Use of modified Escherichia coli and its L-amino acids in fermentation production

Abstract

This paper discloses the use of modified Escherichia coli and its L-amino acid fermentation production. Specifically, it discloses the use of pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase derived from Escherichia coli, branched-chain amino acid transaminase derived from Bacillus subtilis, thiamine phosphate synthase, and maleate dehydrogenase in the construction of genetically modified bacteria that produce L-amino acids. Experiments have shown that by knocking out the genes encoding pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase derived from Escherichia coli, thiamine phosphate synthase, and maleate dehydrogenase in E. coli capable of producing L-amino acids, and introducing the gene encoding branched-chain amino acid transaminase derived from Bacillus subtilis, recombinant bacteria favorable for L-amino acid accumulation can be obtained. These recombinant bacteria can significantly increase L-amino acid production and reduce costs, which is of great significance in accelerating the industrialization process of L-amino acids.

Description

[Technical Field]

[0001] Cross-reference of related applications This application is a Chinese patent application filed with the China Patent Office on June 27, 2023, with application number 202310765899.5, and the title of the invention is "Recombinant Escherichia coli for L-valine fermentation production", and a Chinese patent application filed with the China Patent Office on June 29, 2023, with application number 202310784984.6, and the title of the invention is "Modified Escherichia coli and its use in L-valine fermentation production", and June 2023 Priority is claimed for the Chinese patent application filed with the China Patent Office on 29th of the month, application number 202310784863.1, with the title of the invention "Recombinant bacteria and their use in the production of L-valine", and for the Chinese patent application filed with the China Patent Office on 4th July 2023, application number 202310809284.8, with the title of the invention "Method for improving L-valine production and recombinant bacteria used therein". The entire contents of these four patent applications are incorporated into this application by reference.

[0002] This invention belongs to the field of biotechnology and relates to modified Escherichia coli and its use in the fermentation production of L-amino acids. [Background technology]

[0003] L-valine plays an important role in fields such as food, pharmaceuticals, health foods, and animal feed. In the food sector, L-valine contributes to nutritional supplementation, promotion of physical growth, and energy supply. In the pharmaceutical sector, L-valine is used in intravenous fluids and injections to promote surgical wound healing. Domestic exports of L-valine for animal feed exceed 30,000 tons, with an average annual growth rate of 20%.

[0004] Currently, many companies in Japan are producing L-valine using fermentation methods, and many laboratories are conducting research on the L-valine fermentation process. Researchers have found the following: The process by which intracellular L-valine is secreted outside the cell by permeating the cell membrane requires the involvement of specific transporters. However, in this process, the activity of specific transporters necessary for rapidly permeating the cell membrane and secreting intracellular valine outside the cell is insufficient, leading to the accumulation of L-valine inside the cell. Furthermore, when L-valine synthesized inside the cell is not released promptly and accumulates, acetohydroxy acid synthase, which is present in the bacterial cell and catalyzes L-valine biosynthesis and promotes acid production, is strongly inhibited by L-valine, reducing its catalytic activity. As a result, further L-valine synthesis becomes impossible, limiting further L-valine synthesis and ultimately leading to a decrease in production. Furthermore, research shows that E. coli is highly sensitive to L-valine, and if L-valine is not released in a timely manner and accumulates within the cells of L-valine-producing bacteria, the growth of E. coli is significantly inhibited. As a result, L-valine synthesis within the bacterial cells is inhibited, leading to a decrease in production. [Overview of the Initiative]

[0005] The primary objective of the present invention is to improve the production of L-amino acids. The objectives of the present invention are not limited to the subject matter described herein, and those skilled in the art will clearly understand from the following description other objectives not described herein.

[0006] The present invention first protects the use of protein combinations. Such use may be at least one of A1) to A5): A1) Construction of genetically engineered bacteria that produce L-amino acids, A2) Production of L-amino acids, A3) Control of L-amino acid production, A4) Preparation of products used in the production of L-amino acids, A5) Preparation of foods, feeds, or pharmaceuticals containing L-amino acids.

[0007] In the above use, the protein combination may include at least one of pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase derived from Escherichia coli, branched-chain amino acid transaminase derived from Bacillus subtilis, thiamine phosphate synthase, and malate dehydrogenase.

[0008] In the above use, the protein combination may specifically consist of at least one of the following: pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase derived from Escherichia coli, branched-chain amino acid transaminase derived from Bacillus subtilis, thiamine phosphate synthase, and malate dehydrogenase.

[0009] The pyruvate formate lyase described above may be B1), B2), or B3): B1) A protein having the amino acid sequence shown in SEQ ID No. 13, B2) A protein having 90% or more identity with the protein described in B1) and having similar function, in which amino acid residues are substituted and / or deleted and / or added in the amino acid sequence shown in SEQ ID No. 13. A fusion protein with similar function, obtained by ligating a tag to the N-terminus and / or C-terminus of B3), B1), or B2).

[0010] The alcohol dehydrogenase described in any of the above may be C1), C2), or C3): C1) A protein having the amino acid sequence shown in SEQ ID No. 14, C2) A protein having 90% or more identity with the protein described in C1) and having similar function, in which amino acid residues are substituted and / or deleted and / or added in the amino acid sequence shown in SEQ ID No. 14. A fusion protein with similar function, obtained by ligating a tag to the N-terminus and / or C-terminus of C3), C1), or C2).

[0011] The branched-chain amino acid transaminase derived from Escherichia coli according to any one of the above may be D1), or D2), or D3): D1) A protein having the amino acid sequence shown in SEQ ID No. 15, D2) A protein having 90% or more identity with the protein described in D1) and having a similar function, in which amino acid residues are substituted and / or deleted and / or added in the amino acid sequence shown in SEQ ID No. 15, D3) A fusion protein having a similar function, obtained by linking a tag to the N-terminus and / or C-terminus of D1) or D2).

[0012] The branched-chain amino acid transaminase derived from Bacillus subtilis according to any one of the above may be E1), or E2), or E3): E1) A protein having the amino acid sequence shown in SEQ ID No. 16, E2) A protein having 90% or more identity with the protein described in E1) and having a similar function, in which amino acid residues are substituted and / or deleted and / or added in the amino acid sequence shown in SEQ ID No. 16, E3) A fusion protein having a similar function, obtained by linking a tag to the N-terminus and / or C-terminus of E1) or E2).

[0013] The thiamine phosphate synthase according to any one of the above may be F1), or F2), or F3): F1) A protein having the amino acid sequence shown in SEQ ID No. 17, F2) A protein having 90% or more identity with the protein described in F1) and having a similar function, in which amino acid residues are substituted and / or deleted and / or added in the amino acid sequence shown in SEQ ID No. 17, F3) A fusion protein having a similar function, obtained by linking a tag to the N-terminus and / or C-terminus of F1) or F2).

[0014] The malate dehydrogenase described in any of the above may be G1), or G2), or G3): G1) A protein having the amino acid sequence shown in SEQ ID No. 18, G2) A protein having 90% or more identity with the protein described in G1), in which amino acid residues are substituted and / or deleted and / or added in the amino acid sequence shown in SEQ ID No. 18 and having a similar function, G3) A fusion protein having a similar function, obtained by linking a tag to the N-terminus and / or C-terminus of G1) or G2).

[0015] The present invention also protects the use of a nucleic acid molecule encoding the protein combination described in any of the above. The use may be at least one of A1) to A5): A1) Construction of a genetically engineered bacterium for producing L-amino acids, A2) Production of L-amino acids, A3) Control of the production amount of L-amino acids, A4) Preparation of a product used for the production of L-amino acids, A5) Preparation of a food, feed, or pharmaceutical product containing L-amino acids.

[0016] The present invention further protects the use of a substance that inhibits or reduces the expression level and / or activity of pyruvate formate-lyase described in any of the above, and / or a substance that inhibits or reduces the expression level and / or activity of alcohol dehydrogenase described in any of the above, and / or a substance that inhibits or reduces the expression level and / or activity of branched-chain amino acid transaminase derived from Escherichia coli described in any of the above, and / or a substance that inhibits or reduces the expression level and / or activity of thiamine phosphate synthase described in any of the above, and / or a substance that inhibits or reduces the expression level and / or activity of malate dehydrogenase described in any of the above, and / or a substance that improves or increases the expression level and / or activity of branched-chain amino acid transaminase derived from Bacillus subtilis described in any of the above. The use may be at least one of A1) to A5): A1) Construction of genetically engineered bacteria that produce L-amino acids, A2) Production of L-amino acids, A3) Control of L-amino acid production, A4) Preparation of products used in the production of L-amino acids, A5) Preparation of foods, feeds, or pharmaceuticals containing L-amino acids.

[0017] In the above use, the substance that inhibits or reduces the expression level and / or activity of pyruvate formate lyase described above may be the sgRNAa shown in SEQ ID No. 1.

[0018] In the above use, the substance that inhibits or reduces the expression level and / or activity of any of the alcohol dehydrogenases described above may be the sgRNAb shown in SEQ ID No. 4.

[0019] In the above use, the substance that inhibits or reduces the expression level and / or activity of the branched-chain amino acid transaminase derived from Escherichia coli described above may be the sgRNAc shown in SEQ ID No. 7.

[0020] In the above use, the substance that inhibits or reduces the expression level and / or activity of thiamine phosphate synthase as described above may be the sgRNAd shown in SEQ ID No. 9.

[0021] In the above use, the substance that inhibits or reduces the expression level and / or activity of any of the maleate dehydrogenases described above may be the sgRNAe shown in SEQ ID No. 11.

[0022] In the above use, the substance that improves or increases the expression level and / or activity of branched-chain amino acid transaminase derived from Bacillus subtilis as described above may be a nucleic acid molecule. Furthermore, the nucleic acid molecule may be a coding gene that drives branched-chain amino acid transaminase derived from Bacillus subtilis by ptrc. Moreover, the nucleotide sequence of the nucleic acid molecule may be that shown in SEQ ID No. 8.

[0023] The present invention also protects the use of expression cassettes, recombinant vectors, recombinant bacteria, or recombinant host cells comprising any of the nucleic acid molecules described above and a substance that inhibits or reduces the expression and / or activity of pyruvate formate lyase described above, a substance that inhibits or reduces the expression and / or activity of alcohol dehydrogenase described above, a substance that inhibits or reduces the expression and / or activity of branched-chain amino acid transaminase derived from Escherichia coli described above, a substance that inhibits or reduces the expression and / or activity of thiamine phosphate synthase described above, a substance that inhibits or reduces the expression and / or activity of malate dehydrogenase described above, and / or a substance that improves or increases the expression and / or activity of branched-chain amino acid transaminase derived from Bacillus subtilis described above. Such use may be at least one of A1) to A5): A1) Construction of genetically engineered bacteria that produce L-amino acids, A2) Production of L-amino acids, A3) Control of L-amino acid production, A4) Preparation of products used in the production of L-amino acids, A5) Preparation of foods, feeds, or pharmaceuticals containing L-amino acids.

[0024] The present invention also protects recombinant bacteria, characterized by the following: weak expression or non-expression of at least one of the pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase derived from Escherichia coli, thiamine phosphate synthase, and malate dehydrogenase described above in the starting bacteria; and / or expression or overexpression of the branched-chain amino acid transaminase derived from Bacillus subtilis described above.

[0025] In the recombinant bacteria described above, low expression or non-expression is achieved by reducing the expression level and / or activity of pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase derived from Escherichia coli, thiamine phosphate synthase, and / or malate dehydrogenase described above in the starting bacteria.

[0026] In the recombinant bacteria described above, expression or overexpression is achieved by introducing a gene encoding a branched-chain amino acid transaminase derived from Bacillus subtilis as described above into the starting organism.

[0027] In the recombinant bacteria described above, reducing the expression level and / or activity of any of the pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase derived from Escherichia coli described above, thiamine phosphate synthase, and / or maleate dehydrogenase described above in the starting bacteria can be achieved by genome editing, gene knockout, gene mutation, or gene weakening techniques, which may reduce the expression level, activity, or inactivate the pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase, thiamine phosphate synthase, and / or maleate dehydrogenase described above in the starting bacteria.

[0028] In the recombinant bacteria described above, genome editing is performed by the CRISPR / Cas9 system. The CRISPR / Cas9 system may include a vector expressing sgRNA targeting a gene encoding pyruvate formate lyase as described above, a vector expressing sgRNA targeting a gene encoding alcohol dehydrogenase as described above, a vector expressing sgRNA targeting a gene encoding branched-chain amino acid transaminase derived from Escherichia coli as described above, a vector expressing sgRNA targeting a gene encoding thiamine phosphate synthase as described above, and / or a vector expressing sgRNA targeting a gene encoding maleate dehydrogenase as described above.

[0029] In the recombinant bacteria described above, the sgRNA targeting the gene encoding pyruvate formate lyase may be the one shown in SEQ ID No. 1. The sgRNA targeting the gene encoding alcohol dehydrogenase may be the one shown in SEQ ID No. 4. The sgRNA targeting the gene encoding branched-chain amino acid transaminase derived from Escherichia coli may be the one shown in SEQ ID No. 7. The sgRNA targeting the gene encoding thiamine phosphate synthase may be the one shown in SEQ ID No. 9. The sgRNA targeting the gene encoding malate dehydrogenase may be the one shown in SEQ ID No. 11.

[0030] The recombinant bacteria according to the present invention may specifically be genetically modified bacteria A. This method may involve microorganisms that inhibit or reduce the expression level and / or activity of pyruvate formate lyase described in any of the above descriptions within the body. The microorganisms may produce valine.

[0031] In the genetically modified bacteria described above, inhibition or reduction of the expression level and / or activity of pyruvate formate lyase is achieved by knocking out or knocking down the gene encoding pyruvate formate lyase in the microorganism (i.e., the pflB gene).

[0032] Inhibition or reduction of pyruvate formate lyase expression and / or activity is achieved by introducing the pGRB-pflB sgRNA plasmid and ΔpflB-Up-Down fragments mentioned in the examples into microorganisms.

[0033] Within the genetically modified fungal cells described above, the expression level and / or activity of alcohol dehydrogenase are further inhibited or reduced.

[0034] In the genetically modified bacterium described above, inhibition or reduction of the expression level and / or activity of alcohol dehydrogenase is achieved by knocking out or knocking down the gene encoding alcohol dehydrogenase (i.e., the adhE gene) in the microorganism.

[0035] Inhibition or reduction of alcohol dehydrogenase expression and / or activity is achieved by introducing the pGRB-adhE sgRNA plasmid and ΔadhE-Up-Down fragments mentioned in the examples into microorganisms.

[0036] Within the body of the genetically modified bacterium described above, the expression level and / or activity of branched-chain amino acid transaminase derived from Escherichia coli is further inhibited or reduced, and branched-chain amino acid transaminase derived from Bacillus subtilis is possessed or expressed.

[0037] In the genetically modified bacteria described above, inhibition or reduction of the expression level and / or activity of branched-chain amino acid transaminase derived from Escherichia coli is achieved by knockout or knockdown of the gene encoding branched-chain amino acid transaminase in the microorganism (i.e., the ilvE(E) gene).

[0038] In the genetically modified bacterium described above, the possession or expression of branched-chain amino acid transaminase derived from Bacillus subtilis is achieved by knocking in or introducing the gene encoding branched-chain amino acid transaminase derived from Bacillus subtilis (i.e., the ilvE(B) gene) into the microorganism.

[0039] In the genetically modified bacterium described above, the knock-in or introduction of a gene encoding branched-chain amino acid transaminase derived from Bacillus subtilis into the microorganism is achieved by introducing an expression cassette into the microorganism. The expression cassette contains a promoter and a gene encoding branched-chain amino acid transaminase derived from Bacillus subtilis. The promoter may be an inducible promoter. Specifically, the inducible promoter may be a ptrc promoter.

[0040] Inhibition or reduction of the expression level and / or activity of branched-chain amino acid transaminases derived from Escherichia coli, and possession or expression of branched-chain amino acid transaminases derived from Bacillus subtilis, can be achieved by introducing the pGRB-ilvE sgRNA plasmid and ptrc-ilvE(B)-Up-Down fragments mentioned in the examples into microorganisms.

[0041] Within the genetically modified fungal cells described above, the expression level and / or activity of thiamine phosphate synthase are further inhibited or reduced.

[0042] In the genetically modified bacteria described above, inhibition or reduction of the expression level and / or activity of thiamine phosphate synthase is achieved by knocking out or knocking down the gene encoding thiamine phosphate synthase (i.e., the thiE gene) in the microorganism.

[0043] Inhibition or reduction of the expression level and / or activity of the thiamine phosphate synthase can be achieved by introducing the pGRB-thiE sgRNA plasmid and ΔthiE-Up-Down fragments mentioned in the examples into microorganisms.

[0044] Within the genetically modified fungal cells described above, the expression level and / or activity of maleate dehydrogenase are further inhibited or reduced.

[0045] In the genetically modified bacteria described above, inhibition or reduction of the expression level and / or activity of maleate dehydrogenase is achieved by knocking out or knocking down the gene encoding maleate dehydrogenase (i.e., the mdh gene) in the microorganism.

[0046] Inhibition or reduction of maleate dehydrogenase expression and / or activity can be achieved by introducing the pGRB-mdh sgRNA plasmid and Δmdh-Up-Down fragments mentioned in the examples into microorganisms.

[0047] The recombinant microorganism according to the present invention may specifically be genetically modified microorganism B. This method may involve a microorganism that inhibits or reduces the expression and / or activity of alcohol dehydrogenase in the body. The microorganism can produce valine.

[0048] In the genetically modified bacterium B described above, inhibition or reduction of alcohol dehydrogenase expression and / or activity is achieved by knocking out or knocking down the gene encoding alcohol dehydrogenase (i.e., the adhE gene) in the microorganism.

[0049] Inhibition or reduction of alcohol dehydrogenase expression and / or activity is achieved by introducing the pGRB-adhE sgRNA plasmid and ΔadhE-Up-Down fragments mentioned in the examples into microorganisms.

[0050] Within the body of the genetically modified bacterium B described above, the expression level and / or activity of branched-chain amino acid transaminases derived from microorganisms are further inhibited or reduced, and branched-chain amino acid transaminases derived from Bacillus subtilis are possessed or expressed.

[0051] In the genetically modified bacterium B described above, inhibition or reduction of the expression level and / or activity of the branched-chain amino acid transaminase derived from the microorganism is achieved by knocking out or knocking down the gene encoding the branched-chain amino acid transaminase in the microorganism (i.e., the ilvE(E) gene).

[0052] In the genetically modified bacterium B described above, the possession or expression of the branched-chain amino acid transaminase derived from Bacillus subtilis is achieved by knocking in or introducing the gene encoding the branched-chain amino acid transaminase derived from Bacillus subtilis (i.e., the ilvE(B) gene) into the microorganism.

[0053] In the genetically modified bacterium B described above, the knock-in or introduction of the gene encoding branched-chain amino acid transaminase derived from Bacillus subtilis into the microorganism is achieved by introducing an expression cassette into the microorganism. The expression cassette contains a promoter and the gene encoding branched-chain amino acid transaminase derived from Bacillus subtilis. The promoter may be an inducible promoter. Specifically, the inducible promoter may be a ptrc promoter.

[0054] Inhibition or reduction of the expression level and / or activity of microbial branched-chain amino acid transaminases, and possession or expression of Bacillus subtilis-derived branched-chain amino acid transaminases, can be achieved by introducing the pGRB-ilvE sgRNA plasmid and ptrc-ilvE(B)-Up-Down fragments mentioned in the examples into microorganisms.

[0055] In the genetically modified bacterium B described above, the expression level and / or activity of thiamine phosphate synthase are further inhibited or reduced.

[0056] In the genetically modified bacterium B described above, inhibition or reduction of the expression level and / or activity of thiamine phosphate synthase is achieved by knocking out or knocking down the gene encoding thiamine phosphate synthase (i.e., the thiE gene) in the microorganism.

[0057] Inhibition or reduction of thiamine phosphate synthase expression and / or activity can be achieved by introducing the pGRB-thiE sgRNA plasmid and ΔthiE-Up-Down fragments mentioned in the examples into microorganisms.

[0058] In the genetically modified bacterium B described above, the expression level and / or activity of maleate dehydrogenase are further inhibited or reduced.

[0059] In the genetically modified bacterium B described above, inhibition or reduction of the expression level and / or activity of maleate dehydrogenase is achieved by knockout or knockdown of the gene encoding maleate dehydrogenase (i.e., the mdh gene) in the microorganism.

[0060] Inhibition or reduction of maleate dehydrogenase expression and / or activity can be achieved by introducing the pGRB-mdh sgRNA plasmid and Δmdh-Up-Down fragments mentioned in the examples into microorganisms.

[0061] The recombinant bacteria according to the present invention may specifically be genetically modified bacteria C. This method may involve a microorganism that inhibits or reduces the expression level and / or activity of a branched-chain amino acid transaminase derived from a microorganism in the body, and that possesses or expresses a branched-chain amino acid transaminase derived from Bacillus subtilis. The microorganism can produce valine.

[0062] In the genetically modified bacterium C described above, inhibition or reduction of the expression level and / or activity of the branched-chain amino acid transaminase derived from the microorganism is achieved by knocking out or knocking down the gene encoding the branched-chain amino acid transaminase in the microorganism (i.e., the ilvE(E) gene).

[0063] In the genetically modified bacterium C described above, the possession or expression of branched-chain amino acid transaminase derived from Bacillus subtilis is achieved by knocking in or introducing the gene encoding branched-chain amino acid transaminase derived from Bacillus subtilis (i.e., the ilvE(B) gene) into the microorganism.

[0064] In the genetically modified bacterium C described above, the knock-in or introduction of the gene encoding branched-chain amino acid transaminase derived from Bacillus subtilis into the microorganism is achieved by introducing an expression cassette into the microorganism. The expression cassette includes a promoter and the gene encoding branched-chain amino acid transaminase derived from Bacillus subtilis. The promoter may be an inducible promoter. Specifically, the inducible promoter may be a ptrc promoter.

[0065] Inhibition or reduction of the expression level and / or activity of microbial branched-chain amino acid transaminases, and possession or expression of Bacillus subtilis-derived branched-chain amino acid transaminases, can be achieved by introducing the pGRB-ilvE sgRNA plasmid and ptrc-ilvE(B)-Up-Down fragments mentioned in the examples into microorganisms.

[0066] In the genetically modified bacterium C described above, the expression level and / or activity of thiamine phosphate synthase are further inhibited or reduced.

[0067] In the genetically modified bacterium C described above, inhibition or reduction of thiamine phosphate synthase expression and / or activity is achieved by knockout or knockdown of the gene encoding thiamine phosphate synthase (i.e., the thiE gene) in the microorganism.

[0068] Inhibition or reduction of the expression level and / or activity of the thiamine phosphate synthase can be achieved by introducing the pGRB-thiE sgRNA plasmid and ΔthiE-Up-Down fragments mentioned in the examples into microorganisms.

[0069] In the genetically modified bacterium C described above, the expression level and / or activity of maleate dehydrogenase are further inhibited or reduced.

[0070] In the genetically modified bacterium C described above, inhibition or reduction of the expression level and / or activity of maleate dehydrogenase is achieved by knockout or knockdown of the gene encoding maleate dehydrogenase (i.e., the mdh gene) in the microorganism.

[0071] Inhibition or reduction of maleate dehydrogenase expression and / or activity can be achieved by introducing the pGRB-mdh sgRNA plasmid and Δmdh-Up-Down fragments mentioned in the examples into microorganisms.

[0072] The recombinant bacteria according to the present invention may specifically be genetically modified bacteria. This method may involve microorganisms that inhibit or reduce the expression and / or activity of thiamine phosphate synthase in the body. The microorganisms may produce valine.

[0073] In the genetically modified fungi described above, inhibition or reduction of thiamine phosphate synthase expression and / or activity is achieved by knocking out or knocking down the gene encoding thiamine phosphate synthase (i.e., the thiE gene) in the microorganism.

[0074] Inhibition or reduction of thiamine phosphate synthase expression and / or activity can be achieved by introducing the pGRB-thiE sgRNA plasmid and ΔthiE-Up-Down fragments mentioned in the examples into microorganisms.

[0075] In the genetically modified fungi described above, the expression level and / or activity of maleate dehydrogenase are further inhibited or reduced.

[0076] In the genetically modified fungi described above, inhibition or reduction of the expression level and / or activity of maleate dehydrogenase is achieved by knocking out or knocking down the gene encoding maleate dehydrogenase (i.e., the mdh gene) in the microorganism.

[0077] Inhibition or reduction of maleate dehydrogenase expression and / or activity can be achieved by introducing the pGRB-mdh sgRNA plasmid and Δmdh-Up-Down fragments mentioned in the examples into microorganisms.

[0078] The recombinant bacteria according to the present invention may specifically be genetically modified bacteria. This method may involve microorganisms that inhibit or reduce the expression and / or activity of maleate dehydrogenase in the body. The microorganisms may produce valine.

[0079] In the genetically modified bacteria described above, inhibition or reduction of the expression level and / or activity of maleate dehydrogenase is achieved by knocking out or knocking down the gene encoding maleate dehydrogenase (i.e., the mdh gene) in the microorganism.

[0080] Inhibition or reduction of maleate dehydrogenase expression and / or activity can be achieved by introducing the pGRB-mdh sgRNA plasmid and Δmdh-Up-Down fragments mentioned in the examples into microorganisms.

[0081] The present invention further protects a method for improving the production of L-amino acids. This method includes obtaining recombinant bacteria by reducing the expression level and / or activity of at least one of the pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase derived from Escherichia coli, thiamine phosphate synthase, and malate dehydrogenase described above in a starting organism, and / or increasing the expression level and / or activity of the branched-chain amino acid transaminase derived from Bacillus subtilis described above, wherein the L-amino acid production of the recombinant bacteria is higher than that of the starting organism.

[0082] In the above method, reducing the expression level and / or activity of at least one of the pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase derived from Escherichia coli, thiamine phosphate synthase, and / or maleate dehydrogenase described above in the starting organism may be achieved by genome editing, gene knockout, gene mutation, or gene weakening techniques, which can reduce the expression level, activity, or inactivate the pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase, thiamine phosphate synthase, and / or maleate dehydrogenase described above in the starting organism.

[0083] In the above method, improving the expression level and / or activity of branched-chain amino acid transaminase derived from Bacillus subtilis as described above can be achieved by introducing the gene encoding branched-chain amino acid transaminase derived from Bacillus subtilis into the starting organism.

[0084] In the above method, genome editing may be performed by a CRISPR / Cas9 system. The CRISPR / Cas9 system may include a vector expressing an sgRNA targeting a gene encoding pyruvate formate lyase as described above, a vector expressing an sgRNA targeting a gene encoding alcohol dehydrogenase as described above, a vector expressing an sgRNA targeting a gene encoding branched-chain amino acid transaminase from Escherichia coli as described above, a vector expressing an sgRNA targeting a gene encoding thiamine phosphate synthase as described above, and / or a vector expressing an sgRNA targeting a gene encoding maleate dehydrogenase as described above.

[0085] In the above method, the sgRNA targeting the gene encoding pyruvate formate lyase described in any of the above may be the one shown in SEQ ID No. 1. The sgRNA targeting the gene encoding alcohol dehydrogenase described in any of the above may be the one shown in SEQ ID No. 4. The sgRNA targeting the gene encoding branched-chain amino acid transaminase derived from Escherichia coli described in any of the above may be the one shown in SEQ ID No. 7. The sgRNA targeting the gene encoding thiamine phosphate synthase described in any of the above may be the one shown in SEQ ID No. 9. The sgRNA targeting the gene encoding malate dehydrogenase described in any of the above may be the one shown in SEQ ID No. 11.

[0086] The method for preparing recombinant bacteria according to the present invention may specifically be a method for preparing genetically modified bacteria. This method may include a step (a1) of reducing the expression level and / or activity of pyruvate formate lyase described in any of the above in a microorganism. The microorganism can produce valine.

[0087] In the above preparation method, reducing the expression level and / or activity of pyruvate formate lyase in microorganisms is achieved by knocking out or knocking down the gene encoding pyruvate formate lyase in the microorganism (i.e., the pflB gene).

[0088] Reducing the expression level and / or activity of pyruvate formate lyase in microorganisms is achieved by introducing the pGRB-pflB sgRNA plasmid and ΔpflB-Up-Down fragments mentioned in the examples into the microorganisms.

[0089] The above preparation method further includes, after the completion of step (a1), step (a2) of reducing the expression level and / or activity of any of the alcohol dehydrogenases described above in the microorganism.

[0090] In the above preparation method, reducing the expression level and / or activity of alcohol dehydrogenase in microorganisms is achieved by knocking out or knocking down the gene encoding alcohol dehydrogenase (i.e., the adhE gene) in the microorganisms.

[0091] Reducing the expression level and / or activity of alcohol dehydrogenase in microorganisms is achieved by introducing the pGRB-adhE sgRNA plasmid and ΔadhE-Up-Down fragments mentioned in the examples into the microorganisms.

[0092] The above preparation method further includes, after the completion of step (a2), step (a3) ​​of reducing the expression level and / or activity of branched-chain amino acid transaminase derived from Escherichia coli and expressing branched-chain amino acid transaminase derived from Bacillus subtilis in the microorganism.

[0093] In the above preparation method, reducing the expression level and / or activity of branched-chain amino acid transaminase in microorganisms is achieved by knocking out or knocking down the gene encoding branched-chain amino acid transaminase (i.e., the ilvE(E) gene) in the microorganism. Expressing branched-chain amino acid transaminase derived from Bacillus subtilis in microorganisms is achieved by knocking in or introducing the gene encoding branched-chain amino acid transaminase derived from Bacillus subtilis (i.e., the ilvE(E) gene) into the microorganism. Knocking in or introducing the gene encoding branched-chain amino acid transaminase derived from Bacillus subtilis into microorganisms is achieved by introducing an expression cassette into the microorganism. The expression cassette includes a promoter and the gene encoding branched-chain amino acid transaminase derived from Bacillus subtilis. The promoter may be an inducible promoter. Specifically, the inducible promoter may be a ptrc promoter.

[0094] Decreasing the expression level and / or activity of branched-chain amino acid transaminase derived from Escherichia coli, and expressing branched-chain amino acid transaminase derived from Bacillus subtilis in microorganisms, can be achieved by introducing the pGRB-ilvE sgRNA plasmid and ptrc-ilvE(B)-Up-Down fragments mentioned in the examples into microorganisms.

[0095] The above preparation method further includes a step (a4) after the completion of step (a3), which reduces the expression level and / or activity of any of the thiamine phosphate synthases described above in the microorganism.

[0096] In the above preparation method, reducing the expression level and / or activity of thiamine phosphate synthase in microorganisms is achieved by knocking out or knocking down the gene encoding thiamine phosphate synthase (i.e., the thiE gene) in the microorganisms.

[0097] Reducing the expression level and / or activity of thiamine phosphate synthase in microorganisms can be achieved by introducing the pGRB-thiE sgRNA plasmid and ΔthiE-Up-Down fragments mentioned in the examples into the microorganisms.

[0098] The above preparation method further includes a step (a5) after the completion of step (a4), which reduces the expression level and / or activity of any of the above-described maleate dehydrogenases in the microorganism.

[0099] In the above preparation method, reducing the expression level and / or activity of maleate dehydrogenase in microorganisms is achieved by knocking out or knocking down the gene encoding maleate dehydrogenase (i.e., the mdh gene) in the microorganisms.

[0100] Reducing the expression level and / or activity of maleate dehydrogenase in microorganisms can be achieved by introducing the pGRB-mdh sgRNA plasmid and Δmdh-Up-Down fragments mentioned in the examples into the microorganisms.

[0101] The method for preparing recombinant bacteria according to the present invention may specifically be a method for preparing genetically modified bacteria B. This method may include a step (b1) of reducing the expression level and / or activity of any of the alcohol dehydrogenases described above in the microorganism. The microorganism can produce valine.

[0102] In the above method, reducing the expression level and / or activity of alcohol dehydrogenase in microorganisms is achieved by knocking out or knocking down the gene encoding alcohol dehydrogenase (i.e., the adhE gene) in the microorganism.

[0103] Reducing the expression level and / or activity of alcohol dehydrogenase in microorganisms is achieved by introducing the pGRB-adhE sgRNA plasmid and ΔadhE-Up-Down fragments mentioned in the examples into the microorganisms.

[0104] The above method further includes, after the completion of step (b1), step (b2) of reducing the expression level and / or activity of branched-chain amino acid transaminase derived from microorganisms and expressing branched-chain amino acid transaminase derived from Bacillus subtilis in the microorganisms.

[0105] In the above method, reducing the expression level and / or activity of branched-chain amino acid transaminase in microorganisms is achieved by knocking out or knocking down the gene encoding branched-chain amino acid transaminase (i.e., the ilvE(E) gene) in the microorganism. Expressing branched-chain amino acid transaminase derived from Bacillus subtilis in microorganisms is achieved by knocking in or introducing the gene encoding branched-chain amino acid transaminase derived from Bacillus subtilis (i.e., the ilvE(B) gene) into the microorganism. Knocking in or introducing the gene encoding branched-chain amino acid transaminase derived from Bacillus subtilis into the microorganism is achieved by introducing an expression cassette into the microorganism. The expression cassette includes a promoter and the gene encoding branched-chain amino acid transaminase derived from Bacillus subtilis. The promoter may be an inducible promoter. Specifically, the inducible promoter may be a ptrc promoter.

[0106] Decreasing the expression level and / or activity of microbial branched-chain amino acid transaminases, and expressing Bacillus subtilis-derived branched-chain amino acid transaminases in microorganisms, can be achieved by introducing the pGRB-ilvE sgRNA plasmid and ptrc-ilvE(B)-Up-Down fragments mentioned in the examples into microorganisms.

[0107] The above method further includes step (b3), after step (b2) is completed, a step of reducing the expression level and / or activity of any of the thiamine phosphate synthases described above in the microorganism.

[0108] In the above method, reducing the expression level and / or activity of thiamine phosphate synthase in microorganisms is achieved by knocking out or knocking down the gene encoding thiamine phosphate synthase (i.e., the thiE gene) in the microorganism.

[0109] Reducing the expression level and / or activity of any of the thiamine phosphate synthases described above in microorganisms can be achieved by introducing the pGRB-thiE sgRNA plasmid and ΔthiE-Up-Down fragments mentioned in the examples into the microorganisms.

[0110] The above method further includes step (b4), after step (b3) is completed, a step of reducing the expression level and / or activity of any of the above-described maleate dehydrogenases in the microorganism.

[0111] In the above method, reducing the expression level and / or activity of maleate dehydrogenase in microorganisms is achieved by knocking out or knocking down the gene encoding maleate dehydrogenase (i.e., the mdh gene) in the microorganism.

[0112] Reducing the expression level and / or activity of maleate dehydrogenase in microorganisms can be achieved by introducing the pGRB-mdh sgRNA plasmid and Δmdh-Up-Down fragments mentioned in the examples into the microorganisms.

[0113] The method for preparing recombinant bacteria according to the present invention may specifically be a method for preparing genetically modified bacteria C. This method may include a step (c1) of reducing the expression level and / or activity of a branched-chain amino acid transaminase derived from the microorganism in the microorganism, and expressing a branched-chain amino acid transaminase derived from Bacillus subtilis in the microorganism. The microorganism can produce valine.

[0114] In the above preparation method, reducing the expression level and / or activity of branched-chain amino acid transaminase in microorganisms is achieved by knocking out or knocking down the gene encoding branched-chain amino acid transaminase (i.e., the ilvE(E) gene) in the microorganism. Expressing branched-chain amino acid transaminase derived from Bacillus subtilis in microorganisms is achieved by knocking in or introducing the gene encoding branched-chain amino acid transaminase derived from Bacillus subtilis (i.e., the ilvE(B) gene) into the microorganism. Knocking in or introducing the gene encoding branched-chain amino acid transaminase derived from Bacillus subtilis into microorganisms is achieved by introducing an expression cassette into the microorganism. The expression cassette includes a promoter and the gene encoding branched-chain amino acid transaminase derived from Bacillus subtilis. The promoter may be an inducible promoter. Specifically, the inducible promoter may be a ptrc promoter.

[0115] Decreasing the expression level and / or activity of microbial branched-chain amino acid transaminases in microorganisms, and expressing Bacillus subtilis-derived branched-chain amino acid transaminases in microorganisms, can be achieved by introducing the pGRB-ilvE sgRNA plasmid and ptrc-ilvE(B)-Up-Down fragments mentioned in the examples into the microorganisms.

[0116] The above preparation method further includes a step (c2) after the completion of step (c1) to reduce the expression level and / or activity of the thiamine phosphate synthase described in any of the above descriptions in the microorganism.

[0117] In the above preparation method, reducing the expression level and / or activity of thiamine phosphate synthase in the microorganism is achieved by knocking out or knocking down the gene encoding thiamine phosphate synthase (i.e., the thiE gene) in the microorganism.

[0118] Reducing the expression level and / or activity of any of the thiamine phosphate synthases described above in microorganisms can be achieved by introducing the pGRB-thiE sgRNA plasmid and ΔthiE-Up-Down fragments mentioned in the examples into the microorganisms.

[0119] The above preparation method further includes a step (c3) after the completion of step (c2) to reduce the expression level and / or activity of the maleate dehydrogenase described in any of the above in the microorganism.

[0120] In the above preparation method, reducing the expression level and / or activity of maleate dehydrogenase in microorganisms is achieved by knocking out or knocking down the gene encoding maleate dehydrogenase (i.e., the mdh gene) in the microorganisms.

[0121] Reducing the expression level and / or activity of maleate dehydrogenase in the aforementioned microorganisms can be achieved by introducing the pGRB-mdh sgRNA plasmid and Δmdh-Up-Down fragments mentioned in the examples into the microorganisms.

[0122] The method for preparing recombinant bacteria according to the present invention may specifically be a method for preparing genetically modified bacteria. This method may include a step (d1) of reducing the expression level and / or activity of any of the thiamine phosphate synthases described above in the microorganism. The microorganism can produce valine.

[0123] In the above preparation method, reducing the expression level and / or activity of thiamine phosphate synthase in microorganisms is achieved by knocking out or knocking down the gene encoding thiamine phosphate synthase (i.e., the thiE gene) in the microorganisms.

[0124] Reducing the expression level and / or activity of any of the thiamine phosphate synthases described above in the microorganisms can be achieved by introducing the pGRB-thiE sgRNA plasmid and ΔthiE-Up-Down fragments mentioned in the examples into the microorganisms.

[0125] The above preparation method further includes a step (d2) after the completion of step (d1) to reduce the expression level and / or activity of any of the above-described maleate dehydrogenases in the microorganism.

[0126] In the above preparation method, reducing the expression level and / or activity of maleate dehydrogenase in microorganisms is achieved by knocking out or knocking down the gene encoding maleate dehydrogenase (i.e., the mdh gene) in the microorganisms.

[0127] Reducing the expression level and / or activity of maleate dehydrogenase in microorganisms can be achieved by introducing the pGRB-mdh sgRNA plasmid and Δmdh-Up-Down fragments mentioned in the examples into the microorganisms.

[0128] The method for preparing recombinant bacteria according to the present invention may specifically be a method for preparing genetically modified bacteria. This method may include a step (e1) of reducing the expression level and / or activity of any of the above-described maleate dehydrogenases in the microorganism. The microorganism can produce valine.

[0129] In the above preparation method, reducing the expression level and / or activity of maleate dehydrogenase in microorganisms is achieved by knocking out or knocking down the gene encoding maleate dehydrogenase (i.e., the mdh gene) in the microorganisms.

[0130] Reducing the expression level and / or activity of maleate dehydrogenase in microorganisms can be achieved by introducing the pGRB-mdh sgRNA plasmid and Δmdh-Up-Down fragments mentioned in the examples into the microorganisms.

[0131] The present invention also protects a method for producing L-amino acids. This method may include the steps of fermenting and culturing a recombinant microorganism described in any of the above, collecting the fermentation product, and obtaining an L-amino acid.

[0132] The present invention also protects the use of recombinant bacteria as described in any of the above. Such use may be at least one of A2) to A5): A2) Production of L-amino acids, A3) Control of L-amino acid production, A4) Preparation of products used in the production of L-amino acids, A5) Preparation of foods, feeds, or pharmaceuticals containing L-amino acids.

[0133] In this specification, culture may be carried out according to conventional methods of the art, including but not limited to microplate culture, shaking culture, batch culture, continuous culture, and fed-batch culture, and each culture condition such as temperature, time, and pH value of the medium can be appropriately adjusted according to the actual situation.

[0134] In this specification, L-amino acids may include L-valine, L-isoleucine, L-threonine, L-tryptophan, L-arginine, L-lysine, L-glutamic acid, L-glycine, L-alanine, L-leucine, L-methionine, L-proline, L-serine, L-tyrosine, L-cysteine, L-phenylalanine, L-asparagine, L-glutamine, L-aspartic acid, and / or L-histidine.

[0135] The L-amino acid mentioned in any of the above may specifically be L-valine.

[0136] In this specification, "identity" refers to the identity of amino acid sequences. Amino acid sequence identity can be measured using homology search sites on the internet, such as the BLAST webpage on the NCBI homepage. For example, using Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the Matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), the identity of amino acid sequences can be calculated, and then the identity value (%) can be obtained.

[0137] In this specification, identity of 90% or more may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0138] In this specification, microorganisms may be yeasts, bacteria, algae, or fungi. Here, the bacteria belong to the genera Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., Bacillus sp., Brevibacterium sp., Corynebacterium sp., Aerobacter sp., Enterobacteria sp., Micrococcus sp., Serratia sp., Salmonella sp., Streptomyces sp., and Providencia. It may derive from genera such as sp., but is not limited to these.

[0139] Furthermore, the bacteria may, but are not limited to, Escherichia coli, Corynebacterium glutamicum, Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium pekinense, Brevibacterium ammoniagenes, Corynebacterium crenatum, or Pantoea.

[0140] In this specification, the starting organism may be Escherichia coli.

[0141] In one or more embodiments of the present invention, the microorganism is Escherichia coli. Specifically, the Escherichia coli may be Escherichia coli YP045 CGMCC No. 22721 or Escherichia coli W3110.

[0142] The inventors of this invention have discovered through numerous experiments the following: (1) In Escherichia coli capable of producing valine (e.g., Escherichia coli YP045 CGMCC No. 22721, or Escherichia coli W3110), "knockout of the pflB gene," "knockout of the pflB gene and adhE gene," "knockout of the pflB gene and adhE gene, and insertion of the ilvE(B) gene from Bacillus subtilis driven by the ptrc promoter simultaneously with the knockout of the ilvE(E) gene," and "knockout of the pflB gene, adhE gene, and thiE gene, and simultaneous knockout of the ilvE(E) gene driven by the ptrc promoter." (1) Genetically modified bacteria obtained by "insertion of the ilvE(B) gene derived from Bacillus subtilis" or "knockout of the pflB gene, adhE gene, thiE gene, and mdh gene, and knockout of the ilvE(E) gene, simultaneously with insertion of the ilvE(B) gene derived from Bacillus subtilis driven by the ptrc promoter" can all significantly increase the production of L-amino acids (e.g., L-valine); (2) Escherichia coli capable of producing valine (e.g., Escherichia coli YP045 CGMCC No.In *Escherichia coli* 22721 or *Escherichia coli* W3110), genetically modified bacteria obtained by "knockout of the adhE gene," "knockout of the adhE gene and the ilvE(E) gene, and simultaneous insertion of the ilvE(B) gene from *Bacillus subtilis* driven by the ptrc promoter," "knockout of the adhE and thiE genes, and simultaneous insertion of the ilvE(B) gene from *Bacillus subtilis* driven by the ptrc promoter," or "knockout of the adhE, thiE, and mdh genes, and simultaneous insertion of the ilvE(E) gene from *Bacillus subtilis* driven by the ptrc promoter" can all significantly increase the production of L-amino acids (e.g., L-valine); (3) *Escherichia coli* capable of producing valine (e.g., *Escherichia coli* YP045 CGMCC) In *Escherichia coli* (No. 22721, or *Escherichia coli* W3110), genetically modified bacteria obtained by "knockout of the ilvE(E) gene and simultaneous insertion of the ilvE(B) gene from *Bacillus subtilis* driven by the ptrc promoter," "knockout of the thiE gene and simultaneous insertion of the ilvE(E) gene and simultaneous insertion of the ilvE(B) gene from *Bacillus subtilis* driven by the ptrc promoter," or "knockout of the thiE gene and mdh gene and simultaneous insertion of the ilvE(E) gene and simultaneous insertion of the ilvE(B) gene from *Bacillus subtilis* driven by the ptrc promoter" can all significantly increase the production of L-amino acids (e.g., L-valine); (4) *Escherichia coli* capable of producing valine (e.g., *Escherichia coli* YP045 CGMCC) In *E. coli* species No. 22721 (or *E. coli* W3110), genetically modified bacteria obtained by knocking out the thiE gene, the mdh gene, or both the thiE and mdh genes simultaneously, all exhibit a significant increase in L-amino acid (e.g., L-valine) production. This invention has significant application value.

[0143] [Deposit explanation] Fungal species name: Escherichia coli Latin name: Escherichia coli Species name: Escherichia coli Strain number: YP045 Depository name: Center for Ordinary Microorganisms, China Microbial Species Preservation and Storage Administration Depository abbreviation: CGMCC Depository address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing Deposit date: June 15, 2021 Registration number by the deposit center: CGMCC No.22721 [Modes for carrying out the invention]

[0144] The present invention will be described in more detail below in combination with specific embodiments, but the provided examples are for illustrative purposes only and do not limit the scope of the invention. The following examples can be used as a guideline for those skilled in the art to make further improvements and do not limit the present invention in any way.

[0145] The experimental methods in the following examples are, unless otherwise specified, standard methods in accordance with the techniques or conditions described in the literature of the art or product descriptions. The materials, reagents, etc., used in the following examples are commercially available unless otherwise specified.

[0146] Escherichia coli YP045 can be used for valine production and was deposited on June 15, 2021, with the Center for Ordinary Microorganisms (CGMCC, address: No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing) by the China Microbial Species Preservation and Storage Administration, with deposit number CGMCC No. 22721. The official name of Escherichia coli YP045 is Escherichia coli YP045 CGMCC No. 22721, and it will be hereinafter abbreviated as valine-producing bacterium CGMCC 22721.

[0147] Example 1: Modification of a genetically engineered bacterium starting from the valine-producing bacterium CGMCC 22721. The inventors of this invention conducted numerous experiments and modified the valine-producing bacterium CGMCC 22721 as a starting strain to obtain genetically modified bacteria YPVal-pflB01, YPVal-pflB02, YPVal-pflB03, YPVal-pflB04, and YPVal-pflB05. The genotypes of the genetically modified bacteria YPVal-pflB01, YPVal-pflB02, YPVal-pflB03, YPVal-pflB04, and YPVal-pflB05 are shown in Table 1.

[0148] TIFF2026521933000001.tif55170

[0149] 1. Obtaining the genetically modified bacterium YPVal-pflB01 Based on the genome sequence of Escherichia coli W3110 published by NCBI, the pflB gene in the genome of the valine-producing bacterium CGMCC 22721 was knocked out using CRISPR / Cas9 genome editing technology to obtain the genetically modified bacterium YPVal-pflB01.

[0150] The pflB gene encodes pyruvate formate lyase, has a Gene ID of 945514, and its amino acid sequence is shown as SEQ ID No. 13.

[0151] SEQ ID No. 13 (Pyruvate-formate lyase pflB): MSELNEKLATAWEGFTKGDWQNEVNVRDFIQKNYTPYEGDESFLAGATEATTTLWDKVMEGVKLENRTHAPVDFDTAVASTITSHDAGYINKQLEKIVGLQTEAPLKRALIPFGGIKMIEGSCKAYNRELDPMIKKIFTEYRKTHNQGVFDVYTPDILRCRKSGVLTGLPDAYGRGRIIGDYRRVALYGI DYLMKDKLAQFTSLQADLENGVNLEQTIRLREEIAEQHRALGQMKEMAAKYGYDISGPATNAQEAIQWTYFGYLAAVKSQNGAAMSFGRTSTFLDVYIERDLKAGKITEQEAQEMVDHLVMKLRMVRFLRTPEYDELFSGDPIWATESIGGMGLDGRTLVTKNSFRFLNTLYTMGPSPEPNMTILWSEKL PLNFKKFAAKVSIDTSSLQYENDDLMRPDFNNDDYAIACCVSPMIVGKQMQFFGARANLAKTMLYAINGGVDEKLKMQVGPKSEPIKGDVLNYDEVMERMDHFMDWLAKQYITALNIIHYMHDKYSYEASLMALHDRDVIRTMACGIAGLSVAADSLSAIKYAKVKPIRDEDGLAIDFEIEGEYPQFGNN DPRVDDLAVDLVERFMKKIQKLHTYRDAIPTQSVLTITSNVVYGKKTGNTPDGRRAGAPFGPGANPMHGRDQKGAVASLTSVAKLPFAYAKDGISYTFSIVPNALGKDDEVRKTNLAGLMDGYFHHEASIEGGQHLNVNVMNREMLLDAMENPEKYPQLTIRVSGYAVRFNSLTKEQQQDVITRTFTQSM

[0152] The specific steps are as follows: 1. Construction of the pGRB-pflB sgRNA plasmid Based on the Escherichia coli W3110 genome sequence published by NCBI, a target sequence for sgRNA to knock out the pflB gene, GCGAATTTCTTGAAGTTCAGCGG (shown as SEQ ID No. 1), was designed using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ). Homology arm sequences of a linearized pGRB vector for constructing the sgRNA plasmid were added to the 5' and 3' ends of the target sequence.

[0153] (1) Primer synthesized by Invitrogen: sgRNA-1F:5'- TGACAGCTAGCTCAGTCCTAGGTATAATACTAGT gcgaatttcttgaagttcagcgg GTTTTAGGCTAGAATAGCAAGTTAAAATAAGG -3' (shown in SEQ ID No. 19, the underlined portion is the homology arm sequence of the pGRB vector), primer sgRNA-1R:5'- CCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC ccgctgaacttcaagaaattcgc ACTAGTATTATACCTAGGACTGAGCTAGCTGTCA These are the -3' (shown in SEQ ID No. 20, where the underlined portion is the homology arm sequence of the pGRB vector), the primer sgRNA-PF:5'-GTCTCATGAGCGGATACATATTTG-3' (SEQ ID No. 21), and the primer sgRNA-PR:5'-ATGAGAAAGCGCCACGCT-3' (SEQ ID No. 22).

[0154] (2) After annealing primer sgRNA-1F and primer sgRNA-1R (reaction program: denaturation at 95°C for 5 minutes, annealing at 50°C for 1 minute), the target fragment was recovered using a DNA purification kit, its DNA concentration was measured, and the concentration was diluted to 100 ng / μL to obtain the annealing product. The annealing product contains an sgRNA-1 fragment having the nucleotide sequence shown in SEQ ID No. 1.

[0155] (3) The pGRB vector (Addgene product, catalog number 71539) was digested with the restriction enzyme SpeI (Takara product, catalog number 1631), and a DNA fragment of approximately 2700 bp was recovered using a DNA recovery kit (QIAGEN Gel Extraction Kit). The digestive system was 50 μL and consisted of 5 μL of 10× Buffer (with restriction enzyme SpeI), 2.5 μL of restriction enzyme SpeI, 3000-5000 ng of pGRB vector, and ddH2O. Digestion program: 3 hours at 37°C.

[0156] (4) The DNA fragments recovered in step (3) were subjected to a dephosphorylation reaction (to prevent self-ligation of the pGRB vector), recovered using a DNA recovery kit, and a linearized pGRB vector was obtained. The dephosphorylation system was 50 μL and consisted of 5 μL of 10× Buffer (included with CIAP), 1000-2000 ng of DNA fragments recovered in step (3), 2.5 μL of CIAP (Takara product, catalog number 2250A), and ddH2O. Dephosphorylation program: 1 hour at 37°C.

[0157] (5) Using the Gibson Assembly Kit (New England Biolabs), the linearized pGRB vector obtained in step (4) and the annealing product obtained in step (2) were recombined, and then transformed into E. coli DH5α competent cells (TAKARA) to obtain the pGRB-pflB sgRNA plasmid. The recombinant system consisted of 5 μL of linearized pGRB vector (2 μL), annealing product (0.5 μL), and assembly enzyme (included in the Gibson Assembly Kit) (2.5 μL). Reassembly program: Assemble at 50°C for 30 minutes.

[0158] 2. Obtaining the ΔpflB-Up-Down fragment (1) Based on the genome sequence of Escherichia coli W3110 published by NCBI, a primer pair for amplifying the upstream homology arm and a primer pair for amplifying the downstream homology arm were designed and synthesized by Invitrogen. The primer pair for amplifying the upstream homology arm consists of primer P1: 5'-CGTTGGTGTCCAGACAGGTATG-3' (shown in SEQ ID No. 23) and primer P2: 5'-GACATCCTGCGTTGCCGTAAATGAACCGTGAAATGCTGCTCG-3' (shown in SEQ ID No. 24). The primer pair for amplifying the downstream homology arm consists of primer P3:5'-CGAGCAGCATTTCACGGTTCATTTACGGCAACGCAGGATGTC-3' (shown in SEQ ID No. 25) and primer P4:5'-TTTCTCACCTGACCGTGATG-3' (shown in SEQ ID No. 26).

[0159] (2) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P1 and primer P2, and the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 686 bp upstream homology arm was recovered using a DNA recovery kit.

[0160] (3) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P3 and primer P4, and the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 695 bp downstream homology arm was recovered using a DNA recovery kit.

[0161] (4) The upstream homology arm recovered in step (2) and the downstream homology arm recovered in step (3) were mixed, and using this as a template, overlap PCR was performed using a primer pair consisting of primer P1 and primer P4 to obtain the ΔpflB-Up-Down fragment shown in SEQ ID No. 2. SEQ ID No.2: ΔpflB-Up-Down(1339bp)

[0162] 3. Acquisition of CGMCC 22721-Cas9 strain (1) Plasmid pREDCas9 (Addgene product, catalog number 71541; containing the spectinomycin resistance gene) was used to transform valine-producing bacteria CGMCC 22721 competent cells. These cells were then spread onto 2-YT agar plates containing 100 mg / L spectinomycin and cultured at 32°C to obtain single colonies resistant to spectinomycin. The preparation method for 2-YT agar plates is as follows: Dissolve 16g of tryptone, 10g of yeast extract, 5g of sodium chloride, and 16g of agar in an appropriate amount of water, add water to a total volume of 1L, adjust the pH to 7.0 with sodium hydroxide, and sterilize at 121°C for 20 minutes.

[0163] (2) Using the single colonies obtained in step (1) as templates, PCR amplification was performed using primer pairs consisting of primer pRedCas9-PF:5'-GCAGTGGCGGTTTTCATG-3' (SEQ ID No. 27) and primer pRedCas9-PR:5'-CCTTGGTGATCTCGCCTTTC-3' (SEQ ID No. 28) to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contains a DNA fragment having the nucleotide sequence shown in SEQ ID No. 3, the colony is considered to contain plasmid pREDCas9. The strain of the colony was named CGMCC 22721-Cas9 strain. SEQ ID No.3: PCR amplification sequence (943 bp) of test primer pRedCas9-F / pRedCas9-R GCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGGGGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATGAGGGAAGCGGTGATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAGG

[0164] 4. Obtaining the genetically modified bacterium YPVal-pflB01 (1) Culture of CGMCC 22721-Cas9 strain: CGMCC 22721-Cas9 strain is OD 600nm When the concentration reached 0.1 mM, IPTG was added to bring the IPTG concentration in the system to 0.1 mM, and the culture was continued to induce homologous recombination via λ-Red. The CGMCC 22721-Cas9 strain was OD 600nm When the concentration reached 0.6, the bacterial cells were collected and CGMCC 22721-Cas9 competent cells were prepared.

[0165] (2) CGMCC 22721-Cas9 competent cells were transformed with the pGRB-pflB sgRNA plasmid obtained in step 1 and the ΔpflB-Up-Down fragment obtained in step 2. These cells were then spread onto 2-YT agar plates containing 100 mg / L spectinomycin and 100 mg / L ampicillin, and cultured at 32°C to obtain single colonies.

[0166] (3) Using the single colonies obtained in step (2) as templates, PCR amplification was performed using primer pairs consisting of primer P1 and primer P4 to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contained a 1339 bp DNA fragment, that colony was provisionally determined to be a positive transformant.

[0167] (4) The positive transformants obtained in step (3) were inoculated onto 2-YT agar plates containing 100 mg / L spectinomycin and 0.2% (m / v) arabinose and cultured at 32°C (to remove the pGRB-pflB sgRNA plasmid). Subsequently, they were grown on 2-YT agar plates containing 100 mg / L spectinomycin, and colonies that did not grow on 2-YT agar plates containing 100 mg / L ampicillin were selected. These colonies were then subcultured on 2-YT agar plates and cultured at 42°C (to remove the pREDCas9 plasmid). Finally, colonies that did not grow on 2-YT agar plates containing 100 mg / L spectinomycin but grew on untreated 2-YT agar plates were selected.

[0168] (5) Using the single colony obtained in step (4) as a template, PCR amplification was performed using a primer pair consisting of primer P1 and primer P4 to obtain a PCR amplification product. If the PCR amplification product obtained from a single colony contained a 1339 bp DNA fragment, that colony was determined to be a positive transformant. This positive transformant is one in which the pflB gene in the genome of the valine-producing bacterium CGMCC 22721 has been knocked out, and this positive transformant was named the genetically modified bacterium YPVal-pflB01.

[0169] 2. Obtaining the genetically modified bacterium YPVal-pflB02 Using the genetically modified bacterium YPVal-pflB01 obtained in step one as the starting cell, the adhE gene in the genome of the genetically modified bacterium YPVal-pflB01 was knocked out using CRISPR / Cas9 genome editing technology, based on the genome sequence of Escherichia coli W3110 published by NCBI.

[0170] The adhE gene encodes alcohol dehydrogenase, has a Gene ID of 945837, and its amino acid sequence is shown as SEQ ID No. 14. SEQ ID NO.14 (Alcohol dehydrogenase adhE) MAVTNVAELNALVERVKKAQREYASFTQEQVDKIFRAAALAAADARIPLAKMAVAESGMGIVEDKVIKNHFASEYIYNAYKDEKTCGVLSEDDTFGTITIAEPIGIICGIV PTTNPTSTAIFKSLISLKTRNAIIFSPHPRAKDATNKAADIVLQAAIAAGAPKDLIGWIDQPSVELSNALMHHPDINLILATGGPGMVKAAYSSGKPAIGVGAGNTPVVID ETADIKRAVASVLMSKTFDNGVICASEQSVVVVDSVYDAVRERFATHGGYLLQGKELKAVQDVILKNGALNAAIVGQPAYKIAELAGFSVPENTKILIGEVTVVDESEPFA HEKLSPTLAMYRAKDFEDAVEKAEKLVAMGGIGHTSCLYTDQDNQPARVSYFGQKMKTARILINTPASQGGIGDLYNFKLAPSLTLGCGSWGGNSISENVGPKHLINKKTVA KRAENMLWHKLPKSIYFRRGSLPIALDEVITDGHKRALIVTDRFLFNNGYADQITSVLKAAGVETEVFFEVEADPTLSIVRKGAELANSFKPDVIIALGGGSPMDAAKIMW VMYEHPETHFEELALRFMDIRKRIYKFPKMGVKAKMIAVTTTSGTGSEVTPFAVVTDDATGQKYPLADYALTPDMAIVDANLVMDMPKSLCAFGGLDAVTHAMEAYVSVLAS EFSDGQALQALKLLKEYLPASYHEGSKNPVARERVHSAATIAGIAFANAFLGVCHSMAHKLGSQFHIPHGLANALLICNVIRYNANDNPTKQTAFSQYDRPQARRRYAEIA DHLGLSAPGDRTAAKIEKLLAWLETLKAELGIPKSIREAGVQEADFLANVDKLSEDAFDDQCTGANPRYPLISELKQILLDTYYGRDYVEGETAAKKEAAPAKAEKKAKKSA

[0171] The specific steps are as follows: 1. Construction of the pGRB-adhE sgRNA plasmid Based on the Escherichia coli W3110 genome sequence published by NCBI, a target sequence for sgRNA to knock out the adhE gene, AAGAACCACAACCCAGAGTCAGG (shown as SEQ ID No. 4), was designed using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ). Homology arm sequences of a linearized pGRB vector for constructing the sgRNA plasmid were added to the 5' and 3' ends of the target sequence.

[0172] (1) Primer synthesized by Invitrogen: sgRNA-2F:5'- TGACAGCTAGCTCAGTCCTAGGTATAATACTAGT aagaaccacaacccagagtcagg GTTTTAGGCTAGAATAGCAAGTTAAAATAAGG -3' (shown in SEQ ID No. 29, with the underlined portion being the homology arm sequence of the pGRB vector), and primer sgRNA-2R: 5'- CCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC cctgactctgggttgtggttctt ACTAGTATTATACCTAGGACTGAGCTAGCTGTCA -3' (shown as SEQ ID No. 30, with the underlined portion being the homology arm sequence of the pGRB vector)

[0173] (2) After annealing primer sgRNA-2F and primer sgRNA-2R (reaction program: denaturation at 95°C for 5 minutes, annealing at 50°C for 1 minute), the target fragment was recovered using a DNA purification kit, its DNA concentration was measured, and the concentration was diluted to 100 ng / μL to obtain the annealing product. The annealing product contained an sgRNA-2 fragment having the nucleotide sequence shown in SEQ ID No. 4.

[0174] (3) The pGRB vector was digested with restriction enzyme SpeI, and a DNA fragment of approximately 2700 bp was recovered using a DNA recovery kit (QIAGEN Gel Extraction Kit). The digestive system was 50 μL and consisted of 5 μL of 10× Buffer (with restriction enzyme SpeI), 2.5 μL of restriction enzyme SpeI, 3000-5000 ng of pGRB vector, and ddH2O. Digestion program: 3 hours at 37°C.

[0175] (4) The DNA fragments recovered in step (3) were subjected to a dephosphorylation reaction (to prevent self-ligation of the pGRB vector), recovered using a DNA recovery kit, and a linearized pGRB vector was obtained. The dephosphorylation system was 50 μL and consisted of 5 μL of 10×Buffer (provided with CIAP), 1000-2000 ng of DNA fragments recovered in step (3), 2.5 μL of CIAP, and ddH2O. Dephosphorylation program: 1 hour at 37°C.

[0176] (5) Using the Gibson Assembly Kit, the linearized pGRB vector obtained in step (4) and the annealing product obtained in step (2) were recombined, and then transformed into E. coli DH5α competent cells to obtain the pGRB-adhE sgRNA plasmid. The recombinant system consisted of 5 μL of linearized pGRB vector (2 μL), annealing product (0.5 μL), and assembly enzyme (included in the Gibson Assembly Kit) (2.5 μL). Reassembly program: Assemble at 50°C for 30 minutes.

[0177] 2. Obtaining the ΔadhE-Up-Down fragment (1) Based on the genome sequence of Escherichia coli W3110 published by NCBI, a primer pair for amplifying the upstream homology arm and a primer pair for amplifying the downstream homology arm were designed and synthesized by Invitrogen. The primer pair for amplifying the upstream homology arm consists of primer P5:5'-GTGCCAGTCATCCTTCAGGT-3' (SEQ ID No. 31) and primer P6:5'-CGTTCCGACCACTAACCCGACTTGGGTATTCCGAAATCTATCC-3' (SEQ ID No. 32). The primer pair for amplifying the downstream homology arm consists of primer P7:5'-GGATAGATTTCGGAATACCCAAGTCGGGTTAGTGGTCGGAACG-3' (SEQ ID No. 33) and primer P8:5'-AAGCGATGCTGAAAGGTGTC-3' (SEQ ID No. 34).

[0178] (2) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P5 and primer P6, and the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 765 bp upstream homology arm was recovered using a DNA recovery kit.

[0179] (3) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P7 and primer P8, and the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 643 bp downstream homology arm was recovered using a DNA recovery kit.

[0180] (4) The upstream homology arm recovered in step (2) and the downstream homology arm recovered in step (3) were mixed, and using this as a template, overlap PCR was performed using a primer pair consisting of primer P5 and primer P8 to obtain the ΔadhE-Up-Down fragment shown in SEQ ID No. 5. SEQ ID No.5: ΔadhE-Up-Down(1365bp)

[0181] 3. Obtaining the YPVal-pflB01-Cas9 strain (1) Plasmid pREDCas9 was transformed into competent cells of the genetically modified bacterium YPVal-pflB01, and then spread onto 2-YT agar plates containing 100 mg / L spectinomycin. The plates were then cultured at 32°C to obtain single colonies resistant to spectinomycin.

[0182] (2) Using the single colonies obtained in step (1) as templates, PCR amplification was performed using primer pairs consisting of primer pRedCas9-PF and primer pRedCas9-PR to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contains a DNA fragment having the nucleotide sequence shown in SEQ ID No. 3, then that colony contains plasmid pREDCas9. The strain of that colony was named YPVal-pflB01-Cas9 strain.

[0183] 4. Obtaining the genetically modified bacterium YPVal-pflB02 (1) Culture of YPVal-pflB01-Cas9 strain: YPVal-pflB01-Cas9 strain 600nm When the concentration reached 0.1 mM, IPTG was added to bring the IPTG concentration in the system to 0.1 mM, and the culture was continued to induce homologous recombination via λ-Red. The YPVal-pflB01-Cas9 strain was OD 600nm When the concentration reached 0.6, the bacterial cells were collected and competent cells of the YPVal-pflB01-Cas9 strain were prepared.

[0184] (2) Competent cells of the YPVal-pflB01-Cas9 strain were transformed with the pGRB-adhE sgRNA plasmid obtained in step 1 and the ΔadhE-Up-Down fragment obtained in step 2. These cells were then spread onto 2-YT agar plates containing 100 mg / L spectinomycin and 100 mg / L ampicillin, and cultured at 32°C to obtain single colonies.

[0185] (3) Using the single colonies obtained in step (2) as templates, PCR amplification was performed using primer pairs consisting of primer P5 and primer P8 to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contained a 1365 bp DNA fragment, that colony was provisionally determined to be a positive transformant.

[0186] (4) The positive transformants obtained in step (3) were inoculated onto 2-YT agar plates containing 100 mg / L spectinomycin and 0.2% (m / v) arabinose and cultured at 32°C (to remove the pGRB-adhE sgRNA plasmid). Subsequently, they were grown on 2-YT agar plates containing 100 mg / L spectinomycin, and colonies that did not grow on 2-YT agar plates containing 100 mg / L ampicillin were selected. These colonies were then subcultured on 2-YT agar plates and cultured at 42°C (to remove the pREDCas9 plasmid). Finally, colonies that did not grow on 2-YT agar plates containing 100 mg / L spectinomycin but grew on untreated 2-YT agar plates were selected.

[0187] (5) Using the single colony obtained in step (4) as a template, PCR amplification was performed using a primer pair consisting of primer P5 and primer P8 to obtain a PCR amplification product. If the PCR amplification product obtained from a single colony contained a 1365 bp DNA fragment, that colony was determined to be a positive transformant of the genetically modified bacterium YPVal-pflB01 in which the adhE gene was deleted from the genome. This positive transformant was named the genetically modified bacterium YPVal-pflB02.

[0188] 3. Obtaining the genetically modified bacterium YPVal-pflB03 Based on the genome sequences of Escherichia coli W3110 and Bacillus subtilis subsp. subtilis str.168 published by NCBI, the ilvE(E) gene in the genome of the genetically modified bacterium YPVal-pflB02 obtained in step two was knocked out using CRISPR / Cas9 genome editing technology, while simultaneously knocking in the ilvE gene of Bacillus subtilis driven by the ptrc promoter (i.e., the ptrc-ilvE(B) sequence), thereby obtaining the genetically modified bacterium YPVal-pflB03 (hereinafter sometimes abbreviated as YPVal-pflB03). The nucleotide sequence of the ptrc-ilvE(B) sequence is shown in SEQ ID No. 6. In SEQ ID No. 6, positions 1096-1169 from the 5' end are the nucleotide sequence of the ptrc promoter, and positions 1-1095 are the ilvE(B) gene of Bacillus subtilis. SEQ ID No.6:

[0189] The ilvE(E) gene of the genetically modified bacterium YPVal-pflB02 encodes a branched-chain amino acid transaminase, with Gene ID 948278 and amino acid sequence shown as SEQ ID No. 15. SEQ ID NO.15 (Branched-chain amino acid transaminase ilvE) MTTKKADYIWFNGEMVRWEDAKVHVMSHALHYGTSVFEGIRCYDSHKGPVVFRHREHMQRLHDSAKIYRFPVSQSIDELMEACRDVIRKNNLTSAYIRPLIFVGDVGMGVNPPAGYSTDVIIAAFPWGAYLGAEALEQGIDAMVSSWNRAAPNT IPTAAKAGGNYLSSLLVGSEARRHGYQEGIALDVNGYISEGAGENLFEVKDGVLFTPPFTSSALPGITRDAIIKLAKELGIEVREQVLSRESLYLADEVFMSGTAAEITPVRSVDGIQVGEGRCGPVTKRIQQAFFGLFTGETEDKWGWLDQVNQ

[0190] The ilvE(B) gene of Bacillus subtilis encodes a branched-chain amino acid transaminase, with Gene ID 938420 and amino acid sequence shown as SEQ ID No. 16. SEQ ID NO.16 (Branched-chain amino acid transaminase ilvE) MNKLIEREKTVYYKKDPSSLGFGQYFTDYMFVMDYEEGIGWHHPRIAPYAPLTLDPSSSVFHYGQAVFEGLKAYRTDDGRVLLFRPDQNIKRLNRSCERMSMPPLDEELVLEALTQLVELEKDWVPKEKGTSLYIRPFVIATEPSLGVKASRSYTFMIVLSPVGSYYGDDQLKPVR IYVEDEYVRAVNGGVGFAKTAGNYAASLQAQRKANELGYDQVLWLDAIEKKYVEEVGSMNIFFVINGEAVTPALSGSILSGVTRASAIELIRSWGIPVREERISIDEVYAASARGELTEVFGTGTAAVVTPVGELNIHGKTVIVGDGQIGDLSKKLYETITDIQLGKVKGPFNWTVEV

[0191] The specific steps are as follows: 1. Construction of the pGRB-ilvE sgRNA plasmid (1) Primer synthesized by Invitrogen: sgRNA-3F:5'- TGACAGCTAGCTCAGTCCTAGGTATAATACTAGT gaaagcagcgataatcacgtcgg GTTTTAGGCTAGAATAGCAAGTTAAAATAAGG -3' (shown in SEQ ID No. 35, with the underlined portion being the homology arm sequence of the pGRB vector), and primer sgRNA-3R:5'- CCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC ccgacgtgattatcgctgctttc ACTAGTATTATACCTAGGACTGAGCTAGCTGTCA -3' (shown in SEQ ID No. 36, with the underlined portion being the homology arm sequence of the pGRB vector)

[0192] (2) After annealing primer sgRNA-3F and primer sgRNA-3R (reaction program: denaturation at 95°C for 5 minutes, annealing at 50°C for 1 minute), the target fragment was recovered using a DNA purification kit, its DNA concentration was measured, and the concentration was diluted to 100 ng / μL to obtain the annealing product. The annealing product contained the nucleotide sequence: GAAAGCAGCGATAATCACGTCGG (shown in SEQ ID No. 7).

[0193] (3) The pGRB vector (Addgene product, catalog number 71539) was digested with the restriction enzyme SpeI (Takara product, catalog number 1631), and a DNA fragment of approximately 2700 bp was recovered using a DNA recovery kit (QIAGEN Gel Extraction Kit). The digestive system was 50 μL and consisted of 5 μL of 10× Buffer (with restriction enzyme SpeI), 2.5 μL of restriction enzyme SpeI, 3000-5000 ng of pGRB vector, and ddH2O. Digestion program: 3 hours at 37°C.

[0194] (4) The DNA fragments recovered in step (3) were subjected to a dephosphorylation reaction (to prevent self-ligation of the pGRB vector), recovered using a DNA recovery kit, and a linearized pGRB vector was obtained. The dephosphorylation system was 50 μL and consisted of 5 μL of 10× Buffer (included with CIAP), 1000-2000 ng of DNA fragments recovered in step (3), 2.5 μL of CIAP (Takara product, catalog number 2250A), and ddH2O. Dephosphorylation program: 1 hour at 37°C.

[0195] (5) Using the Gibson Assembly Kit (New England Biolabs), the linearized pGRB vector obtained in step (4) and the annealing product obtained in step (2) were recombined, and then transformed into E. coli DH5α competent cells (TAKARA) to obtain the pGRB-ilvE sgRNA plasmid. The recombinant system consisted of 5 μL of linearized pGRB vector (2 μL), annealing product (0.5 μL), and assembly enzyme (included in the Gibson Assembly Kit) (2.5 μL). Reassembly program: Assemble at 50°C for 30 minutes.

[0196] 2. Obtaining the ptrc-ilvE(B)-Up-Down fragment (1) Based on the genome sequence of Escherichia coli W3110 published by NCBI, primer pairs for amplifying the upstream homology arm and primer pairs for amplifying the downstream homology arm were designed and synthesized by Invitrogen. Based on the genome sequence of Bacillus subtilis subsp. subtilis str.168 published by NCBI, primer pairs for amplifying ptrc-ilvE(B) were designed and synthesized by Invitrogen. The primer pair for amplifying the upstream homology arm consists of primer P9:5'-CAGGCAGTTCATTGAGTTAGCG-3' (SEQ ID No. 37) and primer P10:5'-CACAGTGTATTAAGCAGACGTTAAATACAAAAAATGGGACGGCAC-3' (SEQ ID No. 38). The primer pair for amplifying the downstream homology arm is primer P13:5'- GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAA It consists of TTTTATATTCCTTTTGCGCTC-3' (shown as SEQ ID No. 39, with the underlined portion being part of the ptrc promoter sequence) and primer P14:5'-ACGGTTAGGGATGGTTCGAC-3' (SEQ ID No. 40). The primer pair for amplifying ptrc-ilvE(B) is primer P11:5'-GTGCCGTCCCATTTTTTGTATTTAACGTCTGCTTAATACACTGTG-3' (SEQ ID No. 41) and primer P12:5'- GTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACC It consists of ATGGAACTTTTTAAATATATGGAG-3' (shown as SEQ ID No. 42, with the underlined portion being part of the ptrc promoter sequence).

[0197] (2) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P9 and primer P10, and the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 709 bp upstream homology arm was recovered using a DNA recovery kit.

[0198] (3) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P13 and primer P14 with the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 611 bp downstream homology arm was recovered using a DNA recovery kit.

[0199] (4) Using the genomic DNA of Bacillus subtilis subsp. subtilis str.168 as a template, PCR amplification was performed using a primer pair consisting of primer P11 and primer P12, and the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 1168 bp ilvE(B) fragment was recovered using a DNA recovery kit.

[0200] (5) The upstream homology arm recovered in step (2), the downstream homology arm recovered in step (3), and the ilvE(B) fragment recovered in step (4) were mixed, and using this as a template, overlap PCR was performed using a primer pair consisting of primer P9 and primer P14 to obtain the ptrc-ilvE(B)-Up-Down fragment shown in SEQ ID No. 8. SEQ ID No.8: ptrc-ilvE(B)-Up-Down(2413bp)

[0201] 3. Obtaining the YPVal-pflB02-Cas9 strain (1) Plasmid pREDCas9 was transformed into competent cells of the genetically modified bacterium YPVal-pflB02, and then spread onto 2-YT agar plates containing 100 mg / L spectinomycin. The cells were cultured at 32°C to obtain single colonies resistant to spectinomycin.

[0202] (2) Using the single colonies obtained in step (1) as templates, PCR amplification was performed using primer pairs consisting of primer pRedCas9-PF and primer pRedCas9-PR to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contains a DNA fragment having the nucleotide sequence shown in SEQ ID No. 3, then that colony contains plasmid pREDCas9. The strain of that colony was named YPVal-pflB02-Cas9 strain.

[0203] 4. Obtaining the genetically modified bacterium YPVal-pflB03 (1) Culture of YPVal-pflB02-Cas9 strain: YPVal-pflB02-Cas9 strain is OD 600nm When the concentration reached 0.1 mM, IPTG was added to bring the IPTG concentration in the system to 0.1 mM, and the culture was continued to induce homologous recombination via λ-Red. The YPVal-pflB02-Cas9 strain was OD 600nm When the concentration reached 0.6, the bacterial cells were collected and competent cells of the YPVal-pflB02-Cas9 strain were prepared.

[0204] (2) Competent cells of the YPVal-pflB02-Cas9 strain were transformed with the pGRB-ilvE sgRNA plasmid obtained in step 1 and the ptrc-ilvE(B)-Up-Down fragment obtained in step 2. These cells were then spread onto 2-YT agar plates containing 100 mg / L spectinomycin and 100 mg / L ampicillin, and cultured at 32°C to obtain single colonies.

[0205] (3) Using the single colonies obtained in step (2) as templates, PCR amplification was performed using primer pairs consisting of primer P11 and primer P12 to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contained a DNA fragment of 1168 bp, that colony was provisionally determined to be a positive transformant.

[0206] (4) The positive transformants obtained in step (3) were inoculated onto 2-YT agar plates containing 100 mg / L spectinomycin and 0.2% (m / v) arabinose and cultured at 32°C (to remove the pGRB-ilvE sgRNA plasmid). Subsequently, they were grown on 2-YT agar plates containing 100 mg / L spectinomycin, and colonies that did not grow on 2-YT agar plates containing 100 mg / L ampicillin were selected. These colonies were then subcultured on 2-YT agar plates and cultured at 42°C (to remove the pREDCas9 plasmid). Finally, colonies that did not grow on 2-YT agar plates containing 100 mg / L spectinomycin but grew on untreated 2-YT agar plates were selected.

[0207] (5) Using the single colony obtained in step (4) as a template, PCR amplification was performed using a primer pair consisting of primer P11 and primer P12 to obtain a PCR amplification product. If the PCR amplification product obtained from a single colony contained a 1168 bp DNA fragment, that colony was determined to be a positive transformant. This positive transformant was determined to be a genetically modified bacterium YPVal-pflB03 in which the ilvE(E) gene in the genome of the genetically modified bacterium YPVal-pflB02 was knocked out, and at the same time, the ilvE(B) gene of Bacillus subtilis driven by the ptrc promoter (i.e., the ptrc-ilvE(B) sequence) was knocked in.

[0208] IV. Obtaining the genetically modified bacterium YPVal-pflB04 Using the genetically modified bacterium YPVal-pflB03 as a starting cell, the thiE gene in the genome of the genetically modified bacterium YPVal-pflB03 was knocked out using CRISPR / Cas9 genome editing technology, based on the genome sequence of Escherichia coli W3110 published by NCBI.

[0209] The thiE gene encodes thiamine phosphate synthase, has a Gene ID of 948491, and its amino acid sequence is shown as SEQ ID No. 17. SEQ ID NO.17 (Thiamine Phosphate Synthase thiE) MYQPDFPPVPFRSGLYPVVDSVQWIERLLDAGVRTLQLRIKDRRDEEVEADVVAAIALGRRYNARLFINDYWRLAIKHQAYGVHLGQEDLQATDLNAIRAAGLRL GVSTHDDMEIDVALAARPSYIALGHVFPTQTKQMPSAPQGLEQLARHVERLADYPTVAIGGISLARAPAVIATGVGSIAVVSAITQAADWRLATAQLLEIAGVGDE

[0210] The specific steps are as follows: 1. Construction of the pGRB-thiE sgRNA plasmid Based on the Escherichia coli W3110 genome sequence published by NCBI, a target sequence for sgRNA to knock out the thiE gene, CGCCCCTCTTATATCGCGCTGGG (shown as SEQ ID No. 9), was designed using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ). Homology arm sequences of a linearized pGRB vector for constructing the sgRNA plasmid were added to the 5' and 3' ends of the target sequence.

[0211] (1) Primer synthesized by Invitrogen: sgRNA-4F:5'- TGACAGCTAGCTCAGTCCTAGGTATAATACTAGT cgcccctcttatatcgcgctggg GTTTTAGGCTAGAATAGCAAGTTAAAATAAGG -3' (shown in SEQ ID No. 43, with the underlined portion being the homology arm sequence of the pGRB vector), and primer sgRNA-4R:5'- CCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC cccagcgcgatataagaggggcg ACTAGTATTATACCTAGGACTGAGCTAGCTGTCA -3' (shown in SEQ ID No. 44, with the underlined portion being the homology arm sequence of the pGRB vector)

[0212] (2) After annealing primer sgRNA-4F and primer sgRNA-4R (reaction program: denaturation at 95°C for 5 minutes, annealing at 50°C for 1 minute), the target fragment was recovered using a DNA purification kit, its DNA concentration was measured, and the concentration was diluted to 100 ng / μL to obtain the annealing product. The annealing product contained an sgRNA-4 fragment having the nucleotide sequence shown in SEQ ID No. 9.

[0213] (3) The pGRB vector was digested with restriction enzyme SpeI, and a DNA fragment of approximately 2700 bp was recovered using a DNA recovery kit (QIAGEN Gel Extraction Kit). The digestive system was 50 μL and consisted of 5 μL of 10× Buffer (with restriction enzyme SpeI), 2.5 μL of restriction enzyme SpeI, 3000-5000 ng of pGRB vector, and ddH2O. Digestion program: 3 hours at 37°C.

[0214] (4) The DNA fragments recovered in step (3) were subjected to a dephosphorylation reaction (to prevent self-ligation of the pGRB vector), recovered using a DNA recovery kit, and a linearized pGRB vector was obtained. The dephosphorylation system was 50 μL and consisted of 5 μL of 10×Buffer (provided with CIAP), 1000-2000 ng of DNA fragments recovered in step (3), 2.5 μL of CIAP, and ddH2O. Dephosphorylation program: 1 hour at 37°C.

[0215] (5) Using the Gibson Assembly Kit, the linearized pGRB vector obtained in step (4) and the annealing product obtained in step (2) were recombined, and then transformed into E. coli DH5α competent cells to obtain the pGRB-thiE sgRNA plasmid. The recombinant system consisted of 5 μL of linearized pGRB vector (2 μL), annealing product (0.5 μL), and assembly enzyme (included in the Gibson Assembly Kit) (2.5 μL). Reassembly program: Assemble at 50°C for 30 minutes.

[0216] 2. Obtaining the ΔthiE-Up-Down fragment (1) Based on the genome sequence of Escherichia coli W3110 published by NCBI, a primer pair for amplifying the upstream homology arm and a primer pair for amplifying the downstream homology arm were designed and synthesized by Invitrogen. The primer pair for amplifying the upstream homology arm consists of primer P15:5'-TTCTATTCAGGACGCCAACG-3' (SEQ ID No. 45) and primer P16:5'-GCTATAACGCATAAAGTCACGGCACGCTTCCTCCTTACGCAGG-3' (SEQ ID No. 46). The primer pair for amplifying the downstream homology arm consists of primer P17:5'-CCTGCGTAAGGAGGAAGCGTGCCGTGACTTTATGCGTTATAGC-3' (SEQ ID No. 47) and primer P18:5'-GCCTGCAAAGTGCCCATAACCC-3' (SEQ ID No. 48).

[0217] (2) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P15 and primer P16, and the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 721 bp upstream homology arm was recovered using a DNA recovery kit.

[0218] (3) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P17 and primer P18 with the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 618 bp downstream homology arm was recovered using a DNA recovery kit.

[0219] (4) The upstream homology arm recovered in step (2) and the downstream homology arm recovered in step (3) were mixed, and using this as a template, overlap PCR was performed using a primer pair consisting of primer P15 and primer P18 to obtain the ΔthiE-Up-Down fragment shown in SEQ ID No. 10. SEQ ID No.10: ΔthiE-Up-Down(1296bp)

[0220] 3. Obtaining the YPVal-pflB03-Cas9 strain (1) Plasmid pREDCas9 was transformed into competent cells of the genetically modified bacterium YPVal-pflB03, and then spread onto 2-YT agar plates containing 100 mg / L spectinomycin. The cells were cultured at 32°C to obtain single colonies resistant to spectinomycin.

[0221] (2) Using the single colonies obtained in step (1) as templates, PCR amplification was performed using primer pairs consisting of primer pRedCas9-PF and primer pRedCas9-PR to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contains a DNA fragment having the nucleotide sequence shown in SEQ ID No. 3, then that colony contains plasmid pREDCas9. The strain of that colony was named YPVal-pflB03-Cas9 strain.

[0222] 4. Obtaining the genetically modified bacterium YPVal-pflB04 (1) Culture of YPVal-pflB03-Cas9 strain: YPVal-pflB03-Cas9 strain 600nm When the concentration reached 0.1 mM, IPTG was added to bring the IPTG concentration in the system to 0.1 mM, and the culture was continued to induce homologous recombination via λ-Red. The YPVal-pflB03-Cas9 strain was OD 600nm When the concentration reached 0.6, the bacterial cells were collected and competent cells of the YPVal-pflB03-Cas9 strain were prepared.

[0223] (2) Competent cells of the YPVal-pflB03-Cas9 strain were transformed with the pGRB-thiE sgRNA plasmid obtained in step 1 and the ΔthiE-Up-Down fragment obtained in step 2. These cells were then spread onto 2-YT agar plates containing 100 mg / L spectinomycin and 100 mg / L ampicillin, and cultured at 32°C to obtain single colonies.

[0224] (3) Using the single colonies obtained in step (2) as templates, PCR amplification was performed using primer pairs consisting of primer P15 and primer P18 to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contained a 1296 bp DNA fragment, that colony was provisionally determined to be a positive transformant.

[0225] (4) The positive transformants obtained in step (3) were inoculated onto 2-YT agar plates containing 100 mg / L spectinomycin and 0.2% (m / v) arabinose and cultured at 32°C (to remove the pGRB-thiE sgRNA plasmid). Subsequently, they were grown on 2-YT agar plates containing 100 mg / L spectinomycin, and colonies that did not grow on 2-YT agar plates containing 100 mg / L ampicillin were selected. These colonies were then subcultured on 2-YT agar plates and cultured at 42°C (to remove the pREDCas9 plasmid). Finally, colonies that did not grow on 2-YT agar plates containing 100 mg / L spectinomycin but grew on untreated 2-YT agar plates were selected.

[0226] (5) Using the single colony obtained in step (4) as a template, PCR amplification was performed using a primer pair consisting of primer P15 and primer P18 to obtain a PCR amplification product. If the PCR amplification product obtained from a single colony contained a 1296 bp DNA fragment, that colony was determined to be a positive transformant of the genetically modified bacterium YPVal-pflB03 in which the thiE gene was deleted from the genome. This positive transformant was named the genetically modified bacterium YPVal-pflB04.

[0227] 5. Obtaining the genetically modified bacterium YPVal-pflB05 Using the genetically modified bacterium YPVal-pflB04 as the starting cell, the mdh gene in the genome of the genetically modified bacterium YPVal-pflB04 was knocked out using CRISPR / Cas9 genome editing technology, based on the genome sequence of Escherichia coli W3110 published by NCBI.

[0228] The mdh gene encodes maleate dehydrogenase, has a Gene ID of 947854, and its amino acid sequence is shown as SEQ ID No. 18. SEQ ID NO.18 (Malate dehydrogenase mdh) MKVAVLGAAGGIGQALALLLKTQLPSGSELSLYDIAPVTPGVAVDLSHIPTAVKIKGFSGEDATPALEGADVVLISAGVARKPGMDRSDLLFNVNAGIVKNLVQQVAKTCPKACIGIITNPVNTTVAIAAEVLKKAGVYDKNKLFGVTTLDIIRSNT FVAELKGKQPGEVEVPVIGGHSGVTILPLLSQVPGVSFTEQEVADLTKRIQNAGTEVVEAKAGGGSATLSMGQAAARFGLSLVRALQGEQGVVECAYVEGDGQYARFFSQPLLLGKNGVEERKSIGTLSAFEQNALEGMLDTLKKDIALGEEFVNK

[0229] The specific steps are as follows: 1. Construction of the pGRB-mdh sgRNA plasmid Based on the Escherichia coli W3110 genome sequence published by NCBI, a target sequence for sgRNA to knock out the mdh gene, GCCTTTCAGTTCCGCAACAAAGG (shown as SEQ ID No. 11), was designed using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ). Homology arm sequences of a linearized pGRB vector for constructing the sgRNA plasmid were added to the 5' and 3' ends of the target sequence.

[0230] (1) Primer synthesized by Invitrogen: sgRNA-5F:5'- TGACAGCTAGCTCAGTCCTAGGTATAATACTAGT gcctttcagttccgcaacaaagg GTTTTAGGCTAGAATAGCAAGTTAAAATAAGG-3’ (as shown in SEQ ID No. 49, the underlined part is the homology arm sequence of the pGRB vector), and primer sgRNA-5R: 5’- CCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC cctttgttgcggaactgaaaggc ACTAGTATTATACCTAGGACTGAGCTAGCTGTCA -3’ (as shown in SEQ ID No. 50, the underlined part is the homology arm sequence of the pGRB vector).

[0231] (2) After annealing primer sgRNA-5F and primer sgRNA-5R (reaction program: denaturation at 95°C for 5 minutes, annealing at 50°C for 1 minute), the target fragment was recovered using a DNA purification kit. After measuring its DNA concentration, it was diluted to 100 ng / μL to obtain an annealing product. The annealing product contains the sgRNA-5 fragment having the nucleotide sequence shown in SEQ ID No. 11.

[0232] (3) The pGRB vector was digested with the restriction enzyme SpeI, and a DNA fragment of about 2700 bp was recovered using a DNA recovery kit (QIAGEN Gel Extraction Kit). The digestion system was 50 μL, composed of 5 μL of 10× Buffer (attached to the restriction enzyme SpeI), 2.5 μL of the restriction enzyme SpeI, 3000 - 5000 ng of the pGRB vector, and ddH2O. Digestion program: 37°C for 3 hours.

[0233] (4) The DNA fragment recovered in step (3) was subjected to a dephosphorylation reaction (to prevent self-ligation of the pGRB vector), recovered using a DNA recovery kit, and a linearized pGRB vector was obtained. The dephosphorylation system was 50 μL, composed of 5 μL of 10× Buffer (attached to CIAP), 1000 - 2000 ng of the DNA fragment recovered in step (3), 2.5 μL of CIAP, and ddH2O. Dephosphorylation program: 37°C for 1 hour.

[0234] (5) Using the Gibson Assembly kit, the linearized pGRB vector obtained in step (4) and the annealing product obtained in step (2) were recombined, and then transformed into Escherichia coli DH5α competent cells to obtain the pGRB-mdh sgRNA plasmid. The recombination system was 5 μL, composed of 2 μL of the linearized pGRB vector, 0.5 μL of the annealing product, and 2.5 μL of the assembly enzyme (attached to the Gibson Assembly kit). Recombination program: Assembly at 50 °C for 30 minutes.

[0235] 2. Obtaining the Δmdh-Up-Down fragment (1) Based on the genomic sequence of Escherichia coli W3110 published on NCBI, primer pairs for amplifying the upstream homology arm and primer pairs for amplifying the downstream homology arm were designed and synthesized by Invitrogen. The primer pair for amplifying the upstream homology arm consists of primer P19: 5'-AACTTCCTCCAAACCGATGC-3' (SEQ ID No. 51) and primer P20: 5'-CAATATAATAAGGAGTTTAGGTTGATTAGCGGATAATAAAAAACC-3' (SEQ ID No. 52). The primer pair for amplifying the downstream homology arm consists of primer P21: 5'-GGTTTTTTATTATCCGCTAATCAACCTAAACTCCTTATTATATTG-3' (SEQ ID No. 53) and primer P22: 5'-TCTTCAATGGACTGGAGGTG-3' (SEQ ID No. 54).

[0236] (2) Using the genomic DNA of Escherichia coli W3110 as a template, PCR amplification was performed using the primer pair consisting of primer P19 and primer P20, and the high-fidelity amplification enzyme KAPA HiFi HotStart, and the 590-bp upstream homology arm was recovered using a DNA recovery kit.

[0237] (3) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P21 and primer P22, and the high-fidelity amplification enzyme KAPA HiFi HotStart. A 708 bp downstream homology arm was recovered using a DNA recovery kit.

[0238] (4) The upstream homology arm recovered in step (2) and the downstream homology arm recovered in step (3) were mixed, and using this as a template, overlap PCR was performed using a primer pair consisting of primer P19 and primer P22 to obtain the Δmdh-Up-Down fragment shown in SEQ ID No. 12. SEQ ID No.12: Δmdh-Up-Down(1253bp)

[0239] 3. Obtaining the YPVal-pflB04-Cas9 Strain (1) After transforming the plasmid pREDCas9 into the genetically engineered bacterium YPVal-pflB04 competent cells, it was spread on a 2-YT agar plate containing 100 mg / L spectinomycin and cultured at 32 °C to obtain single colonies showing resistance to spectinomycin.

[0240] (2) Using each of the single colonies obtained in step (1) as a template, PCR amplification was performed using the primer pair consisting of primer pRedCas9-PF and primer pRedCas9-PR to obtain a PCR amplification product. If the PCR amplification product obtained from a certain single colony contains a DNA fragment having the nucleotide sequence shown in SEQ ID No. 3, the colony contains the plasmid pREDCas9. The strain of this colony was named the YPVal-pflB04-Cas9 strain.

[0241] 4. Obtaining the Genetically Engineered Bacterium YPVal-pflB05 (1) Culturing of the YPVal-pflB04-Cas9 strain: When the OD of the YPVal-pflB04-Cas9 strain reached 0.1, IPTG was added so that the IPTG concentration in the system became 0.1 mM, and the culture was continued to induce homologous recombination via λ-Red. When the OD of the YPVal-pflB04-Cas9 strain reached 0.6, the cells were collected and YPVal-pflB04-Cas9 strain competent cells were prepared. 600nm 600nm

[0242] (2) The pGRB-mdh sgRNA plasmid obtained in step 1 and the Δmdh-Up-Down fragment obtained in step 2 were transformed into the YPVal-pflB04-Cas9 strain competent cells, spread on a 2-YT agar plate containing 100 mg / L spectinomycin and 100 mg / L ampicillin, and cultured at 32 °C to obtain single colonies.

[0243] ​​(3) Using the single colonies obtained in step (2) as templates, PCR amplification was performed using primer pairs consisting of primer P19 and primer P22 to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contained a 1253 bp DNA fragment, that colony was provisionally determined to be a positive transformant.

[0244] (4) The positive transformants obtained in step (3) were inoculated onto 2-YT agar plates containing 100 mg / L spectinomycin and 0.2% (m / v) arabinose and cultured at 32°C (to remove the pGRB-thiE sgRNA plasmid). Subsequently, they were grown on 2-YT agar plates containing 100 mg / L spectinomycin, and colonies that did not grow on 2-YT agar plates containing 100 mg / L ampicillin were selected. These colonies were then subcultured on 2-YT agar plates and cultured at 42°C (to remove the pREDCas9 plasmid). Finally, colonies that did not grow on 2-YT agar plates containing 100 mg / L spectinomycin but grew on untreated 2-YT agar plates were selected.

[0245] (5) Using the single colony obtained in step (4) as a template, PCR amplification was performed using a primer pair consisting of primer P19 and primer P22 to obtain a PCR amplification product. If the PCR amplification product obtained from a single colony contained a 1253 bp DNA fragment, that colony was determined to be a positive transformant of the genetically modified bacterium YPVal-pflB04 in which the mdh gene was deleted from the genome. This positive transformant was named the genetically modified bacterium YPVal-pflB05.

[0246] Example 2: Modification of genetically engineered bacteria starting from E. coli W3110 The inventor of the present invention conducted a number of experiments, modified Escherichia coli W3110 as the starting strain, and obtained genetically engineered bacteria YPVal-pflB06, YPVal-pflB07, YPVal-pflB08, YPVal-pflB09, and YPVal-pflB10. The genotypes of the genetically engineered bacteria YPVal-pflB06, YPVal-pflB07, YPVal-pflB08, YPVal-pflB09, and YPVal-pflB10 are shown in Table 2.

[0247] TIFF2026521933000002.tif55170

[0248] I. Obtaining the genetically engineered bacterium YPVal-pflB06 1. Construction of the pGRB-pflB sgRNA plasmid It is the same as 1 in Step 1 of Example 1.

[0249] 2. Obtaining the ΔpflB-Up-Down fragment It is the same as 2 in Step 1 of Example 1.

[0250] 3. Obtaining the W3110-Cas9 strain According to the steps of 3 in Step 1 of Example 1, the valine-producing bacterium CGMCC 22721 competent cells were replaced with Escherichia coli W3110 competent cells, and the other steps remained the same to obtain the W3110-Cas9 strain.

[0251] 4. Obtaining the genetically engineered bacterium YPVal-pflB06 According to the steps of 4 in Step 1 of Example 1, the CGMCC 22721-Cas9 strain was replaced with the W3110-Cas9 strain, and the other steps remained the same to obtain the genetically engineered bacterium YPVal-pflB06.

[0252] [[ID=:33]] II. Obtaining the genetically engineered bacterium YPVal-pflB07 1. Construction of the pGRB-adhE sgRNA plasmid It is the same as 1 in Step 2 of Example 1.

[0253] 2. Obtaining the ΔadhE-Up-Down fragment This is the same as step 2 in Example 1.

[0254] 3. Obtaining the YPVal-pflB06-Cas9 strain In accordance with step 3 of step 2 of Example 1, the genetically modified YPVal-pflB01 competent cells were replaced with genetically modified YPVal-pflB06 competent cells, and the other steps were carried out as is to obtain the YPVal-pflB06-Cas9 strain.

[0255] 4. Obtaining the genetically modified bacterium YPVal-pflB07 In accordance with step 4 of step 2 of Example 1, the YPVal-pflB01-Cas9 strain was replaced with the YPVal-pflB06-Cas9 strain, and the other steps were carried out as is to obtain the genetically modified bacterium YPVal-pflB07.

[0256] 3. Obtaining the genetically modified bacterium YPVal-pflB08 1. Construction of the pGRB-ilvE sgRNA plasmid This is the same as step 3 of Example 1.

[0257] 2. Obtaining the ptrc-ilvE(B)-Up-Down fragment This is the same as step 2 in step 3 of Example 1.

[0258] 3. Obtaining the YPVal-pflB07-Cas9 strain In accordance with step 3 of step 3 of Example 1, the genetically modified YPVal-pflB02 competent cells were replaced with genetically modified YPVal-pflB07 competent cells, and the other steps were carried out as is to obtain the YPVal-pflB07-Cas9 strain.

[0259] 4. Obtaining the genetically modified bacterium YPVal-pflB08 In accordance with step 4 of step 3 of Example 1, the YPVal-pflB02-Cas9 strain was replaced with the YPVal-pflB07-Cas9 strain, and the other steps were carried out as is to obtain the genetically modified bacterium YPVal-pflB08.

[0260] IV. Obtaining the genetically modified bacterium YPVal-pflB09 1. Construction of the pGRB-thiE sgRNA plasmid This is the same as step 4 of Example 1.

[0261] 2. Obtaining the ΔthiE-Up-Down fragment This is the same as step 2 in step four of Example 1.

[0262] 3. Obtaining the YPVal-pflB08-Cas9 strain In accordance with step 3 of step 4 of Example 1, the genetically modified YPVal-pflB03 competent cells were replaced with genetically modified YPVal-pflB08 competent cells, and the other steps were carried out as is to obtain the YPVal-pflB08-Cas9 strain.

[0263] 4. Obtaining the genetically modified bacterium YPVal-pflB09 In accordance with step 4 of step 4 of Example 1, the YPVal-pflB03-Cas9 strain was replaced with the YPVal-pflB08-Cas9 strain, and the other steps were carried out as is to obtain the genetically modified bacterium YPVal-pflB09.

[0264] 5. Obtaining the genetically modified bacterium YPVal-pflB10 1. Construction of the pGRB-mdh sgRNA plasmid This is the same as step 5 of Example 1.

[0265] 2. Obtaining the Δmdh-Up-Down fragment This is the same as step 2 in step 5 of Example 1.

[0266] 3. Obtaining the YPVal-pflB08-Cas9 strain In accordance with step 3 of step 5 of Example 1, the genetically modified YPVal-pflB04 competent cells were replaced with genetically modified YPVal-pflB09 competent cells, and the other steps were carried out as is to obtain the YPVal-pflB09-Cas9 strain.

[0267] 4. Obtaining the genetically modified bacterium YPVal-pflB10 In accordance with step 4 of step 5 of Example 1, the YPVal-pflB04-Cas9 strain was replaced with the YPVal-pflB09-Cas9 strain, and the other steps were carried out as is to obtain the genetically modified bacterium YPVal-pflB10.

[0268] Example 3: L-valine is produced by fermentation of genetically modified bacteria obtained in Examples 1 and 2. 1. The genetically modified bacteria YPVal-pflB01, YPVal-pflB02, YPVal-pflB03, YPVal-pflB04, YPVal-pflB05, YPVal-pflB06, YPVal-pflB07, YPVal-pflB08, YPVal-pflB09, and YPVal-pflB10 obtained by modification in Examples 1 and 2, as well as the starting bacteria, the valine-producing bacterium CGMCC 22721 and Escherichia coli W3110, were each fermented in a fermentation tank (Shanghai Bailun Biotechnology Co., Ltd., model BLBIO-5GC-4-H) to obtain fermentation liquid.

[0269] Each strain was fermented by repeating the process three times. The components of the fermentation medium used during fermentation are shown in Table 3. The fermentation control process is shown in Table 4.

[0270] TIFF2026521933000003.tif97170

[0271] TIFF2026521933000004.tif124170

[0272] 2. High-performance liquid chromatography analysis of L-valine production in each fermentation broth. Table 5 shows the results of three fermentation tests using the valine-producing bacterium CGMCC 22721 and genetically modified strains YPVal-pflB01, YPVal-pflB02, YPVal-pflB03, YPVal-pflB04, and YPVal-pflB05 derived from it (a P-value < 0.01 indicates a highly statistically significant difference). The results show that all of YPVal-pflB01, YPVal-pflB02, YPVal-pflB03, YPVal-pflB04, and YPVal-pflB05 can increase L-valine production compared to the valine-producing bacterium CGMCC 22721. In other words, in the valine-producing bacterium CGMCC 22721, genetically modified bacteria obtained by "knockout of the pflB gene," "knockout of the pflB gene and adhE gene," "knockout of the pflB gene and adhE gene, and knockout of the ilvE(E) gene, simultaneously with insertion of ilvE(B) derived from Bacillus subtilis driven by the ptrc promoter," "knockout of the pflB gene, adhE gene, and thiE gene, and knockout of the ilvE(E) gene, simultaneously with insertion of ilvE(B) derived from Bacillus subtilis driven by the ptrc promoter," or "knockout of the pflB gene, adhE gene, thiE gene, and mdh gene, and knockout of the ilvE gene(E), simultaneously with insertion of ilvE(B) derived from Bacillus subtilis driven by the ptrc promoter" can all significantly increase the production of L-valine.

[0273] TIFF2026521933000005.tif73170

[0274] Table 6 shows the results of three fermentation tests using E. coli W3110 and genetically modified strains YPVal-pflB06, YPVal-pflB07, YPVal-pflB08, YPVal-pflB09, and YPVal-pflB10 derived from it (a P-value < 0.01 indicates a highly statistically significant difference). The results show that all of YPVal-pflB06, YPVal-pflB07, YPVal-pflB08, YPVal-pflB09, and YPVal-pflB10 can improve L-valine production compared to E. coli W3110. In other words, in E. coli W3110, genetically modified bacteria obtained by "knockout of the pflB gene," "knockout of the pflB gene and adhE gene," "knockout of the pflB gene and adhE gene, and simultaneous knockout of the ilvE(E) gene with insertion of ilvE(B) derived from Bacillus subtilis driven by the ptrc promoter," "knockout of the pflB gene, adhE gene, and thiE gene, and simultaneous knockout of the ilvE(E) gene with insertion of ilvE(B) derived from Bacillus subtilis driven by the ptrc promoter," or "knockout of the pflB gene, adhE gene, thiE gene, and mdh gene, and simultaneous knockout of the ilvE(E) gene with insertion of ilvE(B) derived from Bacillus subtilis driven by the ptrc promoter" can all significantly increase L-valine production.

[0275] TIFF2026521933000006.tif73170

[0276] Example 4 Modification of a genetically engineered bacterium starting from the valine-producing bacterium CGMCC 22721 The inventors of this invention conducted numerous experiments and modified the valine-producing bacterium CGMCC 22721 as a starting strain to obtain genetically modified bacteria YPVal-adhE01, YPVal-adhE02, YPVal-adhE03, and YPVal-adhE04. The genotypes of the genetically modified bacteria YPVal-adhE01, YPVal-adhE02, YPVal-adhE03, and YPVal-adhE04 are shown in Table 7.

[0277] TIFF2026521933000007.tif47170

[0278] 1. Obtaining the genetically modified bacterium YPVal-adhE01 Based on the genome sequence of Escherichia coli W3110 published by NCBI, the adhE gene in the genome of the valine-producing bacterium CGMCC 22721 was knocked out using CRISPR / Cas9 genome editing technology.

[0279] The adhE gene encodes alcohol dehydrogenase, has a Gene ID of 945837, and its amino acid sequence is shown as SEQ ID No. 14.

[0280] The specific steps are as follows: 1. Construction of the pGRB-adhE sgRNA plasmid Based on the Escherichia coli W3110 genome sequence published by NCBI, a target sequence for sgRNA to knock out the adhE gene (shown as SEQ ID No. 4) was designed using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ), and homology arm sequences of a linearized pGRB vector for constructing the sgRNA plasmid were added to the 5' and 3' ends of the target sequence.

[0281] (1) Primer synthesized by Invitrogen: sgRNA-2F:5'- TGACAGCTAGCTCAGTCCTAGGTATAATACTAGT aagaaccacaacccagagtcagg GTTTTAGGCTAGAATAGCAAGTTAAAATAAGG -3' (shown in SEQ ID No. 29, the underlined portion is the homology arm sequence of the pGRB vector), primer sgRNA-2R: 5'- CCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC cctgactctgggttgtggttctt ACTAGTATTATACCTAGGACTGAGCTAGCTGTCAThese are the -3' (shown as SEQ ID No. 30, with the underlined portion being the homology arm sequence of the pGRB vector), the primer sgRNA-PF:5'-GTCTCATGAGCGGATACATATTTG-3' (SEQ ID No. 21), and the primer sgRNA-PR:5'-ATGAGAAAGCGCCACGCT-3' (SEQ ID No. 22).

[0282] (2) After annealing primer sgRNA-2F and primer sgRNA-2R (reaction program: denaturation at 95°C for 5 minutes, annealing at 50°C for 1 minute), the target fragment was recovered using a DNA purification kit, its DNA concentration was measured, and the concentration was diluted to 100 ng / μL to obtain the annealing product. The annealing product contained an sgRNA-2 fragment having the nucleotide sequence shown in SEQ ID No. 4.

[0283] (3) The pGRB vector was digested with restriction enzyme SpeI, and a DNA fragment of approximately 2700 bp was recovered using a DNA recovery kit (QIAGEN Gel Extraction Kit). The digestive system was 50 μL and consisted of 5 μL of 10×Buffer (with restriction enzyme SpeI), 2.5 μL of restriction enzyme SpeI, 3000-5000 ng of pGRB vector, and ddH2O. Digestion program: 3 hours at 37°C.

[0284] (4) The DNA fragments recovered in step (3) were subjected to a dephosphorylation reaction (to prevent self-ligation of the pGRB vector), recovered using a DNA recovery kit, and a linearized pGRB vector was obtained. The dephosphorylation system was 50 μL and consisted of 5 μL of 10× Buffer (included with CIAP), 1000-2000 ng of DNA fragments recovered in step (3), 2.5 μL of CIAP (Takara product, catalog number 2250A), and ddH2O. Dephosphorylation program: 1 hour at 37°C.

[0285] (5) Using the Gibson Assembly Kit (New England Biolabs), the linearized pGRB vector obtained in step (4) and the annealing product obtained in step (2) were recombined, and then transformed into E. coli DH5α competent cells (TAKARA) to obtain the pGRB-adhE sgRNA plasmid. The recombinant system consisted of 5 μL of linearized pGRB vector (2 μL), annealing product (0.5 μL), and assembly enzyme (included in the Gibson Assembly Kit) (2.5 μL). Reassembly program: Assemble at 50°C for 30 minutes.

[0286] 2. Obtaining the ΔadhE-Up-Down fragment (1) Based on the genome sequence of Escherichia coli W3110 published by NCBI, a primer pair for amplifying the upstream homology arm and a primer pair for amplifying the downstream homology arm were designed and synthesized by Invitrogen. The primer pair for amplifying the upstream homology arm consists of primer P5:5'-GTGCCAGTCATCCTTCAGGT-3' (SEQ ID No. 31) and primer P6:5'-CGTTCCGACCACTAACCCGACTTGGGTATTCCGAAATCTATCC-3' (SEQ ID No. 32). The primer pair for amplifying the downstream homology arm consists of primer P7:5'-GGATAGATTTCGGAATACCCAAGTCGGGTTAGTGGTCGGAACG-3' (SEQ ID No. 33) and primer P8:5'-AAGCGATGCTGAAAGGTGTC-3' (SEQ ID No. 34).

[0287] (2) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P5 and primer P6, and the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 765 bp upstream homology arm was recovered using a DNA recovery kit.

[0288] (3) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P7 and primer P8, and the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 643 bp downstream homology arm was recovered using a DNA recovery kit.

[0289] (4) The upstream homology arm recovered in step (2) and the downstream homology arm recovered in step (3) were mixed, and using this as a template, overlap PCR was performed using a primer pair consisting of primer P5 and primer P8 to obtain the ΔadhE-Up-Down fragment shown in SEQ ID No. 5.

[0290] 3. Acquisition of CGMCC 22721-Cas9 strain (1) Plasmid pREDCas9 (Addgene, catalog number 71541; containing the spectinomycin resistance gene) was used to transform valine-producing bacteria CGMCC 22721 competent cells. These cells were then spread onto 2-YT agar plates containing 100 mg / L spectinomycin and cultured at 32°C to obtain single colonies resistant to spectinomycin. The preparation method for 2-YT agar plates is as follows: Dissolve 16g of tryptone, 10g of yeast extract, 5g of sodium chloride, and 16g of agar in an appropriate amount of water, add water to a total volume of 1L, adjust the pH to 7.0 with sodium hydroxide, and sterilize at 121°C for 20 minutes.

[0291] (2) Using the single colonies obtained in step (1) as templates, PCR amplification was performed using primer pairs consisting of primer pRedCas9-PF:5'-GCAGTGGCGGTTTTCATG-3' (SEQ ID No. 27) and primer pRedCas9-PR:5'-CCTTGGTGATCTCGCCTTTC-3' (SEQ ID No. 28) to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contains a DNA fragment having the nucleotide sequence shown in SEQ ID No. 3, the colony is considered to contain plasmid pREDCas9. The strain of the colony was named CGMCC 22721-Cas9 strain.

[0292] 4. Obtaining the genetically modified bacterium YPVal-adhE01 (1) Culture of CGMCC 22721-Cas9 strain: CGMCC 22721-Cas9 strain is OD 600nm When the concentration reached 0.1 mM, IPTG was added to bring the IPTG concentration in the system to 0.1 mM, and the culture was continued to induce homologous recombination via λ-Red. The CGMCC 22721-Cas9 strain was OD 600nm When the concentration reached 0.6, the bacterial cells were collected and CGMCC 22721-Cas9 competent cells were prepared.

[0293] (2) CGMCC 22721-Cas9 competent cells were transformed with the pGRB-adhE sgRNA plasmid obtained in step 1 and the ΔadhE-Up-Down fragment obtained in step 2. These cells were then spread onto 2-YT agar plates containing 100 mg / L spectinomycin and 100 mg / L ampicillin, and cultured at 32°C to obtain single colonies.

[0294] (3) Using the single colonies obtained in step (2) as templates, PCR amplification was performed using primer pairs consisting of primer P5 and primer P8 to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contained a 1365 bp DNA fragment, that colony was provisionally determined to be a positive transformant.

[0295] (4) The positive transformants obtained in step (3) were inoculated onto 2-YT agar plates containing 100 mg / L spectinomycin and 0.2% (m / v) arabinose and cultured at 32°C (to remove the pGRB-adhE sgRNA plasmid). Subsequently, they were grown on 2-YT agar plates containing 100 mg / L spectinomycin, and colonies that did not grow on 2-YT agar plates containing 100 mg / L ampicillin were selected. These colonies were then subcultured on 2-YT agar plates and cultured at 42°C (to remove the pREDCas9 plasmid). Finally, colonies that did not grow on 2-YT agar plates containing 100 mg / L spectinomycin but grew on untreated 2-YT agar plates were selected.

[0296] (5) Using the single colony obtained in step (4) as a template, PCR amplification was performed using a primer pair consisting of primer P5 and primer P8 to obtain a PCR amplification product. If the PCR amplification product obtained from a single colony contained a 1365 bp DNA fragment, that colony was determined to be a positive transformant. This positive transformant is one in which the adhE gene in the genome of the valine-producing bacterium CGMCC 22721 has been knocked out, and this positive transformant was named the genetically modified bacterium YPVal-adhE01.

[0297] 2. Obtaining the genetically modified bacterium YPVal-adhE02 Based on the genome sequences of Escherichia coli W3110 and Bacillus subtilis subsp. subtilis str.168 published by NCBI, the ilvE(E) gene in the genome of the genetically modified bacterium YPVal-adhE01 obtained in step two was knocked out using CRISPR / Cas9 genome editing technology, while simultaneously knocking in the ilvE gene of Bacillus subtilis driven by the ptrc promoter (i.e., the ptrc-ilvE(B) sequence), thereby obtaining the genetically modified bacterium YPVal-adhE02 (hereinafter sometimes abbreviated as YPVal-adhE02). The nucleotide sequence of the ptrc-ilvE(B) sequence is shown in SEQ ID No. 6. In SEQ ID No. 6, positions 1096-1169 from the 5' end are the nucleotide sequence of the ptrc promoter, and positions 1-1095 are the ilvE(B) gene of Bacillus subtilis.

[0298] The ilvE(E) gene of the genetically modified bacterium YPVal-adhE01 encodes a branched-chain amino acid transaminase, with Gene ID 948278 and amino acid sequence shown as SEQ ID No. 15.

[0299] The ilvE(B) gene of Bacillus subtilis encodes a branched-chain amino acid transaminase, with Gene ID 938420 and amino acid sequence shown as SEQ ID No. 16.

[0300] The specific steps are as follows: 1. Construction of the pGRB-ilvE sgRNA plasmid (1) Primer synthesized by Invitrogen: sgRNA-3F:5'- TGACAGCTAGCTCAGTCCTAGGTATAATACTAGT gaaagcagcgataatcacgtcgg GTTTTAGGCTAGAATAGCAAGTTAAAATAAGG -3' (shown in SEQ ID No. 35, with the underlined portion being the homology arm sequence of the pGRB vector), and primer sgRNA-3R:5'- CCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC ccgacgtgattatcgctgctttc ACTAGTATTATACCTAGGACTGAGCTAGCTGTCA -3' (shown in SEQ ID No. 36, with the underlined portion being the homology arm sequence of the pGRB vector)

[0301] (2) After annealing primer sgRNA-3F and primer sgRNA-3R (reaction program: denaturation at 95°C for 5 minutes, annealing at 50°C for 1 minute), the target fragment was recovered using a DNA purification kit, its DNA concentration was measured, and the concentration was diluted to 100 ng / μL to obtain the annealing product. The annealing product contains the nucleotide sequence shown in SEQ ID No. 7.

[0302] (3) The pGRB vector (Addgene product, catalog number 71539) was digested with the restriction enzyme SpeI (Takara product, catalog number 1631), and a DNA fragment of approximately 2700 bp was recovered using a DNA recovery kit (QIAGEN Gel Extraction Kit). The digestive system was 50 μL and consisted of 5 μL of 10×Buffer (with restriction enzyme SpeI), 2.5 μL of restriction enzyme SpeI, 3000-5000 ng of pGRB vector, and ddH2O. Digestion program: 3 hours at 37°C.

[0303] (4) The DNA fragments recovered in step (3) were subjected to a dephosphorylation reaction (to prevent self-ligation of the pGRB vector), recovered using a DNA recovery kit, and a linearized pGRB vector was obtained. The dephosphorylation system was 50 μL and consisted of 5 μL of 10× Buffer (included with CIAP), 1000-2000 ng of DNA fragments recovered in step (3), 2.5 μL of CIAP (Takara product, catalog number 2250A), and ddH2O. Dephosphorylation program: 1 hour at 37°C.

[0304] (5) Using the Gibson Assembly Kit (New England Biolabs), the linearized pGRB vector obtained in step (4) and the annealing product obtained in step (2) were recombined, and then transformed into E. coli DH5α competent cells (TAKARA) to obtain the pGRB-ilvE sgRNA plasmid. The recombinant system consisted of 5 μL of linearized pGRB vector (2 μL), annealing product (0.5 μL), and assembly enzyme (included in the Gibson Assembly Kit) (2.5 μL). Reassembly program: Assemble at 50°C for 30 minutes.

[0305] 2. Obtaining the ptrc-ilvE(B)-Up-Down fragment (1) Based on the genome sequence of Escherichia coli W3110 published by NCBI, primer pairs for amplifying the upstream homology arm and primer pairs for amplifying the downstream homology arm were designed and synthesized by Invitrogen. Based on the genome sequence of Bacillus subtilis subsp. subtilis str.168 published by NCBI, primer pairs for amplifying ptrc-ilvE(B) were designed and synthesized by Invitrogen. The primer pair for amplifying the upstream homology arm consists of primer P9:5'-CAGGCAGTTCATTGAGTTAGCG-3' (SEQ ID No. 37) and primer P10:5'-CACAGTGTATTAAGCAGACGTTAAATACAAAAAATGGGACGGCAC-3' (SEQ ID No. 38). The primer pair for amplifying the downstream homology arm is primer P13:5'- GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAA It consists of TTTTATATTCCTTTTGCGCTC-3' (shown as SEQ ID No. 39, with the underlined portion being part of the ptrc promoter sequence) and primer P14:5'-ACGGTTAGGGATGGTTCGAC-3' (SEQ ID No. 40). The primer pair for amplifying ptrc-ilvE(B) is primer P11:5'-GTGCCGTCCCATTTTTTGTATTTAACGTCTGCTTAATACACTGTG-3' (SEQ ID No. 41) and primer P12:5'- GTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACC It consists of ATGGAACTTTTTAAATATATGGAG-3' (shown as SEQ ID No. 42, with the underlined portion being part of the ptrc promoter sequence).

[0306] (2) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P9 and primer P10, and the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 709 bp upstream homology arm was recovered using a DNA recovery kit.

[0307] (3) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P13 and primer P14 with the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 611 bp downstream homology arm was recovered using a DNA recovery kit.

[0308] (4) Using the genomic DNA of Bacillus subtilis subsp. subtilis str.168 as a template, PCR amplification was performed using a primer pair consisting of primer P11 and primer P12, and the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 1168 bp ilvE(B) fragment was recovered using a DNA recovery kit.

[0309] (6) The upstream homology arm recovered in step (2), the downstream homology arm recovered in step (3), and the ilvE(B) fragment recovered in step (4) were mixed, and using this as a template, overlap PCR was performed using a primer pair consisting of primer P9 and primer P14 to obtain the ptrc-ilvE(B)-Up-Down fragment shown in SEQ ID No. 8.

[0310] 3. Obtaining the YPVal-adhE01-Cas9 strain (1) Plasmid pREDCas9 was transformed into competent cells of the genetically modified bacterium YPVal-adhE01, and then spread onto 2-YT agar plates containing 100 mg / L spectinomycin. The cells were cultured at 32°C to obtain single colonies resistant to spectinomycin.

[0311] (2) Using the single colonies obtained in step (1) as templates, PCR amplification was performed using primer pairs consisting of primer pRedCas9-PF and primer pRedCas9-PR to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contains a DNA fragment having the nucleotide sequence shown in SEQ ID No. 3, then that colony contains plasmid pREDCas9. The strain of that colony was named YPVal-adhE01-Cas9 strain.

[0312] 4. Obtaining the genetically modified bacterium YPVal-adhE02 (1) Culture of YPVal-adhE01-Cas9 strain: The YPVal-adhE01-Cas9 strain was OD 600nm When the concentration reached 0.1 mM, IPTG was added to bring the IPTG concentration in the system to 0.1 mM, and the culture was continued to induce homologous recombination via λ-Red. The YPVal-adhE01-Cas9 strain was OD 600nm When the concentration reached 0.6, the bacterial cells were collected and competent cells of the YPVal-adhE01-Cas9 strain were prepared.

[0313] (2) Competent cells of the YPVal-adhE01-Cas9 strain were transformed with the pGRB-ilvE sgRNA plasmid obtained in step 1 and the ptrc-ilvE(B)-Up-Down fragment obtained in step 2. These cells were then spread onto 2-YT agar plates containing 100 mg / L spectinomycin and 100 mg / L ampicillin, and cultured at 32°C to obtain single colonies.

[0314] (3) Using the single colonies obtained in step (2) as templates, PCR amplification was performed using primer pairs consisting of primer P11 and primer P12 to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contained a DNA fragment of 1168 bp, that colony was provisionally determined to be a positive transformant.

[0315] (4) The positive transformants obtained in step (3) were inoculated onto 2-YT agar plates containing 100 mg / L spectinomycin and 0.2% (m / v) arabinose and cultured at 32°C (to remove the pGRB-ilvE sgRNA plasmid). Subsequently, they were grown on 2-YT agar plates containing 100 mg / L spectinomycin, and colonies that did not grow on 2-YT agar plates containing 100 mg / L ampicillin were selected. These colonies were then subcultured on 2-YT agar plates and cultured at 42°C (to remove the pREDCas9 plasmid). Finally, colonies that did not grow on 2-YT agar plates containing 100 mg / L spectinomycin but grew on untreated 2-YT agar plates were selected.

[0316] (5) Using the single colony obtained in step (4) as a template, PCR amplification was performed using a primer pair consisting of primer P11 and primer P12 to obtain a PCR amplification product. If the PCR amplification product obtained from a single colony contained a 1168 bp DNA fragment, that colony was determined to be a positive transformant. This positive transformant was determined to be a genetically modified bacterium YPVal-adhE02 in which the ilvE(E) gene in the genome of the genetically modified bacterium YPVal-adhE01 was knocked out, and at the same time, the ilvE(B) gene of Bacillus subtilis driven by the ptrc promoter (i.e., the ptrc-ilvE(B) sequence) was knocked in.

[0317] 3. Obtaining the genetically modified bacterium YPVal-adhE03 Using the genetically modified bacterium YPVal-adhE02 as the starting cell, the thiE gene in the genome of the genetically modified bacterium YPVal-adhE02 was knocked out using CRISPR / Cas9 genome editing technology, based on the genome sequence of Escherichia coli W3110 published by NCBI.

[0318] The thiE gene encodes thiamine phosphate synthase, has a Gene ID of 948491, and its amino acid sequence is shown as SEQ ID No. 17.

[0319] The specific steps are as follows: 1. Construction of the pGRB-thiE sgRNA plasmid Based on the Escherichia coli W3110 genome sequence published by NCBI, a target sequence for sgRNA to knock out the thiE gene (shown as SEQ ID No. 9) was designed using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ), and homology arm sequences of a linearized pGRB vector for constructing the sgRNA plasmid were added to the 5' and 3' ends of the target sequence.

[0320] (1) Primer synthesized by Invitrogen: sgRNA-4F:5'- TGACAGCTAGCTCAGTCCTAGGTATAATACTAGT cgcccctcttatatcgcgctggg GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG -3' (shown in SEQ ID No. 43, with the underlined portion being the homology arm sequence of the pGRB vector), and primer sgRNA-4R:5'- CCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC cccagcgcgatataagaggggcg ACTAGTATTATACCTAGGACTGAGCTAGCTGTCA -3' (shown in SEQ ID No. 44, with the underlined portion being the homology arm sequence of the pGRB vector)

[0321] (2) After annealing primer sgRNA-4F and primer sgRNA-4R (reaction program: denaturation at 95°C for 5 minutes, annealing at 50°C for 1 minute), the target fragment was recovered using a DNA purification kit, its DNA concentration was measured, and the concentration was diluted to 100 ng / μL to obtain the annealing product. The annealing product contained an sgRNA-4 fragment having the nucleotide sequence shown in SEQ ID No. 9.

[0322] (3) The pGRB vector was digested with restriction enzyme SpeI, and a DNA fragment of approximately 2700 bp was recovered using a DNA recovery kit (QIAGEN Gel Extraction Kit). The digestive system was 50 μL and consisted of 5 μL of 10×Buffer (with restriction enzyme SpeI), 2.5 μL of restriction enzyme SpeI, 3000-5000 ng of pGRB vector, and ddH2O. Digestion program: 3 hours at 37°C.

[0323] (4) The DNA fragments recovered in step (3) were subjected to a dephosphorylation reaction (to prevent self-ligation of the pGRB vector), recovered using a DNA recovery kit, and a linearized pGRB vector was obtained. The dephosphorylation system was 50 μL and consisted of 5 μL of 10×Buffer (provided with CIAP), 1000-2000 ng of DNA fragments recovered in step (3), 2.5 μL of CIAP, and ddH2O. Dephosphorylation program: 1 hour at 37°C.

[0324] (5) Using the Gibson Assembly Kit, the linearized pGRB vector obtained in step (4) and the annealing product obtained in step (2) were recombined, and then transformed into E. coli DH5α competent cells to obtain the pGRB-thiE sgRNA plasmid. The recombinant system consisted of 5 μL of linearized pGRB vector (2 μL), annealing product (0.5 μL), and assembly enzyme (included in the Gibson Assembly Kit) (2.5 μL). Reassembly program: Assemble at 50°C for 30 minutes.

[0325] 2. Obtaining the ΔthiE-Up-Down fragment (1) Based on the genome sequence of Escherichia coli W3110 published by NCBI, a primer pair for amplifying the upstream homology arm and a primer pair for amplifying the downstream homology arm were designed and synthesized by Invitrogen. The primer pair for amplifying the upstream homology arm consists of primer P15:5'-TTCTATTCAGGACGCCAACG-3' (SEQ ID No. 45) and primer P16:5'-GCTATAACGCATAAAGTCACGGCACGCTTCCTCCTTACGCAGG-3' (SEQ ID No. 46). The primer pair for amplifying the downstream homology arm consists of primer P17:5'-CCTGCGTAAGGAGGAAGCGTGCCGTGACTTTATGCGTTATAGC-3' (SEQ ID No. 47) and primer P18:5'-GCCTGCAAAGTGCCCATAACCC-3' (SEQ ID No. 48).

[0326] (2) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P15 and primer P16, and the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 721 bp upstream homology arm was recovered using a DNA recovery kit.

[0327] (3) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P17 and primer P18 with the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 618 bp downstream homology arm was recovered using a DNA recovery kit.

[0328] (4) The upstream homology arm recovered in step (2) and the downstream homology arm recovered in step (3) were mixed, and using this as a template, overlap PCR was performed using a primer pair consisting of primer P15 and primer P18 to obtain the ΔthiE-Up-Down fragment shown in SEQ ID No. 10.

[0329] 3. Obtaining the YPVal-adhE02-Cas9 strain (1) Plasmid pREDCas9 was transformed into competent cells of the genetically modified bacterium YPVal-adhE02, and then spread onto 2-YT agar plates containing 100 mg / L spectinomycin. The cells were cultured at 32°C to obtain single colonies resistant to spectinomycin.

[0330] (2) Using the single colonies obtained in step (1) as templates, PCR amplification was performed using primer pairs consisting of primer pRedCas9-PF and primer pRedCas9-PR to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contains a DNA fragment having the nucleotide sequence shown in SEQ ID No. 3, then that colony contains plasmid pREDCas9. The strain of that colony was named the YPVal-adhE02-Cas9 strain.

[0331] 4. Obtaining the genetically modified bacterium YPVal-adhE03 (1) Culture of YPVal-adhE02-Cas9 strain: The YPVal-adhE02-Cas9 strain was OD 600nm When the concentration reached 0.1 mM, IPTG was added to bring the IPTG concentration in the system to 0.1 mM, and the culture was continued to induce homologous recombination via λ-Red. The YPVal-adhE02-Cas9 strain was OD 600nm When the concentration reached 0.6, the bacterial cells were collected and competent cells of the YPVal-adhE02-Cas9 strain were prepared.

[0332] (2) YPVal-adhE02-Cas9 competent cells were transformed with the pGRB-thiE sgRNA plasmid obtained in step 1 and the ΔthiE-Up-Down fragment obtained in step 2, and the transformed cells were spread on a 2-YT agar plate containing 100 mg / L spectinomycin and 100 mg / L ampicillin, and cultured at 32°C to obtain single colonies.

[0333] (3) Using the single colonies obtained in step (2) as templates, PCR amplification was performed using primer pairs consisting of primer P15 and primer P18 to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contained a 1296 bp DNA fragment, that colony was provisionally determined to be a positive transformant.

[0334] (4) The positive transformants obtained in step (3) were inoculated onto 2-YT agar plates containing 100 mg / L spectinomycin and 0.2% (m / v) arabinose and cultured at 32°C (to remove the pGRB-thiE sgRNA plasmid). Subsequently, they were grown on 2-YT agar plates containing 100 mg / L spectinomycin, and colonies that did not grow on 2-YT agar plates containing 100 mg / L ampicillin were selected. These colonies were then subcultured on 2-YT agar plates and cultured at 42°C (to remove the pREDCas9 plasmid). Finally, colonies that did not grow on 2-YT agar plates containing 100 mg / L spectinomycin but grew on untreated 2-YT agar plates were selected.

[0335] (5) Using the single colony obtained in step (4) as a template, PCR amplification was performed using a primer pair consisting of primer P15 and primer P18 to obtain a PCR amplification product. If the PCR amplification product obtained from a single colony contained a 1296 bp DNA fragment, that colony was determined to be a positive transformant of the genetically modified bacterium YPVal-adhE02 in which the thiE gene was deleted from the genome. This positive transformant was named the genetically modified bacterium YPVal-adhE03.

[0336] IV. Obtaining the genetically modified bacterium YPVal-adhE04 Using the genetically modified bacterium YPVal-adhE03 as the starting cell, the mdh gene in the genome of the genetically modified bacterium YPVal-adhE03 was knocked out using CRISPR / Cas9 genome editing technology, based on the genome sequence of Escherichia coli W3110 published by NCBI.

[0337] The mdh gene encodes maleate dehydrogenase, has a Gene ID of 947854, and its amino acid sequence is shown as SEQ ID No. 18.

[0338] The specific steps are as follows: 1. Construction of the pGRB-mdh sgRNA plasmid Based on the Escherichia coli W3110 genome sequence published by NCBI, a target sequence for sgRNA to knock out the mdh gene (shown as SEQ ID No. 11) was designed using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ), and homology arm sequences of a linearized pGRB vector for constructing the sgRNA plasmid were added to the 5' and 3' ends of the target sequence.

[0339] (1) Primer synthesized by Invitrogen: sgRNA-5F:5'- TGACAGCTAGCTCAGTCCTAGGTATAATACTAGT gcctttcagttccgcaacaaagg GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG -3' (shown in SEQ ID No. 49, with the underlined portion being the homology arm sequence of the pGRB vector), and primer sgRNA-5R:5'- CCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC cctttgttgcggaactgaaaggc ACTAGTATTATACCTAGGACTGAGCTAGCTGTCA -3' (shown in SEQ ID No. 50, with the underlined portion being the homology arm sequence of the pGRB vector)

[0340] (2) After annealing primer sgRNA-5F and primer sgRNA-5R (reaction program: denaturation at 95°C for 5 minutes, annealing at 50°C for 1 minute), the target fragment was recovered using a DNA purification kit, its DNA concentration was measured, and the concentration was diluted to 100 ng / μL to obtain the annealing product. The annealing product contained an sgRNA-5 fragment having the nucleotide sequence shown in SEQ ID No. 11.

[0341] (3) The pGRB vector was digested with restriction enzyme SpeI, and a DNA fragment of approximately 2700 bp was recovered using a DNA recovery kit (QIAGEN Gel Extraction Kit). The digestive system was 50 μL and consisted of 5 μL of 10×Buffer (with restriction enzyme SpeI), 2.5 μL of restriction enzyme SpeI, 3000-5000 ng of pGRB vector, and ddH2O. Digestion program: 3 hours at 37°C.

[0342] (4) The DNA fragments recovered in step (3) were subjected to a dephosphorylation reaction (to prevent self-ligation of the pGRB vector), recovered using a DNA recovery kit, and a linearized pGRB vector was obtained. The dephosphorylation system was 50 μL and consisted of 5 μL of 10×Buffer (provided with CIAP), 1000-2000 ng of DNA fragments recovered in step (3), 2.5 μL of CIAP, and ddH2O. Dephosphorylation program: 1 hour at 37°C.

[0343] (5) Using the Gibson Assembly Kit, the linearized pGRB vector obtained in step (4) and the annealing product obtained in step (2) were recombined, and then transformed into E. coli DH5α competent cells to obtain the pGRB-mdh sgRNA plasmid. The recombinant system consisted of 5 μL of linearized pGRB vector (2 μL), annealing product (0.5 μL), and assembly enzyme (included in the Gibson Assembly Kit) (2.5 μL). Reassembly program: Assemble at 50°C for 30 minutes.

[0344] 2. Obtaining the Δmdh-Up-Down fragment (1) Based on the genome sequence of Escherichia coli W3110 published by NCBI, a primer pair for amplifying the upstream homology arm and a primer pair for amplifying the downstream homology arm were designed and synthesized by Invitrogen. The primer pair for amplifying the upstream homology arm consists of primer P19:5'-AACTTCCTCCAAACCGATGC-3' (SEQ ID No. 51) and primer P20:5'-CAATATAATAAGGAGTTTAGGTTGATTAGCGGATAATAAAAAACC-3' (SEQ ID No. 52). The primer pair for amplifying the downstream homology arm consists of primer P21:5'-GGTTTTTTATTATCCGCTAATCAACCTAAACTCCTTATTATATTG-3' (SEQ ID No. 53) and primer P22:5'-TCTTCAATGGACTGGAGGTG-3' (SEQ ID No. 54).

[0345] (2) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P19 and primer P20, and the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 590 bp upstream homology arm was recovered using a DNA recovery kit.

[0346] (3) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P21 and primer P22, and the high-fidelity amplification enzyme KAPA HiFi HotStart. A 708 bp downstream homology arm was recovered using a DNA recovery kit.

[0347] (4) The upstream homology arm recovered in step (2) and the downstream homology arm recovered in step (3) were mixed, and using this as a template, overlap PCR was performed using a primer pair consisting of primer P19 and primer P22 to obtain the Δmdh-Up-Down fragment shown in SEQ ID No. 12.

[0348] 3. Obtaining the YPVal-adhE03-Cas9 strain (1) Plasmid pREDCas9 was transformed into competent cells of the genetically modified bacterium YPVal-adhE03, and then spread onto 2-YT agar plates containing 100 mg / L spectinomycin. The cells were cultured at 32°C to obtain single colonies resistant to spectinomycin.

[0349] (2) Using the single colonies obtained in step (1) as templates, PCR amplification was performed using primer pairs consisting of primer pRedCas9-PF and primer pRedCas9-PR to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contains a DNA fragment having the nucleotide sequence shown in SEQ ID No. 3, then that colony contains plasmid pREDCas9. The strain of that colony was named the YPVal-adhE03-Cas9 strain.

[0350] 4. Obtaining the genetically modified bacterium YPVal-adhE04 (1) Culture of YPVal-adhE03-Cas9 strain: The YPVal-adhE03-Cas9 strain was OD 600nm When the concentration reached 0.1 mM, IPTG was added to bring the IPTG concentration in the system to 0.1 mM, and the culture was continued to induce homologous recombination via λ-Red. The YPVal-adhE03-Cas9 strain was OD 600nm When the concentration reached 0.6, the bacterial cells were collected and competent cells of the YPVal-adhE03-Cas9 strain were prepared.

[0351] (2) Competent cells of the YPVal-adhE03-Cas9 strain were transformed with the pGRB-mdh sgRNA plasmid obtained in step 1 and the Δmdh-Up-Down fragment obtained in step 2. These cells were then spread onto 2-YT agar plates containing 100 mg / L spectinomycin and 100 mg / L ampicillin, and cultured at 32°C to obtain single colonies.

[0352] (3) Using the single colonies obtained in step (2) as templates, PCR amplification was performed using primer pairs consisting of primer P19 and primer P22 to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contained a 1253 bp DNA fragment, that colony was provisionally determined to be a positive transformant.

[0353] (4) The positive transformants obtained in step (3) were inoculated onto 2-YT agar plates containing 100 mg / L spectinomycin and 0.2% (m / v) arabinose and cultured at 32°C (to remove the pGRB-thiE sgRNA plasmid). Subsequently, they were grown on 2-YT agar plates containing 100 mg / L spectinomycin, and colonies that did not grow on 2-YT agar plates containing 100 mg / L ampicillin were selected. These colonies were then subcultured on 2-YT agar plates and cultured at 42°C (to remove the pREDCas9 plasmid). Finally, colonies that did not grow on 2-YT agar plates containing 100 mg / L spectinomycin but grew on untreated 2-YT agar plates were selected.

[0354] (5) Using the single colony obtained in step (4) as a template, PCR amplification was performed using a primer pair consisting of primer P19 and primer P22 to obtain a PCR amplification product. If the PCR amplification product obtained from a single colony contained a 1253 bp DNA fragment, that colony was determined to be a positive transformant of the genetically modified bacterium YPVal-adhE03 in which the thiE gene was deleted from the genome. This positive transformant was named the genetically modified bacterium YPVal-adhE04.

[0355] Example 5: Modification of genetically engineered bacteria starting from E. coli W3110 The inventors of this invention conducted numerous experiments and modified E. coli W3110 as a starting strain to obtain genetically modified bacteria YPVal-adhE05, YPVal-adhE06, YPVal-adhE07, and YPVal-adhE08. The genotypes of the genetically modified bacteria YPVal-adhE05, YPVal-adhE06, YPVal-adhE07, and YPVal-adhE08 are shown in Table 8.

[0356] TIFF2026521933000008.tif47170

[0357] 1. Obtaining the genetically modified bacterium YPVal-adhE05 1. Construction of the pGRB-adhE sgRNA plasmid This is the same as step 1 in step 1 of Example 4.

[0358] 2. Obtaining the ΔadhE-Up-Down fragment This is the same as step 2 in step 1 of Example 4.

[0359] 3. Obtaining the W3110-Cas9 strain In step 3 of step 1 of Example 4, the valine-producing bacteria CGMCC 22721 competent cells were replaced with E. coli W3110 competent cells, and the other steps were carried out as is to obtain the W3110-Cas9 strain.

[0360] 4. Obtaining the genetically modified bacterium YPVal-adhE05 In accordance with step 4 of step 1 of Example 4, the CGMCC 22721-Cas9 strain was replaced with the W3110-Cas9 strain, and the other steps were carried out as is to obtain the genetically modified bacterium YPVal-adhE05.

[0361] 2. Obtaining the genetically modified bacterium YPVal-adhE06 1. Construction of the pGRB-ilvE sgRNA plasmid This is the same as step 1 in step 2 of Example 4.

[0362] 2. Obtaining the ptrc-ilvE(B)-Up-Down fragment This is the same as step 2 in step 2 of Example 4.

[0363] 3. Obtaining the YPVal-adhE05-Cas9 strain In accordance with step 3 of step 2 of Example 4, the genetically modified YPVal-adhE01 competent cells were replaced with genetically modified YPVal-adhE05 competent cells, and the other steps were carried out as is to obtain the YPVal-adhE05-Cas9 strain.

[0364] 4. Obtaining the genetically modified bacterium YPVal-adhE06 In step 4 of step 2 of Example 4, the YPVal-adhE01-Cas9 strain was replaced with the YPVal-adhE05-Cas9 strain, and the other steps were carried out as is to obtain the genetically modified bacterium YPVal-adhE06.

[0365] 3. Obtaining the genetically modified bacterium YPVal-adhE07 1. Construction of the pGRB-thiE sgRNA plasmid This is the same as step 1 in step 3 of Example 4.

[0366] 2. Obtaining the ΔthiE-Up-Down fragment This is the same as step 2 in step 3 of Example 4.

[0367] 3. Obtaining the YPVal-adhE06-Cas9 strain In accordance with step 3 of step 3 of Example 4, the genetically modified YPVal-adhE02 competent cells were replaced with genetically modified YPVal-adhE06 competent cells, and the other steps were carried out as is to obtain the YPVal-adhE06-Cas9 strain.

[0368] 4. Obtaining the genetically modified bacterium YPVal-adhE07 In step 4 of step 3 of Example 4, the YPVal-adhE02-Cas9 strain was replaced with the YPVal-adhE06-Cas9 strain, and the other steps were carried out as is to obtain the genetically modified bacterium YPVal-adhE07.

[0369] IV. Obtaining the genetically modified bacterium YPVal-adhE08 1. Construction of the pGRB-mdh sgRNA plasmid This is the same as step 1 in step four of Example 4.

[0370] 2. Obtaining the Δmdh-Up-Down fragment This is the same as step 2 in step four of Example 4.

[0371] 3. Obtaining the YPVal-adhE07-Cas9 strain In step 4 of Example 4, following step 3, the genetically modified YPVal-adhE03 competent cells were replaced with genetically modified YPVal-adhE07 competent cells, and the other steps were carried out as is to obtain the YPVal-adhE07-Cas9 strain.

[0372] 4. Obtaining the genetically modified bacterium YPVal-adhE08 In step 4 of step four of Example 4, the YPVal-adhE03-Cas9 strain was replaced with the YPVal-adhE07-Cas9 strain, and the other steps were carried out as is to obtain the genetically modified bacterium YPVal-adhE08.

[0373] Example 6: L-valine is produced by fermentation of the genetically modified bacteria obtained in Examples 4 and 5. 1. The genetically modified bacteria YPVal-adhE01, YPVal-adhE02, YPVal-adhE03, YPVal-adhE04, YPVal-adhE05, YPVal-adhE06, YPVal-adhE07, and YPVal-adhE08 obtained by modification in Examples 4 and 5, as well as the starting bacteria, the valine-producing bacterium CGMCC 22721 and Escherichia coli W3110, were each fermented in a fermentation tank (Shanghai Bailun Biotechnology Co., Ltd., model BLBIO-5GC-4-H) to obtain fermentation liquid.

[0374] Each strain was fermented by repeating the process three times. The components of the fermentation medium used during fermentation are shown in Table 3. The fermentation control process is shown in Table 4.

[0375] 2. High-performance liquid chromatography analysis of L-valine production in each fermentation broth. Table 9 shows the results of three fermentation tests using the valine-producing bacterium CGMCC 22721 and genetically modified strains YPVal-adhE01, YPVal-adhE02, YPVal-adhE03, and YPVal-adhE04 derived from it (a P-value < 0.01 indicates a highly statistically significant difference). The results show that all of YPVal-adhE01, YPVal-adhE02, YPVal-adhE03, and YPVal-adhE04 can increase L-valine production compared to the valine-producing bacterium CGMCC 22721. In other words, in the valine-producing bacterium CGMCC 22721, genetically modified bacteria obtained by "knockout of the adhE gene," "knockout of the adhE gene and the ilvE gene (E) simultaneously with insertion of ilvE(B) derived from Bacillus subtilis driven by the ptrc promoter," "knockout of the adhE gene and the thiE gene, and the ilvE gene (E) simultaneously with insertion of ilvE(B) derived from Bacillus subtilis driven by the ptrc promoter," or "knockout of the adhE gene, the thiE gene, and the mdh gene, and the ilvE gene (E) simultaneously with insertion of ilvE(B) derived from Bacillus subtilis driven by the ptrc promoter" can all significantly increase the production of L-valine.

[0376] TIFF2026521933000009.tif65170

[0377] Table 10 shows the results of three fermentation tests using E. coli W3110 and genetically modified strains YPVal-adhE05, YPVal-adhE06, YPVal-adhE07, and YPVal-adhE08 derived from it (a P-value < 0.01 indicates a highly statistically significant difference). The results show that all of YPVal-adhE05, YPVal-adhE06, YPVal-adhE07, and YPVal-adhE08 can increase L-valine production compared to E. coli W3110. In other words, genetically modified bacteria obtained by "knockout of the adhE gene," "knockout of the adhE gene and the ilvE gene (E) simultaneously with insertion of ilvE(B) derived from Bacillus subtilis driven by the ptrc promoter," "knockout of the adhE gene and the thiE gene, and the ilvE gene (E) simultaneously with insertion of ilvE(B) derived from Bacillus subtilis driven by the ptrc promoter," or "knockout of the adhE gene, the thiE gene, and the mdh gene, and the ilvE gene (E) simultaneously with insertion of ilvE(B) derived from Bacillus subtilis driven by the ptrc promoter" in E. coli W3110 can all significantly increase the production of L-valine.

[0378] TIFF2026521933000010.tif60170

[0379] Example 7 Modified genetically engineered bacteria starting from the valine-producing bacterium CGMCC 22721 The inventors of this invention conducted numerous experiments and modified the valine-producing bacterium CGMCC 22721 as a starting strain to obtain genetically modified bacteria YPVal-ilvE01, YPVal-ilvE02, and YPVal-ilvE03. The genotypes of the genetically modified bacteria YPVal-ilvE01, YPVal-ilvE02, and YPVal-ilvE03 are shown in Table 11.

[0380] TIFF2026521933000011.tif39170

[0381] 1. Obtaining the genetically modified bacterium YPVal-ilvE01 Based on the genome sequences of Escherichia coli W3110 and Bacillus subtilis subsp. subtilis str.168 published by NCBI, the ilvE(E) gene in the genome of the valine-producing bacterium CGMCC 22721 was knocked out using CRISPR / Cas9 genome editing technology, while the ilvE gene of Bacillus subtilis driven by the ptrc promoter (i.e., the ptrc-ilvE(B) sequence) was knocked in to obtain the genetically modified bacterium YPVal-ilvE01 (hereinafter sometimes abbreviated as YPVal-ilvE01). The nucleotide sequence of the ptrc-ilvE(B) sequence is shown in SEQ ID No. 6. In SEQ ID No. 6, positions 1096-1169 from the 5' end are the nucleotide sequence of the ptrc promoter, and positions 1-1095 are the ilvE(B) gene of Bacillus subtilis.

[0382] The ilvE(E) gene of the valine-producing bacterium CGMCC 22721 encodes a branched-chain amino acid transaminase, with Gene ID 948278 and amino acid sequence shown as SEQ ID No. 15.

[0383] The ilvE(B) gene of Bacillus subtilis encodes a branched-chain amino acid transaminase, with Gene ID 938420 and amino acid sequence shown as SEQ ID No. 16.

[0384] The specific steps are as follows: 1. Construction of the pGRB-ilvE sgRNA plasmid (1) Primer synthesized by Invitrogen: sgRNA-3F:5'- TGACAGCTAGCTCAGTCCTAGGTATAATACTAGT gaaagcagcgataatcacgtcgg GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG -3' (shown in SEQ ID No. 35, with the underlined portion being the homology arm sequence of the pGRB vector), and primer sgRNA-3R:5'- CCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC ccgacgtgattatcgctgctttc ACTAGTATTATACCTAGGACTGAGCTAGCTGTCA These are the primers sgRNA-PF:5'-GTCTCATGAGCGGATACATATTTG-3' (SEQ ID No. 21) and sgRNA-PR:5'-ATGAGAAAGCGCCACGCT-3' (SEQ ID No. 22).

[0385] (2) After annealing primer sgRNA-3F and primer sgRNA-3R (reaction program: denaturation at 95°C for 5 minutes, annealing at 50°C for 1 minute), the target fragment was recovered using a DNA purification kit, its DNA concentration was measured, and the concentration was diluted to 100 ng / μL to obtain the annealing product. The annealing product contains the nucleotide sequence shown in SEQ ID No. 7.

[0386] (3) The pGRB vector (Addgene product, catalog number 71539) was digested with the restriction enzyme SpeI (Takara product, catalog number 1631), and a DNA fragment of approximately 2700 bp was recovered using a DNA recovery kit (QIAGEN Gel Extraction Kit). The digestive system was 50 μL and consisted of 5 μL of 10×Buffer (with restriction enzyme SpeI), 2.5 μL of restriction enzyme SpeI, 3000-5000 ng of pGRB vector, and ddH2O. Digestion program: 3 hours at 37°C.

[0387] (4) The DNA fragments recovered in step (3) were subjected to a dephosphorylation reaction (to prevent self-ligation of the pGRB vector), recovered using a DNA recovery kit, and a linearized pGRB vector was obtained. The dephosphorylation system was 50 μL and consisted of 5 μL of 10× Buffer (included with CIAP), 1000-2000 ng of DNA fragments recovered in step (3), 2.5 μL of CIAP (Takara product, catalog number 2250A), and ddH2O. Dephosphorylation program: 1 hour at 37°C.

[0388] (5) Using the Gibson Assembly Kit (New England Biolabs), the linearized pGRB vector obtained in step (4) and the annealing product obtained in step (2) were recombined, and then transformed into E. coli DH5α competent cells (TAKARA) to obtain the pGRB-ilvE sgRNA plasmid. The recombinant system consisted of 5 μL of linearized pGRB vector (2 μL), annealing product (0.5 μL), and assembly enzyme (included in the Gibson Assembly Kit) (2.5 μL). Reassembly program: Assemble at 50°C for 30 minutes.

[0389] 2. Obtaining the ptrc-ilvE(B)-Up-Down fragment (1) Based on the genome sequence of Escherichia coli W3110 published by NCBI, primer pairs for amplifying the upstream homology arm and primer pairs for amplifying the downstream homology arm were designed and synthesized by Invitrogen. Based on the genome sequence of Bacillus subtilis subsp. subtilis str.168 published by NCBI, primer pairs for amplifying ptrc-ilvE(B) were designed and synthesized by Invitrogen. The primer pair for amplifying the upstream homology arm consists of primer P9:5'-CAGGCAGTTCATTGAGTTAGCG-3' (SEQ ID No. 37) and primer P10:5'-CACAGTGTATTAAGCAGACGTTAAATACAAAAAATGGGACGGCAC-3' (SEQ ID No. 38). The primer pair for amplifying the downstream homology arm is primer P13:5'- GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAA It consists of TTTTATATTCCTTTTGCGCTC-3' (shown as SEQ ID No. 39, with the underlined portion being part of the ptrc promoter sequence) and primer P14:5'-ACGGTTAGGGATGGTTCGAC-3' (SEQ ID No. 40). The primer pair for amplifying ptrc-ilvE(B) is primer P11:5'-GTGCCGTCCCATTTTTTGTATTTAACGTCTGCTTAATACACTGTG-3' (SEQ ID No. 41) and primer P12:5'- GTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACC It consists of ATGGAACTTTTTAAATATATGGAG-3' (shown as SEQ ID No. 42, with the underlined portion being part of the ptrc promoter sequence).

[0390] (2) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P9 and primer P10, and the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 709 bp upstream homology arm was recovered using a DNA recovery kit.

[0391] (3) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P13 and primer P14 with the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 611 bp downstream homology arm was recovered using a DNA recovery kit.

[0392] (4) Using the genomic DNA of Bacillus subtilis subsp. subtilis str.168 as a template, PCR amplification was performed using a primer pair consisting of primer P11 and primer P12, and the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 1168 bp ilvE(B) fragment was recovered using a DNA recovery kit.

[0393] (5) The upstream homology arm recovered in step (2), the downstream homology arm recovered in step (3), and the ilvE(B) fragment recovered in step (4) were mixed, and using this as a template, overlap PCR was performed using a primer pair consisting of primer P9 and primer P14 to obtain the ptrc-ilvE(B)-Up-Down fragment shown in SEQ ID No. 8.

[0394] 3. Acquisition of CGMCC 22721-Cas9 strain (1) Plasmid pREDCas9 (Addgene product, catalog number 71541; containing the spectinomycin resistance gene) was used to transform valine-producing bacteria CGMCC 22721 competent cells. These cells were then spread onto 2-YT agar plates containing 100 mg / L spectinomycin and cultured at 32°C to obtain single colonies resistant to spectinomycin.

[0395] The preparation method for 2-YT agar plates is as follows: Dissolve 16g of tryptone, 10g of yeast extract, 5g of sodium chloride, and 16g of agar in an appropriate amount of water, add water to a total volume of 1L, adjust the pH to 7.0 with sodium hydroxide, and sterilize at 121°C for 20 minutes.

[0396] (2) Using the single colonies obtained in step (1) as templates, PCR amplification was performed using primer pairs consisting of primer pRedCas9-PF:5'-GCAGTGGCGGTTTTCATG-3' (SEQ ID No. 27) and primer pRedCas9-PR:5'-CCTTGGTGATCTCGCCTTTC-3' (SEQ ID No. 28) to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contains a DNA fragment having the nucleotide sequence shown in SEQ ID No. 3, the colony is considered to contain plasmid pREDCas9. The strain of the colony was named CGMCC 22721-Cas9 strain.

[0397] 4. Obtaining the genetically modified bacterium YPVal-ilvE01 (1) Culture of CGMCC 22721-Cas9 strain: CGMCC 22721-Cas9 strain is OD 600nm When the concentration reached 0.1 mM, IPTG was added to bring the IPTG concentration in the system to 0.1 mM, and the culture was continued to induce homologous recombination via λ-Red. The CGMCC 22721-Cas9 strain was OD 600nm When the concentration reached 0.6, the bacterial cells were collected and CGMCC 22721-Cas9 competent cells were prepared.

[0398] (2) Competent cells of the CGMCC 22721-Cas9 strain were transformed with the pGRB-ilvE sgRNA plasmid obtained in step 1 and the ptrc-ilvE(B)-Up-Down fragment obtained in step 2. These cells were then spread onto 2-YT agar plates containing 100 mg / L spectinomycin and 100 mg / L ampicillin, and cultured at 32°C to obtain single colonies.

[0399] (3) Using the single colonies obtained in step (2) as templates, PCR amplification was performed using primer pairs consisting of primer P11 and primer P12 to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contained a DNA fragment of 1168 bp, that colony was provisionally determined to be a positive transformant.

[0400] (4) The positive transformants obtained in step (3) were inoculated onto 2-YT agar plates containing 100 mg / L spectinomycin and 0.2% (m / v) arabinose and cultured at 32°C (to remove the pGRB-ilvE sgRNA plasmid). Subsequently, they were grown on 2-YT agar plates containing 100 mg / L spectinomycin, and colonies that did not grow on 2-YT agar plates containing 100 mg / L ampicillin were selected. These colonies were then subcultured on 2-YT agar plates and cultured at 42°C (to remove the pREDCas9 plasmid). Finally, colonies that did not grow on 2-YT agar plates containing 100 mg / L spectinomycin but grew on untreated 2-YT agar plates were selected.

[0401] (5) Using the single colony obtained in step (4) as a template, PCR amplification was performed using a primer pair consisting of primer P11 and primer P12 to obtain a PCR amplification product. If the PCR amplification product obtained from a single colony contained a 1168 bp DNA fragment, that colony was determined to be a positive transformant. This positive transformant was a genetically modified bacterium in which the ilvE(E) gene in the genome of the valine-producing bacterium CGMCC 22721 was knocked out, and at the same time, the ilvE(B) gene of Bacillus subtilis driven by the ptrc promoter (i.e., the ptrc-ilvE(B) sequence) was knocked in, and was named the genetically modified bacterium YPVal-ilvE01.

[0402] 2. Obtaining the genetically modified bacterium YPVal-ilvE02 Using the genetically modified bacterium YPVal-ilvE01 obtained in step one as the starting cell, the thiE gene in the genome of the genetically modified bacterium YPVal-ilvE01 was knocked out using CRISPR / Cas9 genome editing technology, based on the genome sequence of Escherichia coli W3110 published by NCBI.

[0403] The thiE gene encodes thiamine phosphate synthase, has a Gene ID of 948491, and its amino acid sequence is shown as SEQ ID No. 17.

[0404] The specific steps are as follows: 1. Construction of the pGRB-thiE sgRNA plasmid Based on the Escherichia coli W3110 genome sequence published by NCBI, a target sequence for sgRNA to knock out the thiE gene (shown as SEQ ID No. 9) was designed using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ), and homology arm sequences of a linearized pGRB vector for constructing the sgRNA plasmid were added to the 5' and 3' ends of the target sequence.

[0405] (1) Primer synthesized by Invitrogen: sgRNA-4F:5'- TGACAGCTAGCTCAGTCCTAGGTATAATACTAGT cgcccctcttatatcgcgctggg GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG -3' (shown in SEQ ID No. 43, with the underlined portion being the homology arm sequence of the pGRB vector), and primer sgRNA-4R:5'- CCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC cccagcgcgatataagaggggcg ACTAGTATTATACCTAGGACTGAGCTAGCTGTCA -3' (shown in SEQ ID No. 44, with the underlined portion being the homology arm sequence of the pGRB vector)

[0406] (2) After annealing primer sgRNA-4F and primer sgRNA-4R (reaction program: denaturation at 95°C for 5 minutes, annealing at 50°C for 1 minute), the target fragment was recovered using a DNA purification kit, its DNA concentration was measured, and the concentration was diluted to 100 ng / μL to obtain the annealing product. The annealing product contained an sgRNA-4 fragment having the nucleotide sequence shown in SEQ ID No. 9.

[0407] (3) The pGRB vector was digested with restriction enzyme SpeI, and a DNA fragment of approximately 2700 bp was recovered using a DNA recovery kit (QIAGEN Gel Extraction Kit). The digestive system was 50 μL and consisted of 5 μL of 10×Buffer (with restriction enzyme SpeI), 2.5 μL of restriction enzyme SpeI, 3000-5000 ng of pGRB vector, and ddH2O. Digestion program: 3 hours at 37°C.

[0408] (4) The DNA fragments recovered in step (3) were subjected to a dephosphorylation reaction (to prevent self-ligation of the pGRB vector), recovered using a DNA recovery kit, and a linearized pGRB vector was obtained. The dephosphorylation system was 50 μL and consisted of 5 μL of 10×Buffer (provided with CIAP), 1000-2000 ng of DNA fragments recovered in step (3), 2.5 μL of CIAP, and ddH2O. Dephosphorylation program: 1 hour at 37°C.

[0409] (5) Using the Gibson Assembly Kit, the linearized pGRB vector obtained in step (4) and the annealing product obtained in step (2) were recombined, and then transformed into E. coli DH5α competent cells to obtain the pGRB-thiE sgRNA plasmid. The recombinant system consisted of 5 μL of linearized pGRB vector (2 μL), annealing product (0.5 μL), and assembly enzyme (included in the Gibson Assembly Kit) (2.5 μL). Reassembly program: Assemble at 50°C for 30 minutes.

[0410] 2. Obtaining the ΔthiE-Up-Down fragment (1) Based on the genome sequence of Escherichia coli W3110 published by NCBI, a primer pair for amplifying the upstream homology arm and a primer pair for amplifying the downstream homology arm were designed and synthesized by Invitrogen. The primer pair for amplifying the upstream homology arm consists of primer P15:5'-TTCTATTCAGGACGCCAACG-3' (SEQ ID No. 45) and primer P16:5'-GCTATAACGCATAAAGTCACGGCACGCTTCCTCCTTACGCAGG-3' (SEQ ID No. 46). The primer pair for amplifying the downstream homology arm consists of primer P17:5'-CCTGCGTAAGGAGGAAGCGTGCCGTGACTTTATGCGTTATAGC-3' (SEQ ID No. 47) and primer P18:5'-GCCTGCAAAGTGCCCATAACCC-3' (SEQ ID No. 48).

[0411] (2) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P15 and primer P16, and the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 721 bp upstream homology arm was recovered using a DNA recovery kit.

[0412] (3) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P17 and primer P18 with the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 618 bp downstream homology arm was recovered using a DNA recovery kit.

[0413] (4) The upstream homology arm recovered in step (2) and the downstream homology arm recovered in step (3) were mixed, and using this as a template, overlap PCR was performed using a primer pair consisting of primer P15 and primer P18 to obtain the ΔthiE-Up-Down fragment shown in SEQ ID No. 10.

[0414] 3. Obtaining the YPVal-ilvE01-Cas9 strain (1) Plasmid pREDCas9 was transformed into competent cells of the genetically modified bacterium YPVal-ilvE01, and then spread onto 2-YT agar plates containing 100 mg / L spectinomycin. The cells were cultured at 32°C to obtain single colonies resistant to spectinomycin.

[0415] (2) Using the single colonies obtained in step (1) as templates, PCR amplification was performed using primer pairs consisting of primer pRedCas9-PF and primer pRedCas9-PR to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contains a DNA fragment having the nucleotide sequence shown in SEQ ID No. 3, then that colony contains plasmid pREDCas9. The strain of that colony was named YPVal-ilvE01-Cas9 strain.

[0416] 4. Obtaining the genetically modified bacterium YPVal-ilvE02 (1) Culture of YPVal-ilvE01-Cas9 strain: YPVal-ilvE01-Cas9 strain is OD 600nm When the concentration reached 0.1 mM, IPTG was added to bring the IPTG concentration in the system to 0.1 mM, and the culture was continued to induce homologous recombination via λ-Red. The YPVal-ilvE01-Cas9 strain was OD 600nm When the concentration reached 0.6, the bacterial cells were collected and competent cells of the YPVal-ilvE01-Cas9 strain were prepared.

[0417] (2) Competent cells of the YPVal-ilvE01-Cas9 strain were transformed with the pGRB-thiE sgRNA plasmid obtained in step 1 and the ΔthiE-Up-Down fragment obtained in step 2. These cells were then spread onto 2-YT agar plates containing 100 mg / L spectinomycin and 100 mg / L ampicillin, and cultured at 32°C to obtain single colonies.

[0418] (3) Using the single colonies obtained in step (2) as templates, PCR amplification was performed using primer pairs consisting of primer P15 and primer P18 to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contained a 1296 bp DNA fragment, that colony was provisionally determined to be a positive transformant.

[0419] (4) The positive transformants obtained in step (3) were inoculated onto 2-YT agar plates containing 100 mg / L spectinomycin and 0.2% (m / v) arabinose and cultured at 32°C (to remove the pGRB-thiE sgRNA plasmid). Subsequently, they were grown on 2-YT agar plates containing 100 mg / L spectinomycin, and colonies that did not grow on 2-YT agar plates containing 100 mg / L ampicillin were selected. These colonies were then subcultured on 2-YT agar plates and cultured at 42°C (to remove the pREDCas9 plasmid). Finally, colonies that did not grow on 2-YT agar plates containing 100 mg / L spectinomycin but grew on untreated 2-YT agar plates were selected.

[0420] (5) Using the single colony obtained in step (4) as a template, PCR amplification was performed using a primer pair consisting of primer P15 and primer P18 to obtain a PCR amplification product. If the PCR amplification product obtained from a single colony contained a 1296 bp DNA fragment, that colony was determined to be a positive transformant of the genetically modified bacterium YPVal-ilvE01 in which the thiE gene was deleted from the genome. This positive transformant was named the genetically modified bacterium YPVal-ilvE02.

[0421] 3. Obtaining the genetically modified bacterium YPVal-ilvE03 Using the genetically modified bacterium YPVal-ilvE02 obtained in step two as the starting cell, the mdh gene in the genome of the genetically modified bacterium YPVal-ilvE02 was knocked out using CRISPR / Cas9 genome editing technology, based on the genome sequence of Escherichia coli W3110 published by NCBI.

[0422] The mdh gene encodes maleate dehydrogenase, has a Gene ID of 947854, and its amino acid sequence is shown as SEQ ID No. 18.

[0423] The specific steps are as follows: 1. Construction of the pGRB-mdh sgRNA plasmid Based on the Escherichia coli W3110 genome sequence published by NCBI, a target sequence for sgRNA to knock out the mdh gene (shown as SEQ ID No. 11) was designed using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ), and homology arm sequences of a linearized pGRB vector for constructing the sgRNA plasmid were added to the 5' and 3' ends of the target sequence.

[0424] (1) Primer synthesized by Invitrogen: sgRNA-5F:5'- TGACAGCTAGCTCAGTCCTAGGTATAATACTAGT gcctttcagttccgcaacaaagg GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG -3' (shown in SEQ ID No. 49, with the underlined portion being the homology arm sequence of the pGRB vector), and primer sgRNA-5R:5'- CCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC cctttgttgcggaactgaaaggc ACTAGTATTATACCTAGGACTGAGCTAGCTGTCA -3' (shown in SEQ ID No. 50, with the underlined portion being the homology arm sequence of the pGRB vector)

[0425] (2) After annealing primer sgRNA-5F and primer sgRNA-5R (reaction program: denaturation at 95°C for 5 minutes, annealing at 50°C for 1 minute), the target fragment was recovered using a DNA purification kit, its DNA concentration was measured, and the concentration was diluted to 100 ng / μL to obtain the annealing product. The annealing product contained an sgRNA-5 fragment having the nucleotide sequence shown in SEQ ID No. 11.

[0426] (3) The pGRB vector was digested with restriction enzyme SpeI, and a DNA fragment of approximately 2700 bp was recovered using a DNA recovery kit (QIAGEN Gel Extraction Kit). The digestive system was 50 μL and consisted of 5 μL of 10×Buffer (with restriction enzyme SpeI), 2.5 μL of restriction enzyme SpeI, 3000-5000 ng of pGRB vector, and ddH2O. Digestion program: 3 hours at 37°C.

[0427] (4) The DNA fragments recovered in step (3) were subjected to a dephosphorylation reaction (to prevent self-ligation of the pGRB vector), recovered using a DNA recovery kit, and a linearized pGRB vector was obtained. The dephosphorylation system was 50 μL and consisted of 5 μL of 10×Buffer (provided with CIAP), 1000-2000 ng of DNA fragments recovered in step (3), 2.5 μL of CIAP, and ddH2O. Dephosphorylation program: 1 hour at 37°C.

[0428] (5) Using the Gibson Assembly Kit, the linearized pGRB vector obtained in step (4) and the annealing product obtained in step (2) were recombined, and then transformed into E. coli DH5α competent cells to obtain the pGRB-mdh sgRNA plasmid. The recombinant system consisted of 5 μL of linearized pGRB vector (2 μL), annealing product (0.5 μL), and assembly enzyme (included in the Gibson Assembly Kit) (2.5 μL). Reassembly program: Assemble at 50°C for 30 minutes.

[0429] 2. Obtaining the Δmdh-Up-Down fragment (1) Based on the genome sequence of Escherichia coli W3110 published by NCBI, a primer pair for amplifying the upstream homology arm and a primer pair for amplifying the downstream homology arm were designed and synthesized by Invitrogen. The primer pair for amplifying the upstream homology arm consists of primer P19:5'-AACTTCCTCCAAACCGATGC-3' (SEQ ID No. 51) and primer P20:5'-CAATATAATAAGGAGTTTAGGTTGATTAGCGGATAATAAAAAACC-3' (SEQ ID No. 52). The primer pair for amplifying the downstream homology arm consists of primer P21:5'-GGTTTTTTATTATCCGCTAATCAACCTAAACTCCTTATTATATTG-3' (SEQ ID No. 53) and primer P22:5'-TCTTCAATGGACTGGAGGTG-3' (SEQ ID No. 54).

[0430] (2) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P19 and primer P20, and the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 590 bp upstream homology arm was recovered using a DNA recovery kit.

[0431] (3) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P21 and primer P22, and the high-fidelity amplification enzyme KAPA HiFi HotStart. A 708 bp downstream homology arm was recovered using a DNA recovery kit.

[0432] (4) The upstream homology arm recovered in step (2) and the downstream homology arm recovered in step (3) were mixed, and using this as a template, overlap PCR was performed using a primer pair consisting of primer P19 and primer P22 to obtain the Δmdh-Up-Down fragment shown in SEQ ID No. 12.

[0433] 3. Obtaining the YPVal-ilvE02-Cas9 strain (1) Plasmid pREDCas9 was transformed into competent cells of the genetically modified bacterium YPVal-ilvE02, and then spread onto 2-YT agar plates containing 100 mg / L spectinomycin. The cells were cultured at 32°C to obtain single colonies resistant to spectinomycin.

[0434] (2) Using the single colonies obtained in step (1) as templates, PCR amplification was performed using primer pairs consisting of primer pRedCas9-PF and primer pRedCas9-PR to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contains a DNA fragment having the nucleotide sequence shown in SEQ ID No. 3, then the colony contains plasmid pREDCas9. The strain of the colony was named YPVal-ilvE02-Cas9 strain.

[0435] 4. Obtaining the genetically modified bacterium YPVal-ilvE03 (1) Culturing of YPVal-ilvE02-Cas9 strain: When the YPVal-ilvE02-Cas9 strain reached 0.1 at OD600nm, IPTG was added to bring the IPTG concentration in the system to 0.1 mM, and cultivation was continued to induce homologous recombination via λ-Red. When the YPVal-ilvE02-Cas9 strain reached 0.6 at OD600nm, the cells were harvested, and competent cells of the YPVal-ilvE02-Cas9 strain were prepared.

[0436] (2) Competent cells of the YPVal-ilvE02-Cas9 strain were transformed with the pGRB-mdh sgRNA plasmid obtained in step 1 and the Δmdh-Up-Down fragment obtained in step 2. These cells were then spread onto 2-YT agar plates containing 100 mg / L spectinomycin and 100 mg / L ampicillin, and cultured at 32°C to obtain single colonies.

[0437] (3) Using the single colonies obtained in step (2) as templates, PCR amplification was performed using primer pairs consisting of primer P19 and primer P22 to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contained a 1253 bp DNA fragment, that colony was provisionally determined to be a positive transformant.

[0438] (4) The positive transformants obtained in step (3) were inoculated onto 2-YT agar plates containing 100 mg / L spectinomycin and 0.2% (m / v) arabinose and cultured at 32°C (to remove the pGRB-thiE sgRNA plasmid). Subsequently, they were grown on 2-YT agar plates containing 100 mg / L spectinomycin, and colonies that did not grow on 2-YT agar plates containing 100 mg / L ampicillin were selected. These colonies were then subcultured on 2-YT agar plates and cultured at 42°C (to remove the pREDCas9 plasmid). Finally, colonies that did not grow on 2-YT agar plates containing 100 mg / L spectinomycin but grew on untreated 2-YT agar plates were selected.

[0439] (5) Using the single colony obtained in step (4) as a template, PCR amplification was performed using a primer pair consisting of primer P19 and primer P22 to obtain a PCR amplification product. If the PCR amplification product obtained from a single colony contained a 1253 bp DNA fragment, that colony was determined to be a positive transformant of the genetically modified bacterium YPVal-ilvE02 in which the mdh gene was deleted from the genome. This positive transformant was named the genetically modified bacterium YPVal-ilvE03.

[0440] Example 8 Modified genetically engineered bacteria starting from E. coli W3110 The inventors of this invention conducted numerous experiments and modified E. coli W3110 as a starting strain to obtain genetically modified bacteria YPVal-ilvE04, YPVal-ilvE05, and YPVal-ilvE06. The genotypes of the genetically modified bacteria YPVal-ilvE04, YPVal-ilvE05, and YPVal-ilvE06 are shown in Table 12.

[0441] TIFF2026521933000012.tif39170

[0442] 1. Obtaining the genetically modified bacterium YPVal-ilvE04 1. Construction of the pGRB-ilvE sgRNA plasmid This is the same as step 1 in step 1 of Example 7.

[0443] 2. Obtaining the ptrc-ilvE(B)-Up-Down fragment This is the same as step 2 in step 1 of Example 7.

[0444] 3. Obtaining the W3110-Cas9 strain In step 3 of step 1 of Example 7, the valine-producing bacteria CGMCC 22721 competent cells were replaced with E. coli W3110 competent cells, and the other steps were carried out as is to obtain the W3110-Cas9 strain.

[0445] 4. Obtaining the genetically modified bacterium YPVal-ilvE04 In accordance with step 4 of step 1 of Example 7, the CGMCC 22721-Cas9 strain was replaced with the W3110-Cas9 strain, and the other steps were carried out as is to obtain the genetically modified bacterium YPVal-ilvE04.

[0446] 2. Obtaining the genetically modified bacterium YPVal-ilvE05 1. Construction of the pGRB-thiE sgRNA plasmid This is the same as step 1 in step 2 of Example 7.

[0447] 2. Obtaining the ΔthiE-Up-Down fragment This is the same as step 2 in Example 7.

[0448] 3. Obtaining the YPVal-ilvE04-Cas9 strain In accordance with step 3 of step 2 of Example 7, the genetically modified YPVal-ilvE01 competent cells were replaced with genetically modified YPVal-ilvE04 competent cells, and the other steps were carried out as is to obtain the YPVal-ilvE04-Cas9 strain.

[0449] 4. Obtaining the genetically modified bacterium YPVal-ilvE05 In accordance with step 4 of step 2 of Example 7, the YPVal-ilvE01-Cas9 strain was replaced with the YPVal-ilvE04-Cas9 strain, and the other steps were carried out as is to obtain the genetically modified bacterium YPVal-ilvE05.

[0450] 3. Obtaining the genetically modified bacterium YPVal-ilvE06 1. Construction of the pGRB-mdh sgRNA plasmid This is the same as step 1 in step 3 of Example 7.

[0451] 2. Obtaining the Δmdh-Up-Down fragment This is the same as step 2 in step 3 of Example 7.

[0452] 3. Obtaining the YPVal-ilvE05-Cas9 strain In accordance with step 3 of step 3 of Example 7, the genetically modified YPVal-ilvE02 competent cells were replaced with genetically modified YPVal-ilvE05 competent cells, and the other steps were carried out as is to obtain the YPVal-ilvE05-Cas9 strain.

[0453] 4. Obtaining the genetically modified bacterium YPVal-ilvE06 In accordance with step 4 of step 3 of Example 7, the YPVal-ilvE02-Cas9 strain was replaced with the YPVal-ilvE05-Cas9 strain, and the other steps were carried out as is to obtain the genetically modified bacterium YPVal-ilvE06.

[0454] Example 9: L-valine is produced by fermentation of genetically modified bacteria obtained in Examples 7 and 8. 1. The genetically modified bacteria YPVal-ilvE01, YPVal-ilvE02, YPVal-ilvE03, YPVal-ilvE04, YPVal-ilvE05, and YPVal-ilvE06 obtained by modification in Examples 7 and 8, as well as the starting bacteria, the valine-producing bacterium CGMCC 22721 and Escherichia coli W3110, were each fermented in a fermentation tank (Shanghai Bailun Biotechnology Co., Ltd., model BLBIO-5GC-4-H) to obtain fermentation liquid.

[0455] Each strain was fermented by repeating the process three times. The components of the fermentation medium used during fermentation are shown in Table 3. The fermentation control process is shown in Table 4.

[0456] 2. High-performance liquid chromatography analysis of L-valine production in each fermentation broth. Table 13 shows the results of three fermentation tests using the valine-producing bacterium CGMCC 22721 and genetically modified strains YPVal-ilvE01, YPVal-ilvE02, and YPVal-ilvE03 derived from it (a P-value < 0.01 indicates a highly statistically significant difference). The results show that all four strains—YPVal-ilvE01, YPVal-ilvE02, and YPVal-ilvE03—can increase L-valine production compared to the valine-producing bacterium CGMCC 22721. In other words, in the valine-producing bacterium CGMCC 22721, genetically modified bacteria obtained by "knockout of the ilvE(E) gene and simultaneous insertion of ilvE(B) derived from Bacillus subtilis driven by the ptrc promoter," or "knockout of the ilvE gene (E) and simultaneous insertion of ilvE(B) derived from Bacillus subtilis driven by the ptrc promoter, along with knockout of the thiE gene," or "knockout of the ilvE gene (E) and simultaneous insertion of ilvE(B) derived from Bacillus subtilis driven by the ptrc promoter, along with knockout of the thiE and mdh genes" can all significantly increase the production of L-valine.

[0457] TIFF2026521933000013.tif57170

[0458] Table 14 shows the results of three fermentation tests using E. coli W3110 and genetically modified strains YPVal-ilvE04, YPVal-ilvE05, and YPVal-ilvE06 derived from it (a P-value < 0.01 indicates a highly statistically significant difference). The results show that YPVal-ilvE04, YPVal-ilvE05, and YPVal-ilvE06 can all produce higher levels of L-valine compared to E. coli W3110. In other words, in E. coli W3110, the production of any L-valine can be significantly increased by "knockout of the ilvE gene (E) and simultaneous insertion of ilvE(B) from Bacillus subtilis driven by the ptrc promoter," or "knockout of the ilvE gene (E) and simultaneous insertion of ilvE(B) from Bacillus subtilis driven by the ptrc promoter, along with knockout of the thiE gene," or "knockout of the ilvE gene (E) and simultaneous insertion of ilvE(B) from Bacillus subtilis driven by the ptrc promoter, along with knockout of the thiE gene and mdh gene."

[0459] TIFF2026521933000014.tif52170

[0460] Example 10 Modified genetically engineered bacteria starting from the valine-producing bacterium CGMCC 22721 The inventors of this invention conducted numerous experiments and modified the valine-producing bacterium CGMCC 22721 as a starting strain to obtain genetically modified bacteria YPVal-thiE01, YPVal-thiE02, and YPVal-mdh01. The genotypes of the genetically modified bacteria YPVal-thiE01, YPVal-thiE02, and YPVal-mdh01 are shown in Table 15.

[0461] TIFF2026521933000015.tif39170

[0462] 1. Obtaining the genetically modified bacterium YPVal-thiE01 Based on the genome sequence of Escherichia coli W3110 published by NCBI, the thiE gene in the genome of the valine-producing bacterium CGMCC 22721 was knocked out using CRISPR / Cas9 genome editing technology to obtain the genetically modified bacterium YPVal-thiE01 (hereinafter sometimes abbreviated as YPVal-thiE01).

[0463] The thiE gene encodes thiamine phosphate synthase, has a Gene ID of 948491, and its amino acid sequence is shown as SEQ ID No. 17.

[0464] The specific steps are as follows: 1. Construction of the pGRB-thiE sgRNA plasmid Based on the Escherichia coli W3110 genome sequence published by NCBI, a target sequence for sgRNA to knock out the thiE gene (shown as SEQ ID No. 9) was designed using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ), and homology arm sequences of a linearized pGRB vector for constructing the sgRNA plasmid were added to the 5' and 3' ends of the target sequence.

[0465] (1) Primer synthesized by Invitrogen: sgRNA-4F:5'- TGACAGCTAGCTCAGTCCTAGGTATAATACTAGT cgcccctcttatatcgcgctggg GTTTTAGGCTAGAATAGCAAGTTAAAATAAGG -3' (shown in SEQ ID No. 43, with the underlined portion being the homology arm sequence of the pGRB vector), and primer sgRNA-4R:5'- CCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC cccagcgcgatataagaggggcg ACTAGTATTATACCTAGGACTGAGCTAGCTGTCA-3' (shown in SEQ ID No. 44, with the underlined portion being the homology arm sequence of the pGRB vector); primer sgRNA-PF:5'-GTCTCATGAGCGGATACATATTTG-3' (SEQ ID No. 21), and primer sgRNA-PR:5'-ATGAGAAAGCGCCACGCT-3' (SEQ ID No. 22).

[0466] (2) After annealing primer sgRNA-4F and primer sgRNA-4R (reaction program: denaturation at 95°C for 5 minutes, annealing at 50°C for 1 minute), the target fragment was recovered using a DNA purification kit, its DNA concentration was measured, and the concentration was diluted to 100 ng / μL to obtain the annealing product. The annealing product contained an sgRNA-4 fragment having the nucleotide sequence shown in SEQ ID No. 9.

[0467] (3) The pGRB vector was digested with restriction enzyme SpeI, and a DNA fragment of approximately 2700 bp was recovered using a DNA recovery kit (QIAGEN Gel Extraction Kit).

[0468] The digestive system was 50 μL and consisted of 5 μL of 10×Buffer (with restriction enzyme SpeI), 2.5 μL of restriction enzyme SpeI, 3000-5000 ng of pGRB vector, and ddH2O. Digestion program: 3 hours at 37°C.

[0469] (4) The DNA fragments recovered in step (3) were subjected to a dephosphorylation reaction (to prevent self-ligation of the pGRB vector), recovered using a DNA recovery kit, and a linearized pGRB vector was obtained. The dephosphorylation system was 50 μL and consisted of 5 μL of 10×Buffer (provided with CIAP), 1000-2000 ng of DNA fragments recovered in step (3), 2.5 μL of CIAP, and ddH2O. Dephosphorylation program: 1 hour at 37°C.

[0470] (5) Using the Gibson Assembly Kit, the linearized pGRB vector obtained in step (4) and the annealing product obtained in step (2) were recombined, and then transformed into E. coli DH5α competent cells to obtain the pGRB-thiE sgRNA plasmid. The recombinant system consisted of 5 μL of linearized pGRB vector (2 μL), annealing product (0.5 μL), and assembly enzyme (included in the Gibson Assembly Kit) (2.5 μL). Reassembly program: Assemble at 50°C for 30 minutes.

[0471] 2. Obtaining the ΔthiE-Up-Down fragment (1) Based on the genome sequence of Escherichia coli W3110 published by NCBI, a primer pair for amplifying the upstream homology arm and a primer pair for amplifying the downstream homology arm were designed and synthesized by Invitrogen. The primer pair for amplifying the upstream homology arm consists of primer P15:5'-TTCTATTCAGGACGCCAACG-3' (SEQ ID No. 45) and primer P16:5'-GCTATAACGCATAAAGTCACGGCACGCTTCCTCCTTACGCAGG-3' (SEQ ID No. 46). The primer pair for amplifying the downstream homology arm consists of primer P17:5'-CCTGCGTAAGGAGGAAGCGTGCCGTGACTTTATGCGTTATAGC-3' (SEQ ID No. 47) and primer P18:5'-GCCTGCAAAGTGCCCATAACCC-3' (SEQ ID No. 48).

[0472] (2) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P15 and primer P16, and the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 721 bp upstream homology arm was recovered using a DNA recovery kit.

[0473] (3) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P17 and primer P18 with the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 618 bp downstream homology arm was recovered using a DNA recovery kit.

[0474] (4) The upstream homology arm recovered in step (2) and the downstream homology arm recovered in step (3) were mixed, and using this as a template, overlap PCR was performed using a primer pair consisting of primer P15 and primer P18 to obtain the ΔthiE-Up-Down fragment shown in SEQ ID No. 10.

[0475] 3. Acquisition of CGMCC 22721-Cas9 strain (1) Plasmid pREDCas9 (Addgene product, catalog number 71541; containing the spectinomycin resistance gene) was used to transform valine-producing bacteria CGMCC 22721 competent cells. These cells were then spread onto 2-YT agar plates containing 100 mg / L spectinomycin and cultured at 32°C to obtain single colonies resistant to spectinomycin.

[0476] The preparation method for 2-YT agar plates is as follows: Dissolve 16g of tryptone, 10g of yeast extract, 5g of sodium chloride, and 16g of agar in an appropriate amount of water, add water to a total volume of 1L, adjust the pH to 7.0 with sodium hydroxide, and sterilize at 121°C for 20 minutes.

[0477] (2) Using the single colonies obtained in step (1) as templates, PCR amplification was performed using primer pairs consisting of primer pRedCas9-PF:5'-GCAGTGGCGGTTTTCATG-3' (SEQ ID No. 27) and primer pRedCas9-PR:5'-CCTTGGTGATCTCGCCTTTC-3' (SEQ ID No. 28) to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contains a DNA fragment having the nucleotide sequence shown in SEQ ID No. 3, the colony is considered to contain plasmid pREDCas9. The strain of the colony was named CGMCC 22721-Cas9 strain.

[0478] 4. Obtaining the genetically modified bacterium YPVal-thiE01 (1) Culture of CGMCC 22721-Cas9 strain: CGMCC 22721-Cas9 strain is OD 600nm When the concentration reached 0.1 mM, IPTG was added to bring the IPTG concentration in the system to 0.1 mM, and the culture was continued to induce homologous recombination via λ-Red. The CGMCC 22721-Cas9 strain was OD 600nm When the concentration reached 0.6, the bacterial cells were collected and CGMCC 22721-Cas9 competent cells were prepared.

[0479] (2) Competent cells of the CGMCC 22721-Cas9 strain were transformed with the pGRB-thiE sgRNA plasmid obtained in step 1 and the ΔthiE-Up-Down fragment obtained in step 2. These cells were then spread onto 2-YT agar plates containing 100 mg / L spectinomycin and 100 mg / L ampicillin, and cultured at 32°C to obtain single colonies.

[0480] (3) Using the single colonies obtained in step (2) as templates, PCR amplification was performed using primer pairs consisting of primer P15 and primer P18 to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contained a 1296 bp DNA fragment, that colony was provisionally determined to be a positive transformant.

[0481] (4) The positive transformants obtained in step (3) were inoculated onto 2-YT agar plates containing 100 mg / L spectinomycin and 0.2% (m / v) arabinose and cultured at 32°C (to remove the pGRB-thiE sgRNA plasmid). Subsequently, they were grown on 2-YT agar plates containing 100 mg / L spectinomycin, and colonies that did not grow on 2-YT agar plates containing 100 mg / L ampicillin were selected. These colonies were then subcultured on 2-YT agar plates and cultured at 42°C (to remove the pREDCas9 plasmid). Finally, colonies that did not grow on 2-YT agar plates containing 100 mg / L spectinomycin but grew on untreated 2-YT agar plates were selected.

[0482] (5) Using the single colony obtained in step (4) as a template, PCR amplification was performed using a primer pair consisting of primer P15 and primer P18 to obtain a PCR amplification product. If the PCR amplification product obtained from a single colony contained a 1296 bp DNA fragment, that colony was determined to be a positive transformant of the valine-producing bacterium CGMCC 22721 in which the thiE gene was deleted from the genome. This positive transformant was named the genetically modified bacterium YPVal-thiE01.

[0483] 2. Obtaining the genetically modified bacterium YPVal-mdh01 Based on the genome sequence of Escherichia coli W3110 published by NCBI, the mdh gene in the genome of the valine-producing bacterium CGMCC 22721 was knocked out using CRISPR / Cas9 genome editing technology to obtain the genetically modified bacterium YPVal-mdh01 (hereinafter sometimes abbreviated as YPVal-mdh01).

[0484] The mdh gene encodes maleate dehydrogenase, has a Gene ID of 947854, and its amino acid sequence is shown as SEQ ID No. 18.

[0485] The specific steps are as follows: 1. Construction of the pGRB-mdh sgRNA plasmid Based on the Escherichia coli W3110 genome sequence published by NCBI, a target sequence for sgRNA to knock out the mdh gene (shown as SEQ ID No. 11) was designed using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ), and homology arm sequences of a linearized pGRB vector for constructing the sgRNA plasmid were added to the 5' and 3' ends of the target sequence.

[0486] (1) Primer synthesized by Invitrogen: sgRNA-5F:5'- TGACAGCTAGCTCAGTCCTAGGTATAATACTAGT gcctttcagttccgcaacaaagg GTTTTAGGCTAGAATAGCAAGTTAAAATAAGG -3' (shown in SEQ ID No. 49, with the underlined portion being the homology arm sequence of the pGRB vector), and primer sgRNA-5R:5'- CCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC cctttgttgcggaactgaaaggc ACTAGTATTATACCTAGGACTGAGCTAGCTGTCA -3' (shown in SEQ ID No. 50, with the underlined portion being the homology arm sequence of the pGRB vector)

[0487] (2) After annealing primer sgRNA-5F and primer sgRNA-5R (reaction program: denaturation at 95°C for 5 minutes, annealing at 50°C for 1 minute), the target fragment was recovered using a DNA purification kit, its DNA concentration was measured, and the concentration was diluted to 100 ng / μL to obtain the annealing product. The annealing product contained an sgRNA-5 fragment having the nucleotide sequence shown in SEQ ID No. 11.

[0488] (3) The pGRB vector was digested with restriction enzyme SpeI, and a DNA fragment of approximately 2700 bp was recovered using a DNA recovery kit (QIAGEN Gel Extraction Kit). The digestive system was 50 μL and consisted of 5 μL of 10×Buffer (with restriction enzyme SpeI), 2.5 μL of restriction enzyme SpeI, 3000-5000 ng of pGRB vector, and ddH2O. Digestion program: 3 hours at 37°C.

[0489] (4) The DNA fragments recovered in step (3) were subjected to a dephosphorylation reaction (to prevent self-ligation of the pGRB vector), recovered using a DNA recovery kit, and a linearized pGRB vector was obtained. The dephosphorylation system was 50 μL and consisted of 5 μL of 10×Buffer (provided with CIAP), 1000-2000 ng of DNA fragments recovered in step (3), 2.5 μL of CIAP, and ddH2O. Dephosphorylation program: 1 hour at 37°C.

[0490] (5) Using the Gibson Assembly Kit, the linearized pGRB vector obtained in step (4) and the annealing product obtained in step (2) were recombined, and then transformed into E. coli DH5α competent cells to obtain the pGRB-mdh sgRNA plasmid. The recombinant system consisted of 5 μL of linearized pGRB vector (2 μL), annealing product (0.5 μL), and assembly enzyme (included in the Gibson Assembly Kit) (2.5 μL). Reassembly program: Assemble at 50°C for 30 minutes.

[0491] 2. Obtaining the Δmdh-Up-Down fragment (1) Based on the genome sequence of Escherichia coli W3110 published by NCBI, a primer pair for amplifying the upstream homology arm and a primer pair for amplifying the downstream homology arm were designed and synthesized by Invitrogen. The primer pair for amplifying the upstream homology arm consists of primer P19:5'-AACTTCCTCCAAACCGATGC-3' (SEQ ID No. 51) and primer P20:5'-CAATATAATAAGGAGTTTAGGTTGATTAGCGGATAATAAAAAACC-3' (SEQ ID No. 52). The primer pair for amplifying the downstream homology arm consists of primer P21:5'-GGTTTTTTATTATCCGCTAATCAACCTAAACTCCTTATTATATTG-3' (SEQ ID No. 53) and primer P22:5'-TCTTCAATGGACTGGAGGTG-3' (SEQ ID No. 54).

[0492] (2) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P19 and primer P20, and the high-fidelity amplification enzyme KAPA HiFi HotStart, and a 590 bp upstream homology arm was recovered using a DNA recovery kit.

[0493] (3) Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed using a primer pair consisting of primer P21 and primer P22, and the high-fidelity amplification enzyme KAPA HiFi HotStart. A 708 bp downstream homology arm was recovered using a DNA recovery kit.

[0494] (4) The upstream homology arm recovered in step (2) and the downstream homology arm recovered in step (3) were mixed, and using this as a template, overlap PCR was performed using a primer pair consisting of primer P19 and primer P22 to obtain the Δmdh-Up-Down fragment shown in SEQ ID No. 12.

[0495] 3. Acquisition of CGMCC 22721-Cas9 strain This is the same as step 3 in process 1.

[0496] 4. Obtaining the genetically modified bacterium YPVal-mdh01 (1) Culture of CGMCC 22721-Cas9 strain: CGMCC 22721-Cas9 strain is OD 600nm When the concentration reached 0.1 mM, IPTG was added to bring the IPTG concentration in the system to 0.1 mM, and the culture was continued to induce homologous recombination via λ-Red. The CGMCC 22721-Cas9 strain was OD 600nm When the concentration reached 0.6, the bacterial cells were collected and CGMCC 22721-Cas9 competent cells were prepared.

[0497] (2) Competent cells of the CGMCC 22721-Cas9 strain were transformed with the pGRB-mdh sgRNA plasmid obtained in step 1 and the Δmdh-Up-Down fragment obtained in step 2. These cells were then spread onto 2-YT agar plates containing 100 mg / L spectinomycin and 100 mg / L ampicillin, and cultured at 32°C to obtain single colonies.

[0498] (3) Using the single colonies obtained in step (2) as templates, PCR amplification was performed using primer pairs consisting of primer P19 and primer P22 to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contained a 1253 bp DNA fragment, that colony was provisionally determined to be a positive transformant.

[0499] (4) The positive transformants obtained in step (3) were inoculated onto 2-YT agar plates containing 100 mg / L spectinomycin and 0.2% (m / v) arabinose and cultured at 32°C (to remove the pGRB-mdh sgRNA plasmid). Subsequently, they were grown on 2-YT agar plates containing 100 mg / L spectinomycin, and colonies that did not grow on 2-YT agar plates containing 100 mg / L ampicillin were selected. These colonies were then subcultured on 2-YT agar plates and cultured at 42°C (to remove the pREDCas9 plasmid). Finally, colonies that did not grow on 2-YT agar plates containing 100 mg / L spectinomycin but grew on untreated 2-YT agar plates were selected.

[0500] (5) Using the single colony obtained in step (4) as a template, PCR amplification was performed using a primer pair consisting of primer P19 and primer P22 to obtain a PCR amplification product. If the PCR amplification product obtained from a single colony contained a 1253 bp DNA fragment, that colony was determined to be a positive transformant of the valine-producing bacterium CGMCC 22721 in which the mdh gene was deleted from the genome. This positive transformant was named the genetically modified bacterium YPVal-mdh01.

[0501] 3. Obtaining the genetically modified bacterium YPVal-thiE02 Using the genetically modified bacterium YPVal-thiE01 obtained in step one as the starting cell, the mdh gene in the genome of the genetically modified bacterium YPVal-thiE01 was knocked out using CRISPR / Cas9 genome editing technology, based on the genome sequence of Escherichia coli W3110 published by NCBI.

[0502] The mdh gene encodes maleate dehydrogenase, has a Gene ID of 947854, and its amino acid sequence is shown as SEQ ID No. 18.

[0503] The specific steps are as follows: 1. Construction of the pGRB-mdh sgRNA plasmid This is the same as step 1 in step two.

[0504] 2. Obtaining the Δmdh-Up-Down fragment This is the same as step 2 in process two.

[0505] 3. Obtaining the YPVal-thiE01-Cas9 strain (1) Plasmid pREDCas9 was transformed into competent cells of the genetically modified bacterium YPVal-thiE01, and then spread onto 2-YT agar plates containing 100 mg / L spectinomycin. The cells were cultured at 32°C to obtain single colonies resistant to spectinomycin.

[0506] (2) Using the single colonies obtained in step (1) as templates, PCR amplification was performed using primer pairs consisting of primer pRedCas9-PF and primer pRedCas9-PR to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contains a DNA fragment having the nucleotide sequence shown in SEQ ID No. 3, then that colony contains plasmid pREDCas9. The strain of that colony was named YPVal-thiE01-Cas9 strain.

[0507] 4. Obtaining the genetically modified bacterium YPVal-thiE02 (1) Culture of YPVal-thiE01-Cas9 strain: YPVal-thiE01-Cas9 strain 600nm When the concentration reached 0.1 mM, IPTG was added to bring the IPTG concentration in the system to 0.1 mM, and the culture was continued to induce homologous recombination via λ-Red. The YPVal-thiE01-Cas9 strain was OD 600nm When the concentration reached 0.6, the bacterial cells were collected and competent cells of the YPVal-thiE01-Cas9 strain were prepared.

[0508] (2) Competent cells of the YPVal-thiE01-Cas9 strain were transformed with the pGRB-mdh sgRNA plasmid obtained in step 1 and the Δmdh-Up-Down fragment obtained in step 2. These cells were then spread onto 2-YT agar plates containing 100 mg / L spectinomycin and 100 mg / L ampicillin, and cultured at 32°C to obtain single colonies.

[0509] (3) Using the single colonies obtained in step (2) as templates, PCR amplification was performed using primer pairs consisting of primer P19 and primer P22 to obtain PCR amplification products. If the PCR amplification product obtained from a single colony contained a 1253 bp DNA fragment, that colony was provisionally determined to be a positive transformant.

[0510] (4) The positive transformants obtained in step (3) were inoculated onto 2-YT agar plates containing 100 mg / L spectinomycin and 0.2% (m / v) arabinose and cultured at 32°C (to remove the pGRB-mdh sgRNA plasmid). Subsequently, they were grown on 2-YT agar plates containing 100 mg / L spectinomycin, and colonies that did not grow on 2-YT agar plates containing 100 mg / L ampicillin were selected. These colonies were then subcultured on 2-YT agar plates and cultured at 42°C (to remove the pREDCas9 plasmid). Finally, colonies that did not grow on 2-YT agar plates containing 100 mg / L spectinomycin but grew on untreated 2-YT agar plates were selected.

[0511] (5) Using the single colony obtained in step (4) as a template, PCR amplification was performed using a primer pair consisting of primer P19 and primer P22 to obtain a PCR amplification product. If the PCR amplification product obtained from a single colony contained a 1253 bp DNA fragment, that colony was determined to be a positive transformant of the genetically modified bacterium YPVal-thiE01 in which the mdh gene was deleted from the genome. This positive transformant was named the genetically modified bacterium YPVal-thiE02.

[0512] Example 11: Modified genetically engineered bacteria starting from E. coli W3110 The inventors of this invention conducted numerous experiments and modified E. coli W3110 as a starting strain to obtain genetically modified bacteria YPVal-thiE03, YPVal-thiE04, and YPVal-mdh02. The genotypes of the genetically modified bacteria YPVal-thiE03, YPVal-thiE04, and YPVal-mdh02 are shown in Table 16.

[0513] TIFF2026521933000016.tif39170

[0514] 1. Obtaining the genetically modified bacterium YPVal-thiE03 1. Construction of the pGRB-thiE sgRNA plasmid This is the same as step 1 in step 1 of Example 10.

[0515] 2. Obtaining the ΔthiE-Up-Down fragment This is the same as step 2 in step 1 of Example 10.

[0516] 3. Obtaining the W3110-Cas9 strain In accordance with step 3 of step 1 of Example 10, the valine-producing bacteria CGMCC 22721 competent cells were replaced with E. coli W3110 competent cells, and the other steps were carried out as is to obtain the W3110-Cas9 strain.

[0517] 4. Obtaining the genetically modified bacterium YPVal-thiE03 In accordance with step 4 of step 1 of Example 10, the CGMCC 22721-Cas9 strain was replaced with the W3110-Cas9 strain, and the other steps were carried out as is to obtain the genetically modified bacterium YPVal-thiE03.

[0518] 2. Obtaining the genetically modified bacterium YPVal-mdh02 1. Construction of the pGRB-mdh sgRNA plasmid This is the same as step 1 in step 2 of Example 10.

[0519] 2. Obtaining the Δmdh-Up-Down fragment This is the same as step 2 in step 2 of Example 10.

[0520] 3. Obtaining the W3110-Cas9 strain In accordance with step 3 of step 1 of Example 10, the valine-producing bacteria CGMCC 22721 competent cells were replaced with E. coli W3110 competent cells, and the other steps were carried out as is to obtain the W3110-Cas9 strain.

[0521] 4. Obtaining the genetically modified bacterium YPVal-mdh02 In accordance with step 4 of step 2 of Example 10, the CGMCC 22721-Cas9 strain was replaced with the W3110-Cas9 strain, and the other steps were carried out as is to obtain the genetically modified bacterium YPVal-mdh02.

[0522] 3. Obtaining the genetically modified bacterium YPVal-thiE04 1. Construction of the pGRB-mdh sgRNA plasmid This is the same as step 1 in step 2 of Example 10.

[0523] 2. Obtaining the Δmdh-Up-Down fragment This is the same as step 2 in step 2 of Example 10.

[0524] 3. Obtaining the YPVal-thiE03-Cas9 strain In accordance with step 3 of step 3 of Example 10, the genetically modified YPVal-thiE01 competent cells were replaced with genetically modified YPVal-thiE03 competent cells, and the other steps were carried out as is to obtain the YPVal-thiE03-Cas9 strain.

[0525] 4. Obtaining the genetically modified bacterium YPVal-thiE04 In accordance with step 4 of step 3 of Example 10, the YPVal-thiE01-Cas9 strain was replaced with the YPVal-thiE03-Cas9 strain, and the other steps were carried out as is to obtain the genetically modified bacterium YPVal-thiE04.

[0526] Example 12: L-valine is produced by fermentation of genetically modified bacteria obtained in Examples 10 and 11. 1. The genetically modified bacteria YPVal-thiE01, YPVal-thiE02, YPVal-mdh01, YPVal-thiE03, YPVal-thiE04, and YPVal-mdh02 obtained by modification in Examples 10 and 11, as well as the starting bacteria, the valine-producing bacterium CGMCC 22721 and Escherichia coli W3110, were each fermented in a fermentation tank (Shanghai Bailun Biotechnology Co., Ltd., model BLBIO-5GC-4-H) to obtain fermentation liquids.

[0527] Each strain was fermented by repeating the process three times. The components of the fermentation medium used during fermentation are shown in Table 3. The fermentation control process is shown in Table 4.

[0528] 2. High-performance liquid chromatography analysis of L-valine production in each fermentation broth. Table 17 shows the results of three fermentation tests using the valine-producing bacterium CGMCC 22721 and genetically modified strains YPVal-thiE01, YPVal-thiE02, and YPVal-mdh01 derived from it (P-value < 0.01 indicates a highly statistically significant difference). The results show that YPVal-thiE01, YPVal-thiE02, and YPVal-mdh01 all produce higher levels of L-valine compared to the valine-producing bacterium CGMCC 22721. In other words, in the valine-producing bacterium CGMCC 22721, knockout of the thiE gene, knockout of the mdh gene, or simultaneous knockout of the thiE and mdh genes can significantly improve the production of any of these L-valine strains.

[0529] TIFF2026521933000017.tif57170

[0530] Table 18 shows the results of three fermentation tests using E. coli W3110 and genetically modified strains YPVal-thiE03, YPVal-thiE04, and YPVal-mdh02 derived from it (P-value < 0.01 indicates a highly statistically significant difference). The results show that YPVal-thiE03, YPVal-thiE04, and YPVal-mdh02 all produce higher levels of L-valine compared to E. coli W3110. In other words, in E. coli W3110, knockout of the thiE gene, knockout of the mdh gene, or simultaneous knockout of both the thiE and mdh genes can significantly improve the production of any of these L-valine strains.

[0531] TIFF2026521933000018.tif52170

[0532] The present invention has been described in detail above. Those skilled in the art will be able to implement the present invention over a wide range of equivalent parameters, concentrations, and conditions, without departing from the spirit and scope of the invention and without conducting unnecessary experiments. While specific embodiments have been given, it should be understood that further improvements to the present invention are possible. In short, according to the principles of the present invention, this application includes any modifications, uses, or improvements to the present invention, including modifications using ordinary art known in the art, even if they fall outside the scope disclosed herein. The following appended claims also allow for the application of some basic features. [Industrial applicability]

[0533] This invention provides the use of the pflB gene, adhE gene, ilvE(E) gene, ilvE(B) gene, thiE gene, and mdh gene in improving the production of L-amino acids in Escherichia coli. Experiments have shown that in E. coli capable of producing valine, insertion of the ilvE(B) gene derived from Bacillus subtilis driven by the ptrc promoter and / or at least one of the pflB gene, adhE gene, thiE gene, mdh gene and ilvE(E) gene (e.g., "knockout of the pflB gene", "knockout of the pflB gene and adhE gene", "knockout of the pflB gene and adhE gene and knockout of the ilvE(E) gene and simultaneous insertion of the ilvE(B) gene derived from Bacillus subtilis driven by the ptrc promoter", "knockout of the pflB gene, adhE gene and thiE gene and knockout of the ilvE(E) gene and / or insertion of the ilvE(B) gene derived from Bacillus subtilis driven by the ptrc promoter", "knockout of the pflB gene, adhE gene and thiE gene and knockout of the ilvE(E) gene and / or insertion of the ilvE(B) gene derived from Bacillus subtilis driven by the ptrc promoter) "Insertion of the ilvE(B) gene derived from Bacillus subtilis, driven by the ptrc promoter, simultaneously with knockout," "knockout of the adhE gene," "knockout of the adhE gene and knockout of the ilvE(E) gene, simultaneously with insertion of the ilvE(B) gene derived from Bacillus subtilis, driven by the ptrc promoter," "knockout of the adhE gene and thiE gene, and knockout of the ilvE(E) gene, simultaneously with insertion of the ilvE(B) gene derived from Bacillus subtilis, driven by the ptrc promoter," "knockout of the adhE gene, thiE gene and mdh gene, and knockout of the ilvE(E) gene, simultaneously with insertion of the ilvE(B) gene derived from Bacillus subtilis, driven by the ptrc promoter," "knockout of the ilvE(E) gene, simultaneously with insertion of the ilvE(B) gene derived from Bacillus subtilis, driven by the ptrc promoter,"It has been shown that knockout or weakening of the following genes ("knockout of the thiE gene and the ilvE(E) gene, simultaneously with insertion of the ilvE(B) gene from Bacillus subtilis driven by the ptrc promoter"), "knockout of the thiE gene and the mdh gene, and simultaneously with insertion of the ilvE(B) gene from Bacillus subtilis driven by the ptrc promoter"), or knockout of the thiE gene, the mdh gene, or knockout of the thiE gene and the mdh gene simultaneously, is advantageous for L-amino acid accumulation. Based on this, it is possible to construct genetically engineered bacterial strains that produce L-amino acids using the pflB gene, adhE gene, ilvE(E) gene, ilvE(B) gene, thiE gene, and mdh gene, which can significantly increase L-amino acid production, reduce costs, and have significant implications for accelerating the industrialization process of L-amino acids.

Claims

1. At least one of A1) to A5): A1) Construction of genetically engineered bacteria that produce L-amino acids, A2) Production of L-amino acids, A3) Control of L-amino acid production, A4) Preparation of products used in the production of L-amino acids, A5) Preparation of foods, feeds, or pharmaceuticals containing L-amino acids, This is the use of protein combinations, The aforementioned protein combination includes at least one of pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase derived from Escherichia coli, branched-chain amino acid transaminase derived from Bacillus subtilis, thiamine phosphate synthase, and maleate dehydrogenase. The pyruvate formate lyase is B1), or B2), or B3): B1) A protein having the amino acid sequence shown in SEQ ID No. 13, B2) A protein having 90% or more identity with the protein described in B1) and having similar function, wherein amino acid residues are substituted and / or deleted and / or added in the amino acid sequence shown in SEQ ID No.

13. A fusion protein having similar function, obtained by ligating a tag to the N-terminus and / or C-terminus of B3), B1), or B2); The alcohol dehydrogenase is C1), C2), or C3): C1) A protein having the amino acid sequence shown in SEQ ID No. 14, C2) A protein having 90% or more identity with the protein described in C1) and having similar function, wherein amino acid residues are substituted and / or deleted and / or added in the amino acid sequence shown in SEQ ID No.

14. A fusion protein having similar function, obtained by ligating a tag to the N-terminus and / or C-terminus of C3), C1), or C2); The branched-chain amino acid transaminase derived from E. coli is D1), or D2), or D3): D1) A protein having the amino acid sequence shown in SEQ ID No. 15, D2) A protein having 90% or more identity with the protein described in D1) and having similar function, wherein amino acid residues are substituted and / or deleted and / or added in the amino acid sequence shown in SEQ ID No.

15. A fusion protein having similar function, obtained by ligating a tag to the N-terminus and / or C-terminus of D3), D1), or D2); The branched-chain amino acid transaminase derived from Bacillus subtilis is E1), or E2), or E3): E1) A protein having the amino acid sequence shown in SEQ ID No. 16, E2) A protein having 90% or more identity with the protein described in E1) and having similar function, wherein amino acid residues are substituted and / or deleted and / or added in the amino acid sequence shown in SEQ ID No.

16. A fusion protein having similar function, obtained by ligating a tag to the N-terminus and / or C-terminus of E3)E1) or E2); The thiamine phosphate synthase is F1), F2), or F3): F1) A protein having the amino acid sequence shown in SEQ ID No. 17, F2) A protein having 90% or more identity with the protein described in F1) and having similar function, wherein amino acid residues are substituted and / or deleted and / or added in the amino acid sequence shown in SEQ ID No.

17. A fusion protein having similar function, obtained by ligating a tag to the N-terminus and / or C-terminus of F3), F1), or F2); The maleate dehydrogenase is G1), G2), or G3): G1) A protein having the amino acid sequence shown in SEQ ID No. 18, G2) A protein having 90% or more identity with the protein described in G1) and having similar function, wherein amino acid residues are substituted and / or deleted and / or added in the amino acid sequence shown in SEQ ID No.

18. A fusion protein having similar function, obtained by ligating a tag to the N-terminus and / or C-terminus of G3), G1), or G2), Use of protein combinations.

2. At least one of A1) to A5): A1) Construction of genetically engineered bacteria that produce L-amino acids, A2) Production of L-amino acids, A3) Control of L-amino acid production, A4) Preparation of products used in the production of L-amino acids, A5) Preparation of foods, feeds, or pharmaceuticals containing L-amino acids The use of a nucleic acid molecule encoding the protein combination described in claim 1.

3. At least one of A1) to A5): A1) Construction of genetically engineered bacteria that produce L-amino acids, A2) Production of L-amino acids, A3) Control of L-amino acid production, A4) Preparation of products used in the production of L-amino acids, A5) Preparation of foods, feeds, or pharmaceuticals containing L-amino acids The use of a substance that inhibits or reduces the expression and / or activity of pyruvate formate lyase as described in claim 1, and / or a substance that inhibits or reduces the expression and / or activity of alcohol dehydrogenase as described in claim 1, and / or a substance that inhibits or reduces the expression and / or activity of branched-chain amino acid transaminase derived from Escherichia coli as described in claim 1, and / or a substance that inhibits or reduces the expression and / or activity of thiamine phosphate synthase as described in claim 1, and / or a substance that inhibits or reduces the expression and / or activity of malate dehydrogenase as described in claim 1, and / or a substance that improves or increases the expression and / or activity of branched-chain amino acid transaminase derived from Bacillus subtilis as described in claim 1.

4. At least one of A1) to A5): A1) Construction of genetically engineered bacteria that produce L-amino acids, A2) Production of L-amino acids, A3) Control of L-amino acid production, A4) Preparation of products used in the production of L-amino acids, A5) Preparation of foods, feeds, or pharmaceuticals containing L-amino acids The use of an expression cassette, recombinant vector, recombinant bacteria, or recombinant host cell comprising the nucleic acid molecule described in claim 2, a substance in claim 3 that inhibits or reduces the expression level and / or activity of pyruvate formate lyase described in claim 1, a substance in claim 1 that inhibits or reduces the expression level and / or activity of alcohol dehydrogenase described in claim 1, a substance in claim 1 that inhibits or reduces the expression level and / or activity of branched-chain amino acid transaminase derived from Escherichia coli described in claim 1, a substance in claim 1 that inhibits or reduces the expression level and / or activity of thiamine phosphate synthase described in claim 1, a substance in claim 1 that inhibits or reduces the expression level and / or activity of maleate dehydrogenase described in claim 1, and / or a substance in claim 1 that improves or increases the expression level and / or activity of branched-chain amino acid transaminase derived from Bacillus subtilis.

5. Recombinant bacteria obtained by weakly expressing or not expressing at least one of the pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase derived from Escherichia coli, thiamine phosphate synthase, and maleate dehydrogenase described in claim 1 in the starting bacteria, and / or expressing or overexpressing the branched-chain amino acid transaminase derived from Bacillus subtilis described in claim 1.

6. The aforementioned weak expression or non-expression is achieved by reducing the expression levels and / or activity of the pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase derived from E. coli, thiamine phosphate synthase, and / or maleate dehydrogenase in the starting bacteria. The recombinant bacterium according to claim 5, characterized in that the expression or overexpression is achieved by introducing a gene encoding a branched-chain amino acid transaminase derived from Bacillus subtilis into the starting bacterium.

7. Reducing the expression level and / or activity of the pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase derived from Escherichia coli, thiamine phosphate synthase, and / or maleate dehydrogenase in the starting organism is characterized by reducing the expression level, reducing the activity, or inactivating the activity of the pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase derived from Escherichia coli, thiamine phosphate synthase, and / or maleate dehydrogenase in the starting organism by genome editing, gene knockout, gene mutation, or gene weakening technology, as described in claim 6.

8. A method for increasing the production of L-amino acids, The method includes the step of obtaining recombinant bacteria by reducing the expression level and / or activity of at least one of the pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase derived from Escherichia coli, thiamine phosphate synthase, and maleate dehydrogenase described in claim 1 in the starting bacteria, and / or increasing the expression level and / or activity of the branched-chain amino acid transaminase derived from Bacillus subtilis described in claim 1. A method for producing more L-amino acids in recombinant bacteria than in the starting bacteria.

9. Reducing the expression level and / or activity of at least one of the pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase derived from Escherichia coli, thiamine phosphate synthase, and maleate dehydrogenase described in claim 1 in the starting organism means reducing the expression level, reducing the activity, or inactivating the pyruvate formate lyase, alcohol dehydrogenase, branched-chain amino acid transaminase derived from Escherichia coli, thiamine phosphate synthase, and / or maleate dehydrogenase in the starting organism by genome editing, gene knockout, gene mutation, or gene weakening technology. The method according to claim 8, characterized in that the expression level and / or activity of the branched-chain amino acid transaminase derived from Bacillus subtilis described in claim 1 is achieved by introducing the gene encoding the branched-chain amino acid transaminase derived from Bacillus subtilis into the starting organism.

10. A method for producing L-amino acids, comprising the steps of fermenting and culturing a recombinant microorganism according to any one of claims 5 to 7, collecting the fermentation product, and obtaining L-amino acids.

11. The use according to any one of claims 1 to 4, or the method according to claim 8 or 10, characterized in that the L-amino acid is L-valine.

12. The recombinant organism according to any one of claims 5 to 7, characterized in that the starting organism is Escherichia coli, or the method according to claim 8 or 9.

13. At least one of A2) to A5): A2) Production of L-amino acids, A3) Control of L-amino acid production, A4) Preparation of products used in the production of L-amino acids, A5) Preparation of foods, feeds, or pharmaceuticals containing L-amino acids The use of recombinant bacteria according to any one of claims 5 to 7.

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