U.S. patent application number 12/469494 was filed with the patent office on 2009-09-17 for production of polyunsaturated fatty acids in oleaginous yeasts.
This patent application is currently assigned to E. I. DU PONT DE NEMOURS AND COMPANY. Invention is credited to Stephen K. Picataggio, Narendra S. Yadav, Quinn Qun Zhu.
Application Number | 20090233347 12/469494 |
Document ID | / |
Family ID | 33452224 |
Filed Date | 2009-09-17 |
United States Patent
Application |
20090233347 |
Kind Code |
A1 |
Picataggio; Stephen K. ; et
al. |
September 17, 2009 |
PRODUCTION OF POLYUNSATURATED FATTY ACIDS IN OLEAGINOUS YEASTS
Abstract
The present invention relates to methods for the production of
.omega.-3 and/or .omega.-6 fatty acids in oleaginous yeast. Thus,
desaturases and elongases able to catalyze the conversion of
linoleic acid (LA) to .gamma.-linolenic acid (GLA);
.alpha.-linoleic acid (ALA) to stearidonic acid (STA); GLA to
dihomo-.gamma.-linoleic acid (DGLA); STA to eicosatetraenoic acid
(ETA); DGLA to arachidonic acid (ARA); ETA to eicosapentaenoic acid
(EPA); DGLA to ETA; EPA to docosapentaenoic acid (DPA); and ARA to
EPA have been introduced into the genome of Yarrowia for synthesis
of ARA and EPA.
Inventors: |
Picataggio; Stephen K.;
(Gaithersburg, MD) ; Yadav; Narendra S.;
(Wilmington, DE) ; Zhu; Quinn Qun; (West Chester,
PA) |
Correspondence
Address: |
E I DU PONT DE NEMOURS AND COMPANY;LEGAL PATENT RECORDS CENTER
BARLEY MILL PLAZA 25/1122B, 4417 LANCASTER PIKE
WILMINGTON
DE
19805
US
|
Assignee: |
E. I. DU PONT DE NEMOURS AND
COMPANY
Wilmington
DE
|
Family ID: |
33452224 |
Appl. No.: |
12/469494 |
Filed: |
May 20, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11714377 |
Mar 5, 2007 |
7553628 |
|
|
12469494 |
|
|
|
|
10840579 |
May 6, 2004 |
7238482 |
|
|
11714377 |
|
|
|
|
60468677 |
May 7, 2003 |
|
|
|
Current U.S.
Class: |
435/254.2 ;
554/224 |
Current CPC
Class: |
C12P 7/6472 20130101;
C12N 15/815 20130101; C12P 7/6427 20130101 |
Class at
Publication: |
435/254.2 ;
554/224 |
International
Class: |
C12N 1/19 20060101
C12N001/19; C07C 57/00 20060101 C07C057/00 |
Claims
1. A transformed oleaginous yeast comprising genes encoding enzymes
of the .omega.-6 fatty acid biosynthetic pathway.
2. A microbial oil comprising at least one .omega.-6 fatty acid
obtained from the transformed oleaginous yeast of claim 1.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/468,677, filed May 7, 2003.
FIELD OF THE INVENTION
[0002] This invention is in the field of biotechnology. More
specifically, this invention pertains to the production of long
chain polyunsaturated fatty acids (PUFAs) in oleaginous yeasts.
BACKGROUND OF THE INVENTION
[0003] It has long been recognized that certain polyunsaturated
fatty acids, or PUFAs, are important biological components of
healthy cells. For example, such PUFAs are recognized as: [0004]
"Essential" fatty acids that can not be synthesized de novo in
mammals and instead must be obtained either in the diet or derived
by further desaturation and elongation of linoleic acid (LA) or
.alpha.-linolenic acid (ALA); [0005] Constituents of plasma
membranes of cells, where they may be found in such forms as
phospholipids or triglycerides; [0006] Necessary for proper
development, particularly in the developing infant brain, and for
tissue formation and repair; and, [0007] Precursors to several
biologically active eicosanoids of importance in mammals, including
prostacyclins, eicosanoids, leukotrienes and prostaglandins.
[0008] In the 1970's, observations of Greenland Eskimos linked a
low incidence of heart disease and a high intake of long-chain
.omega.-3 PUFAs (Dyerberg, J. et al., Amer. J. Clin Nutr.
28:958-966 (1975); Dyerberg, J. et al., Lancet 2(8081):117-119
(Jul. 15, 1978)). More recent studies have confirmed the
cardiovascular protective effects of .omega.-3 PUFAs (Shimokawa,
H., World Rev Nutr Diet, 88:100-108 (2001); von Schacky, C., and
Dyerberg, J., World Rev Nutr Diet, 88:90-99 (2001)). Further, it
has been discovered that several disorders respond to treatment
with .omega.-3 fatty acids, such as the rate of restenosis after
angioplasty, symptoms of inflammation and rheumatoid arthritis,
asthma, psoriasis and eczema. .gamma.-linolenic acid (GLA, an
.omega.-6 PUFA) has been shown to reduce increases in blood
pressure associated with stress and to improve performance on
arithmetic tests. GLA and dihomo-.gamma.-linolenic acid (DGLA,
another .omega.-6 PUFA) have been shown to inhibit platelet
aggregation, cause vasodilation, lower cholesterol levels and
inhibit proliferation of vessel wall smooth muscle and fibrous
tissue (Brenner et al., Adv. Exp. Med. Biol. 83:85-101 (1976)).
Administration of GLA or DGLA, alone or in combination with
eicosapentaenoic acid (EPA, an .omega.-3 PUFA), has been shown to
reduce or prevent gastrointestinal bleeding and other side effects
caused by non-steroidal anti-inflammatory drugs (U.S. Pat. No.
4,666,701). Further, GLA and DGLA have been shown to prevent or
treat endometriosis and premenstrual syndrome (U.S. Pat. No.
4,758,592) and to treat myalgic encephalomyelitis and chronic
fatigue after viral infections (U.S. Pat. No. 5,116,871). Other
evidence indicates that PUFAs may be involved in the regulation of
calcium metabolism, suggesting that they may be useful in the
treatment or prevention of osteoporosis and kidney or urinary tract
stones. Finally, PUFAs can be used in the treatment of cancer and
diabetes (U.S. Pat. No. 4,826,877; Horrobin et al., Am. J. Clin.
Nutr. 57 (Suppl.): 732S-737S (1993)).
[0009] PUFAs are generally divided into two major classes
(consisting of the .omega.-6 and the .omega.-3 fatty acids) that
are derived by desaturation and elongation of the essential fatty
acids, linoleic acid (LA) and .alpha.-linolenic acid (ALA),
respectively. Despite this common derivation from "essential" fatty
acids, it is becoming increasingly apparent that the ratio of
.omega.-6 to .omega.-3 fatty acids in the diet is important for
maintenance of good health. Due to changes in human dietary habits,
the current ratio of .omega.-6 to .omega.-3 fatty acids is
approximately 10:1, whereas the preferred ratio is 2:1
(Kris-Etherton, P. M. et al., Am. J. Clin. Nutr. 71 (1
Suppl.):179S-88S (2000); Simopoulos, A. P. et al., Ann. Nutr.
Metab. 43:127-130 (1999); Krauss, R. M. et al. AHA Circulation
102:2284-2299 (2000)).
[0010] The main sources of .omega.-6 fatty acids are vegetable oils
(e.g., corn oil, soy oil) that contain high amounts of LA. GLA is
found in the seeds of a number of plants, including evening
primrose (Oenothera biennis), borage (Borago officinalis) and black
currants (Ribes nigrum). Microorganisms in the genera Mortierella
(filamentous fungus), Entomophthora, Pythium and Porphyridium (red
alga) can be used for commercial production of the .omega.-6 fatty
acid, arachidonic acid (ARA). The fungus Mortierella alpina, for
example, is used to produce an oil containing ARA, while U.S. Pat.
No. 5,658,767 (Martek Corporation) teaches a method for the
production of an oil containing ARA comprising cultivating Pythium
insidiuosum in a culture medium containing a carbon and nitrogen
source.
[0011] The .omega.-3 PUFAs of importance include EPA and
docosahexaenoic acid (DHA), both of which are found in different
types of fish oil and marine plankton. U.S. Pat. No. 5,244,921
(Martek Corporation) describes a process for producing an edible
oil containing EPA by cultivating heterotrophic diatoms in a
fermentor, specifically Cyclotella sp. and Nitzschia sp. DHA can be
obtained from cold water marine fish, egg yolk fractions and by
cultivation of certain heterotrophic microalgae of the class
Dinophyceae, specifically, Crypthecodinium sp. such as C. cohnii
(U.S. Pat. No. 5,492,938 and U.S. Pat. No. 5,407,957). Stearidonic
acid (STA), a precursor to EPA and DHA, can be found in marine oils
and plant seeds; its commercial sources include production in the
genera Trichodesma and Echium. Other sources of .omega.-3 acids are
found in flaxseed oil and walnut oil, each containing predominantly
ALA.
[0012] Despite a variety of commercial sources of PUFAs from
natural sources, there are several disadvantages associated with
these methods of production. First, natural sources such as fish
and plants tend to have highly heterogeneous oil compositions. The
oils obtained from these sources therefore can require extensive
purification to separate or enrich one or more of the desired
PUFAs. Fish oils commonly have unpleasant tastes and odors, which
may be impossible to separate economically from the desired product
and can render such products unacceptable as food supplements.
Unpleasant tastes and odors can make medical regimens based on
ingestion of high dosages undesirable, and may inhibit compliance
by the patient. Furthermore, fish may accumulate environmental
pollutants and ingestion of fish oil capsules as a dietary
supplement may result in ingestion of undesired contaminants.
Natural sources are also subject to uncontrollable fluctuations in
availability (e.g., due to weather, disease, or over-fishing in the
case of fish stocks); and, crops that produce PUFAs often are not
competitive economically with hybrid crops developed for food
production. Large-scale fermentation of some organisms that
naturally produce PUFAs (e.g., Porphyridium, Mortierella) can also
be expensive and/or difficult to cultivate on a commercial
scale.
[0013] As a result of the limitations described above, extensive
work has been conducted toward: 1.) the development of recombinant
sources of PUFAs that are easy to produce commercially; and 2.)
modification of fatty acid biosynthetic pathways, to enable
production of desired PUFAs.
[0014] Advances in the isolation, cloning and manipulation of fatty
acid desaturase and elongase genes from various organisms have been
made over the last several years. Knowledge of these gene sequences
offers the prospect of producing a desired fatty acid and/or fatty
acid composition in novel host organisms that do not naturally
produce PUFAs. The literature reports a number of examples in
Saccharomyces cerevisiae, such as: [0015] 1. Domergue, F. et al.
(Eur. J. Biochem. 269:4105-4113 (2002)), wherein two desaturases
from the marine diatom Phaeodactylum tricornutum were cloned into
S. cerevisiae, leading to the production of EPA; [0016] 2. Beaudoin
F., et al. (Proc. Natl. Acad. Sci. U.S.A. 97(12):6421-6 (2000)),
wherein the .omega.-3 and .omega.-6 PUFA biosynthetic pathways were
reconstituted in S. cerevisiae, using genes from Caenorhabditis
elegans; [0017] 3. Dyer, J. M. et al. (Appl. Eniv. Microbiol.,
59:224-230 (2002)), wherein plant fatty acid desaturases (FAD2 and
FAD3) were expressed in S. cerevisiae, leading to the production of
ALA; and [0018] 4. U.S. Pat. No. 6,136,574 (Knutzon et al., Abbott
Laboratories), wherein one desaturase from Brassica napus and two
desaturases from the fungus Mortierella alpina were cloned into S.
cerevisiae, leading to the production of LA, GLA, ALA and STA.
There remains a need, however, for an appropriate microbial system
in which these types of genes can be expressed to provide for
economical production of commercial quantities of one or more
PUFAs. Additionally, a need exists for oils enriched in specific
PUFAs, notably EPA and DHA.
[0019] Many microorganisms (including algae, bacteria, molds and
yeasts) can synthesize oils in the ordinary course of cellular
metabolism. Thus, oil production involves cultivating the
microorganism in a suitable culture medium to allow for oil
synthesis, followed by separation of the microorganism from the
fermentation medium and treatment for recovery of the intracellular
oil. Attempts have been made to optimize production of fatty acids
by fermentive means involving varying such parameters as
microorganisms used, media and conditions that permit oil
production. However, these efforts have proved largely unsuccessful
in improving yield of oil or the ability to control the
characteristics of the oil composition produced.
[0020] One class or microorganisms that has not been previously
examined as a production platform for PUFAs, however, are the
oleaginous yeasts. These organisms can accumulate oil up to 80% of
their dry cell weight. 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
may offer a cost advantage compared to commercial micro-algae
fermentation for production of .omega.-3 or .omega.-6 PUFAs. Whole
yeast cells may also represent a convenient way of encapsulating
.omega.-3 or .omega.-6 PUFA-enriched oils for use in functional
foods and animal feed supplements.
[0021] Despite the advantages noted above, oleaginous yeast are
naturally deficient in .omega.-6 and .omega.-3 PUFAs, since
naturally produced PUFAs in these organisms are limited to 18:2
fatty acids (and less commonly, 18:3 fatty acids). Thus, the
problem to be solved is to develop an oleaginous yeast that
accumulates oils enriched in .omega.-3 and/or .omega.-6 fatty
acids. Toward this end, it is necessary to introduce desaturases
and elongases that allow for the synthesis and accumulation of
.omega.-3 and/or .omega.-6 fatty acids in oleaginous yeasts.
Although advances in the art of genetic engineering have been made,
such techniques have not been developed for oleaginous yeasts.
Thus, one must overcome problems associated with the use of these
particular host organisms for the production of PUFAs.
[0022] Applicants have solved the stated problem by demonstrating
production of PUFAs in the host Yarrowia lipolytica, following the
introduction of a heterologous .omega.-6 and/or .omega.-3
biosynthetic pathway. Specifically, ARA (representative of
.omega.-6 fatty acids) and EPA (representative of .omega.-3 fatty
acids) were produced herein, to exemplify the techniques of the
invention.
SUMMARY OF THE INVENTION
[0023] The present invention provides methods for the expression of
enzymes comprising the .omega.-3/.omega.-6 fatty acid biosynthetic
pathway in an oleaginous yeast host for the production of .omega.-3
and/or .omega.-6 fatty acids. Accordingly, the invention provides a
method for the production of .omega.-3 and/or .omega.-6 fatty acids
comprising: [0024] a) providing an oleaginous yeast comprising a
functional .omega.-3/.omega.-6 fatty acid biosynthetic pathway;
[0025] b) growing the yeast of step (a) in the presence of a
fermentable carbon source whereby an .omega.-3 or .omega.-6 fatty
acid is produced; and [0026] c) optionally recovering the .omega.-3
or .omega.-6 fatty acid.
[0027] In one specific embodiment the invention provides a method
for the production of linoleic acid comprising: [0028] a) providing
an oleaginous yeast comprising: [0029] (i) a gene encoding a
.DELTA.12 desaturase polypeptide; and [0030] (ii) an endogenous
source of oleic acid; [0031] b) growing the yeast of step (a) in
the presence of a suitable fermentable carbon source wherein the
gene encoding a .DELTA.12 desaturase polypeptide is expressed and
the oleic acid is converted to linoleic acid; and [0032] c)
optionally recovering the linoleic acid of step (b).
[0033] In specific embodiments the invention provides for the
production of specific .omega.-6 fatty acids such as linoleic acid
(LA), .gamma.-linolenic acid (GLA), dihomo-.gamma.-linoleic acid
(DGLA) and arachidonic acid (ARA) by de novo biosynthesis or single
step enzymatic reactions from the appropriate precursors. Similarly
the invention provides for the production of specific .omega.-3
fatty acids such as .alpha.-linoleic acid (ALA), stearidonic acid
(STA), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA),
docosapentaenoic acid (DPA) and docosahexaenoic acid by single step
enzymatic reactions from the appropriate precursors.
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS
[0034] FIG. 1 shows a schematic illustration of the biochemical
mechanism for lipid accumulation in oleaginous yeast.
[0035] FIG. 2 illustrates the .omega.-3/.omega.-6 fatty acid
biosynthetic pathway.
[0036] FIG. 3 illustrates the construction of plasmid vector pY5
for gene expression in Yarrowia lipolytica.
[0037] FIG. 4 illustrates the construction of plasmid vectors pY5-4
and pY5-13 for gene expression in Y. lipolytica.
[0038] FIG. 5 is a schematic presentation of the construction of
intermediate vector pYZM5CHPPA.
[0039] FIG. 6 show a comparison between the DNA sequence of the
Saprolegnia diclina .DELTA.17 desaturase gene and the synthetic
gene codon-optimized for expression in Y. lipolytica.
[0040] FIG. 7 illustrates the favored consensus sequences around
the translation initiation codon `ATG` in Y. lipolytica.
[0041] FIG. 8 illustrates the strategy utilized for in vitro
synthesis of the codon-optimized .DELTA.17 desaturase gene.
[0042] FIG. 9 shows plasmids for expression of the synthetic
codon-optimized and wildtype .DELTA.17 desaturase genes in Y.
lipolytica.
[0043] FIGS. 10A and 10B show the results of gas chromatographic
analysis of fatty acids produced in Y. lipolytica transformed with
the wildtype and synthetic codon-optimized .DELTA.17 desaturase
genes, respectively.
[0044] FIG. 11 is a schematic presentation of the construction of
intermediate vector pY24-4.
[0045] FIG. 12 is a schematic presentation of the construction of
intermediate vector pYZV16.
[0046] FIG. 13 is a schematic presentation of the construction of
integration vector pYZM5EL6.
[0047] FIG. 14 is a schematic presentation of the construction of
integration vectors pYZV5EL6 and pYZV5EL6/17.
[0048] FIG. 15 is a chromatogram illustrating the production of ARA
from an engineered Y. lipolytica.
[0049] FIG. 16 is a chromatogram illustrating the production of EPA
from an engineered Y. lipolytica.
[0050] The invention can be more fully understood from the
following detailed description and the accompanying sequence
descriptions, which form a part of this application.
[0051] 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.
[0052] SEQ ID NO:1 shows the DNA sequence of the Mortierella alpina
.DELTA.6 desaturase gene, while SEQ ID NO:2 shows the amino acid
sequence of the M. alpina .DELTA.6 desaturase.
[0053] SEQ ID NO:3 shows the DNA sequence of the Mortierella alpina
.DELTA.5 desaturase gene, while SEQ ID NO:4 shows the amino acid
sequence of the M. alpina .DELTA.5 desaturase.
[0054] SEQ ID NO:5 shows the DNA sequence of the Saprolegnia
diclina .DELTA.17 desaturase gene, while SEQ ID NO:6 shows the
corresponding amino acid sequence of the S. diclina .DELTA.17
desaturase.
[0055] SEQ ID NO:7 shows the DNA sequence of the Mortierella alpina
high affinity elongase gene, while SEQ ID NO:8 shows the amino acid
sequence of the M. alpina high affinity elongase.
[0056] SEQ ID NO:9 shows the DNA sequence of the synthetic
.DELTA.17 desaturase gene codon-optimized for expression in
Yarrowia lipolytica.
[0057] SEQ ID NOs:10-31 correspond to the 11 pairs of
oligonucleotides that together comprise the entire codon-optimized
coding region of the S. diclina .DELTA.17 desaturase gene (e.g.,
D17-1A, D17-1B, D17-2A, D17-2B, D17-3A, D17-3B, D17-4A, D17-4B,
D17-5A, D17-5B, D17-6A, D17-6B, D17-7A, D17-7B, D17-8A, D17-8B,
D17-9A, D17-9B, D17-10A, D17-10B, D17-11A and D17-11B,
respectively).
[0058] SEQ ID NOs:32-37 correspond to primers D17-1, D17-4R, D17-5,
D17-8D, D17-8U and D17-11, respectively, used for PCR amplification
during synthesis of the codon-optimized .DELTA.17 desaturase
gene.
[0059] SEQ ID NOs:38 and 39 correspond to primers TEF5' and TEF3',
respectively, used to isolate the TEF promoter.
[0060] SEQ ID NOs:40 and 41 correspond to primers XPR5' and XPR3',
respectively, used to isolate the XPR2 transcriptional
terminator.
[0061] SEQ ID NOs:42 and 43 correspond to primers YL21A and YL22,
used for amplifying the wild type .DELTA.17 desaturase gene of S.
diclina from plasmid pRSP19.
[0062] SEQ ID NOs:44 and 45 correspond to primers YL53 and YL54,
respectively, used for site-directed mutagenesis to generate
pYSD17M.
[0063] SEQ ID NOs:46 and 47 correspond to primers KU5 and KU3,
respectively, used for amplifying a 1.7 kB DNA fragment (SEQ ID
NO:48; amino acid sequence provided as SEQ ID NO:49) containing the
Yarrowia URA3 gene.
[0064] SEQ ID NOs:50 and 51 correspond to primers KI5 and KI3,
respectively, used for amplifying a 1.1 kB DNA fragment (SEQ ID
NO:52; amino acid sequence provided as SEQ ID NO:53) containing the
conjugase gene of Impatients balsama.
[0065] SEQ ID NOs:54 and 55 correspond to primers KTI5 and KTI3,
respectively, used for amplifying a 1.7 kB DNA fragment (SEQ ID
NO:56; amino acid sequence provided as SEQ ID NO:57) containing a
TEF::conjugase::XPR chimeric gene.
[0066] SEQ ID NOs:58 and 59 correspond to primers KH5 and KH3,
respectively, used for amplifying a 1 kB DNA fragment (SEQ ID
NO:60; amino acid sequence provided as SEQ ID NO:61) containing the
E. coli hygromycin resistance gene.
[0067] SEQ ID NOs:62 and 63 correspond to primers KTH5 and KTH3,
respectively, used for amplifying a 1.6 kB DNA fragment (SEQ ID
NO:64; amino acid sequence provided as SEQ ID NO:65) containing the
TEF::HPT::XPR fusion gene.
[0068] SEQ ID NOs:66 and 67 correspond to the 401 bp of 5'-sequence
and 568 bp of 3'-sequence of the Yarrowia lipolytica URA3 gene,
respectively, used to direct integration of expression cassettes
into the Ura loci of the Yarrowia genome.
[0069] SEQ ID NOs:68-71 correspond to primers YL63, YL64, YL65 and
YL66, respectively, used for site-directed mutagenesis to generate
pY24-4.
[0070] SEQ ID NOs:72 and 73 correspond to primers YL11 and YL12,
respectively, used for amplifying the M. alpina .DELTA.5
desaturase.
[0071] SEQ ID NOs:74-77 correspond to primers YL81, YL82, YL83 and
YL84, respectively, used for site-directed mutagenesis to generate
pYZM5CH.
[0072] SEQ ID NOs:78 and 79 correspond to primers YL105 and YL106,
respectively, used for site-directed mutagenesis to generate
pYZM5CHPP.
[0073] SEQ ID NOs:80 and 81 correspond to primers YL119 and YL120,
respectively, used for site-directed mutagenesis to generate
pYZM5CHPPA.
[0074] SEQ ID NOs:82 and 83 correspond to primers YL121 and YL122,
respectively, used for amplifying 440 bp of 5'-non-coding DNA
sequence (SEQ ID NO:84) upstream from the Y. lipolytica URA3
gene.
[0075] SEQ ID NOs:85 and 86 correspond to primers YL114 and YL115,
respectively, used for site-directed mutagenesis to generate pYZV5
and pYZV5P.
[0076] SEQ ID NO:87 corresponds to a 5.2 kB DNA fragment suitable
for integration and expression of the M. alpina .DELTA.5 desaturase
gene in the Yarrowia lipolytica genome.
[0077] SEQ ID NOs:88-91 correspond to primers YL61, YL62, YL69 and
YL70, respectively, used for site-directed mutagenesis to generate
pY58BH.
[0078] SEQ ID NOs:92-95 correspond to primers YL77, YL78, YL79A and
YL80A, respectively, used for site-directed mutagenesis to generate
pY54PC.
[0079] SEQ ID NO:96 corresponds to a 8.9 kB DNA fragment suitable
for integration and coordinate expression of the M. alpina .DELTA.6
desaturase, M. alpina elongase and M. alpina .DELTA.5 desaturase
genes in the Yarrowia lipolytica genome.
[0080] SEQ ID NOs:97-100 correspond to primers YL101, YL102, YL103
and YL104, respectively, used for site-directed mutagenesis to
generate pYSD17SPC.
[0081] SEQ ID NO:101 corresponds to a 10.3 kB DNA fragment suitable
for integration and coordinate expression of the M. alpina .DELTA.6
desaturase, M. alpina elongase, M. alpina .DELTA.5 desaturase and
codon-optimized .DELTA.17 desaturase genes in the Yarrowia
lipolytica genome.
[0082] SEQ ID NOs:102-113 correspond to primers YL1, YL2, YL3, YL4,
YL5, YL6, YL7, YL8, YL9, YL10, YL23 and YL24, respectively, used
for plasmid construction.
[0083] SEQ ID NO:114 shows the DNA sequence of the Saprolegnia
diclina .DELTA.5 desaturase gene, while SEQ ID NO:115 shows the
amino acid sequence of the S. diclina .DELTA.5 desaturase.
[0084] SEQ ID NOs:116, 117, 120, 121, 124 and 125 correspond to
primers YL13A, YL14, YL19A, YL20, YL15 and YL16B, respectively,
used for cloning various .DELTA.5 desaturases.
[0085] SEQ ID NO:118 shows the DNA sequence of the Isochrysis
galbana .DELTA.5 desaturase gene, while SEQ ID NO:119 shows the
amino acid sequence of the T. galbana .DELTA.5 desaturase.
[0086] SEQ ID NO:122 shows the DNA sequence of the Thraustochytrium
aureum .DELTA.5 desaturase gene, while SEQ ID NO:123 shows the
amino acid sequence of the T. aureum .DELTA.5 desaturase.
[0087] SEQ ID NO:126 corresponds to the codon-optimized translation
initiation site for genes optimally expressed in Yarrowia sp.
DETAILED DESCRIPTION OF THE INVENTION
[0088] In accordance with the subject invention, Applicants provide
methods for the production of .omega.-3 and/or .omega.-6 fatty
acids in oleaginous yeasts. Specifically, Applicants provide
methods for production of linoleic acid, .gamma.-linolenic acid,
dihomo-.gamma.-linolenic acid, arachidonic acid, .alpha.-linolenic
acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic
acid, docosapentaenoic acid and docosahexaenoic acid. This is
accomplished by introduction of functional .omega.-3/.omega.-6
fatty acid biosynthetic pathway encoded by genes conferring
.DELTA.17 desaturase, .DELTA.6 desaturase, .DELTA.5 desaturase,
.DELTA.9 desaturase, .DELTA.12 desaturase, .DELTA.15 desaturase,
.DELTA.4 desaturase and elongase activities into oleaginous yeast
hosts for recombinant expression. Thus, this disclosure
demonstrates that oleaginous yeasts can be engineered to enable
production of any PUFA composition that is desired.
[0089] The subject invention finds many applications. PUFAs, or
derivatives thereof, made by the methodology disclosed herein 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).
[0090] Supplementation of humans or animals with PUFAs produced by
recombinant means can result in increased levels of the added
PUFAs, as well as their metabolic progeny. For example, treatment
with arachidonic acid (ARA) can result not only in increased levels
of ARA, but also downstream products of ARA such as prostaglandins.
Complex regulatory mechanisms can make it desirable to combine
various PUFAs, or add different conjugates of PUFAs, in order to
prevent, control or overcome such mechanisms to achieve the desired
levels of specific PUFAs in an individual.
DEFINITIONS
[0091] In this disclosure, a number of terms and abbreviations are
used. The following definitions are provided.
[0092] "Open reading frame" is abbreviated ORF.
[0093] "Polymerase chain reaction" is abbreviated PCR.
[0094] "American Type Culture Collection" is abbreviated ATCC.
"Polyunsaturated fatty acid(s)" is abbreviated PUFA(s).
[0095] 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.
[0096] Generally, fatty acids are classified as saturated or
unsaturated. The term "saturated fatty acids" refers to those fatty
acids that have no "double bonds" between their carbon backbone. In
contrast, "unsaturated fatty acids" are cis-isomers that have
"double bonds" along their carbon backbones. "Monounsaturated fatty
acids" have only one "double bond" along the carbon backbone (e.g.,
usually between the 9.sup.th and 10.sup.th carbon atom as for
palmitoleic acid (16:1) and oleic acid (18:1)), while
"polyunsaturated fatty acids" (or "PUFAs") have at least two double
bonds along the carbon backbone (e.g., between the 9.sup.th and
10.sup.th, and 12.sup.th and 13.sup.th carbon atoms for linoleic
acid (18:2); and between the 9.sup.th and 10.sup.th, 12.sup.th and
13.sup.th, and 15.sup.th and 16.sup.th for .alpha.-linolenic acid
(18:3)).
[0097] "PUFAs" can be classified into two major families (depending
on the position (n) of the first double bond nearest the methyl end
of the fatty acid carbon chain). Thus, the ".omega.-6 fatty acids"
(.omega.-6 or n-6) have the first unsaturated double bond six
carbon atoms from the omega (methyl) end of the molecule and
additionally have a total of two or more double bonds, with each
subsequent unsaturation occurring 3 additional carbon atoms toward
the carboxyl end of the molecule. In contrast, the ".omega.-3 fatty
acids" (.omega.-3 or n-3) have the first unsaturated double bond
three carbon atoms away from the omega end of the molecule and
additionally have a total of three or more double bonds, with each
subsequent unsaturation occurring 3 additional carbon atoms toward
the carboxyl end of the molecule.
[0098] For the purposes of the present disclosure, the
omega-reference system will be used to indicate the number of
carbons, the number of double bonds and the position of the double
bond closest to the omega carbon, counting from the omega carbon
(which is numbered 1 for this purpose). This nomenclature is shown
below in Table 1, in the column titled "Shorthand Notation". The
remainder of the Table summarizes the common names of .omega.-3 and
.omega.-6 fatty acids, the abbreviations that will be used
throughout the specification and each compounds' chemical name.
TABLE-US-00001 TABLE 1 Nomenclature of Polyunsaturated Fatty Acids
Abbrevi- Shorthand Common Name ation Chemical Name Notation
Linoleic LA cis-9,12-octadecadienoic 18:2 .omega.-6
.gamma.-Linoleic GLA cis-6,9,12- 18:3 .omega.-6 octadecatrienoic
Dihomo-.gamma.- DGLA cis-8,11,14- 20:3 .omega.-6 Linoleic
eicosatrienoic 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 Eicosa- ETA cis-8,11,14,17- 20:4 .omega.-3
tetraenoic eicosatetraenoic Eicosa- EPA cis-5,8,11,14,17- 20:5
.omega.-3 pentaenoic eicosapentaenoic Docosa- DPA
cis-7,10,13,16,19- 22:5 .omega.-3 pentaenoic docosapentaenoic
Docosa- DHA cis-4,7,10,13,16,19- 22:6 .omega.-3 hexaenoic
docosahexaenoic
[0099] The term "essential fatty acid" refers to a particular PUFA
that an organism must ingest in order to survive, being unable to
synthesize the particular essential fatty acid de novo. For
example, mammals can not synthesize the essential fatty acid LA
(18:2, .omega.-6). Other essential fatty acids include GLA
(.omega.-6), DGLA (.omega.-6), ARA (.omega.-6), EPA (.omega.-3) and
DHA (.omega.-3).
[0100] The term "fat" refers to a lipid substance that is solid at
25.degree. C. and usually saturated.
[0101] The term "oil" refers to a lipid substance that is liquid at
25.degree. C. and usually polyunsaturated. PUFAs are found in the
oils of some algae, oleaginous yeasts and filamentous fungi.
"Microbial oils" or "single cell oils" are those oils naturally
produced by microorganisms during their lifespan. Such oils can
contain long chain PUFAs.
[0102] The term "PUFA biosynthetic pathway enzyme" refers to any of
the following enzymes (and genes which encode said enzymes)
associated with the biosynthesis of a PUFA, including: a .DELTA.4
desaturase, a .DELTA.5 desaturase, a .DELTA.6 desaturase, a
.DELTA.12 desaturase, a .DELTA.15 desaturase, a .DELTA.17
desaturase, a .DELTA.9 desaturase and/or an elongase.
[0103] The term ".omega.-3/.omega.-6 fatty acid biosynthetic
pathway" refers to a set of genes which, when expressed under the
appropriate conditions encode enzymes that catalyze the production
of either or both .omega.-3 and .omega.-6 fatty acids. Typically
the genes involved in the .omega.-3/.omega.-6 fatty acid
biosynthetic pathway encode some or all of the following enzymes:
.DELTA.12 desaturase, .DELTA.6 desaturase, elongase, .DELTA.5
desaturase, .DELTA.17 desaturase, .DELTA.15 desaturase, .DELTA.9
desaturase and .DELTA.4 desaturase. A representative pathway is
illustrated in FIG. 2, which demonstrates how both .omega.-3 and
.omega.-6 fatty acids may be produced from a common source. The
term "functional" as used herein in context with the
.omega.-3/.omega.-6 fatty acid biosynthetic pathway means that some
or all of the genes in the pathway express active enzymes. It
should be understood that ".omega.-3/.omega.-6 fatty acid
biosynthetic pathway" or "functional .omega.-3/.omega.-6 fatty acid
biosynthetic pathway" does not imply that all the genes listed in
this paragraph are required as a number of fatty acid products will
only require the expression of a subset of the genes of this
pathway.
[0104] The term "desaturase" refers to a polypeptide component of a
multi-enzyme complex that can desaturate one or more fatty acids to
produce a mono- or polyunsaturated fatty acid or precursor of
interest. Despite use of the omega-reference system throughout the
specification 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 substrate using the delta-system. Of
particular interest herein are: 1.) .DELTA.17 desaturases that
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
which, for example, catalyze the conversion of ARA to EPA and/or
DGLA to ETA; 2.) .DELTA.6 desaturases that catalyze the conversion
of LA to GLA and/or ALA to STA; 3.) .DELTA.5 desaturases that
catalyze the conversion of DGLA to ARA and/or ETA to EPA; 4.)
.DELTA.4 desaturases that catalyze the conversion of DPA to DHA;
5.) .DELTA.12 desaturases that catalyze the conversion of oleic
acid to LA; 6.) .DELTA.15 desaturases that catalyze the conversion
of LA to ALA; and 7.) .DELTA.9 desaturases that catalyze the
conversion of palmitate to palmitoleic acid (16:1) and/or stearate
to oleic acid (18:1).
[0105] The term "elongase" refers to a polypeptide component of a
multi-enzyme complex that can elongate a fatty acid carbon chain to
produce a mono- or polyunsaturated fatty acid that is 2 carbons
longer than the fatty acid substrate that the elongase acts upon.
This process of elongation occurs in a multi-step mechanism in
association with fatty acid synthase, whereby CoA is the acyl
carrier (Lassner et al., The Plant Cell 8:281-292 (1996)). Briefly,
malonyl-CoA is condensed with a long-chain acyl-CoA to yield
CO.sub.2 and a .beta.-ketoacyl-CoA (where the acyl moiety has been
elongated by two carbon atoms). Subsequent reactions include
reduction to .beta.-hydroxyacyl-CoA, dehydration to an enoyl-CoA
and a second reduction to yield the elongated acyl-CoA. Examples of
reactions catalyzed by elongases are the conversion of GLA to DGLA,
STA to ETA and EPA to DPA. Accordingly, elongases can have
different specificities (e.g., a C.sub.16/18 elongase will prefer a
C.sub.16 substrate, a C.sub.18/20 elongase will prefer a C.sub.18
substrate and a C.sub.20/22 elongase will prefer a C.sub.20
substrate).
[0106] The term "high affinity elongase" refers to an elongase
whose substrate specificity is preferably for GLA (with DGLA as a
product of the elongase reaction). One such elongase is described
in WO 00/12720.
[0107] The terms "conversion efficiency" and "percent substrate
conversion" refer to the efficiency by which a particular enzyme
(e.g., a desaturase or elongase) can convert substrate to product.
The conversion efficiency is measured according to the following
formula: ([product]/[substrate+product])*100, where `product`
includes the immediate product and all products in the pathway
derived from it.
[0108] 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)).
[0109] The term "oleaginous yeast" refers to those microorganisms
classified as yeasts 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.
[0110] The term "fermentable carbon source" means a carbon source
that a microorganism will metabolize to derive energy. Typical
carbon sources of the invention include, but are not limited to:
monosaccharides, oligosaccharides, polysaccharides, alkanes, fatty
acids, esters of fatty acids, monoglycerides, diglycerides,
triglycerides, carbon dioxide, methanol, formaldehyde, formate and
carbon-containing amines.
[0111] As used herein, an "isolated nucleic acid fragment" 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 fragment in the form of a polymer
of DNA may be comprised of one or more segments of cDNA, genomic
DNA or synthetic DNA.
[0112] 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 fragment 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 fragment comprising the sequence.
[0113] 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.
[0114] "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.
[0115] "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.
[0116] "Gene" refers to a nucleic acid fragment 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.
"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.
[0117] "Coding sequence" refers to a DNA sequence that codes for a
specific amino acid sequence. "Suitable regulatory sequences" refer
to 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.
[0118] "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.
[0119] 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.
[0120] "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.
[0121] The term "operably linked" refers to the association of
nucleic acid sequences on a single nucleic acid fragment 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.
[0122] The term "expression", as used herein, refers to the
transcription and stable accumulation of sense (mRNA) or antisense
RNA derived from the nucleic acid fragments of the invention.
Expression may also refer to translation of mRNA into a
polypeptide.
[0123] "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 fragments are referred to as "transgenic"
or "recombinant" or "transformed" organisms.
[0124] The terms "plasmid", "vector" and "cassette" refer to an
extra chromosomal element often carrying genes that are not part of
the central metabolism of the cell, and usually in the form of
circular double-stranded DNA fragments. Such elements may be
autonomously replicating sequences, genome integrating sequences,
phage or nucleotide sequences, linear or circular, of a single- or
double-stranded DNA or RNA, derived from any source, in which a
number of nucleotide sequences have been joined or recombined into
a unique construction which is capable of introducing a promoter
fragment and DNA sequence for a selected gene product along with
appropriate 3' untranslated sequence into a cell. "Transformation
cassette" refers to a specific vector containing a foreign gene and
having elements in addition to the foreign gene that facilitates
transformation of a particular host cell. "Expression cassette"
refers to a specific vector containing a foreign gene and having
elements in addition to the foreign gene that allow for enhanced
expression of that gene in a foreign host.
[0125] 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.); 4.) Sequencher (Gene Codes
Corporation, Ann Arbor, Mich.); and 5.) the FASTA program
incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput.
Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992,
111-20. Editor(s): Suhai, Sandor. 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.
[0126] Standard recombinant DNA and molecular cloning techniques
used herein are well known in the art and are described by
Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory: Cold
Spring Harbor, N.Y. (1989) (hereinafter "Maniatis"); by Silhavy, T.
J., Bennan, M. L. and Enquist, L. W., Experiments with Gene
Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.
(1984); and by Ausubel, F. M. et al., Current Protocols in
Molecular Biology, published by Greene Publishing Assoc. and
Wiley-Interscience (1987).
Microbial Biosynthesis of Fatty Acids
[0127] In general, lipid accumulation in oleaginous microorganisms
is triggered in response to the overall carbon to nitrogen ratio
present in the growth medium (FIG. 1). When cells have exhausted
available nitrogen supplies (e.g., when the carbon to nitrogen
ratio is greater than about 40), the depletion of cellular
adenosine monophosphate (AMP) leads to the cessation of
AMP-dependent isocitrate dehydrogenase activity in the mitochondria
and the accumulation of citrate, transport of citrate into the
cytosol and subsequent cleavage of the citrate by ATP-citrate lyase
to yield acetyl-CoA and oxaloacetate. Acetyl-CoA is the principle
building block for de novo biosynthesis of fatty acids. Although
any compound that can effectively be metabolized to produce
acetyl-CoA can serve as a precursor of fatty acids, glucose is the
primary source of carbon in this type of reaction (FIG. 1). Glucose
is converted to pyruvate via glycolysis and pyruvate is then
transported into the mitochondria where it can be converted to
acetyl-CoA by pyruvate dehydrogenase ("PD"). Since acetyl-CoA
cannot be transported directly across the mitochondrial membrane
into the cytoplasm, the two carbons from acetyl-CoA condense with
oxaloacetate to yield citrate (catalyzed by citrate synthase).
Citrate is transported directly into the cytoplasm, where it is
cleaved by ATP-citrate lyase to regenerate acetyl-CoA and
oxaloacetate. The oxaloacetate reenters the tricarboxylic acid
cycle, via conversion to malate.
[0128] The synthesis of malonyl-CoA is the first committed step of
fatty acid biosynthesis, which takes place in the cytoplasm.
Malonyl-CoA is produced via carboxylation of acetyl-CoA by
acetyl-CoA carboxylase ("ACC"). Fatty acid synthesis is catalyzed
by a multi-enzyme fatty acid synthase complex ("FAS") and occurs by
the condensation of eight two-carbon fragments (acetyl groups from
acetyl-CoA) to form a 16-carbon saturated fatty acid, palmitate.
More specifically, FAS catalyzes a series of 7 reactions, which
involve the following (Smith, S. FASEB J., 8(15):1248-59 (1994)):
[0129] 1. Acetyl-CoA and malonyl-CoA are transferred to the acyl
carrier peptide (ACP) of FAS. The acetyl group is then transferred
to the malonyl group, forming .beta.-ketobutyryl-ACP and releasing
CO.sub.2. [0130] 2. The .beta.-ketobutyryl-ACP undergoes reduction
(via .beta.-ketoacyl reductase) and dehydration (via
.beta.-hydroxyacyl dehydratase) to form a trans-monounsaturated
fatty acyl group. [0131] 3. The double bond is reduced by NADPH,
yielding a saturated fatty-acyl group two carbons longer than the
initial one. The butyryl-group's ability to condense with a new
malonyl group and repeat the elongation process is then
regenerated. [0132] 4. When the fatty acyl group becomes 16 carbons
long, a thioesterase activity hydrolyses it, releasing free
palmitate.
[0133] Palmitate (16:0) is the precursor of longer chain saturated
and unsaturated fatty acids (e.g., stearic (18:0), palmitoleic
(16:1) and oleic (18:1) acids) through the action of elongases and
desaturases present in the endoplasmic reticulum membrane.
Palmitate and stearate are converted to their unsaturated
derivatives, palmitoleic (16:1) and oleic (18:1) acids,
respectively, by the action of a .DELTA.9 desaturase.
[0134] Triacylglycerols (the primary storage unit for fatty acids)
are formed by the esterification of two molecules of acyl-CoA to
glycerol-3-phosphate to yield 1,2-diacylglycerol phosphate
(commonly identified as phosphatidic acid) (FIG. 1). The phosphate
is then removed, by phosphatidic acid phosphatase, to yield
1,2-diacylglycerol. Triacylglycerol is formed upon the addition of
a third fatty acid by the action of a diacylglycerol-acyl
transferase.
Biosynthesis of Omega-3 and Omega-6 Fatty Acids
[0135] Simplistically, the metabolic process that converts LA to
GLA, DGLA and ARA (the .omega.-6 pathway) and ALA to STA, ETA, EPA
and DHA (the .omega.-3 pathway) involves elongation of the carbon
chain through the addition of carbon atoms and desaturation of the
molecule through the addition of double bonds (FIG. 2). This
requires a series of special desaturation and elongation enzymes
present in the endoplasmic reticulim membrane.
.omega.-6 Fatty Acids
[0136] Oleic acid is converted to LA (18:2), the first of the
.omega.-6 fatty acids, by the action of a .DELTA.12 desaturase.
Subsequent .omega.-6 fatty acids are produced as follows: 1.) LA is
converted to GLA by the activity of a .DELTA.6 desaturase; 2.) GLA
is converted to DGLA by the action of an elongase; and 3.) DGLA is
converted to ARA by the action of a .DELTA.5 desaturase.
[0137] .omega.-3 Fatty Acids
[0138] Linoleic acid (LA) is converted to ALA, the first of the
.omega.-3 fatty acids, by the action of a .DELTA.15 desaturase.
Subsequent .omega.-3 fatty acids are produced in a series of steps
similar to that for the .omega.-6 fatty acids. Specifically: 1.)
ALA is converted to STA by the activity of a .DELTA.6 desaturase;
2.) STA is converted to ETA by the activity of an elongase; and 3.)
ETA is converted to EPA by the activity of a .DELTA.5 desaturase.
Alternatively, ETA and EPA can be produced from DGLA and ARA,
respectively, by the activity of a .DELTA.17 desaturase. EPA can be
further converted to DHA by the activity of an elongase and a
.DELTA.4 desaturase.
Genes Involved in Omega Fatty Acid Production
[0139] Many microorganisms, including algae, bacteria, molds and
yeasts, can synthesize PUFAs and omega fatty acids in the ordinary
course of cellular metabolism. Particularly well-studied are fungi
including Schizochytrium aggregatm, species of the genus
Thraustochytrium and Morteriella alpina. Additionally, many
dinoflagellates (Dinophyceaae) naturally produce high
concentrations of PUFAs. As such, a variety of genes involved in
oil production have been identified through genetic means and the
DNA sequences of some of these genes are publicly available
(non-limiting examples are shown below in Table 2):
TABLE-US-00002 TABLE 2 Some Publicly Available Genes Involved In
PUFA Production Genbank Accession No. Description AY131238 Argania
spinosa .DELTA.6 desaturase Y055118 Echium pitardii var. pitardii
.DELTA.6 desaturase AY055117 Echium gentianoides .DELTA.6
desaturase AF296076 Mucor rouxii .DELTA.6 desaturase AF007561
Borago officinalis .DELTA.6 desaturase L11421 Synechocystis sp.
.DELTA.6 desaturase NM_031344 Rattus norvegicus .DELTA.6 fatty acid
desaturase AF465283, Mortierella alpina .DELTA.6 fatty acid
desaturase AF465281, AF110510 AF465282 Mortierella isabellina
.DELTA.6 fatty acid desaturase AF419296 Pythium irregulare .DELTA.6
fatty acid desaturase AB052086 Mucor circinelloides D6d mRNA for
.DELTA.6 fatty acid desaturase AJ250735 Ceratodon purpureus mRNA
for .DELTA.6 fatty acid desaturase AF126799 Homo sapiens .DELTA.6
fatty acid desaturase AF126798 Mus musculus .DELTA.6 fatty acid
desaturase AF199596, Homo sapiens .DELTA.5 desaturase AF226273
AF320509 Rattus norvegicus liver .DELTA.5 desaturase AB072976 Mus
musculus D5D mRNA for .DELTA.5 desaturase AF489588 Thraustochytrium
sp. ATCC21685 .DELTA.5 fatty acid desaturase AJ510244 Phytophthora
megasperma mRNA for .DELTA.5 fatty acid desaturase AF419297 Pythium
irregulare .DELTA.5 fatty acid desaturase AF07879 Caenorhabditis
elegans .DELTA.5 fatty acid desaturase AF067654 Mortierella alpina
.DELTA.5 fatty acid desaturase AB022097 Dictyostelium discoideum
mRNA for .DELTA.5 fatty acid desaturase AF489589.1 Thraustochytrium
sp. ATCC21685 .DELTA.4 fatty acid desaturase AY332747 Pavlova
lutheri .DELTA.4 fatty acid desaturase (des1) mRNA AAG36933
Emericella nidulans oleate .DELTA.12 desaturase AF110509,
Mortierella alpina .DELTA.12 fatty acid desaturase mRNA AB020033
AAL13300 Mortierella alpina .DELTA.12 fatty acid desaturase
AF417244 Mortierella alpina ATCC 16266 .DELTA.12 fatty acid
desaturase AF161219 Mucor rouxii .DELTA.12 desaturase mRNA X86736
Spiruline platensis .DELTA.12 desaturase AF240777 Caenorhabditis
elegans .DELTA.12 desaturase AB007640 Chlamydomonas reinhardtii
.DELTA.12 desaturase AB075526 Chlorella vulgaris .DELTA.12
desaturase AP002063 Arabidopsis thaliana microsomal .DELTA.12
desaturase NP_441622, Synechocystis sp. PCC 6803 .DELTA.15
desaturase BAA18302, BAA02924 AAL36934 Perilla frutescens .DELTA.15
desaturase AF338466 Acheta domesticus .DELTA.9 desaturase 3 mRNA
AF438199 Picea glauca desaturase .DELTA.9 (Des9) mRNA E11368
Anabaena .DELTA.9 desaturase E11367 Synechocystis .DELTA.9
desaturase D83185 Pichia angusta DNA for .DELTA.9 fatty acid
desaturase U90417 Synechococcus vulcanus .DELTA.9 acyl-lipid fatty
acid desaturase (desC) gene AF085500 Mortierella alpina .DELTA.9
desaturase mRNA AY504633 Emericella nidulans .DELTA.9 stearic acid
desaturase (sdeB) gene NM_069854 Caenorhabditis elegans essential
fatty acid desaturase, stearoyl-CoA desaturase (39.1 kD) (fat-6)
complete mRNA AF230693 Brassica oleracea cultivar Rapid Cycling
stearoyl-ACP desaturase (.DELTA.9-BO-1) gene, exon sequence
AX464731 Mortierella alpina elongase gene (also WO 02/08401)
NM_119617 Arabidopsis thaliana fatty acid elongase 1 (FAE1)
(At4g34520) mRNA NM_134255 Mus musculus ELOVL family member 5,
elongation of long chain fatty acids (yeast) (Elovl5), mRNA
NM_134383 Rattus norvegicus fatty acid elongase 2 (rELO2), mRNA
NM_134382 Rattus norvegicus fatty acid elongase 1 (rELO1), mRNA
NM_068396, Caenorhabditis elegans fatty acid ELOngation (elo-6),
NM_068392, (elo-5), (elo-2), (elo-3), and (elo-9) mRNA NM_070713,
NM_068746, NM_064685
[0140] Additionally, the patent literature provides many additional
DNA sequences of genes (and/or details concerning several of the
genes above and their methods of isolation) involved in PUFA
production. See, for example: U.S. Pat. No. 5,968,809 (.DELTA.6
desaturases); U.S. Pat. No. 5,972,664 and U.S. Pat. No. 6,075,183
(.DELTA.5 desaturases); WO 91/13972 and U.S. Pat. No. 5,057,419
(.DELTA.9 desaturases); WO 93/11245 (.DELTA.15 desaturases); WO
94/11516, U.S. Pat. No. 5,443,974 and WO 03/099216 (.DELTA.12
desaturases); U.S. 2003/0196217 A1 (.DELTA.17 desaturase); WO
02/090493 (.DELTA.4 desaturases); and, WO 00/12720 and U.S.
2002/0139974A1 (elongases). Each of these patents and applications
are herein incorporated by reference in their entirety.
[0141] As will be obvious to one skilled in the art, the particular
functionalities required to be introduced into a host organism for
production of a particular PUFA final product will depend on the
host cell (and its native PUFA profile and/or desaturase profile),
the availability of substrate and the desired end product(s). As
shown in FIG. 2, LA, GLA, DGLA, ARA, ALA, STA, ETA, EPA, DPA and
DHA may all be produced in oleaginous yeasts, by introducing
various combinations of the following PUFA enzyme functionalities:
a .DELTA.4 desaturase, a .DELTA.5 desaturase, a .DELTA.6
desaturase, a .DELTA.12 desaturase, a .DELTA.15 desaturase, a
.DELTA.17 desaturase, a .DELTA.9 desaturase and/or an elongase. One
skilled in the art will be able to identify various candidate genes
encoding each of the above enzymes, according to publicly available
literature (e.g., GenBank), the patent literature, and experimental
analysis of microorganisms having the ability to produce PUFAs. The
sequences may be derived from any source, e.g., isolated from a
natural source (from bacteria, algae, fungi, plants, animals,
etc.), produced via a semi-synthetic route or synthesized de novo.
In some embodiments, manipulation of genes endogenous to the host
is preferred; for other purposes, it is necessary to introduce
heterologous genes.
[0142] Although the particular source of the desaturase and
elongase genes introduced into the host is not critical to the
invention, considerations for choosing a specific polypeptide
having desaturase or elongase activity include: 1.) the substrate
specificity of the polypeptide; 2.) whether the polypeptide or a
component thereof is a rate-limiting enzyme; 3.) whether the
desaturase or elongase is essential for synthesis of a desired
PUFA; and/or 4.) co-factors required by the polypeptide. The
expressed polypeptide preferably has parameters compatible with the
biochemical environment of its location in the host cell. For
example, the polypeptide may have to compete for substrate with
other enzymes in the host cell. Analyses of the KM and specific
activity of the polypeptide are therefore considered in determining
the suitability of a given polypeptide for modifying PUFA
production in a given host cell. The polypeptide used in a
particular host cell is one that can function under the biochemical
conditions present in the intended host cell but otherwise can be
any polypeptide having desaturase or elongase activity capable of
modifying the desired PUFA.
[0143] Endogenous PUFA Genes
[0144] In some cases, the host organism in which it is desirable to
produce PUFAs will possess endogenous genes encoding some PUFA
biosynthetic pathway enzymes. For example, oleaginous yeast can
typically produce 18:2 fatty acids (and some have the additional
capability of synthesizing 18:3 fatty acids); thus, oleaginous
yeast typically possess native .DELTA.12 desaturase activity and
may also have .DELTA.15 desaturases. In some embodiments,
therefore, expression of the native desaturase enzyme is preferred
over a heterologous (or "foreign") enzyme since: 1.) the native
enzyme is optimized for interaction with other enzymes and proteins
within the cell; and 2.) heterologous genes are unlikely to share
the same codon preference in the host organism. Additionally,
advantages are incurred when the sequence of the native gene is
known, as it permits facile disruption of the endogenous gene by
targeted disruption.
[0145] Heterologous PUFA Genes
[0146] In many instances, the appropriate desaturases and elongases
are not present in the host organism of choice to enable production
of the desired PUFA products. Thus, it is necessary to introduce
heterologous genes.
[0147] For the purposes of the present invention herein, it was
desirable to demonstrate an example of the introduction of an
.omega.-3 and/or .omega.-6 biosynthetic pathway into an oleaginous
host organism; and thus, a Mortierella alpina .DELTA.5 desaturase,
M. alpina .DELTA.6 desaturase, Saprolegnia diclina .DELTA.17
desaturase and M. alpina elongase were introduced into Yarrowia
lipolytica. However, the specific enzymes (and genes encoding those
enzymes) introduced into the host organism and the specific PUFAs
produced are by no means limiting to the invention herein.
[0148] If one desired to produce EPA, as demonstrated herein, it
will be obvious to one skilled in the art that numerous other genes
derived from different sources would be suitable to introduce
.DELTA.5 desaturase, .DELTA.6 desaturase, .DELTA.17 desaturase and
elongase activity into the preferred microbial host. Thus, in one
embodiment of the present invention, other DNAs which are
substantially identical to the M. alpina .DELTA.6 desaturase,
.DELTA.5 desaturase and high-affinity PUFA elongase and the S.
diclina .DELTA.17 desaturase also can be used for production of
.omega.-6 and/or .omega.-3 fatty acids (e.g., EPA) in oleaginous
yeast. By "substantially identical" is intended an amino acid
sequence or nucleic acid sequence exhibiting in order of increasing
preference at least 80%, 90% or 95% homology to the selected
polypeptides, or nucleic acid sequences encoding the amino acid
sequence. For polypeptides, the length of comparison sequences
generally is at least 16 amino acids, preferably at least 20 amino
acids or most preferably 35 amino acids. For nucleic acids, the
length of comparison sequences generally is at least 50
nucleotides, preferably at least 60 nucleotides, more preferably at
least 75 nucleotides, and most preferably 110 nucleotides.
[0149] Homology typically is measured using sequence analysis
software, wherein 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, 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. In general, such
computer software matches similar sequences by assigning degrees of
homology to various substitutions, deletions, and other
modifications.
[0150] Additionally it will be appreciated by one of skill in the
art that polypeptides may have amino acids conservatively
substituted in a manner such that the function of the polypeptide
is not altered or compromised. Polypeptides having the desaturase
and elongase activities as described herein and possessing such
conservative substitutions are considered within the scope of the
present invention. Conservative substitutions typically include
substitutions within the following groups: 1.) glycine and alanine;
2.) valine, isoleucine and leucine; 3.) aspartic acid, glutamic
acid, asparagine and glutamine; 4.) serine and threonine; 5.)
lysine and arginine; and 6.) phenylalanine and tyrosine.
Substitutions may also be made on the basis of conserved
hydrophobicity or hydrophilicity (Kyte and Doolittle, J. Mol. Biol.
157:105-132 (1982)), or on the basis of the ability to assume
similar polypeptide secondary structure (Chou and Fasman, Adv.
Enzymol. 47:45-148 (1978)).
[0151] In alternate embodiments of the invention, other DNAs which,
although not substantially identical to the M. alpina .DELTA.6
desaturase, .DELTA.5 desaturase and high-affinity PUFA elongase and
the S. diclina .DELTA.17 desaturase, also can be used for the
purposes herein (e.g., for production of .omega.-3 and/or .omega.-6
PUFAs such as ARA and EPA). For example, DNA sequences encoding
.DELTA.6 desaturase polypeptides that would be useful for
introduction into an oleaginous yeast according to the teachings of
the present invention may be obtained from microorganisms having an
ability to produce GLA or STA. Such microorganisms include, for
example, those belonging to the genera Mortierella, Conidiobolus,
Pythium, Phytophathora, Penicillium, Porphyridium, Coidosporium,
Mucor, Fusarium, Aspergillus, Rhodotorula and Entomophthora. Within
the genus Porphyridium, of particular interest is P. cruentum.
Within the genus Mortierella, of particular interest are M.
elongata, M. exigua, M. hygrophila, M. ramanniana var. angulispora
and M. alpina. Within the genus Mucor, of particular interest are
M. circinelloides and M. javanicus.
[0152] Alternatively, a related desaturase that is not
substantially identical to the M. alpina .DELTA.6 desaturase, but
which can desaturate a fatty acid molecule at carbon 6 from the
carboxyl end of the molecule would also useful in the present
invention as a .DELTA.6 desaturase, assuming the desaturase can
still effectively convert LA to GLA and/or ALA to STA. As such,
related desaturases and elongases can be identified by their
ability of function substantially the same as the desaturases and
elongase disclosed herein.
[0153] In summary, genes encoding PUFA biosynthetic pathway enzymes
suitable for the purposes herein may be isolated from a variety of
sources. Desaturases for the purposes herein are characterized by
the ability to: 1.) 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 catalyze the conversion of ARA to EPA and DGLA
to ETA (.DELTA.17 desaturases); 2.) catalyze the conversion of LA
to GLA and/or ALA to STA (.DELTA.6 desaturases); 3.) catalyze the
conversion of DGLA to ARA and/or ETA to EPA (.DELTA.5 desaturases);
4.) catalyze the conversion of DPA to DHA (.DELTA.4 desaturases);
5.) catalyze the conversion of oleic acid to LA (.DELTA.12
desaturases); 6.) catalyze the conversion of LA to ALA (.DELTA.15
desaturases); and/or 7.) catalyze the conversion of palmitate to
palmitoleic acid and/or stearate to oleic acid (.DELTA.9
desaturases). In like manner, suitable elongases for the purposes
herein are not limited to those from a specific source; instead,
the enzymes having use for the purposes herein are characterized by
their ability to elongate a fatty acid carbon chain by 2 carbons
relative to the substrate the elongase acts upon, to thereby
produce a mono- or polyunsaturated fatty acid.
Optimization of Omega Fatty Acid Genes for Expression in Particular
Organisms
[0154] Although the particular source of a PUFA desaturase or
elongase is not critical in the invention herein, it will be
obvious to one of skill in the art that heterologous genes will be
expressed with variable efficiencies in an alternate host. Thus,
.omega.-3 and/or .omega.-6 PUFA production may be optimized by
selection of a particular desaturase or elongase whose level of
expression in a heterologous host is preferred relative to the
expression of an alternate desaturase or elongase in the host
organism of interest. Furthermore, it may be desirable to modify
the expression of particular PUFA biosynthetic pathway enzymes to
achieve optimal conversion efficiency of each, according to the
specific PUFA product composition of interest. A variety of genetic
engineering techniques are available to optimize expression of a
particular enzyme. Two such techniques include codon optimization
and gene mutation, as described below. Genes produced by. e.g.,
either of these two methods, having desaturase and/or elongase
activity(s) would be useful in the invention herein for synthesis
of .omega.-3 and/or .omega.-6 PUFAs.
[0155] Codon Optimization
[0156] As will be appreciated by one skilled in the art, it is
frequently useful to modify a portion of the codons encoding a
particular polypeptide that is to be expressed in a foreign host,
such that the modified polypeptide uses codons that are preferred
by the alternate host. Use of host-preferred codons can
substantially enhance the expression of the foreign gene encoding
the polypeptide.
[0157] In general, host-preferred codons can be determined within a
particular host species of interest by examining codon usage in
proteins (preferably those expressed in the largest amount) and
determining which codons are used with highest frequency. Then, the
coding sequence for a polypeptide of interest having desaturase or
elongase activity can be synthesized in whole or in part using the
codons preferred in the host species. All (or portions) of the DNA
also can be synthesized to remove any destabilizing sequences or
regions of secondary structure that would be present in the
transcribed mRNA. All (or portions) of the DNA also can be
synthesized to alter the base composition to one more preferable in
the desired host cell.
[0158] In the present invention, it was desirable to modify a
portion of the codons encoding the polypeptide having .DELTA.17
desaturase activity, to enhance the expression of the gene in
Yarrowia lipolytica. The nucleic acid sequence of the native gene
(e.g., the Saprolegnia diclina .DELTA.17 desaturase) was modified
to employ host-preferred codons. The skilled artisan will
appreciate that this optimization method will be equally applicable
to other genes in the .omega.-3/.omega.-6 fatty acids biosynthetic
pathway (see for example, co-pending U.S. Provisional Application
No. 60/468,718, herein incorporated entirely by reference).
Furthermore, modulation of the S. diclina .DELTA.17 desaturase is
only exemplary.
[0159] Gene Mutation
[0160] Methods for synthesizing sequences and bringing sequences
together are well established in the literature. For example, in
vitro mutagenesis and selection, site-directed mutagenesis, error
prone PCR (Melnikov et al., Nucleic Acids Research, 27(4):1056-1062
(Feb. 15, 1999)), "gene shuffling" or other means can be employed
to obtain mutations of naturally occurring desaturase or elongase
genes. This would permit production of a polypeptide having
desaturase or elongase activity, respectively, in vivo with more
desirable physical and kinetic parameters for function in the host
cell such as a longer half-life or a higher rate of production of a
desired PUFA.
[0161] If desired, the regions of a polypeptide of interest (i.e.,
a desaturase or an elongase) important for enzymatic activity can
be determined through routine mutagenesis, expression of the
resulting mutant polypeptides and determination of their
activities. Mutants may include deletions, insertions and point
mutations, or combinations thereof. A typical functional analysis
begins with deletion mutagenesis to determine the N- and C-terminal
limits of the protein necessary for function, and then internal
deletions, insertions or point mutants are made to further
determine regions necessary for function. Other techniques such as
cassette mutagenesis or total synthesis also can be used. Deletion
mutagenesis is accomplished, for example, by using exonucleases to
sequentially remove the 5' or 3' coding regions. Kits are available
for such techniques. After deletion, the coding region is completed
by ligating oligonucleotides containing start or stop codons to the
deleted coding region after the 5' or 3' deletion, respectively.
Alternatively, oligonucleotides encoding start or stop codons are
inserted into the coding region by a variety of methods including
site-directed mutagenesis, mutagenic PCR or by ligation onto DNA
digested at existing restriction sites. Internal deletions can
similarly be made through a variety of methods including the use of
existing restriction sites in the DNA, by use of mutagenic primers
via site-directed mutagenesis or mutagenic PCR. Insertions are made
through methods such as linker-scanning mutagenesis, site-directed
mutagenesis or mutagenic PCR, while point mutations are made
through techniques such as site-directed mutagenesis or mutagenic
PCR.
[0162] Chemical mutagenesis also can be used for identifying
regions of a desaturase or elongase polypeptide important for
activity. A mutated construct is expressed, and the ability of the
resulting altered protein to function as a desaturase or elongase
is assayed. Such structure-function analysis can determine which
regions may be deleted, which regions tolerate insertions, and
which point mutations allow the mutant protein to function in
substantially the same way as the native desaturase or native
elongase. All such mutant proteins and nucleotide sequences
encoding them that are derived from the codon-optimized gene
described herein are within the scope of the present invention.
[0163] In the present invention, it was desirable to modify a
portion of the codons encoding the polypeptide having .DELTA.17
desaturase activity, to enhance the expression of the gene in
Yarrowia lipolytica. The nucleic acid sequence of the native gene
(e.g., the S. diclina .DELTA.17 desaturase) was modified to employ
host preferred codons. The skilled artisan will appreciate that
these optimization methods will be equally applicable to other
genes in the .omega.-3/.omega.-6 fatty acids biosynthetic pathway
and that modulation of the S. diclina .DELTA.17 desaturase and M.
alpina .DELTA.6 desaturase, .DELTA.5 desaturase are only
exemplary.
Microbial Production of .omega.-3 and/or .omega.-6 Fatty Acids
[0164] Microbial production of .omega.-3 and/or .omega.-6 fatty
acids has several advantages over purification from natural sources
such as fish or plants. For example: [0165] 1.) Many microbes are
known with greatly simplified oil compositions compared with those
of higher organisms, making purification of desired components
easier; [0166] 2.) Microbial production is not subject to
fluctuations caused by external variables, such as weather and food
supply; [0167] 3.) Microbially produced oil is substantially free
of contamination by environmental pollutants; [0168] 4.) Microbes
can provide PUFAs in particular forms which may have specific uses;
and [0169] 5.) Microbial oil production can be manipulated by
controlling culture conditions, notably by providing particular
substrates for microbially expressed enzymes, or by addition of
compounds/genetic engineering to suppress undesired biochemical
pathways. In addition to these advantages, production of .omega.-3
and/or .omega.-6 fatty acids from recombinant microbes provides the
ability to alter the naturally occurring microbial fatty acid
profile by providing new biosynthetic pathways in the host or by
suppressing undesired pathways, thereby increasing levels of
desired PUFAs, or conjugated forms thereof, and decreasing levels
of undesired PUFAs. For example, it is possible to modify the ratio
of .omega.-3 to .omega.-6 fatty acids so produced, produce either
.omega.-3 or .omega.-6 fatty acids exclusively while eliminating
production of the alternate omega fatty acid, or engineer
production of a specific PUFA without significant accumulation of
other PUFA downstream or upstream products.
[0170] Expression Systems, Cassettes and Vectors
[0171] The genes and gene products described herein may be produced
in heterologous microbial host cells, particularly in the cells of
oleaginous yeasts (e.g., Yarrowia lipolytica). Expression in
recombinant microbial hosts may be useful for the production of
various PUFA pathway intermediates, or for the modulation of PUFA
pathways already existing in the host for the synthesis of new
products heretofore not possible using the host.
[0172] Microbial expression systems and expression vectors
containing regulatory sequences that direct high level expression
of foreign proteins are well known to those skilled in the art. Any
of these could be used to construct chimeric genes for production
of any of the gene products of the preferred desaturase and/or
elongase sequences. These chimeric genes could then be introduced
into appropriate microorganisms via transformation to provide
high-level expression of the encoded enzymes.
[0173] Accordingly, it is expected that introduction of chimeric
genes encoding a PUFA biosynthetic pathway (e.g., the .DELTA.5
desaturase, .DELTA.6 desaturase, .DELTA.17 desaturase and elongase
described herein), under the control of the appropriate promoters
will result in increased production of .omega.-3 and/or .omega.-6
fatty acids. It is contemplated that it will be useful to express
various combinations of these PUFA desaturase and elongase genes
together in a host microorganism. It will be obvious to one skilled
in the art that the particular genes included within a particular
expression cassette(s) will depend on the host cell, its ability to
synthesize PUFAs using native desaturases and elongases, the
availability of substrate and the desired end product(s). For
example, it may be desirable for an expression cassette to be
constructed comprising genes encoding one or more of the following
enzymatic activities: a .DELTA.4 desaturase, a .DELTA.5 desaturase,
a .DELTA.6 desaturase, a .DELTA.12 desaturase, a .DELTA.15
desaturase, a .DELTA.17 desaturase, a .DELTA.9 desaturase and/or an
elongase. As such, the present invention encompasses a method of
producing PUFAs comprising exposing a fatty acid substrate to the
PUFA enzyme(s) described herein, such that the substrate is
converted to the desired fatty acid product. Thus, each PUFA gene
and corresponding enzyme product described herein (e.g., a
wildtype, codon-optimized, synthetic and/or mutant enzyme having
appropriate desaturase or elongase activity) can be used directly
or indirectly for the production of PUFAs. Direct production of
PUFAs occurs wherein the fatty acid substrate is converted directly
into the desired fatty acid product without any intermediate steps
or pathway intermediates. For example, production of ARA would
occur in a host cell which produces or which is provided DLGA, by
adding or introducing into said cell an expression cassette that
provides .DELTA.5 desaturase activity.
[0174] In contrast, multiple genes encoding the PUFA biosynthetic
pathway may be used in combination, such that a series of reactions
occur to produce a desired PUFA. For example, expression
cassette(s) encoding elongase, .DELTA.5 desaturase, .DELTA.17
desaturase and .DELTA.4 desaturase activity would enable a host
cell that naturally produces GLA, to instead produce DHA (such that
GLA is converted to DGLA by an elongase; DGLA may then be converted
to ARA by a .DELTA.5 desaturase; ARA is then converted to EPA by a
.DELTA.17 desaturase, which may in turn be converted to DPA by an
elongase; and DPA would be converted to DHA by a .DELTA.4
desaturase). In a preferred embodiment, wherein the host cell is an
oleaginous yeast, expression cassettes encoding each of the enzymes
necessary for PUFA biosynthesis will need to be introduced into the
organism, since naturally produced PUFAs in these organisms are
limited to 18:2 fatty acids (i.e., LA), and less commonly, 18:3
fatty acids (i.e., ALA). Alternatively, substrate feeding may be
required.
[0175] 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.
[0176] Initiation control regions or promoters which are useful to
drive expression of desaturase and/or elongase ORFs in the desired
host cell are numerous and familiar to those skilled in the art.
Virtually any promoter capable of directing expression of these
genes in the selected host cell is suitable for the present
invention. Expression in a host cell can be accomplished in a
transient or stable fashion. Transient expression can be
accomplished by inducing the activity of a regulatable promoter
operably linked to the gene of interest. Stable expression can be
achieved by the use of a constitutive promoter operably linked to
the gene of interest. As an example, when the host cell is yeast,
transcriptional and translational regions functional in yeast cells
are provided, particularly from the host species. The
transcriptional initiation regulatory regions can be obtained, for
example, from: 1.) genes in the glycolytic pathway, such as alcohol
dehydrogenase, glyceraldehyde-3-phosphate-dehydrogenase (see U.S.
Patent Application No. 60/482,263, incorporated herein by
reference), phosphoglycerate mutase (see U.S. Patent Application
No. 60/482,263, incorporated herein by reference),
fructose-bisphosphate aldolase (see U.S. Patent Application No.
60/519,971, incorporated herein by reference),
phosphoglucose-isomerase, phosphoglycerate kinase, etc.; or, 2.)
regulatable genes such as acid phosphatase, lactase,
metallothionein, glucoamylase, the translation elongation factor
EF1-.alpha. (TEF) protein (U.S. Pat. No. 6,265,185), ribosomal
protein S7 (U.S. Pat. No. 6,265,185), etc. Any one of a number of
regulatory sequences can be used, depending upon whether
constitutive or induced transcription is desired, the efficiency of
the promoter in expressing the ORF of interest, the ease of
construction and the like.
[0177] 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 to
obtain optimal gene expression. For expression in yeast, this can
be done by site-directed mutagenesis of an inefficiently expressed
gene by fusing it in-frame to an endogenous yeast gene, preferably
a highly expressed gene. Alternatively, as demonstrated in the
invention herein in Yarrowia lipolytica, one can determine the
consensus translation initiation sequence in the host and engineer
this sequence into heterologous genes for their optimal expression
in the host of interest.
[0178] 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.
[0179] As one of skill in the art is aware, merely inserting a gene
into a cloning vector does not ensure that it will be successfully
expressed at the level needed. In response to the need for a high
expression rate, many specialized expression vectors have been
created by manipulating a number of different genetic elements that
control aspects of transcription, translation, protein stability,
oxygen limitation and secretion from the host cell. More
specifically, some of the molecular features that have been
manipulated to control gene expression include: 1.) the nature of
the relevant transcriptional promoter and terminator sequences; 2.)
the number of copies of the cloned gene and whether the gene is
plasmid-borne or integrated into the genome of the host cell; 3.)
the final cellular location of the synthesized foreign protein; 4.)
the efficiency of translation in the host organism; 5.) the
intrinsic stability of the cloned gene protein within the host
cell; and 6.) the codon usage within the cloned gene, such that its
frequency approaches the frequency of preferred codon usage of the
host cell. Each of these types of modifications are encompassed in
the present invention, as means to further optimize expression of
the PUFA biosynthetic pathway enzymes.
[0180] Transformation of Microbial Hosts
[0181] Once the DNA encoding a desaturase or elongase polypeptide
suitable for expression in an oleaginous yeast has been obtained,
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 within 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.
[0182] 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.
[0183] Constructs comprising the gene 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,
bolistic impact, electroporation, microinjection, or any other
method that introduces the gene of interest into the host cell.
More specific teachings applicable for oleaginous yeasts (i.e.,
Yarrowia lipolytica) include U.S. Pat. Nos. 4,880,741 and 5,071,764
and Chen, D. C. et al. (Appl Microbiol Biotechnol. 48(2):232-235
(1997)).
[0184] For convenience, a host cell that has been manipulated by
any method to take up a DNA sequence (e.g., an expression cassette)
will be referred to as "transformed" or "recombinant" herein. The
transformed host will have at least one copy of the expression
construct and may have two or more, depending upon whether the gene
is integrated into the genome, amplified or is present on an
extrachromosomal element having multiple copy numbers. The
transformed host cell can be identified by selection for a marker
contained on the introduced construct. Alternatively, a separate
marker construct may be co-transformed with the desired construct,
as many transformation techniques introduce many DNA molecules into
host cells. Typically, transformed hosts are selected for their
ability to grow on selective media. Selective media may incorporate
an antibiotic or lack a factor necessary for growth of the
untransformed host, such as a nutrient or growth factor. An
introduced marker gene may confer antibiotic resistance, or encode
an essential growth factor or enzyme, thereby permitting growth on
selective media when expressed in the transformed host. Selection
of a transformed host can also occur when the expressed marker
protein can be detected, either directly or indirectly. The marker
protein may be expressed alone or as a fusion to another protein.
The marker protein can be detected by: 1.) its enzymatic activity
(e.g., .beta.-galactosidase can convert the substrate X-gal
[5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside] to a
colored product; luciferase can convert luciferin to a
light-emitting product); or 2.) its light-producing or modifying
characteristics (e.g., the green fluorescent protein of Aequorea
victoria fluoresces when illuminated with blue light).
Alternatively, antibodies can be used to detect the marker protein
or a molecular tag on, for example, a protein of interest. Cells
expressing the marker protein or tag can be selected, for example,
visually, or by techniques such as FACS or panning using
antibodies. For selection of yeast transformants, any marker that
functions in yeast may be used. Preferred for use herein are
resistance to kanamycin, hygromycin and the amino glycoside G418,
as well as ability to grow on media lacking uracil or leucine.
[0185] Following transformation, substrates suitable for the
recombinantly expressed desaturases and/or elongases (and
optionally other PUFA enzymes that are expressed within the host
cell) may be produced by the host either naturally or
transgenically, or they may be provided exogenously.
Metabolic Engineering of .omega.-3 and/or .omega.-6 Fatty Acid
Biosynthesis in Microbes
[0186] Methods for manipulating biochemical pathways are well known
to those skilled in the art; and, it is expected that numerous
manipulations will be possible to maximize .omega.-3 and/or
.omega.-6 fatty acid biosynthesis in oleaginous yeasts, and
particularly, in Yarrowia lipolytica. This may require metabolic
engineering directly within the PUFA biosynthetic pathway or
additional manipulation of pathways that contribute carbon to the
PUFA biosynthetic pathway.
[0187] In the case of manipulations within the PUFA biosynthetic
pathway, it may be desirable to increase the production of LA to
enable increased production of .omega.-6 and/or .omega.-3 fatty
acids. This may be accomplished by introducing and/or amplifying
genes encoding .DELTA.9 and/or .DELTA.12 desaturases.
[0188] To maximize production of .omega.-6 unsaturated fatty acids,
such as ARA, it is well known to one skilled in the art that
production is favored in a host microorganism that is substantially
free of ALA. Thus, preferably, the host is selected or obtained by
removing or inhibiting .DELTA.15 or .omega.-3 type desaturase
activity that permits conversion of LA to ALA. The endogenous
desaturase activity can be reduced or eliminated by, for example:
1.) providing a cassette for transcription of antisense sequences
to the .DELTA.15 desaturase transcription product; 2.) disrupting
the .DELTA.15 desaturase gene through insertion, substitution
and/or deletion of all or part of the target gene; or 3.) using a
host cell which naturally has [or has been mutated to have] low or
no .DELTA.15 desaturase activity. Inhibition of undesired
desaturase pathways can also be accomplished through the use of
specific desaturase inhibitors such as those described in U.S. Pat.
No. 4,778,630.
[0189] Alternatively, it may be desirable to maximize production of
.omega.-3 fatty acids (and minimize synthesis of .omega.-6 fatty
acids). Thus, one could utilize a host microorganism wherein the
.DELTA.12 desaturase activity that permits conversion of oleic acid
to LA is removed or inhibited, using any of the means described
above (see also, for example, co-pending U.S. Provisional
Application No. 60/484,209, herein incorporated entirely by
reference). Subsequently, appropriate expression cassettes would be
introduced into the host, along with appropriate substrates (e.g.,
ALA) for conversion to .omega.-3 fatty acid derivatives of ALA
(e.g., STA, ETA, EPA, DPA, DHA).
[0190] Beyond the immediate PUFA biosynthetic pathway, it is
expected that manipulation of several other enzymatic pathways
leading to the biosynthesis of precursor fatty acids may contribute
to the overall net biosynthesis of specific PUFAs. Identification
and manipulation of these related pathways will be useful in the
future.
[0191] Techniques to Up-Regulate Desirable Biosynthetic
Pathways
[0192] Additional copies of desaturase and elongase genes may be
introduced into the host to increase the output of .omega.-3 and/or
.omega.-6 fatty acid biosynthetic pathways. Expression of the
desaturase or elongase genes also can be increased at the
transcriptional level through the use of a stronger promoter
(either regulated or constitutive) to cause increased expression,
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). Yet another approach to increase
expression of the desaturase or elongase genes, as demonstrated in
the instant invention, is to increase the translational efficiency
of the encoded mRNAs by replacement of codons in the native gene
with those for optimal gene expression in the selected host
microorganism.
[0193] Techniques to Down-Regulate Undesirable Biosynthetic
Pathways
[0194] Conversely, biochemical pathways competing with the
.omega.-3 and/or .omega.-6 fatty acid biosynthetic pathways for
energy or carbon, or native PUFA biosynthetic pathway enzymes that
interfere with production of a particular PUFA end-product, may be
eliminated by gene disruption or down-regulated by other means
(e.g., antisense mRNA). For gene disruption, a foreign DNA fragment
(typically a selectable marker gene) is inserted into the
structural gene to be disrupted in order to interrupt its coding
sequence and thereby functionally inactivate the gene.
Transformation of the disruption cassette into the host cell
results in replacement of the functional native gene by homologous
recombination with the non-functional disrupted gene (see, for
example: Hamilton et al. J. Bacteriol. 171:4617-4622 (1989); Balbas
et al. Gene 136:211-213 (1993); Gueldener et al. Nucleic Acids Res.
24:2519-2524 (1996); and Smith et al. Methods Mol. Cell. Biol.
5:270-277 (1996)).
[0195] Antisense technology is another method of down-regulating
genes when the sequence of the target gene is known. To accomplish
this, a nucleic acid segment from the desired gene is cloned and
operably linked to a promoter such that the anti-sense strand of
RNA will be transcribed. This construct is then introduced into the
host cell and the antisense strand of RNA is produced. Antisense
RNA inhibits gene expression by preventing the accumulation of mRNA
that encodes the protein of interest. The person skilled in the art
will know that special considerations are associated with the use
of antisense technologies in order to reduce expression of
particular genes. For example, the proper level of expression of
antisense genes may require the use of different chimeric genes
utilizing different regulatory elements known to the skilled
artisan.
[0196] Although targeted gene disruption and antisense technology
offer effective means of down-regulating genes where the sequence
is known, other less specific methodologies have been developed
that are not sequence-based. For example, cells may be exposed to
UV radiation and then screened for the desired phenotype.
Mutagenesis with chemical agents is also effective for generating
mutants and commonly used substances include chemicals that affect
nonreplicating DNA (e.g., HNO.sub.2 and NH.sub.2OH), as well as
agents that affect replicating DNA (e.g., acridine dyes, notable
for causing frameshift mutations). Specific methods for creating
mutants using radiation or chemical agents are well documented in
the art. See, for example: Thomas D. Brock in Biotechnology: A
Textbook of Industrial Microbiology, 2.sup.nd ed. (1989) Sinauer
Associates: Sunderland, Mass.; or Deshpande, Mukund V., Appl.
Biochem. Biotechnol., 36:227 (1992).
[0197] Another non-specific method of gene disruption is the use of
transposable elements or transposons. Transposons are genetic
elements that insert randomly into DNA but can be later retrieved
on the basis of sequence to determine where the insertion has
occurred. Both in vivo and in vitro transposition methods are
known. Both methods involve the use of a transposable element in
combination with a transposase enzyme. When the transposable
element or transposon is contacted with a nucleic acid fragment in
the presence of the transposase, the transposable element will
randomly insert into the nucleic acid fragment. The technique is
useful for random mutagenesis and for gene isolation, since the
disrupted gene may be identified on the basis of the sequence of
the transposable element. Kits for in vitro transposition are
commercially available [see, for example: 1.) The Primer Island
Transposition Kit, available from Perkin Elmer Applied Biosystems,
Branchburg, N.J., based upon the yeast Ty1 element; 2.) The Genome
Priming System, available from New England Biolabs, Beverly, Mass.,
based upon the bacterial transposon Tn7; and 3.) the EZ::TN
Transposon Insertion Systems, available from Epicentre
Technologies, Madison, Wis., based upon the Tn5 bacterial
transposable element].
[0198] Within the context of the present invention, it may be
useful to modulate the expression of the fatty acid biosynthetic
pathway by any one of the methods described above. For example, the
present invention provides methods whereby genes encoding key
enzymes in the biosynthetic pathways are introduced into oleaginous
yeasts for the production of .omega.-3 and/or .omega.-6 fatty
acids. These genes encode one or more of the following: .DELTA.6
desaturase, .DELTA.5 desaturase, .DELTA.12 desaturase, .DELTA.15
desaturase, .DELTA.4 desaturase, .DELTA.17 desaturase, .DELTA.9
desaturase and PUFA elongase. It will be particularly useful to
express these genes in oleaginous yeasts that do not naturally
possess .omega.-3 and/or .omega.-6 fatty acid biosynthetic pathways
and coordinate the expression of these genes, to maximize
production of preferred PUFA products using various means for
metabolic engineering of the host organism.
Preferred Microbial Hosts for Recombinant Production of .omega.-3
and/or .omega.-6 Fatty Acids
[0199] Host cells for production of omega fatty acids may include
microbial hosts that grow on a variety of feedstocks, including
simple or complex carbohydrates, organic acids and alcohols, and/or
hydrocarbons over a wide range of temperature and pH values.
[0200] Preferred microbial hosts, however, are oleaginous yeasts.
These organisms 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
yeasts 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).
[0201] 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)).
[0202] Historically, various strains of Y. lipolytica have been
used for the manufacture and production of: isocitrate lyase
(DD259637); lipases (SU1454852, WO2001083773, DD279267);
polyhydroxyalkanoates (WO2001088144); citric acid (RU2096461,
RU2090611, DD285372, DD285370, DD275480, DD227448, PL160027);
erythritol (EP770683); 2-oxoglutaric acid (DD267999);
.gamma.-decalactone (U.S. Pat. No. 6,451,565, FR2734843);
.gamma.-dodecalatone (EP578388); and pyruvic acid (JP09252790).
Fermentation Processes for PUFA Production
[0203] The transformed microbial host cell is grown under
conditions that optimize desaturase and elongase activities and
produce the greatest and the most economical yield of the preferred
PUFAs. In general, media conditions that may be optimized include
the type and amount of carbon source, the type and amount of
nitrogen source, the carbon-to-nitrogen ratio, the oxygen level,
growth temperature, pH, length of the biomass production phase,
length of the oil accumulation 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.)).
[0204] 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 and 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.
[0205] Nitrogen may be supplied from an inorganic (e.g.,
(NH.sub.4).sub.2SO.sub.4) or organic source (e.g., urea or
glutamate). In addition to appropriate carbon and nitrogen sources,
the fermentation media must also contain suitable minerals, salts,
cofactors, buffers, vitamins and other components known to those
skilled in the art suitable for the growth of the microorganism and
promotion of the enzymatic pathways necessary for PUFA production.
Particular attention is given to several metal ions (e.g.,
Mn.sup.+2, Co.sup.+2, Zn.sup.+2, Mg.sup.+2) that promote synthesis
of lipids and PUFAs (Nakahara, T. et al., Ind. Appl. Single Cell
Oils, D. J. Kyle and R. Colin, eds. pp 61-97 (1992)).
[0206] Preferred growth media in the present invention are common
commercially prepared media, such as Yeast Nitrogen Base (DIFCO
Laboratories, Detroit, Mich.). Other defined or synthetic growth
media may also be used and the appropriate medium for growth of the
particular microorganism will be known by one skilled in the art of
microbiology or fermentation science. A suitable pH range for the
fermentation is typically between about pH 4.0 to pH 8.0, wherein
pH 5.5 to pH 7.0 is preferred as the range for the initial growth
conditions. The fermentation may be conducted under aerobic or
anaerobic conditions, wherein microaerobic conditions are
preferred.
[0207] Typically, accumulation of high levels of PUFAs in
oleaginous yeast cells requires a two-stage process, since the
metabolic state must be "balanced" between growth and
synthesis/storage of fats. Thus, most preferably, a two-stage
fermentation process is necessary for the production of PUFAs in
oleaginous yeast. In this approach, the first stage of the
fermentation is dedicated to the generation and accumulation of
cell mass and is characterized by rapid cell growth and cell
division. In the second stage of the fermentation, it is preferable
to establish conditions of nitrogen deprivation in the culture to
promote high levels of lipid accumulation. The effect of this
nitrogen deprivation is to reduce the effective concentration of
AMP in the cells, thereby reducing the activity of the
NAD-dependent isocitrate dehydrogenase of mitochondria. When this
occurs, citric acid will accumulate, thus forming abundant pools of
acetyl-CoA in the cytoplasm and priming fatty acid synthesis. Thus,
this phase is characterized by the cessation of cell division
followed by the synthesis of fatty acids and accumulation of
oil.
[0208] 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.
[0209] It is contemplated that a variety of fermentation process
designs may be applied, where commercial production of omega fatty
acids using recombinant expression of desaturase and/or elongase
genes is desired. For example, commercial production of PUFAs from
a recombinant microbial host may be produced by a batch, fed-batch
or continuous fermentation process.
[0210] A batch fermentation process is a closed system wherein the
media composition is fixed at the beginning of the process and not
subject to further additions beyond those required for maintenance
of pH and oxygen level during the process. Thus, at the beginning
of the culturing process the media is inoculated with the desired
organism and growth or metabolic activity is permitted to occur
without adding additional sources (i.e., carbon and nitrogen
sources) to the medium. In batch processes the metabolite and
biomass compositions of the system change constantly up to the time
the culture is terminated. In a typical batch process, cells
proceed through a static lag phase to a high-growth log phase and
finally to a stationary phase, wherein the growth rate is
diminished or halted. Left untreated, cells in the stationary phase
will eventually die. A variation of the standard batch process is
the fed-batch process, wherein the carbon source is continually
added to the fermentor over the course of the fermentation process.
A fed-batch process is also suitable in the present invention.
Fed-batch processes are useful when catabolite repression is apt to
inhibit the metabolism of the cells or where it is desirable to
have limited amounts of carbon source in the media at any one time.
Measurement of the carbon source concentration in fed-batch systems
is difficult and therefore may be estimated on the basis of the
changes of measurable factors such as pH, dissolved oxygen and the
partial pressure of waste gases (e.g., CO.sub.2). Batch and
fed-batch culturing methods are common and well known in the art
and examples may be found in Thomas D. Brock in Biotechnology: A
Textbook of Industrial Microbiology, 2.sup.nd ed., (1989) Sinauer
Associates Sunderland, Mass.; or Deshpande, Mukund V., Appl.
Biochem. Biotechnol., 36:227 (1992), herein incorporated by
reference.
[0211] Commercial production of omega fatty acids using recombinant
expression of desaturase and/or elongase genes may also be
accomplished by a continuous fermentation process wherein a defined
media is continuously added to a bioreactor while an equal amount
of culture volume is removed simultaneously for product recovery.
Continuous cultures generally maintain the cells in the log phase
of growth at a constant cell density. Continuous or semi-continuous
culture methods permit the modulation of one factor or any number
of factors that affect cell growth or end product concentration.
For example, one approach may limit the carbon source and allow all
other parameters to moderate metabolism. In other systems, a number
of factors affecting growth may be altered continuously while the
cell concentration, measured by media turbidity, is kept constant.
Continuous systems strive to maintain steady state growth and thus
the cell growth rate must be balanced against cell loss due to
media being drawn off the culture. Methods of modulating nutrients
and growth factors for continuous culture processes, as well as
techniques for maximizing the rate of product formation, are well
known in the art of industrial microbiology and a variety of
methods are detailed by Brock, supra.
Purification of PUFAs
[0212] The PUFAs may be found in the host microorganism 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)).
[0213] In general, means for the purification of PUFAs may include
extraction with organic solvents, sonication, supercritical fluid
extraction (e.g., using carbon dioxide), saponification and
physical means such as presses, or combinations thereof. Of
particular interest is extraction with methanol and chloroform in
the presence of water (E. G. Bligh & W. J. Dyer, Can. J.
Biochem. Physiol. 37:911-917 (1959)). Where desirable, the aqueous
layer can be acidified to protonate negatively-charged moieties and
thereby increase partitioning of desired products into the organic
layer. After extraction, the organic solvents can be removed by
evaporation under a stream of nitrogen. When isolated in conjugated
forms, the products may be enzymatically or chemically cleaved to
release the free fatty acid or a less complex conjugate of
interest, and can then be subject to further manipulations to
produce a desired end product. Desirably, conjugated forms of fatty
acids are cleaved with potassium hydroxide.
[0214] If further purification is necessary, standard methods can
be employed. Such methods may include extraction, treatment with
urea, fractional crystallization, HPLC, fractional distillation,
silica gel chromatography, high-speed centrifugation or
distillation, or combinations of these techniques. Protection of
reactive groups, such as the acid or alkenyl groups, may be done at
any step through known techniques (e.g., alkylation, iodination).
Methods used include methylation of the fatty acids to produce
methyl esters. Similarly, protecting groups may be removed at any
step. Desirably, purification of fractions containing GLA, STA,
ARA, DHA and EPA may be accomplished by treatment with urea and/or
fractional distillation.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0215] The present invention demonstrates the feasibility of
introducing an .omega.-3 and/or .omega.-6 biosynthetic pathway into
oleaginous yeast for the production of PUFAs. Toward this end, ARA
(representative of .omega.-6 fatty acids) and EPA (representative
of .omega.-3 fatty acids) were selected as desirable products to
produce in the oleaginous yeast, Yarrowia lipolytica. Thus, the
synthesis of ARA required the introduction of genes encoding
.DELTA.6 desaturase, elongase and .DELTA.5 desaturase activities
into Yarrowia, whereas the synthesis of EPA required the
introduction of genes encoding .DELTA.6 desaturase, elongase,
.DELTA.5 desaturase and .DELTA.17 desaturase activities into
Yarrowia.
[0216] A variety of publicly available .DELTA.5 desaturases from
different organisms having the ability to convert DGLA to ARA and
ETA to EPA were expressed in Yarrowia lipolytica and screened for
activity, in order to identify the gene demonstrating the highest
level of activity in the alternate host. On this basis, a
Mortierella alpina .DELTA.5 desaturase (SEQ ID NO:4) was selected
as the preferred gene for expression in oleaginous yeast, based on
its ability to convert .about.30% of intracellular DGLA to ARA in a
substrate feeding trial.
[0217] Additional substrate feeding trials were conducted to verify
the enzymatic activities encoded by the following genes: [0218] A
M. alpina .DELTA.6 desaturase (SEQ ID NO:2) converts LA to GLA and
ALA to STA (wherein the percent substrate conversion of LA to GLA
in Y. lipolytica was .about.30%); [0219] A Saprolegnia diclina
.DELTA.17 desaturase (SEQ ID NO:6) converts DGLA to ETA and ARA to
EPA (wherein the percent substrate conversion of ARA to EPA in Y.
lipolytica was .about.23%); and [0220] A M. alpina high affinity
PUFA elongase (SEQ ID NO:8) converts GLA to DGLA, STA to ETA and
EPA to DPA (wherein the percent substrate conversion of GLA to DGLA
in Y. lipolytica was .about.30%). Based on the lower percent
substrate conversion of the S. diclina .DELTA.17 desaturase
(relative to the .DELTA.6 and .DELTA.5 desaturase and the
elongase), this particular gene was codon-optimized to enhance its
expression in Yarrowia. This was accomplished by determining the
codon usage and signature of structural genes in Yarrowia
lipolytica, designing a codon-optimized .DELTA.17 desaturase gene,
and then synthesizing the gene in vitro to enable its increased
efficiency in the alternate host (with respect to the wildtype
gene).
[0221] To enable synthesis of ARA or EPA (and thereby demonstrate
proof-of-concept for the ability of oleaginous hosts to be
engineered for production of .omega.-6 and .omega.-3 fatty acids
(i.e., ARA and EPA)), two different DNA expression constructs were
subsequently prepared: 1.) the first contained the .DELTA.6
desaturase, .DELTA.5 desaturase and high-affinity PUFA elongase;
and 2.) the second contained the .DELTA.6 desaturase, .DELTA.5
desaturase, high-affinity PUFA elongase and codon-optimized
.DELTA.17 desaturase. Both constructs were separately transformed
into Yarrowia lipolytica and integrated into the chromosomal URA3
gene encoding the enzyme orotidine-5'-phosphate decarboxylase (EC
4.1.1.23). GC analysis of the host cells fed with appropriate
substrates detected production of ARA (Example 5) and EPA (Example
6), respectively. Thus, this is the first demonstration of PUFA
biosynthesis in an oleaginous yeast whereby the .omega.-3 and/or
.omega.-6 biosynthetic pathways have been introduced into an
oleaginous yeast.
[0222] On the basis of the teachings and results described herein,
it is expected that one skilled in the art will recognize the
feasability and commercial utility created by using oleaginous
yeast as a production platform for the synthesis of a variety of
.omega.-3 and/or .omega.-6 PUFAs.
EXAMPLES
[0223] 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
[0224] 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) (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).
[0225] 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.
[0226] E. coli (XL1-Blue) competent cells were purchased from the
Stratagene Company (San Diego, Calif.). E. coli strains were
typically grown at 37.degree. C. on Luria Bertani (LB) plates.
[0227] General molecular cloning was performed according to
standard methods (Sambrook et al., supra). Oligonucleotides were
synthesized by Sigma-Genosys (Spring, Tex.). Site-directed
mutagenesis was performed using Stratagene's QuickChange.TM.
Site-Directed Mutagenesis kit, per the manufacturers' instructions.
When polymerase chain reaction (PCR) or site-directed mutagenesis
was involved in subcloning, the constructs were sequenced to
confirm that no errors had been introduced to the sequence.
[0228] PCR products were cloned into Promega's pGEM-T-easy vector
(Madison, Wis.).
[0229] DNA sequence was generated on an ABI Automatic sequencer
using dye terminator technology (U.S. Pat. No. 5,366,860; EP
272,007) using a combination of vector and insert-specific primers.
Sequence editing was performed in Sequencher (Gene Codes
Corporation, Ann Arbor, Mich.). All sequences represent coverage at
least two times in both directions. Comparisons of genetic
sequences were accomplished using DNASTAR software (DNA Star,
Inc.). Alternatively, manipulations of genetic sequences were
accomplished using the suite of programs available from the
Genetics Computer Group Inc. (Wisconsin Package Version 9.0,
Genetics Computer Group (GCG), Madison, Wis.). The GCG program
"Pileup" was used with the gap creation default value of 12, and
the gap extension default value of 4. The GCG "Gap" or "Bestfit"
programs were used with the default gap creation penalty of 50 and
the default gap extension penalty of 3. Unless otherwise stated, in
all other cases GCG program default parameters were used.
[0230] 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).
Cultivation of Yarrowia lipolytica
[0231] Yarrowia lipolytica strains ATCC #76982 and ATCC #90812 were
purchased from the American Type Culture Collection (Rockville,
Md.). Y. lipolytica strains were usually grown at 28.degree. C. on
YPD agar (1% yeast extract, 2% bactopeptone, 2% glucose, 2% agar).
For selection of transformants, minimal medium (0.17% yeast
nitrogen base (DIFCO Laboratories, Detroit, Mich.) without ammonium
sulfate or amino acids, 2% glucose, 0.1% proline, pH 6.1) was used.
Supplements of adenine, leucine, lysine and/or uracil were added as
appropriate to a final concentration of 0.01%.
Fatty Acid Analysis of Yarrowia lipolytica
[0232] For fatty acid analysis, cells were collected by
centrifugation and lipids were extracted as described in Bligh, E.
G. & Dyer, W. J. (Can. J. Biochem. Physiol. 37:911-917 (1959)).
Fatty acid methyl esters were prepared by transesterification of
the lipid extract with sodium methoxide (Roughan, G., and Nishida
I. Arch Biochem Biophys. 276(1):38-46 (1990)) and subsequently
analyzed with a Hewlett-Packard 6890 GC fitted with a
30-m.times.0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard) column. The
oven temperature was from 170.degree. C. (25 min hold) to
185.degree. C. at 3.5.degree. C./min.
[0233] 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 of Plasmids Suitable for Heterologous Gene Expression
in Yarrowia lipolytica
[0234] The plasmid pY5, a derivative of pINA532 (a gift from Dr.
Claude Gaillardin, Insitut National Agronomics, Centre de
biotechnologie Agro-Industrielle, laboratoire de Genetique
Moleculaire et Cellularie INRA-CNRS, F-78850 Thiverval-Grignon,
France), was constructed for expression of heterologous genes in
Yarrowia lipolytica, as diagrammed in FIG. 3.
[0235] First, the partially-digested 3598 bp EcoRI fragment
containing the ARS18 sequence and LEU2 gene of pINA532 was
subcloned into the EcoRI site of pBluescript (Strategene, San
Diego, Calif.) to generate pY2. The TEF promoter (Muller S., et al.
Yeast, 14: 1267-1283 (1998)) was amplified from Yarrowia lipolytica
genomic DNA by PCR using TEF5' (SEQ ID NO:38) and TEF3' (SEQ ID
NO:39) as primers. PCR amplification was carried out in a 50 .mu.l
total volume containing: 100 ng Yarrowia genomic DNA, PCR buffer
containing 10 mM KCl, 10 mM (NH.sub.4).sub.2SO.sub.4, 20 mM
Tris-HCl (pH 8.75), 2 mM MgSO.sub.4, 0.1% Triton X-100, 100
.mu.g/mL BSA (final concentration), 200 .mu.M each
deoxyribonucleotide triphosphate, 10 pmole of each primer and 1
.mu.l of PfuTurbo DNA polymerase (Stratagene, San Diego, Calif.).
Amplification was carried out as follows: initial denaturation at
95.degree. C. for 3 min, followed by 35 cycles of the following:
95.degree. C. for 1 min, 56.degree. C. for 30 sec, 72.degree. C.
for 1 min. A final extension cycle of 72.degree. C. for 10 min was
carried out, followed by reaction termination at 4.degree. C. The
418 bp PCR product was ligated into pCR-Blunt to generate pIP-tef.
The BamHI/EcoRV fragment of pIP-tef was subcloned into the
BamHI/SmaI sites of pY2 to generate pY4.
[0236] The XPR2 transcriptional terminator was amplified by PCR
using pINA532 as template and XPR5' (SEQ ID NO:40) and XPR3' (SEQ
ID NO:41) as primers. The PCR amplification was carried out in a 50
.mu.l total volume, using the components and conditions described
above. The 179 bp PCR product was digested with SacII and then
ligated into the SacII site of pY4 to generate pY5. Thus, pY5
(shown in FIGS. 3 and 4) is useful as a Yarrowia-E. coli shuttle
plasmid containing: [0237] 1.) a Yarrowia autonomous replication
sequence (ARS18); [0238] 2.) a ColE1 plasmid origin of replication;
[0239] 3.) an ampicillin-resistance gene (Amp.sup.R), for selection
in E. coli; [0240] 4.) a Yarrowia LEU2 gene (E.C. 4.2.1.33,
encoding isopropylmalate isomerase), for selection in Yarrowia;
[0241] 5.) the translation elongation promoter (TEF P), for
expression of heterologous genes in Yarrowia; and [0242] 6.) the
extracellular protease gene terminator (XPR2) for transcriptional
termination of heterologous gene expression in Yarrowia.
[0243] pY5-13 (FIG. 4) was constructed as a derivative of pY5 to
faciliate subcloning and heterologous gene expression in Yarrowia
lipolytica. Specifically, pY5-13 was constructed by 6 rounds of
site-directed mutagenesis using pY5 as template. Both SalI and ClaI
sites were eliminated from pY5 by site-directed mutagenesis using
oligonucleotides YL5 and YL6 (SEQ ID NOs:106 and 107) to generate
pY5-5. A SalI site was introduced into pY5-5 between the Leu2 gene
and the TEF promoter by site-directed mutagenesis using
oligonucleotides YL9 and YL10 (SEQ ID NOs:110 and 111) to generate
pY5-6. A PacI site was introduced into pY5-6 between the LEU2 gene
and ARS18 using oligonucleotides YL7 and YL8 (SEQ ID NOs:108 and
109) to generate pY5-8. A NcoI site was introduced into pY5-8
around the translation start codon of the TEF promoter using
oligonucleotides YL3 and YL4 (SEQ ID NOs:104 and 105) to generate
pY5-9. The NcoI site inside the Leu2 gene of pY5-9 was eliminated
using YL1 and YL2 oligonucleotides (SEQ ID NOs:102 and 103) to
generate pY5-12. Finally, a BsiWI site was introduced into pY5-12
between the ColEI and XPR2 region using oligonucleotides YL61 and
YL62 (SEQ ID NOs:88 and 89) to generate pY5-13.
[0244] A second derivative of plasmid pY5 was constructed to
faciliate subcloning. Specifically, pY5-4 (FIG. 4) was constructed
by three rounds of site-directed mutagenesis using pY5 as template.
A NcoI site located inside the Leu2 reporter gene was eliminated
from pY5 using oligonucleotides YL1 and YL2 (SEQ ID NOs:102 and
103) to generate pY5-1. A NcoI site was introduced into pY5-1
between the TEF promoter and XPR2 transcriptional terminator by
site-directed mutagenesis using oligonucleotides YL3 and YL4 (SEQ
ID NOs:104 and 105) to generate pY5-2. A PacI site was then
introduced into pY5-2 between the TEF promoter and XPR2
transcriptional terminator using oligonucleotides YL23 and YL24
(SEQ ID NOs:112 and 113) to generate pY5-4.
Example 2
Selection of .DELTA.6 Desaturase, .DELTA.5 Desaturase, .DELTA.17
Desaturase and High Affinity PUFA Elongase Genes for Expression in
Yarrowia lipolytica
[0245] Prior to the introduction of specific genes encoding an
.omega.-3 and/or .omega.-6 biosynthetic pathway into oleaginous
yeast, it was necessary to confirm the functionality of
heterologous .DELTA.6 desaturase, elongase, .DELTA.5 desaturase and
.DELTA.17 desaturase genes expressed in Yarrowia. This was
accomplished by measuring the conversion efficiency encoded by each
wildtype gene in the alternate host. Specifically, four .DELTA.5
desaturases, a Mortierella alpina .DELTA.6 desaturase, a
Saprolegnia diclina .DELTA.17 desaturase and a M. alpina high
affinity PUFA elongase were separately expressed and screened for
activity in substrate-feeding trials. Based on these results, a M.
alpina .DELTA.5 desaturase gene was selected for use in conjunction
with the .DELTA.6 and .DELTA.17 desaturase and high affinity PUFA
elongase genes.
Construction of Expression Plasmids
[0246] In general, wildtype desaturase or elongase genes were
either isolated by restriction digestion or amplified by PCR and
inserted into appropriate vectors for expression. Each PCR
amplification was carried out in a 50 .mu.l total volume,
comprising PCR buffer containing: 10 ng template, 10 mM KCl, 10 mM
(NH.sub.4).sub.2SO.sub.4, 20 mM Tris-HCl (pH 8.75), 2 mM
MgSO.sub.4, 0.1% Triton X-100, 100 .mu.g/mL BSA (final
concentration), 200 .mu.M each deoxyribonucleotide triphosphate, 10
pmole of each primer and 1 .mu.l of PfuTurbo DNA polymerase
(Stratagene, San Diego, Calif.). Amplification was carried out as
follows (unless otherwise specified): initial denaturation at
95.degree. C. for 3 min, followed by 35 cycles of the following:
95.degree. C. for 1 min, 56.degree. C. for 30 sec, 72.degree. C.
for 1 min. A final extension cycle of 72.degree. C. for 10 min was
carried out, followed by reaction termination at 4.degree. C.
[0247] Wild Type Mortierella alpina (Accession #AF465281) .DELTA.6
Desaturase
[0248] The 1384 bp NcoI/NotI fragment of pCGR5 (U.S. Pat. No.
5,968,809), which contains the M. alpina .DELTA.6 desaturase gene
(SEQ ID NO:1), was inserted into the NcoI/NotI sites of pY5-2
(Example 1) to generate pY54.
[0249] Wild Type Mortierella alpina (Accession #AF067654) .DELTA.5
Desaturase
[0250] The M. alpina .DELTA.5 desaturase gene (SEQ ID NO:3) was
amplified by PCR using oligonucleotides YL11 and YL12 (SEQ ID
NOs:72 and 73) as primers and plasmid pCGR-4 (U.S. Pat. No.
6,075,183) as template. PCR amplification was carried out as
described above, with the exception that the elongation step was
extended to 1.5 min (for cycles 1-35). The 1357 bp PCR product was
digested with NcoI/NotI and ligated to NcoI/NotI-digested pY5-13
(described in Example 1) to generate pYMA5pb (FIG. 5).
[0251] Wild Type Saprolegnia diclina (ATCC #56851) .DELTA.5
Desaturase
[0252] The S. diclina .DELTA.5 desaturase gene (SEQ ID NO:114) was
amplified by PCR using oligonucleotides YL13A and YL14 (SEQ ID
NOs:116 and 117) as primers and plasmid pRSP3 (WO 02/081668) as
template. PCR amplification was carried out as described above,
with the exception that the elongation step was extended to 1.5 min
(for cycles 1-35). The 1.4 kB PCR product was digested with
NcoI/PacI and ligated to NcoI/PacI-digested pY5-4 (FIG. 4;
described in Example 1) to generate pYSD5.
[0253] Wild Type Isochrysis galbana CCMP1323 .DELTA.5
Desaturase
[0254] The I. galbana .DELTA.5-desaturase gene (SEQ ID NO:118) was
amplified by PCR using oligonucleotides YL19A and YL20 (SEQ ID
NOs:120 and 121) as primers and plasmid pRIG-1 (WO 02/081668 A2) as
template. PCR amplification was carried out as described above,
with the exception that the elongation step was extended to 1.5 min
(for cycles 1-35). The 1.4 kB PCR product was digested with
BamHI/PacII and ligated to BamHI/PacII-digested pY5-4 (described in
Example 1) to generate pYIG5.
[0255] Wild Type Thraustochytrium aureum (ATCC #34304) .DELTA.5
Desaturase
[0256] The T. aureum .DELTA.5-desaturase gene (SEQ ID NO:122) was
amplified by PCR using oligonucleotides YL15 and YL16B (SEQ ID
NOs:124 and 125) as primers and plasmid pRTA4 (WO 02/081668 A2) as
template. PCR amplification was carried out as described above,
with the exception that the elongation step was extended to 1.5 min
(for cycles 1-35). The 1.4 kB PCR product was digested with
NcoI/NotI and ligated to NcoI/NotI-digested pY5-2 (described in
Example 1) to generate pYTA5.
[0257] Wild Type Saprolegnia diclina (ATCC #56851) .DELTA.17
Desaturase
[0258] The wild type .DELTA.17 desaturase gene of S. diclina was
amplified from plasmid pRSP19 (US 2003/0196217 A1) by PCR using
oligonucleotides YL21A (SEQ ID NO:42) and YL22 (SEQ ID NO:43) as
primers. The PCR products were digested with NcoI/PacI and then
ligated to NcoI/PacI-digested pY5-4 (FIG. 4; described in Example
1) to generate pYSD17.
[0259] Wild Type Mortierella alpina (Accession #AX464731) High
Affinity Elongase
[0260] The 973 bp NotI fragment of pRPB2 (WO 00/12720), containing
the coding region of a M. alpina high affinity PUFA elongase gene
(SEQ ID NO:7), was inserted into the NotI site of pY5 (described in
Example 1; FIGS. 3 and 4) to generate pY58.
Transformation of Yarrowia lipolytica
[0261] The plasmids pY54, pYMA5pb, pYSD5, pYIG5, pYTA5, pYSD17 and
pY58 were transformed separately into Y. lipolytica ATCC #76982
according to the method of Chen, D. C. et al. (Appl Microbiol
Biotechnol. 48(2):232-235-(1997)).
[0262] Briefly, a leucine auxotroph of Yarrowia was streaked onto a
YPD plate and grown at 30.degree. C. for approximately 18 hr.
Several large loopfuls of cells were scraped from the plate and
resuspended in 1 mL of transformation buffer containing: [0263]
2.25 mL of 50% PEG, average MW 3350; [0264] 0.125 mL of 2 M Li
acetate, pH 6.0; [0265] 0.125 m L of 2M DTT; and [0266] 50 .mu.g
sheared salmon sperm DNA.
[0267] About 500 ng of plasmid DNA were incubated in 100 .mu.l of
resuspended cells, and maintained at 39.degree. C. for 1 hr with
vortex mixing at 15 min intervals. The cells were plated onto
minimal media plates lacking leucine and maintained at 30.degree.
C. for 2 to 3 days.
Determination of Percent Substrate Conversion
[0268] Single colonies of transformant Y. lipolytica containing
pY54, pYMA5pb, pYSD5, pYIG5, pYTA5, pYSD17 or pY58 were each grown
in 3 mL minimal media (20 g/L glucose, 1.7 g/L yeast nitrogen base
without amino acids, 1 g/L L-proline, 0.1 g/L L-adenine, 0.1 g/L
L-lysine, pH 6.1) at 30.degree. C. to an OD.sub.600 .about.1.0. For
substrate feeding, 100 .mu.l of cells were then subcultured in 3 mL
minimal media containing 10 .mu.g of substrate for about 24 hr at
30.degree. C. Cells were subsequently collected by centrifugation,
the lipids were extracted, and fatty acid methyl esters were
prepared by transesterification and subsequently analyzed by GC (as
described in the General Methods). Percent substrate conversion was
determined as: [product/(substrate+product)]*100.
[0269] Percent Substrate Conversion by M. alpina .DELTA.6
Desaturase
[0270] The M. alpina .DELTA.6 desaturase converts LA to GLA and ALA
to STA. Y. lipolytica strains containing pY54 were grown as
described above (no substrate feeding required) and lipids were
analyzed. The results showed that Yarrowia strains with pY54
converted about 30% LA to GLA.
[0271] Percent Substrate Conversion by M. alpina, S. diclina, I.
galbana and T. aureum .DELTA.5 Desaturases
[0272] The .DELTA.5 desaturases from M. alpina, S. diclina, I.
galbana and T. aureum each convert DGLA to ARA and ETA to EPA. Y.
lipolytica strains containing pYMA5pb, pYSD5, pYIG5 or pYTA5 were
grown separately from single colonies, subcultured in minimal media
containing 10 .mu.g of DGLA, and then subjected to lipid analysis
as described above. Yarrowia strains with pYMA5pb (M. alpina)
converted about 30% of intracellular DGLA to ARA; the Yarrowia
strains with pYSD5 (S. diclina) converted about 12%; the Yarrowia
strains with pYIG5 (I. galbana) converted about 7%; and the
Yarrowia strains with pYTA5 (T. aureum) converted about 23% of
intracellular DGLA to ARA.
[0273] Percent Substrate Conversion by S. diclina .DELTA.17
Desaturase
[0274] The S. diclina .DELTA.17 desaturase converts ARA to EPA and
DGLA to ETA. Y. lipolytica strains containing pYSD17 were grown
from single colonies, subcultured in minimal media containing 10
.mu.g of ARA, and subjected to lipid analysis as described above.
The results of the ARA feeding experiments showed that Yarrowia
strains with pYSD17 converted about 23% of intracellular ARA to
EPA.
[0275] Percent Substrate Conversion of Wild Type M. alpina High
Affinity Elongase
[0276] The M. alpina high affinity PUFA elongase converts GLA to
DGLA, STA to ETA and EPA to DPA. Y. lipolytica strains containing
pY58 were grown from single colonies, subcultured in minimal media
containing 10 .mu.g of GLA, and subjected to lipid analysis as
described above. The results of the GLA feeding experiments showed
that Yarrowia strains with pY58 converted about 30% of
intracellular GLA to DGLA.
Example 3
Synthesis and Expression of a Codon-Optimized .DELTA.17 Desaturase
Gene in Yarrowia lipolytica
[0277] Based on the results of Example 2, genes encoding .DELTA.6
desaturase, elongase and .DELTA.5 desaturase activities were
available that each enabled .about.30% substrate conversion in
Yarrowia lipolytica. The .DELTA.17 desaturase from S. diclina,
however, had a maximum percent substrate conversion of only 23%.
Thus, a codon-optimized .DELTA.17 desaturase gene was designed,
based on the Saprolegnia diclina DNA sequence (SEQ ID NO:5),
according to the Yarrowia codon usage pattern, the consensus
sequence around the ATG translation initiation codon and the
general rules of RNA stability (Guhaniyogi, G. and J. Brewer, Gene
265(1-2):11-23 (2001)).
[0278] In addition to modification to the translation initiation
site, 127 bp of the 1077 bp coding region (comprising 117 codons)
were codon-optimized. A comparison between this codon-optimized DNA
sequence (SEQ ID NO:9) and the S. diclina .DELTA.17 desaturase gene
DNA sequence (SEQ ID NO:5) is shown in FIG. 6, wherein nucleotides
in bold text correspond to nucleotides that were modified in the
codon-optimized gene. None of the modifications in the
codon-optimized gene changed the amino acid sequence of the encoded
protein (SEQ ID NO:6).
Determining the Preferred Codon Usage in Yarrowia lipolytica
[0279] Approximately 100 genes of Y. lipolytica were found in the
National Center for Biotechnology Information public database. The
coding regions of these genes, comprising 121,167 bp, were
translated by the Editseq program of DNAStar to the corresponding
40,389 amino acids and were tabulated to determine the Y.
lipolytica codon usage profile shown in Table 3. The column titled
"No." refers to the number of times a given codon encodes a
particular amino acid in the sample of 40,389 amino acids. The
column titled "%" refers to the frequency that a given codon
encodes a particular amino acid. Entries shown in bold text
represent the codons favored in Yarrowia lipolytica.
TABLE-US-00003 TABLE 3 Codon Usage In Yarrowia lipolytica Amino
Codon Acid No. % GCA Ala (A) 359 11.4 GCC Ala (A) 1523 48.1 GCG Ala
(A) 256 8.1 GCU Ala (A) 1023 32.3 AGA Arg (R) 263 13.2 AGG Arg (R)
91 4.6 CGA Arg (R) 1133 56.8 CGC Arg (R) 108 5.4 CGG Arg (R) 209
1.0 CGU Arg (R) 189 9.5 AAC Ans (N) 1336 84.0 AAU Ans (N) 255 16.0
GAC Asp (D) 1602 66.8 GAU Asp (D) 795 33.2 UGC Cys (C) 268 53.2 UGU
Cys (C) 236 46.8 CAA Gln (Q) 307 17.0 CAG Gln (Q) 1490 83.0 GAA Glu
(E) 566 23.0 GAG Glu (E) 1893 77.0 GGA Gly (G) 856 29.7 GGC Gly (G)
986 34.2 GGG Gly (G) 148 5.1 GGU Gly (G) 893 31.0 CAC His (H) 618
65.5 CAU His (H) 326 34.5 AUA Ile (I) 42 2.1 AUC Ile (I) 1106 53.7
AUU Ile (I) 910 44.2 CUA Leu (L) 166 4.7 CUC Leu (L) 1029 29.1 CUG
Leu (L) 1379 38.9 CUU Leu (L) 591 16.7 UUA Leu (L) 54 1.5 UUG Leu
(L) 323 9.1 AAA Lys (K) 344 14.8 AAG Lys (K) 1987 85.2 AUG Met (M)
1002 100 UUC Phe (F) 996 61.1 UUU Phe (F) 621 38.9 CCA Pro (P) 207
9.6 CCC Pro (P) 1125 52.0 CCG Pro (P) 176 8.2 CCU Pro (P) 655 30.2
AGC Ser (S) 335 11.3 AGU Ser (S) 201 6.8 UCA Ser (S) 221 7.5 UCC
Ser (S) 930 31.5 UCG Ser (S) 488 16.5 UCU Ser (S) 779 26.4 UAA Term
38 46.9 UAG Term 30 37.0 UGA Term 13 16.1 ACA Thr (T) 306 12.7 ACC
Thr (T) 1245 51.6 ACG Thr (T) 269 11.1 ACU Thr (T) 595 24.6 UGG Trp
(W) 488 100 UAC Tyr (Y) 988 83.2 UAU Tyr (Y) 200 16.8 GUA Val (V)
118 4.2 GUC Val (V) 1052 37.3 GUG Val (V) 948 33.6 GUU Val (V) 703
24.9
[0280] For further optimization of gene expression in Y.
lipolytica, the consensus sequence around the `ATG` initiation
codon of 79 genes was examined. In FIG. 7, the first `A` of the
underlined ATG translation codon is considered to be +1. Seventy
seven percent of the genes analyzed had an `A` in the -3 position,
indicating a strong preference for `A` at this position. There was
also preference for `A` or `C` at the -4, -2 and -1 positions, an
`A`, `C` or `T` at position +5, and a `G` or `C` at position +6.
Thus, the preferred consensus sequence of the codon-optimized
translation initiation site for optimal expression of genes in Y.
lipolytica is `MAMMATGNHS` (SEQ ID NO:126), wherein the nucleic
acid degeneracy code used is as follows: M=A/C; S=C/G; H=A/C/T; and
N=A/C/G/T.
In Vitro Synthesis of a Codon-Optimized Gene
[0281] The method used to synthesize the codon-optimized .DELTA.17
desaturase gene is illustrated in FIG. 8. First, eleven pairs of
oligonucleotides were designed to extend the entire length of the
codon-optimized coding region of the S. diclina .DELTA.17
desaturase gene (e.g., D17-1A, D17-1B, D17-2A, D17-2B, D17-3A,
D17-3B, D17-4A, D17-4B, D17-5A, D17-5B, D17-6A, D17-6B, D17-7A,
D17-7B, D17-8A, D17-8B, D17-9A, D17-9B, D17-10A, D17-10B, D17-11A
and D17-11B, corresponding to SEQ ID NOs:10-31). Each pair of sense
(A) and anti-sense (B) oligonucleotides were complementary, with
the exception of a 4 bp overhang at each 5'-end. Additionally,
primers D17-1A, D17-4B, D17-5A, D17-8A and D17-8B also introduced
NcoI, BglII and SalI restriction sites for subsequent subcloning,
respectively.
[0282] 100 ng of each oligonucleotide was phosphorylated at
37.degree. C. for 1 hr in a volume of 20 .mu.l containing 50 mM
Tris-HCl (pH 7.5), 10 mM MgCl.sub.2, 10 mM DTT, 0.5 mM spermidine,
0.5 mM ATP and 10 U of T4 polynucleotide kinase. Each pair of sense
and antisense oligonucleotides was mixed and annealed in a
thermocycler using the following parameters: 95.degree. C. (2 min),
85.degree. C. (2 min), 65.degree. C. (15 min), 37.degree. C. (15
min), 24.degree. C. (15 min) and 4.degree. C. (15 min). Thus,
D17-1A (SEQ ID NO:10) was annealed to D17-1B (SEQ ID NO:11) to
produce the double-stranded product "D17-1AB". Similarly, D17-2A
(SEQ ID NO:12) was annealed to D17-2B (SEQ ID NO:13) to produce the
double-stranded product "D17-2AB", etc.
[0283] Three separate pools of annealed, double-stranded
oligonucleotides were then ligated together, as shown below: [0284]
Pool 1: comprised D17-1AB, D17-2AB, D17-3AB and D17-4AB; [0285]
Pool 2: comprised D17-5AB, D17-6AB, D17-7AB and D17-8AB; and [0286]
Pool 3: comprised D17-9AB, D17-10AB and D17-11AB. Each pool of
annealed oligonucleotides was mixed in a volume of 20 .mu.l with 10
U of T4 DNA ligase and the ligation reaction was incubated
overnight at 16.degree. C.
[0287] The product of each ligation reaction was then amplified by
PCR. Specifically, using the ligated "Pool 1" mixture (i.e.,
D17-1AB, D17-2AB, D17-3AB and D17-4AB) as template, and
oligonucleotides D17-1 (SEQ ID NO:32) and D17-4R (SEQ ID NO:33) as
primers, the first portion of the codon-optimized .DELTA.17
desaturase gene was amplified by PCR. The PCR amplification was
carried out in a 50 .mu.l total volume, comprising PCR buffer
containing 10 mM KCl, 10 mM (NH.sub.4).sub.2SO.sub.4, 20 mM
Tris-HCl (pH 8.75), 2 mM MgSO.sub.4, 0.1% Triton X-100, 100
.mu.g/mL BSA (final concentration), 200 .mu.M each
deoxyribonucleotide triphosphate, 10 pmole of each primer and 1
.mu.l of PfuTurbo DNA polymerase (Stratagene, San Diego, Calif.).
Amplification was carried out as follows: initial denaturation at
95.degree. C. for 3 min, followed by 35 cycles of the following:
95.degree. C. for 1 min, 56.degree. C. for 30 sec, 72.degree. C.
for 40 sec. A final extension cycle of 72.degree. C. for 10 min was
carried out, followed by reaction termination at 4.degree. C. The
430 bp PCR fragment was subcloned into the PGEM-T easy vector
(Promega) to generate pT17(1-4).
[0288] Using the ligated "Pool 2" mixture (i.e., D17-5AB, D17-6AB,
D17-7AB and D17-8AB) as template, and oligonucleotides D17-5 (SEQ
ID NO:34) and D17-8D (SEQ ID NO:35) as primers, the second portion
of the codon-optimized .DELTA.17 desaturase gene was amplified
similarly by PCR and cloned into pGEM-T-easy vector to generate
pT17(5-8). Finally, using the "Pool 3" ligation mixture (i.e.,
D17-9AB, D17-10AB and D17-11AB) as template, and oligonucleotides
D17-8U (SEQ ID NO:36) and D17-11 (SEQ ID NO:37) as primers, the
third portion of the codon-optimized .DELTA.17 desaturase gene was
amplified similarly by PCR and cloned into PGEM-T-easy vector to
generate pT17(9-11).
[0289] E. coli was transformed separately with pT17(1-4), pT17(5-8)
and pT17(9-11) and the plasmid DNA was isolated from
ampicillin-resistant transformants. Plasmid DNA was purified and
digested with the appropriate restriction endonucleases to liberate
the 420 bp NcoI/BglII fragment of pT17(1-4), the 400 bp BglII/SalI
fragment of pT17(5-8) and the 300 bp SalI/NotI fragment of
pT17(9-11). These fragments were then combined, ligated together
and used as template for amplification of the entire synthetic
codon-optimized .DELTA.17 desaturase gene using D17-1 (SEQ ID NO:
32) and D17-11 (SEQ ID NO:37) as primers. The PCR amplification was
carried out in a 50 .mu.l total volume, using the conditions
described above for each portion of the .DELTA.17 desaturase gene
and the thermocycling program as follows: initial denaturation at
95.degree. C. for 3 min, followed by 35 cycles of the following:
95.degree. C. for 1 min, 56.degree. C. for 30 sec, 72.degree. C.
for 1.1 min. A final extension cycle of 72.degree. C. for 10 min
was carried out, followed by reaction termination at 4.degree. C.
This generated a 1.1 kB PCR product.
Construction of Plasmid pYSD17s Containing the Codon-Optimized
.DELTA.17 Desaturase
[0290] The 1.1 kB PCR product comprising the entire synthetic
.DELTA.17 desaturase was digested with NcoI/NotI and subcloned into
NcoI/NotI-digested pY5-13 (Example 1) to generate pYSD17S (FIG.
9A).
[0291] As an additional "control", to compare the efficiency of the
wild type and synthetic genes in Yarrowia, the AT-rich PacI site in
pYSDl 7 (comprising the wild-type gene; described in Example 2) was
eliminated by site-directed mutagenesis using YL53 (SEQ ID NO:44)
and YL54 (SEQ ID NO:45) as primers to generate pYSD17M (FIG.
9B).
Transformation of Yarrowia lipolytica with the Codon-Optimized
.DELTA.17 Desaturase Gene
[0292] Plasmids containing the wildtype and codon-optimized
.DELTA.17 desaturase were transformed separately into Y. lipolytica
ATCC #76982 according to the methods described above in Example 2.
Using this technique, transformants were obtained that contained
the following plasmids:
TABLE-US-00004 TABLE 4 Summary Of Plasmids In Transformant Yarrowia
Plasmid Description pYSD17 wildtype .DELTA.17 desaturase pYSD17M
wildtype .DELTA.17 desaturase, minus AT-rich Pacl site pYSD17S
codon-optimized .DELTA.17 desaturase
Percent Substrate Conversion with the Codon-Optimized .DELTA.17
Desaturase Gene
[0293] .DELTA.17 desaturase converts ARA to EPA (see FIG. 2). The
percent substrate conversion ([product]/[substrate+product]*100) of
the wildtype and codon-optimized .DELTA.17 desaturase genes was
determined in Yarrowia lipolytica containing each alternate plasmid
construct, using the methodology described in the General
Methods.
[0294] The results of the ARA feeding experiments showed that
Yarrowia strains with control plasmids pYSD17 or pYSD17M converted
about 23% of intracellular ARA to EPA (FIG. 10A) while those
containing the codon-optimized .DELTA.17 desaturase gene within
pYSD17S converted about 45% of intracellular ARA to EPA (FIG. 10B).
Thus, Yarrowia containing the codon-optimized .DELTA.17 desaturase
converted about 2-fold more ARA than the strains containing the
wild type S. diclina gene.
Example 4
Construction of Plasmids Suitable for the Coordinate Expression of
Multiple Omega Fatty Acid Biosynthesis Genes in Yarrowia
lipolytica
[0295] The present Example describes the synthesis of a variety of
expression plasmids that were required in order to construct: 1.) a
DNA fragment suitable for integration into the Yarrowia genome for
expression of the .DELTA.6 desaturase, PUFA elongase and .DELTA.5
desaturase (for ARA production); and 2.) a DNA fragment suitable
for integration into the Yarrowia genome for expression of the
.DELTA.6 desaturase, PUFA elongase, .DELTA.5 desaturase and
.DELTA.17 desaturase (for EPA production).
Construction of Plasmid pY24
[0296] Plasmid pY24 (FIG. 11) was a parent vector for construction
of expression cassettes suitable for integration into the genome of
Yarrowia lipolytica. pY24 was constructed as follows:
[0297] Using oligonucleotides KU5 and KU3 (SEQ ID NOs:46 and 47) as
primers and Yarrowia genomic DNA as template, a 1.7 kB DNA fragment
(SEQ ID NO:48) containing the Yarrowia URA3 gene was PCR amplified.
The PCR amplification was carried out in a 50 .mu.l total volume
containing: 100 ng Yarrowia genomic DNA, PCR buffer containing 10
mM KCl, 10 mM (NH.sub.4).sub.2SO.sub.4, 20 mM Tris-HCl (pH 8.75), 2
mM MgSO.sub.4, 0.1% Triton X-100, 100 .mu.g/mL BSA (final
concentration), 200 .mu.M each deoxyribonucleotide triphosphate, 10
pmole of each primer and 1 .mu.l of PfuTurbo DNA polymerase
(Stratagene, San Diego, Calif.). Amplification was carried out as
follows: initial denaturation at 95.degree. C. for 3 min, followed
by 35 cycles of the following: 95.degree. C. for 1 min, 56.degree.
C. for 30 sec, 72.degree. C. for 2 min. A final extension cycle of
72.degree. C. for 10 min was carried out, followed by reaction
termination at 4.degree. C. The PCR product was inserted into
pGEM-T easy vector (Promega, Madison, Wis.) to generate pGYUM.
[0298] Using oligonucleotides KI5 and KI3 (SEQ ID NOs:50 and 51), a
1.1 kB DNA fragment (SEQ ID NO:52) containing the conjugase gene
(or "imp H8") of Impatients balsama (clone ids.pk0001.h8; E. I. du
Pont de Nemours and Company, Inc., Wilmington, Del.) was PCR
amplified. The PCR amplification was carried out in a 50 .mu.l
total volume using the components described above, with the
exception that 10 ng plasmid DNA of ids.pk0001.h8 was used as
template. Amplification was carried out as follows: initial
denaturation at 95.degree. C. for 3 min, followed by 35 cycles of
the following: 95.degree. C. for 1.5 min, 56.degree. C. for 30 sec,
72.degree. C. for 1.2 min. A final extension cycle of 72.degree. C.
for 10 min was carried out, followed by reaction termination at
4.degree. C. The PCR products were digested with NotI, and then
inserted into the NotI site of pY5 (FIG. 3) to generate pY9.
[0299] Using oligonucleotides KTI5 and KTI3 (SEQ ID NOs:54 and 55),
a 1.7 kB DNA fragment (SEQ ID NO:56) containing the TEF::IMP
H8::XPR chimeric gene of pY9 was PCR amplified. The PCR
amplification was carried out in a 50 .mu.l total volume as
described above, with the exception that 10 ng plasmid DNA of pGYUM
was used as template. Amplification was carried out as follows:
initial denaturation at 95.degree. C. for 3 min, followed by 35
cycles of the following: 95.degree. C. for 1 min, 56.degree. C. for
30 sec, 72.degree. C. for 2 min. A final extension cycle of
72.degree. C. for 10 min was carried out, followed by reaction
termination at 4.degree. C. The PCR products were inserted into
PCR-Script (Stratagene) to generate pY9R. The 1.7 kB Xho/EcoRV
fragment of pY9R was exchanged with the XhoI/EcoRV fragment of
pGYUM to generate pY21.
[0300] Using oligonucleotides KH5 and KH3 (SEQ ID NOs:58 and 59) as
primers and genomic DNA of KS65 as template, a 1 kB DNA fragment
(SEQ ID NO:60) containing the E. coli hygromycin resistance gene
("HPT"; Kaster, K. R., et al., Nucleic Acids Res. 11:6895-6911
(1983)) was PCR amplified. The PCR amplification was carried out in
a 50 .mu.l total volume using the components described above, with
the exception that 10 ng plasmid DNA of ids.pk0001.h8 was used as
template. Amplification was carried out as follows: initial
denaturation at 95.degree. C. for 3 min, followed by 35 cycles of
the following: 95.degree. C. for 1 min, 56.degree. C. for 30 sec,
72.degree. C. for 1.2 min. A final extension cycle of 72.degree. C.
for 10 min was carried out, followed by reaction termination at
4.degree. C. The PCR products were digested with NotI, and then
inserted into the NotI site of pY5 (FIG. 3) to generate
pTHPT-1.
[0301] Using oligonucleotides KTH5 and KTH3 (SEQ ID NOs:62 and 63)
as primers and pTHPT-1 plasmid DNA as template, a 1.6 kB DNA
fragment (SEQ ID NO:64) containing the TEF::HPT::XPR fusion gene
was amplified as described above. The PCR products were digested
with BglII and then inserted into pY21 to generate pY24.
Construction of pY24-4
[0302] Plasmid pY24 (FIG. 11) was used for construction of
expression cassettes suitable for integration into the Yarrowia
lipolytica genome. The 401 bp of 5'-sequence (SEQ ID NO:66) and the
568 bp of 3'-sequence (SEQ ID NO:67) from the Y. lipolytica URA3
gene in pY24 plasmid were used to direct integration of expression
cassettes into the Ura loci of the Yarrowia genome. Two chimeric
genes (TEF::HPT::XPR and TEF::IMP H8::XPR) were first removed from
pY24 by digestion with BamHI and self-ligation to generate pY24-1.
PacI and BsiWI sites were introduced into pY24-1 by site-directed
mutagenesis using YL63/YL64 (SEQ ID NOs:68 and 69) and YL65/YL66
(SEQ ID NOs:70 and 71) primer pairs, respectively, to generate
pY24-4.
Construction of an Integration Vector for Expression of .DELTA.5
Desaturase
[0303] The 4261 bp PacI/BsiWI fragment of pYMA5pb (comprising the
M. alpina .DELTA.5 desaturase gene; described in Example 2) was
ligated into the PacI/BsiWI sites of pY24-4 (FIG. 11) to generate
pYZM5 (FIG. 5). HindIII and ClaI sites were introduced into pYZM5
by site-directed mutagenesis using primer pairs YL81 and YL82 (SEQ
ID NOs:74 and 75) and YL83 and YL84 (SEQ ID NOs:76 and 77),
respectively, to generate pYZM5CH. A PmeI site was introduced into
pYZM5CH by site-directed mutagenesis using YL105 and YL106 (SEQ ID
NOs:78 and 79) as primers to generate pYZM5CHPP. An AscI site was
introduced into pYZM5CHPP by site-directed mutagenesis using YL119
and YL120 (SEQ ID NOs:80 and 81) as primers to generate pYZM5CHPPA
(FIG. 5).
[0304] To optimize the integration vector, 440 bp of 5'-non-coding
DNA sequence upstream from the Yarrowia lipolytica URA3 gene (SEQ
ID NO:84) was amplified by PCR using YL121 and YL122 (SEQ ID NOs:82
and 83) as primers. The PCR product was digested with AscI and
BsiWI and then exchanged with the AscI/BsiWI fragment of pYZM5CHPPA
(FIGS. 5 and 12) to generate pYZM5UPA (FIG. 12). An AscI site was
introduced into pYZM5UPA by site-directed mutagenesis using
oligonucleotides YL114 and YL115 (SEQ ID NOs:85 and 86) to generate
pYZV5. In order to reduce the size of the 3'-non-coding region of
the URA3 gene in pYZV5, a second PacI site was introduced into the
middle of this region by site-directed mutagenesis using
oligonucleotides YL114 and YL115 (described above) to generate
pYZV5P. The PacI fragment of pYZV5P was excised by digestion with
PacI and religation to generate pYZV16 (FIG. 12). Digestion of
pYZV16 with AscI liberates a 5.2 kB DNA fragment (SEQ ID NO:87)
suitable for integration and expression of the .DELTA.5 desaturase
gene ("MAD5") in the Y. lipolytica genome.
Construction of an Integration Vector for Expression of the High
Affinity Elongase and .DELTA.5 Desaturase
[0305] BsiWI and HindIII sites were introduced into pY58
(containing the coding region of the M. alpina high affinity PUFA
elongase; described in Example 2) by site-directed mutagenesis
using YL61/YL62 (SEQ ID NOs:88 and 89) and YL69/YL70 (SEQ ID NOs:90
and 91) primer pairs, respectively, to generate pY58BH (FIG. 13;
elongase gene labeled as "EL"). The 1.7 kB BsiWI/HindIII fragment
of pY58BH, which contains the TEF::EL::XPR chimeric gene, was
ligated into the BsiWI/HindIII site of pYZM5CHPP (construction
described in FIG. 5) to generate pYZM5EL (FIG. 13). This plasmid is
suitable for integration and coordinate expression of the M. alpina
.DELTA.5 desaturase and high affinity PUFA elongase genes in Y.
lipolytica.
Construction of an Integration Vector for Expression of the
.DELTA.6 Desaturase, High Affinity Elongase and .DELTA.5
Desaturase
[0306] PacI and ClaI sites were introduced into pY54 (containing
the M. alpina .DELTA.6 desaturase; described in Example 2) by
site-directed mutagenesis using YL77/YL78 (SEQ ID NOs:92 and 93)
and YL79A/YL80A (SEQ ID NOs:94 and 95) primer pairs, respectively,
to generate pY54PC (FIG. 13; .DELTA.6 desaturase gene labeled as
"MAD6"). The 2 kB ClaI/PacI DNA fragment of pY54PC, which contains
the TEF::MAD6::XPR chimeric gene, was ligated into the ClaI/PacI
sites of pYZM5EL to generate pYZM5EL6 (FIG. 13). This plasmid is
suitable for integration and coordinate expression of the M. alpina
.DELTA.6 desaturase, .DELTA.5 desaturase and high affinity PUFA
elongase genes in the Y. lipolytica genome.
Construction of a DNA Fragment Suitable for Integration into the
Yarrowia Genome, for Expression of the .DELTA.6 Desaturase, PUFA
Elongase and .DELTA.5 Desaturase
[0307] The plasmid pYZV16 (construction described in FIG. 12) was
used for construction of plasmids containing multiple expression
cassettes.
[0308] First, the 3.5 kB BsiWI/PacI fragment of pYZV16 was ligated
to the 7.9 kB BsiWI/PacI fragment of pYZM5EL6 (construction
described in FIG. 13) to generate pYZV5EL6 (FIG. 14). Digestion of
pYZV5EL6 with AscI liberates a 8.9 kB DNA fragment (SEQ ID NO:96)
suitable for integration and coordinate expression of the .DELTA.6
desaturase, PUFA elongase and .DELTA.5 desaturase genes in the Y.
lipolytica genome.
Construction of a DNA Fragment Suitable for Integration into the
Yarrowia Genome, for Expression of the .DELTA.6 Desaturase, PUFA
Elongase, .DELTA.5 Desaturase and .DELTA.17 Desaturase
[0309] As described in Example 3, the synthetic S. diclina
.DELTA.17 desaturase gene was inserted into the NcoI/NotI sites of
pY5-13 to generate pYSD17S (FIG. 9A). ClaI and PmeI sites were
introduced into pYSD17S by site-directed mutagenesis using YL101
YL102 (SEQ ID NOs:97 and 98) and YL103/YL104 (SEQ ID NOs:99 and
100) primer pairs, respectively, to generate pYSD17SPC (FIG.
14).
[0310] The 347 bp ClaI/PmeI fragment of pYZV5EL6 (FIG. 14) was
exchanged with the 1760 bp ClaI/PmeI fragment from pYSD17SPC
containing the .DELTA.17 desaturase expression cassette to generate
pYZV5E6/17. Digestion of pYZV5E6/17 with AscI liberates a 10.3 kB
DNA fragment (SEQ ID NO:101) suitable for integration and
coordinate expression of the .DELTA.6 desaturase, PUFA elongase,
.DELTA.5 desaturase and .DELTA.17 desaturase genes in the Y.
lipolytica genome.
Example 5
Biosynthesis of .omega.-6 Fatty Acids in Yarrowia lipolytica
Transformants
[0311] pYZV5EL6 (from Example 4, containing the .DELTA.6
desaturase, PUFA elongase and .DELTA.5 desaturase genes) was
digested with the AscI restriction endonuclease and transformed
into Yarrowia lipolytica according to the methodology described in
Example 2.
[0312] Of 52 transformants selected on minimal media lacking
leucine, 34 could not grow on media also lacking uracil, suggesting
that 65% of the transformants contained the 8.9 kB multi-gene
expression cassette integrated into the targeted Yarrowia
lipolytica URA3 locus. Transformants from single colonies were
inoculated in minimal media lacking leucine and were incubated at
30.degree. C. for up to 48 hr.
[0313] The cells were collected by centrifugation, lipids were
extracted, and fatty acid methyl esters were prepared by
transesterification and subsequently analyzed with a
Hewlett-Packard 6890 GC (according to the methodology described in
the General Methods).
[0314] GC analyses showed the presence of arachidonic acid (ARA) in
the transformants containing the 3 chimeric genes (FIG. 15), but
not in the wild type Yarrowia control strain. These data confirmed
that Yarrowia lipolytica was engineered to produce ARA, an
.omega.-6 fatty acid.
Example 6
Biosynthesis of .omega.-3 Fatty Acids in Yarrowia lipolytica
Transformants
[0315] In a manner similar to that in Example 5, pYZV5E6/17 (from
Example 4, containing the .DELTA.6 desaturase, PUFA elongase,
.DELTA.5 desaturase and .DELTA.17 desaturase) was digested with the
AscI restriction endonuclease and transformed into Yarrowia
lipolytica (ATCC #76982). Of 133 transformants selected on minimal
media lacking leucine, 89 could not grow on media also lacking
uracil, suggesting that 67% of the transformants contained the 10.3
kB multi-gene expression cassette integrated into the targeted
Yarrowia lipolytica URA3 locus.
[0316] GC analyses (according to the methodology described in the
General Methods) showed the presence of eicosapentaenoic acid (EPA)
in the transformants containing the 4 chimeric genes (FIG. 16), but
not in the wild-type Yarrowia control strain. These data confirmed
that Yarrowia lipolytica was engineered to produce EPA, an
.omega.-3 fatty acid.
Sequence CWU 1
1
12611374DNAMortierella alpina AF465281 1atggctgctg ctcccagtgt
gaggacgttt actcgggccg aggttttgaa tgccgaggct 60ctgaatgagg gcaagaagga
tgccgaggca cccttcttga tgatcatcga caacaaggtg 120tacgatgtcc
gcgagttcgt ccctgatcat cccggtggaa gtgtgattct cacgcacgtt
180ggcaaggacg gcactgacgt ctttgacact tttcaccccg aggctgcttg
ggagactctt 240gccaactttt acgttggtga tattgacgag agcgaccgcg
atatcaagaa tgatgacttt 300gcggccgagg tccgcaagct gcgtaccttg
ttccagtctc ttggttacta cgattcttcc 360aaggcatact acgccttcaa
ggtctcgttc aacctctgca tctggggttt gtcgacggtc 420attgtggcca
agtggggcca gacctcgacc ctcgccaacg tgctctcggc tgcgcttttg
480ggtctgttct ggcagcagtg cggatggttg gctcacgact ttttgcatca
ccaggtcttc 540caggaccgtt tctggggtga tcttttcggc gccttcttgg
gaggtgtctg ccagggcttc 600tcgtcctcgt ggtggaagga caagcacaac
actcaccacg ccgcccccaa cgtccacggc 660gaggatcccg acattgacac
ccaccctctg ttgacctgga gtgagcatgc gttggagatg 720ttctcggatg
tcccagatga ggagctgacc cgcatgtggt cgcgtttcat ggtcctgaac
780cagacctggt tttacttccc cattctctcg tttgcccgtc tctcctggtg
cctccagtcc 840attctctttg tgctgcctaa cggtcaggcc cacaagccct
cgggcgcgcg tgtgcccatc 900tcgttggtcg agcagctgtc gcttgcgatg
cactggacct ggtacctcgc caccatgttc 960ctgttcatca aggatcccgt
caacatgctg gtgtactttt tggtgtcgca ggcggtgtgc 1020ggaaacttgt
tggcgatcgt gttctcgctc aaccacaacg gtatgcctgt gatctcgaag
1080gaggaggcgg tcgatatgga tttcttcacg aagcagatca tcacgggtcg
tgatgtccac 1140ccgggtctat ttgccaactg gttcacgggt ggattgaact
atcagatcga gcaccacttg 1200ttcccttcga tgcctcgcca caacttttca
aagatccagc ctgctgtcga gaccctgtgc 1260aaaaagtaca atgtccgata
ccacaccacc ggtatgatcg agggaactgc agaggtcttt 1320agccgtctga
acgaggtctc caaggctacc tccaagatgg gtaaggcgca gtaa
13742457PRTMortierella alpina AF465281 2Met Ala Ala Ala Pro Ser Val
Arg Thr Phe Thr Arg Ala Glu Val Leu1 5 10 15Asn Ala Glu Ala Leu Asn
Glu Gly Lys Lys Asp Ala Glu Ala Pro Phe20 25 30Leu Met Ile Ile Asp
Asn Lys Val Tyr Asp Val Arg Glu Phe Val Pro35 40 45Asp His Pro Gly
Gly Ser Val Ile Leu Thr His Val Gly Lys Asp Gly50 55 60Thr Asp Val
Phe Asp Thr Phe His Pro Glu Ala Ala Trp Glu Thr Leu65 70 75 80Ala
Asn Phe Tyr Val Gly Asp Ile Asp Glu Ser Asp Arg Asp Ile Lys85 90
95Asn Asp Asp Phe Ala Ala Glu Val Arg Lys Leu Arg Thr Leu Phe
Gln100 105 110Ser Leu Gly Tyr Tyr Asp Ser Ser Lys Ala Tyr Tyr Ala
Phe Lys Val115 120 125Ser Phe Asn Leu Cys Ile Trp Gly Leu Ser Thr
Val Ile Val Ala Lys130 135 140Trp Gly Gln Thr Ser Thr Leu Ala Asn
Val Leu Ser Ala Ala Leu Leu145 150 155 160Gly Leu Phe Trp Gln Gln
Cys Gly Trp Leu Ala His Asp Phe Leu His165 170 175His Gln Val Phe
Gln Asp Arg Phe Trp Gly Asp Leu Phe Gly Ala Phe180 185 190Leu Gly
Gly Val Cys Gln Gly Phe Ser Ser Ser Trp Trp Lys Asp Lys195 200
205His Asn Thr His His Ala Ala Pro Asn Val His Gly Glu Asp Pro
Asp210 215 220Ile Asp Thr His Pro Leu Leu Thr Trp Ser Glu His Ala
Leu Glu Met225 230 235 240Phe Ser Asp Val Pro Asp Glu Glu Leu Thr
Arg Met Trp Ser Arg Phe245 250 255Met Val Leu Asn Gln Thr Trp Phe
Tyr Phe Pro Ile Leu Ser Phe Ala260 265 270Arg Leu Ser Trp Cys Leu
Gln Ser Ile Leu Phe Val Leu Pro Asn Gly275 280 285Gln Ala His Lys
Pro Ser Gly Ala Arg Val Pro Ile Ser Leu Val Glu290 295 300Gln Leu
Ser Leu Ala Met His Trp Thr Trp Tyr Leu Ala Thr Met Phe305 310 315
320Leu Phe Ile Lys Asp Pro Val Asn Met Leu Val Tyr Phe Leu Val
Ser325 330 335Gln Ala Val Cys Gly Asn Leu Leu Ala Ile Val Phe Ser
Leu Asn His340 345 350Asn Gly Met Pro Val Ile Ser Lys Glu Glu Ala
Val Asp Met Asp Phe355 360 365Phe Thr Lys Gln Ile Ile Thr Gly Arg
Asp Val His Pro Gly Leu Phe370 375 380Ala Asn Trp Phe Thr Gly Gly
Leu Asn Tyr Gln Ile Glu His His Leu385 390 395 400Phe Pro Ser Met
Pro Arg His Asn Phe Ser Lys Ile Gln Pro Ala Val405 410 415Glu Thr
Leu Cys Lys Lys Tyr Asn Val Arg Tyr His Thr Thr Gly Met420 425
430Ile Glu Gly Thr Ala Glu Val Phe Ser Arg Leu Asn Glu Val Ser
Lys435 440 445Ala Thr Ser Lys Met Gly Lys Ala Gln450
45531341DNAMortierella alpina AF067654 3atgggaacgg accaaggaaa
aaccttcacc tgggaagagc tggcggccca taacaccaag 60gacgacctac tcttggccat
ccgcggcagg gtgtacgatg tcacaaagtt cttgagccgc 120catcctggtg
gagtggacac tctcctgctc ggagctggcc gagatgttac tccggtcttt
180gagatgtatc acgcgtttgg ggctgcagat gccattatga agaagtacta
tgtcggtaca 240ctggtctcga atgagctgcc catcttcccg gagccaacgg
tgttccacaa aaccatcaag 300acgagagtcg agggctactt tacggatcgg
aacattgatc ccaagaatag accagagatc 360tggggacgat acgctcttat
ctttggatcc ttgatcgctt cctactacgc gcagctcttt 420gtgcctttcg
ttgtcgaacg cacatggctt caggtggtgt ttgcaatcat catgggattt
480gcgtgcgcac aagtcggact caaccctctt catgatgcgt ctcacttttc
agtgacccac 540aaccccactg tctggaagat tctgggagcc acgcacgact
ttttcaacgg agcatcgtac 600ctggtgtgga tgtaccaaca tatgctcggc
catcacccct acaccaacat tgctggagca 660gatcccgacg tgtcgacgtc
tgagcccgat gttcgtcgta tcaagcccaa ccaaaagtgg 720tttgtcaacc
acatcaacca gcacatgttt gttcctttcc tgtacggact gctggcgttc
780aaggtgcgca ttcaggacat caacattttg tactttgtca agaccaatga
cgctattcgt 840gtcaatccca tctcgacatg gcacactgtg atgttctggg
gcggcaaggc tttctttgtc 900tggtatcgcc tgattgttcc cctgcagtat
ctgcccctgg gcaaggtgct gctcttgttc 960acggtcgcgg acatggtgtc
gtcttactgg ctggcgctga ccttccaggc gaaccacgtt 1020gttgaggaag
ttcagtggcc gttgcctgac gagaacggga tcatccaaaa ggactgggca
1080gctatgcagg tcgagactac gcaggattac gcacacgatt cgcacctctg
gaccagcatc 1140actggcagct tgaactacca ggctgtgcac catctgttcc
ccaacgtgtc gcagcaccat 1200tatcccgata ttctggccat catcaagaac
acctgcagcg agtacaaggt tccatacctt 1260gtcaaggata cgttttggca
agcatttgct tcacatttgg agcacttgcg tgttcttgga 1320ctccgtccca
aggaagagta g 13414446PRTMortierella alpina AF067654 4Met Gly Thr
Asp Gln Gly Lys Thr Phe Thr Trp Glu Glu Leu Ala Ala1 5 10 15His Asn
Thr Lys Asp Asp Leu Leu Leu Ala Ile Arg Gly Arg Val Tyr20 25 30Asp
Val Thr Lys Phe Leu Ser Arg His Pro Gly Gly Val Asp Thr Leu35 40
45Leu Leu Gly Ala Gly Arg Asp Val Thr Pro Val Phe Glu Met Tyr His50
55 60Ala Phe Gly Ala Ala Asp Ala Ile Met Lys Lys Tyr Tyr Val Gly
Thr65 70 75 80Leu Val Ser Asn Glu Leu Pro Ile Phe Pro Glu Pro Thr
Val Phe His85 90 95Lys Thr Ile Lys Thr Arg Val Glu Gly Tyr Phe Thr
Asp Arg Asn Ile100 105 110Asp Pro Lys Asn Arg Pro Glu Ile Trp Gly
Arg Tyr Ala Leu Ile Phe115 120 125Gly Ser Leu Ile Ala Ser Tyr Tyr
Ala Gln Leu Phe Val Pro Phe Val130 135 140Val Glu Arg Thr Trp Leu
Gln Val Val Phe Ala Ile Ile Met Gly Phe145 150 155 160Ala Cys Ala
Gln Val Gly Leu Asn Pro Leu His Asp Ala Ser His Phe165 170 175Ser
Val Thr His Asn Pro Thr Val Trp Lys Ile Leu Gly Ala Thr His180 185
190Asp Phe Phe Asn Gly Ala Ser Tyr Leu Val Trp Met Tyr Gln His
Met195 200 205Leu Gly His His Pro Tyr Thr Asn Ile Ala Gly Ala Asp
Pro Asp Val210 215 220Ser Thr Ser Glu Pro Asp Val Arg Arg Ile Lys
Pro Asn Gln Lys Trp225 230 235 240Phe Val Asn His Ile Asn Gln His
Met Phe Val Pro Phe Leu Tyr Gly245 250 255Leu Leu Ala Phe Lys Val
Arg Ile Gln Asp Ile Asn Ile Leu Tyr Phe260 265 270Val Lys Thr Asn
Asp Ala Ile Arg Val Asn Pro Ile Ser Thr Trp His275 280 285Thr Val
Met Phe Trp Gly Gly Lys Ala Phe Phe Val Trp Tyr Arg Leu290 295
300Ile Val Pro Leu Gln Tyr Leu Pro Leu Gly Lys Val Leu Leu Leu
Phe305 310 315 320Thr Val Ala Asp Met Val Ser Ser Tyr Trp Leu Ala
Leu Thr Phe Gln325 330 335Ala Asn His Val Val Glu Glu Val Gln Trp
Pro Leu Pro Asp Glu Asn340 345 350Gly Ile Ile Gln Lys Asp Trp Ala
Ala Met Gln Val Glu Thr Thr Gln355 360 365Asp Tyr Ala His Asp Ser
His Leu Trp Thr Ser Ile Thr Gly Ser Leu370 375 380Asn Tyr Gln Ala
Val His His Leu Phe Pro Asn Val Ser Gln His His385 390 395 400Tyr
Pro Asp Ile Leu Ala Ile Ile Lys Asn Thr Cys Ser Glu Tyr Lys405 410
415Val Pro Tyr Leu Val Lys Asp Thr Phe Trp Gln Ala Phe Ala Ser
His420 425 430Leu Glu His Leu Arg Val Leu Gly Leu Arg Pro Lys Glu
Glu435 440 44551077DNASaprolegnia diclina (ATCC #56851) 5atgactgagg
ataagacgaa ggtcgagttc ccgacgctca cggagctcaa gcactcgatc 60ccgaacgcgt
gctttgagtc gaacctcggc ctctcgctct actacacggc ccgcgcgatc
120ttcaacgcgt cggcctcggc ggcgctgctc tacgcggcgc gctcgacgcc
gttcattgcc 180gataacgttc tgctccacgc gctcgtttgc gccacctaca
tctacgtgca gggcgtcatc 240ttctggggct tcttcacggt cggccacgac
tgcggccact cggccttctc gcgctaccac 300agcgtcaact ttatcatcgg
ctgcatcatg cactctgcga ttttgacgcc gttcgagagc 360tggcgcgtga
cgcaccgcca ccaccacaag aacacgggca acattgataa ggacgagatc
420ttttacccgc accggtcggt caaggacctc caggacgtgc gccaatgggt
ctacacgctc 480ggcggtgcgt ggtttgtcta cttgaaggtc gggtatgccc
cgcgcacgat gagccacttt 540gacccgtggg acccgctcct ccttcgccgc
gcgtcggccg tcatcgtgtc gctcggcgtc 600tgggccgcct tcttcgccgc
gtacgcgtac ctcacatact cgctcggctt tgccgtcatg 660ggcctctact
actatgcgcc gctctttgtc tttgcttcgt tcctcgtcat tacgaccttc
720ttgcaccaca acgacgaagc gacgccgtgg tacggcgact cggagtggac
gtacgtcaag 780ggcaacctct cgagcgtcga ccgctcgtac ggcgcgttcg
tggacaacct gagccaccac 840attggcacgc accaggtcca ccacttgttc
ccgatcattc cgcactacaa gctcaacgaa 900gccaccaagc actttgcggc
cgcgtacccg cacctcgtgc gcaggaacga cgagcccatc 960atcacggcct
tcttcaagac cgcgcacctc tttgtcaact acggcgctgt gcccgagacg
1020gcgcagatct tcacgctcaa agagtcggcc gcggccgcca aggccaagtc ggactaa
10776358PRTSaprolegnia declina (ATCC #56851) 6Met Ala Glu Asp Lys
Thr Lys Val Glu Phe Pro Thr Leu Thr Glu Leu1 5 10 15Lys His Ser Ile
Pro Asn Ala Cys Phe Glu Ser Asn Leu Gly Leu Ser20 25 30Leu Tyr Tyr
Thr Ala Arg Ala Ile Phe Asn Ala Ser Ala Ser Ala Ala35 40 45Leu Leu
Tyr Ala Ala Arg Ser Thr Pro Phe Ile Ala Asp Asn Val Leu50 55 60Leu
His Ala Leu Val Cys Ala Thr Tyr Ile Tyr Val Gln Gly Val Ile65 70 75
80Phe Trp Gly Phe Phe Thr Val Gly His Asp Cys Gly His Ser Ala Phe85
90 95Ser Arg Tyr His Ser Val Asn Phe Ile Ile Gly Cys Ile Met His
Ser100 105 110Ala Ile Leu Thr Pro Phe Glu Ser Trp Arg Val Thr His
Arg His His115 120 125His Lys Asn Thr Gly Asn Ile Asp Lys Asp Glu
Ile Phe Tyr Pro His130 135 140Arg Ser Val Lys Asp Leu Gln Asp Val
Arg Gln Trp Val Tyr Thr Leu145 150 155 160Gly Gly Ala Trp Phe Val
Tyr Leu Lys Val Gly Tyr Ala Pro Arg Thr165 170 175Met Ser His Phe
Asp Pro Trp Asp Pro Leu Leu Leu Arg Arg Ala Ser180 185 190Ala Val
Ile Val Ser Leu Gly Val Trp Ala Ala Phe Phe Ala Ala Tyr195 200
205Ala Tyr Leu Thr Tyr Ser Leu Gly Phe Ala Val Met Gly Leu Tyr
Tyr210 215 220Tyr Ala Pro Leu Phe Val Phe Ala Ser Phe Leu Val Ile
Thr Thr Phe225 230 235 240Leu His His Asn Asp Glu Ala Thr Pro Trp
Tyr Gly Asp Ser Glu Trp245 250 255Thr Tyr Val Lys Gly Asn Leu Ser
Ser Val Asp Arg Ser Tyr Gly Ala260 265 270Phe Val Asp Asn Leu Ser
His His Ile Gly Thr His Gln Val His His275 280 285Leu Phe Pro Ile
Ile Pro His Tyr Lys Leu Asn Glu Ala Thr Lys His290 295 300Phe Ala
Ala Ala Tyr Pro His Leu Val Arg Arg Asn Asp Glu Pro Ile305 310 315
320Ile Thr Ala Phe Phe Lys Thr Ala His Leu Phe Val Asn Tyr Gly
Ala325 330 335Val Pro Glu Thr Ala Gln Ile Phe Thr Leu Lys Glu Ser
Ala Ala Ala340 345 350Ala Lys Ala Lys Ser Asp3557957DNAMortierella
alpina AX464731 7atggagtcga ttgcgccatt cctcccatca aagatgccgc
aagatctgtt tatggacctt 60gccaccgcta tcggtgtccg ggccgcgccc tatgtcgatc
ctctcgaggc cgcgctggtg 120gcccaggccg agaagtacat ccccacgatt
gtccatcaca cgcgtgggtt cctggtcgcg 180gtggagtcgc ctttggcccg
tgagctgccg ttgatgaacc cgttccacgt gctgttgatc 240gtgctcgctt
atttggtcac ggtctttgtg ggcatgcaga tcatgaagaa ctttgagcgg
300ttcgaggtca agacgttttc gctcctgcac aacttttgtc tggtctcgat
cagcgcctac 360atgtgcggtg ggatcctgta cgaggcttat caggccaact
atggactgtt tgagaacgct 420gctgatcata ccttcaaggg tcttcctatg
gccaagatga tctggctctt ctacttctcc 480aagatcatgg agtttgtcga
caccatgatc atggtcctca agaagaacaa ccgccagatc 540tccttcttgc
acgtttacca ccacagctcc atcttcacca tctggtggtt ggtcaccttt
600gttgcaccca acggtgaagc ctacttctct gctgcgttga actcgttcat
ccatgtgatc 660atgtacggct actacttctt gtcggccttg ggcttcaagc
aggtgtcgtt catcaagttc 720tacatcacgc gctcgcagat gacacagttc
tgcatgatgt cggtccagtc ttcctgggac 780atgtacgcca tgaaggtcct
tggccgcccc ggatacccct tcttcatcac ggctctgctt 840tggttctaca
tgtggaccat gctcggtctc ttctacaact tttacagaaa gaacgccaag
900ttggccaagc aggccaaggc cgacgctgcc aaggagaagg caaggaagtt gcagtaa
9578318PRTMortierella alpina AX464731 8Met Glu Ser Ile Ala Pro Phe
Leu Pro Ser Lys Met Pro Gln Asp Leu1 5 10 15Phe Met Asp Leu Ala Thr
Ala Ile Gly Val Arg Ala Ala Pro Tyr Val20 25 30Asp Pro Leu Glu Ala
Ala Leu Val Ala Gln Ala Glu Lys Tyr Ile Pro35 40 45Thr Ile Val His
His Thr Arg Gly Phe Leu Val Ala Val Glu Ser Pro50 55 60Leu Ala Arg
Glu Leu Pro Leu Met Asn Pro Phe His Val Leu Leu Ile65 70 75 80Val
Leu Ala Tyr Leu Val Thr Val Phe Val Gly Met Gln Ile Met Lys85 90
95Asn Phe Glu Arg Phe Glu Val Lys Thr Phe Ser Leu Leu His Asn
Phe100 105 110Cys Leu Val Ser Ile Ser Ala Tyr Met Cys Gly Gly Ile
Leu Tyr Glu115 120 125Ala Tyr Gln Ala Asn Tyr Gly Leu Phe Glu Asn
Ala Ala Asp His Thr130 135 140Phe Lys Gly Leu Pro Met Ala Lys Met
Ile Trp Leu Phe Tyr Phe Ser145 150 155 160Lys Ile Met Glu Phe Val
Asp Thr Met Ile Met Val Leu Lys Lys Asn165 170 175Asn Arg Gln Ile
Ser Phe Leu His Val Tyr His His Ser Ser Ile Phe180 185 190Thr Ile
Trp Trp Leu Val Thr Phe Val Ala Pro Asn Gly Glu Ala Tyr195 200
205Phe Ser Ala Ala Leu Asn Ser Phe Ile His Val Ile Met Tyr Gly
Tyr210 215 220Tyr Phe Leu Ser Ala Leu Gly Phe Lys Gln Val Ser Phe
Ile Lys Phe225 230 235 240Tyr Ile Thr Arg Ser Gln Met Thr Gln Phe
Cys Met Met Ser Val Gln245 250 255Ser Ser Trp Asp Met Tyr Ala Met
Lys Val Leu Gly Arg Pro Gly Tyr260 265 270Pro Phe Phe Ile Thr Ala
Leu Leu Trp Phe Tyr Met Trp Thr Met Leu275 280 285Gly Leu Phe Tyr
Asn Phe Tyr Arg Lys Asn Ala Lys Leu Ala Lys Gln290 295 300Ala Lys
Ala Asp Ala Ala Lys Glu Lys Ala Arg Lys Leu Gln305 310
31591077DNASaprolegnia declina 9atggctgagg ataagaccaa ggtcgagttc
cctaccctga ctgagctgaa gcactctatc 60cctaacgctt gctttgagtc caacctcgga
ctctcgctct actacactgc ccgagcgatc 120ttcaacgcat ctgcctctgc
tgctctgctc tacgctgccc gatctactcc cttcattgcc 180gataacgttc
tgctccacgc tctggtttgc gccacctaca tctacgtgca gggtgtcatc
240ttctggggtt tctttaccgt cggtcacgac tgtggtcact ctgccttctc
ccgataccac 300tccgtcaact tcatcattgg ctgcatcatg cactctgcca
ttctgactcc cttcgagtcc 360tggcgagtga cccaccgaca ccatcacaag
aacactggca acattgataa ggacgagatc 420ttctaccctc atcggtccgt
caaggacctc caggacgtgc gacaatgggt ctacaccctc 480ggaggtgctt
ggtttgtcta cctgaaggtc ggatatgctc ctcgaaccat gtcccacttt
540gacccctggg accctctcct gcttcgacga gcctccgctg tcatcgtgtc
cctcggagtc 600tgggctgcct tcttcgctgc ctacgcctac ctcacatact
cgctcggctt tgccgtcatg 660ggcctctact actatgctcc tctctttgtc
tttgcttcgt tcctcgtcat tactaccttc 720ttgcatcaca acgacgaagc
tactccctgg tacggtgact cggagtggac ctacgtcaag 780ggcaacctga
gctccgtcga ccgatcgtac ggagctttcg tggacaacct gtctcaccac
840attggcaccc accaggtcca tcacttgttc cctatcattc cccactacaa
gctcaacgaa 900gccaccaagc actttgctgc cgcttaccct cacctcgtga
gacgtaacga cgagcccatc 960attactgcct tcttcaagac cgctcacctc
tttgtcaact acggagctgt gcccgagact 1020gctcagattt tcaccctcaa
agagtctgcc
gctgcagcca aggccaagag cgactaa 107710105DNAArtificial SequencePrimer
D17-1A 10catggctgag gataagacca aggtcgagtt ccctaccctg actgagctga
agcactctat 60ccctaacgct tgctttgagt ccaacctcgg actctcgctc tacta
10511106DNAArtificial SequencePrimer D17-1B 11cagtgtagta gagcgagagt
ccgaggttgg actcaaagca agcgttaggg atagagtgct 60tcagctcagt cagggtaggg
aactcgacct tggtcttatc ctcagc 10612106DNAArtificial SequencePrimer
D17-2A 12cactgcccga gcgatcttca acgcatctgc ctctgctgct ctgctctacg
ctgcccgatc 60tactcccttc attgccgata acgttctgct ccacgctctg gtttgc
10613106DNAArtificial SequencePrimer D17-2B 13gtggcgcaaa ccagagcgtg
gagcagaacg ttatcggcaa tgaagggagt agatcgggca 60gcgtagagca gagcagcaga
ggcagatgcg ttgaagatcg ctcggg 10614105DNAArtificial SequencePrimer
D17-3A 14gccacctaca tctacgtgca gggtgtcatc ttctggggtt tctttaccgt
cggtcacgac 60tgtggtcact ctgccttctc ccgataccac tccgtcaact tcatc
10515105DNAArtificial SequencePrimer D17-3B 15ccaatgatga agttgacgga
gtggtatcgg gagaaggcag agtgaccaca gtcgtgaccg 60acggtaaaga aaccccagaa
gatgacaccc tgcacgtaga tgtag 10516105DNAArtificial SequencePrimer
D17-4A 16attggctgca tcatgcactc tgccattctg actcccttcg agtcctggcg
agtgacccac 60cgacaccatc acaagaacac tggcaacatt gataaggacg agatc
10517105DNAArtificial SequencePrimer D17-4B 17tagaagatct cgtccttatc
aatgttgcca gtgttcttgt gatggtgtcg gtgggtcact 60cgccaggact cgaagggagt
cagaatggca gagtgcatga tgcag 10518105DNAArtificial SequencePrimer
D17-5A 18acgagatctt ctaccctcat cggtccgtca aggacctcca ggacgtgcga
caatgggtct 60acaccctcgg aggtgcttgg tttgtctacc tgaaggtcgg atatg
10519107DNAArtificial SequencePrimer D17-5B 19aggagcatat ccgaccttca
ggtagacaaa ccaagcacct ccgagggtgt agacccattg 60tcgcacgtcc tggaggtcct
tgacggaccg atgagggtag aagatct 10720105DNAArtificial SequencePrimer
D17-6A 20ctcctcgaac catgtcccac tttgacccct gggaccctct cctgcttcga
cgagcctccg 60ctgtcatcgt gtccctcgga gtctgggctg ccttcttcgc tgcct
10521106DNAArtificial SequencePrimer D17-6B 21aggcgtaggc agcgaagaag
gcagcccaga ctccgaggga cacgatgaca gcggaggctc 60gtcgaagcag gagagggtcc
caggggtcaa agtgggacat ggttcg 10622104DNAArtificial SequencePrimer
D17-7A 22acgcctacct cacatactcg ctcggctttg ccgtcatggg cctctactac
tatgctcctc 60tctttgtctt tgcttcgttc ctcgtcatta ctaccttctt gcat
10423103DNAArtificial SequencePrimer D17-7B 23ttgtgatgca agaaggtagt
aatgacgagg aacgaagcaa agacaaagag aggagcatag 60tagtagaggc ccatgacggc
aaagccgagc gagtatgtga ggt 10324106DNAArtificial SequencePrimer
D17-8A 24cacaacgacg aagctactcc ctggtacggt gactcggagt ggacctacgt
caagggcaac 60ctgagctccg tcgaccgatc gtacggagct ttcgtggaca acctgt
10625106DNAArtificial SequencePrimer D17-8B 25gtgagacagg ttgtccacga
aagctccgta cgatcggtcg acggagctca ggttgccctt 60gacgtaggtc cactccgagt
caccgtacca gggagtagct tcgtcg 10626102DNAArtificial SequencePrimer
D17-9A 26ctcaccacat tggcacccac caggtccatc acttgttccc tatcattccc
cactacaagc 60tcaacgaagc caccaagcac tttgctgccg cttaccctca cc
10227102DNAArtificial SequencePrimer D17-9B 27cacgaggtga gggtaagcgg
cagcaaagtg cttggtggct tcgttgagct tgtagtgggg 60aatgataggg aacaagtgat
ggacctggtg ggtgccaatg tg 1022876DNAArtificial SequencePrimer
D17-10A 28tcgtgagacg taacgacgag cccatcatta ctgccttctt caagaccgct
cacctctttg 60tcaactacgg agctgt 762976DNAArtificial SequencePrimer
D17-10B 29cgggcacagc tccgtagttg acaaagaggt gagcggtctt gaagaaggca
gtaatgatgg 60gctcgtcgtt acgtct 763067DNAArtificial SequencePrimer
D17-11A 30gcccgagact gctcagattt tcaccctcaa agagtctgcc gctgcagcca
aggccaagag 60cgactaa 673162DNAArtificial SequencePrimer D17-11B
31ttagtcgctc ttggccttgg ctgcagcggc agactctttg agggtgaaaa tctgagcagt
60ct 623232DNAArtificial SequencePrimer D17-1 32tttccatggc
tgaggataag accaaggtcg ag 323334DNAArtificial SequencePrimer D17-4R
33ccctagaaga tctcgtcctt atcaatgttg ccag 343427DNAArtificial
SequencePrimer D17-5 34cccacgagat cttctaccct catcggt
273524DNAArtificial SequencePrimer D17-8D 35gaaagctccg tacgatcggt
cgac 243624DNAArtificial SequencePrimer D17-8U 36gtcgaccgat
cgtacggagc tttc 243734DNAArtificial SequencePrimer D17-11
37aaagcggccg cttagtcgct cttggccttg gctg 343819DNAArtificial
SequencePrimer TEF5' 38agagaccggg ttggcggcg 193930DNAArtificial
SequencePrimer TEF3' 39ttggatcctt tgaatgattc ttatactcag
304029DNAArtificial SequencePrimer XPR5' 40tttccgcggc ccgagattcc
ggcctcttc 294131DNAArtificial SequencePrimer XPR3' 41tttccgcgga
cacaatatct ggtcaaattt c 314233DNAArtificial SequencePrimer YL21A
42tttccatggc tgaggataag acgaaggtcg agt 334336DNAArtificial
SequencePrimer YL22 43cccttaatta attagtccga cttggccttg gcggcc
364436DNAArtificial SequencePrimer YL53 44gccaagtcgg actaagctgc
taactagagc ggccgc 364536DNAArtificial SequencePrimer YL54
45gcggccgctc tagttagcag cttagtccga cttggc 364635DNAArtificial
SequencePrimer KU5 46tttgcccggg cgagtatctg tctgactcgt cattg
354733DNAArtificial SequencePrimer KU3 47aaagcccggg caaaggcctg
tttctcggtg tac 33481710DNAYarrowia lipolytica 48gtcgacgagt
atctgtctga ctcgtcattg ccgcctttgg agtacgactc caactatgag 60tgtgcttgga
tcactttgac gatacattct tcgttggagg ctgtgggtct gacagctgcg
120ttttcggcgc ggttggccga caacaatatc agctgcaacg tcattgctgg
ctttcatcat 180gatcacattt ttgtcggcaa aggcgacgcc cagagagcca
ttgacgttct ttctaatttg 240gaccgatagc cgtatagtcc agtctatcta
taagttcaac taactcgtaa ctattaccat 300aacatatact tcactgcccc
agataaggtt ccgataaaaa gttctgcaga ctaaatttat 360ttcagtctcc
tcttcaccac caaaatgccc tcctacgaag ctcgagctaa cgtccacaag
420tccgcctttg ccgctcgagt gctcaagctc gtggcagcca agaaaaccaa
cctgtgtgct 480tctctggatg ttaccaccac caaggagctc attgagcttg
ccgataaggt cggaccttat 540gtgtgcatga tcaagaccca tatcgacatc
attgacgact tcacctacgc cggcactgtg 600ctccccctca aggaacttgc
tcttaagcac ggtttcttcc tgttcgagga cagaaagttc 660gcagatattg
gcaacactgt caagcaccag tacaagaacg gtgtctaccg aatcgccgag
720tggtccgata tcaccaacgc ccacggtgta cccggaaccg gaatcattgc
tggcctgcga 780gctggtgccg aggaaactgt ctctgaacag aagaaggagg
acgtctctga ctacgagaac 840tcccagtaca aggagttcct ggtcccctct
cccaacgaga agctggccag aggtctgctc 900atgctggccg agctgtcttg
caagggctct ctggccactg gcgagtactc caagcagacc 960attgagcttg
cccgatccga ccccgagttt gtggttggct tcattgccca gaaccgacct
1020aagggcgact ctgaggactg gcttattctg acccccgggg tgggtcttga
cgacaaggga 1080gacgctctcg gacagcagta ccgaactgtt gaggatgtca
tgtctaccgg aacggatatc 1140ataattgtcg gccgaggtct gtacggccag
aaccgagatc ctattgagga ggccaagcga 1200taccagaagg ctggctggga
ggcttaccag aagattaact gttagaggtt agactatgga 1260tatgtcattt
aactgtgtat atagagagcg tgcaagtatg gagcgcttgt tcagcttgta
1320tgatggtcag acgacctgtc tgatcgagta tgtatgatac tgcacaacct
gtgtatccgc 1380atgatctgtc caatggggca tgttgttgtg tttctcgata
cggagatgct gggtacaagt 1440agctaatacg attgaactac ttatacttat
atgaggcttg aagaaagctg acttgtgtat 1500gacttattct caactacatc
cccagtcaca ataccaccac tgcactacca ctacaccaaa 1560accatgatca
aaccacccat ggacttcctg gaggcagaag aacttgttat ggaaaagctc
1620aagagagaga agccaagata ctatcaagac atgtgtcgca acttcaagga
ggaccaagct 1680ctgtacaccg agaaacaggc ctttgtcgac
171049286PRTYarrowia lipolytica 49Met Pro Ser Tyr Glu Ala Arg Ala
Asn Val His Lys Ser Ala Phe Ala1 5 10 15Ala Arg Val Leu Lys Leu Val
Ala Ala Lys Lys Thr Asn Leu Cys Ala20 25 30Ser Leu Asp Val Thr Thr
Thr Lys Glu Leu Ile Glu Leu Ala Asp Lys35 40 45Val Gly Pro Tyr Val
Cys Met Ile Lys Thr His Ile Asp Ile Ile Asp50 55 60Asp Phe Thr Tyr
Ala Gly Thr Val Leu Pro Leu Lys Glu Leu Ala Leu65 70 75 80Lys His
Gly Phe Phe Leu Phe Glu Asp Arg Lys Phe Ala Asp Ile Gly85 90 95Asn
Thr Val Lys His Gln Tyr Lys Asn Gly Val Tyr Arg Ile Ala Glu100 105
110Trp Ser Asp Ile Thr Asn Ala His Gly Val Pro Gly Thr Gly Ile
Ile115 120 125Ala Gly Leu Arg Ala Gly Ala Glu Glu Thr Val Ser Glu
Gln Lys Lys130 135 140Glu Asp Val Ser Asp Tyr Glu Asn Ser Gln Tyr
Lys Glu Phe Leu Val145 150 155 160Pro Ser Pro Asn Glu Lys Leu Ala
Arg Gly Leu Leu Met Leu Ala Glu165 170 175Leu Ser Cys Lys Gly Ser
Leu Ala Thr Gly Glu Tyr Ser Lys Gln Thr180 185 190Ile Glu Leu Ala
Arg Ser Asp Pro Glu Phe Val Val Gly Phe Ile Ala195 200 205Gln Asn
Arg Pro Lys Gly Asp Ser Glu Asp Trp Leu Ile Leu Thr Pro210 215
220Gly Val Gly Leu Asp Asp Lys Gly Asp Ala Leu Gly Gln Gln Tyr
Arg225 230 235 240Thr Val Glu Asp Val Met Ser Thr Gly Thr Asp Ile
Ile Ile Val Gly245 250 255Arg Gly Leu Tyr Gly Gln Asn Arg Asp Pro
Ile Glu Glu Ala Lys Arg260 265 270Tyr Gln Lys Ala Gly Trp Glu Ala
Tyr Gln Lys Ile Asn Cys275 280 2855035DNAArtificial SequencePrimer
KI5 50agagcggccg catgggagaa gtgggaccca caaac 355138DNAArtificial
SequencePrimer KI3 51gtggcggccg ctcaaatgtc gttattgtac caataaac
38521152DNAImpatients balsama 52atgggagaag tgggacccac aaaccgaacc
aaaaccaagt tggacaagca acaagaatcc 60gaaaacaggg ttcctcacga gccacctcca
ttcacactaa gtgaccttaa gaaagccatc 120ccaccccatt gcttcgagcg
ctccctcgtg aaatcattct accacgtgat tcacgacatt 180atcatcctgt
cctttttcta ctatgtcgcc gccaattaca tccccatgct accccaaaac
240ctccgttacg ttgcatggcc aatttattgg gccatccaag gctgtgtcca
acttggtata 300ttggtcttag gccatgaatg cggccaccac gccttcagcg
actaccaatg ggtagacgac 360atggtcgggt tcgtcctcca ctcgtcccaa
ttgattccct acttctcatg gaaacatagc 420caccgtcgcc accactccaa
cacggcctcc atcgagcgcg acgaggtcta cccgcccgcg 480tacaaaaacg
acctgccgtg gttcgccaaa tacctacgca accccgtcgg tcgtttcctc
540atgattttcg gggcgctact gttcggctgg ccgtcgtacc ttctgttcaa
cgcgaacggc 600cgtctctacg accgcttcgc ttcccactac gacccgcaat
ccccgatctt caacaaccgc 660gagaggctgc aagtgatcgc gtccgacgtc
gggctcgtct tcgcgtactt tgtcctgtac 720aagatcgcgc tggccaaggg
atttgtgtgg ttaatttgtg tgtatggcgt cccgtacgtg 780atcctcaacg
ggcttatcgt cttgatcacg ttcctacagc acacgcaccc gaatctgccc
840cgttacgacc tttccgagtg ggactggctt aggggagccc tgtcgactgt
ggaccgcgat 900tacgggatgt tgaataaggt gttccataac gtgacggaca
cgcacttggt gcatcatttg 960ttcacgacca tgccacatta tcgcgccaag
gaggcgaccg aggtgattaa accgatattg 1020ggagactact ataagtttga
cgacactccg tttctcaaag cgttgtggaa ggacatggga 1080aagtgtattt
atgtggagtc ggacgtgcct ggcaagaaca agggagttta ttggtacaat
1140aacgacattt ga 115253383PRTImpatients balsama 53Met Gly Glu Val
Gly Pro Thr Asn Arg Thr Lys Thr Lys Leu Asp Lys1 5 10 15Gln Gln Glu
Ser Glu Asn Arg Val Pro His Glu Pro Pro Pro Phe Thr20 25 30Leu Ser
Asp Leu Lys Lys Ala Ile Pro Pro His Cys Phe Glu Arg Ser35 40 45Leu
Val Lys Ser Phe Tyr His Val Ile His Asp Ile Ile Ile Leu Ser50 55
60Phe Phe Tyr Tyr Val Ala Ala Asn Tyr Ile Pro Met Leu Pro Gln Asn65
70 75 80Leu Arg Tyr Val Ala Trp Pro Ile Tyr Trp Ala Ile Gln Gly Cys
Val85 90 95Gln Leu Gly Ile Leu Val Leu Gly His Glu Cys Gly His His
Ala Phe100 105 110Ser Asp Tyr Gln Trp Val Asp Asp Met Val Gly Phe
Val Leu His Ser115 120 125Ser Gln Leu Ile Pro Tyr Phe Ser Trp Lys
His Ser His Arg Arg His130 135 140His Ser Asn Thr Ala Ser Ile Glu
Arg Asp Glu Val Tyr Pro Pro Ala145 150 155 160Tyr Lys Asn Asp Leu
Pro Trp Phe Ala Lys Tyr Leu Arg Asn Pro Val165 170 175Gly Arg Phe
Leu Met Ile Phe Gly Ala Leu Leu Phe Gly Trp Pro Ser180 185 190Tyr
Leu Leu Phe Asn Ala Asn Gly Arg Leu Tyr Asp Arg Phe Ala Ser195 200
205His Tyr Asp Pro Gln Ser Pro Ile Phe Asn Asn Arg Glu Arg Leu
Gln210 215 220Val Ile Ala Ser Asp Val Gly Leu Val Phe Ala Tyr Phe
Val Leu Tyr225 230 235 240Lys Ile Ala Leu Ala Lys Gly Phe Val Trp
Leu Ile Cys Val Tyr Gly245 250 255Val Pro Tyr Val Ile Leu Asn Gly
Leu Ile Val Leu Ile Thr Phe Leu260 265 270Gln His Thr His Pro Asn
Leu Pro Arg Tyr Asp Leu Ser Glu Trp Asp275 280 285Trp Leu Arg Gly
Ala Leu Ser Thr Val Asp Arg Asp Tyr Gly Met Leu290 295 300Asn Lys
Val Phe His Asn Val Thr Asp Thr His Leu Val His His Leu305 310 315
320Phe Thr Thr Met Pro His Tyr Arg Ala Lys Glu Ala Thr Glu Val
Ile325 330 335Lys Pro Ile Leu Gly Asp Tyr Tyr Lys Phe Asp Asp Thr
Pro Phe Leu340 345 350Lys Ala Leu Trp Lys Asp Met Gly Lys Cys Ile
Tyr Val Glu Ser Asp355 360 365Val Pro Gly Lys Asn Lys Gly Val Tyr
Trp Tyr Asn Asn Asp Ile370 375 3805434DNAArtificial SequencePrimer
KTI5 54aagctcgaga ccgggttggc ggcgtatttg tgtc 345538DNAArtificial
SequencePrimer KTI3 55ggtctcgaga tctccaccgc ggacacaata tctggtca
38561756DNAArtificial SequenceTEF/conjugase/XPR chimeric gene
56gaccgggttg gcggcgtatt tgtgtcccaa aaaacagccc caattgcccc aattgacccc
60aaattgaccc agtagcgggc ccaaccccgg cgagagcccc cttcacccca catatcaaac
120ctcccccggt tcccacactt gccgttaagg gcgtagggta ctgcagtctg
gaatctacgc 180ttgttcagac tttgtactag tttctttgtc tggccatccg
ggtaacccat gccggacgca 240aaatagacta ctgaaaattt ttttgctttg
tggttgggac tttagccaag ggtataaaag 300accaccgtcc ccgaattacc
tttcctcttc ttttctctct ctccttgtca actcacaccc 360gaaatcgtta
agcatttcct tctgagtata agaatcattc aaaggatcca ctagttctag
420agcggccgca tgggagaagt gggacccaca aaccgaacca aaaccaagtt
ggacaagcaa 480caagaatccg aaaacagggt tcctcacgag ccacctccat
tcacactaag tgaccttaag 540aaagccatcc caccccattg cttcgagcgc
tccctcgtga aatcattcta ccacgtgatt 600cacgacatta tcatcctgtc
ctttttctac tatgtcgccg ccaattacat ccccatgcta 660ccccaaaacc
tccgttacgt tgcatggcca atttattggg ccatccaagg ctgtgtccaa
720cttggtatat tggtcttagg ccatgaatgc ggccaccacg ccttcagcga
ctaccaatgg 780gtagacgaca tggtcgggtt cgtcctccac tcgtcccaat
tgattcccta cttctcatgg 840aaacatagcc accgtcgcca ccactccaac
acggcctcca tcgagcgcga cgaggtctac 900ccgcccgcgt acaaaaacga
cctgccgtgg ttcgccaaat acctacgcaa ccccgtcggt 960cgtttcctca
tgattttcgg ggcgctactg ttcggctggc cgtcgtacct tctgttcaac
1020gcgaacggcc gtctctacga ccgcttcgct tcccactacg acccgcaatc
cccgatcttc 1080aacaaccgcg agaggctgca agtgatcgcg tccgacgtcg
ggctcgtctt cgcgtacttt 1140gtcctgtaca agatcgcgct ggccaaggga
tttgtgtggt taatttgtgt gtatggcgtc 1200ccgtacgtga tcctcaacgg
gcttatcgtc ttgatcacgt tcctacagca cacgcacccg 1260aatctgcccc
gttacgacct ttccgagtgg gactggctta ggggagccct gtcgactgtg
1320gaccgcgatt acgggatgtt gaataaggtg ttccataacg tgacggacac
gcacttggtg 1380catcatttgt tcacgaccat gccacattat cgcgccaagg
aggcgaccga ggtgattaaa 1440ccgatattgg gagactacta taagtttgac
gacactccgt ttctcaaagc gttgtggaag 1500gacatgggaa agtgtattta
tgtggagtcg gacgtgcctg gcaagaacaa gggagtttat 1560tggtacaata
acgacatttg agcggccgcc accgcggccc gagattccgg cctcttcggc
1620cgccaagcga cccgggtgga cgtctagagg tacctagcaa ttaacagata
gtttgccggt 1680gataattctc ttaacctccc acactccttt gacataacga
tttatgtaac gaaactgaaa 1740tttgaccaga tattgt 175657383PRTArtificial
SequenceTEF/conjugase/XPR chimeric protein 57Met Gly Glu Val Gly
Pro Thr Asn Arg Thr Lys Thr Lys Leu Asp Lys1 5 10 15Gln Gln Glu Ser
Glu Asn Arg Val Pro His Glu Pro Pro Pro Phe Thr20 25 30Leu Ser Asp
Leu Lys Lys Ala Ile Pro Pro His Cys Phe Glu Arg Ser35 40 45Leu Val
Lys Ser Phe Tyr His Val Ile His Asp Ile Ile Ile Leu Ser50 55 60Phe
Phe Tyr Tyr Val Ala Ala Asn Tyr Ile Pro Met Leu Pro Gln Asn65 70 75
80Leu Arg Tyr Val Ala Trp Pro Ile Tyr Trp Ala Ile Gln Gly Cys Val85
90 95Gln Leu Gly Ile Leu Val Leu Gly His Glu Cys Gly His His Ala
Phe100 105 110Ser Asp Tyr Gln Trp Val Asp Asp Met Val Gly Phe Val
Leu His Ser115 120 125Ser Gln Leu Ile Pro Tyr
Phe Ser Trp Lys His Ser His Arg Arg His130 135 140His Ser Asn Thr
Ala Ser Ile Glu Arg Asp Glu Val Tyr Pro Pro Ala145 150 155 160Tyr
Lys Asn Asp Leu Pro Trp Phe Ala Lys Tyr Leu Arg Asn Pro Val165 170
175Gly Arg Phe Leu Met Ile Phe Gly Ala Leu Leu Phe Gly Trp Pro
Ser180 185 190Tyr Leu Leu Phe Asn Ala Asn Gly Arg Leu Tyr Asp Arg
Phe Ala Ser195 200 205His Tyr Asp Pro Gln Ser Pro Ile Phe Asn Asn
Arg Glu Arg Leu Gln210 215 220Val Ile Ala Ser Asp Val Gly Leu Val
Phe Ala Tyr Phe Val Leu Tyr225 230 235 240Lys Ile Ala Leu Ala Lys
Gly Phe Val Trp Leu Ile Cys Val Tyr Gly245 250 255Val Pro Tyr Val
Ile Leu Asn Gly Leu Ile Val Leu Ile Thr Phe Leu260 265 270Gln His
Thr His Pro Asn Leu Pro Arg Tyr Asp Leu Ser Glu Trp Asp275 280
285Trp Leu Arg Gly Ala Leu Ser Thr Val Asp Arg Asp Tyr Gly Met
Leu290 295 300Asn Lys Val Phe His Asn Val Thr Asp Thr His Leu Val
His His Leu305 310 315 320Phe Thr Thr Met Pro His Tyr Arg Ala Lys
Glu Ala Thr Glu Val Ile325 330 335Lys Pro Ile Leu Gly Asp Tyr Tyr
Lys Phe Asp Asp Thr Pro Phe Leu340 345 350Lys Ala Leu Trp Lys Asp
Met Gly Lys Cys Ile Tyr Val Glu Ser Asp355 360 365Val Pro Gly Lys
Asn Lys Gly Val Tyr Trp Tyr Asn Asn Asp Ile370 375
3805832DNAArtificial SequencePrimer KH5 58tagagcggcc gcttaaacca
tgaaaaagcc tg 325933DNAArtificial SequencePrimer KH3 59gtggcggccg
ctttaggtac ctcactattc ctt 33601026DNAEscherichia coli 60atgaaaaagc
ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac 60agcgtctccg
acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat
120gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt
ctacaaagat 180cgttatgttt atcggcactt tgcatcggcc gcgctcccga
ttccggaagt gcttgacatt 240ggggaattca gcgagagcct gacctattgc
atctcccgcc gtgcacaggg tgtcacgttg 300caagacctgc ctgaaaccga
actgcccgct gttctgcagc cggtcgcgga ggccatggat 360gcgatcgctg
cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga
420atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc
ccatgtgtat 480cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg
tcgcgcaggc tctcgatgag 540ctgatgcttt gggccgagga ctgccccgaa
gtccggcacc tcgtgcacgc ggatttcggc 600tccaacaatg tcctgacgga
caatggccgc ataacagcgg tcattgactg gagcgaggcg 660atgttcgggg
attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct
720tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc
aggatcgccg 780cggctccggg cgtatatgct ccgcattggt cttgaccaac
tctatcagag cttggttgac 840ggcaatttcg atgatgcagc ttgggcgcag
ggtcgatgcg acgcaatcgt ccgatccgga 900gccgggactg tcgggcgtac
acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc 960tgtgtagaag
tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag 1020gaatag
102661341PRTEscherichia coli 61Met Lys Lys Pro Glu Leu Thr Ala Thr
Ser Val Glu Lys Phe Leu Ile1 5 10 15Glu Lys Phe Asp Ser Val Ser Asp
Leu Met Gln Leu Ser Glu Gly Glu20 25 30Glu Ser Arg Ala Phe Ser Phe
Asp Val Gly Gly Arg Gly Tyr Val Leu35 40 45Arg Val Asn Ser Cys Ala
Asp Gly Phe Tyr Lys Asp Arg Tyr Val Tyr50 55 60Arg His Phe Ala Ser
Ala Ala Leu Pro Ile Pro Glu Val Leu Asp Ile65 70 75 80Gly Glu Phe
Ser Glu Ser Leu Thr Tyr Cys Ile Ser Arg Arg Ala Gln85 90 95Gly Val
Thr Leu Gln Asp Leu Pro Glu Thr Glu Leu Pro Ala Val Leu100 105
110Gln Pro Val Ala Glu Ala Met Asp Ala Ile Ala Ala Ala Asp Leu
Ser115 120 125Gln Thr Ser Gly Phe Gly Pro Phe Gly Pro Gln Gly Ile
Gly Gln Tyr130 135 140Thr Thr Trp Arg Asp Phe Ile Cys Ala Ile Ala
Asp Pro His Val Tyr145 150 155 160His Trp Gln Thr Val Met Asp Asp
Thr Val Ser Ala Ser Val Ala Gln165 170 175Ala Leu Asp Glu Leu Met
Leu Trp Ala Glu Asp Cys Pro Glu Val Arg180 185 190His Leu Val His
Ala Asp Phe Gly Ser Asn Asn Val Leu Thr Asp Asn195 200 205Gly Arg
Ile Thr Ala Val Ile Asp Trp Ser Glu Ala Met Phe Gly Asp210 215
220Ser Gln Tyr Glu Val Ala Asn Ile Phe Phe Trp Arg Pro Trp Leu
Ala225 230 235 240Cys Met Glu Gln Gln Thr Arg Tyr Phe Glu Arg Arg
His Pro Glu Leu245 250 255Ala Gly Ser Pro Arg Leu Arg Ala Tyr Met
Leu Arg Ile Gly Leu Asp260 265 270Gln Leu Tyr Gln Ser Leu Val Asp
Gly Asn Phe Asp Asp Ala Ala Trp275 280 285Ala Gln Gly Arg Cys Asp
Ala Ile Val Arg Ser Gly Ala Gly Thr Val290 295 300Gly Arg Thr Gln
Ile Ala Arg Arg Ser Ala Ala Val Trp Thr Asp Gly305 310 315 320Cys
Val Glu Val Leu Ala Asp Ser Gly Asn Arg Arg Pro Ser Thr Arg325 330
335Pro Arg Ala Lys Glu3406234DNAArtificial SequencePrimer KTH5
62tttagatctc gagaccgggt tggcggcgta tttg 346331DNAArtificial
SequencePrimer KTH3 63tttagatctc caccgcggac acaatatctg g
31641650DNAArtificial SequenceTEF::HPT::XPR fusion 64gaccgggttg
gcggcgtatt tgtgtcccaa aaaacagccc caattgcccc aattgacccc 60aaattgaccc
agtagcgggc ccaaccccgg cgagagcccc cttcacccca catatcaaac
120ctcccccggt tcccacactt gccgttaagg gcgtagggta ctgcagtctg
gaatctacgc 180ttgttcagac tttgtactag tttctttgtc tggccatccg
ggtaacccat gccggacgca 240aaatagacta ctgaaaattt ttttgctttg
tggttgggac tttagccaag ggtataaaag 300accaccgtcc ccgaattacc
tttcctcttc ttttctctct ctccttgtca actcacaccc 360gaaatcgtta
agcatttcct tctgagtata agaatcattc aaaggatcca ctagttctag
420agcggccgct taaaccatga aaaagcctga actcaccgcg acgtctgtcg
agaagtttct 480gatcgaaaag ttcgacagcg tctccgacct gatgcagctc
tcggagggcg aagaatctcg 540tgctttcagc ttcgatgtag gagggcgtgg
atatgtcctg cgggtaaata gctgcgccga 600tggtttctac aaagatcgtt
atgtttatcg gcactttgca tcggccgcgc tcccgattcc 660ggaagtgctt
gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc
720acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc
tgcagccggt 780cgcggaggcc atggatgcga tcgctgcggc cgatcttagc
cagacgagcg ggttcggccc 840attcggaccg caaggaatcg gtcaatacac
tacatggcgt gatttcatat gcgcgattgc 900tgatccccat gtgtatcact
ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc 960gcaggctctc
gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt
1020gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa
cagcggtcat 1080tgactggagc gaggcgatgt tcggggattc ccaatacgag
gtcgccaaca tcttcttctg 1140gaggccgtgg ttggcttgta tggagcagca
gacgcgctac ttcgagcgga ggcatccgga 1200gcttgcagga tcgccgcggc
tccgggcgta tatgctccgc attggtcttg accaactcta 1260tcagagcttg
gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc
1320aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca
gaagcgcggc 1380cgtctggacc gatggctgtg tagaagtact cgccgatagt
ggaaaccgac gccccagcac 1440tcgtccgagg gcaaaggaat agtgaggtac
ctaaagcggc cgccaccgcg gcccgagatt 1500ccggcctctt cggccgccaa
gcgacccggg tggacgtcta gaggtaccta gcaattaaca 1560gatagtttgc
cggtgataat tctcttaacc tcccacactc ctttgacata acgatttatg
1620taacgaaact gaaatttgac cagatattgt 165065341PRTArtificial
SequenceTEF::HPT::XPR fusion 65Met Lys Lys Pro Glu Leu Thr Ala Thr
Ser Val Glu Lys Phe Leu Ile1 5 10 15Glu Lys Phe Asp Ser Val Ser Asp
Leu Met Gln Leu Ser Glu Gly Glu20 25 30Glu Ser Arg Ala Phe Ser Phe
Asp Val Gly Gly Arg Gly Tyr Val Leu35 40 45Arg Val Asn Ser Cys Ala
Asp Gly Phe Tyr Lys Asp Arg Tyr Val Tyr50 55 60Arg His Phe Ala Ser
Ala Ala Leu Pro Ile Pro Glu Val Leu Asp Ile65 70 75 80Gly Glu Phe
Ser Glu Ser Leu Thr Tyr Cys Ile Ser Arg Arg Ala Gln85 90 95Gly Val
Thr Leu Gln Asp Leu Pro Glu Thr Glu Leu Pro Ala Val Leu100 105
110Gln Pro Val Ala Glu Ala Met Asp Ala Ile Ala Ala Ala Asp Leu
Ser115 120 125Gln Thr Ser Gly Phe Gly Pro Phe Gly Pro Gln Gly Ile
Gly Gln Tyr130 135 140Thr Thr Trp Arg Asp Phe Ile Cys Ala Ile Ala
Asp Pro His Val Tyr145 150 155 160His Trp Gln Thr Val Met Asp Asp
Thr Val Ser Ala Ser Val Ala Gln165 170 175Ala Leu Asp Glu Leu Met
Leu Trp Ala Glu Asp Cys Pro Glu Val Arg180 185 190His Leu Val His
Ala Asp Phe Gly Ser Asn Asn Val Leu Thr Asp Asn195 200 205Gly Arg
Ile Thr Ala Val Ile Asp Trp Ser Glu Ala Met Phe Gly Asp210 215
220Ser Gln Tyr Glu Val Ala Asn Ile Phe Phe Trp Arg Pro Trp Leu
Ala225 230 235 240Cys Met Glu Gln Gln Thr Arg Tyr Phe Glu Arg Arg
His Pro Glu Leu245 250 255Ala Gly Ser Pro Arg Leu Arg Ala Tyr Met
Leu Arg Ile Gly Leu Asp260 265 270Gln Leu Tyr Gln Ser Leu Val Asp
Gly Asn Phe Asp Asp Ala Ala Trp275 280 285Ala Gln Gly Arg Cys Asp
Ala Ile Val Arg Ser Gly Ala Gly Thr Val290 295 300Gly Arg Thr Gln
Ile Ala Arg Arg Ser Ala Ala Val Trp Thr Asp Gly305 310 315 320Cys
Val Glu Val Leu Ala Asp Ser Gly Asn Arg Arg Pro Ser Thr Arg325 330
335Pro Arg Ala Lys Glu34066401DNAYarrowia lipolytica 66cgagtatctg
tctgactcgt cattgccgcc tttggagtac gactccaact atgagtgtgc 60ttggatcact
ttgacgatac attcttcgtt ggaggctgtg ggtctgacag ctgcgttttc
120ggcgcggttg gccgacaaca atatcagctg caacgtcatt gctggctttc
atcatgatca 180catttttgtc ggcaaaggcg acgcccagag agccattgac
gttctttcta atttggaccg 240atagccgtat agtccagtct atctataagt
tcaactaact cgtaactatt accataacat 300atacttcact gccccagata
aggttccgat aaaaagttct gcagactaaa tttatttcag 360tctcctcttc
accaccaaaa tgccctccta cgaagctcga g 40167568DNAYarrowia lipolytica
67atcataattg tcggccgagg tctgtacggc cagaaccgag atcctattga ggaggccaag
60cgataccaga aggctggctg ggaggcttac cagaagatta actgttagag gttagactat
120ggatatgtca tttaactgtg tatatagaga gcgtgcaagt atggagcgct
tgttcagctt 180gtatgatggt cagacgacct gtctgatcga gtatgtatga
tactgcacaa cctgtgtatc 240cgcatgatct gtccaatggg gcatgttgtt
gtgtttctcg atacggagat gctgggtaca 300agtagctaat acgattgaac
tacttatact tatatgaggc ttgaagaaag ctgacttgtg 360tatgacttat
tctcaactac atccccagtc acaataccac cactgcacta ccactacacc
420aaaaccatga tcaaaccacc catggacttc ctggaggcag aagaacttgt
tatggaaaag 480ctcaagagag agaagccaag atactatcaa gacatgtgtc
gcaacttcaa ggaggaccaa 540gctctgtaca ccgagaaaca ggcctttg
5686836DNAArtificial SequencePrimer YL63 68ttatgatatc gaattaatta
acctgcagcc cggggg 366936DNAArtificial SequencePrimer YL64
69cccccgggct gcaggttaat taattcgata tcataa 367033DNAArtificial
SequencePrimer YL65 70tacgccgcca acccgtacgt ctcgagcttc gta
337133DNAArtificial SequencePrimer YL66 71tacgaagctc gagacgtacg
ggttggcggc gta 337230DNAArtificial SequencePrimer YL11 72ttttccatgg
gaacggacca aggaaaaacc 307330DNAArtificial SequencePrimer YL12
73tttgcggccg cctactcttc cttgggacgg 307433DNAArtificial
SequencePrimer YL81 74gttatccgct cacaagcttc cacacaacgt acg
337533DNAArtificial SequencePrimer YL82 75cgtacgttgt gtggaagctt
gtgagcggat aac 337633DNAArtificial SequencePrimer YL83 76atttgaatcg
aatcgatgag cctaaaatga acc 337733DNAArtificial SequencePrimer YL84
77ggttcatttt aggctcatcg attcgattca aat 337838DNAArtificial
SequencePrimer YL105 78ccaagcacta acctaccgtt taaacaccac taaaaccc
387938DNAArtificial SequencePrimer YL106 79gggttttagt ggtgtttaaa
cggtaggtta gtgcttgg 388036DNAArtificial SequencePrimer YL119
80cgggaaacct gtcgtggcgc gccagctgca ttaatg 368136DNAArtificial
SequencePrimer YL120 81cattaatgca gctggcgcgc cacgacaggt ttcccg
368234DNAArtificial SequencePrimer YL121 82tttggcgcgc ctatcacatc
acgctctcat caag 348334DNAArtificial SequencePrimer YL122
83tttcgtacga accaccaccg tcagcccttc tgac 3484440DNAYarrowia
lipolytica 84aaccaccacc gtcagccctt ctgactcacg tattgtagcc accgacacag
gcaacagtcc 60gtggatagca gaatatgtct tgtcggtcca tttctcacca actttaggcg
tcaagtgaat 120gttgcagaag aagtatgtgc cttcattgag aatcggtgtt
gctgatttca ataaagtctt 180gagatcagtt tggccagtca tgttgtgggg
ggtaattgga ttgagttatc gcctacagtc 240tgtacaggta tactcgctgc
ccactttata ctttttgatt ccgctgcact tgaagcaatg 300tcgtttacca
aaagtgagaa tgctccacag aacacacccc agggtatggt tgagcaaaaa
360ataaacactc cgatacgggg aatcgaaccc cggtctccac ggttctcaag
aagtattctt 420gatgagagcg tgatgtgata 4408535DNAArtificial
SequencePrimer YL114 85tgatagtatc ttggcgcgcc ttctctctct tgagc
358635DNAArtificial SequencePrimer YL115 86gctcaagaga gagaaggcgc
gccaagatac tatca 35875218DNAArtificial Sequence5218 bp fragment for
integration and expression of the delta-5 desaturase gene
87tatcacatca cgctctcatc aagaatactt cttgagaacc gtggagaccg gggttcgatt
60ccccgtatcg gagtgtttat tttttgctca accataccct ggggtgtgtt ctgtggagca
120ttctcacttt tggtaaacga cattgcttca agtgcagcgg aatcaaaaag
tataaagtgg 180gcagcgagta tacctgtaca gactgtaggc gataactcaa
tccaattacc ccccacaaca 240tgactggcca aactgatctc aagactttat
tgaaatcagc aacaccgatt ctcaatgaag 300gcacatactt cttctgcaac
attcacttga cgcctaaagt tggtgagaaa tggaccgaca 360agacatattc
tgctatccac ggactgttgc ctgtgtcggt ggctacaata cgtgagtcag
420aagggctgac ggtggtggtt cgtacgttgt gtggaagctt gtgagcggat
aacaatttca 480cacaggaaac agctatgacc atgattacgc caagctcgaa
attaaccctc actaaaggga 540acaaaagctg gagctccacc gcggacacaa
tatctggtca aatttcagtt tcgttacata 600aatcgttatg tcaaaggagt
gtgggaggtt aagagaatta tcaccggcaa actatctgtt 660aattgctagg
tacctctaga cgtccacccg ggtcgcttgg cggccgaaga ggccggaatc
720tcgggccgcg gtggcggccg cctactcttc cttgggacgg agtccaagaa
cacgcaagtg 780ctccaaatgt gaagcaaatg cttgccaaaa cgtatccttg
acaaggtatg gaaccttgta 840ctcgctgcag gtgttcttga tgatggccag
aatatcggga taatggtgct gcgacacgtt 900ggggaacaga tggtgcacag
ccggtagttc aagctgccag tgatgctggt ccagaggtgc 960gaatcgtgtg
cgtaatcctg cgtagtctcg acctgcatag ctgcccagtc cttttggatg
1020atcccgttct cgtcaggcaa cggccactga acttcctcaa caacgtggtt
cgcctggaag 1080gtcagcgcca gccagtaaga cgacaccatg tccgcgaccg
tgaacaagag cagcaccttg 1140cccaggggca gatactgcag gggaacaatc
aggcgatacc agacaaagaa agccttgccg 1200ccccagaaca tcacagtgtg
ccatgtcgag atgggattga cacgaatagc gtcattggtc 1260ttgacaaagt
acaaaatgtt gatgtcctga atgcgcacct tgaacgccag cagtccgtac
1320aggaaaggaa caaacatgtg ctggttgatg tggttgacaa accacttttg
gttgggcttg 1380atacgacgaa catcgggctc agacgtcgac acgtcgggat
ctgctccagc aatgttggtg 1440taggggtgat ggccgagcat atgttggtac
atccacacca ggtacgatgc tccgttgaaa 1500aagtcgtgcg tggctcccag
aatcttccag acagtggggt tgtgggtcac tgaaaagtga 1560gacgcatcat
gaagagggtt gagtccgact tgtgcgcacg caaatcccat gatgattgca
1620aacaccacct gaagccatgt gcgttcgaca acgaaaggca caaagagctg
cgcgtagtag 1680gaagcgatca aggatccaaa gataagagcg tatcgtcccc
agatctctgg tctattcttg 1740ggatcaatgt tccgatccgt aaagtagccc
tcgactctcg tcttgatggt tttgtggaac 1800accgttggct ccgggaagat
gggcagctca ttcgagacca gtgtaccgac atagtacttc 1860ttcataatgg
catctgcagc cccaaacgcg tgatacatct caaagaccgg agtaacatct
1920cggccagctc cgagcaggag agtgtccact ccaccaggat ggcggctcaa
gaactttgtg 1980acatcgtaca ccctgccgcg gatggccaag agtaggtcgt
ccttggtgtt atgggccgcc 2040agctcttccc aggtgaaggt ttttccttgg
tccgttccca tggtgaatga ttcttatact 2100cagaaggaaa tgcttaacga
tttcgggtgt gagttgacaa ggagagagag aaaagaagag 2160gaaaggtaat
tcggggacgg tggtctttta tacccttggc taaagtccca accacaaagc
2220aaaaaaattt tcagtagtct attttgcgtc cggcatgggt tacccggatg
gccagacaaa 2280gaaactagta caaagtctga acaagcgtag attccagact
gcagtaccct acgcccttaa 2340cggcaagtgt gggaaccggg ggaggtttga
tatgtggggt gaagggggct ctcgccgggg 2400ttgggcccgc tactgggtca
atttggggtc aattggggca attggggctg ttttttggga 2460cacaaatacg
ccgccaaccc ggtctctcct gaattctgca gatgggctgc aggaattccg
2520tcgtcgcctg agtcgacatc atttatttac cagttggcca caaacccttg
acgatctcgt 2580atgtcccctc cgacatactc ccggccggct ggggtacgtt
cgatagcgct atcggcatcg 2640acaaggtttg ggtccctagc cgataccgca
ctacctgagt cacaatcttc ggaggtttag 2700tcttccacat agcacgggca
aaagtgcgta tatatacaag agcgtttgcc agccacagat 2760tttcactcca
cacaccacat cacacataca accacacaca tccacaatgg aacccgaaac
2820taagaagacc aagactgact ccaagaagat tgttcttctc ggcggcgact
tctgtggccc 2880cgaggtgatt gccgaggccg tcaaggtgct caagtctgtt
gctgaggcct ccggcaccga 2940gtttgtgttt gaggaccgac tcattggagg
agctgccatt
gagaaggagg gcgagcccat 3000caccgacgct actctcgaca tctgccgaaa
ggctgactct attatgctcg gtgctgtcgg 3060aggcgctgcc aacaccgtat
ggaccactcc cgacggacga accgacgtgc gacccgagca 3120gggtctcctc
aagctgcgaa aggacctgaa cctgtacgcc aacctgcgac cctgccagct
3180gctgtcgccc aagctcgccg atctctcccc catccgaaac gttgagggca
ccgacttcat 3240cattgtccga gagctcgtcg gaggtatcta ctttggagag
cgaaaggagg atgacggatc 3300tggcgtcgct tccgacaccg agacctactc
cgttcctgag gttgagcgaa ttgcccgaat 3360ggccgccttc ctggcccttc
agcacaaccc ccctcttccc gtgtggtctc ttgacaaggc 3420caacgtgctg
gcctcctctc gactttggcg aaagactgtc actcgagtcc tcaaggacga
3480attcccccag ctcgagctca accaccagct gatcgactcg gccgccatga
tcctcatcaa 3540gcagccctcc aagatgaatg gtatcatcat caccaccaac
atgtttggcg atatcatctc 3600cgacgaggcc tccgtcatcc ccggttctct
gggtctgctg ccctccgcct ctctggcttc 3660tctgcccgac accaacgagg
cgttcggtct gtacgagccc tgtcacggat ctgcccccga 3720tctcggcaag
cagaaggtca accccattgc caccattctg tctgccgcca tgatgctcaa
3780gttctctctt aacatgaagc ccgccggtga cgctgttgag gctgccgtca
aggagtccgt 3840cgaggctggt atcactaccg ccgatatcgg aggctcttcc
tccacctccg aggtcggaga 3900cttgttgcca acaaggtcaa ggagctgctc
aagaaggagt aagtcgtttc tacgacgcat 3960tgatggaagg agcaaactga
cgcgcctgcg ggttggtcta ccggcagggt ccgctagtgt 4020ataagactct
ataaaaaggg ccctgccctg ctaatgaaat gatgatttat aatttaccgg
4080tgtagcaacc ttgactagaa gaagcagatt gggtgtgttt gtagtggagg
acagtggtac 4140gttttggaaa cagtcttctt gaaagtgtct tgtctacagt
atattcactc ataacctcaa 4200tagccaaggg tgtagtcggt ttattaaagg
aagggagttg tggctgatgt ggatagatat 4260ctttaagctg gcgactgcac
ccaacgagtg tggtggtagc ttgttactgt atattcggta 4320agatatattt
tgtggggttt tagtggtgtt taaacggtag gttagtgctt ggtatatgag
4380ttgtaggcat gacaatttgg aaaggggtgg actttgggaa tattgtggga
tttcaatacc 4440ttagtttgta cagggtaatt gttacaaatg atacaaagaa
ctgtatttct tttcatttgt 4500tttaattggt tgtatatcaa gtccgttaga
cgagctcagt gccttggctt ttggcactgt 4560atttcatttt tagaggtaca
ctacattcag tgaggtatgg taaggttgag ggcataatga 4620aggcaccttg
tactgacagt cacagacctc tcaccgagaa ttttatgaga tatactcggg
4680ttcattttag gctcatcgat tcgattcaaa ttaattaatt cgatatcata
attgtcggcc 4740gaggtctgta cggccagaac cgagatccta ttgaggaggc
caagcgatac cagaaggctg 4800gctgggaggc ttaccagaag attaactgtt
agaggttaga ctatggatat gtcatttaac 4860tgtgtatata gagagcgtgc
aagtatggag cgcttgttca gcttgtatga tggtcagacg 4920acctgtctga
tcgagtatgt atgatactgc acaacctgtg tatccgcatg atctgtccaa
4980tggggcatgt tgttgtgttt ctcgatacgg agatgctggg tacaagtagc
taatacgatt 5040gaactactta tacttatatg aggcttgaag aaagctgact
tgtgtatgac ttattctcaa 5100ctacatcccc agtcacaata ccaccactgc
actaccacta caccaaaacc atgatcaaac 5160cacccatgga cttcctggag
gcagaagaac ttgttatgga aaagctcaag agagagaa 52188833DNAArtificial
SequencePrimer YL61 88acaattccac acaacgtacg agccggaagc ata
338933DNAArtificial SequencePrimer YL62 89tatgcttccg gctcgtacgt
tgtgtggaat tgt 339033DNAArtificial SequencePrimer YL69 90agcccatctg
cagaagcttc aggagagacc ggg 339133DNAArtificial SequencePrimer YL70
91cccggtctct cctgaagctt ctgcagatgg gct 339233DNAArtificial
SequencePrimer YL77 92tagtgagggt taattaatcg agcttggcgt aat
339333DNAArtificial SequencePrimer YL78 93attacgccaa gctcgattaa
ttaaccctca cta 339434DNAArtificial SequencePrimer YL79A
94attcctgcag cccatcgatg cagaattcag gaga 349534DNAArtificial
SequencePrimer YL80A 95tctcctgaat tctgcatcga tgggctgcag gaat
34968894DNAArtificial Sequence8894 bp fragment for integration and
expression of the delta-6 and delta-5 desaturase genes and the
elongase gene 96tatcacatca cgctctcatc aagaatactt cttgagaacc
gtggagaccg gggttcgatt 60ccccgtatcg gagtgtttat tttttgctca accataccct
ggggtgtgtt ctgtggagca 120ttctcacttt tggtaaacga cattgcttca
agtgcagcgg aatcaaaaag tataaagtgg 180gcagcgagta tacctgtaca
gactgtaggc gataactcaa tccaattacc ccccacaaca 240tgactggcca
aactgatctc aagactttat tgaaatcagc aacaccgatt ctcaatgaag
300gcacatactt cttctgcaac attcacttga cgcctaaagt tggtgagaaa
tggaccgaca 360agacatattc tgctatccac ggactgttgc ctgtgtcggt
ggctacaata cgtgagtcag 420aagggctgac ggtggtggtt cgtacgttgt
gtggaattgt gagcggataa caatttcaca 480caggaaacag ctatgaccat
gattacgcca agctcgaaat taaccctcac taaagggaac 540aaaagctgga
gctccaccgc ggacacaata tctggtcaaa tttcagtttc gttacataaa
600tcgttatgtc aaaggagtgt gggaggttaa gagaattatc accggcaaac
tatctgttaa 660ttgctaggta cctctagacg tccacccggg tcgcttggcg
gccgaagagg ccggaatctc 720gggccgcggt ggcggccgct tactgcaact
tccttgcctt ctccttggca gcgtcggcct 780tggcctgctt ggccaacttg
gcgttctttc tgtaaaagtt gtagaagaga ccgagcatgg 840tccacatgta
gaaccaaagc agagccgtga tgaagaaggg gtatccgggg cggccaagga
900ccttcatggc gtacatgtcc caggaagact ggaccgacat catgcagaac
tgtgtcatct 960gcgagcgcgt gatgtagaac ttgatgaacg acacctgctt
gaagcccaag gccgacaaga 1020agtagtagcc gtacatgatc acatggatga
acgagttcaa cgcagcagag aagtaggctt 1080caccgttggg tgcaacaaag
gtgaccaacc accagatggt gaagatggag ctgtggtggt 1140aaacgtgcaa
gaaggagatc tggcggttgt tcttcttgag gaccatgatc atggtgtcga
1200caaactccat gatcttggag aagtagaaga gccagatcat cttggccata
ggaagaccct 1260tgaaggtatg atcagcagcg ttctcaaaca gtccatagtt
ggcctgataa gcctcgtaca 1320ggatcccacc gcacatgtag gcgctgatcg
agaccagaca aaagttgtgc aggagcgaaa 1380acgtcttgac ctcgaaccgc
tcaaagttct tcatgatctg catgcccaca aagaccgtga 1440ccaaataagc
gagcacgatc aacagcacgt ggaacgggtt catcaacggc agctcacggg
1500ccaaaggcga ctccaccgcg accaggaacc cacgcgtgtg atggacaatc
gtggggatgt 1560acttctcggc ctgggccacc agcgcggcct cgagaggatc
gacatagggc gcggcccgga 1620caccgatagc ggtggcaagg tccataaaca
gatcttgcgg catctttgat gggaggaatg 1680gcgcaatcga ctccatgcgg
ccgctctaga actagtggat cctttgaatg attcttatac 1740tcagaaggaa
atgcttaacg atttcgggtg tgagttgaca aggagagaga gaaaagaaga
1800ggaaaggtaa ttcggggacg gtggtctttt atacccttgg ctaaagtccc
aaccacaaag 1860caaaaaaatt ttcagtagtc tattttgcgt ccggcatggg
ttacccggat ggccagacaa 1920agaaactagt acaaagtctg aacaagcgta
gattccagac tgcagtaccc tacgccctta 1980acggcaagtg tgggaaccgg
gggaggtttg atatgtgggg tgaagggggc tctcgccggg 2040gttgggcccg
ctactgggtc aatttggggt caattggggc aattggggct gttttttggg
2100acacaaatac gccgccaacc cggtctctcc tgaagcttgt gagcggataa
caatttcaca 2160caggaaacag ctatgaccat gattacgcca agctcgaaat
taaccctcac taaagggaac 2220aaaagctgga gctccaccgc ggacacaata
tctggtcaaa tttcagtttc gttacataaa 2280tcgttatgtc aaaggagtgt
gggaggttaa gagaattatc accggcaaac tatctgttaa 2340ttgctaggta
cctctagacg tccacccggg tcgcttggcg gccgaagagg ccggaatctc
2400gggccgcggt ggcggccgcc tactcttcct tgggacggag tccaagaaca
cgcaagtgct 2460ccaaatgtga agcaaatgct tgccaaaacg tatccttgac
aaggtatgga accttgtact 2520cgctgcaggt gttcttgatg atggccagaa
tatcgggata atggtgctgc gacacgttgg 2580ggaacagatg gtgcacagcc
tggtagttca agctgccagt gatgctggtc cagaggtgcg 2640aatcgtgtgc
gtaatcctgc gtagtctcga cctgcatagc tgcccagtcc ttttggatga
2700tcccgttctc gtcaggcaac ggccactgaa cttcctcaac aacgtggttc
gcctggaagg 2760tcagcgccag ccagtaagac gacaccatgt ccgcgaccgt
gaacaagagc agcaccttgc 2820ccaggggcag atactgcagg ggaacaatca
ggcgatacca gacaaagaaa gccttgccgc 2880cccagaacat cacagtgtgc
catgtcgaga tgggattgac acgaatagcg tcattggtct 2940tgacaaagta
caaaatgttg atgtcctgaa tgcgcacctt gaacgccagc agtccgtaca
3000ggaaaggaac aaacatgtgc tggttgatgt ggttgacaaa ccacttttgg
ttgggcttga 3060tacgacgaac atcgggctca gacgtcgaca cgtcgggatc
tgctccagca atgttggtgt 3120aggggtgatg gccgagcata tgttggtaca
tccacaccag gtacgatgct ccgttgaaaa 3180agtcgtgcgt ggctcccaga
atcttccaga cagtggggtt gtgggtcact gaaaagtgag 3240acgcatcatg
aagagggttg agtccgactt gtgcgcacgc aaatcccatg atgattgcaa
3300acaccacctg aagccatgtg cgttcgacaa cgaaaggcac aaagagctgc
gcgtagtagg 3360aagcgatcaa ggatccaaag ataagagcgt atcgtcccca
gatctctggt ctattcttgg 3420gatcaatgtt ccgatccgta aagtagccct
cgactctcgt cttgatggtt ttgtggaaca 3480ccgttggctc cgggaagatg
ggcagctcat tcgagaccag tgtaccgaca tagtacttct 3540tcataatggc
atctgcagcc ccaaacgcgt gatacatctc aaagaccgga gtaacatctc
3600ggccagctcc gagcaggaga gtgtccactc caccaggatg gcggctcaag
aactttgtga 3660catcgtacac cctgccgcgg atggccaaga gtaggtcgtc
cttggtgtta tgggccgcca 3720gctcttccca ggtgaaggtt tttccttggt
ccgttcccat ggtgaatgat tcttatactc 3780agaaggaaat gcttaacgat
ttcgggtgtg agttgacaag gagagagaga aaagaagagg 3840aaaggtaatt
cggggacggt ggtcttttat acccttggct aaagtcccaa ccacaaagca
3900aaaaaatttt cagtagtcta ttttgcgtcc ggcatgggtt acccggatgg
ccagacaaag 3960aaactagtac aaagtctgaa caagcgtaga ttccagactg
cagtacccta cgcccttaac 4020ggcaagtgtg ggaaccgggg gaggtttgat
atgtggggtg aagggggctc tcgccggggt 4080tgggcccgct actgggtcaa
tttggggtca attggggcaa ttggggctgt tttttgggac 4140acaaatacgc
cgccaacccg gtctctcctg aattctgcag atgggctgca ggaattccgt
4200cgtcgcctga gtcgacatca tttatttacc agttggccac aaacccttga
cgatctcgta 4260tgtcccctcc gacatactcc cggccggctg gggtacgttc
gatagcgcta tcggcatcga 4320caaggtttgg gtccctagcc gataccgcac
tacctgagtc acaatcttcg gaggtttagt 4380cttccacata gcacgggcaa
aagtgcgtat atatacaaga gcgtttgcca gccacagatt 4440ttcactccac
acaccacatc acacatacaa ccacacacat ccacaatgga acccgaaact
4500aagaagacca agactgactc caagaagatt gttcttctcg gcggcgactt
ctgtggcccc 4560gaggtgattg ccgaggccgt caaggtgctc aagtctgttg
ctgaggcctc cggcaccgag 4620tttgtgtttg aggaccgact cattggagga
gctgccattg agaaggaggg cgagcccatc 4680accgacgcta ctctcgacat
ctgccgaaag gctgactcta ttatgctcgg tgctgtcgga 4740ggcgctgcca
acaccgtatg gaccactccc gacggacgaa ccgacgtgcg acccgagcag
4800ggtctcctca agctgcgaaa ggacctgaac ctgtacgcca acctgcgacc
ctgccagctg 4860ctgtcgccca agctcgccga tctctccccc atccgaaacg
ttgagggcac cgacttcatc 4920attgtccgag agctcgtcgg aggtatctac
tttggagagc gaaaggagga tgacggatct 4980ggcgtcgctt ccgacaccga
gacctactcc gttcctgagg ttgagcgaat tgcccgaatg 5040gccgccttcc
tggcccttca gcacaacccc cctcttcccg tgtggtctct tgacaaggcc
5100aacgtgctgg cctcctctcg actttggcga aagactgtca ctcgagtcct
caaggacgaa 5160ttcccccagc tcgagctcaa ccaccagctg atcgactcgg
ccgccatgat cctcatcaag 5220cagccctcca agatgaatgg tatcatcatc
accaccaaca tgtttggcga tatcatctcc 5280gacgaggcct ccgtcatccc
cggttctctg ggtctgctgc cctccgcctc tctggcttct 5340ctgcccgaca
ccaacgaggc gttcggtctg tacgagccct gtcacggatc tgcccccgat
5400ctcggcaagc agaaggtcaa ccccattgcc accattctgt ctgccgccat
gatgctcaag 5460ttctctctta acatgaagcc cgccggtgac gctgttgagg
ctgccgtcaa ggagtccgtc 5520gaggctggta tcactaccgc cgatatcgga
ggctcttcct ccacctccga ggtcggagac 5580ttgttgccaa caaggtcaag
gagctgctca agaaggagta agtcgtttct acgacgcatt 5640gatggaagga
gcaaactgac gcgcctgcgg gttggtctac cggcagggtc cgctagtgta
5700taagactcta taaaaagggc cctgccctgc taatgaaatg atgatttata
atttaccggt 5760gtagcaacct tgactagaag aagcagattg ggtgtgtttg
tagtggagga cagtggtacg 5820ttttggaaac agtcttcttg aaagtgtctt
gtctacagta tattcactca taacctcaat 5880agccaagggt gtagtcggtt
tattaaagga agggagttgt ggctgatgtg gatagatatc 5940tttaagctgg
cgactgcacc caacgagtgt ggtggtagct tgttactgta tattcggtaa
6000gatatatttt gtggggtttt agtggtgttt aaacggtagg ttagtgcttg
gtatatgagt 6060tgtaggcatg acaatttgga aaggggtgga ctttgggaat
attgtgggat ttcaatacct 6120tagtttgtac agggtaattg ttacaaatga
tacaaagaac tgtatttctt ttcatttgtt 6180ttaattggtt gtatatcaag
tccgttagac gagctcagtg ccttggcttt tggcactgta 6240tttcattttt
agaggtacac tacattcagt gaggtatggt aaggttgagg gcataatgaa
6300ggcaccttgt actgacagtc acagacctct caccgagaat tttatgagat
atactcgggt 6360tcattttagg ctcatcgatg cagaattcag gagagaccgg
gttggcggcg tatttgtgtc 6420ccaaaaaaca gccccaattg ccccaattga
ccccaaattg acccagtagc gggcccaacc 6480ccggcgagag cccccttcac
cccacatatc aaacctcccc cggttcccac acttgccgtt 6540aagggcgtag
ggtactgcag tctggaatct acgcttgttc agactttgta ctagtttctt
6600tgtctggcca tccgggtaac ccatgccgga cgcaaaatag actactgaaa
atttttttgc 6660tttgtggttg ggactttagc caagggtata aaagaccacc
gtccccgaat tacctttcct 6720cttcttttct ctctctcctt gtcaactcac
acccgaaatc gttaagcatt tccttctgag 6780tataagaatc attcaccatg
gctgctgctc ccagtgtgag gacgtttact cgggccgagg 6840ttttgaatgc
cgaggctctg aatgagggca agaaggatgc cgaggcaccc ttcttgatga
6900tcatcgacaa caaggtgtac gatgtccgcg agttcgtccc tgatcatccc
ggtggaagtg 6960tgattctcac gcacgttggc aaggacggca ctgacgtctt
tgacactttt caccccgagg 7020ctgcttggga gactcttgcc aacttttacg
ttggtgatat tgacgagagc gaccgcgata 7080tcaagaatga tgactttgcg
gccgaggtcc gcaagctgcg taccttgttc cagtctcttg 7140gttactacga
ttcttccaag gcatactacg ccttcaaggt ctcgttcaac ctctgcatct
7200ggggtttgtc gacggtcatt gtggccaagt ggggccagac ctcgaccctc
gccaacgtgc 7260tctcggctgc gcttttgggt ctgttctggc agcagtgcgg
atggttggct cacgactttt 7320tgcatcacca ggtcttccag gaccgtttct
ggggtgatct tttcggcgcc ttcttgggag 7380gtgtctgcca gggcttctcg
tcctcgtggt ggaaggacaa gcacaacact caccacgccg 7440cccccaacgt
ccacggcgag gatcccgaca ttgacaccca ccctctgttg acctggagtg
7500agcatgcgtt ggagatgttc tcggatgtcc cagatgagga gctgacccgc
atgtggtcgc 7560gtttcatggt cctgaaccag acctggtttt acttccccat
tctctcgttt gcccgtctct 7620cctggtgcct ccagtccatt ctctttgtgc
tgcctaacgg tcaggcccac aagccctcgg 7680gcgcgcgtgt gcccatctcg
ttggtcgagc agctgtcgct tgcgatgcac tggacctggt 7740acctcgccac
catgttcctg ttcatcaagg atcccgtcaa catgctggtg tactttttgg
7800tgtcgcaggc ggtgtgcgga aacttgttgg ccatcgtgtt ctcgctcaac
cacaacggta 7860tgcctgtgat ctcgaggagg aggcggtcga tatggatttc
ttcacgaagc agatcatcac 7920gggtcgtgat gtccacccgg gtctatttgc
caactggttc acgggtggat tgaactatca 7980gatcgagcac cacttgttcc
cttcgatgcc tcgccacaac ttttcaaaga tccagcctgc 8040tgtcgagacc
ctgtgcaaaa agtacaatgt ccgataccac accaccggta tgatcgaggg
8100aactgcagag gtctttagcc gtctgaacga ggtctccaag gctacctcca
agatgggtaa 8160ggcgcagtaa gcggccgcca ccgcggcccg agattccggc
ctcttcggcc gccaagcgac 8220ccgggtggac gtctagaggt acctagcaat
taacagatag tttgccggtg ataattctct 8280taacctccca cactcctttg
acataacgat ttatgtaacg aaactgaaat ttgaccagat 8340attgtgtccg
cggtggagct ccagcttttg ttccctttag tgagggttaa ttaattcgat
8400atcataattg tcggccgagg tctgtacggc cagaaccgag atcctattga
ggaggccaag 8460cgataccaga aggctggctg ggaggcttac cagaagatta
actgttagag gttagactat 8520ggatatgtca tttaactgtg tatatagaga
gcgtgcaagt atggagcgct tgttcagctt 8580gtatgatggt cagacgacct
gtctgatcga gtatgtatga tactgcacaa cctgtgtatc 8640cgcatgatct
gtccaatggg gcatgttgtt gtgtttctcg atacggagat gctgggtaca
8700agtagctaat acgattgaac tacttatact tatatgaggc ttgaagaaag
ctgacttgtg 8760tatgacttat tctcaactac atccccagtc acaataccac
cactgcacta ccactacacc 8820aaaaccatga tcaaaccacc catggacttc
ctggaggcag aagaacttgt tatggaaaag 8880ctcaagagag agaa
88949731DNAArtificial SequencePrimer YL101 97gagcttggcg taatcgatgg
tcatagctgt t 319831DNAArtificial SequencePrimer YL102 98aacagctatg
accatcgatt acgccaagct c 319936DNAArtificial SequencePrimer YL103
99atgatgactc aggcgtttaa acgacggaat tcctgc 3610036DNAArtificial
SequencePrimer YL104 100gcaggaattc cgtcgtttaa acgcctgagt catcat
3610110328DNAArtificial Sequence10328 bp fragment for integration
and expression of the delta-6, delta-5, and delta-17 desaturase
genes and the elongase gene 101tatcacatca cgctctcatc aagaatactt
cttgagaacc gtggagaccg gggttcgatt 60ccccgtatcg gagtgtttat tttttgctca
accataccct ggggtgtgtt ctgtggagca 120ttctcacttt tggtaaacga
cattgcttca agtgcagcgg aatcaaaaag tataaagtgg 180gcagcgagta
tacctgtaca gactgtaggc gataactcaa tccaattacc ccccacaaca
240tgactggcca aactgatctc aagactttat tgaaatcagc aacaccgatt
ctcaatgaag 300gcacatactt cttctgcaac attcacttga cgcctaaagt
tggtgagaaa tggaccgaca 360agacatattc tgctatccac ggactgttgc
ctgtgtcggt ggctacaata cgtgagtcag 420aagggctgac ggtggtggtt
cgtacgttgt gtggaattgt gagcggataa caatttcaca 480caggaaacag
ctatgaccat gattacgcca agctcgaaat taaccctcac taaagggaac
540aaaagctgga gctccaccgc ggacacaata tctggtcaaa tttcagtttc
gttacataaa 600tcgttatgtc aaaggagtgt gggaggttaa gagaattatc
accggcaaac tatctgttaa 660ttgctaggta cctctagacg tccacccggg
tcgcttggcg gccgaagagg ccggaatctc 720gggccgcggt ggcggccgct
tactgcaact tccttgcctt ctccttggca gcgtcggcct 780tggcctgctt
ggccaacttg gcgttctttc tgtaaaagtt gtagaagaga ccgagcatgg
840tccacatgta gaaccaaagc agagccgtga tgaagaaggg gtatccgggg
cggccaagga 900ccttcatggc gtacatgtcc caggaagact ggaccgacat
catgcagaac tgtgtcatct 960gcgagcgcgt gatgtagaac ttgatgaacg
acacctgctt gaagcccaag gccgacaaga 1020agtagtagcc gtacatgatc
acatggatga acgagttcaa cgcagcagag aagtaggctt 1080caccgttggg
tgcaacaaag gtgaccaacc accagatggt gaagatggag ctgtggtggt
1140aaacgtgcaa gaaggagatc tggcggttgt tcttcttgag gaccatgatc
atggtgtcga 1200caaactccat gatcttggag aagtagaaga gccagatcat
cttggccata ggaagaccct 1260tgaaggtatg atcagcagcg ttctcaaaca
gtccatagtt ggcctgataa gcctcgtaca 1320ggatcccacc gcacatgtag
gcgctgatcg agaccagaca aaagttgtgc aggagcgaaa 1380acgtcttgac
ctcgaaccgc tcaaagttct tcatgatctg catgcccaca aagaccgtga
1440ccaaataagc gagcacgatc aacagcacgt ggaacgggtt catcaacggc
agctcacggg 1500ccaaaggcga ctccaccgcg accaggaacc cacgcgtgtg
atggacaatc gtggggatgt 1560acttctcggc ctgggccacc agcgcggcct
cgagaggatc gacatagggc gcggcccgga 1620caccgatagc ggtggcaagg
tccataaaca gatcttgcgg catctttgat gggaggaatg 1680gcgcaatcga
ctccatgcgg ccgctctaga actagtggat cctttgaatg attcttatac
1740tcagaaggaa atgcttaacg atttcgggtg tgagttgaca aggagagaga
gaaaagaaga 1800ggaaaggtaa ttcggggacg gtggtctttt atacccttgg
ctaaagtccc aaccacaaag 1860caaaaaaatt ttcagtagtc tattttgcgt
ccggcatggg ttacccggat ggccagacaa 1920agaaactagt acaaagtctg
aacaagcgta gattccagac tgcagtaccc tacgccctta 1980acggcaagtg
tgggaaccgg gggaggtttg atatgtgggg tgaagggggc tctcgccggg
2040gttgggcccg ctactgggtc aatttggggt caattggggc aattggggct
gttttttggg 2100acacaaatac gccgccaacc cggtctctcc tgaagcttgt
gagcggataa caatttcaca 2160caggaaacag ctatgaccat gattacgcca
agctcgaaat taaccctcac taaagggaac 2220aaaagctgga gctccaccgc
ggacacaata tctggtcaaa tttcagtttc gttacataaa 2280tcgttatgtc
aaaggagtgt gggaggttaa gagaattatc accggcaaac tatctgttaa
2340ttgctaggta cctctagacg tccacccggg tcgcttggcg gccgaagagg
ccggaatctc
2400gggccgcggt ggcggccgcc tactcttcct tgggacggag tccaagaaca
cgcaagtgct 2460ccaaatgtga agcaaatgct tgccaaaacg tatccttgac
aaggtatgga accttgtact 2520cgctgcaggt gttcttgatg atggccagaa
tatcgggata atggtgctgc gacacgttgg 2580ggaacagatg gtgcacagcc
tggtagttca agctgccagt gatgctggtc cagaggtgcg 2640aatcgtgtgc
gtaatcctgc gtagtctcga cctgcatagc tgcccagtcc ttttggatga
2700tcccgttctc gtcaggcaac ggccactgaa cttcctcaac aacgtggttc
gcctggaagg 2760tcagcgccag ccagtaagac gacaccatgt ccgcgaccgt
gaacaagagc agcaccttgc 2820ccaggggcag atactgcagg ggaacaatca
ggcgatacca gacaaagaaa gccttgccgc 2880cccagaacat cacagtgtgc
catgtcgaga tgggattgac acgaatagcg tcattggtct 2940tgacaaagta
caaaatgttg atgtcctgaa tgcgcacctt gaacgccagc agtccgtaca
3000ggaaaggaac aaacatgtgc tggttgatgt ggttgacaaa ccacttttgg
ttgggcttga 3060tacgacgaac atcgggctca gacgtcgaca cgtcgggatc
tgctccagca atgttggtgt 3120aggggtgatg gccgagcata tgttggtaca
tccacaccag gtacgatgct ccgttgaaaa 3180agtcgtgcgt ggctcccaga
atcttccaga cagtggggtt gtgggtcact gaaaagtgag 3240acgcatcatg
aagagggttg agtccgactt gtgcgcacgc aaatcccatg atgattgcaa
3300acaccacctg aagccatgtg cgttcgacaa cgaaaggcac aaagagctgc
gcgtagtagg 3360aagcgatcaa ggatccaaag ataagagcgt atcgtcccca
gatctctggt ctattcttgg 3420gatcaatgtt ccgatccgta aagtagccct
cgactctcgt cttgatggtt ttgtggaaca 3480ccgttggctc cgggaagatg
ggcagctcat tcgagaccag tgtaccgaca tagtacttct 3540tcataatggc
atctgcagcc ccaaacgcgt gatacatctc aaagaccgga gtaacatctc
3600ggccagctcc gagcaggaga gtgtccactc caccaggatg gcggctcaag
aactttgtga 3660catcgtacac cctgccgcgg atggccaaga gtaggtcgtc
cttggtgtta tgggccgcca 3720gctcttccca ggtgaaggtt tttccttggt
ccgttcccat ggtgaatgat tcttatactc 3780agaaggaaat gcttaacgat
ttcgggtgtg agttgacaag gagagagaga aaagaagagg 3840aaaggtaatt
cggggacggt ggtcttttat acccttggct aaagtcccaa ccacaaagca
3900aaaaaatttt cagtagtcta ttttgcgtcc ggcatgggtt acccggatgg
ccagacaaag 3960aaactagtac aaagtctgaa caagcgtaga ttccagactg
cagtacccta cgcccttaac 4020ggcaagtgtg ggaaccgggg gaggtttgat
atgtggggtg aagggggctc tcgccggggt 4080tgggcccgct actgggtcaa
tttggggtca attggggcaa ttggggctgt tttttgggac 4140acaaatacgc
cgccaacccg gtctctcctg aattctgcag atgggctgca ggaattccgt
4200cgtcgcctga gtcgacatca tttatttacc agttggccac aaacccttga
cgatctcgta 4260tgtcccctcc gacatactcc cggccggctg gggtacgttc
gatagcgcta tcggcatcga 4320caaggtttgg gtccctagcc gataccgcac
tacctgagtc acaatcttcg gaggtttagt 4380cttccacata gcacgggcaa
aagtgcgtat atatacaaga gcgtttgcca gccacagatt 4440ttcactccac
acaccacatc acacatacaa ccacacacat ccacaatgga acccgaaact
4500aagaagacca agactgactc caagaagatt gttcttctcg gcggcgactt
ctgtggcccc 4560gaggtgattg ccgaggccgt caaggtgctc aagtctgttg
ctgaggcctc cggcaccgag 4620tttgtgtttg aggaccgact cattggagga
gctgccattg agaaggaggg cgagcccatc 4680accgacgcta ctctcgacat
ctgccgaaag gctgactcta ttatgctcgg tgctgtcgga 4740ggcgctgcca
acaccgtatg gaccactccc gacggacgaa ccgacgtgcg acccgagcag
4800ggtctcctca agctgcgaaa ggacctgaac ctgtacgcca acctgcgacc
ctgccagctg 4860ctgtcgccca agctcgccga tctctccccc atccgaaacg
ttgagggcac cgacttcatc 4920attgtccgag agctcgtcgg aggtatctac
tttggagagc gaaaggagga tgacggatct 4980ggcgtcgctt ccgacaccga
gacctactcc gttcctgagg ttgagcgaat tgcccgaatg 5040gccgccttcc
tggcccttca gcacaacccc cctcttcccg tgtggtctct tgacaaggcc
5100aacgtgctgg cctcctctcg actttggcga aagactgtca ctcgagtcct
caaggacgaa 5160ttcccccagc tcgagctcaa ccaccagctg atcgactcgg
ccgccatgat cctcatcaag 5220cagccctcca agatgaatgg tatcatcatc
accaccaaca tgtttggcga tatcatctcc 5280gacgaggcct ccgtcatccc
cggttctctg ggtctgctgc cctccgcctc tctggcttct 5340ctgcccgaca
ccaacgaggc gttcggtctg tacgagccct gtcacggatc tgcccccgat
5400ctcggcaagc agaaggtcaa ccccattgcc accattctgt ctgccgccat
gatgctcaag 5460ttctctctta acatgaagcc cgccggtgac gctgttgagg
ctgccgtcaa ggagtccgtc 5520gaggctggta tcactaccgc cgatatcgga
ggctcttcct ccacctccga ggtcggagac 5580ttgttgccaa caaggtcaag
gagctgctca agaaggagta agtcgtttct acgacgcatt 5640gatggaagga
gcaaactgac gcgcctgcgg gttggtctac cggcagggtc cgctagtgta
5700taagactcta taaaaagggc cctgccctgc taatgaaatg atgatttata
atttaccggt 5760gtagcaacct tgactagaag aagcagattg ggtgtgtttg
tagtggagga cagtggtacg 5820ttttggaaac agtcttcttg aaagtgtctt
gtctacagta tattcactca taacctcaat 5880agccaagggt gtagtcggtt
tattaaagga agggagttgt ggctgatgtg gatagatatc 5940tttaagctgg
cgactgcacc caacgagtgt ggtggtagct tgttactgta tattcggtaa
6000gatatatttt gtggggtttt agtggtgttt aaacgacgga attcctgcag
cccatctgca 6060gaattcagga gagaccgggt tggcggcgta tttgtgtccc
aaaaaacagc cccaattgcc 6120ccaattgacc ccaaattgac ccagtagcgg
gcccaacccc ggcgagagcc cccttcaccc 6180cacatatcaa acctcccccg
gttcccacac ttgccgttaa gggcgtaggg tactgcagtc 6240tggaatctac
gcttgttcag actttgtact agtttctttg tctggccatc cgggtaaccc
6300atgccggacg caaaatagac tactgaaaat ttttttgctt tgtggttggg
actttagcca 6360agggtataaa agaccaccgt ccccgaatta cctttcctct
tcttttctct ctctccttgt 6420caactcacac ccgaaatcgt taagcatttc
cttctgagta taagaatcat tcaccatggc 6480tgaggataag accaaggtcg
agttccctac cctgactgag ctgaagcact ctatccctaa 6540cgcttgcttt
gagtccaacc tcggactctc gctctactac actgcccgag cgatcttcaa
6600cgcatctgcc tctgctgctc tgctctacgc tgcccgatct actcccttca
ttgccgataa 6660cgttctgctc cacgctctgg tttgcgccac ctacatctac
gtgcagggtg tcatcttctg 6720gggtttcttt accgtcggtc acgactgtgg
tcactctgcc ttctcccgat accactccgt 6780caacttcatc attggctgca
tcatgcactc tgccattctg actcccttcg agtcctggcg 6840agtgacccac
cgacaccatc acaagaacac tggcaacatt gataaggacg agatcttcta
6900ccctcatcgg tccgtcaagg acctccagga cgtgcgacaa tgggtctaca
ccctcggagg 6960tgcttggttt gtctacctga aggtcggata tgctcctcga
accatgtccc actttgaccc 7020ctgggaccct ctcctgcttc gacgagcctc
cgctgtcatc gtgtccctcg gagtctgggc 7080tgccttcttc gctgcctacg
cctacctcac atactcgctc ggctttgccg tcatgggcct 7140ctactactat
gctcctctct ttgtctttgc ttcgttcctc gtcattacta ccttcttgca
7200tcacaacgac gaagctactc cctggtacgg tgactcggag tggacctacg
tcaagggcaa 7260cctgagctcc gtcgaccgat cgtacggagc tttcgtggac
aacctgtctc accacattgg 7320cacccaccag gtccatcact tgttccctat
cattccccac tacaagctca acgaagccac 7380caagcacttt gctgccgctt
accctcacct cgtgagacgt aacgacgagc ccatcattac 7440tgccttcttc
aagaccgctc acctctttgt caactacgga gctgtgcccg agactgctca
7500gattttcacc ctcaaagagt ctgccgctgc agccaaggcc aagagcgacc
accaccatca 7560ccaccattaa gcggccgcca ccgcggcccg agattccggc
ctcttcggcc gccaagcgac 7620ccgggtggac gtctagaggt acctagcaat
taacagatag tttgccggtg ataattctct 7680taacctccca cactcctttg
acataacgat ttatgtaacg aaactgaaat ttgaccagat 7740attgtgtccg
cggtggagct ccagcttttg ttccctttag tgagggttaa tttcgagctt
7800ggcgtaatcg atgcagaatt caggagagac cgggttggcg gcgtatttgt
gtcccaaaaa 7860acagccccaa ttgccccaat tgaccccaaa ttgacccagt
agcgggccca accccggcga 7920gagccccctt caccccacat atcaaacctc
ccccggttcc cacacttgcc gttaagggcg 7980tagggtactg cagtctggaa
tctacgcttg ttcagacttt gtactagttt ctttgtctgg 8040ccatccgggt
aacccatgcc ggacgcaaaa tagactactg aaaatttttt tgctttgtgg
8100ttgggacttt agccaagggt ataaaagacc accgtccccg aattaccttt
cctcttcttt 8160tctctctctc cttgtcaact cacacccgaa atcgttaagc
atttccttct gagtataaga 8220atcattcacc atggctgctg ctcccagtgt
gaggacgttt actcgggccg aggttttgaa 8280tgccgaggct ctgaatgagg
gcaagaagga tgccgaggca cccttcttga tgatcatcga 8340caacaaggtg
tacgatgtcc gcgagttcgt ccctgatcat cccggtggaa gtgtgattct
8400cacgcacgtt ggcaaggacg gcactgacgt ctttgacact tttcaccccg
aggctgcttg 8460ggagactctt gccaactttt acgttggtga tattgacgag
agcgaccgcg atatcaagaa 8520tgatgacttt gcggccgagg tccgcaagct
gcgtaccttg ttccagtctc ttggttacta 8580cgattcttcc aaggcatact
acgccttcaa ggtctcgttc aacctctgca tctggggttt 8640gtcgacggtc
attgtggcca agtggggcca gacctcgacc ctcgccaacg tgctctcggc
8700tgcgcttttg ggtctgttct ggcagcagtg cggatggttg gctcacgact
ttttgcatca 8760ccaggtcttc caggaccgtt tctggggtga tcttttcggc
gccttcttgg gaggtgtctg 8820ccagggcttc tcgtcctcgt ggtggaagga
caagcacaac actcaccacg ccgcccccaa 8880cgtccacggc gaggatcccg
acattgacac ccaccctctg ttgacctgga gtgagcatgc 8940gttggagatg
ttctcggatg tcccagatga ggagctgacc cgcatgtggt cgcgtttcat
9000ggtcctgaac cagacctggt tttacttccc cattctctcg tttgcccgtc
tctcctggtg 9060cctccagtcc attctctttg tgctgcctaa cggtcaggcc
cacaagccct cgggcgcgcg 9120tgtgcccatc tcgttggtcg agcagctgtc
gcttgcgatg cactggacct ggtacctcgc 9180caccatgttc ctgttcatca
aggatcccgt caacatgctg gtgtactttt tggtgtcgca 9240ggcggtgtgc
ggaaacttgt tggccatcgt gttctcgctc aaccacaacg gtatgcctgt
9300gatctcgaag gaggaggcgg tcgatatgga tttcttcacg aagcagatca
tcacgggtcg 9360tgatgtccac ccgggtctat ttgccaactg gttcacgggt
ggattgaact atcagatcga 9420gcaccacttg ttcccttcga tgcctcgcca
caacttttca aagatccagc ctgctgtcga 9480gaccctgtgc aaaaagtaca
atgtccgata ccacaccacc ggtatgatcg agggaactgc 9540agaggtcttt
agccgtctga acgaggtctc caaggctacc tccaagatgg gtaaggcgca
9600gtaagcggcc gccaccgcgg cccgagattc cggcctcttc ggccgccaag
cgacccgggt 9660ggacgtctag aggtacctag caattaacag atagtttgcc
ggtgataatt ctcttaacct 9720cccacactcc tttgacataa cgatttatgt
aacgaaactg aaatttgacc agatattgtg 9780tccgcggtgg agctccagct
tttgttccct ttagtgaggg ttaattaatt cgatatcata 9840attgtcggcc
gaggtctgta cggccagaac cgagatccta ttgaggaggc caagcgatac
9900cagaaggctg gctgggaggc ttaccagaag attaactgtt agaggttaga
ctatggatat 9960gtcatttaac tgtgtatata gagagcgtgc aagtatggag
cgcttgttca gcttgtatga 10020tggtcagacg acctgtctga tcgagtatgt
atgatactgc acaacctgtg tatccgcatg 10080atctgtccaa tggggcatgt
tgttgtgttt ctcgatacgg agatgctggg tacaagtagc 10140taatacgatt
gaactactta tacttatatg aggcttgaag aaagctgact tgtgtatgac
10200ttattctcaa ctacatcccc agtcacaata ccaccactgc actaccacta
caccaaaacc 10260atgatcaaac cacccatgga cttcctggag gcagaagaac
ttgttatgga aaagctcaag 10320agagagaa 1032810230DNAArtificial
SequencePrimer YL1 102cagtgccaaa agccaaggca ctgagctcgt
3010331DNAArtificial SequencePrimer YL2 103gacgagctca gtgccttggc
ttttggcact g 3110436DNAArtificial SequencePrimer YL3 104gtataagaat
cattcaccat ggatccacta gttcta 3610536DNAArtificial SequencePrimer
YL4 105tagaactagt ggatccatgg tgaatgattc ttatac 3610639DNAArtificial
SequencePrimer YL5 106cccccctcga ggtcgatggt gtcgataagc ttgatatcg
3910739DNAArtificial SequencePrimer YL6 107cgatatcaag cttatcgaca
ccatcgacct cgagggggg 3910837DNAArtificial SequencePrimer YL7
108caaccgattt cgacagttaa ttaataattt gaatcga 3710937DNAArtificial
SequencePrimer YL8 109tcgattcaaa ttattaatta actgtcgaaa tcggttg
3711035DNAArtificial SequencePrimer YL9 110tggtaaataa atgatgtcga
ctcaggcgac gacgg 3511135DNAArtificial SequencePrimer YL10
111ccgtcgtcgc ctgagtcgac atcatttatt tacca 3511236DNAArtificial
SequencePrimer YL23 112atggatccac tagttaatta actagagcgg ccgcca
3611336DNAArtificial SequencePrimer YL24 113tggcggccgc tctagttaat
taactagtgg atccat 361141413DNASaprolegnia diclina (ATCC #56851)
114atggccccgc agacggagct ccgccagcgc cacgccgccg tcgccgagac
gccggtggcc 60ggcaagaagg cctttacatg gcaggaggtc gcgcagcaca acacggcggc
ctcggcctgg 120atcattatcc gcggcaaggt ctacgacgtg accgagtggg
ccaacaagca ccccggcggc 180cgcgagatgg tgctgctgca cgccggtcgc
gaggccaccg acacgttcga ctcgtaccac 240ccgttcagcg acaaggccga
gtcgatcttg aacaagtatg agattggcac gttcacgggc 300ccgtccgagt
ttccgacctt caagccggac acgggcttct acaaggagtg ccgcaagcgc
360gttggcgagt acttcaagaa gaacaacctc catccgcagg acggcttccc
gggcctctgg 420cgcatgatgg tcgtgtttgc ggtcgccggc ctcgccttgt
acggcatgca cttttcgact 480atctttgcgc tgcagctcgc ggccgcggcg
ctctttggcg tctgccaggc gctgccgctg 540ctccacgtca tgcacgactc
gtcgcacgcg tcgtacacca acatgccgtt cttccattac 600gtcgtcggcc
gctttgccat ggactggttt gccggcggct cgatggtgtc atggctcaac
660cagcacgtcg tgggccacca catctacacg aacgtcgcgg gctcggaccc
ggatcttccg 720gtcaacatgg acggcgacat ccgccgcatc gtgaaccgcc
aggtgttcca gcccatgtac 780gcattccagc acatctacct tccgccgctc
tatggcgtgc ttggcctcaa gttccgcatc 840caggacttca ccgacacgtt
cggctcgcac acgaacggcc cgatccgcgt caacccgcac 900gcgctctcga
cgtggatggc catgatcagc tccaagtcgt tctgggcctt ctaccgcgtg
960taccttccgc ttgccgtgct ccagatgccc atcaagacgt accttgcgat
cttcttcctc 1020gccgagtttg tcacgggctg gtacctcgcg ttcaacttcc
aagtaagcca tgtctcgacc 1080gagtgcggct acccatgcgg cgacgaggcc
aagatggcgc tccaggacga gtgggcagtc 1140tcgcaggtca agacgtcggt
cgactacgcc catggctcgt ggatgacgac gttccttgcc 1200ggcgcgctca
actaccaggt cgtgcaccac ttgttcccca gcgtgtcgca gtaccactac
1260ccggcgatcg cgcccatcat cgtcgacgtc tgcaaggagt acaacatcaa
gtacgccatc 1320ttgccggact ttacggcggc gttcgttgcc cacttgaagc
acctccgcaa catgggccag 1380cagggcatcg ccgccacgat ccacatgggc taa
1413115470PRTSaprolegnia diclina (ATCC #56851) 115Met Ala Pro Gln
Thr Glu Leu Arg Gln Arg His Ala Ala Val Ala Glu1 5 10 15Thr Pro Val
Ala Gly Lys Lys Ala Phe Thr Trp Gln Glu Val Ala Gln20 25 30His Asn
Thr Ala Ala Ser Ala Trp Ile Ile Ile Arg Gly Lys Val Tyr35 40 45Asp
Val Thr Glu Trp Ala Asn Lys His Pro Gly Gly Arg Glu Met Val50 55
60Leu Leu His Ala Gly Arg Glu Ala Thr Asp Thr Phe Asp Ser Tyr His65
70 75 80Pro Phe Ser Asp Lys Ala Glu Ser Ile Leu Asn Lys Tyr Glu Ile
Gly85 90 95Thr Phe Thr Gly Pro Ser Glu Phe Pro Thr Phe Lys Pro Asp
Thr Gly100 105 110Phe Tyr Lys Glu Cys Arg Lys Arg Val Gly Glu Tyr
Phe Lys Lys Asn115 120 125Asn Leu His Pro Gln Asp Gly Phe Pro Gly
Leu Trp Arg Met Met Val130 135 140Val Phe Ala Val Ala Gly Leu Ala
Leu Tyr Gly Met His Phe Ser Thr145 150 155 160Ile Phe Ala Leu Gln
Leu Ala Ala Ala Ala Leu Phe Gly Val Cys Gln165 170 175Ala Leu Pro
Leu Leu His Val Met His Asp Ser Ser His Ala Ser Tyr180 185 190Thr
Asn Met Pro Phe Phe His Tyr Val Val Gly Arg Phe Ala Met Asp195 200
205Trp Phe Ala Gly Gly Ser Met Val Ser Trp Leu Asn Gln His Val
Val210 215 220Gly His His Ile Tyr Thr Asn Val Ala Gly Ser Asp Pro
Asp Leu Pro225 230 235 240Val Asn Met Asp Gly Asp Ile Arg Arg Ile
Val Asn Arg Gln Val Phe245 250 255Gln Pro Met Tyr Ala Phe Gln His
Ile Tyr Leu Pro Pro Leu Tyr Gly260 265 270Val Leu Gly Leu Lys Phe
Arg Ile Gln Asp Phe Thr Asp Thr Phe Gly275 280 285Ser His Thr Asn
Gly Pro Ile Arg Val Asn Pro His Ala Leu Ser Thr290 295 300Trp Met
Ala Met Ile Ser Ser Lys Ser Phe Trp Ala Phe Tyr Arg Val305 310 315
320Tyr Leu Pro Leu Ala Val Leu Gln Met Pro Ile Lys Thr Tyr Leu
Ala325 330 335Ile Phe Phe Leu Ala Glu Phe Val Thr Gly Trp Tyr Leu
Ala Phe Asn340 345 350Phe Gln Val Ser His Val Ser Thr Glu Cys Gly
Tyr Pro Cys Gly Asp355 360 365Glu Ala Lys Met Ala Leu Gln Asp Glu
Trp Ala Val Ser Gln Val Lys370 375 380Thr Ser Val Asp Tyr Ala His
Gly Ser Trp Met Thr Thr Phe Leu Ala385 390 395 400Gly Ala Leu Asn
Tyr Gln Val Val His His Leu Phe Pro Ser Val Ser405 410 415Gln Tyr
His Tyr Pro Ala Ile Ala Pro Ile Ile Val Asp Val Cys Lys420 425
430Glu Tyr Asn Ile Lys Tyr Ala Ile Leu Pro Asp Phe Thr Ala Ala
Phe435 440 445Val Ala His Leu Lys His Leu Arg Asn Met Gly Gln Gln
Gly Ile Ala450 455 460Ala Thr Ile His Met Gly465
47011630DNAArtificial SequencePrimer YL13A 116ttggatccgc agacggagct
ccgccagcgc 3011736DNAArtificial SequencePrimer YL14 117cccttaatta
aattagccca tgtggatcgt ggcggc 361181329DNAIsochrysis galbana
CCMP1323 118atggtggcag gcaaatcagg cgctgcggcg cacgtgactc acagctcgac
attgccccgt 60gagtaccatg gcgcgaccaa cgactcgcgc tctgaggcgg ccgacgtcac
cgtctctagc 120atcgatgctg aaaaggagat gatcatcaac ggccgcgtgt
atgacgtgtc gtcatttgtg 180aagcggcacc caggtggctc ggtgatcaag
ttccagctgg gcgccgacgc gagcgacgcg 240tacaacaact ttcacgtccg
ctccaagaag gcggacaaga tgctgtattc gctcccgtcc 300cggccggccg
aggccggcta cgcccaggac gacatctccc gcgactttga gaagctgcgc
360ctcgagctga aggaggaggg ctacttcgag cccaacctgg tgcacgtgag
ctacaggtgt 420gtggaggttc ttgccatgta ctgggctggc gtccagctca
tctggtccgg gtactggttc 480ctcggcgcga tcgtggccgg cattgcgcag
ggccgctgcg gctggctcca gcatgagggt 540gggcactact cgctcaccgg
caacatcaag atcgaccggc atctgcagat ggccatctat 600gggcttggct
gcggcatgtc gggctgctac tggcgcaacc agcacaacaa gcaccacgcc
660acgccgcaga agctcgggac cgaccccgac ctgcagacga tgccgctggt
ggccttccac 720aagatcgtcg gcgccaaggc gcgaggcaag ggcaaggcgt
ggctggcgtg gcaggcgccg 780ctcttctttg gcgggatcat ctgctcgctc
gtctctttcg gctggcagtt cgtgctccac 840cccaaccacg cgctgcgcgt
gcacaatcac ctggagctcg cgtacatggg cctgcggtac 900gtgctgtggc
acctggcctt tggccacctc gggctgctga gctcgctccg cctgtacgcc
960ttttacgtgg ccgtgggcgg cacctacatc ttcaccaact tcgccgtctc
gcacacccac 1020aaggacgtcg tcccgcccac caagcacatc tcgtgggcac
tctactcggc caaccacacg 1080accaactgct ccgactcgcc ctttgtcaac
tggtggatgg cctacctcaa cttccagatc 1140gagcaccacc tcttcccgtc
gatgccgcag tacaaccacc ccaagatcgc cccgcgggtg 1200cgcgcgctct
tcgagaagca cggggtcgag tatgacgtcc ggccatacct ggagtgtttt
1260cgggtcacgt acgtcaacct gctcgccgta ggcaacccgg agcactccta
ccacgagcac 1320acgcactag 1329119442PRTIsochrysis galbana CCMP1323
119Met Val Ala Gly Lys Ser Gly Ala Ala Ala His Val Thr
His Ser Ser1 5 10 15Thr Leu Pro Arg Glu Tyr His Gly Ala Thr Asn Asp
Ser Arg Ser Glu20 25 30Ala Ala Asp Val Thr Val Ser Ser Ile Asp Ala
Glu Lys Glu Met Ile35 40 45Ile Asn Gly Arg Val Tyr Asp Val Ser Ser
Phe Val Lys Arg His Pro50 55 60Gly Gly Ser Val Ile Lys Phe Gln Leu
Gly Ala Asp Ala Ser Asp Ala65 70 75 80Tyr Asn Asn Phe His Val Arg
Ser Lys Lys Ala Asp Lys Met Leu Tyr85 90 95Ser Leu Pro Ser Arg Pro
Ala Glu Ala Gly Tyr Ala Gln Asp Asp Ile100 105 110Ser Arg Asp Phe
Glu Lys Leu Arg Leu Glu Leu Lys Glu Glu Gly Tyr115 120 125Phe Glu
Pro Asn Leu Val His Val Ser Tyr Arg Cys Val Glu Val Leu130 135
140Ala Met Tyr Trp Ala Gly Val Gln Leu Ile Trp Ser Gly Tyr Trp
Phe145 150 155 160Leu Gly Ala Ile Val Ala Gly Ile Ala Gln Gly Arg
Cys Gly Trp Leu165 170 175Gln His Glu Gly Gly His Tyr Ser Leu Thr
Gly Asn Ile Lys Ile Asp180 185 190Arg His Leu Gln Met Ala Ile Tyr
Gly Leu Gly Cys Gly Met Ser Gly195 200 205Cys Tyr Trp Arg Asn Gln
His Asn Lys His His Ala Thr Pro Gln Lys210 215 220Leu Gly Thr Asp
Pro Asp Leu Gln Thr Met Pro Leu Val Ala Phe His225 230 235 240Lys
Ile Val Gly Ala Lys Ala Arg Gly Lys Gly Lys Ala Trp Leu Ala245 250
255Trp Gln Ala Pro Leu Phe Phe Gly Gly Ile Ile Cys Ser Leu Val
Ser260 265 270Phe Gly Trp Gln Phe Val Leu His Pro Asn His Ala Leu
Arg Val His275 280 285Asn His Leu Glu Leu Ala Tyr Met Gly Leu Arg
Tyr Val Leu Trp His290 295 300Leu Ala Phe Gly His Leu Gly Leu Leu
Ser Ser Leu Arg Leu Tyr Ala305 310 315 320Phe Tyr Val Ala Val Gly
Gly Thr Tyr Ile Phe Thr Asn Phe Ala Val325 330 335Ser His Thr His
Lys Asp Val Val Pro Pro Thr Lys His Ile Ser Trp340 345 350Ala Leu
Tyr Ser Ala Asn His Thr Thr Asn Cys Ser Asp Ser Pro Phe355 360
365Val Asn Trp Trp Met Ala Tyr Leu Asn Phe Gln Ile Glu His His
Leu370 375 380Phe Pro Ser Met Pro Gln Tyr Asn His Pro Lys Ile Ala
Pro Arg Val385 390 395 400Arg Ala Leu Phe Glu Lys His Gly Val Glu
Tyr Asp Val Arg Pro Tyr405 410 415Leu Glu Cys Phe Arg Val Thr Tyr
Val Asn Leu Leu Ala Val Gly Asn420 425 430Pro Glu His Ser Tyr His
Glu His Thr His435 44012036DNAArtificial SequencePrimer YL19A
120tttggatccg gcaggcaaat caggcgctgc ggcgca 3612136DNAArtificial
SequencePrimer YL20 121ccttaattaa ctagtgcgtg tgctcgtggt aggagt
361221320DNAThraustochytrium aureum (ATCC #34304) 122atgggacgcg
gcggcgaagg tcaggtgaac agcgcgcagg tggcacaagg cggtgcggga 60acgcgaaaga
cgatcctgat cgagggcgag gtctacgatg tcaccaactt taggcacccc
120ggcgggtcga tcatcaagtt tctcacgacc gacggcaccg aggctgtgga
cgcgacgaac 180gcgtttcgcg agtttcactg ccggtcgggc aaggcggaaa
agtacctcaa gagcctgccc 240aagctcggcg cgccgagcaa gatgaagttt
gacgccaagg agcaggcccg gcgcgacgcg 300atcacgcgag actacgtcaa
gctgcgcgag gagatggtgg ccgagggcct cttcaagccc 360gcgcccctcc
acattgtcta caggtttgcg gagatcgcag ccctgttcgc ggcctcgttc
420tacctgtttt cgatgcgcgg aaacgtgttc gccacgctcg cggccatcgc
agtcgggggc 480atcgcgcagg gccgctgcgg ctggctcatg cacgagtgcg
gacacttctc gatgaccggg 540tacatcccgc ttgacgtgcg cctgcaggag
ctggtgtacg gcgtggggtg ctcgatgtcg 600gcgagctggt ggcgcgttca
gcacaacaag caccacgcga ccccgcagaa actcaagcac 660gacgtcgacc
tcgacaccct gccgctcgtt gcgttcaacg agaagatcgc cgccaaggtg
720cgccccggct cgttccaggc caagtggctc tcggcgcagg cgtacatttt
tgcgccggtg 780tcctgcttcc tggttggtct cttctggacc ctgtttctgc
acccgcgcca catgccgcgc 840acgagccact ttgctgagat ggccgccgtc
gcggtgcgcg tcgtgggctg ggcggcgctc 900atgcactcgt tcgggtacag
cgggagcgac tcgttcggtc tctacatggc cacctttggc 960tttggctgca
cctacatctt caccaacttt gcggtcagcc acacgcacct cgacgtcacc
1020gagccggacg agttcctgca ctgggtcgag tacgccgcgc tgcacacgac
caacgtgtcc 1080aacgactcgt ggttcatcac ctggtggatg tcgtacctca
actttcagat cgagcaccac 1140ctctttccgt cgctgcccca gctcaacgcc
ccgcgcgtcg ccccgcgcgt ccgcgccctc 1200ttcgagaagc acggcatggc
ttacgacgag cgcccgtacc ttaccgcgct tggcgacacg 1260tttgccaacc
tgcacgccgt gggccaaaac gcgggccagg cggcggccaa ggccgcttag
1320123439PRTThraustochytrium aureum (ATCC #34304) 123Met Gly Arg
Gly Gly Glu Gly Gln Val Asn Ser Ala Gln Val Ala Gln1 5 10 15Gly Gly
Ala Gly Thr Arg Lys Thr Ile Leu Ile Glu Gly Glu Val Tyr20 25 30Asp
Val Thr Asn Phe Arg His Pro Gly Gly Ser Ile Ile Lys Phe Leu35 40
45Thr Thr Asp Gly Thr Glu Ala Val Asp Ala Thr Asn Ala Phe Arg Glu50
55 60Phe His Cys Arg Ser Gly Lys Ala Glu Lys Tyr Leu Lys Ser Leu
Pro65 70 75 80Lys Leu Gly Ala Pro Ser Lys Met Lys Phe Asp Ala Lys
Glu Gln Ala85 90 95Arg Arg Asp Ala Ile Thr Arg Asp Tyr Val Lys Leu
Arg Glu Glu Met100 105 110Val Ala Glu Gly Leu Phe Lys Pro Ala Pro
Leu His Ile Val Tyr Arg115 120 125Phe Ala Glu Ile Ala Ala Leu Phe
Ala Ala Ser Phe Tyr Leu Phe Ser130 135 140Met Arg Gly Asn Val Phe
Ala Thr Leu Ala Ala Ile Ala Val Gly Gly145 150 155 160Ile Ala Gln
Gly Arg Cys Gly Trp Leu Met His Glu Cys Gly His Phe165 170 175Ser
Met Thr Gly Tyr Ile Pro Leu Asp Val Arg Leu Gln Glu Leu Val180 185
190Tyr Gly Val Gly Cys Ser Met Ser Ala Ser Trp Trp Arg Val Gln
His195 200 205Asn Lys His His Ala Thr Pro Gln Lys Leu Lys His Asp
Val Asp Leu210 215 220Asp Thr Leu Pro Leu Val Ala Phe Asn Glu Lys
Ile Ala Ala Lys Val225 230 235 240Arg Pro Gly Ser Phe Gln Ala Lys
Trp Leu Ser Ala Gln Ala Tyr Ile245 250 255Phe Ala Pro Val Ser Cys
Phe Leu Val Gly Leu Phe Trp Thr Leu Phe260 265 270Leu His Pro Arg
His Met Pro Arg Thr Ser His Phe Ala Glu Met Ala275 280 285Ala Val
Ala Val Arg Val Val Gly Trp Ala Ala Leu Met His Ser Phe290 295
300Gly Tyr Ser Gly Ser Asp Ser Phe Gly Leu Tyr Met Ala Thr Phe
Gly305 310 315 320Phe Gly Cys Thr Tyr Ile Phe Thr Asn Phe Ala Val
Ser His Thr His325 330 335Leu Asp Val Thr Glu Pro Asp Glu Phe Leu
His Trp Val Glu Tyr Ala340 345 350Ala Leu His Thr Thr Asn Val Ser
Asn Asp Ser Trp Phe Ile Thr Trp355 360 365Trp Met Ser Tyr Leu Asn
Phe Gln Ile Glu His His Leu Phe Pro Ser370 375 380Leu Pro Gln Leu
Asn Ala Pro Arg Val Ala Pro Arg Val Arg Ala Leu385 390 395 400Phe
Glu Lys His Gly Met Ala Tyr Asp Glu Arg Pro Tyr Leu Thr Ala405 410
415Leu Gly Asp Thr Phe Ala Asn Leu His Ala Val Gly Gln Asn Ala
Gly420 425 430Gln Ala Ala Ala Lys Ala Ala43512430DNAArtificial
SequencePrimer YL15 124ttttccatgg gacgcggcgg cgaaggtcag
3012537DNAArtificial SequencePrimer YL16B 125ttttgcggcc gctaagcggc
cttggccgcc gcctggc 3712610DNAYarrowia
lipolyticamisc_feature(8)..(8)n is a, c, g, or t 126mammatgnhs
10
* * * * *