 4320 ttgaccccaa attgacccag tagcgggccc
aaccccggcg agagccccct tcaccccaca 4380
tatcaaacct cccccggttc ccacacttgc cgttaagggc gtagggtact gcagtctgga
4440 atctacgctt gttcagactt tgtactagtt tctttgtctg gccatccggg
taacccatgc 4500 cggacgcaaa atagactact gaaaattttt ttgctttgtg
gttgggactt tagccaaggg 4560 tataaaagac caccgtcccc gaattacctt
tcctcttctt ttctctctct ccttgtcaac 4620 tcacacccga aatcgttaag
catttccttc tgagtataag aatcattcac catggctgcc 4680 gctccctctg
tgcgaacctt tacccgagcc gaggttctga acgctgaggc tctgaacgag 4740
ggcaagaagg acgctgaggc tcccttcctg atgatcatcg acaacaaggt gtacgacgtc
4800 cgagagttcg tccctgacca tcctggaggc tccgtgattc tcacccacgt
tggcaaggac 4860 ggcaccgacg tctttgacac ctttcatccc gaggctgctt
gggagactct cgccaacttc 4920 tacgttggag acattgacga gtccgaccga
gacatcaaga acgatgactt tgccgctgag 4980 gtccgaaagc tgcgaaccct
gttccagtct ctcggctact acgactcctc taaggcctac 5040 tacgccttca
aggtctcctt caacctctgc atctggggac tgtccaccgt cattgtggcc 5100
aagtggggtc agacctccac cctcgccaac gtgctctctg ctgccctgct cggcctgttc
5160 tggcagcagt gcggatggct ggctcacgac tttctgcacc accaggtctt
ccaggaccga 5220 ttctggggtg atctcttcgg agccttcctg ggaggtgtct
gccagggctt ctcctcttcc 5280 tggtggaagg acaagcacaa cactcaccat
gccgctccca acgtgcatgg cgaggatcct 5340 gacattgaca cccaccctct
cctgacctgg tccgagcacg ctctggagat gttctccgac 5400 gtccccgatg
aggagctgac ccgaatgtgg tctcgattca tggtcctgaa ccagacctgg 5460
ttctacttcc ccattctctc cttcgctcga ctgtcttggt gcctccagtc cattctcttt
5520 gtgctgccca acggtcaggc tcacaagccc tccggagctc gagtgcccat
ctccctggtc 5580 gagcagctgt ccctcgccat gcactggacc tggtacctcg
ctaccatgtt cctgttcatc 5640 aaggatcctg tcaacatgct cgtgtacttc
ctggtgtctc aggctgtgtg cggaaacctg 5700 ctcgccatcg tgttctccct
caaccacaac ggtatgcctg tgatctccaa ggaggaggct 5760 gtcgacatgg
atttctttac caagcagatc atcactggtc gagatgtcca tcctggactg 5820
ttcgccaact ggttcaccgg tggcctgaac taccagatcg agcatcacct gttcccttcc
5880 atgcctcgac acaacttctc caagatccag cctgccgtcg agaccctgtg
caagaagtac 5940 aacgtccgat accacaccac tggtatgatc gagggaactg
ccgaggtctt ctcccgactg 6000 aacgaggtct ccaaggccac ctccaagatg
ggcaaggctc agtaagcggc cgcatgagaa 6060 gataaatata taaatacatt
gagatattaa atgcgctaga ttagagagcc tcatactgct 6120 cggagagaag
ccaagacgag tactcaaagg ggattacacc atccatatcc acagacacaa 6180
gctggggaaa ggttctatat acactttccg gaataccgta gtttccgatg ttatcaatgg
6240 gggcagccag gatttcaggc acttcggtgt ctcggggtga aatggcgttc
ttggcctcca 6300 tcaagtcgta ccatgtcttc atttgcctgt caaagtaaaa
cagaagcaga tgaagaatga 6360 acttgaagtg aaggaattta aattgccccg
gagaagacgg ccaggccgcc tagatgacaa 6420 attcaacaac tcacagctga
ctttctgcca ttgccactag gggggggcct ttttatatgg 6480 ccaagccaag
ctctccacgt cggttgggct gcacccaaca ataaatgggt agggttgcac 6540
caacaaaggg atgggatggg gggtagaaga tacgaggata acggggctca atggcacaaa
6600 taagaacgaa tactgccatt aagactcgtg atccagcgac tgacaccatt
gcatcatcta 6660 agggcctcaa aactacctcg gaactgctgc gctgatctgg
acaccacaga ggttccgagc 6720 actttaggtt gcaccaaatg tcccaccagg
tgcaggcaga aaacgctgga acagcgtgta 6780 cagtttgtct taacaaaaag
tgagggcgct gaggtcgagc agggtggtgt gacttgttat 6840 agcctttaga
gctgcgaaag cgcgtatgga tttggctcat caggccagat tgagggtctg 6900
tggacacatg tcatgttagt gtacttcaat cgccccctgg atatagcccc gacaataggc
6960 cgtggcctca tttttttgcc ttccgcacat ttccattgct cggtacccac
accttgcttc 7020 tcctgcactt gccaacctta atactggttt acattgacca
acatcttaca agcggggggc 7080 ttgtctaggg tatatataaa cagtggctct
cccaatcggt tgccagtctc ttttttcctt 7140 tctttcccca cagattcgaa
atctaaacta cacatcacac aatgcctgtt actgacgtcc 7200 ttaagcgaaa
gtccggtgtc atcgtcggcg acgatgtccg agccgtgagt atccacgaca 7260
agatcagtgt cgagacgacg cgttttgtgt aatgacacaa tccgaaagtc gctagcaaca
7320 cacactctct acacaaacta acccagctct ccatggagtc cattgctccc
ttcctgccct 7380 ccaagatgcc tcaggacctg ttcatggacc tcgccagcgc
tatcggtgtc cgagctgctc 7440 cctacgtcga tcccctggag gctgccctgg
ttgcccaggc cgagaagtac attcccacca 7500 ttgtccatca cactcgaggc
ttcctggttg ccgtggagtc tcccctggct cgagagctgc 7560 ctctgatgaa
ccccttccac gtgctcctga tcgtgctcgc ctacctggtc accgtgtttg 7620
tgggtatgca gatcatgaag aactttgaac gattcgaggt caagaccttc tccctcctgc
7680 acaacttctg tctggtctcc atctccgcct acatgtgcgg tggcatcctg
tacgaggctt 7740 atcaggccaa ctatggactg tttgagaacg ctgccgatca
caccttcaag ggtctcccta 7800 tggctaagat gatctggctc ttctacttct
ccaagatcat ggagtttgtc gacaccatga 7860 tcatggtcct caagaagaac
aaccgacaga tttcctttct gcacgtgtac caccactctt 7920 ccatcttcac
catctggtgg ctggtcacct tcgttgctcc caacggtgaa gcctacttct 7980
ctgctgccct gaactccttc atccacgtca tcatgtacgg ctactacttt ctgtctgccc
8040 tgggcttcaa gcaggtgtcg ttcatcaagt tctacatcac tcgatcccag
atgacccagt 8100 tctgcatgat gtctgtccag tcttcctggg acatgtacgc
catgaaggtc cttggccgac 8160 ctggataccc cttcttcatc accgctctgc
tctggttcta catgtggacc atgctcggtc 8220 tcttctacaa cttttaccga
aagaacgcca agctcgccaa gcaggccaag gctgacgctg 8280 ccaaggagaa
ggccagaaag ctccagtaag cggccgcaag tgtggatggg gaagtgagtg 8340
cccggttctg tgtgcacaat tggcaatcca agatggatgg attcaacaca gggatatagc
8400 gagctacgtg gtggtgcgag gatatagcaa cggatattta tgtttgacac
ttgagaatgt 8460 acgatacaag cactgtccaa gtacaatact aaacatactg
tacatactca tactcgtacc 8520 cgggcaacgg tttcacttga gtgcagtggc
tagtgctctt actcgtacag tgtgcaatac 8580 tgcgtatcat agtctttgat
gtatatcgta ttcattcatg ttagttgcgt acgaagtcgt 8640 caatgatgtc
gatatgggtt ttgatcatgc acacataagg tccgacctta tcggcaagct 8700
caatgagctc cttggtggtg gtaacatcca gagaagcaca caggttggtt ttcttggctg
8760 ccacgagctt gagcactcga gcggcaaagg cggacttgtg gacgttagct
cgagcttcgt 8820 aggagggcat tttggtggtg aagaggagac tgaaataaat
ttagtctgca gaacttttta 8880 tcggaacctt atctggggca gtgaagtata
tgttatggta atagttacga gttagttgaa 8940 cttatagata gactggacta
tacggctatc ggtccaaatt agaaagaacg tcaatggctc 9000 tctgggcgtc
gcctttgccg acaaaaatgt gatcatgatg aaagccagca atgacgttgc 9060
agctgatatt gttgtcggcc aaccgcgccg aaaacgcagc tgtcagaccc acagcctcca
9120 acgaagaatg tatcgtcaaa gtgatccaag cacactcata gttggagtcg
tactccaaag 9180 gcggcaatga cgagtcagac agatactcgt cgaccttttc
cttgggaacc accaccgtca 9240 gcccttctga ctcacgtatt gtagccaccg
acacaggcaa cagtccgtgg atagcagaat 9300 atgtcttgtc ggtccatttc
tcaccaactt taggcgtcaa gtgaatgttg cagaagaagt 9360 atgtgccttc
attgagaatc ggtgttgctg atttcaataa agtcttgaga tcagtttggc 9420
gcgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
9480 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
ggcgagcggt 9540 atcagctcac tcaaaggcgg taatacggtt atccacagaa
tcaggggata acgcaggaaa 9600 gaacatgtga gcaaaaggcc agcaaaaggc
caggaaccgt aaaaaggccg cgttgctggc 9660 gtttttccat aggctccgcc
cccctgacga gcatcacaaa aatcgacgct caagtcagag 9720 gtggcgaaac
ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 9780
gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
9840 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
aggtcgttcg 9900 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
gaccgctgcg ccttatccgg 9960 taactatcgt cttgagtcca acccggtaag
acacgactta tcgccactgg cagcagccac 10020 tggtaacagg attagcagag
cgaggtatgt aggcggtgct acagagttct tgaagtggtg 10080 gcctaactac
ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt 10140
taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
10200 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
aagaagatcc 10260 tttgatcttt tctacggggt ctgacgctca gtggaacgaa
aactcacgtt aagggatttt 10320 ggtcatgaga ttatcaaaaa ggatcttcac
ctagatcctt ttaaattaaa aatgaagttt 10380 taaatcaatc taaagtatat
atgagtaaac ttggtctgac agttaccaat gcttaatcag 10440 tgaggcacct
atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 10500
cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
10560 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
ccggaagggc 10620 cgagcgcaga agtggtcctg caactttatc cgcctccatc
cagtctatta attgttgccg 10680 ggaagctaga gtaagtagtt cgccagttaa
tagtttgcgc aacgttgttg ccattgctac 10740 aggcatcgtg gtgtcacgct
cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 10800 atcaaggcga
gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 10860
tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
10920 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
gtgagtactc 10980 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
tgctcttgcc cggcgtcaat 11040 acgggataat accgcgccac atagcagaac
tttaaaagtg ctcatcattg gaaaacgttc 11100 ttcggggcga aaactctcaa
ggatcttacc gctgttgaga tccagttcga tgtaacccac 11160 tcgtgcaccc
aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 11220
aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
11280 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
tcatgagcgg 11340 atacatattt gaatgtattt agaaaaataa acaaataggg
gttccgcgca catttccccg 11400 aaaagtgcca cctgatgcgg tgtgaaatac
cgcacagatg cgtaaggaga aaataccgca 11460 tcaggaaatt gtaagcgtta
atattttgtt aaaattcgcg ttaaattttt gttaaatcag 11520 ctcatttttt
aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac 11580
cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
11640 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
gtgaaccatc 11700 accctaatca agttttttgg ggtcgaggtg ccgtaaagca
ctaaatcgga accctaaagg 11760 gagcccccga tttagagctt gacggggaaa
gccggcgaac gtggcgagaa aggaagggaa 11820 gaaagcgaaa ggagcgggcg
ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac 11880
caccacaccc gccgcgctta atgcgccgct acagggcgcg tccattcgcc attcaggctg
11940 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
gctggcgaaa 12000 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
ggttttccca gtcacgacgt 12060 tgtaaaacga cggccagtga attgtaatac
gactcactat agggcgaatt gggcccgacg 12120 tcgcatgcag tggtggtatt
gtgactgggg atgtagttga gaataagtca tacacaagtc 12180 agctttcttc
gagcctcata taagtataag tagttcaacg tattagcact gtacccagca 12240
tctccgtatc gagaaacaca acaacatgcc ccattggaca gatcatgcgg atacacaggt
12300 tgtgcagtat catacatact cgatcagaca ggtcgtctga ccatcataca
agctgaacaa 12360 gcgctccata cttgcacgct ctctatatac acagttaaat
tacatatcca tagtctaacc 12420 tctaacagtt aatcttctgg taagcctccc
agccagcctt ctggtatcgc ttggcctcct 12480 caataggatc tcggttctgg
ccgtacagac ctcggccgac aattatgata tccgttccgg 12540 tagacatgac
atcctcaaca gttcggtact gctgtccgag agcgtctccc ttgtcgtcaa 12600
gacccacccc gggggtcaga ataagccagt cctcagagtc gcccttaat 12649
<210> SEQ ID NO 28 <211> LENGTH: 957 <212> TYPE:
DNA <213> ORGANISM: Mortierella alpina <220> FEATURE:
<221> NAME/KEY: misc_feature <223> OTHER INFORMATION:
synthetic elongase 1 (codon-optimized) <400> SEQUENCE: 28
atggagtcca ttgctccctt cctgccctcc aagatgcctc aggacctgtt catggacctc
60 gccagcgcta tcggtgtccg agctgctccc tacgtcgatc ccctggaggc
tgccctggtt 120 gcccaggccg agaagtacat tcccaccatt gtccatcaca
ctcgaggctt cctggttgcc 180 gtggagtctc ccctggctcg agagctgcct
ctgatgaacc ccttccacgt gctcctgatc 240 gtgctcgcct acctggtcac
cgtgtttgtg ggtatgcaga tcatgaagaa ctttgaacga 300 ttcgaggtca
agaccttctc cctcctgcac aacttctgtc tggtctccat ctccgcctac 360
atgtgcggtg gcatcctgta cgaggcttat caggccaact atggactgtt tgagaacgct
420 gccgatcaca ccttcaaggg tctccctatg gctaagatga tctggctctt
ctacttctcc 480 aagatcatgg agtttgtcga caccatgatc atggtcctca
agaagaacaa ccgacagatt 540 tcctttctgc acgtgtacca ccactcttcc
atcttcacca tctggtggct ggtcaccttc 600 gttgctccca acggtgaagc
ctacttctct gctgccctga actccttcat ccacgtcatc 660 atgtacggct
actactttct gtctgccctg ggcttcaagc aggtgtcgtt catcaagttc 720
tacatcactc gatcccagat gacccagttc tgcatgatgt ctgtccagtc ttcctgggac
780 atgtacgcca tgaaggtcct tggccgacct ggatacccct tcttcatcac
cgctctgctc 840 tggttctaca tgtggaccat gctcggtctc ttctacaact
tttaccgaaa gaacgccaag 900 ctcgccaagc aggccaaggc tgacgctgcc
aaggagaagg ccagaaagct ccagtaa 957 <210> SEQ ID NO 29
<211> LENGTH: 318 <212> TYPE: PRT <213> ORGANISM:
Mortierella alpina AX464731 <400> SEQUENCE: 29 Met Glu Ser
Ile Ala Pro Phe Leu Pro Ser Lys Met Pro Gln Asp Leu 1 5 10 15 Phe
Met Asp Leu Ala Thr Ala Ile Gly Val Arg Ala Ala Pro Tyr Val 20 25
30 Asp Pro Leu Glu Ala Ala Leu Val Ala Gln Ala Glu Lys Tyr Ile Pro
35 40 45 Thr Ile Val His His Thr Arg Gly Phe Leu Val Ala Val Glu
Ser Pro 50 55 60 Leu Ala Arg Glu Leu Pro Leu Met Asn Pro Phe His
Val Leu Leu Ile 65 70 75 80 Val Leu Ala Tyr Leu Val Thr Val Phe Val
Gly Met Gln Ile Met Lys 85 90 95 Asn Phe Glu Arg Phe Glu Val Lys
Thr Phe Ser Leu Leu His Asn Phe 100 105 110 Cys Leu Val Ser Ile Ser
Ala Tyr Met Cys Gly Gly Ile Leu Tyr Glu 115 120 125 Ala Tyr Gln Ala
Asn Tyr Gly Leu Phe Glu Asn Ala Ala Asp His Thr 130 135 140 Phe Lys
Gly Leu Pro Met Ala Lys Met Ile Trp Leu Phe Tyr Phe Ser 145 150 155
160 Lys Ile Met Glu Phe Val Asp Thr Met Ile Met Val Leu Lys Lys Asn
165 170 175 Asn Arg Gln Ile Ser Phe Leu His Val Tyr His His Ser Ser
Ile Phe 180 185 190 Thr Ile Trp Trp Leu Val Thr Phe Val Ala Pro Asn
Gly Glu Ala Tyr 195 200 205 Phe Ser Ala Ala Leu Asn Ser Phe Ile His
Val Ile Met Tyr Gly Tyr 210 215 220 Tyr Phe Leu Ser Ala Leu Gly Phe
Lys Gln Val Ser Phe Ile Lys Phe 225 230 235 240 Tyr Ile Thr Arg Ser
Gln Met Thr Gln Phe Cys Met Met Ser Val Gln 245 250 255 Ser Ser Trp
Asp Met Tyr Ala Met Lys Val Leu Gly Arg Pro Gly Tyr 260 265 270 Pro
Phe Phe Ile Thr Ala Leu Leu Trp Phe Tyr Met Trp Thr Met Leu 275 280
285 Gly Leu Phe Tyr Asn Phe Tyr Arg Lys Asn Ala Lys Leu Ala Lys Gln
290 295 300 Ala Lys Ala Asp Ala Ala Lys Glu Lys Ala Arg Lys Leu Gln
305 310 315 <210> SEQ ID NO 30 <211> LENGTH: 1374
<212> TYPE: DNA <213> ORGANISM: Mortierella alpina
<220> FEATURE: <221> NAME/KEY: misc_feature <223>
OTHER INFORMATION: synthetic delta-6 desaturase (codon-optimized)
<400> SEQUENCE: 30 atggctgccg ctccctctgt gcgaaccttt
acccgagccg aggttctgaa cgctgaggct 60 ctgaacgagg gcaagaagga
cgctgaggct cccttcctga tgatcatcga caacaaggtg 120 tacgacgtcc
gagagttcgt ccctgaccat cctggaggct ccgtgattct cacccacgtt 180
ggcaaggacg gcaccgacgt ctttgacacc tttcatcccg aggctgcttg ggagactctc
240 gccaacttct acgttggaga cattgacgag tccgaccgag acatcaagaa
cgatgacttt 300 gccgctgagg tccgaaagct gcgaaccctg ttccagtctc
tcggctacta cgactcctct 360 aaggcctact acgccttcaa ggtctccttc
aacctctgca tctggggact gtccaccgtc 420 attgtggcca agtggggtca
gacctccacc ctcgccaacg tgctctctgc tgccctgctc 480 ggcctgttct
ggcagcagtg cggatggctg gctcacgact ttctgcacca ccaggtcttc 540
caggaccgat tctggggtga tctcttcgga gccttcctgg gaggtgtctg ccagggcttc
600 tcctcttcct ggtggaagga caagcacaac actcaccatg ccgctcccaa
cgtgcatggc 660 gaggatcctg acattgacac ccaccctctc ctgacctggt
ccgagcacgc tctggagatg 720 ttctccgacg tccccgatga ggagctgacc
cgaatgtggt ctcgattcat ggtcctgaac 780 cagacctggt tctacttccc
cattctctcc ttcgctcgac tgtcttggtg cctccagtcc 840 attctctttg
tgctgcccaa cggtcaggct cacaagccct ccggagctcg agtgcccatc 900
tccctggtcg agcagctgtc cctcgccatg cactggacct ggtacctcgc taccatgttc
960 ctgttcatca aggatcctgt caacatgctc gtgtacttcc tggtgtctca
ggctgtgtgc 1020 ggaaacctgc tcgccatcgt gttctccctc aaccacaacg
gtatgcctgt gatctccaag 1080 gaggaggctg tcgacatgga tttctttacc
aagcagatca tcactggtcg agatgtccat 1140 cctggactgt tcgccaactg
gttcaccggt ggcctgaact accagatcga gcatcacctg 1200 ttcccttcca
tgcctcgaca caacttctcc aagatccagc ctgccgtcga gaccctgtgc 1260
aagaagtaca acgtccgata ccacaccact ggtatgatcg agggaactgc cgaggtcttc
1320 tcccgactga acgaggtctc caaggccacc tccaagatgg gcaaggctca gtaa
1374 <210> SEQ ID NO 31 <211> LENGTH: 457 <212>
TYPE: PRT <213> ORGANISM: Mortierella alpina AF465281
<400> SEQUENCE: 31 Met Ala Ala Ala Pro Ser Val Arg Thr Phe
Thr Arg Ala Glu Val Leu 1 5 10 15 Asn Ala Glu Ala Leu Asn Glu Gly
Lys Lys Asp Ala Glu Ala Pro Phe 20 25 30 Leu Met Ile Ile Asp Asn
Lys Val Tyr Asp Val Arg Glu Phe Val Pro 35 40 45 Asp His Pro Gly
Gly Ser Val Ile Leu Thr His Val Gly Lys Asp Gly 50 55 60 Thr Asp
Val Phe Asp Thr Phe His Pro Glu Ala Ala Trp Glu Thr Leu 65 70 75 80
Ala Asn Phe Tyr Val Gly Asp Ile Asp Glu Ser Asp Arg Asp Ile Lys 85
90 95 Asn Asp Asp Phe Ala Ala Glu Val Arg Lys Leu Arg Thr Leu Phe
Gln 100 105 110 Ser Leu Gly Tyr Tyr Asp Ser Ser Lys Ala Tyr Tyr Ala
Phe Lys Val 115 120 125 Ser Phe Asn Leu Cys Ile Trp Gly Leu Ser Thr
Val Ile Val Ala Lys 130 135 140 Trp Gly Gln Thr Ser Thr Leu Ala Asn
Val Leu Ser Ala Ala Leu Leu 145 150 155 160 Gly Leu Phe Trp Gln Gln
Cys Gly Trp Leu Ala His Asp Phe Leu His 165 170 175 His Gln Val Phe
Gln Asp Arg Phe Trp Gly Asp Leu Phe Gly Ala Phe 180 185 190 Leu Gly
Gly Val Cys Gln Gly Phe Ser Ser Ser Trp Trp Lys Asp Lys 195 200 205
His Asn Thr His His Ala Ala Pro Asn Val His Gly Glu Asp Pro Asp 210
215 220 Ile Asp Thr His Pro Leu Leu Thr Trp Ser Glu His Ala Leu Glu
Met 225 230 235 240 Phe Ser Asp Val Pro Asp Glu Glu Leu Thr Arg Met
Trp Ser Arg Phe 245 250 255
Met Val Leu Asn Gln Thr Trp Phe Tyr Phe Pro Ile Leu Ser Phe Ala 260
265 270 Arg Leu Ser Trp Cys Leu Gln Ser Ile Leu Phe Val Leu Pro Asn
Gly 275 280 285 Gln Ala His Lys Pro Ser Gly Ala Arg Val Pro Ile Ser
Leu Val Glu 290 295 300 Gln Leu Ser Leu Ala Met His Trp Thr Trp Tyr
Leu Ala Thr Met Phe 305 310 315 320 Leu Phe Ile Lys Asp Pro Val Asn
Met Leu Val Tyr Phe Leu Val Ser 325 330 335 Gln Ala Val Cys Gly Asn
Leu Leu Ala Ile Val Phe Ser Leu Asn His 340 345 350 Asn Gly Met Pro
Val Ile Ser Lys Glu Glu Ala Val Asp Met Asp Phe 355 360 365 Phe Thr
Lys Gln Ile Ile Thr Gly Arg Asp Val His Pro Gly Leu Phe 370 375 380
Ala Asn Trp Phe Thr Gly Gly Leu Asn Tyr Gln Ile Glu His His Leu 385
390 395 400 Phe Pro Ser Met Pro Arg His Asn Phe Ser Lys Ile Gln Pro
Ala Val 405 410 415 Glu Thr Leu Cys Lys Lys Tyr Asn Val Arg Tyr His
Thr Thr Gly Met 420 425 430 Ile Glu Gly Thr Ala Glu Val Phe Ser Arg
Leu Asn Glu Val Ser Lys 435 440 445 Ala Thr Ser Lys Met Gly Lys Ala
Gln 450 455 <210> SEQ ID NO 32 <211> LENGTH: 1001
<212> TYPE: DNA <213> ORGANISM: Yarrowia lipolytica
<220> FEATURE: <221> NAME/KEY: misc_feature <223>
OTHER INFORMATION: Promoter FBA <400> SEQUENCE: 32 taaacagtgt
acgcagtact atagaggaac aattgccccg gagaagacgg ccaggccgcc 60
tagatgacaa attcaacaac tcacagctga ctttctgcca ttgccactag ggggggcctt
120 tttatatggc caagccaagc tctccacgtc ggttgggctg cacccaacaa
taaatgggta 180 gggttgcacc aacaaaggga tgggatgggg ggtagaagat
acgaggataa cggggctcaa 240 tggcacaaat aagaacgaat actgccatta
agactcgtga tccagcgact gacaccattg 300 catcatctaa gggcctcaaa
actacctcgg aactgctgcg ctgatctgga caccacagag 360 gttccgagca
ctttaggttg caccaaatgt cccaccaggt gcaggcagaa aacgctggaa 420
cagcgtgtac agtttgtctt aacaaaaagt gagggcgctg aggtcgagca gggtggtgtg
480 acttgttata gcctttagag ctgcgaaagc gcgtatggat ttggctcatc
aggccagatt 540 gagggtctgt ggacacatgt catgttagtg tacttcaatc
gccccctgga tatagccccg 600 acaataggcc gtggcctcat ttttttgcct
tccgcacatt tccattgctc ggtacccaca 660 ccttgcttct cctgcacttg
ccaaccttaa tactggttta cattgaccaa catcttacaa 720 gcggggggct
tgtctagggt atatataaac agtggctctc ccaatcggtt gccagtctct 780
tttttccttt ctttccccac agattcgaaa tctaaactac acatcacaca atgcctgtta
840 ctgacgtcct taagcgaaag tccggtgtca tcgtcggcga cgatgtccga
gccgtgagta 900 tccacgacaa gatcagtgtc gagacgacgc gttttgtgta
atgacacaat ccgaaagtcg 960 ctagcaacac acactctcta cacaaactaa
cccagctctc c 1001 <210> SEQ ID NO 33 <211> LENGTH: 1434
<212> TYPE: DNA <213> ORGANISM: Fusarium monoliforme
<220> FEATURE: <221> NAME/KEY: misc_feature <223>
OTHER INFORMATION: delta-12 desaturase <400> SEQUENCE: 33
atggcgtcca cttcggctct gcccaagcag aaccctgcgc ttagacgcac cgtcacctca
60 actactgtga cggattctga gtctgccgcc gtctctcctt cagactctcc
ccgccactcg 120 gcctcttcca catcgctctc gtccatgtcc gaggttgata
tcgccaagcc caagtccgag 180 tatggtgtca tgctcgacac ctacggcaac
cagttcgagg ttcccgactt taccatcaag 240 gacatctaca atgccatccc
taagcactgc ttcaagcgct ccgctctcaa gggatacggt 300 tatatcctcc
gcgacattgt cctcctgact accactttca gcatctggta caactttgtg 360
acccccgaat atatcccctc cacccccgcc cgcgctggtc tgtgggccgt gtacaccgtt
420 cttcagggtc ttttcggtac tggtctctgg gttattgccc atgagtgcgg
tcacggtgct 480 ttctccgatt ctcgcatcat caacgacatt actggctggg
ttcttcactc ttccctcctt 540 gtcccctact tcagctggca aatctcccac
cgaaagcacc acaaggccac tggcaacatg 600 gagcgtgaca tggtcttcgt
tccccgaacc cgcgagcagc aggctactcg tctcggaaag 660 atgacccacg
agctcgctca tcttactgag gagacccccg ctttcactct tctcatgctc 720
gtccttcagc agctcgttgg ctggcccaac tacctcatca ccaatgttac cggccacaac
780 taccacgagc gccagcgtga gggtcgcggc aagggcaagc ataacggcct
cggcggtggt 840 gttaaccact tcgatccccg cagccctctg tacgagaaca
gtgacgctaa gctcatcgtc 900 ctcagcgata ttggtatcgg tctgatggcc
actgctctgt acttcctcgt tcagaagttc 960 ggtttctaca acatggccat
ctggtacttt gttccctacc tctgggttaa ccactggctc 1020 gttgccatca
ccttcctcca gcacaccgac cctacccttc cccactacac caacgacgag 1080
tggaacttcg tccgtggtgc cgctgctacc attgaccgtg agatgggctt catcggccgc
1140 caccttctcc acggcatcat cgagactcat gtcctccacc actacgtcag
cagcatcccc 1200 ttctacaacg cggacgaggc caccgaggcc attaagccca
tcatgggcaa gcactaccgg 1260 gctgatgtcc aggatggtcc tcgtggcttc
atccgcgcca tgtaccgcag tgcgcgtatg 1320 tgccagtggg ttgagcccag
cgctggtgcc gagggtgctg gtaagggtgt tctgttcttc 1380 cgcaaccgca
acaacgtggg cacccccccc gctgttatca agcccgttgc ttaa 1434 <210>
SEQ ID NO 34 <211> LENGTH: 477 <212> TYPE: PRT
<213> ORGANISM: Fusarium monoliforme <400> SEQUENCE: 34
Met Ala Ser Thr Ser Ala Leu Pro Lys Gln Asn Pro Ala Leu Arg Arg 1 5
10 15 Thr Val Thr Ser Thr Thr Val Thr Asp Ser Glu Ser Ala Ala Val
Ser 20 25 30 Pro Ser Asp Ser Pro Arg His Ser Ala Ser Ser Thr Ser
Leu Ser Ser 35 40 45 Met Ser Glu Val Asp Ile Ala Lys Pro Lys Ser
Glu Tyr Gly Val Met 50 55 60 Leu Asp Thr Tyr Gly Asn Gln Phe Glu
Val Pro Asp Phe Thr Ile Lys 65 70 75 80 Asp Ile Tyr Asn Ala Ile Pro
Lys His Cys Phe Lys Arg Ser Ala Leu 85 90 95 Lys Gly Tyr Gly Tyr
Ile Leu Arg Asp Ile Val Leu Leu Thr Thr Thr 100 105 110 Phe Ser Ile
Trp Tyr Asn Phe Val Thr Pro Glu Tyr Ile Pro Ser Thr 115 120 125 Pro
Ala Arg Ala Gly Leu Trp Ala Val Tyr Thr Val Leu Gln Gly Leu 130 135
140 Phe Gly Thr Gly Leu Trp Val Ile Ala His Glu Cys Gly His Gly Ala
145 150 155 160 Phe Ser Asp Ser Arg Ile Ile Asn Asp Ile Thr Gly Trp
Val Leu His 165 170 175 Ser Ser Leu Leu Val Pro Tyr Phe Ser Trp Gln
Ile Ser His Arg Lys 180 185 190 His His Lys Ala Thr Gly Asn Met Glu
Arg Asp Met Val Phe Val Pro 195 200 205 Arg Thr Arg Glu Gln Gln Ala
Thr Arg Leu Gly Lys Met Thr His Glu 210 215 220 Leu Ala His Leu Thr
Glu Glu Thr Pro Ala Phe Thr Leu Leu Met Leu 225 230 235 240 Val Leu
Gln Gln Leu Val Gly Trp Pro Asn Tyr Leu Ile Thr Asn Val 245 250 255
Thr Gly His Asn Tyr His Glu Arg Gln Arg Glu Gly Arg Gly Lys Gly 260
265 270 Lys His Asn Gly Leu Gly Gly Gly Val Asn His Phe Asp Pro Arg
Ser 275 280 285 Pro Leu Tyr Glu Asn Ser Asp Ala Lys Leu Ile Val Leu
Ser Asp Ile 290 295 300 Gly Ile Gly Leu Met Ala Thr Ala Leu Tyr Phe
Leu Val Gln Lys Phe 305 310 315 320 Gly Phe Tyr Asn Met Ala Ile Trp
Tyr Phe Val Pro Tyr Leu Trp Val 325 330 335 Asn His Trp Leu Val Ala
Ile Thr Phe Leu Gln His Thr Asp Pro Thr 340 345 350 Leu Pro His Tyr
Thr Asn Asp Glu Trp Asn Phe Val Arg Gly Ala Ala 355 360 365 Ala Thr
Ile Asp Arg Glu Met Gly Phe Ile Gly Arg His Leu Leu His 370 375 380
Gly Ile Ile Glu Thr His Val Leu His His Tyr Val Ser Ser Ile Pro 385
390 395 400 Phe Tyr Asn Ala Asp Glu Ala Thr Glu Ala Ile Lys Pro Ile
Met Gly 405 410 415 Lys His Tyr Arg Ala Asp Val Gln Asp Gly Pro Arg
Gly Phe Ile Arg 420 425 430 Ala Met Tyr Arg Ser Ala Arg Met Cys Gln
Trp Val Glu Pro Ser Ala 435 440 445 Gly Ala Glu Gly Ala Gly Lys Gly
Val Leu Phe Phe Arg Asn Arg Asn 450 455 460 Asn Val Gly Thr Pro Pro
Ala Val Ile Lys Pro Val Ala 465 470 475 <210> SEQ ID NO 35
<211> LENGTH: 819 <212> TYPE: DNA <213> ORGANISM:
Thraustochytrium aureum <220> FEATURE: <221> NAME/KEY:
misc_feature <223> OTHER INFORMATION: synthetic elongase
(codon-optimized) <400> SEQUENCE: 35
atggccaact cctctgtctg ggacgacgtg gtcggacgag tcgagaccgg tgtcgaccag
60 tggatggacg gagctaagcc ctacgctctg accgacggtc tgcccatgat
ggacgtctcc 120 accatgctcg ccttcgaggt cggctacatg gccatgctgc
tcttcggcat tcccatcatg 180 aagcagatgg agaagccctt cgagctgaag
accatcaagc tgctccacaa cctgttcctc 240 ttcggactgt ccctctacat
gtgcgtcgag accatccgac aggctatcct gggtggctac 300 aaggtcttcg
gcaacgacat ggagaagggc aacgagtccc acgctcaggg catgtcccga 360
atcgtctacg tgttctacgt ctccaaggcc tacgagttcc tggacaccgc tatcatgatc
420 ctgtgcaaga agttcaacca ggtctccttc ctgcacgtgt accaccatgc
caccatcttc 480 gccatctggt gggctattgc caagtacgct cctggtggcg
acgcctactt ctccgtcatc 540 ctcaactcct tcgtccacac cgtcatgtac
gcctactact tcttttcctc tcagggcttc 600 ggcttcgtca agcccatcaa
gccctacatc accactctgc agatgaccca gttcatggct 660 atgctggtgc
agtccctgta cgactacctc ttcccctgcg actaccctca ggctctggtc 720
cagctgctcg gcgtgtacat gatcaccctg ctcgctctgt tcggcaactt ctttgtccag
780 tcctacctga agaagcccaa gaagtccaag accaactaa 819 <210> SEQ
ID NO 36 <211> LENGTH: 272 <212> TYPE: PRT <213>
ORGANISM: Thraustochytrium aureum <400> SEQUENCE: 36 Met Ala
Asn Ser Ser Val Trp Asp Asp Val Val Gly Arg Val Glu Thr 1 5 10 15
Gly Val Asp Gln Trp Met Asp Gly Ala Lys Pro Tyr Ala Leu Thr Asp 20
25 30 Gly Leu Pro Met Met Asp Val Ser Thr Met Leu Ala Phe Glu Val
Gly 35 40 45 Tyr Met Ala Met Leu Leu Phe Gly Ile Pro Ile Met Lys
Gln Met Glu 50 55 60 Lys Pro Phe Glu Leu Lys Thr Ile Lys Leu Leu
His Asn Leu Phe Leu 65 70 75 80 Phe Gly Leu Ser Leu Tyr Met Cys Val
Glu Thr Ile Arg Gln Ala Ile 85 90 95 Leu Gly Gly Tyr Lys Val Phe
Gly Asn Asp Met Glu Lys Gly Asn Glu 100 105 110 Ser His Ala Gln Gly
Met Ser Arg Ile Val Tyr Val Phe Tyr Val Ser 115 120 125 Lys Ala Tyr
Glu Phe Leu Asp Thr Ala Ile Met Ile Leu Cys Lys Lys 130 135 140 Phe
Asn Gln Val Ser Phe Leu His Val Tyr His His Ala Thr Ile Phe 145 150
155 160 Ala Ile Trp Trp Ala Ile Ala Lys Tyr Ala Pro Gly Gly Asp Ala
Tyr 165 170 175 Phe Ser Val Ile Leu Asn Ser Phe Val His Thr Val Met
Tyr Ala Tyr 180 185 190 Tyr Phe Phe Ser Ser Gln Gly Phe Gly Phe Val
Lys Pro Ile Lys Pro 195 200 205 Tyr Ile Thr Thr Leu Gln Met Thr Gln
Phe Met Ala Met Leu Val Gln 210 215 220 Ser Leu Tyr Asp Tyr Leu Phe
Pro Cys Asp Tyr Pro Gln Ala Leu Val 225 230 235 240 Gln Leu Leu Gly
Val Tyr Met Ile Thr Leu Leu Ala Leu Phe Gly Asn 245 250 255 Phe Phe
Val Gln Ser Tyr Leu Lys Lys Pro Lys Lys Ser Lys Thr Asn 260 265 270
<210> SEQ ID NO 37 <211> LENGTH: 10945 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Plasmid pDMW232 <400>
SEQUENCE: 37 aattcctgca gcccatcgat caggagagac cgggttggcg gcgtatttgt
gtcccaaaaa 60 acagccccaa ttgccccaat tgaccccaaa ttgacccagt
agcgggccca accccggcga 120 gagccccctt caccccacat atcaaacctc
ccccggttcc cacacttgcc gttaagggcg 180 tagggtactg cagtctggaa
tctacgcttg ttcagacttt gtactagttt ctttgtctgg 240 ccatccgggt
aacccatgcc ggacgcaaaa tagactactg aaaatttttt tgctttgtgg 300
ttgggacttt agccaagggt ataaaagacc accgtccccg aattaccttt cctcttcttt
360 tctctctctc cttgtcaact cacacccgaa atcgttaagc atttccttct
gagtataaga 420 atcattcacc atgggaacgg accaaggaaa aaccttcacc
tgggaagagc tggcggccca 480 taacaccaag gacgacctac tcttggccat
ccgcggcagg gtgtacgatg tcacaaagtt 540 cttgagccgc catcctggtg
gagtggacac tctcctgctc ggagctggcc gagatgttac 600 tccggtcttt
gagatgtatc acgcgtttgg ggctgcagat gccattatga agaagtacta 660
tgtcggtaca ctggtctcga atgagctgcc catcttcccg gagccaacgg tgttccacaa
720 aaccatcaag acgagagtcg agggctactt tacggatcgg aacattgatc
ccaagaatag 780 accagagatc tggggacgat acgctcttat ctttggatcc
ttgatcgctt cctactacgc 840 gcagctcttt gtgcctttcg ttgtcgaacg
cacatggctt caggtggtgt ttgcaatcat 900 catgggattt gcgtgcgcac
aagtcggact caaccctctt catgatgcgt ctcacttttc 960 agtgacccac
aaccccactg tctggaagat tctgggagcc acgcacgact ttttcaacgg 1020
agcatcgtac ctggtgtgga tgtaccaaca tatgctcggc catcacccct acaccaacat
1080 tgctggagca gatcccgacg tgtcgacgtc tgagcccgat gttcgtcgta
tcaagcccaa 1140 ccaaaagtgg tttgtcaacc acatcaacca gcacatgttt
gttcctttcc tgtacggact 1200 gctggcgttc aaggtgcgca ttcaggacat
caacattttg tactttgtca agaccaatga 1260 cgctattcgt gtcaatccca
tctcgacatg gcacactgtg atgttctggg gcggcaaggc 1320 tttctttgtc
tggtatcgcc tgattgttcc cctgcagtat ctgcccctgg gcaaggtgct 1380
gctcttgttc acggtcgcgg acatggtgtc gtcttactgg ctggcgctga ccttccaggc
1440 gaaccacgtt gttgaggaag ttcagtggcc gttgcctgac gagaacggga
tcatccaaaa 1500 ggactgggca gctatgcagg tcgagactac gcaggattac
gcacacgatt cgcacctctg 1560 gaccagcatc actggcagct tgaactacca
ggctgtgcac catctgttcc ccaacgtgtc 1620 gcagcaccat tatcccgata
ttctggccat catcaagaac acctgcagcg agtacaaggt 1680 tccatacctt
gtcaaggata cgttttggca agcatttgct tcacatttgg agcacttgcg 1740
tgttcttgga ctccgtccca aggaagagta ggcagctaag cggccgcatg agaagataaa
1800 tatataaata cattgagata ttaaatgcgc tagattagag agcctcatac
tgctcggaga 1860 gaagccaaga cgagtactca aaggggatta caccatccat
atccacagac acaagctggg 1920 gaaaggttct atatacactt tccggaatac
cgtagtttcc gatgttatca atgggggcag 1980 ccaggatttc aggcacttcg
gtgtctcggg gtgaaatggc gttcttggcc tccatcaagt 2040 cgtaccatgt
cttcatttgc ctgtcaaagt aaaacagaag cagatgaaga atgaacttga 2100
agtgaaggaa tttaaattgc cccggagaag acggccaggc cgcctagatg acaaattcaa
2160 caactcacag ctgactttct gccattgcca ctaggggggg gcctttttat
atggccaagc 2220 caagctctcc acgtcggttg ggctgcaccc aacaataaat
gggtagggtt gcaccaacaa 2280 agggatggga tggggggtag aagatacgag
gataacgggg ctcaatggca caaataagaa 2340 cgaatactgc cattaagact
cgtgatccag cgactgacac cattgcatca tctaagggcc 2400 tcaaaactac
ctcggaactg ctgcgctgat ctggacacca cagaggttcc gagcacttta 2460
ggttgcacca aatgtcccac caggtgcagg cagaaaacgc tggaacagcg tgtacagttt
2520 gtcttaacaa aaagtgaggg cgctgaggtc gagcagggtg gtgtgacttg
ttatagcctt 2580 tagagctgcg aaagcgcgta tggatttggc tcatcaggcc
agattgaggg tctgtggaca 2640 catgtcatgt tagtgtactt caatcgcccc
ctggatatag ccccgacaat aggccgtggc 2700 ctcatttttt tgccttccgc
acatttccat tgctcggtac ccacaccttg cttctcctgc 2760 acttgccaac
cttaatactg gtttacattg accaacatct tacaagcggg gggcttgtct 2820
agggtatata taaacagtgg ctctcccaat cggttgccag tctctttttt cctttctttc
2880 cccacagatt cgaaatctaa actacacatc acacaatgcc tgttactgac
gtccttaagc 2940 gaaagtccgg tgtcatcgtc ggcgacgatg tccgagccgt
gagtatccac gacaagatca 3000 gtgtcgagac gacgcgtttt gtgtaatgac
acaatccgaa agtcgctagc aacacacact 3060 ctctacacaa actaacccag
ctctccatgg gaacggacca aggaaaaacc ttcacctggg 3120 aagagctggc
ggcccataac accaaggacg acctactctt ggccatccgc ggcagggtgt 3180
acgatgtcac aaagttcttg agccgccatc ctggtggagt ggacactctc ctgctcggag
3240 ctggccgaga tgttactccg gtctttgaga tgtatcacgc gtttggggct
gcagatgcca 3300 ttatgaagaa gtactatgtc ggtacactgg tctcgaatga
gctgcccatc ttcccggagc 3360 caacggtgtt ccacaaaacc atcaagacga
gagtcgaggg ctactttacg gatcggaaca 3420 ttgatcccaa gaatagacca
gagatctggg gacgatacgc tcttatcttt ggatccttga 3480 tcgcttccta
ctacgcgcag ctctttgtgc ctttcgttgt cgaacgcaca tggcttcagg 3540
tggtgtttgc aatcatcatg ggatttgcgt gcgcacaagt cggactcaac cctcttcatg
3600 atgcgtctca cttttcagtg acccacaacc ccactgtctg gaagattctg
ggagccacgc 3660 acgacttttt caacggagca tcgtacctgg tgtggatgta
ccaacatatg ctcggccatc 3720 acccctacac caacattgct ggagcagatc
ccgacgtgtc gacgtctgag cccgatgttc 3780 gtcgtatcaa gcccaaccaa
aagtggtttg tcaaccacat caaccagcac atgtttgttc 3840 ctttcctgta
cggactgctg gcgttcaagg tgcgcattca ggacatcaac attttgtact 3900
ttgtcaagac caatgacgct attcgtgtca atcccatctc gacatggcac actgtgatgt
3960 tctggggcgg caaggctttc tttgtctggt atcgcctgat tgttcccctg
cagtatctgc 4020 ccctgggcaa ggtgctgctc ttgttcacgg tcgcggacat
ggtgtcgtct tactggctgg 4080 cgctgacctt ccaggcgaac cacgttgttg
aggaagttca gtggccgttg cctgacgaga 4140 acgggatcat ccaaaaggac
tgggcagcta tgcaggtcga gactacgcag gattacgcac 4200 acgattcgca
cctctggacc agcatcactg gcagcttgaa ctaccaggct gtgcaccatc 4260
tgttccccaa cgtgtcgcag caccattatc ccgatattct ggccatcatc aagaacacct
4320 gcagcgagta caaggttcca taccttgtca aggatacgtt ttggcaagca
tttgcttcac 4380 atttggagca cttgcgtgtt cttggactcc gtcccaagga
agagtaggca gctaagcggc 4440 cgcaagtgtg gatggggaag tgagtgcccg
gttctgtgtg cacaattggc aatccaagat 4500 ggatggattc aacacaggga
tatagcgagc tacgtggtgg tgcgaggata tagcaacgga 4560 tatttatgtt
tgacacttga gaatgtacga tacaagcact gtccaagtac aatactaaac 4620
atactgtaca tactcatact cgtacccggg caacggtttc acttgagtgc agtggctagt
4680 gctcttactc gtacagtgtg caatactgcg tatcatagtc tttgatgtat
atcgtattca 4740 ttcatgttag ttgcgtacgc caccattctg tctgccgcca
tgatgctcaa gttctctctt 4800 aacatgaagc ccgccggtga cgctgttgag
gctgccgtca aggagtccgt cgaggctggt 4860 atcactaccg ccgatatcgg
aggctcttcc tccacctccg aggtcggaga cttgttgcca 4920 acaaggtcaa
ggagctgctc aagaaggagt aagtcgtttc tacgacgcat tgatggaagg 4980
agcaaactga cgcgcctgcg ggttggtcta ccggcagggt ccgctagtgt ataagactct
5040 ataaaaaggg ccctgccctg ctaatgaaat gatgatttat aatttaccgg
tgtagcaacc 5100 ttgactagaa gaagcagatt gggtgtgttt gtagtggagg
acagtggtac gttttggaaa 5160 cagtcttctt gaaagtgtct tgtctacagt
atattcactc ataacctcaa tagccaaggg 5220 tgtagtcggt ttattaaagg
aagggagttg tggctgatgt ggatagatat ctttaagctg 5280 gcgactgcac
ccaacgagtg tggtggtagc ttgttactgt atattcggta agatatattt 5340
tgtggggttt tagtggtgtt tggtaggtta gtgcttggta tatgagttgt aggcatgaca
5400 atttggaaag gggtggactt tgggaatatt gtgggatttc aataccttag
tttgtacagg 5460 gtaattgtta caaatgatac aaagaactgt atttcttttc
atttgtttta attggttgta 5520 tatcaagtcc gttagacgag ctcagtgggc
gcgccagctg cattaatgaa tcggccaacg 5580 cgcggggaga ggcggtttgc
gtattgggcg ctcttccgct tcctcgctca ctgactcgct 5640 gcgctcggtc
gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 5700
atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
5760 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
cccctgacga 5820 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
ccgacaggac tataaagata 5880 ccaggcgttt ccccctggaa gctccctcgt
gcgctctcct gttccgaccc tgccgcttac 5940 cggatacctg tccgcctttc
tcccttcggg aagcgtggcg ctttctcata gctcacgctg 6000 taggtatctc
agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 6060
cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
6120 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
cgaggtatgt 6180 aggcggtgct acagagttct tgaagtggtg gcctaactac
ggctacacta gaagaacagt 6240 atttggtatc tgcgctctgc tgaagccagt
taccttcgga aaaagagttg gtagctcttg 6300 atccggcaaa caaaccaccg
ctggtagcgg tggttttttt gtttgcaagc agcagattac 6360 gcgcagaaaa
aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 6420
gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
6480 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
atgagtaaac 6540 ttggtctgac agttaccaat gcttaatcag tgaggcacct
atctcagcga tctgtctatt 6600 tcgttcatcc atagttgcct gactccccgt
cgtgtagata actacgatac gggagggctt 6660 accatctggc cccagtgctg
caatgatacc gcgagaccca cgctcaccgg ctccagattt 6720 atcagcaata
aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc 6780
cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
6840 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
cgtcgtttgg 6900 tatggcttca ttcagctccg gttcccaacg atcaaggcga
gttacatgat cccccatgtt 6960 gtgcaaaaaa gcggttagct ccttcggtcc
tccgatcgtt gtcagaagta agttggccgc 7020 agtgttatca ctcatggtta
tggcagcact gcataattct cttactgtca tgccatccgt 7080 aagatgcttt
tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 7140
gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
7200 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
ggatcttacc 7260 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
aactgatctt cagcatcttt 7320 tactttcacc agcgtttctg ggtgagcaaa
aacaggaagg caaaatgccg caaaaaaggg 7380 aataagggcg acacggaaat
gttgaatact catactcttc ctttttcaat attattgaag 7440 catttatcag
ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 7500
acaaataggg gttccgcgca catttccccg aaaagtgcca cctgatgcgg tgtgaaatac
7560 cgcacagatg cgtaaggaga aaataccgca tcaggaaatt gtaagcgtta
atattttgtt 7620 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
aaccaatagg ccgaaatcgg 7680 caaaatccct tataaatcaa aagaatagac
cgagataggg ttgagtgttg ttccagtttg 7740 gaacaagagt ccactattaa
agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta 7800 tcagggcgat
ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg 7860
ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
7920 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
ctagggcgct 7980 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
gccgcgctta atgcgccgct 8040 acagggcgcg tccattcgcc attcaggctg
cgcaactgtt gggaagggcg atcggtgcgg 8100 gcctcttcgc tattacgcca
gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg 8160 gtaacgccag
ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac 8220
gactcactat agggcgaatt gggcccgacg tcgcatgcta tcggcatcga caaggtttgg
8280 gtccctagcc gataccgcac tacctgagtc acaatcttcg gaggtttagt
cttccacata 8340 gcacgggcaa aagtgcgtat atatacaaga gcgtttgcca
gccacagatt ttcactccac 8400 acaccacatc acacatacaa ccacacacat
ccacaatgga acccgaaact aagaagacca 8460 agactgactc caagaagatt
gttcttctcg gcggcgactt ctgtggcccc gaggtgattg 8520 ccgaggccgt
caaggtgctc aagtctgttg ctgaggcctc cggcaccgag tttgtgtttg 8580
aggaccgact cattggagga gctgccattg agaaggaggg cgagcccatc accgacgcta
8640 ctctcgacat ctgccgaaag gctgactcta ttatgctcgg tgctgtcgga
ggcgctgcca 8700 acaccgtatg gaccactccc gacggacgaa ccgacgtgcg
acccgagcag ggtctcctca 8760 agctgcgaaa ggacctgaac ctgtacgcca
acctgcgacc ctgccagctg ctgtcgccca 8820 agctcgccga tctctccccc
atccgaaacg ttgagggcac cgacttcatc attgtccgag 8880 agctcgtcgg
aggtatctac tttggagagc gaaaggagga tgacggatct ggcgtcgctt 8940
ccgacaccga gacctactcc gttaattaat ttgaatcgaa tcgatgagcc taaaatgaac
9000 ccgagtatat ctcataaaat tctcggtgag aggtctgtga ctgtcagtac
aaggtgcctt 9060 cattatgccc tcaaccttac catacctcac tgaatgtagt
gtacctctaa aaatgaaata 9120 cagtgccaaa agccaaggca ctgagctcgt
ctaacggact tgatatacaa ccaattaaaa 9180 caaatgaaaa gaaatacagt
tctttgtatc atttgtaaca attaccctgt acaaactaag 9240 gtattgaaat
cccacaatat tcccaaagtc cacccctttc caaattgtca tgcctacaac 9300
tcatatacca agcactaacc taccgtttaa acagtgtacg cagatctggt gtagtggtag
9360 tgcagtggtg gtattgtgac tggggatgta gttgagaata agtcatacac
aagtcagctt 9420 tcttcgagcc tcatataagt ataagtagtt caacgtatta
gcactgtacc cagcatctcc 9480 gtatcgagaa acacaacaac atgccccatt
ggacagatca tgcggataca caggttgtgc 9540 agtatcatac atactcgatc
agacaggtcg tctgaccatc atacaagctg aacaagcgct 9600 ccatacttgc
acgctctcta tatacacagt taaattacat atccatagtc taacctctaa 9660
cagttaatct tctggtaagc ctcccagcca gccttctggt atcgcttggc ctcctcaata
9720 ggatctcggt tctggccgta cagacctcgg ccgacaatta tgatatccgt
tccggtagac 9780 atgacatcct caacagttcg gtactgctgt ccgagagcgt
ctcccttgtc gtcaagaccc 9840 accccggggg tcagaataag ccagtcctca
gagtcgccct taggtcggtt ctgggcaatg 9900 aagccaacca caaactcggg
gtcggatcgg gcaagctcaa tggtctgctt ggagtactcg 9960 ccagtggcca
gagagccctt gcaagacagc tcggccagca tgagcagacc tctggccagc 10020
ttctcgttgg gagaggggac taggaactcc ttgtactggg agttctcgta gtcagagacg
10080 tcctccttct tctgttcaga gacagtttcc tcggcaccag ctcgcaggcc
agcaatgatt 10140 ccggttccgg gtacaccgtg ggcgttggtg atatcggacc
actcggcgat tcggtgacac 10200 cggtactggt gcttgacagt gttgccaata
tctgcgaact ttctgtcctc gaacaggaag 10260 aaaccgtgct taagagcaag
ttccttgagg gggagcacag tgccggcgta ggtgaagtcg 10320 tcaatgatgt
cgatatgggt tttgatcatg cacacataag gtccgacctt atcggcaagc 10380
tcaatgagct ccttggtggt ggtaacatcc agagaagcac acaggttggt tttcttggct
10440 gccacgagct tgagcactcg agcggcaaag gcggacttgt ggacgttagc
tcgagcttcg 10500 taggagggca ttttggtggt gaagaggaga ctgaaataaa
tttagtctgc agaacttttt 10560 atcggaacct tatctggggc agtgaagtat
atgttatggt aatagttacg agttagttga 10620 acttatagat agactggact
atacggctat cggtccaaat tagaaagaac gtcaatggct 10680 ctctgggcgt
cgcctttgcc gacaaaaatg tgatcatgat gaaagccagc aatgacgttg 10740
cagctgatat tgttgtcggc caaccgcgcc gaaaacgcag ctgtcagacc cacagcctcc
10800 aacgaagaat gtatcgtcaa agtgatccaa gcacactcat agttggagtc
gtactccaaa 10860 ggcggcaatg acgagtcaga cagatactcg tcgacctttt
ccttgggaac caccaccgtc 10920 agcccttctg actcacgtat tgtag 10945
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References