Method for increasing intracellular ATP (adenosine triphosphate) content and application of method in production of L-histidine

Abstract

The invention relates to a method for increasing intracellular ATP (adenosine triphosphate) content and application of the method in production of L-histidine, and belongs to the technical field of biology. According to the method, expression of NADH dehydrogenase in the oxidative phosphorylation process is adjusted through an L-histidine response type promoter, the intracellular ATP content is increased by 12.5%, a recombinant strain constructed through the method is fermented in a 5L fermentation tank at the temperature of 37 DEG C, the pH value of 7.0-7.2 and the dissolved oxygen of 25%-35% for 36 h, the yield of L-histidine reaches 45 g / L-55 g / L, the production intensity is 1.2 g / L-1. 5 g / L, and the yield of the L-histidine is increased by 30%. And the conversion rate is 0.19 g / g-0. 24 g / g of glucose. Therefore, by using the method, the synthesis of the L-histidine can be effectively improved.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly to a method for increasing intracellular ATP content and its application in L-histidine production. Background Art

[0002] L-histidine, also known as L-α-amino-β-imidazolepropionic acid, is a basic amino acid with an imidazole group in its side chain. L-histidine is a semi-essential amino acid, which is particularly important for the growth of infants and animals. In addition, its main metabolic pathway in the body is through deamination by histidine deaminase or through decarboxylase to form histamine and aminotransferase reactions. L-histidine is synthesized through ten-step catalytic reactions with adenosine triphosphate (ATP) and 5-phosphoribosyl-1-pyrophosphate (PRPP) as precursors. In addition, ATP is associated with the biosynthesis of purines, pyrimidines, folic acid, and tryptophan in the intermediate metabolism and energy metabolism of cells, and is a general energy donor for the growth and survival of strains. Therefore, ATP supply is crucial for the survival and normal operation of microbial cell factories and the efficient synthesis of L-histidine. The de novo synthesis pathway of purines uses PRPP as a substrate to generate IMP through 10-step enzymatic reactions, and then converts it into ATP or other purine nucleotides. However, for the de novo synthesis of ATP, 5 molecules of ATP are required to synthesize 1 molecule of ATP, which is not cost-effective. Therefore, the use of fast and efficient ATP supply technology to promote intracellular energy synthesis is crucial for the survival of chassis strains and the synthesis of L-histidine.

[0003] Microbial cells synthesize ATP mainly through two mechanisms: substrate-level phosphorylation and oxidative phosphorylation. ATP synthase plays a central role in the process of oxidative phosphorylation and is driven by the proton electrochemical gradient established based on the in vivo redox reaction and electron transfer along the electron transport chain. The electron transport chain consists of different dehydrogenases, coenzyme Q, and oxidases. The expression of dehydrogenases is regulated by respiratory conditions or electron acceptors and donors, and the proton potential generated by different dehydrogenases is different. However, for strains, they tend to select dehydrogenases suitable for high growth rather than those suitable for high L-histidine synthesis. Therefore, it is crucial to select dehydrogenases that contribute to ATP synthesis and L-histidine production. So far, ATP regeneration based on the modification of oxidative phosphorylation has been applied to natural cells to provide energy and the biosynthesis of chemical substances. Gao Cong et al. increased the supply of ATP by regulating the electron transport chain with an ATP-sensing switch, thereby increasing the concentration of uridine triphosphate (recorded in the paper "Multivariate Modular Metabolic Engineering for High Titer Uridine Triphosphate Production in Escherichia coli"). Zhang Xueli et al. regulated dehydrogenases, oxidases, and ATP synthase using five promoters with different strengths to improve the intracellular ATP synthesis and the yield of β-carotene (recorded in the paper "Engineering central metabolic modules of Escherichia coli for improving b-carotene production").

[0004] However, since dehydrogenases are expressed by larger operons, it is difficult to integrate and express them in the genome. Using an L-histidine-responsive promoter with dynamic regulatory function to regulate the expression of operons is an ideal solution. Therefore, screening for L-histidine-responsive promoters and using these promoters to modify the oxidative phosphorylation process is an effective way to increase the intracellular ATP content and the yield of L-histidine. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a method for increasing the intracellular ATP content and its application in L-histidine production. The method for increasing the intracellular ATP content in the present invention uses an L-histidine-responsive promoter to modify NADH dehydrogenase in the oxidative phosphorylation process in the starting strain.

[0006] The present invention is achieved through the following technical solutions:

[0007] The first object of the present invention is to provide an L-histidine-responsive promoter, the nucleotide sequence of which is SEQ ID NO:1, or a nucleotide sequence having a homology of more than 95% with SEQ ID NO:1. With the increase of the L-histidine concentration, the expression intensity of the promoter increases.

[0008] In one embodiment of the present invention, the L-histidine-responsive promoter is derived from Escherichia coli.

[0009] The second object of the present invention is to provide an expression vector containing the L-histidine-responsive promoter.

[0010] The third object of the present invention is to provide a method for increasing the intracellular ATP content, by using the L-histidine-responsive promoter to regulate the expression of the NADH dehydrogenase-encoding gene nuo operon.

[0011] In one embodiment of the present invention, the NADH dehydrogenase is expressed by the native nuo operon gene of Escherichia coli.

[0012] In one embodiment of the present invention, the nuo operon is derived from Escherichia coli.

[0013] In one embodiment of the present invention, the nuo operon contains genes such as nuoA, nuoB, nuoC, nuoE, nuoF, nuoG, nuoH, nuoI, nuoJ, nuoK, nuoL, nuoM, nuoN, etc.

[0014] In one embodiment of the present invention, the nucleotide sequence of the nuo operon gene is as shown in SEQ ID NO:2.

[0015] In one embodiment of the present invention, the intracellular ATP content is increased by 12.5% by using this method, and the effect is remarkable.

[0016] The fourth object of the present invention is to provide a recombinant bacterium for producing L-histidine, which is obtained by integrating the gene hisG* encoding the mutant of Corynebacterium glutamicum ATP phosphoribosyltransferase with the nucleotide sequence shown in SEQ ID NO:3 into the genome of Escherichia coli and enabling its strong expression; and integrating the L-histidine-responsive promoter described in claim 1 upstream of the NADH dehydrogenase-encoding gene nuo operon.

[0017] In one embodiment of the present invention, the Escherichia coli is E. coli W3110.

[0018] In one embodiment of the present invention, the gene hisG* encoding the HisG mutant of Corynebacterium glutamicum ATP phosphoribosyltransferase is integrated at at least three gene loci on the genome and is driven by a strong promoter.

[0019] In one embodiment of the present invention, the gene hisG* encoding the HisG mutant of Corynebacterium glutamicum ATP phosphoribosyltransferase is respectively integrated at the ilvG, ycgH and ygaY gene loci on the genome and is driven by the promoter P trc to initiate.

[0020] The fifth object of the present invention is to provide a method for constructing the recombinant bacterium for producing L-histidine, which is obtained by directionally modifying Escherichia coli by using the CRISPR / Cas 9-mediated gene editing technology, and includes the following steps:

[0021] (1) Integrating the gene hisG* encoding the HisG mutant of Corynebacterium glutamicum ATP phosphoribosyltransferase on the genome of Escherichia coli and strongly expressing it;

[0022] (2) Constructing a plasmid targeting the gene locus;

[0023] (3) Constructing a connecting fragment of the L-histidine-responsive promoter and the upstream and downstream homologous arm fragments;

[0024] (4) Co-electroporating the plasmid obtained in step (2) and the connecting fragment obtained in step (3) into Escherichia coli.

[0025] In one embodiment of the present invention, in step (1), the integration method is: constructing a connecting fragment P trc of the promoter P and the gene hisG* encoding the HisG mutant of Corynebacterium glutamicum ATP phosphoribosyltransferase trc -hisG * and integrating it at the ilvG, ycgH and ygaY gene loci on the genome respectively.

[0026] The sixth object of the present invention is to provide the application of the recombinant bacterium in the fermentative production of L-histidine.

[0027] The starting strain of the present invention uses Escherichia coli W3110 as the chassis strain, and fuses the gene hisG * encoding the HisG mutant with the promoter P trc to obtain the fusion fragment P trc -hisG *It is obtained by integrating them at the ilvG, ycgH, and ygaY gene loci respectively using gene editing. The natural promoter of the nuo operon of NADH dehydrogenase in the starting strain is replaced with an L-histidine-responsive promoter by gene editing.

[0028] In one embodiment of the present invention, the recombinant bacterium can produce 45 g / L - 55 g / L of L-histidine by fermenting in a 5 L fermenter for 36 h, with a production intensity of 1.2 g / L / h - 1.5 g / L / h and a conversion rate of 0.19 g / g - 0.24 g / g of glucose.

[0029] The present invention also provides a method for efficiently producing L-histidine using the above recombinant Escherichia coli, and the method includes the following steps:

[0030] (1) After activating the recombinant bacterium, inoculate it into an LB liquid medium and culture it at a temperature of 37°C and a rotation speed of 120 rpm - 180 rpm for 8 h - 12 h to obtain an activated bacterial liquid;

[0031] (2) Inoculate all of the activated bacterial liquid obtained in step (1) into a 5 L fermenter. The seed medium is: glucose 20 g / L - 40 g / L, yeast extract 3 g / L - 7 g / L, peptone 2 g / L - 6 g / L, citric acid 1 g / L - 4 g / L, (NH4)2SO4 3 g / L - 8 g / L, KH2PO4 1 g / L - 5 g / L, MgSO4·7H2O 1 g / L - 3 g / L, FeSO4·7H2O 10 mg / L - 30 mg / L, MnSO4·H2O 10 mg / L - 30 mg / L, ZnSO4 10 - 30 mg / L, V B1 、V B3 、V B5 、V B12 、V H Each is 1 mg / L - 3 mg / L. Culture it at a temperature maintained at 37°C, a rotation speed maintained at 400 rpm - 600 rpm, and a pH maintained at 7 - 7.2 for 10 h - 14 h to obtain a seed liquid;

[0032] (3) Inoculate the seed liquid obtained in step (2) into a 5 L fermenter at an inoculation amount of 15% - 20%. The fermentation medium is: glucose 10 - 30 g / L, yeast extract 3 g / L - 7 g / L, citric acid 1 g / L - 4 g / L, (NH4)2SO4 1 g / L - 5 g / L, KH2PO4 4 g / L - 10 g / L, MgSO4·7H2O 1 g / L - 3 g / L, FeSO4·7H2O 10 mg / L - 30 mg / L, MnSO4·H2O 10 mg / L - 30 mg / L, ZnSO4 10 mg / L - 30 mg / L, V B1 、VB3 、V B5 、V B12 、V H Each is 1 mg / L - 3 mg / L, the temperature is maintained at 37 °C, the pH is maintained at 7.0 - 7.2, and the dissolved oxygen is maintained at 25% - 35% for fermentation. When the glucose in the culture medium is consumed, an 80% (m / v) glucose solution is added to maintain the glucose concentration at 0.1 g / L - 2 g / L.

[0033] (4) Centrifuge and collect the transformation solution obtained in step (3) to obtain L-histidine.

[0034] The above technical solution of the present invention has the following advantages compared with the prior art:

[0035] (1) The present invention provides a method for increasing the intracellular ATP content. This method uses an L-histidine-responsive promoter to regulate the expression of the NADH dehydrogenase-encoding gene nuo operon. Using this method, the intracellular ATP content of recombinant Escherichia coli is increased by 12.5%.

[0036] (2) The method for increasing the intracellular ATP content of the present invention is applied to the production of L-histidine. The yield of L-histidine in the fermentation broth reaches 45 g / L - 55 g / L of L-histidine, the production intensity is 1.2 g / L / h - 1.5 g / L / h, and the conversion rate is 0.19 g / g - 0.24 g / g of glucose.

[0037] (3) In addition, there is currently no research on increasing intracellular ATP by modifying the oxidative phosphorylation process in the construction of high-yield L-histidine strains. In particular, using an L-histidine-responsive promoter can regulate the expression of dehydrogenases, promote the increase of intracellular ATP content and the synthesis of L-histidine. The present invention for the first time proves that modifying the oxidative phosphorylation process in L-histidine-producing strains helps to greatly promote the supply of precursor ATP and increase the yield of L-histidine, which is an effective technology for increasing the yield of L-histidine-producing strains. Description of the Drawings

[0038] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to the specific embodiments of the present invention in combination with the drawings, wherein,

[0039] Figure 1 : Fluorescence change of the L-histidine-responsive promoter with the increase of L-histidine concentration;

[0040] Figure 2 : ATP content and NADH / NAD of recombinant Escherichia coli and control strains + Change diagram. Detailed Embodiments

[0041] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the exemplified embodiments are not intended to limit the present invention.

[0042] The sources of materials and reagents involved in the following embodiments are as follows:

[0043] L-histidine was purchased from Macklin; the enhanced ATP detection kit (#S0027) was purchased from Beyotime Biotechnology Co., Ltd.; the coenzyme I NAD(H) content detection kit (#BC0310) was purchased from Beijing Solarbio Science & Technology Co., Ltd.; Escherichia coli BL21 was purchased from Sangon Biotech (Shanghai) Co., Ltd.; Escherichia coli W3110 was purchased from BioVector Plasmid Vector Strain Cell Gene Preservation Center.

[0044] The media and required solutions involved in the following embodiments are as follows:

[0045] LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0046] LB solid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar powder 20 g / L.

[0047] 2YT liquid medium: peptone 16 g / L, yeast extract 10 g / L, NaCl 5 g / L.

[0048] The detection methods involved in the following embodiments are as follows:

[0049] The determination of L-histidine content was carried out by high performance liquid chromatography: Agilent C18, 5 μm, 4.6×250 mm chromatographic column; flow rate was 1.0 mL / min; column temperature was 40 °C; detection wavelength was 338 nm; mobile phase: phase A: 3.01 g of sodium acetate (5.0 g of sodium acetate trihydrate) was dissolved in 1000 mL of water, 200 μL of triethylamine was added, the pH was adjusted to 7.20±0.05 with 5% acetic acid, and finally 5 mL of tetrahydrofuran was added; phase B: 3.01 g of sodium acetate was weighed and dissolved in 200 mL of water, the pH was adjusted to 7.20±0.05 with 5% acetic acid, and this solution was added to 400 mL of HPLC-grade methanol and 400 mL of HPLC-grade acetonitrile and mixed.

[0050] The determination of ATP content: The enhanced ATP detection kit purchased from Beyotime Biotechnology Co., Ltd. was used to determine the ATP content of the strains, and the operation procedure was referred to the kit operation manual.

[0051] NADH and NAD+ Determination of content: The coenzyme Ⅰ NAD(H) content detection kit purchased from Beijing Solarbio Science & Technology Co., Ltd. was used to determine the NADH and NAD content of the strain. + For the determination procedure, refer to the operation manual of the kit.

[0052] Determination of fluorescence intensity: The Cytation3 multifunctional microplate reader purchased from Biotek was used to determine the fluorescence intensity.

[0053] Example 1: Screening of L-histidine-responsive promoter

[0054] The specific steps are as follows:

[0055] (1) Universal fluorescence reporter plasmid: Multiple E. coli W3110 endogenous promoters were selected. Using the pTrc99A plasmid as the backbone and the mKate fluorescent protein as the reporter gene, the selected multiple promoters were used for the expression of the fluorescent protein to construct the reporter plasmid.

[0056] (2) Preparation of the PU7-mKate fragment: Using the E. coli W3110 genome as the template, the endogenous promoter PU7 was amplified using the primers (99A-PU7-1 and 99A-PU7-2) designed for the PU7 promoter, and the fluorescent protein expression gene was amplified using the primers (99A-PU7-3 and 99A-PU7-4) designed for the mKate fluorescent protein. The above fragments were obtained as the PU7-mKate fragment by the method of overlap PCR. The fragments with other promoters were constructed by the same procedure.

[0057] (3) Construction of the pTrc99A-PU7-mKate plasmid: The reverse P primers (99A-PU-1 and 99A-PU-2) designed for pTrc99A were used to amplify and remove P trcThe entire plasmid backbone outside the promoter. After homologous recombination of the PU7-mKate fragment with the reverse P backbone fragment of pTrc99A, it was transformed into Escherichia coli BL21 to obtain a transformation product. The transformation product was spread on an LB solid medium containing ampicillin and cultured inverted in a constant temperature incubator at 37°C for 12 h to obtain transformants. Colony PCR verification was performed, and the correctly verified transformants were picked and inoculated into an LB liquid medium (containing 100 μg / mL ampicillin). After shaking flask culture at 37°C and 180 rpm for 12 h, the plasmid was extracted for restriction enzyme verification and sequencing verification. If the verification was correct, the successfully transformed recombinant Escherichia coli BL21 / pTrc99A-PU7-mKate and the recombinant plasmid pTrc99A-PU7-mKate were obtained. The recombinant plasmid pTrc99A-PU7-mKate was transformed into E. coli W3110 competent cells to obtain the final strain E. coli W3110 / pTrc99A-PU7-mKate. Reporter plasmids with other promoters were constructed using the same procedure.

[0058] (4) Measurement of fluorescence intensity: The strain carrying the pTrc99A-PU7-mKate plasmid cultured overnight was transferred to a 24-well plate containing LB medium. After culturing the bacteria to the mid-logarithmic phase at 37°C, L-histidine with final concentrations of 0 g / L, 10 g / L, 20 g / L, and 30 g / L was added, and the fluorescence intensity was measured after continued culture for 6 h. 200 μL of the bacterial solution was taken into a multi-functional microplate, and the OD 600 of the bacteria and the fluorescence intensity (excitation wavelength 588 nm, emission wavelength 640 nm) were measured. After converting the data into the fluorescence intensity per unit OD 600 , the promoter with the fluorescence intensity increasing with the increase in the L-histidine concentration (the fluorescence intensity of the L-histidine-responsive promoter is shown in Figure 1 ) was selected. The fluorescence intensity of other strains was measured using the same procedure. It was found that only the strain carrying the pTrc99A-PU7-mKate plasmid met the conditions, and other promoters did not show a gradient response to the gradient concentration of L-histidine.

[0059] The primers used in the above protocol are shown in Table 1 below:

[0060] Table 1

[0061]

[0062] Example 2: Modifying L-histidine-producing strains using L-histidine-responsive promoters

[0063] The specific steps are as follows:

[0064] (1) The starting strain was obtained by modifying E. coli W3110 as the chassis strain as follows: In the chassis strain, using the CRISPR / Cas9 gene editing technology, the HisG mutant gene hisG * was fused with the promoter P trc to form the fusion fragment P trc -hisG * which was respectively integrated at three gene loci of ilvG, ycgH and ygaY.

[0065] (2) Preparation of the plasmid targeting the gene locus P nuo : The synthesized primers gRNA-nuo-1 and gRNA-nuo-2 were annealed to construct a double-stranded nucleic acid band targeting the P nuo gene locus. Then, it was subjected to homologous recombination with the linearized pGRB plasmid and transformed into Escherichia coli BL21 to obtain the transformation product. The transformation product was spread on the LB solid medium containing ampicillin and incubated upside down in a 37 °C constant temperature incubator for 12 h to obtain the transformants. Colony PCR verification was carried out. The correctly verified transformants were inoculated into the LB liquid medium (containing 100 μg / mL ampicillin) and cultured in a shaker flask at 37 °C and 180 rpm for 12 h, and then the plasmid was extracted for enzyme digestion verification and sequencing verification. After correct verification, the successfully transformed recombinant Escherichia coli E. coli / pGRB-P nuo and the recombinant plasmid pGRB-P nuo were obtained.

[0066] (3) Preparation of the recombinant DNA fragment: Using the E. coli W3110 genome as a template, the upstream homologous arm primers (P nuo -PU7-1 and P nuo -PU7-2) and the downstream homologous arm primers (P nuo -PU7-5 and P nuo -PU7-6) designed for P nuo were used to amplify the upstream and downstream homologous arm fragments, and the primers (P nuo -PU7-3 and P nuo -PU7-4) of the L-histidine-responsive promoter PU7 were used to amplify the L-histidine-responsive promoter PU7. The above fragments were obtained by the method of overlap PCR to obtain the integration fragment of P nuo -PU7.

[0067] (4) Construction of the integrated strain: The overnight cultured starting strain with pREDCas9 was transferred 1 mL to 100 mL of 2YT medium, cultured at 30 °C for 1 h, then IPTG with a final concentration of 0.1 mM was added, and after continuous culture for 1.5 h, competent cells were prepared. pGRB-P nuo and P nuo-PU7 was simultaneously electrotransformed into competent cells, and the transformation products were spread on an LB solid medium containing ampicillin and spectinomycin, and incubated upside down in a constant temperature incubator at 30 °C for 20 h to obtain transformants; P nuo -PU7-1 and P nuo -PU7-4 were used for colony PCR verification. The correctly verified transformants were inoculated into an LB liquid medium (containing 2 g / L L-arabinose and 50 μg / mL spectinomycin), and cultured in a shaking flask at 30 °C and 180 rpm for 12 h to eliminate the pGRB plasmid. An appropriate amount of the bacterial liquid was streaked on an LB plate with spectinomycin resistance, and incubated upside down in a constant temperature incubator at 30 °C for 12 h to 14 h. For the LB plates separately containing only ampicillin and spectinomycin resistance, single colonies that did not grow on the ampicillin plate and grew on the spectinomycin plate were selected. The correctly verified transformants were inoculated into an LB liquid medium (containing 50 μg / mL spectinomycin), and cultured in a shaking flask at 30 °C and 180 rpm for 12 h and then preserved.

[0068] (5) Elimination of the pREDCas9 plasmid: The successfully constructed recombinant bacteria were transferred to an antibiotic-free LB liquid medium and cultured overnight at 42 °C. An appropriate amount of the bacterial liquid was streaked on an antibiotic-free LB plate, and incubated upside down in a constant temperature incubator at 37 °C for 12 h. For the plates containing spectinomycin and antibiotic-free LB, single colonies that did not grow on the spectinomycin-resistant plate and grew on the antibiotic-free plate were inoculated into an LB liquid medium, and cultured in a shaking flask at 37 °C and 180 rpm for 12 h and then preserved.

[0069] Table 2

[0070]

[0071] Example 3: Determination of ATP content and NADH / NAD + in recombinant strains

[0072] The specific steps are as follows:

[0073] (1) Preparation of the fermentation seed liquid: The activated strain was inoculated into a 500 mL Erlenmeyer flask containing 30 mL of the seed medium and cultured at a temperature of 37 °C and 220 rpm for 12 h to obtain the seed liquid. The seed medium was: glucose 30 g / L, yeast extract 5 g / L, peptone 4 g / L, citric acid 2 g / L, (NH4)2SO4 5 g / L, KH2PO4 3 g / L, MgSO4·7H2O 2 g / L, FeSO4·7H2O 10 mg / L, MnSO4·H2O 10 mg / L, ZnSO4 10 mg / L, V B1 、V B3 、V B5 、V B12, V H Each is 1 mg / L.

[0074] (2) Shake flask fermentation: The seed liquid obtained in step (1) was inoculated into a 500 mL Erlenmeyer flask containing 30 mL of fermentation medium at an inoculation amount of 15%-20% to start fermentation. The temperature was maintained at 37 °C, the pH was maintained at neutral, and the culture was carried out for 16 h under the condition of 220 rpm. The fermentation medium was: glucose 20 g / L, yeast extract 4 g / L, citric acid 2 g / L, (NH4)2SO4 5 g / L, KH2PO4 3 g / L, MgSO4·7H2O 2 g / L, FeSO4·7H2O 20 mg / L, MnSO4·H2O 20 mg / L, ZnSO4 20 mg / L, V B1 , V B3 , V B5 , V B12 , V H Each is 2 mg / L.

[0075] The ATP content in the recombinant Escherichia coli was as Figure 2 , relative to the starting strain, using the L-histidine-responsive promoter to regulate the expression of NADH dehydrogenase, promoting the regeneration of ATP in the oxidative phosphorylation process, increasing the intracellular ATP content by 12.5%. At the same time, the intracellular NADH / NAD + significantly decreased, and the effect was significant.

[0076] Example 4: Fermentation production of L-histidine by recombinant strain

[0077] The specific steps are as follows:

[0078] (1) Preparation of fermentation seed liquid: After activating the strain, it was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of LB liquid medium and cultured at 37 °C and 220 rpm for 10 h to obtain the activated bacterial liquid. The activated bacterial liquid was all inoculated into a 5 L fermenter and cultured at 37 °C, a rotation speed of 500 rpm, and a pH of 7-7.2 for 12 h to obtain the seed liquid. The seed medium was: glucose 20 g / L, yeast extract 4 g / L, peptone 4 g / L, citric acid 2 g / L, (NH4)2SO4 5 g / L, KH2PO4 3 g / L, MgSO4·7H2O 2 g / L, FeSO4·7H2O 10 mg / L, MnSO4·H2O 10 mg / L, ZnSO4 10 mg / L, V B1 , V B3 , V B5 , V B12 , V H Each is 1 mg / L.

[0079] (2) Fermentation in a fermenter: The seed liquid obtained in step (1) is inoculated into a 5 L fermenter at an inoculation amount of 15%-20% to start fermentation. The temperature is maintained at 37 °C, the pH is maintained at 7.0 - 7.2, and the dissolved oxygen is maintained at 25%-35% for fermentation. When the glucose in the medium is consumed, an 80% (m / v) glucose solution is added to maintain the glucose concentration at 0.1 g / L - 2 g / L. The fermentation medium is: glucose 20 g / L, yeast extract 4 g / L, citric acid 2 g / L, (NH4)2SO4 3 g / L, KH2PO4 7 g / L, MgSO4·7H2O 1 g / L, FeSO4·7H2O 20 mg / L, MnSO4·H2O 20 mg / L, ZnSO4 20 mg / L, V B1 、V B3 、V B5 、V B12 、V H Each is 2 mg / L.

[0080] After 36 h of fermentation in a 5 L fermenter using the recombinant strain, the yield of L-histidine reaches 45 g / L - 55 g / L, the production intensity is 1.2 g / L / h - 1.5 g / L / h, and the conversion rate is 0.19 g / g - 0.24 g / g glucose.

[0081] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of this invention.

Claims

1. An L-histidine-responsive promoter, characterized in that, The nucleotide sequence of the promoter is as shown in SEQ ID NO:1, or a nucleotide sequence having a homology of more than 95% with SEQ ID NO:

1.

2. An expression vector containing the L-histidine-responsive promoter according to claim 1 or 2.

3. A method for increasing the intracellular ATP content, characterized in that, Utilize the L-histidine-responsive promoter according to claim 1 to regulate the expression of the nuo operon encoding NADH dehydrogenase gene.

4. A recombinant bacterium for producing L-histidine, characterized in that, The recombinant bacterium has integrated the gene hisG* encoding the mutant of Corynebacterium glutamicum ATP phosphoribosyltransferase HisG with a nucleotide sequence as shown in SEQ ID NO:3 on the genome of Escherichia coli and strongly expresses it; the L-histidine-responsive promoter according to claim 1 is integrated upstream of the nuo operon encoding the NADH dehydrogenase gene.

5. The recombinant bacterium according to claim 4, wherein The Escherichia coli is E. coli W3110.

6. The recombinant bacterium according to claim 4, characterized in that, The gene hisG* encoding the mutant of Corynebacterium glutamicum ATP phosphoribosyltransferase HisG is integrated at at least three gene loci on the genome and is driven by a strong promoter.

7. The recombinant bacterium according to claim 4, characterized in that, The coding gene hisG* of the Corynebacterium glutamicum ATP phosphoribosyltransferase HisG mutant is integrated at the genomic ilvG, ycgH, and ygaY gene loci, and is promoted by the promoter P trc promoter.

8. The method for constructing a recombinant bacterium for producing L-histidine according to claim 4, characterized in that, The recombinant bacterium is obtained by directed modification of Escherichia coli using the CRISPR / Cas 9-mediated gene editing technology, and comprises the following steps: (1) Integrate the gene hisG* encoding the mutant of Corynebacterium glutamicum ATP phosphoribosyltransferase HisG on the genome of Escherichia coli and strongly express it; (2) Construct a plasmid targeting the gene locus; (3) Construct a ligation fragment of the L-histidine-responsive promoter and the upstream and downstream homologous arm fragments; (4) Co-electroporate the plasmid obtained in step (2) and the ligation fragment obtained in step (3) into Escherichia coli.

9. The construction method according to claim 7, characterized in that, In step (1), the integration method is as follows: construct a promoter P trc and a ligation fragment P trc -hisG * of the mutant encoding gene hisG* of the ATP phosphoribosyltransferase HisG from Corynebacterium glutamicum, and integrate them at the genomic ilvG, ycgH and ygaY gene loci respectively.

10. Use of the recombinant bacterium according to any one of claims 4-7 in the fermentative production of L-histidine.

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