WO2019196791A1 - 生产神经酸的重组酵母菌株及其应用 - Google Patents

生产神经酸的重组酵母菌株及其应用 Download PDF

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WO2019196791A1
WO2019196791A1 PCT/CN2019/081736 CN2019081736W WO2019196791A1 WO 2019196791 A1 WO2019196791 A1 WO 2019196791A1 CN 2019081736 W CN2019081736 W CN 2019081736W WO 2019196791 A1 WO2019196791 A1 WO 2019196791A1
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gene
seq
nucleotide sequence
fatty acid
endoplasmic reticulum
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French (fr)
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李福利
王士安
樊伟明
孟慧敏
张锴
李家欣
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中国科学院青岛生物能源与过程研究所
浙江震元制药有限公司
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Priority to JP2020556255A priority Critical patent/JP7261815B2/ja
Priority to EP19785189.2A priority patent/EP3778866B1/en
Priority to US17/046,672 priority patent/US11603545B2/en
Publication of WO2019196791A1 publication Critical patent/WO2019196791A1/zh

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Definitions

  • the invention belongs to the field of biotechnology. More specifically, the present invention relates to an engineered recombinant yeast strain capable of efficiently producing a neric acid (cis-15-tetracosole monoenoic acid, alias shark acid, C24:1, ⁇ 15) at a high concentration.
  • a neric acid cis-15-tetracosole monoenoic acid, alias shark acid, C24:1, ⁇ 15
  • Unsaturated fatty acids are mostly essential fatty acids, which have the functions of regulating blood lipids, clearing blood clots, nourishing brain and brain, and relieving inflammation, including monounsaturated fatty acids and polyunsaturated fatty acids.
  • VLCMFA Very long chain monounsaturated fatty acid
  • Eicosenoic acid is common.
  • Ultra-long-chain monounsaturated fatty acids have unique pharmacological effects, health benefits, and industrial uses, but the application of ultra-long-chain monounsaturated fatty acids needs to be strengthened compared to polyunsaturated fatty acids.
  • Nernic acid (cis-15-tetracosole monoenoic acid, alias shark acid, C24:1 ⁇ 15) is an ultralong chain monounsaturated fatty acid closely related to human health.
  • the nervonic acid is mainly present in the form of glycosphingolipids and sphingomyelin in the nerve fibers of the white matter and myelin of the animal's brain, and is an important component of the biofilm.
  • Nernic acid plays an important role in medicine and health care and can be used to treat neurological disorders such as multiple sclerosis.
  • studies have shown that nervonic acid has a positive effect on the development of the nervous system, especially in the growth and development of brain cells and optic nerve cells in infants and young children.
  • the nervous acid needed by the human body mainly depends on exogenous intake.
  • the value of the development and utilization of its resources has become prominent, and the demand for products is gradually expanding.
  • Acer truncatum is a unique Chinese plant rich in nerve acid in nature, and the oil content of garlic seed kernel is about 64.5%, and the nerve acid content is as high as 43.2%, but the cultivation of garlic fruit is difficult.
  • the oleic acid content of Acer truncatum seed oil is about 5.8%, which is the main source of current nervous acid.
  • the growth of Acer truncatum is slow, artificially planted ingots of 4-6 years, and it will enter the fruiting period in 8-10 years.
  • Oil-producing microorganisms can synthesize high-cell fatty acids, which can be genetically engineered to form microbial oils similar in composition to vegetable oils.
  • Yarrowia lipolytica can account for 44-70% of the dry weight of cells, and has a fast growth rate, high cell fermentation density, wide utilization of carbon sources, and genetic manipulation. Simple and so on, it has great potential to develop into a nerve acid cell factory. Most fatty acids in Yarrowia yeast are C16 and C18 fatty acids, and wild strains are unable to synthesize nervonic acid due to the lack of carbon chain elongation enzymes and fatty acid desaturases necessary for the synthesis of ultralong chain monounsaturated fatty acids.
  • fatty acid elongases AtFAE1, BtFAE1 and CgKCS
  • SCD desaturase
  • DGAT1 diacylglycerol acyltransferase
  • the present invention solves the above technical problems by overexpressing a fatty acid elongase, a desaturase, a diacyl acyltransferase and the like, optionally a triglyceride synthesis and decomposition pathway, sphingomyelin synthesis and decomposition of a recombinant yeast strain.
  • the pathway, the synthesis and decomposition pathway of oil sub-cell level and the redox equilibrium pathway are regulated, so that the ability of the recombinant yeast strain to produce nervonic acid is greatly improved.
  • the content of nervonic acid extracted and the total fatty acid content is 39.6. %.
  • the specific technical solutions are as follows:
  • the present invention provides a recombinant yeast strain characterized by overexpression:
  • the recombinant yeast strain overexpresses a fatty acid synthesis expression module, specifically including a fatty acid elongase and a desaturase gene related to sulphuric acid production.
  • a fatty acid elongase gene may be selected from, but not limited to, the Mortierella alpina C16 elongase gene MaLCE1, as set forth in SEQ ID No: 93; Arabidopsis AtFAE1, as set forth in SEQ ID No: 94; African mustard BtFAE1 , as shown in SEQ ID No: 95; C.
  • chinensis CgKCS as shown in SEQ ID No: 96
  • rat fatty acid elongase 2 gene rELO2 as shown in SEQ ID No: 97
  • long-chain fatty acid elongation of Cryptosporidium parvum The enzyme gene CpLCE is shown in SEQ ID No: 98
  • the goat fatty acid elongase 6 gene gELOVL6 is shown in SEQ ID No: 99.
  • the desaturase gene may be selected from, but not limited to, Yarrowia lipolytica SCD, as set forth in SEQ ID No: 84; G. sphaeroides ⁇ 9 fatty acid desaturase gene D9DMB, as SEQ ID No: 100; C.
  • elegans ⁇ 9 fatty acid desaturase gene CeFAT6 as shown in SEQ ID No: 101
  • Mortierella alpina ⁇ 9 fatty acid desaturase gene MaOLE2 as shown in SEQ ID No: 102
  • Arabidopsis AtADS1 such as SEQ ID No: 103
  • Arabidopsis AtADS2 as shown in SEQ ID No: 104.
  • the recombinant yeast strain overexpresses a triglyceride synthesis module, specifically a diacylglycerol acyltransferase gene, which is an enzyme that catalyzes the final reaction of triacylglycerol (TAG) synthesis, and is the only key enzyme in the TAG synthesis process.
  • a triglyceride synthesis module specifically a diacylglycerol acyltransferase gene, which is an enzyme that catalyzes the final reaction of triacylglycerol (TAG) synthesis, and is the only key enzyme in the TAG synthesis process.
  • TAG triacylglycerol
  • rate-limiting enzymes increasing the expression of diglyceride acyltransferase in yeast cells can increase the content of intracellular lipids.
  • the recombinant yeast strain over-expresses the regulation and synthesis of the sub-cell level regulation module of the yeast oil, specifically refers to the regulation of the endoplasmic reticulum level, that is, the endoplasmic reticulum retention signal peptide KDEL is added at the 3' end of the corresponding gene.
  • the yeast strain of the first scheme is Yarrowia yeast.
  • the gene encoding the ⁇ 9 desaturase is a Y. lipolytic yeast SCD gene, the nucleotide sequence of which is shown in SEQ ID No: 84;
  • the four encoded fatty acid elongase genes are respectively the Mortierella alpina C16/18 elongase gene MaLCE1, the nucleotide sequence of which is shown in SEQ ID No: 93; the Arabidopsis AtFAE1 gene, the nucleoside thereof The acid sequence is shown in SEQ ID No: 94; the African mustard BtFAE1 gene has a nucleotide sequence as shown in SEQ ID No: 95; the C. chinensis CgKCS gene has a nucleotide sequence as shown in SEQ ID No: 96;
  • the gene encoding a diglyceride acyltransferase is a Y. lipolytica DGAT1 gene, and the nucleotide sequence thereof is shown in SEQ ID No: 83;
  • the gene encoding a fatty acid elongase that targets the endoplasmic reticulum is a C. crispus CgKCS ER gene with a coding sequence encoding an endoplasmic reticulum signal peptide, the nucleotide sequence of which is set forth in SEQ ID No: 121. ;
  • the gene encoding a diglyceride acyltransferase targeting endoplasmic reticulum is a Y. lipolytica DGAT1 ER gene with a coding sequence encoding an endoplasmic reticulum signal peptide, the nucleotide sequence of which is SEQ. ID No: 122;
  • the gene encoding a ⁇ 9 desaturase targeting endoplasmic reticulum is a Y. lipolytic yeast SCD ER gene with a sequence encoding an endoplasmic reticulum signal peptide, the nucleotide sequence of which is SEQ ID No. :123 is shown.
  • the invention provides a recombinant yeast strain for the production of nervonic acid, which strain can be further overexpressed on the basis of the strain involved in Scheme 1:
  • the two genes encoding the endoplasmic reticulating fatty acid elongase are a C. crispus CgKCS ER gene with a targeting endoplasmic reticulum signal peptide coding sequence, and the nucleotide sequence thereof is SEQ ID No: 121; an African mustard BtFAE1 ER gene having a targeting endoplasmic reticulum signal peptide coding sequence, the nucleotide sequence of which is shown in SEQ ID No: 124;
  • the two genes encoding the peroxisome-derived fatty acid elongase are respectively the C. chinensis CgKCS PTS gene with a peroxisome signal peptide coding sequence, and the nucleotide sequence thereof is as follows. SEQ ID No: 125; an African mustard BtFAE1 PTS gene carrying a peroxisome signal peptide coding sequence, the nucleotide sequence of which is shown in SEQ ID No: 126.
  • the invention provides a recombinant yeast strain for the production of nervonic acid, which strain can be further overexpressed on the basis of the strain involved in Scheme 1:
  • the recombinant yeast species further includes a regulatory module for redox balance, which is involved in maintaining a gene related to reducing power NADPH regeneration and oxidative stress defense during nervic acid synthesis.
  • the aldehyde dehydrogenase gene is preferably an Escherichia coli EcAldH gene having a nucleotide sequence as shown in SEQ ID No: 105; the glucose-6-phosphate dehydrogenase gene is preferably a Saccharomyces cerevisiae ScZwf gene, the nucleotide sequence of which is As shown in SEQ ID No: 106, the glutathione disulfide reductase gene is preferably a Yarrowia lipolytica ylGSR gene having a nucleotide sequence as shown in SEQ ID No: 91, glutathione peroxide
  • the enzyme gene is preferably the Yarrowia lipolytica ylGPO gene, and the nucleotide sequence thereof is shown in SEQ ID No: 92.
  • the recombinant yeast strain comprises a regulation module for synthesis and decomposition of sphingomyelin, specifically relates to a phospholipase A2 (PLA2) gene, and the PLA2 is a hydrolase capable of catalyzing a diacyl group on the phospholipid molecule, and the overexpression thereof can increase the nerve. Supply of substrate during acid synthesis.
  • the encoding phospholipase A2 gene may be selected from, but not limited to, PLA2-1 as shown in SEQ ID No: 85, PLA2-2 as shown in SEQ ID No: 86, and PLA2-3 as shown in SEQ ID No: 87.
  • PLA2-4 is shown in SEQ ID No: 88
  • PLA2-5 is shown in SEQ ID No: 89
  • PLA2-6 is shown in SEQ ID No: 90.
  • the gene encoding the ⁇ 9 desaturase is a Y. lipolytic yeast SCD gene having a nucleotide sequence as shown in SEQ ID No: 84;
  • the three genes encoding the fatty acid elongase are the Arabidopsis thaliana AtFAE1 gene, and the nucleotide sequence thereof is SEQ ID No: 94, the African mustard BtFAE1 gene, and the nucleotide sequence thereof is SEQ ID No: 95, the C. crispus CgKCS gene, the nucleotide sequence of which is shown in SEQ ID No: 96;
  • the gene encoding the diacyl acyltransferase is the Y. lipolytica DGAT1 gene, and the nucleotide sequence thereof is shown in SEQ ID No: 83.
  • the yeast strain is preferably Yarrowia lipolytica
  • the peroxisome-targeting fatty acid elongase gene is a C. chinensis CgKCS PTS gene carrying a peroxisome signal peptide coding sequence, and the nucleotide sequence thereof is SEQ ID No: 125 Shown
  • the fatty acid elongase gene is the M. alpina C16/18 elongase gene MaLCE1 gene, and the nucleotide sequence thereof is shown in SEQ ID No: 93;
  • the gene of the endoplasmic reticulum-targeting fatty acid elongase is a C. crispus CgKCS ER having a coding sequence encoding the endoplasmic reticulum signal peptide, and the nucleotide selection sequence thereof is shown in SEQ ID No: 121;
  • the endoplasmic reticulum-targeting diacyl acyltransferase gene is a Y. lipolytica DGAT1 ER gene with a targeting endoplasmic reticulum signal peptide coding sequence, the nucleotide sequence of which is SEQ ID No. :122;
  • the ⁇ 9 desaturase gene targeting the endoplasmic reticulum is a Y. lipolytic yeast SCD ER gene with a targeting endoplasmic reticulum signal peptide coding sequence, and the nucleotide sequence thereof is SEQ ID No: 123 Shown.
  • the yeast strain is preferably Yarrowia lipolytica
  • the gene encoding the ⁇ 9 desaturase is a M. alpina ⁇ 9 fatty acid desaturase MaOLE2 gene, the nucleotide sequence of which is shown in SEQ ID No: 102;
  • the encoded fatty acid elongase is a sheep fatty acid elongase 6g ELOVL6 gene, and the nucleotide sequence thereof is shown in SEQ ID No: 99;
  • the fatty acid elongase gene encoding the endoplasmic reticulum is a C. crispus CgKCS ER having a coding sequence encoding the endoplasmic reticulum signal peptide, and the nucleotide sequence thereof is shown in SEQ ID No: 121;
  • the fatty acid elongase gene encoding the mitochondria is a C. crispus CgKCS MTS having a targeting mitochondrial signal peptide coding sequence, the nucleotide sequence of which is shown in SEQ ID No: 127.
  • the yeast strain is Yarrowia yeast
  • the two coding ⁇ 9 desaturase genes are respectively Y. lipolytica SCD gene, the nucleotide sequence thereof is shown as SEQ ID No: 84; the Arabidopsis AtADS1 gene has a nucleotide sequence such as SEQ ID No: 103; or the two coding ⁇ 9 desaturase genes are the Y. lipolytic yeast SCD gene, the nucleotide sequence of which is shown in SEQ ID No: 84; the Arabidopsis AtADS2 gene, Its nucleotide sequence is shown as SEQ ID No: 104;
  • the three encoded fatty acid elongase genes are Arabidopsis thaliana AtFAE1 gene, the nucleotide sequence thereof is shown in SEQ ID No: 94; the African mustard BtFAE1 gene has a nucleotide sequence of SEQ ID No: 95. Shown; the C. crispus CgKCS gene, the nucleotide sequence of which is shown in SEQ ID No: 96;
  • the encoding diglyceride acyltransferase is Yarrowia lipolytica DGAT1 as set forth in SEQ ID No:83.
  • the present invention provides a recombinant yeast strain characterized in that the expression of the peroxisome biogenesis factor 10 of the strain is down-regulated and further overexpressed:
  • the recombinant yeast strain includes a triglyceride decomposition module, specifically a peroxisome biogenesis factor 10 gene knockout module, and the knockout of the gene can reduce the decomposition of long-chain fatty acids.
  • the yeast strain is Yarrowia yeast
  • the down-regulated peroxisome biogenesis factor 10 is a pex10 gene, and the nucleotide sequence thereof is represented by SEQ ID No: 120;
  • the gene encoding the peroxisome-derived fatty acid elongase is a C. crispus CgKCS PTS gene carrying a peroxisome signal peptide coding sequence, and the nucleotide sequence thereof is SEQ ID No. :125;
  • the gene encoding the fatty acid elongase is the M. alpina C16/18 elongase MaLCE1 gene, and the nucleotide sequence thereof is shown in SEQ ID No: 93;
  • the gene encoding a fatty acid elongase that targets the endoplasmic reticulum is a C. crispus CgKCS ER gene with a coding sequence encoding an endoplasmic reticulum signal peptide, the nucleotide sequence of which is set forth in SEQ ID No: 121. ;
  • the gene encoding a diglyceride acyltransferase targeting endoplasmic reticulum is a Y. lipolytica DGAT1 ER gene with a coding sequence encoding an endoplasmic reticulum signal peptide, the nucleotide sequence of which is SEQ. ID No: 122;
  • the ⁇ 9 desaturase gene encoding the endoplasmic reticulum is a Y. lipolytic yeast SCD ER gene with a targeting endoplasmic reticulum signal peptide coding sequence, the nucleotide sequence of which is SEQ ID No: 123 shows.
  • the yeast is Yarrowia yeast.
  • the present invention provides the use of any of the recombinant yeast strains constructed in the above schemes for the preparation of microbial oil or nervonic acid. Specifically included, but not limited to, infant formulas containing microbial oil or nervonic acid, functional foods, medical foods, medical supplements, dietary supplements, pharmaceutical compositions, animal feeds, and personal care products.
  • the present invention provides a method for producing microbial oil and/or nervic acid using any recombinant yeast strain constructed in the above technical scheme, and specifically includes, but is not limited to, cultivation of microorganisms, optimization and control of fermentation conditions. Optimization of the fermentation conditions includes optimization of different carbon sources, carbon to nitrogen ratios, and erythrose addition induction at different growth stages, including, but not limited to, temperature, pH, fermentation time, dissolved oxygen, and feed mode. Wait for control.
  • the extraction process of the microbial oil or/and nervonic acid includes, but is not limited to, separation, fragmentation, and organic solvent extraction processes of the strain.
  • the method for preparing a microbial oil comprises:
  • step (b) recovering the microbial oil of step (a).
  • the method for preparing a nervonic acid comprises:
  • step (b) recovering the microbial oil of the step (a) and extracting the nervonic acid.
  • the method of the invention relates to the metabolic pathway and fermentation regulation of the nervonic acid synthesis system, obtaining a high-quality recombinant Yeluvian yeast strain, increasing the yield of the microbial oil, and preparing the nervonic acid
  • the content is 39.6% of total fatty acid content
  • the concentration of nerve acid is 16g/L, which has good industrial application prospects.
  • FIG. 1 is a diagram of a neural acid synthesis strategy provided by an embodiment of the present invention.
  • FIG. 2 is a PCR identification of yeast transformants according to an embodiment of the present invention.
  • A PCR verification results of CgKCS genes of different transformants when strain YL1 was constructed.
  • B PCR verification results of the MaLCE1 gene of different transformants when strain YL2 was constructed.
  • C PCR verification results of CgKCS genes of different transformants when strain YL2-1 was constructed.
  • D PCR verification results of the BtFAE1 gene of different transformants when strain YL2-2 was constructed.
  • E PCR verification results of CgKCS genes of different transformants when strain YL2-3 was constructed.
  • F PCR validation results of the ScZwf gene of different transformants when strain YL2-4 was constructed.
  • G PCR verification results of CgKCS genes of different transformants when strain YL3 was constructed.
  • H PCR verification results of PLA2-1 gene of different transformants when strain YL4-1 was constructed.
  • I PCR verification results of the PLA2-2 gene of different transformants when strain YL4-2 was constructed.
  • J PCR verification results of PLA2-3 gene of different transformants when strain YL4-3 was constructed.
  • K PCR verification results of PLA2-4 gene of different transformants when strain YL4-4 was constructed.
  • L PCR verification results of the PLA2-5 gene of different transformants when strain YL4-5 was constructed.
  • M PCR verification results of PLA2-6 genes of different transformants when strain YL4-6 was constructed.
  • N PCR verification results of gELOVL6 gene of different transformants when strain YL5 was constructed.
  • O PCR verification results of CgKCS genes of different transformants when strain YL6 was constructed.
  • P PCR verification results of the AtADS1 gene of different transformants when strain YL7 was constructed.
  • Q PCR validation results of the AtADS2 gene of different transformants when strain YL8 was constructed.
  • R PCR verification results of the pex10 gene of different transformants when strain YL9 was constructed.
  • S PCR verification results of CgKCS genes of different transformants when strain YL10 was constructed.
  • T PCR validation results of the DGAT1 gene of different transformants when strain YL11 was constructed.
  • FIG. 3 is a verification diagram of six gene expression expressions in the YL2-3 strain according to an embodiment of the present invention.
  • FIG. 4 is a diagram showing the positional specificity of nervonic acid in TAG by using the TLC method according to an embodiment of the present invention.
  • Figure 5 is a graph showing the analysis of fatty acid components provided by an embodiment of the present invention.
  • 6 is a growth curve of a strain of YL2-3 under shaking flask fermentation conditions according to an embodiment of the present invention.
  • Figure 7 is a graph showing the content of nervonic acid in different strains under shake flask fermentation conditions according to an embodiment of the present invention.
  • Figure 8 is a graph showing the intracellular aldehyde levels in the Po1g and YL2-4 strains provided by the examples of the present invention.
  • FIG. 9 is a diagram showing the fatty acid composition of the YL2-3 strain under the condition of fermentation fermentation tank of the embodiment of the present invention.
  • a desaturase refers to a polypeptide that can be desaturated (ie, introduced into a double bond) to produce a fatty acid or precursor of interest in one or more fatty acids, using a delta-system to count the decarboxylase from the carboxy terminus of the substrate.
  • Activity Preferably, the present invention is a ⁇ 9 desaturase desaturase, which in the molecule are numbered to the carboxy terminus of saturated fatty acids of carbon atoms between 9 th and 10 th of, for example, the catalytic substrate fatty acid (C18: 0 ) Formation of oleic acid (C18:1).
  • a fatty acid elongase refers to a polypeptide which is capable of extending a fatty acid carbon chain to produce an acid having 2 carbon atoms longer than the fatty acid substrate to which the elongase acts.
  • the fatty acid elongases of the invention include, but are not limited to, C16/18 elongase, C18/20 elongase, C20/22 elongase, and C22/24 elongase.
  • the C16/18 elongase will utilize a C16 substrate such as the Mortierella alpina C16/18 elongase gene MaLCE1, a sheep fatty acid elongase 6 gene gELOVL6.
  • Some elongases have broad specificity and thus a single elongase can catalyze several elongase reactions, such as C. chinensis CgKCS not only has substrate specificity for C18 and C20 fatty acids, but can continue to use C22 fatty acids as a substrate, so CgKCS Activity with C18/20, C20/22 and C22/24 elongases.
  • Diglyceride acyltransferase is the enzyme that catalyzes the final reaction of triacylglycerol (TAG) synthesis. It is also the only key enzyme and rate-limiting enzyme in the process of TAG synthesis. It can increase the expression of diglyceride acyltransferase in yeast cells. Increase the amount of oil in the cells.
  • TAG triacylglycerol
  • Endoplasmic reticulum, peroxisomes, and mitochondria refer to organelles that are ubiquitous in all eukaryotic cells. Enzymes targeting endoplasmic reticulum, peroxisomes, and mitochondria require the addition of the endoplasmic reticulum retention signal peptide KDEL, the peroxisome targeting signal peptide SKL, and the mitochondrial targeting signal peptide at the 3' end of the corresponding gene. CoxIV (MLSLRQSIRFFKPATRTLCSSRYLL).
  • Peroxisome biosynthesis factor protein peroxisome protein, Pex protein, refers to peroxisome biosynthesis and/or participates in the passage of cellular proteins through the peroxisome membrane by ATP hydrolysis. Process of protein.
  • An expression cassette refers to a DNA fragment comprising a coding sequence of a selected gene and a regulatory sequence preceding the coding sequence (5' non-coding sequence) and after (3' non-coding sequence) required for expression of the selected gene product.
  • Expression cassettes are typically included in a vector to facilitate cloning and transformation. Different expression cassettes can be transformed into different organisms including bacterial, yeast, plant and mammalian cells, as long as the correct regulatory sequences can be used for each host.
  • the expression cassette usually consists of the following sequence:
  • a promoter sequence such as GPAT, TEF1, EXP1, EYK1, GPD, etc.;
  • a 3' untranslated region i.e., terminator which typically comprises polyadenylation sites in eukaryotic cells, such as XPR2, LIP1t and PQX3t.
  • Microbial oil refers to a large amount of oil produced by bacteria, such as yeast, mold, bacteria and algae under certain conditions, using carbohydrates, hydrocarbons or common fats and oils as a carbon source.
  • the main components are triglycerides and Free fatty acids.
  • the microbial oil of the present invention is produced by fermentation of Y. lipolytica yeast, and is regulated by a metabolic pathway and a fermentation process, and the ability to obtain a microbial oil produced by a high-quality strain is greatly improved, and the amount of the obtained nervous acid is total.
  • other fatty acids include, but are not limited to, palmitoleic acid, oleic acid, linoleic acid, palmitic acid, octadecanoic acid, and tetracosanoic acid.
  • FIG. 1 A schematic diagram of a neural acid synthesis strategy provided by an embodiment of the present invention is shown in FIG.
  • the Y. lipolytic yeast strain (strain number polg, purchased from Yeastern Biotech Company, Taiwan) was used in YPD medium (YPD medium component was glucose 20 g/L, peptone 20 g/L, yeast extract 10 g/L).
  • YPD medium component was glucose 20 g/L, peptone 20 g/L, yeast extract 10 g/L.
  • CTAB hexadecyltrimethylammonium bromide, cetyltrimethylammonium bromide
  • the appropriate amount of bacteria was added to liquid nitrogen for freezing, ground into powder, and an appropriate amount of 2 ⁇ CTAB extraction buffer (100 mmol/L Tris-HCl, pH 8.0, 20 mmol/L EDTA, 1.4 mol/L NaCl, 2% (w/v) was added.
  • CTAB 40 mmol/L mercaptoethanol
  • TE buffer 100 mM Tris-HCl, 10 mM EDTA pH 8.0
  • the total DNA was partially digested with Sau3AI, and the digested DNA fragment was purified by electrophoresis. A fragment of about 2 to 6 kb was recovered by a gel recovery and purification kit, and the recovered DNA was dissolved in 10 mmol/L of Tris-HCl (pH 8. In 0), it is stored at -20 °C.
  • the genomic DNA of Y. lipolytica was used as a template, and the gene was amplified by KAPA HiFi high-fidelity DNA polymerase (purchased from KAPA Biosystems) using SEQ ID No: 1-20 as a primer sequence, and PCR amplification was performed separately (Polymerase). Chain Reaction, also known as polymerase chain reaction).
  • the amplification system was 25ul, specifically 2 ⁇ KAPA Mix, 12.5ul; 10uM primers each 0.5ul; template 1ul; added water to 25ul; amplification conditions: pre-denaturation at 95°C for 3 minutes; 98°C denaturation for 20 seconds, 60 Anneal at -72 ° C for 15 seconds, 72 ° C extension, the extension time is calculated according to 30 seconds per kb, the number of cycles is 29-35; 72 ° C extension for 10 minutes.
  • Each gene sequence DGAT1 is obtained as shown in SEQ ID No: 83, SCD is represented by SEQ ID No: 84, PLA2-1 is represented by SEQ ID No: 85, PLA2-2 is represented by SEQ ID No: 86, and PLA2- 3, as shown in SEQ ID No: 87, PLA2-4 as shown in SEQ ID No: 88, PLA2-5 as shown in SEQ ID No: 89, PLA2-6 as shown in SEQ ID No: 90, and ylGSR as SEQ ID No: 91 and ylGPO are shown as SEQ ID No: 92.
  • the gene encoding the exogenous fatty acid desaturase D9DMB is shown in SEQ ID No: 100
  • CeFAT6 is shown in SEQ ID No: 101
  • MaOLE2 is shown in SEQ ID No: 102
  • AtADS1 is shown in SEQ ID No: 103.
  • AtADS2 is represented by SEQ ID No: 104
  • EcAldH is represented by SEQ ID No: 105
  • ScZwf is represented by SEQ ID No. 106, and is obtained by gene synthesis by Wuxi Qinglan Biotechnology Co., Ltd.
  • the above sequence can be subjected to PCR amplification using SEQ ID No: 35-48 as a primer sequence.
  • the genomic DNA of Y. lipolytica was extracted by the above CTAB method, and the genomic DNA of Y. lipolytica was used as a template, and SEQ ID No: 49-58 was used as a primer sequence, and amplified by KAPA HiFi high-fidelity DNA polymerase.
  • the promoter was separately subjected to PCR amplification.
  • the amplification system was 25 ul, and the amplification conditions and the amount of the amplification system were the same as those described in the above step 1).
  • the promoter gene GPAT was obtained as shown in SEQ ID No: 107, TEF1 as shown in SEQ ID No: 108, EXP1 as shown in SEQ ID No: 109, EYK1 as shown in SEQ ID No: 110, and GPD as SEQ ID No: 111 is shown.
  • the genomic DNA of Y. lipolytica was used as a template, and the SEQ ID No: 59-64 was used as a primer sequence, and the terminator was amplified by KAPA HiFi high-fidelity DNA polymerase, and PCR amplification was performed separately. .
  • the amplification system was 25 ul, and the amplification conditions and the amount of the amplification system were the same as those described in the above step 1).
  • the terminator sequence XPR2 was obtained as shown in SEQ ID No: 112, LIP1t is shown in SEQ ID No: 113, and PQX3t is shown in SEQ ID No: 114.
  • the hygromycin (Hgr) resistance screening marker gene was obtained by PCR amplification using plasmid pAG32 (purchased from EUROSCARF) as a template and SEQ ID No: 65-66 as a primer sequence using KAPA HiFi high-fidelity DNA polymerase.
  • the amplification system was 25 ul, and the amplification conditions and the amount of the amplification system were the same as those described in the above step 1).
  • the hygromycin (Hgr) resistance screening marker gene was obtained by PCR amplification as shown in SEQ ID No: 115.
  • LEU leucine synthesis gene
  • UUA3 uracil synthase key gene
  • genomic DNA of Y. lipolytica was extracted by the above CTAB method to obtain genomic DNA as a template, and SEQ ID No: 67-70 was used as a primer sequence, and PCR amplification was carried out using KAPA HiFi high-fidelity DNA polymerase.
  • the amplification system was 25 ul, and the amplification conditions and the amount of the amplification system were the same as those described in the above step 1).
  • PCR amplification yielded the gene LEU as SEQ ID No: 116 and the gene URA3 as SEQ ID No: 117, respectively.
  • SEQ ID No: 71-78 was used as the primer sequence, and KAPA HiFi high-fidelity DNA polymerase, 25 ul reaction system, PCR amplification were used.
  • the homologous recombination fragment pex10-up was obtained as shown in SEQ ID No: 118 and pex10-dow as shown in SEQ ID No: 119.
  • the amplification conditions were: pre-denaturation at 95 ° C for 3 minutes; denaturation at 98 ° C for 20 seconds, annealing at 60-72 ° C for 15 seconds, extension at 72 ° C, extension time according to 15 seconds per kb, cycle number 29-35; 72 ° C extension 6 minute.
  • plasmid pYLEX1 purchased from Yeastern Biotech Company, Taiwan
  • KAPA HiFi high-fidelity DNA polymerase 25 ul reaction system
  • PCR amplification of plasmid basic skeleton fragments target genes, promoters, terminators
  • the marker gene was screened using the Gibson Assembly method (Gibson DG. Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides. Nucleic Acids Research.
  • kit purchased from New England Biolabs assembled the pYLEX1 plasmid backbone with the gene of interest, promoter, terminator and selection marker gene into a complete plasmid (see Table 1).
  • Each plasmid contains a selection marker gene, one to three genes of interest, each of which carries a promoter and a terminator.
  • plasmid pYLEX1 as a basic skeleton, using pYLEX1 and Y. lipolytica genomic DNA as templates, and SEQ ID No: 51-52, SEQ ID No: 59-60, SEQ ID No: 79-80 as primer sequences, respectively.
  • the plasmid backbone fragment, the TEF promoter fragment and the XPR2 terminator fragment were amplified by PCR, and the three DNA fragments were assembled by Gibson Assembly method to obtain plasmid pYLEX1-P TEF1 -T XPR2 .
  • the DNA fragment concentration was controlled at 100-200 ng per reaction, the reaction system was 10 ⁇ l, and the assembly conditions were 50 ° C for 1 hour. After the reaction, 2 ⁇ l of transformed DH5 ⁇ competent cells (purchased from TransGen Biotech) were taken, and positive clones were obtained by colony PCR and DNA sequencing verification.
  • the construction of the plasmid described in Table 1 was similar to the assembly of the pDGAT1 plasmid by integrating the gene of interest, promoter, terminator and selection marker gene into a plasmid using the Gibson Assembly method.
  • Targeting genes expressed in the endoplasmic reticulum, peroxisomes and mitochondria requires the addition of the endoplasmic reticulum retention signal peptide KDEL, the peroxisome targeting signal peptide SKL and the mitochondrial targeting signal at the 3' end of the corresponding gene.
  • Peptide CoxIV (MLSLRQSIRFFKPATRTLCSSRYLL).
  • the marker in Table 1 is a marker.
  • Example 2 Construction of an engineered strain of Yarrowia yeast
  • the plasmids pDS, pAB, pCgKCS, pCgKCS ER pCgKCS PTS , pCgKCS MTS , pC ER C MTS , pMCSD, pCB, pPLA2-1, pPLA2- described in Table 1 were digested with NotI endonuclease (purchased from Thermo Fisher Scientific), respectively. 2.
  • the specific digestion system was: 10 ⁇ FD Green Buffer, 2 ul; NotI, 1 ul; plasmid, ⁇ 1 ug; ddH 2 O was added to 20 ul.
  • the digestion product was purified and recovered by using the Cycle Pure Kit (purchased from OMEGA bio-tek). The recovery procedure was as follows: the digestion product was added to 4-5 volumes of buffer CP; after mixing, it was transferred to a DNA adsorption column and centrifuged at 13,000 g at room temperature.
  • the PCR verification method was as follows: the DNA of the corresponding Y. lipolytica transformant was used as a template, and the corresponding primers were used for PCR amplification.
  • the amplification system was 25 ul, specifically 2 ⁇ Taq Mix, 12.5 ul; 10 uM primers each 0.5 Ul; template 1ul; add water to 25ul; amplification conditions: 94 ° C pre-denaturation 5 minutes; 94 ° C denaturation 30 seconds, 60-72 ° C annealing 30 seconds, 72 ° C extension, extension time according to 1 minute per kb calculation, cycle The number was 30; the extension was carried out for 10 minutes at 72 ° C, and 1% agarose gel electrophoresis was performed after the amplification.
  • the method for verifying gene expression levels by RT-PCR was as follows: Total RNA of the above strains was extracted by TRizol method, and the concentration was measured by a nucleic acid analyzer ND-1000, and 1% agarose gel electrophoresis was used to detect whether the RNA was degraded. Real-time PCR specific primers were designed based on each gene sequence. The detected RNA was reverse transcribed into cDNA and subjected to Real time-PCR. The specific steps of the Real time-PCR method can be found in Biotium. Master Mixes for qPCR Quantitative Assay Kit. Real time-PCR amplification was performed by the Light Cycler 480 real-time PCR system manufactured by Roche, USA. Three replicate wells were made for each sample, and three replicates of different samples were performed, and the Actin gene of Yarrowia yeast was used as an internal reference. Finally, the relative expression amount was calculated according to the 2 - ⁇ Ct method.
  • the strain YL1 was obtained by transforming the expression cassettes DGAT1-SCD-Hgr, AtFAE1-BtFAE1-LEU and CgKCS-URA derived from the plasmids pDS, pBA and pCgKCS, that is, the overexpressed genes DGAT1 and SCD were overexpressed.
  • YL1 YL2 on the basis of the strain, transformed by the expression cassette is obtained MaLCE1-CgKCS ER -DGAT1 ER -SCD ER -URA pMCSD derived plasmid, i.e., through the expression of the gene further strain YL2 MaLCE1 on the basis YL1, CgKCS ER, DGAT1 ER And SCD ER .
  • the strain YL2-1 was obtained on the basis of YL2 by transformation of the expression cassette CgKCS ER- URA derived from the plasmid pCgKCS ER , that is, the strain YL2-1 further overexpressed the gene CgKCS ER on the basis of YL2.
  • strain YL2-2 was transformed from plasmid pCB-derived expression cassette CgKCS ER -BtFAE1 ER -CgKCS PTS -BtFAE1 PTS -URA, ie strain YL2-2 was further differentiated in different subcellular cells based on YL2 The genes CgKCS and BtFAE1 were further overexpressed (endoplasmic reticulum, peroxisome).
  • strain YL2-3 was transformed from the expression cassette MaCCE1-CgKCS ER -DGAT1 ER -SCD ER -URA derived from plasmid pMCSD, ie strain YL2-3 further overexpressed gene MaLCE1, CgKCS on the basis of YL2 ER , DGAT1 ER and SCD ER .
  • the strain is deposited in the General Microbial Culture Collection and Management Center of China, and the deposit number is CGMCC NO.15309.
  • strain YL2-4 was transformed from the expression cassettes EcAldH-URA, ScZwf-URA, ylGSR-URA and ylGPO-URA derived from plasmids pEcAldH, pScZwf, pylGSR and pylGPO, ie strain YL2-4 in YL2 Further overexpressed genes EcAldH, ScZwf, ylGSR and ylGPO.
  • the strain YL4-1 was obtained on the basis of YL1 by transformation of the expression cassette PLA2-1-URA derived from the plasmid pPLA2-1, that is, the strain YL4-1 further overexpressed the gene PLA2-1 on the basis of YL1.
  • the strain YL4-2 was obtained on the basis of YL1 by transformation of the expression cassette PLA2-2-URA derived from the plasmid pPLA2-2, that is, the strain YL4-2 further overexpressed the gene PLA2-2 on the basis of YL1.
  • the strain YL4-3 was obtained on the basis of YL1 by transformation of the expression cassette PLA2-3-URA derived from the plasmid pPLA2-3, that is, the strain YL4-3 further overexpressed the gene PLA2-3 on the basis of YL1.
  • the strain YL4-4 was obtained on the basis of YL1 by transformation of the expression cassette PLA2-4-URA derived from the plasmid pPLA2-4, that is, the strain YL4-4 further overexpressed the gene PLA2-4 on the basis of YL1.
  • the strain YL4-5 was obtained on the basis of YL1 by transformation of the expression cassette PLA2-5-URA derived from the plasmid pPLA2-5, that is, the strain YL4-5 further overexpressed the gene PLA2-5 on the basis of YL1.
  • the strain YL4-6 was obtained on the basis of YL1 by transformation of the expression cassette PLA2-6-URA derived from the plasmid pPLA2-6, that is, the strain YL4-6 further overexpressed the gene PLA2-6 on the basis of YL1.
  • Strain YL5 was obtained by transformation of expression cassettes gELOVL6-URA and MaOLE2-URA derived from plasmids pgELOVL6 and pMaOLE2, i.e., overexpressing the foreign genes gELOVL6 and MaOLE2.
  • the strain YL6 was obtained on the basis of YL5 by transformation of the expression cassette CgKCS ER- CgKCS MTS- URA derived from the plasmid pC ER C MTS , that is, the strain YL6 further overexpressed the genes CgKCS ER and CgKCS MTS on the basis of YL5.
  • the strain YL7 was obtained on the basis of YL1 by transformation of the expression cassette AtADS1-URA derived from the plasmid pAtADS1, that is, the strain YL7 further overexpressed the gene AtADS1 on the basis of YL1.
  • the strain YL8 was obtained on the basis of YL1 by transformation of the expression cassette AtADS2-URA derived from the plasmid pAtADS2, that is, the strain YL8 further overexpressed the gene AtADS2 on the basis of YL1.
  • the strain YL9 was obtained by transformation of the expression cassette ⁇ pex10-URA derived from the plasmid p ⁇ pex10, that is, the knockout gene pex10.
  • the strain YL10 was obtained on the basis of YL9 by transformation of the expression cassette CgKCS PTS- URA derived from the plasmid pCgKCS PTS , that is, the strain YL10 further overexpressed the gene CgKCS PTS on the basis of YL9.
  • the strain YL11 YL10 basis, transformed by the expression cassette is obtained MaLCE1-CgKCS ER -DGAT1 ER -SCD ER -URA pMCSD derived plasmid, i.e., through the expression of the gene further strain YL11 MaLCE1 on the basis YL10, CgKCS ER, DGAT1 ER And SCD ER .
  • Example 3 Culture of strains to produce nervonic acid
  • the strains po1g and YL2-3 were activated on YPD solid plates, respectively, and cultured at 28 ° C for 1 day. Single colonies were picked and inoculated into 250 ml shake flasks containing 50 ml of YPD medium, and cultured at 28 ° C for 1 day as a seed culture solution.
  • the seed culture solution was separately inoculated into a 250 ml shake flask containing 50 ml of YNB medium to have an initial OD 600 of 0.2, and cultured at 28 ° C for 6 days, and was used.
  • the composition of YNB medium is YNB1.7g/L, glucose 80g/L, yeast extract 1.5g/L, uracil 20mg/L, leucine 100mg/L.
  • the seed culture medium cultured in the above method was inoculated into a 250 ml shake flask containing 50 ml of induction medium, and the initial OD 600 was 0.2, and cultured at 28 ° C for 6 days, and was used.
  • the induction medium is YNB containing 10g/L glucose, and after the culture for 1d, the glucose is basically consumed completely, and then erythritol is added as a carbon source, and the glucose is further cultured for 2 days.
  • the strain YL2-3 obtained above was activated as a seed liquid, and 3 L of medium YNBF was added to a 5 L fermentor, and the dissolved oxygen was controlled to be greater than 20% (growth period: 0-48 h) and 0-5% (stability period).
  • the pH during the fermentation was constantly controlled at 5.5 until the end of the fermentation.
  • the temperature was controlled at 28 ° C for 6 days.
  • the composition of the YNBF medium was 3.4 g/L yeast nitrogen source without amino acid and ammonium sulfate, 150 g/L glucose, 2 g/L yeast extract and 8.8 g/L ammonium sulfate.
  • the inoculation amount is 10%.
  • the total lipids of the starting strain and the engineered strain were cultured for 6 days by the above-mentioned acid heat method.
  • the position of the nervic acid in the TAG was detected by lipase digestion.
  • the specific steps are as follows: 10 mg of fat and 10 mg of immobilized 1,3 specific lipase were added to 3 ml of methanol solution, and reacted at 30 ° C for 8 h.
  • the fatty acid methyl ester and 2-MAG were purified by TLC plate, and gas phase detection showed that only the nervonic acid was present in the free fatty acid layer, that is, the nervonic acid was located at the sn-1, 3 position of TAG (Fig. 4).
  • methyl nerate content was determined by GC method.
  • the column was HP-5 (30 m x 0.32 mm x 0.25 ⁇ m) using an Agilent 7890B-GC instrument.
  • Injection temperature 250 ° C; detector temperature: 250 ° C; injection volume: 1 ⁇ L; initial column temperature of 140 ° C, for 1 min, 10 ° C / min to 180 ° C, for 2 min, 5 ° C / min to The temperature was maintained at 210 ° C for 4 min and 5 ° C / min to 250 ° C for 4 min.
  • the ratio of other fatty acids in strain YL2-3 to oil and fat was: C16:0 was 5.3%, C16:1 was 10.9%, C18:0 was 1.5%, C18:1 was 28.7%, and C18:2 was 9.1%. , C24:0 is 2.8%.
  • strain YL2-3 was amplified in a 500 L fermentor.
  • the activated seed solution was inoculated at a seeding rate of 3%, the culture temperature was 28 ° C, the aeration rate was 5-8 L/min, the stirring speed was 300 r/min, and the fermentation pH (5.5) was adjusted with a 3 M NaOH solution.
  • a total of three batches of fermentation were carried out.
  • the results showed that the biomass of strain YL2-3 was 126.56 g/L on average, the content of nervonic acid was 39.6% of the total fatty acid content, and the fat content was about 39.3 g/L.

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Abstract

本发明公开了生产神经酸的工程化酵母菌株，所述酵母菌株过表达脂肪酸延长酶、去饱和酶、甘油二酯酰基转移酶等长链不饱和脂肪酸合成过程所需酶的相关基因，并且任选地进一步对菌株的甘油三酯合成与分解途径、鞘磷脂合成与分解途径、油脂亚细胞水平合成与分解途径以及氧化还原平衡途径进行调控。该重组酵母菌株能够产生微生物油，制备得到神经酸的含量占总脂肪酸含量的39.6％。

Description

生产神经酸的重组酵母菌株及其应用 技术领域
本发明属于生物技术领域。更具体地，本发明涉及工程化重组酵母菌株，其能够以高浓度有效地制备神经酸(顺-15-二十四碳单烯酸，别名鲨鱼酸，C24:1,Δ15)。
背景技术
不饱和脂肪酸多为人体必需脂肪酸，具有调节血脂、清理血栓、补脑健脑、缓解炎症等作用，主要包括单不饱和脂肪酸和多不饱和脂肪酸。其中，超长链单不饱和脂肪酸(Very long chain monounsaturated fatty acid,VLCMFA)为主碳链上碳原子数大于18，并且仅有一个双键的不饱和脂肪酸，常见的有鳕油酸(Eicosenoic acid,C20:1Δ11)、芥酸(Erucic acid,C22:1Δ13)、神经酸(Nervonic acid,C24:1Δ15)和西门木烯酸(Ximenynic acid,C26:1Δ17)。超长链单不饱和脂肪酸具有独特的药效、保健功效和工业用途等，但是相比多不饱和脂肪酸，超长链单不饱和脂肪酸的应用推广亟待加强。
神经酸(顺-15-二十四碳单烯酸，别名鲨鱼酸，C24:1Δ15)是与人类健康关系密切的超长链单不饱和脂肪酸。神经酸主要以鞘糖脂和鞘磷脂的形式存在于动物大脑白质和髓鞘的神经纤维中，是生物膜的重要组成成分。神经酸在医学和保健方面具有重要的作用，可用于治疗多发性硬化症等神经紊乱病症。此外，研究表明神经酸对神经系统的发育具有促进作用，尤其是在婴幼儿脑神经细胞和视神经细胞生长与发育过程具有重要的作用。人体所需的神经酸主要依靠外源摄取。近年，随着对神经酸医学和保健功效认识的不断深入，其资源的开发和利用价值凸显，产品需求正逐步扩大。
神经酸有多种天然来源。目前发现的富含神经酸的动植物及微生物有鲨鱼、蒜头果、元宝枫、碎米芥、微藻、少数霉菌等。蒜头果是自然界富含神经酸的中国特有植物，并且蒜头果种仁含油量在64.5％左右，其中神经酸含量高达43.2％，但是蒜头果的种植困难。元宝枫籽油的神经酸含量约5.8％，为目前神经酸的主要来源。元宝枫生长缓慢，人工种植元宝枫4-6年挂果，8-10年才进入盛果期。因此，由元宝枫提取神经酸存在生长周期长、原料供应受季节约束、产量低等弊端。产油微生物能够合成高细胞含量的脂肪酸，经过遗传工程改造， 可形成与植物油脂组成相似的微生物油脂。
解脂耶鲁维亚酵母(Yarrowia lipolytica)作为一种产油微生物，其油脂积累可占细胞干重的44～70％，并具有生长速率快、细胞发酵密度高、碳源利用范围广、遗传操作简单等特点，具有发展为神经酸细胞工厂的极大潜力。解脂耶鲁维亚酵母中大多数的脂肪酸为C16和C18脂肪酸，由于缺少合成超长链单不饱和脂肪酸所必需的碳链延长酶和脂肪酸去饱和酶，野生菌株无法合成神经酸。前期研究通过基因工程手段将脂肪酸延长酶(AtFAE1，BtFAE1和CgKCS)、去饱和酶(SCD)以及甘油二酯酰基转移酶(DGAT1)导入解脂耶鲁维亚酵母，构建的重组酵母细胞可产生神经酸，但其含量仅占细胞中总油脂含量的1.5％，难以满足工业需求。
发明内容
本发明为解决上述技术问题，通过过表达脂肪酸延长酶、去饱和酶、甘油二酯酰基转移酶等相关基因，任选地对重组酵母菌株的甘油三酯合成与分解途径、鞘磷脂合成与分解途径、油脂亚细胞水平合成与分解途径以及氧化还原平衡途径进行调控，使得构建的重组酵母菌株生产神经酸的能力大大提高，经发酵优化后，提取获得神经酸的含量占总脂肪酸的含量为39.6％。具体技术方案如下：
方案一、本发明提供了一种重组酵母菌株，其特征在于，过表达：
(a)一种编码Δ9去饱和酶的基因；
(b)至少四种编码脂肪酸延长酶的基因；
(c)一种编码甘油二酯酰基转移酶的基因；
(d)一种编码靶向内质网的脂肪酸延长酶的基因；
(e)一种编码靶向内质网的甘油二酯酰基转移酶的基因；和/或
(f)一种编码靶向内质网的Δ9去饱和酶基因。
所述重组酵母菌株中过表达脂肪酸合成表达模块，具体包括神经酸生产相关的脂肪酸延长酶和去饱和酶基因。其中所述脂肪酸延长酶基因可选自，但不限于高山被孢霉C16延长酶基因MaLCE1，如SEQ ID No：93所示；拟南芥AtFAE1，如SEQ ID No：94所示；非洲芥菜BtFAE1，如SEQ ID No：95所示；碎米芥CgKCS，如SEQ ID No：96所示；大鼠脂肪酸延长酶2基因rELO2，如SEQ ID No：97所示；微小隐孢子虫长链脂肪酸延长酶基因CpLCE，如SEQ ID No：98所示；羊脂肪酸延长酶6基因gELOVL6，如SEQ ID No：99所示。其中所述去 饱和酶基因可选自，但不限于解脂耶鲁维亚酵母SCD，如SEQ ID No：84所示；刺孢小克银汉霉Δ9脂肪酸去饱和酶基因D9DMB，如SEQ ID No：100所示；线虫Δ9脂肪酸去饱和酶基因CeFAT6，如SEQ ID No：101所示；高山被孢霉Δ9脂肪酸去饱和酶基因MaOLE2，如SEQ ID No：102所示；拟南芥AtADS1，如SEQ ID No：103所示；拟南芥AtADS2，如SEQ ID No：104所示。
同时，所述重组酵母菌株中过表达甘油三酯合成模块，具体指二酰基甘油酰基转移酶基因，是催化三酰甘油(TAG)合成最后一步反应的酶，也是TAG合成过程中唯一的关键酶和限速酶，提高酵母细胞中甘油二酯酰基转移酶的表达量可以提高细胞内油脂的含量。
同时，所述重组酵母菌株中过表达酵母油脂合成与分解亚细胞水平调节模块，具体是指内质网水平的调控，即在相应基因的3'末端添加内质网滞留信号肽KDEL。
优选的，方案一所述酵母菌株为解脂耶鲁维亚酵母。
优选的，所述编码Δ9去饱和酶的基因为解脂耶鲁维亚酵母SCD基因，其核苷酸序列如SEQ ID No：84所示；
优选的，所述的四种编码脂肪酸延长酶基因分别为高山被孢霉C16/18延长酶基因MaLCE1，其核苷酸序列如SEQ ID No：93所示；拟南芥AtFAE1基因，其核苷酸序列如SEQ ID No：94所示；非洲芥菜BtFAE1基因，其核苷酸序列如SEQ ID No：95所示；碎米芥CgKCS基因，其核苷酸序列如SEQ ID No：96所示；
优选的，所述编码甘油二酯酰基转移酶的基因为解脂耶鲁维亚酵母DGAT1基因，其核苷酸序列如SEQ ID No：83所示；
优选的，所述编码靶向内质网的脂肪酸延长酶的基因为带有靶向内质网信号肽编码序列的碎米芥CgKCS ER基因，其核苷酸序列如SEQ ID No：121所示；
优选的，所述编码靶向内质网的甘油二酯酰基转移酶的基因为带有靶向内质网信号肽编码序列的解脂耶鲁维亚酵母DGAT1 ER基因，其核苷酸序列如SEQ ID No：122所示；
优选的，所述编码靶向内质网的Δ9去饱和酶的基因为带有靶向内质网信号肽编码序列的解脂耶鲁维亚酵母SCD ER基因，其核苷酸序列如SEQ ID No：123所示。
在另一个实施方案中，本发明提供用于生产神经酸的重组酵母菌株，所述菌株在方案一所涉及菌株的基础上可进一步过表达：
(a)两种编码靶向内质网的脂肪酸延长酶的基因；和/或
(b)两种编码靶向过氧化物酶体的脂肪酸延长酶的基因。
优选的，所述两种编码靶向内质网的脂肪酸延长酶的基因分别为带有靶向内质网信号肽编码序列的碎米芥CgKCS ER基因，其核苷酸序列如SEQ ID No：121所示；带有靶向内质网信号肽编码序列的非洲芥菜BtFAE1 ER基因，其核苷酸序列如SEQ ID No：124所示；
优选的，所述两种编码靶向过氧化物酶体的脂肪酸延长酶的基因分别为带有靶向过氧化物酶体信号肽编码序列的碎米芥CgKCS PTS基因，其核苷酸序列如SEQ ID No：125所示；带有靶向过氧化物酶体信号肽编码序列的非洲芥菜BtFAE1 PTS基因，其核苷酸序列如SEQ ID No：126所示。
在另一个实施方案中，本发明提供用于生产神经酸的重组酵母菌株，所述菌株在方案一所涉及菌株的基础上可进一步过表达：
(a)一种编码醛脱氢酶的基因；
(b)一种编码葡萄糖-6-磷酸脱氢酶的基因；
(c)一种编码谷胱甘肽二硫化物还原酶的基因；和/或
(d)一种编码谷胱甘肽过氧化物酶的基因。
所述重组酵母菌种中进一步包括氧化还原平衡的调控模块，涉及维持神经酸合成过程中还原力NADPH再生和氧化应激防御相关的基因。
所述醛脱氢酶基因优选为大肠杆菌EcAldH基因，其核苷酸序列如SEQ ID No：105所示；葡萄糖-6-磷酸脱氢酶基因优选为酿酒酵母ScZwf基因，其核苷酸序列如SEQ ID No：106所示，谷胱甘肽二硫化物还原酶基因优选为解脂耶鲁维亚酵母ylGSR基因，其核苷酸序列如SEQ ID No：91所示，谷胱甘肽过氧化物酶基因优选为解脂耶鲁维亚酵母ylGPO基因，其核苷酸序列如SEQ ID No：92所示。
方案二、本发明提供了一种重组酵母菌株，其特征在于，过表达：
(a)一种编码Δ9去饱和酶的基因；
(b)至少三种编码脂肪酸延长酶的基因；
(c)一种编码甘油二酯酰基转移酶的基因；和/或
(d)一种编码磷脂酶A2的基因。
所述重组酵母菌株包括鞘磷脂合成与分解的调控模块，具体涉及磷脂酶A2(phospholipaseA2，PLA2)基因，PLA2是一种能催化磷脂甘油分子上二位酰 基的水解酶，其过表达可以增加神经酸合成过程中底物的供应。所述编码磷脂酶A2基因可选自，但不限于PLA2-1如SEQ ID No：85所示，PLA2-2如SEQ ID No：86所示，PLA2-3如SEQ ID No：87所示，PLA2-4如SEQ ID No：88所示，PLA2-5如SEQ ID No：89所示，PLA2-6如SEQ ID No：90所示。
优选的，所述编码Δ9去饱和酶基因为解脂耶鲁维亚酵母SCD基因，其核苷酸序列如SEQ ID No：84所示；
优选的，所述三种编码脂肪酸延长酶的基因分别为拟南芥AtFAE1基因，其核苷酸序列如SEQ ID No：94所示、非洲芥菜BtFAE1基因，其核苷酸序列如SEQ ID No：95所示、碎米芥CgKCS基因，其核苷酸序列如SEQ ID No：96所示；
优选的，所述编码甘油二酯酰基转移酶基因为解脂耶鲁维亚酵母DGAT1基因，其核苷酸序列如SEQ ID No：83所示。
方案三、本发明提供了一种重组酵母菌株，其特征在于，过表达：
(a)一种编码靶向过氧化物酶体的脂肪酸延长酶的基因；
(b)一种编码脂肪酸延长酶的基因；
(c)一种编码靶向内质网的脂肪酸延长酶的基因；
(d)一种编码靶向内质网的甘油二酯酰基转移酶的基因；和/或
(e)一种编码靶向内质网的Δ9去饱和酶基因。
优选的，所述酵母菌株优选为解脂耶鲁维亚酵母；
优选的，所述靶向过氧化物酶体的脂肪酸延长酶基因为带有靶向过氧化物酶体信号肽编码序列的碎米芥CgKCS PTS基因，其核苷酸序列如SEQ ID No：125所示；
优选的，所述脂肪酸延长酶基因为高山被孢霉C16/18延长酶基因MaLCE1基因，其核苷酸序列如SEQ ID No：93所示；
优选的，所述靶向内质网的脂肪酸延长酶的基因为带有靶向内质网信号肽编码序列的碎米芥CgKCS ER，其核苷选序列如SEQ ID No：121所示；
优选的，所述靶向内质网的甘油二酯酰基转移酶基因为带有靶向内质网信号肽编码序列的解脂耶鲁维亚酵母DGAT1 ER基因，其核苷酸序列如SEQ ID No：122所示；
优选的，所述靶向内质网的Δ9去饱和酶基因为带有靶向内质网信号肽编码序列的解脂耶鲁维亚酵母SCD ER基因，其核苷酸序列如SEQ ID No：123所示。
方案四、本发明提供了一种重组酵母菌株，其特征在于，过表达：
(a)一种编码Δ9去饱和酶的基因；
(b)一种编码脂肪酸延长酶的基因；
(c)一种编码靶向内质网的脂肪酸延长酶的基因；和/或
(d)一种编码靶向线粒体的脂肪酸延长酶的基因。
优选的，所述酵母菌株优选为解脂耶鲁维亚酵母；
优选的，所述编码Δ9去饱和酶基因为高山被孢霉Δ9脂肪酸去饱和酶MaOLE2基因，其核苷酸序列如SEQ ID No：102所示；
优选的，所述编码脂肪酸延长酶为羊脂肪酸延长酶6gELOVL6基因，其核苷酸序列如SEQ ID No：99所示；
优选的，所述编码靶向内质网的脂肪酸延长酶基因为带有靶向内质网信号肽编码序列的碎米芥CgKCS ER，其核苷酸序列如SEQ ID No：121所示；
优选的，所述编码靶向线粒体的脂肪酸延长酶基因为带有靶向线粒体信号肽编码序列的碎米芥CgKCS MTS，其核苷酸序列如SEQ ID No：127所示。
方案五、本发明提供了一种重组酵母菌株，其特征在于，过表达：
(a)两种编码Δ9去饱和酶的基因；
(b)三种编码脂肪酸延长酶的基因；和/或
(c)一种编码甘油二酯酰基转移酶的基因。
优选的，所述酵母菌株为解脂耶鲁维亚酵母；
优选的，所述两种编码Δ9去饱和酶基因分别为解脂耶鲁维亚酵母SCD基因，其核苷酸序列如SEQ ID No：84所示；拟南芥AtADS1基因，其核苷酸序列如SEQ ID No：103所示；或者所述两种编码Δ9去饱和酶基因分别为解脂耶鲁维亚酵母SCD基因，其核苷酸序列如SEQ ID No：84所示；拟南芥AtADS2基因，其核苷酸序列如SEQ ID No：104所示；
优选的，所述三种编码脂肪酸延长酶基因分别为拟南芥AtFAE1基因，其核苷酸序列如SEQ ID No：94所示；非洲芥菜BtFAE1基因，其核苷酸序列如SEQ ID No：95所示；碎米芥CgKCS基因，其核苷酸序列如SEQ ID No：96所示；
优选的，所述编码甘油二酯酰基转移酶为解脂耶鲁维亚酵母DGAT1如SEQ ID No：83所示。
方案六、本发明提供了一种重组酵母菌株，其特征在于，所述菌株的过氧 化物酶体生物发生因子10的表达被下调，并进一步过表达：
(a)一种编码靶向过氧化物酶体的脂肪酸延长酶的基因；
(b)一种编码脂肪酸延长酶的基因；
(c)一种编码靶向内质网的脂肪酸延长酶的基因；
(d)一种编码靶向内质网的甘油二酯酰基转移酶的基因；和/或
(e)一种编码靶向内质网的Δ9去饱和酶的基因。
所述重组酵母菌株中包括甘油三酯分解模块，具体涉及过氧化物酶体生物发生因子10基因敲除模块，该基因的敲除可减少长链脂肪酸的分解。
优选的，所述酵母菌株为解脂耶鲁维亚酵母；
优选的，所述表达下调的过氧化物酶体生物发生因子10为pex10基因，其核苷酸序列如SEQ ID No：120所示；
优选的，所述编码靶向过氧化物酶体的脂肪酸延长酶的基因为带有靶向过氧化物酶体信号肽编码序列的碎米芥CgKCS PTS基因，其核苷酸序列如SEQ ID No：125所示；
优选的，所述编码脂肪酸延长酶的基因为高山被孢霉C16/18延长酶MaLCE1基因，其核苷酸序列如SEQ ID No：93所示；
优选的，所述编码靶向内质网的脂肪酸延长酶的基因为带有靶向内质网信号肽编码序列的碎米芥CgKCS ER基因，其核苷酸序列如SEQ ID No：121所示；
优选的，所述编码靶向内质网的甘油二酯酰基转移酶的基因为带有靶向内质网信号肽编码序列的解脂耶鲁维亚酵母DGAT1 ER基因，其核苷酸序列如SEQ ID No：122所示；
优选的，所述编码靶向内质网的Δ9去饱和酶基因为带有靶向内质网信号肽编码序列的解脂耶鲁维亚酵母SCD ER基因，其核苷酸序列如SEQ ID No：123所示。
优选的，所述酵母为解脂耶鲁维亚酵母。
本发明提供了利用上述方案中构建的任一重组酵母菌株制备微生物油或神经酸的应用。具体包括，但不限于含有微生物油或神经酸的婴儿代乳品、功能性食物、医疗食物、医疗营养品、饮食补充剂、药物组合物、动物饲料、和个人护理产品等。
本发明提供了利用上述技术方案中构建的任一重组酵母菌株制备微生物 油和/或神经酸的方法，具体包括，但不限于微生物的培养、发酵条件的优化及控制。所述发酵条件的优化包括不同碳源、碳氮比和不同生长时期添加赤藓糖诱导的优化，所述发酵条件的控制包括，但不限于温度、pH、发酵时间、溶氧、补料方式等的控制。所述微生物油或/和神经酸的提取工艺包括，但不限于菌株的分离、破碎以及有机溶剂提取过程。
优选的，所述制备微生物油的方法，包括：
(a)培养本发明方案一、方案二、方案三、方案四、方案五和/或方案六中所述的任一重组酵母菌株，其中产生包含神经酸的微生物油；以及
(b)回收所述步骤(a)的微生物油。
优选的，所述制备神经酸的方法，包括：
(a)培养本发明方案一、方案二、方案三、方案四、方案五和/或方案六中所述的任一重组酵母菌株，产生微生物油；以及
(b)回收所述步骤(a)的微生物油，提取神经酸。
相比现有技术，本发明的有益效果：本发明所述方法涉及神经酸合成系统的代谢通路和发酵调控，获得优质的重组耶鲁维亚酵母菌株，微生物油的产量提高，制备得到神经酸的含量占总脂肪酸含量的39.6％，神经酸的浓度为16g/L，具有良好的工业应用前景。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为本发明实施例提供的神经酸合成策略图。
图2为本发明实施例提供的PCR鉴定酵母转化子。
A：构建菌株YL1时不同转化子的CgKCS基因的PCR验证结果。B：构建菌株YL2时不同转化子的MaLCE1基因的PCR验证结果。C：构建菌株YL2-1时不同转化子的CgKCS基因的PCR验证结果。D：构建菌株YL2-2时不同转化子的BtFAE1基因的PCR验证结果。E：构建菌株YL2-3时不同转化子的CgKCS基因的PCR验证结果。F：构建菌株YL2-4时不同转化子的ScZwf基因的PCR验证结 果。G：构建菌株YL3时不同转化子的CgKCS基因的PCR验证结果。H：构建菌株YL4-1时不同转化子的PLA2-1基因的PCR验证结果。I：构建菌株YL4-2时不同转化子的PLA2-2基因的PCR验证结果。J：构建菌株YL4-3时不同转化子的PLA2-3基因的PCR验证结果。K：构建菌株YL4-4时不同转化子的PLA2-4基因的PCR验证结果。L：构建菌株YL4-5时不同转化子的PLA2-5基因的PCR验证结果。M：构建菌株YL4-6时不同转化子的PLA2-6基因的PCR验证结果。N：构建菌株YL5时不同转化子的gELOVL6基因的PCR验证结果。O：构建菌株YL6时不同转化子的CgKCS基因的PCR验证结果。P：构建菌株YL7时不同转化子的AtADS1基因的PCR验证结果。Q：构建菌株YL8时不同转化子的AtADS2基因的PCR验证结果。R：构建菌株YL9时不同转化子的pex10基因的PCR验证结果。S：构建菌株YL10时不同转化子的CgKCS基因的PCR验证结果。T：构建菌株YL11时不同转化子的DGAT1基因的PCR验证结果。
图3为本发明实施例提供的YL2-3菌株中6种基因表达验证图。
图4为本发明实施例提供的应用TLC法分析神经酸在TAG中的位置特异性。
图5为本发明实施例提供的脂肪酸组分分析图。
图6为本发明实施例提供的摇瓶发酵条件下YL2-3菌株的生长曲线。
图7为本发明实施例提供的摇瓶发酵条件下不同菌株神经酸的含量图。
图8为本发明实施例提供的Po1g和YL2-4菌株中的细胞内醛水平。
图9为本发明实施例提供的发酵罐放大发酵条件下YL2-3菌株的脂肪酸组分。
具体实施方式
如下为本发明中所涉及术语的定义。
去饱和酶是指可以在一种或多种脂肪酸中去饱和(即引入双键)而产生所关注的脂肪酸或前体的多肽，使用Δ-系统从底物的羧基端计数来表示去饱和酶的活性。优选的，本发明所述去饱和酶为Δ9去饱和酶，它在分子羧基末端编号为9 th和10 th的碳原子之间去饱和脂肪酸，例如可催化底物脂肪酸硬脂酸(C18:0)生成油酸(C18:1)。
脂肪酸延长酶是指能延长脂肪酸碳链从而产生比该延长酶作用于其上的脂肪酸底物长2个碳原子的酸的多肽。优选的，本发明所述脂肪酸延长酶包括， 但不限于C16/18延长酶、C18/20延伸酶、C20/22延伸酶和C22/24延伸酶。通常，C16/18延长酶将利用C16底物，如高山被孢霉C16/18延长酶基因MaLCE1、羊脂肪酸延长酶6基因gELOVL6。一些延长酶具有广泛的特异性并因而单个延长酶可以催化几种延伸酶反应，如碎米芥CgKCS不仅对C18和C20脂肪酸具有底物特异性，而且可以继续以C22脂肪酸为底物，因此CgKCS具有C18/20、C20/22和C22/24延伸酶的活性。
甘油二酯酰基转移酶是催化三酰基甘油酯(TAG)合成最后一步反应的酶，也是TAG合成过程中唯一的关键酶和限速酶，提高酵母细胞中甘油二酯酰基转移酶的表达量可以提高细胞内油脂的含量。
内质网、过氧化物酶体以及线粒体是指普遍存在于所有真核细胞中的细胞器。酶靶向内质网、过氧化物酶体、线粒体进行表达，需要在相应基因的3'末端添加内质网滞留信号肽KDEL、过氧化物酶体靶向信号肽SKL和线粒体靶向信号肽CoxIV(MLSLRQSIRFFKPATRTLCSSRYLL)。
过氧化物酶体生物合成因子蛋白即过氧化物酶体蛋白、Pex蛋白，是指涉及过氧化物酶体生物合成的和/或参与通过ATP水解使细胞蛋白穿过过氧化物酶体膜的过程的蛋白。
表达盒是指包含如下序列的DNA片段：所选基因的编码序列和所选基因产物表达所需的位于编码序列之前(5’非编码序列)和之后(3’非编码序列)的调控序列。表达盒通常包含于载体中以有利于克隆和转化。可以将不同表达盒转化进包括细菌、酵母、植物和哺乳动物细胞在内的不同生物体中，只要能针对每种宿主使用正确的调控序列。表达盒通常由如下序列构成：
1)一种启动子序列，如GPAT、TEF1、EXP1、EYK1和GPD等；
2)一种编码序列；和
3)一种3’不翻译区域(即，终止子)，它在真核细胞中通常包含聚腺苷酸位点，如XPR2、LIP1t和PQX3t。
微生物油是指由酵母、霉菌、细菌和藻类等微生物在一定的条件下，利用碳水化合物、碳氢化合物或普通油脂作为碳源，在菌体内产生的大量油脂，其主要成分为甘油三酯和游离脂肪酸。优选的，本发明所述微生物油是由解脂耶鲁维亚酵母发酵产生的，经过代谢通路和发酵工艺的调控，获得优质菌株产微 生物油的能力大大提高，并且制备得到神经酸的量占总脂肪酸含量的39.6％，其他脂肪酸包括，但不限于棕榈油酸、油酸、亚油酸、十六烷酸、十八烷酸以及二十四烷酸等。
下面的具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如(Sambrook和Russell等人，分子克隆：实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)(2001)CSHL出版社)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例中使用的标准的重组DNA技术和分子克隆技术是本领域所熟知的(Ausubel，F.M等人，Current Protocols in Molecular Biology,Greene Publishing Assoc.和Wiley-Interscience出版)，适用于微生物生长的材料和方法是本领域熟知的。主要化学试剂购自KAPA Biosystems，New England Biolabs，TransGen Biotech，Thermo Fisher Scientific，OMEGA bio-tek等。
下面结合具体实施例对本发明进行详细说明。
本发明实施例提供的神经酸合成策略图见图1。
实施例1、质粒构建
1.1基因元件的克隆
1)基因DGAT1、SCD、PLA2-1、PLA2-2、PLA2-3、PLA2-4、PLA2-5、PLA2-6、ylGSR和ylGPO的获得：
将解脂耶鲁维亚酵母菌株(菌株号polg，购买自Yeastern Biotech Company,台湾)在YPD培养基(YPD培养基成分为葡萄糖20g/L、蛋白胨20g/L、酵母提取物10g/L))中进行培养，采用CTAB(hexadecyltrimethylammonium bromide,十六烷基三甲基溴化铵)法提取高纯度的基因组总DNA。将适量菌体加入液氮冷冻，研磨成粉，加入适量2×CTAB提取缓冲液(100mmol/L Tris-HCl，pH8.0，20mmol/L EDTA，1.4mol/L NaCl，2％(w/v)CTAB，40mmol/L巯基乙醇)，65℃ 保温10分钟，间歇摇动。随后加入等体积的氯仿/异戊醇，轻缓颠倒离心管混匀，室温下，12000rpm离心10min，将上清液转入另一离心管中，加入等体积的氯仿/异戊醇，颠倒离心管混匀，室温，12000rpm离心10分钟。将上层水相转入新的离心管中，加入等体积异丙醇混匀，室温放置30分钟。4000rpm离心10分钟，移去上清液，用70％乙醇漂洗，风干后加入20μl的TE缓冲液(100mM Tris-HCl，10mM EDTA pH8.0)溶解DNA，-20℃保存备用。采用Sau3AI对总DNA进行部分酶切，酶切后的DNA片段通过电泳进行纯化，采用胶回收纯化试剂盒回收大约2～6kb的片段，回收的DNA溶解于10mmol/L的Tris-HCl(pH8.0)中，置于-20℃保藏。
以解脂耶鲁维亚酵母的基因组DNA作为模板，以SEQ ID No：1-20作为引物序列，采用KAPA HiFi高保真DNA聚合酶(购自KAPA Biosystems)扩增基因，分别进行PCR扩增(Polymerase Chain Reaction，又称多聚酶链式反应)。扩增体系皆为25ul，具体为2×KAPA Mix,12.5ul；10uM引物各0.5ul；模板1ul；加水补至25ul；扩增条件为：95℃预变性3分钟；98℃变性20秒、60-72℃退火15秒、72℃延伸，延伸时间按照30秒每kb计算，循环数为29-35；72℃延伸10分钟。得到各基因序列DGAT1如SEQ ID No：83所示、SCD如SEQ ID No：84所示、PLA2-1如SEQ ID No：85所示、PLA2-2如SEQ ID No：86所示、PLA2-3如SEQ ID No：87所示、PLA2-4如SEQ ID No：88所示、PLA2-5如SEQ ID No：89所示、PLA2-6如SEQ ID No：90所示、ylGSR如SEQ ID No：91所示和ylGPO如SEQ ID No：92所示。
2)编码外源脂肪酸延长酶的基因MaLCE如SEQ ID No：93所示、AtFAE1如SEQ ID No：94所示、BtFAE1如SEQ ID No：95所示、CgKCS如SEQ ID No：96所示、rELO2如SEQ ID No：97所示、CpLCE如SEQ ID No：98所示、gELOVL6如SEQ ID No：99所示，均有由无锡青兰生物科技有限公司通过基因合成获得。以SEQ ID No：21-34作为引物序列可对上述序列进行PCR扩增。
3)编码外源脂肪酸去饱和酶的基因D9DMB如SEQ ID No：100所示、CeFAT6如SEQ ID No：101所示、MaOLE2如SEQ ID No：102所示、AtADS1如SEQ ID No：103所示、AtADS2如SEQ ID No：104所示、EcAldH如SEQ ID No：105所示和ScZwf如SEQ ID No：106所示，均有由无锡青兰生物科技有限公司 通过基因合成获得。以SEQ ID No：35-48作为引物序列可对上述序列进行PCR扩增。
1.2启动子和终止子元件的克隆
1)GPAT、TEF1、EXP1、EYK1和GPD基因启动子的克隆：
采用上述CTAB法提取解脂耶鲁维亚酵母的基因组DNA，以解脂耶鲁维亚酵母的基因组DNA作为模板，以SEQ ID No：49-58作为引物序列，采用KAPA HiFi高保真DNA聚合酶扩增启动子，分别进行PCR扩增。扩增体系皆为25ul，扩增条件以及扩增体系的用量与上述步骤1)中记载的一致。得到启动子基因GPAT如SEQ ID No：107所示、TEF1如SEQ ID No：108所示、EXP1如SEQ ID No：109所示、EYK1如SEQ ID No：110所示和GPD如SEQ ID No：111所示。
2)XPR2、LIP1t和PQX3t终止子的克隆：
与启动子的克隆类似，以解脂耶鲁维亚酵母的基因组DNA为模板，以SEQ ID No：59-64作为引物序列，采用KAPA HiFi高保真DNA聚合酶扩增终止子，分别进行PCR扩增。扩增体系皆为25ul，扩增条件以及扩增体系的用量与上述步骤1)中记载的一致。得到终止子序列XPR2如SEQ ID No：112所示、LIP1t如SEQ ID No：113所示和PQX3t如SEQ ID No：114所示。
1.3筛选标记基因元件的克隆
1)潮霉素抗性基因的克隆
潮霉素(Hgr)抗性筛选标记基因的获得是以质粒pAG32(购自EUROSCARF)为模板，以SEQ ID No：65-66作为引物序列，采用KAPA HiFi高保真DNA聚合酶进行PCR扩增。扩增体系为25ul，扩增条件以及扩增体系的用量与上述步骤1)中记载的一致。PCR扩增得到潮霉素(Hgr)抗性筛选标记基因如SEQ ID No：115所示。
2)亮氨酸合成基因(LEU)和尿嘧啶合成酶关键基因(URA3)的获得
采用上述CTAB法提取解脂耶鲁维亚酵母的基因组DNA，以获得基因组DNA为模板，以SEQ ID No：67-70作为引物序列，采用KAPA HiFi高保真DNA聚合酶进行PCR扩增。扩增体系为25ul，扩增条件以及扩增体系的用量与上述步骤1)中记载的一致。PCR扩增分别得到基因LEU如SEQ ID No：116和基因URA3如SEQ ID No：117。
1.4DNA同源重组片段的克隆
pex10低表达水平的调控，采用同源替换(Verbeke J,Beopoulos A,Nicaud JM.Efficient homologous recombination with short length flanking fragments in Ku70deficient Yarrowia lipolytica strains.Biotechnology Letters,2013,35(4):571-576.)的方法对基因进行敲除。根据解脂耶鲁维亚酵母基因组序列，查找pex10基因的DNA序列，选择目的基因的上下游序列(1000bp左右)。采用CTAB法提取解脂耶鲁维亚酵母的基因组DNA，以获得基因组DNA为模板，以SEQ ID No：71-78作为引物序列，采用KAPA HiFi高保真DNA聚合酶，25ul反应体系，PCR扩增分别得到同源重组片段pex10-up如SEQ ID No：118所示和pex10-dow如SEQ ID No：119所示。
扩增条件为：95℃预变性3分钟；98℃变性20秒、60-72℃退火15秒、72℃延伸，延伸时间按照15秒每kb计算，循环数为29-35；72℃延伸6分钟。
1.5质粒的组装构建
所有质粒的构建以质粒pYLEX1(购买自Yeastern Biotech Company,台湾)为基本骨架，采用KAPA HiFi高保真DNA聚合酶，25ul反应体系，PCR扩增质粒基本骨架片段、目的基因、启动子、终止子、筛选标记基因，应用Gibson Assembly方法(Gibson DG.Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides.Nucleic Acids Research.2009,37(20):6984-6990.)和试剂盒(购自New England Biolabs)将pYLEX1质粒骨架与目的基因、启动子、终止子和筛选标记基因组装成完整质粒(参见表1)。每个质粒包含一种筛选标记基因、一至三个目的基因，每个目的基因带有一个启动子和一个终止子。
以构建pDGAT1质粒为例：
1)以质粒pYLEX1为基本骨架，分别以pYLEX1和解脂耶鲁维亚酵母基因组DNA为模板，以SEQ ID No：51-52，SEQ ID No：59-60，SEQ ID No：79-80作为引物序列，PCR扩增质粒骨架片段、TEF启动子片段和XPR2终止子片段，将三个DNA片段应用Gibson Assembly方法组装在一起，获得质粒pYLEX1-P TEF1-T XPR2。DNA片段浓度控制在100-200ng每个反应，反应体系为10微升，装配条件为50℃，1小时。反应结束后，取2微升转化DH5α感受态细胞(购 自TransGen Biotech)，阳性克隆由菌落PCR和DNA测序验证筛选获得。
2)分别以pYLEX1-P TEF1-T XPR2和解脂耶鲁维亚酵母基因组DNA为模板，以SEQ ID No：1-2，SEQ ID No：81-82作为引物序列，PCR扩增带有启动子和终止子的质粒骨架片段和DGAT1基因，将两个DNA片段应用Gibson Assembly方法组装在一起，得到质粒pYLEX1-PT-DGAT1，记为pDGAT1。
表1所记载质粒的构建与pDGAT1质粒的组装类似，即应用Gibson Assembly方法将目的基因、启动子、终止子和筛选标记基因整合到一个质粒上。靶向内质网、过氧化物酶体和线粒体中表达的基因，需要在相应基因的3'末端添加内质网滞留信号肽KDEL、过氧化物酶体靶向信号肽SKL和线粒体靶向信号肽CoxIV(MLSLRQSIRFFKPATRTLCSSRYLL)。
表1、本发明构建质粒描述
Figure PCTCN2019081736-appb-000001
Figure PCTCN2019081736-appb-000002
Figure PCTCN2019081736-appb-000003
注：表1中marker为标记。
实施例2、解脂耶鲁维亚酵母工程菌的构建
2.1表达盒的获得
应用NotI内切酶(购自Thermo Fisher Scientific)分别酶切表1所记载的质粒pDS、pAB、pCgKCS、pCgKCS ER pCgKCS PTS、pCgKCS MTS、pC ERC MTS、pMCSD、pCB、pPLA2-1、pPLA2-2、pPLA2-3、pPLA2-4、pPLA2-5、pPLA2-6、pylGSR、pylGPO、prELO2、pCpLCE、pgELOVL6、pMaKCS、pD9DMB、pCeFAT6、pMaOLE2、pAtADS1、pAtADS2、pEcAldH、pScZwf和p△pex10。
具体酶切体系为：10×FD Green Buffer,2ul；NotI,1ul；质粒,<1ug；ddH 2O补至20ul。酶切产物利用Cycle Pure Kit(购自OMEGA bio-tek)进行纯化回收，回收步骤如下：酶切产物加入4-5倍体积buffer CP；混匀后转移至DNA吸附柱，室温下13,000g离心1分钟；弃滤液并加入700μL DNA洗涤缓冲液，13,000g离心1分钟；弃滤液重复洗涤一次；弃滤液，将空的吸附柱柱13,000g离心2分钟，干燥柱子；将吸附柱转移到干净的1.5mL离心管中，加30-50μL洗脱缓冲液，13,000g离心洗脱DNA。
获得表达盒DGAT1-SCD-Hgr或AtFAE1-BtFAE1-LEU或CgKCS-URA或Cg KCS ER-URA或CgKCS PTS-URA或CgKCS MTS-URA、CgKCS ER-CgKCS MTS-URA或MaLCE1-CgKCS ER-DGAT1 ER-SCD ER-URA或CgKCS ER-BtFAE1 ER-CgKCS PTS-BtF AE1 PTS-URA或PLA2-1-URA或PLA2-2-URA或PLA2-3-URA或PLA2-4-URA或P LA2-5-URA或PLA2-6-URA或ylGSR-URA或ylGPO-URA或rELO2-URA或CpL CE-URA或gELOVL6-URA或MaKCS-URA或D9DMB-URA或CeFAT6-URA或M aOLE2-URA或AtADS1-URA或AtADS2-URA或EcAldH-URA或ScZwf-URA或△pex10-URA。
2.2解脂耶鲁维亚酵母转化
(1)培养。从YPD平板培养基上挑取菌株po1g单菌落，接种于含50ml YPD培养液的250ml摇瓶(YPD培养基成分为葡萄糖20g/L、蛋白胨20g/L、酵母提取物10g/L)，28℃培养过夜。将上述培养的菌液接种于含50ml YPD的250ml摇瓶 中，至终浓度为OD 600＝0.5，而后于28℃培养至OD 600为1.0，约需4h。
(2)转化。取4ml上述细胞，5000rpm离心3min，弃上清，加入1μg线性化的基因表达盒DNA，分别为DGAT1-SCD-Hgr或AtFAE1-BtFAE1-LEU或CgKCS-URA或CgKCS ER-URA或CgKCS PTS-URA或CgKCS MTS-URA、CgKCS ER-CgKCS MTS-URA或MaLCE1-CgKCS ER-DGAT1 ER-SCD ER-URA或CgKCS ER-BtFAE1 ER-CgKCS PTS-BtFAE1 PTS-URA或PLA2-1-URA或PLA2-2-URA或PLA2-3-URA或PLA2-4-URA或PLA2-5-URA或PLA2-6-URA或ylGSR-URA或ylGPO-URA或rELO2-URA或CpLCE-URA或gELOVL6-URA或MaKCS-URA或D9DMB-URA或CeFAT6-URA或MaOLE2-URA或AtADS1-URA或AtADS2-URA或EcAldH-URA或ScZwf-URA或△pex10-URA，同时加入90μl 50％PEG3350，5μl 2M LiAC，5μl 2M DTT，2μl DMSO，2.5μl Salman liner DNA(10mg/ml)，在30℃水浴中孵育1h后涡旋震荡，然后39℃水浴10min，直接取50μl转化体系的混合液涂布筛选平板。YPD筛选平板分别为Hgr(150μg/ml)、URA选择缺陷型培养基和LEU选择缺陷型培养基。
2.3工程菌株
从筛选平板上挑取单菌落，利用PCR验证转化结果(图2)，RT-PCR验证基因表达水平(图3和表2)，筛选阳性转化子，获得工程菌株YL1、YL2、YL2-1、YL2-2、YL2-3、YL2-4、YL3、YL4-1、YL4-2、YL4-3、YL4-4、YL4-5、YL4-6、YL6、YL7、YL8和YL11。
PCR验证方法如下：以相应解脂耶鲁维亚酵母转化子的DNA为模板，用相应的引物进行PCR扩增，扩增体系皆为25ul，具体为2×Taq Mix,12.5ul；10uM引物各0.5ul；模板1ul；加水补至25ul；扩增条件为：94℃预变性5分钟；94℃变性30秒、60-72℃退火30秒、72℃延伸，延伸时间按照1分钟每kb计算，循环数为30；72℃延伸10分钟，扩增结束后1％琼脂糖凝胶电泳进行检测。
RT-PCR验证基因表达水平方法如下：采用TRizol法抽提上述菌株总RNA，利用核酸测定仪ND-1000检测浓度，同时1％的琼脂糖凝胶电泳检测RNA是否降解。根据各个基因序列，设计Real-time PCR特异引物。检测合格的RNA反转录成cDNA后进行Real time-PCR。Real time-PCR方法具体步骤见Biotium的
Figure PCTCN2019081736-appb-000004
Master Mixes for qPCR定量检测试剂盒。Real time-PCR扩增由美国 Roche公司生产的Light Cycler 480real-time PCR system完成。每个样本做三个复孔，不同样本重复三次，解脂耶鲁维亚酵母Actin基因作为内参。最后根据2 -ΔΔCt方法计算相对表达量。
表2.稳定期不同菌株中基因的表达水平。
Figure PCTCN2019081736-appb-000005
注：()内数字表示该基因上调的倍数；+表明该基因上调。
菌株YL1由上述可知其是经转化由质粒pDS、pBA和pCgKCS来源的表达盒DGAT1-SCD-Hgr、AtFAE1-BtFAE1-LEU和CgKCS-URA而获得，即过量表达基因DGAT1和SCD，过表达外源基因AtFAE1、BtFAE1和CgKCS。
菌株YL2在YL1基础上，经转化由质粒pMCSD来源的表达盒MaLCE1-CgKCS ER-DGAT1 ER-SCD ER-URA而获得，即菌株YL2在YL1的基础上 进一步过表达基因MaLCE1、CgKCS ER、DGAT1 ER和SCD ER。
菌株YL2-1在YL2基础上，经转化由质粒pCgKCS ER来源的表达盒CgKCS ER-URA而获得，即菌株YL2-1在YL2的基础上进一步过表达基因CgKCS ER。
菌株YL2-2在YL2基础上，经转化由质粒pCB来源的表达盒CgKCS ER-BtFAE1 ER-CgKCS PTS-BtFAE1 PTS-URA而获得，即菌株YL2-2在YL2的基础上进一步在不同的亚细胞(内质网、过氧化物酶体)中进一步过表达基因CgKCS和BtFAE1。
菌株YL2-3在YL2基础上，经转化由质粒pMCSD来源的表达盒MaLCE1-CgKCS ER-DGAT1 ER-SCD ER-URA而获得，即菌株YL2-3在YL2的基础上进一步过表达基因MaLCE1、CgKCS ER、DGAT1 ER和SCD ER。该菌株保藏于中国普通微生物菌种保藏管理中心，保藏号为CGMCC NO.15309。
菌株YL2-4在YL2基础上，经转化由质粒pEcAldH、pScZwf、pylGSR和pylGPO来源的表达盒EcAldH-URA、ScZwf-URA、ylGSR-URA和ylGPO-URA而获得，即菌株YL2-4在YL2的基础上进一步过表达基因EcAldH、ScZwf、ylGSR和ylGPO。
菌株YL3由上述可知其是经转化由质粒pCgKCS PTS和pMCSD来源的表达盒CgKCS PTS-URA和MaLCE1-CgKCS ER-DGAT1 ER-SCD ER-URA而获得，即过表达基因CgKCS PTS、MaLCE1、CgKCS ER、DGAT1 ER和SCD ER。
菌株YL4-1在YL1基础上，经转化由质粒pPLA2-1来源的表达盒PLA2-1-URA而获得，即菌株YL4-1在YL1的基础上进一步过表达基因PLA2-1。
菌株YL4-2在YL1基础上，经转化由质粒pPLA2-2来源的表达盒PLA2-2-URA而获得，即菌株YL4-2在YL1的基础上进一步过表达基因PLA2-2。
菌株YL4-3在YL1基础上，经转化由质粒pPLA2-3来源的表达盒PLA2-3-URA而获得，即菌株YL4-3在YL1的基础上进一步过表达基因PLA2-3。
菌株YL4-4在YL1基础上，经转化由质粒pPLA2-4来源的表达盒PLA2-4-URA而获得，即菌株YL4-4在YL1的基础上进一步过表达基因PLA2-4。
菌株YL4-5在YL1基础上，经转化由质粒pPLA2-5来源的表达盒PLA2-5-URA而获得，即菌株YL4-5在YL1的基础上进一步过表达基因PLA2-5。
菌株YL4-6在YL1基础上，经转化由质粒pPLA2-6来源的表达盒PLA2-6-URA而获得，即菌株YL4-6在YL1的基础上进一步过表达基因PLA2-6。
菌株YL5其是经转化由质粒pgELOVL6和pMaOLE2来源的表达盒gELOVL6-URA和MaOLE2-URA而获得，即过表达外源基因gELOVL6和MaOLE2。
菌株YL6在YL5基础上，经转化由质粒pC ERC MTS来源的表达盒CgKCS ER-CgKCS MTS-URA而获得，即菌株YL6在YL5的基础上进一步过表达基因CgKCS ER和CgKCS MTS。
菌株YL7在YL1基础上，经转化由质粒pAtADS1来源的表达盒AtADS1-URA而获得，即菌株YL7在YL1的基础上进一步过表达基因AtADS1。
菌株YL8在YL1基础上，经转化由质粒pAtADS2来源的表达盒AtADS2-URA而获得，即菌株YL8在YL1的基础上进一步过表达基因AtADS2。
菌株YL9由上述可知其是经转化由质粒pΔpex10来源的表达盒Δpex10-URA而获得，即敲除基因pex10。
菌株YL10在YL9基础上，经转化由质粒pCgKCS PTS来源的表达盒CgKCS PTS-URA而获得，即菌株YL10在YL9的基础上进一步过表达基因CgKCS PTS。
菌株YL11在YL10基础上，经转化由质粒pMCSD来源的表达盒MaLCE1-CgKCS ER-DGAT1 ER-SCD ER-URA而获得，即菌株YL11在YL10的基础上进一步过表达基因MaLCE1、CgKCS ER、DGAT1 ER和SCD ER。
实施例3、菌种培养生产神经酸
3.1菌种摇瓶培养及诱导调控
a.在YPD固体平板上分别活化菌株po1g和YL2-3，28℃培养1天。挑取单菌落分别接种于装有50ml YPD培养基的250ml摇瓶，28℃培养1天，作为种子培养液。将种子培养液分别接种于含50ml YNB培养基的250ml摇瓶，使其起始OD 600为0.2，28℃培养6天，待用。
其中，YNB培养基的组成为YNB1.7g/L，葡萄糖80g/L，酵母提取物1.5g/L，尿嘧啶20mg/L，亮氨酸100mg/L。
b.上述方法培养好的种子培养液，分别接种于含50ml诱导培养基的250ml摇瓶，使其起始OD 600为0.2，28℃培养6天，待用。
其中，诱导培养基为含10g/L葡萄糖的YNB，培养1d后待葡萄糖基本消耗完全，再补加赤藓糖醇作为碳源，再培养2d继续补加葡萄糖。
3.2菌种发酵罐培养(以菌株YL2-3为例)
将上述获得的菌株YL2-3活化后作为种子液，在5L发酵罐中加入3L培养基YNBF，发酵控制溶氧大于20％(生长期：0-48h)和0-5％(稳定期)。发酵过程中pH值恒定地控制在5.5，直到发酵结束。控制温度为28℃培养6天。
其中，YNBF培养基的成分为3.4g/L无氨基酸和硫酸铵的酵母氮源，150g/L葡萄糖，2g/L酵母提取物和8.8g/L硫酸铵。接种量为10％。
3.3微生物油脂的提取
取上述获得的5ml培养液，离心，1g湿菌体加入4mol/L盐酸10mL，振荡均匀，室温放置30min-1h；沸水浴6-8min，立即放于-20℃速冷30min；然后加入氯仿-甲醇(1:1，v/v)20mL充分混合，4000r/min离心10min；分离下层氯仿并称量体积，加入等体积浓度为0.15％的氯化钠，4000r/min离心10min；将下层氯仿层收集转移到锥形瓶中，70℃干燥2h冷却称重计算微生物油脂的产量，待GC分析用。
3.4微生物油脂中神经酸的位置特异性分析
利用上述酸热法提取出发菌株和工程菌株发酵培养6d的总脂。采用脂肪酶消化法检测神经酸在TAG中的位置。具体步骤如下：在3ml甲醇溶液中加入10mg油脂和10mg固定化的1,3位专一性脂肪酶，30℃反应8h。脂肪酸甲酯和2-MAG经TLC板进行纯化，气相检测显示只在游离脂肪酸层中存在神经酸，即神经酸位于TAG的sn-1,3位(图4)。
3.5神经酸占总脂肪酸含量的测定
微生物称重后，在玻璃管中添加2.6ml的甲醇：硫酸＝98：2溶液，并在85℃中反应3h。之后在冰箱内冷却，添加lml饱和NaCl和lml正己烷。震荡后进行高速离心(5000rpm)，进行5min。吸取上清，并用有机溶剂滤膜进行过滤，添加到气相色谱小瓶中。
神经酸甲酯含量测定应用GC方法测定。使用Agilent7890B-GC仪器，色谱 柱为HP-5(30m×0.32mm×0.25μm)。进样温度：250℃；检测器温度：250℃；进样体积：1μL；起始柱温为140℃，保持1min，以10℃/min升温至180℃，保持2min，5℃/min升温至210℃，保持4min，5℃/min升温至250℃，保持4min。
在上述升温条件下，GC检测的神经酸甲酯出峰时间为23.775min(图5)。在摇瓶培养条件下，菌株YL1、YL2、YL2-1、YL2-2、YL2-3、YL2-4、YL3、YL4-1、YL4-2、YL4-3、YL4-4、YL4-5、YL4-6、YL6、YL7、YL8和YL11的神经酸含量占总脂肪酸含量的百分比分别为2.40％、11.09％、10.66％、11.90％、17.57％、15.12％、9.63％、4.02％、4.36％、4.62％、4.57％、4.82％、4.19％、8.62％、8.12％、9.12％和11.12％(图7)。17株菌摇瓶批次培养的OD 600(以YL2-3为例，如图6)、生物量和油脂含量相差不大，生物量在20g/L左右，油脂含量约在8.2g/L。在发酵罐培养条件下，菌株YL2-3的最大神经酸含量占总脂肪酸含量的30.6％，最大生物量为82.6g/L。菌株YL2-3中其他脂肪酸的含量占油脂的比例分别为：C16:0为5.3％，C16:1为10.9％，C18:0为1.5％，C18:1为28.7％，C18:2为9.1％，C24:0为2.8％。
3.6细胞内醛水平的测定
为了验证氧化还原平衡的调控，对菌株中的反应性醛物质进行定量分析，步骤如下：通过冷冻离心机收集po1g和YL2-4细胞沉淀并重悬于PBS缓冲液中。在振荡器上使酵母沉淀物均质化，在4℃下离心，取上清。根据Sigma的荧光醛测定试剂盒(MAK141-1KT)所述的说明从上清液中测量细胞内活性醛。结果发现：相比po1g菌株，YL2-4菌株中的总脂含量提高了2.5倍，活性醛的量明显减少，其中在50h时，YL2-4菌株中活性醛的量比po1g菌株下降了大约4倍(图8)。
实施例4、神经酸发酵
根据上述的优化结果，将菌株YL2-3在500L发酵罐中进行放大。活化后的种子液按3％的接种量进行接种，培养温度为28℃，通气量5-8L/min，搅拌转速为300r/min，发酵pH(5.5)用3M的NaOH溶液调节。一共进行3批发酵，结果发现菌株YL2-3的生物量平均为126.56g/L，神经酸含量占总脂肪酸含量的39.6％，油脂含量约在39.3g/L。其他脂肪酸的含量占总脂肪酸含量的比例分别为：C16:0为4.3％，C16:1为7.9％，C18:0为3.5％，C18:1为25.7％，C18:2为4.9％， C24:0为4.8％(图9)。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (21)

  1. 一种重组酵母菌株，其特征在于，过表达：
    (a)一种编码Δ9去饱和酶的基因；
    (b)至少四种编码脂肪酸延长酶的基因；
    (c)一种编码甘油二酯酰基转移酶的基因；
    (d)一种编码靶向内质网的脂肪酸延长酶的基因；
    (e)一种编码靶向内质网的甘油二酯酰基转移酶的基因；和/或
    (f)一种编码靶向内质网的Δ9去饱和酶基因。
  2. 根据权利要求1所述的重组酵母菌株，其特征在于，
    所述编码Δ9去饱和酶的基因为解脂耶鲁维亚酵母SCD基因，其核苷酸序列如SEQ ID No：84所示；
    所述的四种编码脂肪酸延长酶基因分别为高山被孢霉C16/18延长酶基因MaLCE1，其核苷酸序列如SEQ ID No：93所示；拟南芥AtFAE1基因，其核苷酸序列如SEQ ID No：94所示；非洲芥菜BtFAE1基因，其核苷酸序列如SEQ ID No：95所示；碎米芥CgKCS基因，其核苷酸序列如SEQ ID No：96所示；
    所述编码甘油二酯酰基转移酶的基因为解脂耶鲁维亚酵母DGAT1基因，其核苷酸序列如SEQ ID No：83所示；
    所述编码靶向内质网的脂肪酸延长酶的基因为带有靶向内质网信号肽编码序列的碎米芥CgKCS ER基因，其核苷酸序列如SEQ ID No：121所示；
    所述编码靶向内质网的甘油二酯酰基转移酶的基因为带有靶向内质网信号肽编码序列的解脂耶鲁维亚酵母DGAT1 ER基因，其核苷酸序列如SEQ ID No：122所示；
    所述编码靶向内质网的Δ9去饱和酶的基因为带有靶向内质网信号肽编码序列的解脂耶鲁维亚酵母SCD ER基因，其核苷酸序列如SEQ ID No：123所示。
  3. 根据权利要求1所述的重组酵母菌株，其特征在于，进一步过表达：
    (a)两种编码靶向内质网的脂肪酸延长酶的基因；和/或
    (b)两种编码靶向过氧化物酶体的脂肪酸延长酶的基因。
  4. 根据权利要求3所述的重组酵母菌株，其特征在于，
    所述两种编码靶向内质网的脂肪酸延长酶的基因分别为带有靶向内质网信号肽编码序列的碎米芥CgKCS ER基因，其核苷酸序列如SEQ ID No：121所示；带有靶向内质网信号肽编码序列的非洲芥菜BtFAE1 ER基因，其核苷酸序列如SEQ ID No：124所示；
    所述两种编码靶向过氧化物酶体的脂肪酸延长酶的基因分别为带有靶向过氧化物酶体信号肽编码序列的碎米芥CgKCS PTS基因，其核苷酸序列如SEQ ID No：125所示；带有靶向过氧化物酶体信号肽编码序列的非洲芥菜BtFAE1 PTS基因，其核苷酸序列如SEQ ID No：126所示。
  5. 根据权利要求1所述的重组酵母菌株，其特征在于，进一步过表达：
    (a)一种编码醛脱氢酶的基因；
    (b)一种编码葡萄糖-6-磷酸脱氢酶的基因；
    (c)一种编码谷胱甘肽二硫化物还原酶的基因；和/或
    (d)一种编码谷胱甘肽过氧化物酶的基因。
  6. 根据权利要求5所述的重组酵母菌株，其特征在于，
    所述编码醛脱氢酶的基因为大肠杆菌EcAldH基因，其核苷酸序列如SEQ ID No：105所示；
    所述编码葡萄糖-6-磷酸脱氢酶的基因为酿酒酵母ScZwf基因，其核苷酸序列如SEQ ID No：106所示；
    所述谷胱甘肽二硫化物还原酶的基因为解脂耶鲁维亚酵母ylGSR基因，其核苷酸序列如SEQ ID No：91所示；
    所述谷胱甘肽过氧化物酶的基因为解脂耶鲁维亚酵母ylGPO基因，其核苷酸序列如SEQ ID No：92所示。
  7. 一种重组酵母菌株，其特征在于，过表达：
    (a)一种编码Δ9去饱和酶的基因；
    (b)至少三种编码脂肪酸延长酶的基因；
    (c)一种编码甘油二酯酰基转移酶的基因；和/或
    (d)一种编码磷脂酶A2的基因。
  8. 根据权利要求7所述的重组酵母菌株，其特征在于，
    所述编码Δ9去饱和酶基因为解脂耶鲁维亚酵母SCD基因，其核苷酸序列如 SEQ ID No：84所示；
    所述三种编码脂肪酸延长酶的基因分别为拟南芥AtFAE1基因，其核苷酸序列如SEQ ID No：94所示、非洲芥菜BtFAE1基因，其核苷酸序列如SEQ ID No：95所示、碎米芥CgKCS基因，其核苷酸序列如SEQ ID No：96所示；
    所述编码甘油二酯酰基转移酶基因为解脂耶鲁维亚酵母DGAT1基因，其核苷酸序列如SEQ ID No：83所示；
    所述编码磷脂酶A2基因为PLA2-1基因，其核苷酸序列如SEQ ID No：85所示；或PLA2-2基因，其核苷酸序列如SEQ ID No：86所示；或PLA2-3基因，其核苷酸序列如SEQ ID No：87所示；或PLA2-4基因，其核苷酸序列如SEQ ID No：88所示；或PLA2-5基因，其核苷酸序列如SEQ ID No：89所示；或PLA2-6基因，其核苷酸序列如SEQ ID No：90所示。
  9. 一种重组酵母菌株，其特征在于，过表达：
    (a)一种编码靶向过氧化物酶体的脂肪酸延长酶的基因；
    (b)一种编码脂肪酸延长酶的基因；
    (c)一种编码靶向内质网的脂肪酸延长酶的基因；
    (d)一种编码靶向内质网的甘油二酯酰基转移酶的基因；和/或
    (e)一种编码靶向内质网的Δ9去饱和酶基因。
  10. 根据权利要求9所述的重组酵母菌株，其特征在于，
    所述靶向过氧化物酶体的脂肪酸延长酶基因为带有靶向过氧化物酶体信号肽编码序列的碎米芥CgKCS PTS基因，其核苷酸序列如SEQ ID No：125所示；
    所述脂肪酸延长酶基因为高山被孢霉C16/18延长酶基因MaLCE1基因，其核苷酸序列如SEQ ID No：93所示；
    所述靶向内质网的脂肪酸延长酶的基因为带有靶向内质网信号肽编码序列的碎米芥CgKCS ER基因，其核苷酸序列如SEQ ID No：121所示；
    所述靶向内质网的甘油二酯酰基转移酶基因为带有靶向内质网信号肽编码序列的解脂耶鲁维亚酵母DGAT1 ER基因，其核苷酸序列如SEQ ID No：122所示；
    所述靶向内质网的Δ9去饱和酶基因为带有靶向内质网信号肽编码序列的解脂耶鲁维亚酵母SCD ER基因，其核苷酸序列如SEQ ID No：123所示。
  11. 一种重组酵母菌株，其特征在于，过表达：
    (a)一种编码Δ9去饱和酶的基因；
    (b)一种编码脂肪酸延长酶的基因；
    (c)一种编码靶向内质网的脂肪酸延长酶的基因；和/或
    (d)一种编码靶向线粒体的脂肪酸延长酶的基因。
  12. 根据权利要求11所述的重组酵母菌株，其特征在于，
    所述编码Δ9去饱和酶基因为高山被孢霉Δ9脂肪酸去饱和酶MaOLE2基因，其核苷酸序列如SEQ ID No：102所示；
    所述编码脂肪酸延长酶为羊脂肪酸延长酶6gELOVL6基因，其核苷酸序列如SEQ ID No：99所示；
    所述编码靶向内质网的脂肪酸延长酶基因为带有靶向内质网信号肽编码序列的碎米芥CgKCS ER，其核苷酸序列如SEQ ID No：121所示；
    所述编码靶向线粒体的脂肪酸延长酶基因为带有靶向线粒体信号肽编码序列的碎米芥CgKCS MTS，其核苷酸序列如SEQ ID No：127所示。
  13. 一种重组酵母菌株，其特征在于，过表达：
    (a)两种编码Δ9去饱和酶的基因；
    (b)三种编码脂肪酸延长酶的基因；和/或
    (c)一种编码甘油二酯酰基转移酶的基因。
  14. 根据权利要求13所述的重组酵母菌株，其特征在于，
    所述两种编码Δ9去饱和酶基因分别为解脂耶鲁维亚酵母SCD基因，其核苷酸序列如SEQ ID No：84所示；拟南芥AtADS1基因，其核苷酸序列如SEQ ID No：103所示；或者所述两种编码Δ9去饱和酶基因分别为解脂耶鲁维亚酵母SCD基因，其核苷酸序列如SEQ ID No：84所示；拟南芥AtADS2基因，其核苷酸序列如SEQ ID No：104所示；
    所述三种编码脂肪酸延长酶基因分别为拟南芥AtFAE1基因，其核苷酸序列如SEQ ID No：94所示；非洲芥菜BtFAE1基因，其核苷酸序列如SEQ ID No：95所示；碎米芥CgKCS基因，其核苷酸序列如SEQ ID No：96所示；
    所述编码甘油二酯酰基转移酶为解脂耶鲁维亚酵母DGAT1如SEQ ID No：83所示。
  15. 一种重组酵母菌株，其特征在于，该菌株中过氧化物酶体生物发生因子10 的表达被下调，并且进一步过表达：
    (a)一种编码靶向过氧化物酶体的脂肪酸延长酶的基因；
    (b)一种编码脂肪酸延长酶的基因；
    (c)一种编码靶向内质网的脂肪酸延长酶的基因；
    (d)一种编码靶向内质网的甘油二酯酰基转移酶的基因；和/或
    (e)一种编码靶向内质网的Δ9去饱和酶基因。
  16. 根据权利要求15所述的重组酵母菌株，其特征在于，
    所述表达下调的过氧化物酶体生物发生因子10为pex10基因，其核苷酸序列如SEQ ID No：120所示；
    所述编码靶向过氧化物酶体的脂肪酸延长酶的基因为带有靶向过氧化物酶体信号肽编码序列的碎米芥CgKCS PTS基因，其核苷酸序列如SEQ ID No：125所示；
    所述编码脂肪酸延长酶的基因为高山被孢霉C16/18延长酶MaLCE1基因，其核苷酸序列如SEQ ID No：93所示；
    所述编码靶向内质网的脂肪酸延长酶的基因为带有靶向内质网信号肽编码序列的碎米芥CgKCS ER基因，其核苷酸序列如SEQ ID No：121所示；
    所述编码靶向内质网的甘油二酯酰基转移酶的基因为带有靶向内质网信号肽编码序列的解脂耶鲁维亚酵母DGAT1 ER基因，其核苷酸序列如SEQ ID No：122所示；
    所述编码靶向内质网的Δ9去饱和酶基因为带有靶向内质网信号肽编码序列的解脂耶鲁维亚酵母SCD ER基因，其核苷酸序列如SEQ ID No：123所示。
  17. 根据权利要求1-16所述的任一重组酵母菌株，其中所述酵母为解脂耶鲁维亚酵母。
  18. 利用权利要求1-16所述的任一重组酵母菌株制备微生物油的应用。
  19. 利用权利要求1-16所述的任一重组酵母菌株制备微生物油的方法：
    (a)培养权利要求1-16所述的任一重组酵母菌株，其中产生包含神经酸的微生物油；以及
    (b)回收所述步骤(a)的微生物油。
  20. 利用权利要求1-16所述的任一重组酵母菌株制备神经酸的应用。
  21. 利用权利要求1-16所述的任一重组酵母菌株制备神经酸的方法，包括：
    (a)培养权利要求1-16所述的任一重组酵母菌株，产生微生物油；以及
    (b)回收所述步骤(a)的微生物油，提取神经酸。
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