Method for producing L-valine as well as genetically engineered bacterium and application thereof

Abstract

The invention relates to a method for producing L-valine and a genetically engineered bacterium and application thereof, and belongs to the technical field of metabolic engineering. According to the invention, E. coliW3110 is taken as an original strain, and a system metabolism engineering means is utilized to enhance the L-valine synthesis flux, improve the reducing power supply, enhance the precursor accumulation, regulate the balance of auxiliary factors and enhance the product efflux, so that a series of L-valine production strains are constructed. The L-valine is used as a raw material, fed-batch fermentation is carried out in a 5 L fermentation tank, the fermentation period is 26 h, the concentration of the L-valine in fermentation liquor reaches 131.3 g / L, and the conversion rate reaches 58.1%. The adopted fermentation process is simple, easy to control, low in production cost and beneficial to popularization and application of industrial production. Compared with the prior art, the engineering strain and the fermentation process thereof have the advantages that the fermentation period is short, and the yield and conversion rate of L-valine are high.

Description

Technical Field

[0001] This invention relates to a method for producing L-valine, its genetically engineered strain, and its applications, belonging to the field of metabolic engineering technology. Background Technology

[0002] L-valine, a branched-chain amino acid (BCAA), is an essential amino acid for the human body. It promotes normal growth, repairs tissues, regulates blood sugar, and provides the energy needed by the body, exhibiting broad application prospects and significant commercial value. In industrial production, L-valine is widely used in pharmaceuticals, food, and animal feed. Particularly in the animal feed industry, L-valine can enhance the animal's immune system by influencing organ performance, significantly improving the production performance of livestock and poultry. Simultaneously, L-valine is also the fourth limiting amino acid in crude protein feed for piglets and broilers. With the development of the times and social progress, the demand for L-valine is increasing year by year, making the development of efficient L-valine production methods a focus of attention.

[0003] Currently, the main method for producing L-valine is microbial fermentation. In recent years, with the rapid development of synthetic biology and genetic engineering, recombinant engineered bacteria with clear genetic backgrounds and easy cultivation can be obtained through mutagenesis, high-throughput screening breeding, and synthetic biology methods. However, the yield and conversion rate of existing L-valine engineered strains are low, severely limiting their further large-scale production and application. *Escherichia coli*, due to its clear genetic background, simple gene editing technology, and the ability to be easily and inexpensively grown and cultured in a laboratory environment, is widely used in amino acid production and is an ideal industrial chassis cell. Summary of the Invention

[0004] To overcome the shortcomings of existing L-valine-producing strains, such as low acid production and low conversion rate, this invention provides a genetically engineered strain with high L-valine synthesis efficiency and a method for directly fermenting L-valine using this strain.

[0005] The present invention solves the above-mentioned technical problems by adopting the following technical approach:

[0006] One of the technical solutions provided by this invention is an L-valine-producing strain, wherein the strain is derived from *Escherichia coli* and the gene encoding the lactose operon repressor protein is knocked out in the genome. lacI ; and overexpress the gene encoding acetolactate synthase, which relieves feedback inhibition, on the genome. ilvBN M1 , ilvBN M2 or ilvIH MAny one of them; Furthermore, based on the above gene editing, the gene encoding branched-chain amino acid aminotransferases was also knocked out. ilvE Overexpression of the leucine dehydrogenase encoding gene bcd ; Gene encoding NADH-preferred hydroxy acid reductase ilvC M Or the gene encoding a NADPH-preferred hydroxy acid reductase. ilvC ; Furthermore, based on the above gene editing, continue to perform any one or more of the following gene editing methods (1)-(10); (1) Knockout of the gene encoding fumarate reductase frdB pyruvate-formate lyase encoding gene pflB lactate dehydrogenase encoding gene ldhA Ethanol dehydrogenase encoding gene adhE pyruvate dehydrogenase encoding gene poxB Acetylkinase encoding gene ackA Or the gene encoding alanine transaminase. yfbQ Any one or more of the following; (2) Overexpression of the gene encoding pyridine nucleotide transhydrogenase pntAB ; Furthermore, the promoter used for the overexpression is P. trc or P pntAB promoter; (3) Overexpression of the glucose-6-phosphate dehydrogenase encoding gene zwf 6-phosphoglucate dehydratase encoding gene edd and the gene encoding 2-keto-3-deoxy-6-phosphate glucuronide aldolase eda ; (4) Knockout of the gene encoding phosphoenolpyruvate synthase pps ; (5) Overexpression of the pyruvate kinase encoding gene pykA or pykF ; (6) Knockout of the gene encoding valine-alanine aminotransferase avtA ; (7) Overexpression of the gene encoding the nonphosphotransferase-dependent glucose uptake protein galP ; and / or glucokinase encoding genes glk ; (8) Overexpression of the gene encoding glyceraldehyde-3-phosphate dehydrogenase from Escherichia coli. apA The gene encoding glyceraldehyde-3-phosphate dehydrogenase from Clostridium acetone-butanol is g. apC The gene encoding glyceraldehyde-3-phosphate dehydrogenase from Streptococcus pyogenes is g.apN Or the gene encoding glyceraldehyde-3-phosphate dehydrogenase from Streptococcus canis. apN Sc ; (9) Overexpression of genes encoding branched chain amino acid export proteins ygaZH or L-valine export protein encoding gene brnEF ; (10) Knockout of genes encoding substrate-binding proteins of branched-chain amino acid transport systems livJ Branched-chain amino acid transporter encoding genes brnQ or genes encoding branched-chain amino acid transporters yhjE Any one or more of the following; Preferably, the starting strain is *Escherichia coli* (E. coli). Escherichia coli W3110; More preferably, the high-yield L-valine strain is *Escherichia coli* (E. coli). Escherichia coli W3110 was the starting strain, and the following gene editing was performed on its genome to obtain the strain: knockout of the gene encoding the lactose operon repressor protein. lacI Overexpression of the gene encoding acetolactate synthase, which relieves feedback inhibition, on the genome. ilvBN M1 Knockout of the gene encoding branched-chain amino acid aminotransferases ilvE And overexpression of the leucine dehydrogenase encoding gene of Bacillus subtilis bcd ; Gene encoding NADH-preferred hydroxy acid reductase ilvC M Knock out the genes encoding fumarate reductase, pyruvate-formate lyase, lactate dehydrogenase, alcohol dehydrogenase, pyruvate dehydrogenase, acetate kinase, and alanine transaminase. frdB , pflB , ldhA , adhE , poxB , ackA and yfbQ Overexpression of the gene encoding pyridine nucleotide transhydrogenase pntAB Overexpression of the glucose-6-phosphate dehydrogenase encoding gene zwf、 6-phosphoglucate dehydratase encoding gene edd And the gene encoding 2-keto-3-deoxy-6-phosphate glucuronide aldolase. eda Knockout of the gene encoding phosphoenolpyruvate synthase pps Overexpression of the gene encoding pyruvate kinase pykF Knockout of the gene encoding valine-alanine aminotransferase avtA Overexpression of the D-galactose transporter encoding gene galP and glucokinase encoding gene glkOverexpression of the g gene encoding glyceraldehyde-3-phosphate dehydrogenase from Streptococcus pyogenes apN Overexpression of the gene encoding the L-valine export protein of Corynebacterium glutamicum brnEF Knockout of genes encoding substrate-binding proteins of branched-chain amino acid transport systems livJ Branched-chain amino acid transporter encoding genes brnQ and branched-chain amino acid transporter encoding genes yhjE ; Furthermore, the genotype of the L-valine-producing strain is as follows: E. coli W3110 lacIyncI ::P trc - ilvBN M1 -T trc Δ ilvE ycgh ::P trc - bcd -T trc yeeL ::P trc - ilvC M -T trc Δ frdB Δ pflB Δ ldhA Δ adhE Δ poxB Δ ackA Δ yfbQyjgX ::P pntAB - pntAB -T trc gapC ::P trc - zwf-edd-eda -T trc Δ ppsyjiT ::P trc - pykF -T trc Δ avtA P galP ::P trc yeeP ::P trc - glK -T trc yciQ ::P trc - gapN -T trc rph ::P trc - brnEF -T trc Δ livJ Δ brnQ Δ yhjE .

[0007] Furthermore, the lactose operon repressor protein is derived from Escherichia coli W3110, Protein ID: BAE76127.1 in NCBI; Furthermore, the acetolactate synthase encoding gene ilvBN M1 Depend on ilvB and ilvN M1 composition, ilvB The nucleotide sequence is shown in SEQ ID NO.5. ilvN M1 The nucleotide sequence is shown in SEQ ID NO.7; Furthermore, the acetolactate synthase encoding gene ilvBN M2 Depend on ilvB and ilvN M2 composition, ilvB The nucleotide sequence is shown in SEQ ID NO.5. ilvN M2 The nucleotide sequence is shown in SEQ ID NO.8; Furthermore, the gene encoding the acetolactate synthase that relieves feedback inhibition... ilvIH M Reference: Metabolic engineering of Escherichia coli for the production of L-valine based on transcriptome analysis and in silico gene knockout simulation; the encoding gene ilvIH M Include ilvI and ilIH M The nucleotide sequences are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively; Furthermore, the branched-chain amino acid aminotransferase was derived from Escherichia coli W3110, Protein ID: BAE77527.1 in NCBI; Furthermore, the leucine dehydrogenase is derived from Bacillus subtilis, Protein ID: NP_390288.1 in NCBI; Furthermore, the NADH-preferred hydroxy acid reductase encoding gene ilvC M Its nucleotide sequence is shown in SEQ ID NO.11 of the sequence listing; Furthermore, the NADPH-preferred hydroxy acid reductase is derived from Escherichia coli W3110, Protein ID: BAE77523.1 in NCBI; Furthermore, the promoter P trc It is derived from Escherichia coli W3110, and its nucleotide sequence is shown in SEQ ID NO. 12 of the sequence listing; Furthermore, the fumarate reductase was derived from Escherichia coli W3110, Protein ID: BAE78157.1 in NCBI; Furthermore, the pyruvate-formate lyase was derived from Escherichia coli W3110, Protein ID: BAA35638.1 in NCBI; Furthermore, the lactate dehydrogenase was derived from Escherichia coli W3110, Protein ID: BAA14990.1 in NCBI; Furthermore, the alcohol dehydrogenase was derived from Escherichia coli W3110, Protein ID: BAA36121.2 in NCBI; Furthermore, the pyruvate dehydrogenase was derived from Escherichia coli W3110, Protein ID: BAA35585.1 in NCBI; Furthermore, the acetate kinase was derived from Escherichia coli W3110, Protein ID: BAA16135.1 in NCBI; Furthermore, the alanine-synthesizing transaminase was derived from Escherichia coli W3110, Protein ID: BAA16127.1 in NCBI; Furthermore, the promoter P pntAB For Escherichia coli W3110 pntAB The promoter of the gene, whose nucleotide sequence is shown in SEQ ID NO.13 of the sequence listing; Furthermore, the pyridine nucleotide transhydrogenase was derived from Escherichia coli W3110, with Protein IDs BAA15342.1 and BAA15336.1 in NCBI. Furthermore, the glucose-6-phosphate dehydrogenase was derived from Escherichia coli W3110, Protein ID: BAA15660.1 in NCBI; Furthermore, the 6-phosphoglucate dehydratase was derived from Escherichia coli W3110, Protein ID: BAA15659.1 in NCBI; Furthermore, the 2-keto-3-deoxy-6-phosphate glucuronide aldolase was derived from Escherichia coli W3110, Protein ID: BAA15658.1 in NCBI; Furthermore, the phosphoenolpyruvate synthase was derived from Escherichia coli W3110, Protein ID: BAA15471.1 in NCBI; Furthermore, the pyruvate kinase is derived from Escherichia coli W3110, with Protein ID BAA15445.2 or BAA15662.1 in NCBI; Furthermore, the valine-alanine aminotransferase has the Protein ID BAE77721.1 in NCBI. Furthermore, the D-galactose transporter protein was derived from Escherichia coli W3110, Protein ID: BAE77006.1 in NCBI; Furthermore, the glucokinase was derived from Escherichia coli W3110, Protein ID: BAA16258.1 in NCBI; Furthermore, the 3-phosphoglyceraldehyde dehydrogenases were derived from Escherichia coli W3110, Clostridium acetobutyricum, Streptococcus mutans, and Streptococcus canis, respectively, with Protein IDs in NCBI of BAA15576.1, AAK78686.1, AAN58410.1, and VEE25119.1, respectively. Furthermore, the L-valine export protein is from Corynebacterium glutamicum, with Protein IDs CAF18829.1 and CAF18830.1 in NCBI; Furthermore, the branched-chain amino acid transporter-export protein was derived from Escherichia coli W3110, with ProteinIDs BAE76784.1 and BAA16545.1 in NCBI. Furthermore, the substrate-binding protein of the branched-chain amino acid transport system is derived from Escherichia coli W3110, Protein ID: BAE77833.1 in NCBI; Furthermore, the branched-chain amino acid transporter protein is derived from Escherichia coli W3110, with Protein ID BAE76181.1 or Protein ID BAE77771.1 in NCBI.

[0008] The second technical solution provided by the present invention is the application of the strain described in technical solution one in the production of L-valine.

[0009] Furthermore, the method for producing L-valine using the above-mentioned strain through fermentation is as follows: The seed culture was inoculated into the fermentation medium at an inoculum rate of 10-20% (v / v) for fermentation culture. The pH was maintained at 6.8-7.2 and the culture temperature was 35-37℃. The residual sugar concentration was maintained at 0.1-0.5 g / L. The dissolved oxygen was maintained at 20-50% for the first 12 hours and then at 0-15%. The fermentation cycle was 18-26 hours, and the L-valine concentration in the fermentation broth reached 109.3-131.3 g / L.

[0010] Beneficial effects: (1) The present invention is based on E. coli Using strain W3110 as the starting strain, a new L-valine-producing strain was constructed by employing systems metabolic engineering techniques to enhance L-valine synthesis flux, increase reducing power supply, improve precursor accumulation, regulate cofactor balance, and strengthen product efflux. Adaptive evolution was then performed using this strain to obtain a high-yield L-valine strain. Fed-batch fermentation was conducted in a 5 L fermenter for 26 hours, achieving an L-valine concentration of 131.3 g / L in the fermentation broth and a conversion rate of 58.1%.

[0011] (2) The fermentation process used in this invention is simple, easy to control, and has low production costs, which is conducive to the promotion and application of industrial production. Compared with the prior art, the engineered strains and fermentation process obtained in this invention have a short fermentation cycle and high L-valine yield and conversion rate.

[0012] (3) The acetolactate synthase mutant IlvBN provided by the present invention M1 and IlvBN M2 It exhibits the following characteristics: the feedback inhibition effects of L-valine, L-leucine, and L-isoleucine were eliminated; the enzyme activity showed no significant change under conditions where the concentrations of L-valine, L-leucine, and L-isoleucine were 0-30 mmol / L; and the acetolactate synthase mutant IlvBN under conditions where the concentrations of L-valine, L-leucine, and L-isoleucine were 0-30 mmol / L... M1 and IlvBN M2 The enzyme activity was not significantly reduced compared to wild-type acetolactate synthase IlvBN at L-valine, L-leucine, and L-isoleucine concentrations of 0 mmol / L. This mutant can be widely used in the synthesis of L-valine, L-leucine, and L-isoleucine. Attached Figure Description

[0013] Figure 1 Wild-type acetolactate synthase IlvBN and acetolactate synthase mutant IlvBN M1 and IlvBN M2 Comparison of enzyme activities in the absence of branched-chain amino acids.

[0014] Figure 2 Branched-chain amino acid pairs for wild-type acetolactate synthase IlvBN and acetolactate synthase mutant IlvBN M1 and IlvBN M2 Effects on activity; In the figure, Figure A represents L-valine; Figure B represents L-leucine; and Figure C represents L-isoleucine. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of this patent clearer, the following detailed description is provided in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of this patent and are not intended to limit the scope of the invention.

[0016] The gene sequences involved in the embodiments of the present invention can be encoded and synthesized according to the sequence information corresponding to the Protein ID number in NCBI provided in the foregoing invention content section, or amplified using the microbial genome of the corresponding source as a template; The method for detecting substances during the fermentation process of this invention is as follows: (1) L-valine detection method L-valine was quantitatively analyzed using high-performance liquid chromatography (HPLC). The sample was derivatized with 2,4-dinitrofluorobenzene to make it detectable by the HPLC signal analyzer; the specific procedure is as follows: Sample derivatization method: Centrifuge 1 ml of fermentation broth at 13000 g for 3 min, collect the supernatant, dilute it 10 times with deionized water, and then perform derivatization reaction with 0.8% (V / V) 2,4-dinitrofluorobenzene (pass the supernatant through a 0.22 μm organic membrane for later use. Add 200 μL of derivatization buffer, 10 μL of the membrane-passed supernatant, and 300 μL of derivatizing agent to a 1.5 mL EP tube, shake well, and place in a 65°C water bath in the dark for 60 min. After cooling, take 690 μL of volume-adjusting buffer and make up to 1.2 mL, mix well, and pass through a membrane). Filter into the sample cell, using 50% acetonitrile (filtered by ultrasonication) and 4.1 g / L sodium acetate as the organic and inorganic phases, respectively. Adjust the column temperature to 33°C. When the baseline is 0 and the pressure line is horizontal, start the determination of the sample. The detection conditions were: Agilent AAA (4.6 mm × 150 mm, 5-Micron), acetonitrile / sodium acetate binary gradient elution, flow rate 1 mL / min, column temperature 33℃, and detection wavelength 360 nm.

[0017] (2) Detection of bacterial cell concentration and other products OD of the strain was measured by spectrophotometer. 600Its growth was monitored. The concentration of glucose during fermentation was detected using an SBA biosensor instrument (SBA-40C; Shandong Academy of Sciences Institute of Biology, Jinan).

[0018] The primer sequences used in the embodiments of the present invention are shown in Table 1 below.

[0019] Table 1

[0020] The strain genotypes in the embodiments of this invention are shown in Table 2 below.

[0021] Table 2

[0022] It should be noted that the gene editing order can be adjusted according to actual needs during the construction of the strain of the present invention, and the order of editing will not affect the final strain's ability to produce L-valine.

[0023] The present invention will be further explained and illustrated below through specific embodiments.

[0024] Example 1: The gene encoding acetolactate synthase that relieves branched-chain amino acid feedback inhibition ilvBN M1 and ilvBN M2 The acquisition 1. Identification of the acetolactate synthase mutant of the present invention The mutated amino acid in the acetolactate synthase mutant is represented by "original amino acid + position + substituted amino acid". For example, G20D indicates that the amino acid at position 20 is replaced by Asp by Gly in the wild-type acetolactate synthase. The position number corresponds to the amino acid sequence number of the regulatory subunit IlvN of the wild-type acetolactate synthase in SEQ ID NO.2.

[0025] In this invention, IlvBN represents wild-type acetolactate synthase, IlvB represents the catalytic subunit of wild-type acetolactate synthase, and IlvN represents the regulatory subunit of wild-type acetolactate synthase.

[0026] In this invention, ilvBN The gene encoding wild-type acetolactate synthase IlvBN. ilvB The gene encoding the wild-type acetolactate synthase catalytic subunit IlvB. ilvNThe gene encoding the wild-type acetolactate synthase regulatory subunit IlvN; ilvN M1 Represents the acetolactate synthase mutant IlvBN M1 Regulatory subunit IlvN M1 The encoding gene, ilvN M2 Represents the acetolactate synthase mutant IlvBN M2 Regulatory subunit IlvN M2 The gene that encodes it.

[0027] In this invention, the acetolactate synthase mutant IlvBN M1 Composed of catalytic subunit IlvB and regulatory subunit IlvN M1 The composition, the amino acid sequence of the catalytic subunit is shown in SEQ ID NO.1, and the regulatory subunit IlvN M1 It was obtained by mutations of G20D and S23F in the wild-type acetolactate synthase regulatory subunit IlvN shown in SEQ ID NO.2. M1 The amino acid sequence is shown in SEQ ID NO.3.

[0028] In this invention, the acetolactate synthase mutant IlvBN M2 Composed of catalytic subunit IlvB and regulatory subunit IlvN M2 The composition, the amino acid sequence of the catalytic subunit is shown in SEQ ID NO.1, and the regulatory subunit IlvN M2 It was obtained by mutations of G20E and S23F in the wild-type acetolactate synthase regulatory subunit IlvN shown in SEQ ID NO.2. M2 The amino acid sequence is shown in SEQ ID NO.4.

[0029] The details are shown in Table 3 below.

[0030] Table 3

[0031] In this invention, the source is C. glutamicum The wild-type acetolactate synthase IlvBN of ATCC13032 is composed of a catalytic subunit IlvB and a regulatory subunit IlvN. The amino acid sequence of the catalytic subunit IlvB is shown in SEQ ID NO.1, and the amino acid sequence of the regulatory subunit IlvN is shown in SEQ ID NO.2. In this invention, the acetolactate synthase mutant IlvBN M1 Composed of catalytic subunit IlvB and regulatory subunit IlvN M1Composition: The amino acid sequence of the catalytic subunit IlvB is shown in SEQ ID NO.1, and the regulatory subunit IlvN... M1 The amino acid sequence is shown in SEQ ID NO.3; In this invention, the acetolactate synthase mutant IlvBN M2 Composed of catalytic subunit IlvB and regulatory subunit IlvN M2 Composition: The amino acid sequence of the catalytic subunit IlvB is shown in SEQ ID NO.1, and the regulatory subunit IlvN... M2 The amino acid sequence is shown in SEQ ID NO.4; SEQ ID NO.1: MNVAASQQPTPATVASRGRSAAPERMTGAKAIVRSLEELNADIVFGIPGGAVLPVYDPLYSSTKVRHVLVRHEQGAGHAATGYAQVTGRVGVCIATSGPGATNLVTPIADANLDSVPMVAITGQVGSGLLGTDAFQEADIRGITMPVTKHNF MVTNPNDIPQALAEAFHLAITGRPGPVLVDIPKDVQNAELDFVWPPKIDLPGYRPVSTPHARQIEQAVKLIGEAKKPVLYVGGGVIKADAHEELRAFAEYTGIPVVTTLMALGTFPESHELHMGMPGMHGTVSAVGALQRSDLLIAIGSRFDDRVTGD VDTFAPDAKIIHADIDPAEIGKIKQVEVPIVGDAREVLARLLETTKASKAETEDISEWVDYLKGLKARFPRGYDEQPGDLLAPQFVIETLSKEVGPDAIYCAGVGQHQMWAAQFVDFEKPRTWLNSGGLGTMGYAVPAALGAKAGAPDKEVWAIDGDG CFQMTNQELTTAAVEGFPIKIALINNGNLGMVRQWQTLFYEGRYSNTKLRNQGEYMPDFVTLSEGLGCVAIRVTKAEEVLPAIQKAREINDRPVVIDFIVGEDAQVWPMVSAGSSNSDIQYALGLRPFFDGDESAAEDPADIHEAVSDIDAAVESTEA SEQ ID NO.2: MANSDVTRHILSVLVQDVDGIISRVSGMFTRRAFNLVSLVSAKTETHGINRITVVVDADELNIEQITKQLNKLIPVLKVVRLDEETTIARAIMLVKVSADSTNRPQIVDAANIFRARVVDVAPDSVVIESTGTPGKLRALLDVMEPFGIRELIQSGQIALNRGPKTMAPAKI SEQ ID NO.3: MANSDVTRHILSVLVQDVDDIIFRVSGMFTRRAFNLVSLVSAKTETHGINRITVVVDADELNIEQITKQLNKLIPVLKVVRLDEETTIARAIMLVKVSADSTNRPQIVDAANIFRARVVDVAPDSVVIESTGTPGKLRALLDVMEPFGIRELIQSGQIALNRGPKTMAPAKI SEQ ID NO.4: MANSDVTRHILSVLVQDVDEIIFRVSGMFTRRAFNLVSLVSAKTETHGINRITVVVDADELNIEQITKQLNKLIPVLKVVRLDEETTIARAIMLVKVSADSTNRPQIVDAANIFRARVVDVAPDSVVIESTGTPGKLRALLDVMEPFGIRELIQSGQIALNRGPKTMAPAKI 2. Assay of acetyllactate synthase activity (1) Definition of specific enzyme activity: The amount of sodium pyruvate consumed per minute per milligram of total protein containing acetolactate synthase (nmol).

[0032] (2) Enzyme activity assay: 100 μL of crude enzyme solution was added to 800 μL of 100 mmol / L potassium phosphate buffer (pH 7.8, containing 10 mmol / L sodium pyruvate, 1 mmol / L MgCl2, and 0.2 mmol / L thiamine pyrophosphate), and reacted at 37℃ for 1 h. 100 μL of 3 mol / L H2SO4 was added to terminate the enzymatic reaction. After the reaction was completed, the amount of sodium pyruvate consumed was determined by high performance liquid chromatography.

[0033] (3) The detection method of sodium pyruvate is as follows: After centrifuging the reaction solution at 8000×g for 10min, the supernatant is taken. The detection conditions are: chromatographic column REzex RoA-organic Acid H+, mobile phase 5 mmol / L H2SO4, flow rate 0.5mL / min, column temperature 30℃, detection wavelength 215nm, and injection volume 20μL.

[0034] (4) Formula for calculating specific enzyme activity: Specific enzyme activity = (ΔC) Pyr × V_total) / (t × C) TP × V TP ) C Pyr : The concentration of sodium pyruvate decreased (nmol / L); Vtotal: Total volume of the reaction solution (L); t represents the reaction time: 60 min; C TP Total protein concentration in crude enzyme solution (mg / L); V TP : Crude enzyme solution volume (L).

[0035] 3. Obtaining mutants wild-type Corynebacterium glutamicum C. glutamicum Using the ATCC13032 genome as a template, error-prone PCR (ready-to-use error-prone PCR kit, Beijing Tianenze Gene Technology Co., Ltd.) was performed using primers ilvBN-1 / ilvBN-2 for amplification. ilvBN Mutant library.

[0036] Error-prone PCR conditions are: 95℃ pre-denaturation for 5 min; 95℃ for 30 s, 54℃ for 30 s, 72℃ for 3 min, 40 cycles; 72℃ for 10 min.

[0037] use Eco pSTV28 was digested with RI enzyme and recovered, then combined with the above-mentioned recombinant kit ClonExpress II One Step Cloning Kit (Nanjing Novizan Medical Technology Co., Ltd.) ilvBN Mutant library recombination ligation and transformation to E. coli After revival, DH5α competent cells were plated onto solid selection medium containing 100 μg / mL chloramphenicol and incubated at 37°C. The next day, 50 larger single colonies were randomly selected from the plates and transferred to 96-well cell culture plates containing 100 μg / mL chloramphenicol liquid selection medium, and cultured at 37°C with shaking for 24 h.

[0038] OD was measured using an enzyme-linked immunosorbent assay (ELISA) reader. 600 Select OD600 The top 8 strains were used to extract plasmids for analysis. ilvBN The mutants were sequenced. Two types of mutants were found, that is, relative to those from... C. glutamicum ATCC13032 wild-type acetolactate synthase, these mutants have the following amino acid mutation: IlvBN M1 (G20D / S23F) and IlvBN M2 (G20E / S23F), whose encoding genes are named respectively. ilvBN M1 and ilvBN M2 The plasmids containing the above genes were named pST- ilvBN M1 and pST- ilvBN M2 Will contain wild type ilvBN The plasmid for the gene was named pST- ilvBN .

[0039] Solid screening medium: glucose 10 g / L, MgSO4 0.24 g / L, KH2PO4 2.5 g / L, (NH4)2SO4 5 g / L, FeSO4 2 g / L, 2-hydroxy-3-methylbutyric acid 50 mg / L, agar 20 g / L, deionized water 1000 mL, pH 6.5-7.0.

[0040] Liquid screening medium: glucose 10 g / L, MgSO4 0.24 g / L, KH2PO4 2.5 g / L, (NH4)2SO4 5 g / L, FeSO4 2 g / L, 2-hydroxy-3-methylbutyric acid 50 mg / L, deionized water 1000 mL, pH 6.5-7.0.

[0041] The principles of the above solid-state and liquid-state screening are as follows: branched-chain amino acids bind to acetolactate synthase, altering its spatial structure and thus inhibiting its activity. Since the binding of branched-chain amino acids to acetolactate synthase is reversible, this inhibition is also reversible and has no lethal effect on bacterial cells. Therefore, it cannot be used to screen for acetolactate synthase mutants.

[0042] 2-Hydroxy-3-methylbutyric acid (2-HMA) is a structural analog of branched-chain amino acids and can also bind to acetolactate synthase (ASY), but this binding is irreversible and lethal to bacterial cells. Therefore, recombinants expressing wild-type or non-positively mutated ASY do not grow in media containing 2-HMA. Some acetolactate synthases mutate and do not bind to 2-HMA, thus strains containing these mutants can grow in media containing 2-HMA. Since 2-HMA is a structural analog of branched-chain amino acids, this means that these mutants also do not bind to branched-chain amino acids, thereby relieving the feedback inhibition of branched-chain amino acids. Using this principle, resistant bacterial colonies are first obtained in a solid medium containing 2-HMA (initial screening), and then these colonies are transferred to a liquid medium containing 2-HMA. Growth is rapid (OD). 600 The presence of high OD values ​​in strains indicates strong resistance to 2-hydroxy-3-methylbutyric acid stress; therefore, strains with high OD values ​​were selected. 600 High strains (secondary screening).

[0043] Example 2: Enzymatic Characteristics Analysis of Acetolactate Synthase 1. Preparation of crude enzyme solution Cultured in LB liquid medium containing pST- ilvBN pST- ilvBN M1 and ilvBN M2 of E. coli After 12 hours of DH5α culture, 20 mL of the culture was centrifuged at 10,000 g for 1 min at 4°C to collect the bacterial cells. The precipitate was washed three times with 1 mL of buffer (200 mmol / L Tris-HCl, pH 8.1) and then resuspended in 1 mL of buffer. The bacterial suspension was then sonicated using an ultrasonic homogenizer under the following conditions: 350 W power, 5 s working time, 10 s interval, 5 cycles, operated on ice. The homogenate was centrifuged at 8,000 g at 4°C, and the supernatant was collected as IlvBN. M1 and IlvBN M2 The total protein concentration of the crude enzyme solution was determined using the BCA Protein Quantification Kit (Nanjing Novizan Medical Technology Co., Ltd.).

[0044] 2. Enzyme activity assay IlvBN, IlvBN M1 and IlvBN M2The enzyme activity was determined as follows: 100 μL of crude enzyme solution was added to 800 μL of 100 mmol / L potassium phosphate buffer (pH 7.8, containing 10 mmol / L sodium pyruvate, 1 mmol / L MgCl2, and 0.2 mmol / L thiamine pyrophosphate), and the reaction was carried out at 37℃ for 1 h. 100 μL of 3 mol / L H2SO4 was then added to terminate the enzymatic reaction. After the reaction was complete, the amount of sodium pyruvate consumed was determined by high-performance liquid chromatography (HPLC). The specific enzyme activity was then calculated using the aforementioned method.

[0045] The results are as follows Figure 1 As shown, IlvBN, IlvBN M1 and IlvBN M2 The specific enzyme activities were 6.1, 6.2, and 5.9 nmol / (min·mg total protein), respectively, indicating that in the absence of branched-chain amino acids, the mutant IlvBN... M1 and IlvBN M2 There was no significant difference in enzyme activity compared to wild-type IlvBN.

[0046] 3. Branched-chain amino acids on IlvBN, IlvBN M1 and IlvBN M2 The effect of enzyme activity Branched chain amino acids for IlvBN, IlvBN M1 and IlvBN M2 The method for determining the effect of enzyme activity is as follows: Take 100 μL of acetyllactate synthase IlvBN or its mutant IlvBN respectively M1 or IlvBN M2 The crude enzyme solution was added to 800 μL of 100 mmol / L potassium phosphate buffer (pH 7.8, containing 10 mmol / L sodium pyruvate, 1 mmol / L MgCl2, and 0.2 mmol / L thiamine pyrophosphate). Then, 0, 5, 10, 15, 20, 25, and 30 mmol / L of branched-chain amino acids (including L-valine, L-leucine, and L-isoleucine) were added to the above reaction solution. The reaction was incubated at 37°C for 1 h, and 100 μL of 3 mol / L H2SO4 was added to terminate the enzymatic reaction. After the reaction was complete, the consumption of sodium pyruvate was determined by high-performance liquid chromatography (HPLC). This was to investigate the effects of IlvBN and IlvBN. M1 and IlvBN M2 To relieve the feedback inhibition of branched-chain amino acids.

[0047] IlvBN and IlvBN were added at a concentration of 0 with branched-chain amino acids. M1 and IlvBN M2IlvBN and IlvBN were defined as having specific enzyme activities of 100% and the concentrations of the remaining branched chain amino acids, respectively. M1 and IlvBN M2 The relative enzyme activity is the enzyme activity compared to its 100% enzyme activity. The results are as follows: Figure 2 As shown, the relative enzyme activity of IlvBN decreases rapidly with increasing branched-chain amino acid concentration, and is almost non-existent when the branched-chain amino acid concentration is above 10 mmol / L, indicating that this wild-type enzyme is subject to feedback inhibition by branched-chain amino acids; while the mutant IlvBN M1 and IlvBN M2 The relative activity of the compound did not change significantly with the increase of branched-chain amino acid concentration, indicating that it relieved the feedback inhibition effect of branched-chain amino acids.

[0048] Based on the above results, the acetolactate synthase mutant IlvBN M1 and IlvBN M2 The feedback inhibition of branched-chain amino acids was eliminated. Furthermore, in the absence of branched-chain amino acids, the enzyme activity of the mutants was not significantly reduced compared to the wild-type IlvBN.

[0049] Example 3: lacI Knockout strains E. coli W3110 lacI Construction The promoters used in this invention are mostly P trc , lacI The gene encoding LacI represses transcription by this promoter. However, the gene expressed in this invention requires constitutive expression, therefore it must be knocked out first. lacI .

[0050] (1) Overlapping segment U lacI -D lacI Construction wild-type Escherichia coli E. coli Using the W3110 genome as a template, primers U- were used respectively. lacI -S / U- lacI -A and D- lacI -S / D- lacI -A amplification lacI The upstream and downstream homologous arms were identified, and then overlap PCR was used to obtain... lacI Upstream and downstream homologous arm fusion fragment U lacI -D lacI .

[0051] (2) pGRB- lacI plasmid construction according to lacISequence design and synthesis of 20 bp forward and reverse sequences pGRB- of gRNA. lacI -S / pGRB- lacI -A, after annealing, both were ligated to plasmid pGRB using the ClonExpress II One Step Cloning Kit (Nanjing Novizan Medical Technology Co., Ltd.), and then transformed. E. coli The recombinant plasmid pGRB- was obtained by screening and sequencing on LB solid medium containing DH5α and 100 μg / mL ampicillin. lacI .

[0052] (3) lacI Knockout strains E. coli W3110 lacI Construction The recombinant plasmid pGRB- lacI and fusion fragment U lacI -D lacI Electroconversion to plasmids containing pREDcas9 E. coli W3110 competent cells were revived and plated on LB agar containing 100 μg / mL spectinomycin and ampicillin, and cultured overnight at 32°C. The following day, primers were used... lacI -1 / lacI -4. Colony PCR identification was performed to screen for positive transformants. The transformants were activated, and arabinose was added to a final concentration of 0.2 mmol / L. The mixture was then incubated overnight at 32°C with shaking to allow pGRB- to develop. lacI The pREDcas9 plasmid was lost; then the strain was cultured overnight at 42°C with shaking to induce its loss, thus obtaining the strain. E. coli W3110 lacI .

[0053] Example 4: ilvBN , ilvBN M1 and ilvBN M2 Construction of overexpression strains VAL-CK, VAL-1-M1 and VAL-1-M2 Acetolactate synthase is a key enzyme in the synthesis of L-valine, and overexpression of this enzyme facilitates L-valine synthesis. In Corynebacterium glutamicum and Escherichia coli, acetolactate synthase is expressed by […]. ilvBN and ilvIH The coding of L-valine is subject to feedback inhibition by L-valine. Therefore, overexpression of acetolactate synthase, which relieves feedback inhibition, is necessary to enhance L-valine synthesis.

[0054] (1) Overlapping segment U yncI -Ptrc - ilvBN M1 -T trc -D yncI and U yncI -P trc - ilvBN M2 -T trc -D yncI Construction With Escherichia coli E. coli Using the W3110 genome as a template, primers U- were used respectively. yncI -S / U- yncI -A and D- yncI -S / D- yncI -A amplification yncI The upstream and downstream homologous arms; respectively with C. glutamicum ATCC13032 genome, artificially synthesized ilvBN M1 and ilvBN M2 Using primers as templates ilvBN -S and ilvBN -A amplification includes P trc promoter, T trc Termination and ilvBN , ilvBN M1 or ilvBN M2 gene segment P trc - ilvBN -T trc P trc - ilvBN M1 -T trc and P trc - ilvBN M2 -T trc (P) trc Promoters and T trc The terminator has been designed into the primer. ilvBN -S and ilvBN -A (in the middle). After PCR product recovery, primer U- yncI -S / D- yncI -A was obtained via overlap PCR and contained yncI Upstream and downstream homologous arms and P trc - ilvBN -T trc P trc - ilvBN M1 -T trc or P trc - ilvBN M2-T trc fusion fragment U yncI -P trc - ilvBN -T trc -D yncI U yncI -P trc - ilvBN M1 -T trc -D yncI and U yncI -P trc - ilvBN M2 -T trc -D yncI .

[0055] (2) pGRB- yncI plasmid construction according to yncI Sequence design and synthesis of 20 bp forward and reverse sequences pGRB- of gRNA. yncI -S and pGRB- yncI -A, The recombinant plasmid pGRB- was constructed using the same method as step (2) in Example 3. yncI .

[0056] (3) Construction of L-valine engineered strains VAL-CK, VAL-1-M1 and VAL-1-M2 The recombinant plasmid pGRB- yncI and fusion fragment U yncI -P trc - ilvBN -T trc -D yncI Transformed into plasmid containing pREDcas9 E. coli W3110 lacI In competent cells, using primer U- yncI -S / D- yncI -A identifies positive transformants. The plasmid was then lost using the same method as step (3) in Example 3 to obtain strain VAL-CK.

[0057] The recombinant plasmid pGRB- yncI and fusion fragment U yncI -P trc - ilvBN M1 -T trc -D yncI Transformed into plasmids containing pREDcas9 E. coli W3110 wideI In competent cells, using primer U- yncI -S / D- yncI -A identifies positive transformants. The plasmid was then lost using the same method as step (3) in Example 3 to obtain strain VAL-1-M1.

[0058] The recombinant plasmid pGRB- was processed using the same method. yncI and fusion fragment U yncI -P trc - ilvBN M2 -T trc -D yncI Transformed into plasmids containing pREDcas9 E. coli W3110 wideI strain VAL-1-M2 was obtained from competent cells.

[0059] Example 5: ilvIH M Construction of overexpression strain VAL-2 (1) Overlapping segment U yncI -P trc - ilvIH M -T trc -D yncI Construction With Escherichia coli E. coli Using the W3110 genome as a template, primers U- were used respectively. yncI -S / U- yncI -A and D- yncI -S / D- yncI -A amplification yncI The upstream and downstream homologous arms; artificially synthesized ilvIH M Using the fragment as a template, primers were used. ilvIH M -S and ilvIH M -A amplification includes P trc promoter, T trc Termination and ilvIH M gene segment P trc - ilvIH M -T trc After PCR product recovery, primer U- yncI -S / D- yncI -A was obtained via overlap PCR and contained yncI Upstream and downstream homologous arms, P trc - ilvIH M -T trc fusion fragment UyncI -P trc - ilvIH M -T trc -D yncI .

[0060] (2) pGRB- yncI plasmid construction according to yncI Sequence design and synthesis of 20 bp forward and reverse sequences pGRB- of gRNA. yncI -S and pGRB- yncI -A, The recombinant plasmid pGRB- was constructed using the same method as step (2) in Example 3. yncI .

[0061] (3) Construction of L-valine engineered strain VAL-2 The recombinant plasmid pGRB- yncI and fusion fragment U yncI -P trc - ilvIH M -T trc -D yncI Transformed into plasmids containing pREDcas9 E. coli W3110 wideI In competent cells, using primer U- yncI -S / D- yncI -A identifies positive transformants. The plasmid was then lost using the same method as step (3) in Example 3 to obtain strain VAL-2.

[0062] Example 6: Shake-flask fermentation of L-valine strains (1) Seed culture The strains were respectively E. coli W3110 wideI VAL-CK, VAL-1-M1, VAL-1-M2, and VAL-2 were inoculated onto LB solid medium slant and cultured at 37°C for 12 h. Then, they were inoculated into 30 mL of seed medium and cultured at 37°C with shaking at 220 rpm for 6-8 h.

[0063] (2) Shake flask fermentation Inoculate the fermentation medium with a 1% inoculum and incubate at 37°C. Shake at 220 rpm for 12 h, then incubate statically for another 12 h. During fermentation, supplement with 60% glucose 2-3 times to maintain a residual sugar concentration of 0.1-0.5 g / L, adding 1 mL each time. Adjust the pH to approximately 7.0 with ammonia.

[0064] (3) Detection of L-valine in fermentation broth After centrifuging the fermentation broth at 8000 × g for 10 min, the supernatant was collected and diluted 10 times with deionized water. The fermentation broth was then derivatized using 0.8% (v / v) 2,4-dinitrofluorobenzene, and the L-valine content was determined by high-performance liquid chromatography (HPLC). The L-valine yield of the strain is shown in Table 4 below.

[0065] Table 4 Expression ilvBN、ilvBN M and ilvIH M Effects on L-valine synthesis

[0066] (4) Culture medium The seed culture medium consisted of: 20 g / L glucose, 10 g / L yeast extract, 6 g / L peptone, 1.2 g / L KH2PO4, 1 g / L MgSO4·7H2O, 4 mg / L FeSO4·7H2O, 4 mg / L MnSO4·7H2O, 20 mL / L phenol red, with the remainder being water. The pH was 7.0-7.2, and the medium was autoclaved at 115℃ for 15 min.

[0067] The fermentation medium consisted of: 20 g / L glucose, 2 g / L yeast extract, 2 g / L peptone, 1 g / L KH₂PO₄, 1.2 g / L MgSO₄·7H₂O, 15 mg / L FeSO₄·7H₂O, 12 mg / L MnSO₄·7H₂O, and 0.8 mg / L MgSO₄·7H₂O. B1 0.2 mg / LV H 20 mL / L phenol red, the remainder being water, pH 7.0-7.2, autoclaved at 115℃ for 15 min.

[0068] The results showed that VAL-1-M1 had a slightly higher L-valine content, so VAL-1-M1 was used as the chassis for further modification.

[0069] Example 7: Construction of leucine dehydrogenase knockout strain Depend on ilvE The encoded branched-chain amino acid aminotransferase catalyzes the transamination of 3-methyl-2-oxobutyrate to L-valine. This reaction uses L-glutamate as the amino donor, and since L-glutamate synthesis requires NADPH, this reaction indirectly consumes NADPH. Leucine dehydrogenase can also catalyze the production of L-valine from 3-methyl-2-oxobutyrate, using NH3 as the amino donor. In *E. coli*, ilvE and ilvD(Dihydroxy acid dehydratase) forms the operon, and both are involved in L-valine synthesis. In this example, the knockout enzyme is first... ilvE Then, the gene encoding leucine dehydrogenase derived from Bacillus subtilis was overexpressed. bcd .

[0070] (1) VAL-1-M1 ilvE Knockout Using the same method as step (1) in Example 3, with Escherichia coli E. coli Using the W3110 genome as a template, primers U- were used respectively. ilvE -S / U- ilvE -A and D- ilvE -S / D- ilvE -A was obtained via PCR and overlap PCR. ilvE -D ilvE Using the same method as step (2) in Example 3, primer pGRB- ilvE -S / pGRB- ilvE -A Construct recombinant plasmid pGRB- ilvE Using the same method as step (3) in Example 3, pGRB- ilvE with U ilvE -D ilvE The cells were transformed into VAL-1-M1 containing the pREDcas9 plasmid. Following the same procedure as step (3) in Example 3, the cells were screened, identified, and plasmids were removed to obtain... ilvE Knockout strain VAL-3.

[0071] (2) bcd Construction of overexpression strains Using the same method as step (1) in Example 4, with Escherichia coli E. coli Using the W3110 genome as a template, primers U- were used respectively. ycgh -S / U- ycgh -A、D- ycgh -S / D- ycgh -A amplification ycgh Upstream and downstream homologous arms; artificially synthesized bcd Using the fragment as a template, primers were used. bcd- S / bcd- Amplification bcd U was obtained by overlap PCR ycgH -P trc - bcd -T trc -D ycgH Using the same method as step (2) in Example 4, primer pGRB- ycgh -S / pGRB- ycgh -A Construct recombinant plasmid pGRB- ycgh Using the same method as step (3) in Example 4, pGRB- ycgh with U ycgH -P trc - bcd -T trc -D ycgH Transformed into VAL-3 containing the pREDcas9 plasmid. After screening, identification, and plasmid removal in the same manner as step (3) of Example 4, strain VAL-4 was obtained.

[0072] (4) VAL-4 fermentation performance test Shake-flask fermentation was performed using the same method as in Example 6, with VAL-1-M1 as a control. The results showed that knocking out... ilvE and overexpression bcd This resulted in a 21.1% increase in L-valine levels.

[0073] Table 5 Overexpression bcd Effects on L-valine synthesis

[0074] Example 8: ilvC and ilvC M Construction of overexpression strains and testing of their fermentation performance wild type ilvC The encoded NADH-dependent keto-alcohol reductase is an NADPH-dependent enzyme. Since microbial intracellular NADPH concentrations are low, overexpression of the NADH-dependent keto-alcohol reductase encoding gene is recommended. ilvC M (S34G, L48E, R49F) may benefit L-valine synthesis. Comparison with overexpression of wild-type... ilvC and ilvC M The effect on L-valine production.

[0075] (1) ilvC and ilvC M Construction of overexpression strains Using the same method as step (1) in Example 4, with Escherichia coli E. coli Using the W3110 genome as a template, primers U- were used respectively. yesL -S / U- yesL -A、D- yesL -S / D- yesL -A amplification yeel Upstream and downstream homologous arms; artificially synthesized ilvC M Fragments and E. coli E. coli Using the W3110 genome as a template, primers were used... ilvC M -S / ilvC M - Amplification P trc - ilvC M -T trc Using primers ilvC -S / ilvC- Amplification P trc - ilvC -T trc Then, U was obtained through overlap PCR. yeeL -P trc - ilvC M -T trc -D yeeL and U yeeL -P trc - ilvC -T trc -D yeeL Using the same method as step (2) in Example 4, primer pGRB- yesL -S / pGRB- yesL -A Construct recombinant plasmid pGRB- yesL Using the same method as step (3) in Example 4, pGRB- yesL with U yeeL -P trc - ilvC M -T trc -D yeeL or U yeeL -P trc - ilvC -T trc -D yeeL Transformed into VAL-4 containing the pREDcas9 plasmid. After screening, identification, and plasmid removal in the same manner as step (3) of Example 4, strains VAL-5 and VAL-6 were obtained.

[0076] (3) Fermentation performance test of VAL-5 and VAL-6 Shake-flask fermentation was performed using the same method as in Example 6, with VAL-4 as a control. The results showed that overexpression... ilvC and ilvC M Both can increase L-valine production, but ilvC M The effect was better, with an improvement of 32.8% compared to the control.

[0077] Table 6 Fermentation parameters of L-valine engineered strains

[0078] Example 9: Knockout frdB , pflB , ldhA , adhe , poxB , ackA and yfbQ Construction of knockout strains and testing of their fermentation performance frdB , pflB , ldhA ... poxB , ackA and yfbQ The encoded fumarate reductase, pyruvate-formate lyase, lactate dehydrogenase, alcohol dehydrogenase, pyruvate dehydrogenase, and acetate kinase directly or indirectly catalyze the synthesis of succinate, formic acid, lactate, ethanol, acetic acid (both pyruvate dehydrogenase and acetate kinase can catalyze the synthesis of acetic acid), and L-alanine from pyruvate. Pyruvate is a precursor for L-valine synthesis; therefore, knocking out these genes is expected to increase L-valine production.

[0079] (1) frdB , pflB , ldhA , adhe , poxB , ackA and yfbQ Construction of single knockout strains Using the same method as step (1) in Example 3, wild-type Escherichia coli E. coli Using the W3110 genome as a template, primers U- were used respectively. frdB -S / U- frdB -A、D- frdB -S / D- frdB -A, U- pflB -S / U- pflB -A、D- pflB -S / D- pflB -A, U- ldhA -S / U- ldhA -A、D- ldhA -S / D- ldhA -A, U- adhe -S / U- adhe -A、D- adhe -S / D- adhe -A U- poxB -S / U- poxB -A、D- poxB -S / D- poxB -A、U- ackA -S / U- ackA -A、D- ackA -S / D- ackA -A and U- yfbQ -S / U- yfbQ -A、D- yfbQ -S / D- yfbQ -A fusion fragment U was obtained via PCR and overlap PCR. frdB -D frdB U flB -D flB U ldhA -D ldhA U adhE -D adhE U poxB -D poxB U ackA -D ackA and U yfbQ -D yfbQ .

[0080] Using the same method as step (2) in Example 3, using primer pGRB- frdB -S / pGRB- frdB -A、pGRB- pflB -S / pGRB- pflB -A、pGRB- ldhA -S / pGRB- ldhA -A、pGRB- adhe -S / pGRB- adhe -A、pGRB- poxB -S / pGRB- poxB -A、pGRB- ackA -S / pGRB- ackA -A and pGRB- yfbQ -S / pGRB- yfbQ -A Construct recombinant plasmid pGRB- frdB pGRB- pflB pGRB- ldhA pGRB- adhe pGRB- poxB pGRB- ackA and pGRB- yfbQ .

[0081] Using the same method as step (3) of Example 3, the above-mentioned pGRB plasmid and the corresponding fusion fragment were transformed into VAL-5 containing the pREDcas9 plasmid. After screening, identification, and plasmid removal using the same method as step (3) of Example 3, the pGRB plasmid was obtained. frdB , pflB , ldhA , adhe , poxB , ackA and yfbQSingle knockout strains VAL-7, VAL-8, VAL-9, VAL-10, VAL-11, VAL-12 and VAL-13.

[0082] Knocking out some or all of the above genes in the VAL-5 combination yields VAL-14 (VAL-5Δ) frdB Δ pflB Δ ldhA Δ adhe VAL-15 (VAL-5Δ) frdB Δ pflB Δ ldhA Δ adhe Δ poxB VAL-16 (VAL-5Δ) frdB Δ pflB Δ ldhA Δ adhe Δ poxB Δ ackA VAL-17 (VAL-5Δ) frdB Δ pflB Δ ldhA Δ adhe Δ poxB Δ ackA Δ yfbQ ).

[0083] (2) frdB , pflB , ldhA , adhe , poxB , ackA and yfbQ Fermentation performance test of knockout strains Shake-flask fermentation was performed using the same method as in Example 6, with VAL-5 as a control. The results showed that knocking out... frdB , pflB , ldhA , adhe , poxB , ackA and yfbQ All of them can increase L-valine production, and significantly reduce the corresponding byproducts. After combined knockout, the increase in L-valine production is more significant, and the corresponding byproducts are significantly reduced.

[0084] Table 7. L-valine yield and byproduct concentration of strains

[0085] Example 10: pntAB Construction of overexpression strains and testing of their fermentation performance The synthesis of one molecule of L-valine requires two molecules of NADPH, which is derived from... pntABThe encoded pyridine nucleotide transhydrogenase catalyzes the conversion of NADH to NADPH. It utilizes its own promoter P... pntAB and P trc Overexpression of this gene enhances NADPH supply.

[0086] (1) pntAB Construction of overexpression strains Using the same method as step (1) in Example 4, with Escherichia coli E. coli Using the W3110 genome as a template, primers U- were used respectively. yjgX -S / U- yjgX -A and D- yjgX -S / D- yjgX -A amplification yjgX Upstream and downstream homologous arms, respectively utilizing P trc - pntAB -S / P trc - pntAB- A and P pntAB - pntAB -S / P pntAB - pntAB- Amplification P trc - pntAB -T trc and P pntAB - pntAB -T pntAB The fusion fragment U was obtained by overlap PCR. yjgX -P trc - pntAB -T trc -D yjgX and U yjgX -P pntAB - pntAB -T pntAB -D yjgX Using the same method as step (2) in Example 4, primer pGRB- yjgX -S / pGRB- yjgX -A Construct recombinant plasmid pGRB- yjgX Using the same method as step (3) in Example 4, pGRB- yjgX with U yjgX -P trc - pntAB -T trc -D yjgX or U yjgX -P pntAB - pntAB -T pntAB -D yjgXTransformed into VAL-5 containing the pREDcas9 plasmid. After screening, identification and plasmid removal in the same manner as step (3) of Example 4, strains VAL-18 and VAL-19 were obtained.

[0087] (2) pntAB Fermentation performance test of overexpression strains Shake-flask fermentation was performed using the same method as in Example 6, with VAL-5 as a control. The results showed that... pntAB Overexpression strains all increased L-valine production and intracellular NADPH concentration, utilizing their own promoters for expression. pntAB The VAL-19 has better performance.

[0088] Table 8 Overexpression pntAB Effects on L-valine production and NADPH concentration

[0089] Example 11: Construction of ED pathway enhanced strains and testing of their fermentation performance Glucose can be converted into pyruvate via the EMP and ED pathways. The former generates 2 molecules of NADH, while the latter generates 1 molecule of NADPH and 1 molecule of NADH. Therefore, strengthening ED can increase the supply of NADPH, thereby promoting the synthesis of L-valine. zwf (Encoding glucose-6-phosphate dehydrogenase) edd (Encoding 6-phosphoglucate dehydratase) and sister It is a key enzyme in the ED pathway, and zwf - edd - sister Construct an operator. Utilize P trc Overexpression of this operon can enhance the ED pathway.

[0090] (1) Construction of ED pathway enhanced strains Using the same method as step (1) in Example 4, with Escherichia coli E. coli Using the W3110 genome as a template, primers U- were used respectively. gapC -S / U- gapC -A、D- gapC -S / D- gapC -A and ED-S / ED-A amplification gapC Upstream and downstream homologous arms and P trc - zwf - edd - sister -T trc The fusion fragment U was obtained by overlap PCR. gapC -P trc - zwf - edd - sister -Ttrc -D gapC Using the same method as step (2) in Example 4, primer pGRB- gapC -S / pGRB- gapC -A Construct recombinant plasmid pGRB- gapC Using the same method as step (3) in Example 4, pGRB- gapC with U gapC -P trc - zwf - edd - sister -T trc -D gapC Transformed into VAL-5 containing the pREDcas9 plasmid. After screening, identification, and plasmid removal using the same method as step (3) in Example 4, strain VAL-20 was obtained.

[0091] (2) Fermentation performance test of strains enhanced by ED pathway Shake-flask fermentation was performed using the same method as in Example 6, with VAL-5 as a control. The results showed that enhancing the ED pathway significantly increased L-valine production and intracellular NADPH concentration.

[0092] Table 9. Effects of ED pathway enhancement on L-valine production

[0093] Example 14: pps Construction of knockout strains and testing of their fermentation performance Depend on pps The encoded phosphoenolpyruvate synthase catalyzes the conversion of pyruvate to phosphoenolpyruvate. Therefore, knocking out this gene strongly reduces pyruvate consumption, providing more pyruvate for L-valine synthesis.

[0094] (1) pps Construction of knockout strains Using the same method as step (1) in Example 3, wild-type Escherichia coli E. coli Using the W3110 genome as a template, primers U- were used respectively. pps -S / U- pps -A and D- pps -S / D- pps -A fusion fragment U was obtained via PCR and overlap PCR. pps -D pps Using the same method as step (2) of Example 3, using primer pGRB- pps -S / pGRB- pps -A Construct recombinant plasmid pGRB- ppsUsing the same method as step (3) of Example 3, the above pGRB plasmid and U... pps -D pps The cells were transformed into VAL-5 containing the pREDcas9 plasmid. Following the same procedure as step (3) in Example 3, the cells were screened, identified, and plasmids were removed to obtain... pps Knockout strain VAL-21.

[0095] (2) pps Fermentation performance test of knockout strains Shake-flask fermentation was performed using the same method as in Example 6, with VAL-5 as a control. The results showed that... pps The knockout strain produced significantly more L-valine than the control strain.

[0096] Table 10 pps Effect of knockout on L-valine production

[0097] Example 13: Construction of a strain overexpressing the pyruvate kinase encoding gene and its fermentation performance test Pyruvate kinase catalyzes the synthesis of pyruvate from phosphoenolpyruvate; therefore, enhancing this reaction favors pyruvate synthesis, thus providing more pyruvate for L-valine synthesis. *E. coli* contains two genes encoding pyruvate kinase (…). pykA and pykF ), using P trc These two genes are expressed separately.

[0098] (1) Construction of strains overexpressing pyruvate kinase encoding gene Using the same method as step (1) in Example 4, with Escherichia coli E. coli Using the W3110 genome as a template, primers U- were used respectively. yjiT -S / U- yjiT -A、D- yjiT -S / D- yjiT -A amplification yjiT Upstream and downstream homologous arms; respectively utilizing pykA -S / pykA -A and pykF -S / pykF -A amplification P trc - pykA -T trc and P trc - pykF -T trc The fusion fragment U was obtained by overlap PCR. yjiT -P trc - pykA -T trc -D yjiT and UyjiT -P trc - pykF -T trc -D yjiT Using the same method as step (2) in Example 4, primer pGRB- yjiT -S / pGRB- yjiT -A Construct recombinant plasmid pGRB- yjiT Using the same method as step (3) in Example 4, pGRB- yjiT with U yjiT -P trc - pykA -T trc -D yjiT or U yjiT -P trc - pykF -T trc -D yjiT Transformed into VAL-5 containing the pREDcas9 plasmid. After screening, identification and plasmid removal in the same manner as step (3) of Example 4, strains VAL-22 and VAL-23 were obtained.

[0099] (3) Fermentation performance test of strains overexpressing pyruvate kinase encoding gene Shake-flask fermentation was performed using the same method as in Example 6, with VAL-5 as a control. The results showed that the L-valine production of the strains overexpressing the pyruvate kinase encoding gene was significantly higher than that of the control strains. pykF It works even better.

[0100] Table 11 Effects of overexpression of the pyruvate kinase encoding gene on L-valine production

[0101] Example 14: avtA Construction of knockout strains and testing of their fermentation performance L-valine can be converted to L-alanine via valine-alanine aminotransferase. Knockout avtA (Encoding valine-alanine aminotransferase) to reduce L-valine consumption.

[0102] (1) avtA Construction of knockout strains Using the same method as step (1) in Example 3, wild-type Escherichia coli E. coli Using the W3110 genome as a template, primers U- were used respectively. avtA -S / U- avtA -A、D- avtA -S / D- avtA -A, fusion fragment U was obtained via PCR and overlap PCR. avtA-D avtA Using the same method as step (2) of Example 3, using primer pGRB- avtA -S / pGRB- avtA -A Construct recombinant plasmid pGRB- avtA Using the same method as step (3) in Example 3, pGRB- avtA plasmids and U avtA -D avtA The cells were transformed into VAL-5 containing the pREDcas9 plasmid. Following the same procedure as step (3) in Example 3, the cells were screened, identified, and plasmids were removed to obtain... avtA Construction of the knockout strain VAL-24.

[0103] (2) avtA Fermentation performance test of knockout strains Shake-flask fermentation was performed using the same method as in Example 6, with VAL-5 as a control. Results showed... avtA The L-valine production of the knockout strain VAL-24 was significantly higher than that of the control strain, and the combined knockout effect was even better.

[0104] Table 12 Effects of L-valine competition or degradation pathway knockout on L-valine yield

[0105] Example 15: Construction of a glucose uptake-enhancing strain and its fermentation performance test Escherichia coli possesses two glucose uptake systems: a phosphotransferase-dependent system and a non-phosphotransferase-dependent system. The phosphotransferase-dependent system transports glucose while phosphorylating it to glucose-6-phosphate, which then directly enters the EMP pathway. In contrast, the non-phosphotransferase-dependent system (which is...) galP The gene encodes D-galactose transporter, which first transfers glucose into the cell, and then glucose is transported by glucokinase (which...). glk (Gene-encoded) phosphorylation to glucose-6-phosphate. Because galP Gene repression means that only the phosphotransferase-dependent system functions. galP Replace the promoter with P trc To remove its repressive effect and integrate a copy of P trc - glk This is to enhance glucose intake and increase pyruvate synthesis.

[0106] (1) Construction of glucose uptake enhanced strains Using the same method as step (1) in Example 4, with Escherichia coli E. coli Using the W3110 genome as a template, primers U- were used respectively. galP -S / U- galP -A、D- galP -S / D- galP -A amplification galP Upstream and downstream homologous arms were used to obtain the fusion fragment U via overlap PCR. galP -P trc -D galP Using primer U- yesP -S / U- yesP -A、D- yesP -S / D- yesP -A and glk-S / glk-A amplification yesP Upstream and downstream homologous arms and P trc - glk -T trc The fusion fragment U was obtained by overlap PCR. yeeP -P trc - glk -T trc -D yeeP Using the same method as step (2) in Example 4, primer pGRB- galP -S / pGRB- galP -A and pGRB- yesP -S / pGRB- yesP -A Construct recombinant plasmid pGRB- galP and pGRB- yesP Using the same method as step (3) in Example 4, pGRB- galP with U galP -P trc -D galP pGRB- yesP with U yeeP -P trc - glk -T trc -D yeeP Transformed into VAL-5 containing the pREDcas9 plasmid. Following the same method as step (3) in Example 4, strains VAL-25 and VAL-26 were obtained through screening, identification, and plasmid removal. pGRB- yesP with U yeeP -P trc - glk -T trc -D yeeP The cells were transformed into VAL-25 containing the pREDcas9 plasmid to obtain VAL-27.

[0107] (2) Fermentation performance test of the constructed glucose uptake enhanced strain Shake-flask fermentation was performed using the same method as in Example 6, with VAL-5 as a control. The results showed that the glucose-enhanced strain exhibited significantly increased L-valine production and overexpression. galP and glk Even better results.

[0108] Table 13 Effects of enhanced glucose intake on L-valine production

[0109] Example 16: Construction of a strain overexpressing the glyceraldehyde-3-phosphate dehydrogenase gene and its fermentation performance test 3-Glyceraldehyde-3-phosphate dehydrogenase is a key enzyme in the EMP pathway, and overexpression of the gene encoding this enzyme can enhance pyruvate synthesis. These genes include those from *E. coli* g... apA Clostridium acetone-butanol g apC Streptococcus pyogenes g apN and canine streptococcus g apN Sc .

[0110] (1) Construction of strains overexpressing the gene encoding glyceraldehyde-3-phosphate dehydrogenase Using the same method as step (1) in Example 4, with Escherichia coli E. coli Using the W3110 genome as a template, primers U- were used respectively. yciQ -S / U- yciQ -A、D- yciQ -S / D- yciQ -A amplification yciQ Upstream and downstream homologous arms were used to amplify P using primers gapA-S / gapA-A. trc - gapA -T trc . respectively using artificially synthesized g apC g apN and g apN Sc Using gapC-S / gapC-A, gapN-S / gapN-A, and gapNsc-S / gapNsc-A as templates, P was amplified using primers gapC-S / gapC-A, gapN-S / gapNsc-A, and gapNsc-S / gapNsc-A, respectively. trc - gapC -T trc P trc - gapN -T trc and P trc -g apN Sc -T trc U was obtained by overlap PCR. yicQ -P trc - gapA -T trc -D yciQ U yicQ -P trc - gapC -T trc-D yciQ U yicQ -P trc - gapN -T trc -D yciQ and U yicQ -P trc - gapN Sc -T trc -D yciQ Using the same method as step (2) in Example 4, primer pGRB- yciQ -S / pGRB- yciQ -A Construct recombinant plasmid pGRB- yciQ Using the same method as step (3) in Example 4, pGRB- yciQ with U yicQ -P trc - gapA -T trc -D yciQ U yicQ -P trc - gapC -T trc -D yciQ U yicQ -P trc - gapN -T trc -D yciQ or U yicQ -P trc - gapN Sc -T trc -D yciQ Transformed into VAL-5 containing the pREDcas9 plasmid. After screening, identification and plasmid removal in the same manner as step (3) of Example 4, strains VAL-28, VAL-29, VAL-30 and VAL-31 were obtained.

[0111] (2) Fermentation performance test of strains overexpressing the glyceraldehyde-3-phosphate dehydrogenase encoding gene Shake-flask fermentation was performed using the same method as in Example 6, with VAL-5 as a control. The results showed that overexpression of the gene encoding glyceraldehyde-3-phosphate dehydrogenase increased L-valine yield, g. apN and g apN Sc Even better results.

[0112] Table 14 Effects of overexpression of the gene encoding glyceraldehyde-3-phosphate dehydrogenase on L-valine production

[0113] Example 17: Construction of L-valine export-enhancing strain and its fermentation performance test brnEF This is the gene encoding the L-valine export protein from Corynebacterium glutamicum. ygaZH This is a gene encoding a branched-chain amino acid export protein derived from *E. coli*. One copy of P is integrated into each gene. trc - brnEF and P trc - ygaZH To enhance L-valine output.

[0114] (1) Construction of L-valine export enhancement strains Using the same method as step (1) in Example 4, with Escherichia coli E. coli Using the W3110 genome as a template, primers U- were used respectively. rph -S / U- rph -A and D- rph -S / D- rph -A amplification obtained rph Upstream and downstream homologous arms, using primers ygaZH -S / ygaZH -A amplification yields P trc - ygaZH -T trc Corynebacterium glutamicum Corynebacterium glutamicum Using the genome as a template, primers brnEF- S / brnEF -A amplification yields P trc - brnEF -T trc U was obtained by overlap PCR. rph -P trc - ygaZH -T trc -D rph and U rph -P trc - brnEF -T trc -D rph Using the same method as step (2) in Example 4, primer pGRB- rph -S / pGRB- rph -A Construct recombinant plasmid pGRB- rph Using the same method as step (3) in Example 4, pGRB- rph with U rph -P trc - ygaZH -T trc -D rph U rph -P trc - brnEF-T trc -D rph Transformed into VAL-5 containing the pREDcas9 plasmid. After screening, identification and plasmid removal in the same manner as step (3) of Example 4, strains VAL-32 and VAL-33 were obtained.

[0115] (2) Fermentation performance test of L-valine export-enhancing strains Shake-flask fermentation was performed using the same method as in Example 6, with VAL-5 as a control. The results showed that the L-valine-exporting strain significantly increased L-valine production. brnFE Even better results.

[0116] Table 15. Effect of enhanced L-valine output on its yield

[0117] Example 18: Construction of L-valine uptake gene knockout strain and its fermentation performance test LivJ, BrnQ, and YhjE are responsible for L-valine uptake; knocking out their coding genes colorJ , brnQ and yhjE To reduce L-valine loss.

[0118] (1) Construction of L-valine uptake gene knockout strain Using the same method as step (1) in Example 3, wild-type Escherichia coli E. coli Using the W3110 genome as a template, primers U- were used respectively. colorJ -S / U- colorJ -A、D- colorJ -S / D- colorJ -A, U- brnQ -S / U- brnQ -A、D- brnQ -S / D- brnQ -A, U- yhjE -S / U- yhjE -A、D- yhjE -S / D- yhjE -A, fusion fragment U was obtained via PCR and overlap PCR. livJ -D livJ U brnQ -D brnQ and U yhjE -D yhjE Using the same method as step (2) of Example 3, using primer pGRB- colorJ -S / pGRB- colorJ -A、pGRB- brnQ -S / pGRB- brnQ -A and pGRB- yhjE -S / pGRB- yhjE -A Construct recombinant plasmid pGRB- livJ、 pGRB- brnQ and pGRB- yhjE Using the same method as step (3) of Example 3, the above pGRB plasmid and U... livJ -D livJ U brnQ -D brnQ and U yhjE -D yhjE Transformed into VAL-5 containing the pREDcas9 plasmid. After screening, identification and plasmid removal in the same manner as step (3) of Example 3, L-valine competition or degradation pathway knockout strains VAL-34, VAL-35 and VAL-36 were obtained.

[0119] Knocking out the above genes in the VAL-5 combination yielded VAL-37 (VAL-5Δ) colorJ Δ brnQ VAL-38 (VAL-5Δ) colorJ Δ yhjE VAL-39 (VAL-5Δ) brnQ Δ yhjE ) and VAL-40 (VAL-5Δ colorJ Δ brnQ Δ yhjE ).

[0120] (2) Fermentation performance test of L-valine uptake gene knockout strain Shake-flask fermentation was performed using the same method as in Example 6, with VAL-5 as a control. The results showed that the L-valine uptake gene knockout strain produced significantly higher L-valine yields than the control strain. colorJ and brnQ The effect is better than yhjE Combined knockout is superior to single knockout; simultaneous knockout... colorJ , brnQ and yhjE Knocking the fruit is the best way to prepare it.

[0121] Table 16. Effects of knocking out the L-valine uptake gene on its yield.

[0122] Example 19: Construction of L-valine synthase strain and its fermentation performance test Using VAL-5 as the starting strain, the strategies in Examples 5-17 were integrated to obtain strain VAL-41 ( E. coli W3110 lacIyncI::P trc - ilvBN M1 -T trc Δ ilvE ycgh ::P trc - bcd -T trc yesL ::P trc - ilvC M -T trc Δ frdB Δ pflB Δ ldhA Δ adhe Δ poxB Δ ackA Δ yfbQyjgX ::P pntAB - pntAB -T trc gapC ::P trc - zwf- edges -T trc Δ ppsyjiT ::P trc - pykF -T trc Δ avtA P galP ::P trc yeeP ::P trc - glK -T trc yciQ ::P trc - gapN -T trc rph ::P trc - brnEF -T trc Δ livJ Δ brnQ Δ yhjE Using the method described in Example 6, the L-valine yield reached 41.3 g / L, which is 3.8 times higher than that of VAL-5 (8.6 g / L).

[0123] Example 20: Fermentation of L-valine engineered bacteria in a 5 L fermenter (1) Seed culture Using an inoculation loop, 3-5 tubes of L-valine engineered bacteria VAL-5 and VAL-41, activated with fresh LB slant medium, were inoculated into a 5 L fermenter containing 2.5 L of seed medium. 25% ammonia was added to adjust the pH of the fermentation broth to 6.8-7.2, dissolved oxygen was maintained at 20%, and aeration was maintained at 2-4 m³ / h. 3 / h, stirring speed 200-800 rpm, incubate at 37℃ for 6 h.

[0124] (2) Fermentation culture The seed culture from step (1) was inoculated at a 10% inoculum into a 5 L fermenter containing 3 L of fermentation medium for fermentation at 35℃. Aeration was maintained at 2-4 m³ / h before fermentation. 3 / h, stirring speed 300-900 rpm, dissolved oxygen maintained at 30-40%; after 12 h, ventilation rate 0.02 m 3 The stirring speed is 300-400 rpm, and the dissolved oxygen is maintained at 10-15%. During fermentation, 80% (w / v) glucose solution is added to maintain the residual sugar concentration at 0.1-0.5 g / L, and 25% ammonia water is added to adjust the pH of the fermentation broth to 6.8-7.2.

[0125] (3) Detection of L-valine in fermentation broth After 26 h of fermentation, the fermentation broth was centrifuged at 8000 × g for 10 min, and the supernatant was collected and diluted 10 times with deionized water. The fermentation broth was then derivatized using 0.8% (V / V) 2,4-dinitrofluorobenzene, and the contents of L-valine, alanine, and organic acids were determined by high performance liquid chromatography. The fermentation parameters are shown in Table 17 below.

[0126] Table 17 Fermentation parameters of L-valine engineered strains

[0127] After 26 hours of fermentation, the genetically engineered strain VAL-41 produced 109.3 g / L of L-valine, with a conversion rate of 52.7%, which were 5.58 and 2.63 times higher than those of VAL-5, respectively.

[0128] (4) Culture medium The seed culture medium consisted of: 30 g / L glucose, 5 g / L yeast extract, 2 g / L peptone, 2 g / L KH₂PO₄, 1.5 g / L MgSO₄·7H₂O, 10 mg / L FeSO₄·7H₂O, 10 mg / L MnSO₄·7H₂O, and 1.3 mg / L MgSO₄·7H₂O. B1 0.3 mg / LV H 8 mL / L corn steep liquor, 2 g / L isoleucine, the remainder being water, pH 7.0-7.2, autoclaved at 115℃ for 15 min.

[0129] The fermentation medium consisted of: 10 g / L glucose, 3 g / L yeast extract, 1 g / L glutamic acid, 3 g / L KH2PO4, 1.8 g / L MgSO4·7H2O, 2 g / L sodium citrate dihydrate, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4·7H2O, and 2 mg / L LnSO4·7H2O. B1 0.2 mg / LV H 8 mL / L corn steep liquor, the remainder being water, pH 7.0-7.2, autoclaved at 115℃ for 15 min.

[0130] Example 21: Fermentation of L-valine engineered bacteria VAL-41 in a 5L fermenter (1) Seed culture Same as step (1) in Example 20. (2) Fermentation culture The seed culture from step (1) was inoculated at a 15% inoculum into a 5 L fermenter containing 3 L of fermentation medium for fermentation culture at 35℃. Aeration was maintained at 2-3 m³ / h before 12 hours. 3 / h, stirring speed 300-800 rpm, dissolved oxygen maintained at 20-30%; after 12 h, ventilation rate 0.01 m 3 The stirring speed is 100-200 rpm, and the dissolved oxygen is maintained at 0-5%. During fermentation, 80% (w / v) glucose solution is added to maintain the residual sugar concentration at 0.1-0.5 g / L, and 25% ammonia water is added to adjust the pH of the fermentation broth to 6.8-7.2.

[0131] (3) Detection of L-valine in fermentation broth Same as step (3) in Example 20. After 26 h of fermentation, the L-valine engineered bacteria VAL-41 produced 121.3 g / L of L-valine, with a conversion rate of 53.6%.

[0132] (4) Culture medium The seed culture medium consisted of: 30 g / L glucose, 5 g / L yeast extract, 2 g / L peptone, 2 g / L KH₂PO₄, 1.5 g / L MgSO₄·7H₂O, 12 mg / L FeSO₄·7H₂O, 10 mg / L MnSO₄·7H₂O, and 1.5 mg / L MgSO₄·7H₂O. B1 0.5 mg / LV H 8 mL / L corn steep liquor, 2 g / L isoleucine, the remainder being water, pH 7.0-7.2, autoclaved at 115℃ for 15 min.

[0133] The fermentation medium consisted of: 10 g / L glucose, 3 g / L yeast extract, 2 g / L glutamic acid, 3 g / L KH2PO4, 1.8 g / L MgSO4·7H2O, 2 g / L sodium citrate dihydrate, 10 mg / L FeSO4·7H2O, 15 mg / L MnSO4·7H2O, and 2 mg / L L... B1 0.2 mg / LV H 10 mL / L corn steep liquor, the remainder being water, pH 7.0-7.2, autoclaved at 115℃ for 15 min.

[0134] Example 22: Fermentation of L-valine engineered bacteria VAL-41 in a 5L fermenter (1) Seed culture Same as step (1) in Example 20. (2) Fermentation culture The seed culture from step (1) was inoculated at a 20% inoculum into a 5 L fermenter containing 3 L of fermentation medium for fermentation at 37℃. Aeration was maintained at 3-4 m³ / h for 12 hours prior to fermentation. 3 / h, stirring speed 300-900 rpm, dissolved oxygen maintained at 40-50%; after 12 h, ventilation rate 0.01 m 3 The stirring speed is 200-300 rpm, and the dissolved oxygen is maintained at 5-10%. During fermentation, 80% (w / v) glucose solution is added to maintain the residual sugar concentration at 0.1-0.5 g / L, and 25% ammonia water is added to adjust the pH of the fermentation broth to 6.8-7.2.

[0135] (3) Detection of L-valine in fermentation broth Same as step (3) in Example 20. After 26 h of fermentation, the L-valine engineered strain VAL-41 produced 131.3 g / L of L-valine, with a conversion rate of 58.1%.

[0136] (4) Culture medium The seed culture medium consisted of: 30 g / L glucose, 5 g / L yeast extract, 2 g / L peptone, 2 g / L KH₂PO₄, 1.5 g / L MgSO₄·7H₂O, 12 mg / L FeSO₄·7H₂O, 10 mg / L MnSO₄·7H₂O, and 1.5 mg / L MgSO₄·7H₂O. B1 0.5 mg / LV H 8 mL / L corn steep liquor, 2 g / L isoleucine, the remainder being water, pH 7.0-7.2, autoclaved at 115℃ for 15 min.

[0137] The fermentation medium consisted of: 20 g / L glucose, 4 g / L yeast extract, 2 g / L glutamic acid, 3 g / L KH₂PO₄, 2 g / L MgSO₄·7H₂O, 2.5 g / L sodium citrate dihydrate, 20 mg / L FeSO₄·7H₂O, 15 mg / L MnSO₄·7H₂O, and 2 mg / L LnSO₄·7H₂O. B1 0.2 mg / LV H 20 mL / L corn steep liquor, the remainder being water, pH 7.0-7.2, autoclaved at 115℃ for 15 min.

[0138] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that those skilled in the art can make various modifications, combinations, and improvements to the above embodiments without departing from the concept of this patent, and these modifications and combinations all fall within the scope of protection of this patent. Therefore, the scope of protection of this patent should be determined by the claims.

Claims

1. An L-valine-producing strain, characterized in that, The strain used *Escherichia coli* as the starting strain, and the gene encoding the lactose operon repressor protein was knocked out in the genome. lacI ; and overexpress the gene encoding acetolactate synthase, which relieves feedback inhibition. ilvBN M1 or ilvBN M2 ; The acetolactate synthase encoding gene ilvBN M1 Depend on ilvB and ilvN M1 composition, ilvB The nucleotide sequence is shown in SEQ ID NO.

5. ilvN M1 The nucleotide sequence is shown in SEQ ID NO.7; The acetolactate synthase encoding gene ilvBN M2 Depend on ilvB and ilvN M2 composition, ilvB The nucleotide sequence is shown in SEQ ID NO.

5. ilvN M2 The nucleotide sequence is shown in SEQ ID NO.

8.

2. The L-valine-producing strain as described in claim 1, characterized in that, The gene encoding branched-chain amino acid aminotransferases was also knocked out. ilvE Overexpression of the leucine dehydrogenase encoding gene bcd ; Gene encoding NADH-preferred hydroxy acid reductase ilvC M Or the gene encoding a NADPH-preferred hydroxy acid reductase. ilvC .

3. The L-valine-producing strain as described in claim 2, characterized in that, Based on the above gene editing, continue to perform any one or more of the following gene editing (1)-(10); (1) Knockout of the gene encoding fumarate reductase frdB pyruvate-formate lyase encoding gene pflB lactate dehydrogenase encoding gene ldhA Ethanol dehydrogenase encoding gene adhE pyruvate dehydrogenase encoding gene poxB Acetylkinase encoding gene ackA Or the gene encoding alanine transaminase. yfbQ Any one or more of the following; (2) Overexpression of the gene encoding pyridine nucleotide transhydrogenase pntAB ; (3) Overexpression of the glucose-6-phosphate dehydrogenase encoding gene zwf 6-phosphoglucate dehydratase encoding gene edd and the gene encoding 2-keto-3-deoxy-6-phosphate glucuronide aldolase eda ; (4) Knockout of the gene encoding phosphoenolpyruvate synthase pps ; (5) Overexpression of the pyruvate kinase encoding gene pykA or pykF ; (6) Knockout of the gene encoding valine-alanine aminotransferase avtA ; (7) Overexpression of the gene encoding D-galactose transporter galP ; and / or glucokinase encoding genes glk ; (8) Overexpression of the gene encoding glyceraldehyde-3-phosphate dehydrogenase from Escherichia coli. apA The gene encoding glyceraldehyde-3-phosphate dehydrogenase from Clostridium acetone-butanol is g. apc The gene encoding glyceraldehyde-3-phosphate dehydrogenase from Streptococcus pyogenes is g. apN Or the gene encoding glyceraldehyde-3-phosphate dehydrogenase from Streptococcus canis. apN Sc ; (9) Overexpression of genes encoding branched chain amino acid export proteins ygaZH or L-valine export protein encoding gene brnEF ; (10) Knockout of genes encoding substrate-binding proteins of branched-chain amino acid transport systems livJ Branched-chain amino acid transporter encoding genes brnQ or genes encoding branched-chain amino acid transporters yhjE Any one or more of them.

4. The L-valine-producing strain according to any one of claims 1-3, characterized in that, The starting strain was Escherichia coli (E. coli) Escherichia coli W3110.

5. The L-valine-producing strain according to any one of claims 1-3, characterized in that, The L-valine-producing strain is *Escherichia coli* (Escherichia coli) Escherichia coli W3110 was the starting strain, and the following gene editing was performed on its genome to obtain the strain: knockout of the gene encoding the lactose operon repressor protein. lacI Overexpression of the gene encoding acetolactate synthase, which relieves feedback inhibition, on the genome. ilvBN M1 Knockout of the gene encoding branched-chain amino acid aminotransferases ilvE And overexpression of the leucine dehydrogenase encoding gene of Bacillus subtilis bcd ; Gene encoding NADH-preferred hydroxy acid reductase ilvC M Knock out the genes encoding fumarate reductase, pyruvate-formate lyase, lactate dehydrogenase, alcohol dehydrogenase, pyruvate dehydrogenase, acetate kinase, and alanine transaminase. frdB , pflB , ldhA , adhE , poxB , ackA and yfbQ Overexpression of the gene encoding pyridine nucleotide transhydrogenase pntAB Overexpression of the glucose-6-phosphate dehydrogenase encoding gene zwf、 6-phosphoglucate dehydratase encoding gene edd And the gene encoding 2-keto-3-deoxy-6-phosphate glucuronide aldolase. eda Knockout of the gene encoding phosphoenolpyruvate synthase pps Overexpression of the gene encoding pyruvate kinase pykF Knockout of the gene encoding valine-alanine aminotransferase avtA Overexpression of the D-galactose transporter encoding gene galP and glucokinase encoding gene glk Overexpression of the g gene encoding glyceraldehyde-3-phosphate dehydrogenase from Streptococcus pyogenes apN Overexpression of the gene encoding the L-valine export protein of Corynebacterium glutamicum brnEF Knockout of genes encoding substrate-binding proteins of branched-chain amino acid transport systems livJ Branched-chain amino acid transporter encoding genes brnQ and branched-chain amino acid transporter encoding genes yhjE .

6. The L-valine-producing strain according to claim 3, characterized in that, The lactose operon repressor protein was derived from Escherichia coli W3110, Protein ID: BAE76127.1 in NCBI; The branched-chain amino acid aminotransferase was derived from Escherichia coli W3110, Protein ID: BAE77527.1 in NCBI; The leucine dehydrogenase was derived from Bacillus subtilis, Protein ID: NP_390288.1 in NCBI; The NADH-preferred hydroxy acid reductase encoding gene ilvC M Its nucleotide sequence is shown in SEQ ID NO.11 of the sequence listing; The NADPH-preferred hydroxy acid reductase isomerase was derived from Escherichia coli W3110, Protein ID: BAE77523.1 in NCBI. The fumarate reductase was derived from Escherichia coli W3110, Protein ID: BAE78157.1 in NCBI; The pyruvate-formate lyase was derived from Escherichia coli W3110, Protein ID: BAA35638.1 in NCBI; The lactate dehydrogenase was derived from Escherichia coli W3110, Protein ID: BAA14990.1 in NCBI; The alcohol dehydrogenase was derived from Escherichia coli W3110, Protein ID: BAA36121.2 in NCBI; The pyruvate dehydrogenase was derived from Escherichia coli W3110, Protein ID: BAA35585.1 in NCBI; The acetate kinase was derived from Escherichia coli W3110, Protein ID: BAA16135.1 in NCBI; The alanine transaminase was derived from Escherichia coli W3110, Protein ID: BAA16127.1 in NCBI; The pyridine nucleotide transhydrogenases were derived from Escherichia coli W3110, with Protein IDs BAA15342.1 and BAA15336.1 in NCBI. The glucose-6-phosphate dehydrogenase was derived from Escherichia coli W3110, Protein ID: BAA15660.1 in NCBI; The 6-phosphoglucate dehydratase was derived from Escherichia coli W3110, Protein ID: BAA15659.1 in NCBI; The 2-keto-3-deoxy-6-phosphate gluconate aldolase was derived from Escherichia coli W3110, Protein ID: BAA15658.1 in NCBI; The phosphoenolpyruvate synthase was derived from Escherichia coli W3110, Protein ID: BAA15471.1 in NCBI; The pyruvate kinase was derived from Escherichia coli W3110, with Protein ID BAA15445.2 or BAA15662.1 in NCBI. The valine-alanine aminotransferase was obtained from Escherichia coli W3110, Protein ID in NCBI: BAE77721.1; The D-galactose transporter was derived from Escherichia coli W3110, Protein ID: BAE77006.1 in NCBI; The glucokinase was derived from Escherichia coli W3110, Protein ID: BAA16258.1 in NCBI; The 3-phosphoglyceraldehyde dehydrogenases were derived from Escherichia coli W3110, Clostridium acetobutyricum, Streptococcus mutans, or Streptococcus canis, with Protein IDs in NCBI of BAA15576.1, AAK78686.1, AAN58410.1, and VEE25119.1, respectively. The L-valine export protein was derived from Corynebacterium glutamicum ATCC13032, with Protein IDs CAF18829.1 and CAF18830.1 in NCBI. The branched-chain amino acid transporter-export protein was derived from Escherichia coli W3110, with Protein IDs BAE76784.1 and BAA16545.1 in NCBI. The substrate-binding protein of the branched-chain amino acid transport system is from Escherichia coli W3110, Protein ID: BAE77833.1 in NCBI; The branched-chain amino acid transporter was derived from Escherichia coli W3110, with Protein ID BAE76181.1 or Protein ID BAE77771.1 in NCBI.

7. The use of the strain according to any one of claims 1-3 in the production of L-valine.

8. The application as described in claim 7, characterized in that, The method for producing L-valine using the strain via fermentation is as follows: Inoculate the seed culture into the fermentation medium at an inoculum rate of 10-20% for fermentation culture, maintain the pH at 6.8-7.2, and the culture temperature at 35-37℃; maintain the residual sugar concentration at 0.1-0.5 g / L; maintain the dissolved oxygen at 20-50% for the first 12 hours, and then maintain the dissolved oxygen at 0-15%.

9. The application as described in claim 8, characterized in that, Fermentation cycle: 18-26 hours.

Images