L-histidine transporter mutant and method for increasing yield of L-histidine

Abstract

The invention discloses an L-histidine transporter mutant and a method for increasing the yield of L-histidine, and the mutant has any one mutation of I20A, V28L, I20A-V28L and I20A-V21L-T28S relative to an escherichia coli wild type yeaS. The yield of the L-histidine of the recombinant escherichia coli containing the yeaS mutant protein is remarkably increased, it is indicated that the yeaS mutant protein and the coding gene of the yeaS mutant protein can be used for increasing the yield of the L-histidine, and the mutant can lay a good foundation for industrial efficient production of the L-histidine and downstream metabolites.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an L-histidine transporter mutant and a method for increasing L-histidine. Background Art

[0002] L-histidine is one of the 20 standard amino acids. Although it is not completely necessary in the daily nutrition of adults, it is classified as an indispensable amino acid for growing children in nutrition. In addition, L-histidine plays a role in multiple key physiological processes, including anti-oxidation and immune regulation. 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] To achieve high-yield amino acid cell factories, transport is a key factor. Overexpression of the yeaS gene, which encodes a protein belonging to the RhtB transporter family, confers resistance to glycyl-L-leucine, leucine analogs, several amino acids and their analogs. Overexpression of yeaS promoted the accumulation of leucine and, to a lesser extent, methionine and histidine in the respective production strains.

[0005] These studies have demonstrated that modifying histidine-related transporters through genetic engineering is of great significance for obtaining L-histidine production strains with higher yields. Summary of the invention

[0006] In order to construct a genetically engineered strain with a higher L-histidine yield, the present invention uses genetic engineering technology to transform Escherichia coli that produces L-histidine. The present invention performs a series of site-directed mutations on yeaS, transfers the mutant yeaS gene into a host bacterium, and screens to obtain a yeaS mutant that increases the L-histidine yield. The obtained yeaS mutant can timely discharge the produced histidine to the extracellular space, avoid accumulation in the cell, and increase the fermentation yield of histidine.

[0007] The present invention first provides an L-histidine transporter mutant (ie, yeaS mutant), which is any one of the following 1)-4): 1) The isoleucine at position 20 of the L-histidine transporter is mutated to alanine, and the amino acid sequence thereof is shown in SEQ ID No. 3 in the sequence listing; 2) mutating the threonine at position 28 of the L-histidine transporter to serine, and the amino acid sequence thereof is shown in SEQ ID No. 7 in the sequence listing; 3) the isoleucine at position 20 of the L-histidine transporter is mutated to alanine and the threonine at position 28 is mutated to serine, and the amino acid sequence thereof is shown in SEQ ID No. 11 in the sequence listing; 4) The isoleucine at position 20 of the L-histidine transporter is mutated to alanine, the valine at position 21 is mutated to leucine, and the threonine at position 28 is mutated to serine, and the amino acid sequence is shown in SEQ ID No. 15 in the sequence listing.

[0008] The amino acid sequence of the L-histidine transporter, namely the yeaS protein, is shown in SEQ ID No.1.

[0009] The present invention also provides a nucleic acid molecule encoding the yeaS mutant.

[0010] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0011] Specifically, the nucleotide sequence of the nucleic acid molecule may be shown as SEQ ID No.4, SEQ ID No.8, SEQ ID No.12 or SEQ ID No.16.

[0012] The invention also provides an expression vector containing the nucleic acid molecule.

[0013] The expression vector may be a plasmid, cosmid, phage or viral vector.

[0014] The recombinant engineered bacteria containing the yeaS mutant, the nucleic acid molecule or the expression vector also fall within the protection scope of the present invention.

[0015] The above-mentioned recombinant engineering bacteria can be Escherichia coli.

[0016] In one embodiment of the present invention, the Escherichia coli is Escherichia coli W3110.

[0017] The nucleic acid molecule can be expressed in the recombinant engineering bacteria.

[0018] The starting strain of the recombinant engineering bacteria can relieve the feedback inhibition of ATP phosphoribosyl transferase.

[0019] Specifically, the aspartic acid at position 84 of the amino acid sequence of ATP phosphoribosyltransferase as shown in SEQ ID No. 17 is mutated to alanine, and the serine at position 205 is mutated to histidine to release the feedback inhibition of ATP phosphoribosyltransferase.

[0020] The gene encoding the mutated ATP phosphoribosyltransferase after the feedback inhibition of ATP phosphoribosyltransferase is released is shown in SEQ ID No.19.

[0021] The use of the recombinant engineered bacteria in the preparation of L-histidine also falls within the protection scope of the present invention. The above application is to inoculate the recombinant engineering bacteria into a culture medium for culturing to produce L-histidine.

[0022] The present invention also protects a method for increasing the yield of L-histidine, which comprises inoculating the recombinant engineering bacteria into a culture medium for culturing to produce L-histidine.

[0023] The culture medium only needs to be able to culture the recombinant engineered bacteria.

[0024] In one embodiment of the present invention, the recombinant engineered bacteria is fermented in the following fermentation medium: 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, ammonia water was added every 4h during the fermentation process to the pH of 7.2-7.4, and the balance was water.

[0025] The fermentation culture may be carried out at 35-40°C, such as 37°C.

[0026] The fermentation culture can be carried out on a shaker.

[0027] During the fermentation process, ammonia water can be used to control the pH of the system at 7.

[0028] The recombinant engineered bacteria can be first cultured in a seed culture medium and then transferred to a fermentation medium.

[0029] In one embodiment of the present invention, the seed culture medium formula is as follows (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, and the balance is water.

[0030] The present invention performs a series of site-directed mutations on yeaS, transfers the mutant yeaS gene into a host bacterium, and screens to obtain a yeaS mutant protein that increases the yield of L-histidine. The L-histidine yield of the recombinant Escherichia coli containing the yeaS mutant protein of the present invention is significantly increased, indicating that the yeaS mutant protein of the present invention and its encoding gene can be used to increase the yield of L-histidine, and have broad prospects for industrial development and application.

[0031] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way. DETAILED DESCRIPTION

[0032] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. The quantitative tests in the following examples were all repeated at least three times, and the results were averaged. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.

[0033] Table 1. Strains and plasmids used in the present invention

[0034] Table 2. Primers used in the present invention

[0035] Example 1: Construction of recombinant strains HIS002 and HIS003 Starting from Escherichia coli W3110, the ATP phosphoribosyltransferase gene hisG was knocked out by a two-step homologous recombination method, and the hisG gene of Acinetobacter baumannii (hisG-Ab) or the modified hisG-D84A-S205H gene of Acinetobacter baumannii (hisG-Ab-D84A-S205H) was inserted. The specific steps are as follows: (1) Knock out the hisG gene of the W3310 strain and insert the hisG gene of Acinetobacter baumannii In the first step, pRE112 plasmid DNA was used as a template and primers hisG-cs-up / hisG-cs-down were used to perform PCR amplification to obtain a 3593 bp DNA fragment I for the first step of homologous recombination.

[0036] The above DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into Escherichia coli W3110 by electroporation to obtain the recombinant bacteria W3110-pKD46; then the DNA fragment I was electroporated into Escherichia coli W3110 carrying pKD46 (ie, W3110-pKD46).

[0037] The electroporation conditions were as follows: 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. 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 at 30°C overnight, select a single colony for PCR verification. The primers used are XZ-hisG-up / XZ-hisG-down. The correct colony amplification product is a fragment of 3715 bp. Select a single colony with the correct sequence and name it HIS001.

[0038] The DNA changes of HIS001 were as follows: the DNA fragment hisG-cs was introduced into E. coli W3110 containing the pKD46 plasmid.

[0039] In the second step, the plasmid PUC19-hisG-Ab synthesized by Hongxun was used as a template and the primers hisG-mut-cs-up / hisG-mut-cs-down were used to amplify the 812 bp DNA fragment II for the second homologous recombination.

[0040] The pKD46 plasmid was transformed into HIS001 by electroporation to obtain the recombinant bacterium HIS001-pKD46; then the DNA fragment II was transformed into HIS001 carrying the pKD46 plasmid (ie, HIS001-pKD46) by electroporation.

[0041] The electroporation conditions are as follows: first prepare electroporation competent cells of HIS001 with pKD46 plasmid; 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 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 an LB plate 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 930 bp fragment. A single colony with the correct sequence was selected and named HIS002.

[0042] The DNA changes of HIS002 are as follows: the hisG-cs fragment in HIS001 was replaced with hisG from Acinetobacter baumannii.

[0043] (2) Knock out the hisG gene of the W3310 strain and insert the hisG-D84A-S205H gene of Acinetobacter baumannii Starting from Escherichia coli W3110, the ATP phosphoribosyltransferase gene hisG was knocked out by a two-step homologous recombination method, and the hisG-D84A-S205H gene of Acinetobacter baumannii (whose nucleotide sequence is shown in SEQ ID No.19, encoding the hisG-D84A-S205H protein shown in SEQ ID No.18) was inserted. The specific steps are as follows: The first step is the same as step (1) the first step.

[0044] In the second step, the plasmid PUC19-hisG-Ab-D84A-S205H synthesized by Hongxun was used as a template and primers hisG-mut-cs-up / hisG-mut-cs-down were used to amplify the 812 bp DNA fragment II for the second homologous recombination.

[0045] The pKD46 plasmid was transformed into HIS001 by electroporation to obtain the recombinant bacterium HIS001-pKD46.

[0046] DNA fragment II was transformed into HIS001 with pKD46 plasmid (i.e. HIS001-pKD46) by electroporation. The electroporation conditions were as follows: first, prepare electroporation competent cells of HIS001 with pKD46 plasmid; place 50μl HIS001-pKD46 competent cells on ice, add 50ng DNA fragment II, place on ice for 2 minutes, and transfer to a 0.2cm Bio-Rad electroporation cup. Use MicroPulser (Bio-Rad) electroporator with an electroporation parameter of 2.5kv. After electroporation, quickly transfer 1ml LB liquid culture medium to the electroporation cup, pipette 5 times, transfer to a test tube, and incubate at 75 rpm and 30°C for 4 hours. The bacterial liquid was transferred to LB liquid medium without sodium chloride containing 10% sucrose (50 ml medium in a 250 ml flask), and after culturing for 24 hours, it was streaked on an LB plate 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 930 bp fragment. A correct single colony was selected and named HIS003.

[0047] The DNA changes of HIS003 are as follows: the hisG-cs fragment in HIS001 was replaced with hisG-D84A-S205H from Acinetobacter baumannii.

[0048] Example 2, yeaS transporter gene yeaS - Genomic insertion of I20A Starting from Escherichia coli HIS003, a two-step homologous recombination method was used to knock out the transporter gene yeaS (The protein sequence is shown in SEQ ID No.1, and the gene sequence is shown in SEQ ID No.2), insert yeaS -I20A gene (protein sequence is shown in SEQ ID No.3, and its gene sequence is shown in SEQ ID No.4), the specific steps are as follows: In the first step, pRE112 plasmid DNA was used as a template and primers yeaS-cs-up / yeaS-cs-down were used to perform PCR amplification to obtain a 3575 bp DNA fragment I for the first step of homologous recombination.

[0049] The above DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into HIS003 by electroporation to obtain the recombinant bacterium HIS003-pKD46; then the DNA fragment I was electroporated into HIS003 carrying pKD46 (ie, HIS003-pKD46).

[0050] 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 at 30°C overnight, select a single colony for PCR verification. The primers used are XZ-yeaS-up / XZ-yeaS-down. The correct colony amplification product is a 4084 bp fragment. Select a correct single colony and name it HIS004.

[0051] The DNA changes of HIS004 are as follows: yeaS -cs was introduced into E. coli HIS003 containing the pKD46 plasmid.

[0052] In the second step, the W3110 genome was used as a template and primers yeaS-mut1-cs-up / yeaS-mut1-cs-down were used to amplify the 739 bp DNA fragment II for the second homologous recombination.

[0053] The pKD46 plasmid was transformed into HIS004 by electroporation to obtain the recombinant bacterium HIS004-pKD46; then the DNA fragment II was transformed into HIS004 with the pKD46 plasmid (i.e. HIS004-pKD46) by electroporation. The electroporation conditions were: First, prepare the electroporation competent cells of HIS004 with pKD46 plasmid; 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, 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 transfer to a test tube, 75 rpm, 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-yeaS-up / XZ-yeaS-down, and the correct colony amplification product was a 1254bp fragment. A single colony with the correct sequence was selected and named HIS005.

[0054] The DNA changes of HIS005 are as follows: yeaS - I20A gene replaces HIS004 yeaS -cs fragment.

[0055] Example 3, yeaS transporter gene yeaS -Genome insertion of V21L Starting from Escherichia coli HIS004 and using the W3110 genome as a template, the 739 bp DNA fragment II was amplified using primers yeaS-mut2-cs-up / yeaS-mut1-cs-down for the second homologous recombination.

[0056] The pKD46 plasmid was transformed into HIS004 by electroporation to obtain the recombinant bacterium HIS004-pKD46; then the DNA fragment II was transformed into HIS004 carrying the pKD46 plasmid (ie, HIS004-pKD46) by electroporation.

[0057] The electroporation conditions are as follows: first prepare electroporation competent cells of HIS004 with pKD46 plasmid; 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 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-yeaS-up / XZ-yeaS-down, and the correct colony amplification product was a 1254 bp fragment. A correct single colony was selected and named HIS006.

[0058] The DNA changes of HIS006 are as follows: yeaS -V21L gene replacement in HIS004 yeaS -cs fragment. yeaS The -V21L gene sequence is shown in SEQ ID No.6, encoding the yeaS-V21L protein shown in SEQ ID No.5.

[0059] Example 4, yeaS transporter gene yeaS -Genome insertion of T28S Starting from Escherichia coli HIS004 and using the W3110 genome as a template, the 739 bp DNA fragment II was amplified using primers yeaS-mut3-cs-up / yeaS-mut1-cs-down for the second homologous recombination.

[0060] The pKD46 plasmid was transformed into HIS004 by electroporation to obtain the recombinant bacterium HIS004-pKD46; then the DNA fragment II was transformed into HIS004 carrying the pKD46 plasmid (ie, HIS004-pKD46) by electroporation.

[0061] The electroporation conditions are as follows: first prepare electroporation competent cells of HIS004 with pKD46 plasmid; 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 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-yeaS-up / XZ-yeaS-down, and the correct colony amplification product was a 1254bp fragment. A correct single colony was selected and named HIS007.

[0062] The DNA changes of HIS007 are as follows: yeaS -T28S gene replacement in HIS004 yeaS -cs fragment. yeaS The -T28S gene sequence is shown in SEQ ID No.8, encoding the yeaS-T28S protein shown in SEQ ID No.7.

[0063] Example 5, yeaS transporter gene yeaS - Genomic insertion of I20A-V21L Starting from Escherichia coli HIS004 and using the W3110 genome as a template, the 739 bp DNA fragment II was amplified using primers yeaS-mut4-cs-up / yeaS-mut1-cs-down for the second homologous recombination.

[0064] The pKD46 plasmid was transformed into HIS004 by electroporation to obtain the recombinant bacterium HIS004-pKD46; then the DNA fragment II was transformed into HIS004 carrying the pKD46 plasmid (ie, HIS004-pKD46) by electroporation.

[0065] The electroporation conditions are as follows: first prepare electroporation competent cells of HIS004 with pKD46 plasmid; 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 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-yeaS-up / XZ-yeaS-down, and the correct colony amplification product was a 1254 bp fragment. A correct single colony was selected and named HIS008.

[0066] The DNA changes of HIS008 are as follows: yeaS - I20A-V21L gene replacement in HIS004 yeaS -cs fragment. yeaS The -I20A-V21L gene sequence is shown in SEQ ID No.10, encoding the yeaS-I20A-V21L protein shown in SEQ ID No.9.

[0067] Example 6, yeaS transporter gene yeaS - Genomic insertion of I20A-T28S Starting from Escherichia coli HIS004 and using the W3110 genome as a template, the 739 bp DNA fragment II was amplified using primers yeaS-mut5-cs-up / yeaS-mut1-cs-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 recombinant bacterium HIS004-pKD46; then the DNA fragment II was transformed into HIS004 carrying the pKD46 plasmid (ie, HIS004-pKD46) by electroporation.

[0069] The electroporation conditions are as follows: first prepare electroporation competent cells of HIS004 with pKD46 plasmid; 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 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-yeaS-up / XZ-yeaS-down, and the correct colony amplification product was a 1254bp fragment. A correct single colony was selected and named HIS009.

[0070] The DNA changes of HIS009 are as follows: yeaS - I20A-T28S gene replacement in HIS004 yeaS -cs fragment. yeaS The -I20A-T28S gene sequence is shown in SEQ ID No.12, encoding the yeaS-I20A-T28S protein shown in SEQ ID No.11.

[0071] Example 7, yeaS transporter gene yeaS -Genome insertion of V21L-T28S Starting from Escherichia coli HIS004 and using the W3110 genome as a template, the 739 bp DNA fragment II was amplified using primers yeaS-mut6-cs-up / yeaS-mut1-cs-down for the second homologous recombination.

[0072] The pKD46 plasmid was transformed into HIS004 by electroporation to obtain the recombinant bacterium HIS004-pKD46; then the DNA fragment II was transformed into HIS004 carrying the pKD46 plasmid (ie, HIS004-pKD46) by electroporation.

[0073] The electroporation conditions are as follows: first prepare electroporation competent cells of HIS004 with pKD46 plasmid; 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 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-yeaS-up / XZ-yeaS-down, and the correct colony amplification product was a 1254 bp fragment. A correct single colony was selected and named HIS010.

[0074] The DNA changes of HIS0010 are as follows: yeaS - V21L-T28S gene replacement in HIS004 yeaS -cs fragment. yeaS The -V21L-T28S gene sequence is shown in SEQ ID No.14, encoding the yeaS-V21L-T28S protein shown in SEQ ID No.13.

[0075] Example 8, yeaS transporter gene yeaS - Genomic insertion of I20A-V21L-T28S Starting from Escherichia coli HIS004 and using the W3110 genome as a template, the 739 bp DNA fragment II was amplified using primers yeaS-mut7-cs-up / yeaS-mut1-cs-down for the second homologous recombination.

[0076] The pKD46 plasmid was transformed into HIS004 by electroporation to obtain the recombinant bacterium HIS004-pKD46; then the DNA fragment II was transformed into HIS004 carrying the pKD46 plasmid (ie, HIS004-pKD46) by electroporation.

[0077] The electroporation conditions are as follows: first prepare electroporation competent cells of HIS004 with pKD46 plasmid; 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 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-yeaS-up / XZ-yeaS-down, and the correct colony amplification product was a 1254 bp fragment. A correct single colony was selected and named HIS011.

[0078] The DNA changes of HIS0011 are as follows: yeaS - I20A-V21L-T28S gene replacement in HIS004 yeaS -cs fragment. yeaS The -I20A-V21L-T28S gene sequence is shown in SEQ ID No.16, encoding the yeaS-I20A-V21L-T28S protein shown in SEQ ID No.15.

[0079] Example 9: Fermentation of recombinant strains to produce L-histidine The obtained recombinant bacteria HIS003, HIS005, HIS006, HIS007, HIS008, HIS009, HIS010, and HIS011 are used to produce L-histidine by fermentation, comprising the following steps: (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.

[0080] (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.

[0081] Analytical method: Agilent (Agilent-1260) high performance liquid chromatograph was used to determine the L-histidine content in the fermentation broth after fermentation for 3 days. L-histidine was determined using the amino acid analysis column ZORBAX Eclipse AAA 4.6mm×75mm3.5-Micron. Detection wavelength: 338nm. Flow rate 1.0mL / min; injection volume: 10μl; detector: UV 206nm; mobile phase: acetonitrile / phosphate mixed solution. Weigh 0.05 M potassium dihydrogen phosphate and dissolve it in ultrapure water, adjust the pH to 3.0 with phosphoric acid, filter with a 0.22μm filter membrane, and add the corresponding volume of acetonitrile in the ratio of acetonitrile: 0.05M potassium dihydrogen phosphate = 3:1. Mix well and ultrasonicate for 20 minutes until there are no bubbles.

[0082] The experiment was repeated 3 times, and the average values ​​of the analytical results are presented in Table 1 below.

[0083] The culture medium used was as follows: LB medium: 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride. (Add 20g / L agar powder to solid medium.) 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, sodium hydroxide to adjust the pH between 7.0-7.2, and the balance is water.

[0084] 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, ammonia water was added every 4h during the fermentation process to the pH of 7.2-7.4, and the balance was water.

[0085] Table 3. Comparison of L-histidine production capacity

[0086] As shown in Table 3, compared with the control HIS003 strain, the HIS005, HIS007, HIS009, and HIS0011 strains containing mutants all had improved histidine productivity, especially the HIS009 strain containing the yeaS-I20A-T28S mutant, whose L-histidine production capacity increased by 2.2 times, indicating that this strain is more conducive to the production of histidine.

[0087] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. An L-histidine transporter mutant, characterized in that: It is any one of the following 1)-4): 1) The isoleucine at position 20 of the L-histidine transporter is mutated to alanine, and the amino acid sequence thereof is shown in SEQ ID No. 3 in the sequence listing; 2) mutating the threonine at position 28 of the L-histidine transporter to serine, and the amino acid sequence thereof is shown in SEQ ID No. 7 in the sequence listing; 3) the isoleucine at position 20 of the L-histidine transporter is mutated to alanine and the threonine at position 28 is mutated to serine, and the amino acid sequence thereof is shown in SEQ ID No. 11 in the sequence listing; 4) The isoleucine at position 20 of the L-histidine transporter is mutated to alanine, the valine at position 21 is mutated to leucine, and the threonine at position 28 is mutated to serine, and the amino acid sequence is shown in SEQ ID No. 15 in the sequence listing.

2. A nucleic acid molecule encoding the mutant according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID No.4, SEQ ID No.8, SEQ ID No.12 or SEQ ID No.

16.

4. An expression vector containing the nucleic acid molecule according to claim 2 or 3.

5. A recombinant engineered bacterium containing the mutant according to claim 1, the nucleic acid molecule according to claim 2 or 3, or the expression vector according to claim 4.

6. The recombinant engineered bacterium according to claim 5, characterized in that The recombinant engineering bacteria is Escherichia coli.

7. The recombinant engineered bacterium according to claim 6, characterized in that The starting strain of the recombinant engineering bacteria relieves the feedback inhibition of ATP phosphoribosyltransferase.

8. The recombinant engineered bacterium according to claim 7, characterized in that The aspartic acid at position 84 of the amino acid sequence of ATP phosphoribosyltransferase as shown in SEQ ID No. 17 is mutated to alanine, and the serine at position 205 is mutated to histidine to release the feedback inhibition of ATP phosphoribosyltransferase.

9. Use of the recombinant engineered bacteria according to any one of claims 5 to 8 in the preparation of L-histidine.

10. A method for increasing the yield of L-histidine, characterized in that: The method comprises inoculating the recombinant engineered bacteria as claimed in any one of claims 5 to 8 into a culture medium for culturing to produce L-histidine.

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