Glutamic acid-pyruvate aminotransferase mutant and application thereof

Abstract

The invention discloses a glutamic acid-pyruvate aminotransferase mutant and application thereof.The mutant is obtained by replacing any one or more of 15th, 276th, 292nd and 296th amino acid residues on the basis of a wild-type glutamic acid-pyruvate aminotransferase amino acid sequence, compared with a wild-type glutamic acid-pyruvate aminotransferase amino acid sequence, the mutant has the advantages that the enzyme activity is reduced, and the activity of the glutamic acid-pyruvate aminotransferase mutant is improved; the mutant is introduced into valine producing bacteria to regulate and control metabolic synthesis of alanine, the alanine synthesis amount is obviously reduced, meanwhile, normal growth of the bacterial strain is not influenced, and a foundation can be laid for producing valine by utilizing a microbial fermentation method.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a glutamate-pyruvate aminotransferase mutant and its application. Background Art

[0002] As one of the three branched-chain amino acids (BCAAs), L-valine is an essential amino acid that cannot be synthesized in humans and animals and can only be obtained by supplementation from the outside. L-valine has the functions of promoting protein synthesis, inhibiting protein decomposition, and enhancing the body's immune protection. It can be used as a food additive, nutritional supplement, flavoring agent, and antibiotic, and is widely used in the fields of food, medicine, cosmetics, feed, etc., with good market prospects.

[0003] The main production methods of L-valine include microbial fermentation, enzymatic catalysis, chemical synthesis, and protein hydrolysis extraction. Among them, microbial fermentation has the advantages of low raw material cost, mild production conditions, high fermentation yield, and easy large-scale production. Currently, L-valine is mainly produced by microbial fermentation worldwide. The L-valine-producing strains used for fermentation are mostly obtained by mutagenesis, and the original strains mainly include Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium flavum, etc. For example, Chen Ning et al. used Brevibacterium flavum as the original strain, and by using the strategy of ultraviolet mutagenesis of protoplasts combined with DES chemical mutagenesis, a high-yield L-valine strain TV2564 was screened, and the L-valine yield was as high as 29.39 g / L. However, the strains obtained by traditional mutagenesis have strong randomness, unclear genetic background, and the regulatory networks such as a large amount of intracellular feedback inhibition also greatly limit the production capacity of wild cells. It is difficult to screen high-yield strains, and there are many by-products accumulated during fermentation, resulting in high purification costs, which further hinders the industrial production of valine. Therefore, to obtain fermentation strains that can efficiently produce L-valine, it is necessary to effectively regulate the metabolic network in microorganisms.

[0004] Currently, in the biosynthesis of L-valine, pyruvate is an important precursor in the anabolic pathway. After four steps of reactions, pyruvate synthesizes L-valine. However, as a precursor of most compounds, pyruvate will also produce other impurity amino acids, such as L-alanine and isoleucine, while synthesizing L-valine. In Escherichia coli cells, there are three natural synthesis pathways for L-alanine, including Pathway 1: formed by the catalysis of L-valine by valine-pyruvate aminotransferase (AvtA); Pathway 2: formed by the catalysis of pyruvate by glutamate-pyruvate aminotransferase (AlaA, alaC); Pathway 3: formed by the catalysis of L-cysteine by cysteine desulfurase (iscS). Among them, Pathway 1 and 2 are the main synthesis pathways of L-alanine. It can be seen that in the engineering strains for producing L-valine, the synthesis of alanine is inevitable, which also increases the difficulty of separating and purifying the L-valine fermentation broth. Currently, reducing the accumulation of L-alanine basically takes blocking the L-alanine synthesis route as the main means. However, as L-alanine is necessary to maintain the normal physiological metabolism of the strain, completely blocking the corresponding synthesis pathway, such as deleting any one of the key genes in the two pathways (AvtA, AlaA, alaC) in the wild-type strain, will not affect the normal growth and metabolism of the strain. But if the ΔAvtA mutant strain deletes any one of the alaA and alaC genes again, it will cause the growth condition of the strain to deteriorate, and then lead to a decrease in the yield of the target product L-valine, which is not worth the loss; even the mutant strain with the deletion of three genes must be supplemented with exogenous L-alanine to maintain the production of the strain, which undoubtedly increases the cost and complexity of industrial production. Therefore, it is necessary to find a catalytic enzyme with appropriate synthesis intensity so that the synthesis amount of L-alanine can not only meet the growth requirements of the strain but also not synthesize too much to affect the purification cost of L-valine. Summary of the Invention

[0005] To solve the above technical problems, this patent combines gene editing technology and gene shuffling technology to develop a glutamate-pyruvate aminotransferase mutant, which is a key enzyme for alanine synthesis. Introducing this mutant into the valine-producing bacteria can regulate the metabolic synthesis of alanine, significantly reducing the synthesis amount of alanine while not affecting the normal growth of the strain, laying a foundation for the production of valine by microbial fermentation.

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

[0007] In the first aspect, the present invention provides a protein, which is a glutamate-pyruvate aminotransferase mutant with reduced enzyme activity obtained by replacing any one or more (two or more) amino acid residues at positions 15, 276, 292, and 296 based on the amino acid sequence of the wild-type glutamate-pyruvate aminotransferase shown in SEQ ID NO:1.

[0008] Among them, the substitution is to mutate amino acid residues; the ways of mutating each amino acid residue are as follows:

[0009] The tyrosine Y at position 15 is mutated to arginine R;

[0010] The leucine L at position 276 is mutated to isoleucine I;

[0011] The glycine G at position 292 is mutated to arginine R;

[0012] The isoleucine I at position 296 is mutated to serine S.

[0013] In one embodiment of the present invention, the glutamate-pyruvate aminotransferase mutant contains amino acid mutations at any one or more than two of the following sites on the basis of the glutamate-pyruvate aminotransferase amino acid sequence shown in SEQ ID NO: 1: Y15R, L276I, G292R, I296S.

[0014] Any one of the following amino acid mutations A1)-A5):

[0015] A1) Amino acid mutation at 1 site: Y15R;

[0016] A2) Amino acid mutation at 1 site: L276I;

[0017] A3) Amino acid mutations at 2 sites: Y15R, I296S;

[0018] A4) Amino acid mutations at 2 sites: L276I, G292R;

[0019] A5) Amino acid mutations at 4 sites: Y15R, L276I, G292R, I296S.

[0020] In one embodiment of the present invention, the glutamate-pyruvate aminotransferase mutant contains an amino acid mutation at the following one site on the basis of the wild-type glutamate-pyruvate aminotransferase amino acid sequence shown in SEQ ID NO: 1: Y15R.

[0021] In one embodiment of the present invention, the glutamate-pyruvate aminotransferase mutant undergoes an amino acid mutation at the following one site on the basis of the wild-type glutamate-pyruvate aminotransferase amino acid sequence shown in SEQ ID NO: 1: Y15R.

[0022] In one embodiment of the present invention, the glutamate-pyruvate aminotransferase mutant contains an amino acid mutation at the following one site on the basis of the wild-type glutamate-pyruvate aminotransferase amino acid sequence shown in SEQ ID NO: 1: L276I.

[0023] In one embodiment of the present invention, the glutamate-pyruvate aminotransferase mutant has an amino acid mutation at the following one site on the basis of the wild-type glutamate-pyruvate aminotransferase amino acid sequence shown in SEQ ID NO: 1: L276I.

[0024] In one embodiment of the present invention, the glutamate-pyruvate aminotransferase mutant contains amino acid mutations at the following two sites on the basis of the wild-type glutamate-pyruvate aminotransferase amino acid sequence shown in SEQ ID NO: 1: Y15R, I296S.

[0025] In one embodiment of the present invention, the glutamate-pyruvate aminotransferase mutant has amino acid mutations at the following two sites on the basis of the wild-type glutamate-pyruvate aminotransferase amino acid sequence shown in SEQ ID NO: 1: Y15R, I296S.

[0026] In one embodiment of the present invention, the glutamate-pyruvate aminotransferase mutant contains amino acid mutations at the following two sites on the basis of the glutamate-pyruvate aminotransferase amino acid sequence shown in SEQ ID NO: 1: L276I, G292R.

[0027] In one embodiment of the present invention, the glutamate-pyruvate aminotransferase mutant has amino acid mutations at the following two sites on the basis of the glutamate-pyruvate aminotransferase amino acid sequence shown in SEQ ID NO: 1: L276I, G292R.

[0028] In one embodiment of the present invention, the glutamate-pyruvate aminotransferase mutant contains amino acid mutations at the following four sites on the basis of the glutamate-pyruvate aminotransferase amino acid sequence shown in SEQ ID NO: 1: Y15R, L276I, G292R, I296S.

[0029] In one embodiment of the present invention, the glutamate-pyruvate aminotransferase mutant has amino acid mutations at the following four sites on the basis of the glutamate-pyruvate aminotransferase amino acid sequence shown in SEQ ID NO: 1: Y15R, L276I, G292R, I296S, and the mutated amino acid sequence is SEQ ID NO: 3.

[0030] In the second aspect, the present invention provides a nucleic acid molecule encoding any one of the proteins described in the first aspect.

[0031] In one embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 4.

[0032] In a third aspect, the present invention provides an expression cassette or a recombinant vector containing the nucleic acid molecule described in the second aspect.

[0033] Further, the expression cassette refers to a nucleic acid molecule capable of expressing the protein described in the first aspect in a host bacterium. This nucleic acid molecule may not only include a promoter that initiates the transcription of the mutant coding gene sequence, but also include a terminator that terminates the transcription of the mutant coding gene sequence. Even further, the expression cassette may also include an enhancer sequence.

[0034] In a fourth aspect, the present invention provides a recombinant microorganism expressing any one of the proteins described in the first aspect.

[0035] In one embodiment of the present invention, the recombinant microorganism is obtained by introducing the nucleic acid molecule described in the second aspect, or the expression cassette or recombinant vector described in the third aspect, into a host bacterium.

[0036] In one embodiment of the present invention, the host bacterium is Escherichia coli.

[0037] In one embodiment of the present invention, the reduction or inactivation of the activity of valine-pyruvate aminotransferase AvtA is achieved by knocking out the AvtA gene.

[0038] In a preferred embodiment of the present invention, the host bacterium is Escherichia coli Sval031 (CGMCC No. 19456, deposit date March 6, 2020; see CN113278641B), Escherichia coli Sval049 (deposit number CGMCC No. 19457, deposit date March 6, 2020; see CN113278568B), or Escherichia coli Sval065 (deposit number CGMCC No. 19458, deposit date March 6, 2020; see CN113278655B).

[0039] In a fifth aspect, the present invention provides the use of the protein described in the first aspect in any one of the following B1)-B6);

[0040] or, the use of the nucleic acid molecule described in the second aspect in any one of the following B1)-B6);

[0041] or, the use of the expression cassette or recombinant vector described in the third aspect in any one of the following B1)-B6);

[0042] or, the use of the recombinant microorganism described in the fourth aspect in any one of the following B1), B3)-B6):

[0043] B1) Preparing glutamate-pyruvate aminotransferase;

[0044] B2) Construct a recombinant engineered bacterium that produces L-valine, L-leucine or L-isoleucine;

[0045] B3) Produce L-valine, L-leucine or L-isoleucine;

[0046] B4) Increase the yield of L-valine, L-leucine or L-isoleucine;

[0047] B5) Reduce the accumulation of L-alanine;

[0048] B6) Increase the yield of L-valine, L-leucine or L-isoleucine and reduce the accumulation of L-alanine.

[0049] In a preferred embodiment of the present invention, the present invention provides the application of the protein described in the first aspect in any one of B1)-B6) below;

[0050] Or, the application of the nucleic acid molecule described in the second aspect in any one of B1)-B6) below;

[0051] Or, the application of the expression cassette or recombinant vector described in the third aspect in any one of B1)-B6) below;

[0052] Or, the application of the recombinant microorganism described in the fourth aspect in any one of B1), B3)-B6) below:

[0053] B1) Prepare glutamate-pyruvate aminotransferase;

[0054] B2) Construct a recombinant engineered bacterium that produces L-valine;

[0055] B3) Produce L-valine;

[0056] B4) Increase the yield of L-valine;

[0057] B5) Reduce the accumulation of L-alanine;

[0058] B6) Increase the yield of L-valine and reduce the accumulation of L-alanine.

[0059] In a preferred embodiment of the present invention, the glutamate-pyruvate aminotransferase can be prepared by cell disruption and centrifugation of the recombinant microorganism described in the fourth aspect and collection.

[0060] In a preferred embodiment of the present invention, the recombinant microorganism is a strain obtained by introducing the nucleic acid molecule described in the second aspect or the expression cassette or recombinant vector described in the third aspect into a host bacterium, and the host bacterium is Escherichia coli Sval031 (CGMCC No. 19456, deposit date: March 6, 2020; see CN113278641B for details), Escherichia coli Sval049 (deposit number: CGMCC No. 19457, deposit date: March 6, 2020; see CN113278568B for details), or Escherichia coli Sval065 (deposit number: CGMCC No. 19458, deposit date: March 6, 2020; see CN113278655B for details); the above three host bacteria are all obtained by genetically modifying Escherichia coli ATCC 8739, and can all ferment and produce L-valine, L-leucine or L-isoleucine, and all use pyruvate as a precursor substance, and L-alanine impurities will inevitably be produced during the fermentation process.

[0061] In the sixth aspect, the present invention provides a production method for producing L-valine, including:

[0062] (1) Fermenting and culturing the recombinant microorganism described in the fourth aspect;

[0063] (2) Separating and harvesting L-valine.

[0064] Compared with the prior art, the present invention has the following beneficial technical effects: The present invention provides a glutamate-pyruvate aminotransferase mutant. By introducing this mutant to replace the wild-type glutamate-pyruvate aminotransferase into an engineered bacterium, a valine-producing strain is obtained. When it is used for fermenting and producing L-valine, the accumulation of the by-product L-alanine during the fermentation process is significantly reduced, the purification cost of the valine product is reduced, and at the same time, the growth and activity of the strain are not affected, providing strong support for the industrial production of valine products. Specific Embodiments

[0065] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0066] The experimental methods in the following embodiments, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions, such as "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).

[0067] The materials, reagents, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0068] 1. The information on the kits and culture media involved in the following examples is as follows:

[0069] PCR SuperMix(+dye) was purchased from TransGen Biotech, catalog number: AS111-11.

[0070] PCR amplification uses GXL DNA Polymerase kit, purchased from TaKaRa, CodeNo.R050A.

[0071] The PCR product was purified using the SanPrep column DNA gel extraction kit, purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number B518131-0050.

[0072] Amplite Fluorometric L-Alanine Detection Kit, purchased from Xi'an Biying Biotechnology Co., Ltd., catalog number 13825-AAT.

[0073] LB plate: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract, 20 g / L agar, with water as the solvent.

[0074] LB liquid medium: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract, with water as the solvent.

[0075] AM1 liquid medium: composed of glucose, inorganic salts, trace elements and water;

[0076] Preferably, AM1 liquid medium: 50 g / L glucose, 2.63 g / L (NH4)2HPO4, 0.87 g / L NH4H2PO4, 0.37 g / L KCl, 0.37 g / L MgSO4·7H2O, 0.154 g / L betaine hydrochloride, 1 mL of trace elements, with water as the solvent.

[0077] AMI solid medium: composed of glucose, inorganic salts, trace elements, agar and water; preferably: 50 g / L glucose, 2.63 g / L (NH4)2HPO4, 0.87 g / L NH4H2PO4, 0.37 g / L KCl, 0.37 g / L MgSO4·7H2O, 0.154 g / L betaine hydrochloride, 1 mL of trace elements, 20 g / L agar, with water as the solvent.

[0078] LB liquid medium containing 10% sucrose (without sodium chloride): 10 g / L sucrose, 10 g / L peptone, 5 g / L yeast extract, with water as the solvent.

[0079] LB solid medium containing 10% sucrose (without sodium chloride): 10 g / L sucrose, 10 g / L peptone, 5 g / L yeast extract, 20 g / L agar, with water as the solvent.

[0080] LB solid medium containing 6% sucrose (without sodium chloride): 6 g / L sucrose, 10 g / L peptone, 5 g / L yeast extract, 20 g / L agar, with water as the solvent.

[0081] Synthetic medium: 6 g / L glycerol, 4.5 g / L diammonium hydrogen phosphate, 0.37 g / L magnesium sulfate heptahydrate, 0.25 g / L betaine hydrochloride, 0.005 g / L vitamin B1, 10 g / L potassium dihydrogen phosphate, 1 mL / L trace element mother liquor, adjusted to pH 7.2 - 7.5 with sodium hydroxide;

[0082] Among them, the composition of the trace element mother liquor is: 0.0021 g / L ferric chloride hexahydrate, 0.00027 g / L cobalt chloride hexahydrate, 0.00013 g / L copper chloride dihydrate, 0.00042 g / L zinc acetate dihydrate, 0.00027 g / L sodium molybdate dihydrate, 0.00007 g / L boric acid, 0.00044 g / L manganese chloride tetrahydrate.

[0083] Fermentation medium: 120 g / L glucose, 0.37 g / L magnesium sulfate heptahydrate, 0.25 g / L betaine hydrochloride, 0.13 g / L potassium dihydrogen phosphate, 0.005 g / L vitamin B1, 1 g / L pure phosphoric acid, 1 mL / L trace element mother liquor;

[0084] Among them, the composition of the trace element mother liquor is: 0.0021 g / L ferric chloride hexahydrate, 0.00027 g / L cobalt chloride hexahydrate, 0.00013 g / L copper chloride dihydrate, 0.00042 g / L zinc acetate dihydrate, 0.00027 g / L sodium molybdate dihydrate, 0.00007 g / L boric acid, 0.00044 g / L manganese chloride tetrahydrate.

[0085] Working concentrations of the antibiotics used: ampicillin (concentration 50 μg / mL), chloramphenicol (concentration 50 μg / mL).

[0086] 2. The strain and plasmid information involved in the following examples is shown in Table 1, and the primers used are shown in Table 2.

[0087] Table 1: Strains and plasmids used in the present invention

[0088]

[0089]

[0090] Table 2: Primers used in the present invention

[0091]

[0092] 3. Detection method for the contents of L-valine and L-alanine:

[0093] Take 2 mL of the fermentation broth, centrifuge at 12,000 rpm for 2 min to collect the supernatant. Transfer 100 μL of the supernatant to 900 μL of sterile water, dilute 10 times, filter the supernatant using a 0.22 μm inorganic filter membrane, and analyze the contents of L-valine and L-alanine using high performance liquid chromatography (HPLC).

[0094] Conditions for detecting L-alanine by high performance liquid chromatography (HPLC): The chromatographic column is Primesep 100 4.6*250 mm, 5 μm; mobile phase A is 30% acetonitrile + 0.3% trifluoroacetic acid solution, and the rinsing mobile phase B is 75% acetonitrile + 0.1% formic acid solution; the column oven is controlled at 35 °C, the flow rate is 0.5 mL / min, the injection volume is 10 μL, and the detection wavelength is 210 nm;

[0095] Conditions for detecting L-valine by high performance liquid chromatography (HPLC): The chromatographic column is Primesep 100 4.6*250 mm, 5 μm; mobile phase 0.04 mol / L potassium dihydrogen phosphate aqueous solution: acetonitrile = 1:3 (V / V), and the rinsing mobile phase B is 75% acetonitrile; the column oven is controlled at 30 °C, the flow rate is 1 mL / min, the injection volume is 10 μL, and the detection wavelength is 205 nm;

[0096] Among them, the retention time of the characteristic peak of L-alanine is 20.6 min, and the retention time of the characteristic peak of L-valine is 10.6 min.

[0097] In the present invention, by constructing and screening AlaA mutants, mutants with low efficiency in catalyzing the synthesis of alanine and meeting the growth of the bacterial cells are obtained. Combining with the means of optimizing the metabolic pathway, the metabolic flux is maximally directed to the valine synthesis metabolic pathway, and the alanine content is reduced to meet the requirements of industrial production of valine.

[0098] Example 1: Construction of an AlaA mutant library test strain

[0099] The synthesis of alanine mainly occurs through the aforementioned Pathways 1 and 2. It is found that blocking any one of the pathways has no effect, while blocking both pathways seriously affects the growth of the bacterial strain. In the present invention, on the basis of the L-valine-producing bacterium Sval031, Pathway 1 (L-valine is catalyzed by valine-pyruvate aminotransferase AvtA to form L-alanine) is blocked first to form the test chassis bacterium Sval031-1.

[0100] The specific steps include:

[0101] 1.1. Construction of AvtA-CS fragment

[0102] Using the pRE112 plasmid as a template and AvtA-CS-F / R as primers, amplify the AvtA-CS fragment, send it to Sangon Biotech for sequencing. After the sequencing is verified to be correct, purify and recover the amplified fragment, name it AvtA-CS, and store it at -20°C for later use after purification.

[0103] Amplification system (50 μL): 10 μL of 5×PrimeSTAR GXL Buffer (Mg2+ plus), 4 μL of dNTP Mixture (2.5 mM each dNTP), 1 μL of template (20 ng / μL), 1 μL each of primers (10 μM), 0.5 μL of PrimeSTAR GXL DNA Polymerase (1.25 U / μL), 32.5 μL of distilled water, with a total volume of 50 μL;

[0104] Amplification program: Pre-denaturation at 98°C for 2 minutes (1 cycle); denaturation at 98°C for 10 seconds, annealing at 54°C for 15 seconds, extension at 68°C for 70 seconds (30 cycles); extension at 68°C for 5 minutes (1 cycle).

[0105] 1.2. Construction of knockout fragment

[0106] Using the Sval031 genome as a template and UP-AvtA-F / R, DN-AvtA-F / R as primers to amplify the upstream and downstream homologous fragments. Using the SanPrep Column DNA Gel Extraction Kit, with the amplified and purified upstream and downstream homologous fragments as templates, use the primers UP-AvtA-F / DN-AvtA-R to amplify by overlap pcr. The size of the amplified knockout fragment is 1027 bp, named ΔAvtA. The knockout fragment is purified using the SanPrep Column DNA Gel Extraction Kit, and the purified product is stored at -20°C.

[0107] Among them, the amplification system and amplification program for amplifying the upstream and downstream homologous fragments are the same as those in 1.1

[0108] The overlap pcr amplification system and amplification program are as follows:

[0109] 25 μL of PCR SuperMix (+dye), 1 μL each of the upstream and downstream homologous fragment templates, 1 μL each of the upstream and downstream primers (10 μM), 21 μL of distilled water, with a total volume of 50 μL.

[0110] Amplification program: Pre-denaturation at 94°C for 5 minutes (1 cycle); denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 70 seconds (30 cycles); extension at 72°C for 5 minutes (1 cycle).

[0111] 1.3. Knock out the AvtA gene

[0112] (1) Prepare electrocompetent Sval031 cells (for details, refer to the Cold Spring Harbor Molecular Cloning Manual). Electrotransform the pKD46 plasmid into Sval031 to obtain the Sval031-pkD46 strain;

[0113] (2) Refer to the method for preparing electrocompetent Sval031 cells to prepare electrocompetent Sval031-pkD46 strain cells. Electrotransform the AvtA-CS fragment prepared in step 1.1 into the electrocompetent Sval031-pkD46 strain cells, and incubate them in a constant temperature shaker at 30 °C and 220 rpm for 4 h. After incubation, spread them on an LB plate containing chloramphenicol and ampicillin for culture. Verify the single colonies growing on the plate with the AvtA-YZ-up / down primers and send them for sequencing. Preserve the single colonies with correct sequencing results to obtain the Sval031-CS strain

[0114] (3) Place 80 μL of electrocompetent Sval031-CS strain cells on ice, add 100 ng of DNA fragment (ΔAvtA), place it on ice for 2 minutes, and transfer it to a 2 mm Bio-Rad electroporation cuvette. Use a MicroPulser (BioRad) electroporator with an electroporation parameter of 2.5 kV voltage. Immediately after electroporation, transfer 1 mL of LB medium to the electroporation cuvette, pipette 3 times and then transfer it to a test tube, and incubate it overnight at 75 rpm and 37 °C. Transfer the bacterial solution to an LB liquid medium containing 10% sucrose (without sodium chloride) (50 mL of medium in a 250 mL flask), culture for 24 hours and then streak it on an LB solid medium containing 10% sucrose (without sodium chloride). Pick single colonies on an LB solid medium containing 6% sucrose (without sodium chloride) and streak them for rescreening on an LB plate and an LB plate containing chloramphenicol respectively, and incubate them overnight in a 37 °C constant temperature incubator. Pick the single colonies that grow on the LB plate but not on the LB plate containing chloramphenicol for PCR verification. The primers used are AvtA-YZ-up / down primers. Select three single colonies with correct band sizes and send them to Shanghai Sangon Biotech Co., Ltd. for sequencing. Select the single colonies with correct sequencing results for bacterial preservation and name them Sval031-1.

[0115] Among them, the method for preparing electrocompetent Escherichia coli Sval031 strain cells includes:

[0116] 1) Take the activated bacterial solution and inoculate it into a 250 mL Erlenmeyer flask containing 25 mL of LB liquid medium at a ratio of 1%, and culture it at 37 °C and 220 rpm until the OD 550 is approximately 0.4 - 0.6, and ice-bath for 20 min;

[0117] 2) Transfer it into a 50 mL sterile centrifuge tube and centrifuge at 4°C and 6000 rpm for 5 min;

[0118] 3) Discard the supernatant, resuspend the precipitate with 30 mL of sterile water, and centrifuge at 4°C and 6000 rpm for 5 min;

[0119] 4) Discard the supernatant, resuspend the precipitate with 30 mL of 10% glycerol sterile water, and centrifuge at 4°C and 6000 rpm for 5 min;

[0120] 5) Repeat the operation in the fourth step;

[0121] 6) Discard the supernatant, add 100 mL of 10% glycerol sterile water, gently tap to mix evenly, and store at -80°C for later use.

[0122] Among them, the preparation method of the Sval031-pkD46 strain includes:

[0123] Prepare Sval031 electrocompetent cells and place them on ice. Mix 50 μL of pKD46 plasmid with Sval031 electrocompetent cells, place on ice for 2 min, and transfer to a 2 mm Bio-Rad electroporation cuvette; use a MicroPulser (Bio-Rad) electroporator with the electroporation parameters of voltage 2.5 kv. Immediately transfer 1 mL of LB liquid medium to the electroporation cuvette after electroporation, pipette 3 times and then transfer to a test tube, incubate at 75 rpm and 30°C for 1 h; take 200 μL of the incubated bacterial solution and spread it on an LB plate containing ampicillin, and culture overnight at 30°C. The growing monoclonal strain is the Sval031-pkD46 strain.

[0124] Among them, the commercial pKD46 plasmid is transformed into BL21 electrocompetent cells. The obtained recombinant bacteria are inoculated into an LB liquid medium containing ampicillin and cultured overnight, and then the pKD46 plasmid is obtained by plasmid extraction and stored at -20°C for later use.

[0125] Example 2: Screening of glutamate-pyruvate aminotransferase mutants

[0126] 2.1. Amplify the gene AlaA encoding glutamate-pyruvate aminotransferase

[0127] Using the genome of the Sval031 strain as a template, perform PCR amplification with the primer pair AlaA-F / AlaA-R to obtain the gene fragment AlaA encoding glutamate-pyruvate aminotransferase. The nucleotide sequence of the glutamate-pyruvate aminotransferase is shown in SEQ ID NO:2 in the sequence listing, and the amino acid sequence is shown in SEQ ID NO:1 in the sequence listing.

[0128] Using the primer pair AlaA-F / R, error-prone PCR was used to mutate the above-mentioned gene AlaA encoding glutamate-pyruvate aminotransferase;

[0129] Among them, the kit used for amplification was GXL DNA Polymerase, and the amplification system and procedure were as follows:

[0130] The amplification system was: 10 μL of 5×PrimeSTAR GXL Buffer (Mg2+ plus), 4 μL of dNTP Mixture (2.5 mM each of dNTP), 1 μL of the genomic template of strain Sval031 (20 ng / μL), 1 μL each of the primers (10 μM), 0.5 μL of PrimeSTAR GXL DNA Polymerase (1.25 U / μL), and 32.5 μL of distilled water, with a total volume of 50 μL;

[0131] The amplification procedure was: pre-denaturation at 98°C for 2 minutes (1 cycle); denaturation at 98°C for 10 seconds, annealing at 54°C for 15 seconds, extension at 68°C for 70 seconds (30 cycles); extension at 68°C for 5 minutes (1 cycle).

[0132] The target mutant gene obtained by PCR amplification was 1218 bp. After amplification, the PCR product was recovered using agarose gel, and the PCR product was named AlaA-Sep-X. The product was a combination of different mutant genes; the purified PCR product was electrotransformed and integrated into the Sval031-1-CS strain to form the mutant strain library Sval031-1-Sep-X; among them, the electrotransformation method was referred to as described in 1.3.

[0133] Among them, using the pRE112 plasmid as the template and AlaA-CS-F / R as the primers, the AlaA-CS fragment was amplified, and then sent to Sangon for sequencing. After the sequencing was verified to be correct, the amplified fragment was purified and recovered. This fragment was named AvtA-CS, and the product was electrotransformed and integrated into the Sval031-1 strain to obtain the strain Sval031-1-CS. The electrotransformation method was referred to as described in 1.3.

[0134] 2.2. Primary screening of AlaA mutants

[0135] The cells Sval031-1-Sep-X transformed in step 2.1 were spread on the AMI solid medium and cultured overnight at 37°C. The cloned strains carrying the mutant of glutamate-pyruvate aminotransferase with intact catalytic function could grow normally, while the cloned strains carrying the mutant of glutamate-pyruvate aminotransferase with reduced activity grew slowly, and the colony diameters of the corresponding cloned strains were relatively small. The strains containing the AlaA mutant were initially screened using the Amplite Fluorometric L-Alanine Detection Kit and re-screened using high performance liquid chromatography (HPLC).

[0136] The initial screening method is as follows:

[0137] The mutant strains were cultured using a 96-well deep well plate, with the cloned strain Sval031-1 containing the original glutamate-pyruvate aminotransferase as a control. Monoclonal colonies containing mutants and smaller than the control group were selected. The specific steps are as follows:

[0138] 96-well deep well plate culture: The cloned strain of the control group and the mutant clones with smaller colony diameters were picked and inoculated into a 96-well deep well plate containing AM1 liquid medium (500 μL medium, 2000 μL volume) respectively. At the same time, the strains were inoculated on an LB plate as a backup and cultured overnight at 37°C and 800 rpm.

[0139] The 96-well deep well plate containing the culture solution after culture was centrifuged to obtain the supernatant, and the supernatant was added to a 95-well plate, and the samples were detected according to the usage method of the Amplite Fluorometric L-Alanine Detection Kit. During the use of the kit, the detection conditions of the microplate reader were: detecting the absorbance value at 575 nm. The mutant clones with absorbance values smaller than the control group were selected, and the corresponding mutants were subjected to colony PCR amplification. The primers used were AlaA-YZ-up / down, and the samples were sent to Youkang Biology for sequencing analysis to determine the mutant sequences.

[0140] 2.3 Construction of the AlaA mutant library using DNA shuffling

[0141] 2.3.1 Obtaining AlaA mutant fragments using DNA shuffling

[0142] The sequencing results of the monoclonal colonies obtained in step 2.2 were compared with the wild-type AlaA gene, and AlaA-Sep-X containing different mutant genes was selected as the template, and the AlaA-mT mutant fragment was amplified using the primer pair AlaA-mT-F / R:

[0143] The amplification system was: 25 μL of PCR SuperMix (+dye), 1 μL of template, 1 μL of each upstream and downstream primer (10 μM), 22 μL of distilled water, and the total volume was 50 μL.

[0144] The amplification procedure was as follows: pre-denaturation at 94°C for 5 minutes (1 cycle); denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 70 seconds (30 cycles); extension at 72°C for 5 minutes (1 cycle).

[0145] The PCR amplification products were detected by agarose gel electrophoresis. The correct clone had a DNA band of 1218 bp. All amplified fragments were recovered using agarose gel electrophoresis, and different mutant fragments were mixed in equal amounts for DNA shuffling experiments.

[0146] The experimental steps of DNA shuffling refer to the reference "Ness, J. E., Kim, S., Gottman, A., Pak, R., Krebber, A., Borchert, T. V., Govindarajan, S., Mundorff, E. C., & Minshull, J. (2002). Synthetic shuffling expands functional protein diversity by allowing aminoacids to recombine independently. Nature Biotechnology, 20(12), 1251–1255. https: / / doi.org / 10.1038 / nbt754."

[0147] 2.3.2 Construction of DNA shuffling library and re-screening

[0148] The gene fragments obtained after shuffling were transformed into the test strain Sval031-1 to construct a DNA shuffling library of AlaA, and the Sval031-1-mT-X strain was obtained; the preliminary screening steps of the shuffling library were the same as those in 2.2.

[0149] For the strains passed the primary screening, AM1 liquid medium was used as the fermentation medium for parallel shake-flask fermentation re-screening. After fermentation, the fermentation broth was detected by liquid phase to determine the content of L-alanine in the fermentation broth, so as to screen the "dominant" mutants of glutamate-pyruvate aminotransferase AlaA, that is, the mutant strains with low L-alanine content in the fermentation broth.

[0150] The host strains Sval031, Sval031-1 (Sval031ΔAvtA), and the mutant strains containing glutamate-pyruvate transferase AlaA mutants (Sval031-2, Sval031-3, Sval031-4, Sval031-5, Sval031-6) were tested and compared for valine production in a 5 L fermenter. The main measured data were valine yield, L-alanine content, and the growth of the strain OD 600Value

[0151] The specific steps are as follows:

[0152] 1) Seed culture: Pick a monoclonal to be tested and inoculate it into a small test tube (15 mm × 100 mm) containing 3 mL of LB liquid medium, and culture it overnight at 37 °C and 220 rpm to obtain a seed solution.

[0153] 2) Scale-up culture: Transfer the seed culture medium at 1% (V / V) to 200 mL of synthetic medium (in a 1000 mL Erlenmeyer flask) for culture, and culture it overnight at 37 °C and 220 rpm. Wait until the OD 600 reaches 3 - 5 for inoculation.

[0154] 3) Fermentation culture: Inoculate the culture solution from step 2) at 2% (V / V) into a 5 L fermenter medium, and perform anaerobic fermentation at 37 °C and 300 rpm for 50 - 61 h (taking the consumption of glucose as the end point of fermentation).

[0155] 4) Detection and analysis: Take the fermented broth after the culture in step (3) to measure the OD 600 , and use HPLC to detect the contents of L-valine and L-alanine in the fermented broth.

[0156] 2.4 Performance evaluation of glutamate-pyruvate aminotransferase AlaA mutant

[0157] Five glutamate-pyruvate aminotransferase AlaA "dominant" mutants screened from the DNA shuffling library in step 2.3, namely AlaA-mT-1, AlaA-mT-2, AlaA-mT-3, AlaA-mT-4, AlaA-mT-5 (see Table 3), and the corresponding strains Sval031-2, Sval031-3, Sval031-4, Sval031-5, Sval031-6 (see Table 4) containing the mutants were subjected to 5 L fermenter fermentation, and the fermentation conditions were the same as in step 3) of 2.3.2; The Sval031-1 strain was used as the control strain, and the L-valine yield, L-pyruvate content, and strain growth status (OD 600 ) of the fermented broth were measured.

[0158] Table 3: Sequence changes of glutamate-pyruvate aminotransferase mutant AlaA-mT

[0159]

[0160] Table 4: Comparison of fermentation results of strains after introducing mutants in the present invention

[0161]

[0162] Table 5: Results of repeated fermentation of the mutant strain Sval031-6 in the present invention

[0163]

[0164] As can be seen from Table 4, using Sval031 containing wild-type glutamate-pyruvate aminotransferase AlaA as the starting strain, the L-alanine yields of the 5 AlaA mutants (Sval031-2, Sval031-3, Sval031-4, Sval031-5, Sval031-6) obtained by combining gene editing technology and gene shuffling technology are all lower than that of the starting strain. However, the growth conditions of each mutant are basically the same, without significant reduction, which lays a foundation for the production of valine by microbial fermentation method; among them, the content of L-alanine in the fermentation broth of the mutant Sval031-6 is the lowest, reaching 0.03±0.01 g / L (500 ppm), which is 10 times lower than that of the starting gene AlaA. And as can be seen from Table 5, the fermentation is stable and more suitable for industrial production of valine.

[0165] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although the present invention gives specific embodiments, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modification, use or improvement of the present invention, including changes made by using conventional techniques known in the art that depart from the scope disclosed in this application. Some basic features can be applied according to the scope of the appended claims below.

Claims

1. A protein, characterized in that it is A glutamate-pyruvate aminotransferase mutant with reduced enzyme activity obtained by replacing any one or more of the amino acid residues at positions 15, 276, 292, and 296 based on the wild-type glutamate-pyruvate aminotransferase amino acid sequence shown in SEQ ID NO:

1.

2. The protein according to claim 1, characterized in that The replacement is to mutate the amino acid residues; the mutation methods for each amino acid residue are as follows: The tyrosine Y at position 15 is mutated to arginine R; The leucine L at position 276 is mutated to isoleucine I; The glycine G at position 292 is mutated to arginine R; The isoleucine I at position 296 is mutated to serine S.

3. The protein according to claim 2, characterized in that The glutamate-pyruvate aminotransferase mutant contains any one of the following amino acid mutations A1)-A5) based on the wild-type glutamate-pyruvate aminotransferase amino acid sequence shown in SEQ ID NO:1: A1) Amino acid mutation at 1 site: Y15R; A2) Amino acid mutation at 1 site: L276I; A3) Amino acid mutations at two sites: Y15R, I296S; A4) Amino acid mutations at two sites: L276I, G292R; A5) Amino acid mutations at four sites: Y15R, L276I, G292R, I296S.

4. A nucleic acid molecule encoding the protein according to any one of claims 1-3.

5. An expression cassette or recombinant vector containing the nucleic acid molecule according to claim 4.

6. A recombinant microorganism expressing the protein according to any one of claims 1-3.

7. The recombinant microorganism according to claim 6, characterized in that Obtained by introducing the nucleic acid molecule according to claim 5 or the expression cassette or recombinant vector according to claim 6 into a host bacterium; the host bacterium is Escherichia coli.

8. The recombinant microorganism according to claim 7, characterized in that The recombinant microorganism also has a valine-pyruvate aminotransferase AvtA with reduced or inactivated activity.

9. Use of the protein according to any one of claims 1-3 in any one of the following B1)-B6); Or, use of the nucleic acid molecule according to claim 4 in any one of the following B1)-B6); Or, use of the expression cassette or recombinant vector according to claim 5 in any one of the following B1)-B6); Or, use of the recombinant microorganism according to any one of claims 6-8 in any one of the following B1), B3)-B6): B1) Preparing glutamate-pyruvate aminotransferase; B2) Constructing a recombinant engineering bacterium producing L-valine, L-leucine or L-isoleucine; B3) Producing L-valine, L-leucine or L-isoleucine; B4) Increasing the yield of L-valine, L-leucine or L-isoleucine; B5) Reducing the accumulation of L-alanine; B6) Increasing the yield of L-valine, L-leucine or L-isoleucine and reducing the accumulation of L-alanine.

10. A production method for producing L-valine, characterized in that, Including: (1) Fermentatively culturing the recombinant microorganism according to any one of claims 6-8; (2) Separating and harvesting L-valine.

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