L-histidinol phosphate aminotransferase mutant, encoding gene, plasmid, genetically engineered bacteria and application

Abstract

The application discloses an L-homoserine phosphotransferase mutant, a coding gene, a plasmid, a genetically engineered bacterium and application, and belongs to the technical field of bioengineering, wherein a cysteine at the 223th position of L-homoserine phosphotransferase is mutated into a glycine single mutation or a double mutation of mutating isoleucine at the 180th position into serine on the basis of the single mutation, namely the L-homoserine phosphotransferase mutant, a corresponding coding gene is obtained, and then a corresponding plasmid and genetically engineered bacterium are obtained; the enzyme mutant not only has high enzyme activity, but also is resistant to feedback inhibition of alpha-ketoglutarate; compared with simply improving enzyme activity, the application can further promote the synthesis of L-homoserine by affecting the reversible reaction direction. The application is suitable for applying the enzyme mutant to industrial fermentation production of L-homoserine, can significantly improve production efficiency, and can improve the yield of L-homoserine by about 60% by adding L-glutamine.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to an L-histamine phosphate aminotransferase mutant, specifically an L-histamine phosphate aminotransferase mutant, its encoding gene, plasmid, genetically engineered bacteria, and its applications. Background Technology

[0002] L-histidine is one of the 20 amino acids that make up proteins. Infants cannot synthesize it themselves and must obtain it from food; it is a semi-essential amino acid. L-histidine can act as both a proton donor and acceptor, and is often located at the active site of biological enzymes. It is mainly used in food, medicine, and animal feed, and is frequently used in comprehensive amino acid preparations as an adjunct treatment for anemia, arthritis, gastric ulcers, and duodenal ulcers. In recent years, research has found that L-histidine, as a component of carnosine, has broad market prospects in antioxidant health care. According to Hengzhou Bozhi's forecast, global consumption of histidine will reach over 3,100 tons in 2023.

[0003] Currently, the main producers of L-histidine are concentrated in China and Japan. Represented by well-known companies such as Kyowa Hakko and Ajinomoto, the main production methods include hydrolysis and fermentation. Fermentation has become the mainstream method for producing L-histidine due to its high efficiency and low cost, but some problems still exist.

[0004] Firstly, early genetically engineered strains used for L-histidine fermentation production primarily employed *Serratia marcescens* and *Corynebacterium glutamicum*, resulting in low yields. For instance, in its Korean invention patent (KR102204917B1), CJ Corporation of South Korea used *Corynebacterium glutamicum* KCCM11759P for L-histidine fermentation, achieving a yield of only 1.6 g / L. Later, with the intervention of genetic engineering techniques, while L-histidine yields improved, even in large-scale fermentation in fermenters, yields generally remained below 10 g / L. Furthermore, limited research into the genetic background of *Serratia marcescens* and *Corynebacterium glutamicum* further hampered efforts to increase yield.

[0005] In recent years, obtaining high-yield L-histidine strains by modifying *E. coli* strains with more thorough genetic background studies has become a research hotspot. In 2020, Chinese invention patent application CN111996155B disclosed a method to improve the production capacity of L-histidine-producing bacteria. This method involves mutating ATP phosphoribosyltransferase from *E. coli*, increasing the strain's L-histidine production capacity to more than 3.5 times that of the wild type. This technology improves the ability of *E. coli* to produce L-histidine through fermentation to some extent. The principle behind this increase in L-histidine production is eliminating the feedback inhibition of ATP phosphoribosyltransferase by the final product L-histidine. However, this method limits the modification of *E. coli* solely to this rate-limiting enzyme, severely restricting further pathways to increase L-histidine production.

[0006] Therefore, in order to meet the market's diverse demand for high-yield L-histidine strains and to broaden the path for future development, it is necessary to construct a genetically engineered strain for high-yield L-histidine production based on different modification principles. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide an L-histidine phosphate aminotransferase mutant. Through genetic engineering technology, the enzyme mutant is constructed and its encoding gene is obtained, so as to improve the catalytic activity of L-histidine phosphate aminotransferase and enable it to be used for the efficient fermentation production of L-histidine.

[0008] Another objective of this invention is to provide the application of strains carrying the above-mentioned L-histidine phosphate aminotransferase mutant encoding gene in the fermentation production of L-histidine.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A mutant of L-histamine phosphoaminotransferase, a single mutant in which cysteine ​​at position 223 of L-histamine phosphoaminotransferase is mutated to glycine, or

[0011] The L-histidine phosphate aminotransferase mutant is a double mutant in which cysteine ​​at position 223 is mutated to glycine and isoleucine at position 180 is mutated to serine.

[0012] The amino acid sequence of the L-histamine phosphate aminotransferase is shown in SEQ ID NO.1.

[0013] The nucleotide sequence of L-histidine phosphate transaminase is registered in NCBI under the number NC_000913.3:2092398-2093468. In L-histidine synthesis, this enzyme catalyzes a reversible reaction between imidazole pyruvate and glutamate to produce L-histidine phosphate and α-ketoglutarate, with L-histidine phosphate serving as a precursor to L-histidine.

[0014] SEQ ID NO.1:

[0015] MSTVTITDLARENVRNLTPYQSARRLGGNGDVWLNANEYPTAVEFQLTQQTLNRYPECQPKAVIENYAQYAGVKPEQVLVSRGADEGIELLIRAFCEPGKDAILYCPPTYGMYSVSAETIGVECRTVPTLDNWQLDLQGISDKLDGVKVVYVCSPNNPTGQLINPQDFRTLLELTRGK AIVVADEAYIEFCPQASLAGWLAEYPHLAILRTLSKAFALAGLRCGFTLANEEVINLLMKVIAPYPLSTPVADIAAQALSPQGIVAMRERVAQIIAEREYLIAALKEIPCVEQVFDSETNYILARFKASSAVFKSLWDQGIILRDQNKQPSLSGCLRITVGTREESQRVIDALRAEQV

[0016] As a limitation of the present invention, the amino acid sequence of the single mutant of L-histidine phosphoaminotransferase with cysteine ​​at position 223 mutated to glycine is shown in SEQ ID NO.2.

[0017] SEQ ID NO.2:

[0018] MSTVTITDLARENVRNLTPYQSARRLGGNGDVWLNANEYPTAVEFQLTQQTLNRYPECQPKAVIENYAQYAGVKPEQVLVSRGADEGIELLIRAFCEPGKDAILYCPPTYGMYSVSAETIGVECRTVPTLDNWQLDLQGISDKLDGVKVVYVCSPNNPTGQLINPQDFRTLLELTRGK AIVVADEAYIEFCPQASLAGWLAEYPHLAILRTLSKAFALAGLRGGFTLANEEVINLLMKVIAPYPLSTPVADIAAQALSPQGIVAMRERVAQIIAEREYLIAALKEIPCVEQVFDSETNYILARFKASSAVFKSLWDQGIILRDQNKQPSLSGCLRITVGTREESQRVIDALRAEQV

[0019] As a limitation of this invention, a double mutant of L-histidine phosphoaminotransferase, with cysteine ​​at position 223 mutated to glycine and isoleucine at position 180 mutated to serine, is shown in SEQ ID NO.3. The sequence SEQ ID NO.3 is the amino acid sequence of mut3 hereinafter referred to as mut3.

[0020] SEQ ID NO.3:

[0021] MSTVTITDLARENVRNLTPYQSARRLGGNGDVWLNANEYPTAVEFQLTQQTLNRYPECQPKAVIENYAQYAGVKPEQVLVSRGADEGIELLIRAFCEPGKDAILYCPPTYGMYSVSAETIGVECRTVPTLDNWQLDLQGISDKLDGVKVVYVCSPNNPTGQLINPQDFRTLLELTRGK ASVVADEAYIEFCPQASLAGWLAEYPHLAILRTLSKAFALAGLRGGFTLANEEVINLLMKVIAPYPLSTPVADIAAQALSPQGIVAMRERVAQIIAEREYLIAALKEIPCVEQVFDSETNYILARFKASSAVFKSLWDQGIILRDQNKQPSLSGCLRITVGTREESQRVIDALRAEQV

[0022] The gene sequence encoding the L-histidine phosphoaminotransferase mutant mut3 is shown in SEQ ID NO. 6:

[0023]

[0024]

[0025] According to common knowledge, the sequence of L-histamine phosphoamyltransferase mutants may also include point mutations, additions of various tags before and after the sequence, conserved substitutions at other positions, and amino acid truncations, which do not affect the active site of L-histamine phosphoamyltransferase mutants.

[0026] The present invention also provides a coding gene, which is a gene encoding the L-histamine phosphoaminotransferase mutant described in any of the preceding claims.

[0027] As a further limitation of the present invention, the expression host of the L-histamine phosphate aminotransferase mutant encoding gene is a genetically engineered bacterium used for gene expression; including hosts commonly used in the art, such as Escherichia coli BL21(DE3), Escherichia coli BL21, Escherichia coli M15, Bacillus subtilis, yeast, Aspergillus, Streptomyces, etc.; the expression vector of the L-histamine phosphate aminotransferase mutant, in addition to the pTrc99a vector commonly used in prokaryotes, also includes vectors that are compatible with each host system, and the integration mode of the vector with the host includes not only free existence within the host, but also integration at a specific location in the genome and integration at a random location; the expression mode of the L-histamine phosphate aminotransferase mutant is not limited to expression within the host or secretion.

[0028] The present invention also provides a plasmid carrying the coding gene for an L-histamine phosphate aminotransferase mutant.

[0029] As a limitation of the present invention, the plasmid is expressed either in free form or through genome integration.

[0030] The present invention also provides a genetically engineered bacterium, using Escherichia coli as a host, to express the encoding gene of an L-histamine phosphate aminotransferase mutant through free expression or genome integration.

[0031] This invention also provides the application of the above-mentioned genetically engineered bacteria in the production of L-histidine through fermentation.

[0032] As a limitation of the present invention, the fermentation broth used for the fermentation production of L-histidine contains 0.1-2 g / L of L-glutamine or 0.1-2 g / L of L-glutamic acid.

[0033] The fermentation temperature is 35-38℃, and the pH is 6.8-7.2.

[0034] The principle of this invention is based on the analysis of the L-histidine synthesis pathway. By mutating cysteine ​​at position 223 and / or isoleucine at position 180 of L-histidine phosphoaminotransferase to other amino acids, this L-histidine phosphoaminotransferase mutant not only has high enzyme activity, but also has a certain anti-feedback inhibition effect on α-ketoglutarate. Compared with simply increasing enzyme activity, it can further promote the synthesis of L-histidine by affecting the direction of reversible reaction.

[0035] By adopting the above technical solution, the technical progress achieved by this invention compared with the prior art is as follows:

[0036] ① The L-histidine phosphate aminotransferase mutant of the present invention increases the enzyme activity to about 1.5 times that of the wild type; at the same time, the cysteine ​​mutation at position 223 has a certain anti-feedback inhibition effect on high concentrations of α-ketoglutarate, which can enhance the forward reaction activity of catalyzing L-histidine synthesis and reduce the reverse reaction activity, thereby further promoting the synthesis of L-histidine.

[0037] ② The L-histidine phosphate aminotransferase mutant of this invention promotes the synthesis of L-histidine by enhancing L-histidine phosphate aminotransferase activity and altering the reversible reaction to a favorable direction. In particular, the yield is even higher with the addition of small amounts of L-glutamine and L-glutamic acid. In the shake-flask production stage, the L-histidine yield can be increased to 23-59%, demonstrating good applicability for industrial-scale L-histidine production. It also has a significant promoting effect on the production of the final product L-histidine, by 130%-158%.

[0038] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0039] Figure 1 This is a schematic diagram illustrating the role of L-histidine phosphate aminotransferase in the synthesis of L-histidine in an embodiment of the present invention. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the described embodiments are only for explaining the present invention and do not limit the present invention.

[0041] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available. Experimental methods not specifically described in the embodiments are generally performed under standard conditions or as recommended by the manufacturer.

[0042] The molecular biology experiments described in this example, including plasmid construction, enzyme digestion, competent cell preparation, and transformation, were mainly performed in accordance with *Molecular Cloning: A Laboratory Manual* (3rd Edition), edited by J. Sambrook and DW. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002. For example, the competent cell method used for transformation and the method for preparing competent cells were both performed according to Chapter 1, page 96 of *Molecular Cloning: A Laboratory Manual* (3rd Edition). Specific experimental conditions can be determined through simple experiments if necessary.

[0043] Example 1 Construction of L-histamine phosphoaminotransferase mutant

[0044] This embodiment includes the following steps performed sequentially:

[0045] Construction of wild-type expression strain: Based on the NCBI reference nucleotide sequence of L-histamine phosphoaminotransferase NC_000913.3:2092398-2093468 of the wild-type strain (Escherichia coli str.K-12substr.MG1655), primers as shown in Table 1 were designed. The enzyme was cloned by PCR, and Nco1 and Sal1 were selected as double restriction sites. The enzyme was directionally constructed into the pTrc99a vector sequence. The verified positive plasmid was transformed into E. coli BL21(DE3) to obtain the wild-type expression strain.

[0046] The PCR system consisted of 50 μL of RandomMut buffer (10X) 5 μL, Mutation enhancer (10X) 5 μL, RandomMut DNA polymerase 1 μL, dNTPs (2.5 mM each) 5 μL, template fragment supply of 120 pg / μL 1 μL, P1 and P2 primer mixture (10 μM each) 1 μL, and water to make up the remaining volume. The PCR program was as follows: initial cycle at 94℃ for 3 min; followed by 94℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min; for a total of 30 cycles; and finally extension at 72℃ for 10 min.

[0047] Table 1 Primer sequences

[0048] Primer name Serial Number 5'-3' sequence P1 SEQ ID NO.4 CATGCCATGGGCAGCACCGTGACTATTACCG P2 SEQ ID NO.5 ACGCGTCGACTCAAACTTGCTCCGCACG

[0049] Construction of mutant expression strains: Using the constructed wild-type expression strain expression vector as a template, random mutation was initiated using a gene random mutation kit. The obtained random mutated fragment was constructed into the pTrc99a vector using the above construction method. After transformation into E. coli BL21(DE3), the clones were screened by Amp plates. The obtained single clones were picked one by one to obtain L-histamine phosphoaminotransferase mutant expression strains: mut1 expression strain and mut2 expression strain.

[0050] Sequencing revealed that the amino acid sequence of the L-histamine phosphate aminotransferase mutant mut1 in the mut1 expression strain was based on the wild type, with the cysteine ​​at position 223 of L-histamine phosphate aminotransferase mutated to glycine.

[0051] In the mut2 expression strain, the amino acid sequence of the L-histamine phosphate aminotransferase mutant mut2 is based on the wild type, with the isoleucine at position 180 of L-histamine phosphate aminotransferase mutated to serine.

[0052] Furthermore, through genetic engineering design, a mut3 expression strain was constructed. The amino acid sequence of the L-histamine phosphoaminotransferase mutant mut3 in this strain is based on the wild-type sequence, with cysteine ​​at position 223 mutated to glycine and isoleucine at position 180 mutated to serine. While the L-histamine enhancement effect described in this invention is based on mut3, mut1 and mut2 can also enhance L-histamine production to some extent, and therefore are also within the scope of this invention.

[0053] The amino acid sequence of the L-histamine phosphoaminotransferase mutant mut3 is SEQ ID NO.3, and its encoding gene sequence is shown in SEQ ID NO.6:

[0054]

[0055]

[0056] In addition to the above-described embodiments, those skilled in the art can also, based on the amino acid sequence of wild-type L-histamine phosphate aminotransferase, design primers using genetic engineering techniques to mutate cysteine ​​at position 223 of L-histamine phosphate aminotransferase to glycine, thereby obtaining L-histamine phosphate aminotransferase mutant mut1, or mutate isoleucine at position 180 to serine, thereby obtaining L-histamine phosphate aminotransferase mutant mut2.

[0057] Those skilled in the art can also construct L-histamine phosphate aminotransferase mutants by using genetic engineering techniques to mutate cysteine ​​at position 223 and / or isoleucine at position 180 of L-histamine phosphate aminotransferase to other amino acids based on the amino acid sequence of wild-type L-histamine phosphate aminotransferase.

[0058] like Figure 1 As shown, L-histidine phosphate aminotransferase participates in the synthesis of L-histidine. In L-histidine synthesis, L-histidine phosphate aminotransferase catalyzes the reaction of imidazole pyruvate and glutamate to produce L-histidine phosphate and α-ketoglutarate via a reversible reaction, where L-histidine phosphate is a precursor of L-histidine. Therefore, the L-histidine phosphate aminotransferase mutant of this invention can regulate L-histidine production by affecting the direction of the reversible reaction catalyzed by L-histidine phosphate aminotransferase.

[0059] Example 2 Activity detection of L-histamine phosphate aminotransferase mutant

[0060] In this embodiment, the forward and reverse reaction enzyme activities of the wild-type, mut1-expressing, mut2-expressing, and mut3-expressing strains obtained in Example 1 were measured. The specific process is as follows:

[0061] Wild-type, mut1, mut2, and mut3 expression strains were inoculated into 2 mL of LB medium (final Amp concentration 100 mg / L) and incubated overnight at 37°C and 220 rpm. Then, 1% of the bacterial culture was inoculated into 100 mL of LB medium and incubated at 37°C and 220 rpm for 3 hours, followed by overnight incubation at 30°C. The cells were collected by centrifugation at 5000 rpm for 10 min, placed on ice, and sonicated using 50 mM PBNa buffer (pH 7.0). The sonication was performed for 15 min (3 s working, 7 s rest), followed by centrifugation at 12000 rpm for 2 min. The supernatant was collected and stored at -80°C for viability determination.

[0062] Forward enzyme activity assay system: An enzymatic reaction method was used to determine the reaction activity of L-histidine phosphoaminotransferase catalyzing the reaction of imidazole pyruvate phosphate and glutamate to produce L-histidine phosphate and α-ketoglutarate. 1 mL of the reaction system contained the following components at final concentrations: 50 mM PBNa (pH 7.0), 75 mM L-glutamate, 0.2 g / L PLP, and 100 μL enzyme solution. The reaction was carried out at 37℃ and 220 rpm for 10 min. α-ketoglutarate in the reaction product was then detected. One unit of enzyme activity was defined as the production of 1 μM α-ketoglutarate per minute under these conditions.

[0063] Reverse enzyme activity assay system: A high-concentration α-ketoglutarate reverse experiment was conducted to determine the reaction activity of L-histidine phosphorammonium transferase catalyzing the reaction of L-histidine phosphoric acid and α-ketoglutarate to produce imidazole pyruvate phosphoric acid and glutamate. 1 mL of the reaction system contained the following components at final concentrations: 50 mM sodium phosphate buffer (PBNa) pH 7.0, 75 mM L-histidine phosphoric acid, 150 mM α-ketoglutarate, 0.2 g / L pyridoxal phosphate (PLP), and 100 μL enzyme solution. The reaction was carried out at 37℃ and 220 rpm for 10 min. The production of glutamate in the reaction product was detected. The enzyme activity unit was defined as 1 μM of glutamate produced per minute by histidine phosphorammonium transferase under these conditions.

[0064] Table 2. Vitality Measurement Results

[0065]

[0066] The results are shown in Table 2. The mut1 and mut3 expression strains containing the C223G mutation exhibited some anti-feedback inhibition against high concentrations of α-ketoglutarate, with a positive-to-negative reaction ratio of 1.69. I180S showed no significant inhibitory effect on the reverse reaction, similar to the wild type, with ratios of 1.18 and 1.13, respectively. The double mutation of C223G and I180S retained some anti-feedback inhibition, with a positive-to-negative reaction ratio of 1.56. This indicates that the mut1, mut2, and mut3 expression strains can all increase L-histidine production by enhancing enzyme activity, with the mut1 and mut3 expression strains showing particularly better results.

[0067] Example 3: L-histidine phosphate aminotransferase mutant used for fermentation production of L-histidine

[0068] Example 3 describes the fermentation of L-histidine using two methods: free expression in the production strain and integrated expression in the genome of the wild-type and L-histidine phosphoaminotransferase mutant mut3. The effects of adding L-glutamine or L-glutamate to the fermentation broth on the L-histidine yield were also investigated.

[0069] (1) Free expression of producing strain: The plasmid containing the L-histidine phosphate aminotransferase mutant mut3 was extracted and transformed into the L-histidine producing strain (the L-histidine phosphate aminotransferase expression strain with Escherichia coli as the host, denoted as L-His) by chemical transformation. The strain was plated on Amp plates to obtain a single clone, which was then preserved and labeled as L-His+mut3A for L-histidine yield testing.

[0070] (2) Genomic integration expression of the producing strain: A plasmid containing the L-histamine phosphoaminotransferase mutant mut3 was extracted and recombined into the L-histidine producing strain using the CRISPR method. The integration system used was a transposon system for genome integration expression. The specific method was as follows: The integration site is as shown in SEQ ID NO.7, which is the genomic site carried by 5'-TGGCACGGCTGGGACGGAAGTCGCTGTCGTTC-3'.

[0071] The pDonor-mut3 vector and three plasmids, pTnsABC, were co-transformed into an L-histidine-producing strain. The strain was screened at 37°C on LB agar plates containing ampicillin, kanamycin, and streptomycin. Single clones were obtained by shaking and mixing. 100 μL of the stock solution was plated onto agar plates containing 1 mM IPTG and induced to transposonize. After PCR identification, the plasmid was ablated using a standard plasmid ablation program (see ACS Synth. Biol. 2020, 9, 1998-2008 for details). The successfully integrated strain was named L-His+mut3.

[0072] (3) Fermentation production method: The yield of L-histidine was determined under shake-flask conditions. The activation medium was LB medium. The shake-flask fermentation medium (H1) consisted of: glucose 25 g / L, yeast extract 4 g / L, peptone 3 g / L, sodium citrate 1.5 g / L, MgSO4·7H2O 2 g / L, KH2PO4 2.5 g / L, FeSO4·7H2O 12 mg / L, MnSO4·H2O 8 mg / L, VB1, VB5, and VB6. 12 Administer 1.5 mg / mL of each of VH and adjust the pH to 6.8-7.2;

[0073] The culture medium was activated at 37℃ and 220 rpm for 24 h. The culture medium was then inoculated into shake flasks at an inoculum size of 2%. The temperature was changed to 35℃ and the culture was shaken for 10 h. L-glutamine (L-Gln) and L-glutamic acid (L-Glu) were then added. The L-histidine yield was measured after 48 h.

[0074] (4) L-histidine production detection method: The L-histidine in the shake-flask fermentation medium was quantitatively detected by a modified colorimetric method. 0.5 mL of the test liquid reaction solution of different concentrations was taken, and 0.3 mL of 1% p-aminobenzenesulfonic acid solution and 0.3 mL of 5% sodium nitrite solution were added. The mixture was allowed to stand at 37℃ for 10 min. 1 mL of 10% sodium carbonate solution and 2 mL of 20% ethanol solution were added and mixed well for color development. The absorbance of the standard and sample of different concentrations was measured at 510 nm. The L-histidine production results were calculated by comparing with the standard curve, as shown in Table 3.

[0075] Table 3 L-histidine production results

[0076]

[0077] This example compares the effects of various factors on L-histidine production, narrowing the comparison to a shake-flask system to examine production trends. Table 3 shows that free expression significantly increases L-histidine production more than genome integration. Overall, L-His+mut3A can increase L-histidine production to a maximum of 5.7 g / L during the shake-flask stage, which is over 33% of the wild-type. Adding certain concentrations of L-Gln and L-Glu after 10 hours of culture significantly further stimulates L-histidine production, with L-Gln showing a more pronounced effect. Furthermore, at 48 hours, the highest L-histidine production exceeded 7 g / L, an increase of approximately 59% compared to the original level.

[0078] The reaction system was scaled up to 10L, and industrial-scale production was carried out at a fermentation temperature of 35-38℃ and a pH of 6.8-7.2. Although the yield of the control wild-type strain L-His was only about 4.3g / L in shake flask, it could show a yield of more than 40g / L in the fed-batch fermentation of the fermenter. After adding L-Gln, the L-histidine yield of L-His+mut3A increased by about 60% compared with the original.

[0079] Example 4-11 Method for Fermentation Production of L-Histidine

[0080] Examples 4-11 are methods for producing L-histidine by adding L-glutamine or L-glutamic acid to the fermentation broth of L-His+mut3A.

[0081] Example 4 involved taking L-His+mut3A strain, activating it in LB medium at 37℃ and 220 rpm for 24 h, and then inoculating it into shake-flask fermentation medium H1 at pH 6.8 at an inoculum size of 2%. The temperature was changed to 35℃, and after shaking-flask culture for 10 h, 0.1 g / L of L-glutamine was added. The culture was continued for 48 h, and samples were taken to detect the L-histidine yield, which was 6.22 g / L.

[0082] Example 5 involved taking L-His+mut3A strain, activating it in LB medium at 37℃ and 220 rpm for 24 h, and then inoculating it into shake-flask fermentation medium H1 at pH 7.2 at an inoculum size of 2%. The temperature was changed to 35℃, and after shaking-flask culture for 10 h, 0.5 g / L of L-glutamine was added. The culture was continued for 48 h, and samples were taken to detect the L-histidine yield, which was 6.53 g / L.

[0083] Example 6 involved taking L-His+mut3A strain, activating it in LB medium at 37°C and 220 rpm for 24 h, and then inoculating it into shake-flask fermentation medium H1 at pH 6.9 at an inoculum size of 2%. The temperature was changed to 38°C, and after shaking-flask culture for 10 h, 1.5 g / L of L-glutamine was added. The culture was continued for 48 h, and samples were taken to detect the L-histidine yield, which was 6.89 g / L.

[0084] Example 7 involved taking L-His+mut3A strain, activating it in LB medium at 37°C and 220 rpm for 24 h, and then inoculating it into shake-flask fermentation medium H1 at pH 7.1 at an inoculum size of 2%. The temperature was changed to 36°C, and after shaking-flask culture for 10 h, 2 g / L of L-glutamine was added. The culture was continued for 48 h, and samples were taken to detect the L-histidine yield, which was 7.01 g / L.

[0085] Example 8 involved taking L-His+mut3A strain, activating it in LB medium at 37°C and 220 rpm for 24 h, and then inoculating it into shake-flask fermentation medium H1 at pH 7.2 at an inoculum size of 2%. The temperature was changed to 37°C, and after shaking-flask culture for 10 h, 1.5 g / L of L-glutamic acid was added. The culture was continued for 48 h, and samples were taken to detect the L-histidine yield, which was 5.89 g / L.

[0086] Example 9 involved taking L-His+mut3A strain, activating it in LB medium at 37°C and 220 rpm for 24 h, and then inoculating it into shake-flask fermentation medium H1 at pH 6.8 at an inoculum size of 2%. The temperature was changed to 37°C, and after shaking-flask culture for 10 h, 1.2 g / L of L-glutamic acid was added. After culturing for another 48 h, samples were taken to detect the L-histidine yield, which was 5.73 g / L.

[0087] Example 10 involved taking L-His+mut3A strain, activating it in LB medium at 37°C and 220 rpm for 24 h, and then inoculating it into shake-flask fermentation medium H1 at pH 7.2 at an inoculum size of 2%. The temperature was changed to 37°C, and after shaking-flask culture for 10 h, 2 g / L of L-glutamic acid was added. The culture was continued for 48 h, and samples were taken to detect the L-histidine yield, which was 5.65 g / L.

[0088] Example 11 involved taking L-His+mut3A strain, activating it in LB medium at 37°C and 220 rpm for 24 h, and then inoculating it into shake-flask fermentation medium H1 at pH 7.0 at an inoculum size of 2%. The temperature was changed to 37°C, and after shaking-flask culture for 10 h, 0.1 g / L of L-glutamic acid was added. The culture was continued for 48 h, and samples were taken to detect the L-histidine yield, which was 5.59 g / L.

[0089] The above embodiments show that L-histidine can be produced by adding L-glutamine or L-glutamic acid to the fermentation broth of L-His+mut3A in a shake flask system and then fermenting. The addition of L-glutamine can further increase the yield of L-histidine, indicating that the present invention has the prospect of high-yield industrial production of L-histidine.

[0090] It should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. An L-histamine phosphoaminotransferase mutant, characterized in that, It is a single mutant of L-histamine phosphoaminotransferase with a cysteine ​​at position 223 mutated to glycine, or a double mutant of L-histamine phosphoaminotransferase with a cysteine ​​at position 223 mutated to glycine and an isoleucine at position 180 mutated to serine. The amino acid sequence of the single mutant is shown in SEQ ID NO.2; The amino acid sequence of the double mutant is shown in SEQ ID NO.

3.

2. A gene encoding a gene, characterized in that, The encoding gene is the gene encoding the L-histamine phosphate aminotransferase mutant as described in claim 1.

3. The encoding gene according to claim 2, characterized in that, The host for the expression of the encoded gene is a genetically engineered bacterium.

4. A plasmid, characterized in that, The plasmid carries the encoding gene as described in claim 3.

5. The plasmid according to claim 4, characterized in that, The plasmid is expressed either in free form or through genome integration.

6. A genetically engineered bacterium, characterized in that, Express the plasmid as described in claim 4 or 5.

7. The application of a genetically engineered bacterium, characterized in that, L-histidine is produced by fermentation using the genetically engineered bacteria described in claim 6.

8. The application of the genetically engineered bacteria according to claim 7, characterized in that, The fermentation broth contains 0.1-2 g / L of L-glutamine or 0.1-2 g / L of L-glutamic acid; The fermentation temperature is 35-38 ℃, and the pH is 6.8-7.2.

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