An isopropylmalate synthase mutant and its application

Abstract

The present invention belongs to the field of biotechnology, and particularly relates to an isopropylmalate synthase mutant and its application. The isopropylmalate synthase mutant is randomly mutated based on the error-prone PCR technology, greatly improving its enzyme activity; the L-leucine yield of the recombinant strain constructed with the mutant is significantly increased, having great practical value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an isopropylmalate synthase mutant and its application. Background Art

[0002] L-Leucine is an essential branched-chain amino acid, which is crucial for human health and has wide applications in the fields of medicine, food, cosmetics, etc. In the medical field, it is used in amino acid injection solutions to promote insulin secretion, maintain the nutrition of critically ill patients, and treat various diseases, including the synthesis of anticancer complexes; in the food industry, as a food additive, it enhances flavor, improves nutrition, and promotes muscle recovery after exercise; in the cosmetics field, leucine, as a natural moisturizing factor, helps skin nutrition supply and improves skin and hair health; in the agricultural field, L-leucine is used as a fertilizer component to promote crop growth and improve soil.

[0003] With the continuous growth of the demand for leucine, the development of its production technology has attracted increasing attention. Currently, the industrial production methods of leucine mainly include protein hydrolysis method, chemical synthesis method, and enzymatic conversion method. However, these methods have some limitations, such as complex processes, difficult separation and purification, and serious environmental pollution. In contrast, although the enzymatic conversion method has the advantages of simple process and high product purity, it is limited by factors such as expensive precursor substances and low conversion rate, which restricts its large-scale industrial production. The microbial fermentation method is considered to be a more favorable production method for large-scale industrial application due to its advantages of environmental friendliness, low cost, and easy reaction control. However, the metabolic network of microbial synthesis of leucine is complex, and problems such as poor specificity and low conversion rate of the key rate-limiting enzymes in the synthesis pathway limit its industrialization process. Therefore, improving the conversion rate of microbial synthesis of leucine and solving the specificity problem of the key enzymes in the synthesis pathway are the technical problems that need to be solved urgently at present.

[0004] α-Isopropylmalate synthase (IPMS), encoded by the leuA gene, is the core rate-limiting enzyme in the L-leucine biosynthesis pathway. This enzyme is responsible for catalyzing the aldol condensation reaction between α-ketoisovalerate and acetyl coenzyme A to generate α-isopropylmalate, which is a key step in the synthesis of L-leucine. As L-leucine accumulates during the synthesis process, the activity of IPMS will be inhibited by its metabolites, and this feedback inhibition will gradually increase. Therefore, in order to increase the yield of L-leucine, a key strategy is to weaken or eliminate the inhibitory effect of leucine on IPMS, thereby relieving its metabolic control on L-leucine synthesis. We modified IPMS to make it less sensitive to the feedback inhibition of L-leucine, thereby improving the biosynthesis efficiency of L-leucine. This method can promote the accumulation of L-leucine, which is of great significance for improving production efficiency and reducing costs. Summary of the Invention

[0005] As the key rate-limiting enzyme in the L-leucine synthesis pathway, the specificity and catalytic efficiency of leuA play a decisive role in the biosynthesis of L-leucine. To solve the feedback inhibition of leucine on LeuA and thereby improve the yield and conversion rate of L-leucine, the present invention performs error-prone PCR-mediated random mutagenesis on the leuA gene to obtain mutants that are less sensitive to the feedback inhibition of leucine, and then constructs a series of strains containing the mutants; subsequently, fermentation tests are carried out to verify these strains containing the leuA mutants. The specific research method is, taking SvalM029 bacteria as the chassis strain (CN117304283A) as an example, first blocking the production of valine, that is, knocking out the ilvE, avtA, and leuDH genes to construct a recombinant strain; the second step is to perform random mutagenesis on the leuA gene using error-prone PCR technology; the third step is to construct a strain containing the leuA mutant, and through fermentation tests, screen for leuA mutant strains with increased yield and conversion rate of L-leucine.

[0006] Through this method, the present invention is proposed by successfully obtaining a LeuA mutant that can significantly improve the yield and conversion rate of L-leucine.

[0007] The present invention provides an isopropylmalate synthase mutant, which has a substitution mutation at at least one of the following sites in the amino acid sequence compared to the wild-type isopropylmalate synthase: R308, H390, T407, or G462.

[0008] Specifically, the amino acid sequence of the wild-type isopropylmalate synthase is SEQ ID NO:2, and its nucleotide sequence is SEQ ID NO:1.

[0009] Specifically, it has a substitution mutation at at least one of the following sites in the amino acid sequence compared to the wild-type isopropylmalate synthase: R308S, H390N, T407I, or G462D.

[0010] More specifically, it has the following substitution mutations in the amino acid sequence compared to the wild-type isopropylmalate synthase: G462D; R308S; H390N; T407I; R308S and G462D; H390N and G462D; T407I and G462D; R308S and T407I; R308S, T407I, and G462D.

[0011] The present invention also provides the coding nucleic acid of the isopropylmalate synthase mutant.

[0012] The present invention also provides a recombinant vector containing the coding nucleic acid.

[0013] The present invention also provides a recombinant bacterium expressing the isopropylmalate synthase mutant. Preferably, it is a Klebsiella strain, Corynebacterium glutamicum, Bacillus subtilis, Brevibacterium flavum, yeast or Escherichia coli.

[0014] Specifically, the coding nucleic acid of the isopropylmalate synthase mutant can exist in the initial strain in any suitable manner known in the art, such as in the form of a vector (a recombinant vector containing the coding nucleic acid) or integrated into the genome, to obtain a recombinant bacterium expressing the isopropylmalate synthase mutant.

[0015] The present invention also provides the use of the isopropylmalate synthase mutant, the coding nucleic acid of the isopropylmalate synthase mutant, the recombinant expression vector containing the coding nucleic acid, or the recombinant bacterium expressing the isopropylmalate synthase mutant in the preparation of L-leucine.

[0016] The present invention further provides a recombinant bacterium for producing L-leucine, which is obtained by the following method:

[0017] S1: Construct a general-purpose chassis bacterium for the initial strain, specifically by blocking the production of valine and / or isoleucine;

[0018] S2: Introduce the coding nucleic acid of the isopropylmalate synthase mutant into the general-purpose chassis bacterium to obtain a recombinant bacterium for producing L-valine;

[0019] Preferably, the initial strain is Escherichia coli;

[0020] More preferably, the blocking of valine production is achieved by knocking out or weakening the ilvE, avtA and leuDH genes.

[0021] The present invention further provides a method for preparing L-leucine, which includes the step of fermenting and culturing the recombinant bacterium to obtain L-leucine, and optionally further includes the step of separating the L-leucine.

[0022] Specifically, the conditions for the fermentation culture are culturing at 37°C and 200 r / min in a fermentation medium.

[0023] The present invention further provides the use of the isopropylmalate synthase mutant, the coding nucleic acid of the isopropylmalate synthase mutant, the recombinant expression vector containing the coding nucleic acid, or the recombinant bacterium expressing the isopropylmalate synthase mutant in increasing the yield and / or conversion rate of L-leucine.

[0024] Specifically, the conversion rate is the conversion rate of the sugar source to L-leucine, such as the conversion rate of glucose to L-leucine (molar conversion rate or mass conversion rate).

[0025] Based on error-prone PCR technology, the present invention randomly mutated to obtain mutant strains containing leuA mutants. Among them, the Ls004 strain had the best effect, with a shake-flask L-leucine yield reaching 1.29 g / L and a conversion rate reaching 10%. Detailed implementation methods

[0026] Materials, reagents, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0027] 1. The strains and plasmids constructed in this study are shown in Table 1 in detail, and the primers used are shown in Table 2 in detail.

[0028] Table 1. Strains and plasmids used in the present invention

[0029]

[0030]

[0031] Table 2. Primers used in the present invention

[0032]

[0033]

[0034] 2. Biological materials involved in the following examples:

[0035] 2×Phanta Mix high-fidelity enzyme is a product of Novizan Company, catalog number: P525-01.

[0036] DH5α competent cells are products of Novizan Company, catalog number C502-03.

[0037] QuickMutation TM The gene random mutation kit is a product of Beyotime Company, number D0219S.

[0038] Example 1. Construction of recombinant strains Ls001, Ls002 and Ls003

[0039] 1. Construction of Ls001 strain: SvalM029ΔilvE

[0040] Using specific primers ΔilvE-up-F / ΔilvE-up-R, with the SvalM029 genome as a template, the upstream homologous arm UP fragment was amplified by PCR technology;

[0041] Using specific primers ΔilvE-down-F / ΔilvE-down-R, with the SvalM029 genome as a template, the downstream homologous arm DOWN fragment was amplified by PCR;

[0042] The UP and DOWN were ligated using the overlap PCR technique to obtain the homologous recombination fragment UP-DOWN (ΔilvE-donor).

[0043] Using the specific primers ΔilvE-sgRNA-F / ΔilvE-sgRNA-R, with the pTarget plasmid as the template, the pTarget-ΔilvE-sgRNA fragment was amplified and transformed into DH5α competent cells to obtain the pTarget-ΔilvE-sgRNA targeting plasmid.

[0044] 1) The PCR reaction system (50 μL) was as follows: 20 μL of ddH2O, 2 μL of upstream primer (10 mM), 2 μL of downstream primer (10 mM), 1 μL of genomic DNA template, and 25 μL of 2×Phanta Mix high-fidelity enzyme;

[0045] The PCR reaction program was as follows: Step 1: 95°C, 3 min; Step 2: 95°C, 15 s, 60°C, 10 s; Step 3: 72°C, 1 min; this step was repeated 30 cycles, 72°C, 5 min. Step 4: Store at 4°C.

[0046] The ΔilvE-UP, ΔilvE-DOWN, and pTarget-ΔilvE-sgRNA fragments were obtained by PCR amplification.

[0047] 2) The overlap PCR reaction system (50 μL) was as follows: 25 μL of 2×Phanta Mix high-fidelity enzyme, 1 μL each of the ΔilvE-UP and ΔilvE-DOWN fragments, 2 μL each of ΔilvE-up-F (10 mM) and ΔilvE-down-R (10 mM), and 19 μL of ddH2O;

[0048] The overlap PCR reaction program was as follows: Step 1: 95°C, 3 min; Step 2: 95°C, 15 s, 60°C, 10 s; Step 3: 72°C, 2 min; this step was repeated 30 cycles, 72°C, 5 min. Step 4: Store at 4°C. The ΔilvE-donor fragment was obtained.

[0049] 3) Transformation: The pTarget-ΔilvE-sgRNA fragment obtained by PCR amplification was transformed into DH5α competent cells to obtain the pTarget-ΔilvE-sgRNA targeting plasmid.

[0050] 4) Obtaining the Ls001 recombinant strain: SvalM029ΔilvE

[0051] a. Preparation of SvalM029 Escherichia coli competent cells: Pick a monoclonal colony of the SvalM029 Escherichia coli strain and inoculate it into a 500 mL Erlenmeyer flask containing 100 mL of LB liquid medium. Incubate at 37 °C and 200 r / min until the OD550 is approximately 0.5 - 0.6. Ice-bath for 20 min. Transfer to a 50 mL sterile centrifuge tube and centrifuge at 4 °C and 5000 r / min for 6 min. Discard the supernatant, resuspend the pellet in 20 mL of 10% glycerol, centrifuge at 4 °C and 5000 r / min for 6 min. Repeat the above step twice. Finally, resuspend the cells in 1 mL of 10% glycerol, aliquot 100 μL per tube, and store at -80 °C for later use.

[0052] b. Transformation of pECas9 plasmid: Take 1 μL of pECas9 plasmid and 100 μL of the above-mentioned SvalM029 competent cells and add them to an electroporation cuvette. Mix well and place on ice for 5 min. Dry the electroporation cuvette, perform electroporation at 2.5 KV and immediately add 1 mL of pre-cooled LB medium. Pipette a few times to mix well and transfer to a test tube. Incubate at 37 °C and 200 r / min for 1 - 2 h. Take 100 μL of the bacterial solution and spread it evenly on a solid LB plate containing ampicillin antibiotic (final concentration 100 μg / mL). Incubate at 37 °C overnight.

[0053] c. Preparation of SvalM029 / pECas9 strain competent cells: Pick a monoclonal colony of the SvalM029 / pECas9 strain and inoculate it into 100 mL of LB medium containing 5% L-arabinose (Amp resistant). Incubate at 37 °C and 200 rpm for 3 - 4 h until OD600 = 0.5 - 0.6. Ice-bath for 20 min. Transfer to a 50 mL sterile centrifuge tube and centrifuge at 4 °C and 5000 r / min for 6 min. Discard the supernatant, resuspend the pellet in 20 mL of 10% glycerol, centrifuge at 4 °C and 5000 r / min for 6 min. Repeat the above step twice. Finally, resuspend the cells in 1 mL of 10% glycerol, aliquot 100 μL per tube, and store at -80 °C for later use.

[0054] d. Transformation of pTarget-ΔilvE-sgRNA plasmid and ΔilvE-donor fragment: Take 2 μL each of the above-constructed pTarget-ΔilvE-sgRNA plasmid and ΔilvE-donor fragment and 100 μL of SvalM029 / pECas9 strain competent cells and add them to an electroporation cuvette. Mix well and ice-bath for 5 min. Incubate at 37 °C and 200 r / min for 1 - 2 h. Centrifuge and spread all on a solid LB plate containing ampicillin antibiotic and spectinomycin (final concentrations are 100 μg / mL and 100 μg / mL respectively). Incubate at 37 °C overnight.

[0055] e. Use ilvE-up-F / ilvE-down-R verification primers for single colony PCR verification, and send the strains with correct verification for sequencing. The strains with correct sequencing are the strains with successful gene editing, named Ls001.

[0056] 2. Construction of Ls002 strain: SvalM029ΔilvEΔleuDH

[0057] Use specific primers ΔleuDH-up-F / ΔleuDH-up-R, using the SvalM029 genome as a template, and amplify the upstream homologous arm UP fragment by PCR technology;

[0058] Use specific primers ΔleuDH-down-F / ΔleuDH-down-R, using the SvalM029 genome as a template, and PCR amplify the downstream homologous arm DOWN fragment;

[0059] Adopt overlapping PCR technology to connect UP and DOWN to obtain the homologous recombination fragment UP-DOWN (ΔleuDH-donor).

[0060] Use specific primers ΔleuDH-sgRNA-F / ΔleuDH-sgRNA-R, using the pTarget plasmid as a template, amplify the pTarget-ΔleuDH-sgRNA fragment, and transform it into DH5α competent cells to obtain the pTarget-ΔleuDH-sgRNA targeting plasmid.

[0061] 1) The PCR reaction system (50 μL) is: 20 μL of ddH2O, 2 μL of upstream primer (10 mM), 2 μL of downstream primer (10 mM), 1 μL of genomic DNA template, and 25 μL of 2×Phanta Mix high-fidelity enzyme;

[0062] The PCR reaction program is: Step 1: 95°C, 3 min; Step 2: 95°C, 15 s, 60°C, 10 s; Step 3: 72°C, 1 min; This step is repeated 30 cycles, 72°C, 5 min. Step 4: Store at 4°C.

[0063] PCR amplification obtains the ΔleuDH-UP and ΔleuDH-DOWN and pTarget-ΔleuDH-sgRNA fragments.

[0064] 2) The overlapping PCR reaction system (50 μL) is: 25 μL of 2×Phanta Mix high-fidelity enzyme, 1 μL each of the ΔleuDH-UP and ΔleuDH-DOWN fragments, 2 μL each of ΔleuDH-up-F (10 mM) and ΔleuDH-down-R (10 mM), and 19 μL of ddH2O;

[0065] The overlapping PCR reaction procedure is as follows: Step 1: 95°C, 3 min; Step 2: 95°C, 15 s, 60°C, 10 s; Step 3: 72°C, 2 min; this step is repeated 30 cycles, 72°C, 5 min. Step 4: Store at 4°C. The ΔleuDH-donor fragment is obtained.

[0066] 3) Transformation: The pTarget-ΔleuDH-sgRNA fragment obtained by PCR amplification is transformed into DH5α competent cells to obtain the pTarget-ΔleuDH-sgRNA targeting plasmid.

[0067] 4) Obtaining the Ls002 recombinant strain:

[0068] a. Preparation of Ls001 competent cells: Pick a single colony of the Ls001 strain and inoculate it into a 500 mL Erlenmeyer flask containing 100 mL of LB liquid medium. Culture at 37°C and 200 r / min until the OD550 is about 0.5 - 0.6. Ice bath for 20 min; transfer to a 50 mL sterile centrifuge tube, centrifuge at 4°C and 5000 r / min for 6 min; discard the supernatant, resuspend the pellet with 20 mL of 10% glycerol, centrifuge at 4°C and 5000 r / min for 6 min; repeat the above operation twice, and finally resuspend the cells with 1 mL of 10% glycerol. Aliquot 100 μL per tube and store at -80°C for later use.

[0069] b. Transformation of the pECas9 plasmid: Take 1 μL of the pECas9 plasmid and 100 μL of Ls001 competent cells and add them to an electroporation cuvette. After mixing evenly, place it on ice for 5 min; dry the electroporation cuvette, perform electroporation at 2.5 KV and immediately add 1 mL of pre-cooled LB medium and pipette several times to mix evenly and transfer it to a test tube; culture at 37°C and 200 r / min for 1 - 2 h; take 100 μL of the bacterial solution and spread it evenly on a solid LB plate with ampicillin antibiotic (final concentration 100 μg / mL) and culture at 37°C overnight.

[0070] c. Preparation of Ls001 / pECas9 competent cells: Pick a single colony of the Ls001 / pECas9 strain and inoculate it into 100 mL of LB medium containing 5% L-arabinose (Amp resistant). Culture at 37°C and 200 rpm for 3 - 4 h until the OD600 = 0.5 - 0.6; ice bath for 20 min; transfer to a 50 mL sterile centrifuge tube, centrifuge at 4°C and 5000 r / min for 6 min; discard the supernatant, resuspend the pellet with 20 mL of 10% glycerol, centrifuge at 4°C and 5000 r / min for 6 min; repeat the above operation twice, and finally resuspend the cells with 1 mL of 10% glycerol. Aliquot 100 μL per tube and store at -80°C for later use.

[0071] Transformation of pTarget-ΔleuDH-sgRNA plasmid and ΔleuDH-donor fragment: Take 2 μL of the above-constructed pTarget-ΔleuDH-sgRNA plasmid and 100 μL of the ΔleuDH-donor fragment and add them to an electroporation cup containing 100 μL of Ls001 / pECas9 competent cells. After mixing, incubate on ice for 5 min; culture at 37 °C and 200 r / min for 1 - 2 h; centrifuge and spread all on a solid LB plate with ampicillin antibiotic and spectinomycin (final concentrations are 100 μg / mL and 100 μg / mL respectively), and culture overnight at 37 °C.

[0072] e. Use leuDH-up-F / leuDH-down-R verification primers for single colony PCR verification, and send the strains with correct verification for sequencing. The strains with correct sequencing are the strains with successful gene editing, named Ls002.

[0073] 3. Construction of Ls003 strain: SvalM029ΔilvEΔleuDHΔavtA

[0074] Use specific primers ΔavtA-up-F / ΔavtA-up-R, and use the SvalM029 genome as a template to amplify the upstream homologous arm UP fragment by PCR technology;

[0075] Use specific primers ΔavtA-down-F / ΔavtA-down-R, and use the SvalM029 genome as a template to PCR amplify the downstream homologous arm DOWN fragment;

[0076] Use overlap PCR technology to connect UP and DOWN to obtain the homologous recombination fragment UP-DOWN (ΔavtA-donor).

[0077] Use specific primers ΔavtA-sgRNA-F / ΔavtA-sgRNA-R, and use the pTarget plasmid as a template to amplify the pTarget-ΔavtA-sgRNA fragment, and transform it into DH5α competent cells to obtain the pTarget-ΔavtA-sgRNA targeting plasmid.

[0078] 1) The PCR reaction system (50 μL) is: 20 μL of ddH2O, 2 μL of upstream primer (10 mM), 2 μL of downstream primer (10 mM), 1 μL of genomic DNA template, and 25 μL of 2×Phanta Mix high-fidelity enzyme;

[0079] The PCR reaction procedure is as follows: Step 1: 95°C for 3 min; Step 2: 95°C for 15 s, 60°C for 10 s; Step 3: 72°C for 1 min; this step is repeated 30 cycles, and then 72°C for 5 min. Step 4: Store at 4°C.

[0080] The ΔavtA-UP, ΔavtA-DOWN, and pTarget-ΔavtA-sgRNA fragments were obtained by PCR amplification.

[0081] 2) The overlapping PCR reaction system (50 μL) is as follows: 25 μL of 2×Phanta Mix high-fidelity enzyme, 1 μL each of the ΔavtA-UP and ΔavtA-DOWN fragments, 2 μL each of ΔavtA-up-F (10 mM) and ΔavtA-down-R (10 mM), and 19 μL of ddH2O;

[0082] The overlapping PCR reaction procedure is as follows: Step 1: 95°C for 3 min; Step 2: 95°C for 15 s, 60°C for 10 s; Step 3: 72°C for 2 min; this step is repeated 30 cycles, and then 72°C for 5 min. Step 4: Store at 4°C. The ΔavtA-donor fragment was obtained.

[0083] 3) Transformation: The pTarget-ΔavtA-sgRNA fragment obtained by PCR amplification was transformed into DH5α competent cells to obtain the pTarget-ΔavtA-sgRNA targeting plasmid.

[0084] 4) Obtaining the Ls003 recombinant strain:

[0085] a. Preparation of Ls002 competent cells: Pick a single colony of the Ls002 strain and inoculate it into a 500 mL Erlenmeyer flask containing 100 mL of LB liquid medium. Culture at 37°C and 200 r / min until the OD550 is about 0.5 - 0.6, then ice-bath for 20 min; Transfer to a 50 mL sterile centrifuge tube, centrifuge at 4°C and 5000 r / min for 6 min; Discard the supernatant, resuspend the pellet with 20 mL of 10% glycerol, centrifuge at 4°C and 5000 r / min for 6 min; Repeat the above operation twice, and finally resuspend the cells with 1 mL of 10% glycerol. Aliquot 100 μL per tube and store at -80°C for later use.

[0086] b. Transformation of pECas9 plasmid: Take 1 μL of pECas9 plasmid and 100 μL of Ls002 competent cells and add them to an electroporation cup. After mixing evenly, place it on ice for 5 min; dry the electroporation cup, perform electroporation at 2.5 KV and immediately add 1 mL of pre-cooled LB medium, pipette several times to mix evenly, and transfer it into a test tube; culture at 37 °C and 200 r / min for 1 - 2 h; take 100 μL of the bacterial solution and spread it evenly on a solid LB plate with ampicillin antibiotic (final concentration 100 μg / mL), and culture at 37 °C overnight.

[0087] c. Preparation of competent cells of Ls002 / pECas9 strain: Pick a single colony of Ls002 / pECas9 strain and inoculate it into 100 mL of LB medium containing 5% L - arabinose (Amp resistant), culture at 37 °C and 200 rpm for 3 - 4 h until OD600 = 0.5 - 0.6; ice bath for 20 min; transfer it into a 50 mL sterile centrifuge tube, centrifuge at 4 °C and 5000 r / min for 6 min; discard the supernatant, resuspend the precipitate with 20 mL of 10% glycerol, centrifuge at 4 °C and 5000 r / min for 6 min; repeat the above operation twice, finally add 1 mL of 10% glycerol to resuspend the bacteria, aliquot 100 μL per tube and store it at -80 °C for later use.

[0088] d. Transformation of pTarget - ΔavtA - sgRNA plasmid and ΔavtA - donor fragment: Take 2 μL of each of the above - constructed pTarget - ΔavtA - sgRNA plasmid and ΔavtA - donor fragment and 100 μL of Ls002 / pECas9 competent cells and add them to an electroporation cup. After mixing evenly, place it on ice for 5 min; culture at 37 °C and 200 r / min for 1 - 2 h; centrifuge and spread all on a solid LB plate with ampicillin antibiotic and spectinomycin (final concentrations are 100 μg / mL and 100 μg / mL respectively), and culture at 37 °C overnight.

[0089] e. Use avtA - up - F / avtA - down - R verification primers for single - colony PCR verification, and send the strains with correct verification for sequencing. The strains with correct sequencing are the strains with successful gene editing, named Ls003.

[0090] Example 2. Construction of leuA mutant strain

[0091] 1. Construction of a fragment containing the leuA mutant:

[0092] The NCBI accession number of the amino acid sequence encoded by the leuA gene is WP_000082850.1 (SEQ ID NO: 2); the NCBI accession number of its nucleotide sequence is NC_000913.3 81958-83529 (-) (SEQ ID NO: 1). Using the QuickMutation TM Gene Random Mutation Kit from Beyotime, with the leuA-F / leuA-R primers, and using the SvalM029 genome as a template, PCR amplified the fragment containing the leuA mutant.

[0093] The PCR reaction system (50 μL) was: 30 μL of ddH2O, 2 μL of upstream primer (10 mM), 2 μL of downstream primer (10 mM), 1 μL of DNA template, 5 μL of RandomMut buffer (10X), 4 μL of Mutation enhancer (10X), 5 μL of dNTP (2.5 mM each), 1 μL of RandomMut DNA polymerase;

[0094] The PCR reaction program was: Step 1: 94 °C, 3 min; Step 2: 94 °C, 30 s, 55 °C, 30 s; Step 3: 72 °C, 1.5 min; this step was repeated 30 cycles, 72 °C, 10 min. Step 4: Store at 4 °C. The fragment containing the leu mutant was obtained by PCR amplification.

[0095] 2. Construct the leuA mutant editing element for genome integration: Using the specific primers ΔycjV-up-F / ΔycjV-up-R, with the SvalM029 genome as a template, the upstream homologous arm UP fragment was amplified by PCR technology; using the specific primers ΔycjV-down-F / ΔycjV-down-R, with the SvalM029 genome as a template, the downstream homologous arm DOWN fragment was PCR amplified; the UP, the leuA fragment containing the mutant, and the DOWN were connected by overlap PCR to obtain the homologous recombination fragment UP-leuA-DOWN (ΔycjV::leuA-donor). The pTarget-ΔycjV-sgRNA fragment was amplified using the ΔycjV-sgRNA-F / ΔycjV-sgRNA-R primers and transformed into DH5α competent cells to obtain the pTarget-ΔycjV-sgRNA targeting plasmid.

[0096] 1) The PCR reaction system (50 μL) was: 20 μL of ddH2O, 2 μL of upstream primer (10 mM), 2 μL of downstream primer (10 mM), 1 μL of plasmid template, 25 μL of 2×Phanta Mix high-fidelity enzyme;

[0097] The PCR reaction procedure is as follows: Step 1: 95°C, 3 min; Step 2: 95°C, 15 s, 60°C, 10 s; Step 3: 72°C, 3 min; this step is repeated 30 cycles, 72°C, 5 min. Step 4: Store at 4°C. ΔycjV-UP and ΔycjV-DDOWN are obtained by PCR amplification.

[0098] 2) The overlapping PCR reaction system (50 μL) is as follows: 25 μL of 2×Phanta Mix high-fidelity enzyme, 1 μL each of ΔycjV-UP, the fragment containing the leuA mutant, and the ΔycjV-DOWN fragment, 2 μL each of ΔycjV-up-F (10 mM) and ΔycjV-down-R (10 mM), 18 μL of ddH2O; The overlapping PCR reaction procedure is as follows: Step 1: 95°C, 3 min; Step 2: 95°C, 15 s, 60°C, 10 s; Step 3: 72°C, 2 min; this step is repeated 30 cycles, 72°C, 5 min. Step 4: Store at 4°C. The ΔycjV-donor fragment is obtained.

[0099] 3) Transformation: The pTarget-ΔycjV-sgRNA fragment obtained by PCR amplification is transformed into DH5α competent cells to obtain the pTarget-ΔycjV-sgRNA targeting plasmid.

[0100] 4) Obtaining the recombinant strain containing the leuA mutant: The constructed pTarget-ΔycjV-sgRNA plasmid and the ΔycjV::leuA-donor fragment are electrotransformed into the strain Ls003 for gene editing. All the single colonies grown on the plate are stored in a -80°C refrigerator for later 48-well deep plate testing.

[0101] Example 3: Fermentation of mutants in 48-well deep plates

[0102] The bacteria containing the leuA mutant and the control strain Leu003 are inoculated into a 48-well deep plate containing fermentation medium and cultured at 37°C, 220 r / min for 48 h. Then, the fermentation broth is taken out, and the content of L-leucine is detected by HPLC. The strains with leucine accumulation are subjected to leuA gene sequencing.

[0103] The leuA mutation sites corresponding to the mutant strains of the present invention are as follows:

[0104] Leucine strain Ls004: leuA (R308S, T407I, and G462D)

[0105] Leucine strain Ls005: leuA (H390N and G462D)

[0106] Leucine strain Ls006: leuA(H390N)

[0107] Leucine strain Ls007: leuA(G462D)

[0108] Leucine strain Ls008: leuA(R308S)

[0109] Leucine strain Ls009: leuA(T407I)

[0110] Leucine strain Ls010: leuA(R308S and G462D)

[0111] Leucine strain Ls011: leuA(T407I and G462D)

[0112] Leucine strain Ls012: leuA(R308S and T407I).

[0113] Among them, the HPLC detection method for L-leucine: After the sample is diluted 10 times, it is centrifuged at 12000 r / min for 10 min, and the supernatant is filtered through a filter membrane and then measured by HPLC.

[0114] Chromatographic column: primesep 100 chromatographic column

[0115] Mobile phase: 0.05% sulfuric acid, 35% acetonitrile, 64.95% water;

[0116] Detector: UV detector

[0117] The column temperature of the chromatographic column is 35 °C

[0118] Detection wavelength: 200 nm

[0119] Fermentation medium: glucose 20 g / L, ammonium sulfate 3 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 2 g / L, betaine 0.2 g / L, valine 0.2 g / L, isoleucine 0.2 g / L, 1X trace element mother liquor: 1 mL, biotin mother liquor (0.4 g / L): 15 mL.

[0120] Table 3. Formulation table of trace element mother liquor

[0121]

[0122] Example 4. Shake flask fermentation of control bacteria and recombinant bacteria containing leuA mutants

[0123] The above-mentioned strains Ls004-Ls012 containing mutants and the control strain Ls003 (containing wild-type leuA) were inoculated into an LB liquid test tube and cultured overnight at 37°C and 200 r / min. The above-mentioned culture broth was transferred to 50 mL of fermentation medium at an inoculation amount of 1% respectively, and after culturing at 37°C and 200 r / min for 48 h, 1 mL of the fermentation broth was taken, and the L-leucine yield was detected by HPLC method. The results are shown in Table 4. Among them, the sugar-acid conversion rate is the mass conversion rate of glucose converted into L-leucine.

[0124] Table 4. Fermentation results of L-leucine

[0125]

[0126] The fermentation results showed that the leucine yields of the mutant strains containing leuA were all increased. Among them, the mutant strain Ls004 had the best effect, and the L-leucine yield in the shake flask reached 1.29 g / L, and the conversion rate reached 10%.

Claims

1. An isopropylmalate synthase mutant, characterized in that, It is any one of the following substitution mutations (1)-(4) made to the wild-type isopropylmalate synthase with the amino acid sequence shown in SEQ ID NO: 2: (1) R308S; (2) R308S and G462D; (3) R308S and T407I; (4) R308S, T407I and G462D.

2. The coding nucleic acid of the isopropylmalate synthase mutant according to claim 1.

3. A recombinant vector containing the coding nucleic acid according to claim 2.

4. A recombinant bacterium expressing the isopropylmalate synthase mutant according to claim 1.

5. The recombinant bacterium according to claim 4, characterized in that, It is a Klebsiella strain, Corynebacterium glutamicum, Bacillus subtilis, Brevibacterium flavum, yeast or Escherichia coli.

6. The application of the isopropylmalate synthase mutant according to claim 1, the coding nucleic acid according to claim 2, the recombinant vector according to claim 3, or the recombinant bacterium according to claim 4 or 5 in the preparation of L-leucine.

7. A recombinant bacterium for producing L-leucine, characterized in that, Obtained by the following method: S1: Construct a general chassis bacterium by blocking the production of valine and / or isoleucine in the initial strain; S2: Introduce the coding nucleic acid according to claim 2 into the general chassis bacterium to obtain an L-leucine-producing recombinant bacterium.

8. The recombinant bacterium for producing L-leucine according to claim 7, wherein The initial strain is Escherichia coli.

9. The recombinant bacterium for producing L-leucine according to claim 7, characterized in that, The blocking of valine production is achieved by knocking out or weakening the ilvE, avtA and leuDH genes.

10. A method for preparing L-leucine, characterized in that, It includes the step of fermenting and culturing the recombinant bacterium according to any one of claims 7 to 9 to obtain L-leucine.

11. The method according to claim 10, wherein It further includes the step of separating the L-leucine.

12. The application of the isopropylmalate synthase mutant according to claim 1, the coding nucleic acid according to claim 2, the recombinant vector according to claim 3, or the recombinant bacterium according to claim 4 or 5 in increasing the yield and / or conversion rate of L-leucine.

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