Method for improving l-leucine production of strain, mutant and application thereof

Abstract

This invention relates to the field of agricultural biotechnology, specifically to a method for increasing L-leucine yield in bacterial strains, as well as mutants and applications. According to the technical solution of this invention, the leucine dehydrogenase mutant Asleudh... M65L and Asleudh M65L / N69D The relative activity against KIC / KIV was significantly enhanced compared to wild-type leucine dehydrogenase, resulting in a significant increase in Asleudh activity. M65L and Asleudh M65L / N69D Expression of the gene encoding L-leucine in recombinant strains significantly increased L-leucine production and decreased L-valine production.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to a method for increasing the L-leucine yield of bacterial strains, as well as mutants and applications. Background Technology

[0002] Leucine dehydrogenase (LeuDH, EC1.4.1.9) is a NADH-dependent oxidoreductase with wide availability. It reversibly catalyzes the reductive amination and oxidative deamination reactions between α-keto acids and their corresponding chiral amino acids. It is primarily used for the efficient and selective production of various branched-chain amino acids and chiral amines. Leucine dehydrogenases from different sources exhibit significant differences in function, substrate preference, and catalytic properties, allowing for their rational design and modification through enzyme engineering.

[0003] The chemical formula of L-leucine is C6H 13 NO2 is one of the three essential branched-chain amino acids (BCAAs). It directly participates in the biosynthesis of proteins in the body and is a key nutrient signaling molecule that regulates muscle growth and metabolic balance. It is widely used in feed, food, medicine and chemical industries. However, at present, the production level and annual output of L-leucine are far lower than those of other amino acids such as L-valine.

[0004] Currently, the main method for large-scale industrial production of L-leucine is microbial fermentation. In the synthesis of L-leucine using glucose as a substrate, glucose first produces pyruvate, which is then converted to α-ketoisovaleric acid, subsequently to α-ketoisovaleric acid, and finally L-leucine is produced under the catalysis of branched-chain amino acid transaminases (BCAT) or leucine dehydrogenases. BCAT uses glutamate as an ammonia donor (organic ammonia), while leucine dehydrogenase uses inorganic ammonia such as NH3. Therefore, leucine dehydrogenases have a greater advantage in production. Furthermore, due to the structural similarity of the three branched amino acids, both BCAT and leucine dehydrogenases exhibit catalytic activity towards both α-ketoisovaleric acid and α-ketoisocalcic acid, leading to the production of a large amount of L-valine during L-leucine synthesis. Therefore, screening and modifying leucine dehydrogenases with a higher substrate preference for the L-leucine precursor α-ketoisocalcic acid is of great significance. Summary of the Invention

[0005] One of the objectives of this invention is to provide a leucine dehydrogenase mutant with a high substrate preference for α-ketoisocaproic acid.

[0006] Another object of the present invention is to provide a gene encoding the above-mentioned leucine dehydrogenase mutant with high substrate preference for α-ketoisocaproic acid.

[0007] Another object of the present invention is to provide the application of the above-mentioned leucine dehydrogenase mutant.

[0008] Another object of the present invention is to provide a method for increasing the substrate preference of leucine dehydrogenase for α-ketoisocaproic acid.

[0009] Another object of the present invention is to provide a method for increasing the L-leucine production of the strain.

[0010] The leucine dehydrogenase mutant of the present invention, exhibiting a higher substrate preference for α-ketoisocaproic acid, is obtained by mutating methionine at position 65 of the protein shown in SEQ ID NO: 1 to leucine (M65L) and asparagine at position 69 to aspartic acid (N69D). The leucine dehydrogenase mutant Asleudh... M65L The amino acid sequence is as shown in SEQ ID NO: 3, and the leucine dehydrogenase mutant Asl...

[0011] Furthermore, based on the amino acid sequence of the leucine dehydrogenase mutant provided above, those skilled in the art can obtain the sequence of its encoding gene. Due to codon degeneracy, there is more than one gene sequence encoding the above amino acid sequence, and all genes capable of encoding the above leucine dehydrogenase mutant are within the protection scope of this invention.

[0012] The expression vectors described in this article refer to recombinant DNA molecules constructed by linking exogenous target genes with vectors in vitro. They can be constructed in any suitable manner, as long as the constructed expression vector can carry the exogenous target gene into the recipient strain or cell and provide the exogenous target gene with the ability to replicate, integrate, amplify and / or express in the recipient strain or cell.

[0013] Furthermore, the expression vector described in this application may contain the encoding Asleudh M65L protein or Asleudh M65L / N69D An overexpression vector for a protein's DNA molecule. Furthermore, the expression vector described in this application may be an overexpression vector for the DNA molecule shown in SEQ ID NO: 4 or SEQ ID NO: 6.

[0014] According to the technical solution of this application, a recombinant microorganism is obtained by introducing the coding sequence of a protein with an amino acid sequence such as SEQ ID NO: 3 or SEQ ID NO: 5 into a target microorganism; or

[0015] Recombinant microorganisms are obtained by introducing gene sequences with coding sequences such as SEQ ID NO:4 or SEQ ID NO:6 into the target microorganism.

[0016] The introduction can be either by integrating a foreign gene into the host chromosome or by expressing it extrachromosomally using a plasmid.

[0017] This invention provides for any of the following applications of the leucine dehydrogenase mutant or the gene encoding the leucine dehydrogenase mutant:

[0018] Regulate the production of L-leucine or L-valine in microorganisms;

[0019] Construct L-leucine-producing genetically engineered bacteria.

[0020] The fourth objective of this invention is to provide a method for increasing the yield of L-leucine from recombinant microorganisms, the method comprising the following steps:

[0021] The encoding sequence of the protein expressing the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:5.

[0022] Advantages of the technical solution of the present invention:

[0023] In the pathway of L-leucine synthesis via microbial fermentation using glucose as a substrate, the carbon metabolic flux first passes through α-ketoisovalerate (KIV), a precursor of L-valine, and then generates α-ketoisocaproate (KIC), another precursor of L-leucine. Finally, L-leucine is generated under the catalysis of leucine dehydrogenase. However, due to the structural similarity between KIV and KIC, and the fact that KIV is usually more abundant in cells than KIC, and that wild-type leucine dehydrogenase has a weak catalytic preference and specificity for KIC, the expression of leucine dehydrogenase can lead to the catalysis of KIV to form L-valine, resulting in a decrease in the yield and conversion rate of L-leucine. According to the technical solution of the present invention, the leucine dehydrogenase mutant Asleudh... M65L and Asleudh M65L / N69D The relative activity (activity against KIC / activity against KIV) of Asleudh was significantly enhanced compared to wild-type leucine dehydrogenase. M65L and Asleudh M65L / N69D Expression of the gene encoding this gene in recombinant strains significantly increased L-leucine production and decreased L-valine production. Attached Figure Description

[0024] Figure 1 Asleudh protein in Example 1 of this invention, Asleudh M65L Protein, Asleudh M65A Protein, Asleudh M65F Protein, Asleudh M65L / N69D Asleudh M65L / N69A Protein and AsleudhM65L / N69L SDS-PAGE results of purified protein;

[0025] Figure 2 Asleudh protein in Example 1 of this invention, Asleudh M65L Protein, Asleudh M65A Protein, Asleudh M65F Protein, Asleudh M65L / N69D Asleudh M65L / N69A Protein and Asleudh M65L / N69L Results of relative enzyme activity assay for proteins;

[0026] Figure 3 The production of L-leucine and L-valine by the engineered strain BW1-BW4 provided in Example 2 of this invention;

[0027] Figure 4 The ratio of L-leucine to L-valine concentrations in the fermentation broth of the engineered strain BW1-BW4 provided in Example 2 of this invention. Detailed Implementation

[0028] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0030] This invention provides a leucine dehydrogenase mutant with enhanced substrate affinity for α-ketoisocaproic acid (KIC), said mutant being derived from microorganisms. Anoxybacillus sp. Using BCO1 leucine dehydrogenase (Asleudh) as the parent, a mutant enzyme was obtained by mutating the amino acid at position 65 and / or 69. Specifically, the amino acid at position 65 of the parent was mutated from methionine to leucine, and named Asleudh. M65L Alternatively, the 65th amino acid in the parent enzyme can be mutated from methionine to alanine to obtain a mutant enzyme, named Asleudh. M65A Alternatively, the 65th amino acid in the parent enzyme can be mutated from methionine to phenylalanine to obtain a mutant enzyme, named Asleudh. M65FAlternatively, by mutating amino acid position 65 of the parent enzyme from methionine to leucine and simultaneously mutating amino acid position 69 from asparagine to aspartic acid, a mutant enzyme can be obtained, named Asleudh. M65L / N69D Alternatively, by mutating amino acid position 65 of the parent enzyme from methionine to leucine and simultaneously mutating amino acid position 69 from asparagine to alanine, a mutant enzyme can be obtained, named Asleudh. M65L / N69A Alternatively, by mutating amino acid position 65 of the parent enzyme from methionine to leucine and simultaneously mutating amino acid position 69 from asparagine to leucine, a mutant enzyme can be obtained, named Asleudh. M65L / N69L Among them, the mutant Asleudh M65L and Asleudh M65L / N69D The relative activity is significantly improved, and it has a better effect when used for L-leucine production.

[0031] The culture media involved in the following examples are as follows:

[0032] LB liquid medium: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L; pH 7.0, sterilized at 121℃ for 21 min;

[0033] LB solid medium: Agar powder with a mass concentration of 20 g / L is added to the liquid medium.

[0034] Fermentation medium FM: 5 g / L yeast extract, 20 g / L glucose, 14 g / L KH2PO4, 4 g / L (NH4)2HPO4, 0.6 g / L MgSO4·7H2O, 2 g / L Na2SO4, 1.8 g / L citric acid, and trace elements (13 mg / L Zn(CH3COO)2·2H2O, 10 mg / L ferric citrate, 8.4 mg / L EDTA, 3 mg / L H3BO3, 2.5 mg / L Na2MoO4·2H2O, 2.5 mg / L CoCl2·6H2O, 1.5 mg / L CuCl2·2H2O, 0.9 mg / L VB1, 0.15 mg / L MnCl2·4H2O), prepared with distilled water, pH adjusted to 7.0.

[0035] Table 1. List of engineered Escherichia coli strains constructed in various embodiments of the present invention.

[0036] .

[0037] The following embodiments involve the following sequences:

[0038] Derived from microorganisms Anoxybacillus sp.Amino acid sequence of leucine dehydrogenase Asleudh of BCO1, SEQ ID NO: 1: MEIFKYMEQYDYEQLVFCQDKESGLKAIIAIHDTTLGPALGGTRMWMYESEDAAIEDALRLARGMTYKNAAAGLNLGGGKAVIIGDPRKDKNEAMFRAFGRFIQGLNGRYITAEDVGTTVADMDIIYEETDYVTGISPAFGSSGNPSPVTAYGVYRGMKAAAKEAFGSDSLEGKVIAVQGVGNVAYNLCRHLHEEGAKLIVTDINKEAVQRVVEEFGAQAVDPNDIYSVDCDIFAPCALGGVINDQTIPQLKAKVIAGAANNQLREARHGDIIHEMGIVYAPDYVINAGGVINVADELYGYNRERAMKKVEQIYNNIEKVIEIAKRDGIPTYQAADRLAEERIAKMRQSRSQFLQNGQHILSRRRTR*;

[0039]

[0040] Amino acid sequence of the M65L mutant of Asleudh, SEQ ID NO: 3: MEIFKYMEQYDYEQLVFCQDKESGLKAIIAIHDTTLGPALGGTRMWMYESEDAAIEDALRLARGLTYKNAAAGLNLGGGKAVIIGDPRKDKNEAMFRAFGRFIQGLNGRYITAEDVGTTVADMDIIYEETDYVTGISPAFGSSGNPSPVTAYGVYRGMKAAAKEAFGSDSLEGKVIAVQGVGNVAYNLCRHLHEEGAKLIVTDINKEAVQRVVEEFGAQAVDPNDIYSVDCDIFAPCALGGVINDQTIPQLKAKVIAGAANNQLREARHGDIIHEMGIVYAPDYVINAGGVINVADELYGYNRERAMKKVEQIYNNIEKVIEIAKRDGIPTYQAADRLAEERIAKMRQSRSQFLQNGQHILSRRRTR*;

[0041]

[0042] The amino acid sequence of the Asleudh mutant M65L / N69D, SEQ ID NO: 5:

[0043] MEIFKYMEQYDYEQLVFCQDKESGLKAIIAIHDTTLGPALGGTRMWMYESEDAAIEDALRLARGLTYKDAAAGLNLGGGKAVIIGDPRKDKNEAMFRAFGRFIQGLNGRYITAEDVGTTVADMDIIYEETDYVTGISPAFGSSGNPSPVTAYGVYRGMKAAAKEAFGSDSLEGKVIAVQGVGNV AYNLCRHLHEEGAKLIVTDINKEAVQRVVEEFGAQAVDPNDIYSVDCDIFAPCALGGVINDQTIPQLKAKVIAGAANNQLREARHGDIIHEMGIVYAPDYVINAGGVINVADELYGYNRERAMKKVEQIYNNIEKVIEIAKRDGIPTYQAADRLAEERIAKMRQSRSQFLQNGQHILSRRRTR*;

[0044] The coding sequence of mutant M65L / N69D, SEQ ID NO: 6:

[0045]

[0046] SEQ ID NO: 7 leA r

[0047]

[0048] Example 1: Construction of a leucine dehydrogenase mutant

[0049] Wild-type leucine dehydrogenase is active for both α-ketoisovaleric acid (KIV) and α-ketoisocaproic acid (KIC) intermediates and exhibits virtually no substrate preference, leading to a significant increase in L-valine production during L-leucine synthesis. Mutagenesis of wild-type leucine dehydrogenase can enhance its relative activity (activity against KIC / activity against KIV), thereby increasing L-leucine yield and reducing byproducts.

[0050] I. Construction and purification of leucine dehydrogenase mutant

[0051] Artificially synthesized coding sequences such as SEQ ID No: 2 ( Asleudh SEQ ID No: 4 Asleudh M65L ), SEQ ID No: 6 ( Asleudh M65L / N69D ), Asleudh M65A The encoded sequence, Asleudh M65F The encoded sequence, Asleudh M65L / N69A The encoded sequence and Asleudh M65L / N69L The coding sequence was obtained by PCR amplification of the corresponding DNA fragment, which was then ligated to the pET28a(+) plasmid digested with EcoRI and HindIII to construct the plasmid pET28a(+)- Asleudh pET28a(+)- Asleudh M65L pET28a(+)- Asleudh M65A pET28a(+)- Asleudh M65F pET28a(+)- Asleudh M65L / N69D pET28a(+)- Asleudh M65L / N69A and pET28a(+)- Asleudh M65L / N69L The obtained plasmids were then transferred into... E. coli In BL21 (DE3) commercial competent cells, the transformation process was carried out according to the instructions for use of the commercial competent cells.

[0052] The obtained transformants were inoculated into LB liquid medium containing 50 µg / mL kanamycin and cultured overnight at 37 °C on a shaking incubator. Then, at a 1% inoculation rate, they were transferred to 400 mL of fresh LB medium containing 50 µg / mL kanamycin and cultured at 37 °C and 200 rpm until OD (outcome limit) was reached. 600 When the pH reaches 0.6-0.8, 0.5 mM IPTG is added for induction. After induction at 37 ℃ for 6 hours, the induced bacterial culture is centrifuged at 5,500 rpm for 7 min, the bacterial cells are collected, and the cells are washed with 20 mL of 1×PBS solution (pH 7.4). The cells are then resuspended in 20 mL of Tris-HCl solution (10 mM, pH 7.4), and then sonicated. The supernatant is collected by centrifugation at 12,000 rpm for 60 min at 4 ℃.

[0053] The obtained supernatant was used to purify the protein using nickel affinity chromatography. The eluent was 0.4 M imidazole, 50 mM Tris-HCl, and 0.5 M NaCl. The eluent was collected and analyzed by SDS-PAGE. Asleudh protein, Asleudh... M65L Protein, Asleudh M65A Protein, Asleudh M65F Protein, Asleudh M65L / N69D Asleudh M65L / N69A Protein and Asleudh M65L / N69L The relative molecular weights of the proteins are all around 40 kDa. SDS-PAGE images of each protein are shown below. Figure 1 The gel imaging results showed that all proteins were correctly expressed and successfully purified.

[0054] II. Determination of relative enzyme activity of leucine dehydrogenase mutants

[0055] In the process of leucine dehydrogenase catalyzing the reductive amination of α-keto acids to produce L-amino acids, NADH is required as a coenzyme to initiate the reaction. Since NADH has a specific absorption at 340 nm while other substances do not have absorption peaks, the relative activity parameter of the enzyme is calculated from the change in absorbance at 340 nm, specifically the ratio of the changes in absorbance at 340 nm for reaction systems containing KIC or KIV within 1 min.

[0056] In this embodiment, the relative enzyme activity of leucine dehydrogenase was determined in an NH4Cl / NH3·H2O buffer (0.5 M, pH 9.5) containing a certain amount of pure enzyme, 2 mM substrate (KIC or KIV), and 0.1 mM NADH. The reaction system was 1 mL, and the temperature was 30 °C. After incubating NADH and other components separately for 2 min, 0.1 mM NADH was added to the reaction system to start the reaction for 1 min. The change in absorbance at 340 nm was measured, and the ratio of the absorbance change with KIC as substrate to the absorbance change with KIV as substrate was calculated as the relative enzyme activity. The relative enzyme activity of wild-type Asleudh was defined as 1. The experimental results are as follows: Figure 2 As shown. The results indicate that the optimal single-point mutant is Asleudh. M65L The protein had a relative enzyme activity of 10.9, which was 9.9 times higher than that of wild-type Asleudh; the optimal two-point mutant was Asleudh. M65L / N69D The protein has a relative enzyme activity of 11.4, which is 10.4 times higher than that of wild-type Asleudh.

[0057] Example 2: Construction of an engineered strain containing a leucine dehydrogenase mutant gene

[0058] Asleudh M65L Protein and Asleudh M65L / N69D The protein exhibits enhanced relative enzyme activity against KIC and KIV, which can be used to increase L-leucine production in L-leucine-producing strains and improve the L-leucine / L-valine ratio.

[0059] I. Construction of Escherichia coli strain BW1 for producing L-leucine

[0060] The *Escherichia coli* strain ILE-5 (J. Agric. Food Chem. 2025, 73, 19, 11900–11911) is a high-yield L-isoleucine producer. This strain was isolated by knocking out the methylmalate synthase gene. how much And knock in the anti-feedback inhibitory isopropyl malate synthase gene leuA r This allows the strain to be modified into an L-leucine-producing strain. Using *Escherichia coli* ILE-5 as the starting strain, the CRISPR-associated transposases gene editing method was employed to... how much Gene replacement leuA r The gene (whose DNA sequence is shown in SEQ ID NO: 7) is as follows:

[0061] (1) Preparation of Escherichia coli electrotransformation competent cells

[0062] 40 μL of *E. coli* ILE-5 glycerol stock culture was inoculated into 40 mL of LB medium and incubated overnight at 37 °C on a shaking incubator. The inoculum was then transferred to 40 mL of fresh LB medium at a 1% inoculation rate and incubated at 37 °C on a shaking incubator at 220 rpm until OD (open-circuit retrieval). 600 The culture concentration should reach 0.6-0.8. After incubating the cultured bacterial suspension on ice for 30 min, centrifuge to collect the cells (4 ℃, 5,500 rpm, 5 min). Resuspend the cells in 25 mL of pre-chilled double-distilled water. Centrifuge again under the same conditions and resuspend the cells in double-distilled water. Centrifuge as above and resuspend the cells in 25 mL of pre-chilled 10% glycerol. Finally, centrifuge at 4 ℃, 5,500 rpm for 8 min to collect the cells. Add 0.7 mL of pre-chilled 10% glycerol to suspend the cells. Aliquot into 100 μL vials and store at -80 ℃.

[0063] (2) Electroporation of Escherichia coli and screening of transformants

[0064] 500 ng of plasmid pTetQCas (containing tniQ and cas876 (gene), 500 ng plasmid pRE57-tns-Ter- leuA r (contain tnsABC Genes and right end- leuA r The gene-left end sequence and 500 ng of plasmid pUC-spacer-cimA (containing the spacer sequence tactgtgtcacaaacacaaactctgtctgctc) were mixed and added to ILE-5 electroporation competent cells. After mixing, the mixture was transferred to a 1 mm electroporation cuvette, and the three plasmids were transformed into ILE-5 cells using an electroporator. Then, 0.6 mL of antibiotic-free LB medium was added, and the cells were incubated at 37 °C with shaking for 1 h. After recovery, the cells were centrifuged at 5,500 rpm for 5 min, collected, and plated on LB agar plates containing 50 µg / mL kanamycin, 100 µg / mL ampicillin, and 25 µg / mL streptomycin. The plates were incubated overnight at 37 °C. After colonies appeared, single colonies were picked with a sterile toothpick for PCR verification, confirming that all three plasmids had been successfully transformed into ILE-5 cells.

[0065] (3) Construction of engineered strain BW1

[0066] The above contains pTetQCas, pRE57-tns-Ter- leuA rILE-5 transformants containing the pUC-spacer-cimA plasmid were inoculated into LB liquid medium containing 50 µg / mL kanamycin, 100 µg / mL ampicillin, and 25 µg / mL streptomycin, and cultured with shaking at 37 °C. 600 When the expression level reached approximately 0.6, 100 ng / mL of adeoxytetracycline was added for induction, stimulating the expression of proteins related to the CRISPR-associated transposases gene editing system. The culture was incubated overnight at 37°C with shaking. The induced culture medium was diluted and plated onto LB agar plates containing the three antibiotics and adeoxytetracycline inducer, and incubated at 37°C for 15 h. After single clones appeared, cloning PCR and sequencing verification were performed. how much Genes are blocked and leuA r The PCR product band of the successfully knocked-in mutant strain was 3770 bp larger than that of the wild-type strain. The obtained positive clones were inoculated into fresh LB liquid medium without antibiotics.

[0067] To remove the three edited plasmids from the obtained positive strains, the positive strains containing the three edited plasmids were passaged in antibiotic-free liquid LB medium at an inoculum size of 1%. After five passages at 45 °C, the bacterial culture was diluted and spread onto antibiotic-free LB agar plates and incubated at 37 °C for 15 h. Once single clones appeared, they were randomly picked with a sterile toothpick and spotted onto four different LB agar plates: antibiotic-free, containing kanamycin, ampicillin, and streptomycin. The single clones on the four plates were matched one-to-one, and the plates were incubated at 37 °C for 15 h. Single clones that grew on antibiotic-free LB agar plates but not on the other three antibiotic-containing plates were selected for further verification using clonal PCR. how much The gene was replaced with leuA r Genetic information: positive clones are Escherichia coli BW1 engineered strains (Table 1).

[0068] II. Constructing a structure containing Asleudh and Asleudh M65L and Asleudh M65L / N69D Recombinant strain BW2-BW4

[0069] Based on the engineered strain BW1, the original strain was knocked out. Lsleudh Genes, and integrate them separately by knocking them in. Asleudh , Asleudh M65L and Asleudh M65L / N69D Genes were used to construct recombinant strains BW2, BW3, and BW4.

[0070] Using the methods described above for preparing, electrotransforming, and screening transformants of *E. coli* electrotransformation competent cells, engineered strain BW1 was prepared as electrotransformation competent cells. These cells were first electrotransformed into two plasmids: pTetQCas and pUC-spacer-Lsleudh (containing the spacer sequence tacgcgacagaagaaaatgcgattgaggatgc). Subsequently, the constructed plasmid pRE57-tns-Ter- Asleudh pRE57-tns-Ter- Asleudh M65L and pRE57-tns-Ter- Asleudh M65L / N69D The cells were transformed into BW1 cells containing the pTetQCas and pUC-spacer-Lsleudh plasmids, respectively. Following tetracycline induction, mutant selection, and plasmid removal, the desired mutants were finally obtained. Lsleudh Gene knockout and Asleudh Gene, Asleudh M65L Gene or Asleudh M65L / N69D Recombinant engineered strains BW2, BW3, and BW4, whose genes were successfully integrated into the genome (Table 1).

[0071] III. Production of L-Leucine from Recombinant Strains Cultivated in a 3L Fermenter

[0072] The engineered strain BW1-BW4 constructed above was fermented in a 3 L fermenter using FM fermentation medium. The temperature was maintained at 37 °C throughout the fermentation process, and the pH was kept at approximately 7.0 by automatic ammonia replenishment. Dissolved oxygen levels were controlled between 10% and 30%. Once the initial glucose was depleted, a peristaltic pump was used to replenish the glucose concentration to below 10 g / L, with the replenished glucose being 700 g / L. Fermentation lasted for 36 h, and cell density (OD) was measured periodically. 600 The fermentation broth contained glucose. After fermentation, the contents of L-leucine and L-valine in the culture medium were separated and determined using an amino acid analysis method with a high-performance liquid chromatography-ultraviolet detector. The fermentation results are as follows: Figure 3 (Concentrations of L-leucine and L-valine in the fermentation broths of BW1-BW4) and Figure 4 (The ratio of L-leucine to L-valine concentrations in the fermentation broths of BW1-BW4 is shown).

[0073] Depend on Figure 3 and Figure 4 The fermentation results shown indicate that, compared to the engineered strain BW1 (containing...), Lsleudh Genes), engineered strain BW2 (containing AsleudhThe L-leucine production of the gene increased by 69.7%, while L-valine production decreased by 59.6%, and the L-leucine / L-valine ratio also increased by 3.2 times, indicating that... Asleudh Gene ratio Lsleudh The gene is more conducive to the synthesis of L-leucine. Compared to strain BW2, the engineered strain BW3 (containing...) Asleudh M65L (genes) and BW4 (containing) Asleudh M65L / N69D The L-leucine production of the gene increased by 42.9% and 23.2%, respectively, and the L-leucine / L-valine ratio increased by 0.3% and 1.4 times, respectively, indicating that... Asleudh M65L and Aseuldh M65L / N69D mutant ratio Asleudh Wild-type genes are more conducive to the synthesis of L-leucine in the intracellular environment.

[0074] The above embodiments are only used to understand the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. A leucine dehydrogenase mutant having high substrate preference for a-ketoisocaproic acid, characterized in that, The amino acid sequence of the leucine dehydrogenase mutant with high substrate preference for α-ketoisocaproic acid is shown in SEQ ID NO: 3 or SEQ ID NO:

5.

2. A leucine dehydrogenase gene characterized in that, The leucine dehydrogenase gene encodes the leucine dehydrogenase mutant with a high substrate preference for α-ketoisocaproic acid as described in claim 1.

3. The leucine dehydrogenase gene of claim 2, wherein, The nucleotide sequence of the leucine dehydrogenase gene is shown in SEQ ID NO: 4 or SEQ ID NO:

6.

4. A recombinant expression vector, characterized in that, The recombinant expression vector contains the leucine dehydrogenase gene as described in claim 2.

5. A recombinant bacterial strain, characterized in that, The recombinant strain contains the leucine dehydrogenase gene as described in claim 2.

6. A method for improving the substrate preference of leucine dehydrogenase for α-ketoisocaproic acid, characterized in that, The method includes the step of mutating a leucine dehydrogenase with an amino acid sequence as shown in SEQ ID NO: 1 as follows: The amino acid at position 65 is mutated from methionine to leucine; or The amino acid at position 65 is mutated from methionine to leucine, while the amino acid at position 69 is mutated from asparagine to aspartic acid.

7. A method for improving the production of L-leucine by a recombinant microorganism, characterized by, The method includes the following steps: expressing in an engineered strain a gene encoding a leucine dehydrogenase mutant with high substrate preference for α-ketoisocaproic acid as described in claim 1, wherein the engineered strain is an Escherichia coli engineered strain.

8. The method for improving L-leucine production of a recombinant microorganism according to claim 7, wherein, The engineered Escherichia coli strain is an Escherichia coli strain that has the methylmalate synthase gene knocked out and expresses the isopropylmalate synthase gene.

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