L-histidine transporter mutant and method for increasing L-histidine production

Abstract

The present invention discloses an L-histidine transporter mutant and a method for increasing L-histidine production. The mutant has any one of the following mutations: I20A, V28L, I20A-V28L, or I20A-V21L-T28S, relative to wild-type yeaS in Escherichia coli. The recombinant Escherichia coli containing the yeaS mutant of the present invention significantly increases L-histidine production, indicating that the yeaS mutant and its encoding gene can be used to increase L-histidine production. The mutant provided by the present invention lays a good foundation for the industrialized and efficient production of L-histidine and downstream metabolites.

Description

Technical Field

[0001] The present 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. While not fully essential in adults' daily nutrition, it is nutritionally classified as essential for growing children. Furthermore, L-histidine plays a role in several key physiological processes, including antioxidant and immunomodulatory functions. Therefore, it is widely used in the medical industry in the preparation of medications for gastric ulcer treatments, as a raw material for cardiovascular drugs, and as an amino acid infusion.

[0003] Histidine is mainly found in hemoglobin, and its traditional preparation method is to produce it by protein hydrolysis and extraction using blood powder as raw material. However, this method has disadvantages such as low efficiency and environmental pollution. There are reports in the prior art that the production of L-histidine can be achieved by microbial fermentation, but it has not yet been realized in large-scale industry. This is because the histidine metabolic pathway is long, involving multiple key enzymes, and the current yield of histidine production using 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] Transport is a key factor in achieving high-yield amino acid factories. 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 promotes 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-producing strains with higher yields. Summary of the Invention

[0006] To construct a genetically engineered strain with increased L-histidine production, the present invention utilizes genetic engineering techniques to modify L-histidine-producing Escherichia coli. By performing a series of site-directed mutagenesis on yeaS, the mutant yeaS gene was transferred into the host bacterium and screened to obtain yeaS mutants with increased L-histidine production. The resulting yeaS mutants are able to excrete the produced histidine to the extracellular space, preventing intracellular accumulation and thereby increasing the fermentation yield of histidine.

[0007] The present invention first provides an L-histidine transporter mutant (ie, a yeaS mutant), which is any one of the following 1) to 4):

[0008] 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;

[0009] 2) mutating the threonine at position 28 of the L-histidine transporter to serine, the amino acid sequence of which is shown in SEQ ID No. 7 in the sequence listing;

[0010] 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;

[0011] 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. The amino acid sequence is shown in SEQ ID No. 15 in the sequence listing.

[0012] The amino acid sequence of the L-histidine transporter, ie, the yeaS protein, is shown in SEQ ID No. 1.

[0013] The present invention also provides a nucleic acid molecule encoding the yeaS mutant.

[0014] 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.

[0015] 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.

[0016] The present invention also provides an expression vector containing the nucleic acid molecule.

[0017] The expression vector can be a plasmid, cosmid, phage or viral vector.

[0018] The recombinant engineered bacteria containing the yeaS mutant, the nucleic acid molecule or the expression vector also fall within the scope of protection of the present invention.

[0019] The above-mentioned recombinant engineered bacteria can be Escherichia coli.

[0020] In one embodiment of the present invention, the Escherichia coli is Escherichia coli W3110.

[0021] The nucleic acid molecule can be expressed in the recombinant engineering bacteria.

[0022] The starting strain of the recombinant engineering bacteria can relieve the feedback inhibition of ATP phosphoribosyltransferase.

[0023] 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.

[0024] The gene encoding the mutated ATP phosphoribosyltransferase after the feedback inhibition of ATP phosphoribosyltransferase is released is shown in SEQ ID No. 19.

[0025] The use of the recombinant engineered bacteria in the preparation of L-histidine also falls within the scope of protection of the present invention.

[0026] The above application is to inoculate the recombinant engineered bacteria into a culture medium for culturing to produce L-histidine.

[0027] The present invention also protects a method for increasing the yield of L-histidine, which comprises inoculating the recombinant engineered bacteria into a culture medium for culturing to produce L-histidine.

[0028] The culture medium only needs to be able to culture the recombinant engineered bacteria.

[0029] In one embodiment of the present invention, the recombinant engineered bacteria is fermented in the following fermentation medium:

[0030] Fermentation medium (1 L): 20 g glucose, 4 g yeast extract, 3 g peptone, 2 g sodium citrate monohydrate, 2 g potassium dihydrogen phosphate, 2 g magnesium sulfate·7H2O, 20 mg ferrous sulfate·7H2O, 20 mg manganese sulfate monohydrate, 2 mg VB1, 2 mg VB3, 2 mg VB5, 2 mg VB12, and 2 mg VH. Sodium hydroxide was used to adjust the pH to 7.0-7.2. Aqueous ammonia was added every 4 h during the fermentation process to a pH of 7.2-7.4. The balance was water.

[0031] The fermentation culture can be carried out at 35-40°C, such as 37°C.

[0032] The fermentation culture can be carried out on a shaker.

[0033] During the fermentation process, ammonia water can be used to control the pH of the system at 7.

[0034] The recombinant engineered bacteria can be cultured in a seed culture medium first and then transferred to a fermentation medium.

[0035] In one embodiment of the present invention, the seed culture medium formula is as follows (1 L): 20 g glucose, 4 g yeast extract, 3 g peptone, 1.2 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate·7H2O, 10 mg ferrous sulfate·7H2O, 10 mg manganese sulfate monohydrate, 1 mg VB1, 1 mg VB3, 1 mg VB5, 1 mg VB12, 1 mg VH, sodium hydroxide is used to adjust the pH between 7.0 and 7.2, and the balance is water.

[0036] The present invention involves performing a series of site-directed mutagenesis on yeaS, transferring the mutant yeaS gene into host bacteria, and screening to obtain yeaS mutant proteins that enhance L-histidine production. The recombinant Escherichia coli containing the yeaS mutant proteins exhibited significantly increased L-histidine production, demonstrating that the yeaS mutant proteins and their encoding genes can be used to enhance L-histidine production and have broad prospects for industrial development and application.

[0037] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way. DETAILED DESCRIPTION

[0038] The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels. 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.

[0039] Table 1. Strains and plasmids used in the present invention

[0040]

[0041] Table 2. Primers used in the present invention

[0042]

[0043] Example 1: Construction of recombinant strains HIS002 and HIS003

[0044] Starting from Escherichia coli W3110, a two-step homologous recombination method was used to knock out the ATP phosphoribosyltransferase gene hisG and insert the hisG gene of Acinetobacter baumannii (hisG-Ab) or the modified hisG-D84A-S205H gene of Acinetobacter baumannii (hisG-Ab-D84A-S205H). The specific steps are as follows:

[0045] (1) Knock out the hisG gene of the W3310 strain and insert the hisG gene of Acinetobacter baumannii

[0046] In the first step, pRE112 plasmid DNA was used as a template and primers hisG-cs-up / hisG-cs-down were used to amplify a 3593 bp DNA fragment I for the first step of homologous recombination.

[0047] 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 (i.e., W3110-pKD46).

[0048] Electroporation conditions were as follows: W3110-pKD46 competent cells were prepared 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 cuvette. Electroporation was performed using a MicroPulser (Bio-Rad) at a voltage of 2.5 kV. After electroporation, 1 ml of LB medium was quickly transferred to the cuvette, pipetted five times, and then transferred to a test tube and 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 incubation at 30°C, a single colony was selected for PCR verification using primers XZ-hisG-up / XZ-hisG-down. The correct colony amplified product was a 3715 bp fragment. A single colony with the correct sequence was selected and named HIS001.

[0049] The DNA changes of HIS001 were as follows: the DNA fragment hisG-cs was introduced into Escherichia coli W3110 containing the pKD46 plasmid.

[0050] 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.

[0051] 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.

[0052] Electroporation conditions were as follows: First, prepare electroporation competent cells for HIS001 harboring the pKD46 plasmid; place 50 μl of competent cells on ice, add 50 ng of DNA fragment II, incubate on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad cuvette. Electroporation was performed using a MicroPulser (Bio-Rad) at 2.5 kV. Immediately after electroporation, transfer 1 ml of LB liquid medium to the cuvette, pipette five times, transfer to a test tube, incubate at 75 rpm, and incubate at 30°C for 4 hours. The bacterial suspension was transferred to LB liquid medium containing 10% sucrose and no sodium chloride (50 ml in a 250 ml flask). After 24 hours of incubation, the cells were streaked onto LB plates containing 6% sucrose and no 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.

[0053] The DNA changes in HIS002 are as follows: the hisG-cs fragment in HIS001 was replaced with hisG from Acinetobacter baumannii.

[0054] (2) Knock out the hisG gene of the W3310 strain and insert the hisG-D84A-S205H gene of Acinetobacter baumannii

[0055] Starting from Escherichia coli W3110, a two-step homologous recombination method was used to knock out the ATP phosphoribosyltransferase gene hisG and insert the hisG-D84A-S205H gene of Acinetobacter baumannii (the nucleotide sequence of which is shown in SEQ ID No. 19, encoding the hisG-D84A-S205H protein shown in SEQ ID No. 18). The specific steps are as follows:

[0056] The first step is the same as step (1) the first step.

[0057] In the second step, the plasmid PUC19-hisG-Ab-D84A-S205H 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.

[0058] The pKD46 plasmid was transformed into HIS001 by electroporation to obtain the recombinant bacterium HIS001-pKD46.

[0059] DNA fragment II was transformed into HIS001 harboring the pKD46 plasmid (i.e., HIS001-pKD46) by electroporation. The electroporation conditions were as follows: First, prepare electroporation competent cells of HIS001 harboring the pKD46 plasmid. Add 50 μl of HIS001-pKD46 competent cells to the culture medium on ice, add 50 ng of DNA fragment II, incubate on ice for 2 minutes, and transfer the culture to a 0.2 cm Bio-Rad cuvette. Electroporation was performed using a MicroPulser (Bio-Rad) at a voltage of 2.5 kV. Immediately after electroporation, transfer 1 ml of LB liquid medium to the cuvette, pipette five times, and then transfer the culture to a test tube. Incubate at 75 rpm and 30°C for 4 hours. The bacterial suspension was transferred to LB liquid medium (50 ml in a 250 ml flask) containing 10% sucrose and no sodium chloride. After 24 hours of incubation, the culture was streaked onto LB plates containing 6% sucrose and no sodium chloride. PCR verification confirmed that the primers used were XZ-hisG-up / XZ-hisG-down. The correct amplification product was a 930 bp fragment. A single correct colony was selected and named HIS003.

[0060] The DNA changes in HIS003 are as follows: the hisG-cs fragment in HIS001 was replaced with hisG-D84A-S205H from Acinetobacter baumannii.

[0061] Example 2, yeaS transporter gene yeaS -Genome insertion of I20A

[0062] 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 its 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:

[0063] In the first step, pRE112 plasmid DNA was used as a template and primers yeaS-cs-up / yeaS-cs-down were used to amplify a 3575 bp DNA fragment I for the first step of homologous recombination.

[0064] 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 bacteria HIS003-pKD46; then the DNA fragment I was electroporated into HIS003 carrying pKD46 (i.e., HIS003-pKD46).

[0065] Electroporation conditions were as follows: HIS003-pKD46 competent cells were prepared 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 cuvette. Electroporation was performed using a MicroPulser (Bio-Rad) at a voltage of 2.5 kV. After electroporation, 1 ml of LB medium was quickly transferred to the cuvette, pipetted five times, and then transferred to a test tube and incubated at 75 rpm and 30°C for 2 hours. 200 μl of bacterial solution was spread on LB plates containing ampicillin (final concentration 100 μg / ml) and chloramphenicol (final concentration 34 μg / ml). After overnight incubation at 30°C, a single colony was selected for PCR verification using primers XZ-yeaS-up / XZ-yeaS-down. The correct amplification product was a 4084 bp fragment. A correct single colony was selected and named HIS004.

[0066] The DNA changes of HIS004 are as follows: yeaS -cs was introduced into Escherichia coli HIS003 containing the pKD46 plasmid.

[0067] 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 a 739 bp DNA fragment II for the second homologous recombination.

[0068] The pKD46 plasmid was transformed into HIS004 by electroporation to obtain the recombinant strain HIS004-pKD46. DNA fragment II was then transformed into HIS004 carrying the pKD46 plasmid (i.e., HIS004-pKD46) by electroporation. The electroporation conditions were:

[0069] First, prepare electroporation competent cells for HIS004 harboring the pKD46 plasmid. Place 50 μl of competent cells on ice, add 50 ng of DNA fragment II, incubate on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad cuvette. Electroporate using a MicroPulser (Bio-Rad) at 2.5 kV. Immediately after electroporation, transfer 1 ml of LB liquid medium to the cuvette, pipette five times, transfer to a test tube, and incubate at 75 rpm at 30°C for 4 hours. Transfer the culture to LB liquid medium without sodium chloride containing 10% sucrose (50 ml per 250 ml flask). After 24 hours of incubation, streak the culture onto 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 single colony with the correct sequence was selected and named HIS005.

[0070] The DNA changes of HIS005 are as follows: yeaS -I20A gene replaces HIS004 yeaS -cs fragment.

[0071] Example 3, yeaS transporter gene yeaS -Genome insertion of V21L

[0072] Starting from Escherichia coli HIS004 and using the W3110 genome as a template, a 739 bp DNA fragment II was amplified using primers yeaS-mut2-cs-up / yeaS-mut1-cs-down for the second homologous recombination.

[0073] 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.

[0074] Electroporation conditions were as follows: First, prepare electroporation competent cells for HIS004 harboring the pKD46 plasmid; place 50 μl of competent cells on ice, add 50 ng of DNA fragment II, incubate on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad cuvette. Electroporation was performed using a MicroPulser (Bio-Rad) at 2.5 kV. Immediately after electroporation, transfer 1 ml of LB liquid medium to the cuvette, pipette five times, transfer to a test tube, incubate at 75 rpm, and incubate at 30°C for 4 hours. The bacterial suspension was transferred to LB liquid medium without sodium chloride containing 10% sucrose (50 ml per 250 ml flask). After 24 hours of incubation, the cells were streaked onto 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.

[0075] 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.

[0076] Example 4, yeaS transporter gene yeaS -T28S genomic insertion

[0077] Starting from Escherichia coli HIS004 and using the W3110 genome as a template, a 739 bp DNA fragment II was amplified using primers yeaS-mut3-cs-up / yeaS-mut1-cs-down for the second homologous recombination.

[0078] 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.

[0079] Electroporation conditions were as follows: First, prepare electroporation competent cells for HIS004 harboring the pKD46 plasmid; place 50 μl of competent cells on ice, add 50 ng of DNA fragment II, incubate on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad cuvette. Electroporation was performed using a MicroPulser (Bio-Rad) at 2.5 kV. Immediately after electroporation, transfer 1 ml of LB liquid medium to the cuvette, pipette five times, transfer to a test tube, incubate at 75 rpm, and incubate at 30°C for 4 hours. The bacterial suspension was transferred to LB liquid medium without sodium chloride containing 10% sucrose (50 ml per 250 ml flask). After 24 hours of incubation, the cells were streaked onto 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 HIS007.

[0080] 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.

[0081] Example 5, yeaS transporter gene yeaS -Genome insertion of I20A-V21L

[0082] Starting from Escherichia coli HIS004 and using the W3110 genome as a template, a 739 bp DNA fragment II was amplified using primers yeaS-mut4-cs-up / yeaS-mut1-cs-down for the second homologous recombination.

[0083] 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.

[0084] Electroporation conditions were as follows: First, prepare electroporation competent cells for HIS004 harboring the pKD46 plasmid; place 50 μl of competent cells on ice, add 50 ng of DNA fragment II, incubate on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad cuvette. Electroporation was performed using a MicroPulser (Bio-Rad) at 2.5 kV. Immediately after electroporation, transfer 1 ml of LB liquid medium to the cuvette, pipette five times, transfer to a test tube, incubate at 75 rpm, and incubate at 30°C for 4 hours. The bacterial suspension was transferred to LB liquid medium without sodium chloride containing 10% sucrose (50 ml per 250 ml flask). After 24 hours of incubation, the cells were streaked onto 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.

[0085] 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.

[0086] Example 6, yeaS transporter gene yeaS -Genome insertion of I20A-T28S

[0087] Starting from Escherichia coli HIS004 and using the W3110 genome as a template, a 739 bp DNA fragment II was amplified using primers yeaS-mut5-cs-up / yeaS-mut1-cs-down. The amplification system was the same as that in the first step and was used for the second homologous recombination.

[0088] 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.

[0089] Electroporation conditions were as follows: First, prepare electroporation competent cells for HIS004 harboring the pKD46 plasmid; place 50 μl of competent cells on ice, add 50 ng of DNA fragment II, incubate on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad cuvette. Electroporation was performed using a MicroPulser (Bio-Rad) at 2.5 kV. Immediately after electroporation, transfer 1 ml of LB liquid medium to the cuvette, pipette five times, transfer to a test tube, incubate at 75 rpm, and incubate at 30°C for 4 hours. The bacterial suspension was transferred to LB liquid medium without sodium chloride containing 10% sucrose (50 ml per 250 ml flask). After 24 hours of incubation, the cells were streaked onto 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 HIS009.

[0090] 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.

[0091] Example 7, yeaS transporter gene yeaS -Genome insertion of V21L-T28S

[0092] Starting from Escherichia coli HIS004 and using the W3110 genome as a template, a 739 bp DNA fragment II was amplified using primers yeaS-mut6-cs-up / yeaS-mut1-cs-down for the second homologous recombination.

[0093] 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.

[0094] Electroporation conditions were as follows: First, prepare electroporation competent cells for HIS004 harboring the pKD46 plasmid; place 50 μl of competent cells on ice, add 50 ng of DNA fragment II, incubate on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad cuvette. Electroporation was performed using a MicroPulser (Bio-Rad) at 2.5 kV. Immediately after electroporation, transfer 1 ml of LB liquid medium to the cuvette, pipette five times, transfer to a test tube, incubate at 75 rpm, and incubate at 30°C for 4 hours. The bacterial suspension was transferred to LB liquid medium without sodium chloride containing 10% sucrose (50 ml per 250 ml flask). After 24 hours of incubation, the cells were streaked onto 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.

[0095] 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.

[0096] Example 8, yeaS transporter gene yeaS -Genome insertion of I20A-V21L-T28S

[0097] Starting from Escherichia coli HIS004 and using the W3110 genome as a template, a 739 bp DNA fragment II was amplified using primers yeaS-mut7-cs-up / yeaS-mut1-cs-down for the second homologous recombination.

[0098] 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.

[0099] Electroporation conditions were as follows: First, prepare electroporation competent cells for HIS004 harboring the pKD46 plasmid; place 50 μl of competent cells on ice, add 50 ng of DNA fragment II, incubate on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad cuvette. Electroporation was performed using a MicroPulser (Bio-Rad) at 2.5 kV. Immediately after electroporation, transfer 1 ml of LB liquid medium to the cuvette, pipette five times, transfer to a test tube, incubate at 75 rpm, and incubate at 30°C for 4 hours. The bacterial suspension was transferred to LB liquid medium without sodium chloride containing 10% sucrose (50 ml per 250 ml flask). After 24 hours of incubation, the cells were streaked onto 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.

[0100] 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.

[0101] Example 9: Fermentation production of L-histidine by recombinant strains

[0102] 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:

[0103] (1) Seed Culture: A fresh single colony from the LB plate was inoculated into a test tube containing 4 ml of seed medium and cultured overnight at 37°C with shaking at 250 rpm. Subsequently, the culture was transferred to a 250 ml Erlenmeyer flask containing 30 ml of seed medium at a 2% (v / v) inoculum volume and cultured at 37°C with shaking at 250 rpm for 12 hours to obtain a seed culture solution for inoculation of the fermentation medium.

[0104] (2) Fermentation: The fermentation medium volume was 250 ml in a 500 ml fermenter. The seed culture was inoculated into the fermentation medium at a final concentration of OD550 = 0.1. The culture was fermented at 37°C, 150 rpm, for 3 days to obtain a fermentation broth. The neutralizer was 5 M aqueous ammonia, and the pH of the fermenter was controlled at 7.0.

[0105] Analytical Method: L-histidine content in fermentation broth after 3 days of fermentation was 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 column. Detection wavelength: 338 nm. Flow rate: 1.0 mL / min; injection volume: 10 μl; detector: UV 206 nm; mobile phase: acetonitrile / phosphate mixture. Weigh 0.05 M potassium dihydrogen phosphate and dissolve it in ultrapure water. Adjust the pH to 3.0 with phosphoric acid. Filter through a 0.22 μm filter. Then, add the corresponding volume of acetonitrile in a ratio of 3:1:acetonitrile:0.05 M potassium dihydrogen phosphate. Mix thoroughly and sonicate for 20 minutes until no bubbles appear.

[0106] The experiment was repeated 3 times, and the average values ​​of the analysis results are presented in Table 1 below.

[0107] The culture medium used was as follows:

[0108] 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.)

[0109] Seed culture medium (1 L): 20 g glucose, 4 g yeast extract, 3 g peptone, 1.2 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate·7H2O, 10 mg ferrous sulfate·7H2O, 10 mg manganese sulfate monohydrate, 1 mg VB1, 1 mg VB3, 1 mg VB5, 1 mg VB12, 1 mg VH, sodium hydroxide to adjust the pH between 7.0 and 7.2, and the balance is water.

[0110] Fermentation medium (1 L): 20 g glucose, 4 g yeast extract, 3 g peptone, 2 g sodium citrate monohydrate, 2 g potassium dihydrogen phosphate, 2 g magnesium sulfate·7H2O, 20 mg ferrous sulfate·7H2O, 20 mg manganese sulfate monohydrate, 2 mg VB1, 2 mg VB3, 2 mg VB5, 2 mg VB12, and 2 mg VH. Sodium hydroxide was used to adjust the pH to 7.0-7.2. Aqueous ammonia was added every 4 h during the fermentation process to a pH of 7.2-7.4. The balance was water.

[0111] Table 3. Comparison of L-histidine production capacity

[0112]

[0113] 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.

[0114] 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 further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using 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, the amino acid sequence of which 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. 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 engineered 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 L-histidine production, characterized in that: The method comprises inoculating the recombinant engineered bacteria according to any one of claims 5 to 8 into a culture medium for culturing to produce L-histidine.

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