CN110499259B - Yarrowia lipolytica YW100-1 and application thereof - Google Patents

Abstract

The yarrowia lipolytica YW100-1 is obtained by over-expressing glycerol kinase GUT1, glycerol kinase GUT2, glycerol dehydrogenase enzyme GCY1, dihydroxyacetone kinase DAK1, dihydroxyacetone kinase DAK2 and NADH kinase POS5 in glycerol metabolism in yarrowia lipolytica and simultaneously knocking out diacylglycerol acyltransferase gene DGA2 and 3-phosphoglycerol dehydrogenase gene GPD2 in the yarrowia lipolytica. When the strain takes glycerol as a carbon source, the yield of pyruvic acid is increased by 66.2 percent and reaches 121.2g/L, and the strain provides an excellent strain for industrial production of pyruvic acid.

Description

Yarrowia lipolytica YW100-1 and application thereof

(I) technical field

The invention relates to a construction method and application of yarrowia lipolytica engineering bacteria, belonging to the technical field of biological engineering.

(II) background of the invention

Pyruvic acid is used as an intermediate in microbial metabolic pathways, is also one of important organic acids, and has wide functions in the fields of biochemical engineering, pharmacy, food and science. In the medical industry, pyruvic acid is an important medical intermediate, can be used for synthesizing levodopa, anti-inflammatory analgesic sinkefen, antituberculosis drugs such as isoniazid calcium pyruvate, thiaimidazole drugs and the like, and in addition, pyruvate salts (such as calcium pyruvate, potassium pyruvate, pyruvate creatine) are widely applied to weight-losing health care products and drugs. In the food industry, pyruvic acid is used as a food additive and has natural preservative and fresh-keeping effects. In the chemical industry, ethyl pyruvate is used as a skin whitening agent, can inhibit the formation of melanin in the skin and has good skin whitening effect. In addition, pyruvic acid is used as a precursor of a new generation of biofuel, the market demand at home and abroad is rapidly increased, and the pyruvic acid has wide application value.

At present, the methods for producing pyruvic acid mainly comprise three major methods, namely a chemical synthesis method, an enzyme conversion method and a microbial fermentation method. The chemical synthesis method mainly uses tartaric acid as a raw material to chemically synthesize pyruvic acid, but the method has heavy pollution and high cost, and the industrial production is limited. The enzyme conversion method mainly utilizes a dehydrogenase system in microorganisms to convert lactic acid into pyruvic acid, but the method has low conversion rate and high cost, and industrial production is not realized at present. The microbial fermentation method mainly uses microorganisms and uses glucose or glycerol and the like as cheap carbon sources to directly produce pyruvic acid through microbial fermentation. Currently used strains for microbial fermentation are Saccharomyces cerevisiae (Saccharomyces cerevisiae), Escherichia coli (Escherichia coli), Torulopsis glabrata (Torulopsis glabrata), Yarrowia lipolytica (Yarrowia lipolytica), and the like. Compared with chemical synthesis and enzyme conversion, it has the advantages of less pollution, high conversion rate, low cost, etc. and is also the main industrial process.

Although yarrowia lipolytica has a good pyruvate production capacity, the conversion rate and synthesis rate of pyruvate synthesis from glycerol still need to be improved, and the rapid degradation rate of pyruvate results in the failure of large-scale accumulation. Two glycerol catabolic pathways exist in yarrowia lipolytica, one is the glycerol-3-phosphate pathway, in which glycerol is phosphorylated to glycerol-3-phosphate in the cytoplasm by the action of glycerol kinase (encoded by GUT 1), followed by shuttling into the mitochondria, oxidation of glycerol-3-phosphate to dihydroxyacetone phosphate by the action of glycerol-3-phosphate dehydrogenase (encoded by GUT 2), which is then transported back into the cytoplasm into the glycolytic metabolic pathway. The other is the Dihydroxyacetone (DHA) pathway in which glycerol dehydrogenase (encoded by GCY 1) oxidizes glycerol to DHA, which is subsequently phosphorylated to dihydroxyacetone phosphate by dihydroxyacetone kinase (encoded by DAK1 and DAK 2) into the glycolytic metabolic pathway. Yarrowia lipolytica, a high-producing lipid yeast, diacylglycerol acyltransferase (encoded by DGA 2) can synthesize triacylglycerol, an important component of lipid, using dihydroxyacetone phosphate. Glycerol synthesis glycerol was synthesized by conversion to glycerol-3-phosphate via dihydroxyacetone phosphate followed by glycerol-3-phosphate dehydrogenase (encoded by GPD1, GPD 2). It has been shown that the knockout of GPD2 results in the accumulation of intracellular NADH, thereby breaking the NADH/NADPH balance and affecting the growth of the thallus, while Pos5, as a NADH kinase, can phosphorylate NADH to NADPH using ATP.

In view of the above, the present invention enhances the glycerol catabolism pathway by enhancing the glycerol-3-phosphate pathway and the dihydroxyacetone pathway to accelerate the glycerol decomposition and simultaneously weaken the glycerol synthesis pathway, thereby realizing the massive accumulation of pyruvic acid in cells.

Disclosure of the invention

The invention aims to provide yarrowia lipolytica engineering bacteria for high yield of pyruvic acid, and construction and application thereof, and provides excellent bacteria for industrial production of pyruvic acid.

The technical scheme adopted by the invention is as follows:

the invention provides a Yarrowia lipolytica YW100-1 with high pyruvate yield, wherein the Yarrowia lipolytica YW100-1 is a Yarrowia lipolytica Yarrowia engineering bacterium with high pyruvate yield, and the Yarrowia lipolytica YW100-1 is a Yarrowia lipolytica strain which is obtained by over-expressing a glycerol kinase GUT1, a glycerol kinase GUT2, a glycerol dehydrogenase enzyme GCY1, a dihydroxyacetone kinase DAK1 and a dihydroxyacetone kinase DAK2 in the glycerol metabolism of glycerol, simultaneously knocking out a diacylglycerol acyltransferase gene DGA2 in the Yarrowia lipolytica to enhance the catabolism of the glycerol, knocking out a 3-phosphate glycerol dehydrogenase gene GPD2 and over-expressing an NADH kinase POS5 to reduce the anabolism of the glycerol.

Further, the yarrowia lipolytica YW100-1 was constructed as follows:

(1) GUT1 gene, GUT2 gene and pEXP1 fragment of a promoter from yarrowia lipolytica are overlapped and extended to obtain GUT1-pEXP1-GUT2, and the GUT1-pEXP1-GUT2 is connected to a vector JMP113 to obtain a vector E14;

(2) carrying out enzyme digestion on the constructed vector E14 by Not I, and transferring the vector E14 into wild yarrowia lipolytica AS2.1405 to obtain transformed yarrowia lipolytica engineering bacteria ZS 102;

(3) transferring three large fragments, namely 5 'KU 70-URA3-pTEF-DAK1, DAK1-pEXP1-DAK2-pGPD and pGPD-GYC 1-3' KU70, of GCY1, DAK1 and DAK2 genes, promoters pTEF, pEXP1 and pGPD derived from yarrowia lipolytica, and 5 'end and 3' end of a homologous arm KU70 for integration into ZS102 through overlapping extension to obtain transformed yarrowia lipolytica engineering bacteria ZS 104;

(4) transferring a POS5 gene and pTEF derived from yarrowia lipolytica, a 5 'GPD 2 fragment and a 3' GPD2 fragment for knocking out GPD2 to ZS104 by overlapping and extending to obtain a 5 'GPD 2-URA3-pTEF-POS5 fragment and a POS 5-3' GPD2 fragment, and obtaining a yarrowia lipolytica engineering bacterium ZS106 after homologous recombination;

(5) the 5 ' and 3 ' DGA2 used for knockout were transferred to ZS106 by overlapping extension to obtain 5 ' DGA2-URA3-DGA2 fragment, and yarrowia lipolytica YW100-1 was obtained.

Further, the nucleotide sequence of the glycerol kinase GUT1 gene is shown as SEQ ID NO.1, the nucleotide sequence of the glycerol kinase GUT2 gene is shown as SEQ ID NO.2, the nucleotide sequence of the dihydroxyacetone kinase DAK1 gene is shown as SEQ ID NO.3, the nucleotide sequence of the dihydroxyacetone kinase DAK2 gene is shown as SEQ ID NO.4, the nucleotide sequence of the glycerol dehydrogenase GCY1 gene is shown as SEQ ID NO.5, the nucleotide sequence of the NADH kinase POS5 gene is shown as SEQ ID NO.6, the nucleotide sequence of the diacylglycerol acyltransferase 5 'DGA 2 gene is shown as SEQ ID NO.7, the nucleotide sequence of the diacylglycerol acyltransferase 3' DGA2 gene is shown as SEQ ID NO.8, the nucleotide sequence of the promoter pEXP1 is shown as SEQ ID NO.9, the nucleotide sequence of the promoter KU70 is shown as SEQ ID NO.10, the nucleotide sequence of Loxp-URA3-Loxp p nucleotide NO.11, the nucleotide sequence of the promoter pTEF NO.12, the nucleotide sequence of the promoter pGPD is shown as SEQ ID NO.13, the nucleotide sequence of the 3 ' KU70 is shown as SEQ ID NO.14, the nucleotide sequence of the 3-phosphoglycerol dehydrogenase 5 ' GPD2 gene is shown as SEQ ID NO.15, and the nucleotide sequence of the 3 ' GPD2 gene is shown as SEQ ID NO. 16.

The Yarrowia lipolytica YW100-1 is preserved in China center for type culture Collection with the preservation date of 2019, 3 months and 18 days and the preservation number of CCTCC NO: m2019168, address: wuhan university, Wuhan, China, zip code 430072.

The invention also provides an application of the yarrowia lipolytica YW100-1 in preparing pyruvic acid by fermenting glycerol, wherein the application is one of the following methods: (1) and (3) shaking flask fermentation: inoculating yarrowia lipolytica YW100-1 into YPD culture medium, culturing at 30 deg.C and 200rpm for 24 hr to obtain seed solution; at an initial cell concentration OD₆₀₀Inoculation of 0.05 ═Fermenting and culturing at 30 deg.C and 200rpm in YNG medium to OD₆₀₀At 4.0-5.0 (preferably 4.5), obtaining fermentation liquor containing pyruvic acid; the YPD liquid culture medium comprises the following components in parts by weight: 1% of yeast extract, 2% of glucose, 2% of peptone and distilled water as a solvent, wherein the pH value is natural; YNG culture medium comprises the following components by mass: 0.67% YNB (containing ammonium sulfate and yeast basic nitrogen source without amino acids), 50g/L glycerol, distilled water as solvent, and Na₂HPO₄-adjusting the pH to 4.0 with a citric acid buffer;

(2) fermentation in a fermentation tank: inoculating yarrowia lipolytica YW100-1 into a fermentation tank containing a fermentation culture medium, fermenting at 30-32 ℃, pH value of 4-4.5 and dissolved oxygen of 40-50% to obtain fermentation liquor containing pyruvic acid, and separating and purifying to obtain pyruvic acid; the fermentation medium comprises the following components: 60g/L of glycerin, 10g/L of (NH)₄)₂SO₄、1.4g/L MgSO₄·7H₂O、2g/L KH₂PO₄、0.8g/L CaCl₂0.5g/L NaCl, 1 mu g/L vitamin B1, and the solvent is distilled water, and the pH value is natural.

Further, during fermentation in the fermentation tank, glycerol is added in a batch mode, 60g/L is added for the first time, and after the glycerol is exhausted, the glycerol is supplemented, 40g/L is supplemented each time, and 2 times are preferred.

Further, before fermentation in the fermentation tank, yarrowia lipolytica YW100-1 is subjected to seed expansion culture, and then a seed solution is inoculated to the fermentation medium in an inoculation amount with a volume concentration of 10%, wherein the seed culture comprises the following steps: inoculating yarrowia lipolytica YW100-1 to a seed culture medium, and performing shake-flask culture at 30 ℃ for 18h to obtain a seed solution; the seed culture medium: 2g/L glycerol, 0.4g/L tryptone, 0.2g/L yeast extract, 0.24g/L KH₂PO₄、1.7g/L K₂HPO₄·3H₂O, the solvent is distilled water, and the pH value is natural.

Compared with the prior art, the invention has the following beneficial effects:

compared with the original strain yarrowia lipolytica AS2.1405, the yarrowia lipolytica YW100-1 (the preservation number is CCTCC NO: M2019168) obtained by the invention has the advantages that when glycerol is used AS a carbon source, the yield of pyruvic acid is increased by 66.2 percent and reaches 121.2g/L, and an excellent strain is provided for industrial production of pyruvic acid.

(IV) description of the drawings

FIG. 1 is a scheme showing the construction of yarrowia lipolytica genetically engineered bacteria.

FIG. 2 is a comparison of the pyruvic acid yield and the glycerol residue of the recombinant strain YW100-1 and the starting strain AS2.1405 in the flask fermentation.

FIG. 3 is a comparison of the pyruvic acid yields of the recombinant strain YW100-1 and the starting strain AS2.1405 fermented and cultured in a 20L fermentation tank.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

the methods in the following examples are well known unless otherwise specified.

The YPD liquid culture medium comprises the following components in parts by weight: 1% of yeast extract, 2% of glucose, 2% of peptone and distilled water as a solvent, and the pH value is natural.

YNG culture medium comprises the following components by mass: 0.67% YNB (containing ammonium sulfate and yeast basic nitrogen source without amino acids), 50g/L glycerol, distilled water as solvent, and Na₂HPO₄The pH was adjusted to 4.0 with a citrate buffer.

The YPD solid medium was prepared by adding agar at a concentration of 2g/L to a YPD liquid medium.

The LB culture medium comprises the following components in percentage by mass: peptone 1%, yeast extract 0.5%, sodium chloride 0.5%, distilled water as solvent, and natural pH.

Example 1: construction of yarrowia lipolytica gene engineering bacteria for high yield of pyruvic acid

The procedure is shown in FIG. 1, and the yarrowia lipolytica YW100-1 was constructed as follows:

1. construction of E14 plasmid

Amplification of GUT 1: GUT1, which is about 1.5kb in size and has the nucleotide sequence shown in SEQ ID NO.1, is amplified by designing primers YW238 and YW239 and taking the genome of wild type yarrowia lipolytica AS2.1405 (purchased from Guangdong collection of microorganisms and strains, with the number of GIM 2.187) AS a template.

Amplification of promoter pEXP 1: through designing primers YW405 and YW240, using a wild yarrowia lipolytica AS2.1405 genome AS a template, pEXP1 with the size of about 1kb and a nucleotide sequence shown in SEQ ID NO.9 is amplified.

Amplification of GUT 2: by designing primers YW241 and YW242, GUT2 with the size of about 2kb and the nucleotide sequence shown in SEQ ID NO.2 is amplified by taking the wild type yarrowia lipolytica AS2.1405 genome AS a template.

The PCR amplification conditions of the GUT1, pEXP1 and GUT2 are pre-denaturation at 98 ℃ for 3 minutes; denaturation at 98 ℃ for 10 seconds, annealing at 58 ℃ for 10 seconds, and extension at 72 ℃ for 2 minutes (30 cycles), and re-extension at 72 ℃ for 10 minutes.

Overlapping extension to construct GUT1-pEXP1-GUT2 fragment: overlap extension was performed using 2 rounds of PCR amplification. A first round: the amplification system was 25. mu.l (2 XPrimeSTAR Max DNA polymerase (from Beijing Baorizimen, Co., Ltd.) 12.5. mu.l, GUT1, pEXP1 and GUT2 fragments mixed in a molar ratio of 1:3:1, supplemented with distilled water to 25. mu.l), pre-denatured at 95 ℃ for 3 minutes, denatured at 98 ℃ for 10 seconds, annealed at 58 ℃ for 15 seconds, extended at 72 ℃ for 2 minutes (15 cycles), and re-extended at 72 ℃ for 10 minutes. And a second round: the amplification system was 50. mu.l (2 XPrimeSTAR Max DNA polymerase 25. mu.l, 2. mu.l PCR product from the first amplification step, 1. mu.l YW238+ 1. mu.l YW243 and distilled water supplemented to 50. mu.l), pre-denatured at 95 ℃ for 3 min, denatured at 98 ℃ for 10 sec, annealed at 56 ℃ for 15 sec, extended at 72 ℃ for 2 min (30 cycles), and re-extended at 72 ℃ for 10 min.

The GUT1-pEXP1-GUT2 fragment obtained as described above was recovered with a gel recovery kit (available from Beijing Baoji doctor Co., Ltd.), and cloned into JMP113 vector (fillers P, Le Dall MT, Garllarin C, ThonJM P, Nicaud. New dispersion cassettes for rapid gene dispersion and marker recovery in the yeast Yarrowia lipolytica. J. Microbiol methods.2003.55(3):727-37.) using a one-step cloning kit (available from Nanjing Nozam Biotech Co., Ltd.). Cloning system: 2. mu.l of 5 XCE MultiS buffer, 3. mu.l of GUT1-pEXP1-GUT2 fragment, 2. mu.l of BamH I linearized JMP113, 1. mu.l of Exnase TM MultiS enzyme, 2. mu.l of distilled water. After reaction at 37 ℃ for half an hour, 10. mu.l of the mixture was added to 100. mu.l of E.coli DH 5. alpha. competent cells for 30 minutes in ice bath after 5 minutes. Heat shock was carried out at 42 ℃ for 30 seconds and immediately placed on ice for 2 minutes. 1mL of LB medium was added and incubated at 37 ℃ for 1 hour at 200 rpm. And (3) centrifugally collecting bacterial liquid, coating the bacterial liquid on an LB plate containing 50 mu g/mL kanamycin, carrying out overnight culture at 37 ℃ until a transformant grows out, identifying the transformant by using primers YW244 and YW245, screening 4 positive single colonies by PCR (polymerase chain reaction), inoculating the colonies in an LB culture medium for culture, extracting positive clone plasmids for sequencing verification, and indicating that the construction of the vector E14 is successful by a sequencing result.

TABLE 1 primer List

2. Construction of recombinant yarrowia lipolytica ZS102

The Not I is used for enzyme digestion of the plasmid E14, the pTEF-GUT1-pEXP1-GUT2 fragment is recovered by gel cutting, and the fragment is transferred into wild yarrowia lipolytica AS2.1405 competent cells by an electrotransfer method to obtain the recombinant yarrowia lipolytica engineering bacteria ZS102, which comprises the following specific steps:

inoculating wild type yarrowia lipolytica AS2.1405 in YPD liquid medium, culturing overnight at 30 deg.C, transferring to 50mL fresh YPD liquid medium to make initial OD₆₀₀Culturing at 0.1 deg.C for 4-6 hr to obtain OD₆₀₀Up to 1.0. The supernatant was removed by centrifugation, and the cells were suspended in a buffer (0.6M sorbitol, 10mM Tris-HCl, 25mM DTT, 150mM LiAc, pH 7.5) and then allowed to stand for 1 hour. Centrifuge again, and use 1M sorbitol three times. After removing the supernatant, the cells were suspended in 150mL of 1M sorbitol, i.e., the wild-type yarrowia lipolytica AS2.1405 competent cells. 80 μ l of competent cells were mixed with 200ng of pTEF-GUT1-pEXP1-GUT2 fragment, and subjected to electroporation at 1.5kV (E ═ 12.4kV/cm), 200. omega. and 25. mu.F, 1mL of 1M aqueous sorbitol solution was rapidly added after the electroporation, the mixture was plated on YND solid medium and subjected to inverted culture for 2 to 3 days, and the transformants were identified by PCR using primers YW253+ YW 254.

3. Construction of yarrowia lipolytica ZS104

Amplification of 5' KU 70: by designing primers YW600 and YW601, the 5' upstream of KU70, which is about 1.2kb in size and has the nucleotide sequence shown in SEQ ID NO.10, is amplified by using the wild type yarrowia lipolytica AS2.1405 genome AS a template.

Amplification of Loxp-URA 3-Loxp: by designing primers YW602 and YW603 and taking a plasmid JMP113 as a template, Loxp-URA3-Loxp with the size of about 1.8kb and the nucleotide sequence shown in SEQ ID NO.11 are amplified.

Amplification of pTEF: by designing primers YW604 and YW605, and taking the genome of wild-type yarrowia lipolytica AS2.1405 AS a template, a promoter pTEF with the size of about 0.4kb and the nucleotide sequence shown in SEQ ID NO.12 is amplified.

Amplification of DAK 1: by designing primers YW606 and YW607 and taking the genome of wild-type yarrowia lipolytica AS2.1405 AS a template, a promoter DAK1 with the size of about 2.2kb and the nucleotide sequence shown in SEQ ID NO.3 is amplified.

Amplification of pEXP 1: by designing primers YW608 and YW609, a promoter pEXP1 with the size of about 1kb and the nucleotide sequence shown AS SEQ ID NO.9 is amplified by taking the genome of wild type yarrowia lipolytica AS2.1405 AS a template.

Amplification of DAK 2: by designing primers YW610 and YW611, and taking the genome of wild-type yarrowia lipolytica AS2.1405 AS a template, a promoter DAK2 with the size of about 2.2kb and the nucleotide sequence shown in SEQ ID NO.4 is amplified.

Amplification of pGPD: by designing primers YW612 and YW613, and taking the genome of wild-type yarrowia lipolytica AS2.1405 AS a template, a promoter pGPD with the size of about 0.94kb and the nucleotide sequence shown in SEQ ID NO.13 is amplified.

GYC1 amplification: through designing primers YW614 and YW615, a promoter GYC1 with the size of about 1.5kb and the nucleotide sequence shown in SEQ ID NO.5 is amplified by taking the genome of wild type yarrowia lipolytica AS2.1405 AS a template.

Amplification of 3' KU 70: by designing primers YW616 and YW617 and using the wild type yarrowia lipolytica AS2.1405 genome AS a template, the 3' downstream of KU70 was amplified to be about 1kb in size and the nucleotide sequence shown in SEQ ID NO. 14.

The PCR amplification conditions of the above 5 'KU 70, Loxp-URA3-Loxp, pTEF, DAK1, pEXP1, DAK2, pGPD, GYC1, 3' KU70 are the same as those in step 1.

The DNA fragments obtained above were assembled into three large fragments, 5 'KU 70-URA3-pTEF-DAK1, DAK1-pEXP1-DAK2-pGPD, pGPD-GYC 1-3' KU70, by overlap extension.

The 5' KU70-URA3-pTEF-DAK1 fragment was constructed by overlap extension. A first round: 5' KU70, URA3, pTEF, DAK1 fragments were mixed at a molar ratio of 1:3:3:1, and the amplification conditions were the same as above. And a second round: PCR amplification was performed by adding 1. mu.l of YW600+ 1. mu.l of YW607 to the PCR product obtained in the first round of amplification as a template.

The overlap extension constructed DAK1-pEXP1-DAK2-pGPD fragment. A first round: DAK1, pEXP1, DAK2 and pGPD fragments were mixed at a molar ratio of 1:3:3:1, and the amplification conditions were the same as above. And a second round: using the PCR product obtained in the first round of amplification as a template, 1. mu.l of YW618+ 1. mu.l of YW613 was added to perform PCR amplification.

The pGPD-GYC 1-3' KU70 fragment was constructed by overlap extension. A first round: pGPD, GYC1, 3' KU70 fragment were mixed at a molar ratio of 1:3:1, and the amplification conditions were the same as above. And a second round: using the PCR product obtained in the first round of amplification as a template, 1. mu.l of YW612+ 1. mu.l of YW617 were added to perform PCR amplification.

Three large fragment gels of 5 'KU 70-URA3-pTEF-DAK1, DAK1-pEXP1-DAK2-pGPD and pGPD-GYC 1-3' KU70 obtained by the above overlap extension are recovered, 50ng of each fragment is mixed and transferred into yarrowia lipolytica recombinant engineering bacteria ZS102, and transformants are respectively identified by PCR with primers YW619+ YW620, YW621+ YW622 and YW226+ YW 227.

TABLE 2 primer List

4. Construction of yarrowia lipolytica ZS106

Amplification of 5' GPD 2: by designing primers YW626 and YW627, using the wild type yarrowia lipolytica AS2.1405 genome AS a template, 5' GPD2 was amplified, the size was about 1kb, and the nucleotide sequence is shown in SEQ ID NO. 15.

Amplification of Loxp-URA 3-Loxp: by designing primers YW628 and YW603 and taking a plasmid JMP113 as a template, Loxp-URA3-Loxp with the size of about 1.8kb and the nucleotide sequence shown in SEQ ID NO.11 are amplified.

Amplification of pTEF: by designing primers YW604 and YW629 and using the genome of wild-type yarrowia lipolytica AS2.1405 AS a template, a promoter pTEF with the size of about 0.4kb and the nucleotide sequence shown in SEQ ID NO.12 is amplified.

Amplification of POS 5: by designing primers YW630 and YW631, a wild yarrowia lipolytica AS2.1405 genome is used AS a template, and a promoter POS5 with the size of about 1.2kb and the nucleotide sequence shown in SEQ ID NO.6 is amplified.

Amplification of 3' GPD 2: by designing primers YW632 and YW633 and taking the wild yarrowia lipolytica AS2.1405 genome AS a template, 3' GPD2 with the size of about 1.2kb and the nucleotide sequence shown in SEQ ID NO.16 is amplified.

The PCR amplification conditions for 5 'GPD 2, Loxp-URA3-Loxp, pTEF, POS5 and 3' GPD2 are the same as those in step 1.

The DNA fragments obtained above were assembled into two large fragments, 5 'GPD 2-URA3-pTEF-POS5, POS 5-3' GPD2, by overlap extension.

The 5' GPD2-URA3-pTEF-POS5 fragment was constructed by overlap extension. A first round: 5' GPD2, URA3, pTEF and POS5 fragments were mixed at a molar ratio of 1:3:3:1, and the amplification conditions were the same as above. And a second round: using the PCR product obtained in the first round of amplification as a template, 1. mu.l of YW626+ 1. mu.l of YW631 was added to perform PCR amplification.

Overlapping extension constructed the POS 5-3' GPD2 fragment. A first round: POS5 and 3' GUT2 fragments were mixed at a molar ratio of 1:1, and the amplification conditions were as above. And a second round: using the PCR product obtained in the first round of amplification as a template, 1. mu.l of YW630+ 1. mu.l of YW633 was added to perform PCR amplification.

Two large fragment gels of 5 'GPD 2-URA3-pTEF-POS5 and POS 5-3' GPD2 obtained by the overlapping extension are recovered, 50ng of each fragment is mixed and transferred into yarrowia lipolytica recombinant engineering bacteria ZS104, and transformants are respectively identified by PCR by using primers YW634+ YW635 and YW636+ YW 637.

TABLE 3 primer List

5. Construction of yarrowia lipolytica engineered bacterium YW100-1

Amplification of 5' DGA 2: by designing primers YW646 and YW647 and taking the wild type yarrowia lipolytica AS2.1405 genome AS a template, 5' DGA2 with the size of about 1kb and the nucleotide sequence shown in SEQ ID NO.7 is amplified.

Amplification of Loxp-URA 3-Loxp: by designing primers YW648 and YW649 and taking a plasmid JMP113 as a template, Loxp-URA3-Loxp with the size of about 1.8kb and the nucleotide sequence shown in SEQ ID NO.11 are amplified.

Amplification of 3' DGA 2: by designing primers YW650 and YW651 and using the wild type yarrowia lipolytica AS2.1405 genome AS a template, 3' DGA2 was amplified, the size was about 1kb, and the nucleotide sequence was shown in SEQ ID NO. 8.

The PCR amplification conditions of 5 'DGA 2, Loxp-URA3-Loxp, and 3' DGA2 are the same as those in step 1.

The DNA fragments obtained above were assembled into a large fragment 5 'DGA 2-URA 3-3' DGA2 using overlap extension. The 5 'DGA 2-URA 3-3' DGA2 fragment gel obtained by the overlapping extension is recovered and then transferred into yarrowia lipolytica recombinant engineering bacterium ZS106, and the transformant is subjected to PCR identification by using primers YW652+ YW 653.

TABLE 4 primer List

The GUT1-pEXP1-GUT2 fragment obtained by overlapping extension is connected with the BamH I linearized plasmid JMP113 plasmid to construct E14 by a one-step cloning method, and then NotI enzyme cuts the E14 plasmid and transfers the E14 plasmid into yarrowia lipolytica AS2.1405 competent cells to construct ZS 102. The large fragments of 5 'KU 70-URA3-pTEF-DAK1, DAK1-pEXP1-DAK2-pGPD and pGPD-GYC 1-3' KU70 obtained by overlap extension were transferred into yarrowia lipolytica ZS102 competent cells to construct ZS 104. The 5 'GPD 2-URA3-pTEF-POS5, POS 5-3' GPD2 large fragment obtained by overlap extension was transferred into yarrowia lipolytica ZS104 competent cells to construct ZS 106. The 5 'DGA 2-URA 3-3' DGA2 large fragment obtained by overlapping extension is transferred into yarrowia lipolytica ZS106 competent cells to construct recombinant engineering bacteria YW100-1 (yarrowia lipolytica YW100-1), which is preserved in China center for type culture Collection in 3.18.2019 with the preservation number of CCTCC NO: M2019168, the address of university of Wuhan, China, postal code 430072.

Example 2: comparison of Shake flask fermentations of wild type AS2.1405 and recombinant engineered Strain YW100-1

Respectively inoculating a wild yarrowia lipolytica strain AS2.1405 and a recombinant engineering bacterium YW100-1 into a YPD liquid culture medium, and culturing at 30 ℃ and 200rpm in a 250mL triangular flask for 24h to obtain a seed solution; at an initial cell concentration OD₆₀₀Inoculating 0.05 into YNG culture medium, respectively, performing three-group parallel analysis of each strain, and performing fermentation culture at 30 deg.C and 200rpm for 16h to OD₆₀₀About 4.5, 10mL of the fermentation broth was used for determination of the concentrations of pyruvic acid and glycerol by high performance liquid chromatography, and the results are shown in FIG. 2.

Determination of the pyruvate concentration: 10mL of fermentation liquor is taken, centrifuged at 6000rpm for 5min, the supernatant is collected, and the content of pyruvic acid is measured by using a C18 column. Mobile phase: 0.1% phosphoric acid; flow rate: 1ml min^-1(ii) a Sample introduction temperature: 28 ℃; sample introduction amount: 10 μ l.

As shown in FIG. 2, the wild type strain was fermented in YNG medium for 16 hours, the yield of pyruvic acid was about 3.5g/L, and the amount of residual glycerol was about 10.2 g/L. The yield of pyruvic acid of the engineering bacteria YW100-1 is about 5.95g/L, and the residual quantity of glycerol is about 7.4 g/L. Therefore, the yield of pyruvic acid produced by fermentation of the engineering bacterium YW100-1 is obviously higher than that of a wild starting bacterium.

Example 3: comparison of wild type AS2.1405 and engineering bacterium YW100-1 for producing pyruvic acid by fermentation in 20L fermentation tank

Seed culture medium: 2g/L glycerol, 0.4g/L tryptone, 0.2g/L yeast extract, 0.24g/L KH₂PO₄、1.7g/L K₂HPO₄·3H₂O, the solvent is distilled water, and the pH value is natural.

Fermentation medium groupThe composition is as follows: 60g/L of glycerin, 10g/L of (NH)₄)₂SO₄、1.4g/L MgSO₄·7H₂O、2g/L KH₂PO₄、0.8g/L CaCl₂0.5g/L NaCl, 1 mu g/L vitamin B1, and the solvent is distilled water, and the pH value is natural.

Wild-type yarrowia lipolytica strain AS2.1405 and engineered strain YW100-1 were inoculated into YPD liquid medium, respectively, and cultured overnight at 30 ℃, then 200ml was inoculated into 3L seed medium, shake-flask culture was carried out at 30 ℃ for 18h, then inoculation was carried out at 10% volume concentration in 20L fermentor containing 12L fermentation medium, and fermentation culture was carried out for 1 h. In the whole fermentation culture process, 20% NaOH is added to regulate pH value to 4.0, dissolved oxygen is kept at about 40%, and fermentation temperature is controlled at 30 ℃. 40g/L of glycerol was added to the fermentor at 50 and 64 hours, respectively, to add 1440g of glycerol.

The process of producing pyruvic acid by glycerol fermentation of two strains is shown in FIG. 3. The wild strain is fermented for 120 hours, the yield of the pyruvic acid reaches the maximum value of 72.9g/L, and the conversion rate of the pyruvic acid/glycerol is about 0.52 g/g. The engineering bacteria YW100-1 is fermented for 120h, the yield of the pyruvic acid reaches the maximum value of 121.2g/L, and the conversion rate of the pyruvic acid/glycerol reaches 0.865 g/g. Compared with the fermentation strain, the fermentation pyruvic acid yield of the engineering strain YW100-1 is improved by 66.2 percent and is obviously higher than other gene engineering strains with the highest reported yield. Therefore, the yarrowia lipolytica recombinant strain YW100-1 can provide an excellent strain for industrial production of pyruvic acid.

Sequence listing

<110> Zhejiang industrial university

<120> yarrowia lipolytica YW100-1 and application thereof

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1512

<212> DNA

<213> Unknown (Unknown)

<400> 1

atgtcttcct acgtaggagc tctcgaccag ggtaccacct ccacccgttt cattctcttt 60

tcgcctgacg gcaagcccgt ggcatcccac cagatcgaat tcacccagat ctacccccac 120

cccggatggg tggagcacga ccccgaggag ctcgtgagct cgtgtctgga gtgcatgtcg 180

tcggtggcca aggaaatgcg aacccagggc atcaaggtgg ccgacgtgaa ggcgatcgga 240

atcaccaacc agcgagaaac caccgtgctt tgggacattg agaccggcca gcccctgtac 300

aacgccattg tgtggtccga cgcccgaacc ggcgacaccg tcaagaagct cgaggcccag 360

cccggcgctg acgaaatccc caagctctgt ggcctgcccc tgtccaccta ctttgccgga 420

gtcaaggtcc gatggatcct ggataacgtc aaggaggccc gagagtgcta cgatcgaggc 480

aagctggcct tctccaccat cgactcgtgg ctgctctaca acctcacggg cggcctcaac 540

ggcggcgccc acatcaccga cacctccaac gcctcccggt ccatgttcat gaacattgag 600

accctcaagt acgacgagaa gctcatcaag ttctttggcg tcgagaagct cattctcccc 660

aagattgtct cgtccgccga ggtctacggc cgaatcggaa ccggcccctt cgccaacatc 720

cccctggccg gctgtctcgg tgaccagtcc gccgccctcg tcggccagaa ggcctttgag 780

cccggccagg ccaagaacac atatggaacc ggctgcttcc tgctctacaa cgccggcgag 840

aagcccatca tctccaacaa cggcctgctg accaccgtcg gctaccactt caagggccag 900

aagcccgtct acgctctgga gggctccatc tccgtcgccg gctcgtgcat caagtggctg 960

cgagacaaca ttggtctcat tgagtcttcc gagcagattg gagagcttgc ctcccaggtc 1020

gacgactctg ccggcgtggt gtttgtcacc gctctgtccg gcctgtttgc cccctactgg 1080

cgaaccgacg cccgaggcac cattctgggt ctcactcagt tcaccaccaa ggcccacatt 1140

tgccgagccg ccctggaggc tacttgtttc cagacccggg ccattctcga cgccatggcc 1200

aaggactctg gtaagccctt caccaagctg cgagtcgacg gaggaatgac caactcggac 1260

attgctatgc agatccaggc cgacattctt ggcattgagg tcgagcgacc cgccatgcga 1320

gagaccaccg ctctgggtgc cgccattgct gccggctttg ccgttggcgt gtggaagtcc 1380

attgaggatc ttaaggacat caacaccgag ggcatgaccg agtttgcttc caagaccaac 1440

gaggaggagc gggccgccat gatgaagcag tggaaccggg gcattgagcg agctgttggc 1500

tggcttgagt aa 1512

<210> 2

<211> 1839

<212> DNA

<213> Unknown (Unknown)

<400> 2

atgttcagaa ccattcgaaa acccgcgtgg gctgctgccg ccgccgtggc agccgctggc 60

gctggagccg tcgccctgtc tgtgcctgcc caggcccagg aggagctcca caagaagcac 120

aaattcacag tgccccccgt ggccgccgag cccccctctc gagccgccca gctcgagaag 180

atgaagaccg aggagtttga tctcgtcgtt gttggtggag gagctaccgg atccggtatc 240

gccctcgacg ctgtcacacg aggcctcaag gttgctctgg tcgagcgaga cgatttctcc 300

tgcggaacct cgtcccgatc caccaagctc atccacggag gtgtccgata cctcgagaag 360

gctgtgtgga acctcgacta caaccagtac gagctggtca aggaggccct gcacgagcga 420

aaggtcttcc tcgacattgc tccccacctc acctttgctc tgcccatcat gatccccgtc 480

tacacctggt ggcagcttcc ctacttctgg atgggtgtca agtgctacga tctgcttgcc 540

ggccgacaga acctcgagtc ctcttacatg ctctcccgat cccgtgctct cgatgccttc 600

cccatgcttt ccgatgacaa gctcaagggc gccattgtct actatgatgg ctcccagaac 660

gactctcgaa tgaacgtttc tcttattatg actgctgttg agaagggtgc caccatcctg 720

aaccattgcg aggtcaccga gctcaccaag ggcgccaatg gccagctcaa cggtgttgtt 780

gccaaggata ctgacggaaa cgctggatcc ttcaacatca aggccaagtg tgtcgttaat 840

gctactggac ccttcactga ctctctgcga cagatggacg acaagaacac caaggagatc 900

tgtgctcctt cctccggtgt tcacatcatt ctccccggtt actactcccc caagaagatg 960

ggactccttg accccgctac ttctgacggc cgagttatct tcttcctccc ctggcaggga 1020

aacacccttg ccggtactac tgaccagcct accaagatca ctgctaaccc tatcccctcc 1080

gaggaggaca ttgacttcat tctcaacgag gtccgacact acgttgaggg caaggttgat 1140

gtgcgacgag aggacgttct ggccgcctgg tccggaatcc gaccccttgt ccgggacccc 1200

cacgccaaga acaccgagtc tcttgtccga aaccatctca tcacctactc cgagtctggt 1260

cttgtcacca ttgctggcgg aaagtggacc acttaccgac agatggctga ggagactgtc 1320

gatgcctgca ttgccaagtt cggtctcaag cctgaaatct ccgccaaggc cgtcacccga 1380

gacgtcaagc tcatcggtgc taaggactgg actcctctca cttacattga tctgatccag 1440

caggaggacc ttgaccccga ggttgctaag cacctttctg agaactacgg atctcgagct 1500

ttcaccgttg cttctcttgc tgagatgccc acccccgaac ccggtgtgat cccccagtct 1560

actctcacaa agggtaagcg aatcctgtac ccctacccct acctcgatgc cgagtgcaag 1620

tactctatga agtacgagta tgccaccacc gccatcgact tccttgctcg acgaactcgt 1680

cttgctttcc ttaacgccgc tgccgcctac gaggctctcc ctgaggtcat tgagatcatg 1740

gccaaggagc tccagtggga cgaggctcga aaggagcagg aattcaacac cggtgtcgag 1800

tacctctact ccatgggcct tacccccaag gacaaataa 1839

<210> 3

<211> 1014

<212> DNA

<213> Unknown (Unknown)

<400> 3

atgttccggt cagtatataa acgggggcga atcctgtcca aaatccccat caaacaacac 60

atccagatca cccacaaaca cacaatgact tctctcgact ttactctcaa caacggcaag 120

accatccctg ccatcggtct tggaacctgg aagtccacca ccgaggaggt ggctggcgcc 180

gtcgagtgcg ccctcaccga gggaggctac cgacacattg acaccgcctt caactaccga 240

aacgaagacg ccgtcggact cggaatcaag cgagccatgg agaagggtgt caagcgagaa 300

gacatcttcg tcaccaccaa gatctgggtc acctaccacg accgagtcga ggagaacctc 360

gacatgtctc tggagcgact gggtcttgac tacgtcgaca tgctcctcat ccactggccc 420

gttcccctca accctaacgg taacgacccc gtctaccccc tgcgacccga tggctctcga 480

gacattgacg agtccggctc ccagcccaag acctggaagc agatggaggc tgttctgaag 540

accggcaaga ccaagtctat cggtgtctcc aacttctcca tcccttacct cgaggagctg 600

ctcaaggagg ccgaggttgt ccccgccgtc aaccaggtcg agctccaccc tctgctgccc 660

cagctcgagc tcatggaatt ctgcaagaag aacaacattg tcatgaccgc cttctctccc 720

tttggctctg tcggtggccc tctgctcaag aacgagctcg tcgtctctct ggccgacaag 780

tacaacacct ctcccggagg aatcctcacc tcctaccaca ttggtaacgg caccgtggtc 840

atccccaagt ctgtcaccaa ctctcgaatc gtcgagaacg gaaagtccgc cgtcaccctg 900

tcccaggagg acctcaaggc tctgaacgac ctccacaaga ccgagggtat ccaccgaacc 960

tccaagccca agtggggtgt tgacctcgga ttccccgact ttgacttctg ctaa 1014

<210> 4

<211> 1719

<212> DNA

<213> Unknown (Unknown)

<400> 4

atgaccacta aacagttcca attcgactcg gatccgctca attctgccct tgccgccacc 60

gcggaggcct caggcctcgc ttacctcccc aagagcaagg tcatctacta ccctctgacc 120

aacgacaagg tgacgttgat ttcaggtgga ggagctggcc acgagcctgc tcagaccggg 180

tttgtgggtc ccggactgct ggatgcggcc gtgtcgggcc agatctttgc ctcaccttcc 240

accaaacaga tcattgccgg agtcaatgcc gtcaagtcgc aacggggctc catcattatc 300

gtcatgaact acactggcga tgtgatccac tttggaatgg ccgccgagca gctgcggtcc 360

cgatatgact accacgccga actggtgtcc attggcgacg acatttccgt caacaagaag 420

gccggacgac gaggtctggc aggaaccgtt cttgttcaca agatcgcagg ccatcttgcc 480

cgagatggct gggacgtcgg agtgcttgct gaagctctgc gaaccaccgc cgccaacctg 540

gccaccgtgg ctgcgtctct ggaacactgc actgtacctg gcagaaagtt cgagaccgaa 600

ctggcggccg atgagatgga gattggcatg ggtatccaca acgagcccgg tgtcaagacc 660

atcaagattg gcaaggttga gtctctgctg gacgaattgg tcgacaagtt cgagccctcc 720

aagcaggact ttgtgccctt caacaagggc gacgaggtgg tgctgctggt caattccctc 780

ggaggagtct cttctctgga actccacgcc attgccaaca ttgcccagac aaagttcgag 840

aaggtgctgg gcgtcaagac cgtgcgactt attgttggca acttcatggc tgccttcaac 900

ggtcctggct tctctttgac tctgctcaac gtcaccacga ccgccaagaa gggcaacttt 960

gacgttctgg gagccctgga cgctcccgtg tccaccgccg cctggccctc tctgcagcag 1020

aaggacaagc ctgccaacgg cggtgtccag gaggagaagg agaccgactc ggacaagcct 1080

gctgagccta ctggaatcaa ggccgacgga aagctgttca aggccatgat tgagagtgct 1140

gttgacgatc tcaagaagga ggagccccag attaccaaat acgacactat tgctggcgat 1200

ggagactgtg gagagactct gttggctgga ggcgacggta ttctggacgc tatcaagaac 1260

aagaagattg accttgatga tgccgctgga gtggctgata tttctcacat cgtcgagaac 1320

tccatgggag gcacctcggg aggtctctac tccatcttct tctccggtct cgtggtcggt 1380

atcaaggaga ccaaggccaa ggagctgtct gtcgatgtgt ttgccaaggc atgtgagact 1440

gctctggaga ctctttctaa gtacacccag gcccgagtcg gcgaccgaac cctcatggac 1500

gcacttgttc cctttgtaga gaccctcagc aagaccaagg acttcgccaa ggccgtagag 1560

gctgctcgga agggcgccga cgagacttcc aagctgcctg ccaattttgg ccgtgcctcg 1620

tatgtgaacg aggagggatt ggagaacatt cctgaccctg gagctcttgg actggccgtc 1680

attttcgaag gtcttctcaa ggcctgggag aagaagtag 1719

<210> 5

<211> 1692

<212> DNA

<213> Unknown (Unknown)

<400> 5

atgaaacact tcgccaaaac gaatctcgtc aatctctacc tcgaatctct cctggctagc 60

aacccgcaac tgggtctagt ggaagatcag agaatcattt attacaagaa gaaaaagtcg 120

gacaaggtcc gagttatttc gggcggcgga tcgggccatg agcccagctg gagcggtctt 180

gtgggctcgg gactactaga tgctgctgtc tgtggagaca tttttgcatc tccttcggcc 240

agacaggtta tggccggtat cagagcctcg gaacccgaca gtggcgttat tctggcaatc 300

acaaactaca cgggcgacaa gctgcacttt ggactggccc aagagaagtt ccaggccgag 360

tctggaggca tgcaggtggc agtcatccct gtgactgatg atgtcgctct cggacgaacc 420

cggtcctcaa aggtcggaag acggggactg gcgggaaacc tgcttgttct caagtccatg 480

ggagcctgtg ctgaggctgg aggttccttt gatcacgtct ccaatgtagg acgggcagtg 540

aacgacggac tggtcacggt gggatgttct ctggaccact gcagtgttcc tggtcgaaca 600

gacgtggact ttcatatccc tcatgacaag gctgtacttg gaatgggtat tcacaacgaa 660

cgaggacttg ttgaggtcga cattcccgaa cggcctgaag atctcatcaa acagatgttg 720

actcttttgc tagaccccaa cgacaaggag cgagcctttg tgtccttcaa ggagaaggac 780

gaggttattc tgctggtcaa caactttggt gggctgtcta atctcgagaa tggagcccta 840

actcaagtgg ccctgtctgt tctggagcag gattataaca ttgttccctg tcgagtcctg 900

tctggagcct ttgagacgtc gctagacggc ccaggctttt caatcactct ttacaaccct 960

tcatactctg caactctcgt tgaaaaatta tctagcaaac agcttctaga gctcatcgat 1020

gctccaactg atgctcctgc ttggccaagg gtcggtgtca acgagcccaa gaaacagaag 1080

gtgctctcca agcaggagga gctagcggcc aaggactgcg aagagtcgcc ttatgacgag 1140

cttgtttcgc gtatttgcaa acatgtcatt tcaatcgagc cttctctcac cacctgggat 1200

actgtaatgg gtgatggaga ctgcggtatg gcagccaaag acgcggcact tcacattcaa 1260

aaggagtgga attcccgcaa gcagtcttct ttaaagggaa ctcttaatct cctctcgtcc 1320

tgcctggatg acatgggtgg ctctctggga gccattctgg gcatctttgt tagtgctctc 1380

atctacaacc tgcaaaagga aggagttgaa caggctccaa aggcggttgg attggcttca 1440

aaatctctcc agacacacac acaggctcgc aagggtgacc gaacggtcat ggactctctg 1500

attcccttct gtgaagtcta cgcctcgtct ggaagtcttc aacatgcagc caaagccgct 1560

caggagggag cagaaagcac aaagaccctc aaagctcagt atggacgagc cagttatgtt 1620

tcaaagaccg cagatgttcc cgatcccgga gcctggctgt ttgccgcagt tgttgaccag 1680

ctttccaagt ag 1692

<210> 6

<211> 1581

<212> DNA

<213> Unknown (Unknown)

<400> 6

atggaagtcc gacgacgaaa aatcgacgtg ctcaaggccc agaaaaacgg ctacgaatcg 60

ggcccaccat ctcgacaatc gtcgcagccc tcctcaagag catcgtccag aacccgcaac 120

aaacactcct cgtccaccct gtcgctcagc ggactgacca tgaaagtcca gaagaaacct 180

gcgggacccc cggcgaactc caaaacgcca ttcctacaca tcaagcccgt gcacacgtgc 240

tgctccacat caatgctttc gcgcgattat gacggctcca accccagctt caagggcttc 300

aaaaacatcg gcatgatcat tctcattgtg ggaaatctac ggctcgcatt cgaaaactac 360

ctcaaatacg gcatttccaa cccgttcttc gaccccaaaa ttactccttc cgagtggcag 420

ctctcaggct tgctcatagt cgtggcctac gcacatatcc tcatggccta cgctattgag 480

agcgctgcca agctgctgtt cctctctagc aaacaccact acatggccgt ggggcttctg 540

cataccatga acactttgtc gtccatctcg ttgctgtcct acgtcgtcta ctactacctg 600

cccaaccccg tggcaggcac aatagtcgag tttgtggccg ttattctgtc tctcaaactc 660

gcctcatacg ccctcactaa ctcggatctc cgaaaagccg caattcatgc ccagaagctc 720

gacaagacgc aagacgataa cgaaaaggaa tccacctcgt cttcctcttc ttcagatgac 780

gcagagactt tggcagacat tgacgtcatt cctgcatact acgcacagct gccctacccc 840

cagaatgtga cgctgtcgaa cctgctgtac ttctggtttg ctcccacact ggtctaccag 900

cccgtgtacc ccaagacgga gcgtattcga cccaagcacg tgatccgaaa cctgtttgag 960

ctcgtctctc tgtgcatgct tattcagttt ctcatcttcc agtacgccta ccccatcatg 1020

cagtcgtgtc tggctctgtt cttccagccc aagctcgatt atgccaacat ctccgagcgc 1080

ctcatgaagt tggcctccgt gtctatgatg gtctggctca ttggattcta cgctttcttc 1140

cagaacggtc tcaatcttat tgccgagctc acctgttttg gaaacagaac cttctaccag 1200

cagtggtgga attcccgctc cattggccag tactggactc tatggaacaa gccagtcaac 1260

cagtacttta gacaccacgt ctacgtgcct cttctcgctc ggggcatgtc gcggttcaat 1320

gcgtcggtgg tggttttctt tttctccgcc gtcatccatg aactgcttgt cggcatcccc 1380

actcacaaca tcatcggagc cgccttcttc ggcatgatgt cgcaggtgcc tctgatcatg 1440

gctactgaga accttcagca tattaactcc tctctgggcc ccttccttgg caactgtgca 1500

ttctggttca cctttttcct gggacaaccc acttgtgcat tcctttatta tctggcttac 1560

aactacaagc agaaccagta g 1581

<210> 7

<211> 1838

<212> DNA

<213> Unknown (Unknown)

<400> 7

atgttcagaa ccattcgaaa acccgcgtgg gctgctgccg ccgccgtggc agccgctggc 60

gctggagccg tcgccctgtc tgtgcctgcc caggcccagg aggagctcca caagaagcac 120

aaattcacag tgccccccgt ggccgccgag cccccctctc gagccgccca gctcgagaag 180

atgaagaccg aggagtttga tctcgtcgtt gttggtggag gagctaccgg atccggtatc 240

gccctcgacg ctgtcacacg aggcctcagg ttgctctggt cgagcgagac gatttctcct 300

gcggaacctc gtcccgatcc accaagctca tccacggagg tgtccgatac ctcgagaagg 360

ctgtgtggaa cctcgactac aaccagtacg agctggtcaa ggaggccctg cacgagcgaa 420

aggtcttcct cgacattgct ccccacctca cctttgctct gcccatcatg atccccgtct 480

acacctggtg gcagcttccc tacttctgga tgggtgtcaa gtgctacgat ctgcttgccg 540

gccgacagaa cctcgagtcc tcttacatgc tctcccgatc ccgtgctctc gatgccttcc 600

ccatgctttc cgatgacaag ctcaagggcg ccattgtcta ctatgatggc tcccagaacg 660

actctcgaat gaacgtttct cttattatga ctgctgttga gaagggtgcc accatcctga 720

accattgcga ggtcaccgag ctcaccaagg gcgccaatgg ccagctcaac ggtgttgttg 780

ccaaggatac tgacggaaac gctggatcct tcaacatcaa ggccaagtgt gtcgttaatg 840

ctactggacc cttcactgac tctctgcgac agatggacga caagaacacc aaggagatct 900

gtgctccttc ctccggtgtt cacatcattc tccccggtta ctactccccc aagaagatgg 960

gactccttga ccccgctact tctgacggcc gagttatctt cttcctcccc tggcagggaa 1020

acacccttgc cggtactact gaccagccta ccaagatcac tgctaaccct atcccctccg 1080

aggaggacat tgacttcatt ctcaacgagg tccgacacta cgttgagggc aaggttgatg 1140

tgcgacgaga ggacgttctg gccgcctggt ccggaatccg accccttgtc cgggaccccc 1200

acgccaagaa caccgagtct cttgtccgaa accatctcat cacctactcc gagtctggtc 1260

ttgtcaccat tgctggcgga aagtggacca cttaccgaca gatggctgag gagactgtcg 1320

atgcctgcat tgccaagttc ggtctcaagc ctgaaatctc cgccaaggcc gtcacccgag 1380

acgtcaagct catcggtgct aaggactgga ctcctctcac ttacattgat ctgatccagc 1440

aggaggacct tgaccccgag gttgctaagc acctttctga gaactacgga tctcgagctt 1500

tcaccgttgc ttctcttgct gagatgccca cccccgaacc cggtgtgatc ccccagtcta 1560

ctctcacaaa gggtaagcga atcctgtacc cctaccccta cctcgatgcc gagtgcaagt 1620

actctatgaa gtacgagtat gccaccaccg ccatcgactt ccttgctcga cgaactcgtc 1680

ttgctttcct taacgccgct gccgcctacg aggctctccc tgaggtcatt gagatcatgg 1740

ccaaggagct ccagtgggac gaggctcgaa aggagcagga attcaacacc ggtgtcgagt 1800

acctctactc catgggcctt acccccaagg acaaataa 1838

<210> 8

<211> 1200

<212> DNA

<213> Unknown (Unknown)

<400> 8

atgcgactac tcatccgccg aaccggtata acacggcccc acagcgtgca agcgcgccga 60

tccacatgga ttcggcttct ctcgaccgag atattgcatg cagaactgct tcccgaccgc 120

cagtcgcccc actacgtcca ggagtcgacc tctctgtcat ctctggtgtg ggacaagcct 180

ctggaaaacg ttctgatcgt caaaaaaccc tgggaccaca atgtgcgcga gtcgctcatc 240

cagatggcat ctcacatcca gcgccggtac ccccgagtca acattctggt ggaggaacat 300

gtggccgacg aggtccagaa gcagattgga gccgcaggcg tgaccgccat ccacacgggg 360

ccaggagagg tgctgagaaa caagacggat ctgctcgtga ctctgggagg cgacggaact 420

attctacatg ccacctccat gtttgcttcc ggagaagtgc cgccggtgct gtccttttcg 480

ctggggactc tgggtttcct gctgccgttt gatttcaagg acttcaaaac tgcattcgac 540

atggtgtact cgtcgcaggc ctcggtggtc aaccgcgccc gcctagcatg tcagaaaatg 600

tccattcgca aggaaatcac ccacttgccc tcccaatcgc acattgaaca caactcaacc 660

catgtctacg gcaatcccga cgactacaat cttagcccac taacctacgc catgaacgac 720

atcaacatcc accgtggagc tgagccgcat ctcaccaagc tcgacatcca cgttgacggc 780

gagttcatca cccgagccat tgctgacggt gtcaccatcg ccacacccac gggctccacg 840

gcctactcgc tgtcgtctgg cggctccatt gtgcatcccc gagtcgcctg cattctgctg 900

acccccatct gtccgcgatc gctgtcattc cggcctctca ttttcccagc cacctccaaa 960

atatgcatca ccgcctcgtc cgaatctcga ggtagaggcg ccgagctgtc tgtcgacgga 1020

atcgccaagg gtctggttcg acccagcgac aagattctgg tcgaaagcga aaccggccac 1080

aactcgggca tctggtgcgt ggccaagaca gacagagact gggtcagtgg cctcaacggg 1140

ttactgggct tcaatagcag ttttggcaag ggcggggagg cgtcaggcga tgttgcttag 1200

<210> 9

<211> 1000

<212> DNA

<213> Unknown (Unknown)

<400> 9

ggagtttggc gcccgttttt tcgagcccca cacgtttcgg tgagtatgag cggcggcaga 60

ttcgagcgtt tccggtttcc gcggctggac gagagcccat gatgggggct cccaccacca 120

gcaatcaggg ccctgattac acacccacct gtaatgtcat gctgttcatc gtggttaatg 180

ctgctgtgtg ctgtgtgtgt gtgttgtttg gcgctcattg ttgcgttatg cagcgtacac 240

cacaatattg gaagcttatt agcctttcta ttttttcgtt tgcaaggctt aacaacattg 300

ctgtggagag ggatggggat atggaggccg ctggagggag tcggagaggc gttttggagc 360

ggcttggcct ggcgcccagc tcgcgaaacg cacctaggac cctttggcac gccgaaatgt 420

gccacttttc agtctagtaa cgccttacct acgtcattcc atgcatgcat gtttgcgcct 480

tttttccctt gcccttgatc gccacacagt acagtgcact gtacagtgga ggttttgggg 540

gggtcttaga tgggagctaa aagcggccta gcggtacact agtgggattg tatggagtgg 600

catggagcct aggtggagcc tgacaggacg cacgaccggc tagcccgtga cagacgatgg 660

gtggctcctg ttgtccaccg cgtacaaatg tttgggccaa agtcttgtca gccttgcttg 720

cgaacctaat tcccaatttt gtcacttcgc acccccattg atcgagccct aacccctgcc 780

catcaggcaa tccaattaag ctcgcattgt ctgccttgtt tagtttggct cctgcccgtt 840

tcggcgtcca cttgcacaaa cacaaacaag cattatatat aaggctcgtc tctccctccc 900

aaccacactc acttttttgc ccgtcttccc ttgctaacac aaaagtcaag aacacaaaca 960

accaccccaa cccccttaca cacaagacat atctacagca 1000

<210> 10

<211> 1193

<212> DNA

<213> Unknown (Unknown)

<400> 10

caatgaatta agtctccgtg ttaccatctt agatatgaac actaaatatg ccaagttctc 60

tttcccctac atgattcata gcacttgccc aaagacgagg agactttctt cttacgacat 120

atacatcaat agaccattca tggcaagaaa gaagtactcg aacataggcc cataaagtac 180

gagtacagta cactacacta cactacttgt accattctac ccggggtctg ccggcttgta 240

cacaccgaca gcactcgtac tctcccacga atgctccggc tgccgacatc aacacgatct 300

caaaagcgca tactgagctt cctttcctag ctcttccttc cttcaactcg ataaatacat 360

tggatatata catgtgtggc gactgtcgac ttgatgttta gagtgtccag atccgcaaga 420

tcggctcgca cttgtgttgt gttgtttcaa atcagcctgt cgttttgtgt cgtttgagat 480

cattctgtct cactcttagg ctcgcttaga accgacaacg gagaatccgg gctcggtttt 540

tcggtcggcc ttgatctggg ccttggactt gtactggtcg gccatctcca cgttgaccag 600

ctccttgacc ttgtagagct gaccggcgat accaggagac accttgtagt acttctggga 660

gccgaccttg cccagaccga gggtcttgag cacgtcacgt gttctccacg gcattcgcag 720

gatagatcgg acctgtgtga ctttgtagaa catggcgttt caggtggttg cgtgagtgtg 780

taaaatcgtg tctttcagaa gttacaaatt tcaccgcatt tagagtttat gcagatgggc 840

ggtgtgtggt tgggagttcg atttccgtgc gtgcatttga tcttgatgaa ttggatttgt 900

acatgaggaa gagcacgtca agcaccgcct actgcaaact cgtgaatatt gagattattg 960

aggaaattca aggaaaattc agatcagatt tgagagcaaa gtccaacaat actacacaat 1020

ccctttcctg tattcttcca ccatcgtcat cgtcgtctgt cttctcttca gctttttaat 1080

ttcactcccc acaaacccaa atttagctgc atcattcatc aacctccaat tataactata 1140

catcgcgaca cgaacacgaa acacgaacca cgaaccgccg ctttttgaaa atg 1193

<210> 11

<211> 1726

<212> DNA

<213> Unknown (Unknown)

<400> 11

gcactttgct agatagagtc gagaattacc ctgttatccc tagataactt cgtatagcat 60

acattatacg aagttattct gaattccgag aaacacaaca acatgcccca ttggacagac 120

catgcggata cacaggttgt gcagtaccat acatactcga tcagacaggt cgtctgacca 180

tcatacaagc tgaacagcgc tccatacttg cacgctctct atatacacag ttaaattaca 240

tatccatagt ctaacctcta acagttaatc ttctggtaag cctcccagcc agccttctgg 300

tatcgcttgg cctcctcaat aggatctcgg ttctggccgt acagacctcg gccgacaatt 360

atgatatccg ttccggtaga catgacatcc tcaacagttc ggtactgctg tccgagagcg 420

tctcccttgt cgtcaagacc caccccgggg gtcagaataa gccagtcctc agagtcgccc 480

ttaggtcggt tctgggcaat gaagccaacc acaaactcgg ggtcggatcg ggcaagctca 540

atggtctgct tggagtactc gccagtggcc agagagccct tgcaagacag ctcggccagc 600

atgagcagac ctctggccag cttctcgttg ggagagggga ctaggaactc cttgtactgg 660

gagttctcgt agtcagagac gtcctccttc ttctgttcag agacagtttc ctcggcacca 720

gctcgcaggc cagcaatgat tccggttccg ggtacaccgt gggcgttggt gatatcggac 780

cactcggcga ttcggtgaca ccggtactgg tgcttgacag tgttgccaat atctgcgaac 840

tttctgtcct cgaacaggaa gaaaccgtgc ttaagagcaa gttccttgag ggggagcaca 900

gtgccggcgt aggtgaagtc gtcaatgatg tcgatatggg tcttgatcat gcacacataa 960

ggtccgacct tatcggcaag ctcaatgagc tccttggtgg tggtaacatc cagagaagca 1020

cacaggttgg ttttcttggc tgccacgagc ttgagcactc gagcggcaaa ggcggacttg 1080

tggacgttag ctcgagcttc gtaggagggc attttggtgg tgaagaggag actgaaataa 1140

atttagtctg cagaactttt tatcggaacc ttatctgggg cagtgaagta tatgttatgg 1200

taatagttac gagttagttg aacttataga tagactggac tatacggcta tcggtccaaa 1260

ttagaaagaa cgtcaatggc tctctgggcg gaattcgtat aacttcgtat agcaggagtt 1320

atccgaagcg ataattaccc tgttatccct agaatcgata gagaccgggt tggcggcgca 1380

tttgtgtccc aaaaaacagc cccaattgcc ccaattgacc ccaaattgac ccagtagcgg 1440

acccaacccc ggcgagagcc cccttcaccc cacatatcaa acctcccccg gttcccacac 1500

ttgccgttaa gggcgtaggg tactgcagtc tggaatctac gcttgttcag actttgtact 1560

agtttctttg tctggccatc cgggtaaccc atgccggacg caaaatagac tactgaaaat 1620

ttttttgctt tgtggttggg actttagcca agggtataaa agaccaccgt ccccgaatta 1680

cctttcctct tcttttctct ctctccttgt caactcacac ccgaag 1726

<210> 12

<211> 444

<212> DNA

<213> Unknown (Unknown)

<400> 12

gcaaaggagg ggctggtgag ggcgtctgga agtcgaccag agaccgggtt ggcggcgcat 60

ttgtgtccca aaaaacagcc ccaattgccc caattgaccc caaattgacc cagtagcggg 120

cccaaccccg gcgagagccc ccttctcccc acatatcaaa cctcccccgg ttcccacact 180

tgccgttaag ggcgtagggt actgcagtct ggaatctacg cttgttcaga ctttgtacta 240

gtttctttgt ctggccatcc gggtaaccca tgccggacgc aaaatagact actgaaaatt 300

tttttgcttt gtggttggga ctttagccaa gggtataaaa gaccaccgtc cccgaattac 360

ctttcctctt cttttctctc tctccttgtc aactcacacc cgaaatcgtt aagcatttcc 420

ttctgagtat aagaatcatt caaa 444

<210> 13

<211> 941

<212> DNA

<213> Unknown (Unknown)

<400> 13

ataaaccgga cgcagtagga tgtcctgcac gggtcttttt gtggggtgtg gagaaagggg 60

tgcttggaga tggaagccgg tagaaccggg ctgcttgggg ggatttgggg ccgctgggct 120

ccaaagaggg gtaggcattt cgttggggtt acgtaattgc ggcatttggg tcctgcgcgc 180

atgtcccatt ggtcagaatt agtccggata ggagacttat cagccaatca cagcgccgga 240

tccacctgta ggttgggttg ggtgggagca cccctccaca gagtagagtc aaacagcagc 300

agcaacatga tagttggggg tgtgcgtgtt aaaggaaaaa aaaagaagct tgggttatat 360

tcccgctcta tttagaggtt gcgggataga cgccgacgga gggcaatggc gccatggaac 420

cttgcggata tcgatacgcc gcggcggact gcgtccgaac cagctccagc agcgtttttt 480

ccgggccatt gagccgactg cgaccccgcc aacgtgtctt ggcccacgca ctcatgtcat 540

gttggtgttg ggaggccact ttttaagtag cacaaggcac ctagctcgca gcaaggtgtc 600

cgaaccaaag aagcggctgc agtggtgcaa acggggcgga aacggcggga aaaagccacg 660

ggggcacgaa ttgaggcacg ccctcgaatt tgagacgagt cacggcccca ttcgcccgcg 720

caatggctcg ccaacgcccg gtcttttgca ccacatcagg ttaccccaag ccaaaccttt 780

gtgttaaaaa gcttaacata ttataccgaa cgtaggtttg ggcgggcttg ctccgtctgt 840

ccaaggcaac atttatataa gggtctgcat cgccggctca attgaatctt ttttcttctt 900

ctcttctcta tattcattct tgaattaaac acacatcaac a 941

<210> 14

<211> 1219

<212> DNA

<213> Unknown (Unknown)

<400> 14

ctagggaggc acatctaaac gaataacgaa tattaatgat accatcatat ctcagaacat 60

gtatgactgc tgcttccaaa cgatatgagg atgagtcctc tttcagatta agatagagta 120

caaatatatt atctatatac tggtgtctgt gcgatgtcgt atgagcggtg aatcatgtga 180

ctgtcacgtg gtttggccca agttacaccg tagctacgcc tttcttgacc gtctccatgg 240

tcttctgggc gggttgacag tttccactgg atgagcgtcc gcctcctgtt cctgtcgttg 300

tccctgcagc tcagcctcaa tcttctgacc gagctcggag tccagggaaa tgccaacagg 360

ttgtccaagc aacatcatgg tttggtgggc agccgtgatc tcatcgtcgt tggataccat 420

tcggtacttg gcctcaatct gcacaaagta gcggtaccac tggtttcgag caaaccgctc 480

caattgagcc tctccgtcga gagagagagt aggtgattgc tccaacttgc ggccaaaatg 540

aagttctcga ctcacctttt tgaagcggtt cttcttgccc atcttggtgg cgaaagtagt 600

ggctagtggt ggatgacttt gtataatgta ccgatgaaga gggttgtatt tgctcagtaa 660

gaagtagcga gtgaaatcag atgacttaac gagagcaaag ggcaatggaa tacctgctgc 720

ctgattaaca acagcttctg tgtcgtttct ctcttgtgaa tgagtgtgtt gctagaggta 780

ggttggcact ccaatgttac gacacacaat agtctataga gcactacaaa gggctatatc 840

gtcaactgct ctattgtagc tacagtacag tacataccat caagtgaaca atggaccacc 900

aaactcggca ctaagccaat agaacctttg cggcctcctt tatcacgttt ctatatacct 960

tgtccattta tgtgccaccc tttagtcttg gtcgttcact tcagctcaac ttcagccatg 1020

atagcaagat gatctgaagg atacatgtca atgcgaggct gaccactggg ctcgggcccc 1080

atatcctcct caaggggcat cttcaacaga ctcttgacct ggacctcatc gctgttgttg 1140

gacgaaacga aaatgtagtc caaaagaccc ctccaggcgt gggcccagtt actgaagcga 1200

ggctccttct tgtgcttgc 1219

<210> 15

<211> 1034

<212> DNA

<213> Unknown (Unknown)

<400> 15

agcggtttgt tttctatggc atgttgttga cgcatgctgc caacggctat tcaacggtga 60

caacggatga tgctgtcaca tgacgccatt ttttatgttg tatccaacag cacggtacta 120

aaacaggcca tttgtaaagg cctcactcag ctcacacacg ctcaacggtc acgataaggt 180

cgcactagag gcgttagttg gtttcaagaa tagtggttat tggtcttggg atacgggttg 240

gacaatatac aaatgggctc gcgtacactt atacagtcct accattctgt cgccctctga 300

ttctccgcca catcagccac gccgcaacgt ctcctcctca tccccctcct gctcttccac 360

tcgcaaaacg tccaaactca attgtgtcaa aattggaggt tctcttcgtt tgagcctacc 420

attttcaatt ttttagttgc gacagcggcc cggtcagagg ttcacaacaa ggtctagaga 480

cactttgtca tggggccgag aaggaccata aaaaccaaac gatggtcacg tcaggtcaat 540

tactgaccag tctcacatcc gacccctcgc gtgctcgacc ggaggatttc tctgcactcg 600

tccttgcata cctcggctag cgggatttat tcaccaatca cacagccgag agtttttccg 660

gacccttcat ccaacagctt agagttgcat gagtcagtag caacgtagac tttgagcctt 720

tgtgacagat gtccaagtgc agcacgttgt aggaaaataa ggtgaaggat tggccaatgt 780

gaacagaggc gacaagagtc cgtctggagg gcttgttgta gtcaattgcc cgcgcaattg 840

attgacctca tcgtttctgc cggaaccccc ccacaagccc ggataaatag acacgcccca 900

caagccgttc gtctggtctg ctcacagcac acttccattt aaaattcaaa caaagcgcac 960

caccgcaaag catacttaac ccactcaatg tagacgtcgc ggaacttctc tttcctaccc 1020

accaccccaa acaa 1034

<210> 16

<211> 1240

<212> DNA

<213> Unknown (Unknown)

<400> 16

tacctctact ccatgggcct tacccccaag gacaaataac tgtatagtaa aagcgtatag 60

ccaataagat aatcacttga atgaaggagc agcaactcgt atgtttagca cttcaacgga 120

ctatttcccc gcagcaaaga gactattgct gagttgttga gtatctgctt tacaataatg 180

gggtatggac acacaaggag gggtcttagt gagaagttag ataggtctag catacatgag 240

atcaatgtgg tcttacctat atcgtttgtt atcatttatc ttggtttgaa ttgataacac 300

gagttgttca ttgaagtgat ggcaccgggt ctcacacgca acagttggcg aacaggtcgt 360

attgttcctt agatacgacg ctcttttgga catgatggga agtgaaacta caattacagt 420

agctacatag cttggctaac tagaccgctt acagaaccag tagtcgtcac aagaccacca 480

cgaacaaagt ccaactaccc cactcccacc actcgtattt acttaccgca gatcacacgc 540

ttcggtgtat ctccgtgggg catcgtgggg cattgttcta agttttccgt atggtgcaca 600

gtcggtacgt gctttgacta accagtagaa gttaggctac tgtagtggag attgagcaat 660

gaaacgatga caggaagacc ccaaaatgcg accacctcaa ctatacacgg cttgttgcta 720

ttgccgcctt gcactccaca cagcaaacat gcacacgata tgcactcaag tcttaaccga 780

atgaaggtaa aagtagcaac caacaagcga gagttactgt atacttacaa gttatacgac 840

agtctcactt atcaccaatt ggcaacttga ccgcacagac aaacacccta caatgacctt 900

cctcaatgtg ctctactacg tgctgctggc tgccatcatg atcggcaccg gctacttcta 960

ctacctgtgg ttcactgaga ccaacgacca aaccgagaag atcatcagag ctgcgcttgg 1020

agtctttgat atcgccatct ggtacattct aggtatctcc acctccttta agatcctcac 1080

ccagatgatt ctcgcctgtt tccttgtggt tctggctggt cttaagatct acatcaaccc 1140

tcgagttgga ggggctcttc tggccggcag tctgctattt gtggctgctg tctggttcgg 1200

attccgccga gacggtcgag aggcccgaga cgatcttaac 1240

Claims (9)

1. Yarrowia lipolytica (yarrowia) for producing pyruvic acidYarrowialipolytica) YW100-1 genetically engineered bacterium, characterized in that said yarrowia lipolytica (Yersinia esterica) ((R))Yarrowialipolytica) YW100-1 is a glycerol kinase in the overexpression of glycerol metabolism in wild-type yarrowia lipolyticaGUT1Glycerol kinaseGUT2Glycerol dehydrogenaseGCY1Dihydroxyacetone kinaseDAK1Dihydroxyacetone kinaseDAK2And NADH kinasePOS5Simultaneous knock-out of diacylglycerol acyltransferase gene in yarrowia lipolyticaDGA2And glycerol-3-phosphate dehydrogenase geneGPD2Obtaining; the glycerol kinaseGUT1The gene nucleotide sequence is shown as SEQ ID NO.1, and the glycerol kinaseGUT2The gene nucleotide sequence is shown in SEQ ID NO.2, and the dihydroxyacetone kinaseDAK1The gene nucleotide sequence is shown in SEQ ID NO.3, and the dihydroxyacetone kinaseDAK2The nucleotide sequence of the gene is shown as SEQ ID NO.4, and the glycerol dehydrogenaseGCY1The gene nucleotide sequence is shown as SEQ ID NO.5, NADH kinasePOS5The nucleotide sequence of the gene is SEQID NO. 6; diacylglycerol acyltransferase5’DGA2The nucleotide sequence of the gene is shown as SEQ ID NO.7, and diacylglycerol acyltransferase3’DGA2The gene nucleotide sequence is shown in SEQ ID NO. 8; 3-Glycerol phosphate dehydrogenase 5'GPD2The nucleotide sequence of the gene is shown as SEQ ID NO.15 and 3'GPD2The gene nucleotide sequence is shown in SEQ ID NO. 16.

2. The yarrowia lipolytica YW100-1 of claim 1, wherein said yarrowia lipolytica YW100-1 is constructed by:

(1) derived from yarrowia lipolyticaGUT1Gene, gene,GUT2Genes, and promoterspEXP1Fragments obtained by overlap extensionGUT1-pEXP1-GUT2To vector JMP113 to give vector E14;

(2) the constructed vector E14 was usedNot I, enzyme digestion is carried out, and the enzyme digestion is carried out and transferred into wild yarrowia lipolytica GIM2.187 to obtain transformed yarrowia lipolytica engineering bacteria ZS 102;

(3) derived from yarrowia lipolyticaGCY1，DAK1，DAK2Gene, promoterpTEF，pEXP1，pGPDAnd homology arms for integrationKU70Respectively 5 ' and 3 ' by overlapping extension to obtain 5 'KU70-URA3-pTEF-DAK1，DAK1-pEXP1-DAK2-pGPD，pGPD-GYC1-3’KU70Three large fragments are transferred into ZS102 to obtain transformed yarrowia lipolytica engineering bacteria ZS 104;

(4) derived from yarrowia lipolyticaPOS5Genes andpTEFand for knock-outGPD25' end of (3)GPD2And 3' endGPD2Obtained by overlapping extensions5’GPD2-URA3-pTEF-POS5AndPOS5-3’GPD2two fragments are transferred into ZS104 to obtain the yarrowia lipolytica engineering bacteria ZS106 after homologous recombination;

(5) 5 'and 3' ends to be used for knockdownDGA2, obtained by overlap extension5’DGA2-URA3-DGA2 fragment, transferred into ZS106 to obtain yarrowia lipolytica YW 100-1.

3. The yarrowia lipolytica of claim 1 (ii)Yarrowia lipolytica) YW100-1, which is preserved in China center for type culture Collection with the preservation date of 2019, 3 and 18 months and the preservation number of CCTCC NO: m2019168, address: wuhan university, Wuhan, China, zip code 430072.

4. Use of yarrowia lipolytica YW100-1 of claim 1 for the fermentation of glycerol to pyruvate.

5. Use according to claim 4, wherein the fermentation is a shake flask fermentation, and the method comprises: inoculating yarrowia lipolytica YW100-1 into YPD culture medium, culturing at 30 deg.C and 200rpm for 24 hr to obtain seed solution; at an initial cell concentration OD₆₀₀ = 0.05 inoculating YNG culture medium, fermenting culture at 30 deg.C and 200rpm to OD₆₀₀When the concentration is 4.0-5.0, fermentation liquor containing pyruvic acid is obtained.

6. Use according to claim 4, wherein the fermentation is a fermenter fermentation, the method comprising: inoculating yarrowia lipolytica YW100-1 into a fermentation tank containing a fermentation culture medium, fermenting at 30-32 ℃, pH value of 4-4.5 and dissolved oxygen of 40-50% to obtain fermentation liquor containing pyruvic acid, and separating and purifying to obtain pyruvic acid; the fermentation medium comprises the following components: 60g/L of glycerin, 10g/L of (NH)₄)₂SO₄、1.4 g/L MgSO₄·7H₂O、2 g/L KH₂PO₄、0.8 g/L CaCl₂0.5g/L NaCl, 1 mug/L vitamin B1, and the solvent is distilled water, and the pH value is natural.

7. The use of claim 6, wherein the glycerol is added in batch mode during fermentation in the fermenter, wherein the glycerol is added for the first time at 60g/L and is supplemented after the glycerol is exhausted, and the glycerol is supplemented at 40g/L each time.

8. Use according to claim 7, characterized in that glycerol is supplemented 2 times.

9. The use of claim 6, wherein before fermentation in said fermentor, yarrowia lipolytica YW100-1 is subjected to seed expansion culture, and the seed solution is inoculated into the fermentation medium at a volume concentration of 10%, said seed culture being: inoculating yarrowia lipolytica YW100-1 to a seed culture medium, and performing shake-flask culture at 30 ℃ for 18h to obtain a seed solution; the seed culture medium: 2g/L glycerol, 0.4g/L tryptone, 0.2g/L yeast extract, 0.24g/L KH₂PO₄、1.7 g/L K₂HPO₄·3H₂O, the solvent is distilled water, and the pH value is natural.

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