ATP Phosphoribosyltransferase Mutant and Its Application in Histidine Production

Abstract

The present invention provides an ATP phosphoribosyltransferase mutant and its application in histidine production. The mutant is obtained by performing point mutations at positions 84 and / or 205 on the amino acid sequence of the wild-type ATP phosphoribosyltransferase derived from Acinetobacter baumannii. The mutant, the nucleic acid molecule encoding the mutant, the expression cassette containing the nucleic acid molecule, the recombinant vector or the recombinant microorganism are used for the production of L-histidine, and the yield of L-histidine is significantly increased, laying a good foundation for the large-scale industrial production of L-histidine.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to an ATP phosphoribosyltransferase mutant and its application in histidine production. Background Art

[0002] L-Histidine is one of the 20 amino acids that make up proteins. Infants and young children cannot synthesize it autonomously and need to obtain it from food, belonging to semi-essential amino acids. L-Histidine can act as both a proton donor and a proton acceptor, and is often located in the active center of biological enzymes, participating in important physiological processes, such as antioxidant, vasodilation, improving gastric function, alleviating rheumatoid arthritis symptoms, etc. Therefore, it is widely used in the fields of food, medicine, feed, etc.

[0003] At present, there are mainly two methods for the preparation of L-histidine: in China, it is mainly prepared by protein hydrolysis method (see Chinese Journal of Biochemical Drugs, 1982(02):12 - 17; CN101125831B); abroad, it is mainly prepared by fermentation method using Corynebacterium glutamicum or Escherichia coli mutants to obtain high-yield L-histidine strains (US8071339; CA02319283; CN1749390A; CN102286562A).

[0004] In the L-histidine biosynthesis pathway of Escherichia coli, a key enzyme - ATP phosphoribosyltransferase (HisG) is inhibited by different intermediate metabolites and end products, especially the end product L-histidine. Eliminating the feedback inhibition of HisG is an important step in histidine breeding strains. ZL201810118782.7 discloses a recombinant bacterium for producing L-histidine. This strain uses Corynebacterium glutamicum as the starting bacterium, and improves the histidine synthesis ability of the strain by increasing the expression of adenylosuccinate synthase PurA, weakening the expression of 5'-nucleotidase UshA, and increasing the expression of ATP phosphoribosyl kinase HisG (by amino acid mutations N215R / G233H / T235Q of HisG from Corynebacterium glutamicum), but the effect of only mutating HisG is unknown; Doroshenko et al. weakened the feedback inhibition of L-histidine on HisG by amino acid mutation E271K of HisG, but the weakening degree is limited, and the L-histidine yield only reaches 0.2 ± 0.1 g / L. Therefore, in order to realize the industrial application of microbial fermentation production of L-histidine, a more suitable mutation method for ATP phosphoribosyltransferase HisG needs to be found to improve its ability to resist histidine feedback inhibition, and then improve the L-histidine fermentation yield of the engineering bacterium. Summary of the Invention

[0005] The object of the present invention is to provide an ATP phosphoribosyltransferase mutant by studying the structure of the HisG gene derived from Acinetobacter baumannii, introducing mutation sites into HisG, and expressing the mutant in a host bacterium to improve the L-histidine fermentation yield of the strain.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides an ATP phosphoribosyltransferase mutant, which is a point mutation including the 84th and / or 205th positions based on the amino acid sequence of the ATP phosphoribosyltransferase shown in SEQ ID NO:1.

[0008] In one embodiment of the present invention, the ATP phosphoribosyltransferase mutant is any one of the following:

[0009] (1) Mutating the aspartic acid at the 84th position of the ATP phosphoribosyltransferase sequence shown in SEQ ID NO:1 to alanine, and the mutated sequence is as shown in SEQ ID NO:2;

[0010] (2) Mutating the serine at the 205th position of the ATP phosphoribosyltransferase sequence shown in SEQ ID NO:1 to histidine, and the mutated sequence is as shown in SEQ ID NO:3;

[0011] (3) Mutating the aspartic acid at the 84th position of the ATP phosphoribosyltransferase sequence shown in SEQ ID NO:1 to alanine and mutating the serine at the 205th position to histidine, and the mutated sequence is as shown in SEQ ID NO:4.

[0012] In a second aspect, the present invention provides a nucleic acid molecule encoding the above ATP phosphoribosyltransferase mutant.

[0013] In one embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

[0014] In a third aspect, the present invention provides an expression cassette containing the above nucleic acid molecule.

[0015] In a fourth aspect, the present invention provides a recombinant vector containing the above nucleic acid molecule.

[0016] In a fifth aspect, the present invention provides a recombinant microorganism expressing the above ATP phosphoribosyltransferase mutant.

[0017] In one embodiment of the present invention, the recombinant microorganism contains the above nucleic acid molecule, expression cassette, or recombinant vector.

[0018] In a sixth aspect, the present invention provides the use of the above-mentioned ATP phosphoribosyltransferase mutant in any one of the following A2)-A3);

[0019] or, the use of the above-mentioned nucleic acid molecule in any one of the following A1)-A3);

[0020] or, the use of the above-mentioned expression cassette in any one of the following A1)-A3);

[0021] or, the use of the above-mentioned recombinant vector in any one of the following A1)-A3);

[0022] or, the use of the above-mentioned recombinant microorganism in any one of the following A1)-A3);

[0023] A1) preparing ATP phosphoribosyltransferase;

[0024] A2) producing histidine, preferably L-histidine;

[0025] A3) increasing the yield of histidine, preferably L-histidine.

[0026] In a seventh aspect, a method for producing L-histidine is characterized by using the above-mentioned recombinant microorganism to ferment and produce L-histidine.

[0027] Compared with the prior art, the present invention has the following beneficial technical effects: based on the wild-type ATP phosphoribosyltransferase, the present invention obtains a recombinant bacterium containing the ATP phosphoribosyltransferase derived from Acinetobacter baumannii through exogenous introduction and replacement, improving its L-histidine acid production ability; and further obtaining a mutant of the ATP phosphoribosyltransferase derived from Acinetobacter baumannii through site-directed mutagenesis technology, using it to construct a recombinant bacterium, and using the recombinant engineering bacterium expressing the ATP phosphoribosyltransferase mutant for fermentative production of L-histidine, and the yield of L-histidine is increased by 6.5 times, having broad prospects for industrial development and application. Description of the Drawings

[0028] Figure 1 is the molecular docking diagram of HisG-Ab with L-histidine as the substrate in AutoDock Vina. Detailed Embodiments

[0029] The present invention will be further described below in conjunction with specific embodiments, but any embodiment or its combination should not be construed as a limitation on the protection scope or implementation manner of the present invention. Any method transformation made by those of ordinary skill in the art according to these implementation manners is included in the protection scope of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, such as those described in "Molecular Cloning: A Laboratory Manual" (J. Sambrook, D.W. Russell, translated by Huang Peitang, Wang Jiaxi, Zhu Houchu, etc. 3rd Edition, Beijing: Science Press, 2002); the reagents and materials used, unless otherwise specified, can be obtained from commercial sources.

[0031] The information on the culture media and reagents involved in the following examples is as follows:

[0032] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, with deionized water as the solvent, pH 7;

[0033] LB plate: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar powder, with deionized water as the solvent, pH 7;

[0034] Seed medium: 20 g / L glucose, 4 g / L yeast extract, 3 g / L peptone, 1.2 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 10 mg / L ferrous sulfate heptahydrate, 10 mg / L manganese sulfate monohydrate, 1 mg / L vitamin B1, 1 mg / L vitamin B3, 1 mg / L vitamin B5, 1 mg / L vitamin B12, 1 mg / L vitamin H, adjusted to pH 7.0 - 7.2 with sodium hydroxide;

[0035] Fermentation medium: 20 g / L glucose, 4 g / L yeast extract, 3 g / L peptone, 2 g / L sodium citrate monohydrate, 2 g / L potassium dihydrogen phosphate, 2 g / L magnesium sulfate heptahydrate, 20 mg / L ferrous sulfate heptahydrate, 20 mg / L manganese sulfate monohydrate, 2 mg / L vitamin B1, 2 mg / L vitamin B3, 2 mg / L vitamin B5, 2 mg / L vitamin B12, and 2 mg / L vitamin H, adjusted to pH 7.0 - 7.2 with sodium hydroxide; Ammonia water is added every 4 h during fermentation to adjust the pH to 7.2 - 7.4.

[0036] Reagents: Phusion 5X buffer was purchased from NEB; Phusion TM High-fidelity DNA polymerase was purchased from Thermo Scientific TM .

[0037] The information on the strains, plasmids and primers involved in the following examples is as follows:

[0038] Table 1 Strains and plasmids used in the present invention

[0039]

[0040]

[0041] Table 2 Primers used in the present invention

[0042]

[0043]

[0044] Example 1: Bioinformatics analysis of HisG gene structure - AutoDock Vina molecular docking

[0045] ATP phosphoribosyltransferase (HisG) uses L-histidine as a substrate for molecular docking in AutoDock Vina to observe their interactions. As Figure 1 shown, L-histidine forms hydrogen bonds with the S205 and D84 residues of HisG, and the above residues are key residues of the active pocket. Therefore, site-directed mutagenesis of the above key residues was selected.

[0046] Example 2: Construction of recombinant bacterium HIS002 containing HisG-Ab

[0047] Starting from Escherichia coli W3110, the original ATP phosphoribosyltransferase gene HisG in Escherichia coli W3110 was knocked out by a two-step homologous recombination method, and the HisG gene derived from Acinetobacter baumannii was inserted. The specific steps are as follows:

[0048] In the first step, using pRE112 plasmid DNA as a template, a 3593 bp DNA fragment I was amplified using primers HisG-cs-up / HisG-cs-down for the first-step homologous recombination.

[0049] The amplification system was: 10 μl of Phusion 5X buffer (New England Biolabs), 1 μl of dNTP (10 mM each of each dNTP), 20 ng of DNA template, 2 μl each of primers (10 μM), Phusion TM high-fidelity DNA polymerase (2.5 U / μl, purchased from Thermo Scientific TM , catalog number: F530S) 0.5 μl, and made up to 50 μl with distilled water.

[0050] The amplification conditions were: pre-denaturation at 98 °C for 2 minutes (1 cycle); denaturation at 98 °C for 10 seconds, annealing at 56 °C for 10 seconds, extension at 72 °C for 4 minutes (30 cycles); extension at 72 °C for 10 minutes (1 cycle).

[0051] Use the above DNA fragment I for the first homologous recombination: First, transform the pKD46 plasmid into Escherichia coli W3110 by electroporation, and then electroporate DNA fragment I into Escherichia coli W3110 carrying pKD46 to obtain the recombinant strain W3110-pKD46.

[0052] Prepare the recombinant strain W3110-pKD46 into competent cells by the CaCl2 method; then place 50 μl of W3110-pKD46 competent cells on ice, add 50 ng of DNA fragment I, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporator with an electroporation parameter of 2.5 kV voltage. Immediately after electroporation, transfer 1 ml of LB liquid medium to the electroporation cuvette, pipette 5 times and then transfer to a test tube, incubate at 75 rpm and 30 °C for 2 hours. Take 200 μl of the bacterial solution and spread it on an LB plate containing ampicillin (final concentration 100 μg / ml) and chloramphenicol (final concentration 34 μg / ml), culture overnight at 30 °C, pick single colonies for PCR verification, using primers XZ-HisG-up / XZ-HisG-down. The correct colony amplification product is a 3715 bp fragment. Pick one correct single colony and name it HIS001.

[0053] In the second step, use the plasmid PUC19-HisG synthesized by Synbio Technologies as a template, and amplify an 812 bp DNA fragment II with primers HisG-mut-cs-up / HisG-mut-cs-down. The amplification system is the same as that in the first step. DNA fragment II is used for the second homologous recombination.

[0054] The pKD46 plasmid was transformed into HIS001 (HIS001-pKD46) by electroporation, and competent cells were prepared by the CaCl2 method. 50 μl of HIS001-pKD46 competent cells were placed on ice, 50 ng of DNA fragment II was added, and the mixture was placed on ice for 2 minutes and then transferred to a 0.2 cm Bio-Rad electroporation cuvette. Using a MicroPulser (Bio-Rad) electroporator, the electroporation parameters were a voltage of 2.5 kV. Immediately after electroporation, 1 ml of LB liquid medium was transferred to the electroporation cuvette, pipetted 5 times, and then transferred to a test tube. The cells were incubated at 75 rpm and 30 °C for 4 hours. The bacterial solution was transferred to LB liquid medium without sodium chloride containing 10% sucrose (50 ml of medium in a 250 ml flask), and after culturing for 24 hours, it was streaked on an LB plate 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 correct single colony was selected to obtain a recombinant bacterium containing the HisG gene of Acinetobacter baumannii-derived ATP phosphoribosyltransferase, which was named HIS002.

[0055] Example 3: Construction of the recombinant bacterium HIS003 containing HisG-Ab-D84A

[0056] Starting from HIS001-pKD46, the original ATP phosphoribosyltransferase gene HisG in Escherichia coli W3110 was knocked out by a two-step homologous recombination method, and the HisG-Ab-D84A gene from Acinetobacter baumannii was inserted. The specific steps are as follows:

[0057] In the first step, using the plasmid PUC19-HisG-D84A synthesized by Synbio Technologies as a template, a 812 bp DNA fragment II was amplified using the primers HisG-mut-cs-up / HisG-mut-cs-down. The amplification system and conditions were the same as in Example 1. The DNA fragment II was used for the second homologous recombination.

[0058] Place 50 μl of HIS001-pKD46 competent cells (prepared according to the protocol in Example 1) on ice, add 50 ng of the above DNA fragment II, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporator with an electroporation parameter of 2.5 kV voltage. Immediately after electroporation, transfer 1 ml of LB liquid medium to the electroporation cuvette, pipette 5 times and then transfer to a test tube, incubate at 75 rpm and 30 °C for 4 hours. Transfer the bacterial solution to an LB liquid medium without sodium chloride containing 10% sucrose, culture for 24 hours and then streak on an LB plate 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. Select a correct single colony to obtain a recombinant bacterium containing the mutant gene HisG-Ab-D84A of ATP phosphoribosyltransferase from Acinetobacter baumannii, and name it HIS003.

[0059] Example 4: Construction of recombinant bacterium HIS004 containing HisG-Ab-S205H

[0060] Starting from HIS001-pKD46, the ATP phosphoribosyltransferase gene HisG was knocked out by a two-step homologous recombination method, and the HisG-Ab-S205H gene from Acinetobacter baumannii was inserted. The specific steps are as follows:

[0061] In the first step, using the plasmid PUC19-HisG-S205H synthesized by Synbio Technologies as a template, a DNA fragment II of 812 bp was amplified using primers HisG-mut-cs-up / HisG-mut-cs-down. The amplification system and conditions were the same as in Example 1. The DNA fragment II was used for the second homologous recombination.

[0062] Place 50 μl of HIS001-pKD46 competent cells (prepared according to the same protocol as in Example 1) on ice, add 50 ng of the above DNA fragment II, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporator with an electroporation parameter of 2.5 kV voltage. Immediately after electroporation, transfer 1 ml of LB liquid medium to the electroporation cuvette, pipette 5 times and then transfer to a test tube, incubate at 75 rpm and 30 °C for 4 hours. Transfer the bacterial solution to LB liquid medium without sodium chloride containing 10% sucrose, culture for 24 hours and then streak on an LB plate containing 6% sucrose and no sodium chloride. After PCR verification, the primers used are XZ-HisG-up / XZ-HisG-down, and the correct colony amplification product is a 930 bp fragment. Select a correct single colony to obtain a recombinant bacterium containing the mutant gene HisG-Ab-S205H of ATP phosphoribosyltransferase from Acinetobacter baumannii, and name it HIS003.

[0063] Example 5: Construction of recombinant bacterium HIS004 containing HisG-Ab-D84A-S205H

[0064] Starting from HIS001-pKD46, the ATP phosphoribosyltransferase gene HisG was knocked out by a two-step homologous recombination method, and the HisG-Ab-D84A-S205H gene from Acinetobacter baumannii was inserted. The specific steps are as follows:

[0065] In the first step, using the plasmid PUC19-HisG-D84A-S205H synthesized by Hongxun Biotech as a template, a 812 bp DNA fragment II was amplified using primers HisG-mut-cs-up / HisG-mut-cs-down. The amplification system and conditions are the same as in Example 1. The DNA fragment II is used for the second homologous recombination.

[0066] Place 50 μl of HIS001-pKD46 competent cells (prepared according to the protocol in Example 1) on ice, add 50 ng of the above DNA fragment II, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporator with an electroporation parameter of 2.5 kV voltage. Immediately after electroporation, transfer 1 ml of LB liquid medium to the electroporation cuvette, pipette 5 times and then transfer to a test tube, incubate at 75 rpm and 30 °C for 4 hours. Transfer the bacterial solution to LB liquid medium without sodium chloride containing 10% sucrose, culture for 24 hours and then streak on an LB plate containing 6% sucrose and no sodium chloride. After PCR verification, the primers used are XZ-HisG-up / XZ-HisG-down, and the correct colony amplification product is a 930 bp fragment. Select a correct single colony to obtain a recombinant bacterium containing the mutant gene HisG-Ab-D84A-S205H of ATP phosphoribosyltransferase from Acinetobacter baumannii, and name it HIS005.

[0067] Example 6: Fermentation production of L-histidine by recombinant strains

[0068] Use Escherichia coli W3110, recombinant bacteria HIS002, HIS003, HIS004, HIS005 to ferment and produce L-histidine, including the following steps:

[0069] (1) Seed culture: Inoculate a fresh recombinant bacterial monoclonal on an LB plate into a test tube containing 4 ml of seed medium, and culture overnight at 37 °C with shaking at 250 rpm. Then, transfer the culture to a 250 ml Erlenmeyer flask containing 30 ml of seed medium at an inoculation amount of 2% (V / V), and culture with shaking at 37 °C and 250 rpm for 12 hours to obtain a seed culture solution for inoculating the fermentation medium.

[0070] (2) Fermentation culture: Load 250 ml of fermentation medium into a 500 ml fermenter, inoculate the seed culture solution into the fermentation medium at an inoculation amount with a final concentration of OD550 = 0.1, ferment at 37 °C and 150 rpm for 3 days to obtain a fermentation broth. The neutralizing agent is 5 M ammonia water to control the pH of the fermenter at 7.0. Keep the dissolved oxygen at 25 - 35% during the culture process.

[0071] Analysis method: Use an Agilent (Agilent-1260) high performance liquid chromatograph to measure the components in the fermentation broth after 3 days of fermentation. For L-histidine determination, use an amino acid analysis column ZORBAX Eclipse AAA 4.6 mm × 75 mm 3.5-Micron. Detection wavelength: 338 nm.

[0072] The experiment was repeated three times, and the average value of the analysis results is presented in Table 1 below.

[0073] Table 1 Comparison of L-histidine production capacity

[0074] Strain Characteristic L-Histidine Concentration (g / L) W3110 W3110 (containing wild-type HisG gene) 0.01 HIS002 W3110ΔHisG::HisG-Ab 0.15 HIS003 W3110ΔHisG::HisG-Ab-D84A 0.27 HIS004 W3110ΔHisG::HisG-Ab-S205H 0.31 HIS005 W3110ΔHisG::HisG-Ab-D84A-S205H 1.12

[0075] As shown in Table 1, Escherichia coli W3110 has poor ability to ferment L-histidine. By exogenously introducing the wild-type HisG-Ab (HIS002) from Acinetobacter baumannii, the L-histidine fermentation level of the strain is greatly improved; compared with the HIS002 strain, the strains HIS003 (HisG-Ab-D84A), HIS004 (HisG-Ab-S205H), and HIS005 (HisG-Ab-D84A-S205H) containing the HisG-Ab mutant all have increased histidine production rates. In particular, the HIS005 strain containing the HisG-Ab-D84A-S205H mutant has a 6.5-fold increase in the L-histidine production level; it is speculated that introducing the D84A and / or S205H mutations into HisG-Ab relieves the feedback inhibition of L-histidine on HisG.

[0076] The above are only the preferred embodiments of the present invention. The protection scope of the present invention is not limited to the above embodiments. All technical solutions within the concept of the present invention belong to the protection scope of the present invention. It should be noted that for those of ordinary skill in the art, several improvements and refinements made without departing from the principle of the present invention should be regarded as within the protection scope of the present invention.

Claims

1. An ATP phosphoribosyltransferase mutant, characterized in that The ATP phosphoribosyltransferase mutant is any one of the following: (1) mutating the aspartic acid at position 84 of the ATP phosphoribosyltransferase sequence shown in SEQ ID NO: 1 to alanine; (2) mutating the serine at position 205 of the ATP phosphoribosyltransferase sequence shown in SEQ ID NO: 1 to histidine; (3) The aspartic acid at position 84 of the ATP phosphoribosyltransferase sequence shown in SEQ ID NO: 1 was mutated to alanine, and the serine at position 205 was mutated to histidine.

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

3. An expression cassette containing the nucleic acid molecule according to claim 2.

4. A recombinant vector containing the nucleic acid molecule according to claim 2.

5. A recombinant microorganism expressing the ATP phosphoribosyltransferase mutant of claim 1.

6. The recombinant microorganism according to claim 5, characterized in that The recombinant microorganism contains the nucleic acid molecule according to claim 2, the expression cassette according to claim 3, or the recombinant vector according to claim 4.

7. Use of the ATP phosphoribosyltransferase mutant according to claim 1 in any one of A1) to A2); Or, use of the nucleic acid molecule according to claim 2 in any one of A1)-A2); Or, use of the expression cassette of claim 3 in any one of A1)-A2); Or, use of the recombinant vector according to claim 4 in any one of A1)-A2); Or, use of the recombinant microorganism according to any one of claims 5 to 6 in any one of A1) to A2); A1) production of histidine; A2) Increase histidine production.

8. A method for producing L-histidine, characterized in that: L-histidine is produced by fermentation using the recombinant microorganism described in claim 5 or 6.

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