Genetically modified bacteria that produce L-valine, method for constructing the same, and its use.

Abstract

This invention provides genetically modified bacteria that produce L-valine, as well as a method for constructing and using such bacteria. By knocking out the yjiT gene of E. coli to express brnF and brnE, knocking out the yjiV gene to express ilvE and ilvD, knocking out the trpR gene to express the ilvH mutant protein, knocking out the lacI and lacZ genes to express DNA polymerase, and / or knocking out the ycgH gene to express ilvC, the resulting genetically modified bacteria can increase L-valine production. These genetically modified bacteria can be used for L-valine production and have promising application prospects.

Description

Technical Field

[0001] Cross-reference to Related Applications This application claims the priority of five Chinese patent applications with a filing date of July 4, 2023 (the application numbers are 2023108092706, 2023108092710, 2023108092744, 2023108092778, and 2023108092814 respectively), and the entire contents of the five patent applications are incorporated herein by reference.

[0002] The present invention relates to a genetically engineered bacterium for producing L-valine in the field of biotechnology, a method for constructing the same, and its use.

Background Art

[0003] In microorganisms, L-valine (a type of branched-chain amino acid) is biosynthesized from pyruvate via acetolactate, dihydroxyisovalerate, and ketoisovalerate. These intermediate metabolites are produced by catalytic reactions of acetohydroxyacid synthase, acetohydroxyacid reductoisomerase, dihydroxyacid dehydratase, and transaminase B. However, these enzymes are also involved in the biosynthesis of L-isoleucine from ketobutyrate and pyruvate. Furthermore, L-leucine is biosynthesized from the intermediate metabolite ketoisovalerate via 2-isopropylmalate, 3-isopropylmalate, and ketoisocaproate. Therefore, since the same enzymes are used in the biosynthesis process of branched-chain amino acids (i.e., L-valine, L-isoleucine, L-leucine), it is known that it is difficult to industrially produce a single type of branched-chain amino acid by fermentation. In addition, there is a problem that industrial mass production is limited due to feedback inhibition by the final product L-valine or its derivatives.

[0004] Escherichia coli has a clear genetic background and is an attractive industrial production strain in amino acid production. However, compared with Corynebacterium glutamicum, there are few reports on Escherichia coli strains that produce L-valine, which is thought to be due to the more complex regulatory mechanism of L-valine biosynthesis in Escherichia coli. Acetohydroxyacid synthase (AHAS) is the rate-limiting enzyme in the biosynthesis of L-valine, and there are three types of AHAS isozymes encoded by ilvBN, ilvGM, and ilvIH in Escherichia coli, each with different characteristics and regulatory mechanisms. Park et al. reported L-valine-producing strains obtained by modifying Escherichia coli W3110 and Escherichia coli W through systems metabolic engineering, with a final L-valine yield of 60.7 g / L and a sugar-acid conversion rate of 0.22 g / g. In addition to mutagenesis breeding and modification by conventional metabolic engineering, cofactor balance is also considered an important bottleneck in improving the yield of L-valine. Intracellular cofactors affect metabolic networks, signal transduction, and substance transport, and further affect the physiological functions of microbial cells. Therefore, in the production of chemicals by microbial fermentation, the titer and yield of chemicals are often limited by cofactor imbalance. This is mainly caused by the unbalanced expression of cofactor-dependent enzymes in the synthetic pathway. Savrasova, Stoynova et al. constructed an L-valine genetically engineered strain using Escherichia coli MG1655 as the starting strain by replacing the natural NADPH-dependent transaminase with a heterologous NADH-dependent leucine dehydrogenase. Under microaerobic conditions, the sugar-acid conversion rate (0.23 g / g) of this strain was only 35.4% of the theoretical maximum yield of 0.65 g / g. The development of a high-throughput screening method using biosensors, the introduction of a regeneration pathway for exogenous coenzymes to balance intracellular cofactors, and the construction of efficient industrial production strains are important scientific issues to be solved.

Summary of the Invention

Problems to be Solved by the Invention

[0006] To solve the above technical problems, the present invention provides a genetically modified bacterium for valine production. This genetically modified bacterium is a recombinant bacterium obtained by modifying a recipient bacterium, and the modifications are A1), A2), A3), A4), or A5): A1) includes A11), A12), and A13): A11) Knock out the yjiT gene in the receptor bacterium to suppress the expression of the yjiT gene, or to suppress the activity of the protein encoded by the yjiT gene. A12) To increase the content of the protein encoded by the brnF gene in the receptor bacteria, or to enhance the activity of the protein encoded by the brnF gene. A13) Increasing the content of the protein encoded by the brnE gene in the receptor bacteria, or enhancing the activity of the protein encoded by the brnE gene; A2) includes the following A21), A22), and A23): A21) Knock out the yjiV gene in the receptor bacterium, suppress the expression of the yjiV gene, or suppress the activity of the protein encoded by the yjiV gene. A22) To increase the content of the protein encoded by the ilvE gene in the receptor bacteria, or to enhance the activity of the protein encoded by the ilvE gene. A23) To increase the content of the protein encoded by the ilvD gene in the receptor bacteria, or to enhance the activity of the protein encoded by the ilvD gene; A3) includes A31) and A32): A31) Knocking out the trpR gene in the receptor bacterium, thereby suppressing the expression of the trpR gene, or suppressing the activity of the protein encoded by the trpR gene. A32) ilvH in receptor bacteria G14D、S17F To increase the content of the gene-encoded protein, or ilvH G14D、S17FTo enhance the activity of a gene-encoded protein, here, ilvH G14D、S17F The gene is obtained by substituting the glycine codon at position 14 of the ilvH gene with an aspartic acid codon and the serine codon at position 17 with a phenylalanine codon; A4) includes A41), A42), and A43): A41) Knock out the lacI gene in the receptor bacterium to suppress the expression of the lacI gene, or suppress the activity of the protein encoded by the lacI gene. A42) Knocking out the lacZ gene in the receptor bacterium to suppress the expression of the lacZ gene, or to suppress the activity of the protein encoded by the lacZ gene. A43) To increase the DNA polymerase content of receptor bacteria or to enhance the activity of DNA polymerase; A5) includes A51) and A52): A51) Knock out the ycgH gene in the receptor bacterium to suppress the expression of the ycgH gene, or suppress the activity of the protein encoded by the ycgH gene. A52) To increase the content of the protein encoded by the ilvC gene in the receptor bacteria, or to enhance the activity of the protein encoded by the ilvC gene; Includes, The receptor bacterium is Escherichia coli.

[0007] Specifically, genetically modified bacteria for valine production have the following characteristics: B1), B2), B3), B4), or B5): B1) includes B11), B12), and B13): B11) Not expressing or weakly expressing the yjiT gene, or reducing or losing the activity of the protein encoded by the yjiT gene. B12) Increase in the content of the protein encoded by the brnF gene, or enhancement of the activity of the protein encoded by the brnF gene. B13) Increase in the content of the protein encoded by the brnE gene, or enhancement of the activity of the protein encoded by the brnE gene; B2) includes B21), B22), and B23): B21) Not expressing or weakly expressing the yjiV gene, or reducing or losing the activity of the protein encoded by the yjiV gene. B22) Increase in the content of the protein encoded by the ilvE gene, or enhancement of the activity of the protein encoded by the ilvE gene. B23) Increased content of the protein encoded by the ilvD gene, or enhancement of the activity of the protein encoded by the ilvD gene; B3) includes B31) and B32): B31) Not expressing or weakly expressing the trpR gene, or reducing or losing the activity of the protein encoded by the trpR gene. B32)ilvH G14D、S17F Increased content of gene-encoded proteins, or ilvH G14D、S17F Enhancement of the activity of gene-encoded proteins, here, ilvH G14D、S17F The gene is obtained by substituting the glycine codon at position 14 of the ilvH gene with an aspartic acid codon and the serine codon at position 17 with a phenylalanine codon; B4) includes B41), B42), and B43): B41) Not expressing or weakly expressing the lacI gene, or suppressing the decrease or loss of activity of the protein encoded by the lacI gene. B42) Not expressing or weakly expressing the lacZ gene, or suppressing the decrease or loss of activity of the protein encoded by the lacZ gene. B43) Increase in DNA polymerase content or enhancement of DNA polymerase activity; B5) includes B51) and B52): B51) Not expressing or weakly expressing the ycgH gene, or reducing or losing the activity of the protein encoded by the ycgH gene. B52) Increase in the content of the protein encoded by the ilvC gene, or enhancement of the activity of the protein encoded by the ilvC gene; It has, The genetically modified bacterium is Escherichia coli.

[0008] Here, the receptor bacteria contain the yjiT gene (GeneID:945056, updated on 2023-04-14), the yjiV gene (GeneID:2847669, updated on 2023-04-14), the trpR gene (GeneID:948917, updated on 2023-04-14), the lacI gene (GeneID:945007, updated on 2023-04-14), the lacZ gene (GeneID:945006, updated on 2023-04-14), and the ycgH gene (GeneID:2847703, updated on 2023-04-14).

[0009] In the genetically modified bacteria described above, the brnF and brnE genes may be derived from Corynebacterium glutamicum.

[0010] Furthermore, the brnF gene can encode the protein shown in SEQ ID No. 2 of the sequence listing. The brnE gene can encode the protein shown as SEQ ID No. 3 in the sequence listing.

[0011] Furthermore, the brnF gene may be the DNA molecule located at positions 832-1587 of SEQ ID No. 1 in the sequence listing. The brnE gene may be the DNA molecule located at positions 1584-1910 of SEQ ID No. 1 in the sequence listing.

[0012] In the genetically modified bacteria described above, the ilvE gene may be derived from Bacillus subtilis, and the ilvD gene may be derived from Escherichia coli.

[0013] Furthermore, the ilvE gene can encode the protein indicated by SEQ ID No. 6 in the sequence listing. The ilvD gene can encode the protein indicated by SEQ ID No. 5 in the sequence listing.

[0014] Furthermore, the ilvE gene may be the DNA molecule located at positions 808-1902 of SEQ ID No. 4 in the sequence listing. The ilvD gene may be the DNA molecule located at positions 1977-3827 of SEQ ID No. 4 in the sequence listing.

[0015] In the genetically modified bacteria described above, the ilvH gene may be derived from Escherichia coli.

[0016] Furthermore, ilvH G14D、S17F The gene can encode the protein shown in sequence ID No. 8 of the sequence listing.

[0017] Furthermore, ilvH G14D、S17F The gene may be the DNA molecule located at positions 835-1326 of SEQ ID No. 7 in the sequence listing.

[0018] In the genetically modified bacteria described above, the DNA polymerase may be derived from Escherichia coli.

[0019] Furthermore, DNA polymerase may be the protein shown as SEQ ID No. 10 in the sequence listing.

[0020] Furthermore, the gene encoding DNA polymerase may be the DNA molecule shown at positions 701-3352 of SEQ ID No. 9 in the sequence listing.

[0021] In the genetically modified bacteria described above, the ilvC gene may be derived from Escherichia coli.

[0022] Furthermore, the ilvC gene can encode the protein shown in SEQ ID No. 12 of the sequence listing.

[0023] Furthermore, the ilvC gene can be the DNA molecule shown at positions 794 to 2269 of SEQ ID No. 11 in the sequence listing.

[0024] The brnF gene, brnE gene, ilvE gene, ilvD gene, ilvH G14D、S17F gene, the gene encoding DNA polymerase, and the expression of the ilvC gene are driven by a promoter that can drive the expression of the corresponding gene in the genetically engineered bacterium, including but not limited to strong promoters and constitutive promoters.

[0025] In one embodiment of the present invention, the expression of the brnF gene and the brnE gene is driven by thePtrc promoter, and thePtrc promoter is the DNA molecule shown at positions 758 to 831 of SEQ ID No. 1.

[0026] In one embodiment of the present invention, the expression of the ilvE gene is driven by thePtrc promoter, and thePtrc promoter is the DNA molecule shown at positions 1903 to 1976 of SEQ ID No. 4.

[0027] In another embodiment of the present invention, the expression of the ilvD gene is driven by the PilvD promoter, and the PilvD promoter is the DNA molecule shown at positions 3828 to 3893 of SEQ ID No. 4.

[0028] In another embodiment of the present invention, ilvH G14D、S17F the expression of the gene is driven by thePtrc promoter, and thePtrc promoter is the DNA molecule shown at positions 761 to 834 of SEQ ID No. 7.

[0029] In another embodiment of the present invention, the expression of the gene encoding DNA polymerase is driven by the PxylF promoter, which is the DNA molecule located at positions 3353-3617 of SEQ ID No. 9.

[0030] In another embodiment of the present invention, the expression of the ilvC gene is driven by the Ptrc promoter, which is a DNA molecule located at positions 2270-2343 of SEQ ID No. 11.

[0031] Specifically, A1) can be achieved by genome editing using the CRISPR / Cas9 system, which involves using an sgRNA targeting the yjiT gene and a DNA fragment indicated by donor SEQ ID No. 1. A2) can be achieved by genome editing using the CRISPR / Cas9 system, targeting the yjiV gene with sgRNA and the donor DNA fragment indicated by SEQ ID No. 4. A3) can be achieved by genome editing using the CRISPR / Cas9 system, employing an sgRNA targeting the trpR gene and a donor DNA fragment indicated by SEQ ID No. 7. A4) can be achieved by genome editing using the CRISPR / Cas9 system, targeting sgRNAs for the lacI and lacZ genes, and the donor DNA fragment indicated by SEQ ID No. 9. A5) can be achieved by genome editing using the CRISPR / Cas9 system, targeting the ycgH gene with sgRNA and the donor DNA fragment indicated by SEQ ID No. 11.

[0032] The recipient bacteria of the present invention include, but are not limited to, Escherichia coli. Any bacteria containing the yjiT gene, yjiV gene, trpR gene, lacI and lacZ genes, and ycgH gene, and capable of synthesizing L-valine, can be used to prepare recombinant bacteria and produce L-valine by the method of the present invention. The bacteria may be Escherichia coli, Corynebacterium glutamicum, Brevibacterium lactofermentum, Corynebacterium pekinense, Brevibacterium ammoniagenes, Corynebacterium crenatum, Pantoea, Pantoea ananatis, Bacillus brevis, Brevibacterium lactofermentum, or Brevibacterium flavum. The yeast may belong to the genus Saccharomyces sp. or Pichia sp.

[0033] In one embodiment of the present invention, Escherichia coli is Escherichia coli YP045 or Escherichia coli W3110.

[0034] In one embodiment of the present invention, the recombinant bacteria obtained are recombinant CGMCC22721-yjiT and recombinant W3110-yjiT. Recombinant CGMCC22721-yjiT and recombinant W3110-yjiT are recombinant bacteria obtained by knocking out a portion of the coding region of the yjiT gene in the genomes of the L-valine-producing bacterium CGMCC22721 and the wild-type Escherichia coli W3110, respectively, while inserting a brnF-brnE gene (derived from Corynebacterium glutamicum ATCC13032) driven by the Ptrc promoter, and retaining other nucleotides in the genome without modification.

[0035] In one embodiment of the present invention, the obtained recombinant bacteria are recombinant CGMCC22721-yjiV and recombinant W3110-yjiV. Recombinant CGMCC22721-yjiV and recombinant W3110-yjiV are recombinant bacteria obtained by knocking out a portion of the coding region of the yjiV gene in the genomes of the L-valine-producing bacterium CGMCC22721 and the wild-type Escherichia coli W3110, respectively, and inserting the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168) driven by the Ptrc promoter and the ilvD gene (derived from Escherichia coli W3110) driven by the PilvD promoter, while retaining other nucleotides in the genome without modification.

[0036] In one embodiment of the present invention, the obtained recombinant bacteria are recombinant CGMCC22721-trpR and recombinant W3110-trpR. Recombinant CGMCC22721-trpR and recombinant W3110-trpR have a portion of the coding region of the trpR gene on the genome of the L-valine-producing bacterium CGMCC22721 and the wild-type E. coli W3110 knocked out, and the ilvH gene driven by the Ptrc promoter is also knocked out. G14D、S17F This is a recombinant bacterium obtained by inserting a gene while retaining other nucleotides in the genome without alteration.

[0037] In one embodiment of the present invention, the recombinant bacteria obtained are recombinant CGMCC22721-lacIZ and recombinant W3110-lacIZ. Recombinant CGMCC22721-lacIZ and recombinant W3110-lacIZ are recombinant bacteria obtained by knocking out a portion of the coding region of the lacI-lacZ gene in the genomes of the L-valine-producing bacterium CGMCC22721 and the wild-type Escherichia coli W3110, respectively, while inserting a DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter, and retaining other nucleotides in the genome without modification.

[0038] In one embodiment of the present invention, the recombinant bacteria obtained are recombinant CGMCC22721-ycgH and recombinant W3110-ycgH. Recombinant CGMCC22721-ycgH and recombinant W3110-ycgH are recombinant bacteria obtained by knocking out a portion of the coding region of the ycgH gene in the genomes of the L-valine-producing bacterium CGMCC22721 and the wild-type Escherichia coli W3110, respectively, while inserting the ilvC gene (derived from Escherichia coli W3110) driven by the Ptrc promoter, and retaining other nucleotides in the genome without modification.

[0039] In the genetically modified bacteria described above, the modifications may include any two, any three, any four, or any five of A1), A2), A3), A4), and A5) described above.

[0040] Specifically, the modifications are as follows: A1) and A2), A1) and A3), A1) and A4), A1) and A5), A2) and A3), A2) and A4), A2) and A5), A3) and A4), A3) and A5), A4) and A5), A1), A2) and A3), A1), A2) and A4), A1), A2) and A5), A1), A3) and A4), A1), A3) and A5), A1), A 4) and A5), A2), A3) and A4), A2), A3) and A5), A2), A4) and A5), A3), A4) and A5), A1), A2), A3) and A4), A1), A2), A3) and A5), A1), A2), A4) and A5), A1), A3), A4) and A5), A1), A3), A4) and A5), A2), A3), A4) and A5), A1), A2), A3), A4) and A5).

[0041] Specifically, genetically modified bacteria for valine production further possess any two, three, four, or five of the characteristics listed in B1), B2), B3), B4), and B5) above.

[0042] In other embodiments of the present invention, the recombinant bacteria are recombinant CGMCC22721-yjiT-yjiV and recombinant W3110-yjiT-yjiV. Recombinant bacteria CGMCC22721-yjiT-yjiV and recombinant bacteria W3110-yjiT-yjiV are recombinant bacteria obtained by knocking out a portion of the coding region of the yjiT gene in the genomes of the L-valine-producing bacterium CGMCC22721 and wild-type Escherichia coli W3110, respectively, and inserting the brnF-brnE gene (derived from Corynebacterium glutamicum ATCC13032) driven by the Ptrc promoter, while knocking out a portion of the coding region of the yjiV gene and inserting the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168) driven by the Ptrc promoter and the ilvD gene (derived from Escherichia coli W3110) driven by the PilvD promoter, while retaining other nucleotides in the genome without modification.

[0043] In other embodiments of the present invention, the recombinant bacteria are recombinant CGMCC22721-yjiV-trpR and recombinant W3110-yjiV-trpR. Recombinant CGMCC22721-yjiV-trpR and recombinant W3110-yjiV-trpR are obtained by knocking out a portion of the coding region of the yjiV gene in the genomes of L-valine-producing bacteria CGMCC22721 and wild-type Escherichia coli W3110, respectively, and inserting the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168) driven by the Ptrc promoter and the ilvD gene (derived from Escherichia coli W3110) driven by the PilvD promoter, as well as knocking out a portion of the coding region of the trpR gene and inserting the mutant ilvH gene driven by the Ptrc promoter. G14D、S17F This is a recombinant bacterium obtained by inserting a gene while retaining other nucleotides in the genome without alteration.

[0044] In other embodiments of the present invention, the recombinant bacteria are recombinant CGMCC22721-trpR-lacIZ and recombinant W3110-trpR-lacIZ. Recombinant CGMCC22721-trpR-lacIZ and recombinant W3110-trpR-lacIZ have a portion of the coding region of the trpR gene on the genome of the L-valine-producing bacterium CGMCC22721 and the wild-type E. coli W3110 knocked out, respectively, and have a Ptrc promoter-driven mutant ilvH G14D、S17F This recombinant bacterium was obtained by inserting a gene, knocking out a portion of the coding region of the lacI-lacZ gene, inserting a DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter, and maintaining other nucleotides in the genome without modification.

[0045] In other embodiments of the present invention, the recombinant bacteria are recombinant CGMCC22721-lacIZ-ycgH and recombinant W3110-lacIZ-ycgH. Recombinant CGMCC22721-lacIZ-ycgH and recombinant W3110-lacIZ-ycgH are recombinant bacteria obtained by knocking out a portion of the coding region of the lacI-lacZ gene in the genome of the L-valine-producing bacterium CGMCC22721 and the wild-type Escherichia coli W3110, respectively, inserting a DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter, and knocking out a portion of the coding region of the ycgH gene, inserting an ilvC gene (derived from Escherichia coli W3110) driven by the Ptrc promoter, while retaining other nucleotides in the genome without modification.

[0046] In other embodiments of the present invention, the recombinant bacteria are recombinant CGMCC22721-yjiT-yjiV-trpR and recombinant W3110-yjiT-yjiV-trpR. Recombinant strains CGMCC22721-yjiT-yjiV-trpR and W3110-yjiT-yjiV-trpR were created by knocking out a portion of the coding region of the yjiT gene in the genomes of L-valine-producing bacterium CGMCC22721 and wild-type E. coli W3110, respectively, and inserting the brnF-brnE gene (derived from Corynebacterium glutamicum ATCC13032) driven by the Ptrc promoter, knocking out a portion of the coding region of the yjiV gene, and inserting the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168) driven by the Ptrc promoter and the ilvD gene (derived from Escherichia coli) driven by the PilvD promoter. A mutant ilvH gene (derived from coli)W3110 is inserted, and a portion of the coding region of the trpR gene is knocked out, resulting in a Ptrc promoter-driven mutant ilvH. G14D、S17FThis is a recombinant bacterium obtained by inserting a gene while retaining other nucleotides in the genome without alteration.

[0047] In other embodiments of the present invention, the recombinant bacteria are recombinant CGMCC22721-yjiV-trpR-lacIZ and recombinant W3110-yjiV-trpR-lacIZ. Recombinant CGMCC22721-yjiV-trpR-lacIZ and recombinant W3110-yjiV-trpR-lacIZ are obtained by knocking out a portion of the coding region of the yjiV gene in the genomes of L-valine-producing bacteria CGMCC22721 and wild-type Escherichia coli W3110, respectively, inserting the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168) driven by the Ptrc promoter and the ilvD gene (derived from Escherichia coli W3110) driven by the PilvD promoter, knocking out a portion of the coding region of the trpR gene, and inserting the mutant ilvH gene (derived from Escherichia coli W3110) driven by the Ptrc promoter. G14D、S17F This recombinant bacterium was obtained by inserting a gene, knocking out a portion of the coding region of the lacI-lacZ gene, inserting a DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter, and maintaining other nucleotides in the genome without modification.

[0048] In other embodiments of the present invention, the recombinant bacteria are recombinant CGMCC22721-trpR-lacIZ-ycgH and recombinant W3110-trpR-lacIZ-ycgH. Recombinant CGMCC22721-trpR-lacIZ-ycgH and recombinant W3110-trpR-lacIZ-ycgH respectively have a portion of the coding region of the trpR gene on the genome of the L-valine-producing bacterium CGMCC22721 and the wild-type E. coli W3110 knocked out, and a Ptrc promoter-driven mutant ilvH G14D、S17FThis recombinant bacterium was obtained by inserting genes, knocking out a portion of the coding region of the lacI-lacZ gene, inserting a DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter, knocking out a portion of the coding region of the ycgH gene, inserting an ilvC gene (derived from Escherichia coli W3110) driven by the Ptrc promoter, and retaining other nucleotides in the genome.

[0049] In other embodiments of the present invention, the recombinant bacteria are recombinant CGMCC22721-yjiT-yjiV-trpR-lacIZ and recombinant W3110-yjiT-yjiV-trpR-lacIZ. Recombinant strains CGMCC22721-yjiT-yjiV-trpR-lacIZ and W3110-yjiT-yjiV-trpR-lacIZ were created by knocking out a portion of the coding region of the yjiT gene in the genomes of L-valine-producing bacterium CGMCC22721 and wild-type Escherichia coli W3110, respectively, and inserting the brnF-brnE gene (derived from Corynebacterium glutamicum ATCC13032) driven by the Ptrc promoter, knocking out a portion of the coding region of the yjiV gene, and inserting the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168) driven by the Ptrc promoter and the ilvD gene (derived from Escherichia coli) driven by the PilvD promoter. A mutant ilvH gene was created by inserting a gene from coli (W3110), knocking out a portion of the coding region of the trpR gene, and driving the Ptrc promoter. G14D、S17F This recombinant bacterium was obtained by inserting a gene, knocking out a portion of the coding region of the lacI-lacZ gene, inserting a DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter, and maintaining other nucleotides in the genome without modification.

[0050] In other embodiments of the present invention, the recombinant bacteria are recombinant CGMCC22721-yjiV-trpR-lacIZ-ycgH and recombinant W3110-yjiV-trpR-lacIZ-ycgH. Recombinant bacteria CGMCC22721-yjiV-trpR-lacIZ-ycgH and W3110-yjiV-trpR-lacIZ-ycgH were created by knocking out a portion of the coding region of the yjiV gene in the genomes of L-valine-producing bacteria CGMCC22721 and wild-type Escherichia coli W3110, respectively, inserting the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168) driven by the Ptrc promoter and the ilvD gene (derived from Escherichia coli W3110) driven by the PilvD promoter, knocking out a portion of the coding region of the trpR gene, and creating a mutant ilvH gene driven by the Ptrc promoter. G14D、S17F This recombinant bacterium was obtained by inserting a gene, knocking out a portion of the coding region of the lacI-lacZ gene, inserting a DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter, knocking out a portion of the coding region of the ycgH gene, inserting an ilvC gene (derived from Escherichia coli W3110) driven by the Ptrc promoter, and maintaining other nucleotides in the genome without modification.

[0051] In other embodiments of the present invention, the recombinant bacteria are recombinant CGMCC22721-yjiT-yjiV-trpR-lacIZ-ycgH and recombinant W3110-yjiT-yjiV-trpR-lacIZ-ycgH. Recombinant strains CGMCC22721-yjiT-yjiV-trpR-lacIZ-ycgH and W3110-yjiT-yjiV-trpR-lacIZ-ycgH were created by knocking out a portion of the coding region of the yjiT gene in the genomes of L-valine-producing strain CGMCC22721 and wild-type Escherichia coli W3110, respectively, and inserting the brnF-brnE gene (derived from Corynebacterium glutamicum ATCC13032) driven by the Ptrc promoter, knocking out a portion of the coding region of the yjiV gene, and inserting the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168) driven by the Ptrc promoter and the ilvD gene (derived from Escherichia coli) driven by the PilvD promoter. A mutant ilvH gene was created by inserting a gene from coli (W3110), knocking out a portion of the coding region of the trpR gene, and driving the Ptrc promoter. G14D、S17F This recombinant bacterium was obtained by inserting a gene, knocking out a portion of the coding region of the lacI-lacZ gene, inserting a DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter, knocking out a portion of the coding region of the ycgH gene, inserting an ilvC gene (derived from Escherichia coli W3110) driven by the Ptrc promoter, and maintaining other nucleotides in the genome without modification.

[0052] The present invention also provides a method for preparing genetically modified bacteria for valine production. This method involves modifying a recipient bacterium according to A1), A2), A3), A4), or A5) above to obtain the desired genetically modified bacterium. The recipient bacterium is Escherichia coli.

[0053] The above method involves making any two, three, four, or five modifications of A1), A2), A3), A4), and A5) above to the recipient bacteria.

[0054] The present invention also provides a method for preparing L-valine. This method includes culturing genetically modified bacteria to obtain L-valine.

[0055] In the above method, the culture of genetically modified bacteria may be carried out using a culture medium capable of growing genetically modified bacteria. And / or, the cultivation of genetically modified bacteria may be carried out using conditions that enable the growth of genetically modified bacteria.

[0056] The present invention also provides a product for L-valine production. This product is one of P1), P2), P3), P4), P5), or a product consisting of any two, any three, any four, or any five of these. P1) is a substance that realizes A11), A12), and A13) individually or simultaneously: A11) Knocking out the yjiT gene in the receptor bacterium, or suppressing the expression of the yjiT gene, or suppressing the activity of the protein encoded by the yjiT gene, A12) To increase the content of the protein encoded by the brnF gene in the receptor bacteria, or to enhance the activity of the protein encoded by the brnF gene. A13) Increasing the content of the protein encoded by the brnE gene in the receptor bacteria, or enhancing the activity of the protein encoded by the brnE gene; P2) is a substance that realizes A21), A22), and A23) individually or simultaneously: A21) Knocking out the yjiV gene in the receptor bacterium, or suppressing the expression of the yjiV gene, or suppressing the activity of the protein encoded by the yjiV gene, A22) To increase the content of the protein encoded by the ilvE gene in the receptor bacteria, or to enhance the activity of the protein encoded by the ilvE gene. A23) Increasing the content of the protein encoded by the ilvD gene in the receptor bacteria, or enhancing the activity of the protein encoded by the ilvD gene; P3) is a substance that realizes A31) and A32) individually or simultaneously: A31) Knocking out the trpR gene of the receptor bacterium, or suppressing the expression of the trpR gene, or suppressing the activity of the protein encoded by the trpR gene, A32) ilvH in receptor bacteria G14D、S17F Increasing the content of gene-encoded proteins, or ilvH G14D、S17F To enhance the activity of genes-encoded proteins; P4) is a substance that realizes A41), A42), and A43) individually or simultaneously: A41) Knocking out the lacI gene of the receptor bacterium, or suppressing the expression of the lacI gene, or suppressing the activity of the protein encoded by the lacI gene, A42) Knocking out the lacZ gene of the receptor bacterium, or suppressing the expression of the lacZ gene, or suppressing the activity of the protein encoded by the lacZ gene, A43) Increasing the DNA polymerase content in receptor bacteria, or enhancing the activity of DNA polymerase; P5) is a substance that realizes A51) and A52) individually or simultaneously: A51) Knocking out the ycgH gene in the receptor bacterium, or suppressing the expression of the ycgH gene, or suppressing the activity of the protein encoded by the ycgH gene, A52) To increase the content of the protein encoded by the ilvC gene in the receptor bacteria, or to enhance the activity of the protein encoded by the ilvC gene.

[0057] The use of genetically modified bacteria or products in the production of L-valine, or in the preparation of products for L-valine production, or in the preparation of L-valine-containing foods, feeds, or pharmaceuticals is also within the scope of protection of this invention.

[0058] The genetically modified bacteria and method for preparing the same, as well as related products of the present invention, can be used to produce a variety of products (including, but not limited to, valine in the examples). The products to be produced may also include glutamic acid, threonine, tryptophan, arginine, lysine, glycine, alanine, leucine, isoleucine, methionine, proline, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, aspartic acid, histidine, shikimic acid, protocatechuic acid, succinic acid, α-ketoglutaric acid, citric acid, ornithine, citrulline, and the like.

[0059] The present invention will be described in detail below with reference to specific embodiments, but the embodiments shown are merely for illustrative purposes and do not limit the scope of the present invention. The embodiments provided below can be used by those skilled in the art as a guide for further improvements, but do not limit the present invention in any way.

[0060] Deposit information for biomaterials Classification name: Escherichia coli Strain number: YP045 Depository name: Center for Ordinary Microorganisms, China Microbial Species Preservation and Storage Administration Abbreviation for depositary institution: CGMCC Depository address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Postal Code: 100101 Deposit date: June 15, 2021 Deposit Center Registration Number: CGMCC No.22721 [Brief explanation of the drawing]

[0061]

Figure 1

Figure 2

Figure 3

[0062] The experimental methods in the following examples are conventional methods unless otherwise specified, and are carried out in accordance with the techniques or conditions described in the literature in this art, or in accordance with the product instructions. The materials, reagents, and equipment used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples are all performed by setting up three repeated experiments and taking the average value as the result. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5' nucleotide of the corresponding DNA / RNA, and the last position is the 3' nucleotide of the corresponding DNA / RNA.

[0063] In the following example, the L-valine-producing bacterium CGMCC22721 is Escherichia coli YP045. This strain was deposited with the Ordinary Microbial Center of the China Microbial Species Preservation and Storage Administration on June 15, 2021, with deposit number CGMCC No. 22721. Ningxia Yipin Biotechnology Co., Ltd., the depositor of Escherichia coli YP045, authorized Heilongjiang Yipin Biotechnology Co., Ltd. to use this strain.

[0064] Example 1: Construction of a genetically modified bacterium in which the yjiT gene is deleted from the genome and the brnF-brnE gene is overexpressed. Based on the genome sequence of Escherichia coli W3110 published by NCBI, CRISPR / Cas9 genome editing technology was used to knock out the yjiT gene (sequencing analysis confirmed that the complete yjiT gene (GeneID: 945056, updated 2023-04-14)) in the genomes of L-valine-producing strain CGMCC22721 and wild-type E. coli W3110, and to insert the brnF-brnE gene (branched-chain amino acid exporter), driven by the Ptrc promoter, derived from Corynebacterium glutamicum ATCC13032. This allowed for a more detailed study of the effects of these genes on L-valine synthesis.

[0065] 1. Construction of sgRNA Based on the Escherichia coli W3110 genome sequence published by NCBI, sgRNA target sequences were designed using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ). After selecting appropriate sgRNA target sequences, homology arm sequences of a linearized pGRB cloning vector were added to the 5' and 3' ends of the target sequences, and complete sgRNA plasmids were formed by recombination.

[0066] When amplifying sgRNA fragments, no template is required; only PCR annealing is necessary. The reaction system and program are as follows: PCR reaction system: sgRNA-1F 10 μL, sgRNA-1R 10 μL; PCR reaction program: Denaturate at 95°C for 5 minutes, anneal at 50°C for 1 minute. After annealing was complete, the target DNA fragment of sgRNA was recovered using a DNA purification kit, its DNA concentration was measured, and then it was diluted to a concentration of 100 ng / μL.

[0067] The pGRB plasmid was digested with Spe I enzyme and then dephosphorylated. Recombination of the sgRNA with the dephosphorylated linear plasmid was performed using the Gibson Assembly Kit (New England). Recombination reaction system: 2.5 μL of enzyme for NEB assembly, 2 μL of dephosphorylated linear plasmid, and 0.5 μL of sgRNA target DNA fragment. After a recombination reaction at 50°C for 30 minutes, the product was transformed into DH5α-competent cells, the plasmid was extracted, and sequencing was performed using the sequencing primers sgRNA-PF / sgRNA-PR. Plasmids that matched the sequencing results were named pGRB-sgRNA-1.

[0068] The primers used in this experiment are as follows (synthesized by Shanghai Invitrogen). Underlined nucleotides represent the homology arm sequences of the pGRB cloning vector, and bolded nucleotides represent the sgRNA sequences: sgRNA-1F:5'- TGACAGCTAGCTCAGTCCTAGGTATAATACTAGT GTACGCGTTGCCAATTCTAT GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG -3' (SEQ ID No. 14), sgRNA-1R:5'- CCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC ATAGAATTGGCAACGCGTAC ACTAGTATTATACCTAGGACTGAGCTAGCTGTCA -3' (SEQ ID No. 15), sgRNA-PF:5'-GTCTCATGAGCGGATACATATTTG-3' (SEQ ID No.16), sgRNA-PR:5'-GCGTCAGGTGCATAAACAGA-3' (SEQ ID No.17).

[0069] 2. PCR amplification of homologous recombination fragments Based on the genome sequence of Escherichia coli W3110 published by NCBI, primers for knocking out the yjiT gene and primers for inserting the Ptrc-brnF-brnE gene sequence were designed and synthesized. Using CRISPR / Cas9 genome editing techniques, the yjiT gene was knocked out in the genomes of L-valine-producing bacterium CGMCC22721 and wild-type E. coli W3110, and the brnF-brnE gene (derived from Corynebacterium glutamicum ATCC13032), driven by the Ptrc promoter, was inserted.

[0070] The primers are as follows (synthesized by Shanghai Invitrogen): P1:5'-GAGTGATGAGCGGTTGAAG-3' (SEQ ID No.18), P2:5'-CTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAAAAACAGGCAGCAAAGTCC-3'(SEQ ID No.19), P3:5'-CGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCGTGCAAAAAACGCAAGAG-3'(SEQ ID No.20), P4:5'-CTTCACAGGTAGTGCTTTTAGTTAGAAAAGATTCACCAGTCCAAC-3' (SEQ ID No.21), P5:5'-GTGAATCTTTTCTAACTAAAAGCACTACCTGTGAAGG-3' (SEQ ID No.22), P6:5'-CTGCGGCAATAATCAACG-3' (SEQ ID No. 23).

[0071] Using the genomic DNA of *E. coli* W3110 as a template, PCR amplification was performed with primers P1 / P2, P5 / P6, and KAPA HiFi HotStart (Shanghai Huaya Sichuang Biotechnology Co., Ltd., KK2601) to obtain upstream and downstream homology arm fragments with sizes of 801 bp and 684 bp, respectively. Using the genomic DNA of *Corynebacterium glutamicum* ATCC13032 as a template, PCR amplification was performed with primers P3 / P4 and KAPA HiFi HotStart to obtain a Ptrc-brnF-brnE gene fragment with a size of 1153 bp. After the completion of the PCR reaction, each was recovered by agarose gel electrophoresis using a column-type DNA gel recovery kit. The recovered DNA was subjected to overlap PCR with primers P1 / P6 to obtain the homologous recombination DNA fragment ΔyjiT-Ptrc-brnF-brnE (SEQ ID No. 1) 2579 bp.

[0072] In SEQ ID No. 1, positions 758-831 represent the Ptrc promoter, and positions 832-1910 represent the brnF-brnE genes (the genes encoding the brnF protein shown in SEQ ID No. 2 (the coding sequence is positions 832-1587 in SEQ ID No. 1), and the genes encoding the brnE protein shown in SEQ ID No. 3 (the coding sequence is positions 1584-1910 in SEQ ID No. 1)).

[0073] 3. Preparation and transformation of competent cells The pREDCas9 plasmid (containing the spectinomycin resistance gene) was extracted and transformed into competent cells of L-valine-producing bacteria CGMCC22721 and E. coli W3110, respectively. These cells were then spread on 2-YT agar plates containing spectinomycin (100 mg / L) and cultured at 32°C. Single colonies showing resistance to spectinomycin (100 mg / L) were selected and identified by PCR using primers pRedCas9-PF / pRedCas9-PR. Cells yielding 943 bp were designated as CGMCC22721-Cas9 and W3110-Cas9 transformants containing the pREDCas9 plasmid.

[0074] The primers are as follows (synthesized by Shanghai Invitrogen): pRedCas9-PF:5'-GCAGTGGCGGTTTTCATG-3' (SEQ ID No.24), pRedCas9-PR:5'-CCTTGGTGATCTCGCCTTTC-3' (SEQ ID No. 25).

[0075] Competent cells of CGMCC22721-Cas9 and W3110-Cas9 were prepared. The bacterial cells were OD 600 Once the cells had grown to 0.1 mM, a final concentration of 0.1 mM IPTG was added to induce homologous recombination via λ-Red. 600 When the ratio reached 0.4, the bacterial cells were harvested and competent cells were prepared. These were transformed with the pGRB-sgRNA-1 plasmid and the homologous recombination DNA fragment ΔyjiT-Ptrc-brnF-brnE, respectively, and spread on 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L), and incubated at 32°C for 12 hours. After culturing, the obtained single colonies were subcultured and then PCR identified using primers P7 / P8. Fragments of size 1720 bp that were amplified by PCR were considered positive transformants.

[0076] The primers are as follows (synthesized by Shanghai Invitrogen): P7:5'-GCTGTATTCCTTATGTGGACC-3' (SEQ ID No.26), P8:5'-GCAGGAATCCAAAGTCAGC-3' (SEQ ID No.27).

[0077] Positive transformants were seeded in 2-YT medium containing spectinomycin (100 mg / L) and 0.2% arabinose, and the plasmid pGRB-sgRNA-1 was removed. Colonies that grew with spectinomycin (100 mg / L) but not with ampicillin (100 mg / L) were selected, and these colonies were re-seed in 2-YT medium and cultured at 42°C to remove the pREDCas9 plasmid. Colonies that did not grow with spectinomycin (100 mg / L) but grew in antibiotic-free 2-YT were selected, and PCR identification was performed again with primers P1 / P6. Strains from which a fragment of size 2579 bp (SEQ ID No. 1) was amplified were designated as positive strains. Sequence analysis was performed on these positive strains, and the strains with correct results were named CGMCC22721-yjiT and W3110-yjiT, respectively.

[0078] Recombinant strains CGMCC22721-yjiT and W3110-yjiT both lack a yjiT gene and overexpress the brnF-brnE gene (derived from Corynebacterium glutamicum ATCC13032), which is driven by the Ptrc promoter. Specifically, CGMCC22721-yjiT and W3110-yjiT are recombinant strains obtained by knocking out a portion of the coding region of the yjiT gene in the genomes of L-valine-producing strain CGMCC22721 and wild-type Escherichia coli W3110, respectively, while inserting the brnF-brnE gene (derived from Corynebacterium glutamicum ATCC13032), which is driven by the Ptrc promoter, and retaining other nucleotides in the genome without modification.

[0079] Example 2: Construction of a genetically modified bacterium in which the yjiV gene is deleted from the genome and the ilvE-ilvD genes are overexpressed. Based on the genome sequence of Escherichia coli W3110 published by NCBI, the yjiV gene (GeneID: 2847669, updated 2023-04-14) was knocked out in the genomes of L-valine-producing strain CGMCC22721 and wild-type Escherichia coli W3110 using CRISPR / Cas9 genome editing technology. (Sequencing analysis confirmed that these strains retain the complete yjiV gene on their chromosomes.) The ilvE gene (branched-chain amino-acid aminotransferase, derived from Bacillus subtilis subsp. subtilis str.168), driven by the Ptrc promoter, and the ilvD gene (dihydroxyacid dehydratase, derived from Escherichia coli W3110), driven by the PilvD promoter, were inserted. This allowed researchers to study in more detail the effects of these genes on L-valine synthesis.

[0080] 1. Construction of sgRNA Based on the Escherichia coli W3110 genome sequence published by NCBI, sgRNA target sequences were designed using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ). After selecting appropriate sgRNA target sequences, homology arm sequences of a linearized pGRB cloning vector were added to the 5' and 3' ends of the target sequences, and complete sgRNA plasmids were formed by recombination.

[0081] When amplifying sgRNA fragments, no template is required; only PCR annealing is necessary. The reaction system and program are as follows: PCR reaction system: sgRNA-2F 10 μL, sgRNA-2R 10 μL; PCR reaction program: Denaturate at 95°C for 5 minutes, anneal at 50°C for 1 minute. After annealing was complete, the target DNA fragment of sgRNA was recovered using a DNA purification kit, its DNA concentration was measured, and then it was diluted to a concentration of 100 ng / μL.

[0082] The pGRB plasmid was digested with Spe I enzyme and then dephosphorylated. Recombination of the sgRNA with the dephosphorylated linear plasmid was performed using the Gibson Assembly Kit (New England). Recombination reaction system: 2.5 μL of enzyme for NEB assembly, 2 μL of dephosphorylated linear plasmid, and 0.5 μL of sgRNA target DNA fragment. After a recombination reaction at 50°C for 30 minutes, the product was transformed into DH5α-competent cells, the plasmid was extracted, and sequencing was performed using the sequencing primers sgRNA-PF / sgRNA-PR. Plasmids that matched the sequencing results were named pGRB-sgRNA-2.

[0083] The primers used in this experiment are as follows (synthesized by Shanghai Invitrogen). Underlined nucleotides represent the homology arm sequences of the pGRB cloning vector, and bolded nucleotides represent the sgRNA sequences: sgRNA-2F:5'- TGACAGCTAGCTCAGTCCTAGGTATAATACTAGT CTATTGATATTATCAATACA GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG -3' (SEQ ID No. 28), sgRNA-2R:5'- CCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC TGTATTGATAATATCAATAG ACTAGTATTATACCTAGGACTGAGCTAGCTGTCA -3' (SEQ ID No. 29).

[0084] 2. PCR amplification of homologous recombination fragments Based on the genome sequence of Escherichia coli W3110 published by NCBI, primers for knocking out the yjiV gene and primers for inserting the Ptrc-ilvE gene and the PilvD-ilvD gene were designed and synthesized. Using CRISPR / Cas9 genome editing techniques, the yjiV gene was knocked out in the genomes of L-valine-producing strain CGMCC22721 and wild-type Escherichia coli W3110, and the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168) driven by the Ptrc promoter and the ilvD gene (derived from Escherichia coli W3110) driven by the PilvD promoter were inserted.

[0085] The primers are as follows (synthesized by Shanghai Invitrogen): P9:5'-TGAATGGACTGCTATGCG-3' (SEQ ID No.30), P10:5'-CACAGTGTATTAAGCAGACGTTAACAACGCAGTACTTCCTGCTG-3'(SEQ ID No.31), P11:5'-GAAGTACTGCGTTGTTAACGTCTGCTTAATACACTGTG-3' (SEQ ID No.32), P12:5'-GTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGGAACTTTTTAAATATATGGAG-3'(SEQ ID No.33), P13:5'-CTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATTAACCCCCCAGTTTCGATTTAT-3'(SEQ ID No.34), P14:5'-CCACCAGCACATCCGTTGAAATACAAAAAATGGGAC-3'(SEQ ID No.35), P15:5'-TCCCATTTTTTGTATTTCAACGGATGTGCTGGTGG-3' (SEQ ID No.36), P16:5'-TGAAGACACGCTGGCTAAC-3' (SEQ ID No. 37).

[0086] Using the genomic DNA of *E. coli* W3110 as a template, PCR amplification was performed with primers P9 / P10, P13 / P14, P15 / P16, and KAPA HiFi HotStart to obtain fragments of the upstream homology arm, the PilvD-ilvD gene, and the downstream homology arm, with sizes of 831 bp, 1979 bp, and 856 bp, respectively. Using the genomic DNA of *Bacillus subtilis* subsp. subtilis str.168 as a template, PCR amplification was performed with primers P11 / P12 and KAPA HiFi HotStart to obtain a fragment of the Ptrc-ilvE gene with a size of 1161 bp. After the completion of the PCR reaction, each fragment was recovered by agarose gel electrophoresis using a column-type DNA gel recovery kit. The recovered DNA was subjected to overlap PCR using primers P9 / P16 to obtain a homologous recombination DNA fragment ΔyjiV-Ptrc-ilvE-PilvD-ilvD (SEQ ID No. 4) of 4732 bp.

[0087] In SEQ ID No. 4, positions 3828-3893 represent the PilvD promoter, positions 1977-3827 represent the ilvD gene (the gene encoding the ilvD protein shown in SEQ ID No. 5), positions 1903-1976 represent the Ptrc promoter, and positions 808-1902 represent the ilvE gene (the gene encoding the ilvE protein shown in SEQ ID No. 6).

[0088] 3. Preparation and transformation of competent cells Competent cells of CGMCC22721-Cas9 and W3110-Cas9 were prepared in Example 1. 600Once the cells had grown to 0.1 mM, a final concentration of 0.1 mM IPTG was added to induce homologous recombination via λ-Red. 600 When the ratio reached 0.4, the bacterial cells were harvested and competent cells were prepared. These were transformed with the pGRB-sgRNA-2 plasmid and the homologous recombination DNA fragment ΔyjiV-Ptrc-ilvE-PilvD-ilvD, respectively. These were then spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L) and cultured at 32°C for 12 hours. After culturing, the obtained single colonies were subcultured and then PCR identification was performed using primers P17 / P18. Fragments of size 1543 bp that were amplified by PCR were considered positive transformants.

[0089] The primers are as follows (synthesized by Shanghai Invitrogen): P17:5'-AGCGAGTTTCCAATACCG-3' (SEQ ID No.38), P18:5'-ATTTCTCCTGCTTTCGGC-3' (SEQ ID No. 39).

[0090] Positive transformants were seeded in 2-YT medium containing spectinomycin (100 mg / L) and 0.2% arabinose, and the plasmid pGRB-sgRNA-2 was removed. Colonies that grew with spectinomycin (100 mg / L) but not with ampicillin (100 mg / L) were selected, and these colonies were re-seeded in 2-YT medium and cultured at 42°C to remove the pREDCas9 plasmid. Colonies that did not grow with spectinomycin (100 mg / L) but grew in antibiotic-free 2-YT were selected, and PCR identification was performed again with primers P9 / P16. Strains from which a fragment of size 4732 bp (SEQ ID No. 4) was amplified were designated as positive strains. Sequence analysis was performed on these positive strains, and the strains with correct results were named CGMCC22721-yjiV and W3110-yjiV, respectively.

[0091] Both recombinant strains CGMCC22721-yjiV and W3110-yjiV lack the yjiV gene and overexpress the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168), which is driven by the Ptrc promoter, and the ilvD gene (derived from Escherichia coli W3110), which is driven by the PilvD promoter. Specifically, recombinant bacteria CGMCC22721-yjiV and W3110-yjiV are recombinant bacteria obtained by knocking out a portion of the coding region of the yjiV gene in the genomes of the L-valine-producing bacterium CGMCC22721 and the wild-type Escherichia coli W3110, respectively, while inserting the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168) driven by the Ptrc promoter and the ilvD gene (derived from Escherichia coli W3110) driven by the PilvD promoter, while retaining other nucleotides in the genome without modification.

[0092] Example 3: Construction of a genetically modified bacterium in which the trpR gene is deleted from the genome and the ilvH gene is overexpressed. Based on the genome sequence of Escherichia coli W3110 published by NCBI, CRISPR / Cas9 genome editing technology was used to knock out the trpR gene in the genomes of L-valine-producing strain CGMCC22721 and wild-type E. coli W3110 (sequencing analysis confirmed that the complete trpR gene (DNA-binding transcriptional repressor; GeneID: 948917, updated 2023-04-14) is retained on the chromosomes of these strains), and the Ptrc promoter-driven ilvH G14D、S17F We inserted the gene (acetolactate synthase). This allowed us to study in more detail the effects of these genes on L-valine synthesis.

[0093] 1. Construction of sgRNA Based on the Escherichia coli W3110 genome sequence published by NCBI, sgRNA target sequences were designed using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ). After selecting appropriate sgRNA target sequences, homology arm sequences of a linearized pGRB cloning vector were added to the 5' and 3' ends of the target sequences, and complete sgRNA plasmids were formed by recombination.

[0094] When amplifying sgRNA fragments, no template is required; only PCR annealing is necessary. The reaction system and program are as follows: PCR reaction system: sgRNA-3F 10 μL, sgRNA-3R 10 μL; PCR reaction program: Denaturate at 95°C for 5 minutes, anneal at 50°C for 1 minute. After annealing was complete, the target DNA fragment of sgRNA was recovered using a DNA purification kit, its DNA concentration was measured, and then it was diluted to a concentration of 100 ng / μL.

[0095] The pGRB plasmid was digested with Spe I enzyme and then dephosphorylated. Recombination of the sgRNA with the dephosphorylated linear plasmid was performed using the Gibson Assembly Kit (New England). Recombination reaction system: 2.5 μL of enzyme for NEB assembly, 2 μL of dephosphorylated linear plasmid, and 0.5 μL of sgRNA target DNA fragment. After a recombination reaction at 50°C for 30 minutes, the product was transformed into DH5α-competent cells, the plasmid was extracted, and sequencing was performed using the sequencing primers sgRNA-PF / sgRNA-PR. Plasmids that matched the sequencing results were named pGRB-sgRNA-3.

[0096] The primers used in this experiment are as follows (synthesized by Shanghai Invitrogen). Underlined nucleotides represent the homology arm sequences of the pGRB cloning vector, and bolded nucleotides represent the sgRNA sequences: sgRNA-3F:5'- TGACAGCTAGCTCAGTCCTAGGTATAATACTAGT CGTGAGTTAAAAAATGAACT GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG -3' (SEQ ID No. 40), sgRNA-3R:5'- CCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC AGTTCATTTTTTAACTCACG ACTAGTATTATACCTAGGACTGAGCTAGCTGTCA -3' (SEQ ID No. 41).

[0097] 2. PCR amplification of homologous recombination fragments Based on the Escherichia coli W3110 genome sequence published by NCBI, primers for knocking out the trpR gene and Ptrc-ilvH G14D、S17F We designed and synthesized primers for inserting gene sequences. Using CRISPR / Cas9 genome editing techniques, we knocked out the trpR gene in the genomes of L-valine-producing bacterium CGMCC22721 and wild-type E. coli W3110, and also knocked out ilvH, which is driven by the Ptrc promoter. G14D、S17F A gene was inserted.

[0098] The primers are as follows (synthesized by Shanghai Invitrogen): P19:5'-CCAATCTGGTGAAGAGCAAG-3' (SEQ ID No.42), P20:5'-CTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATGCTGAATAGGGTGATTGTTG-3' (SEQ ID No.43), P21:5'-CAATTTCACACAGGAAACAGACCATGCGCCGGATATTATCAGTCTTACTCGAAAATGAATCAGACGCGTTATTCCGC-3' (SEQ ID No.44), P22:5'-GTCTTATCATGCCTACCAAATCAACGCATTATTTTATCG-3'(SEQ ID No.45), P23:5'-CGATAAAATAATGCGTTGATTTGGTAGGCATGATAAGAC-3'(SEQ ID No.56), P24:5'-GTGCGTCCTAAATCGCTAC-3' (SEQ ID No.47).

[0099] Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed with primers P19 / P20, P21 / P22, P23 / P24 and KAPA HiFi HotStart, resulting in upstream homology arms of sizes 831 bp, 535 bp, and 801 bp, respectively, and Ptrc-ilvH. G14D、S17F Gene and downstream homology arm fragments were obtained. After the PCR reaction was complete, each was recovered by agarose gel electrophoresis using a column-type DNA gel recovery kit. The recovered DNA was subjected to overlap PCR with primers P19 / P24 to obtain the homologous recombination DNA fragment ΔtrpR-Ptrc-ilvH (SEQ ID No. 7) 2108 bp.

[0100] In SEQ ID No. 7, the Ptrc promoter is shown at positions 761-834, and the ilvH promoter is shown at positions 835-1326. G14D、S17F Gene (ilvH, indicated by SEQ ID No. 8) G14D、S17F It is a gene that codes for a protein.

[0101] 3. Preparation and transformation of competent cells Competent cells of CGMCC22721-Cas9 and W3110-Cas9 were prepared in Example 1. 600 Once the cells had grown to 0.1 mM, a final concentration of 0.1 mM IPTG was added to induce homologous recombination via λ-Red. 600When the ratio reached 0.4, the bacterial cells were harvested and competent cells were prepared. These were transformed with the pGRB-sgRNA-3 plasmid and the homologous recombinant DNA fragment ΔtrpR-Ptrc-ilvH, respectively, and spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L). The cells were incubated at 32°C for 12 hours. After culturing, the single colonies obtained were subcultured and then PCR identification was performed using primers P25 / P26. Fragments of size 1437 bp that were amplified by PCR were considered positive transformants.

[0102] The primers are as follows (synthesized by Shanghai Invitrogen): P25:5'-CATCGGCGAAGAGTATGAG-3' (SEQ ID No.48), P26:5'-AAAGTCCGACCACACCAGAG-3' (SEQ ID No.49).

[0103] Positive transformants were seeded in 2-YT medium containing spectinomycin (100 mg / L) and 0.2% arabinose, and the plasmid pGRB-sgRNA-3 was removed. Colonies that grew with spectinomycin (100 mg / L) but not with ampicillin (100 mg / L) were selected, and these colonies were re-seeded in 2-YT medium and cultured at 42°C to remove the pREDCas9 plasmid. Colonies that did not grow with spectinomycin (100 mg / L) but grew in antibiotic-free 2-YT were selected, and PCR identification was performed again with primers P19 / P24. Strains from which a fragment of size 2108 bp (SEQ ID No. 7) was amplified were designated as positive strains. Sequence analysis was performed on these positive strains, and the strains with correct results were named CGMCC22721-trpR and W3110-trpR, respectively.

[0104] Recombinant strains CGMCC22721-trpR and W3110-trpR both lack a trpR gene and are driven by the Ptrc promoter, and also exhibit ilvH G14D、S17FThe gene is overexpressed. Specifically, recombinant bacteria CGMCC22721-trpR and W3110-trpR knock out a portion of the coding region of the trpR gene in the genomes of L-valine-producing bacteria CGMCC22721 and wild-type E. coli W3110, respectively, and also overexpress ilvH, which is driven by the Ptrc promoter. G14D、S17F This is a recombinant bacterium obtained by inserting a gene while retaining other nucleotides in the genome without alteration.

[0105] Example 4: Construction of a genetically modified bacterium in which the lacI-lacZ gene is deleted from the genome and the DNA polymerase gene is overexpressed. Based on the genome sequence of Escherichia coli W3110 published by NCBI, CRISPR / Cas9 genome editing technology was used to knock out the lacI-lacZ genes (sequencing analysis confirmed that these strains retain complete lacI-lacZ genes (DNA-binding transcriptional repressor; beta-D-galactosidase; lacI GeneID:945007, updated 2023-04-14; lacZ GeneID:945006, updated 2023-04-14)) in the genomes of the L-valine-producing strain CGMCC22721 and the wild-type Escherichia coli W3110, while inserting a DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter. This allowed for a more detailed study of the effects of these genes on L-valine synthesis.

[0106] 1. Construction of sgRNA Based on the Escherichia coli W3110 genome sequence published by NCBI, sgRNA target sequences were designed using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ). After selecting appropriate sgRNA target sequences, homology arm sequences of a linearized pGRB cloning vector were added to the 5' and 3' ends of the target sequences, and complete sgRNA plasmids were formed by recombination.

[0107] When amplifying sgRNA fragments, no template is required; only PCR annealing is necessary. The reaction system and program are as follows: PCR reaction system: sgRNA-4F 10 μL, sgRNA-4R 10 μL; PCR reaction program: Denaturate at 95°C for 5 minutes, anneal at 50°C for 1 minute. After annealing was complete, the target DNA fragment of sgRNA was recovered using a DNA purification kit, its DNA concentration was measured, and then it was diluted to a concentration of 100 ng / μL.

[0108] The pGRB plasmid was digested with Spe I enzyme and then dephosphorylated. Recombination of the sgRNA with the dephosphorylated linear plasmid was performed using the Gibson Assembly Kit (New England). Recombination reaction system: 2.5 μL of enzyme for NEB assembly, 2 μL of dephosphorylated linear plasmid, and 0.5 μL of sgRNA target DNA fragment. After a recombination reaction at 50°C for 30 minutes, the product was transformed into DH5α-competent cells, the plasmid was extracted, and sequencing was performed using the sequencing primers sgRNA-PF / sgRNA-PR. Plasmids that matched the sequencing results were named pGRB-sgRNA-4.

[0109] The primers used in this experiment are as follows (synthesized by Shanghai Invitrogen). Underlined nucleotides represent the homology arm sequences of the pGRB cloning vector, and bolded nucleotides represent the sgRNA sequences: sgRNA-4F:5'- TGACAGCTAGCTCAGTCCTAGGTATAATACTAGT TCACTGCCCGCTTTCCAGTC GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG -3' (SEQ ID No. 50), sgRNA-4R:5'- CCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC GACTGGAAAGCGGGCAGTGA ACTAGTATTATACCTAGGACTGAGCTAGCTGTCA -3' (SEQ ID No. 51).

[0110] 2. PCR amplification of homologous recombination fragments Based on the genome sequence of Escherichia coli W3110 published by NCBI, primers were designed and synthesized to knock out the lacI-lacZ gene sequence and to insert the PxylF-DNA polymerase gene sequence. Using CRISPR / Cas9 genome editing techniques, the lacI-lacZ gene was knocked out in the genomes of L-valine-producing strain CGMCC22721 and wild-type Escherichia coli W3110, and a DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter was inserted.

[0111] The primers are as follows (synthesized by Shanghai Invitrogen): P27:5'-CGGTAATAATCCACAGCAGG-3'(SEQ ID No.52), P28:5'-GACTTCGCGTTCGCGTAACAGGTAGCAGAGCGGGTA-3' (SEQ ID No.53), P29:5'-TACCCGCTCTGCTACCTGTTACGCGAACGCGAAGTC-3' (SEQ ID No.54), P30:5'-CTAACTACAGAAGGCCCTACACCATGAACACGATTAACATCGC-3'(SEQ ID No.55), P31:5'-GCGATGTTAATCGTGTTCATGGTGTAGGGCCTTCTGTAGTTAG-3' (SEQ ID No.56), P32:5'-CATTAAGTTCTGTCTCGGCGGAGATAATTCACAAGTGTGCG-3'(SEQ ID No.57), P33:5'-CGCACACTTGTGAATTATCTCCGCCGAGACAGAACTTAATG-3' (SEQ ID No.58), P34:5'-GGTTACGGACAGAACTACCG-3' (SEQ ID No.59).

[0112] Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed with primers P27 / P28, P31 / P32, P33 / P34, and KAPA HiFi HotStart to obtain fragments of the upstream homology arm, PxylF promoter, and downstream homology arm, with sizes of 718 bp, 305 bp, and 928 bp, respectively. Using the genomic DNA of E. coli BL21 as a template, PCR amplification was performed with primers P29 / P30 and KAPA HiFi HotStart to obtain a fragment of the DNA polymerase gene with a size of 2693 bp. After the completion of the PCR reaction, each was recovered by agarose gel electrophoresis using a column-type DNA gel recovery kit. The recovered DNA was subjected to overlap PCR with primer P27 / P34 to obtain the homologous recombination DNA fragment ΔlacIZ-PxylF-DNA polymerase (SEQ ID No. 9) 4524 bp.

[0113] In SEQ ID No. 9, positions 3353-3617 represent the PxylF promoter, and positions 701-3352 represent the DNA polymerase gene (the gene encoding DNA polymerase shown in SEQ ID No. 10).

[0114] 3. Preparation and transformation of competent cells Competent cells of CGMCC22721-Cas9 and W3110-Cas9 were prepared in Example 1. 600 Once the cells had grown to 0.1 mM, a final concentration of 0.1 mM IPTG was added to induce homologous recombination via λ-Red. 600 When the ratio reached 0.4, the bacterial cells were harvested and competent cells were prepared. These were transformed with the pGRB-sgRNA-4 plasmid and the homologous recombination DNA fragment ΔlacIZ-PxylF-DNA polymerase, respectively. These cells were then spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L) and cultured at 32°C for 12 hours. After culture, the obtained single colonies were subcultured and then PCR identification was performed using primers P35 / P36. Cells that amplified a fragment of size 1655 bp were considered positive transformants.

[0115] The primers are as follows (synthesized by Shanghai Invitrogen): P35:5'-TTCGCCCATTGTCGTTAC-3' (SEQ ID No.60), P36:5'-AGTTCCGCTTACAGCCTACC-3' (SEQ ID No.61).

[0116] Positive transformants were seeded in 2-YT medium containing spectinomycin (100 mg / L) and 0.2% arabinose, and the plasmid pGRB-sgRNA-4 was removed. Colonies that grew with spectinomycin (100 mg / L) but not with ampicillin (100 mg / L) were selected, and these colonies were re-seeded in 2-YT medium and cultured at 42°C to remove the pREDCas9 plasmid. Colonies that did not grow with spectinomycin (100 mg / L) but grew in antibiotic-free 2-YT were selected, and PCR identification was performed again with primers P27 / P34. Strains from which a fragment of size 4524 bp (SEQ ID No. 9) was amplified were designated as positive strains. Sequence analysis was performed on these positive strains, and the strains with correct results were named CGMCC22721-lacIZ and W3110-lacIZ, respectively.

[0117] Recombinant strains CGMCC22721-lacIZ and W3110-lacIZ both lack the lacI-lacZ gene and overexpress a DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter. Specifically, recombinant strains CGMCC22721-lacIZ and W3110-lacIZ were obtained by knocking out a portion of the coding region of the lacI-lacZ gene in the genomes of the L-valine-producing strain CGMCC22721 and the wild-type Escherichia coli W3110, respectively, while inserting a DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter, and retaining other nucleotides in the genome without modification.

[0118] Example 5: Construction of a genetically modified bacterium in which the ycgH gene is deleted and the ilvC gene is overexpressed in the genome. Based on the genome sequence of Escherichia coli W3110 published by NCBI, CRISPR / Cas9 genome editing technology was used to knock out the ycgH gene (sequencing analysis confirmed that the complete ycgH gene (GeneID: 2847703, updated 2023-04-14)) in the genomes of L-valine-producing strain CGMCC22721 and wild-type Escherichia coli W3110, while simultaneously inserting the ilvC gene (ketol-acid reductoisomerase, derived from Escherichia coli W3110) driven by the Ptrc promoter. This allowed for a more detailed study of the effects of these genes on L-valine synthesis.

[0119] 1. Construction of sgRNA Based on the Escherichia coli W3110 genome sequence published by NCBI, sgRNA target sequences were designed using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ). After selecting appropriate sgRNA target sequences, homology arm sequences of a linearized pGRB cloning vector were added to the 5' and 3' ends of the target sequences, and complete sgRNA plasmids were formed by recombination.

[0120] When amplifying sgRNA fragments, no template is required; only PCR annealing is necessary. The reaction system and program are as follows: PCR reaction system: sgRNA-5F 10 μL, sgRNA-5R 10 μL; PCR reaction program: Denaturate at 95°C for 5 minutes, anneal at 50°C for 1 minute. After annealing was complete, the target DNA fragment of sgRNA was recovered using a DNA purification kit, its DNA concentration was measured, and then it was diluted to a concentration of 100 ng / μL.

[0121] The pGRB plasmid was digested with Spe I enzyme and then dephosphorylated. Recombination of the sgRNA with the dephosphorylated linear plasmid was performed using the Gibson Assembly Kit (New England). Recombination reaction system: 2.5 μL of enzyme for NEB assembly, 2 μL of dephosphorylated linear plasmid, and 0.5 μL of sgRNA target DNA fragment. After a recombination reaction at 50°C for 30 minutes, the product was transformed into DH5α-competent cells, the plasmid was extracted, and sequencing was performed using the sequencing primers sgRNA-PF / sgRNA-PR. Plasmids that matched the sequencing results were named pGRB-sgRNA-5.

[0122] The primers used in this experiment are as follows (synthesized by Shanghai Invitrogen). Underlined nucleotides represent the homology arm sequences of the pGRB cloning vector, and bolded nucleotides represent the sgRNA sequences: sgRNA-5F:5'- TGACAGCTAGCTCAGTCCTAGGTATAATACTAGT CCCGTCCTGCGCCCCGAAGC GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG -3' (SEQ ID No. 62), sgRNA-5R:5'- CCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC GCTTCGGGGCGCAGGACGGG ACTAGTATTATACCTAGGACTGAGCTAGCTGTCA -3' (SEQ ID No. 63).

[0123] 2. PCR amplification of homologous recombination fragments Based on the genome sequence of Escherichia coli W3110 published by NCBI, primers for knocking out the ycgH gene and primers for inserting the Ptrc-ilvC gene were designed and synthesized. Using CRISPR / Cas9 genome editing techniques, the ycgH gene was knocked out in the genomes of L-valine-producing strain CGMCC22721 and wild-type Escherichia coli W3110, and the ilvC gene (derived from Escherichia coli W3110) driven by the Ptrc promoter was inserted.

[0124] The primers are as follows (synthesized by Shanghai Invitrogen): P37:5'-AAATGGAGGGATAACAGCC-3'(SEQ ID No.64), P38:5'-GCTGTTGCGGGTTAACGCTCCATTTAGAATAGAGTCGGACGAAAT-3' (SEQ ID No.65), P39:5'-CTAAATGGAGCGTTAACCCGCAACAGCAATAC-3'(SEQ ID No.66), P40:5'-GCCATTTACGCCAAGTTTTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAG-3' (SEQ ID No.67), P41:5'-CAGTGTATTGAAGTAGTTAGCCATGGTCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATAC-3' (SEQ ID No.68), P42:5'-TTGCCTTCGGGCTTATCTC-3' (SEQ ID No.69).

[0125] Using the genomic DNA of E. coli W3110 as a template, PCR amplification was performed with primers P37 / P38, P39 / P40, P41 / P42, and KAPA HiFi HotStart to obtain fragments of the upstream homology arm, the Ptrc-ilvC gene, and the downstream homology arm, with sizes of 808 bp, 1580 bp, and 944 bp, respectively. After the PCR reaction was complete, each fragment was recovered by agarose gel electrophoresis using a column-type DNA gel recovery kit. The recovered DNA was subjected to overlap PCR with primer P37 / P42 to obtain the homologous recombination DNA fragment ΔycgH-Ptrc-ilvC (SEQ ID No. 11) 3189 bp.

[0126] In SEQ ID No. 11, the Ptrc promoter is located at positions 2270-2343, and the ilvC gene (the gene encoding the ilvC protein shown in SEQ ID No. 12) is located at positions 794-2269.

[0127] IV. Preparation and transformation of competent cells Competent cells of CGMCC22721-Cas9 and W3110-Cas9 were prepared in Example 1. 600 Once the cells had grown to 0.1 mM, a final concentration of 0.1 mM IPTG was added to induce homologous recombination via λ-Red. 600When the ratio reached 0.4, the bacterial cells were harvested and competent cells were prepared. These were transformed with the pGRB-sgRNA-5 plasmid and the homologous recombinant DNA fragment ΔycgH-Ptrc-ilvC, respectively, and spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L). The cells were incubated at 32°C for 12 hours. After culturing, the single colonies obtained were subcultured and then PCR identified using primers P43 / P44. Fragments of size 1669 bp that were amplified by PCR were considered positive transformants.

[0128] The primers are as follows (synthesized by Shanghai Invitrogen): P43:5'-AAGACCTCTGGAAGCGTATC-3' (SEQ ID No.70), P44:5'-CGTTGCGGAAGTGAAATC-3' (SEQ ID No.71).

[0129] Positive transformants were seeded in 2-YT medium containing spectinomycin (100 mg / L) and 0.2% arabinose, and the plasmid pGRB-sgRNA-5 was removed. Colonies that grew with spectinomycin (100 mg / L) but not with ampicillin (100 mg / L) were selected, and these colonies were reseeded in 2-YT medium and cultured at 42°C to remove the pREDCas9 plasmid. Colonies that did not grow with spectinomycin (100 mg / L) but grew in antibiotic-free 2-YT were selected, and PCR identification was performed again with primers P37 / P42. Strains from which a fragment of size 3189 bp (SEQ ID No. 11) was amplified were designated as positive strains. Sequence analysis was performed on these positive strains, and the strains with correct results were named CGMCC22721-ycgH and W3110-ycgH, respectively.

[0130] Recombinant strains CGMCC22721-ycgH and W3110-ycgH both lack a ycgH gene and overexpress the ilvC gene (derived from Escherichia coli W3110), which is driven by the Ptrc promoter. Specifically, recombinant strains CGMCC22721-ycgH and W3110-ycgH were obtained by knocking out a portion of the coding region of the ycgH gene in the genomes of the L-valine-producing strain CGMCC22721 and the wild-type Escherichia coli W3110, respectively, while inserting the ilvC gene (derived from Escherichia coli W3110), which is driven by the Ptrc promoter, and retaining other nucleotides in the genome without modification.

[0131] Example 6: Construction of genetically modified bacteria in which the yjiV gene is deleted and the ilvE-ilvD genes are overexpressed in CGMCC22721-yjiT and W3110-yjiT. Based on the genome sequence of Escherichia coli W3110 published by NCBI, the yjiV gene (the complete yjiV gene was confirmed to be present on the chromosomes of these strains by sequencing analysis) was knocked out using CRISPR / Cas9 genome editing technology. Simultaneously, the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168), driven by the Ptrc promoter, and the ilvD gene (derived from Escherichia coli W3110), driven by the PilvD promoter, were inserted. This allowed for a more detailed study of the effects of these genes on L-valine synthesis.

[0132] 1. PCR amplification of homologous recombination fragments Based on the genome sequence of Escherichia coli W3110 published by NCBI, primers for knocking out the yjiV gene and primers for inserting the Ptrc-ilvE gene and the PilvD-ilvD gene were designed and synthesized. Using CRISPR / Cas9 genome editing techniques, the yjiV gene was knocked out in the genomes of L-valine-producing strain CGMCC22721-yjiT and wild-type E. coli W3110-yjiT, and the ilvE gene (derived from Bacillus subtilis) driven by the Ptrc promoter and the ilvD gene driven by the PilvD promoter were inserted.

[0133] The primers are as follows (synthesized by Shanghai Invitrogen): P45:5'-AGTTCGCCCTTTGCTCTCTC-3' (SEQ ID No.72).

[0134] Using the genomic DNA of *E. coli* W3110-yjiT as a template, PCR amplification was performed with primers P45 / P10, P13 / P14, P15 / P16, and KAPA HiFi HotStart to obtain fragments of the upstream homology arm, the PilvD-ilvD gene, and the downstream homology arm, with sizes of 812 bp, 1979 bp, and 856 bp, respectively. Using the genomic DNA of *Bacillus subtilis* subsp. subtilis str.168 as a template, PCR amplification was performed with primers P11 / P12 and KAPA HiFi HotStart to obtain a fragment of the Ptrc-ilvE gene with a size of 1161 bp. After the completion of the PCR reaction, each fragment was recovered by agarose gel electrophoresis using a column-type DNA gel recovery kit. The recovered DNA was subjected to overlap PCR using primers P45 / P16 to obtain the homologous recombination DNA fragment ΔyjiV-Ptrc-ilvE-PilvD-ilvD-2 (SEQ ID No. 13) 4713 bp.

[0135] In SEQ ID No. 13, positions 3809-3874 represent the PilvD promoter, positions 1958-3808 represent the ilvD gene (the gene encoding the ilvD protein shown in SEQ ID No. 5), positions 1884-1957 represent the Ptrc promoter, and positions 789-1883 represent the ilvE gene (the gene encoding the ilvE protein shown in SEQ ID No. 6).

[0136] 2. Preparation and transformation of competent cells The pREDCas9 plasmid (containing the spectinomycin resistance gene) was extracted and transformed into competent cells of CGMCC22721-yjiT and W3110-yjiT from Example 1, respectively. These transformed cells were then spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and cultured at 32°C. Single colonies showing resistance to spectinomycin (100 mg / L) were selected and identified by PCR using primers pRedCas9-PF / pRedCas9-PR. Cells yielding 943 bp were designated as CGMCC22721-yjiT-Cas9 and W3110-yjiT-Cas9 transformants containing the pREDCas9 plasmid.

[0137] Competent cells of CGMCC22721-yjiT-Cas9 and W3110-yjiT-Cas9 were prepared. The bacterial cells were OD 600 Once the cells had grown to 0.1 mM, a final concentration of 0.1 mM IPTG was added to induce homologous recombination via λ-Red. 600 When the ratio reached 0.4, the bacterial cells were harvested and competent cells were prepared. These were transformed with the pGRB-sgRNA-2 plasmid and homologous recombinant DNA fragment ΔyjiV-Ptrc-ilvE-PilvD-ilvD-2 from Example 2, respectively. These cells were then spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L) and cultured at 32°C for 12 hours. After culturing, the obtained single colonies were subcultured and then PCR identified using primers P46 / P18. Fragments with a size of 1561 bp that were amplified by PCR were considered positive transformants.

[0138] The primers are as follows (synthesized by Shanghai Invitrogen): P46:5'-AGCGTTTCACTCCTACTGGG-3' (SEQ ID No.73).

[0139] Positive transformants were seeded in 2-YT medium containing spectinomycin (100 mg / L) and 0.2% arabinose, and the plasmid pGRB-sgRNA-2 was removed. Colonies that grew with spectinomycin (100 mg / L) but not with ampicillin (100 mg / L) were selected, and these colonies were re-seeded in 2-YT medium and cultured at 42°C to remove the pREDCas9 plasmid. Colonies that did not grow with spectinomycin (100 mg / L) but grew in antibiotic-free 2-YT were selected, and PCR identification was performed again using primers P45 / P16. Amplified fragments of size 4713 bp (SEQ ID No. 13) were identified as positive strains. Sequence analysis was performed on the positive bacterial strains, and the strains whose results were correct were named CGMCC22721-yjiT-yjiV and W3110-yjiT-yjiV, respectively.

[0140] Recombinant strains CGMCC22721-yjiT-yjiV and W3110-yjiT-yjiV both lack the yjiT gene and overexpress the brnF-brnE gene (derived from Corynebacterium glutamicum ATCC13032), which is driven by the Ptrc promoter. They also lack the yjiV gene and overexpress the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168), which is driven by the Ptrc promoter, and the ilvD gene (derived from Escherichia coli W3110), which is driven by the PilvD promoter. Specifically, recombinant bacteria CGMCC22721-yjiT-yjiV and W3110-yjiT-yjiV are recombinant bacteria obtained by knocking out a portion of the coding region of the yjiT gene in the genomes of the L-valine-producing bacterium CGMCC22721 and wild-type Escherichia coli W3110, respectively, and inserting the brnF-brnE gene (derived from Corynebacterium glutamicum ATCC13032) driven by the Ptrc promoter, while knocking out a portion of the coding region of the yjiV gene and inserting the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168) driven by the Ptrc promoter and the ilvD gene (derived from Escherichia coli W3110) driven by the PilvD promoter, while retaining other nucleotides in the genome without modification.

[0141] Example 7: Construction of genetically modified bacteria in which the trpR gene is deleted and the ilvH gene is overexpressed in CGMCC22721-yjiT-yjiV and W3110-yjiT-yjiV. Based on the genome sequence of Escherichia coli W3110 published by NCBI, CRISPR / Cas9 genome editing technology was used to knock out the trpR gene in the genomes of L-valine-producing strain CGMCC22721-yjiT-yjiV and wild-type E. coli W3110-yjiT-yjiV (sequencing analysis confirmed that the complete trpR gene is retained on the chromosomes of these strains), as well as the Ptrc promoter-driven ilvH G14D、S17F Genes were inserted. This allowed for a more detailed study of the effects of these genes on L-valine synthesis.

[0142] The pREDCas9 plasmid (containing the spectinomycin resistance gene) was extracted and transformed into competent cells of CGMCC22721-yjiT-yjiV and W3110-yjiT-yjiV from Example 6, respectively. These transformed cells were then spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and cultured at 32°C. Single colonies showing resistance to spectinomycin (100 mg / L) were selected and identified by PCR using primers pRedCas9-PF / pRedCas9-PR. Cells yielding 943 bp were designated as CGMCC22721-yjiT-yjiV-Cas9 and W3110-yjiT-yjiV-Cas9 transformants containing the pREDCas9 plasmid.

[0143] Competent cells of CGMCC22721-yjiT-yjiV-Cas9 and W3110-yjiT-yjiV-Cas9 were prepared. 600 Once the cells had grown to 0.1 mM, a final concentration of 0.1 mM IPTG was added to induce homologous recombination via λ-Red. 600When the ratio reached 0.4, the bacterial cells were harvested and competent cells were prepared. These were transformed with the pGRB-sgRNA-3 plasmid and homologous recombinant DNA fragment ΔtrpR-Ptrc-ilvH from Example 3, respectively, and spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L). The cells were incubated at 32°C for 12 hours. After culturing, the single colonies obtained were subcultured and then PCR identified using primers P25 / P26. Fragments of size 1437 bp that were amplified by PCR were considered positive transformants.

[0144] Positive transformants were seeded in 2-YT medium containing spectinomycin (100 mg / L) and 0.2% arabinose, and the plasmid pGRB-sgRNA-3 was removed. Colonies that grew with spectinomycin (100 mg / L) but not with ampicillin (100 mg / L) were selected, and these colonies were re-seed in 2-YT medium and cultured at 42°C to remove the pREDCas9 plasmid. Colonies that did not grow with spectinomycin (100 mg / L) but grew in antibiotic-free 2-YT were selected, and PCR identification was performed again with primers P19 / P24. Amplified fragments of size 2108 bp (SEQ ID No. 7) were identified as positive strains. Sequence analysis was performed on the positive bacterial strains, and the strains whose results were correct were named CGMCC22721-yjiT-yjiV-trpR and W3110-yjiT-yjiV-trpR, respectively.

[0145] Recombinant strains CGMCC22721-yjiT-yjiV-trpR and W3110-yjiT-yjiV-trpR both lack the yjiT gene and overexpress the brnF-brnE gene (derived from Corynebacterium glutamicum ATCC13032) driven by the Ptrc promoter, while also lacking the yjiV gene and overexpressing the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168) driven by the Ptrc promoter and the ilvD gene (derived from Escherichia coli W3110) driven by the PilvD promoter. Furthermore, both strains lack the trpR gene and overexpress the ilvH gene (derived from Escherichia coli W3110) driven by the Ptrc promoter. G14D、S17F The gene is overexpressed. Specifically, recombinant bacteria CGMCC22721-yjiT-yjiV-trpR and W3110-yjiT-yjiV-trpR were created by knocking out a portion of the coding region of the yjiT gene in the genomes of L-valine-producing bacteria CGMCC22721 and wild-type E. coli W3110, respectively, and inserting the brnF-brnE gene (derived from Corynebacterium glutamicum ATCC13032) driven by the Ptrc promoter, knocking out a portion of the coding region of the yjiV gene, and inserting the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168) driven by the Ptrc promoter and the ilvD gene (derived from Escherichia coli) driven by the PilvD promoter. A mutant ilvH gene (derived from coli)W3110 is inserted, and a portion of the coding region of the trpR gene is knocked out, resulting in a Ptrc promoter-driven mutant ilvH. G14D、S17F This is a recombinant bacterium obtained by inserting a gene while retaining other nucleotides in the genome without alteration.

[0146] Example 8: Construction of genetically modified bacteria in which the lacI-lacZ gene is deleted and the DNA polymerase gene is overexpressed on CGMCC22721-yjiT-yjiV-trpR and W3110-yjiT-yjiV-trpR. Based on the genome sequence of Escherichia coli W3110 published by NCBI, CRISPR / Cas9 genome editing technology was used to knock out the lacI-lacZ gene (the complete lacI-lacZ gene was confirmed to be present on the chromosomes of these strains by sequencing analysis) in the genomes of L-valine-producing strain CGMCC22721-yjiT-yjiV-trpR and wild-type E. coli W3110-yjiT-yjiV-trpR, while inserting a DNA polymerase gene (derived from E. coli BL21) driven by the PxylF promoter. This allowed for a more detailed study of the effects of these genes on L-valine synthesis.

[0147] The pREDCas9 plasmid (containing the spectinomycin resistance gene) was extracted and transformed into competent cells of CGMCC22721-yjiT-yjiV-trpR and W3110-yjiT-yjiV-trpR from Example 7, respectively. These cells were then spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and cultured at 32°C. Single colonies showing resistance to spectinomycin (100 mg / L) were selected and identified by PCR using primers pRedCas9-PF / pRedCas9-PR. Cells yielding 943 bp were designated as CGMCC22721-yjiT-yjiV-trpR-Cas9 and W3110-yjiT-yjiV-trpR-Cas9 transformants containing the pREDCas9 plasmid.

[0148] Competent cells of CGMCC22721-yjiT-yjiV-trpR-Cas9 and W3110-yjiT-yjiV-trpR-Cas9 were prepared. The bacterial cells were OD 600 Once the cells had grown to 0.1 mM, a final concentration of 0.1 mM IPTG was added to induce homologous recombination via λ-Red.600 When the ratio reached 0.4, the bacterial cells were harvested and competent cells were prepared. These were transformed with the pGRB-sgRNA-4 plasmid and homologous recombinant DNA fragment ΔlacIZ-PxylF-DNA polymerase from Example 4, respectively. These were then spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L) and cultured at 32°C for 12 hours. After culturing, the obtained single colonies were subcultured and then PCR identification was performed using primers P35 / P36. Fragments of size 1655 bp that were amplified by PCR were considered positive transformants.

[0149] Positive transformants were seeded in 2-YT medium containing spectinomycin (100 mg / L) and 0.2% arabinose, and the plasmid pGRB-sgRNA-4 was removed. Colonies that grew with spectinomycin (100 mg / L) but not with ampicillin (100 mg / L) were selected, and these colonies were re-seeded in 2-YT medium and cultured at 42°C to remove the pREDCas9 plasmid. Colonies that did not grow with spectinomycin (100 mg / L) but grew in antibiotic-free 2-YT were selected, and PCR identification was performed again using primers P27 / P34. Amplified fragments of size 4524 bp (SEQ ID No. 9) were identified as positive strains. Sequence analysis was performed on the positive bacterial strains, and the strains whose results were correct were named CGMCC22721-yjiT-yjiV-trpR-lacIZ and W3110-yjiT-yjiV-trpR-lacIZ, respectively.

[0150] Recombinant strains CGMCC22721-yjiT-yjiV-trpR-lacIZ and W3110-yjiT-yjiV-trpR-lacIZ both lack the yjiT gene and overexpress the brnF-brnE gene (derived from Corynebacterium glutamicum ATCC13032) driven by the Ptrc promoter, while also lacking the yjiV gene and overexpressing the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168) driven by the Ptrc promoter and the ilvD gene (derived from Escherichia coli W3110) driven by the PilvD promoter, while both lack the trpR gene and overexpress the ilvH gene (derived from Escherichia coli W3110) driven by the Ptrc promoter. G14D、S17F The gene is overexpressed, with a deletion of the lacI-lacZ gene, and the DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter is also overexpressed. Specifically, recombinant bacteria CGMCC22721-yjiT-yjiV-trpR-lacIZ and W3110-yjiT-yjiV-trpR-lacIZ were created by knocking out a portion of the coding region of the yjiT gene in the genomes of L-valine-producing bacteria CGMCC22721 and wild-type E. coli W3110, respectively, and inserting the brnF-brnE gene (derived from Corynebacterium glutamicum ATCC13032) driven by the Ptrc promoter, knocking out a portion of the coding region of the yjiV gene, and inserting the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168) driven by the Ptrc promoter and the ilvD gene (derived from Escherichia coli) driven by the PilvD promoter. A mutant ilvH gene was created by inserting a gene from coli (W3110), knocking out a portion of the coding region of the trpR gene, and driving the Ptrc promoter. G14D、S17FThis recombinant bacterium was obtained by inserting a gene, knocking out a portion of the coding region of the lacI-lacZ gene, inserting a DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter, and maintaining other nucleotides in the genome without modification.

[0151] Example 9: Construction of genetically modified bacteria in which the ycgH gene is deleted and the ilvC gene is overexpressed on CGMCC22721-yjiT-yjiV-trpR-lacIZ and W3110-yjiT-yjiV-trpR-lacIZ. Based on the genome sequence of Escherichia coli W3110 published by NCBI, the ycgH gene (the complete ycgH gene was confirmed to be present on the chromosomes of these strains by sequencing analysis) was knocked out using CRISPR / Cas9 genome editing technology in the genomes of the L-valine-producing strain CGMCC22721-yjiT-yjiV-trpR-lacIZ and the wild-type E. coli W3110-yjiT-yjiV-trpR-lacIZ. Simultaneously, the ilvC gene (derived from E. coli W3110) driven by the Ptrc promoter was inserted. This allowed for a more detailed study of the effects of these genes on L-valine synthesis.

[0152] The pREDCas9 plasmid (containing the spectinomycin resistance gene) was extracted and transformed into competent cells of CGMCC22721-yjiT-yjiV-trpR-lacIZ and W3110-yjiT-yjiV-trpR-lacIZ from Example 8, respectively. These cells were then spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and cultured at 32°C. Single colonies showing resistance to spectinomycin (100 mg / L) were selected and identified by PCR using primers pRedCas9-PF / pRedCas9-PR. Cells yielding 943 bp were designated as CGMCC22721-yjiT-yjiV-trpR-lacIZ-Cas9 and W3110-yjiT-yjiV-trpR-lacIZ-Cas9 transformants containing the pREDCas9 plasmid.

[0153] Competent cells of CGMCC22721-yjiT-yjiV-trpR-lacIZ-Cas9 and W3110-yjiT-yjiV-trpR-lacIZ-Cas9 were prepared. The bacterial cells were OD 600 Once the cells had grown to 0.1 mM, a final concentration of 0.1 mM IPTG was added to induce homologous recombination via λ-Red. 600 When the ratio reached 0.4, the bacterial cells were harvested and competent cells were prepared. These were transformed with the pGRB-sgRNA-5 plasmid and homologous recombinant DNA fragment ΔycgH-Ptrc-ilvC from Example 5, respectively, and spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L). The cells were incubated at 32°C for 12 hours. After culturing, the single colonies obtained were subcultured and then PCR identified using primers P43 / P44. Fragments of size 1669 bp that were amplified by PCR were considered positive transformants.

[0154] Positive transformants were seeded in 2-YT medium containing spectinomycin (100 mg / L) and 0.2% arabinose, and the plasmid pGRB-sgRNA-5 was removed. Colonies that grew with spectinomycin (100 mg / L) but not with ampicillin (100 mg / L) were selected, and these colonies were re-seed in 2-YT medium and cultured at 42°C to remove the pREDCas9 plasmid. Colonies that did not grow with spectinomycin (100 mg / L) but grew in antibiotic-free 2-YT were selected, and PCR identification was performed again with primers P37 / P42. Amplified fragments of size 3189 bp (SEQ ID No. 11) were identified as positive strains. Sequence analysis was performed on the positive bacterial strains, and the strains whose results were correct were named CGMCC22721-yjiT-yjiV-trpR-lacIZ-ycgH and W3110-yjiT-yjiV-trpR-lacIZ-ycgH, respectively.

[0155] Recombinant strains CGMCC22721-yjiT-yjiV-trpR-lacIZ-ycgH and W3110-yjiT-yjiV-trpR-lacIZ-ycgH both lack the yjiT gene and overexpress the brnF-brnE gene (derived from Corynebacterium glutamicum ATCC13032), which is driven by the Ptrc promoter; they lack the yjiV gene and overexpress the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168), which is driven by the Ptrc promoter, and the ilvD gene (derived from Escherichia coli W3110), which is driven by the PilvD promoter; and they both lack the trpR gene and overexpress the ilvH gene (derived from Escherichia coli W3110), which is driven by the Ptrc promoter. G14D、S17FThe following genes are overexpressed: the lacI-lacZ gene is deleted, the DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter is overexpressed, the ycgH gene is deleted, and the ilvC gene (derived from Escherichia coli W3110) driven by the Ptrc promoter is overexpressed. Specifically, recombinant bacteria CGMCC22721-yjiT-yjiV-trpR-lacIZ-ycgH and W3110-yjiT-yjiV-trpR-lacIZ-ycgH were created by knocking out a portion of the coding region of the yjiT gene in the genomes of L-valine-producing bacteria CGMCC22721 and wild-type Escherichia coli W3110, respectively, and inserting the brnF-brnE gene (derived from Corynebacterium glutamicum ATCC13032) driven by the Ptrc promoter, knocking out a portion of the coding region of the yjiV gene, and inserting the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168) driven by the Ptrc promoter and the ilvD gene (derived from Escherichia coli) driven by the PilvD promoter. A mutant ilvH gene was created by inserting a gene from coli (W3110), knocking out a portion of the coding region of the trpR gene, and driving the Ptrc promoter. G14D、S17F This recombinant bacterium was obtained by inserting a gene, knocking out a portion of the coding region of the lacI-lacZ gene, inserting a DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter, knocking out a portion of the coding region of the ycgH gene, inserting an ilvC gene (derived from Escherichia coli W3110) driven by the Ptrc promoter, and maintaining other nucleotides in the genome without modification.

[0156] Example 10: Construction of genetically modified bacteria in which the trpR gene is deleted and the ilvH gene is overexpressed in CGMCC22721-yjiV and W3110-yjiV. Based on the genome sequence of Escherichia coli W3110 published by NCBI, CRISPR / Cas9 genome editing technology was used to knock out the trpR gene in the genomes of L-valine-producing strain CGMCC22721-yjiV and wild-type E. coli W3110-yjiV (sequencing analysis confirmed that the complete trpR gene is preserved on the chromosomes of these strains), as well as the Ptrc promoter-driven ilvH G14D、S17F Genes were inserted. This allowed for a more detailed study of the effects of these genes on L-valine synthesis.

[0157] The pREDCas9 plasmid (containing the spectinomycin resistance gene) was extracted and transformed into competent cells of CGMCC22721-yjiV and W3110-yjiV from Example 2, respectively. These transformed cells were then spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and cultured at 32°C. Single colonies showing resistance to spectinomycin (100 mg / L) were selected and identified by PCR using primers pRedCas9-PF / pRedCas9-PR. Those yielding 943 bp were designated as CGMCC22721-yjiV-Cas9 and W3110-yjiV-Cas9 transformants containing the pREDCas9 plasmid.

[0158] Competent cells of CGMCC22721-yjiV-Cas9 and W3110-yjiV-Cas9 were prepared. The bacterial cells were OD 600 Once the cells had grown to 0.1 mM, a final concentration of 0.1 mM IPTG was added to induce homologous recombination via λ-Red. 600When the ratio reached 0.4, the bacterial cells were harvested and competent cells were prepared. These were transformed with the pGRB-sgRNA-3 plasmid and homologous recombinant DNA fragment ΔtrpR-Ptrc-ilvH from Example 3, respectively, and spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L). The cells were incubated at 32°C for 12 hours. After culturing, the single colonies obtained were subcultured and then PCR identified using primers P25 / P26. Fragments of size 1437 bp that were amplified by PCR were considered positive transformants.

[0159] Positive transformants were seeded in 2-YT medium containing spectinomycin (100 mg / L) and 0.2% arabinose, and the plasmid pGRB-sgRNA-3 was removed. Colonies that grew with spectinomycin (100 mg / L) but not with ampicillin (100 mg / L) were selected, and these colonies were re-seed in 2-YT medium and cultured at 42°C to remove the pREDCas9 plasmid. Colonies that did not grow with spectinomycin (100 mg / L) but grew in antibiotic-free 2-YT were selected, and PCR identification was performed again with primers P19 / P24. Amplified fragments of size 2108 bp (SEQ ID No. 7) were identified as positive strains. Sequence analysis was performed on the positive bacterial strains, and the strains whose results were correct were named CGMCC22721-yjiV-trpR and W3110-yjiV-trpR, respectively.

[0160] Recombinant strains CGMCC22721-yjiV-trpR and W3110-yjiV-trpR both lack the yjiV gene and overexpress the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168) driven by the Ptrc promoter and the ilvD gene (derived from Escherichia coli W3110) driven by the PilvD promoter, while both lack the trpR gene and overexpress the ilvH gene (derived from Escherichia coli W3110) driven by the Ptrc promoter. G14D、S17FGene overexpression is occurring. Specifically, recombinant bacteria CGMCC22721-yjiV-trpR and W3110-yjiV-trpR have a portion of the coding region of the yjiV gene in the genomes of L-valine-producing bacteria CGMCC22721 and wild-type Escherichia coli W3110, respectively, knocked out, and inserted the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168) driven by the Ptrc promoter and the ilvD gene (derived from Escherichia coli W3110) driven by the PilvD promoter, as well as a portion of the coding region of the trpR gene, and a mutant ilvH gene driven by the Ptrc promoter. G14D、S17F This is a recombinant bacterium obtained by inserting a gene while retaining other nucleotides in the genome without alteration.

[0161] Example 11: Construction of genetically modified bacteria in which the lacI-lacZ gene is deleted and the DNA polymerase gene is overexpressed on CGMCC22721-yjiV-trpR and W3110-yjiV-trpR. Based on the genome sequence of Escherichia coli W3110 published by NCBI, CRISPR / Cas9 genome editing technology was used to knock out the lacI-lacZ gene (the complete lacI-lacZ gene was confirmed to be present on the chromosomes of these strains by sequencing analysis) in the genomes of L-valine-producing strain CGMCC22721-yjiV-trpR and wild-type E. coli W3110-yjiV-trpR, while inserting a DNA polymerase gene (derived from E. coli BL21) driven by the PxylF promoter. This allowed for a more detailed study of the effects of these genes on L-valine synthesis.

[0162] The pREDCas9 plasmid (containing the spectinomycin resistance gene) was extracted and transformed into competent cells of CGMCC22721-yjiV-trpR and W3110-yjiV-trpR from Example 10, respectively. These transformed cells were then spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and cultured at 32°C. Single colonies showing resistance to spectinomycin (100 mg / L) were selected and identified by PCR using primers pRedCas9-PF / pRedCas9-PR. Cells yielding 943 bp were designated as CGMCC22721-yjiV-trpR-Cas9 and W3110-yjiV-trpR-Cas9 transformants containing the pREDCas9 plasmid.

[0163] Competent cells of CGMCC22721-yjiV-trpR-Cas9 and W3110-yjiV-trpR-Cas9 were prepared. 600 Once the cells had grown to 0.1 mM, a final concentration of 0.1 mM IPTG was added to induce homologous recombination via λ-Red. 600 When the ratio reached 0.4, the bacterial cells were harvested and competent cells were prepared. These were transformed with the pGRB-sgRNA-4 plasmid and homologous recombinant DNA fragment ΔlacIZ-PxylF-DNA polymerase from Example 4, respectively. These were then spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L) and cultured at 32°C for 12 hours. After culturing, the obtained single colonies were subcultured and then PCR identification was performed using primers P35 / P36. Fragments of size 1655 bp that were amplified by PCR were considered positive transformants.

[0164] Positive transformants were seeded in 2-YT medium containing spectinomycin (100 mg / L) and 0.2% arabinose, and the plasmid pGRB-sgRNA-4 was removed. Colonies that grew with spectinomycin (100 mg / L) but not with ampicillin (100 mg / L) were selected, and these colonies were re-seeded in 2-YT medium and cultured at 42°C to remove the pREDCas9 plasmid. Colonies that did not grow with spectinomycin (100 mg / L) but grew in antibiotic-free 2-YT were selected, and PCR identification was performed again using primers P27 / P34. Amplified fragments of size 4524 bp (SEQ ID No. 9) were identified as positive strains. Sequence analysis was performed on the positive bacterial strains, and the strains whose results were correct were named CGMCC22721-yjiV-trpR-lacIZ and W3110-yjiV-trpR-lacIZ, respectively.

[0165] Recombinant strains CGMCC22721-yjiV-trpR-lacIZ and W3110-yjiV-trpR-lacIZ both lack the yjiV gene and overexpress the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168) driven by the Ptrc promoter and the ilvD gene (derived from Escherichia coli W3110) driven by the PilvD promoter, while both lack the trpR gene and overexpress the ilvH gene (derived from Escherichia coli W3110) driven by the Ptrc promoter. G14D、S17FThe genes are overexpressed, with the lacI-lacZ genes deleted and the DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter overexpressed. Specifically, recombinant CGMCC22721-yjiV-trpR-lacIZ and W3110-yjiV-trpR-lacIZ have a portion of the coding region of the yjiV gene in the genomes of L-valine-producing bacterium CGMCC22721 and wild-type Escherichia coli W3110 knocked out, and the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168) driven by the Ptrc promoter and the ilvD gene (derived from Escherichia coli W3110) driven by the PlvD promoter inserted, a portion of the coding region of the trpR gene knocked out, and the mutant ilvH gene (driven by the Ptrc promoter) inserted. G14D、S17F This recombinant bacterium was obtained by inserting a gene, knocking out a portion of the coding region of the lacI-lacZ gene, inserting a DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter, and maintaining other nucleotides in the genome without modification.

[0166] Example 12: Construction of genetically modified bacteria in which the ycgH gene is deleted and the ilvC gene is overexpressed on CGMCC22721-yjiV-trpR-lacIZ and W3110-yjiV-trpR-lacIZ. Based on the genome sequence of Escherichia coli W3110 published by NCBI, the ycgH gene (the complete ycgH gene was confirmed to be present on the chromosomes of these strains by sequencing analysis) was knocked out using CRISPR / Cas9 genome editing technology in the genomes of L-valine-producing strain CGMCC22721-yjiV-trpR-lacIZ and wild-type E. coli W3110-yjiV-trpR-lacIZ. Simultaneously, the ilvC gene (derived from Escherichia coli W3110), driven by the Ptrc promoter, was inserted. This allowed for a more detailed study of the effects of these genes on L-valine synthesis.

[0167] The pREDCas9 plasmid (containing the spectinomycin resistance gene) was extracted and transformed into competent cells of CGMCC22721-yjiV-trpR-lacIZ and W3110-yjiV-trpR-lacIZ from Example 11, respectively. These cells were then spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and cultured at 32°C. Single colonies showing resistance to spectinomycin (100 mg / L) were selected and identified by PCR using primers pRedCas9-PF / pRedCas9-PR. Cells yielding 943 bp were designated as CGMCC22721-yjiV-trpR-lacIZ-Cas9 and W3110-yjiV-trpR-lacIZ-Cas9 transformants containing the pREDCas9 plasmid.

[0168] Competent cells of CGMCC22721-yjiV-trpR-lacIZ-Cas9 and W3110-yjiV-trpR-lacIZ-Cas9 were prepared. The bacterial cells were OD 600 Once the cells had grown to 0.1 mM, a final concentration of 0.1 mM IPTG was added to induce homologous recombination via λ-Red. 600When the ratio reached 0.4, the bacterial cells were harvested and competent cells were prepared. These were transformed with the pGRB-sgRNA-5 plasmid and homologous recombinant DNA fragment ΔycgH-Ptrc-ilvC from Example 5, respectively, and spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L). The cells were incubated at 32°C for 12 hours. After culturing, the single colonies obtained were subcultured and then PCR identified using primers P43 / P44. Fragments of size 1669 bp that were amplified by PCR were considered positive transformants.

[0169] Positive transformants were seeded in 2-YT medium containing spectinomycin (100 mg / L) and 0.2% arabinose, and the plasmid pGRB-sgRNA-5 was removed. Colonies that grew with spectinomycin (100 mg / L) but not with ampicillin (100 mg / L) were selected, and these colonies were re-seed in 2-YT medium and cultured at 42°C to remove the pREDCas9 plasmid. Colonies that did not grow with spectinomycin (100 mg / L) but grew in antibiotic-free 2-YT were selected, and PCR identification was performed again with primers P37 / P42. Amplified fragments of size 3189 bp (SEQ ID No. 11) were identified as positive strains. Sequence analysis was performed on the positive bacterial strains, and the strains whose results were correct were named CGMCC22721-yjiV-trpR-lacIZ-ycgH and W3110-yjiV-trpR-lacIZ-ycgH, respectively.

[0170] Recombinant strains CGMCC22721-yjiV-trpR-lacIZ-ycgH and W3110-yjiV-trpR-lacIZ-ycgH both lack a yjiV gene and overexpress the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168) driven by the Ptrc promoter and the ilvD gene (derived from Escherichia coli W3110) driven by the PilvD promoter, while both lack a trpR gene and overexpress the ilvH gene (derived from Escherichia coli W3110) driven by the Ptrc promoter. G14D、S17F The following genes are overexpressed: the lacI-lacZ gene is deleted, the DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter is overexpressed, the ycgH gene is deleted, and the ilvC gene (derived from Escherichia coli W3110) driven by the Ptrc promoter is overexpressed. Specifically, recombinant CGMCC22721-yjiV-trpR-lacIZ-ycgH and W3110-yjiV-trpR-lacIZ-ycgH were created by knocking out a portion of the coding region of the yjiV gene in the genomes of the L-valine-producing bacterium CGMCC22721 and wild-type Escherichia coli W3110, respectively, inserting the ilvE gene (derived from Bacillus subtilis subsp. subtilis str.168) driven by the Ptrc promoter and the ilvD gene (derived from Escherichia coli W3110) driven by the PilvD promoter, knocking out a portion of the coding region of the trpR gene, and creating a mutant ilvH gene driven by the Ptrc promoter. G14D、S17FThis recombinant bacterium was obtained by inserting a gene, knocking out a portion of the coding region of the lacI-lacZ gene, inserting a DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter, knocking out a portion of the coding region of the ycgH gene, inserting an ilvC gene (derived from Escherichia coli W3110) driven by the Ptrc promoter, and maintaining other nucleotides in the genome without modification.

[0171] Example 13: Construction of genetically modified bacteria in which the lacI-lacZ gene is deleted and the DNA polymerase gene is overexpressed on CGMCC22721-trpR and W3110-trpR. Based on the genome sequence of Escherichia coli W3110 published by NCBI, CRISPR / Cas9 genome editing technology was used to knock out the lacI-lacZ gene (the complete lacI-lacZ gene was confirmed to be present on the chromosomes of these strains by sequencing analysis) in the genomes of L-valine-producing strain CGMCC22721-trpR and wild-type E. coli W3110-trpR, and to insert a DNA polymerase gene (derived from E. coli BL21) driven by the PxylF promoter. This allowed for a more detailed study of the effects of these genes on L-valine synthesis.

[0172] The pREDCas9 plasmid (containing the spectinomycin resistance gene) was extracted and transformed into competent cells of CGMCC22721-trpR and W3110-trpR from Example 3, respectively. These transformed cells were then spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and cultured at 32°C. Single colonies showing resistance to spectinomycin (100 mg / L) were selected and identified by PCR using primers pRedCas9-PF / pRedCas9-PR. Cells yielding 943 bp were designated as CGMCC22721-trpR-Cas9 and W3110-trpR-Cas9 transformants containing the pREDCas9 plasmid.

[0173] Competent cells of CGMCC22721-trpR-Cas9 and W3110-trpR-Cas9 were prepared. 600 Once the cells had grown to 0.1 mM, a final concentration of 0.1 mM IPTG was added to induce homologous recombination via λ-Red. 600 When the ratio reached 0.4, the bacterial cells were harvested and competent cells were prepared. These were transformed with the pGRB-sgRNA-4 plasmid and homologous recombinant DNA fragment ΔlacIZ-PxylF-DNA polymerase from Example 4, respectively. These were then spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L) and cultured at 32°C for 12 hours. After culturing, the obtained single colonies were subcultured and then PCR identification was performed using primers P35 / P36. Fragments of size 1655 bp that were amplified by PCR were considered positive transformants.

[0174] Positive transformants were seeded in 2-YT medium containing spectinomycin (100 mg / L) and 0.2% arabinose, and the plasmid pGRB-sgRNA-4 was removed. Colonies that grew with spectinomycin (100 mg / L) but not with ampicillin (100 mg / L) were selected, and these colonies were re-seeded in 2-YT medium and cultured at 42°C to remove the pREDCas9 plasmid. Colonies that did not grow with spectinomycin (100 mg / L) but grew in antibiotic-free 2-YT were selected, and PCR identification was performed again using primers P27 / P34. Amplified fragments of size 4524 bp (SEQ ID No. 9) were identified as positive strains. Sequence analysis was performed on the positive bacterial strains, and the strains whose results were correct were named CGMCC22721-trpR-lacIZ and W3110-trpR-lacIZ, respectively.

[0175] Recombinant strains CGMCC22721-trpR-lacIZ and W3110-trpR-lacIZ both lack a trpR gene and are driven by the Ptrc promoter, and also lack ilvH G14D、S17F The genes are overexpressed, with a deletion of the lacI-lacZ gene and overexpression of the DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter. Specifically, recombinant CGMCC22721-yjiV-trpR-lacIZ and W3110-yjiV-trpR-lacIZ have knockouts in a portion of the coding region of the trpR gene on the genomes of L-valine-producing bacterium CGMCC22721 and wild-type E. coli W3110, respectively, and have mutations driven by the Ptrc promoter, such as ilvH. G14D、S17F This recombinant bacterium was obtained by inserting a gene, knocking out a portion of the coding region of the lacI-lacZ gene, inserting a DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter, and maintaining other nucleotides in the genome without modification.

[0176] Example 14: Construction of genetically modified bacteria in which the ycgH gene is deleted and the ilvC gene is overexpressed on CGMCC22721-trpR-lacIZ and W3110-trpR-lacIZ. Based on the genome sequence of Escherichia coli W3110 published by NCBI, the ycgH gene (the complete ycgH gene was confirmed to be present on the chromosomes of these strains by sequencing analysis) was knocked out using CRISPR / Cas9 genome editing technology in the genomes of the L-valine-producing strain CGMCC22721-trpR-lacIZ and the wild-type E. coli W3110-trpR-lacIZ. Simultaneously, the ilvC gene (derived from E. coli W3110) driven by the Ptrc promoter was inserted. This allowed for a more detailed study of the effects of these genes on L-valine synthesis.

[0177] The pREDCas9 plasmid (containing the spectinomycin resistance gene) was extracted and transformed into competent cells of CGMCC22721-trpR-lacIZ and W3110-trpR-lacIZ from Example 13, respectively. These transformed cells were then spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and cultured at 32°C. Single colonies showing resistance to spectinomycin (100 mg / L) were selected and identified by PCR using the primers pRedCas9-PF / pRedCas9-PR. Cells yielding 943 bp were designated as CGMCC22721-trpR-lacIZ-Cas9 and W3110-trpR-lacIZ-Cas9 transformants containing the pREDCas9 plasmid.

[0178] Competent cells of CGMCC22721-trpR-lacIZ-Cas9 and W3110-trpR-lacIZ-Cas9 were prepared. The bacterial cells were OD 600 Once the cells had grown to 0.1 mM, a final concentration of 0.1 mM IPTG was added to induce homologous recombination via λ-Red. 600When the ratio reached 0.4, the bacterial cells were harvested and competent cells were prepared. These were transformed with the pGRB-sgRNA-5 plasmid and homologous recombinant DNA fragment ΔycgH-Ptrc-ilvC from Example 5, respectively, and spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L). The cells were incubated at 32°C for 12 hours. After culturing, the single colonies obtained were subcultured and then PCR identified using primers P43 / P44. Fragments of size 1669 bp that were amplified by PCR were considered positive transformants.

[0179] Positive transformants were seeded in 2-YT medium containing spectinomycin (100 mg / L) and 0.2% arabinose, and the plasmid pGRB-sgRNA-5 was removed. Colonies that grew with spectinomycin (100 mg / L) but not with ampicillin (100 mg / L) were selected, and these colonies were re-seed in 2-YT medium and cultured at 42°C to remove the pREDCas9 plasmid. Colonies that did not grow with spectinomycin (100 mg / L) but grew in antibiotic-free 2-YT were selected, and PCR identification was performed again with primers P37 / P42. Amplified fragments of size 3189 bp (SEQ ID No. 11) were identified as positive strains. Sequence analysis was performed on the positive bacterial strains, and the strains whose results were correct were named CGMCC22721-trpR-lacIZ-ycgH and W311-trpR-lacIZ-ycgH, respectively.

[0180] Recombinant bacteria CGMCC22721-trpR-lacIZ-ycgH and W3110-trpR-lacIZ-ycgH both lack a trpR gene and are driven by the Ptrc promoter, ilvH G14D、S17FThe genes are overexpressed, with the lacI-lacZ gene being deleted, the DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter being overexpressed, the ycgH gene being deleted, and the ilvC gene (derived from Escherichia coli W3110) driven by the Ptrc promoter being overexpressed. Specifically, recombinant CGMCC22721-trpR-lacIZ-ycgH and W3110-trpR-lacIZ-ycgH have a portion of the coding region of the trpR gene on the genomes of L-valine-producing strain CGMCC22721 and wild-type Escherichia coli W3110 knocked out, respectively, and the mutant ilvH gene driven by the Ptrc promoter is overexpressed. G14D、S17F This recombinant bacterium was obtained by inserting a gene, knocking out a portion of the coding region of the lacI-lacZ gene, inserting a DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter, knocking out a portion of the coding region of the ycgH gene, inserting an ilvC gene (derived from Escherichia coli W3110) driven by the Ptrc promoter, and maintaining other nucleotides in the genome without modification.

[0181] Example 15: Construction of genetically modified bacteria in which the ycgH gene is deleted and the ilvC gene is overexpressed on CGMCC22721-lacIZ and W3110-lacIZ. Based on the genome sequence of Escherichia coli W3110 published by NCBI, the ycgH gene (the complete ycgH gene was confirmed to be present on the chromosomes of these strains by sequencing analysis) was knocked out using CRISPR / Cas9 genome editing technology, and the ilvC gene, driven by the Ptrc promoter, was inserted. This allowed for a more detailed study of the effects of these genes on L-valine synthesis.

[0182] The pREDCas9 plasmid (containing the spectinomycin resistance gene) was extracted and transformed into competent cells of CGMCC22721-lacIZ and W3110-lacIZ from Example 4, respectively. These transformed cells were then spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and cultured at 32°C. Single colonies showing resistance to spectinomycin (100 mg / L) were selected and identified by PCR using primers pRedCas9-PF / pRedCas9-PR. Cells yielding 943 bp were designated as CGMCC22721-lacIZ-Cas9 and W3110-lacIZ-Cas9 transformants containing the pREDCas9 plasmid.

[0183] Competent cells of CGMCC22721-lacIZ-Cas9 and W3110-lacIZ-Cas9 were prepared. The bacterial cells were OD 600 Once the cells had grown to 0.1 mM, a final concentration of 0.1 mM IPTG was added to induce homologous recombination via λ-Red. 600 When the ratio reached 0.4, the bacterial cells were harvested and competent cells were prepared. These were transformed with the pGRB-sgRNA-5 plasmid and homologous recombinant DNA fragment ΔycgH-Ptrc-ilvC from Example 5, respectively, and spread onto 2-YT agar plates containing spectinomycin (100 mg / L) and ampicillin (100 mg / L). The cells were incubated at 32°C for 12 hours. After culturing, the single colonies obtained were subcultured and then PCR identified using primers P43 / P44. Fragments of size 1669 bp that were amplified by PCR were considered positive transformants.

[0184] Positive transformants were seeded in 2-YT medium containing spectinomycin (100 mg / L) and 0.2% arabinose, and the plasmid pGRB-sgRNA-5 was removed. Colonies that grew with spectinomycin (100 mg / L) but not with ampicillin (100 mg / L) were selected, and these colonies were re-seed in 2-YT medium and cultured at 42°C to remove the pREDCas9 plasmid. Colonies that did not grow with spectinomycin (100 mg / L) but grew in antibiotic-free 2-YT were selected, and PCR identification was performed again with primers P37 / P42. Amplified fragments of size 3189 bp (SEQ ID No. 11) were identified as positive strains. Sequence analysis was performed on the positive bacterial strains, and the strains whose results were correct were named CGMCC22721-lacIZ-ycgH and W311-lacIZ-ycgH, respectively.

[0185] Recombinant strains CGMCC22721-lacIZ-ycgH and W3110-lacIZ-ycgH both lack the lacI-lacZ gene and overexpress the DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter, while both lack the ycgH gene and overexpress the ilvC gene (derived from Escherichia coli W3110) driven by the Ptrc promoter. Specifically, recombinant bacteria CGMCC22721-lacIZ-ycgH and W311-lacIZ-ycgH are recombinant bacteria obtained by knocking out a portion of the coding region of the lacI-lacZ gene in the genomes of the L-valine-producing bacterium CGMCC22721 and wild-type Escherichia coli W3110, respectively, inserting a DNA polymerase gene (derived from Escherichia coli BL21) driven by the PxylF promoter, and knocking out a portion of the coding region of the ycgH gene (derived from Escherichia coli W3110) driven by the Ptrc promoter, while retaining other nucleotides in the genome without modification.

[0186] Example 16: Fermentation experiment of L-valine The strains constructed in Examples 1-15 above, the valine-producing bacterium CGMCC22721 and the wild-type E. coli W3110, were subjected to fermentation experiments in a BLBIO-5GC-4-H fermentation tank (Shanghai Bailun Biotechnology Co., Ltd.) using the culture medium shown in Figure 1 and the control process shown in Figure 2. The results of three repetitions for each strain are shown in Figure 3. From these fermentation results, it was shown that both the L-valine-producing bacterium CGMCC22721 and the model strain, wild-type E. coli W3110, contributed to an improvement in L-valine production after being modified in Examples 1-15.

[0187] The present invention has been described in detail above. Those skilled in the art will be able to implement the present invention over a wider range of parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without performing unnecessary experiments. While the present invention has shown specific examples, it should be understood that further improvements can be made to the present invention. In short, based on the principles of the present invention, this application is intended to include any modifications, uses, or improvements to the present invention, including those that deviate from the scope disclosed herein and are made using prior art known in the art. [Industrial applicability]

[0188] Experiments have demonstrated that the genetically modified bacteria of the present invention can increase L-valine production, can be used for L-valine production, and have good prospects for application. [Sequence Listing Free Text]

[0189] SEQ ID No.1: Homologous recombination DNA fragment ΔyjiT-Ptrc-brnF-brnE sequence (2579 bp), artificial sequence. 1 GAGTGATGAG CGGTTGAAGG ATATCGGGTT ACGCAGGGAG GATGTGGAGT GAGGGGGGAT 61 ATAGATTTAT ATATAATAAA ACGTTTTTAT GTTTTTAAAT TAAGTTATAA AAATTTTCCC 121 GAGACAATTC ATCAATAGGT ATGGAGTGTA CAGGAATATC TTCTTCACTA ACCTCTTTAA 181 ACTCATGTCG ATAACGGTCA AAAAACGCAC AGTGATTACT TGTGTTTTTT GACTGACTTT 241 ACAACTCTCA TTATTCGCTA TTGTGCAGTT TCTCTAATTG TTTTATACCC TGGAAAGTTA 301 AATGTCAGCT ACTGAATACT TTTTGATTGT TTGAGATTTA TTTTCATTTG AAATTATAAA 361 ATCAGGTGAT AAATGAGTTG TGATTAATAG ATGGTTGATA TCATTTTTAT CTAAAATTGA 421 TTTATAGTAT CGACCTGAAA AAATAGTTGT TGCCGCCTGA GTAACTATAC AATATTCTGA 481 AAGGTTTTCT TTCAAATTAG AAATGTTGTG GGGTTTTGTT TTTGTATCTT TTATCTCTAA 541 GGAGCTTGCT TTGGGTCAAT CAGAATACAT TTCATGGGTA AAATGTACTT CCTGGCTAAG 601 TAACTTTGTG AATCTTAGAG GGTTGAGACA ACCGGATGGC CGTCCTCTTT ATGAATATCA 661 TGCAACCAAT GATGAATATA CCCAATTAAC GCAGCTACTC CGTGCAGTCG GTCAATCACA 721 ATCTAATATA TGTAATAGGG ACTTTGCTGC CTGTTTTTTG ACAATTAATC ATCCGGCTCG 781 TATAATGTGT GGAATTGTGA GCGGATAACA ATTTCACACA GGAAACAGAC CGTGCAAAAA 841 ACGCAAGAGA TTCATTCAAG CCTGGAGGTG TCGCCATCCA AGGCAGCCCT GGAACCAGAT 901 GATAAAGGTT ATCGGCGCTA CGAAATCGCG CAAGGTCTAA AAACCTCCCT TGCTGCAGGT 961 TTGGGCATGT ACCCGATTGG TATTGCGTTT GGTCTCTTGG TTATTCAATA CGGCTACGAA 1021 TGGTGGGCAG CCCCACTGTT TTCCGGCCTG ATTTTCGCGG GCTCCACCGA AATGCTGGTC 1081 ATCGCCCTCG TTGTGGGCGC AGCGCCCCTG GGCGCCATCG CGCTCACCAC ATTGCTGGTG 1141 AACTTCCGCC ACGTATTCTA TGCGTTTTCA TTCCCGCTGC ATGTGGTCAA AAACCCCATT 1201 GCCCGTTTCT ATTCGGTTTT CGCGCTTATC GACGAAGCCT ACGCAGTCAC TGCGGCCAGG 1261 CCCGCAGGCT GGTCGGCGTG GCGACTTATC TCAATGCAAA TAGCGTTTCA CTCCTACTGG 1321 GTATTCGGCG GTCTCACCGG AGTGGCGATC GCAGAGTTGA TTCCTTTTGA AATTAAGGGC 1381 CTCGAGTTCG CCCTTTGCTC TCTCTTTGTC ACGCTGACTT TGGATTCCTG CCGAACGAAA 1441 AAGCAGATCC CTTCTCTGCT GCTCGCAGGT TTGAGCTTCA CCATTGCTCT TGTGGTAATT 1501 CCAGGTCAGG CCCTATTTGC GGCGCTGCTG ATCTTCTTGG GTCTGTTGAC CATCCGGTAC 1561 TTCTTCTTGG GAAAGGCTGC TAAATGACAA CTGATTTCTC CTGTATTCTC CTTGTTGTCG 1621 CAGTATGTGC AGTCATTACT TTTGCGCTCC GGGCGGTTCC GTTCTTAATC CTTAAGCCCC 1681 TACGTGAATC ACAATTTGTG GGCAAAATGG CGATGTGGAT GCCAGCAGGA ATCCTTGCCA 1741 TTTTGACCGC ATCAACGTTT CGCAGCAATG CGATAGATCT GAAGACTCTA ACCTTTGGTC 1801 TCATTGCCGT TGCGATTACA GTGGTGGCGC ATCTTCTTGG CGGTCGACGC ACCTTGTTGA 1861 GCGTTGGCGC TGGCACCATC GTTTTTGTTG GACTGGTGAA TCTTTTCTAA CTAAAAGCAC 1921 TACCTGTGAA GGGATGTCAG GACGTTACAG TTACAGCAAA TGAAACTTAT CGGATTCGCA 1981 CCGGAAGAGA ACAAATCAGC ATCGGAAGGT TTGCTCTAAA TGGAAAGCGT GCAAGCTGGG 2041 TTTGTCATCC AGATGAAACA TTTATTGGTG TACCAAAAGT CATTTCTACA CTACCGGATA 2101 TTCAAAGCAT TGATGTAACG CGTTACACGT GCTGACAAAA CAGCATTACA GCCAGCAGGA 2161 AGTACTGCGT TGGATCGATG TCTGTTCAGG GACTCAACCT AATGCAAAGG ATCCCGCATT 2221 TCTTAAAGTC AGGGCGCATA TCTTCCAGCG TAATACCTAG GGAATATGGG CTTGTGTTGA 2281 TAAAGATTGC AGATTAAAGC ACGGTACACC GCTCGACAAA GGCTGGCCCT TTGGCTATGT 2341 GTATGTGAAC CAGCGACAAA ATTGTGACTG TGGAAGCCCT GTATACGAAG TTGCATTCTG 2401 TAATGATTGT AATGAGCCTC ATCTTCTGGC ACGGGACAAA AAGGGCAAAC TAGTCCAGTG 2461 GGAAAATAAA GGTGGCGATG AATTCTCTTT GCAGGATGA GTACCTGTTG AACATGACGC 2521 TACAGAAGAA AAAGTCGAAA AAGAGAACAG TTTTCAGCCT CCGTTGATTA TTGCCGCAG SEQ ID No.2:brnF fragmentase(Corynebacterium glutamicum) ATCC13032 transcript) . 1 VQKTQEIHSS SCHOOL OF LIFE EPDDKGYRRY EIAQGLKTSL AAGLGMYPIG IAFGLLVIQY 61 GYEWWAAPLF SGLIFAGSTE MLVIALVVGA APLGAIALTT LLVNFRHVFY AFSFPLHVVK 121 NPIARFYSVF ALIDEAYAVT AARPAGWSAW RLISMQIAFH SYWVFGGLTG VAIAELIPFE 181 IKGLEFALCS LFVTLTLDSC RTKKQIPSLL LAGLSFTIAL VVIPGQALFA ALLIFLGLLT 241 IRYFFLGKAA K SEQ ID No.3:brnE scaffold(Corynebacterium glutamicum) ATCC13032 transcript) . 1 MTTDFSCILL VVAVCAVITF ALRAVPFLIL KPLRESQFVG KMAMWMPAGI LAILTASTFR 61 SNAIDLKTLT FGLIAVAITV VAHLLGGRRT LLSVGAGTIV FVGLVNLF SEQ ID No.4:Acquisition construct consisting of DNA polymerase chain yjiV-Ptrc-ilvE-PilvD-ilvD fragment(4732bp) (Artificial sequence); 1 TGAATGGACT GCTATGCGCA GCCTCTGTAG AGGCTAAGCC TCGACTACAA AAAAATAAGA 61 GTACGCGTTG CCAATTCTAT TGGTCAGAAA AGCATCCAGA TGAGCTTAGG GTGATAGTAT 121 CTCTTCCGGA CGAAGTTTCC TTTCCTGTAA CAAGCGAGCC GTCAACTACG CGCTTTGAAC 181 TTGCCATTTG TGAAGATGGT GAGGAAGCTCT CTGGCCTTGG GCCAGCCTAT GCTTCTCTGG 241 AAAACAGACA GGCAACAGTT CGATTACGTA AAAGCGAAGT GAGATTTGGC AGGCAAAATC 301 CATCGGCAGG TTTGTCGTTA GTGGCTCGTG CTGGAGGGAT GATTGTTGGG AGCATTAAAC 361 TTGATGACAG TGAAATTGCC ATTGGTGAGG TGCCGTTAAC CTTCATCGTT GATGCAGATC 421 AATGGCTGTT ACAGGGACAG GCTTCTTGCA GTGTGCGAAG CAGCGATGTT CTGATTGTGC 481 TCCCTCGGGA TAATAGCAAT GTTGCTGGTT TTGATGGCCA ATCGAGGGCA GTAAACGTAT 541 TAGGACTAAA AGCACTACCT GTGAAGGGAT GTCAGGACGT TACAGTTACA GCAAATGAAA 601 CTTATCGGAT TCGCACCGGA AGAGAACAAA TCAGCATCGG AAGGTTTGCT CTAAATGGAA 661 AGCGTGCAAG CTGGGTTTGT CATCCAGATG AAACATTTAT TGGTGTCACCA AAAGTCTATT 721 CTACACTACC GGATATTCAA AGCATTGATG TAACGCGTTA CACGTGCTGA CAAAACAGCA 781 TTACAGCCAG CAGGAAGTAC TGCGTTGTTA ACGTCTGCTT AATACACTGT GGCCGTTTTG 841 CAAAAACTGG CTTCTTGAGC GGCGCATGCG TTCAATCCGT TCCTCTGCTA AGCGGTCAGC 901 CGCTAAATAT GCAGGAATGC CGTCACGCTG AGAAATCTCA AGTACACGCT CGATATTGCC 961 GTAAATGCCT TCAACTTTTT TCAATGCACG TTCTGCATTA TAGCCGTAAA GCTCATCTGC 1021 CACGTTGATG ACACCGCCCG CGTTAATCAC GTAATCCGGT GCATAAACGA TGCCCATTTC 1081 GTGAATTTGA TCACCATGGC GTGTCTCTTT TAATTGGTTG TTAGCCGCAC CTGCGATCAC 1141 TTTCGCCTTC AGCTGTTTAA TGGTGTCGTC GTTAATAGTC GCACCAAGGG CACACGGCGC 1201 ATAAATATCG CAGTCTTGTG AATAAATGTC ATCAGGATCT ACCGCACGGG CGCCAAAATC 1261 TTCAACTGCA CGCTGTACAG ATTGTTTGTT GATATCCGTA ACGATTAAGT TTGCTCCTTC 1321 TTCATGCAGG TGGCGGCAAA GGTTATAGGC TACGTTCCCA ACACCCTGTA CAGCAATGGT 1381 TTTTCCTTCA AGAGAGTCGG TTCCGAAAGC AGCTTTAGCT GCTGCCTTCA TTCCTCTGTA 1441 CACCCCGTAC GCTGTGACTG GGGACGGATT TCCAGAAGAG CCGAAAGCAG GAGAAATCCC 1501 TGTGACATAG TCTGTCTCAT CATGAATGAT ATCCATATCC TCGACCGTTG TGCCCACATC 1561 TTCAGCCGTG ATGTATCTGC CATTCAGTCC TTGAATATAG CGGCCAAACG CGCGGAACAT 1621 TTCCTCATTT TTGTCTTTGC GCGGATCGCC GATAATGACT GTTTTTCCGC CGCCAAGGTT 1681 TAAGCCTGCC GCCGCGTTCT TATAGGTCAT GCCTCTTGCC AATCTGAGCG CATCTTCAAT 1741 TGCCGCTTCT TCATTTTCAT ATGTCCACAT TCTCGTTCCG CCAAGCGCCG GACCAAGCGT 1801 TGTATCATGA ATGGCGATAA TCGCTTTTAA TCCAGATTGT TCATCCTGGC AGAATACCAA 1861 TTGTTCGTAA TCGTATTTCT CCATATATTT AAAAAGTTCC ATGGTCTGTT TCCTGTGTGA 1921 AATTGTTATC CGCTCACAAT TCCACACATT ATACGAGCCG GATGATTAAT TGTCAATTAA 1981 CCCCCCAGTT TCGATTTATC GCGCACCGCG CCTTTGTCGG CGCTGGTTGC CAGGCTGGCA 2041 TAAGCACGCA GGGCAAAGGA GACCTGACGT TCACGATTTT TCGGCGTCCA GGCTTTGTCA 2101 CCTCGAGCGT CCTGCGCTTC ACGACGCGCC GCCAGTTCGG CATCGCTTAC CTGTAACTGA 2161 ATGCCACGGT TCGGGATGTC GATAGCGATC AGGTCACCAT CTTCAATCAG GCCAATGCTG 2221 CCGCCGCTTG CCGCTTCCGG TGAGACGTGG CCGATGGAAA GACCAGAGGT GCCACCAGAG 2281 AAACGACCGT CGGTGATCAG CGCACAGGCT TTGCCGAGAC CCATTGATTT CAGGAAGCTG 2341 GTTGGGTAGA GCATTTCCTG CATCCCCGGA CCGCCTTTCG GGCCTTCATA GCGAATTACT 2401 ACCACATCTC CGGCGACAAC TTTACCGCCG AGAATCGCTT CTACCGCATC GTCCTGGCTT 2461 TCGTACACTT TCGCCGGGCC GGTGAATTTG AGGATGCTGT CATCGACGCC TGCCGTTTTC 2521 ACGATGCAGC CGTTTTCCGC AAAGTTACCG TAGAGCACCG CCAGGCCGCC GTCTTTGCTG 2581 TAGGCGTGTT CCAGCGAGCG GATACAGCCA TTGGCGCGAT CGTCGTCCAG CGTATCCCAA 2641 CGGCAATCTT GCGAGAATGC CTGTGTGGTA CGAATGCCTG CAGGACCTGC GCGGAACATA 2701 TTTTTTACCG CGTCATCCTG GGTCAGCATA ACGTCGTATT GTTCCAGCGT TTGCGGCAAC 2761 GTCAGGCCAA GTACGTTTTT CACATCACGG TTCAGTAACC CCGCGCGATC CAGTTCGCCG 2821 AGAATACCGA TAACACCACC AGCACGGTGA ACATCTTCCA TATGGTATTT CTGGGTGCTC 2881 GGCGCAACTT TACACAGCTG TGGAACCTTG CGGGAAAGCT TATCGATATC ACTCATGGTG 2941 AAGTCGATTT CCGCTTCCTG CGCCGCCGCC AGCAGGTGAA GTACGGTGTT AGTCGATCCA 3001 CCCATCGCGA TATCCAGCGT CATGGCGTTT TCAAACGCCG CCTTACTGGC GATATTACGC 3061 GGCAGTGCAC TTTCGTCGTT TTGCTCGTAA TAACGTTTGG TCAATTCAAC AATGCGTTTA 3121 CCAGCATTAA GGAACAGCTG CTTACGGTCG GCGTGGGTTG CCAGCAGCGA GCCGTTGCCC 3181 GGCTGCGACA GGCCCAGCGC TTCGGTCAGG CAGTTCATTG AGTTAGCGGT AAACATCCCG 3241 GAGCAGGAAC CGCAGGTCGG ACACGCGGAA CGTTCAACCT GATCGCTCTG GGAGTCAGAT 3301 ACTTTCGGGT CTGCGCCCTG GATCATCGCA TCAACCAGAT CGAGCTTGAT GATCTGATCG 3361 GAAAGTTTGG TTTTCCCGGC CTCCATCGGG CCGCCGGAAA CAAAGATCAC CGGAATATTC 3421 AGGCGCAGGG AAGCCATCAG CATCCCCGGG GTGATTTTGT CGCAGTTAGA GATGCAGACC 3481 ATGGCGTCGG CGCAGTGGGC GTTGACCATA TACTCAACGG AATCAGCGAT CAGTTCGCGA 3541 GATGGCAGTG AATAAAGCAT CCCCCCGTGG CCCATGGCAA TCCCATCATC CACCGCAATG 3601 GTGTTGAACT CTTTGGCAAC GCCGCCAGCC GCTTCAATTT GTTCGGCGAC CAGTTTACCG 3661 AGATCGCGCA GATGGACGTG ACCCGGTACA AATTGGGTGA ACGAGTTCAC AACCGCGATA 3721 ATCGGCTTAC CGAAATCGGC GTCGGTCATT CCGGTGGCGC GCCACAGCGC ACGAGCACCC 3781 GCCATATTAC GACCATGAGT GGTGGTGGCG GAACGGTACT TAGGCATACT TTATTTACTC 3841 CCAGTGTCTG TCTCGTAAAT GGGACGGTGC GTGCCGTCCC ATTTTTTGTA TTTCAACGGA 3901 TGTGCTGGTG GAGGTGATCG CCTCCTGATG ATGAGCCGCT CCCGATGTGG TGTCGGGAGC 3961 GGTATTTTCT ATAAAACTTA CCGCTTATTT GAGATATTCA TCGAAAATGT CGAGTAATTC 4021 TTGATGTATA CACGGCCATT CCTGACCTAA ATTGACGGTA CACAAGCCAA TATCGAAGCC 4081 ATTAATTTTA TAACGATGTT TCACTGCGGT ATCTACGTGG GGATATATTA ATAACCCCCC 4141 TATGTTTTCG CCATTTTCAG GCTTTAACGA CCATAAGTAA TTCATCAGTT GATAAAGATT 4201 TTGCGAATGA AATTTTTCTG TTCCCATTCG TCGTGAAAAA ATGCTCTTAT AGTATTTGGC 4261 GTCAACGATA AGTATTTTTT CTGATGAGCG AATGGTGATG TCAGTTTCCA TTCGAGGTAA 4321 CAAATTAAGT GACTGATCCG ATATACTCGA TGCATCCCAT TTTAAATAAG AGCGGGTTGT 4381 GTTTGCAGAC GTTAATTCAC GACGGCAAAA TTCATAAAGA AACTTTTGAT AAAGTAATGA 4441 CATCTCTTTT TCGTTTCTTT CAAAATCATA GAAACGGTAG TGTCCTTTGT TTTGACCTGG 4501 AATAGAATTA TTGACGATGA ATTTGCAGAC ACTGATAACG AATTTATAAT AACGCGTATT 4561 TTTTCCGCCA TTCAGATAGC TGAAATGCTG CGGAGTTAAA TGAAGAGTGC TAATGCCCGG 4621 TAATTTTCTA TAAAGTGAAC GAGCTTCATC TCTGATAGTT GAATTTAACT TTTCATGCTT 4681 AATTAATATG GCTAATGTGC TTTTTATAAT TCGGTTAGCC AGCGTGTCTT CA SEQ ID No.5: ilvD protein (derived from Escherichia coli W3110) 1 MPKYRSATTT HGRNMAGARA LWRATGMTDA DFGKPIIAVV NSFTQFVPGH VHLRDLGKLV 61 AEQIEAAGGV AKEFNTIAVD DGIAMGHGGM LIGHTING RELIE ADSVEYMVNA HCADAMVCIS 121 NCDKITPGML MASLRLNIPV IFVSGGPMEA GKTKLSDQII KLDLVDAMIQ GADPKVSDSQ 181 SDQVERSACP TCGSCSGMFT ANSMNCLTEA LGLSQPGNGS LLATHADRKQ LFLNAGKRIV 241 ELECTRICAL DESALPRNIA SKAAFENAMT LDIAMGGSTN TVLHLLAAAQ EAEIDFTMSD 301 IDKLSRKVPQ LCKVAPSTQK YHMEDVHRAG GVIGILGELD RAGLLNRDVK NVLGLTLPQT 361 LEQYDVMLTQ DDAVKNMFRA GPAGIRTTQA FSQDCRWDTL DDDRANGCIR SLEHAYSKDG 421 GLAVLYGNFA ENGCIVKTAG VDDSILKFTG PAKVYESQDD AVEAILGGKV VAGDVVVIRY 481 EGPKGGPGMQ EMLYPTSFLK SMGLGKACAL ITDGRFSGGT SGLSIGHVSP EAASGGSIGL 541 IEDGDLIAID IPNRGIQLQV SDAELAARRE AQDARGDKAW TPKNRERQVS FALRAYASLA 601 TSADKGAVRD KSKLGG SEQ ID No.6:ilvE strain(Bacillus subtilis subsp. subtilis str.168) sequence) 1 MELFKYMEKY DYEQLVFCQD EQSGLKAIIA IHDTTLGPAL GGTRMWTYEN EEAAIEDALR 61 LARGMTYKNA AAGLNLGGGK TVIIGDPRKD KNEEMFRAFG RYIQGLNGRY ITAEDVGTTV 121 EDMDIIHDET DYVTGISPAF GSSGNPSPVT AYGVYRGMKA AAKAAFGTDS LEGKTIAVQG 181 VGNVAYNLCR HLHEEGANLI VTDINKQSVQ RAVEDFGARA VDPDDIYSQD CDIYAPCALG 241 ATINDDTIKQ LKAKVIAGAA NNQLKETRHG DQIHEMGIVY APDYVINAGG VINVADELYG 301 YNAERALKKV EGIYGNIERV LEISQRDGIP AYLAADRLAE ERIERMRRSR SQFLQNGHSV 361 LSRR SEQ ID No.7: Artificial sequence (Artificial sequence) 1 CCAATCTGGT GAAGAGCAAG TCAAAAACAG AGCAGGCTCA ACTGGCGCGG TATGCTTTCA 61 ACAACCAATG GTGGGATCTT AGCGTTCAGG CAACGATCGC CGGGAAGCTG TGGGATCATC 121 TGGAAGAGCG ATTCCCGCTG GCTTACAACG ATCTTTTCAA ACGCTACACC AGCGGTAAGG 181 AGATCCCGCA AAGCTATGCG ATGGCGATTG CTCGTCAGGA GAGCGCCTGG AATCCGAAAG 241 TGAAATCACC GGTAGGGGCC AGCGGCTTGA TGCAGATTAT GCCTGGTACA GCGACCCATA 301 CGGTGAAGAT GTTCTCTATT CCCGGTTATA GCAGTCCTGG GCAATTGCTG GATCCGGAAA 361 CGAATATCAA CATTGGCACC AGTTACCTGC AATATGTTTA TCAGCAGTTT GGCAATAATC 421 GTATTTTCTC CTCAGCAGCT TATAACGCCG GACCAGGGCG GGTGCGAACC TGGCTTGGCA 481 ACAGCGCCGG GCGTATCGAC GCAGTGGCAT TTGTCGAGAG TATTCCATTC TCCGAGACGC 541 GCGGTTATGT GAAGAACGTG CTGGCTTATG ACGCTTACTA CCGCTTATTTC ATGGGGGATA 601 AACCGACGTT GATGAGCGCC ACGGAATGGG GACGTCGTTA CTGATCCGCA CGTTATGAT 661 ATGCTATCGT ACTCTTTAGC GAGTACAACC GGGGGAGGCA TTTTGCTTCC CCCGCTAACA 721 ATGGCGACAT ATTATGGCCC AACAATCACC CTATTCAGCA TTGACAATTA ATCATCCGGC 781 TCGTATAATG TGTGGAATTG TGAGCGGATA ACAATTTCAC ACAGGAAACA GACCATGCGC 841 CGGATATTAT CAGTCTTACT CGAAAATGAA TCAGACGCGT TATTCCGCGT GATTGGCCTT 901 TTTTCCCAGC GTGGCTACAA CATTGAAAGC CTGACCGTTG CGCCAACCGA CGATCCGACA 961 TTATCGCGTA TGACCATCCA GACCGTGGGC GATGAAAAAG TACTTGAGCA GATCGAAAAG 1021 CAATTACACA AACTGGTCGA TGTCTTGCGC GTGAGTGAGT TGGGGCAGGG CGCGCATGTT 1081 GAGCGGGAAA TCATGCTGGT GAAAATTCAG GCCAGCGGTT ACGGGCGTGA CGAAGTGAAA 1141 CGTAATACGG AAATATTCCG TGGGCAAATT ATCGATGTCA CACCCTCGCT TTATACCGTT 1201 CAATTAGCAG GCACCAGCGG TAAGCTTGAT GCATTTTTAG CATCGATTCG CGATGTGGCG 1261 AAAATTGTGG AGGTTGCTCG CTCTGGTGTG GTCGGACTTT CGCGCGGCGA TAAAATAATG 1321 CGTTGATTTG GTAGGCATGA TAAGACGCGG CAGCGTCGCA TCAGGCGCTT AATACACGGC 1381 ATTATGAAAC GGACTCAGCG CCAGGATCAC CGCCTGGTGA TAGACGCTGG CGCGAGTGAG 1441 TTTCCCGGCG GTAAACACGC CGATCGCCCC TTCCTTACGA CCAATCTCAT CAATACCGGT 1501 ATAACGCGAC ATCACGGGAC CAAGCGCCTC ACCTTCACGC ACTTTTTCCA GAATCACCGC 1561 AGGCAACGGC AAAGTAGCCG AACGCGCCTC GCCGCGCTGG CTGGCGTTTT CAATCACCAC 1621 CCAACTGAAA GTGCTGTCAC CATCGATGCC AGCTTCAATC GCCACCCAAA AATCAGCCTC 1681 TGGAAGTAAA CGGCGGGCAT TGGCTACCCG ATTTCGTGCG CCAGCGCGCG TTTCCTCACT 1741 GCCAAAGGGC TGTTCCGGTA CACCGCTCTC GACGGCAACG GATGCAATAT GGCAGGATCC 1801 TTCGCCGAAG ATCTCGTGAA ATGCCTGCAG AATGGCCTGA ATTTTAGCGG GATTGGTGGT 1861 CGCACAGACA ACTTGGTGCA TAATCAGCAT TACTCAGAAA ATTAACGTTA CAGCAGTATA 1921 CGGAAAAAAA GCATGTTACA GGTATACCTA GTCCGCCACG GTGAAACGCA GTGGAACGCC 1981 GAGCGACGTA TTCAGGGCCA GTCTGACAGC CCGCTGACCG CCAAAGGTGA GCAACAGGCG 2041 ATGCAGGTGG CAACCCGTGC CAAAGAGCTT GGCATTACGC ATATCATCAG TAGCGATTTA 2101 GGACGCAC SEQ ID No.8:ilvH G14D、S17F Gene-encoded amino acid sequence (163aa), artificial sequence 1 MRRILSVLLE NESDALFRVI GLFSQRGYNI ESLTVAPTDD PTLSRMTIQT VGDEKVLEQI 61 EKQLHKLVDV LRVSELGQGA HVEREIMLVK IQASGYGRDE VKRNTEIFRG QIIDVTPSLY 121 TVQLAGTSGK LDAFLASIRD VAKIVEVARS GVVGLSRGDK IMR SEQ ID No.9: Homologous recombination DNA fragment ΔlacIZ-PxylF-DNA polymerase sequence (4524 bp), artificial sequence. 1 CGGTAATAAT CCACAGCAGG TATTTGCGCA GCCCGAGTTT GTCAGAAAGC AGACCAAACA 61 GCGGTTGGAA TAATAGCGAG AACAGAGAAA TAGCGGCAAA AATAATACCC GTATCACTTT 121 TGCTGATATG GTTGATGTCA TGTAGCCAAA TCGGGAAAAA CGGGAAGTAG GCTCCCATGA 181 TAAAAAAGTA AAAAAAAAG AATAAACCGA ACATCCAAAA GTTTGTGTTT TTTAAATAGT 241 ACATAATGGA TTTCCTTACG CGAAATACGG GCAGACATGG CCTGCCCGGT TATTATTATT 301 TTTGACACCA GACCAACTGG TAATGGTAGC GACCGGCGCT CAGCTGGAAT TCCGCCGATA 361 CTGACGGGCT CCAGGAGTCG TCGCCACCAA TCCCCATATG GAAACCGTCG ATATTCAGCC 421 ATGTGCCTTC TTCCGCGTGC AGCAGATGGC GATGGCTGGT TTCCATCAGT TGCTGTTGAC 481 TGTAGCGGCT GATGTTGAAC TGGAAGTCGC CGCGCCACTG GTGTGGGCCA TAATTCAATT 541 CGCGCGTCCC GCAGCGCAGA CCGTTTTCGC TCGGGAAGAC GTACGGGGTA TACATGTCTG 601 ACAATGGCAG ATCCCAGCGG TCAAAACAGG CGGCAGTAAG GCGGTCGGGA TAGTTTTCTT 661 GCGGCCCTAA TCCGAGCCAG TTTACCCGCT CTGCTACCTG TTACGCGAAC GCGAAGTCCG 721 ACTCTAAGAT GTCACGGAGG TTCAAGTTAC CTTTAGCCGG AAGTGCTGGC ATTTTGTCCA 781 ATTGAGACTC GTGCAACTGG TCAGCGAACT GGTCGTAGAA ATCAGCCAGT ACATCACAAG 841 ACTCATATGT GTCAACCATA GTTTCGCGCA CTGCTTTGAA CAGGTTCGCA GCGTCAGCCG 901 GAATGGTACC GAAGGAGTCG TGAATCAGTG CAAAAGATTC GATTCCGTAC TTCTCGTGTG 961 CCCACACTAC AGTCTTACGA AGGTGGCTAC CGTCTTGGCT GTGTACAAAG TTAGGAGCGA 1021 TACCAGACTC CTGTTTGTGT GCATCAATCT CGCTATCTTT GTTGGTGTTA ATGGTAGGCT 1081 GTAAGCGGAA CTGACCGAGG AACATCAGGT TCAAGCGCGT CTGAATAGGC TTCTTGTATT 1141 CCTGCCACAC AGGGAAACCA TCAGGAGTTA CCCAATGCAC AGCGCAACGC TTGCGAAGAA 1201 TCTCTCCAGT CTTCTTATCT TTGACCTCAG CAGCCAGCAG CTTAGCAGCA GACTTAAGCC 1261 AGTTCATTGC TTCAACCGCA GCTACCACCG TCACGCTCAC AGATTCCCAA ATCAGCTTAG 1321 CCATGTATCC AGCAGCCTGA TTCGGCTGAG TGAACATCAG ACCCTTGCCG GAATCAATAG 1381 CTGGCTGAAT GGTATCTTCC AGCACTTGTT GACGGAAGCC GAACTCTTTG GACCCGTAAG 1441 CCAGCGTCAT GACTGAACGC TTAGTCACAC TGCGAGTAAC ACCGTAAGCC AGCCATTGAC 1501 CAGCCAGTGC CTTAGTGCCC AGCTTGACTT TCTCAGAGAT TTCACCAGTG TTCTCATCGG 1561 TCACGGTAAC TACTTCGTTA TCGGTCCCAT TGATTGCGTC TGCTTGTAGA ATCTCGTTGA 1621 CTTTCTTAGC AACAATCCCG TAGATGTCCT GAACGGTTTC ACTAGGAAGC AAGTTAACCG 1681 CGCGACCACC TACCTCATCT CGGAGCATCG CGGAGAAGTG CTGGATGCCA GAGCAAGACC 1741 CGTCAAACGC CAGCGGAAGG GAGCAGTTAT AGCTCAGGCC GTGGTGCTGT ACCCCAGCGT 1801 ACTCAAAGCA GAACGCAAGG AAGCAGAACG GAGAATCTTG CTCAGCCCAC CAAGTGTTCT 1861 CCAGTGGAGA CTTAGCGCAA GCCATGATGT TCTCGTGGTT TTCCTCAATG AACTTGATGC 1921 GCTCAGGGAA CGGAACCTTA TCGACACCCG CACAGTTTGC ACCGTGGATT TTCAGCCAGT 1981 AGTAACCTTC CTTACCGATT GGTTTACCTT TCGCCAGCGT AAGCAGTCCT TTGGTCATAT 2041 CGTTACCTTG CGGGTTGAAC ATTGACACAG CGTAAACACG ACCGCGCCAG TCCATGTTGT 2101 AAGGGAACCA GATGGCCTTA TGGTTAGCAA ACTTATTGGC TTGCTCAAGC ATGAACTCAA 2161 GGCTGATACG GCGAGACTTG CGAGCCTTGT CCTTGCGGTA CACAGCAGCG GCAGCACGTT 2221 TCCACGCGGT GAGAGCCTCA GGATTCATGT CGATGTCTTC CGGTTTCATC GGGAGTTCTT 2281 CACGCTCAAT CGCAGGGATG TCCTCGACCG GACAATGCTT CCACTTGGTG ATTACGTTGG 2341 CGACCGCTAG GACTTTCTTG TTGATTTTCC ATGCGGTGTT TTGCGCAATG TTAATCGCTT 2401 TGTACACCTC AGGCATGTAA ACGTCTTCGT AGCGCATCAG TGCTTTCTTA CTGTGAGTAC 2461 GCACCAGCGC CAGAGGACGA CGACCGTTAG CCCAATAGCC ACCACCAGTA ATGCCAGTCC 2521 ACGGCTTAGG AGGAACTACG CAAGGTTGGA ACATCGGAGA GATGCCAGCC AGCGCACCTG 2581 CACGGGTTGC GATAGCCTCA GCGTATTCAG GTGCGAGTTC GATAGTCTCA GAGTCTTGAC 2641 CTACTACGCC AGCATTTTGG CGGTGTAAGC TAACCATTCC GGTTGACTCA ATGAGCATCT 2701 CGATGCAGCG TACTCCTACA TGAATAGAGT CTTCCTTATG CCACGAAGAC CACGCCTCGC 2761 CACCGAGTAG ACCCTTAGAG AGCATGTCAG CCTCGACAAC TTGCATAAAT GCTTTCTTGT 2821 AGACGTGCCC TACGCGCTTG TTGAGTTGTT CCTCAACGTT TTTCTTGAAG TGCTTAGCTT 2881 CAAGGTCACG GATACGACCG AAGCGAGCCT CGTCCTCAAT GGCCCGACCG ATTGCGCTTG 2941 CTACAGCCTG AACGGTTGTA TTGTCAGCAC TGGTTAGGCA AGCCAGAGTG GTCTTAATGG 3001 TGATGTACGC TACGGCTTCC GGCTTGATTT CTTGCAGGAA CTGGAAGGCT GTCGGGCGCT 3061 TGCCGCGCTT AGCTTTCACT TCCTCAAACC AGTCGTTGAT GCGTGCAATC ATCTTAGGGA 3121 GTAGGGTAGT GATGAGAGGC TTGGCGGCAG CGTTATCCGC AACCTCACCA GCTTTAAGTT 3181 GACGCTCAAA CATCTTGCGG AAGCGTGCTT CACCCATCTC GTAAGACTCA TGCTCAAGGG 3241 CCAACTGTTC GCGAGCTAAA CGCTCACCGT AATGGTCAGC CAGAGTGTTG AACGGGATAG 3301 CAGCCAGTTC GATGTCAGAG AAGTCGTTCT TAGCGATGTT AATCGTGTTC ATGGTGTAGG 3361 GCCTTCTGTA GTTAGAGGAC AGTTTTAATA AGTAACAATC ACCGCGATAA ACGTAACCAA 3421 TTTTTAGCAA CTAAACAGGG GAAAACAATT ACAGATTTTT ATCTTTCGAT TACGATTTTT 3481 GGTTTATTTC TTGATTTATG ACCGAGATCT TACTTTTGTT GCGCAATTGT ACTTATTGCA 3541 TTTTTCTCTT CGAGGAATTA CCCAGTTTCA TCATTCCATT TTATTTTGCG AGCGAGCGCA 3601 CACTTGTGAA TTATCTCCGC CGAGACAGAA CTTAATGGGC CCGCTAACAG CGCGATTTGC 3661 TGGTGACCCA ATGCGACCAG ATGCTCCACG CCCAGTCGCG TACCGTCTTC ATGGGAGAAA 3721 ATAATACTGT TGATGGGTGT CTGGTCAGAG ACATCAAGAA ATAACGCCGG AACATTAGTG 3781 CAGGCAGCTT CCACAGCAAT GGCATCCTGG TCATCCAGCG GATAGTTAAT GATCAGCCCA 3841 CTGACGCGTT GCGCGAGAAG ATTGTGCACC GCCGCTTTAC AGGCTTCGAC GCCGCTTCGT 3901 TCTACCATCG ACACCACCAC GCTGGCACCC AGTTGATCGG CGCGAGATTT AATCGCCGCG 3961 ACAATTTGCG ACGGCGCGTG CAGGGCCAGA CTGGAGGTGG CAACGCCAAT CAGCAACGAC 4021 TGTTTGCCCG CCAGTTGTTG TGCCACGCGG TTGGGAATGT AATTCAGCTC CGCCATCGCC 4081 GCTTCCACTT TTTCCCGCGT TTTCGCAGAA ACGTGGCTGG CCTGGTTCAC CACGCGGGAA 4141 ACGGTCTGAT AAGAGACACC GGCATACTCT GCGACATCGT ATAACGTTAC TGGTTTCACA 4201 TTCACCACCC TGAATTGACT CTCTTCCGGG CGCTATCATG CCATACCGCG AAAGGTTTTG 4261 CGCCATTCGA TGGTGTCAAC GTAAATGCAT GCCGCTTCGC CTTCCGGCCA CCAGAATAGC 4321 CTGCGATTCA ACCCCTCTT CGATCTGTTT TGCTACCCGT TGTAGCGCCG GAAGATGCTT 4381 TTCCGCTGCC TGTTCAATGG TCATTGCGCT CGCCATATAC ACCAGATTCA GACAGCCAAT 4441 CACCCGTTGT TCACTGCGCA GCGGTACGGC PRICEGGCG ATCTTCTCCT CCTGATCCCA 4501 GCCGCGGTAG TTCTGTCCGT AACC SEQ ID No.10:DNA Enrichment Fragment Framework (Product (Escherichia coli)BL21 Strain) 1 MNTINIAKND FSDIELAAIP FNTLADHYGE RLAREQLALE HESYEMGEAR FRKMFERQLK 61 AGEVADNAAA KPLITTLLPK MIARINDWFE EVKAKRGKRP TAFQFLQEIK PEAVAYITIK 121 TTLACLTSAD NTTVQAVASA IGRAIEDEAR FGRIRDLEAK HFKKNVEEQL NKRVGHVYKK 181 AFMQVVEADM LSKGLLGGEA WSSWHKEDSI HVGVRCIEML IESTGMVSLH RQNAGVVGQD 241 AEAIATRAGA LAGISPMFQP CVVPPKPWTG ITGGGYWANG RRPLALVRTH 301 SKKALMRYED VYMPEVYKAI NIAQNTAWKI NKKVLAVANV ITKWKHCPVE DIPAIEREEL 361 PMKPEDIDMN PEALTAWKRA AAAVYRKDKA RKSRRISLEF MLEQANKFAN HKAIWFPYNM 421 DWRGRVYAVS MFNPQGNDMT KGLLTLAKGK PIGKEGYYWL KIHGANCAGV DKVPFPERIK 481 FIEENHENIM ACAKSPLENT WWAEQDSPFC FLAFCFEYAG VQHHGLSYNC SPLAFDGSC 541 SGIQHFSAML RDEVGGRAVN LLPSETVQDI YGIVAKKVNE ILQADAINGT DNEVVTVTDE 601 NTGEISEKVK LGTKALAGQW LAYGVTRSVT KRSVMTLAYG SKEFGFRQQV LEDTIQPAID 661 SGKGLMFTQP NQAAGYMAKL IWESVSVTVV AAVEAMNWLK SAAKLLAAEV KDKKTGEILR 721 KRCAVHWVTP DGFPVWQEYK KPIQTRLNLM FLGQFRLQPT INTNKDSEID AHKQESGIAP 781 NFVHSQDGSH LRKTVVWAHE KYGIESFALI HDSFGTIPAD AANLFKAVRE TMVDTYESCD 841 VLADFYDQFA DQLHESQLDK MPALPAKGNL NLRDILESDF AFA SEQ ID No.11: Artificial sequence (3189bp) DNA sequence. 1 AAATGGAGGG ATAACAGCCA GCGCAGTAGT CAGTGAATTT GGTGGCACCA TCTTTATGAA 61 TGGTGATAAT TCAGTCGAGT CGGGTGGGGC ATATTCAGCG GGACTTTTAA GCCAGGTTAA 121 TGATTCTGAA AAGATGGTAA ATAACACCCG TCTTGAAACC ACAGATAAAA CGAACATTGT 181 TACCTCTGGG GAAAATGCAG TAGGTGTTCT TGCATGTTCA AGTCCTGGAG AGTCTCGAAC 241 ATGTGTCGAT GCTGTAGATG ATGAAGTTAG TGATTCTAAC ATGAAGTAAG TTATTAGCCG 301 TGCTGATTTA AAAATGAATG GTGGTTCCAT AACAACTAAT GGCATTAATA GCTATGGTGC 361 TTATGCTAAT GGGAAAAAAG CATATATTAA TTTAGATTAT GTGGCACTTG AAACTGTGGC 421 TGATGGAAGT TATGCAGTTG CTATTCGACA AGGTAACATT GATATAAAAA ATAGTTCTAT 481 TACAACAACA GGCACTAAAG CCCCCATTGC AAAAATATAC AATGGTGGAG AGTTATTTTT 541 TTCCAATGTC ACCGCGGTAT CAAAACAAGA TAAGGAATA TCAATTGATG CATCAAATAT 601 CGATTCTCAA GCCAAAATAG CACTATTAAG TGTTGAACTT TCAAGTGCTT TGGATAGTAT 661 TGATGTTAAC AAAACTACAA CGGATGTAAG TATCCTTAAT CGAAGTATTA TCACACCTGG 721 FATHERGTT CTGGTTAATACTGGAGG TGACTTAAAC FATHERATTCGT CCGACTCTAT 781 TCTAAATGGA GCGTTAACCC GCAACAGCAA TACGTTTCAT ATCTGTCATA TAGCCGCGCA 841 GTTTCTTACC TACCTGCTCA ATCGCATGGC TGCGAATCGC TTCGTTCACA TCACGCAGTT 901 GCCCGTTATC TACCGCGCCT TCCGGAATAG CTTTACCCAG GTCGCCCGGT TGCAGCTCTG 961 CCATAAACGG TTTCAGCAAC GGCACACAAG CGTAAGAGAA CAGATAGTTA CCGTACTCAG 1021 CGGTATCAGA GATAACCACG TTCATTTCGT ACAGACGCTT ACGGGCGATG GTGTTGGCAA 1081 TCAGCGGCAG CTCGTGCAGT GATTCATAAT ATGCAGACTC TTCAATGATG CCGGAATCGA 1141 CCATGGTTTC GAACGCCAGT TCAACGCCCG CTTTCACCAT CGCAATCATC AGTACGCCTT 1201 TATCGAAGTA CTCCTGCTCG CCGATTTTGC CTTCATACTG CGGCGCGGTT TCAAACGCGG 1261 TTTTGCCGGT CTCTTCACGC CAGGTCAGCA GTTTCTTATC ATCGTTGGCC CAGTCCGCCA 1321 TCATACCGGA AGAGAATTCG CCGGAGATGA TGTCGTCCAT ATGTTTCTGG AACAGGGGTG 1381 CCATGATCTC TTTCAGCTGT TCAGAAAGCG CATAAGCACG CAGTTTCGCC GGGTTAGAGA 1441 GACGGTCCAT CATCAGGGTG ATGCCGCCCT GTTTCAGTGC TTCGGTGATG GTTTCCCAAC 1501 CGAACTGAAT CAGTTTTTCT GCGTATGCTG GATCGGTACC TTCTTCCACC AGCTTGTCGA 1561 AGCACAGCAG AGAGCCAGCC TGCAACATAC CGCACAGGAT GGTTTGCTCG CCCATCAGGT 1621 CAGATTTCAC TTCCGCAACG AAGGACGATT CCAGCACACC CGCACGGTGA CCACCGGTTG 1681 CAGCCGCCCA GGCTTTGGCA ATCGCCATGC CTTCGCCTTT CGGATCGTTT TCCGGGTGAA 1741 CGGCAATCAG CGTCGGTACG CCGAACCCAC GTTTGTACTC TTCACGCACT TCGGTGCCTG 1801 GGCATTTCGG CGCAACCATC ACTACGGTGA TATCTTTACG GATCTGCTCG CCCACTTCGA 1861 CGATGTTGAA ACCGTGCGAG TAGCCCAGCG CCGCGCCGTC TTTCATCAGT GGCTGTACGG 1921 TGCGCACTAC ATCAGAGTGC TGCTTGTCCG GCGTCAGGTT AATCACCAGA TCCGCCTGTG 1981 GGATCAGTTC TTCGTAAGTA CCCACTTTAA AACCATTTTC GGTCGCTTTA CGCCAGGACG 2041 CGCGCTTCTC GGCAATCGCT TCTTTACGCA GAGCGTAGGA GATATCGAGA CCAGAATCAC 2101 GCATGTTCAG GCCCTGGTTC AGACCCTGTG CGCCACAGCC GACGATGACT ACTTTTTTAC 2161 CCTGAAGGTA GCTCGCGCCA TCGGCGAATT CATCGCGGCC CATAAAGCGA CATTTGCCCA 2221 GCTGTGCCAG CTGCTGGCGC AGATTCAGTG TATTGAAGTA GTTAGCCATG GTCTGTTTCC 2281 TGTGTGAAAT TGTTATCCGC TCACAATTCC ACACATTATA CGAGCCGGAT GATTAATTGT 2341 CAAAAACTTG GCGTAAATGG CAACCTGAAT CCAGCGGCCA GCGTCTGGGG AATGTGGGCG 2401 TGCAGCTGGG TGATAATGGC TACAATGACA CCGCAGTGAT GGTGGGCCTG AAATATAAGT 2461 TCTGATCCCG CCGTTAGCTA AAAAACCGCG TCGTATTCAT CGACGCGGTA CATATGAAAT 2521 ATTATTTTTT GCCGATAGCA CGCATGGTGT CATCAATTGC CGTGATCAAC AGCATTTGCG 2581 GGTCTTTAGC GCAAACCTGA TTCAGTTTTT CTACCACTTT GGCGCTCAGT TCCGGAGATT 2641 GCTCAATTTT TAAATCACGG CTGGCAACGC TGGCATTACC CATTACCGCA ACAATTTCTG 2701 CAACCTGTGC GCTGTCAGTT TTTGCCATTT CGTTGGCTTC TGCGCAAGTA ATATAGGTTT 2761 CTGACGGCAA ACCGTTTTTA ATATTGTAGT CCTGCGCCCA GGTCATTGGT GCGAAAAACAA 2821 ACAGGCCCGC CAGTAAAGCA AATTTTTTCA TCATCATTCC TTATTTCATT TTACCCAGAA 2881 TTGCACCACC CGTACCGCCA ATCACGGCAC CTTTAATCGC CCCTTCGAGG CCATTGCCGG 2941 TCAGAACGCC AGTGACAGCA CCAACGGCGG CACCCACTTT TGCACCTTTA CGCGCATTTT 3001 TACCGTCGCG GCCTTTTTCT GTTACTGCAC CAACACCAGC GCCAACAGCT GCACCTTTCA 3061 GTACGCCATT AACACCATTG CCAGTAAGTA AACCAACGCC TGCGCCCTAGC AATGCACCTT 3121 TCGTGGTGCG GTTCATATCC GCCATCGCTG GCGTGGAGCA GAACAATGCT GAGATAAGCC 3181 CGAAGGCAA SEQ ID No.12:ilvC scaffold fragment (protein (Escherichia coli)W3110 strain) . 1 MANYFNTLNL RQQLAQLGKC RFMGRDEFAD GASYLQGKKV VIVGCGAQGL NQGLNMRDSG 61 LDISYALRKE AIAEKRASWR KATENGFKVG TYEELIPQAD LVINLTPDKQ HSDVVRTVQP 121 LMKDGAALGY SHGFLEVVG EQIRKDITVV MVAPKCPGTE VREEYKRGFG VPTLIAVHPE 181 NDPKGEGMAI AKAWAAATGG HRAGVLESSF VAEVKSDLMG EQTILCGMLQ AGSLLCFDKL 241 VEEGTDPAYA EKLIQFGWET ITEALKQGGI TLMMDRLSNP ACCREDITATION QLKEIMAPLF 301 QKHMDDIISG EFSSGMMADW ANDDKKLLTW REETGKTAFE LOSS EQEYFDKGVL 361 MIAMVKAGVE LAFE™VDSG IIEESAYYES LHELPLIANT IARKRLYEMN VVISDTAEYG 421 NYLFSYACVP LLKPFMAELQ PGDLGKAIPE GAVDNGQLRD VNEAIRSHAI EQVGKKLRGY 481 MTDMKRIAVA G. SEQ ID No.13: Artificial sequence (4713bp) 1 AGTTCGCCCT TTGCTCTCTC TTTGTCACGC TGACTTTGGA TTCCTGCCGA ACGAAAAAGC 61 AGATCCCTTC TCTGCTGCTC GCAGGTTTGA GCTTCACCAT TGCTCTTGTG GTAATTCCAG 121 GTCAGGCCCT ATTTGCGGCG CTGCTGATCT TCTTGGGTCT GTTGACCATC CGGTACTTCT 181 TCTTGGGAAA GGCTGCTAAA TGACAACTGA TTTCTCCTGT ATTCTCCTTG TTGTCGCAGT 241 ATGTGCAGTC ATTACTTTTG CGCTCCGGGC GGTTCCGTTC TTAATCCTTA AGCCCCTACG 301 TGAATCACAA TTTGTGGGCA AAATGGCGAT GTGGATGCCA GCAGGAATCC TTGCCATTTT 361 GACCGCATCA ACGTTTCGCA GCAATGCGAT AGATCTGAAG ACTCTAACCT TTGGTCTCAT 421 TGCCGTTGCG ATTACAGTGG TGGCGCATCT TCTTGGCGGT CGACGCACCT TGTTGAGCGT 481 TGGCGCTGGC ACCATCGTTT TTGTTGGACT GGTGAATCTT TTCTAACTAA AAGCACTACC 541 TGTGAAGGGA TGTCAGGACG TTACAGTTAC AGCAAATGAA ACTTATCGGA TTCGCACCGG 601 AAGAGAACAA ATCAGCATCG GAAGGTTTGC TCTAAATGGA AAGCGTGCAA GCTGGGTTTG 661 TCATCCAGAT GAAACATTTA TTGGTGTACC AAAAGTCATT TCTACACTAC CGGATATTCA 721 AAGCATTGAT GTAACGCCGTT ACACGTGCTG ACAAAACAGC ATTACAGCCA GCAGGAAGTA 781 CTGCGTTGTT AACGTCTGCT TAATACACTG TGGCCGTTTT GCAAAAACTG GCTTCTTGAG 841 CGGCGCATGC GTTCAATCCG TTCCTCTGCT AAGCGGTCAG CCGCTAAATA TGCAGGAATG 901 CCGTCACGCT GAGAAATCTC AAGTACACGC TCGATATTGC CGTAAATGCC TTCAACTTTT 961 TTCAATGCAC GTTCTGCATT ATAGCCGTAA AGCTCATCTG CCACGTTGAT GACACCGCCC 1021 GCGTTAATCA CGTAATCCGG TGCATAAACG ATGCCCATTT CGTGAATTTG ATCACCATGG 1081 CGTGTCTCTT TTAATTGGTT GTTAGCCGCA CCTGCGATCA CTTTCGCCTT CAGCTGTTTA 1141 ATGGTGTCGT CGTTAATAGT CGCACCAAGG GCACACGGCG CATAAATATC GCAGTCTTGT 1201 GAATAAATGT CATCAGGATC TACCGCACGG GCGCCAAAAT CTTCAACTGC ACGCTGTACA 1261 GATTGTTTGT TGATATCCGT AACGATTAAG TTTGCTCCTT CTTCATGCAG GTGGCGGCAA 1321 AGGTTATAGG CTACGTTCCC AACACCCTGT ACAGCAATGG TTTTTCCTTC AAGAGAGTCG 1381 GTTCCGAAAG CAGCTTTAGC TGCTGCCTTC ATTCCTCTGT ACACCCCGTA CGCTGTGACT 1441 GGGGACGGAT TTCCAGAAGA GCCGAAAGCA GGGAAATCC CTGTGACATA GTCTGTCTCA 1501 TCATGAATGA TATCCATATC CTCGACCGTT GTGCCCACAT CTTCAGCCGT GATGTATCTG 1561 CCATTCAGTC CTTGAATATA GCGGCCAAAC GCGCGGAACA TTTCCTCATT TTTTGTCTTTG 1621 CGCGGATCGC CGATAATGAC TGTTTTTCCG CCGCCAAGGT TTAAGCCTGC CGCCGCGTTC 1681 TTATAGGTCA TGCCTCTTGC CAATCTGAGC GCATCTTCAA TTGCCGCTTC TTCATTTTCA 1741 TATGTCCACA TTCTCGTTCC GCCAAGCGCC GGACCAAGCG TTGTATCATG AATGGCGATA 1801 ATCGCTTTTA ATCCAGATTG TTCATCCTGG CAGAATACCA ATTGTTCGTA ATCGTATTTC 1861 TCCATATATT TAAAAAGTTC CATGGTCTGT TTCCTGTGTG AAATTGTTAT CCGCTCACAA 1921 TTCCACACAT TATACGAGCC GGATGATTAA TTGTCAATTA ACCCCCCAGT TTCGATTTAT 1981 CGCGCACCGC GCCTTTGTCG GCGCTGGTTG CCAGGCTGGC ATAAGCACGC AGGGCAAAGG 2041 AGACCTGACG TTCACGATTT TTCGGCGTCC AGGCTTTGTC ACCTCGAGCG TCCTGCGCTT 2101 CACGACGCGC CGCCAGTTCG GCATCGCTTA CCTGTAACTG AATGCCACGG TTCGGGATGT 2161 CGATAGCGAT CAGGTCACCA TCTTCAATCA GGCCAATGCT GCCGCCGCTT GCCGCTTCCG 2221 GTGAGACGTG GCCGATGGAA AGACCAGAGG TGCCACCAGA GAAACGACCG TCGGTGATCA 2281 GCGCACAGGC TTTGCCGAGA CCCATTGATT TCAGGAAGCT GGTTGGGTAG AGCATTTCCT 2341 GCATCCCCGG ACCGCCTTTC GGGCCTTCAT AGCGAATTAC TACCACATCT CCGGCGACAA 2401 CTTTACCGCC GAGAATCGCT TCTACCGCAT CGTCCTGGCT TTCGTACACT TTCGCCGGGC 2461 CGGTGAATTT GAGGATGCTG TCATCGACGC CTGCCGTTTT CACGATGCAG CCGTTTTCCG 2521 CAAAGTTACC GTAGAGCACC GCCAGGCCGC CGTCTTTGCT GTAGGCGTGT TCCAGCGAGC 2581 GGATACAGCC ATTGGCGCGA TCGTCGTCCA GCGTATCCCA ACGGCAATCT TGCGAGAATG 2641 CCTGTGTGGT ACGAATGCCT GCAGGACCTG CGCGGAACAT ATTTTTTACC GCGTCATCCT 2701 GGGTCAGCAT AACGTCGTAT TGTTCCAGCG TTTGCGGCAA CGTCAGGCCA AGTACGTTTT 2761 TCACATCACG GTTCAGTAAC CCCGCGCGAT CCAGTTCGCC GAGAATACCG ATAACACCAC 2821 CAGCACGGTG AACATCTTCC ATATGGTATT TCTGGGTGCT CGGCGCAACT TTACACAGCT 2881 GTGGAACCTT GCGGGAAAGC TTATCGATAT CACTCATGGT GAAGTCGATT TCCGCTTCCT 2941 GCGCCGCCGC CAGCAGGTGA AGTACGGTGT TAGTCGATCC ACCCATCGCG ATATCCAGCG 3001 TCATGGCGTT TTCAAACGCC GCCTTACTGG CGATATTACG CGGCAGTGCA CTTTCGTCGT 3061 TTTGCTCGTA ATAACGTTTG GTCAATTCAA CAATGCGTTT ACCAGCATTA AGGAACAGCT 3121 GCTTACGGTC GGCGTGGGTT GCCAGCAGCG AGCCGTTGCC CGGCTGCGAC AGGCCCAGCG 3181 CTTCGGTCAG GCAGTTCATT GAGTTAGCGG TAAACATCCC GGAGCAGGAA CCGCAGGTCG 3241 GACACGCGGA ACGTTCAACC TGATCGCTCT GGGAGTCAGA TACTTTCGGG TCTGCGCCCT 3301 GGATCATCGC ATCAACCAGA TCGAGCTTGA TGATCTGATC GGAAAGTTTG GTTTTCCCGG 3361 CCTCCATCGG GCCGCCGGAA ACAAAGATCA CCGGAATATT CAGGCGCAGG GAAGCCATCA 3421 GCATCCCCGG GGTGATTTTG TCGCAGTTAG AGATGCAGAC CATGGCGTCG GCGCAGTGGG 3481 CGTTGACCAT ATACTCAACG GAATCAGCGA TCAGTTCGCG AGATGGCAGT GAATAAAGCA 3541 TCCCCCCGTG GCCCATGGCA ATCCCATCAT CCACCGCAAT GGTGTTGAAC TCTTTGGCAA 3601 CGCCGCCAGC CGCTTCAATT TGTTCGGCGA CCAGTTTACC GAGATCGCGC AGATGGACGT 3661 GACCCGGTAC AAATTGGGTG AACGAGTTCA CAACCGCGAT AATCGGCTTA CCGAAATCGG 3721 CGTCGGTCAT TCCGGTGGCG CGCCACAGCG CACGAGCACC CGCCATATTA CGACCATGAG 3781 TGTGTGGTGGC GGAACGGTAC TTAGGCATAC TTTATTTACT CCCAGTGTCT GTCTCGTAAA 3841 TGGGACGGTG CGTGCCGTCC CATTTTTTGT ATTTCAACGG ATGTGCTGGT GGAGGTGATC 3901 GCCTCCTGAT GATGAGCCGC TCCCGATGTG GTGTCGGGAG CGGTATTTTC TATAAAACTT 3961 ACCGCTTATT TGAGATATTC ATCGAAAATG TCGAGTAATT CTTGATGTAT ACACGGCCAT 4021 TCCTGACCTA AATTGACGGT ACACAAGCCA ATATCGAAGC CATTAATTTT ATAACGATGT 4081 TTCACTGCGG TATCTACGTG GGGATATATT AATAACCCCC CTATGTTTTC GCCATTTTCA 4141 GGCTTTAACG ACCATAAGTA ATTCATCAGT TGATAAAGAT TTTGCGAATG AAATTTTTCT 4201 GTTCCCATTC GTCGTGAAAA AATGCTCTTA TAGTATTTGG CGTCAACGAT AAGTATTTTT 4261 TCTGATGAGC GAATGGTGAT GTCAGTTTCC ATTCGAGGTA ACAAATTAAG TGACTGATCC 4321 GATATACTCG ATGCATCCCA TTTTAAATAA GAGCGGGTTG TGTTTGCAGA CGTTAATTCA 4381 CGACGGCAAA ATTCATAAAG AAACTTTTGA TAAAGTAATG ACATCTCTTT TTCGTTTCTT 4441 TCAAAATCAT AGAAACGGTA GTGTCCTTTG TTTTGACCTG GAATAGAATT ATTGACGATG 4501 AATTTGCAGA CACTGATAAC GAATTTATAA TAACGCGTAT TTTTTCCGCC ATTCAGATAG 4561 CTGAAATGCT GCGGAGTTAA ATGAAGAGTG CTAATGCCCG GTAATTTTCT ATAAAGTGAA 4621 CGAGCTTCAT CTCTGATAGT TGAATTTAAC TTTTCATGCT TAATTAATAT GGCTAATGTG 4681 CTTTTTATAA TTCGGTTAGC CAGCGTGTCT TCA

Claims

1. Genetically modified bacteria for valine production, characterized by B1), B2), B3), B4), or B5): B1) includes B11), B12), and B13): B11) Not expressing or weakly expressing the yjiT gene, or reducing or losing the activity of the protein encoded by the yjiT gene. B12) Increase in the content of the protein encoded by the brnF gene, or enhance the activity of the protein encoded by the brnF gene. B13) Increasing the content of the protein encoded by the brnE gene, or enhancing the activity of the protein encoded by the brnE gene; B2) includes B21), B22), and B23): B21) Not expressing or weakly expressing the yjiV gene, or reducing or losing the activity of the protein encoded by the yjiV gene, B22) Increase in the content of the protein encoded by the ilvE gene, or enhance the activity of the protein encoded by the ilvE gene. B23) Increasing the content of the protein encoded by the ilvD gene, or enhancing the activity of the protein encoded by the ilvD gene; B3) includes B31) and B32): B31) Not expressing or weakly expressing the trpR gene, or reducing or losing the activity of the protein encoded by the trpR gene, B32) ilvH Ｇ１４Ｄ、Ｓ１７Ｆ An increase in the content of the gene-encoded protein, or the ilvH Ｇ１４Ｄ、Ｓ１７Ｆ Enhancement of the activity of the gene-encoded protein, where ilvH Ｇ１４Ｄ、Ｓ１７Ｆ The gene is obtained by substituting the glycine codon at position 14 of the ilvH gene with the aspartic acid codon and the serine codon at position 17 with the phenylalanine codon; B4) includes B41), B42), and B43): B41) Not expressing or weakly expressing the lacI gene, or suppressing the decrease or loss of activity of the protein encoded by the lacI gene, B42) Not expressing or weakly expressing the lacZ gene, or suppressing the decrease or loss of activity of the protein encoded by the lacZ gene, B43) Increasing the content of DNA polymerase, or enhancing the activity of the DNA polymerase; B5) includes B51) and B52): B51) Not expressing or weakly expressing the ycgH gene, or reducing or losing the activity of the protein encoded by the ycgH gene, B52) Increase in the content of the protein encoded by the ilvC gene, or enhance the activity of the protein encoded by the ilvC gene, It has, The aforementioned genetically modified bacterium is Escherichia coli.

2. A genetically modified bacterium for valine production, which is a recombinant bacterium obtained by modifying the recipient bacterium, wherein the modification is one of the following: A1), A2), A3), A4), or A5): A1) includes A11), A12), and A13): A11) Knock out the yjiT gene of the receptor bacterium, suppress the expression of the yjiT gene, or suppress the activity of the protein encoded by the yjiT gene. A12) To increase the content of the protein encoded by the brnF gene in the receptor bacteria, or to enhance the activity of the protein encoded by the brnF gene. A13) Increasing the content of the protein encoded by the brnE gene in the receptor bacteria, or enhancing the activity of the protein encoded by the brnE gene; A2) includes A21), A22), and A23): A21) Knock out the yjiV gene of the receptor bacterium, suppress the expression of the yjiV gene, or suppress the activity of the protein encoded by the yjiV gene. A22) To increase the content of the protein encoded by the ilvE gene in the receptor bacteria, or to enhance the activity of the protein encoded by the ilvE gene. A23) Increasing the content of the protein encoded by the ilvD gene in the receptor bacteria, or enhancing the activity of the protein encoded by the ilvD gene; A3) includes A31) and A32): A31) Knock out the trpR gene of the receptor bacterium to suppress the expression of the trpR gene, or suppress the activity of the protein encoded by the trpR gene. A32) ilvH in the aforementioned receptor bacteria Ｇ１４Ｄ、Ｓ１７Ｆ To increase the content of the gene-encoded protein, or the ilvH Ｇ１４Ｄ、Ｓ１７Ｆ To enhance the activity of a gene-encoded protein, where ilvH Ｇ１４Ｄ、Ｓ１７Ｆ The gene is obtained by substituting the glycine codon at position 14 of the ilvH gene with the aspartic acid codon and the serine codon at position 17 with the phenylalanine codon; A4) includes A41), A42), and A43): A41) Knock out the lacI gene of the receptor bacterium to suppress the expression of the lacI gene, or suppress the activity of the protein encoded by the lacI gene. A42) Knock out the lacZ gene of the receptor bacterium to suppress the expression of the lacZ gene, or suppress the activity of the protein encoded by the lacZ gene. A43) Increasing the content of DNA polymerase in the receptor bacteria or enhancing the activity of the DNA polymerase; A5) includes A51) and A52): A51) Knock out the ycgH gene of the receptor bacterium to suppress the expression of the ycgH gene, or suppress the activity of the protein encoded by the ycgH gene. A52) To increase the content of the protein encoded by the ilvC gene in the receptor bacteria, or to enhance the activity of the protein encoded by the ilvC gene. Includes, The aforementioned recipient bacterium is a genetically modified strain of Escherichia coli.

3. The genetically modified bacterium according to claim 1 or 2, characterized in that the brnF gene and the brnE gene are derived from Corynebacterium glutamicum.

4. The aforementioned brnF gene encodes the protein shown in SEQ ID No. 2 of the sequence listing, The genetically modified bacterium according to claim 3, characterized in that the brnE gene encodes a protein shown in SEQ ID No. 3 of the sequence listing.

5. The aforementioned brnF gene is a DNA molecule located at positions 832-1587 of SEQ ID No. 1 in the sequence listing. The genetically modified bacterium according to claim 4, characterized in that the brnE gene is a DNA molecule located at positions 1584-1910 of SEQ ID No. 1 in the sequence listing.

6. The aforementioned ilvE gene originates from Bacillus subtilis, The genetically modified bacterium according to any one of claims 1 to 5, characterized in that the ilvD gene is derived from Escherichia coli.

7. The ilvE gene codes for the protein shown in SEQ ID No. 6 of the sequence listing. The genetically modified bacterium according to claim 6, characterized in that the ilvD gene encodes a protein shown in SEQ ID No. 5 of the sequence listing.

8. The aforementioned ilvE gene is a DNA molecule located at positions 808-1902 of SEQ ID No. 4 in the sequence listing. The genetically modified bacterium according to claim 7, characterized in that the ilvD gene is a DNA molecule located at positions 1977 to 3827 of SEQ ID No. 4 in the sequence listing.

9. The genetically modified bacterium according to any one of claims 1 to 8, characterized in that the ilvH gene is derived from Escherichia coli.

10. The aforementioned ilvH Ｇ１４Ｄ、Ｓ１７Ｆ The genetically modified bacterium according to claim 9, characterized in that the gene encodes a protein indicated by SEQ ID No. 8 in the sequence listing.

11. The aforementioned ilvH Ｇ１４Ｄ、Ｓ１７Ｆ The genetically modified bacterium according to claim 10, characterized in that the gene is a DNA molecule located at positions 835 to 1326 of SEQ ID No. 7 in the sequence listing.

12. The genetically modified bacterium according to any one of claims 1 to 11, characterized in that the DNA polymerase is derived from Escherichia coli.

13. The genetically modified bacterium according to claim 12, characterized in that the DNA polymerase is the protein shown as SEQ ID No. 10 in the sequence listing.

14. The genetically modified bacterium according to claim 13, characterized in that the gene encoding the DNA polymerase is a DNA molecule shown at positions 701 to 3352 of SEQ ID No. 9 in the sequence listing.

15. The genetically modified bacterium according to any one of claims 1 to 14, characterized in that the ilvC gene is derived from Escherichia coli.

16. The genetically modified bacterium according to claim 15, characterized in that the ilvC gene encodes a protein shown in SEQ ID No. 12 of the sequence listing.

17. The genetically modified bacterium according to claim 16, characterized in that the ilvC gene is a DNA molecule located at positions 794 to 2269 of SEQ ID No. 11 in the sequence listing.

18. The genetically modified bacterium according to any one of claims 1, 3 to 17, characterized in that the genetically modified bacterium further has any two, any three, any four, or any five characteristics of B1), B2), B3), B4), and B5).

19. The genetically modified bacterium according to any one of claims 2 to 17, characterized in that the modification further comprises any two, any three, any four, or any five of A1), A2), A3), A4), and A5).

20. A method for preparing genetically modified bacteria for valine production, comprising modifying a recipient bacterium according to any one of claims 2 to 17 (A1), A2), A3), A4), or A5), to obtain the desired genetically modified bacterium, wherein the recipient bacterium is Escherichia coli.

21. The method according to claim 20, further comprising making any two, any three, any four, or any five modifications of A1), A2), A3), A4), and A5) described in any of claims 2 to 17 to the receptor bacterium.

22. A method for preparing L-valine, comprising culturing a genetically modified bacterium according to any one of claims 1 to 19 to obtain L-valine.

23. Use of the genetically modified bacteria according to any one of claims 1 to 19 in the production of L-valine, or in the preparation of a product for the production of L-valine, or in the preparation of food, feed, or pharmaceuticals containing L-valine.

Images