Recombinant cerevisiae yeast with high polylactic acid production, construction method and application thereof

Abstract

The invention discloses a recombinant saccharomyces cerevisiae for synthesizing polylactic acid from scratch. The saccharomyces cerevisiae engineering bacteria is obtained by knocking out the pyruvate decarboxylase gene and the alcohol dehydrogenase gene in saccharomyces cerevisiae BY4741, over-expressing the D-lactate dehydrogenase gene from Escherichia coli, the PHA synthase gene from brown nitrogen-fixing bacteria, and the acetate acetyl-CoA transferase mutant YDIF from Escherichia coli. F199Y‑D358L The obtained recombinant strain was constructed using glucose as a substrate and achieved a polylactic acid production of 133 mg / g DCW in shake flask fermentation, which was significantly higher than that of other similar polylactic acids.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant engineering strain of Saccharomyces cerevisiae, and also relates to a construction method of the recombinant strain and its application in the production of polylactic acid. Background Art

[0002] Polylactic acid (PLA), also known as poly(lactic acid), is a polyester polymer obtained by polymerizing lactic acid as the main raw material. It is a new type of biodegradable material. Due to its non-toxic and harmless characteristics, it is widely used in the fields of disposable tableware and food packaging materials. Polylactic acid also has the characteristics of high safety and tissue absorbability, and can be applied to materials such as dissolvable surgical sutures, tissue repair materials, and drug sustained-release packaging agents in the field of medical technology. Therefore, polylactic acid has broad application prospects in the field of degradable plastics.

[0003] The synthesis raw material of polylactic acid is lactic acid. Saccharomyces cerevisiae has the characteristics of high acid tolerance and high yield, making it an excellent candidate for synthesizing polylactic acid. It is reported that Zhu et al. [Zhu P, Luo R, Li Y, Chen X. Metabolic Engineering and Adaptive Evolution for Efficient Production of l-Lactic Acid in Saccharomyces cerevisiae. Microbiol Spectr. 2022 Dec 21;10(6):e0227722] rationally modified the lactic acid metabolic pathway of Saccharomyces cerevisiae and carried out adaptive evolution, and obtained 121.5 g / L of lactic acid through fed-batch fermentation. Liu et al. [Liu T, Sun L, Zhang C, Liu Y, Li J, Du G, Lv X, Liu L. Combinatorial metabolic engineering and process optimization enables highly efficient production of L-lactic acid by acid-tolerant Saccharomyces cerevisiae. Bioresour Technol. 2023 Jul;379:129023] down-regulated the glycerol and ethanol synthesis pathways to further increase energy supply and redox balance, enabling the engineered strain to produce 192.3 g / L of L-lactic acid.

[0004] There are two main methods for preparing polylactic acid: chemical synthesis and biosynthesis. The chemical synthesis method is complex, costly, and highly polluting, which is not conducive to sustainable development strategies. With the vigorous development of synthetic biology, biological synthesis of polylactic acid has become a very promising strategy. The biosynthesis method mainly focuses on genetic engineering of microorganisms to increase the production of polylactic acid. In Chinese invention patent application CN 114196609 A, Escherichia coli biotransforms lactic acid to produce polylactic acid, and the final fermented polylactic acid accounts for 16% of the cell dry weight; Tan et al. [Chunlin Tan, Fei Tao, Ping Xu. Direct carbon capture for the production of high-performance biodegradable plastics by cyanobacterial cell factories [J]. Green Chemistry, 2022, 24 (11): 4470-4483] used cyanobacteria as host cells, overexpressed PCT and PHA genes, and the molecular weight of polylactic acid reached 50KDa, producing 36.7mg / g DCW of polylactic acid. Although biosynthesis has been used to produce polylactic acid, the low yield has limited the widespread application of microbial production of polylactic acid to a certain extent. With the rapid development of synthetic biology, the use of genetic engineering technology to modify enzyme molecules to obtain high-yield polylactic acid engineering bacteria has become an important research direction. SUMMARY OF THE INVENTION

[0005] The purpose of the present invention is to overcome the defects of the prior art and improve the polylactic acid production capacity of the engineering strain of Saccharomyces cerevisiae through genetic engineering technology.

[0006] The idea of ​​the present invention is to use Saccharomyces cerevisiae as the starting strain, delete the genes of the ethanol synthesis pathway of Saccharomyces cerevisiae, introduce the genes of polylactic acid synthesis, and open up the polylactic acid synthesis pathway as the basic engineering strain for subsequent experiments. On the other hand, gene mining is carried out on the key gene of polylactic acid synthesis, the acetate coenzyme A transferase gene YDIF, to investigate the effect of YDIF from different sources on the polylactic acid production capacity of the recombinant bacteria, and to attempt to perform site-directed mutation on the acetate coenzyme A transferase gene in order to further increase the yield of polylactic acid.

[0007] For the above purpose, the present invention provides a recombinant Saccharomyces cerevisiae engineering bacterium with high lactic acid production. The recombinant Saccharomyces cerevisiae engineering bacterium expresses the D-lactate dehydrogenase gene LDHA, the acetyl-CoA transferase gene YDIF, and the PHA synthase gene PHAC, and deletes the pyruvate decarboxylase gene PDC1 and the alcohol dehydrogenase gene ADH1. The D-lactate dehydrogenase gene LDHA is derived from Escherichia coli, the acetyl-CoA transferase gene YDIF is derived from Escherichia coli, Serratia fonticola, Bacillus sp. oxB-1, or Escherichia albertii, and the PHA synthase gene PHAC is derived from Azotobacter vinelandii.

[0008] In the present invention, the nucleic acid sequence of the D-lactate dehydrogenase gene LDHA is as shown in SEQ ID NO.1; the nucleic acid sequence of the PHA synthase gene PHAC is as shown in SEQ ID NO.2; the nucleic acid sequence of the pyruvate decarboxylase gene PDC1 is as shown in SEQ ID NO.3; the nucleic acid sequence of the alcohol dehydrogenase gene ADH1 is as shown in SEQ ID NO.4; the nucleic acid sequence of the acetyl-CoA transferase gene derived from Escherichia coli is as shown in SEQ ID NO.5; the nucleic acid sequence of the acetyl-CoA transferase gene derived from Serratia fonticola is as shown in SEQ ID NO.7; the nucleic acid sequence of the acetyl-CoA transferase gene derived from Bacillus sp. oxB-1 is as shown in SEQ ID NO.8; the nucleic acid sequence of the acetyl-CoA transferase gene derived from Escherichia albertii is as shown in SEQ ID NO.9.

[0009] In the present invention, each expression cassette is composed of a promoter-gene-terminator. The promoter is one of P TEF and P GPD and the terminator is one of T ADH1 and T CYC1

[0010] ​In particular, the present invention also provides a genetically engineered Saccharomyces cerevisiae strain with high poly(lactic acid) productivity. The genetically engineered Saccharomyces cerevisiae strain expresses the D-lactate dehydrogenase gene LDHA, the acetate-CoA transferase gene YDIF mutant, and the PHA synthase gene PHAC, and lacks the pyruvate decarboxylase gene PDC1 and the alcohol dehydrogenase gene ADH1. The D-lactate dehydrogenase gene LDHA is derived from Escherichia coli, and the PHA synthase gene PHAC is derived from Azotobacter vinelandii. The acetate-CoA transferase gene YDIF mutant is obtained by mutating the 199th phenylalanine residue of the acetate-CoA transferase gene YDIF derived from Serratia fonticola to tyrosine, or mutating the 358th aspartic acid residue to tyrosine, or simultaneously mutating the 199th phenylalanine residue of the YDIF gene derived from Serratia fonticola to tyrosine and the 358th aspartic acid residue to tyrosine.

[0011] In the present invention, the host strain for the recombinant genetically engineered Saccharomyces cerevisiae strain can be BY4741, INVSC1, or YPH499.

[0012] The present invention also provides the use of the acetate-CoA transferase gene YDIF in improving the poly(lactic acid) productivity of a genetically engineered Saccharomyces cerevisiae strain. The acetate-CoA transferase gene YDIF is derived from Escherichia coli, Serratia fonticola, Bacillus sp. oxB-1, or Escherichia albertii.

[0013] As a particularly preferred embodiment, the present invention also provides the use of the acetate-CoA transferase gene YDIF mutant in improving the poly(lactic acid) productivity of a genetically engineered Saccharomyces cerevisiae strain. The acetate-CoA transferase gene YDIF mutant is obtained by mutating the 199th phenylalanine residue of the acetate-CoA transferase gene YDIF to tyrosine, or mutating the 358th aspartic acid residue to tyrosine, or simultaneously mutating the 199th phenylalanine residue of the YDIF gene to tyrosine and the 358th aspartic acid residue to tyrosine.

[0014] In addition, the present invention also provides the use of the above recombinant Saccharomyces cerevisiae in the fermentation production of poly(lactic acid).

[0015] In the present invention, an engineered bacterium with high poly(lactic acid) productivity is inoculated into 50 ml of SD-URA liquid medium and cultured at 37 °C and 200 rpm for 72 h to form a seed culture. The seed culture is inoculated into 150 ml of fermentation medium at an inoculation amount of 2% by volume and subjected to aerobic growth at 200 rpm and 30 °C for 48 h, and then transferred to a low-oxygen supply acid production stage, and fermented at 30 °C and 100 rpm for 96 h to obtain poly(lactic acid).

[0016] On the other hand, the present invention also provides a method for constructing a recombinant Saccharomyces cerevisiae with high poly(lactic acid) productivity, and the method includes the following steps:

[0017] (1) Construction of poly(lactic acid) pathway plasmid

[0018] Amplify the D-lactate dehydrogenase gene LDHA from Escherichia coli, insert the amplified fragment into the SpeⅠ site of pY26TEF-GPD to obtain pY26TEF-L; artificially synthesize the PHA synthase PHAC gene sequence from Azotobacter vinelandii shown in SEQ ID No.2, amplify the GAP promoter and CYC1 terminator of pY26TEF-GPD, and insert the synthesized gene fragments PHAC gene, GAP promoter and CYC1 terminator into the EcoRⅠ site of plasmid pY26TEF-L to obtain plasmid pY26TEF-LP;

[0019] Artificially synthesize the mutants YDIF, YDIF, and YDIF of the acetyl-CoA transferase gene YDIF from Serratia fonticola shown in SEQ ID.No.10, 11, and 12 F199Y 、YDIF D358L and YDIF F199Y-D358L gene sequences, and insert the synthesized gene sequences into the NotⅠ site of plasmid pY26TEF-LP respectively to obtain plasmids pY26TEF-LP-YDIF F199Y 、pY26TEF-LP-YDIF D358L and pY26TEF-LP-YDIF F199Y-D358L ;

[0020] (2) Construct a Saccharomyces cerevisiae engineering strain BY4741-2 with PDC1 and ADH1 genes knocked out and LDHA gene transferred

[0021] Using the genome of Saccharomyces cerevisiae BY4841 as a template, PCR amplify the upstream and downstream homologous arms of the PCD1 and ADH1 genes. The obtained upstream homologous arm fragment is ligated to the ploxpura3loxp vector digested with ApaⅠ and XbaⅠ, and the downstream homologous arm is ligated to the ploxpura3loxp vector digested with NdeⅠ and MluⅠ to obtain plasmids ploxpura3loxp-PDC1 and ploxpura3loxp-ADH1 respectively;

[0022] The Cre gene as shown in SEQ ID NO.13 was synthesized onto the pUC57 vector to obtain the vector pUC57-Cre. The Cre gene was amplified using the pUC57-Cre vector as a template, and the resulting gene fragment was ligated to the JN44-GFP vector digested with HindⅢ and SmaⅠ to obtain the JN44-Cre vector;

[0023] The ploxpura3loxp-PDC1 linearized with ApaⅠ was transformed into Saccharomyces cerevisiae BY4741. The successfully transformed single colonies were screened using SD-URA defective medium, and the strain with the lowest ethanol production was selected to remove the URA marker and the JN44-Cre plasmid. After the resulting strain was verified correctly by PCR, it was named BY4741-1, and BY4741-1 is the Saccharomyces cerevisiae BY4741 strain with the PDC1 gene knocked out;

[0024] The ploxpura3loxp-ADH1 linearized with ApaⅠ was transferred into BY4741-1. After screening with defective medium and fermentation, the URA marker and the JN44-Cre plasmid were removed. The resulting colonies with correct PCR were named BY4741-2, and BY4741-2 is the Saccharomyces cerevisiae BY4741 strain with the PDC1 and ADH1 genes knocked out;

[0025] (3) Construction of recombinant Saccharomyces cerevisiae BY4741-9, BY4741-11 and BY4741-12 with high poly(lactic acid) production

[0026] The pY26TEF-LP-YDIF in step (1) F199Y , pY26TEF-LP-YDIF D358L and pY26TEF-LP-YDIF F199Y-D358L plasmids were separately transferred into the BY4741-2 strain. The resulting strains were spread on SD-URA solid medium, and the strains verified correctly by amplification were named recombinant Saccharomyces cerevisiae BY4741-9, BY4741-11 and BY4741-12.

[0027] In the present invention, by deleting the pyruvate decarboxylase gene PDC1 and the alcohol dehydrogenase gene ADH1, and introducing the D-lactate dehydrogenase gene LDHA, the acetyl-CoA transferase mutant gene SfYDIF F199Y or SfYDIF D358L or SfYDIF F199Y-D358L and the PHA synthase gene PHAC, the biosynthesis of poly(lactic acid) using glucose as a raw material was achieved. In the shake flask experiment, a fermentation medium containing 30% glucose was used, and finally, up to 133 mg / g DCW of poly(lactic acid) was obtained at most.

[0028] Compared with Escherichia coli and Lactobacillus, Saccharomyces cerevisiae has characteristics such as food safety, clear genetic background, and strong acid environment tolerance. Selecting Saccharomyces cerevisiae as the host bacterium and introducing the lactic acid synthesis pathway by genetic engineering means is beneficial to obtaining higher lactic acid production. In the present invention, the lactic acid production of Saccharomyces cerevisiae reaches 154 g / L, providing sufficient substrate for the synthesis of polylactic acid.

[0029] The present invention verifies that acetyl-CoA transferase has good catalytic effect in Saccharomyces cerevisiae, and can effectively catalyze lactic acid into lactoyl-CoA to enter the next reaction to produce polylactic acid. The present invention first introduces the polylactic acid synthesis pathway into Saccharomyces cerevisiae, and uses the acetyl-CoA transferase gene SfYDIF from Serratia fonticola as the key gene of the pathway, synthesizes polylactic acid with lactic acid as the substrate, and obtains the recombinant Saccharomyces cerevisiae with a polylactic acid production of 87 mg / g DCW.

[0030] The present invention also performs saturation mutagenesis on the key gene SfYDIF for polylactic acid synthesis by bioinformatics means, and experimentally verifies and screens out the SfYDIF F199Y-D358L mutant, which can make the polylactic acid production of Saccharomyces cerevisiae reach 133 mg / g DCW. Compared with Tan et al. [Chunlin Tan, Fei Tao, Ping Xu. Direct carbon capture for the production of high-performance biodegradable plastics by cyanobacterial cell factories[J]. Green Chemistry, 2022, 24(11): 4470-4483], the polylactic acid production increases by 262.39%. Description of the Drawings

[0031] Figure 1 is the polylactic acid biosynthesis pathway;

[0032] Figure 2 is the comparison of lactic acid and polylactic acid production of strains with acetyl-CoA transferase from different sources;

[0033] Figure 3 is the comparison of polylactic acid production of BY4741 and its derivative strains;

[0034] Figure 4 is the comparison of lactic acid and polylactic acid production of INVSC1 and YPH499 and their derivative strains; Detailed Embodiments

[0035] The following examples are used to explain the technical solutions of the present invention non-restrictively.

[0036] In the present invention, unless otherwise specified, "%" for indicating concentration is the mass percentage.

[0037] The bacterial strains and plasmids involved in the examples are shown in Table 1, and the synthesized primer sequences are shown in Table 2.

[0038] Table 1 Bacterial strains and plasmids involved in the present invention

[0039]

[0040]

[0041]

[0042] Table 2 Primers involved in the present invention

[0043]

[0044]

[0045] The present invention relates to the following culture media:

[0046] YPD liquid medium: 2% glucose, 1% yeast extract, 2% peptone; 2% agar powder is added to the solid medium.

[0047] SD-LEU liquid medium: 2% glucose, 0.17% YNB (yeast nitrogen base, without amino acids and ammonium sulfate), 0.5% (NH4) 2 SO 4 SO

[0048] SD-URA liquid medium: 2% glucose, 0.17% YNB (yeast nitrogen base, without amino acids and ammonium sulfate), 0.5% (NH4) 2 SO 4 SO

[0049] In the present invention, unless otherwise specified, the fermentation method of each recombinant yeast is as follows:

[0050] The recombinant yeast is cultured in 50 ml of SD-URA liquid medium at 200 rpm and 30 °C for 72 h to obtain a seed solution. Then, 5% of the seed solution is transferred to 150 ml of fermentation medium and grown at 200 rpm and 30 °C for 48 h, and then continued to ferment at 100 rpm and 30 °C for 96 h to obtain the fermentation broth to be tested.

[0051] The method for lactic acid determination is as follows:

[0052] Centrifuge the fermentation broth to be tested at 12,000 rpm, collect the supernatant, filter it through a 0.22 μm filter membrane, and then detect it by HPLC. The detection chromatographic column is Bio-Rad HPX 87H; mobile phase: 5 mM H 2 SO 4 ; flow rate: 0.5 mL / min; column temperature: 30 °C; the detector is a UV (210 nm) detector. Quantitative analysis is carried out using a D-lactic acid standard.

[0053] The method for ethanol determination is the same as that for lactic acid detection, and the detector is changed to a differential refractometer.

[0054] The method for polylactic acid determination is as follows:

[0055] Centrifuge the fermentation broth to be tested at 12,000 rpm, collect the cell precipitate, and freeze-dry it using a low-temperature freeze dryer to obtain a sample. Accurately weigh 10 mg of polylactic acid and place it in an esterification tube. Add 2 mL of analytical pure chloroform and 2 mL of esterification solution (1 g / L benzoic acid and 3%

[0056] (v / v) concentrated sulfuric acid) are added to 500 mL of anhydrous methanol). Seal the esterification tube, mix it evenly by shaking, and then carry out esterification at a constant temperature of 100 °C for 4 h. Cool to room temperature, carefully open the bottle cap, add 1 mL of deionized water, shake until completely mixed, and then let it stand for layering. After the aqueous phase and the organic phase are completely separated, take the lower layer (chloroform phase) solution, filter it through a 0.22 μm filter membrane, and carry out GC-MS analysis. Qualitative and quantitative analysis are carried out using a polylactic acid standard solution.

[0057] GC-MS detection conditions: The initial temperature is 50 °C, hold for 5 min, increase the temperature at a rate of 7.5 °C / min to 230 °C, hold for 0 min, increase the temperature at a rate of 10 °C / min to 260 °C, hold for 5 min, the inlet temperature is 240 °C, and the detector temperature is 250 °C.

[0058] Example 1 Construction of a Saccharomyces cerevisiae engineering bacterium with knockout of PDC1 and ADH1 genes and transfer of LDHA gene

[0059] The sequences of the target genes, pyruvate decarboxylase gene PDC1 (GeneID: 850733) and alcohol dehydrogenase gene ADH1 (GeneID: 854068), were found on NCBI. Using the Saccharomyces cerevisiae S288C genome (GenBank: GCA_000146045.2) as a template, upstream and downstream homologous arm amplification primers PDC1-up-F / R, PDC1-down-F / R, ADH1-up-F / R, and ADH1-down-F / R were designed. The upstream and downstream homologous arms of the PCD1 and ADH1 genes were amplified by PCR using the Saccharomyces cerevisiae BY4841 genome as a template. The obtained upstream homologous arm fragments were ligated to the ploxpura3loxp vector digested with ApaⅠ and XbaⅠ using the Gibson Assembly kit, and the downstream homologous arms were ligated to the ploxpura3loxp vector digested with NdeⅠ and MluⅠ using the Gibson Assembly kit to obtain plasmids ploxpura3loxp-PDC1 and ploxpura3loxp-ADH1.

[0060] The Cre gene sequence is shown in SEQ ID NO.13. The Cre gene was sent to GenScript Biotech Corporation for synthesis into the pUC57 vector. Using the pUC57-Cre vector as a template, upstream and downstream homologous arm amplification primers Cre-F / R were designed and the Cre gene was amplified. The gene fragment was recovered by gel extraction. The obtained gene fragment was ligated to the JN44-GFP vector digested with HindⅢ and SmaⅠ using the Gibson Assembly kit to obtain the JN44-Cre vector.

[0061] Using a yeast transformation kit, linearized ploxpura3loxp-PDC1 with ApaⅠ was transformed into Saccharomyces cerevisiae BY4741 (purchased from Miaoling Biology). Single colonies with successful transformation were screened out on SD-URA defective medium. Different single colonies were picked for fermentation verification. Compared with the control strain BY4741, the strain with the lowest ethanol production was selected to remove the URA marker.

[0062] Among them, the method for removing the URA marker is as follows: The constructed JN44-Cre plasmid is transferred into the strain to be marked and removed, cultured in SD-LEU liquid medium, and the strain containing JN44-Cre is obtained through screening. Single colonies are picked into SD-LEU liquid medium to induce the expression of Cre protein. The expressed protein cleaves the LOXP site of the ploxpura3loxp-PDC1 plasmid to remove the URA marker. The successfully removed strains are subcultured 3 times in YPD liquid medium. The bacterial solution of the third subculture is spread on plates of SD-LEU solid medium and YPD solid medium for culture. The strains that do not grow on the SD-LEU solid medium plate but grow on the YPD solid medium plate are the strains with the JN44-Cre plasmid removed. The colonies with correct colony PCR verification are named BY4741-1 (i.e., the Saccharomyces cerevisiae BY4741 strain with the PDC1 gene knocked out);

[0063] Similarly, the ploxpura3loxp-ADH1 linearized by ApaⅠ is transferred into BY4741-1. After screening with defective medium and fermentation, the URA marker and the JN44-Cre plasmid are removed. The strain with correct colony PCR is named BY4741-2 (i.e., the Saccharomyces cerevisiae BY4741 strain with the PDC1 and ADH1 genes knocked out).

[0064] Using the genome of Escherichia coli K12 series strain BW25113 (NCBI Reference Sequence: NZ_CP009273.1) as a template, the lactate dehydrogenase gene sequence (LDHA, protein sequence number: AAC74462.1) amplified with LDHA-F / R as primers is ligated to the vector pY26TEF-GPD (purchased from Miaoling Biology) digested with SpeⅠ to obtain the vector pY26TEF-L. The plasmid pY26TEF-L is transferred into the Saccharomyces cerevisiae BY4741-2 using a yeast transformation kit, and the engineered strain BY4741-3 producing D-lactic acid (the Saccharomyces cerevisiae BY4741 strain with the PDC1 and ADH1 genes knocked out and the LDHA gene transferred in) is obtained through screening with SD-URA medium.

[0065] Example 2 compares the effects of transferring YDIF genes from different sources on the polylactic acid production ability of engineered Saccharomyces cerevisiae

[0066] The transcription initiation and termination of the PHAC gene were initiated and terminated by the GAP promoter and the CYC1 terminator, respectively. Using the pY26TEF-GPD plasmid purchased from Miaoling as a template, the GAP promoter and the CYC1 terminator were amplified with Pgap-F / R and Tcyc1-F / R as primers. Using the plasmid containing the synthetic gene sequence PHAC (protein sequence number: ASL26409.1) as a template, the PHA synthase gene sequence PHAC was amplified with PHAC-F / R as primers. The GAP promoter, the CYC1 terminator, and the PHAC gene were ligated to the pY26TEF-L linear vector digested with EcoRⅠ using Gibson Assembly to obtain the recombinant plasmid pY26TEF-LP. The transcription initiation and termination of the acetyl-CoA transferase gene from different sources were initiated and terminated by the TEF1 promoter and the ADH1 terminator, respectively. The acetyl-CoA gene sequences from Escherichia coli (EcYDIF, nucleic acid sequence as shown in SEQ ID NO.5), Citrobacter werkmanii (CwYDIF, nucleic acid sequence as shown in SEQ ID NO.6), Serratia fonticola (SfYDIF, nucleic acid sequence as shown in SEQ ID NO.7), Bacillus sp. (BsYDIF, nucleic acid sequence as shown in SEQ ID NO.8), and Escherichia albertii (EaYDIF, nucleic acid sequence as shown in SEQ ID NO.9) were obtained and synthesized. The vector pY26TEF-LP was digested with NotⅠ, and the YDIF genes from different sources were ligated to the linear vector pY26TEF-LP, respectively, to obtain the recombinant plasmids pY26TEF-LP-EcYDIF, pY26TEF-LP-CwYDIF, pY26TEF-LP-SfYDIF, pY26TEF-LP-BsYDIF, and pY26TEF-LP-EaYDIF. The above plasmids were transformed into Saccharomyces cerevisiae BY4741-2, respectively. After screening with SD-URA medium, the strains verified correctly by PCR were named BY4741-4, BY4741-5, BY4741-6, BY4741-7, and BY4741-8, which had correctly inserted the EcYDIF, CwYDIF, SfYDIF, BsYDIF, or EaYDIF gene, respectively.

[0067] By detecting the lactic acid and polylactic acid contents in the fermentation broth, recombinant strains with high polylactic acid production were screened, with BY4741-3 as a control. The fermentation results are as Figure 2 shown.

[0068] The results showed that the lactic acid production of BY4741-3, BY4741-4, BY4741-5, BY4741-6, BY4741-7, and BY4741-7 were 154.34 g / L, 141.28 g / L, 152.96 g / L, 137.73 g / L, 143.11 g / L, and 147.54 g / L, respectively, and the polylactic acid production was 0.03 mg / L, 62.16 mg / L, 5.01 mg / L, 87.56 mg / L, 59.94 mg / L, and 47.16 mg / L, respectively. The lactic acid concentration in the fermentation broth of each strain was high, while the polylactic acid production was significantly different. Among them, the recombinant bacteria from Serratia juquan had the highest polylactic acid production, so the SfYDIF gene from Serratia juquan was selected as the best acetate coenzyme A transferase gene.

[0069] Example 3 Comparison of the effects of different SfYDIF gene mutants on the polylactic acid capacity of Saccharomyces cerevisiae engineering bacteria

[0070] Use AlphaFold to model the acetate-CoA transferase SfYDIF of Serratia cirrhosa from scratch and determine the best model:

[0071] Download the substrate D-lactic acid molecule from PubChem (PubChem CID: 61503), use the SfYDIF enzyme molecule modeled by AlphaFold as the receptor and the D-lactic acid molecule as the ligand, use Autoduck 4.0 to hydrogenate SfYDIF and D-lactic acid and balance them, use the docking algorithm to complete the molecular docking, and determine the grid according to the docking results box range, and then docked again. The results showed that the D-lactic acid molecule had good spatial complementary characteristics with the sfYDIF enzyme binding pocket, and the hydrogen bonds formed with the carbonyl groups on F199 and V210 respectively interacted with each other. At the same time, the ligand formed strong van der Waals interactions with R241 and D358. Since non-covalent interactions such as hydrogen bonds are the main interaction forces between proteins and ligand molecules, the ligand and receptor are stably bound. Finally, the possible active centers of the sfYDIF enzyme catalyzing the D-lactic acid molecule were F199, Y205, L206, D207, A208, L209, V210, R241, I242 and D358. Using Discovery Studio software, alanine scanning mutagenesis was performed on all amino acids within the 6A° range of the active center, and it was found that the affinity of the F199, V210 and D358 sites changed greatly.

[0072] F199, V210 and D358 were subjected to saturation mutagenesis, and F199Y, V210E and D358L with the largest affinity changes were selected for experimental verification:

[0073] Using the synthesized SfYDIF gene as a template, amplify with SfYDIF-F / SfYDIF(199)-R, SfYDIF(199)-F / SfYDIF-R, SfYDIF-F / SfYDIF(210)-R, SfYDIF(210)-F / SfYDIF-R, and SfYDIF-F / SfYDIF(358)-R, SfYDIF(358)-F / SfYDIF-R as primers respectively to obtain mutant fragments YDIF(199)-U / YDIF(199)-D, YDIF(210)-U / YDIF(210)-D, YDIF(358)-U / YDIF(358)-D. Digest the vector pY26TEF-LP with NotⅠ alone, and ligate the mutant fragments YDIF(199)-U / YDIF(199)-D, YDIF(210)-U / YDIF(210)-D, YDIF(358)-U / YDIF(358)-D to the linear vector pY26TEF-LP respectively to obtain recombinant plasmids pY26TEF-LP-SfYDIF F199Y 、pY26TEF-LP-SfYDIF V210E 、pY26TEF-LP-SfYDIF D358L 。The above plasmids were respectively transformed into Saccharomyces cerevisiae BY4741-2, and screened with SD-URA medium. The strains verified correctly by PCR were named BY4741-9, BY4741-10, BY4741-11, which inserted mutants SfYDIF F199Y 、SfYDIF V210E and SfYDIF D358L 。

[0074] Fermentation experiments were carried out with Saccharomyces cerevisiae BY4741-6, BY4741-9, BY4741-10, BY4741-11 strains, and the fermentation results are as Figure 3 shown.

[0075] By comparing the polylactic acid content of each mutant strain, it was found that compared with BY4741-6, the polylactic acid yield of the SfYDIF V210E mutant decreased by 4.9%, while the SfYDIF F199Y and SfYDIF D358L mutants increased the polylactic acid yield by 24% and 16% respectively. Therefore, the two mutants were combined subsequently to study the synergistic effect of the mutations.

[0076] Using the synthesized SfYDIF gene as a template, amplify with SfYDIF-F / SfYDIF(199)-R, SfYDIF(199)-F / SfYDIF(358)-R, and SfYDIF(358)-F / SfYDIF-R as primers respectively to obtain mutant fragments YDIF(199)-U, YDIF(199 / 358)-M / , and YDIF(358)-D. Digest the vector pY26TEF-LP with NotⅠ alone, and ligate the mutant fragments YDIF(199)-U, YDIF(199 / 358)-M / , and YDIF(358)-D to the linear vector pY26TEF-LP respectively to obtain the recombinant plasmid pY26TEF-LP-SfYDIF F199Y-D358L Transfer the plasmid into Saccharomyces cerevisiae BY4741-2, screen with SD-URA medium, and name the strain with correct PCR verification as BY4741-12.

[0077] Perform fermentation experiments with Saccharomyces cerevisiae BY4741-6, BY4741-9, BY4741-11, and BY4741-12 strains respectively. The results are as Figure 3 shown. By comparing the polylactic acid content of each mutant strain, it is known that the SfYDIF V210E-D358L yield of the mutant polylactic acid is 133 mg / g DCW. Compared with BY4741-6, the polylactic acid yield increases by 34%.

[0078] Example 4 Production of Polylactic Acid by Saccharomyces cerevisiae INVSC1 and YPH499 Yeast

[0079] After linearizing the knockout plasmid ploxpura3loxp-PDC1 with ApaⅠ, transfer it into Saccharomyces cerevisiae INVSC1 (purchased from Mingzhou Biology) and YPH499 (purchased from Mingzhou Biology) respectively. Screen the transformants containing the linearized plasmid with SD-URA defective medium, pick different single colonies for fermentation verification, and select the strains with the lowest ethanol content to remove the URA marker compared with the control strains INVSC1 and YPH499 respectively. The method for removing URA is the same as that in Example 1, and the strains with correct colony PCR verification are named INVSC1-1 (i.e., Saccharomyces cerevisiae INVSC1 with the PDC1 gene knocked out) and YPH499-1 (i.e., Saccharomyces cerevisiae YPH499 with the PDC1 gene knocked out);

[0080] Similarly, the ApaⅠ-linearized ploxpura3loxp-ADH1 was separately transformed into INVSC1-1 and YPH499-1. After screening with defective media and fermentation, the URA and JN44-Cre plasmids were removed. The strains with correct colony PCR results were named INVSC1-2 (i.e., Saccharomyces cerevisiae INVSC1 with PDC1 and ADH1 genes knocked out) and YPH499-2 (i.e., Saccharomyces cerevisiae YPH499 with PDC1 and ADH1 genes knocked out).

[0081] The plasmids pY26TEF-LP-SfYDIF and pY26TEF-LP-SfYDIF constructed in Example 3 F199Y-D358L were separately transformed into Saccharomyces cerevisiae INVSC1-2, and the correct strains verified by PCR were screened using SD-URA medium and named INVSC1-3 and INVSC1-4 respectively; pY26TEF-LP-SfYDIF and pY26TEF-LP-SfYDIF F199Y-D358L were separately transformed into Saccharomyces cerevisiae YPH499-2, and the correct strains verified by PCR were screened using SD-URA medium and named YPH499-3 and YPH499-4 respectively.

[0082] Fermentation experiments were carried out using Saccharomyces cerevisiae strains INVSC1-2, INVSC1-3, INVSC1-4, YPH499-2, YPH499-3, and YPH499-4 respectively. The results are as Figure 4 shown. By comparing the polylactic acid production of each strain, it was found that Saccharomyces cerevisiae INVSC1-2 and YPH499-2 did not produce polylactic acid by themselves, but could produce 90.21 mg / g DCW and 85.30 mg / g DCW of polylactic acid respectively after introducing the polylactic acid pathway. Introducing the acetate-CoA transferase mutant increased the polylactic acid production by 27.8% and 30.1% respectively.

[0083] From the above experimental results, it can be seen that not all YDIF genes of acetate-CoA transferase from any source can improve the ability of Saccharomyces cerevisiae to produce polylactic acid. Among them, the YDIF genes of acetate-CoA transferase from Escherichia coli, Serratia fonticola, Bacillus sp.oxB-1, and Escherichia albertii can significantly enhance the polylactic acid production capacity of Saccharomyces cerevisiae. Among them, the YDIF gene of acetate-CoA transferase from Serratia fonticola has the strongest enhancing effect.

[0084] Furthermore, by mutating the acetyl-CoA transferase YDIF gene from a preferred source, the poly(lactic acid) production capacity of Saccharomyces cerevisiae can be further improved, especially by mutating the 199th position of the YDIF gene from phenylalanine to tyrosine or the 358th position from aspartic acid to tyrosine, when the YDIF gene is from Serratia fonticola.

[0085] In addition, simultaneously mutating the 199th position of the YDIF gene from phenylalanine to tyrosine and the 358th position from aspartic acid to tyrosine can further increase the production capacity on the basis of single-site mutation.

[0086] In the present invention, the pyruvate decarboxylase PDC1 gene and alcohol dehydrogenase ADH1 gene of Saccharomyces cerevisiae were knocked out, and the D-lactate dehydrogenase LDHA gene and PHA synthase PHAC gene were introduced as engineering strains. Then, the effects of acetyl-CoA transferase YDIF genes from different sources on the poly(lactic acid) production ability of Saccharomyces cerevisiae were compared, and a recombinant Saccharomyces cerevisiae BY4741-6 with high poly(lactic acid) production was successfully constructed. The poly(lactic acid) yield of this strain was 87 mg / g DCW. On this basis, the present invention used a bioinformatics analysis method to find the catalytic active center of YDIF, mutated and experimented on the active site to verify different mutants SfYDIF F199Y 、SfYDIF D358L 、SfYDIF F199Y-D358L on the lactic acid yield, so as to select the recombinant strain BY4741-12 containing the SfYDIF F199Y -D358L mutant with the highest yield. The poly(lactic acid) yield of this strain reached 133 mg / g DCW in a shake flask, which was significantly higher than that of the prior art. The recombinant Saccharomyces cerevisiae BY4741-12 of the present invention can efficiently synthesize poly(lactic acid).

Claims

1. A polylactic acid-producing yeast strain, characterized in that The brewer's yeast engineering bacterium is an engineering bacterium that uses brewer's yeast BY4741, INVSC1 or YPH499 as a starting strain to express a D-lactate dehydrogenase gene LDHA, an acetate coenzyme A transferase gene YDIF mutant and a PHA synthase gene PHAC, and lacks a pyruvate decarboxylase gene PDC1 and an alcohol dehydrogenase gene ADH1; the D-lactate dehydrogenase gene LDHA is derived from Escherichia coli, the PHA synthase gene PHAC is derived from brown nitrogen-fixing bacteria, the acetate coenzyme A transferase gene YDIF mutant is obtained by mutating the 199th position of the acetate coenzyme A transferase gene YDIF gene derived from Serratia juquani as shown in SEQ ID No.7 from phenylalanine to tyrosine, and the nucleic acid sequence thereof is shown in SEQ ID NO.10, or mutating the 358th position from aspartic acid to tyrosine, and the nucleic acid sequence thereof is shown in SEQ ID As shown in SEQ ID NO.11, or simultaneously mutating position 199 of the YDIF gene from Serratia avium from phenylalanine to tyrosine and position 358 from aspartic acid to tyrosine, the nucleic acid sequence of which is shown in SEQ ID NO.

12.

2. Application of an acetate coenzyme A transferase gene YDIF mutant in improving the polylactic acid production capacity of an engineered yeast strain of saccharomyces cerevisiae, wherein the engineered yeast strain is an engineered strain that uses saccharomyces cerevisiae BY4741, INVSC1 or YPH499 as a starting strain to express a D-lactate dehydrogenase gene LDHA, an acetate coenzyme A transferase gene YDIF mutant and a PHA synthase gene PHAC, and lacks a pyruvate decarboxylase gene PDC1 and an alcohol dehydrogenase gene ADH1; the D-lactate dehydrogenase gene LDHA is derived from Escherichia coli, the PHA synthase gene PHAC is derived from brown nitrogen-fixing bacteria, and the acetate coenzyme A transferase gene YDIF mutant is obtained by replacing the amino acid residue of SEQ ID The acetate CoA transferase gene YDIF from Salmonella juvenile shown in No. 7 is obtained by mutating position 199 from phenylalanine to tyrosine, or mutating position 358 from aspartic acid to tyrosine, or by mutating position 199 from phenylalanine to tyrosine and position 358 from aspartic acid to tyrosine at the same time.

3. Use of the engineered yeast Saccharomyces cerevisiae according to claim 1 in fermentation to produce polylactic acid.

4. A method for constructing a recombinant Saccharomyces cerevisiae that produces polylactic acid, the method comprising the following steps: (1) Construction of PLA pathway plasmid The D-lactate dehydrogenase gene LDHA from Escherichia coli was amplified, and the amplified fragment was inserted into the SpeⅠ site of pY26TEF-GPD to obtain pY26TEF-L; the PHA synthase PHAC gene sequence from brown nitrogen-fixing bacteria shown in SEQ ID No. 2 was artificially synthesized, the GAP promoter and CYC1 terminator of pY26TEF-GPD were amplified, and the synthesized gene fragment PHAC gene, GAP promoter and CYC1 terminator were inserted into the EcoR Ⅰ site of plasmid pY26TEF-L to obtain plasmid pY26TEF-LP; Synthetic synthesis of the acetate coenzyme A transferase gene YDIF mutant YDIF from Serratia juquanensis as shown in SEQ ID. No. 10, 11, 12 F199Y 、YDIF D358L and YDIF F199Y-D358L The synthesized gene sequences were inserted into the NotⅠ site of plasmid pY26TEF-LP to obtain plasmids pY26TEF-LP-YDIF F199Y 、pY26TEF-LP-YDIF D358L and pY26TEF-LP-YDIF F199Y-D358L ; (2) Construction of the engineered yeast BY4741-2 with PDC1 and ADH1 genes knocked out and LDHA gene introduced The upstream and downstream homology arms of PCD1 and ADH1 genes were amplified by PCR using the genome of Saccharomyces cerevisiae BY4841 as a template. The upstream homology arm fragment was ligated to the ploxpura3loxp vector digested with ApaⅠ and XbaⅠ, and the downstream homology arm was ligated to the ploxpura3loxp vector digested with NdeⅠ and MluⅠ, to obtain plasmids ploxpura3loxp-PDC1 and ploxpura3loxp-ADH1, respectively. The Cre gene as shown in SEQ ID NO.13 was synthesized into the pUC57 vector to obtain the vector pUC57-Cre, the Cre gene was amplified using the pUC57-Cre vector as a template, and the obtained gene fragment was ligated to the JN44-GFP vector digested with HindⅢ and SmaⅠ to obtain the JN44-Cre vector; The ploxpura3loxp-PDC1 linearized by ApaⅠ was transformed into Saccharomyces cerevisiae BY4741, and the single colonies with successful transformation were screened by SD-URA-deficient medium. The strain with the lowest ethanol production was selected to remove the URA marker and JN44-Cre plasmid. The obtained strain was named BY4741-1 after PCR verification. BY4741-1 is the Saccharomyces cerevisiae BY4741 strain with the PDC1 gene knocked out. The ploxpura3loxp-ADH1 linearized by ApaⅠ was transferred into BY4741-1, and the URA marker and JN44-Cre plasmid were removed after screening by defective medium and fermentation. The strain with the correct colony PCR was named BY4741-2, which is the BY4741 strain of Saccharomyces cerevisiae with PDC1 and ADH1 genes knocked out. (3) Construction of recombinant Saccharomyces cerevisiae BY4741-9, BY4741-11 and BY4741-12 producing polylactic acid Step (1) pY26TEF-LP-YDIF F199Y 、pY26TEF-LP-YDIF D358L and pY26TEF-LP-YDIF F199Y-D358L The plasmids were transferred into BY4741-2 strain respectively, and the obtained strains were coated with SD-URA flat solid medium. The correct strains verified by amplification were named recombinant Saccharomyces cerevisiae BY4741-9, BY4741-11 and BY4741-12.

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