A production strain of fermented l-histidine and application thereof

Abstract

The application discloses a fermentation L-histidine production strain and application thereof, and belongs to the technical field of bioengineering.The application takes E.coli CICC 10243 as a starting strain, integrates histidine synthesis operon genes hisDCB-cg2302-cg2301-HA-impA-FI, hisN and hisEG from corynebacterium glutamicum on the genome, introduces a pyruvate dehydrogenase AceE mutant coding gene, and inactivates the E.coli transcription factor SlyA coding gene slyA.The application prepares a new strain E.coli His08 for fermentation production of L-histidine, and the histidine yield reaches 58.39 g / L in a 5L fermenter for 60 h, and the sugar acid conversion rate is 0.15 g / g, so the application has important application value.

Description

Technical Field

[0001] This invention relates to a strain for producing L-histidine through fermentation and its application, belonging to the field of bioengineering technology. Background Technology

[0002] L-histidine (“histidine”) is one of the 20 standard protein amino acids found in proteins of all living organisms. Its side chain is an imidazole ring, giving it aromatic properties. Because its side chain has a pKa value of 6.0, histidine is the only amino acid whose side chain can transition from a deprotonated state to a protonated state under neutral pH conditions.

[0003] Histidine is a semi-essential amino acid that plays an important role in regulating human physiological functions and in industrial applications. In the pharmaceutical field, histidine is a precursor to histamine, participates in immune regulation and anti-allergic reactions, and is also an adjunct component in the treatment of rheumatoid arthritis and anemia. In the food industry, histidine can be used as a natural preservative and flavor enhancer in functional foods and sports nutrition supplements. In the daily chemical industry, its derivatives, such as imidazole alanine, are often used in skin care products to exert antioxidant and moisturizing functions.

[0004] The main production processes for histidine include chemical synthesis, enzymatic catalysis, and microbial fermentation. Chemical synthesis uses asymmetric catalytic reactions to produce L-histidine, but suffers from low chiral purity and numerous byproducts. Enzymatic catalysis utilizes biocatalysts such as histidine dehydrogenases, but raw material costs are high and enzyme stability is limited. Microbial fermentation involves metabolic engineering of *Escherichia coli* or *Corynebacterium glutamicum*, employing systems metabolic engineering control strategies such as enhanced glucose uptake, phosphoribosyl pyrophosphate (PRPP) synthesis, removal of natural feedback inhibition, and promotion of histidine efflux to increase yield.

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

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

[0007] To address the aforementioned problems, this invention enhances the metabolic flux of the histidine synthesis pathway by introducing the histidine operon gene of Corynebacterium glutamicum into histidine-producing bacteria. Furthermore, through protein engineering, a double mutant is obtained by mutating alanine at position 432 of the aceE gene encoding pyruvate dehydrogenase (AceE) to threonine and glycine at position 636 to glutamine, thereby strengthening the bacterial ATP synthesis capacity. Finally, by inactivating the taa terminator by mutating glutamine at position 125 of the slyA gene encoding the SlyA transcription factor from Escherichia coli, its function is improved by enhancing the physiological characteristics and fermentation performance of the bacteria.

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

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

[0010] A second objective of this invention is to provide a polynucleotide encoding the aforementioned pyruvate dehydrogenase mutant.

[0011] A third objective of this invention is to provide a plasmid vector carrying the aforementioned polynucleotides or cells expressing the aforementioned pyruvate dehydrogenase mutant.

[0012] A fourth objective of this invention is to provide a recombinant Escherichia coli strain expressing the aforementioned 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 derived from Corynebacterium glutamicum, wherein hisDCB-cg2302-cg2301-HA-impA-FI are hisD, hisC, hisB, cg2302, cg2301, hisH, hisA, impA, hisF, hisI in sequence, and hisEG is hisE and hisG in sequence;

[0014] Among them, hisEG is integrated into the ldhA, poxB and yaeQ gene sites in the genome, and its start codon is changed from GTG to ATG;

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

[0016] hisN is integrated into the ydfT gene locus in the Escherichia 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 as shown in SEQ ID NO. 8~20.

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

[0019] In one embodiment, the recombinant Escherichia 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 expresses AceE using the E. coli endogenous promoter P. aceE SlyA is expressed 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) Construct the linker fragment PJ23119-hisEG of promoter PJ23119 and gene hisEG with nucleotide sequence as shown in SEQ ID NO.1, and change its start codon from GTG to ATG, and knock it into the yaeQ, ldhA and poxB gene sites of Escherichia coli genome respectively.

[0024] (2) Construct the promoter PJ23119 and the gene hisDCB-cg2302-cg2301-HA-impA-FI linker PJ23119-hisDCB-cg2302-cg2301-HA-impA-FI with the nucleotide sequence shown in SEQ ID NO.2 (the underlined part is the promoter sequence), and integrate it into the yghE gene site of the Escherichia coli genome in two consecutive steps;

[0025] (3) Construct a linker fragment PJ23119-hisN between the promoter PJ23119 and the gene hisN with the nucleotide sequence shown in SEQ ID NO.3, and knock it into the ydfT gene site of the Escherichia coli genome;

[0026] (4) Construct the gene aceE* linker fragment P, which is the nucleotide sequence of the endogenous aceE promoter of E. coli as shown in SEQ ID NO.6. aceE -aceE*, and knock it into the aceE gene locus of the E. coli genome;

[0027] (5) Construct the linker fragment P with the nucleotide sequence shown in SEQ ID NO.7. slyA -slyA*, and knocked it into the slyA gene locus in the E. coli genome.

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

[0029] A sixth object of the present invention is to provide a method for fermenting L-histidine production, using any of the above-described recombinant Escherichia coli strains to ferment L-histidine, comprising the steps of:

[0030] Inoculate any of the above-mentioned recombinant Escherichia coli strains into seed culture medium and culture at 34-36℃ with shaking for 10-14 h. Inoculate into fermentation medium at an inoculation rate of 15-20%, control the pH at 6.7-7.0 with dilute sulfuric acid and ammonia, aerate at 0.8-1.5 vvm, stir at 300-800 rpm, and add 800 g / L glucose monohydrate solution during fermentation to control the glucose concentration of the fermentation broth at 0.5-3.0 g / L.

[0031] In one embodiment, the production of histidine using the *E. coli* His08 fermentation method includes: inoculating the *E. coli* His08 strain into a seed culture medium, incubating it at 34-36°C with shaking for 10-14 h, inoculating it into a fermentation culture medium at an inoculation rate of 15-20%, controlling the pH at 6.7-7.0 with dilute sulfuric acid and ammonia, aerating the medium at 0.8-1.5 vvm, stirring at 300-800 rpm, and adding 800 g / L glucose monohydrate solution during fermentation to control the glucose concentration in the fermentation broth at 0.5-3.0 g / L.

[0032] In one embodiment, the seed culture medium comprises: 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: glucose 15-25 g / L, yeast powder 5-10 g / L, (NH4)2SO4 3-5 g / L, KH2PO4 3-5 g / L, and MgSO4·7H2O 1.0-1.5 g / L.

[0034] Advantages of this invention:

[0035] This invention enhances the metabolic flux of the histidine synthesis pathway by introducing the histidine operon gene of Corynebacterium glutamicum into histidine-producing bacteria; it also strengthens the bacterial ATP synthesis capacity by modifying the aceE gene encoding pyruvate dehydrogenase (ACEE) to a double mutant, where alanine at position 432 is mutated to threonine and glycine at position 636 is mutated to glutamine; and it improves the physiological characteristics and fermentation performance of the bacteria by inactivating the taa terminator of the slyA gene encoding the SlyA transcription factor of Escherichia coli.

[0036] The above metabolic engineering and protein engineering strategies improved the histidine concentration in the fermentation broth of E. coli with glucose as the single carbon source. The recombinant strain E. coli His08 was fermented in a 5L fermenter for 60 h, and the histidine yield reached 58.39 g / L with a sugar-acid conversion rate of 0.15 g / g. Attached Figure Description

[0037] Figure 1 A graph showing the yield variation during the fermentation process of histidine production by recombinant strain E.coli His08. Detailed Implementation

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

[0039] In the following examples, Escherichia coli (Escherichia coli) Escherichia coli The starting strain was purchased from CICC, number 10243, and is also applicable to other Escherichia coli that can produce amino acids.

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

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

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

[0043] raw material:

[0044] Activation culture medium components: 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 g / L, NaCl 5-10 g / L;

[0046] Fermentation medium composition: 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 chassis strain, recombinant bacteria E.coliHis01, E.coli His02, E.coli His03, E.coli His04, E.coli His05, E.coli His06, E.coliHis07 and E.coli His08 were constructed, and their genotypes are shown in Table 1.

[0049] Table 1

[0050]

[0051] The recombinant bacteria described in Table 1 are constructed as follows:

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

[0053] Based on the genome sequence of *Escherichia coli* CICC 10243 obtained from NCBI, primers for upper and lower homologous arms (500 bp upstream and downstream of the yaeQ gene) were designed (primer sequences are shown in Table 6). The upper and lower homologous arm fragments (UH-hisEG(yaeQ), DH-hisEG(yaeQ)) were amplified by PCR. Primers for amplification of the hisEG gene (nucleotide sequence as shown in SEQ ID NO.1, underlined portion is the promoter sequence) were designed, with the PJ23119 promoter designed in the downstream primer of the upper homologous arm and the upstream primer of hisEG. The hisEG gene fragment was obtained by PCR. The upper and lower homologous arm fragments and the hisEG gene fragment were then used to obtain the integration frame by fusion PCR. The pTargetF sgRNA-yaeQ plasmid was obtained by circular PCR using primers N20-yaeQ-F and N20-yaeQ-R. This plasmid, along with the hisEG integration frame, was electroporated into E. coli CICC 10243 (containing pCas9). Single colonies were selected on LB agar plates protected with kanamycin and spectinomycin for colony PCR identification and sequencing to obtain the correct recombinant bacteria. After removing the plasmid, the strain E. coli His01 was obtained.

[0054] HisEG was integrated into the ldhA site (constructing E. coli His02):

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

[0056] HisEG was integrated into the poxB site (constructing E. coli His03):

[0057] Based on the genome sequence of *Escherichia coli* CICC 10243 obtained from NCBI, primers for upper and lower homologous arms (500 bp upstream and downstream of the poxB gene) were designed (primer sequences are shown in Table 6). The upper and lower homologous arm fragments (UH-hisEG(poxB), DH-hisEG(poxB)) were amplified by PCR. Primers for amplification were designed based on the hisEG gene (nucleotide sequence as shown in SEQ ID NO. 1). The PJ23119 promoter was designed in the downstream primer of the upper homologous arm and the upstream primer of hisEG. The hisEG gene fragment was obtained by PCR. The upper and lower homologous arm fragments and the hisEG gene fragment were then used to obtain the integration frame by fusion PCR. The pTargetF sgRNA-poxB plasmid was obtained by circular PCR using primers N20-poxB-F and N20-poxB-R. This plasmid, along with the hisEG integration frame, was electroporated into E. coli His02 (containing pCas9). Single colonies were selected on LB agar plates protected with kanamycin and spectinomycin for colony PCR identification and sequencing to obtain the correct recombinant bacteria. After removing the manipulated plasmid, strain E. coli His03 was obtained.

[0058] HisDCB-cg2302-cg2301-HA-impA-FI was integrated into the yghE site (E.coli His04 was constructed, and the first half of the yghE sequence was replaced with hisDCB-cg2302-cg2301):

[0059] The hisDCB-cg2302-cg2301-HA-impA-FI gene was divided into two segments, hisDCB-cg2302-cg2301 and hisHA-impA-FI, which were integrated into the yhgE site in two separate steps. Based on the genome sequence of Escherichia coli CICC 10243 obtained from NCBI, homologous arm primers were designed 500 bp upstream and downstream of the first half (first 430 bp) of the yghE gene (primer sequences are shown in Table 6). The homologous arm fragments (UH-hisDCB-cg2302-cg2301 (yhgE) and DH-hisDCB-cg2302-cg2301 (yhgE)) were amplified by PCR. Based on the hisDCB-cg2302-cg2301 gene (i.e., the first half of SEQ ID NO.2, nucleotide sequence as shown in SEQ ID NO.2), the homologous arm fragments were amplified. Primers were designed for amplification (NO.4, underlined portion is the promoter sequence). The PJ23119 promoter was designed in the downstream primer of the upper homologous arm and the upstream primer of hisDCB-cg2302-cg2301. The hisDCB-cg2302-cg2301 gene fragment was obtained by PCR. The upper and lower homologous arm fragments and the hisDCB-cg2302-cg2301 gene fragment were used to obtain the integration frame by fusion PCR. The pTargetFsgRNA-yghE1 plasmid was obtained by circular PCR using primers N20-yghE1-F and N20-yghE1-R. This plasmid, along with the hisDCB-cg2302-cg2301 integration frame, was electroporated into E. coli His03 (containing pCas9). Single colonies were selected on LB agar plates protected with kanamycin and spectinomycin for colony PCR identification and sequencing to obtain the correct recombinant bacteria. After removing the manipulated plasmid, strain E. coli His04 was obtained.

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

[0061] Primers for upper and lower homologous arms (primer sequences shown in Table 6) were designed 500 bp upstream and downstream of the latter half (431-861 bp) of the yghE gene in E. coli His04 strain. The upper and lower homologous arm fragments (UH-hisHA-impA-FI(yhgE) and DH-hisHA-impA-FI(yhgE)) were amplified by PCR. Primers for amplification based on the hisHA-impA-FI gene (nucleotide sequence as shown in SEQ ID NO. 5, i.e., the latter half of SEQ ID NO. 2) were designed, with the PJ23119 promoter designed in the downstream primer of the upper homologous arm and the upstream primer of hisHA-impA-FI. The hisHA-impA-FI gene fragment was obtained by PCR. The upper and lower homologous arm fragments and the hisHA-impA-FI gene fragment were then used to obtain the integration frame by fusion PCR. The pTargetF sgRNA-yghE2 plasmid was obtained by circular PCR using primers N20-yghE2-F and N20-yghE2-R. This plasmid, along with the hisHA-impA-FI integration frame, was electroporated into E. coli His04 (containing pCas9). Single colonies were selected on LB agar plates protected with kanamycin and spectinomycin for colony PCR identification and sequencing to obtain the correct recombinant bacteria. After removing the manipulated plasmid, the strain E. coli His05 was obtained.

[0062] HisN was integrated into the ydfT site (constructing E.coli His06):

[0063] Based on the genome sequence of *Escherichia coli* CICC 10243 obtained from NCBI, primers for upper and lower homologous arms (500 bp upstream and downstream of the ydfT gene) were designed (primer sequences are shown in Table 6). The upper and lower homologous arm fragments (UH-hisN(ydfT), DH-hisN(ydfT)) were amplified by PCR. Primers for amplification of the hisN gene (nucleotide sequence as shown in SEQ ID NO.3, underlined portion is the promoter sequence) were designed, with the PJ23119 promoter designed in the downstream primer of the upper homologous arm and the upstream primer of hisN. The hisN gene fragment was obtained by PCR. The upper and lower homologous arm fragments and the hisN gene fragment were then used to obtain the integration frame by fusion PCR. The pTargetF sgRNA-ydfT plasmid was obtained by circular PCR using primers N20-ydfT-F and N20-ydfT-R. This plasmid, along with the hisN integration frame, was electroporated into E. coli His05 (containing pCas9). Single colonies were selected on LB agar plates protected with kanamycin and spectinomycin for colony PCR identification and sequencing to obtain the correct recombinant bacteria. After removing the manipulated plasmid, strain E. coli His06 was obtained.

[0064] Replace endogenous aceE in E. coli with aceE* (construct E. coli His07):

[0065] Based on the genome sequence of *Escherichia coli* CICC 10243 and the aceE* gene (nucleotide sequence as shown in SEQ ID NO. 6 (underlined part is the promoter sequence), amino acid sequence as shown in SEQ ID NO. 21) obtained from NCBI, amplification primers were designed. At amino acid position 432 of the aceE gene, a downstream primer for the upper homologous arm and an upstream primer for the middle fragment were designed, moving forward and backward respectively. The A432T mutation site was designed in the downstream primer for the upper homologous arm and the upstream primer for the middle fragment. At amino acid position 636 of the aceE gene, a downstream primer for the middle fragment and an upstream primer for the lower homologous arm were designed, moving forward and backward respectively. The G636Q mutation site was designed in the downstream primer for the middle fragment and the upstream primer for the lower homologous arm. A 100 bp upstream primer for the upper homologous arm and a downstream primer for the lower homologous arm were designed upstream and downstream of the aceE gene (primer sequences are shown in Table 6). PCR amplification was performed on the upper and lower homologous arms and the middle fragment (UH-aceE*, DH-aceE*). The aceE* integration frame fragment was obtained through 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 frame, was electroporated into E. coli His06 (containing pCas9). Single colonies were selected on LB agar plates protected with kanamycin and spectinomycin for colony PCR identification and sequencing to obtain the correct recombinant bacteria. After removing the plasmid, strain E. coli His07 was obtained (the aceE mutant aceE-A432T / G636Q was constructed, with the amino acid sequence shown in SEQ ID NO.22).

[0066] Replace endogenous slyA in E. coli with slyA* (to construct E. coli His08 and E. coli His09):

[0067] According to the E. coli query from NCBI ( Escherichia coliPrimers were designed based on the CICC 10243 genome sequence and the slyA* gene (nucleotide sequence as shown in SEQ ID NO.7, the underlined part is the promoter sequence). Downstream primers for the upper homologous arm and upstream primers for the lower homologous arm were designed forward and backward at amino acid position 125 of the slyA gene, respectively. The Q125* (mutated to the terminator taa) mutation site was designed in the downstream primers for the upper homologous arm and the upstream primers for the lower homologous arm. Upstream primers for the upper homologous arm and downstream primers for the lower homologous arm were designed 100 bp upstream and downstream of slyA (primer sequences are shown in Table 6). The upper and lower homologous arm fragments (UH-slyA*, DH-slyA*) were amplified by PCR, 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. This plasmid, along with the slyA* integration frame, was electroporated into E. coli His07 (containing pCas9). Single colonies were selected on LB agar plates protected with kanamycin and spectinomycin for colony PCR identification and sequencing to obtain the correct recombinant bacteria. After removing the manipulated plasmid, strain E. coli His08 was obtained.

[0068] The pTargetF sgRNA-slyA and slyA* integration frames were electroporated into E. coli His06 (containing pCas9). Single colonies were selected on kanamycin and spectinomycin double antibody LB agar plates for colony PCR identification and sequencing 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. Constructing the integration frame of the target gene

[0071] The gene fragment used for integration consists of an upstream homologous arm, a target gene, and a downstream homologous arm. Bacterial genomes, plasmids, or single-clone colonies are used as templates 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 95 ℃, 1 min; denaturation 95 ℃, 15 s; annealing 55-60 ℃, 15 s; extension 72 ℃, 30 s-2 min (depending on fragment length, 2 kb / min), 34 cycles. After obtaining each fragment, an integration frame was constructed by overlap fusion PCR. The PCR fusion system is shown in Table 3.

[0073] Table 2

[0074]

[0075] Table 3

[0076]

[0077] 2. Construct pTargetF sgRNA plasmid

[0078] The pTargetF sgRNA plasmid can transcribe the gRNA of the target gene, which forms a complex with the Cas9 protein. This complex guides the Cas9 protein to recognize and cleave the target gene, causing a DSB (Device Stranding) at the target gene site in the genome. Subsequently, the target gene is integrated into the bacterial genome through the bacteria's own homologous recombination system. The specific construction steps are as follows:

[0079] (1) Design of the guiding sequence

[0080] Design target gene guide sequences using the CRISPR RGEN Tools website.

[0081] (2) Plasmid circularization PCR

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

[0083] Table 4

[0084]

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

[0086] (1) Transformation of pCas9 plasmid

[0087] The pCas9 plasmid was electroporated into E. coli CICC 10243 electroporated competent cells, incubated at 30 ℃ for 45 min, plated on LB agar plates containing kanamycin, and incubated at 30 ℃ for 24 h. Positive single colonies were selected for the next step of the experiment.

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

[0089] E. coli containing the pCas9 plasmid were inoculated into 30 mL of liquid LB medium (with 15 μL kanamycin, concentration 100 mg / mL) and cultured at 30 ℃ and 220 rpm for 12 h. The culture was then transferred to 50 mL of liquid LB medium (with 25 μL kanamycin and 1 mL arabinose, concentration 400 g / L) and cultured at 30 ℃ until OD500 was reached. 600 Competent cells were prepared when the ratio was 0.6 to 0.8.

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

[0091] The target gene integration frame constructed in step 1 and the pTargetF sgRNA plasmid constructed in step 2 were simultaneously transformed into E. coli electrotransformation competent cells containing the pCas9 plasmid. After incubation at 30°C for 45 min, the bacterial cells were plated on LB agar plates containing kanamycin and spectinomycin and incubated at 30°C for 20–24 h. Validation primers were designed 100 bp upstream of the homologous arm and 100 bp downstream of the homologous arm upstream of the gene editing site in the recombinant strain for colony PCR screening of positive transformants. The PCR system is shown in Table 5. The genes of the positive transformants were sequenced.

[0092] Table 5

[0093]

[0094] 4. Eliminate tool plasmids

[0095] (1) Elimination of pTargrtF sgRNA plasmid

[0096] Strains with correct gene sequencing were inoculated into LB liquid medium containing 0.05% kanamycin and 0.1% IPTG and incubated at 30°C for 12 h. A loopful of the inoculated strain was streaked onto an LB agar plate containing kanamycin resistance and incubated at 30°C for 12 h. Twelve single colonies were then picked from the kanamycin resistance plate and streaked sequentially onto LB agar plates containing kanamycin, spectinomycin dual resistance, and kanamycin monoclonal resistance and incubated at 30°C for 12 h. Colonies that could not grow on the kanamycin and spectinomycin dual resistance plates but could grow on the kanamycin monoclonal resistance plates were those colonies with pTargetF sgRNA plasmid 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 incubated at 42 °C for 12 h. One loopful of bacteria was then streaked onto an antibiotic-free LB agar plate and incubated at 30 °C for 12 h. Twelve single colonies were then picked from the antibiotic-free plate and streaked sequentially onto LB agar plates containing kanamycin monoclonal antibody and those without. The colonies were incubated at 30 °C for 12 h. The colonies that could not grow on the kanamycin-resistant plate but could grow on the antibiotic-free plate were those with 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] Example 2: Histidine production performance test of recombinant strain E. coli His08

[0103] The recombinant strain constructed in Example 1 was used to test its histidine production performance.

[0104] Strain activation: In a sterile environment, use an inoculation loop to take a loopful of recombinant strain from the glycerol tube and streak it on the three zones of an activation plate. Incubate at 32-36 ℃ for 18-24 h. Then, pick a single colony of suitable size and streak it densely on a slant culture medium. Incubate at 32-36 ℃ for 18-24 h.

[0105] Its production performance was investigated in a 5 L fermenter:

[0106] The recombinant strain was washed off the activated slant and inoculated into seed culture medium, and cultured with shaking at 34–36 °C for 10–14 h. Then, it was inoculated into fermentation medium at a 15% inoculum size. During fermentation, the pH was controlled at 6.7–7.0 using dilute sulfuric acid and ammonia, the aeration rate was 0.8–1.5 vvm, and the stirring speed was 300–800 rpm. During fermentation, 800 g / L glucose monohydrate solution was added to maintain the glucose concentration in the fermentation broth at 0.5–3.0 g / L.

[0107] The results of three fermentation experiments using recombinant strains E. coli His06, E. coli His07, E. coli His08, and E. coli His09, derived from E. coli CICC 10243, are shown in Table 7 (P < 0.01 indicates highly significant difference). The results showed that compared with the starting strain E. coli CICC 10243, the L-histidine production of E. coli His06, E. coli His07, E. coli His08, and E. coli His09 was increased. This indicates that overexpression of the histidine pathway gene of Corynebacterium glutamicum and expression of the highly active pyruvate dehydrogenase double mutant aceE*, while simultaneously inactivating the transcription factor slyA, can increase L-histidine production in E. coli CICC 10243.

[0108] Fermentation results of E. coli His08 strain are as follows: Figure 1 After 60 h of fermentation, the L-histidine content 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 non-aceE mutated strain E. coli His09.

[0109] Table 7

[0110]

[0111] The sequence list used in this invention

[0112] hisEG nucleotide sequence SEQ ID NO.1

[0113] TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCAAAGAGGAGAAATACTAG

[0114] hisDCB-cg2302-cg2301-HA-impA-FI nucleotide sequence SEQ ID NO.2

[0115] TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCAAAGAGGAGAAATACTAG

[0116] The hisN nucleotide sequence SEQ ID NO.3

[0117] TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCAAAGAGGAGAAATACTAG atgagcaaatatgcagacgatttagccttagccctcgaacttgccgaacttgccgattccatcaccctcgaccgcttcgaagcctctgacctggaagtatcctccaagccagacatgactcccgtcagcgatgccgacctggcgaccgaagaagcactccgtgagaaaatcgccaccgcccgccccgccgactccatcctcggtgaagaattcggtggcgacgtagaattcagcggccgccagtggatcatcgaccccatcgacggcaccaaaaactacgtccgcggcgtccccgtatgggcaaccctgatcgcgctgctcgacaacggcaaacccgtcgcaggtgtcatctccgcacccgcactggctaggcgttggtgggcatccgaaggggccggcgcatggcgcaccttcaacggcagctccccacgcaaactgtccgtgtcccaggtgtccaagcttgacgacgcctccctctccttctcctccctctccggctgggccgaacgagatttgcgcgatcagttcgtctccctaactgataccacctggcgactccgcggctacggcgacttcttctcctactgcctcgtcgccgaaggtgccgtcgatatcgccgctgaaccagaagtcagcctctgggatcttgctcccctgtccatcctggtcaccgaagccggaggaaagttcacctcactggctggcgtcgatggaccacacggtggcgatgcagtagccaccaacggcatcctgcacgatgagacgctggatcgtttaaaatag

[0118] The hisDCB-cg2302-cg2301 nucleotide sequence SEQ ID NO.4

[0119] TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCAAAGAGGAGAAATACTAG

[0120] hisHA-impA-FI nucleotide sequence SEQ ID NO.5

[0121]

[0122] aceE* promoter and nucleotide sequence SEQ ID NO.6

[0123] TCTTATTGAGCTTTCCGGCGAGAGTTCAATGGGACAGGTTCCAGAAAACTCAACGTTATTAGATAGAT AAGGAATAACCC

[0124] The slyA* promoter and nucleotide sequence SEQ ID NO.7

[0125] TTTGTTTTAGCAATACAATTGCTGCACACTATTCTAAAAGCCGCATAATATCTTAGCAAGCTAATTAT AAGGAGATGAAA ttggaatcgccactaggttctgatctggcacggttggtgcgcatatggcgtgctctgatagaccatcgcctgaaaccgctggagttaacacaaacccattgggttacgttacacaatatccatcagttacctccagaccagtcgcaaattcaactggcaaaagcgattggcatcgagcagccatcactggtccgtactctggaccaactggaagaaaaagggttaatttcgcgtcaaacttgtgccagcgatcgtcgggctaaacgtattaaactgacggaaaaggcagagccgctgatcagcgaaatggaagctgttattaacaaaacccgcgcggaaatattacatggcatctccgcagaggaactggagtaactgattacgctcatcgcaaaacttgagcataatatcattgagttacaggccaaagggtga

[0126] The HisD amino acid sequence SEQ ID NO.8

[0127] MLNVTDLRGQTPSKSDIRRALPRGGTDVWSVLPIVQPVVEDVQNRGAEAALDYGEKFDHIRPASVRVPAEVIAAAENTLDPLVRESIEESIRRVRKVHAEQKPSEHTTELSPGGTVTERFMPIDRVGLYVPGGNAVYPSSVIMNTVPAQEAGVNSLVVASPPQAEHGGWPHPTILAACSILGVDEVWAVGGGQAVALLAYGDDAAGLEPVDMITGPGNIFVTAAKRLVRGVVGTDSEAGPTEIAVLADASANAVNVAYDLISQAEHDVMAASVLITDSEQLAKDVNREIEARYSITRNAERVAEALRGAQSGIVLVDDISVGIQVADQYAAEHLEIHTENARAVAEQITNAGAIFVGDFSPVPLGDYSAGSNHVLPTSGSARFSAGLSTHTFLRPVNLIEYDEAALKDVSQVVINFANAEDLPAHGEAIRARFENLPTTDEA*

[0128] Amino acid sequence of HisC SEQ ID NO.9

[0129] MTKITLSDLPLREELRGEHAYGAPQLNVDIRLNTNENPYPPSEALVADLVATVDKIATELNRYPERDAVELRDELAAYITKQTGVAVTRDNLWAANGSNEILQQLLQAFGGPGRTALGFQPSYSMHPILAKGTHTEFIAVSRGADFRIDMDVALEEIRAKQPDIVFVTTPNNPTGDVTSLDDVERIINVAPGIVIVDEAYAEFSPSPSATTLLEKYPTKLVVSRTMSKAFDFAGGRLGYFVANPAFIDAVMLVRLPYHLSALSQAAAIVALRHSADTLGTVEKLSVERVRVAARLEELGYAVVPSESNFVFFGDFSDQHAAWQAFLDRGVLIRDVGIAGHLRTTIGVPEENDAFLDAAAEIIKLNL*

[0130] Amino acid sequence of HisB SEQ ID NO.10

[0131] MTVAPRIGTATRTTSESDITVEINLDGTGKVDIDTGLPFFDHMLTAFGVHGSFDLKVHAKGDIEIDAHHTVEDTAIVLGQALLDAIGDKKGIRRFASCQLPMDEALVESVVDISGRPYFVISGEPDHMITSVIGGHYATVINEHFFETLALNSRITLHVICHYGRDPHHITEAEYKAVARALRGAVEMDPRQTGIPSTKGAL*

[0132] Cg2302 amino acid sequence SEQ ID NO.11

[0133] MNSSPISDMVTAAVQNEPDAGDRWFIYGLFLIAGLFFGGAWSAYKSENKILMVAAGLIAVLAVAGGILWLLGEMT*

[0134] Cg2301 amino acid sequence SEQ ID NO.12

[0135] MWKSPGFVAVLVAVAAAFGSWSLLLPVVPLAVLNNGGSSAVAGATTGIFMAATVITQIFTPAALRKIGYTPVMAFAAFMLGVPAIGYIFSVEPIPVLVVSALRGIGFGALTVAESALVAELVPVRFLGKASGMLGVFIGLSQMLFLPAGLALGDQFGYNVVYVLGAVIALVAAVMCLRIPQVKAAAKQQPQVSEQERSVSTWKLVLVPSLAVTSLSMTFGAVSSFLPAAVIELDPGLGAALAGIILSITGGSSMVFRYLSGVIADRRGVPGTTMIPAQIIGFLGVVLITVTIFQGWSVWLLIIGAVMFGGAFGMVQNEALLSMFFRLPRTRVSEASAIWNIAFDSGTGIGSFLLGIVAASLAYSGAFGSGAVVILFGIVLTTADRIIGRHRITEYNNTRARLRQVPVARRAVQGLRNRRKDR*

[0136] HisH amino acid sequence SEQ ID NO.13

[0137] MTKTVALLDYGSGNLRSAQRALERAGAEVIVSSDPEVCTNADGLLVPGVGAFDACMKGLKNVFGHRIIGQRLAGGRPVMGICVGMQILFDEGDEHGIKSAGCGEWPGKVERLQAEILPHMGWNTLEMPTNSPMFEGISPDERFYFVHSYGVRKWTLETDDLTTPPEVVWAKHENDRFVAAVENGTLWATQFHPEKSGDAGAQLLRNWINYI*

[0138] HisA amino acid sequence SEQ ID NO.14

[0139] MTFTILPAVDVVNGQAVRLDQGEAGTEKSYGTPLESALKWQEQGAKWLHFVDLDAAFNRGSNHEMMAEIVGKLDVDVELTGGIRDDESLERALATGARRVNIGTAALEKPEWIASAIQRYGEKIAVDIAVRLEDGEWRTRGNGWVSDGGDLWEVLERLDSQGCARFVVTDVSKDGTLSGPNVELLREVAAATDAPIVASGGISVLEDVLELAKYQDEGIDSVIIGKALYEHKFTLEEALAAVEKLG*

[0140] ImpA amino acid sequence SEQ ID NO.15

[0141] MDARGMLAIAEAVVDDAEALFMQGFGAAPAHMKSPGDFATEVDMAIESHMRSMLNMMTGIAVIGEEGGGATSGTRWVIDPIDGTANFAASNPMSAILVSLLVDDQPVLGITSMPMLGKRLTAFEGSPLMINGEPQEPLQEQSSLVSHIGFSSMASPRNTAFPVELRRDLLTELTESYLRPRITGSVGVDLAFTAQGIFGACVSFSPHVWDNSAGVMLMRAAGAQVTDTEGHPWAPGRGVVAGTKRAHDVLLSKIEKVRLMHADAGNDQSLNEEYK*

[0142] HisF amino acid sequence SEQ ID NO.16

[0143] MGVAIRVIPCLDVDNGRVVKGVNFENLRDAGDPVELAKRYDEEGADELTFLDVTASKHGRGTMLDVVRRTADQVFIPLTVGGGVRSEEDVDQLLRAGADKVSVNTSAIARPELLSELSKRFGAQCIVLSVDARRVPEGGTPQPSGFEVTTHGGSKSAELDAIEWAKRGEELGVGEILLNSMDGDGTKNGFDLELLEKVRAAVSIPVIASGGAGKAEHFPPAVAAGANAVLAATIFHFREVTIAEVKGAIKDAGFEVRK*

[0144] HisI amino acid sequence SEQ ID NO.17

[0145] MSDNPQEYELDWDVEKRLKLNDAGLVPAIVQADGTNEVLMMAWMDTHALAYTLATRRGTYFSRSRNEYWIKGLTSGNVQEVTGLALDCDGDTVLLTVKQTGGACHTGAHTCFDNDVLL*

[0146] HisN amino acid sequence SEQ ID NO.18

[0147] MSKYADDLALALELAELADSITLDRFEASDLEVSSKPDMTPVSDADLATEEALREKIATARPADSILGEEFGGDVEFSGRQWIIDPIDGTKNYVRGVPVWATLIALLDNGKPVAGVISAPALARRWWASEGAGAWRTFNGSSPRKLSVSQVSKLDDASLSFSSLSGWAERDLRDQFVSLTDTTWRLRGYGDFFSYCLVAEGAVDIAAEPEVSLWDLAPLSILVTEAGGKFTSLAGVDGPHGGDAVATNGILHDETLDRLK*

[0148] HisE amino acid sequence SEQ ID NO.19

[0149] MKTFDSLYEELLNRAQTRPEGSGTVAALDKGIHHLGKKVIEEAGEVWIAAEYETDEELAGEISQLIYWTQVIMVARGLKPEDIYKNL*

[0150] HisG amino acid sequence SEQ ID NO.20

[0151] MLKIAVPNKGSLSERAMEILAEAGYAGRGDSKSLNVFDEANNVEFFFLRPKDIAIYVAGGQLDLGITGRDLARDSQADVHEVLSLGFGSSTFRYAAPADEEWSIEKLDGKRIATSYPNLVRDDLAARGLSAEVLRLDGAVEVSIKLGVADAIADVVSTGRTLRQQGLAPFGEVLCTSEAVIVGRKDEKVTPEQQILLRRIQGILHAQNFLMLDYNVDRDNLDAATAVTPGLSGPTVSPLARDNWVAVRAMVPRRSANAIMDKLAGLGAEAILASEIRIARI*

[0152] Amino acid sequence of AceE SEQ ID NO.21

[0153] MSERFPNDVDPIETRDWLQAIESVIREEGVERAQYLIDQLLAEARKGGVNVAAGTGISNYINTIPVEEQPEYPGNLELERRIRSAIRWNAIMTVLRASKKDLELGGHMASFQSSATIYDVCFNHFFRARNEQDGGDLVYFQGHISPGVYARAFLEGRLTQEQLDNFRQEVHGNGLSSYPHPKLMPEFWQFPTVSMGLGPIGAIYQAKFLKYLEHRGLKDTSKQTVYAFLGDGEMDEPESKGAITIATREKLDNLVFVINCNLQRLDGPVTGNGKIINELEGIFEGAGWNVIKVMWGSRWDELLRKDTSGKLIQLMNETVDGDYQTFKSKDGAYVREHFFGKYPETAALVADWTDEQIWALNRGGHDPKKIYAAFKKAQETKGKATVILAHTIKGYGMGDAAEGKNIAHQVKKMNMDGVRHIRDRFNVPVSDADIEKLPYITFPEGSEEHTYLHAQRQKLHGYLPSRQPNFTEKLELPSLQDFGALLEEQSKEISTTIAFVRALNVMLKNKSIKDRLVPIIADEARTFGMEGLFRQIGIYSPNGQQYTPQDREQVAYYKEDEKGQILQEGINELGAGCSWLAAATSYSTNNLPMIPFYIYYSMFGFQRIGDLCWAAGDQQARGFLIGGTSGRTTLNGEGLQHEDGHSHIQSLTIPNCISYDPAYAYEVAVIMHDGLERMYGEKQENVYYYITTLNENYHMPAMPEGAEEGIRKGIYKLETIEGSKGKVQLLGSGSILRHVREAAEILAKDYGVGSDVYSVTSFTELARDGQDCERWNMLHPLETPRVPYIAQVMNDAPAVASTDYMKLFAEQVRTYVPADDYRVLGTDGFGRSDSRENLRHHFEVDASYVVVAALGELAKRGEIDKKVVADAIAKFNIDADKVNPRLA*

[0154] Amino acid sequence of AceE mutant, SEQ ID NO.22

[0155] MSERFPNDVDPIETRDWLQAIESVIREEGVERAQYLIDQLLAEARKGGVNVAAGTGISNYINTIPVEEQPEYPGNLELERRIRSAIRWNAIMTVLRASKKDLELGGHMASFQSSATIYDVCFNHFFRARNEQDGGDLVYFQGHISPGVYARAFLEGRLTQEQLDNFRQEVHGNGLSSYPHPKLMPEFWQFPTVSMGLGPIGAIYQAKFLKYLEHRGLKDTSKQTVYAFLGDGEMDEPESKGAITIATREKLDNLVFVINCNLQRLDGPVTGNGKIINELEGIFEGAGWNVIKVMWGSRWDELLRKDTSGKLIQLMNETVDGDYQTFKSKDGAYVREHFFGKYPETAALVADWTDEQIWALNRGGHDPKKIYAAFKKAQETKGKATVILAHTIKGYGMGDAAEGKNIAHQVKKMNMDGVRHIRDRFNVPVSDTDIEKLPYITFPEGSEEHTYLHAQRQKLHGYLPSRQPNFTEKLELPSLQDFGALLEEQSKEISTTIAFVRALNVMLKNKSIKDRLVPIIADEARTFGMEGLFRQIGIYSPNGQQYTPQDREQVAYYKEDEKGQILQEGINELGAGCSWLAAATSYSTNNLPMIPFYIYYSMFGFQRIGDLCWAAGDQQARGFLIGGTSGRTTLNQEGLQHEDGHSHIQSLTIPNCISYDPAYAYEVAVIMHDGLERMYGEKQENVYYYITTLNENYHMPAMPEGAEEGIRKGIYKLETIEGSKGKVQLLGSGSILRHVREAAEILAKDYGVGSDVYSVTSFTELARDGQDCERWNMLHPLETPRVPYIAQVMNDAPAVASTDYMKLFAEQVRTYVPADDYRVLGTDGFGRSDSRENLRHHFEVDASYVVVAALGELAKRGEIDKKVVADAIAKFNIDADKVNPRLA*

[0156] SlyA amino acid sequence SEQ ID NO.23

[0157] LESPLGSDLARLVRIWRALIDHRLKPLELTQTHWVTLHNIHQLPPDQSQIQLAKAIGIEQPSLVRTLDQLEEKGLISRQTCASDRRAKRIKLTEKAEPLISEMEAVINKTRAEILHGISAEELEQLITLIAKLEHNIIELQAKG*

[0158] SlyA mutant amino acid sequence SEQ ID NO.24

[0159] LESPLGSDLARLVRIWRALIDHRLKPLELTQTHWVTLHNIHQLPPDQSQIQLAKAIGIEQPSLVRTLDQLEEKGLISRQTCASDRRAKRIKLTEKAEPLISEMEAVINKTRAEILHGISAEELE*

[0160] Although the present invention has been disclosed above with reference to preferred embodiments, it should be specifically 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 final scope of rights is subject to the statements in the claims. Any equivalent substitutions, adjustments to process parameters, or modifications to the implementation methods made by those skilled in the art to the raw material components, proportioning parameters, etc., described in the embodiments without departing from the core technology of the present invention and the definition of the claims, shall fall within the patent protection scope of the present invention.

Claims

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

2. A polynucleotide encoding the pyruvate dehydrogenase mutant of claim 1.

3. A plasmid vector carrying the polynucleotide of claim 2.

4. Cells expressing the pyruvate dehydrogenase mutant of claim 1.

5. A recombinant strain of *Escherichia coli*, characterized in that, The recombinant Escherichia coli expressed the pyruvate dehydrogenase mutant of claim 1.

6. The recombinant Escherichia coli according to claim 5, characterized in that, The recombinant Escherichia coli also overexpressed the histidine operon genes hisEG, hisDCB-cg2302-cg2301-HA-impA-FI and hisN derived from Corynebacterium glutamicum. hisDCB-cg2302-cg2301-HA-impA-FI are hisD, hisC, hisB, cg2302, cg2301, hisH, hisA, impA, hisF, and hisI in sequence; hisEG is hisE and hisG in sequence. Among them, hisEG is integrated into the ldhA, poxB and yaeQ gene sites in the genome, and the start codon of the histidine operon gene hisEG is changed from GTG to ATG. hisDCB-cg2302-cg2301-HA-impA-FI is integrated into the yghE gene locus of the Escherichia coli genome; hisN is integrated into the ydfT gene locus in the Escherichia coli genome.

7. The recombinant Escherichia coli according to claim 6, characterized in that, hisEG, hisDCB-cg2302-cg2301-HA-impA-FI and hisN were expressed using the promoter PJ23119, respectively.

8. The recombinant Escherichia coli according to claim 6, characterized in that, The amino acid sequence of transcription factor SlyA in the recombinant Escherichia coli is shown in SEQ ID NO.

24.

9. The recombinant Escherichia coli according to any one of claims 5 to 8, characterized in that, The recombinant Escherichia coli strain is E. coli CICC 10243, E. coli MG1655, E. coli W3110 or E. coli BL21.

10. The use of the pyruvate dehydrogenase mutant of claim 1, the polynucleotide of claim 2, the plasmid vector of claim 3, the cell of claim 4, or the recombinant Escherichia coli of any one of claims 5 to 9 in the preparation of L-histidine.

11. A method for producing L-histidine by fermentation, characterized in that, The production of L-histidine by fermentation using recombinant Escherichia coli according to any one of claims 5 to 9 includes the following steps: The recombinant Escherichia coli strain described in any one of claims 5 to 9 is inoculated into a seed culture medium and cultured with shaking at 34 to 36°C for 10 to 14 h. The inoculated strain is then inoculated into a fermentation culture medium at an inoculation rate of 15 to 20% v / v. The pH is controlled at 6.7 to 7.0 with dilute sulfuric acid and ammonia. The aeration rate is 0.8 to 1.5 vvm. The mixture is stirred at 300 to 800 rpm. During the fermentation process, 800 g / L glucose monohydrate solution is added to maintain the glucose concentration in the fermentation broth at 0.5 to 3.0 g / L.

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