Transport protein lysE mutant and application thereof in increasing yield of L-histidine

Abstract

The invention relates to an application of a transporter lysE mutant in increasing the yield of L-histidine. Specifically, the invention provides a transporter lysE mutant, and the transporter lysE mutant has the following amino acid substitution relative to a wild transporter lysE as shown in SEQ ID NO: 1: valine at the 197th site is substituted by alanine, or / and serine at the 205th site is substituted by alanine or glycine. The invention also provides an application of the transporter lysE mutant in preparation of L-histidine or an L-histidine derivative. Compared with a strain containing wild type lysE, the strain containing the lysE mutant has the advantage that the histidine yield is improved.

Description

Technical Field

[0001] The invention relates to the field of L-histidine production, and in particular to the preparation of L-histidine through a microbial pathway. Background Art

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

[0003] Histidine is mainly present in hemoglobin, and its traditional preparation method is to produce it by protein hydrolysis and extraction method using blood powder as raw material. However, this method has disadvantages such as low efficiency and environmental pollution. It is reported in the prior art that the production of L-histidine can be achieved by microbial fermentation, but it has not yet been achieved in large-scale industry. This is because the histidine metabolic pathway is long, involving multiple key enzymes, and the current yield of histidine produced by Escherichia coli is low, which may be due to the negative feedback inhibition caused by the accumulation of histidine in the organism, limiting its further synthesis in the organism.

[0004] In order to realize a cell factory with high amino acid production, the transport of L-histidine is a key factor. CN110184230A discloses a genetically engineered bacterium with high L-histidine production, in which the coding gene lysE of the arginine / lysine transporter derived from Corynebacterium glutamicum is integrated into the genome and the strong promoter Ptrc is used to make it strongly expressed. The application first confirms that the arginine / lysine transporter LysE introduced into Corynebacterium glutamicum helps to improve the production of histidine, and is an important target for the transformation of histidine strains. The transformation of histidine-related transporters by genetic engineering means is of great significance for obtaining a higher yield of L-histidine production strains. Summary of the invention

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

[0006] In microorganisms such as Escherichia coli, the main pathway for L-histidine biosynthesis is the pentose phosphate pathway. When glucose is used as the carbon source, the L-histidine synthesis precursor phosphoribosyl pyrophosphate (PRPP) is generated through the pentose phosphate pathway. Subsequently, the PRPP and adenosine triphosphate (ATP) are catalyzed by ATP phosphoribosyltransferase, and then undergo a series of reactions including ring opening, isomerization and amide transfer to generate L-histidine. Among them, ATP phosphoribosyltransferase (HisG) is the first key enzyme in this biosynthetic pathway. Its activity is inhibited by different intermediate metabolites and end products, especially the end product L-histidine, thereby limiting the production of L-histidine. In the early research and development work, the inventors found that site mutation of HisG (D84A + S205H) can relieve its feedback inhibition.

[0007] To achieve high L-histidine production, efficient intracellular L-histidine transport is a key factor. As can be seen from the above, the arginine / lysine transporter lysE from Corynebacterium glutamicum (its encoding gene is called lysE gene (GeneID: 1019243)) helps to increase histidine production and is an important target for the transformation of L-histidine-producing strains.

[0008] In order to construct a genetically engineered strain with higher L-histidine production, the present invention utilizes genetic engineering technology to transform the L-histidine-producing Escherichia coli. The present invention studies the structure of the lysE gene, performs a series of site-directed mutations on lysE, transfers the mutated lysE gene into a host bacterium, and screens to obtain a mutant with improved histidine transport effect, which can timely discharge the produced histidine to the extracellular space to avoid accumulation in the cell, and therefore, relative to the wild-type lysE transporter, the mutant of the present application increases the fermentation yield of L-histidine.

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

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

[0011] Further, as can be seen from the above, ATP phosphoribosyl transferase (HisG) is a key enzyme in the histidine biosynthesis pathway. Therefore, in a preferred embodiment of the present application, in order to further increase the yield of histidine, the applicant also introduced an exogenous hisG gene in the chassis strain and performed D84A and S205H mutations according to previous studies (see patent application number CN202410091107.5) to relieve the feedback inhibition of histidine on ATP phosphoribosyl transferase (HisG). It will be appreciated by those skilled in the art that the introduction of an exogenous hisG gene in the chassis strain and the D84A and S205H mutations are only the preferred technical solutions of the present application; in strains that do not perform the above operations, the lysE mutant of the present application still has the effect of increasing the yield of L-histidine relative to the wild-type lysE.

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

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

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

[0015] 3. A nucleic acid molecule encoding the transporter lysE mutant 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. Use of the transporter protein lysE mutant according to any one of items 1-2, the nucleic acid molecule according to item 3, the expression vector according to item 4 or the host cell according to item 5 in the preparation of L-histidine or an L-histidine derivative.

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

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

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

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

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

[0024] Beneficial Effects

[0025] In order to construct a genetically engineered strain with higher L-histidine production, the present invention studies the structure of the lysE gene, performs a series of site-directed mutations on the lysE gene, transfers the mutated lysE gene into a host bacterium, and screens to obtain a lysE variant with increased histidine production. The variant can more efficiently discharge the generated histidine to the extracellular space to avoid accumulation in the cell, and relative to the wild-type lysE transporter, it increases the fermentation yield of histidine, and has broad industrial development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The molecular docking results of the wild-type lysE transporter using L-histidine as substrate in AutoDock Vina software are shown. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

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

[0029] The strains, plasmids and primers used in this application can be found in Tables 1 and 2.

[0030] Table 1 Strains and plasmids used in the present invention

[0031]

[0032]

[0033] Table 2 Primers used in the present invention

[0034]

[0035] Example 1: Bioinformatics analysis of lysE gene structure - AutoDock Vina molecular docking

[0036] The lysE transporter protein uses L-histidine as a substrate, and molecular docking is performed in AutoDock Vina to observe the interaction between the lysE transporter protein and L-histidine. The AutoDock Vina is a conventional molecular simulation software in the prior art, which is usually used to study the interaction between biological macromolecules (such as proteins) and small molecules (such as ligands). The results are as follows Figure 1 As shown, L-histidine forms hydrogen bonds with the V197, A201, and S205 residues of the lysE transporter, which are key residues of the active pocket. Therefore, in order to obtain a strain with higher histidine production, the present application performs point mutations on the above key residues, and obtains lysE mutants that may increase histidine production, namely V197A, V197F, A201F, A201G, S205A, and S205G, through analysis.

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

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

[0039] Starting from Escherichia coli W3110, the ATP phosphoribosyltransferase gene hisG (GeneID: 66396838) was knocked out by a two-step homologous recombination method, and the modified Acinetobacter baumannii (also known as AB bacteria) hisG-D84A-S205H gene (whose sequence is shown in SEQ ID NO: 11) was inserted. The specific steps are as follows:

[0040] 1. Preparation of HIS001 strain

[0041] Using pRE112 plasmid (purchased from Miaoling Plasmid Platform, product number: P1702, which contains cat-sacB gene) DNA as template, primers hisG-cs-up / hisG-cs-down were used to amplify 3593bp DNA fragment I (i.e., cat-sacB gene fragment) for the first step of homologous recombination.

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

[0043] Amplification conditions were as follows: pre-denaturation at 98°C for 2 minutes (1 cycle); denaturation at 98°C for 10 seconds, annealing at 56°C for 10 seconds, extension at 72°C for 4 minutes (30 cycles); extension at 72°C 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 Escherichia coli Collection Center of Yale University, USA, CGSC#7739) was transformed into Escherichia coli W3110 by electroporation to obtain strain W3110-pKD46, and then the DNA fragment I (i.e., the cat-sacB gene fragment) was electroporated into Escherichia coli W3110 carrying pKD46 (i.e., electroporated into strain W3110-pKD46).

[0045] The electroporation conditions were as follows: strain W3110-pKD46 was prepared as competent cells by the CaCl2 method; then 50 μL of W3110-pKD46 competent cells were placed on ice, 50 ng of DNA fragment I was added, placed on ice for 2 minutes, and transferred to a 0.2 cm Bio-Rad electroporation cup. MicroPulser (Bio-Rad) electroporator was used, and the electroporation parameters were 2.5 kV. After electroporation, 1 ml of LB medium was quickly transferred to the electroporation cup, pipetted 5 times, and then transferred to a test tube, incubated at 75 rpm and 30°C for 2 hours. 200 μL of bacterial solution was spread on LB plates containing ampicillin (final concentration of 100 μg / ml) and chloramphenicol (final concentration of 34 μg / ml). After overnight culture 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 with successful electroporation of DNA fragment I) was selected and named HIS001 (also known as W3110△hisG::cat-sacB, i.e., the ATP phosphoribosyltransferase gene hisG of Escherichia coli W3110 was knocked out and the cs (cat-sacB) gene fragment of the pRE112 plasmid was successfully electroporated).

[0046] 2. Preparation of HIS003 strain

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

[0048] Using plasmid PUC19-hisG-D84A-S205H as a template, primers hisG-mut-cs-up / hisG-mut-cs-down were used to amplify the 812 bp DNA fragment II. The amplification system was the same as 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 to strain HIS004-pKD46. The specific operation was as follows: prepare electroporation competent cells of HIS001-pKD46 strain; place 50 μL competent cells on ice, add 50 ng DNA fragment II, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cup. Use MicroPulser (Bio-Rad) electroporator, and the electroporation parameter is 2.5 kV. After electroporation, quickly transfer 1 ml LB liquid culture medium to the electroporation cup, blow 5 times and then transfer to a test tube, 75 rpm, and incubate at 30°C for 4 hours. Transfer the bacterial liquid to LB liquid culture medium without sodium chloride containing 10% sucrose (50 ml culture medium in a 250 ml flask), and after culturing for 24 hours, streak culture on LB plates containing 6% sucrose and without sodium chloride. After PCR verification, 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, lysE transporter gene genome insertion

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

[0053] 1. Preparation of HIS004 strain

[0054] Using pRE112 plasmid (purchased from Miaoling Plasmid Platform, product number: P1702, which contains cat-sacB gene) DNA as template, primers yjiP-cs-up / yjiP-cs-down were used to amplify 3588bp DNA fragment I (i.e., cat-sacB gene fragment) for the first step of homologous recombination.

[0055] The amplification system was the same as the first step system in 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 (obtaining the strain HIS003-pKD46), and then the DNA fragment I was electroporated into HIS003 carrying pKD46.

[0057] The electroporation conditions were as follows: HIS003-pKD46 was prepared as competent cells by the CaCl2 method; then 50 μL of HIS003-pKD46 competent cells were placed on ice, 50 ng of DNA fragment I was added, placed on ice for 2 minutes, and transferred to a 0.2 cm Bio-Rad electroporation cup. MicroPulser (Bio-Rad) electroporator was used, and the electroporation parameters were 2.5 kV. After electroporation, 1 ml of LB medium was quickly transferred to the electroporation cup, pipetted 5 times, and then transferred to a test tube, incubated at 75 rpm and 30°C for 2 hours. Take 200 μL of bacterial solution and spread it on LB plate containing ampicillin (final concentration of 100 μg / ml) and chloramphenicol (final concentration of 34 μg / ml). After culturing overnight at 30°C, select a single colony for PCR verification. The primers used are XZ-lysE-up / XZ-lysE-down. The amplification product of the correct colony is a fragment of 4099 bp. Select a correct single colony and name it 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 PUC19-lysE original plasmid as a template, the primers yjiP-lysE-up / yjiP-lysE-down were used to amplify the 833 bp DNA fragment II (i.e., the lysE gene fragment). The amplification system was the same as the first step and was used for the second homologous recombination.

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

[0061] DNA fragment II was electroporated to strain HIS004-pKD46. The electroporation conditions were as follows: first, prepare electroporation competent cells of strain HIS004-pKD46; place 50 μL competent cells on ice, add 50 ng DNA fragment II, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cup. Use MicroPulser (Bio-Rad) electroporator, and the electroporation parameters are 2.5 kV. After electroporation, quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, blow 5 times, transfer to a test tube, 75 rpm, and incubate at 30°C for 4 hours. Transfer the bacterial liquid to LB liquid culture medium without sodium chloride containing 10% sucrose (50 ml culture medium in a 250 ml flask), and after culturing for 24 hours, streak culture on an LB plate containing 6% sucrose and without sodium chloride. After PCR verification, 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 (a strain obtained by replacing the cat-sacB gene fragment in HIS004 with lysE from Corynebacterium glutamicum).

[0062] Example 4, lysE transporter gene lysE-V197A genome insertion

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

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

[0065] DNA fragment II was electroporated to strain HIS004-pKD46. The electroporation conditions were as follows: first, prepare electroporation competent cells of strain HIS004-pKD46; place 50 μL competent cells on ice, add 50 ng DNA fragment II, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cup. Use MicroPulser (Bio-Rad) electroporator, and the electroporation parameters are 2.5 kV. After electroporation, quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, blow 5 times, transfer to a test tube, 75 rpm, and incubate at 30°C for 4 hours. Transfer the bacterial liquid to LB liquid culture medium without sodium chloride containing 10% sucrose (50 ml culture medium in a 250 ml flask), and after culturing for 24 hours, streak culture on an LB plate containing 6% sucrose and without sodium chloride. After PCR verification, 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 (a strain obtained by replacing the cat-sacB gene fragment in HIS004 with lysE-V197A from Corynebacterium glutamicum).

[0066] Example 5, lysE transporter gene lysE-V197F genome insertion

[0067] Starting from Escherichia coli HIS004, using the PUC19-lysE-V197F plasmid as a template, the 833 bp 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 and was used for the second homologous recombination.

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

[0069] DNA fragment II was electroporated to strain HIS004-pKD46. The electroporation conditions were as follows: first, prepare electroporation competent cells of HIS004-pKD46 strain; place 50 μL competent cells on ice, add 50 ng DNA fragment II, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cup. Use MicroPulser (Bio-Rad) electroporator, and the electroporation parameters are 2.5 kV. After electroporation, quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, blow 5 times, transfer to a test tube, 75 rpm, and incubate at 30°C for 4 hours. Transfer the bacterial liquid to LB liquid culture medium without sodium chloride containing 10% sucrose (50 ml culture medium in a 250 ml flask), and after culturing for 24 hours, streak culture on LB plates containing 6% sucrose and without sodium chloride. After PCR verification, 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 (a strain obtained by replacing the cat-sacB gene fragment in HIS004 with lysE-V197F from Corynebacterium glutamicum).

[0070] Example 6, lysE transporter gene lysE-A201F genome insertion

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

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

[0073] DNA fragment II was electroporated to strain HIS004-pKD46. The electroporation conditions were as follows: first, prepare electroporation competent cells of strain HIS004-pKD46; place 50 μL competent cells on ice, add 50 ng DNA fragment II, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cup. Use MicroPulser (Bio-Rad) electroporator, and the electroporation parameters are 2.5 kV. After electroporation, quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, blow 5 times, transfer to a test tube, 75 rpm, and incubate at 30°C for 4 hours. Transfer the bacterial liquid to LB liquid culture medium without sodium chloride containing 10% sucrose (50 ml culture medium in a 250 ml flask), and after culturing for 24 hours, streak culture on an LB plate containing 6% sucrose and without sodium chloride. After PCR verification, 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 (a strain obtained by replacing the cat-sacB gene fragment in HIS004 with lysE-A201F from Corynebacterium glutamicum).

[0074] Example 7, lysE transporter gene lysE-A201G genome insertion

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

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

[0077] DNA fragment II was electroporated to strain HIS004-pKD46. The electroporation conditions were as follows: first, prepare electroporation competent cells of HIS004-pKD46 strain; place 50 μL competent cells on ice, add 50 ng DNA fragment II, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cup. Use MicroPulser (Bio-Rad) electroporator, and the electroporation parameters are 2.5 kV. After electroporation, quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, blow 5 times, transfer to a test tube, 75 rpm, and incubate at 30°C for 4 hours. Transfer the bacterial liquid to LB liquid culture medium without sodium chloride containing 10% sucrose (50 ml culture medium in a 250 ml flask), and after culturing for 24 hours, streak culture on an LB plate containing 6% sucrose and without sodium chloride. After PCR verification, 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 (a strain obtained by replacing the cat-sacB gene fragment in HIS004 with lysE-A201G from Corynebacterium glutamicum).

[0078] Example 8, lysE transporter gene lysE-S205A genome insertion

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

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

[0081] DNA fragment II was electroporated to strain HIS004-pKD46. The electroporation conditions were as follows: first, prepare electroporation competent cells of strain HIS004-pKD46; place 50 μL competent cells on ice, add 50 ng DNA fragment II, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cup. Use MicroPulser (Bio-Rad) electroporator, and the electroporation parameters are 2.5 kV. After electroporation, quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, blow 5 times, transfer to a test tube, 75 rpm, and incubate at 30°C for 4 hours. Transfer the bacterial liquid to LB liquid culture medium without sodium chloride containing 10% sucrose (50 ml culture medium in a 250 ml flask), and after culturing for 24 hours, streak culture on an LB plate containing 6% sucrose and without sodium chloride. After PCR verification, 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 (a strain obtained by replacing the cat-sacB gene fragment in HIS004 with lysE-S205A from Corynebacterium glutamicum).

[0082] Example 9, lysE transporter gene lysE-S205G genome insertion

[0083] Starting from Escherichia coli HIS004, using the PUC19-lysE-S205G plasmid as a template, the 833 bp 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 and was used for the second homologous recombination.

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

[0085] DNA fragment II was electroporated to strain HIS004-pKD46. The electroporation conditions were as follows: first, prepare electroporation competent cells of strain HIS004-pKD46; place 50 μL competent cells on ice, add 50 ng DNA fragment II, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cup. Use MicroPulser (Bio-Rad) electroporator, and the electroporation parameters are 2.5 kV. After electroporation, quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, blow 5 times, transfer to a test tube, 75 rpm, and incubate at 30°C for 4 hours. Transfer the bacterial liquid to LB liquid culture medium without sodium chloride containing 10% sucrose (50 ml culture medium in a 250 ml flask), and after culturing for 24 hours, streak culture on an LB plate containing 6% sucrose and without sodium chloride. After PCR verification, 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 (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 examples are fermented to produce L-histidine, comprising the following steps:

[0088] (1) Seed culture: A fresh single clone on the LB plate was inoculated into a test tube containing 4 ml of seed culture medium and cultured overnight at 37°C, 250 rpm with shaking. Then, the culture was transferred to a 250 ml Erlenmeyer flask containing 30 ml of seed culture medium at an inoculum volume of 2% (V / V) and cultured at 37°C, 250 rpm with shaking for 12 hours to obtain a seed culture solution for inoculation of the fermentation medium.

[0089] (2) Fermentation culture: The volume of the fermentation medium in a 500 ml fermenter is 250 ml. The seed culture solution is inoculated into the fermentation medium at a final concentration of OD550 = 0.1, and fermented at 37°C, 150 rpm for 3 days to obtain a fermentation solution. The neutralizer is 5 M ammonia water, and the pH of the fermenter is controlled at 7.0.

[0090] Analysis method: Agilent-1260 high performance liquid chromatograph was used to determine the components in the fermentation broth after 3 days of fermentation. L-histidine was determined using an amino acid analysis column ZORBAX Eclipse AAA 4.6 mm×75 mm3.5-Micron. Detection wavelength: 338.

[0091] The experiment was repeated 3 times and the average values ​​of the analytical results are presented in Table 3 below.

[0092] Main culture medium:

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

[0094] Shake flask fermentation seed 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, 1mgVB5, 1mg VB12, 1mg VH, sodium hydroxide to adjust the pH between 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, sodium hydroxide was used to adjust the pH to 7.0-7.2, and ammonia water was added every 4h of fermentation to a pH of 7.2-7.4.

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

[0097]

[0098] The results showed (see Table 3 for details) that compared with the HIS005 strain containing wild-type lysE, for the 197 mutation, the histidine production of HIS006 (HIS003△yjiP::lysE-V197A) was increased, while the histidine production of HIS007 (HIS003△yjiP::lysE-V197F) was reduced, so the V197A mutation was selected for subsequent experiments; for the 201 mutation, the histidine production of HIS008 (HIS003△yjiP::lysE-A201F) and HIS009 (HIS003△yjiP::lysE-A201G) were both reduced, so the 201 mutation could not achieve the effect of increasing the histidine production; for the 205 mutation, HIS010 (HIS003△yjiP::lysE-S205A) and HIS011 The histidine production of (HIS003△yjiP::lysE-S205G) was improved, so both can be used for subsequent experiments.

[0099] Example 11, lysE transporter gene lysE-V197A-S205A genome insertion

[0100] In this example, strains containing combinations of mutations were further constructed.

[0101] Starting from Escherichia coli HIS004, using the PUC19-lysE-V197A-S205A plasmid as a template, the 833 bp 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 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 the strain HIS004-pKD46.

[0103] DNA fragment II was electroporated to strain HIS004-pKD46. The electroporation conditions were as follows: first, prepare electroporation competent cells of HIS004-pKD46 strain; place 50 μL competent cells on ice, add 50 ng DNA fragment II, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cup. Use MicroPulser (Bio-Rad) electroporator, and the electroporation parameters are 2.5 kV. After electroporation, quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, blow 5 times and transfer to a test tube, incubate at 75 rpm and 30°C for 4 hours. Transfer the bacterial liquid to LB liquid culture medium without sodium chloride containing 10% sucrose (50 ml culture medium in a 250 ml flask), and after culturing for 24 hours, streak culture on an LB plate containing 6% sucrose and without sodium chloride. After PCR verification, 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 (a strain obtained by replacing the cat-sacB gene fragment in HIS004 with the lysE-V197A-S205A gene from Corynebacterium glutamicum).

[0104] Example 12, lysE transporter gene lysE-V197A-S205G genome insertion

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

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

[0107] DNA fragment II was electroporated to strain HIS004-pKD46. The electroporation conditions were as follows: first, prepare electroporation competent cells of HIS004-pKD46 strain; place 50 μL competent cells on ice, add 50 ng DNA fragment II, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cup. Use MicroPulser (Bio-Rad) electroporator, and the electroporation parameters are 2.5 kV. After electroporation, quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, blow 5 times, transfer to a test tube, 75 rpm, and incubate at 30°C for 4 hours. Transfer the bacterial liquid to LB liquid culture medium without sodium chloride containing 10% sucrose (50 ml culture medium in a 250 ml flask), and after culturing for 24 hours, streak culture on an LB plate containing 6% sucrose and without sodium chloride. After PCR verification, 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 (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] According to the operation of Example 10, the recombinant bacteria HIS012, HIS013 and the HIS005 strain containing wild-type lysE obtained above were fermented to produce L-histidine, and the average values ​​of the analysis results are presented in Table 4 below.

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

[0111]

[0112] As shown in Table 3 and Table 4, compared with the HIS005 strain containing wild-type lysE, the HIS006, HIS010, HIS011, HIS0012, HIS0013 strains containing mutants (i.e., strains containing mutants lysE-V197A, lysE-S205A, lysE-S205G, lysE-V197A-S205A, lysE-V197A-S205G, respectively) all have the histidine productivity that improves, and the HIS013 strain that particularly contains the lysE-V197A-S205G mutant, its L-histidine production capacity has increased by 1.34 times. It has been shown that the lysE mutant obtained by the present application screening has a higher histidine transport capacity relative to wild-type lysE, thereby being able to improve the output of L-histidine.

[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 lysE transporter V197A mutant

[0117] MVIMEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFLFIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLG GSAVATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLNPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLIWFPLAGFGAAALSRPLSSPKVWRWINVVVAVVMTALAIKLMLMG

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

[0119] MVIMEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFLFIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLG GSAVATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLNPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLIWFPLFGFGAAALSRPLSSPKVWRWINVVVAVVMTALAIKLMLMG

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

[0121] MVIMEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFLFIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLG GSAVATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLNPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLIWFPLVGFGFAALSRPLSSPKVWRWINVVVAVVMTALAIKLMLMG

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

[0123] MVIMEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFLFIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLG GSAVATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLNPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLIWFPLVGFGGAALSRPLSSPKVWRWINVVVAVVMTALAIKLMLMG

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

[0125] MVIMEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFLFIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLG GSAVATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLNPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLIWFPLVGFGAAALARPLSSPKVWRWINVVVAVVMTALAIKLMLMG

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

[0127] MVIMEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFLFIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLG GSAVATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLNPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLIWFPLVGFGAAALGRPLSSPKVWRWINVVVAVVMTALAIKLMLMG

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

[0129] MVIMEIFITGLLLGASLLLSIGPQNVLVIKQGIKREGLIAVLLVCLISDVFLFIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEAPQIIEETEPTVPDDTPLG GSAVATDTRNRVRVEVSVDKQRVWVKPMLMAIVLTWLNPNAYLDAFVFIGGVGAQYGDTGRWIFAAGAFAASLIWFPLAGFGAAALARPLSSPKVWRWINVVVAVVMTALAIKLMLMG

[0130] SEQ ID NO: 9: Amino acid sequence of 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 wild-type lysE gene, artificial sequence):

[0139] ATGGTGATCATGGAAATCTTCATTACAGGTCTGCTTTTGGGGGCCAGTCTTTTACTGTCCATCGGACCGCAGAATGTACTGGTGATTAAACAAGGAATTAAGCGCGAAGGACTCATTGCGGTTCTTCTCGTGTGTTTAATTTCTGACGTCTTTTTGTTCATCGCCGGCACCTTGGGCGTTGATCTTTTGTCCAATGCCGCGCCGATCGTGCTCGATATTATGCGCTGGGGTGGCATCGCTTACCTGTTATGGTTTGCCGTCATGGCAGCGAAAGACGCCATGACAAACAAGGTGGAAGCGCCACAGATCATTGAAGAAACAGAACCAACCGTGCCCGATGACACGCCTTTGGGCGGTTCGGCGGTGGCCACTGACACGCGCAACCGGGTGCGGGTGGAGGTGAGCGTCGATAAGCAGCGGGTTTGGGTAAAGCCCATGTTGATGGCAATCGTGCTGACCTGGTTGAACCCGAATGCGTATTTGGACGCGTTTGTGTTTATCGGCGGCGTCGGCGCGCAATACGGCGACACCGGACGGTGGATTTTCGCCGCTGGCGCGTTCGCGGCAAGCCTGATCTGGTTCCCGCTGGTGGGTTTCGGCGCAGCAGCATTGTCACGCCCGCTGTCCAGCCCCAAGGTGTGGCGCTGGATCAACGTCGTCGTGGCAGTTGTGATGACCGCATTGGCCATCAAACTGATGTTGATGGGTTAG

[0140] SEQ ID NO:14 (DNA sequence of 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 objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is 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 in the scope of protection of the present invention.

Claims

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

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

13.

3. nucleic acid molecules, encoding the translocator lysE mutant according to any one of claims 1-2. An expression vector comprising the nucleic acid molecule according to claim 3 . A host cell comprising the nucleic acid molecule of claim 3 or the expression vector of claim 4.

6. The use of the transporter lysE mutant 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 an L-Histidine derivative.

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 contains the nucleic acid molecule of claim 3 or the expression vector of claim 4.

8. The method according to claim 7, wherein: The bacteria is Escherichia coli or Corynebacterium glutamicum.

9. The method according to claim 7 or 8, wherein: Feedback inhibition of ATP phosphoribosyltransferase in the bacteria is relieved.

10. The method according to claim 9, wherein: Integrating the gene hisG-D84A-S205H of the ATP phosphoribosyltransferase mutant with double mutations of D84A and S205H from Acinetobacter baumannii into the bacterial genome or replacing the hisG gene of the bacterial genome to release the feedback inhibition of the ATP phosphoribosyltransferase; Preferably, in the bacterium, a nucleic acid molecule encoding the transporter protein lysE mutant is integrated into the yjiP gene site on the bacterial genome.

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