Transporter lysE mutants and their use in increasing l-histidine production

Abstract

This application relates to the use of a lysE mutant transporter in increasing L-histidine production. Specifically, this application provides a lysE mutant transporter with the following amino acid substitutions relative to the wild-type lysE transporter shown in SEQ ID NO:1: valine at position 197 is replaced by alanine, and / or serine at position 205 is replaced by alanine or glycine. This application also provides the use of the lysE mutant transporter in the preparation of L-histidine or L-histidine derivatives. Compared to strains containing wild-type lysE, strains containing the lysE mutant exhibit increased histidine production.

Description

Technical Field

[0001] This invention relates to the field of L-histidine production, specifically to the preparation of L-histidine via a microbial pathway. Background Technology

[0002] L-histidine plays a role in several key physiological processes, including antioxidant and immunomodulatory functions. Therefore, it is widely used in the medical industry to prepare drugs for treating gastric ulcers, raw materials for cardiovascular drugs, and amino acid infusions.

[0003] Histidine is mainly found in hemoglobin, and its traditional preparation method is protein hydrolysis extraction using blood meal as raw material. However, this method has drawbacks such as low efficiency and environmental pollution. Existing technologies have reported the production of L-histidine through microbial fermentation, but this has not yet been achieved on a large industrial scale. This is because the histidine metabolic pathway is long, involving multiple key enzymes, and the current yield of histidine production using *E. coli* is low, possibly due to negative feedback inhibition caused by the accumulation of histidine in the body, limiting its further synthesis.

[0004] L-histidine transport is a key factor in achieving high-yield amino acid production in cells. CN110184230A discloses a genetically engineered bacterium that produces high levels of L-histidine. The gene encoding the arginine / lysine transporter from *Corynebacterium glutamicum*, *lysE*, is integrated into the bacterium's genome and strongly expressed using the strong promoter Ptrc. This application is the first to demonstrate that introducing the arginine / lysine transporter *LysE* from *Corynebacterium glutamicum* contributes to increased histidine production and represents an important target for histidine-producing bacterial strains. Modifying histidine-related transporters through genetic engineering is significant for obtaining higher-yield L-histidine-producing strains. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a LysE mutant that can be used to obtain higher yields of L-histidine.

[0006] In microorganisms such as *Escherichia coli*, the main biosynthesis pathway for L-histidine is the pentose phosphate pathway. When glucose is used as the carbon source, the pentose phosphate pathway generates the L-histidine precursor phosphoribosyl pyrophosphate (PRPP). Subsequently, PRPP and adenosine triphosphate (ATP) undergo a series of reactions, including ring opening, isomerization, and amide transfer, catalyzed by ATP phosphoribosyltransferase, to produce L-histidine. ATP phosphoribosyltransferase (HisG), as the first key enzyme in this biosynthetic pathway, has its activity inhibited by various intermediate metabolites and end products, especially the end product L-histidine, thus limiting L-histidine production. In their previous research, the inventors discovered that a site mutation in HisG (D84A + S205H) could relieve this feedback inhibition.

[0007] Efficient intracellular transport of L-histidine is a key factor for achieving high L-histidine production. As shown above, the arginine / lysine transporter lysE protein (encoding gene lysE gene (GeneID: 1019243)) from Corynebacterium glutamicum contributes to increased histidine production and is an important target for the modification of L-histidine-producing bacterial strains.

[0008] To construct genetically engineered strains with higher L-histidine production, this invention utilizes genetic engineering technology to modify L-histidine-producing *E. coli*. Through structural studies of the lysE gene, this invention performs a series of site-directed mutagenesis on lysE, transfers the mutated lysE gene into the host bacteria, and screens for improved histidine transport efficiency. This mutant can promptly release the produced histidine extracellularly, preventing its accumulation within the cell. Therefore, compared to the wild-type lysE transporter, the mutant of this application increases the fermentation yield of L-histidine.

[0009] Specifically, this application provides a mutant of the transporter protein lysE, which, relative to the wild-type transporter protein lysE shown in SEQ ID NO: 1, has valine at position 197 of the amino acid sequence replaced by alanine, and / or serine at position 205 replaced by alanine or glycine.

[0010] In a specific embodiment of this application, the coding gene of the transporter protein lysE mutant can be directly integrated into the genome of a microorganism (e.g., Escherichia coli or Corynebacterium glutamicum), or replace the original lysE gene in the genome of a microorganism (e.g., Corynebacterium glutamicum), which can be done by conventional means in the prior art, such as gene editing technology.

[0011] Furthermore, as mentioned above, ATP phosphoribosyltransferase (HisG) is a key enzyme in the histidine biosynthesis pathway. Therefore, in the preferred embodiment of this application, to further increase histidine production, the applicant, based on previous research (see patent application number CN202410091107.5), introduced an exogenous hisG gene into the chassis strain and mutated it with D84A and S205H to relieve the feedback inhibition of histidine on ATP phosphoribosyltransferase (HisG). Those skilled in the art will understand that introducing an exogenous hisG gene into the chassis strain and mutating it with D84A and S205H is only a preferred technical solution of this application; in strains without the above operations, the lysE mutant of this application still has the effect of increasing L-histidine production compared to the wild-type lysE.

[0012] Specifically, this application provides the following technical solutions:

[0013] 1. A mutant of the transporter protein lysE, which, relative to the wild-type transporter protein lysE shown in SEQ ID NO: 1, has the following amino acid substitutions: valine at position 197 is replaced by alanine, and / or serine at position 205 is replaced by alanine or glycine.

[0014] 2. The lysE mutant of the transporter protein according to Project 1, wherein the coding sequence of the wild-type transporter protein lysE is shown in SEQ ID NO: 13.

[0015] 3. A nucleic acid molecule encoding a mutant of the transport protein lysE according to any one of items 1-2.

[0016] 4. An expression vector comprising the nucleic acid molecule described in item 3.

[0017] 5. A host cell comprising the nucleic acid molecule described in item 3 or the expression vector described in item 4.

[0018] 6. The use of the lysE mutant of the transporter protein as described in any one of Items 1-2, the nucleic acid molecule as described in Item 3, the expression vector as described in Item 4, or the host cell as described in Item 5 in the preparation of L-histidine or L-histidine derivatives.

[0019] 7. A method for producing L-histidine or an L-histidine derivative, the method comprising culturing bacteria in a culture medium to produce L-histidine, wherein the bacteria contain the nucleic acid molecule described in item 3 or the expression vector described in item 4.

[0020] 8. The method according to Project 7, wherein the bacteria is Escherichia coli or Corynebacterium glutamicum.

[0021] 9. The method according to item 7 or 8, wherein feedback inhibition of ATP phosphoribosyltransferase in the bacteria is relieved.

[0022] 10. The method according to Project 9, wherein the hisG-D84A-S205H gene of the ATP phosphoribosyltransferase mutant with double-site mutations of D84A and S205H from Acinetobacter baumannii is integrated into the bacterial genome or the hisG gene of the bacterial genome is replaced to relieve the feedback inhibition of ATP phosphoribosyltransferase.

[0023] Preferably, in the bacteria, a nucleic acid molecule encoding the lysE mutant of the transporter protein is integrated into the yjiP gene site on the bacterial genome.

[0024] Beneficial effects

[0025] To construct genetically engineered strains with higher L-histidine production, this invention involved structural studies of the lysE gene and a series of site-directed mutagenesis. The mutated lysE gene was then transferred into host bacteria and screened to obtain a lysE variant with increased histidine production. This variant can more efficiently release the produced histidine extracellularly, avoiding intracellular accumulation. Compared to the wild-type lysE transporter, it increases histidine fermentation yield and has broad prospects for industrial development and application. Attached Figure Description

[0026] Figure 1 The results of molecular docking of wild-type lysE transporter protein with L-histidine as substrate in AutoDock Vina software are shown. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0028] Unless otherwise specified, the methods used in the following examples are conventional methods, and the reagents used are commercially available reagents.

[0029] For details of the strains, plasmids and primers used in this application, please refer to Tables 1 and 2.

[0030] Table 1. Strains and plasmids used in this invention

[0031]

[0032]

[0033] Table 2 Primers used in this invention

[0034]

[0035] Example 1: Bioinformatics Analysis of lysE Gene Structure - AutoDock Vina Molecular Docking

[0036] The lysE transporter protein was used as a substrate for molecular docking in AutoDock Vina to observe the interaction between the lysE transporter protein and L-histidine. AutoDock Vina is a conventional molecular simulation software commonly used to study the interactions between complexes of biomacromolecules (e.g., proteins) and small molecules (e.g., ligands). Results are as follows: Figure 1 As shown, L-histidine forms hydrogen bonds with residues at positions V197, A201, and S205 of the lysE transporter, and these residues are key residues in the active pocket. Therefore, in order to obtain strains with higher histidine production, this application performed point mutations on the above key residues and obtained lysE mutants that may increase histidine production, namely V197A, V197F, A201F, A201G, S205A, and S205G.

[0037] Example 2: Genomic insertion of the ATP phosphoribosyltransferase gene hisG-D84A-S205H

[0038] ATP phosphoribosyltransferase (HisG) is a key enzyme in the L-histidine biosynthesis pathway. The applicant previously (see patent application number CN202410091107.5) studied ATP phosphoribosyltransferase (HisG) by introducing an exogenously modified hisG gene mutant to relieve the inhibitory effect of the final product on this enzyme. The specific operation is as follows.

[0039] Starting with *Escherichia coli* W3110, a two-step homologous recombination method was used to knock out the ATP phosphoribosyltransferase gene hisG (GeneID: 66396838) and insert it into the modified *Acinetobacter baumannii* (also known as AB bacteria) hisG-D84A-S205H gene (its sequence is shown in SEQ ID NO: 11). The specific steps are as follows:

[0040] 1. Preparation of HIS001 strain

[0041] Using pRE112 plasmid (purchased from Miaoling Plasmid Platform, catalog number: P1702, containing the cat-sacB gene) DNA as a template, a 3593bp DNA fragment I (i.e., the cat-sacB gene fragment) was amplified using primers hisG-cs-up / hisG-cs-down for the first step of homologous recombination.

[0042] The amplification system consisted of: 10 μL of Phusion 5X buffer (New England Biolabs), 1 μL of dNTPs (10 mM each), 20 ng of DNA template, 2 μL of primers (10 μM each), 0.5 μL of Phusion High-Fidelity DNA polymerase (2.5 U / μL), and 33.5 μL of distilled water, for a total volume of 50 μL.

[0043] The amplification conditions were: 98℃ pre-denaturation for 2 minutes (1 cycle); 98℃ denaturation for 10 seconds, 56℃ annealing for 10 seconds, and 72℃ extension for 4 minutes (30 cycles); 72℃ extension for 10 minutes (1 cycle).

[0044] The above DNA fragment I was used for the first homologous recombination: First, the pKD46 plasmid (purchased from the CGSC E. coli Collection Center at Yale University, CGSC#7739) was transformed into E. coli W3110 by electroporation to obtain strain W3110-pKD46. Then, DNA fragment I (i.e., the cat-sacB gene fragment) was electroporated into E. coli W3110 carrying pKD46 (i.e., electroporated into strain W3110-pKD46).

[0045] The electroporation conditions were as follows: W3110-pKD46 cells were prepared as competent cells using the CaCl2 method. Then, 50 μL of W3110-pKD46 competent cells were placed on ice, 50 ng of DNA fragment I was added, and the cells were incubated on ice for 2 minutes before being transferred to a 0.2 cm Bio-Rad electroporation cuvette. A MicroPulser (Bio-Rad) electroporator was used with a voltage of 2.5 kV. After electroporation, 1 ml of LB medium was quickly transferred to the cuvette, and the cells were pipetted 5 times before being transferred to a test tube. The cells were incubated at 75 rpm and 30°C for 2 hours. 200 μL of bacterial culture was plated on an LB agar plate containing ampicillin (final concentration 100 μg / ml) and chloramphenicol (final concentration 34 μg / ml). After overnight incubation at 30°C, single colonies were selected for PCR verification. The primers used were XZ-hisG-up / XZ-hisG-down. The correct colony amplification product was a 3715 bp fragment. A correct single colony (i.e., the strain that successfully electroporated DNA fragment I) was selected and named HIS001 (also known as W3110△hisG::cat-sacB, i.e., the cs(cat-sacB) gene fragment of E. coli W3110 with the ATP phosphoribosyltransferase gene hisG knocked out and successfully electroporated with pRE112 plasmid).

[0046] 2. Preparation of HIS003 strain

[0047] Starting from strain HIS001-pKD46, the ATP phosphoribosyltransferase gene hisG was knocked out using a two-step homologous recombination method, and then inserted into the hisG-D84A-S205H gene of Acinetobacter baumannii. The specific steps are as follows:

[0048] Using plasmid PUC19-hisG-D84A-S205H as a template, an 812bp DNA fragment II was amplified using primers hisG-mut-cs-up / hisG-mut-cs-down. The amplification system was the same as that in the first step and was used for the second homologous recombination.

[0049] The pKD46 plasmid was transformed into the HIS001 strain by electroporation to obtain the HIS001-pKD46 strain.

[0050] DNA fragment II was electroporated into strain HIS004-pKD46 as follows: Prepare competent cells for electroporation of strain HIS001-pKD46; place 50 μL of competent cells on ice, add 50 ng of DNA fragment II, incubate on ice for 2 minutes, and then transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporator with an electroporation voltage of 2.5 kV. Immediately after electroporation, transfer 1 ml of LB broth to the electroporation cuvette, pipette 5 times, and then transfer to a test tube. Incubate at 75 rpm and 30°C for 4 hours. Transfer the bacterial culture to LB broth (50 ml in a 250 ml flask) containing 10% sucrose and sodium chloride-free medium. After culturing for 24 hours, streak the culture onto LB agar plates containing 6% sucrose and sodium chloride-free medium. PCR verification confirmed that the primers used were XZ-hisG-up / XZ-hisG-down, and the correct colony amplification product was a 930bp fragment. A correct single colony was selected and named HIS003 (also known as W3110△hisG::hisG-Ab-D84A-S205H, which is a strain obtained by knocking out hisG in Escherichia coli W3110 and successfully electroporating the hisG-D84A-S205H gene from Acinetobacter baumannii).

[0051] Example 3: Genomic insertion of the lysE transporter gene

[0052] Starting from Escherichia coli HIS003, the yjiP gene was knocked out using a two-step homologous recombination method, and the lysE gene (GeneID: 1019243, whose nucleotide sequence is shown in SEQ ID NO: 13) was inserted at the yjiP gene site. The specific steps are as follows:

[0053] 1. Preparation of strain HIS004

[0054] Using pRE112 plasmid (purchased from Miaoling Plasmid Platform, catalog number: P1702, containing the cat-sacB gene) DNA as a template, a 3588bp DNA fragment I (i.e., the cat-sacB gene fragment) was amplified using primers yjiP-cs-up / yjiP-cs-down for the first step of homologous recombination.

[0055] The amplification system was the same as the system in the first step of Example 2. The amplification conditions were: 98°C pre-denaturation for 2 minutes (1 cycle); 98°C denaturation for 10 seconds, 56°C annealing for 10 seconds, 72°C extension for 4 minutes (30 cycles); 72°C extension for 10 minutes (1 cycle).

[0056] The above DNA fragment I was used for the first homologous recombination: First, the pKD46 plasmid was transformed into HIS003 by electroporation (to obtain strain HIS003-pKD46), and then DNA fragment I was electroporated into HIS003 containing pKD46.

[0057] The electroporation conditions were as follows: HIS003-pKD46 cells were prepared as competent cells using the CaCl2 method; then, 50 μL of HIS003-pKD46 competent cells were placed on ice, 50 ng of DNA fragment I was added, and the cells were incubated on ice for 2 minutes before being transferred to a 0.2 cm Bio-Rad electroporation cuvette. A MicroPulser (Bio-Rad) electroporator was used with an electroporation voltage of 2.5 kV. After electroporation, 1 ml of LB medium was quickly transferred to the electroporation cuvette, and the cells were pipetted 5 times before being transferred to a test tube and incubated at 75 rpm and 30°C for 2 hours. 200 μL of bacterial culture was plated on an LB agar plate containing ampicillin (final concentration 100 μg / ml) and chloramphenicol (final concentration 34 μg / ml). After overnight incubation at 30°C, single colonies were selected for PCR verification. The primers used were XZ-lysE-up / XZ-lysE-down. The correct colony amplification product was a 4099 bp fragment. One correct single colony was selected and named HIS004 (which is a strain obtained by inserting the cat-sacB gene fragment into the yjiP gene site in HIS003).

[0058] 2. Preparation of HIS005 strain

[0059] Using the original plasmid PUC19-lysE as a template, an 833bp DNA fragment II (i.e., the lysE gene fragment) was amplified using primers yjiP-lysE-up / yjiP-lysE-down. The amplification system was the same as that in the first step and was used for the second homologous recombination.

[0060] The pKD46 plasmid was transformed into HIS004 by electroporation (to obtain strain HIS004-pKD46).

[0061] DNA fragment II was electroporated into strain HIS004-pKD46. The electroporation conditions were as follows: First, prepare competent cells for electroporation of strain HIS004-pKD46. Place 50 μL of competent cells on ice, add 50 ng of DNA fragment II, incubate on ice for 2 minutes, and then transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporator with a voltage of 2.5 kV. Immediately after electroporation, transfer 1 ml of LB broth to the cuvette, pipette five times, and then transfer to a test tube. Incubate at 75 rpm and 30°C for 4 hours. Transfer the bacterial culture to LB broth (50 ml in a 250 ml flask) containing 10% sucrose and sodium chloride-free medium. After culturing for 24 hours, streak the culture onto LB agar plates containing 6% sucrose and sodium chloride-free medium. PCR verification showed that the primers used were XZ-lysE-up / XZ-lysE-down, and the correct colony amplification product was a 1341bp fragment. A correct single colony was selected and named HIS005 (which is a strain obtained by replacing the cat-sacB gene fragment in HIS004 with lysE from Corynebacterium glutamicum).

[0062] Example 4: Genomic insertion of the lysE transporter gene lysE-V197A

[0063] Starting from Escherichia coli HIS004, using the PUC19-lysE-V197A plasmid as a template, an 833bp DNA fragment II (i.e., the lysE-V197A gene fragment) was amplified using primers yjiP-lysE-up / yjiP-lysE-down. The amplification system was the same as the first step system and was used for the second homologous recombination.

[0064] The pKD46 plasmid was transformed into HIS004 by electroporation (to obtain strain HIS004-pKD46).

[0065] DNA fragment II was electroporated into strain HIS004-pKD46. The electroporation conditions were as follows: First, prepare competent cells for electroporation of strain HIS004-pKD46. Place 50 μL of competent cells on ice, add 50 ng of DNA fragment II, incubate on ice for 2 minutes, and then transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporator with a voltage of 2.5 kV. Immediately after electroporation, transfer 1 ml of LB broth to the cuvette, pipette five times, and then transfer to a test tube. Incubate at 75 rpm and 30°C for 4 hours. Transfer the bacterial culture to LB broth (50 ml in a 250 ml flask) containing 10% sucrose and sodium chloride-free medium. After culturing for 24 hours, streak the culture onto LB agar plates containing 6% sucrose and sodium chloride-free medium. PCR verification showed that the primers used were XZ-lysE-up / XZ-lysE-down, and the correct colony amplification product was a 1341bp fragment. A correct single colony was selected and named HIS006 (which is a strain obtained by replacing the cat-sacB gene fragment in HIS004 with lysE-V197A from Corynebacterium glutamicum).

[0066] Example 5: Genomic insertion of the lysE transporter gene lysE-V197F

[0067] Starting from Escherichia coli HIS004, using the PUC19-lysE-V197F plasmid as a template, an 833bp DNA fragment II (i.e., the lysE-V197F gene fragment) was amplified using primers yjiP-lysE-up / yjiP-lysE-down. The amplification system was the same as the first step system and was used for the second homologous recombination.

[0068] The pKD46 plasmid was transformed into HIS004 by electroporation to obtain strain HIS004-pKD46.

[0069] DNA fragment II was electroporated into strain HIS004-pKD46. The electroporation conditions were as follows: First, prepare competent cells for electroporation of strain HIS004-pKD46. Place 50 μL of competent cells on ice, add 50 ng of DNA fragment II, incubate on ice for 2 minutes, and then transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporator with a voltage of 2.5 kV. Immediately after electroporation, transfer 1 ml of LB broth to the cuvette, pipette five times, and then transfer to a test tube. Incubate at 75 rpm and 30°C for 4 hours. Transfer the bacterial culture to LB broth (50 ml in a 250 ml flask) containing 10% sucrose and sodium chloride-free medium. After culturing for 24 hours, streak the culture onto LB agar plates containing 6% sucrose and sodium chloride-free medium. PCR verification showed that the primers used were XZ-lysE-up / XZ-lysE-down, and the correct colony amplification product was a 1341bp fragment. A correct single colony was selected and named HIS007 (which is a strain obtained by replacing the cat-sacB gene fragment in HIS004 with lysE-V197F from Corynebacterium glutamicum).

[0070] Example 6: Genomic insertion of the lysE transporter gene lysE-A201F

[0071] Starting from Escherichia coli HIS004, using the PUC19-lysE-A201F plasmid as a template, an 833bp DNA fragment II (i.e., the lysE-A201F gene fragment) was amplified using primers yjiP-lysE-up / yjiP-lysE-down. The amplification system was the same as the first step system and was used for the second homologous recombination.

[0072] The pKD46 plasmid was transformed into HIS004 by electroporation to obtain strain HIS004-pKD46.

[0073] DNA fragment II was electroporated into strain HIS004-pKD46. The electroporation conditions were as follows: First, prepare competent cells for electroporation of strain HIS004-pKD46. Place 50 μL of competent cells on ice, add 50 ng of DNA fragment II, incubate on ice for 2 minutes, and then transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporator with a voltage of 2.5 kV. Immediately after electroporation, transfer 1 ml of LB broth to the cuvette, pipette five times, and then transfer to a test tube. Incubate at 75 rpm and 30°C for 4 hours. Transfer the bacterial culture to LB broth (50 ml in a 250 ml flask) containing 10% sucrose and sodium chloride-free medium. After culturing for 24 hours, streak the culture onto LB agar plates containing 6% sucrose and sodium chloride-free medium. PCR verification showed that the primers used were XZ-lysE-up / XZ-lysE-down, and the correct colony amplification product was a 1341bp fragment. A correct single colony was selected and named HIS008 (which is a strain obtained by replacing the cat-sacB gene fragment in HIS004 with lysE-A201F from Corynebacterium glutamicum).

[0074] Example 7: Genomic insertion of the lysE transporter gene lysE-A201G

[0075] Starting from Escherichia coli HIS004, using the PUC19-lysE-A201G plasmid as a template, an 833bp DNA fragment II (i.e., the lysE-A201G gene fragment) was amplified using primers yjiP-lysE-up / yjiP-lysE-down. The amplification system was the same as the first step system and was used for the second homologous recombination.

[0076] The pKD46 plasmid was transformed into HIS004 by electroporation to obtain strain HIS004-pKD46.

[0077] DNA fragment II was electroporated into strain HIS004-pKD46. The electroporation conditions were as follows: First, prepare competent cells for electroporation of strain HIS004-pKD46. Place 50 μL of competent cells on ice, add 50 ng of DNA fragment II, incubate on ice for 2 minutes, and then transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporator with a voltage of 2.5 kV. After electroporation, quickly transfer 1 ml of LB broth to the cuvette, pipette five times, and then transfer to a test tube. Incubate at 75 rpm and 30°C for 4 hours. Transfer the bacterial culture to LB broth (50 ml in a 250 ml flask) containing 10% sucrose and sodium chloride-free medium. After culturing for 24 hours, streak the culture onto LB agar plates containing 6% sucrose and sodium chloride-free medium. PCR verification showed that the primers used were XZ-lysE-up / XZ-lysE-down, and the correct colony amplification product was a 1341bp fragment. A correct single colony was selected and named HIS009 (which is a strain obtained by replacing the cat-sacB gene fragment in HIS004 with lysE-A201G from Corynebacterium glutamicum).

[0078] Example 8: Genomic insertion of the lysE transporter gene lysE-S205A

[0079] Starting from Escherichia coli HIS004, using the PUC19-lysE-S205A plasmid as a template, an 833bp DNA fragment II (i.e., the lysE-S205A gene fragment) was amplified using primers yjiP-lysE-up / yjiP-lysE-down. The amplification system was the same as the first step system and was used for the second homologous recombination.

[0080] The pKD46 plasmid was transformed into HIS004 by electroporation to obtain strain HIS004-pKD46.

[0081] DNA fragment II was electroporated into strain HIS004-pKD46. The electroporation conditions were as follows: First, prepare competent cells for electroporation of strain HIS004-pKD46. Place 50 μL of competent cells on ice, add 50 ng of DNA fragment II, incubate on ice for 2 minutes, and then transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporator with a voltage of 2.5 kV. Immediately after electroporation, transfer 1 ml of LB broth to the cuvette, pipette five times, and then transfer to a test tube. Incubate at 75 rpm and 30°C for 4 hours. Transfer the bacterial culture to LB broth (50 ml in a 250 ml flask) containing 10% sucrose and sodium chloride-free medium. After culturing for 24 hours, streak the culture onto LB agar plates containing 6% sucrose and sodium chloride-free medium. PCR verification showed that the primers used were XZ-lysE-up / XZ-lysE-down, and the correct colony amplification product was a 1341bp fragment. A correct single colony was selected and named HIS010 (which is a strain obtained by replacing the cat-sacB gene fragment in HIS004 with lysE-S205A from Corynebacterium glutamicum).

[0082] Example 9: Genomic insertion of the lysE transporter gene lysE-S205G

[0083] Starting from Escherichia coli HIS004, using the PUC19-lysE-S205G plasmid as a template, an 833bp DNA fragment II (i.e., the lysE-S205G gene fragment) was amplified using primers yjiP-lysE-up / yjiP-lysE-down. The amplification system was the same as the first step system and was used for the second homologous recombination.

[0084] The pKD46 plasmid was transformed into HIS004 by electroporation to obtain strain HIS004-pKD46.

[0085] DNA fragment II was electroporated into strain HIS004-pKD46. The electroporation conditions were as follows: First, prepare competent cells for electroporation of strain HIS004-pKD46. Place 50 μL of competent cells on ice, add 50 ng of DNA fragment II, incubate on ice for 2 minutes, and then transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporator with a voltage of 2.5 kV. Immediately after electroporation, transfer 1 ml of LB broth to the cuvette, pipette five times, and then transfer to a test tube. Incubate at 75 rpm and 30°C for 4 hours. Transfer the bacterial culture to LB broth (50 ml in a 250 ml flask) containing 10% sucrose and sodium chloride-free medium. After culturing for 24 hours, streak the culture onto LB agar plates containing 6% sucrose and sodium chloride-free medium. PCR verification showed that the primers used were XZ-lysE-up / XZ-lysE-down, and the correct colony amplification product was a 1341bp fragment. A correct single colony was selected and named HIS011 (which is a strain obtained by replacing the cat-sacB gene fragment in HIS004 with lysE-S205G from Corynebacterium glutamicum).

[0086] Example 10: Production of L-histidine by strains containing wild-type lysE or lysE mutants

[0087] The strains containing wild-type lysE or lysE mutants constructed in the above embodiments were fermented to produce L-histidine, including the following steps:

[0088] (1) Seed culture: Fresh single clones from LB plates were inoculated into test tubes containing 4 ml of seed culture medium and cultured overnight at 37°C and 250 rpm with shaking. Then, the culture was transferred to 250 ml Erlenmeyer flasks containing 30 ml of seed culture medium at an inoculation rate of 2% (V / V) and cultured at 37°C and 250 rpm with shaking for 12 hours to obtain the seed culture solution, which was used for inoculation of fermentation medium.

[0089] (2) Fermentation culture: The volume of fermentation medium in a 500ml fermenter is 250ml. The seed culture is inoculated into the fermentation medium at an inoculation rate of OD550 = 0.1. Fermentation is carried out at 37℃ and 150 rpm for 3 days to obtain the fermentation broth. The neutralizing agent is 5M ammonia water to maintain the pH of the fermenter at 7.0.

[0090] Analytical methods: Components in the fermentation broth after 3 days of fermentation were determined using an Agilent 1260 high-performance liquid chromatograph. L-histidine was determined using a ZORBAX Eclipse AAA 4.6 mm × 75 mm 3.5-Micron amino acid analysis column. Detection wavelength: 338 nm.

[0091] The experiment was repeated three times, and the average value of the analysis results is shown in Table 3 below.

[0092] Main culture medium:

[0093] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride. (For solid medium, add 20 g / L agar powder.)

[0094] Shake-flask fermentation seed culture medium (1L): 20g glucose, 4g yeast extract, 3g peptone, 1.2g potassium dihydrogen phosphate, 0.5g magnesium sulfate·7H2O, 10mg ferrous sulfate·7H2O, 10mg manganese sulfate monohydrate, 1mg VB1, 1mg VB3, 1mg VB5, 1mg VB12, 1mg VH, with sodium hydroxide to adjust pH to 7.0-7.2.

[0095] Shake flask fermentation medium (1L): 20g glucose, 4g yeast extract, 3g peptone, 2g sodium citrate monohydrate, 2g potassium dihydrogen phosphate, 2g magnesium sulfate·7H2O, 20mg ferrous sulfate·7H2O, 20mg manganese sulfate monohydrate, 2mg VB1, 2mg VB3, 2mg VB5, 2mg VB12, and 2mg VH. Adjust the pH to 7.0-7.2 with sodium hydroxide. Add ammonia water every 4 hours during fermentation to bring the pH to 7.2-7.4.

[0096] Table 3 Comparison of L-histidine production capacity

[0097]

[0098] The results (see Table 3) showed that, compared with the HIS005 strain containing wild-type lysE, for the mutation at position 197, the histidine production of HIS006 (HIS003△yjiP::lysE-V197A) was increased, while that of HIS007 (HIS003△yjiP::lysE-V197F) was decreased; therefore, the V197A mutation was chosen for subsequent experiments. For the mutation at position 201, the histidine production of both HIS008 (HIS003△yjiP::lysE-A201F) and HIS009 (HIS003△yjiP::lysE-A201G) was decreased; therefore, the mutation at position 201 could not increase histidine production. For the mutation at position 205, the histidine production of HIS010 (HIS003△yjiP::lysE-S205A) and HIS011 was decreased. Both (HIS003△yjiP::lysE-S205G) showed increased histidine production, therefore both can be used for subsequent experiments.

[0099] Example 11: Genomic insertion of the lysE transporter gene lysE-V197A-S205A

[0100] In this embodiment, a strain containing mutant combinations was further constructed.

[0101] Starting from Escherichia coli HIS004, using the plasmid PUC19-lysE-V197A-S205A as a template, an 833bp DNA fragment II (i.e., the lysE-V197A-S205A gene fragment) was amplified using primers yjiP-lysE-up / yjiP-lysE-down. The amplification system was the same as the first step system and was used for the second homologous recombination.

[0102] The electroporation conditions were as follows: the pKD46 plasmid was transformed into HIS004 by electroporation to obtain strain HIS004-pKD46.

[0103] DNA fragment II was electroporated into strain HIS004-pKD46. The electroporation conditions were as follows: First, prepare competent cells for electroporation of strain HIS004-pKD46. Place 50 μL of competent cells on ice, add 50 ng of DNA fragment II, incubate on ice for 2 minutes, and then transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporator with a voltage of 2.5 kV. Immediately after electroporation, transfer 1 ml of LB broth to the cuvette, pipette five times, and then transfer to a test tube. Incubate at 75 rpm and 30°C for 4 hours. Transfer the bacterial culture to LB broth (50 ml in a 250 ml flask) containing 10% sucrose and sodium chloride-free medium. After culturing for 24 hours, streak the culture onto LB agar plates containing 6% sucrose and sodium chloride-free medium. PCR verification showed that the primers used were XZ-lysE-up / XZ-lysE-down, and the correct colony amplification product was a 1341bp fragment. A correct single colony was selected and named HIS012 (which is a strain obtained by replacing the cat-sacB gene fragment in HIS004 with the lysE-V197A-S205A gene from Corynebacterium glutamicum).

[0104] Example 12: Genomic insertion of the lysE transporter gene lysE-V197A-S205G

[0105] Starting from Escherichia coli HIS004, using the plasmid PUC19-lysE-V197A-S205G as a template, an 833bp DNA fragment II (i.e., the lysE-V197A-S205G gene fragment) was amplified using primers yjiP-lysE-up / yjiP-lysE-down. The amplification system was the same as the first step system and was used for the second homologous recombination.

[0106] The pKD46 plasmid was transformed into HIS004 (HIS004-pKD46) by electroporation to obtain strain HIS004-pKD46.

[0107] DNA fragment II was electroporated into strain HIS004-pKD46. The electroporation conditions were as follows: First, prepare competent cells for electroporation of strain HIS004-pKD46. Place 50 μL of competent cells on ice, add 50 ng of DNA fragment II, incubate on ice for 2 minutes, and then transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporator with a voltage of 2.5 kV. After electroporation, quickly transfer 1 ml of LB broth to the cuvette, pipette five times, and then transfer to a test tube. Incubate at 75 rpm and 30°C for 4 hours. Transfer the bacterial culture to LB broth (50 ml in a 250 ml flask) containing 10% sucrose and sodium chloride-free medium. After culturing for 24 hours, streak the culture onto LB agar plates containing 6% sucrose and sodium chloride-free medium. PCR verification showed that the primers used were XZ-lysE-up / XZ-lysE-down, and the correct colony amplification product was a 1341bp fragment. A correct single colony was selected and named HIS013 (which is a strain obtained by replacing the cat-sacB gene fragment in HIS004 with lysE-V197A-S205G from Corynebacterium glutamicum).

[0108] Example 13: Fermentation production of L-histidine by recombinant strains

[0109] Following the procedure in Example 10, the recombinant strains HIS012, HIS013, and HIS005 containing wild-type lysE obtained above were fermented to produce L-histidine. The average values ​​of the analytical results are shown in Table 4 below.

[0110] Table 4 Comparison of L-histidine production capacity

[0111]

[0112] As shown in Tables 3 and 4, compared with the wild-type lysE-containing HIS005 strain, the mutant strains HIS006, HIS010, HIS011, HIS0012, and HIS0013 (i.e., strains containing the mutants lysE-V197A, lysE-S205A, lysE-S205G, lysE-V197A-S205A, and lysE-V197A-S205G, respectively) all exhibited increased histidine yields. In particular, the HIS013 strain containing the lysE-V197A-S205G mutant showed a 1.34-fold increase in L-histidine production. This demonstrates that the lysE mutants obtained in this application have a higher histidine transport capacity than wild-type lysE, thereby increasing L-histidine production.

[0113] sequence

[0114] SEQ ID NO: 1, the original amino acid sequence of the wild-type lysE transporter.

[0115] MVIMEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFLFIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLG GSAVATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLNPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLIWFPLVGFGAAALSRPLSSPKVWRWINVVVAVVMTALAIKLMLMG

[0116] SEQ ID NO: 2: Amino acid sequence of the lysE transporter V197A mutant

[0117] MVIMEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFLFIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLG GSAVATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLNPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLIWFPLAGFGAAALSRPLSSPKVWRWINVVVAVVMTALAIKLMLMG

[0118] SEQ ID NO: 3: Amino acid sequence of the lysE transporter V197F mutant

[0119] MVIMEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFLFIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLG GSAVATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLNPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLIWFPLFGFGAAALSRPLSSPKVWRWINVVVAVVMTALAIKLMLMG

[0120] SEQ ID NO: 4: Amino acid sequence of the lysE transporter A201F mutant

[0121] MVIMEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFLFIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLG GSAVATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLNPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLIWFPLVGFGFAALSRPLSSPKVWRWINVVVAVVMTALAIKLMLMG

[0122] SEQ ID NO: 5: Amino acid sequence of the lysE transporter A201G mutant

[0123] MVIMEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFLFIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLG GSAVATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLNPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLIWFPLVGFGGAALSRPLSSPKVWRWINVVVAVVMTALAIKLMLMG

[0124] SEQ ID NO: 6: Amino acid sequence of the lysE transporter S205A mutant

[0125] MVIMEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFLFIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLG GSAVATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLNPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLIWFPLVGFGAAALARPLSSPKVWRWINVVVAVVMTALAIKLMLMG

[0126] SEQ ID NO: 7: Amino acid sequence of the lysE transporter S205G mutant

[0127] MVIMEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFLFIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLG GSAVATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLNPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLIWFPLVGFGAAALGRPLSSPKVWRWINVVVAVVMTALAIKLMLMG

[0128] SEQ ID NO: 8: Amino acid sequence of the lysE transporter V197A-S205A mutant

[0129] MVIMEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFLFIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLG GSAVATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLNPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLIWFPLAGFGAAALARPLSSPKVWRWINVVVAVVMTALAIKLMLMG

[0130] SEQ ID NO: 9: Amino acid sequence of the lysE transporter V197A-S205G mutant

[0131] MVIMEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFLFIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLG GSAVATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLNPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLIWFPLAGFGAAALGRPLSSPKVWRWINVVVAVVMTALAIKLMLMG

[0132] SEQ ID NO: 10 (amino acid sequence of hisG-D84A-S205H, artificial sequence):

[0133] MNDVRNDDPNFNVMGNFDHGLTLALSKGRILKETLPLLATAGINLLEDPEKSRKLIFPTTHKQVRILILRASDVPTYVENGAAALGVAGKDVLMEHGAQHVYELLDLQIAKCK LMTAGKVGMERPKGRLKIATKYVNLTRQYYASLGEQVDVIKLYGSMELAPLVGLGDYIVDVVDTGNTLRANGLEPLEEICKVSSRLIVNKAHFKRKQVLLNPIISQLEQAVQSR

[0134] SEQ ID NO:11 (DNA sequence of hisG-D84A-S205H, artificial sequence):

[0135] ATGAATGACGTAAGAAACGATGATCCTAATTTTAACGTAATGGGTAATTTTGATCATGGTTTGACCTTGGCACTGAGTAAAGGACGTATTTTAAAAGAAACTTTACCTTTACTTGCTACAGCCGGTATTAACTTGCTTGAAGACCCGGAAAAATCGCGTAAGTTAATTTTTCCAACCACACATAAGCAGGTTCGTATTTTAATTTTACGTGCATCTGATGTGCCAACTTATGTTGAGAATGGTGCAGCTGCCTTAGGTGTAGCTGGTAAAGATGTACTTATGGAACATGGCGCTCAGCATGTCTATGAGTTATTGGACCTGCAGATTGCGAAATGTAAATTAATGACTGCTGGTAAAGTAGGAATGGAACGCCCAAAAGGTCGTTTAAAAATTGCCACTAAGTATGTCAATCTTACACGTCAATATTATGCGAGCTTAGGCGAGCAGGTGGATGTTATTAAACTTTATGGTTCTATGGAGTTAGCACCATTAGTTGGTTTGGGCGATTATATTGTTGACGTAGTGGATACTGGAAATACTTTACGAGCAAATGGCCTTGAGCCATTAGAAGAAATTTGCAAAGTATCTTCACGTCTTATTGTCAATAAAGCGCACTTTAAGCGTAAACAGGTTTTATTGAACCCAATCATTTCCCAACTTGAACAAGCTGTTCAATCACGTTAA

[0136] SEQ ID NO:12 (PUC19 sequence, artificial sequence)

[0137]

[0138] SEQ ID NO: 13 (DNA sequence of the wild-type lysE gene, artificial sequence):

[0139] ATGGTGATCATGGAAATCTTCATTACAGGTCTGCTTTTGGGGGCCAGTCTTTTACTGTCCATCGGACCGCAGAATGTACTGGTGATTAAACAAGGAATTAAGCGCGAAGGACTCATTGCGGTTCTTCTCGTGTGTTTAATTTCTGACGTCTTTTTGTTCATCGCCGGCACCTTGGGCGTTGATCTTTTGTCCAATGCCGCGCCGATCGTGCTCGATATTATGCGCTGGGGTGGCATCGCTTACCTGTTATGGTTTGCCGTCATGGCAGCGAAAGACGCCATGACAAACAAGGTGGAAGCGCCACAGATCATTGAAGAAACAGAACCAACCGTGCCCGATGACACGCCTTTGGGCGGTTCGGCGGTGGCCACTGACACGCGCAACCGGGTGCGGGTGGAGGTGAGCGTCGATAAGCAGCGGGTTTGGGTAAAGCCCATGTTGATGGCAATCGTGCTGACCTGGTTGAACCCGAATGCGTATTTGGACGCGTTTGTGTTTATCGGCGGCGTCGGCGCGCAATACGGCGACACCGGACGGTGGATTTTCGCCGCTGGCGCGTTCGCGGCAAGCCTGATCTGGTTCCCGCTGGTGGGTTTCGGCGCAGCAGCATTGTCACGCCCGCTGTCCAGCCCCAAGGTGTGGCGCTGGATCAACGTCGTCGTGGCAGTTGTGATGACCGCATTGGCCATCAAACTGATGTTGATGGGTTAG

[0140] SEQ ID NO: 14 (DNA sequence of the lysE-V197A mutant, artificial sequence):

[0141] ATGGTGATCATGGAAATCTTCATTACAGGTCTGCTTTTGGGGGCCAGTCTTTTACTGTCCATCGGACCGCAGAATGTACTGGTGATTAAACAAGGAATTAAGCGCGAAGGACTCATTGCGGTTCTTCTCGTGTGTTTAATTTCTGACGTCTTTTTGTTCATCGCCGGCACCTTGGGCGTTGATCTTTTGTCCAATGCCGCGCCGATCGTGCTCGATATTATGCGCTGGGGTGGCATCGCTTACCTGTTATGGTTTGCCGTCATGGCAGCGAAAGACGCCATGACAAACAAGGTGGAAGCGCCACAGATCATTGAAGAAACAGAACCAACCGTGCCCGATGACACGCCTTTGGGCGGTTCGGCGGTGGCCACTGACACGCGCAACCGGGTGCGGGTGGAGGTGAGCGTCGATAAGCAGCGGGTTTGGGTAAAGCCCATGTTGATGGCAATCGTGCTGACCTGGTTGAACCCGAATGCGTATTTGGACGCGTTTGTGTTTATCGGCGGCGTCGGCGCGCAATACGGCGACACCGGACGGTGGATTTTCGCCGCTGGCGCGTTCGCGGCAAGCCTGATCTGGTTCCCGCTGGcaGGTTTCGGCGCAGCAGCATTGTCACGCCCGCTGTCCAGCCCCAAGGTGTGGCGCTGGATCAACGTCGTCGTGGCAGTTGTGATGACCGCATTGGCCATCAAACTGATGTTGATGGGTTAG

[0142] SEQ ID NO:15 (DNA sequence of lysE-V197F mutant, artificial sequence):

[0143] ATGGTGATCATGGAAATCTTCATTACAGGTCTGCTTTTGGGGGCCAGTCTTTTACTGTCCATCGGACCGCAGAATGTACTGGTGATTAAACAAGGAATTAAGCGCGAAGGACTCATTGCGGTTCTTCTCGTGTGTTTAATTTCTGACGTCTTTTTGTTCATCGCCGGCACCTTGGGCGTTGATCTTTTGTCCAATGCCGCGCCGATCGTGCTCGATATTATGCGCTGGGGTGGCATCGCTTACCTGTTATGGTTTGCCGTCATGGCAGCGAAAGACGCCATGACAAACAAGGTGGAAGCGCCACAGATCATTGAAGAAACAGAACCAACCGTGCCCGATGACACGCCTTTGGGCGGTTCGGCGGTGGCCACTGACACGCGCAACCGGGTGCGGGTGGAGGTGAGCGTCGATAAGCAGCGGGTTTGGGTAAAGCCCATGTTGATGGCAATCGTGCTGACCTGGTTGAACCCGAATGCGTATTTGGACGCGTTTGTGTTTATCGGCGGCGTCGGCGCGCAATACGGCGACACCGGACGGTGGATTTTCGCCGCTGGCGCGTTCGCGGCAAGCCTGATCTGGTTtCCGCTGGTGGGTTTCGGCGCAGCAGCATTGTCACGCCCGCTGTCCAGCCCCAAGGTGTGGCGCTGGATCAACGTCGTCGTGGCAGTTGTGATGACCGCATTGGCCATCAAACTGATGTTGATGGGTTAG

[0144] SEQ ID NO: 16 (DNA sequence of lysE-A201F mutant, artificial sequence):

[0145] ATGGTGATCATGGAAATCTTCATTACAGGTCTGCTTTTGGGGGCCAGTCTTTTACTGTCCATCGGACCGCAGAATGTACTGGTGATTAAACAAGGAATTAAGCGCGAAGGACTCATTGCGGTTCTTCTCGTGTGTTTAATTTCTGACGTCTTTTTGTTCATCGCCGGCACCTTGGGCGTTGATCTTTTGTCCAATGCCGCGCCGATCGTGCTCGATATTATGCGCTGGGGTGGCATCGCTTACCTGTTATGGTTTGCCGTCATGGCAGCGAAAGACGCCATGACAAACAAGGTGGAAGCGCCACAGATCATTGAAGAAACAGAACCAACCGTGCCCGATGACACGCCTTTGGGCGGTTCGGCGGTGGCCACTGACACGCGCAACCGGGTGCGGGTGGAGGTGAGCGTCGATAAGCAGCGGGTTTGGGTAAAGCCCATGTTGATGGCAATCGTGCTGACCTGGTTGAACCCGAATGCGTATTTGGACGCGTTTGTGTTTATCGGCGGCGTCGGCGCGCAATACGGCGACACCGGACGGTGGATTTTCGCCGCTGGCGCGTTCGCGGCAAGCCTGATCTGGTTCCCGCTGGTGGGTTTCGGCtttGCAGCATTGTCACGCCCGCTGTCCAGCCCCAAGGTGTGGCGCTGGATCAACGTCGTCGTGGCAGTTGTGATGACCGCATTGGCCATCAAACTGATGTTGATGGGTTAG

[0146] SEQ ID NO: 17 (DNA sequence of lysE - A201G mutant, artificial sequence):

[0147] ATGGTGATCATGGAAATCTTCATTACAGGTCTGCTTTTGGGGGCCAGTCTTTTACTGTCCATCGGACCGCAGAATGTACTGGTGATTAAACAAGGAATTAAGCGCGAAGGACTCATTGCGGTTCTTCTCGTGTGTTTAATTTCTGACGTCTTTTTGTTCATCGCCGGCACCTTGGGCGTTGATCTTTTGTCCAATGCCGCGCCGATCGTGCTCGATATTATGCGCTGGGGTGGCATCGCTTACCTGTTATGGTTTGCCGTCATGGCAGCGAAAGACGCCATGACAAACAAGGTGGAAGCGCCACAGATCATTGAAGAAACAGAACCAACCGTGCCCGATGACACGCCTTTGGGCGGTTCGGCGGTGGCCACTGACACGCGCAACCGGGTGCGGGTGGAGGTGAGCGTCGATAAGCAGCGGGTTTGGGTAAAGCCCATGTTGATGGCAATCGTGCTGACCTGGTTGAACCCGAATGCGTATTTGGACGCGTTTGTGTTTATCGGCGGCGTCGGCGCGCAATACGGCGACACCGGACGGTGGATTTTCGCCGCTGGCGCGTTCGCGGCAAGCCTGATCTGGTTCCCGCTGGTGGGTTTCGGCGgtGCAGCATTGTCACGCCCGCTGTCCAGCCCCAAGGTGTGGCGCTGGATCAACGTCGTCGTGGCAGTTGTGATGACCGCATTGGCCATCAAACTGATGTTGATGGGTTAG

[0148] SEQ ID NO: 18 (DNA sequence of lysE-S205A mutant, artificial sequence):

[0149] ATGGTGATCATGGAAATCTTCATTACAGGTCTGCTTTTGGGGGCCAGTCTTTTACTGTCCATCGGACCGCAGAATGTACTGGTGATTAAACAAGGAATTAAGCGCGAAGGACTCATTGCGGTTCTTCTCGTGTGTTTAATTTCTGACGTCTTTTTGTTCATCGCCGGCACCTTGGGCGTTGATCTTTTGTCCAATGCCGCGCCGATCGTGCTCGATATTATGCGCTGGGGTGGCATCGCTTACCTGTTATGGTTTGCCGTCATGGCAGCGAAAGACGCCATGACAAACAAGGTGGAAGCGCCACAGATCATTGAAGAAACAGAACCAACCGTGCCCGATGACACGCCTTTGGGCGGTTCGGCGGTGGCCACTGACACGCGCAACCGGGTGCGGGTGGAGGTGAGCGTCGATAAGCAGCGGGTTTGGGTAAAGCCCATGTTGATGGCAATCGTGCTGACCTGGTTGAACCCGAATGCGTATTTGGACGCGTTTGTGTTTATCGGCGGCGTCGGCGCGCAATACGGCGACACCGGACGGTGGATTTTCGCCGCTGGCGCGTTCGCGGCAAGCCTGATCTGGTTCCCGCTGGTGGGTTTCGGCGgtGCAGCATTGTCACGCCCGCTGTCCAGCCCCAAGGTGTGGCGCTGGATCAACGTCGTCGTGGCAGTTGTGATGACCGCATTGGCCATCAAACTGATGTTGATGGGTTAG

[0150] SEQ ID NO: 19 (DNA sequence of lysE-S205G mutant, artificial sequence):

[0151] ATGGTGATCATGGAAATCTTCATTACAGGTCTGCTTTTGGGGGCCAGTCTTTTACTGTCCATCGGACCGCAGAATGTACTGGTGATTAAACAAGGAATTAAGCGCGAAGGACTCATTGCGGTTCTTCTCGTGTGTTTAATTTCTGACGTCTTTTTGTTCATCGCCGGCACCTTGGGCGTTGATCTTTTGTCCAATGCCGCGCCGATCGTGCTCGATATTATGCGCTGGGGTGGCATCGCTTACCTGTTATGGTTTGCCGTCATGGCAGCGAAAGACGCCATGACAAACAAGGTGGAAGCGCCACAGATCATTGAAGAAACAGAACCAACCGTGCCCGATGACACGCCTTTGGGCGGTTCGGCGGTGGCCACTGACACGCGCAACCGGGTGCGGGTGGAGGTGAGCGTCGATAAGCAGCGGGTTTGGGTAAAGCCCATGTTGATGGCAATCGTGCTGACCTGGTTGAACCCGAATGCGTATTTGGACGCGTTTGTGTTTATCGGCGGCGTCGGCGCGCAATACGGCGACACCGGACGGTGGATTTTCGCCGCTGGCGCGTTCGCGGCAAGCCTGATCTGGTTCCCGCTGGTGGGTTTCGGCGCAGCAGCATTGggtCGCCCGCTGTCCAGCCCCAAGGTGTGGCGCTGGATCAACGTCGTCGTGGCAGTTGTGATGACCGCATTGGCCATCAAACTGATGTTGATGGGTTAG

[0152] SEQ ID NO: 20 (DNA sequence of lysE-V197A-S205A mutant, artificial sequence):

[0153] ATGGTGATCATGGAAATCTTCATTACAGGTCTGCTTTTGGGGGCCAGTCTTTTACTGTCCATCGGACCGCAGAATGTACTGGTGATTAAACAAGGAATTAAGCGCGAAGGACTCATTGCGGTTCTTCTCGTGTGTTTAATTTCTGACGTCTTTTTGTTCATCGCCGGCACCTTGGGCGTTGATCTTTTGTCCAATGCCGCGCCGATCGTGCTCGATATTATGCGCTGGGGTGGCATCGCTTACCTGTTATGGTTTGCCGTCATGGCAGCGAAAGACGCCATGACAAACAAGGTGGAAGCGCCACAGATCATTGAAGAAACAGAACCAACCGTGCCCGATGACACGCCTTTGGGCGGTTCGGCGGTGGCCACTGACACGCGCAACCGGGTGCGGGTGGAGGTGAGCGTCGATAAGCAGCGGGTTTGGGTAAAGCCCATGTTGATGGCAATCGTGCTGACCTGGTTGAACCCGAATGCGTATTTGGACGCGTTTGTGTTTATCGGCGGCGTCGGCGCGCAATACGGCGACACCGGACGGTGGATTTTCGCCGCTGGCGCGTTCGCGGCAAGCCTGATCTGGTTCCCGCTGGcaGGTTTCGGCGCAGCAGCATTGgCACGCCCGCTGTCCAGCCCCAAGGTGTGGCGCTGGATCAACGTCGTCGTGGCAGTTGTGATGACCGCATTGGCCATCAAACTGATGTTGATGGGTTAG

[0154] SEQ ID NO: 21 (DNA sequence of lysE-V197A-S205G mutant, artificial sequence):

[0155] ATGGTGATCATGGAAATCTTCATTACAGGTCTGCTTTTGGGGGCCAGTCTTTTACTGTCCATCGGACCGCAGAATGTACTGGTGATTAAACAAGGAATTAAGCGCGAAGGACTCATTGCGGTTCTTCTCGTGTGTTTAATTTCTGACGTCTTTTTGTTCATCGCCGGCACCTTGGGC GTTGATCTTTTGTCCAATGCCGCCGATCGTGCTCGATATTATGCGCTGGGGTGGCATCGCTTACCTGTTATGGTTTGCCGTCATGGCAGCGAAAGACGCCATGACAAACAAGGTGGAAGCGCCACAGATCATTGAAGAAACAGAACCAACCGTGCCCGATGACACGCCTTTGGGCG GTTCGGCGGTGGCCACTGACACGCGCAACCGGGTGCGGGTGGAGGTGAGCGTCGATAAGCAGCGGGTTTGGGTAAAGCCCATGTTGATGGCAATCGTGCTGACCTGGTTGAACCCGAATGCGTATTTGGACGCGTTTGTGTTTATCGGCGGCGTCGGCGCGCAATACGGCGACACCGG ACGGTGGATTTTCGCCGCTGGCGCGTTCGCGGCAAGCCTGATCTGGTTCCCGCTGGcaGGTTTCGGCGCAGCAGCATTGggtCGCCCGCTGTCCAGCCCCAAGGTGTGGCGCTGGATCAACGTCGTCGTGGCAGTTGTGATGACCGCATTGGCCATCAAACTGATGTTGATGGGTTAG

[0156] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A mutant of the transporter protein lysE, which, relative to the wild-type transporter protein lysE shown in SEQ ID NO: 1, has the following amino acid substitutions: valine at position 197 is replaced by alanine, and / or serine at position 205 is replaced by alanine or glycine.

2. The lysE mutant transporter protein according to claim 1, wherein, The coding sequence of the wild-type transporter lysE is shown in SEQ ID NO:

13.

3. A nucleic acid molecule encoding a mutant of the transporter protein lysE according to any one of claims 1-2.

4. An expression vector comprising the nucleic acid molecule of claim 3.

5. A host cell comprising the nucleic acid molecule of claim 3 or the expression vector of claim 4.

6. The use of the lysE mutant of the transporter protein as described in any one of claims 1-2, the nucleic acid molecule as described in claim 3, the expression vector as described in claim 4, or the host cell as described in claim 5 in the preparation of L-histidine or L-histidine derivatives.

7. A method for producing L-histidine or an L-histidine derivative, the method comprising culturing bacteria in a culture medium to produce L-histidine, wherein the bacteria contain the nucleic acid molecule of claim 3 or the expression vector of claim 4; in, The bacteria are *Escherichia coli*. The hisG gene of the bacterial genome is replaced with the hisG-D84A-S205H gene of an ATP phosphoribosyltransferase mutant derived from *Acinetobacter baumannii*, which has dual mutations at both D84A and S205H sites, to relieve the feedback inhibition of ATP phosphoribosyltransferase. Furthermore, the yjiP gene of the bacteria is knocked out, and a nucleic acid molecule encoding the transporter protein lysE mutant is inserted at the yjiP gene site. The sequence of the gene hisG-D84A-S205H is shown in SEQ ID NO: 11.

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