Search

EP-4741491-A1 - ENGINEERING BACTERIUM FOR PRODUCING L-VALINE, CONSTRUCTION METHOD, AND USE

EP4741491A1EP 4741491 A1EP4741491 A1EP 4741491A1EP-4741491-A1

Abstract

The disclosure discloses an engineering bacterium producing L-valine-producing, a construction method thereof, and a use thereof. This disclosure finds that after knocking out the yjiT gene in Escherichia coli and expressing brnF and brnE, followed by knocking out the yjiV gene and expressing ilvE and ilvD, knocking out the trpR gene and expressing the ilvH mutant protein, knocking out the lacI and lacZ genes and expressing DNA polymerase, and/or, knocking out the ycgH gene and expressing ilvC, the resulting engineering bacterium can enhance L-valine production. The engineering bacterium of this disclosure can be used for producing L-valine and holds promising application prospects.

Inventors

  • WEI, Aiying
  • WANG, Pan
  • WANG, JIWEI
  • MENG, GANG
  • ZHAO, Chunguang
  • ZHOU, XIAOQUN
  • ZHANG, YING
  • BI, Guodong
  • SU, Houbo
  • YANG, Lipeng
  • ZHANG, XIAOQIN

Assignees

  • Heilongjiang Eppen Biotech Co., Ltd.

Dates

Publication Date
20260513
Application Date
20240624

Claims (20)

  1. An engineering bacterium producing valine, comprising characteristics of B1), B2), B3), B4), or B5); B1) comprising B11), B12), and B13): B11) a yjiT gene is not expressed or weakly expressed, or activity of protein encoded by the yjiT gene is reduced or lost; B12) content of protein encoded by a brnF gene is increased or activity of protein encoded by the brnF gene is enhanced; B13) content of protein encoded by a brnE gene is increased or activity of the protein encoded by the brnE gene is enhanced; B2) comprising B21), B22), and B23): B21) a yjiV gene is not expressed or weakly expressed, or activity of the protein encoded by the yjiV gene is reduced or lost; B22) content of protein encoded by an ilvE gene is increased or activity of protein encoded by the ilvE gene is enhanced; B23) content of protein encoded by an ilvD gene is increased or activity of protein encoded by the ilvD gene is enhanced; B3) comprising B31) and B32): B31) a trpR gene is not expressed or weakly expressed, or activity of protein encoded by the trpR gene is reduced or lost; B32) content of protein encoded by an ilvH G14D , S17F genes is increased or activity of protein encoded the ilvH G14D , S17F genes is enhanced; the ilvH G14D , S17F genes are obtained by replacing a glycine codon at position 14 of an ilvH gene with an aspartic acid codon and a serine codon at position 17 with a phenylalanine codon; B4) comprising B41), B42), and B43): B41) a lacI gene is not expressed or weakly expressed, or inhibitory activity of protein encoded by the lacI gene is reduced or lost; B42) a lacZ gene is not expressed or weakly expressed, or inhibitory activity of protein encoded by the lacZ gene is reduced or lost; B43) content of DNA polymerase is increased or activity of DNA polymerase is enhanced; B5) comprising B51) and B52): b51) a ycgH gene is not expressed or weakly expressed, or activity of the protein encoded by the ycgH gene is reduced or lost; b52) content of the protein encoded by an ilvC gene is increased or activity of protein encoded by the ilvC gene is enhanced; the engineered bacterium is Escherichia coli.
  2. An engineering bacterium producing valine, which is a recombinant bacterium obtained by modifying a recipient bacterium; the modifying the recipient bacterium comprises A1), A2), A3), A4), or A5); A1) comprising A11), A12), and A13): A11) knocking out a yjiT gene of the recipient bacterium, inhibiting expression of the yjiT gene, or inhibiting activity of protein encoded by the yjiT gene; A12) increasing content of protein encoded by a brnF gene in the recipient bacterium or enhancing activity of protein encoded by the brnF gene; A13) increasing content of protein encoded by a brnE gene in the recipient bacterium or enhancing activity of protein encoded by the brnE gene; A2) comprising A21), A22), and A23): A21) knocking out a yjiV gene of the recipient bacterium, inhibiting expression of a yjiV gene, or inhibiting activity of protein encoded by the yjiV gene; A22) increasing content of protein encoded by an ilvE gene in the recipient bacterium or enhancing activity of protein encoded by the ilvE gene; A23) increasing content of protein encoded by an ilvD gene in the recipient bacterium or enhancing activity of protein encoded by the ilvD gene; A3) comprising A31) and A32): A31) knocking out a trpR gene of the recipient bacterium, inhibiting expression of a trpR gene, or inhibiting activity of protein encoded by the trpR gene; A32) increasing content of protein encoded by ilvH G14D , S17F genes in the recipient bacterium or enhancing activity of protein encoded by the ilvH G14D , S17F genes; the ilvH G14D , S17F genes are obtained by replacing a glycine codon at position 14 of an ilvH gene with an aspartic acid codon and a serine codon at position 17 with a phenylalanine codon; A4) comprising A41), A42), and A43): A41) knocking out a lacI gene of the recipient bacterium, inhibiting expression of the lacI gene, or inhibiting activity of protein encoded by the lacI gene; A42) knocking out a lacZ gene of the recipient bacterium, inhibiting expression of a lacZ gene, or inhibiting activity of protein encoded by the lacZ gene; A43) increasing content of DNA polymerase in the recipient bacterium or enhancing activity of DNA polymerase; A5) comprising A51) and A52): A51) knocking out a ycgH gene of the recipient bacterium, inhibiting expression of the ycgH gene, or inhibiting activity of protein encoded by the ycgH gene; A52) increasing content of protein encoded by an ilvC gene in the recipient bacterium or enhancing activity of protein encoded by the ilvC gene; the recipient bacterium is Escherichia coli.
  3. The engineering bacterium according to claim 1 or 2, wherein the brnF gene and the brnE gene are derived from Corynebacterium glutamicum.
  4. The engineering bacterium according to claim 3, wherein the brnF gene encodes the protein set forth in SEQ ID No.2 in a sequence listing; the brnE gene encodes protein set forth in SEQ ID No.3 in the sequence listing.
  5. The engineering bacterium according to claim 4, wherein the brnF gene is a DNA molecule shown at positions 832-1587 of SEQ ID No.1 in the sequence listing; the brnE gene is a DNA molecule shown at positions 1584-1910 of SEQ ID No.1 in the sequence listing.
  6. The engineering bacterium according to any one of claims 1-5, wherein the ilvE gene is derived from Bacillus subtilis; the ilvD gene is derived from Escherichia coli.
  7. The engineering bacterium according to claim 6, wherein the ilvE gene encodes protein set forth in SEQ ID No.6 in the sequence listing; the ilvD gene encodes protein set forth in SEQ ID No.5 in the sequence listing.
  8. The engineering bacterium according to claim 7, wherein the ilvE gene is a DNA molecule shown at positions 808-1902 of SEQ ID No.4 in the sequence listing; the ilvD gene is a DNA molecule shown at positions 1977-3827 of SEQ ID No.4 in the sequence listing.
  9. The engineering bacterium according to any one of claims 1-8, wherein the ilvH gene is derived from Escherichia coli.
  10. The engineering bacterium according to claim 9, wherein the ilvH G14D , S17F gene encodes protein set forth in SEQ ID No.8 in the sequence listing.
  11. The engineering bacterium according to claim 10, wherein the ilvH G14D , S17F gene is a DNA molecule shown at positions 835-1326 of SEQ ID No.7 in the sequence listing.
  12. The engineering bacterium according to any one of claims 1-11, wherein the DNA polymerase is derived from Escherichia coli.
  13. The engineering bacterium according to claim 12, wherein the DNA polymerase is protein set forth in SEQ ID No.10 in the sequence listing.
  14. The engineering bacterium according to claim 13, wherein a coding gene of the DNA polymerase is a DNA molecule shown at positions 701-3352 of SEQ ID No.9 in the sequence listing.
  15. The engineering bacterium according to any one of claims 1-14, wherein the ilvC gene is derived from Escherichia coli.
  16. The engineering bacterium according to claim 15, wherein the ilvC gene encodes protein set forth in SEQ ID No.12 in the sequence listing.
  17. The engineering bacterium according to claim 16, wherein the ilvC gene is a DNA molecule shown at positions 794-2269 of SEQ ID No.11 in the sequence listing.
  18. The engineering bacterium according to any one of claims 1, 3-17, wherein the engineering bacterium also comprises any two, any three, any four, or five of characteristics of B1), B2), B3), B4), and B5).
  19. The engineering bacterium according to any one of claims 2-17, wherein the modification also comprises any two, any three, any four, or five of a1), A2), A3), A4), and A5).
  20. A method for preparing an engineering bacterium for producing valine, comprising: modifying a recipient bacterium according to any one of A1), A2), A3), A4), or A5) described in claims 2-17 to obtain a target engineering bacterium, the recipient bacterium is Escherichia coli.

Description

CROSS REFERENCE TO THE RELATED APPLICATION This application claims priority to five Chinese patent applications (with application numbers 2023108092706, 2023108092710, 2023108092744, 2023108092778, and 2023108092814 respectively), all filed on July 4, 2023. The entire contents of these five patent applications are hereby incorporated by reference into this document. TECHNICAL FIELD The present disclosure relates to the field of biotechnology, specifically to an engineering bactierium for L-valine production, a construction method thereof, and use thereof. BACKGROUND In microorganisms, L-valine (a type of branched-chain amino acid) is biosynthesized starting from pyruvate, proceeding via acetolactate, dihydroxyisovalerate, and ketoisovalerate. These intermediate metabolites are produced through reactions catalyzed by acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, dihydroxyacid dehydratase, and transaminase B. However, these enzymes are also involved in the biosynthesis of L-isoleucine starting from 2-oxobutyrate and pyruvate, and L-leucine is biosynthesized starting from ketoisovalerate (an intermediate metabolite), proceeding via 2-isopropylmalate, 3-isopropylmalate, and ketoisocaproate. Therefore, because branched-chain amino acids (namely L-valine, L-isoleucine, and L-leucine) utilize the same enzymes for their biosynthesis processes, it is known to be challenging to industrially produce one type of branched-chain amino acid through fermentation. Additionally, there is a problem that industrial mass production is limited by feedback inhibition caused by L-valine (the end product) or its derivatives. Escherichia coli, with its well-defined genetic background, is an attractive industrial production chassis for amino acid production. However, compared to Corynebacterium glutamicum, there are fewer reports on E. coli strains producing L-valine, possibly due to the more complex regulatory mechanisms of L-valine biosynthesis in E. coli. Acetohydroxyacid synthase (AHAS) is the rate-limiting enzyme in L-valine biosynthesis, and E. coli has three AHAS isozymes encoded by ilvBN, ilvGM, and ilvIH, with different properties and regulatory mechanisms. Park et al. reported the engineering of L-valine-producing strains through systematic metabolic engineering of Escherichia coli W3110 and Escherichia coli W, achieving a final L-valine yield of 60.7 g/L and a sugar-to-acid conversion rate of 0.22 g/g. In addition to mutagenesis breeding and conventional metabolic engineering modifications, cofactor balance is also considered a key bottleneck in improving L-valine yield. Since intracellular cofactors affect the metabolic network, signal transduction, and substance transport, thereby influencing the physiological functions of microbial cells. During the microbial fermentation production of chemicals, the potency and yield of chemicals are often limited by cofactor imbalance, primarily caused by the imbalanced expression of cofactor-dependent enzymes in the synthetic pathway. Savrasova and Stoynova et al. constructed an L-valine-producing engineering bacterium of E. coli MG1655 by replacing the native NADPH-dependent transaminase with a heterologous NADH-dependent leucine dehydrogenase. Under microaerobic conditions, the sugar-to-acid conversion rate (0.23 g/g) of this strain is only 35.4% of the maximum theoretical yield of 0.65 g/g. Developing high-throughput screening methods using biosensors, introducing exogenous cofactor regeneration pathways to balance intracellular cofactors, and constructing efficient industrial chassis production strains are key scientific issues that need to be addressed. SUMMARY The technical problem to be solved by the present disclosure is how to improve the yield of L-valine. In order to address the aforementioned technical problem, the present disclosure first provides an engineering bacterium for producing valine, which is a recombinant strain obtained by modifying a recipient bacterium; the modification comprises A1), A2), A3), A4), or A5); A1) comprises A11), A12), and A13): A11) knocking out the yjiT gene of the recipient bacterium, inhibiting the expression of the yjiT gene, or inhibiting the activity of the protein encoded by the yjiT gene;A12) increasing the content or enhancing the activity of the protein encoded by the brnF gene in the recipient bacterium;A13) increasing the content or enhancing the activity of the protein encoded by the brnE gene in the recipient bacterium;A2) comprises A21), A22), and A23); A21) knocking out the yjiV gene of the recipient bacterium, inhibiting the expression of the yjiV gene, or inhibiting the activity of the protein encoded by the yjiV gene;A22) increasing the content or enhancing the activity of the protein encoded by the ilvE gene in the recipient bacterium;A23) increasing the content or enhancing the activity of the protein encoded by the ilvD gene in the recipient bacterium;A3) comprises A31) and A32): A31) knocking out the trp