Construction and application of escherichia coli for producing L-valine

Abstract

The invention discloses construction and application of escherichia coli for producing L-valine, and belongs to the technical field of biological engineering. According to the escherichia coli capable of producing the L-valine, the escherichia coli capable of producing the L-valine is obtained through rational transformation of metabolic engineering, the L-valine yield can reach 95.0 g / L or above when the escherichia coli is fermented in a 5L fermentation tank for 40 h, the glucose conversion rate reaches 50% or above, and the escherichia coli has good genetic stability and is suitable for industrial production. The mdh promoter with weakened expression intensity is also screened and identified, and the concentration of the byproduct succinic acid in the fermentation liquor can be effectively reduced.

Description

Technical Field

[0001] This invention relates to the construction and application of an L-valine-producing Escherichia coli strain, belonging to the field of bioengineering technology. Background Technology

[0002] L-valine is a branched-chain amino acid (BCAA) that, along with isoleucine and leucine, forms this family. As one of the eight essential amino acids for humans, L-valine cannot be synthesized by the body and must be obtained from external sources. It is currently widely used in the feed, pharmaceutical, and food industries. In industrial production, microbial fermentation has become the main method for preparing L-valine due to its stable yield, mild reaction conditions, and low cost.

[0003] Escherichia coli ( Escherichia coli Due to its clear genetic background, simple cultivation, and non-spore-producing characteristics, *L-valine* is widely used in the industrial fermentation production of amino acids such as L-valine. The synthesis pathway of L-valine in this microorganism is basically clear: glucose is converted to pyruvate via the EMP pathway, and pyruvate is then converted to L-valine through a four-step enzymatic reaction. Pyruvate is catalyzed by acetohydroxyacid synthase (AHAS) or catalytic acetolactate synthase (CALS) to form 2-acetolactate; 2-acetolactate is catalyzed by acetohydroxyacid isomeroreductase (AHAIR) to form 2,3-dihydroxyisovalerate; then, 2,3-dihydroxyisovalerate is catalyzed by dihydroxyacid dehydratase (DHAD) to form 2-ketoisovalerate; finally, L-valine is catalyzed by branched-chain amino acid transaminase (TA). L-valine is ultimately transported out of the cell via transport proteins. L-valine also exhibits feedback inhibition of the rate-limiting enzyme acetylhydroxyl synthase.

[0004] Although various high-yield L-valine strains have been successfully constructed through metabolic engineering strategies, current industrial production still faces challenges such as limited acid production efficiency, low sugar-acid conversion rate, and insufficient production intensity, making it difficult to fully meet the needs of large-scale industrial production. Therefore, further improving the overall performance of L-valine-producing strains remains a key research focus in the field of fermentation engineering, and is of great significance for reducing production costs and expanding application scope. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides the construction and application of an L-valine-producing *Escherichia coli* strain. This invention involves introducing the gene encoding malate dehydrogenase (MDH) into the valine-producing strain. mdh promoter P mdh Replace with promoter P mdh* Through mutation modification mdh The promoter, weaken mdh The aim of reducing the expression level of succinic acid by reducing the accumulation of the byproduct succinic acid is to address the problems that are still prevalent in current industrial production, such as limited acid production efficiency, low sugar-acid conversion rate, and insufficient production intensity.

[0006] The first technical solution provided in this invention is a mutant promoter P. mdh* The mutant promoter is the promoter P whose sequence is shown in SEQ ID NO. 1. mdh The 15th base is mutated.

[0007] In one embodiment, the nucleotide sequence of the mutant promoter is shown in any one of SEQ ID NO. 5-7.

[0008] The second technical solution provided by the present invention is a recombinant cell containing the mutant promoter described in the first technical solution.

[0009] In one embodiment, the recombinant cells are recombinant Escherichia coli that produce L-valine.

[0010] The third technical solution provided by this invention is a method for constructing recombinant Escherichia coli that produces L-valine, by knocking out the gene encoding lactose repressor protein in Escherichia coli. lacI Gene encoding lactate dehydrogenase ldhA Gene encoding formate acetyltransferase pflB Gene encoding acetate kinase ackA Encoding pyruvate oxidase poxB and the gene encoding valine-pyruvate transaminase avtA It integrates and expresses transketolase tkt and acetylhydroxylase. ilvBN M Acetylhydroxy acid reductase C SM NADH-dependent leucine dehydrogenase (leuDH), membrane-bound pyridine nucleotide transhydrogenase (pntAB), and L-valine transporter (brnFE).

[0011] In one embodiment, the transketolase tkt and acetylhydroxyl synthase ilvBN M Acetylhydroxy acid reductase ilvC SMThe expression of NADH-dependent leucine dehydrogenase leuDH, membrane-bound pyridine nucleotide transhydrogenase pntAB, and L-valine transporter brnFE is driven by the promoter pJ23119.

[0012] In one implementation, lacI , ldhA , pflB , ackA , poxB and avtA Each of the six sites integrates one copy of the gene pJ23119- encoding transketolase. tkt、 One copy of pJ23119-, tandemly expressing the genes for acetylhydroxyl synthase and acetylhydroxyl reductase. ilvBN M C SM Gene (from Corynebacterium glutamicum), 1 copy of the tandemly expressed acetylhydroxyl synthase and acetylhydroxyl reductase gene pJ23119- ilvBN M C SM Gene (from Corynebacterium glutamicum), encoding NADH-dependent leucine dehydrogenase gene pJ23119- leuDH (Source: Bacillus spheroidae), encoding membrane-bound pyridine nucleotide transhydrogenase gene pJ23119- pntAB and the gene pJ23119- encoding the L-valine transporter brnFE (Source: Corynebacterium glutamicum).

[0013] In one embodiment, the GeneID encoding the ketolase tkt gene is 947711; the GeneID encoding the membrane-bound pyridine nucleotide transhydrogenase pntAB gene is 945939; and the NCBI accession numbers encoding the L-valine transporter brnFE gene are NCgl0258 and NCgl0254.

[0014] In one embodiment, the ilvBN M and ilvC SM The nucleotide sequences are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0015] In one embodiment, the NADH-dependent leucine dehydrogenase encoding *Bacillus spheroidae* is described. ls The nucleotide sequence of the leuDH gene is as follows: leuDH As shown in SEQ ID NO.4. In one embodiment, the recombinant Escherichia coli is the model strain W3110 or MG1655.

[0016] The fourth technical solution provided by this invention is a recombinant Escherichia coli constructed using the method described in the third technical solution.

[0017] The fifth technical solution provided by the present invention is a method for producing L-valine by fermentation, wherein the method utilizes the recombinant Escherichia coli described in the fourth technical solution to produce L-valine in a glucose-containing fermentation system.

[0018] In one embodiment, the fermentation system contains 30 g / L glucose.

[0019] In one embodiment, the fermentation system further contains ammonium sulfate 2.5 g / L, dipotassium hydrogen phosphate 4.5 g / L, magnesium sulfate heptahydrate 1.5 g / L, yeast extract powder 7.0 g / L, citric acid 3.2 g / L; ferrous sulfate 2.8 mg / L, manganese sulfate 1.2 mg / L, biotin 0.5 mg / L, vitamin B1 1.5 mg / L, vitamin B3 1 mg / L, vitamin B5 1.5 mg / L, vitamin B12 0.5 mg / L, and defoamer 0.2 g / L.

[0020] In one embodiment, the reaction is a two-stage fermentation: Aerobic fermentation stage: initial tank pressure 0.03 MPa, air volume 2.0 L / min, rotation speed 400 r / min, temperature 35℃; residual sugar controlled at 0.05%-0.1%; pH controlled in stages according to OD600 value: pH 7.0 when OD600 value is below 30, pH 6.3 when OD value is ≥30; when dissolved oxygen is below 30%, increase rotation speed to a maximum of 800 r / min, without adjusting air volume or tank pressure; Anaerobic fermentation stage: When the OD600 value reaches 18-25, it begins to switch to microanaerobic fermentation, with a tank pressure of 0.03 MPa, an air volume of 2.0 L / min, and a rotation speed of 300-400 r / min. An antifoaming agent is added in the later stages of fermentation to prevent foam overflow.

[0021] The sixth technical solution provided by the present invention is the application of the mutant promoter described in the first technical solution, or the recombinant cell described in the second technical solution, or the method described in the third technical solution, or the recombinant Escherichia coli described in the fourth technical solution, or the method described in the fifth technical solution in the preparation of L-valine or products containing L-valine.

[0022] The present invention also provides the application of the recombinant bacteria in the production of products containing L-valine or its derivatives in the fields of feed, pharmaceuticals, food or chemicals.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is based on E.coli Using W3110 as the starting strain, a recombinant strain capable of efficiently fermenting and producing L-valine was obtained through metabolic engineering. This strain was named... E.coli MMEC-VAL09. Fermentation in a 5 L fermenter for 40 h yields L-valine production exceeding 95.0 g / L and glucose conversion exceeding 50%, exhibiting good genetic stability and suitability for industrial production. Detailed Implementation

[0024] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0025] Test method: Valine was detected using high-performance liquid chromatography (HPLC). The fermentation broth was centrifuged at 12000 rpm for 10 min, and the supernatant was diluted a certain factor. The HPLC detection conditions were as follows: Aglient ZORBAX SB column. Aq (250 × 4.6 mm, 5 μm) was used to determine the amino acid content using a pre-column online derivatization method. The derivatizing agent was o-phthalaldehyde (o-phthalaldehyde). The solvent used was phthalaldehyde (OPA). Mobile phase A was 0.01 mol / L KH₂PO₄, and mobile phase B was acetonitrile:methanol:mobile phase A = 5:3:1. The pH was adjusted to 5.3. The flow rate was 1.0 mL / min, gradient elution was used, the column temperature was 35℃, and the detector was a UV detector at 254 nm. If the product reached its upper solubility limit and precipitated, reconstitution by heating or dilution was performed.

[0026] Succinic acid was detected by high-performance liquid chromatography (HPLC). The fermentation broth was centrifuged at 12000 rpm for 10 min, and the supernatant was diluted a certain factor. An Aminex HPX-87 H column was used; the mobile phase was 5 mM dilute sulfuric acid; the flow rate was set to 0.6 mL / min; the detector was a UV detector with a detection wavelength of 210 nm; and the column temperature was 52℃.

[0027] Glucose determination method: Analysis was performed using the Shenzhen Silman Biosensor Analyzer M-100.

[0028] Calculation of glucose yield: Glucose yield (%) = Maximum valine production (g / L) / Glucose consumption (g / L) × 100.

[0029] Raw materials used in the examples: The culture media involved in the following examples are as follows: In the following embodiments, the technical solutions used are all implemented based on conventional techniques in the art, and the materials used are all commercially available. Tryptone was OXOID brand LP0042B, yeast extract was OXOID brand LP0021B, and reagents such as glucose, ammonium sulfate, citric acid, and magnesium sulfate heptahydrate were Sinopharm Shanghai Test & Test Analytical Grade reagents. Biotin, vitamin B1, vitamin B3, vitamin B5, and vitamin B12 were purchased from Shanghai Sangon Biotech, and the defoamer was Dow brand DF103. In the following examples, Escherichia coli (Escherichia coli) Escherichia coli The type strain is either W3110 or MG1655.

[0030] Activation medium components: yeast extract 2~6 g / L, tryptone 5~10 g / L, NaCl 5 g / L; Seed culture medium composition: base sugar 30 g / L, yeast extract powder 5 g / L, dipotassium hydrogen phosphate trihydrate 2.5 g / L, ammonium sulfate 2.5 g / L, citric acid 2.5 g / L, magnesium sulfate heptahydrate 1.5 g / L, ferrous sulfate 3.0 mg / L, manganese sulfate 2.0 mg / L, biotin 0.5 mg / L, vitamin B1 1.5 mg / L, vitamin B3 1 mg / L, vitamin B5 1.5 mg / L, vitamin B12 0.5 mg / L; Fermentation medium composition: ammonium sulfate 2.5 g / L, dipotassium hydrogen phosphate 4.5 g / L, magnesium sulfate heptahydrate 1.5 g / L, yeast extract powder 7.0 g / L, citric acid 3.2 g / L; ferrous sulfate 2.8 mg / L, manganese sulfate 1.2 mg / L, biotin 0.5 mg / L, vitamin B1 1.5 mg / L, vitamin B3 1 mg / L, vitamin B5 1.5 mg / L, vitamin B12 0.5 mg / L, defoamer 0.2 g / L.

[0031] Example 1: Construction of L-valine-producing strain With Escherichia coli E.coli MG1655 was used as the modified chassis strain to construct recombinant bacteria. E.coli MMEC-VAL01 E.coli MMEC-VAL 02 E.coli MMEC-VAL 03 E.coli MMEC-VAL 04 E.coli MMEC-VAL 05 E.coli MMEC-VAL 06 E.coli MMEC-VAL 07 E.coli MMEC-VAL 08 and E.coliThe genotypes of strain MMEC-VAL 09 are shown in Table 1.

[0032] Table 1 Genotypes of Recombinant Strains

[0033] The recombinant bacteria described in Table 1 are constructed as follows: pJ23119- tkt Integration lacI Site (construction) E.coli VAL01): According to E. coli data from NCBI E.coli The MG1655 genome sequence, in lacI Primers for the upper and lower homologous arms of the gene (GeneID: 945006) were designed with a 500 bp overlap (primer sequences are shown in Table 2). The upper and lower homologous arm primers UH- tkt ( lacI )-F / R and DH- tkt ( lacI )-F / R amplifies upstream and downstream homologous arm fragments via PCR, based on tkt Designed amplification primers (tkt(lacI)-F and tkt(lacI)-R) for gene (GeneID: 947711), and downstream primers for the PJ23119 promoter on the upper homologous arm. tkt The upstream primer was obtained by PCR. tkt Gene fragments. Homologous arm fragments and... tkt Gene fragments were obtained into integration frames via fusion PCR. This was achieved using primer N20. -lacI -F and N20- lacI -R was used for circular PCR to obtain pTargetF sgRNA- lacI plasmid, and tkt Integrated frame for electro-conversion E.coli MG1655 (containing pCas9) was used to select single colonies on LB agar plates containing kanamycin and spectinomycin for colony PCR identification and sequencing to obtain the correct recombinant bacteria. After removing the manipulation plasmid, the strain was obtained. E.coli VAL01.

[0034] pJ23119-cg ilvBN M C SM Integration at ldhA site (construction) E.coli VAL02): According to E. coli data from NCBI E.coli The MG1655 genome sequence, in ldhAPrimers for the upper and lower homologous arms of the gene (GeneID: 945717) were designed with a 500 bp primer sequence (see Table 2). The upper and lower primers were UH-cgilvBN. M C SM (ldhA)-F / R and DH-cg ilvBN M C SM (ldhA)-F / R fragments with upper and lower homologous arms were amplified by PCR, according to ilvBN M C SM Design amplification primers (cg ilvBN) for genes (SEQ ID NO.2, SEQ ID NO.3). M C SM (ldhA)-F / R), PJ23119 promoter design in upstream homologous arm downstream primer and ilvBN M C SM The upstream primer was obtained by PCR. ilvBN M C SM Gene fragments. Homologous arm fragments and... ilvBN M C SM Gene fragments were obtained as integration frames via fusion PCR. Circularization PCR was performed using primers N20-ldhA-F and N20-ldhA-R to obtain the pTargetF sgRNA-ldhA plasmid, which was then combined with… ilvBN M C SM Integrated frame for electro-conversion E.coli VAL01 (containing pCas9) was used to select single colonies on LB agar plates containing kanamycin and spectinomycin for colony PCR identification and sequencing to obtain the correct recombinant bacteria. After removing the manipulation plasmid, the strain was obtained. E.coli VAL02.

[0035] pJ23119-Cg ilvBN M C SM Integration pflB Site (constructing E.coli VAL03): According to E. coli data from NCBI E.coli The MG1655 genome sequence, in pflB Primers for the upstream and downstream homologous arms of the gene (GeneID: 945234) were designed with a length of 500 bp (primer sequences are shown in Table 2). The upstream and downstream primers were UH-cgilvBN.M C SM (pflB)-F / R and DH-cg ilvBN M C SM (pflB)-F / R was amplified by PCR to extract the upper and lower homologous arm fragments, according to... ilvBN M C SM Design amplification primers (cg ilvBN) for genes (SEQ ID NO.2, SEQ ID NO.3). M C SM (pflB)-F / R), PJ23119 promoter design in upstream homologous arm downstream primer and ilvBN M C SM In the upstream primer, the upstream and downstream homologous arm fragments and ilvBN M C SM Gene fragments were obtained into integration frames via fusion PCR. This was achieved using primer N20- pflB -F and N20- pflB -R was used for circular PCR to obtain pTargetF sgRNA- pflB plasmid, and ilvBN M C SM Integrated frame for electro-conversion E.coli VAL02 (containing pCas9) was obtained by selecting single colonies on LB agar plates with kanamycin and spectinomycin for colony PCR identification and sequencing to obtain the correct recombinant bacteria. After removing the manipulation plasmid, strain E. coli VAL03 was obtained.

[0036] pJ23119- ls leuDH Integration ackA Site (constructing E.coli VAL04): According to E. coli data from NCBI E.coli The MG1655 genome sequence, in ackA Primers for the upper and lower homologous arms of the gene (GeneID: 946627) were designed with a 500 bp primer sequence (primer sequences are shown in Table 2). The upper and lower homologous arm fragments were amplified by PCR using the upstream and downstream primers UH-ls leuDH(ackA)-F / R and DH-ls leuDH(ackA)-F / R. ls leuDH Amplification primers (ls leuDH (ackA)-F / R) were designed for the gene (SEQ ID NO.4), and downstream primers were designed for the PJ23119 promoter on the upper homologous arm. ls leuDHThe upstream primer was obtained by PCR. ls leuDH Gene fragments. Homologous arm fragments and... ls leuDH Gene fragments were obtained into integration frames via fusion PCR. This was achieved using primer N20- ackA -F and N20- ackA -R was used for circular PCR to obtain pTargetF sgRNA- ackA plasmid, and ls leuDH Integrated frame for electro-conversion E.coli VAL03 (containing pCas9) was used to select single colonies on LB agar plates containing kanamycin and spectinomycin for colony PCR identification and sequencing to obtain the correct recombinant bacteria. After removing the manipulation plasmid, the strain was obtained. E.coli VAL04.

[0037] pJ23119- pntAB Integration poxB Site (construction) E.coli VAL05): According to E. coli data from NCBI E.coli The MG1655 genome sequence, in poxB Primers for the upper and lower homologous arms of the gene (GeneID: 948421) were designed with a 500 bp primer sequence (see Table 2). The upper and lower homologous arm fragments were amplified by PCR using the upstream and downstream primers UH-pntAB(poxB)-F / R and DH-pntAB(poxB)-F / R. pntAB Gene (GeneID: 945939) amplification primers (pntAB (poxB)-F / R), PJ23119 promoter downstream primers designed on the upper homologous arm and pntAB The upstream primer was obtained by PCR. pntAB Gene fragments. Homologous arm fragments and... pntAB Gene fragments were obtained into integration frames via fusion PCR. This was achieved using primer N20- poxB -F and N20- poxB -R was used for circular PCR to obtain pTargetF sgRNA- poxB plasmid, and pntAB The integrated frame was electroporated into E. coli VAL O4 (containing pCas9). Single colonies were selected on kanamycin and spectinomycin-protected LB agar plates for colony PCR identification and sequencing to obtain the correct recombinant bacteria. After removing the manipulation plasmid, the strain was obtained. E.coli VAL05.

[0038] pJ23119- cg brnFE Integration avtA Site (construction)E.coli VAL06): According to E. coli data from NCBI E.coli The MG1655 genome sequence, in avtA Primers for the upper and lower homologous arms (500 bp upstream and downstream of gene (GeneID: 948426) were designed (primer sequences are shown in Table 2). The upper and lower homologous arm fragments were amplified by PCR using primers UH-cg brnFE (avtA)-F / R and DH-cg brnFE (avtA)-F / R. Based on *Corynebacterium glutamicum*... brnFE Primers (cg brnFE (avtA)-F / R) were designed for gene (NCgl0258 and NCgl0254), and primers downstream of the PJ23119 promoter on the upper homologous arm were designed. cg brnFE The upstream primer was obtained by PCR. cg brnFE Gene fragments. Homologous arm fragments and... cg brnFE Gene fragments were obtained into integration frames via fusion PCR. This was achieved using primer N20- avtA -F and N20- avtA -R was used for circular PCR to obtain pTargetF sgRNA- avtA plasmid, and cgbrnFE The integrated frame was electroporated into E. coli VAL 05 (containing pCas9). Single colonies were selected on kanamycin and spectinomycin-protected LB agar plates for colony PCR identification and sequencing to obtain the correct recombinant bacteria. After removing the manipulation plasmid, the strain was obtained. E.coli VAL06.

[0039] Will mdh The promoter of the gene is replaced with (construction) E.coli VAL07 E.coli VAL08 E.coli VAL09)P mdh-1、 P mdh-2、 P According to E. coli data from NCBI mdh-3 The MG1655 genome sequence, in E.coli Homologous arm primers (500 bp upstream and downstream of gene SEQ ID NO.1) were designed (primer sequences are shown in Table 2). The upstream and downstream homologous arm fragments were amplified by PCR using primers UH-Pmdh-F, UH-Pmdh-1-R, DH-Pmdh-1-F, UH-Pmdh-2-R, DH-Pmdh-2-F, UH-Pmdh-3-R, and DH-Pmdh-3-F. -1 (SEQ ID NO.5), P mdh -2 (SEQ ID NO.6), P mdh-3 (SEQ ID NO.7) is P mdh The three promoters were obtained by mutating the 15th base G to A, T, and C, respectively, and were designed in [locations not specified]. mdh In homologous arm primers, obtained by PCR mdh Integration box. Using primer N20- mdh F and N20- mdh -R was used for circular PCR to obtain pTargetF sgRNA- mdh- plasmid, and mdh Integrated frame for electro-conversion mdh His05 (containing pCas9) was used to select single colonies on LB agar plates containing kanamycin and spectinomycin for colony PCR identification and sequencing to obtain the correct recombinant bacteria. After removing the manipulation plasmid, the strains could be obtained. mdh VAL07 E.coli VAL08 and E.coli VAL09.

[0040] Table 2 Primer Sequences

[0041] Example 2: Production of valine by fermentation of recombinant strains The recombinant strain obtained was fermented in a 5 L fermenter, and the specific steps are as follows: (1) Seed activation and culture Plate activation: Inoculate one loopful of the strain from the preservation tube onto a solid LB plate and incubate at 37°C for 24 h; Primary seed culture: Pick a well-grown single colony from the plate and inoculate it into a 500mL Erlenmeyer flask containing 50mL LB medium. Incubate at 37℃ for approximately 10-12 hours until the OD of the primary seed culture is reached. 600 The value is between 6 and 8.

[0042] Secondary seed culture: Inoculate the primary seed culture at a rate of 5-10% into a 500mL Erlenmeyer flask containing 50mL of fermentation medium, and incubate at 37℃ for approximately 8-10 hours until the OD of the secondary seed culture reaches the specified level. 600 The value is between 12 and 16.

[0043] (2) Fermentation culture Aerobic fermentation stage: initial tank pressure 0.03 MPa, air volume 2.0 L / min, rotation speed 400 r / min, temperature 35℃; residual sugar controlled at 0.05%-0.1%; pH adjusted according to OD. 600 Value segmentation control: OD 600When the dissolved oxygen value is below 30, control the pH to 7.0; when the dissolved oxygen value is ≥30, control the pH to 6.3. When the dissolved oxygen value is below 30%, increase the rotation speed to a maximum of 700-800 r / min, without adjusting the air volume or tank pressure.

[0044] Anaerobic fermentation stage: When the OD600 value reaches 18-25, microanaerobic fermentation begins, with a tank pressure of 0.03 MPa, an air volume of 2.0 L / min, and a rotation speed of 300-400 r / min. An antifoaming agent is added in the later stages of fermentation to prevent foam overflow. Samples were taken during the process to detect the accumulation of valine and the byproduct succinic acid (Table 3). Introducing a promoter mutant of the mdh gene significantly reduced the accumulation of the byproduct succinic acid and improved glucose conversion. The optimal strain was identified. E.coli E.coli E. coli VAL 09, fermented in a 5 L fermenter for 40 h, yielded an L-valine production of over 95.2 g / L and a glucose conversion rate of 50.4%.

[0045] Table 3 Fermentation status of recombinant Escherichia coli

[0046] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A mutant promoter, characterized in that, The mutant promoter is the promoter P whose sequence is shown in SEQ ID NO.

1. mdh The 15th base is mutated.

2. Recombinant cells containing the mutant promoter of claim 1.

3. The recombinant cell according to claim 2, characterized in that, The recombinant cells are recombinant Escherichia coli that produce L-valine.

4. A method for constructing a recombinant Escherichia coli strain producing L-valine, characterized in that, The gene encoding lactose repressor protein in E. coli was knocked out. lacI Gene encoding lactate dehydrogenase ldhA Gene encoding formate acetyltransferase pflB Gene encoding acetate kinase ackA Encoding pyruvate oxidase poxB and the gene encoding valine-pyruvate transaminase avtA It integrates and expresses transketolase tkt and acetylhydroxyl synthase ilvBN. M Acetylhydroxy acid reductase ilvC SM NADH-dependent leucine dehydrogenase (leuDH), membrane-bound pyridine nucleotide transhydrogenase (pntAB), and L-valine transporter (brnFE).

5. The method according to claim 4, characterized in that, The Gene ID encoding the ketolase tkt gene is 947711; the Gene ID encoding the membrane-bound pyridine nucleotide transhydrogenase pntAB gene is 945939; the NCBI accession numbers for the L-valine transporter brnFE gene are NCgl0258 and NCgl0254; and the gene encoding the acetylhydroxyl synthase ilvBN... M and acetylhydroxy acid reductase ilvC SM The nucleotide sequences of the gene are shown in SEQ ID NO.2 and SEQ ID NO.3, and the nucleotide sequence encoding the NADH-dependent leucine dehydrogenase leuDH gene is shown in SEQ ID NO.

4.

6. The method according to claim 4, characterized in that, The recombinant Escherichia coli is the model strain W3110 or MG1655.

7. Recombinant Escherichia coli constructed using the method described in any one of claims 4 to 6.

8. A method for producing L-valine by fermentation, characterized in that, The method involves using the recombinant Escherichia coli described in claim 7 to produce L-valine in a glucose-containing fermentation system.

9. The method according to claim 8, characterized in that, The reaction is a two-stage fermentation, including an aerobic fermentation stage and an anaerobic fermentation stage.

10. The use of the mutant promoter of claim 1, or the recombinant cell of any one of claims 2-3, or the method of any one of claims 4-6, or the recombinant Escherichia coli of claim 7, or the method of claim 8 or 9 in the preparation of L-valine or products containing L-valine.

Images