Atp-transphosphoribosylase mutant, encoding gene, recombinant vector, transgenic cell and application thereof

Abstract

The application relates to the technical field of genetic engineering, and discloses an ATP phosphoribosyltransferase mutant, a coding gene of the ATP phosphoribosyltransferase mutant, a recombinant carrier, a transgenic cell and application of the ATP phosphoribosyltransferase mutant. The mutant is a mutant generated by mutating at least one amino acid in the ATP phosphoribosyltransferase mutant with an amino acid sequence as shown in SEQ ID NO:1; and according to the ATP phosphoribosyltransferase with the amino acid sequence as shown in SEQ ID NO:1 as a benchmark, the mutation site of the mutant is the 214th position and / or the 215th position. The ATP phosphoribosyltransferase mutant can effectively remove the feedback inhibition of histidine, maintain high catalytic activity, and significantly improve the biosynthesis efficiency of histidine.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to an ATP transphosphoribosylase mutant and its encoding gene, recombinant vector, transgenic cells, and their applications. Background Technology

[0002] L-histidine is one of the eight essential amino acids that the human body cannot synthesize. It is a precursor for the synthesis of many drugs and can be used to treat various diseases such as ulcers, kidney failure, rheumatoid arthritis, and Alzheimer's disease. It has wide applications in medicine, industry, food, cosmetics, and animal husbandry, and has significant economic and social value.

[0003] Currently, there are three main methods for producing L-histidine: biosynthesis, chemical synthesis, and protein hydrolysis. Among them, biosynthesis has advantages such as a wide range of raw material sources, relatively simple production process, relatively small environmental impact, and high product purity, and has now become the mainstream method for L-histidine production.

[0004] In the L-histidine biosynthesis pathway, the first enzymatic reaction is the condensation reaction of 5-phosphoribose-1-pyrophosphate (PRPP) and adenosine triphosphate (ATP) catalyzed by ATP transphosphoribosylase encoded by the HisG gene. The effect of HisG on L-histidine synthesis is as follows: Figure 1 As shown, HisG, a key enzyme in L-histidine biosynthesis, is subject to feedback inhibition by the end product L-histidine. Furthermore, adenosine monophosphate (AMP) synergistically enhances the feedback inhibition effect of L-histidine on HisG enzymes, thereby precisely regulating the metabolic flux of the intracellular L-histidine biosynthesis pathway.

[0005] Therefore, developing ATP transphosphoribosylases unaffected by L-histidine feedback inhibition is crucial for the efficient biosynthesis of L-histidine. However, existing ATP transphosphoribosylases exhibit weak tolerance to L-histidine feedback inhibition, resulting in less than ideal biosynthetic efficiency for L-histidine. Summary of the Invention

[0006] The purpose of this invention is to overcome the problem that existing technologies have weak tolerance to feedback inhibition of L-histidine by ATP transphosphoribosylase, resulting in low biosynthetic efficiency of L-histidine. This invention provides an ATP transphosphoribosylase mutant, its encoding gene, recombinant vector, transgenic cells, and their applications. This ATP transphosphoribosylase mutant can effectively relieve the feedback inhibition of histidine, maintain high catalytic activity, and significantly improve the biosynthetic efficiency of histidine.

[0007] To achieve the above objectives, the first aspect of the present invention provides an ATP transphosphoribosylase mutant, wherein the mutant is a mutant generated by mutating at least one amino acid of the ATP transphosphoribosylase with the amino acid sequence as shown in SEQ ID NO:1; wherein, based on the ATP transphosphoribosylase with the amino acid sequence as shown in SEQ ID NO:1, the mutation site of the mutant is position 214 and / or position 215.

[0008] Preferably, the mutant has its tyrosine Y at position 214 mutated to aspartic acid D, glutamic acid E, phenylalanine F, lysine K, methionine M, threonine T, or tryptophan W, and more preferably, the tyrosine Y at position 214 mutated to aspartic acid D, phenylalanine F, or tryptophan W.

[0009] Preferably, the 215th position of asparagine N in the mutant is mutated to alanine A, aspartic acid D, glutamic acid E, phenylalanine F, glycine G, isoleucine I, lysine K, leucine L, methionine M, glutamine Q, serine S, threonine T, valine V, or tyrosine Y, and more preferably, the 215th position of asparagine N is mutated to glutamic acid E, phenylalanine F, isoleucine I, leucine L, glutamine Q, or serine S.

[0010] Preferably, the mutant is a single-point mutation mutant, and the mutation site of the mutant is position 215.

[0011] Preferably, the mutant is a mutant N215E in which asparagine N at position 215 is mutated to glutamic acid E, and its amino acid sequence is shown in SEQ ID NO:2;

[0012] Alternatively, the mutant is a mutant N215F in which asparagine N at position 215 is mutated to phenylalanine F, and its amino acid sequence is shown in SEQ ID NO:3;

[0013] Alternatively, the mutant is a mutant N215Q in which asparagine N at position 215 is mutated to glutamine Q, and its amino acid sequence is shown in SEQ ID NO:4.

[0014] A second aspect of the present invention provides a coding gene capable of encoding the ATP transphosphorylated ribosylase mutant as described above.

[0015] Preferably, the encoding gene has a nucleotide sequence encoding a mutant with an amino acid sequence as shown in SEQ ID NO:2, and more preferably, a nucleotide sequence as shown in SEQ ID NO:5;

[0016] Alternatively, the coding gene has a nucleotide sequence encoding a mutant with an amino acid sequence as shown in SEQ ID NO:3, preferably a nucleotide sequence as shown in SEQ ID NO:6;

[0017] Alternatively, the coding gene may have a nucleotide sequence encoding a mutant with an amino acid sequence as shown in SEQ ID NO:4, preferably a nucleotide sequence as shown in SEQ ID NO:7.

[0018] A third aspect of the present invention provides a recombinant vector containing the coding gene as described above.

[0019] Preferably, the expression vector of the recombinant vector is the pSTV28 plasmid.

[0020] A fourth aspect of the present invention provides a transgenic cell containing the coding gene as described above or the recombinant vector as described above.

[0021] Preferably, the host strain of the transgenic cells is a prokaryotic cell, preferably *Escherichia coli*, and more preferably *Escherichia coli* HCBHIS1. The inventors have found that this preferred embodiment is beneficial for further improving the expression efficiency of the HisG mutant.

[0022] The fifth aspect of the present invention provides the use of at least one of the ATP transphosphoribosylase mutant, the coding gene, the recombinant vector, and the transgenic cell as described above in the production of histidine.

[0023] The sixth aspect of the present invention provides a method for producing histidine, the method comprising the step of culturing transgenic cells as described above.

[0024] Preferably, the culture medium used for the culture contains: glucose 10-20 g / L, yeast extract 1-3 g / L, ammonium salt 1-3 g / L, potassium salt 4-8 g / L, magnesium salt 1-2 g / L, iron salt 15-20 mg / L, manganese salt 15-20 mg / L, citric acid 1-3 g / L, methionine 1-3 g / L, vitamin B1, vitamin B3, vitamin B5, and vitamin B6. 12 The culture medium contains 1-3 mg / L of vitamin H and 30-40 g / L of 3-(N-morpholino)propanesulfonic acid; the pH of the culture medium is 7.0-7.2.

[0025] Preferably, the culture conditions include at least the following: a temperature of 30-45°C and a rotation speed of 180-220 rpm.

[0026] Through the above technical solution, the ATP transphosphoribosylase mutant provided by the present invention can effectively relieve the feedback inhibition effect of L-histidine, maintain high catalytic activity in the presence of L-histidine, which is beneficial to increase the flux of the L-histidine synthesis pathway and improve the synthesis efficiency of L-histidine.

[0027] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0028] Figure 1 This is a schematic diagram illustrating the effect of ATP-transphosphoribosylase HisG on L-histidine synthesis;

[0029] Figure 2 These are the spectra of the pSTV28 plasmid and the recombinant vector pSTV28-HisG from Example 1;

[0030] Figure 3 This is a data graph of saturated fermentation of recombinant strain E. coli HCBHIS1-pSTV28-HisG-N215;

[0031] Figure 4 This is a data graph of saturated fermentation of recombinant strain E. coli HCBHIS1-pSTV28-HisG-N214. Detailed Implementation

[0032] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0033] The first aspect of the present invention provides an ATP transphosphoribosylase mutant, wherein the mutant is a mutant generated by mutating at least one amino acid of the ATP transphosphoribosylase with the amino acid sequence as shown in SEQ ID NO:1; wherein, based on the ATP transphosphoribosylase with the amino acid sequence as shown in SEQ ID NO:1, the mutation site of the mutant is position 214 and / or position 215.

[0034] Based on the amino acid sequence shown in SEQ ID NO:1, an ATP transphosphoribosylase mutant was used as the wild-type enzyme (hereinafter referred to as HisG wild-type enzyme). An amino acid residue substitution was performed at position 214 and / or 215 of the amino acid sequence of the wild-type enzyme. The resulting ATP transphosphoribosylase mutant (HisG mutant) can effectively relieve the feedback inhibition effect of L-histidine, has strong histidine tolerance, and maintains high catalytic activity in the presence of L-histidine. This is beneficial to increasing the flux of the L-histidine synthesis pathway in cell metabolism and improving the synthesis efficiency of L-histidine. This has important research value for the industrial biosynthesis of L-histidine.

[0035] In this invention, the amino acid sequence of the HisG wild-type enzyme shown in SEQ ID NO:1 is as follows:

[0036] MLKIAVPNKGSLSERAMEILAEAGYAGRGDSKSLNVFDEANNVEFFFLRPKDIAIYVAGGQLDLGITGRDLARDSQADVHEVLSLGFGSSTFRYAAPADEEWSIEKL DGKRIATSYPNLVRDDLAARGLSAEVLRLDGAVEVSIKLGVADAIADVVSTGRTLRQQGLAPFGEVLCTSEAVIVGRKDEKVTPEQQILLRRIQGILHAQNFLMDY N VDRDNLDAATAVTPGLSGPTVSPLARDNWVAVRAMVPRRSANAIMDKLAGLGAEAILASEIRIARI (SEQ ID NO: 1).

[0037] According to the present invention, preferably, the mutant has its tyrosine Y at position 214 mutated to aspartic acid D, glutamic acid E, phenylalanine F, lysine K, methionine M, threonine T, or tryptophan W. More preferably, the mutant is a mutant Y214D with its tyrosine Y at position 214 mutated to aspartic acid D, or a mutant Y214F with its tyrosine Y at position 214 mutated to phenylalanine F, or a mutant Y214W with its tyrosine Y at position 214 mutated to tryptophan W. The inventors have found that, under this preferred embodiment, it is beneficial to further enhance the mutant's ability to relieve histidine feedback inhibition and improve the biosynthetic efficiency of histidine.

[0038] Any HisG mutant with a mutation at position 215 of the amino acid sequence shown in SEQ ID NO:1 is included in this invention. Preferably, the mutant has the asparagine N at position 215 mutated to alanine A, aspartic acid D, glutamic acid E, phenylalanine F, glycine G, isoleucine I, lysine K, leucine L, methionine M, glutamine Q, serine S, threonine T, valine V, or tyrosine Y; more preferably, the asparagine N at position 215 mutated to glutamic acid E, phenylalanine F, isoleucine I, leucine L, glutamine Q, or serine S. The inventors have found that, under this preferred embodiment, it is beneficial to further maintain the catalytic activity of the mutant in the presence of histidine and improve the biosynthetic efficiency of histidine.

[0039] In this invention, the mutant can be a mutant formed by mutating position 214 of the HisG wild-type enzyme, a mutant formed by mutating position 215 of the HisG wild-type enzyme, or a mutant formed by simultaneously mutating positions 214 and 215 of the HisG wild-type enzyme. Preferably, the mutant is a single-point mutation. More preferably, the mutation site of the mutant is position 215.

[0040] More preferably, the mutant is a mutant N215E in which asparagine N at position 215 is mutated to glutamic acid E, and its amino acid sequence is shown in SEQ ID NO:2;

[0041] MLKIAVPNKGSLSERAMEILAEAGYAGRGDSKSLNVFDEANNVEFFFLRPKDIAIYVAGGQLDLGITGRDLARDSQADVHEVLSLGFGSSTFRYAAPADEEWSIEKL DGKRIATSYPNLVRDDLAARGLSAEVLRLDGAVEVSIKLGVADAIADVVSTGRTLRQQGLAPFGEVLCTSEAVIVGRKDEKVTPEQQILLRRIQGILHAQNFLMDY E VDRDNLDAATAVTPGLSGPTVSPLARDNWVAVRAMVPRRSANAIMDKLAGLGAEAILASEIRIARI(SEQ ID NO:2);

[0042] Alternatively, the mutant is a mutant N215F in which asparagine N at position 215 is mutated to phenylalanine F, and its amino acid sequence is shown in SEQ ID NO:3;

[0043] MLKIAVPNKGSLSERAMEILAEAGYAGRGDSKSLNVFDEANNVEFFFLRPKDIAIYVAGGQLDLGITGRDLARDSQADVHEVLSLGFGSSTFRYAAPADEEWSIEKL DGKRIATSYPNLVRDDLAARGLSAEVLRLDGAVEVSIKLGVADAIADVVSTGRTLRQQGLAPFGEVLCTSEAVIVGRKDEKVTPEQQILLRRIQGILHAQNFLMDY F VDRDNLDAATAVTPGLSGPTVSPLARDNWVAVRAMVPRRSANAIMDKLAGLGAEAILASEIRIARI(SEQ ID NO:3);

[0044] Alternatively, the mutant is a mutant N215Q in which asparagine N at position 215 is mutated to glutamine Q, and its amino acid sequence is shown in SEQ ID NO:4;

[0045] MLKIAVPNKGSLSERAMEILAEAGYAGRGDSKSLNVFDEANNVEFFFLRPKDIAIYVAGGQLDLGITGRDLARDSQADVHEVLSLGFGSSTFRYAAPADEEWSIEKL DGKRIATSYPNLVRDDLAARGLSAEVLRLDGAVEVSIKLGVADAIADVVSTGRTLRQQGLAPFGEVLCTSEAVIVGRKDEKVTPEQQILLRRIQGILHAQNFLMDY Q VDRDNLDAATAVTPGLSGPTVSPLARDNWVAVRAMVPRRSANAIMDKLAGLGAEAILASEIRIARI (SEQ ID NO: 4).

[0046] According to the present invention, there are no particular limitations on the source of the ATP transphosphoribosylase mutant, as long as an ATP transphosphoribosylase mutant with the above amino acid sequence can be obtained. For example, the ATP transphosphoribosylase mutant can be obtained artificially, or its encoding gene can be obtained from the amino acid sequence of the ATP transphosphoribosylase mutant, and then obtained through corresponding biological expression.

[0047] A second aspect of the present invention provides a coding gene capable of encoding the ATP transphosphorylated ribosylase mutant as described above.

[0048] In this invention, any gene capable of encoding the HisG mutant described above is within the scope of protection of this invention. It is well known to those skilled in the art that genetic codons are degenerate; therefore, given the amino acid sequence of the HisG mutant described above, those skilled in the art can obtain genes with different nucleotide sequences that can encode the HisG mutant described above using conventional techniques. For example, based on the coding gene of the HisG wild-type enzyme (e.g., the coding gene with the nucleotide sequence shown in SEQ ID NO: 8), the codon encoding the amino acid at the mutation site can be adjusted accordingly. Furthermore, the coding gene can be optimized based on the characteristics of the expression host to improve the expression level and / or expression efficiency of the HisG mutant.

[0049] According to the present invention, preferably, the encoding gene has a nucleotide sequence encoding a mutant with an amino acid sequence as shown in SEQ ID NO:2, and more preferably, the nucleotide sequence is as shown in SEQ ID NO:5;

[0050] Alternatively, the coding gene has a nucleotide sequence encoding a mutant with an amino acid sequence as shown in SEQ ID NO:3, preferably a nucleotide sequence as shown in SEQ ID NO:6;

[0051] Alternatively, the coding gene may have a nucleotide sequence encoding a mutant with an amino acid sequence as shown in SEQ ID NO:4, preferably a nucleotide sequence as shown in SEQ ID NO:7.

[0052] The inventors have discovered that, under this preferred embodiment, it is beneficial to further improve the ability of HisG mutants to relieve the feedback inhibition effect of L-histidine and improve the synthesis efficiency of L-histidine.

[0053] The nucleotide sequences provided by this invention can typically be obtained using polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis. Once the relevant nucleotide sequence is obtained, the relevant amino acid sequence can be obtained in large quantities using recombination. The obtained nucleotide sequence is usually cloned into an expression vector, then transformed into an expression host, and then the relevant nucleotide sequence is isolated from the proliferated host cells using conventional methods.

[0054] A third aspect of the present invention provides a recombinant vector containing the coding gene as described above.

[0055] In this invention, the expression vector used in the recombinant vector can be any vector known in the art, such as various commercially available plasmids, granules, bacteriophages, and retroviruses. The preferred expression vector in this invention is the pSTV28 plasmid. The recombinant vector can be constructed using various methods commonly used in the art, such as ligating the target gene into the expression vector after enzyme digestion.

[0056] A fourth aspect of the present invention provides a transgenic cell containing the coding gene as described above or the recombinant vector as described above.

[0057] The method for constructing the transgenic cells can be accomplished using conventional techniques in the art, such as by introducing and inserting the recombinant vector as described above into the corresponding strain.

[0058] In this invention, any engineered bacteria or other host cells capable of expressing exogenous genes can be used for the construction of transgenic cells provided by this invention, as long as they can express the HisG mutant described in this invention. The host cells of the transgenic cells can be prokaryotic or eukaryotic cells, preferably prokaryotic cells, such as *Escherichia coli*, *Bacillus subtilis*, etc.; more preferably *Escherichia coli*. There are no special requirements for the type of *Escherichia coli* used to construct the transgenic cells; it can be any *Escherichia coli* commonly used in the art capable of expressing the target gene, more preferably *Escherichia coli* HCBHIS1. The inventors have found that this preferred embodiment is beneficial for improving the expression efficiency of the HisG mutant.

[0059] The ATP transphosphoribosylase mutant provided by this invention can effectively relieve the feedback inhibition effect of L-histidine and maintain high catalytic activity in the presence of L-histidine. A fifth aspect of this invention provides the use of at least one of the ATP transphosphoribosylase mutant, the encoding gene, the recombinant vector, and the transgenic cells described above in the production of histidine.

[0060] The sixth aspect of the present invention provides a method for producing histidine, the method comprising the step of culturing transgenic cells as described above.

[0061] In this invention, the transgenic cells can be cultured using conventional methods to achieve large-scale proliferation. Generally, the culture process includes: first, performing seed culture on the transgenic cells to obtain seed solution, and then inoculating the seed solution into the fermentation medium for fermentation culture.

[0062] According to the present invention, preferably, the method for preparing the seed culture includes: inoculating the colonies of the transgenic cells into a seed culture medium for seed culture to obtain the seed culture. In this invention, the colonies of the transgenic cells can be selected from freshly prepared transgenic cells (obtained by liquid culture after scraping from LB plates) or cryopreserved transgenic cells. If transgenic cells preserved in glycerol tubes are used for inoculation, the inoculum in each glycerol tube is inoculated into 50 mL of seed culture medium.

[0063] This invention does not impose any particular limitations on the seed culture method, as long as the method can activate and proliferate the transgenic cells; the parameters used in seed culture, such as temperature, pH, rotation speed, and time, can be conventional settings within the art. Preferably, the seed culture conditions include: a temperature of 30-45℃, a rotation speed of 180-220 rpm, and a time of 10-12 hours.

[0064] In this invention, there are no particular limitations on the seed culture medium, and it can be any seed culture medium conventionally used in the art. Preferably, when the starting strain is Escherichia coli, the seed culture medium contains: peptone, yeast extract, and NaCl; more preferably, the seed culture medium contains: 1-5 g / L peptone, 1-5 g / L yeast extract, and 1-2.5 g / L NaCl; the pH is 7.0-7.2.

[0065] In this invention, there are no particular limitations on the fermentation method. Appropriate parameters such as temperature, pH, rotation speed, and time can be selected based on the type of originating strain of the transgenic cells. Preferably, the fermentation conditions include at least the following: an inoculum size of 5-15% (v / v), specifically 5%, 7%, 9%, 11%, 13%, 15%, or any value between these two values; a temperature of 30-45℃, specifically 30℃, 33℃, 36℃, 39℃, 42℃, 45℃, or any value between these two values; and a rotation speed of 180-220 rpm, specifically 180 rpm, 190 rpm, 200 rpm, 210 rpm, 220 rpm, 230 rpm, 240 rpm, 250 rpm, or any value between these two values.

[0066] According to the present invention, preferably, when the starting strain is *Escherichia coli*, the fermentation medium contains: glucose, yeast extract, ammonium salt, potassium salt, magnesium salt, iron salt, manganese salt, citric acid, methionine, vitamin B1, vitamin B3, vitamin B5, and vitamin B6. 12Vitamin H, 3-(N-morpholino)propanesulfonic acid; more preferably, the fermentation medium used for the culture contains: glucose 10-20 g / L, yeast extract 1-3 g / L, ammonium salt 1-3 g / L, potassium salt 4-8 g / L, magnesium salt 1-2 g / L, iron salt 15-20 mg / L, manganese salt 15-20 mg / L, citric acid 1-3 g / L, methionine 1-3 g / L, vitamin B1, vitamin B3, vitamin B5, vitamin B6. 12 The culture medium contains 1-3 mg / L of vitamin H and 30-40 g / L of 3-(N-morpholino)propanesulfonic acid; the pH of the culture medium is 7.0-7.2.

[0067] The ammonium salt can be at least one of ammonium sulfate, ammonium chloride and ammonium nitrate; the potassium salt can be potassium dihydrogen phosphate and / or dipotassium hydrogen phosphate, preferably dipotassium hydrogen phosphate; the magnesium salt can be at least one of magnesium sulfate, magnesium chloride and magnesium nitrate; the iron salt can be at least one of ferric sulfate, ferric chloride and ferric nitrate; and the manganese salt can be at least one of manganese sulfate, manganese chloride and manganese nitrate.

[0068] The present invention will be described in detail below through embodiments.

[0069] In the following examples, the pSTV28 plasmid was purchased from Shanghai Zeye Biotechnology Co., Ltd., product number ZY-64369. Unless otherwise specified, other reagents and raw materials were conventional commercial products.

[0070] In the following examples, experimental methods without specific conditions were performed under conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual, or as recommended by the manufacturers of the relevant biological reagents.

[0071] In the following examples, E. coli HCBHIS1 is a strain with histidine synthesis ability obtained by performing multiple rounds of adaptive evolutionary screening on wild-type E. coli MG1655 in a medium containing different histidine concentrations.

[0072] The solid culture medium for the activated plates consisted of: 3 g / L peptone, 3 g / L yeast extract, 1.5 g / L NaCl, 18 g / L agar, and the remainder was water, with a pH of 7.1.

[0073] The seed culture medium consisted of 3 g / L peptone, 3 g / L yeast extract, 1.5 g / L NaCl, and the remainder was water, with a pH of 7.1.

[0074] The liquid chromatography method for detecting L-histidine in the fermentation broth was as follows: C18 liquid chromatography column, single-pump isocratic elution, and ultraviolet detection.

[0075] The mobile phase was prepared by diluting 2.73 g sodium heptanesulfonate, 4.5 g ammonium sulfate, 155 μL concentrated hydrochloric acid, and 100 mL pure acetonitrile to a final volume of 1 L.

[0076] The flow rate was 0.5 mL / min, the column temperature was 30℃, the UV detection wavelength was 205 nm, and the retention time was 15 min.

[0077] Example 1

[0078] 1. Construction of recombinant plasmid pSTV28-HisG and recombinant strain E. coli DH5α-pSTV28-HisG

[0079] First, using the pSTV28 plasmid as a template, a linearized vector was obtained by reverse PCR using primer pSTV28-S / A (specific sequences shown in Table 1). Then, the HisG gene fragment (HisG wild-type enzyme, amino acid sequence as shown in SEQ ID NO:1, nucleotide sequence as shown in SEQ ID NO:8) was obtained by PCR using primer HisG-S / A (specific sequences shown in Table 1). The pSTV28 linearized vector and the HisG gene fragment were then... The IIOne Step Cloning Kit series of homologous recombinases (purchased from Nanjing Novizan Biotechnology Co., Ltd.) were used to ligate and obtain the recombinant vector pSTV28-HisG;

[0080] The PCR reaction system consisted of: a total volume of 50 μL, 19 μL of sterile water, 2 μL of upstream primer, 2 μL of downstream primer, 2 μL of template, and 25 μL of Primestar DNA Polymerase.

[0081] The PCR reaction program was as follows: 98℃ for 30s, 98℃ for 10s, 57℃ for 30s, 72℃ for 5s / kb, 72℃ for 10min, 16℃ ± ∞, 30 cycles.

[0082] The homologous recombination reaction conditions were: 37℃, 10 min; the reaction system consisted of: total volume 10 μL, 5×CE II Buffer 2 μL, linearized cloning vector 1 μL, and insert fragment 1 μL. II 1μL, ddH2O 5μL.

[0083] The recombinant vector pSTV28-HisG ligation system was transformed into E. coli DH5α by CaCl2 chemical transformation and then plated on LB agar plates containing 100 mg / L chloramphenicol. The plates were incubated upside down at 37°C overnight. After single colonies grew on the plates, colony PCR was performed using primers JD-pSTV28-S / A to identify the recombinant strain E. coli DH5α-pSTV28-HisG.

[0084] 2. Construction of E. coli HCBHIS1-pSTV28-HisG saturated mutant strain

[0085] Based on the codon preference of E. coli, the recombinant expression vector was used as the backbone of pSTV28 to verify the potential highly active HisG mutant protein. Two complementary primers were designed for each of the 19 amino acids that were changed from aspartic acid N at position 215 (the specific sequences of some primers are shown in Table 1) to construct the single-point mutant at position 215 of the HisG wild-type enzyme.

[0086] The plasmid pSTV28-HisG was extracted from the recombinant strain E. coli DH5α-pSTV28-HisG using the OMEGA DNA Extraction Kit (Omega, catalog number D3350-01). Using pSTV28-HisG as a template, a saturation mutation was performed at position 215 of the amino acid sequence of the HisG wild-type enzyme via whole-plasmid PCR to obtain a recombinant expression vector containing the ATP transphosphorylated ribosylase mutant (HisG mutant). The whole-plasmid PCR reaction system was as described above. After verification by agarose gel electrophoresis, the product was digested with restriction endonuclease Q.Cut Dpn I at 37°C for 1 h.

[0087] The digestion system consisted of: a total volume of 50 μL, 43 μL of PCR product, 5 μL of Cutsmart Buffer, and 2 μL of restriction endonuclease Q.Cut Dpn I.

[0088] After purification and recovery, the digestion products were chemically transformed into *E. coli* HCBHIS1, a strain selected through adaptive evolution to possess histidine synthesis capabilities. After single-cell colony growth on plates, colony PCR identification yielded 19 recombinant strains containing nucleotide sequences encoding different ATP transphosphorylribosylase mutants: *E. coli* HCBHIS1-pSTV28-HisG-N215A, *E. coli* HCBHIS1-pSTV28-HisG-N215C, *E. coli* HCBHIS1-pSTV28-HisG-N215D, *E. coli* HCBHIS1-pSTV28-HisG-N215E, *E. coli* HCBHIS1-pSTV28-HisG-N215F, *E. coli* HCBHIS1-pSTV28-HisG-N215G ... HCBHIS1-pSTV28-HisG-N215H, E.coli HCBHIS1-pSTV28-HisG-N215I, E.coli HCBHIS1-pSTV28-HisG-N215K, E.coli HCBHIS1-pSTV28-HisG-N215M, E.coli HCBHIS1-pSTV28-HisG-N215P, E.coli HCBHIS1-pSTV28-HisG-N215Q, E.coli HCBHIS1-pSTV28-HisG-N215R, E.coli HCBHIS1-pSTV28-HisG-N215S, E.coli HCBHIS1-pSTV28-HisG-N215T, E.coli HCBHIS1-pSTV28-HisG-N215V, E.coli HCBHIS1-pSTV28-HisG-N215W, E.coli HCBHIS1-pSTV28-HisG-N215Y.

[0089] The recombinant vector pSTV28-HisG was chemically transformed into the recombinant strain E. coli HCBHIS1, which was screened for histidine synthesis through adaptive evolution, to obtain the recombinant strain E. coli HCBHIS1-pSTV28-HisG as a control strain.

[0090] Table 1

[0091]

[0092]

[0093] Example 2

[0094] The recombinant strains obtained in Example 1 were cultured and fermented as follows.

[0095] Plate culture: Recombinant bacterial strains stored at -80℃ were streaked and inoculated onto activation plates, and cultured at 37℃ for 12 hours to obtain slant seed culture;

[0096] Test tube culture: Use an inoculation loop to scrape a loop of slanted seeds and inoculate them into a test tube containing 5 mL of seed culture medium. Culture at 37℃ and 200 rpm for 11 h to obtain primary seed culture.

[0097] 24-well plate seed culture: The primary seed culture was inoculated into a 24-well plate containing 2.7 mL of seed culture medium (final volume 3 mL) at an inoculation rate of 10% (v / v) of the seed culture medium. The plate was then shaken and cultured for 11 h at 37 °C and 200 rpm to obtain the secondary seed culture.

[0098] 24-well plate fermentation culture: The secondary seed culture was inoculated at a rate of 10% (v / v) into 24-well plates containing 2.7 mL of fermentation medium (final volume 3 mL). The plates were then cultured with shaking at 37℃ and 200 rpm for 22 h to obtain the fermentation broth. The fermentation medium consisted of: glucose 15 g / L, yeast extract 2 g / L, (NH4)2SO4 2 g / L, K2HPO4·3H2O 6 g / L, citric acid 2 g / L, methionine 2 g / L, MgSO4·7H2O 1.5 g / L, FeSO4·7H2O 18 mg / L, MnSO4·3H2O 18 mg / L, and VB1, VB3, VB5, and VB6. 12 V H Each of the following was 2 mg / L: 3-(N-morpholino)propanesulfonic acid (Mops) 35 g / L, with the remainder being water, and the pH was 7.1.

[0099] The concentration of L-histidine in the fermentation broth of each recombinant strain obtained in Example 1 was detected, and the results are shown in Table 2 and... Figure 3 As shown.

[0100] Table 2

[0101]

[0102]

[0103] From Table 2 and Figure 3The results showed that, compared with the control strain E. coli HCBHIS1-pSTV28-HisG, the L-histidine concentration in the fermentation broth of the 19 recombinant strains corresponding to the HisG mutant at position 215 was increased. In particular, the L-histidine concentration in the recombinant strain E. coli HCBHIS1-pSTV28-HisG-N215Q increased from 0.23 g / L to 1.76 g / L, and the L-histidine yield increased significantly by 6.6 times; the L-histidine concentration in E. coli HCBHIS1-pSTV28-HisG-N215F was 1.68 g / L, and the L-histidine yield increased by 6.3 times; and the L-histidine concentration in E. coli HCBHIS1-pSTV28-HisG-N215E was 1.49 g / L, and the L-histidine yield increased by 5.4 times.

[0104] It is evident that mutating the 215th amino acid residue of ATP transphosphoribosylase (encoded by the hisG gene) is beneficial to enhancing the catalytic activity of the enzyme and can significantly improve the L-histidine production performance of the strain.

[0105] Example 3

[0106] 1. Construction of recombinant plasmid pSTV28-HisG and recombinant strain E. coli DH5α-pSTV28-HisG

[0107] First, using the pSTV28 plasmid as a template, a linearized vector was obtained by reverse PCR using primer pSTV28-S / A (specific sequences shown in Table 2). Then, the HisG gene fragment (HisG wild-type enzyme, amino acid sequence as shown in SEQ ID NO:1, nucleotide sequence as shown in SEQ ID NO:8) was obtained by PCR using primer HisG-S / A (specific sequences shown in Table 2). The pSTV28 linearized vector and the HisG gene fragment were then... The IIOne Step Cloning Kit series of homologous recombinases (purchased from Nanjing Novizan Biotechnology Co., Ltd.) were used to ligate and obtain the recombinant vector pSTV28-HisG;

[0108] The PCR reaction system consisted of: a total volume of 50 μL, 19 μL of sterile water, 2 μL of upstream primer, 2 μL of downstream primer, 2 μL of template, and 25 μL of Primestar DNA Polymerase.

[0109] The PCR reaction program was as follows: 98℃ for 30s, 98℃ for 10s, 57℃ for 30s, 72℃ for 5s / kb, 72℃ for 10min, 16℃ ± ∞, 30 cycles.

[0110] The homologous recombination reaction conditions were: 37℃, 10 min; the reaction system consisted of: total volume 10 μL, 5×CE II Buffer 2 μL, linearized cloning vector 1 μL, and insert fragment 1 μL. II 1μL, ddH2O 5μL.

[0111] The recombinant vector pSTV28-HisG ligation system was transformed into E. coli DH5α by CaCl2 chemical transformation. The transformed strain was then plated on LB agar plates containing 100 mg / L chloramphenicol and incubated upside down at 37°C overnight. After single colonies grew on the plates, colony PCR was performed using primers JD-pSTV28-S / A (specific sequences are shown in Table 2) to identify the recombinant strain E. coli DH5α-pSTV28-HisG.

[0112] 2. Construction of E. coli HCBHIS1-pSTV28-HisG saturated mutant strain

[0113] Based on the codon preference of E. coli, the recombinant expression vector was used as the backbone of pSTV28 to verify the potential highly active HisG mutant protein. Two complementary primers were designed for each of the 18 other amino acids when the tyrosine Y at position 214 was mutated (the specific sequences of some primers are shown in Table 3) to construct the single-point mutant at position 214 of the HisG wild-type enzyme.

[0114] The plasmid pSTV28-HisG was extracted from the recombinant strain E. coli DH5α-pSTV28-HisG using the OMEGA DNA Extraction Kit (Omega, catalog number D3350-01). Using pSTV28-HisG as a template, a saturation mutation was performed at position 214 of the amino acid sequence of the HisG wild-type enzyme via whole-plasmid PCR to obtain a recombinant expression vector containing the ATP transphosphorylated ribosylase mutant (HisG mutant). The whole-plasmid PCR reaction system was as described above. After verification by agarose gel electrophoresis, the product was digested with restriction endonuclease Q.Cut Dpn I at 37°C for 1 h.

[0115] The digestion system consisted of: a total volume of 50 μL, 43 μL of PCR product, 5 μL of Cutsmart Buffer, and 2 μL of restriction endonuclease Q.Cut Dpn I.

[0116] After purification and recovery, the digestion products were chemically transformed into *E. coli* HCBHIS1, a strain selected through adaptive evolution to possess histidine synthesis capabilities. After single-cell colony growth on plates, colony PCR identification yielded 18 recombinant strains containing nucleotide sequences encoding different ATP-transferase mutants: *E. coli* HCBHIS1-pSTV28-HisG-Y214A, *E. coli* HCBHIS1-pSTV28-HisG-Y214C, *E. coli* HCBHIS1-pSTV28-HisG-Y214D, *E. coli* HCBHIS1-pSTV28-HisG-Y214E, *E. coli* HCBHIS1-pSTV28-HisG-Y214F, *E. coli* HCBHIS1-pSTV28-HisG-Y214G ...G, *E. coli* HCBHI HCBHIS1-pSTV28-HisG-Y214H, E.coli HCBHIS1-pSTV28-HisG-Y214K, E.coli HCBHIS1-pSTV28-HisG-Y214L, E.coli HCBHIS1-pSTV28-HisG-Y214N, E.coli HCBHIS1-pSTV28-HisG-Y214P, E.coli HCBHIS1-pSTV28-HisG-Y214Q, E.coli HCBHIS1-pSTV28-HisG-Y214R, E.coli HCBHIS1-pSTV28-HisG-Y214S, E.coli HCBHIS1-pSTV28-HisG-Y214T, E. coli HCBHIS1-pSTV28-HisG-Y214V, E. coli HCBHIS1-pSTV28-HisG-Y214W.

[0117] The recombinant vector pSTV28-HisG was chemically transformed into the recombinant strain E. coli HCBHIS1, which was screened for histidine synthesis through adaptive evolution, to obtain the recombinant strain E. coli HCBHIS1-pSTV28-HisG as a control strain.

[0118] Table 3

[0119] Y214D-S GGATGATAACGTCGACCGCGACAACCTGGACGC SEQ ID NO:27 Y214D-A GTCGACGTTATCATCCAGCATGAGGAAGTTCTGCGC SEQ ID NO:28 Y214F-S GGATTTTAACGTCGACCGCGACAACCTGGACGC SEQ ID NO:29 Y214F-A GTCGACGTTAAAATCCAGCATGAGGAAGTTCTGCGC SEQ ID NO:30 Y214W-S GGATTGGAACGTCGACCGCGACAACCTGGACGC SEQ ID NO:31 Y214W-A GTCGACGTTCCAATCCAGCATGAGGAAGTTCTGCGC SEQ ID NO:32

[0120] Example 4

[0121] The recombinant strains obtained in Example 3 were cultured and fermented as follows.

[0122] Plate culture: Recombinant bacterial strains stored at -80℃ were streaked and inoculated onto activation plates, and cultured at 37℃ for 12 hours to obtain slant seed culture;

[0123] Test tube culture: Use an inoculation loop to scrape a loop of slanted seeds and inoculate them into a test tube containing 5 mL of seed culture medium. Culture at 37℃ and 200 rpm for 11 h to obtain primary seed culture.

[0124] 24-well plate seed culture: The primary seed culture was inoculated into a 24-well plate containing 2.7 mL of seed culture medium (final volume 3 mL) at an inoculation rate of 10% (v / v) of the seed culture medium. The plate was then shaken and cultured for 11 h at 37 °C and 200 rpm to obtain the secondary seed culture.

[0125] 24-well plate fermentation culture: The secondary seed culture was inoculated at a rate of 10% (v / v) into 24-well plates containing 2.7 mL of fermentation medium (final volume 3 mL). The plates were then cultured with shaking at 37℃ and 200 rpm for 22 h to obtain the fermentation broth. The fermentation medium consisted of: glucose 15 g / L, yeast extract 2 g / L, (NH4)2SO4 2 g / L, K2HPO4·3H2O 6 g / L, citric acid 2 g / L, methionine 2 g / L, MgSO4·7H2O 1.5 g / L, FeSO4·7H2O 18 mg / L, MnSO4·3H2O 18 mg / L, and VB1, VB3, VB5, and VB6. 12 V H Each of the following was 2 mg / L: 3-(N-morpholino)propanesulfonic acid (Mops) 35 g / L, with the remainder being water, and the pH was 7.1.

[0126] The L-histidine content in the fermentation broth of each recombinant strain obtained in Example 3 was detected, and the results are shown in Table 4. Figure 4 As shown.

[0127] Table 4

[0128]

[0129] From Table 4 and Figure 4The results showed that, compared with the control strain E. coli HCBHIS1-pSTV28-HisG, the L-histidine concentration in the fermentation broth of the 18 recombinant strains corresponding to the HisG mutant at position 214 was increased. In particular, the L-histidine concentration in the recombinant strain E. coli HCBHIS1-pSTV28-HisG-Y214D increased from 0.23 g / L to 0.57 g / L, and the L-histidine yield increased by 1.4 times. In the recombinant strain E. coli HCBHIS1-pSTV28-HisG-Y214F, the L-histidine concentration was 0.46 g / L, and the L-histidine yield increased by 1 time.

[0130] It is evident that mutating the 214th amino acid residue of ATP transphosphoribosylase (encoded by the hisG gene) can also enhance the catalytic activity of the enzyme in the presence of histidine feedback inhibition, thereby improving the L-histidine production performance of the strain.

[0131] Example 5

[0132] The recombinant strains E. coli HCBHIS1-pSTV28-HisG-N215Q, E. coli HCBHIS1-pSTV28-HisG-N215F, and E. coli HCBHIS1-pSTV28-HisG-N215E obtained in Example 1 were subjected to the following fermentation cultures:

[0133] Plate culture: Recombinant bacterial strains stored at -80℃ were streaked and inoculated onto activation plates, and cultured at 37℃ for 12 hours to obtain slant seed culture;

[0134] Test tube culture: Use an inoculation loop to scrape a loop of slanted seeds and inoculate them into a test tube containing 5 mL of seed culture medium. Culture at 37℃ and 200 rpm for 11 h to obtain primary seed culture.

[0135] 24-well plate seed culture: The primary seed culture was inoculated into a 24-well plate containing 2.7 mL of seed culture medium (final volume 3 mL) at an inoculation rate of 10% (v / v) of the seed culture medium. The plate was then shaken and cultured for 11 h at 37 °C and 200 rpm to obtain the secondary seed culture.

[0136] 24-well plate fermentation culture: The secondary seed culture was inoculated at a rate of 10% (v / v) into 24-well plates containing 2.7 mL of fermentation medium (final volume 3 mL). The plates were then cultured with shaking at 37℃ and 200 rpm for 22 h to obtain the fermentation broth. The fermentation medium consisted of: glucose 20 g / L, yeast extract 3 g / L, (NH4)2SO4 1 g / L, K2HPO4·3H2O 4 g / L, citric acid 1 g / L, methionine 3 g / L, MgSO4·7H2O 1 g / L, FeSO4·7H2O 15 mg / L, MnSO4·3H2O 20 mg / L, and VB1, VB3, VB5, and VB6. 12 V H Each of the following was 3 mg / L: 3-(N-morpholino)propanesulfonic acid (Mops) 30 g / L, with the remainder being water, and the pH was 7.2.

[0137] The concentration of L-histidine in the fermentation broth of each recombinant strain was measured, and the results are shown in Table 5.

[0138] Example 6

[0139] The recombinant strains E. coli HCBHIS1-pSTV28-HisG-N215Q, E. coli HCBHIS1-pSTV28-HisG-N215F, and E. coli HCBHIS1-pSTV28-HisG-N215E were fermented according to the method in Example 5, except that the fermentation medium was replaced with: glucose 10 g / L, yeast extract 1 g / L, (NH4)2SO4 3 g / L, K2HPO4·3H2O 8 g / L, citric acid 3 g / L, methionine 1 g / L, MgSO4·7H2O 2 g / L, FeSO4·7H2O 20 mg / L, MnSO4·3H2O 15 mg / L, VB1, VB3, VB5, and VB6. 12 V H Each of the following was 1 mg / L: 3-(N-morpholino)propanesulfonic acid (Mops) 40 g / L, with the remainder being water, and the pH being 7.0.

[0140] The concentration of L-histidine in the fermentation broth of each recombinant strain was measured, and the results are shown in Table 5.

[0141] Example 7

[0142] The recombinant strains E. coli HCBHIS1-pSTV28-HisG-N215Q, E. coli HCBHIS1-pSTV28-HisG-N215F, and E. coli HCBHIS1-pSTV28-HisG-N215E were fermented according to the method in Example 5, except that the fermentation medium was replaced with: glucose 40 g / L, yeast extract 5 g / L, (NH4)2SO4 3 g / L, K2HPO4·3H2O 8 g / L, citric acid 3 g / L, methionine 1 g / L, MgSO4·7H2O 2 g / L, FeSO4·7H2O 20 mg / L, MnSO4·3H2O 15 mg / L, VB1, VB3, VB5, and VB6. 12 V H Each of the following was 1 mg / L: 3-(N-morpholino)propanesulfonic acid (Mops) 10 g / L, with the remainder being water, and the pH being 7.0.

[0143] The concentration of L-histidine in the fermentation broth of each recombinant strain was measured, and the results are shown in Table 5.

[0144] Example 8

[0145] The recombinant strains E. coli HCBHIS1-pSTV28-HisG-N215Q, E. coli HCBHIS1-pSTV28-HisG-N215F, and E. coli HCBHIS1-pSTV28-HisG-N215E were fermented according to the method in Example 5, except that the fermentation medium was replaced with: glucose 20 g / L, yeast extract 3 g / L, (NH4)2SO4 1 g / L, K2HPO4·3H2O 4 g / L, MgSO4·7H2O 1 g / L, FeSO4·7H2O 15 mg / L, MnSO4·3H2O 20 mg / L, with the remainder being water, and the pH being 7.2.

[0146] The concentration of L-histidine in the fermentation broth of each recombinant strain was measured, and the results are shown in Table 5.

[0147] Table 5

[0148] E.coli HCBHIS1-pSTV28-HisG-N215Q 1.93g / L 1.73g / L 1.34g / L 0.98g / L E.coli HCBHIS1-pSTV28-HisG-N215F 1.74g / L 1.59g / L 1.26g / L 0.91g / L E.coli HCBHIS1-pSTV28-HisG-N215E 1.71g / L 1.37g / L 1.14g / L 0.85g / L

[0149] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combining the various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. An ATP transphosphorylribosylase mutant, wherein, The mutant is N215E, which is a mutant in which asparagine N is mutated to glutamic acid E at position 215, and its amino acid sequence is shown in SEQ ID NO:2; Alternatively, the mutant is a mutant N215F in which asparagine N at position 215 is mutated to phenylalanine F, and its amino acid sequence is shown in SEQ ID NO:3; Alternatively, the mutant is a mutant N215Q in which asparagine N at position 215 is mutated to glutamine Q, and its amino acid sequence is shown in SEQ ID NO:

4.

2. A gene encoding a gene, wherein, The encoding gene can encode the ATP transphosphoribosylase mutant of claim 1.

3. The encoding gene according to claim 2, wherein, The nucleotide sequence of the encoding gene is shown in SEQ ID NO:6; Alternatively, the nucleotide sequence of the encoding gene is shown in SEQ ID NO:

7.

4. A recombinant vector, wherein, The recombinant vector contains the coding gene as described in claim 2 or 3.

5. The recombinant vector according to claim 4, wherein, The expression vector for the recombinant vector is the pSTV28 plasmid.

6. A recombinant cell, wherein, The recombinant cells contain the coding gene as described in claim 2 or 3, or the recombinant vector as described in claim 4 or 5.

7. The recombinant cell according to claim 6, wherein, The host strain of the recombinant cells is a prokaryotic cell.

8. The recombinant cell according to claim 7, wherein, The host strain of the recombinant cells is Escherichia coli.

9. The recombinant cell according to claim 8, wherein, The host strain of the recombinant cells was Escherichia coli HCBHIS1.

10. The use of at least one of the ATP transphosphoribosylase mutant of claim 1, the encoding gene of claim 2 or 3, the recombinant vector of claim 4 or 5, and the recombinant cell of any one of claims 6-9 in the production of histidine.

11. A method for producing histidine, wherein, The method includes the following steps: culturing the recombinant cells according to any one of claims 6-9.

12. The method according to claim 11, wherein, The culture medium used in the culture contains: glucose 10-20 g / L, yeast extract 1-3 g / L, ammonium salt 1-3 g / L, potassium salt 4-8 g / L, magnesium salt 1-2 g / L, iron salt 15-20 mg / L, manganese salt 15-20 mg / L, citric acid 1-3 g / L, methionine 1-3 g / L, vitamin B1, vitamin B3, vitamin B5, and vitamin B6. 12 Vitamin H 1-3 mg / L, 3-(N-morpholino)propanesulfonic acid 30-40 g / L; the pH of the culture medium is 7.0-7.2; And / or, the culture conditions include at least: a temperature of 30-45°C and a rotation speed of 180-220 rpm.

Images