DK2087105T3 - Delta 17-desaturase og anvendelse heraf ved fremstilling af flerumættede fedtsyrer - Google Patents

Description

DESCRIPTION

FIELD OF THE INVENTION

[0001] This invention is in the field of biotechnology. More specifically, this invention pertains to the identification of a nucleic acid fragment encoding a Δ17 fatty acid desaturase enzyme and the use of this desaturase in making long-chain polyunsaturated fatty acids (PUFAs).

BACKGROUND OF THE INVENTION

[0002] The importance of PUFAs is undisputed. For example, certain PUFAs are important biological components of healthy cells and are recognized as: "essential" fatty acids that cannot be synthesized de now in mammals and instead must be obtained either in the diet or derived by further desaturation and elongation of linoleic acid (LA;18:2 ω-6) or α-linolenic acid (ALA; 18:3 ω-3); constituents of plasma membranes of cells, where they may be found in such forms as phospholipids or triacylglycerols; necessary for proper development (particularly in the developing infant brain) and for tissue formation and repair; and, precursors to several biologically active eicosanoids of importance in mammals (e.g., prostacyclins, eicosanoids, leukotrienes, prostaglandins). Additionally, a high intake of long-chain ω-3 PUFAs produces cardiovascular protective effects ( Dyerberg, J. et al., Amer. J. Clin. Nutr., 28:958-966 (1975); Dyerberg, J. et at, Lancet, 2(8081):117-119 (July 15, 1978); Shimokawa, H„ World Rev. Nutr. Diet, 88:100-108 (2001); von Schacky, C. and Dyerberg, J., World Rev. Nutr. Diet, 88:90-99 (2001 )). Numerous other studies document wide-ranging health benefits conferred by administration of ω-3 and/or ω-6 PUFAs against a variety of symptoms and diseases (e g., asthma, psoriasis, eczema, diabetes, cancer).

[0003] A variety of different hosts including plants, algae, fungi and yeast are being investigated as means for commercial PUFA production. Genetic engineering has demonstrated that the natural abilities of some hosts (even those natively limited to LA and ALA fatty acid production) can be substantially enhanced to produce high levels of e.g., γ-linolenic acid (GLA; 18:3 ω-6), dihomo-γ-linolenic acid (DGLA; 20:3 ω-6), arachidonic acid (ARA; 20:4 ω-6), eicosapentaenoic acid (EPA; 20:5 ω-3), docosapentaenoic acid (DPA; 22:5 ω-3) and docosahexaenoic acid (DHA; 22:6 ω-3).

[0004] Whether ω-3/ω-6 PUFA production is the result of natural abilities or recombinant technology, both strategies may require conversion of ω-6 PUFAs into their ω-3 counterparts. Specifically, a Δ15 desaturase is responsible for the conversion of LA to ALA, while a Δ17 desaturase is responsible for the conversion of ARA to EPA (although some Δ17 desaturases can also use DGLA as a substrate to produce eicosatetraenoic acid (ETA; 20:4 ω-3)). Both of these enzymes have a role in the Δ6 desaturase/Δδ elongase pathway (which is predominantly found in algae, mosses, fungi, nematodes and humans and which is characterized by the production of GLA and/or stearidonic acid (STA; 18:4 ω-3)) and the Δ9 elongase/Δβ desaturase pathway (which operates in some organisms, such as euglenoid species and which is characterized by the production of eicosadienoic acid (EDA; 20:2 ω-6) and/or eicosatrienoic acid (ETrA; 20:3 ω-3)) (Figure 1).

[0005] Because of the role Δ17 desaturase enzymes play in enabling the synthesis of ω-3 fatty acids, there has been considerable effort to identify and characterize these enzymes from various sources. However, only a few Δ17 desaturases are presently known and these have been isolated from only two different taxonomic genera. Specifically, Patent Publication No. US 2003/0190733 describes a Δ17 desaturase from Saprolegnia diclina (see also GenBank Accession No. AY373823). PCT Publication No. WO 2005/083053 describes a Phytophthora infestans "ω3 desaturase" (see also GenBank Accession No. CAJ30870), while PCT Publication No. WO 2006/100241 describes a Phytophthora sojae "ω3 desaturase", both of which appear to function as Δ17 desaturases. Also, commonly owned, co-pending application having U.S. Patent Application No. 11/787772 (filed April 18, 2007) discloses nucleic acid and amino acid sequences for Δ17 desaturases from Phytophthora sojae and Phytophthora ramorum. Thus, there is need for the identification and isolation of additional genes encoding Δ17 desaturases that will be suitable for heterologous expression in a variety of host organisms for use in the production of ω-3 fatty acids.

[0006] Applicants have solved the stated problem by isolating the gene encoding Δ17 desaturase from the oomycete, Pythium aphanidermatum.

SUMMARY OF THE INVENTION

[0007] The present invention relates to new genetic constructs encoding polypeptides having Δ17 desaturase activity, and their use in plants, bacteria, algae, fungi and yeast for the production of PUFAs and particularly ω-3 fatty acids.

[0008] Accordingly, the invention provides an isolated nucleic acid molecule which encodes: a. ) a Δ17 desaturase enzyme, wherein said enzyme has the amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:3; or b. ) an isolated nucleotide molecule that is completely complementary to (a).

[0009] In another embodiment the invention provides isolated nucleic acid molecules encoding Δ17 desaturase enzyme, selected from the group consisting of SEQ ID NO:1 and 4 or isolated nucleic acid molecules which encode a Δ17 desaturase enzyme as set forth in SEQ ID NO:2, wherein at least 175 codons are codon-optimized for expression in Yarrowia. An isolated nucleic acid molecule is also disclosed herein comprising a first nucleotide sequence encoding a Δ17 desaturase enzyme of at least 359 amino acids that has at least 75.3% identity based on Clustal W algorithms when compared to a polypeptide having the sequence as set forth in SEQ ID NO:2; or a second nucleotide sequence comprising the complement of the first nucleotide sequence.

[0010] In other embodiments the invention provides chimeric genes comprising the isolated nucleic acid molecules of the invention operably linked to suitable regulatory sequences and transformed host cells comprising the isolated nucleic acid molecules of the invention.

[0011] A method is disclosed herein for the production of eicosapentaenoic acid comprising: 1. a.) providing a host cell comprising: 1. (i) an isolated nucleotide molecule encoding a bifunctional Δ17/ Δ15 desaturase polypeptide having at least 75.3% identity when compared to a polypeptide having the amino acid sequence as set forth in SEQ ID NO:2, based on the Clustal W method of alignment; and, 2. (ii) a source of arachidonic acid; 2. b.) growing the host cell of step (a) under conditions wherein the nucleic acid molecule encoding the a bifunctional Δ17/ Δ15 desaturase polypeptide is expressed and the arachidonic acid is converted to eicosapentaenoic acid; and, 3. c.) optionally recovering the eicosapentaenoic acid of step (b).

Similarly a method is disclosed herein for the production of eicosatetraenoic acid comprising: 1. a.) providing a host cell comprising: 1. (i) an isolated nucleotide molecule encoding a bifunctional Δ17/ Δ15 desaturase polypeptide having at least 75.3% identity when compared to a polypeptide having the amino acid sequence as set forth in SEQ ID NO:2, based on the Clustal W method of alignment; and, 2. (ii) a source of dihomo-y-linolenic acid; 2. b.) growing the host cell of step (a) under conditions wherein the nucleic acid molecule encoding the a bifunctional Δ17/Δ15 desaturase polypeptide is expressed and the dihomo-y-linolenic acid is converted to eicosatetraenoic acid; and, 3. c.) optionally recovering the eicosatetraenoic acid of step (c).

[0012] The invention also provides a method for the production of polyunsaturated fatty acids comprising: 1. a) providing a host cell comprising: 1. i) an isolated nucleotide molecule encoding a bifunctional Δ17/ Δ15 desaturase polypeptide having the amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:3; and, 2. ii) a source of at least one fatty acid selected from the group consisting of: linoleic acid, eicosadienoic acid, dihomo-y-linolenic acid and arachidonic acid; 2. b) growing the host cell of step (a) under conditions wherein the nucleic acid molecule encoding the bifunctional Δ17/ Δ15 desaturase polypeptide is expressed and the at least one fatty acid is converted to at least one product fatty acid, wherein the reaction is selected from the group consisting of: linoleic acid is converted to α-linolenic acid, eicosadienoic acid is converted to eicosatrienoic acid; dihomo-y-linolenic acid is coverted to eicosatetraenoic acid, and arachidonic acid is converted to eicosapentaenoic acid; and, 3. c) optionally recovering the at least one product fatty acid of step (b).

[0013] Another isolated nucleic acid fragment is disclosed herein comprising a nucleic acid sequence encoding a Δ17 desaturase polypeptide comprising at least one amino acid sequence motifs selected from the group consisting of: 1. a) FTXGHDXGH (SEQ ID NO:96); 2. b) HRHHHKNTG (SEQ ID NO:97); and, 3. c) IGTHQXHHLFP (SEQ ID NO:98); wherein Xcan be any amino acid, and wherein the Δ17 desaturase polypeptide does not have the amino acid sequence as set forth in SEQ ID NOs:43 and 95.

[0014] ΑΔ17 desaturase polypeptide comprising at least one amino acid motif selected from the group consisting of SEQ ID NO:96-98 is also disclosed herein.

[0015] In other embodiments the invention provides methods for the identification and isolation of a Δ17 desaturase polypeptide comprising: 1. a) probing a genomic library with: 1. i) an isolated nucleic acid fragment encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 96-98; or, 2. ii) an isolated nucleic acid fragment that is complementary to (i); 2. b) identifying a DNA clone that hybridizes with the nucleic acid fragment of step (a); and, 3. c) sequencing the genomic fragment that comprises the clone identified in step (b); wherein the sequenced genomic fragment encodes a Δ17 desaturase polypeptide, or alternatively, 1. a) synthesizing at least one oligonucleotide primer corresponding to a portion of an isolated nucleic acid sequence encoding an amino acid motif selected from the group consisting of SEQ ID NOs:96-98; and, 2. b) amplifying an insert present in a cloning vector using the oligonucleotide primer of step (a); wherein the amplified insert encodes a portion of an amino acid sequence encoding a Δ17 desaturase enzyme.

BIOLOGICAL DEPOSITS

[0016] The following biological material has been deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209, and bears the following designation, accession number and date of deposit.

[0017] The biological material listed above was deposited under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The listed deposit will be maintained in the indicated international depository for at least 30 years and will be made available to the public upon the grant of a patent disclosing it. The availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by government action.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS

[0018]

Figure 1 illustrates the ω-3/ω-6 fatty acid biosynthetic pathway.

Figure 2 shows a pairwise alignment of the amino acid sequences of the Phytophthora sojae Δ17 desaturase (SEQ ID NO:45) and the Phytophthora ramorum Δ17 desaturase (SEQ ID NO:47), created using default parameters of Vector NTI®s AlignX program (Invitrogen Corporation, Carlsbad, CA).

Figure 3 provides plasmid maps for the following: (A) pKUNFmkF2; (B) pDMW287F; (C) pDMW214; and, (D) pFmD8S.

Figure 4A diagrams the development of Yarrowia lipolytica strain Y2047, producing 11%ARAin the total lipid fraction. Figure 4B provides a plasmid map for pKUNF12T6E, while Figure 4C provides a plasmid map for pDMW271.

Figures 5A and 5B show a comparison of the DNA sequence of the Phytophthora aphanidermatum Δ17 desaturase gene (designated as "PaD17"; SEQ ID NO:1) and the synthetic gene (designated as “PaD17S"; SEQ ID NO:4) codon-optimized for expression in Yarrowia lipolytica.

Figure 6Adiagrams the development of Yarrowia lipolytica strain Y4070, producing 12% ARAin the total lipid fraction. Figure 6B provides a plasmid map for pZKLeuN-29E3, while Figure 6C provides a plasmid map for pY116.

Figure 7 provides plasmid maps for the following: (A) pK02UF8289; and, (B) pZKSL-555R.

Figure 8 provides plasmid maps for the following: (A) pFBAIN-MOD-1; and, (B) pY6.GPD.Leu2.

Figure 9 shows a comparison of the DNA sequence of the Phytophthora sojae Δ17 desaturase gene (designated as “PsD17“; SEQ ID NO:44) and the synthetic gene (designated as "PsD17S"; SEQ ID NO:81) codon-optimized for expression in Y. lipolytica.

Figure 10 shows a comparison of the DNA sequence of the Phytophthora ramorum Δ17 desaturase gene (designated as ”PrD17”; SEQ ID NO:46) and the synthetic gene (designated as "PrD17S"; SEQ ID NO:84) codon-optimized for expression in Y. lipolytica.

Figure 11 provides plasmid maps for the following: (A) pY130; (B) pY138; (C) pY139; and, (D) pY140.

Figure 12 provides plasmid maps for the following: (A) pY137; and, (B) pY117.

Figure 13 is a graph showing the ω-6 fatty acid substrate specificity of the following ω-3 desaturases: Fusarium moniliforme Δ15 desaturase (FmD15; SEQ ID NOs:86 and 87); a synthetic Δ17 desaturase derived from Phytopthora ramorum, codon-optimized for expression in Yarrowia lipolytica (PrD17S; SEQ ID NOs:84 and 47); a synthetic Δ17 desaturase derived from Phytopthora sojae, codon-optimized for expression in Yarrowia lipolytica (PsD17S; SEQ ID NOs:81 and 82); and the synthetic Δ17 desaturase derived from Pythium aphanidermatum, codon-optimized for expression iri Yarrowia lipolytica (PaD17S; SEQ ID NOs:4 and 2).

Figure 14 shows a Clustal V alignment (with default parameters) of the of the following ω-3 desaturases: Phytophthora infestans Δ17 desaturase (PiD17; SEQ ID NO:43); Phytopthora ramorum Δ17 desaturase (PrD17; SEQ ID NO:47); synthetic Δ17 desaturase derived from Phytopthora sojae, codon-optimized for expression in Yarrowia lipolytica (PsD17S; SEQ ID NO:82); Saprolegnia diclina Δ17 desaturase, (SdD17; SEQ ID NO:95); and the Pythium aphanidermatum Δ17 desaturase of the instant invention (PaD17S; SEQ ID NO:2). Sequence regions shown in boxes correspond to delta-17 motifs #1, #2 and #3, respectively. The invention can be more fully understood from the following detailed description and the accompanying sequence descriptions, which form a part of this application.

[0019] The following sequences comply with 37 C.F.R. §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. §1.822.

[0020] SEQ ID NOs:1-8, 42-53, 56-95 and 102 are ORFs encoding genes or proteins or plasmids, as identified in Table 1.

Table 1

[0021] SEQ ID NOs:9-11 correspond to SMART™ IV oligonucleotide primer, CDSIII/3' PCR primer and 5-PCR primer, respectively, used for Pythium aphanidermatum cDNA synthesis.

[0022] SEQ ID NO:12 corresponds to degenerate oligonucleotide primer PD17-F1, which encodes the peptide set forth in SEQ ID NO:13.

[0023] SEQ ID NOs:14 and 15 correspond to degenerate oligonucleotide primers PD17-F2 and PD17-F3, respectively, both of which encode the peptide set forth in SEQ ID NO: 16.

[0024] SEQ ID NOs:17 and 18 correspond to degenerate oligonucleotide primers PD17-F4 and PD17-F5, respectively, both of which encode the peptide set forth in SEQ ID NO: 19.

[0025] SEQ ID NOs:20 and 21 correspond to degenerate oligonucleotide primers PD17-F6 and PD17-F7, respectively, both of which encode the peptide set forth in SEQ ID NO:22.

[0026] SEQ ID NOs:23 and 24 correspond to degenerate oligonucleotide primers PD17-R1 and PD17-R2, respectively, both of which encode the peptide set forth in SEQ ID NO:25.

[0027] SEQ ID NOs:26 and 27 correspond to degenerate oligonucleotide primers PD17-R3 and PD17-R4, respectively, both of which encode the peptide set forth in SEQ ID NO:28.

[0028] SEQ ID NOs:29 and 30 correspond to degenerate oligonucleotide primers PD17-R5 and PD17-R6, respectively, both of which encode the peptide set forth in SEQ ID NO: 31.

[0029] SEQ ID NO:32 corresponds to degenerate oligonucleotide primer PD17-R7, which encodes the peptide set forth in SEQ ID NO:33.

[0030] SEQ ID NOs:34 and 35 correspond to the Universal GenomeWalker™ adaptor.

[0031] SEQ ID NOs:36, 37, 38 and 39 correspond to primers PUD17-5-1, Universal GenomeWalker TM primer AP1, PUD17-5-3 and Universal GenomeWalker™ primer AP2, respectively, used for PCR amplification of the 5'-end of genomic DNA encoding the Pythium aphanidermatum Δ17 desaturase.

[0032] SEQ ID NOs:40 and 41 correspond to primers PUD17-3-1 and PUD17-3-2, respectively, used for PCR amplification of the 3'-end of cDNA encoding the Pythium aphanidermatum Δ17 desaturase.

[0033] SEQ ID NOs:54 and 55 correspond to primers PUD17-F and PUD17-R, respectively, used for amplification of the full length cDNA encoding the Pythium aphanidermatum hM desaturase.

[0034] SEQ ID NOs:96-98 correspond to Δ17 desaturase motif #1, Δ17 desaturase motif #2 and Δ17 desaturase motif #3, respectively.

[0035] SEQ ID NOs:99-101 correspond to His-rich motifs that are featured in membrane-bound fatty acid desaturases belonging to a super-family of membrane di-iron proteins.

DETAILED DESCRIPTION OF THE INVENTION

[0036] Patents, patent applications, and publications cited herein include the following Applicants' Assignee's co-pending applications: U.S. Patent 7,125,672, U.S. Patent 7,189,559, U.S. Patent 7,192,762, U.S. Patent 7,198,937, U.S. Patent 7,202,356, U.S. Patent Applications No. 10/840579 and No. 10/840325 (filed May 6, 2004), U.S. Patent Application No. 10/869630 (filed June 16, 2004), U.S. Patent Application No. 10/882760 (filed July 1, 2004), U.S. Patent Applications No. 10/985254 and No. 10/985691 (filed November 10, 2004), U.S. Patent Application No. 11/024544 (filed December 29, 2004), U.S. Patent Application No. 11/166993 (filed June 24, 2005), U.S. Patent Application No. 11/183664 (filed July 18, 2005), U.S. Patent Application No. 11/185301 (filed July 20, 2005) , U.S. Patent Application No. 11/190750 (filed July 27 2005), U.S. Patent Application No. 11/198975 (filed August 8, 2005), U.S. Patent Application No. 11/225354 (filed September 13, 2005), U.S. Patent Application No. 11/253882 (filed October 19, 2005), U.S. Patent Applications No. 11/264784 and No. 11/264737 (filed November 1,2005), U.S. Patent Application No. 11/265761 (filed November 2, 2005), U.S. Patent Application No. 60/853563 (filed October 23, 2006), U.S. Patent Application No. 60/855177 (filed October 30, 2006), U.S. Patent Applications No. 11/601563 and No. 11/601564 (filed November 16, 2006), U.S. Patent Application No. 11/635258 (filed December 7, 2006), U.S. Patent Application No. 11/613420 (filed December 20, 2006), U.S. Patent Application No. 60/909790 (filed April 3, 2007), U.S. Patent Application No. 60/910831 (filed April 10, 2007), U.S. Patent Application No. 60/911925 (filed April 16, 2007), U.S. Patent Application No. 11/787772 (filed April 18, 2007), U.S. Patent Application No. 11/737772 (filed April 20, 2007), U.S. Patent Application No. 11/740298 (filed April 26, 2007), U.S. Patent Application No. 60/915733 (filed May 3, 2007) and U.S. Patent Applications No. 11/748629 and No. 11/748637 (filed May 15, 2007).

[0037] The invention provides a novel Oomycota Δ17 desaturase enzyme and gene encoding the same that may be used for the manipulation of biochemical pathways for the production of healthful PUFAs.

[0038] 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).

[0039] 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 EPAcan result not only in increased levels of EPA, but also downstream products of EPA such as eicosanoids (i.e., prostaglandins, leukotrienes, thromboxanes). 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 [0040] In this disclosure, a number of terms and abbreviations are used. The following definitions are provided.

[0041] "Open reading frame" is abbreviated ORF.

[0042] "Polymerase chain reaction" is abbreviated PCR.

[0043] "American Type Culture Collection" is abbreviated ATCC.

[0044] "Polyunsaturated fatty acid(s)" is abbreviated PUFA(s).

[0045] "Triacylglycerols" are abbreviated TAGs.

[0046] The term "fatty acids” refers to long-chain aliphatic acids (alkanoic acids) of varying chain lengths, from about C-|2 to C22 (although both longer and shorter chain-length acids are known). The predominant chain lengths are between C-|6 and C22· The structure of a fatty acid is represented by a simple notation system of "XY", where X is the total number of carbon (C) atoms in the particular fatty acid and Y is the number of double bonds. Additional details concerning the differentiation between "saturated fatty acids" versus "unsaturated fatty acids", "monounsaturated fatty acids" versus "polyunsaturated fatty acids" (or "PUFAs"), and "omega-6 fatty acids" (ω-6 or n-6) versus "omega-3 fatty acids" (ω-3 or n-3) are provided in PCT Publication No. WO 2004/101757.

[0047] Nomenclature used to describe PUFAs in the present disclosure is shown below in Table 2. In the column titled "Shorthand Notation", the omega-reference system is used to indicate the number of carbons, the number of double bonds and the position of the double bond closest to the omega carbon, counting from the omega carbon (which is numbered 1 for this purpose). The remainder of the Table summarizes the common names of ω-3 and ω-6 fatty acids and their precursors, the abbreviations that will be used throughout the specification and each compounds' chemical name.

Table 2

[0048] The terms "triacylglycerol", "oil" and "TAGs" refer to neutral lipids composed of three fatty acyl residues esterified to a glycerol molecule' (and such terms will be used interchangeably throughout the present disclosure herein). Such oils can contain long chain PUFAs. as well as shorter saturated and unsaturated fatty acids and longer chain saturated fatty acids. Thus, "oil biosynthesis" generically refers to the synthesis of TAGs in the cell. "Microbial oils" or "single cell oils" are those oils naturally produced by microorganisms during their lifespan.

[0049] "Percent (%) PUFAs in the total lipid and oil fractions" refers to the percent of PUFAs relative to the total fatty acids in those fractions. The term "total lipid fraction" or "lipid fraction" both refer to the sum of all lipids (i.e., neutral and polar) within an oleaginous organism, thus including those lipids that are located in the phosphatidylcholine (PC) fraction, phosphatidyletanolamine (PE) fraction and triacylglycerol (TAG or oil) fraction. However, the terms "lipid" and "oil" will be used interchangeably throughout the specification.

[0050] A metabolic pathway, or biosynthetic pathway, in a biochemical sense, can be regarded as a series of chemical reactions occurring within a cell, catalyzed by enzymes, to achieve either the formation of a metabolic product to be used or stored by the cell, or the initiation of another metabolic pathway (then called a flux generating step). Many of these pathways are elaborate, and involve a step by step modification of the initial substance to shape it into a product having the exact chemical structure desired.

[0051] The term "PUFA biosynthetic pathway" refers to a metabolic process that converts oleic acid to LA, EDA, GLA, DGLA, ARA, ALA, STA, ETrA, ETA, EPA, DPAand DHA. This process is well described in the literature (e.g., see PCT Publication No. WO 2006/052870). Briefly, this process involves elongation of the carbon chain through the addition of carbon atoms and desaturation of the molecule through the addition of double bonds, via a series of special desaturation and elongation enzymes (i.e., "PUFA biosynthetic pathway enzymes") present in the endoplasmic reticulim membrane. More specifically, "PUFA biosynthetic pathway enzymes" refer to any of the following enzymes (and genes which encode said enzymes) associated with the biosynthesis of a PUFA, including: a Δ4 desaturase, a Δ5 desaturase, a Δ6 desaturase, a Δ12 desaturase, a Δ15 desaturase, a Δ17 desaturase, a Δ9 desaturase, a Δ8 desaturase, a Δ9 elongase, a C-14/16 elongase, a C16/I8 elongase, a C-|8/20 elongase and/or a C20/22 elongase.

[0052] The term "ω-3/ω-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 ω-3 and ω-6 fatty acids. Typically the genes involved in the ω-3/ω-6 fatty acid biosynthetic pathway encode PUFA biosynthetic pathway enzymes. A representative pathway is illustrated in Figure 1, providing for the conversion of myristic acid through various intermediates to DHA, which demonstrates how both ω-3 and ω-6 fatty acids may be produced from a common source. The pathway is naturally divided into two portions where one portion will generate ω-3 fatty acids and the other portion, only ω-6 fatty acids. That portion that only generates ω-3 fatty acids will be referred to herein as the ω-3 fatty acid biosynthetic pathway, whereas that portion that generates only ω-6 fatty acids will be referred to herein as the ω-6 fatty acid biosynthetic pathway.

[0053] The term "functional" as used herein in context with the ω-3/ω-6 fatty acid biosynthetic pathway means that some (or all) of the genes in the pathway express active enzymes, resulting in in mo catalysis or substrate conversion. It should be understood that "ω-3/ω-6 fatty acid biosynthetic pathway" or "functional ω-3/ω-6 fatty acid biosynthetic pathway" does not imply that all the genes in the above paragraph are required, as a number of fatty acid products will only require the expression of a subset of the genes of this pathway.

[0054] The term "desaturase" refers to a polypeptide that can desaturate, i.e., introduce a double bond, in one or more fatty acids to produce a 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 interest herein are: 1) Δ8 desaturases that will catalyze the conversion of EDA to DGLA and/or ETrA to ETA; 2.) Δ5 desaturases that catalyze the conversion of DGLA to ARA and/or ETA to EPA; 3.) Δ6 desaturases that catalyze the conversion of LA to GLA and/or ALA to STA; 4.) Δ4 desaturases that catalyze the conversion of DPAto DHA; 5.) Δ12 desaturases that catalyze the conversion of oleic acid to LA; 6.) Δ15 desaturases that catalyze the conversion of LA to ALA and/or GLA to STA; and, 7.) Δ9 desaturases that catalyze the conversion of palmitate to palmitoleic acid (16:1) and/or stearate to oleic acid (18:1).

[0055] Of particular interest herein are Δ17 desaturases that desaturate a fatty acid between the 17111 and 18th carbon atom numbered from the carboxyl-terminal end of the molecule and which, for example, catalyze the conversion of ARA to EPA (and optionally DGLA to ETA). In the art, Δ17 desaturases (and also Δ15 desaturases) are also occasionally referred to as "omega-3 desaturases", "w-3 desaturases", and/or "ω-3 desaturases", based on their ability to convert ω-6 fatty acids into their ω-3 counterparts (e.g., conversion of LA into ALA or DGLA into ETA and ARA into EPA, respectively).

[0056] Some desaturases have activity on two or more substrates. Based on this ability, these enzymes can be further classified with respect to their desaturase activities as being either "monofunctional" or "bifunctional". In some embodiments, it is most desirable to empirically determine the specificity of a fatty acid desaturase by transforming a suitable host with the gene for the fatty acid desaturase and determining its effect on the fatty acid profile of the host.

[0057] More specifically, Δ17 desaturases are defined herein as those fatty acid desaturases having monofunctional or bifunctional Δ17 desaturase activity, wherein Δ17 desaturase activity is the conversion of ARAto EPA and/or DGLAto ETA. The term "monofunctional Δ17 desaturase", "monofunctional Δ17 desaturase activity" or "exclusive Δ17 desaturase activity" refers to a Δ17 desaturase that is capable of converting ARA to EPA and/or DGLA to ETA but not LA to ALA. In contrast, "bifunctional Δ17 desaturase", "bifunctional Δ17 desaturase activity" or "primary Δ17 desaturase activity" refers to a Δ17 desaturase that preferentially converts ARA to EPA and/or DGLAto ETA but additionally has limited ability to convert LA into ALA (thus exhibiting primarily Δ17 desaturase activity and limited Δ15 desaturase activity).

[0058] It should be noted that Δ17 desaturases can have specificities other than Δ17 and Δ15 desaturation that are not relevant in this classification.

[0059] For the purposes herein, the term "PaD17" refers to a Δ17 desaturase enzyme (SEQ ID NO:2) isolated from Pythium aphanidermatum, encoded by SEQ ID NO:1. Similarly, the term "PaD17*" refers to a Δ17 desaturase enzyme (SEQ ID NO:3) comprising up to (and including) two conservative amino acid mutations (i.e., 155S to P and 351Ato T) with respect to SEQ ID NO:2. In contrast, the term "PaD17S" refers to a synthetic Δ17 desaturase derived from Pythium aphanidermatum that is codon-optimized for expression in Yarrowia lipolytica (i.e., SEQ ID NOs:4 and 2). Based on analyses described herein, PaD17 and PaD17S are further classified as bifunctional Δ17 desaturases.

[0060] For the purposes herein, the term "PsD17" refers to a Δ17 desaturase enzyme (SEQ ID NO:45) isolated from Phytophthora sojae, encoded by SEQ ID NO:44. In contrast, the term "PsD17S" refers to a synthetic Δ17 desaturase derived from Phytophthora sojae that is codon-optimized for expression in Yarrowia lipolytica (i.e., SEQ ID NOs:81 and 82). Based on analyses described herein, PsD17 and PsD17S are further classified as bifunctional Δ17 desaturases.

[0061] Similarly, the term "PrD17" refers to a Δ17 desaturase enzyme (SEQ ID NO:47) isolated from Phytophthora ramorum, encoded by SEQ ID NO:46. In contrast, the term "PrD17S" refers to a synthetic Δ17 desaturase derived from Phytophthora ramonum that is codon-optimized for expression in Yarrowia lipolytica (i.e., SEQ ID NOs:84 and 47). Previous analyses described in U.S. Patent Application No. 11/787772 classified PrD17 and PrD17S as monofunctional Δ17 desatu rases; however, based on analyses described herein, PrD17 and PrD17S are now identified as bifunctional Δ17 desaturases.

[0062] Relatedly, the term "PiD17" refers to a Δ17 desaturase enzyme (SEQ ID NO:43) isolated from Phytophthora infestans, encoded by SEQ ID NO:42.

[0063] The terms "conversion efficiency" and "percent substrate conversion" refer to the efficiency by which a particular enzyme (e g., a desaturase) 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.

[0064] The term "elongase" refers to a polypeptide that can elongate a fatty acid carbon chain to produce an 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, as described in PCT Publication No. WO 2004/101757. Examples of reactions catalyzed by elongase systems are the conversion of GLA to DGLA, STA to ETA and EPA to DPA. In general, the substrate selectivity of elongases is somewhat broad but segregated by both chain length and the degree and type of unsaturation. For example: a C-14/16 elongase will utilize a C-14 substrate (e.g., myristic acid); a C16/18 elongase will utilize a C-|6 substrate (e.g., palmitate); a C-|8/20 elongase (also known as a Δ6 elongase as the terms can be used interchangeably) will utilize a C-|8 substrate (e.g., GLA, STA); and, a C20/22 elongase will utilize a C20 substrate (e.g., EPA). In like manner, a Δ9 elongase is able to catalyze the conversion of LA and ALA to EDA and ETrA, respectively. It is important to note that some elongases have broad specificity and thus a single enzyme may be capable of catalyzing several elongase reactions (e.g., thereby acting as both a C-|6/18 elongase and a C-|8/20 elongase).

[0065] The term "oomycetes" refers to a group of heterotrophic organisms generally known as the water molds and downy mildews. They are filamentous protists that must absorb their food from the surrounding water or soil, or may invade the body of another organism to feed. As such, oomycetes play an important role in the decomposition and recycling of decaying matter. Although oomycetes have similarities to fungi through convergent evolution, they are not fungi (as previously thought); instead, the oomycetes are part of the kingdom Stramenopiles and are thereby distinct from plants, fungi and animals. Diatoms and golden-brown and brown algae (e.g., kelp) are also included within kingdom Stramenopiles.

[0066] Pythium is a genus of the oomycetes, comprising about eighty-five species. Pythium species are common pathogens causing disease in plants and fishes. The species of this genus are among the most destructive plant pathogens, inflicting serious economic losses of crops by destroying seed, storage organs, roots and other plant tissues. Members of the genus Pythium have been described as "aquatic fungi".

[0067] The term "oleaginous" refers to those organisms that tend to store their energy source in the form of lipid (Weete, In: Fungal Lipid Biochemistry, 2nd Ed., Plenum, 1980). The term "oleaginous yeast" refers to those microorganisms classified as yeasts that can make oil. Generally, the cellular oil or TAG content of oleaginous 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)). It is not uncommon for oleaginous microorganisms to accumulate in excess of about 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.

[0068] The term "amino acid" will refer to the basic chemical structural unit of a protein or polypeptide. Amino acids are identified by either the one-letter code or the three-letter codes for amino acids, in conformity with the IUPAC-IYUB standards described in Nucleic Acids Research, 13:3021-3030 (1985) and in the Biochemical Journal, 219(2):345-373 (1984).

[0069] The term "conservative amino acid substitution" refers to a substitution of an amino acid residue in a given protein with another amino acid, without altering the chemical or functional nature of that protein. For example, it is well known in the art that alterations in a gene that result in the production of a chemically equivalent amino acid at a given site (but that do not affect the structural and functional properties of the encoded, folded protein) are common. For the purposes of the present invention, "conservative amino acid substitutions" are defined as exchanges within one of the following five groups: 1. 1. Small aliphatic, nonpolar or slightly polar residues: Ala [A], Ser [S], Thr [Γ] (Pro [P], Gly [G]); 2. 2. Polar, negatively charged residues and their amides: Asp [D], Asn [N], Glu [E], Gin [Q]; 3. 3. Polar, positively charged residues: Fis [H], Arg [R], Lys [K]; 4. 4. Large aliphatic, nonpolar residues: Met [M], Leu [L], Ile [I], Val [V] (Cys [C]); and, 5. 5. Large aromatic residues: Phe [F], Tyr [Y], Trp [W],

Conservative amino acid substitutions generally maintain: 1.) the structure of the polypeptide backbone in the area of the substitution; 2.) the charge or hydrophobicity of the molecule at the target site; or 3.) the bulk of the side chain. Additionally, in many cases, alterations of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein.

[0070] The term "non-conservative amino acid substitution" refers to an amino acid substitution that is generally expected to produce the greatest change in protein properties. Thus, for example, a non-conservative amino acid substitution would be one whereby: 1.) a hydrophilic residue is substituted for/by a hydrophobic residue (e.g., Ser or Thr for/by Leu, Ile, Val); 2.) a Cys or Pro is substituted for/by any other residue; 3.) a residue having an electropositive side chain is substituted for/by an electronegative residue (e.g., Lys, Arg or His for/by Asp or Glu); or, 4.) a residue having a bulky side chain is substituted for/by one not having a side chain (e.g., Phe for/by Gly). Sometimes, non-conservative amino acid substitutions between two of the five groups will not affect the activity of the encoded protein.

[0071] As used herein, an "isolated nucleic acid fragment" or "isolated nucleic acid molecule" will be used interchangeably and refers to 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.

[0072] A nucleic acid fragment is "hybridizable" to another nucleic acid fragment, such as a cDNA, genomic DNA, or RNA molecule, when a single-stranded form of the nucleic acid fragment can anneal to the other nucleic acid fragment under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989), particularly Chapter 11 and Table 11.1 therein. The conditions of temperature and ionic strength determine the "stringency" of the hybridization. Stringency conditions can be adjusted to screen for moderately similar fragments (such as homologous sequences from distantly related organisms), to highly similar fragments (such as genes that duplicate functional enzymes from closely related organisms). Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6X SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2X SSC, 0.5% SDS at 45 °C for 30 min, and then repeated twice with 0.2X SSC, 0.5% SDS at 50 °C for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2X SSC, 0.5% SDS was increased to 60 °C. Another preferred set of highly stringent conditions uses two final washes in 0.1 X SSC, 0.1% SDS at 65 °C. An additional set of stringent conditions include hybridization at 0.1XSSC, 0.1% SDS, 65 °C and washes with 2XSSC, 0.1% SDS followed by 0.1 XSSC, 0.1% SDS, for example.

[0073] Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of 1m for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNADNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et a I., supra, 9.50-9.51). For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). In one embodiment the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least about 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.

[0074] 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. R, 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 putatively identify 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. The instant specification teaches the complete amino acid and nucleotide sequence encoding a particular oomycete protein. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above are disclosed herein.

[0075] The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the isolated nucleic acid fragments that are complementary to the complete sequences as reported in the accompanying Sequence Listing, as well as those substantially similar nucleic acid sequences are also disclosed herein.

[0076] The terms "homology" and "homologous" are used interchangeably and refer to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. It is therefore understood, as those skilled in the art will appreciate, that the invention as defined in the claims encompasses more than the specific exemplary sequences.

[0077] Moreover, the skilled artisan recognizes that homologous nucleic acid sequences may be defined by their ability to hybridize, under moderately stringent conditions (e.g., 0.5 XSSC, 0.1% SDS, 60 °C) with the sequences exemplified herein, or to any portion of the present nucleotide sequences and which are functionally equivalent to any of the nucleic acid sequences disclosed herein.

[0078] "Codon degeneracy" refers to the nature in the genetic code permitting variation of the nucleotide sequence without effecting 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.

[0079] "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.

[0080] "Gene" refers to a nucleic acid fragment that expresses a specific protein, and that may refer to the coding region alone or may include 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 that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, native genes introduced into a new location within the native host, 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.

[0081] "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.

[0082] "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.

[0083] The terms "3' non-coding sequences" and "transcription terminator" refer 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.

[0084] "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. Patent No. 5,107,065; PCT Publication No. 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.

[0085] 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.

[0086] 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.

[0087] "Mature" protein refers to a post-translationally processed polypeptide, i.e., one from which any pre- or propeptides present in the primary translation product have been removed. "Precursor" protein refers to the primary product of translation of mRNA, i.e., with pre- and propeptides still present. Pre- and propeptides may be (but are not limited to) intracellular localization signals.

[0088] "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.

[0089] 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 DNAfragments. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell. "Expression cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.

[0090] The term "percent identity", as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in: 1.) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.) Biocomputing: Informatics and Genome Projects (Smith, D. W, Ed.) Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and, 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991).

[0091] Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wl). Multiple alignment of the sequences is performed using the "Clustal method of alignment" which encompasses several varieties of the algorithm including the "Clustal V method of alignment" corresponding to the alignment method labeled Clustal V (described by Higgins and Sharp, CABIOS, 5:151-153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal V method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences using the Clustal V program, it is possible to obtain a "percent identity" by viewing the "sequence distances" table in the same program. Additionally the "Clustal W method of alignment" is available and corresponds to the alignment method labeled Clustal W (described by Higgins and Sharp, CABIOS, 5:151-153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in the MegAlign™ v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). Default parameters for multiple alignment correspond to GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series and DNA Weight Matrix=IUB. After alignment of the sequences using the Clustal W program, it is possible to obtain a "percent identity" by viewing the "sequence distances" table in the same program.

[0092] " BLASTN method of alignment" is an algorithm provided by the National Center for Biotechnology Information (NCBI) to compare nucleotide sequences using default parameters.

[0093] It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides, from other species, wherein such polypeptides have the same or similar function or activity. Nucleic acid fragments (isolated polynucleotides) may encode polypeptides that are at least about 70% identical, preferably at least about 75% identical, and more preferably at least about 80% identical to the amino acid sequences reported herein. Nucleic acid fragments may also encode amino acid sequences that are at least about 85% identical to the amino acid sequences reported herein.

Other nucleic acid fragments may encode amino acid sequences that are at least about 90% identical to the amino acid sequences reported herein. Still further nucleic acid fragments may encode amino acid sequences that are at least about 95% identical to the amino acid sequences reported herein. Indeed, any integer amino acid identity from 70% to 100% may be useful in describing nucleic acid fragments disclosed herein, such as 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Nucleic acid fragments not only may have the above homologies but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids.

[0094] 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, Wl); 2.) BLASTP, BLASTN, BLASTX ( Altschul et al., J. Mol. Biol., 215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wl); 4.) Sequencher (Gene Codes Corporation, Ann Arbor, Ml); and, 5.) the FASTA program incorporating the Smith-V\faterman algorithm ( W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, NY ). 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.

[0095] The term "conserved domain" or "motif means a set of amino acids conserved at specific positions along an aligned sequence of evolutionarily related proteins. While amino acids at other positions can vary between homologous proteins, amino acids that are highly conserved at specific positions indicate amino acids that are essential in the structure, the stability, or the activity of a protein. Because they are identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers, or "signatures", to determine if a protein with a newly determined sequence belongs to a previously identified protein family. For the purposes herein, the following Table describes motifs which are indicative of a protein having Δ17 desaturase activity.

Table 3

[0096] The term "His Box" refers to a histidine box having a motif selected from the group consisting of: H(X)3H (SEQ ID NO:99), H(X)2HH (SEQ ID NO:100) and H/Q(X)2HH (SEQ ID NO:101).

[0097] Standard recombinant DNAand 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, NY (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, NY (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc, and Wiley-lnterscience, Hoboken, NJ (1987).

An Overview: Microbial Biosynthesis Of Fatty Acids And Triacvlalvcerols [0098] In general, lipid accumulation in oleaginous microorganisms is triggered in response to the overall carbon to nitrogen ratio present in the growth medium. This process, leading to the de novo synthesis of free palmitate (16:0) in oleaginous microorganisms, is described in detail in PCT Publication No. WO 2004/101757. Palmitate is the precursor of longer-chain saturated and unsaturated fatty acid derivates, which are formed through the action of elongases and desaturases (Figure 1).

[0099] TAGs (the primary storage unit for fatty acids) are formed by a series of reactions that involve: 1.) the esterification of one molecule of acyl-CoAto glycerol-3-phosphate via an acyltransferase to produce lysophosphatidic acid; 2.) the esterification of a second molecule of acyl-CoA via an acyltransferase to yield 1,2-diacylglycerol phosphate (commonly identified as phosphatidic acid); 3.) removal of a phosphate by phosphatidic acid phosphatase to yield 1,2-diacylglycerol (DAG); and, 4.) the addition of a third fatty acid by the action of an acyltransferase to form TAG. A wide spectrum of fatty acids can be incorporated into TAGs, including saturated and unsaturated fatty acids and short-chain and long-chain fatty acids.

Biosynthesis Of Omega Fatty Acids [0100] The metabolic process wherein oleic acid is converted to ω-3/ω-6 fatty acids involves elongation of the carbon chain through the addition of carbon atoms and desaturation of the molecule through the addition of double bonds. This requires a series of special desaturation and elongation enzymes present in the endoplasmic reticulim membrane. However, as seen in Figure 1 and as described below, there are often multiple alternate pathways for production of a specific ω-3/ω-6 fatty acid.

[0101] Specifically, all pathways require the initial conversion of oleic acid to LA, the first of the ω-6 fatty acids, by a Δ12 desaturase. Then, using the "Δ6 desaturase/Δβ elongase pathway", ω-6 fatty acids are formed as follows: (1) LA is converted to GLA by a Δ6 desaturase; (2) GLA is converted to DGLA by a C 18/20 elongase; and (3) DGLA is converted to ARA by a Δ5 desaturase. Alternatively, the "Δ6 desaturase/Δβ elongase pathway" can be utilized for formation of ω-3 fatty acids as follows: (1) LA is converted to ALA, the first of the ω-3 fatty acids, by a Δ15 desaturase; (2) ALA is converted to STAby a Δ6 desaturase; (3) STAis converted to ETA by a C 18/20 elongase; (4) ETA is converted to EPA by a Δ5 desaturase;(5) EPA is converted to DPAby a C20/22 elongase; and, (6) DPAis converted to DHAby a Δ4 desaturase. Optionally, ω-6 fatty acids may be converted to ω-3 fatty acids; for example, ETA and EPA are produced from DGLA and ARA, respectively, by Δ17 desaturase activity.

[0102] Alternate pathways for the biosynthesis of ω-3/ω-6 fatty acids utilize the Δ9 elongase/Δβ desaturase biosynthetic pathway. More specifically, LA and ALA may be converted to EDA and ETrA, respectively, by a Δ9 elongase; then, a Δ8 desaturase converts EDA to DGLA and/or ETrA to ETA.

[0103] It is contemplated that the particular functionalities required to be expressed in a specific host organism for production of ω-3/ω-6 fatty acids will depend on the host cell (and its native PUFA profile and/or desaturase/elongase profile), the availability of substrate, and the desired end product(s). One skilled in the art will be able to identify various candidate genes encoding each of the enzymes desired for ω-3/ω-6 fatty acid biosynthesis. Useful desaturase and elongase sequences may be derived from any source, e.g., isolated from a natural source (from bacteria, algae, fungi, oomycetes, plants, animals, etc.), produced via a semisynthetic route or synthesized de novo. Although the particular source of the desaturase and elongase genes introduced into the host is not critical, 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; 4.) co-factors required by the polypeptide; and/or, 5.) whether the polypeptide is modified after its production (e.g., by a kinase). The expressed polypeptide preferably has parameters compatible with the biochemical environment of its location in the host cell (see PCT Publication No. WO 2004/101757 for additional details).

[0104] In additional embodiments, it will also be useful to consider the conversion efficiency of each particular desaturase and/or elongase. More specifically, since each enzyme rarely functions with 100% efficiency to convert substrate to product, the final lipid profile of un-purified oils produced in a host cell will typically be a mixture of various PUFAs consisting of the desired ω-3/ω-6 fatty acid, as well as various upstream intermediary PUFAs. Thus, consideration of each enzyme's conversion efficiency is also a variable to consider when optimizing biosynthesis of a desired fatty acid.

[0105] With each of the considerations above in mind, candidate genes having the appropriate desaturase and elongase activities (e.g., Δ6 desaturases, C18/20 elongases, Δ5 desaturases, Δ17 desaturases, Δ15 desaturases, Δ9 desaturases, Δ12 desaturases, C14/16 elongases, C16/I8 elongases, Δ9 elongases, Δ8 desaturases, Δ4 desaturases and C20/22 elongases) can be identified according to publicly available literature (e.g., GenBank), the patent literature, and experimental analysis of organisms having the ability to produce PUFAs. These genes will be suitable for introduction into a specific host organism, to enable or enhance the organism's synthesis of PUFAs.

Identification Of A Novel Δ17 Desaturase [0106] In the present invention, a nucleotide sequence has been isolated from Pythium aphanidermatum encoding a Δ17 desaturase, designated herein as "PaD17".

[0107] Comparison of the PaD17 nucleotide base and deduced amino acid sequences to public databases reveals that the most similar known sequences are about 75.3% identical to the amino acid sequence of PaD17 reported herein over a length of 359 amino acids using the Clustal W method of alignment algorithms.

[0108] PaD17 encoding nucleic acid sequences corresponding to the instant Δ17 desaturase ORF are those encoding active proteins with an amino acid sequence as set forth in SEQ ID NO:2 or 3 and which are at least about 70%-85% identical to the nucleic acid sequences of PaD17 reported herein, where those sequences that are at least about 85%-90% identical are particularly suitable and those sequences that are at least about 90%-95% identical are most preferred.

[0109] In alternate embodiments, the instant PaD17 sequence can be codon-optimized for expression in a particular host organism. As is well known in the art, this can be a useful means to further optimize the expression of the enzyme in the alternate host, since use of host-preferred codons can substantially enhance the expression of the foreign gene encoding the polypeptide. In general, host-preferred codons can be determined within a particular host species of interest by examining codon usage in proteins (preferably those expressed in the largest amount) and determining which codons are used with highest frequency. Then, the coding sequence for a polypeptide of interest having e.g., desaturase activity can be synthesized in whole or in part using the codons preferred in the host species.

[0110] In one preferred embodiment of the invention, PaD17 was codon-optimized for expression in Yarrowia lipolytica. This was possible by first determining the Y. lipolytica codon usage profile (see PCT Publication No. WO 04/101757; U.S. Patent 7,125,672) and identifying those codons that were preferred. Further optimization of gene expression in Y. lipolytica was achieved by determining the consensus sequence around the 'ATG' initiation codon. This optimization resulted in modification of 188 bp of the 1080 bp coding region (17.4%) and optimization of 175 codons (48.6%). None of the modifications in the codon-optimized gene ("PaD17S"; SEQ ID NO:4) changed the amino acid sequence of the encoded protein (SEQ ID NO:2). As described in Example 10, the codon-optimized gene was more efficient desaturating ARAto EPAthan the wildtype gene, when expressed in Y. lipolytica.

[0111] One skilled in the art would be able to use the teachings herein to create various other codon-optimized Δ17 desaturase proteins suitable for optimal expression in alternate hosts (i.e., other than Yarrowia lipolytica), based on the wildtype PaD17 sequence (i.e., SEQ ID NO:2) or a variant thereof as set forth in SEQ ID NO:3. Accordingly, the instant invention relates to any codon-optimized Δ17 desaturase protein encoding sequence that is derived from either SEQ ID NO:2 or SEQ ID NO:3. This includes, but is not limited to, the nucleotide sequence set forth in SEQ ID NO:4, which encodes a synthetic Δ17 desaturase protein (i.e., PaD17S) that was codon-optimized for expression in Yarrowia lipolytica.

[0112] Upon identification of the Oomycete polypeptide described above, the activity of the wildtype and codon-optimized fatty acid desaturase was determined by transformation into a suitable host (i.e., Yarrowia lipolytica) and determination of its effect on the fatty acid profile of the host (Examples 7, 10 and 17). As expected, PaD17 and PaD17S both possessed Δ17 desaturase activity, such that the enzyme was capable of catalyzing conversion of ARA to EPA. Specifically, the ARA to EPA conversion efficiency of PaD17 ranged from 18.4-19.5%, while the ARAto EPA conversion efficiency of PaD17S ranged from 54.1-55.8% (based on determination in two different strains of Y. lipolytica and under different growth conditions). Conversion efficiency was 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.

[0113] Unexpectedly, however, PaD17S additionally possessed limited Δ15 desaturase activity (i.e., the LA to ALA conversion efficiency was 34.6%) (Example 17). Thus, the Pythium aphanidermatum desaturase is defined herein as a bifunctional Δ17 desaturase.

[0114] Further analysis with PaD17S revealed that the enzyme demonstrated broad catalytic promiscuity, based on greater than 25% conversion efficiency using the ω-6 fatty acid substrates EDA and DGLA (Example 17). Thus, the ω-6 fatty acid substrate specificity of PaD17S is similar to that of the synthetic Δ17 desaturase derived from Phytopthora sojae and codon-optimized for expression in Yarrowia lipolytica (i.e., PsD17S; U.S. Patent Application No. 11/787772 and Example 17 herein) and the synthetic Δ17 desaturase derived from Phytopthora ramorum and codon-optimized for expression in Yarrowia lipolytica (i.e., PrD17S; U.S. Patent Application No. 11/787772 and Example 17 herein). These results are in contrast to those demonstrated for the related ω-3 desaturase of Saprolegnia diclina, which has been reported to function exclusively on C20 ω-6 fatty acid substrates as a monofunctional Δ17 desaturase (Pereira, S.L. et. al., Biochem. J., 378:665 (2004)) [0115] An isolated nucleic acid fragment is disclosed herein comprising a nucleic acid sequence encoding a Δ17 desaturase, excluding SEQ ID NO:43 (i.e., "PiD17", the ω-3 desaturase from Phytophthora infestans (GenBank Accession No. CAJ30870)) and SEQ ID NO:95 (i.e., "SdD17", the Δ17 desaturase from Saprolegnia diclina (GenBank Accession No. AAR20444)), wherein the amino acid sequence comprising said Δ17 desaturase contains at least one of the following amino acid sequence motifs selected from the group consisting of: 1. a) FTXGHDXGH (Δ17 Desaturase Motif #1; SEQ ID NO:96); 2. b) HRHHHKNTG(A17 Desaturase Motif #2; SEQ ID NO:97); and, 3. c) IGTHQXHHLFP (Δ17 Desaturase Motif #3; SEQ ID NO:98); wherein Xcan be any amino acid.

[0116] The underlined amino acids represent histidine residues that are part of the desaturase His Box motifs. The His Box motifs are described as: H(X)3H (SEQ ID NO:99), H(X)2HH (SEQ ID NO: 100) and H/Q(X)2HH (SEQ ID NO: 101). Figure 14 sets forth a comparison of the Δ17 desaturase of the present invention with other publicly disclosed Δ17 desaturases using a Clustal V alignment (with default parameters). Specifically, SEQ ID NO:2 (PaD17), SEQ ID NO:43 (PiD17), SEQ ID NO:47 (PrD17), SEQ ID NO:82 (PsD17S) and SEQ ID NO:95 (SdD17) were compared. Regions comprising the motifs described above (i.e., Δ17 Desaturase Motif #1, Δ17 Desaturase Motif #2 and Δ17 Desaturase Motif #3, respectively) are shown in boxes.

Identification And Isolation Of Homologs [0117] Any of the instant desaturase sequences (i.e., PaD17, PaD17*, PaD17S) or portions thereof (i.e., Δ17 Desaturase Motif #1, Δ17 Desaturase Motif #2 and/or Δ17 Desaturase Motif #3) may be used to search for Δ17 desaturase homologs in the same or other bacterial, algal, fungal, Oomycete or plant species using sequence analysis software. In general, such computer software matches similar sequences by assigning degrees of homology to various substitutions, deletions and other modifications.

[0118] Alternatively, any of the instant desaturase sequences or portions thereof may also be employed as hybridization reagents for the identification of Δ17 homologs. The basic components of a nucleic acid hybridization test include a probe, a sample suspected of containing the gene or gene fragment of interest and a specific hybridization method. Probes are typically single-stranded nucleic acid sequences that are complementary to the nucleic acid sequences to be detected. Probes are "hybridizable” to the nucleic acid sequence to be detected. Although the probe length can vary from 5 bases to tens of thousands of bases, typically a probe length of about 15 bases to about 30 bases is suitable. Only part of the probe molecule need be complementary to the nucleic acid sequence to be detected. In addition, the complementarity between the probe and the target sequence need not be perfect. Hybridization does occur between imperfectly complementary molecules with the result that a certain fraction of the bases in the hybridized region are not paired with the proper complementary base.

[0119] Hybridization methods are well defined. Typically the probe and sample must be mixed under conditions that will permit nucleic acid hybridization. This involves contacting the probe and sample in the presence of an inorganic or organic salt under the proper concentration and temperature conditions. The probe and sample nucleic acids must be in contact for a long enough time that any possible hybridization between the probe and sample nucleic acid may occur. The concentration of probe or target in the mixture will determine the time necessary for hybridization to occur. The higher the probe or target concentration, the shorter the hybridization incubation time needed. Optionally, a chaotropic agent may be added (e.g., guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate, potassium iodide, cesium trifluoroacetate). If desired, one can add formamide to the hybridization mixture, typically 30-50% (v/v).

[0120] Various hybridization solutions can be employed. Typically, these comprise from about 20 to 60% volume, preferably 30%, of a polar organic solvent. A common hybridization solution employs about 30-50% v/v formamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 M buffers (e.g., sodium citrate, Tris-HCI, PIPES or HEPES (pH range about 6-9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), or between 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kdal), polyvinylpyrrolidone (about 250-500 kdal), and serum albumin. Also included in the typical hybridization solution will be unlabeled carrier nucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA(e.g., calf thymus or salmon sperm DNA, or yeast RNA), and optionally from about 0.5 to 2% wt/vol glycine. Other additives may also be included, such as volume exclusion agents that include a variety of polar water-soluble or swellable agents (e.g., polyethylene glycol), anionic polymers (e.g., polyacrylate or polymethylacrylate) and anionic saccharidic polymers (e.g., dextran sulfate).

[0121] Nucleic acid hybridization is adaptable to a variety of assay formats. One of the most suitable is the sandwich assay format. The sandwich assay is particularly adaptable to hybridization under non-denaturing conditions. A primary component of a sandwch-type assay is a solid support. The solid support has adsorbed to it or covalently coupled to it immobilized nucleic acid probe that is unlabeled and complementary to one portion of the sequence.

[0122] Any of the Δ17 desaturase nucleic acid fragments described herein (or any homologs identified thereof) may be used to isolate genes encoding homologous proteins from the same or other bacterial, algal, fungal, oomycete or plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to: 1.) methods of nucleic acid hybridization; 2.) methods of DNAand RNA amplification, as exemplified by various uses of nucleic acid amplification technologies [e.g., polymerase chain reaction (PCR), Mullis et al., U.S. Patent 4,683,202; ligase chain reaction (LCR), Tabor, S. et al., Proc. Natl. Acad. Sci. U.S.A., 82:1074 (1985); or strand displacement amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci. U.S.A., 89:392 (1992)]; and 3.) methods of library construction and screening by complementation.

[0123] For example, genes encoding similar proteins or polypeptides to the Δ17 desaturases described herein could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired yeast, fungus or oomycete using methodology well known to those skilled in the art (wherein those yeast or fungus producing EPA [or derivatives thereof] would be preferred). Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis, supra). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan (e.g., random primers DNA labeling, nick translation or end-labeling techniques), or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part of (or full-length of) the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full-length DNA fragments under conditions of appropriate stringency.

[0124] Typically, in PCR-type amplification techniques, the primers have different sequences and are not complementary to each other. Depending on the desired test conditions, the sequences of the primers should be designed to provide for both efficient and faithful replication of the target nucleic acid. Methods of PCR primer design are common and well known in the art (Thein and Wallace, "The use of oligonucleotide as specific hybridization probes in the Diagnosis of Genetic Disorders", in Human Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp 33-50, IRL: Herndon, VA; and Rychlik, W., In Methods in Molecular Biology. White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols: Current Methods and Applications. Humania: Totowa, NJ).

[0125] Generally two short segments of the instant desaturase sequences may be used in PCR protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. PCR may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding eukaryotic genes.

[0126] Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al., Proc. Natl. Acad. Sci. U.S.A., 85:8998 (1988)) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3' or 5' end. Primers oriented in the 3' and 5' directions can be designed from the instant sequences. Using commercially available 3' RACE or 5' RACE systems (Gibco/BRL, Gaithersburg, MD), specific 3' or 5' cDNA fragments can be isolated (Ohara et al., Proc. Natl. Acad. Sci. U.S.A, 86:5673 (1989); Loh et al., Science, 243:217 (1989)).

[0127] Any of the Δ17 desaturase nucleic acid fragments described herein (or any homologs identified thereof) may be used for creation of new and improved fatty acid desaturases. As is wall known in the art, in vitro mutagenesis and selection, chemical mutagenesis, "gene shuffling" methods or other means can be employed to obtain mutations of naturally occurring desaturase genes. Alternatively, improved fatty acids may be synthesized by domain swapping, wherein a functional domain from any of the Δ17 desaturase nucleic acid fragments described herein are exchanged with a functional domain in an alternate desaturase gene to thereby result in a novel protein.

Methods For Production Of Various ω-3 And/Or ω-6 Fatty Acids [0128] It is expected that introduction of chimeric genes encoding the Δ17 desaturases described herein (i.e., PaD17, PaD17*, PaD17S or other mutant enzymes, codon-optimized enzymes or homologs thereof), under the control of the appropriate promoters will result in increased production of EPA in the transformed host organism, respectively. As such, a method for the direct production of PUFAs is disclosed herein comprising exposing a fatty acid substrate (i.e., ARA) to the desaturase enzymes described herein (e.g., PaD17, PaD17*, PaD17S), such that the substrate is converted to the desired fatty acid product (i.e., EPA).

[0129] More specifically, a method is disclosed herein for the production of EPA in a host cell (e.g., oleaginous yeast), wherein the host cell comprises: 1. a.) an isolated nucleotide molecule encoding a Δ17 desaturase polypeptide having at least 75.3% identity when compared to a polypeptide having the amino acid sequence as set forth in SEQ ID NO:2, based on the Clustal W method of alignment; and, 2. b.) a source of ARA; 3. c.) growing the host cell of step (a) under conditions wherein the nucleic acid molecule encoding the Δ17 desaturase polypeptide is expressed and the ARA is converted to EPA; and, 4. d.) optionally recovering the EPA of step (c).

[0130] The person of skill in the art will recognize that the broad substrate range of the Δ17 desaturase will allow for the use of the enzyme for the conversion of DGLAto ETA. Accordingly, a further method is disclosed herein for the production of ETA in a host cell, wherein the host cell comprises: 1. a.) an isolated nucleotide molecule encoding a Δ17 desaturase polypeptide having at least 75.3% identity when compared to a polypeptide having the amino acid sequence as set forth in SEQ ID NO:2, based on the Clustal W method of alignment; and, 2. b.) a source of DGLA; 3. c.) growing the host cell of step (a) under conditions wherein the nucleic acid molecule encoding the Δ17 desaturase polypeptide is expressed and the DGLA is converted to ETA; and, 4. d.) optionally recovering the ETA of step (c).

[0131] Based on the bifunctionality of the Pythium aphanidermatum Δ17 desaturases, it is an object of the present invention to provide a method for the production of polyunsaturated fatty acids in a host cell (e.g., oleaginous yeast), wherein the host cell comprises: 1. a.) an isolated nucleotide molecule encoding a bifunctional Δ17/Δ15 desaturase polypeptide having the amino acid sequence as set forth in SEQ ID NO:2 or 3; and, 2. b.) a source of at least one fatty acid selected from the group consisting of: linoleic acid, eicosadienoic acid, dihomo-y-linolenic acid and arachidonic acid; wherein the host cell is grown under conditions wherein the nucleic acid molecule encoding the bifunctional Δ17/Δ15 desaturase polypeptide is expressed and the at least one substrate fatty acid is converted to at least one product fatty acid, wherein the reaction is selected form the group consisting of: linoleic acid is converted to a-linolenic acid, eicosadienoic acid is converted to eicosatrienoic acid, dihomo-y-linolenic acid is converted to eicosatetraenoic acid, and arachidonic acid is coinverted to eicosapentaenoic acid; and, said fatty acid is then optionally recovered.

[0132] Substrate feeding may be required in any of the methods described above.

[0133] Alternatively, the Δ17 desaturase gene and its corresponding enzyme product described herein can be used indirectly for the production of ω-3 fatty acids (see PCT Publications No. WO 2004/101757 and No. WO 2006/052870). Indirect production of ω-3/ω-6 PUFAs occurs wherein the fatty acid substrate is converted indirectly into the desired fatty acid product, via means of an intermediate step(s) or pathway intermediate(s). Thus, it is contemplated that the Δ17 desaturases described herein (e.g., PaD17, PaD17*, PaD17S or other mutant enzymes, codon-optimized enzymes or homologs thereof) may be expressed in conjunction with additional genes encoding enzymes of the PUFA biosynthetic pathway (e.g., Δ6 desaturases, C18/20 elongases, Δ5 desaturases, Δ15 desaturases, Δ9 desaturases, Δ12 desaturases, C14/16 elongases, C16/I8 elongases, Δ9 elongases, Δ8 desaturases, Δ4 desaturases, C20/22 elongases) to result in higher levels of production of longer-chain ω-3 fatty acids (e.g., EPA, DPA and DHA). The particular genes included within a particular expression cassette will depend on the host cell (and its PUFA profile and/or desaturase/elongase profile), the availability of substrate and the desired end product(s).

[0134] In alternative embodiments, it may be useful to disrupt a host organism's native Δ17 desaturase, based on the complete sequences described herein, the complement of those complete sequences, substantial portions of those sequences, codon-optimized desaturases derived therefrom and those sequences that are substantially homologous thereto. For example, the targeted disruption of the Δ17 desaturase (and optionally a Δ15 desaturase) in a host organism produces a mutant strain that has diminished ability to synthesize ω-3 fatty acids. This mutant strain could be useful for the production of "pure" ω-6 fatty acids (without co-synthesis of ω-3 fatty acids).

Expression Systems. Cassettes And Vectors [0135] The genes and gene products of the instant sequences described herein may be expressed in heterologous host cells. Expression in recombinant 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.

[0136] 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 instant sequences. These chimeric genes could then be introduced into appropriate host cells via transformation to provide high-level expression of the encoded enzymes.

[0137] 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 (e g., a promoter) and a region 3' of the DNA fragment that controls transcriptional termination (i.e., a terminator). 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.

[0138] Initiation control regions or promoters which are useful to drive expression of the instant Δ17 desaturase 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 (e.g., see PCT Publication No. WO 2006/052870 [Patent Publication US 2006-0115881-A1] for preferred transcriptional initiation regulatory regions for use in Yarrowia lipolytica). 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.

[0139] 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. 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.

[0140] 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 and correct folding of the protein in the host organism; 5.) the intrinsic stability of the mRNAand protein of the cloned gene 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 means to further optimize expression of the Δ17 desaturases described herein.

Transformation Of Host Cells [0141] Once the DNA encoding a polypeptide suitable for expression in an appropriate host cell has been obtained, it is placed in a plasmid vector capable of autonomous replication in the host cell, or it is directly integrated into the genome of the host cell. Integration of expression cassettes can occur randomly within the host genome or can be targeted through the use of constructs containing regions of homology with the host genome sufficient to target recombination with the host locus. Where constructs are targeted to an endogenous locus, all or some of the transcriptional and translational regulatory regions can be provided by the endogenous locus.

[0142] 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 construct(s) 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(s) can be experimentally determined so that all introduced genes are expressed at the necessary levels to provide for synthesis of the desired products.

[0143] 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, biolistic impact, electroporation, microinjection, or any other method that introduces the gene of interest into the host cell.

[0144] For convenience, a host cell that has been manipulated by any method to take up a DNA sequence (e.g., an expression cassette) will be referred to as "transformed" or "recombinant" herein. The transformed host will have at least one copy of the expression construct and may have two or more, depending upon whether the gene is integrated into the genome, amplified, or is present on an extrachromosomal element having multiple copy numbers. The transformed host cell can be identified by various selection techniques, as described in PCT Publications No. WO 2004/101757, No. WO 2005/003310 and No. WO 2006/052870.

[0145] Following transformation, substrates suitable for the instant Δ17 desaturases (and, optionally other PUFA enzymes that are co-expressed within the host cell) may be produced by the host either naturally or transgenically, or they may be provided exogenously.

Metabolic Engineering Of ω-3 And/Or ω-6 Fatty Acid Biosynthesis [0146] Knowledge of the sequences of the present Δ17 desaturases will be useful for manipulating ω-3 and/or ω-6 fatty acid biosynthesis in various host cells. This may require metabolic engineering directly within the PUFA biosynthetic pathway or additional manipulation of pathways that contribute carbon to the PUFA biosynthetic pathway. Methods useful for up-regulating desirable biochemical pathways and down-regulating undesirable biochemical pathways are well known to those skilled in the art. For example, biochemical pathways competing with the ω-3 and/or ω-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 downregulated by other means (e.g., antisense mRNA and zinc-finger targeting technologies).

[0147] Detailed discussion of manipulations within the PUFA biosynthetic pathway as a means to increase ARA, EPAor DHA (and associated techniques thereof) are presented in PCT Publication No. WO 2006/055322 [Patent Publication No. US 2006-0094092-A1], PCT Publication No. WO 2006/052870 [Patent Publication No. US 2006-0115881-A1] and PCT Publication No. WO 2006/052871 [Patent Publication No. US 2006-0110806-A1], respectively, as are desirable manipulations in the TAG biosynthetic pathway and the TAG degradation pathway (and associated techniques thereof).

Preferred Hosts For Recombinant Expression Of Δ17 Desaturases [0148] Host cells for expression of the instant genes and nucleic acid fragments may include hosts that grow on a variety of feedstocks, including simple or complex carbohydrates, fatty acids, organic acids, oils and alcohols, and/or hydrocarbons over a wide range of temperature and pH values. Based on the needs of the Applicants' Assignee, the genes described in the instant invention were initially isolated for expression in an oleaginous yeast (and in particular Yarrowia lipolytica)] however, it is contemplated that because transcription, translation and the protein biosynthetic apparatus are highly conserved, any plant, bacteria, yeast, algae, oomycete and/or filamentous fungus will be a suitable host for expression of the present nucleic acid fragments.

[0149] Preferred hosts are oleaginous organisms, such as oleaginous yeast. These oleaginous 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 yeast include: Rhodosporidium toruloides, Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulchemima, C. tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorula glutinus, R. graminis and Yarrowia lipolytica (formerly classified as Candida lipolytica).

[0150] Most preferred is the oleaginous yeast Yarrowia lipolytica; and, in a further embodiment, most preferred are the Y lipolytica strains designated as ATCC #76982, ATCC #20362, ATCC #8862, ATCC #18944 and/or LGAM S(7)1 (Papanikolaou S., and Aggelis G., Bioresour. Technol., 82(1):43-9 (2002)).

[0151] Specific teachings applicable for engineering EPAand DHA in Y. lipolytica are provided in U.S. Patent Applications No. 11/265761 (PCT Publication No. WO 2006/052870; Patent Publication No. US 2006-0115881-A1) and No. 11/264737 (PCT Publication No. WO 2006/052871; Patent Publication No. US 2006-0110806-A1), respectively. Detailed means for the synthesis and transformation of expression vectors comprising Δ17 desaturases in oleaginous yeast (i.e., Yarrowia lipolytica) are provided in PCT Publications No. WO 2004/101757 and No. WO 2006/052870. The preferred method of expressing genes in this yeast is by integration of linear DNA into the genome of the host; and, integration into multiple locations within the genome can be particularly useful when high level expression of genes are desired [e.g., in the Ura3 locus (GenBank Accession No. AJ306421), the Leu2 gene locus (GenBank Accession No. AF260230), the Lys5 gene locus (GenBank Accession No. M34929), the Aco2 gene locus (GenBank Accession No. AJ001300), the Pox3 gene locus (Pox3: GenBank Accession No. XP_503244; or, Aco3: GenBank Accession No. AJ001301), the Δ12 desaturase gene locus (PCT Publication No. WO 2004/104167), the Lip 1 gene locus (GenBank Accession No. Z50020) and/or the Lip2 gene locus (GenBank Accession No. AJ012632)].

[0152] Preferred selection methods for use in Yarrowia lipolytica are resistance to kanamycin, hygromycin and the amino glycoside G418, as well as ability to grow on media lacking uracil, leucine, lysine, tryptophan or histidine. In alternate embodiments, 5-fluoroorotic acid (5-fluorouracil-6-carboxylic acid monohydrate; "5-FOA") is used for selection of yeast Ura' mutants. The compound is toxic to yeast cells that possess a functioning URA3 gene encoding orotidine 5-monophosphate decarboxylase (OMP decarboxylase); thus, based on this toxicity, 5-FOA is especially useful for the selection and identification of Ura' mutant yeast strains (Bartel, P.L. and Fields, S., Yeast 2-Hybrid System, Oxford University: New York, v. 7, pp 109-147, 1997).

[0153] Other preferred microbial hosts include oleaginous bacteria, algae, Oomycetes and other fungi; and, within this broad group of microbial hosts, of particular interest are microorganisms that synthesize ω-3/ω-6 fatty acids (or those that can be genetically engineered for this purpose [e.g., other yeast such as Saccharomyces cerevisiae]). Thus, for example, transformation of Mortierella alpina (which is commercially used for production of ARA) with any of the present Δ17 desaturase genes under the control of inducible or regulated promoters could yield a transformant organism capable of synthesizing ERA The method of transformation of M. alpina is described by Mackenzie et al. (Appl. Environ. Microbiol., 66:4655 (2000)). Similarly, methods for transformation of Thraustochytriales microorganisms are disclosed in U.S. Patent 7,001,772.

[0154] No matter what particular host is selected for expression of them Δ17 desaturases described herein, it is preferable if multiple transformants are screened in order to obtain a strain displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA blots (Southern, J. Mol. Biol., 98:503 (1975)), Northern analysis of mRNA expression (Kroczek, J. Chromatogr. Biomed. Appl., 618 (1-2):133-145 (1993)), Western and/or Elisa analyses of protein expression, phenotypic analysis or GC analysis of the PUFA products.

Fermentation Processes For Omega Fatty Acid Production [0155] The transformed host cell is grown under conditions that optimize expression of chimeric desaturase genes and produce the greatest and most economical yield of desired 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 amount of different mineral ions, the oxygen level, growth temperature, pH, length of the biomass production phase, length of the oil accumulation phase and the time and method of cell harvest. Yarrowia lipolytica are generally 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, Ml)).

[0156] Fermentation media for use in the methods of the present invention must contain a suitable carbon source. Suitable carbon sources are taught in PCT Publication No. WO 2004/101757. Although it is contemplated that the source of carbon utilized in the methods of the present invention may encompass a wide variety of carbon-containing sources, preferred carbon sources are sugars, glycerol and/or fatty acids. Most preferred is glucose and/or fatty acids containing between 10-22 carbons.

[0157] Nitrogen may be supplied from an inorganic (e.g., (NH4)2SC>4) or organic (e.g., urea or glutamate) source. 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 oleaginous host and promotion of the enzymatic pathways necessary for PUFA production. Particular attention is given to several metal ions (e.g., Fe+2, Cu+2,

Mn+2, Co+2, Zn+2, Mg+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)).

[0158] Preferred growth media for use in the methods of the present invention are common commercially prepared media, such as Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Ml). Other defined or synthetic growth media may also be used and the appropriate medium for growth of the transformant host cells 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 7.5 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.

[0159] 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 Yarrowia lipolytica. This approach is described in PCT Publication No. WO 2004/101757, as are various suitable fermentation process designs (i.e., batch, fed-batch and continuous) and considerations during growth.

Oils For Use In Foodstuffs. Health Food Products. Pharmaceuticals And Animal Feeds [0160] The market place currently supports a large variety of food and feed products, incorporating ω-3 and/or ω-6 fatty acids (particularly ALA, GLA, ARA, EPA, DPAand DHA). It is contemplated that the oils disclosed herein comprising long-chain PUFAs will function in food and feed products to impart the health benefits of current formulations. More specifically, oils containing ω-3 and/or ω-6 fatty acids will be suitable for use in a variety of food and feed products including, but not limited to: food analogs, drinks, meat products, cereal products, baked foods, snack foods and dairy products (see Patent Publication No. US 2006/0094092 for details).

[0161] Additionally oils may be used in formulations to impart health benefits in medical foods including medical nutritionals, dietary supplements, infant formula as well as pharmaceutical products. One of skill in the art of food processing and food formulation will understand how the amount and composition of the present oils may be added to the food or feed product. Such an amount will be referred to herein as an "effective" amount and will depend on the food or feed product, the diet that the product is intended to supplement or the medical condition that the medical food or medical nutritional is intended to correct or treat.

EXAMPLES

[0162] 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 scope thereof as defined in the appended claims, can make various changes and modifications of the invention to adapt it to various usages and conditions.

GENERAL METHODS

[0163] Standard recombinant DNAand 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, NY (1989) (Maniatis); 2.) T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1984); and, 3.)Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc, and Wiley-lnterscience, Hoboken, NJ (1987).

[0164] 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, 2nd ed., Sinauer Associates: Sunderland, MA (1989). All reagents, restriction enzymes and materials used for the growth and maintenance of microbial cells were obtained from Aldrich Chemicals (Milwaukee, Wl), DIFCO Laboratories (Detroit, Ml), GIBCO/BRL (Gaithersburg, MD), or Sigma Chemical Company (St. Louis, MO), unless otherwise specified. E. coli strains were typically grown at 37 °C on Luria Bertani (LB) plates.

[0165] General molecular cloning was performed according to standard methods (Sambrook et al., supra) DNA sequence was generated on an ABI Automatic sequencer using dye terminator technology (U.S. Patent 5,366,860; EP 272,007) using a combination of vector and insert-specific primers. Comparisons of genetic sequences were accomplished using DNASTAR software (DNA Star, Inc.).

[0166] Unless otherwise specified, BLAST (Basic Local Alignment Search Tool; Altschul, S. E, et al., J. Mol. Biol., 215:403-410 (1993) and Nucleic Acids Res., 25:3389-3402 (1997)) searches were conducted to identity isolated sequences having similarity to sequences contained in the BLAST "nr" database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the SWISS-PROT protein sequence database, EMBL and DDBJ databases). Query sequences were analyzed for similarity to all publicly available DNA sequences contained in the "nr" database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). Sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the "nr" database, using the BLASTX algorithm (Gish, W. and States, D. J. Nature Genetics, 3:266-272 (1993)) provided by the NCBI. The results of BLAST comparisons summarizing the sequence to which a query sequence had the most similarity are reported according to the % identity, % similarity, and Expectation value. "% Identity" is defined as the percentage of amino acids that are identical between the two proteins. "% Similarity" is defined as the percentage of amino acids that are identical or conserved between the two proteins. "Expectation value" estimates the statistical significance of the match, specifying the number of matches, with a given score, that are expected in a search of a database of this size absolutely by chance.

[0167] The meaning of abbreviations is as follows: "sec" means second(s), "min" means minute(s), "h" means hour(s), "d" means day(s), "pL" means microliter(s). "mL" means milliliter(s), "L" means liter(s), "μΜ" means micromolar, "mM" means millimolar, "M" means molar, "mmol" means millimole(s), "pmole" mean micromole(s), "g" means gram(s), "pg" means microgram(s), "ng" means nanogram(s), "U" means unit(s), "bp" means base pair(s) and "kB" means kilobase(s).

Transformation And Cultivation Of Yarrowia lioolvtica [0168] Yarrowia lipolytica strain ATCC #20362 was purchased from the American Type Culture Collection (Rockville, MD). Y. lipolytica strains were usually grown at 28 °C on YPD agar (1% yeast extract, 2% bactopeptone, 2% glucose, 2% agar).

[0169] Transformation of Y lipolytica was performed according to the method of Chen, D. C. et al. (Appl. Microbiol Biotechnol., 48(2):232-235 (1997)), unless otherwise noted. Briefly, Yarrowia was streaked onto a YPD plate and grown at 30 °C for approximately 18 hr. Several large loopfuls of cells were scraped from the plate and resuspended in 1 mL of transformation buffer containing: 2.25 mL of 50% PEG, average MW 3350; 0.125 mL of 2 M Li acetate, pH 6.0; and, 0.125 mL of 2 M DTT. Then, approximately 500 ng of linearized plasmid DNA was incubated in 100 pi of resuspended cells, and maintained at 39 °C for 1 hr with vortex mixing at 15 min intervals. The cells were plated onto selection media plates and maintained at 30 °C for 2 to 3 days.

[0170] For selection of transformants, minimal medium ("MM") was generally used; the composition of MM is as follows: 0.17% yeast nitrogen base (DIFCO Laboratories, Detroit, Ml) without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, pH 6.1. Supplements of leucine, lysine and/or uracil were added as appropriate to a final concentration of 0.01% (thereby producing "MMLeu", "MMLys" and "MMU" selection media, each prepared with 20 g/L agar).

[0171] Alternatively, transformants were selected on 5-fluoroorotic acid ("FOA"; also 5-fluorouracil-6-carboxylic acid monohydrate) selection media, comprising: 0.17% yeast nitrogen base (DIFCO Laboratories) without ammonium sulfate or amino acids, 2% glucose, 0.1 % proline, 75 mg/L uracil, 75 mg/L uridine, 900 mg/L FOA(Zymo Research Corp., Orange, CA) and 20 g/L agar.

[0172] Finally, High Glucose Media f'HGM") was prepared as follows, as a means to promote conditions of oleaginy: 6.3 g/L KH2PO4, 27 g/L K2HP04 and 80 g/L glucose (pH 7.5).

[0173] The methodology used to create the strains identified herein as Y4001U1, Y4036U and L38 relied on site-specific recombinase systems. Briefly, the site-specific recombination system consists of two elements: (1) a recombination site having a characteristic DNA sequence [e.g., LoxP]; and, (2) a recombinase enzyme that binds to the DNA sequence specifically and catalyzes recombination (i.e., excision) between DNA sequences when two or more of the recombination sites are oriented in the same direction at a given interval on the same DNA molecule [e.g., Cre], For the purposes herein, an integration construct was created comprising a target gene that was desirable to insert into the host genome (i.e., a first selection marker [i.e., Ura3 or Leu2]) that was flanked by recombination sites. Following transformation and selection of the transformants, the first selection marker was excised from the chromosome by the introduction of a replicating plasmid carrying a second selection marker (i.e., Leu2 or sulfonylurea resistance [AHAS]) and a recombinase suitable to recognize the site-specific recombination sites introduced into the genome (i.e., Cre). Upon selection of those transformants carrying the second marker, the replicating plasmid was then cured from the host in the absence of selection and excision of the first selection marker from the cured strain's host genome was confirmed by loss of Ura or Leu prototrophy. This produced a transformant that possessed neither the first nor second selection marker, and thus the cured strain was available for another round of transformation using the first selection marker. Additional details concerning site-specific recombinase based methodology for use in Yarrowia lipolytica is described in PCT Publication No. WO 2006/052870.

[0174] The second selection marker gene utilized in pY117 (Example 16) was a native Yarrowia lipolytica acetohydroxyacid synthase (AHAS or acetolactate synthase; E.C. 4.1.3.18; GenBank Accession No. XM_501277) containing a single amino acid change (W497L) that confers sulfonyl urea herbicide resistance (SUR; described in PCT Publication No. WO 2006/052870). AHAS is the first common enzyme in the pathway for the biosynthesis of branched-chain amino acids and it is the target of the sulfonylurea and imidazolinone herbicides.

Fatty Acid Analysis Of Yarrowia lipolytica [0175] 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 X 0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard) column. The oven temperature was from 170 °C (25 min hold) to 185 °C at 3.5 °C/min.

[0176] 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 pH 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 NaCI and 400 pi hexane, the sample was vortexed and spun. The upper layer was removed and analyzed by GC as described above. EXAMPLE 1

Pvthium aohanidermatum Lipid Profile. Total RNA Isolation And Genomic DNA Isolation [0177] A Pythium aphanidermatum strain was obtained from Lisa Hoffman (E.l. duPont de Nemours, Inc., Wlimington, DE).

[0178] The strain was grown on malt extract agar medium (Difco Laboratories, Detroit, Ml) at room temperature for 3 days. Cells were scraped off the plate and resuspended in 600 pi of sodium methoxide dissolved in methanol. The sample was shaken for 20 min, and 50 μΙ of 1 M NaCI was added. After mixing, 600 μΙ of heptane was added. The sample was vortexed and centrifuged in an Eppendorf microfuge for 1 min. The upper layer was carefully separated from the lower layer and placed in a glass vial for GC analysis. The results of the analysis are shown below in Table 4. Fatty acids are identified as 16:0 (palmitate), 16:1 (palmitoleic acid), 18:0, 18:1 (oleic acid), 18:2, GLA, 20:1,20:2, DGLA, ARA, EPAand DHA; and the composition of each is presented as a % of the total fatty acids.

Tahle 4

[0179] Based on the presence of ARA and EPA, it was concluded that the P. aphanidermatum strain likely had both a Δ5 desaturase (capable of converting DGLA to ARA) and a Δ17 desaturase (capable of converting ARA to EPA).

[0180] Total RNA and genomic DNA were isolated from cells scraped off a malt extract agar plate using the Trizol reagent (Invitrogen, Carlsbad, CA). Specifically, scraped cells were resuspended in 1 mL water and centrifuged for 30 sec in an Eppendorf microfuge. The cell pellet was resuspended in 0.75 mL Trizol reagent, mixed with 0.75 mL of 0.5 mm glass beads, and homogenized in a Biospec mini beadbeater (Bartlesville, OK) at the highest setting for 3 min. The mixture was centrifuged in an Eppendorf centrifuge for 30 sec at 14,000 rpm to remove debris and glass beads. The supernatant was extracted with 150 μΙ of 24:1 chloroform:isoamyl alcohol (Invitrogen). The upper aqueous phase was used for RNA isolation and the lower organic phase for DNA isolation.

[0181] For RNA isolation, the aqueous phase was mixed with 0.375 mL of isopropyl alcohol and allowed to incubate at room temperature for 5 min. Precipitated RNA was collected by centrifugation at 8000 rpm and 4 °C for 5 min. The pellet was washed once with 0.7 mL of 80% ethanol and air-dried. Total RNA (59 pg) was obtained (i.e., 200 μΙ of sample at 29.5 pg/μΙ).

[0182] For genomic DNA isolation, the lower organic phase of the sample was mixed with 225 μΙ of ethanol and incubated at room temperature for 5 min. The sample was then centrifuged at 5000 rpm for 2 min in an Eppendorf centrifuge. The pellet was washed with 0.75 mL of 0.1 M sodium citrate/10% ethanol twice. Each time the sample was incubated for 15 min at room temperature in the wash solution, followed by centrifugation at 5000 rpm for 5 min at 4 °C in an Eppendorf centrifuge. The pellet was air dried and re-dissolved in 300 μΙ of 8 mM NaOH. The pH of the sample was adjusted to 7.5 with 1 M HEPES, and then further purified with a Qiagen PCR purification kit exactly as described in the manufacturer's protocol. A total of 7.2 pg of P. aphanidermatum genomic DNA was obtained. EXAMPLE 2

Pvthium aphanidermatum cDNA Synthesis [0183] Double-stranded cDNA was synthesized directly from the Pythium aphanidermatum total RNA using the BD-Clontech Creator™ Smart™ cDNA library kit (Mississauga, ON, Canada). Specifically, 3 μΙ of total RNA sample (0.9 pg) was mixed with 1 μΙ of SMART™ IV oligonucleotide (SEQ ID NO:9) and 1 μΙ CDSIII/3'PCR primer (SEQ ID NO: 10). The mixture was heated to 75 °C for 5 min, and cooled,on ice for 5 min. Two (2) μΙ of 5Xfirst strand buffer, 1 μΙ of 20 mM DTT, 1 μΙ of dNTP mix(10 mM each of dATP, dCTP, dGTP and dTTP) and 1 μΙ of PowerScript reverse transcriptase were added to the mixture. The sample was incubated at 42 C for 1 hr.

[0184] The resulting first strand cDNA synthesis mixture was then used as template for PCR amplification. The reaction mixture contained 2 μΙ of the above first strand cDNA sample, 80 μΙ of water, 10 μΙ of 10XAdvantage 2 PCR buffer, 2 μΙ 50XdNTP mix (10 mM each of dATP, dCTP, dGTP and dTTP), 2 μΙ of 5' PCR primer (SEQ ID NO: 11). 2 μΙ CDSIII/3' PCR primer (SEQ ID NO: 10) and 2 μΙ 50XAdvantage 2 polymerase mix The thermocycler conditions were set for 95 °C for 1 min and then 20 cycles of 95 °C for 10 sec and 68 °C for 6 min.

[0185] Amplification product was purified with a Qiagen PCR purification kit following the manufacturer's protocol exactly. Purified cDNA product was eluted with 50 pi of water. EXAMPLE 3

Isolation Of A Portion Of The Coding Region Of The Pvthium aphanidermatum Δ17 Desaturase Gene [0186] The present Example describes the identification of a portion of the Pythium aphanidermatum gene encoding Δ17 desaturase (designated herein as "PaD17" (SEQ ID NOs:1 and 2)), by use of primers derived from conserved regions of other known Δ17 desaturase sequences.

[0187] The P. aphanidermatum cDNA sample from Example 2 was used as template for PCR using degenerated primers designed to amplify portions of the potential Δ17 desaturase gene, based on the Δ17 fatty acid desaturase sequences of Phytophthora sojae (SEQ ID NO:45; U S. Patent Application No. 1,1/787772, filed April 18, 2007; see also Example 11. infra) and Phytophthora ramorum (SEQ ID NO:47; U S. Patent Application No. 11/787772, filed April 18, 2007 ; see also Example 13, infra). Based on the alignments provided herein as Figure 2, degenerate primers were designed as shown in Table 5 (location of primers with respect to SEQ ID NOs:45 and 47 are shown as dotted boxes on Figure 2).

Table 5

[0188] A total of 49 different PCR amplification reactions were performed, using all possible combinations of the 7 forward and 7 reverse primers. Each reaction mixture contained 1 μΙ of 1:10 diluted P. aphanidermatum cDNA, 5 μΙ each of the forward and reverse primers (20 μΜ), 14 μΙ water and 25 μΙ of TaKaRa ExTaq 2X premix (TaKaRa Bio, Mountain View, CA). The thermocycler conditions were set for 94 °C for 1 min, then 30 cycles of 94 °C for 20 sec, 55 °C for 20 sec and 72 °C for 1 min, followed by a final extension at 72 °C for 7 min. PCR products were analyzed by electrophoresis on standard agarose gels, and putative Δ17 desaturase fragments were detected as shown below in Table 6.

Table 6

[0189] Each of the fragments described above in Table 6 were purified with a Qiagen PCR purification kit (Valencia, CA), cloned into pCR2.1-TOPO (Invitrogen) and sequenced.

[0190] BLAST sequence analysis showed that each of the fragments were from a single gene that showed extensive homology to the known Δ17 desaturases from other organisms. The sequences were assembled into a 614 bp contig (SEQ ID NO:5), which was assumed to encode a putative Δ17 desaturases from P. aphanidermatum. EXAMPLE 4

Isolation Of The Full-Length Δ17 Desaturase From Pvthium aphanidermatum [0191] Primers were designed to isolate the 5' and 3' ends of the putative Δ17desaturase gene from cDNAand genomic DNA samples of P. aphanidermatum, based on the partial sequence set forth in SEQ ID NO:5 and described in Example 3.

[0192] The 5' region of the putative Δ17 desaturase from P. aphanidermatum was isolated by genome walking using the Universal GenomeWalker™ kit (BD Biosciences Clonetech, Palo Alto, CA), according to the manufacturer's protocol. First, genomic DNA from P. aphanidermatum (1 pg per digestion) was digested with Oral, EcoRV, Pvull and Stu\ separately. Digested DNA samples were purified with Qiagen enzyme reaction clean-up kits according to the manufacturer's protocol and each sample was eluted with 20 μΙ of water.

[0193] The digested genomic DNA samples were ligated with Universal GenomeWalker™ adaptor (SEQ ID NOs:34 [top strand] and 35 [bottom strand]), as shown below: 5'-GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT-3’ 3’-H2N-CCCGACCA-5'

Specifically, 4 μΙ each of the digested DNA was mixed with 1.9 μΙ of 25 μΜ GenomeWalker™ adaptor, 1.6 μΙ of 10Xligation buffer and 0.5 μΙ of T4 DNAIigase. The reaction was carried out overnight at 16 °C. After heating at 70 °C for 5 min, 72 μΙ of 10 mM Tris, 1 mM EDTA, pH 7.4 buffer was added to each reaction mixture. These reaction mixtures were then used as template for PCR amplification.

[0194] For the first round of PCR, primers PUD17-5-1 (SEQ ID NO:36) and Universal GenomeWalker™ primer AP1 (SEQ ID NO:37) from the kit were used. The reaction mixture contained 1 μΙ of each primer at 10 μΜ, 2 μΙ of the purified ligation products as template, 21 μΙ water and 25 μΙ of TaKaRa ExTaq 2X premix The thermocycler conditions were set for 94 °C for 90 sec, then 30 cycles at 94 °C for 20 sec, 55 °C for 20 sec and 72 °C for 2 min, followed by a final extension at 72 °C for 5 min.

[0195] PCR products were diluted 1:20, and 1 μΙ of diluted PCR product was used as template for a second round of PCR using primers PUD17-5-3 (SEQ ID NO:38) and Universal GenomeWalker™ primer AP2 (SEQ ID NO:39). PCR components and amplification conditions were as described above.

[0196] A-750 bp DNA fragment was generated from the second-round of PCR. This fragment was purified with a Qiagen PCR purification kit, cloned into pCR2.1-TOPO (Invitrogen) and sequenced. Subsequent sequence analysis showed that this fragment contained the 5' end of the putative Δ17 desaturase gene, including the translation initiation codon and 387 bp of additional untranslated 5' sequence. The 5' fragment (SEQ ID NO:6) shared significant homology to the Saprolegnia diclina Δ17 desaturase (GenBank Accession No. AAR20444; SEQ ID NO:95).

[0197] The 3' region of the putative Δ17 desaturase was isolated by PCR amplification using R aphanidermatum cDNA as template. Primers PUD17-3-1 (SEQ ID NO:40 and CDSIII/3' PCR primer (SEQ ID NO: 10; from BD-Clontech Creator™ Smart™ cDNA library construction kit, see Example 1) ware used for the first round of amplification. The reaction mixture contained 1 pi of each primer (10 μΜ), 1 μΙ of P. aphanidermatum cDNA, 22 μΙ water and 25 μΙ TaKaRa ExTaq 2X premix. The thermocycler conditions were set for 94 °C for 90 sec, then 30 cycles at 94 °C for 30 sec, 55 °C for 30 sec and 72 °C for 30 sec, followed by a final extension at 72 °C for 5 min.

[0198] PCR product was diluted 1:20, and 1 μΙ of the diluted product was used as template for a second round of PCR using PUD17-3-2 (SEQ ID NO:41) and CDSIII/3' PCR primer (SEQ ID NO: 10), using components and amplification conditions as described above. The second round PCR generated a -550 bp DNA fragment. This was purified with a Qiagen PCR purification kit, cloned into pCR2.1-TOPO and sequenced. Sequence analysis showed that this fragment contained the 3'-region of the putative Δ17 desaturase cDNA, including the polyA tail. The 3' fragment (SEQ ID NO:7) shared significant homology to the Saprolegnia diclina Δ17 desaturase (GenBank Accession No. AAR20444; SEQ ID NO:95).

[0199] Assembly of the 5' genomic region (SEQ ID NO:6), the original partial cDNA sequence (SEQ ID NO:5) and the 3' cDNA sequence (SEQ ID NO:7) resulted in a 1533 bp contig (SEQ ID NO:8), comprising the complete sequence of the putative Δ17 desaturase from P. aphanidermatum and additional untranslated 5' and 3' ends. The coding region of SEQ ID NO:8, which is set forth as SEQ ID NO:1, is 1080 bp long (corresponding to bases 388-1467 of SEQ ID NO:8) and encodes a peptide of 359 amino acids (SEQ ID NO:2). The coding sequence of Pythium aphanidermatum was designated herein as "PaD17”.

[0200] The results of BLAST searches using the full length PaD17 gene (i.e., SEQ ID NO:1) as the query sequence showed that it shared 58% identity and 71% similarity with the amino acid sequence of the Δ17 desaturase of Saprolegnia diclina (GenBank Accession No. AAR20444), with an Expectation value of e-121; additionally, it shared identity and similarity with other omega-3 desaturases.

[0201] Similarly, pairwise comparison between and among Δ17 desaturase proteins from Phytophthora infestans ("PiD17"; SEQ ID NO:43), Phytophthora sojae ("PsD17”; SEQ ID NO:45), Phytophthora ramorum ("PrD17"; SEQ ID NO:47) and Pythium aphanidermatum ("PaD17"; SEQ ID NO:2) using a Clustal W analysis (MegAlign™ program of DNASTAR software) resulted in the following percent similarities: 74.5% between PiD17 and PaD17; 75.0% between PrD17 and PaD17; and 75.3% between PsD17 and PaD17.

EXAMPLES

Generation Of Yarrowia lioolvtica Expression Vectors Comprising The Pvthium aphanidermatum Δ17 Desaturase ("PaD17"1 [0202] The present Example describes the construction of plasmids pFmD17-1, pFmD17-2. pFmD17-3 and pFmD17-4, each comprising a chimeric FBAINm::PaD17*::XPR gene, wherein PaD17* (SEQ ID NO:3) comprises up to (and including) 2 amino acid mutations with respect to SEQ ID NO:2. Plasmids pFmD17-1, pFmD17-2, pFmD17-3 and pFmD17-4 were utilized to test functional expression of PaD17*, as described in Example 7, infra.

[0203] Plasmids pFmD17-1, pFmD17-2, pFmD17-3 and pFmD17-4 were constructed by three-way ligation using fragments from plasmid pFmD8S, a 5' portion of PaD17 and a 3' portion of PaD17. Plasmid pFmD8S (SEQ ID NO:51; Figure 3D) was constructed by three-way ligation using fragments from plasmids pKUNFmkF2, pDMW287F and pDMW214.

Plasmid pKUNFmkF2 [0204] pKUNFmkF2 (SEQ ID NO:48; Figure 3A; PCT Publication No. WO 2006/012326) is a construct comprising a chimeric FBAINm::F.D12::Lip2 gene (wherein "FBAINmK" is the Yarrowia lipolytica FBAINkn promoter [PCT Publication No. WO 2005/049805; U.S. Patent 7,202,356], "F.D12" is the Fusarium moniliforme Δ12 desaturase [PCT Publication No. WO 2005/047485], and "Lip2" is the Yarrowia lipolytica Lip2 terminator sequence (GenBank Accession No. AJ012632)).

Plasmid dDMW267F

[0205] pDMW287F (SEQ ID NO:49; Figure 3B; PCT Publication No. WO 2006/012326) is a construct comprising a synthetic Δ8 desaturase ("EgDeS"; SEQ ID NO:52 herein), derived from wildtype Euglena gracilis, and codon-optimized for expression in Yarrowia lipolytica (wherein EgD8S is identified as "D8SF" in the Figure). The desaturase gene is flanked by a Yarrowia lipolytica FBAIN promoter (PCT Publication No. WO 2005/049805; U.S. Patent 7,202,356; identified as "FBA1+intron" in the Figure) and a Pex16 terminator sequence of the Yarrowia Pex16 gene (GenBank Accession No. U75433).

Plasmid PDMW214 [0206] pDMW214 (SEQ ID NO:150; Figure 3C; PCT Publication No. WO 2005/049805; U.S. Patent 7,202,356) is a shuttle plasmid that replicates both in E. coli and Yarrowia lipolytica. It contained the following components:

Table 7

Plasmid pFmD8S

[0207] The Pme\INco\ fragment of plasmid pKUNFmkF2 (Figure 3A; comprising the FBAINm promoter) and the Nco\INot\ fragment of plasmid pDMW287F (Figure 3B; comprising the synthetic Δ8 desaturase gene ”EgD8S”) were used directionally to replace the PmeVNoti fragment of pDMW214 (Figure 3C). This resulted in generation of pFmD8S (SEQ ID NO:51; Figure 3D), comprising a chimeric FBAIINm::EgD8S::XPR gene. Thus, the components of pFmD8S are as described in Table 8 below.

Table 8

Generation Of Plasmids pFmD17-1. pFmD17-2. pFmD17-3 And pFmD17-4 [0208] The P. aphanidermatum Δ17 desaturase was amplified from cDNA via a reaction mixture that contained: 1 pi of 20 μΜ forward primer PUD17-F (SEQ ID NO:54), 1 μΙ of 20 μΜ reverse primer PUD17-R (SEQ ID NO:55), 1 μΙ P. aphanidermatum cDNA, 10 μΙ 5X PCR buffer, 1 μΙ dNTP mix (10 μΜ each), 35 μΙ water and 1 μΙ Phusion polymerase (New England Biolabs). The thermocycler conditions were set for 98°C for 1 min, then 30 cycles at 98 °C for 10 sec, 55 °C for 10 sec and 72 °C for 30 sec, followed by a final extension at 72 °C for 5 min.

[0209] The PCR product was cloned into pCR2.1-TOPO (Invitrogen) and 8 individual clones were sequenced. Based on the sequence results, 2 clones (i.e., clone 2 and clone 4) were used to construct the final expression plasmid. Clone 2 contained a 351Ato T mutation with respect to SEQ ID NO:2, while clone 4 contained a 155S to P mutation with respect to SEQ ID NO:2; thus, they differed from one another by two conservative amino acid substitutions and they each differed from the wildtype cDNA PaD17 sequence set forth in SEQ ID NO:2 by one conservative amino acid substitution.

[0210] Each clone was digested with Nco\ and Bg/ll to generate a -370 bp fragment that contained the 5' region of the Δ17 desaturase cDNA; and, each clone was also digested with Bg/ll and Not\ to generate a 710 bp fragment that contained the 3' region of the cDNA. The -370 bp fragment comprising the 5' region of the Δ17 desaturase and the 710 bp fragment comprising the 3' region of the Δ17 desaturase were ligated into pFmD8S predigested with Λ/col and Not\ (such that the codon-optimized Δ8 desaturase gene ["EgD8S"] was excised from the plasmid) in a three-way ligation reaction. The reaction mixture contained 10 μΙ 2X ligation buffer and 1 μΙ T4 DNA ligase (Promega), 4 μΙ each of the 5' and the 3' Δ17 desaturase fragments (-300 ng each) and 1 μΙ pFmD8S (-150 ng).

[0211] Using the above methodology, the components of the newly created expression plasmids pFmD17-1, pFmD17-2, pFmD17-3 and pFmD17-4 are identical to those described in Table 8 for pFmDBS (SEQ ID NO:51), with the exception that the pFmD17 vectors possessed chimeric FBAINm::PaD17*::XPR genes instead of the chimeric FBAINm::EgD8S::XPR gene within pFmD8S. The notation of "PaD17*" corresponds to the below mutations with respect to SEQ ID NO:2 (i.e., the amino acid of PaD17 as described in Example 4). The null mutation, 155S to P mutation, 351 A to T mutation, and 155S to P and 351Ato T mutations are each encompassed in SEQ ID NO:3, hereinafter referred to as PaD17*. Based of the combination of the two clones, the four variant expression plasmids contained the following mutations, as shown below in Table 9.

Table 9

[0212] Each reaction mixture was incubated at room temperature for 2 hrs and used to transform E. coli Top10 competent cells. Plasmid DNA from transformants was recovered with Qiagen Miniprep kits. EXAMPLE 6

Generation Of Yarrowia lioolvtica Strain Y2047 To Produce About 11%ARA0f Total Lipids Via The Δ6 Desaturase/A6 Elonaase Pathway [0213] The present Example describes the construction of strain Y2047, derived from Yarrowia lipolytica ATCC #20362, capable of producing 11 % ARA relative to the total lipids via expression of a Δ6 desaturase/Δβ elongase pathway (Figure 4A). This strain was utilized to test the functional expression of PaD17* in Example 7, Infra.

[0214] Yarrowia lipolytica strain Y2047 has been deposited under the terms of the Budapest Treaty and bears the ATCC number PTA-7186. Additionally, construction of Y2047 has been described in co-pending U.S. Patent Application No. 11/265761 (Patent Publication No. US 2006-0115881 A1 and PCT Publication No. WO 2006/052870).

[0215] The development of strain Y2047 first required the construction of strain M4 (producing 8% DGLA).

Generation Of M4 Strain To Produce About 8%DGLAOf Total Lipids [0216] Construct pKUNF12T6E (Figure 4B; SEQ ID NO:56) was generated to integrate four chimeric genes (comprising a Δ12 desaturase, a Δ6 desaturase and two C-I8/20 elongases) into the Ura3 loci of wild type Yarrowia strain ATCC #20362, to thereby enable production of DGLA. The pKUNF12T6E plasmid contained the following components:

Table 10

[0217] The pKUNF12T6E plasmid was digested with Asc\ISph\, and then used for transformation of wild type Y lipolytica ATCC #20362 according to the General Methods. The transformant cells were plated onto FOA selection media plates and maintained at 30 °C for 2 to 3 days. The FOA resistant colonies were picked and streaked onto MM and MMU selection plates. The colonies that could grow on MMU plates but not on MM plates were selected as Ura- strains. Single colonies of Ura- strains were then inoculated into liquid MMU at 30 °C and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Flewlett-Packard 6890 GC.

[0218] GC analyses showed the presence of DGLAin the transformants containing the 4 chimeric genes of pKUNF12T6E, but not in the wild type Yarrowia control strain. Most of the selected 32 Ura' strains produced about 6% DGLAof total lipids. There were 2 strains (i.e., strains M4 and 13-8) that produced about 8% DGLAof total lipids.

Generation Of Y2047 Strain To Produce About 11%ARAOf Total Lipids [0219] Construct pDMW271 (Figure 4C; SEQ ID NO:59) was generated to integrate three Δ5 Chimeric genes into the Leu2 gene of Yarrowia strain M4. Plasmid pDMW271 contained the following components, as described in Table 11:

Table 11

[0220] Plasmid pDMW271 was digested with Asc\ISph\, and then used to transform strain M4 according to the General Methods. Following transformation, the cells were plated onto MMLeu plates and maintained at 30 °C for 2 to 3 days. The individual colonies grown on MMLeu plates were picked and streaked onto MM and MMLeu plates. Those colonies that could grow on MMLeu plates but not on MM plates were selected as Leu2~ strains. Single colonies of Leu2~ strains were then inoculated into liquid MMLeu media at 30 °C and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.

[0221] GC analyses showed the presence of ARA in pDMW271 transformants, but not in the parental M4 strain. Specifically, among the 48 selected Leu2T transformants with pDMW271, there were 35 strains that produced less than 5% ARA of total lipids, 12 strains that produced 6-8% ARA, and 1 strain that produced about 11 % ARA of total lipids in the engineered Yarrowia. The strain that produced 11 % ARA was named Ύ2047". EXAMPLE 7

Functional Analysis Of The Pvthium aphanidermatum Δ17 Desaturase f"PaD17*"> In Yarrowia lipolvtica Strain Y2047 [0222] The present Example describes functional analysis of PaD17* in Yarrowia lipolytica strain Y2047 (Example 6). Thus, following transformation of the variant pFmD17 plasmids comprising PaD17* (from Example 5), lipid profiles within the transformant organisms were compared.

Transformation Of Yarrowia lioolvtica [0223] Plasmids pFmD17-1, pFmD17-2, pFm17-3 and pFmD17-4 (comprising the chimeric FBAINm::PaD17*::XPR genes) were transformed into Yarrowia lipolytica strain Y2047 as described in the General Methods. The transformant cells were plated onto MM plates lacking uracil and maintained at 30 °C for 2 to 3 days. Then, single colonies of transformant Yarrowia lipolytica were patched onto fresh MM plates lacking uracil and allowed to grow at 30 °C for 1 day. The patches were then used to inoculate 3 mL MM liquid medium. Cells were grown for 2 days in MM medium and then 4 days in HGM medium. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC, as described in the General Methods.

[0224] As shown in Table 12, GC analyses demonstrated conversion of ARA to EPA in each of the clones comprising pFmD17-1, pFmD17-2, pFmD17-3 and pFmD17-4, respectively. Composition of ARA and EPA are presented as a % of the total fatty acids. The conversion efficiency ("Conv. Effic.") was 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.

Table 12

[0225] The conversion efficiency whereby PaD17* converted ARA to EPA ranged from 18.4 to 24.6%. More specifically, the experimental data demonstrated that the cloned cDNAfrom P. aphanidermatum (SEQ ID NO:2; PaD17) that was present in vector pFmD17-3 functioned as a Δ17 desaturase, efficiently desaturating ARA to EPA (conversion efficiency ranged from 18.4% to 19.52%); however, neither the Ser at amino acid position 155 of SEQ ID NO:2 nor the Ala at amino acid position 351 of SEQ ID NO:2 were required for enzyme activity. The PaD17* variants encoded by SEQ ID NO:3 comprising the 155S to P mutation, the 351 A to T mutation, or both mutations (expressed in pFmD17-2, pFmD17-1 and pFmD17-4, respectively) all had greater conversion efficiency than that of PaD17 (SEQ ID NO:2) in pFmD17-3. Transformant cells demonstrating the highest Δ17 desaturase conversion efficiency were those expressing vector pFmD17-4, comprising the PaD17* variant with the S155 to P and A351 to T mutations (SEQ ID NO:3). EXAMPLE 8

Synthesis Of A Codon-Optimized Δ17 Desaturase Gene Of Pvthium aphanidermatum f"PaD17S"1 For Yarrowia lioolvtica [0226] The codon usage of the Δ17 desaturase gene of Pythium aphanidermatum (SEQ ID NOs:1 and 2) was optimized for expression in Yarrowia lipolytica, in a manner similar to that described in PCT Publication No. WO 2004/101753 and U.S. Patent 7,125,672. Specifically, a codon-optimized Δ17 desaturase gene of Pythium aphanidermatum (designated "PaD17S", SEQ ID NO:4) was designed based on the coding sequence of PaD17, according to the Yarrowia codon usage pattern (PCT Publication No. WO 2004/101753), 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)). In addition to modification of the translation initiation site, 188 bp of the 1080 bp coding region (including the stop codon) were modified (17.4%; Figure 5Aand 5B) and 175 codons were optimized (48.6%). The GC content was reduced from 61.8% within the wild type gene (i.e., PaD17) to 54.5% within the synthetic gene (i.e., PaD17S). A Ncol site and a NotI site were incorporated around the translation initiation codon and after the stop codon of PaD17S, respectively. None of the modifications in the codon-optimized gene changed the amino acid sequence of the encoded protein (SEQ ID NO:2). The designed PaD17S gene (SEQ ID NO:4) was synthesized by GenScript Corporation (Piscataway, NJ) and cloned into pUC57 (GenBank Accession No. Y14837) to generate pPaD17S (SEQ ID NO:62). EXAMPLE 9

Generation Of Yarrowia lipoMica Strain Y4070 To Produce About 12% ARA Of Total Lipids Via The Δ9 Elonaase/A8 Desaturase Pathway [0227] The present Example describes Yarrowia lipolytica strain Y4070, derived from Yarrowia lipolytica ATCC #20362, capable of producing about 12% ARA relative to the total lipids via expression of a Δ9 elongase/Δβ desaturase pathway (Figure 6A). Strain Y4070 was utilized to test the functional expression of PaD17S in Example 10, infra.

[0228] The development of strain Y4070 required the construction of strain Y2224 (a FOA resistant mutant from an autonomous mutation of the Ura3 gene of wildtype Yarrowia strain ATCC #20362), strain Y4001 (producing 17% EDA with a Leu- phenotype), strain Y4001 U (producing 17% EDA with a Leu- and Ura- phenotype), strain Y4036 (producing 18% DGLA with a Leu-phenotype) and strain Y4036U (producing 18% DGLA with a Leu- and Ura- phenotype).

Generation Of Strain Y2224 [0229] Strain Y2224 was isolated in the following manner: Yarrowia lipolytica ATCC #20362 cells from a YPD agar plate (1% yeast extract, 2% bactopeptone, 2% glucose, 2% agar) were streaked onto a MM plate (75 mg/L each of uracil and uridine, 6.7 g/L YNB with ammonia sulfate, without amino acid, and 20 g/L glucose) containing 250 mg/L 5-FOA (Zymo Research). Plates were incubated at 28 °C and four of the resulting colonies were patched separately onto MM plates containing 200 mg/mL 5-FOA and MM plates lacking uracil and uridine to confirm uracil Ura3 auxotrophy.

Generation Of Strain Y4001 To Produce About 17%EDA Of Total Lipids [0230] Strain Y4001 was created via integration of construct pZKLeuN-29E3 (Figure 6B). This construct, comprising four chimeric genes (i.e., a Δ12 desaturase, a Ci©/18 elongase and two Δ9 elongases), was integrated into the Leu2 loci of strain Y2224 to thereby enable production of EDA.

[0231] Construct pZKLeuN-29E3 contained the components shown in Table 13. TABLE 13

[0232] Plasmid pZKLeuN-29E3 was digested with Ase IISph I, and then used for transformation of V. lipolytica strain Y2224 (i.e., ATCC #20362 Ura3-) according to the General Methods. The transformant cells were plated onto MMLeu media plates and maintained at 30 °C for 2 to 3 days. The colonies were picked and streaked onto MM and MMLeu selection plates. The colonies that could grow on MMLeu plates but not on MM plates were selected as Leu- strains. Single colonies of Leu- strains were then inoculated into liquid MMLeu at 30 °C and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.

[0233] GC analyses showed the presence of EDA in the transformants containing the 4 chimeric genes of pZKLeuN-29E3, but not in the Yarrowia Y2224 control strain. Most of the selected 36 Leu- strains produced about 12 to 16.9% EDA of total lipids. There were 3 strains (i.e., strains #11, #30 and #34) that produced about 17.4%, 17% and 17.5% EDA of total lipids; they were designated as strains Y4001, Y4002 and Y4003, respectively.

Generation Of Strain Y4001U (Leu-. UraA To Produce About 17%EDA Of Total Lipids [0234] Strain Y4001U was created via temporary expression of the Cre recombinase enzyme in plasmid pY116 (Figure 6C) within strain Y4001 to produce a Leu- and Ura- phenotype. Construct pY116 contained the following components:

Tahlfi 14

[0235] Plasmid pY116 was used for transformation of freshly grown Y4001 cells according to the General Methods. The transformants were plated onto MMLeu + Ura plates (MMU plus Leucine) containing 280 pg/mL sulfonylurea and maintained at 30 °C for 3 to 4 days. Four colonies were picked, inoculated into 3 mL liquid YPD media at 30 °C and shaken at 250 rpm/min for 1 day. The cultures were diluted to 1:50,000 with liquid MMLeu + Lira media, and 100 pL was plated onto new YPD plates and maintained at 30 °C for 2 days. Colonies were picked and streaked onto MMLeu and MMLeu+Ura selection plates. The colonies that could grow on MMLeu+Ura plates but not on MMLeu plates were selected and analyzed by GC to confirm the presence of C20:2 (EDA). One strain, having a Leu- and Ura- phenotype, produced about 17% EDA of total lipids and was designated as Y4001 U.

Generation Of Y4036 Strain To Produce About 18%DGLA Of Total Lipids [0236] Construct pK02UF8289 (Figure 7A; SEQ ID NO:70) was generated to integrate four chimeric genes (comprising a Δ12 desaturase, one Δ9 elongase and two mutant Δ8 desaturases) into the Δ12 loci of strain Y4001 U1, to thereby enable production of DGLA. Construct pK02UF8289 contained the following components:

Table 15

[0237] The pK02UF8289 plasmid was digested with AscAISpM, and then used for transformation of strain Y4001U1 according to the General Methods. The transformants were plated onto MMLeu plates and maintained at 30 °C for 2 to 3 days. The colonies were picked and streaked onto MMLeu selection plates at 30 °C for 2 days. These cells were then inoculated into liquid MMLeu at 30 °C and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.

[0238] GC analyses showed the presence of DGLA in the transformants containing the 4 chimeric genes of pK02UF8289, but not in the parent Y4001 U1 strain. Most of the selected 96 strains produced between 7 and 13% DGLA of total lipids. There were 6 strains (i.e., #32, #42, #60, #68, #72 and #94) that produced about 15%, 13.8%, 18.2%, 13.1%, 15.6% and 13.9% DGLA of total lipids. These six strains were designated as Y4034, Y4035, Y4036, Y4037, Y4038 and Y4039, respectively.

Generation Of Strain Y4036U (Leu·. Ura3-) To Produce About 18%DGLA Of Total Lipids [0239] Construct pY116 (Figure 6C; SEQ ID NO:69) was utilized to temporarily express a Cre recombinase enzyme in strain Y4036. This released the LoxP sandwiched Ura3 gene from the genome.

[0240] Plasmid pY116 was used to transform strain Y4036 according to the General Methods. Following transformation, the cells were plated onto MMLeu+Ura plates (MMU plus Leucine) and maintained at 30 °C for 2 to 3 days. The individual colonies grown on MMLeu+Ura plates were picked, and streaked into YPD liquid media at 30 °C and shaken at 250 rpm/min for 1 day to cure the pY116 plasmid. The grown cultures were streaked on MMLeu+Ura u plates. After two days at 30 °C, the individual colonies were re-streaked on MMLeu+Ura, MMU and MMLeu plates. Those colonies that could grow on MMLeu+Ura, but not on MMU or MMLeu plates were selected. One of these strains with Leu- and Ura- phenotypes was designated as Y4036U (Ura-, Leu-).

Generation Of Y4070 Strain To Produce About 12%ARA Of Total Lipids [0241] Construct pZKSL-555R (Figure 7B; SEQ ID NO:74) was generated to integrate three Δ5 desaturase genes into the Lys loci of strain Y4036U, to thereby enable production of ARA. The pZKSL-555R plasmid contained the following components:

Table 16

[0242] The pZKSL'-555R plasmid was digested with Asc\ISph\, and then used for transformation of strain Y4036U according to the General Methods. The transformant cells were plated onto MMLeuLys plates (MMLeu plus Lysine) and maintained at 30 °C for 2 to 3 days. Single colonies were then re-streaked onto MMLeuLys plates, and then inoculated into liquid MMLeuLys at 30 °C and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.

[0243] GC analyses showed the presence of ARAin the transformants containing the 3 chimeric genes of pZKSL-555R, but not in the parent Y4036U strain. Most of the selected 96 strains produced -10% ARAof total lipids. There were 4 strains (i.e., #57, #58, #69 and #75) that produced about 11.7%, 11.8%, 11.9% and 11.7% ARAof total lipids. These four strains were designated as Y4068, Y4069, Y4070 and Y4071, respectively. Further analyses showed that the three chimeric genes of pZKSL-555R were not integrated into the Lys5 site in the Y4068, Y4069, Y4070 and Y4071 strains. All strains possessed a Lys+ phenotype.

[0244] The final genotype of strain Y4070 with respect to wildtype Yarrowla lipolytica ATCC #20362 was Ura3-, Leu+, Lys+, GPD::F.D12::Pex20, YAT::F.D12::OCT, YAT::ME3S::Pex16, GPAT::EgD9e::Lip2, Exp::EgD9eS::Lip1, FBAINm::EgD9eS::Lip2, FBAINm::EgD8M:;Pex20, EXP::EgD8M::Pex16, FBAIN::EgD5WT::Aco, EXP::EgD5S::Pex20, YAT::RD5S::OCT. EXAMPLE 10

Generation Of Construct pFBAINPaD17S (Comprising The Codon-Optimized Δ17 Desaturase Gene "PaD17S"1 And Expression In Yarrowia lioolvtica [0245] The present Example describes functional analysis of PaD17S in Yarrowia lipolytica strain Y4070 (Example 9). Thus, following construction of plasmid pFBAINPaD17S (SEQ ID NO:102) comprising a chimeric FBAINm::PaD17S::Pex20 gene and transformation, lipid profiles within the transformant organisms were compared.

[0246] Specifically, plasmid pFBAINPaD17S was constructed by three-way ligation using 5' PaD17S and 3' PaD17S fragments from plasmid pPaD17S (Example 8; wherein the 5' PaD17S fragment was generated by Nco\ and Sg/ll digestion and wherein the 3' PaD17S fragment was generated by Sg/ll and Nott digestion, as described in Example 5) and plasmid pFBAIN-MOD-1 (SEQ ID NO:80; Figure 8A) predigested with Λ/col and Λ/oil. Thus, PaD17S was operably linked with the Yarrowia lipolytica FBAINm promoter (PCT Publication No. WO 2005/049805; U.S. Patent 7,202,356) and the PEX20-3' terminator region of the Yarrowia Pex20 gene (GenBank Accession No. A054613).

[0247] Plasmid pFBAINPaD17S (SEQ ID NO: 102) was transformed into Yarrowia lipolytica strain Y4070 and transformants were selected on SD-Ura plates (comprising: 20 g/L agar; 6.7 g/L YNB without amino acids but with ammonium sulfate; 20 g/L glucose; 20 mg/L each of adenine sulfate, L-tryptophan, L-histidine-HCI, L-arginine-HCI, L-methionine; 30 mg/L each of L-tyrosine, L-leucine, L-isoleucine, L-lysine-HCI; 50 mg/L L-phenylalanine; 100 mg/mL each of L-glutamic acid, L-aspartic acid; 150 mg/L L-valine; 200 mg/L L-threonine; and 400 mg/L L-serine).

[0248] The fatty acid profile and conversion efficiency of four transformants were determined as described in Example 7. The results of GC analysis are shown in Table 17; composition of ARA and EPA are presented as a % of the total fatty acids.

Table 17

[0249] The GC results demonstrated production of ARA and EPA in the transformants carrying pFBAINPaD17S, but only production of ARA in transformants carrying the control plasmid pFBAIN-MOD-1 (Figure 8A, vector only). The conversion efficiency of the codon-optimized P. aphanidermatum Δ17 desaturase (PaD17S; SEQ ID NO:4) ranged between 54.1% to 55.6%, compared with 18.4 to 19.5% conversion efficiency for the wild-type PaD17 (SEQ ID NO:2). EXAMPLE 11

Identification Of A Phvtoohthora soiae Gene Encoding Δ17 Desaturase [0250] The present Example, disclosed in U.S. Patent Application No. 11/787772, describes the identification of a Δ17 desaturase from Phytophthora sojae (SEQ ID NOs:44 and 45).

[0251] The U.S. Department of Energy's Joint Genome Institute ("JGI"; Walnut Creek, CA) created version 1.0 of the Phytophthora sojae genome (estimated genome size is 95 Mbp). This genomic sequence was generated using a whole genome shotgun strategy and comprises a total of 19,276 gene models.

[0252] Using the amino acid sequence of the Δ17 desaturase of Phytophthora infestans (GenBank Accession No. CAJ30870; designated as "PiD17" herein and corresponding to SEQ ID NO:43) as a query sequence, a TBLASTN (BLAST protein versus translated nucleotide) search was conducted against JGI's Phytophthora sojae database (using the default parameters available from JGI). One P. sojae ORF located on scaffold 17:338148-339167 was found to share extensive homology with PiD17 (i.e., 91.8% identity and 95.6% similarity, with an Expectation value of 0). Based on this homology, the P. sojae ORF was tentatively identified as a Δ17 desaturase and was designated as "PsD17". When the 1092 bp DNAsequence of PsD17 (SEQ ID NO:44) was retrieved from the database, it was found to encode a polypeptide of 363 amino acids in length (SEQ ID NO:45). Amino acid sequence alignment using a Clustal W analysis (MegAlign™ program of DNASTAR software) showed that there was 90.9% identity between PiD17 and PsD17; in contrast, the nucleotide sequences shared only 86.6% identity.

[0253] The sequence homology of PsD17 to all publicly available protein sequences contained in the "nr" database (see General Methods) was also determined by conducting protein-protein BLAST searches using PsD17 (SEQ ID NO:45) as the query sequence. Based on this analysis, PsD17 was found to share the most homology with the omega-3 fatty acid desaturase of Saprolegnia diclina (GenBank Accession No. AAR20444); specifically, PsD17 had 60% identity and 74% similarity with the amino acid sequence of GenBank Accession No. AAR20444 with an Expectation value of 7E-117. Additionally, PsD17 had 39% identity and 57% similarity with the amino acid sequence of the fatty acid desaturase of Anabaena variabilis ATCC #29413 (GenBank Accession No. ABA23809), with an Expectation value of 4E-57. EXAMPLE 12

Syntheses Of A Codon-Optimized Δ17 Desaturase Gene ("PsD17S"1 For Yarrowia lictolvtica [0254] The present Example, disclosed in U S. Patent Application No: 11/787772, describes the creation of a synthetic Δ17 desaturase, derived from Phytophthora sojae (SEQ ID NOs:44 and 45) and codon-optimized for expression in Yarrowia lipolytica (SEQ ID NOs:81 and 82).

[0255] The codon usage of the Δ17 desaturase gene of Phytophthora sojae was optimized for expression in Yarrowia lipolytica, in a manner similar to that described in U S. Patent 7,125,672. Specifically, a codon-optimized Δ17 desaturase gene (designated "PsD17S". SEQ ID NOs:81 and 82) was designed based on the coding sequence of PsD17 (SEQ ID NOs:44 and 45), according to the Yarrowia codon usage pattern (PCT Publication No. WO 2004/101753), the consensus sequence around the 'ATG' translation initiation codon, and the general rules of RNAstability (Guhaniyogi, G. and J. Brewer, Gene, 265(1-2):11-23 (2001)). In addition to modification of the translation initiation site, 175 bp of the 1092 bp coding region were modified (16.0%) and 168 codons were optimized (46.2%). The GC content was reduced from 65.1% within the wild type gene (i.e., PsD17) to 54.5% within the synthetic gene (i.e., PsD17S). A Nco\ site and Not\ sites were incorporated around the translation initiation codon and after the stop codon of PsD17S (SEQ ID NO:81), respectively. Figure 9 shows a comparison of the nucleotide sequences of PsD17 and PsD17S. At the amino acid level, PsD17S lacked the third and forth amino acid, as compared with the wild type PsD17; thus, the total length of PsD17S is 361 amino acids (SEQ ID NO:82). The designed PsD17S gene was synthesized by GenScript Corporation (Piscataway, NJ) and cloned into pUC57 (GenBank Accession No. Y14837) to generate pPsD17S (SEQ ID NO:83). EXAMPLE 13

Identification Of A Phytophthora ramorum Gene Encoding Δ17 Desaturase [0256] The present Example, disclosed in U.S. Patent Application No. 11/787772, describes the identification of a Δ17 desaturase from Phytophthora ramorum (SEQ ID NOs:46 and 47).

[0257] The U.S. Department of Energy's Joint Genome Institute ("JGI"; Walnut Creek, CA) created version 1.0 of the Phytophthora ramorum genome (estimated genome size is 65 Mbp). This genomic sequence was generated using a whole genome shotgun strategy and comprises a total of 16,066 gene models.

[0258] In a manner similar to that described in Example 11, the amino acid sequence of PiD17 (SEQ ID NO:43) was used as a query sequence to perform a TBLASTN search against JGI's Phytophthora ramorum database (using the default parameters available from JGI).

[0259] Two ORFs were found to share extensive homology with PiD17 in the genome sequence of Phytophthora ramorum. Specifically, ORF 80222 shared 89% identity and 94% similarity with SEQ ID NO:43, with an Expectation value of 0. Similarly, ORF48790 shared up to 40% identity and 61% similarity with SEQ ID NO:43, with an Expectation value of 6E-44. Based on these results, ORF 80222 was tentatively identified as a Δ17 desaturase and was designated as "PrD17".

[0260] When the 1086 bp DNA sequence of PrD17 (SEQ ID NO:46) was retrieved from the database, it was found to encode a polypeptide of 361 amino acids in length (SEQ ID NO:47). Amino acid sequence alignment using a Clustal W analysis (MegAlign ™ program of DNASTAR software) showed that there was 89.5% identity between PiD17 and PrD17; in contrast, the nucleotide sequences shared only 85.7% identity.

[0261] The sequence homology of PrD17 was in turn compared with all publicly available protein sequences contained in the "nr" database (see General Methods) by conducting protein-protein BLAST searches using PrD17 (SEQ ID NO:47) as the query sequence. The sequence that showed the highest degree of similarity was that of the omega-3 fatty acid desaturase of Saprolegnia diclina (GenBank Accession No. AAR20444), sharing 59% identity and 74% similarity, with an Expectation value of E-124. Additionally, PrD17 had 38% identity and 57% similarity with the amino acid sequence of the fatty acid desaturase of Anabaena variabilis ATCC #29413 (GenBank Accession No. ABA23809), with an Expectation value of 6E-61. EXAMPLE 14

Synthesis Of A Codon-Optimized Δ17 Desaturase Gene ("PrD17S"1 For Yarrowia lioolvtica [0262] The present Example, disclosed in U.S. Patent Application No. 11/787772, describes the creation of a synthetic Δ17 desaturase, derived from Phytophthora ramorum (SEQ ID NOs:46 and 47) and codon-optimized for expression in Yarrowia lipolytica (SEQ ID NOs:84 and 47).

[0263] The codon usage of the Δ17 desaturase gene of Phytophthora ramorum was optimized for expression in Yarrowia lipolytica, in a manner similar to that described in U.S. Patent 7,125,672. Specifically, a codon-optimized Δ17 desaturase gene (designated "PrD17S", SEQ ID NO:84) was designed based on the coding sequence of PrD17 (SEQ ID NOs:46 and 47), according to the Yarrowia codon usage pattern (PCT Publication No. WO 2004/101753), 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)). In addition to modification of the translation initiation site, 168 bp of the 1086 bp coding region were modified (15.5%) and 160 codons were optimized (44.2%). The GC content was reduced from 64.4% within the wild type gene (i.e., PrD17) to 54.5% within the synthetic gene (i.e., PrD17S). A Nco\ site and Not\ sites were incorporated around the translation initiation codon and after the stop codon of PrD17S (SEQ ID NO:84), respectively. Figure 10 shows a comparison of the nucleotide sequences of PrD17 and PrD17S. None of the modifications in the codon-optimized gene changed the amino acid sequence of the encoded protein (SEQ ID NO:47). The designed PrD17S gene was synthesized by GenScript Corporation (Piscataway, NJ) and cloned into pUC57 (GenBank Accession No. Y14837) to generate pPrD17S (SEQ ID NO:85). EXAMPLE 15

Generation Of Constructs pY130. pY138. pY139 And pY140 (Comprising A Fusarium moniliforme Δ15 Desaturase. PrD17S. PsD17S And PaD17S1 For Comparison Of Omeaa-6 Fatty Acid Substrate Specificity [0264] The present Example, and related Examples 16 and 17 (infra) describe comparison of the substrate specificity of a Fusarium moniliforme Δ15 desaturase (FmD15; SEQ ID NOs:86 and 87) to that of PaD17S (SEQ ID NOs:4 and 2), PrD17S (SEQ ID NOs:84 and 47) and PsD17S (SEQ ID NOs:81 and 82) in Yarrowia lipolytica.

[0265] This work included the following steps: (1) construction of Yarrowia expression vectors pY130 (comprising FmD15), pY138 (comprising PrD17S), pY139 (comprising PsD17S) and pY140 (comprising PaD17S), as described in Example 15 herein; (2) construction of a Δ12 desaturase-disrupted strain of Yarrowia lipolytica ATCC #76982, identified as strain L38, as described in Example 16; 3.) transformation of pY130, pY138, pY139 and pY140 into wildtype Yarrowia and Yarrowia strain L38, as described in Example 17; and, 4.) comparison of lipid profiles within transformant organisms comprising of pY130, pY138, pY139 or pY140 after feeding fatty acid substrates, as described in Example 17.

Experimental Basis [0266] Omega-3 desaturases, which include both Δ15 desaturases that act on C18 fatty acids substrates and Δ17 desaturases that act on C20 fatty acids substrates, play an important role in the biosynthesis of long chain PUFAs by converting ω-6 fatty acids into their ω-3 counterparts (Figure 1). It is well known that some fungal ω-3 desaturases show broad catalytic promiscuity. For example, the Δ15 desaturases of Fusarium moniliforme (GenBank Accession No. DQ272516.1) and Magnaporthe grisea (GenBank Accession No. XP362963) both additionally have limited Δ17 desaturase activity (PCT Publications No. WO 2005/047485 and No. WO 2005/047480; U.S. Patent Application No. 11/740298).

[0267] Similarly, the synthetic Δ17 desaturase derived from Phytopthora sojae and codon-optimized for expression in Yarrowia lipolytica (i.e., PsD17S) was previously demonstrated in U.S. Patent Application No. 11/787772 to have both Δ17 and Δ15 desaturase activities. More specifically, PsD17S displayed "bifunctional Δ17 desaturase activity" or "primary Δ17 desaturase activity", wherein the desaturase preferentially converts ARA to EPA and/or DGLA to ETA but additionally has limited ability to convert LA into ALA (thus exhibiting primarily Δ17 desaturase activity and limited Δ15 desaturase activity).

[0268] Despite the broad catalytic promiscuity described above, not all ω-3 desaturases possess bifunctional activity. For example, the Saprolegnia diclina Δ17 desaturase functions exclusively on C20 ω6 fatty acid substrates (Pereira, S.L. et. al., Biochem. J., 378:665 (2004)).

[0269] The purpose of the following Examples was to compare the relative ω-6 fatty acid substrate specificities of Δ17 desaturases from Phytopthora sojae (PsD17S; SEQ ID NOs:81 and 82), Phytopthora ramorum (PrD17S; SEQ ID NOs:84 and 47) and Pythium aphanidermatum (PaD17S; SEQ ID NOs:4 and 2) with that of the previously characterized Fusarium moniliforme Δ15 desaturase (FmD15: SEQ ID NOs:86 and 87). In contrast to previous work performed with PsD17S and PrD17S in U.S. Patent Application No. 11/787772, the ω-3 desaturases ware expressed herein in Yarrowia lipolytica strains lacking desaturases and elongases involved in converting LA to EPA, since their presence allows alternative routes for long-chain PUFA biosynthesis (Figure 1). As a result, interpretation concerning ω-6 substrate specificity in PrD17S, PsD17S and PaD17S is much clearer than in previous work.

Construction Of Yarrowia Expression Vector pY130. Comprising FmD15 [0270] Plasmid pY6.GPD.Leu2 (SEQ ID NO:88) is a shuttle plasmid that can replicate both in E. coli and Yarrowia lipolytica, containing the following: a Yarrowia autonomous replication sequence (ARS18; GenBank Accession No. M91600); a ColE1 plasmid origin of replication; an E. coli f1 origin of replication; an ampicillin-resistance gene (AmpR) for selection in E. coli; a Yarrowia Leu2 gene (GenBank Accession No. AF260230) for selection in Yarrowia; and, a chimeric GPD::/\fcol/A/oil::XPR cassette. The Yarrowia "GPD promoter" refers to the 5' upstream untranslated region in front of the 'ATG' translation initiation codon of a protein encoded by the Yarrowia lipolytica glyceraldehyde-3-phosphate dehydrogenase (GPD) gene and that is necessary for expression (PCT Publication No. WO 2005/003310). "XPR" refers to -100 bp of the 3' region of the Yarrowia Xpr gene (GenBank Accession No. M17741). Although the construction of plasmid pY6.GPD.Leu2 is not described herein in detail, it was derived from pY28 GPD.YID12d (previously described in U.S. Patent Application No. 11/740298, filed April 26, 2007, and comprising a chimeric GPD:: Yarrowia lipolytica Δ12 desaturase (Yld12d)::Lip1 gene cassette).

[0271] The Fusarium moniliforme Δ15 desaturase was derived from plasmid pY34 which was previously described in PCT Publication No. WO 2005/047485, first by a single bp substitution at position 180 of the FmD15 desaturase ORF. This C180T "silent" mutation resulted in the loss of the NcoI site in the ORF for cloning convenience. Then, the modified sequence was used to PCR the ORF using 5' and 3' PCR primers with Λ/col and Λ/ofl restriction sites, and the resultant Nco\-Noti fragment containing the FmD15 desaturase ORF (SEQ ID NO:86) was used to replace the Yld12d ORF in plasmid pY28 described supra using A/col and Not I sites to produce pY130 (SEQ ID NO:89; Figure 11A [labeled as "pY130.GPD.Fmd15" therein]).

[0272] The 9048 bp sequence of expression vector pY130 containing the chimeric GPD::FmD15::Lip1 gene is disclosed in SEQ ID NO:89 and described in the table below.

Table 18

Construction Of Yarrowia Expression Vectors pY138 (Comprising PrD17SL pY139 (Comprising PsD17S> And dY140 (Comprising PaD17S1 [0273] The A/col-Λ/οίΙ fragment comprising FmD15 in pY130 was replaced by similarly digested fragments comprising the synthetic Δ17 desaturase ORFs of Phytopthora ramorum and Phytopthora sojae that had been codon-optimized for expression in Yarrowia (i.e., PrD17S and PsD17S, respectively) from the source plasmids pPrD17S (SEQ ID NO:85; Example 14, supra) and pPsD17S (SEQ ID NO:83; Example 12, supra). This produced plasmids pY138 (SEQ ID NO:90; Figure 11B [labeled as "pY138 GPD-PrD17" therein]) and pY139 (SEQ ID NO:91; Figure 11C [labeled as "pY139 GPD PsD17" therein]), respectively.

[0274] A similar strategy was used to substitute the FmD15 ORF in pY130 with the synthetic Δ17 desaturase ORF of Pythium aphanidermatum from the source plasmid pPaD17S (SEQ ID NO:62; Example 8, supra)', however, since PaD17S contained an internal Nco\ site, this was achieved by a three-way ligation of the Nco\-Bgl\\ and Bgl\\-Not\ fragments of PaD17S into the pY130 vector backbone. This resulted in formation of plasmid pY140 (SEQ ID NO:92), as shown in Figure 11D (labeled as ”pY140 GPD-PaD17" therein). EXAMPLE 16

Generation Of Yarrowia lipolvtica Δ12 Knockout Strain L38 [0275] The present Example, disclosed in U.S. Patent Application No. 11/740298, describes the creation of a Δ12 desaturase-disrupted [Δ12 knockout (KO)] strain of Yarrowia lipolytica ATCC #76982, identified as strain L38 and referred to generically as a "d12KO" strain. The only native Δ12 desaturase gene of this strain was disrupted by replacement with a disrupted version via homologous recombination.

[0276] The methodology used to create the d12KO strain identified herein as L38 relied on site-specific recombinase systems, as described in the General Methods.

Experimental Methodology [0277] Yarrowia lipolytica ATCC #76982 was transformed with Sph\ and Asd linearized plasmid pY137. The sequence of plasmid pY137 (labeled as pY137.YID12ko.Leu2 in Figure 12A) is disclosed as SEQ ID NO:93 and pY137 Is described in the table below. Table 19

[0278] Eleven LEU prototrophic pY137 transformants were analyzed by GC and four were identified as Δ12 knockout (d12KO) strains by the absence of detectable 18:2 (LA) upon GC analysis. One of these was designated strain L37.

[0279] The LEU2 gene in d12KO strain L37 was excised by transient expression of Cre recombinase under the control of the Yarrowia glycerol-3-phosphate acyltransferase (GPAT) promoter. Specifically, strain L37 was transformed with plasmid pY117. The mutated Yarrowia AHAS enzyme in plasmid pY117 conferred SUR, which was used as a positive screening marker.

[0280] Plasmid pY117 was derived from plasmid pY116 (described in Table 14 herein and in U.S. Patent Application No. 11/635258) by inserting the mutant AHAS gene flanked by Pad-Swal sites into Pad-S\røl digested pY116 thereby replacing the LEU selectable marker with the sulfonylurea marker. Plasmid pY117 (SEQ ID NO:94) is represented in Figure 12B (labeled therein as pY117.Cre.AHASw497L) and is described in Table 20 below.

Table 20

[0281] L37 transformed by pY117 were plated on minimal plates containing Leu and 280 pg/mL sulfonyurea (chlorimuron ethyl, E. I. duPont de Nemours & Co., Inc., Wilmington, DE). To cure the strains of pY117, two SU^ colonies were used to inoculate 3 mL YPD. After overnight growth at 30 °C, 100 pi of 1:250,000 diluted cultures were plated on YPD plates. After overnight growth at 30 °C, 6 single colonies were streaked on both YPD and MM plates. All grew on YPD but not on MM plates, confirming their Leu auxotrophy. One of these was designated as strain L38.

Example 17

Expression Of Constructs pY130. pY138. pY139 And pY140 (Comprising FmD15. PrD17S. PsD17S And PaD17S> In Yarrowia lioolvtica Strains For Comparison Of Omeaa-6 Fatty Acid Substrate Specificity [0282] The present Example describes transformation of expression plasmids pY130, pY138, pY139 and pY140 into Yarrowia lipolytica ATCC #76982, followed by comparison of lipid profiles within transformant organisms.

Transformation [0283] The following expression plasmids were transformed into wild type (WT) Yarrowia lipolytica ATCC #76982 and its Δ12 desaturase-disrupted derivative (Δ12 KO) strain L38 (Example 16), as described in the General Methods: 1.) plasmid pY130 (comprising FmD15); 2.) plasmid pY138 (comprising PrD17S); 3.) plasmid pY139 (comprising PsD17S); 4.) plasmid pY140 (comprising PaD17S); and, 5.) plasmid pY6.GPD.Leu2 (empty vector control lacking any desaturase ORF; also referred to as plasmid "pY6").

Comparison Of Lipid Profiles Without Substrate Feeling [0284] Three independent transformants from each transformation were streaked on MM plates. Fresh cultures were used to separately inoculate 3 mL MM in triplicate. After growth in a shaker at 30 °C for 2 days, cells from 2 mL aliquots of each 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.

[0285] The fatty acid profiles for Yarrowia lipolytica expressing pY6 (SEQ ID NO:88), pY130 (SEQ ID NO:89), pY138 (SEQ ID NO:90), pY139 (SEQ ID N0:91) and pY140 (SEQ ID NO:92) are shown below in Table 21. In Table 21, fatty acids are identified as 16:0 (palmitate), 16:1, 18:0 (stearic acid), 18:1 (oleic acid), 18:2 (LA) and ALA. Fatty acid compositions were expressed as the weight percent (wt. %) of total fatty acids. The conversion efficiency ("CE") was 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. Thus, Δ12 activity (i.e., "d12d CE”) was calculated according to the following formula: ([LA]/ [oleic acid+LA])*100 and represents percent substrate conversion to LA. "Δ15 Activity" (i.e., "d15d CE") was calculated according to the following formula: ([ALA]/[LA+ALA])*100 and represents percent substrate conversion to ALA. Standard deviation is abbreviated "SD", while "nd" is not detected.

Table 21

Comparison Of Lipid Profiles With Substrate Feeding [0286] To study the relative substrate specificities of the different ω-3 desaturases on ω6 substrates other than LA, d12 KO strains transformed with the different plasmids (i.e., pY6, pY130, pY138, pY139 and pY140) were fed a mixture of different FAs, For this, the strains were streaked onto MM plates and fresh cultures were used to inoculate 3 mL MM. After overnight growth at 30 °C, all cultures were diluted to an ODqoo of 0.5 before aliquoting them into three 3-mL cultures. After growth for another 6 hrs, the cultures were harvested and resuspended in 3 mL MM containing 1% Tergitol and 0.5 mM each of GLA, EDA and ARAand allowed to grow for 24 hr at which time they were harvested, washed once with 12 mL 0.5% Triton X-100, and once with 12 mL distilled water. The pellets were analyzed for fatty acid composition, as described above.

[0287] The fatty acid profiles for d 12 KO Yarrowia lipolytica expressing pY6 (SEQ ID NO:88), pY130 (SEQ ID NO:89), pY138 (SEQ ID NO:90), pY139 (SEQ ID NO:91) and pY140 (SEQ ID NO:92) are shown below in Table 22. In the Table, fatty acids are identified as GLA(oj-6), ΕϋΑ(ω-6), DG LA (ω-6), ARA (ω-6), ALA (ω-3), STA(co-3), ETrA(u)-3), ΕΤΑ(ω-3) and ΕΡΑ(ω-3). Fatty acid compositions were expressed as the weight percent (wt. %) of total fatty acids. The ω-3 desaturase conversion efficiency ("Conv. Effic ") of the ω-6 substrates GLA, EDA, DGLA, and ARAto their ω-3 products, STA, ETrA, ETA, and EPA, respectively, was calculated according to the following formula: [product/(substrate+product)]*100. Standard deviation is abbreviated "SD”, while "nd" is not detected,

Table 22

[0288] Results concerning ω-6 fatty acid substrate specificity of FmD15, PsD17S, PrD17S and PaD17S are visually summarized in Figure 13. Specifically, data relating to LA is from wild type Y. lipolytica transformants, as shown in Table 21; all other data are from A12-desaturase disrupted (d12KO) Yarrowia lipolytica strains fed different ω-6 fatty acid substrates, as shown in Table 22. The fatty acid DGLA is abbreviated as "HGLA" in the Figure.

[0289] Based on the data presented herein, FmD15 had the highest Δ15 desaturase activity as compared to PsD17S, PrD17S and PaD17S (Table 21, Figure 13). Unlike FmD15 (which has bifunctional Δ12/Δ15 desaturase activity), however, none of the tested three Δ17 desaturases possessed any detectable Δ12 desaturase activity on oleate (Table 21). Growth in the presence of ω-6 fatty acid substrates showed that all Δ17 desaturases had the strongest preference for ARA, relatively lower activities on EDA and DGLA, and least activity on GLA. PaD17S had the strongest activity on ARA. The Δ17 desaturase had significant Δ15 desaturase activity on the C18 substrate LA, wherein the activity was comparable to the Δ17 desaturase activity on the C20 substrates EDA and DGLA(PsD17S and PrD17S also displayed significant Δ15 desaturase activity on LA, although activity was slightly diminished with respect to the Δ17 desaturase activity on C20 substrates). The broad catalytic promiscuity of the three Δ17 desaturases distinguishes them from the Saprolegnia diclina Δ17 desaturase that works exclusively on C20 ω-6 fatty acid substrates.

SEQUENCE LISTING

[0290] <110> E. I. du Pont de Neinours &amp; Co., Inc.

<120> DELTA-17 DESATURASE AND ITS USE IN MAKING POLYUNSATURATED FATTY ACIDS <130> CL3548 <150> US 60/855,177 <151 >2006-10-30 <160> 102 <170> Patentln version 3.4 <210> 1 <211 > 1080

<212> DNA <213> Pythium aphanidermatum <400> 1 atggcttctt ccactgttgc tgegccgtac gagttcccga cgctgacgga gatcaagcgc 60 tcgctgccag cgcactgctt tgaggcctcg gtcccgtggt cgctctacta caccgtgcgc 120 gcgctgggca tcgccggctc gctcgcgctc ggcctctact acgcgcgcgc gctcgcgatc 180 gtgcaggagt ttgccctgct ggatgcggtg ctctgcacgg ggtacattct gctgcagggc 240 atcgtattct gggggttctt caccatcggc catgactgcg gccacggcgc gttcccgcgt 300 tcgcacctgc tcaacttcag cgtcggcacg ctcattcact cgatcatcct cacgccgtac 360 gagtcatgga. agatctcgca ccgccaccac cacaagaaca cgggcaacat cgacaaggac 420 gagattttct acccgcagcg cgaggccgac tcgcacccac tgtcccgaca catggtgatc 480 tcgctcggct cggcctggtt cgcgtacctc gttgcgggct tccctcctcg caaggtgaac 540 cacttcaacc cttgggaacc gttgtacctg cgccgcatgt ctgccgtcat catctcactc 600 ggctcgctcg tggcgttcgc gggcttgtat gcgtatctca cctacgtcta tggccttaag 660 accatggcgc tgtactactt cgcccctctc ttcgggttcg ccacgatgct cgtggtcact 720 acctttttgc accacaatga cgaggaaacg ccatggtacg ccgactcgga gtggacgtac 780 gtcaagggca acctctcgtc cgtggaccgc tcgtacggcg cgctcatcga caacctgagc 840 cacaacatcg gcacgcacca gatccaccac ctgtttccga tcatcccgca ctacaagctg 900 aacgaggcga cggcagcgtt cgcgcaggcg ttcccggagc tcgtgcgcaa gagcgcgtcg 960 ccgatcatcc cgacgttcat ccgcatcggg ctcatgtacg ccaagtacgg cgtcgtggac 1020 aaggacgcca agatgtttac gctcaaggag gccaaggccg ccaagaccaa ggccaactag 1080

<210> 2 <211 > 359 <212> PRT <213> Pythium aphanidermatum <220> <221 > MISC_FEATURE <223> delta-17 desaturase <400>2

Met Ala Ser Ser Thr val Ala Ala Pro Tyr Glu Phe Pro Thr Leu Thr 1 5 10 15

Glu Ile Lys Arg Ser Leu Pro Ala His Cys Phe Glu Ala Ser val Pro 20 25 30

Trp Ser Leu Tyr Tyr Thr val Arg Ala Leu Gly lie Ala Gly Ser Leu 35 40 45

Ala Leu Gly Leu Tyr Tyr Ala Arg Ala Leu Ala lie Val Gin Glu Phe 50 55 60

Ala Leu Leu Asp Ala val Leu cys Thr Gly Tyr lie Leu Leu Gin Gly 65 70 75 80 lie Val Phe Trp Gly Phe Phe Thr lie Gly His Asp Cys Gly His Gly 85 90 95

Ala Phe ser Arg ser His Leu Leu Asn Phe ser val Gly Thr Leu lie 100 105 110

His Ser lie He Leu Thr Pro Tyr Glu Ser Trp Lys lie Ser His Arg 115 120 125

His His His Lys Asn Thr Gly Asn lie Asp Lys Asp Glu lie Phe Tyr 130 135 140

Pro Gin Arg Glu Ala Asp Ser His Pro Leu ser Arg His Met Val lie 145 150 155 160

Ser Leu Gly Ser Ala Trp Phe Ala Tyr Leu Val Ala Gly Phe Pro Pro 165 170 175

Arg Lys val Asn His Phe Asn Pro Trp Glu Pro Leu Tyr Leu Arg Arg 180 185 190

Met Ser Ala val lie He Ser Leu Gly ser Leu val Ala Phe Ala Gly 19S 200 205

Leu Tyr Ala Tyr Leu Thr Tyr val Tyr Gly Leu Lys Thr Met Ala Leu 210 215 220

Tyr Tyr Phe Ala Pro Leu Phe Gly Phe Ala Thr Met Leu val val Thr 225 230 235 240

Thr Phe Leu His His Asn Asp Glu Glu Thr Pro Trp Tyr Ala Asp ser 245 250 255

Glu Trp Thr Tyr Val Lys Gly Asn Leu Ser Ser Val Asp Arg Ser Tyr 260 265 270

Gly Ala Leu lie Asp Asn Leu ser His Asn lie Gly Thr His Gin lie 275 * 280 285

His His Leu Phe Pro lie lie Pro His Tyr Lys Leu Asn Glu Ala Thr 290 295 300

Ala Ala Phe Ala Gin Ala Phe Pro Glu Leu val Arg Lys ser Ala ser 305 310 315 320

Pro lie lie Pro Thr Phe lie Arg lie Gly Leu Met Tyr Ala Lys Tyr 325 330 335

Gly val val Asp Lys Asp Ala Lys Met Phe Thr Leu Lys Glu Ala Lys 340 345 350

Ala Ala Lys Thr Lys Ala Ash 355

<2103 <211 > 359 <212> PRT <213> Pythium aphanidermatum <220 <221 > MISC_FEATURE <223> delta-17 desaturase <220 <221 > MISC_FEATURE <222> (155)..(155) <223> xaa = ser or Pro <220> <221 > MISC_FEATURE <222> (351)..(351) <223> Xaa = Ala or Thr <400>3

Met Ala Ser ser Thr val Ala Ala Pro Tyr Glu Phe Pro Thr Leu Thr 1 5 10 15

Glu Ile Lys Arg Ser Leu Pro Ala His Cys Phe Glu Ala Ser val Pro 20 25 30

Trp Ser Leu Tyr Tyr Thr val Arg Ala Leu Gly ile Ala Gly ser Leu 35 40 45

Ala Leu Gly Leu Tyr Tyr Ala Arg Ala Leu Ala ile val Gin Glu Phe 50 55 60

Ala Leu Leu Asp Ala Val Leu Cys Thr Gly Tyr ile Leu Leu Gin Gly 65 70 75 80 ile val Phe Trp Gly Phe Phe Thr ile Gly His Asp Cys Gly His Gly 85 90 95

Ala Phe Ser Arg Ser His Leu Leu Asn Phe Ser val Gly Thr Leu ile 100 105 110

His Ser Ile Ile Leu Thr pro Tyr Glu ser Trp Lys ile ser His Arg 115 120 125

His His His Lys Asn Thr Gly Asn ile Asp Lys Asp Glu Ile Phe Tyr 130 135 140

Pro Gin Arg Glu Ala Asp ser His Pro Leu xaa Arg His Met val ile 145 150 155 160

Ser Leu Gly Ser Ala Trp phe Ala Tyr Leu val Ala Gly Phe Pro Pro 165 170 175

Arg Lys val Asn His Phe Ash Pro Trp Glu Pro Leu Tyr Leu Arg Arg 180 185 190

Met Ser Ala val ile ile Ser Leu Gly Ser Leu val Ala Phe Ala Gly 195 200 205

Leu Tyr Ala Tyr Leu Thr Tyr val Tyr Gly Leu Lys Thr Met Ala Leu 210 215 220

Tyr Tyr Phe Ala Pro Leu Phe Gly Phe Ala Thr Met Leu val val Thr 225 230 235 240

Thr Phe Leu His His Asn Asp Glu Glu Thr Pro Trp Tyr Ala Asp ser 245 250 255

Glu Trp Thr Tyr val Lys Gly Asn Leu Ser Ser val Asp Arg Ser Tyr 260 265 270

Gly Ala Leu ile Asp Asn Leu Ser His Asn ile Gly Thr His Gin ile 275 280 285

His His Leu Phe Pro ile Ile Pro His Tyr Lys Leu Asn Glu Ala Thr 290 295 300

Ala Ala Phe Ala Gin Ala Phe Pro Glu Leu val Arg Lys Ser Ala Ser 305 310 315 320

Pro ile Ile Pro Thr Phe Ile Arg Ile Gly Leu Met Tyr Ala Lys Tyr 325 330 " 335

Gly val val Asp Lys Asp Ala Lys Met Phe Thr Leu Lys Glu xaa Lys 340 345 350

Ala Ala Lys Thr Lys Ala Asn 355 <2104 <211 > 1080

<212> DNA <213> Pythium aphanidermatum <220 <221 > misc_feature <223> syntthetic delta-17 desaturase, codon-optimized forYarrowia lipolytica <400>4 atggcttcct ctaccgttgc cgctccctac gagttcccta ctctcaccga gatcaagcga 60 tccctgcctg cccactgctt cgaagcctct gttccctggt ccctctacta taccgtgcga 120 gctctgggca ttgccggttc ccttgctctc ggactgtact atgctcgagc ccttgctatc 180 gtgcaggagt ttgcactgct cgatgccgtc ctttgcactg gctacattct gctccagggt 240 atcgtgttct ggggattctt taccatcggt cacgactgtg gacatggtgc cttctcgcga 300 tcccacctgc tcaacttctc tgttggcaca ctcattcact ccatcattct gactccctac 360 gagtcgtgga agatcagcca tcgacaccat cacaagaaca ccggcaacat cgacaaggat 420 gagatcttct accctcagcg agaagccgac tctcatcccc tgtcccgaca catggtcatc 480 tcccttggtt cggcttggtt tgcctacctc gttgctggat ttcctccccg aaaggtcaac 540 cacttcaatc cctgggagcc tctctacctg cgaagaatgt ctgccgtcat catttccctc 600 ggctctctcg tggcctttgc tggtctgtac gcctacctta cctacgtcta cggcctcaag 660 accatggctc tgtattactt cgcacctctc tttggartcg ccaccatgct ggttgtcacr 720 accttccccc atcacaacga cgaggaaact ccctggtacg ccgattcgga gtggacctat 780 gtcaagggca acttgtcctc tgtggaccga agctacggag ccctcatcga caacctgtcc 840 cacaacattg gtacacatca gatccaccat ctgtttccca tcattcctca ctacaagctc 900 aacgaggcca ctgctgcctt cgctcaggcc tttcccgaac tggtgcgaaa gtcggcttct 960 cccatcattc ccaccttcat ccgaattggt cttatgtacg ccaagtacgg cgtggtcgac 1020 aaggatgcca agatgtttac cctcaaggag gccaaggctg ccaagaccaa agccaactaa 1080

<210>5 <211 > 614 <212> DNA <213> Pythium aphanidermatum <400>5 ttttgggggt tcttcaccgt cggccatgac tgcggccacggcgcgttctc gcgttcgcac 60 ctgctcaact tcagcgtcgg cacgctcatt cactcgatca tcctcacgcc gtacgagtca 120 tggaagatct cgcaccgcca ccaccacaag aacacgggca acatcgacaa ggacgagatt 180 ttctacccgc agcgcgaggc cgactcgcac ccactgtccc gacacatggt gatctcgctc 240 ggctcggcct ggttcgcgta cctcgttgcg ggcttccctc ctcgcatggt gaaccacttc 300 aacccttggg aaccgttgta cctgcgccgc atgtctgccg tcatcatctc actcggctcg 360 ctcgtggcgt tcgcgggctt gtatgcgtat ctcacctacg tctatggcct taagaccatg 420 gcgctgtact acttcgcccc tctctttggg ttcgccacga tgctcgtggt cactaccttt 480 ttgcaccaca atggcgagga aacgccatgg tacgccgact cggagtggac gtacgtcaag 540 ggcaacctct cgtccgtgga ccgctcgtac ggcgcgctca tcgacaacct gagccacaac 600 atcggcacgc acaa $14

<210> 6 <211> 739 <212> DNA <213> Pythium aphanidermatum <220> <221 > misc_feature <222> (132)..(132) <223> n is a, c, g, or t <400>6 actatagggc acgcgtggtc gacggcccgg gctggtatca aatacttttt ctaatrtaat 60 atctacgaaa cgtttttttg ctatgattgg cacctattca atgctcatga atctgatgat 120 agtatttgca tnacttcatc ctctcttcca ttttatgctg actcaaacct ctttcgcgct 180 cggtttcaaa gggttacact actcgtgcgt ggtaccgagt gtaaccagca gcaaaaccgc 240 tccatacaac cgccaagtgt gaatgagggg cagacactgc gcgtgatctt gttctatgcg 300 cagccagcca gtggaggtct ctcccgggcg tggacctcac ttcagcttga gccgcggacc 360 gcgcagacca cccgacccgc acccgccatg gcttcttcca ctgttgctgc gccgtacgag 420 ttcccgacgc tgacggagat caagcgctcg ctgccagcgc actgctttga ggcctcggtc 480 ccgtggtcgc tctactacac cgtgcgcgcg ctgggcatcg ccggctcgct cgcgctcggc 540 ctctactacg cgcgcgcgct cgcgatcgtg caggagtttg cccxgcxgga xgcggxgctc 600 xgcacggggx acaxxctgcx gcagggcaxc gxaxxcxggg ggxxcxxcac caxcggccax 660 gacxgcggcc acggcgcgtt cxcgcgxxcg caccxgcxca acttcagcgt cggcacgcxc 720 axtcacxcga tcaxccxca 739

<210>7 <211 > 512 <212> DNA <213> Pythium aphanidermatum <400>7 ctgtactact xcgccccxcx cxxxgggxxc gccacgatgc tcgtggtcac xaccxxxxxg 60 caccacaaxg acgaggaaac gccaxggxac gccgacxcgg agxggacgxa cgxcaagggc 120 aaccxcxcgx ccgxggaccg cxcgxacggc gcgcxcatxg acaacctgag ccacaacatc 180 ggcacgcacc agatccacca ccxgxxxccg atcaxcccgc acxacaagcx gaacgaggcg 240 acggcagcgt tcgcgcaggc gttcccggag ctcgxgcgca agagcgcgxc gccgaxcatc 300 ccgacgttca xccgcatcgg geteatgtac gccaagxacg gcgtcgtgga caaggacgcc 360 aagaxgxxxa cgcxcaagga ggccaaggcc gccaagacca aggccaacxa ggcagaggca 420 aacaaggaag agaagxxgxg xaxaggctcg xaaxgaacax gcgggxxxxx tgtttttwww 480 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 512

<210> 8 <211>1533 <212> DNA <213> Pythium aphanidermatum <220> <221 > misc_feature <223> delta-17 desaturase contig (CDS corresponds to nucleotides 388-1467) <220> <221 > misc_feature <222> (132)..(132) <223> n is a, c, g, or t <400>8 acxaxagggc acgcgtggtc gacggcccgg gctggtatca aatacttttt ctaatttaat 60 atctacgaaa cgtttttttg ctatgattgg cacctattca atgctcatga atctgatgat 120 agtatttgca tnacttcatc ctctcttcca ttttatgctg actcaaacct ctttcgcgct 180 cggtttcaaa gggttacact actcgtgcgt ggtaccgagt gtaaccagca gcaaaaccgc 240 tccatacaac cgccaagtgt gaatgagggg cagacactgc gcgtgatctt gttctatgcg 300 cagccagcca gtggaggtct ctcccgggcg tggacctcac ttcagcttga gccgcggacc 360 gcgcagacca cccgacccgc acccgccatg gcttcttcca ctgttgctgc gccgtacgag 420 ttcccgacgc tgacggagat caagcgctcg ctgccagcgc actgctttga ggcctcggtc 480 ccgtggtcgc tctactacac cgtgcgcgcg ctgggcatcg ccggctcgct cgcgctcggc 540 ctctactacg cgcgcgcgct cgcgatcgtg caggagtttg ccctgctgga tgcggtgctc 600 tgcacggggt acattctgct gcagggcatc gtattctggg ggttcttcac catcggccat 660 gactgcggcc acggcgcgtt ctcgcgttcg cacctgctca acttcagcgt cggcacgctc 720 attcactcga tcatcctcac gccgtacgag tcatggaaga tctcgcaccg ccaccaccac 780 aagaacacgg gcaacatcga caaggacgag attttctacc cgcagcgcga ggccgactcg 840 cacccactgt cccgacacat ggtgatctcg ctcggctcgg cctggttcgc gtacctcgtt 900 gcgggcttcc ctcctcgcat ggtgaaccac ttcaaccctt gggaaccgtt gtacctgcgc 960 cgcatgtctg ccgtcatcat ctcactcggc tcgctcgtgg cgttcgcggg cttgtatgcg 1020 tatctcacct acgtctatgg ccttaagacc atggcgctgt actacttcgc ccctctcttt 1080 gggttcgcca cgatgctcgt ggtcactacc tttttgcacc acaatgrcga ggaaacgcca 1140 tggtacgccg actcggagtg gacgtacgtc aagggcaacc tctcgtccgt ggaccgctcg 1200 tacggcgcgc tcattgacaa cctgagccac aacatcggca cgcaccagat ccaccacctg 1260 tttccgatca tcccgcacta caagctgaac gaggcgaegg cagcgttcgc gcaggcgttc 1320 ccggagctcg cgcgcaagag cgcgtcgccg atcatcccga cgttcatccg catcgggctc 1380 atgtacgcca agtacggcgt cgtggacaag gacgccaaga tgtttacgct caaggaggcc 1440 aaggccgcca agaccaaggc caactaggca gaggcaaaca aggaagagaa gttgtgtata 1500 ggctcgtaat gaacatgcgg gttttttgtt ttt 1533

<210>9 <211> 39 <212> DNA <213> Artificial sequence <220> <223> SMART IV oligonucleotide <400>9 aagcagtggt atcaacgcag agtggccatt acggccggg 39

<210> 10 <211> 59 <212> DNA <213> Artificial sequence <220> <223> CDSIII/3'PCR primer <220> <221 > misc_feature <222> (28)..(57) <223> thymidine (dT); see BD Biosciences Clontech's SMART cDNA technology <220> <221 > misc_feature <222> (59)..(59) <223> n is a, c, g, or t <400> 10 attctagagg ccgaggcggc cgacatgttt tttttttttt tttttttttt tttttttvn 59

<210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 5'-PCR primer <400> 11 aagcagtggt atcaacgcag agt 23

<210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer PD17-F1 <220> <221 > misc_feature <222> (9)..(9) <223> n is a, c, g, or t <220> <221 > misc_feature <222> (18)..(18) <223> n is a, c, g, or t <400> 12 ttytggggnt tyttyacngt 20

<210> 13 <211 > 7 <212> PRT <213> Artificial sequence <220> <223> Translation of primer PD17-F1 <400> 13

Phe Trp Gly phe Phe Thr Tyr 1 5

<210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer PD17-F2 <220> <221 > misc_feature <222> (9)..(9) <223> n is a, c, g, or t <220> <221 > misc_feature <222> (12)..(12) <223> n is a, c, g, or t <220> <221 > misc_feature <222> (15)..(15) <223> n is a, c, g, or t <400> 14 ttcttyacng tnggncayga 20

<210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer PD17-F3 <220> <221 > misc_feature <222> (9)..(9) <223> n is a, c, g, or t <220> <221 > misc_feature <222> (12)..(12) <223> n is a, c, g, or t <220> <221 > misc_feature <222> (15)..(15) <223> n is a, c, g, or t <400> 15 tttttyacng tnggncayga 20

<210> 16 <211 > 7 <212> PRT <213> Artificial Sequence <220> <223> Translation of primers PD17-F2 and PD17-F3 <400> 16

Phe Phe Thr val Gly His Asp 1 5

<210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer PD17-F4 <220> <221 > misc_feature <222> (3)..(3) <223> n is a, c, g, or t <220> <221 > misc_feature <222> (9)..(9) <223> n is a, c, g, or t <400> 17 acncaycgnc aycaycayaa 20

<210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer PD17-F5 <220> <221 > misc_feature <222> (3)..(3) <223> n is a, c, g, or t <400> 18 acncayagrc aycaycayaa 20 <210> 19 <211 > 7

<212> PRT <213> Artificial Sequence <220> <223> Translation of primers PD17-F4 and PD17-F5 <400> 19

Thr His Arg His His His Lys 1 5

<210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer PD17-F6 <220> <221 > misc_feature <222> (9)..(9) <223> n is a, c, g, or t <220> <221 > misc_feature <222> (12)..(12) <223> n is a, c, g, or t <400> 20 aaraayacng gnaayatyga 20

<210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer PD17-F7 <220> <221 > misc_feature <222> (9)..(9) <223> n is a, c, g, or t <220> <221 > misc_feature <222> (12)..(12) <223> n is a, c, g, or t <400> 21 aaraayacng gnaayataga 20

<210> 22 <211 > 7 <212> PRT <213> Artificial Sequence <220> <223> Translation of primers PD17-F6 and PD17-F7 <400> 22

Lys Asn Thr Gly Asn Ile Asp 1 5 <210> 23

<211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer PD17-R1 <220> <221 > misc_feature <222> (15)..(15) <223> n is a, c, g, or t <400> 23 tcrtcrttrt grtgnagraa 20

<210> 24 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primer PD17-R2 <400> 24 tcrtcrttrt grtgyaaraa 20

<210> 25 <211 > 7 <212> PRT <213> Artificial sequence <220> <223> Translation of primers PD17-R1 and PD17-R2 <400> 25

Phe Leu His His Asn Asp Glu 1 5

<210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer PD17-R3 <220> <221 > misc_feature <222> (18)..(18) <223> n is a, c, g, or t <400> 26 aaraargcyt tdatdatngg 20

<210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer PD17-R4 <220> <221 > misc_feature <222> (18)..(18) <223> n is a, c, g, or t <400> 27 aaraaygcyt tdatdatngg 20

<210> 28 <211 > 7 <212> PRT <213> Artificial Sequence <220> <223> Translation of primers PD17-R3 and PD17-R4 <400> 28 pro Ile Ile Lys Ala Phe Phe 1 5

<210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer PD17-R5 <220> <221 > misc_feature <222> (6)..(6) <223> n is a, c, g, or t <220> <221 > misc_feature <222> (9)..(9) <223> n is a, c, g, or t <400> 29 ttrtgngtnc cdatrttatg 20

<210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer PD17-R6 <220> <221 > misc_feature <222> (6)..(6) <223> n is a, c, g, or t <220> <221 > misc_feature <222> (9)..(9) <223> n is a, c, g, or t <400> 30 ttrtgngtnc cdatrttgtg 20

<210> 31 <211 > 7 <212> PRT <213> Artificial Sequence <220> <223> Translation of primers PD17-R5 and PD17-R6 <400> 31

His Asn Ile Gly Thr His Gin 1 S

<210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer PD17-R7 <220> <221 > misc_feature <222> (6)..(6) <223> n is a, c, g, or t <220> <221 > misc_feature <222> (12)..(12) <223> n is a, c, g, or t <400> 32 ccyttnacrt angtccaytc 20

<210> 33 <211 > 7 <212> PRT <213> Artificial Sequence <220> <223> Translation of primer PD17-R7 <400> 33

Glu rrp Thr Tyr val Lys Gly 1 5

<210> 34 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Genome walker adaptor-1 <400> 34 gtaatacgac tatagggcac gcgtggtcga cggcccgggc tggt 44

<210> 35 <211 > 8 <212> DNA <213> Artificial Sequence <220> <223> Genome walker adaptor-2 <220> <221 > misc_feature <222> (1)..(1) <223> 5' end is associated with a -P04 group <220> <221 > misc_feature <222> (8)..(8) <223> 3' end is associated with a -H2N group <400> 35 accagccc 8

<210> 36 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer PUD17-5-1 <400> 36 aatctcgtcc ttgtcgatgt tg 22

<210> 37 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer AP1 <400> 37 gtaatacgac tcactatagg gc 22

<210> 38 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer PUD17-5-3 <400> 38 tgaggatgat cgagtgaatg ag 22

<210> 39 <211 > 19 <212> DNA <213> Artificial Sequence <220> <223> Primer AP2 <400> 39 actatagggc acgcgtggt 19

<210>40 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer PUD17-3-1 <400> 40 cacctacgtc tatggcctta ag 22

<210>41 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer PUD17-3-2 <400> 41 ctgtactact tcgcccctct ct 22

<210> 42 <211 > 1085 <212> DNA <213> Phytophthora infestans (GenBank Accession No. CAJ30870) <400> 42 atggcgacga aggaggcgta tgtgttcccc actctgacgg agatcaagcg gtcgctacct 60 aagactgttt cgaggcttcg gtgcctctgt cgctctacta caccgtgcgt tgtctggtga 120 tcgcggtggc tctaaccctc ggtctcaact acgctcgcgc tctgeccgag gtcgagagct 180 tctgggctct ggacgccgca ctctgcacgg gctacatctt gctgcagggc atcgtgttct 240 ggggcttctt cacggtgggc cacgatgccg gccacggcgc cttctcgcgc taccacctgc 300 ttaacttcgt ggtgggcact ttcatgcact cgctcatcct cacgcccttc gagtcgtgga 360 agctcacgca ecgtcaccac cacaagaaca cgggcaacat tgaccgtgac gaggtcttct 420 acccgcaacg caaggccgac gaccacccgc tgtctcgcaa cctgattctg gcgctcgggg 480 cagcgtggct cgcctatttg gtcgagggct tccctcctcg taaggtcaac cacttcaacc 540 cgttcgagcc tctgttcgtg cgtcaggtgt cagctgtggt aatctctctt ctcgcccact 600 tcttcgtggc cggactctcc atctatctga gcctccagct gggccttaag acgatggcaa 660 tctactacca rgqacctqrt tttgtgttcg gcagcatgct qqtcattacc accttcccac 720 accacaatga tgaggagacc ccatggtacg ccgactcgga gtggacgtac gtcaagggca 780 acctctcgtc cgtggaccga tcgtacggcg cgctcattga caacctgagc cacaacatcg 840 gcacgcacca gatccaccac cttttcccta tcattccgca ctacaaactc aagaaagcca 900 ctgcggcctt ccaccaggct ttccctgagc tcgtgcgcaa gagcgacgag ccaattatca 960 aggctttctt ccgggttgga cgtctctacg caaactacgg cgttgtggac caggaggcga 1020 agctcttcac gctaaaggaa gccaaggcgg cgaccgaggc ggcggccaag accaagtcca 1080 cgtaa 1085

<210> 43 <211 > 361 <212> PRT <213> Phytophthora infestans <300> <302> METHOD FOR PRODUCING UNSATURATED Omega3 FATTY ACIDS IN TRANSGENIC ORGANISMS <308> CAJ30870 <309> 2005-09-21 <310> WO 2005083053 <311 >2005-02-23 <312> 2005-09-09 <313> (1)..(361) <400> 43

Met Ala Thr Lys Glu Ala Tyr val Phe Pro Thr Leu Thr Glu ile Lys 15 10 IS

Arg Ser Leu Pro Lys Asp Cys Phe Glu Ala ser val Pro Leu Ser Leu 20 25 30

Tyr Tyr Thr val Arg Cys Leu val lie Ala val Ala Leu Thr Phe Gly 35 40 45

Leu Asn Tyr Ala Arg Ala Leu Pro Glu val Glu ser Phe Trp Ala Leu 50 55 60

Asp Ala Ala Leu Cys Thr Gly Tyr lie Leu Leu Gin Gly He Val Phe 65 70 75 80

Trp Gly Phe Phe Thr val Gly His Asp Ala Gly His Gly Ala Phe ser 85 90 95

Arg Tyr His Leu Leu Asn Phe val val Gly Thr Phe Met His ser Leu 100 105 110 lie Leu Thr Pro Phe Glu ser Trp Lys Leu Thr His Arg His His His 115 120 125

Lys Asn Thr Gly Asn lie Asp Arg Asp Glu val Phe Tyr Pro Gin Arg 130 135 140

Lys Ala Asp Asp His Pro Leu ser Arg Asn Leu lie Leu Ala Leu Gly 145 150 155 160

Ala Ala Trp Leu Ala Tyr Leu val Glu Gly Phe Pro Pro Arg Lys Val

165 170 17S

Asn His Phe Asn Pro Phe Glu Pro Leu Phe val Arg Gin val Ser Ala 180 185 190 val val lie Ser Leu Leu Ala His Phe Phe val Ala Gly Leu ser lie 195 200 205

Tyr Leu Ser Leu Gin Leu Gly Leu Lys Thr Met Ala lie Tyr Tyr Tyr 210 215 220

Gly Pro val Phe val Phe Gly ser Met Leu Val lie Thr Thr Phe Leu 225 230 235 240

His His Asn Asp Glu Glu Thr Pro Trp Tyr Ala Asp ser Glu Trp Thr 245 250 255

Tyr val Lys Gly Asn Leu Ser Ser val Asp Arg ser Tyr Gly Ala Leu 260 265 270 lie Asp Asn Leu ser His Asn He Gly Thr His Gin lie His His Leu 275 280 285

Phe Pro lie lie Pro His Tyr Lys Leu Lys Lys Ala Thr Ala Ala Phe 290 295 300

His Gin Ala Phe Pro Glu Leu val Arg Lys ser Asp Glu Pro lie He 305 310 315 320

Lys Ala Phe Phe Arg val Gly Arg Leu Tyr Ala Asn Tyr Gly val val 325 330 335

Asp Gin Glu Ala Lys Leu Phe Thr Leu Lys Glu Ala Lys Ala Ala Thr 340 345 "350

Glu Ala Ala Ala Lys Thr Lys ser Thr 355 360

<210 44 <211> 1092 <212> DNA <213> Phytophthora sojae (us Patent Application No. 11/787772; filed 4/18/2007) <400> 44 atggcgtcca agcaggagca gccgtaccag ttcccgacgc tgacggagat caagcgctcg 60 ctgcccagcg agtgtttcga ggcgtccgtg ccgctctcgc tctactacac ggtgcgctgc 120 ctggtgatcg ccgtgtcgct ggccttcggg ctccaccacg cgcgctcgct gcccgtggtc 180 gagggcctct gggcgctgga cgccgcgctc tgcacgggct acgrgctgct gcagggcatc 240 gtgttctggg gcttcttcac cgtgggccat gacgccggcc acggcgcctt ctcgcgctac 300 cacctgctca acttcgtgat cggcaccttc atccaetcgc teatcctgac gcccttcgag 360 tcgtggaagc tcacgcaccg ccaccaccac aagaacacgg gcaacatcga ccgcgacgag 420 atcttctacc cgcagcgcaa ggccgacgac cacccgctct cgcgtaacct catcctggcg 480 ctgggcgccg cgtggttcgc ctacctggtc gagggcttcc cgccgcgcaa ggtcaaccac 540 ttcaacccgt tcgagccgct gttcgtccgc caggtgtccg ccgtggtcat ctcgctggCc 600 gcgcacttcg gcgtggccgc gctgtccatc tacctgagcc tgcagttcgg cttcaagacc 660 atggctatct actactacgg gcccgtgttc gtgttcggca gcatgctggt catcaccacc 720 ttcctgcacc acaacgacga ggagaccccc tggtacgccg actcggagtg gacctacgtc 780 aagggcaacc tctcgtcggt cgaccgctcc tacggcgcgc tcatcgacaa cctgagccac 840 aacatcggca cgcaccagat ccaccacctc ttccccatca tcccgcacta taagctcaag 900 cgcgccaccg aggccttcca ccaggcgttc cccgagctcg tgcgcaagag cgacgagccc 960 atcattaagg ccttcttccg cgtcggccgc ctctacgcca actacggcgt cgtggactcg 1020 gacgccaagc tcttcacgct caaggaggcc aaggccgtgt ccgaggcggc gaccaagact 1080 aaggccaact ga 1092

<210> 45 <211 > 363 <212> PRT <213> Phytophthora sojae (US Patent Application No. 11/787772; filed 4/18/2007) <400> 45

Met Ala ser Lys Gin Glu Gin Pro Tyr Gin Phe Pro Thr Leu Thr Glu 15 10 15

Ile Lys Arg ser Leu Pro Ser Glu Cys Phe Glu Ala Ser Val Pro Leu 20 25 30

Ser Leu Tyr Tyr Thr val Arg Cys Leu val Ile Al a val "ser Leu Ala 35 40 45

Phe Gly Leu His His Ala Arg ser Leu Pro val Val Glu Gly Leu Trp 50 55 60

Ala Leu Asp Ala Ala Leu Cys Thr Gly Tyr val Leu Leu Gin Gly ile 65 70 75 80 val Phe Trp Gly Phe Phe Thr val Gly His Asp Ala Gly His Gly Ala 85 90 95

Phe Ser Arg Tyr His Leu Leu Asn Phe val ile Gly Thr Phe Ile His 100 105 110 ser Leu Ile Leu Thr Pro Phe Glu ser Trp Lys Leu Thr His Arg His 115 120 125

His His Lys Asn Thr Gly Asn ile Asp Arg Asp Glu ile Phe Tyr Pro 130 135 140

Gin Arg Lys Ala Asp Asp His Pro Leu Ser Arg Asn Leu ile Leu Ala 145 150 155 160

Leu Gly Ala Ala Trp Phe Ala Tyr Leu Val Glu Gly Phe Pro Pro Arg 165 170 175

Lys val Asn His Phe Asn Pro Phe Glu Pro Leu Phe val Arg Gin val 180 18S 190 ser Ala Val val ile ser Leu Ala Ala His Phe Gly val Ala Ala Leu 195 200 205 ser ile Tyr Leu ser Leu Gin Phe Gly Phe Lys Thr Met Ala ile Tyr 210 215 220

Tyr Tyr Gly Pro Val Phe Val Phe Gly Ser Met Leu val Ile Thr Thr 225 230 235 240

Phe Leu His His Asn Asp Glu Glu Thr Pro Trp Tyr Ala Asp ser Glu 245 250 255

Trp Thr Tyr val Lys Gly Asn Leu ser ser val Asp Arg ser Tyr Gly 260 265 270

Ala Leu Ile Asp Asn Leu Ser His Asn Ile Gly Thr His Gin Ile His 275 280 285

His Leu Phe Pro ile ile Pro His Tyr Lys Leu Lys Arg Ala Thr Glu 290 295 300

Ala Phe His Gin Ala Phe Pro Glu Leu Val Arg Lys Ser Asp Glu Pro 305 310 315 320

Ile Ile Lys Ala Phe Phe Arg val Gly Arg Leu Tyr Ala Asn Tyr Gly 325 330 335 val val Asp Ser Asp Ala Lys Leu Phe Thr Leu Lys Glu Ala Lys Ala 340 345 350 val Ser Glu Ala Ala Thr Lys Thr Lys Ala Asn 355 360

<210 46 <211 > 1086 <212> DNA <213> Phytophthora ramorum (US Patent Application No. 11/787772; filed 4/18/2007) <400 46 atggcgacta agcagccgta ccagttcccg accctgacgg agatcaagcg gtcgctgccc 60 agcgagtgct ttgaggcctc ggtgccgctg tcgctctact acacggtgcg catcgtggcc 120 atcgccgtgg cgctggcgtt cggcctcaac tacgcgcgcg cgctgcccgt ggccgagagc 180 ttgtgggcgc tggacgctgc gctctgctgc ggttacgtgc tgctgcaggg catcgtgttc 240 tggggcttcc tcacggtggg ccatgacgcc ggccacggcg ccttctcgcg ttaccacctg 300 ctcaacttcg tggtgggcac cttcatccac tcgctcatcc tcacgccctt cgagtcgtgg 360 aagctcacgc accgccacca ccacaagaac acgggcaaca ttgaccgcga cgagatcttc 420 tacccgcagc gcaaggccga cgaccacccg ctgtcgcgca acctcgtgct ggcgctcggc 480 gccgcgtggc tcgcctacct ggtcgagggc ttcccgcccc gcaaggtcaa ccacttcaac 540 ccattcgagc cgctgtttgt gcgccaggtg gccgccgtcg tcatctcgct ctccgcgcac 600 ttcgccgtgt tggcgctgtc cgtgtatctg agcttccagt tcggtctcaa gaccarggcg 660 ctctactact acggccccgt cttcgtgttc ggcagcatgc ttgtgatcac caccttcctg 720 catcacaatg acgaggagac cccatggtac ggagactccg actggaccta cgtcaagggc 780 aacctgtcgt ccgtggaccg gtcctacggc gcgttcatcg acaacctgag ccacaacatc 840 ggcacgcacc agatccacca cctcttcccc atcatcccgc actacaagct caaccgcgct 900 acggcggcat tccaccaggc cttccccgag ctcgtgcgca agagcgacga gccgatcctc 960 aaggccttct ggcgcgtcgg ccgactgtac gccaactacg gcgtcgtgga cccggacgcc 1020 aagctcttca cgctcaagga ggccaaggcg gcgtccgagg cggcgaccaa gaccaaggcc 1080 acctaa 1086

<210> 47 <211 > 361 <212> PRT <213> Phytophthora ramorum (us Patent Application No. 11/787772; filed <400> 47

Met Ala Thr Lys Gin Pro Tyr Gin Phe Pro Thr Leu Thr Glu Ile Lys 15 10 15

Arg ser Leu pro Ser Glu Cys Phe Glu Ala Ser val Pro Leu Ser Leu 20 25 30

Tyr Tyr Thr val Arg Ile val Ala Ile Ala val Ala Leu Ala Phe Gly 35 40 45

Leu Asn Tyr Ala Arg Ala Leu Pro val Val Glu Ser Leu Trp Ala Leu 50 55 60

Asp Ala Ala Leu cys Cys Gly Tyr val Leu Leu Gin Gly Ile val Phe 65 70 75 80

Trp Gly Phe Phe Thr val Gly His Asp Ala Gly His Gly Ala Phe Ser 85 90 95

Arg Tyr His Leu Leu Asn Phe val val Gly Thr Phe Ile His Ser Leu 100 105 110 ile Leu Thr pro Phe Glu Ser Trp Lys Leu Thr His Arg His His His 115 120 125

Lys Asn Thr Gly Asn Ile Asp Arg Asp Glu Ile Phe Tyr pro Gin Arg 130 135 140

Lys Ala Asp Asp His Pro Leu Ser Arg Asn Leu val Leu Ala Leu Gly 145 150 155 160

Ala Ala Trp Phe Ala Tyr Leu val Glu Gly Phe Pro pro Arg Lys val 165 170 175

Asn His Phe Asn Pro Phe Glu Pro Leu Phe Val Arg Gin Val Ala Ala 180 185 190 val val Ile ser Leu Ser Ala His Phe Ala val Leu Ala Leu ser val 195 200 205

Tyr Leu Ser Phe Gin Phe Gly Leu Lys Thr Met Ala Leu Tyr Tyr Tyr 210 2IS 220

Gly Pro val Phe val Phe Gly Ser Met Leu val Ile Thr Thr Phe Leu 225 230 235 240

His His Asn Asp Glu Glu Thr Pro Trp Tyr Gly Asp Ser Asp Trp Thr 245 250 255

Tyr val Lys Gly Asn Leu Ser Ser val Asp Arg ser Tyr Gly Ala Phe 260 26S 270

Ile Asp Asn Leu Ser His Asn Ile Gly Thr His Gin Ile His His Leu 275 280 285

Phe Pro Ile Ile Pro His Tyr Lys Leu Asn Arg Ala Thr Ala Ala Phe 290 295 300

His Gin Ala Phe Pro Glu Leu val Arg Lys Ser Asp Glu Pro Ile Leu 305 310 315 320

Lys Ala Phe Trp Arg val Gly Arg Leu Tyr Ala Asn Tyr Gly val val 325 330 335 asp pro asp Ala Lys Leu Phe Thr Leu Lys Glu Ala Lys Ala Ala Ser 340 345 350

Glu Ala Ala Thr Lys Thr Lys Ala Thr 355 360

<210> 48 <211 > 7145 <212> DNA <213> Artificial Sequence <220> <223> Plasmid pKUNFmkF2 <220> <221 > misc_feature <222> (692)..(695) <223> n is a, c, g, or t <220> <221 > misc_feature <222> (700)..(700) <223> n is a, c, g, or t <400> 48 catggcgtcc acttcggctc tgcccaagca gaaccctgcg cttagacgca ccgtcacctc 60 aaetactgtg acggattctg agtctgccgc cgtctctcct tcagactctc cccgccactc 120 ggcctcttcc acatcgctet cgtccatgtc cgaggttgat atcgccaagc ccaagtccga 180 gtatggtgtc atgctcgaca cctacggcaa ccagttcgag gttcccgact ttaccatcaa 240 ggacatctac aatgccatcc ctaagcactg cttcaagcgc tccgctctca agggatacgg 300 ttatatcctc cgcgacattg tcctcctgac taccactttc agcatctggt acaactttgt 360 gacccccgaa tatatcccct ccacccccgc ccgcgctggt ctgtgggccg tgtacaccgt 420 tcttcagggt cttttcggta ctggtctctg ggttattgcc catgagtgcg gtcacggtgc 480 tttctccgat tctcgcatca tcaacgacat tactggctgg gttcttcact cttccctcct 540 tgtcccctac ttcagctggc aaatctccca ccgaaagcac cacaaggcca ctggcaacat 600 ggagcgtgac atggtcttcg ttccccgaac ccgcgagcag caggctactc gtctcggaaa 660 gatgacccac gagctcgctc atcttactga gnnnntcgtn ggctggccca actacctcat 720 caccaatgtt accggccaca actaccacga gcgccagcgt gagggtcgcg gcaagggcaa 780 gcataacggc ctcggcggtg gtgttaacca cttcgatccc cgcagccctc tgtacgagaa 840 cagtgacgct aagctcatcg tcctcagcga tattggtatc ggtctgatgg ccactgctct 900 gtacttcctc gttcagaagt tcggtttcta caacatggcc atctggtact ttgttcccta 960 cctctgggtt aaccactggc tcgttgccat caccttcctc cagcacaccg accctaccct 1020 tccccactac accaacgacg agtggaactt cgtccgtggt gccgctgcta ccattgaccg 1080 tgagatgggc ttcatcggcc gccaccttct ccacggcatc atcgagactc atgtcctcca 1140 ccactacgTc agcagcatcc ccttctacaa cgcggacgag gccaccgagg ccattaagcc 1200 catcatgggc aagcactacc gggctgatgt ccaggatggt cctcgtggct tcatccgcgc 1260 catgtaccgc agtgcgcgta tgtgccagtg ggttgagccc agcgctggtg ccgagggtgc 1320 tggtaagggt gttctgttct tccgcaaccg caacaacgtg ggcacccccc ccgctgttat 1380 caagcccgtt gcttaagtag gcgcggccgc tatttatcac tctttacaac ttctacctca 1440 actatctact ttaataaatg aatatcgttt attctctatg attactgtat atgcgttcct 1500 ctaagacaaa tcgaaaccag catgtgatcg aacggcatac aaaagrttcr tccgaagttg 1560 atcaatgtcc tgatagtcag gcagcttgag aagattgaca caggtggagg ccgtagggaa 1620 ccgatcaacc tgtctaccag cgttacgaat ggcaaatgac gggttcaaag ccttgaatcc 1680 ttgcaatggt gccttggata ctgatgtcac aaacttaaga agcagccgct tgtcctcttc 1740 ctcgatcgat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac 1800 aacgtacgaa gtcgtcaatg atgtcgatat gggttttgat catgcacaca taaggtccga 1860 ccttatcggc aagctcaatg agctccttgg tggtggtaac atccagagaa gcacacaggt 1920 tggttttctt ggctgccacg agcttgagca ctcgagcggc aaaggcggac ttgtggacgt 1980 tagctcgagc ttcgtaggag ggcattttgg tggtgaagag gagactgaaa taaatttagt 2040 ctgcagaact ttttatcgga accttatctg gggcagtgaa gtatatgtta tggtaatagt 2100 tacgagttag ttgaacttat agatagactg gactatacgg ctatcggtcc aaattagaaa 2160 gaacgtcaat ggctctctgg gcgtcgcctt tgccgacaaa aatgtgatca tgatgaaagc 2220 cagcaatgac gttgcagctg atattgttgt cggccaaccg cgccgaaaac gcagctgtca 2280 gacccacagc ctccaacgaa gaatgtatcg tcaaagtgat ccaagcacac tcatagttgg 2340 agtcgtactc caaaggcggc aatgacgagt cagacagata ctcgtcgacc ttttccttgg 2400 gaaccaccac cgtcagccct tctgactcac gtattgtagc caccgacaca ggcaacagtc 2460 cgtggatagc agaatatgtc ttgtcggtcc atttctcacc aactttaggc gtcaagtgaa 2520 tgttgcagaa gaagtatgtg ccttcattga gaatcggtgt tgctgatttc aataaagtct 2580 tgagatcagt ttggcgcgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 2640 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 2700 gctgcggcga gcggtatcag crcactcaaa ggcggtaata cggttatcca cagaatcagg 2760 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 2820 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 2880 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 2940 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 3000 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 3060 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 3120 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 3180 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 3240 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc 3300 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 3360 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 3420 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 3480 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 3S40 ctaaaaatga agttttåaat caatctaaag tatatatgag taaacttggt ctgacagtta 3600 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 3660 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 3720 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 3780 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 3840 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 3900 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 3960 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 4020 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcacrcat 4080 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 4140 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagctgctc 4200 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 4260 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 4320 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 4380 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 4440 gaaatgttga atacccatac tcttcctttt tcaatattat tgaagcattt atcagggtta 4500 ttgtctcatg agcggataca tattrgaarg tattraqaaa aataaacaaa taggggttcc 4560 gcgcacattt ccccgaaaag tgccacctga tgcggtgtga aataccgcac agatgcgtaa 4620 ggagaaaata ccgcatcagg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa 4680 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa 4740 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact 4800 axtaaagaac gxggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc 4860 artacgtgaa ccaccaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa 4920 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc 4980 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt 5040 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat 5100 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta 5160 cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggxaac gccagggttt 5220 tcccagtcac gacgttgtaa aacgacggcc agtgaattgt aatacgactc actatagggc 5280 gaattgggcc cgacgtcgca tgcagtggtg gtattgtgac tggggatgta gttgagaata 5340 agtcatacac aagtcagctt tcttcgagcc tcatataagt ataagtagtt caacgtatta 5400 gcactgtacc cagcatctcc gtatcgagaa acacaacaac atgccccatt ggacagatca 5460 tgcggataca caggttgtgc agtatcatac atactcgatc agacaggtcg tctgaccatc 5520 atacaagctg aacaagcgct ccatacttgc acgctctcta tatacacagt taaattacat 5580 atccatagtc taacctctaa cagttaatct tctggtaagc ctcccagcca gccttctggt 5640 atcgcttggc ctcctcaata ggatctcggt tctggccgta cagacctcgg ccgacaatta 5700 tgatatccgt tccggtagac atgacatcct caacagttcg gtactgctgt ccgagagcgt 5760 ctcccttgtc gtcaagaccc accccggggg tcagaataag ccagtcctca gagtcgccct 5820 taattaattt gaatcgaatc gatgagccta aaatgaaccc gagtatatct cataaaattc 5880 tcggtgagag gtctgtgact gtcagtacaa ggtgccttca ttatgccetc aaccttacca 5940 tacctcactg aatgtagtgt acctctaaaa atgaaataca gtgccaaaag ccaaggcact 6000 gagcccgtct aacggacttg atatacaacc aattaaaaca aatgaaaaga aatacagttc 6060 tttgtatcat ttgtaacaat taccetgtac aaactaaggt attgaaatcc cacaatattc 6120 ccaaagtcca cccctttcca aattgtcatg cctacaactc atataccaag cactaaccta 6180 ccgtttaaac agtgtacgca gatctactat agaggaacat ttaaattgcc ccggagaaga 6240 cggccaggcc gcctagatga caaattcaac aactcacagc tgactttctg ccattgecac 6300 tagggggggg cctttttata tggccaagcc aagctctcca cgtcggttgg gctgcaccca 6360 acaataaatg ggtagggttg caccaacaaa gggatgggat ggggggtaga agatacgagg 6420 ataacggggc tcaatggcac aaataagaac gaatactgcc attaagactc gtgatccagc 6480 gactgacacc attgcatcat ctaagggcct caaaactacc tcggaactgc tgcgctgatc 6540 tggacaccac agaggttccg agcactttag gttgcaccaa atgtcccacc aggtgcaggc 6600 agaaaacgct ggaacagcgt gtacagtttg tcttaacaaa aagtgagggc gctgaggtcg 6660 agcagggtgg tgtgacttgt tatagccttt agagctgcga aagcgcgtat ggatttggct 6720 catcaggcca gatxgagggt ctgtggacac atgtcatgtt agtgtacttc aatcgccccc 6780 tggatatagc cccgacaata ggccgtggcc tcattttttt gccttccgca catttccatt 6840 gctcgatacc cacaccttgc ttctcctgca cttgccaacc ttaatactgg tttacattga 6900 ccaacatctt acaagcgggg ggcttgtcta gggtatatat aaacagtggc tctcccaate 6960 ggttgccagt ctcttttttc cttxctttcc ccacagaxxc gaaatctaaa ctacacatca 7020 cagaattccg agccgtgagt axccacgaca agatcagtgt cgagacgacg cgxxxxgxgx 7080 aatgacacaa tccgaaagtc gctagcaaca cacactctct acacaaacta acccagctct 7140 ggtac 7145

<210> 49 <211 >5473 <212> DNA <213> Artificial sequence <220> <223> Plasmid pDMW287F <400> 49 ggccgcattg atgattggaa acacacacat gggttatatc taggtgagag ttagttggac 60 agttatatat taaatcagct atgccaacgg taacttcatt catgtcaacg aggaaccagt 120 gactgcaagt aatatagaat ttgaccacct tgccattctc ttgcactcct ttactatatc 180 tcatttattt cttatataca aatcacttct tcttcccagc atcgagctcg gaaacctcat 240 gagcaataac atcgtggatc tcgtcaatag agggcttttt ggactccttg ctgttggcca 300 ccttgtcctt gctgtctggc tcattctgtt tcaacgcctt ttaattaatc gagcttggcg 360 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 420 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 480 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 540 taaxgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 600 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtaxc agcxcactca 660 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 720 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 780 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 840 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 900 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 960 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 1020 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 1080 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 1140 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 1200 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 1260 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 1320 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 1380 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1440 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1500 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcaccratc 1560 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 1620 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 1680 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 1740 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 1800 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 1860 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 1920 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 1980 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 2040 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattic 2100 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 2160 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 2220 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 2280 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 2340 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 2400 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 2460 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 2520 gacgcgccct gtagcggcgc attaagcgcg gcgggxgtgg tggtcacgcg cagcgtgacc 2580 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 2640 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 2700 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 2760 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 2820 ggactcrcgt tccaaactgg aacaacaccc aaccctatct cggtctattc ttttgattta 2880 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 2940 aacgcgaatt ttaacaaaat attaacgctt acaatttcca ttcgccattc aggctgcgca 3000 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 3060 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt: ttcccagtca cgacgttgta 3120 aaacgacggc cagtgaattg taatacgact cactataggg cgaatcgggt accgggcccc 3180 ccctcgaggt cgacgtttaa acagtgtacg cagtactata gaggaacatc gattgccccg 3240 gagaagacgg ccaggccgcc tagatgacaa attcaacaac tcacagctga ctttctgcca 3300 ttgccactag gggggggcct ttttatatgg ccaagccaag ctctccacgt cggttgggct 3360 gcacccaaca ataaatgggt agggttgcac caacaaaggg atgggatggg gggtagaaga 3420 tacgaggata acggggctca atggcacaaa taagaacgaa tactgccatt aagactcgtg 3480 atccagcgac tgacaccatt gcatcatcta agggcctcaa aactacctcg gaactgctgc 3540 gctgatctgg acaccacaga ggttccgagc actttaggtt gcaccaaatg tcccaccagg 3600 tgcaggcaga aaacgctgga acagcgtgta cagtttgtct taacaaaaag tgagggcgct 3660 gaggtcgagc agggtggtgt gacttgttat agcctttaga gctgcgaaag cgcgtatgga 3720 tttggctcat caggccagat tgagggtctg tggacacatg tcatgttagt gtacttcaat 3780 cgccccctgg atatagcccc gacaataggc cgtggcctca tttttttgcc ttccgcacat 3840 ttccattgct cggtacccac accttgcttc tcctgcactt gccaacctta atactggttt 3900 acattgacea acatcttaca agcggggggc ttgtctaggg tatatataaa cagtggctct 3960 cccaatcggt tgccagtctc ctttttcctt tctttcccca cagattcgaa atctaaacta 4020 cacatcacac aatgcctgrc actgacgtcc ttaagcgaaa gtccggtgtc atcgteggcg 4080 acgatgtccg agccgtgagt atccacgaca agatcagtgt cgagacgacg cgttttgtgt 4140 aatgacacaa tccgaaagtc gctagcaaca cacactctct acacaaacta acccagctct 4200 ccatggtgaa gtccaagcga caggctctgc ccctcaccat cgacggaact acctacgacg 4260 tctccgcttg ggtgaacttc caccctggtg gagctgaaat cattgagaac taccagggac 4320 gagatgctac tgacgccttc atggttatgc actctcagga agccttcgac aagctcaagc 4380 gaatgcccaa gatcaacccc tcctccgagc tgcctcccca ggctgccgtc aacgaagctc 4440 aggaggattt ecgaaagctc cgagaagagc tgatcgccac tggcatgttt gacgcctctc 4500 ccctctggta ctcgtacaag atctccacca ccctgggtct tggcgtgctt ggatacttcc 4560 tgatggtcca gtaccagatg tacttcattg gtgctgtgct gctcggtatg cactaccagc 4620 aaatgggatg gctgtctcat gacatctgcc accaccagac cttcaagaac cgaaactgga 46B0 ataacctcgt gggtctggtc tttggcaacg gactccaggg cctctccgtg acctggtgga 4740 aggacagaca caacgcccat cattctgcta ccaacgttca gggtcacgat cccgacattg 4800 ataacctgcc tctgctcgcc tggtccgagg acgatgtcac tcgagcttct cccatctccc 4860 gaaagctcat tcagttccaa cagtactatt tcctggtcat ctgtattctc ctgcgattca 4920 tctggtgttt ccagtctgtg ctgaccgttc gatcccrcaa ggaccgagac aaccagttct 4980 accgatctca gtacaagaaa gaggccattg gactcgctct gcactggact ctcaagaccc 5040 tgttccacct cttctttatg ccctccatcc tgacctcgct cctggtgttc tttgtttccg 5100 agctcgtcgg tggcttcgga attgccatcg tggtcttcat gaaccactac cctctggaga 5160 agatcggtga ttccgtctgg gacggacatg gcttctctgt gggtcagatc catgagacca 5220 tgaacattcg acgaggcatc attactgact ggttctttgg aggcctgaac taccagatcg 5280 agcaccatct ctggcccacc ctgcctcgac acaacctcac tgccgtttcc taccaggtgg 5340 aacagctgtg ccagaagcac aacctcccct accgaaaccc tctgccccat gaaggtctcg 5400 tcatcctgct ccgatacctg gccgtgttcg ctcgaatggc cgagaagcag cccgctggca 5460 aggctctcta age 5473

<210> 50 <211> 9513 <212> DNA <213> Artificial Sequence <220> <223> Plasmid pDMW214 <400> 50 ggtggagcrc cagcttttgt tccctttagt gagggttaat ttcgagcttg gcgtaatcat 60 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag 120 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 180 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 240 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 300 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 360 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 420 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 480 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 540 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 600 tgccgctcac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 660 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 720 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 780 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 840 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 900 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 960 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 1020 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggc 1080 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 1140 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 1200 atgagtaaac ttggtctgac agttaccaat gctraatcag tgaggcacct atctcagcga 1260 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 1320 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 1380 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 1440 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 1500 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 1560 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 1620 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 1680 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 1740 tgccatcegt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 1800 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 1860 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 1920 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 1980 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 2040 caaaaaaggg aataagggcg acacggaaat gttgaatact cataetettc ctttttcaat 2100 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 2160 agaaaaataa acaaataggg gtcccgcgca catttccccg aaaagtgcca cctgacgcgc 2220 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 2280 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 2340 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 2400 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 2460 cctgacagac ggtttttcgc cctttgacgt tggagcccac gttctttaat agtggactct 2520 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 2580 ttttgccgac ttcggcctat cggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 2640 attttaacaa aatattaacg cttacaattt ccattcgcca ttcaggctgc gcaactgttg 2700 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc 2760 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac 2820 ggccagtgaa ttgtaatacg actcactata gggcgaattg ggtaccgggc cccccctcga 2880 ggtcgatggt gtcgataagc ttgatatcga attcatgtca cacaaaccga tcttcgcctc 2940 aaggaaacct aattctacat ccgagagact gccgagatcc agtctacact gattaatttt 3000 cgggccaara atttaaaaaa atcgtgttat ataatattac atgtattata tatatacatc 3060 atgatgatac tgacagtcat gtcccattgc taaatagaca gactccatct gccgcctcca 3120 actgatgttc tcaatattta aggggtcatc tcgcattgtt taataataaa cagactccat 3180 ctaccgcctc caaatgatgt tctcaaaata tattgtatga acttattttt attacttagt 3240 attattagac aacttacttg ctttatgaaa aacacttcct atttaggaaa caatttataa 3300 tggcagttcg ttcatttaac aatttatgta gaataaatgt tataaatgcg tatgggaaat 3360 cttaaatatg gatagcataa atgatatctg cattgcctaa ttcgaaatca acagcaacga 3420 aaaaaatccc ttgtacaaca taaatagtca tcgagaaata tcaactatca aagaacagct 3480 attcacacgt tactattgag attattattg gacgagaatc acacactcaa ctgtctttct 3540 ctcttctaga aatacaggta caagtatgta ctattctcat tgttcatact tctagtcatt 3600 tcatcccaca tattccttgg atttctctcc aatgaatgac attctatctt gcaaattcaa 3660 caattataat aagatatacc aaagtagcgg tatagtggca atcaaaaagc ttctctggtg 3720 tgcttctcgt atttattttt attctaatga tccattaaag gtatatattt atttcttgtt 3780 atataatcct tttgtttatt acatgggctg gatacataaa ggtattttga tttaattttt 3840 tgcttaaatt caatcccccc tcgttcagtg tcaactgtaa tggtaggaaa ttaccatact 3900 tttgaagaag caaaaaaaat gaaagaaaaa aaaaatcgta tttccaggtt agacgttccg 3960 cagaatctag aatgcggtat gcggtacatt gttcttcgaa cgtaaaagtt gcgctccctg 4020 agatattgta catttttgct tttacaagta caagtacatc gtacaactat gtactactgt 4080 tgatgcatcc acaacagttt gttttgtttt tttttgtttt ttttttttct aatgattcat 4140 taccgctatg tatacctact tgtacttgta gtaagccggg ttattggcgt tcaattaatc 4200 atagacttat gaatctgcac ggtgtgcgct gcgagttact tttagcttat gcatgctact 4260 tgggtgtaat attgggatct gttcggaaat caacggatgc tcaaccgatt tcgacagtaa 4320 taatttgaat cgaatcggag cctaaaatga acccgagtat atctcataaa attctcggtg 4380 agaggtctgt gactgtcagt acaaggtgcc ttcattatgc cctcaacctt accatacctc 4440 actgaatgta gtgtacctct aaaaatgaaa tacagtgcca aaagccaagg cactgagctc 4500 gtctaacgga cttgatatac aaccaattaa aacaaatgaa aagaaataca gttctttgta 4560 tcatttgtaa caattaccct gtacaaacta aggtattgaa atcccacaat attcccaaag 4620 tccacccctt tccaaattgt catgcctaca actcatatac caagcactaa cctaccaaac 4680 accacxaaaa ccccacaaaa tatatcttac cgaatataca gtaacaagct accaccacac 4740 tcgttgggtg cagtcgccag cttaaagata tctatccaca tcagccacaa ctcccttcct 4800 ttaataaacc gactacaccc ttggctattg aggttatgag tgaatatact gtagacaaga 4860 cactttcaag aagactgttt ccaaaacgta ecactgtcct ccactacaaa cacacccaat 4920 ctgcttcttc tagtcaaggt tgctacaccg gtaaattata aatcatcatt tcattagcag 4980 ggcagggccc tttttataga gtcttataca ctagcggacc ctgccggtag accaacccgc 5040 aggcgcgtca gtttgctcct tccatcaatg cgtcgtagaa acgacttact ccttcttgag 5100 cagctccttg accttgttgg caacaagtct ccgacctcgg aggtggagga agagcctccg 5160 atatcggcgg tagtgatacc agcctcgacg gactccttga cggcagcctc aacagcgtca 5220 ccggcgggct tcatgttaag agagaacttg ageatcatgg cggcagacag aatggtggca 5280 atggggttga ccttctgctt gccgagatcg ggggcagatc cgtgacaggg ctcgtacaga 5340 ccgaacgcct cgttggtgtc gggcagagaa gccagagagg cggagggcag cagacccaga 5400 gaaccgggga tgacggaggc ctcgtcggag atgatatcgc caaacatgtt ggtggtgatg 5460 atgataccat tcatcttgga gggctgcttg atgaggatca tggcggccga gtcgatcagc 5520 tggtggttga gctcgagctg ggggaattcg tccttgagga ctcgagtgac agtctttcgc 5580 caaagtcgag aggaggccag cacgttggcc ttgtcaagag accacacggg aagagggggg 5640 ttgtgctgaa gggccaggaa ggcggccatt cgggcaattc gctcaacctc aggaacggag 5700 taggtctcgg tgtcggaagc gacgccagat ccgtcatcct cctttcgctc tccaaagtag 5760 atacctccga cgagctctcg gacaatgatg aagtcggtgc cctcaacgtt tcggatgggg 5820 gagagatcgg cgagctcggg cgacagcagc tggcagggtc gcaggttggc gtacaggttc 5880 aggtcctttc gcagcttgag gagaccctgc tcgggtcgca cgtcggttcg tccgtcggga 5940 gtggtccata cggtgttggc agcgcctccg acagcaccga gcataataga gtcagccttt 6000 cggcagatgt cgagagtagc gtcggtgatg ggetcgccct ccttctcaat ggcagctcct 6060 ccaatgagtc ggtcctcaaa cacaaactcg gtgccggagg cctcagcaac agacttgagc 6120 accttgacgg cctcggcaat cacctcgggg ccacagaagt cgccgccgag aagaacaatc 6180 ttcttggagt cagtcttggt cttcttagtt tcgggttcca ttgtggatgt gtgtggttgt 6240 atgtgtgatg tggtgtgtgg agtgaaaatc tgtggctggc aaacgctctt gtatatatac 6300 gcacttttgc ccgtgctatg tggaagacta aacctccgaa gattgtgact caggtagtgc 6360 ggtatcggct agggacccaa accttgtcga tgccgatagc gctatcgaac gtaccccagc 6420 cggccgggag tatgtcggag gggacatacg agatcgtcaa gggtttgtgg ccaactggta 6480 aataaatgat gncgacgttt aaacagtgta cgcagtacta tagaggaaca attgccccgg 6540 agaagacggc caggccgcct agatgacaaa ttcaacaact cacagctgac tttctgccat 6600 tgccactagg ggggggcctt tttatatggc caagccaagc tctccacgtc ggttgggctg 6660 cacccaacaa taaatgggta gggttgcacc aacaaaggga cgggatgggg ggtagaagat 6720 acgaggataa cggggctcaa tggcacaaat aagaacgaat actgccatxa agactcgtga 6780 tccagcgact gacaccattg catcatctaa gggcctcaaa actacctcgg aactgctgcg 6840 ctgatccgga caccacagag gttccgagca ctttaggttg caccaaatgt cccaccaggt 6900 gcaggcagaa aacgctggaa cagcgtgtac agxttgtctt aacaaaaagt gagggcgctg 6960 aggtcgagca gggtggtgtg acttgttata gcctttagag ctgcgaaagc gcgtatggat 7020 ttggctcatc aggccagatt gagggtctgt ggacacatgt catgttagtg tacttcaatc 7080 gccccctgga tatagccccg acaataggcc gtggcctcat ttttttgcct tccgcacatt 7140 tccattgctc ggtacccaca ccttgcttct cctgcacttg ccaaccttaa tactggttta 7200 cattgaccaa catcttacaa gcggggggct tgtctagggt atatataaac agtggctctc 7260 ccaatcggtt gccagtctct tttttccttt ctttccccac agattcgaaa tctaaactac 7320 acatcacaca atgcctgtta ctgacgtcct taagcgaaag tccggtgtca tcgtcggcga 7380 cgatgtccga gccgtgagta tccacgacaa gatcagtgtc gagacgacgc gttttgtgta 7440 atgacacaat ccgaaagtcg ctagcaacac acactctcta cacaaactaa cccagctctc 7500 catggcatgg atggtacgtc ctgtagaaac cccaacccgt gaaatcaaaa aactcgacgg 7560 cctgtgggca ttcagtctgg atcgcgaaaa ctgtggaatt gatcagcgtt ggtgggaaag 7620 cgcgttacaa gaaagccggg caattgctgt gccaggcagt tttaacgatc agttcgccga 7680 tgcagatatt cgtaattatg cgggcaacgt ctggtatcag cgcgaagtct ttataccgaa 7740 aggttgggca ggccagcgta tcgtgctgcg tttcgatgcg grcactcatt acggcaaagt 7800 gtgggtcaat aatcaggaag tgatggagca tcagggcggc tatacgccat ttgaagccga 7860 tgtcacgccg tatgttattg ccgggaaaag tgtacgtatc accgtttgtg tgaacaacga 7920 actgaactgg cagactatcc cgccgggaat ggtgattacc gacgaaaacg gcaagaaaaa 7980 gcagtcttac ttccatgatt tctttaacta tgccgggatc catcgcagcg taatgctcta 8040 caccacgccg aacacctggg tggacgatat caccgtggtg acgcatgtcg cgcaagactg 8100 taaccacgcg tctgttgact ggcaggtggt ggccaatggt gatgtcagcg ttgaactgcg 8160 tgatgcggat caacaggtgg ttgcaactgg acaaggcact agcgggactt tgcaagtggt 8220 gaatccgcac ctctggcaac cgggtgaagg ttatctctat gaactgtgcg tcacagccaa 8280 aagccagaca gagtgtgata tctacccgct tcgcgtcggc atccggtcag tggcagtgaa 8340 gggcgaacag ttcctgatta accacaaacc gttctacttt actggctttg gtcgtcatga 8400 agatgcggac ttacgtggca aaggattcga taacgtgctg atggtgcacg accacgcatt 8460 aatggactgg attggggcca actcctaccg tacctcgcat tacccttacg ctgaagagat 8S20 gctcgactgg gcagatgaac atggcatcgt ggtgattgat gaaactgctg ctgtcggctt 8580 taacctctct ttaggcattg gtttcgaagc gggcaacaag ccgaaagaac tgtacagcga 8640 agaggcagtc aacggggaaa ctcagcaagc gcacttacag gcgattaaag agctgatagc 8700 gcgtgacaaa aaccacccaa gcgtggtgat gtggagtatt gccaacgaac cggatacccg 8760 tccgcaagtg cacgggaata tttcgccact ggcggaagca acgcgtaaac tcgacccgac 8820 gcgtccgatc acctgcgtca atgtaatgtt ctgcgacgct cacaccgata ccatcagcga 8880 tctctttgat gtgctgtgcc tgaaccgtta ttacggatgg tatgtccaaa gcggcgattt 8940 ggaaacggca gagaaggtac tggaaaaaga acttctggcc tggcaggaga aactgcatca 9000 gccgattatc atcaccgaat acggcgrgga tacgttagcc gggctgcact caatgtacac 9060 cgacatgtgg agcgaagagt atcagtgtgc atggctggat atgtatcacc gcgtctttga 9120 tcgcgtcagc gccgtcgtcg gtgaacaggt atggaatttc gccgattttg cgacctcgca 9180 aggcatattg cgcgttggcg gtaacaagaa agggatcttc actcgcgacc gcaaaccgaa 9240 gtcggcggct tttctgctgc aaaaacgctg gactggcatg aacttcggtg aaaaaccgca 9300 gcagggaggc aaacaatgat taattaacta gagcggccgc caccgcggcc cgagattccg 9360 gcctcttcgg ccgccaagcg acccgggtgg acgtctagag gtacctagca atraacagat 9420 agtttgccgg tgataattct cttaacctcc cacactcctt tgacataacg atttatgtaa 9480 cgaaactgaa atttgaccag atattgtgtc cgc 9513

<210>51 <211> 8910 <212> DNA <213> Artificial Sequence <220> <223> Plasmid pFmD8S <400> 51 catggtgaag tccaagcgac aggctctgcc cctcaccatc gacggaacta cctacgacgt 60 ctccgcttgg gtgaacttcc accctggtgg agctgaaatc attgagaact accagggacg 120 agatgctact gacgccttca tggttatgca ctctcaggaa gccttcgaca agctcaagcg 180 aatgcccaag atcaacccct cctccgagct gcctccccag gctgccgtca acgaagctca 240 ggaggatttc cgaaagctcc gagaagagct gatcgccact ggcatgtttg acgcctctcc 300 cctctggtac tcgtacaaga tctccaccac cctgggtctt ggcgtgcttg gatacttcct 360 gatggtccag taccagatgt acttcattgg tgctgtgctg ctcggtatgc actaccagca 420 aatgggatgg ctgtctcatg acatctgcca ccaccagacc ttcaagaaee gaaactggaa 480 taacctcgtg ggtctggtct ttggcaacgg actccagggc ttctccgtga cctggtggaa 540 ggacagacac aacgcccatc attctgctac caacgttcag ggtcacgatc ccgacattga 600 taacctgcct ctgctcgcct ggtccgagga cgatgtcact cgagcttctc ccatctcccg 660 aaagctcatt cagttccaac agtactattt cctggtcatc tgtattctcc tgcgattcat 720 crggtgtttc cagtctgtgc tgaccgttcg atccctcaag gaccgagaca accagttcta 780 ccgatctcag tacaagaaag aggccattgg actcgctctg cactggactc tcaagaccct 840 gttccacctc ttctttatgc cctccatcct gacctcgctc ctggtgttct ttgtttccga 900 gctcgtcggt ggcttcggaa ttgccatcgt ggtcttcatg aaccactacc ctctggagaa 960 gatcggtgat tccgtctggg acggacatgg cttctctgcg ggtcagatcc atgagaccat 1020 gaacattcga cgaggcatca ttactgactg gttctttgga ggcctgaact accagatcga 1080 gcaccatctc tggcccaccc tgcctcgaca caacctcact gccgtttcct accaggtgga 1140 acagctgtgc cagaagcaca acctccccta ccgaaaccct ctgccccatg aaggtctcgt 1200 catcctgctc cgatacctgg ccgtgttcgc tcgaatggcc gagaagcagc ccgctggcaa 1260 ggctctctaa gcggccgcca ccgcggcccg agattccggc ctcttcggcc gccaagcgac 1320 ccgggtggac gtctagaggt acctagcaat taacagatag tttgccggtg ataattctct 1380 taacctccca cactcctttg acataacgat ttatgtaacg aaactgaaat ttgaccagat 1440 attgtgtccg cggtggagct ccagcttttg trccctttag tgagggttaa tttcgagctt 1500 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 1560 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 1620 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 1680 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 1740 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 1800 ctcaaaggcg gtaatacggt tatccacaga arcaggggat aacgcaggaa agaacatgtg 1860 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 1920 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 1980 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 2040 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 2100 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 2160 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 2220 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 2280 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 2340 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 2400 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 2460 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 2520 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 2580 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagct ttaaatcaat 2640 ctaaagtata tatgagtaaa cttggtctga cagtcaccaa tgcttaatca gtgaggcacc 2700 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 2760 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 2820 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 2880 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 2940 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 3000 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 3060 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 3120 Tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 3180 tcttactgtc atgccatccg taagatgcct ttctgtgact ggtgagtact caaccaagtc 3240 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 3300 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 3360 aaaactctca aggatcttac cgctgttgag atccagttcg acgtaaccca ctcgtgcacc 3420 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 3480 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 3540 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 3600 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttccce gaaaagtgcc 3660 acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 3720 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct 3780 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg 3840 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag 3900 tgggccatcg ccctgataga cggtttttcg ccctttgacg trggagtcca cgttctttaa 3960 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga 4020 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa 4080 atttaacgcg aattttaaca aaatattaac gcttacaatt tccattcgcc attcaggctg 4140 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa 4200 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt 4260 tgcaaaacga cggccagtga attgtaatac gactcactat agggcgaatt gggtaccggg 4320 ccccccctcg aggtcgatgg tgtcgataag cttgatatcg aattcatgtc acacaaaccg 4380 atcttcgcct caaggaaacc taattctaca tccgagagac tgccgagatc cagtctacac 4440 tgattaattt tcgggccaat aatttaaaaa aatcgtgtta tataatatta tatgtattat 4500 atatatacat catgatgata ctgacagtca tgtcccattg ctaaatagac agactccatc 4560 tgccgcctcc aactgatgtt ctcaatattt aaggggtcat ctcgcattgt ttaataataa 4620 acagactcca tctaccgcct ccaaatgatg ttctcaaaat atattgtatg aacttatttt 4680 tattacttag tattattaga caacttactt gctttatgaa aaacacttcc tatttaggaa 4740 acaatttata atggcagttc gttcattraa caatttatgt agaacaaatg ttataaatgc 4800 gtatgggaaa tcttaaatat ggatagcata aatgatatct gcattgccta attcgaaatc 4860 aacagcaacg aaaaaaatcc ctitgtacaac ataaatagtc atcgagaaat atcaactatc 4920 aaagaacagc tattcacacg ttactattga gattattatt ggacgagaat cacacactca 4980 actgtctttc tctcttctag aaatacaggt acaagtatgt actattctca ttgttcatac 5040 ttctagtcat ttcatcccac atattccttg gatttctctc caatgaatga cattctatct 5100 tgcaaattca acaattataa taagatatac caaagtagcg gtatagtggc aatcaaaaag 5160 cttctctggt gtgcttctcg tatttatttt tattctaatg atccattaaa ggtatatatt 5220 tatttcttgt tatataatcc ttttgtttat tacatgggct ggatacataa aggtattttg 5280 atttaatttt ttgcttaaat tcaatccccc ctcgttcagt gtcaactgta atggtaggaa 5340 attaccatac ttttgaagaa gcaaaaaaaa tgaaagaaaa aaaaaatcgt atttccaggt 5400 tagacgtccc gcagaatcta gaatgcggta tgcggtacat tgttcttcga acgtaaaagt 5460 tgcgctccct gagatattgt acatttttgc ttfcacaagt acaagtacat cgtacaacca 5520 tgtactactg ttgatgcatc cacaacagtt tgttttgttt'ttttttgttt tttttttttc 5580 taatgattca ttaccgctat gtatacctac ttgtacttgt agtaagccgg gttattggcg 5640 ttcaattaat catagactta tgaatctgca cggtgtgcgc tgcgagttac ttttagctta S700 tgcatgctac ttgggtgtaa tattgggatc tgttcggaaa tcaacggatg ctcaaccgat 5760 ttcgacagta ataatttgaa tcgaatcgga gcctaaaatg aacccgagta tatctcataa 5820 aattctcggt gagaggtctg tgactgtcag tacaaggtgc cttcattatg ccctcaacct 5880 taccatacct cactgaatgt agtgtacctc taaaaatgaa atacagtgcc aaaagccaag 5940 gcactgagct cgtctaacgg acttgatata caaccaatta aaacaaatga aaagaaatac 6000 agttctrtgt atcatttgta acaattaccc tgtacaaact aaggtattga aatcccacaa 6060 tattcccaaa gtccacccct ttccaaattg tcatgcctac aactcatata ccaagcacta 6120 acctaccaaa caccactaaa accccacaaa atatatctta ccgaatatac agtaacaagc 6180 taccaccaca ctcgttgggt gcagtcgcca gcttaaagat atctatccac atcagccaca 6240 actcccttcc tttaataaac cgactacacc cttggctatt gaggttatga gtgaatatac 6300 tgtagacaag acactttcaa gaagaccgtt tccaaaacgt accactgtcc tccactacaa 6360 acacacccaa tctgcttctt ctagtcaagg ttgctacacc ggtaaattat aåatcatcat 6420 ttcartagca gggcagggcc ctttttatag agtcttatac actagcggac cctgccggta 6480 gaccaacccg caggcgcgtc agtttgctcc ttccatcaat gcgtcgtaga aacgacttac 6540 tccttcttga gcagctcctt gaccttgttg gcaacaagtc tccgacctcg gaggtggagg 6600 aagagcctcc gatatcggcg gtagtgatac cagcctcgac ggactccttg acggcagcct 6660 caacagcgtc accggcgggc ttcatgttaa gagagaactt gagcatcatg gcggcagaca 6720 gaatggtggc aatggggttg accttctgct tgccgagatc gggggcagat ccgtgacagg 6780 gctcgtacag accgaacgcc tcgttggtgt cgggcagaga agccagagag gcggagggca 6840 gcagacccag agaaccgggg atgacggagg cctcgtcgga gatgatatcg ccaaacatgt 6900 tggtggtgat gatgatacca ttcaccttgg agggctgctt gatgaggatc atggcggccg 6960 agtcgatcag ctggtggttg agctcgagct gggggaattc gtccttgagg actcgagtga 7020 cagcctttcg ccaaagtcga gaggaggcca gcacgttggc cttgtcaaga gaccacacgg 7080 gaagaggggg gttgtgctga agggccagga aggcggccat tcgggcaatt cgctcaacct 7140 caggaacgga gtaggtctcg gtgtcggaag cga'cgccaga tccgtcatcc tcctttcgct 7200 ctccaaagta gatacctccg acgagctctc ggacaatgat gaagtcggtg cccncaacgt 7260 rtcggatggg ggagagatcg gcgagcttgg gcgacagcag ctggcagggt cgcaggttgg 7320 cgtacaggtt caggtccttt cgcagcttga ggagaccctg ctcgggtcgc acgtcggttc 7380 gtccgtcggg agtggtccat acggtgttgg cagcgcctcc gacagcaccg agcataatag 7440 agtcagcctt tcggcagatg tcgagagtag cgtcggtgat gggctcgccc tccttctcaa 7500 tggcagctcc tccaatgagt cggtcctcaa acacaaactc ggtgccggag gcctcagcaa 7S60 cagacttgag caccttgacg gcctcggcaa tcacctcggg gccacagaag tcgccgccga 7620 gaagaacaat cttcttggag tcagtcttgg tcttcttagt ttcgggttcc attgtggatg 7680 tgtgtggttg tatgtgtgat gtggtgtgtg gagtgaaaat ctgtggctgg caaacgctct 7740 tgtatatata egeacttttg cccgtgctat gtggaagact aaacctccga agattgtgac 7800 tcaggtagtg cggtatcggc tagggaccca aaccttgtcg atgccgatag cgctatcgaa 7860 cgtaccccag ccggccggga gtatgtcgga ggggacatac gagatcgtca agggttcgtg 7920 gccaactggt aaataaatga tgtcgacgtt taaacagtgt acgcagatct actatagagg 7980 aacatttaaa ttgccccgga gaagacggcc aggccgccta gatgacaaat tcaacaactc 8040 acagctgact ttctgccatt gccactaggg gggggccttt ttatatggcc aagccaagct 8100 ctccacgtcg gttgggctgc acccaacaat aaatgggtag ggttgcacca acaaagggat 8160 gggatggggg gtagaagata cgaggataac ggggctcaat ggcacaaata agaacgaata 8220 ctgccattaa gactcgtgat ccagcgactg acaccattgc atcatctaag ggcctcaaaa 8280 ctacctcgga actgctgcgc tgatctggac accacagagg ttccgagcac tttaggttgc 8340 accaaatgtc ccaccaggtg caggcagaaa acgctggaac agcgtgtaca gtttgtctta 8400 acaaaaagtg agggcgctga ggtcgagcag ggtggtgtga cttgttatag cccttagagc 8460 tgcgaaagcg cgtatggatt tggctcatca ggccagattg agggtctgtg gacacatgtc 8520 atgttagtgt acttcaatcg ccccctggat atagccccga caataggccg tggcctcatt 8580 tttttgcctt ccgcacattt ccattgctcg gtacccacac cttgcttctc ctgcacttgc 8640 caacctrtaat actggtttac attgaccaac atcttacaag cggggggctt gtctagggta 8700 tatataaaca gtggctctcc caatcggttg ccagtctctt ttttcctttc tttccccaca 8760 gattcgaaat ctaaactaca catcacagaa ttccgagccg tgagtatcca cgacaagatc 8820 agtgtcgaga cgacgcgttt tgtgtaatga cacaatccga aagtcgctag caacacacac 8880 tcrctacaca aactaaccca gctctggtac 8910 <210> 52 <211> 1272

<212> DNA <213> Euglena gracilis <220 <221 > misc_feature <222> (2)..(1270) <223> synthetic delta-8 desaturase CDS, codon-optimized for Yarrowia lipolytica ("D8SF" or "EgD8S”) <300

<302> DELTA-8 DESATURASE AND ITS USE IN MAKING POLYUNSATURATED FATTY ACIDS <310> WO 2006/012325 and WO 2006/012326 <311 >2005-06-24 <312> 2006-02-02 <313> (1)..(1272) <300>

<302> DELTA-8 DESATURASE AND ITS USE IN MAKING POLYUNSATURATED FATTY ACIDS <310 US-2005-0287652-A1 <311 >2005-06-24 <312> 2005-12-29 <313> (1)..(1272) <400> 52 catggtgaag tccaagcgac aggctctgcc cctcaccatc gacggaacta cctacgacgt 60 ctccgcttgg gtgaacttcc accctggtgg agctgaaatc attgagaact accagggacg 120 agatgctact gacgccttca tggttatgca ctctcaggaa gccttcgaca agctcaagcg 180 aatgcccaag atcaacccct cctccgagct gcctccccag gctgccgtca acgaagctca 240 ggaggatttc cgaaagctcc gagaagagct gatcgccact ggcatgtttg acgcctctcc 300 cctctggtac tcgtacaaga tctccaccac cctgggtctt ggcgtgcttg gatacttcct 360 gatggtccag taccagatgt acttcattgg tgctgtgctg ctcggtatgc actaccagca 420 aatgggatgg ctgtctcatg acatctgcca ccaccagacc ttcaagaacc gaaactggaa 480 taacctcgtg ggtctggtct ttggcaacgg aetceagggc ttctccgtga cctggtggaa 540 ggacagacac aacgcccatc attctgctac caacgttcag ggtcacgatc ccgacactga 600 taacctgcct ctgctcgcct ggtccgagga cgatgtcact cgagcttctc ccatctcccg 660 aaagctcatt cagttccaac agtactattt cctggtcatc tgtattctcc tgcgattcat 720 ctggtgtttc cagtctgtgc tgaccgttcg atccctcaag gaccgagaca accagttcta 780 ccgatctcag tacaagaaag aggccattgg actcgctctg cactggactc Tcaagaccct 840 gttccacctc ttctttatgc cctccatcct gacctcgctc ctggtgttct ttgtttccga 900 gctcgtcggt ggcttcggaa ttgccatcgt ggtcttcatg aaccactacc ctctggagaa 960 gatcggrgat tccgtctggg acggacatgg cttctctgtg ggtcagatcc atgagaccat 1020 gaacattcga cgaggcatca ttactgactg gttctttgga ggcetgaact accagatcga 1080 gcaccatctc tggcccaccc tgcctcgaca caacctcact grrgrt t rrt accaggtgga 1140 acagctgtgc cagaagcaca acctccccta ccgaaaccct ctgccccatg aaggtctcgt 1200 catcctgctc cgatacctgg ccgtgttcgc tcgaatggcc gagaagcagc ccgctggcaa 1260 ggctctctaa gc 1272

<210> 53 <211> 422 <212> PRT <213> Euglena gracilis <220>

<221 > MISC FEATURE <223> synthetic delta-8 desaturase codon-optimized for Yarrowia lipolytica (“D8SF" or "EgD8S") <300>

<302> DELTA-8 DESATURASE AND ITS USE IN MAKING POLYUNSATURATED FATTY ACIDS <310> WO 2006/012325 and WO 2006/012326 <311 >2005-06-24 <312> 2006-02-02 <313> (1)..(422) <300>

<302> DELTA-8 DESATURASE AND ITS USE IN MAKING POLYUNSATURATED FATTY ACIDS <310> US-2005-0287652-A1 <311 >2005-06-24 <312> 2005-12-29 <313> (1)..(422) <400> 53

Met val Lys Ser Lys Arg Gin Ala Leu Pro Leu Thr lie Asp Gly Thr 15 10 15

Thr Tyr Asp val Ser Ala Trp val Asn Phe His Pro Gly Gly Ala Glu 20 25 30

Ile Ile Glu Asn Tyr Gin Gly Arg Asp Ala Thr Asp Ala Phe Met Val 35 40 45

Met His ser Gin Glu Ala Phe Asp Lys Leu Lys Arg Met Pro Lys lie 50 55 60

Asn Pro ser ser Glu Leu Pro Pro Gin Ala Ala val Asn Glu Ala Gin 65 70 75 80

Glu Asp Phe Arg Lys Leu Arg Glu Glu Leu lie Ala Thr Gly Met Phe 85 90 95

Asp Ala Ser Pro Leu Trp Tyr ser Tyr Lys lie Ser Thr Thr Leu Gly 100 105 110

Leu Gly val Leu Gly Tyr Phe Leu Met val Gin Tyr Gin Met Tyr Phe 115 120 125 lie Gly Ala val Leu Leu Gly Met His Tyr Gin Gin Met Gly Trp Leu 130 13S 140

Ser His Asp lie Cys His His Gin Thr Phe Lys Asn Arg Asn Trp Asn 145 150 155 160

Asn Leu val Gly Leu val Phe Gly Asn Gly Leu Gin Gly Phe ser val 165 170 175

Thr Trp Trp Lys Asp Arg His Asn Ala His His Ser Ala Thr Asn val 180 185 190

Gin Gly His Asp Pro Asp lie Asp Asn Leu Pro Leu Leu Ala Trp Ser 195 200 205

Glu Asp Asp val Thr Arg Ala Ser Pro Ile Ser Arg Lys Leu Ile Gin 210 21S 220 phe Gin Gin Tyr Tyr Phe Leu val Ile cys Ile Leu Leu Arg Phe Ile 225 230 235 240

Trp Cys Phe Gin ser val Leu Thr val Arg ser Leu Lys Asp Arg Asp 245 250 255

Asn Gin Phe Tyr Arg Ser Gin Tyr Lys Lys Glu Ala Ile Gly Leu Ala 260 265 270

Leu His Trp Thr Leu Lys Thr Leu Phe His Leu Phe Phe Met pro ser 275 280 285

Ile Leu Thr ser Leu Leu val Phe Phe val ser Glu Leu val Gly Gly 290 295 300

Phe Gly Ile Ala Ile val Val Phe Met Asn His Tyr Pro Leu Glu Lys 305 310 315 320 ile Gly Asp ser val Trp Asp Gly His Gly Phe Ser val Gly Gin Ile 325 330 335

His Glu Thr Met Asn Ile Arg Arg Gly Ile Ile Thr Asp Trp Phe Phe 340 345 350

Gly Gly Leu Asn Tyr Gin Ile Glu His His Leu Trp Pro Thr Leu Pro 355 360 365

Arg His Asn Leu Thr Ala val Ser Tyr Gin val Glu Gin Leu Cys Gin 370 375 380

Lys His Asn Leu Pro Tyr Arg Asn Pro Leu pro His Glu Gly Leu val 385 390 395 400

Ile Leu Leu Arg Tyr Leu Ala val Phe Ala Arg Met Ala Glu Lys Gin 405 410 415 pro Ala Gly Lys Ala Leu 420

<210> 54 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer PUD17-F <400> 54 aagatcccat ggcttcttcc actgttg 27

<210> 55 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer PUD17-R <400> 55 atcatcgcgg ccgcctagtt ggccttggtc ttg 33

<210> 56 <211> 12649 <212> DNA <213> Artificial Sequence <220>

<223> Plasmid PKUNF12T6E <220> <221 > misc_feature <222> (2507)..(2507) <223> n is a, c, g, or t <220> <221 > misc_feature <222> (2512)..(2515) <223> n is a, c, g, or t <400> 56 taaccctcac taaagggaac aaaagctgga gctccaccgc ggacacaata tctggtcaaa 60 tttcagtttc gttacataaa tcgttatgtc aaaggagtgt gggaggttaa gagaattatc 120 accggcaaac tatctgttaa ttgctaggta cctctagacg tccacccggg tcgcttggcg 180 gccgaagagg ccggaatctc gggccgcggt ggcggccgct tagttggtct tggacttctt 240 gggcttctte aggtaggact ggacaaagaa gttgccgaac agagcgagea gggtgatcat 300 gtacacgccg agcagctgga ccagagcctg agggtagtcg caggggaaga ggtagtcgta 360 cagggactgc accagcatag ccatgaactg ggtcatctgc agagtggtga tgtagggctt 420 gatgggcttg acgaagccga agccctgaga ggaaaagaag tagtaggcgt acatgacggt 480 gtggacgaag gagttgagga tgacggagaa gtaggcgtcg ccaccaggag cgtactcggc 540 aatagcccac cagatggcga agatggtggc atggtggtac acgtgcagga aggagacctg 600 gttgaacttc ttgcacagga tcatgatagc ggtgtccagg aactcgtagg ccttggagac 660 gtagaacacg tagacgattc gggacatgcc ctgagcgtgg gactcgttgc ccttctccat 720 gtcgttgccg aagaccttgt agccacccag gatagcctgt cggatggtct cgacgcacat 780 gtagagggac agtccgaaga ggaacaggtt gtggagcagc ttgatggtct tcagctcgaa 840 gggcttctcc atctgcttca tgatgggaat gccgaagagc agcatggcca tgtagccgac 900 ctcgaaggcg agcatggtgg agacgtccat catgggcaga ccgtcggtca gagcgtaggg 960 cttagctccg tccatccact ggtcgacacc ggtctcgact cgtccgacca cgtcgtccca 1020 gacagaggag ttggccatgg tgaatgattc ttatactcag aaggaaatgc ttaacgattt 1080 cgggtgtgag ttgacaagga gagagagaaa agaagaggaa aggtaattcg gggacggtgg 1140 tcttttatac ccttggctaa agtcccaacc acaaagcaaa aaaattttca gtagtctatt 1200 ttgcgtccgg catgggttac ccggatggcc agacaaagaa actagtacaa agtctgaaca 1260 agcgtagatt ccagactgca gtaccctacg cccttaacgg caagtgtggg aaccggggga 1320 ggtttgatat gtggggtgaa gggggctctc gccggggttg ggcccgctac tgggtcaatt 1380 tggggtcaat tggggcaatt ggggctgttt tttgggacac aaatacgccg ccaacccggt 1440 ctctcctgaa ttctgcatcg atcgaggaag aggacaagcg gctgcttctt aagtttgtga 1500 catcagtatc caaggcacca ttgcaaggat tcaaggcttt gaacccgtca tttgccattc 1560 gtaacgctgg tagacaggtt gatcggttcc ctacggcctc cacctgtgtc aatcttctca 1620 agctgcctga ctatcaggac attgatcaac ttcggaagaa acttttgtat gccattcgat 1680 cacatgctgg tttcgatttg tcttagagga acgcatatac agtaatcata gagaataaac 1740 gatattcatt tattaaagta gatagttgag gtagaagttg taaagagtga taaatagcgg 1800 ccgcgcctac ttaagcaacg ggcttgataa cagcgggggg ggtgcccacg ttgttgcggt 1860 tgcggaagaa cagaacaccc ttaccagcac cctcggcacc agcgctgggc tcaacccact 1920 ggcacatacg cgcactgcgg tacatggcgc ggatgaagcc acgaggacca tcctggacat 1980 cagcccggta gtgcttgccc atgatgggct taatggcctc ggtggcctcg tccgcgttgt 2040 agaaggggat gctgctgacg tagtggtgga ggacatgagt ctcgatgatg ccgtggagaa 2100 ggtggcggcc gatgaagccc atctcacggt caatggtagc agcggcacca cggacgaagt 2160 tccactcgtc gttggtgtag tggggaaggg tagggtcggt gtgctggagg aaggtgatgg 2220 caacgagcca gtggttaacc cagaggtagg gaacaaagta ccagatggcc atgttgtaga 2280 aaccgaactt ctgaacgagg aagtacagag cagtggccat cagaccgata ccaatatcgc 2340 tgaggacgat gagcttagcg tcactgttct cgtacagagg gctgcgggga tcgaagtggt 2400 taacaccacc gccgaggccg ttatgcttgc ccttgccgcg accctcacgc tggcgctcgt 2460 ggtagttgtg gccggtaaca ttggtgatga ggtagttggg ccagccnacg annnnctcag 2520 taagatgagc gagctcgtgg gtcatctttc cgagacgagt agcctgctgc tcgcgggttc 2580 ggggaacgaa gaccatgtca cgctccatgt tgccagtggc cttgtggtgc tttcggtggg 2640 agatttgcca gctgaagtag gggacaagga gggaagagtg aagaacccag ccagtaatgt 2700 cgttgatgat gcgagaatcg gagaaagcac cgtgaccgca ctcatgggca ataacccaga 2760 gaccagtacc gaaaagaccc tgaagaacgg tgtacacggc ccacagacca gcgcgggcgg 2820 gggtggaggg gatatattcg ggggtcacaa agttgtacca gatgctgaaa gtggtagtca 2880 ggaggacaat gtcgcggagg atataaccgt atcccttgag agcggagcgc ttgaagcagt 2940 gcttagggat ggcattgtag atgtccttga tggtaaagtc gggaacctcg aactggttgc 3000 cgtaggtgtc gagcatgaca ccatactcgg acttgggctt ggcgatatca acctcggaca 3060 tggacgagag cgatgtggaa gaggccgagt ggcggggaga gtctgaagga gagacggcgg 3120 cagactcaga atccgtcaca gtagttgagg tgacggtgcg tctaagcgca gggttctgct 3180 tgggcagagc cgaagtggac gccatggaga gctgggttag tttgtgtaga gagtgtgtgt 3240 tgctagcgac tttcggattg tgtcattaca caaaacgcgt cgtctcgaca ctgatcttgt 3300 cgtggatact cacggctcgg acatcgtcgc cgacgatgac accggacttt cgcttaagga 3360 cgtcagtaac aggcattgtg tgatgtgtag tttagatttc gaatctgtgg ggaaagaaag 3420 gaaaaaagag actggcaacc gattgggaga gccactgttt atatataccc tagacaagcc 3480 ccccgcttgt aagatgttgg tcaatgtaaa ccagtattaa ggttggcaag tgcaggagaa 3540 gcaaggtgtg ggtaccgagc aatggaaatg tgcggaaggc aaaaaaatga ggccacggcc 3600 tattgtcggg gctatatcca gggggcgatt gaagtacact aacatgacat gtgtccacag 3660 accctcaatc tggcctgatg agccaaatcc atacgcgctt tcgcagctct aaaggctata 3720 acaagtcaca ccaccctgct cgacctcagc gccctcactt tttgttaaga caaactgtac 3780 acgctgttcc agcgttttct gcctgcacct ggtgggacat ttggtgcaac ctaaagtgct 3840 cggaacctct gtggtgtcca gatcagcgca gcagttccga ggtagttttg aggcccttag 3900 atgatgcaat ggtgtcagtc gctggatcac gagtcttaat ggcagtattc gttcttattt 3960 gtgccattga gccccgttat cctcgtatct tctacccccc atcccatccc tttgttggtg 4020 caaccctacc catttattgt tgggtgcagc ccaaccgacg tggagagctt ggcttggcca 4080 tataaaaagg ccccccccta gtggcaatgg cagaaagtca gctgtgagtt gttgaatttg 4140 tcatctaggc ggcctggccg tcttctccgg ggcaattgtt cctctatagt actgcgtaca 4200 ctgtttaaac agtgtacgca gatctgcgac gacggaattc ctgcagccca tctgcagaat 4260 tcaggagaga ccgggttggc ggcgtatttg tgtcccaaaa aacagcccca attgccccaa 4320 ttgaccccaa attgacccag tagcgggccc aaccccggcg agagccccct tcaccccaca 4380 tatcaaacct cccccggtrc ccacacttgc cgttaagggc gtagggtact gcagtccgga 4440 atctacgctt gttcagactt tgtactagtt tctittgtctg gccatccggg taacccatgc 4500 cggacgcaaa atagactact gaaaattttt ttgctttgtg gttgggactt tagccaaggg 4560 tataaaagac caccgtcccc gaattacctt tcctcttctt ttctctctct ccttgtcaac 4620 tcacacccga aatcgttaag catttccttc tgagtataag aatcattcac catggctgcc 4680 gctccctctg tgcgaacctt tacccgagcc gaggttctga acgctgaggc tctgaacgag 4740 ggcaagaagg acgctgaggc tcccttcctg atgatcatcg acaacaaggt gtacgacgtc 4800 cgagagttcg tccctgacca tcctggaggc tccgtgattc tcacccacgt tggcaaggac 4860 ggcaccgacg tctttgacac ctttcatccc gaggctgctt gggagactct cgccaacttc 4920 tacgttggag acattgacga gtccgaccga gacatcaaga acgatgactt tgccgctgag 4980 gtccgaaagc tgcgaaccct gttccagtct ctcggctact acgactcctc taaggcctac 5040 tacgccttca aggtctcctt caacctctgc atctggggac tgtccaccgt cattgtggcc 5100 aagtggggtc agacctccac cctcgccaac gtgctctctg ctgccctgct cggcctgttc 5160 tggcagcagt gcggatggct ggctcacgac tttctgcacc accaggtctt ccaggaccga 5220 ttctggggtg atctcttcgg agccttcctg ggaggtgtct gccagggctt ctcctcttcc 5280 tggtggaagg acaagcacaa cactcaccat gccgctccca acgtgcatgg cgaggatcct 5340 gacattgaca cccaccctct cctgacctgg tccgagcacg ctctggagat gttctccgac S400 gtccccgatg aggagctgac ccgaatgtgg tctcgattca tggtcctgaa ccagacctgg 5460 ttctacttcc ccattctctc cttcgctcga ctgtcttggt gcctccagtc cattctcttt 5520 gtgctgccca acggtcaggc ccacaagccc tccggagctc gagtgcccat ctccctggtc 5580 gagcagctgt cccccgccat gcactggacc tggtacctcg ctaccatgtt cctgttcatc 5640 aaggatcctg tcaacatgct cgtgtacttc ctggtgtctc aggctgtgtg cggaaacctg 5700 ctcgccatcg tgttctccct caaccacaac ggtatgcctg tgatctccaa ggaggaggct 5760 gtcgacatgg atttctttac caagcagatc atcactggtc gagatgtcca tcctggactg 5820 ttcgccaact ggttcaccgg tggcctgaac taccagatcg agcatcacct gttcccttcc 5880 atgcctcgac acaacttctc caagacccag cctgccgtcg agaccctgtg caagaagtac 5940 aacgtccgat accacaccac tggtacgatc gagggaactg ccgaggtctt ctcccgactg 6000 aacgaggtct ccaaggccac ctccaagatg ggcaaggctc agtaagcggc cgcatgagaa 6060 gataaatata taaatacatt gagatattaa atgcgctaga ttagagagcc tcatactgct 6120 cggagagaag ccaagacgag tactcaaagg ggattacacc atccatatcc acagacacaa 6180 gctggggaaa ggttctatat acactttccg gaataccgta gtttccgatg ttatcaatgg 6240 gggcagccag gatttcaggc acttcggtgt ctcggggtga aatggcgttc ttggcctcca 6300 tcaagtcgta ccatgtcttc atttgcctgt caaagtaaaa cagaagcaga tgaagaatga 6360 acttgaagtg aaggaattta aattgccccg gagaagacgg ccaggccgec tagatgacaa 6420 attcaacaac tcacagctga ctttctgcca ttgccactag gggggggcct ttttatatgg 6480 ccaagccaag ctctccacgt cggttgggct geacccaaca ataaatgggt agggttgcac 6540 caacaaaggg atgggatggg gggtagaaga tacgaggata acggggctca atggcacaaa 6600 taagaacgaa tactgccatt aagactcgtg atccagcgac tgacaccatt gcatcateta 6660 agggcctcaa aactacctcg gaactgctgc gctgatctgg acaccacaga ggttccgagc 6720 actttaggtt gcaccaaatg tcccaccagg tgcaggcaga aaacgctgga acagcgtgta 6780 cagtttgtct taacaaaaag tgagggcgct gaggtcgagc agggtggtgt gacttgtmat 6840 agcctttaga gctgcgaaag cgcgtatgga tttggctcat caggccagat tgagggtctg 6900 .tggacacatg tcatgttagt gtacttcaat cgccccctgg atatagcccc gacaataggc 6960 cgtggcctca tttttttgcc ttccgcacat ttccattgct cggtacccac accttgcttc 7020 tcctgcactt gccaacctta atactggttt acattgacca acatcttaca agcggggggc 7080 ttgtctaggg tatatataaa cagtggctct cccaatcggt tgccagtctc ttttttcctt 7140 tctttcccca cagattcgaa atctaaacta cacatcacac aatgcctgtt actgacgtcc 7200 ttaagcgaaa gtccggtgtc atcgtcggcg acgatgtccg agccgtgagt atccacgaca 7260 agatcagtgt cgagacgacg cgttttgtgt aatgacacaa tccgaaagtc gctagcaaca 7320 cacactctct acacaaacta acccagctct ccatggagtc cattgctccc ttcctgccct 7380 ccaagatgcc tcaggacctg ttcatggacc tcgccagcgc tatcggtgtc cgagctgctc 7440 cctacgtcga tcccctggag gctgccctgg ttgcccaggc cgagaagtac attcccacca 7500 ttgtccatca cactcgaggc ttcctggttg ccgtggagtc tcccctggct cgagagctgc 7560 ctctgatgaa ccccttccac gtgctcctga tcgtgctcgc ctacctggtc accgtgtttg 7620 tgggtatgca gatcatgaag aactttgaac gattcgaggt caagaccttc tccctcctgc 7680 acaacttctg tctggtctcc atctccgcct acatgtgcgg tggcatcctg tacgaggctt 7740 atcaggccaa ctatggactg tttgagaacg ctgccgatca caccttcaag ggtctcccta 7800 tggccaagat gatctggctc ttctacttct ccaagatcat ggagtttgtc gacaccatga 7860 tcatggtcct caagaagaac aaccgacaga tttcctttct gcacgtgtac caccactctt 7920 ccatcttcac catctggtgg ctggtcacct tcgttgctcc caacggtgaa gcctacttct 7980 ctgctgccct gaactccttc atccacgtca tcatgtacgg ctactacttt ctgtctgccc 8040 tgggcttcaa gcaggtgtcg ttcatcaagt tctacatcac tcgatcccag atgacccagt 8100 tctgcatgat gtctgtccag tcttcctggg acatgtacgc catgaaggtc cttggccgac 8160 ctggataccc cttcttcatc accgctctgc tctggttcta catgtggacc atgctcggtc 8220 tcttctacaa cttttaccga aagaacgcca agctcgccaa gcaggccaag gctgacgctg 8280 ccaaggagaa ggccagaaag ctccagtaag cggccgcaag tgtggatggg gaagtgagtg 8340 cccggttctg tgtgcacaat tggcaatcca agatggatgg attcaacaca gggatatagc 8400 gagctacgtg gtggtgcgag gatatagcaa cggatattta tgtttgacac ttgagaatgt 8460 acgatacaag cactgtccaa gtacaatact aaacatactg tacatactca tactcgtacc 8520 cgggcaacgg tttcacttga gtgcagtggc tagtgctctt actcgtacag tgtgcaatac 8580 tgcgtatcat agtctttgat gtatatcgta ttcattcatg ttagttgcgt acgaagtcgt 8640 caatgatgtc gatatgggtt ttgatcatgc acacataagg fccgacctta tcggcaagct 8700 caatgagctc cttggtggtg gtaacatcca gagaagcaca caggttggtt ttcttggctg 8760 ccacgagcrc gagcactcga gcggcaaagg cggactrgtg gacgttagct cgagcttcgt 8820 aggagggcat tttggtggtg aagaggagac tgaaaraaat ttagtctgca gaacttttta 8880 tcggaacctt atctggggca gtgaagtata tgttatggta atagttacga gttagttgaa 8940 cttatagata gactggacta tacggctatc ggtccaaatt agaaagaacg tcaatggctc 9000 tctgggcgtc gcctttgccg acaaaaatgt gatcatgatg aaagccagca atgacgttgc 9060 agctgatatt gttgtcggcc aaccgcgccg aaaacgcagc tgtcagaccc acagcctcca 9120 acgaagaatg tatcgtcaaa gtgatccaag cacactcata gttggagtcg tactccaaag 9180 gcggcaatga cgagtcagac agatactcgt cgaccttttc cttgggaacc accaccgtca 9240 gcccttctga ctcacgtatt gtagccaccg acacaggcaa cagtccgtgg atagcagaat 9300 atgtcttgtc ggtccatttc tcaccaactt taggcgtcaa gtgaatgttg cagaagaagt 9360 atgtgccttc attgagaatc ggtgttgctg atttcaataa agtcttgaga tcagtttggc 9420 gcgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 9480 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 9540 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 9600 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 9660 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 9720 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 9780 gcgctctcct gttccgaccc tgccgettae cggatacctg tccgcctttc tcccttcggg 9840 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 9900 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 9960 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 10020 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 10080 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt 10140 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 10200 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 10260 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 10320 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 10380 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 10440 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 10500 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 10560 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 10620 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 10680 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 10740 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 10800 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 10860 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 10920 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 10980 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 11040 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 11X00 ttcggggcga aaactctcaa ggatcttacc gctgttgåga tccagttcga tgtaacccac 11160 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 11220 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 11280 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 11340 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 11400 aaaagtgcca cctgatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca 11460 tcaggaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag 11520 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac 11580 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga 11640 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc 11700 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg 11760 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa 11820 gaaagcgaaa ggagcgggcg ctagggcgcr ggcaagtgta gcggtcacgc tgcgcgtaac 11880 caccacaccc gccgcgctta atgcgccgct acagggcgcg tccattcgcc attcaggctg 11940 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa 12000 gggggatgtg ctgcaaggcg attaagttgg gtaaegccag ggttttccca gtcacgacgt 12060 tgtaaaacga eggceagtga attgtaatac gactcactat agggcgaatt gggcccgacg 12120 tcgcatgcag tggtggtatt gtgactgggg atgtagttga gaataagtca tacacaagtc 12180 agctttcttc gagcctcata taagtataag tagctcaacg tattagcact gtacccagca 12240 tctccgtatc gagaaacaca acaacatgcc ccattggaca gatcatgcgg atacacaggt 12300 Cgtgcagtat catacatact cgatcagaca ggtcgtctga ccatcataca agctgaacaa 12360 gcgctccata cttgcacgct ctctatatac acagttaaat tacatatcca tagtctaacc 12420 tctaacagtt aatcttctgg taagcctccc agccagcctt ctggtatcgc ttggcctcct 12480 caataggatc tcggttctgg ccgtacagac ctcggccgac aattatgata tccgttccgg 12S40 tagacatgac atcctcaaca gttcggtact gctgtccgag agcgtctccc ttgtcgtcaa 12600 gacccacccc gggggtcaga ataagccagt cctcagagtc gcccttaat 12649

<210> 57 <211> 819 <212> DNA <213> Thraustochytrium aureum <220> <221 > misc_feature <223> synthetic elongase (codon-optimized) <400> 57 atggccaact cctctgtctg ggacgacgtg gtcggacgag tcgagaccgg tgtcgaccag 60 tggatggacg gagctaagcc ctacgctctg accgacggtc tgcccatgat ggacgtctcc 120 accatgctcg ccttcgaggt cggctacatg gccatgctgc tcttcggcat tcccatcatg 180 aagcagatgg agaagccctt cgagctgaag accatcaagc tgctccacaa cctgttcctc 240 ttcggactgt ccctctacat gtgcgtcgag accatccgac aggctatcct gggtggctac 300 aaggtcttcg gcaacgacat ggagaagggc aacgagtccc acgctcaggg catgtcccga 360 atcgtctacg tgttctacgt ctccaaggcc tacgagttcc tggacaccgc tatcatgatc 420 ctgtgcaaga agttcaacca ggtctccttc ctgcacgtgt accaccatgc caccatcttc 480 gccatctggt gggctattgc caagtacgct cctggtggcg acgcctactt ctccgtcatc 540 ctcaactcct tcgtccacac cgtcatgtac gcctactact tcttttcctc tcagggcttc 600 ggcttcgtca agcccatcaa gccctacatc accactctgc agatgaccca gttcatggct 660 atgctggtgc agtccctgta cgactacctc ttcccctgcg actaccctca ggctctggtc 720 cagctgctcg gcgtgtacat gatcaccctg ctcgctctgt tcggcaactt ctttgtccag 780 tcctacctga agaagcccaa gaagtccaag accaactaa 819

<210> 58 <211 > 272 <212> PRT <213> Thraustochytrium aureum <400> 58

Met Ala Asn ser ser Val Trp Asp Asp val val Gly Arg Val Glu Thr 1 5 . 10 15

Gly Val Asp Gin Trp Met Asp Gly Ala Lys Pro Tyr Ala Leu Thr Asp 20 25 30

Gly Leu Pro Met Met Asp val ser Thr Met Leu Ala Phe Glu val Gly 35 40 45

Tyr Met Ala Met Leu Leu Phe Gly lie Pro lie Met Lys Gin Met Glu 50 55 60

Lys Pro Phe Glu Leu Lys Thr lie Lys Leu Leu His Asn Leu Phe Leu 65 70 75 80

Phe Gly Leu Ser Leu Tyr Met Cys val Glu Thr ile Arg Gin Ala lie 85 90 95

Leu Gly Gly Tyr Lys val Phe Gly Asn Asp Met Glu Lys Gly Asn Glu 100 105 110

Ser His Ala Gin Gly Met Ser Arg lie val Tyr val Phe Tyr val Ser 115 120 125

Lys Ala Tyr Glu Phe Leu Asp Thr Ala lie Met lie Leu Cys Lys Lys 130 135 140

Phe Asn Gin val Ser Phe Leu His val Tyr His His Ala Thr lie Phe 145 150 155 160

Ala lie Trp Trp Ala lie Ala tys Tyr Ala Pro Gly Gly Asp Ala Tyr 165 170 175

Phe Ser val lie Leu Asn Ser Phe Val His Thr Val Met Tyr Ala Tyr 180 185 190

Tyr Phe Phe Ser Ser Gin Gly Phe Gly Phe val Lys Pro lie Lys Pro 195 200 205

Tyr lie Thr Thr Leu Gin Met Thr Gin phe Met Ala Met Leu Val Gin 210 215 220

Ser Leu Tyr Asp Tyr Leu Phe Pro Cys Asp Tyr Pro Gin Ala Leu Val 225 230 235 240

Gin Leu Leu Gly val Tyr Met lie Thr Leu Leu Ala Leu Phe Gly Asn 245 250 255

Phe Phe Val Gin ser Tyr Leu Lys Lys Pro Lys Lys Ser Lys Thr Asn 260 265 270

<210> 59 <211 > 13034 <212> DNA <213> Artificial Sequence <220> <223> Plasmid pDMW271 <400> 59 cgatgcagaa ttcaggagag accgggttgg cggcgtattt gtgtcccaaa aaacagcccc 60 aattgcccca attgacccca aattgaccca gtagcgggcc caaccccggc gagagccccc 120 ttcaccccac atatcaaacc tcccccggtt cccacacttg ccgttaaggg cgtagggtac 180 tgcagtctgg aatctacgct tgttcagact ttgtactagt ttctttgtct ggccatccgg 240 gtaacccatg ccggacgcaa aatagactac tgaaaatttt tttgctttgt ggttgggact 300 ttagccaagg gtataaaaga ccaccgtccc cgaattacct ttcctcttct tttctctctc 360 tccttgtcaa ctcacacccg aaatcgttaa gcatttcctt ctgagtataa gaatcattca 420 ccatggatgg ctcccgaccc tgtcgctgcc gagaccgctg cccagggtcc cactccccga 480 tacttcacct gggacgaggt cgcccagcga tccggttgcg aggaacgatg gctggtcatc 540 gaccgaaagg tgtacaacat ctctgagttc acccgacgac atcccggtgg ctcccgagtg 600 atctcgcact acgctggaca ggacgccact gaccccttcg ttgcctttca cattaacaag 660 ggcctggtta agaagtacat gaactccctg ctcattggag agctgtctcc cgaacagcct 720 tcgtttgagc ctaccaagaa caaggagctg accgacgagt ttcgagagct ccgagccacc 780 gttgagcgaa tgggactgat gaaggccaac catgtcttct ttctgctcta cctgctccac 840 attcttctcc ttgacggagc tgcctggctt accctgtggg tcttcggcac ttcctttctg 900 ccctttcttc tctgcgccgt cctgctctcc gccgtgcagg ctcaggctgg ttggcttcag 960 catgactttg gtcacctttc cgtgttctct acctccaagt ggaaccacct gctccatcac 1020 ttcgtgatcg gccacctcaa gggtgctcct gcctcgtggt ggaaccacat gcatttccag 1080 caccatgcca agcccaactg ttttcgaaag gatcccgaca tcaacatgca ccccttcttt 1140 ttcgctcttg gcaagatcct gtccgtcgag ctcggaaagc agaagaagaa gtacatgccc 1200 tacaaccacc agcacaagta cttcttcctg attggacctc ccgctctcct gcctctttac 1260 tttcagtggt acatctttta ctttgttatt cagcgaaaga agtgggttga tcttgcctgg 1320 atgatcacct tctacgtccg attcttcctg acctacgtcc ctctccttgg actgaaggcc 1380 tttctcggtc tgttctttat cgtccgattc ctggagtcca actggttcgt gtgggtgacc 1440 cagatgaacc acattcccat gcacattgac catgatcgaa acatggactg ggtgtcgact 1500 cagctgcagg ccacctgcaa cgttcacaag tctgctttca acgactggtt ttccggtcac 1S60 ctcaactttc agattgagca ccatctgttt cccaccatgc ctcgacacaa ctaccacaag 1620 gttgctcccc tggtccagtc gctctgtgcc aagcatggca tcgagtacca gtecaagccc 1680 ctgctctctg ccttcgctga catcattcac tcgctgaagg aatctggcca gctctggctc 1740 gatgcctacc tgcaccagta agcggccgca ttgatgattg gaaacacaca catgggttat 1800 atctaggtga gagttagttg gacagttata tattaaatca gctatgccaa cggtaacttc 1860 attcatgtca acgaggaacc agtgactgca agtaatatag aatttgacca ccttgccatt 1920 ctcttgcact cctttactat atctcattta tttcttatat acaaatcact tcttcttccc 1980 agcatcgagc tcggaaacct catgagcaat aacatcgtgg atctcgtcaa tagagggctt 2040 tttggactcc ttgctgttgg ccaccttgtc cttgctgtct ggctcattct gtttcaacgc 2100 cttttaatta acggagtagg tctcggtgtc ggaagcgacg ccagatccgt catcctcctt 2160 tcgctctcca aagtagatac ctccgacgag ctctcggaca atgatgaagt cggtgccctc 2220 aacgtttcgg atgggggaga gatcggcgag cttgggcgac agcagctggc agggtcgcag 2280 gttggcgtac aggttcaggt cctttcgcag cttgaggaga ccctgctcgg gtcgcacgtc 2340 ggttcgtccg tcgggagcgg tccatacggt gttggcagcg cctccgacag caccgagcat 2400 aatagagtca gcctttcggc agatgtcgag agtagcgtcg gtgatgggct cgccctcctt 2460 ctcaatggca gctcctccaa tgagtcggtc ctcaaacaca aactcggtgc cggaggcctc 2520 agcaacagac ctgagcacct tgacggcctc ggcaaccacc tcggggccac agaagtcgcc 2580 gccgagaaga acaatcttct tggagtcagt cttggtcttc ttagtttcgg gttccattgt 2640 ggatgtgtgt ggttgtatgt gtgatgtggt gtgtggagtg aaaatctgtg gctggcaaac 2700 gctcttgtat atatacgcac ttttgcccgt gctatgtgga agactaaacc tccgaagatt 2760 gtgactcagg tagtgcggta tcggctaggg acccaaacct tgtcgatgcc gatagcatgc 2820 gacgtcgggc ccaattcgcc ctatagtgag tcgtattaca attcactggc cgtcgtttta 2880 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc 2940 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg 3000 cgcagcctga atggcgaatg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 3060 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt 3120 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc 3180 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg 3240 gtgatggttc acgnagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg 3300 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct 3360 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg 3420 agctgattta acaaaaatct aacgcgaart rcaacaaaat attaacgctt acaatttcct 3480 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatca ggtggcactt 3540 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 3600 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 3660 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 3720 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 3780 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 3840 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 3900 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 3960 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 4020 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 4080 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 4140 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 4200 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 4260 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 4320 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 4380 gcggtaccat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 4440 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 4S00 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagartgatt 4560 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 4620 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 4680 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 4740 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 4800 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag 4860 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 4920 cagtggctgc tgccagtggc gataagtcgt grcttaccgg gttggactca agacgatagt 4980 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 5040 agcgaacgac ctacaccgaa ctgagatacc racagcgtga gctatgagaa agcgccacgc 5100 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 5160 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 5220 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 5280 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 5340 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 5400 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 5460 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 5520 gcgcccactg agctcgtcta acggacttga tatacaacca attaaaacaa atgaaaagaa 5580 atacagttct ttgtatcatt tgtaacaatt accctgtaca aactaaggta ttgaaatcce 5640 acaatattcc caaagtccac ccctttccaa attgtcatgc ctacaactca tataccaagc 5700 actaacctac caaacaccac taaaacccca caaaatatat cttaccgaat atacagtaac 5760 aagctaccac cacactcgtt gggtgcagtc gccagcttaa agatatctat ccacatcagc 5820 cacaactccc ttcctttaat aaaccgacta cacccttggc tattgaggtt atgagtgaat 5880 atactgtaga caagacactt tcaagaagac tgtttccaaa acgtaccact gtcctccact S940 acaaacacac ccaatctgct tcttctagtc aaggttgcta caccggtaaa ttataaatca 6000 tcatttcatt agcagggcag ggcccttttt acagagtctt atacactagc ggaccctgcc 6060 ggtagaccaa cccgcaggcg cgrcagtttg ctccttccat caacgcgtcg tagaaacgac 6120 ttactccttc ttgagcagct ccttgacctt gttggcaaca agtctccgac ctcggaggtg 6180 gaggaagagc ctccgatatc ggcggtagtg ataccagcct cgacggactc cttgacggca 6240 .gcctcaacag cgtcaccggc gggcttcatg ttaagagaga acttgagcat catggcggca 6300 gacagaatgg tggcgtacgc aactaacatg aatgaatacg atatacatca aagactatga 6360 tacgcagtat tgcacactgt acgagtaaga gcactagcca ctgcactcaa gtgaaaccgt 6420 tgcccgggta cgagtatgag tatgtacagt atgtttagta ttgtacttgg acagtgcttg 6480 tatcgtacat tctcaagtgt caaacataaa tatccgttgc tatatcctcg caccaccacg 6540 tagctcgcta tatccctgtg ttgaatccat ccatcttgga ttgccaattg tgcacacaga 6600 accgggcact cacttcccca tccacacttg cggccgctta gctgcctact cttccttggg 6660 acggagtcca agaacacgca agtgctccaa atgtgaagca aatgcttgcc aaaacgtatc 6720 cttgacaagg tatggaacct tgtactcgct gcaggtgttc ttgatgatgg ccagaatatc 6780 gggataatgg tgctgcgaca cgttggggaa cagatggtgc acagcctggt agttcaagct 6840 gccagtgatg ctggtccaga ggtgcgaatc gtgtgcgtaa tcctgcgtag tctcgacctg 6900 catagctgcc cagtcctttt ggatgatccc gttctcgtca ggcaacggcc actgaacttc 6960 ctcaacaacg tggttcgcct ggaaggtcag cgecagceag taagacgaca ccatgtccgc 7020 gaccgtgaac aagagcagca ccttgcccag gggcagatac tgcaggggaa caatcaggcg 7080 ataccagaca aagaaagcct tgccgcccca gaacatcaca gtgtgccatg tcgagatggg 7140 attgacacga atagcgtcat tggtcttgac aaagtacaaa atgttgatgt cctgaatgcg 7200 caccttgaac gccagcagtc cgtacaggaa aggaacaaac atgtgctggt tgatgtggtt 7260 gacaaaccac ttttggttgg gcttgatacg acgaacatcg ggctcagacg tcgacacgtc 7320 gggatctgct ccagcaatgt tggtgtaggg gtgatggccg agcatatgtt ggtacatcca 7380 caccaggtac gatgctccgt tgaaaaagtc gtgcgtggct cccagaatct tccagacagt 7440 9999ttgtgg gtcactgaaa agtgagacgc atcatgaaga gggttgagtc cgacttgtgc 7500 gcacgcaaat cccatgatga ttgcaaacac cacctgaagc catgtgcgtt cgacaacgaa 7S60 aggcacaaag agctgcgcgt agtaggaagc gatcaaggat ccaaagataa gagcgtatcg 7620 tccccagatc tctggtctat tcttgggatc aatgttccga tccgtaaagt agccctcgac 7680 tctcgtcttg atggttttgt ggaacaccgt tggctccggg aagatgggca gctcattcga 7740 gaccagtgta ccgacatagt acttcttcat aatggcatct gcagccccaa acgcgtgata 7800 catctcaaag accggagtaa catctcggcc agctccgagc aggagagtgt ccactccacc 7860 aggatggcgg ctcaagaact ttgtgacatc gtacaccctg ccgcggatgg ccaagagtag 7920 gtcgtccttg gtgttatggg ccgccagctc ttcccaggtg aaggtttttc cttggtccgt 7980 tcccatggag agctgggtta gtttgtgtag agagtgtgtg ttgctagcga ctttcggatt 8040 gtgtcattac acaaaacgcg tcgtctcgac actgatcttg tcgtggatac tcacggctcg 8100 gacatcgtcg ccgacgatga caccggactt tcgcttaagg acgtcagtaa caggcattgt 8160 gtgatgtgta gtttagattt cgaatctgtg gggaaagaaa ggaaaaaaga gactggcaac 8220 cgattgggag agccactgtt tatatatacc ctagacaagc cccccgcttg taagatgttg 8280 gtcaatgtaa accagtatta aggttggcaa gtgcaggaga agcaaggtgt gggtaccgag 8340 caatggaaat gtgcggaagg caaaaaaatg aggccacggc ctattgtcgg ggctatatcc 8400 agggggcgat tgaagtacac taacatgaca tgtgtccaca gaccctcaat ctggcctgat 8460 gagccaaatc catacgcgct ttcgcagctc taaaggctat aacaagtcac accaccctgc 8520 tcgacctcag cgccctcact ttttgttaag acaaactgta cacgctgttc cagcgttttc 8580 tgcctgcacc tggtgggaca tttggtgcaa cctaaagtgc tcggaacctc tgtggtgtcc 8640 agatcagcgc agcagttccg aggtagtttt gaggccctta gatgatgcaa tggtgtcagt 8700 cgctggatca cgagtcttaa tggcagtatt cgttcttatt tgtgccattg agccccgtta 8760 tcctcgtatc ttctaccccc catcccatcc ctttgttggt gcaaccctac ccatttattg 8820 ttgggtgcag cccaaccgac gtggagagct tggcttggcc atataaaaag gcecccccct 8880 agtggcaatg gcagaaagtc agctgtgagt tgttgaattt gtcatctagg cggcctggcc 8940 gtcttctccg gggcaattta aattcctrca cttcaagttc attctrcatc tgcttctgtt 9000 ttactttgac aggcaaatga agacatggta cgacttgatg gaggccaaga acgccatttc 9060 accccgagac accgaagtgc ctgaaatcct ggctgccccc attgataaca tcggaaacta 9120 cggtattccg gaaagtgtat atagaacctt tccccagctt gtgtctgtgg atatggatgg 9180 tgtaatcccc tttgagtact cgtcttggct tctctccgag cagtatgagg ctctctaatc 9240 tagcgcattt aatatctcaa tgtatttata tatttatctt ctcatgcggc cgcttagctg 9300 cctactcttc cttgggacgg agtccaagaa cacgcaagtg ctccaaatgt gaagcaaatg 9360 cttgccaaaa cgtatccttg acaaggtatg gaaccttgta ctcgctgcag gtgttcttga 9420 tgatggccag aatatcggga taatggtgct gcgacacgtt ggggaacaga tggtgcacag 9480 cctggtagtt caagctgcca gtgatgctgg tccagaggtg cgaatcgcgt gcgtaatcct 9540 gcgtagtctc gacctgcata gctgcccagt ccttttggat gatcccgttc tcgtcaggca 9600 acggccactg aacttcctca acaacgtggt tcgcctggaa ggtcagcgcc agccagtaag 9660 acgacaccat gtccgcgacc gtgaacaaga gcagcacctt gcccaggggc agatactgca 9720 ggggaacaat caggcgatac cagacaaaga aagccttgcc gccccagaac atcacagtgt 9780 gccatgtcga gatgggattg acacgaatag cgtcattggt cttgacaaag tacaaaatgt 9840 tgatgtcctg aatgcgcacc ttgaacgcca gcagtccgca caggaaagga acaaacatgt 9900 gctggttgat gtggttgaca aaccactttt ggttgggctt gatacgacga acatcgggct 9960 cagacgtcga cacgtcggga tctgctccag caatgttggt gtaggggtga tggccgagca 10020 targcrggta catccacacc aggtacgatg ctccgttgaa aaagtcgtgc gtggctccca 10080 gaatcttcca gacagtgggg ttgtgggtca ctgaaaagtg agacgeatca tgaagagggt 10140 tgagtccgac ttgtgcgcac gcaaatccca tgatgattgc aaacaccacc tgaagccatg 10200 tgcgttcgac aacgaaaggc acaaagagct gcgcgtagta ggaagcgatc aaggatccaa 10260 agataagagc gtatcgtccc cagatctctg gtctattctt gggatcaatg ttccgatccg 10320 taaagtagcc ctcgactctc gtcttgatgg ttttgtggaa caccgttggc tccgggaaga 10380 tgggcagctc attcgagacc agtgtaccga catagtactt cttcataatg gcatctgeag 10440 ccccaaacgc gtgatacatc tcaaagaccg gagtaacatc tcggccagct ccgagcagga 10500 gagtgtccac tccaccagga tggcggctca agaactttgt gacatcgtac accctgccgc 10560 ggatggccaa gagtaggtcg tccttggtgt tatgggccgc cagctcttce caggtgaagg 10620 tttttccttg gtccgttccc atggtgaatg attcttatac tcagaaggaa atgcttaacg 10680 atttcgggtg tgagttgaca aggagagaga gaaaagaaga ggaaaggtaa ttcggggacg 10740 gtggtctttt atacccttgg ctaaagtccc aaccacaaag caaaaaaatt ttcagtagtc 10800 tattttgcgt ccggcatggg ttacccggat ggccagacaa agaaactagt; acaaagtctg 10860 aacaagcgta gattccagac tgcagtaccc tacgccctta acggcaagtg tgggaaccgg 10920 gggaggtttg atatgtgggg tgaagggggc tctcgccggg gttgggcccg ctactgggtc 10980 aatttggggt caattggggc aattggggct gttttttggg acacaaatac gccgccaacc 11040 cggtctctcc tgatcgatgg gctgcaggaa ttctacaata cgtgagtcag aagggctgac 11100 ggtggtggtt cccaaggaaa aggtcgacga gtatctgtct gactcgtcat tgecgccttt 11160 ggagtacgac tccaactatg agtgtgcttg gatcactttg acgatacatt cttcgttgga 11220 ggctgtgggt ctgacagctg cgttttcggc gcggttggcc gacaacaata tcagctgcaa 11280 cgtcattgct ggctttcatc atgatcacat ctttgtcggc aaaggcgacg cccagagagc 11340 cattgacgtt ctttctaatt tggaccgata gccgtatagt ccagtctatc tataagttca 11400 actaactcgt aactattacc ataacatata cttcactgcc ccagataagg ttccgataaa 11460 aagttctgca gactaaatct atttcagtct cctcttcacc accaaaatgc cctcctacga 11520 agctcgagct aacgtccaca agtccgcctt tgccgctcga gtgctcaagc tcgtggcagc 11580 caagaaaacc aacctgtgtg cttctctgga tgttaccacc accaaggagc tcattgagct 11640 tgccgataag gtcggacctt atgtgtgcat gatcaaaacc catatcgaca tcattgacga 11700 cttcacctac gccggcactg tgctccccct caaggaactt gctcttaagc acggtttctt 11760 cctgttcgag gacagaaagt tcgcagatat tggcaacact gtcaagcacc agtaccggtg 11820 tcaccgaatc gccgagtggc ccgatatcac caacgcccac ggtgtacccg gaaccggaat 11880 cattgctggc ctgcgagctg gtgccgagga aactgtctct gaacagaaga aggaggacgt 11940 ctctgactac gagaactccc agtacaagga gttcctagtc ccctctccca acgagaagct 12000 ggccagaggt ctgctcatgc tggccgagct gtcttgcaag ggctccctgg ccactggcga 12060 gtactccaag cagaccattg agcttgcccg atccgacccc gagtttgtgg ttggcttcat 12120 tgcccagaac cgacctaagg gcgactctga ggactggctt attctgaccc ccggggtggg 12180 tcttgacgac aagggagacg ctctcggaca gcagtaccga actgttgagg atgtcatgtc 12240 taccggaacg gatatcataa ttgtcggccg aggtctgtac ggccagaacc gagatcctat 12300 tgaggaggcc aagcgatacc agaaggctgg ctgggaggct taccagaaga ttaactgtta 12360 gaggttagac tatggatatg taatttaact gtgtatatag agagcgtgca agtatggagc 12420 gcttgttcag cttgtatgat ggtcagacga cctgtctgat cgagtatgta tgatactgca 12480 caacctgtgt atccgcatga tctgtccaat ggggcatgtt gttgtgtttc tcgatacgga 12540 gatgctgggt acagtgctaa cacgttgaac cacttatact tatatgaggc tcgaagaaag 12600 ctgacttgtg tatgacttat tctcaactac atccccagtc acaataccac cactgcacta 12660 ccactacacc agatctgcgt acactgttta aacggtaggt tagtgcttgg tatatgagtt 12720 gtaggcatga caatttggaa aggggtggac tttgggåata ttgtgggatt tcaatacctt 12780 agtttgtaca gggtaattgt tacaaatgat acaaagaact gtatttcttt tcatttgttt 12840 taattggttg tatatcaagt ccgttagacg agctcagtgc cttggctttt ggcaccgtar 12900 ttcattttta gaggtacact acattcagtg aggtatggta aggttgaggg cataatgaag 12960 gcaccttgta ctgacagtca cagacctctc accgagaatt ttatgagata tactcgggtt 13020 cattttaggc tcat 13034 <210> 60 <211 > 1335

<212> DNA <213> Homo sapiens <220> <221 > misc_feature <223> synthetic delta-5 desaturase (codon-optimized) <400> 60 atggctcccg accctgtcgc tgccgagacc gctgcccagg gtcccactcc ccgatacttc 60 acctgggacg aggtcgccca gcgatccggt tgcgaggaac gatggctggt catcgaccga 120 aaggtgtaca acatctctga gttcacccga cgacatcccg gtggctcccg agtgatctcg 180 cactacgctg gacaggacgc cactgacccc ttcgttgcct ttcacattaa caagggcctg 240 gttaagaagt acatgaactc cctgctcatt ggagagctgt ctcccgaaca gccttcgttt 300 gagcctacca agaacaagga gctgaccgac gagtttcgag agctccgagc caccgttgag 360 cgaatgggac tgatgaaggc caaccatgtc ttctttctgc tctacctgct ccacattctt 420 ctccttgacg gagctgcctg gcttaccctg tgggtcttcg gcacttcctt tctgcccttt 480 cttctctgcg ccgtcctgct ctctgccgtg caggctcagg ctggttggct tcagcatgac 540 tttggtcacc tttccgtgtt ctctacctcc aagtggaacc acctgctcca tcacttcgtg 600 atcggccacc tcaagggtgc tcctgcctcg tggtggaacc acatgcattt ccagcaccat 660 gccaagccca actgttttcg aaaggatccc gacatcaaca tgcacccctt ctttttcgct 720 cttggcaaga tcctgtccgt cgagctcgga aagcagaaga agaagtacat gccctacaac 780 caccagcaca agtacttctt cctgattgga cctcccgctc tcctgcctct ttactttcag 840 tggtacatct tttactttgt tattcagcga aagaagtggg ttgatcttgc ctggatgatc 900 accttctacg tccgattctt cctgacctac gtccctctcc ttggactgaa ggcctttctc 960 ggtctgttct ttatcgtccg attcctggag tccaactggt tcgtgtgggt gacccagatg 1020 aaccacattc ccatgcacat tgaccatgat cgaaacatgg actgggtgtc gactcagctg 1080 caggccacct gcaacgttca caagtctgct ttcaacgact ggttttccgg tcacctcaac 1140 tttcagattg agcaccatct gtttcccacc atgcctcgac acaactacca caaggttgct 1200 cccctggtcc agtcgctctg tgccaagcat ggcatcgagt accagtccaa gcccctgctc 1260 tctgccttcg ctgacatcat tcactcgctg aaggaatctg gccagctctg gctcgatgcc 1320 tacctgcacc agtaa 1335 <210>61 <211 > 444

<212> PRT <213> Homo sapiens <400> 61

Met Ala Pro Asp Pro val Ala Ala Glu Thr Ala Ala Gin Gly Pro Thr 15 10 15

Pro Arg Tyr Phe Thr Trp Asp Glu val Ala Gin Arg ser Gly Cys Glu 20 25 30

Glu Arg Trp Leu val ile Asp Arg Lys val Tyr Asn ile ser Glu phe 35 40 45

Thr Arg Arg His Pro Gly Gly ser Arg val ile Ser His Tyr Ala Gly 50 55 60

Gin Asp Ala Thr Asp Pro Phe val Ala Phe His Ile Asn Lys Gly Leu 65 70 75 80 val Lys Lys Tyr Met Asn ser Leu Leu ile Gly Glu Leu Ser Pro Glu 85 90 95

Gin pro Ser Phe Glu Pro Thr Lys Asn Lys Glu Leu Thr Asp Glu Phe 100 105 110

Arg Glu Leu Arg Ala thr val Glu Arg Met Gly Leu Met Lys Ala Asn 115 120 125

His Val Phe Phe Leu Leu Tyr Leu Leu His Ile Leu Leu Leu Asp Gly 130 135 140

Ala Ala Trp Leu Thr Leu Trp val Phe Gly Thr Ser phe Leu Pro Phe 145 150 155 160

Leu Leu Cys Ala val Leu Leu Ser Ala val Gin Ala Gin Ala Gly Trp 165 170 175

Leu Gin His Asp Phe Gly His Leu ser val Phe Ser Thr ser Lys Trp 180 185 190

Asn His Leu Leu His His Phe val ile Gly His Leu Lys Gly Ala pro 195 200 205

Ala ser Trp Trp Asn His Met His Phe Gin His His Ala Lys Pro Asn 210 215 220

Cys Phe Arg Lys Asp pro Asp Ile Asn Met His Pro Phe Phe Phe Ala 225 230 235 240

Leu Gly Lys Ile Leu ser val Glu Leu Gly Lys Gin Lys Lys Lys Tyr 245 250 255

Met Pro Tyr Asn His Gin His Lys Tyr Phe Phe Leu ile Gly Pro Pro 260 265 270

Ala Leu Leu Pro Leu Tyr Phe Gin Trp Tyr rle Phe Tyr Phe val Ile 275 280 285

Gin Arg Lys Lys Trp val Asp Leu Ala Trp Met ile Thr Phe Tyr val 290 295 300

Arg Phe Phe Leu Thr Tyr val Pro Leu Leu Gly Leu Lys Ala Phe Leu 305 310 315 320

Gly Leu Phe Phe Ile val Arg Phe Leu Glu Ser Asn Trp Phe val Trp 325 330 335 val Thr Gin Met Asn His ile Pro Met His ile Asp His Asp Arg Asn 340 345 350

Met Asp Trp Val Ser Thr Gin Leu Gin Ala Thr Cys Asn Val His Lys 355 360 365 ser Ala Phe Asn Asp Trp phe Ser Gly His Leu Asn Phe Gin ile Glu 370 375 380

His His Leu Phe Pro Thr Met Pro Arg His Asn Tyr His Lys val Ala 385 390 395 400

Pro Leu val Gin Ser Leu cys Ala Lys His Gly ile Glu Tyr Gin Ser 405 410 415

Lys Pro Leu Leu ser Ala phe Ala Asp Ile Ile His ser Leu Lys Glu 420 425 430

Ser Gly Gin Leu Trp Leu Asp Ala Tyr Leu His Gin 435 440

<210> 62 <211 >3800 <212> DNA <213> Artificial sequence <220> <223> Plasmid pPaD17S <400> 62 ggccgcatcg gatcccgggc ccgtcgactg cagaggcctg catgcaagct tggcgtaatc 60 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 120 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 180 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg 240 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct 300 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 360 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 420 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 480 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 540 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 600 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 660 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 720 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 780 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 840 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 900 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 960 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 1020 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 1080 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 1140 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 1200 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc 1260 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat 1320 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc 1380 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc 1440 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag 1500 ttcgccagtt aatagtttgc gcaacgttgt tgccattgcr acaggcatcg tggtgtcacg 1560 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg 1620 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag 1680 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt 1740 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 1800 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc 1860 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc 1920 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc 1980 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggeaaaatgc 2040 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 'ctcatactct tcctttttca 2X00 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat 2160 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt 2220 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt 2280 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac 2340 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc 2400 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga tcgtactgag 2460 agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag 2520 gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc 2580 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc 2640 agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattcgagc tcggtacctc 2700 gcgaatgcat ctagatccat ggcttcctct accgttgccg ctccctacga gttccctact 2760 ctcaccgaga tcaagcgatc cctgcctgcc cactgcttcg aagcctctgt tccctggtcc 2820 ctctactata ccgtgcgagc tctgggcatt gccggttccc ttgctctcgg actgtactat 2880 gctcgagccc ttgctatcgt gcaggagttt gcactgctcg atgccgtcct ttgcactggc 2940 tacattctgc tccagggtat cgtgttctgg ggattcttta ccatcggtca cgactgtgga 3000 catggtgcct tctcgcgatc ccacctgctc aacttctctg ttggcacact cattcactcc 3060 atcattctga ctccctacga gtcgtgg&amp;ag atcagccatc gacaccatca caagaacacc 3120 ggcaacatcg acaaggatga gatcttctac cctcagcgag aagccgactc tcatcccctg 3180 tcccgacaca tggtcatctc ccttggttcg gcttggtttg cctacctcgt cgctggattt 3240 cctccccgaa aggtcaacca cttcaatccc tgggagcctc tctacctgcg aagaatgtct 3300 gccgtcatca tttccctcgg ctctctcgtg gcctttgctg gtctgtacgc ctaccttacc 3360 tacgtctacg gcctcaagac catggctctg tattacttcg cacctctctt tggattcgcc 3420 accatgctgg ttgtcactae cttcctccat cacaacgacg aggaaactcc ctggtacgcc 3480 gattcggagt ggacctatgt caagggcaac ttgtcctctg tggaccgaag ctacggagcc 3540 ctcatcgaca acctgtccca caacattggt acacatcaga tccaccatct gtttcccatc 3600 attcctcact acaagctcaa cgaggccact gctgccttcg ctcaggcctt tcccgaactg 3660 gtgcgaaagt cggcttctcc catcattccc accttcatcc gaattggtct tatgtacgcc 3720 aagtacggcg tggtcgacaa ggatgccaag atgtttaccc tcaaggaggc caaggctgcc 3780 aagaccaaag ccaactaagc 3800

<210> 63 <211> 14655 <212> DNA <213> Artificial Sequence <220> <223> Plasmid pZKLeuN-29E3 <220> <221 > misc_feature <222> (8822)..(8822) <223> n is a, c, g, or t <220> <221 > misc_feature <222> (8827)..(8830) <223> n is a, c, g, or t <400> 63 cgattgttgt ctactaacta tcgtacgata acttcgtata gcatacatta tacgaagtta 60 tcgcgtcgac gagtatctgt ctgactcgtc attgccgcct ttggagtacg actccaacta 120 tgagtgtgct tggatcactt tgacgataca ttcttcgttg gaggctgtgg gtctgacagc 180 tgcgttttcg gcgcggttgg ccgacaacaa tatcagctgc aacgtcattg ctggctttca 240 tcatgatcac atttttgtcg gcaaaggcga cgcccagaga gccattgacg ttctttctaa 300 tttggaccga tagccgtata gtccagtcta tctataagtt caactaactc gtaactatta 360 ccataacata tacttcactg ccccagataa ggtcccgata aaaagttctg cagactaaat 420 ttatttcagt ctcctcttca ccaccaaaat gccctcctac gaagctcgag ctaacgtcca 480 caagtccgcc tttgccgctc gagtgctcaa gctcgtggca gccaagaaaa ccaacctgtg S40 tgcttctctg gatgttacca ccaccaagga gctcattgag cttgccgata aggtcggacc 600 ttatgtgtgc atgatcaaaa cccatatcga catcattgac gacttcacct acgccggcac 660 tgtgctcccc ctcaaggaac ttgctcttaa gcacggtttc ttcctgttcg aggacagaaa 720 gttcgcagat attggcaaca ctgtcaagca ccagtaccgg tgtcaccgaa tcgccgagtg 780 gtccgatatc accaacgccc acggtgtacc cggaaccgga atcattgctg gcctgcgagc 840 tggtgccgag gaaactgtct ctgaacagaa gaaggaggac gtctctgact acgagaactc 900 ccagtacaag gagttcctag tcccctctcc caacgagaag ctggccagag gtctgctcat 960 gctggccgag ctgtcrtgca agggctctct ggccactggc gagtactcca agcagaccat; 1020 tgagcttgcc cgatcegacc ccgagtttgt ggttggcttc attgcccaga accgacctaa 1080 gggcgactct gaggactggc ttattctgac ccccggggtg ggtcttgacg acaagggaga 1140 cgctctcgga cagcagtacc gaactgttga ggatgtcatg tctaccggaa cggatatcat 1200 aattgtcggc cgaggtctgt acggccagaa ccgagatcct attgaggagg ccaagcgata 1260 ccagaaggct ggctgggagg cttaccagaa gattaactgt tagaggttag actatggata 1320 tgtaatttaa ctgtgtatat agagagcgtg caagtatgga gcgcttgttc agcttgtatg 1380 atggtcagac gacctgtctg atcgagtatg tatgatactg cacaacctgt gtatccgcat 1440 gatctgtcca atggggcatg ttgttgtgtt tctcgatacg gagatgctgg gtacagtgct 1S00 aatacgttga actacttata cttatatgag gctcgaagaa agctgacttg tgtatgaett 1560 attctcaact acatccccag tcacaatacc accactgcac taccactaca ccaaaaccat 1620 gatcaaacca cccatggact tcctggaggc agaagaactt gttatggaaa agctcaagag 1680 agagatcata acttcgtata gcatacatca tacgaagtta tcctgcaggt aaaggaattc 1740 tggagtttct gagagaaaaa ggcaagatac gtatgtaaca aagcgacgca tggtacaata 1800 ataccggagg catgtatcat agagagttag tggttcgatg atggcactgg tgcctggtat 1860 gactttatac ggctgactac atatttgtcc tcagacatac aattacagtc aagcacttac 1920 ccttggacat ctgtaggtac cccccggcca agacgatctc agcgtgtcgt atgtcggatt 1980 ggcgtagctc cctcgctcgt caattggctc ccatctactt tcttctgctt ggctacaccc 2040 agcatgtctg ctatggctcg ttttcgtgcc ttatctatcc tcccagtatt accaactcta 2100 aatgacatga tgtgattggg tctacacttt catatcagag ataaggagta gcacagttgc 2160 ataaaaagcc caactctaat cagcttcttc ctttcttgta attagtacaa aggtgattag 2220 cgaaatctgg aagcttagtt ggccctaaaa aaatcaaaaa aagcaaaaaa cgaaaaacga 2280 aaaaccacag ttctgagaac agggaggtaa cgaaggatcg tatatatata tatatatata 2340 tatacccacg gatcccgaga ccggcctttg attcttccct acaaccaacc attctcacca 2400 ccctaattca caaccatgga gtctggaccc atgcctgctg gcattccctt ccctgagtac 2460 tatgacttct ttatggactg gaagactccc ctggccatcg ctgccaccta cactgctgcc 2520 gtcggtctct tcaaccccaa ggttggcaag gtctcccgag tggttgccaa gtcggctaac 2580 gcaaagcctg ccgagcgaac ccagtccgga gctgccatga ctgccttcgt ctttgtgcac 2640 aacctcattc tgtgtgtcta ctctggcatc accttctact acatgtttcc tgctatggte 2700 aagaacttcc gaacccacac actgcacgaa gcctactgcg acacggatca gtccctctgg 2760 aacaacgcac ttggctactg gggttacctc ttctacctgt ccaagttcta cgaggtcatt 2820 gacaccatca tcatcatcct gaagggacga cggtcctcgc tgcttcagac ctaccaccat 2880 gctggagcca tgattaccat gtggtctggc atcaactacc aagccacrcc catttggatc 2940 tttgtggtct tcaactcctt cattcacacc atcatgtact gttactatgc cttcacctct 3000 atcggattcc atcctcctgg caaaaagtac ctgacttcga tgcagattac tcagtttctg 3060 gtcggtatca ccattgccgt grcctacctc ttcgttcctg gctgcatccg aacacccggt 3120 gctcagatgg ctgtctggat caacgtcggc tacctgtttc ccttgaccta tctgttcgtg 3180 gactttgcca agcgaaccta ctccaagcga tctgccattg ccgctcagaa aaaggctcag 3240 taagcggccg cattgargat tggaaacaca cacatgggtt atatctaggt gagagttagt 3300 tggacagtta tatattaaat cagctatgcc aacggtaact tcattcatgt caacgaggaa 3360 ccagtgactg caagtaatat agaatttgac caccttgcca ttctcttgca ctcctttact 3420 atatctcatt tatttcttat atacaaatca cttcttcttc ccagcatcga gctcggaaac 3480 ctcatgagca ataacatcgt ggatctcgtc aatagagggc tttttggact ccttgctgtt 3540 ggccaccttg tccttgctgt ctggctcatt ctgtttcaac gccttttaat taacggagta 3600 ggtctcggtg tcggaagcga cgccagatcc gtcatcctcc tttcgctctc caaagtagat 3660 acctccgacg agctctcgga caatgatgaa gtcggtgccc tcaacgtttc ggatggggga 3720 gagatcggcg agcttgggcg acagcagctg gcagggtcgc aggttggcgt acaggttcag 3780 gtcctttcgc agcttgagga gaccctgctc gggtcgcacg tcggttcgtc cgtcgggagt 3840 ggtccatacg gtgttggcag cgcctccgac agcaccgagc ataatagagt cagcctttcg 3900 gcagatgtcg agagtagcgt cggtgatggg ctcgccctcc ttctcaatgg cagctcctcc 3960 aatgagtcgg tcctcaaaca caaactcggt gccggaggcc tcagcaacag acttgagcac 4020 cttgacggcc tcggcaatca cctcggggcc acagaagtcg ccgccgagaa gaacaatctt 4080 cttggagtca gtcttggtct tcttagtttc gggttccatt gtggatgtgt gtggttgtat 4140 gtgtgatgtg gtgtgtggag tgaaaatctg tggctggcaa acgctcttgt atatatacgc 4200 acttttgccc gtgctatgtg gaagactaaa cctccgaaga ttgtgactca ggtagtgcgg 4260 tatcggctag ggacccaaac cttgtcgatg ccgatagcat gcgacgtcgg gcccaattcg 4320 ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg tgactgggaa 4380 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt 4440 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa 4500 tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 4560 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 4620 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 4680 ttagtgcttt acggcacctc gaccccaaaa aactcgatta gggtgatggt tcacgtagtg 4740 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 4800 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt 4860 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat 4920 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc ctgatgcggt attttxtcct 4980 tacgcatctg tgcggtattt cacaccgcat caggtggcac ttttcgggga aatgtgcgcg 5040 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat 5100 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc 5160 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa 5220 cgctggtgaa agtaaaagat gctgaagatc agtrgggrgc acgagtgggt tacatcgaac 5280 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga 5340 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag 5400 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca 5460 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca S520 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa 5580 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 5640 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa 5700 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag 5760 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct 5820 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac 5880 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 5940 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 6000 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat 6060 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 6120 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 6180 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 6240 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 6300 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact 6360 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 6420 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 6480 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 6540 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 6600 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 6660 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 6720 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 6780 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 6840 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc 6900 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac 6960 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcgegcceac tgagctcgtc 7020 taacggactt gatatacaac caattaaaac aaatgaaaag aaatacagtt ctttgtatca 7080 tttgtaacaa ttaccctgta caaactaagg tattgaaatc ccacaatatt cccaaagtcc 7140 acccctttcc aaattgtcat gcctacaact catataccaa gcactaacct accaaacacc 7200 actaaaaccc cacaaaatat atcttaccga atatacagta acaagctacc accacactcg 7260 ttgggtgcag tcgccagctt aaagatatct atccacatca gccacaactc ccttccttta 7320 ataaaccgac tacacccttg gctattgagg ttatgagtga atatactgta gacaagacac 7380 tttcaagaag actgtttcca aaacgtacca ctgtcctcca ctacaaacac acccaatctg 7440 cttcttctag tcaaggttgc tacaccggxa aattataaat catcatttca ttagcagggc 7500 agggcccttt ttatagagtc ttatacacta gcggaccctg ccggtagacc aacccgcagg 7560 cgcgtcagtt tgctccttcc atcaatgcgt cgtagaaacg acttactcct tcttgagcag 7620 ctccttgacc ttgttggcaa caagtctccg acctcggagg tggaggaaga gcctccgata 7680 tcggcggtag tgataccagc ctcgacggac tccttgacgg cagcctcaac agcgtcaccg 7740 gcgggcttca tgrtaagaga gaacttgagc atcatggcgg cagacagaat ggtggcgtac 7800 gcaactaaca tgaatgaata cgatatacat caaagactat gatacgcagt attgcacact 7860 gtacgagtaa gagcactagc cactgcactc aagtgaaacc gttgcccggg tacgagtatg 7920 agtatgtaca gtatgtttag tattgtactt ggacagtgct tgtatcgtac attctcaagt 7980 gtcaaacata aatatccgtt gctatatcct cgcaccacca cgtagctcgc tatatccctg 8040 tgttgaatcc atccatcttg gattgccaat tgtgcacaca gaaccgggca ctcacttccc 8100 catccacact tgcggccgcg cctacttaag caacgggctt gataacagcg gggggggtgc 8160 ccacgttgtt gcggttgcgg aagaacagaa cacccttacc agcaccctcg gcaccagcgc 8220 tgggctcaac ccactggcac atacgcgcac tgcggtacat ggcgcggatg aagccacgag 8280 gaccatcctg gacatcagcc cggtagtgct tgcccatgat gggcttaatg gcctcggtgg 8340 cctcgtccgc gttgtagaag gggatgctgc tgacgtagtg gtggaggaca tgagtctcga 8400 tgatgccgtg gagaaggtgg cggccgatga agcccatctc acggtcaatg gtagcagcgg 8460 caccacggac gaagttccac tcgtcgttgg tgtagtgggg aagggtaggg tcggtgtgct 8S20 ggaggaaggt gatggcaacg agccagtggt taacccagag gtagggaaca aagtaccaga 8580 tggccatgtt gtagaaaccg aacttctgaa cgaggaagta cagagcagtg gccatcagac 8640 egataccaat atcgctgagg acgatgagct tagcgtcact gttctcgtac agagggctgc 8700 ggggatcgaa gtggttaaca ccaccgccga ggccgttatg cttgcccttg ccgcgaccct 8760 cacgctggcg ctcgtggtag ttgtggccgg taacattggt gatgaggtag ttgggccagc 8820 cnacgannnn ctcagtaaga tgagcgagct cgtgggtcat ctttccgaga cgagtagcct 8880 gctgctcgcg ggttcgggga acgaagacca tgtcacgctc catgttgcca gtggccttgt 8940 ggtgctttcg gtgggagatt tgccagctga agtaggggac aaggagggaa gagtgaagaa 9000 cccagccagt aatgtcgttg atgatgcgag aatcggagaa agcaccgtga ccgcactcat 9060 gggcaataac ccagagacca gtaccgaaaa gaccctgaag aacggtgtac acggcccaca 9120 gaccagcgcg ggcgggggtg gaggggatat attcgggggt cacaaagttg taccagatgc 9180 tgaaagtggt agtcaggagg acaatgtcgc ggaggatata accgtatccc ttgagagcgg 9240 agcgcttgaa gcagtgctta gggatggcat tgtagatgtc cttgatggta aagtcgggaa 9300 cctcgaactg gttgccgtag gtgtcgagca tgacaccata ctcggacttg ggcttggcga 9360 tatcaacctc ggacatggac gagagcgatg tggaagaggc cgagtggcgg ggagagtctg 9420 aaggagagac ggcggcagac tcagaatccg tcacagtagt tgaggtgacg gtgcgtctaa 9480 gcgcagggtt ctgcttgggc agagccgaag tggacgccat ggttgatgtg tgtttaattc 9540 aagaatgaat atagagaaga gaagaagaaa aaagattcaa ttgagccggc gatgcagacc 9600 cttatataaa tgttgccttg gacagacgga gcaagcccgc ccaaacctac gttcggtata 9660 atatgttaag ctttttaaca caaaggtttg gcttggggta acctgatgtg gtgcaaaaga 9720 ccgggcgttg gcgagccatt gcgcgggcga atggggccgt gactcgtctc aaattcgagg 9780 gcgtgcctca attcgtgccc ccgtggcttt ttcccgccgt rtccgccccg tttgcaccac 9840 tgcagccgct tctttggttc ggacaccttg ctgcgagcta ggtgccttgt gctacttaaa 9900 aagtggcctc ccaacaccaa catgacatga gtgcgtgggc caagacacgt tggcggggtc 9960 gcagtcggct caatggcccg gaaaaaacgc tgctggagct ggttcggacg cagtccgccg 10020 cggcgtatgg atatccgcaa ggttccatag cgccattgcc ctccgtcggc gtctatcccg 10080 caacctctaa atagagcggg aatataaccc aagcttcttt tttttccttt aacacgcaca 10140 cccccaacta tcatgttgct gctgctgttt gactctactc tgtggagggg tgctcccacc 10200 caacccaacc tacaggtgga tccggcgctg tgattggctg ataagtctcc tatccggact 10260 aattctgacc aatgggacat gcgcgcagga cccaaatgcc gcaattacgt aaccccaacg 10320 aaatgcctac ccctctttgg agcccagcgg ccccaaatcc ccccaagcag eccggttcta 10380 ccggcttcca tctccaagca caagcagccc ggttctaccg gcttccatct ccaagcaccc 10440 ctttctccac accccacaaa aagacccgtg caggacatcc tactgcgtcg acatcattta 10500 aattccttca cttcaagttc attcttcatc tgcttctgtt ttactttgac aggcaaatga 10560 agacatggta cgacttgatg gaggccaaga acgccatttc accccgagac accgaagtgc 10620 ctgaaatcct ggctgccccc attgataaca tcggaaacta cggtattccg gaaagtgtat 10680 atagaacctt tccccagctt gtgtctgtgg atatggatgg tgtaateecc tttgagtact 10740 cgtcttggcc tctctccgag cagtatgagg ctctctaatc tagcgcattt aatatctcaa 10800 tgtatttata tatttatctt ctcatgcggc cgctcactga atctttttgg ctcccttgtg 10860 cttcctgacg atatacgttt gcacatagaa attcaagaac aaacacaaga ctgtgccaac 10920 ataaaagtaa ttgaagaacc agccaaacat cctcatccca tcttggcgat aacagggaat 10980 gttcctgtac ttccagacaa tgtagaaacc aacattgaat tgaatgatct gcattgatgt 11040 aatcagggat tttggcatgg ggaacttcag cttgatcaat ctggtccaat aataaccgta 11100 catgatccag tggacgaaac cattcaacag cacaaaaatc caaacagctt catttcggta 11160 attatagaac agccacatat ccatcggtgc ccccaaatga tggaagaatt gcaaccaggt 11220 cagaggcttg cccatcagtg gcaaatagaa ggagtcaata tactccagga acttgctcaa 11280 acagaacaac tgcgtggtga tcctgaagac gttgttgtca aaagccttct cgcagttgtc 11340 agacataaca ccgatggtgt acatggcata tgccattgag aggaatgatc ccaacgaata 11400 aatggacatg agaaggttgt aattggtgaa aacaaacttc atacgagacc gaccttttgg 11460 accaaggggg ccaagagtga acttcaagat gacaaatgcg atggacaagt aaagcacctc 11520 acagtgactg gcatcactcc agagttgggc ataatcaact ggttgggtaa aacttcctgc 11580 ccaattgaga ctatttcatt caccacctcc atggccattg ctgtagatat gtcttgtgtg 11640 taagggggtt ggggtggttg tttgtgttct tgacttttgt gttagcaagg gaagacgggc 11700 aaaaaagtga gtgtggttgg gagggagaga cgagccttat atataatgct tgtttgtgtt 11760 tgtgcaagtg gacgccgaaa cgggcaggag ccaaactaaa caaggcagac aatgcgagct 11820 taattggatt gcctgatggg caggggttag ggctcgatca atgggggtgc gaagtgacaa 11880 aattgggaat taggttcgca agcaaggctg acaagacttt ggcccaaaca tttgtacgcg 11940 gtggacaaca ggagccaccc atcgtctgtc acgggctagc cggtcgtgcg tcctgtcagg 12000 ctccacctag gctccatgcc actccataca atcccactag tgtaccgcta ggccgctttt 12060 agctcccatc taagaccccc ccaaaacctc cactgtacag tgcactgtac tgtgtggcga 12120 tcaagggcaa gggaaaaaag gcgcaaacat gcacgcatgg aatgacgtag gtaaggcgtt 12180 actagactga aaagtggcac atttcggcgt gccaaagggt cctaggtgcg tttcgcgagc 12240 tgggcgccag gccaagccgc tccaaaacgc ctctccgact ccctccagcg gcctccatar 12300 ccccatccct ctccacagca atgttgttaa gccttgcaaa cgaaaaaata gaaaggctaa 12360 taagcttcca atattgtggt gtacgctgca taacgcaaca atgagcgcca aacaacacac 12420 acacacagca cacagcagca ttaaccacga rgaacagcat gacattacag gtgggtgtgt 12480 aatcagggcc ctgattgctg gtggtgggag cccccatcat gggcagatct gcgtacactg 12540 tttaaacagt gtacgcagat ctaetataga ggaacattta aattgccccg gagaagacgg 12600 ccaggccgcc tagatgacaa attcaacaac tcacagctga ctttctgcca ttgccactag 12660 9999999cct ttttatatgg ccaagccaag ctctccacgt cggttgggct gcacccaaca 12720 ataaatgggt agggttgcac caacaaaggg atgggatggg gggtagaaga tacgaggata 12780 acggggctca atggcacaaa taagaacgaa tactgccatt aagactcgtg atccagcgac 12840 tgacaccatt gcatcatcta agggcctcaa aactacctcg gaactgctgc gctgatctgg 12900 acaccacaga ggttccgagc actttaggtt gcaccaaatg tcccaccagg tgcaggcaga 12960 aaacgctgga acagcgtgta cagtttgtct taacaaaaag tgagggcgct gaggtegage 13020 agggtggtgt gacttgttat agcctttaga gctgcgaaag cgcgcatgga tttggctcat 13080 caggccagat tgagggtctg tggacacatg tcatgttagt gtacttcaat cgccccctgg 13140 atatagcccc gacaacaggc cgtggcctca tttttttgcc ttccgcacat ttccattgct 13200 cgatacccac accttgcttc tcctgcactt gccaacctta atactggttt acattgacca 13260 acatcttaca agcggggggc ttgtctaggg tatatataaa cagtggctct cccaatcggt 13320 tgccagtctc ttttttcctt tctttcccca cagattcgaa atctaaacta cacatcacag 13380 aattccgagc cgtgagtatc cacgacaaga tcagtgtcga gacgacgcgt tttgtgtaat 13440 gacacaatcc gaaagtcgct agcaacacac actctctaca caaactaacc cagctctggt 13500 accatggagg tcgtgaacga aatcgtctcc attggccagg aggttcttcc caaggtcgac 13560 tatgctcagc tctggtctga tgcctcgcac tgcgaggcgc tgtacctctc catcgccttc 13620 gtcatccrga agttcaccct tggtcccctc ggacccaagg gtcagtctcg aangaagttt 13680 gtgttcacca actacaacct gctcatgtcc atctactcgc tgggctcctt cctctctatg 13740 gcctacgcca tgtacaccat tggtgtcatg tccgacaact gcgagaaggc tttcgacaac 13800 aatgtcttcc gaatcaccac tcagctgttc tacctcagca agttcctcga gtacattgac 13860 tccttctatc rgcccctcat gggcaagcct ctgacctggt tgcagttctt tcaccatctc 13920 ggagctccta tggacatgtg gctgttctac aactaccgaa acgaagccgt ttggatcttt 13980 gtgctgctca acggcttcat tcactggatc atgtacggct actattggac ccgactgatc 14040 aagctcaagt tccctatgcc caagtccctg attacttcta tgcagatcat tcagttcaac 14100 gttggcttct acaccgtctg gaagtaccgg aacattccct gctaccgaca agatggaatg 14160 agaatgtttg gctggttttt caactacttc tacgttggta ctgtcctgtg tctgttcctc 14220 aacttctacg tgcagaccta catcgtccga aagcacaagg gagccaaaaa gattcagtga 14280 gcggccgcat gtacacacaa gattatttat agaaatgaat cgcgatcgaa caaagagtac 14340 gagtgtacga gtaggggatg atgataaaag tggaagaagt tccgcatctt tggatttatc 14400 aacgtgtagg acgatacttc ctgtaaaaat gcaatgtctt taccataggt tctgctgtag 14460 atgttattaa ctaccattaa catgtctact tgtacagttg cagaccagtt ggagtataga 14520 atggtacact taccaaaaag tgttgatggt tgtaactacg atatataaaa ctgttgacgg 14580 gatccccgct gatatgccta aggaacaatc aaagaggaag atattaattc agaatgctag 14640 tatacagtta gggat 14655 <210> 64 <211> 777

<212> DNA <213> Euglena gracilis <220> <221 > misc_feature <223> synthetic delta-9 elongase (codon-optimized) <300>

<302> DELTA-9 ELONGASES AND THEIR USE IN MAKING POLYUNSATURATED FATTY ACIDS <310> WO 2007/061742 <311> 2006-11-16 <312> 2007-05-31 <313> (1)..(777) <400> 64 atggaggtcg tgaacgaaat cgtctccatt ggccaggagg ttcttcccaa ggtcgactat 60 gctcagctct ggtctgatgc ctcgcactgc gaggtgctgt acctctccat cgccttcgtc 120 atcctgaagt tcacccttgg tcctctcgga cccaagggtc agtctcgaat gaagtttgtg 180 ttcaccaact acaacctgct catgtccatc tactcgctgg gctccttcct ctctatggcc 240 tacgccatgt acaccattgg tgtcatgtcc gacaactgcg agaaggcttt cgacaacaat 300 gtcttccgaa tcaccactca gctgttctac ctcagcaagt tcctcgagta cattgactcc 360 ttctatctgc ccctcatggg caagcctctg acctggttgc 'agttctttca ccatctcgga 420 gctcctatgg acatgtggct gttctacaac taccgaaacg aagccgtttg gatctttgtg 480 ctgctcaacg gcttcattca ctggatcatg tacggctact attggacccg actgatcaag S40 ctcaagttcc ctatgcccaa gtccctgatt acttctatgc agatcattca gttcaacgtt 600 ggcttctaca tcgtctggaa gtaccggaac attccctgct accgacaaga tggaatgaga 660 atgtttggct ggtttttcaa ctacttctac gttggtactg tcctgrgtct gttcctcaac 720 ttetacgtgc agacctacat cgtccgaaag cacaagggag ccaaaaagat tcagtga 777

<210> 65 <211 > 258 <212> PRT <213> Euglena gracilis <220> <221 > MISC_FEATURE <222> (1)..(258) <223> delta-9 elongase (EgD9e) <300>

<302> DELTA-9 ELONGASES AND THEIR USE IN MAKING POLYUNSATURATED FATTY ACIDS <310> WO 2007/061742 <311> 2006-11-16 <312> 2007-05-31 <313> (1)..(258) <400> 65

Met Glu Val Val Asn Glu Ile Val Ser Ile Gly GIn Glu Val Leu Pro 1 5 10 15

Lys val Asp Tyr Ala Gin Leu Trp ser Asp Ala Ser His Cys Glu Val 20 25 30

Leu Tyr Leu Ser lie Ala Phe val lie Leu Lys Phe Thr Leu Gly Pro 35 40 45

Leu Gly Pro Lys Gly Gin Ser Arg Met Lys Phe val Phe Thr Asn Tyr 50 55 60

Asn Leu Leu Met ser lie Tyr Ser Leu Gly ser Phe Leu ser Met Ala 65 70 75 80

Tyr Ala Met Tyr Thr lie Gly val Met ser Asp Asn cys Glu Lys Ala 85 90 95

Phe Asp Asn Asn val Phe Arg lie Thr Thr Gin Leu Phe Tyr Leu Ser 100 105 110

Lys Phe Leu Glu Tyr lie Asp Ser Phe Tyr Leu Pro Leu Met Gly Lys 115 120 125 pro Leu Thr Trp Leu Gin Phe Phe His His Leu Gly Ala Pro Met Asp 130 135 140

Met Trp Leu Phe Tyr Asn Tyr Arg Asn Glu Ala val Trp lie Phe val 145 150 155 160

Leu Leu Asn Gly Phe lie His Trp lie Met Tyr Gly Tyr Tyr Trp Thr 165 170 175

Arg Leu Ile Lys Leu Lys Phe Pro Met Pro Lys Ser Leu Ile Thr ser 180 185 190

Met Gin lie lie Gin Phe Asn val Gly Phe Tyr lie val Trp Lys Tyr 195 200 205

Arg Asn lie Pro Cys Tyr Arg Gin Asp Gly Met Arg Met Phe Gly Trp 210 215 220

Phe Phe Asn Tyr Phe Tyr val Gly Thr val Leu Cys Leu Phe Leu Asn 225 230 235 240 phe Tyr val Gin Thr Tyr lie val Arg Lys His Lys Gly Ala Lys Lys 245 250 255 lie Gin <210> 66 <211 > 34

<212> DNA <213> Escherichia coli <400> 66 ataacttcgt ataatgtatg ctatacgaag ttat 34

<210> 67 <211 > 828 <212> DNA <213> Mortierella alpina <220> <221 > CDS <222> (1)..(828) <223> synthetic C16/18 elongase (codon-optimized) <300>

<302> A MORTIERELLA ALPINA C16/18 FATTY ACID ELONGASE <310> US 2007/0087420-A1 <311> 2005-10-19 <312> 2007-04-19 <313> (1)..(828) <400> 67 atg gag tct gga ccc atg cct get ggc att ccc ttc cct gag tac tat 48

Met Glu ser Gly Pro Met Pro Ala Gly lie Pro Phe Pro Glu Tyr Tyr 1 5 10 ' 15 gac ttc ttt atg gac tgg aag act ccc ctg gcc ate get gee ace tac 96

Asp Phe Phe Met Asp Trp Lys Thr Pro Leu Ala Ile Ala Ala Thr Tyr 20 25 30 act get gcc gtc gat etc ttc aac ccc aag gtt ggc aag gtc tee ega 144

Thr Ala Ala val Giy Leu Phe Asn Pro Lys val GTy Lys val Ser Arg 35 40 45 gtg gtt gcc aag teg get aac gca aag cct gcc gag ega acc cag tee 192 vaT val Ala Lys ser Ala Asn Ala Lys Pro Ala Glu Arg Thr Gin ser 50 55 60 gga get gcc atg act gcc ttc gtc ttt gtg cac aac etc att ctg tgt 240

Gly Ala Ala Met Thr Ala Phe val Phe VaT His Asn Leu lie Leu Cys 65 70 75 80 gtc tac tet ggc ate acc ttc tac tac atg ttt cct get atg gtc aag 288 val Tyr ser GTy He Thr Phe Tyr Tyr Met Phe Pro Ala Met val Lys 85 90 95 aac ttc ega acc cac aca ctg cac gaa gee tac tgc gac aeg gat cag 336

Asn Phe Arg Thr His Thr Leu His Glu Ala Tyr Cys Asp Thr Asp Gin 100 105 110 tee etc tgg aac aac gca ett ggc tac tgg ggt tac etc ttc tac ctg 384

Ser Leu Trp Asn Asn Ala Leu GTy Tyr Trp GTy Tyr Leu Phe Tyr Leu 115 120 125 tee aag ttc tac gag gtc att gac acc ate ate ate ate ctg aag gga 432

Ser Lys Phe Tyr Glu val lie Asp Thr lie lie lie lie Leu Lys GTy 130 135 140 ega egg tee teg ctg ett cag acc tac cac cat get gga gcc atg att 480

Arg Arg Ser Ser Leu Leu Gin Thr Tyr His His Ala GTy Ala Met lie 145 150 155 160 acc atg tgg tet ggc ate aac tac caa gcc act ccc att tgg ate ttt 528

Thr Met Trp ser GTy He Asn Tyr Gin Ala Thr Pro lie Trp lie Phe 165 170 175 gtg gtc ttc aac tee ttc att cac acc ate atg tac tgt tac tat gcc 576

VaT val Phe Asn Ser Phe lie His Thr lie Met Tyr cys Tyr Tyr Ala 180 185 190 ttc acc tet ate gga ttc cat cct cct ggc aaa aag tac ctg act teg 624

Phe Thr Ser lie GTy Phe His Pro Pro GTy Lys Lys Tyr Leu Thr ser 195 200 205 atg cag att act cag ttt ctg gtc ggt ate acc att gcc gtg tee tac 672

Met Gin lie Thr Gin Phe Leu Val GTy lie Thr lie Ala VaT Ser Tyr 210 215 220 etc ttc gtt cct ggc tgc ate ega aca ccc ggt get cag atg get gtc 720

Leu Phe val Pro Gly cys lie Arg Thr Pro Giy Ala Gin Met Ala val 225 230 235 240 tgg ate aac gtc ggc tac ctg ttt ccc ttg acc tat ctg ttc gtg gac 768

Trp lie Asn val GTy Tyr Leu Phe Pro Leu Thr Tyr Leu Phe vaT Asp 245 250 255 ttt gee aag ega acc tac tee aag ega tet gcc att gcc get cag aaa 816

Phe Ala Lys Arg Thr Tyr ser Lys Arg ser Ala lie Ala Ala Gin Lys 260 265 270 aag get cag taa 828 n-./,a 75

Lys Ala Gin 275

<210> 68 <211 > 275 <212> PRT <213> Mortierella alpina <400> 68

Met Glu ser Gly Pro Met pro Ala Gly ile Pro Phe pro Glu Tyr Tyr 1 5 10 15

Asp Phe Phe Met Asp Trp Lys Thr Pro Leu Ala Ile Ala Ala Thr Tyr 20 25 30

Thr Ala Ala val Gly Leu Phe Asn Pro Lys val Gly Lys val Ser Arg 35 40 45 val val Ala Lys Ser Ala Asn Ala Lys Pro Ala Glu Arg Thr Gin ser 50 55 60

Gly Ala Ala Met Thr Ala Phe val Phe val His Asn Leu ile Leu cys 65 70 75 80

Val Tyr ser Gly ile Thr Phe Tyr Tyr Met Phe Pro Ala Met val Lys 85 90 95

Asn Phe Arg Thr His Thr Leu His Glu Ala Tyr Cys Asp Thr Asp Gin 100 105 110

Ser Leu Trp Asn Asn Ala Leu Gly Tyr Trp Gly Tyr Leu Phe Tyr Leu 115 120 125

Ser Lys Phe Tyr Glu val Ile Asp Thr ile ile ile ile Leu tys Gly 130 135 140

Arg Arg Ser Ser Leu Léu Gin Thr Tyr His His Ala Gly Ala Met ile 145 150 155 160

Thr Met Trp Ser Gly Ile Asn Tyr Gin Ala Thr Pro ile Trp ile Phe 165 170 175 val val Phe Asn Ser Phe Ile His Thr ile Met Tyr cys Tyr Tyr Ala 180 185 190

Phe Thr ser ile Gly Phe His Pro Pro Gly Lys Lys Tyr Leu Thr ser 195 200 205

Met Gin Ile Thr Gin Phe Leu val Gly Ile Thr ile Ala Val Ser Tyr 210 215 220

Leu Phe val Pro Gly Cys Ile Arg Thr Pro Gly'Ala Gln^Met Ala val 225 230 235 240

Trp ile Asn val Gly Tyr Leu Phe Pro Leu Thr Tyr Leu Phe Val Asp 245 250 255

Phe Ala Lys Arg Thr Tyr Ser Lys Arg ser Ala ile Ala Ala Gin Lys 260 265 270

Lys Ala Gin 275

<210> 69 <211 >8739 <212> DNA <213> Artificial Sequence <220> <223> Plasmid pY116 <400> 69 ggccgccacc gcggcccgag attccggcct cttcggccgc caagcgaccc gggtggacgt 60 ctagaggtac ctagcaatta acagatagtt tgccggtgat aattctctta acctcccaca 120 ctcctttgac ataacgattt atgtaacgaa actgaaattt gaccagatat tgtgtccgcg 180 gtggagctcc agcttttgtt ccctttagtg agggtttaaa cgagcttggc gtaatcatgg 240 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa cgtacgagcc 300 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg 360 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc 420 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact 480 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta S40 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 600 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 660 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 720 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 780 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 840 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 900 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 960 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 1020 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 1080 aggacagtat ttggtatctg cgctctgctg aagccagtta cettcggaaa aagagttggt 1140 agcccctgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 1200 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 1260 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 1320 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 1380 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 1440 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 1500 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 1560 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 1620 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 1680 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg 1740 tcgtttggta tggcttcatt cagctccggt tcceaacgat eaaggcgagt tacatgatcc 1800 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 1860 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 1920 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 1980 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaarac gggataatac cgcgccacar 2040 agcagaactt taaaagtgct catcattgga aaacgtrctt cggggcgaaa actctcaagg 2100 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 2160 gcatctttta cttteaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 2220 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 2280 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 2340 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgcgccc 2400 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 2460 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 2520 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 2580 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc 2640 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 2700 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt 2760 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 2820 tttaacaaaa tattaacgct tacaatttcc attcgccatt caggctgcgc aactgttggg 2880 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg 2940 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg 3000 ccagtgaatt gtaatacgac tcactatagg gcgaattggg taccgggccc cccctcgagg 3060 tcgatggtgt cgataagctt gatatcgaat tcatgtcaca caaaccgatc ttcgcctcaa 3120 ggaaacctaa ttctacatcc gagagactgc cgagatccag tctacactga ttaattttcg 3180 ggccaataat ttaaaaaaat cgtgttatat aatattatat gtattatata tatacatcat 3240 gatgatactg acagtcatgt cccattgcta aatagacaga ctccatctgc cgcctccaac 3300 tgatgttctc aatatttaag gggtcatctc gcattgttta ataataaaca gactccatct 3360 accgcctcca aatgatgttc tcaaaatata ttgtatgaac ttatttttat tacttagtat 3420 tattagacaa cttacttgct ttatgaaaaa cacttcctat ttaggaaaca atttataatg 3480 gcagttcgtt catttaacaa tttatgraga araaatgtta taaatgcgta tgggaaatct 3540 taaatatgga tagcataaat gatatctgca ttgcctaatt cgaaatcaac agcaacgaaa 3600 aaaatccctt gtacaacata aatagtcatc gagaaatatc aactatcaaa gaacagctat 3660 tcacacgtta ctattgagat tattattgga cgagaatcac acactcaact gtctttctct 3720 cttctagaaa tacaggtaca agtatgtact attctcattg ttcatacttc tagtcatttc 3780 atcccacata ttccttggat ttctctccaa tgaatgacat tctatcttgc aaattcaaca 3840 attataataa gatataccaa agtagcggta tagtggcaat caaaaagctt ctctggtgtg 3900 cttctcgtat ctatttttat tctaatgatc cattaaaggt atatatttat ttcttgttat 3960 ataatccttt tgtttattac atgggctgga tacataaagg tattttgatt taattttttg 4020 cttaaattca atcccccctc gttcagtgtc aactgtaatg gtaggaaatt accatacttt 4080 tgaagaagca aaaaaaatga aagaaaaaaa aaatcgtatt tccaggttag acgttccgca 4140 gaatctagaa tgcggtatgc ggtacattgt tcttcgaacg caaaagttgc gctccctgag 4200 atattgtaca tttttgcttt tacaagtaca agtacatcgt acaactatgt actactgttg 4260 atgcatccac aacagtttgt tttgtttttt tttgtttttt ttttttctaa tgattcatta 4320 ccgctatgta tacctacttg tacttgtagt aagccgggtt attggcgttc aattaatcat 4380 agacttatga atctgeacgg tgtgcgctgc gagttacttt tagcttatgc atgctacttg 4440 ggtgtaatat tgggatctgt tcggaaatca acggatgctc aaccgatttc gacagtaatt 4500 aattaatttg aatcgaatcg gagcctaaaa tgaacccgag tatatctcat aaaattctcg 4560 gtgagaggtc tgtgactgtc agtacaaggt gccttcatta tgccctcaac cttaccatac 4620 ctcactgaat gtagtgtacc tctaaaaatg aaatacagtg ccaaaagcca aggcactgag 4680 ctcgtctaac ggacttgata tacaaccaat taaaacaaat gaaaagaaat acagttcttt 4740 gtatcatttg taacaattac cctgtacaaa ctaaggtatt gaaatcccac aatattccca 4800 aagtccaccc ctttccaaat tgtcatgcct acaactcata taccaagcac taacctacca 4860 aacaccacta aaaccccaca aaatatatct taccgaatat acagtaacaa gctaccacca 4920 cactcgttgg gtgcagtcgc cagcttaaag atatctatcc acatcagcca caactccctt 4980 cctttaataa accgactaca cccttggcta ttgaggttat gagtgaatat actgtagaca S040 agacactttc aagaagactg tttccaaaac gtaccactgt cctccactac aaacacaccc 5100 aatctgcttc ttctagtcaa ggttgctaca ccggtaaatt ataaatcatc atttcattag 5160 cagggcaggg ccctttttat agagtcttat acactagcgg accctgccgg tagaccaacc 5220 cgcaggcgcg tcagtttgct ccttccatca atgcgtcgta gaaacgactt actccttctt 5280 gagcagctcc ttgaccttgt tggcaacaag tctccgacct cggaggtgga ggaagagcct 5340 ccgatatcgg cggtagtgat accagcctcg acggactcct tgacggcagc ctcaacagcg 5400 tcaccggcgg gcttcatgtt aagagagaac ttgagcatca tggcggcaga cagaatggtg 5460 gcaatggggt tgaccttctg cttgccgaga tcgggggcag atccgtgaca gggctcgtac 5520 agaccgaacg cctcgttggt gtcgggcaga gaagccagag aggcggaggg cagcagaecc 5580 agagaaccgg ggatgacgga ggcctcgtcg gagatgatat cgccaaacat gttggtggtg 5640 atgatgatac cattcatctt ggagggctgc ttgatgagga tcatggcggc cgagtcgatc 5700 agctggtggt tgagctcgag ctgggggaat tcgtccttga ggarccgagt gacagtcttt 5760 cgccaaagtc gagaggaggc cagcacgtrg gccttgtcaa gagaccacac gggaagaggg 5820 gggttgtgct gaagggccag gaaggcggcc attcgggcaa ttcgctcaac ctcaggaacg 5880 gagtaggtct cggtgtcgga agcgacgcca gatccgtcat cctcctttcg ctctccaaag S940 tagatacctc cgacgagctc tcggacaatg atgaagtcgg tgcccrcaac gtttcggatg 6000 ggggagagat cggcgagctt gggcgacagc agctggcagg gtcgcaggtt ggcgtacagg 6060 ttcaggtcct ttcgcagctt gaggagaccc tgctcgggtc gcacgtcggt tcgtccgtcg 6120 ggagtggtcc atacggtgtt ggcagcgcct ccgacagcac cgagcataat agagtcagcc 6180 tttcggcaga tgtcgagagt agcgtcggtg argggcccgc cctccctctc aacggcagct 6240 cctccaatga gtcggtcctc aaacacaaac tcggtgccgg aggcctcagc aacagacttg 6300 agcaccttga cggcctcggc aatcacctcg gggccacaga agtcgccgcc gagaagaaca 6360 atcttcttgg agtcagtctt ggtcttctta gtttcgggtt ccattgtgga tgtgtgtggt 6420 tgtatgtgtg atgtggtgtg tggagtgaaa atctgtggct ggcaaacgct crtgtatata 6480 tacgcacttt tgcccgtgct acgtggaaga ctaaacctcc gaagactgtg actcaggtag 6540 tgcggtatcg gctagggacc caaaccttgt cgatgccgat agcgctatcg aacgtacccc 6600 agecggccgg gagtatgtcg gaggggacat acgagatcgt caagggtttg tggccaactg 6660 gtatttaaat gtagctaacg gtagcaggcg aactactggt acatacctcc cccggaatat 6720 gtacaggcat aatgcgtatc tgtgggacat gtggtcgttg cgccattatg taagcagcgt 6780 gtactcccct gactgtccat atggtttgct ccatctcacc ctcatcgttt tcattgttca 6840 caggcggcca caaaaaaact gtcttctctc cttctctctt cgccttagtc tactcggacc 6900 agttttagtt tagcttggcg ccactggata aatgagacct caggccttgt gatgaggagg 6960 tcacttatga agcatgttag gaggtgcttg tatggataga gaagcaccca aaataataag 7020 aataataata aaacaggggg cgttgtcatt tcatatcgtg rtttcaccat caatacacct 7080 ccaaacaatg cccttcatgt ggccagcccc aatattgtcc tgtagttcaa ctctatgcag 7140 ctcgtatctt attgagcaag taaaactctg tcagccgata ttgcccgacc cgcgacaagg 7200 grcaacaagg tggtgtaagg ccttcgcaga agtcaaaact gtgccaaaca aacatctaga 7260 gtctctttgg tgtttctcgc atatatttwa tcggctgtct tacgtatttg cgcctcggta 7320 ccggactaat ttcggatcat ccccaatacg ctttttcttc gcagctgtca acagtgtcca 7380 tgatctatcc acctaaatgg gtcatatgag gcgtataatt tcgtggtgct gataataatt 7440 cccatatatt tgacacaaaa cttccccccc tagacataca tctcacaatc tcacttcttg 7500 tgcttctgtc acacatctcc tccagctgac ttcaactcac acctctgccc cagttggtct 7560 acagcggtat aaggtttctc cgcatagagg tgcaccactc ctcccgatac ttgtttgtgt 7620 gacttgtggg tcacgacata tatatctaca cacattgcgc caccctttgg ttcttccagc 7680 acaacaaaaa cacgacacgc taaecatggc caatttactg accgtacacc aaaatttgcc 7740 tgcattaccg gtcgatgcaa cgagtgatga ggttcgcaag aacctgatgg acatgttcag 7800 ggatcgccag gcgttttctg agcatacctg gaaaatgctt ctgtccgttt gccggtcgtg 7860 ggcggcatgg tgcaagttga ataaccggaa atggtttccc gcagaacctg aagatgttcg 7920 cgartatctt ctatatcttc aggcgcgcgg tctggcagta aaaactatcc agcaacattt 7980 gggccagcta aacatgcttc atcgtcggtc cgggctgcca cgaccaagtg acagcaatgc 8040 tgtttcactg gttatgcggc ggatccgaaa agaaaacgtt gatgccggtg aacgtgcaaa 8100 acaggctcta gcgttcgaac gcactgattt cgaccaggtt cgttcactca tggaaaatag 8160 cgatcgctgc caggatatac gtaatctggc atttetgggg attgcttata acaccctgtt 8220 acgtatagcc gaaattgcca ggatcagggt taaagatatc tcacgtactg acggtgggag 8280 aatgttaatc catattggca gaacgaaaac gctggttagc accgcaggtg tagagaaggc 8340 acttagcctg ggggtaacta aactggtcga gcgatggatt tccgtctctg gtgtagctga 8400 tgatccgaat aactacctgt tttgccgggt cagaaaaaat ggtgttgccg cgccatctgc 8460 caccagccag ctatcaactc gcgccctgga agggattttt gaagcaactc atcgattgat 8520 ttacggcgct aaggatgact ctggtcagag atacctggcc tggtctggac acagtgcccg 8580 tgtcggagcc gcgcgagata tggcccgcgc tggagtttca ataccggaga tcatgcaagc 8640 tggtggctgg accaatgtaa atattgtcat gaactatatc cgtaacctgg atagtgaaac 8700 aggggcaatg gtgcgcctgc tggaagatgg cgattaagc 8739 <210> 70

<211> 15304 <212> DNA <213> Artificial Sequence <220> <223> Plasmid pK02UF8289 <220> <221 > misc_feature <222> (5601)..(5601) <223> n is a, c, g, or t <220> <221 > misc_feature <222> (5606)..(5609) <223> n is a, c, g, or t <400> 70 cgatcgagga agaggacaag cggctgcttc ttaagtttgt gacatcagta tccaaggcac 60 cattgcaagg attcaaggct ttgaacccgt catttgccat tcgtaacgct ggtagacagg 120 ttgatcggtt ccctacggcc tccacctgtg tcaatcttct caagctgcct gactatcagg 180 acattgatca acttcggaag aaacttttgt atgccattcg atcacatgct ggtttcgatt 240 tgtcttagag gaacgcatat acagtaatca tagagaataa acgatattca tttattaaag 300 tagatagttg aggtagaagt tgtaaagagt gataaatagc ggccgctcac tgaatctttt 360 tggctccctt gtgcttcctg acgatatacg tttgcacata gaaattcaag aacaaacaca 420 agactgtgcc aacataaaag caattgaaga accagccaaa catcctcatc ccacctcggc 450 gataacaggg aatgttcctg tacttccaga caatgtagaa accaacattg aattgaatga 540 tctgcattga tgtaatcagg gattttggca tggggaactt cagcttgatc aatctggtcc 600 aataataacc gtacatgatc cagtggatga aaccattcaa cagcacaaaa atccaaacag 660 cttcatttcg gtaattatag aacagccaca tatccatcgg tgcccccaaa tgatggaaga 720 attgcaacca ggtcagaggc ttgcccatca gtggcaaata gaaggagtca atatactcca 780 ggaacttgct caaatagaac aactgcgtgg tgatcctgaa gacgttgttg tcaaaagcct 840 tctcgcagtt gtcagacata acaccgatgg tgtacatggc atatgccatt gagaggaatg 900 atcccaacga ataaatggac atgagaaggt tgtaattggt gaaaacaaac ttcatacgag 960 actgaccttt tggaccaagg gggccaagag tgaacttcaa gatgacaaat gcgatggaca 1020 agtaaagcac ctcacagtga ctggcatcac tccagagttg ggcataatca actggttggg 1080 taaaacttcc tgcccaattg agactatttc attcaccacc tccatggtta gcgtgtcgtg 1140 tttttgttgt gctggaagaa ccaaagggtg gcgcaatgtg tgtagatata tatgtcgtga 1200 cccacaagcc acacaaacaa gtatcgggag gagtggtgca cctctatgcg gagaaacctt 1260 ataccgctgt agaccaactg gggcagaggt gtgagttgaa gtcagctgga ggagatgtgt 1320 gacagaagca caagaagtga gattgtgaga tgtatgtcta gggggggaag ttttgtgtca 1380 aatatatggg aattattatc agcaccacga aattatacgc ctcatatgac ccatttaggt 1440 ggatagatca tggacactgt tgacagctgc gaagaaaaag cgtattgggg atgatccgaa 1500 attagtccgg taccgaggcg caaatacgta agacagccga twaaatatat gcgagaaaca 1560 ccaaagagac tctagatgtt tgtttggcac agttttgact tctgcgaagg ccttacacca 1620 ccttgttgac ccttgtcgcg ggtcgggcaa tatcggctga cagagtttta cttgctcaat 1680 aagatacgag ctgcatagag ttgaaccaca ggacaatatt ggggctggcc acatgaaggg 1740 cattgtttgg aggtgtattg atggtgaaaa cacgatatga aatgacaacg ccccctgttt 1800 tattattatt cttattattt tgggtgcttc tctatccata caagcacctc ctaacatgct 1860 tcataagcga cccccccarc acaaggcctg aggtctcatt catceagtgg cgccaagcta 1920 aactaaaact ggtccgagta gactaaggcgåagagagaag ’gagagaagac agtttttttg 1980 tggccgcctg tgaacaatga aaacgatgag ggtgagatgg agcaaaccat atggtttaaa 2040 cagtcagagg agtacaeget gcttacataa tggcgcaacg accacatgtc ccacagatac 2100 gcatcgattc gattcaaatt aattaaaagg cgttgaaaca gaatgagcca gacagcaagg 2160 acaaggtggc caacagcaag gagtccaaaa agccctctat tgacgagatc cacgatgtta 2220 ttgctcatga ggtttccgag ctcgatgctg ggaagaagaa gtgatttgta tataagaaat 2280 aaatgagata tagtaaagga gtgcaagaga atggcaaggt ggtcaaattc tatattactt 2340 gcagtcactg gttcctcgtt gacatgaatg aagttaccgt tggcatagct gatttaatat 2400 ataactgtcc aactaactct cacctagata taacccatgt gtgtgtttcc aarcatcaat 2460 gcggccgctt actgagcctt ggcaccgggc tgcttctcgg ccattcgagc gaactgggac 2520 aggtatcgga gcaggatgac gagaccttca tggggcagag ggtttcggta ggggaggttg 2580 tgcttctggc acagctgttc cacctggtag gaaacggcag tgaggttgtg tcgaggcagg 2640 gtgggccaga gatggtgctc gatctggtag ttcaggcctc caaagaacca gicagtaatg 2700 atgcctcgtc gaatgttcat ggtctcatgg auctgaccca cagagaagcc atgtccgtcc 2760 cagacggaat caccgatctt ctccagaggg tagtggttca tgaagaccac gatggcaatt 2820 ccgaagccac cgacgagctc ggaaacaaag aacaccagca tcgaggtcag gatggagggc 2880 ataaagaaga ggtggaacag ggtcttgaga gtccagtgca gagcgagtcc aatggcctct 2940 ttcttgtact gagatcggta gaactggttg tctcggtcct tgagggatcg aacggtcagc 3000 acagactgga aacaccagat gaatcgcagg agaatacaga tgaccaggaa atagtactgt 3060 tggaactgaa tgagctttcg ggagatggga gaagcrcgag tgacatcgtc ctcggaccag 3120 gcgagcagag gcaggttatc aatgtcggga tcgtgaccct gaacgttggt agcagaatga 3180 tgggcgttgt gtctgtcett ccaccaggtc acggagaagc cctggagtcc gttgccaaag 3240 accagaccca ggacgttatt ccagtttcgg ttcttgaagg tctggtggtg gcagatgtca 3300 tgagacagcc atcccatttg ctggtagtgc ataccgagca cgagagcacc aatgaagtac 3360 aggtggtact ggaccagcat gaagaaggca agcacgccaa gacccagggt ggtcaagatc 3420 ttgtacgagt accagagggg agaggcgtca aacatgccag tggcgatcag ctcttctcgg 3480 agctttcgga aatcctcctg agcttcgttg acggcagcct ggggaggcag ctcggaagcc 3540 tggttgatct tgggcattcg cttgagcttg tcgaaggctt cctgagagtg cacaaccatg 3600 aaggcgtcag-tagcatctcg tccctggtag ttctcaatga tttcagctcc accagggtgg 3660 aagttcaccc aagcggagac gtcgtacacc tttccgtcga tgacgagggg cagagcctgt 3720 cgagaagcct tcaccatggc cattgctgta gatatgtctt gtgtgtaagg gggttggggt 3780 ggttgtttgt gttcttgact tttgtgttag caagggaaga cgggcaaaaa agtgagtgtg 3840 gttgggaggg agagacgagc cttatatata atgcttgttt gtgtttgtgc aagtggacgc 3900 cgaaacgggc aggagccaaa ctaaacaagg cagacaatgc gagcttaatt ggattgcctg 3960 atgggcaggg gttagggctc gatcaatggg ggtgcgaagt gacaaaattg ggaattaggt 4020 tcgcaagcaa ggctgacaag actttggccc aaacatttgt acgcggtgga caacaggagc 4080 cacccatcgt ctgtcacggg ctagccggtc gtgcgtcctg tcaggctcca cctaggctcc 4140 atgccactcc atacaarccc actagtgtac cgcxaggccg cttttagctc ccatctaaga 4200 cccccccaaa acctccactg tacagtgcac tgtactgtgt ggcgatcaag ggcaagggaa 4260 aaaaggcgca aacatgcacg catggaatga cgtaggtaag gcgttactag actgaaaagt 4320 ggcacatttc ggcgtgccaa agggtcctag gtgcgtttcg cgagctgggc gccaggccaa 4380 gccgctccaa aacgcctctc cgactccctc cagcggcctc catatcccca tccctctcca 4440 cagcaatgtt gttaagcctt gcaaacgaaa aaatagaaag gctaataagc ttccaatatt 4500 gtggtgtacg ctgcataacg caacaatgag cgccaaacaa cacacacaca cagcacacag 4S60 cagcattaac cacgatgttt aaacagtgta cgcagatccc gtcaacagtt ttatatatcg 4620 tagttacaac catcaacact ttttggtaag tgtaccattc tatactccaa ctggtctgca 4680 actgtacaag tagacatgtt aatggtagtt aataacatct acagcagaac ctatggtaaa 4740 gacattgcat ttttacagga agtatcgtcc tacacgttga taaatccaaa gatgcggaac 4800 ttcttccact tttatcatca tcccctactc gtacactcgt actctttgtt cgatcgcgat 4860 tcatttctat aaataatctt gtatgtacat gcggccgcgc ctacttaagc aacgggcttg 4920 ataacagcgg ggggggtgcc cacgttgttg cggttgcgga agaacagaac acccttacca 4980 gcaccctcgg caccagcgct gggctcaacc cactggcaca tacgcgcact gcggtacatg 5040 gcgcggatga agccacgagg aecatcctgg acatcagccc ggtagtgctt gcccatgatg 5100 ggcttaatgg cctcggtggc ctcgtccgcg ttgtagaagg ggatgctgct gacgtagtgg S160 tggaggacat gagtctcgat gatgccgtgg agaaggtggc ggccgatgaa gcccatctca 5220 cggtcaatgg tagcagcggc accacggacg aagttccact cgtcgttggt gtagtgggga S280 agggtagggt cggtgtgctg gaggaaggtg atggcaacga gccagtggtt aacccagagg 5340 tagggaacaa agtaccagat ggccatgttg tagaaaccga acttctgaac gaggaagtac 5400 agagcagtgg ccatcagacc gataccaata tcgctgagga cgatgagctt agcgtcactg 5460 ttctcgtaca gagggctgcg gggatcgaag tggttaacac caccgccgag gccgttatgc 5520 ttgcccttgc cgcgaccctc acgctggcgc tcgtggtagt tgtggceggt aacattggtg 5580 atgaggtagt tgggccagcc nacgannnnc tcagtaagac gagcgagctc gtgggtcatc 5640 tttccgagac gagtagcctg ctgctcgcgg gttcggggaa cgaagaccat gtcacgctcc 5700 atgxxgccag tggccttgtg gtgctttcgg tgggagattt gccagctgaa gtaggggaca 5760 aggagggaag agtgaagaac ccagccagta atgtcgctga tgatgcgaga atcggagaaa 5820 gcaccgtgac cgcactcatg ggcaataacc cagagaccag taccgaaaag accctgaaga 5880 acggtgtaca cggcccacag accagcgcgg gcgggggtgg aggggatata ttcgggggtc 5940 acaaagttgt accagatgct gaaagtggta gtcaggagga caatgtcgcg gaggatataa 6000 ccgtatccct tgagagcgga gcgcttgaag cagtgcttag ggatggcatt gtagatgtcc 6060 ttgatggtaa agtcgggaac ctcgaactgg ttgccgtagg tgtcgagcat gacaccatac 6120 tcggacttgg gcttggcgat atcaacctcg gacatggacg agagcgatgt ggaagaggcc 6180 gagtggcggg gagagtctga aggagagacg gcggcagact cagaatccgt cacagtagtx 6240 gaggtgacgg tgcgtctaag cgcagggttc tgcttgggca gagccgaagt ggacgccatg 6300 gttgtgaatt agggtggtga gaatggttgg ttgtagggaa gaatcaaagg ccggtctcgg 6360 gatccgtggg tatatatata tatatatata tatacgatcc ttcgttacct ccctgttctc 6420 aaaactgtgg tttttcgttt ttcgtttttt gctttttttg atttttttag ggccaactaa 6480 gcttccagat ttcgctaatc acctttgtac taattacaag aaaggaagaa gctgattaga 6540 gttgggcttt ttatgcaact gtgctactcc ttatctctga tatgaaagtg tagacccaat 6600 cacateatgt eatttagagt tggtaatact gggaggatag ataaggcacg aaaacgagcc 6660 atagcagaca tgctgggtgt agccaagcag aagaaagtag atgggagcca attgacgagc 6720 gagggagcta cgccaatccg acatacgaca cgctgagacc gtcrtggccg gggggtacct 6780 acagatgtcc aagggtaagt gcttgactgt aattgtatgt ctgaggacaa atatgtagtc 6840 agccgtataa agtcatacca ggcaccagtg ccatcatcga accactaact ctctatgata 6900 catgcctccg gtattattgt accatgcgtc gctttgttac atacgtatct tgcctttttc 6960 tctcagaaac tccagacttt ggctattggt cgagataagc ccggaccata gtgagtcttt 7020 cacactctac atttctccct tgctccaact atttaaattg ccccggagaa gacggccagg 7080 ccgcctagat gacaaattca acaactcaca gctgactttc tgccattgcc actagggggg 7140 ggccttttta tatggccaag ccaagctctc cacgtcggtt gggctgcacc caacaataaa 7200 tgggtagggt tgcaccaaca aagggatggg atggggggta gaagatacga ggataacggg 7260 gctcaatggc acaaataaga acgaatactg ccattaagac tcgtgatcca gcgactgaca 7320 ccattgcatc atctaagggc ctcaaaacta cctcggaact gctgcgctga tctggacacc 7380 acagaggttc cgagcacttt aggttgcacc aaatgtccca ccaggtgcag gcagaaaacg 7440 ctggaacagc gtgtacagtt tgtcttaaca aaaagtgagg gcgctgaggt cgagcagggt 7500 ggtgtgactt gttatagcct ttagagctgc gaaagcgcgt atggatttgg ctcatcaggc 7560 cagattgagg gtctgtggac acatgtcatg ttagtgtact tcaatcgccc cctggatata 7620 gccccgacaa taggccgtgg cctcattttt ttgccttccg cacatttcca ttgctcggta 7680 cccacacctt gcttctcctg cacttgccaa ccttaatact ggtttacatt gaccaacatc 7740 ttacaagcgg ggggcttgtc tagggtatat ataaacagtg gctctcccaa tcggttgcca 7800 gtctcftttt tcctttcttt ccccacagat tcgaaatcta aactacacat cacagaattc 7860 cgagccgtga gtatccacga caagatcagt gtcgagacga cgcgttttgt gtaatgacac 7920 aatccgaaag ccgctagcaa cacacactct ctacacaaac taacccagct ctggtaccat 7980 ggtgaaggct tctcgacagg ctctgcccct cgtcatcgac ggaaaggtgt acgacgtctc 8040 cgcttgggtg aacttccacc ctggtggagc tgaaatcatt gagaactacc agggacgaga 6100 tgctactgac gccttcatgg ttatgcactc tcaggaagcc ttcgacaagc tcaagcgaat 8160 gcccaagatc aaccaggctt ccgagctgcc tccccaggct gccgtcaacg aagctcagga 8220 ggatttccga aagctccgag aagagctgat cgccactggc atgtttgacg cctctcccct 8280 ccggtactcg tacaagatct tgaccaccct gggtcttggc gtgcttgcct tcrccatgct 8340 ggtccagtac cacctgtact tcattggtgc tctcgtgctc ggtatgcact accagcaaat 8400 gggatggctg tctcatgaca tctgccacca ccagaccttc aagaaccgaa actggaataa 8460 cgtcctgggt etggtctttg gcaacggact ccagggcttc tccgtgacct ggtggaagga 8520 cagacacaac gcccatcatt ctgctaccaa cgttcagggt cacgatcccg acattgataa 8580 cctgcctctg ctcgcctggt ccgaggacga tgtcactcga gcttctccca tctcccgaaa 8640 gctcattcag ttccaacagt actatttcct ggtcarctgt attctcctgc gattcatctg 8700 gtgtttccag tctgtgctga ccgttcgatc cctcaaggac cgagacaacc agttctaccg 8760 atctcagtac aagaaagagg ccattggact cgctctgcac tggactctca agaccctgtt 8820 ccacctcttc tttatgccct ccatcctgac ctcgatgctg gtgttctttg tttccgagct 8880 cgtcggtggc ttcggaattg ccatcgtggt cttcatgaac cactaccctc tggagaagat 8940 cggtgattcc gtctgggacg gacatggctt ctctgtgggt cagatccatg agaccatgaa 9000 cattcgacga ggcatcatta ctgactggtt ctttggaggc ctgaactacc agatcgagca 9060 ccatctctgg cccaccctgc ctcgacacaa cctcactgcc gtttcctacc aggtggaaca 9120 gctgtgceag aagcaeaaec tcccctaccg aaaccctctg ccccatgaag gtctcgtcat 9180 cctgctccga tacctgtccc agttcgctcg aatggccgag aagcagcccg gtgccaaggc 9240 tcagtaagcg gccgcaagtg tggatgggga agtgagtgcc cggttctgtg tgcacaattg 9300 gcaatccaag atggatggat tcaacacagg gatatagcga gctacgtggt ggtgcgagga 9360 tatagcaacg gatatttatg tttgacactt gagaatgtac gatacaagca ctgtccaagt 9420 acaatactaa acatactgta catactcata ctcgtacccg ggcaacggtt tcacttgagt 9480 gcagtggcta gtgctcttac tcgtacagtg tgcaatactg cgtatcatag tctttgatgt 9540 atatcgtatt cattcatgtt agttgcgtac gggtgaagct tccactggtc ggcgtggtag 9600 tggggcagag tggggtcggt gtgctgcagg taggtgatgg ccacgagcca gtggttgacc 9660 cacaggtagg ggatcaggta gtagagggtg acggaagcca ggccccatcg gttgatggag 9720 tatgcgatga cggacatggt gataccaata ccgacgttag agatccagat gttgaaccag 9780 tccttcttct caaacagcgg ggcgttgggg ttgaagtggt tgacagccca tttgttgagc 9840 ttggggtact tctgtccggt aacgtaagac agcagataca gaggccatcc aaacacctgc 9900 tgggtgatga ggccgtagag ggtcacgagg ggagcgtcct cagcaagctc agaccagtca 9960 tgggcgcctc ggttctccat aaactccttt cggtccttgg gcacaaacac catatcacgg 10020 gtgaggtgac cagtggactt gtggtgcatg gagtgggtca gcttccaggc gtagtaaggg 10080 accagcatgg aggagtgcag aacccatccg gtgacgttgt tgacggtgtt agagtcggag 10140 aaagcagagt ggccacactc gtgggcaaga acccacagae cggtgccaaa cagaccctgg 10200 acaatggagt acatggccca ggccacagct cggccggaag ccgagggaat aagaggcagg 10260 tacgcgtagg ccatgtaggc aaaaacggcg ataaagaagc aggcgcgcca gctgcattaa 10320 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 10380 cteactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 10440 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 10500 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 10560 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 10620 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 10680 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 10740 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 10800 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 10860 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 10920 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 10980 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 11040 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 11100 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 11160 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 11220 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 11280 atatatgagt aaacttggtc tgacagttac eaatgcttaa tcagtgaggc acctatctca 11340 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 11400 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 11460 ccggccccag atttatcagc aaraaaccag ccagccggaa gggccgagcg cagaagtggt 11520 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 11580 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 11640 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 11700 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 11760 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 11820 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 11880 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 11940 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 12000 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 12060 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 12120 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 12180 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 12240 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgat 12300 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc 12360 gttaatattt tgttaaaatt cgcgttaaac ttttgttaaa tcagctcatt ttttaaccaa 12420 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt 12480 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg 12540 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt 12600 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga 12660 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg 12720 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg 12780 cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag 12840 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa 12900 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca 12960 gtgaattgta atacgactca ctatagggcg aattgggccc gacgtcgcat gcttgaatct 13020 acaagtagga gggttggagt gattaagtga aacttcttta acggctctat gccagttcta 13080 ttgatatccg aaacatcagt atgaaggtct gataagggtg acttcttccc acagattcgt 13140 atcagtacga gtacgagacc ggtacttgta acagtattga tactaaaggg aaactacaac 13200 ggttgtcagc gtaatgtgac ttcgcccatg aacgcagaca cgcagtgccg agtgcggtga 13260 tatcgcctac tcgttacgtc catggactac acaacccctc ggcttcgctt ggcttagcct 13320 cgggetcggt gctgttcagt taaaacacaa tcaaataaca tttctacttt ttagaaggca 13380 ggccgtcagg agcaaccccg actccattga cgtttctaaa catctgaatg ccttccttac 13440 cttcaacaaa ctggcaggtt cgggegacag tgtaaagaga cttgatgaag ttggtgtcgt 13500 cgtgtcggta gtgcttgccc atgaccttct tgatcttctc agtggcgatt cgggcgtcgt 13560 agaagggaat tcctttacct gcaggataac ttcgtataat gtatgctata cgaagttatg 13620 atctctctct tgagcttttc cataacaagt tcttctgcct ccaggaagtc catgggtggt 13680 ttgaccacgg tttcggtgta gtggtagtgc agtggtggta ttgtgactgg ggatgtagtt 13740 gagaataagt catacacaag tcagctttct tcgagcctca tataagtata agtagttcaa 13800 cgtattagca ctgtacccag catctccgta tcgagaaaca caacaacatg ccccattgga 13860 cagatcatgc ggatacacag gttgtgcagt atcatacata ctcgatcaga caggrcgtct 13920 gaccatcata caagctgaac aagcgctcca tacttgcacg ctctctatat acacagttaa 13980 attacatatc catagtctaa cctctaacag ttaatcttct ggtaagcctc ccagccagcc 14040 ttctggtatc gcttggcctc ctcaatagga tctcggttct ggccgtacag acctcggccg 14100 acaattatga tatccgttcc ggtagacatg acatcctcaa cagttcggta ctgctgtccg 14160 agagcgtctc ccttgtcgtc aagacccacc ccgggggtca gaataagcca gtcctcagag 14220 tcgcccttag gtcggttctg ggcaatgaag ccaaccacaa actcggggtc ggatcgggca 14280 agctcaatgg tctgcttgga gtactcgcca gtggccagag agcccttgca agacagctcg 14340 gccagcatga gcagacctct ggccagcttc tcgttgggag aggggactag gaactccttg 14400 tactgggagt tctcgtagtc agagacgtcc tccttcttct gttcagagac agtttcctcg 14460 gcaccagctc gcaggccagc aatgattccg gttccgggta caccgtgggc gttggtgata 14520 tcggaccact cggcgattcg gtgacaccgg tactggtgct tgacagtgtt gccaatatct 14580 gcgaactttc tgtcctcgaa caggaagaaa ccgtgcttaa gagcaagttc cttgaggggg 14640 agcacagtgc cggcgtaggt gaagtcgtca atgatgtcga tatgggtttt gatcatgcac 14700 acataaggtc cgaccttatc ggcaagctca atgagctcct tggtggtggt aacatccaga 14760 gaagcacaca ggttggtttt cttggctgcc acgagcttga gcactcgagc ggcaaaggcg 14820 gacttgtgga cgttagctcg agcttcgtag gagggcattt tggtggtgaa gaggagactg 14880 aaataaattt agtctgcaga actttttatc ggaaccttat etggggcagt gaagtatatg 14940 ttatggtaat agttacgagt tagttgaact tatagataga ctggactata cggctatcgg 15000 tccaaattag aaagaacgtc aatggctctc tgggcgtcgc ctttgccgac aaaaatgtga 15060 tcatgatgaa agccagcaat gacgttgcag ctgatattgt tgtcggccaa ccgcgccgaa 15120 aacgcagctg tcagacccac agcctccaac gaagaatgta tcgtcaaagt gatccaagca 15180 cactcatagt tggagtcgta ctccaaaggc ggcaatgacg agtcagacag atactcgtcg 15240 acgcgataac ttcgtataat gtatgctata cgaagttatc gtacgatagt tagtagacaa 15300 caat 15304

<210>71 <211> 1272 <212> DNA <213> Artificial Sequence <220> <223> mutant EgD8S-23 <220> <221 > misc_feature <222> (2)..(1270) <223> mutant EgD8S-23 delta-8 desaturase CDS <400> 71 catggtgaag gcttctcgac aggctctgcc cctcgtcatc gacggaaagg tgtacgacgt 60 ctccgcttgg gtgaacttcc accctggtgg agctgaaatc attgagaact accagggacg 120 agatgctact gacgccttca tggttatgca ctctcaggaa gccttcgaca agctcaagcg 180 aatgcccaag atcaaccagg cttccgagct gcctccccag gctgccgtca acgaagctca 240 ggaggatttc cgaaagctcc gagaagagct gatcgccact ggcatgtttg acgcctctcc 300 cctctggtac tcgtacaaga tcttgaccac cctgggtctt ggcgtgcttg ccttcttcat 360 gctggtccag taccacctgt acttcattgg tgctctcgtg ctcggtatgc actaccagca 420 aatgggatgg ctgtctcatg acatctgcca ccaccagacc ttcaagaacc gaaactggaa 480 taacgtcctg ggtctggtct ttggcaacgg actccagggc ttctccgtga cctggtggaa 540 ggacagacac aacgcccatc attctgctac caacgttcag ggtcacgatc ccgacattga 600 taacctgcct ctgctcgcct ggtccgagga cgatgtcact cgagcttctc ccatctcccg 660 aaagctcatt cagttccaac agtactattt cctggtcatc tgtattctcc tgcgattcat 720 ctggtgtttc cagtctgtgc tgaccgttcg atccctcaag gaccgagaca accagttcta 780 ccgatctcag tacaagaaag aggccattgg actcgctctg cactggactc tcaagaccct 840 gttccacctc ttctttatgc cctccatcct gacctcgatg ctggtgttct ttgtttccga 900 gctcgtcggt ggcttcggaa ttgccatcgt ggtcttcatg aaccactacc etctggagaa 960 gaccggtgat tccgtctggg acggacatgg cttctctgtg ggtcagatcc atgagaccat 1020 gaacactcga cgaggcatca ttactgactg gttctttgga ggcctgaact accagatcga 1080 gcaccatcte tggcccaccc tgcctcgaca caacctcact gccgtttcct accaggtgga 1140 acagctgtgc cagaagcaca acctccccta ccgaaaccct ctgccccatg aaggtctcgt 1200 catcctgctc cgatacctgt cccagttcgc tcgaatggcc gagaagcagc ccggtgccaa 1260 ggctcagtaa gc 1272

<210> 72 <211 > 422 <212> PRT <213> Artificial sequence <220> <223> mutant EgD8S-23, comprising M1, M2, M3, M8, M12, M15, M16, M18, M21, M26, M45, M46, M68 and M70 mutation sites <400> 72

Mer val Lys Ala Ser Arg Gin Ala Leu Pro Leu val Ile Asp Gly Lys 15 10 15 val Tyr Asp Val Ser Ala Trp val Asn Phe His Pro Gly Gly Ala Glu 20 25 30

Ile Ile Glu Asn Tyr Gin Gly Arg Asp Ala Thr Asp Ala Phe Met val 35 40 45

Met His Ser Gin Glu Ala Phe Asp Lys Leu Lys Arg Met pro Lys Ile 50 55 60

Asn Gin Ala ser Glu Leu pro Pro Gln’Ala Ala’val ÅsrTGlu Ala Gin 65 70 75 80

Glu Asp Phe Arg Lys Leu Arg Glu Glu Leu Ile Ala Thr Gly Met Phe 85 90 95

Asp Ala Ser Pro Leu Trp Tyr Ser Tyr Lys Ile Leu Thr Thr Leu Gly 100 105 110

Leu Gly val Leu Ala Phe Phe Met Leu val Gin Tyr His Leu Tyr Phe 115 120 125

Ile Gly Ala Leu Val Leu Gly Met His Tyr Gin Gin Met Gly Trp Leu 130 135 140 ser His Asp ile Cys His His Gin Thr phe Lys Asn Arg Asn Trp Asn 145 150 155 160

Asn val Leu Gly Leu Val Phe Gly Asn Gly Leu Gin Gly Phe Ser val 165 170 175

Thr Trp Trp Lys Asp Arg His Asn Ala His His ser Ala Thr Åsn Val 180 185 190

Gin Gly His Asp Pro Asp Ile Asp Asn Leu Pro teu Leu Ala Trp ser 195 200 205

Glu Asp Asp Val Thr Arg Ala ser Pro ile ser Arg Lys Leu Ile Gin 210 215 220

Phe Gin Gin Tyr Tyr Phe Leu Val ile cys ile Leu Leu Arg Phe Ile 225 230 235 240

Trp cys Phe Gin Ser Val Leu Thr val Arg ser Leu Lys Asp Arg Asp 245 250 255

Asn Gin Phe Tyr Arg Ser Gin Tyr Lys Lys Glu Ala Ile Gly Leu Ala 260 265 270

Leu His Trp Thr Leu Lys Thr Leu Phe His Leu Phe Phe Met Pro ser 275 280 2*5

Ile Leu Thr Ser Met Leu val Phe Phe val ser Glu Leu val Gly Gly 290 295 300

Phe Gly ile Ala ile Val val Phe Met Asn His Tyr Pro Leu Glu Lys 305 310 315 320

Ile Gly Asp Ser val Trp asp Gly His Gly Phe ser val Gly Gin ile 32S 330 335

His Glu Thr Met Asn Ile Arg Arg Gly Ile Ile Thr Asp Trp Phe Phe 340 345 350

Gly Gly Leu Asn Tyr Gin ile Glu His His Leu Trp Pro Thr Leu Pro 355 360 365

Arg His Asn Leu Thr Ala val ser Tyr Gin val Glu Gin Leu Cys Gin 370 375 380

Lys Kis Asn Leu Pro Tyr Arg Asn Pro Leu Pro His Glu Gly Leu Val 385 390 395 400

Ile Leu Leu Arg Tyr Leu ser Gin Phe Ala Arg Met Ala Glu Lys Gin 405 410 415

Pro Gly Ala Lys Ala Gin 420 <210> 73 <211> 777

<212> DNA <213> Euglena gracilis <220> <221 > misc_feature <223> delta-9 elongase <300>

<302> DELTA-9 ELONGASES AND THEIR USE IN MAKING POLYUNSATURATED FATTY ACIDS <310> WO 2007/061742 <311> 2006-11-16 <312> 2007-05-31 <313> (1)..(777) <400> 73 atggaggtgg tgaatgaaat agtctcaatt gggcaggaag ttttacccaa agttgattat 60 gcccaactct ggagtgatgc cagtcactgt gaggtgcttt acttgtccat cgcatrtgtc 120 atcttgaagt tcactcttgg cccccttggt ccaaaaggtc agtctcgtat gaagtttgtt 180 ttcaccaatt acaaccttct catgtccatt tattcgttgg gatcattcct ctcaatggca 240 tatgccatgt acaccatcgg tgttatgtct gacaactgcg agaaggcttt tgacaacaac 300 gtcttcagga tcaccacgca gttgttctat ttgagcaagt tcctggagta tattgactcc 360 ttctatttgc cactgatggg caagcctctg acctggttgc aattcttcca tcatttgggg 420 gcaccgatgg atatgtggct gttctataat taccgaaatg aagccgtttg gatttttgtg 480 ctgttgaatg gtttcatcca ctggatcatg tacggttatt attggaccag attgatcaag 540 ctgaagttcc ccatgccaaa atccctgatt acatcaatgc agatcattca attcaatgtt 600 ggtttctaca ttgtctggaa gtacaggaac attccctgtt atcgccaaga tgggatgagg 660 atgtttggct ggttcttcaa ttacttttat gttggcacag tcttgtgttt gttcttgaat 720 ttctatgtgc aaacgtatat cgtcaggaag cacaagggag ccaaaaagat tcagtga 777

<210> 74 <211 > 13707 <212> DNA <213> Artificial sequence <220> <223> Plasmid pZKSL-555R <400> 74 aaacagtgta cgcagatctg cccatgatgg gggctcccac caccagcaat cagggccctg 60 attacacacc cacctgtaat gtcatgctgt tcatcgtggt taatgctgct gtgtgctgtg 120 tgtgtgtgtt gtttggcgct cattgttgcg ttatgcagcg tacaccacaa tattggaagc 180 ttattagcct ttctattttt tcgtttgcaa ggcttaacaa cattgctgtg gagagggatg 240 gggatatgga ggccgctgga gggagtcgga gaggcgtttt ggagcggctt ggcctggcgc 300 ccagctcgcg aaacgcacct aggacccttt ggcacgccga aatgtgccac ttttcagtct 360 agtaacgcct tacctacgtc attccatgcg tgcatgtttg cgcctttttt cccttgccct 420 tgatcgccac acagtacagc gcactgtaca gtggaggttt tgggggggtc ttagatggga 480 gctaaaagcg gcctagcggt aeactagtgg gattgtatgg agtggcatgg agcctaggtg 540 gagcctgaca ggacgcacga ccggctagcc cgtgacagac gatgggtggc tcctgttgtc 600 caccgcgtac aaatgtctgg gccaaagtct tgtcagcctt gcttgcgaac ctaattccca 660 attttgtcac ttcgcacccc cattgatcga gccctaaccc ctgcccatca ggcaatccaa 720 ttaagctcgc attgtctgcc ttgtttagtt tggctcctgc ccgtttcggc gtccacttgc 780 acaaacacaa acaagcatta tatataaggc tcgtctctcc ctcccaacca cactcacttt 840 tttgcccgtc ttcccttgct aacacaaaag tcaagaacac aaacaaccac cccaaccccc 900 ttacacacaa gacatatcta cagcaatggc catggctctc tcccttacta ccgagcagct 960 gctcgagcga cccgacctgg ttgccatcga cggcattctc tacgatctgg aaggtcttgc 1020 caaggtccat cccggaggcg acttgatcct cgcttctggt gcctccgatg cttctcctct 1080 gttctactcc atgcaccctt acgtcaagcc cgagaactcg aagctgcttc aacagttcgt 1140 gcgaggcaag cacgaccgaa cctccaagga cattgtctac acctacgact ctccctttgc 1200 acaggacgtc aagcgaacta tgcgagaggt catgaaaggt cggaactggt atgccacacc 1260 tggattctgg ctgcgaaccg ttggcatcat tgctgtcacc gccttttgcg agtggcactg 1320 ggctactacc ggaatggtgc tgtggggtct cttgactgga ttcatgcaca tgcagatcgg 1380 cctgtccatt cagcacgatg cctctcatgg tgccatcagc aaaaagccct gggtcaacgc 1440 tctctttgcc tacggcatcg acgtcattgg atcgtccaga tggatctggc tgcagtctca 1500 catcatgcga catcacaccr acaccaatca gcatggtctc gacctggatg ccgagtccgc 1560 agaaccattc cttgtgttcc acaactaccc tgctgccaac actgctcgaa agtggtttca 1620 ccgattccag gcctggtaca tgtacctcgt gcttggagcc'tacggcgttt cgctggtgta 1680 caaccctctc tacatcttcc gaatgcagca caacgacacc attcccgagt ctgtcacagc 1740 catgcgagag aacggctttc tgcgacggta ccgaaccctt gcattcgtta tgcgagcttt 1800 cttcatcttt cgaaccgcct tcttgccctg gtatctcact ggaacctccc tgctcatcac 1860 cattcctctg gtgcccactg ctaccggtgc cttcctcacc ttctttttca tcttgtctca 1920 caacttcgat ggctcggagc gaatccccga caagaactgc aaggtcaaga gctccgagaa 1980 ggacgttgaa gccgatcaga tcgactggta cagagctcag gtggagacct cttccaccta 2040 cggtggaccc attgccatgt tctttactgg cggtctcaac ttccagatcg agcatcacct 2100 ctttcctcga atgtcgtctt ggcactatcc cttcgtgcag caagctgtcc gagagtgttg 2160 cgaacgacac ggagttcggt acgtcttcta ccctaccatt gtgggcaaca tcatttccac 2220 cctcaagtac atgcacaaag tcggtgtggt tcactgtgtc aaggacgctc aggattccta 2280 agcggccgca agtgtggatg gggaagtgag tgcccggttc tgtgtgcaca attggcaatc 2340 caagatggat ggattcaaca cagggatata gcgagctacg tggtggtgcg aggatatagc 2400 aacggatatt tatgtttgac acttgagaat gracgataca agcactgtcc aagtacaata 2460 ctaaacatac tgtacatact catactcgta cccgggcaac ggtttcactt gagtgcagtg 2520 gctagtgctc ttactcgtae agtgtgcaat actgcgtatc atagtctttg atgtatatcg 2580 tattcattca tgttagttgc gtacgctgtg ttgttgtatg tggtgaagct tgacaatgga 2640 tggtgtgtcg tatcaggctg gggaacaatt gtgcttaagt atgctgcagt tgagtaagag 2700 tcatcgctcc accaaaataa agtttgccat tagggttgga gagagagatg gtggctggaa 2760 gaattaaatg acatcaagct gaggattgtg ggtgtgcaat aacacatgtt aggggtgacc 2820 tgtggctcga aatctgataa ttattttgta actttatgat tattcttaga ttttttaata 2880 ttcctctata taacacataa gtagctgtcg tctagttgtt catagcctga ctcctgcaat 2940 agatcagtgc agagtgattt tgtgcaattg agagccacgg ttgagtcaag tgactttgtg 3000 tgtgaagtca tcttacgttt caagtctcac aggttactca attggttggt tgtctgccct 3060 ttacagatat ttacagtacc tgagcgtaaa gccgctcatc cacggaatga ctgttcctgt 3120 cacgcagtca tgatcatgga tgtggctggt caggaaccat cttggatagg agacttaggg 3180 attggactat tattgaaaaa actgagccga atatgatata gttctatttg aatgcagaac 3240 ttctgatggt caattcactt atttcaggca tatcggtcat ggtggcagct gccacgatgt 3300 tatctcgttg gaaacctcgg cgcgccagct gcattaatga atcggccaac gcgcggggag 3360 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 3420 cgttcggctg cggcgagcgg tatcagctca cccaaaggcg gtaatacggt tatccacaga 3480 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 3540 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 3600 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 3660 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 3720 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 3780 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 3840 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 3900 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 3960 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat 4020 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 4080 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 4140 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 4200 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 4260 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 4320 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 4380 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 4440 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 4500 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 4S60 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 4620 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 4680 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 4740 agcggttagc cccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 4800 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 4860 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 4920 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 4980 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 5040 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 5100 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 5160 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 5220 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 5280 ggttccgcgc acatttcccc gaaaagtgcc acctgatgcg gtgtgaaata cegcacagat 5340 gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt aatattttgt taaaattcgc 5400 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc 5460 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag 5520 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga 5580 tggcccacta cgtgaaecat caccctaatc aagttttttg gggtcgaggt gccgtaaagc 5640 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa 5700 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt 5760 agcggccacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc 5820 gtccattcgc cattcaggct; gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg 5880 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca 5940 gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta 6000 tagggcgaat tgggcccgac gtcgcatgca ttccatagcc acacctttgc ctatggcttc 6060 acaaccgaag gcaattcgag aggtcgcgct tatggaatcg actcgtataa agctgaaggg 6120 aaagggagac gttccgagcg ctcagatgca atagtcgtcc agctaatgtg gattcaaaaa 6180 caaccccaac agtaatcttg aaaatttgaa cggatcaatc tgaacactct tgctccaggt 6240 cattcttcta acgcacatcc ccagagtcta gagggagttg tgttgtgaac atcctaataa 6300 acaatgcaat ggattcggga tatcttctgt ctcgccccct actcgatgtc gagtaaaccg 6360 atcaccaact aacaatactc ctccgcgttc tgccattgac tctcaaacag acatcgctat 6420 caacggaaca gcatatttta gcttcttagg acaataaata ttgataatgc cggctctccc 6480 tcggtatatt aagcaatcca ttcatacact cattcatcag gttaatttta tatatataat 6540 ttgtctattc aaacaccgta aattactggt accatcatct cctccttttc aaatacacgt 6600 ctatttgcat taatgaaatt actcgccaat tcgcagaacg tgtttgtcga acagagcctt 6660 agctcgggtc cagacaggag cagtgtctcg ctgaggaagc tgcaggagag ttaattaact 6720 cacctgcagg attgagacta tgaatggatt cccgtgcccg tattactcta ctaatttgat 6780 cttggaacgc gaaaatacgt ttctaggact ccaaagaatc tcaactcttg tccttactaa 6840 atatactacc catagttgat ggtttacttg aacagagagg acatgttcac ttgacccaaa 6900 gtttctcgca tctcttggat atttgaacaa cggcgtccac tgaccgtcag ttatccagtc 6960 acaaaacccc cacattcata cattcccatg tacgtttaca aagttctcaa ttccatcgtg 7020 caaatcaaaa tcacatetat tcattcatca tatataaacc catcatgtct actaacactc 7080 acaactccat agaaaacatc gactcagaac acacgctcca tgcggccgct taggaatcct 7140 gtgcgtcctt cacgcagtgg acgacaccca ccttatgcat gtacttcagg gtggagatga 7200 tgttgccgac gåtggtaggg tagaaaacat atcgcactcc atgtcgttcg caacactccc 7260 ggaccgcctg ctggacgaag gggtagtgcc aagacgacat ccggggaaag aggtggtgct 7320 cgatctggaa attgagaccg ccagtgaaga acatggcgat ggggccaccg tatgtggagg 7380 acgtctccac ctgcgcccga taccagtcaa tttggtcagc ctcaacgtcc ttctcagatc 7440 gcttaacctt gcagttcttg tcggggatcc gttcggagcc atcaaaattg tgggacaaaa 7500 tgaagaagaa cgtcaagaag gcaccagttg cggxgggcac cagaggaatg gtgatcagca 7560 atgaggtccc agtgaggtac cagggcaaga atgcggtccg gaagatgaag aaagctcgca 7620 tcacgaatgc aagtgtgcgg tagcgccgca gaaagccatt ttcccgcatg gccgtgacag 7680 actctgggat ggtgtcattg tgctgcatcc ggaaaatgta gagcgggttg tacaccagcg 7740 ataccccgta tgcccccagc acaaggtaca tgtaccaagc ctggaagcgg tggaaccact 7800 ttcgggcggt gtttgcggcg gggtagttgt ggaacaccag gaacggctct gccgactccg 7860 catccaggtc gaggccgrgc tggttggtgr aggtgtggtg ccgcatgatg tgcgactgca 7920 gccaaatcca ccgggacgat ccgatgacgt caatgccgta ggcgaagagg gcgttgaccc 7980 aaggcttctt gctgatggcc ccgtgggacg catcatgctg gatggataag ccgatctgca 8040 tgtgcatgaa tccagtcaac aggccccaca gcaccatccc cgtggtagcc cagtgccact 8100 cgcaaaaggc cgtcacggcg atgatcccaa cggtgcgcag ccagaagcca ggggttgcgt 8160 accagttcct ccctttcatc acctcgcgca ttgtccgctt aacgtcttgt gcgaagggag 8220 aatcatacgt gtagacaatg tccttcgagg tgcggtcatg cttccctcgg acgaactgtt 8280 gaagcaattt ggagttctcc ggtttgacgt atggatgcat tgaataaaag agaggggagg 8340 catcagaggc accagaagcg agaatcaaat ctcctcctgg atgaactttg gcaagccctt 8400 caaggtcgta gaggatgcca tcaatcgcaa ccaaatcagg gcgttctaac agctgttctg 8460 tggtaagact gagagccatg gagagctggg ttagtttgtg tagagagtgt gtgttgctag 8520 cgactttcgg attgtgtcat tacacaaaac gcgtcgtctc gacactgatc ttgtcgtgga 8580 tactcacggc tcggacatcg tcgccgacga tgacaccgga ctttcgctta aggacgtcag 8640 taacaggcat tgtgtgatgt gtagtttaga tttcgaatct gtggggaaag aaaggaaaaa 8700 agagactggc aaccgattgg gagagccact gtttatatat accctagaca agccccccgc 8760 ttgtaagatg ttggtcaatg taaaccagta ttaaggttgg caagtgcagg agaagcaagg 8820 tgtgggtacc gagcaatgga aatgtgcgga aggcaaaaaa atgaggccac ggcctattgt 8880 CQ999Ctata tccagggggc gattgaagta cactaacatg acatgtgtcc acagaccctc 8940 aatctggcct gatgagccaa atccatacgc gctttcgcag ctctaaaggc tataacaagt 9000 cacaccaccc tgctcgacct cagcgccctc actttttgtt aagacaaact gtacacgctg 9060 ttccagcgtt ttctgcctgc acctggtggg acatttggtg caacctaaag tgctcggaac 9120 ctctgtggtg tccagatcag cgcagcagtt ccgaggtagt tttgaggccc ttagatgatg 9160 caatggtgtc agtcgctgga tcacgagtct taatggcagt attcgttctt atttgtgcca 9240 ttgagccccg ttatcctcgt atcttctacc ccccatccca tccctttgtt ggtgcaaccc 9300 tacccattta ttgttgggtg cagcccaacc gacgtggaga gcttggcttg gccatataaa 9360 aaggcccccc cctagtggca atggcagaaa gtcagctgtg agttgttgaa tttgtcatct 9420 aggcggcctg gccgtcttct ccggggcaat tggggctgtt ttttgggaca caaatacgcc 9480 gccaacccgg tctctcctga attccgtcgt cgcctgagtc gacatcattt atttaccagt 9540 tggccacaaa cccttgacga tctcgtatgt cccctccgac atactcccgg ccggctgggg 9600 tacgttcgat agcgctatcg gcatcgacaa ggtttgggtc ectagccgat accgcactac 9660 ctgagtcaca atctteggag gtttagtett ccacatagca cgggcaaåag tgegtatata 9720 tacaagagcg tttgccagcc aeagatttte actccacaca ccacatcaca catacaacca 9780 cacacatcca caatggaacc cgaaactaag aagaccaaga ctgactccaa gaagattgtt 9840 cttctcggcg gcgacttctg tggccccgag gtgattgccg aggccgtcaa ggtgctcaag 9900 tctgttgctg aggcctccgg caccgagttt gtgtttgagg accgactcac tggaggaget 9960 gccattgaga aggagggega gcccatcacc gacgctactc tcgacatctg ccgaaaggct 10020 gaetetatta tgctcggtgc tgteggagge gctgccaaca ccgtatggac cactcccgac 10080 ggaegaaeeg acgtgcgacc egageagggt ctcctcaagc tgcgaaagga cctgaacctg 10140 tacgccaacc tgcgaccctg ccagctgctg tcgcccaagc tcgccgatct ctcccccatc 10200 cgaaacgttg agggcaccga cttcatcatt gtccgagagc tegteggagg tatetaettt 10260 ggagagcgaa aggaggatga eggatetgge gtcgcttccg acaccgagac ctactccgtt 10320 cctgaggttg agcgaattgc ccgaatggcc gccttcctgg cccttcagca caacccccct 10380 cttcccgtgt ggtctcttga caaggccaac gtgctggcct cctctcgact ttggcgaaag 10440 actgtcactc gagtcctcaa ggacgaactc ccccagctcg agctcaacca ccagctgatc 10500 gactcggccg ccatgatcct catcaagcag ccctccaaga tgaatggtat catcatcacc 10560 accaacatgt ttggcgatat catctccgac gaggcctccg tcatccccgg ttctctgggt 10620 ctgctgccct ccgcctctct ggcttctctg cccgacacca aegaggegtt eggtetgtae 10680 gagccctgtc aeggatetge ccccgatctc ggcaageaga aggteaaeec cattgccacc 10740 attctgtctg ccgccatgat gctcaagttc tetettaaea tgaagcccgc eggtgaeget 10800 gttgaggctg ccgtcaagga gtccgtcgag gctggtatca ctaccgccga tateggagge 10860 tctccctcca cccccgaggt eggagaettg ttgccaacaa ggtcaaggag ctgctcaaga 10920 aggagtaagt egtttetaeg aegeaetgat ggaaggagca aaetgaegeg cctgcgggtt 10980 ggtctaccgg cagggtccgc tagtgtataa gaetetataa aaagggccct gccctgctaa 11040 tgaaatgatg atttataatt taccggtgta gcaaccttga ctagaagaag cagattgggt 11100 gtgtttgtag tggaggacag tggtacgttt tggaaacagt cttcttgaaa gtgtcttgtc 11160 taeagtatat tcactcataa cctcaatagc caagggtgta gteggtttat taaaggaagg 11220 gagttgtggc tgatgtggat ategatagtt ggagcaaggg agaaatgtag agtgtgaaag 11280 actcactatg gtccgggctt atctcgacca atagccaaag tctggagttt ctgagagaaa 11340 aaggeaagat acgtatgtaa caaagcgacg catggtacaa taataccgga ggcatgtatc 11400 atagagagtt agtggttega tgatggcact ggtgcctggt atgaetttat aeggetgaet 11460 acatatttgt cctcagacat acaattacag teaageaett: acccttggac atetgtaggt 11520 accccccggc caagacgatc teagegtgtre gtatgtcgga ttggcgtagc tccctcgctc 11580 gtcaattggc tcccatctac tttcttctgc ttggctacac ccagcatgtc tgctatggct 11640 egttttegtg ccttatctat cctcccagta ttaccaactc taaatgacat gatgtgattg 11700 ggtctacact ttcatatcag agataaggag tagcacagtt gcataaaaag cccaactcta 11760 atcagcttct tcctttcttg taattagtac aaaggtgatt agcgaaatct ggaagcctag 11820 ttggccctaa aaaaatcaaa aaaagcaaaa aacgaaaaac gaaaaaccac agttttgaga 11880 acagggaggt aacgaaggat cgtatatata tatatatata tatataccca cggatcccga 11940 gaccggcctt tgattcttcc ctacaaccaa ccattctcac caccctaatt cacaaccatg 12000 gctcccgacg ccgacaagct gcgacagcga aaggctcagt ccatccagga cactgccgat 12060 tctcaggcta ccgagctcaa gattggcacc ctgaagggtc tccaaggcac cgagatcgtc 12120 attgatggcg acatctacga catcaaagac ttcgatcacc ctggaggcga atccatcatg 12180 acctttggtg gcaacgacgt tactgccacc tacaagatga ttcatcccta ccactcgaag 12240 caccacctgg agaagatgaa aaaggtcggt cgagtgcccg actacacctc cgagtacaag 12300 ttcgatactc ccttcgaacg agagatcaaa caggaggtct tcaagattgt gcgaagaggt 12360 cgagagtttg gaacacctgg ctacttcttx cgagccttct gctacatcgg tctcttcttt 12420 tacctgcagt atctctgggt taccactcct accactttcg cccttgctat cttctacggt 12480 gtgtctcagg ccttcattgg cctgaacgtc cagcacgacg ccaaccacgg agctgcctcc 12540 aaaaagccct ggaccaacaa tttgctcggc ctgggtgccg actttatcgg aggctccaag 12600 tggctctgga tgaaccagca ctggacccax cacacttaca ccaaccatca cgagaaggat 12660 cccgacgccc tgggtgcaga gcctatgctg ctcctcaacg actatccctt gggtcacccc 12720 aagcgaaccc teattcatca cttccaagcc ttctactatc tgtttgtcct tgctggctac 12780 tgggtgtctt cggtgttcaa ccctcagatc ctggacetce agcaccgagg tgcccaggct 12840 gtcggcatga agatggagaa cgactacatt gccaagtctc gaaagtacgc tatcttcctg 12900 cgactcctgt acatctacac caacattgtg gctcccatcc agaaccaagg cttttcgctc 12960 accgtcgttg ctcacattct tactatgggt gtcgcctcca gcctgaccct cgctactctg 13020 ttcgccctct cccacaactt cgagaacgca gatcgggatc ccacctacga ggctcgaaag 13080 ggaggcgagc ctgtctgttg gttcaagtcg caggtggaaa cctcctctac ttacggtggc 13140 ttcatttccg gttgccttac aggcggactc aactttcagg tcgagcatca cctgtttcct 13200 cgaatgtcct ctgcctggta cccctacatc gctcctaccg ttcgagaggt ctgcaaaaag 13260 cacggcgtca agtacgccta ctatccctgg gtgtggcaga acctcatctc gaccgtcaag 13320 tacctgcatc agtccggaac tggctcgaac tggaagaacg gtgccaatcc ctactctggc 13380 aagctgtaag cggccgcatg tacatacaag attatttata gaaatgaatc gcgatcgaac 13440 aaagagtacg agtgtacgag taggggatga tgataaaagt ggaagaagtt ccgcatcttt 13500 ggatttatca acgtgtagga cgatacttcc tgtaaaaatg caatgtcttt accataggtt 13560 ctgctgcaga tgttattaac taccattaac atgtctaett gtacagttgc agaccagttg 13620 gagtatagaa tggcacactt accaaaaagt gttgatggtt gtaactacga tatataaaac 13680 tgttgacggg atctgcgtac actgttt ' 13707 <210> 75 <211 > 1350 <212> DNA <213> Euglena gracilis <220> <221 > misc_feature <223> synthetic delta-5 desaturase (codon-optimized) for Yarrowia lipolytica <400> 75 atggctctct cccttactac cgagcagctg ctcgagcgac ccgacctggt tgccatcgac 60 ggcattctct acgatctgga aggtcttgcc aaggtccatc ccggaggcga cttgatcctc 120 gcttctggtg cctccgatgc ttctcctctg ttctactcca tgcaccctta cgtcaagccc 180 gagaacccga agctgcttca acagttcgtg cgaggcaagc acgaccgaac ctccaaggac 240 attgtctaca cctacgactc tccctttgca caggacgtca agcgaactat gcgagaggtc 300 atgaaaggtc ggaactggta tgccacacct ggattctggc tgcgaaccgt tggcatcatt 360 gctgtcaccg ccttttgcga gtggcactgg gctactaccg gaatggtgct gtggggtctc 420 -ttgactggat tcatgcacat gcagatcggc ctgtccattc agcacgatgc ctctcatggt 480 gccatcagca aaaagccctg ggtcaacgct ctctttgcct acggcatcga cgtcattgga 540 tcgtccagat ggatctggct gcagtctcac atcatgcgac atcacaccta caccaatcag 600 catggtctcg acctggatgc cgagtccgca gaaccattcc ttgtgttcca caacraccct 660 gctgccaaca ctgctcgaaa gtggtttcac cgattccagg cctggtacat gtacctcgtg 720 cttggagcct acggcgtttc gctggtgtac aaccctctct acatcttccg aatgcagcac 780 aacgacacca ttcccgagtc tgtcacagcc atgegagaga acggctttct gcgacggtac 840 cgaacccttg cattcgttat gcgagctttc ttcatctttc gaaccgcctt cttgccctgg 900 tatctcactg gaacctccct gctcatcacc attcctctgg tgcccactgc taccggtgcc 960 ttcctcacct tctttttcat cttgtctcac aacttcgatg gctcggagcg aatccccgac 1020 aagaactgca aggtcaagag ctccgagaag gacgttgaag ccgatcagat cgactggtac 1080 agagctcagg tggagacctc ttccacctac ggtggaccca ttgccatgtt ctttactggc 1140 ggtctcaact tccagatcga gcatcacctc tttcctcgaa tgtcgtcttg gcactatccc 1200 ttcgtgcagc aagctgtccg agagtgttgc gaacgacacg gagttcggta cgtcttctac 1260 cctaccattg tgggcaacat catttccacc ctcaagtaca tgcacaaagt cggtgtggtt 1320 cactgtgtca aggacgctca ggattcctaa 1350

<210> 76 <211 > 449 <212> PRT <213> Euglena gracilis <400> 76

Met Ala Leu ser Leu Thr Thr Glu Gin Leu Leu Glu Arg Pro Asp Leu 15 10 15

Val Ala lie Asp Gly lie Leu Tyr Asp Leu Glu Gly Leu Ala Lys val 20 25 30

His Pro Gly Gly Asp Leu lie Leu Ala Ser Gly Ala ser Asp Ala ser 35 40 45

Pro Leu Phe Tyr Ser Met His Pro Tyr val Lys Pro Glu Asn ser Lys SO 55 60

Leu Leu Gin Gin Phe val Arg Gly Lys His Asp Arg Thr ser Lys Asp 65 70 75 80

He val Tyr Thr Tyr Asp Ser Pro Phe Ala Gin Asp val Lys Arg Thr 85 90 95

Met Arg Glu val Met Lys Gly Arg Asn Trp Tyr Ala Thr Pro Gly Phe 100 105 110 .

Trp Leu Arg Thr val Gly lie lie Ala val Thr Ala Phe cys Glu Trp IIS 120 125

His Trp Ala Thr Thr Gly Met val Leu Trp Gly Leu Leu Thr Gly Phe 130 135 140

Met His Met Gin lie Gly Leu ser lie Gin His Asp Ala ser His Gly 145 150 155 160

Ala lie ser Lys Lys Pro Trp val Asn Ala Leu Phe Ala Tyr Gly lie 165 170 175

Asp Val lie Gly ser Ser Arg Trp lie Trp Leu Gin Ser His lie Met 180 185 190

Arg His His Thr Tyr Thr Asn Gin His Gly Leu Asp Leu Asp Ala Glu 195 200 205 ser Ala Glu Pro Phe Leu val Phe His Asn Tyr Pro Ala Ala Asn Thr 210 215 220

Ala Arg Lys Trp Phe His Arg Phe Gin Ala Trp Tyr Met Tyr Leu val 225 230 235 240

Leu Gly Ala Tyr Gly val Ser Leu Val Tyr Asn Pro Leu Tyr lie Phe 245 250 255

Arg Met Gin His Asn Asp Thr lie Pro Glu Ser val Thr Ala Met Arg 260 265 '270

Glu Asn Gly Phe Leu Arg Arg Tyr Arg Thr Leu Ala Phe val Met Arg 275 280 285

Ala Phe Phe lie Phe Arg Thr Ala Phe Leu Pro Trp Tyr Leu Thr Gly 290 295 300

Thr Ser Leu Leu ile Thr He Pro Leu val Pro Thr Ala Thr Gly Ala 305 310 315 320

Phe Leu Thr Phe Phe Phe lie Leu Ser His Asn Phe Asp Gly Ser Glu 325 330 335

Arg He Pro Asp Lys Asn cys Lys Val Lys Ser Ser Glu Lys Aisp val 340 345 350

Glu Ala Asp Gin lie Asp Trp Tyr Arg Ala Gin val Glu Thr ser ser 355 360 365

Thr Tyr Gly Gly Pro lie Ala Met Phe Phe Thr Gly Gly Leu Asn Phe 370 375 380

Gin lie Glu His His Leu Phe Pro Arg Met ser Ser Trp His Tyr pro 385 390 395 400 phe val Gin Gin Ala val Arg Glu Cys Cys Glu Arg His Gly val Arg 405 410 415

Tyr val Phe Tyr Pro Thr lie val Gly Asn lie lie ser Thr Leu Lys 420 425 430

Tyr Met His Lys val Gly val val His Cys val Lys Asp Ala Gin Asp 435 440 445

Ser <210> 77

<211 > 1392 <212> DNA <213> Peridinium sp. CCMP626 <220> <221 > misc_feature <223> synthetic delta-5 desaturase (codon-optimized) for Yarrowia lipolytica <400> 77 atggctcccg acgccgacaa gctgcgacag cgaaaggctc agtccatcca ggacactgcc 60 gattctcagg ctaccgagct caagattggc accctgaagg gtctccaagg caccgagatc 120 gtcattgatg gcgacatcta cgacatcaaa gacttcgatc accctggagg cgaatccatc 180 atgacctttg gtggcaacga cgttactgcc acctacaaga tgattcatcc ctaccactcg 240 aagcatcacc tggagaagat gaaaaaggtc ggtcgagtgc ccgactacac ctccgagtac 300 aagttcgata ctcccttcga acgagagatc aaacaggagg tcttcaagat tgtgcgaaga 360 ggtcgagagt ttggaacacc tggctacttc tttcgagcct tctgctacat cggtctcttc 420 ttttacctgc agtatctctg ggttaccact cctaccactt tcgcccttgc tatcttctac 480 ggtgtgtctc aggccttcat tggcctgaac gtccagcacg acgccaacca cggagctgcc 540 tccaaaaagc cctggatcaa caatttgctc ggcctgggtg ccgactttat cggaggctcc 600 aagtggctct ggatgaacca gcactggacc catcacactt acaccaacca tcacgagaag 660 gateccgacg ccctgggtgc agagcctatg ctgctcttca acgactatcc cttgggtcac 720 cccaagcgaa ccctcattca trarrrccaa gcctrctact atctgtttgt ccttgctggc 780 tactgggtgt cttcggtgtt caaccctcag atcctggacc tccagcaccg aggtgcccag 840 gctgtcggca tgaagatgga gaacgactac attgccaagt ctcgaaagta cgctatcttc 900 ctgcgactcc tgtacatcta caccaacatt gtggctccca tccagaacca aggcttttcg 960 ctcaccgtcg ttgctcacat tcttactatg ggtgtcgcct ccagcctgac cctcgctact 1020 ctgttcgccc tctcccacaa cttcgagaac gcagatcggg atcccaccta cgaggctcga 1080 aagggaggcg agcctgtctg ttggttcaag tcgcaggtgg aaacctcctc tacttacggt 1140 ggcttcattt ccggttgcct tacaggcgga ctcaactttc aggtcgagca tcacctgttt 1200 cctcgaatgt cctctgcctg gtacccctac atcgctccta ccgttcgaga ggtctgcaaa 1260 aagcacggcg tcaagtacgc ctactatccc tgggtgtggc agaacctcat ctcgaccgtc 1320 aagtacctgc atcagtccgg aactggctcg aactggaaga acggtgccaa tccctactct 1380 ggcaagctgt aa 1392

<210> 78 <211> 463 <212> PRT <213> Peridinium sp. CCMP626 <400> 78

Met Ala Pro Asp Ala Asp Lys Leu Arg Gin Arg Lys Ala Gin ser Ile 15 10 15

Gin Asp Thr Ala Asp ser Gin Ala Thr Glu Leu Lys ile Gly Thr Leu 20 25 30

Lys Gly Leu Gin Gly Thr Glu Ile val Ile Asp Gly Asp Ile Tyr Asp 35 40 45

Ile Lys Asp Phe Asp His Pro Gly Gly Glu ser ile Met Thr phe Gly 50 55 60 G ly Asn Asp val Thr Ala Thr Tyr Lys Met Ile His Pro Tyr His Ser 65 70 75 80

Lys His His Leu Glu Lys Met Lys Lys val Gly Arg val Pro Asp Tyr 85 90 95

Thr Ser Glu Tyr Lys Phe Asp Thr pro Phe Glu Arg Glu ile Lys Gin 100 105 110

Glu val Phe Lys ile val Arg Arg Gly Arg Glu Phe Gly Thr Pro Gly 115 120 125

Tyr Phe Phe Arg Ala Phe Cys Tyr ile Gly Leu Phe Phe Tyr Leu Gin 130 135 140

Tyr Leu Trp val Thr Thr Pro Thr Thr Phe Ala Leu Ala Ile Phe Tyr 145 150 15S 160

Gly val Ser Gin Ala Phe ile Gly Leu Asn val Gin His Asp Ala Asn 165 170 175

His Gly Ala Ala ser Lys Lys Pro Trp Ile Asn Asn Leu Leu Gly Leu 180 185 190

Gly Ala Asp Phe Ile Gly Gly Ser Lys Trp Leu Trp Met Asn Gin His 195 200 205

Trp Thr His His Thr Tyr Thr Asn His His Glu Lys Asp Pro Asp Ala 210 215 220

Leu Gly Ala Glu Pro Met Leu Leu Phe Asn Asp Tyr Pro Leu Gly His 225 230 235 240

Pro Lys Arg Thr Leu ile His His Phe Gin Ala phe Tyr Tyr Leu Phe

245 250 25S val Leu Ala Gly Tyr Trp val ser ser val Phe Asn Pro Gin ile Leu 260 265 270

Asp Leu Gin His Arg Gly Ala Gin Ala Val Gly Met Lys Met Glu Asn 275 280 285

Asp Tyr ile Ala Lys Ser Arg Lys Tyr Ala Ile Phe Leu Arg Leu Leu 290 295 300

Tyr ile Tyr Thr Asn ile val Ala Pro ile Gin Asn Gin Gly Phe Ser 305 310 315 320

Leu Thr Val val Ala His Ile Leu Thr Met Gly val Ala Ser Ser Leu

325 330 33S

Thr Leu Ala Thr Leu Phe Ala Leu Ser His Asn Phe Glu Asn Ala Asp 340 345 350

Arg Asp Pro Thr Tyr Glu Ala Arg Lys Gly Gly Glu Pro val Cys Trp 355 360 365

Phe Lys Ser Gin val Glu Thr Ser ser Thr Tyr Gly Gly Phe Ile Ser 370 375 380

Gly cys Leu Thr Gly Gly Leu Asn Phe Gin val Glu His His Leu Phe 385 390 395 400

Pro Arg Met Ser ser Ala Trp Tyr pro Tyr ile Ala Pro Thr Val Arg 405 410 415

Glu val Cys Lys Lys His Gly val Lys Tyr Ala Tyr Tyr Pro Trp val 420 425 430

Trp Gin Asn Leu Ile Ser Thr val Lys Tyr Leu His Gin Ser Gly Thr 435 440 445

Gly ser Asn Trp Lys Asn Gly Ala Asn Pro Tyr ser Gly Lys Leu 450 455 460 <210> 79 <211 > 1350

<212> DNA <213> Euglena gracilis <220> <221 > misc_feature <222> (1)..(1350) <223> delta-5 desaturase <400> 79 atggctctca gtcttaccac agaacagctg ttagaacgcc ctgatttggt tgcgattgat 60 ggcatcctct acgaccttga agggcttgcc aaagttcatc caggaggaga tttgattcte 120 gctcctggtg cctctgatgc ctcccctctc ttttattcaa tgcatccata cgtcaaaccg 180 gagaattcca aattgcttca acagttcgtc cgagggaagc atgaccgcac ctcgaaggac 240

attgtctaca cgtatgattc tcccttcgca caagacgtta agcggacaat gcgcgaggtg BOO atgaaaggga ggaactggta cgcaacccct ggcttctggc tgcgcaccgt tgggatcatc 360 gccgtgacgg ccttttgcga gtggcactgg gctaccacgg ggatggtgct gtggggcctg 420 ttgactggat tcatgcacat gcagatcggc ttatccatcc agcatgatgc gtcccacggg 480 gccatcagca agaagccttg ggtcaacgcc ctcttcgcct acggcattga cgtcatcgga S40 tcgtcccggt ggatttggct gcagtcgcac atcatgcggc accacaccta caccaaccag 600 cacggcctcg acctggatgc ggagtcggca gagccgttcc tggtgttcca eaactaeeec 660 gccgcaaaca ccgcccgaaa gtggttccac cgcttccaag cttggtacat gtaccttgtg 720 ctgggggcat acggggtatc gctggtgtac aacccgctct acattttccg gatgcagcac 780 aatgacacca tcccagagtc tgtcacggcc atgcgggaga atggctttct gcggcgctac 840 cgcacacttg cattcgtgat gcgagctttc ttcatcttcc ggaccgcatt cttgccctgg 900 tacctcactg ggacctcatt gctgatcacc attectctgg tgcccactgc aactggtgcc 960 ttcttgacgt tcttcttcat tttgtcccac aattttgatg gctccgaacg gatccccgac 1020 aagaactgca aggttaagag ctctgagaag gacgtcgagg ctgaccaaat tgactggtat 1080 cgggcgcagg tggagacgtc ctccacatac ggtggcccca tcgccatgtt cttcactggc 1140 ggtctcaatt tccagatcga gcaccacctc tttccccgga tgtcgtcttg gcactacccc 1200 ttcgtccagc aggcggtccg ggagtgttgc gaacgccatg gagtgcgata tgttttctac 1260 cctaccatcg tcggcaacat catctccacc ctgaagtaca tgcataaggt gggtgtcgtc 1320 cactgcgtga aggacgcaca ggattcctga 1350

<210> 80 <211> 7222 <212> DNA <213> Artificial Sequence <220> <223> Plasmid pFBAIn-MOD-1 <400> 80 catggatcca ggcctgttaa cggccattac ggcctgcagg atccgaaaaa acctcccaca 60 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc 120 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt 180 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgcgg 240 ccgcaagtgt ggatggggaa gtgagtgccc ggttctgtgt gcacaattgg caatccaaga 300 tggatggatt caacacaggg atatagcgag ctacgtggtg gtgcgaggat atagcaacgg 360 atatttatgt ttgacacttg agaatgtacg atacaagcac tgtccaagta caatactaaa 420 catactgtac atactcatac tegtacccgg gcaacggttt cacttgagtg cagtggctag 480 tgctcttact cgtacagtgt gcaatactgc gtatcatagt ctttgatgta tatcgtattc 540 attcatgtra gttgcgtacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 600 qrgagctaac tcacattaat tgcqttqcqc tcactgcccg ctttccagtc gggaaacctg 660 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gegtattggg 720 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 780 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 840 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc.gtaaaaaggc cgcgrrgctg 900 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 960 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 1020 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 1080 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 1140 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 1200 ggtaaccatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 1260 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 1320 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca 1380 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 1440 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 1500 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 1560 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatra aaaatgaagr 1620 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 1680 agtgaggcac ctatctcagc gatctgtcta trtcgttcat ccatagttgc ctgactcccc 1740 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 1800 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 1860 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 1920 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 1980 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 2040 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 2100 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 2160 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 2220 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 2280 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 2340 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 2400 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggcgagca 2460 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 2520 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 2580 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 2640 cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 2700 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc 2760 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 2820 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggrgat 2880 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc 2940 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc 3000 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg 3060 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttccattcgc 3120 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc 3180 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc 3240 agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat 3300 tgggtaccgg gccccccctc gaggtcgatg gtgtcgataa gcttgatatc gaattcatgt 3360 cacacaaacc gatcttcgcc tcaaggaaac ctaattctac atccgagaga ctgccgagat 3420 ccagtctaca ctgattaart ttcgggccaa taatttaaaa aaatcgtgtt atataatatt 3480 atatgtatta tatatataca tcatgatgat actgacagtc atgtcccatt gctaaataga 3540 cagactccat ctgccgcctc caactgatgt tctcaatatt taaggggtca tctcgcattg 3600 tttaataata aacagactcc atctaccgcc tccaaatgat grtctcaaaa tatattgtat 3660 gaacttattt ttattactta gtattattag acaacttact tgctttatga aaaacacttc 3720 ctatttagga aacaatttat aatggcagtt cgttcattta acaatttatg tagaataaat 3780 gttataaatg cgtatgggaa atcctaaata tggatagcat aaatgatatc tgcattgcct 3840 aattcgaaat caaeagcaac gaaaaaaatc CCttgtacaa cataaatagt catcgagaaa 3900 tatcaactat caaagaacag ctattcacac gttactattg agattattat tggacgagaa 3960 tcacacactc aactgtcttt ctctcttcta gaaatacagg tacaagtatg tactattctc 4020 attgttcata cttctagtca tttcatccca catattcctt ggatttctct ccaatgaatg 4080 acattctatc ttgcaaattc aacaattata ataagatata ccaaagtagc ggtatagtgg 4140 caatcaaaaa gcttctctgg tgtgcttctc gtatttattt ttattctaat gatccattaa 4200 aggtatatat ttatttcttg ttatataatc cttttgttta ttacatgggc tggatacata 4260 aaggtatttt gatttaattt tttgcttaaa ttcaatcccc cctcgttcag tgtcaactgt 4320 aatggtagga aattaccata cttttgaaga agcaaaaaaa atgaaagaaa aaaaaaatcg 4380 tatttccagg ttagacgttc cgcagaatct agaatgcggt atgcggtaca ttgttcttcg 4440 aacgtaaaag ttgcgctccc tgagatattg tacatttttg cttttacaag tacaagtaca 4500 tcgtacaact atgtactact gttgatgcat ccacaacagt ttgttttgtt tttttttgtt 4560 tttttttttt ctaatgattc attaccgcta tgtataccta cttgtacttg tagtaagccg 4620 ggttattggc gttcaattaa tcatagactt atgaatctgc acggtgtgcg ctgcgagtta 4680 cttttagctt atgcatgcta cttgggtgta atattgggat ctgttcggaa atcaacggat 4740 gctcaatcga tttcgacagt aattaattaa gtcatacaca agtcagcttt cttcgagcct 4800 catataagta taagtagttc aacgtattag cactgtaccc agcacctccg tatcgagaaa 4860 cacaacaaca tgccccattg gacagatcat gcggatacac aggttgtgca gtatcataca 4920 tactcgatca gacaggtcgt ctgaccatca tacaagctga acaagcgctc catacttgca 4980 cgctctctat atacacagtt aaattacata tccatagtct’aacctctåac agttaatctt 5040 ctggtaagcc tcccagccag ccttctggta tcgcttggcc tcctcaatag gatctcggtt 5100 ctggccgtac agacctcggc cgacaattat gatatccgtt ccggtagaca tgacatcctc 5160 aacagttcgg tactgctgtc cgagagcgtc tcccttgtcg tcaagaccca ccccgggggt 5220 cagaataagc cagtcctcag agtcgccctt aggtcggttc tgggcaatga agccaaccac 5280 aaactcgggg tcggatcggg caagctcaat ggtctgcttg gagtactcgc cagtggecag 5340 agagcccttg caagacagct cggccagcat gagcagacct ctggccagct tctcgttggg 5400 agaggggact aggaactCCt tgtactggga gttctegtag tcagagacgt cctccttctt 5460 ctgttcagag acagtttcct cggcaccagc tcgcaggcca gcaatgattc cggttccggg 5520 tacaccgtgg gcgttggtga tatcggacca ctcggcgatt cggtgacacc ggtactggtg 5580 cttgacagtg ttgccaatat ctgcgaactt tctgtcctcg aacaggaaga aaccgtgctt 5640 aagagcaagt tccttgaggg ggagcacagt gccggcgtag gtgaagtcgt caatgatgcc 5700 gatatgggtt ttgatcatgc acacataagg tccgacctta tcggcaagct caatgagctc 5760 cttggtggtg gtaacatcca gagaagcaca caggttggtt ttcttggctg ccacgagctt 5820 gagcactcga gcggcaaagg cggacttgtg gacgttagct cgagcttcgt aggagggcat 5880 tttggtggtg aagaggagac tgaaataaat ttagtctgca gaacttttta tcggaacctt 5940 atctggggca gtgaagtata tgttatggta atagttacga gttagttgaa cttatagata 6000 gactggacta tacggctatc ggtccaaatt agaaagaacg tcaatggctc tctgggegtc 6060 gcctttgccg acaaaaatgt gatcatgaxg aaagccagca atgacgttgc agctgatatt 6120 gttgtcggcc aaccgcgccg aaaacgcagc tgtcagaccc acagcctcca acgaagaatg 6180 tatcgtcaaa gtgatccaag cacactcata gttggagtcg tactccaaag gcggcaatga 6240 cgagtcagac agatactcgt cgaaaacagt gtacgcagat ctactataga ggaacattta 6300 aattgccccg gagaagacgg ccaggccgcc tagatgacaa attcaacaac tcacagetga 6360 crttctgcca ccgccactag gggggggcct trttatatgg ccaagccaag ctctccacgt 6420 cggttgggct gcacccaaca ataaatgggt agggttgcac caacaaaggg atgggatggg 6480 gggtagaaga tacgaggata acggggctca atggcacaaa taagaacgaa tactgccatt 6540 aagactcgtg atccagcgac tgacaccatt gcatcatcta agggcctcaa aactacctcg 6600 gaactgctgc gctgatctgg acaccacaga ggttccgagc actttaggtt gcaccaaatg 6660 tcccaccagg tgcaggcaga aaacgctgga acagcgtgta cagtttgtct taacaaaaag 6720 tgagggcgct gaggtcgagc agggtggtgt gacttgttat agcctttaga gctgcgaaag 6780 cgcgtatgga tttggctcat caggccagat tgagggtctg tggacacatg tcatgttagt 6840 gtacttcaat cgccccctgg atatagcccc gacaataggc cgtggcctca tttttttgcc 6900 ttccgcacat ttccattgct cggtacccac accttgcttc tcctgcactt gccaacctta 6960 atactggttt acattgacca acatcttaca agcggggggc ttgtctaggg tatatataaa 7020 cagtggctct cccaatcggt tgccagtctc ttttttcctt tctttcccca cagattcgaa 7080 atctaaacta cacatcacag aattccgagc cgtgagtatc cacgacaaga tcagtgtcga 7140 gacgacgcgt tttgtgtaat gacacaatcc gaaagtcgct agcaacacac actctctaca 7200 caaactaacc cagctctggt ac 7222

<210>81 <211> 1086 <212> DNA <213> Phytophthora sojae <220> <221 > misc_feature <223> synthetic delta-17 desaturase (codon-optimized) <400> 81 atggctacca agcagcccta ccagttccct actctgaccg agatcaagcg atctctgccc 60 tccgagtgtt tcgaggcctc cgtgcctctc tctctgtact acaccgttcg atgcctggtc 120 attgctgtgt cgctcgcctt cggacttcac catgcacgat ctctgcccgt tgtcgaaggc 180 ctctgggctc tggatgccgc tctctgcacc ggttacgtgc tgctccaggg catcgtcttc 240 tggggattct ttactgttgg tcacgacgct ggacatggtg ccttctcccg ataccacctg 300 ctcaactttg tcatcggaac cttcattcac tctctcatcc ttacaccctt cgagtcctgg 360 aagcccaccc acagacacca tcacaagaac actggcaaca tcgaccgaga cgaaatcttc 420 taccctcaac gaaaggccga cgatcatcct ctgtctcgaa acctcattct ggctttgggt 480 gcagcctggt ttgcctacct ggtcgaaggc tttcctcccc gaaaggtcaa ccacttcaac 540 cccttcgagc ctctctttgt tcgacaggtc tctgccgtgg tcatttcgct ggctgcgcac 600 tttggagtgg ctgccctgtc catctacctc agcctgcagt tcggcttcaa gactatggcc 660 atctactact atggtcccgt ctttgtgttc ggatccatgc tcgtcattac tacctttctt 720 catcacaacg acgaagagac accttggtac gcagattcgg agtggaccta cgtcaaaggc 780 aacctgtcct ctgtcgaccg atcccacggt gccctcatcg acaacctttc tcacaacatc 840 ggaacccacc agattcatca cctctttccc atcattcctc actacaagct caagcgagct 900 accgaggcct tccatcaagc ctttcccgag ctggttcgaa agtccgacga acccatcatc 960 aaggcctttt tcagagtcgg ccgactctac gcaaactacg gtgtggtcga ctcggatgcc 1020 aagctgttca ctctcaagga ggccaaggct gtttccgaag ccgctaccaa gactaaggcc 1080 acctaa 1086

<210> 82 <211 > 361 <212> PRT <213> Phytophthora sojae <220>

<221 > MISC_FEATURE <223> synthetic delta-17 desaturase (codon-optimized) <400> 82

Met Ala Thr Lys Gin Pro Tyr Gin Phe Pro Thr Leu Thr Glu ile Lys 15 10 15

Arg ser Leu Pro Ser Glu Cys Phe Glu Ala Ser Val Pro Leu ser Leu 20 25 30

Tyr Tyr Thr val Arg Cys Leu val lie Ala Val Ser Leu Ala Phe Gly 35 40 45

Leu His His Ala Arg ser Leu Pro val val Glu Gly Leu Trp Ala Leu 50 55 60

Asp Ala Ala Leu cys Thr Gly Tyr Val Leu Leu Gin Gly He val Phe 65 70 75 80

Trp Gly Phe Phe Thr val Gly His Asp Ala Gly His Gly Ala Phe Ser 85 90 95

Arg Tyr His Leu Leu Asn Phe val lie Gly Thr phe lie His ser Leu 100 105 110 lie Leu Thr Pro Phe Glu ser Trp Lys Leu Thr His Arg His His His 115 120 125

Lys Asn Thr Gly Asn lie Asp Arg Asp Glu lie Phe Tyr Pro Gin Arg 130 135 140

Lys Ala Asp Asp His Pro Leu Ser Arg Asn Leu lie Leu Ala Leu Gly 145 150 155 160

Ala Ala Trp Phe Ala Tyr Leu val Glu Gly Phe ProrPro Arg Lys val 165 170 175

Asn His Phe Asn Pro Phe Glu Pro Leu Phe val Arg Gin val ser Ala 180 18S 190

Val val lie Ser Leu Ala Ala His Phe Gly val Ala Ala Leu Ser lie 195 200 205

Tyr Leu Ser Leu Gin Phe Gly Phe Lys Thr Met Ala lie Tyr Tyr Tyr 210 215 220

Gly Pro val Phe val Phe Gly Ser Met Leu val lie Thr Thr Phe Leu 225 230 235 240

His His Asn asp Glu Glu Thr pro Trp Tyr Ala Asp ser Glu Trp Thr 245 250 255

Tyr Val Lys Gly Asn Leu Ser Ser val Asp Arg Ser Tyr Gly Ala Leu 260 265 270 lie Asp Asn Leu Ser His Asn lie Gly Thr His Gin lie His His Leu 275 280 285

Phe Pro lie lie Pro His Tyr Lys Leu Lys Arg Ala Thr Glu Ala Phe 290 295 300

His Gin Ala Phe Pro Glu Leu val Arg Lys Ser Asp Glu Pro lie lie 305 310 315 320

Lys Ala Phe Phe Arg val Gly Arg Leu Tyr Ala Asn Tyr Gly val val 325 330 335

Asp Ser Asp Ala Lys Leu Phe Thr Leu Lys Glu Ala Lys, Ala Val Ser 340 345 350

Glu Ala Ala Thr Lys Thr Lys Ala Thr 355 360

<210> 83 <211 >3806 <212> DNA <213> Artificial Sequence <220> <223> Plasmid pPSD17S <400> 83 atcggatccc gggcccgtcg actgcagagg cctgcatgca agcttggcgt aatcatggtc 60 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 120 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 180 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 240 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 300 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 360 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 420 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 480 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 540 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 600 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 660 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 720 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 780 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatra gcagagcgag 840 gtatgtaggc ggcgctacag agttcttgaa gtggtggcct aactacggct acactagaag 900 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 960 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 1020 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 1080 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 1140 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 1200 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 1260 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 1320 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 1380 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 1440 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 1500 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 1560 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 1620 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 1680 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 1740 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 1800 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 1860 cagaacttta aaagtgctca ccattggaaa acgttcttcg gggcgaaaac tctcaaggat 1920 ctcaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 1980 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 2040 aaagggaata agggcgacac ggaaatgrtg aatactcata ctcttccttt ttcaatatta 2100 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 2160 aaataaacaa acaggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga 2220 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct 2280 cgcgcgttxc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac 2340 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt 2400 tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca 2460 ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca 2520 ctcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt 2580 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt 2640 ttcccagtca cgacgttgta aaacgacggc cagtgaattc gagctcggca cctcgcgaat 2700 gcatctagat ccatggctac caagcagccc taccagttcc ctactctgac cgagatcaag 2760 cgatctctgc cctccgagtg tttcgaggcc tccgtgcctc tctctctgta ctacaccgtt 2820 cgatgcctgg tcattgctgt gtcgctcgcc ttcggacttc accatgcacg atctctgccc 2880 gttgtcgaag gcctctgggc tctggatgcc gctctctgca ccggttacgt gctgctccag 2940 ggcatcgtct tctggggatt ctttactgtt ggtcacgacg ctggacatgg tgccttctcc 3000 cgataccacc tgctcaactt tgtcatcgga accttcattc actctctcat ccttacaccc 3060 ttcgagtcct ggaagctcac ccacagacac catcacaaga acactggcaa catcgaccga 3120 gacgaaatct tctaccctca acgaaaggcc gacgatcatc ctctgtctcg aaacctcatt 3180 ctggctttgg gtgcagcctg gtttgcctac ctggtcgaag gctttcctcc ccgaaaggtc 3240 aaccacttca accccttcga gcctctcttt gttcgacagg tctctgccgt ggtcatttcg 3300 ctggctgcgc actttggagt ggctgccctg tccatctacc tcagcctgca gttcggcttc 3360 aagactatgg ccacctacta ctatggtccc gcctttgtgt tcggatccat gctcgtcatt 3420 actacctttc ttcatcacaa cgacgaagag acaccttggt acgcagattc ggagtggacc 3480 tacgtcaaag gcaacctgtc ctctgtcgac cgatcctacg gtgccctcat cgacaacctt 3540 tctcacaaca tcggaaccca ccagattcat cacctctttc ccatcattcc tcactacaag 3600 ctcaagcgag ctaccgaggc cttccatcaa gcctttcccg agctggttcg aaagtccgac 3660 gaacccatca tcaaggcctt tttcagagtc ggccgactct acgcaaacta cggtgtggtc 3720 gactcggatg ccaagctgtt cactctcaag gaggccaagg ctgtttccga agccgctacc 3780 aagactaagg ccacctaagc ggccgc 3806

<210> 84 <211> 1086 <212> DNA <213> Phytophthora ramorum <220> <221 > misc_feature <223> synthetic delta-17 desaturase (codon-optimized) <400> 84 atggctacca agcagcccta ccagttccct aetctgaccg agatcaagcg atctcttccc 60 tccgagtgct ttgaagcctc ggtccctctg tccttgtact acaccgtgcg aatcgtcgct 120 attgccgttg ctctggcctt cggactcaac tacgctcgag cccttcccgt ggtcgagtct 180 ctgtgggcac tcgacgctgc cctttgttgc ggttacgttc tgctccaagg cattgtcttc 240 tggggattct ttaccgcggg tcacgatgct ggacatggtg ccttctctcg ataccacctg 300 clcaactttg tcgttggcac ctttatccac tccctcattc ttactccctt cgagtcgtgg 360 aagctcacac atcgacacca tcacaagaac accggaaaca tcgaccgaga cgaaatcttc 420 taccctcagc gaaaggccga cgatcatcct ctgtctcgaa acctcgtcct ggctctcggt 480 geegettggt ttgectacet tgtcgagggc tttcctcccc gaaaggtcaa ccacttcaac 540 cccttcgaac ctctgtttgt gcqacaggtg gctgccgttg tcatttccct ctctgctcac 600 ttcgccgtcc tggcactgtc cgtgtatctg agctttcagt tcggtctcaa gacaatggct 660 ctgtactact atggacccgt cttcgtgxtc ggctccatgc tcgtcattac tacctttctg 720 catcacaatg acgaggaaac tccttggtac ggagattccg actggaccta cgtcaagggc 780 aacttgtctt ccgtggaccg atcttacggt gccttcatcg acaacctctc gcacaacatt 840 ggcacacacc agatccacca tctgtttccc atcattcctc actacaagct caaccgagcc 900 accgctgcct tccaccaggc ctttcccgaa cttgtccgaa agagcgacga gcccattctc 960 aaggctttct ggagagttgg tcgactttac gccaactacg gagtcgtgga tcccgacgca 1020 aagctgttta ctctcaagga ggccaaagct gcctccgagg ctgccaccaa gaccaaggct 1080 acttaa 1086

<210> 85 <211 >3806 <212> DNA <213> Artificial Sequencec <220> <223> Plasmid pPrD17s <400> 85 ggccgcarcg garcccgggc ccgtcgactg cagaggcctg catgcaagct rggcgtaatc 60 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 120 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 180 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg 240 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct 300 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 360 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 420 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 480 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 540 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 600 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 660 tagctcacgc tgtaggtatc tcagttcggt gtaggxcgtt cgctccaagc tgggctgcgt 720 gcacgaaccc cccgttcagc ccgaccgctg cgcctxatcc ggtaactatc gtctxgagxc 780 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 840 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 900 tagaagaaca gtatttggta tctgcgotct gctgaagcca gttaccttcg gaaaaagagt 960 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 1020 gcagcagarc acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 1080 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattaxcaaa 1140 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 1200 ataxgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc 1260 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat 1320 acgggagggc ttaccatcrg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc 1380 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc 1440 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag 1500 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg 1560 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg 1620 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag 1680 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt 1740 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 1800 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata atåccgcgcc 1860 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc 1920 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc 1980 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc 2040 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca 2100 atattattga agcatttatc agggxxaxtg tctcatgagc ggatacatat ttgaatgtat 2160 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt 2220 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgcatca cgaggccctt 2280 tcgtctcgcg cgttxcggtg atgacggtga aaacctctga cacatgcagc tcccggagac 2340 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc 2400 gggcgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag 2460 agtgcaccat axgcggxgxg aaataccgca cagatgcgta aggagaaaat accgcatcag 2S20 gcgccattcg ccattcaggc tgegcaactg ttgggaaggg cgatcggtgc gggcctcttc 2580 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc 2640 agggttttcc cagtcacgac gttgxaaaac gacggccagt gaattcgagc tcggtacctc 2700 gcgaatgcat ctagatccat ggctaccaag cagccccacc agttccctac tctgaccgag 2760 atcaagcgat ctcttccctc cgagtgcttt gaagccccgg tccctctgtc cttgtactac 2820 accgtgcgaa tcgtcgctat tgccgttgct ctggccttcg gactcaacta cgctcgagcc 2880 cttcccgtgg tcgagtctct gtgggcactc gacgctgccc tttgttgcgg ttacgttctg 2940 ctccaaggca ttgtcttctg gggattcttt accgtgggtc acgatgctgg acatggtgcc 3000 ttctctcgat accacctgct caactttgtc gttggcacct ttatccactc cctcattctt 3060 actcccttcg agtcgtggaa gctcacacat cgacaccatc acaagaacac cggaaacatc 3120 gaccgagacg aaatcttcta ccctcagcga aaggccgacg atcatcctct gtctcgaaac 3180 ctcgtcctgg ctetcggtgc cgcttggttt gcctaccttg tcgagggctt tcctccccga 3240 aaggtcaacc acttcaaccc cttcgaacct ctgtttgtgc gacaggtggc tgccgttgtc 3300 atttccctct ctgctcactt cgccgtcctg gcactgtccg tgtatctgag ctttcagttc 3360 ggtctcaaga caatggctcr gtactactat ggacccgtct tcgtgctcgg ctccatgctc 3420 gtcattacta cctttctgca tcacaatgac gaggaaacte cttggtacgg agattccgac 3480 tggacctacg tcaagggcaa cttgrctrcc gtggaccgat cttacggtgc cttcatcgac 3540 aacctctcgc acaacattgg cacacaccag atccaccatc tgtttcccat cattcctcac 3600 tacaagctca accgagccac cgctgccttc caccaggcct ttcccgaact tgtccgaaag 3660 agcgacgagc ccattctcaa ggctttctgg agagttggtc gactttacgc caactacgga 3720 gtcgtggatc ccgacgcaaa gctgtttact ctcaaggagg ccaaagctgc ctccgaggct 3780 gccaccaaga ccaaggctac ttaagc 3806

<210 86 <211>1209 <212> DNA <213> Fusarium monoliforme <300

<302> DELTA-15 DESATURASES SUITABLE FOR ALTERING LEVELS OF POLYUNSATURATED FATTY ACIDS IN OLEAGINOUS PLANTS AND YEAST <310> WO 2005/047480 <311> 2004-11-10 <312> 2005-05-26 <313> (1)..(1209) <400> 86 atggcgactc gacagcgaac tgccaccact gttgtggtcg aggaccttcc caaggtcact 60 cttgaggcca agtctgaacc tgtgttcccc gatatcaaga ccatcaagga tgccattccc 120 gcgcactgct tccagccctc gctcgtcacc tcattctact acgtcttccg cgattttgct 180 atggtctctg ccctcgtctg ggctgctctc acctacatcc ccagcatccc cgaccagacc 240 ctccgcgtcg eagcttggat ggtctacggc ttcgtccagg gtctgttctg caccggtgtc 300 tggattctcg gccatgagtg cggccacggt gctttctctc tccacggaaa ggtcaacaat 360 gtgaccggct ggttcctcca ctcgttcctc ctcgtccoct acttcagctg gaagtactct 420 caccaccgcc accaccgctt caccggccac atggatctcg acatggcttt cgtccccaag 480 actgagccca agccctccaa gtcgctcatg attgctggca ttgacgtcgc cgagcttgtt 540 gaggacaccc ccgctgctca gatggtcaag ctcatcttcc accagctttt cggatggcag 600 gcgtacctct tcttcaacgc tagctctggc aagggcagca agcagtggga gcccaagact 660 ggcctctcca agtggttccg agtcagtcac ttcgagccta ccagcgctgt cttccgcccc 720 aacgaggcca tcttcatcct catctccgat atcggtcttg ctctaatggg aactgctctg 780 tactttgctt ccaageaagt tggtgtttcg accattctct tcctctacct tgttccctac 840 ctgtgggttc accactggct cgttgccatt acctacctcc accaccacca caccgagctc 900 cctcactaca ccgctgaggg ctggacctac gtcaagggag crcrcgccac tgtcgaccgt 960 gagtttggct: tcatcggaaa gcacctcttc cacggtatca ttgagaagca cgttgttcac 1020 catctcttcc ctaagatccc ctictacaag gctgacgagg ccaccgaggc catcaagccc 1080 gtcattggcg accactactg ccacgacgac cgaagcttcc tgggccagct gtggaccatc 1140 ttcggcacgc tcaagtacgt cgagcacgac cctgcccgac ccggtgccat gcgatggaac 1200 aaggactag 1209

<210> 87 <211 >402 <212> PRT <213> Fusarium monoliforme <300>

<302> DELTA-15 DESATURASES SUITABLE FOR ALTERING LEVELS OF POLYUNSATURATED FATTY ACIDS IN OLEAGINOUS PLANTS AND YEAST <310> wo 2005/047480 <311> 2004-11-10 <312> 2005-05-26 <313> (1)..(402) <400> 87

Met Ala Thr Arg Gin Arg Thr Ala Thr Thr val val val Glu Asp Leu 1 5 10 15

Fro Lys val Thr Leu Glu Ala Lys ser Glu Pro val Phe Pro Asp He 20 25 30

Lys Thr Ile Lys Asp Ala lie Pro Ala His cys Phe Gin Pro ser Leu 35 40 45 val Thr ser Phe Tyr Tyr val Phe Arg Asp Phe Ala Met val ser Ala 50 55 60

Leu val Trp Ala Ala Leu Thr Tyr lie Pro Ser lie Pro Asp Gin Thr 65 70 75 80

Leu Arg Val Ala Ala Trp Met Val Tyr Gly Phe val Gin Gly Leu Phe 85 90 95

Cys Thr Gly val Trp lie Leu Gly His Glu Cys Gly His Gly Ala Phe 100 105 110

Ser Leu His Gly Lys val Asn Asn val Thr Gly Trp Phe Leu His Ser 115 120 125

Phe Leu Leu val Pro Tyr Phe Ser Trp Lys Tyr ser His His Arg His 130 135 140

His Arg Phe Thr Gly His Met Asp Leu Asp Met Ala Phe Val Pro Lys 145 150 155 160

Thr Glu Pro Lys Pro Ser Lys Ser Leu Met lie Ala Gly lie Asp val 165 170 ' 175

Ala Glu Leu val Glu Asp Thr Pro Ala Ala Gin Met val Lys Leu lie 180 185 190

Phe His Gin Leu Phe Gly Trp Gin Ala Tyr Leu Phe Phe Asn Ala Ser 195 200 205

Ser Gly Lys Gly Ser Lys Gin Trp Glu Pro Lys Thr Gly Leu Ser Lys 210 215 220

Trp Phe Arg val Ser His Phe Glu Pro Thr Ser Ala val Phe Arg Pro 225 230 235 240

Asn Glu Ala lie Phe lie Leu lie Ser Asp lie Gly Leu Ala Leu Met 245 250 255

Gly Thr Ala Leu Tyr Phe Ala Ser Lys Gin val Gly Val Ser Thr lie 260 265 270

Leu Phe Leu Tyr Leu val Pro Tyr Leu Trp val His His Trp Leu val 275 280 285

Ala lie Thr Tyr Leu His His His His Thr Glu Leu Pro His Tyr Thr 290 295 300

Ala Glu Gly Trp Thr Tyr val Lys Gly Ala Leu Ala Thr val Asp Arg 305 310 315 320

Glu Phe Gly Phe lie Gly Lys His Leu Phe His Gly lie lie Glu Lys 325 330 335

His val val His His Leu Phe Pro Lys lie Pro Phe Tyr Lys Ala Asp 340 345 350

Glu Ala Thr Glu Ala lie Lys Pro val lie Gly Asp His Tyr cys His 355 360 365

Asp Asp Arg ser Phe Leu Gly Gin Leu Trp Thr He Phe Gly Thr Leu 370 375 380

Lys Tyr val Glu His Asp Pro Ala Arg Pro Gly Ala Met Arg Trp Asn 385 390 395 400

Lys Asp

<210> 88 <211 >7668 <212> DNA <213> Artificial Sequence <220> <223> Plasmid pY6.GPD.Leu2 <400> 88 gtacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 60 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 120 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 180 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 240 aaggcggtaa tacggrtatc cacagaatca ggggataacg caggaaagaa catgtgagca 300 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 360 ctccgccccc ctgacgagca tcaeaaaaat cgacgctCåa gtcagaggtg gcgaaacccg 420 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 480 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 540 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 600 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 660 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 720 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 780 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 840 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 900 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 960 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1020 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagrtttaa atcaatctaa 1080 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1140 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 1200 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 1260 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 1320 ggtccigcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 1380 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 1440 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 1500 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 1560 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 1620 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 1680 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 1740 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttettc ggggcgaaaa 1800 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 1860 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 1920 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 1980 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 2040 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 2100 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 2160 gctacacttg ccagcgccct agcgcccgct cctttcgcct tcttcccttc ctttctcgcc 2220 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 2280 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 2340 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agcccacgtt ctttaatagt 2400 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 2460 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 2S20 aacgcgaått ttaacaaaat attaacgctt acaatttcca ttcgccattc aggctgcgca 2580 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 2640 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta 2700 aaacgacggc cagtgaattg taatacgact cactataggg cgaattgggt åccgggcccc 2760 ccctcgaggt cgatggtgtc gataagcttg atatcgaatt catgtcacac aaaccgatct 2820 tcgcctcaag gaaacctaat tctacatccg agagactgcc gagatccagt ctacactgat 2880 taattttcgg gccaataatt taaaaaaatc gtgttatata atattatatg tattatatat 2940 atacatcatg atgatactga cagtcatgtc ccattgctaa atagacagac tccatctgcc 3000 gcctccaact gatgttctca atatttaagg ggtcatctcg cattgtttaa taataaacag 3060 actccatcta ccgcctccaa atgatgttct caaaatatat tgtatgaact tatttttatt 3120 acttagtatt attagacaac ttacttgctt tatgaaaaac acttcctatt taggaaacaa 3180 tttataatgg cagttcgttc atttaacaat ttatgtagaa taaatgttat aaatgcgtat 3240 gggaaatctt aaatatggat agcataaatg atatccgcat tgcctaattc gaaatcaaca 3300 gcaacgaaaa aaatcccttg tacaacataa atagtcatcg agaaatatca actatcaaag 3360 aacagctatt cacacgttac tattgagatt attattggac gagaatcaca cactcaactg 3420 tctttctctc ttctagaaat acaggtacaa gtatgtacta ttctcattgt tcatacrrct 3480 agtcatttca tcccacatat tccttggatt tctctccaat gaatgacatt ctatcttgca 3540 aattcaacaa ttataataag atataccaaa gtagcggtat agtggcaatc aaaaagcttc 3600 tctggtgtgc ttctcgtatt tatttttatt ctaatgatcc attaaaggta tatatttatt 3660 tcttgttata taatcctttt gtttattaca tgggctggat acataaaggt attttgattt 3720 aattttttgc ttaaattcaa tcccccctcg ttcagtgtca actgtaatgg taggaaatta 3780 ccatactttt gaagaagcaa aaaaaatgaa agaaaaaaaa aatcgtattt ccaggttaga 3840 cgttccgcag aatctagaat gcggtatgcg gtacattgtt cttcgaacgt aaaagttgcg 3900 ctccctgaga tattgtacat ttttgctttt acaagtacaa gtacatcgta caactatgta 3960 ctactgttga tgcatccaca acagtttgtt ttgttttttt ttgttttttt tttttctaar 4020 gattcattac cgctatgtat acctacttgt acttgtagta agccgggtta ttggcgttca 4080 attaatcata gacttatgaa tctgcacggt gtgcgctgcg agttactttt agcttatgca 4140 tgctacttgg gtgtaatatt gggatctgtt cggaaatcaa cggatgctca accgatttcg 4200 acagtaatta attaatttga atcgaatcgg agcctaaaat gaacccgagt atatctcata 4260 aaattctcgg tgagaggtct gtgactgtca gtacaaggtg ccttcattat gccctcaacc 4320 ttaccatacc tcactgaatg tagtgtacct ctaaaaatga aatacagtgc caaaagccaa 4380 ggcactgagc tcgtctaacg gacttgatat acaaccaatt aaaacaaatg aaaagaaata 4440 cagttctttg tatcatttgt aacaattacc ctgtacaaac taaggtattg aaatcccaca 4500 atattcccaa agtccacccc tttccaaatt gtcatgccta caactcatat accaagcact 4560 aacctaccaa acaccactaa aaccccacaa aacatatctt accgaatata cagtaacaag 4620 ctaccaccac actcgttggg tgcagtcgcc agcttaaaga tatctatcca catcagccac 4680 aactcccttc ctttaataaa ccgactacac ccttggctat tgaggttatg agtgaatata 4740 ctgtagacaa gacactttca agaagactgt ttccaaaacg taccactgtc ctccactaca 4800 aacacaccca atctgcttct tctagtcaag gttgctacac cggtaaatta taaatcatca 4860 tttcattagc agggcagggc cctttttata gagtcttata cactagcgga ccctgccggt 4920 agaccaaccc gcaggcgcgt cagtttgctc cttccatcaa tgcgtcgtag aaacgactta 4980 ctccttcttg agcagctcct tgaccttgtt ggcaacaagt ctccgacctc ggaggtggag 5040 gaagagcctc cgatatcggc ggtagtgata ccagcctcga cggactcctt gacggcagcc 5100 tcaacagcgt caccggcggg cttcatgtta agagagaact tgagcatcat ggcggcagac 5160 agaatggtgg caatggggtt gaccttctgc ttgccgagat cgggggcaga tccgtgacag 5220 ggctcgcaca gaccgaacgc ctcgttggtg rcgggcagag aagccagaga ggcggagggc $280 agcagaccca gagaaccggg gatgacggag gcctcgtcgg agatgatatc gccaaacatg 5340 ttggtggtga tgatgatacc attcatcttg gagggctgct tgatgaggat catggcggcc 5400 gagtcgatca gctggtggtt gagctcgagc tgggggaart cgtccttgag gactcgagtg 5460 acagtctttc gccaaagtcg agaggaggcc agcacgttgg ccttgtcaag agaccacacg 5520 ggaagagggg ggttgtgctg aagggccagg aaggcggcca ttcgggcaat tcgctcaacc 5580 tcaggaacgg agtaggtctc ggtgtcggaa gcgacgccag atccgtcatc ctcctttcgc 5640 tctccaaagt agatacctcc gacgagctct cggacaatga tgaagtcggt gccctcaacg 5700 tttcggatgg gggagagatc ggcgagcttg ggcgacagca gctggcaggg tcgcaggttg 5760 gcgtacaggt tcaggtcctt tcgcagcttg aggagaccct gctcgggtcg cacgtcggtt 5820 cgtccgtcgg gagtggtcca tacggtgttg gcagcgcctc cgacagcacc gagcataata 5880 gagtcagcct ttcggcagat gtcgagagta gcgtcggtga tgggctcgcc ctccttctca 5940 atggcagctc ctccaatgag tcggtcctca aacacaaact cggtgccgga ggcctcagca 6000 acagacttga gcaccttgac ggcctcggca atcacctcgg ggccacagaa gtcgccgccg 6060 agaagaacaa tcttcttgga gtcagtcttg gtcttcttag tttcgggttc cattgtggat 6120 gtgtgtggtt gtatgtgtga tgtggtgtgt ggagtgaaaa tctgtggctg gcaaacgctc 6180 ttgtatatat acgcactttt gcccgtgcta tgtggaagac taaacctccg aagattgtga 6240 ctcaggtagt gcggtatcgg ctagggaccc aaaccttgtc gatgccgata gcgctatcga 6300 acgtacccca gccggccggg agtatgtcgg aggggacata cgagatcgtc aagggtttgt 6360 ggccaactgg taaataaatg atgtcgacgc agtaggatgt cctgcacggg tctttttgtg 6420 gggtgtggag aaaggggtgc ttggagatgg aagccggtag aaccgggctg cttgtgcttg 6480 gagatggaag ccggtagaac cgggctgctt ggggggattt ggggccgctg ggctccaaag 6540 aggggtaggc atttcgttgg ggttacgtaa ttgcggcatt tgggtcctgc gcgcatgtcc 6600 cattggtcag aattagtccg gataggagac ttatcagcca atcacagcgc cggatccacc 6660 tgtaggttgg gttgggtggg agcacccctc cacagagtag agtcaaacag cagcagcaac 6720 atgatagttg ggggtgtgcg tgttaaagga aaaaaaagaa gcttgggtta tattcccgct 6780 ctatttagag gttgcgggat agacgccgac ggagggcaat ggcgctatgg aaccttgcgg 6840 atatccatac gccgcggcgg actgcgtccg aaccagctcc agcagcgttt tttccgggcc 6900 attgagccga ctgcgacccc gccaacgtgt cttggcccac gcactcatgt catgttggtg 6960 ttgggaggcc actttttaag tagcacaagg cacctagctc gcagcaaggt gtccgaacca 7020 aagaagcggc tgcagtggtg caaacggggc ggaaacggcg ggaaaaagcc acgggggcac 7080 gaattgaggc acgccctcga atttgagacg agtcacggcc ccattcgccc gcgcaatggc 7140 tcgccaacgc ccggtctttt gcaccacatc aggtracccc aagccaaacc tttgtgttaa 7200 aaagettaae atattatacc gaacgtaggt ttgggcgggc ttgctccgtc tgtccaaggc 7260 aacatttata taagggtctg catcgccggc tcaattgaat cttttttctt cttctcttct 7320 ctatattcat tcttgaatta aacacacatc aaccatggat ccactagttc tagagcggcc 7380 gccaccgcgg cccgagattc cggcctcttc ggccgccaag cgacccgggt ggacgtctag 7440 aggtacctag caattaacag atagtttgcc ggtgataatt ctcttaacct cccaeactcc 7500 tttgacataa cgatttatgt aacgaaactg aaatttgacc agatattgtg tccgcggtgg 7560 agctccagct tttgttccct ttagtgaggg ttaatttcga gcttggcgca arcatggrca 7620 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaac 7668

<210> 89 <211 > 9048 <212> DNA <213> Artificial Sequence <220> <223> Plasmid pY130 <400> 89 gtacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 60 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 120 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 180 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 240 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 300 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 360 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 420 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 480 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 540 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 600 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 660 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 720 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 780 tacactagaa ggacagtatt tggtatctgc gctctgccga agccagttac cttcggaaaa 840 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 900 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 960 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1020 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1080 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1140 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 1200 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 1260 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 1320 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 1380 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 1440 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 1500 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 1560 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 1620 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 1680 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 1740 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 1800 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 1860 tgatcttcag catcttttac tttcaccagc gctrcctgggt gagcaaaaac aggaaggcaa 1920 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 1980 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 2040 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 2100 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 2160 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 2220 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 2280 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 2340 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 2400 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 2460 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 2520 aacgcgaatt ttaacaaaat attaacgctt acaatttcca ttcgccattc aggctgcgca 2580 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 2640 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta 2700 aaacgacggc cagtgaattg taatacgact cactataggg cgaattgggt accgggcccc 2760 ccctcgaggt cgatggtgtc gataagctcg atatcgaatt catgtcacac aaaccgatct 2820 ccgcctcaag gaaacctaat tctacatccg agagactgcc gagatccagt ctacactgat 2880 taattttcgg gccaataatt taaaaaaatc gtgttatata atattatatg tattatatat 2940 atacatcatg atgatactga cagtcatgtc ccattgctaa atagacagac tccatctgcc 3000 gcctccaact gatgttctca atatttaagg ggtcatctcg cattgtttaa taataaacag 3060 actccatcta ccgcctccaa atgatgttct caaaatatat tgtatgaact tatttttatt 3120 acttagtatt attagacaac ttacttgctt tatgaaaaac acttcctatt taggaaacaa 3180 tttataatgg cagttcgttc atttaacaat ttatgtagaa taaatgttat aaatgcgtat 3240 gggaaatctt aaatatggat agcataaatg atatctgcat tgcctaattc gaaatcaaca 3300 gcaacgaaaa aaatcccttg tacaacataa atagtcatcg agaaatatca actatcaaag 3360 aacagctatt cacacgttac tattgagatt attattggac gagaatcaca cactcaactg 3420 tctttctctc ttctagaaat acaggtacaa gtatgtacta ttctcattgt tcatacttct 3480 agtcatttca tcccacatat tccttggatt tctctccaat gaatgacatt ctatcttgca 3540 aattcaacaa ttataataag atataccaaa gtagcggtat agtggcaatc aaaaagcttc 3600 tctggtgtgc ttctcgtatt tatttttatt ctaatgatcc attaaaggta tatatttatt 3660 tcttgttata taatcctttt gtttattaca tgggctggat acataaaggt attttgattt 3720 aattttttgc ttaaattcaa tcccccctcg ttcagtgtca actgtaatgg taggaaatta 3780 ccatactttt gaagaagcaa aaaaaatgaa agaaaaaaaa aatcgtattt ccaggttaga 3840 cgttccgcag aatctagaat gcggtatgcg gtacattgtt cttcgaacgt aaaagttgcg 3900 ctccctgaga tattgtacat ttttgctttt acaagtacaa gtacatcgta caactatgta 3960 ctactgttga tgcatccaca acagtttgtt ttgttttttt ttgttttttt tttttctaat 4020 gattcattac cgctatgtat acctacttgt acttgtagta agccgggtta ttggcgttca 4080 attaatcata gacttatgaa tctgcacggt gtgcgctgcg agttactttt agcttatgca 4140 tgctacttgg gtgtaatatt gggatctgtt cggaaatcaa cggatgctca accgatttcg 4200 acagtaataa tttgaatcga atcggagcct aaaatgaacc cgagtatatc tcataaaatt 4260 ctcggtgaga ggtctgtgac tgtcagtaca aggtgccttc attatgccct caaccttacc 4320 atacctcact gaatgtagrg tacctctaaa aatgaaatac agtgccaaaa gccaaggcac 4380 tgagctcgtc taacggactt gatatacaac caattaaaac aaatgaaaag aaatacagtt 4440 ctttgtatca tttgtaacaa ttaccctgta caaaccaagg tattgaaatc ccacaatatt 4500 cccaaagtcc acccctttcc aaattgtcat gcctacaact catataccaa gcactaacct 4560 accaaacacc actaaaaccc cacaaaatat atcttaccga atatacagta acaagctacc 4620 accacactcg ttgggtgcag tcgccagctt aaagatatct atccacatca gccacaactc 4680 ccttccttta ataaaccgac tacacccttg gctattgagg ttatgagtga atatactgta 4740 gacaagacac tttcaagaag actgtttcca aaacgtacca ctgtcctcca ctacaaacac 4800 acccaatctg cttcttctag tcaaggttgc tacaccggta aattataaat catcatttca 4860 ttagcagggc agggcccttt ttatagagtc ttatacacta gcggaccctg ccggtagaec 4920 aacccgcagg cgcgtcagtt tgctccttcc atcaatgcgt cgtagaaacg acttactcct 4980 tcttgagcag ctccttgacc ttgttggcaa caagtctccg acctcggagg tggaggaaga S040 gcctccgata tcggcggtag tgataccagc ctcgacggac tccttgacgg cagcctcaac 5100 agcgtcaccg gcgggcttca tgttaagaga gaacttgagc atcatggcgg cagacagaat 5160 ggtggcaatg gggttgacct tctgcttgcc gagatcgggg gcagatccgt gacagggctc 5220 gtacagaccg aacgcctcgt tggtgtcggg cagagaagcc agagaggcgg agggcagcag 5280 acccagagaa ccggggatga cggaggcctc gtcggagatg atatcgccaa acatgttggt S340 ggtgatgatg ataccattca tcttggaggg ctgcttgatg aggatcatgg cggccgagtc 5400 gatcagctgg tggttgagct cgagctgggg gaattcgtcc ttgaggactc gagtgacagt 5460 ctttcgccaa agtcgagagg aggccagcac gttggccttg tcaagagacc acacgggaag 5520 aggggggttg tgctgaaggg ccaggaaggc ggccattcgg gcaattcgct caacctcagg 5580 aacggagtag gtctcggtgt cggaagcgac gccagatccg tcatcctcct ttcgctctcc 5640 aaagtagata cctccgacga gctctcggac aatgatgaag tcggtgccct caacgtttcg S700 gatgggggag agatcggcga gcttgggcga cagcagctgg cagggtcgca ggttggcgta 5760 caggttcagg tcctttcgca gcttgaggag accctgctcg ggtcgcacgt cggttcgtcc 5820 gtcgggagtg gtccatacgg tgttggcagc gcctccgaca gcaccgagca taatagagtc 5880 agcctttcgg cagatgtcga gagtagcgtc ggtgatgggc tcgccctcct tctcaatggc 5940 agctcctcca atgagtcggt cctcaaacac aaactcggtg ccggaggcct cagcaacaga 6000 cttgagcacc ttgacggcct cggcaatcac ctcggggcca cagaagtcgc cgccgagaag 6060 aacaatcttc ttggagtcag tcttggtctt cttagtttcg ggttccattg tggatgtgtg 6120 t99ttgtatg tgtgatgtgg tgtgtggagt gaaaatctgt ggctggcaaa cgctcttgta 6180 tatatacgca cttttgcccg tgctatgtgg aagactaaac ctccgaagat tgtgacteag 6240 gtagtgcggt atcggctagg gacccaaacc ttgtcgatgc cgatagcgct atcgaacgta 6300 ccccagccgg ccgggagtat gtcggagggg acatacgaga tcgtcaaggg tttgtggcca 6360 actggtattt aaatgatgtc gacgcagtag gatgtcctgc acgggtcttt ttgtggggtg 6420 tggagaaagg ggtgcttgga gatggaagcc ggtagaaccg ggctgcttgt gcttggagat 6480 ggaagccggt agaaccgggc tgcctggggg gatttggggc cgccgggctc caaagagggg 6540 taggcatttc gttggggtta cgtaattgcg gcatttgggt cctgcgcgca tgtcccatcg 6600 gtcagaatta gtccggatag gagacttatc agccaatcac agcgccggat ccacctgtag 6660 gttgggttgg gtgggagcac ccctccacag agtagagtca aacagcagca gcaacatgat 6720 agttgggggt gtgcgtgtta aaggaaaaaa aagaagcttg ggttatattc ccgctctatt 6780 tagaggttgc gggatagacg ccgacggagg gcaatggcgc tatggaacct tgcggatatc 6840 catacgccgc ggcggactgc gtccgaacca gctccagcag cgttttttcc gggccattga 6900 gccgactgcg accccgccaa cgtgtcttgg cccacgcact catgtcargt tggtgttggg 6960 aggccacttt ttaagtagca caaggcacct agctcgcagc aaggtgtccg aaccaaagaa 7020 gcggctgcag tggtgcaaac ggggcggaaa cggcgggaaa aagccacggg ggcacgaatt 7080 gaggcacgcc ctcgaatttg agacgagtca cggccccatt cgcccgcgca atggctcgcc 7140 aacgcccggt cttttgcacc acatcaggtt accccaagcc aaacctttgt gttaaaaagc 7200 ttaacatatt ataccgaacg taggtttggg cgggcttgct ccgtctgtcc aaggcaacat 7260 ttataraagg gtctgcatcg ccggctcaat tgaatcrttt ttcttcttct cttctccata 7320 ttcattcttg aattaaacac acatcaacca tggcgactcg acagcgaact gccaccactg 7380 ttgtggtcga ggaccttccc aaggtcactc ttgaggccaa gtctgaacct gtgttccccg 7440 atatcaagac catcaaggat gccattcccg cgcactgctt ccagccctcg ctcgtcacct 7500 cattctacta cgtcttccgc gattttgcta tggtctctgc cctcgtctgg gccgctctca 7560 cctacatccc cagcatcccc gaccagaccc tccgcgtcgc agcttggatg gtctacggct 7620 tcgtccaggg tctgttctgc accggtgtct ggattctcgg ccatgagtgc ggccacggtg 7680 ctttctctct ccacggaaag gtcaacaatg tgaccggctg gttcctccac tcgttcctcc 7740 tcgtccccta cttcagctgg aagtactctc accaccgcca ccaccgcttc accggccaca 7800 tggatctcga catggctttc gtccccaaga ctgagcccaa gccccccaag tcgctcatga 7860 ttgctggcat tgacgtcgcc gagcttgttg aggacacccc cgctgctcag atggtcaagc 7920 tcatcttcca ccagcttttc ggatggcagg cgtacctctt crtcaacgct agctctggca 7980 agggcagcaa gcagcgggag cccaagactg gcctctccaa gtggttccga gtcagtcact 8040 tcgagcctac cagcgctgtc ttccgcccca acgaggccat ettcatcctc atctccgata 8100 tcggtcttgc tctaatggga actgctctgt actttgcttc caagcaagtt ggtgtttcga 8160 ccattctctt cctctacctt gttccctacc tgtgggttca ccactggctc gttgccatta 8220 cctacctcqa ccaccaccac accgagctcc ctcactacac cgctgagggc tggacctacg 8280 tcaagggagc tctcgccact gtcgaccgtg agtttggctt catcggaaag cacctcttcc 8340 acggtatcat tgagaagcac gttgttcacc atctcttccc taagatcccc ttctacaagg 8400 ctgacgaggc caccgaggcc atcaagcccg tcattggcga ccactactgc cacgacgacc 8460 gaagcttcct gggccagctg tggaccatct tcggcacgct caagtacgtc gagcacgacc 8520 ccgcccgacc cggcgccatg cgatggaaca aggactaggc ggccgcatga gaagataaat 8580 atataaatac attgagatat taaatgcgct agattagaga gcctcatact gctcggagag 8640 aagccaagac gagtactcaa aggggattac accatccata tccacagaca caagctgggg 8700 aaaggttcta tatacacttt ccggaatacc gtagtttccg atgttatcaa tgggggcagc 8760 caggatttca ggcacttcgg tgtctcgggg tgaaatggcg ttcttggcct ccatcaagtc 8820 gtaccatgtc ttcatttgcc tgtcaaagta aaacagaagc agatgaagaa tgaacttgaa 8880 gtgaaggaat ttaaatgtaa cgaaactgaa atttgaccag atattgtgtc cgcggtggag 8940 ctccagcttt tgttcccttt agtgagggtt aatttcgagc ttggcgtaat catggtcata 9000 gctgtttcct gtgtgaaatt gttatccgct cacaagcttc cacacaac 9048

<210> 90 <211 >8925 <212> DNA <213> Artificial Sequence <220> <223> Plasmid p138 <400> 90 gtacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 60 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 120 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 180 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 240 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca B00 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgct tttccatagg 360 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 420 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 480 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 540 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 600 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct'tatccggtaa ctatcgtctt 660 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 720 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 780 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 840 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 900 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatcettt gatcttttct 960 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1020 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1080 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1140 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 1200 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 1260 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 1320 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 1380 agtagttcgc cagttaatag tttgcgcaac grrgttgcca ttgctacagg carcgtggrg 1440 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 1500 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 1560 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 1620 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 1680 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 1740 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 1800 ctctcaagga tettaecgct gttgagatcc agttcgatgt aacccactcg tgcacccaac I860 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 1920 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 1980 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 2040 tgtatttaga aaaacaaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 2100 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 2160 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 2220 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 2280 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 2340 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 2400 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 2460 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 2520 aacgcgaatt ttaacaaaat atraacgctt acaatttcca ttcgccattc aggctgcgca 2580 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 2640 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta 2700 aaacgacggc cagtgaattg taatacgact cactataggg cgaattgggt accgggcccc 2760 ccctcgaggt cgatggtgtc gataagcttg atatcgaatt catgtcacac aaaccgatct 2820 tcgcctcaag gaaacctaat cctacatccg agagactgcc gagatccagt ctacactgat 2880 taattttcgg gccaataatt taaaaaaatc gtgttatata atattatatg tattatatat 2940 atacatcatg atgatactga cagtcatgtc ccattgctaa atagacagac tccatctgcc 3000 gcctccaact gatgttctca atatttaagg ggtcatctcg cattgtttaa taataaacag 3060 actccatcta ccgcctccaa atgatgttct caaaatatat tgtatgaact tatttttatt 3120 acttagtatt attagacaac ccacttgctt tatgaaaaac acttcctatt taggaaacaa 3180 tttataatgg cagttcgttc atttaacaat ttatgtågaa taaatgttat aaatgcgtat 3240 gggaaatctt aaatatggat agcataaatg atatctgcat tgcctaattc gaaatcaaca 3300 gcaacgaaaa aaatcccttg tacaacataa atagtcatcg agaaatatca actatcaaag 3360 aacagctatt cacacgttac tattgagatt attattggac gagaatcaca cactcaactg 3420 tctttctctc ttctagaaat acaggtacaa gtatgtacta ttctcattgt tcatacttct 3480 agtcatttca tcccacatat tccttggatt tctctccaat gaatgacatt ctatcttgca 3540 aattcaacaa ttataataag atataccaaa gtagcggtat agtggcaatc aaaaagcttc 3600 tctggtgtgc ttctcgtatt tatttttatt ctaatgatcc attaaaggta tatatttatt 3660 tcttgttata taatcctttt gtttattaca tgggetggat acataaaggt attttgattt 3720 aattttttgc ttaaattcaa tcccccctcg ttcagtgtca actgtaatgg taggaaatta 3780 ccatactttt gaagaagcaa aaaaaatgaa agaaaaaaaa aatcgtattt ccaggttaga 3840 cgttccgcag aatctagaat gcggtatgcg gtacattgtt cttcgaacgt aaaagttgcg 3900 ctccctgaga tattgtacat ttttgctttt acaagtacaa gtacatcgta eaactatgta 3960 ctactgttga tgcatccaca acagtttgtt ttgttttttt ttgttttttt tttttctaat 4020 gattcattac cgctatgtat acctacttgt acttgtagta agccgggtta ttggcgttca 4080 attaatcata gacttatgaa tctgcacggt gtgcgctgcg agttactttt agcttatgca 4140 tgctacttgg gtgtaatatt gggatctgtt cggaaatcaa cggatgctca accgatttcg 4200 acagtaataa tttgaatcga atcggagcct aaaatgaacc cgagtatatc tcataaaatt 4260 ctcggtgaga ggtctgtgac tgtcagtaca aggtgccttc attatgccct caaccttacc 4320 atacctcact gaatgtagtg tacctctaaa aatgaaatac agtgccaaaa gccaaggcac 4380 tgagctcgtc taacggactt gatatacaac caattaaaac aaatgaaaag aaatacagtt 4440 ctttgtatca tttgtaacaa ttaccctgta caaactaagg tattgaaatc ccacaatatt 4500 cccaaagtcc acccctttcc aaattgtcat gcctacaact catataccaa gcactaacct 4S60 accaaacacc actaaaaccc cacaaaatat atcttaccga atatacagta acaagctacc 4620 accacactcg ttgggtgcag tcgccagctt aaagatatct atccacatca gccacaactc 4680 ccttccttta ataaaccgac tacacccttg gctattgagg ttatgagtga atatactgta 4740 gacaagacac tttcaagaag actgtttcca aaacgtacca ctgtcctcca ctacaaacac 4800 acccaatxtg cttcttctag tcaaggttgc tacaccggta aattataaat catcatttca 4860 ttagcagggc agggcccttt ttatagagtc ttatacacta gcggaccctg ccggtagacc 4920 aacccgcagg cgcgtcagtt tgctccttcc atcaatgcgt cgtagaaacg acttactcct 4980 tcttgagcag ctccttgacc ttgttggcaa caagtctccg acctcggagg tggaggaaga 5040 gcctccgata tcggcggtag tgataccagc ctcgacggac tccCtgacgg cagcctcaac 5100 agcgtcaccg gcgggcttca tgttaagaga gaacttgagc atcatggcgg cagacagaat 5160 ggtggcaatg gggttgaect tctgcttgcc gagatcgggg gcagatccgt gacagggctc 5220 gtacagaccg aacgcctcgt tggtgtcggg cagagaagcc agagaggcgg agggcagcag 5280 acccagagaa ccggggatga cggaggcctc gtcggagatg atatcgccaa acatgttggt 5340 ggtgatgatg ataccattca tcttggaggg ctgcttgatg aggatcatgg cggccgagtc 5400 gatcagctgg tggttgagct cgagctgggg gaartcgtcc ttgaggactc gagtgacagt 5460 ctttcgccaa agtcgagagg aggccagcac gttggccttg tcaagagacc acacgggaag 5520 aggggggttg tgctgaaggg ccaggaaggc ggccattcgg gcaattcgct caacctcagg 5580 aacggagtag gtctcggtgt cggaagcgac gccagatccg tcatcctcct ttcgctctcc 5640 aaagtagata cctccgacga gctctcggac aatgatgaag tcggtgccct caacgcttcg 5700 gatgggggag agatcggcga gcttgggcga cagcagctgg cagggtcgca ggrcggcgta 5760 caggttcagg tcctttcgca gcttgaggag accctgctcg ggtcgcacgt cggttcgtcc 5820 gtcgggagtg gtccatacgg tgttggcagc gcctccgaea gcaccgagca taatagagtc 5880 agcctttcgg cagatgtcga gagtagcgtc ggtgatgggc tcgccctcct tctcaatggc 5940 agctcctcca atgagtcggt cctcaaacac aaactcggtg ccggaggcct cagcaacaga 6000 cttgagcacc ttgacggcct cggcaatcac ctcggggcca cagaagtcgc cgccgagaag 6060 aacaatcttc ttggagtcag tcttggtctt cttagtttcg ggttccattg tggatgtgtg 6120 tggttgtatg tgtgatgtgg tgtgtggagt gaaaatctgt ggctggcaaa cgctcttgta 6180 tatatacgca cttttgcccg tgctatgtgg aagactaaac ctccgaagat tgtgactcag 6240 gtagtgcggt atcggctagg gacccaaacc ttgtcgatgc cgatagcgct atcgaacgta 6300 ccccagccgg ccgggagtat gtcggagggg acatacgaga tcgtcaaggg tttgtggcca 6360 actggtattt aaatgatgtc gacgcagtag gatgtcctgc acgggtcttt ttgtggggtg 6420 tggagaaagg ggtgcttgga gatggaagcc ggtagaaccg ggctgcttgt gcttggagat 6480 ggaagccggt agaaccgggc tgcttggggg gatttggggc cgctgggctc caaagagggg 6540 taggcatttc gttggggtta cgtaattgcg gcatttgggt cctgcgcgca tgtcccattg 6600 gtcagaatta grccggatag gagacttatc agccaatcac agcgccggat ccacctgtag 6660 gttgggttgg gtgggagcac ccctccacag agtagagtca aacagcagca gcaacatgat 6720 agttgggggt gtgcgtgtta aaggaaaaaa aagaagcttg ggttatattc ccgctctatt 6780 tagaggttgc gggatagacg ccgacggagg gcaatggcgc tatggaacct tgcggatatc 6840 catacgccgc ggcggactgc gtccgaacca gctccagcag cgttttttcc gggccattga 6900 gccgactgcg accccgccaa cgtgtcttgg cccacgcact catgtcatgt tggtgttggg 6960 aggccacttt ttaagtagca caaggcacct agctcgcagc aaggtgtccg aaccaaagaa 7020 gcggctgcag tggtgcaaac ggggcggaaa cggcgggaaa aagccacggg ggcacgaatt 7080 gaggcacgcc ctcgaatttg agacgagtca cggccccatt cgcccgcgca atggctcgcc 7140 aacgcccggt cttttgcacc acatcaggtt accccaagcc aaacctttgt gttaaaaagc 7200 ttaacatatt ataccgaacg taggtttggg cgggcttgct ccgtctgtcc aaggcaacat 7260 ttatataagg gtctgcatcg ccggctcaat tgaatctttt ttcttcttct cttctctata 7320 ttcattcttg aattaaacac acatcaacca tggctaccaa gcagccctac cagttcccta 7380 . ctctgaccga gatcaagcga tctcttccct ccgagtgctt tgaagcctcg gtccctctgt 7440 ccttgtacta caccgtgcga atcgtcgcta ttgccgttgc tctggccttc ggactcaact 7500 acgctcgagc ccttcccgtg gtcgagtctc tgtgggcact cgacgctgcc ctttgttgcg 7560 gttacgttct gctccaaggc attgtcttct ggggattctt taccgtgggt cacgatgctg 7620 gacatggtgc cttctctcga taccacctgc tcaactttgt cgttggcacc tttatccact 7680 ccctcattct tactcccttc gagtcgtgga agctcacaca tcgacaccat caeaagaaca 7740 ccggaaacat cgaccgagac gaaatcttct accctcagcg aaaggccgac gatcatcctc 7800 tgtctcgaaa cctcgtcctg gctctcggtg ccgcttggtt tgcctacctt gtcgagggct 7860 ttcctccccg aaaggtcaac caettcaacc ccttcgaacc tctgtttgtg cgacaggtgg 7920 ctgccgttgt catttccctc tctgctcact tcgccgtcct ggcactgtcc gtgtatctga 7980 gctttcagtt cggtctcaag acaatggctc tgtactacta tggacccgtc tccgtgttcg 8040 gctccatgct cgtcattact acctttctgc atcacaatga cgaggaaact ccttggtacg 8100 gagattccga ctggacctac gtcaagggca acttgtcttc cgtggaccga tcttacggtg 8160 ccttcatcga caacctctcg cacaacattg gcacacacca gatccaccat ctgtttccca 8220 tcattcctca ctacaagctc aaccgagcca ccgctgcctt ccaccaggcc tttcccgaac 8280 ttgcccgaaa gagcgacgag cccattctca aggctttctg gagagttggt cgactttacg 8340 ccaactacgg agtcgtggat cccgacgcaa agctgtttac tctcaaggag gccaaagctg 8400 cctccgaggc tgccaccaag accaaggcta cttaagcggc cgcatgagaa gataaatata 8460 taaatacatt gagatattaa atgcgctaga ttagagagcc tcatactgct cggagagaag 8S20 ccaagacgag tactcaaagg ggattacacc atccatatcc acagacacaa gctggggaaa 8580 ggttctatat acactttccg gaataccgta gtttccgatg ttatcaatgg gggcagccag 8640 gatttcaggc acttcggtgt ctcggggtga aatggcgttc ttggcctcca tcaagtcgta 8700 ccatgtcttc atttgcctgt caaagtaaaa cagaagcaga tgaagaatga acttgaagtg 8760 aaggaattta aatgtaacga aactgaaatt tgaccagata ttgtgtccgc ggtggagctc 8820 cagcttttgt tccctttagt gagggttaat ttcgagcttg gcgtaatcat ggtcatagct 8880 gtttcctgtg tgaaattgtt atccgctcac aagcttccac acaac 8925

<210> 91 <211 >8925 <212> DNA <213> Artificial Sequence <220> <223> Plasmid p139 <400> 91 gtacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 60 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 120 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 180 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 240 aaggcggtaa cacggttatc cacagaatca ggggataacg caggaaagaa cacgtgagca B00 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 360 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 420 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 480 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 540 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 600 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 660 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 720 agcagagcga ggtatgcagg cggtgctaca gagttcttga agtggtggcc taactacggc 780 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 840 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 900 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 960 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1020 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1080 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1140 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 1200 acgatacggg agggcttacc atctggcccc ag-tgctgcaa tgataccgcg agacccacgc 1260 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 1320 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 1380 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 1440 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 1500 acatgatccc ccatgttgtg caaaaaagcg grtagctcct tcggtcctcc gatcgttgtc 1560 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 1620 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 1680 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 1740 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 1800 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 1860 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 1920 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt I960 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 2040 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 2100 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 2160 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 2220 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 2280 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 2340 ccatcgccct gatagacggt ttttcgccct rrgacgttgg agtccacgtt ctttaatagt 2400 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 2460 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 2520 aacgcgaatt ttaacaaaat attaacgctt acaatttcca ttcgccattc aggctgcgca 2580 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 2640 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta 2700 aaacgacggc cagtgaattg taatacgact cactataggg cgaattgggt accgggcccc 2760 ccctcgaggt cgatggtgtc gataagcttg atatcgaatt catgtcacac aaaccgatct 2820 tcgcctcaag gaaacctaat tctacatccg agagactgcc gagatccagt ctacactgat 2880 taattttcgg gccaataatt taaaaaaatc gtgttatata atattatatg tattatatat 2940 atacatcatg atgatactga cagtcatgtc ccattgctaa atagacagac tccatctgcc 3000 gcctccaact gatgttctca atatttaagg ggtcatctcg cattgtttaa taataaacag 3060 actccatcta ccgcctccaa atgatgttct caaaatatat tgtatgaact tatttttatt 3120 acttagtatt attagacaac ttacttgctt tatgaaaaac acttcctatt taggaaacaa 3180 tttataatgg cagttcgttc atttaacaat ttatgtagaa taaatgttat aaatgcgtat 3240 gggaaatctt aaatatggat agcataaatg atatctgcat tgcctaattc gaaatcaaca 3300 gcaacgaaaa aaatcccttg tacaacataa atagtcatcg agaaatatca actatcaaag 3360 aacagctatt cacacgttac tattgagatt attattggac gagaatcaca cactcaactg 3420 tctttctctc ttctagaaat acaggtacaa gtatgtacta ttctcattgt tcatacttct 3480 agtcatttca tcccacatat tccttggatt tctctccaat gaatgacatt ctatcttgca 3540 aattcaacaa ttataataag atataccaaa gtagcggtat agtggcaatc aaaaagcttc 3600 tctggtgtgc ttctcgtatt tatttttatt ctaatgatcc attaaaggta tatatttatt 3660 tcttgttata taatcctttt gtttattaca tgggctggat acataaaggt attttgattt 3720 aattttttgc ttaaattcaa tcccccctcg ttcagcgtca actgtaatgg taggaaatta 3780 ccatactttt gaagaagcaa aaaaaatgaa agaaaaaaaa aatcgtattt ccaggttaga 3840 cgttccgcag aatctagaat gcggtatgcg gtacatrgtt cttcgaacgt aaaagttgcg 3900 ctccctgaga tattgtacat ttttgctttt acaagtacaa gtacatcgta caactatgta 3960 ctactgttga tgcatccaca acagtttgtt ttgttttttt ttgttttttt tttttctaat 4020 gattcattac cgctatgtat acctacttgt acttgtagta agccgggtta rtggcgttca 4080 attaatcata gacttatgaa tctgcacggt gtgcgctgcg agttactttt agcttatgca 4140 tgctacttgg gtgtaatatt gggatctgtt cggaaatcaa cggatgctca accgatttcg 4200 acagtaataa tttgaatcga atcggagcct aaaatgaacc cgagtatatc tcataaaatt 4260 ctcggtgaga ggtctgtgac tgtcagtaca aggtgccttc attatgccct caaccttacc 4320 atacctcact gaatgtagtg tacctctaaa aatgaaatac agtgccaaaa gccaaggcac 4380 tgagctcgtc taacggactt gatatacaac caattaaaac aaatgaaaag aaatacagtt 4440 ctttgtatca tttgtaacaa ttaccctgta caaactaagg tattgaaatc ccacaatatt 4500 cccaaagtcc acccctttcc aaattgtcat gcctacaact catataccaa gcactaacct 4560 accaaacacc actaaaaccc cacaaaatat atcttaccga atatacagta acaagctacc 4620 accacactcg ttgggtgcag tcgccagctt aaagatatct atccacatca gccacaactc 4680 ccttccttta ataaaccgac tacacccttg gctattgagg ttatgagtga atatactgta 4740 gacaagaeac tttcaagaag actgtttcca aaacgtacca ctgtcctcea ctacaaacac 4800 acccaatctg cttcttctag tcaaggttgc tacaccggta aattataaat cateatttca 4860 ttagcagggc agggcccttt ttatagagtc ttatacacta gcggaccctg ccggtagacc 4920 aaecegcagg cgcgtcagtt tgctccttcc atcaatgcgt cgtagaaacg acttactcct 4980 tcttgagcag ctccttgacc ttgttggcaa caagtctccg acctcggagg tggaggaaga 5040 gcctccgata tcggcggtag tgataccagc ctcgacggac tccttgacgg cagcctcaac 5100 agcgtcaccg gcgggcttca tgttaagaga gaacttgagc atcatggcgg cagacagaat 5160 ggtggcaatg gggttgacct tctgcttgcc gagatcgggg gcagatccgt gacagggctc 5220 gtacagaccg aacgcctcgt tggtgtcggg cagagaagcc agagaggcgg agggcagcag 5280 acccagagaa ccggggatga cggaggcctc gtcggagatg atatcgccaa acatgttggt 5340 ggtgatgatg ataccattca tcttggaggg ctgcttgatg aggatcatgg cggccgagtc 5400 gatcagccgg tggttgagct cgagctgggg gaattcgtcc ttgaggactc gagtgacagt 5460 ctttcgccaa agtcgagagg aggccagcac gttggccttg tcaagagacc acacgggaag 5520 aggggggttg tgctgaaggg ccaggaaggc ggccattcgg gcaattcgct caacctcagg 5580 aacggagtag gtctcggtgt cggaagcgac gccagatccg tcatcctcct ttcgctctcc 5640 aaagtagata cctccgacga gctctcggac aatgatgaag tcggtgccct caacgtttcg 5700 gatgggggag agatcggcga gcttgggcga cagcagctgg cagggtcgca ggttggcgta 5760 caggttcagg tcctttcgca gcttgaggag accctgctcg ggtcgcacgt eggttcgtcc 5820 gtcgggagtg gtccatacgg tgttggcagc gcctccgaca gcaccgagca taatagagtc 5880 agcctttcgg cagatgtcga gagtagcgtc ggtgatgggc tcgccctcct tctcaatggc 5940 agctcctcca atgagtcggt cctcaaacac aaactcggtg ccggaggcct cagcaacaga 6000 cttgagcacc ttgacggcct cggcaatcac ctcggggcca cagaagtcgc cgccgagaag 6060 aacaatcttc ttggagtcag tcttggtctt cttagtttcg ggttccattg tggatgtgtg 6120 tggttgtatg tgtgatgtgg tgtgtggagt gaaaatctgt ggctggcaaa cgctcttgta 6180 tatatacgca cttttgcccg tgctatgtgg aagactaaac ctccgaagat tgtgactcag 6240 gtagtgcggt atcggctagg gacccaaacc ttgtcgatgc cgatagcgct atcgaacgta 6300 ccccagccgg ccgggagtat gtcggagggg acatacgaga tcgtcaaggg tttgtggcca 6360 accggtattt aaatgatgtc gacgcagtag gatgtcctgc acgggtcttt ttgtggggtg 6420 tggagaaagg ggtgcttgga gatggaagcc ggtagaaccg ggctgcttgt gcctggagat 6480 ggaagccggt agaaccgggc tgcttggggg gatttggggc cgctgggctc caaagagggg 6S40 taggcatttc gttggggtta cgtaattgcg gcatttgggt cctgcgcgca tgtcccattg 6600 gtcagaatta gtccggatag gagacttatc ågccaatcac agcgccggat ccacctgtag 6660 gttgggttgg gtgggagcac ccctccacag agtagagtca aacagcagca gcaacatgat 6720 agttgggggt gtgegtgtta aaggaaaaaa aagaagcttg ggttatattc ccgctctatt 6780 tagaggttgc gggatagacg ccgacggagg gcaatggcgc tatggaacct tgcggatatc 6840 catacgccgc ggcggactgc gtccgaacca gctccagcag cgttttttcc gggccattga 6900 gccgactgcg accccgccaa cgtgtcttgg cccacgcact catgtcatgt tggtgttggg 6960 aggccacttt ttaagtagca caaggcacct agctcgcagc aaggtgtccg aaccaaagaa 7020 gcggctgcag tggtgcaaac ggggcggaaa cggcgggaaa aagccacggg ggcacgaatt 7080 gaggcacgcc ctcgaatttg agacgagtca cggccccatt cgcccgcgca atggctcgcc 7140 aacgcccggt cttttgcacc acatcaggtt accccaagcc aaacctttgt gttaaaaagc 7200 ttaacatatt ataccgaacg taggtttggg cgggcttgct ccgtctgtcc aaggcaacat 7260 ttatataagg gtctgcatcg ccggctcaat tgaatctttt ttcttcttct cttctctata 7320 ttcattcttg aattaaacac acatcaacca tggctaccaa gcagccctac cagctcccta 7380 ctctgaccga gatcaagcga tctctgccct ccgagtgttt cgaggcctcc gtgcctctct 7440 ctctgtacta caccgttcga tgcctggtca ttgctgtgtc gctcgccttc ggacttcacc 7500 atgcacgatc tctgcccgtt gtcgaaggcc tctgggctct ggatgccgct ctctgcaccg 7560 gttacgtgct gctccagggc atcgtcttct ggggattctt tactgttggt cacgacgctg 7620 gacatggtgc cttctcccga taccacctgc tcaactttgt catcggaacc ttcattcact 7680 ctctcatcct tacacccttc gagtcctgga agctcaccca cagacaccat cacaagaaca 7740 ctggeaacat cgaccgagac gaaatcttct accctcaacg aaaggccgac gatcatcctc 7800 tgtctcgaaa ccrcattctg gctttgggtg cagcctggtt tgcctacctg gtcgaaggct 7860 ttcctccccg aaaggtcaac cacttcaacc ccttcgagcc tctctttgtt cgacaggtct 7920 ctgccgtggt catttcgctg gctgcgcact ttggagtggc tgccctgtcc atctacctea 7980 gcctgcagtt cggctccaag actatggcca tctactacta tggrcccgtc tttgtgttcg 8040 gatccatgct cgtcattact acctttcttc atcacaacga cgaagagaca ccttggtacg 8100 cagattcgga gtggacctac gtcaaaggca acctgtcctc tgtcgaccga tcctacggtg 8160 ccctcatcga caacctttct cacaacatcg gaacccacca gattcatcac ctctttccca 8220 tcattcctca ctacaagctc aagcgagcta ccgaggcctt ccatcaagcc tttcccgagc 8280 tggttcgaaa gtccgacgaa cccatcatca aggccttttt cagagtcggc cgactctacg 8340 caaactacgg tgtggtcgac tcggatgcca agctgttcac tctcaaggag gccaaggctg 8400 tttccgaagc cgctaccaag actaaggcca cctaagcggc cgcatgagaa gataaatata 8460 taaatacatt gagatattaa atgcgctaga ttagagagcc tcatactgct cggagagaag 8520 ccaagacgag tactcaaagg ggattacacc atccatatcc acagacacaa gctggggaaa 8580 ggttctatat acactttccg gaataccgta gtttccgatg ttatcaatgg gggcagccag 8640 gatttcaggc acttcggtgt ctcggggtga aatggcgttc ttggcctcca tcaagtcgta 8700 ccatgtcttc atttgcctgt caaagtaaaa cagaagcaga tgaagaatga acttgaagtg 8760 aaggaattta aatgtaacga aactgaaatt tgaccagata ttgtgtccgc ggtggagctc 8820 cagcttttgt tcccrttagt gagggttaat ttcgagcttg gcgtaatcat ggtcatagct 8880 gtttcctgtg tgaaattgtt atccgctcac aagcttccac acaac 8925 <210> 92

<211> 8919 <212> DNA <213> Artificial Sequence <220> <223> Plasmid pY140 <400> 92 gtacgagccg gaagcataaa gtgtaaagce tggggtgcct aatgagtgag ctaactcaca 60 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 120 taatgaatcg geeaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttcegcttcc 180 tcgctcactg actcgctgcg ctcggtcgtc cggctgcggc gagcggtatc agctcactca 240 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 300 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 360 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 420 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 480 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 540 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 600 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 660 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 720 agcagagcga ggtatgtagg cggtgctaca gagrccttga agtggtggcc taactacggc 780 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 840 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 900 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 960 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1020 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1080 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1140 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 1200 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 1260 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 1320 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 1380 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 1440 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 1500 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 1560 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 1620 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 1680 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 1740 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 1800 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 1860 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 1920 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 1980 tttcaatatt attgaagcat ttatcagggt tattgtctea tgagcggata catatttgaa 2040 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 2100 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 2160 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 2220 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 2280 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 2340 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 2400 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 2460 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 2520 aacgcgaatt ttaacaaaat attaacgctt acaatttcca ttcgccattc aggctgcgca 2580 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 2640 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta 2700 aaacgacggc cagtgaattg taatacgact cactataggg cgaattgggt accgggcccc 2760 ccctcgaggt cgatggtgtc gataagcttg atatcgaatt catgtcacac aaaccgatct 2820 tcgcctcaag gaaacctaat tctacatccg agagactgcc gagatccagt ctacactgat 2880 taattttcgg gccaataatt taaaaaaatc gtgttatata atattatatg tattatatat 2940 atacatcatg atgatactga cagtcatgtc ccattgctaa atagacagac tccatctgcc 3000 gcctccaact gatgttctca atatttaagg ggtcatctcg cattgtttaa taataaacag 3060 actccatcta ccgcctccaa atgatgttct caaaatatat tgtatgaact tatttttatt 3120 acttagtatt attagacaac ttacttgctt cacgaaaaac acttcctatt taggaaacaa 3180 tttataatgg cagttcgttc atttaacaat ttatgtagaa taaatgttat aaatgcgtat 3240 gggaaatctt aaatatggat agcataaatg atatctgcat tgcctaattc gaaatcaaca 3300 gcaacgaaaa aaatcccttg tacaacataa atagtcatcg agaaatatca actatcaaag 3360 aacagctatt cacacgttac tattgagatt attattggac gagaatcaca cactcaactg 3420 tctttctctc ttctagaaat acaggtacaa gtatgtacta ttctcattgt tcatacttct 3480 agtcatttca tcccacatat tccttggatt tcirctccaat gaatgacatt ctatcttgca 3540 aattcaacaa ttataataag atataccaaa gtagcggtat agtggcaatc aaaaagcttc 3600 tctggtgtgc ttctcgtatt tatttttatt ctaatgatcc attaaaggta tatatttatt 3660 tcttgttata taatcctttt gtttattaca tgggctggat acataaaggt attttgattt 3720 aattttttgc ttaaattcaa tcccccctcg ttcagtgtca actgtaatgg taggaaatta 3780 ccatactttt gaagaagcaa aaaaaatgaa agaaaaaaaa aatcgtattt ccaggttaga 3840 cgttccgcag aatctagaat gcggtatgcg gtacattgtt cttcgaacgt aaaagttgcg 3900 ctccctgaga tattgtacat ttttgctttt acaagtacaa gtacatcgta caactatgta 3960 ctactgttga tgcatccaca acagtttgtt ttgttttttt ttgttttttt tttttctaat 4020 gattcattac cgctatgtat acctacttgt acttgtagta agccgggtta ttggcgttca 4080 attaatcata gacttatgaa tctgcacggt gtgcgctgcg agttactttt agcttatgca 4140 tgctacttgg gtgtaatatt gggatctgtt cggaaatcaa cggatgctca accgatttcg 4200 acagtaataa tttgaatcga atcggagcct aaaatgaacc cgagtatatc tcataaaatt 4260 ctcggtgaga ggtctgtgac tgtcagtaca aggtgccttc attatgccct caaccttacc 4320 atacctcact gaatgtagtg tacctctaaa aatgaaatac agtgccaaaa gccaaggcac 4380 tgagctcgtc taacggactt gatatacaac caattaaaac aaatgaaaag aaatacagtt 4440 ctttgtatca tttgtaacaa ttaccctgta caaactaagg tattgaaatc ccacaatatt 4500 cccaaagtcc acccctttcc aaattgtcat gcctacaact catataccaa gcactaacct 4560 accaaacacc actaaaaccc cacaaaatat atrcttaccga atatacagta acaagctacc 4620 accacactcg ttgggtgcag ccgccagctt aaagatatct atccacatca gccacaactc 4680 ccttccttta ataaaccgac tacacccttg gctattgagg ttatgagtga atatactgta 4740 gacaagacac tttcaagaag actgtttcca aaacgtacca ctgtcctcca ctacaaacac 4800 acccaatctg cttcttctag tcaaggttgc tacaccggta aattataaat catcatttea 4860 ttagcagggc agggcccttt ttatagagtc ttatacacta gcggaccctg ccggtagacc 4920 aacccgcagg cgcgtcagtt tgctccttcc atcaatgcgt cgtagaaacg acttactcct 4980 tcttgagcag ctccttgacc ttgttggcaa caagtctccg acctcggagg tggaggaaga 5040 gcctccgata tcggcggtag tgataccagc ctcgacggac tccttgacgg cagcctcaac 5100 agcgtcaccg gcgggcttca tgttaagaga gaacttgagc atcatggcgg cagacagaat 5160 ggtggcaatg gggttgacct tctgcttgcc gagatcgggg gcagatccgt gacagggctc 5220 gtacagaccg aacgcctcgt tggtgtcggg cagagaagcc agagaggcgg agggcagcag 5280 acccagagaa ccggggatga cggaggcctc gtcggagatg atatcgccaa acatgttggt 5340 ggtgatgatg ataccattca tcttggaggg ctgcttgatg aggatcatgg cggccgagtc 5400 gatcagctgg tggttgagct cgagctgggg gaattcgtcc ttgaggactc gagtgacagt 5460 ctttcgccaa agtcgagagg aggccagcac gttggccttg tcaagagacc acacgggaag 5520 aggggggttg tgctgaaggg ccaggaaggc ggccattcgg gcaattcgct caacctcagg 5580 aacggagtag gtctcggtgt cggaagcgac gccagatccg tcatcctcct ttcgctctcc 5640 aaagtagata cctccgacga gctctcggac aatgatgaag tcggtgccct caacgtttcg 5700 gatgggggag agatcggcga gcttgggcga cagcagctgg cagggtcgca ggttggcgta 5760 caggttcagg tcctttcgca gcttgaggag accctgctcg ggtcgcacgt cggttcgtcc 5820 gtcgggagtg gcccatacgg tgttggcagc gcctccgaca gcaccgagca taatagagtc 5880 agcctttcgg cagatgtcga gagtagcgtc ggtgatgggc tcgccctcct tctcaatggc 5940 agctcctcca atgagtcggt cctcaaacac aaactcggtg ccggaggcct cagcaacaga 6000 cttgagcacc ttgacggcct cggcaatcac ctcggggcca cagaagtcgc cgccgagaag 6060 aacaatcttc ttggagtcag tcttggtctt cttagtttcg ggttccattg tggatgtgtg 6120 tggttgtatg tgtgatgtgg tgtgtggagt gaaaatctgt ggctggcaaa cgctcttgta 6180 tatatacgca cttttgcccg tgctatgtgg aagactaaac ctccgaagat tgtgactcag 6240 gtagtgcggt atcggctagg gacccaaacc ttgtcgatgc cgatagcgct atcgaacgta 6300 ccccagccgg ccgggagtat gtcggagggg acatacgaga tcgtcaaggg tttgtggcca 6360 actggtattt aaatgatgtc gacgcagtag gatgtcctgc acgggtcttt ttgtggggtg 6420 tggagaaagg ggtgcttgga gatggaagcc ggtagaaccg ggctgcttgt gcttggagat 6480 ggaagccggt agaaccgggc tgcttggggg gatttggggc cgctgggctc caaagagggg 6540 taggcatttc gttggggtta cgtaattgcg gcatttgggt cctgcgcgca tgtcccattg 6600 gtcagaatta gtccggatag gagacttatc agccaatcac agcgccggat ccacctgtag 6660 gttgggttgg gtgggagcac ccctccacag agtagagtca aacagcagca gcaacatgat 6720 agtcgggggt gtgcgtgtta aaggaaaaaa aagaagcttg ggttatattc ccgctctatt 6780 tagaggttgc gggatagacg ccgacggagg gcaatggcgc tatggaacct tgcggatatc 6840 catacgccgc ggcggactgc gtccgaacca gctccagcag cgttttttcc gggccattga 6900 gccgactgcg accccgccaa cgtgtcttgg cccacgcact catgtcatgt tggtgttggg 6960 aggccacttt ttaagtagca caaggcacct agctcgcagc aaggtgtccg aaccaaagaa 7020 gcggctgcag tggtgcaaac ggggcggaaa cggcgggaaa aagccacggg ggcacgaatt 7080 gaggcacgcc ctcgaatttg agacgagtca cggccccatt cgcccgcgca atggctcgcc 7140 aacgcccggt cttttgcacc acatcaggtt accccaagcc aaacctttgt gttaaaaagc 7200 ttaacatatt ataccgaacg taggtttggg cgggcttgct ccgtctgtcc aaggcaacat 7260 ttatataagg gtctgcatcg ccggctcaat tgaatctttt ttcttcttct cttctctata 7320 ttcattcttg aattaaacac acatcaacca tggcttcctc taccgttgcc gctccctacg 7380 agttccctac tctcaccgag atcaagcgat ccctgcctgc ccactgcttc gaagcctctg 7440 ttccctggtc cctctactat accgtgcgag ctctgggcat tgccggttcc cttgctctcg 7500 gactgtacca tgcrcgagcc cttgctatcg tgcaggagtt tgcactgctc gatgccgtcc 7560 tttgcactgg ctacattctg ctccagggta tcgtgttctg gggatccttt accatcggtc 7620 acgactgtgg acatggtgcc ttctcgcgat cccacctgct caacttctct gttggcacac 7680 tcattcactc catcattctg actccctacg agtcgtggaa gatcagccat cgacaccatc 7740 acaagaacac cggcaacatc gacaaggatg agatcttcta ccctcagcga gaagccgact 7800 ctcatcccct gtcccgacac atggtcatct cccttggttc ggcttggttt gcctacctcg 7860 ttgciggatt tcctccccga aaggtcaacc acttcaatcc ctgggagcct ctctacctgc 7920 gaagaatgtc tgccgtcatc atttccctcg gctctctcgt ggcctttgct ggtctgtacg 7980 cctaccttac ctacgtctac ggcctcaaga ccatggctct gtattacttc gcacctctct 8040 ttggattcgc caccatgctg gttgtcacta ccttcctcca tcacaacgac gaggaaactc 8100 cctggtacgc cgattcggag tggacctatg tcaagggcaa cttgtcctct gtggaccgaa 8160 gctacggagc cctcatcgac aacctgtccc acaacattgg tacacatcag atccaccatc 8220 tgtttcccat cattcctcac tacaagctca acgaggccac tgctgccttc gctcaggcct 8280 ttcccgaact ggtgcgaaag tcggcttctc ccatcattcc caccttcatc cgaattggtc 8340 ttatgtacgc caagtacggc gtggtcgaca aggatgccaa gatgtttacc ctcaaggagg 8400 ccaaggctgc caagaccaaa gccaactaag cggccgcatg agaagataaa tatataaata 8460 cattgagata ttaaatgcgc tagattagag agcctcatac tgctcggaga gaagccaaga 8520 cgagtactca aaggggatta caccatccat atccacagac acaagctggg gaaaggttct 8580 atatacactt tccggaatac cgtagtttcc gatgttatca atgggggcag ccaggatttc 8640 aggcacttcg gtgtctcggg gtgaaatggc gttcttggcc tccatcaagt cgtaccatgt 8700 cttcatttgc ctgtcaaagt aaaacagaag cagatgaaga atgaacttga agtgaaggaa 8760 tttaaatgta acgaaactga aatttgacca gatattgtgt ccgcggtgga gctccagctt 8820 ttgttccctt tagtgagggt taatttcgag cttggcgtaa tcatggtcat agctgtttcc 8880 tgtgtgaaat tgttatccgc tcacaagctt ccacacaac 8919

<210> 93 <211 >6267 <212> DNA <213> Artificial sequence <220> <223> Plasmid pY137 <400> 93 taactttggc cggcctttac ctgcaggata acttcgtata atgtatgcta tacgaagtta 60 tgaattctgt aatattggga tctgttcgga aatcaacgga tgctcaaccg atttcgacag 120 taataatttg aatcgaatcg gagcctaaaa tgaacccgag tatatctcat aaaattctcg 180 gtgagaggtc tgtgactgtc agtacaaggt gccttcatta tgccctcaac cttaccatac 240 ctcactgaat gtagtgtacc tctaaaaatg aaatacagtg ccaaaagcca aggcactgag 300 ctcgtctaac ggacttgata tacaaccaat taaaacaaat gaaaagaaat acagttcttt 360 gtatcatttg taacaattac cctgtacaaa ctaaggtatt gaaatcccac aatattccca 420 aagtccaccc ctttccaaat tgtcatgcct acaactcata taccaagcac taacctacca 480 aacaccacta aaaccccaca aaatatatct taccgaatat acagtaacaa gctaccacca S40 cactcgttgg gtgcagtcgc cagcttaaag atatctatcc acatcagcca caactccctt 600 cctttaataa accgactaca cccttggcta ttgaggttat gagtgaatat actgtagaca 660 agacactttc aagaagactg tttccaaaac gtaccactgt cctccactac aaacacaccc 720 aatctgcttc ttctagtcaa ggttgctaca ccggtaaatt ataaatcatc atttcattag 780 cagggcaggg ccctttttat agagtcttat acactagcgg accctgccgg tagaccaacc 840 cgcaggcgcg tcagtttgct ccttccatca atgcgtcgta gaaacgactt actccttctt 900 gagcagctcc ttgaccttgt tggcaacaag tctccgacct cggaggtgga ggaagagcct 960 ccgatatcgg cggtagtgat accagcctcg acggactcct tgacggcagc ctcaacagcg 1020 tcaccggcgg gcttcatgtt aagagagaac ttgagcatca tggcggcaga cagaatggtg 1080 gcaatggggt tgaccttctg cttgccgaga tcgggggcag atccgtgaca gggctcgtac 1140 agaccgaacg cctcgttggt gtcgggcaga gaagccagag aggcggaggg cagcagaccc 1200 agagaaccgg ggatgacgga ggcctcgtcg gagatgatat cgccaaacat gttggtggtg 1260 atgatgatac cattcatctt ggagggctgc ttgatgagga tcatggcggc cgagtcgatc 1320 agctggtggt tgagctcgag ccgggggaat tcgtccttga ggactcgagt gacagtcttt 1380 cgccaaagtc gagaggaggc cagcacgttg gccttgtcaa gagaccacac gggaagaggg 1440 gggttgtgct gaagggccag gaaggcggcc attcgggcaa ttcgctcaac ctcaggaacg 1500 gagtaggtct cggtgtcgga agcgacgcca gatccgtcat cctcctttcg ctctccaaag 1560 tagatacctc cgacgagctc tcggacaatg atgaagtcgg tgccctcaac gtttcggatg 1620 ggggagagat cggcgagctt gggcgacagc agctggcagg gtcgcaggtt ggcgtacagg 1680 ttcaggtcct ttcgcagctt gaggagaccc tgctcgggtc gcacgtcggt tcgtccgtcg 1740 ggagtggtcc atacggtgtt ggcagcgcct ccgacagcac cgagcataat agagtcagcc 1800 tttcggcaga tgtcgagagt agcgtcggtg atgggctcgc cctccttctc aatggcagct 1860 cctccaatga gtcggtcctc aaacacaaac tcggtgccgg aggcctcagc aacagacttg 1920 agcaccttga cggcctcggc aatcacctcg gggccacaga agtcgccgcc gagaagaaca 1980 atcttcttgg agtcagtctt ggtcttctta gtttcgggtt ccattgtgga tgtgtgtggt 2040 tgtatgtgtg atgtggtgtg tggagtgaaa atctgtggct ggcaaacgct cttgtatata 2100 tacgcacttt tgcccgtgct atgtggaaga ctaaacctcc gaagattgtg actcaggtag 2160 tgcggtatcg gctagggacc caaaccttgt cgatgccgat agcgctatcg aacgtacccc 2220 agccggccgg gagtatgtcg gaggggacat acgagatcgt caagggtttg tggccaactg 2280 gtatttaaat gatgtcgact catcgatata acttcgtata atgtatgcta tacgaagtta 2340 tcctaggtat agatctgtta ccggacagaa gtaccccaag ctcaacaaat gggctgtcaa 2400 ccacttcaac cccaacgccc cgctgtttga gaagaaggac tggttcaaca tctggatctc 2460 taacgtcggt attggtatca ccatgtccgt catcgcatac tccatcaacc gatggggcct 2520 ggcttccgtc accctctact acctgatccc ctacctgtgg gtcaaccact ggctcgtggc 2580 catcacctac ctgcagcaca ccgaccccac tctgccccac taccacgccg accagtggaa 2640 cttcacccga ggagccgccg ccaccatcga ccgagagttt ggcttcatcg gctccttctg 2700 cttccatgac atcatcgaga cccacgttct gcaccactac gtgtctcgaa ttcccttcta 2760 caacgcccga atcgccactg agaagatcaa gaaggtcatg ggcaagcact accgacacga 2820 cgacaccaac ttcatcaagt ctctttacac tgtcgcccga acctgccagt ttgttgaagg 2880 taaggaaggc attcagatgt ttagaaacgt caatggagtc ggagttgctc ctgacggcct 2940 gccttctaaa ggcgcgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt 3000 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 3060 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 3120 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 3180 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 3240 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 3300 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 3360 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 3420 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 3480 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 3540 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 3600 tcttgaagtg gtggcctaac tacggccaca ctagaagaac agtatttggt atctgcgctc 3660 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 3720 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 3780 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 3840 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt 3900 aaaaacgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 3960 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 4020 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg 4080 etgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 4140 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta 4200 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 4260 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 4320 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 4380 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 4440 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagargc ttttctgtga 4500 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 4560 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 4620 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt 4680 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 4740 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 4800 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt 4860 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 4920 gcacatttcc ccgaaaagtg ccacctgatg cggtgtgaaa taccgcacag atgcgtaagg 4980 agaaaatacc gcatcaggaa attgtaagcg ttaatatttt gttaaaarrc gcgttaaatt ' 5040 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat 5100 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat 5160 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac 5220 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc S280 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga 5340 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca 5400 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgegcc gctacagggc gcgtccattc 5460 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg SS20 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc 5580 ceagtcacga cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tatagggcga 5640 attgggcccg acgtcgcatg catggattcg accacgcaga ccaacaccgg caccggcaag 5700 gtggccgtgc agccccccac ggccttcatt aagcccattg agaaggtgtc cgagcccgtc 5760 tacgacacct ttggcaacga gttcactcct ccagactact ctatcaagga tattctggat 5820 gccattcccc aggagtgcta caagcggtcc tacgttaagt cctactcgta cgtggcccga 5880 gactgcttct ttatcgccgt ttttgcctac atggcctacg cgtacctgcc tcttattccc 5940 tcggcttccg gccgagctgt ggcctgggcc atgtactcea ttgtccaggg tctgtttggc 6000 accggtctgt gggtrcttgc ccacgagtgt ggccactctg crttctccga ctctaacacc 6060 gtcaacaacg tcaccggatg ggttctgcac tcctccatgc tggtccctta ctacgcctgg 6120 aagctgaccc actccatgca ccacaagtcc actggtcacc tcacccgtga tatggtgttt 6180 gtgcccaagg accgaaagga gtttatggag aaccgaggcg cccatgactg gtctgagctt 6240 gctgaggacg ctcccctcat gattaat 6267

<210> 94 <211> 9570 <212> DNA <213> Artificial Sequence <220> <223> Plasmid pY117 <400> 94 ggccgccacc gcggcccgag attccggcct cttcggccgc caagcgaccc gggtggacgt 60 ctagaggtac ctagcaatta acagatagtt tgccggtgat aattctctta acctcccaca 120 ctcctttgac ataacgattt atgtaacgaa actgaaattt gaccagatat tgtgtccgcg 180 gtggagctcc agcttttgtt ccctttagtg agggtttaaa cgagcttggc gtaatcatgg 240 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa cgtacgagcc 300 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg 360 ttgcgctcac tgcccgcttt ccagtcggga aacetgtcgt gccagctgea ttaatgaatc 420 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact 480 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 540 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 600 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 660 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 720 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 780 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 840 tcacgctgta ggtatctcag ttcggtgtag gtcgtccgct ccaagctggg ctgtgtgcac 900 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 960 ccggtaagac acgacttatc geeactggca gcagccactg gtaacaggat tagcagagcg 1020 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 1080 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 1140 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 1200 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 1260 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atqaaaaagg 1320 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 1380 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 1440 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 1500 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 1560 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 1620 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 1680 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg 1740 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 1800 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 1860 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 1920 ccatccgtaa gatgcttttc cgtgactggt gagtactcaa ccaagtcatt ctgagaatag 1980 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat 2040 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 2100 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 2160 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 2220 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 2280 tattgaagca rttatcaggg ttattgtctc atgagcggat acatatttga atgratttag 2340 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgceacc tgacgcgccc 2400 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 2460 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 2520 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 2580 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc 2640 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 2700 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt 2760 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 2820 tttaacaaaa tattaacgct tacaatttcc attcgccatt caggctgcgc aactgttggg 2880 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg 2940 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg 3000 ccagtgaatt gtaatacgac tcactatagg gcgaattggg taccgggccc cccctcgagg 3060 tcgatggtgt cgataagctt gatatcgaat tcatgtcaca caaaccgatc ttcgcctcaa 3120 ggaaacctaa ttctacatcc gagagaccgc cgagatccag tctacactga ttaattttcg 3180 ggccaataat ttaaaaaaat cgtgttatat aatattatat gtattatata tatacatcat 3240 gatgatactg acagtcatgt cccattgcta aatagacaga ctccatctgc cgcctccaac 3300 tgatgttctc aatatttaag gggtcatctc gcattgttta ataataaaca gactccatct 3360 accgcctcca aatgatgttc tcaaaatata ttgtatgaac ttatttttat tacttagtat 3420 tattagacaa cttacttgct ttatgaaaaa cacttcctat ttaggaaaca atttataatg 3480 gcagttcgtt catttaacaa tttatgtaga ataaatgtta taaatgcgta tgggaaatct 3540 taaatatgga tagcataaat gatatctgca ttgcctaatt cgaaatcaac agcaacgaaa 3600 aaaatccctt gtacaacata aatagtcatc gagaaatatc aactaccaaa gaacagctat 3660 tcacacgtta ctattgagat tattattgga cgagaatcac acactcaact gtctttctct 3720 cttctagaaa tacaggtaca agtatgtact attctcattg ttcatacttc tagtcatttc 3780 atcccacata ttccttggat ttctctccaa tgaatgacat tctatcttgc aaattcaaca 3840 attataataa gatataccaa agtagcggta tagtggcaat caaaaagctt ctctggtgtg 3900 cttctcgtat ttatttttat tctaatgatc cattaaaggt atatatttat ttcttgttat 3960 ataatccttt tgtttattac atgggctgga tacataaagg tattttgatt taattttttg 4020 cttaaattca atcccccctc gctcagtgtc aactgtaatg gtaggaaatt accatacttt 4080 tgaagaagca aaaaaaatga aagaaaaaaa aaatcgtatt tccaggttag acgttccgca 4140 gaatctagaa tgcggtatgc ggtacattgt tcttcgaacg taaaagttgc gctccctgag 4200 atattgtaca tttttgcttt tacaagtaca agtacatcgt acaactatgt actactgttg 4260 atgcatccac aacagtttgt tttgtttttt tttgtttttt ttttttctaa tgattcatta 4320 ccgctatgta tacctacttg tacttgtagt aagccgggtt attggcgttc aattaatcat 4380 agacttatga atctgcacgg tgtgcgctgc gagttacttt tagcttatgc atgctacttg 4440 ggtgtaatat tgggatctgt tcggaaatca acggatgctc aaccgatttc gacagtaatt 4500 aattaattcc ctagtcccag tgtacacccg ccgatatcgc ttaccctgca gccggattaa 4560 9Qttggcaat ttttcacgtc cttgtctccg caattactca ccgggtggtt tataagattg 4620 caagcgtctt gatttgtctc tgtatactaa catgcaatcg cgactcgccc gacgggccac 4680 taacctggcc agaatctcca gatccaagta ttctcttggt ctgcgatatg tttccaacac 4740 aaaagcccct gctgcccagc cggcaactgc tgagtgagta ttccttgcca taaacgaccc 4800 agaaccactg tatagtgttt ggaagcacta gtcagaagac cagcgaaaac aggtggaaaa 4860 aactgagacg aaaagcaacg accagaaatg taatgtgtgg aaaagcgaca cacacagagc 4920 agataaagag gtgacaaata acgacaaatg aaatatcagt atcttcccac aatcactacc 4980 tctcagctgt ctgaaggtgc ggctgatata tccatcccac gtctaacgta tggagtgtga 5040 tagaatatga cgacacaagc atgagaactc gctctctatc caaccaccga aacactgtca 5100 ctacagccgt tcttgttgct ccattcgctt ttgtgattcc atgccttctc tggtgactga 5160 caacattcct tccttttctc cagccctgtt gttatctgct catgacctac ggccactctc S220 tatcgcatac taacatagac gatcccagcc cgctccccac ttccagggca ccgttggcaa 5280 gcctcctatc ctcaagaagg ctgaggctgc caacgctgac atggacgagt ccttcatcgg 5340 aatgtctgga ggagagatct tccacgagat gatgctgcga cacaacgtcg acactgtctt 5400 cggttacccc ggtggagcca ttctccccgt ctttgacgcc attcacaact ctgagtactt 5460 caactttgtg ctccctcgac acgagcaggg tgccggccac atggccgagg gctacgctcg 5520 agcctctggt aagcccggtg tcgttctcgt cacctctggc cccggtgcca ccaacgtcat 5580 cacccccatg caggacgctc tttccgatgg tacccccatg gttgtcttca ccggtcaggt 5640 cctgacctcc gttatcggca ctgacgcctt ccaggaggcc gatgttgtcg gcatctcccg 5700 atcttgcacc aagtggaacg tcatggtcaa gaacgttgct gagctccccc gacgaatcaa 5760 cgaggccttt gagattgcta cttccggccg acccggtccc gttctcgtcg atctgcccaa 5820 ggatgttact getgeeatce tgcgagagcc catccceace aagtccacca ttccctcgca 5880 ttctctgacc aacctcacct ctgccgccgc caccgagttc cagaagcagg ctatccagcg 5940 agccgccaac ctcatcaacc agtccaagaa gcccgtcctt tacgtcggac agggtatcct 6000 tggctccgag gagggtccta agctgcttaa ggagctggct gagaaggccg agattcccgt 6060 caccactact ctgcagggtc ttggtgcctt tgacgagcga gaccccaagt ctctgcacat 6120 gctcggtatg cacggttccg gctacgccaa catggccatg cagaacgctg actgtatcat 6180 tgctctcggc gcccgatttg atgaccgagt taccggctcc atccccaagt ttgcccccga 6240 ggctcgagcc gctgcccttg agggtcgagg tggtattgtt cactttgaga tccaggccaa 6300 gaacatcaac aaggttgttc aggccaccga agccgttgag ggagacgtta ccgagtctgt 6360 ccgacagctc atccccctca tcaacaaggt ctctgccgct gagcgagctc cctggactga 6420 gactatccag tcctggaagc agcagttccc cttcctcttc gaggctgaag gtgaggatgg 6480 tgttatcaag ccccagtccg tcattgctct gctctctgac ctgacagaga acaacaagga 6540 caagaccatc atcaccaccg gtgttggtca gcatcagatg tggactgccc agcatttccg 6600 atggcgacac cctcgaacca tgatcacttc tggtggtctt ggaactatgg gttacggcct 6660 gcccgccgct atcggcgcca aggttgcccg acctgactgc gacgtcattg acatcgatgg 6720 tgacgcttct ttcaacatga ctctgaccga gctgtccacc gccgttcagt tcaacattgg 6780 cgtcaaggct attgtcctca acaacgagga acagggtatg gtcacccagc tgcagtctct 6840 cttctacgag aaccgatact gccacactca tcagaagaac cccgacttca tgaagctggc 6900 cgagtccatg ggcatgaagg gtatccgaat cactcacatt gaccagctgg aggccggtct 6960 caaggagatg ctcgcataca agggccctgt gctcgttgag gttgttgtcg acaagaagat 7020 ccccgttctt cccatggttc ccgctggtaa ggctttgcat gagttccttg tcracgacgc 7080 tgacgccgag gctgcttctc gacccgatcg actgaagaat gcccccgccc ctcacgtcca 7140 ccagaccacc tttgagaact aagtggaaag gaacacaagc aatccgaacc aaaaataatt 7200 ggggtcccgt gcccacagag tctagtgcag acctaaaatg accacagtaa attatagctg 7260 ttattaaaca tgagattttg accaacaaga gcgtaggaat gttattagct actacttgta 7320 catacacagc atttgtttta aataatgttg cctccagggg cagtgagatc aggacccaga 7380 tccgtggcca gctctctgac ttcagaccgc ttgtacttaa gcagctcgca acactgttgt 7440 cgaggattga acttgccata ttcgattttg tggtcatgaa tccagcacac ctcatttaaa 7500 tgtagctaac ggtagcaggc gaactactgg tacatacctc ccccggaata tgtacaggca 7560 taatgcgtat ctgtgggaca tgtggtcgtt gcgccattat gtaagcagcg tgtactcctc 7620 tgactgtcca tatggtttgc tccatctcac cctcatcgtt ttcattgttc acaggcggcc 7680 acaaaaaaac tgtcttctct ccttctctct tcgccttagt ctactcggac cagttttagt 7740 ttagcttggc gccactggat aaatgagacc tcaggccttg tgatgaggag gtcacttatg 7800 aagcatgtta ggaggtgctt gtatggatag agaagcaccc aaaataataa gaataataat 7860 aaaacagggg gcgttgtcat ttcatatcgt gttttcacca tcaatacacc tccaaacaat 7920 gcccttcatg tggccagccc caatattgtc ctgtagttca actctatgca gctcgtatct 7980 tattgagcaa gtaaaactct gtcagccgat attgcccgac ccgcgacaag ggtcaacaag 8040 gtggtgtaag gccttcgcag aagtcaaaac tgtgccaaac aaacatctag agtctctttg 8100 gtgtttctcg catatatttw atcggctgtc ttacgtattt gcgcctcggt accggactaa 8160 tttcggatca tccccaatac gctttttctt cgcagctgtc aacagtgtcc atgatctatc 8220 cacctaaatg ggtcatatga ggcgtataat ttcgtggtgc tgataataat tcccatatat 8280 ttgacacaaa acttcccccc ctagacatac atctcacaat ctcacttctt gtgcttctgt 8340 cacacatctc ctccagctga cttcaactca cacctctgcc ccagttggtc tacagcggta 8400 taaggtttct ccgcatagag gtgcaccact cctcccgata cttgtttgtg tgacttgtgg 8460 gtcacgacat atatatctac acacattgcg ccaccctttg gttcttccag cacaacaaaa 8520 acacgacacg ctaaccatgg ccaatttact gaccgtacac caaaatttgc ctgcattacc 8580 ggtcgatgca acgagtgatg aggttcgcaa gaacctgatg gacatgttca gggatcgcca 8640 ggcgttttct gagcatacct ggaaaatgct tctgtccgtt tgccggtcgt gggcggcatg 8700 gtgcaagttg aataaccgga aatggtttcc cgcagaacct gaagatgttc gcgattatct 8760 tctatatctt caggcgcgcg gtctggcagt aaaaactatc cagcaacatt tgggccagct 8820 aaacatgctt catcgtcggt ccgggctgcc acgaccaagt gacagcaatg ctgtttcact 6880 ggttatgcgg cggatccgaa aagaaaacgt tgatgccggt gaacgtgcaa aacaggctct 8940 agcgttcgaa cgcactgatt tcgaccaggt tcgttcactc atggaaaata gcgatcgctg 9000 ccaggatata cgtaatctgg catttctggg gattgcttat aacaccctgt tacgtatagc 9060 cgaaattgcc aggatcaggg ttaaagatat ctcacgtact gacggtggga gaatgttaat 9120 ccatattggc agaacgaaaa cgctggttag caccgcaggt gtagagaagg cacttagcct 9180 gggggtaact aaactggtcg agcgatggat ttccgtctct ggtgtagctg atgatccgaa 9240 taactacctg ttttgccggg tcagaaaaaa tggtgttgcc gcgccatctg ccaccagcca 9300 gctatcaact cgcgccctgg aagggatttt tgaagcaact catcgattga ttcacggcgc 9360 taaggatgac tctggtcaga gatacctggc ctggtctgga cacagtgccc gtgtcggagc 9420 cgcgcgagat atggcccgcg ctggagtttc aataccggag atcatgcaag ctggtggctg 9480 gaccaatgta aatattgtca tgaactatat ccgtaacctg gatagtgaaa caggggcaat 9540 gQtQcgcctg ctggaagatg gcgattaagc 9570

<210> 95 <211 > 358 <212> PRT <213> saprolegnia diclina (GenBank Accession No. AAR20444) <400> 95

Met Thr Giu Asp Lys Thr Lys val Glu Phe Pro Thr Leu Thr Glu Leu 15 10 15

Lys His Ser lie Pro Asn Ala Cys Phe Glu Ser Asn Leu Gly Leu Ser 20 25 30

Leu Tyr Tyr Thr Ala Arg Ala lie Phe Asn Ala ser Ala Ser Ala Ala 35 40 45

Leu Leu Tyr Ala Ala Arg ser Thr Pro Phe lie Ala Asp Asn val Leu 50 55 60

Leu His Ala Leu val cys Ala Thr Tyr lie Tyr val Gin Gly val lie 65 70 75 60

Phe Trp Gly Phe Phe Thr val Gly His Asp Cys Gly His Ser Ala Phe 85 90 95

Ser Arg Tyr His Ser val Asn Phe lie lie Gly cys lie Met His ser 100 105 110

Ala lie Leu Thr Pro Phe Glu Ser Trp Arg val Thr His Arg His His 115 120 125

His Lys Asn Thr Gly Asn ile Asp Lys Asp Glu lie Phe Tyr Pro His 130 135 140

Arg ser val Lys Asp Leu Gin Asp val Arg Gin Trp val Tyr Thr Leu 145 150 155 160

Gly Gly Ala Trp Phe val Tyr Leu Lys val Gly Tyr Ala Pro Arg Thr 165 170 175

Met Ser His Phe Asp Pro Trp Asp Pro Leu Leu Leu Arg Arg Ala Ser 180 185 190

Ala val lie val ser Leu Gly val Trp Ala Ala Phe Phe Ala Ala Tyr 195 200 205

Ala Tyr Leu Thr Tyr Ser Leu Gly Phe Ala Val Met Gly Leu Tyr Tyr 210 215 220

Tyr Ala Pro Leu Phe val Phe Ala ser Phe Leu Val lie Thr Thr Phe 225 230 235 240

Leu His His Asn Asp Glu Ala Thr Pro Trp Tyr Gly Asp ser Glu Trp 245 250 255

Thr Tyr val Lys Gly Asn Leu Ser Ser Val Asp Arg Ser Tyr Gly Ala 260 265 270

Phe val Asp Asn Leu Ser His His lie Gly Thr His Gin val His His 275 280 285

Leu Phe Pro lie lie Pro His Tyr Lys Leu Asn Glu Ala Thr Lys His 290 295 300

Phe Ala Ala Ala Tyr Pro His Leu val Arg Arg Asn Asp Glu Pro lie 305 310 315 320 lie Thr Ala Phe Phe Lys Thr Ala His Leu Phe val Asn Tyr Gly Ala 325 330 335 val Pro Glu Thr Ala Gin lie Phe Thr Leu Lys Glu Ser Ala Ala Ala 340 345 350

Ala Lys Ala Lys ser Asp 355

<210> 96 <211 > 9 <212> PRT <213> Artificial Sequence <220> <223> Delta-17 Desaturase Motif #1 <220> <221 > misc_feature <222> (3)..(3) <223> Xaa can be any naturally occurring amino acid <220> <221 > misc_feature <222> (7)..(7) <223> Xaa can be any naturally occurring amino acid <400> 96

Phe Thr Xaa Gly His Asp Xaa Gly His 1 5

<210> 97 <211 > 9 <212> PRT <213> Artificial Sequence <220> <223> Delta-17 Desaturase Motif #2 <400> 97

His Arg His His His Lys Asn Thr Gly

<210> 98 <211 > 11 <212> PRT <213> Artificial Sequence <220> <223> Delta-17 Desaturase Motif #3 <220> <221 > misc_feature <222> (6)..C6) <223> xaa can be any naturally occurring amino acid <400> 98 lie Gly Thr His Gin xaa His His Leu Phe Pro 15 10

<210> 99 <211 > 5 <212> PRT <213> Artificial sequence <220> <223> His-rich motif <220> <221 > misc_feature <222> (2)..(4) <223> xaa can be any naturally occurring amino acid <400> 99

His xaa xaa xaa His 1 5

<210> 100 <211 > 5 <212> PRT <213> Artificial Sequence <220> <223> His-rich motif <220> <221 > misc_feature <222> (2)..(3) <223> Xaa can be any naturally occurring amino acid <400>100

His xaa xaa His His 1 5

<210> 101 <211 > 5 <212> PRT <213> Artificial sequence <220> <223> His-rich motif <220>

<221 > MISCFEATURE <222> (1)..(1) <223> Xaa = His [H] or Gin [Q] <220> <221 > misc_feature <222> (2)..(3) <223> Xaa can be any naturally occurring amino acid <400> 101

Xaa xaa Xaa His His 1 S

<210> 102 <211 >8067 <212> DNA <213> Artificial Sequence <220> <223> Plasmid pFBAINPaD175 <400> 102 ggccgcaagt gtggatgggg aagtgagtgc ccggttctgt gtgcacaatt ggcaatccaa 60 gatggatgga ttcaacacag ggatatagcg agctacgtgg tggtgcgagg atatagcaac 120 ggatatttat gtttgacact tgagaatgta cgatacaagc actgtccaag tacaatacta 180 aacatactgt acatactcat actcgtaccc gggcaacggt ttcacttgag tgcagtggct 240 agtgctctta ctcgtacagt gtgcaatact gcgtatcata gtctttgatg tatatcgtat 300 tcattcatgt tagttgcgta cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 360 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 420 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 480 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag S40 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 600 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 660 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 720 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 780 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 840 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 900 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 960 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 1020 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 1080 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 1140 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 1200 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 1260 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 1320 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 1380 gtrttaaatc aatctaaagt atatatgagt aaactcggtc tgacagttac caatgcttaa 1440 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 1S00 ccgtcgtgta garaactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 1560 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 1620 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 1680 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 1740 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 1800 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 1860 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 1920 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 1980 actcaaccaa gtcattctga gaacagtgta tgcggcgacc gagttgctct tgcccggcgt 2040 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 2100 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 2160 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 2220 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 2280 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 2340 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 2400 cccgaaaagt gccacctgac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 2460 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct 2520 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 2580 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg 2640 atggttcacg tagtgggcca tcgceetgat agacggtttt tcgccctttg acgttggagt 2700 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 2760 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc 2820 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttccattc 2880 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg 2940 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc 3000 ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tacagggcga 3060 attgggtacc gggccccccc tcgaggtcga tggtgtcgat aagcttgata tcgaartcat 3120 gtcacacaaa ccgatcttcg cctcaaggaa acctaattct acatccgaga gactgccgag 3180 atccagtcta cactgattaa ttttcgggcc aataatttaa aaaaatcgtg ttatataata 3240 ttatatgtat tatacatata catcatgatg atactgacag tcatgtccca ttgctaaata 3300 gacagactcc atctgccgcc tccaactgat gttctcaata tttaaggggt catctcgcat 3360 tgtttaataa taaacagact ccatctaccg cctccaaatg atgttctcaa aatatattgt 3420 atgaacttat ttttattact tagtattatt agacaactta cttgctttat gaaaaacact 3480 tcccatttag gaaacaattt ataatggcag ttcgttcatt taacaattta tgtagaataa 3540 atgttataaa tgcgtatggg aaatcttaaa tatggatagc ataaatgata tctgcattgc 3600 ctaattcgaa atcaacagca acgaaaaaaa tceettgtac aacataaata gtcatcgaga 3660 aatatcaact atcaaagaac agctattcac acgttactat tgagattatt attggacgag 3720 aatcacacac tcaactgrct ttctctcttc tagaaataca ggtacaagta tgtactattc 3780 tcattgttca tacttctagt catrrcatcc cacatattcc ttggatttct ctccaatgaa 3840 tgacattcta tcttgcaaat tcaacaatta taataagata taccaaagta gcggtatagt 3900 ggcaatcaaa aagcttctct ggtgtgcttc tcgtatttat ttttattcta atgatccatt 3960 aaaggtatat atttatttct tgttatataa tccttttgtt tattacatgg gctggataca 4020 taaaggtatt ttgatttaat tttttgctta aattcaatcc cccctcgttc agtgtcaact 4080 gtaatggtag gaaattacca tacttttgaa gaagcaaaaa aaatgaaaga aaaaaaaaat 4140 cgtatttcca ggttagacgt tccgcagaat ctagaatgcg gtatgcggta cattgttctt 4200 cgaacgtaaa agttgcgctc cctgagatat tgtacatttt tgcttttaca agtacaagta 4260 catcgtacaa ctatgtacta ctgttgatgc atccacaaca gtttgttttg tttttttttg 4320 tttttttttt ttctaatgat tcattaccgc tatgtatacc tacttgtact tgtagtaagc 4380 cgggttattg gcgttcaatt aatcatagac ttatgaatct gcacggtgtg cgctgcgagt 4440 tacttttagc ttatgcatgc tacttgggtg taatattggg atctgttcgg aaatcaacgg 4500 atgctcaatc gatttcgaca gtaattaatt aagtcataca caagtcagct ttcttcgagc 4560 ctcatacaag tataagtagr tcaacgtatt agcactgtac ccagcatctc cgtatcgaga 4620 aacacaacaa catgccccat tggacagatc atgcggatac acaggttgtg cagtatcata 4680 catactcgat cagacaggtc gtctgaccat catacaagct gaacaagcgc tccatacttg 4740 cacgctctct atatacacag ttaaattaca tatccatagt ctaacctcta acagttaatc 4800 ttctggtaag cctcccagcc agccttctgg tatcgcttgg cctcctcaat aggatctcgg 4860 ttcrggccgc acagacctcg gccgacaatt atgatatccg ttccggtaga catgacatcc 4920 tcaacagttc ggtactgctg tccgagagcg tctcccttgt cgtcaagacc caccccgggg 4980 gtcagaataa gccagtcctc agagtcgccc ttaggtcggc tctgggcaat gaagccaacc 5040 acaaactcgg ggtcggatcg ggcaagctca atggtctgct tggagtactc gccagtggcc 5100 agagagccct tgcaagacag ctcggccagc atgagcagac ctctggccag cttctcgttg 5160 ggagagggga ctaggaactc cttgtactgg gagttctcgt agtcagagac gtcctccttc 5220 ttctgttcag agacagtttc ctcggcacca gctcgcaggc cagcaatgat tccggttccg 5280 ggtaeacegt gggcgttggt gatatcggac cactcggcga ttcggtgaea ccggtactgg 5340 tgcttgacag tgttgccaat atctgcgaac tttctgtcct cgaacaggaa gaaaccgtgc 5400 ttaagagcaa gttccttgag ggggagcaca gtgccggcgt aggtgaagtc gtcaatgatg 5460 tcgatatggg ttttgatcat gcacacataa ggtccgacct tatcggcaag ctcaatgagc 5520 tccttggtgg tggtaacatc cagagaagca cacaggttgg ttttcttggc tgccacgagc 5580 ttgagcactc gagcggcaaa ggcggacttg tggacgttag ctcgagcttc gtaggagggc 5640 attttggtgg tgaagaggag actgaaataa atttagtctg cagaactttt tatcggaacc 5700 ttatctgggg cagtgaagta tatgttatgg taatagttac gagttagttg aacttataga 5760 tagactggac tatacggcta tcggtccaaa ttagaaagaa cgtcaatggc tctctgggcg 5820 tcgcctttgc cgacaaaaat gtgatcatga tgaaagccag caatgacgtt gcagctgata 5880 ttgttgtcgg ccaaccgcgc cgaaaacgca gcrgtcagac ccacagcctc caacgaagaa 5940 tgtatcgtca aagtgatcca agcacactca tagttggagt cgtactccaa aggcggcaat 6000 gacgagtcag acagatactc gtcgaaaaca gtgtacgcag atctactata gaggaacatt 6060 taaattgccc cggagaagac ggccaggccg cctagatgac aaattcaacå actcacagct 6120 gactttctgc cattgccact aggggggggc ctttttatat ggccaagcca agctctccac 6180 gtcggttggg ctgcacccaa caataaatgg gtagggttgc accaacaaag ggatgggatg 6240 gggggtagaa gatacgagga taacggggct caatggcaca aataagaacg aatactgcca 6300 ttaagactcg tgatccagcg actgacacca trgcatcatc taagggcctc aaaactacct 6360 cggaactgct gcgctgatct ggacaccaca gaggttccga gcactttagg ttgcaccaaa 6420 tgtcccacca ggtgcaggca gaaaacgctg gaacagcgtg tacagtttgt cttaacaaaa 6480 agtgagggcg ctgaggtcga gcagggtggt gtgacttgtt atagccttta gagctgcgaa 6S40 agcgcgtatg gatttggctc atcaggccag attgagggtc tgtggacaca tgtcatgtta 6600 gtgtacttca atcgccccct ggatatagcc ccgacaatag gccgtggcct catttttttg 6660 ccttccgcac atttccattg ctcggtaccc acaccttgct tctcctgcac ttgccaacct 6220 taatactggt ttacattgac caacatctta caagcggggg gcttgtctag ggtatatata 6780 aacagtggct ctcccaatcg gttgccagtc tcttttttcc tttcrttccc cacagattcg 6840 aaatctaaac tacacatcac agaattccga gccgtgagta tccacgacaa gatcagtgtc 6900 gagacgacgc gttttgtgta atgacacaat ccgaaagtcg ctagcaacac acactctcta 6960 caeaaactaa cccagctctg gtaccatggc ttcctctacc gttgccgctc cctacgagtt 7020 ccctactctc accgagatca agcgatccct gcctgcccac tgcttcgaag cctctgttcc 7080 ctggtccctc tactataccg tgcgagctct gggcattgcc ggttcccttg ctctcggact 7140 gtactatgct cgagcccttg ctatcgtgca ggagtttgca ctgctcgatg ccgtcctttg 7200 cactggctac attctgctcc agggtatcgt gttctgggga ttctttacca tcggtcacga 7260 ctgtggacat ggtgccttct cgcgatccca cctgctcaac ttctctgttg gcacactcat 7320 tcactccatc attctgactc cctacgagtc gtggaagatc agccatcgac accatcacaa 7380 gaacaccggc aacatcgaca aggatgagat cttetaccct cagcgagaag ccgactctca 7440 tcccctgtcc cgacacatgg tcatctccct tggttcggct tggtttgcct acctcgttgc 7500 tggatttcct ccccgaaagg teaaccactt caatccctgg gagcctctct acctgcgaag 7560 aatgtctgcc gtcatcattt ccctcggctc tctcgtggcc tttgctggtc tgtacgccta 7620 ccttacctac gtctacggcc tcaagaccat ggcrctgtat tacttcgcac ctctcttrgg 7680 attcgccacc atgctggttg tcactacctt cctccatcac aacgacgagg aaactccctg 7740 gtacgccgat tcggagtgga cctatgtcaa gggcaacttg tcctctgtgg accgaagcta 7800 cggagccctc atcgacaacc tgtcccacaa catrtggtaca catcagatcc accatctgtt 7860 tcccatcatt cctcactaca agctcaacga ggccactgct gccttcgctc aggcctttcc 7920 cgaactggtg cgaaagtcgg cttctcccat cattcccacc ttcatccgaa ttggtcttat 7980 gtacgccaag tacggcgtgg tcgacaagga tgccaagatg tttaccctca aggaggccaa 8040 ggctgccaag accaaagcca actaagc 8067

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This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

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Claims (10)

1. DELTA 17-DESATURASE OG ANVENDELSE HERAF VED FREMSTILLING AF FLERUMÆTTEDE FEDTSYRER

   1. Isoleret nukleinsyremolekyle, der koder for: a) et A17-desaturaseenzym, hvor enzymet har aminosyresekvensen ifølge SEQ ID NO: 2 eller SEQ ID NO: 3; eller b) isoleret nukleotidmolekyle, der er fuldt komplementært til a).
2. 2. Isoleret nukleinsyremolekyle ifølge krav 1, hvor det isolerede nukleinsyremolekyle er udvalgt fra gruppen bestående af SEQ ID NO: 1 og SEQ ID NO: 4.
3. 3. Kimært gen omfattende det isolerede nukleinsyremolekyle ifølge krav 1, der er operativt forbundet med egnede regulatoriske sekvenser.
4. 4. Transformeret værtscelle omfattende det isolerede nukleinsyremolekyle ifølge krav 1.
5. 5. Transformeret værtscelle ifølge krav 4, hvor værtscellen er udvalgt fra gruppen bestående af alger, bakterier, gær, olieholdig gær, ægsporesvampe, svampe, Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon og Lipomyces.
6. 6. Fremgangsmåde til fremstilling af flerumættede fedtsyrer, hvilken fremgangsmåde omfatter: a) tilvejebringelse af en værtscelle omfattende: (i) et isoleret nukleotidmolekyle, der koder for et bifunktionelt A17/A15-desaturase-polypeptid med aminosyresekvensen ifølge SEQ ID NO: 2 eller SEQ ID NO: 3; og (ii) en kilde af mindst én substratfedtsyre, der er udvalgt fra gruppen bestående af: linolsyre, eicosadiensyre, dihomo-y-linolensyre og arachidonsyre: b) dyrkning af værtscellen fra trin (a) under forhold, hvor nukleinsyremolekylet, der koder for det bifunktionelle A17/A15-desaturase-polypeptid, udtrykkes og den mindst ene substratfedtsyre omdannes til mindst én produktfedtsyre, hvor reaktionen er udvalgt fra gruppen bestående af: linolsyre omdannes til (X-linolensyre, eicosadiensyre omdannes til eicosatriensyre, dihomo-y-linolensyre omdannes til eicosatetraensyre, og arachidonsyre omdannes til eicosapentaensyre; og c) eventuel genvinding af den mindst ene produktfedtsyre fra trin (b). 7 Fremgangsmåde ifølge krav 6, hvor: a) det isolerede nukleinsyremolekyle har en nukleinsyresekvens, der er udvalgt fra gruppen bestående af SEQ ID NO: 1 og SEQ ID NO: 4; og b) værtscellen er Yarrowia lipolytica.
7. 8. Fremgangsmåde ifølge krav 6, hvor værtscellen er udvalgt fra gruppen bestående af: alger, bakterier, gær, ægsporesvampe og svampe.
8. 9. Fremgangsmåde ifølge krav 8, hvor værtscellen er udvalgt fra gruppen bestående af Thraustochytrium sp., Schizochytrium sp., Mortierella sp., en olieholdig gær, Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon og Lipomyces.
9. 10. Fremgangsmåde til identifikation og isolering af et Δ17-dcsaturasepolypeptid, hvilken fremgangsmåde omfatter: a) undersøgelse af et genomisk bibliotek med: i) et isoleret nukleinsyrefragment, der koder for en aminosyresekvens udvalgt fra gruppen bestående af SEQ ID NO: 96-98; eller ii) et isoleret nukleinsyrefragment, der er komplementært til (i); b) identifikation af en DNA-klon, der hybridiserer med nukleinsyrefragmentet fra trin (a); og c) sekvensering af det genomiske fragment, der omfatter klonet identificeret i trin (b); hvor det sekvenserede genomiske fragment koder for et A17-desaturase-polypeptid.
10. 11. Fremgangsmåde til identifikation og isolering af et A17-desaturase-polypeptid, hvilken fremgangsmåde omfatter: a) syntetisering af mindst én oligonukleotid-primer svarende til en del af en isoleret nukleinsyresekvens, der koder for et aminosyremønster udvalgt fra gruppen bestående af SEQ ID NO: 96-98; og b) amplifikation af en indsætning til stede i en kloningsvektor ved anvendelse af oligonukleotid-primeren fra trin (a); hvor den amplificerede indsætning koder for en del af en aminosyresekvens, der koder for et Δ17-desaturase-enzym.