Leucine dehydrogenase for preparing l-tert-leucine

Abstract

This invention discloses a leucine dehydrogenase mutant for the preparation of L-tert-leucine, which is a derivative polypeptide with enhanced enzyme activity formed by mutation of sites E114 and T43 (i.e., the E114 / T43 combination) of the leucine dehydrogenase Leudh, whose amino acid sequence is as shown in SEQ ID NO:1. Leucine dehydrogenase Leudh and its mutant can form a Leudh / ADH dual-enzyme coupled coenzyme cycle system with alcohol dehydrogenase ADH, catalyzing the reaction of the substrate trimethylpyruvate and isopropanol with an amino donor to obtain L-tert-leucine with high optical purity, thus promoting the industrial application of enzymatic synthesis methods for L-tert-leucine.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology and relates to a leucine dehydrogenase mutant for the preparation of L-tertiary leucine and its uses. Background Technology

[0002] L-tert-leucine is a non-natural chiral amino acid and an important pharmaceutical intermediate. As a sterically hindered branched-chain amino acid, it is widely used in the synthesis of antiviral, antitumor, and hypoglycemic drugs. For example, L-tert-leucine is a chiral compound used in the synthesis of the anti-AIDS drug atazanavir, and the anti-hepatitis C drugs terprevir and bosporine.

[0003] Currently, the enzymatic preparation of L-tert-leucine generally uses trimethylpyruvate as a substrate and is catalyzed by a two-enzyme system. For example, publications CN103981229A, CN111676256A, CN117050961A, CN119662583A, and CN119913122A have reported the synthesis of L-tert-leucine by catalyzing the reaction of trimethylpyruvate, isopropanol, and an amino donor using a two-enzyme coupling system composed of leucine dehydrogenase and alcohol dehydrogenase. Summary of the Invention

[0004] Leucine dehydrogenase (Leudh) belongs to the amino acid dehydrogenase family and is a type of NAD+ / NADH-dependent oxidoreductase. It can asymmetricly reduce and amination prochiral α-keto acid compounds and is an important enzyme in the L-amino acid biosynthesis pathway.

[0005] In our previous study on the synthesis of L-2-aminobutyric acid (L-2-aminobutyric acid) from L-threonine and ammonium formate using a combined enzyme, we screened a thermostable leucine dehydrogenase derived from the thermostable bacterium *Heyndrickxia sporothermodurans*, with the amino acid sequence shown in SEQ ID NO: 1 (NCBI accession number WP_108073426.1, abbreviated as Leudh or Hsleudh in this paper). We further modified its amino acid sequence using mutagenesis, obtaining a mutant with enhanced enzyme activity. This enzyme can catalyze the reaction of threonine and ammonium formate to produce L-2-aminobutyric acid at high reaction temperatures of 40-55℃, in conjunction with threonine deaminase and formate dehydrogenase. Considering that leucine dehydrogenase can also co-catalyze the reaction of trimethylpyruvate and isopropanol with an amino donor to synthesize L-tert-leucine, we further expanded the application of this leucine dehydrogenase and its mutants in the enzymatic preparation of L-tert-leucine, achieving experimental success and confirming that this leucine dehydrogenase possesses the expected function. Specifically, the present invention includes the following technical solutions.

[0006] The first aspect of this invention provides a leucine dehydrogenase mutant for the preparation of L-tertiary leucine, which is a derivative polypeptide of the leucine dehydrogenase Leudh (Hsleudh, 2021 NCBI accession number WP_108073426.1) derived from the thermostable bacterium *Heyndrickxia sporothermodurans*, with the amino acid sequence shown in SEQ ID NO: 1:

[0007] A derivative polypeptide with a mutation at the E114 site of the amino acid sequence SEQ ID NO: 1 and increased enzyme activity;

[0008] A derivative polypeptide with a mutation at the T43 site of the amino acid sequence SEQ ID NO: 1 and increased enzyme activity; or

[0009] A derivative polypeptide with simultaneous mutations at both E114 and T43 sites in the amino acid sequence SEQ ID NO: 1, resulting in increased enzyme activity.

[0010] The enzyme activity refers to the enzyme activity catalyzing the reaction of trimethylpyruvate with ammonium chloride, specifically determined by the following method: Take 1.7 ml of 0.2 M ammonium chloride buffer (pH 7.5) and 2 ml of 1 M trimethylpyruvate solution (pH 7.5), preheat in a 37°C water bath, then add to a cuvette. Next, add 100 μl of 1% NADH solution and 100 μl of enzyme solution sequentially. Measure the absorbance change at 340 nm using a spectrophotometer and calculate the enzyme activity. A larger absorbance change indicates higher enzyme activity.

[0011] Preferably, the above-mentioned leucine dehydrogenase mutant is a polypeptide derived from the leucine dehydrogenase Leudh as follows:

[0012] The E114C mutant of leucine dehydrogenase SEQ ID NO: 1, whose amino acid sequence is shown in SEQ ID NO: 3, is named E114C in this paper:

[0013] MKIFEYMGKYDYEQLLFCQDEQSGLKAIIAIHDTTLGPALGTRMWTYASEDAAIEDALRLARGMTYKNAAAGLNLGGGKTVIIGDPRKDKNEEMFRAFGRFIQGLNGRYITACDVGTTVADMDLIHEETNFVTGISPAFGSSGNPSPVTAYGCYVGMKAAANEAFGTDSLEGLTVSVQGVGNV AFELCRYLHEEGAKLIVTDINKEAVQRAVEQFGAEVVDPNDIYGVNSDIFAPCALGAIINDETIPQLKAKVIAGSANNQLKETRHGDQIHEMGIVYAPDYVINAGGVINVADELYGYNPERAMKKVEGIYNNVAKVIEISKRDGIPTYVAADRMAEERIAKVSKSRSQFLLNNRHILSYRA (SEQ IDNO: 3);

[0014] The E114C / T43M mutant of leucine dehydrogenase SEQ ID NO: 1, whose amino acid sequence is shown in SEQ ID NO: 5, is named E114C / T43M in this paper:

[0015] MKIFEYMGKYDYEQLLFCQDEQSGLKAIIAIHDTTLGPALGGMRMWTYASEDAAIEDALRLARGMTYKNAAAGLNLGGGKTVIIGDPRKDKNEEMFRAFGRFIQGLNGRYITACDVGTTVADMDLIHEETNFVTGISPAFGSSGNPSPVTAYGCYVGMKAAANEAFGTDSLEGLTVSVQGVGNV AFELCRYLHEEGAKLIVTDINKEAVQRAVEQFGAEVVDPNDIYGVNSDIFAPCALGAIINDETIPQLKAKVIAGSANNQLKETRHGDQIHEMGIVYAPDYVINAGGVINVADELYGYNPERAMKKVEGIYNNVAKVIEISKRDGIPTYVAADRMAEERIAKVSKSRSQFLLNNRHILSYRA (SEQ IDNO: 5); or

[0016] The E114C / T43S mutant of leucine dehydrogenase SEQ ID NO: 1, whose amino acid sequence is shown in SEQ ID NO: 7, is named E114C / T43S in this paper.

[0017] MKIFEYMGKYDYEQLLFCQDEQSGLKAIIAIHDTTLGPALGGSRMWTYASEDAAIEDALRLARGMTYKNAAAGLNLGGGKTVIIGDPRKDKNEEMFRAFGRFIQGLNGRYITACDVGTTVADMDLIHEETNFVTGISPAFGSSGNPSPVTAYGCYVGMKAAANEAFGTDSLEGLTVSVQGVGNV AFELCRYLHEEGAKLIVTDINKEAVQRAVEQFGAEVVDPNDIYGVNSDIFAPCALGAIINDETIPQLKAKVIAGSANNQLKETRHGDQIHEMGIVYAPDYVINAGGVINVADELYGYNPERAMKKVEGIYNNVAKVIEISKRDGIPTYVAADRMAEERIAKVSKSRSQFLLNNRHILSYRA (SEQ IDNO: 7).

[0018] A second aspect of the present invention provides a gene encoding the above-mentioned leucine dehydrogenase mutant SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7.

[0019] In one embodiment, the nucleotide sequence of the gene encoding the leucine dehydrogenase mutant SEQ ID NO: 3 is shown in SEQ ID NO: 4; the nucleotide sequence of the gene encoding the leucine dehydrogenase mutant SEQ ID NO: 5 is shown in SEQ ID NO: 6; and the nucleotide sequence of the gene encoding the leucine dehydrogenase mutant SEQ ID NO: 7 is shown in SEQ ID NO: 8.

[0020] A third aspect of the present invention provides a DNA molecule comprising the gene encoding as described above.

[0021] For example, the DNA molecule contains a coding gene with a nucleotide sequence such as SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 and an upstream promoter such as the T7 promoter and / or a downstream terminator such as the T7 terminator, for example, an expression cassette / expression box of a leucine dehydrogenase mutant SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7.

[0022] A fourth aspect of the present invention provides a recombinant plasmid, which is an expression plasmid formed by cloning the coding gene as described above on a plasmid vector.

[0023] Optionally, the above-mentioned plasmid vectors may be selected from the pET series (e.g., pET22b, pET24a, pET28a), pMAL series, pGEX series, pQE series, pBAD series, pCAI series, pSH series, pRSFDuet series, or other vectors.

[0024] The fifth aspect of this invention provides a microbial engineered bacterium that expresses the coding gene as described above.

[0025] In one embodiment, the aforementioned engineered microbial bacteria are transformants that have been transformed with the recombinant plasmids described above; or positive recombinant bacteria that have cloned the gene encoding the above-described gene into the host genome using gene editing technology.

[0026] The host bacteria of the aforementioned engineered microorganisms are preferably microorganisms with rapid proliferation rates and suitable for expressing exogenous recombinant proteins, such as Bacillus subtilis, Lactobacillus brevis, Escherichia coli, sodium-dependent Vibrio, Candida magnolius, Pichia pastoris, and Saccharomyces cerevisiae. Escherichia coli is a preferred microorganism, and more preferably Escherichia coli BL21(DE3).

[0027] In one embodiment, the transformation of the recombinant plasmid can be performed by conventional chemical transformation or electrotransformation into competent cells; the gene editing technology is selected from the following group: homologous double crossover, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, MuGENT (multiplex genome editing by natural transformation), etc.

[0028] The sixth aspect of the present invention provides a method for the enzyme-catalyzed synthesis of L-tert-leucine, comprising the following steps: using trimethylpyruvate, isopropanol and an amino donor as substrates, using leucine dehydrogenase Leudh or its mutant as described above, or microbial engineered bacteria as described above, and alcohol dehydrogenase (ADH), a dual-enzyme catalytic reaction is carried out in the presence of coenzyme NAD+ to obtain L-tert-leucine.

[0029] The amino donor in the above reaction system can be ammonia or an ammonium salt. The ammonium salt can be an inorganic ammonium salt, such as ammonium chloride, ammonium sulfate, or ammonium carbonate; or an organic ammonium salt, such as ammonium formate or ammonium acetate.

[0030] Preferably, a zinc ion donor, such as zinc acetate, is added to the above reaction system as an activator of alcohol dehydrogenase.

[0031] In one embodiment, the alcohol dehydrogenase is a thermostable alcohol dehydrogenase, preferably an alcohol dehydrogenase with a half-life of not less than 3 hours, more preferably not less than 4 hours, and more preferably not less than 5 hours in an aqueous solution at 50°C with a pH of 8.0.

[0032] Preferably, the alcohol dehydrogenase is selected from the group consisting of: alcohol dehydrogenase derived from Rhodococcus ruber DSM 44541, alcohol dehydrogenase derived from Bacillus stearothermophilus LLD-R, and alcohol dehydrogenase derived from Microbacterium chocolatum (GenBank: WP_053546729.1).

[0033] The pH value of the above reaction system is 7.0-9.0, preferably 7.5-8.5, and more preferably around 8.0.

[0034] Furthermore, the reaction temperature is 35℃~55℃, preferably 37℃~52℃, more preferably 40℃~50℃, and even more preferably around 45℃.

[0035] It should be understood that in this article, when describing numerical characteristics, the terms "approximately" or "around" refer to the fact that the represented number may have an error range or fluctuation range of ±10%, ±9%, ±8%, ±7%, ±6%, or ±5%. The range of numerals appearing in this article includes the numeral itself and any number within that range.

[0036] Optionally, both enzymes in the above reaction system are in enzyme form to improve reaction rate and production efficiency, as homogeneous reactions are known to be the fastest in chemistry. The enzymes may be in the form of free enzymes or immobilized enzymes; these enzymes can be purified enzymes or crude enzymes.

[0037] This invention expands the application of thermostable leucine dehydrogenases and their mutants screened for high-temperature synthesis of L-2-aminobutyric acid. Experiments have shown that these enzymes can also catalyze the reaction of trimethylpyruvate with alcohol dehydrogenase at 45℃-55℃ to synthesize L-tert-leucine with high optical purity (ee value of over 99%), effectively shortening the reaction time and significantly improving the production efficiency of L-tert-leucine. This helps to improve the economic efficiency of L-tert-leucine production and has prospects for industrial application. Attached Figure Description

[0038] Figure 1 The plasmid map of the wild-type leucine dehydrogenase gene expression plasmid / pET24a-leudh is shown.

[0039] Figure 2The HPLC chromatogram of the L-tert-leucine preparation catalyzed by the leucine dehydrogenase mutant E114C and alcohol dehydrogenase in the example at 0.5 h is shown.

[0040] Figure 3 The hydrogen nuclear magnetic resonance spectrum of the product L-tert-leucine prepared in the examples is shown.

[0041] Figure 4 The mass spectrum of the product L-tert-leucine prepared in the examples is shown. Detailed Implementation

[0042] Given that the rates and temperatures of chemical and biological reactions are closely related, higher reaction temperatures can significantly shorten reaction times. However, the limiting factor for enzymatic reactions at high temperatures, such as 40°C-50°C, is the heat tolerance of the enzymes themselves, which are proteins. In our previous process development for the enzyme-catalyzed reaction of threonine to produce L-aminobutyric acid, we screened a leucine dehydrogenase derived from the thermostable bacterium *Heyndrickxia sporothermodurans*, which maintained its activity even at 55°C.

[0043] We further expanded the application of this heat-resistant leucine dehydrogenase by attempting to use it to catalyze the reaction of trimethylpyruvate, isopropanol, and an amino donor to prepare L-tert-leucine. We found that it still retains its catalytic function and maintains its enzyme activity at reaction temperatures of 40℃-50℃.

[0044] Next, in order to improve its enzyme activity, we modified the amino acid sequence of the wild-type enzyme SEQ ID NO: 1 using error-prone PCR and site-directed mutagenesis to obtain some mutants with enhanced enzyme activity, including the E114C mutant with the amino acid sequence shown in SEQ ID NO: 3, the E114C / T43M mutant with the amino acid sequence shown in SEQ ID NO: 5, and the E114C / T43S mutant with the amino acid sequence shown in SEQ ID NO: 7.

[0045] When wild-type leucine dehydrogenase and its two mutants, E114C and E114C / T43M, are used in combination with alcohol dehydrogenase to catalyze the reaction of trimethylpyruvate, isopropanol, and an amino donor to synthesize L-tert-leucine, they also exhibit catalytic activity. Moreover, the enzyme activity of the mutants is higher than that of the wild-type enzyme, and the product L-tert-leucine has a high optical purity, with an ee value of over 99%.

[0046] On the other hand, leucine dehydrogenase mutants suitable for synthesizing L-tert-leucine are obviously not limited to E114C and E114C / T43M mutants, but also include mutants with enhanced enzyme activity at E114, T43 sites, and E114C and E114 / T43 dual sites.

[0047] As used herein, the terms “(enzyme activity) increase,” “enhancement,” or “increase” mean an increase of at least 50% or more compared to a reference level, such as wild-type, or an increase of at least 80%, at least about 1, at least about 2, at least about 3, at least about 4, or at least about 5 times compared to a reference level.

[0048] In this article, the terms "wild-type leucine dehydrogenase," "wild-type enzyme," and "primitive enzyme" have the same meaning, for example, referring to the enzyme with the amino acid sequence SEQ ID NO: 1. Leucine dehydrogenase (Named Leudh in the example).

[0049] Correspondingly, the terms "leucine dehydrogenase mutant," "mutant leucine dehydrogenase," "mutant," and "mutant enzyme" have the same meaning, all referring to mutants of leucine dehydrogenase with increased enzyme activity, such as SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7. For the sake of brevity and convenience, wild-type leucine dehydrogenase and its mutants may be collectively referred to as "leucine dehydrogenase" in this article, as long as it is not confused with the wild-type enzyme SEQ ID NO: 1.

[0050] The term "mutation" includes, but is not limited to, the substitution, deletion, insertion, or chemical modification of amino acid residues, preferably a positive mutation, i.e., a mutation that increases enzyme activity. The substitution can be a non-conservative substitution, a conserved substitution, or a combination of both. A "conservative" amino acid substitution or mutation refers to the interchangeability of residues with similar side chains, and therefore generally includes the substitution of amino acids in a polypeptide with amino acids from the same or similar amino acid definition class. However, as used herein, if a conserved mutation can alternatively be an aliphatic to aliphatic, nonpolar to nonpolar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or restriction residue to restriction residue substitution, then a conserved mutation does not include hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or small residue to small residue substitution. As is known in this technical field, common examples of conservative substitutions include: substitutions between aromatic amino acids F, W, and Y; substitutions between hydrophobic amino acids L, I, and V; substitutions between polar amino acids Q and N; substitutions between basic amino acids K, R, and H; substitutions between acidic amino acids D and E; and substitutions between hydroxyl amino acids S and T. Furthermore, A, V, L, or I can be conservatively mutated to another aliphatic residue or another nonpolar residue. Exemplary conservative substitutions can be performed according to the table below.

[0051] The initial residues Representative substitution Preferred replacement Ala (A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu (L) Ile; Val; Met; Ala; Phe Ile Lys (K) Arg;Gln;Asn Arg Met (M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro (P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala Leu

[0052] "Non-conservative substitution" refers to the substitution or mutation of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitution can be performed between, rather than within, the amino acids defined above. In one embodiment, a non-conservative mutation affects (a) the structure of the peptide backbone in the substituted region (e.g., proline replacing glycine), (b) charge or hydrophobicity, or (c) side chain volume.

[0053] "Deletion" refers to a modification of a peptide by removing one or more amino acids from a reference peptide. Deletion can include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids constituting the reference enzyme, while preserving enzyme activity and / or the modified properties of engineered leucine dehydrogenase (Leudh). Deletion can target the interior and / or ends of the peptide. In several embodiments, the deletion can comprise a continuous segment or can be discontinuous.

[0054] "Insertion" refers to a modification of a polypeptide by adding one or more amino acids to a reference polypeptide. In some embodiments, modified engineered leucine dehydrogenases (Leudh) include inserting one or more amino acids into naturally occurring leucine dehydrogenases (Leudh) and into other modified leucine dehydrogenase (Leudh) polypeptides. Insertion can be internal to the polypeptide, or at the carboxyl or amino terminus. Insertions as used herein include fusion proteins as known in the art. Insertion can be a continuous amino acid segment or separated by one or more amino acids in a naturally occurring polypeptide.

[0055] The leucine dehydrogenase and its mutants of the present invention contain 365 amino acids with a well-defined sequence. Therefore, those skilled in the art can easily obtain its encoding gene, expression cassettes (DNA molecules) containing these genes, plasmids, and transformants containing the plasmids.

[0056] These genes, expression cassettes, plasmids, and transformants can be obtained through genetic engineering construction methods well known to those skilled in the art.

[0057] In order to optimally express leucine dehydrogenase SEQ ID NO: 1 and its mutants SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 in microbial hosts such as Escherichia coli, which is most commonly used in genetic engineering, the present invention has optimized the codons of its expression gene.

[0058] Codon optimization is a technique used to maximize protein expression in an organism by increasing the translation efficiency of genes of interest. Different organisms often exhibit a particular preference for one of a set of codons encoding the same amino acid due to mutational predisposition and natural selection. For example, in fast-growing microorganisms such as *E. coli*, optimized codons reflect the composition of their respective genomic tRNA repertoires. Thus, in fast-growing microorganisms, low-frequency codons for amino acids can be used for high-frequency codon substitutions of the same amino acid. Consequently, the expression of optimized DNA sequences is improved in fast-growing microorganisms.

[0059] After codon optimization, the coding gene for wild-type leucine dehydrogenase SEQ ID NO: 1 can be the nucleotide sequence SEQ ID NO: 2, while the coding gene for leucine dehydrogenase mutant SEQ ID NO: 3 can be the nucleotide sequence SEQ ID NO: 4; the coding gene for mutant SEQ ID NO: 5 can be the nucleotide sequence SEQ ID NO: 6; and the coding gene for mutant SEQ ID NO: 7 can be the nucleotide sequence SEQ ID NO: 8.

[0060] As used herein, “DNA molecule” and “expression cassette” refer to a gene expression system containing all the necessary elements required to express a target leucine dehydrogenase, such as SEQ ID NO: 5. Typically, it includes the following elements: a promoter, a gene sequence encoding a leucine dehydrogenase, such as SEQ ID NO: 5, SEQ ID NO: 6, and a terminator; additionally, it may optionally include a signal peptide coding sequence, an enhancer sequence, etc.; these biological elements are operatively linked.

[0061] As used herein, “operable linking” refers to the functional spatial arrangement of two or more nucleic acid regions or sequences. For example, a promoter region is placed at a specific position relative to the target gene nucleic acid sequence SEQ ID NO: 4, 6, or 8, such that transcription of the nucleic acid sequence is guided by the promoter region, thereby “operable linking” the promoter region to the nucleic acid sequence.

[0062] Preferably, the recombinant plasmid (or nucleic acid construct) includes one or more copies, two or more copies, preferably four or more copies, six or more copies, of the coding gene for leucine dehydrogenase, such as SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7.

[0063] When used as biocatalysts to prepare L-tert-leucine, the leucine dehydrogenase and alcohol dehydrogenase of the present invention can be in the form of enzymes or in the form of bacterial cells. The enzyme form includes free enzymes and immobilized enzymes, including purified enzymes, crude enzymes, fermentation broth, enzymes immobilized on carriers, etc.; the bacterial cell form includes live cells and dead cells.

[0064] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0065] Example

[0066] The examples involve the addition amount, content and concentration of various substances, and unless otherwise specified, the percentage content refers to the mass percentage content.

[0067] In the embodiments described herein, unless otherwise specified, the temperature generally refers to room temperature (15-30°C).

[0068] Materials and methods

[0069] In the embodiments, the whole gene synthesis, primer synthesis and sequencing were all commissioned to Nanjing GenScript Biotech Co., Ltd.

[0070] The molecular biology experiments in the examples included plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, and culture medium preparation, etc., mainly referring to "Molecular Cloning: A Laboratory Manual" (4th Edition), edited by M.R. Green and J. Sambrook (USA), translated by He Fuchu, Science Press, Beijing, 2017. Specific experimental conditions can be determined through simple experiments if necessary.

[0071] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0072] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2 (LB solid medium with an additional 20 g / L agar powder).

[0073] TB medium: 24 g / L yeast extract, 12 g / L tryptone, 16.43 g / L K2HPO4·3H2O, 2.31 g / L KH2PO4, 5 g / L glycerol, pH 7.0-7.5.

[0074] HPLC method for the detection of trimethylpyruvate and L-tert-leucine

[0075] Agilent 1260 high performance liquid chromatograph;

[0076] Mobile phase A:

[0077] 30% methanol, 70% sodium acetate

[0078] Mobile phase B: Acetonitrile

[0079] Flow rate: 1.0 ml / min

[0080] Chromatographic column: SB-C18 (250mm*4.5)

[0081] Column temperature: 30℃

[0082] Detection wavelength: 334nm

[0083] gradient:

[0084] T 0 5 13 18 18.1 25 A% 95 95 0 0 95 95 B% 5 5 100 100 5 5

[0085] Derivatizing reagents: 8 ml / L mercaptopropionic acid, 10 g / L o-phthalaldehyde

[0086] Retention time: L-tert-leucine 6.2 min

[0087] For ease of description, in the embodiments, the strain number, plasmid number, enzyme number, and enzyme-encoding gene number can share the same number, that is, the same number can refer to different biological forms in different descriptive scenarios.

[0088] Example 1: Construction of an expression strain for wild-type leucine dehydrogenase (Leudh)

[0089] Based on the amino acid sequence SEQ ID NO: 1 of the wild-type leucine dehydrogenase Leudh from the microorganism Heyndrickxia sporothermodurans, codon optimization was performed according to the codon preference of E. coli to obtain the coding gene sequence SEQ ID NO: 2 suitable for expression in E. coli. The whole genome of SEQ ID NO: 2 was synthesized by Nanjing GenScript Biotech Co., Ltd., and restriction endonuclease sites NdeI and BamHI were designed at both ends of the gene. The gene was subcloned into the corresponding sites of the vector pET24a to obtain the recombinant plasmid pET24a-leudh, the structure of which is shown below. Figure 1 As shown.

[0090] The recombinant plasmid was transformed into competent Escherichia coli BL21(DE3) cells expressing wild-type leucine dehydrogenase using a chemical transformation method. Positive clones were screened to obtain recombinant Escherichia coli BL21(DE3) / pET24a-leudh expressing wild-type leucine dehydrogenase.

[0091] Example 2: Error-prone PCR and construction of random mutant libraries

[0092] Using pET24a-leudh as a template, a random mutant library was constructed using error-prone PCR and large primer PCR.

[0093] PCR primer sequences include:

[0094] Forward primer err-F: 5'-GTTTAACTTTAAGAAGGAGATATAC-3',

[0095] Reverse primer err-R: 5'-CTTGTCGACGGAGCTCGAAT-3'.

[0096] 100 μL error-prone PCR system: 50 ng plasmid template, 0.2 μM primers, 1×Taq buffer, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, 1 mM dTTP, 7 mM MgCl2, 0.2, 0.3, and 0.4 mM MnCl2, 1 U Taq enzyme.

[0097] PCR program: 95℃ for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, 40 cycles; 72℃ for 7 min. The approximately 1.3 kb mutant fragment was recovered from the gel and used as a large primer. MegaPrimer PCR was performed using KOD FX neo DNA polymerase: 94℃ for 2 min, 68℃ for 10 min; 98℃ for 10 s, 55℃ for 30 s, 68℃ for 3 min, 25 cycles; 68℃ for 10 min.

[0098] DpnI was added to the PCR product, and the plasmid template was removed by digestion at 37°C. After purification and recovery, the plasmid was electroporated into E. coli BL21(DE3), and 1 mL of LB broth was added for recovery at 37°C for 1 h. The plating was then plated on Kansas and incubated overnight at 37°C to obtain more than 10 4 Random mutations in clones.

[0099] Example 3: High-throughput screening of mutant libraries

[0100] 1. The enzyme activity of leucine dehydrogenase was determined using trimethylpyruvate as a substrate, as follows.

[0101] Preparation of substrate reaction solution: 0.1M trimethylpyruvate at pH 7.5, 0.2M ammonium chloride, 0.25g / l NADH.

[0102] Strains from the mutant library were transferred to 200 μL of LB 96-well plates containing Kan resistance and incubated at 37°C for 6 hours. Then, 200 μL of LB broth containing the inducer IPTG and Kan resistance was added, and the plate was cooled to 28°C and incubated overnight. The next morning, 100 μL of the culture was transferred to a 96-well deep-well plate and frozen at -70°C for 2 hours, followed by thawing at 37°C for 30 minutes. Then, 30 μL of the culture was transferred to a microplate with 170 μL of reaction solution, and incubated at 37°C for 10 minutes. The absorbance at OD340 nm was then read. A greater decrease in absorbance indicates higher enzyme activity.

[0103] 2. The catalytic ability of the screened mutants to the substrate was compared, and the steps are as follows.

[0104] Shake-flask fermentation of leucine dehydrogenase expression strains: Liquid culture medium TB (pH 7.0-7.5) was prepared, containing 12 g / L peptone, 24 g / L imported yeast extract, 5 g / L glycerol, 2.13 g / L KH₂PO₄, and 16.43 g / L K₂HPO₄•3H₂O. TB was dispensed into 1000 mL Erlenmeyer flasks, each containing 200 mL of the medium, and then autoclaved at 121 °C for 20 min. Several loops of bacterial cells from leucine dehydrogenase and mutant expression strains were inoculated into the TB flasks using an inoculation loop from plates containing these expression strains. Before inoculation, 100 μg / mL kanamycin was added to the TB medium. The cultures were incubated at 37 °C and 220 rpm on a shaker until OD reached the target value. 600 = 5-6, add 0.2mM IPTG and induce at 28℃ for about 24h.

[0105] Preparation of crude enzyme solution: Take 50 mL of fermentation broth and place it in a centrifuge tube; centrifuge to obtain bacterial cells, add purified water at 200 g / L to resuspend, and then sonicate to disrupt: cool the suspended bacterial cells in an ice bath and sonicate to disrupt (voltage 400 W, sonication time 3 s, interval time 5 s, number of cycles 80).

[0106] The enzyme activity assay, using trimethylpyruvate as a substrate, proceeds as follows: Take 1.7 ml of 0.2 M ammonium chloride buffer (pH 7.5) and 2 ml of 1 M trimethylpyruvate solution (pH 7.5), preheat in a 37°C water bath, then add to a cuvette. Next, add 100 μl of 1% NADH solution and 100 μl of 2% crude enzyme solution sequentially. Measure the absorbance change at 340 nm using a spectrophotometer and calculate the enzyme activity. A larger decrease in absorbance indicates higher enzyme activity.

[0107] Strains exhibiting enhanced enzyme activity were selected, plasmids were extracted, and nucleic acid sequencing was performed by Nanjing GenScript Biotech Co., Ltd. The relevant fragments of the leucine dehydrogenase gene in the genome were compared with SEQ ID NO: 2 to determine the amino acid sequence changes of leucine dehydrogenase. The preferred strains with the highest improved enzyme activity were selected as the starting strains for the next round of random mutant library construction. Table 1 shows the activity comparison between some mutants and the wild type.

[0108] Table 1. Partial Results of High-Throughput Screening of Error-Prone PCR Random Mutant Libraries

[0109] mutant Enzyme activity U / ml Wild-type control 158.6 E114Q 182.3 Y67L 150.5 N163M 149.8 T43S 175.9 H193F 155.5 K265D 130.7 T43M 169.3

[0110] Using error-prone PCR1, sites E114 and T43 were identified as promising, suggesting they are key sites within the enzyme pocket structure. Therefore, site-directed saturation mutagenesis was performed on site E114.

[0111] Example 4: Site-directed mutagenesis at amino acid sequences E114 and T43

[0112] Site-directed saturation mutations were generated at sites E114 and T43 in the leucine dehydrogenase sequence using degenerate primers to construct a mutant library.

[0113] The site-directed saturation mutation sites and primer design are shown in Table 2 below.

[0114] Table 2. PCR primers for site-directed saturation mutagenesis of E114 and T43

[0115] mutation site Mutant primers Primer sequence (5'-3') T43 T43-F acctgcattaggtgggNNKaggatgtggac E114 E114-F gctatattactgcaNNKgatgtaggtacaa err-F GTTTAACTTTAAGAAGGAGATATAC err-R GACGGAGCTCGAATTCGGAT

[0116] Degenerate primer codes: M = (A / C), K = (G / T), N = (A / C / G / T). In the table, F represents the forward primer; R represents the reverse primer; err-F / R: common mutation primer. Uppercase NNK represents the mutation site. Using pET24a-leudh as a template, PCR amplification was performed using the forward primer E114-F and the common reverse primer err-R shown in Table 2, employing KOD DNA polymerase (94℃ 2 min; 98℃ 10 s, 55℃ 30 s, 68℃ 45 s; 30 cycles; 68℃ 10 min), yielding a DNA fragment containing the E114 mutation site. The DNA fragment was recovered by gel electrophoresis and used as a large primer for MegaPrimer PCR (94℃ 2 min; 98℃ 10 s, 55℃ 30 s, 68℃ 3 min; 25 cycles; 68℃ 10 min). The recombinant plasmid was digested with DpnI, electroporated into E. coli BL21(DE3) competent cells, and plated on LB agar plates containing kanamycin to construct a saturated mutant library of the Leudh / E114 site, which included a series of mutants.

[0117] Following the method described in Example 3, a site-directed mutant library of E114 was screened. Strains exhibiting enhanced enzyme activity were selected, plasmids were extracted, and nucleic acid sequencing was performed by Nanjing GenScript Biotech Co., Ltd. The relevant fragments of the leucine dehydrogenase gene in the genome were compared with SEQ ID NO: 2 to determine the amino acid sequence changes of leucine dehydrogenase. Some mutants showed increased enzyme activity (see Table 2).

[0118] Table 3. Partial Results of Site-Specific Saturation Mutant Library Screening for Site E114

[0119] mutant Enzyme activity u / ml wild type 160.08 E114H 184.61 E114Q 181.83 E114L 118.69 E114C 229.94 E114T 117.86 E114R 156.28 E114S 183.33 E114V 217.41

[0120] As shown in Table 3, the enzyme activities of mutants E114H, E114Q, E114V, E114C, and E114S were increased compared to the wild-type enzyme. Site-directed saturation mutagenesis was then performed at the T43 site at these sites, and a series of site-directed saturation mutant libraries were constructed using the same method. The enzyme activities of some of the selected two-site mutants are shown in Table 4.

[0121] Table 4. Enzyme activities of some E114 / T43 double-site mutants

[0122] mutant Enzyme activity u / ml wild type 159.2 E114C / T43M 310.1 E114C / T43N 194.5 E114C / T43S 322.3 E114H / T43V 143.4 E114H / T43A 184.2 E114H / T43K 158.2 E114Q / T43M 191.8 E114V / T43G 182.6 E114V / T43A 199.4 E114V / T43S 232.2 E114V / T43T 214.0 E114V / T43C 183.6 E114V / T43I 191.1 E114V / T43M 253.0

[0123] As can be seen from Table 4, the enzyme activity of the two-site mutants E114C / T43M and E114C / T43S increased the most, while the enzyme activity of some E114 / T43 two-site mutants even decreased.

[0124] Example 5: Enzyme thermostableness experiment

[0125] To withstand reaction temperatures of 40°C or higher, the enzymes used must possess thermal stability. We investigated the thermostability of wild-type leucine dehydrogenase and its mutants.

[0126] The enzyme activity of wild-type leucine dehydrogenase and its mutants at 50°C was determined according to the "Enzyme Activity Assay Method" in Example 3, except that a 50°C water bath was used for the reaction, and streptomycin 10 μg / mL was added to the reaction solution to avoid bacterial contamination. Residual enzyme activity was measured, and the enzyme half-life was calculated. The results are shown in Table 5.

[0127] Table 5. Comparison of half-life between wild-type enzymes and mutants at 50°C

[0128] mutant Half-life at 50°C (h) wild type 13 E114C 13 T43S 13.5 E114C / T43M 13 E114C / T43S 13

[0129] Experiments show that wild-type leucine dehydrogenase and its mutants have high thermal stability, sufficient to withstand reaction temperatures above 45°C.

[0130] Example 6: Dual-enzyme combination catalyzes the conversion of trimethylpyruvate to L-tert-leucine.

[0131] L-tert-leucine was prepared by combining leucine dehydrogenase and alcohol dehydrogenase to catalyze the reaction of trimethylpyruvate and isopropanol at a reaction temperature of 45℃. Taking a laboratory-scale 100ml system as an example, the method is as follows: Accurately weigh 7.80g of trimethylpyruvate substrate, 6.60g of isopropanol, and 0.01g of zinc acetate. Measure 75ml of water and add it to a 500ml Erlenmeyer flask. Adjust the pH to approximately 8.0 with 25% ammonia. Preheat the shaker to 45℃. Add 6000U of thermostable alcohol dehydrogenase enzyme solution (crude enzyme solution of the alcohol dehydrogenase expression strain constructed in the literature https: / / doi.org / 10.1002 / elsc.200620902), 800U of wild-type or mutant leucine dehydrogenase enzyme solution, and 0.5g of 2% NAD+ solution to start the reaction. Shake at 45℃, 150rpm, and pH 8.0. Take samples at 0.5h, 1h, and 1.5h, and detect the amount of L-tert-leucine generated in the reaction system by HPLC. Figure 2 The HPLC chromatograms of the reaction catalyzed by the leucine dehydrogenase mutant E114C / T43S are shown. Table 6 compares the catalytic abilities of the wild-type Leudh enzyme and the mutant for the reaction with trimethylpyruvate.

[0132] Table 6. Comparison of the ability of Leudh wild-type enzyme and mutant to catalyze the trimethylpyruvate reaction.

[0133] mutant molar conversion rate Wild-type control 92.56 E114C 96.72 E114C / T43S 99.33 E114C / T43M 99.04

[0134] The results in Table 6 show that the Leudh wild-type enzyme and its mutants E114C, E114C / T43M and E114C / T43S all maintain high catalytic activity at high temperatures.

[0135] Furthermore, after 2 hours of the co-catalytic reaction involving the mutant E114C / T43S, we extracted and purified the product, and identified it using liquid chromatography-mass spectrometry (LC-MS) and proton nuclear magnetic resonance (NMR) spectroscopy. (See attached image.) Figure 3 and Figure 4 The product was confirmed to be L-tert-leucine, with an optical purity ee value of 99.4%.

[0136] 1H NMR (400 MHz, D2O, ppm) δ:: 0.93 (s, 9H), 3.31 (s, 1H).

[0137] MS (ESI): m / z 132.2 [M+H] + .

[0138] The above embodiments demonstrate that the leucine dehydrogenase Leudh and some mutants from the thermostable bacterium Heyndrickxia sporothermodurans screened in this invention are tolerant to higher reaction temperatures, which is beneficial for combining with thermostable alcohol dehydrogenases for the biosynthesis of L-tert-leucine, shortening the production cycle of L-tert-leucine, and improving the economic efficiency of producing high-quality L-tert-leucine.

[0139] It should be understood that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A leucine dehydrogenase mutant for the preparation of L-tertiary leucine, characterized in that, It is a derivative polypeptide of the leucine dehydrogenase Leudh, derived from the thermostable bacterium *Heyndrickxia sporothermodurans*, with the amino acid sequence shown in SEQ ID NO: 1: A derivative polypeptide with a mutation at the E114 site of the amino acid sequence SEQ ID NO: 1 and increased enzyme activity; A derivative polypeptide with a mutation at the T43 site of the amino acid sequence SEQ ID NO: 1 and increased enzyme activity; or A derivative polypeptide with simultaneous mutations at both E114 and T43 sites in the amino acid sequence SEQ ID NO: 1, resulting in increased enzyme activity.

2. The leucine dehydrogenase mutant as described in claim 7, characterized in that, It is the following derivative polypeptide of the leucine dehydrogenase: The E114C mutant of leucine dehydrogenase SEQ ID NO: 1, whose amino acid sequence is shown in SEQ ID NO: 3; The E114C / T43M mutant of leucine dehydrogenase SEQ ID NO: 1, whose amino acid sequence is shown in SEQ ID NO: 5; or The E114C / T43S mutant of leucine dehydrogenase SEQ ID NO: 1 has the amino acid sequence shown in SEQ ID NO:

7.

3. The gene encoding the leucine dehydrogenase mutant SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 as described in claim 1.

4. The gene as described in claim 7, characterized in that, The nucleotide sequence of the gene encoding leucine dehydrogenase mutant SEQ ID NO: 3 is shown in SEQ ID NO: 4; the nucleotide sequence of the gene encoding leucine dehydrogenase mutant SEQ ID NO: 5 is shown in SEQ ID NO: 6; and the nucleotide sequence of the gene encoding leucine dehydrogenase mutant SEQ ID NO: 7 is shown in SEQ ID NO:

8.

5. A DNA molecule, characterized in that, The DNA molecule contains the gene as described in claim 4.

6. A recombinant plasmid, characterized in that, The recombinant plasmid is an expression plasmid formed by cloning the gene as described in claim 4 on a plasmid vector.

7. A type of engineered microbial bacteria, characterized in that, It expresses the gene as described in claim 4.

8. A method for enzyme-catalyzed synthesis of L-tert-leucine, characterized in that, The method includes the following steps: using trimethylpyruvate, isopropanol, and an amino donor as substrates, and employing leucine dehydrogenase Leudh as described in claim 1 or 2 or its mutant, or the microbial engineered bacteria as described in claim 7, and alcohol dehydrogenase (ADH), a dual-enzyme catalytic reaction is carried out in the presence of coenzyme NAD+ to obtain L-tert-leucine.

9. The method as described in claim 8, characterized in that, The reaction system also includes zinc ion donors, such as zinc acetate, as activators of alcohol dehydrogenase.

10. The method as described in claim 8, characterized in that, The alcohol dehydrogenase is an alcohol dehydrogenase with a half-life of not less than 3 hours, preferably not less than 4 hours, and more preferably not less than 5 hours in an aqueous solution at 50°C with a pH of 8.0.

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