WO2018219171A1 - 一株生产dha和epa的细菌、该细菌基因组中的6个基因片段及它们的应用 - Google Patents

一株生产dha和epa的细菌、该细菌基因组中的6个基因片段及它们的应用 Download PDF

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WO2018219171A1
WO2018219171A1 PCT/CN2018/087613 CN2018087613W WO2018219171A1 WO 2018219171 A1 WO2018219171 A1 WO 2018219171A1 CN 2018087613 W CN2018087613 W CN 2018087613W WO 2018219171 A1 WO2018219171 A1 WO 2018219171A1
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protein
sequence
seq
sequence listing
amino acid
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PCT/CN2018/087613
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English (en)
French (fr)
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陈礼毅
钟惠昌
陈水荣
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厦门汇盛生物有限公司
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Priority claimed from CN201710398286.7A external-priority patent/CN106987528B/zh
Priority claimed from CN201711102734.0A external-priority patent/CN109776663B/zh
Application filed by 厦门汇盛生物有限公司 filed Critical 厦门汇盛生物有限公司
Priority to JP2019566735A priority Critical patent/JP7039625B2/ja
Priority to EP18810206.5A priority patent/EP3628679A4/en
Publication of WO2018219171A1 publication Critical patent/WO2018219171A1/zh
Priority to US16/696,331 priority patent/US10941185B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C12N1/125—Unicellular algae isolates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/405—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from algae
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C12P7/6432—Eicosapentaenoic acids [EPA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C12P7/6434—Docosahexenoic acids [DHA]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/89—Algae ; Processes using algae

Definitions

  • the invention relates to the field of industrial microorganisms, food and feed industries, in particular to a strain producing DHA and/or EPA, 6 gene fragments in the bacterial genome and their applications, wherein 6 gene fragments are also associated with DHA and/or Related to EPA synthesis.
  • Polyunsaturated fatty acids are a class of linear fatty acids containing two or more double bonds, typically having a carbon chain length of 18-22 carbon atoms. According to the position of the double bond, polyunsaturated fatty acids can be divided into omega-3 and omega-6. In the multi-unsaturated fatty acid molecule, the double bond at the farthest end from the carboxyl group is called omega on the third last carbon atom. -3, on the sixth carbon atom is called omega-6.
  • Polyunsaturated fatty acids are indispensable fatty acids in the human body, mainly docosahexaenoic acid (DHA), docosapentenoic acid (DPA), eicosapentaenoic acid ( Eicosapentaenoic Acid, EPA), etc.
  • DHA docosahexaenoic acid
  • DPA docosapentenoic acid
  • EPA eicosapentaenoic Acid
  • DHA is the most important class of polyunsaturated fatty acids. In molecular structure, DHA is a linear fatty acid containing 22 carbon atoms and 6 double bonds. Because its first double bond appears on the 3rd carbon atom of the methyl end of the fatty acid chain, it belongs to the omega-3 series of fatty acids. (OMEGA-3). DHA is mainly found in the brain and retina of the human body and has important physiological functions such as promoting nervous system development, improving retinal function, improving vision, preventing cardiovascular disease, treating cardiovascular disease, resisting inflammation and suppressing allergic reactions. Since the body itself cannot synthesize enough DHA, it is mainly obtained by food intake. Because DHA is often insufficient in daily diets, supplementing DHA or adding DHA to food or milk powder is important for humans, especially infants and the elderly.
  • DHA DHA
  • traditional DHA which is extracted from the tissues of marine fish (mainly including carp, carp, salmon and sardine)
  • the quality of fish oil obtained by extraction will follow Fish species, seasons and changes in fishing grounds are affected, and the quality of fish oil is also affected by the growing shortage of fish resources and environmental pollution
  • the emerging DHA production method which uses marine microbes for fermentation to produce DHA.
  • the method has the advantages of short cycle, no influence on objective conditions, no fishy smell, and the like, and has broad prospects.
  • the marine microorganisms used for fermentative production of DHA are mainly Schizochytrium, but the Schizochytrium strains currently used for fermentative production of DHA are limited by their own total fatty acid content, DHA content, growth rate and other technical indicators. Increase production and reduce costs.
  • the DHA biosynthetic pathway is catalyzed by a series of enzymes in the relevant anabolic pathway.
  • Excavation, transformation and heterologous expression of genes related to the DHA biosynthetic pathway will provide favorable conditions for further increasing the yield of DHA. Therefore, obtaining new key genes in the DHA biosynthetic pathway will facilitate the transformation and process optimization of DHA production strains.
  • DHA synthesis pathways There are two DHA synthesis pathways in nature: (1) elongation-desaturation pathway (ED pathway), which is based on the fatty acid synthesis pathway and further synthesizes DHA by prolonging the action of enzymes and desaturases; 2) Polyketide synthase pathway (PKS pathway), mainly under the action of polyketide synthase, acetyl CoA and malonyl CoA as precursors to synthesize DHA.
  • ED pathway elongation-desaturation pathway
  • PKS pathway Polyketide synthase pathway
  • the synthesis of DHA mainly adopts the PKS pathway. It is currently believed that the synthesis of DPA and EPA also has an E-D pathway and a PKS pathway.
  • the object of the invention is to prepare DPA and/or EPA.
  • the present invention first protects the protein combination used to prepare DPA and/or EPA, which may be as follows (X1) or (X2) or (X3) or (X4):
  • (X1) includes protein 1, protein 2, protein 3, protein 4, protein 5, and protein 6;
  • (X2) consists of protein 1, protein 2, protein 3, protein 4, protein 5 and protein 6;
  • (X3) consisting of any two, any three, any four or any five of the protein 1, the protein 2, the protein 3, the protein 4, the protein 5, and the protein 6 composition;
  • the protein 1 may be a1) or a2) or a3) or a4) or a5):
  • amino acid sequence is the protein shown by SEQ ID NO: 9 in the sequence listing;
  • A2 a fusion protein obtained by ligating the N-terminus or/and C-terminus of the protein shown in SEQ ID NO: 9 in the Sequence Listing;
  • A3 a protein having the same function obtained by subjecting the amino acid sequence shown by SEQ ID NO: 9 in the sequence listing to substitution and/or deletion and/or addition of one or several amino acid residues;
  • A5 A protein having the amino acid sequence shown by SEQ ID NO: 9 in the Sequence Listing.
  • the protein 2 may be b1) or b2) or b3) or b4) or b5):
  • the protein 3 may be c1) or c2) or c3) or c4) or c5):
  • C3 a protein having the same function obtained by subjecting the amino acid sequence shown in SEQ ID NO: 11 of the sequence listing to substitution and/or deletion and/or addition of one or several amino acid residues;
  • the protein 4 may be d1) or d2) or d3) or d4) or d5):
  • D3 a protein having the same function obtained by subjecting the amino acid sequence shown by SEQ ID NO: 12 in the sequence listing to substitution and/or deletion and/or addition of one or several amino acid residues;
  • D4 having 80% or more of the identity with the amino acid sequence defined by SEQ ID NO: 12 in the sequence listing, a protein derived from Schizochytrium and associated with polyunsaturated fatty acid synthesis;
  • the protein 5 may be e1) or e2) or e3) or e4) or e5):
  • E2 a fusion protein obtained by ligating the N-terminus or/and C-terminus of the protein shown in SEQ ID NO: 13 in the Sequence Listing;
  • E3 a protein having the same function obtained by subjecting the amino acid sequence shown by SEQ ID NO: 13 in the sequence listing to substitution and/or deletion and/or addition of one or several amino acid residues;
  • E4 having 80% or more of the amino acid sequence defined by the sequence 13 in the sequence listing, derived from Schizochytrium and associated with polyunsaturated fatty acid synthesis;
  • the protein may be f1) or f2) or f3) or f4) or f5):
  • F2 a fusion protein obtained by ligating the N-terminus or/and C-terminus of the protein shown in SEQ ID NO: 14 in the Sequence Listing;
  • F3 a protein having the same function obtained by subjecting the amino acid sequence shown in SEQ ID NO: 14 of the Sequence Listing to substitution and/or deletion and/or addition of one or several amino acid residues;
  • F4 having 80% or more of the identity with the amino acid sequence defined by SEQ ID NO: 14 in the sequence listing, a protein derived from Schizochytrium and associated with polyunsaturated fatty acid synthesis;
  • F5 A protein having the amino acid sequence shown in SEQ ID NO: 14 in the Sequence Listing.
  • Sequence 9 in the sequence listing consists of 669 amino acid residues, and sequence 10 in the sequence listing consists of 1193 amino acid residues. Sequence 11 in the sequence listing consists of 773 amino acid residues. Sequence 12 in the sequence listing consists of 2189 amino acids. The residue consists of a sequence 13 consisting of 1672 amino acid residues in the sequence listing, and sequence 14 in the sequence listing consisting of 21 amino acid residues.
  • a label as shown in Table 1 may be attached to the amino terminus or carboxy terminus of the protein shown in SEQ ID NO:9 in the Sequence Listing.
  • a label as shown in Table 1 may be attached to the amino terminus or carboxy terminus of the protein shown in SEQ ID NO: 10 in the Sequence Listing.
  • a label as shown in Table 1 may be attached to the amino terminus or the carboxy terminus of the protein shown in SEQ ID NO: 11 in the Sequence Listing.
  • a label as shown in Table 1 may be attached to the amino terminus or carboxy terminus of the protein shown in SEQ ID NO: 12 in the Sequence Listing.
  • a label as shown in Table 1 may be attached to the amino terminus or carboxy terminus of the protein shown in SEQ ID NO: 13 in the Sequence Listing.
  • a label as shown in Table 1 may be attached to the amino terminus or carboxy terminus of the protein shown in SEQ ID NO: 14 in the Sequence Listing.
  • Substitutions and/or deletions and/or additions of one or several amino acid residues are substitutions and/or deletions and/or additions of no more than 10 amino acid residues.
  • Protein 1 in a3), protein 2 in b3), protein 3 in c3), protein 4 in d3), protein 5 in e3), and protein 6 in f3) Synthetic, it is also possible to synthesize its coding gene and then obtain the biological expression.
  • the gene encoding the protein 1 in the above a3) can be obtained by deleting a codon of one or several amino acid residues in the DNA sequence shown in the sequence 9 in the sequence listing, and/or performing one or several base pairs.
  • the mutation, and/or the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end is obtained.
  • the gene encoding the protein 2 in the above b3) can be obtained by deleting a codon of one or several amino acid residues in the DNA sequence shown in the sequence 10 in the sequence listing, and/or performing one or several base pair mismatches.
  • the mutation, and/or the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end is obtained.
  • the gene encoding the protein 3 in the above c3) can be obtained by deleting a codon of one or several amino acid residues in the DNA sequence shown in the sequence 11 in the sequence listing, and/or performing one or several base pair misclassifications.
  • the mutation, and/or the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end is obtained.
  • the gene encoding the protein 4 in the above d3) can be obtained by deleting a codon of one or several amino acid residues in the DNA sequence shown in the sequence 12 in the sequence listing, and/or performing a one or several base pair misses.
  • the mutation, and/or the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end is obtained.
  • the gene encoding the protein 5 in the above e3) can be obtained by deleting a codon of one or several amino acid residues in the DNA sequence shown in the sequence 13 in the sequence listing, and/or performing one or several base pairs.
  • the mutation, and/or the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end is obtained.
  • the gene encoding the protein 6 in the above f3) can be obtained by deleting a codon of one or several amino acid residues in the DNA sequence shown in the sequence 14 in the sequence listing, and/or performing one or several base pair mismatches.
  • the mutation, and/or the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end is obtained.
  • identity refers to sequence similarity to a native amino acid sequence. “Identity” includes amino acid sequences having 80%, or 85% or more, or 90% or more, or 95% or more identity to the amino acid sequence of the protein provided by the present invention.
  • Nucleic acid molecules encoding such protein combinations are also within the scope of the invention.
  • the nucleic acid molecule encoding the protein 1 may be a DNA molecule as shown in A1) or A2) or A3) or A4):
  • the coding region is a DNA molecule represented by the 1044th to 3050th position of the sequence 3 from the 5' end in the sequence listing;
  • nucleotide sequence is the DNA molecule shown in SEQ ID NO: 3 in the Sequence Listing;
  • A3) having 75% or more of the identity with the nucleotide sequence defined by A1) or A2), a DNA molecule derived from Schizochytrium and encoding the protein 1;
  • A4) hybridizing under stringent conditions to a nucleotide sequence defined by A1) or A2) and encoding the DNA molecule of said protein 1.
  • the nucleic acid molecule encoding the protein 2 may be a DNA molecule as shown in B1) or B2) or B3) or B4) below:
  • the B1) coding region is the DNA molecule of sequence 4 from positions 568 to 2737 and positions 3254 to 5162 from the 5' end;
  • the B2) nucleotide sequence is the DNA molecule shown in SEQ ID NO: 4 in the Sequence Listing;
  • B3 having 75% or more of the identity of the nucleotide sequence defined by B1) or B2), derived from Schizochytrium and encoding the DNA molecule of said protein 2;
  • B4 hybridizing under stringent conditions to a nucleotide sequence defined by B1) or B2) and encoding the DNA molecule of said protein 2.
  • the nucleic acid molecule encoding the protein 3 may be a DNA molecule as shown by C1) or C2) or C3) or C4):
  • the C1) coding region is a DNA molecule represented by the 10th to 34th positions of the sequence 5 from the 5' end in the sequence listing;
  • nucleotide sequence is the DNA molecule shown in SEQ ID NO: 5 in the Sequence Listing;
  • C3 having 75% or more of the identity of the nucleotide sequence defined by C1) or C2), a DNA molecule derived from Schizochytrium and encoding said protein 3;
  • C4 A nucleotide sequence which hybridizes under stringent conditions to a nucleotide sequence defined by C1) or C2) and which encodes said protein 3.
  • the nucleic acid molecule encoding the protein 4 may be a DNA molecule as shown in D1) or D2) or D3) or D4):
  • the D1) coding region is a DNA molecule of sequence 1 from position 5409 to position 5409, positions 7004 to 7234 and positions 7700 to 10399 from the 5' end;
  • nucleotide sequence is the DNA molecule shown in SEQ ID NO: 6 in the Sequence Listing;
  • D3 having 75% or more of the identity of the nucleotide sequence defined by D1) or D2), derived from Schizochytrium and encoding the DNA molecule of said protein 4;
  • D4 hybridizing under stringent conditions to a nucleotide sequence defined by D1) or D2) and encoding the DNA molecule of said protein 4.
  • the nucleic acid molecule encoding the protein 5 may be a DNA molecule as shown by E1) or E2) or E3) or E4):
  • the E1) coding region is the DNA molecule shown in positions 1473 to 6488 of the sequence 7 from the 5' end in the sequence listing;
  • the E2) nucleotide sequence is the DNA molecule shown in SEQ ID NO: 7 in the Sequence Listing;
  • E3 having 75% or more of the identity of the nucleotide sequence defined by E1) or E2), a DNA molecule derived from Schizochytrium and encoding the protein 5;
  • E4 A DNA sequence which hybridizes under stringent conditions to a nucleotide sequence defined by E1) or E2) and which encodes said protein 5.
  • the nucleic acid molecule encoding the protein 6 may be a DNA molecule as shown by F1) or F2) or F3) or F4):
  • the F1) coding region is a DNA molecule of SEQ ID NO: 953 to 991 and 1063 to 1090 from Sequence 5 in Sequence Listing;
  • nucleotide sequence is the DNA molecule shown in SEQ ID NO: 8 in the Sequence Listing;
  • F3 having 75% or more of the identity of the nucleotide sequence defined by F1) or F2), a DNA molecule derived from Schizochytrium and encoding said protein 6;
  • F4 hybridizing under stringent conditions to a nucleotide sequence defined by F1) or F2) and encoding the DNA molecule of said protein 6.
  • 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.
  • the nucleic acid molecule can be a nucleic acid molecule formed by a gene encoding the protein combination and its regulatory sequences.
  • sequence 3 in the sequence listing is composed of 4100 nucleotides, and the nucleotide sequence shown in the sequence 3 in the sequence listing encodes the amino acid sequence shown in SEQ ID NO: 9 in the sequence listing.
  • sequence 4 in the sequence listing consists of 6200 nucleotides, and the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing encodes the amino acid sequence shown in SEQ ID NO: 10 in the sequence listing.
  • the sequence 5 in the sequence listing consists of 4,500 nucleotides, and the nucleotide sequence shown in the sequence 5 in the sequence listing encodes the amino acid sequence shown in SEQ ID NO: 11 in the sequence listing.
  • Sequence 6 in the Sequence Listing consists of 11100 nucleotides, and the nucleotide sequence shown in Sequence 6 in the Sequence Listing encodes the amino acid sequence shown in SEQ ID NO: 12 in the Sequence Listing.
  • Sequence 7 in the sequence listing consists of 7767 nucleotides, and the nucleotide sequence shown in SEQ ID NO: 7 in the sequence listing encodes the amino acid sequence shown in SEQ ID NO: 13 in the Sequence Listing.
  • Sequence 8 in the sequence listing consists of 7800 nucleotides, and the nucleotide sequence shown in SEQ ID NO: 8 in the sequence listing encodes the amino acid sequence shown in SEQ ID NO: 14 in the Sequence Listing.
  • nucleotide sequences encoding the protein combinations of the present invention can readily mutate the nucleotide sequences encoding the protein combinations of the present invention using known methods, such as directed evolution and point mutation methods.
  • Those artificially modified nucleotides having 75% or more homology to the nucleotide sequence combined with the protein of the present invention are derived from the nucleosides of the present invention as long as they encode a protein combination and are derived from Schizochytrium.
  • the acid sequence is identical to the sequence of the invention.
  • identity refers to sequence similarity to a native nucleic acid sequence. "Identity" includes 75% or more, 80% or more, or 85% or more, or 90% or more, or 95% or more of the nucleotide sequence combined with the encoded protein of the present invention.
  • the nucleotide sequence of identity includes 75% or more, 80% or more, or 85% or more, or 90% or more, or 95% or more of the nucleotide sequence combined with the
  • Expression cassettes, recombinant vectors, recombinant microorganisms or transgenic cell lines containing the nucleic acid molecules are also within the scope of the invention.
  • the recombinant vector may be a nucleic acid molecule encoding the protein 1 (ie, a DNA molecule represented by SEQ ID NO: 3 in the sequence listing), a nucleic acid molecule encoding the protein 2 (ie, a DNA molecule represented by SEQ ID NO: 4 in the Sequence Listing) a nucleic acid molecule encoding the protein 3 (ie, a DNA molecule represented by SEQ ID NO: 5 in the Sequence Listing), a nucleic acid molecule encoding the protein 4 (ie, a DNA molecule represented by SEQ ID NO: 6 in the Sequence Listing), encoding the protein 5
  • the nucleic acid molecule i.e., the DNA molecule shown in SEQ ID NO: 7 in the Sequence Listing
  • the nucleic acid molecule encoding the protein 6 i.e., the DNA molecule shown in SEQ ID NO: 8 in the Sequence Listing
  • the recombinant microorganism can be obtained by introducing the recombinant vector into a starting microorganism.
  • the starting microorganism can be a yeast, a bacterium, an alga or a fungus.
  • the fungus may be a Schizochytrium.
  • the Schizochytrium may specifically be the strain Schizochytrium limacinum Hyundai et Yokochi ATCC MYA-1381.
  • the recombinant microorganism may specifically be the GS-C06 strain mentioned in the examples.
  • the invention also protects the recombinant bacteria B, and the preparation method thereof can be as follows: the expression and/or activity of the protein combination in the starting bacteria is improved, and the obtained recombinant bacteria is the recombinant bacteria B.
  • the "increasing the expression and/or activity of the protein combination in the starting bacteria” is achieved by introducing into the starting bacteria a substance that increases the expression and/or activity of the protein combination.
  • the "substance introduced into the starting bacteria to increase the expression and/or activity of the protein combination” is realized by introducing a nucleic acid molecule encoding the protein combination into the starting bacteria.
  • the starting bacteria may be a Schizochytrium.
  • the Schizochytrium may specifically be the strain Schizochytrium limacinum Hyundai et Yokochi ATCC MYA-1381.
  • the recombinant bacteria B may specifically be the GS-C06 strain mentioned in the examples.
  • the invention also protects a method of producing DHA and/or EPA, which in turn may comprise the following steps:
  • the recombinant strain A may be the recombinant bacteria B.
  • the "improving the expression and/or activity of the protein combination in the starting bacteria” is achieved by introducing a substance which increases the expression and/or activity of the protein combination into the starting bacteria.
  • the "substance introduced into the starting bacteria to increase the expression and/or activity of the protein combination” is realized by introducing a nucleic acid molecule encoding the protein combination into the starting bacteria.
  • the starting bacteria may be a Schizochytrium.
  • the Schizochytrium may specifically be the strain Schizochytrium limacinum Hyundai et Yokochi ATCC MYA-1381.
  • the recombinant strain A may specifically be the GS-C06 strain mentioned in the examples.
  • the "introduction of a nucleic acid molecule encoding the protein combination into a starting bacterium” can be achieved by introducing a recombinant vector into a starting bacterium;
  • the recombinant vector can be a nucleic acid molecule encoding the protein 1 inserted into the expression vector ( That is, the DNA molecule shown by the sequence 3 in the sequence table), the nucleic acid molecule encoding the protein 2 (ie, the DNA molecule shown by the sequence 4 in the sequence listing), and the nucleic acid molecule encoding the protein 3 (ie, the sequence 5 in the sequence listing) a DNA molecule shown), a nucleic acid molecule encoding the protein 4 (ie, a DNA molecule represented by SEQ ID NO: 6 in the Sequence Listing), a nucleic acid molecule encoding the protein 5 (ie, a DNA molecule represented by SEQ ID NO: 7 in the Sequence Listing) And a nucleic acid molecule encoding the protein 6 (
  • the present invention also protects a bacterium producing docosahexaenoic acid and/or EPA, which is Schizoochytrium limacinum HS01, which is deposited with the General Microbiology Center of the Chinese Collection of Microorganisms and Cultures. For CGMCC No. 13746.
  • the present invention also provides a microbial agent comprising the Schizoochytrium limacinum HS01 CGMCC No. 13746, or the recombinant B of any of the above.
  • the present invention also contemplates a method of producing docosahexaenoic acid and/or EPA, the method comprising fermenting the Schizoochytrium limacinum HS01 CGMCC No. 13746, or any of the above Recombinant B, a step of obtaining docosahexaenoic acid and/or EPA.
  • the fermentation medium solute used in the fermentation culture and the solubility thereof may be: glucose 20-120 g/L (such as 20-60 g/L, 60-120 g/L, 20 g/L, 60 g/L or 120 g).
  • glutamic acid or sodium glutamate 5 ⁇ 15g / L (such as 5 ⁇ 10g / L, 10 ⁇ 15g / L, 5g / L, 10g / L or 15g / L), corn syrup dry powder 3 ⁇ 15g /L (such as 3 ⁇ 10g / L, 10 ⁇ 15g / L, 3g / L, 10g / L or 15g / L), NaSO 4 5 ⁇ 24g / L (such as 5 ⁇ 14g / L, 14 ⁇ 24g / L, 5g / L, 14g / L or 24g / L), KCl 0.1 ⁇ 1.0g / L (such as 0.1 ⁇ 0.5g / L, 0.5 ⁇ 1.0g / L, 0.1g / L, 0.5g / L or 1.0g / L ), MgSO 4 1.0 to 3.0 g / L (such as 1.0 ⁇ 2.0g / L, 2.0 ⁇ 3.0g / L (
  • the fermentation medium may specifically be: 60 g of glucose, 10 g of glutamic acid or sodium glutamate, 10 g of corn syrup dry powder, 14 g of NaSO 4 , 0.5 g of KCl, 2.0 g of MgSO 4 , 1.0 g of K 2 SO 4 , KH 2 PO 4 1.0 g, (NH 4 ) 2 SO 4 1.0 g and CaCl 2 0.5 g were dissolved in 1 L of distilled water to adjust the pH to 6.0.
  • the initial biomass in the fermentation culture may be 1.0 ⁇ 10 8 to 2.5 ⁇ 10 8 cfu/mL (for example, 1.0 ⁇ 10 8 to 1.5 ⁇ 10 8 cfu/mL, 1.5 ⁇ 10 8 to 2.5 ⁇ 10 8 ). Cfu/mL, 1.0 ⁇ 10 8 cfu/mL, 1.5 ⁇ 10 8 cfu/mL or 2.5 ⁇ 10 8 cfu/mL).
  • the initial biomass in the fermentation culture may be 5.0 ⁇ 10 8 to 3.0 ⁇ 10 9 cfu/mL (for example, 5.0 ⁇ 10 8 to 1.0 ⁇ 10 9 cfu/mL, 1.0 ⁇ 10 9 to 3.0 ⁇ 10 9 ). Cfu/mL, 5.0 ⁇ 10 8 cfu/mL, 1.0 ⁇ 10 9 cfu/mL or 3.0 ⁇ 10 9 cfu/mL).
  • the fermentation culture inoculum may be 3 to 10% (e.g., 3 to 5%, 5 to 10%, 3%, 5% or 10%).
  • the culture condition of the fermentation culture may be 22 to 28 ° C (eg, 22 to 25 ° C, 25 to 28 ° C, 22 ° C, 25 ° C or 28 ° C) for 72 to 120 h (eg, 72 to 100 h, 100 to). 120h, 72h, 100h or 120h), the dissolved oxygen concentration is 5 to 80% (such as 5 to 50%, 50 to 80%, 5%, 50% or 80%).
  • the "fermentation culture of the Schizoochytrium limacinum HS01 CGMCC No. 13746, or the recombinant bacteria B described in any of the above” may further comprise preparing a first-stage seed liquid and/or preparing two Grade seed liquid and/or preparation of fermentation primary seed liquid and/or preparation of fermentation secondary seed liquid;
  • the step of preparing the first-stage seed liquid may be as follows: cultivating the Schizoochytrium limacinum HS01 CGMCC No. 13746, or the recombinant bacteria B according to any of the above, to obtain a first-stage seed liquid;
  • the step of preparing the second-stage seed liquid can be as follows: shaking the first-stage seed liquid in a shake flask to obtain a second-stage seed liquid;
  • the step of preparing the fermentation first-stage seed liquid may be as follows: fermenting the secondary seed liquid to obtain a fermentation first-stage seed liquid;
  • the step of preparing the fermented secondary seed liquid can be as follows: fermenting and fermenting the first-stage seed liquid to obtain a fermented second-stage seed liquid.
  • the shake flask culture medium solute used in the shake flask culture and the solubility thereof may be: glucose 10 to 90 g/L (eg, 10 to 50 g/L, 50 ⁇ 90g / L, 10g / L, 50g / L or 90g / L), yeast powder 5 ⁇ 25g / L (such as 5 ⁇ 15g / L, 15 ⁇ 25g / L, 5g / L, 15g / L or 25g / L); the solute can be water; the pH is natural.
  • the culture condition of the "shake flask culture” is 22 to 28 ° C (such as 22 to 25 ° C, 25 to 28 ° C, 22 ° C, 25 ° C or 28 ° C), 150 to 250 rpm / min (such as 150 to 200 rpm / min, The culture is carried out for 24 to 48 hours (e.g., 24-36h, 36-48h, 24h, 36h or 48h) at 200 to 250 rpm/min, 150 rpm/min, 200 rpm/min or 250 rpm/min.
  • the inoculum amount of the shake flask culture is 3 to 10% (e.g., 3 to 5%, 5 to 10%, 3%, 5%, or 10%).
  • the shake flask medium may specifically be: 50 g of glucose and 15 g of yeast powder are dissolved in 1 L of distilled water, and the pH is natural.
  • the seed culture solute used in the fermentation culture and the solubility thereof may be: glucose 20-100 g/L (such as 20-60 g/L, 60 ⁇ 120g / L, 20g / L, 60g / L or 120g / L), yeast powder 5 ⁇ 15g / L (such as 5 ⁇ 10g / L, 10 ⁇ 15g / L, 5g / L, 10g / L or 15g / L), NaSO 4 5 ⁇ 24g/L (such as 5 ⁇ 10g/L, 10 ⁇ 24g/L, 5g/L, 10g/L or 24g/L), KCl 0.1 ⁇ 1.0g/L (such as 0.1 ⁇ 0.5g) /L, 0.5 to 1.0 g/L, 0.1 g/L, 0.5 g/L or 1.0 g/L), and MgSO 4 1.0 to 3.0 g/L (for example, 1.0 to 2.0 g/L, 2.0
  • the culture condition of the "fermentation culture” is cultured at 22 to 28 ° C (for example, 22 to 25 ° C, 25 to 28 ° C, 22 ° C, 25 ° C or 28 ° C) for 24 to 48 hours (eg, 24 to 36 hours, 36 to 48 hours, 24 hours). , 36h or 48h), the dissolved oxygen concentration is 10 to 80% (such as 10 to 50%, 50 to 80%, 10%, 50% or 80%).
  • the inoculum amount of the fermentation culture is 3 to 10% (e.g., 3 to 5%, 5 to 10%, 3%, 5% or 10%).
  • the seed medium may specifically be: glucose 60g, yeast powder 10g, NaSO 4 10g, KCl 0.5g, MgSO 4 2.0g, K 2 SO 4 1.0g, KH 2 Po 4 1.0g, (NH 4 ) 2 SO 4 1.0 g and CaCl 2 0.5 g were dissolved in 1 L of distilled water to adjust the pH to 6.0.
  • the fermentation broth is obtained by fermenting Schizoochytrium limacinum HS01 or recombinant bacteria B according to any of the above.
  • the results showed that DHA accounted for 45.0%-60.0% of the oil in the fermentation broth, and EPA accounted for 0.2%-1.0% of the oil.
  • the use of the Schizoochytrium limacinum HS01 provided by the present invention can produce DHA and/or EPA, and has important application value.
  • the present invention provides a set of gene fragments related to DHA and EPA synthesis, consisting of gene fragment 1 to gene fragment 6, and the nucleotide sequence is sequentially shown as sequence 3 to sequence 8 in the sequence listing.
  • gene fragment 1 to gene fragment 6 are introduced into Schizochytrium sp. MYA-1381 to obtain a recombinant strain; the ability of the recombinant strain to produce DHA and EPA is greatly enhanced. Therefore, the six gene fragments provided by the present invention, the proteins encoded by the six gene fragments, and the vector, cell or organism containing the six gene fragments have important application value in the production of DHA and EPA.
  • Figure 1 shows the colony morphology of Schizochytrium sp. HS01.
  • Figure 2 shows the bacterial morphological characteristics of Schizochytrium sp. HS01.
  • Wort agar medium 150 g of malt dipping powder is dissolved in 1 L of a mixed solution (mixed from 1 part by volume of natural sea water and 1 volume of distilled water), and the pH is natural; then agar powder is added to a concentration of 15 g/100 mL. Medium.
  • Screening of solid medium Agar powder was added to the screening liquid medium to a concentration of 15 g/100 mL, and the resulting medium was obtained.
  • Screening plates A solid medium plate was prepared by pouring a screening solid medium of about 55 ° C into a Petri dish and cooling.
  • Shake flask medium 50 g of glucose and 15 g of yeast powder were dissolved in 1 L of distilled water, and the pH was natural.
  • Seed medium glucose 60g, yeast powder 10g, NaSO4 10g, KCl 0.5g, MgSO4 2.0g, K2SO4 1.0g, KH2Po4 1.0g, (NH4)2SO4 1.0g and CaCl2 0.5g dissolved in 1L distilled water, adjust the pH to 6.0 .
  • Fermentation medium 60 g of glucose, 10 g of glutamic acid or sodium glutamate, 10 g of corn syrup dry powder, 14 g of NaSO 4 , 0.5 g of KCl, 2.0 g of MgSO 4 , 1.0 g of K 2 SO 4 , 1.0 g of KH 2 PO 4 , (NH 4 ) 2 SO 4 1.0 g and CaCl 2 0.5 g were dissolved in 1 L of distilled water to adjust the pH to 6.0.
  • the corn syrup dry powder is the product of Beijing Suo Laibao Technology Co., Ltd., and the catalog number is FA0010.
  • the yeast powder is a product of Angel Yeast Co., Ltd., and the catalog number is LMO2.
  • the yeast genome extraction kit is a product of Tiangen Biochemical Technology Co., Ltd., and the product catalogue is DP307.
  • the high-fidelity TransStart FastPfu DNA polymerase is Beijing Quanjin Biotechnology Co., Ltd., and the catalogue is AP221.
  • the agarose gel DNA recovery kit is a product of Tiangen Biochemical Technology Co., Ltd., and the product catalogue is DP210.
  • the pEASY-Blunt carrier is a product of Beijing Quanjin Biotechnology Co., Ltd.
  • the catalogue is CB301-01.
  • the strain Schizochytrium limacinum Honda et Yokochi ATCC MYA-1381 is deposited in the American Model Culture Collection (ATCC, address: American Type Culture Collection (ATCC) 10801 University Boulevard Manassas, VA 20110 USA), and the public can be cultured from the US model. The strain was obtained from the stock.
  • the strain Schizochytrium limacinum Hyundai et Yokochi ATCC MYA-1381 is hereinafter referred to as MYA-1381.
  • the inventor of the present application collected Schizochytrium from a plurality of mangroves in Yunxiao County, Zhangzhou City, Fujian province, and mixed to obtain a mixed solution; inoculating 0.5 mL of the mixture into 5 mL of screening liquid medium, then 25 ° C, 200 rpm / min After culturing for 2 days, a culture broth was obtained.
  • the culture broth obtained in the step 1 was evenly spread on a selection plate, and statically cultured at 25 ° C for 2 days to produce a single colony.
  • step 3 single colonies were picked and inoculated into 5 mL of fermentation medium, and then cultured at 25 ° C, 200 rpm / min for 2 days to obtain a culture bacterial solution.
  • the culture liquid obtained in the step 3 was centrifuged at 4 ° C, 2000 rpm for 5 min, and the cells were collected.
  • the upper organic phase was placed in a glass weighing dish (which has been dried and weighed), and the glass was weighed. Place the dish on a boiling water bath in a fume hood to fully evaporate the organic phase (must be fully evaporated) and the liquid phase is grease.
  • step 6 Take the oil extracted in step 5, test the DHA content according to GB 26400-2011 national food safety standard, and test the composition and content of fatty acid according to the method of AOAC996.06.
  • the strain with a higher DHA content was selected and repeatedly purified 24 times.
  • One of the screened Schizochytrium strains was named as Schizochytrium HS01.
  • the Schizochytrium sp. HS01 was inoculated into the fermentation medium for 12 passages and the DHA content was measured according to the above procedure. The results showed that the stability of DHA produced by Schizochytrium sp. HS01 was good.
  • Schizochytrium sp. HS01 was inoculated onto the wort agar medium, and cultured at 25 ° C in the dark. After 5 days, the morphology of the colonies was observed and the morphological characteristics of the cells were observed by high-resolution transmission electron microscopy.
  • the partial sequence of the 18s rDNA of Schizochytrium sp. HS01 is shown in SEQ ID NO:1 in the sequence listing.
  • Schizochytrium genus HS01 is Schizoochytrium limacinum.
  • Schizoochytrium limacinum HS01 was deposited on March 10, 2017 at the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC, Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing). The deposit number is CGMCC No.13746.
  • the Schizochytrium genus HS01 is referred to as Schizoochytrium limacinum HS01 CGMCC No. 13746, abbreviated as Schizochytrium HS01.
  • the oil of the fermentation liquid is extracted, and then the DHA content is detected according to the GB26400-2011 national food safety standard, and the composition and content of the fatty acid are detected according to the method of AOAC996.06.
  • composition(%) Lauric acid 0 to 1.0 Myristic acid 0.5 to 1.0 Palmitic acid 22 ⁇ 32 Stearic acid 1.0 to 2.5 Di-gamma-linolenic acid 0.1 to 0.3 Arachidonic acid 0 to 0.8 EPA 0.2 to 1.0 DPA 9.0 ⁇ 17.0 DHA 45.0 ⁇ 60.0
  • the DHA algal oil crude oil extracted in the step 1 is refined (the method of refining is described in the Chinese patent document CN 103865642 B).
  • Example 3 Large-scale fermentation of DHA by Schizoochytrium limacinum HS01
  • the fermented secondary seed solution Take the fermented secondary seed solution and inoculate it to a fermenter containing 30,000 L of fermentation medium at a seeding rate of 5 to 15% (v/v) (the fermenter size is 75000 L; the initial biomass after inoculation is 5.0 ⁇ 108 ⁇ In 3.0 ⁇ 109 cfu/mL), the cells were cultured at 22 to 28 ° C for 72 to 120 hours (dissolved oxygen was 5 to 80%) to obtain a fermentation liquid.
  • the fermentation broth contains DHA.
  • the fatty acid composition in the fermentation broth was analyzed according to the method of the second step in Example 2. The results showed that the content of DHA in the fermentation broth was 35.0-60.0%.
  • step 5 in the first embodiment the oil of the fermentation liquid is extracted, and then the DHA content is detected according to the GB26400-2011 national food safety standard, and the DPA content is tested according to the GB28404-2012 national food safety standard according to GB5009.168-2016.
  • the national standard for food safety measures EPA content and the composition and content of fatty acids are measured according to the method of AOAC996.06.
  • composition(%) Lauric acid 0 to 1.0 Myristic acid 0.5 to 1.0 Palmitic acid 22 ⁇ 32 Stearic acid 1.0 to 2.5 Di-gamma-linolenic acid 0.1 to 0.3 Arachidonic acid 0 to 0.8 EPA 0.2 to 1.0 DPA 9.0 ⁇ 17.0 DHA 45.0 ⁇ 60.0
  • step 1 the "Schizochytrium HS01" was replaced with "MYA-1381", and the other steps were unchanged.
  • DHA accounted for 12% to 23% of oil and fat
  • DPA accounted for 20% to 39% of oil and fat
  • EPA accounted for 0.5% to 3% of oil.
  • Schizochytrium HS01 is a high-yield strain for the synthesis of DHA and EPA
  • MYA-1381 is a low-yield strain for the synthesis of DHA and EPA.
  • the genomic DNA of Schizochytrium sp. HS01 and MYA-1381 were extracted by yeast genome extraction kit, and then genome-wide sequencing was performed by Beijing Nuohe Zhiyuan Technology Co., Ltd. using PacBio RS II and Illumina HiSeq4000.
  • Schizochytrium HS01 contained six unique gene fragments, which were named gene fragment 1, gene fragment 2, gene fragment 3, gene fragment 4, gene fragment 5 and gene fragment. 6. The nucleotide sequence thereof is sequentially shown as Sequence 3 to Sequence 8 in the Sequence Listing.
  • Sequence 3 in the Sequence Listing encodes protein 1 from positions 1044 to 3050 from the 5' end, and the amino acid sequence of protein 1 is shown in SEQ ID NO: 9 in the Sequence Listing.
  • Sequence 4 in the Sequence Listing encodes protein 2 from positions 1068 to 2737 and positions 3254 to 5262 from the 5' end, and the amino acid sequence of protein 2 is shown in SEQ ID NO: 10 in the Sequence Listing.
  • Sequence 5 in the Sequence Listing encodes protein 3 from positions 5094 to 3415 from the 5' end, and the amino acid sequence of protein 3 is shown in SEQ ID NO: 11 in the Sequence Listing.
  • Sequence 6 in the Sequence Listing encodes protein 4 from positions 1409 to 5044, positions 7004 to 7234 and 7700 to 10399 from the 5' end, and the amino acid sequence of protein 4 is shown in SEQ ID NO: 12 in the Sequence Listing.
  • Sequence 7 in the Sequence Listing encodes protein 5 from positions 1473 to 6488 from the 5' end, and the amino acid sequence of protein 5 is shown in SEQ ID NO: 13 in the Sequence Listing.
  • Sequence 8 in the Sequence Listing encodes protein 6 from positions 953 to 991 and 1063 to 1090 from the 5' end, and the amino acid sequence of protein 6 is shown in SEQ ID NO: 14 in the Sequence Listing.
  • the nucleotide sequences of the upstream primer and the downstream primer constituting each primer pair are shown in Table 5.
  • step 2 the PCR amplification product is recovered using an agarose gel DNA recovery kit.
  • step 2 the recovered PCR amplification product is ligated to the pEASY-Blunt vector to obtain a recombinant plasmid.
  • step 3 the recombinant plasmid is sequenced.
  • the sequencing results showed that the nucleotide sequence of the PCR amplification product amplified by primer pair HS01-1 was as shown in the sequence 3 in the sequence listing (ie, gene fragment 1), and the PCR amplification using primer pair HS01-2 amplification was carried out.
  • the nucleotide sequence of the amplified product is shown in SEQ ID NO: 4 in the sequence listing (ie, gene fragment 2), and the nucleotide sequence of the PCR amplification product amplified by primer pair HS01-3 is shown in SEQ ID NO: 5 in the sequence listing ( That is, the gene fragment 3), the nucleotide sequence of the PCR amplification product amplified by the primer pair HS01-4 is as shown in the sequence 6 in the sequence listing (ie, the gene fragment 4), and amplified by the primer pair HS01-5.
  • the nucleotide sequence of the PCR amplification product is shown in SEQ ID NO: 7 in the sequence listing (ie, gene fragment 5), and the nucleotide sequence of the PCR amplification product amplified by the primer pair HS01-6 is as shown in the sequence 8 of the sequence listing. Show (ie gene fragment 6). Therefore, 6 gene fragments can be amplified using the primers in Table 2.
  • nucleotide sequences of the primers involved are shown in Table 6.
  • the recombinant plasmid pUC57-LZ was synthesized by Nanjing Jinshirui Biotechnology Co., Ltd.
  • the recombinant plasmid pUC57-LZ was obtained by ligating the nucleotide sequence shown in SEQ ID NO: 15 in the sequence listing with the pUC57 vector.
  • the 25th to 58th positions from the 5' end are Lox66 sequences
  • the 626th to 997th positions are zeocin resistance genes
  • the 2293th to 2326th positions are Lox71 sequences.
  • the genomic DNA of Schizochytrium sp. HS01 was extracted using the yeast genome extraction kit.
  • the recombinant plasmid pUC57-LZ synthesized in step 1 was used as a template, and Zeo-F and Zeo-R were used as primers for PCR amplification to obtain a PCR amplification product of about 2350 bp (nucleotide sequence such as sequence 15 in the sequence listing) Shown), the PCR amplification product is a Zeo fragment.
  • the HS1-1 upstream homologous fragment AU, HS01-1 downstream homologous fragment AD and Zeo fragment were used as templates, and HS01-1-UF and HS01-1-DR were used as primers for overlap amplification, and about 6450 bp PCR was obtained.
  • the PCR amplification product was recovered using an agarose gel DNA recovery kit to obtain a targeting fragment HS01-1-Zeo.
  • step 2 repeat the following steps twice: take the polypropylene tube, add 10 mL of pre-cooled sterile water to clean the cells, centrifuge at 4 ° C, 4472 g for 10 min, and collect the cells.
  • step 2 the polypropylene tube was taken, resuspended by adding 10 mL of pre-cooled 1 mol/L sorbitol aqueous solution, and centrifuged at 4 ° C, 5000 r / min for 10 min, and the cells were collected.
  • step 3 the polypropylene tube was taken and resuspended by adding 10 mL of pre-cooled 1 mol/L sorbitol aqueous solution to obtain pretreated MYA-1381.
  • step 2 After completing step 1, take the electric shock cup, add 1mL seed culture medium, incubate at 30°C, 200r/min for 1h, then centrifuge at 10°C and 5000r/min for 10min, mix and evenly coat the bacteria and a small amount of supernatant. On the resistant plate, incubate for 48 h at 30 ° C to obtain a pseudo-transformant.
  • Resistant plate Zeocin was added to a screening solid medium at about 55 ° C to a concentration of 200 ⁇ g / mL, and then poured into a Petri dish, and the resulting solid plate was cooled.
  • the genomic DNA of the transformant was extracted by yeast genome extraction kit and used as a template.
  • the PCR amplification products were obtained by using HS01-1-F and HS01-1-R as primers to obtain PCR amplification products.
  • the pseudo-transformant is a positive transformant.
  • the pSH65 plasmid (product of Biovector Inc.; this plasmid containing Cre enzyme) was introduced into a positive transformant, and then the Zeo gene was eliminated according to the procedure of the pSH65 plasmid instruction to obtain transformant GS-C01.
  • the transformant GS-C06 is the GS-C06 strain.
  • the strain to be tested is Schizochytrium HS01, MYA-1381 or GS-C06 strain.
  • step 5 in the first embodiment the oil of the fermentation liquid is extracted, and then the DHA content is detected according to the national food safety standard GB5009.168-2016, and the DPA content is tested according to the national food safety standard GB28404-2012, according to GB5009.168 -2016 National Food Safety Standards test EPA content.
  • the experimental results are shown in Table 7. As a result, it was revealed that the six gene fragments obtained by the present invention were transformed into MYA-1381, and a high-yield strain for synthesizing DHA and EPA (i.e., GS-C06 strain) was obtained. Therefore, the six gene fragments provided by the present invention, the proteins encoded by the six gene fragments, and the vector, cell or organism containing the six gene fragments have important application value in the production of DHA and EPA. By engineering the proteins encoded by these six gene segments in the starting strain, engineered strains of high yield DHA and EPA can be constructed.
  • the Schizoochytrium limacinum HS01 provided by the present invention has high production value for producing DHA and/or EPA.
  • a high-yield strain for synthesizing DHA and/or EPA can be obtained by transforming the six gene fragments obtained by the present invention into a low-yield strain of synthetic DHA and/or EPA. Therefore, the six gene fragments provided by the present invention, the proteins encoded by the six gene fragments, and the vector, cell or organism containing the six gene fragments have important application value in the production of DHA and/or EPA.
  • Engineered strains of high yield DHA and/or EPA can be constructed by engineering the proteins encoded by these six gene segments in the starting strain.

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Abstract

本发明公开了一株生产DHA和/或EPA的细菌、该细菌基因组中的6个基因片段及它们的应用。该细菌为裂殖壶菌HS01，其在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC No.13746。6个基因片段由基因片段1至基因片段6组成，核苷酸序列依次如序列表中序列3至序列8所示。实验证明，发酵培养裂殖壶菌HS01，得到含有DHA和EPA的发酵液；向裂殖壶菌MYA-1381中导入基因片段1至基因片段6，得到重组菌；该重组菌生产DHA和EPA的能力大大提高。本发明提供的细菌，6个基因片段，6个基因片段编码的蛋白质，含有这6个基因片段的载体、细胞或生物体均具有重要的应用价值。

Description

一株生产DHA和EPA的细菌、该细菌基因组中的6个基因片段及它们的应用 技术领域
本发明涉及工业微生物、食品和饲料工业领域，具体涉及一株生产DHA和/或EPA的细菌、该细菌基因组中的6个基因片段及它们的应用，其中6个基因片段也与DHA和/或EPA合成有关。
背景技术
[根据细则91更正 29.06.2018]　
多不饱和脂肪酸是一类含有两个或两个以上双键的直链脂肪酸，一般碳链长度为18-22个碳原子。根据双键的位置，多不饱和脂肪酸可分为ω-3和ω-6，在多不饱合脂肪酸分子中，距羧基最远端的双键在倒数第3个碳原子上的称为ω-3，在第6个碳原子上的则称为ω-6。多不饱和脂肪酸是人体不可缺少的一类脂肪酸，主要有二十二碳六烯酸(Docosahexaenoic acid，DHA)、二十二碳五烯酸(Docosapentenoic acid，DPA)、二十碳五烯酸(Eicosapentaenoic Acid，EPA)等。
[根据细则91更正 29.06.2018]　
DHA是最主要的一类多不饱和脂肪酸。在分子结构上，DHA为含有22个碳原子和6个双键的直链脂肪酸，因其第1个双键出现在脂肪酸链甲基端的第3位碳原子上，故属于ω-3系列脂肪酸(OMEGA-3)。DHA主要存在于人体的大脑、视网膜中，具有重要的生理功能，例如促进神经系统发育、改善视网膜功能、提高视力、预防心血管疾病、治疗心血管疾病、抗炎症和抑制过敏反应等等。由于人体自身不能合成足够的DHA，主要通过从食物摄取获得。由于日常饮食中的DHA往往含量不足，因此补充DHA或在食品或奶粉中添加DHA对人类尤其是婴幼儿和老年人具有重要意义。
目前，生产DHA的方法主要有两种：一种是传统DHA来源，即从海洋鱼类(主要包括鲑鱼、鲭鱼、三文鱼和沙丁鱼)组织中提取，但通过提取所获得鱼油的质量会随着鱼的种类、季节和捕捞地的变化而受到影响，并且鱼油的质量还受到日益严重的鱼资源匮乏以及环境污染等影响；另一种是新兴的DHA生产方法，即利用海洋微生物进行发酵生产DHA，该方法具有周期短、不受客观条件影响、没有鱼腥味等优势，具有广泛的前景。用于发酵生产DHA的海洋微生物主要为裂殖壶菌(Schizochytrium)，但目前用于发酵生产DHA的裂殖壶菌，由于自身总脂肪酸含量、DHA含量、生长速率等技术指标受限，无法进一步提高产量、降低成本。
在DHA生产菌株中，DHA生物合成途径是由相关合成代谢途径中的一系列酶催化实现的。对DHA生物合成途径的相关基因进行挖掘、改造、异源表达，将为进一步提高DHA的产量提供有利条件。因此获得DHA生物合成途径中新的关键基因将有利于DHA生产菌株的改造和工艺优化。自然界中存在两种DHA合成途径：(1)延长—去饱和途径(elongation-desaturation pathway，E-D途径)，主要是以脂肪酸合成途径为基础，进一步通过延长酶和去饱和酶的作用合成DHA；(2)聚酮合酶途径(Polyketide synthase pathway，PKS途径)，主要是在聚酮合酶的作用下，以乙酰CoA和丙二酸单酰CoA为前体合成DHA。其中，在裂殖壶菌(Schizochytrium)中，DHA的合成主要采用PKS途径。目前认为，DPA和EPA的合成也都具有E-D途径和PKS途径。
发明公开
本发明的目的是制备DPA和/或EPA。
本发明首先保护用于制备DPA和/或EPA的蛋白质组合，该蛋白质组合可为如下(X1)或(X2)或(X3)或(X4)：
(X1)包括蛋白质1、蛋白质2、蛋白质3、蛋白质4、蛋白质5和蛋白质6；
(X2)由蛋白质1、蛋白质2、蛋白质3、蛋白质4、蛋白质5和蛋白质6组成；
(X3)由所述蛋白质1、所述蛋白质2、所述蛋白质3、所述蛋白质4、所述蛋白质5和所述蛋白质6中的任意两个、任意三个、任意四个或任意五个组成；
(X4)所述蛋白质1、所述蛋白质2、所述蛋白质3、所述蛋白质4、所述蛋白质5或所述蛋白质6。
所述蛋白质1可为a1)或a2)或a3)或a4)或a5)：
a1)氨基酸序列是序列表中序列9所示的蛋白质；
a2)在序列表中序列9所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
a3)将序列表中序列9所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
a4)与序列表中序列9限定的氨基酸序列具有80％或80％以上同一性，来源于裂殖壶菌且与多不饱和脂肪酸合成相关的蛋白质；
a5)具有序列表中序列9所示的氨基酸序列的蛋白质。
所述蛋白质2可为b1)或b2)或b3)或b4)或b5)：
b1)氨基酸序列是序列表中序列10所示的蛋白质；
b2)在序列表中序列10所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
b3)将序列表中序列10所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
b4)与序列表中序列10限定的氨基酸序列具有80％或80％以上同一性，来源于裂殖壶菌且与多不饱和脂肪酸合成相关的蛋白质；
b5)具有序列表中序列10所示的氨基酸序列的蛋白质。
所述蛋白质3可为c1)或c2)或c3)或c4)或c5)：
c1)氨基酸序列是序列表中序列11所示的蛋白质；
c2)在序列表中序列11所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
c3)将序列表中序列11所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
c4)与序列表中序列11限定的氨基酸序列具有80％或80％以上同一性，来源于裂殖壶菌且与多不饱和脂肪酸合成相关的蛋白质；
c5)具有序列表中序列11所示的氨基酸序列的蛋白质。
所述蛋白质4可为d1)或d2)或d3)或d4)或d5)：
d1)氨基酸序列是序列表中序列12所示的蛋白质；
d2)在序列表中序列12所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
d3)将序列表中序列12所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
d4)与序列表中序列12限定的氨基酸序列具有80％或80％以上同一性，来源于裂殖壶菌且与多不饱和脂肪酸合成相关的蛋白质；
d5)具有序列表中序列12所示的氨基酸序列的蛋白质。
所述蛋白质5可为e1)或e2)或e3)或e4)或e5)：
e1)氨基酸序列是序列表中序列13所示的蛋白质；
e2)在序列表中序列13所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
e3)将序列表中序列13所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
e4)与序列表中序列13限定的氨基酸序列具有80％或80％以上同一性，来源于裂殖壶菌且与多不饱和脂肪酸合成相关的蛋白质；
e5)具有序列表中序列13所示的氨基酸序列的蛋白质。
所述蛋白质可6为f1)或f2)或f3)或f4)或f5)：
f1)氨基酸序列是序列表中序列14所示的蛋白质；
f2)在序列表中序列14所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
f3)将序列表中序列14所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
f4)与序列表中序列14限定的氨基酸序列具有80％或80％以上同一性，来源于裂殖壶菌且与多不饱和脂肪酸合成相关的蛋白质；
f5)具有序列表中序列14所示的氨基酸序列的蛋白质。
其中，序列表中序列9由669个氨基酸残基组成，序列表中序列10由1193个氨基酸残基组成，序列表中序列11由773个氨基酸残基组成，序列表中序列12由2189个氨基酸残基组成，序列表中序列13由1672个氨基酸残基组成，序列表中序列14由21个氨基酸残基组成。
为了使a1)中的蛋白质便于纯化，可在序列表中序列9所示的蛋白质的氨基末端或羧基末端连接上如表1所示的标签。为了使b1)中的蛋白质便于纯化，可在序列表中序列10所示的蛋白质的氨基末端或羧基末端连接上如表1所示的标签。为了使c1)中的蛋白质便于纯化，可在序列表中序列11所示的蛋白质的氨基末端或羧基末端连接上如表1所示的标签。为了使d1)中的蛋白质便于纯化，可在序列表中序列12所示的蛋白质的氨基末端或羧基末端连接上如表1所示的标签。为了使e1)中的蛋白质便于纯化，可在序列表中序列13所示的蛋白质的氨基末端或羧基末端连接上如表1所示的标签。为了使f1)中的蛋白质便于纯化，可在序列表中序列14所示的蛋白质的氨基末端或羧基末端连接上如表1所示的标签。
表1.标签的序列
标签 残基 序列
Poly-Arg 5-6(通常为5个) RRRRR
Poly-His 2-10(通常为6个) HHHHHH
FLAG 8 DYKDDDDK
Strep-tag II 8 WSHPQFEK
c-myc 10 EQKLISEEDL
上述a3)中的蛋白质1，上述b3)中的蛋白质2，上述c3)中的蛋白质3，上述d3)中的蛋白质4，上述e3)中的蛋白质5，上述f3)中的蛋白质6，所述一个或几个氨基酸残基的取代和/或缺失和/或添加为不超过10个氨基酸残基的取代和/或缺失和/或添加。
上述a3)中的蛋白质1，上述b3)中的蛋白质2，上述c3)中的蛋白质3，上述d3)中的蛋白质4，上述e3)中的蛋白质5，上述f3)中的蛋白质6，均可人工合成，也可先合成其编码基因，再进行生物表达得到。
上述a3)中的蛋白质1的编码基因可通过将序列表中序列9所示的DNA序列中缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变，和/或在其5′端和/或3′端连上表1所示的标签的编码序列得到。
上述b3)中的蛋白质2的编码基因可通过将序列表中序列10所示的DNA序列中缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变，和/或在其5′端和/或3′端连上表1所示的标签的编码序列得到。
上述c3)中的蛋白质3的编码基因可通过将序列表中序列11所示的DNA序列中缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变，和/或在其5′端和/或3′端连上表1所示的标签的编码序列得到。
上述d3)中的蛋白质4的编码基因可通过将序列表中序列12所示的DNA序列中缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变，和/或在其5′端和/或3′端连上表1所示的标签的编码序列得到。
上述e3)中的蛋白质5的编码基因可通过将序列表中序列13所示的DNA序列中缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变，和/或在其5′端和/或3′端连上表1所示的标签的编码序列得到。
上述f3)中的蛋白质6的编码基因可通过将序列表中序列14所示的DNA序列中缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变，和/或在其5′端和/或3′端连上表1所示的标签的编码序列得到。
上文中，使用的术语“同一性”指与天然氨基酸序列的序列相似性。“同一性”包括与本发明提供的蛋白质的氨基酸序列具有80％，或85％或更高，或90％或更高，或95％或更高同一性的氨基酸序列。
编码所述蛋白质组合的核酸分子也属于本发明的保护范围。
编码所述蛋白质1的核酸分子可为如下A1)或A2)或A3)或A4)所示的DNA分子：
A1)编码区是序列表中序列3自5’末端起第1044至3050位所示的DNA分子；
A2)核苷酸序列是序列表中序列3所示的DNA分子；
A3)与A1)或A2)限定的核苷酸序列具有75％或75％以上同一性，来源于裂殖壶菌且编码所述蛋白质1的DNA分子；
A4)在严格条件下与A1)或A2)限定的核苷酸序列杂交，且编码所述蛋白质1的DNA分子。
编码所述蛋白质2的核酸分子可为如下B1)或B2)或B3)或B4)所示的DNA分子：
B1)编码区是序列表中序列4自5’末端起第1068至2737位和第3254至5162位所示的DNA分子；
B2)核苷酸序列是序列表中序列4所示的DNA分子；
B3)与B1)或B2)限定的核苷酸序列具有75％或75％以上同一性，来源于裂殖壶菌且编码所述蛋白质2的DNA分子；
B4)在严格条件下与B1)或B2)限定的核苷酸序列杂交，且编码所述蛋白质2的DNA分子。
编码所述蛋白质3的核酸分子可为如下C1)或C2)或C3)或C4)所示的DNA分子：
C1)编码区是序列表中序列5自5’末端起第1094至3415位所示的DNA分子；
C2)核苷酸序列是序列表中序列5所示的DNA分子；
C3)与C1)或C2)限定的核苷酸序列具有75％或75％以上同一性，来源于裂殖壶菌且编码所述蛋白质3的DNA分子；
C4)在严格条件下与C1)或C2)限定的核苷酸序列杂交，且编码所述蛋白质3的DNA分子。
编码所述蛋白质4的核酸分子可为如下D1)或D2)或D3)或D4)所示的DNA分子：
D1)编码区是序列表中序列6自5’末端起第1409至5044位、第7004至7234位和第7700至10399位所示的DNA分子；
D2)核苷酸序列是序列表中序列6所示的DNA分子；
D3)与D1)或D2)限定的核苷酸序列具有75％或75％以上同一性，来源于裂殖壶菌且编码所述蛋白质4的DNA分子；
D4)在严格条件下与D1)或D2)限定的核苷酸序列杂交，且编码所述蛋白质4的DNA分子。
编码所述蛋白质5的核酸分子可为如下E1)或E2)或E3)或E4)所示的DNA分子：
E1)编码区是序列表中序列7自5’末端起第1473至6488位所示的DNA分子；
E2)核苷酸序列是序列表中序列7所示的DNA分子；
E3)与E1)或E2)限定的核苷酸序列具有75％或75％以上同一性，来源于裂殖壶菌且编码所述蛋白质5的DNA分子；
E4)在严格条件下与E1)或E2)限定的核苷酸序列杂交，且编码所述蛋白质5的DNA分子。
编码所述蛋白质6的核酸分子可为如下F1)或F2)或F3)或F4)所示的DNA分子：
F1)编码区是序列表中序列8自5’末端起第953至991位和第1063至1090位所示的DNA分子；
F2)核苷酸序列是序列表中序列8所示的DNA分子；
F3)与F1)或F2)限定的核苷酸序列具有75％或75％以上同一性，来源于 裂殖壶菌且编码所述蛋白质6的DNA分子；
F4)在严格条件下与F1)或F2)限定的核苷酸序列杂交，且编码所述蛋白质6的DNA分子。
其中，所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。所述核酸分子可为编码所述蛋白质组合的基因及其调控序列形成的核酸分子。
其中，序列表中序列3由4100个核苷酸组成，序列表中序列3所示的核苷酸编码序列表中序列9所示的氨基酸序列。序列表中序列4由6200个核苷酸组成，序列表中序列4所示的核苷酸编码序列表中序列10所示的氨基酸序列。序列表中序列5由4500个核苷酸组成，序列表中序列5所示的核苷酸编码序列表中序列11所示的氨基酸序列。序列表中序列6由11100个核苷酸组成，序列表中序列6所示的核苷酸编码序列表中序列12所示的氨基酸序列。序列表中序列7由7767个核苷酸组成，序列表中序列7所示的核苷酸编码序列表中序列13所示的氨基酸序列。序列表中序列8由7800个核苷酸组成，序列表中序列8所示的核苷酸编码序列表中序列14所示的氨基酸序列。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的编码蛋白质组合的核苷酸序列进行突变。那些经过人工修饰的，具有与本发明的蛋白质组合的核苷酸序列75％或者更高同源性的核苷酸，只要编码蛋白质组合且来源于裂殖壶菌，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的编码蛋白质组合的核苷酸序列具有75％或更高，80％或更高，或85％或更高，或90％或更高，或95％或更高同一性的核苷酸序列。
含有所述核酸分子的表达盒、重组载体、重组微生物或转基因细胞系也属于本发明的保护范围。
所述重组载体可为将编码所述蛋白质1的核酸分子(即序列表中序列3所示的DNA分子)、编码所述蛋白质2的核酸分子(即序列表中序列4所示的DNA分子)、编码所述蛋白质3的核酸分子(即序列表中序列5所示的DNA分子)、编码所述蛋白质4的核酸分子(即序列表中序列6所示的DNA分子)、编码所述蛋白质5的核酸分子(即序列表中序列7所示的DNA分子)和编码所述蛋白质6的核酸分子(即序列表中序列8所示的DNA分子)插入出发质粒，得到的重组质粒。
所述重组微生物可通过将所述重组载体导入出发微生物得到。所述出发微生物可为酵母、细菌、藻类或真菌。所述真菌可为裂殖壶菌。所述裂殖壶菌具体可为菌株Schizochytrium limacinum Honda et Yokochi ATCC MYA-1381。所述重组微生物具体可为实施例中提及的GS-C06菌株。
上述任一所述蛋白质组合，或，上述任一所述核酸分子，或，上述任一含有所述核酸分子的表达盒、重组载体、重组微生物或转基因细胞系，在生产DHA和/或EPA中的应用也属于本发明的保护范围。
本发明还保护重组菌乙，其制备方法可如下：使出发菌中所述蛋白质组合的表达和/或活性提高，得到的重组菌即为重组菌乙。所述“使出发菌中所述蛋白质组合的表达和/或活性提高”通过向出发菌中导入提高所述蛋白质组合的表达和/或活性的物质实现。所述“向出发菌中导入提高蛋白质组合的表达和/或活性的物质”通过向出发菌中导入编码所述蛋白质组合的核酸分子实现。所述出发菌可为裂殖壶菌。所述裂殖壶菌具体可为菌株Schizochytrium limacinum Honda et Yokochi ATCC MYA-1381。
所述重组菌乙具体可为实施例中提及的GS-C06菌株。
本发明还保护一种生产DHA和/或EPA的方法，依次可包括如下步骤：
(1)使出发菌中所述蛋白质组合的表达和/或活性提高，得到的重组菌甲；与所述出发菌相比，重组菌甲生产DHA和/或EPA的能力提高；
(2)发酵培养重组菌甲，得到DHA和/或EPA。
上述方法中，所述重组菌甲可为所述重组菌乙。
上述方法中，所述“使出发菌中所述蛋白质组合的表达和/或活性提高”通过向出发菌中导入提高蛋白质组合的表达和/或活性的物质实现。
上述方法中，所述“向出发菌中导入提高蛋白质组合的表达和/或活性的物质”通过向出发菌中导入编码所述蛋白质组合的核酸分子实现。
上述方法中，所述出发菌可为裂殖壶菌。所述裂殖壶菌具体可为菌株Schizochytrium limacinum Honda et Yokochi ATCC MYA-1381。
上述方法中，所述重组菌甲具体可为实施例中提及的GS-C06菌株。
上文中，所述“向出发菌中导入编码所述蛋白质组合的核酸分子”可通过向出发菌中导入重组载体实现；所述重组载体可为向表达载体插入编码所述蛋白质1的核酸分子(即序列表中序列3所示的DNA分子)、编码所述蛋白质2的核酸分子(即序列表中序列4所示的DNA分子)、编码所述蛋白质3的核酸分子(即序列表中序列5所示的DNA分子)、编码所述蛋白质4的核酸分子(即序列表中序列6所示的DNA分子)、编码所述蛋白质5的核酸分子(即序列表中序列7所示的DNA分子)和编码所述蛋白质6的核酸分子(即序列表中序列8所示的DNA分子)，得到的重组质粒。
本发明还保护一株生产二十二碳六烯酸和/或EPA的的细菌，该细菌为裂殖壶菌(Schizoochytrium limacinum)HS01，其在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC No.13746。
本发明还提供了一种菌剂，该菌剂含有所述裂殖壶菌(Schizoochytrium limacinum)HS01 CGMCC No.13746，或，上述任一所述的重组菌乙。
所述裂殖壶菌(Schizoochytrium limacinum)HS01 CGMCC No.13746、或、上述任一所述的重组菌乙、或、所述菌剂在生产二十二碳六烯酸和/或EPA中的应用也属于本发明的保护范围。
本发明还保护一种生产二十二碳六烯酸和/或EPA的方法，该方法包括发酵 培养所述裂殖壶菌(Schizoochytrium limacinum)HS01 CGMCC No.13746、或、上述任一所述的重组菌乙，得到二十二碳六烯酸和/或EPA的步骤。
上述方法中，所述发酵培养使用的发酵培养基溶质及其溶度可为：葡萄糖20～120g/L(如20～60g/L、60～120g/L、20g/L、60g/L或120g/L)、谷氨酸或谷氨酸钠5～15g/L(如5～10g/L、10～15g/L、5g/L、10g/L或15g/L)、玉米浆干粉3～15g/L(如3～10g/L、10～15g/L、3g/L、10g/L或15g/L)、NaSO 45～24g/L(如5～14g/L、14～24g/L、5g/L、14g/L或24g/L)、KCl 0.1～1.0g/L(如0.1～0.5g/L、0.5～1.0g/L、0.1g/L、0.5g/L或1.0g/L)、MgSO 4 1.0～3.0g/L(如1.0～2.0g/L、2.0～3.0g/L、1.0g/L、2.0g/L或3.0g/L)、K 2SO 4 0.3～1.5g/L(如0.3～1.0g/L、1.0～1.5g/L、0.3g/L、1.0g/L或1.5g/L)、KH 2PO 4 0.5～1.5g/L(如0.5～1.0g/L、1.0～1.5g/L、0.5g/L、1.0g/L或1.5g/L)、(NH 4) 2SO 4 0.5～1.5g/L(如0.5～1.0g/L、1.0～1.5g/L、0.5g/L、1.0g/L或1.5g/L)、CaCl 2 0.1～1.0g/L(如0.1～0.5g/L、0.5～1.0g/L、0.1g/L、0.5g/L或1.0g/L)；溶质可为水；pH值可为5.0～6.5(如5.0、6.0或6.5)。所述发酵培养基具体可为：将葡萄糖60g、谷氨酸或谷氨酸钠10g、玉米浆干粉10g、NaSO 4 14g、KCl 0.5g、MgSO 4 2.0g、K 2SO 4 1.0g、KH 2PO 4 1.0g、(NH 4) 2SO 4 1.0g和CaCl 2 0.5g溶于1L蒸馏水，调节pH值至6.0。
上述方法中，所述发酵培养中初始生物量可为1.0×10 8～2.5×10 8cfu/mL(如1.0×10 8～1.5×10 8cfu/mL、1.5×10 8～2.5×10 8cfu/mL、1.0×10 8cfu/mL、1.5×10 8cfu/mL或2.5×10 8cfu/mL)。
上述方法中，所述发酵培养中初始生物量可为5.0×10 8～3.0×10 9cfu/mL(如5.0×10 8～1.0×10 9cfu/mL、1.0×10 9～3.0×10 9cfu/mL、5.0×10 8cfu/mL、1.0×10 9cfu/mL或3.0×10 9cfu/mL)。
上述方法中，所述发酵培养接种量可为3～10％(如3～5％、5～10％、3％、5％或10％)。
上述方法中，所述发酵培养的培养条件可为22～28℃(如22～25℃、25～28℃、22℃、25℃或28℃)培养72～120h(如72～100h、100～120h、72h、100h或120h)，溶氧浓度为5～80％(如5～50％、50～80％、5％、50％或80％)。
上述方法中，所述“发酵培养所述裂殖壶菌(Schizoochytrium limacinum)HS01 CGMCC No.13746、或、上述任一所述的重组菌乙”还可包括制备一级种子液和/或制备二级种子液和/或制备发酵一级种子液和/或制备发酵二级种子液；
制备一级种子液的步骤可如下：摇瓶培养所述裂殖壶菌(Schizoochytrium limacinum)HS01 CGMCC No.13746、或、上述任一所述的重组菌乙，得到一级种子液；
制备二级种子液的步骤可如下：摇瓶培养一级种子液，得到二级种子液；
制备发酵一级种子液的步骤可如下：发酵培养二级种子液，得到发酵一级种子液；
制备发酵二级种子液的步骤可如下：发酵培养发酵一级种子液，得到发酵二级种子液。
所述制备一级种子液和所述制备二级种子液中，所述摇瓶培养使用的摇瓶培 养基溶质及其溶度可为：葡萄糖10～90g/L(如10～50g/L、50～90g/L、10g/L、50g/L或90g/L)、酵母粉5～25g/L(如5～15g/L、15～25g/L、5g/L、15g/L或25g/L)；溶质可为水；pH自然。所述“摇瓶培养”的培养条件为22～28℃(如22～25℃、25～28℃、22℃、25℃或28℃)、150～250rpm/min(如150～200rpm/min、200～250rpm/min、150rpm/min、200rpm/min或250rpm/min)培养24～48h(如24～36h、36～48h、24h、36h或48h)。所述摇瓶培养的接种量为3～10％(如3～5％、5～10％、3％、5％或10％)。所述摇瓶培养基具体可为：将葡萄糖50g和酵母粉15g溶于1L蒸馏水，pH值自然。
所述制备发酵一级种子液和所述制备发酵二级种子液中，所述发酵培养使用的种子培养基溶质及其溶度可为：葡萄糖20～100g/L(如20～60g/L、60～120g/L、20g/L、60g/L或120g/L)、酵母粉5～15g/L(如5～10g/L、10～15g/L、5g/L、10g/L或15g/L)、NaSO 4 5～24g/L(如5～10g/L、10～24g/L、5g/L、10g/L或24g/L)、KCl 0.1～1.0g/L(如0.1～0.5g/L、0.5～1.0g/L、0.1g/L、0.5g/L或1.0g/L)、MgSO 4 1.0～3.0g/L(如1.0～2.0g/L、2.0～3.0g/L、1.0g/L、2.0g/L或3.0g/L)、K 2SO 4 0.3～1.5g/L(如0.3～1.0g/L、1.0～1.5g/L、0.3g/L、1.0g/L或1.5g/L)、KH 2Po 4 0.5～1.5g/L(如0.5～1.0g/L、1.0～1.5g/L、0.5g/L、1.0g/L或1.5g/L)、(NH 4) 2SO 4 0.5～1.5g/L(如0.5～1.0g/L、1.0～1.5g/L、0.5g/L、1.0g/L或1.5g/L)、CaCl 2 0.1～1.0g/L(如0.1～0.5g/L、0.5～1.0g/L、0.1g/L、0.5g/L或1.0g/L)；溶质可为水；pH值可为5.0～6.5(如5.0、6.0或6.5)。所述“发酵培养”的培养条件为22～28℃(如22～25℃、25～28℃、22℃、25℃或28℃)培养24～48h(如24～36h、36～48h、24h、36h或48h)，溶氧浓度为10～80％(如10～50％、50～80％、10％、50％或80％)。所述发酵培养的接种量为3～10％(如3～5％、5～10％、3％、5％或10％)。所述种子培养基具体可为：将葡萄糖60g、酵母粉10g、NaSO 4 10g、KCl 0.5g、MgSO 4 2.0g、K 2SO 4 1.0g、KH 2Po 4 1.0g、(NH 4) 2SO 4 1.0g和CaCl 2 0.5g溶于1L蒸馏水，调节pH值至6.0。
发酵培养裂殖壶菌(Schizoochytrium limacinum)HS01、或、上述任一所述的重组菌乙，得到发酵液。结果表明，该发酵液中DHA占油脂的含量为45.0％～60.0％，EPA占油脂的含量为0.2％～1.0％。
因此，利用本发明提供的裂殖壶菌(Schizoochytrium limacinum)HS01可以生产DHA和/或EPA，具有重要的应用价值。本发明提供的一组与DHA和EPA合成有关的基因片段，由基因片段1至基因片段6组成，核苷酸序列依次如序列表中序列3至序列8所示。实验证明，向裂殖壶菌MYA-1381中导入基因片段1至基因片段6，得到重组菌；该重组菌生产DHA和EPA的能力大大提高。因此，本发明提供的6个基因片段，这6个基因片段编码的蛋白质，和，含有这6个基因片段的载体、细胞或生物体，在生产DHA和EPA中具有重要的应用价值。
附图说明
图1为裂殖壶菌HS01的菌落形态特征。
图2为裂殖壶菌HS01的菌体形态特征。
保藏说明
菌种名称：裂殖壶菌
拉丁名：Schizoochytrium limacinum
菌株编号：HS01
保藏机构：中国微生物菌种保藏管理委员会普通微生物中心
保藏机构简称：CGMCC
地址：北京市朝阳区北辰西路1号院3号
保藏日期：2017年3月10日
保藏中心登记入册编号：CGMCC No.13746
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
下述实施例中所用的培养基如下：
麦芽汁琼脂培养基：将150g麦芽浸粉溶于1L混合液(由1体积份天然海水和1体积蒸馏水混合而成)，pH值自然；然后加入琼脂粉至其浓度为15g/100mL，得到的培养基。
筛选液体培养基：将葡萄糖50g和酵母粉15g溶于1L混合液(由1体积份天然海水和1体积蒸馏水混合而成)，pH值自然。
筛选固体培养基：向筛选液体培养基中加入琼脂粉至其浓度为15g/100mL，得到的培养基。
筛选平板：将约55℃的筛选固体培养基倒入培养皿，冷却后得到的固体平板。
摇瓶培养基：将葡萄糖50g和酵母粉15g溶于1L蒸馏水，pH值自然。
种子培养基：葡萄糖60g、酵母粉10g、NaSO4 10g、KCl 0.5g、MgSO4 2.0g、K2SO4 1.0g、KH2Po4 1.0g、(NH4)2SO4 1.0g和CaCl2 0.5g溶于1L蒸馏水，调节pH值至6.0。
发酵培养基：将葡萄糖60g、谷氨酸或谷氨酸钠10g、玉米浆干粉10g、NaSO 4 14g、KCl 0.5g、MgSO 4 2.0g、K 2SO 4 1.0g、KH 2PO 4 1.0g、(NH 4) 2SO 4 1.0g和CaCl 2 0.5g溶于1L蒸馏水，调节pH值至6.0。
玉米浆干粉为北京索莱宝科技有限公司的产品，产品目录号为FA0010。酵母粉为安琪酵母股份有限公司的产品，产品目录号为LMO2。酵母基因组提取试剂盒为天根生化科技有限公司的产品，产品目录为DP307。高保真TransStart FastPfu DNA聚合酶为北京全式金生物技术有限公司，产品目录为AP221。琼脂糖凝胶DNA回收试剂盒为天根生化科技有限公司的产品，产品目录为DP210。pEASY-Blunt载体为北京全式金生物技术有限公司的产品，产品目录为CB301-01。
菌株Schizochytrium limacinum Honda et Yokochi ATCC MYA-1381保藏于美国模式培养物集存库(简称ATCC，地址：American Type Culture Collection(ATCC)10801 University Boulevard Manassas，VA 20110 USA)，公众可从美国模式培养物集存库获得该菌株。菌株Schizochytrium limacinum Honda et Yokochi ATCC MYA-1381在下文中简称MYA-1381。
实施例1、裂殖壶菌(Schizoochytrium limacinum)HS01 CGMCC No.13746的分离、鉴定和保藏
一、裂殖壶菌HS01的分离
1、本申请的发明人从福建省漳州市云霄县红树林多处采集裂殖壶菌，混合，得到混合液；将0.5mL混合液接种至5mL筛选液体培养基，然后25℃、200rpm/min培养2d，得到培养菌液。
2、将步骤1得到的培养菌液均匀涂布于筛选平板上，25℃静置培养2d，产生单菌落。
3、完成步骤2后，分别挑取单菌落接种至5mL发酵培养基，然后25℃、200rpm/min培养2d，得到培养菌液。
4、取步骤3得到的培养菌液，4℃、2000rpm离心5min，收集菌体。
5、取菌体1.0～2.0g至具塞量筒(规格为100mL)中，先加入15mL浓度为8.3mol/L的HCl水溶液，盖好盖子，置于70～80℃水浴中水解50～60min(期间每10min置于漩涡混合器上振荡具塞量筒1次)；待冷却到室温后，先加入10mL 95％(v/v)乙醇水溶液，充分振摇均匀后，再加入20mL无水乙醚充分振摇萃取1～2min，最后加入20mL石油醚，充分振摇萃取1～2min，静置分层，将上层有机相置于玻璃称量皿(已烘干称过空重)，将该玻璃称量皿置于通风橱中的沸水浴上使有机相充分蒸发干净(务必充分挥发干净)，液相即为油脂。
6、取步骤5提取的油脂，按照GB 26400-2011食品安全国家标准检测DHA含量，按照AOAC996.06的方法检测脂肪酸的组成及含量。
挑选DHA含量较高的菌株，反复纯化24次。将筛选到的一株裂殖壶菌菌株命名为裂殖壶菌HS01。
将裂殖壶菌HS01单克隆接种至发酵培养基连续传代12代并按照上述步骤检测DHA含量。结果表明，裂殖壶菌HS01生产DHA的稳定性良好。
二、裂殖壶菌HS01的鉴定
1、形态学鉴定
将裂殖壶菌HS01接种至麦芽汁琼脂培养基上，25℃暗培养，5d后观察菌落的形态并通过高分辨率透射电镜分析观察菌体的形态特征。
实验结果见图1和图2。结果表明，裂殖壶菌HS01的菌落直径为2～4.3mm，白色(后期浅橙色)，边缘不整齐；菌体以裂殖方式进行增殖，细胞壁薄，球形，无色或浅橙色，透明，大小为4.5～15.5μm，游动孢子及外质网未见。
2、18s rDNA序列同源性分析
裂殖壶菌HS01的18s rDNA的部分序列如序列表中的序列1所示。
裂殖壶菌HS01的18s rDNA的部分序列如序列表中的序列2所示。
综合上述各个鉴定结果，裂殖壶菌HS01为裂殖壶菌(Schizoochytrium limacinum)。
三、裂殖壶菌HS01的保藏
裂殖壶菌(Schizoochytrium limacinum)HS01已于2017年03月10日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址为：北京市朝阳区北辰西路1号院3号)，保藏编号为CGMCC No.13746。裂殖壶菌HS01的全称为裂殖壶菌(Schizoochytrium limacinum)HS01 CGMCC No.13746，简称为裂殖壶菌HS01。
实施例2、裂殖壶菌(Schizoochytrium limacinum)HS01发酵生产DHA
一、裂殖壶菌(Schizoochytrium limacinum)HS01发酵生产DHA
1、将裂殖壶菌(Schizoochytrium limacinum)HS01单克隆接种至装有2mL摇瓶培养基的摇瓶(规格为10mL)中，22～28℃、150～250rpm/min培养24～48h，得到一级种子液。
2、取一级种子液，以3～10％(v/v)的接种量接种至装有250mL摇瓶培养基的摇瓶(摇瓶规格为1L)中，22～28℃、150～250rpm/min培养24～48h，得到二级种子液。
3、取二级种子液，以3～10％(v/v)的接种量接种至装有3L种子培养基的发酵罐(发酵罐规格为5L)中，22～28℃培养24～48h(溶解氧为10～80％)，得到发酵一级种子液。
4、取发酵一级种子液，以3～10％(v/v)的接种量接种至装有50L发酵培养基的发酵罐(发酵罐规格为100L；接种后初始生物量为1.0×10 8～2.5×10 8cfu/mL)中，22～28℃培养72～120h(溶解氧为5～80％)，得到发酵液。发酵液中即含有DHA。
二、发酵液中的脂肪酸成分的分析
按照实施例1步骤一中5的方法，提取发酵液的油脂，然后按照GB26400-2011食品安全国家标准检测DHA含量，按照AOAC996.06的方法检测脂肪酸的组成及含量。
实验结果见表2。结果表明，DHA占油脂的含量为45.0％～60.0％。
表2
名称 组成(％)
月桂酸 0～1.0
豆蔻酸 0.5～1.0
棕榈酸 22～32
硬脂酸 1.0～2.5
二高-γ-亚麻酸 0.1～0.3
花生四烯酸 0～0.8
EPA 0.2～1.0
DPA 9.0～17.0
DHA 45.0～60.0
三、发酵液中DHA的分离和质量鉴定
1、取步骤一得到的发酵液，依次进行裂殖壶菌的细胞破壁和DHA藻油粗油的提取(进行裂殖壶菌的细胞破壁和DHA藻油粗油的提取的方法记载于中国发明专利文献CN 101817738 B)。
2、对步骤1提取的DHA藻油粗油进行精炼(精炼的方法记载于中国发明专利文献CN 103865642 B)。
DHA藻油粗油经过精炼后的质量指标见表3。
表3
Figure PCTCN2018087613-appb-000001
实施例3、裂殖壶菌(Schizoochytrium limacinum)HS01大规模发酵生产DHA
1、将裂殖壶菌(Schizoochytrium limacinum)HS01单菌落接种至装有20mL摇瓶培养基的摇瓶(摇瓶规格为250mL)中，22～28℃、150～250rpm/min培养24～48h，得到一级种子液。
2、取一级种子液，以3～10％(v/v)的接种量接种至装有250mL摇瓶培养基的摇瓶(摇瓶规格为2L)中，22～28℃、150～250rpm/min培养24～48h，得到二级种子液。
3、取二级种子液，以3～10％(v/v)的接种量接种至装有500L种子培养基的发酵罐(发酵罐规格为1000L)中，22～28℃培养24～48h(溶解氧为10～80％)，得到生物量在15～30g/L的发酵一级种子液。
4、取发酵一级种子液，以5～15％(v/v)的接种量接种至装有5000L种子培养基的发酵罐(发酵罐规格为8000～10000L)中，22～28℃培养24～48h(溶解氧为10～80％)，得到生物量在15～30g/L的发酵二级种子液。
5、取发酵二级种子液，以5～15％(v/v)的接种量接种至装有30000L发酵培养基的发酵罐(发酵罐规格为75000L；接种后初始生物量为5.0×108～3.0×109cfu/mL)中，22～28℃培养72～120h(溶解氧为5～80％)，得到发酵液。发酵液中即含有DHA。
按照实施例2中步骤二的方法，分析发酵液中的脂肪酸成分。结果表明，该发酵液中DHA占油脂的含量为35.0～60.0％。
实施例4、与DHA和EPA合成有关的基因片段的发现
一、裂殖壶菌HS01发酵生产多不饱和脂肪酸
1、将裂殖壶菌HS01单克隆接种至装有2mL摇瓶培养基的摇瓶(规格为10mL)中，22～28℃、150～250rpm/min培养24～48h，得到一级种子液。
2、取一级种子液，以3～10％(v/v)的接种量接种至装有250mL摇瓶培养基的摇瓶(摇瓶规格为1L)中，22～28℃、150～250rpm/min培养24～48h，得到二级种子液。
3、取二级种子液，以3～10％(v/v)的接种量接种至装有3L种子培养基的发酵罐(发酵罐规格为5L)中，22～28℃培养24～48h(溶解氧为10～80％)，得到发酵一级种子液。
4、取发酵一级种子液，以3～10％(v/v)的接种量接种至装有50L发酵培养基的发酵罐(发酵罐规格为100L；接种后初始生物量为1.0×10 8～2.5×10 8cfu/mL)中，22～28℃培养72～120h(溶解氧为5～80％)，得到发酵液。
5、按照实施例1步骤一中5的方法，提取发酵液的油脂，然后按照GB26400-2011食品安全国家标准检测DHA含量，按照GB28404-2012食品安全国家标准检测DPA含量，按照GB5009.168-2016食品安全国家标准检测EPA含量，按照AOAC996.06的方法检测脂肪酸的组成及含量。
实验结果见表4。结果表明，DHA占油脂的含量为45.0％～60.0％，DPA占油脂的含量为9.0％～17.0％，EPA占油脂的含量为0.2％～1.0％。
表4
名称 组成(％)
月桂酸 0～1.0
豆蔻酸 0.5～1.0
棕榈酸 22～32
硬脂酸 1.0～2.5
二高-γ-亚麻酸 0.1～0.3
花生四烯酸 0～0.8
EPA 0.2～1.0
DPA 9.0～17.0
DHA 45.0～60.0
二、MYA-1381发酵生产多不饱和脂肪酸
按照步骤一的方法，将“裂殖壶菌HS01”替换为“MYA-1381”，其它步骤均不变。结果表明，DHA占油脂的含量为12％～23％，DPA占油脂的含量为20％～39％，EPA占油脂的含量为0.5％～3％。
根据上述结果，裂殖壶菌HS01为合成DHA和EPA的高产菌株，MYA-1381为合成DHA和EPA的低产菌株。
三、与DHA和EPA合成有关的基因片段的发现
采用酵母基因组提取试剂盒分别提取裂殖壶菌HS01和MYA-1381的基因组DNA，然后由北京诺禾致源科技股份有限公司采用PacBio RS II和Illumina HiSeq4000进行全基因组测序。
结果表明，与MYA-1381相比，裂殖壶菌HS01中含有6个独有的基因片段，分别命名为基因片段1、基因片段2、基因片段3、基因片段4、基因片段5和基因片段6，其核苷酸序列依次如序列表中序列3至序列8所示。
序列表中序列3自5’末端起第1044至3050位编码蛋白质1，蛋白质1的氨基酸序列如序列表中序列9所示。序列表中序列4自5’末端起第1068至2737位和第3254至5162位编码蛋白质2，蛋白质2的氨基酸序列如序列表中序列10所示。序列表中序列5自5’末端起 第1094至3415位编码蛋白质3，蛋白质3的氨基酸序列如序列表中序列11所示。序列表中序列6自5’末端起第1409至5044位、第7004至7234位和第7700至10399位编码蛋白质4，蛋白质4的氨基酸序列如序列表中序列12所示。序列表中序列7自5’末端起第1473至6488位编码蛋白质5，蛋白质5的氨基酸序列如序列表中序列13所示。序列表中序列8自5’末端起第953至991位和第1063至1090位编码蛋白质6，蛋白质6的氨基酸序列如序列表中序列14所示。
实施例5、6个基因片段的扩增及其相应的引物的合成
1、采用酵母基因组提取试剂盒提取裂殖壶菌HS01的基因组DNA并以其为模板，采用高保真TransStart FastPfu DNA聚合酶和引物对(引物对HS01-1、引物对HS01-2、引物对HS01-3、引物对HS01-4、引物对HS01-5、引物对HS01-6)进行PCR扩增，得到PCR扩增产物。
组成各个引物对的上游引物和下游引物的核苷酸序列见表5。
反应程序：98℃2min；98℃30s，56℃30s，72℃3min，30个循环；72℃5min。
表5
Figure PCTCN2018087613-appb-000002
2、完成步骤1后，采用琼脂糖凝胶DNA回收试剂盒回收PCR扩增产物。
3、完成步骤2后，将回收的PCR扩增产物和pEASY-Blunt载体连接，得到重组菌质粒。
4、完成步骤3后，对重组菌质粒进行测序。
测序结果表明，采用引物对HS01-1扩增得到的PCR扩增产物的核苷酸序列如序列表中序列3所示(即基因片段1)，采用引物对HS01-2扩增得到的PCR扩增产物的核苷酸序列如序列表中序列4所示(即基因片段2)，采用引物对HS01-3扩增得到的PCR扩增产物的核苷酸序列如序列表中序列5所示(即基因片段3)，采用引物对HS01-4扩增得到的PCR扩增产物的核苷酸序列如序列表中序列6所示(即基因片段4)，采用引物对HS01-5扩增得到的PCR扩增产物的核苷酸序列如序列表中序列7所示(即基因片段5)，采用引物对HS01-6扩增得到的PCR扩增产物的核苷酸序列如序列表中序列8所示(即基因片段6)。因此，采用表2中的引物可以扩增得到6个基因片段。
实施例6、6个基因片段在生产DHA和EPA中的应用
本实施例中，涉及的引物的核苷酸序列见表6。
表6
引物名称 核苷酸序列(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、GS-C06菌株的获得
一、打靶片段HS01-1-Zeo的制备
1、由南京金士瑞生物科技有限公司合成重组质粒pUC57-LZ。重组质粒pUC57-LZ是将序列表中序列15所示的核苷酸序列和pUC57载体连接得到的。序列表中序列15中，自5’末端起第25至58位为Lox66序列，第626至997位为zeocin抗性基因，第2293至2326位为Lox71序列。
2、采用酵母基因组提取试剂盒提取裂殖壶菌HS01的基因组DNA。
3、以裂殖壶菌HS01的基因组DNA为模板，以HS01-1-UF和HS01-1-UR为引物进行PCR扩增，得到约3100bp的PCR扩增产物，该PCR扩增产物即为HS01-1上游同源片段AU。
4、以裂殖壶菌HS01的基因组DNA为模板，以HS01-1-DF和HS01-1-DR为引物进行PCR扩增，得到约1000bp的PCR扩增产物，该PCR扩增产物即为HS01-1下游同源片段AD。
5、以步骤1合成的重组质粒pUC57-LZ为模板，以Zeo-F和Zeo-R为引物进行PCR扩增，得到约2350bp的PCR扩增产物(核苷酸序列如序列表中序列15所示)，该PCR扩增产物即为Zeo片段。
6、以HS01-1上游同源片段AU、HS01-1下游同源片段AD和Zeo片段为模板，以 HS01-1-UF和HS01-1-DR为引物进行overlap扩增，获得约6450bp的PCR扩增产物。采用琼脂糖凝胶DNA回收试剂盒回收该PCR扩增产物，即获得打靶片段HS01-1-Zeo。
二、预处理的MYA-1381的获得
1、取无菌预冷的聚丙烯管(规格为50mL)，加入10mLMYA-1381菌液(浓度为1×10 8cfu/mL)，4℃、5000r/min离心10min，弃上清，收集菌体。
2、完成步骤1后，重复如下步骤两次：取所述聚丙烯管，加入10mL预冷的无菌水清洗菌体，4℃、4472g离心10min，收集菌体。
3、完成步骤2后，取所述聚丙烯管，加入10mL预冷的1mol/L山梨醇水溶液重悬，4℃、5000r/min离心10min，收集菌体。
4、完成步骤3后，取所述聚丙烯管，加入10mL预冷的1mol/L山梨醇水溶液重悬，得到预处理的MYA-1381。
三、电转化
1、取30μL预处理的MYA-1381，加入1μg打靶片段HS01-1-Zeo，轻轻混匀，冰浴静置5min，然后转移到冰预冷的电击杯中电击(电击参数为0.75KV，50μF)。
2、完成步骤1后，取所述电击杯，加入1mL种子培养基，30℃、200r/min培养1h，然后4℃、5000r/min离心10min，将菌体和少量上清混合并均匀涂布在抗性平板上，30℃倒置培养48h，得到拟转化子。
抗性平板：向约55℃的筛选固体培养基中加入Zeocin并使其浓度为200μg/mL，然后倒入培养皿，冷却后得到的固体平板。
四、阳性转化子的获得及鉴定
采用酵母基因组提取试剂盒分别提取拟转化子的基因组DNA并以其作为模板，以HS01-1-F和HS01-1-R为引物进行PCR扩增，得到PCR扩增产物。
如果某拟转化子的PCR扩增产物的大小为4100bp(或其核苷酸序列如序列表中序列3所示)，则该拟转化子为阳性转化子。
五、转化子GS-C01的获得
将pSH65质粒(Biovector Inc.公司的产品；该质粒含有Cre酶)导入阳性转化子，然后按照pSH65质粒说明书的步骤消除Zeo基因，获得转化子GS-C01。
按照上述步骤一至五，将“HS01-1-UF”替换为“HS01-2-UF”，“HS01-1-UR”替换为“HS01-2-UR”，“HS01-1-DF”替换为“HS01-2-DF”，“HS01-1-DR”替换为“HS01-2-DR”，“HS01-1-F”替换为“HS01-2-F”，“HS01-1-R”替换为“HS01-2-R”，“MYA-1381”替换为“转化子GS-C01”，其它步骤均不变，得到转化子GS-C02。
按照上述步骤一至五，将“HS01-1-UF”替换为“HS01-3-UF”，“HS01-1-UR”替换为“HS01-3-UR”，“HS01-1-DF”替换为“HS01-3-DF”，“HS01-1-DR”替换为“HS01-3-DR”，“HS01-1-F”替换为“HS01-3-F”，“HS01-1-R”替换为“HS01-3-R”，“MYA-1381”替换为“转化子GS-C02”，其它步骤均不变，得到转化子GS-C03。
按照上述步骤一至五，将“HS01-1-UF”替换为“HS01-4-UF”，“HS01-1-UR”替换为“HS01-4-UR”，“HS01-1-DF”替换为“HS01-4-DF”，“HS01-1-DR”替换为“HS01-4-DR”， “HS01-1-F”替换为“HS01-4-F”，“HS01-1-R”替换为“HS01-4-R”，“MYA-1381”替换为“转化子GS-C03”，其它步骤均不变，得到转化子GS-C04。
按照上述步骤一至五，将“HS01-1-UF”替换为“HS01-5-UF”，“HS01-1-UR”替换为“HS01-5-UR”，“HS01-1-DF”替换为“HS01-5-DF”，“HS01-1-DR”替换为“HS01-5-DR”，“HS01-1-F”替换为“HS01-5-F”，“HS01-1-R”替换为“HS01-5-R”，“MYA-1381”替换为“转化子GS-C04”，其它步骤均不变，得到转化子GS-C05。
按照上述步骤一至五，将“HS01-1-UF”替换为“HS01-6-UF”，“HS01-1-UR”替换为“HS01-6-UR”，“HS01-1-DF”替换为“HS01-6-DF”，“HS01-1-DR”替换为“HS01-6-DR”，“HS01-1-F”替换为“HS01-6-F”，“HS01-1-R”替换为“HS01-6-R”，“MYA-1381”替换为“转化子GS-C05”，其它步骤均不变，得到转化子GS-C06。
转化子GS-C06即为GS-C06菌株。
B、6个基因片段在生产DHA和EPA中的应用
待测菌为裂殖壶菌HS01、MYA-1381或GS-C06菌株。
1、将待测菌的单克隆接种至装有2mL摇瓶培养基的摇瓶(规格为10mL)中，22～28℃、150～250rpm/min培养24～48h，得到一级种子液。
2、取一级种子液，以3～10％(v/v)的接种量接种至装有50mL摇瓶培养基的摇瓶(摇瓶规格为500mL)中，22～28℃、150～250rpm/min培养24～48h，得到二级种子液。
3、取二级种子液，以3～10％(v/v)的接种量接种至装有500mL种子培养基的发酵罐(发酵罐规格为1L)中，22～28℃培养24～48h(溶解氧为10～80％)，得到三级种子液。
4、取三级种子液，以3～10％(v/v)的接种量接种至装有5L发酵培养基的发酵罐(发酵罐规格为10L；接种后初始生物量为0.3×10 8～0.5×10 8cfu/mL)中，22～28℃培养72～120h(溶解氧为5～80％)，得到发酵液。发酵液中即含有DHA、DPA和EPA。
5、按照实施例1步骤一中5的方法，提取发酵液的油脂，然后按照GB5009.168-2016食品安全国家标准检测DHA含量，按照GB28404-2012食品安全国家标准检测DPA含量，按照GB5009.168-2016食品安全国家标准检测EPA含量。
实验结果见表7。结果表明，向MYA-1381转化本发明获得的6个基因片段，可获得合成DHA和EPA的高产菌株(即GS-C06菌株)。因此，本发明提供的6个基因片段，这6个基因片段编码的蛋白质，和，含有这6个基因片段的载体、细胞或生物体，在生产DHA和EPA中具有重要的应用价值。通过对出发菌株中这6个基因片段编码的蛋白质的改造，可构建出高产的DHA和EPA的工程菌株。
表7
MYA-1381 裂殖壶菌HS01 GS-C06菌株
油脂中DHA的含量(％) 12.38 45.02 30.50
油脂中DPA的含量(％) 25.26 12.74 17.50
油脂中EPA的含量(％) 0.50 1.30 0.71
工业应用
本发明提供的裂殖壶菌(Schizoochytrium limacinum)HS01生产DHA和/或EPA具有较高的生产价值。向合成DHA和/或EPA的低产菌株转化本发明获得的6个基因片段，可获 得合成DHA和/或EPA的高产菌株。因此，本发明提供的6个基因片段，这6个基因片段编码的蛋白质，和，含有这6个基因片段的载体、细胞或生物体，在生产DHA和/或EPA中具有重要的应用价值。通过对出发菌株中这6个基因片段编码的蛋白质的改造，可构建出高产的DHA和/或EPA的工程菌株。

Claims (15)

  1. 蛋白质组合，为如下(X1)或(X2)或(X3)或(X4)：
    (X1)包括蛋白质1、蛋白质2、蛋白质3、蛋白质4、蛋白质5和蛋白质6；
    (X2)由所述蛋白质1、所述蛋白质2、所述蛋白质3、所述蛋白质4、所述蛋白质5和所述蛋白质6组成；
    (X3)由所述蛋白质1、所述蛋白质2、所述蛋白质3、所述蛋白质4、所述蛋白质5和所述蛋白质6中的任意两个、任意三个、任意四个或任意五个组成；
    (X4)所述蛋白质1、所述蛋白质2、所述蛋白质3、所述蛋白质4、所述蛋白质5或所述蛋白质6；
    所述蛋白质1为a1)或a2)或a3)或a4)或a5)：
    a1)氨基酸序列是序列表中序列9所示的蛋白质；
    a2)在序列表中序列9所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
    a3)将序列表中序列9所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
    a4)与序列表中序列9限定的氨基酸序列具有80％或80％以上同一性，来源于裂殖壶菌且与多不饱和脂肪酸合成相关的蛋白质；
    a5)具有序列表中序列9所示的氨基酸序列的蛋白质；
    所述蛋白质2为b1)或b2)或b3)或b4)或b5)：
    b1)氨基酸序列是序列表中序列10所示的蛋白质；
    b2)在序列表中序列10所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
    b3)将序列表中序列10所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
    b4)与序列表中序列10限定的氨基酸序列具有80％或80％以上同一性，来源于裂殖壶菌且与多不饱和脂肪酸合成相关的蛋白质；
    b5)具有序列表中序列9所示的氨基酸序列的蛋白质；
    所述蛋白质3为c1)或c2)或c3)或c4)或c5)：
    c1)氨基酸序列是序列表中序列11所示的蛋白质；
    c2)在序列表中序列11所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
    c3)将序列表中序列11所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
    c4)与序列表中序列11限定的氨基酸序列具有80％或80％以上同一性，来源于裂殖壶菌且与多不饱和脂肪酸合成相关的蛋白质；
    c5)具有序列表中序列11所示的氨基酸序列的蛋白质；
    所述蛋白质4为d1)或d2)或d3)或d4)或d5)：
    d1)氨基酸序列是序列表中序列12所示的蛋白质；
    d2)在序列表中序列12所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
    d3)将序列表中序列12所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
    d4)与序列表中序列12限定的氨基酸序列具有80％或80％以上同一性，来源于裂殖壶菌且与多不饱和脂肪酸合成相关的蛋白质；
    d5)具有序列表中序列12所示的氨基酸序列的蛋白质；
    所述蛋白质5为e1)或e2)或e3)或e4)或e5)：
    e1)氨基酸序列是序列表中序列13所示的蛋白质；
    e2)在序列表中序列13所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
    e3)将序列表中序列13所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
    e4)与序列表中序列13限定的氨基酸序列具有80％或80％以上同一性，来源于裂殖壶菌且与多不饱和脂肪酸合成相关的蛋白质；
    e5)具有序列表中序列13所示的氨基酸序列的蛋白质；
    所述蛋白质6为f1)或f2)或f3)或f4)或f5)：
    f1)氨基酸序列是序列表中序列14所示的蛋白质；
    f2)在序列表中序列14所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
    f3)将序列表中序列14所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
    f4)与序列表中序列14限定的氨基酸序列具有80％或80％以上同一性，来源于裂殖壶菌且与多不饱和脂肪酸合成相关的蛋白质；
    f5)具有序列表中序列14所示的氨基酸序列的蛋白质。
  2. 编码权利要求1所述蛋白质组合的核酸分子。
  3. 如权利要求2所述的核酸分子，其特征在于：
    编码所述蛋白质1的核酸分子为如下A1)或A2)或A3)或A4)所示的DNA分子：
    A1)编码区是序列表中序列3自5’末端起第1044至3050位所示的DNA分子；
    A2)核苷酸序列是序列表中序列3所示的DNA分子；
    A3)与A1)或A2)限定的核苷酸序列具有75％或75％以上同一性，来源于裂殖壶菌且编码权利要求1中所述蛋白质1的DNA分子；
    A4)在严格条件下与A1)或A2)限定的核苷酸序列杂交，且编码权利要求1中所述蛋白质1的DNA分子；
    编码所述蛋白质2的核酸分子为如下B1)或B2)或B3)或B4)所示的DNA分子：
    B1)编码区是序列表中序列4自5’末端起第1068至2737位和第3254至5162位所示的DNA分子；
    B2)核苷酸序列是序列表中序列4所示的DNA分子；
    B3)与B1)或B2)限定的核苷酸序列具有75％或75％以上同一性，来源于裂殖壶菌且编码权利要求1中所述蛋白质2的DNA分子；
    B4)在严格条件下与B1)或B2)限定的核苷酸序列杂交，且编码权利要求 1中所述蛋白质2的DNA分子；
    编码所述蛋白质3的核酸分子为如下C1)或C2)或C3)或C4)所示的DNA分子：
    C1)编码区是序列表中序列5自5’末端起第1094至3415位所示的DNA分子
    C2)核苷酸序列是序列表中序列5所示的DNA分子；
    C3)与C1)或C2)限定的核苷酸序列具有75％或75％以上同一性，来源于裂殖壶菌且编码权利要求1中所述蛋白质3的DNA分子；
    C4)在严格条件下与C1)或C2)限定的核苷酸序列杂交，且编码权利要求1中所述蛋白质3的DNA分子；
    编码所述蛋白质4的核酸分子为如下D1)或D2)或D3)或D4)所示的DNA分子：
    D1)编码区是序列表中序列6自5’末端起第1409至5044位、第7004至7234位和第7700至10399位所示的DNA分子；
    D2)核苷酸序列是序列表中序列6所示的DNA分子；
    D3)与D1)或D2)限定的核苷酸序列具有75％或75％以上同一性，来源于裂殖壶菌且编码权利要求1中所述蛋白质4的DNA分子；
    D4)在严格条件下与D1)或D2)限定的核苷酸序列杂交，且编码权利要求1中所述蛋白质4的DNA分子；
    编码所述蛋白质5的核酸分子为如下E1)或E2)或E3)或E4)所示的DNA分子：
    E1)编码区是序列表中序列7自5’末端起第1473至6488位所示的DNA分子；
    E2)核苷酸序列是序列表中序列7所示的DNA分子；
    E3)与E1)或E2)限定的核苷酸序列具有75％或75％以上同一性，来源于裂殖壶菌且编码权利要求1中所述蛋白质5的DNA分子；
    E4)在严格条件下与E1)或E2)限定的核苷酸序列杂交，且编码权利要求1中所述蛋白质5的DNA分子；
    编码所述蛋白质6的核酸分子为如下F1)或F2)或F3)或F4)所示的DNA分子：
    F1)编码区是序列表中序列8自5’末端起第953至991位和第1063至1090位所示的DNA分子；
    F2)核苷酸序列是序列表中序列8所示的DNA分子；
    F3)与F1)或F2)限定的核苷酸序列具有75％或75％以上同一性，来源于裂殖壶菌且编码权利要求1中所述蛋白质6的DNA分子；
    F4)在严格条件下与F1)或F2)限定的核苷酸序列杂交，且编码权利要求1中所述蛋白质6的DNA分子。
  4. 含有权利要求2或3所述核酸分子的表达盒、重组载体、重组微生物或转基因细胞系。
  5. 权利要求1所述蛋白质组合，或，权利要求2或3所述核酸分子，或，含有权利要求2或3所述核酸分子的表达盒、重组载体、重组微生物或转基因细胞系，在生产DHA和/或EPA中的应用。
  6. 重组菌乙，其制备方法如下：使出发菌中权利要求1所述蛋白质组合的表达和/或活性提高，得到的重组菌即为重组菌乙。
  7. 一种生产DHA和/或EPA的方法，依次包括如下步骤：
    (1)使出发菌中权利要求1所述蛋白质组合的表达和/或活性提高，得到的重组菌甲；与所述出发菌相比，重组菌甲生产DHA和/或EPA的能力提高；
    (2)发酵培养重组菌甲，得到DHA和/或EPA。
  8. 如权利要求6所述的重组菌乙、或权利要求7所述的方法，其特征在于：“使出发菌中所述蛋白质组合的表达和/或活性提高”通过向出发菌中导入提高蛋白质组合的表达和/或活性的物质实现。
  9. 如权利要求6或8所述的重组菌乙、或、权利要求7或8所述的方法，其特征在于：所述出发菌为裂殖壶菌。
  10. 如权利要求9所述的重组菌乙或权利要求9所述的方法，其特征在于：所述裂殖壶菌为菌株Schizochytrium limacinum Honda et Yokochi ATCC MYA-1381。
  11. 裂殖壶菌(Schizoochytrium limacinum)HS01，其在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC No.13746。
  12. 一种菌剂，其特征在于：所述菌剂含有权利要求11所述裂殖壶菌(Schizoochytrium limacinum)HS01 CGMCC No.13746，或，权利要求6、8、9或10所述的重组菌乙。
  13. 权利要求11所述的裂殖壶菌(Schizoochytrium limacinum)HS01 CGMCC No.13746，或，权利要求6、8、9或10所述的重组菌乙，或，权利要求12所述菌剂在生产二十二碳六烯酸和/或EPA中的应用。
  14. 一种生产二十二碳六烯酸和/或EPA的方法，包括发酵培养权利要求11所述裂殖壶菌(Schizoochytrium limacinum)HS01 CGMCC No.13746，或，权利要求6、8、9或10所述的重组菌乙，得到二十二碳六烯酸和/或EPA的步骤。
  15. 如权利要求14所述的方法，其特征在于：所述发酵培养使用的发酵培养基溶质及其溶度为：葡萄糖20～120g/L、谷氨酸或谷氨酸钠5～15g/L、玉米浆干粉3～15g/L、NaSO 4 5～24g/L、KCl 0.1～1.0g/L、MgSO 4 1.0～3.0g/L、K 2SO 4 0.3～1.5g/L、KH 2PO 4 0.5～1.5g/L、(NH 4) 2SO 4 0.5～1.5g/L、CaCl 2 0.1～1.0g/L；溶质为水；pH5.0～6.5。
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