CN110499259A - A kind of Yarrowia Yarrowia YW100-1 and its application - Google Patents

Abstract

The invention discloses a kind of solution ester Ye Shi yeast YW100-1 and its applications; the solution ester Ye Shi yeast YW100-1 is to be overexpressed glycerokinase GUT1 in glycerol metabolism in solution ester Ye Shi yeast; glycerokinase GUT2; glycerol dehydrogenase enzyme GCY1; dihydroxyacetone kinase DAK1; dihydroxyacetone kinase DAK2 and NADH kinases POS5, while knocking out what diacylglycerol acyltransferase gene DGA2 and glycerol-3-phosphate gene GPD2 in solution ester Ye Shi yeast was obtained.When bacterial strain of the present invention is using glycerol as carbon source, output of pyruvic acid increases 66.2%, reaches 121.2g/L, provides strain excellent for pyruvic acid industrialized production.

Description

A kind of Yarrowia Yarrowia YW100-1 and its application

(1) Technical field

The invention relates to a construction method and application of a Yarrowia esterilytica engineering bacterium, and belongs to the technical field of bioengineering.

(2) Background technology

As an intermediate in the metabolic pathway of microorganisms, pyruvate is also one of the important organic acids, which has a wide range of roles in biochemical, pharmaceutical, food and scientific fields. In the pharmaceutical industry, pyruvic acid is an important pharmaceutical intermediate, which can be used to synthesize levodopa, anti-inflammatory and analgesic cincophene, anti-tuberculosis drug isoniazid calcium pyruvate, thiimidazole drugs, etc. In addition, pyruvate ( Such as calcium pyruvate, potassium pyruvate, creatine pyruvate, etc.) are widely used in weight loss health care products. In the food industry, pyruvic acid, as a food additive, has natural antiseptic and fresh-keeping effects. In the chemical industry, ethyl pyruvate, as a skin whitening agent, can inhibit the formation of melanin in the skin and has a good skin whitening effect. In addition, as the precursor of a new generation of biofuels, pyruvic acid has a rapidly growing domestic and foreign market demand and has broad application value.

At present, the production methods of pyruvate mainly include three categories: chemical synthesis method, enzymatic conversion method, and microbial fermentation method. The chemical synthesis method mainly uses tartaric acid as a raw material to chemically synthesize pyruvic acid, but this method has heavy pollution, high cost and limited industrial production. The enzymatic conversion method mainly uses the dehydrogenase system in the microorganism to convert lactic acid into pyruvate, but this method has low conversion rate and high cost, and has not yet been industrialized. The microbial fermentation method mainly uses microorganisms to directly produce pyruvic acid through microbial fermentation with inexpensive carbon sources such as glucose or glycerol. At present, the strains used in the microbial fermentation method include Saccharomyces cerevisiae, Escherichia coli, Torulopsis glabrata, Yarrowia lipolytica and the like. Compared with chemical synthesis method and enzymatic conversion method, it has the advantages of less pollution, high conversion rate and low cost, and is also the main method of industrialization at present.

Although Yarrowia esterolytica has good pyruvate production capacity, the conversion rate and synthesis rate of pyruvic acid synthesis from glycerol still need to be improved, and the pyruvate degradation rate is too fast, so that it cannot accumulate in large quantities. There are two glycerol catabolism pathways in Yarrowia esterolyticum, one is the glycerol-3-phosphate pathway, in the cytoplasm, glycerol is phosphorylated to glycerol-3-phosphate by the action of glycerol kinase (encoded by GUT1), and then shuttled into In mitochondria, glycerol-3-phosphate dehydrogenase (encoded by GUT2) oxidizes glycerol-3-phosphate to dihydroxyacetone phosphate, which is then transported back to the cytoplasm to enter the glycolytic metabolic pathway. The other is the dihydroxyacetone (DHA) pathway, in which glycerol dehydrogenase (encoded by GCY1) oxidizes glycerol to DHA, which is subsequently phosphorylated to phosphate by dihydroxyacetone kinases (encoded by DAK1 and DAK2) Dihydroxyacetone enters the glycolytic metabolic pathway. As a high-yielding oil yeast, Yarrowia sterolytica, diacylglycerol acyltransferase (encoded by DGA2) can synthesize triacylglycerol, an important component of oil, using dihydroxyacetone phosphate. The synthesis of glycerol is first converted into glycerol 3-phosphate by dihydroxyacetone phosphate, and then glycerol is synthesized by glycerol 3-phosphate dehydrogenase (encoded by GPD1, GPD2). Studies have shown that the knockout of GPD2 will cause the accumulation of intracellular NADH, thereby breaking the NADH/NADPH balance and affecting the growth of bacteria, while Pos5, as a NADH kinase, can use ATP to phosphorylate NADH to NADPH.

In view of this, the present invention will strengthen the catabolic pathway of glycerol by enhancing the glycerol-3-phosphate pathway and the dihydroxyacetone pathway to accelerate the decomposition of glycerol, while weakening the synthesis pathway of glycerol, thereby achieving a large accumulation of pyruvate in cells.

(3) Contents of the invention

The purpose of the present invention is to provide a high-yielding pyruvic acid Yarrowia esterilytica engineering bacteria, its construction and application, and to provide excellent strains for the industrial production of pyruvic acid.

The technical scheme adopted in the present invention is:

The invention provides a high-yielding pyruvate Yarrowia lipolytica YW100-1, wherein the Yarrowia lipolytica YW100-1 overexpresses glycerol kinase GUT1 in glycerol metabolism in Yarrowia lipolytica, glycerol Kinase GUT2, glycerol dehydrogenase enzyme GCY1, dihydroxyacetone kinase DAK1 and dihydroxyacetone kinase DAK2, while knocking out the diacylglycerol acyltransferase gene DGA2 in Yarrowia esterolytica to enhance glycerol catabolism, knocking out 3- Glycerol phosphate dehydrogenase gene GPD2 and NADH kinase POS5 were overexpressed to reduce glycerol anabolism, and a high-yielding pyruvate-producing Yarrowia saccharomyces was obtained.

Further, described Yarrowia esterolytica YW100-1 is constructed as follows:

(1) GUT1 gene, GUT2 gene and promoter pEXP1 fragment derived from Yarrowia esterolyticum were obtained by overlapping extension to obtain GUT1-pEXP1-GUT2, and then connected to vector JMP113 to obtain vector E14;

(2) The constructed vector E14 was digested with Not I enzyme, and transferred into wild-type Yarrowia esterolytica AS2.1405 to obtain the transformed Yarrowia esterolyticum engineering strain ZS102;

(3) The GCY1, DAK1, DAK2 genes, promoters pTEF, pEXP1, pGPD, and the 5' end and 3' end of the homology arm KU70 for integration were obtained by overlapping extension, respectively. Three large fragments, 5'KU70-URA3-pTEF-DAK1, DAK1-pEXP1-DAK2-pGPD, and pGPD-GYC1-3'KU70, were transferred into ZS102 to obtain the transformed Yarrowia esterolyticum engineering strain ZS104;

(4) The POS5 gene and pTEF derived from Yarrowia esterolytica, as well as the 5'-end GPD2 and 3'-end GPD2 for knocking out GPD2, were obtained by overlapping extension to obtain 5'GPD2-URA3-pTEF-POS5 and POS5- The two fragments of 3'GPD2 were transferred into ZS104 to obtain the Yarrowia esterolyticum engineering strain ZS106 after homologous recombination;

(5) The 5'-end and 3'-end DGA2 for knockout were obtained by overlapping extension to obtain a 5'DGA2-URA3-DGA2 fragment, which was transferred into ZS106 to obtain Yarrowia esterolytica YW100-1.

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

The Yarrowia lipolytica YW100-1 of the present invention is preserved in the China Center for Type Culture Collection, the preservation date is March 18, 2019, and the preservation number is CCTCC NO: M 2019168, and the address is: Wuhan, China Wuhan University, zip code 430072.

The present invention also provides an application of the Yarrowia esterolytica YW100-1 in fermenting glycerol to prepare pyruvic acid, and the application is one of the following methods: (1) Shake flask fermentation: Yarrowia esterolytica YW100-1 Inoculated in YPD medium, cultured at 30°C, 200rpm for 24h to obtain seed liquid; inoculated in YNG medium with initial cell concentration _OD600 =0.05, fermented at 30°C, 200rpm to _OD600 of 4.0-5.0 ( When 4.5 is preferred, a fermentation broth containing pyruvate is obtained; YPD liquid medium quality composition: 1% yeast extract, 2% glucose, 2% peptone, distilled water as solvent, natural pH value; YNG medium quality composition: 0.67% YNB (a basic nitrogen source for yeast containing ammonium sulfate and no amino acids), 50 g/L glycerol, distilled water as the solvent, and adjusted to pH 4.0 with Na ₂ HPO ₄ -citric acid buffer;

(2) Fermentation in a fermentor: inoculate Yarrowia esterolytica YW100-1 into a fermenter containing a fermentation medium, and ferment at 30-32° C., pH value of 4-4.5, and dissolved oxygen content of 40-50%. , obtain a fermentation broth containing pyruvate, separate and purify to obtain pyruvate; the fermentation medium is composed of: 60g/L glycerol, 10g/L (NH ₄ ) ₂ SO ₄ , 1.4g/L MgSO ₄ ·7H ₂ O , 2g/LKH ₂ PO ₄ , 0.8g/L CaCl ₂ , 0.5g/L NaCl, 1μg/L vitamin B1, the solvent is distilled water, and the pH value is natural.

Further, when the fermentation tank is fermented, glycerol is added in batches, 60 g/L is added for the first time, and when the glycerol is exhausted, glycerol is added, and 40 g/L is added each time, preferably twice.

Further, before the fermentation in the fermenter, Yarrowia esterolytica YW100-1 is firstly subjected to seed expansion culture, and then the seed liquid is inoculated into the fermentation medium with an inoculum volume concentration of 10%, and the seed culture is: Saccharomyces cerevisiae YW100-1 was inoculated into the seed medium, cultured in a shake flask at 30°C for 18 hours to obtain seed liquid; the seed medium: 2g/L glycerol, 0.4g/L tryptone, yeast extract 0.2g/L, 0.24g /L KH ₂ PO ₄ , 1.7g/LK ₂ HPO ₄ ·3H ₂ O, the solvent is distilled water, and the pH value is natural.

Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

Compared with the starting strain Yarrowia esterolytica YW100-1 (the deposit number is CCTCC NO: M 2019168) obtained by the present invention, when glycerol is used as the carbon source, the yield of pyruvate increases. 66.2%, reaching 121.2g/L, providing an excellent strain for the industrial production of pyruvic acid.

(4) Description of drawings

Fig. 1 is the construction roadmap of the genetically engineered bacteria of Yarrowia esterolyticum.

Figure 2 is a comparison of the pyruvate yield and glycerol residue of the shake flask fermentation recombinant strain YW100-1 and the starting strain AS2.1405.

Figure 3 is a comparison of the pyruvate production of recombinant strain YW100-1 and starting strain AS2.1405 in a 20L fermenter.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but the protection scope of the present invention is not limited to this:

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

The quality composition of YPD liquid medium: 1% yeast extract, 2% glucose, 2% peptone, the solvent is distilled water, and the pH value is natural.

Quality composition of YNG medium: 0.67% YNB (basic nitrogen source for yeast containing ammonium sulfate and no amino acids), 50 g/L glycerol, distilled water as solvent, and pH 4.0 adjusted with Na ₂ HPO ₄ -citric acid buffer.

YPD solid medium is the addition of 2g/L agar to YPD liquid medium.

The quality composition of LB medium: 1% peptone, 0.5% yeast extract, 0.5% sodium chloride, the solvent is distilled water, and the pH value is natural.

Example 1: Construction of a genetically engineered strain of Yarrowia esterolytica with high production of pyruvate

The flow chart is shown in Figure 1, and the construction steps of Yarrowia esterolytica YW100-1 are as follows:

1. Construction of E14 plasmid

Amplification of GUT1: By designing primers YW238 and YW239, using the genome of wild-type Yarrowia esterolytica AS2.1405 (purchased from the Guangdong Microbial Culture Collection Center, No. GIM 2.187) as a template, GUT1 was amplified, with a size of about 1.5kb, the nucleotide sequence is shown in SEQ ID NO.1.

Amplification of promoter pEXP1: By designing primers YW405 and YW240, using the genome of wild-type Yarrowia esterolytica AS2.1405 as a template, amplifying pEXP1, the size is about 1kb, and the nucleotide sequence is shown in SEQ ID NO.9. Show.

Amplification of GUT2: By designing primers YW241 and YW242, and using the genome of wild-type Yarrowia esterolytica AS2.1405 as a template, GUT2 was amplified with a size of about 2kb, and the nucleotide sequence is shown in SEQ ID NO.2.

The PCR amplification conditions for the above GUT1, pEXP1, GUT2 were pre-denaturation at 98°C for 3 minutes; denaturation at 98°C for 10 seconds, annealing at 58°C for 10 seconds, extension at 72°C for 2 minutes (30 cycles), and extension at 72°C for 10 minutes .

Overlap extension to construct GUT1-pEXP1-GUT2 fragment: 2 rounds of PCR amplification were used for overlap extension. The first round: the amplification system was 25 μl (2×PrimeSTAR Max DNA polymerase (purchased from Beijing Baori Doctor Bio Co., Ltd.) 12.5 μl, GUT1, pEXP1 and GUT2 fragments mixed in a molar ratio of 1:3:1, supplemented Distilled water to 25 μl), pre-denatured at 95°C for 3 minutes, denatured at 98°C for 10 seconds, annealed at 58°C for 15 seconds, extended at 72°C for 2 minutes (15 cycles), and extended again at 72°C for 10 minutes. The second round: the amplification system was 50 μl (25 μl of 2×PrimeSTAR Max DNA polymerase, 2 μl of PCR product obtained in the first step, 1 μl of YW238+1 μl of YW243 and supplemented with distilled water to 50 μl), pre-denatured at 95°C for 3 minutes, Denaturation at 98°C for 10 seconds, annealing at 56°C for 15 seconds, extension at 72°C for 2 minutes (30 cycles), and extension again at 72°C for 10 minutes.

The GUT1-pEXP1-GUT2 fragment obtained above was recovered by a gel recovery kit (purchased from Beijing Baori Doctor Biotechnology Co., Ltd.), and cloned into JMP113 using a one-step cloning kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.). Vector (Fickers P, Le Dall MT, Gaillardin C, Thonart P, Nicaud JM. New disruptioncassettes for rapid gene disruption and marker rescue in the yeast Yarrowialipolytica. J Microbiol Methods. 2003.55(3):727-37.). Cloning system: 2 μl 5×CEMultiS buffer, 3 μl GUT1-pEXP1-GUT2 fragment, 2 μl BamH I linearized JMP113, 1 μl Exnase TMMultiS enzyme, 2 μl distilled water. The reaction was carried out at 37° C. for half an hour, placed on ice for 5 minutes, and then 10 μl was added to 100 μl of E. coli DH5α competent cells for another 30 minutes on ice. Heat shock at 42°C for 30 seconds and immediately place on ice for 2 minutes. Add 1 mL of LB medium and incubate for 1 hour at 37°C at 200 rpm. The bacterial solution collected by centrifugation was smeared on LB plates containing 50 μg/mL kanamycin, and cultured at 37°C overnight. After the transformants were grown, primers YW244+YW245 were used for identification, and 4 positive single colonies were screened by PCR and inoculated into LB medium. The positive cloned plasmids were extracted and verified by sequencing. The sequencing results showed that the vector E14 was successfully constructed.

Table 1 List of primers

2. Construction of recombinant Yarrowia esterolyticum engineering strain ZS102

The plasmid E14 was digested with Not I enzyme, and the pTEF-GUT1-pEXP1-GUT2 fragment was recovered by cutting the gel, and transferred into wild-type Yarrowia esterolytica AS2.1405 competent cells by electroporation to obtain recombinant Yarrowia esterolyticum engineering bacteria ZS102, the specific steps are:

Inoculate wild-type Yarrowia esterolytica AS2.1405 in YPD liquid medium, cultivate overnight at 30°C, transfer to 50 mL of fresh YPD liquid medium so that the initial OD ₆₀₀ is 0.1, and culture at 30° C for 4-6 hours until the OD ₆₀₀ to 1.0. The supernatant was removed by centrifugation, and the cells were suspended in a buffer (0.6 M sorbitol, 10 mM Tris-HCl, 25 mM DTT, 150 mM LiAc, pH 7.5) and left to stand for 1 hour. Centrifuge again with 1M sorbitol three times. After removing the supernatant, suspend the cells with 150 mL of 1 M sorbitol, that is, the wild-type Yarrowia esterolytica AS2.1405 competent cells. Take 80 μl of competent cells and mix with 200 ng of pTEF-GUT1-pEXP1-GUT2 fragment, and perform electroporation under the conditions of 1.5kV (E=12.4kV/cm), 200Ω, and 25μF. After electroporation, 1 mL of 1M sorbitol aqueous solution was added immediately, and the plate was spread. Inverted cultured in YND solid medium for 2-3 days, the transformants were identified by PCR with primers YW253+YW254.

3. Construction of Yarrowia Esterolyticum Engineering Bacteria ZS104

Amplification of 5'KU70: By designing primers YW600 and YW601, and using the genome of wild-type Yarrowia esterolytica AS2.1405 as a template, the 5' upstream of KU70 was amplified, with a size of about 1.2kb, and the nucleotide sequence is SEQ ID NO.10.

Amplification of Loxp-URA3-Loxp: By designing primers YW602 and YW603, and using plasmid JMP113 as a template, Loxp-URA3-Loxp was amplified with a size of about 1.8kb, and the nucleotide sequence is shown in SEQ ID NO.11.

Amplification of pTEF: By designing primers YW604 and YW605, and using the genome of wild-type Yarrowia esterolytica AS2.1405 as a template, the promoter pTEF was amplified, with a size of about 0.4kb and the nucleotide sequence of SEQ ID NO.12 shown.

Amplification of DAK1: By designing primers YW606 and YW607, using the genome of wild-type Yarrowia esterolytica AS2.1405 as a template, the promoter DAK1 was amplified, with a size of about 2.2kb and the nucleotide sequence of SEQ ID NO.3 shown.

Amplification of pEXP1: By designing primers YW608 and YW609, and using the genome of wild-type Yarrowia esterolytica AS2.1405 as a template, the promoter pEXP1 was amplified, with a size of about 1 kb and the nucleotide sequence shown in SEQ ID NO.9 Show.

Amplification of DAK2: By designing primers YW610 and YW611, and using the genome of wild-type Yarrowia esterolytica AS2.1405 as a template, the promoter DAK2 was amplified, with a size of about 2.2kb and the nucleotide sequence of SEQ ID NO.4 shown.

Amplification of pGPD: By designing primers YW612 and YW613, and using the genome of wild-type Yarrowia esterolytica AS2.1405 as a template, the promoter pGPD was amplified with a size of about 0.94kb and the nucleotide sequence is SEQ ID NO.13 shown.

Amplification of GYC1: By designing primers YW614 and YW615, and using the genome of wild-type Yarrowia esterolytica AS2.1405 as a template, the promoter GYC1 was amplified with a size of about 1.5kb and the nucleotide sequence of SEQ ID NO.5 shown.

Amplification of 3'KU70: By designing primers YW616 and YW617, using the genome of wild-type Yarrowia esterolytica AS2.1405 as a template, the 3' downstream of KU70 was amplified, with a size of about 1kb, and the nucleotide sequence is SEQ ID NO.14 shows.

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

The DNA fragments obtained above were assembled into three large fragments 5'KU70-URA3-pTEF-DAK1, DAK1-pEXP1-DAK2-pGPD, and pGPD-GYC1-3'KU70 by overlapping extension.

The 5' KU70-URA3-pTEF-DAK1 fragment was constructed by overlapping extension. The first round: Mix 5'KU70, URA3, pTEF, DAK1 fragments in a molar ratio of 1:3:3:1, and the amplification conditions are the same as above. The second round: using the PCR product obtained in the first round of amplification as a template, add 1 μl YW600+1 μl YW607 for PCR amplification.

Overlap extension constructs the DAK1-pEXP1-DAK2-pGPD fragment. The first round: DAK1, pEXP1, DAK2, and pGPD fragments were mixed in a molar ratio of 1:3:3:1, and the amplification conditions were the same as above. The second round: using the PCR product obtained in the first round of amplification as a template, add 1 μl YW618+1 μl YW613 for PCR amplification.

The pGPD-GYC1-3'KU70 fragment was constructed by overlapping extension. The first round: mix pGPD, GYC1, and 3'KU70 fragments in a molar ratio of 1:3:1, and the amplification conditions are the same as above. The second round: using the PCR product obtained in the first round of amplification as a template, add 1 μl YW612+1 μl YW617 for PCR amplification.

The three large fragments of 5'KU70-URA3-pTEF-DAK1, DAK1-pEXP1-DAK2-pGPD, and pGPD-GYC1-3'KU70 obtained by the above overlapping extension were recovered, mixed with 50ng of each fragment and transferred to In Yarrowia esterolyticum recombinant engineering strain ZS102, the transformants were identified by PCR with primers YW619+YW620, YW621+YW622, YW226+YW227, respectively.

Table 2 List of primers

4. Construction of Yarrowia Esterolyticum Engineering Bacteria ZS106

Amplification of 5'GPD2: By designing primers YW626 and YW627, using the wild-type Yarrowia esterolytica AS2.1405 genome as a template, amplify 5'GPD2, the size is about 1kb, and the nucleotide sequence is SEQ ID NO. 15 shown.

Amplification of Loxp-URA3-Loxp: By designing primers YW628 and YW603 and using plasmid JMP113 as a template, Loxp-URA3-Loxp was amplified with a size of about 1.8kb, and the nucleotide sequence is shown in SEQ ID NO.11.

Amplification of pTEF: By designing primers YW604 and YW629, and using the genome of wild-type Yarrowia esterolytica AS2.1405 as a template, the promoter pTEF was amplified with a size of about 0.4kb and the nucleotide sequence of SEQ ID NO.12 shown.

Amplification of POS5: By designing primers YW630 and YW631, and using the genome of wild-type Yarrowia esterolytica AS2.1405 as a template, the promoter POS5 was amplified, with a size of about 1.2kb and the nucleotide sequence of SEQ ID NO.6 shown.

Amplification of 3'GPD2: By designing primers YW632 and YW633, using the genome of wild-type Yarrowia esterolytica AS2.1405 as a template, amplify 3'GPD2, the size is about 1.2kb, and the nucleotide sequence is SEQ ID NO .16 shown.

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

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

The 5'GPD2-URA3-pTEF-POS5 fragment was constructed by overlapping extension. The first round: Mix 5'GPD2, URA3, pTEF, and POS5 fragments in a molar ratio of 1:3:3:1, and the amplification conditions are the same as above. The second round: using the PCR product obtained in the first round of amplification as a template, add 1 μl YW626+1 μl YW631 for PCR amplification.

Overlap extension constructs the POS5-3'GPD2 fragment. The first round: Mix POS5 and 3'GUT2 fragments according to a 1:1 molar ratio, and the amplification conditions are the same as above. The second round: using the PCR product obtained in the first round of amplification as a template, add 1 μl YW630+1 μl YW633 for PCR amplification.

After the two large fragments of 5'GPD2-URA3-pTEF-POS5 and POS5-3'GPD2 obtained by the above overlapping extension were recovered, 50ng of each fragment was mixed and transferred to Yarrowia esterolytica recombinant engineering bacteria ZS104 , the transformants were identified by PCR with primers YW634+YW635 and YW636+YW637 respectively.

Table 3 List of primers

5. Construction of Yarrowia esterilytica engineering strain YW100-1

Amplification of 5'DGA2: By designing primers YW646 and YW647, using the genome of wild-type Yarrowia esterolytica AS2.1405 as a template, amplify 5'DGA2, the size is about 1kb, and the nucleotide sequence is SEQ ID NO. 7 is shown.

Amplification of Loxp-URA3-Loxp: By designing primers YW648 and YW649, and using plasmid JMP113 as a template, Loxp-URA3-Loxp was amplified with a size of about 1.8kb, and the nucleotide sequence is shown in SEQ ID NO.11.

Amplification of 3'DGA2: By designing primers YW650 and YW651, and using the genome of wild-type Yarrowia esterolytica AS2.1405 as a template, 3'DGA2 was amplified, with a size of about 1kb and the nucleotide sequence of SEQ ID NO. 8 shown.

The PCR amplification conditions for the above 5'DGA2, Loxp-URA3-Loxp, 3'DGA2 are the same as those shown in step 1.

The DNA fragments obtained above were assembled into a large fragment 5'DGA2-URA3-3'DGA2 using overlap extension. The 5'DGA2-URA3-3'DGA2 fragments obtained by the above overlapping extension were recovered and transferred into Yarrowia esterolytica recombinant engineering bacteria ZS106, and the transformants were identified by PCR with primers YW652+YW653.

Table 4 List of primers

The GUT1-pEXP1-GUT2 fragment obtained by overlapping extension was connected with the BamH I linearized plasmid JMP113 plasmid. E14 was constructed by one-step cloning method, and then the E14 plasmid was digested with Not I and transferred into Yarrowia esterolytica AS2.1405 competent cells , build ZS102. The 5'KU70-URA3-pTEF-DAK1, DAK1-pEXP1-DAK2-pGPD, and pGPD-GYC1-3'KU70 large fragments obtained by overlapping extension were transferred into Yarrowia esterolytica ZS102 competent cells to construct ZS104. The 5'GPD2-URA3-pTEF-POS5, POS5-3'GPD2 large fragment obtained by overlapping extension was transferred into Yarrowia esterolytica ZS104 competent cells to construct ZS106. The 5'DGA2-URA3-3'DGA2 large fragment obtained by overlapping extension was transferred into Yarrowia esterolytica ZS106 competent cells to construct recombinant engineering strain YW100-1 (Yarrowia esterolytica YW100-1), which has been published in It was deposited in the China Center for Type Culture Collection on March 18, 2019, with the deposit number CCTCC NO:M 2019168, address Wuhan University, Wuhan, China, zip code 430072.

Example 2: Comparison of Shake Flask Fermentation of Wild Type AS2.1405 and Recombinant Engineered Bacteria YW100-1

The wild-type Yarrowia esterolytica strain AS2.1405 and the recombinant engineering strain YW100-1 were respectively inoculated into YPD liquid medium, and cultured in a 250mL conical flask at 30°C and 200rpm for 24h to obtain seed liquid; The concentration of OD ₆₀₀ = 0.05 was respectively inoculated into YNG medium, each strain was analyzed in parallel in three groups, fermented and cultured at 30 ° C, 200 rpm for 16 h until OD ₆₀₀ was about 4.5, 10 mL of fermentation broth was taken and acetone was determined by high performance liquid chromatography The concentration of acid and glycerol, the results are shown in Figure 2.

Determination of pyruvic acid concentration: take 10 mL of fermentation broth, centrifuge at 6000 rpm for 5 min, collect the supernatant, and use a C18 column to measure the content of pyruvate. Mobile phase: 0.1% phosphoric acid; flow rate: 1 ml min ⁻¹ ; injection temperature: 28° C.; injection volume: 10 μl.

As shown in Figure 2, the wild-type strain was fermented in YNG medium for 16 h, the yield of pyruvate was about 3.5 g/L, and the residual amount of glycerol was about 10.2 g/L. The yield of pyruvic acid of engineering bacteria YW100-1 was about 5.95g/L, and the residual amount of glycerol was about 7.4g/L. It can be seen that the yield of pyruvic acid produced by fermentation of engineered bacteria YW100-1 was significantly higher than that of wild-type bacteria.

Example 3: Comparison of wild-type AS2.1405 and engineering bacteria YW100-1 in 20L fermenter for pyruvic acid production

Seed medium: 2g/L glycerol, 0.4g/L tryptone, 0.2g/L yeast extract, 0.24g/L KH ₂ PO ₄ , 1.7g/L K ₂ HPO ₄ ·3H ₂ O, the solvent is distilled water, pH Value is natural.

Composition of fermentation medium: 60 g/L glycerol, 10 g/L (NH ₄ ) ₂ SO ₄ , 1.4 g/L MgSO ₄ ·7H ₂ O, 2 g/L KH ₂ PO ₄ , 0.8 g/L CaCl ₂ , 0.5 g/L L NaCl, 1 μg/L vitamin B1, the solvent is distilled water, and the pH value is natural.

The wild-type Yarrowia esterolytica strain AS2.1405 and the engineered strain YW100-1 were respectively inoculated in YPD liquid medium, cultured at 30°C overnight, and then transferred to 3L seed medium with 200ml of the strain, cultured in a shaker flask at 30°C for 18h, and then The inoculum was inoculated into a 20L fermenter containing 12L fermentation medium with a volume concentration of 10%, and the fermentation culture was carried out for 1h. During the whole fermentation and cultivation process, the pH value was adjusted to 4.0 by flow addition of 20% NaOH, the dissolved oxygen was maintained at about 40%, and the fermentation temperature was controlled to 30°C. According to the residual concentration of glycerol in the fermenter, 40 g/L glycerol was added at 50 and 64 hours respectively, and a total of 1440 g of glycerol was added.

The process of glycerol fermentation to produce pyruvate by the two strains is shown in Figure 3. The wild-type strain was fermented for 120h, the yield of pyruvate reached the maximum value of 72.9g/L, and the conversion rate of pyruvate/glycerol was about 0.52g/g. The engineering bacteria YW100-1 was fermented for 120h, the yield of pyruvate reached the maximum value of 121.2g/L, and the conversion rate of pyruvate/glycerol reached 0.865g/g. Compared with the starting strain, the fermentative pyruvate yield of the engineered strain YW100-1 increased by 66.2%, which was significantly higher than that of other genetically engineered strains with the highest yield reported. It can be seen that the recombinant Yarrowia esterolyticum YW100-1 can provide an excellent strain for the industrial production of pyruvate.

sequence listing

<110> Zhejiang University of Technology

<120> A kind of Yarrowia Yarrowia YW100-1 and its application

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1512

<212> DNA

<213> 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

<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

<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

<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

<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

<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 ccttttttcct gggacaaccc acttgtgcat tcctttatta tctggcttac 1560

aactacaagc agaaccagta g 1581

<210> 7

<211> 1838

<212> DNA

<213> 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

<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

<400> 9

ggagttttggc 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

ttttttccctt 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

<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

<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

<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

ttttttgcttt 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

<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

<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

<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

<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 (10)

1. a high-yielding pyruvic acid Yarrowia lipolytica YW100-1, is characterized in that the Yarrowia lipolytica (Yarrowialipolytica) YW100-1 is overexpressing glycerol kinase GUT1 in glycerol metabolism in Yarrowia lipolytica, Glycerol kinase GUT2, glycerol dehydrogenase enzyme GCY1, dihydroxyacetone kinase DAK1, dihydroxyacetone kinase DAK2 and NADH kinase POS5, simultaneously knock out the diacylglycerol acyltransferase gene DGA2 and 3-phosphate glycerol dehydrogenase in Yarrowia estradiolae Hydrogenase gene GPD2 was obtained. 2. as claimed in claim 1, it is characterized in that described Yarrowia esterolytica YW100-1 is constructed as follows: (1) GUT1 gene, GUT2 gene and promoter pEXP1 fragment derived from Yarrowia esterolyticum were obtained by overlapping extension to obtain GUT1-pEXP1-GUT2, and then connected to vector JMP113 to obtain vector E14; (2) The constructed vector E14 was digested with NotI, and transferred into wild-type Yarrowia esterolytica AS2.1405 to obtain the transformed Yarrowia esterolyticum engineering strain ZS102; (3) The GCY1, DAK1, DAK2 genes, promoters pTEF, pEXP1, pGPD, and the 5' end and 3' end of the homology arm KU70 for integration were obtained by overlapping extension, respectively. Three large fragments of 5'KU70-URA3-pTEF-DAK1, DAK1-pEXP1-DAK2-pGPD, and pGPD-GYC1-3'KU70 were transferred into ZS102 to obtain the transformed Yarrowia esterolyticum engineering strain ZS104; (4) The POS5 gene and pTEF derived from Yarrowia esterolytica, as well as the 5'-end GPD2 and 3'-end GPD2 for knocking out GPD2, were obtained by overlapping extension to obtain 5'GPD2-URA3-pTEF-POS5 and POS5- The two fragments of 3'GPD2 were transferred into ZS104 to obtain the Yarrowia esterolyticum engineering strain ZS106 after homologous recombination; (5) The 5'-end and 3'-end DGA2 for knockout were obtained by overlapping extension to obtain a 5'DGA2-URA3-DGA2 fragment, which was transferred into ZS106 to obtain Yarrowia esterolytica YW100-1. 3. Yarrowia esterilytica YW100-1 as claimed in claim 1, characterized in that the glycerol kinase GUT1 gene nucleotide sequence is shown in SEQ ID NO.1, and the glycerol kinase GUT2 gene nucleotide sequence is SEQ ID As shown in NO.2, the nucleotide sequence of the dihydroxyacetone kinase DAK1 gene is shown in SEQ ID NO.3, the nucleotide sequence of the dihydroxyacetone kinase DAK2 gene is shown in SEQID NO.4, and the glycerol dehydrogenase GCY1 gene nucleus The nucleotide sequence is shown in SEQ ID NO.5, and the nucleotide sequence of the NADH kinase POS5 gene is shown in SEQ ID NO.6. 4. Yarrowia lipolytica YW100-1 according to claim 1, preserved in China Type Culture Collection, preservation date is March 18, 2019, preservation number is CCTCC NO: M 2019168, The address is: Wuhan University, Wuhan, China, 430072. 5. an application of the described Yarrowia esterolytica YW100-1 of claim 1 in the preparation of pyruvic acid by fermenting glycerol. 6. The application according to claim 5, wherein when the fermentation is shake flask fermentation, the method is as follows: Yarrowia esterolytica YW100-1 is inoculated in a YPD medium, cultivated at 30° C. and 200 rpm for 24 hours, and obtained Seed liquid; inoculated into YNG medium with initial cell concentration OD ₆₀₀ =0.05, and fermented and cultured at 30° C. and 200 rpm until OD ₆₀₀ was 4.0-5.0 to obtain a pyruvate-containing fermentation broth. 7. application as claimed in claim 5, is characterized in that when fermentation is fermentor fermentation, described method is: inoculate Yarrowia esterolytica YW100-1 in the fermentor containing fermentation medium, at 30-32 Fermentation is carried out under the conditions of ℃, pH value of 4-4.5, and dissolved oxygen content of 40-50% to obtain a fermentation broth containing pyruvate, which is separated and purified to obtain pyruvic acid; the fermentation medium is composed of: 60g/L glycerol, 10g/L L(NH ₄ ) ₂ SO ₄ , 1.4g/L MgSO ₄ ·7H ₂ O, 2g/L KH ₂ PO ₄ , 0.8g/L CaCl ₂ , 0.5g/L NaCl, 1μg/L vitamin B1, the solvent is distilled water, pH is natural. 8. application as claimed in claim 7, is characterized in that during described fermentation tank fermentation, glycerol adopts batch mode to add, adds 60g/L for the first time, when glycerol is exhausted, add glycerol, add 40g/L each time . 9. application as claimed in claim 8 is characterized in that glycerol is supplemented 2 times. 10. application as claimed in claim 7, it is characterized in that before described fermentor fermentation, Yarrowia esterolytica YW100-1 carries out seed expansion culture first, and then seed liquid is inoculated to fermentation with the inoculum of volume concentration 10% culture medium, the seed culture is as follows: Yarrowia esterolytica YW100-1 is inoculated into the seed medium, cultured in a shake flask at 30°C for 18 hours to obtain seed liquid; the seed medium: 2g/L glycerol, 0.4g/L pancreas Peptone, yeast extract 0.2g/L, 0.24g/L KH ₂ PO ₄ , 1.7g/L K ₂ HPO ₄ ·3H ₂ O, the solvent is distilled water, and the pH value is natural.