CN109776663B - Gene fragments related to DHA and EPA synthesis and application thereof - Google Patents

Abstract

The invention discloses a group of gene fragments related to DHA and EPA synthesis and application thereof. The group of gene segments related to DHA and EPA synthesis provided by the invention consists of gene segments 1 to 6, and the nucleotide sequences are sequentially shown as sequences 3 to 8 in a sequence table. Experiments prove that a recombinant bacterium is obtained by introducing gene segments 1 to 6 into schizochytrium MYA-1381; the recombinant strain has greatly improved DHA and EPA producing capacity. Therefore, the 6 gene segments provided by the invention, the proteins coded by the 6 gene segments, and the vectors, cells or organisms containing the 6 gene segments have important application values in the production of DHA and EPA.

Description

Gene fragments related to DHA and EPA synthesis and application thereof

Technical Field

The invention relates to the field of industrial microorganisms and food and feed industries, in particular to a group of gene segments related to DHA and EPA synthesis and application thereof.

Background

Polyunsaturated fatty acids are linear fatty acids containing two or more double bonds, typically with a carbon chain length of 18-22 carbon atoms. Polyunsaturated fatty acids can be classified into omega-3 and omega-6 depending on the position of the double bond, and in the polyunsaturated fatty acid molecule, the double bond furthest from the carboxyl group is designated omega-3 at the 3 rd last carbon atom and omega-6 at the 6 th carbon atom. Polyunsaturated fatty acids are essential fatty acids for human body, and mainly include Docosahexaenoic Acid (DHA), docosapentaenoic Acid (DPA), Eicosapentaenoic Acid (EPA), and the like.

DHA is the most predominant class of polyunsaturated fatty acids. In terms of molecular structure, DHA is a straight-chain fatty acid having 22 carbon atoms and 6 double bonds, and belongs to OMEGA-3 series fatty acids (OMEGA-3) because the 1 st double bond is present at the 3 rd carbon atom on the methyl end of the fatty acid chain. DHA is mainly present in the brain and retina of the human body and has important physiological functions, such as promoting development of the nervous system, improving retinal function, improving vision, preventing cardiovascular diseases, treating cardiovascular diseases, anti-inflammation, and suppressing allergic reactions, etc. Since the human body cannot synthesize sufficient DHA by itself, it is obtained mainly by ingestion from food. Because the DHA content in daily diet is often insufficient, the supplement of DHA or the addition of DHA to foods or milk powder is of great significance to humans, especially infants and the elderly.

At present, there are two main methods for producing DHA: one is a conventional DHA source, i.e., extracted from tissues of marine fishes (mainly including salmon, mackerel, salmon and sardine), but the quality of fish oil obtained by extraction is affected by the variety, season and place of fishing of the fishes, and the quality of fish oil is also affected by the increasingly serious shortage of fish resources and environmental pollution; the other is a novel DHA production method, namely DHA is produced by fermenting marine microorganisms, and the method has the advantages of short period, no objective condition influence, no fishy smell and the like, and has wide prospect. The marine microorganism used for fermentation production of DHA is mainly Schizochytrium (Schizochytrium), but the current Schizochytrium used for fermentation production of DHA cannot further improve yield and reduce cost due to the limitation of technical indexes such as self total fatty acid content, DHA content, growth rate and the like.

In DHA-producing strains, the DHA biosynthetic pathway is catalyzed by a series of enzymes in the relevant anabolic pathway. Mining, modifying and heterogeneously expressing related genes in a DHA biosynthesis pathway, and providing favorable conditions for further improving the yield of DHA. Therefore, the acquisition of the new key genes in the DHA biosynthesis pathway is beneficial to the modification and process optimization of the DHA producing strain. Two DHA synthesis pathways exist in nature: (1) the elongation-desaturation pathway (E-D pathway) is mainly based on the fatty acid synthesis pathway, and DHA is further synthesized by the action of elongases and desaturases; (2) the Polyketide synthase pathway (PKS pathway) is mainly used for the synthesis of DHA from acetyl CoA and malonyl CoA as precursors by Polyketide synthase. Wherein, in Schizochytrium limacinum

In Schizochytrium, the synthesis of DHA mainly adopts PKS pathway. It is currently believed that both DPA and EPA synthesis have E-D pathways and PKS pathways.

Disclosure of Invention

The technical problem to be solved by the present invention is how to prepare DPA and/or EPA.

To solve the above technical problems, the present invention firstly provides a combination of proteins for the preparation of DPA and/or EPA, which may be (X1) or (X2) or (X3) or (X4) as follows:

(X1) includes protein 1, protein 2, protein 3, protein 4, protein 5 and protein 6;

(X2) consisting of protein 1, protein 2, protein 3, protein 4, protein 5 and protein 6;

(X3) consists of any two, any three, any four, or any five of said protein 1, said protein 2, said protein 3, said protein 4, said protein 5, and said protein 6;

(X4) said protein 1, said protein 2, said protein 3, said protein 4, said protein 5, or said protein 6;

the protein 1 may be a1) or a2) or a3) or a4) or a 5):

a1) the amino acid sequence is protein shown as a sequence 9 in a sequence table;

a2) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 9 in the sequence table;

a3) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 9 in the sequence table;

a4) protein which has 80 percent or more than 80 percent of identity with the amino acid sequence limited by the sequence 9 in the sequence table, is derived from schizochytrium and is related to the synthesis of polyunsaturated fatty acid;

a5) a protein having an amino acid sequence shown in a sequence 9 in a sequence table;

the protein 2 may be b1) or b2) or b3) or b4) or b 5):

b1) the amino acid sequence is protein shown as a sequence 10 in a sequence table;

b2) a fusion protein obtained by connecting labels to the N terminal or/and the C terminal of the protein shown in the sequence 10 in the sequence table;

b3) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 10 in the sequence table;

b4) protein which has 80 percent or more than 80 percent of identity with the amino acid sequence limited by the sequence 10 in the sequence table, is derived from schizochytrium and is related to the synthesis of polyunsaturated fatty acid;

b5) a protein having an amino acid sequence shown in a sequence 10 in a sequence table;

the protein 3 may be c1) or c2) or c3) or c4) or c 5):

c1) the amino acid sequence is a protein shown as a sequence 11 in a sequence table;

c2) a fusion protein obtained by connecting labels to the N terminal or/and the C terminal of the protein shown in the sequence 11 in the sequence table;

c3) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 11 in the sequence table;

c4) protein which has 80 percent or more than 80 percent of identity with the amino acid sequence limited by the sequence 11 in the sequence table, is derived from schizochytrium and is related to the synthesis of polyunsaturated fatty acid;

c5) a protein having an amino acid sequence shown in a sequence 11 in a sequence table;

the protein 4 may be d1) or d2) or d3) or d4) or d 5):

d1) the amino acid sequence is protein shown as a sequence 12 in a sequence table;

d2) a fusion protein obtained by connecting labels to the N terminal or/and the C terminal of the protein shown in the sequence 12 in the sequence table;

d3) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 12 in the sequence table;

d4) protein which has 80 percent or more than 80 percent of identity with the amino acid sequence limited by the sequence 12 in the sequence table, is derived from schizochytrium and is related to the synthesis of polyunsaturated fatty acid;

d5) a protein having an amino acid sequence shown as a sequence 12 in a sequence table;

the protein 5 may be e1) or e2) or e3) or e4) or e 5):

e1) the amino acid sequence is protein shown as a sequence 13 in a sequence table;

e2) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 13 in the sequence table;

e3) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 13 in the sequence table;

e4) protein which has 80 percent or more than 80 percent of identity with the amino acid sequence limited by the sequence 13 in the sequence table, is derived from schizochytrium and is related to the synthesis of polyunsaturated fatty acid;

e5) a protein having an amino acid sequence shown as a sequence 13 in a sequence table;

the protein may 6 be f1) or f2) or f3) or f4) or f 5):

f1) the amino acid sequence is protein shown as a sequence 14 in a sequence table;

f2) a fusion protein obtained by connecting labels to the N terminal or/and the C terminal of the protein shown in the sequence 14 in the sequence table;

f3) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 14 in the sequence table;

f4) protein which has 80 percent or more than 80 percent of identity with the amino acid sequence defined by the sequence 14 in the sequence table, is derived from schizochytrium and is related to the synthesis of polyunsaturated fatty acid;

f5) a protein having an amino acid sequence shown as a sequence 14 in a sequence table.

Wherein, sequence 9 in the sequence table is composed of 669 amino acid residues, sequence 10 in the sequence table is composed of 1193 amino acid residues, sequence 11 in the sequence table is composed of 773 amino acid residues, sequence 12 in the sequence table is composed of 2189 amino acid residues, sequence 13 in the sequence table is composed of 1672 amino acid residues, and sequence 14 in the sequence table is composed of 21 amino acid residues.

In order to facilitate the purification of the protein in a1), the amino terminal or the carboxyl terminal of the protein shown in the sequence 9 in the sequence table can be connected with a label shown in the table 1. In order to facilitate the purification of the protein in b1), the amino terminal or the carboxyl terminal of the protein shown in the sequence 10 in the sequence table can be connected with a label shown in the table 1. In order to facilitate the purification of the protein in c1), the amino terminal or the carboxyl terminal of the protein shown in the sequence 11 in the sequence table can be connected with a label shown in the table 1. In order to facilitate the purification of the protein in d1), the amino terminal or the carboxyl terminal of the protein shown in the sequence 12 in the sequence table can be connected with the label shown in the table 1. In order to facilitate the purification of the protein in e1), the amino terminal or the carboxyl terminal of the protein shown in the sequence 13 in the sequence table can be connected with a label shown in the table 1. In order to facilitate the purification of the protein in f1), the amino terminal or the carboxyl terminal of the protein shown in sequence 14 in the sequence table can be connected with a label shown in Table 1.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein 1 in a3) above, the protein 2 in b3) above, the protein 3 in c3) above, the protein 4 in d3) above, the protein 5 in e3) above, and the protein 6 in f3) above, wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

Protein 1 in a3) above, protein 2 in b3) above, protein 3 in c3) above, protein 4 in d3) above, protein 5 in e3) above, and protein 6 in f3) above can be artificially synthesized, or can be obtained by synthesizing the coding genes thereof and then performing biological expression.

The gene encoding protein 1 in a3) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence 9 in the sequence table, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 above at the 5 'end and/or 3' end.

The gene encoding protein 2 in b3) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence No. 10 in the sequence table, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 above at the 5 'end and/or 3' end.

The gene encoding protein 3 in c3) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence 11 in the sequence table, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 above at the 5 'end and/or 3' end.

The gene encoding protein 4 in d3) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence 12 in the sequence table, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 above at the 5 'end and/or 3' end.

The gene encoding protein 5 in e3) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence 13 in the sequence table, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 above at the 5 'end and/or 3' end.

The gene encoding protein 6 in f3) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence 14 in the sequence table, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 above at the 5 'end and/or 3' end.

The term "identity" as used above refers to sequence similarity to a native amino acid sequence. "identity" includes amino acid sequences that are 80%, or 85% or more, or 90% or more, or 95% or more identical to the amino acid sequence of a protein provided by the present invention.

Nucleic acid molecules encoding such combinations of proteins are also within the scope of the invention.

The nucleic acid molecule encoding the protein 1 can be a DNA molecule shown in the following A1) or A2) or A3) or A4):

A1) the coding region is a DNA molecule shown in the 1044 th to the 3050 th site from the 5' end of a sequence 3 in a sequence table;

A2) the nucleotide sequence is a DNA molecule shown in a sequence 3 in a sequence table;

A3) a DNA molecule which has 75% or more 75% identity with the nucleotide sequence defined by A1) or A2), is derived from Schizochytrium limacinum and encodes the protein 1;

A4) a DNA molecule which hybridizes with the nucleotide sequence limited by A1) or A2) under strict conditions and codes for the protein 1.

The nucleic acid molecule encoding the protein 2 can be a DNA molecule shown in the following B1) or B2) or B3) or B4):

B1) the coding region is a DNA molecule shown in 1068 th to 2737 th and 3254 th to 5162 th from the 5' end of a sequence 4 in the sequence table;

B2) the nucleotide sequence is a DNA molecule shown as a sequence 4 in the sequence table;

B3) a DNA molecule having 75% or more 75% identity to the nucleotide sequence defined in B1) or B2), derived from Schizochytrium sp, and encoding said protein 2;

B4) a DNA molecule which hybridizes with the nucleotide sequence defined by B1) or B2) under strict conditions and codes for the protein 2.

The nucleic acid molecule encoding the protein 3 can be a DNA molecule shown in the following C1) or C2) or C3) or C4):

C1) the coding region is a DNA molecule shown in 1094 to 3415 th sites from the 5' end of a sequence 5 in a sequence table;

C2) the nucleotide sequence is a DNA molecule shown as a sequence 5 in a sequence table;

C3) a DNA molecule derived from Schizochytrium limacinum and encoding the protein 3, having 75% or more 75% identity with the nucleotide sequence defined by C1) or C2);

C4) a DNA molecule which hybridizes with the nucleotide sequence limited by C1) or C2) under strict conditions and codes for the protein 3.

The nucleic acid molecule encoding the protein 4 can be a DNA molecule shown in D1) or D2) or D3) or D4) as follows:

D1) the coding region is a DNA molecule shown in 1409 th to 5044 th, 7004 th to 7234 th and 7700 th to 10399 th positions of the 5' end of a sequence 6 in the sequence table;

D2) the nucleotide sequence is a DNA molecule shown as a sequence 6 in a sequence table;

D3) a DNA molecule derived from Schizochytrium limacinum and encoding the protein 4, having 75% or more 75% identity with the nucleotide sequence defined by D1) or D2);

D4) a DNA molecule which hybridizes with the nucleotide sequence limited by D1) or D2) under strict conditions and codes for the protein 4.

The nucleic acid molecule encoding the protein 5 can be a DNA molecule shown in the following E1) or E2) or E3) or E4):

E1) the coding region is a DNA molecule shown in 1473 th to 6488 th positions from the 5' end of a sequence 7 in a sequence table;

E2) the nucleotide sequence is a DNA molecule shown as a sequence 7 in a sequence table;

E3) a DNA molecule derived from Schizochytrium limacinum and encoding said protein 5, having 75% or more 75% identity with the nucleotide sequence defined in E1) or E2);

E4) a DNA molecule which hybridizes with the nucleotide sequence defined by E1) or E2) under strict conditions and codes for the protein 5.

The nucleic acid molecule encoding the protein 6 can be a DNA molecule shown in the following F1) or F2) or F3) or F4):

F1) the coding region is a DNA molecule shown in 953 th to 991 th and 1063 th to 1090 th positions from the 5' end of a sequence 8 in a sequence table;

F2) the nucleotide sequence is a DNA molecule shown as a sequence 8 in a sequence table;

F3) a DNA molecule derived from Schizochytrium limacinum and encoding said protein 6, having 75% or more 75% identity with the nucleotide sequence defined by F1) or F2);

F4) a DNA molecule which hybridizes with the nucleotide sequence defined by F1) or F2) under strict conditions and codes for the protein 6.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc. The nucleic acid molecule may be a nucleic acid molecule formed by genes encoding the combination of proteins and their regulatory sequences.

Wherein, the sequence 3 in the sequence table consists of 4100 nucleotides, and the nucleotide shown in the sequence 3 in the sequence table codes an amino acid sequence shown in the sequence 9 in the sequence table. The sequence 4 in the sequence table is composed of 6200 nucleotides, and the nucleotides shown in the sequence 4 in the sequence table encode an amino acid sequence shown in a sequence 10 in the sequence table. Sequence 5 in the sequence table consists of 4500 nucleotides, and the nucleotide shown in sequence 5 in the sequence table encodes an amino acid sequence shown in sequence 11 in the sequence table. The sequence 6 in the sequence table is composed of 11100 nucleotides, and the nucleotide shown in the sequence 6 in the sequence table codes an amino acid sequence shown in a sequence 12 in the sequence table. The sequence 7 in the sequence table consists of 7767 nucleotides, and the nucleotide shown in the sequence 7 in the sequence table encodes an amino acid sequence shown in the sequence 13 in the sequence table. Sequence 8 in the sequence table consists of 7800 nucleotides, and the nucleotides shown in sequence 8 in the sequence table encode the amino acid sequence shown in sequence 14 in the sequence table.

The nucleotide sequence encoding the combination of proteins of the present invention can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which have been artificially modified to have 75% or more homology with the nucleotide sequence of the protein combination of the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode the protein combination and are derived from Schizochytrium. The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, 80% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence that encodes the protein combination of the present invention.

Expression cassettes, recombinant vectors, recombinant microorganisms or transgenic cell lines containing said nucleic acid molecules also belong to the scope of protection of the present invention.

The recombinant vector can be a recombinant plasmid obtained by inserting a nucleic acid molecule encoding the protein 1 (namely, a DNA molecule shown in a sequence 3 in a sequence table), a nucleic acid molecule encoding the protein 2 (namely, a DNA molecule shown in a sequence 4 in the sequence table), a nucleic acid molecule encoding the protein 3 (namely, a DNA molecule shown in a sequence 5 in the sequence table), a nucleic acid molecule encoding the protein 4 (namely, a DNA molecule shown in a sequence 6 in the sequence table), a nucleic acid molecule encoding the protein 5 (namely, a DNA molecule shown in a sequence 7 in the sequence table) and a nucleic acid molecule encoding the protein 6 (namely, a DNA molecule shown in a sequence 8 in the sequence table) into a starting plasmid.

The recombinant microorganism can be obtained by introducing the recombinant vector into the starting microorganism.

The starting microorganism may be a yeast, bacterium, algae or fungus. The fungus may be schizochytrium limacinum. The Schizochytrium limacinum can be specifically the strain Schizochytrium limacinum Honda et Yokochi ATCC MYA-1381.

The recombinant microorganism may specifically be the GS-C06 strain mentioned in the examples.

The use of any of the above combinations of proteins, or any of the above nucleic acid molecules, or any of the above expression cassettes, recombinant vectors, recombinant microorganisms or transgenic cell lines comprising said nucleic acid molecules, for the production of DHA and/or EPA is also within the scope of the present invention.

The invention also protects a recombinant bacterium B, and the preparation method can be as follows: and introducing a substance which improves the expression and/or activity of the protein combination into the initial strain to obtain a recombinant strain B. The "introduction of a substance which increases the expression and/or activity of a combination of proteins" into an outgrowth bacterium is effected by introducing a nucleic acid molecule encoding said combination of proteins into the outgrowth bacterium. The outgrowing bacteria may be schizochytrium limacinum. The Schizochytrium limacinum can be specifically the strain Schizochytrium limacinum Honda et Yokochi ATCC MYA-1381.

The recombinant strain B can be GS-C06 strain mentioned in the examples.

The invention also relates to a method for producing DHA and/or EPA, which comprises the following steps in sequence:

(1) introducing a substance that increases the expression and/or activity of the combination of proteins into the initial bacteria to obtain recombinant bacteria A; the recombinant bacterium A has an improved ability to produce DHA and/or EPA compared to the starting bacterium;

(2) fermenting and culturing the recombinant bacterium A to obtain DHA and/or EPA.

In the above method, the recombinant bacterium A may be the recombinant bacterium B.

In the above method, the "introducing a substance that increases the expression and/or activity of a combination of proteins" into the microorganism is carried out by introducing a nucleic acid molecule encoding the combination of proteins into the microorganism.

In the above method, the outbreak fungus may be schizochytrium limacinum.

In the above method, the Schizochytrium limacinum Honda et Yokochi ATCC MYA-1381 may be specifically mentioned.

In the above method, the recombinant bacterium A may specifically be GS-C06 strain mentioned in the examples.

As used herein above, the "introduction of a nucleic acid molecule encoding said combination of proteins into the starting bacterium" can be achieved by introducing a recombinant vector into the starting bacterium; the recombinant vector can be a recombinant plasmid obtained by inserting a nucleic acid molecule encoding the protein 1 (namely, a DNA molecule shown in a sequence 3 in a sequence table), a nucleic acid molecule encoding the protein 2 (namely, a DNA molecule shown in a sequence 4 in the sequence table), a nucleic acid molecule encoding the protein 3 (namely, a DNA molecule shown in a sequence 5 in the sequence table), a nucleic acid molecule encoding the protein 4 (namely, a DNA molecule shown in a sequence 6 in the sequence table), a nucleic acid molecule encoding the protein 5 (namely, a DNA molecule shown in a sequence 7 in the sequence table) and a nucleic acid molecule encoding the protein 6 (namely, a DNA molecule shown in a sequence 8 in the sequence table) into an expression vector.

The group of gene segments related to DHA and EPA synthesis provided by the invention consists of gene segments 1 to 6, and the nucleotide sequences are sequentially shown as sequences 3 to 8 in a sequence table. Experiments prove that a recombinant bacterium is obtained by introducing gene segments 1 to 6 into schizochytrium MYA-1381; the recombinant strain has greatly improved DHA and EPA producing capacity. Therefore, the 6 gene segments provided by the invention, the proteins coded by the 6 gene segments, and the vectors, cells or organisms containing the 6 gene segments have important application values in the production of DHA and EPA.

Drawings

FIG. 1 shows the colony morphology characteristics of Schizochytrium HS 01.

FIG. 2 shows morphological characteristics of Schizochytrium HS 01.

Deposit description

The strain name is as follows: schizochytrium limacinum (Fr.) Kuntze

Latin name: schizochytrium limacinum

The strain number is as follows: HS01

The preservation organization: china general microbiological culture Collection center

The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)

Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district

The preservation date is as follows: 3 and 10 months in 2017

Registration number of the preservation center: CGMCC No.13746

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The media used in the following examples are as follows:

wort agar medium: dissolving 150g of malt extract powder in 1L of mixed solution (prepared by mixing 1 volume of natural seawater and 1 volume of distilled water), wherein the pH value is natural; agar powder was then added to a concentration of 15g/100mL to obtain a medium.

Screening a liquid culture medium: 50g of glucose and 15g of yeast powder are dissolved in 1L of mixed solution (formed by mixing 1 volume of natural seawater and 1 volume of distilled water), and the pH value is natural.

Screening a solid culture medium: agar powder was added to the screening liquid medium to a concentration of 15g/100mL to obtain a medium.

Screening the plates: the screened solid medium at about 55 ℃ is poured into a petri dish and cooled to obtain a solid plate.

Shake flask culture medium: dissolving glucose 50g and yeast powder 15g in 1L distilled water, and adjusting pH to natural value.

Seed culture medium: 60g of glucose, 10g of yeast powder and NaSO₄ 10g、KCl 0.5g、MgSO₄ 2.0g、K₂SO₄1.0g、KH₂Po₄ 1.0g、(NH₄)₂SO₄1.0g and CaCl₂0.5g of this solution was dissolved in 1L of distilled water, and the pH was adjusted to 6.0.

Fermentation medium: mixing glucose 60g, glutamic acid or sodium glutamate 10g, corn steep liquor dry powder 10g, and NaSO₄ 14g、KCl 0.5g、MgSO₄ 2.0g、K₂SO₄ 1.0g、KH₂PO₄ 1.0g、(NH₄)₂SO₄1.0g and CaCl₂0.5g of this solution was dissolved in 1L of distilled water, and the pH was adjusted to 6.0.

The corn steep liquor dry powder is a product of Beijing Sorlebao science and technology Limited, and the product catalog number is FA 0010. The yeast powder is a product of Angel Yeast GmbH, and the product catalog number is LMO 2. The yeast genome extraction kit is a product of Tiangen Biotechnology Ltd, and the catalog of the product is DP 307. The high fidelity TransStart FastPfu DNA polymerase is Beijing Quanji Biotechnology, Inc., and the catalog is AP 221. The agarose gel DNA recovery kit is a product of Tiangen Biochemical technology Ltd, and the catalog of the product is DP 210. The pEASY-Blunt vector is a product of Beijing Quanjin Biotechnology Co., Ltd, and the product catalog is CB 301-01.

The strain Schizochytrium limacinum Honda et Yokochi ATCC MYA-1381 is deposited in American Type Culture Collection (ATCC, address: American Type Culture Collection (ATCC)10801University Boulevard Manassas, VA 20110USA), and is publicly available from the American Type Culture Collection. The strain Schizochytrium limacinum Honda et Yokochi ATCC MYA-1381 is hereinafter referred to as MYA-1381.

Example 1 isolation, identification and preservation of Schizochytrium limacinum HS01CGMCC No.13746

Separation of Schizochytrium HS01

1. The inventor collects schizochytrium limacinum from a plurality of mangrove forests in Yuanxiao county of Zhangzhou city in Fujian province, and mixes the schizochytrium limacinum and the mangrove forests to obtain mixed liquid; inoculating 0.5mL of the mixed solution to 5mL of a screening liquid culture medium, and then culturing at 25 ℃ and 200rpm/min for 2d to obtain a culture solution.

2. And (3) uniformly coating the culture bacterial liquid obtained in the step (1) on a screening plate, and performing static culture at 25 ℃ for 2d to generate a single bacterial colony.

3. After the step 2 is completed, single colonies are respectively selected and inoculated to 5mL of fermentation culture medium, and then cultured for 2d at 25 ℃ and 200rpm/min to obtain culture bacteria liquid.

4. And (4) centrifuging the culture solution obtained in the step (3) at 4 ℃ and 2000rpm for 5min, and collecting thalli.

5. Taking 1.0-2.0 g of the thalli to a measuring cylinder with a plug (the specification is 100mL), firstly adding 15mL of HCl aqueous solution with the concentration of 8.3mol/L, covering a cover, and hydrolyzing in a water bath at 70-80 ℃ for 50-60 min (during the hydrolysis, placing the measuring cylinder with the plug on a vortex mixer for 1 time every 10 min); after cooling to room temperature, firstly adding 10mL of 95% (v/v) ethanol aqueous solution, sufficiently and uniformly shaking, then adding 20mL of anhydrous ether, sufficiently shaking for extraction for 1-2 min, finally adding 20mL of petroleum ether, sufficiently shaking for extraction for 1-2 min, standing for layering, placing the upper organic phase in a glass weighing dish (dried and weighed), placing the glass weighing dish on a boiling water bath in a ventilation cabinet to sufficiently evaporate the organic phase completely (sufficiently and completely volatilize completely), and obtaining the liquid phase as the grease.

6. And (3) taking the grease extracted in the step (5), detecting the DHA content according to the national standard of GB 26400-2011 food safety, and detecting the composition and the content of the fatty acid according to the method of AOAC 996.06.

And selecting strains with higher DHA content, and repeatedly purifying for 24 times. The screened schizochytrium limacinum strain is named as schizochytrium limacinum HS 01.

And (3) inoculating the schizochytrium HS01 monoclonal to a fermentation medium for continuous passage of 12 generations, and detecting the DHA content according to the steps. The result shows that the stability of DHA produced by schizochytrium HS01 is good.

II, identification of Schizochytrium HS01

1. Morphological identification

The schizochytrium HS01 is inoculated on a wort agar culture medium, dark culture is carried out at 25 ℃, the morphology of a bacterial colony is observed after 5 days, and the morphological characteristics of the bacterial body are analyzed and observed by a high-resolution transmission electron microscope.

The results of the experiment are shown in FIGS. 1 and 2. The result shows that the colony diameter of the schizochytrium HS01 is 2-4.3 mm, the schizochytrium HS01 is white (light orange at the later stage), and the edge is irregular; the thallus is proliferated in a fission mode, the cell wall is thin, spherical, colorless or light orange, transparent and 4.5-15.5 mu m in size, and zoospores and an exoplasmic reticulum are not seen.

2. 18s rDNA sequence homology analysis

The partial sequence of the 18s rDNA of the schizochytrium HS01 is shown as the sequence 1 in the sequence table.

The partial sequence of the 18s rDNA of the schizochytrium HS01 is shown as the sequence 2 in the sequence table.

Combining the above identification results, the Schizochytrium HS01 is Schizochytrium limacinum (Schizochytrium limacinum).

Third, preservation of Schizochytrium HS01

Schizochytrium limacinum HS01 has been deposited in China general microbiological culture Collection center (CGMCC, address: No. 3, Xilu No.1, Beijing area, Chaoyang area, China) in 2017, 03 and 10 days, and the deposit number is CGMCC No. 13746. The schizochytrium limacinum HS01 is called schizochytrium limacinum HS01CGMCC No.13746, abbreviated as schizochytrium HS 01.

Example 2 discovery of Gene fragments involved in DHA and EPA Synthesis

Production of polyunsaturated fatty acid by fermentation of schizochytrium limacinum HS01

1. The schizochytrium HS01 is inoculated into a shake flask (with the specification of 10mL) filled with 2mL of shake flask culture medium in a single clone mode, and the culture is carried out for 24-48 h at the temperature of 22-28 ℃ and at the speed of 150-250 rpm/min, so as to obtain first-level seed liquid.

2. Taking the first-stage seed solution, inoculating the first-stage seed solution into a shake flask (the shake flask specification is 1L) filled with 250mL of shake flask culture medium in an inoculation amount of 3-10% (v/v), and culturing at 22-28 ℃ at 150-250 rpm/min for 24-48 h to obtain a second-stage seed solution.

3. Inoculating the second-stage seed solution into a fermentation tank (5L in the specification of the fermentation tank) filled with 3L of seed culture medium in an inoculation amount of 3-10% (v/v), and culturing at 22-28 ℃ for 24-48 h (10-80% of dissolved oxygen) to obtain a first-stage fermentation seed solution.

4. Inoculating the first-stage fermentation seed liquid with 3-10% (v/v) of inoculum size to a fermentation tank (100L fermentation tank specification; initial biomass is 1.0 × 10 after inoculation)⁸～2.5×10⁸cfu/mL) at 22-28 ℃ for 72-120 h (dissolved oxygen of 5-80%) to obtain a fermentation broth.

The fermentation broth contains DHA.

5. According to the method in the step one of the embodiment 1, the method comprises the steps of extracting grease from fermentation liquor, detecting DHA content according to GB 26400-2011 national food safety standard, detecting DPA content according to GB28404-2012 national food safety standard, detecting EPA content according to GB5009.168-2016 national food safety standard, and detecting the composition and content of fatty acid according to AOAC 996.06.

The results are shown in Table 2. The results show that DHA accounts for 45.0-60.0% of the grease, DPA accounts for 9.0-17.0% of the grease, and EPA accounts for 0.2-1.0% of the grease.

TABLE 2

Name (R)	Composition (%)
		Lauric acid	0～1.0
Myristic acid	0.5～1.0
		Palmitic acid	22～32
Stearic acid	1.0～2.5
		Dihomo-gamma-linolenic acid	0.1～0.3
Arachidonic acid	0～0.8
		EPA	0.2～1.0
DPA	9.0～17.0
		DHA	45.0～60.0

Fermentation production of polyunsaturated fatty acid by using MYA-1381

According to the method of the first step, the schizochytrium HS01 is replaced by MYA-1381, and other steps are not changed. The results show that DHA accounts for 12-23% of the grease, DPA accounts for 20-39% of the grease, and EPA accounts for 0.5-3% of the grease.

According to the above results, Schizochytrium HS01 is a high-producing strain synthesizing DHA and EPA, and MYA-1381 is a low-producing strain synthesizing DHA and EPA.

Third, discovery of gene fragment related to DHA and EPA synthesis

The genome DNA of Schizochytrium HS01 and MYA-1381 were extracted with a yeast genome extraction kit, and whole genome sequencing was performed by PacBio RS II and Illumina HiSeq 4000 from Beijing Nuo Poa-induced science and technology Ltd.

Results show that compared with MYA-1381, the schizochytrium HS01 contains 6 unique gene segments which are respectively named as gene segment 1, gene segment 2, gene segment 3, gene segment 4, gene segment 5 and gene segment 6, and the nucleotide sequences of the gene segments are sequentially shown as a sequence 3 to a sequence 8 in a sequence table.

The 1044 th to 3050 th positions of a sequence 3 in the sequence table from the 5' terminal encode protein 1, and the amino acid sequence of the protein 1 is shown as a sequence 9 in the sequence table.

The 1068 th to 2737 th site and the 3254 th to 5162 th site of the sequence 4 in the sequence table from the 5' end encode protein 2, and the amino acid sequence of the protein 2 is shown as a sequence 10 in the sequence table.

The 1094 th to 3415 th positions of the sequence 5 in the sequence table code protein 3 from the 5' end, and the amino acid sequence of the protein 3 is shown as the sequence 11 in the sequence table.

The amino acid sequence of the protein 4 is shown as the sequence 12 in the sequence table from 1409 th to 5044 th, 7004 th to 7234 th and 7700 th to 10399 th of the sequence 6 in the sequence table from the 5' end.

The 1473 th to 6488 th positions of the sequence 7 in the sequence table from the 5' end code protein 5, and the amino acid sequence of the protein 5 is shown as the sequence 13 in the sequence table.

The 953 th to 991 th sites and the 1063 th to 1090 th sites of the sequence 8 in the sequence table from the 5' end encode protein 6, and the amino acid sequence of the protein 6 is shown as a sequence 14 in the sequence table.

EXAMPLE 3 amplification of 6 Gene fragments and Synthesis of primers therefor

1. Extracting the genomic DNA of Schizochytrium HS01 by using a yeast genome extraction kit, taking the genomic DNA as a template, and carrying out PCR amplification by using high-fidelity TransStart FastPfu DNA polymerase and primer pairs (a primer pair HS01-1, a primer pair HS01-2, a primer pair HS01-3, a primer pair HS01-4, a primer pair HS01-5 and a primer pair HS01-6) to obtain a PCR amplification product.

The nucleotide sequences of the forward and reverse primers constituting each primer pair are shown in Table 3.

Reaction procedure: 2min at 98 ℃; 30 cycles of 98 ℃ for 30s, 56 ℃ for 30s and 72 ℃ for 3 min; 5min at 72 ℃.

TABLE 3

2. After step 1 was completed, the PCR amplification product was recovered using agarose gel DNA recovery kit.

3. And (3) after the step 2 is completed, connecting the recovered PCR amplification product with a pEASY-Blunt vector to obtain a recombinant bacterium plasmid.

4. After step 3 was completed, the recombinant plasmid was sequenced.

The sequencing result shows that the nucleotide sequence of the PCR amplification product obtained by amplification with the primer pair HS01-1 is shown as sequence 3 in the sequence table (namely, gene fragment 1), the nucleotide sequence of the PCR amplification product obtained by amplification with the primer pair HS01-2 is shown as sequence 4 in the sequence table (namely, gene fragment 2), the nucleotide sequence of the PCR amplification product obtained by amplification with the primer pair HS01-3 is shown as sequence 5 in the sequence table (namely, gene fragment 3), the nucleotide sequence of the PCR amplification product obtained by amplification with the primer pair HS01-4 is shown as sequence 6 in the sequence table (namely, gene fragment 4), the nucleotide sequence of the PCR amplification product obtained by amplification with the primer pair HS01-5 is shown as sequence 7 in the sequence table (namely, gene fragment 5), and the nucleotide sequence of the PCR amplification product obtained by amplification with the primer pair HS01-6 is shown as sequence 8 in the sequence table (namely, gene fragment 6). Therefore, 6 gene fragments can be amplified using the primers in Table 2.

Example 4, 6 applications of Gene fragments in the production of DHA and EPA

In this example, the nucleotide sequences of the primers involved are shown in Table 4.

TABLE 4

Primer name	Nucleotide sequence (5 '-3')
		HS01-1-UF	CACATTCGCTACAAAACGCCGCAGTTTCTA
HS01-1-UR	ACGGTAGAGCGCTTTTGAAGCTGGGGTGGGGTGCGAGGAAGTTGCGTATCCCAGGCTCTC
		HS01-1-DF	GGTAAGGAGGATATTCTCGAGACTAGTCTGACGCTCCCATCAATCTTTGGACACTACGAC
HS01-1-DR	CGCAAACTATTTGCTAACCTATTTATCGTA
		HS01-2-UF	CTGCTGCTACTTCAACATCACTTTGCTCGT
HS01-2-UR	ACGGTAGAGCGCTTTTGAAGCTGGGGTGGGTTGCGATGAATAGCAAACCCCAGAAGTGTG
		HS01-2-DF	GGTAAGGAGGATATTCTCGAGACTAGTCTGGCGAATCCGAGACTCCTTTAAATAGCCAAG
HS01-2-DR	ACTGTAAGTTTATTAAATTGGTCGAGGATG
		HS01-3-UF	ACCGTGGGCCAAGCTGGCCGCCCCAAGACG
HS01-3-UR	ACGGTAGAGCGCTTTTGAAGCTGGGGTGGGGTGTGAGGCCACTTGTATCAACAGAGGTAA
		HS01-3-DF	GGTAAGGAGGATATTCTCGAGACTAGTCTGTACAATTGAAGAGCCATTGGATAAGTTCGA
HS01-3-DR	CTTATCTTTGAGGGTAAGAAGGTCTGGTAT
		HS01-4-UF	CATTGATTGATTGCAGATGATCTTGGGCAA
HS01-4-UR	ACGGTAGAGCGCTTTTGAAGCTGGGGTGGGCCTACAAGGTGTGTTGGTTCGGAAGTTGGT
		HS01-4-DF	GGTAAGGAGGATATTCTCGAGACTAGTCTGATTACAACCACAACTTTCTATAAATAGTGC
HS01-4-DR	CTTTCGCCGTTAGAGAAAAAACCCAAACGA
		HS01-5-UF	TATTGCTATTACTTGAATTTGAATTTGAAT
HS01-5-UR	ACGGTAGAGCGCTTTTGAAGCTGGGGTGGGGTATGATATGTTATGTACTCGAGGAATGTA
		HS01-5-DF	GGTAAGGAGGATATTCTCGAGACTAGTCTGATCAAAGAAATTAAAAAGAAAACAAACATT
HS01-5-DR	CAGCAACTTTCACTCGCCCATTCAATCAAT
		HS01-6-UF	CCACATAATTTGAAAGAAACATTGACCACG
HS01-6-UR	ACGGTAGAGCGCTTTTGAAGCTGGGGTGGGAAATATTCAATCGAAATAAATGCACTGTTT
		HS01-6-DF	GGTAAGGAGGATATTCTCGAGACTAGTCTGCCTGATCATCCTTTCGTTACTTCTCAACTC
HS01-6-DR	GTGCACCGTTCTTATGCATATTTTAAAATC
		Zeo-F	CCCACCCCAGCTTCAAAAGCGCTCTACCGT
Zeo-R	CAGACTAGTCTCGAGAATATCCTCCTTACC

A. Acquisition of GS-C06 Strain

Preparation of targeting fragment HS01-1-Zeo

1. The recombinant plasmid pUC57-LZ was synthesized by Nanjing Kinshire Biotech Ltd. The recombinant plasmid pUC57-LZ was obtained by ligating the nucleotide sequence shown by sequence 15 in the sequence listing with the pUC57 vector. In the sequence 15 in the sequence table, the 25 th to 58 th positions from the 5' end are Lox66 sequences, the 626 th to 997 th positions are zeocin resistance genes, and the 2293 th to 2326 th positions are Lox71 sequences.

2. The yeast genome extraction kit is adopted to extract the genome DNA of the schizochytrium HS 01.

3. PCR amplification is carried out by taking genome DNA of schizochytrium HS01 as a template and HS01-1-UF and HS01-1-UR as primers to obtain a PCR amplification product of about 3100bp, wherein the PCR amplification product is HS01-1 upstream homologous fragment AU.

4. The genome DNA of the schizochytrium HS01 is used as a template, and HS01-1-DF and HS01-1-DR are used as primers to carry out PCR amplification to obtain a PCR amplification product of about 1000bp, wherein the PCR amplification product is the downstream homologous fragment AD of HS 01-1.

5. And (2) carrying out PCR amplification by using the recombinant plasmid pUC57-LZ synthesized in the step (1) as a template and Zeo-F and Zeo-R as primers to obtain a PCR amplification product of about 2350bp (the nucleotide sequence is shown as a sequence 15 in the sequence table), wherein the PCR amplification product is a Zeo fragment.

6. Taking HS01-1 upstream homologous fragment AU, HS01-1 downstream homologous fragment AD and Zeo fragment as templates, and HS01-1-UF and HS01-1-DR as primers to perform overlap amplification, and obtaining PCR amplification product of about 6450 bp. And recovering the PCR amplification product by using an agarose gel DNA recovery kit to obtain the targeting fragment HS 01-1-Zeo.

II, obtaining pretreated MYA-1381

1. Adding 10 mM LYMA-1381 bacterial liquid (concentration of 1 × 10) into sterile pre-cooled polypropylene tube (50 mL)⁸cfu/mL), centrifuging at 4 deg.C and 5000r/min for 10min, discarding supernatant, and collecting thallus.

2. After completing step 1, repeating the following steps twice: and (3) taking the polypropylene tube, adding 10mL of precooled sterile water to clean the thalli, centrifuging for 10min at 4 ℃ under 4472g, and collecting the thalli.

3. And (3) after the step 2 is finished, taking the polypropylene tube, adding 10mL of precooled 1mol/L sorbitol aqueous solution for resuspension, centrifuging for 10min at 4 ℃ at 5000r/min, and collecting thalli.

4. And (3) after the step 3 is finished, adding 10mL of precooled 1mol/L sorbitol aqueous solution into the polypropylene tube for resuspension to obtain the pretreated MYA-1381.

III, electric conversion

1. 30 mu L of pretreated MYA-1381 is taken, added with 1 mu g of targeting fragment HS01-1-Zeo, mixed gently, stood for 5min in ice bath, and then transferred into an ice-precooled electric shock cup for electric shock (the electric shock parameters are 0.75KV and 50 mu F).

2. And (3) after the step 1 is finished, adding 1mL of seed culture medium into the electric shock cup, culturing for 1h at 30 ℃ and 200r/min, centrifuging for 10min at 4 ℃ and 5000r/min, mixing thalli and a small amount of supernatant, uniformly coating the mixture on a resistant plate, and performing inverted culture for 48h at 30 ℃ to obtain a pseudotransformant.

Resistant plates: zeocin was added to the screening solid medium at about 55 ℃ to a concentration of 200. mu.g/mL, and then poured into a petri dish, and the resulting solid plate was cooled.

Fourth, obtaining and identifying positive transformant

And respectively extracting the genomic DNA of the pseudotransformant by using a yeast genome extraction kit, and performing PCR amplification by using the genomic DNA as a template and HS01-1-F and HS01-1-R as primers to obtain a PCR amplification product.

If the size of the PCR amplification product of a pseudotransformant is 4100bp (or the nucleotide sequence of the PCR amplification product is shown as a sequence 3 in a sequence table), the pseudotransformant is a positive transformant.

Fifthly, obtaining a transformant GS-C01

The pSH65 plasmid (a product of Biovector Inc.; the plasmid contains Cre enzyme) was introduced into the positive transformant, and then the Zeo gene was deleted according to the procedures described in the specification for the pSH65 plasmid to obtain a transformant GS-C01.

According to the steps from one to five, replacing 'HS 01-1-UF' with 'HS 01-2-UF', 'HS 01-1-UR' with 'HS 01-2-UR', 'HS 01-1-DF' with 'HS 01-2-DF', 'HS 01-1-DR' with 'HS 01-2-DR', 'HS 01-1-F' with 'HS 01-2-F', 'HS 01-1-R' with 'HS 01-2-R', 'MYA-1381' with 'transformant GS-C01', and the other steps are not changed to obtain the transformant GS-C02.

According to the steps from one to five, replacing 'HS 01-1-UF' with 'HS 01-3-UF', 'HS 01-1-UR' with 'HS 01-3-UR', 'HS 01-1-DF' with 'HS 01-3-DF', 'HS 01-1-DR' with 'HS 01-3-DR', 'HS 01-1-F' with 'HS 01-3-F', 'HS 01-1-R' with 'HS 01-3-R', 'MYA-1381' with 'transformant GS-C02', and the other steps are not changed to obtain the transformant GS-C03.

According to the steps from one to five, replacing 'HS 01-1-UF' with 'HS 01-4-UF', 'HS 01-1-UR' with 'HS 01-4-UR', 'HS 01-1-DF' with 'HS 01-4-DF', 'HS 01-1-DR' with 'HS 01-4-DR', 'HS 01-1-F' with 'HS 01-4-F', 'HS 01-1-R' with 'HS 01-4-R', 'MYA-1381' with 'transformant GS-C03', and the other steps are not changed to obtain the transformant GS-C04.

According to the steps from one to five, replacing 'HS 01-1-UF' with 'HS 01-5-UF', 'HS 01-1-UR' with 'HS 01-5-UR', 'HS 01-1-DF' with 'HS 01-5-DF', 'HS 01-1-DR' with 'HS 01-5-DR', 'HS 01-1-F' with 'HS 01-5-F', 'HS 01-1-R' with 'HS 01-5-R', 'MYA-1381' with 'transformant GS-C04', and the other steps are not changed to obtain the transformant GS-C05.

According to the steps from one to five, replacing 'HS 01-1-UF' with 'HS 01-6-UF', 'HS 01-1-UR' with 'HS 01-6-UR', 'HS 01-1-DF' with 'HS 01-6-DF', 'HS 01-1-DR' with 'HS 01-6-DR', 'HS 01-1-F' with 'HS 01-6-F', 'HS 01-1-R' with 'HS 01-6-R', 'MYA-1381' with 'transformant GS-C05', and the other steps are not changed to obtain the transformant GS-C06.

The transformant GS-C06 is the GS-C06 strain.

B. Application of 6 gene segments in production of DHA and EPA

The bacteria to be detected are schizochytrium HS01, MYA-1381 or GS-C06 strains.

1. And (3) inoculating the single clone of the bacteria to be detected into a shake flask (with the specification of 10mL) filled with 2mL of shake flask culture medium, and culturing at the temperature of 22-28 ℃ and at the speed of 150-250 rpm/min for 24-48 h to obtain a first-stage seed solution.

2. Taking the first-stage seed solution, inoculating the first-stage seed solution into a shake flask (500 mL in shake flask specification) filled with 50mL of shake flask culture medium in an inoculation amount of 3-10% (v/v), and culturing at 22-28 ℃ and 150-250 rpm/min for 24-48 h to obtain a second-stage seed solution.

3. Inoculating the second-stage seed solution into a fermentation tank (1L of the fermentation tank specification) filled with 500mL of seed culture medium in an inoculation amount of 3-10% (v/v), and culturing at 22-28 ℃ for 24-48 h (10-80% of dissolved oxygen) to obtain a third-stage seed solution.

4. Inoculating the third-stage seed solution to a fermentation tank (10L in specification) containing 5L fermentation medium at an inoculation amount of 3-10% (v/v), wherein the initial biomass is 0.3 × 10⁸～0.5×10⁸cfu/mL) at 22-28 ℃ for 72-120 h (dissolved oxygen of 5-80%) to obtain a fermentation broth. The fermentation liquor contains DHA, DPA and EPA.

5. According to the method in the step one of the embodiment 1 and the step 5, the grease of the fermentation liquor is extracted, and then the DHA content is detected according to the GB5009.168-2016 food safety national standard, the DPA content is detected according to the GB28404-2012 food safety national standard, and the EPA content is detected according to the GB5009.168-2016 food safety national standard.

The results are shown in Table 5. The results show that 6 gene fragments obtained by the invention are transformed into MYA-1381, and a high-yield strain (namely GS-C06 strain) for synthesizing DHA and EPA can be obtained. Therefore, the 6 gene segments provided by the invention, the proteins coded by the 6 gene segments, and the vectors, cells or organisms containing the 6 gene segments have important application values in the production of DHA and EPA. By modifying the protein coded by the 6 gene segments in the original strain, the engineering strain with high DHA and EPA yield can be constructed.

TABLE 5

	MYA-1381	Schizochytrium HS01	GS-C06 strain
				DHA content in fat (%)	12.38	45.02	30.50
DPA content (%) in fats and oils	25.26	12.74	17.50
				EPA content (%) in fat and oil	0.50	1.30	0.71

<110> Xiamen Huishi Biometrics Ltd

<120> a group of gene fragments related to DHA and EPA synthesis and application thereof

<160> 15

<170> PatentIn version 3.5

<210> 1

<211> 207

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 1

agccatgcat gtgtaagtat aagcgattgt actgtgagac tgcgaacggc tcattatatc 60

agtaataatt tcttcggtag tttcttttat atggatacct gcagtaattc tggaaataat 120

acatgctgta agagccctgt atggggctgc acttattaga ttgaagccga ttttattggt 180

gaatcatgat aattgagcag attgact 207

<210> 2

<211> 239

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 2

gagttctgcc tctgtccaaa aattaatcca aacagaaaca tcccatggtt tcatcggacc 60

gttcaatcgg taggtgcgac gggcggtgtg tacaaagggc agggacgtat tcaatgcaag 120

ctgatgactt gcgtttacta ggaattcctc gttggagatt aataattgca aaaatctagc 180

cccagcacga tgagcgttcc aaggattagc caggccttcc gaccaagcac tcaattcca 239

<210> 3

<211> 4100

<212> DNA

<213> Schizochytrium limacinum (Schizoochytrium limacinum)

<400> 3

cacattcgct acaaaacgcc gcagtttcta tggtttgatt cctttgaccc ctaaagaatt 60

gttcgaaagg gtgtgttctc ctggagagga catcaccgat tcttcgttag atgccgactc 120

cgtagagacg gcagaaagcg agccttctgc agagcccaag aacaagcaag atggggttca 180

tcgtgccgag gttgctactt tggatggatt cgggtaagtt catgcggtta atctttgttt 240

gggatcgtgt gatcaatcat tccacgaatt gtgttattga gatagaggga atcctaactt 300

tttgcttcta attttattac tccttcaccc tgctcgtttg cttacgtctt cttattcgcc 360

tcagagaacg gccggatgga gcgagcgtga gtttcgctga tgtggtgcag cttgccaaaa 420

acgtctcgaa cttgcgcgcg gaccgaggcg gggtcagagg aaactccctg cagactggga 480

ttgccttgct tgaggaattt attcgaccgg cgatccaggg ctactttggc caagtgaagt 540

tgcgacaggt ctccgctgac atcaagaact ccaaacagtg tcttgatgca ctacaaggcc 600

agattcgtgc aagctctgca aatgagttgc ccagtctcat cgaaaagcgg aatgctgaac 660

gagaccgcct acgtttacta atgcaagtcg tgtacccatg gactcgcgca aacctctgcg 720

aaagtgatga ggatgtagtc cgcgatttca aaatgagaat tgaaagtttg gtacacgatt 780

taaagatcac tcttgcaccg gttactgaaa agaccagcac agatgacttc aaacactgcc 840

aagaactcgt tgctcgggct gtccaatgcg tcgaaggata caagagaaat ggagatggcc 900

gaccaaacaa cattggcgaa cgaagccaac aagctgcaaa atgcgtcgtc tctcgattgt 960

ctgagctaga acttggtgat ggattatggg ttgctcttag gatacctgag caggccatga 1020

atctcggacc cgaattatcc gagcttgagc gtgctcttca agaagagatt gaagtgctac 1080

aaggagatac acttagtgcc cacttatcta agatactttg cgcaataagt tcaacattcg 1140

ctcgtttcga atcatactgt gaaaacactg tgctgaatga agcgagcaag tgcacaccac 1200

aagagctctc tgaactatat gattatacat cactgggttt gaaagaacct tccgtcttag 1260

atgttgataa attgtatcag aaacttggca agtcgaggga agccacaaag agtttgaaaa 1320

gagctagaga agagctcgaa gctgaggacc tggaagagga agaacgagaa tattttagaa 1380

atcgaatcaa gaaacagaag cgcattcttt ctaaaggcaa ccctatcgag ctcagcaaag 1440

tttttcgtca taagcttgct gaaactctgc aacacgcgca ggaacattat cctgagttgt 1500

tgcaagatag agcttggcta gaaaagcttc acgtagctgc agagggtgtt tttgaagtag 1560

catgttctga cctttggttg acaaatgtga cagtttcgga ctttcagcgt gtcggtgagc 1620

ccctatcctt tagaggtggc aagaaagtcc aaaaggttct tgacaacgtt ggaaaaacta 1680

tggttctcaa agagtttcaa cttgggcatc ctagtcaatc gaaaactttc taccagcagg 1740

ttgccaatct gggcaaagtg gtgtcccagc acgtaatacg tatcactggt gcttttgtgg 1800

acatgacaca tggagagtca cgtggttgta tcgtcatgcc tttctacgag caaggtgacc 1860

ttgccaagtg gattgagagt catcctaacg aaggcaaaga agcacgtgac cgacttgctg 1920

tgggtctgct tgtcggtgtg gcggatttac atgcccactg cattgtgcat tgtgacatca 1980

aaccagaaaa cattttcctc acaagcaacg gaacaccatt gatcggtgac tttgacggca 2040

tcaaggttgt aaactacact gcaacataca catcacttca ggcaacgcca cggtacattg 2100

cccctgagtt gcagaatggg cctgtacata ggtttgaaac agcaatggac atgtactctg 2160

tgggcataag cttaaaagag ctgtatccca ctgaacggac tgctgcaatg cagacactta 2220

tcgaagctct taccgcgaga gacccaagtc aacgtccaag cgctcgtcaa gcactccaac 2280

atcaagcttt tggagctcca caaatccctc tcatggaatg tcttgtatgt ttcgagaagc 2340

atcggctttc tgagggcacg agctgcgagg agggggactt cctttgcaga ggctgtatcg 2400

aatctgcagt agaagcagcg gcacaaccac ttgctaacgt cagagtggat gctgatggaa 2460

cgatggcatg catgaaacct gaatgtagtg gaaaaatatc aggtcaagaa atcacccgtc 2520

ttgcaccaac tgccttgaat catcttttgt tgattgcgaa gatgaaggca gaaagtgagg 2580

cagcagtttg ggctgagaaa gagatccaaa ggcgcatagc ggaagttgta cgtgcagaag 2640

aacaaaatag tgacgcacgg tggcatttac tgcatattca agagaacatc ttgagcacat 2700

gttgcccgaa ctgtaaggct tatgtacacg actttgacgg ttgctgcgct gtaaaatgtg 2760

gtaataatgc ttgtggacac ctcttctgcg cttggtgtct tgggtatagt tctcaggatg 2820

gagatgtttg ccatgctcat gtgagaacct gttcaagaag acttgggcaa gatctatatt 2880

tccctggttc actcgagcag gtttgtgaag cgtggcggtt gctccgcgca gaacgtctgc 2940

gtgagtactg gaatgcacaa attcaggata gaaatcttcg actcgttctg aaagacttat 3000

tgtcaccttt gctaacacca gatattgttg gcgcagattt tagattggaa taattaaaaa 3060

taaacgaatc tctgcaactc taaatctggc gatttgtttc catttcttta cacctccttg 3120

caaccaattt atatttttgg acatctgatc gctgctccaa tctaccatca gagagcctgg 3180

gatacgcaac ttcctcgcac acgctcccat caatctttgg acactacgac acgaaggttg 3240

tggagttttg cgcttttcta attttgcaca aggacttcat taaacgtaga gaccagtgta 3300

gtatcacaga cacaacactt tctccgaaat cgttacgtac tctttaacag gttattgcat 3360

attagcttgt gtagtagagt gtacataggt agtctgacaa gaaaatggcg aactcagatg 3420

aagtaatgaa gtgaagcctg tcgacgaagg tgaaagtaaa aaggatgaga cttggttctt 3480

gatgttctag gatgaatgta ccatgttgag gcaaagagtg tcgggctgta gagttaggtt 3540

agtttaaatt gaggtagggt aaggcaccta gctgaacatt acttcgaaat aggatataat 3600

ctttgtttta gtctatgtaa tattacacaa tagaacaaat atataagtag caagctctga 3660

ccaaaagctt ttgcattcca accactgtag gacgctgtaa tgagtctgcc aggttcttct 3720

gcttatttat accctcacaa agtggtttcc tccttcactt tcttaagcca tatattcaga 3780

tctactacta gcaataaatc gacggactct tatacagaga gccatatatt tgccacaagt 3840

tgacccaacc aatctccaaa aggcaaaagg ctacagctct gcaatttatt attcaaagtg 3900

cttgtagaat caaacagagc aacgacacgc aaagtgatga ggagaattca accatgcata 3960

cgtgcttgct caaaacgtac accaagattt ctgatcctgt attgatccgc aatgcctgtg 4020

caagagaaca tccgccgctc gaagtaattc tattgttatt caagctgcaa tacgataaat 4080

aggttagcaa atagtttgcg 4100

<210> 4

<211> 6200

<212> DNA

<213> Schizochytrium limacinum (Schizoochytrium limacinum)

<400> 4

ctgctgctac ttcaacatca ctttgctcgt ttgtttacgt ctttttgttc acctcagaga 60

tttgacggat agagcgaacg tgcgtttcgc taatgtggtg cggcttgcca aggaagtctc 120

ggacttgcgc gcggaccgag gcggggtcag agaaaactcc ctgcagactg ggattgcctt 180

gcttgaggaa ttcgtagggc cggcgattca ggactacctt gcccaagtga agttgcgaca 240

ggtttccgct gacatcaaga actccaaaga gagtcttgac gcactacaag gccagattcg 300

tgcaagctct gcaaatgact tgcccagcct catcgaaaag cggaacgccg aacggaggca 360

cctgcattca cttttgcagg tcgtgtaccc atgggttcgt gcaaaccttt gcgaaagtga 420

tgaggatgta gtgcgcgaat tcaaagtgag agttgaaagt actattttta aatatcaatc 480

ttgcaccagt cactgaagag acaagcgcag atggtttcca acagtgccga gaactcgttg 540

tgcaggctgc caggtgcgtc gaagaataca agaagaacgg agaaaacaga ccaaacaacg 600

ttggcgagca aagccaacaa gctgaaaaac gcgccctctc tcaattgtct gatctagaac 660

ttggtgataa attatcgggt cccaagagca tgacacctga gcaggccatg aagctagaac 720

ccgaattatc taaactggag agtgctctcc aggaagagct cgaagttcta caaggagata 780

cacttagtgg cccattgtcc agaatacttt gcgcgataag ttcaacattt gttcgtttgg 840

aaaataactc tgcaagtgct gtgttgaatg gagcgagcaa gtgcacgcca caagagatct 900

ctgaactgca tgactatgta gtactgggtc taggaaagcc aagtacctca gatgttgagg 960

agctatacca gaaacttgac aagtcgagga aagccacaga aagtctaaag aaagccaaag 1020

aacttgaaac agaggacttg gacgaggagg ttcgagcata ttttagggat cgaatcaatg 1080

aacaagagct cattctctct caaggcaatc ctatcgagct tagcaaagcc tttcgtcata 1140

agcttgctga aactctgcaa catgcgcagg aacattatcc tgagctactg caagatagag 1200

cctggctgaa aaggcttcac ataagcgcag agggtgtttt tgaagtagct tgttctgatc 1260

tttggctggc gaacgtcgag atttcagact tcaagcctgt tggagaactc ctagcttcca 1320

gaggaggtaa gactgtccag aaggttcttg atggagatgg cagaactcta gttctcaaac 1380

agtttcagct tgggcataca agtcaatcca aaaccttcta caaacaagtt gccaatctgg 1440

gcaaagttgt ttctcagaac gtgatccgta tcactggtgc ttttgtggac atgactcatg 1500

gagcaccgcg tggctgtatt gtcatgcctt tctacgagca aggtgacctt gccaagtgga 1560

ttgagtctca tcctaacgaa ggcaaagcag cgcgcgaccg acttgctatg ggcctgctca 1620

tcggtatggc ggatttacat acccatggca ttgtgcattg tgacatcaaa ccagaaaaca 1680

ttttcctcac aagcaacggg acaccattaa tcggcgattt tgacggaatt aaggttgcag 1740

actacactgc aacgtacaca tcacttcaag taacgccaaa gtatcttgcc cccgagttgc 1800

agaatgggcc tgtttataag tttgaaacag cgatggacat gtattccgtg ggtgtaagct 1860

taaaggagct gtatcacact gaacggactg ctgcgatgca gacacttatc gatgctctta 1920

ccgcaacaga tcctggtcag cgtccaaccg cgcgccaagc actccaacat gaagcttttg 1980

gagctcctaa gatccctgtc aagatatgtc tagtatgcat ggaggagtat cagctttccg 2040

aaggcacgag ctgcgaggag ggagacttcc tttgtagaga ctgtatcgaa tctgcagtag 2100

aagcggcggc acagccacta gctaacgtaa gagtcgatgc tgatggaacg atggagtgca 2160

tgaaacctga atgtgttgga agaatatcag gtcaagaaat cactcgtctt gcaccaagtg 2220

ctttaaatca tcttttgttg attgcgaaga cgaaggcaga aattgaagca gcagtttggg 2280

ctgagcaaga gatccaaagg cgcatagcag aagctttgcg tgcagatgga cagaatcgcg 2340

acgcacagcg gcatttactt catattcaag agaagatctt gagcacatgt tgtccgaact 2400

gcaaggcata tgtacacgat tttgacggtt gttgcgctgt agagtgtggt aatgatggtt 2460

gtggtcatag tttctgcgct tggtgtcttg agtttagttc tcaggattca caagcttgtc 2520

atgctcatgt gctagtctgc tcaagaaatc ttagcaataa taagtcatat ttcgctgagt 2580

cattcgagca ggttcgtgag gcgtggcagt tgttacgcgc agaacgccta cacgagtact 2640

ggaatgcgaa tattcaagat gaaaatcttg gacatgctct cgaacagcag cttgtgccgc 2700

tgctaacgcc agacatcgtt ggcccagagt ttaaactgtg aaaactttta aaaaatgatt 2760

ttagtaacta agctttaggt gcattgtgct ttttgtgtag atgcctaggc ttgggtaaga 2820

gcaaggtttg atctttcttg cttaaaatga aatttgctac taccaagcgt tacaatagct 2880

caatattgtc atgcacaaat cctacttact atgatacaag tttagagaca aatatcagaa 2940

atttactgat attgtgaaaa gcttcattct acctgtcacg aattgtttgt gtgtctaaag 3000

tatcgtggta aaagtagaag cagactattc tctcgcttct ttaaatgttc ttagaagcac 3060

aacatctctg gctttaggat gcacttctgt ggtatctgct gatcaaattc taagcgaaac 3120

tgtctttgat cacattttaa ttcatgaata ttgaactcaa ggcctcagcc tctagtttta 3180

gtctttccta aggacacttc gtcggcgttg gcctccccgg gatctgcttc gccataggac 3240

ctaaactgtg cagcatcgag gatgttctgc caagtcacta ctgtagtatc tccgccacgg 3300

ttcccaccac taccactact accaccacca ccgctatcgt ccgaggtgta ggcttcgtcg 3360

ctgccacctt cgtactcact attgccgagc aaccgtccac gaccgtcgca ataggctttg 3420

tccttcttca accacttgct tacgttttgc atgcaccagt ccgagttttt caatacctca 3480

acgaatctgg aaagaaaaat tggtttgatg agccaacaca agaaatgatc gagttcctct 3540

taagtgaaaa cgaagcaatc ctcgccgaag gtcgcgccga aggtcgcgcc aaagttctcg 3600

ccgagttcat tgaacccctc ttggcggggc ttagaaagcc cctgtttctc aaagttacgc 3660

ttcgtgttca tgcatcaact gctgcgacac cttcggcatg gggtcttgag ttcatatccc 3720

tcaagctatg gcggggtttt acaagtgaaa ttataaaccc gtttctcagt gcaaatttac 3780

tcaacatgca aatacgcaat acttcgtttc gaacctcgct ctatccacta aacattagcg 3840

gaaacaccaa tgcaatggct agtttgttta ataattggtg ctgcaggcca tttgttggac 3900

ttcacactga ggttgcaaat tttcctaaag tcatcgtacg catagaagac gatatcggcc 3960

tttcagccaa gcctgatctc accttggtca aaattgatga tcaaagaaat gagactgtct 4020

tctctattgt tgaagttaaa catcctaatc attttttgcc cttgtctgag aaccccaatt 4080

ggaccgacag gcgaaacgcc gctgcagctg cgggaattgc tatttttaca tctagaggcc 4140

aaccgagaca agctcttgaa caattacatg gtaatatgct tatccacggg gttaaatacg 4200

caattctgac atcagtagaa ctcacttatt ttgttaagcg ggataacaag ggtaacatgc 4260

ggatcacccg tggtttctac ggtgctgaaa ctgggggcgc aagagtaata agcaccaacg 4320

aggcaatggc tgcttttata tatattgcgt ccagggatcc tgacaatgga aagtttagac 4380

caagatcaaa tagcacgagc acagaaatca gggcttattc tgaacgtatc cagacaaaat 4440

ttggtggtcg ttcgataagt gatagagcca cagagaagtt caaagatagg gtgggcgaaa 4500

cagccttaca attcatgaaa ggtctaaacc aactcgcagt aaatatagag gagctacgca 4560

gcaatcttat tgaagtttta cctttcaagg atgaagctta ccttttcatg ccgctgccat 4620

ggtccaaaga ctaccgactt gtcacaagtt atgtggttcg gattccctgg gtttcaaaca 4680

aagacttcaa ctgggatatc tttgtgaaga ccatggcggt ctctaaagac tgggcgagag 4740

agacatatga gttcgaaaaa tccttctttc tcaacgaagt taagctgtac cttggccccc 4800

tgcgcctgct ccaaggtaaa cacgcgccct ttttggtgta cggcggcaca tttcataaac 4860

gaatcatcat tgccacaacc ttctctgggg agaccgcaac aaaagaactt attcttgcta 4920

acagtagctg ggcaattaca gccattcgag catcactttc cgcgctgcac caggtcggtg 4980

tgctccatgg cgacatcgcg ctgcgtaata tcgccatcga caccactacc aagaccgccc 5040

acttgatcga ttttggccgc tcctcacaag acaaaaccac caagaaaaac agggacacag 5100

aaatgaaaga actgaacaca cttctggggt ttgctattca tcgcaagcga atccgagact 5160

cctaactgaa cacacttctg gggtttgcta ttcatcgcaa gcgaatccga gactccttta 5220

aatagccaag acgacagaat acagaatcaa atgaccttct gaacgagtaa acaaatatat 5280

atattttttt taagactaca aatttcttac accaaaatca gcaaagaagg tcttctactg 5340

aaaaaagtgc ctgacgttgc aggatttaat ccaagaaaga ccggtagcat tttgggatct 5400

cttagacgag atatgatttt gtctaaccca acataaaata ctacttaagt ttttttataa 5460

tcggttgata gggaagtagc caagcacatg acccaaaaga aagttaaaag caaggtttcg 5520

cctcatcagt ggctggaatt tctttagttg ctcttggggg ttgacacttt tgtcccttgt 5580

ttcatctttt gcagtgatgt ttgcttcaga agttccccaa ttatttccat ctcctagtac 5640

tacataatgt agtgcggcta ctactggtac ctttatggat aggtaggtaa tttttagtca 5700

attcaatgag gtttggcgcg catcaaaatt tatgctgacg cttcttaata tatatgattc 5760

aatgtgaagt ggggaaatgc gggtaaatga tctgctaaaa tccaatagag tggaactttt 5820

ctagataggc ctaatttatt attgagtatt agccactctg aataaacatc cgaggataga 5880

taagagcttg gaagaaaacg taagaaatga agaaaatcaa ttcatgaacc tgaccgaatg 5940

ggactgactt aggaaataat aatcatttcg ctaatcctgc agatgctgaa gaatgaatgg 6000

aaaaagatga cctccaggtc tgtgaaagta atgttgttgt tgatatcaga cactcattta 6060

catcttcagg gcggaacata atttcaacga atgatcatcg cagcaagaga taaacaatca 6120

caaagagttc tttaatgcat ggcctccgtt gtactccaaa gtatgcgatg catcctcgac 6180

caatttaata aacttacagt 6200

<210> 5

<211> 4500

<212> DNA

<213> Schizochytrium limacinum (Schizoochytrium limacinum)

<400> 5

accgtgggcc aagctggccg ccccaagacg atcactggct tccagactca ctcgactcct 60

gtgctcatga gctttggcga ccgtgctgag ctcgtctctg atgactacga accattgaca 120

agcgtcttgg agggcgttgt tcttctcaag aagaagaaga agcgttcctc caaggatcgt 180

gatggtgatg tggaaatggc aaaataaata attcagactt gcattatctt aaaaaaaaac 240

aaagtgcagc ggtaacaacg actctgttgt tgcgtctact tctaccccga cttattcttt 300

aagaagttcg ttcatagaaa ggctggggtg tgagagagag agcatgtttt ttaactcaac 360

atggataagt aaaaaaaagg ccaggaacat caaggagaag cttcaaatga ctgttaggta 420

gctgttgtag ctccttagca tattcttctt cttactactt aacatctctg ctttcggccc 480

caaatcgctc gcttgcttgt tcgctcgtcc cgatagacgc ttctcattcc tatagcattc 540

tctaaaaaaa taatctaaac attacacctc tcaaaataga aaatctatct tccacacgtt 600

gatctaaatt ctccggcttt tgtatctttg tatcacagtc ctgccaacat tcagcatgtt 660

cactcagccc cgcggattgg tctctctctc cctaaggctg agcctgagca gatctaaacg 720

gacgtcgtga attcatcata tcattgctaa taatttaccg taagttaagc actagaaccc 780

tcttattgtg cctcatatca ttgtaacggg cttggggact catagaggtt gtccgggtaa 840

tgcggggaat atagcgacct aacgaccgct ttaacaaagg gaaataatta ggcagtacgt 900

aaagagtact actggtacct agttatgtaa caaggtaatg aattgtcaat gattcaatgc 960

ttggtaagtt tgttcaagtt cgttccgaag cgaagcaaac tcgtgcaagc ggggtcgcgg 1020

agacgagctc gtcccgcgga cgtacgcacg caggaggttg ggaaggtggt caagtaaagg 1080

ttttgacagc aagatgctct ggccggcaga acacctgcat gaaacctacg agcacattcg 1140

taacgatgcc cttggtagtg ggtgctcagt tgtgatttat gtggcgcttg actgcgatgc 1200

gctatgcgct gtgaagatct tgacaagttt gttgaaagcg gataatgtag cgtataagct 1260

gagacccgtt cgaggatata gtgatatttt agaggatttc cgcgagacat caaaggcaga 1320

agcgattaag tccatcatta tgatcaactg cggtggtgat gtcaatgcac aggaaatgtt 1380

taacttggac gacggcatga catgctatat cattgactcg gcgaggccct acaatcatgc 1440

aaacctactc cggtctcata tgcacacgat tgtgtttgca gatgacttca tgaaggagga 1500

agatctcgtt aaggaggcag aactgctcga gcacctggat gaggataacg tagatgaact 1560

cctgggatcg aacgacaacg acgatgacga cgatgacgac gatgacgacg atagcgatga 1620

tggtaaagga gaggtcgagg acgggaatga tgcaaactct gctcgtaacg ggcaagaagg 1680

agagattgat tccgacgaag aacacgaatt cgatggaggt aaccctcagc agaatgcctc 1740

agatagtgat gacgatgaaa atgaagatgc caataagcag actagcaaca acacaaacaa 1800

taagacaagc aaagcaaagt ctgcatctca acttctgaca gagcaagagg aggcagagat 1860

cgagcgtgag cttgctgcag aaaatggcac ccgaaagaag cggaggcgga cttcttcctc 1920

ctcatcctct tcacaaaacg acaaaaacaa cgatagtggc gatgatgatg atgatgatga 1980

tgatgacgat gataaggatg atgatagtct tcctaaacgg aaggtccgtg aaaaggttga 2040

cgctgaggaa cccatggatg acaaagcacg tgtagcaaag tactacgctg ggtcattccg 2100

gggtacatcg gcagcacatg tcctgttttc cctctcacag cgtctcaaca aagatcaaaa 2160

gatttttctt tggcttgcca tcgttgggac tactcaccac ttcatcaatt ctgaactcag 2220

cgaggaggac taccttctcc gtgtactcac ataccaggat ctcgttaaag accgctccat 2280

gtctcgtcga gcaggccaac atacagtcac agaagatggt gctgaggtgc ctcttatgga 2340

aggccaaagc atggaattca ttgaagaact tcgtcttatg ctgcatcgcc actggtcgtt 2400

gcacgaggcg tttctgtact ctgactacat tgctgccaag cttggaatct ggaaaaatga 2460

cggtgaagcc aagcttcgca catttttcgc caagatggga attagtcgga aagaagcaga 2520

gcaaaagtat tctttcatga acatgagtgt taagagggct ctcaaagata aaattggtgc 2580

tcatggagcg gacttttctc ttgacgaaag cttcgtctac gcatcattcc agttccgggc 2640

tggctttggg caccaactat cagctgctga taccgcatac tgtatggctg ctttgctcga 2700

gagtgcagct acacacccta cagtttttgc cgatgattct ggtgctgatg ctacagagga 2760

tgctactgaa atccccgagg ttgaccttga cggggacgac cttgattatg ctggtgaaga 2820

tggagcaaat gggcaaaagg aaaatagcaa ggcaattctg gcagcttcac tttggaaaca 2880

aagcttcaat gcagcttatg atgctctctc tttctcacaa acaactcgca accagcatct 2940

cctggaacaa ggccttaacc tcgccaaagc tctgcaaaag gctattattc aggagggtgg 3000

aaatataatt tcaggaacaa agattgccag tgcaggttct tttcgctact gtattctgga 3060

aggtttgcca cccaagctgg tgagtatttt ttcccagcct gatacacttc tccgccttgc 3120

caaattcatc atgctcgcgt acacaagtgc cggcaagtgg tcgggcctgg gcgccaaacc 3180

ccttgttctt ggtgtcaaaa acctcaagac aaacaaagct cattttgttg gcctccccca 3240

cccaactttt ggagatgacg cgattgcaaa gaaccccttt ggcagatatt ttcgtgtggc 3300

cgcccaaaaa gtgggcacgg cccacatcca tgttggcttt agctcggcat gtatcgagat 3360

ccccctcgag cttgtgcaga aattcttagt ggcattgcat gaaataacag cttgaataaa 3420

tggttgatta tatgctttaa ggttcctttt cctctttgtt cccattgctt ttacctctgt 3480

tgatacaagt ggcctcacac tacaattgaa gagccattgg ataagttcga ttattaatta 3540

gagaatgagt ttccactctc aattctttct attaggcagt gttgattatt acatgagcat 3600

acgtgcatca tcgtcattat atcggcaaat acgaatatct agatgtgcta accgaaagag 3660

ttccaagatt acagtgatat tgttggaagc atatatatga attaatatgc tgcagaatct 3720

ttgaacccgc cgggtgtgtt atcgttctat tggaactggc agtggccctg atgttggcta 3780

ttaagtaggg gagaaggttt ctactggaag tgaatgcatc gctctctaag agcgcgcact 3840

tcaaatgtta cttccgattt cagcgttgtt tgctaggttc tcattatgcg tttatactcg 3900

cttttgagct cttgaggatg accataccga gctacgggct gcacttgtgc tgtcaagagg 3960

ctcaaaatcg gggtctggtt cacggtaatc gaggtcgtga agaagatagc ggtatagttg 4020

atcgcgcatt gcttgattct tgtagttatt gtctgcaaat tgcatttgca tcgcaaaggt 4080

ccgctctaga ttctgaacag catgttgcgt cattccaggg atgaaaacaa tttcaccttc 4140

tttctgtacg cagtgaagaa caacagaatc atcttcaagc tcttcggtta ctttctttgc 4200

cttaattttg cttgcaaacg cgcctgcgcc aaacttactt tgaagttcgg cgtttttctc 4260

aagcttgggg agtttctctc caggcttcgc tttcgattta aacttcttga tcaagtacgt 4320

ctgctcttca tgagccttac agacagggtc gtgatccaga aatctcgagc agtatgtctc 4380

ctctagactt ggtgcgggct caagaagctc tacctcatct tgggcttcca actcagaata 4440

tccagcaacc acgcggttag ataccttcat ataccagacc ttcttaccct caaagataag 4500

<210> 6

<211> 11100

<212> DNA

<213> Schizochytrium limacinum (Schizoochytrium limacinum)

<400> 6

cattgattga ttgcagatga tcttgggcaa cgcgcgtcag cttgagcgag gaatgctttg 60

gacttcaggt tcttcgcttc tgtgtttcat tctttctcga agaaagaaag aatgaaagaa 120

agagagaaag aaagaaagaa agaaagaaag aaagaaagaa agaaagaatg aatgaatgaa 180

agaaagagag aaagaaagaa cgaatgaaag aaagagagaa agaatcaaag agaaagcgca 240

ttcgcagttc ttcttcgtga aagaaaagga aaagagaggc gatggtaggc tctgatctca 300

tcatttctgg tttctctgtt gtacctgtac tctgtgcttg tggccttgcg aaggctgaag 360

acgccatgca gacaaccacg cctccgcaga gactttgcgg gaaagcagag ggcttctcgc 420

cactctcgaa gaaacgagct cgccagtttt cggggttgtt ctcagaattg cgagtgttgg 480

ctttatatgg gatgatggta tggcacttcg tcatcgttac tctcgctcgc ttgcttacga 540

agattttcaa aagggcgaaa gaagtgctca gcttttaaaa taaagtcaca ccaaagacta 600

ggccgcatag cagaaagcta aagtaaaccc aatctgtctg aagagagtgt cgtggttaga 660

tacttacgca agagtttaaa agctgtaaat agtacaggaa caaaaacaaa taaatatata 720

tatattcttt tttattagta aaacatgaaa ccaaaaaact cctttaaaat aaaataaaat 780

aaaataaaat aaaataaaat aaaataaatt tactactata tatacatata tatatacaat 840

aaataaaaac aactttttca gaccagaaaa agactgagaa aaaaggaaac taatgactct 900

cgagcaccga gagcgatata agagtggatt atatttgcta ggcccaccac gagtgagtcc 960

cctaggagga agcgccctct gagacaggag cagaggcgtc gctggtgctc caaaaagcga 1020

cggcgaatgg aaagcaaaac cctttcgagg gaggcttgtg gccgtgacta ttcaaatctc 1080

cagcatctca gctccagcac agcagaagct acctcgcttc tcagctctag ctatcacatc 1140

gatcgcagca tctagctcgt agacagctag cgccgcacct tcccccaaat caacttgggc 1200

aacttaactc ttttttcacc agaactcctc ttttccttta atcttcgaaa agaagacgaa 1260

taaaagagat aatcctctgc cgcagcacat tctaaaagaa aagcggcata ctggcgtagg 1320

caagactttc aagctcttcc tcgcctccac cccgtatttc cctgttcatc tttgtgaaac 1380

gaggaaacaa gaaattttat aggacaagat ggctcaacgt gagaaccgtc tcgaggccaa 1440

catggatacc cgcatcgctg tgatcggcat gtccgccatc ctcccctgcg gtaccaccgt 1500

tcgtgagtct tgggaggcta tccgcgatgg tatcgactgc ctcagtgatc tccccgagga 1560

ccgcgtcgat gtgaccgcct acttcgaccc ggtcaagacc accaaggata agatctactg 1620

caaacgtggt ggattcatcc ctgagtacga cttcgacgcc cgtgagttcg gcctcaacat 1680

gtttcagatg gaggactccg acgcaaacca aaccgtcacc ctcctcaagg tcaaggaggc 1740

cctcgaggac gctggcatcg aagccctcag caaggaaaag aagaacattg gatgtgttct 1800

cggtatcggt ggtggccaga agtccagcca cgagttctac tcccgcttaa actatgttgt 1860

cgttgagaag gtccttcgca agatgggcat gcctgaggag gatgttcaag ctgctgttga 1920

gaagtacaag gccaacttcc ctgagtggcg ccttgactcc ttccccggtt tcctcggcaa 1980

cgttactgcc ggtcgctgta ccaacacctt caacctcgat ggtatgaact gtgtcgtcga 2040

tgctgcctgt gctagttctc tcatcgccgt taaggttgcc attgatgagc ttctccacgg 2100

agactgtgac atgatgatca ctggtgctac ctgcacggat aactccatcg gtatgtacat 2160

ggccttctcc aagaccccgg tgttctctac cgaccctagc gtccgcgcat acgatgagaa 2220

gaccaagggt atgcttattg gcgaaggctc tgccatgctt gtgcttaaac gttacgccga 2280

cgctgttcgt gatggtgacg agattcacgc tgtcattcgc ggctgcgcct cttcctctga 2340

cggtaaggcc tccggtattt acaccccgac catctctggt caagaggagg ctcttcgccg 2400

tgcctacatg cgcgctaacg tcgatcccgc caccgtcact cttgttgagg gccacggtac 2460

cggtaccccc gttggtgacc gtattgagct caccgctctc cgtaacctct tcgacagtgc 2520

ctacggcaac gagaaggaga aggtcgctgt tggcagcatt aagtccaaca tcggtcacct 2580

caaggctgtc gccggtcttg ccggtatgat caaggtcatc atggccctca agcataagac 2640

tcttccggcc accatcaacg ttgatgagcc ccctaagctt tacgacaaca ctcccatcac 2700

cgactcatcg ctgtacatta acacgatgaa ccgtccgtgg ttccctgctc cgggtgtgcc 2760

ccgtcgcgct ggtatctcca gtttcggttt tggtggtgcc aactaccacg ccgttcttga 2820

ggaagccgag cccgagcacc agaaggctta ccgtctcaac aaacgccccc agccggtgct 2880

tctgatggca tcttcaaccc aggctcttgc ttccctctgt gaagcccagc ttaaggaatt 2940

cgagaaggct atcgaggaga acaagaccgt caagaacact gcttacatca agtgcgtcga 3000

cttctgtgag aagttcaagt tccctggatc tatcccgagc tctaacgctc gcctcggttt 3060

tcttgtcaag gaggccgatg atgccaccga gaccctccgt gccatcgttg cccagttcca 3120

aaagtcagct ggcaaggatt cttggcacct tccccgccag ggtgtgagct ttcgtgctca 3180

gggcatcaac accactggtg gtgtcgctgc cctcttctct ggccagggtg ctcagtacac 3240

ccacatgttc agcgaggtcg ccatgaactg gcctcagttc cgtgagagca tctctgacat 3300

ggatcgtgcc caggctaagg ttgctggcgc tgacaaggac tacgagcgtg tctcccaagt 3360

cctctacccg cgtaagcctt ataactctga gcccgagcag gaccacaaga agatctccct 3420

gacctcatac tctcagccct ctaccctcgc ctgcgctctt ggtgcctacg agatcttcaa 3480

gcaggctggt ttcaagcccg acttcgctgc cggtcactct ctcggtgagt ttgcggccct 3540

ctacgctgct gactgcgtca accgtgacga cctctttgag ctcgtgtgcc gtcgtgcccg 3600

catcatgggt ggcaaggatg cacctgctac ccccaaggga tgcatggctg ctgtcattgg 3660

acccaatgcc gagaagatcc agattcgcac tgctgatgtc tggctcggca actgcaactc 3720

cccttcgcag actgtcatca ccggctctgt tgagggtatc aagaaggagt ccgagcttct 3780

ccagagtgag ggcttccgtg ttgtccccct cgcctgcgag agtgccttcc actcaccgca 3840

gatgcaaaac gcctcctctg ccttcaagga tgttctctcc aaggttgcct tccgtcagcc 3900

tagcgcccag accaagctct tcagcaacgt gtctggcgag acctactcca acaatgccca 3960

ggacctcctt aaggagcaca tgaccagcag tgttaagttc atctctcagg ttcgcaacat 4020

gcactctgct ggtgctcgca tctttgtcga gtttggcccc aagcaggtgc tctctaagct 4080

tgtttccgag accctcaagg acgatccttc cattatcact atctctgtca acccttcctc 4140

tggcaaggat gccgatattc agcttcgcga ggctgctgtg cagctcgttg ttgctggagt 4200

caaccttcag ggcttcgaca agtgggacgc acctgacgcc acccgccttc agccgattaa 4260

gaagaagaag actactcttc gtctctcggc tgccacttac gtgtctgaca agaccaagaa 4320

ggctcgcgag gctgccatga acgacggccg catgctcagc tgtgtcagca aggtcatcgc 4380

cccccctgac gccaagccca ttgtggacac caaggctcag gaggaggttg ctcgtctcca 4440

gaagcagctt caggatgccc aggcccagat ccagaaggcc aaggccgatg ctgctgaggc 4500

tgacaagaag cttgccgctg ctaaggatga ggccaagcgt gccgccgctt ctgcacctgt 4560

gcagaagcag gttgacacca ccattgttga taagcaccgt gctatcctca agtctatgct 4620

tgctgagctt gactgctact ccactcctgg tgctgtgtcc agctctttcc aggcacctgt 4680

tgctgctacc cctgctccgg tcgctgcgcc tgttgcagct gctcctgctc cggctgtcaa 4740

caatgctctc cttgccaagg ctgagtctgt tgtcatggag gttcttgccg ccaagactgg 4800

ttacgagact gacatgatcg agcccgacat ggagctcgag actgagctcg gcattgactc 4860

tatcaagcgt gtcgagattc tctctgaggt ccaggcccag ctcaacgttg aggccaagga 4920

tgttgatgct cttagccgca cccgcaccgt tggtgaggtt gtcaacgcca tgaaggctga 4980

gatcgctggc agctctggtg ctgccgctgc tgccccggcc cctgttgctg ctgctcccgc 5040

tgcccctgcc cctgctgtca acagcgctct tcttgccaag gctgagactg ttgtcatgga 5100

ggttcttgcc gccaagactg gttacgagac tgacatgatt gagcccgaca tggagctcga 5160

gactgagctc ggcattgact ccatcaagcg tgtcgagatt ctctctgagg ttcaggccca 5220

gctcaacgtt gaggccaagg atgttgatgc tcttagccgc acccgcaccg ttggtgaggt 5280

tgtcaacgcc atgaaggctg agatcgctgg cagctctggt gctgccgctg ctgccccggc 5340

ccctgttgct gctgctccgg cgcccgtcgc tgccgctgcc cctgctgtca gcagcgctct 5400

ccttgagaag gctgagtctg ttgtcatgga ggttcttgcc gccaagactg gttacgagac 5460

tgacatgatt gaggccgaca tggagctcga gactgagctc ggcattgact ccatcaagcg 5520

tgtcgagatt ctctctgagg tccaggccca gctcaacgtc gaggccaagg atgtcgatgc 5580

tcttagccgc acccgcaccg ttggtgaggt tgtcaacgcc atgaaggctg agatcgctgg 5640

cagctctggt gctgctgccc cggccccggt cgctgcggcc cctgctccgg tcgctgccgc 5700

tgcccctgct gtcaacagcg ctcttcttga gaaggctgag actgttgtca tggaggttct 5760

tgccgccaag actggttacg agactgacat gatcgagccc gacatggagc tcgagactga 5820

gctcggcatt gactctatca agcgtgtcga gattctctct gaggtccagg cccagctcaa 5880

cgttgaggcc aaggatgttg atgctcttag ccgcacccgc accgttggtg aggttgtcaa 5940

cgccatgaag gctgagatcg ctggcagctc tggtgctgcc gctgctgccc cggccccggt 6000

tgctgctgct cccgctcccg tcgctgcccc tgctgtcagc agcgctctcc ttgagaaggc 6060

tgagtctgtc gtcatggagg ttcttgccgc caagactggt tacgagactg acatgattga 6120

ggccgacatg gagctcgaga ctgagctcgg cattgactcc atcaagcgtg tcgagattct 6180

ctctgaggtc caggcccagc tcaacgttga ggccaaggat gtcgatgctc ttagccgcac 6240

ccgcaccgtt ggtgaggttg tcaacgccat gaaggctgag atcgctggca gctctggtgc 6300

tgccgctgct gccccggccc ctgttgctgc ctctcccgct cccgtcgctg ccgctgcccc 6360

tgctgtcagc agcgctctcc ttgagaaggc cgaatctgtt gtcatggagg ttctcgccgc 6420

caagactggt tacgagactg acatgattga ggctgacatg gagctcgaga ctgagctcgg 6480

cattgactct atcaagcgtg tcgagattct ctctgaggtc caggctatgc ttaacgttga 6540

ggccaaggat gttgatgctc ttagccgcac ccgcaccgtt ggtgaggttg tcaacgccat 6600

gaaggctgag atcgctggca gctctggtgc cgccgctgct gccccggccc cggttgctgc 6660

tgctccggcg cccgtcactg ccgctgcccc tgctgtcagc agcgctctcc ttgagaaggc 6720

cgaatctgtt gtcatggagg ttctcgccgc caagactggt tacgagactg acatgattga 6780

ggccgacatg gagctcgaga ctgagcttgg cattgactcc atcaagcgtg tcgagattct 6840

ctctgaggtc caggctatgc ttaacgtcga ggccaaggat gttgatgctc ttagccgcac 6900

ccgcaccgtt ggtgaggttg tcaacgccat gaaggctgag attgctagca gctctggtgc 6960

tgctgcccct gctccggctg ctgccgttgc accggcccct gctgctgccc ctgctgtcag 7020

cagcgctctc cttgagaagg ccgaatctgt tgtcatggag gttctcgccg ccaagactgg 7080

ttacgagact gacatgattg aggccgacat ggagctcgag actgagcttg gcattgactc 7140

catcaagcgt gtcgagattc tctctgaggt ccaggctatg cttaacgtcg aggccaagga 7200

tgttgatgct cttagccgca cccgcaccgt tggtgaggtt gtcaacgcca tgaaggctga 7260

gattgctagc agctctggtg ctgctgcccc tgctcctgct gctgccgctg caccggcccc 7320

tgctgctgcc cctgctgtca gcagcgctct tcttgagaag gctgagtctg ttgtcatgga 7380

ggttctcgcc gccaagactg gttacgagac tgacatgatt gaggccgaca tggagctcga 7440

gactgagctt ggcattgact ccatcaagcg tgtcgagatt ctctctgagg tccaggctat 7500

gcttaacgtt gaggccaagg atgttgatgc tcttagccgc acccgcaccg ttggtgaggt 7560

tgtcaacgcc atgaaggctg agattgctag cagctctggt gctgctgccc ctgctcctgc 7620

tgctgccgct gcaccggccc ctgctgctgc ccctgctgtc agcagcgctc ttcttgagaa 7680

ggctgagtct gttgtcatgg aggttctcgc cgccaagact ggttacgaga ctgacatgat 7740

tgaggccgac atggagctcg agactgagct tggcattgac tccatcaagc gtgtcgagat 7800

tctctctgag gtccaggcta tgcttaacgt tgaggccaag gatgttgatg ctcttagccg 7860

cacccgcacc gttggtgagg ttgtcaacgc catgaaggct gagatcgctg gcagctctgg 7920

tgctgctact gcctctgccc ctgctgctgc agctgccgcc cctgctatca agatctccac 7980

tgttcacggt gctgactgcg atgacctctc tgtgatgtct gctgagcttg tcgacattcg 8040

tcgcgctgat gagctccttc ttgagcgccc tgagaaccgc ccggtcctta ttgtcgatga 8100

tggtaccgag ctcacctctg ctctggttcg tgttcttggt gctggtgctg tagttcttac 8160

ctttgacggt cttcagttgg ctcagcgtgc tggtgctgct gttcgccatg tccaggtgaa 8220

ggacctctcc gctgagagtg ccgagaaggc tatcaaggag gctgagcaac gcttcggcca 8280

gcttggaggc ttcatctctc agcaggctga gcgctttgcc cctgctgaca ttcttggttt 8340

caccctcatg tgcgctaagt ttgccaaggc ttccctctgc acccctgtgc agggtggccg 8400

tgccttcttc attggtgtgg cccgtcttga cggtcgcctt ggtttcacct cccagggatc 8460

tactgactcc ctcacacgtg cccagcgtgg tgctatcttc ggcctctgca agaccattgg 8520

ccttgagtgg tctgctaacg aagtgttcgc ccgcggtatt gatattgctc gtgaggtcca 8580

ccctgaagat gctgccgtcg ccatcactcg cgaaatgtcc tgcgctgaca accgtatccg 8640

cgaggtcggc attggcctca accagaagcg ctgcaccatc cgtgctgtgg acctcaagcc 8700

gggtgccccc aagatccaga tcagccagga tgacgttctc cttgtgtctg gtggtgctcg 8760

tggtattact cctctctgca tccgtgagat cacccgtcag gtccgcggtg gtaagtacat 8820

tctcctcggt cgctccaagg tccctgctgg tgagcctgct tggtgcaacg gtgtttctga 8880

tgacgatctt ggcaaggctg ctatgcagga gctgaagcgt gctttctccg ccggtgaggg 8940

ccccaagccc accccgatga cccacaagaa gctcgttggc actattgctg gtgcccgtga 9000

ggttcgttcc tcaattgcta acattgaggc tctcggtggc aaggcaatct actcctcttg 9060

tgatgtgaac tctgctgctg atgtcgccaa ggctgttcgc gaggctgagg ctcagcttgg 9120

cgcccgtgta actggtgtcg tccacgcttc tggtgtcctt cgtgaccgcc tcattgagca 9180

gaagcgcccc gatgagtttg atgctgtctt cggcaccaag gtgactggtc tcgagaacct 9240

ctttggtgcc attgacatgg ccaaccttaa gcacctcgtc ctcttcagct ctcttgctgg 9300

tttccacggc aacattggtc agtctgacta cgccatggct aacgaggccc tcaacaagat 9360

gggtcttgag ctctctgacc gtgtgtccgt gaagtctatt tgcttcggcc cctgggatgg 9420

tggcatggtt accccccagc tcaagaagca gttccagtct atgggtgttc agatcatccc 9480

ccgtgagggt ggtgccgata ctgtggctcg cattgtcctc ggctcctccc ctgctgagat 9540

ccttgttggc aactggacca ctcccaccaa gaaggttggc agtgagcccg ttgtgatcca 9600

ccgcaagatc agcgctgcat ccaacccttt tcttaaggac cacgtcatcc agggtcgctg 9660

tgtgctcccc atgaccattg ctgtgggctg ccttgctgag acctgcctgg gtcagttccc 9720

tggatactcc ctctgggcta ttgaggatgc tcaactcttc aagggtgtca ccgttgacgg 9780

tgatgtcaac tgtgagatca ctctcaagcc ttcccagggt actgccggcc gcgttatgat 9840

tcaggccacc ctgaagacct tcgctagcgg caagcttgtt ccggcttacc gtgccgtgat 9900

cgttctctcc actcagggaa agccccctgc tgctactact tcccagaccc cctctctcca 9960

ggctgatcct gctgcccgtg gcaaccctta cgacggcaag accctcttcc acggccctgc 10020

cttccagggt cttaaggaga tcatctcttg caacaagtct cagcttgtcg ccgagtgcac 10080

cttcattccg tcttccgaga gcgctggtga gttcgcttct gactacgagt cccacaaccc 10140

tttcgtcaac gacattgctt tccaggccat gctcgtctgg attcgccgca ccctcggcca 10200

ggctgccctc cccaactcta tccagcgcat tgtgcagcac cgtgctcttc cccaggacaa 10260

gcccttctac ttgaccctca agagcaacag cgcgagtggc cactctcagc acaagacctc 10320

cgttcagttt cacaacgagc agggtgacct cttcgtggac atccaggctt ccgtcacctc 10380

ttctgactcc cttgccttct aaagttgtga ggctgtcttg tcttgtcagt cgcgaaagtg 10440

taagcaagaa ctttgtcata caaagaagca accaacttcc gaaccaacac accttgtagg 10500

attacaacca caactttcta taaatagtgc gcaagaataa ccagtaagct atccttcgtg 10560

tacctgttac aacaacgaca tttttacttg atcttcctac ttgtgatggg tagtcccggc 10620

ttgtactgac agtgatgcca cagcagagta gatcactgtg aataagtaaa taagcctact 10680

tattatattc ccaaagtact cgctgggata ttattagtat cacgaaaagt gatatgtttt 10740

ataactcgct tgtcttgcca agatctaacc tttttttttt aaatggccaa aaagtcgcca 10800

gaacacatct tacaataaac aaaaatttag attatatcgt atgtataatg tataatatat 10860

tatattatta tatacatacg atataatcta aagccattcc agacttattc ggtgatgaaa 10920

aatgctttcc cagctttata caaactattc aaaaagttgc atgacccatt ttcagatata 10980

tttaatagta taagattatg tccatttgtt ttcaaagtta ttcaagagtt tacatcttga 11040

agtttcatcc ctttactact acactgtttt tcgtttgggt tttttctcta acggcgaaag 11100

<210> 7

<211> 7767

<212> DNA

<213> Schizochytrium limacinum (Schizoochytrium limacinum)

<400> 7

tattgctatt acttgaattt gaatttgaat ttgaattcag gtagacaata aaataagatt 60

agcaaaacat tttgagagga agcagaggat atgcagtgca aaaggaggtc ccgagtttcg 120

atcttctttg cacctgctac gtatctagtg cacgtagagc aagaaagaat gaaagaaaga 180

acgaaagaaa gaaagagaga gagagagaga gagagagaga gaaagcgaag atgatagcgg 240

agagaactct tcttcgcagt cactctgttt ctcagtcagt cccgcaacca ataacaactc 300

gaactcgcag cagtgttctt cggagtgcca gcgctcgctc gcactgcgtc ggcacagcag 360

cagcagcagc aggccccgcg ctcgctgcac tcagcccggg caggagcaac agctgctgag 420

cagctgaggc cagctggctg gcggctcgcc tcgcctcgcc tcgcgtcgcg tcgcgagaga 480

aagcgatcga ccaactgtca atcgattatt cgagtccttc gagcgcttta tagggcactg 540

attgatcact cattgattca ttgactcatt tattctttgc gtggtcagcc aaacggcgtt 600

agcattgggc aaagcgggtc tttgctttgc tctaaaatag atttgctcgc gagagtacgt 660

acttgcagga gtaggtaggc tctgcctagt acctgggcat ttgaatattt gaacttcgaa 720

cttcgttgag tatctgaata tttgaatatc tgaatatttg aatttcgaaa gtttgaatat 780

ttgaatattt gaattttgga atattggaat agctgggttt ggagataaga cttactaagc 840

taagcgccga cgtaagagcg gcgagtaaat ccacacacaa gagagaggca gagagagagg 900

gagggagaca actcgcgcag gcaagctgag cccactggac gcacggggcg cgtcccccct 960

gacgggcgct ctggtggtgg cgtgtttggg agggttttgc atgcttgtga taggggctct 1020

ggcgcgggct ctgtacggtg cttggagatg cacgggcagg gcgagagagg ggacgggttc 1080

ccgggaggcg ctgcttggag gtgctgagag ggagggagaa ggcgtgcttt gcgatgcgcg 1140

gggcgaccta ggcgctgctg cgcggtgcag cagcagggac ctcggacgtg agtcgaagcc 1200

gtctgcagag gagatggtag aagggccgcg gattggtagc agagaagagg aaatagaaga 1260

agaagaagaa atagaagaag aagaaataga agaagaagaa atagaagaag aagaggagga 1320

cgggcaggcg ggaaagatgg agaaaggact cgcggcggga aaacaagaga atgtgaactt 1380

gggcttgaac tttggtttga atttgaatgt ggagaacgag gggttgaatt tgagtttgaa 1440

tttgaaagaa aacttacgga aagaaagttt agttgaaaaa agaaaaagag aaagaaaaag 1500

agaaagaaaa agagaaagaa aaagagaaag aaaaagagaa agaaaaagag aaagaaaaag 1560

agaaagaaaa agagaaagaa aaagaagaag aaaaagaaga agaaaaagag aaagaaaaag 1620

agaaagaaaa agagaaagaa aaagaagaag gagatttaaa aagttgttta gttgaaaaag 1680

gagaaggagg aagaagcagc gacagcggca gaagaagaag tagttgttgt aagaggggaa 1740

cggaggcagt agcagtggag caggcggagg cgacagcaaa cctcgaactc gaccccgtcg 1800

agccgcagca agaacaagag cccgaccagg tggacgagga cgaggtccgc ttgttgtcag 1860

gaacaacaga agttgcagga ctagccgaga gtgctaccac tgcaattctt agatccacag 1920

acgcaagagc agaaaactta caactgctcg ccacaacaca agaaccacct tcagatacaa 1980

ccaggttcga gaactccaca agtctagaag cagcaacagc tctagcagat aatcaaacag 2040

gtccagaaaa agctacgact agaagagaaa ttatcgagtc gcaacttgca accatggcca 2100

ctcgcgtgaa gaccaacaag aaaccatgct gggagatgac caaggaggag ctcaccagcg 2160

gcaagaacgt cgttttcgac tatgacgagc tccttgagtt cgccgagggt gacatcagca 2220

aggtcttcgg ccccgaattc agccagatcg accagtacaa gcgtcgcgtt cgtctccccg 2280

cccgcgagta cctcctcgtc acccgcgtca ccctcatgga cgccgaggtc aacaactacc 2340

gcgtcggtgc ccgcatggtc actgagtacg acctccccgt caacggtgag ctctctgagg 2400

gtggtgactg cccctgggcc gtgctcgtcg agagtggtca gtgtgatctc atgctcatct 2460

cctacatggg tattgacttc cagaacaaga gcgaccgcgt ctaccgtctg ctcaacacca 2520

ccctcacctt ctacggtgtt gcccaggagg gcgagaccct ggagtacgac atccgcgtga 2580

ccggcttcgc caagcgtctc gacggtgaca tctccatgtt cttcttcgag tacgactgct 2640

acgtcaacgg ccgtctcctc atcgagatgc gcgacggctg tgccggtttc ttcaccaacg 2700

aggagctcgc cgccggcaag ggtgtcgtct ttacccgcgc tgatctcctc gcccgcgaga 2760

agaccaagaa gcaggacatc accccgtacg ccattgcccc gcgtcttaac aagaccgttc 2820

tcaacgagac tgagatgcag tccctcgtgg acaagaactg gaccaaggtt ttcggccccg 2880

agaacggcat ggaccagatc aactacaaac tctgcgcccg taagatgctc atgattgacc 2940

gcgtcaccaa gattgactac accggtggcc cctacggcct tggtcttctc gttggtgaga 3000

agatcctcga gcgcgaccac tggtactttc cgtgccactt cgtcggagac caggtcatgg 3060

ctggatccct cgtgtctgac ggctgcagcc agctcctcaa gatgtacatg ctctggctcg 3120

gcctccacct taagaccggt cccttcgact tccgccccgt caacggccac cccaacaagg 3180

tccgctgccg tggccagatc tccccgcaca agggtaagct cgtatacgtc atggagatca 3240

aggagatggg ctacgacgag gctggtgacc cgtacgccat cgccgatgtc aacattctcg 3300

acattgactt cgagaagggc cagactttcg accttgccaa cctccacgag tacggcaagg 3360

gcgacctcaa caagaagatc gtcgtcgact tcaagggtat tgccctcaag ctccagaagc 3420

gctctggccc tgccgttgtc gctcccgaga agcccctcgc tctcaacaag gacctttgcg 3480

ccccggctgt tgaggccatc cctgagcaca tcctcaaggg cgatgctctt gcccctaacc 3540

agatgacctg gcacccgatg tccaagatcg ctggcaaccc cacgccctcg ttctctccct 3600

cggcctaccc tccccgtccc atcaccttca ccccgttccc cggcaacaag aacgacaaca 3660

accacgtgcc cggcgagatg ccgctctcgt ggtacaacat ggctgagttc atggccggca 3720

aggtcagcct ctgcctcggc cctgagttcg ccaagttcga tgactccaac accagccgca 3780

gccctgcatg ggaccttgct cttgtgactc gtgtggtctc cgtttctgac atggagtggg 3840

tccagtggaa gaacgtggac tgcaacccgt ccaagggaac catggttggc gagttcgact 3900

gccccatcga cgcctggttc ttccagggat cttgtaacga cggccacatg ccgtactcca 3960

tcctcatgga gatcgccctc cagacctctg gtgtcctcac ctctgtgctc aaggccccgc 4020

tcaccatgga gaagaaggac attctcttcc gcaaccttga cgccaacgcc gagatggttc 4080

gctctgatat tgacctccgc ggcaagacca tccacaacct caccaagtgt accggctaca 4140

gcatgctcgg agacatgggt gtccaccgct tcagcttcga gctctctgtt gatggtgtag 4200

tcttctacaa gggtaccacc tccttcggct ggttcgtccc tgaggtcttc atctcccaga 4260

ctggtctcga caacggtcgc cgcacccagc cctggcacat tgagtccaag gtgccttccg 4320

cccaggtcct cacctacgac gttaccccca acggtgccgg tcgcacccag ctctacgcca 4380

acgcccccaa gggcgctcag ctcactcgcc gctggaacca gtgccagtac cttgacacca 4440

tcgaccttgt ggtcgccggt ggctccgccg gtcttggcta cggtcatggc cgcaagcagg 4500

tgaaccccaa ggactggttc ttctcgtgcc acttctggtt cgactccgtc atgcccggct 4560

cgctcggtgt ggagtctatg ttccagctcg tcgagtccat cgctgtcaag caggacctcg 4620

ccggcaagta cggcatcacc aacccgacct tcgctcatgc tccgggcaag atctcctgga 4680

agtaccgtgg tcagctcacc cccacctcca agttcatgga ctccgaggcc cacattgtct 4740

ccatcgaggc ccacgacggc gtcgtcgaca tcgttgccaa tggtaacctc tgggctgatg 4800

gcctccgcgt ctacaacgtc agcaacatcc gtgtgcgcat tgttgctggc gccgcccctg 4860

ctgctgctgc tgctgctgct gctgttgctg ctccggctgc cgcccctgct ccggttgctg 4920

catctggccc tgcccagacc atcaccctca agcagctcaa ggctgagctt cttgacgttg 4980

agaagcctct ctacatctcc tccagcaacg gccaggtcaa gaagcacgcc gatgtggctg 5040

gtggccaggc caccattgtg caggcttgca gcctcagtga cctcggtgat gaaggcttca 5100

tgaagaccta cggtgttgtg gctcctctct acaccggtgc catggccaag ggtattgcct 5160

ctgctgacct tgtgattgcc actggtaagc gcaagatcct cggttccttc ggtgctggcg 5220

gtctccccat gcacattgtc cgtgccgctg ttgagaagat ccaggctgag ctcccgaacg 5280

gccccttcgc cgtcaacctc atccactccc ccttcgatag caaccttgag aagggcaacg 5340

ttgacctctt cctcgagaag ggcgttactg tcgtcgaggc ctccgccttc atgaccttga 5400

ccccgcaagt cgtccgctac cgtgctgctg gtctttcccg taacgctgat ggctccatta 5460

acatcaagaa ccgcatcatc ggtaaggtct cccgtaccga gctcgctgag atgttcatcc 5520

gccctgcccc gcagaacctc ctcgacaagc tcatccagtc tggtgagatt accaaggagc 5580

aggctgagct tgccaagctc gtccccgtcg ccgacgacat cgccgtcgag gccgactctg 5640

gtggccacac cgacaaccgc cccatccacg tcatcctccc ccttatcatc aacctccgca 5700

accgcctcca caaggagtgc ggctaccccg ctcacctccg cgtgcgcgtt ggagctggtg 5760

gtggtgttgg atgcccccag gccgctgccg ctgctctcgc tatgggtgct gccttccttg 5820

ttaccggcac tgtcaaccag gtcgccaagc agtccggcac ctgcgacaat gtccgcaagc 5880

agctctgcat ggccacctac tctgacgtct gcatggctcc cgctgctgac atgttcgagg 5940

agggcgtcaa gctccaggtc ctcaagaagg gaaccatgtt cccgtccagg gctaacaagc 6000

tctacgagct cttctgcaag tacgactcct tcgagtccat gcctgccaca gagctcgagc 6060

gtgttgagaa gcgcatcttc cagtgccctc ttgctgatgt ctgggctgag acctccgact 6120

tctacatcaa ccgcctccac aacccggaga agatcacccg tgccgagcgt gaccccaagc 6180

tcaagatgtc tctctgcttc cgctggtacc ttggtcttgc ctctcgctgg gccaacaccg 6240

gtgaggctgg acgcgtcatg gactaccagg tctggtgtgg ccctgccatt ggagccttca 6300

acgacttcat caagggctcc taccttgacc cggccgtctc tggtgagtac ccggacgtcg 6360

tgcagatcaa cttgcagatc cttcgcggtg cctgctacct ccgccgtctc aatgtcatcc 6420

gcaacgaccc gcgtgtcagc attgaggtcg aggatgctga gttcgtctac gagcccacca 6480

acgccctcta agcgagttat atctgtctag aaaacttggc atggctagca atttatgtct 6540

agctattcca tacacacggt aatgccagta gcctgttagt tatagctctt ttggttgttg 6600

tctcacaata cactgacatc agcagaacaa aatgaaaggg gccttggcta ccatgaaatc 6660

aatacttcaa aaggtctctt ggtttcttta ctcgcatgtc gctatttact tacattcctc 6720

gagtacataa catatcatac atcaaagaaa ttaaaaagaa aacaaacatt caaatatgca 6780

ttactttccc tactgtacta gtaagtacgt ttctggtatt aagttgtttt ttctcaaaag 6840

aacaatgtgc ttacttgtaa aatccacagc tgcttacttg taagcctcaa ctagttagtg 6900

atgtgattat cataaaatgt tcgacactgt acctcctttc cagctatctt cctacacctc 6960

ctctgacgca ggttgacgga ggaggcgtgg gggttgattg aagtgcaaca caacgttttg 7020

tttaagatat tccttgcctt ggccgactcc aaatggatag cacagaagcc taatgataat 7080

ttgaattaat tttatttcga gcttatttaa tgctcttatc agagtccgta ggtatctctt 7140

ttcctactaa ttgttgaaaa aggatgtttt ggacatagca ggtcatcata ctatttggtt 7200

ccatcaaatt catatccatt tctttcgttc aagtgcttcc cttcctactt attatatata 7260

ttatatatcc ataaatgtaa aagagacgat tacgaatact ttgcatacat gtatagcgaa 7320

acagagatgg tagcaaaagt tcaccttcac taatctaaga atctctccac gtgggtaaaa 7380

acttcagcag taagattgta aatgatgtcc aagaacaaaa cgtcatgcta gtccaggggt 7440

tactgagcta acgattaata atgtttcgta gtcttcctaa ttgcaccatc aaaacttgtc 7500

tgcacaagtt ttaaagtatt ggagccttta ctgaagaatc agaggacata gatggggcac 7560

gttcgccttg aaaaaaatag tcttctttac ctgcatggtg ttacaaacaa aaacgagttg 7620

aaaatagctg tgcaaggagg caaacatgat tggaaaagaa aaacgagggg acccttatac 7680

aggagggcgc cacatagtag aatgagtaga ttgttagagt agggtacgct ttatgtgatt 7740

gattgaatgg gcgagtgaaa gttgctg 7767

<210> 8

<211> 7800

<212> DNA

<213> Schizochytrium limacinum (Schizoochytrium limacinum)

<400> 8

ccacataatt tgaaagaaac attgaccacg cagccagtca tcgtgttgga aatcaccacc 60

gtgtgcccca cgacactact gcgaagcgct cgacacctcg tcgacaccaa gcacgagagt 120

ctgcgtgtaa gaaaaataaa tgaaagagct tttgtgtgat ggtacattcg ctgcgcttct 180

tcgcaacttg aagatggccc agtaataagc tcgggcatgc tgcaccaaac agaagtgggg 240

gtatcatgca gtcaagacct cataacaggg acacaaacgg ctgtctcctc gcgcgcctcg 300

gtacgtatcc aagtcccaaa ccgtgagaaa ttttcatgtt gatactggca tgatacgcct 360

cttgtcggac agatgggcgt tctcaacatt ggttctcatg tctgaatctt agggtttaat 420

ataatagtac tctaggtatg tagctattac agaaggtaag aggactagta gttctagtac 480

atgataggta gtgtcaggta ccagtgtgaa aaaaaaatgg aaggccattt tcgatgtccc 540

aggtcgatcc atataaattg tcgtggataa tgacagcaat ggagtttcgg attggctctt 600

gtcattactg tggaccacca ggtttctcgt gatgtaaact aaccctctag cactttttaa 660

ccttcatcga cctggtttta gatctcagaa ttcaatagga agtggcgttg tcaaagttgt 720

tggttctatc gttttgagta cttaatagca ggtactgcct aagcaagtct cagcttccgg 780

cactgtagct gttctttcgg ggaggggaga ttctgctgtt ctttgaagta gtattcttca 840

cgagagtttg tttgttacct tttctgattc acaaatgaga aagaagagca caggataagt 900

tgtaaaaata tcctggtatt cattcttcac gggaaaaccc aaacgagtaa taatgtccac 960

catcaggcta agccatcatg caaatgcaaa gtatgatgaa ttatccctca atgctttaat 1020

gagggtcttt agcataacat gaatggtaca aatcaaatat aggtgtttag gaagctctca 1080

aaaatgctaa ctcccatgta cccaaacacg cgaaacttcc ggcatcttga agaattcgcc 1140

gttgactcac caaccaatga tggtcagtgg aaacaacaac tgcacgatct aatctcgtcc 1200

tgggcagaca cgactggctt gacatgcatc ttcaccaaca acgcacaact agaactattg 1260

taattgagac tttaaaataa ctaagcttcc taggcgttta agcaacaaca aatccctttt 1320

attcttgcga cattgaaata gcagcgacta caagtaaaag cgccctgtgc caatactagt 1380

gtttgcggaa agctcatgtt gtggtcttca gaaaatgagc cttggcacac ggcaaacttt 1440

gccaaggtca tacaatcaaa tggtgaagag gaaatagcca aagagactca ccttgatctt 1500

gaagacgtgc gagataatgt tgttcggtac gcatcagaat ctccctctca acgtgttatc 1560

atatggcttg atgataaagg aaacgaaagt tctgttctta ctcgtgaaag tctagtcaag 1620

agcgctcagg gtcttgcaga ggcaatgaga acccagtgga agatgaaaac aggcgagaaa 1680

gttctccttg tgtttttgcc aggtacagat tttatcattg ctatgatggc atgtcttttt 1740

ggaggcttcg tcgcggttcc agtctaccca ccagatccaa acagtccaaa ggcaggactt 1800

ttaaaactag gtaaagcggt tgaagttgca caggtgtcta ctgtgatttc tcacaccaaa 1860

tttctccatc tgcgtcggtt agtgattggc gttaagaagt ttccgcgagg actgaaatgg 1920

cataacatga gcaaagtgaa accagctgct tctattggcg tcacaatgcc tgtagccagc 1980

catgaattag ccttcataca attttcgagt ggaagcacag gcgaccccaa gggtgtttgt 2040

gtaacacatg gaaacctcaa cagcaatctt gcaagtatta agacgtctat gggcatgagt 2100

gaagacacag ttggtgtcac atgggtccca ttttatcatg acatgggcct ggtaggtgca 2160

attcttacta ccctgtattc tggcgcaatc atggtagcca tgtctccgat tagcttttta 2220

caaaatccat ttgtgtggtt aaatgctatc tcaaagtatc gtggcacgac cacttgcgcc 2280

cccaactttg cgtatgtgct tgcagtacgt aaagttccag tagccaaaag gaaggagctc 2340

gacctatcat gctgtaagcg ctattcgaat ggcgctgagc cagttcgtgc agaaacctta 2400

tgggcctttg cagaggcata cgagaagcaa ggtgtgaaag ctgagtactt ggatccattt 2460

tatggtatgg cagagaacgt tgttcttatt tccacgcatc gaggtccaca tatgcctctc 2520

atgttgcggc tcaaggctgg gttttccaat aaacctggct ctaaaatagt tcttgtgcat 2580

cccgaagaag atggacaaga tttagtaggc aacggccctc ctggcgtagg tatcgaggtc 2640

tcggttgtac acccagagac aagggtacta ttagaagatg atactatcgg tgaactctgg 2700

gtaacaggtc catccaaagt cagtgaatac cttggcaagc ctgctctgtc ggaagaagaa 2760

ctcaatgcca agatttcaac ccgctctgac gaacgaacct ttctccgcac aggtgacatg 2820

ggtttcgttc ataaaggcga aatttttgtg tgtggacgca tcaaagacat gatgattgtt 2880

cgtggacgta acattttccc ccaagacgtt gaaagcactg tagatcaagc aagtactaag 2940

attcgccctg gctgttgtgc tgttttcacg cttgaaggaa ctgaaaatgt tgttgttgtt 3000

gctgaagtcc gtgatggaaa acttgctaac gaagctggtg gcttggcaga cctgattcgc 3060

gctcgcatca tgactgaaca cagttttcga ccttttgctg tgattctaat caagccacga 3120

acgattccta agaccacaag cggcaagatc caacgccgag ctgctcgaga agcgtatgtt 3180

caaggagagc tatcagttct tgtcgaggct cgtgatgaaa gctgtttacc agaacgccgt 3240

tctgcaatgc agaggcgtgc aagtcttgcc gctttggatg acctccttga tgagttagag 3300

gaaatggact tatggacagg gcctgcacag atgcgagcgc ttgtggaaga gatgaattta 3360

gcaggataca cagtctttga tgagcttgct tgtttgcgca tacctgtcag tttgtctcct 3420

cttgttaaac agcttcgtga gttgttagtt catcgcccaa ttcttcgtaa ctttgctgcc 3480

gacttagcca tcggctgcaa tgaggagcta cgaagtagtc gggtcgtggg tccgttgagc 3540

cttgcatatt tccttcatat ttgcattgat gcactgcaag atcgggagtt gtttgttcag 3600

attcttcctc aagtccttga ttcccttgtt gaaaacttta gcgtccctgc caagcgtacc 3660

tggtttggtc atgtacaagc tgggcttgca gcctgggcag caaagctgca acttcagcaa 3720

ggtgggttac gttgtacaga tgagaaccat gctcagttcc aactaggact gggtgtttta 3780

gcgtcggtgg gggctgaaat gcttcatggt gttaaaaagc ccacgttatc tgctctttgt 3840

ggtgctagct tatatgaggt tataagtcac gagactcacc gtggtcacac acgaagtgcc 3900

attgttgaat cttttgttgc atggaatttt gcatttttag aaaaatacaa gcccttcaag 3960

gatatcaccc gggatctgcc atacgaagtt ctactgttgg tgccaagcct cgtaagtgca 4020

cagtcgctag acgagctgta ccataatcgc atgataggac tcttgctgct tatgggcgca 4080

aaacagtcaa aattatttgc caatacagaa agaagtattc agttccgagc aggtaccatc 4140

gatgatgcaa tttgccaggt catgatttca ttaaatgctc agtgtgcaga gcgcctggcc 4200

aatagtatac ctcattattg tggtcctcgc gaaatgagtg ctgcgcccga atcattgttt 4260

tctaggagta agtatgctct tcgaaacttg cagcgagcct ctgttttgcc agacaatctt 4320

ttcgataaca catggagtga gttggtggaa aatatggaga gtcatggtga ggttcgtgct 4380

ggtgagaacc ctgacgaacg tcttcgctgg cttttgcaat gcgtacgtca aagcattgaa 4440

gctccttcct taggtcttga tgacgatctc ttcgaagcgg gtatgagttc catggcaatg 4500

attgaagtga atgctcatgt cgagaatttg attggtttca acgagacagt cgagccagat 4560

ttcattgcca agtatcgtac agtgcggcgt ctggcggagg ctgttgaaca gaaacgtatg 4620

gctcatgttg agctagagga agtcaaatac tcaaatcaac ctgccctgcc atatcatgtg 4680

gcaaccacaa tccaatttct cggtatttgg gtgctttttt tcatgatgtc cgcagcgatt 4740

ttacccgcct atcactatgg actatgggtt cggtacaaat cgggacttcc atctccttgg 4800

tcacactata gagttaccag cacgctcagc atgtttgggc tattagtgcc ttttgtgata 4860

cctttgtgga ttgcttcatt ttcgctgctg gttcttgttg caaagtggat cgtagtgggg 4920

aaatggcgtc catgcgaagt gaaaatcaac tcgtttccgt atttacgctg gtggttcgta 4980

gaccgtgcta cccatttgtg ggagatagct gtgggtcgat ggtttcttaa cacgccatta 5040

ctgaacatat tttatagggc tatggggggg caagtgtctt tcacagccaa ggttaaagtt 5100

aatcttcggg atcttgacct cgtgcatatt ggcgctgggg cagaggtgcg cggaacattg 5160

gtgtgccgag tctttgatgc caaagaaaga actttgcgtt ttcgacgagt caaacttggc 5220

caaggtagcc ggctgcgtac aggttcagtc gtgatgcctg gatgctgggt aggtaaaggt 5280

gcagaagtgg tacctttaca agtgctcaac gagggtgaga aagttgctga aaacattgag 5340

gctatagaga cttctcattc tagcattaat gctaccgtgc gtgagctatt tgcaaaattg 5400

ttcatcatgt gctctttcct gttgttgtat tttaacactt cagtcatcat tggcttgatc 5460

tgggatgcgg cgggttaccc tgaaaacttt cggtatgtgg aattggtcta ttgggcatca 5520

tcttatctcg tgacagcatg tataatgtcg tttgtagctg tcggtatgaa atggatactg 5580

attggtcgtc tcaaaacagg tgctattaaa ggctctagtt tgcggatttg ggcggctgac 5640

tatttgttca cactagtttg ggtgacttgg attcaatttt tagatgaaac acgttctata 5700

ttgggttatg cacgcggttt aggggcttcc atcgggccag gttcatttgt atctgcaata 5760

aagtggattc agccatcaaa tgcagacatg gtgaagatgg gagcctcgtc catggcaagt 5820

ctaattctca ttgagacaga aagtgaagga attcgagggc gcgtagaaat aggagactac 5880

tgtgaggtga gttacctggc attactaggt cgaggatcca caatgcaatc acgctcgtat 5940

ttgggaagtt tgagtgttct catggaaggc gagaccttgc ctacagggta ccgacaattt 6000

cgcggagcga cctttcctgt ggccactaag gaaggtgagg tagatatgga aatctggagc 6060

acaacagatg agattttagg acttttgatg cgcgtggtgt tgattgttct tgtggtcttc 6120

tcgctcatcc cagcgtatga gtttgcaaat tttgttttgt ttgatgctga tatggataga 6180

aatgctgcca ttcttctttt agctgtaagc ttctggctag cgatggtaag catgatggta 6240

gcacaaaagc taatggcaac tgctgtgttg gggtttggta aagagttgca agagcttcct 6300

tgtcgacttt ctttttactg ctcatatcag ctgtttacat actttttcga aaatcttttg 6360

ctcgtggttc tacatgggac tccattttat aattcatttg cgcgctttat gggtgcatgc 6420

gtgggacgtc gggtgatttt actcacgggt ggtcttcgag agcaccaact tatcacgcta 6480

gagtatggcg cagttgtaga caatgcgcaa ctcactggtc accaggcgtg cgatggagcg 6540

tttgaactag gtccttcact tgttggcgca gagtcagtac ttcagcctgg tgctgtatgt 6600

ctttcgaatg gaattgttca cgctggtact gttattcacg ctcgttcctt tgcaccaaag 6660

cgtacagggg ctgcaatata tggcaagccg tctcctgttt gcattgctcc cgaggttgca 6720

tagtttccta catattgtct actatcattc ccggattgtg gactcatgtt aaacagtgca 6780

tttatttcga ttgaatattt cctgatcatc ctttcgttac ttctcaactc tatgtgaata 6840

catagagata ctttgtttgt gcatccaaca ggctggaaag tatgtacggc ctaagtaaat 6900

tactgattgt aagtaaaaaa tcaatgataa aaatgaaagt cggtctgtgt caattgatca 6960

ccggtgcgct ttcaattttc agatgaatac aaattgcttt caaaattact acgagaacga 7020

aagacacgaa ccccttttta cataaaaaca ccgcaacaat ccataataag tgcactttga 7080

gcaaaatatg aaatctagta aataaatgtg aacatatttg ataaagttgt cactgcagga 7140

tcttactctg attgcttgta tcactccttc tatagtttta aacaatggta tgtcctttat 7200

gttaacataa accctttaca tagcacataa atttgcattg cataaagaag ggtaggttta 7260

cttttattat ttgaaatcag caatcaaaga aacgcgtaaa gctctgatca gctaggaagg 7320

atgaaagcaa gatgcttgag aatcttgaag atgttttaag aaggcactat aaacgtgaac 7380

gtttagtgtt aggaatctct tcgcctttta aaaatgaaat ttgcgagcga ctgatttgta 7440

ttgtatttag taaacttgag caagtttgac ttgtattaaa cacaatataa aaggctagga 7500

tttacccttc caaatcatct tgaaaaatgc aattctcgag tccctttgct tcacatatct 7560

gattatgtac tttttatctt acaccaatct aaatagttca gaacctacaa tggtgtgtca 7620

agcaaggact ctcacaatgt cttgttcgca tgacaacttt aattgtaggt cttaacatgt 7680

agcaagggta cactagacaa ctcaggaata ctatgacagg cttaaaaaac tgcaggttct 7740

aattcatagc aatcaagtct aaaaccgtgt gattttaaaa tatgcataag aacggtgcac 7800

<210> 9

<211> 669

<212> PRT

<213> Schizochytrium limacinum (Schizoochytrium limacinum)

<400> 9

Leu Glu Arg Ala Leu Gln Glu Glu Ile Glu Val Leu Gln Gly Asp Thr

1 5 10 15

Leu Ser Ala His Leu Ser Lys Ile Leu Cys Ala Ile Ser Ser Thr Phe

20 25 30

Ala Arg Phe Glu Ser Tyr Cys Glu Asn Thr Val Leu Asn Glu Ala Ser

35 40 45

Lys Cys Thr Pro Gln Glu Leu Ser Glu Leu Tyr Asp Tyr Thr Ser Leu

50 55 60

Gly Leu Lys Glu Pro Ser Val Leu Asp Val Asp Lys Leu Tyr Gln Lys

65 70 75 80

Leu Gly Lys Ser Arg Glu Ala Thr Lys Ser Leu Lys Arg Ala Arg Glu

85 90 95

Glu Leu Glu Ala Glu Asp Leu Glu Glu Glu Glu Arg Glu Tyr Phe Arg

100 105 110

Asn Arg Ile Lys Lys Gln Lys Arg Ile Leu Ser Lys Gly Asn Pro Ile

115 120 125

Glu Leu Ser Lys Val Phe Arg His Lys Leu Ala Glu Thr Leu Gln His

130 135 140

Ala Gln Glu His Tyr Pro Glu Leu Leu Gln Asp Arg Ala Trp Leu Glu

145 150 155 160

Lys Leu His Val Ala Ala Glu Gly Val Phe Glu Val Ala Cys Ser Asp

165 170 175

Leu Trp Leu Thr Asn Val Thr Val Ser Asp Phe Gln Arg Val Gly Glu

180 185 190

Pro Leu Ser Phe Arg Gly Gly Lys Lys Val Gln Lys Val Leu Asp Asn

195 200 205

Val Gly Lys Thr Met Val Leu Lys Glu Phe Gln Leu Gly His Pro Ser

210 215 220

Gln Ser Lys Thr Phe Tyr Gln Gln Val Ala Asn Leu Gly Lys Val Val

225 230 235 240

Ser Gln His Val Ile Arg Ile Thr Gly Ala Phe Val Asp Met Thr His

245 250 255

Gly Glu Ser Arg Gly Cys Ile Val Met Pro Phe Tyr Glu Gln Gly Asp

260 265 270

Leu Ala Lys Trp Ile Glu Ser His Pro Asn Glu Gly Lys Glu Ala Arg

275 280 285

Asp Arg Leu Ala Val Gly Leu Leu Val Gly Val Ala Asp Leu His Ala

290 295 300

His Cys Ile Val His Cys Asp Ile Lys Pro Glu Asn Ile Phe Leu Thr

305 310 315 320

Ser Asn Gly Thr Pro Leu Ile Gly Asp Phe Asp Gly Ile Lys Val Val

325 330 335

Asn Tyr Thr Ala Thr Tyr Thr Ser Leu Gln Ala Thr Pro Arg Tyr Ile

340 345 350

Ala Pro Glu Leu Gln Asn Gly Pro Val His Arg Phe Glu Thr Ala Met

355 360 365

Asp Met Tyr Ser Val Gly Ile Ser Leu Lys Glu Leu Tyr Pro Thr Glu

370 375 380

Arg Thr Ala Ala Met Gln Thr Leu Ile Glu Ala Leu Thr Ala Arg Asp

385 390 395 400

Pro Ser Gln Arg Pro Ser Ala Arg Gln Ala Leu Gln His Gln Ala Phe

405 410 415

Gly Ala Pro Gln Ile Pro Leu Met Glu Cys Leu Val Cys Phe Glu Lys

420 425 430

His Arg Leu Ser Glu Gly Thr Ser Cys Glu Glu Gly Asp Phe Leu Cys

435 440 445

Arg Gly Cys Ile Glu Ser Ala Val Glu Ala Ala Ala Gln Pro Leu Ala

450 455 460

Asn Val Arg Val Asp Ala Asp Gly Thr Met Ala Cys Met Lys Pro Glu

465 470 475 480

Cys Ser Gly Lys Ile Ser Gly Gln Glu Ile Thr Arg Leu Ala Pro Thr

485 490 495

Ala Leu Asn His Leu Leu Leu Ile Ala Lys Met Lys Ala Glu Ser Glu

500 505 510

Ala Ala Val Trp Ala Glu Lys Glu Ile Gln Arg Arg Ile Ala Glu Val

515 520 525

Val Arg Ala Glu Glu Gln Asn Ser Asp Ala Arg Trp His Leu Leu His

530 535 540

Ile Gln Glu Asn Ile Leu Ser Thr Cys Cys Pro Asn Cys Lys Ala Tyr

545 550 555 560

Val His Asp Phe Asp Gly Cys Cys Ala Val Lys Cys Gly Asn Asn Ala

565 570 575

Cys Gly His Leu Phe Cys Ala Trp Cys Leu Gly Tyr Ser Ser Gln Asp

580 585 590

Gly Asp Val Cys His Ala His Val Arg Thr Cys Ser Arg Arg Leu Gly

595 600 605

Gln Asp Leu Tyr Phe Pro Gly Ser Leu Glu Gln Val Cys Glu Ala Trp

610 615 620

Arg Leu Leu Arg Ala Glu Arg Leu Arg Glu Tyr Trp Asn Ala Gln Ile

625 630 635 640

Gln Asp Arg Asn Leu Arg Leu Val Leu Lys Asp Leu Leu Ser Pro Leu

645 650 655

Leu Thr Pro Asp Ile Val Gly Ala Asp Phe Arg Leu Glu

660 665

<210> 10

<211> 1193

<212> PRT

<213> Schizochytrium limacinum (Schizoochytrium limacinum)

<400> 10

Asp Arg Ile Asn Glu Gln Glu Leu Ile Leu Ser Gln Gly Asn Pro Ile

1 5 10 15

Glu Leu Ser Lys Ala Phe Arg His Lys Leu Ala Glu Thr Leu Gln His

20 25 30

Ala Gln Glu His Tyr Pro Glu Leu Leu Gln Asp Arg Ala Trp Leu Lys

35 40 45

Arg Leu His Ile Ser Ala Glu Gly Val Phe Glu Val Ala Cys Ser Asp

50 55 60

Leu Trp Leu Ala Asn Val Glu Ile Ser Asp Phe Lys Pro Val Gly Glu

65 70 75 80

Leu Leu Ala Ser Arg Gly Gly Lys Thr Val Gln Lys Val Leu Asp Gly

85 90 95

Asp Gly Arg Thr Leu Val Leu Lys Gln Phe Gln Leu Gly His Thr Ser

100 105 110

Gln Ser Lys Thr Phe Tyr Lys Gln Val Ala Asn Leu Gly Lys Val Val

115 120 125

Ser Gln Asn Val Ile Arg Ile Thr Gly Ala Phe Val Asp Met Thr His

130 135 140

Gly Ala Pro Arg Gly Cys Ile Val Met Pro Phe Tyr Glu Gln Gly Asp

145 150 155 160

Leu Ala Lys Trp Ile Glu Ser His Pro Asn Glu Gly Lys Ala Ala Arg

165 170 175

Asp Arg Leu Ala Met Gly Leu Leu Ile Gly Met Ala Asp Leu His Thr

180 185 190

His Gly Ile Val His Cys Asp Ile Lys Pro Glu Asn Ile Phe Leu Thr

195 200 205

Ser Asn Gly Thr Pro Leu Ile Gly Asp Phe Asp Gly Ile Lys Val Ala

210 215 220

Asp Tyr Thr Ala Thr Tyr Thr Ser Leu Gln Val Thr Pro Lys Tyr Leu

225 230 235 240

Ala Pro Glu Leu Gln Asn Gly Pro Val Tyr Lys Phe Glu Thr Ala Met

245 250 255

Asp Met Tyr Ser Val Gly Val Ser Leu Lys Glu Leu Tyr His Thr Glu

260 265 270

Arg Thr Ala Ala Met Gln Thr Leu Ile Asp Ala Leu Thr Ala Thr Asp

275 280 285

Pro Gly Gln Arg Pro Thr Ala Arg Gln Ala Leu Gln His Glu Ala Phe

290 295 300

Gly Ala Pro Lys Ile Pro Val Lys Ile Cys Leu Val Cys Met Glu Glu

305 310 315 320

Tyr Gln Leu Ser Glu Gly Thr Ser Cys Glu Glu Gly Asp Phe Leu Cys

325 330 335

Arg Asp Cys Ile Glu Ser Ala Val Glu Ala Ala Ala Gln Pro Leu Ala

340 345 350

Asn Val Arg Val Asp Ala Asp Gly Thr Met Glu Cys Met Lys Pro Glu

355 360 365

Cys Val Gly Arg Ile Ser Gly Gln Glu Ile Thr Arg Leu Ala Pro Ser

370 375 380

Ala Leu Asn His Leu Leu Leu Ile Ala Lys Thr Lys Ala Glu Ile Glu

385 390 395 400

Ala Ala Val Trp Ala Glu Gln Glu Ile Gln Arg Arg Ile Ala Glu Ala

405 410 415

Leu Arg Ala Asp Gly Gln Asn Arg Asp Ala Gln Arg His Leu Leu His

420 425 430

Ile Gln Glu Lys Ile Leu Ser Thr Cys Cys Pro Asn Cys Lys Ala Tyr

435 440 445

Val His Asp Phe Asp Gly Cys Cys Ala Val Glu Cys Gly Asn Asp Gly

450 455 460

Cys Gly His Ser Phe Cys Ala Trp Cys Leu Glu Phe Ser Ser Gln Asp

465 470 475 480

Ser Gln Ala Cys His Ala His Val Leu Val Cys Ser Arg Asn Leu Ser

485 490 495

Asn Asn Lys Ser Tyr Phe Ala Glu Ser Phe Glu Gln Val Arg Glu Ala

500 505 510

Trp Gln Leu Leu Arg Ala Glu Arg Leu His Glu Tyr Trp Asn Ala Asn

515 520 525

Ile Gln Asp Glu Asn Leu Gly His Ala Leu Glu Gln Gln Leu Val Pro

530 535 540

Leu Leu Thr Pro Asp Ile Val Gly Pro Glu Phe Lys Leu Ile Glu Asp

545 550 555 560

Val Leu Pro Ser His Tyr Cys Ser Ile Ser Ala Thr Val Pro Thr Thr

565 570 575

Thr Thr Thr Thr Thr Thr Thr Ala Ile Val Arg Gly Val Gly Phe Val

580 585 590

Ala Ala Thr Phe Val Leu Thr Ile Ala Glu Gln Pro Ser Thr Thr Val

595 600 605

Ala Ile Gly Phe Val Leu Leu Gln Pro Leu Ala Tyr Val Leu His Ala

610 615 620

Pro Val Arg Val Phe Gln Tyr Leu Asn Glu Ser Gly Lys Lys Asn Trp

625 630 635 640

Phe Asp Glu Pro Thr Gln Glu Met Ile Glu Phe Leu Leu Ser Glu Asn

645 650 655

Glu Ala Ile Leu Ala Glu Gly Arg Ala Glu Gly Arg Ala Lys Val Leu

660 665 670

Ala Glu Phe Ile Glu Pro Leu Leu Ala Gly Leu Arg Lys Pro Leu Phe

675 680 685

Leu Lys Val Thr Leu Arg Val His Ala Ser Thr Ala Ala Thr Pro Ser

690 695 700

Ala Trp Gly Leu Glu Phe Ile Ser Leu Lys Leu Trp Arg Gly Phe Thr

705 710 715 720

Ser Glu Ile Ile Asn Pro Phe Leu Ser Ala Asn Leu Leu Asn Met Gln

725 730 735

Ile Arg Asn Thr Ser Phe Arg Thr Ser Leu Tyr Pro Leu Asn Ile Ser

740 745 750

Gly Asn Thr Asn Ala Met Ala Ser Leu Phe Asn Asn Trp Cys Cys Arg

755 760 765

Pro Phe Val Gly Leu His Thr Glu Val Ala Asn Phe Pro Lys Val Ile

770 775 780

Val Arg Ile Glu Asp Asp Ile Gly Leu Ser Ala Lys Pro Asp Leu Thr

785 790 795 800

Leu Val Lys Ile Asp Asp Gln Arg Asn Glu Thr Val Phe Ser Ile Val

805 810 815

Glu Val Lys His Pro Asn His Phe Leu Pro Leu Ser Glu Asn Pro Asn

820 825 830

Trp Thr Asp Arg Arg Asn Ala Ala Ala Ala Ala Gly Ile Ala Ile Phe

835 840 845

Thr Ser Arg Gly Gln Pro Arg Gln Ala Leu Glu Gln Leu His Gly Asn

850 855 860

Met Leu Ile His Gly Val Lys Tyr Ala Ile Leu Thr Ser Val Glu Leu

865 870 875 880

Thr Tyr Phe Val Lys Arg Asp Asn Lys Gly Asn Met Arg Ile Thr Arg

885 890 895

Gly Phe Tyr Gly Ala Glu Thr Gly Gly Ala Arg Val Ile Ser Thr Asn

900 905 910

Glu Ala Met Ala Ala Phe Ile Tyr Ile Ala Ser Arg Asp Pro Asp Asn

915 920 925

Gly Lys Phe Arg Pro Arg Ser Asn Ser Thr Ser Thr Glu Ile Arg Ala

930 935 940

Tyr Ser Glu Arg Ile Gln Thr Lys Phe Gly Gly Arg Ser Ile Ser Asp

945 950 955 960

Arg Ala Thr Glu Lys Phe Lys Asp Arg Val Gly Glu Thr Ala Leu Gln

965 970 975

Phe Met Lys Gly Leu Asn Gln Leu Ala Val Asn Ile Glu Glu Leu Arg

980 985 990

Ser Asn Leu Ile Glu Val Leu Pro Phe Lys Asp Glu Ala Tyr Leu Phe

995 1000 1005

Met Pro Leu Pro Trp Ser Lys Asp Tyr Arg Leu Val Thr Ser Tyr

1010 1015 1020

Val Val Arg Ile Pro Trp Val Ser Asn Lys Asp Phe Asn Trp Asp

1025 1030 1035

Ile Phe Val Lys Thr Met Ala Val Ser Lys Asp Trp Ala Arg Glu

1040 1045 1050

Thr Tyr Glu Phe Glu Lys Ser Phe Phe Leu Asn Glu Val Lys Leu

1055 1060 1065

Tyr Leu Gly Pro Leu Arg Leu Leu Gln Gly Lys His Ala Pro Phe

1070 1075 1080

Leu Val Tyr Gly Gly Thr Phe His Lys Arg Ile Ile Ile Ala Thr

1085 1090 1095

Thr Phe Ser Gly Glu Thr Ala Thr Lys Glu Leu Ile Leu Ala Asn

1100 1105 1110

Ser Ser Trp Ala Ile Thr Ala Ile Arg Ala Ser Leu Ser Ala Leu

1115 1120 1125

His Gln Val Gly Val Leu His Gly Asp Ile Ala Leu Arg Asn Ile

1130 1135 1140

Ala Ile Asp Thr Thr Thr Lys Thr Ala His Leu Ile Asp Phe Gly

1145 1150 1155

Arg Ser Ser Gln Asp Lys Thr Thr Lys Lys Asn Arg Asp Thr Glu

1160 1165 1170

Met Lys Glu Leu Asn Thr Leu Leu Gly Phe Ala Ile His Arg Lys

1175 1180 1185

Arg Ile Arg Asp Ser

　　1190

<210> 11

<211> 773

<212> PRT

<213> Schizochytrium limacinum (Schizoochytrium limacinum)

<400> 11

Leu Trp Pro Ala Glu His Leu His Glu Thr Tyr Glu His Ile Arg

1 5 10 15

Asn Asp Ala Leu Gly Ser Gly Cys Ser Val Val Ile Tyr Val Ala Leu

20 25 30

Asp Cys Asp Ala Leu Cys Ala Val Lys Ile Leu Thr Ser Leu Leu Lys

35 40 45

Ala Asp Asn Val Ala Tyr Lys Leu Arg Pro Val Arg Gly Tyr Ser Asp

50 55 60

Ile Leu Glu Asp Phe Arg Glu Thr Ser Lys Ala Glu Ala Ile Lys Ser

65 70 75 80

Ile Ile Met Ile Asn Cys Gly Gly Asp Val Asn Ala Gln Glu Met Phe

85 90 95

Asn Leu Asp Asp Gly Met Thr Cys Tyr Ile Ile Asp Ser Ala Arg Pro

100 105 110

Tyr Asn His Ala Asn Leu Leu Arg Ser His Met His Thr Ile Val Phe

115 120 125

Ala Asp Asp Phe Met Lys Glu Glu Asp Leu Val Lys Glu Ala Glu Leu

130 135 140

Leu Glu His Leu Asp Glu Asp Asn Val Asp Glu Leu Leu Gly Ser Asn

145 150 155 160

Asp Asn Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Ser Asp Asp

165 170 175

Gly Lys Gly Glu Val Glu Asp Gly Asn Asp Ala Asn Ser Ala Arg Asn

180 185 190

Gly Gln Glu Gly Glu Ile Asp Ser Asp Glu Glu His Glu Phe Asp Gly

195 200 205

Gly Asn Pro Gln Gln Asn Ala Ser Asp Ser Asp Asp Asp Glu Asn Glu

210 215 220

Asp Ala Asn Lys Gln Thr Ser Asn Asn Thr Asn Asn Lys Thr Ser Lys

225 230 235 240

Ala Lys Ser Ala Ser Gln Leu Leu Thr Glu Gln Glu Glu Ala Glu Ile

245 250 255

Glu Arg Glu Leu Ala Ala Glu Asn Gly Thr Arg Lys Lys Arg Arg Arg

260 265 270

Thr Ser Ser Ser Ser Ser Ser Ser Gln Asn Asp Lys Asn Asn Asp Ser

275 280 285

Gly Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Lys Asp Asp Asp

290 295 300

Ser Leu Pro Lys Arg Lys Val Arg Glu Lys Val Asp Ala Glu Glu Pro

305 310 315 320

Met Asp Asp Lys Ala Arg Val Ala Lys Tyr Tyr Ala Gly Ser Phe Arg

325 330 335

Gly Thr Ser Ala Ala His Val Leu Phe Ser Leu Ser Gln Arg Leu Asn

340 345 350

Lys Asp Gln Lys Ile Phe Leu Trp Leu Ala Ile Val Gly Thr Thr His

355 360 365

His Phe Ile Asn Ser Glu Leu Ser Glu Glu Asp Tyr Leu Leu Arg Val

370 375 380

Leu Thr Tyr Gln Asp Leu Val Lys Asp Arg Ser Met Ser Arg Arg Ala

385 390 395 400

Gly Gln His Thr Val Thr Glu Asp Gly Ala Glu Val Pro Leu Met Glu

405 410 415

Gly Gln Ser Met Glu Phe Ile Glu Glu Leu Arg Leu Met Leu His Arg

420 425 430

His Trp Ser Leu His Glu Ala Phe Leu Tyr Ser Asp Tyr Ile Ala Ala

435 440 445

Lys Leu Gly Ile Trp Lys Asn Asp Gly Glu Ala Lys Leu Arg Thr Phe

450 455 460

Phe Ala Lys Met Gly Ile Ser Arg Lys Glu Ala Glu Gln Lys Tyr Ser

465 470 475 480

Phe Met Asn Met Ser Val Lys Arg Ala Leu Lys Asp Lys Ile Gly Ala

485 490 495

His Gly Ala Asp Phe Ser Leu Asp Glu Ser Phe Val Tyr Ala Ser Phe

500 505 510

Gln Phe Arg Ala Gly Phe Gly His Gln Leu Ser Ala Ala Asp Thr Ala

515 520 525

Tyr Cys Met Ala Ala Leu Leu Glu Ser Ala Ala Thr His Pro Thr Val

530 535 540

Phe Ala Asp Asp Ser Gly Ala Asp Ala Thr Glu Asp Ala Thr Glu Ile

545 550 555 560

Pro Glu Val Asp Leu Asp Gly Asp Asp Leu Asp Tyr Ala Gly Glu Asp

565 570 575

Gly Ala Asn Gly Gln Lys Glu Asn Ser Lys Ala Ile Leu Ala Ala Ser

580 585 590

Leu Trp Lys Gln Ser Phe Asn Ala Ala Tyr Asp Ala Leu Ser Phe Ser

595 600 605

Gln Thr Thr Arg Asn Gln His Leu Leu Glu Gln Gly Leu Asn Leu Ala

610 615 620

Lys Ala Leu Gln Lys Ala Ile Ile Gln Glu Gly Gly Asn Ile Ile Ser

625 630 635 640

Gly Thr Lys Ile Ala Ser Ala Gly Ser Phe Arg Tyr Cys Ile Leu Glu

645 650 655

Gly Leu Pro Pro Lys Leu Val Ser Ile Phe Ser Gln Pro Asp Thr Leu

660 665 670

Leu Arg Leu Ala Lys Phe Ile Met Leu Ala Tyr Thr Ser Ala Gly Lys

675 680 685

Trp Ser Gly Leu Gly Ala Lys Pro Leu Val Leu Gly Val Lys Asn Leu

690 695 700

Lys Thr Asn Lys Ala His Phe Val Gly Leu Pro His Pro Thr Phe Gly

705 710 715 720

Asp Asp Ala Ile Ala Lys Asn Pro Phe Gly Arg Tyr Phe Arg Val Ala

725 730 735

Ala Gln Lys Val Gly Thr Ala His Ile His Val Gly Phe Ser Ser Ala

740 745 750

Cys Ile Glu Ile Pro Leu Glu Leu Val Gln Lys Phe Leu Val Ala Leu

755 760 765

His Glu Ile Thr Ala

　　 770

<210> 12

<211> 2189

<212> PRT

<213> Schizochytrium limacinum (Schizoochytrium limacinum)

<400> 12

Ala Gln Arg Glu Asn Arg Leu Glu Ala Asn Met Asp Thr Arg Ile

1 5 10 15

Ala Val Ile Gly Met Ser Ala Ile Leu Pro Cys Gly Thr Thr Val Arg

20 25 30

Glu Ser Trp Glu Ala Ile Arg Asp Gly Ile Asp Cys Leu Ser Asp Leu

35 40 45

Pro Glu Asp Arg Val Asp Val Thr Ala Tyr Phe Asp Pro Val Lys Thr

50 55 60

Thr Lys Asp Lys Ile Tyr Cys Lys Arg Gly Gly Phe Ile Pro Glu Tyr

65 70 75 80

Asp Phe Asp Ala Arg Glu Phe Gly Leu Asn Met Phe Gln Met Glu Asp

85 90 95

Ser Asp Ala Asn Gln Thr Val Thr Leu Leu Lys Val Lys Glu Ala Leu

100 105 110

Glu Asp Ala Gly Ile Glu Ala Leu Ser Lys Glu Lys Lys Asn Ile Gly

115 120 125

Cys Val Leu Gly Ile Gly Gly Gly Gln Lys Ser Ser His Glu Phe Tyr

130 135 140

Ser Arg Leu Asn Tyr Val Val Val Glu Lys Val Leu Arg Lys Met Gly

145 150 155 160

Met Pro Glu Glu Asp Val Gln Ala Ala Val Glu Lys Tyr Lys Ala Asn

165 170 175

Phe Pro Glu Trp Arg Leu Asp Ser Phe Pro Gly Phe Leu Gly Asn Val

180 185 190

Thr Ala Gly Arg Cys Thr Asn Thr Phe Asn Leu Asp Gly Met Asn Cys

195 200 205

Val Val Asp Ala Ala Cys Ala Ser Ser Leu Ile Ala Val Lys Val Ala

210 215 220

Ile Asp Glu Leu Leu His Gly Asp Cys Asp Met Met Ile Thr Gly Ala

225 230 235 240

Thr Cys Thr Asp Asn Ser Ile Gly Met Tyr Met Ala Phe Ser Lys Thr

245 250 255

Pro Val Phe Ser Thr Asp Pro Ser Val Arg Ala Tyr Asp Glu Lys Thr

260 265 270

Lys Gly Met Leu Ile Gly Glu Gly Ser Ala Met Leu Val Leu Lys Arg

275 280 285

Tyr Ala Asp Ala Val Arg Asp Gly Asp Glu Ile His Ala Val Ile Arg

290 295 300

Gly Cys Ala Ser Ser Ser Asp Gly Lys Ala Ser Gly Ile Tyr Thr Pro

305 310 315 320

Thr Ile Ser Gly Gln Glu Glu Ala Leu Arg Arg Ala Tyr Met Arg Ala

325 330 335

Asn Val Asp Pro Ala Thr Val Thr Leu Val Glu Gly His Gly Thr Gly

340 345 350

Thr Pro Val Gly Asp Arg Ile Glu Leu Thr Ala Leu Arg Asn Leu Phe

355 360 365

Asp Ser Ala Tyr Gly Asn Glu Lys Glu Lys Val Ala Val Gly Ser Ile

370 375 380

Lys Ser Asn Ile Gly His Leu Lys Ala Val Ala Gly Leu Ala Gly Met

385 390 395 400

Ile Lys Val Ile Met Ala Leu Lys His Lys Thr Leu Pro Ala Thr Ile

405 410 415

Asn Val Asp Glu Pro Pro Lys Leu Tyr Asp Asn Thr Pro Ile Thr Asp

420 425 430

Ser Ser Leu Tyr Ile Asn Thr Met Asn Arg Pro Trp Phe Pro Ala Pro

435 440 445

Gly Val Pro Arg Arg Ala Gly Ile Ser Ser Phe Gly Phe Gly Gly Ala

450 455 460

Asn Tyr His Ala Val Leu Glu Glu Ala Glu Pro Glu His Gln Lys Ala

465 470 475 480

Tyr Arg Leu Asn Lys Arg Pro Gln Pro Val Leu Leu Met Ala Ser Ser

485 490 495

Thr Gln Ala Leu Ala Ser Leu Cys Glu Ala Gln Leu Lys Glu Phe Glu

500 505 510

Lys Ala Ile Glu Glu Asn Lys Thr Val Lys Asn Thr Ala Tyr Ile Lys

515 520 525

Cys Val Asp Phe Cys Glu Lys Phe Lys Phe Pro Gly Ser Ile Pro Ser

530 535 540

Ser Asn Ala Arg Leu Gly Phe Leu Val Lys Glu Ala Asp Asp Ala Thr

545 550 555 560

Glu Thr Leu Arg Ala Ile Val Ala Gln Phe Gln Lys Ser Ala Gly Lys

565 570 575

Asp Ser Trp His Leu Pro Arg Gln Gly Val Ser Phe Arg Ala Gln Gly

580 585 590

Ile Asn Thr Thr Gly Gly Val Ala Ala Leu Phe Ser Gly Gln Gly Ala

595 600 605

Gln Tyr Thr His Met Phe Ser Glu Val Ala Met Asn Trp Pro Gln Phe

610 615 620

Arg Glu Ser Ile Ser Asp Met Asp Arg Ala Gln Ala Lys Val Ala Gly

625 630 635 640

Ala Asp Lys Asp Tyr Glu Arg Val Ser Gln Val Leu Tyr Pro Arg Lys

645 650 655

Pro Tyr Asn Ser Glu Pro Glu Gln Asp His Lys Lys Ile Ser Leu Thr

660 665 670

Ser Tyr Ser Gln Pro Ser Thr Leu Ala Cys Ala Leu Gly Ala Tyr Glu

675 680 685

Ile Phe Lys Gln Ala Gly Phe Lys Pro Asp Phe Ala Ala Gly His Ser

690 695 700

Leu Gly Glu Phe Ala Ala Leu Tyr Ala Ala Asp Cys Val Asn Arg Asp

705 710 715 720

Asp Leu Phe Glu Leu Val Cys Arg Arg Ala Arg Ile Met Gly Gly Lys

725 730 735

Asp Ala Pro Ala Thr Pro Lys Gly Cys Met Ala Ala Val Ile Gly Pro

740 745 750

Asn Ala Glu Lys Ile Gln Ile Arg Thr Ala Asp Val Trp Leu Gly Asn

755 760 765

Cys Asn Ser Pro Ser Gln Thr Val Ile Thr Gly Ser Val Glu Gly Ile

770 775 780

Lys Lys Glu Ser Glu Leu Leu Gln Ser Glu Gly Phe Arg Val Val Pro

785 790 795 800

Leu Ala Cys Glu Ser Ala Phe His Ser Pro Gln Met Gln Asn Ala Ser

805 810 815

Ser Ala Phe Lys Asp Val Leu Ser Lys Val Ala Phe Arg Gln Pro Ser

820 825 830

Ala Gln Thr Lys Leu Phe Ser Asn Val Ser Gly Glu Thr Tyr Ser Asn

835 840 845

Asn Ala Gln Asp Leu Leu Lys Glu His Met Thr Ser Ser Val Lys Phe

850 855 860

Ile Ser Gln Val Arg Asn Met His Ser Ala Gly Ala Arg Ile Phe Val

865 870 875 880

Glu Phe Gly Pro Lys Gln Val Leu Ser Lys Leu Val Ser Glu Thr Leu

885 890 895

Lys Asp Asp Pro Ser Ile Ile Thr Ile Ser Val Asn Pro Ser Ser Gly

900 905 910

Lys Asp Ala Asp Ile Gln Leu Arg Glu Ala Ala Val Gln Leu Val Val

915 920 925

Ala Gly Val Asn Leu Gln Gly Phe Asp Lys Trp Asp Ala Pro Asp Ala

930 935 940

Thr Arg Leu Gln Pro Ile Lys Lys Lys Lys Thr Thr Leu Arg Leu Ser

945 950 955 960

Ala Ala Thr Tyr Val Ser Asp Lys Thr Lys Lys Ala Arg Glu Ala Ala

965 970 975

Met Asn Asp Gly Arg Met Leu Ser Cys Val Ser Lys Val Ile Ala Pro

980 985 990

Pro Asp Ala Lys Pro Ile Val Asp Thr Lys Ala Gln Glu Glu Val Ala

995 1000 1005

Arg Leu Gln Lys Gln Leu Gln Asp Ala Gln Ala Gln Ile Gln Lys

1010 1015 1020

Ala Lys Ala Asp Ala Ala Glu Ala Asp Lys Lys Leu Ala Ala Ala

1025 1030 1035

Lys Asp Glu Ala Lys Arg Ala Ala Ala Ser Ala Pro Val Gln Lys

1040 1045 1050

Gln Val Asp Thr Thr Ile Val Asp Lys His Arg Ala Ile Leu Lys

1055 1060 1065

Ser Met Leu Ala Glu Leu Asp Cys Tyr Ser Thr Pro Gly Ala Val

1070 1075 1080

Ser Ser Ser Phe Gln Ala Pro Val Ala Ala Thr Pro Ala Pro Val

1085 1090 1095

Ala Ala Pro Val Ala Ala Ala Pro Ala Pro Ala Val Asn Asn Ala

1100 1105 1110

Leu Leu Ala Lys Ala Glu Ser Val Val Met Glu Val Leu Ala Ala

1115 1120 1125

Lys Thr Gly Tyr Glu Thr Asp Met Ile Glu Pro Asp Met Glu Leu

1130 1135 1140

Glu Thr Glu Leu Gly Ile Asp Ser Ile Lys Arg Val Glu Ile Leu

1145 1150 1155

Ser Glu Val Gln Ala Gln Leu Asn Val Glu Ala Lys Asp Val Asp

1160 1165 1170

Ala Leu Ser Arg Thr Arg Thr Val Gly Glu Val Val Asn Ala Met

1175 1180 1185

Lys Ala Glu Ile Ala Gly Ser Ser Gly Ala Ala Ala Ala Ala Pro

1190 1195 1200

Ala Pro Val Ala Ala Ala Pro Ala Ala Ala Ala Pro Ala Val Ser

1205 1210 1215

Ser Ala Leu Leu Glu Lys Ala Glu Ser Val Val Met Glu Val Leu

1220 1225 1230

Ala Ala Lys Thr Gly Tyr Glu Thr Asp Met Ile Glu Ala Asp Met

1235 1240 1245

Glu Leu Glu Thr Glu Leu Gly Ile Asp Ser Ile Lys Arg Val Glu

1250 1255 1260

Ile Leu Ser Glu Val Gln Ala Met Leu Asn Val Glu Ala Lys Asp

1265 1270 1275

Val Asp Ala Leu Ser Arg Thr Arg Thr Val Gly Glu Val Leu Ala

1280 1285 1290

Ala Lys Thr Gly Tyr Glu Thr Asp Met Ile Glu Ala Asp Met Glu

1295 1300 1305

Leu Glu Thr Glu Leu Gly Ile Asp Ser Ile Lys Arg Val Glu Ile

1310 1315 1320

Leu Ser Glu Val Gln Ala Met Leu Asn Val Glu Ala Lys Asp Val

1325 1330 1335

Asp Ala Leu Ser Arg Thr Arg Thr Val Gly Glu Val Val Asn Ala

1340 1345 1350

Met Lys Ala Glu Ile Ala Gly Ser Ser Gly Ala Ala Thr Ala Ser

1355 1360 1365

Ala Pro Ala Ala Ala Ala Ala Ala Pro Ala Ile Lys Ile Ser Thr

1370 1375 1380

Val His Gly Ala Asp Cys Asp Asp Leu Ser Val Met Ser Ala Glu

1385 1390 1395

Leu Val Asp Ile Arg Arg Ala Asp Glu Leu Leu Leu Glu Arg Pro

1400 1405 1410

Glu Asn Arg Pro Val Leu Ile Val Asp Asp Gly Thr Glu Leu Thr

1415 1420 1425

Ser Ala Leu Val Arg Val Leu Gly Ala Gly Ala Val Val Leu Thr

1430 1435 1440

Phe Asp Gly Leu Gln Leu Ala Gln Arg Ala Gly Ala Ala Val Arg

1445 1450 1455

His Val Gln Val Lys Asp Leu Ser Ala Glu Ser Ala Glu Lys Ala

1460 1465 1470

Ile Lys Glu Ala Glu Gln Arg Phe Gly Gln Leu Gly Gly Phe Ile

1475 1480 1485

Ser Gln Gln Ala Glu Arg Phe Ala Pro Ala Asp Ile Leu Gly Phe

1490 1495 1500

Thr Leu Met Cys Ala Lys Phe Ala Lys Ala Ser Leu Cys Thr Pro

1505 1510 1515

Val Gln Gly Gly Arg Ala Phe Phe Ile Gly Val Ala Arg Leu Asp

1520 1525 1530

Gly Arg Leu Gly Phe Thr Ser Gln Gly Ser Thr Asp Ser Leu Thr

1535 1540 1545

Arg Ala Gln Arg Gly Ala Ile Phe Gly Leu Cys Lys Thr Ile Gly

1550 1555 1560

Leu Glu Trp Ser Ala Asn Glu Val Phe Ala Arg Gly Ile Asp Ile

1565 1570 1575

Ala Arg Glu Val His Pro Glu Asp Ala Ala Val Ala Ile Thr Arg

1580 1585 1590

Glu Met Ser Cys Ala Asp Asn Arg Ile Arg Glu Val Gly Ile Gly

1595 1600 1605

Leu Asn Gln Lys Arg Cys Thr Ile Arg Ala Val Asp Leu Lys Pro

1610 1615 1620

Gly Ala Pro Lys Ile Gln Ile Ser Gln Asp Asp Val Leu Leu Val

1625 1630 1635

Ser Gly Gly Ala Arg Gly Ile Thr Pro Leu Cys Ile Arg Glu Ile

1640 1645 1650

Thr Arg Gln Val Arg Gly Gly Lys Tyr Ile Leu Leu Gly Arg Ser

1655 1660 1665

Lys Val Pro Ala Gly Glu Pro Ala Trp Cys Asn Gly Val Ser Asp

1670 1675 1680

Asp Asp Leu Gly Lys Ala Ala Met Gln Glu Leu Lys Arg Ala Phe

1685 1690 1695

Ser Ala Gly Glu Gly Pro Lys Pro Thr Pro Met Thr His Lys Lys

1700 1705 1710

Leu Val Gly Thr Ile Ala Gly Ala Arg Glu Val Arg Ser Ser Ile

1715 1720 1725

Ala Asn Ile Glu Ala Leu Gly Gly Lys Ala Ile Tyr Ser Ser Cys

1730 1735 1740

Asp Val Asn Ser Ala Ala Asp Val Ala Lys Ala Val Arg Glu Ala

1745 1750 1755

Glu Ala Gln Leu Gly Ala Arg Val Thr Gly Val Val His Ala Ser

1760 1765 1770

Gly Val Leu Arg Asp Arg Leu Ile Glu Gln Lys Arg Pro Asp Glu

1775 1780 1785

Phe Asp Ala Val Phe Gly Thr Lys Val Thr Gly Leu Glu Asn Leu

1790 1795 1800

Phe Gly Ala Ile Asp Met Ala Asn Leu Lys His Leu Val Leu Phe

1805 1810 1815

Ser Ser Leu Ala Gly Phe His Gly Asn Ile Gly Gln Ser Asp Tyr

1820 1825 1830

Ala Met Ala Asn Glu Ala Leu Asn Lys Met Gly Leu Glu Leu Ser

1835 1840 1845

Asp Arg Val Ser Val Lys Ser Ile Cys Phe Gly Pro Trp Asp Gly

1850 1855 1860

Gly Met Val Thr Pro Gln Leu Lys Lys Gln Phe Gln Ser Met Gly

1865 1870 1875

Val Gln Ile Ile Pro Arg Glu Gly Gly Ala Asp Thr Val Ala Arg

1880 1885 1890

Ile Val Leu Gly Ser Ser Pro Ala Glu Ile Leu Val Gly Asn Trp

1895 1900 1905

Thr Thr Pro Thr Lys Lys Val Gly Ser Glu Pro Val Val Ile His

1910 1915 1920

Arg Lys Ile Ser Ala Ala Ser Asn Pro Phe Leu Lys Asp His Val

1925 1930 1935

Ile Gln Gly Arg Cys Val Leu Pro Met Thr Ile Ala Val Gly Cys

1940 1945 1950

Leu Ala Glu Thr Cys Leu Gly Gln Phe Pro Gly Tyr Ser Leu Trp

1955 1960 1965

Ala Ile Glu Asp Ala Gln Leu Phe Lys Gly Val Thr Val Asp Gly

1970 1975 1980

Asp Val Asn Cys Glu Ile Thr Leu Lys Pro Ser Gln Gly Thr Ala

1985 1990 1995

Gly Arg Val Met Ile Gln Ala Thr Leu Lys Thr Phe Ala Ser Gly

2000 2005 2010

Lys Leu Val Pro Ala Tyr Arg Ala Val Ile Val Leu Ser Thr Gln

2015 2020 2025

Gly Lys Pro Pro Ala Ala Thr Thr Ser Gln Thr Pro Ser Leu Gln

2030 2035 2040

Ala Asp Pro Ala Ala Arg Gly Asn Pro Tyr Asp Gly Lys Thr Leu

2045 2050 2055

Phe His Gly Pro Ala Phe Gln Gly Leu Lys Glu Ile Ile Ser Cys

2060 2065 2070

Asn Lys Ser Gln Leu Val Ala Glu Cys Thr Phe Ile Pro Ser Ser

2075 2080 2085

Glu Ser Ala Gly Glu Phe Ala Ser Asp Tyr Glu Ser His Asn Pro

2090 2095 2100

Phe Val Asn Asp Ile Ala Phe Gln Ala Met Leu Val Trp Ile Arg

2105 2110 2115

Arg Thr Leu Gly Gln Ala Ala Leu Pro Asn Ser Ile Gln Arg Ile

2120 2125 2130

Val Gln His Arg Ala Leu Pro Gln Asp Lys Pro Phe Tyr Leu Thr

2135 2140 2145

Leu Lys Ser Asn Ser Ala Ser Gly His Ser Gln His Lys Thr Ser

2150 2155 2160

Val Gln Phe His Asn Glu Gln Gly Asp Leu Phe Val Asp Ile Gln

2165 2170 2175

Ala Ser Val Thr Ser Ser Asp Ser Leu Ala Phe

　　 2180 2185

<210> 13

<211> 1672

<212> PRT

<213> Schizochytrium limacinum (Schizoochytrium limacinum)

<400> 13

Leu Lys Lys Glu Lys Glu Lys Glu Lys Glu Lys Glu Lys Glu Lys Glu

1 5 10 15

Lys Glu Lys Glu Lys Glu Lys Glu Lys Glu Lys Glu Lys Glu Lys Glu

20 25 30

Lys Glu Lys Glu Lys Glu Glu Glu Lys Glu Glu Glu Lys Glu Lys Glu

35 40 45

Lys Glu Lys Glu Lys Glu Lys Glu Lys Glu Glu Gly Asp Leu Lys Ser

50 55 60

Cys Leu Val Glu Lys Gly Glu Gly Gly Arg Ser Ser Asp Ser Gly Arg

65 70 75 80

Arg Arg Ser Ser Cys Cys Lys Arg Gly Thr Glu Ala Val Ala Val Glu

85 90 95

Gln Ala Glu Ala Thr Ala Asn Leu Glu Leu Asp Pro Val Glu Pro Gln

100 105 110

Gln Glu Gln Glu Pro Asp Gln Val Asp Glu Asp Glu Val Arg Leu Leu

115 120 125

Ser Gly Thr Thr Glu Val Ala Gly Leu Ala Glu Ser Ala Thr Thr Ala

130 135 140

Ile Leu Arg Ser Thr Asp Ala Arg Ala Glu Asn Leu Gln Leu Leu Ala

145 150 155 160

Thr Thr Gln Glu Pro Pro Ser Asp Thr Thr Arg Phe Glu Asn Ser Thr

165 170 175

Ser Leu Glu Ala Ala Thr Ala Leu Ala Asp Asn Gln Thr Gly Pro Glu

180 185 190

Lys Ala Thr Thr Arg Arg Glu Ile Ile Glu Ser Gln Leu Ala Thr Met

195 200 205

Ala Thr Arg Val Lys Thr Asn Lys Lys Pro Cys Trp Glu Met Thr Lys

210 215 220

Glu Glu Leu Thr Ser Gly Lys Asn Val Val Phe Asp Tyr Asp Glu Leu

225 230 235 240

Leu Glu Phe Ala Glu Gly Asp Ile Ser Lys Val Phe Gly Pro Glu Phe

245 250 255

Ser Gln Ile Asp Gln Tyr Lys Arg Arg Val Arg Leu Pro Ala Arg Glu

260 265 270

Tyr Leu Leu Val Thr Arg Val Thr Leu Met Asp Ala Glu Val Asn Asn

275 280 285

Tyr Arg Val Gly Ala Arg Met Val Thr Glu Tyr Asp Leu Pro Val Asn

290 295 300

Gly Glu Leu Ser Glu Gly Gly Asp Cys Pro Trp Ala Val Leu Val Glu

305 310 315 320

Ser Gly Gln Cys Asp Leu Met Leu Ile Ser Tyr Met Gly Ile Asp Phe

325 330 335

Gln Asn Lys Ser Asp Arg Val Tyr Arg Leu Leu Asn Thr Thr Leu Thr

340 345 350

Phe Tyr Gly Val Ala Gln Glu Gly Glu Thr Leu Glu Tyr Asp Ile Arg

355 360 365

Val Thr Gly Phe Ala Lys Arg Leu Asp Gly Asp Ile Ser Met Phe Phe

370 375 380

Phe Glu Tyr Asp Cys Tyr Val Asn Gly Arg Leu Leu Ile Glu Met Arg

385 390 395 400

Asp Gly Cys Ala Gly Phe Phe Thr Asn Glu Glu Leu Ala Ala Gly Lys

405 410 415

Gly Val Val Phe Thr Arg Ala Asp Leu Leu Ala Arg Glu Lys Thr Lys

420 425 430

Lys Gln Asp Ile Thr Pro Tyr Ala Ile Ala Pro Arg Leu Asn Lys Thr

435 440 445

Val Leu Asn Glu Thr Glu Met Gln Ser Leu Val Asp Lys Asn Trp Thr

450 455 460

Lys Val Phe Gly Pro Glu Asn Gly Met Asp Gln Ile Asn Tyr Lys Leu

465 470 475 480

Cys Ala Arg Lys Met Leu Met Ile Asp Arg Val Thr Lys Ile Asp Tyr

485 490 495

Thr Gly Gly Pro Tyr Gly Leu Gly Leu Leu Val Gly Glu Lys Ile Leu

500 505 510

Glu Arg Asp His Trp Tyr Phe Pro Cys His Phe Val Gly Asp Gln Val

515 520 525

Met Ala Gly Ser Leu Val Ser Asp Gly Cys Ser Gln Leu Leu Lys Met

530 535 540

Tyr Met Leu Trp Leu Gly Leu His Leu Lys Thr Gly Pro Phe Asp Phe

545 550 555 560

Arg Pro Val Asn Gly His Pro Asn Lys Val Arg Cys Arg Gly Gln Ile

565 570 575

Ser Pro His Lys Gly Lys Leu Val Tyr Val Met Glu Ile Lys Glu Met

580 585 590

Gly Tyr Asp Glu Ala Gly Asp Pro Tyr Ala Ile Ala Asp Val Asn Ile

595 600 605

Leu Asp Ile Asp Phe Glu Lys Gly Gln Thr Phe Asp Leu Ala Asn Leu

610 615 620

His Glu Tyr Gly Lys Gly Asp Leu Asn Lys Lys Ile Val Val Asp Phe

625 630 635 640

Lys Gly Ile Ala Leu Lys Leu Gln Lys Arg Ser Gly Pro Ala Val Val

645 650 655

Ala Pro Glu Lys Pro Leu Ala Leu Asn Lys Asp Leu Cys Ala Pro Ala

660 665 670

Val Glu Ala Ile Pro Glu His Ile Leu Lys Gly Asp Ala Leu Ala Pro

675 680 685

Asn Gln Met Thr Trp His Pro Met Ser Lys Ile Ala Gly Asn Pro Thr

690 695 700

Pro Ser Phe Ser Pro Ser Ala Tyr Pro Pro Arg Pro Ile Thr Phe Thr

705 710 715 720

Pro Phe Pro Gly Asn Lys Asn Asp Asn Asn His Val Pro Gly Glu Met

725 730 735

Pro Leu Ser Trp Tyr Asn Met Ala Glu Phe Met Ala Gly Lys Val Ser

740 745 750

Leu Cys Leu Gly Pro Glu Phe Ala Lys Phe Asp Asp Ser Asn Thr Ser

755 760 765

Arg Ser Pro Ala Trp Asp Leu Ala Leu Val Thr Arg Val Val Ser Val

770 775 780

Ser Asp Met Glu Trp Val Gln Trp Lys Asn Val Asp Cys Asn Pro Ser

785 790 795 800

Lys Gly Thr Met Val Gly Glu Phe Asp Cys Pro Ile Asp Ala Trp Phe

805 810 815

Phe Gln Gly Ser Cys Asn Asp Gly His Met Pro Tyr Ser Ile Leu Met

820 825 830

Glu Ile Ala Leu Gln Thr Ser Gly Val Leu Thr Ser Val Leu Lys Ala

835 840 845

Pro Leu Thr Met Glu Lys Lys Asp Ile Leu Phe Arg Asn Leu Asp Ala

850 855 860

Asn Ala Glu Met Val Arg Ser Asp Ile Asp Leu Arg Gly Lys Thr Ile

865 870 875 880

His Asn Leu Thr Lys Cys Thr Gly Tyr Ser Met Leu Gly Asp Met Gly

885 890 895

Val His Arg Phe Ser Phe Glu Leu Ser Val Asp Gly Val Val Phe Tyr

900 905 910

Lys Gly Thr Thr Ser Phe Gly Trp Phe Val Pro Glu Val Phe Ile Ser

915 920 925

Gln Thr Gly Leu Asp Asn Gly Arg Arg Thr Gln Pro Trp His Ile Glu

930 935 940

Ser Lys Val Pro Ser Ala Gln Val Leu Thr Tyr Asp Val Thr Pro Asn

945 950 955 960

Gly Ala Gly Arg Thr Gln Leu Tyr Ala Asn Ala Pro Lys Gly Ala Gln

965 970 975

Leu Thr Arg Arg Trp Asn Gln Cys Gln Tyr Leu Asp Thr Ile Asp Leu

980 985 990

Val Val Ala Gly Gly Ser Ala Gly Leu Gly Tyr Gly His Gly Arg Lys

995 1000 1005

Gln Val Asn Pro Lys Asp Trp Phe Phe Ser Cys His Phe Trp Phe

1010 1015 1020

Asp Ser Val Met Pro Gly Ser Leu Gly Val Glu Ser Met Phe Gln

1025 1030 1035

Leu Val Glu Ser Ile Ala Val Lys Gln Asp Leu Ala Gly Lys Tyr

1040 1045 1050

Gly Ile Thr Asn Pro Thr Phe Ala His Ala Pro Gly Lys Ile Ser

1055 1060 1065

Trp Lys Tyr Arg Gly Gln Leu Thr Pro Thr Ser Lys Phe Met Asp

1070 1075 1080

Ser Glu Ala His Ile Val Ser Ile Glu Ala His Asp Gly Val Val

1085 1090 1095

Asp Ile Val Ala Asn Gly Asn Leu Trp Ala Asp Gly Leu Arg Val

1100 1105 1110

Tyr Asn Val Ser Asn Ile Arg Val Arg Ile Val Ala Gly Ala Ala

1115 1120 1125

Pro Ala Ala Ala Ala Ala Ala Ala Ala Val Ala Ala Pro Ala Ala

1130 1135 1140

Ala Pro Ala Pro Val Ala Ala Ser Gly Pro Ala Gln Thr Ile Thr

1145 1150 1155

Leu Lys Gln Leu Lys Ala Glu Leu Leu Asp Val Glu Lys Pro Leu

1160 1165 1170

Tyr Ile Ser Ser Ser Asn Gly Gln Val Lys Lys His Ala Asp Val

1175 1180 1185

Ala Gly Gly Gln Ala Thr Ile Val Gln Ala Cys Ser Leu Ser Asp

1190 1195 1200

Leu Gly Asp Glu Gly Phe Met Lys Thr Tyr Gly Val Val Ala Pro

1205 1210 1215

Leu Tyr Thr Gly Ala Met Ala Lys Gly Ile Ala Ser Ala Asp Leu

1220 1225 1230

Val Ile Ala Thr Gly Lys Arg Lys Ile Leu Gly Ser Phe Gly Ala

1235 1240 1245

Gly Gly Leu Pro Met His Ile Val Arg Ala Ala Val Glu Lys Ile

1250 1255 1260

Gln Ala Glu Leu Pro Asn Gly Pro Phe Ala Val Asn Leu Ile His

1265 1270 1275

Ser Pro Phe Asp Ser Asn Leu Glu Lys Gly Asn Val Asp Leu Phe

1280 1285 1290

Leu Glu Lys Gly Val Thr Val Val Glu Ala Ser Ala Phe Met Thr

1295 1300 1305

Leu Thr Pro Gln Val Val Arg Tyr Arg Ala Ala Gly Leu Ser Arg

1310 1315 1320

Asn Ala Asp Gly Ser Ile Asn Ile Lys Asn Arg Ile Ile Gly Lys

1325 1330 1335

Val Ser Arg Thr Glu Leu Ala Glu Met Phe Ile Arg Pro Ala Pro

1340 1345 1350

Gln Asn Leu Leu Asp Lys Leu Ile Gln Ser Gly Glu Ile Thr Lys

1355 1360 1365

Glu Gln Ala Glu Leu Ala Lys Leu Val Pro Val Ala Asp Asp Ile

1370 1375 1380

Ala Val Glu Ala Asp Ser Gly Gly His Thr Asp Asn Arg Pro Ile

1385 1390 1395

His Val Ile Leu Pro Leu Ile Ile Asn Leu Arg Asn Arg Leu His

1400 1405 1410

Lys Glu Cys Gly Tyr Pro Ala His Leu Arg Val Arg Val Gly Ala

1415 1420 1425

Gly Gly Gly Val Gly Cys Pro Gln Ala Ala Ala Ala Ala Leu Ala

1430 1435 1440

Met Gly Ala Ala Phe Leu Val Thr Gly Thr Val Asn Gln Val Ala

1445 1450 1455

Lys Gln Ser Gly Thr Cys Asp Asn Val Arg Lys Gln Leu Cys Met

1460 1465 1470

Ala Thr Tyr Ser Asp Val Cys Met Ala Pro Ala Ala Asp Met Phe

1475 1480 1485

Glu Glu Gly Val Lys Leu Gln Val Leu Lys Lys Gly Thr Met Phe

1490 1495 1500

Pro Ser Arg Ala Asn Lys Leu Tyr Glu Leu Phe Cys Lys Tyr Asp

1505 1510 1515

Ser Phe Glu Ser Met Pro Ala Thr Glu Leu Glu Arg Val Glu Lys

1520 1525 1530

Arg Ile Phe Gln Cys Pro Leu Ala Asp Val Trp Ala Glu Thr Ser

1535 1540 1545

Asp Phe Tyr Ile Asn Arg Leu His Asn Pro Glu Lys Ile Thr Arg

1550 1555 1560

Ala Glu Arg Asp Pro Lys Leu Lys Met Ser Leu Cys Phe Arg Trp

1565 1570 1575

Tyr Leu Gly Leu Ala Ser Arg Trp Ala Asn Thr Gly Glu Ala Gly

1580 1585 1590

Arg Val Met Asp Tyr Gln Val Trp Cys Gly Pro Ala Ile Gly Ala

1595 1600 1605

Phe Asn Asp Phe Ile Lys Gly Ser Tyr Leu Asp Pro Ala Val Ser

1610 1615 1620

Gly Glu Tyr Pro Asp Val Val Gln Ile Asn Leu Gln Ile Leu Arg

1625 1630 1635

Gly Ala Cys Tyr Leu Arg Arg Leu Asn Val Ile Arg Asn Asp Pro

1640 1645 1650

Arg Val Ser Ile Glu Val Glu Asp Ala Glu Phe Val Tyr Glu Pro

1655 1660 1665

Thr Asn Ala Leu

　　1670

<210> 14

<211> 21

<212> PRT

<213> Schizochytrium limacinum (Schizoochytrium limacinum)

<400> 14

Met Ser Thr Ile Arg Leu Ser His His Ala Asn Ala Lys Cys Leu Gly

1 5 10 15

Ser Ser Gln Lys Cys

　　　　　　20

<210> 15

<211> 2352

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 15

cccaccccag cttcaaaagc gctctaccgt tcgtataatg tatgctatac gaagttattc 60

tctctccttg tcaactcaca cccgaaatcg ttaagcattt ccttctgagt ataagaatca 120

ttcaaaggcg cgccacagga ctagtcccac acaccatagc ttcaaaatgt ttctactcct 180

tttttactct tccagatttt ctcggactcc gcgcatcgcc gtaccacttc aaaacaccca 240

agcacagcat actaaatttt ccctctttct tcctctaggg tgtcgttaat tacccgtact 300

aaaggtttgg aaaagaaaaa agagaccgcc tcgtttcttt ttcttcgtcg aaaaaggcaa 360

taaaaatttt tatcacgttt ctttttcttg aaattttttt ttttagtttt tttctctttc 420

agtgacctcc attgatattt aagttaataa acggtcttca atttctcaag tttcagtttc 480

atttttcttg ttctattaca acttttttta cttcttgttc attagaaaga aagcatagca 540

atctaatcta aggggcggtg ttgacaatta atcatcggca tagtatatcg gcatagtata 600

atacgacaag gtgaggaact aaaccatggc caagttgacc agtgccgttc cggtgctcac 660

cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt tctcccggga 720

cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc 780

ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc gcggcctgga 840

cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg cctccgggcc 900

ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc gcgacccggc 960

cggcaactgc gtgcacttcg tggccgagga gcaggactga caattgaagc acgctagcct 1020

catgtaatta gttatgtcac gcttacattc acgccctccc tccacatccg ctctaaccga 1080

aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta tagttatgtt 1140

agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca gacgcgtgta 1200

cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct cgaaggcttt 1260

aatttgcaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 1320

tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 1380

gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 1440

tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 1500

ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 1560

cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 1620

gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 1680

tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 1740

agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 1800

tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 1860

cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg 1920

ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 1980

ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 2040

ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 2100

ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 2160

cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 2220

gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 2280

atcctttgat ctataacttc gtataatgta tgctatacga acggtaagga ggatattctc 2340

gagactagtc tg 2352

Claims (19)

1. A combination of proteins as follows (X1) or (X2):

(X1) includes protein 1, protein 2, protein 3, protein 4, protein 5 and protein 6;

(X2) consisting of protein 1, protein 2, protein 3, protein 4, protein 5 and protein 6;

the protein 1 is a1) or a 2):

a1) the amino acid sequence is protein shown as a sequence 9 in a sequence table;

a2) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 9 in the sequence table;

the protein 2 is b1) or b 2):

b1) the amino acid sequence is protein shown as a sequence 10 in a sequence table;

b2) a fusion protein obtained by connecting labels to the N terminal or/and the C terminal of the protein shown in the sequence 10 in the sequence table;

the protein 3 is c1) or c 2):

c1) the amino acid sequence is a protein shown as a sequence 11 in a sequence table;

c2) a fusion protein obtained by connecting labels to the N terminal or/and the C terminal of the protein shown in the sequence 11 in the sequence table;

the protein 4 is d1) or d 2):

d1) the amino acid sequence is protein shown as a sequence 12 in a sequence table;

d2) a fusion protein obtained by connecting labels to the N terminal or/and the C terminal of the protein shown in the sequence 12 in the sequence table;

the protein 5 is e1) or e 2):

e1) the amino acid sequence is protein shown as a sequence 13 in a sequence table;

e2) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 13 in the sequence table;

the protein 6 is f1) or f 2):

f1) the amino acid sequence is protein shown as a sequence 14 in a sequence table;

f2) and (b) fusion protein obtained by connecting labels to the N terminal or/and the C terminal of the protein shown in the sequence 14 in the sequence table.

2. Nucleic acid molecules encoding the combination of proteins of claim 1.

3. The nucleic acid molecule of claim 2, wherein:

the nucleic acid molecule for coding the protein 1 is a DNA molecule shown as A1) or A2):

A1) the coding region is a DNA molecule shown in the 1044 th to the 3050 th site from the 5' end of a sequence 3 in a sequence table;

A2) the nucleotide sequence is a DNA molecule shown in a sequence 3 in a sequence table;

the nucleic acid molecule for coding the protein 2 is a DNA molecule shown as the following B1) or B2):

B1) the coding region is a DNA molecule shown in 1068 th to 2737 th and 3254 th to 5162 th from the 5' end of a sequence 4 in the sequence table;

B2) the nucleotide sequence is a DNA molecule shown as a sequence 4 in the sequence table;

the nucleic acid molecule for coding the protein 3 is a DNA molecule shown as the following C1) or C2):

C1) the coding region is a DNA molecule shown in 1094 to 3415 th sites from the 5' end of a sequence 5 in a sequence table;

C2) the nucleotide sequence is a DNA molecule shown as a sequence 5 in a sequence table;

the nucleic acid molecule for coding the protein 4 is a DNA molecule shown in the following D1) or D2):

D1) the coding region is a DNA molecule shown in 1409 th to 5044 th, 7004 th to 7234 th and 7700 th to 10399 th positions of the 5' end of a sequence 6 in the sequence table;

D2) the nucleotide sequence is a DNA molecule shown as a sequence 6 in a sequence table;

the nucleic acid molecule for coding the protein 5 is a DNA molecule shown as the following E1) or E2):

E1) the coding region is a DNA molecule shown in 1473 th to 6488 th positions from the 5' end of a sequence 7 in a sequence table;

E2) the nucleotide sequence is a DNA molecule shown as a sequence 7 in a sequence table;

the nucleic acid molecule for coding the protein 6 is a DNA molecule shown as F1) or F2):

F1) the coding region is a DNA molecule shown in 953 th to 991 th and 1063 th to 1090 th positions from the 5' end of a sequence 8 in a sequence table;

F2) the nucleotide sequence is a DNA molecule shown as a sequence 8 in a sequence table.

4. An expression cassette comprising the nucleic acid molecule of claim 2 or 3.

5. A recombinant vector comprising the nucleic acid molecule of claim 2 or 3.

6. A recombinant microorganism comprising the nucleic acid molecule of claim 2 or 3.

7. A transgenic cell line comprising the nucleic acid molecule of claim 2 or 3.

8. Use of a combination of proteins according to claim 1 for the production of DHA and/or EPA.

9. Use of the nucleic acid molecule according to claim 2 or 3 for the production of DHA and/or EPA.

10. Use of an expression cassette comprising a nucleic acid molecule according to claim 2 or 3 for the production of DHA and/or EPA.

11. Use of a recombinant vector comprising a nucleic acid molecule according to claim 2 or 3 for the production of DHA and/or EPA.

12. Use of a recombinant microorganism comprising a nucleic acid molecule according to claim 2 or 3 for the production of DHA and/or EPA.

13. Use of a transgenic cell line comprising a nucleic acid molecule according to claim 2 or 3 for the production of DHA and/or EPA.

14. The preparation method of the recombinant bacterium B comprises the following steps: introducing nucleic acid molecule combination into the spawn to obtain recombinant spawn B; the combination of nucleic acid molecules consisting of nucleic acid molecules encoding each of the proteins of the combination of proteins of claim 1.

15. The recombinant bacterium b according to claim 14, wherein: the outbreak fungus is schizochytrium limacinum.

16. The recombinant bacterium b according to claim 15, wherein: the Schizochytrium limacinum Honda et Yokochi ATCC MYA-1381 is a strain Schizochytrium limacinum Honda.

17. A process for the production of DHA and/or EPA comprising the following steps in sequence:

(1) introducing a nucleic acid molecule combination into the fermentation bacteria to obtain recombinant bacteria A; the combination of nucleic acid molecules consisting of nucleic acid molecules encoding each of the proteins of the combination of proteins of claim 1; the recombinant bacterium A has an improved ability to produce DHA and/or EPA compared to the starting bacterium;

(2) fermenting and culturing the recombinant bacterium A to obtain DHA and/or EPA.

18. The method of claim 17, wherein: the outbreak fungus is schizochytrium limacinum.

19. The method of claim 18, wherein: the Schizochytrium limacinum Honda et Yokochi ATCC MYA-1381 is a strain Schizochytrium limacinum Honda.

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