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CN-122012362-A - Method for producing L-valine, genetically engineered bacterium and application thereof

CN122012362ACN 122012362 ACN122012362 ACN 122012362ACN-122012362-A

Abstract

The invention relates to a method for producing L-valine, genetically engineered bacteria and application thereof, and belongs to the technical field of metabolic engineering. The invention uses E. coliW3110 as an initial strain, and utilizes a systematic metabolic engineering means to strengthen the synthesis flux of L-valine, improve the supply of reducing force, enhance the accumulation of precursors, adjust the balance of auxiliary factors and strengthen the discharge of products, thus constructing a series of L-valine production strains. Fed-batch fermentation is carried out in a 5L fermentation tank, the fermentation period is 26 h, the concentration of L-valine in the fermentation liquid reaches 131.3 g/L, and the conversion rate reaches 58.1%. The fermentation process adopted by the invention is simple, easy to control and low in production cost, and is beneficial to popularization and application of industrial production. Compared with the prior art, the engineering strain obtained by the invention has short fermentation period and high L-valine yield and conversion rate in the fermentation process.

Inventors

  • ZHANG CHENGLIN
  • Yin Yaqun
  • WEI MINHUA

Assignees

  • 天津科技大学

Dates

Publication Date
20260512
Application Date
20260413

Claims (9)

  1. 1. The L-valine production strain is characterized in that the strain takes escherichia coli as an initial strain, and a lactose operon repressor protein coding gene lacI is knocked out on a genome, and an acetolactate synthase coding gene ilvBN M1 or ilvBN M2 for releasing feedback inhibition is overexpressed; The acetolactate synthase encoding gene ilvBN M1 consists of ilvB and ilvN M1 , wherein the nucleotide sequence of ilvB is shown as SEQ ID NO.5, and the nucleotide sequence of ilvN M1 is shown as SEQ ID NO. 7; The acetolactate synthase encoding gene ilvBN M2 consists of ilvB and ilvN M2 , the nucleotide sequence of the ilvB is shown as SEQ ID NO.5, and the nucleotide sequence of the ilvN M2 is shown as SEQ ID NO. 8.
  2. 2. The L-valine producing strain according to claim 1, wherein the branched-chain amino acid aminotransferase encoding gene ilvE, the leucine dehydrogenase encoding gene bcd, the NADH-favored hydroxy acid reductase encoding gene ilvC M or the NADPH-favored hydroxy acid reductase encoding gene ilvC are further knocked out.
  3. 3. The L-valine producing strain according to claim 2, wherein any one or more of the following (1) to (10) gene edits are continued on the basis of the above gene edits; (1) Knocking out any one or more of a fumaric acid reductase encoding gene frdB, a pyruvic acid-formate lyase encoding gene pflB, a lactic acid dehydrogenase encoding gene ldhA, an ethanol dehydrogenase encoding gene adhE, a pyruvic acid dehydrogenase encoding gene poxB, an acetate kinase encoding gene ackA or an alanine synthesis aminotransferase encoding gene yfbQ; (2) Overexpression of pyridine nucleotide transhydrogenase encoding gene pntAB; (3) Overexpressing a glucose-6-phosphate dehydrogenase encoding gene zwf, a 6-phosphogluconate dehydratase encoding gene edd, and a 2-keto-3-deoxy-6-phosphogluconate aldolase encoding gene eda; (4) Knocking out phosphoenolpyruvate synthase encoding gene pps; (5) Overexpression of the pyruvate kinase coding gene pykA or pykF; (6) Knocking out a valine-alanine aminotransferase encoding gene avtA; (7) Overexpressing the D-galactose transporter-encoding gene galP, and/or the glucokinase-encoding gene glk; (8) Overexpressing a glyceraldehyde-3-phosphate dehydrogenase-encoding gene gapA derived from E.coli, a glyceraldehyde-3-phosphate dehydrogenase-encoding gene gapC derived from Clostridium acetobutylicum, a glyceraldehyde-3-phosphate dehydrogenase-encoding gene gapN derived from Streptococcus pyogenes, or a glyceraldehyde-3-phosphate dehydrogenase-encoding gene gapN Sc derived from Streptococcus canis; (9) Over-expressing a branched chain amino acid exporter encoding gene ygaZH or an L-valine exporter encoding gene brnEF; (10) Knocking out any one or more of a branched amino acid transport system substrate binding protein coding gene livJ, a branched amino acid transport protein coding gene brnQ or a branched amino acid transport protein coding gene yhjE.
  4. 4. The L-valine producing strain according to any one of claims 1 to 3, wherein the starting strain is Escherichia coli (ESCHERICHIA COLI) W3110.
  5. 5. The L-valine-producing strain according to any one of claims 1 to 3, wherein the L-valine-producing strain is obtained by gene editing on a genome of E.coli (ESCHERICHIA COLI) W3110 as an initial strain, by knocking out lactose operon repressor gene lacI, by overexpressing on a genome the feedback inhibition-releasing acetolactate synthase-encoding gene ilvBN M1 , by knocking out branched-chain amino acid aminotransferase-encoding gene ilvE and by overexpressing the leucine dehydrogenase-encoding gene bcd of Bacillus subtilis, by overexpressing the NADH-preferred hydroxyacid reductase-encoding gene ilvC M , by knocking out fumaric acid reductase, pyruvate formate lyase, lactate dehydrogenase, alcohol dehydrogenase, pyruvate dehydrogenase, acetate kinase and alanine synthase-encoding genes frdB, pflB, ldhA, adhE, poxB, ackA and yfbQ, by overexpressing the pyridine nucleotide transhydrogenase-encoding gene pntAB, by overexpressing the glucose-6-phosphate dehydrogenase-encoding gene zwf, the 6-phosphate gluconate dehydratase-encoding gene edd, and 2-keto-3-deoxy-6-phosphogluconate aldolase encoding gene eda, knocking out phosphoenolpyruvate synthase encoding gene pps, overexpressing pyruvate kinase encoding gene pykF, knocking out valine-alanine aminotransferase encoding gene avtA, overexpressing D-galactose transporter encoding gene galP and glucokinase encoding gene glk, overexpressing glyceraldehyde 3-phosphate dehydrogenase encoding gene gapN of Streptococcus pyogenes, overexpressing L-valine output protein encoding gene brnEF of Corynebacterium glutamicum, knocking out branched amino acid transport system substrate binding protein encoding gene livJ, branched amino acid transporter encoding gene brnQ and branched amino acid transporter encoding gene yhjE.
  6. 6. The L-valine producing strain of claim 3, wherein the lactose operon repressor Protein is derived from E.coli W3110, protein ID in NCBI: BAE76127.1; The branched-chain amino acid aminotransferase is derived from Escherichia coli W3110, protein ID in NCBI: BAE77527.1; The leucine dehydrogenase is derived from bacillus subtilis, and Protein ID in NCBI is NP-390288.1; The nucleotide sequence of the NADH preferred hydroxy acid reductase encoding gene ilvC M is shown in a sequence table SEQ ID NO. 11; The NADPH-preferred hydroxy acid reductase from E.coli W3110, protein ID in NCBI: BAE77523.1; The fumaric acid reductase is derived from Escherichia coli W3110, protein ID in NCBI: BAE78157.1; the pyruvic acid-formate lyase is derived from Escherichia coli W3110, protein ID in NCBI: BAA35638.1; The lactic dehydrogenase is derived from Escherichia coli W3110, protein ID in NCBI: BAA14990.1; the alcohol dehydrogenase is derived from Escherichia coli W3110, protein ID in NCBI: BAA36121.2; The pyruvate dehydrogenase is from escherichia coli W3110, protein ID in NCBI: BAA35585.1; The acetate kinase is derived from Escherichia coli W3110, protein ID in NCBI: BAA16135.1; the alanine synthetic aminotransferase is derived from E.coli W3110, protein ID in NCBI: BAA16127.1; The pyridine nucleotide transhydrogenase is derived from escherichia coli W3110, protein ID in NCBI is BAA15342.1 and BAA15336.1; the glucose-6-phosphate dehydrogenase is derived from Escherichia coli W3110, protein ID in NCBI: BAA15660.1; The 6-phosphogluconate dehydratase is derived from Escherichia coli W3110, protein ID in NCBI: BAA15659.1; The 2-keto-3-deoxy-6-phosphogluconate aldolase is derived from Escherichia coli W3110, protein ID in NCBI: BAA15658.1; The phosphoenolpyruvate synthase is from Escherichia coli W3110, protein ID in NCBI: BAA15471.1; The pyruvate kinase is from Escherichia coli W3110, protein ID in NCBI: BAA15445.2 or BAA15662.1; the valine-alanine aminotransferase is E.coli W3110, protein ID in NCBI: BAE77721.1; the D-galactose transporter is derived from Escherichia coli W3110, protein ID in NCBI: BAE77006.1; The glucokinase is derived from Escherichia coli W3110, protein ID in NCBI: BAA16258.1; the glyceraldehyde-3-phosphate dehydrogenase is respectively from escherichia coli W3110, clostridium acetobutylicum, streptococcus mutans or streptococcus canis, and Protein IDs in NCBI are BAA15576.1, AAK78686.1, AAN58410.1 and VEE25119.1; The L-valine export Protein is derived from Corynebacterium glutamicum ATCC13032, protein ID in NCBI is CAF18829.1 and CAF18830.1; The branched chain amino acid transport export Protein is derived from E.coli W3110, protein ID in NCBI: BAE76784.1 and BAA16545.1; The branched chain amino acid transport system substrate binding Protein is derived from E.coli W3110, protein ID in NCBI: BAE77833.1; the branched chain amino acid transporter is derived from E.coli W3110, protein ID in NCBI: BAE76181.1 or Protein ID in NCBI: BAE77771.1.
  7. 7. Use of a strain according to any one of claims 1-3 for the production of L-valine.
  8. 8. The use according to claim 7, wherein the method for producing L-valine by fermentation using the strain comprises the steps of: Inoculating the seed culture into fermentation medium at 10-20% inoculation amount, fermenting at pH of 6.8-7.2 and culture temperature of 35-37deg.C, maintaining residual sugar concentration of 0.1-0.5 g/L, maintaining dissolved oxygen at 20-50% before 12-h, and maintaining dissolved oxygen at 0-15%.
  9. 9. The use according to claim 8, wherein the fermentation period is 18-26 hours.

Description

Method for producing L-valine, genetically engineered bacterium and application thereof Technical Field The invention relates to a method for producing L-valine, genetically engineered bacteria and application thereof, and belongs to the technical field of metabolic engineering. Background L-valine is taken as a branched chain amino acid (branch chain amino acid, BCAs), belongs to essential amino acids of human body, can promote normal growth of the organism, repair tissues, regulate blood sugar and provide energy required by the organism, and has wide application prospect and great commercial value. In industrial production, L-valine is widely used in the fields of pharmacy, food, animal feed and the like. In particular in the animal feed industry, L-valine can enhance the immune system of animals by affecting the performance of animal organs, and can significantly enhance the productivity of livestock and poultry. Meanwhile, L-valine is also a fourth limiting amino acid of crude protein feeds for piglets and broilers. With the development of the age and the social progress, the demand for L-valine is increasing year by year, and the development of a high-efficiency L-valine production process is the focus of attention. The production method of L-valine at the present stage is mainly a microbial fermentation method. In recent years, with rapid development of synthetic biology and genetic engineering, recombinant engineering bacteria which can efficiently produce L-valine, have clear genetic background and are easy to culture can be obtained through methods such as mutagenesis, high-throughput screening breeding, synthetic biology and the like. The existing L-valine engineering strain has low yield and conversion rate, and severely limits the further large-scale production and application of the strain. Coli is widely used for amino acid production due to its clear genetic background and simple gene editing technology, and can be simply and cheaply grown and cultured in laboratory environment, thus being an ideal industrialized chassis cell. Disclosure of Invention In order to overcome the defects of low acid production rate, low conversion rate and the like of the existing L-valine production strain, the invention provides a genetically engineered bacterium with high synthesis efficiency for producing L-valine and a method for directly fermenting and producing L-valine by adopting the genetically engineered bacterium. The invention solves the technical problems and adopts the following technical route: One of the technical schemes provided by the invention is an L-valine production strain, which takes escherichia coli as an initial strain, knocks out lactose operon repressor protein coding gene lacI on a genome, and overexpresses any one of feedback inhibition released acetolactate synthase coding genes ilvBN M1、ilvBNM2 or ilvIH M on the genome; Further, on the basis of the above gene editing, the branched-chain amino acid aminotransferase encoding gene ilvE is knocked out, and the leucine dehydrogenase encoding gene bcd is overexpressed; Further, on the basis of the above-mentioned gene editing, any one or more of the following (1) to (10) gene editing is continued; (1) Knocking out any one or more of a fumaric acid reductase encoding gene frdB, a pyruvic acid-formate lyase encoding gene pflB, a lactic acid dehydrogenase encoding gene ldhA, an ethanol dehydrogenase encoding gene adhE, a pyruvic acid dehydrogenase encoding gene poxB, an acetate kinase encoding gene ackA or an alanine synthesis aminotransferase encoding gene yfbQ; (2) Overexpression of pyridine nucleotide transhydrogenase encoding gene pntAB; Further, the promoter adopted by the over-expression is a P trc or P pntAB promoter; (3) Overexpressing a glucose-6-phosphate dehydrogenase encoding gene zwf, a 6-phosphogluconate dehydratase encoding gene edd, and a 2-keto-3-deoxy-6-phosphogluconate aldolase encoding gene eda; (4) Knocking out phosphoenolpyruvate synthase encoding gene pps; (5) Overexpression of the pyruvate kinase coding gene pykA or pykF; (6) Knocking out a valine-alanine aminotransferase encoding gene avtA; (7) Overexpressing the gene galP encoding a non-phosphotransferase dependent glucose uptake protein and/or the gene glk encoding a glucokinase; (8) Overexpressing a glyceraldehyde-3-phosphate dehydrogenase-encoding gene gapA derived from E.coli, a glyceraldehyde-3-phosphate dehydrogenase-encoding gene gapC derived from Clostridium acetobutylicum, a glyceraldehyde-3-phosphate dehydrogenase-encoding gene gapN derived from Streptococcus pyogenes, or a glyceraldehyde-3-phosphate dehydrogenase-encoding gene gapN Sc derived from Streptococcus canis; (9) Over-expressing a branched chain amino acid exporter encoding gene ygaZH or an L-valine exporter encoding gene brnEF; (10) Knocking out any one or more of a branched amino acid transport system substrate binding protein coding gene livJ, a branched amino acid transport protein coding g