ATP (adenosine triphosphate) ribosyltransferase mutant and application thereof in histidine production

Abstract

The invention provides an ATP (adenosine triphosphate) ribosyltransferase mutant and application thereof in histidine production. The mutant is obtained by performing point mutation including 84th site and / or 205th site on an amino acid sequence of wild ATP ribosyltransferase derived from acinetobacter baumannii; when 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 producing the L-histidine, the yield of the L-histidine is remarkably increased, and a good foundation is laid for large-scale industrial production of the L-histidine.

Description

Technical Field

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

[0002] L-histidine is one of the 20 amino acids that make up protein. Infants and young children cannot synthesize it independently and need to obtain it from food. It is a semi-essential amino acid. L-histidine can act as both a proton donor and a proton acceptor. It is often located in the active center of biological enzymes and participates in important physiological processes, such as anti-oxidation, vasodilation, improvement of gastric function, and relief of rheumatoid arthritis symptoms. Therefore, it is widely used in food, medicine, feed and other fields.

[0003] At present, there are two main methods for preparing L-histidine: domestically, it is mainly prepared by protein hydrolysis (see Chinese Journal of Biochemical Pharmacology, 1982(02):12-17; CN101125831B); abroad, it is mainly prepared by fermentation using high-yield L-histidine strains obtained by mutagenesis of Corynebacterium glutamicum or Escherichia coli (US8071339; CA02319283; CN1749390A; CN102286562A).

[0004] In the L-histidine biosynthesis pathway of Escherichia coli, a key enzyme, ATP phosphoribosyltransferase (HisG), is inhibited by various intermediate metabolites and end products, especially the end product L-histidine. Eliminating the feedback inhibition of HisG is an important step in histidine breeding. ZL201810118782.7 discloses a recombinant bacterium for producing L-histidine, which uses Corynebacterium glutamicum as the starting bacterium, and improves the histidine synthesis ability of the strain by increasing the expression of adenylosuccinate synthetase PurA, reducing the expression of 5'-nucleotidase UshA, and increasing the expression of ATP phosphoribosyltransferase HisG (through the amino acid mutation N215R / G233H / T235Q of HisG from Corynebacterium glutamicum), etc., but the effect of only HisG mutation transformation is unknown; Doroshenko et al. weakened the feedback inhibition of L-histidine on HisG through the amino acid mutation E271K of HisG, but the degree of weakening was limited, and the L-histidine production was only 0.2±0.1g / L. Therefore, in order to realize the industrial application of microbial fermentation production of L-histidine, it is necessary to find a more suitable mutation method for ATP phosphoribosyltransferase HisG to improve its anti-histidine feedback inhibition ability, thereby increasing the L-histidine fermentation yield of the engineered bacteria. Summary of the invention

[0005] The invention aims to introduce mutation sites into HisG by studying the structure of HisG gene from Acinetobacter baumannii, provide ATP phosphoribosyltransferase mutants, and express the mutants in host bacteria 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, wherein the ATP phosphoribosyltransferase mutant is a point mutation comprising position 84 and / or position 205 based on the ATP phosphoribosyltransferase amino acid sequence 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 position 84 of the ATP phosphoribosyltransferase sequence shown in SEQ ID NO:1 to alanine, and the sequence after mutation is shown in SEQ ID NO:2;

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

[0011] (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. The sequence after mutation is shown in SEQ ID NO:4.

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

[0013] In one embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule is 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-mentioned ATP phosphoribosyltransferase mutant.

[0017] In one embodiment of the present invention, the recombinant microorganism contains the above-mentioned 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, use of the above nucleic acid molecule in any of the following A1)-A3);

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

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

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

[0023] A1) preparing ATP phosphoribosyltransferase;

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

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

[0026] In a seventh aspect, a method for producing L-histidine is provided, characterized in that L-histidine is produced by fermentation using the above-mentioned recombinant microorganism.

[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 ATP phosphoribosyltransferase derived from Acinetobacter baumannii by exogenous introduction and replacement, thereby improving its L-histidine acid production capacity; and further obtains an ATP phosphoribosyltransferase mutant derived from Acinetobacter baumannii by site-directed mutagenesis technology, which is used to construct a recombinant bacterium, and the recombinant engineered bacterium expressing the ATP phosphoribosyltransferase mutant is used for fermentation production of L-histidine, and the L-histidine yield is increased by 6.5 times, which has broad prospects for industrial development and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the molecular docking diagram of HisG-Ab using L-histidine as substrate in AutoDock Vina. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with specific embodiments, but any embodiment or combination thereof should not be construed as limiting the scope of protection or implementation of the present invention. Any method changes made by a person of ordinary skill in the art based on these implementations are all included in the scope of protection of the present invention.

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

[0031] The culture medium and reagent information involved in the following examples are as follows:

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

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

[0034] Seed culture medium: 20g / L glucose, 4g / L yeast extract, 3g / L peptone, 1.2g / L potassium dihydrogen phosphate, 0.5g / L magnesium sulfate heptahydrate, 10mg / L ferrous sulfate heptahydrate, 10mg / L manganese sulfate monohydrate, 1mg / L vitamin B1, 1mg / L vitamin B3, 1mg / L vitamin B5, 1mg / L vitamin B12, 1mg / L vitamin H, pH adjusted to 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, adjust pH to 7.0-7.2 with sodium hydroxide; add ammonia water every 4 hours during fermentation to pH 7.2-7.4.

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

[0037] The strains, plasmids and primers involved in the following examples are 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) was docked with L-histidine as substrate in AutoDock Vina to observe the interaction. Figure 1 As shown, L-histidine forms hydrogen bonds with the S205 and D84 residues of HisG, which are key residues of the active pocket. Therefore, point mutations were selected for the key residues.

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

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

[0048] In the first step, pRE112 plasmid DNA was used as a template and primers HisG-cs-up / HisG-cs-down were used to amplify a 3593 bp DNA fragment I for the first step of homologous recombination.

[0049] The amplification system was as follows: Phusion 5X buffer (New England Biolabs) 10 μl, dNTP (10 mM each) 1 μl, DNA template 20 ng, primers (10 μM) 2 μl each, Phusion TM High-fidelity DNA polymerase (2.5 U / μl, purchased from Thermo Scientific TM , Catalog No.: F530S) 0.5μl, add distilled water to make up to 50μl.

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

[0051] The above DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into Escherichia coli W3110 by electroporation, and then the DNA fragment I was electroporated into Escherichia coli W3110 carrying pKD46 to obtain W3110-pKD46 recombinant bacteria.

[0052] The recombinant bacteria W3110-pKD46 was prepared as competent cells by the CaCl2 method; then 50 μl of W3110-pKD46 competent cells were placed on ice, 50 ng of DNA fragment I was added, placed on ice for 2 minutes, and transferred to a 0.2 cm Bio-Rad electroporation cup. MicroPulser (Bio-Rad) electroporator was used, and the electroporation parameters were 2.5 kV. After electroporation, 1 ml of LB liquid culture medium was quickly transferred to the electroporation cup, pipetted 5 times, and then transferred to a test tube, incubated at 75 rpm and 30°C for 2 hours. Take 200 μl of bacterial solution and spread it on LB plate containing ampicillin (final concentration of 100 μg / ml) and chloramphenicol (final concentration of 34 μg / ml). After culturing overnight at 30°C, select a single colony for PCR verification. The primers used are XZ-HisG-up / XZ-HisG-down. The correct colony amplification product is a fragment of 3715 bp. Select a correct single colony and name it HIS001.

[0053] In the second step, the plasmid PUC19-HisG synthesized by Hongxun Biotechnology was used as a template and the primers HisG-mut-cs-up / HisG-mut-cs-down were used to amplify the 812bp DNA fragment II. The amplification system was the same as the first step, and the DNA fragment II was used for the second homologous recombination.

[0054] The pKD46 plasmid was transformed into HIS001 (HIS001-pKD46) by electroporation, and prepared into competent cells by CaCl2 method. 50 μl HIS001-pKD46 competent cells were placed on ice, 50 ng DNA fragment II was added, placed on ice for 2 minutes, and transferred to a 0.2 cm Bio-Rad electroporation cup. MicroPulser (Bio-Rad) electroporator was used, and the electroporation parameters were 2.5 kV. After electroporation, 1 ml of LB liquid culture medium was quickly transferred to the electroporation cup, and after blowing 5 times, it was transferred to a test tube, 75 rpm, and incubated at 30°C for 4 hours. The bacterial liquid was transferred to LB liquid culture medium without sodium chloride containing 10% sucrose (50 ml culture medium in a 250 ml flask), and after culturing for 24 hours, it was streaked and cultured on an LB plate containing 6% sucrose and without sodium chloride. After PCR verification, the primers used were XZ-HisG-up / XZ-HisG-down, and the correct colony amplification product was a 930bp fragment. A correct single colony was selected to obtain a recombinant bacterium containing the ATP phosphoribosyltransferase gene HisG from Acinetobacter baumannii, which was named HIS002.

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

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

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

[0058] Place 50 μl HIS001-pKD46 competent cells (preparation scheme is the same as 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 cup. Use MicroPulser (Bio-Rad) electroporator, the electroporation parameter is 2.5 kV. After electroporation, quickly transfer 1 ml LB liquid culture medium to the electroporation cup, blow 5 times and then transfer to a test tube, 75 rpm, incubate at 30°C for 4 hours. Transfer the bacterial liquid to LB liquid culture medium without sodium chloride containing 10% sucrose, and after culturing for 24 hours, streak culture on LB plates containing 6% sucrose and without sodium chloride. After PCR verification, the primers used were XZ-HisG-up / XZ-HisG-down, and the correct colony amplification product was a 930bp fragment. A correct single colony was selected to obtain a recombinant bacterium containing the ATP phosphoribosyltransferase mutant gene HisG-Ab-D84A from Acinetobacter baumannii, which was named HIS003.

[0059] Example 4: Construction of recombinant bacteria 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, the plasmid PUC19-HisG-S205H synthesized by Hongxun Biotechnology was used as a template and primers HisG-mut-cs-up / HisG-mut-cs-down were used to amplify the 812 bp DNA fragment II. The amplification system and conditions were the same as those in Example 1. The DNA fragment II was used for the second homologous recombination.

[0062] Place 50 μl HIS001-pKD46 competent cells (preparation scheme is the same as 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 cup. Use MicroPulser (Bio-Rad) electroporator, the electroporation parameter is 2.5 kV. After electroporation, quickly transfer 1 ml LB liquid culture medium to the electroporation cup, blow 5 times and then transfer to a test tube, 75 rpm, incubate at 30°C for 4 hours. Transfer the bacterial liquid to LB liquid culture medium without sodium chloride containing 10% sucrose, and after culturing for 24 hours, streak culture on LB plates containing 6% sucrose and without sodium chloride. After PCR verification, the primers used were XZ-HisG-up / XZ-HisG-down, and the correct colony amplification product was a 930bp fragment. A correct single colony was selected to obtain a recombinant bacterium containing the ATP phosphoribosyltransferase mutant gene HisG-Ab-S205H from Acinetobacter baumannii, which was named HIS003.

[0063] Example 5: Construction of recombinant bacteria 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, the plasmid PUC19-HisG-D84A-S205H synthesized by Hongxun Biotechnology was used as a template and primers HisG-mut-cs-up / HisG-mut-cs-down were used to amplify the 812 bp DNA fragment II. The amplification system and conditions were the same as those in Example 1. The DNA fragment II was used for the second homologous recombination.

[0066] Place 50 μl HIS001-pKD46 competent cells (preparation scheme is the same as 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 cup. Use MicroPulser (Bio-Rad) electroporator, the electroporation parameter is 2.5 kV. After electroporation, quickly transfer 1 ml LB liquid culture medium to the electroporation cup, blow 5 times and then transfer to a test tube, 75 rpm, incubate at 30°C for 4 hours. Transfer the bacterial liquid to LB liquid culture medium without sodium chloride containing 10% sucrose, and after culturing for 24 hours, streak culture on LB plates containing 6% sucrose and without sodium chloride. After PCR verification, the primers used were XZ-HisG-up / XZ-HisG-down, and the correct colony amplification product was a 930bp fragment. A correct single colony was selected to obtain a recombinant bacterium containing the ATP phosphoribosyltransferase mutant gene HisG-Ab-D84A-S205H from Acinetobacter baumannii, which was named HIS005.

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

[0068] The method of producing L-histidine by fermentation using Escherichia coli W3110, recombinant bacteria HIS002, HIS003, HIS004, and HIS005 comprises the following steps:

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

[0070] (2) Fermentation culture: 250 ml of fermentation medium was placed in a 500 ml fermentation tank, and the seed culture solution was inoculated into the fermentation medium at a final concentration of OD550 = 0.1, and fermented at 37°C, 150 rpm for 3 days to obtain a fermentation solution. The neutralizer was 5 M ammonia water, and the pH of the fermentation tank was controlled at 7.0. The dissolved oxygen was maintained at 25-35% during the culture process.

[0071] Analysis method: Agilent (Agilent-1260) high performance liquid chromatograph was used to determine the components in the fermentation broth after fermentation for 3 days. L-histidine was determined using 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 3 times, and the average values ​​of the analytical results are 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, the fermentation ability of Escherichia coli W3110 to produce L-histidine is poor. By exogenously introducing 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 HIS003 (HisG-Ab-D84A), HIS004 (HisG-Ab-S205H), and HIS005 (HisG-Ab-D84A-S205H) strains containing HisG-Ab mutants all have improved histidine yields, especially the HIS005 strain containing the HisG-Ab-D84A-S205H mutant, whose L-histidine production level increased by 6.5 times; it is speculated that the introduction of D84A and / or S205H mutations into HisG-Ab relieves the feedback inhibition of L-histidine on HisG.

[0076] The above are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should be regarded as the protection scope of the present invention.

Claims

1. An ATP phosphoribosyltransferase mutant, characterized in that The ATP phosphoribosyltransferase is prepared by subjecting the amino acid sequence of the ATP phosphoribosyltransferase shown in SEQ ID NO: 1 to point mutations at position 84 and / or position 205.

2. The ATP phosphoribosyltransferase mutant according to claim 1, characterized in that The ATP phosphoribosyltransferase 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.

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

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

5. A recombinant vector containing the nucleic acid molecule according to claim 3.

6. A recombinant microorganism expressing the ATP phosphoribosyltransferase mutant according to claim 1 or 2.

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

8. Use of the ATP phosphoribosyltransferase mutant according to any one of claims 1 to 2 in any one of A2) to A3); Or, use of the nucleic acid molecule according to claim 3 in any one of A1)-A3); Or, use of the expression cassette of claim 4 in any one of A1)-A3); Or, use of the recombinant vector according to claim 5 in any one of A1) to A3); Or, use of the recombinant microorganism according to any one of claims 6 to 7 in any one of A1) to A3); A1) preparing ATP phosphoribosyltransferase; A2) production of histidine; A3) Increase histidine production.

9. A method for producing L-histidine, characterized in that: L-histidine is produced by fermentation using the recombinant microorganism described in claim 6 or 7.

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