Isopropyl malic acid synthase mutant and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an isopropyl malic acid synthase mutant and application thereof. The isopropyl malic acid synthase mutant is obtained based on random mutation of an error-prone PCR technology, and the enzyme activity of the isopropyl malic acid synthase mutant is greatly improved; the L-leucine yield of a recombinant strain constructed by the mutant is remarkably improved, and the mutant has a relatively high practical value.

Description

Technical Field

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

[0002] Leucine (L-Leucine) is an essential branched-chain amino acid, which is vital to human health and has a wide range of applications in the fields of medicine, food, cosmetics, etc. In the field of medicine, it is used in amino acid injections to promote insulin secretion, maintain nutrition for critically ill patients, and treat a variety of diseases, including the synthesis of anti-cancer complexes; in the food industry, it is used as a food additive to enhance flavor, improve nutrition, and promote muscle recovery after exercise; in the field of cosmetics, leucine, as a natural moisturizing factor, helps to replenish skin nutrition and improve skin and hair health; in the field of agriculture, L-leucine is used as a fertilizer component to promote crop growth and improve soil.

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

[0004] α-Isopropylmalate synthase (IPMS), encoded by the leuA gene, is the core rate-limiting enzyme in the L-leucine biosynthetic pathway. This enzyme is responsible for catalyzing the aldol condensation reaction between α-ketoisovalerate and acetyl-CoA to produce α-isopropylmalate, which is a key step in the synthesis of L-leucine. As L-leucine accumulates during the synthesis process, IPMS activity is inhibited by its metabolites, and this feedback inhibition gradually increases. 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 over L-leucine synthesis. We modified IPMS to make it less sensitive to feedback inhibition by L-leucine, thereby improving the biosynthetic 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, leuA has a decisive effect on the biosynthesis of L-leucine with its specificity and catalytic efficiency. In order to solve the feedback inhibition of leucine to LeuA, and then improve the yield and conversion rate of L-leucine, the present invention performs random mutations mediated by error-prone PCR on the leuA gene, obtains mutants that are not so sensitive to the feedback inhibition of leucine, and then constructs a series of strains containing mutants; fermentation tests are then performed to verify these strains containing leuA mutants. The specific research method is to use SvalM029 bacteria as a chassis strain (CN117304283A) as an example, first block the generation of valine, that is, knock out ilvE, avtA and leuDH genes, and construct a recombinant strain; the second step is to use error-prone PCR technology to perform random mutations on the leuA gene; the third step is to construct a strain containing a leuA mutant, and through a fermentation test, screen the leuA mutant strain with improved yield and conversion rate of L-leucine.

[0006] By this method, a LeuA mutant capable of significantly improving the production and conversion rate of L-leucine was successfully obtained, and the present invention was proposed.

[0007] The present invention provides an isopropylmalate synthase mutant, which has a substitution mutation at at least one of the following positions in the amino acid sequence corresponding 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 the nucleotide sequence thereof is SEQ ID NO:1.

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

[0010] More specifically, it is the amino acid sequence corresponding to the wild-type isopropylmalate synthase, and the following substitution mutations exist: 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 encoding nucleic acid of the isopropylmalate synthase mutant.

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

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

[0014] Specifically, the encoding nucleic acid of the isopropylmalate synthase mutant can be present 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 encoding 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 encoding nucleic acid of the isopropylmalate synthase mutant, the recombinant expression vector containing the encoding nucleic acid or the recombinant bacteria 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: constructing universal chassis bacteria for the initial strains, specifically blocking the production of valine and / or isoleucine;

[0018] S2: introducing the encoding nucleic acid of the isopropylmalate synthase mutant into a universal base bacteria to obtain an L-valine-producing recombinant bacterium;

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

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

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

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

[0023] The present invention further provides an application of the isopropylmalate synthase mutant, the encoding nucleic acid of the isopropylmalate synthase mutant, the recombinant expression vector containing the encoding nucleic acid, or the recombinant bacteria expressing the isopropylmalate synthase mutant in improving 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 (molar conversion rate or mass conversion rate) of glucose to L-leucine.

[0025] The invention obtains mutant strains containing leuA mutants by random mutation based on error-prone PCR technology, wherein the Ls004 strain has the best effect, the L-leucine yield in a shake flask reaches 1.29 g / L, and the conversion rate reaches 10%. DETAILED DESCRIPTION

[0026] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

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

[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. The following embodiments involve biological materials:

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

[0036] DH5α competent cells are a product of Novozymes, catalog number C502-03.

[0037] QuickMutation TM The gene random mutagenesis kit is a product of Bio-Time, with the serial number D0219S.

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

[0039] 1. Construction of Ls001 strain: SvalM029ΔilvE

[0040] The upstream homology arm UP fragment was amplified by PCR using specific primers ΔilvE-up-F / ΔilvE-up-R and SvalM029 genome as template;

[0041] The downstream homology arm DOWN fragment was amplified by PCR using specific primers ΔilvE-down-F / ΔilvE-down-R and SvalM029 genome as template;

[0042] Overlapping PCR technology was used to connect UP and DOWN to obtain the homologous recombinant fragment UP-DOWN (ΔilvE-donor).

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

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

[0045] 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, 1 min; this step is repeated for 30 cycles, 72°C, 5 min. Step 4: Store at 4°C.

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

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

[0048] 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 for 30 cycles, 72°C, 5 min. Step 4: stored at 4°C. The ΔilvE-donor fragment is obtained.

[0049] 3) Transformation: The pTarget-ΔilvE-sgRNA obtained by PCR amplification was fragmented and 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 competent E. coli: Pick a single clone of the SvalM029 E. coli strain, inoculate it into a 500mL Erlenmeyer flask containing 100mL LB liquid medium, culture at 37°C, 200r / min until OD550 is about 0.5-0.6, and ice bath for 20min; transfer to a 50mL sterile centrifuge tube, centrifuge at 4°C, 5000r / min for 6min; discard the supernatant, resuspend the precipitate with 20mL 10% glycerol, and centrifuge at 4°C, 5000r / min for 6min; repeat the above step twice, and finally add 1mL 10% glycerol to resuspend the bacteria, dispense 100μL / tube and store it at -80°C for later use.

[0052] b. Transformation of pECas9 plasmid: Take 1 μL of pECas9 plasmid and 100 μL of SvalM029 competent cells mentioned above and add them to the electroporation cup, mix well and place on ice for 5 min; wipe the electroporation cup dry, shock it with 2.5KV and immediately add 1 mL of pre-cooled LB medium and blow it several times, mix well and transfer it to a test tube; culture at 37°C, 200 r / min for 1-2 h; take 100 μL of bacterial solution and evenly spread it on a solid LB plate with ampicillin antibiotic (final concentration 100 μg / mL), and culture it at 37°C overnight.

[0053] c. Preparation of competent state of SvalM029 / pECas9 strain: pick a single clone of SvalM029 / pECas9 strain, inoculate it into LB medium containing 100mL 5% L-arabinose (Amp resistance), culture at 37°C, 200rpm for 3-4h, until OD600 = 0.5-0.6; ice bath for 20min; transfer to a 50mL sterile centrifuge tube, centrifuge at 4°C, 5000r / min for 6min; discard the supernatant, resuspend the precipitate with 20mL 10% glycerol, centrifuge at 4°C, 5000r / min for 6min; repeat the above step twice, and finally add 1mL 10% glycerol to resuspend the bacteria, divide it into 100μL / tube, and store it at -80°C for later use.

[0054] d. Transformation of pTarget-ΔilvE-sgRNA plasmid and ΔilvE-donor fragment: Take 2 μL of the pTarget-ΔilvE-sgRNA plasmid and 100 μL of the ΔilvE-donor fragment constructed above and add them to the electroporation cup. Mix well and then bathe on ice for 5 min; culture at 37°C, 200 r / min for 1-2 h; centrifuge and spread all on a solid LB plate with ampicillin and spectinomycin (final concentrations of 100 μg / mL and 100 μg / mL, respectively), and culture at 37°C overnight.

[0055] e. Single colony PCR verification was performed using ilvE-up-F / ilvE-down-R verification primers, and the verified strains were sent for sequencing. The strains that were sequenced correctly were the strains with successful gene editing and were named Ls001.

[0056] 2. Construction of Ls002 strain: SvalM029ΔilvEΔleuDH

[0057] The upstream homology arm UP fragment was amplified by PCR using specific primers ΔleuDH-up-F / ΔleuDH-up-R and SvalM029 genome as template;

[0058] The downstream homology arm DOWN fragment was amplified by PCR using specific primers ΔleuDH-down-F / ΔleuDH-down-R and SvalM029 genome as template;

[0059] Overlap PCR technology was used to connect UP and DOWN to obtain the homologous recombinant fragment UP-DOWN (ΔleuDH-donor).

[0060] Using specific primers ΔleuDH-sgRNA-F / ΔleuDH-sgRNA-R and pTarget plasmid as template, the pTarget-ΔleuDH-sgRNA fragment was amplified and transformed into DH5α competent cells to obtain the pTarget-ΔleuDH-sgRNA targeting plasmid.

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

[0062] 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, 1 min; this step is repeated for 30 cycles, 72°C, 5 min. Step 4: Store at 4°C.

[0063] ΔleuDH-UP, ΔleuDH-DOWN and pTarget-ΔleuDH-sgRNA fragments were obtained by PCR amplification.

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

[0065] The overlapping PCR reaction program 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 for 30 cycles, 72°C, 5 min. Step 4: stored at 4°C. The ΔleuDH-donor fragment is obtained.

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

[0067] 4) Obtaining the Ls002 recombinant strain:

[0068] a. Make competent cells of Ls001: Pick a single clone of Ls001 strain, inoculate it into a 500mL Erlenmeyer flask containing 100mL LB liquid medium, culture at 37℃, 200r / min until OD550 is about 0.5-0.6, and place on ice for 20min; transfer to a 50mL sterile centrifuge tube, centrifuge at 4℃, 5000r / min for 6min; discard the supernatant, resuspend the precipitate with 20mL 10% glycerol, and centrifuge at 4℃, 5000r / min for 6min; repeat the above operation twice, and finally add 1mL 10% glycerol to resuspend the bacteria, dispense 100μL / tube and store it at -80℃ for later use.

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

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

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

[0072] e. Single colony PCR verification was performed using leuDH-up-F / leuDH-down-R verification primers, and the verified strains were sent for sequencing. The strains that were sequenced correctly were the strains with successful gene editing and were named Ls002.

[0073] 3. Construction of Ls003 strain: SvalM029ΔilvEΔleuDHΔavtA

[0074] The upstream homology arm UP fragment was amplified by PCR using specific primers ΔavtA-up-F / ΔavtA-up-R and SvalM029 genome as template;

[0075] The downstream homology arm DOWN fragment was PCR amplified using specific primers ΔavtA-down-F / ΔavtA-down-R and SvalM029 genome as template;

[0076] Overlapping PCR technology was used to connect UP and DOWN to obtain the homologous recombinant fragment UP-DOWN (ΔavtA-donor).

[0077] Using specific primers ΔavtA-sgRNA-F / ΔavtA-sgRNA-R and the pTarget plasmid as a template, the pTarget-ΔavtA-sgRNA fragment was amplified and transformed into DH5α competent cells to obtain the pTarget-ΔavtA-sgRNA targeting plasmid.

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

[0079] 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, 1 min; this step is repeated for 30 cycles, 72°C, 5 min. Step 4: Store at 4°C.

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

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

[0082] 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 for 30 cycles, 72°C, 5 min. Step 4: stored at 4°C. The ΔavtA-donor fragment is obtained.

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

[0084] 4) Obtaining the Ls003 recombinant strain:

[0085] a. Make competent cells of Ls002: Pick a single clone of Ls002 strain, inoculate it into a 500mL Erlenmeyer flask containing 100mL LB liquid medium, culture at 37℃, 200r / min until OD550 is about 0.5-0.6, and place on ice for 20min; transfer to a 50mL sterile centrifuge tube, centrifuge at 4℃, 5000r / min for 6min; discard the supernatant, resuspend the precipitate with 20mL 10% glycerol, and centrifuge at 4℃, 5000r / min for 6min; repeat the above operation twice, and finally add 1mL 10% glycerol to resuspend the bacteria, dispense 100μL / tube and store it at -80℃ 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 the electroporation cup, mix well and place on ice for 5 min; wipe the electroporation cup dry, shock it with 2.5KV and immediately add 1 mL of pre-cooled LB medium and blow it several times, mix well and transfer it to a test tube; culture at 37°C, 200 r / min for 1-2 h; take 100 μL of bacterial solution and evenly spread it on a solid LB plate with ampicillin antibiotic (final concentration 100 μg / mL), and culture it at 37°C overnight.

[0087] c. Preparation of competent state of Ls002 / pECas9 strain: pick a single clone of Ls002 / pECas9 strain, inoculate it into LB medium containing 100mL 5% L-arabinose (Amp resistance), culture at 37°C, 200rpm for 3-4h, until OD600 = 0.5-0.6; ice bath for 20min; transfer to a 50mL sterile centrifuge tube, centrifuge at 4°C, 5000r / min for 6min; discard the supernatant, resuspend the precipitate with 20mL 10% glycerol, centrifuge at 4°C, 5000r / min for 6min; repeat the above step twice, and finally add 1mL 10% glycerol to resuspend the bacteria, divide it into 100μL / 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 the pTarget-ΔavtA-sgRNA plasmid and ΔavtA-donor fragment constructed above and 100 μL of Ls002 / pECas9 competent cells and add them to the electroporation cup, mix well and bathe on ice for 5 min; culture at 37°C, 200 r / min for 1-2 h; centrifuge and spread all on a solid LB plate with ampicillin and spectinomycin (final concentrations of 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 to perform single colony PCR verification, and send the verified strains for sequencing. The strains with correct sequencing are the strains with successful gene editing and are named Ls003.

[0090] Example 2: Construction of leuA mutant strain

[0091] 1. Construction of fragments containing leuA mutants:

[0092] The amino acid sequence encoded by the leuA gene is NCBI accession number WP_000082850.1 (SEQ ID NO: 2); the nucleotide sequence is NCBI accession number NC_000913.3 81958-83529 (-) (SEQ ID NO: 1). TM The gene random mutagenesis kit was used, and the leuA-F / leuA-R primers were used to PCR amplify the fragment containing the leuA mutant using the SvalM029 genome as a template.

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

[0094] The PCR reaction procedure is as follows: 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 is repeated for 30 cycles, 72°C, 10 min. Step 4: Store at 4°C. PCR amplification obtains a fragment containing the leu mutant.

[0095] 2. Construction of editing elements for genome integration of leuA mutations: Using specific primers ΔycjV-up-F / ΔycjV-up-R, with SvalM029 genome as template, PCR was used to amplify the upstream homology arm UP fragment; using specific primers ΔycjV-down-F / ΔycjV-down-R, with SvalM029 genome as template, PCR was used to amplify the downstream homology arm DOWN fragment; overlapping PCR technology was used to connect UP, the mutant-containing leuA fragment and DOWN to obtain the homologous recombination fragment UP-leuA-DOWN (ΔycjV::leuA-donor). Using ΔycjV-sgRNA-F / ΔycjV-sgRNA-R primers to amplify the pTarget-ΔycjV-sgRNA fragment, it was transformed into DH5α competent cells to obtain the pTarget-ΔycjV-sgRNA targeting plasmid.

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

[0097] The PCR reaction procedure was 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 was repeated for 30 cycles, 72°C, 5 min. Step 4: stored at 4°C. ΔycjV-UP and ΔycjV-DDOWN were obtained by PCR amplification.

[0098] 2) Overlap PCR reaction system (50 μL): 2×Phanta Mix high-fidelity enzyme 25 μL, ΔycjV-UP, leuA mutant fragment and ΔycjV-DOWN fragment 1 μL each, ΔycjV-up-F (10 mM) and ΔycjV-down-R (10 mM) 2 μL each, ddH2O 18 μL; Overlap PCR reaction program: 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 for 30 cycles, 72°C, 5 min. Step 4: Store at 4°C. Obtain ΔycjV-donor fragment.

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

[0100] 4) Obtaining recombinant strains containing leuA mutants: The pTarget-ΔycjV-sgRNA plasmid and ΔycjV::leuA-donor fragment constructed above were electroporated into strain Ls003 for gene editing. All single colonies grown on the plate were stored in a -80℃ refrigerator for bacteria preservation and tested in 48 deep-well plates later.

[0101] Example 3: 48-deep-well plate fermentation of mutants

[0102] The above bacteria containing the leuA mutant and the control strain Leu003 were inoculated into a 48-deep-well plate containing a fermentation medium, and cultured at 37°C and 220 r / min for 48 hours. The fermentation broth was taken out and the content of L-leucine was detected by HPLC. The strain with leucine accumulation was sequenced for leuA gene.

[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 of L-leucine: after the sample is diluted 10 times, it is centrifuged at 12000r / min for 10min, the supernatant is filtered with a filter membrane and then tested by HPLC.

[0114] Chromatographic column: Primesep 100 column

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

[0116] Detector: UV detector

[0117] The column temperature was 35°C

[0118] Detection wavelength: 200nm

[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 solution: 1 mL, biotin mother solution (0.4 g / L): 15 mL.

[0120] Table 3. Trace element mother solution formula

[0121]

[0122] Example 4: Shake flask fermentation of control bacteria and recombinant bacteria containing leuA mutant

[0123] The mutant strains Ls004-Ls012 and the control strain Ls003 (containing wild-type leuA) were inoculated into LB liquid test tubes and cultured overnight at 37°C and 200 r / min. The culture solution was transferred to 50 mL of fermentation medium at a 1% inoculum, and cultured at 37°C and 200 r / min for 48 hours. 1 mL of the fermentation solution was taken and the L-leucine production was detected by HPLC. The results are shown in Table 4. The sugar-acid conversion rate is the mass conversion rate of glucose into L-leucine.

[0124] Table 4. L-leucine fermentation results

[0125]

[0126] The fermentation results showed that the leucine production of the mutant strains containing leuA was improved, among which the mutant strain Ls004 had the best effect, with the L-leucine production in the shake flask reaching 1.29 g / L and the conversion rate reaching 10%.

Claims

1. An isopropylmalate synthase mutant, characterized in that: Compared with the wild-type isopropylmalate synthase, at least one of the following positions in the amino acid sequence has a substitution mutation: R308, H390, T407 or G462.

2. The isopropylmalate synthase mutant according to claim 1, characterized in that Compared with the wild-type isopropylmalate synthase, at least one of the following positions of the amino acid sequence has a substitution mutation: R308S, H390N, T407I or G462D.

3. The isopropylmalate synthase mutant according to claim 1, characterized in that The amino acid sequence of the isopropylmalate synthase corresponding to the wild type has the following substitution mutations: G462D; R308S; H390N; T407I; R308S and G462D; H390N and G462D; T407I and G462D; R308S and T407I; R308S, T407I and G462D.

4. A nucleic acid encoding the isopropylmalate synthase mutant according to any one of claims 1 to 3.

5. A recombinant vector containing the encoding nucleic acid as claimed in claim 4.

6. A recombinant bacterium expressing the isopropylmalate synthase mutant according to any one of claims 1 to 3, preferably, a Klebsiella strain, Corynebacterium glutamicum, Bacillus subtilis, Brevibacterium flavum, yeast or Escherichia coli.

7. Use of the isopropylmalate synthase mutant according to any one of claims 1 to 3, the encoding nucleic acid according to claim 4, the recombinant expression vector according to claim 5 or the recombinant bacterium according to claim 6 in the preparation of L-leucine.

8. A recombinant bacterium for producing L-leucine, characterized in that: Obtained by the following method: S1: constructing universal chassis bacteria for the initial strains, specifically blocking the production of valine and / or isoleucine; S2: introducing the encoding nucleic acid as described in claim 4 into a universal chassis bacterium to obtain an L-valine-producing recombinant bacterium; Preferably, the initial strain is Escherichia coli; More preferably, the blocking of valine production is achieved by knocking out or weakening ilvE, avtA and leuDH genes.

9. A method for preparing L-leucine, characterized in that: The method comprises the step of obtaining L-leucine by fermenting and culturing the recombinant bacteria as claimed in claim 8, and optionally, further comprises the step of separating the L-leucine.

10. Use of the isopropylmalate synthase mutant according to any one of claims 1 to 3, the encoding nucleic acid according to claim 4, the recombinant expression vector according to claim 5 or the recombinant bacterium according to claim 6 in improving the yield and / or conversion rate of L-leucine.

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