Method for directly preparing L-methionine from L-homoserine

Abstract

The invention relates to a method for preparing L-methionine by catalyzing homoserine and methyl mercaptan to react through an enzyme. The method does not include a step of producing O-acyl-L-homoserine, and / or the method does not use O-acyl-L-homoserine.

Description

A method for directly preparing L-methionine from L-homoserine Technical Field

[0001] The present invention belongs to the field of biochemical engineering and specifically relates to a method for directly enzymatically preparing L-methionine using L-homoserine as a substrate. Background Art

[0002] Methionine is one of the 20 naturally occurring amino acids found in proteins and is a precursor for the synthesis of S-adenosylmethionine. S-adenosylmethionine acts as a methyl group donor in the human body and participates in numerous important metabolic processes. Humans cannot synthesize methionine and must obtain it through, for example, dietary intake. Methionine is also a feed additive used extensively in livestock, poultry, and fish farming.

[0003] Methionine can be produced by chemical synthesis. However, chemically synthesized methionine is a racemic form, a D / L mixed form, and D-methionine is difficult to utilize in vivo.

[0004] Optically pure L-methionine can also be obtained by biosynthesis to achieve higher bioavailability. Biosynthesis of methionine is usually carried out by introducing enzymes involved in the methionine biosynthetic metabolic pathway (see Figure 1) into host cells (see, for example, CN 105886449 B, CN 106906176 A, CN 103781913 B, and CN 114787369 A). However, biosynthesis requires the introduction of a large number of exogenous genes into the host cell, requiring complex genetic engineering operations; and because the methionine anabolic pathway is controlled by a complex regulatory network, it is difficult to achieve high yields.

[0005] A method has also been developed for synthesizing methionine by sulfiding and methylating O-acetyl homoserine using a methyl-terminated thioether, such as dimethyl disulfide, as a source of sulfur and methyl groups via fermentation with Corynebacterium glutamicum or a mutant of Escherichia coli (see CN 101223280 B). However, in this method, methyl mercaptan produced by the decomposition of the thioether has a strong toxic effect on microorganisms, affecting their growth and hindering the synthesis of methionine.

[0006] A method for synthesizing L-methionine by reacting O-succinylhomoserine with sodium methyl mercaptan using O-succinylsulfhydryltransferase (see CN 113215124 A) and a method for synthesizing L-methionine by reacting O-acylhomoserine, such as O-acetylhomoserine or O-succinylhomoserine, with methyl mercaptan using O-acylhomoserine sulfhydrylase (see WO 2008 / 013432 A1) have also been developed. However, these methods require the production of O-acylhomoserine (e.g., by fermentation) first, which is limited by i) the accumulation of O-acylhomoserine; ii) the instability of O-acylhomoserine (to acid, alkali, and heating); iii) the inhibition of enzyme activity by byproduct organic acids; and iv) the increase in production cost, process complexity, and product separation difficulty if the pH is adjusted during the reaction.

[0007] Therefore, it is necessary to develop a new method for preparing L-methionine, using readily available stable substrates, reducing the inhibition of the product on the reaction, simplifying the process (including production and separation) and reducing costs.

[0008] Surprisingly, the inventors discovered that L-homoserine can react with methyl mercaptan under enzyme catalysis to generate L-methionine and water (as shown in Formula I).

[0009] L-homoserine is a more readily available and stable compound than O-acylhomoserine, and the reaction of the present invention does not produce organic acid byproducts that inhibit enzyme activity.

[0010] Therefore, the present invention provides a method for preparing L-methionine, comprising the following steps:

[0011] a) providing a reaction medium comprising L-homoserine or a salt, ester, amide or anhydride thereof, methyl mercaptan or a salt thereof and an enzyme selected from the group consisting of a transferase that transfers an alkyl group other than a methyl group or an aromatic group, an ammonia lyase and a lyase that catalyzes the cleavage of a carbon-sulfur bond;

[0012] b) incubating the reaction medium to produce a reaction product comprising L-methionine or a salt, ester or amide thereof; and optionally

[0013] c) recovering L-methionine or its salt, ester or amide from the reaction product.

[0014] In some embodiments, the enzyme is selected from the group consisting of enzymes with EC numbers EC 2.5.1.-, EC 4.3.1.-, and EC 4.4.1.-.

[0015] In some embodiments, the reaction medium further comprises pyridoxal phosphate or a salt thereof.

[0016] In some embodiments, the enzyme is selected from the group consisting of EC numbers EC 2.5.1.47, EC 2.5.1.48, EC 2.5.1.49, EC 2.5.1.51, EC 2.5.1.52, EC 2.5.1.65, EC 2.5.1.76, EC 2.5.1.113, EC 2.5.1.134, EC 2.5.1.140, EC 2.5.1.144, EC 4.3.1.15, EC 4.3.1.17, EC 4.3.1.18, EC 4.3.1.19, EC 4.4.1.1, EC 4.4.1.2, EC 4.4.1.9, EC 4.4.1.10, EC 4.4.1.11, EC 4.4.1.13, EC Enzymes of EC 4.4.1.15, EC 4.4.1.16, EC 4.4.1.25, EC 4.4.1.28 and EC 4.4.1.35.

[0017] In some embodiments, the enzyme is derived from a bacterium selected from Thermus thermophiles, Lysinibacillus sphaericus, Clostridioides difficile, Clostridium novyi, Geobacillus stearothermophilus, Thermotoga maritima, Pseudomonas aeruginosa, Nonlabens dokdonensis, Leucaena leucocephala, Aeropyrum pernix, Methanosarcina acetivorans, Mycobacterium tuberculosis, Bacillus subtilis, Staphylococcus aureus, Pseudomonas fluorescens, Escherichia coli. coli), Candida maltosa, Streptomyces phaeochromogenes, Neurospora crassa, Spinacia oleracea, Cyanobacteria bacterium, Pseudomonas putida, Lactococcus lactis, Ruegeria pomeroyi, Methanococcus maripaludis, and Camelina sativa.

[0018] In some embodiments, the enzyme is selected from the group consisting of cysteine ​​synthase (EC 2.5.1.47) from Thermus thermophiles, cystathionine gamma-synthase (EC 2.5.1.48) from Lysinibacillus sphaericus, O-acetylhomoserine aminocarboxypropyltransferase (EC 2.5.1.49) from Clostridium difficile, O-acetylhomoserine aminocarboxypropyltransferase from Clostridium novyi, O-acetylhomoserine aminocarboxypropyltransferase from Geobacillus stearothermophilus, O-acetylhomoserine aminocarboxypropyltransferase from Thermocystis maritima, O-acetylhomoserine aminocarboxypropyltransferase from Pseudomonas aeruginosa, beta-pyrazoloylalanine synthase (EC 2.5.1.51) from Acinetobacter dokshimaensis. synthase), L-mimosine synthase from Leucaena leucaena (EC 2.5.1.52), O-phosphoserine sulfhydrylase from Aeropyrum pernix (EC 2.5.1.65), cysteine ​​synthase from Methanosarcina acetivorans (EC 2.5.1.76), [CysO sulfur-carrier protein]-thiocarboxylate-dependent cysteine ​​synthase from Mycobacterium tuberculosis (EC 2.5.1.113), cystathionine beta-synthase from Bacillus subtilis (EC 2.5.1.134), N-(2-amino-2-carboxyethyl)-L-glutamate synthase from Staphylococcus aureus (EC 2.5.1.136), and N-(2-amino-2-carboxyethyl)-L-glutamate synthase from Staphylococcus aureus (EC 2.5.1.140). 2.5.1.140-N-(2-amino-2-carboxyethyl)-L-glutamate synthase), S-sulfo-L-cysteine ​​synthase from Pseudomonas fluorescens (EC 2.5.1.144-S-sulfo-L-cysteine ​​synthase), diaminopropionate ammonia lyase from Escherichia coli (EC 4.3.1.15-Diaminopropionate ammonia-lyase), L-serine ammonia-lyase from Escherichia coli (EC 4.3.1.17-L-serine ammonia-lyase), D-serine ammonia-lyase from Escherichia coli (EC 4.3.1.18-D-serine ammonia-lyase), threonine ammonia-lyase from Candida maltosa (EC4.3.1.19-threonine ammonia-lyase), cystathionine gamma-lyase from Streptomyces fuscae (EC 4.4.1.1-cystathionine gamma-lyase), cystathionine gamma-lyase from Neurospora crassa (EC 4.4.1.1-cystathionine gamma-lyase), homocysteine ​​desulfhydrase from Bacillus subtilis (EC 4.4.1.2-homocysteine ​​desulfhydrase), L-3-cyanoalanine synthase from spinach (EC 4.4.1.9 - L-3-cyanoalanine synthase), cysteine ​​lyase from Cyanobacteria bacterium (EC 4.4.1.10 - cysteine ​​lyase), methionine gamma-lyase from Pseudomonas putida (EC 4.4.1.11 - methionine gamma-lyase), cysteine ​​S-conjugate beta-lyase from Lactococcus lactis (EC 4.4.1.13 - cysteine-S-conjugate beta-lyase), D-cysteine ​​desulfhydrase from Escherichia coli (EC 4.4.1.15 - D-cysteine ​​desulfhydrase), selenocysteine ​​lyase from Escherichia coli (EC 4.4.1.16 - selenocysteine ​​lyase), L-cysteine ​​sulfide lyase from Ruegeria pomeroyi (EC 4.4.1.25 - L-cysteine sulfo-lyase), L-cysteine ​​desulfidase from Methanococcus maripaludis (EC 4.4.1.28-L-cysteine ​​desulfidase), and L-cystine beta-lyase from Camelina sativa (EC 4.4.1.35-L-cystine beta-lyase).

[0019] In some embodiments, the enzyme comprises an amino acid sequence selected from SEQ ID NOs: 1-31.

[0020] In some embodiments, the method is a cell-free method.

[0021] In some embodiments, the reaction medium in step a) comprises a host cell expressing the enzyme. In some embodiments, the method of the present invention further comprises the step of culturing the host cell.

[0022] In some embodiments, in step b), the reaction medium is incubated at 30-40°C, 32-39°C, 34-38°C, 36-38°C, or 37°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a schematic diagram of the methionine biosynthesis pathway (see Brewster, JL, et al., Structures and kinetics of Thermotoga maritima MetY reveal new insights into the predominant sulfurylation enzyme of bacterial methionine biosynthesis. The Journal of biological chemistry, 2021, 296, 100797).

[0024] Detailed Description of the Invention

[0025] The present invention provides a novel method for preparing L-methionine. Unless otherwise specified, the terms used herein have the meanings generally understood by those skilled in the art.

[0026] Prior art reports indicate that 4-hydroxy-activated L-homoserine (such as O-acylhomoserine) can react with methyl mercaptan under enzyme catalysis to produce L-methionine.

[0027] Surprisingly, the inventors discovered that L-homoserine can react with methyl mercaptan under enzyme catalysis to generate L-methionine and water (as shown in Formula I).

[0028] L-homoserine is a more readily available and stable compound than O-acylhomoserine, and the reaction of formula I does not produce organic acid byproducts that inhibit enzyme activity.

[0029] Therefore, the present invention provides a method for preparing L-methionine, comprising the following steps:

[0030] a) providing a reaction medium comprising an L-homoserine donor, a methylthio group donor and an enzyme selected from the group consisting of a transferase that transfers an alkyl group other than a methyl group or an aromatic group, an ammonia lyase and a lyase that catalyzes the cleavage of a carbon-sulfur bond;

[0031] b) incubating the reaction medium to produce a reaction product comprising L-methionine or a salt, ester or amide thereof; and optionally

[0032] c) recovering L-methionine or its salt, ester or amide from the reaction product.

[0033] 1. Substrates and Products

[0034] As used herein, "L-homoserine" refers to L-2-amino-4-hydroxy-1-acid with a molecular formula of C4H9NO3. As used herein, L-homoserine donors include, but are not limited to, L-homoserine or its salts, esters, or amides, and anhydrides formed between L-homoserine and L-homoserine or other acids. L-homoserine donors also include derivatives in which one or two hydrogen atoms on the amino group are substituted. Esters of L-homoserine refer to compounds produced by the reaction of the carboxyl group of L-homoserine with an alcohol or phenol. L-homoserine amides refer to compounds produced by the reaction of the carboxyl group of L-homoserine with an amine compound.

[0035] Herein, the L-homoserine donor does not include a derivative in which the hydrogen atom of the 4-hydroxyl group is substituted with an acyl group, that is, O-acyl homoserine.

[0036] In step b), L-homoserine is the direct substrate of the enzyme. The method of the present invention does not include a step of producing O-acyl-L-homoserine, and the method does not use O-acyl-L-homoserine.

[0037] As used herein, "methylthio donor" refers to a compound capable of donating a methylthio group (CH3S-), including but not limited to methylthiol or a salt thereof and a methyl-terminated sulfide or disulfide, such as dimethyl disulfide. In some embodiments, the methylthio donor is methylthiol or a salt thereof, including but not limited to sodium methylthiolate and potassium methylthiolate.

[0038] 2. Enzymes

[0039] As described above, the reaction of the present invention is carried out under the catalysis of an enzyme, that is, an enzymatic reaction.

[0040] As used herein, the term "enzyme" refers to a protein or polypeptide that can specifically catalyze or promote a chemical or biochemical reaction. The enzyme of the present invention can be a complete native protein or polypeptide or a catalytically active portion thereof. The enzyme of the present invention can also be a modified variant of a native protein or polypeptide. The enzyme of the present invention can be isolated from an organism, recombinantly produced, or synthetically produced. Those skilled in the art can obtain the enzyme by conventional methods.

[0041] As used herein, the term "peptide" refers to a chain of at least two amino acids linked by peptide bonds. The term "polypeptide" is used interchangeably herein with the term "protein" to refer to a chain of ten or more amino acid residues. All peptide and polypeptide formulas or sequences herein are written from left to right, indicating the direction from the amino terminus to the carboxyl terminus.

[0042] The term "amino acid" includes naturally occurring amino acids and non-natural amino acids in proteins. The single-letter and three-letter nomenclature for naturally occurring amino acids in proteins adopts the names commonly used in the art and can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0043] As used herein, the term "modification" refers to any modification of a polypeptide consisting of the polypeptide of the present invention or a homologous sequence thereof, including but not limited to substitution, deletion, insertion and / or addition of one or more amino acids.

[0044] Some enzymes require the participation of coenzymes for their catalytic activity. The term "coenzyme" is a general term for a broad class of organic cofactors essential for enzyme-catalyzed redox reactions, group transfer reactions, and isomerization reactions. They function by transferring electrons, atoms, or groups in enzyme-catalyzed reactions.

[0045] The inventors have found that the enzyme used in the present invention only uses pyridoxal phosphate as a coenzyme. Therefore, in some embodiments, the reaction medium further comprises pyridoxal phosphate or a salt thereof.

[0046] In some embodiments, the enzyme is selected from the group consisting of enzymes with EC numbers EC 2.5.1.-, EC 4.3.1.-, and EC 4.4.1.-.

[0047] In some embodiments, the enzyme is selected from the group consisting of EC numbers EC 2.5.1.47, EC 2.5.1.48, EC 2.5.1.49, EC 2.5.1.51, EC 2.5.1.52, EC 2.5.1.65, EC 2.5.1.76, EC 2.5.1.113, EC 2.5.1.134, EC 2.5.1.140, EC 2.5.1.144, EC 4.3.1.15, EC 4.3.1.17, EC 4.3.1.18, EC 4.3.1.19, EC 4.4.1.1, EC 4.4.1.2, EC 4.4.1.9, EC 4.4.1.10, EC 4.4.1.11, EC 4.4.1.13, EC Enzymes of EC 4.4.1.15, EC 4.4.1.16, EC 4.4.1.25, EC 4.4.1.28 and EC 4.4.1.35.

[0048] In some embodiments, the enzyme is selected from cysteine ​​synthase (EC 2.5.1.47-cysteine ​​synthase), cystathionine gamma-synthase (EC 2.5.1.48-cystathionine gamma-synthase), O-acetylhomoserine aminocarboxypropyltransferase (EC 2.5.1.49-O-acetylhomoserine aminocarboxypropyltransferase), beta-pyrazolylalanine synthase (EC 2.5.1.51-beta-pyrazolylalanine synthase), L-mimosine synthase (EC 2.5.1.52-L-mimosine synthase), O-phosphoserine sulfhydrylase (EC 2.5.1.65-O-phosphoserine sulfhydrylase), cysteine ​​synthase (EC 2.5.1.76-cysteate synthase), [CysO sulfur carrier protein]-thioester-dependent cysteine ​​synthase (EC EC 2.5.1.113-[CysO sulfur-carrier protein]-thiocarboxylate-dependent cysteine ​​synthase), cystathionine beta synthase (EC 2.5.1.134-cystathionine beta-synthase), N-(2-amino-2-carboxyethyl)-L-glutamate synthase (EC 2.5.1.140-N-(2-amino-2-carboxyethyl)-L-glutamate synthase), S-sulfo-L-cysteine ​​synthase (EC 2.5.1.144-S-sulfo-L-cysteine ​​synthase), diaminopropionate ammonia-lyase (EC 4.3.1.15-Diaminopropionate ammonia-lyase), L-serine ammonia-lyase (EC 4.3.1.17-L-serine ammonia-lyase), D-serine ammonia-lyase (EC 4.3.1.18-D-serine ammonia-lyase), threonine ammonia-lyase (EC 4.3.1.19-threonine ammonia-lyase), cystathionine gamma-lyase (EC 4.4.1.1-cystathionine gamma-lyase), homocysteine ​​desulfhydrase (EC 4.4.1.2-homocysteine ​​desulfhydrase), L-3-cyanoalanine synthase (EC 4.4.1.9-L-3-cyanoalanine synthase), cysteine ​​lyase (EC 4.4.1.10-cysteine ​​lyase), methionine gamma-lyase (EC 4.4.1.11-methionine gamma-lyase), cysteine ​​S-conjugate beta-lyase (EC 4.4.1.13-cysteine-S-conjugate beta-lyase), D-cysteine ​​desulfhydrase (EC 4.4.1.15-D-cysteine ​​desulfhydrase), selenocysteine ​​lyase (EC 4.4.1.16-selenocysteine ​​lyase), L-cysteine ​​sulfo-lyase (EC 4.4.1.25-L-cysteate sulfo-lyase), L-cysteine ​​desulfidase (EC 4.4.1.28-L-cysteine ​​desulfidase) and L-cysteine ​​beta-lyase (EC 4.4.1.35-L-cystine beta-lyase). .

[0049] The enzymes of the present invention may be derived from any suitable organism, including prokaryotes and eukaryotes, such as, but not limited to, archaea, actinomycetes, bacteria, fungi, animals, plants, algae, and yeast. In some embodiments, the enzyme is derived from a bacterium selected from Thermus thermophiles, Lysinibacillus sphaericus, Clostridioides difficile, Clostridium novyi, Geobacillus stearothermophilus, Thermotoga maritima, Pseudomonas aeruginosa, Nonlabens dokdonensis, Leucaena leucocephala, Aeropyrum pernix, Methanosarcina acetivorans, Mycobacterium tuberculosis, Bacillus subtilis, Staphylococcus aureus, Pseudomonas fluorescens, Escherichia coli, coli), Candida maltosa, Streptomyces phaeochromogenes, Neurospora crassa, Spinacia oleracea, Cyanobacteria bacterium, Pseudomonas putida, Lactococcus lactis, Ruegeria pomeroyi, Methanococcus maripaludis, and Camelina sativa.

[0050] In some embodiments, the enzyme is selected from the group consisting of cysteine ​​synthase (EC 2.5.1.47) from Thermus thermophiles, cystathionine gamma-synthase (EC 2.5.1.48) from Lysinibacillus sphaericus, O-acetylhomoserine aminocarboxypropyltransferase (EC 2.5.1.49) from Clostridium difficile, O-acetylhomoserine aminocarboxypropyltransferase from Clostridium novyi, O-acetylhomoserine aminocarboxypropyltransferase from Geobacillus stearothermophilus, O-acetylhomoserine aminocarboxypropyltransferase from Thermocystis maritima, O-acetylhomoserine aminocarboxypropyltransferase from Pseudomonas aeruginosa, beta-pyrazoloylalanine synthase (EC 2.5.1.51) from Acinetobacter dokshimaensis. synthase), L-mimosine synthase from Leucaena leucaena (EC 2.5.1.52), O-phosphoserine sulfhydrylase from Aeropyrum pernix (EC 2.5.1.65), cysteine ​​synthase from Methanosarcina acetivorans (EC 2.5.1.76), [CysO sulfur-carrier protein]-thiocarboxylate-dependent cysteine ​​synthase from Mycobacterium tuberculosis (EC 2.5.1.113), cystathionine beta-synthase from Bacillus subtilis (EC 2.5.1.134), N-(2-amino-2-carboxyethyl)-L-glutamate synthase from Staphylococcus aureus (EC 2.5.1.136), and N-(2-amino-2-carboxyethyl)-L-glutamate synthase from Staphylococcus aureus (EC 2.5.1.140). 2.5.1.140-N-(2-amino-2-carboxyethyl)-L-glutamate synthase), S-sulfo-L-cysteine ​​synthase from Pseudomonas fluorescens (EC 2.5.1.144-S-sulfo-L-cysteine ​​synthase), diaminopropionate ammonia lyase from Escherichia coli (EC 4.3.1.15-Diaminopropionate ammonia-lyase), L-serine ammonia-lyase from Escherichia coli (EC 4.3.1.17-L-serine ammonia-lyase), D-serine ammonia-lyase from Escherichia coli (EC 4.3.1.18-D-serine ammonia-lyase), threonine ammonia-lyase from Candida maltosa (EC4.3.1.19-threonine ammonia-lyase), cystathionine gamma-lyase from Streptomyces fuscae (EC 4.4.1.1-cystathionine gamma-lyase), cystathionine gamma-lyase from Neurospora crassa (EC 4.4.1.1-cystathionine gamma-lyase), homocysteine ​​desulfhydrase from Bacillus subtilis (EC 4.4.1.2-homocysteine ​​desulfhydrase), L-3-cyanoalanine synthase from spinach (EC 4.4.1.9 - L-3-cyanoalanine synthase), cysteine ​​lyase from Cyanobacteria bacterium (EC 4.4.1.10 - cysteine ​​lyase), methionine gamma-lyase from Pseudomonas putida (EC 4.4.1.11 - methionine gamma-lyase), cysteine ​​S-conjugate beta-lyase from Lactococcus lactis (EC 4.4.1.13 - cysteine-S-conjugate beta-lyase), D-cysteine ​​desulfhydrase from Escherichia coli (EC 4.4.1.15 - D-cysteine ​​desulfhydrase), selenocysteine ​​lyase from Escherichia coli (EC 4.4.1.16 - selenocysteine ​​lyase), L-cysteine ​​sulfide lyase from Ruegeria pomeroyi (EC 4.4.1.25 - L-cysteine The enzymes include L-cysteine ​​desulfidase (EC 4.4.1.28-L-cysteine ​​desulfidase) from Methanococcus maripaludis and L-cystine beta-lyase (EC 4.4.1.35-L-cystine beta-lyase) from Camelina sativa. In some embodiments, the enzyme is O-acetylhomoserine aminocarboxylpropyltransferase from Clostridium novyi.

[0051] In some embodiments, the enzyme comprises an amino acid sequence selected from SEQ ID NOs: 1-31. In some embodiments, the enzyme is a modified variant.

[0052] As used herein, the term "variant" refers to a polypeptide or nucleic acid that has a certain sequence identity compared to the amino acid or nucleotide sequence of a given polypeptide or nucleic acid.

[0053] For the present invention, in order to determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for the purpose of optimal comparison (for example, a gap may be introduced in the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at this position. The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage of identity = number of identical positions / total number of positions (i.e., overlapping positions) × 100). Preferably, the two sequences are of the same length.

[0054] Those skilled in the art are aware that various computer programs can be used to determine the identity between two sequences.

[0055] "Percent amino acid identity" or "percent amino acid sequence identity" means that when the amino acids of two polypeptides are compared, the two polypeptides, when optimally aligned, have approximately the specified percentage of identical amino acids. For example, "95% amino acid identity" means that when the amino acids of two polypeptides are compared, the two polypeptides, when optimally aligned, have 95% of the same amino acids.

[0056] In some embodiments, the enzyme comprises an amino acid sequence having at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99%, and most preferably at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-31, and the enzyme has activity in catalyzing the reaction of formula I.

[0057] In some embodiments, the amino acid sequence of the enzyme differs from the amino acid sequence selected from SEQ ID NOs: 1-31 in that it has one or more amino acid substitutions, deletions, insertions, and / or additions, and the enzyme has activity in catalyzing the reaction of Formula I. In some embodiments, the amino acid sequence of the enzyme differs from the amino acid sequence selected from SEQ ID NOs: 1-31 in that it has one or more conservative amino acid substitutions, and the enzyme has activity in catalyzing the reaction of Formula I. In some embodiments, the amino acid sequence of the enzyme differs from the amino acid sequence selected from SEQ ID NOs: 1-31 in that it has one or more amino acid insertions or deletions, and the enzyme has activity in catalyzing the reaction of Formula I.

[0058] The term "conservative substitution", also known as substitution by a "homologous" amino acid residue, refers to substitutions in which the amino acid residue is replaced by an amino acid residue having a similar side chain, for example, amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), non-charged polar side chain amino acids (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chain amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chain amino acids (e.g., threonine, valine, isoleucine), and aromatic side chain amino acids (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0059] Conservative amino acid substitutions generally have minimal effects on the activity of the resulting protein. Such substitutions are described below. Conservative substitutions replace an amino acid with one that is similar in size, hydrophobicity, charge, polarity, steric properties, aromaticity, etc. These substitutions are often made when fine-tuning the properties of a protein is desired.

[0060] As used herein, "homologous" amino acid residues refer to amino acid residues having similar chemical properties, such as hydrophobicity, charge, polarity, steric characteristics, aromatic characteristics, etc. Examples of amino acids that are homologous to each other include positively charged lysine, arginine, and histidine, negatively charged glutamic acid and aspartic acid, hydrophobic glycine, alanine, valine, leucine, isoleucine, proline, and phenylalanine, polar serine, threonine, cysteine, methionine, tryptophan, tyrosine, asparagine, and glutamine, aromatic phenylalanine, tyrosine, and tryptophan, chemically similar side chain groups of serine and threonine, or glutamine and asparagine, or leucine and isoleucine.

[0061] Examples of conservative amino acid substitutions in proteins include: Ser for Ala, Lys for Arg, Gln or His for Asn, Glu for Asp, Ser for Cys, Asn for Gln, Asp for Glu, Pro for Gly, Asn or Gln for His, Leu or Val for Ile, Ile or Val for Leu, Arg or Gln for Lys, Leu or Ile for Met, Met, Leu or Tyr for Phe, Thr for Ser, Ser for Thr, Tyr for Trp, Trp or Phe for Tyr, and Ile or Leu for Val.

[0062] 3. Preparation conditions

[0063] The enzyme of the present invention may be provided in the form of an isolated polypeptide.

[0064] In some embodiments, the method is a cell-free method, i.e., the reaction medium in step a) comprises an isolated enzyme. The enzyme can be recombinantly produced or synthesized. In some embodiments, the reaction medium is an aqueous medium, preferably an aqueous buffer, such as a phosphate buffered saline (PBS).

[0065] In some embodiments, the reaction medium in step a) comprises a host cell expressing the enzyme. In some embodiments, the method of the present invention further comprises the step of culturing the host cell. In some embodiments, the reaction medium comprises a culture medium for culturing the host cell. In some embodiments, the reaction medium is an aqueous medium, preferably an aqueous buffer, for example a phosphate buffered saline (PBS), such as PBS.

[0066] In some embodiments, the host cell comprises an expression vector encoding the enzyme. In some embodiments, the host cell is Escherichia coli, for example, Escherichia coli BL21 (DE3). In some embodiments, the vector is a prokaryotic expression vector, such as pET-29b (+).

[0067] In some embodiments, in step b), the reaction medium is incubated at 30-40°C, 32-39°C, 34-38°C, 36-38°C, or 37°C.

[0068] In some embodiments, the pH of the reaction medium is from about 5 to about 8, preferably from about 6 to about 7.8, such as 7.5. Example

[0069] Through the following examples, those skilled in the art will more clearly understand the present invention. It should be understood that the examples are only for illustration, not for limiting the scope of the present invention.

[0070] Example 1. Materials and methods

[0071] Unless otherwise specified, the experimental methods used in the present invention are conventional methods. For details of gene cloning procedures, please refer to Sambrook et al., 1989.

[0072] i) Reagents:

[0073] -Isopropyl-β-D-thiogalactopyranoside (IPTG) was purchased from Biyuntian Biotechnology Co., Ltd.;

[0074] -L-homoserine was purchased from Jiangsu Aikang Biopharmaceutical Research and Development Co., Ltd.;

[0075] -L-methionine was purchased from Sigma-Aldrich;

[0076] -Sodium methanethiolate, pyridoxal phosphate (PLP), chromatographic grade disodium hydrogen phosphate, sodium tetraborate, methanol, and acetonitrile were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0077] -Sodium dihydrogen phosphate, sodium hydroxide, and Triton-X100 were purchased from Sangon Biotech (Shanghai) Co., Ltd.;

[0078] -OPA reagent was purchased from Agilent Technologies (China) Co., Ltd.

[0079] ii) Vectors and strains:

[0080] The expression vector used in the examples was pET-29b(+), purchased from Sangon Biotech (Shanghai) Co., Ltd.;

[0081] The host cells used in the examples were Escherichia coli BL21 (DE3), purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0082] iii) Gene cloning:

[0083] The nucleic acid sequences encoding the enzymes in Table 1 were submitted to Sangon Biotech (Shanghai) Co., Ltd. for synthesis to obtain pET29b(+) expression vectors containing the nucleic acid sequences encoding the enzymes.

[0084] Table 1

[0085] iv) Recombinant host cell preparation

[0086] Each expression vector was transformed into competent E. coli BL21(DE3) cells, plated on LB agar medium (containing 50 mg / L kanamycin), and incubated overnight at 37°C. A single colony was then selected and cultured in liquid LB medium (containing 50 mg / L kanamycin). The correctness of the synthesized sequence was verified by sequencing. Verified clones were stored at -80°C for subsequent experiments.

[0087] v) Protein expression and preparation of whole-cell catalysts:

[0088] The stored clones were activated on LB agar medium. A single colony was then inoculated into LB liquid medium (containing 50 mg / L kanamycin) and incubated at 37°C with shaking for 12 h. 400 μL of the culture was transferred to 20 mL of fresh LB liquid medium (containing 50 mg / L kanamycin) and incubated at 37°C with shaking until the OD600 reached approximately 0.6. IPTG (final concentration 0.4 mM) was added and incubated at 25°C for 16 h to induce protein expression.

[0089] After incubation, the culture was centrifuged at 4,000 g for 10 min at 4°C, and the supernatant was discarded to collect the E. coli cells. The collected E. coli cells were resuspended in 15 mL of pre-chilled 50 mM PBS, pH 7.0, to obtain an E. coli suspension containing the recombinant enzyme as a whole-cell catalyst.

[0090] vi) Enzyme preparation

[0091] Disrupt the E. coli cells by ultrasonication at 4°C in 7.5 mL of the E. coli suspension prepared in v). Centrifuge the cell suspension at 6,000 g at 4°C for 15 min to remove the precipitate. The resulting supernatant is a crude enzyme solution containing the recombinant enzyme. Purify the crude enzyme solution using the AKTA protein purification system to obtain purified enzyme.

[0092] vii) Purified enzyme-catalyzed reactions

[0093] To a 100 mM L-homoserine solution in PBS, sodium thiomethoxide and PLP were added, and the pH of the solution was adjusted to 7.5 with phosphoric acid. The final concentrations of L-homoserine, sodium thiomethoxide, and PLP were 169.7 mM, 419.74 mM, and 0.2 mM, respectively. The purified enzyme prepared in step vi) was added to the solution to a final concentration of 8 mg / mL. The mixture was shaken continuously (220 rpm) at 37°C for 12 hours. Samples were collected and analyzed by high-performance liquid chromatography with OPA pre-column derivatization to determine the catalytic activity of the pure enzyme.

[0094] viii) Whole-cell catalytic reactions

[0095] Sodium thiomethoxide, PLP, and Triton X-100 were added to a 100 mM L-homoserine solution in PBS, and the pH of the solution was adjusted to 7.5 with phosphoric acid. The final concentrations of L-homoserine, sodium thiomethoxide, PLP, and Triton X-100 were 169.7 mM, 419.74 mM, 0.2 mM, and 0.2% respectively. The cell suspension prepared in step v) was added to the above solution, and the final concentration of whole cells was 25 OD 600 The cells were shaken at 37°C for 12 hours on a shaker (220 rpm), and samples were taken and the amount of L-methionine produced was measured by OPA pre-column derivatization high performance liquid chromatography to determine the catalytic activity of the whole cells.

[0096] Example 2: Preparation and Detection of MetY from Clostridium knowlesi (CnMetY)

[0097] The clone expressing CnMetY (SEQ ID NO: 4) was activated, and the purified enzyme was prepared according to the method of Example 1, and the amount of L-methionine produced by the purified enzyme was measured. After detection, the pure CnMetY enzyme catalyzed the production of 12.13 g / L of L-methionine.

[0098] Example 3: Preparation and detection of whole cells containing sulfhydrylase (MetY) and cystathionine gamma lyase (CGL)

[0099] Escherichia coli clones expressing CnMetY (SEQ ID NO: 4), CdMetY (SEQ ID NO: 3), GsMetY (SEQ ID NO: 5), TmMetY (SEQ ID NO: 6), PaMetY (SEQ ID NO: 7), NaCGL (SEQ ID NO: 21), and SpCGL (SEQ ID NO: 20) were activated, and whole cells were prepared according to the method of Example 1. The amount of L-methionine catalytically produced by the whole cells was measured. The measured amount of L-methionine catalytically produced by the whole cells is shown in Table 2.

[0100] Table 2

[0101] Example 4 Preparation and detection of whole cells containing enzymes other than sulfhydrylase (MetY) and cystathionine gamma lyase (CGL) in Table 1

[0102] E. coli clones expressing the enzymes listed in Table 1, except for sulfhydrylase (MetY) and cystathionine gamma lyase (CGL), were activated and whole cells were prepared according to the method of Example 1. The amount of L-methionine produced by the whole cells was measured, and the catalytic activity of the enzymes was evaluated based on the amount of L-methionine produced. The catalytic activity of the enzymes is shown in Table 3, where the catalytic activity of the enzyme is represented by a ★, with more ★s indicating stronger catalytic activity.

[0103] Table 3

[0104] sequence

Claims

1. A method for preparing L-methionine, comprising the following steps: a) providing a reaction medium, which comprises L-homoserine or a salt, ester or amide thereof, a methylthio group donor and an enzyme selected from a transferase that transfers an alkyl group other than a methyl group or an aromatic group, an ammonia lyase and a lyase that catalyzes the cleavage of a carbon-sulfur bond; b) incubating the reaction medium to produce a reaction product comprising L-methionine or a salt, ester or amide thereof; and optionally c) recovering L-methionine or its salt, ester or amide from the reaction product.

2. The method of claim 1, wherein the enzyme is selected from the group consisting of enzymes with EC numbers EC 2.5.1.-, EC 4.3.1.- and EC 4.4.1.-.

3. The process of claim 1 or 2, wherein the reaction medium further comprises pyridoxal phosphate or a salt thereof.

4. The method of any one of claims 1 to 3, wherein the enzyme is selected from the group consisting of EC numbers EC 2.5.1.47, EC 2.5.1.48, EC 2.5.1.49, EC 2.5.1.51, EC 2.5.1.52, EC 2.5.1.65, EC 2.5.1.76, EC 2.5.1.113, EC 2.5.1.134, EC 2.5.1.140, EC 2.5.1.144, EC 4.3.1.15, EC 4.3.1.17, EC 4.3.1.18, EC 4.3.1.19, EC 4.4.1.1, EC 4.4.1.2, EC 4.4.1.9, EC 4.4.1.10, EC 4.4.1.11, EC Enzymes of EC 4.4.1.13, EC 4.4.1.15, EC 4.4.1.16, EC 4.4.1.25, EC 4.4.1.28 and EC 4.4.1.

35.

5. The method of any one of claims 1 to 4, wherein the enzyme is derived from a member selected from the group consisting of Thermus thermophiles, Lysinibacillus sphaericus, Clostridioides difficile, Clostridium novyi, Geobacillus stearothermophilus, Thermotoga maritima, Pseudomonas aeruginosa, Nonlabens dokdonensis, Leucaena leucocephala, Aeropyrum pernix, Methanosarcina acetivorans, Mycobacterium tuberculosis, Bacillus subtilis, Staphylococcus aureus, Pseudomonas fluorescens, fluorescens, Escherichia coli, Candida maltosa, Streptomyces phaeochromogenes, Neurospora crassa, Spinacia oleracea, Cyanobacteria bacterium, Pseudomonas putida, Lactococcus lactis, Ruegeria pomeroyi, Methanococcus maripaludis, and Camelina sativa.

6. The method of any one of claims 1 to 5, wherein the enzyme is selected from the group consisting of cysteine ​​synthase derived from Thermus thermophiles, cystathionine gamma synthase derived from Lysinibacillus sphaericus, O-acetylhomoserine aminocarboxypropyltransferase derived from Clostridium difficile, O-acetylhomoserine aminocarboxypropyltransferase derived from Clostridium novyi, O-acetylhomoserine aminocarboxypropyltransferase derived from Geobacillus stearothermophilus, O-acetylhomoserine aminocarboxypropyltransferase derived from Thermocystis maritima, O-acetylhomoserine aminocarboxypropyltransferase derived from Pseudomonas aeruginosa, β-pyrazoloylalanine synthase derived from Acinetobacter dokdoshima, L-mimosine synthase derived from Acacia leucaena, O-phosphoserine sulfhydrylase derived from Aeropyrum pernix, O-phosphoserine sulfhydrylase derived from Methanosarcina acetivorans, [CysO sulfur carrier protein]-thioester-dependent cysteine ​​synthase from Mycobacterium tuberculosis, cystathionine β-synthase from Bacillus subtilis, N-(2-amino-2-carboxyethyl)-L-glutamate synthase from Staphylococcus aureus, S-sulfur-L-cysteine ​​synthase from Pseudomonas fluorescens, diaminopropionate ammonia lyase from Escherichia coli, L-serine ammonia lyase from Escherichia coli, D-serine ammonia lyase from Escherichia coli, threonine ammonia lyase from Candida maltosa, cystathionine γ-lyase from Streptomyces fuscatae, cystathionine γ-lyase from Neurospora crassa, homocysteine ​​desulfhydrase from Bacillus subtilis, L-3-cyanoalanine synthase from Spinacia serrata, bacterium, methionine gamma-lyase from Pseudomonas putida, cysteine ​​S-conjugate beta-lyase from Lactococcus lactis, D-cysteine ​​desulfhydrylase from Escherichia coli, selenocysteine ​​lyase from Escherichia coli, L-cysteine ​​sulfhydrylase from Ruegeria pomeroyi, L-cysteine ​​desulfidase from Methanococcus maripaludis and L-cystine beta-lyase from Camelina sativa.

7. The method of any one of claims 1-6, wherein the enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-31.

8. The method of any one of claims 1 to 7, wherein the method is a cell-free method.

9. The method of any one of claims 1 to 7, wherein the reaction medium in step a) comprises a host cell expressing the enzyme.

10. The method of claim 9, further comprising the step of culturing the host cell.

11. The method of any one of claims 1 to 10, wherein in step b), the reaction medium is incubated at 30-40°C, 32-39°C, 34-38°C, 36-38°C or 37°C.

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