Pyruvate high-yield strain and construction method thereof

Abstract

This invention discloses a high-yield pyruvate strain and its construction method, belonging to the field of microbial genetic engineering technology. The construction method includes the following steps: using *Escherichia coli* CGMCC No. 25786 as the starting strain, and utilizing CRIPSR-Cas9 technology, the gene *ldhA* of lactate dehydrogenase and the gene *zwf* of glucose-6-phosphate dehydrogenase are knocked out; the gene *pntAB* of membrane-bound transhydrogenase is overexpressed; the activity of the gene *aceE* of pyruvate dehydrogenase is reduced; and the genes *prk* and *cbbLS* of the RuBisCO carbon fixation pathway are overexpressed. This invention constructs a high-yield pyruvate strain PYR-6 by reducing by-product generation, enhancing pyruvate synthesis flux, balancing reducing power, and reducing tricarboxylic acid cycle flux. The construction method of this invention can significantly improve the production efficiency and final concentration of pyruvate, and has the potential for industrial application in pyruvate production.

Description

Technical Field

[0001] This invention relates to the field of microbial genetic engineering technology, specifically to a high-yield pyruvate strain and its construction method. Background Technology

[0002] Pyruvate is a three-carbon molecule and a key intermediate in multiple metabolic pathways of central carbon metabolism. It can be utilized under both aerobic and anaerobic conditions, thus playing an indispensable role in the energy metabolism of organisms. Pyruvate is not only the final product of glycolysis but also a precursor to the synthesis of acetyl-CoA, acetic acid, lactic acid, and branched-chain amino acids. It can be converted into various substances, such as carbohydrates (through gluconeogenesis), alanine or ethanol (through fermentation), and fatty acids (through the tricarboxylic acid cycle). It is an important carbon node in oxidative metabolism, amino acid and fatty acid synthesis, and the production of various related metabolites. Pyruvate and its derivatives possess anti-inflammatory and antioxidant properties and can be widely used in chemical, pharmaceutical, and food industries.

[0003] Currently, pyruvate production mainly involves chemical synthesis, enzymatic catalysis, and microbial fermentation. Chemical synthesis suffers from high costs, complex operation, low recovery rates, low selectivity, a lack of suitable substrates, and high energy consumption and pollution, failing to align with green chemistry principles and meet the expanding market demand. Enzymatic catalysis significantly reduces costs compared to chemical synthesis, offers milder reaction conditions, and produces fewer byproducts. However, the specificity of enzyme reactions limits the availability of suitable substrates and catalysts, restricting its application in large-scale pyruvate production. Generally, microbial fermentation has a slightly lower substrate conversion efficiency than enzymatic catalysis, but it offers high substrate selectivity, low cost and environmental friendliness, a wide variety of readily available and easily cultivated strains, stable and controllable fermentation processes, and the production of bioactive substances, gradually gaining dominance in pyruvate and pyruvate production. However, pyruvate, as one of the hubs of microbial energy metabolism, can be synthesized in large quantities through multiple metabolic pathways. At the same time, it is consumed in large quantities as a precursor for the synthesis of substances such as intracellular amino acids, formic acid, lactic acid, and acetyl-CoA. Under natural conditions, it is difficult to accumulate in large quantities, which leads to the problem of low yield when producing pyruvate by microbial fermentation.

[0004] Pyruvate is a key intermediate in microbial metabolic pathways and is readily converted into other products, making it difficult to accumulate in large quantities intracellularly. While existing literature and patents have explored various strategies to increase yield, the accumulation rate remains limited. For example, Chinese patent CN111100832B discloses a genetically engineered bacterium for synthesizing pyruvate and D-alanine, its construction method, and its applications. By blocking key pathways for further pyruvate metabolism and knocking out pyruvate transport proteins, a yield of 32 g / L is achieved. Chinese patent CN117887651A discloses a strain for synthesizing pyruvate or L-tyrosine, which uses the CRISPR / dCas9 interference system (CRISPRi) to inhibit the expression of the pyruvate dehydrogenase complex E1 gene (aceE), increasing the strain yield to 2-35 g / L. However, a comprehensive evaluation of existing technologies shows that the highest accumulation rate of pyruvate has not yet exceeded 35 g / L, meaning that efficient and large-scale accumulation of pyruvate has not yet been achieved. Therefore, developing a new high-yield strain capable of efficiently and massively accumulating pyruvate is of significant practical importance and urgent need. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a high-yield pyruvate strain and its construction method. Using CRISPR / Cas9 technology, and with D-lactic acid-producing Escherichia coli (strain preservation number: CGMCC No. 25786) as the chassis, a highly efficient engineered strain for producing pyruvate is obtained through metabolic engineering.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is as follows: A high-pyruvate-producing strain, named *Escherichia coli* PYR-6, and classified as *Escherichia coli*. Escherichia coli PYR-6, accession number CCTCC NO: M 20261914, accession date August 18, 2026, deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0007] A method for constructing the aforementioned high-yield pyruvate strain includes the following steps: using Escherichia coli CGMCC No. 25786 as the starting strain, using CRIPSR-Cas9 technology, knocking out the lactate dehydrogenase gene ldhA, knocking out the glucose-6-phosphate dehydrogenase gene zwf, overexpressing the membrane-bound transhydrogenase gene pntAB, reducing the activity of the pyruvate dehydrogenase gene aceE, and overexpressing the RuBisCO carbon fixation pathway genes prk and cbbLS; Preferably, the Escherichia coli CGMCC No. 25786 is a D-lactic acid producing bacterium, and its construction method refers to Chinese Patent CN116004467B, which describes a heat-resistant Escherichia coli strain and its application in the production of D-lactic acid. Preferably, knocking out the lactate dehydrogenase gene ldhA includes the following steps: S11. Construction of recombinant plasmid pECgRNA-N20-ldhA: Based on the ldhA gene sequence, an N20 sequence was set. Based on the N20 sequence, forward oligonucleotides N20-ldhA-F and reverse oligonucleotides N20-ldhA-R were designed. The forward and reverse oligonucleotides N20-ldhA-F and N20-ldhA-R were mixed and annealed to form double-stranded DNA. The plasmid was digested with enzymes to obtain a linear plasmid. The double-stranded DNA and the linear plasmid were ligated to obtain a ligation product. The ligation product was transformed and screened to obtain the recombinant plasmid pECgRNA-N20-ldhA. S12. Constructing the ldhA-K fragment: Using the genomic DNA of the starting strain as a template, the target fragment is amplified with primers ldhA1 / ldhA2 and ldhA3 / ldhA4, respectively. After purification, two purified fragments are obtained. Using the two purified fragments as a mixed template, overlap extension PCR is performed with primers ldhA1 / ldhA4 to obtain the ldhA-K fragment. S13. Constructing a strain with ldhA knocked out: Transform the plasmid pEcCAS into the starting strain to obtain the recombinant strain D-Lac / pEcCAS; Transform the ldhA-K fragment and the recombinant plasmid pECgRNA-N20-ldhA into the recombinant strain D-Lac / pEcCAS, screen to eliminate plasmids pEcCAS and pECgRNA-N20-ldhA, and obtain strain PYR-1; More preferably, in S11, the nucleotide sequence of the N20 sequence is as shown in SEQ ID No. 1 in the sequence listing; More preferably, in S11, the nucleotide sequence of the forward oligonucleotide N20-ldhA-F is as shown in SEQ ID No. 2 in the sequence listing; More preferably, in S11, the nucleotide sequence of the reverse oligonucleotide N20-ldhA-R is as shown in SEQ ID No. 3 in the sequence listing; More preferably, in S11, when the plasmid is digested with enzymes to obtain a linear plasmid, the plasmid is plasmid pECgRNA; More preferably, in S11, when the plasmid is digested with an enzyme to obtain a linear plasmid, the enzyme digestion is BsaI digestion; More preferably, in S11, when transforming and screening the ligation product, the method is to transform the ligation product into Escherichia coli DH5α competent cells, identify it as a positive clone by DNA sequencing, and extract the plasmid. More preferably, in S12 and S13, the starting strain is Escherichia coli CGMCC No.25786, and its construction method refers to Chinese Patent CN116004467B A thermoresistant Escherichia coli and its application in the production of D-lactic acid; More preferably, in S12, the nucleotide sequence of ldhA1 is as shown in SEQ ID No. 4 in the sequence listing, the nucleotide sequence of ldhA2 is as shown in SEQ ID No. 5 in the sequence listing, the nucleotide sequence of ldhA3 is as shown in SEQ ID No. 6 in the sequence listing, and the nucleotide sequence of ldhA4 is as shown in SEQ ID No. 7 in the sequence listing; More preferably, in S13, electroporation is used when transforming plasmid pEcCAS into the starting strain and when transforming the ldhA-K fragment and recombinant plasmid pECgRNA-N20-ldhA into the recombinant strain D-Lac / pEcCAS. More preferably, in S13, the screening is performed using a combination of LB solid medium plates containing spectinomycin and kanamycin dual resistance and PCR verification. Preferably, knocking out the glucose-6-phosphate dehydrogenase gene zwf includes the following steps: S21. Construction of recombinant plasmid pECgRNA-N20-zwf: The N20 sequence is set according to the zwf gene sequence. Forward oligonucleotides N20-zwf-F and reverse oligonucleotides N20-zwf-R are designed based on the N20 sequence. The forward and reverse oligonucleotides N20-zwf-F and N20-zwf-R are mixed and annealed to form double-stranded DNA. The plasmid is digested with enzymes to obtain a linear plasmid. The double-stranded DNA and the linear plasmid are ligated to obtain a ligation product. The ligation product is transformed and screened to obtain the recombinant plasmid pECgRNA-N20-zwf. S22. Constructing the zwf-K fragment: Using the genomic DNA of the starting strain as a template, the target fragment is amplified with primers zwf1 / zwf2 and zwf3 / zwf4 respectively. After purification, two purified fragments are obtained. Using the two purified fragments as a mixed template, overlap extension PCR is performed with primers zwf1 / zwf4 to obtain the zwf-K fragment. S23. Constructing a zwf-knockout strain: Transform the plasmid pEcCAS into strain PYR-1 to obtain the recombinant strain PYR-1 / pEcCAS; Transform the zwf-K fragment and the recombinant plasmid pECgRNA-N20-zwf into the recombinant strain PYR-1 / pEcCAS, screen to eliminate plasmids pEcCAS and pECgRNA-N20-zwf, and obtain strain PYR-2; More preferably, in S21, the nucleotide sequence of the N20 sequence is as shown in SEQ ID No. 8 in the sequence listing; More preferably, in S21, the nucleotide sequence of the forward oligonucleotide N20-zwf-F is as shown in SEQ ID No. 9 in the sequence listing; More preferably, in S21, the nucleotide sequence of the reverse oligonucleotide N20-zwf-R is shown in SEQ ID No. 10 of the sequence listing; More preferably, in S21, when the plasmid is digested with enzymes to obtain a linear plasmid, the plasmid is plasmid pECgRNA; More preferably, in S21, when the plasmid is digested with an enzyme to obtain a linear plasmid, the enzyme digestion is BsaI digestion; More preferably, in S21, when transforming and screening the ligation product, the method is to transform the ligation product into Escherichia coli DH5α competent cells, identify it as a positive clone by DNA sequencing, and extract the plasmid. More preferably, in S22, the starting strain is Escherichia coli CGMCC No.25786, and its construction method refers to Chinese Patent CN116004467B A thermoresistant Escherichia coli and its application in the production of D-lactic acid; More preferably, in S22, the nucleotide sequence of zwf1 is as shown in SEQ ID No. 11 in the sequence listing, the nucleotide sequence of zwf2 is as shown in SEQ ID No. 12 in the sequence listing, the nucleotide sequence of zwf3 is as shown in SEQ ID No. 13 in the sequence listing, and the nucleotide sequence of zwf4 is as shown in SEQ ID No. 14 in the sequence listing; More preferably, in S23, when transforming plasmid pEcCAS into strain PYR-1 to obtain recombinant strain PYR-1 / pEcCAS, and when transforming zwf-K fragment and recombinant plasmid pECgRNA-N20-zwf into recombinant strain PYR-1 / pEcCAS, electroporation is used. More preferably, in S23, the screening is performed using a combination of LB solid medium plates containing spectinomycin and kanamycin dual resistance and PCR verification. Preferably, overexpression of the membrane-binding transhydrogenase gene pntAB includes the following steps: S31. Construction of recombinant plasmid pECgRNA-N20-ldhA-2: The N20 sequence was set based on the sequence of the unknocked portion of the ldhA gene. Based on the N20 sequence, forward oligonucleotides 2-N20-ldhA-F and reverse oligonucleotides 2-N20-ldhA-R were designed. The forward and reverse oligonucleotides 2-N20-ldhA-F and 2-N20-ldhA-R were mixed and annealed to form double-stranded DNA. The plasmid was digested with enzymes to obtain a linear plasmid. The double-stranded DNA and the linear plasmid were ligated to obtain a ligation product. The ligation product was transformed and screened to obtain the recombinant plasmid pECgRNA-N20-ldhA-2. S32. Constructing the pntAB knock-in fragment of a moderate-strength promoter: Using the genomic DNA of the starting strain as a template, the pntAB knock-in fragment was constructed using primers pntAB::ldhA1 / P J23118 -pntAB::ldhA2、P J23118 The target fragment was amplified using pntAB::ldhA3 / pntAB::ldhA4 and pntAB::ldhA5 / pntAB::ldhA6, purified, and three purified fragments were obtained. Using these three purified fragments as a mixed template, overlap extension PCR was performed using primers pntAB::ldhA1 / pntAB::ldhA6 to obtain P... J23118 -pntAB::ldhA segment; S33. Construct a strain overexpressing the promoter pntAB: Transform the plasmid pEcCAS into strain PYR-2 to obtain the recombinant strain PYR-2 / pEcCAS; J23118 The pntAB::ldhA fragment and the recombinant plasmid pECgRNA-N20-ldhA-2 were transformed into the recombinant strain PYR-2 / pEcCAS. After screening, plasmids pEcCAS and pECgRNA-N20-ldhA-2 were eliminated to obtain strain PYR-4. More preferably, in S31, the nucleotide sequence of the non-knockout portion of the ldhA gene is shown in SEQ ID No. 57 of the sequence listing; More preferably, in S31, the nucleotide sequence of the N20 sequence is as shown in SEQ ID No. 15 in the sequence listing; More preferably, in S31, the nucleotide sequence of the forward oligonucleotide 2-N20-ldhA-F is as shown in SEQ ID No. 16 in the sequence listing; More preferably, in S31, the nucleotide sequence of the reverse oligonucleotide 2-N20-ldhA-R is as shown in SEQ ID No. 17 in the sequence listing; More preferably, in S31, when the plasmid is digested with enzymes to obtain a linear plasmid, the plasmid is plasmid pECgRNA; More preferably, in S31, when the plasmid is digested with an enzyme to obtain a linear plasmid, the enzyme digestion is BsaI digestion; More preferably, in S31, when transforming and screening the ligation product, the method is to transform the ligation product into Escherichia coli DH5α competent cells, identify it as a positive clone by DNA sequencing, and extract the plasmid. More preferably, in S32, the starting strain is Escherichia coli CGMCC No.25786, and its construction method refers to Chinese Patent CN116004467B A thermoresistant Escherichia coli and its application in the production of D-lactic acid; More preferably, in S32, the nucleotide sequence of pntAB::ldhA1 is as shown in SEQ ID No. 18 of the sequence listing; P J23118 The nucleotide sequence of -pntAB::ldhA2 is shown in SEQ ID No. 24 of the sequence listing; P J23118 The nucleotide sequence of pntAB::ldhA3 is shown in SEQ ID No. 25 of the sequence listing; the nucleotide sequence of pntAB::ldhA4 is shown in SEQ ID No. 21 of the sequence listing; the nucleotide sequence of pntAB::ldhA5 is shown in SEQ ID No. 22 of the sequence listing; and the nucleotide sequence of pntAB::ldhA6 is shown in SEQ ID No. 23 of the sequence listing. More preferably, in S32, P J23118 The nucleotide sequence of the -pntAB::ldhA fragment is shown in SEQ ID No. 59 of the sequence listing; More preferably, in S33, when transforming plasmid pEcCAS into strain PYR-2, and P J23118 When the -pntAB::ldhA fragment and the recombinant plasmid pECgRNA-N20-ldhA-2 were transformed into the recombinant strain PYR-2 / pEcCAS, electroporation was used. More preferably, in S33, the screening is performed using a combination of LB solid medium plates containing spectinomycin and kanamycin dual resistance and PCR verification. Preferably, reducing the activity of the pyruvate dehydrogenase gene aceE includes the following steps: S41. Construction of recombinant plasmid pECgRNA-N20-aceE: The N20 sequence is set according to the aceE gene. Forward oligonucleotides N20-aceE-F and reverse oligonucleotides N20-aceE-R are designed based on the N20 sequence. The forward and reverse oligonucleotides N20-aceE-F and N20-aceE-R are mixed and annealed to form double-stranded DNA. The plasmid is digested with enzymes to obtain a linear plasmid. The double-stranded DNA and the linear plasmid are ligated to obtain a ligation product. The ligation product is transformed and screened to obtain the recombinant plasmid pECgRNA-N20-aceE. S42. Constructing a point-mutated aceE knock-in fragment: Using the genomic DNA of the starting strain as a template, the target fragment was amplified using primers aceE1 / aceE(107V)2, aceE(107V)3 / aceE4, and aceE5 / aceE6, respectively. After purification, three purified fragments were obtained. Using the three purified fragments as a mixed template, overlap extension PCR was performed using primers aceE1 / aceE6 to obtain the aceE(107V) fragment. S43. Constructing an aceE mutant strain: Transform plasmid pEcCAS into strain PYR-4 to obtain recombinant strain PYR-4 / pEcCAS; Transform the aceE (107V) fragment and recombinant plasmid pECgRNA-N20-aceE into recombinant strain PYR-4 / pEcCAS, screen to eliminate plasmids pEcCAS and pECgRNA-N20-aceE, and obtain strain PYR-4V; More preferably, in S41, the nucleotide sequence of the N20 sequence is as shown in SEQ ID No. 28 in the sequence listing; More preferably, in S41, the nucleotide sequence of the forward oligonucleotide N20-aceE-F is as shown in SEQ ID No. 29 in the sequence listing; More preferably, in S41, the nucleotide sequence of the reverse oligonucleotide N20-aceE-R is as shown in SEQ ID No. 30 in the sequence listing; More preferably, in S41, when the plasmid is digested with enzymes to obtain a linear plasmid, the plasmid is plasmid pECgRNA; More preferably, in S41, when the plasmid is digested with an enzyme to obtain a linear plasmid, the enzyme digestion is BsaI digestion; More preferably, in S41, when transforming and screening the ligation product, the method is to transform the ligation product into Escherichia coli DH5α competent cells, identify it as a positive clone by DNA sequencing, and extract the plasmid. More preferably, in S42, the starting strain is Escherichia coli CGMCC No.25786, and its construction method refers to Chinese Patent CN116004467B A thermoresistant Escherichia coli and its application in the production of D-lactic acid; More preferably, in S42, the nucleotide sequence of aceE1 is as shown in SEQ ID No. 31 of the sequence listing; the nucleotide sequence of aceE(107V)2 is as shown in SEQ ID No. 37 of the sequence listing; the nucleotide sequence of aceE(107V)3 is as shown in SEQ ID No. 38 of the sequence listing; the nucleotide sequence of aceE4 is as shown in SEQ ID No. 34 of the sequence listing; the nucleotide sequence of aceE5 is as shown in SEQ ID No. 35 of the sequence listing; and the nucleotide sequence of aceE6 is as shown in SEQ ID No. 36 of the sequence listing. More preferably, in S42, the nucleotide sequence of the aceE(107V) fragment is as shown in SEQ ID No. 62 in the sequence listing; More preferably, in S43, electroporation is used when transforming plasmid pEcCAS into strain PYR-4 and when transforming the aceE(107V) fragment with recombinant plasmid pECgRNA-N20-aceE into recombinant strain PYR-4 / pEcCAS. More preferably, in S43, the screening is performed using a combination of LB solid medium plates containing spectinomycin and kanamycin dual resistance and PCR verification. Preferably, overexpression of the RuBisCO carbon fixation pathway gene prk and the gene cbbLS includes the following steps: S51. Construction of recombinant plasmid pECgRNA-N20-poxB: Based on the poxB gene, the poxB N20 sequence was set. Based on the poxB N20 sequence, forward oligonucleotides N20-poxB-F and reverse oligonucleotides N20-poxB-R were designed. The forward and reverse oligonucleotides N20-poxB-F and N20-poxB-R were mixed and annealed to form double-stranded DNA. The plasmid was digested with enzymes to obtain a linear plasmid. The double-stranded DNA and the linear plasmid were ligated to obtain a ligation product. The ligation product was transformed and screened to obtain the recombinant plasmid pECgRNA-N20-poxB. S52. Construction of recombinant plasmid pECgRNA-N20-yghX: Based on the yghX gene, the yghX N20 sequence was set. Based on the yghX N20 sequence, forward oligonucleotides N20-yghX-F and reverse oligonucleotides N20-yghX-R were designed. The forward and reverse oligonucleotides N20-yghX-F and N20-yghX-R were mixed and annealed to form double-stranded DNA. The plasmid was digested with enzymes to obtain a linear plasmid. The double-stranded DNA and the linear plasmid were ligated to obtain a ligation product. The ligation product was transformed and screened to obtain the recombinant plasmid pECgRNA-N20-yghX. S53. Constructing the overexpression prk gene knock-in fragment: Using the genomic DNA of the starting strain as a template, and using primers prk::yghX1 / P... J23119 -prk::yghX2, prk::yghX5 / prk::yghX6 amplify the target fragment; use the synthesized prk gene sequence as a template, and primer P J23119 -prk::yghX3 / prk::yghX4 amplifies the target fragment; after purifying the amplified fragments, three purified fragments are obtained. Using the three purified fragments as a mixed template, overlap extension PCR is performed with primers prk::yghX1 / prk::yghX6 to obtain P J23119 -prk::yghX fragment; S54. Constructing a strain overexpressing the prk gene: Transform plasmid pEcCAS into strain PYR-4V to obtain the recombinant strain PYR-4V / pEcCAS; J23119 The -prk::yghX fragment and the recombinant plasmid pECgRNA-N20-yghX were transformed into the recombinant strain PYR-4V / pEcCAS. After screening, plasmids pEcCAS and pECgRNA-N20-yghX were eliminated to obtain strain PYR-5OE. S55. Constructing the overexpression of the cbbLS gene: Using the genomic DNA of the starting strain as a template, and with primers cbbLS::poxB1 / P... Trc -cbbLS::poxB2, cbbLS::poxB5 / cbbLS::poxB6 amplify the target fragment; use the synthesized cbbLS gene sequence as a template, and primer P Trc -cbbLS::poxB3 / P Trc -cbbLS::poxB4 amplifies the target fragment; after purifying the amplified fragment, three purified fragments are obtained. Using the three purified fragments as a mixed template, overlap extension PCR is performed with primers cbbLS::poxB1 / cbbLS::poxB6 to obtain P. Trc -cbbLS::poxB fragment; S56. Constructing a strain overexpressing the cbbLS gene: Transform plasmid pEcCAS into strain PYR-5OE to obtain the recombinant strain PYR-5OE / pEcCAS; Trc The -cbbLS::poxB fragment and the recombinant plasmid pECgRNA-N20-poxB were transformed into the recombinant strain PYR-5OE / pEcCAS. After screening, the plasmids pEcCAS and pECgRNA-N20-poxB were eliminated to obtain strain PYR-6. More preferably, in S51, the nucleotide sequence of the poxB N20 sequence is as shown in SEQ ID No. 39 in the sequence listing; More preferably, in S51, the nucleotide sequence of N20-poxB-F is as shown in SEQ ID No. 40 in the sequence listing; More preferably, in S51, the nucleotide sequence of N20-poxB-R is as shown in SEQ ID No. 41 in the sequence listing; More preferably, in S51, when the plasmid is digested with enzymes to obtain a linear plasmid, the plasmid is plasmid pECgRNA; More preferably, in S51, when the plasmid is digested with an enzyme to obtain a linear plasmid, the enzyme digestion is BsaI digestion; More preferably, in S51, when transforming and screening the ligation product, the method is to transform the ligation product into Escherichia coli DH5α competent cells, identify it as a positive clone by DNA sequencing, and extract the plasmid. More preferably, in S52, the nucleotide sequence of the yghX N20 sequence is as shown in SEQ ID No. 42 in the sequence listing; More preferably, in S52, the nucleotide sequence of N20-yghX-F is as shown in SEQ ID No. 43 in the sequence listing; More preferably, in S52, the nucleotide sequence of N20-yghX-R is as shown in SEQ ID No. 44 in the sequence listing; More preferably, in S52, when the plasmid is digested with enzymes to obtain a linear plasmid, the plasmid is plasmid pECgRNA; More preferably, in S52, when the plasmid is digested with an enzyme to obtain a linear plasmid, the enzyme digestion is BsaI digestion; More preferably, in S52, when transforming and screening the ligation product, the method is to transform the ligation product into Escherichia coli DH5α competent cells, identify it as a positive clone by DNA sequencing, and extract the plasmid. More preferably, in S53 and S55, the starting strain is Escherichia coli CGMCC No.25786, and its construction method refers to Chinese Patent CN116004467B A thermoresistant Escherichia coli and its application in the production of D-lactic acid; More preferably, in S53, the nucleotide sequence of the synthesized prk gene is shown as SEQ ID No. 63 in the sequence listing; More preferably, in S53, the nucleotide sequence of prk::yghX1 is as shown in SEQ ID No. 45 of the sequence listing; P J23119 The nucleotide sequences of -prk::yghX2 are shown in SEQ ID No. 46 of the sequence listing; the nucleotide sequences of prk::yghX5 are shown in SEQ ID No. 49 of the sequence listing; the nucleotide sequences of prk::yghX6 are shown in SEQ ID No. 50 of the sequence listing; P J23119 The nucleotide sequence of -prk::yghX3 is shown in SEQ ID No. 47 of the sequence listing; the nucleotide sequence of prk::yghX4 is shown in SEQ ID No. 48 of the sequence listing; More preferably, in S53, P J23119 The nucleotide sequence of the -prk:: yghX fragment is shown in SEQ ID No. 64 of the sequence listing; More preferably, in S54, when transforming plasmid pEcCAS into strain PYR-4V, and P J23119 When the -prk::yghX fragment and the recombinant plasmid pECgRNA-N20-yghX were transformed into the recombinant strain PYR-4V / pEcCAS, electroporation was used. More preferably, in S54, the screening is performed using a combination of LB solid medium plates containing spectinomycin and kanamycin dual resistance and PCR verification. More preferably, in S55, the nucleotide sequence of the synthesized cbbLS gene is shown in SEQ ID No. 65 of the sequence listing; More preferably, in S55, the nucleotide sequence of cbbLS::poxB1 is as shown in SEQ ID No. 51 of the sequence listing; P Trc The nucleotide sequences of -cbbLS::poxB2 are shown in SEQ ID No. 52 of the sequence listing; the nucleotide sequences of cbbLS::poxB5 are shown in SEQ ID No. 55 of the sequence listing; the nucleotide sequences of cbbLS::poxB6 are shown in SEQ ID No. 56 of the sequence listing; P Trc The nucleotide sequence of -cbbLS::poxB3 is shown in SEQ ID No. 53 of the sequence listing; P TrcThe nucleotide sequence of -cbbLS::poxB4 is shown in SEQ ID No. 54 of the sequence listing; More preferably, in S55, P Trc The nucleotide sequence of the -cbbLS::poxB fragment is shown in SEQ ID No. 66 of the sequence listing; More preferably, in S56, when transforming plasmid pEcCAS into strain PYR-5OE, and P Trc When the -cbbLS::poxB fragment and the recombinant plasmid pECgRNA-N20-poxB were transformed into the recombinant strain PYR-5OE / pEcCAS, electroporation was used. More preferably, in S56, the screening process combines screening on LB solid medium plates containing both spectinomycin and kanamycin antibiotics with PCR verification.

[0008] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention involves metabolic engineering of a D-lactic acid-producing bacterium with the strain preservation number CGMCC No. 25786. During the modification, CRISPR / Cas9 gene editing technology was used to construct a high-yield pyruvate strain, PYR-6. Compared with existing strains, strain PYR-6 exhibits significant advantages in sugar-acid conversion rate and production efficiency, showing promising prospects for industrial application. The high-yield pyruvate strain PYR-6 constructed in this invention, in a 5L fermenter, can produce 67.5 g / L of pyruvate after 30 hours of fermentation, achieving a sugar-acid conversion rate of 0.703 g / g and a production efficiency of 2.25 g / (L•h).

[0009] (2) This invention constructs a high-yield pyruvate strain PYR-6 by reducing byproduct generation, enhancing pyruvate synthesis flux, balancing reducing power, and reducing tricarboxylic acid cycle flux. The strain PYR-6 is a non-nutritive Escherichia coli that can effectively block the accumulation of lactic acid and the pentose phosphate pathway in the chassis strain, enhance the activity of key enzymes to supplement NADPH, reduce glucose consumption in the TCA cycle, and introduce a new carbon fixation pathway. Ultimately, it achieves a high concentration of carbon source flow to pyruvate, significantly improving the production efficiency and final concentration of pyruvate. Therefore, the construction method of this invention has the potential for industrial application in the production of pyruvate. Detailed Implementation

[0010] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, specific embodiments of the present invention are now described.

[0011] The components of the culture medium involved in the embodiments of the present invention are as follows: (1) LB solid medium: Tryptone 10g / L, Yeast Extract 5g / L, NaCl 10g / L, Agar 20g / L, pH 7.0.

[0012] (2) LB liquid medium: Tryptone 10g / L, Yeast Extract 5g / L, NaCl 10g / L, pH 7.0.

[0013] (3) LBS solid culture medium: Tryptone 10g / L, Yeast Extract 5g / L, NaCl 10g / L, sucrose 10% (w / v, i.e., 100g / L), agar 20g / L, pH 7.0.

[0014] (4) LBS liquid culture medium: Tryptone 10g / L, Yeast Extract 5g / L, NaCl 10g / L, sucrose 10% (w / v, i.e., 100g / L), pH 7.0.

[0015] In the embodiments of the present invention, the fermentation results obtained by the same strain in different fermentation experiments are inconsistent because there are acceptable normal process fluctuations in the composition of the culture medium and the fermentation conditions during fermentation, making it difficult to guarantee complete reproducibility of the fermentation results.

[0016] In the embodiments of the present invention, both pECgRNA plasmid and pEcCAS plasmid were purchased from addgene.

[0017] Example 1 1. Block the formation of the byproduct lactic acid: The D-lactic acid producing bacterium *Escherichia coli* (strain preservation number CGMCC No. 25786, constructed by the applicant, the construction method of which has been disclosed in Chinese patent CN116004467B, "A Thermoresistant *E. coli* and its Application in D-Lactic Acid Production") was used as the starting strain. Since this starting strain accumulates large amounts of D-lactic acid, the lactate dehydrogenase gene *ldhA* was knocked out to block lactate accumulation, reduce the generation of the byproduct lactate, and redirect the carbon source to pyruvate production. The specific method is as follows: (1) Construction of recombinant plasmid pECgRNA-N20-ldhA (The purpose of constructing recombinant plasmid pECgRNA-N20-ldhA is to use recombinant plasmid pECgRNA-N20-ldhA to locate the ldhA position in the genome, guide Cas cutting protein to cut DNA, and achieve homologous recombination) Based on the ldhA gene sequence published by NCBI (https: / / www.ncbi.nlm.nih.gov / nuccore / NZ_CP022959.1?report=genbank&from=2508042&to=2509031) (the protein_id of ldhA is WP_000762229.1), the N20 sequence was established. The nucleotide sequence of N20 is shown in SEQ ID No. 1 of the sequence listing. The downstream of the N20 sequence is immediately adjacent to the PAM sequence (NGG). A BsaI sticky terminator sequence was added to the 5' end of N20. TAGT was added to the nucleotide sequence of the forward oligonucleotide (N20-ldhA-F) (the nucleotide sequence of N20-ldhA-F is shown in SEQ ID No. 2 of the sequence listing), and AAAC was added to the nucleotide sequence of the reverse oligonucleotide (N20-ldhA-R) (the nucleotide sequence of N20-ldhA-R is shown in SEQ ID No. 3 of the sequence listing).

[0018] After synthesizing the above-mentioned forward and reverse oligonucleotides, double-stranded DNA (dsDNA) with BsaI sticky ends was formed by annealing. The annealing program was as follows: denaturation at 95℃ for 5 min, cooling to 16℃ at a rate of 0.12℃ / s and holding for 10 min, and then storing the product at -20℃ for later use.

[0019] The pECgRNA plasmid was digested with BsaI at 37℃ for 2 h. The digestion product was purified by gel extraction to obtain a linearized plasmid, which was ligated with the above dsDNA at 16℃ for 30 min. The ligation product was then transformed into E. coli DH5α competent cells. The transformation method was as follows: E. coli DH5α competent cells were thawed on ice (the volume after thawing was 90 μL), 10 μL of ligation product was added, and the cells were incubated on ice for 30 min, heat-shocked at 37℃ for 5 min, immediately incubated on ice for 2 min, and 500 μL of LB liquid medium was added. The cells were then incubated at 37℃ and 1000 rpm for 45 min. After resuscitation, centrifuge at 5000 rpm for 5 min and discard 500 μL of supernatant. Mix the remaining 100 μL of bacterial culture by pipetting and spread it onto LB solid medium containing 50 μg / mL spectinomycin. Incubate overnight at 37°C. Pick single clones and inoculate them into LB liquid medium containing 50 μg / mL spectinomycin for amplification. DNA sequencing identifies positive clones. Extract the plasmid to obtain the recombinant plasmid pECgRNA-N20-ldhA. Store the recombinant plasmid pECgRNA-N20-ldhA with glycerol for later use.

[0020] (2) Construction of ldhA-K fragment Primers were designed based on the ldhA gene sequence. ldhA1: The nucleotide sequence is shown in SEQ ID No. 4 of the sequence listing; ldhA2: The nucleotide sequence is shown in SEQ ID No. 5 of the sequence listing; ldhA3: The nucleotide sequence is shown in SEQ ID No. 6 of the sequence listing; ldhA4: The nucleotide sequence is shown in SEQ ID No. 7 of the sequence listing; Using the genomic DNA of *Escherichia coli* (CGMCC No. 25786, constructed by the applicant, the construction method of which has been disclosed in Chinese Patent CN116004467B, "A Thermoresistant *Escherichia coli* and its Application in D-Lactic Acid Production") as a template, the target fragment was amplified using primers ldhA1 / ldhA2 and ldhA3 / ldhA4, yielding DNA products of lengths 418 bp and 421 bp, respectively. The two fragments had a 40 bp overlap at the ligation site. After separation and purification by agarose gel electrophoresis, the purified two fragments were used as a mixed template for overlap extension PCR using primers ldhA1 and ldhA4, successfully amplifying a fusion fragment of length 799 bp, named the ldhA-K fragment.

[0021] (3) Construction of strains with ldhA knocked out The plasmid pEcCAS carrying a kanamycin resistance marker was electroporated into competent cells of D-lactic acid-producing Escherichia coli (strain preservation number CGMCC No. 25786, constructed by the applicant, the construction method of which has been disclosed in Chinese patent CN116004467B, "A thermoresistant Escherichia coli and its application in D-lactic acid production"). After screening on 50 μg / ml kanamycin-resistant LB solid medium plates, positive single clones were selected and cultured to obtain the recombinant strain D-Lac / pEcCAS.

[0022] The constructed ldhA-K fragment was co-electrotransformed with the recombinant plasmid pECgRNA-N20-ldhA into competent cells of the recombinant strain D-Lac / pEcCAS. After screening on LB agar plates with 50 μg / ml spectinomycin and 50 μg / ml kanamycin, colony PCR was performed on single colonies using primers ldhA1 / ldhA4. Transformants amplifying a 799 bp DNA fragment were considered positive. The positive transformants obtained above were inoculated into LB liquid medium containing 10 mM rhamnose and 50 μg / mL kanamycin for induction culture to eliminate plasmids pEcCAS and pECgRNA-N20-ldhA. Subsequently, the bacterial culture was plated on LB agar plates. Single colonies were picked and inoculated onto LB agar plates containing 50 μg / mL spectinomycin, 50 μg / mL kanamycin, and no antibiotics, respectively. Colonies that did not grow on antibiotic-containing plates but grew on non-antibiotic plates were screened, and the resulting strain was named strain PYR-1.

[0023] (4) PYR-1 shake flask fermentation experiment Streak strain PYR-1 on LB solid medium, inoculate single colonies into 5 mL LB liquid medium shake tubes, and incubate overnight at 37°C and 200 rpm for 16 h to obtain activated bacterial culture. Use 500 mL Erlenmeyer flasks and seal with sealing film; the volume of the shake flask should be 100 mL. The shake flask fermentation medium formulation is shown in Table 1. Inoculate 1 mL of the activated bacterial culture into the shake flask.

[0024] Table 1: Formula of fermentation medium in shake flasks

[0025] Fermentation conditions: 37℃, initial pH 7.0, agitation speed of 200 rpm, culture period of 48 h, with no additional glucose added during the fermentation, and residual sugar measured every 24 h. Fermentation results are shown in Table 2.

[0026] Table 2: Results of pyruvate fermentation experiment (D-Lac in Table 2 is the starting strain D-lactic acid producing bacteria Escherichia coli CGMCC No. 25786)

[0027] The results showed that, compared with the starting strain D-Lac, the lactate synthesis pathway of strain PYR-1 was completely blocked, and it entered the pyruvate accumulation stage, confirming the feasibility of the strategy of blocking lactate production by knocking out lactate dehydrogenase; however, the pyruvate accumulation and conversion rate of this strain were still at a low level and further optimization is needed.

[0028] 2. Blocking the pentose phosphate pathway After knocking out the lactate dehydrogenase gene ldhA, strain PYR-1 was obtained. Although lactate no longer accumulated during fermentation in strain PYR-1, the pyruvate conversion rate remained low. The pentose phosphate pathway is another sugar breakdown pathway in organisms, in which glucose is oxidized and decarboxylated to produce carbon dioxide. To reduce carbon loss and improve conversion rate, the glucose-6-phosphate dehydrogenase gene zwf in the PYR-1 pentose phosphate pathway was knocked out (glucose-6-phosphate dehydrogenase is the first reaction in the pentose phosphate pathway and is an irreversible process). Blocking the pentose phosphate pathway reduces glucose loss.

[0029] (1) Construction of recombinant plasmid pECgRNA-N20-zwf Based on the zwf gene sequence published by NCBI (https: / / www.ncbi.nlm.nih.gov / nuccore / NZ_CP022959.1?report=genbank&from=1979597&to=1981072) (the protein_id of zwf is WP_000301727.1), the N20 sequence was constructed. The nucleotide sequence of the N20 sequence is shown in SEQ ID No. 8 of the sequence listing. The downstream of the N20 sequence is immediately adjacent to the PAM sequence (NGG). A BsaI sticky terminator sequence was added to the 5' end of N20. TAGT was added to the nucleotide sequence of the forward oligonucleotide (N20-zwf-F) (the nucleotide sequence of N20-zwf-F is shown in SEQ ID No. 9 of the sequence listing), and AAAC was added to the reverse oligonucleotide (N20-zwf-R) (the nucleotide sequence of N20-zwf-R is shown in SEQ ID No. 10 of the sequence listing).

[0030] After synthesizing the two oligonucleotides mentioned above, double-stranded DNA (dsDNA) with BsaI sticky ends was formed by annealing. The annealing program was as follows: denaturation at 95℃ for 5 min, cooling to 16℃ at a rate of 0.12℃ / s and holding for 10 min, and then storing the product at -20℃ for later use.

[0031] The pECgRNA plasmid was digested with BsaI at 37℃ for 2 h. The digestion product was purified by gel extraction to obtain a linearized plasmid, which was ligated with the above dsDNA at 16℃ for 30 min. The ligation product was then transformed into E. coli DH5α competent cells. The transformation method was as follows: E. coli DH5α competent cells were thawed on ice (the volume after thawing was 90 μL), 10 μL of ligation product was added, and the cells were incubated on ice for 30 min, heat-shocked at 37℃ for 5 min, immediately incubated on ice for 2 min, and 500 μL of LB liquid medium was added. The cells were then incubated at 37℃ and 1000 rpm for 45 min. After resuscitation, centrifuge at 5000 rpm for 5 min and discard 500 μL of supernatant. Mix the remaining 100 μL of bacterial culture by pipetting and spread it onto LB solid medium containing 50 μg / mL spectinomycin. Incubate overnight at 37°C. Pick single clones and inoculate them into LB liquid medium containing 50 μg / mL spectinomycin for amplification. DNA sequencing identifies positive clones. Extract the plasmid to obtain the recombinant plasmid pECgRNA-N20-zwf. Store the recombinant plasmid pECgRNA-N20-zwf for later use using glycerol.

[0032] (2) Construction of zwf-K Primers were designed based on the zwf gene sequence. zwf1: The nucleotide sequence is shown in SEQ ID No. 11 of the sequence listing; zwf2: The nucleotide sequence is shown in SEQ ID No. 12 of the sequence listing; zwf3: The nucleotide sequence is shown in SEQ ID No. 13 of the sequence listing; zwf4: The nucleotide sequence is shown in SEQ ID No. 14 of the sequence listing; Using the genomic DNA of *Escherichia coli* (CGMCC No. 25786, constructed by the applicant, the construction method of which has been disclosed in Chinese Patent CN116004467B, "A Thermoresistant *Escherichia coli* and its Application in D-Lactic Acid Production") as a template, the target fragment was amplified using primers zwf1 / zwf2 and zwf3 / zwf4, yielding DNA products of lengths 486 bp and 551 bp, respectively. The two fragments have a 41 bp overlap region at the promoter mutation site. After separation and purification by agarose gel electrophoresis, the purified two fragments were used as a mixed template for overlap extension PCR with primers zwf1 / zwf4, successfully amplifying a fusion fragment of length 996 bp, named the zwf-K fragment.

[0033] (3) Construction of strains with zwf knockout The plasmid pEcCAS carrying a kanamycin resistance marker was electroporated into competent cells of strain PYR-1. After screening with kanamycin resistance plates, positive single clones were selected and cultured to obtain the recombinant strain PYR-1 / pEcCAS.

[0034] The constructed zwf-K fragment was co-electrotransformed with the recombinant plasmid pECgRNA-N20-zwf into competent cells of the recombinant strain PYR-1 / pEcCAS. After screening on LB agar plates containing 50 μg / mL spectinomycin and 50 μg / mL kanamycin, single colonies were verified by colony PCR using primers zwf1 / zwf4. Transformants amplifying a 996 bp DNA fragment were considered positive. The positive transformants were inoculated into LB liquid medium containing 10 mM rhamnose and 50 μg / mL kanamycin for induction culture to eliminate plasmids pEcCAS and pECgRNA-N20-zwf. Subsequently, the bacterial culture was plated on LB agar plates. Single colonies were picked and inoculated onto LB agar plates containing 50 μg / mL spectinomycin, 50 μg / mL kanamycin, and no antibiotics. Colonies that did not grow on antibiotic-containing plates but grew on non-antibiotic plates were selected. The resulting strain was named strain PYR-2.

[0035] (4) PYR-2 shake flask fermentation experiment Streak the PYR-2 strain on LB solid medium, inoculate single colonies into 5 mL LB liquid medium shake tubes, and incubate overnight at 37°C and 200 rpm for 16 h to obtain activated bacterial culture. Use 500 mL Erlenmeyer flasks and seal with sealing film; fill each flask with 100 mL of the activated bacterial culture. The shake flask fermentation medium formulation is shown in Table 1. Inoculate 1 mL of the activated bacterial culture into the shake flask.

[0036] Fermentation conditions: 37℃, initial pH 7.0, agitation speed of 200 rpm, culture period of 48 h, with no additional glucose added during the fermentation, and residual sugar measured every 24 h. Fermentation results are shown in Table 3.

[0037] Table 3: Results of Pyruvic Acid Fermentation Experiment

[0038] The results showed that after knocking out glucose-6-phosphate dehydrogenase zwf and blocking the pentose phosphate pathway, the pyruvate conversion rate of strain PYR-1 increased from 0.49 g / g to 0.59 g / g, but the sugar consumption rate decreased significantly. This indicates that blocking the pentose phosphate pathway can reduce carbon loss. However, the pentose phosphate pathway is the main way to produce NADPH, which is crucial for biological growth. This may be the main reason for the significantly slower sugar consumption rate in PYR-2.

[0039] 3. Overexpression of membrane-bound transhydrogenase pntAB The membrane-bound transhydrogenase pntAB catalyzes the following reaction: NADH + NADP + +H + (outer membrane) ⇌NAD + +NADPH+H + (Inner membrane side). This reaction directly regulates the intracellular NADH / NAD ratio. + With NADPH / NADP + The ratio of NADH to NADPH needs to be balanced. Overexpression of pntAB not only replenishes NADPH for strain PYR-2, but also consumes excess NADH produced during pyruvate production. To better balance the NADH / NAD ratio... + With NADPH / NADP + The ratio is expressed using three promoters of different strengths, J23119, J23118, and J23117, to represent pntAB.

[0040] (1) Construction of recombinant plasmid pECgRNA-N20-ldhA-2 The N20 sequence was constructed based on the nucleotide sequence of the non-knockout portion of the ldhA gene (nucleotide sequence shown in SEQ ID No. 57 of the sequence listing), and the nucleotide sequence of the N20 sequence is shown in SEQ ID No. 15 of the sequence listing. The PAM sequence (NGG) is immediately downstream of the N20 sequence. A BsaI sticky terminator sequence was added to the 5' end of the N20 sequence. TAGT was added to the nucleotide sequence of the forward oligonucleotide (2-N20-ldhA-F) (nucleotide sequence of 2-N20-ldhA-F is shown in SEQ ID No. 16), and AAAC was added to the nucleotide sequence of the reverse oligonucleotide (2-N20-ldhA-R) (nucleotide sequence of 2-N20-ldhA-R is shown in SEQ ID No. 17 of the sequence listing).

[0041] After synthesizing the two oligonucleotides mentioned above, double-stranded DNA (dsDNA) with BsaI sticky ends was formed by annealing. The annealing program was as follows: denaturation at 95℃ for 5 min, cooling to 16℃ at a rate of 0.12℃ / s and holding for 10 min, and then storing the product at -20℃ for later use.

[0042] The pECgRNA plasmid was digested with BsaI at 37℃ for 2 h. The digestion product was purified by gel extraction to obtain a linearized plasmid, which was ligated with the above dsDNA at 16℃ for 30 min. The ligation product was then transformed into E. coli DH5α competent cells. The transformation method was as follows: E. coli DH5α competent cells were thawed on ice (the volume after thawing was 90 μL), 10 μL of ligation product was added, and the cells were incubated on ice for 30 min, heat-shocked at 37℃ for 5 min, immediately incubated on ice for 2 min, 500 μL of LB medium was added, and the cells were incubated at 37℃ and 1000 rpm for 45 min. After resuscitation, centrifuge at 5000 rpm for 5 min and discard 500 μL of supernatant. Mix the remaining 100 μL of bacterial culture by pipetting and spread it onto LB solid medium containing 50 μg / mL spectinomycin. Incubate overnight at 37°C. Pick single clones and inoculate them into LB liquid medium containing 50 μg / mL spectinomycin for amplification. DNA sequencing identifies positive clones. Extract the plasmid to obtain the recombinant plasmid pECgRNA-N20-ldhA-2. Store the recombinant plasmid pECgRNA-N20-ldhA-2 with glycerol for later use.

[0043] (2) Construction of pntAB knock-in fragments with promoters of different strengths Primers were designed based on the pntAB gene sequence and promoter sequence. The pntAB::ldhA1:nucleotide sequence is shown in SEQ ID No. 18 of the sequence listing; P J23119 -pntAB::ldhA2: The nucleotide sequence is shown in SEQ ID No. 19 of the sequence listing; P J23119 -pntAB::ldhA3: The nucleotide sequence is shown in SEQ ID No. 20 of the sequence listing; The pntAB::ldhA4:nucleotide sequence is shown in SEQ ID No. 21 of the sequence listing; The pntAB::ldhA5:nucleotide sequence is shown in SEQ ID No. 22 of the sequence listing; The pntAB::ldhA6:nucleotide sequence is shown in SEQ ID No. 23 of the sequence listing; P J23118 -pntAB::ldhA2: The nucleotide sequence is shown in SEQ ID No. 24 of the sequence listing; P J23118 The -pntAB::ldhA3:nucleotide sequence is shown in SEQ ID No. 25 of the sequence listing; P J23117-pntAB::ldhA2: The nucleotide sequence is shown in SEQ ID No. 26 of the sequence listing; P J23117 -pntAB::ldhA3: The nucleotide sequence is shown in SEQ ID No. 27 of the sequence listing; Using the genomic DNA of *Escherichia coli* (CGMCC No. 25786, constructed by the applicant, the construction method of which has been disclosed in Chinese Patent CN116004467B, "A Thermoresistant *Escherichia coli* and its Application in D-Lactic Acid Production") as a template, primers pntAB::ldhA1 / P were used. J23119 -pntAB::ldhA2、P J23119 -pntAB::ldhA3 / pntAB::ldhA4, pntAB::ldhA5 / pntAB::ldhA6, pntAB::ldhA1 / P J23118 -pntAB::ldhA2、P J23118 -pntAB::ldhA3 / pntAB::ldhA4, pntAB::ldhA1 / P J23117 -pntAB::ldhA2、P J23117 The target fragment was amplified using pntAB::ldhA3 / pntAB::ldhA4. After separation and purification by agarose gel electrophoresis, fragment P was obtained. J23119 -pntAB::ldhA12、P J23119 -pntAB::ldhA34, pntAB::ldhA56, P J23118 -pntAB::ldhA12、P J23118 -pntAB::ldhA34、P J23117 -pntAB::ldhA12、P J23117 -pntAB::ldhA34. With purified P J23119 -pntAB::ldhA12、P J23119 Using the three fragments pntAB::ldhA34, pntAB::ldhA56 as a mixed template, overlap extension PCR was performed using primers pntAB::ldhA1 and pntAB::ldhA6, successfully amplifying a fusion fragment of 3342 bp, named P. J23119 -pntAB::ldhA fragment. With purified P J23118 -pntAB::ldhA12、P J23118Using the three fragments -pntAB::ldhA34, pntAB::ldhA56 as a mixed template, and with primers pntAB::ldhA1 / pntAB::ldhA6, overlap extension PCR was performed, successfully amplifying a fusion fragment of 3342 bp, named P. J23118 -pntAB::ldhA fragment. With purified P J23117 -pntAB::ldhA12、P J23117 Using the three fragments pntAB::ldhA34, pntAB::ldhA56 as a mixed template, overlap extension PCR was performed using primers pntAB::ldhA1 and pntAB::ldhA6, successfully amplifying a fusion fragment of 3342 bp, named P. J23117 -pntAB::ldhA fragment. P J23119 The nucleotide sequence of the -pntAB::ldhA fragment is shown in SEQ ID No. 58 of the sequence listing; P J23118 The nucleotide sequence of the -pntAB::ldhA fragment is shown in SEQ ID No. 59 of the sequence listing; P J23117 The nucleotide sequence of the -pntAB::ldhA fragment is shown in SEQ ID No. 60 of the sequence listing.

[0044] (3) Construction of strains overexpressing the pntAB promoter with different strengths The plasmid pEcCAS carrying a kanamycin resistance marker was electroporated into competent cells of strain PYR-2. After screening with kanamycin resistance plates, positive single clones were selected and cultured to obtain the recombinant strain PYR-2 / pEcCAS.

[0045] The constructed P J23119 -pntAB::ldhA、P J23118 -pntAB::ldhA、P J23117The pntAB::ldhA fragment was co-electrotransformed with the recombinant plasmid pECgRNA-N20-ldhA-2 into PYR-2 / pEcCAS competent cells. After screening on LB agar plates with 50 μg / mL spectinomycin and 50 μg / mL kanamycin, single colonies were verified by colony PCR using primers pntAB::ldhA1 and pntAB::ldhA6. Transformants amplifying a 3342 bp DNA fragment were considered positive. The positive transformants were inoculated into LB liquid medium containing 10 mM rhamnose and 50 μg / mL kanamycin to eliminate plasmids pEcCAS and pECgRNA-N20-ldhA-2. Subsequently, the bacterial culture was plated on LB agar plates. Single colonies were picked and inoculated onto LB agar plates containing 50 μg / mL spectinomycin, 50 μg / mL kanamycin, and no antibiotics. Colonies that did not grow on antibiotic-containing plates but grew on antibiotic-free plates were screened. PCR amplification of the colonies was performed using primers pntAB::ldhA1 and pntAB::ldhA6, and the P colonies were recovered. J23119 -pntAB::ldhA、P J23118 -pntAB::ldhA、P J23117 The -pntAB::ldhA fragment was sequenced, and the results showed that no mutations occurred during gene editing. The resulting strains were named PYR-3, PYR-4, and PYR-5, respectively (strains PYR-3, PYR-4, and PYR-5 correspond to P...). J23119 -pntAB::ldhA、P J23118 -pntAB::ldhA、P J23117 -pntAB::ldhA).

[0046] (4) Shake-flask fermentation experiments of strains PYR-3, PYR-4, and PYR-5 Strains PYR-3, PYR-4, and PYR-5 were streaked onto LB solid medium, and single colonies were inoculated into LB liquid medium in shake tubes and incubated overnight at 37°C and 200 rpm for 16 h to obtain activated bacterial suspensions. 500 mL Erlenmeyer flasks were used for shake flask fermentation, sealed with sealing film, with a flask volume of 100 mL. The shake flask fermentation medium formulation is shown in Table 1. 1 mL of the activated bacterial suspension was inoculated into the shake flask.

[0047] Fermentation conditions: 37℃, initial pH 7.0, agitation speed of 200 rpm, culture period of 48 h, with no additional glucose added during the fermentation, and residual sugar measured every 24 h. Fermentation results are shown in Table 4.

[0048] Table 4: Results of Pyruvic Acid Fermentation Experiment

[0049] The results showed that overexpression of pntAB significantly increased the sugar consumption rate and pyruvate production of the strain, but the sugar-acid conversion rate did not change significantly. Among them, strain PYR-4, which overexpressed pntAB using the moderate-strength promoter J23118, showed the best effect, with pyruvate production increasing by 17% compared to PYR-2.

[0050] 4. Reduces pyruvate dehydrogenase (ACEE) activity The oxidative decarboxylation of pyruvate to acetyl-CoA is the link between glycolysis and the citric acid cycle, catalyzed by the pyruvate dehydrogenase complex. To further improve the pyruvate conversion rate in the strain, the activity of pyruvate dehydrogenase aceE can be reduced, decreasing the flux of pyruvate to the citric acid cycle. Therefore, two point mutations, H107V and H107M, were designed to reduce the activity of the pyruvate dehydrogenase aceE in PYR-4.

[0051] (1) Construction of recombinant plasmid pECgRNA-N20-aceE The N20 sequence was constructed based on the aceE gene sequence published by NCBI (https: / / www.ncbi.nlm.nih.gov / nuccore / NZ_CP022959.1?report=genbank&from=3873886&to=3876549) (the protein_id of aceE is WP_000003820.1). The nucleotide sequence of N20 is shown in SEQ ID No. 28 of the sequence listing. The downstream of the N20 sequence is immediately adjacent to the PAM sequence (NGG). Adaptor sequences for BsaI sticky ends were added to the 5' end of N20. TAGT was added to the nucleotide sequence of the forward oligonucleotide (N20-aceE-F) (the nucleotide sequence of N20-aceE-F is shown in SEQ ID No. 29 of the sequence listing), and AAAC was added to the nucleotide sequence of the reverse oligonucleotide (N20-aceE-R) (the nucleotide sequence of N20-aceE-R is shown in SEQ ID No. 30 of the sequence listing).

[0052] After synthesizing the two oligonucleotides mentioned above, double-stranded DNA (dsDNA) with BsaI sticky ends was formed by annealing. The annealing program was as follows: denaturation at 95℃ for 5 min, cooling to 16℃ at a rate of 0.12℃ / s and holding for 10 min, and then storing the product at -20℃ for later use.

[0053] The pECgRNA plasmid was digested with BsaI at 37℃ for 2 h. The digestion product was purified by gel extraction to obtain a linearized plasmid, which was ligated with the above dsDNA at 16℃ for 30 min. The ligation product was then transformed into E. coli DH5α competent cells. The transformation method was as follows: E. coli DH5α competent cells were thawed on ice (the volume after thawing was 90 μL), 10 μL of ligation product was added, and the cells were incubated on ice for 30 min, heat-shocked at 37℃ for 5 min, immediately incubated on ice for 2 min, 500 μL of LB medium was added, and the cells were incubated at 37℃ and 1000 rpm for 45 min. After resuscitation, centrifuge at 5000 rpm for 5 min and discard 500 μL of supernatant. Mix the remaining 100 μL of bacterial culture by pipetting and spread it onto LB solid medium containing 50 μg / mL spectinomycin. Incubate overnight at 37°C. Pick single clones and inoculate them into LB liquid medium containing 50 μg / mL spectinomycin for amplification. DNA sequencing identifies positive clones. Extract the plasmid to obtain the recombinant plasmid pECgRNA-N20-aceE. Preserve the recombinant plasmid pECgRNA-N20-aceE using glycerol for later use.

[0054] (2) Construction of aceE knock-in fragments with different point mutations Primers were designed based on the aceE gene sequence and promoter sequence. aceE1: The nucleotide sequence is shown in SEQ ID No. 31 of the sequence listing; aceE(107M)2: The nucleotide sequence is shown in SEQ ID No. 32 of the sequence listing; aceE(107M)3: The nucleotide sequence is shown in SEQ ID No. 33 of the sequence listing; aceE4: The nucleotide sequence is shown in SEQ ID No. 34 of the sequence listing; aceE5: The nucleotide sequence is shown in SEQ ID No. 35 of the sequence listing; aceE6: The nucleotide sequence is shown in SEQ ID No. 36 of the sequence listing; aceE(107V)2: The nucleotide sequence is shown in SEQ ID No. 37 of the sequence listing; aceE(107V)3: The nucleotide sequence is shown in SEQ ID No. 38 of the sequence listing; Using the genomic DNA of *Escherichia coli* (CGMCC No. 25786, constructed by the applicant, the construction method of which has been disclosed in Chinese Patent CN116004467B, "A Thermoresistant *Escherichia coli* and its Application in D-Lactic Acid Production") as a template, the target fragment was amplified using primers aceE1 / aceE(107M)2, aceE(107M)3 / aceE4, aceE5 / aceE6, aceE1 / aceE(107V)2, and aceE(107V)3 / aceE4, respectively. After separation and purification by agarose gel electrophoresis, fragments aceE(107M)12, aceE(107M)34, aceE56, aceE(107V)12, and aceE(107V)34 were obtained. Using purified aceE(107M)12, aceE(107M)34, and aceE56 fragments as a mixed template, overlap extension PCR was performed using primers aceE1 and aceE6, successfully amplifying a fusion fragment of 2339 bp, named the aceE(107M) fragment. Using purified aceE(107V)12, aceE(107V)34, and aceE56 fragments as a mixed template, overlap extension PCR was performed using primers aceE1 / aceE6, successfully amplifying a fusion fragment of 2339 bp, named the aceE(107V) fragment. The nucleotide sequence of the aceE(107M) fragment is shown in SEQ ID No. 61 of the sequence listing, and the nucleotide sequence of the aceE(107V) fragment is shown in SEQ ID No. 62 of the sequence listing.

[0055] (3) Construction of aceE mutant strain The plasmid pEcCAS carrying a kanamycin resistance marker was electroporated into competent cells of strain PYR-4. After screening with kanamycin resistance plates, positive single clones were selected and cultured to obtain the recombinant strain PYR-4 / pEcCAS.

[0056] The constructed aceE (107M) and aceE (107V) fragments were co-electrotransformed with the recombinant plasmid pECgRNA-N20-aceE into competent cells of the recombinant strain PYR-4 / pEcCAS. After screening on LB agar plates containing 50 μg / mL spectinomycin and 50 μg / mL kanamycin, single colonies were amplified by colony PCR using primers aceE1 and aceE6. The fragments were purified by agarose gel electrophoresis, and sequencing revealed mutations, indicating positive transformants. The obtained positive transformants were inoculated into LB liquid medium containing 10 mM rhamnose and 50 μg / mL kanamycin for induction culture to eliminate plasmids pEcCAS and pECgRNA-N20-aceE. Subsequently, the bacterial culture was plated on LB agar plates. Single colonies were picked and inoculated onto LB agar plates containing 50 μg / mL spectinomycin, 50 μg / mL kanamycin, and no antibiotics. Colonies that did not grow on antibiotic-containing plates but grew on non-antibiotic plates were selected. The resulting strains were named strains PYR-4M and PYR-4V.

[0057] (4) Shake-flask fermentation experiments of strains PYR-4M and PYR-4V Strawberries PYR-4M and PYR-4V were streaked onto LB solid medium, and single colonies were inoculated into LB liquid medium shake tubes and incubated overnight at 37°C and 200 rpm for 16 h to obtain activated bacterial suspension. 500 mL Erlenmeyer flasks were used for shake flask fermentation, sealed with sealing film, with a volume of 100 mL in each flask. The shake flask fermentation medium formulation is shown in Table 1. 1 mL of the activated bacterial suspension was inoculated into the shake flask.

[0058] Fermentation conditions: 37℃, initial pH 7.0, agitation speed of 200 rpm, culture period of 48 h, with no additional glucose added during the fermentation, and residual sugar measured every 24 h. Fermentation results are shown in Table 5.

[0059] Table 5: Results of Pyruvic Acid Fermentation Experiment

[0060] The results showed that reducing the activity of aceE through point mutation effectively decreased the flux of pyruvate to the citric acid cycle, thereby improving the sugar-acid conversion rate, without significantly affecting the sugar consumption rate. The two different aceE point-mutated strains showed similar effects on improving pyruvate yield and sugar-acid conversion rate; compared to PYR-4, strains PYR-4M and PYR-4V both increased pyruvate yield by 14.6% after 48 h of fermentation.

[0061] 5. Increase carbon sequestration pathways The reaction catalyzed by RuBisCO (ribulose-1,5-bisphosphate carboxylase / oxygenase) is the first step in CO2 fixation in the Calvin cycle. Theoretically, introducing the RuBisCO carbon fixation pathway can increase the theoretical conversion rate of pyruvate from 2 mol / mol glucose to 2.4 mol / mol glucose. To further improve the pyruvate conversion rate, the genes prk and cbbLS were overexpressed in strain PYR-4V to introduce the RuBisCO carbon fixation pathway.

[0062] (1) Construction of recombinant plasmids pECgRNA-N20-poxB and pECgRNA-N20-yghX Based on the poxB gene (https: / / www.ncbi.nlm.nih.gov / nuccore / NZ_CP022959.1?report=genbank&from=3000185&to=3001903, poxB protein_id is WP_000815337.1) and yghX gene (https: / / www.ncbi.nlm.nih.gov / nuccore / NZ_CP022959.1?report=genbank&from=747601&to=748486) published by NCBI, poxB N20 was sequenced according to the gene sequence. The nucleotide sequence of poxB N20 is shown as SEQ ID No. 39 in the sequence listing. The poxB N20 sequence is immediately downstream of the PAM sequence (NGG). A BsaI sticky terminator sequence was added to the 5' end of poxB N20. TAGT was added to the nucleotide sequence of the forward oligonucleotide (N20-poxB-F) (the nucleotide sequence of N20-poxB-F is shown in SEQ ID No. 40 of the sequence listing), and AAAC was added to the nucleotide sequence of the reverse oligonucleotide (N20-poxB-R) (the nucleotide sequence of N20-poxB-R is shown in SEQ ID No. 41 of the sequence listing). The nucleotide sequence of yghX N20 is shown in SEQ ID No. 42 of the sequence listing. The yghX N20 sequence is immediately downstream of the PAM sequence (NGG). A BsaI sticky terminator sequence was added to the 5' end of yghX N20. TAGT (nucleotide sequence shown in SEQ ID No. 43 in the sequence listing) was added to the nucleotide sequence of the forward oligonucleotide (N20-yghX-F), and AAAC (nucleotide sequence shown in SEQ ID No. 44 in the sequence listing) was added to the sequence listing of the reverse oligonucleotide (N20-yghX-R).

[0063] After synthesizing the two oligonucleotides (forward and reverse oligonucleotides), they were annealed to form double-stranded DNA (dsDNA) with BsaI sticky ends. The annealing program was as follows: denaturation at 95°C for 5 min, cooling to 16°C at a rate of 0.12°C / s and holding for 10 min, and then storing the product at -20°C for later use.

[0064] The pECgRNA plasmid was digested with BsaI at 37℃ for 2 h. The digestion product was purified by gel extraction to obtain linearized plasmids, which were then ligated with the above dsDNA at 16℃ for 30 min. The ligation products were then transformed into E. coli DH5α competent cells. The transformation method was as follows: E. coli DH5α competent cells were thawed on ice (the volume after thawing was 90 μL), 10 μL of ligation product was added, and the cells were incubated on ice for 30 min, then heat-shocked at 37℃ for 5 min, immediately incubated on ice for 2 min, and 500 μL of LB medium was added. The cells were then incubated at 37℃ and 1000 rpm for 45 min. After resuscitation, centrifuge at 5000 rpm for 5 min and discard 500 μL of supernatant. Mix the remaining 100 μL of bacterial culture by pipetting and spread it onto LB solid medium containing 50 μg / mL spectinomycin. Incubate overnight at 37°C. Pick single clones and inoculate them into LB liquid medium containing 50 μg / mL spectinomycin for amplification. DNA sequencing identifies positive clones. Extract plasmids to obtain recombinant plasmids pECgRNA-N20-poxB and pECgRNA-N20-yghX. Store the recombinant plasmids pECgRNA-N20-poxB and pECgRNA-N20-yghX with glycerol for later use.

[0065] (2) Construction of overexpression prk gene knock-in fragment Primers were designed based on the prk gene sequence and promoter sequence. The prk::yghX1:nucleotide sequence is shown in SEQ ID No. 45 of the sequence listing; P J23119 The -prk::yghX2:nucleotide sequence is shown in SEQ ID No. 46 of the sequence listing; P J23119 The -prk::yghX3:nucleotide sequence is shown in SEQ ID No. 47 of the sequence listing; The prk::yghX4:nucleotide sequence is shown in SEQ ID No. 48 of the sequence listing; The prk::yghX5 nucleotide sequence is shown in SEQ ID No. 49 of the sequence listing; The prk::yghX6:nucleotide sequence is shown in SEQ ID No. 50 of the sequence listing; Using the genomic DNA of *Escherichia coli* (CGMCC No. 25786, constructed by the applicant, the construction method of which has been disclosed in Chinese Patent CN116004467B, "A Thermoresistant *Escherichia coli* and its Application in D-Lactic Acid Production") as a template, primers prk::yghX1 / P were used. J23119 -prk::yghX2, prk::yghX5 / prk::yghX6 amplify the target fragment. Using the synthesized prk gene sequence (nucleotide sequence shown in SEQ ID No. 63 in the sequence listing) as a template, primer P... J23119 The target fragment was amplified using the prk::yghX3 / prk::yghX4 method. After separation and purification by agarose gel electrophoresis, fragments prk::yghX12, prk::yghX56, and prk::yghX34 were obtained. Using the purified prk::yghX12, prk::yghX56, and prk::yghX34 as a mixed template, overlap extension PCR was performed using primers prk::yghX1 / prk::yghX6, successfully amplifying a fusion fragment of 2043 bp, named P. J23119 -prk::yghX fragment, P J23119 The nucleotide sequence of the -prk::yghX fragment is shown in SEQ ID No. 64 of the sequence listing.

[0066] (3) Construction of strains overexpressing the prk gene The plasmid pEcCAS carrying the kanamycin resistance marker was electroporated into competent cells of strain PYR-4V. After screening with kanamycin resistance plates, positive single clones were selected and cultured to obtain the recombinant strain PYR-4V / pEcCAS.

[0067] The constructed P J23119The prk::yghX fragment and the recombinant plasmid pECgRNA-N20-yghX were co-electrotransformed into competent cells of the recombinant strain PYR-4V / pEcCAS. After screening on LB agar plates containing 50 μg / mL spectinomycin and 50 μg / mL kanamycin, single colonies were verified by colony PCR using primers prk::yghX1 and prk::yghX6. Transformants amplifying a 2043 bp DNA fragment were identified as positive transformants. These positive transformants were inoculated into LB liquid medium containing 10 mM rhamnose and 50 μg / mL kanamycin for induction culture to eliminate plasmids pEcCAS and pECgRNA-N20-yghX. Subsequently, the bacterial culture was plated on LB agar plates. Single colonies were picked and inoculated onto LB agar plates containing 50 μg / mL spectinomycin, 50 μg / mL kanamycin, and no antibiotics, respectively. Colonies that did not grow on antibiotic-containing plates but grew on non-antibiotic plates were selected. The resulting strain was named strain PYR-5OE.

[0068] (4) Construction of the cbbLS gene knock-in fragment overexpression Primers were designed based on the cbbLS gene sequence and promoter sequence. The cbbLS::poxB1: nucleotide sequence is shown in SEQ ID No. 51 of the sequence listing; P Trc The -cbbLS::poxB2: nucleotide sequence is shown in SEQ ID No. 52 of the sequence listing; P Trc The -cbbLS::poxB3: nucleotide sequence is shown in SEQ ID No. 53 of the sequence listing; The cbbLS::poxB4 nucleotide sequence is shown in SEQ ID No. 54 of the sequence listing; The cbbLS::poxB5 nucleotide sequence is shown in SEQ ID No. 55 of the sequence listing; The cbbLS::poxB6 nucleotide sequence is shown in SEQ ID No. 56 of the sequence listing; Using the genomic DNA of *Escherichia coli* (CGMCC No. 25786, constructed by the applicant, the construction method of which has been disclosed in Chinese Patent CN116004467B, "A Thermoresistant *Escherichia coli* and its Application in D-Lactic Acid Production") as a template, primers cbbLS::poxB1 / P were used. Trc-cbbLS::poxB2, cbbLS::poxB5 / cbbLS::poxB6 amplify the target fragment. Using the synthesized cbbLS gene sequence (nucleotide sequence shown in SEQ ID No. 65 in the sequence listing) as a template, primers P and P were used respectively. Trc -cbbLS::poxB3 / P Trc The target fragment was amplified using cbbLS::poxB4. After separation and purification by agarose gel electrophoresis, fragments cbbLS::poxB12, cbbLS::poxB56, and cbbLS::poxB34 were obtained. Using the purified cbbLS::poxB12, cbbLS::poxB56, and cbbLS::poxB34 fragments as a mixed template, overlap extension PCR was performed using primers cbbLS::poxB1 / cbbLS::poxB6, successfully amplifying a fusion fragment of 2809 bp, named P. Trc -cbbLS::poxB fragment, P Trc The nucleotide sequence of the -cbbLS::poxB fragment is shown in SEQ ID No. 66 of the sequence listing.

[0069] (5) Construction of strains overexpressing the cbbLS gene The plasmid pEcCAS carrying a kanamycin resistance marker was electroporated into competent cells of strain PYR-5OE. After screening with kanamycin resistance plates, positive single clones were selected and cultured to obtain the recombinant strain PYR-5OE / pEcCAS.

[0070] The constructed P Trc The cbbLS::poxB fragment and recombinant plasmid pECgRNA-N20-poxB were co-electrotransformed into PYR-5OE / pEcCAS competent cells. After selection on LB agar plates containing 50 μg / mL spectinomycin and 50 μg / mL kanamycin, single colonies were verified by colony PCR using primers cbbLS::poxB1 and cbbLS::poxB6. Transformants amplifying a 2809 bp DNA fragment were identified as positive transformants. These positive transformants were inoculated into LB liquid medium containing 10 mM rhamnose and 50 μg / mL kanamycin for induction culture to eliminate plasmids pEcCAS and pECgRNA-N20-poxB. Subsequently, the bacterial culture was plated on LB agar plates. Single colonies were picked and inoculated onto LB agar plates containing 50 μg / mL spectinomycin, 50 μg / mL kanamycin, and no antibiotics, respectively. Colonies that did not grow on antibiotic-containing plates but grew on non-antibiotic plates were selected. The resulting strain was named strain PYR-6.

[0071] (6) Fermentation test of PYR-4V and PYR-6 fermenters Strawberries PYR-4V and PYR-6 were streaked onto LB solid medium, and single colonies were inoculated into 500 mL LB liquid flasks (100 mL volume). The flasks were incubated overnight at 37°C and 200 rpm for 16 h to obtain activated bacterial cultures. The activated bacterial cultures were then inoculated at a 10% inoculum into a 5 L fermenter (3 L volume of medium). Fermentation conditions are shown in Table 6. During fermentation, the airflow was 0.5 vvm, the tank pressure was 0.1 MPa, the fermenter rotation speed was 400 rpm, and the pH was maintained at 7.0. The pH was adjusted using a 30% sodium hydroxide solution and maintained at 7.0 throughout the fermentation process. The fermentation results are shown in Table 7.

[0072] Table 6: Fermenter Culture Medium Formulation

[0073] Table 7: Results of Pyruvic Acid Fermentation Experiment

[0074] The results showed that after introducing the RuBisCO carbon fixation pathway, strain PYR-6 significantly increased the sugar consumption rate of strain PYR-4V, shortening the fermentation time from 38h to 30h. Residual sugar was monitored during fermentation, and the fermentation was stopped when the bottom sugar was exhausted. No additional glucose was added during the process. The pyruvate yield was 67.5g / L, and the sugar-acid conversion rate was 0.703g / g, which was basically the same as that of strain PYR-4V. The production efficiency increased from 1.77g / (L•h) to 2.25g / (L•h).

[0075] The strain PYR-6 was preserved and classified as Escherichia coli. Escherichia coli PYR-6, accession number CCTCC NO: M 20261914, accession date August 18, 2026, deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China.

Claims

1. A high-yield pyruvate-producing strain, characterized in that, The high-pyruvate-producing strain was classified as Escherichia coli. Escherichia coli PYR-6, with accession number CCTCC NO: M 20261914, was deposited on August 18, 2026, and the depositary institution is the China Center for Type Culture Collection.

2. A method for constructing a high-yield pyruvate strain according to claim 1, characterized in that, The procedure includes the following steps: using Escherichia coli CGMCC No. 25786 as the starting strain, and employing CRIPSR-Cas9 technology, the gene ldhA for lactate dehydrogenase was knocked out, the gene zwf for glucose-6-phosphate dehydrogenase was knocked out, the gene pntAB for membrane-bound transhydrogenase was overexpressed, the activity of the gene aceE for pyruvate dehydrogenase was reduced, and the genes prk and cbbLS for the RuBisCO carbon fixation pathway were overexpressed.

3. The method for constructing a high-yield pyruvate strain according to claim 2, characterized in that, Knocking out the lactate dehydrogenase gene ldhA involves the following steps: S11. Construction of recombinant plasmid pECgRNA-N20-ldhA: Based on the ldhA gene sequence, an N20 sequence was set. Based on the N20 sequence, forward oligonucleotides N20-ldhA-F and reverse oligonucleotides N20-ldhA-R were designed. The forward and reverse oligonucleotides N20-ldhA-F and N20-ldhA-R were mixed and annealed to form double-stranded DNA. The pECgRNA plasmid was digested with enzymes to obtain a linear plasmid. The double-stranded DNA and the linear plasmid were ligated to obtain a ligation product. The ligation product was transformed and screened to obtain the recombinant plasmid pECgRNA-N20-ldhA. S12. Constructing the ldhA-K fragment: Using the genomic DNA of the starting strain as a template, the target fragment is amplified with primers ldhA1 / ldhA2 and ldhA3 / ldhA4, respectively. After purification, two purified fragments are obtained. Using the two purified fragments as a mixed template, overlap extension PCR is performed with primers ldhA1 / ldhA4 to obtain the ldhA-K fragment. S13. Constructing a strain with ldhA knocked out: Transform the plasmid pEcCAS into the starting strain to obtain the recombinant strain D-Lac / pEcCAS; Transform the ldhA-K fragment and the recombinant plasmid pECgRNA-N20-ldhA into the recombinant strain D-Lac / pEcCAS, screen to eliminate plasmids pEcCAS and pECgRNA-N20-ldhA, and obtain strain PYR-1; In S11, the nucleotide sequence of N20 is shown in SEQ ID No. 1 of the sequence listing; In S11, the nucleotide sequence of the forward oligonucleotide N20-ldhA-F is shown in SEQ ID No. 2 of the sequence listing; In S11, the nucleotide sequence of the reverse oligonucleotide N20-ldhA-R is shown in SEQ ID No. 3 of the sequence listing; In S11, when the plasmid is digested with enzymes to obtain a linear plasmid, the plasmid is plasmid pECgRNA; In S11, when the plasmid is digested with an enzyme to obtain a linear plasmid, the enzyme digestion is BsaI digestion; In S11, when transforming and screening the ligation product, the method is to transform the ligation product into E. coli DH5α competent cells, identify positive clones by DNA sequencing, and extract plasmids. In S12 and S13, the starting strain is Escherichia coli CGMCC No.25786; In S12, the nucleotide sequence of ldhA1 is shown as SEQ ID No. 4 in the sequence listing, the nucleotide sequence of ldhA2 is shown as SEQ ID No. 5 in the sequence listing, the nucleotide sequence of ldhA3 is shown as SEQ ID No. 6 in the sequence listing, and the nucleotide sequence of ldhA4 is shown as SEQ ID No. 7 in the sequence listing; In S13, electroporation was used when transforming plasmid pEcCAS into the starting strain and when transforming the ldhA-K fragment with recombinant plasmid pECgRNA-N20-ldhA into recombinant strain D-Lac / pEcCAS. In S13, the screening process combined LB solid medium plates containing both spectinomycin and kanamycin antibiotics with PCR verification.

4. The method for constructing a high-yield pyruvate strain according to claim 2, characterized in that, Knocking out the glucose-6-phosphate dehydrogenase gene zwf includes the following steps: S21. Construction of recombinant plasmid pECgRNA-N20-zwf: The N20 sequence is set according to the zwf gene sequence. Forward oligonucleotides N20-zwf-F and reverse oligonucleotides N20-zwf-R are designed based on the N20 sequence. The forward and reverse oligonucleotides N20-zwf-F and N20-zwf-R are mixed and annealed to form double-stranded DNA. The plasmid is digested with enzymes to obtain a linear plasmid. The double-stranded DNA and the linear plasmid are ligated to obtain a ligation product. The ligation product is transformed and screened to obtain the recombinant plasmid pECgRNA-N20-zwf. S22. Constructing the zwf-K fragment: Using the genomic DNA of the starting strain as a template, the target fragment is amplified with primers zwf1 / zwf2 and zwf3 / zwf4 respectively. After purification, two purified fragments are obtained. Using the two purified fragments as a mixed template, overlap extension PCR is performed with primers zwf1 / zwf4 to obtain the zwf-K fragment. S23. Constructing a zwf-knockout strain: Transform the plasmid pEcCAS into strain PYR-1 to obtain the recombinant strain PYR-1 / pEcCAS; Transform the zwf-K fragment and the recombinant plasmid pECgRNA-N20-zwf into the recombinant strain PYR-1 / pEcCAS, screen to eliminate plasmids pEcCAS and pECgRNA-N20-zwf, and obtain strain PYR-2; In S21, the nucleotide sequence of N20 is shown in SEQ ID No. 8 of the sequence listing; In S21, the nucleotide sequence of the forward oligonucleotide N20-zwf-F is shown in SEQ ID No. 9 of the sequence listing; In S21, the nucleotide sequence of the reverse oligonucleotide N20-zwf-R is shown in SEQ ID No. 10 of the sequence listing; In S21, when the plasmid is digested with enzymes to obtain a linear plasmid, the plasmid is plasmid pECgRNA; In S21, when the plasmid is digested with an enzyme to obtain a linear plasmid, the enzyme digestion is BsaI digestion; In S21, when transforming and screening the ligation product, the method is to transform the ligation product into E. coli DH5α competent cells, identify positive clones by DNA sequencing, and extract plasmids. In S22, the starting strain is Escherichia coli CGMCC No.25786; In S22, the nucleotide sequence of zwf1 is shown as SEQ ID No. 11 in the sequence listing, the nucleotide sequence of zwf2 is shown as SEQ ID No. 12 in the sequence listing, the nucleotide sequence of zwf3 is shown as SEQ ID No. 13 in the sequence listing, and the nucleotide sequence of zwf4 is shown as SEQ ID No. 14 in the sequence listing; In S23, electroporation was used when transforming plasmid pEcCAS into strain PYR-1 to obtain recombinant strain PYR-1 / pEcCAS, and when transforming zwf-K fragment with recombinant plasmid pECgRNA-N20-zwf into recombinant strain PYR-1 / pEcCAS. In S23, the screening process combined LB solid medium plates containing both spectinomycin and kanamycin antibiotics with PCR verification.

5. The method for constructing a high-yield pyruvate strain according to claim 2, characterized in that, Overexpression of the membrane-binding transhydrogenase gene pntAB includes the following steps: S31. Construction of recombinant plasmid pECgRNA-N20-ldhA-2: The N20 sequence was set based on the sequence of the unknocked portion of the ldhA gene. Based on the N20 sequence, forward oligonucleotides 2-N20-ldhA-F and reverse oligonucleotides 2-N20-ldhA-R were designed. The forward and reverse oligonucleotides 2-N20-ldhA-F and 2-N20-ldhA-R were mixed and annealed to form double-stranded DNA. The plasmid was digested with enzymes to obtain a linear plasmid. The double-stranded DNA and the linear plasmid were ligated to obtain a ligation product. The ligation product was transformed and screened to obtain the recombinant plasmid pECgRNA-N20-ldhA-2. S32. Constructing the pntAB knock-in fragment of a moderate-strength promoter: Using the genomic DNA of the starting strain as a template, and using primers pntAB::ldhA1 / P... J23118 -pntAB::ldhA2、P J23118 The target fragment was amplified using pntAB::ldhA3 / pntAB::ldhA4 and pntAB::ldhA5 / pntAB::ldhA6, purified, and three purified fragments were obtained. Using these three purified fragments as a mixed template, overlap extension PCR was performed using primers pntAB::ldhA1 / pntAB::ldhA6 to obtain P... J23118 -pntAB::ldhA segment; S33, constructing a strain overexpressing the promoter pntAB: the plasmid pEcCAS was transformed into the strain PYR-2 to obtain the recombinant strain PYR-2 / pEcCAS; P J23118 The pntAB::ldhA fragment was transformed into the recombinant strain PYR-2 / pEcCAS with the recombinant plasmid pECgRNA-N20-ldhA-2, and screening was performed to eliminate the plasmids pEcCAS and pECgRNA-N20-ldhA-2, thereby obtaining the strain PYR-4; In S31, the nucleotide sequence of the non-knockout portion of the ldhA gene is shown in SEQ ID No. 57 of the sequence listing; In S31, the nucleotide sequence of the N20 sequence is shown in SEQ ID No. 15 of the sequence listing; In S31, the nucleotide sequence of the forward oligonucleotide 2-N20-ldhA-F is shown in SEQ ID No. 16 of the sequence listing; In S31, the nucleotide sequence of the reverse oligonucleotide 2-N20-ldhA-R is shown in SEQ ID No. 17 of the sequence listing; In S31, when the plasmid is digested with enzymes to obtain a linear plasmid, the plasmid is plasmid pECgRNA; In S31, when the plasmid is digested with an enzyme to obtain a linear plasmid, the enzyme digestion is BsaI digestion; In S31, when transforming and screening the ligation product, the method is to transform the ligation product into E. coli DH5α competent cells, identify positive clones by DNA sequencing, and extract plasmids. In S32, the starting strain is Escherichia coli CGMCC No.25786; In S32, the nucleotide sequence of pntAB::ldhAl is shown in SEQ ID No. 18 of the Sequence Listing; P J23118 the nucleotide sequence of pntAB::ldhA2 is shown in SEQ ID No. 24 of the Sequence Listing; P J23118 the nucleotide sequence of pntAB::ldhA3 is shown in SEQ ID No. 25 of the Sequence Listing; the nucleotide sequence of pntAB::ldhA4 is shown in SEQ ID No. 21 of the Sequence Listing; the nucleotide sequence of pntAB::ldhA5 is shown in SEQ ID No. 22 of the Sequence Listing; the nucleotide sequence of pntAB::ldhA6 is shown in SEQ ID No. 23 of the Sequence Listing; In S32, P J23118 The nucleotide sequence of the -pntAB::ldhA fragment is shown in SEQ ID No. 59 of the sequence listing; In S33, plasmid pEcCAS was transformed into strain PYR-2, and P... J23118 When the -pntAB::ldhA fragment and the recombinant plasmid pECgRNA-N20-ldhA-2 were transformed into the recombinant strain PYR-2 / pEcCAS, electroporation was used. In S33, the screening process combined LB solid medium plates containing both spectinomycin and kanamycin antibiotics with PCR verification.

6. The method for constructing a high-yield pyruvate strain according to claim 2, characterized in that, To reduce the activity of the aceE gene, which is an enzyme in the pyruvate dehydrogenase, the following steps are included: S41. Construction of recombinant plasmid pECgRNA-N20-aceE: The N20 sequence is set according to the aceE gene. Forward oligonucleotides N20-aceE-F and reverse oligonucleotides N20-aceE-R are designed based on the N20 sequence. The forward and reverse oligonucleotides N20-aceE-F and N20-aceE-R are mixed and annealed to form double-stranded DNA. The plasmid is digested with enzymes to obtain a linear plasmid. The double-stranded DNA and the linear plasmid are ligated to obtain a ligation product. The ligation product is transformed and screened to obtain the recombinant plasmid pECgRNA-N20-aceE. S42. Constructing a point-mutated aceE knock-in fragment: Using the genomic DNA of the starting strain as a template, the target fragment was amplified using primers aceE1 / aceE(107V)2, aceE(107V)3 / aceE4, and aceE5 / aceE6, respectively. After purification, three purified fragments were obtained. Using the three purified fragments as a mixed template, overlap extension PCR was performed using primers aceE1 / aceE6 to obtain the aceE(107V) fragment. S43. Constructing an aceE mutant strain: Transform plasmid pEcCAS into strain PYR-4 to obtain recombinant strain PYR-4 / pEcCAS; Transform the aceE (107V) fragment and recombinant plasmid pECgRNA-N20-aceE into recombinant strain PYR-4 / pEcCAS, screen to eliminate plasmids pEcCAS and pECgRNA-N20-aceE, and obtain strain PYR-4V; In S41, the nucleotide sequence of the N20 sequence is shown in SEQ ID No. 28 of the sequence listing; In S41, the nucleotide sequence of the forward oligonucleotide N20-aceE-F is shown in SEQ ID No. 29 of the sequence listing; In S41, the nucleotide sequence of the reverse oligonucleotide N20-aceE-R is shown in SEQ ID No. 30 of the sequence listing; In S41, when the plasmid is digested with enzymes to obtain a linear plasmid, the plasmid is plasmid pECgRNA; In S41, when the plasmid is digested with an enzyme to obtain a linear plasmid, the enzyme digestion is BsaI digestion; In S41, when transforming and screening the ligation product, the method is to transform the ligation product into E. coli DH5α competent cells, identify positive clones by DNA sequencing, and extract plasmids. In S42, the starting strain is Escherichia coli CGMCC No.25786; In S42, the nucleotide sequence of aceE1 is shown in SEQ ID No. 31 of the sequence listing; the nucleotide sequence of aceE(107V)2 is shown in SEQ ID No. 37 of the sequence listing; the nucleotide sequence of aceE(107V)3 is shown in SEQ ID No. 38 of the sequence listing; the nucleotide sequence of aceE4 is shown in SEQ ID No. 34 of the sequence listing; the nucleotide sequence of aceE5 is shown in SEQ ID No. 35 of the sequence listing; and the nucleotide sequence of aceE6 is shown in SEQ ID No. 36 of the sequence listing. In S42, the nucleotide sequence of the aceE(107V) fragment is shown in SEQ ID No. 62 of the sequence listing; In S43, electroporation was used when transforming plasmid pEcCAS into strain PYR-4 and when transforming the aceE(107V) fragment with recombinant plasmid pECgRNA-N20-aceE into recombinant strain PYR-4 / pEcCAS. In S43, the screening process combined LB solid medium plates containing both spectinomycin and kanamycin antibiotics with PCR verification.

7. The method for constructing a high-yield pyruvate strain according to claim 2, characterized in that, Overexpression of the RuBisCO carbon fixation pathway genes prk and cbbLS includes the following steps: S51. Construction of recombinant plasmid pECgRNA-N20-poxB: Based on the poxB gene, the poxB N20 sequence was set. Based on the poxB N20 sequence, forward oligonucleotides N20-poxB-F and reverse oligonucleotides N20-poxB-R were designed. The forward and reverse oligonucleotides N20-poxB-F and N20-poxB-R were mixed and annealed to form double-stranded DNA. The plasmid was digested with enzymes to obtain a linear plasmid. The double-stranded DNA and the linear plasmid were ligated to obtain a ligation product. The ligation product was transformed and screened to obtain the recombinant plasmid pECgRNA-N20-poxB. S52. Construction of recombinant plasmid pECgRNA-N20-yghX: Based on the yghX gene, the yghX N20 sequence was set. Based on the yghX N20 sequence, forward oligonucleotides N20-yghX-F and reverse oligonucleotides N20-yghX-R were designed. The forward and reverse oligonucleotides N20-yghX-F and N20-yghX-R were mixed and annealed to form double-stranded DNA. The plasmid was digested with enzymes to obtain a linear plasmid. The double-stranded DNA and the linear plasmid were ligated to obtain a ligation product. The ligation product was transformed and screened to obtain the recombinant plasmid pECgRNA-N20-yghX. S53. Constructing the overexpression prk gene knock-in fragment: Using the genomic DNA of the starting strain as a template, and using primers prk::yghX1 / P... J23119 -prk::yghX2, prk::yghX5 / prk::yghX6 amplify the target fragment; use the synthesized prk gene sequence as a template, and primer P J23119 -prk::yghX3 / prk::yghX4 amplifies the target fragment; after purifying the amplified fragments, three purified fragments are obtained. Using the three purified fragments as a mixed template, overlap extension PCR is performed with primers prk::yghX1 / prk::yghX6 to obtain P J23119 -prk::yghX fragment; S54. Constructing a strain overexpressing the prk gene: Transform plasmid pEcCAS into strain PYR-4V to obtain the recombinant strain PYR-4V / pEcCAS; J23119 The -prk::yghX fragment and the recombinant plasmid pECgRNA-N20-yghX were transformed into the recombinant strain PYR-4V / pEcCAS. After screening, plasmids pEcCAS and pECgRNA-N20-yghX were eliminated to obtain strain PYR-5OE. S55. Constructing overexpression of the cbbLS gene: Using the genomic DNA of the starting strain as a template, and with primers cbbLS::poxB1 / P... Trc -cbbLS::poxB2, cbbLS::poxB5 / cbbLS::poxB6 amplify the target fragment; using the synthesized cbbLS gene sequence as a template, primer P Trc -cbbLS::poxB3 / P Trc -cbbLS::poxB4 amplifies the target fragment; after purifying the amplified fragment, three purified fragments are obtained. Using the three purified fragments as a mixed template, overlap extension PCR is performed with primers cbbLS::poxB1 / cbbLS::poxB6 to obtain P. Trc -cbbLS::poxB fragment; S56. Constructing a strain overexpressing the cbbLS gene: Transform plasmid pEcCAS into strain PYR-5OE to obtain the recombinant strain PYR-5OE / pEcCAS; Trc The -cbbLS::poxB fragment and the recombinant plasmid pECgRNA-N20-poxB were transformed into the recombinant strain PYR-5OE / pEcCAS. After screening, the plasmids pEcCAS and pECgRNA-N20-poxB were eliminated to obtain strain PYR-6. In S51, the nucleotide sequence of the poxB N20 sequence is shown in SEQ ID No. 39 of the sequence listing; In S51, the nucleotide sequence of N20-poxB-F is shown in SEQ ID No. 40 of the sequence listing; In S51, the nucleotide sequence of N20-poxB-R is shown in SEQ ID No. 41 of the sequence listing; In S51, when the plasmid is digested with enzymes to obtain a linear plasmid, the plasmid is plasmid pECgRNA; In S51, when the plasmid is digested with an enzyme to obtain a linear plasmid, the enzyme digestion is BsaI digestion; In S51, when transforming and screening the ligation product, the method is to transform the ligation product into E. coli DH5α competent cells, identify positive clones by DNA sequencing, and extract plasmids. In S52, the nucleotide sequence of the yghX N20 sequence is shown in SEQ ID No. 42 of the sequence listing; In S52, the nucleotide sequence of N20-yghX-F is shown in SEQ ID No. 43 of the sequence listing; In S52, the nucleotide sequence of N20-yghX-R is shown in SEQ ID No. 44 of the sequence listing; In S52, when the plasmid is digested with enzymes to obtain a linear plasmid, the plasmid is plasmid pECgRNA; In S52, when the plasmid is digested with an enzyme to obtain a linear plasmid, the enzyme digestion is BsaI digestion; In S52, when transforming and screening the ligation product, the method is to transform the ligation product into E. coli DH5α competent cells, identify positive clones by DNA sequencing, and extract plasmids. In S53 and S55, the starting strain is Escherichia coli CGMCC No.25786; In S53, the nucleotide sequence of the synthesized prk gene is shown in SEQ ID No. 63 of the sequence listing; In S53, the nucleotide sequence of prk::yghX1 is shown in SEQ ID No. 45 of the sequence listing; P J23119 The nucleotide sequences of -prk::yghX2 are shown in SEQ ID No. 46 of the sequence listing; the nucleotide sequences of prk::yghX5 are shown in SEQ ID No. 49 of the sequence listing; the nucleotide sequences of prk::yghX6 are shown in SEQ ID No. 50 of the sequence listing; P J23119 The nucleotide sequence of -prk::yghX3 is shown in SEQ ID No. 47 of the sequence listing; the nucleotide sequence of prk::yghX4 is shown in SEQ ID No. 48 of the sequence listing; In S53, P J23119 The nucleotide sequence of the -prk::yghX fragment is shown in SEQ ID No. 64 of the sequence listing; In S54, plasmid pEcCAS was transformed into strain PYR-4V, and P... J23119 When the -prk:: yghX fragment and the recombinant plasmid pECgRNA-N20-yghX were transformed into the recombinant strain PYR-4V / pEcCAS, electroporation was used. In S54, the screening process combined LB solid medium plates containing both spectinomycin and kanamycin antibiotics with PCR verification. In S55, the nucleotide sequence of the synthesized cbbLS gene is shown in SEQ ID No. 65 of the sequence listing; In S55, the nucleotide sequence of cbbLS::poxB1 is shown in SEQ ID No. 51 of the sequence listing; P Trc The nucleotide sequences of -cbbLS::poxB2 are shown in SEQ ID No. 52 of the sequence listing; the nucleotide sequences of cbbLS::poxB5 are shown in SEQ ID No. 55 of the sequence listing; the nucleotide sequences of cbbLS::poxB6 are shown in SEQ ID No. 56 of the sequence listing; P Trc The nucleotide sequence of -cbbLS::poxB3 is shown in SEQ ID No. 53 of the sequence listing; P Trc The nucleotide sequence of -cbbLS::poxB4 is shown in SEQ ID No. 54 of the sequence listing; In S55, P Trc The nucleotide sequence of the -cbbLS::poxB fragment is shown in SEQ ID No. 66 of the sequence listing; In S56, plasmid pEcCAS was transformed into strain PYR-5OE, and P... Trc When the -cbbLS::poxB fragment and the recombinant plasmid pECgRNA-N20-poxB were transformed into the recombinant strain PYR-5OE / pEcCAS, electroporation was used. In S56, the screening process combined LB solid medium plates containing both spectinomycin and kanamycin antibiotics with PCR verification.

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