Production strain for fermenting L-histidine and application thereof

Abstract

The invention discloses a production strain for fermenting L-histidine and application of the production strain, and belongs to the technical field of biological engineering. According to the invention, escherichia coli E.coli CICC 10243 is taken as an original strain, histidine synthesis operon genes hisDCB-cg2302-cg2301-HA-impA-FI, hisN and hisEG from corynebacterium glutamicum are integrated on a genome of the escherichia coli E.coli CICC 10243, a pyruvate dehydrogenase AceE mutant coding gene is introduced, and an escherichia coli transcription factor SlyA coding gene slyA is inactivated. The novel strain E.coli His08 for fermentation production of L-histidine is prepared and fermented in a 5L fermentation tank for 60 h, the histidine yield reaches 58.39 g / L, the sugar-acid conversion rate is 0.15 g / g, and the strain E.coli His08 has important application value.

Description

Technical Field

[0001] The invention relates to a production strain of L-histidine by fermentation and application thereof, belonging to the technical field of bioengineering. Background Art

[0002] L-histidine ("histidine") is one of the 20 standard proteinogenic amino acids found in all living proteins. Its side chain is an imidazole ring, which exhibits aromatic properties. With a pKa value of 6.0, histidine is the only amino acid whose side chain can transition from a non-protonated state to a protonated state at neutral pH.

[0003] Histidine is a semi-essential amino acid that plays a crucial role in regulating human physiological functions and in industrial applications. In medicine, histidine is a precursor to histamine, participating in immune regulation and anti-allergic responses. It is also an adjunct ingredient in medications for the treatment of rheumatoid arthritis and anemia. In the food industry, histidine serves as a natural preservative and flavor enhancer in functional foods and sports supplements. In the cosmetics sector, histidine derivatives, such as imidazole alanine, are commonly used in skincare products for their antioxidant and moisturizing properties.

[0004] The production processes for histidine primarily include chemical synthesis, enzymatic catalysis, and microbial fermentation. Chemical synthesis produces L-histidine through asymmetric catalytic reactions, but this method suffers from low chiral purity and numerous byproducts. Enzymatic catalysis utilizes biocatalysts such as histidine dehydrogenase, but this results in high raw material costs and limited enzyme stability. Microbial fermentation, through metabolic engineering of Escherichia coli or Corynebacterium glutamicum, increases production through systematic metabolic engineering strategies such as enhanced glucose uptake, phosphoribosylpyrophosphate (PRPP) synthesis, relief of natural feedback inhibition, and promotion of histidine export.

[0005] The strain with the highest reported histidine fermentation yield is E. coli WHY3-1 (CN 111321102 A). This strain enhances the activity of HisG, a key enzyme in histidine synthesis, and the expression of the E. coli native histidine operon gene hissDBCHAFI. It also integrates the lysE gene encoding the arginine / lysine transporter from Corynebacterium glutamicum and the rocG gene encoding the glutamate dehydrogenase from Bacillus subtilis. In a 5L fermentor for 36-48 hours, the fermentation broth can accumulate 50-65g / L of histidine, with an average production rate of 1.5-2.0g / L / h and a sugar-acid conversion rate of 0.2-0.24g / g. However, histidine synthesis in E. coli is subject to its own rigorous and complex regulation, consumes a lot of ATP, and the accumulation of intermediate metabolites (such as imidazole glycerol phosphate) may inhibit cell activity.

[0006] To improve the efficiency of histidine synthesis, heterologous expression of efficient pathway genes, enhanced ATP supply, and improved cellular physiological properties are feasible strategies. Therefore, developing a more efficient histidine production method has extremely high practical and economic value. Summary of the Invention

[0007] To solve the above problems, the present invention introduces the histidine operon gene of Corynebacterium glutamicum into histidine-producing bacteria to enhance the metabolic flux of the histidine synthesis pathway; through protein engineering, a double mutant is obtained in which the alanine at position 432 of the aceE gene encoding the pyruvate dehydrogenase AceE is mutated to threonine, and the glycine at position 636 is mutated to glutamine, thereby enhancing the ATP synthesis capacity of the bacteria; and the glutamine at position 125 of the slyA gene encoding the Escherichia coli transcription factor SlyA is mutated to a taa terminator to inactivate its function, thereby improving the physiological characteristics and fermentation performance of the bacteria.

[0008] The first object of the present invention is to provide a pyruvate dehydrogenase (aceE) mutant, wherein the pyruvate dehydrogenase mutant is based on the amino acid sequence of SEQ ID NO. 21, wherein the alanine at position 432 is mutated to threonine, and the glycine at position 636 is mutated to glutamine.

[0009] In one embodiment, the amino acid sequence of the pyruvate dehydrogenase mutant (ie, aceE-A432T / G636Q) is shown in SEQ ID NO. 22.

[0010] The second object of the present invention is to provide a polynucleotide encoding the above-mentioned pyruvate dehydrogenase mutant.

[0011] The third object of the present invention is to provide a plasmid vector carrying the above-mentioned polynucleotide or a cell expressing the above-mentioned pyruvate dehydrogenase mutant.

[0012] The fourth object of the present invention is to provide a recombinant Escherichia coli bacterium, wherein the recombinant Escherichia coli bacterium expresses the above-mentioned pyruvate dehydrogenase mutant.

[0013] In one embodiment, the recombinant Escherichia coli also overexpresses the histidine operon genes hisEG, hisDCB-cg2302-cg2301-HA-impA-FI and hisN from Corynebacterium glutamicum, wherein hisDCB-cg2302-cg2301-HA-impA-FI is hisD, hisC, hisB, cg2302, cg2301, hisH, hisA, impA, hisF, hisI arranged in sequence, and hisEG is hisE and hisG arranged in sequence;

[0014] Among them, hisEG was integrated into the genomic ldhA, poxB, and yaeQ gene sites, and their start codons were changed from GTG to ATG;

[0015] hisDCB-cg2302-cg2301-HA-impA-FI was integrated into the yghE gene locus of the E. coli genome;

[0016] hisN is integrated into the ydfT gene site of the E. coli genome.

[0017] In one embodiment, the amino acid sequences of hisD, hisC, hisB, cg2302, cg2301, hisH, hisA, impA, hisF, hisI, hisN, hisE, and hisG are shown in SEQ ID NOs. 8 to 20.

[0018] In one embodiment, promoter PJ23119 is used to express hisEG, hisDCB-cg2302-cg2301-HA-impA-FI, and hisN, respectively.

[0019] In one embodiment, the recombinant E. coli also mutates glutamine at position 125 of the transcription factor SlyA encoding gene slyA to a taa terminator (the amino acid sequence before mutation is shown in SEQ ID NO. 23; the amino acid sequence after mutation is shown in SEQ ID NO. 24).

[0020] In one embodiment, the recombinant E. coli bacteria express AceE using the E. coli endogenous promoter P aceE , expressing SlyA using the E. coli endogenous promoter P slyA .

[0021] In one embodiment, the host of the recombinant Escherichia coli is E. coli CICC 10243, E. coli MG1655, E. coli W3110 or E. coli BL21.

[0022] In one embodiment, the method for constructing the recombinant Escherichia coli is as follows:

[0023] (1) constructing a linker fragment PJ23119-hisEG, which is a fragment of the promoter PJ23119 and the gene hisEG whose nucleotide sequence is shown in SEQ ID NO. 1, replacing its start codon from GTG to ATG, and knocking it into the yaeQ, ldhA, and poxB gene sites of the Escherichia coli genome, respectively;

[0024] (2) Constructing a fragment PJ23119-hisDCB-cg2302-cg2301-HA-impA-FI linker between the promoter PJ23119 and the gene hisDCB-cg2302-cg2301-HA-impA-FI as shown in SEQ ID NO. 2 (the underlined portion is the promoter sequence), and integrating it into the yghE gene site of the Escherichia coli genome in two consecutive steps;

[0025] (3) constructing a linker fragment PJ23119-hisN consisting of the promoter PJ23119 and the gene hisN whose nucleotide sequence is shown in SEQ ID NO. 3, and knocking it into the ydfT gene site of the Escherichia coli genome;

[0026] (4) Construction of the endogenous aceE promoter of Escherichia coli and the aceE* gene nucleotide sequence shown in SEQ ID NO.6 aceE -aceE*, and knocked it into the aceE gene site of the Escherichia coli genome;

[0027] (5) Construct the nucleotide sequence of the connecting fragment P shown in SEQ ID NO.7 slyA -slyA* and knocked it into the slyA gene site of the Escherichia coli genome.

[0028] A fifth object of the present invention is to provide the use of the aforementioned pyruvate dehydrogenase mutant, the aforementioned polynucleotide, the aforementioned plasmid vector, the aforementioned cell, or any of the aforementioned recombinant Escherichia coli bacteria in the preparation of histidine.

[0029] A sixth object of the present invention is to provide a method for producing L-histidine by fermentation, using the recombinant Escherichia coli bacteria according to any one of claims 4 to 8 to produce L-histidine, comprising the steps of:

[0030] Any of the above-mentioned recombinant Escherichia coli bacteria is inoculated into a seed culture medium, cultured with shaking at 34-36°C for 10-14 hours, and then inoculated into a fermentation medium at a 15-20% inoculum size. The pH is controlled at 6.7-7.0 with dilute sulfuric acid and ammonia water, the ventilation rate is 0.8-1.5 vvm, and the stirring is 300-800 rpm. During the fermentation process, 800 g / L monohydrate glucose solution is added to control the glucose concentration of the fermentation liquid to be 0.5-3.0 g / L.

[0031] In one embodiment, the method of producing histidine by fermenting E. coli His08 comprises: inoculating the E. coli His08 strain into a seed culture medium, culturing with shaking at 34-36° C. for 10-14 hours, inoculating the fermentation medium at a 15-20% inoculum size, controlling the pH at 6.7-7.0 with dilute sulfuric acid and ammonia water, aerating at 0.8-1.5 vvm, stirring at 300-800 rpm, and adding 800 g / L monohydrate glucose solution during the fermentation process to control the glucose concentration of the fermentation liquid at 0.5-3.0 g / L.

[0032] In one embodiment, the components of the seed culture medium include: 30-40 g / L glucose, 2-6 g / L yeast extract, 5-10 g / L tryptone, and 5-10 g / L NaCl.

[0033] In one embodiment, the fermentation medium comprises: 15-25 g / L glucose, 5-10 g / L yeast powder, 3-5 g / L (NH4)2SO4, 3-5 g / L KH2PO4, and 1.0-1.5 g / L MgSO4·7H2O.

[0034] The benefits of the present invention are:

[0035] The present invention introduces the histidine operon gene of Corynebacterium glutamicum into histidine-producing bacteria, thereby enhancing the metabolic flux of the histidine synthesis pathway; obtains a double mutant by protein engineering in which the alanine at position 432 of the aceE gene encoding pyruvate dehydrogenase (AceE) is mutated to threonine and the glycine at position 636 is mutated to glutamine, thereby enhancing the ATP synthesis capacity of the bacteria; and inactivates the function of the terminator by mutating the glutamine at position 125 of the slyA gene encoding the Escherichia coli transcription factor SlyA to a taa terminator, thereby improving the physiological characteristics and fermentation performance of the bacteria.

[0036] The above metabolic engineering and protein engineering strategies were used to increase the histidine concentration in the fermentation broth of Escherichia coli using glucose as the sole carbon source. The recombinant strain E. coli His08 was fermented in a 5L fermentor for 60 hours, and the histidine production reached 58.39g / L, with a sugar-acid conversion rate of 0.15g / g. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a curve of the yield change during the fermentation process of histidine production by the recombinant strain E. coli His08. DETAILED DESCRIPTION

[0038] In the following embodiments, the technical solutions adopted are all implemented based on conventional technical means in the field, and the implementation materials are all commercially available.

[0039] In the following examples, Escherichia coli was used as the starting strain, purchased from CICC, No. 10243, and is also applicable to other Escherichia coli that can produce amino acids.

[0040] Amino acid detection method: conventional high performance liquid chromatography detection was used.

[0041] Glucose determination method: Glucose was analyzed using an SBA-40 biosensor analyzer (Institute of Biology, Shandong Academy of Sciences).

[0042] Calculation of sugar-acid conversion rate: Sugar-acid conversion rate = total histidine accumulation in fermentation broth (g) / total glucose consumption in fermentation (g).

[0043] raw material:

[0044] Activation medium ingredients: yeast extract 2-6 g / L, tryptone 5-10 g / L, NaCl 5-10 g / L, beef extract 5-10 g / L;

[0045] Seed culture medium composition: glucose 30-40 g / L, yeast extract 2-6 g / L, tryptone 5-10 h / L, NaCl 5-10 g / L;

[0046] Fermentation medium ingredients: glucose 15-25 g / L, yeast powder 5-10 g / L, (NH4)2SO4 3-5 g / L, KH2PO4 3-5 g / L, MgSO4·7H2O 1.0-1.5 g / L.

[0047] Example 1: Construction of recombinant E. coli His08

[0048] Using Escherichia coli CICC 10243 as the transformation chassis strain, recombinant bacteria E. coli His01, E. coli His02, E. coli His03, E. coli His04, E. coli His05, E. coli His06, E. coli His07 and E. coli His08 were constructed. The strain genotypes are shown in Table 1.

[0049] Table 1

[0050] Engineered bacteria genotype E.coli His01 E.coli CICC 10243-ΔyaeQ::hisEG E.coli His02 E.coli His01-ΔldhA::hisEG E.coli His03 E.coli His02-ΔpoxB::hisEG E.coli His04 E.coli His03-ΔyghE::hisDCB-cg2302-cg2301 E.coli His05 E.coli His04::hisHA-impA-hisFI E.coli His06 E.coli His05-ΔydfT::hisN E.coli His07 E.coli His06-ΔaceE::aceE* E.coli His08 E.coli His07-ΔslyA::slyA* E. coli His09 (control) E.coli His06-ΔslyA::slyA*

[0051] The construction of the recombinant bacteria described in Table 1 is as follows:

[0052] Integrate hisEG at the yaeQ site (construct E. coli His01):

[0053] Based on the Escherichia coli CICC 10243 genome sequence retrieved from NCBI, upper and lower homology arm primers were designed 500 bp upstream and downstream of the yaeQ gene (primer sequences are shown in Table 6). PCR amplification of the upper and lower homology arm fragments (UH-hisEG(yaeQ) and DH-hisEG(yaeQ)) was performed. Amplification primers were designed based on the hisEG gene (nucleotide sequence shown in SEQ ID NO. 1; the underlined portion represents the promoter sequence). The PJ23119 promoter was incorporated into the upper homology arm downstream primer and the hisEG upstream primer. The hisEG gene fragment was obtained by PCR. The upper and lower homology arm fragments and the hisEG gene fragment were fused to form an integration cassette by PCR. The pTargetF sgRNA-yaeQ plasmid was obtained by circular PCR using primers N20-yaeQ-F and N20-yaeQ-R, and it was electroporated into E. coli CICC 10243 (containing pCas9) together with the hisEG integration frame. Monoclonal colonies were selected on LB agar plates containing kanamycin and spectinomycin, and colony PCR identification and sequencing were performed to obtain the correct recombinant bacteria. After removing the manipulation plasmid, the strain E. coli His01 was obtained.

[0054] Integration of hisEG at the ldhA site (construction of E. coli His02):

[0055] Based on the Escherichia coli CICC 10243 genome sequence retrieved from NCBI, upper and lower homology arm primers were designed 500 bp upstream and downstream of the ldhA gene (primer sequences are shown in Table 6). PCR amplification of the upper and lower homology arm fragments (UH-hisEG(ldhA) and DH-hisEG(ldhA)) was performed. Amplification primers were designed based on the hisEG gene (nucleotide sequence shown in SEQ ID NO. 1). The PJ23119 promoter was designed into the upper homology arm downstream primer and the hisEG upstream primer. The hisEG gene fragment was obtained by PCR. The upper and lower homology arm fragments and the hisEG gene fragment were fused by PCR to obtain an integration cassette. The pTargetF sgRNA-ldhA plasmid was obtained by circular PCR using primers N20-ldhA-F and N20-ldhA-R, and it was electroporated into E. coli His01 (containing pCas9) together with the hisEG integration frame. Monoclonal colonies were selected on LB agar plates containing kanamycin and spectinomycin, and colony PCR identification and sequencing were performed to obtain the correct recombinant bacteria. After removing the manipulation plasmid, the strain E. coli His02 was obtained.

[0056] Integration of hisEG at the poxB site (construction of E. coli His03):

[0057] Based on the Escherichia coli CICC 10243 genome sequence retrieved from NCBI, upper and lower homology arm primers were designed 500 bp upstream and downstream of the poxB gene (primer sequences are shown in Table 6). PCR amplification of the upper and lower homology arm fragments (UH-hisEG(poxB) and DH-hisEG(poxB)) was performed. Amplification primers were designed based on the hisEG gene (nucleotide sequence shown in SEQ ID NO. 1). The PJ23119 promoter was designed into the upper homology arm downstream primer and the hisEG upstream primer. The hisEG gene fragment was obtained by PCR. The upper and lower homology arm fragments and the hisEG gene fragment were fused by PCR to obtain an integration cassette. The pTargetF sgRNA-poxB plasmid was obtained by circular PCR using primers N20-poxB-F and N20-poxB-R, and it was electroporated into E. coli His02 (containing pCas9) together with the hisEG integration frame. Monoclonal colonies were selected on LB agar plates containing kanamycin and spectinomycin, and colony PCR identification and sequencing were performed to obtain the correct recombinant bacteria. After removing the operating plasmid, the strain E. coli His03 was obtained.

[0058] Integrate hisDCB-cg2302-cg2301-HA-impA-FI at the yghE site (construct E. coli His04, replacing the first half of the yghE sequence with hisDCB-cg2302-cg2301):

[0059] The hisDCB-cg2302-cg2301-HA-impA-FI was divided into two segments, namely hisDCB-cg2302-cg2301 and hisHA-impA-FI, and integrated into the yhgE site twice. According to the Escherichia coli CICC 10243 genome sequence queried by NCBI, upper and lower homology arm primers were designed 500 bp upstream and downstream of the first half of the yghE gene (the first 430 bp bases) (primer sequences are shown in Table 6), and the upper and lower homology arm fragments (UH-hisDCB-cg2302-cg2301 (yhgE), DH-hisDCB-cg2302-cg2301 (yhgE)) were PCR amplified. According to the hisDCB-cg2302-cg2301 gene (i.e., the first half of SEQ ID NO. 2, the nucleotide sequence is as shown in SEQ ID NO. Amplification primers were designed using primers from the pJ23119 promoter (NO. 4, the underlined portion is the promoter sequence). The PJ23119 promoter was incorporated into the downstream primer for the upper homology arm and the upstream primer for the hisDCB-cg2302-cg2301 gene fragment by PCR. The upper and lower homology arm fragments and the hisDCB-cg2302-cg2301 gene fragment were then fused by PCR to create the integration cassette. The pTargetF sgRNA-yghE1 plasmid was obtained by circular PCR using primers N20-yghE1-F and N20-yghE1-R, and it was electroporated into E. coli His03 (containing pCas9) together with the hisDCB-cg2302-cg2301 integration frame. Monoclonal colonies were selected on LB agar plates containing kanamycin and spectinomycin, and colony PCR identification and sequencing were performed to obtain the correct recombinant bacteria. After removing the manipulation plasmid, the strain E. coli His04 was obtained.

[0060] Integrate hisHA-impA-hisFI into yghE (construct E. coli His05, replace the second half of the yghE sequence with hisHA-impA-hisFI):

[0061] Upper and lower homology arm primers were designed 500bp upstream and downstream of the second half of the yhgE gene (base 431-861bp) of the strain E. coli His04 strain (primer sequences are shown in Table 6), and the upper and lower homology arm fragments (UH-hisHA-impA-FI (yhgE), DH-hisHA-impA-FI (yhgE)) were PCR amplified. Amplification primers were designed based on the hisHA-impA-FI gene (nucleotide sequence such as SEQ ID NO.5, i.e., the second half of SEQ ID NO.2). The PJ23119 promoter was designed in the upper homology arm downstream primer and the hisHA-impA-FI upstream primer, and the hisHA-impA-FI gene fragment was obtained by PCR. The upper and lower homology arm fragments and the hisHA-impA-FI gene fragment were integrated by fusion PCR to obtain an integration frame. The pTargetF sgRNA-yghE2 plasmid was obtained by circular PCR using primers N20-yghE2-F and N20-yghE2-R, and it was electroporated into E. coli His04 (containing pCas9) together with the hisHA-impA-FI integration frame. Monoclonal colonies were selected on LB agar plates containing kanamycin and spectinomycin, and colony PCR identification and sequencing were performed to obtain the correct recombinant bacteria. After removing the operating plasmid, the strain E. coli His05 was obtained.

[0062] Integrate hisN at the ydfT site (construct E. coli His06):

[0063] Based on the Escherichia coli CICC 10243 genome sequence retrieved from NCBI, upper and lower homology arm primers were designed 500 bp upstream and downstream of the ydfT gene (primer sequences are shown in Table 6). PCR amplification of the upper and lower homology arm fragments (UH-hisN(ydfT) and DH-hisN(ydfT)) was performed. Amplification primers were designed based on the hisN gene (nucleotide sequence shown in SEQ ID NO. 3; the underlined portion represents the promoter sequence). The PJ23119 promoter was incorporated into the upper homology arm downstream primer and the hisN upstream primer. The hisN gene fragment was obtained by PCR. The upper and lower homology arm fragments and the hisN gene fragment were fused by PCR to generate an integration cassette. The pTargetF sgRNA-ydfT plasmid was obtained by circular PCR using primers N20-ydfT-F and N20-ydfT-R, and it was electroporated into E. coli His05 (containing pCas9) together with the hisN integration frame. Monoclonal colonies were selected on LB agar plates resistant to kanamycin and spectinomycin for colony PCR identification and sequencing to obtain the correct recombinant bacteria. After removing the manipulation plasmid, the strain E. coli His06 was obtained.

[0064] aceE* replaces endogenous aceE in E. coli (constructing E. coli His07):

[0065] Amplification primers were designed based on the Escherichia coli CICC 10243 genome sequence and aceE* gene (nucleotide sequence shown in SEQ ID NO. 6 (the underlined portion is the promoter sequence), and amino acid sequence shown in SEQ ID NO. 21) obtained from NCBI. Upper homology arm downstream primers and middle fragment upstream primers were designed forward and backward at amino acid position 432 of the aceE gene, respectively. The A432T mutation site was designed in the upper homology arm downstream primer and middle fragment upstream primer. At amino acid position 636 of the aceE gene, middle fragment downstream primers and lower homology arm upstream primers were designed forward and backward, respectively. The G636Q mutation site was designed in the middle fragment downstream primer and lower homology arm upstream primer. Upper homology arm upstream primers and lower homology arm downstream primers were designed 100 bp upstream and downstream of the aceE gene (primer sequences are shown in Table 6). PCR was performed to amplify the upper and lower homology arms and the middle fragment (UH-aceE*, DH-aceE*), and the aceE* integration frame fragment was obtained by fusion PCR. The pTargetF sgRNA-aceE plasmid was obtained by circular PCR using primers N20-aceE-F and N20-aceE-R. This plasmid, along with the aceE* integration cassette, was electroporated into E. coli His06 (containing pCas9). Monoclonal colonies were selected on LB agar plates resistant to kanamycin and spectinomycin, and identified by colony PCR and sequencing to obtain the correct recombinant bacteria. After removing the manipulation plasmid, the strain E. coli His07 (the aceE mutant aceE-A432T / G636Q was constructed, with the amino acid sequence shown in SEQ ID NO. 22) was obtained.

[0066] slyA* replaces endogenous slyA in E. coli (constructing E. coli His08 and E. coli His09):

[0067] Amplification primers were designed based on the Escherichia coli CICC 10243 genome sequence and the slyA* gene (nucleotide sequence as SEQ ID NO. 7, the underlined portion is the promoter sequence) queried from NCBI. Upper homology arm downstream primers and lower homology arm upstream primers were designed forward and backward at amino acid position 125 of the slyA gene, respectively. The Q125* mutation site (mutated to the terminator taa) was designed in the upper homology arm downstream primer and the lower homology arm upstream primer. Upper homology arm upstream primers and lower homology arm downstream primers were designed 100 bp upstream and downstream of slyA (primer sequences are shown in Table 6). PCR amplified the upper and lower homology arm fragments (UH-slyA*, DH-slyA*), and the slyA* integration frame fragment was obtained by fusion PCR. The pTargetF sgRNA-slyA plasmid was obtained by circular PCR using primers N20-slyA-F and N20-slyA-R, and the pTargetF sgRNA-slyA plasmid was electroporated into E. coli His07 (containing pCas9) together with the slyA* integration frame. Monoclonal colonies were selected on LB agar plates containing kanamycin and spectinomycin, and colony PCR identification and sequencing were performed to obtain the correct recombinant bacteria. After removing the manipulation plasmid, the strain E. coli His08 was obtained.

[0068] The pTargetF sgRNA-slyA and slyA* integration frame were electroporated into E. coli His06 (containing pCas9). Monoclonal colonies were selected on LB agar plates containing both kanamycin and spectinomycin, and colony PCR identification and sequencing were performed to obtain the correct recombinant bacteria. After removing the manipulation plasmid, the strain E. coli His09 without the mutant aceE* was obtained.

[0069] The specific steps for constructing the recombinant strain are as follows:

[0070] 1. Construction of target gene integration cassette

[0071] The gene fragment for integration consists of an upstream homology arm, the target gene, and a downstream homology arm. A bacterial genome, plasmid, or monoclonal colony was used as a template for amplification of the target fragment. The PCR amplification system is shown in Table 2. Upstream and downstream primers were designed using SnapGene software and synthesized by Yixin (Shanghai) Biotechnology Co., Ltd.

[0072] PCR reaction conditions: pre-denaturation at 95°C for 1 min; denaturation at 95°C for 15 s; annealing at 55-60°C for 15 s; extension at 72°C for 30 s-2 min (depending on fragment length, 2 kb / min), cycled 34 times. After obtaining each fragment, the integration cassette was constructed by overlapping fusion PCR. The PCR fusion system is shown in Table 3.

[0073] Table 2

[0074] Components Volume / μL Upstream primer 1 Downstream primer 1 template 1 2X phanta UniFi Master Mix 25 <![CDATA[ddH2O]]> 22

[0075] Table 3

[0076] Components Volume / μL Upper arm upstream primer 1 Lower arm downstream primer 1 Upstream homology arm 1 target gene 1 Downstream homology arm 1 2X phanta UniFi Master Mix 25 <![CDATA[ddH2O]]> 20

[0077] 2. Construction of pTargetF sgRNA plasmid

[0078] The pTargetF sgRNA plasmid can transcribe the gRNA of the target gene, which forms a complex with the Cas9 protein, guiding the Cas9 protein to recognize and cut the target gene, causing a DSB in the genome at the target gene, and then integrating the target gene into the bacterial genome through the bacteria's own homologous recombination system. The specific construction steps are as follows:

[0079] (1) Guide sequence design

[0080] The target gene guide sequence was designed using the CRISPR RGEN Tools website.

[0081] (2) Plasmid circularization PCR

[0082] Reverse PCR was performed using upstream and downstream primers containing the target gene guide sequence. The PCR system is shown in Table 4. The PCR reaction conditions were: initial denaturation at 95°C for 1 minute; denaturation at 95°C for 15 seconds; annealing at 55-60°C for 15 seconds; and extension at 72°C for 1 minute, for 34 cycles. The PCR product was digested with DpnI and transformed into E. coli DH5α competent cells, plated, and incubated at 37°C for 12 hours. Positive single colonies were selected for sequencing.

[0083] Table 4

[0084] Components Volume / μL Upstream primer 1 Downstream primer 1 plasmids 1 2X phanta UniFi Master Mix 25 <![CDATA[ddH2O]]> 22

[0085] 3. Transformation of target gene integration frame and plasmid

[0086] (1) Transformation of pCas9 plasmid

[0087] The pCas9 plasmid was electroporated into E. coli CICC 10243 electroporation competent cells, cultured at 30°C for 45 min, spread on LB agar plates containing kanamycin, cultured at 30°C for 24 h, and positive monoclonal colonies were selected for the next experiment.

[0088] (2) Preparation of competent cells for electroporation of Escherichia coli containing pCas9 plasmid

[0089] Escherichia coli containing pCas9 plasmid was inoculated into 30 mL of liquid LB medium (added with 15 μL of kanamycin, concentration of 100 mg / mL), cultured at 30°C, 220 rpm, for 12 h, transferred to 50 mL of liquid LB medium (added with 25 μL of kanamycin, 1 mL of arabinose, concentration of 400 g / L), and cultured at 30°C until OD 600 =0.6-0.8 when the competent state is prepared.

[0090] (3) Transformation of pTargetF sgRNA plasmid and integration cassette

[0091] The target gene integration cassette constructed in 1 and the pTargetF sgRNA plasmid constructed in 2 were simultaneously transformed into electroporated E. coli containing the pCas9 plasmid. After incubation at 30°C for 45 minutes, the cells were plated on LB agar plates containing kanamycin and spectinomycin and incubated at 30°C for 20-24 hours. Verification primers were designed 100 bp upstream of the upstream homology arm and 100 bp downstream of the downstream homology arm of the gene editing site in the recombinant strain, respectively. Colony PCR was performed to screen for positive transformants. The PCR system is shown in Table 5. Positive transformants were then sequenced.

[0092] Table 5

[0093] Components Volume / μL Upstream verification primers 0.4 Downstream validation primers 0.4 template Single colony 2X phanta UniFi Master Mix 10 <![CDATA[ddH2O]]> 9.2

[0094] 4. Eliminate tool plasmids

[0095] (1) Elimination of pTargrtF sgRNA plasmid

[0096] The strain with correct gene sequencing was inoculated into LB liquid medium containing 0.05% kanamycin and 0.1% IPTG at 30°C and cultured for 12 hours. A sterile inoculation loop was used to streak a ring on an LB agar plate containing kanamycin resistance and cultured at 30°C for 12 hours. Twelve single colonies were picked from the kanamycin-resistant plate and streaked on LB agar plates containing kanamycin, spectinomycin double resistance and kanamycin single resistance, and cultured at 30°C for 12 hours. The colonies that could not grow on the kanamycin and spectinomycin double resistance plates but could grow on the kanamycin single resistance plate were the colonies in which the pTargetF sgRNA plasmid had been removed.

[0097] (2) Elimination of pCas9 plasmid

[0098] The bacteria with the pTargetF sgRNA plasmid removed were inoculated into 50 mL of liquid LB medium and cultured at 42°C for 12 h. A sterile inoculation loop was used to streak a ring on an LB agar plate without antibiotics. The plate was cultured at 30°C for 12 h. Twelve single colonies were picked from the non-resistant plate and streaked onto LB agar plates containing kanamycin monoclonal antibody and non-resistant bacteria, respectively. The plates were cultured at 30°C for 12 h. The colonies that could not grow on the kanamycin-resistant plate but could grow on the non-resistant plate were the colonies that had the pCas9 plasmid removed.

[0099] All primers designed during the construction of the above strains are shown in Table 6.

[0100] Table 6

[0101]

[0102]

[0103]

[0104] Example 2: Histidine production performance test of recombinant strain E. coli His08

[0105] The recombinant strain constructed in Example 1 was taken to test its histidine production performance.

[0106] Strain activation: In a sterile environment, use an inoculating loop to dip a loop of the recombinant strain from the glycerol tube and streak it on three zones of the activation plate. Incubate at 32-36°C for 18-24 hours. Then, pick a single colony of appropriate size and streak it densely on a slant culture medium and incubate at 32-36°C for 18-24 hours.

[0107] The production performance was investigated in a 5L fermenter:

[0108] The recombinant strain was washed from the activated slant and inoculated into a seed culture medium. The culture was shaken at 34-36°C for 10-14 hours, and then inoculated into a fermentation medium at a 15% inoculum. During the fermentation process, the pH was controlled at 6.7-7.0 using dilute sulfuric acid and aqueous ammonia. The aeration rate was 0.8-1.5 vvm, and the agitation was 300-800 rpm. During the fermentation process, 800 g / L of glucose monohydrate solution was added to maintain a glucose concentration of 0.5-3.0 g / L in the fermentation broth.

[0109] Table 7 shows the results of three fermentations of recombinant strains (E. coli His06, E. coli His07, E. coli His08, and E. coli His09) derived from E. coli CICC 10243 (P values ​​< 0.01 indicate extremely significant differences). The results showed that compared with the starting strain, E. coli CICC 10243, E. coli His06, E. coli His07, E. coli His08, and E. coli His09 all showed increased L-histidine production. This suggests that overexpressing histidine pathway genes from Corynebacterium glutamicum, expressing the highly active pyruvate dehydrogenase double mutant aceE*, and simultaneously inactivating the transcription factor slyA in E. coli CICC 10243 can increase L-histidine production.

[0110] The fermentation results of E.coli His08 strain are as follows Figure 1 After 60 h of fermentation, the L-histidine content in the fermentation broth of E. coli His08 reached 58.39 g / L, and the sugar-acid conversion rate was 0.15 g / g, which was significantly better than that of the E. coli His09 strain without aceE mutation.

[0111] Table 7

[0112]

[0113] Sequence Listing used in the present invention

[0114] hisEG nucleotide sequence SEQ ID NO.1

[0115] TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCAAAGAGGAGAAATACTAG

[0116] hisDCB-cg2302-cg2301-HA-impA-FI nucleotide sequence SEQ ID NO.2

[0117] TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCAAAGAGGAGAAATACTAG

[0118] hisN nucleotide sequence SEQ ID NO.3

[0119] TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCAAAGAGGAGAAATACTAG atgagcaaatatgcagacgatttagccttagccctcgaacttgccgaacttgccgattccatcaccctcgaccgcttcgaagcctctgacctggaagtatcctccaagccagacatgactcccgtcagcgatgccgacctggcgaccgaagaagcactccgtgagaaaatcgccaccgcccgccccgccgactccatcctcggtgaagaattcggtggcgacgtagaattcagcggccgccagtggatcatcgaccccatcgacggcaccaaaaactacgtccgcggcgtccccgtatgggcaaccctgatcgcgctgctcgacaacggcaaacccgtcgcaggtgtcatctccgcacccgcactggctaggcgttggtgggcatccgaaggggccggcgcatggcgcaccttcaacggcagctccccacgcaaactgtccgtgtcccaggtgtccaagcttgacgacgcctccctctccttctcctccctctccggctgggccgaacgagatttgcgcgatcagttcgtctccctaactgataccacctggcgactccgcggctacggcgacttcttctcctactgcctcgtcgccgaaggtgccgtcgatatcgccgctgaaccagaagtcagcctctgggatcttgctcccctgtccatcctggtcaccgaagccggaggaaagttcacctcactggctggcgtcgatggaccacacggtggcgatgcagtagccaccaacggcatcctgcacgatgagacgctggatcgtttaaaatag

[0120] hisDCB-cg2302-cg2301 nucleotide sequence SEQ ID NO.4

[0121] TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCAAAGAGGAGAAATACTAG

[0122] hisHA-impA-FI nucleotide sequence SEQ ID NO.5

[0123]

[0124] aceE* promoter and nucleotide sequence SEQ ID NO.6

[0125] TCTTATTGAGCTTTCCGGCGAGAGTTCAATGGGACAGGTTCCAGAAAACTCAACGTTATTAGATAGAT AAGGAATAACCC

[0126] TTTGTTTTAGCAATACAATTGCTGCACACTATTCTAAAAGCCGCATAATATCTTAGCAAGCTAATTAT AAGGAGATGAAA ttggaatcgccactaggttctgatctggcacggttggtgcgcatatggcgtgctctgatagaccatcgcctgaaaccgctggagttaacacaaacccattgggttacgttacacaatatccatcagttacctccagaccagtcgcaaattcaactggcaaaagcgattggcatcgagcagccatcactggtccgtactctggaccaactggaagaaaaagggttaatttcgcgtcaaacttgtgccagcgatcgtcgggctaaacgtattaaactgacggaaaaggcagagccgctgatcagcgaaatggaagctgttattaacaaaacccgcgcggaaatattacatggcatctccgcagaggaactggagtaactgattacgctcatcgcaaaacttgagcataatatcattgagttacaggccaaagggtga

[0127] The amino acid sequence of HisD SEQ ID NO.8

[0128] MLNVTDLRGQTPSKSDIRRALPRGGTDVWSVLPIVQPVVEDVQNRGAEAALDYGEKFDHIRPASVRVPAEVIAAAENTLDPLVRESIEESIRRVRKVHAEQKPSEHTTELSPGGTVTERFMPIDRVGLYVPGGNAVYPSSVIMNTVPAQEAGVNSLVVASPPQAEHGGWPHPTILAACSILGVDEVWAVGGGQAVALLAYGDDAAGLEPVDMITGPGNIFVTAAKRLVRGVVGTDSEAGPTEIAVLADASANAVNVAYDLISQAEHDVMAASVLITDSEQLAKDVNREIEARYSITRNAERVAEALRGAQSGIVLVDDISVGIQVADQYAAEHLEIHTENARAVAEQITNAGAIFVGDFSPVPLGDYSAGSNHVLPTSGSARFSAGLSTHTFLRPVNLIEYDEAALKDVSQVVINFANAEDLPAHGEAIRARFENLPTTDEA*

[0129] The amino acid sequence of HisC SEQ ID NO.9

[0130] MTKITLSDLPLREELRGEHAYGAPQLNVDIRLNTNENPYPPSEALVADLVATVDKIATELNRYPERDAVELRDELAAYITKQTGVAVTRDNLWAANGSNEILQQLLQAFGGPGRTALGFQPSYSMHPILAKGTHTEFIAVSRGADFRIDMDVALEEIRAKQPDIVFVTTPNNPTGDVTSLDDVERIINVAPGIVIVDEAYAEFSPSPSATTLLEKYPTKLVVSRTMSKAFDFAGGRLGYFVANPAFIDAVMLVRLPYHLSALSQAAAIVALRHSADTLGTVEKLSVERVRVAARLEELGYAVVPSESNFVFFGDFSDQHAAWQAFLDRGVLIRDVGIAGHLRTTIGVPEENDAFLDAAAEIIKLNL*

[0131] The amino acid sequence of HisB SEQ ID NO.10

[0132] MTVAPRIGTATRTTSESDITVEINLDGTGKVDIDTGLPFFDHMLTAFGVHGSFDLKVHAKGDIEIDAHHTVEDTAIVLGQALLDAIGDKKGIRRFASCQLPMDEALVESVVDISGRPYFVISGEPDHMITSVIGGHYATVINEHFFETLALNSRITLHVICHYGRDPHHITEAEYKAVARALRGAVEMDPRQTGIPSTKGAL*

[0133] Cg2302 amino acid sequence SEQ ID NO.11

[0134] MNSSPISDMVTAAVQNEPDAGDRWFIYGLFLIAGLFFGGAWSAYKSENKILMVAAGLIAVLAVAGGILWLLGEMT*

[0135] Cg2301 amino acid sequence SEQ ID NO.12

[0136] MWKSPGFVAVLVAVAAAFGSWSLLLPVVPLAVLNNGGSSAVAGATTGIFMAATVITQIFTPAALRKIGYTPVMAFAAFMLGVPAIGYIFSVEPIPVLVVSALRGIGFGALTVAESALVAELVPVRFLGKASGMLGVFIGLSQMLFLPAGLALGDQFGYNVVYVLGAVIALVAAVMCLRIPQVKAAAKQQPQVSEQERSVSTWKLVLVPSLAVTSLSMTFGAVSSFLPAAVIELDPGLGAALAGIILSITGGSSMVFRYLSGVIADRRGVPGTTMIPAQIIGFLGVVLITVTIFQGWSVWLLIIGAVMFGGAFGMVQNEALLSMFFRLPRTRVSEASAIWNIAFDSGTGIGSFLLGIVAASLAYSGAFGSGAVVILFGIVLTTADRIIGRHRITEYNNTRARLRQVPVARRAVQGLRNRRKDR*

[0137] HisH amino acid sequence SEQ ID NO.13

[0138] MTKTVALLDYGSGNLRSAQRALERAGAEVIVSSDPEVCTNADGLLVPGVGAFDACMKGLKNVFGHRIIGQRLAGGRPVMGICVGMQILFDEGDEHGIKSAGCGEWPGKVERLQAEILPHMGWNTLEMPTNSPMFEGISPDERFYFVHSYGVRKWTLETDDLTTPPEVVWAKHENDRFVAAVENGTLWATQFHPEKSGDAGAQLLRNWINYI*

[0139] HisA amino acid sequence SEQ ID NO.14

[0140] MTFTILPAVDVVNGQAVRLDQGEAGTEKSYGTPLESALKWQEQGAKWLHFVDLDAAFNRGSNHEMMAEIVGKLDVDVELTGGIRDDESLERALATGARRVNIGTAALEKPEWIASAIQRYGEKIAVDIAVRLEDGEWRTRGNGWVSDGGDLWEVLERLDSQGCARFVVTDVSKDGTLSGPNVELLREVAAATDAPIVASGGISVLEDVLELAKYQDEGIDSVIIGKALYEHKFTLEEALAAVEKLG*

[0141] ImpA amino acid sequence SEQ ID NO.15

[0142] MDARGMLAIAEAVVDDAEALFMQGFGAAPAHMKSPGDFATEVDMAIESHMRSMLNMMTGIAVIGEEGGGATSGTRWVIDPIDGTANFAASNPMSAILVSLLVDDQPVLGITSMPMLGKRLTAFEGSPLMINGEPQEPLQEQSSLVSHIGFSSMASPRNTAFPVELRRDLLTELTESYLRPRITGSVGVDLAFTAQGIFGACVSFSPHVWDNSAGVMLMRAAGAQVTDTEGHPWAPGRGVVAGTKRAHDVLLSKIEKVRLMHADAGNDQSLNEEYK*

[0143] HisF amino acid sequence SEQ ID NO.16

[0144] MGVAIRVIPCLDVDNGRVVKGVNFENLRDAGDPVELAKRYDEEGADELTFLDVTASKHGRGTMLDVVRRTADQVFIPLTVGGGVRSEEDVDQLLRAGADKVSVNTSAIARPELLSELSKRFGAQCIVLSVDARRVPEGGTPQPSGFEVTTHGGSKSAELDAIEWAKRGEELGVGEILLNSMDGDGTKNGFDLELLEKVRAAVSIPVIASGGAGKAEHFPPAVAAGANAVLAATIFHFREVTIAEVKGAIKDAGFEVRK*

[0145] HisI amino acid sequence SEQ ID NO.17

[0146] MSDNPQEYELDWDVEKRLKLNDAGLVPAIVQADGTNEVLMMAWMDTHALAYTLATRRGTYFSRSRNEYWIKGLTSGNVQEVTGLALDCDGDTVLLTVKQTGGACHTGAHTCFDNDVLL*

[0147] HisN amino acid sequence SEQ ID NO.18

[0148] MSKYADDLALALELAELADSITLDRFEASDLEVSSKPDMTPVSDADLATEEALREKIATARPADSILGEEFGGDVEFSGRQWIIDPIDGTKNYVRGVPVWATLIALLDNGKPVAGVISAPALARRWWASEGAGAWRTFNGSSPRKLSVSQVSKLDDASLSFSSLSGWAERDLRDQFVSLTDTTWRLRGYGDFFSYCLVAEGAVDIAAEPEVSLWDLAPLSILVTEAGGKFTSLAGVDGPHGGDAVATNGILHDETLDRLK*

[0149] HisE amino acid sequence SEQ ID NO.19

[0150] MKTFDSLYEELLNRAQTRPEGSGTVAALDKGIHHLGKKVIEEAGEVWIAAEYETDEELAGEISQLIYWTQ VIMVARGLKPEDIYKNL*

[0151] HisG amino acid sequence SEQ ID NO.20

[0152] MLKIAVPNKGSLSERAMEILAEAGYAGRGDSKSLNVFDEANNVEFFFLRPKDIAIYVAGGQLDLGITGRDLARDSQADVHEVLSLGFGSSTFRYAAPADEEWSIEKLDGKRIATSYPNLVRDDLAARGLSAEVLRLDGAVEVSIKLGVADAIADVVSTGRTLRQQGLAPFGEVLCTSEAVIVGRKDEKVTPEQQILLRRIQGILHAQNFLMLDYNVDRDNLDAATAVTPGLSGPTVSPLARDNWVAVRAMVPRRSANAIMDKLAGLGAEAILASEIRIARI*

[0153] Amino acid sequence of AceE SEQ ID NO.21

[0154] MSERFPNDVDPIETRDWLQAIESVIREEGVERAQYLIDQLLAEARKGGVNVAAGTGISNYINTIPVEEQPEYPGNLELERRIRSAIRWNAIMTVLRASKKDLELGGHMASFQSSATIYDVCFNHFFRARNEQDGGDLVYFQGHISPGVYARAFLEGRLTQEQLDNFRQEVHGNGLSSYPHPKLMPEFWQFPTVSMGLGPIGAIYQAKFLKYLEHRGLKDTSKQTVYAFLGDGEMDEPESKGAITIATREKLDNLVFVINCNLQRLDGPVTGNGKIINELEGIFEGAGWNVIKVMWGSRWDELLRKDTSGKLIQLMNETVDGDYQTFKSKDGAYVREHFFGKYPETAALVADWTDEQIWALNRGGHDPKKIYAAFKKAQETKGKATVILAHTIKGYGMGDAAEGKNIAHQVKKMNMDGVRHIRDRFNVPVSDADIEKLPYITFPEGSEEHTYLHAQRQKLHGYLPSRQPNFTEKLELPSLQDFGALLEEQSKEISTTIAFVRALNVMLKNKSIKDRLVPIIADEARTFGMEGLFRQIGIYSPNGQQYTPQDREQVAYYKEDEKGQILQEGINELGAGCSWLAAATSYSTNNLPMIPFYIYYSMFGFQRIGDLCWAAGDQQARGFLIGGTSGRTTLNGEGLQHEDGHSHIQSLTIPNCISYDPAYAYEVAVIMHDGLERMYGEKQENVYYYITTLNENYHMPAMPEGAEEGIRKGIYKLETIEGSKGKVQLLGSGSILRHVREAAEILAKDYGVGSDVYSVTSFTELARDGQDCERWNMLHPLETPRVPYIAQVMNDAPAVASTDYMKLFAEQVRTYVPADDYRVLGTDGFGRSDSRENLRHHFEVDASYVVVAALGELAKRGEIDKKVVADAIAKFNIDADKVNPRLA*

[0155] Amino acid sequence of AceE mutant SEQ ID NO.22

[0156] MSERFPNDVDPIETRDWLQAIESVIREEGVERAQYLIDQLLAEARKGGVNVAAGTGISNYINTIPVEEQPEYPGNLELERRIRSAIRWNAIMTVLRASKKDLELGGHMASFQSSATIYDVCFNHFFRARNEQDGGDLVYFQGHISPGVYARAFLEGRLTQEQLDNFRQEVHGNGLSSYPHPKLMPEFWQFPTVSMGLGPIGAIYQAKFLKYLEHRGLKDTSKQTVYAFLGDGEMDEPESKGAITIATREKLDNLVFVINCNLQRLDGPVTGNGKIINELEGIFEGAGWNVIKVMWGSRWDELLRKDTSGKLIQLMNETVDGDYQTFKSKDGAYVREHFFGKYPETAALVADWTDEQIWALNRGGHDPKKIYAAFKKAQETKGKATVILAHTIKGYGMGDAAEGKNIAHQVKKMNMDGVRHIRDRFNVPVSDTDIEKLPYITFPEGSEEHTYLHAQRQKLHGYLPSRQPNFTEKLELPSLQDFGALLEEQSKEISTTIAFVRALNVMLKNKSIKDRLVPIIADEARTFGMEGLFRQIGIYSPNGQQYTPQDREQVAYYKEDEKGQILQEGINELGAGCSWLAAATSYSTNNLPMIPFYIYYSMFGFQRIGDLCWAAGDQQARGFLIGGTSGRTTLNQEGLQHEDGHSHIQSLTIPNCISYDPAYAYEVAVIMHDGLERMYGEKQENVYYYITTLNENYHMPAMPEGAEEGIRKGIYKLETIEGSKGKVQLLGSGSILRHVREAAEILAKDYGVGSDVYSVTSFTELARDGQDCERWNMLHPLETPRVPYIAQVMNDAPAVASTDYMKLFAEQVRTYVPADDYRVLGTDGFGRSDSRENLRHHFEVDASYVVVAALGELAKRGEIDKKVVADAIAKFNIDADKVNPRLA*

[0157] The amino acid sequence of SlyA SEQ ID NO.23

[0158] LESPLGSDLARLVRIWRALIDHRLKPLELTQTHWVTLHNIHQLPPDQSQIQLAKAIGIEQPSLVRTLDQLEEKGLISRQTCASDRRAKRIKLTEKAEPLISEMEAVINKTRAEILHGISAEELEQLITLIAKLEHNIIELQAKG*

[0159] SlyA mutant amino acid sequence SEQ ID NO.24

[0160] LESPLGSDLARLVRIWRALIDHRLKPLELTQTHWVTLHNIHQLPPDQSQIQLAKAIGIEQPSLVRTLDQLEEKGLISRQTCASDRRAKRIKLTEKAEPLISEMEAVINKTRAEILHGISAEELE*

[0161] Although the present invention has been disclosed as above with preferred embodiments, it should be noted that the specific embodiments in the specification are only used to illustrate the technical principles and do not constitute a limitation on the scope of protection of the present invention. The ultimate scope of rights shall be subject to the description of the claims. Those skilled in the art, without departing from the technical core of the present invention and the definition of the claims, may make equivalent substitutions, adjust process parameters, or modify implementation methods for the raw material components, ratio parameters, and other elements described in the embodiments, which fall within the scope of patent protection of the present invention.

Claims

1. A pyruvate dehydrogenase mutant, characterized in that: The pyruvate dehydrogenase mutant is based on the amino acid sequence of SEQ ID NO. 21, with the alanine at position 432 mutated to threonine and the glycine at position 636 mutated to glutamine.

2. A polynucleotide encoding the pyruvate dehydrogenase mutant according to claim 1.

3. A plasmid vector carrying the polynucleotide of claim 2 or a cell expressing the pyruvate dehydrogenase mutant of claim 1.

4. A recombinant Escherichia coli, characterized in that The recombinant Escherichia coli expresses the pyruvate dehydrogenase mutant according to claim 1.

5. The recombinant Escherichia coli according to claim 4, characterized in that The recombinant Escherichia coli also overexpresses the histidine operon genes hisEG, hisDCB-cg2302-cg2301-HA-impA-FI and hisN derived from Corynebacterium glutamicum; hisDCB-cg2302-cg2301-HA-impA-FI is the sequential arrangement of hisD, hisC, hisB, cg2302, cg2301, hisH, hisA, impA, hisF, and hisI; hisEG is the sequential arrangement of hisE and hisG; Among them, hisEG was integrated into the genomic ldhA, poxB, and yaeQ gene sites, and their start codons were changed from GTG to ATG; hisDCB-cg2302-cg2301-HA-impA-FI was integrated into the yghE gene locus of the E. coli genome; hisN is integrated into the ydfT gene site of the E. coli genome.

6. The recombinant Escherichia coli according to claim 5, characterized in that Promoter PJ23119 was used to express hisEG, hisDCB-cg2302-cg2301-HA-impA-FI, and hisN, respectively.

7. The recombinant Escherichia coli according to claim 5, characterized in that The recombinant E. coli bacteria also mutates the 125th glutamine of the transcription factor SlyA encoding gene slyA into a taa terminator.

8. The recombinant Escherichia coli according to any one of claims 4 to 7, characterized in that The host of the recombinant E. coli bacteria is E. coli CICC 10243, E. coli MG1655, E. coli W3110 or E. coli BL21.

9. Use of the pyruvate dehydrogenase mutant according to claim 1, the polynucleotide according to claim 2, the plasmid vector or cell according to claim 3, or the recombinant Escherichia coli according to any one of claims 4 to 8 in the preparation of histidine.

10. A method for producing L-histidine by fermentation, characterized in that: The method of fermenting and producing L-histidine using the recombinant Escherichia coli bacteria according to any one of claims 4 to 8 comprises the following steps: The recombinant Escherichia coli bacteria according to any one of claims 4 to 8 are inoculated into a seed culture medium, cultured with shaking at 34 to 36° C. for 10 to 14 hours, and then inoculated into a fermentation medium at a 15 to 20% v / v inoculum. The pH is controlled at 6.7 to 7.0 with dilute sulfuric acid and ammonia water, the ventilation rate is 0.8 to 1.5 vvm, and the stirring is 300 to 800 rpm. During the fermentation process, 800 g / L of monohydrate glucose solution is added to control the glucose concentration of the fermentation liquid to be 0.5 to 3.0 g / L.

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