Microorganism producing eicosapentaenoic acid and method for producing eicosapentaenoic acid

Abstract

The object of the present invention is to provide a microorganism that efficiently produces EPA and a method for producing EPA using the microorganism. The present invention relates to the following microorganisms, etc.: It is a microorganism capable of producing docosahexaenoic acid (DHA), which comprises a protein (mutant OrfB) composed of an amino acid sequence in which at least one of the amino acid residues at positions 6, 65, 230, 231 and 275 in the amino acid sequence represented by SEQ ID NO: 2 is replaced by other amino acid residues, and can produce eicosapentaenoic acid.

Description

Technical Field

[0001] The present invention relates to a microorganism producing eicosapentaenoic acid and a method for producing eicosapentaenoic acid using the microorganism. Background Art

[0002] Long-chain fatty acids having multiple unsaturated bonds in the molecule, such as docosahexaenoic acid (hereinafter referred to as DHA), eicosapentaenoic acid (hereinafter referred to as EPA), arachidonic acid (hereinafter referred to as ARA), and docosapentaenoic acid (hereinafter referred to as DPA), are referred to as polyunsaturated fatty acids (hereinafter referred to as PUFA). PUFA is known to have various physiological functions such as prevention of arteriosclerosis and hyperlipidemia (Non-Patent Documents 1 and 2).

[0003] As biosynthetic pathways for PUFA, two types are known: an aerobic pathway and an anaerobic pathway using polyunsaturated fatty acid polyketide synthase (hereinafter referred to as PUFA-PKS). The aerobic pathway is a pathway for synthesizing PUFA by introducing double bonds of long-chain fatty acids such as palmitic acid synthesized by fatty acid synthase by various desaturases or extending carbon chains by chain elongases, and is a synthetic pathway that has long been known to exist in many organisms (Non-Patent Document 3).

[0004] On the other hand, the PUFA-PKS-based anaerobic pathway is a pathway for synthesizing PUFA from malonyl-CoA (CoA), and it is known that some marine bacteria and myxomycete eukaryotic organisms have this pathway (Non-Patent Documents 4 and 5).

[0005] PUFA-PKS is a complex enzyme composed of a plurality of proteins (hereinafter also referred to as a protein complex), and each protein contains a plurality of functional domains involved in the synthesis of PUFA.

[0006] The functional domains present in PUFA-PKS include: a β-ketoacyl-acyl carrier protein synthase domain (hereinafter referred to as KS domain) that is believed to be involved in the condensation of malonyl ACP and acyl ACP; an acyl carrier protein domain (hereinafter referred to as ACP domain) that is believed to function as a site for fatty acid synthesis by binding to the acyl group via a thioester bond with the phosphopantetheine group; a ketoreductase domain (hereinafter referred to as KR domain) that is believed to reduce the carbonyl group generated by the condensation; and a DH domain that is believed to dehydrate the hydroxyl group generated by the KR domain to form a double bond. domain; a chain elongation factor domain (hereinafter referred to as the CLF domain) believed to be involved in the elongation of the carbon chain; an enoyl reductase domain (hereinafter referred to as the ER domain) believed to reduce the resulting double bond; an acyltransferase domain (hereinafter referred to as the AT domain) and a malonyl-CoA:acyltransferase domain (hereinafter referred to as the MAT domain) believed to be involved in the transfer of the acyl group; and a phosphopantetheinyltransferase domain (hereinafter referred to as the PPT domain) believed to activate the ACP domain. It is believed that these multiple domains extend the carbon chain of fatty acids by acting synergistically.

[0007] It is known that the types of PUFA produced vary depending on the type of PUFA-PKS. For example, PUFA-PKS derived from Schizochytrium sp., Aurantiochytrium sp., and Moritella marina produce DHA as a main product, PUFA-PKS derived from Shewanella oneidensis and Photobacterium profundum produce EPA as a main product, and PUFA-PKS derived from Aureispira marina produces ARA as a main product, and produces almost no other PUFA, or even if other PUFA is produced, it is a small amount compared to the main product.

[0008] As can be seen, PUFA-PKS has high product specificity, and many studies have been conducted so far with the goal of analyzing the functions of PUFA-PKS. In Non-Patent Documents 4 and 6, studies were conducted on cloning PUFA-PKS genes from Shewanella bacteria or stramenopile eukaryotic organisms and expressing them in heterologous organisms to produce PUFA.

[0009] In non-patent document 7, through research using the pfaB gene, which is a constituent gene of PUFA-PKS derived from Moritella marina that produces DHA, and the pfaB gene that constitutes PUFA-PKS derived from Shewanella pneumatophori that produces EPA, it is disclosed that the pfaB gene encoding the AT domain is related to the type of PUFA produced.

[0010] Non-Patent Document 8 discloses that when the DH domain of PUFA-PKS derived from Thraustochytrium is introduced into Escherichia coli, the production of fatty acids increases and the ratio of unsaturated fatty acids increases.

[0011] As an industrial production method for EPA, purification from fish oil and the like is known, but there is a problem of producing many by-products (Patent Document 2).

[0012] Prior art literature

[0013] Patent Literature

[0014] Patent Document 1: International Publication No. 2008 / 144473

[0015] Patent Document 2: Japanese Patent Application Publication No. 2013-055893

[0016] Non-patent literature

[0017] Non-patent document 1: Annu. Nutr. Metabol., 1991, 35, 128-131

[0018] Non-patent document 2: J. Am. Coll. Nutr., 1994, 13, 658-664

[0019] Non-patent document 3: Ann. Rev. Biochem., 1983, 52, 537-579

[0020] Non-patent document 4: Science, 2001, 293, 290-293

[0021] Non-patent document 5: PLoS One, 2011, 6, e20146

[0022] Non-patent document 6: Plant Physiol. Biochem., 2009, 47, 472-478

[0023] Non-patent document 7: FEMS Microbiol. Lett., 2009, 295, 170-176

[0024] Non-patent literature 8: Appl. Microbiol. Biotechnol., 2018, 847-856 Summary of the invention

[0025] Problems to be solved by the invention

[0026] As described above, as an industrial production method for EPA, a method of purifying EPA from fish oil is used, but there are problems such as a large number of by-products and poor production efficiency, and therefore, an efficient production method for EPA is desired.

[0027] Therefore, an object of the present invention is to provide a microorganism that efficiently produces EPA and a method for producing EPA using the microorganism.

[0028] Methods used to solve problems

[0029] The present inventors have found that PUFA containing EPA at a high concentration can be produced by expressing OrfB in which a mutation is introduced into a specific amino acid residue in a microorganism capable of producing DHA, thereby completing the present invention.

[0030] The present invention relates to the following contents.

[0031] 1. A microorganism having the ability to produce DHA, comprising a protein consisting of an amino acid sequence in which at least one of the amino acid residues at positions 6, 65, 230, 231 and 275 in the amino acid sequence represented by SEQ ID NO: 2 is replaced by other amino acid residues (hereinafter referred to as mutant OrfB), and capable of producing eicosapentaenoic acid (hereinafter referred to as EPA).

[0032] 2. A microorganism having the ability to produce DHA, comprising a protein consisting of the following amino acid sequence (hereinafter referred to as a mutant OrfB homolog), and capable of producing EPA, wherein the amino acid sequence is an amino acid sequence in which, when the amino acid sequence of the OrfB homolog is compared with the amino acid sequence represented by sequence number 2, the amino acid residue corresponding to at least one of the amino acid residues at positions 6, 65, 230, 231 and 275 of sequence number 2 is replaced by another amino acid residue in the amino acid sequence of a homologous protein of the protein consisting of the amino acid sequence represented by sequence number 2 (hereinafter referred to as an OrfB homolog).

[0033] 3. The microorganism according to 1 or 2 above, wherein the microorganism having the ability to produce DHA is a Labyrinthus microorganism.

[0034] 4. The microorganism as described in 3 above, wherein the myxomycete microorganism is a myxomycete microorganism belonging to the genus Aurantiochytrium, the genus Thraustochytrium, the genus Ulkenia, the genus Parietichytrium, the genus Labyrinthula, the genus Aplanochytrium, the genus Oblongichytrium or the genus Schizochytrium.

[0035] 5. The microorganism according to 1 or 2 above, wherein the microorganism having the ability to produce DHA is a microorganism in which genes encoding the following domains (a) to (j) having an activity of synthesizing DHA are introduced into a microorganism not having a DHA metabolic pathway.

[0036] (a) KS domain

[0037] (b) MAT domain

[0038] (c) ACP domain

[0039] (d) KR domain

[0040] (e) Polyketide synthase dehydratase (hereinafter referred to as PS-DH) domain

[0041] (f) CLF domain

[0042] (g) AT domain

[0043] (h) FabA-like β-hydroxyacyl-ACP dehydratase (hereinafter referred to as FabA-DH) domain

[0044] (i) ER domain

[0045] (j) PPT domain

[0046] 6. The microorganism as described in 5 above, wherein the microorganism that does not have a DHA metabolic pathway is a microorganism belonging to the genus Escherichia, Bacillus, Corynebacterium, Yarrowia, Saccharomyces, Candida or Pichia.

[0047] 7. A method for producing EPA or a composition containing EPA, wherein the microorganism described in any one of 1 to 6 above is cultured in a culture medium, EPA or a composition containing EPA is produced and accumulated in the culture, and EPA or a composition containing EPA is collected from the culture.

[0048] 8. A method for producing EPA or a composition containing EPA, comprising using the following microorganism (I) or (II) capable of producing EPA.

[0049] (I) A microorganism capable of producing DHA, comprising a mutant OrfB consisting of an amino acid sequence in which at least one of the amino acid residues at positions 6, 65, 230, 231 and 275 in the amino acid sequence represented by SEQ ID NO: 2 is substituted with another amino acid residue, and capable of producing EPA

[0050] (II) A microorganism capable of producing DHA, comprising a mutant OrfB homolog consisting of the following amino acid sequence, wherein, in the amino acid sequence of the OrfB homolog, when the amino acid sequence of the OrfB homolog is compared with the amino acid sequence represented by SEQ ID NO: 2, at least one of the amino acid residues at positions 6, 65, 230, 231 and 275 of SEQ ID NO: 2 is substituted with another amino acid residue, and capable of producing EPA

[0051] Effects of the Invention

[0052] The microorganism of the present invention can efficiently produce EPA by expressing a mutant OrfB in which a mutation is introduced into a specific amino acid residue to change the specificity for the substrate in a microorganism capable of producing DHA. The method for producing EPA of the present invention can produce EPA at a low cost and high efficiency by expressing the mutant OrfB in a microorganism capable of producing DHA at an industrial level, and can be applied to the industrial-level production of EPA. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Schematic diagram showing the structure of the PUFA-PKS of Aurantiochytrium sp.

[0054] Figure 2 An example of the result of alignment of the amino acid sequences of OrfB and OrfB homologs is shown. DETAILED DESCRIPTION

[0055] In the present invention, "polyunsaturated fatty acid (PUFA)" refers to a long-chain fatty acid with a carbon chain length of 18 or more and an unsaturated bond number of 2 or more. In addition, in this specification, "domain" refers to a part of a protein consisting of a continuous amino acid sequence, which is a region in the protein having a specific biological activity or function.

[0056] In the present invention, "PUFA-PKS" has the same meaning as PUFA synthase. PUFA synthase refers to an enzyme group that uses malonyl-CoA and the like as a carbon source to synthesize specific long-chain unsaturated fatty acids, and contains the domains of KS, MAT, ACP, KR, PS-DH, CLF, AT, FabA-DH, ER, and PPTase (ACOS Lipid Library: PUFA synthase; Science, 2001, 293, 290-293; PLoS One, 2011, 6, e20146, etc.).

[0057] The KS domain is a domain possessed by a protein constituting a protein complex having PUFA-PKS activity, and refers to a domain involved in the condensation of malonyl-ACP and acyl-ACP.

[0058] The MAT domain and the AT domain are domains possessed by proteins constituting a protein complex having PUFA-PKS activity, and refer to domains involved in the transfer of acyl groups.

[0059] The ACP domain is a domain possessed by proteins constituting a protein complex having PUFA-PKS activity, and is a domain necessary for PUFA-PKS activity that functions as a site for fatty acid synthesis by binding to an acyl group via a phosphopantetheine group via a thioester bond.

[0060] The KR domain is a domain contained in a protein constituting a protein complex having PUFA-PKS activity, and refers to a domain involved in the reduction of a keto group generated by condensation.

[0061] The PS-DH domain and the FabA-DH domain, which are DH domains, are domains possessed by proteins constituting a protein complex having PUFA-PKS activity, and are domains involved in the dehydration of a hydroxyl group generated by the reduction of a keto group.

[0062] The CLF domain is a domain contained in a protein constituting a protein complex having PUFA-PKS activity, and refers to a domain involved in the extension of a carbon chain.

[0063] The ER domain refers to a domain possessed by a protein constituting a protein complex having PUFA-PKS activity.

[0064] PPTase is an enzyme constituting a protein complex having PUFA-PKS activity, and refers to an enzyme involved in the activation of the ACP domain.

[0065] In this specification, the identity of an amino acid sequence or a nucleotide sequence can be determined using the algorithm BLAST (Pro. Natl. Acad. Sci. USA, 1993, 90, 5873) or FASTA (Methods Enzymol., 1990, 183, 63) of Karlin and Altschul. Based on the algorithm BLAST, a program called BLASTN or BLASTX was developed (J. Mol. Biol., 1990, 215, 403). In the case where BLASTN is used to analyze a nucleotide sequence based on BLAST, the parameter is set to, for example, score = 100 and word length = 12. In addition, in the case where BLASTX is used to analyze an amino acid sequence based on BLAST, the parameter is set to, for example, score = 50 and word length = 3. In the case of using BLAST and Gapped BLAST programs, the default parameters of each program are used. The specific methods of these analytical methods are well known (reference www.ncbi.nlm.nih.gov).

[0066] In this specification, "exogenous" means non-endogenous and derived from a foreign species, and is used to indicate that the gene based on the present invention is introduced into the host organism when the host organism before transformation does not have the gene to be introduced by the present invention, when the protein encoded by the gene is not substantially expressed, and when the amino acid sequence of the protein is encoded by a different gene but the activity of the endogenous protein is not expressed after transformation.

[0067] [microorganism]

[0068] The microorganism of the present invention is a microorganism having the ability to produce docosahexaenoic acid (DHA), characterized in that it comprises a protein (mutant OrfB) composed of an amino acid sequence in which at least one of the amino acid residues at positions 6, 65, 230, 231 and 275 in the amino acid sequence represented by sequence number 2 is replaced by other amino acid residues, and is capable of producing eicosapentaenoic acid (EPA).

[0069] Examples of microorganisms capable of producing DHA include the following (1) and (2).

[0070] (1) Microorganisms capable of metabolizing DHA.

[0071] (2) A microorganism having the ability to produce DHA by using a microorganism that does not have the ability to metabolize DHA as a host organism and introducing into the host organism genes encoding the KS domain, MAT domain, ACP domain, KR domain, PS-DH domain, CLF domain, AT domain, FabA-DH domain, ER domain and PPT domain, which are domains constituting a PUFA-PKS having the activity of biosynthesizing DHA.

[0072] In this specification, "host organism" refers to an original organism to be subjected to genetic modification, transformation, etc. When the original organism to be subjected to transformation by gene introduction is a microorganism, it is also referred to as a parent strain or a host strain.

[0073] As (1) microorganisms having the ability to metabolize DHA, microorganisms belonging to the genus Myxomycetes can be cited. As microorganisms belonging to the genus Myxomycetes, for example, microorganisms of the genus Aurantiochytrium, the genus Thraustochytrium, the genus Ulkenia, the genus Parietichytrium, the genus Labyrinthula, the genus Aplanochytrium, the genus Oblongichytrium, or the genus Schizochytrium can be cited. Preferred examples include Aurantiochytrium limacinum, Thraustochytrium aureum, etc., but as long as they naturally have a DHA metabolic pathway, they are not limited to these.

[0074] Specifically, microorganisms having the ability to metabolize DHA are preferably microorganisms belonging to the genus Aurantiochytrium, such as Aurantiochytrium OH4 strain (Deposit No. FERM BP-11524). Microorganisms having the ability to produce DHA which are mutants thereof may also be used.

[0075] The above-mentioned Aurochrysalis OH4 strain is deposited in the Patent Microorganisms Depository of the National Institute of Technology for Evaluation (NITE), located at 1-1-Higashi, Tsukuba City, Ibaraki Prefecture, Japan (zip code 305-8566). The acceptance date (deposit date) is January 11, 2013 (Gregorian calendar), and the deposit number is FERM BP-11524.

[0076] (2) Microorganisms that do not have the ability to metabolize DHA refer to microorganisms that do not naturally have the ability to produce DHA. Examples of microorganisms that do not have the ability to metabolize DHA include bacteria, microalgae, fungi, protozoa, and protozoa.

[0077] Examples of the bacteria include microorganisms belonging to a genus selected from the group consisting of Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, and Aureispira. Among them, preferably selected from the group consisting of Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No. 49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli BL21 codon plus (manufactured by Stratagene), Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium immariophilum ATCC14068, Brevibacterium saccharolyticum ATCC14066, Corynebacterium ammoniagenes, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC13869, Corynebacterium acetoacidophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354, Pseudomonas sp. D-0110 and Aureispira marina JCM23201.

[0078] Examples of microalgae include Euglenophyceae [e.g., Euglena and Peranema], Chrysophyceae [e.g., Ochromonas], Dinobryaceae [e.g., Dinobryon, Platychrysis, and Chrysochromulina], Dinophyceae [e.g., Crypthecodinium], and Chrysochromulina. , Gymnodinium, Peridinium, Ceratium, Gyrodinium, and Oxyrrhis], Cryptophyceae [e.g., Cryptomonas and Rhodomonas], Xanthophyceae [e.g., Olisthodiscus] [and, including the Rhizochloridaceae and Aphanochaete] pascheri, Bumilleriastigeoclonium, and Vaucheria geminata], Eustigmatophyceae, and Prymnesiopyceae [including, for example, the genera Prymnesium and Diacronema].

[0079] Preferred species in these genera are not particularly limited, and examples thereof include Nannochlorops isoculata, Crypthecodinium cohnii, and Euglena gracilis.

[0080] Examples of the fungus include: Saccharomyces [e.g., yeasts including Saccharomyces cerevisiae and Saccharomyces carlsbergensis], or other yeasts of the genera Yarrowia, Candida, Pichia, and Kluyveromyces; or other fungi, such as fibrous fungi of the genera Aspergillus, Neurospora, and Penicillium.

[0081] Cell strains that can be used as host cells may be wild-type in the usual sense, or may be auxotrophic mutants, antibiotic-resistant mutants, or may be transformed with various marker genes, such as strains that are resistant to antibiotics such as chloramphenicol, ampicillin, kanamycin, and tetracycline.

[0082] As genes encoding the various domains constituting the PUFA-PKS having the activity of biosynthesizing DHA for enabling (2) microorganisms that do not have the ability to metabolize DHA to acquire the ability to produce DHA, genes encoding the various domains (KS domain, MAT domain, ACP domain, KR domain, PS-DH domain, CLF domain, AT domain, FabA-DH domain, ER domain and PPT domain) constituting the PUFA-PKS having the activity of biosynthesizing DHA possessed by the above-mentioned (1) microorganisms having the ability to metabolize DHA are preferred.

[0083] The domains constituting PUFA-PKS are not limited as long as the domains act synergistically to produce DHA, and examples thereof include the domains possessed by known PUFA-PKS.

[0084] In this specification, "synergistic effect" means that when a certain protein coexists with other proteins, they become one with each other and undergo a specific reaction. In particular, in this specification, it means that when multiple domains required for PUFA-PKS activity coexist, they become one with other domains and exhibit PUFA-PKS activity.

[0085] In the present specification, the “known PUFA-PKS” preferably includes PUFA-PKS originally possessed by a microorganism belonging to a genus selected from the group consisting of Aurantiochytrium, Thraustochytrium, Ulkenia, Parietichytrium, Labyrinthula, Aplanochytrium, Oblongichytrium or Schizochytrium, and more preferably includes PUFA-PKS originally possessed by a microorganism selected from the group consisting of Aurantiochytrium limacinum ATCC MYA-1381, Schizochytrium sp. ATCC 20888, and Thraustochytrium aureum ATCC 34304.

[0086] That the PUFA-PKS composed of each domain has DHA synthesis activity can be confirmed as follows: a microorganism transformed with a gene encoding each domain is produced, the microorganism is cultured in a culture medium, DHA is produced and accumulated in the culture, and the DHA accumulated in the culture is measured by gas chromatography.

[0087] PUFA-PKS is a protein complex (complex enzyme) composed of a plurality of proteins having the above-mentioned domains, and OrfB is a protein constituting PUFA-PKS. Figure 1 Schematic diagram of the domain structure of the protein complex constituting PUFA-PKS in a microorganism belonging to the genus Aurantiochytrium sp. is shown in FIG. OrfB contains a KS domain, a CLF domain, an AT domain, and an ER domain.

[0088] Examples of mutant OrfB include the proteins described in (a) or (b) below.

[0089] (a) A protein consisting of an amino acid sequence in which at least one of the amino acid residues at positions 6, 65, 230, 231 and 275 in the amino acid sequence represented by SEQ ID NO: 2 is substituted with another amino acid residue

[0090] (b) A protein consisting of the following amino acid sequence, wherein, in the amino acid sequence of an OrfB homolog, when the amino acid sequence is aligned with the amino acid sequence represented by SEQ ID NO: 2, at least one of the amino acid residues corresponding to the amino acid residues at positions 6, 65, 230, 231 and 275 of the amino acid sequence represented by SEQ ID NO: 2 is substituted with another amino acid residue.

[0091] Regarding the protein of (a) above, it is preferred that at least the amino acid residue at position 230 in the amino acid sequence represented by SEQ ID NO: 2 is replaced by another amino acid residue; more preferably, in addition to the amino acid residue at position 230, at least one of the amino acid residues selected from positions 6, 65, 231 and 275 is further replaced by another amino acid residue; particularly preferably, the amino acid residues at positions 6 and 230, the amino acid residues at positions 65 and 230, the amino acid residues at positions 6, 65 and 230, or the amino acid residues at positions 65, 230, 231 and 275 are replaced by other amino acid residues.

[0092] In addition, regarding the protein of (b) above, it is preferred that, in the amino acid sequence of the OrfB homolog, when the amino acid sequence of the OrfB homolog is aligned with the amino acid sequence represented by SEQ ID NO: 2, at least the amino acid residue corresponding to the amino acid residue at position 230 of the amino acid sequence represented by SEQ ID NO: 2 is substituted with another amino acid residue; more preferably, in addition to the amino acid residue corresponding to the amino acid residue at position 230, at least one selected from the group consisting of the amino acid residues corresponding to the amino acid residues at positions 6, 65, 231 and 275 is substituted with another amino acid residue; particularly preferably, the amino acid residues corresponding to the amino acid residues at positions 6 and 230, the amino acid residues at positions 65 and 230, the amino acid residues at positions 6, 65 and 230, or the amino acid residues at positions 65, 230, 231 and 275 are substituted with other amino acid residues.

[0093] An OrfB homolog refers to a protein present in an organism in nature as described below, which is composed of an amino acid sequence that is highly homologous to the amino acid sequence represented by sequence number 2, and has a structure and function similar to OrfB having the amino acid sequence represented by sequence number 2, and therefore it is believed that the gene encoding the protein has the same evolutionary origin as the gene encoding the original protein.

[0094] Specific examples of OrfB homologs include PhoC derived from Photobacterium profundum represented by sequence number 27, EpaC derived from Shewanella oneidensis represented by sequence number 28, DhaC derived from Moritella marina represented by sequence number 29, AraC derived from Aureispira marina represented by sequence number 30, and OrfB derived from Schizochytrium sp. (ATCC20888) represented by sequence number 31. Figure 2 An example of the result of the alignment of the amino acid sequences of OrfB and OrfB homologs is shown in FIG.

[0095] The alignment of amino acid sequences can be prepared using the known alignment program ClustalW [Nucleic Acids Research 22, 4673, (1994)]. ClustalW is available from http: / / www.ebi.ac.uk / clustalw / (European Bioinformatics Institute). When preparing the alignment using ClustalW, for example, the default parameters are used.

[0096] More preferably, the mutant OrfB includes a protein in which at least one of the following amino acid residues is substituted in the amino acid sequence of the protein described in (a) or (b).

[0097] (i) the amino acid residue at position 6 of the amino acid sequence of SEQ ID NO: 2 or the amino acid residue corresponding to the amino acid residue in the amino acid sequence of an OrfB homolog is substituted with serine;

[0098] (ii) the amino acid residue at position 65 of the amino acid sequence of SEQ ID NO: 2 or the amino acid residue corresponding to the amino acid residue in the amino acid sequence of an OrfB homolog is substituted with leucine;

[0099] (iii) the amino acid residue at position 230 of the amino acid sequence of SEQ ID NO: 2 or the amino acid residue corresponding to the amino acid residue in the amino acid sequence of an OrfB homolog is substituted with leucine, L-tryptophan, L-asparagine, glycine, L-aspartic acid or L-alanine;

[0100] (iv) the amino acid residue at position 231 of the amino acid sequence of SEQ ID NO: 2 or the amino acid residue corresponding to the amino acid residue in the amino acid sequence of an OrfB homolog is substituted with threonine;

[0101] (v) The amino acid residue at position 275 of the amino acid sequence of SEQ ID NO: 2 or the amino acid residue corresponding to the amino acid residue in the amino acid sequence of an OrfB homolog is substituted with glycine.

[0102] The amino acid residues after the above substitution may be amino acids that can substitute for each other. Examples of amino acids that can substitute for each other are shown below. Amino acids included in the same group can substitute for each other.

[0103] Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutyric acid, methionine, o-methylserine, tert-butylglycine, tert-butylalanine, cyclohexylalanine

[0104] Group B: Aspartic acid, Glutamic acid, Isoaspartic acid, Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid

[0105] Group C: Asparagine, Glutamine

[0106] Group D: lysine, arginine, ornithine, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid

[0107] Group E: Proline, 3-hydroxyproline, 4-hydroxyproline

[0108] Group F: serine, threonine, homoserine

[0109] Group G: Phenylalanine, Tyrosine

[0110] The above-mentioned substituted amino acid can be natural or non-natural. As natural amino acids, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine ​​etc. can be listed.

[0111] [Method for producing microorganisms]

[0112] Examples of methods for causing a microorganism having the ability to produce DHA to express mutant OrfB or a mutant OrfB homolog include the following (I) and (II).

[0113] (I) An exogenous gene encoding a mutant OrfB or a mutant OrfB homolog is introduced into a microorganism having the ability to produce DHA.

[0114] (II) A mutation is introduced into a gene encoding endogenous OrfB or an OrfB homolog in a microorganism having the ability to produce DHA.

[0115] Regarding the above (I), the introduction of an exogenous gene encoding a mutant OrfB or a mutant OrfB homolog includes: the presence of the exogenous gene in the cell of the host organism in the form of a plasmid capable of autonomous replication, the replacement of the gene to be replaced in the cell with the corresponding exogenous gene, and the integration of the exogenous gene encoding a mutant OrfB or a mutant OrfB homolog into a region of the chromosome DNA in the cell different from the gene encoding OrfB. It should be noted that when introducing an exogenous gene, it is preferred to optimize the sequence with reference to the codon usage frequency of the host microorganism.

[0116] Regarding the above (II), for example, mutations can be introduced into the gene encoding endogenous OrfB or an OrfB homolog by introducing site-directed mutations using the site-directed mutagenesis method described in Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001) (hereinafter referred to as Molecular Cloning 3rd Edition), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) (hereinafter referred to as Molecular Biology Laboratory Manual), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985), etc.

[0117] In this specification, "gene" refers to DNA that may include transcriptional regulatory regions, promoter regions, and terminator regions in addition to protein coding regions. When a prokaryotic organism such as bacteria is used as a parent strain as the host organism, a plasmid in which the Shine-Dalgarno sequence as a ribosome binding region and the start codon are adjusted to an appropriate distance (e.g., 6 to 18 bases) is preferably used as the DNA. In the DNA, a transcription termination factor is not essential for the expression of the DNA, but a transcription termination sequence is preferably configured immediately downstream of the structural gene.

[0118] As the gene introduced into the host organism, for example, it can be introduced into the host cell by making a recombinant gene inserted into the downstream of the promoter of a suitable expression vector. The expression vector may also include a promoter, a transcription termination signal, a selectable marker gene for selecting a transformant (e.g., a drug resistance gene such as a kanamycin resistance gene, a streptomycin resistance gene, a carboxin resistance gene, a bleomycin resistance gene, a hygromycin resistance gene, a gene complementary to an amino acid defective mutation such as leucine, histidine, methionine, arginine, tryptophan, lysine, etc., a gene complementary to a nucleic acid base defective mutation such as uracil, adenine, etc.). In the case of a uracil defective strain, as a marker gene, for example, orotidine-5'-phosphate decarboxylase gene (ura3 gene) or orotate pyrophosphorylase gene (ura5 gene) may be cited.

[0119] As promoter, no matter it is structural promoter or regulating promoter, it is defined as the base sequence of DNA that RNA polymerase is combined with DNA and starts RNA synthesis. Strong promoter refers to the promoter that starts mRNA synthesis with high frequency, and is preferably used. The main operator and promoter region of lac system, trp system, TAC or TRC system, lambda phage, fd coating protein regulatory region, for glycolytic enzyme (such as 3-phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase), glutamate decarboxylase A, serine hydroxymethyl transferase promoter etc. can be used according to the property of its host cell etc.

[0120] In addition to promoter and terminator sequences, other regulatory elements include, for example, selection markers, amplification signals, replication origins, etc. Preferred regulatory sequences include, for example, sequences described in "Gene Expression Technology: Methods in Enzymology 185", Academic Press (1990).

[0121] The vector is not particularly limited as long as it can express the target gene. The reagents used to construct the vector, such as restriction enzymes and ligases, are not particularly limited either, and commercially available products can be used as appropriate.

[0122] The promoter used when using a Labyrinthus microorganism as a host organism is not particularly limited as long as it is a promoter that functions in cells of the Labyrinthus microorganism, and examples thereof include an actin promoter, a tubulin promoter, an elongation factor Tu promoter, and a promoter for expression of a glycolytic system gene.

[0123] When a microorganism belonging to the genus Escherichia is used as the parent strain, examples of expression vectors include pColdI (manufactured by Takara Bio), pET21a, pCOLADuet-1, pACYCDuet-1, pCDF-1b, pRSF-1b (all manufactured by Novagen), pMAL-c2x (manufactured by New England Biolabs), pGEX-4T-1 (manufactured by GE Healthcare Biosciences), pTrcHis (manufactured by Invitrogen), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-30 (manufactured by Qiagen), pET-3 (manufactured by Novagen), pTrc99A (manufactured by GE Healthcare Biosciences), pTrcHis (manufactured by Invitrogen), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-30 (manufactured by Qiagen), pET-3 (manufactured by Novagen), and pTrc99A (manufactured by GE Healthcare Biosciences). Biosciences), pKYP10 (Japanese Patent Application Laid-Open No. 58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci., USA, 82, 4306 (1985)], pBluescriptII SK (+), pBluescriptII KS (-) (Stratagene), pTrS30 [prepared from Escherichia coli JM109 / pTrS30 (Ferm BP-5407)], pTrS32 [prepared from Escherichia coli JM109 / pTrS32 (Ferm BP-5408)], pTK31 [APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2007, Vol. 73, No. 20, p6378-6385], pPAC31 (International Publication No. 98 / 12343), pUC19 [Gene, 33, 103 (1985)], pSTV28 (manufactured by Takara Bio), pUC118 (manufactured by Takara Bio), pPA1 (Japanese Patent Application Publication No. 63-233798), pHSG298 (manufactured by Takara Bio), and pUC18 (manufactured by Takara Bio).

[0124] The promoter used in the expression vector is not particularly limited as long as it functions in cells of microorganisms belonging to the genus Escherichia, and examples thereof include promoters derived from Escherichia coli or bacteriophages, such as trp promoter (Ptrp), lac promoter (Plac), PL promoter, PR promoter, PSE promoter, and T7 promoter. In addition, examples thereof include promoters artificially designed and modified, such as a promoter formed by connecting two Ptrp promoters in series, a tac promoter, a trc promoter, a lacT7 promoter, and a letI promoter.

[0125] When a coryneform bacterium is used as the parent strain, examples of expression vectors include pCG1 (Japanese Patent Application Publication No. 57-134500), pCG2 (Japanese Patent Application Publication No. 58-35197), pCG4 (Japanese Patent Application Publication No. 57-183799), pCG11 (Japanese Patent Application Publication No. 57-134500), pCG116, pCE54, pCB101 (all Japanese Patent Application Publication No. 58-105999), pCE51, pCE52, pCE53 [all Molecular and General Genetics, 196, 175 (1984)], and the like.

[0126] The promoter used in the expression vector is not particularly limited as long as it functions in cells of coryneform bacteria, and examples thereof include the P54-6 promoter [Appl. Microbiol. Biotechnol., 53, 674-679 (2000)].

[0127] When a yeast strain is used as the parent strain, examples of the expression vector include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp51 (ATCC37419), pHS19, and pHS15.

[0128] The promoter used in the expression vector is not particularly limited as long as it functions in cells of yeast strains, and examples thereof include PH05 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter, MFα1 promoter, CUP1 promoter and the like.

[0129] As a method for integrating a recombinant gene into the chromosome of a host organism, a homologous recombination method can be used. As a homologous recombination method, for example, a method for introducing a recombinant gene using a homologous recombination system can be cited, which can be prepared by connecting with a plasmid DNA having a drug resistance gene that cannot replicate autonomously in the parent strain to be introduced. As a method for utilizing homologous recombination frequently used in Escherichia coli, a method for introducing a recombinant gene using the homologous recombination system of λ phage can be cited [Proc. Natl. Acad. Sci. USA, 97, 6641-6645 (2000)].

[0130] In addition, a selection method utilizing the fact that Escherichia coli is made sucrose sensitive by integrating Bacillus subtilis levansucrase into the chromosome together with the recombinant gene, a selection method utilizing the fact that Escherichia coli is made streptomycin sensitive by integrating the wild-type rpsL gene into Escherichia coli with a mutant rpsL gene having streptomycin resistance [Mol.Microbiol., 55, 137 (2005), Biosci.Biotechnol.Biochem., 71, 2905 (2007)], etc. can be used to obtain a microorganism in which the target region on the chromosomal DNA of the parent strain is replaced with recombinant DNA.

[0131] In addition, homologous recombination methods include, for example, the ATMT method using Agrobacterium [Appl. Environ. Microbiol., (2009), vol. 75, p. 5529-5535]. In addition, as long as a transformant stably retaining the target trait can be obtained, it may include improved methods of the ATMT method, but is not limited to these.

[0132] Examples of methods for introducing a gene to be introduced in the form of a plasmid that can autonomously replicate in a host organism include a method using calcium ions [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], a protoplast method (Japanese Patent Application Laid-Open No. 63-248394), and an electroporation method [Nucleic Acids Res., 16, 6127 (1988)].

[0133] That the microorganism obtained by the above method is the target microorganism can be confirmed by culturing the microorganism and detecting EPA accumulated in the culture by gas chromatography.

[0134] The EPA / DHA ratio in the final product (PUFA) produced by the microorganism of the present invention when cultured at 20°C for 48 hours is preferably 0.1 or more, more preferably 0.2 or more, and even more preferably 0.5 or more, when measured by gas chromatography-mass spectrometry as described later in Examples.

[0135] [Method for producing EPA or a composition containing EPA]

[0136] The present invention includes a method for producing EPA or a composition containing EPA (hereinafter referred to as the production method of the present invention), which is characterized in that the above-produced microorganisms are cultured in a culture medium, EPA or a composition containing EPA is generated and accumulated in the culture, and EPA or a composition containing EPA is collected from the culture.

[0137] Examples of the composition containing EPA include oils and fats containing EPA or phospholipids containing EPA, and preferably oils and fats containing EPA. The culture of the microorganism can be obtained by inoculating the microorganism into an appropriate medium and culturing it according to a conventional method.

[0138] As a culture medium, any known culture medium containing a carbon source, a nitrogen source, and an inorganic salt can be used. For example, as a carbon source, in addition to carbohydrates such as glucose, fructose, and galactose, oils and fats such as oleic acid and soybean oil, glycerol, sodium acetate, etc. can also be exemplified. These carbon sources can be used, for example, at a concentration of 20 to 300 g per liter of culture medium. According to a particularly preferred embodiment, the culture can be continued by supplying a carbon source after the initial carbon source is consumed. By culturing under such conditions, the amount of the consumed carbon source can be increased, and the production of the composition containing EPA can be increased.

[0139] In addition, examples of nitrogen sources include organic nitrogen such as yeast extract, corn steep liquor, polypeptone, sodium glutamate, and urea, and inorganic nitrogen such as ammonium acetate, ammonium sulfate, ammonium chloride, sodium nitrate, ammonium nitrate, and ammonia. As inorganic salts, potassium phosphate and the like can be used in combination as appropriate.

[0140] The culture medium containing the above-mentioned components is preferably used after the pH is adjusted to a range of 4.0 to 9.5 by adding an appropriate acid or alkali and sterilized by autoclave. The culture temperature is usually 10 to 45°C, preferably 20 to 37°C. The culture temperature is preferably controlled to a culture temperature that can produce a composition containing EPA. The pH during culture is usually 3.5 to 9.5, preferably 4.5 to 9.5. The particularly preferred pH varies depending on the purpose, and for mass production of oils and fats, the pH is 5.0 to 8.0.

[0141] The culture time can be set to, for example, 2 to 7 days, and the culture can be carried out by aeration and stirring culture, etc. The method of separating the culture solution and the microorganisms from the culture can be carried out by conventional methods known to those skilled in the art, for example, by centrifugation or filtration, etc. The microorganisms separated from the above culture are crushed by, for example, ultrasound or a dyno mill, and then solvent extracted by, for example, chloroform, hexane, butanol, etc., to obtain a composition containing EPA.

[0142] The EPA-containing composition produced by the above-mentioned production method can be concentrated by methods such as low-temperature solvent fractionation [Takahashi Taro, Oil Chemistry, 40: 931-941 (1991)] or a method of freeing short-chain fatty acids using hydrolases such as lipase [Takahashi Taro, Oil Chemistry, 40: 931-941 (1991)] to obtain a composition containing EPA with a high EPA content.

[0143] EPA can be produced by separating and collecting EPA from a composition containing EPA. For example, EPA can be produced by preparing a mixed fatty acid containing EPA from a composition containing EPA by a hydrolysis method, and then separating and collecting EPA by, for example, a urea addition method, a cooling separation method, a high performance liquid chromatography method, or a supercritical chromatography method.

[0144] Alternatively, EPA alkyl esters can be produced by separating and collecting EPA alkyl esters from a composition containing EPA. The EPA alkyl ester is not particularly limited as long as it is an EPA alkyl ester, but preferably EPA ethyl ester is used.

[0145] In order to separate and collect EPA alkyl esters from a composition containing EPA, for example, a mixed fatty acid alkyl ester containing EPA alkyl esters can be prepared from the composition containing EPA by alcoholysis, and then the EPA alkyl esters can be separated and collected by, for example, a urea addition method, a cooling separation method, a high performance liquid chromatography method or a supercritical chromatography method.

[0146] Example

[0147] Although Examples are shown below, the present invention is not limited to the following Examples.

[0148] [Example 1]

[0149] Production of EPA using Escherichia coli producing mutant OrfB-1

[0150] (1) Preparation of expression plasmids

[0151] [Preparation of OrfA protein expression plasmid]

[0152] By the same method as Lin et al. (Sci. Rep., 2016, 6, 35441), an expression plasmid pET21-orfA containing DNA (DNA consisting of the base sequence represented by SEQ ID NO: 4) encoding the OrfA protein derived from Schizochytrium sp. (ATCC20888) strain was obtained.

[0153] [Preparation of OrfC protein expression plasmid]

[0154] Using the genomic DNA of Aurantiochytrium sp. OH4 strain extracted by a conventional method as a template, PCR was performed using the primers represented by sequence numbers 7 and 8 to obtain a DNA fragment containing a DNA encoding the OrfC protein (DNA consisting of the base sequence represented by sequence number 3). The obtained DNA and the Escherichia coli vector pCOLADuet-1 (manufactured by Merck Millipore) were treated with restriction enzymes NdeI and MfeI, respectively, and the obtained restriction enzyme-treated fragments were ligated to obtain an expression plasmid pCOLA-OH4_orfC for the OrfC protein derived from Aurantiochytrium sp. OH4 strain.

[0155] [Preparation of HetI protein expression plasmid]

[0156] By the same method as Lin et al. (Sci. Rep., 2016, 6, 35441), an expression plasmid pSTV-hetI containing DNA (DNA consisting of the base sequence represented by SEQ ID NO: 5) encoding the HetI protein derived from Nostoc sp. PCC7120 (ATCC27893) strain was obtained.

[0157] (2) Construction of a DNA library encoding mutant OrfB

[0158] [Preparation of wild-type OrfB expression plasmid]

[0159] The OrfB expression plasmid pCDF-orfB1 (Sci. Rep., 2016, 6, 35441) derived from Schizochytrium sp. (ATCC20888) was treated with AgeI to obtain an AgeI-treated fragment, and the ends of the AgeI-treated fragment were blunted using a Blunting high kit (manufactured by Toyobo Co., Ltd.) and then self-ligated. Thus, pCDF-orfB1' in which the AgeI recognition sequence downstream of the T7 terminator of pCDF-orfB1 was deleted was obtained.

[0160] Next, using the genomic DNA of Aurantiochytrium sp. OH4 strain extracted by a conventional method as a template, overlap extension PCR was performed using primers represented by sequence numbers 9, 10, 11, and 12 to amplify a DNA fragment containing the DNA encoding OrfB (DNA consisting of the base sequence represented by sequence number 1). In the amplified DNA fragment, the base at position 4713 of the coding region was replaced from adenine to thymine, and the NdeI recognition sequence (base sequence at positions 4712 to 4717) was deleted. The obtained DNA fragment and pCDF-orfB1' were treated with restriction enzymes NdeI and EcoRI, respectively, and the obtained restriction enzyme-treated fragments were ligated to obtain pCDF-OH4_orfB.

[0161] Next, using pCDF-OH4_orfB as a template, overlap extension PCR was performed using the primers represented by sequence numbers 9, 12, 13, and 14 to amplify a DNA fragment containing a DNA encoding OrfB. In this DNA fragment, the base at position 2625 of the coding region was replaced from guanine to adenine, and an SphI recognition sequence was introduced into the base sequence at positions 2623 to 2628. The obtained DNA fragment and pCDF-orfB1' were treated with restriction enzymes NdeI and EcoRI, respectively, and the obtained restriction enzyme-treated fragments were ligated to obtain a plasmid pCDF-OH4_orfBs expressing wild-type OrfB derived from Aurantiochytrium sp. OH4 strain.

[0162] [Construction of a DNA library encoding mutant OrfB]

[0163] Next, error-prone PCR was performed using pCDF-OH4_orfBs as a template and the primers represented by SEQ ID NOs: 15 and 16, using TaKaRaTaq Hot Start Version (manufactured by Takara Bio). In the error-prone PCR, the concentration of MgCl in the PCR reaction solution was set to 5 mM in order to induce mutation.

[0164] The DNA fragment obtained by error-prone PCR was purified, treated with restriction enzymes NdeI and AgeI, and ligated with pCDF-OH4_orfBs treated with the same restriction enzymes, thereby constructing a DNA library encoding mutant OrfB.

[0165] (3) EPA Productivity Evaluation

[0166] By the same method as Lin et al. (Sci. Rep., 2016, 6, 35441), an Escherichia coli BLR (DE3) ΔfadE strain in which the gene encoding acyl-CoA dehydrogenase FadE (a protein consisting of the amino acid sequence represented by SEQ ID NO: 6) was deleted was prepared.

[0167] Escherichia coli BLR(DE3)ΔfadE strain was transformed with DNA libraries encoding pET21-orfA, pCOLA-OH4_orfC and pSTV-hetI, and pCDF-OH4_orfBs or mutant OrfB.

[0168] The obtained Escherichia coli was inoculated into 2 mL of Terrific Broth medium (Becton, Dickinson and Company) containing 100 mg / L of ampicillin, 20 mg / L of kanamycin, 30 mg / L of chloramphenicol, and 20 mg / L of streptomycin, and cultured with shaking at 30°C for 16 hours.

[0169] 1 mL of the obtained culture solution was inoculated into a 200 mL paddle flask containing 20 mL of freshly prepared premium broth medium (manufactured by Becton, Dickinson and Company) containing 100 mg / L of ampicillin, 20 mg / L of kanamycin, 30 mg / L of chloramphenicol, 20 mg / L of streptomycin, and 1 mM IPTG, and cultured at 230 rpm and 20°C for 48 hours.

[0170] After the culture, the culture fluid was collected, and lipids were extracted using the Bligh-Dyer method [Bligh, eG and Dyer, WJ (1959) Can. J. Biochem. Physiol. 37, 911-917], and the fatty acids were methylated using a boron trifluoride-methanol solution and analyzed using gas chromatography-mass spectrometry. The abundance of DHA and EPA in the culture fluid was calculated from the area of ​​the peaks corresponding to DHA methyl ester and EPA methyl ester using gas chromatography-mass spectrometry, and the abundance ratio of EPA to DHA was further calculated.

[0171] As a result, Escherichia coli producing wild-type OrfB did not produce EPA, whereas strains producing EPA were confirmed among Escherichia coli transformed with the DNA library encoding mutant OrfB.

[0172] The base sequence of the DNA encoding the mutant OrfB produced by the EPA-producing Escherichia coli was determined. As a result, L-phenylalanine at position 230 in the amino acid sequence of OrfB was substituted with L-leucine.

[0173] (4) Obtaining further mutant OrfB

[0174] Furthermore, error-prone PCR was performed using the same method as above using a DNA encoding a mutant OrfB consisting of an amino acid sequence in which L-phenylalanine at position 230 was replaced by L-leucine as a template, and the mutant was introduced into the Escherichia coli BLR(DE3)ΔfadE strain using the same method as above to confirm the productivity of EPA.

[0175] As a result, a strain with a further improved EPA productivity was confirmed compared with the Escherichia coli producing the mutant OrfB having an amino acid sequence in which L-phenylalanine at position 230 was substituted with L-leucine obtained above.

[0176] The base sequence of the DNA encoding the mutant OrfB expressed by Escherichia coli that improves the productivity of EPA was determined. As a result, in the amino acid sequence of OrfB, L-phenylalanine at position 230 was replaced by L-leucine, L-asparagine at position 6 was replaced by L-serine, and L-phenylalanine at position 65 was replaced by L-leucine.

[0177] Table 1 summarizes the results of measuring EPA, DHA, and DPA in the above culture solution.

[0178] [Table 1]

[0179]

[0180] As shown in Table 1, it can be seen that by using Escherichia coli producing a mutant OrfB in which the amino acid residue at position 230 in the amino acid sequence of OrfB is substituted with L-leucine, or a mutant OrfB in which the amino acid residue at position 6 is substituted with L-serine, the amino acid residue at position 65 is substituted with L-leucine, and the amino acid residue at position 230 is substituted with L-leucine, EPA can be produced more efficiently than when using Escherichia coli producing wild-type OrfB.

[0181] [Example 2]

[0182] Production of EPA using Escherichia coli producing mutant OrfB-2

[0183] (1) Preparation of expression plasmids

[0184] Using pCDF-OH4_orfB obtained in Example 1 (2) as a template and the primers represented by SEQ ID NOs: 9 and 17, PCR was performed to amplify a DNA fragment containing DNA encoding the N-terminal region of the KS domain of OrfB.

[0185] In addition, PCR was performed using pCDF-OH4_orfB as a template and primers represented by sequence numbers 16 and 18, 19, 20, 21 or 22 to amplify a DNA fragment containing the C-terminal region of the KS domain of a mutant OrfB in which the amino acid residue at position 230 in the amino acid sequence encoding OrfB was replaced with L-tryptophan, L-asparagine, glycine, L-aspartic acid or L-alanine.

[0186] Overlapping extension PCR was performed using the obtained DNA fragment encoding the N-terminal region or C-terminal region of the KS domain and the primers represented by sequence numbers 9 and 16 to obtain a DNA fragment containing the full length of the KS domain of a mutant OrfB in which the amino acid residue at position 230 in the amino acid sequence encoding OrfB was replaced with L-tryptophan, L-asparagine, glycine, L-aspartic acid or L-alanine.

[0187] The DNA fragment and pCDF-OH4_orfBs were treated with restriction enzymes NdeI and AgeI, respectively, and the resulting restriction enzyme-treated fragments were ligated to obtain pCDF-OH4_orfB-F230W, pCDF-OH4_orfB-F230N, pCDF-OH4_orfB-F230G, pCDF-OH4_orfB-F230D and pCDF-OH4_orfB-F230A.

[0188] In addition, a plasmid pCDF-OH4_orfB-F230L containing a DNA encoding a mutant OrfB in which the amino acid residue at position 230 in the amino acid sequence of OrfB is substituted with L-leucine was obtained from the E. coli obtained in Example 1(3).

[0189] (2) Production of EPA

[0190] Escherichia coli BLR(DE3)ΔfadE strain was transformed using pET21-orfA, pCOLA-OH4_orfC and pSTV-hetI, as well as expression plasmids of wild-type OrfB or six mutant OrfBs (pCDF-OH4_orfBs, pCDF-OH4_orfB-F230L, pCDF-OH4_orfB-F230W, pCDF-OH4_orfB-F230N, pCDF-OH4_orfB-F230G, pCDF-OH4_orfB-F230D or pCDF-OH4_orfB-F230A).

[0191] The obtained Escherichia coli was inoculated into 2 mL of premium broth medium (Becton, Dickinson and Company) containing 100 mg / L of ampicillin, 20 mg / L of kanamycin, 30 mg / L of chloramphenicol, and 20 mg / L of streptomycin, and cultured with shaking at 30°C for 16 hours.

[0192] 1 mL of the obtained culture solution was inoculated into a 200 mL paddle flask containing 20 mL of freshly prepared premium broth medium (manufactured by Becton, Dickinson and Company) containing 100 mg / L of ampicillin, 20 mg / L of kanamycin, 30 mg / L of chloramphenicol, 20 mg / L of streptomycin, and 1 mM IPTG, and cultured at 230 rpm and 20°C for 48 hours.

[0193] After the culture, the culture medium was collected, lipids were extracted by the Bligh-Dyer method, fatty acids were methylated using a boron trifluoride-methanol solution, and analyzed by gas chromatography-mass spectrometry.

[0194] Table 2 shows the results of measuring EPA, DHA, and DPA in the culture solution.

[0195] [Table 2]

[0196]

[0197] As shown in Table 2, similar to the case where a mutant OrfB in which the amino acid residue at position 230 in the amino acid sequence of OrfB is substituted with L-leucine is used, when Escherichia coli producing a mutant OrfB in which the amino acid residue at position 230 in the amino acid sequence of OrfB is substituted with L-tryptophan, L-asparagine, glycine, L-aspartic acid or L-alanine is used, EPA can be efficiently produced compared to the case where Escherichia coli producing wild-type OrfB is used.

[0198] [Example 3]

[0199] Production of EPA using Escherichia coli producing mutant OrfB-3

[0200] (1) Preparation of expression plasmids

[0201] [Preparation of pCDF-OH4_orfB-N6S-F230L]

[0202] Using pCDF-OH4_orfB-F230L as a template, PCR was performed using the primers represented by SEQ ID NOs: 23 and 16 to obtain a DNA fragment containing a DNA encoding the KS domain of OrfB. The obtained DNA fragment and pCDF-OH4_orfB-F230L were treated with restriction enzymes NdeI and AgeI, respectively, and the obtained restriction enzyme-treated fragments were ligated to obtain pCDF-OH4_orfB-N6S-F230L.

[0203] pCDF-OH4_orfB-N6S-F230L has a DNA encoding an amino acid sequence in which the amino acid residue at position 6 is substituted with L-serine and the amino acid residue at position 230 is substituted with L-leucine in the amino acid sequence of OrfB derived from Aurantiochytrium sp. OH4 strain.

[0204] [Preparation of pCDF-OH4_orfB-F65L-F230L]

[0205] Using pCDF-OH4_orfB-F230L as a template, overlap extension PCR was performed using primers represented by sequence numbers 24, 25, 26 and 16 to obtain a DNA fragment containing a DNA encoding the KS domain of OrfB. The obtained DNA fragment and pCDF-OH4_orfB-F230L were treated with restriction enzymes NdeI and AgeI, respectively, and the obtained restriction enzyme-treated fragments were ligated to obtain pCDF-OH4_orfB-F65L-F230L.

[0206] pCDF-OH4_orfB-F65L-F230L has a DNA encoding an amino acid sequence in which the amino acid residue at position 65 and the amino acid residue at position 230 of the amino acid sequence of OrfB derived from Aurantiochytrium sp. OH4 strain are substituted with L-leucine.

[0207] [Preparation of pCDF-OH4_orfB-N6S-F65L-F230L]

[0208] The plasmid was extracted from the Escherichia coli obtained in Example 1 (4), which produces OrfB in which the L-phenylalanine at position 230 was replaced by L-leucine, the L-asparagine at position 6 was replaced by L-serine, and the L-phenylalanine at position 65 was replaced by L-leucine, to obtain pCDF-OH4_orfB-N6S-F65L-F230L.

[0209] pCDF-OH4_orfB-N6S-F65L-F230L has a DNA having an amino acid sequence in which the amino acid residue at position 6 is substituted with L-serine, the amino acid residue at position 65 is substituted with L-leucine, and the amino acid residue at position 230 is substituted with L-leucine, encoding the amino acid sequence of OrfB derived from Aurantiochytrium sp. OH4 strain.

[0210] (2) Production of EPA

[0211] Escherichia coli BLR(DE3)ΔfadE strain was transformed using pET21-orfA, pCOLA-OH4_orfC and pSTV-hetI, as well as expression plasmids of wild-type OrfB or four mutant OrfBs (pCDF-OH4_orfBs, pCDF-OH4_orfB-F230L, pCDF-OH4_orfB-N6S-F230L, pCDF-OH4_orfB-F65L-F230L or pCDF-OH4_orfB-N6S-F65L-F230L).

[0212] The obtained Escherichia coli was inoculated into 2 mL of premium broth medium (Becton, Dickinson and Company) containing 100 mg / L of ampicillin, 20 mg / L of kanamycin, 30 mg / L of chloramphenicol, and 20 mg / L of streptomycin, and cultured with shaking at 30°C for 16 hours.

[0213] 1 mL of the obtained culture solution was inoculated into a 200 mL paddle flask containing 20 mL of freshly prepared premium broth medium (manufactured by Becton, Dickinson and Company) containing 100 mg / L of ampicillin, 20 mg / L of kanamycin, 30 mg / L of chloramphenicol, 20 mg / L of streptomycin, and 1 mM IPTG, and cultured at 230 rpm and 20°C for 48 hours.

[0214] After the culture, the culture medium was collected, lipids were extracted by the Bligh-Dyer method, fatty acids were methylated using a boron trifluoride-methanol solution, and analyzed by gas chromatography-mass spectrometry.

[0215] Table 3 shows the results of measuring EPA, DHA, and DPA in the culture solution.

[0216] [Table 3]

[0217]

[0218] As shown in Table 3, when Escherichia coli is used to produce a mutant OrfB in which the amino acid residue at position 6 and / or the amino acid residue at position 65 is replaced with L-serine and L-leucine, respectively, on the basis of the replacement of the amino acid residue at position 230 in the amino acid sequence of OrfB, EPA can be produced more efficiently than when Escherichia coli is used to produce a mutant OrfB in which the amino acid residue at position 230 in the amino acid sequence of OrfB is replaced with L-leucine.

[0219] [Example 4]

[0220] Production of EPA using Escherichia coli producing mutant OrfB-4

[0221] Using the DNA obtained in Example 3, encoding the mutant OrfB consisting of an amino acid sequence in which L-phenylalanine at position 230 was substituted with L-leucine and L-phenylalanine at position 65 was substituted with L-leucine, as a template, error-prone PCR was carried out by the same method as in Example 1(2), and the DNA was introduced into the Escherichia coli BLR(DE3)ΔfadE strain by the same method as in Example 1(3), and the productivity of EPA was confirmed.

[0222] As a result, a strain with further improved EPA productivity was confirmed compared with Escherichia coli producing mutant OrfB consisting of an amino acid sequence in which L-phenylalanine at position 230 was substituted with L-leucine and L-phenylalanine at position 65 was substituted with L-leucine.

[0223] The base sequence of the DNA encoding the mutant OrfB expressed by Escherichia coli that improves the productivity of EPA was determined. As a result, in the amino acid sequence of OrfB, L-phenylalanine at position 230 was substituted with L-leucine, L-phenylalanine at position 65 was substituted with L-leucine, L-isoleucine at position 231 was substituted with L-threonine, and L-aspartic acid at position 275 was substituted with L-glycine.

[0224] Table 4 summarizes the results of measuring EPA, DHA, and DPA in the above culture solution.

[0225] [Table 4]

[0226]

[0227] As shown in Table 4, when Escherichia coli is used to produce a mutant OrfB in which the amino acid residue at position 231 is replaced with L-threonine and the amino acid residue at position 275 is replaced with glycine on the basis of replacement of the amino acid residue at position 230 and the amino acid residue at position 65 in the amino acid sequence of OrfB, EPA can be produced more efficiently than when Escherichia coli is used to produce a mutant OrfB in which the amino acid residue at position 230 and the amino acid residue at position 65 in the amino acid sequence of OrfB are replaced with L-leucine, respectively.

[0228] The present invention is described in detail with reference to a specific mode, but it is obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention. It should be noted that this application is based on the Japanese patent application (Japanese patent application 2018-151234) filed on August 10, 2018, and its entire contents are cited by reference. In addition, all references cited herein are incorporated into this specification as a whole. Sequence Listing <110> Kyowa Hakko Biochemical Co., Ltd. <120> Microorganism producing eicosapentaenoic acid and method for producing eicosapentaenoic acid <130> W527738 <140> JP2018-151234 <141> 2018-08-10 <160> 31 <170> PatentIn version 3.5 <210> 1 <211> 6105 <212> DNA <213> Aurantiochytrium sp. OH4 <220> <221> CDS <222> (1)..(6105) <400> 1 atg gcc tct cgc aag aat gtg agc gct gct cac gaa atg cac gac gag 48 Met Ala Ser Arg Lys Asn Val Ser Ala Ala His Glu Met His Asp Glu 1 5 10 15 aag cgc att gcc gtg gtg ggc atg gcc gtg caa tac gcg ggc tgc aaa 96 Lys Arg Ile Ala Val Val Gly Met Ala Val Gln Tyr Ala Gly Cys Lys 20 25 30 gac aag gaa gag ttc tgg aaa gta gtc atg ggc ggt gag gct gca tgg 144 Asp Lys Glu Glu Phe Trp Lys Val Val Met Gly Gly Glu Ala Ala Trp 35 40 45 act aag att agc gat aaa cgc ctc gga tcc aac aag cga gcc gag cac 192 Thr Lys Ile Served Asp Lys Arg Leu Gly Served Asn Lys Arg Ala Glu His 50 55 60 ttc aaa gca gag cgt agc aaa ttt gca gat acc ttt tgc aac gag aac 240 Phe Lys Ala Glu Arg Ser Lys Phe Ala Asp Thr Phe Cys Asn Glu Asn 65 70 75 80 tac ggc tgc gtc gat gac tc gtc gat aac gaa cac gag ctt ctc ctc 288 Tyr Gly Cys Val Asp Ser Val Asp Asn Glu His Glu Leu Leu Leu 85 90 95 aag ctc tcc aag aag gct ctc tcc gag aca tcg gtc tcc gac tct aca Lys Leu Will Be Lys Lys Ala Leu Will Be Glu Thr Will Be Val Be Asp Will Be Thr 100 105 110 agg tgc gtt att gtg agc gga tgc ctg tcc ttc ccc atg gac aac ctc 384 Arg Cys Gly Ile Val Ser Gly Cys Leu Ser Phe Pro Met Asp Asn Leu 115 120 125 cag ggc gaa ctc ctc aat gtg tac CAAAC cac gtc gaa aag aaa ctc 432 Gln Gly Glu Lion Asn Will Tyr Gln Asn His Will Glu Lys Lys 130 135 140 ggc gct cgc gtc ttc aag gat gcc tcc aag tgg tcc gag cgt gag cag 480 Gly Ala Arg Val Phe Lys Asp Ala Ser Lys Trp Ser Glu Arg Glu Gln 145 150 155 160 tcg cag aac ccc gag gct ggt gac cgc cgc atc ttt atg gac ccg gca 528 Ser Gln Asn Pro Glu Ala Gly Asp Arg Arg Ile Phe Met Asp Pro Ala 165 170 175 tcc ttc gta gca gaa gag ctt aac ctc ggt cct ctt cac tac tct gtc 576 Ser Phe Val Ala Glu Glu Leu Asn Leu Gly Pro Leu His Tyr Ser Val 180 185 190 gat gct gcc tgt gcc acc gcc ctt tac gtc ctt cgc ctc gcc cag gac 624 Asp Ala Ala Cys Ala Thr Ala Leu Tyr Val Leu Arg Leu Ala Gln Asp 195 200 205 cac ctc gtt tcc ggt gct gct gat gtc atg ctc gct ggt gca act tgc 672 His Leu Val Ser Gly Ala Ala Asp Val Met Leu Ala Gly Ala Thr Cys 210 215 220 ttc ccg gag ccc ttt ttc att ctc tcc gga ttc tcc act ttc cag gcc 720 Phe Pro Glu Pro Phe Phe Ile Leu Ser Gly Phe Ser Thr Phe Gln Ala 225 230 235 240 atg cct gta tcg gga gac ggc atc tcg tac ccg ctt cac aag gac agt 768 Met Pro Val Ser Gly Asp Gly Ile Ser Tyr Pro Leu His Lys Asp Ser 245 250 255 cag ggt ctc acc cct ggt gaa ggt ggt gcc att atg gtt ctc aag cgc 816 Gln Gly Leu Thr Pro Gly Glu Gly Gly Ala Ile Met Val Leu Lys Arg 260 265 270 ctt gac gac gct att cgc gat gga gac cac att tac ggt act ctg ctc 864 Leu Asp Asp Ala Ile Arg Asp Gly Asp His Ile Tyr Gly Thr Leu Leu 275 280 285 ggt gct acc atc agc aat gct ggc tgt ggt ctt ccc ctc aag cca cac 912 Gly Ala Thr Ile Ser Asn Ala Gly Cys Gly Leu Pro Leu Lys Pro His 290 295 300 ttg ccc agc gag aag tcc tgc ctc att gat acc tac aag cgc gtc aac 960 Leu Pro Ser Glu Lys Ser Cys Leu Ile Asp Thr Tyr Lys Arg Val Asn 305 310 315 320 gtg cac ccg cac aag atc cag tac gtc gag tgc cac gca acg ggt act 1008 Val His Pro His Lys Ile Gln Tyr Val Glu Cys His Ala Thr Gly Thr 325 330 335 ccc cag gga gac cgc gtt gag att gat gcc gtc aag gct tgc ttc gag 1056 Pro Gln Gly Asp Arg Val Glu Ile Asp Ala Val Lys Ala Cys Phe Glu 340 345 350 ggc aag gtg cct cgc ttt gga agc tcc aag ggt aac ttt ggc cac aca 1104 Gly Lys Val Pro Arg Phe Gly Ser Ser Lys Gly Asn Phe Gly His Thr 355 360 365 ctc gtt gca gct ggt ttc gca ggc atg tgc aag gta ctc ctt gcc atg 1152 Leu Val Ala Ala Gly Phe Ala Gly Met Cys Lys Val Leu Leu Ala Met 370 375 380 aag cat ggt gtg atc ccg ccc act cct ggt gtc gat gga tct tcc caa 1200 Lys His Gly Val Ile Pro Pro Thr Pro Gly Val Asp Gly Ser Ser Gln 385 390 395 400 atg gac ccg ctt gtg gtc tct gag ccc atc cca tgg ccc gac act gag 1248 Met Asp Pro Leu Val Val Ser Glu Pro Ile Pro Trp Pro Asp Thr Glu 405 410 415 ggc gag ccc aag cgc gct ggt ctc tcc gct ttc ggc ttt ggt ggc acc 1296 Gly Glu Pro Lys Arg Ala Gly Leu Ser Ala Phe Gly Phe Gly Gly Thr 420 425 430 aac gcc cac gca gtc ttt gag gag ttt gac cgc tcc aag gct gcc tgt 1344 Asn Ala His Ala Val Phe Glu Glu Phe Asp Arg Ser Lys Ala Ala Cys 435 440 445 gcc acc cac gat agc atc agt tcc ctc agc tca cgt tgt ggc ggg gag 1392 Ala Thr His Asp Ser Ile Ser Ser Leu Ser Ser Arg Cys Gly Gly Glu 450 455 460 ggc aac atg cgc att gct att acc ggt atg gat gcc acc ttc ggc tcc 1440 Gly Asn Met Arg Ile Ala Ile Thr Gly Met Asp Ala Thr Phe Gly Ser 465 470 475 480 ctc aag ggc ctg gac gcc ttt gag cgt gcc atc tac aat ggc caa cat 1488 Leu Lys Gly Leu Asp Ala Phe Glu Arg Ala Ile Tyr Asn Gly Gln His 485 490 495 ggt gct gtg cca ttg cct gag aag cgc tgg cgt ttc ctt ggt aaa gac 1536 Gly Ala Val Pro Leu Pro Glu Lys Arg Trp Arg Phe Leu Gly Lys Asp 500 505 510 aag gac ttt ttg gac ctg tgc ggc gtc aag gag gtg ccc cac gga tgc 1584 Lys Asp Phe Leu Asp Leu Cys Gly Val Lys Glu Val Pro His Gly Cys 515 520 525 tac att gag gac gtc gag gtg gac ttt agc cgc ctg cgc acg ccc atg 1632 Tyr Ile Glu Asp Val Glu Val Asp Phe Ser Arg Leu Arg Thr Pro Met 530 535 540 acg cca gac gac atg ttg cgc ccc atg cag cta ctt gct gtc aca acc 1680 Thr Pro Asp Asp Met Leu Arg Pro Met Gln Leu Leu Ala Val Thr Thr 545 550 555 560 atc gac cgt gcc att ctc aac tct ggc ctc aag aag gga ggt aag gtc 1728 Ile Asp Arg Ala Ile Leu Asn Ser Gly Leu Lys Lys Gly Gly Lys Val 565 570 575 gct gtc ttc gtc ggc ctt ggc act gac ctt gag ctc tac cgt cac cgc 1776 Ala Val Phe Val Gly Leu Gly Thr Asp Leu Glu Leu Tyr Arg His Arg 580 585 590 gcc cgc gtt gcc ctc aag gag cgt gct cgt ccc gaa gcc gct gca gcc 1824 Ala Arg Val Ala Leu Lys Glu Arg Ala Arg Pro Glu Ala Ala Ala Ala 595 600 605 ctc aat gat atg atg tcc tac atc aac gat tgc ggt acc gct acc tcg 1872 Leu Asn Asp Met Met Ser Tyr Ile Asn Asp Cys Gly Thr Ala Thr Ser 610 615 620 tac aca tcc tac atc ggc aac ctc gtg gcc acc cgc gtg tct tca caa 1920 Tyr Thr Ser Tyr Ile Gly Asn Leu Val Ala Thr Arg Val Ser Ser Gln 625 630 635 640 tgg ggt ttc gag ggt cct tct ttc acc atc aca gag ggc aac aac tcc 1968 Trp Gly Phe Glu Gly Pro Ser Phe Thr Ile Thr Glu Gly Asn Asn Ser 645 650 655 gtc tac cgt tgc gca gag ttg ggc aag tac ttg ctc gag act ggc gag 2016 Val Tyr Arg Cys Ala Glu Leu Gly Lys Tyr Leu Leu Glu Thr Gly Glu 660 665 670 gtc gag gcc gta gtg atc gcc ggt gtg gat ctt tgc gcc agc gct gag 2064 Val Glu Ala Val Val Ile Ala Gly Val Asp Leu Cys Ala Ser Ala Glu 675 680 685 aat ctc tac gtg aag tcg cgt cgt ttc aag gtc tcg gag cag gag agc 2112 Asn Leu Tyr Val Lys Ser Arg Arg Phe Lys Val Ser Glu Gln Glu Ser 690 695 700 ccg cgg gcc agc ttc gac tcc ggc gct gac ggc tac ttt gtt ggt gag 2160 Pro Arg Ala Ser Phe Asp Ser Gly Ala Asp Gly Tyr Phe Val Gly Glu 705 710 715 720 gga tgt ggt gcc ctc gtc ctc aag cgc gag agc gac tgc acc aag gac 2208 Gly Cys Gly Ala Leu Val Leu Lys Arg Glu Ser Asp Cys Thr Lys Asp 725 730 735 gaa cgc att tac gcc tgc atg gac gct atc gtg ccc gggc aac atg ccg 2256 Glu Arg Ile Tyr Ala Cys Met Asp Ala Ile Val Pro Gly Asn Met Pro 740,745,750 gca gcc tgc atg gag gag gct cctc gcc cag gct cgc gtc aac ccc aag 2304 Ala Ala Cys Met Glu Glu Ala Leu Ala Gln Ala Arg Val Asn Pro Lys 755,760,765 gac gtt gag atg ctc gag ctc tcc gct gac tct gcc cgc cac ctc aag 2352 Asp Val Glu Met Leu Glu Leu Ser Ala Asp Ser Ala Arg His Leu Lys 770,775,780 aac ccc tcc gtt ctg cct aag gaa ctc act gct gag gag gaa atc ​​cgc 2400 Asn Pro Ser Val Leu Pro Lys Glu Leu Thr Ala Glu Glu Glu Ile Arg 785,790,795,800 ggc att gag gcc att ctc agc cag cgc tct agc aac gaa gct gtg gag 2448 Gly Ile Glu Ala Ile Leu Ser Gln Arg Ser Ser Asn Glu Ala Val Glu 805 810 815 ccc cac aac gtc gct gtc agc agc gtc aag tcc act gtc ggt gac acc 2496 Pro His Asn Val Ala Val Ser Ser Val Lys Ser Thr Val Gly Asp Thr 820 825 830 ggc tac gcc tca gga gct gcc agt ctc atc aag acg gct ctc tgt ctg 2544 Gly Tyr Ala Ser Gly Ala Ala Ser Leu Ile Lys Thr Ala Leu Cys Leu 835 840 845 tac aac cgc tac ttg ccc tca aac ggc gcc tcc tgg gag gag cct gca 2592 Tyr Asn Arg Tyr Leu Pro Ser Asn Gly Ala Ser Trp Glu Glu Pro Ala 850 855 860 cct gag aca cag tgg ggc aag tct ctg tac gcg tgc cag tcc tcg cgg 2640 Pro Glu Thr Gln Trp Gly Lys Ser Leu Tyr Ala Cys Gln Ser Ser Arg 865 870 875 880 gcc tgg ttg aag aac cct gga gct cgc cgc cac gca gct gtc tca ggt 2688 Ala Trp Leu Lys Asn Pro Gly Ala Arg Arg His Ala Ala Val Ser Gly 885 890 895 gtt tcc gag acc cgt tca tgc tac acg gtg ctg ctc tct gat gtg gag 2736 Val Ser Glu Thr Arg Ser Cys Tyr Thr Val Leu Leu Ser Asp Val Glu 900 905 910 ggc cac cac gag acc aag agc cgc att tcg ctc gat gac gat gcc gtc 2784 Gly His His Glu Thr Lys Ser Arg Ile Ser Leu Asp Asp Asp Ala Val 915 920 925 aaa ctc ctc gta atc cgc gga gac tcc cac gac gct atc acg cag cgt 2832 Lys Leu Leu Val Ile Arg Gly Asp Ser His Asp Ala Ile Thr Gln Arg 930 935 940 gtt gac aag ctc cgc gag cgc ctc gcc cag cct agc gct aat gta cgt 2880 Val Asp Lys Leu Arg Glu Arg Leu Ala Gln Pro Ser Ala Asn Val Arg 945 950 955 960 ctt gct ttt atg gag ttg ctc ggc gag agc att gcc cag gag acc aag 2928 Leu Ala Phe Met Glu Leu Leu Gly Glu Ser Ile Ala Gln Glu Thr Lys 965 970 975 acc ccg ttg ccg gcc ttc gct ctg tgc ctg gtg acc tct cct agt aag 2976 Thr Pro Ser Lys 980,985,990 ctc cag aag gag ctt gaa ctc gcc tcc aag ggc atc ccg cgg agt ctt 3024 Leu Gln Lys Glu Leu Glu Leu Ala Ser Lys Gly Ile Pro Arg Ser Leu 995 1000 1005 aag atg ggc cgc gac tgg aca tca ccc tcg agc cac ttt gca 3069 Lys Met Gly Arg Asp Trp Thr Ser Pro Ser Gly Ser His Phe Ala 1010 1015 1020 ccc aag cca ctg tca agc gat cgc gtt gcg ttt atg tac ggc gaa 3114 Pro Lys Pro Leu Ser Ser Asp Arg Val Ala Phe Met Tyr Gly Glu 1025 1030 1035 ggc cga agc cct tac tat ggt atc ggc ctt gac att cac cgc atc 3159 Gly Arg Ser Pro Tyr Tyr Gly Ile Gly Leu Asp Ile His Arg Ile 1040 1045 1050 tgg ccc gaa ctt cac gag ttt gta aac gcc aag acc aac aag ctt 3204 Trp Pro Glu Leu His Glu Phe Val Asn Lys Thr Asn Lys Leu 1055 1060 1065 tgg gat caa ggc gac aga tgg ttg atc ccg cgc gcc tcg acg aag 3249 Trp Asp Gln Gly Asp Arg Trp Leu Ile Pro Arg Ala Ser Thr Lys 1070 1075 1080 gag gag ctt aag gcg cag gaa gat gag ttc agc cgc aac cag gtg 3294 Glu Glu Leu Lys Ala Gln Glu Asp Glu Phe Ser Arg Asn Gln Val 1085 1090 1095 gag atg ttc cga ctc ggt att ctc atg tcc atg tgc ttc acc cac 3339 Glu Met Phe Arg Leu Gly Ile Leu Met Ser Met Cys Phe Thr His 1100 1105 1110 atc gct cgt gac gtg ctt ggc atc cag ccc aag gct gct ttc gga 3384 Ile Ala Arg Asp Val Leu Gly Ile Gln Pro Lys Ala Ala Phe Gly 1115 1120 1125 ctg agc ctt gga gag att tcc atg gtt ttt gcc ttt tct gag aag 3429 Leu Ser Leu Gly Glu Ile Ser Met Val Phe Ala Phe Ser Glu Lys 1130 1135 1140 aac ggc ctt gtc tct gag gag ctg aca act aaa ctc cgc aac tcg 3474 Asn Gly Leu Val Ser Glu Glu Leu Thr Thr Lys Leu Arg Asn Ser 1145 1150 1155 gag gtc tgg cgt aag gcc ctc gct gtt gag ttt gac gcc ctc cgc 3519 Glu Val Trp Arg Lys Ala Leu Ala Val Glu Phe Asp Ala Leu Arg 1160 1165 1170 aag gcc tgg aat att ccc caa gat acc cct gtc agc gag ttc tgg 3564 Lys Ala Trp Asn Ile Pro Gln Asp Thr Pro Val Ser Glu Phe Trp 1175 1180 1185 caa gga tac gtg gta cgt gga acc cgc gag gcc gtt gaa gcg gcc 3609 Gln Gly Tyr Val Val Arg Gly Thr Arg Glu Ala Val Glu Ala Ala 1190 1195 1200 atc ggc ccc aac aat aag tac gtg cac ttg acc att gtc aac gat 3654 Ile Gly Pro Asn Asn Lys Tyr Val His Leu Thr Ile Val Asn Asp 1205 1210 1215 gcc aac agt gct ctc atc agt ggc aag cct gaa gat tgc aag gct 3699 Ala Asn Ser Ala Leu Ile Ser Gly Lys Pro Glu Asp Cys Lys Ala 1220 1225 1230 gcc att gct cgc ctg agc agc aac ctc cct gct ttg ccc gtg gac 3744 Ala Ile Ala Arg Leu Ser Ser Asn Leu Pro Ala Leu Pro Val Asp 1235 1240 1245 ctt ggt atg tgt ggc cac tgc ccc gtg gtc gag ccg tac ggc aag 3789 Leu Gly Met Cys Gly His Cys Pro Val Val Glu Pro Tyr Gly Lys 1250 1255 1260 cag atc gct gag atc cat agc gtc ctc gag att ccc gag gtt gcc 3834 Gln Ile Ala Glu Ile His Ser Val Leu Glu Ile Pro Glu Val Ala 1265 1270 1275 ggc ctt gac ctg tac acg agc gtc aac cag aag aag ctt gtt aac 3879 Gly Leu Asp Leu Tyr Thr Ser Val Asn Gln Lys Lys Leu Val Asn 1280 1285 1290 aag tcc act gga gcc agc gac gag tac gca ccc agc ttt ggt gaa 3924 Lys Ser Thr Gly Ala Ser Asp Glu Tyr Ala Pro Ser Phe Gly Glu 1295 1300 1305 tac gca gca cag ctg tac act gtt cag gca gac ttt cct aag atc 3969 Tyr Ala Ala Gln Leu Tyr Thr Val Gln Ala Asp Phe Pro Lys Ile 1310 1315 1320 gcc aag acc gtt agc gac aag aac ttt gac gtc ttt gtt gag act 4014 Ala Lys Thr Val Ser Asp Lys Asn Phe Asp Val Phe Val Glu Thr 1325 1330 1335 ggt ccc aac gct cac cgt agc gcc gca att cgc gcc acc ctt gga 4059 Gly Pro Asn Ala His Arg Ser Ala Ala Ile Arg Ala Thr Leu Gly 1340 1345 1350 aat agc aag cct ttt gtc acc gga tcc atg gac cgc cag aac gag 4104 Asn Ser Lys Pro Phe Val Thr Gly Ser Met Asp Arg Gln Asn Glu 1355 1360 1365 aat gct tgg aca acc atg gtc aag ctg gtt gcc tct ctc caa gcc 4149 Asn Ala Trp Thr Thr Met Val Lys Leu Val Ala Ser Leu Gln Ala 1370 1375 1380 cac cgc gtg cct ggc gtg aag gtc tcc cct ctg tac cac ccc gag 4194 His Arg Val Pro Gly Val Lys Val Ser Pro Leu Tyr His Pro Glu 1385 1390 1395 act gtt gag gag gct acg cag agt tac aac gat atg gtg gct ggc 4239 Thr Val Glu Glu Ala Thr Gln Ser Tyr Asn Asp Met Val Ala Gly 1400 1405 1410 aag aag cct act aag aac aag ttc ttg cgt aag att gtg gtc aat 4284 Lys Lys Pro Thr Lys Phe Leu Arg Lys Ile Val Val Asn 1415 1420 1425 ggt cgc tat gac ccc aaa aag cag ctc gtg ccg ccc cag gtg cta 4329 Gly Arg Tyr Asp Pro Lys Lys Gln Leu Val Pro Pro Gln Val Leu 1430 1435 1440 gct aag ctt cct cct gcg gac ccc aag atc gag gct ctt atc cag 4374 Lys Leu Pro Pro Wing Asp Pro Lys With Glu Wing Leu With Gln 1445 1450 1455 gct cgc aag atg cag cct att gcc ccc aag ttc atg gag cgt ctc 4419 Ala Pro Lys Phe Met Gln Pro Ile Ala Pro Lys Phe Met Glu Arg Leu 1460 1465 1470 gac att cag gag caa gac gcc aca cgc gac cct att ctc aac aag 4464 Asp Ile Gln Glu Gln Asp Ala Thr Arg Asp Pro Ile Leu Asn Lys 1475 1480 1485 gat aac aaa cct tcc gct gct cct gcc ctt gtc cct gct gct ccg 4509 Asp Asn Lys Pro Ser Ala Ala Pro Ala Leu Val Pro Ala Ala Pro 1490 1495 1500 gcc cct gct ccg gcc cgc agc gcc tcc gga gct gtt gtg gct tcc 4554 Ala Pro Ala Pro Ala Arg Ser Ala Ser Gly Ala Val Val Ala Ser 1505 1510 1515 tct gag gct ctc cgt gcc aaa ctt ttg gag ctc aac agc act ttg 4599 Ser Glu Ala Leu Arg Ala Lys Leu Leu Glu Leu Asn Ser Thr Leu 1520 1525 1530 atg ctt ggt gtc aac gcc aac ggt gat ctc gtt gaa gca agc cca 4644 Met Leu Gly Val Asn Ala Asn Gly Asp Leu Val Glu Ala Ser Pro 1535 1540 1545 agt gaa gca tct att gtt gtg ccc aag tgc gat atc aag gat ctt 4689 Ser Glu Ala Ser Ile Val Val Pro Lys Cys Asp Ile Lys Asp Leu 1550 1555 1560 ggc agc cgt gcc ttc atg gag aca tat ggt gta tcc gcc ccc atg 4734 Gly Ser Arg Ala Phe Met Glu Thr Tyr Gly Val Ser Ala Pro Met 1565 1570 1575 tac acc ggc gcc atg gca aag ggc att gca tcc gct gag atg gtt 4779 Tyr Thr Gly Ala Met Ala Lys Gly Ile Ala Ser Ala Glu Met Val 1580 1585 1590 atc gct gcc gga aag cgc ggc atc ctt ggt tct ctc ggt gct ggt 4824 Ile Ala Ala Gly Lys Arg Gly Ile Leu Gly Ser Leu Gly Ala Gly 1595 1600 1605 ggt ctt cct atc gcc acc gta cgc aag gct ctc gaa gct atc cag 4869 Gly Leu Pro Ile Ala Thr Val Arg Lys Ala Leu Glu Ala Ile Gln 1610 1615 1620 gct gaa ctg ccc aag ggc cct tac gct gtc aac ctc atc cac tct 4914 Ala Glu Leu Pro Lys Gly Pro Tyr Ala Val Asn Leu Ile His Ser 1625 1630 1635 ccc ttc gac agc aac ctc gag aag ggt aac gtc gac ctc ttc ctc 4959 Pro Phe Asp Ser Asn Leu Glu Lys Gly Asn Val Asp Leu Phe Leu 1640 1645 1650 gag aag ggc gtc act gtc gtt gaa gcc tcc gcc ttt atg acc ttg 5004 Glu Lys Gly Val Thr Val Val Glu Ala Ser Ala Phe Met Thr Leu 1655 1660 1665 acc ccg cag ctc gtg cgc tac cgt gct gca ggt ctc tct cgc gct 5049 Thr Pro Gln Leu Val Arg Tyr Arg Ala Ala Gly Leu Ser Arg Ala 1670 1675 1680 gct gat ggc tcc acg gtt att aag aac cgc gtc atc ggt aag gtt 5094 Ala Asp Gly Ser Thr Val Ile Lys Asn Arg Val Ile Gly Lys Val 1685 1690 1695 tct cgc aca gag ctt gcc gca atg ttt atc cgt ccc gcg ccc gag 5139 Ser Arg Thr Glu Leu Ala Ala Met Phe Ile Arg Pro Ala Pro Glu 1700 1705 1710 aat ctc ctc gag aag ctg ctg aag tcc ggc gag atc acc caa gag 5184 Asn Leu Leu Glu Lys Leu Leu Lys Ser Gly Glu Ile Thr Gln Glu 1715 1720 1725 cag gct gct ctc gca cgc aca gtg cct gtg gca gac gac att gcc 5229 Gln Ala Ala Leu Ala Arg Thr Val Pro Val Ala Asp Asp Ile Ala 1730 1735 1740 gtt gag gcg gac tcc ggt ggc cac acc gat aac cgc ccc atc cac 5274 Val Glu Ala Asp Ser Gly Gly His Thr Asp Asn Arg Pro Ile His 1745 1750 1755 gtc atc ctc cct ctc att gtc aac ctc cgt gat cgt ctg cac aag 5319 Valley Island Pro Leo Valley Asn Leo Arg Asp Arg Leo His Lys 1760 1765 1770 gag tgc ggc tac cct gcc cac ctt cgc gtt cgc gtt gtt gct gct ggt 5364 Glu Cys Gly Tyr Pro Ala His Leu Arg Val Arg Val Gly Ala Gly 1775 1780 1785 ggt ggc att gga tgc cct cag gcc gcc att gcc acc ttc aac atg 5409 Gly Gly Ile Gly Cys Pro Gln Ala Ala Ile Ala Thr Phe Asn Met 1790 1795 1800 ggc gcg gcc ttc atc gtc act ggt acc gta aac cag atg agt aag 5454 Gly Ala Ala Phe Ile Val Thr Gly Thr Val Asn Gln Met Ser Lys 1805 1810 1815 caa gct gga acc tgt gac acc gtt cgc aag cag ctc tca caa gcc 5499 Gln Ala Gly Thr Cys Asp Thr Val Arg Lys Gln Leu Ser Gln Ala 1820 1825 1830 acc tac tcc gac atc tgc atg gcc cca gca gct gac atg ttt gag 5544 Thr Tyr Ser Asp Ile Cys Met Ala Pro Ala Ala Asp Met Phe Glu 1835 1840 1845 gaa ggt gtc aag ctc cag gtg ctc aag aag gga act atg ttc ccc 5589 Glu Gly Val Lys Leu Gln Val Leu Lys Lys Gly Thr Met Phe Pro 1850 1855 1860 tcg cgt gcc aac aag ctc tat gag ctc ttc gtc aag tat gac tcc 5634 Ser Arg Ala Asn Lys Leu Tyr Glu Leu Phe Val Lys Tyr Asp Ser 1865 1870 1875 ttt gag tcc atg gct cct gga gag ctg gaa cgt gtg gag aag cgc 5679 Phe Glu Ser Met Ala Pro Gly Glu Leu Glu Arg Val Glu Lys Arg 1880 1885 1890 att ttc aag aag tct ctg tca gag gtt tgg gaa gag acc aag gac 5724 Phe Lys Lys Ser Leu Ser Glu Val Trp Glu Glu Thr Lys Asp 1895 1900 1905 ttc tac atc aac agg ttg cag aac ccg gag aag att gag cgc gcg 5769 Phe Tyr with Arg Asn Leu Gln Asn Pro Glu Lys With Glu Arg Ala 1910 1915 1920 gag cgt gac ccc aag ctt aag atg tcc ttg tgc ttc cgc tgg tac 5814 Glu Arg Asp Pro Lys Leu Lys Met Ser Cys Phe Arg Trp Tyr 1925 1930 1935 ctt ggt ttg gcg agc ttc tgg gca aac gct ggc atc ccg gac cgt 5859 Leu Gly Leu Ala Ser Phe Trp Ala Asn Ala Gly Ile Pro Asp Arg 1940 1945 1950 gcc atg gac tac cag gtt tgg tgt ggc cca gcg att gga tct ttc 5904 Ala Met Asp Tyr Gln Val Trp Cys Gly Pro Ala Ile Gly Ser Phe 1955 1960 1965 aac gac ttc atc aag ggt acc tac ctt gac ccc gcc gtt gcc aac 5949 Asn Asp Phe Ile Lys Gly Thr Tyr Leu Asp Pro Ala Val Ala Asn 1970 1975 1980 gag tac ccc gat gtt gtg caa atc aac ttg cag atc ctc cgt ggt 5994 Glu Tyr Pro Asp Val Val Gln Ile Asn Leu Gln Ile Leu Arg Gly 1985 1990 1995 gcc tgc ttc ttg cgc cgc ctc gaa gct gtc cgt aat gcc ccg ctg 6039 Ala Cys Phe Leu Arg Arg Leu Glu Ala Val Arg Asn Ala Pro Leu 2000 2005 2010 aag gct aac gcc aag cag gtt gct gcc gag att gat gac atc tac 6084 Lys Ala Asn Ala Lys Gln Val Ala Ala Glu Ile Asp Asp Ile Tyr 2015 2020 2025 gtg ccc act gag cgc ctg taa 6105 Val Pro Thr Glu Arg Leu 2030 <210> 2 <211> 2034 <212> PRT <213> Aurantiochytrium sp. OH4 <400> 2 Met Ala Ser Arg Lys Asn Val Ser Ala Ala His Glu Met His Asp Glu 1 5 10 15 Lys Arg Ile Ala Val Val Gly Met Ala Val Gln Tyr Ala Gly Cys Lys 20 25 30 Asp Lys Glu Glu Phe Trp Lys Val Val Met Gly Gly Glu Ala Ala Trp 35 40 45 Thr Lys Ile Ser Asp Lys Arg Leu Gly Ser Asn Lys Arg Ala Glu His 50 55 60 Phe Lys Ala Glu Arg Ser Lys Phe Ala Asp Thr Phe Cys Asn Glu Asn 65 70 75 80 Tyr Gly Cys Val Asp Asp Ser Val Asp Asn Glu His Glu Leu Leu Leu 85 90 95 Lys Leu Ser Lys Lys Ala Leu Ser Glu Thr Ser Val Ser Asp Ser Thr 100 105 110 Arg Cys Gly Ile Val Ser Gly Cys Leu Ser Phe Pro Met Asp Asn Leu 115 120 125 Gln Gly Glu Leu Leu Asn Val Tyr Gln Asn His Val Glu Lys Lys Leu 130 135 140 Gly Ala Arg Val Phe Lys Asp Ala Ser Lys Trp Ser Glu Arg Glu Gln 145 150 155 160 Ser Gln Asn Pro Glu Ala Gly Asp Arg Arg Ile Phe Met Asp Pro Ala 165 170 175 Ser Phe Val Ala Glu Glu Leu Asn Leu Gly Pro Leu His Tyr Ser Val 180 185 190 Asp Ala Ala Cys Ala Thr Ala Leu Tyr Val Leu Arg Leu Ala Gln Asp 195 200 205 His Leu Val Ser Gly Ala Ala Asp Val Met Leu Ala Gly Ala Thr Cys 210 215 220 Phe Pro Glu Pro Phe Phe Ile Leu Ser Gly Phe Ser Thr Phe Gln Ala 225 230 235 240 Met Pro Val Ser Gly Asp Gly Ile Ser Tyr Pro Leu His Lys Asp Ser 245 250 255 Gln Gly Leu Thr Pro Gly Glu Gly Gly Ala Ile Met Val Leu Lys Arg 260 265 270 Leu Asp Asp Ala Ile Arg Asp Gly Asp His Ile Tyr Gly Thr Leu Leu 275 280 285 Gly Ala Thr Ile Ser Asn Ala Gly Cys Gly Leu Pro Leu Lys Pro His 290 295 300 Leu Pro Ser Glu Lys Ser Cys Leu Ile Asp Thr Tyr Lys Arg Val Asn 305 310 315 320 Val His Pro His Lys Ile Gln Tyr Val Glu Cys His Ala Thr Gly Thr 325 330 335 Pro Gln Gly Asp Arg Val Glu Ile Asp Ala Val Lys Ala Cys Phe Glu 340 345 350 Gly Lys Val Pro Arg Phe Gly Ser Ser Lys Gly Asn Phe Gly His Thr 355 360 365 Leu Val Ala Ala Gly Phe Ala Gly Met Cys Lys Val Leu Leu Ala Met 370 375 380 Lys His Gly Val Ile Pro Pro Thr Pro Gly Val Asp Gly Ser Ser Gln 385 390 395 400 Met Asp Pro Leu Val Val Ser Glu Pro Ile Pro Trp Pro Asp Thr Glu 405 410 415 Gly Glu Pro Lys Arg Ala Gly Leu Ser Ala Phe Gly Phe Gly Gly Thr 420 425 430 Asn Ala His Ala Val Phe Glu Glu Phe Asp Arg Ser Lys Ala Ala Cys 435 440 445 Ala Thr His Asp Ser Ile Ser Ser Leu Ser Ser Arg Cys Gly Gly Glu 450 455 460 Gly Asn Met Arg Ile Ala Ile Thr Gly Met Asp Ala Thr Phe Gly Ser 465 470 475 480 Leu Lys Gly Leu Asp Ala Phe Glu Arg Ala Ile Tyr Asn Gly Gln His 485 490 495 Gly Ala Val Pro Leu Pro Glu Lys Arg Trp Arg Phe Leu Gly Lys Asp 500 505 510 Lys Asp Phe Leu Asp Leu Cys Gly Val Lys Glu Val Pro His Gly Cys 515 520 525 Tyr Ile Glu Asp Val Glu Val Asp Phe Ser Arg Leu Arg Thr Pro Met 530 535 540 Thr Pro Asp Asp Met Leu Arg Pro Met Gln Leu Leu Ala Val Thr Thr 545 550 555 560 Ile Asp Arg Ala Ile Leu Asn Ser Gly Leu Lys Lys Gly Gly Lys Val 565 570 575 Ala Val Phe Val Gly Leu Gly Thr Asp Leu Glu Leu Tyr Arg His Arg 580 585 590 Ala Arg Val Ala Leu Lys Glu Arg Ala Arg Pro Glu Ala Ala Ala Ala 595 600 605 Leu Asn Asp Met Met Ser Tyr Ile Asn Asp Cys Gly Thr Ala Thr Ser 610 615 620 Tyr Thr Ser Tyr Ile Gly Asn Leu Val Ala Thr Arg Val Ser Ser Gln 625 630 635 640 Trp Gly Phe Glu Gly Pro Ser Phe Thr Ile Thr Glu Gly Asn Asn Ser 645 650 655 Val Tyr Arg Cys Ala Glu Leu Gly Lys Tyr Leu Leu Glu Thr Gly Glu 660 665 670 Val Glu Ala Val Val Ile Ala Gly Val Asp Leu Cys Ala Ser Ala Glu 675 680 685 Asn Leu Tyr Val Lys Ser Arg Arg Phe Lys Val Ser Glu Gln Glu Ser 690 695 700 Pro Arg Ala Ser Phe Asp Ser Gly Ala Asp Gly Tyr Phe Val Gly Glu 705 710 715 720 Gly Cys Gly Ala Leu Val Leu Lys Arg Glu Ser Asp Cys Thr Lys Asp 725 730 735 Glu Arg Ile Tyr Ala Cys Met Asp Ala Ile Val Pro Gly Asn Met Pro 740 745 750 Ala Ala Cys Met Glu Glu Ala Leu Ala Gln Ala Arg Val Asn Pro Lys 755 760 765 Asp Val Glu Met Leu Glu Leu Ser Ala Asp Ser Ala Arg His Leu Lys 770 775 780 Asn Pro Ser Val Leu Pro Lys Glu Leu Thr Ala Glu Glu Glu Ile Arg 785 790 795 800 Gly Ile Glu Ala Ile Leu Ser Gln Arg Ser Ser Asn Glu Ala Val Glu 805 810 815 Pro His Asn Val Ala Val Ser Ser Val Lys Ser Thr Val Gly Asp Thr 820 825 830 Gly Tyr Ala Ser Gly Ala Ala Ser Leu Ile Lys Thr Ala Leu Cys Leu 835 840 845 Tyr Asn Arg Tyr Leu Pro Ser Asn Gly Ala Ser Trp Glu Glu Pro Ala 850 855 860 Pro Glu Thr Gln Trp Gly Lys Ser Leu Tyr Ala Cys Gln Ser Ser Arg 865 870 875 880 Ala Trp Leu Lys Asn Pro Gly Ala Arg Arg His Ala Ala Val Ser Gly 885 890 895 Val Ser Glu Thr Arg Ser Cys Tyr Thr Val Leu Leu Ser Asp Val Glu 900 905 910 Gly His His Glu Thr Lys Ser Arg Ile Ser Leu Asp Asp Asp Ala Val 915 920 925 Lys Leu Leu Val Ile Arg Gly Asp Ser His Asp Ala Ile Thr Gln Arg 930 935 940 Val Asp Lys Leu Arg Glu Arg Leu Ala Gln Pro Ser Ala Asn Val Arg 945 950 955 960 Leu Ala Phe Met Glu Leu Leu Gly Glu Ser Ile Ala Gln Glu Thr Lys 965 970 975 Thr Pro Leu Pro Ala Phe Ala Leu Cys Leu Val Thr Ser Pro Ser Lys 980 985 990 Leu Gln Lys Glu Leu Glu Leu Ala Ser Lys Gly Ile Pro Arg Ser Leu 995 1000 1005 Lys Met Gly Arg Asp Trp Thr Ser Pro Ser Gly Ser His Phe Ala 1010 1015 1020 Pro Lys Pro Leu Ser Ser Asp Arg Val Ala Phe Met Tyr Gly Glu 1025 1030 1035 Gly Arg Ser Pro Tyr Tyr Gly Ile Gly Leu Asp Ile His Arg Ile 1040 1045 1050 Trp Pro Glu Leu His Glu Phe Val Asn Ala Lys Thr Asn Lys Leu 1055 1060 1065 Trp Asp Gln Gly Asp Arg Trp Leu Ile Pro Arg Ala Ser Thr Lys 1070 1075 1080 Glu Glu Leu Lys Ala Gln Glu Asp Glu Phe Ser Arg Asn Gln Val 1085 1090 1095 Glu Met Phe Arg Leu Gly Ile Leu Met Ser Met Cys Phe Thr His 1100 1105 1110 Ile Ala Arg Asp Val Leu Gly Ile Gln Pro Lys Ala Ala Phe Gly 1115 1120 1125 Leu Ser Leu Gly Glu Ile Ser Met Val Phe Ala Phe Ser Glu Lys 1130 1135 1140 Asn Gly Leu Val Ser Glu Glu Leu Thr Thr Lys Leu Arg Asn Ser 1145 1150 1155 Glu Val Trp Arg Lys Ala Leu Ala Val Glu Phe Asp Ala Leu Arg 1160 1165 1170 Lys Ala Trp Asn Ile Pro Gln Asp Thr Pro Val Ser Glu Phe Trp 1175 1180 1185 Gln Gly Tyr Val Val Arg Gly Thr Arg Glu Ala Val Glu Ala Ala 1190 1195 1200 Ile Gly Pro Asn Asn Lys Tyr Val His Leu Thr Ile Val Asn Asp 1205 1210 1215 Ala Asn Ser Ala Leu Ile Ser Gly Lys Pro Glu Asp Cys Lys Ala 1220 1225 1230 Ala Ile Ala Arg Leu Ser Ser Asn Leu Pro Ala Leu Pro Val Asp 1235 1240 1245 Leu Gly Met Cys Gly His Cys Pro Val Val Glu Pro Tyr Gly Lys 1250 1255 1260 Gln Ile Ala Glu Ile His Ser Val Leu Glu Ile Pro Glu Val Ala 1265 1270 1275 Gly Leu Asp Leu Tyr Thr Ser Val Asn Gln Lys Lys Leu Val Asn 1280 1285 1290 Lys Ser Thr Gly Ala Ser Asp Glu Tyr Ala Pro Ser Phe Gly Glu 1295 1300 1305 Tyr Ala Ala Gln Leu Tyr Thr Val Gln Ala Asp Phe Pro Lys Ile 1310 1315 1320 Ala Lys Thr Val Ser Asp Lys Asn Phe Asp Val Phe Val Glu Thr 1325 1330 1335 Gly Pro Asn Ala His Arg Ser Ala Ala Ile Arg Ala Thr Leu Gly 1340 1345 1350 Asn Ser Lys Pro Phe Val Thr Gly Ser Met Asp Arg Gln Asn Glu 1355 1360 1365 Asn Ala Trp Thr Thr Met Val Lys Leu Val Ala Ser Leu Gln Ala 1370 1375 1380 His Arg Val Pro Gly Val Lys Val Ser Pro Leu Tyr His Pro Glu 1385 1390 1395 Thr Val Glu Glu Ala Thr Gln Ser Tyr Asn Asp Met Val Ala Gly 1400 1405 1410 Lys Lys Pro Thr Lys Asn Lys Phe Leu Arg Lys Ile Val Val Asn 1415 1420 1425 Gly Arg Tyr Asp Pro Lys Lys Gln Leu Val Pro Pro Gln Val Leu 1430 1435 1440 Ala Lys Leu Pro Pro Ala Asp Pro Lys Ile Glu Ala Leu Ile Gln 1445 1450 1455 Ala Arg Lys Met Gln Pro Ile Ala Pro Lys Phe Met Glu Arg Leu 1460 1465 1470 Asp Ile Gln Glu Gln Asp Ala Thr Arg Asp Pro Ile Leu Asn Lys 1475 1480 1485 Asp Asn Lys Pro Ser Ala Ala Pro Ala Leu Val Pro Ala Ala Pro 1490 1495 1500 Ala Pro Ala Pro Ala Arg Ser Ala Ser Gly Ala Val Val Ala Ser 1505 1510 1515 Ser Glu Ala Leu Arg Ala Lys Leu Leu Glu Leu Asn Ser Thr Leu 1520 1525 1530 Met Leu Gly Val Asn Ala Asn Gly Asp Leu Val Glu Ala Ser Pro 1535 1540 1545 Ser Glu Ala Ser Ile Val Val Pro Lys Cys Asp Ile Lys Asp Leu 1550 1555 1560 Gly Ser Arg Ala Phe Met Glu Thr Tyr Gly Val Ser Ala Pro Met 1565 1570 1575 Tyr Thr Gly Ala Met Ala Lys Gly Ile Ala Ser Ala Glu Met Val 1580 1585 1590 Ile Ala Ala Gly Lys Arg Gly Ile Leu Gly Ser Leu Gly Ala Gly 1595 1600 1605 Gly Leu Pro Ile Ala Thr Val Arg Lys Ala Leu Glu Ala Ile Gln 1610 1615 1620 Ala Glu Leu Pro Lys Gly Pro Tyr Ala Val Asn Leu Ile His Ser 1625 1630 1635 Pro Phe Asp Ser Asn Leu Glu Lys Gly Asn Val Asp Leu Phe Leu 1640 1645 1650 Glu Lys Gly Val Thr Val Val Glu Ala Ser Ala Phe Met Thr Leu 1655 1660 1665 Thr Pro Gln Leu Val Arg Tyr Arg Ala Ala Gly Leu Ser Arg Ala 1670 1675 1680 Ala Asp Gly Ser Thr Val Ile Lys Asn Arg Val Ile Gly Lys Val 1685 1690 1695 Ser Arg Thr Glu Leu Ala Ala Met Phe Ile Arg Pro Ala Pro Glu 1700 1705 1710 Asn Leu Leu Glu Lys Leu Leu Lys Ser Gly Glu Ile Thr Gln Glu 1715 1720 1725 Gln Ala Ala Leu Ala Arg Thr Val Pro Val Ala Asp Asp Ile Ala 1730 1735 1740 Val Glu Ala Asp Ser Gly Gly His Thr Asp Asn Arg Pro Ile His 1745 1750 1755 Val Ile Leu Pro Leu Ile Val Asn Leu Arg Asp Arg Leu His Lys 1760 1765 1770 Glu Cys Gly Tyr Pro Ala His Leu Arg Val Arg Val Gly Ala Gly 1775 1780 1785 Gly Gly Ile Gly Cys Pro Gln Ala Ala Ile Ala Thr Phe Asn Met 1790 1795 1800 Gly Ala Ala Phe Ile Val Thr Gly Thr Val Asn Gln Met Ser Lys 1805 1810 1815 Gln Ala Gly Thr Cys Asp Thr Val Arg Lys Gln Leu Ser Gln Ala 1820 1825 1830 Thr Tyr Ser Asp Ile Cys Met Ala Pro Ala Ala Asp Met Phe Glu 1835 1840 1845 Glu Gly Val Lys Leu Gln Val Leu Lys Lys Gly Thr Met Phe Pro 1850 1855 1860 Ser Arg Ala Asn Lys Leu Tyr Glu Leu Phe Val Lys Tyr Asp Ser 1865 1870 1875 Phe Glu Ser Met Ala Pro Gly Glu Leu Glu Arg Val Glu Lys Arg 1880 1885 1890 Ile Phe Lys Lys Ser Leu Ser Glu Val Trp Glu Glu Thr Lys Asp 1895 1900 1905 Phe Tyr Ile Asn Arg Leu Gln Asn Pro Glu Lys Ile Glu Arg Ala 1910 1915 1920 Glu Arg Asp Pro Lys Leu Lys Met Ser Leu Cys Phe Arg Trp Tyr 1925 1930 1935 Leu Gly Leu Ala Ser Phe Trp Ala Asn Ala Gly Ile Pro Asp Arg 1940 1945 1950 Ala Met Asp Tyr Gln Val Trp Cys Gly Pro Ala Ile Gly Ser Phe 1955 1960 1965 Asn Asp Phe Ile Lys Gly Thr Tyr Leu Asp Pro Ala Val Ala Asn 1970 1975 1980 Glu Tyr Pro Asp Val Val Gln Ile Asn Leu Gln Ile Leu Arg Gly 1985 1990 1995 Ala Cys Phe Leu Arg Arg Leu Glu Ala Val Arg Asn Ala Pro Leu 2000 2005 2010 Lys Ala Asn Ala Lys Gln Val Ala Ala Glu Ile Asp Asp Ile Tyr 2015 2020 2025 Val Pro Thr Glu Arg Leu 2030 <210> 3 <211> 4302 <212> DNA <213> Aurantiochytrium sp. OH4 <220> <221> CDS <222> (1)..(4302) <400> 3 atg gcc act cgc gtg aag acc aac aag aaa cca tgc tgg gag atg acc 48 Met Ala Thr Arg Val Lys Thr Asn Lys Lys Pro Cys Trp Glu Met Thr 1 5 10 15 aag gag gag ctc acc agc ggc aag aac gtc gtt ttc gac tat gac gag 96 Lys Glu Glu Leu Thr Ser Gly Lys Asn Val Val Phe Asp Tyr Asp Glu 20 25 30 ctc ctt gag ttc gcc gag ggt gac atc agc aag gtc ttc ggc ccc gaa 144 Leu Leu Glu Phe Ala Glu Gly Asp Ile Ser Lys Val Phe Gly Pro Glu 35 40 45 ttc agc cag atc gac cag tac aag cgt cgc gtt cgt ctc ccc gcc cgc 192 Phe Ser Gln Ile Asp Gln Tyr Lys Arg Arg Val Arg Leu Pro Ala Arg 50 55 60 gag tac ctc ctc gtc acc cgc gtc acc ctc atg gac gcc gag gtc aac 240 Glu Tyr Leu Leu Val Thr Arg Val Thr Leu Met Asp Ala Glu Val Asn 65 70 75 80 aac tac cgc gtc ggt gcc cgc atg gtc act gag tac gac ctc ccc gtc 288 Asn Tyr Arg Val Gly Ala Arg Met Val Thr Glu Tyr Asp Leu Pro Val 85 90 95 aac ggt gag ctc tct gag ggt ggt gac tgc ccc tgg gcc gtg ctc gtc 336 Asn Gly Glu Leu Ser Glu Gly Gly Asp Cys Pro Trp Ala Val Leu Val 100 105 110 gag agt ggc cag tgt gat ctc atg ctc atc tcc tac atg ggt att gac 384 Glu Ser Gly Gln Cys Asp Leu Met Leu Ile Ser Tyr Met Gly Ile Asp 115 120 125 ttc cag aac aag agc gac cgc gtc tac cgt ctg ctc aac acc acc ctc 432 Phe Gln Asn Lys Ser Asp Arg Val Tyr Arg Leu Leu Asn Thr Thr Leu 130 135 140 acc ttc tac ggt gtt gcc cag gag ggc gag acc ctg gag tac gat atc 480 Thr Phe Tyr Gly Val Ala Gln Glu Gly Glu Thr Leu Glu Tyr Asp Ile 145 150 155 160 cgc gtg acc ggc ttc gcc aag cgt ctc gac ggt gac atc tcc atg ttc 528 Arg Val Thr Gly Phe Ala Lys Arg Leu Asp Gly Asp Ile Ser Met Phe 165 170 175 ttc ttc gag tac gac tgc tac gtc aac ggc cgt ctc ctc atc gag atg 576 Phe Phe Glu Tyr Asp Cys Tyr Val Asn Gly Arg Leu Leu Ile Glu Met 180 185 190 cgc gac ggc tgt gcc ggt ttc ttc acc aac gag gag ctc gcc gcc ggc 624 Arg Asp Gly Cys Ala Gly Phe Phe Thr Asn Glu Glu Leu Ala Ala Gly 195 200 205 aag ggt gtc gtc ttt acc cgc gct gat ctc ctc gcc cgc gag aag acc 672 Lys Gly Val Val Phe Thr Arg Ala Asp Leu Leu Ala Arg Glu Lys Thr 210 215 220 aag aag cag gac atc acc ccg tac gcc att gcc ccg cgt ctt aac aag 720 Lys Lys Gln Asp Ile Thr Pro Tyr Ala Ile Ala Pro Arg Leu Asn Lys 225 230 235 240 acc gtt ctc aac gag act gag atg cag tcc ctc gtg gac aag aac tgg 768 Thr Val Leu Asn Glu Thr Glu Met Gln Ser Leu Val Asp Lys Asn Trp 245 250 255 acc aag gtt ttc ggc ccc gag aac ggc atg gac cag atc aac tac aaa 816 Thr Lys Val Phe Gly Pro Glu Asn Gly Met Asp Gln Ile Asn Tyr Lys 260 265 270 ctc tgc gcc cgt aag atg ctc atg att gac cgc gtc acc aag att gac 864 Leu Cys Ala Arg Lys Met Leu Met Ile Asp Arg Val Thr Lys Ile Asp 275 280 285 tac acc ggt ggc ccc tac ggc ctt ggt ctc ctc gtt ggt gag aag atc 912 Tyr Thr Gly Gly Pro Tyr Gly Leu Gly Leu Leu Val Gly Glu Lys Ile 290 295 300 ctc gag cgc gac cac tgg tac ttc ccg tgc cac ttc gtc gga gac cag 960 Leu Glu Arg Asp His Trp Tyr Phe Pro Cys His Phe Val Gly Asp Gln 305 310 315 320 gtc atg gct gga tcc ctc gtg tct gac ggc tgc agc cag ctc ctc aag 1008 Val Met Ala Gly Ser Leu Val Ser Asp Gly Cys Ser Gln Leu Leu Lys 325 330 335 atg tac atg ctc tgg ctc ggc ctc cac ctt aag acc ggt ccc ttc gac 1056 Met Tyr Met Leu Trp Leu Gly Leu His Leu Lys Thr Gly Pro Phe Asp 340 345 350 ttc cgc ccc gtc aac ggc cac ccc aac aag gtc cgc tgc cgt ggc cag 1104 Phe Arg Pro Val Asn Gly His Pro Asn Lys Val Arg Cys Arg Gly Gln 355 360 365 atc tcc ccg cac aag ggt aag ctc gtc tac gtc atg gag atc aag gag 1152 Ile Ser Pro His Lys Gly Lys Leu Val Tyr Val Met Glu Ile Lys Glu 370 375 380 atg gga tac gac gag gct ggt gac ccg tac gcc att gcc gat gtc aac 1200 Met Gly Tyr Asp Glu Ala Gly Asp Pro Tyr Ala Ile Ala Asp Val Asn 385 390 395 400 att ctc gac att gac ttc gag aag ggc cag act ttc gac ctt gcc aac 1248 I Leu Asp Ile Asp Phe Glu Lys Gly Gln Thr I Phe Asp Leu Ala Asn 405 410 415 ctc cac gag tac ggc aag ggc gac ctc aac aag aag atc gtc gtc gac 1296 Lew His Glu Tyr Gly Lys Gly Asp Lion Asn Lys Lys Ile Val Val Asp 420 425 430 ttc aag gtt att gcc ctc aag ctc cag aag cgc tct ggc cct gcc gtt 1344 Phe Lys Gly Ile Ala Leu Lys Leu Gln Lys Arg Ser Gly Pro Ala Val 435 440 445 gtc gct ccc gag aag ccc ctc gct ctc aac aag gac ctt tgc gcc ccg 1392 Val Ala Pro Glu Lys Pro Leu Ala Leu Asn Lys Asp Leu Cys Ala Pro 450 455 460 gct gtt gag gcc atc cct gag cac atc ctc aag ggc gat gct ctt gcc 1440 Wing Val Glu Wing Wing Pro Glu His Wing Leu Lys Gly Asp Wing Leu Wing 465 470 475 480 cct aac cag atg acc tgg cac ccg atg tcc aag atc gct ggc aac ccc 1488 Pro Asn Gln Met Thr Trp His Pro Met Ser Lys Ile Ala Gly Asn Pro 485,490,495 acg ccc tcg ttc tct ccc tcg gcc tac cct ccc cgt ccc atc acc ttc 1536 Thr Pro Ser Phe Ser Pro Ser Ala Tyr Pro Pro Arg Pro Ile Thr Phe 500 505 510 acc ccg ttc ccc ggc aac aag aac gac aac aac cac gtg ccc ggc gag 1584 Thr Pro Phe Pro Gly Asn Lys Asn Asp Asn Asn His Val Pro Gly Glu 515 520 525 atg ccg ctc tcg tgg tac aac atg gct gag ttc atg gcc ggc aag gtc 1632 Met Pro Leu Ser Trp Tyr Asn Met Ala Glu Phe Met Ala Gly Lys Val 530 535 540 agc ctc tgc ctc ggc cct gag ttc gcc aag ttc gat gac tcc aac acc 1680 Ser Leu Cys Leu Gly Pro Glu Phe Ala Lys Phe Asp Asp Ser Asn Thr 545 550 555 560 agc cgc agc cct gca tgg gat ctt gct ctt gtg act cgt gtg gtc tcc 1728 Ser Arg Ser Pro Ala Trp Asp Leu Ala Leu Val Thr Arg Val Val Ser 565 570 575 gtt tct gac atg gag tgg gtc cag tgg aag aac gtg gac tgc aac ccg 1776 Val Ser Asp Met Glu Trp Val Gln Trp Lys Asn Val Asp Cys Asn Pro 580 585 590 tcc aag gga acc atg gtt ggc gag ttc gac tgc ccc atc gac gcc tgg 1824 Ser Lys Gly Thr Met Val Gly Glu Phe Asp Cys Pro Ile Asp Ala Trp 595 600 605 ttc ttc cag gga tct tgt aac gac ggc cac atg ccg tac tcc atc ctc 1872 Phe Phe Gln Gly Ser Cys Asn Asp Gly His Met Pro Tyr Ser Ile Leu 610 615 620 atg gag atc gcc ctc cag acc tct ggt gtc ctc acc tct gtg ctc aag 1920 Met Glu Ile Ala Leu Gln Thr Ser Gly Val Leu Thr Ser Val Leu Lys 625 630 635 640 gcc ccg ctc acc atg gag aag aag gac att ctc ttc cgc aac ctt gac 1968 Ala Pro Leu Thr Met Glu Lys Lys Asp Ile Leu Phe Arg Asn Leu Asp 645 650 655 gcc aac gcc gag atg gtt cgc tct gat att gac ctc cgc ggc aag acc 2016 Ala Asn Ala Glu Met Val Arg Ser Asp Ile Asp Leu Arg Gly Lys Thr 660 665 670 atc cac aac ctc acc aag tgt acc ggc tac agc atg ctc gga gac atg 2064 Ile His Asn Leu Thr Lys Cys Thr Gly Tyr Ser Met Leu Gly Asp Met 675 680 685 ggt gtc cac cgc ttc agc ttc gag ctc tct gtt gat ggt gta gtc ttc 2112 Gly Val His Arg Phe Ser Phe Glu Leu Ser Val Asp Gly Val Val Phe 690 695 700 tac aag ggt acc acc tcc ttc ggc tgg ttc gtc cct gag gtc ttc atc 2160 Tyr Lys Gly Thr Thr Ser Phe Gly Trp Phe Val Pro Glu Val Phe Ile 705 710 715 720 tcc cag act ggt ctc gac aac ggt cgc cgc acc cag ccc tgg cac att 2208 Ser Gln Thr Gly Leu Asp Asn Gly Arg Arg Thr Gln Pro Trp His Ile 725 730 735 gag tcc aag gtg cct tcc gcc cag gtc ctc acc tac gac gtt acc ccc 2256 Glu Ser Lys Val Pro Ser Ala Gln Val Leu Thr Tyr Asp Val Thr Pro 740 745 750 aac ggt gcc ggt cgc acc cag ctc tac gcc aac gct ccc aag ggt gct 2304 Asn Gly Ala Gly Arg Thr Gln Leu Tyr Ala Asn Ala Pro Lys Gly Ala 755 760 765 cag ctc agt cgc cgc tgg aac cag tgc cag tac ctt gac acc atc gac 2352 Gln Leu Ser Arg Arg Trp Asn Gln Cys Gln Tyr Leu Asp Thr Ile Asp 770 775 780 ctt gtg gtc gcc ggt ggc tcc gcc ggt ctt ggc tac ggt cat ggc cgc 2400 Leu Val Val Ala Gly Gly Ser Ala Gly Leu Gly Tyr Gly His Gly Arg 785 790 795 800 aag cag gtg aac ccc aag gac tgg ttc ttc tcg tgc cac ttc tgg ttc 2448 Lys Gln Val Asn Pro Lys Asp Trp Phe Phe Ser Cys His Phe Trp Phe 805 810 815 gac tcc gtc atg ccc ggc tcg ctc ggt gtg gag tct atg ttc cag ctc 2496 Asp Ser Val Met Pro Gly Ser Leu Gly Val Glu Ser Met Phe Gln Leu 820 825 830 gtc gag tcc atc gct gtc aag cag gac ctc gcc ggc aag tac ggc atc 2544 Val Glu Ser Ile Ala Val Lys Gln Asp Leu Ala Gly Lys Tyr Gly Ile 835 840 845 acc aac ccg acc ttc gct cat gct ccg ggc aag atc tcc tgg aag tac 2592 Thr Asn Pro Thr Phe Ala His Ala Pro Gly Lys Ile Ser Trp Lys Tyr 850 855 860 cgt ggt cag ctc acc ccc acc tcc aag ttc atg gac tcc gag gcc cac 2640 Arg Gly Gln Leu Thr Pro Thr Ser Lys Phe Met Asp Ser Glu Ala His 865 870 875 880 att gtc tcc atc gag gcc cac gac ggc gtc gtc gac atc gtt gcc aat 2688 Ile Val Ser Ile Glu Ala His Asp Gly Val Val Asp Ile Val Ala Asn 885 890 895 ggt aac ctc tgg gct gat ggc ctc cgc gtc tac aac gtc agc aac atc 2736 Gly Asn Leu Trp Ala Asp Gly Leu Arg Val Tyr Asn Val Ser Asn Ile 900 905 910 cgt gtt cgc att acc atc acc ctc aag cag ctc aag gct gag ctt ctt 2784 Arg Val Arg Ile Thr Ile Thr Leu Lys Gln Leu Lys Ala Glu Leu Leu 915 920 925 gac gtt gag aag cct ctc tac atc tcc tcc agc aac ggc cag gtc aag 2832 Asp Val Glu Lys Pro Leu Tyr Ile Ser Ser Ser Asn Gly Gln Val Lys 930,935,940 aag cac gcc gat gtg gct ggt ggc cag gcc acc att gtg cag gct tgc 2880 Lys His Ala Asp Val Ala Gly Gly Gln Ala Thr Ile Val Gln Ala Cys 945 950 955 960 agc ctc agt gac ctc ggt gat gaa ggc ttc atg aag acc tac ggt gtt 2928 Ser Leu Ser Asp Leu Gly Asp Glu Gly Phe Met Lys Thr Tyr Gly Val 965,970,975 gtg gct cct ctc tac acc ggt gcc atg gcc aag ggt att gcc tct gct 2976 Val Ala Pro Leu Tyr Thr Gly Ala Met Ala Lys Gly Ile Ala Ser Ala 980,985,990 gac ctt gtg att gcc act ggt aag cgt aag atc ctc ggt tcc ttc ggt 3024 Asp Leu Val Ile Ala Thr Gly Lys Arg Lys Ile Leu Gly Ser Phe Gly 995 1000 1005 gct ggt ggt ctc ccc atg cac att gtc cgt gcc gct gtt gag aag 3069 Ala Gly Gly Leu Pro Met His Ile Val Arg Ala Ala Val Glu Lys 1010 1015 1020 atc cag gct gag ctc ccg aac ggc ccc ttc gcc gtc aac ctc atc 3114 Ile Gln Ala Glu Leu Pro Asn Gly Pro Phe Ala Val Asn Leu Ile 1025 1030 1035 cac tcc ccc ttc gat agc aac ctt gag aag ggc aac gtt gac ctc 3159 His Ser Pro Phe Asp Ser Asn Leu Glu Lys Gly Asn Val Asp Leu 1040 1045 1050 ttc ctc gag aag ggc gtc act gtc gtc gag gcc tcc gcc ttc atg 3204 Phe Leu Glu Lys Gly Val Thr Val Val Glu Ala Ser Ala Phe Met 1055 1060 1065 acc ttg acc ccg caa gtc gtc cgc tac cgt gct gct ggt ctt tcc 3249 Thr Leu Thr Pro Gln Val Val Arg Tyr Arg Ala Ala Gly Leu Ser 1070 1075 1080 cgt aac gct gat ggc tcc att aac atc aag aac cgc atc atc ggt 3294 Arg Asn Ala Asp Gly Ser Ile Asn Ile Lys Asn Arg Ile Ile Gly 1085 1090 1095 aag gtc tcc cgt acc gag ctc gct gag atg ttc atc cgc cct gcc 3339 Lys Val Ser Arg Thr Glu Leu Ala Glu Met Phe Ile Arg Pro Ala 1100 1105 1110 ccg cag aac ctc ctc gac aag ctc atc cag tct ggt gag att acc 3384 Pro Gln Asn Leu Leu Asp Lys Leu Ile Gln Ser Gly Glu Ile Thr 1115 1120 1125 aag gag cag gct gag ctt gcc aag ctc gtc ccc gtc gcc gac gat 3429 Lys Glu Gln Ala Glu Leu Ala Lys Leu Val Pro Val Ala Asp Asp 1130 1135 1140 atc gcc gtc gag gcc gac tct ggt ggc cac acc gac aac cgc ccc 3474 Ile Ala Val Glu Ala Asp Ser Gly Gly His Thr Asp Asn Arg Pro 1145 1150 1155 atc cac gtc atc ctc ccc ctt atc atc aac ctc cgc aac cgc ctc 3519 Ile His Val Ile Leu Pro Leu Ile Ile Asn Leu Arg Asn Arg Leu 1160 1165 1170 cac aag gag tgc ggc tac ccc gct cac ctc cgc gtg cgc gtt gga 3564 His Lys Glu Cys Gly Tyr Pro Ala His Leu Arg Val Arg Val Gly 1175 1180 1185 gct ggt ggt ggt gtt gga tgc ccc cag gcc gct gcc gct gct ctc 3609 Ala Gly Gly Gly Val Gly Cys Pro Gln Ala Ala Ala Ala Ala Leu 1190 1195 1200 gct atg ggt gct gcc ttc ctt gtt acc ggc act gtc aac cag gtc 3654 Ala Met Gly Ala Ala Phe Leu Val Thr Gly Thr Val Asn Gln Val 1205 1210 1215 gcc aag cag tcc ggc acc tgc gac aat gtc cgc aag cag ctc tgc 3699 Ala Lys Gln Ser Gly Thr Cys Asp Asn Val Arg Lys Gln Leu Cys 1220 1225 1230 atg gcc acc tac tct gac gtc tgc atg gct ccc gct gct gac atg 3744 Met Ala Thr Tyr Ser Asp Val Cys Met Ala Pro Ala Ala Asp Met 1235 1240 1245 ttc gag gag ggc gtc aag ctc cag gtc ctc aag aag gga acc atg 3789 Phe Glu Glu Gly Val Lys Leu Gln Val Leu Lys Lys Gly Thr Met 1250 1255 1260 ttc ccg tcc agg gct aac aag ctc tac gag ctc ttc tgc aag tac 3834 Phe Pro Ser Arg Ala Asn Lys Leu Tyr Glu Leu Phe Cys Lys Tyr 1265 1270 1275 gac tcc ttc gag tcc atg cct gcc gca gag ctc gag cgt gtt gag 3879 Asp Ser Phe Glu Ser Met Pro Ala Ala Glu Leu Glu Arg Val Glu 1280 1285 1290 aag cgc atc ttc cag tgc cct ctt gct gat gtc tgg gct gag acc 3924 Lys Arg Ile Phe Gln Cys Pro Leu Ala Asp Val Trp Ala Glu Thr 1295 1300 1305 tcc gac ttc tac atc aac cgc ctc cac aac ccg gag aag atc acc 3969 Ser Asp Phe Tyr Ile Asn Arg Leu His Asn Pro Glu Lys Ile Thr 1310 1315 1320 cgt gcc gag cgt gac ccc aag ctc aag atg tct ctc tgc ttc cgc 4014 Arg Ala Glu Arg Asp Pro Lys Leu Lys Met Ser Leu Cys Phe Arg 1325 1330 1335 tgg tac ctt ggt ctt gcc tct cgc tgg gcc aac acc ggt gag gct 4059 Trp Tyr Leu Gly Leu Ala Ser Arg Trp Ala Asn Thr Gly Glu Ala 1340 1345 1350 gga cgc gtc atg gac tac cag gtc tgg tgt ggc cct gcc att gga 4104 Gly Arg Val Met Asp Tyr Gln Val Trp Cys Gly Pro Ala Ile Gly 1355 1360 1365 gcc ttc aac gac ttc atc aag ggc tcc tac ctt gac ccg gcc gtc 4149 Ala Phe Asn Asp Phe Ile Lys Gly Ser Tyr Leu Asp Pro Ala Val 1370 1375 1380 tct ggt gag tac ccg gac gtc gtg cag atc aac ttg cag atc ctt 4194 Ser Gly Glu Tyr Pro Asp Val Val Gln Ile Asn Leu Gln Ile Leu 1385 1390 1395 cgc ggt gcc tgc tac ctc cgc cgt ctc aat gcc atc cgc aac gac 4239 Arg Gly Ala Cys Tyr Leu Arg Arg Leu Asn Ala Ile Arg Asn Asp 1400 1405 1410 ccg cgt gtc agc att gag gtc gag gat gct gag ttc gtc tac gag 4284 Pro Arg Val Ser Ile Glu Val Glu Asp Ala Glu Phe Val Tyr Glu 1415 1420 1425 ccc acc aac gcc ctc taa 4302 Pro Thr Asn Ala Leu 1430 <210> 4 <211> 8733 <212> DNA <213> Schizochytrium sp. <220> <221> CDS <222> (1)..(8733) <400> 4 atg gcg gcc cgt ctg cag gag caa aag gga ggc gag atg gat acc cgc 48 Met Ala Ala Arg Leu Gln Glu Gln Lys Gly Gly Glu Met Asp Thr Arg 1 5 10 15 att gcc atc atc ggc atg tcg gcc atc ctc ccc tgc ggc acg acc gtg 96 Ile Ala Ile Ile Gly Met Ser Ala Ile Leu Pro Cys Gly Thr Thr Val 20 25 30 cgc gag tcg tgg gag acc atc cgc gcc ggc atc gac tgc ctg tcg gat 144 Arg Glu Ser Trp Glu Thr Ile Arg Ala Gly Ile Asp Cys Leu Ser Asp 35 40 45 ctc ccc gag gac cgc gtc gac gtg acg gcg tac ttt gac ccc gtc aag 192 Leu Pro Glu Asp Arg Val Asp Val Thr Ala Tyr Phe Asp Pro Val Lys 50 55 60 acc acc aag gac aag atc tac tgc aag cgc ggt ggc ttc att ccc gag 240 Thr Thr Lys Asp Lys Ile Tyr Cys Lys Arg Gly Gly Phe Ile Pro Glu 65 70 75 80 tac gac ttt gac gcc cgc gag ttc gga ctc aac atg ttc cag atg gag 288 Tyr Asp Phe Asp Ala Arg Glu Phe Gly Leu Asn Met Phe Gln Met Glu 85 90 95 gac tcg gac gca aac cag acc atc tcg ctt ctc aag gtc aag gag gcc 336 Asp Ser Asp Ala Asn Gln Thr Ile Ser Leu Leu Lys Val Lys Glu Ala 100 105 110 ctc cag gac gcc ggc atc gac gcc ctc ggc aag gaa aag aag aac atc 384 Gln Asp With Gly Ile Asp With Leu Gly Lys Glu Lys Asn Ile 115 120 125 ggc tgc gtg ctc ggc att ggc ggc ggc caa aag tcc agc cac gag ttc 432 Gly Cys Val Leu Gly Ile Gly Gly Gly Gln Lys Ser Ser His Glu Phe 130 135 140 tac tcg cgc ctt aat tat gtt gtc gtg gag aag gtc ctc cgc aag atg 480 Tyr Ser Arg Leu Asn Tyr Val Val Val Glu Lys Val Leu Arg Lys Met 145 150 155 160 ggc atg ccc gag gag gac gtc aag gtc gcc gtc gaa aag tac aag gcc 528 Gly Met Pro Glu Glu Asp Val Lys Val Ala Val Glu Lys Tyr Lys Ala 165 170 175 aac ttc ccc gag tgg cgc ctc gac tcc ttc cct ggc ttc ctc ggc aac 576 Asn Phe Pro Glu Trp Arg Leu Asp Ser Phe Pro Gly Phe Leu Gly Asn 180 185 190 gtc acc gcc ggt cgc tgc acc aac acc ttc aac ctc gac ggc atg aac 624 Val Thr Ala Gly Arg Cys Thr Asn Thr Phe Asn Leu Asp Gly Met Asn 195 200 205 tgc gtt gtc gac gcc gca tgc gcc tcg tcc ctc atc gcc gtc aag gtc 672 Cys Val Val Asp Ala Ala Cys Ala Ser Ser Leu Ile Ala Val Lys Val 210 215 220 gcc atc gac gag ctg ctc tac ggt gac tgc gac atg atg gtc acc ggt 720 Ala Ile Asp Glu Leu Leu Tyr Gly Asp Cys Asp Met Met Val Thr Gly 225 230 235 240 gcc acc tgc acg gat aac tcc atc ggc atg tac atg gcc ttc tcc aag 768 Ala Thr Cys Thr Asp Asn Ser Ile Gly Met Tyr Met Ala Phe Ser Lys 245 250 255 acc ccc gtg ttc tcc acg gac ccc agc gtg cgc gcc tac gac gaa aag 816 Thr Pro Val Phe Ser Thr Asp Pro Ser Val Arg Ala Tyr Asp Glu Lys 260 265 270 aca aag ggc atg ctc atc ggc gag ggc tcc gcc atg ctc gtc ctc aag 864 Thr Lys Gly Met Leu Ile Gly Glu Gly Ser Ala Met Leu Val Leu Lys 275 280 285 cgc tac gcc gac gcc gtc cgc gac ggc gat gag atc cac gct gtt att 912 Arg Tyr Ala Asp Ala Val Arg Asp Gly Asp Glu Ile His Ala Val Ile 290 295 300 cgc ggc tgc gcc tcc tcc agt gat ggc aag gcc gcc ggc atc tac acg 960 Arg Gly Cys Ala Ser Ser Ser Asp Gly Lys Ala Ala Gly Ile Tyr Thr 305 310 315 320 ccc acc att tcg ggc cag gag gag gcc ctc cgc cgc gcc tac aac cgc 1008 Pro Thr Ile Ser Gly Gln Glu Glu Ala Leu Arg Arg Ala Tyr Asn Arg 325 330 335 gcc tgt gtc gac ccg gcc acc gtc act ctc gtc gag ggt cac ggc acc 1056 Ala Cys Val Asp Pro Ala Thr Val Thr Leu Val Glu Gly His Gly Thr 340 345 350 ggt act ccc gtt ggc gac cgc atc gag ctc acc gcc ttg cgc aac ctc 1104 Gly Thr Pro Val Gly Asp Arg Ile Glu Leu Thr Ala Leu Arg Asn Leu 355 360 365 ttt gac aag gcc tac ggc gag ggc aac acc gaa aag gtc gct gtg ggc 1152 Phe Asp Lys Ala Tyr Gly Glu Gly Asn Thr Glu Lys Val Ala Val Gly 370 375 380 agc atc aag tcc agc atc ggc cat ctc aag gcc gtc gcc ggt ctc gcc 1200 Ser Ile Lys Ser Ser Ile Gly His Leu Lys Ala Val Ala Gly Leu Ala 385 390 395 400 ggt atg atc aag gtc atc atg gcg ctc aag cac aag act ctc ccg ggc 1248 Gly Met Ile Lys Val Ile Met Ala Leu Lys His Lys Thr Leu Pro Gly 405 410 415 acc atc aac gtc gac aac cca ccc aac ctc tac gac aac acg ccc atc 1296 Thr Ile Asn Val Asp Asn Pro Pro Asn Leu Tyr Asp Asn Thr Pro Ile 420 425 430 aac gag tcc tcg ctc tac att aac acc atg aac cgc ccc tgg ttc ccg 1344 Asn Glu Ser Ser Leu Tyr Ile Asn Thr Met Asn Arg Pro Trp Phe Pro 435 440 445 ccc cct ggt gtg ccc cgc cgc gcc ggc att tcg agc ttt ggc ttt ggt 1392 Pro Pro Gly Val Pro Arg Arg Ala Gly Ile Ser Ser Phe Gly Phe Gly 450 455 460 ggc gcc aac tac cac gcc gtc ctc gag gag gcc gag ccc gag cac acg 1440 Gly Ala Asn Tyr His Ala Val Leu Glu Glu Ala Glu Pro Glu His Thr 465 470 475 480 acc gcg tac cgc ctc aac aag cgc ccg cag ccc gtg ctc atg atg gcc 1488 Thr Ala Tyr Arg Leu Asn Lys Arg Pro Gln Pro Val Leu Met Met Ala 485 490 495 gcc acg ccc gcg gcc ctc cag tcg ctc tgc gag gcc cag ctc aag gag 1536 Ala Thr Pro Ala Ala Leu Gln Ser Leu Cys Glu Ala Gln Leu Lys Glu 500 505 510 ttc gag gcc gcc atc aag gag aac gag acc gtc aag aac acc gcc tac 1584 Phe Glu Ala Ala Ile Lys Glu Asn Glu Thr Val Lys Asn Thr Ala Tyr 515 520 525 atc aag tgc gtc aag ttc ggc gag cag ttc aaa ttc cct ggc tcc atc 1632 Ile Lys Cys Val Lys Phe Gly Glu Gln Phe Lys Phe Pro Gly Ser Ile 530 535 540 ccg gcc aca aac gcg cgc ctc ggc ttc ctc gtc aag gat gct gag gat 1680 Pro Ala Thr Asn Ala Arg Leu Gly Phe Leu Val Lys Asp Ala Glu Asp 545 550 555 560 gcc tgc tcc acc ctc cgt gcc atc tgc gcc caa ttc gcc aag gat gtc 1728 Ala Cys Ser Thr Leu Arg Ala Ile Cys Ala Gln Phe Ala Lys Asp Val 565 570 575 acc aag gag gcc tgg cgc ctc ccc cgc gag ggc gtc agc ttc cgc gcc 1776 Thr Lys Glu Ala Trp Arg Leu Pro Arg Glu Gly Val Ser Phe Arg Ala 580 585 590 aag ggc atc gcc acc aac ggc gct gtc gcc gcg ctc ttc tcc ggc cag 1824 Lys Gly Ile Ala Thr Asn Gly Ala Val Ala Ala Leu Phe Ser Gly Gln 595 600 605 ggc gcg cag tac acg cac atg ttt agc gag gtg gcc atg aac tgg ccc 1872 Gly Ala Gln Tyr Thr His Met Phe Ser Glu Val Ala Met Asn Trp Pro 610 615 620 cag ttc cgc cag agc att gcc gcc atg gac gcc gcc cag tcc aag gtc 1920 Gln Phe Arg Gln Ser Ile Ala Ala Met Asp Ala Ala Gln Ser Lys Val 625 630 635 640 gct gga agc gac aag gac ttt gag cgc gtc tcc cag gtc ctc tac ccg 1968 Ala Gly Ser Asp Lys Asp Phe Glu Arg Val Ser Gln Val Leu Tyr Pro 645 650 655 cgc aag ccg tac gag cgt gag ccc gag cag gac cac aag aag atc tcc 2016 Arg Lys Pro Tyr Glu Arg Glu Pro Glu Gln Asp His Lys Lys Ile Ser 660 665 670 ctc acc gcc tac tcg cag ccc tcg acc ctg gcc tgc gct ctc ggt gcc 2064 Leu Thr Ala Tyr Ser Gln Pro Ser Thr Leu Ala Cys Ala Leu Gly Ala 675 680 685 ttt gag atc ttc aag gag gcc ggc ttc acc ccg gac ttt gcc gcc ggc 2112 Phe Glu Ile Phe Lys Glu Ala Gly Phe Thr Pro Asp Phe Ala Ala Gly 690 695 700 cat tcg ctc ggt gag ttc gcc gcc ctc tac gcc gcg ggc tgc gtc gac 2160 His Ser Leu Gly Glu Phe Ala Ala Leu Tyr Ala Ala Gly Cys Val Asp 705 710 715 720 cgc gac gag ctc ttt gag ctt gtc tgc cgc cgc gcc cgc atc atg ggc 2208 Arg Asp Glu Leu Phe Glu Leu Val Cys Arg Arg Ala Arg Ile Met Gly 725 730 735 ggc aag gac gca ccg gcc acc ccc aag ggc tgc atg gcc gcc gtc att 2256 Gly Lys Asp Ala Pro Ala Thr Pro Lys Gly Cys Met Ala Ala Val Ile 740 745 750 ggc ccc aac gcc gag aac atc aag gtc cag gcc gcc aac gtc tgg ctc 2304 Gly Pro Asn Ala Glu Asn Ile Lys Val Gln Ala Ala Asn Val Trp Leu 755 760 765 ggc aac tcc aac tcg cct tcg cag acc gtc atc acc ggc tcc gtc gaa 2352 Gly Asn Ser Asn Ser Pro Ser Gln Thr Val Ile Thr Gly Ser Val Glu 770 775 780 ggt atc cag gcc gag agc gcc cgc ctc cag aag gag ggc ttc cgc gtc 2400 Gly Ile Gln Ala Glu Ser Ala Arg Leu Gln Lys Glu Gly Phe Arg Val 785 790 795 800 gtg cct ctt gcc tgc gag agc gcc ttc cac tcg ccc cag atg gag aac 2448 Val Pro Leu Ala Cys Glu Ser Ala Phe His Ser Pro Gln Met Glu Asn 805 810 815 gcc tcg tcg gcc ttc aag gac gtc atc tcc aag gtc tcc ttc cgc acc 2496 Ala Ser Ser Ala Phe Lys Asp Val Ile Ser Lys Val Ser Phe Arg Thr 820 825 830 ccc aag gcc gag acc aag ctc ttc agc aac gtc tct ggc gag acc tac 2544 Pro Lys Ala Glu Thr Lys Leu Phe Ser Asn Val Ser Gly Glu Thr Tyr 835 840 845 ccc acg gac gcc cgc gag atg ctt acg cag cac atg acc agc agc gtc 2592 Pro Thr Asp Ala Arg Glu Met Leu Thr Gln His Met Thr Ser Ser Val 850 855 860 aag ttc ctc acc cag gtc cgc aac atg cac cag gcc ggt gcg cgc atc 2640 Lys Phe Leu Thr Gln Val Arg Asn Met His Gln Ala Gly Ala Arg Ile 865 870 875 880 ttt gtc gag ttc gga ccc aag cag gtg ctc tcc aag ctt gtc tcc gag 2688 Phe Val Glu Phe Gly Pro Lys Gln Val Leu Ser Lys Leu Val Ser Glu 885 890 895 acc ctc aag gat gac ccc tcg gtt gtc acc gtc tct gtc aac ccg gcc 2736 Thr Leu Lys Asp Asp Pro Ser Val Val Thr Val Ser Val Asn Pro Ala 900 905 910 tcg ggc acg gat tcg gac atc cag ctc cgc gac gcg gcc gtc cag ctc 2784 Ser Gly Thr Asp Ser Asp Ile Gln Leu Arg Asp Ala Ala Val Gln Leu 915 920 925 gtt gtc gct ggc gtc aac ctt cag ggc ttt gac aag tgg gac gcc ccc 2832 Val Val Ala Gly Val Asn Leu Gln Gly Phe Asp Lys Trp Asp Ala Pro 930 935 940 gat gcc acc cgc atg cag gcc atc aag aag aag cgc act acc ctc cgc 2880 Asp Ala Thr Arg Met Gln Ala Ile Lys Lys Lys Arg Thr Thr Leu Arg 945 950 955 960 ctt tcg gcc gcc acc tac gtc tcg gac aag acc aag aag gtc cgc gac 2928 Leu Ser Ala Ala Thr Tyr Val Ser Asp Lys Thr Lys Lys Val Arg Asp 965 970 975 gcc gcc atg aac gat ggc cgc tgc gtc acc tac ctc aag ggc gcc gca 2976 Ala Ala Met Asn Asp Gly Arg Cys Val Thr Tyr Leu Lys Gly Ala Ala 980 985 990 ccg ctc atc aag gcc ccg gag ccc gtt gtc gac gag gcc gcc aag cgc 3024 Pro Leu Ile Lys Ala Pro Glu Pro Val Val Asp Glu Ala Ala Lys Arg 995 1000 1005 gag gcc gag cgt ctc cag aag gag ctt cag gat gcc cag cgc cag 3069 Glu Ala Glu Arg Leu Gln Lys Glu Leu Gln Asp Ala Gln Arg Gln 1010 1015 1020 ctc gac gac gcc aag cgc gcc gcc gcc gag gcc aac tcc aag ctc 3114 Leu Asp Asp Ala Lys Arg Ala Ala Ala Glu Ala Asn Ser Lys Leu 1025 1030 1035 gcc gct gcc aag gag gag gcc aag acc gcc gct gct tcg gcc aag 3159 Ala Ala Ala Lys Glu Glu Ala Lys Thr Ala Ala Ala Ser Ala Lys 1040 1045 1050 ccc gca gtt gac act gct gtt gtc gaa aag cat cgt gcc atc ctc 3204 Pro Ala Val Asp Thr Ala Val Val Glu Lys His Arg Ala Ile Leu 1055 1060 1065 aag tcc atg ctc gcg gag ctc gat ggc tac gga tcg gtc gac gct 3249 Lys Ser Met Leu Ala Glu Leu Asp Gly Tyr Gly Ser Val Asp Ala 1070 1075 1080 tct tcc ctc cag cag cag cag cag cag cag acg gcc ccc gcc ccg 3294 Ser Ser Leu Gln Gln Gln Gln Gln Gln Gln Thr Ala Pro Ala Pro 1085 1090 1095 gtc aag gct gct gcg cct gcc gcc ccc gtt gcc tcg gcc cct gcc 3339 Val Lys Ala Ala Ala Pro Ala Ala Pro Val Ala Ser Ala Pro Ala 1100 1105 1110 ccg gct gtc tcg aac gag ctt ctt gag aag gcc gag act gtc gtc 3384 Pro Ala Val Ser Asn Glu Leu Leu Glu Lys Ala Glu Thr Val Val 1115 1120 1125 atg gag gtc ctc gcc gcc aag acc ggc tac gag acc gac atg atc 3429 Met Glu Val Leu Ala Ala Lys Thr Gly Tyr Glu Thr Asp Met Ile 1130 1135 1140 gag gct gac atg gag ctc gag acc gag ctc ggc att gac tcc atc 3474 Glu Ala Asp Met Glu Leu Glu Thr Glu Leu Gly Ile Asp Ser Ile 1145 1150 1155 aag cgt gtc gag atc ctc tcc gag gtc cag gcc atg ctc aat gtc 3519 Lys Arg Val Glu Ile Leu Ser Glu Val Gln Ala Met Leu Asn Val 1160 1165 1170 gag gcc aag gat gtc gat gcc ctc agc cgc act cgc act gtt ggt 3564 Glu Ala Lys Asp Val Asp Ala Leu Ser Arg Thr Arg Thr Val Gly 1175 1180 1185 gag gtt gtc aac gcc atg aag gcc gag atc gct ggc agc tct gcc 3609 Glu Val Val Asn Ala Met Lys Ala Glu Ile Ala Gly Ser Ser Ala 1190 1195 1200 ccg gcg cct gct gcc gct gct ccg gct ccg gcc aag gct gcc cct 3654 Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala Lys Ala Ala Pro 1205 1210 1215 gcc gcc gct gcg cct gct gtc tcg aac gag ctt ctc gag aag gcc 3699 Ala Ala Ala Ala Pro Ala Val Ser Asn Glu Leu Leu Glu Lys Ala 1220 1225 1230 gag acc gtc gtc atg gag gtc ctc gcc gcc aag act ggc tac gag 3744 Glu Thr Val Val Met Glu Val Leu Ala Ala Lys Thr Gly Tyr Glu 1235 1240 1245 act gac atg atc gag tcc gac atg gag ctc gag act gag ctc ggc 3789 Thr Asp Met Ile Glu Ser Asp Met Glu Leu Glu Thr Glu Leu Gly 1250 1255 1260 att gac tcc atc aag cgt gtc gag atc ctc tcc gag gtt cag gcc 3834 Ile Asp Ser Ile Lys Arg Val Glu Ile Leu Ser Glu Val Gln Ala 1265 1270 1275 atg ctc aac gtc gag gcc aag gac gtc gac gct ctc agc cgc act 3879 Met Leu Asn Val Glu Ala Lys Asp Val Asp Ala Leu Ser Arg Thr 1280 1285 1290 cgc act gtg ggt gag gtc gtc aac gcc atg aag gct gag atc gct 3924 Arg Thr Val Gly Glu Val Val Asn Ala Met Lys Ala Glu Ile Ala 1295 1300 1305 ggt ggc tct gcc ccg gcg cct gcc gcc gct gcc cca ggt ccg gct 3969 Gly Gly Ser Ala Pro Ala Pro Ala Ala Ala Ala Pro Gly Pro Ala 1310 1315 1320 gct gcc gcc cct gcg cct gcc gcc gcc gcc cct gct gtc tcg aac 4014 Ala Ala Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Val Ser Asn 1325 1330 1335 gag ctt ctt gag aag gcc gag acc gtc gtc atg gag gtc ctc gcc 4059 Glu Leu Leu Glu Lys Ala Glu Thr Val Val Met Glu Val Leu Ala 1340 1345 1350 gcc aag act ggc tac gag act gac atg atc gag tcc gac atg gag 4104 Ala Lys Thr Gly Tyr Glu Thr Asp Met Ile Glu Ser Asp Met Glu 1355 1360 1365 ctc gag acc gag ctc ggc att gac tcc atc aag cgt gtc gag att 4149 Leu Glu Thr Glu Leu Gly Ile Asp Ser Ile Lys Arg Val Glu Ile 1370 1375 1380 ctc tcc gag gtc cag gcc atg ctc aac gtc gag gcc aag gac gtc 4194 Leu Ser Glu Val Gln Ala Met Leu Asn Val Glu Ala Lys Asp Val 1385 1390 1395 gac gct ctc agc cgc acc cgc act gtt ggc gag gtc gtc gat gcc 4239 Asp Ala Leu Ser Arg Thr Arg Thr Val Gly Glu Val Val Asp Ala 1400 1405 1410 atg aag gcc gag atc gct ggt ggc tct gcc ccg gcg cct gcc gcc 4284 Met Lys Ala Glu Ile Ala Gly Gly Ser Ala Pro Ala Pro Ala Ala 1415 1420 1425 gct gct cct gct ccg gct gct gcc gcc cct gcg cct gcc gcc cct 4329 Ala Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala Ala Pro 1430 1435 1440 gcg cct gct gtc tcg agc gag ctt ctc gag aag gcc gag act gtc 4374 Ala Pro Ala Val Ser Ser Glu Leu Leu Glu Lys Ala Glu Thr Val 1445 1450 1455 gtc atg gag gtc ctc gcc gcc aag act ggc tac gag act gac atg 4419 Val Met Glu Val Leu Ala Ala Lys Thr Gly Tyr Glu Thr Asp Met 1460 1465 1470 atc gag tcc gac atg gag ctc gag acc gag ctc ggc att gac tcc 4464 Ile Glu Ser Asp Met Glu Leu Glu Thr Glu Leu Gly Ile Asp Ser 1475 1480 1485 atc aag cgt gtc gag att ctc tcc gag gtc cag gcc atg ctc aac 4509 Ile Lys Arg Val Glu Ile Leu Ser Glu Val Gln Ala Met Leu Asn 1490 1495 1500 gtc gag gcc aag gac gtc gac gct ctc agc cgc acc cgc act gtt 4554 Val Glu Ala Lys Asp Val Asp Ala Leu Ser Arg Thr Arg Thr Val 1505 1510 1515 ggc gag gtc gtc gat gcc atg aag gcc gag atc gct ggt ggc tct 4599 Gly Glu Val Val Asp Ala Met Lys Ala Glu Ile Ala Gly Gly Ser 1520 1525 1530 gcc ccg gcg cct gcc gcc gct gct cct gct ccg gct gct gcc gcc 4644 Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala Ala Ala Ala 1535 1540 1545 cct gcg cct gcc gcc cct gcg cct gcc gcc cct gcg cct gct gtc 4689 Pro Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala Pro Ala Val 1550 1555 1560 tcg agc gag ctt ctc gag aag gcc gag act gtc gtc atg gag gtc 4734 Ser Ser Glu Leu Leu Glu Lys Ala Glu Thr Val Val Met Glu Val 1565 1570 1575 ctc gcc gcc aag act ggc tac gag act gac atg att gag tcc gac 4779 Leu Ala Ala Lys Thr Gly Tyr Glu Thr Asp Met Ile Glu Ser Asp 1580 1585 1590 atg gag ctc gag acc gag ctc ggc att gac tcc atc aag cgt gtc 4824 Met Glu Leu Glu Thr Glu Leu Gly Ile Asp Ser Ile Lys Arg Val 1595 1600 1605 gag att ctc tcc gag gtt cag gcc atg ctc aac gtc gag gcc aag 4869 Glu Ile Leu Ser Glu Val Gln Ala Met Leu Asn Val Glu Ala Lys 1610 1615 1620 gac gtc gac gct ctc agc cgc act cgc act gtt ggt gag gtc gtc 4914 Asp Val Asp Ala Leu Ser Arg Thr Arg Thr Val Gly Glu Val Val 1625 1630 1635 gat gcc atg aag gct gag atc gct ggc agc tcc gcc tcg gcg cct 4959 Asp Ala Met Lys Ala Glu Ile Ala Gly Ser Ser Ala Ser Ala Pro 1640 1645 1650 gcc gcc gct gct cct gct ccg gct gct gcc gct cct gcg ccc gct 5004 Ala Ala Ala Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala 1655 1660 1665 gcc gcc gcc cct gct gtc tcg aac gag ctt ctc gag aaa gcc gag 5049 Ala Ala Ala Pro Ala Val Ser Asn Glu Leu Leu Glu Lys Ala Glu 1670 1675 1680 act gtc gtc atg gag gtc ctc gcc gcc aag act ggc tac gag act 5094 Thr Val Val Met Glu Val Leu Ala Ala Lys Thr Gly Tyr Glu Thr 1685 1690 1695 gac atg atc gag tcc gac atg gag ctc gag act gag ctc ggc att 5139 Asp Met Ile Glu Ser Asp Met Glu Leu Glu Thr Glu Leu Gly Ile 1700 1705 1710 gac tcc atc aag cgt gtc gag atc ctc tcc gag gtt cag gcc atg 5184 Asp Ser Ile Lys Arg Val Glu Ile Leu Ser Glu Val Gln Ala Met 1715 1720 1725 ctc aac gtc gag gcc aag gac gtc gat gcc ctc agc cgc acc cgc 5229 Leu Asn Val Glu Ala Lys Asp Val Asp Ala Leu Ser Arg Thr Arg 1730 1735 1740 act gtt ggc gag gtt gtc gat gcc atg aag gcc gag atc gct ggt 5274 Thr Val Gly Glu Val Val Asp Ala Met Lys Ala Glu Ile Ala Gly 1745 1750 1755 ggc tct gcc ccg gcg cct gcc gcc gct gcc cct gct ccg gct gcc 5319 Gly Ser Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala Ala 1760 1765 1770 gcc gcc cct gct gtc tcg aac gag ctt ctc gag aag gcc gag act 5364 Ala Ala Pro Ala Val Ser Asn Glu Leu Leu Glu Lys Ala Glu Thr 1775 1780 1785 gtc gtc atg gag gtc ctc gcc gcc aag act ggc tac gag acc gac 5409 Val Val Met Glu Val Leu Ala Ala Lys Thr Gly Tyr Glu Thr Asp 1790 1795 1800 atg atc gag tcc gac atg gag ctc gag acc gag ctc ggc att gac 5454 Met Ile Glu Ser Asp Met Glu Leu Glu Thr Glu Leu Gly Ile Asp 1805 1810 1815 tcc atc aag cgt gtc gag att ctc tcc gag gtt cag gcc atg ctc 5499 Ser Ile Lys Arg Val Glu Ile Leu Ser Glu Val Gln Ala Met Leu 1820 1825 1830 aac gtc gag gcc aag gac gtc gat gct ctc agc cgc act cgc act 5544 Asn Val Glu Ala Lys Asp Val Asp Ala Leu Ser Arg Thr Arg Thr 1835 1840 1845 gtt ggc gag gtc gtc gat gcc atg aag gct gag atc gcc ggc agc 5589 Val Gly Glu Val Val Asp Ala Met Lys Ala Glu Ile Ala Gly Ser 1850 1855 1860 tcc gcc ccg gcg cct gcc gcc gct gct cct gct ccg gct gct gcc 5634 Ser Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala Ala Ala 1865 1870 1875 gct cct gcg ccc gct gcc gct gcc cct gct gtc tcg agc gag ctt 5679 Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Val Ser Ser Glu Leu 1880 1885 1890 ctc gag aag gcc gag acc gtc gtc atg gag gtc ctc gcc gcc aag 5724 Leu Glu Lys Ala Glu Thr Val Val Met Glu Val Leu Ala Ala Lys 1895 1900 1905 act ggc tac gag act gac atg att gag tcc gac atg gag ctc gag 5769 Thr Gly Tyr Glu Thr Asp Met Ile Glu Ser Asp Met Glu Leu Glu 1910 1915 1920 act gag ctc ggc att gac tcc atc aag cgt gtc gag atc ctc tcc 5814 Thr Glu Leu Gly Ile Asp Ser Ile Lys Arg Val Glu Ile Leu Ser 1925 1930 1935 gag gtt cag gcc atg ctc aac gtc gag gcc aag gac gtc gat gcc 5859 Glu Val Gln Ala Met Leu Asn Val Glu Ala Lys Asp Val Asp Ala 1940 1945 1950 ctc agc cgc acc cgc act gtt ggc gag gtt gtc gat gcc atg aag 5904 Leu Ser Arg Thr Arg Thr Val Gly Glu Val Val Asp Ala Met Lys 1955 1960 1965 gcc gag atc gct ggt ggc tct gcc ccg gcg cct gcc gcc gct gcc 5949 Ala Glu Ile Ala Gly Gly Ser Ala Pro Ala Pro Ala Ala Ala Ala 1970 1975 1980 cct gct ccg gct gcc gcc gcc cct gct gtc tcg aac gag ctt ctt 5994 Pro Ala Pro Ala Ala Ala Ala Pro Ala Val Ser Asn Glu Leu Leu 1985 1990 1995 gag aag gcc gag acc gtc gtc atg gag gtc ctc gcc gcc aag act 6039 Glu Lys Ala Glu Thr Val Val Met Glu Val Leu Ala Ala Lys Thr 2000 2005 2010 ggc tac gag acc gac atg atc gag tcc gac atg gag ctc gag acc 6084 Gly Tyr Glu Thr Asp Met Ile Glu Ser Asp Met Glu Leu Glu Thr 2015 2020 2025 gag ctc ggc att gac tcc atc aag cgt gtc gag att ctc tcc gag 6129 Glu Leu Gly Ile Asp Ser Ile Lys Arg Val Glu Ile Leu Ser Glu 2030 2035 2040 gtt cag gcc atg ctc aac gtc gag gcc aag gac gtc gac gct ctc 6174 Val Gln Ala Met Leu Asn Val Glu Ala Lys Asp Val Asp Ala Leu 2045 2050 2055 agc cgc act cgc act gtt ggc gag gtc gtc gat gcc atg aag gct 6219 Ser Arg Thr Arg Thr Val Gly Glu Val Val Asp Ala Met Lys Ala 2060 2065 2070 gag atc gct ggt ggc tct gcc ccg gcg cct gcc gcc gct gct cct 6264 Glu Ile Ala Gly Gly Ser Ala Pro Ala Pro Ala Ala Ala Ala Pro 2075 2080 2085 gcc tcg gct ggc gcc gcg cct gcg gtc aag att gac tcg gtc cac 6309 Ala Ser Ala Gly Ala Ala Pro Ala Val Lys Ile Asp Ser Val His 2090 2095 2100 ggc gct gac tgt gat gat ctt tcc ctg atg cac gcc aag gtg gtt 6354 Gly Ala Asp Cys Asp Asp Leu Ser Leu Met His Ala Lys Val Val 2105 2110 2115 gac atc cgc cgc ccg gac gag ctc atc ctg gag cgc ccc gag aac 6399 Asp Ile Arg Arg Pro Asp Glu Leu Ile Leu Glu Arg Pro Glu Asn 2120 2125 2130 cgc ccc gtt ctc gtt gtc gat gac ggc agc gag ctc acc ctc gcc 6444 Arg Pro Val Leu Val Val Asp Asp Gly Ser Glu Leu Thr Leu Ala 2135 2140 2145 ctg gtc cgc gtc ctc ggc gcc tgc gcc gtt gtc ctg acc ttt gag 6489 Leu Val Arg Val Leu Gly Ala Cys Ala Val Val Leu Thr Phe Glu 2150 2155 2160 ggt ctc cag ctc gct cag cgc gct ggt gcc gct gcc atc cgc cac 6534 Gly Leu Gln Leu Ala Gln Arg Ala Gly Ala Ala Ala Ile Arg His 2165 2170 2175 gtg ctc gcc aag gat ctt tcc gcg gag agc gcc gag aag gcc atc 6579 Val Leu Ala Lys Asp Leu Ser Ala Glu Ser Ala Glu Lys Ala Ile 2180 2185 2190 aag gag gcc gag cag cgc ttt ggc gct ctc ggc ggc ttc atc tcg 6624 Lys Glu Ala Glu Gln Arg Phe Gly Ala Leu Gly Gly Phe Ile Ser 2195 2200 2205 cag cag gcg gag cgc ttc gag ccc gcc gaa atc ctc ggc ttc acg 6669 Gln Gln Ala Glu Arg Phe Glu Pro Ala Glu Ile Leu Gly Phe Thr 2210 2215 2220 ctc atg tgc gcc aag ttc gcc aag gct tcc ctc tgc acg gct gtg 6714 Leu Met Cys Ala Lys Phe Ala Lys Ala Ser Leu Cys Thr Ala Val 2225 2230 2235 gct ggc ggc cgc ccg gcc ttt atc ggt gtg gcg cgc ctt gac ggc 6759 Ala Gly Gly Arg Pro Ala Phe Ile Gly Val Ala Arg Leu Asp Gly 2240 2245 2250 cgc ctc gga ttc act tcg cag ggc act tct gac gcg ctc aag cgt 6804 Arg Leu Gly Phe Thr Ser Gln Gly Thr Ser Asp Ala Leu Lys Arg 2255 2260 2265 gcc cag cgt ggt gcc atc ttt ggc ctc tgc aag acc atc ggc ctc 6849 Ala Gln Arg Gly Ala Ile Phe Gly Leu Cys Lys Thr Ile Gly Leu 2270 2275 2280 gag tgg tcc gag tct gac gtc ttt tcc cgc ggc gtg gac att gct 6894 Glu Trp Ser Glu Ser Asp Val Phe Ser Arg Gly Val Asp Ile Ala 2285 2290 2295 cag ggc atg cac ccc gag gat gcc gcc gtg gcg to gtg cgc gag 6939 Gln Gly Met His Pro Glu Asp Ala Ala Val Ala Ile Val Arg Glu 2300 2305 2310 atg gcg tgc gct gac att cgc att cgc gag gtc ggc att ggc gca 6984 Met Ala Cys Ala Asp Ile Arg Ile Arg Glu Val Gly Ile Gly Ala 2315 2320 2325 aac cag cag cgc tgc acg atc cgt gcc gcc aag ctc gag acc ggc 7029 Asn Gln Gln Arg Cys Thr Ile Arg Ala Ala Lys Leu Glu Thr Gly 2330 2335 2340 aac ccg cag cgc cag atc gcc aag gac gac gtg ctg ctc gtt tct 7074 Asn Pro Gln Arg Gln Ile Ala Lys Asp Asp Val Leu Leu Val Ser 2345 2350 2355 ggc ggc gct cgc ggc atc acg cct ctt tgc atc cgg gag atc acg 7119 Gly Gly Ala Arg Gly Ile Thr Pro Leu Cys Ile Arg Glu Ile Thr 2360 2365 2370 cgc cag atc gcg ggc ggc aag tac att ctg ctt ggc cgc agc aag 7164 Arg Gln Ile Ala Gly Gly Lys Tyr Ile Leu Leu Gly Arg Ser Lys 2375 2380 2385 gtc tct gcg agc gaa ccg gca tgg tgc gct ggc atc act gac gag 7209 Val Ser Ala Ser Glu Pro Ala Trp Cys Ala Gly Ile Thr Asp Glu 2390 2395 2400 aag gct gtg caa aag gct gct acc cag gag ctc aag cgc gcc ttt 7254 Lys Ala Val Gln Lys Ala Ala Thr Gln Glu Leu Lys Arg Ala Phe 2405 2410 2415 agc gct ggc gag ggc ccc aag ccc acg ccc cgc gct gtc act aag 7299 Ser Ala Gly Glu Gly Pro Lys Pro Thr Pro Arg Ala Val Thr Lys 2420 2425 2430 ctt gtg ggc tct gtt ctt ggc gct cgc gag gtg cgc agc tct att 7344 Leu Val Gly Ser Val Leu Gly Ala Arg Glu Val Arg Ser Ser Ile 2435 2440 2445 gct gcg att gaa gcg ctc ggc ggc aag gcc atc tac tcg tcg tgc 7389 Ala Ala Ile Glu Ala Leu Gly Gly Lys Ala Ile Tyr Ser Ser Cys 2450 2455 2460 gac gtg aac tct gcc gcc gac gtg gcc aag gcc gtg cgc gat gcc 7434 Asp Val Asn Ser Ala Ala Asp Val Ala Lys Ala Val Arg Asp Ala 2465 2470 2475 gag tcc cag ctc ggt gcc cgc gtc tcg ggc atc gtt cat gcc tcg 7479 Glu Ser Gln Leu Gly Ala Arg Val Ser Gly Ile Val His Ala Ser 2480 2485 2490 ggc gtg ctc cgc gac cgt ctc atc gag aag aag ctc ccc gac gag 7524 Gly Val Leu Arg Asp Arg Leu Ile Glu Lys Lys Leu Pro Asp Glu 2495 2500 2505 ttc gac gcc gtc ttt ggc acc aag gtc acc ggt ctc gag aac ctc 7569 Phe Asp Ala Val Phe Gly Thr Lys Val Thr Gly Leu Glu Asn Leu 2510 2515 2520 ctc gcc gcc gtc gac cgc gcc aac ctc aag cac atg gtc ctc ttc 7614 Leu Ala Ala Val Asp Arg Ala Asn Leu Lys His Met Val Leu Phe 2525 2530 2535 agc tcg ctc gcc ggc ttc cac ggc aac gtc ggc cag tct gac tac 7659 Ser Ser Leu Ala Gly Phe His Gly Asn Val Gly Gln Ser Asp Tyr 2540 2545 2550 gcc atg gcc aac gag gcc ctt aac aag atg ggc ctc gag ctc gcc 7704 Ala Met Ala Asn Glu Ala Leu Asn Lys Met Gly Leu Glu Leu Ala 2555 2560 2565 aag gac gtc tcg gtc aag tcg atc tgc ttc ggt ccc tgg gac ggt 7749 Lys Asp Val Ser Val Lys Ser Ile Cys Phe Gly Pro Trp Asp Gly 2570 2575 2580 ggc atg gtg acg ccg cag ctc aag aag cag ttc cag gag atg ggc 7794 Gly Met Val Thr Pro Gln Leu Lys Lys Gln Phe Gln Glu Met Gly 2585 2590 2595 gtg cag atc atc ccc cgc gag ggc ggc gct gat acc gtg gcg cgc 7839 Val Gln Ile Ile Pro Arg Glu Gly Gly Ala Asp Thr Val Ala Arg 2600 2605 2610 atc gtg ctc ggc tcc tcg ccg gct gag atc ctt gtc ggc aac tgg 7884 Ile Val Leu Gly Ser Ser Pro Ala Glu Ile Leu Val Gly Asn Trp 2615 2620 2625 cgc acc ccg tcc aag aag gtc ggc tcg gac acc atc acc ctg cac 7929 Arg Thr Pro Ser Lys Lys Val Gly Ser Asp Thr Ile Thr Leu His 2630 2635 2640 cgc aag att tcc gcc aag tcc aac ccc ttc ctc gag gac cac gtc 7974 Arg Lys Ile Ser Ala Lys Ser Asn Pro Phe Leu Glu Asp His Val 2645 2650 2655 atc cag ggc cgc cgc gtg ctg ccc atg acg ctg gcc att ggc tcg 8019 Ile Gln Gly Arg Arg Val Leu Pro Met Thr Leu Ala Ile Gly Ser 2660 2665 2670 ctc gcg gag acc tgc ctc ggc ctc ttc ccc ggc tac tcg ctc tgg 8064 Leu Ala Glu Thr Cys Leu Gly Leu Phe Pro Gly Tyr Ser Leu Trp 2675 2680 2685 gcc att gac gac gcc cag ctc ttc aag ggt gtc act gtc gac ggc 8109 Ala Ile Asp Asp Ala Gln Leu Phe Lys Gly Val Thr Val Asp Gly 2690 2695 2700 gac gtc aac tgc gag gtg acc ctc acc ccg tcg acg gcg ccc tcg 8154 Asp Val Asn Cys Glu Val Thr Leu Thr Pro Ser Thr Ala Pro Ser 2705 2710 2715 ggc cgc gtc aac gtc cag gcc acg ctc aag acc ttt tcc agc ggc 8199 Gly Arg Val Asn Val Gln Ala Thr Leu Lys Thr Phe Ser Ser Gly 2720 2725 2730 aag ctg gtc ccg gcc tac cgc gcc gtc atc gtg ctc tcc aac cag 8244 Lys Leu Val Pro Ala Tyr Arg Ala Val Ile Val Leu Ser Asn Gln 2735 2740 2745 ggc gcg ccc ccg gcc aac gcc acc atg cag ccg ccc tcg ctc gat 8289 Gly Ala Pro Pro Ala Asn Ala Thr Met Gln Pro Pro Ser Leu Asp 2750 2755 2760 gcc gat ccg gcg ctc cag ggc tcc gtc tac gac ggc aag acc ctc 8334 Ala Asp Pro Ala Leu Gln Gly Ser Val Tyr Asp Gly Lys Thr Leu 2765 2770 2775 ttc cac ggc ccg gcc ttc cgc ggc atc gat gac gtg ctc tcg tgc 8379 Phe His Gly Pro Ala Phe Arg Gly Ile Asp Asp Val Leu Ser Cys 2780 2785 2790 acc aag agc cag ctt gtg gcc aag tgc agc gct gtc ccc ggc tcc 8424 Thr Lys Ser Gln Leu Val Ala Lys Cys Ser Ala Val Pro Gly Ser 2795 2800 2805 gac gcc gct cgc ggc gag ttt gcc acg gac act gac gcc cat gac 8469 Asp Ala Ala Arg Gly Glu Phe Ala Thr Asp Thr Asp Ala His Asp 2810 2815 2820 ccc ttc gtg aac gac ctg gcc ttt cag gcc atg ctc gtc tgg gtg 8514 Pro Phe Val Asn Asp Leu Ala Phe Gln Ala Met Leu Val Trp Val 2825 2830 2835 cgc cgc acg ctc ggc cag gct gcg ctc ccc aac tcg atc cag cgc 8559 Arg Arg Thr Leu Gly Gln Ala Ala Leu Pro Asn Ser Ile Gln Arg 2840 2845 2850 atc gtc cag cac cgc ccg gtc ccg cag gac aag ccc ttc tac att 8604 Ile Val Gln His Arg Pro Val Pro Gln Asp Lys Pro Phe Tyr Ile 2855 2860 2865 acc ctc cgc tcc aac cag tcg ggc ggt cac tcc cag cac aag cac 8649 Thr Leu Arg Ser Asn Gln Ser Gly Gly His Ser Gln His Lys His 2870 2875 2880 gcc ctt cag ttc cac aac gag cag ggc gat ctc ttc att gat gtc 8694 Ala Leu Gln Phe His Asn Glu Gln Gly Asp Leu Phe Ile Asp Val 2885 2890 2895 cag gct tcg gtc atc gcc acg gac agc ctt gcc ttc taa 8733 Gln Ala Ser Val Ile Ala Thr Asp Ser Leu Ala Phe 2900 2905 2910 <210> 5 <211> 714 <212> DNA <213> Nostoc sp. PCC7120 <220> <221> CDS <222> (1)..(714) <400> 5 atg ctg caa cat acc tgg ctg ccg aaa ccg cct aat ctg acc ctg ctg 48 Met Leu Gln His Thr Trp Leu Pro Lys Pro Pro Asn Leu Thr Leu Leu 1 5 10 15 agt gat gaa gtt cat ctg tgg cgt att ccg ctg gat cag ccg gaa agc 96 Ser Asp Glu Val His Leu Trp Arg Ile Pro Leu Asp Gln Pro Glu Ser 20 25 30 cag ctg caa gac ctg gca gca acc ctg agc agt gat gaa ctg gca cgt 144 Gln Leu Gln Asp Leu Ala Ala Thr Leu Ser Ser Asp Glu Leu Ala Arg 35 40 45 gca aat cgt ttc tat ttt ccg gaa cat cgt cgt cgt ttt acc gca ggt 192 Ala Asn Arg Phe Tyr Phe Pro Glu His Arg Arg Arg Phe Thr Ala Gly 50 55 60 cgt ggt att ctg cgt agc att ctg ggt ggt tat ctg ggt gtt gaa ccg 240 Arg Gly Ile Leu Arg Ser Ile Leu Gly Gly Tyr Leu Gly Val Glu Pro 65 70 75 80 ggt cag gtt aaa ttt gat tat gaa agc cgt ggt aaa ccg att ctg ggc 288 Gly Gln Val Lys Phe Asp Tyr Glu Ser Arg Gly Lys Pro Ile Leu Gly 85 90 95 gat cgt ttt gca gaa agc ggt ctg ctg ttt aat ctg agc cat agc cag 336 Asp Arg Phe Ala Glu Ser Gly Leu Leu Phe Asn Leu Ser His Ser Gln 100 105 110 aat ctg gca ctg tgt gca gtg aat tat acc cgt cag att ggt atc gat 384 Asn Leu Ala Leu Cys Ala Val Asn Tyr Thr Arg Gln Ile Gly Ile Asp 115 120 125 ctg gaa tat ctg cgt ccg acc agt gat ctg gaa agc ctg gcc aaa cgt 432 Leu Glu Tyr Leu Arg Pro Thr Ser Asp Leu Glu Ser Leu Ala Lys Arg 130 135 140 ttt ttt ctg cct cgt gaa tat gaa ctg ctg cgt agc ctg ccg gat gaa 480 Phe Phe Leu Pro Arg Glu Tyr Glu Leu Leu Arg Ser Leu Pro Asp Glu 145 150 155 160 cag aaa cag aaa atc ​​ttt ttt cgt tat tgg acc tgc aaa gaa gcc tat 528 Gln Lys Gln Lys Ile Phe Phe Arg Tyr Trp Thr Cys Lys Glu Ala Tyr 165 170 175 ctg aaa gca acc ggt gat ggt att gca aaa ctg gaa gaa at gaa att 576 Leu Lys Ala Thr Gly Asp Gly Ile Ala Lys Leu Glu Glu Ile Glu Ile 180 185 190 gca ctg acc ccg acc gaa ccg gca aaa ctg caa acc gca ccg gca tgg 624 Ala Leu Thr Pro Thr Glu Pro Ala Lys Leu Gln Thr Ala Pro Ala Trp 195 200 205 tca ctg ctg gaa ctg gtt ccg gat gat aat tgt gtt gca gcc gtt gca 672 Ser Leu Leu Glu Leu Val Pro Asp Asp Asn Cys Val Ala Ala Val Ala 210 215 220 gtt gca ggt ttt ggt tgg cag ccg aaa ttt tgg cat tat taa 714 Val Ala Gly Phe Gly Trp Gln Pro Lys Phe Trp His Tyr 225 230 235 <210> 6 <211> 814 <212> PRT <213> Escherichia coli <400> 6 Met Met Ile Leu Ser Ile Leu Ala Thr Val Val Leu Leu Gly Ala Leu 1 5 10 15 Phe Tyr His Arg Val Ser Leu Phe Ile Ser Ser Leu Ile Leu Leu Ala 20 25 30 Trp Thr Ala Ala Leu Gly Val Ala Gly Leu Trp Ser Ala Trp Val Leu 35 40 45 Val Pro Leu Ala Ile Ile Leu Val Pro Phe Asn Phe Ala Pro Met Arg 50 55 60 Lys Ser Met Ile Ser Ala Pro Val Phe Arg Gly Phe Arg Lys Val Met 65 70 75 80 Pro Pro Met Ser Arg Thr Glu Lys Glu Ala Ile Asn Ala Gly Thr Thr 85 90 95 Trp Trp Glu Gly Asp Leu Phe Gln Gly Lys Pro Asp Trp Lys Lys Leu 100 105 110 His Asn Tyr Pro Gln Pro Arg Leu Thr Ala Glu Glu Gln Ala Phe Leu 115 120 125 Asp Gly Pro Val Glu Glu Ala Cys Arg Met Ala Asn Asp Phe Gln Ile 130 135 140 Thr His Glu Leu Ala Asp Leu Pro Pro Glu Leu Trp Ala Tyr Leu Lys 145 150 155 160 Glu His Arg Phe Phe Ala Met Ile Ile Lys Lys Glu Tyr Gly Gly Leu 165 170 175 Glu Phe Ser Ala Tyr Ala Gln Ser Arg Val Leu Gln Lys Leu Ser Gly 180 185 190 Val Ser Gly Ile Leu Ala Ile Thr Val Gly Val Pro Asn Ser Leu Gly 195 200 205 Pro Gly Glu Leu Leu Gln His Tyr Gly Thr Asp Glu Gln Lys Asp His 210 215 220 Tyr Leu Pro Arg Leu Ala Arg Gly Gln Glu Ile Pro Cys Phe Ala Leu 225 230 235 240 Thr Ser Pro Glu Ala Gly Ser Asp Ala Gly Ala Ile Pro Asp Thr Gly 245 250 255 Ile Val Cys Met Gly Glu Trp Gln Gly Gln Gln Val Leu Gly Met Arg 260 265 270 Leu Thr Trp Asn Lys Arg Tyr Ile Thr Leu Ala Pro Ile Ala Thr Val 275 280 285 Leu Gly Leu Ala Phe Lys Leu Ser Asp Pro Glu Lys Leu Leu Gly Gly 290 295 300 Ala Glu Asp Leu Gly Ile Thr Cys Ala Leu Ile Pro Thr Thr Thr Pro 305 310 315 320 Gly Val Glu Ile Gly Arg Arg His Phe Pro Leu Asn Val Pro Phe Gln 325 330 335 Asn Gly Pro Thr Arg Gly Lys Asp Val Phe Val Pro Ile Asp Tyr Ile 340 345 350 Ile Gly Gly Pro Lys Met Ala Gly Gln Gly Trp Arg Met Leu Val Glu 355 360 365 Cys Leu Ser Val Gly Arg Gly Ile Thr Leu Pro Ser Asn Ser Thr Gly 370 375 380 Gly Val Lys Ser Val Ala Leu Ala Thr Gly Ala Tyr Ala His Ile Arg 385 390 395 400 Arg Gln Phe Lys Ile Ser Ile Gly Lys Met Glu Gly Ile Glu Glu Pro 405 410 415 Leu Ala Arg Ile Ala Gly Asn Ala Tyr Val Met Asp Ala Ala Ala Ser 420 425 430 Leu Ile Thr Tyr Gly Ile Met Leu Gly Glu Lys Pro Ala Val Leu Ser 435 440 445 Ala Ile Val Lys Tyr His Cys Thr His Arg Gly Gln Gln Ser Ile Ile 450 455 460 Asp Ala Met Asp Ile Thr Gly Gly Lys Gly Ile Met Leu Gly Gln Ser 465 470 475 480 Asn Phe Leu Ala Arg Ala Tyr Gln Gly Ala Pro Ile Ala Ile Thr Val 485 490 495 Glu Gly Ala Asn Ile Leu Thr Arg Ser Met Met Ile Phe Gly Gln Gly 500 505 510 Ala Ile Arg Cys His Pro Tyr Val Leu Glu Glu Met Glu Ala Ala Lys 515 520 525 Asn Asn Asp Val Asn Ala Phe Asp Lys Leu Leu Phe Lys His Ile Gly 530 535 540 His Val Gly Ser Asn Lys Val Arg Ser Phe Trp Leu Gly Leu Thr Arg 545 550 555 560 Gly Leu Thr Ser Ser Thr Pro Thr Gly Asp Ala Thr Lys Arg Tyr Tyr 565 570 575 Gln His Leu Asn Arg Leu Ser Ala Asn Leu Ala Leu Leu Ser Asp Val 580 585 590 Ser Met Ala Val Leu Gly Gly Ser Leu Lys Arg Arg Glu Arg Ile Ser 595 600 605 Ala Arg Leu Gly Asp Ile Leu Ser Gln Leu Tyr Leu Ala Ser Ala Val 610 615 620 Leu Lys Arg Tyr Asp Asp Glu Gly Arg Asn Glu Ala Asp Leu Pro Leu 625 630 635 640 Val His Trp Gly Val Gln Asp Ala Leu Tyr Gln Ala Glu Gln Ala Met 645 650 655 Asp Asp Leu Leu Gln Asn Phe Pro Asn Arg Val Val Ala Gly Leu Leu 660 665 670 Asn Val Val Ile Phe Pro Thr Gly Arg His Tyr Leu Ala Pro Ser Asp 675 680 685 Lys Leu Asp His Lys Val Ala Lys Ile Leu Gln Val Pro Asn Ala Thr 690 695 700 Arg Ser Arg Ile Gly Arg Gly Gln Tyr Leu Thr Pro Ser Glu His Asn 705 710 715 720 Pro Val Gly Leu Leu Glu Glu Ala Leu Val Asp Val Ile Ala Ala Asp 725 730 735 Pro Ile His Gln Arg Ile Cys Lys Glu Leu Gly Lys Asn Leu Pro Phe 740 745 750 Thr Arg Leu Asp Glu Leu Ala His Asn Ala Leu Val Lys Gly Leu Ile 755 760 765 Asp Lys Asp Glu Ala Ala Ile Leu Val Lys Ala Glu Glu Ser Arg Leu 770 775 780 Arg Ser Ile Asn Val Asp Asp Phe Asp Pro Glu Glu Leu Ala Thr Lys 785 790 795 800 Pro Val Lys Leu Pro Glu Lys Val Arg Lys Val Glu Ala Ala 805 810 <210> 7 <211> 40 <212> Ms <213> Artificial sequence <220> <223> Synthetic DNA <400> 7 aaaaaacata tggccactcg cgtgaagacc aacaagaaac 40 <210> 8 <211> 44 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 8 aaacaattgt tagagggcgt tggtgggctc gtagacgaac tcag 44 <210> 9 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 9 aaaaaacata tggcctctcg caagaatgtg agcgctgc 38 <210> 10 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 10 aaagaattct tacaggcgct cagtgggcac gtagatgtc 39 <210> 11 <211> 41 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 11 gccttcatgg agacttatgg tgtatccgcc cccatgtaca c 41 <210> 12 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 12 acaccataag tctccatgaa ggcacggc 28 <210> 13 <211> 37 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 13 gtctctgtac gcatgccagt cctcgcgggc ctggttg 37 <210> 14 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 14 gaggactggc atgcgtacag agacttgccc cactgtgtc 39 <210> 15 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 15 ctctcttccg ggcgctatca tgccatacc 29 <210> 16 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 16 gcacagcacc atgttggcca ttgtagatg 29 <210> 17 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 17 aaagggctcc gggaagcaag ttgc 24 <210> 18 <211> 51 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 18 gcaacttgct tcccggagcc cttttggatt ctctccggat tctccacttt c 51 <210> 19 <211> 51 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 19 gcaacttgct tcccggagcc ctttaatatt ctctccggat tctccacttt c 51 <210> 20 <211> 51 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 20 gcaacttgct tcccggagcc ctttgggatt ctctccggat tctccacttt c 51 <210> twenty one <211> 51 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> twenty one gcaacttgct tcccggagcc ctttgacatt ctctccggat tctccacttt c 51 <210> twenty two <211> 51 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> twenty two gcaacttgct tcccggagcc ctttgctatt ctctccggat tctccacttt c 51 <210> twenty three <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> twenty three gatatacata tggcctctcg caagagtgtg agcgctgctc acgaaatg 48 <210> twenty four <211> 29 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> twenty four ctctcttccg ggcgctatca tgccatacc 29 <210> 25 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 25 ttgaggtgct cggctcgctt gttg 24 <210> 26 <211> 37 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 26 cgagccgagc acctcaaagc agagcgtagc aaatttg 37 <210> 27 <211> 2006 <212> PRT <213> Photobacterium profundum <400> 27 Met His Cys Pro Val Asn Tyr Ala Pro Asn Thr Ala Val Thr Phe Ser 1 5 10 15 Pro Ala Ser Arg Val Arg Arg Thr Ser Glu Cys Thr Glu Ile Thr Gln 20 25 30 Cys Ile Val Ser Glu Tyr His Thr Glu His Tyr Thr Arg Arg Ala Ser 35 40 45 Val Ser Ser Gln His Ser Thr Ala Ser Gln Ser Asn Lys Ile Ala Ile 50 55 60 Val Gly Leu Ala Asn Gln Tyr Pro Asp Ala Asp Thr Pro Lys Asp Phe 65 70 75 80 Trp Gln Asn Leu Leu Ala Lys Lys Asp Ser Arg Thr Thr Leu Ser Pro 85 90 95 Asp Lys Leu Gly Ala Asn Pro Asp Ala Tyr Gln Gly Ile Gln Gly Glu 100 105 110 Ser Asp Arg Phe Tyr Cys Asp Lys Gly Gly Tyr Ile Gln Asn Phe Ser 115 120 125 Phe Asp Ser Asn Gly Tyr Arg Leu Pro Ala Glu Thr Phe Glu Gly Leu 130 135 140 Asp Glu Ser Phe Leu Trp Ala Leu Asp Thr Ser Arg Lys Ala Leu Ala 145 150 155 160 Asp Ala Gly Ile Pro Leu Asp Asp Ala Val Leu Glu Arg Thr Gly Val 165 170 175 Ile Met Gly Ala Leu Ser Phe Pro Thr Lys Arg Ser Asn Asp Leu Phe 180 185 190 Leu Pro Ile Tyr His Ser Ala Val Glu Lys Ala Leu Gln Thr Lys Leu 195 200 205 Gly Asn Glu His Phe Thr Leu Thr Pro Ser Asn Ala Asn Ile Thr Ser 210 215 220 Leu Asn Pro Ala Asn Gly Ser Ala Ala His Asn Ala Ser Arg Leu Val 225 230 235 240 Ala Asp Ala Leu Gly Leu Gly Ser Val Gln Leu Ser Leu Asp Ala Ala 245 250 255 Cys Ala Ser Ser Val Tyr Ser Leu Lys Leu Ala Cys Asp Tyr Leu Asn 260 265 270 Thr Gly Lys Ala Asp Met Met Leu Ala Gly Ala Val Ser Gly Ala Asp 275 280 285 Pro Phe Phe Ile Asn Met Gly Phe Ser Ile Phe His Ala Tyr Pro Asp 290 295 300 His Gly Val Ser Val Pro Phe Asp Thr Asn Ser Lys Gly Leu Phe Ala 305 310 315 320 Gly Glu Gly Ala Gly Val Leu Ile Leu Lys Arg Leu Glu Asp Ala Glu 325 330 335 Arg Asp Gly Asp Asn Ile Tyr Ala Val Val Ser Gly Ile Gly Leu Ser 340 345 350 Asn Asp Gly Arg Gly Gln Phe Val Leu Ser Pro Asn Ser Lys Gly Gln 355 360 365 Val Gln Ala Phe Glu Arg Ala Tyr Glu Ala Thr Asp Leu Ser Pro Glu 370 375 380 Ser Ile Glu Val Ile Glu Cys His Ala Thr Gly Thr Pro Leu Gly Asp 385 390 395 400 Lys Val Glu Met Thr Ser Met Glu Arg Phe Phe Ala Asp Lys Leu Asn 405 410 415 Gly Ser Gln Ala Pro Leu Ile Gly Ser Ala Lys Ser Asn Leu Gly His 420 425 430 Leu Leu Thr Ala Ala Gly Met Pro Gly Ile Met Lys Met Ile Phe Ala 435 440 445 Met Lys Glu Gly Val Leu Pro Pro Ser Ile Asn Leu Ser Thr Pro Leu 450 455 460 Ser Ser Pro Glu Gly Leu Phe Gly Ser His Thr Leu Pro Thr Gln Val 465 470 475 480 Gln Ala Trp Pro Asp Lys Ala Gly Asn Thr Glu Arg Cys Ala Gly Val 485 490 495 Ser Val Phe Gly Phe Gly Gly Cys Asn Ala His Leu Leu Leu Glu Ala 500 505 510 His Ser Ala Asn Ser Ala Arg Asn Ser Pro Ala Ala Asn Ser Pro Ala 515 520 525 Lys Pro Ala Val Ser Ala Pro Leu Lys Val Thr Gly Leu Ala Ser His 530 535 540 Phe Gly Ser Leu Lys Thr Ile Asn Ala Leu His Asn Ala Ile Thr Thr 545 550 555 560 Gly Ala Asp Ala Phe Val Ala Leu Pro Lys Lys Arg Trp Lys Gly Leu 565 570 575 Asp Gln His Pro Glu Leu Leu Ser Gln Phe Gly Leu Asp Ala Ile Pro 580 585 590 His Gly Ala Tyr Ile Asp Gln Phe Glu Leu Asp Phe Leu Arg Phe Lys 595 600 605 Val Pro Pro Asn Glu Asp Asp Arg Leu Ile Ser Gln Gln Leu Leu Leu 610 615 620 Met Lys Val Ala Asp Glu Ala Ile Arg Asp Ala Lys Leu Glu Val Gly 625 630 635 640 Gln Lys Val Ala Val Leu Val Ala Met Glu Thr Glu Leu Glu Met His 645 650 655 Gln Phe Arg Gly Arg Val Asn Leu His Thr Gln Leu Ala Asp Ser Leu 660 665 670 Glu Asn Met Gly Val Gln Leu Thr Asp Ser Glu Tyr Gln Ala Leu Glu 675 680 685 Ala Ile Ala Met Asp Ser Val Leu Asp Ala Ala Lys Leu Asn Gln Tyr 690 695 700 Thr Ser Phe Ile Gly Asn Ile Met Ala Ser Arg Ile Ala Ser Leu Trp 705 710 715 720 Asp Phe Asn Gly Pro Ala Phe Thr Ile Ser Ala Ala Glu Gln Ser Val 725 730 735 Ala Arg Cys Ile Asp Val Ala Gln Asn Leu Met Ser Gln Glu Ser Leu 740 745 750 Asp Ala Val Val Ile Ala Ala Val Asp Leu Ser Gly Ser Val Glu Gln 755 760 765 Ile Ile Leu Lys Asn Ser Val Thr Pro Val Ala Leu His Pro Gln Asp 770 775 780 Ser Gly Trp Asn Val Gly Glu Gly Ala Gly Ala Ile Val Leu Val Asp 785 790 795 800 Thr Asp Asn Ala Ser Thr Lys Asn Ser Tyr Gly Glu Ile Thr Ala Leu 805 810 815 Asp Phe Gly Ser Val Ala Gln Ser Asn Ile Thr Ser Asp Arg Leu Leu 820 825 830 Thr Thr Ala Gly Ile Thr Ala Asn Asn Val Ser Leu Leu Glu Leu Asn 835 840 845 Gln Ala Pro Glu Ser Val Glu Thr Val Gln Phe Pro Leu Pro Ser Ala 850 855 860 Thr His Ile Gln Ala Asn Gln Arg Leu Gly His Cys Tyr Ala Ala Ser 865 870 875 880 Gly Met Ala Ser Ile Leu His Gly Leu Leu Ser Leu Asn Ala Ile Pro 885 890 895 Lys Gln Thr Thr Ile Pro Ser Leu Asn Thr Ser Val Thr Ala Ala Val 900 905 910 Thr Lys Ala Ala Ile Val Ala Asn Val Ser Glu Asn Gln Cys Ser Gln 915 920 925 Leu Leu Leu Thr Gln Thr Ser Thr Glu Thr Gln Ser Leu Thr Ala Arg 930 935 940 Leu Asn Ser Glu Leu Ala Asn Asp Ser Lys Arg Gln Leu Ile Lys Gln 945 950 955 960 Val Thr Leu Gly Gly Arg Asp Ile Tyr Gln His Ile Val Thr Ala Glu 965 970 975 Leu Ser Asp Ile Gln His Ile Gln Gln Lys Val Ala Asn Thr Lys Pro 980 985 990 Leu Val Gln Lys Gln Asn Ile Lys Gln Pro Arg Ile Gln Ala Ile Ala 995 1000 1005 Lys Pro Val Ala Gln Pro Gln Ser Ile Ala Gln Pro Thr Ala Ile 1010 1015 1020 Pro Ser Val Ser Pro Ile Leu Thr Thr Pro Arg Gln Thr Pro Ile 1025 1030 1035 Thr Gly Ile Gln Ser Asn Met Thr Asn Val Leu Ser Ala Lys Asn 1040 1045 1050 Lys His Asp Leu Thr Ala Phe Gln Ser Ser Ala Phe Val Glu Asn 1055 1060 1065 Gln Gln Leu Ala Gln Gln Val His Gln Ala Phe Leu Gln Asn Arg 1070 1075 1080 Glu Gln Gly Leu Lys Met Ala Asp Ala Leu Leu Lys Ala Gln Leu 1085 1090 1095 Asn Glu Val Thr Ala Gln Met Asn Ala Ala Thr Gly Gln Val Phe 1100 1105 1110 Asp His Gln Leu Ala Pro Ser Pro Ala Leu Thr Glu Ala Ser Thr 1115 1120 1125 Ser Val Pro Val Asn Val Ala Ala Thr Pro Ala Ile Val Asn Pro 1130 1135 1140 Ile Arg Lys Pro Cys Ile Trp Asp Tyr Glu Asp Leu Val Glu Tyr 1145 1150 1155 Ala Glu Gly Asp Ile Ala Asn Val Phe Gly Pro Asp Tyr Ala Val 1160 1165 1170 Ile Asp Asn Tyr Ser Arg Arg Val Arg Leu Pro Thr Thr Asp Tyr 1175 1180 1185 Leu Leu Val Ser Arg Val Thr Lys Leu Asp Ala Thr Met Leu Glu 1190 1195 1200 Tyr Lys Pro Ser Thr Met Thr Thr Glu Tyr Asp Ile Pro Val Asp 1205 1210 1215 Ala Pro Tyr Leu Val Asp Gly Gln Ile Pro Trp Ala Val Ala Val 1220 1225 1230 Glu Ser Gly Gln Cys Asp Leu Met Leu Ile Ser Tyr Leu Gly Ile 1235 1240 1245 Asp Phe Glu Asn Lys Gly Glu Arg Val Tyr Arg Leu Leu Asp Cys 1250 1255 1260 Thr Leu Thr Phe Leu Gly Asp Leu Pro Arg Gly Gly Asp Thr Leu 1265 1270 1275 Arg Tyr Asp Ile Ser Ile Asn Asn Phe Ala Arg Asn Gly Asp Thr 1280 1285 1290 Leu Leu Phe Phe Phe Ser Tyr Glu Cys Phe Val Gly Asp Lys Met 1295 1300 1305 Val Leu Lys Met Asp Asn Gly Cys Ala Gly Phe Phe Thr Asp Glu 1310 1315 1320 Glu Leu Ser Asp Gly Lys Gly Val Ile Arg Thr Glu Asp Glu Ile 1325 1330 1335 Lys Ser Arg Asn Leu Ala Val Lys Gln Arg Phe Asn Pro Leu Leu 1340 1345 1350 His Cys Gln Lys Thr Gln Phe Asp Tyr Gln Thr Leu His Asn Leu 1355 1360 1365 Leu Asp Ala Asn Ile Ala Gly Cys Phe Gly Glu Ser His Ile Ser 1370 1375 1380 Asp Arg His Gln Pro Ser Leu Cys Phe Ser Ser Asp Lys Phe Met 1385 1390 1395 Met Ile Glu Gln Ile Ser His Val Asp Pro Gln Gly Gly Thr Trp 1400 1405 1410 Gly Leu Gly Leu Ile Glu Gly His Lys Gln Leu Glu Ala Asp His 1415 1420 1425 Trp Tyr Phe Pro Cys His Phe Lys Asp Asp Ser Val Met Ala Gly 1430 1435 1440 Ser Leu Met Ala Glu Gly Cys Gly Gln Leu Leu Gln Phe Phe Met 1445 1450 1455 Met Tyr Leu Gly Met His Thr Gln Val Glu Asn Gly Arg Phe Gln 1460 1465 1470 Pro Leu Glu Asn Ala Pro Gln Gln Val Arg Cys Arg Gly Gln Val 1475 1480 1485 Leu Pro Gln Ser Ala Val Leu Thr Tyr Arg Met Glu Val Thr Glu 1490 1495 1500 Ile Gly Leu Ser Pro Arg Pro Tyr Ala Lys Ala Asn Ile Asp Ile 1505 1510 1515 Leu Leu Asp Gly Lys Val Val Val Asp Phe Gln Asn Leu Gly Val 1520 1525 1530 Met Ile Lys Glu Glu Ser Glu Cys Thr Arg Tyr Leu Gly Ser Ser 1535 1540 1545 Asp Phe Asp Ser Ala Ser Leu Ala Ser Ser Ser Tyr Val Ala Glu 1550 1555 1560 Ser Ala Pro Ala Gln Ala Asp Val Ile Thr Pro Val Glu Ala Pro 1565 1570 1575 Ile Ser Gln Gln Ala Ser Ala Asn Ala Pro Leu Met Ala Gln Ile 1580 1585 1590 Pro Asp Leu Asn Thr Ala Pro Asn Lys Gly Val Ile Pro Leu Gln 1595 1600 1605 His Ile Glu Ala Pro Ile Val Pro Asp Tyr Gln Asn Arg Thr Pro 1610 1615 1620 Asp Thr Val Pro Phe Thr Pro Tyr His Met Phe Glu Phe Ala Thr 1625 1630 1635 Gly Asp Ile Glu Lys Cys Phe Gly Pro Asp Phe Ser Ile Tyr Arg 1640 1645 1650 Gly Met Ile Pro Pro Arg Thr Pro Cys Gly Asp Leu Gln Leu Thr 1655 1660 1665 Thr Arg Val Ile Glu Val Asn Gly Thr Arg Gly Asp Phe Lys Thr 1670 1675 1680 Pro Ser Ser Cys Ile Ala Glu Tyr Glu Val Pro Glu Asn Ala Trp 1685 1690 1695 Tyr Phe Asp Glu Asn Ser His Ser Ser Leu Met Pro Tyr Ser Val 1700 1705 1710 Leu Met Glu Ile Ser Leu Gln Pro Asn Gly Phe Ile Ser Gly Tyr 1715 1720 1725 Met Gly Thr Thr Leu Gly Phe Pro Gly Leu Glu Leu Phe Phe Arg 1730 1735 1740 Asn Leu Asp Gly Ser Gly Lys Met Leu Arg Asn Val Asp Leu Arg 1745 1750 1755 Gly Lys Thr Ile Val Asn Asp Ser Arg Leu Leu Ser Thr Val Met 1760 1765 1770 Met Gly Thr Asn Ile Val Gln Ser Phe Ser Phe Glu Leu Ser Thr 1775 1780 1785 Asp Gly Val Pro Phe Tyr Glu Gly Thr Ala Val Phe Gly Tyr Phe 1790 1795 1800 Lys Gly Ala Ala Leu Lys Asp Gln Leu Gly Leu Asp Asn Gly Gln 1805 1810 1815 Val Thr Tyr Pro Trp His Val Asn Asn Asn Arg Thr Pro Asp Val 1820 1825 1830 Ser Ile Asn Leu Leu Asp Lys Glu Ser Arg Tyr Phe Asn Ala Pro 1835 1840 1845 Leu Ser Ala Thr Gly Glu Ala Gln Pro His Tyr Gln Leu Ala Gly 1850 1855 1860 Gly Arg Leu Asn Phe Ile Asp Lys Val Asp Ile Thr Ser Asp Gly 1865 1870 1875 Gly Lys Ala Gly Leu Gly Tyr Leu Tyr Ala Glu Arg Thr Ile Asp 1880 1885 1890 Pro Ser Asp Trp Phe Phe Gln Phe His Phe His Gln Asp Pro Val 1895 1900 1905 Met Pro Gly Ser Leu Gly Val Glu Ala Ile Ile Glu Leu Met Gln 1910 1915 1920 Thr Tyr Ala Leu Asn Lys Asp Leu Gly Ala Gly Phe Arg Ser Pro 1925 1930 1935 Lys Phe Gly Gln Ile Gln Ser Glu Val Lys Trp Lys Tyr Arg Gly 1940 1945 1950 Gln Ile Asn Pro Leu Asn Lys Gln Met Ser Leu Asp Val His Ile 1955 1960 1965 Thr Ala Ile Lys Asp Glu Asp Gly Lys Arg Ile Ile Val Gly Asp 1970 1975 1980 Ala Asn Leu Ser Lys Asp Gly Leu Arg Ile Tyr Glu Val Lys Asp 1985 1990 1995 Ile Ala Ile Cys Ile Glu Glu Ala 2000 2005 <210> 28 <211> 1963 <212> PRT <213> Shewanella oneidensis <400> 28 Met Ser Ser Gln Met His Thr His Pro Thr Leu Gln Asp Ser Ala Ala 1 5 10 15 Val Pro Asn Asp Gln Arg Gln Thr Leu Lys Ala Met Pro Lys Ile Ala 20 25 30 Ile Val Gly Leu Ala Val Gln Tyr Pro Asp Ala Asp Thr Pro Glu Gln 35 40 45 Phe Trp Gln Asn Leu Leu Asp Lys Lys Asp Ser Arg Ser Gln Ile Asp 50 55 60 Ala Ala Lys Leu Asn Ala Asn Pro Ala Asp Tyr Gln Gly Ile Gln Gly 65 70 75 80 Gln Ala Asp Arg Phe Tyr Cys Asp Lys Gly Gly Tyr Ile Arg Asn Phe 85 90 95 Arg Phe Asp Pro Gln Gly Tyr Gln Leu Leu Pro Ala Thr Phe Ala Gly 100 105 110 Leu Asp Glu Ser Phe Leu Trp Ala Leu Asp Cys Ser Lys Lys Ala Leu 115 120 125 Leu Asn Ala Gly Val Asp Leu Thr Ala Pro Leu Leu Glu Arg Thr Gly 130 135 140 Ile Val Met Gly Thr Leu Ser Phe Pro Thr Ala Arg Ser Asn Glu Leu 145 150 155 160 Phe Leu Pro Ile Tyr His Gln Ala Val Glu Lys Ala Leu Lys Thr Lys 165 170 175 Leu Asn Gln Pro Gln Phe Ala Leu Ala Pro Phe Ala Asn Ala Ser Ile 180 185 190 Ala Gly Ser Gln Leu Ala Ala Asn Gly Val Ile Ala His Thr Ala Ser 195 200 205 Lys Leu Leu Ser Asp Ala Leu Gly Leu Gly Gly Ala Gln Leu Ser Leu 210 215 220 Asp Ala Ala Cys Ala Ser Ser Val Tyr Ala Leu Lys Leu Ala Cys Asp 225 230 235 240 Tyr Leu Thr Thr Gly Lys Ala Asp Met Met Leu Ala Gly Ala Val Ser 245 250 255 Gly Ala Asp Pro Phe Phe Ile Asn Met Gly Phe Ser Ile Phe His Ala 260 265 270 Tyr Pro Asp His Gly Ile Ser Ala Pro Phe Asp Ser Asn Ser Lys Gly 275 280 285 Leu Phe Ala Gly Glu Gly Ala Gly Val Leu Val Leu Lys Arg Leu Glu 290 295 300 Asp Ala Glu Arg Asp Gly Asp Asn Ile Tyr Ala Val Val Ser Gly Ile 305 310 315 320 Gly Leu Ser Asn Asp Gly Lys Gly Gln Phe Val Leu Ser Pro Asn Ser 325 330 335 Lys Gly Gln Val Gln Ala Phe Glu Arg Ala Tyr Ala Ala Ala Asn Thr 340 345 350 His Pro Ser Asn Ile Glu Val Ile Glu Cys His Ala Thr Gly Thr Pro 355 360 365 Leu Gly Asp Lys Val Glu Leu Thr Ser Met Glu Arg Phe Phe Glu Asp 370 375 380 Lys Leu Asp Gly Thr Lys Ala Pro Leu Ile Gly Ser Ala Lys Ser Asn 385 390 395 400 Leu Gly His Leu Leu Thr Ala Ala Gly Met Pro Gly Ile Met Lys Met 405 410 415 Ile Phe Ala Met Arg Ser Gly His Leu Pro Pro Ser Ile Asn Leu Thr 420 425 430 Ala Pro Ile Ser Ser Pro Lys Gly Leu Phe Ser Val Asn Asn Leu Pro 435 440 445 Thr Gln Arg Gln Ala Trp Pro Asp Lys Ala Gly Asn Asp Arg Arg His 450 455 460 Ala Gly Val Ser Val Phe Gly Phe Gly Gly Cys Asn Ala His Leu Leu 465 470 475 480 Leu Glu Ser Tyr Gln Pro Thr Ala His Ser Ala Glu Lys Gln Ala Asn 485 490 495 Lys Pro Val Tyr Gln Gln Gln Ala Leu Thr Val Ile Gly Met Ala Ser 500 505 510 His Phe Gly Pro Leu Ala Ser Ile Asn Ala Leu Asp Lys Ala Leu Ile 515 520 525 Ala Gln Thr Asp Ala Phe Ile Pro Leu Pro Pro Lys Arg Trp Lys Gly 530 535 540 Leu Asp Lys His Pro Asp Ile Leu Gln Gln Phe Gly Leu Asn Arg Ala 545 550 555 560 Pro Lys Gly Ala Tyr Ile Glu Gln Phe Asp Phe Asp Phe Leu Arg Phe 565 570 575 Lys Val Pro Pro Asn Glu Asp Asp Arg Leu Ile Ser Gln Gln Leu Leu 580 585 590 Leu Ile Lys Val Ala Asp Glu Ala Ile Arg Asp Ala Lys Leu Thr Ala 595 600 605 Gly Ser Lys Val Ala Val Leu Val Ala Met Glu Thr Glu Leu Glu Leu 610 615 620 His Gln Phe Arg Gly Arg Val Asn Leu His Thr Gln Leu Ala Asp Ser 625 630 635 640 Leu Lys Lys Gln Gly Val His Leu Ser Asn Asp Glu Tyr Leu Ala Leu 645 650 655 Glu Ala Ile Ala Met Asp Ser Val Leu Asp Ala Ala Lys Leu Asn Gln 660 665 670 Tyr Thr Ser Phe Ile Gly Asn Ile Met Ala Ser Arg Ile Ala Ser Leu 675 680 685 Trp Asp Phe Asn Gly Pro Ala Phe Thr Ile Ser Ala Ala Glu Gln Ser 690 695 700 Val Ala Arg Cys Ile Asp Val Ala Gln Asn Leu Leu Ser Lys Glu Ala 705 710 715 720 Leu Asp Gly Val Val Ile Ala Ala Val Asp Leu Ser Gly Ser Val Glu 725 730 735 Gln Val Ile Leu Lys Asn Ala Gln Val Ala Val Asp Leu Asp Ala Asn 740 745 750 Ser Ala Asn Pro Gln Trp Lys Val Gly Glu Gly Ala Gly Ala Ile Val 755 760 765 Leu Thr Asn Gln Gln Ala Ser Asn Ser Gln Gln Ala Gly Tyr Gly Gln 770 775 780 Ile Arg Gly Gln Ala Phe Gly Thr Asn His Gln Leu Pro Lys Leu Leu 785 790 795 800 Asp Ser Leu Ile Thr Glu Thr Ala Ile Ala Asn Pro Ser Met Pro Thr 805 810 815 Ala Ile His Met Ile Glu Gln Cys Ile Ala Pro Glu Glu Gln Leu Pro 820 825 830 Ala Glu His Leu Leu Ala Gln Leu Asn Leu Leu Gly Thr Ser Cys Asn 835 840 845 Arg Val Ala Asn Thr Leu Gly His Asn Phe Ala Ala Ala Gly Met Ala 850 855 860 Ser Leu Leu Ser Ala Leu Leu Ser Leu Lys Asn Arg Ser Ala Asn Ser 865 870 875 880 Asp Lys Asn Ala Glu Lys Gln Ala Leu Val Ser Thr Gln Ser Gln Gly 885 890 895 Val Ser Ser Leu Leu Leu Leu Ser Gln Thr Ala Thr Gln Ala Ala Gln 900 905 910 Leu Glu Leu Arg Leu Ala Gln Asp Leu Thr Leu Ser Glu Gln Lys His 915 920 925 Leu Ile Lys Pro Val Thr Leu Gly Gly Arg Asp Ile Tyr Gln His Ile 930 935 940 Val Asp Thr Pro Leu Pro Ala Leu Ala Ala Ile Gln Gly Lys Met Arg 945 950 955 960 Gln Leu Gln Pro Leu Ala Ser Gln Ala Thr Gln Thr Lys Pro Ala Val 965 970 975 Gly Ala Ala Leu Asp Ile Thr Ala Glu Asn Ala Thr Pro Leu Ala Ala Ala 980 985 990 Glu Ser Gly Met Ser Ser Asn Ala Pro Leu Gln Phe Glu Thr Thr Ala 995 1000 1005 Ser Ala Gln Asp Ser Ala Ala Leu Leu Gln Asn Gln Gln Leu Ala 1010 1015 1020 Arg Glu Ala His Leu Ala Phe Leu Gln Ser Arg Glu Gln Gly Leu 1025 1030 1035 Lys Leu Ala Asp Ala Leu Leu Lys Ala Gln Leu Ser Gln Thr Thr 1040 1045 1050 Gln Met Gly Ala Val Ala Ala His Val Ala Thr Ser Ala Asn Val 1055 1060 1065 Ala Glu Thr Lys Ala Gln Gln Ala Val Ser Ile Pro Glu Leu Met 1070 1075 1080 Pro Asn His Ala Pro Asn His Ala Arg Val Pro Pro Tyr Thr Pro 1085 1090 1095 Pro Ile Pro Ala Ala Lys Pro Cys Ile Trp Asn Tyr Gln Asp Leu 1100 1105 1110 Val Glu Tyr Ala Glu Gly Asp Ile Ala Lys Val Phe Gly Ala Asp 1115 1120 1125 Tyr Ala Ile Ile Asp Ser Tyr Ala Arg Arg Val Arg Leu Pro Thr 1130 1135 1140 Ser Asp Tyr Leu Leu Val Ser Arg Val Thr Lys Leu Asn Ala Gln 1145 1150 1155 Met Asn Arg Tyr Gln Pro Ser Ser Met Thr Thr Glu Tyr Asp Ile 1160 1165 1170 Pro Val Asp Ala Pro Phe Leu Val Asp Gly Gln Ile Pro Trp Ala 1175 1180 1185 Val Ala Val Glu Ser Gly Gln Cys Asp Leu Met Leu Ile Ser Tyr 1190 1195 1200 Leu Gly Ile Asp Phe Glu Asn Lys Gly Glu Arg Val Tyr Arg Leu 1205 1210 1215 Leu Asp Cys Thr Leu Thr Phe Leu Gly Asp Leu Pro Arg Gly Gly 1220 1225 1230 Asp Thr Leu Arg Tyr Asp Ile Ser Ile Asn His Phe Ala Arg Asn 1235 1240 1245 Gly Asp Thr Leu Leu Phe Phe Phe Ser Tyr Glu Cys Phe Val Gly 1250 1255 1260 Asp Lys Leu Ile Leu Lys Met Asp Gly Gly Cys Ala Gly Phe Phe 1265 1270 1275 Thr Asp Lys Glu Leu Ala Asp Gly Lys Gly Val Ile Arg Thr Glu 1280 1285 1290 Val Glu Ile Lys Val Arg Glu Gln Ala Gln Ile Ala Leu Ala Asn 1295 1300 1305 Glu Tyr Thr Arg Asn Gly Asn Lys Pro Arg Phe Thr Pro Leu Leu 1310 1315 1320 Asn Cys Ala Gln Thr Ala Phe Ser Tyr Gly Gln Ile His Arg Leu 1325 1330 1335 Leu Ser Ala Asp Ile Gly Gly Cys Phe Gly Gly Glu His Ala Ala 1340 1345 1350 His Gln Ala Lys Phe Gly Leu Gln Pro Ser Leu Cys Phe Ala Ser 1355 1360 1365 Glu Lys Phe Leu Met Ile Glu Gln Val Ser Lys Leu Glu Val His 1370 1375 1380 Gly Gly Ala Trp Gly Leu Gly Leu Ile Glu Gly His Lys Gln Leu 1385 1390 1395 Ala Pro Asp His Trp Tyr Phe Pro Cys His Phe Lys Gly Asp Gln 1400 1405 1410 Val Met Ala Gly Ser Leu Met Ala Glu Gly Cys Gly Gln Leu Leu 1415 1420 1425 Gln Phe Phe Met Leu His Ile Gly Met His Ala Asn Thr Gln Ala 1430 1435 1440 Gly Gly Val Thr Asn Gly Arg Phe Gln Pro Leu Glu Asn Ala Ser 1445 1450 1455 Gln Lys Val Arg Cys Arg Gly Gln Val Leu Pro Gln Ser Gly Thr 1460 1465 1470 Leu Thr Tyr Arg Met Glu Val Thr Glu Ile Gly Met Ser Pro Arg 1475 1480 1485 Pro Tyr Ala Lys Ala Asn Ile Asp Ile Leu Leu Asn Gly Lys Val 1490 1495 1500 Val Val Asp Phe Gln Asn Leu Gly Val Met Ile Lys Glu Glu Ala 1505 1510 1515 Asp Cys Thr Arg Tyr Ser Gln Ser His Ser Ser Gln Gly Asn His 1520 1525 1530 Thr Gln Ala Ala Asn Ile Glu Ser Leu Ala Glu Gln Ala Pro Leu 1535 1540 1545 Met Ala Gln Ile Pro Asp Val Ala Ala Pro Val Asn Lys Gly Val 1550 1555 1560 Val Pro Leu Lys His Val Ser Ala Pro Ile Ala Pro Ala Gly Ser 1565 1570 1575 Lys Tyr Ala Asn Arg Val Pro Asp Thr Leu Pro Phe Thr Pro Tyr 1580 1585 1590 His Leu Phe Glu Phe Ala Thr Gly Asp Ile Glu Asn Cys Phe Gly 1595 1600 1605 Pro Asp Phe Ser Ile Tyr Arg Gly Leu Ile Pro Pro Arg Thr Pro 1610 1615 1620 Cys Gly Asp Leu Gln Leu Thr Thr Arg Val Val Ala Ile Glu Gly 1625 1630 1635 Lys Arg Gly Glu Leu Lys Lys Pro Ser Thr Cys Ile Ala Glu Tyr 1640 1645 1650 Glu Val Pro Ser Asn Ala Trp Tyr Tyr Arg Lys Thr Ser His Pro 1655 1660 1665 Ser Val Met Pro Tyr Ser Val Leu Met Glu Ile Ser Leu Gln Pro 1670 1675 1680 Asn Gly Phe Ile Ser Gly Tyr Met Gly Thr Thr Leu Gly Phe Pro 1685 1690 1695 Gly Gln Glu Leu Phe Phe Arg Asn Leu Asp Gly Ser Gly Lys Leu 1700 1705 1710 Leu Arg Glu Val Asp Leu Arg Gly Lys Thr Ile Val Asn Asp Ser 1715 1720 1725 Arg Leu Leu Ser Thr Val Ile Ala Gly Ser Asn Ile Ile Gln Asn 1730 1735 1740 Phe Ser Phe Glu Leu Ser Cys Asp Gly Glu Pro Phe Tyr Arg Gly 1745 1750 1755 Asn Ala Val Phe Gly Tyr Phe Lys Ala Asp Ala Leu Lys Asn Gln 1760 1765 1770 Leu Gly Ile Asp Asn Gly Lys Ile Thr Gln Ala Trp His Leu Glu 1775 1780 1785 Arg Gly Ile Lys Ala Asp Cys Gln Ile Asn Leu Leu Asp Lys Asn 1790 1795 1800 Gly Arg Ser Phe Val Ala Pro Leu Gly Lys Pro His Tyr Arg Leu 1805 1810 1815 Ala Gly Gly Gln Leu Asn Phe Ile Asp Lys Ala Glu Ile Val Lys 1820 1825 1830 Thr Gly Gly Lys Lys Gly Leu Gly Tyr Leu Tyr Ala Glu Arg Thr 1835 1840 1845 Ile Asp Pro Ser Asp Trp Phe Phe Gln Phe His Phe His Gln Asp 1850 1855 1860 Pro Val Met Pro Gly Ser Leu Gly Val Glu Ala Ile Ile Glu Leu 1865 1870 1875 Leu Gln Thr Tyr Ala Ile Asp Gln Asp Leu Gly Ala Gly Phe Asn 1880 1885 1890 Asn Pro Lys Phe Gly Gln Ile Leu Ser Glu Ile Lys Trp Lys Tyr 1895 1900 1905 Arg Gly Gln Ile Asn Pro Leu Asn Lys Gln Met Ser Leu Asp Val 1910 1915 1920 His Ile Thr Ser Ile Glu Asp Lys Asp Gly Lys Arg Ile Ile Lys 1925 1930 1935 Gly Asp Ala Asn Leu Ser Lys Asp Gly Leu Arg Ile Tyr Glu Val 1940 1945 1950 Thr Asp Ile Ala Ile Cys Ile Glu Glu Ala 1955 1960 <210> 29 <211> 2011 <212> PRT <213> Moritella marina <400> 29 Met Glu Asn Ile Ala Val Val Gly Ile Ala Asn Leu Phe Pro Gly Ser 1 5 10 15 Gln Ala Pro Asp Gln Phe Trp Gln Gln Leu Leu Glu Gln Gln Asp Cys 20 25 30 Arg Ser Lys Ala Thr Ala Val Gln Met Gly Val Asp Pro Ala Lys Tyr 35 40 45 Thr Ala Asn Lys Gly Asp Thr Asp Lys Phe Tyr Cys Val His Gly Gly 50 55 60 Tyr Ile Ser Asp Phe Asn Phe Asp Ala Ser Gly Tyr Gln Leu Asp Asn 65 70 75 80 Asp Tyr Leu Ala Gly Leu Asp Asp Leu Asn Gln Trp Gly Leu Tyr Val 85 90 95 Thr Lys Gln Ala Leu Thr Asp Ala Gly Tyr Trp Gly Ser Thr Ala Leu 100 105 110 Glu Asn Cys Gly Val Ile Leu Gly Asn Leu Ser Phe Pro Thr Lys Ser 115 120 125 Ser Asn Gln Leu Phe Met Pro Leu Tyr His Gln Val Val Asp Asn Ala 130 135 140 Leu Lys Ala Val Leu His Pro Asp Phe Gln Leu Thr His Tyr Thr Ala 145 150 155 160 Pro Lys Lys Thr His Ala Asp Asn Ala Leu Val Ala Gly Tyr Pro Ala 165 170 175 Ala Leu Ile Ala Gln Ala Ala Gly Leu Gly Gly Ser His Phe Ala Leu 180 185 190 Asp Ala Ala Cys Ala Ser Ser Cys Tyr Ser Val Lys Leu Ala Cys Asp 195 200 205 Tyr Leu His Thr Gly Lys Ala Asn Met Met Leu Ala Gly Ala Val Ser 210 215 220 Ala Ala Asp Pro Met Phe Val Asn Met Gly Phe Ser Ile Phe Gln Ala 225 230 235 240 Tyr Pro Ala Asn Asn Val His Ala Pro Phe Asp Gln Asn Ser Gln Gly 245 250 255 Leu Phe Ala Gly Glu Gly Ala Gly Met Met Val Leu Lys Arg Gln Ser 260 265 270 Asp Ala Val Arg Asp Gly Asp His Ile Tyr Ala Ile Ile Lys Gly Gly 275 280 285 Ala Leu Ser Asn Asp Gly Lys Gly Glu Phe Val Leu Ser Pro Asn Thr 290 295 300 Lys Gly Gln Val Leu Val Tyr Glu Arg Ala Tyr Ala Asp Ala Asp Val 305 310 315 320 Asp Pro Ser Thr Val Asp Tyr Ile Glu Cys His Ala Thr Gly Thr Pro 325 330 335 Lys Gly Asp Asn Val Glu Leu Arg Ser Met Glu Thr Phe Phe Ser Arg 340 345 350 Val Asn Asn Lys Pro Leu Leu Gly Ser Val Lys Ser Asn Leu Gly His 355 360 365 Leu Leu Thr Ala Ala Gly Met Pro Gly Met Thr Lys Ala Met Leu Ala 370 375 380 Leu Gly Lys Gly Leu Ile Pro Ala Thr Ile Asn Leu Lys Gln Pro Leu 385 390 395 400 Gln Ser Lys Asn Gly Tyr Phe Thr Gly Glu Gln Met Pro Thr Thr Thr 405 410 415 Val Ser Trp Pro Thr Thr Pro Gly Ala Lys Ala Asp Lys Pro Arg Thr 420 425 430 Ala Gly Val Ser Val Phe Gly Phe Gly Gly Ser Asn Ala His Leu Val 435 440 445 Leu Gln Gln Pro Thr Gln Thr Leu Glu Thr Asn Phe Ser Val Ala Lys 450 455 460 Pro Arg Glu Pro Leu Ala Ile Ile Gly Met Asp Ser His Phe Gly Ser 465 470 475 480 Ala Ser Asn Leu Ala Gln Phe Lys Thr Leu Leu Asn Asn Asn Gln Asn 485 490 495 Thr Phe Arg Glu Leu Pro Glu Gln Arg Trp Lys Gly Met Glu Ser Asn 500 505 510 Ala Asn Val Met Gln Ser Leu Gln Leu Arg Lys Ala Pro Lys Gly Ser 515 520 525 Tyr Val Glu Gln Leu Asp Ile Asp Phe Leu Arg Phe Lys Val Pro Pro 530 535 540 Asn Glu Lys Asp Cys Leu Ile Pro Gln Gln Leu Met Met Met Gln Val 545 550 555 560 Ala Asp Asn Ala Ala Lys Asp Gly Gly Leu Val Glu Gly Arg Asn Val 565 570 575 Ala Val Leu Val Ala Met Gly Met Glu Leu Glu Leu His Gln Tyr Arg 580 585 590 Gly Arg Val Asn Leu Thr Thr Gln Ile Glu Asp Ser Leu Leu Gln Gln 595 600 605 Gly Ile Asn Leu Thr Val Glu Gln Arg Glu Glu Leu Thr Asn Ile Ala 610 615 620 Lys Asp Gly Val Ala Ser Ala Ala Gln Leu Asn Gln Tyr Thr Ser Phe 625 630 635 640 Ile Gly Asn Ile Met Ala Ser Arg Ile Ser Ala Leu Trp Asp Phe Ser 645 650 655 Gly Pro Ala Ile Thr Val Ser Ala Glu Glu Asn Ser Val Tyr Arg Cys 660 665 670 Val Glu Leu Ala Glu Asn Leu Phe Gln Thr Ser Asp Val Glu Ala Val 675 680 685 Ile Ile Ala Ala Val Asp Leu Ser Gly Ser Ile Glu Asn Ile Thr Leu 690 695 700 Arg Gln His Tyr Gly Pro Val Asn Glu Lys Gly Ser Val Ser Glu Cys 705 710 715 720 Gly Pro Val Asn Glu Ser Ser Ser Val Thr Asn Asn Ile Leu Asp Gln 725 730 735 Gln Gln Trp Leu Val Gly Glu Gly Ala Ala Ala Ile Val Val Lys Pro 740 745 750 Ser Ser Gln Val Thr Ala Glu Gln Val Tyr Ala Arg Ile Asp Ala Val 755 760 765 Ser Phe Ala Pro Gly Ser Asn Ala Lys Ala Ile Thr Ile Ala Ala Asp 770 775 780 Lys Ala Leu Thr Leu Ala Gly Ile Ser Ala Ala Asp Val Ala Ser Val 785 790 795 800 Glu Ala His Ala Ser Gly Phe Ser Ala Glu Asn Asn Ala Glu Lys Thr 805 810 815 Ala Leu Pro Thr Leu Tyr Pro Ser Ala Ser Ile Ser Ser Val Lys Ala 820 825 830 Asn Ile Gly His Thr Phe Asn Ala Ser Gly Met Ala Ser Ile Ile Lys 835 840 845 Thr Ala Leu Leu Leu Asp Gln Asn Thr Ser Gln Asp Gln Lys Ser Lys 850 855 860 His Ile Ala Ile Asn Gly Leu Gly Arg Asp Asn Ser Cys Ala His Leu 865 870 875 880 Ile Leu Ser Ser Ser Ala Gln Ala His Gln Val Ala Pro Ala Pro Val 885 890 895 Ser Gly Met Ala Lys Gln Arg Pro Gln Leu Val Lys Thr Ile Lys Leu 900 905 910 Gly Gly Gln Leu Ile Ser Asn Ala Ile Val Asn Ser Ala Ser Ser Ser 915 920 925 Leu His Ala Ile Lys Ala Gln Phe Ala Gly Lys His Leu Asn Lys Val 930 935 940 Asn Gln Pro Val Met Met Asp Asn Leu Lys Pro Gln Gly Ile Ser Ala 945 950 955 960 His Ala Thr Asn Glu Tyr Val Val Thr Gly Ala Ala Asn Thr Gln Ala 965 970 975 Ser Asn Ile Gln Ala Ser His Val Gln Ala Ser Ser His Ala Gln Glu 980 985 990 Ile Ala Pro Asn Gln Val Gln Asn Met Gln Ala Thr Ala Ala Ala Val 995 1000 1005 Ser Ser Pro Leu Ser Gln His Gln His Thr Ala Gln Pro Val Ala 1010 1015 1020 Ala Pro Ser Val Val Gly Val Thr Val Lys His Lys Ala Ser Asn 1025 1030 1035 Gln Ile His Gln Gln Ala Ser Thr His Lys Ala Phe Leu Glu Ser 1040 1045 1050 Arg Leu Ala Ala Gln Lys Asn Leu Ser Gln Leu Val Glu Leu Gln 1055 1060 1065 Thr Lys Leu Ser Ile Gln Thr Gly Ser Asp Asn Thr Ser Asn Asn 1070 1075 1080 Thr Ala Ser Thr Ser Asn Thr Val Leu Thr Asn Pro Val Ser Ala 1085 1090 1095 Thr Pro Leu Thr Leu Val Ser Asn Ala Pro Val Val Ala Thr Asn 1100 1105 1110 Leu Thr Ser Thr Glu Ala Lys Ala Gln Ala Ala Ala Thr Gln Ala 1115 1120 1125 Gly Phe Gln Ile Lys Gly Pro Val Gly Tyr Asn Tyr Pro Pro Leu 1130 1135 1140 Gln Leu Ile Glu Arg Tyr Asn Lys Pro Glu Asn Val Ile Tyr Asp 1145 1150 1155 Gln Ala Asp Leu Val Glu Phe Ala Glu Gly Asp Ile Gly Lys Val 1160 1165 1170 Phe Gly Ala Glu Tyr Asn Ile Ile Asp Gly Tyr Ser Arg Arg Val 1175 1180 1185 Arg Leu Pro Thr Ser Asp Tyr Leu Leu Val Thr Arg Val Thr Glu 1190 1195 1200 Leu Asp Ala Lys Val His Glu Tyr Lys Lys Ser Tyr Met Cys Thr 1205 1210 1215 Glu Tyr Asp Val Pro Val Asp Ala Pro Phe Leu Ile Asp Gly Gln 1220 1225 1230 Ile Pro Trp Ser Val Ala Val Glu Ser Gly Gln Cys Asp Leu Met 1235 1240 1245 Leu Ile Ser Tyr Ile Gly Ile Asp Phe Gln Ala Lys Gly Glu Arg 1250 1255 1260 Val Tyr Arg Leu Leu Asp Cys Glu Leu Thr Phe Leu Glu Glu Met 1265 1270 1275 Ala Phe Gly Gly Asp Thr Leu Arg Tyr Glu Ile His Ile Asp Ser 1280 1285 1290 Tyr Ala Arg Asn Gly Glu Gln Leu Leu Phe Phe Phe His Tyr Asp 1295 1300 1305 Cys Tyr Val Gly Asp Lys Lys Val Leu Ile Met Arg Asn Gly Cys 1310 1315 1320 Ala Gly Phe Phe Thr Asp Glu Glu Leu Ser Asp Gly Lys Gly Val 1325 1330 1335 Ile His Asn Asp Lys Asp Lys Ala Glu Phe Ser Asn Ala Val Lys 1340 1345 1350 Ser Ser Phe Thr Pro Leu Leu Gln His Asn Arg Gly Gln Tyr Asp 1355 1360 1365 Tyr Asn Asp Met Met Lys Leu Val Asn Gly Asp Val Ala Ser Cys 1370 1375 1380 Phe Gly Pro Gln Tyr Asp Gln Gly Gly Arg Asn Pro Ser Leu Lys 1385 1390 1395 Phe Ser Ser Glu Lys Phe Leu Met Ile Glu Arg Ile Thr Lys Ile 1400 1405 1410 Asp Pro Thr Gly Gly His Trp Gly Leu Gly Leu Leu Glu Gly Gln 1415 1420 1425 Lys Asp Leu Asp Pro Glu His Trp Tyr Phe Pro Cys His Phe Lys 1430 1435 1440 Gly Asp Gln Val Met Ala Gly Ser Leu Met Ser Glu Gly Cys Gly 1445 1450 1455 Gln Met Ala Met Phe Phe Met Leu Ser Leu Gly Met His Thr Asn 1460 1465 1470 Val Asn Asn Ala Arg Phe Gln Pro Leu Pro Gly Glu Ser Gln Thr 1475 1480 1485 Val Arg Cys Arg Gly Gln Val Leu Pro Gln Arg Asn Thr Leu Thr 1490 1495 1500 Tyr Arg Met Glu Val Thr Ala Met Gly Met His Pro Gln Pro Phe 1505 1510 1515 Met Lys Ala Asn Ile Asp Ile Leu Leu Asp Gly Lys Val Val Val 1520 1525 1530 Asp Phe Lys Asn Leu Ser Val Met Ile Ser Glu Gln Asp Glu His 1535 1540 1545 Ser Asp Tyr Pro Val Thr Leu Pro Ser Asn Val Ala Leu Lys Ala 1550 1555 1560 Ile Thr Ala Pro Val Ala Ser Val Ala Pro Ala Ser Ser Pro Ala 1565 1570 1575 Asn Ser Ala Asp Leu Asp Glu Arg Gly Val Glu Pro Phe Lys Phe 1580 1585 1590 Pro Glu Arg Pro Leu Met Arg Val Glu Ser Asp Leu Ser Ala Pro 1595 1600 1605 Lys Ser Lys Gly Val Thr Pro Ile Lys His Phe Glu Ala Pro Ala 1610 1615 1620 Val Ala Gly His His Arg Val Pro Asn Gln Ala Pro Phe Thr Pro 1625 1630 1635 Trp His Met Phe Glu Phe Ala Thr Gly Asn Ile Ser Asn Cys Phe 1640 1645 1650 Gly Pro Asp Phe Asp Val Tyr Glu Gly Arg Ile Pro Pro Arg Thr 1655 1660 1665 Pro Cys Gly Asp Leu Gln Val Val Thr Gln Val Val Glu Val Gln 1670 1675 1680 Gly Glu Arg Leu Asp Leu Lys Asn Pro Ser Ser Cys Val Ala Glu 1685 1690 1695 Tyr Tyr Val Pro Glu Asp Ala Trp Tyr Phe Thr Lys Asn Ser His 1700 1705 1710 Glu Asn Trp Met Pro Tyr Ser Leu Ile Met Glu Ile Ala Leu Gln 1715 1720 1725 Pro Asn Gly Phe Ile Ser Gly Tyr Met Gly Thr Thr Leu Lys Tyr 1730 1735 1740 Pro Glu Lys Asp Leu Phe Phe Arg Asn Leu Asp Gly Ser Gly Thr 1745 1750 1755 Leu Leu Lys Gln Ile Asp Leu Arg Gly Lys Thr Ile Val Asn Lys 1760 1765 1770 Ser Val Leu Val Ser Thr Ala Ile Ala Gly Gly Ala Ile Ile Gln 1775 1780 1785 Ser Phe Thr Phe Asp Met Ser Val Asp Gly Glu Leu Phe Tyr Thr 1790 1795 1800 Gly Lys Ala Val Phe Gly Tyr Phe Ser Gly Glu Ser Leu Thr Asn 1805 1810 1815 Gln Leu Gly Ile Asp Asn Gly Lys Thr Thr Asn Ala Trp Phe Val 1820 1825 1830 Asp Asn Asn Thr Pro Ala Ala Asn Ile Asp Val Phe Asp Leu Thr 1835 1840 1845 Asn Gln Ser Leu Ala Leu Tyr Lys Ala Pro Val Asp Lys Pro His 1850 1855 1860 Tyr Lys Leu Ala Gly Gly Gln Met Asn Phe Ile Asp Thr Val Ser 1865 1870 1875 Val Val Glu Gly Gly Gly Lys Ala Gly Val Ala Tyr Val Tyr Gly 1880 1885 1890 Glu Arg Thr Ile Asp Ala Asp Asp Trp Phe Phe Arg Tyr His Phe 1895 1900 1905 His Gln Asp Pro Val Met Pro Gly Ser Leu Gly Val Glu Ala Ile 1910 1915 1920 Ile Glu Leu Met Gln Thr Tyr Ala Leu Lys Asn Asp Leu Gly Gly 1925 1930 1935 Lys Phe Ala Asn Pro Arg Phe Ile Ala Pro Met Thr Gln Val Asp 1940 1945 1950 Trp Lys Tyr Arg Gly Gln Ile Thr Pro Leu Asn Lys Gln Met Ser 1955 1960 1965 Leu Asp Val His Ile Thr Glu Ile Val Asn Asp Ala Gly Glu Val 1970 1975 1980 Arg Ile Val Gly Asp Ala Asn Leu Ser Lys Asp Gly Leu Arg Ile 1985 1990 1995 Tyr Glu Val Lys Asn Ile Val Leu Ser Ile Val Glu Ala 2000 2005 2010 <210> 30 <211> 2241 <212> PRT <213> Aureispira marina <400> 30 Met Lys Ile Ala Ile Ile Gly Leu Ser Gly Leu Phe Pro Gly Ser Ser 1 5 10 15 Thr Asn Glu Glu Phe Trp Gln Asn Leu Leu Asp Glu Lys Asp Leu Thr 20 25 30 Asn Leu Ala Asn Leu Glu Asp Phe Gly Ala Asp Pro Ala Leu Phe Tyr 35 40 45 Glu Asp Lys Lys Gly Ala Val Asp Arg Cys Tyr Ser Leu Arg Gly Gly 50 55 60 Tyr Ile Arg Asp Phe Asp Phe Asp Pro Thr Gly Tyr Gln Leu Ser Ala 65 70 75 80 Asp Phe Leu Ala Gln Gln Asp Lys Leu Tyr Gln Trp Ser Leu Tyr Val 85 90 95 Ala Lys Thr Ala Leu Glu Glu Ser Gly Tyr Ala His Asn Lys Glu Val 100 105 110 Leu Ala Lys Cys Gly Leu Ile Leu Gly Asn Leu Ser Phe Pro Thr Gly 115 120 125 Ser Ser His Lys Leu Leu Ala Asp Leu Tyr Thr Lys Thr Thr Glu Lys 130 135 140 Ala Leu Gln Glu Leu Leu Glu Asp Lys Asn Phe Lys Ile Pro Ala Ser 145 150 155 160 Gln Leu Pro Ile Pro Asn Asn Glu Val Leu Ala Asp Thr Pro Ser Gln 165 170 175 Met Val Ala Lys Gly Leu Gly Leu Gly Gly Gly His Tyr Ala Leu Asp 180 185 190 Ala Ala Cys Ala Thr Ser Leu Tyr Ala Ile Lys Leu Ala Cys Asp Glu 195 200 205 Leu Ile Thr Gly Lys Ala Asp Leu Met Leu Ala Gly Ala Val Cys Gly 210 215 220 Ser Asp Gln Leu Phe Ile His Met Gly Phe Ser Ile Phe His Ala Tyr 225 230 235 240 Ala Pro His Gly Glu Lys Phe Ala Pro Leu Asp Lys Ala Ser Gly Gly 245 250 255 Leu Val Ser Ala Glu Gly Ala Gly Met Val Val Leu Lys Arg Leu Glu 260 265 270 Asp Ala Glu Arg Asp Gly Asp Asn Ile Leu Gly Leu Ile Gly Gly Ile 275 280 285 Gly Leu Ser Asn Asp Gly Ser Gly Lys Phe Leu Leu Ser Pro Asn Pro 290 295 300 Lys Gly Gln Arg Leu Ala Phe Glu Arg Ala Tyr Asp Leu Glu Glu Val 305 310 315 320 Leu Pro Gln Asn Thr Ser Tyr Leu Glu Cys His Ala Thr Gly Thr Pro 325 330 335 Leu Gly Asp Val Thr Glu Met Asn Ser Ile Ser Asp Phe Phe Ala Gln 340 345 350 His Gln Thr Lys Pro Leu Leu Gly Ser Val Lys Ser Asn Met Gly His 355 360 365 Leu Leu Thr Ala Ala Gly Met Ser Gly Leu Phe Lys Val Leu Leu Ser 370 375 380 Met Gln Lys Gly Ile Ile Pro Pro Asn Ile Asn Leu Glu Ser Ala Val 385 390 395 400 Gln Ala Asn Asn Gln Trp Ile Gln Asp Glu Gln Ile Ile Lys Lys Thr 405 410 415 Thr Pro Trp Lys Gly Asp Gln Ala Gly Ile Asn Ser Phe Gly Phe Gly 420 425 430 Gly Thr Asn Ala His Met Val Val Gln Lys Pro Thr Ser Ser Thr Leu 435 440 445 Lys Glu Lys Lys Ala Tyr Gln Ala Gln Glu Leu Leu Pro Leu Ala Ile 450 455 460 Val Gly Met Asp Ala His Phe Gly Ser Cys Glu Asn Leu Glu Asp Phe 465 470 475 480 Tyr Ala Ala Ile Tyr Asn Gly Asn Gln Asp Phe Lys Pro Leu Pro Pro 485 490 495 Lys Arg Trp Lys Gly Phe Asp Ala Asp Gln Asp Leu Leu Lys Arg Tyr 500 505 510 Gly Phe Lys Asp Gly Leu Ala Pro Lys Gly Ala Tyr Ile Asp Gln Phe 515 520 525 Asp Ile Asp Leu Leu Arg Tyr Lys Ile Gln Pro Lys Glu Ala Glu Thr 530 535 540 Leu Glu Pro Gln Gln Ala Leu Ile Leu Lys Val Ala Asp Lys Ala Leu 545 550 555 560 Gln Asp Ala Gln Ile Ser Pro Ser Gln Asn Ile Ala Val Leu Ile Ala 565 570 575 Met Glu Ser Glu Leu Ala Ile His His Tyr Leu Ala Arg Trp Asp Ser 580 585 590 Val Trp Gln Leu Asp Lys Ala Leu Glu Gln Ser Gly Leu Ser Leu Ser 595 600 605 Glu Glu Lys Lys Thr Ala Leu Lys Glu Tyr Ser Lys Asn Ala Leu Tyr 610 615 620 Phe Arg Glu Gly Ser Gln Thr Pro Ser Gln His Thr Ser Phe Val Gly 625 630 635 640 Asn Ile Met Ala Ser Arg Ile Ala Ala Leu Trp Asp Phe Ser Gly Pro 645 650 655 Ala Phe Thr Val Ser Cys Gly Asp Asn Ala Val Phe Lys Ala Leu Glu 660 665 670 Val Ala Gln Asn Ile Leu Ser Leu Gly Glu Val Asp Ala Val Val Val 675 680 685 Gly Gly Val Asp Phe Cys Gly Gly Leu Glu Asn Val Leu Leu Arg Gln 690 695 700 Glu Lys Glu Ala Ser Ser Gln Asn Ile Ala Pro Ser Leu Ser Leu Asn 705 710 715 720 Gln Gly Gln Lys Gly Trp Leu Val Gly Glu Gly Ala Gly Ala Val Val 725 730 735 Leu Lys Arg Gln Ile Asp Leu Gln Lys Gln Asp Asn Val Tyr Ala Val 740 745 750 Leu Glu His Ile Gly Gln Ala Ser Glu Gln Leu Asn Val Gly Tyr Gln 755 760 765 Glu Leu Val Ser Ser Gly Tyr Ala Ala Gln Asp His Gln Glu Leu Lys 770 775 780 Gln Leu Leu Ala Thr Gln Leu Glu Gln Lys Thr Ala Leu Gly Ser Val 785 790 795 800 Lys Thr Ser Phe Gly His Thr Gly Ala Ala Ser Gly Ile Ala Ala Leu 805 810 815 Ile Lys Thr Ala Leu Cys Leu His His Lys Phe Ile Pro Gly Ile Pro 820 825 830 Asn Trp Glu Ala Pro Gln Glu Ala Thr Ala Phe Ala Lys Thr Lys Tyr 835 840 845 Tyr Phe Pro Met Ala Ser Arg Pro Trp Leu Leu Asn Ala Gly Glu Lys 850 855 860 Arg Lys Ala Ala Ile Asn Gly Leu Glu Gly Leu Gln Ile His Leu Ser 865 870 875 880 Glu Gly Val Arg Ser Ser Pro Ala Pro Ser Pro Leu Leu Gln Gly Arg 885 890 895 Val Gly Ser Leu Phe Val Leu Lys Gly Asn Thr Glu Thr Ala Leu Arg 900 905 910 Glu Ala Leu Ala Leu Leu Leu Glu Asp Leu Ala Gly Lys Ser Ser Leu 915 920 925 Pro Glu Leu Ala Ala Arg Leu Tyr Tyr Asn His Gln Ala Lys Pro Ser 930 935 940 Ser Tyr Thr Ile Val Leu Leu Ala Asn Ser Lys Lys Asn Leu Gln Gln 945 950 955 960 Glu Ile Arg Phe Met Gln Val Gly Leu Glu Ala Ala Leu Thr Glu Asn 965 970 975 Lys Val Leu Lys Thr Pro Arg Gly Ser Tyr Phe Thr Ala Lys Pro Leu 980 985 990 Gly Lys Thr Gly Lys Ile Ala Phe Ser Tyr Pro Gly Ser Ala Thr Ala 995 1000 1005 Tyr Arg Gly Leu Gly Gln Asp Ile Phe Gln Leu Phe Pro Ser Leu 1010 1015 1020 His Glu His Phe Gly Gln Lys Leu Glu Asp Ile Ala Asp Phe Val 1025 1030 1035 Gly Ser Ser Tyr Leu His Pro Lys Leu Gln Ser Arg Gln Glu Glu 1040 1045 1050 Ala Pro Ser Ile Gln Thr Asp Ala Val Ser Met Met Cys Ala Gly 1055 1060 1065 Val Phe Ser Ser Ala Ile Tyr Thr His Leu Leu Lys Asp Lys Phe 1070 1075 1080 Gly Leu Lys Pro Asp Leu Ala Phe Gly Tyr Ser Met Gly Glu Ser 1085 1090 1095 Ala Gly Met Trp Tyr Ser Phe Asp Val Trp Asn Pro Asp Asn Thr 1100 1105 1110 Ala Val Phe Arg Asn Ser Asp Leu Phe Ala Asn Gln Leu Ser Gly 1115 1120 1125 Asp Leu Arg Leu Leu Ala Glu Thr Trp Gly Ile Ser Ser Glu Glu 1130 1135 1140 Ala Lys Ala Arg Trp Ile Ser Leu Ile Leu Leu Ala Asp Lys Glu 1145 1150 1155 Ala Val Gln Asn Leu Val Ala Gln Glu Asp Arg Cys Tyr Leu Ser 1160 1165 1170 Phe Ile Asn Thr Pro Gln Glu Val Ile Ile Ser Gly Asp Lys Glu 1175 1180 1185 Ala Cys Asn Arg Val Val Gln Gln Leu Gly Cys Pro Ala Val Glu 1190 1195 1200 Val Pro Phe Gln Asn Val Ile His His Asp Phe Cys Lys Lys Val 1205 1210 1215 Gln Glu Glu Leu Tyr Asp Met His His Phe Pro Leu Glu Thr Gln 1220 1225 1230 Pro Asn Ile Asp Phe Tyr Ser Ser Leu Ser Leu Ala Pro Leu Pro 1235 1240 1245 Met Asp Ser Gly Val Ile Ala Gln Asn Ser Thr Gln Val Cys Phe 1250 1255 1260 Gln Pro Val Asp Tyr Pro Thr Thr Ile Gln Gln Leu Tyr Asn Asp 1265 1270 1275 Gly Ala Arg Ile Phe Ile Glu Leu Gly Ala Gly Asn Thr Cys Thr 1280 1285 1290 Gln Trp Thr Ser Ser Ile Leu Gly Gln Gln Ala His Leu Ala Val 1295 1300 1305 Ser Cys Thr Gln Lys Gly Lys Pro Glu Gly Thr Ala Leu Leu Gln 1310 1315 1320 Ala Leu Ala Gln Leu Leu Ser His Gly Val Ala Leu Asp Leu Gln 1325 1330 1335 Pro Leu Phe Ala Ala Asp Leu Leu Ala Pro Ser Pro Arg Ala Phe 1340 1345 1350 Tyr Lys Ala Ile Val Ser Gly Gly Ala Arg Ile Phe Asp Tyr Leu 1355 1360 1365 Leu Gln Pro Gln Thr Lys Lys Gln Phe Ala Gly Val Thr Lys Thr 1370 1375 1380 Ala Leu Val Gln Gln Leu Glu Pro Ala Leu Ala Ser Asn Ser Arg 1385 1390 1395 Glu Tyr Ser Phe Thr Ser Thr Lys Thr Thr Thr Val Asp Thr Thr 1400 1405 1410 Gln Ser Ile Pro Ser Pro Ser Gln Lys Val Leu Leu Gly Glu Asn 1415 1420 1425 Gly Leu Lys Leu Gln Asp Phe Asn Asp Pro Asn His Leu Gln Gly 1430 1435 1440 Lys Thr Ile Ile Phe Ser Gln Glu Asp Leu Glu Glu Phe Ala Thr 1445 1450 1455 Gly Lys Ile Ala Lys Val Phe Gly Glu Glu Tyr Ser Ile Ile Asp 1460 1465 1470 Thr Tyr Lys Arg Arg Val Met Leu Pro Met Ala Pro Tyr Leu Leu 1475 1480 1485 Val Ser Arg Val Thr Gly Leu Asp Ala Lys Arg Gly Glu Phe Lys 1490 1495 1500 Pro Ser Thr Met Gln Thr Glu Tyr Asp Ile Pro Tyr Asn Ala Trp 1505 1510 1515 Phe Thr Thr Asp Gly Gln Ile Pro Trp Ala Val Ser Val Glu Ser 1520 1525 1530 Gly Gln Cys Asp Leu Leu Leu Ile Ser Tyr Leu Gly Ile Asp Phe 1535 1540 1545 Glu Asn Lys Gly Asp Leu Val Tyr Arg Leu Leu Asp Cys Thr Leu 1550 1555 1560 Thr Phe Val Asp Asp Leu Pro Phe Glu Gly Gln Thr Leu Arg Tyr 1565 1570 1575 Asp Ile Ser Ile Asn Ser Phe Val Arg Asn Gly Asp Asn Leu Leu 1580 1585 1590 Phe Phe Phe Ser Tyr Asn Cys Tyr Val Glu Asp Arg Leu Val Leu 1595 1600 1605 Lys Met Arg Asn Gly Cys Ala Gly Phe Phe Thr Asp Glu Gln Leu 1610 1615 1620 Glu Glu Gly Leu Gly Val Val Tyr Ser Lys Glu Glu Leu Glu Ala 1625 1630 1635 Lys Thr Asn Ala Lys Lys Pro Ala Phe Thr Pro Leu Leu Asn Thr 1640 1645 1650 Lys Lys Thr Ser Phe Ser Lys Glu Asp Leu His His Leu Ile Glu 1655 1660 1665 Gly Asn Met Glu Leu Cys Phe Asp Ser Pro Ala Tyr Phe Ala Asn 1670 1675 1680 Gly Arg Asn Pro Ser Leu Arg Leu Pro Pro Glu Gln Ile Leu Met 1685 1690 1695 Ile Asp Arg Ile Val Ser Val Asp Leu Lys Gly Gly Ala Tyr Gly 1700 1705 1710 Leu Gly Tyr Val Ile Ala Glu Lys Asp Leu Ala Pro Glu Asp Trp 1715 1720 1725 Tyr Phe Pro Cys His Phe Arg Asp Asp Glu Val Leu Ala Gly Ser 1730 1735 1740 Leu Gln Ala Glu Gly Gly Gly Asn Leu Leu Arg Phe Phe Met Leu 1745 1750 1755 Met Leu Gly Leu Gln Arg Leu Thr Lys Asp Ala Arg Tyr Gln Pro 1760 1765 1770 Ile Phe Asp Leu Pro Gln Lys Val Arg Cys Arg Lys Gln Val Thr 1775 1780 1785 Pro Ser Lys Asp Thr Lys Leu Val Tyr Lys Leu Glu Val Lys Glu 1790 1795 1800 Ile Gly Leu Val Pro Asn Pro Tyr Val Ile Ala Asp Leu Glu Ile 1805 1810 1815 Val Ser Asp Gly Val Ile Thr Val His Phe Glu Asn Leu Gly Leu 1820 1825 1830 Gln Leu Arg Glu Lys Asp Asn Pro Arg Tyr Leu Glu Gln Gln Lys 1835 1840 1845 Gly Val His Ile Ser Pro Arg Ser Lys Asp Ala Leu Leu Thr Glu 1850 1855 1860 Leu Asp Ile Thr Asn Phe Ala Leu Asn Asn Leu Ser Val Ala Phe 1865 1870 1875 Gly Pro Asp Phe Ala Cys Tyr Asp Gly Arg Thr Val Ser Arg Gln 1880 1885 1890 Pro Asn Thr Asp Leu Gln Leu Ile Ser Arg Val Leu Lys Ile Glu 1895 1900 1905 Gly Glu Arg Leu Asn Phe Lys Gln Pro Ser Thr Ile Tyr Ala Glu 1910 1915 1920 Tyr Asp Val Pro Glu Asp Ala Trp Tyr Tyr Gln Gln Asn Ala Ser 1925 1930 1935 Met Thr Met Pro Tyr Ser Val Leu Met Glu Ile Ala Leu Gln Pro 1940 1945 1950 Cys Gly Leu Leu Gly Ala Tyr Leu Gly Ser Thr Leu Pro Phe Ser 1955 1960 1965 Asp Lys Asn Leu Phe Phe Arg Asn Leu Asp Gly Thr Gly Glu Met 1970 1975 1980 Leu Glu Leu Pro Met Gly Thr Asp Trp Arg Gly Lys Thr Ile His 1985 1990 1995 Asn Lys Ala Val Leu Ala Ser Ser Val Ala Leu Gly Gly Thr Val 2000 2005 2010 Leu Gln Asn Tyr Thr Phe Glu Leu Ser Ile Asp Gly Gln Val Phe 2015 2020 2025 Tyr Lys Gly Lys Ser Ser Phe Gly Phe Phe Pro Ala Glu Ala Leu 2030 2035 2040 Ala Gln Gln Val Gly Leu Asp Asn Gly Thr Ala Val Ala Pro Trp 2045 2050 2055 Tyr Gln Gln Gln Asn Leu Ala Gln Lys Asp Tyr Met Ser Ile Lys 2060 2065 2070 Leu Asp Ser Leu Tyr Gly Lys Met Lys Leu Phe Lys Ala Pro Ala 2075 2080 2085 Asn Lys Pro His Tyr His Leu Ser Gly Glu Gln Leu Ser Leu Leu 2090 2095 2100 Asn Asn Leu Lys Ile Val Lys Asp Gly Gly Gln Tyr Gly Lys Gly 2105 2110 2115 Tyr Ile Tyr Gly His Gln Ala Ile Asn Leu Tyr Asp Trp Phe Phe 2120 2125 2130 Thr Cys His Phe Tyr Gln Asp Pro Val Met Pro Gly Ser Leu Gly 2135 2140 2145 Val Glu Ala Ile Leu Gln Ala Met Gln Thr Phe Ala Leu Gln Gln 2150 2155 2160 Asp Leu Gly Lys Asp Phe Lys Ser Pro Arg Phe Val Gln Val Pro 2165 2170 2175 Gln His Thr Thr Val Trp Lys Tyr Arg Gly Gln Ile Leu Gln Gly 2180 2185 2190 Val Glu Asn Met His Cys Glu Val His Phe Lys Ser Ile Glu Lys 2195 2200 2205 Lys Gly Glu Gln Leu Val Ile Val Gly Asp Ala Tyr Leu Trp Asn 2210 2215 2220 Glu Asp Thr Arg Ile Tyr Gln Ile Thr Asp Leu Ala Leu Gly Ile 2225 2230 2235 Glu Glu Ala 2240 <210> 31 <211> 2059 <212> PRT <213> Schizochytrium sp <400> 31 Met Ala Ala Arg Asn Val Ser Ala Ala His Glu Met His Asp Glu Lys 1 5 10 15 Arg Ile Ala Val Val Gly Met Ala Val Gln Tyr Ala Gly Cys Lys Thr 20 25 30 Lys Asp Glu Phe Trp Glu Val Leu Met Asn Gly Lys Val Glu Ser Lys 35 40 45 Val Ile Ser Asp Lys Arg Leu Gly Ser Asn Tyr Arg Ala Glu His Tyr 50 55 60 Lys Ala Glu Arg Ser Lys Tyr Ala Asp Thr Phe Cys Asn Glu Thr Tyr 65 70 75 80 Gly Thr Leu Asp Glu Asn Glu Ile Asp Asn Glu His Glu Leu Leu Leu 85 90 95 Asn Leu Ala Lys Gln Ala Leu Ala Glu Thr Ser Val Lys Asp Ser Thr 100 105 110 Arg Cys Gly Ile Val Ser Gly Cys Leu Ser Phe Pro Met Asp Asn Leu 115 120 125 Gln Gly Glu Leu Leu Asn Val Tyr Gln Asn His Val Glu Lys Lys Leu 130 135 140 Gly Ala Arg Val Phe Lys Asp Ala Ser His Trp Ser Glu Arg Glu Gln 145 150 155 160 Ser Asn Lys Pro Glu Ala Gly Asp Arg Arg Ile Phe Met Asp Pro Ala 165 170 175 Ser Phe Val Ala Glu Glu Leu Asn Leu Gly Ala Leu His Tyr Ser Val 180 185 190 Asp Ala Ala Cys Ala Thr Ala Leu Tyr Val Leu Arg Leu Ala Gln Asp 195 200 205 His Leu Val Ser Gly Ala Ala Asp Val Met Leu Cys Gly Ala Thr Cys 210 215 220 Leu Pro Glu Pro Phe Phe Ile Leu Ser Gly Phe Ser Thr Phe Gln Ala 225 230 235 240 Met Pro Val Gly Thr Gly Gln Asn Val Ser Met Pro Leu His Lys Asp 245 250 255 Ser Gln Gly Leu Thr Pro Gly Glu Gly Gly Ser Ile Met Val Leu Lys 260 265 270 Arg Leu Asp Asp Ala Ile Arg Asp Gly Asp His Ile Tyr Gly Thr Leu 275 280 285 Leu Gly Ala Asn Val Ser Asn Ser Gly Thr Gly Leu Pro Leu Lys Pro 290 295 300 Leu Leu Pro Ser Glu Lys Lys Cys Leu Met Asp Thr Tyr Thr Arg Ile 305 310 315 320 Asn Val His Pro His Lys Ile Gln Tyr Val Glu Cys His Ala Thr Gly 325 330 335 Thr Pro Gln Gly Asp Arg Val Glu Ile Asp Ala Val Lys Ala Cys Phe 340 345 350 Glu Gly Lys Val Pro Arg Phe Gly Thr Thr Lys Gly Asn Phe Gly His 355 360 365 Thr Leu Val Ala Ala Gly Phe Ala Gly Met Cys Lys Val Leu Leu Ser 370 375 380 Met Lys His Gly Ile Ile Pro Pro Thr Pro Gly Ile Asp Asp Glu Thr 385 390 395 400 Lys Met Asp Pro Leu Val Val Ser Gly Glu Ala Ile Pro Trp Pro Glu 405 410 415 Thr Asn Gly Glu Pro Lys Arg Ala Gly Leu Ser Ala Phe Gly Phe Gly 420 425 430 Gly Thr Asn Ala His Ala Val Phe Glu Glu His Asp Pro Ser Asn Ala 435 440 445 Ala Cys Thr Gly His Asp Ser Ile Ser Ala Leu Ser Ala Arg Cys Gly 450 455 460 Gly Glu Ser Asn Met Arg Ile Ala Ile Thr Gly Met Asp Ala Thr Phe 465 470 475 480 Gly Ala Leu Lys Gly Leu Asp Ala Phe Glu Arg Ala Ile Tyr Thr Gly 485 490 495 Ala His Gly Ala Ile Pro Leu Pro Glu Lys Arg Trp Arg Phe Leu Gly 500 505 510 Lys Asp Lys Asp Phe Leu Asp Leu Cys Gly Val Lys Ala Thr Pro His 515 520 525 Gly Cys Tyr Ile Glu Asp Val Glu Val Asp Phe Gln Arg Leu Arg Thr 530 535 540 Pro Met Thr Pro Glu Asp Met Leu Leu Pro Gln Gln Leu Leu Ala Val 545 550 555 560 Thr Thr Ile Asp Arg Ala Ile Leu Asp Ser Gly Met Lys Lys Gly Gly 565 570 575 Asn Val Ala Val Phe Val Gly Leu Gly Thr Asp Leu Glu Leu Tyr Arg 580 585 590 His Arg Ala Arg Val Ala Leu Lys Glu Arg Val Arg Pro Glu Ala Ser 595 600 605 Lys Lys Leu Asn Asp Met Met Gln Tyr Ile Asn Asp Cys Gly Thr Ser 610 615 620 Thr Ser Tyr Thr Ser Tyr Ile Gly Asn Leu Val Ala Thr Arg Val Ser 625 630 635 640 Ser Gln Trp Gly Phe Thr Gly Pro Ser Phe Thr Ile Thr Glu Gly Asn 645 650 655 Asn Ser Val Tyr Arg Cys Ala Glu Leu Gly Lys Tyr Leu Leu Glu Thr 660 665 670 Gly Glu Val Asp Gly Val Val Val Ala Gly Val Asp Leu Cys Gly Ser 675 680 685 Ala Glu Asn Leu Tyr Val Lys Ser Arg Arg Phe Lys Val Ser Thr Ser 690 695 700 Asp Thr Pro Arg Ala Ser Phe Asp Ala Ala Ala Asp Gly Tyr Phe Val 705 710 715 720 Gly Glu Gly Cys Gly Ala Phe Val Leu Lys Arg Glu Thr Ser Cys Thr 725 730 735 Lys Asp Asp Arg Ile Tyr Ala Cys Met Asp Ala Ile Val Pro Gly Asn 740 745 750 Val Pro Ser Ala Cys Leu Arg Glu Ala Leu Asp Gln Ala Arg Val Lys 755 760 765 Pro Gly Asp Ile Glu Met Leu Glu Leu Ser Ala Asp Ser Ala Arg His 770 775 780 Leu Lys Asp Pro Ser Val Leu Pro Lys Glu Leu Thr Ala Glu Glu Glu 785 790 795 800 Ile Gly Gly Leu Gln Thr Ile Leu Arg Asp Asp Asp Lys Leu Pro Arg 805 810 815 Asn Val Ala Thr Gly Ser Val Lys Ala Thr Val Gly Asp Thr Gly Tyr 820 825 830 Ala Ser Gly Ala Ala Ser Leu Ile Lys Ala Ala Leu Cys Ile Tyr Asn 835 840 845 Arg Tyr Leu Pro Ser Asn Gly Asp Asp Trp Asp Glu Pro Ala Pro Glu 850 855 860 Ala Pro Trp Asp Ser Thr Leu Phe Ala Cys Gln Thr Ser Arg Ala Trp 865 870 875 880 Leu Lys Asn Pro Gly Glu Arg Arg Tyr Ala Ala Val Ser Gly Val Ser 885 890 895 Glu Thr Arg Ser Cys Tyr Ser Val Leu Leu Ser Glu Ala Glu Gly His 900 905 910 Tyr Glu Arg Glu Asn Arg Ile Ser Leu Asp Glu Glu Ala Pro Lys Leu 915 920 925 Ile Val Leu Arg Ala Asp Ser His Glu Glu Ile Leu Gly Arg Leu Asp 930 935 940 Lys Ile Arg Glu Arg Phe Leu Gln Pro Thr Gly Ala Ala Pro Arg Glu 945 950 955 960 Ser Glu Leu Lys Ala Gln Ala Arg Arg Ile Phe Leu Glu Leu Leu Gly 965 970 975 Glu Thr Leu Ala Gln Asp Ala Ala Ser Ser Gly Ser Gln Lys Pro Leu 980 985 990 Ala Leu Ser Leu Val Ser Thr Pro Ser Lys Leu Gln Arg Glu Val Glu 995 1000 1005 Leu Ala Ala Lys Gly Ile Pro Arg Cys Leu Lys Met Arg Arg Asp 1010 1015 1020 Trp Ser Ser Pro Ala Gly Ser Arg Tyr Ala Pro Glu Pro Leu Ala 1025 1030 1035 Ser Asp Arg Val Ala Phe Met Tyr Gly Glu Gly Arg Ser Pro Tyr 1040 1045 1050 Tyr Gly Ile Thr Gln Asp Ile His Arg Ile Trp Pro Glu Leu His 1055 1060 1065 Glu Val Ile Asn Glu Lys Thr Asn Arg Leu Trp Ala Glu Gly Asp 1070 1075 1080 Arg Trp Val Met Pro Arg Ala Ser Phe Lys Ser Glu Leu Glu Ser 1085 1090 1095 Gln Gln Gln Glu Phe Asp Arg Asn Met Ile Glu Met Phe Arg Leu 1100 1105 1110 Gly Ile Leu Thr Ser Ile Ala Phe Thr Asn Leu Ala Arg Asp Val 1115 1120 1125 Leu Asn Ile Thr Pro Lys Ala Ala Phe Gly Leu Ser Leu Gly Glu 1130 1135 1140 Ile Ser Met Ile Phe Ala Phe Ser Lys Lys Asn Gly Leu Ile Ser 1145 1150 1155 Asp Gln Leu Thr Lys Asp Leu Arg Glu Ser Asp Val Trp Asn Lys 1160 1165 1170 Ala Leu Ala Val Glu Phe Asn Ala Leu Arg Glu Ala Trp Gly Ile 1175 1180 1185 Pro Gln Ser Val Pro Lys Asp Glu Phe Trp Gln Gly Tyr Ile Val 1190 1195 1200 Arg Gly Thr Lys Gln Asp Ile Glu Ala Ala Ile Ala Pro Asp Ser 1205 1210 1215 Lys Tyr Val Arg Leu Thr Ile Ile Asn Asp Ala Asn Thr Ala Leu 1220 1225 1230 Ile Ser Gly Lys Pro Asp Ala Cys Lys Ala Ala Ile Ala Arg Leu 1235 1240 1245 Gly Gly Asn Ile Pro Ala Leu Pro Val Thr Gln Gly Met Cys Gly 1250 1255 1260 His Cys Pro Glu Val Gly Pro Tyr Thr Lys Asp Ile Ala Lys Ile 1265 1270 1275 His Ala Asn Leu Glu Phe Pro Val Val Asp Gly Leu Asp Leu Trp 1280 1285 1290 Thr Thr Ile Asn Gln Lys Arg Leu Val Pro Arg Ala Thr Gly Ala 1295 1300 1305 Lys Asp Glu Trp Ala Pro Ser Ser Phe Gly Glu Tyr Ala Gly Gln 1310 1315 1320 Leu Tyr Glu Lys Gln Ala Asn Phe Pro Gln Ile Val Glu Thr Ile 1325 1330 1335 Tyr Lys Gln Asn Tyr Asp Val Phe Val Glu Val Gly Pro Asn Asn 1340 1345 1350 His Arg Ser Thr Ala Val Arg Thr Thr Leu Gly Pro Gln Arg Asn 1355 1360 1365 His Leu Ala Gly Ala Ile Asp Lys Gln Asn Glu Asp Ala Trp Thr 1370 1375 1380 Thr Ile Val Lys Leu Val Ala Ser Leu Lys Ala His Leu Val Pro 1385 1390 1395 Gly Val Thr Ile Ser Pro Leu Tyr His Ser Lys Leu Val Ala Glu 1400 1405 1410 Ala Glu Ala Cys Tyr Ala Ala Leu Cys Lys Gly Glu Lys Pro Lys 1415 1420 1425 Lys Asn Lys Phe Val Arg Lys Ile Gln Leu Asn Gly Arg Phe Asn 1430 1435 1440 Ser Lys Ala Asp Pro Ile Ser Ser Ala Asp Leu Ala Ser Phe Pro 1445 1450 1455 Pro Ala Asp Pro Ala Ile Glu Ala Ala Ile Ser Ser Arg Ile Met 1460 1465 1470 Lys Pro Val Ala Pro Lys Phe Tyr Ala Arg Leu Asn Ile Asp Glu 1475 1480 1485 Gln Asp Glu Thr Arg Asp Pro Ile Leu Asn Lys Asp Asn Ala Pro 1490 1495 1500 Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 1505 1510 1515 Pro Ser Pro Ala Pro Ser Ala Pro Val Gln Lys Lys Ala Ala Pro 1520 1525 1530 Ala Ala Glu Thr Lys Ala Val Ala Ser Ala Asp Ala Leu Arg Ser 1535 1540 1545 Ala Leu Leu Asp Leu Asp Ser Met Leu Ala Leu Ser Ser Ala Ser 1550 1555 1560 Ala Ser Gly Asn Leu Val Glu Thr Ala Pro Ser Asp Ala Ser Val 1565 1570 1575 Ile Val Pro Pro Cys Asn Ile Ala Asp Leu Gly Ser Arg Ala Phe 1580 1585 1590 Met Lys Thr Tyr Gly Val Ser Ala Pro Leu Tyr Thr Gly Ala Met 1595 1600 1605 Ala Lys Gly Ile Ala Ser Ala Asp Leu Val Ile Ala Ala Gly Arg 1610 1615 1620 Gln Gly Ile Leu Ala Ser Phe Gly Ala Gly Gly Leu Pro Met Gln 1625 1630 1635 Val Val Arg Glu Ser Ile Glu Lys Ile Gln Ala Ala Leu Pro Asn 1640 1645 1650 Gly Pro Tyr Ala Val Asn Leu Ile His Ser Pro Phe Asp Ser Asn 1655 1660 1665 Leu Glu Lys Gly Asn Val Asp Leu Phe Leu Glu Lys Gly Val Thr 1670 1675 1680 Phe Val Glu Ala Ser Ala Phe Met Thr Leu Thr Pro Gln Val Val 1685 1690 1695 Arg Tyr Arg Ala Ala Gly Leu Thr Arg Asn Ala Asp Gly Ser Val 1700 1705 1710 Asn Ile Arg Asn Arg Ile Ile Gly Lys Val Ser Arg Thr Glu Leu 1715 1720 1725 Ala Glu Met Phe Met Arg Pro Ala Pro Glu His Leu Leu Gln Lys 1730 1735 1740 Leu Ile Ala Ser Gly Glu Ile Asn Gln Glu Gln Ala Glu Leu Ala 1745 1750 1755 Arg Arg Val Pro Val Ala Asp Asp Ile Ala Val Glu Ala Asp Ser 1760 1765 1770 Gly Gly His Thr Asp Asn Arg Pro Ile His Val Ile Leu Pro Leu 1775 1780 1785 Ile Ile Asn Leu Arg Asp Arg Leu His Arg Glu Cys Gly Tyr Pro 1790 1795 1800 Ala Asn Leu Arg Val Arg Val Gly Ala Gly Gly Gly Ile Gly Cys 1805 1810 1815 Pro Gln Ala Ala Leu Ala Thr Phe Asn Met Gly Ala Ser Phe Ile 1820 1825 1830 Val Thr Gly Thr Val Asn Gln Val Ala Lys Gln Ser Gly Thr Cys 1835 1840 1845 Asp Asn Val Arg Lys Gln Leu Ala Lys Ala Thr Tyr Ser Asp Val 1850 1855 1860 Cys Met Ala Pro Ala Ala Asp Met Phe Glu Glu Gly Val Lys Leu 1865 1870 1875 Gln Val Leu Lys Lys Gly Thr Met Phe Pro Ser Arg Ala Asn Lys 1880 1885 1890 Leu Tyr Glu Leu Phe Cys Lys Tyr Asp Ser Phe Glu Ser Met Pro 1895 1900 1905 Pro Ala Glu Leu Ala Arg Val Glu Lys Arg Ile Phe Ser Arg Ala 1910 1915 1920 Leu Glu Glu Val Trp Asp Glu Thr Lys Asn Phe Tyr Ile Asn Arg 1925 1930 1935 Leu His Asn Pro Glu Lys Ile Gln Arg Ala Glu Arg Asp Pro Lys 1940 1945 1950 Leu Lys Met Ser Leu Cys Phe Arg Trp Tyr Leu Ser Leu Ala Ser 1955 1960 1965 Arg Trp Ala Asn Thr Gly Ala Ser Asp Arg Val Met Asp Tyr Gln 1970 1975 1980 Val Trp Cys Gly Pro Ala Ile Gly Ser Phe Asn Asp Phe Ile Lys 1985 1990 1995 Gly Thr Tyr Leu Asp Pro Ala Val Ala Asn Glu Tyr Pro Cys Val 2000 2005 2010 Val Gln Ile Asn Lys Gln Ile Leu Arg Gly Ala Cys Phe Leu Arg 2015 2020 2025 Arg Leu Glu Ile Leu Arg Asn Ala Arg Leu Ser Asp Gly Ala Ala 2030 2035 2040 Ala Leu Val Ala Ser Ile Asp Asp Thr Tyr Val Pro Ala Glu Lys 2045 2050 2055 Lion

Claims

1. A microorganism having the ability to produce docosahexaenoic acid (DHA), comprising a mutant OrfB and capable of producing eicosapentaenoic acid (EPA), wherein the mutant OrfB is any one of the proteins selected from the following [1] to [5]: [1] A protein consisting of an amino acid sequence in which the amino acid residue at position 230 in the amino acid sequence set forth in SEQ ID NO: 2 is substituted with leucine, asparagine, glycine, aspartic acid or alanine, [2] A protein consisting of an amino acid sequence in which the amino acid residue at position 230 is substituted with leucine and the amino acid residue at position 6 is substituted with serine in the amino acid sequence set forth in SEQ ID NO: 2, [3] a protein consisting of an amino acid sequence in which the amino acid residue at position 230 is substituted with leucine and the amino acid residue at position 65 is substituted with leucine in the amino acid sequence set forth in SEQ ID NO: 2, [4] a protein consisting of an amino acid sequence in which the amino acid residue at position 230 is substituted with leucine, the amino acid residue at position 6 is substituted with serine, and the amino acid residue at position 65 is substituted with leucine in the amino acid sequence set forth in SEQ ID NO: 2, [5] A protein consisting of an amino acid sequence in which the amino acid residue at position 230 is substituted with leucine, the amino acid residue at position 65 is substituted with leucine, the amino acid residue at position 231 is substituted with threonine, and the amino acid residue at position 275 is substituted with glycine in the amino acid sequence shown in SEQ ID NO:

2.

2. The microorganism according to claim 1, wherein The microorganisms capable of producing DHA are Labyrinthus microorganisms.

3. The microorganism according to claim 2, wherein The microorganisms of the web-myxomycetes are microorganisms of the web-myxomycetes belonging to the genus Aurantiochytrium, the genus Thraustochytrium, the genus Ulkenia, the genus Parietichytrium, the genus Labyrinthula, the genus Aplanochytrium, the genus Oblongichytrium or the genus Schizochytrium.

4. The microorganism according to claim 1, wherein The microorganism having the ability to produce DHA is a microorganism in which genes encoding the following domains (a) to (j) having the activity of synthesizing DHA are introduced into a microorganism that does not have a DHA metabolic pathway. (a) β-ketoacyl-ACP synthase, i.e., KS domain; (b) malonyl-CoA:ACP acyltransferase, i.e., MAT domain; (c) ACP domain; (d) ketoreductase or KR domain; (e) polyketide synthase dehydratase, i.e., PS-DH domain; (f) chain elongation factor, i.e., CLF domain; (g) acyltransferase or AT domain; (h) FabA-like β-hydroxyacyl-ACP dehydratase, i.e., FabA-DH domain; (i) Enoyl ACP reductase, i.e., the ER domain; (j) Phosphopantetheinyl transferase or PPT domain.

5. The microorganism according to claim 4, wherein The microorganism that does not have a DHA metabolic pathway is a microorganism belonging to the genus Escherichia, Bacillus, Corynebacterium, Yarrowia, Saccharomyces, Candida or Pichia.

6. A method for producing EPA or a composition containing EPA, wherein: The microorganism according to any one of claims 1 to 5 is cultured in a culture medium to produce and accumulate EPA or a composition containing EPA in the culture, and EPA or a composition containing EPA is collected from the culture.

7. A method for producing EPA or a composition containing EPA, wherein: Using the following (I) microorganism capable of producing EPA, (I) A microorganism capable of producing DHA, comprising a mutant OrfB and capable of producing EPA, wherein the mutant OrfB is any one of the proteins selected from the following [1] to [5]: [1] A protein consisting of an amino acid sequence in which the amino acid residue at position 230 in the amino acid sequence set forth in SEQ ID NO: 2 is substituted with leucine, asparagine, glycine, aspartic acid or alanine, [2] A protein consisting of an amino acid sequence in which the amino acid residue at position 230 is substituted with leucine and the amino acid residue at position 6 is substituted with serine in the amino acid sequence set forth in SEQ ID NO: 2, [3] a protein consisting of an amino acid sequence in which the amino acid residue at position 230 is substituted with leucine and the amino acid residue at position 65 is substituted with leucine in the amino acid sequence set forth in SEQ ID NO: 2, [4] a protein consisting of an amino acid sequence in which the amino acid residue at position 230 is substituted with leucine, the amino acid residue at position 6 is substituted with serine, and the amino acid residue at position 65 is substituted with leucine in the amino acid sequence set forth in SEQ ID NO: 2, [5] A protein consisting of an amino acid sequence in which the amino acid residue at position 230 is substituted with leucine, the amino acid residue at position 65 is substituted with leucine, the amino acid residue at position 231 is substituted with threonine, and the amino acid residue at position 275 is substituted with glycine in the amino acid sequence shown in SEQ ID NO: 2.

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