Process for preparing L-glufosinate directly from L-homoserine

Abstract

A process for preparing L-glufosinate by the enzymatically catalyzed reaction of homoserine and methylphosphinic acid is provided. The process according to the present disclosure does not include a step of producing O-acyl-L-homoserine, and O-acyl-L-homoserine is not used in the process.

Description

[Technical Field]

[0001] The present disclosure is in the field of biochemical engineering and, more particularly, relates to a process for the direct enzymatic preparation of L-glufosinate using L-homoserine as a substrate. [Background technology]

[0002] Glufosinate (also known as 4-[hydroxyl(methyl)phosphono]-D,L-homoalanine) is the second-best-selling herbicide in the world, and genetically modified crops are tolerant to it. Glufosinate is a broad-spectrum contact herbicide that inhibits the activity of L-glutamine synthase in plants, causing disorders in nitrogen metabolism and ultimately killing the plants. Compared with glyphosate, glufosinate has significant advantages, such as a wide range of application, rapid onset of effect, long-lasting effect, low toxicity, safety, and the like. Therefore, sales of glufosinate are growing rapidly, and there is a prospect of a huge market and demand in the future.

[0003] However, the complex preparation process of glufosinate poses high technical difficulties in its production. Its high price prevents glufosinate from quickly replacing glyphosate. In addition, chemically synthesized glufosinate is a racemic mixture containing equal amounts of two optical isomers (D,L-glufosinate), of which only the L-configuration is physiologically active.

[0004] Recently, there have been many reports on the preparation of L-glufosinate from D,L-glufosinate, for example, D-glufosinate is oxidized to 2-carbonyl-4-(hydroxylmethylphosphono)butyric acid (PPO), which is then reduced or transaminated to give L-glufosinate.

[0005] However, the process of chemically synthesizing D / L-glufosinate and then converting D-glufosinate to L-glufosinate via enzymatic methods is complicated and expensive. Therefore, there remains a need for a more efficient process for synthesizing L-glufosinate.

[0006] WO 2022 / 207543 reports a process for synthesizing L-glufosinate or its phosphonates using activated homoserine (O-acetylhomoserine or O-succinylhomoserine) and methylphosphinic acid or its ester as substrates under enzymatic catalysis (hydrogen sulfide lyase or cystathionine gamma synthase).

[0007] However, this process requires the initial production of O-acylhomoserine (e.g., via fermentation), and such production is limited by: i) accumulation of O-acylhomoserine; ii) instability of O-acylhomoserine (toward acid, base, and heat); iii) inhibition of enzyme activity by by-product organic acids; and iv) increased production costs, process complexity, and difficulty in product separation when pH is adjusted during the reaction process.

[0008] Therefore, there is a need to develop a novel process for preparing L-glufosinate that uses readily available, stable substrates, reduces reaction inhibition of the product, simplifies the process (including purification and separation), and reduces costs. Summary of the Invention

[0009] Surprisingly, the present inventors have found that L-homoserine can react with methylphosphinic acid or its ester under enzyme catalysis to produce L-glufosinate or its phosphonate and water (as shown in Formula I).

[0010] [ka]

[0011] L-homoserine is a more readily available and stable compound than O-acylhomoserine, and the disclosed reaction does not produce organic acid by-products that inhibit enzyme activity.

[0012] Thus, there is provided a process for preparing L-glufosinate, comprising: a) providing a reaction medium comprising L-homoserine, or a salt, ester, amide, or anhydride thereof, methylphosphinic acid or an ester thereof, and an enzyme selected from the group consisting of a transferase that transfers alkyl or aryl groups other than methyl, 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-glufosinate or a phosphonate thereof; and Optionally, c) recovering L-glufosinate or its phosphonate from the reaction medium. A process is provided, including:

[0013] In some embodiments, the enzyme is selected from the group consisting of enzymes having the following EC numbers: EC 2.5.1.-, EC 4.3.1.-, and EC 4.4.1.-.

[0014] In some embodiments, the reaction medium further comprises phosphopyridoxal or a salt thereof.

[0015] In some embodiments, the enzyme is 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 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 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.

[0016] In some embodiments, the enzyme is 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, and the like. pernix, Methanosarcina acetivorans, Mycobacterium tuberculosis, Bacillus subtilis, Staphylococcus aureus, Pseudomonas fluorescens, Escherichia coli, Candida maltosa, Streptomyces phaeochromogenes, Neurospora crassa, Spinacia oleracea, Cyanobacteria bacterium, Pseudomonas putida, Lactococcus lactis lactis, Ruegeria pomeroyi, Methanococcus maripaludis, and Camelina sativa.

[0017] In some embodiments, the enzyme is selected from the group consisting of cysteine ​​synthase from Thermus thermophilus (EC 2.5.1.47 - cysteine ​​synthase), cystathionine gamma synthase from Lysinibacillus sphaericus (EC 2.5.1.48 - cystathionine gamma synthase), O-acetylhomoserine aminocarboxypropyltransferase from Clostridioides difficile (EC 2.5.1.49 - O-acetylhomoserine aminocarboxypropyltransferase from Clostridium novyi, O-acetylhomoserine aminocarboxypropyltransferase from Geobacillus stearothermophilus, O-acetylhomoserine aminocarboxypropyltransferase from Thermotoga maritima, O-acetylhomoserine aminocarboxypropyltransferase from Pseudomonas aeruginosa, β-pyrazolylalanine synthase (EC2.5.1.51- beta-pyrazolylalanine synthase) from Nonlavens dokudnensis, L-mimosine synthase (EC2.5.1.52- L-mimosine synthase) from Leucaena leucineum, O-phosphoserine sulfhydrylase (EC2.5.1.65- O-phosphoserine sulfhydrylase), cysteine ​​synthase from Methanosarcina acetivorans (EC 2.5.1.76 - cysteate synthase), [CysO sulfur carrier protein]-thiocarboxylate-dependent cysteine ​​synthase from Mycobacterium tuberculosis (EC 2.5.1.113 - [CysO sulfur carrier protein]-thiocarboxylate-dependent cysteine ​​synthase), cystathionine β-synthase from Bacillus subtilis (EC 2.5.1.134 - cystathionine β-synthase), N-(2-amino-2-carboxyethyl)-L-glutamate synthase from Staphylococcus aureus (EC 2.5.1.140 - N-(2-amino-2-carboxyethyl)-L-glutamate synthase), and 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 (EC 4.3.1.19-threonine ammonia-lyase), cystathionine gamma-lyase from Streptomyces phaeochromogenes (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 (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-cysteate sulfolyase from Rueggeria pomeroi (EC 4.4.1.25- 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).

[0018] In some embodiments, the enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-31.

[0019] In some embodiments, the process is a cell-free process.

[0020] In some embodiments, the reaction medium in step a) comprises a host cell that expresses an enzyme. In some embodiments, the process further comprises culturing the host cell.

[0021] 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 explanation of the drawings]

[0022] [Figure 1] LC-MS results of the reaction products catalyzed by purified GsMetY: A: Detection result of the reaction product sample, B: Detection result after adding an internal standard. [Figure 2] LC-MS results of the reaction products catalyzed by E. coli expressing GsMetY: A: detection result of the sample, B: detection result after adding an internal standard. [Figure 3] 1 shows the results of LC-MS of the reaction products catalyzed by E. coli expressing SpCGL, A: detection result of the sample, B: detection result after adding an internal standard. DETAILED DESCRIPTION OF THE INVENTION

[0023] A novel process for preparing L-glufosinate is provided. Unless otherwise specified, the terms used herein have the common understanding and interpretation of those skilled in the art.

[0024] It has been reported in the prior art that 4-hydroxyl activated L-homoserine (such as O-acylhomoserine) can react with methylphosphinic acid or its ester under enzyme catalysis to give L-glufosinate or its phosphonate ester.

[0025] Surprisingly, the present inventors have found that L-homoserine can react with methylphosphinic acid or its ester under enzyme catalysis to produce L-glufosinate or its phosphonate and water (as shown in Formula I).

[0026] [ka]

[0027] L-homoserine is a more readily available and stable compound than O-acylhomoserine, and the reaction of formula I does not produce organic acid by-products that inhibit enzyme activity.

[0028] therefore, a) providing a reaction medium comprising an L-homoserine donor, methylphosphinic acid or an ester thereof, and an enzyme selected from the group consisting of a transferase that transfers alkyl or aryl groups other than methyl, 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-glufosinate or a phosphonate thereof; and Optionally, c) recovering L-glufosinate or its phosphonate from the reaction medium A process for preparing L-glufosinate is provided, comprising:

[0029] I. Substrate As used herein, "L-homoserine" refers to L-2-amino-4-hydroxyl-1-butanoic acid, which has the molecular formula CHNO. As used herein, L-homoserine donors include, but are not limited to, L-homoserine, or a salt, ester, or amide thereof, and an anhydride formed from L-homoserine and L-homoserine or another acid. L-homoserine donors also include derivatives in which one or two hydrogens are substituted at the amino group. An ester of L-homoserine refers to a compound formed by reacting the carboxyl group of L-homoserine with an alcohol or phenol. An amide of L-homoserine refers to a compound formed by reacting the carboxyl group of L-homoserine with an amine compound.

[0030] As used herein, L-homoserine donor does not include derivatives in which the 4-hydroxyl hydrogen atom is replaced by acyl, i.e., O-acylhomoserine.

[0031] In step b), L-homoserine is a direct substrate for the enzyme. The process according to the present disclosure does not include a step of generating O-acyl-L-homoserine, and O-acyl-L-homoserine is not used in the process.

[0032] Methylphosphinic acid or its ester is as shown in Formula II.

[0033] [ka]

[0034] wherein R is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, hydroxyalkyl, and aryl.

[0035] In some embodiments, the reaction medium comprises methyl phosphinate. In some embodiments, the process further comprises hydrolyzing the L-glufosinate phosphonate.

[0036] II. Enzymes As noted above, the reactions of the process of the present invention are carried out under enzyme catalysis, i.e., as enzymatic reactions.

[0037] As used herein, the term "enzyme" refers to a protein or peptide that can specifically catalyze or promote a chemical or biochemical reaction. An enzyme according to the present disclosure can be an intact, naturally occurring protein or polypeptide or a portion thereof that has catalytic activity. An enzyme according to the present disclosure can also be an engineered variant of a naturally occurring protein or polypeptide. An enzyme according to the present disclosure can be isolated from an organism or can be produced recombinantly or synthetically. One skilled in the art can obtain enzymes through conventional methods.

[0038] As used herein, the term "peptide" refers to a chain of at least two amino acids joined by a peptide bond. The term "polypeptide," which may be used interchangeably with the term "protein," refers to a chain of 10 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.

[0039] The term "amino acid" includes naturally occurring amino acids in proteins and unnatural amino acids. The one-letter and three-letter names of naturally occurring amino acids in proteins are those commonly used in the art, see Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Amino Acid One-Letter Code Three-Letter Code Alanine A Ala Arginine R Arg Asparagine N Asn Aspartic Acid D Asp Cysteine ​​C Cys Glutamine Q Gln Glutamic Acid E Glu Glycine G Gly Histidine H His Isoleucine I Ile Leucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe Proline P Pro Serine S Threonine T Thr Tryptophan W Trp Tyrosine Y Tyr Valin V Val

[0040] As used herein, the term "modification" refers to any modification of a polypeptide according to the present disclosure or a peptide composed of its homologous sequence, including, but not limited to, substitution, deletion, insertion, and / or addition of one or more amino acids.

[0041] Some enzymes require the participation of a coenzyme for catalysis. The term "coenzyme" is a general term for a type of organic cofactor that is an essential factor for enzyme-catalyzed oxidation-reduction, group transfer, and isomerization reactions. Coenzymes are involved in the function of transferring electrons, atoms, or groups in enzyme-catalyzed reactions.

[0042] The inventors have found that the enzymes used in the present disclosure use only phosphopyridoxal as a coenzyme, and therefore, in some embodiments, the reaction medium further comprises phosphopyridoxal or a salt thereof.

[0043] In some embodiments, the enzyme is selected from the group consisting of enzymes having the following EC numbers: EC 2.5.1.-, EC 4.3.1.-, and EC 4.4.1.-.

[0044] In some embodiments, the enzyme is 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 4.3.1.20, EC 4.3.1.21, EC 4.3.1.22, EC 4.3.1.23, EC 4.3.1.24, EC 4.3.1.25, EC 4.3.1.26, EC 4.3.1.27, EC 4.3.1.28, EC 4.3.1.29, EC 4.3.1.30, EC 4.3.1.31, EC 4.3.1.32, EC 4.3.1.33, EC 4.3.1.34, EC 4.3.1.35, EC 4.3.1.36, EC 4.3.1.37, EC 4.3.1.38, EC 4.3.1.39, EC 4.3.1.40, EC 4.3.1.41, EC 4.3.1.42, EC 4.3.1.43, EC 4.3.1.44, EC 4.3.1.45, EC 4.3.1.46, EC 4.3.1 EC 4.4.1.18, 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 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.

[0045] In some embodiments, the enzyme is 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 (EC2.5.1.76 - cysteate synthase), [CysO sulfur carrier protein]-thiocarboxylate-dependent cysteine ​​synthase (EC2.5.1.113 - [CysO sulfur carrier protein]-thiocarboxylate-dependent cysteine ​​synthase), cystathionine beta synthase (EC2.5.1.134 - cystathionine beta synthase), N-(2-amino-2-carboxyethyl)-L-glutamate synthase (EC2.5.1.140 - N-(2-amino-2-carboxyethyl)-L-glutamate synthase), S-sulfo-L-cysteine ​​synthase (EC2.5.1.144 - 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 γ-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 ​​sulfolyase (EC 4.4.1.25-L-cysteine ​​sulfolyase), L-cysteine ​​desulfidase (EC 4.4.1.28-L-cysteine ​​desulfidase), and L-cystine beta lyase (EC 4.4.1.35-L-cystine beta lyase).

[0046] Enzymes according to the present disclosure may be derived from any suitable organism, including prokaryotes and eukaryotes, for example, but not limited to, archaea, actinomycetes, bacteria, fungi, animals, plants, algae, and yeasts. In some embodiments, the enzyme is derived from an organism selected from the group consisting of Thermus thermophilus, Lysinibacillus sphaericus, Clostridioides difficile, Clostridium novyi, Geobacillus stearothermophilus, Thermotoga maritima, Pseudomonas aeruginosa, Nonravens docdonensis, Leucaena leucine, Aeropyrum pernix, Methanosarcina acetivorans, Mycobacterium tuberculosis, Bacillus subtilis, Staphylococcus aureus, Pseudomonas fluorescens, Escherichia coli, Candida maltosa, Streptomyces phaeochromogenes, Neurospora crassa, Spinach, Cyanobacterium bacterium, Pseudomonas putida, Lactococcus lactis, Rueggeria pomeroi, Methanococcus maripaludis, and Camelina sativa.

[0047] In some embodiments, the enzyme is selected from the group consisting of cysteine ​​synthase from Thermus thermophilus (EC 2.5.1.47 - cysteine ​​synthase), cystathionine gamma synthase from Lysinibacillus sphaericus (EC 2.5.1.48 - cystathionine gamma synthase), O-acetylhomoserine aminocarboxypropyltransferase from Clostridioides difficile (EC 2.5.1.49 - O-acetylhomoserine aminocarboxypropyltransferase from Clostridium novyi, O-acetylhomoserine aminocarboxypropyltransferase from Geobacillus stearothermophilus, O-acetylhomoserine aminocarboxypropyltransferase from Thermotoga maritima, O-acetylhomoserine aminocarboxypropyltransferase from Pseudomonas aeruginosa, β-pyrazolylalanine synthase (EC2.5.1.51- beta-pyrazolylalanine synthase) from Nonlavens dokudnensis, L-mimosine synthase (EC2.5.1.52- L-mimosine synthase) from Leucaena leucineum, O-phosphoserine sulfhydrylase (EC2.5.1.65- O-phosphoserine sulfhydrylase), cysteine ​​synthase from Methanosarcina acetivorans (EC 2.5.1.76 - cysteate synthase), [CysO sulfur carrier protein]-thiocarboxylate-dependent cysteine ​​synthase from Mycobacterium tuberculosis (EC 2.5.1.113 - [CysO sulfur carrier protein]-thiocarboxylate-dependent cysteine ​​synthase), cystathionine β-synthase from Bacillus subtilis (EC 2.5.1.134 - cystathionine β-synthase), N-(2-amino-2-carboxyethyl)-L-glutamate synthase from Staphylococcus aureus (EC 2.5.1.140 - N-(2-amino-2-carboxyethyl)-L-glutamate synthase), and 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 (EC 4.3.1.19-threonine ammonia-lyase), cystathionine gamma-lyase from Streptomyces phaeochromogenes (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 (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-cysteate sulfolyase from Rueggeria pomeroi (EC 4.4.1.25- 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). In some embodiments, the enzyme is O-acetylhomoserine aminocarboxypropyltransferase from Clostridium novyi.

[0048] In some embodiments, the enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 31. In some embodiments, the enzyme is an engineered variant.

[0049] As used herein, the term "variant" refers to a peptide or nucleic acid that has a certain sequence identity compared to the amino acid or nucleotide sequence of a given peptide or nucleic acid.

[0050] In the present disclosure, to determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps can be introduced into a first amino acid sequence or nucleic acid sequence for optimal alignment with a second amino acid sequence or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. If the same amino acid residue or nucleotide is present at that position in the first sequence as at the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage identity = number of identical positions / total number of positions (i.e., overlapping positions) × 100). Preferably, the two sequences are of the same length.

[0051] Those skilled in the art know that various computer programs can be used to determine identity between two sequences.

[0052] "Percentage amino acid identity" or "percentage amino acid sequence identity" refers to a comparison of the amino acids of two polypeptides, where, when optimally aligned, the two polypeptides have approximately the specified percentage of identical amino acids. For example, "95% amino acid identity" means that when two polypeptides are compared for amino acids in optimal alignment, the two polypeptides have 95% identical amino acids.

[0053] 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 the group consisting of SEQ ID NOs: 1-31, and the enzyme has activity to catalyze the reaction of Formula I.

[0054] In some embodiments, the amino acid sequence of the enzyme differs from an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-31 by having one or more amino acid substitutions, deletions, insertions, and / or additions, and the enzyme has activity to catalyze the reaction of Formula I. In some embodiments, the amino acid sequence of the enzyme differs from an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-31 by having one or more amino acid conservative substitutions, and the enzyme has activity to catalyze the reaction of Formula I. In some embodiments, the amino acid sequence of the enzyme differs from an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-31 by having one or more amino acid insertions or deletions, and the enzyme has activity to catalyze the reaction of Formula I.

[0055] The term "conservative substitution," also known as substitution with a "homologous" amino acid residue, refers to substitutions in which one amino acid residue is replaced with another amino acid residue having a similar side chain, e.g., amino acids with basic side chains (such as lysine, arginine, and histidine), acidic side chains (such as aspartic acid and glutamic acid), uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine), nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan), β-branched side chains (such as threonine, valine and isoleucine), and aromatic side chains (such as tyrosine, phenylalanine, tryptophan and histidine).

[0056] Conservative amino acid substitutions typically have minimal effect on the activity of the resulting protein. Such substitutions are described below. A conservative substitution is the replacement of one amino acid with another amino acid that has similar size, hydrophobicity, charge, polarity, spatial characteristics, aromaticity, or the like. When it is desired to fine-tune the properties of a protein, substitutions are typically conservative.

[0057] As used herein, "homologous" amino acid residues refer to amino acid residues that have similar chemical properties, such as hydrophobicity, charge, polarity, spatial characteristics, aromaticity, or the like. 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; and serine and threonine, or glutamine and asparagine, or leucine and isoleucine, that have chemically similar side chain groups.

[0058] 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.

[0059] III. Preparation conditions Enzymes according to the present disclosure may be provided in the form of isolated polypeptides.

[0060] In some embodiments, the process is a cell-free process, i.e., the reaction medium in step a) comprises an isolated enzyme. The enzyme can be recombinantly or synthetically produced. In some embodiments, the reaction medium is an aqueous medium, preferably an aqueous buffer, such as a phosphate buffer, e.g., PBS.

[0061] In some embodiments, the reaction medium in step a) comprises a host cell expressing the enzyme. In some embodiments, the process according to the present disclosure further comprises a 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 solution, such as a phosphate buffer solution, e.g., PBS.

[0062] In some embodiments, the host cell comprises an expression vector encoding the enzyme. In some embodiments, the host cell is E. coli, such as E. coli BL21(DE3). In some embodiments, the expression vector is a prokaryotic expression vector, such as pET-29b(+).

[0063] 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.

[0064] In some embodiments, the reaction medium has a pH of about 5 to about 8, preferably about 6 to about 7.8, for example, 7.5. [Example]

[0065] Those skilled in the art will understand the present disclosure more clearly through the following examples. It should be noted that the examples are provided for illustrative purposes only, rather than to limit the scope of the present disclosure.

[0066] Example 1 Materials and Methods Unless otherwise stated, the laboratory methods used herein are conventional and specific gene cloning procedures can be found in Sambrook et al., 1989, supra.

[0067] i) Reagents: Isopropyl-β-D-thiogalactoside (IPTG) was purchased from Beyotime Biotechnology Co., Ltd., L-homoserine was purchased from Jiangsu Aikon Biomedical Research & Development Co., Ltd., butyl methylphosphinate was synthesized in-house according to the process reported in China Patent No. 106674275, and L-glufosinate, phosphopyridoxal (PLP), and acetonitrile were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and Triton X100 was purchased from Sangon Biotechnology (Shanghai) Co., Ltd.

[0068] ii) Vectors and strains: The expression vector used in the examples was pET-29b(+), which was purchased from Sangon Biotechnology (Shanghai) Co., Ltd.

[0069] The host cells used in the examples were Escherichia coli BL21 (DE3), purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0070] iii) Gene cloning The nucleic acid sequences encoding each enzyme in Table 1 were provided to Sangon Biotechnology (Shanghai) Co., Ltd. for synthesis, and pET29b(+) expression vectors carrying the nucleic acid sequences encoding each enzyme were obtained.

[0071] [Table 1-1] [Table 1-2]

[0072] iv) Preparation of recombinant host cells The expression vectors were transformed into E. coli BL21 (DE3) competent cells, which were then spread onto LB agar plates (containing 50 mg / L kanamycin) and incubated overnight at 37°C. Single colonies were then selected, cultured in LB broth (containing 50 mg / L kanamycin), and sequenced to confirm the accuracy of the synthesized sequences. Correct clones were stored at -80°C for further experiments.

[0073] v) Protein expression and whole cell catalyst preparation: The stored clones were activated on LB agar medium. A single colony was then inoculated into LB broth (containing 50 mg / L kanamycin) and incubated at 37°C with shaking for 12 hours. 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 incubation was continued at 25°C for 16 hours to induce protein expression.

[0074] After incubation, the culture was centrifuged at 4000 g for 10 minutes at 4°C, the supernatant was discarded, and the E. coli cells were harvested. The harvested 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.

[0075] vi) Enzyme Preparation 7.5 mL of the E. coli suspension prepared in v) was collected, and the E. coli cells were sonicated at 4°C. The cell disruption solution was centrifuged at 6000 g for 15 minutes at 4°C to remove the precipitate, and the resulting supernatant was obtained as a crude enzyme solution containing the recombinant enzyme. The crude enzyme solution was purified using the AKTA protein purification system to obtain the purified enzyme.

[0076] vii) Catalysis with purified enzymes Butyl methylphosphinate and PLP were added to an L-homoserine solution in Tris (20 mM), and the solution was adjusted to pH 7.4 with hydrochloric acid. The final concentrations of L-homoserine, butyl methylphosphinate, and PLP in the solution were 84 mM, 168 mM, and 0.3 mM, respectively. The purified enzyme prepared in (vi) was added to the above solution to a final concentration of 0.5 mg / mL. The system was shaken (220 rpm) in an oscillator at 37°C for 12 hours. A sample was taken and hydrolyzed with 6 N HCl in a metal bath at 100°C for 2 hours. After hydrolysis, the pH was adjusted to 7.0. LC-MS was used to detect the hydrolyzed sample, and 1 mM L-glufosinate was added as an internal standard. The peak with a molecular weight of 182 was extracted in the positive mode to determine the production of L-glufosinate.

[0077] viii) Whole-cell catalytic reactions Butylmethylphosphinate, PLP, and Triton X-100 were added sequentially to an L-homoserine solution in Tris (20 mM), and the solution was adjusted to pH 7.4 with hydrochloric acid. In the solution, the final concentrations of L-homoserine, butylmethylphosphinate, PLP, and Triton X-100 were 84 mM, 168 mM, 0.3 mM, and 0.2%. The whole cells prepared according to the procedure in (v) were added to the above solutions, respectively, to a final total cell concentration of 30 OD. 600 The system was shaken at 37°C for 12 hours using an oscillator (220 rpm). Samples were taken and hydrolyzed with 6N HCl in a metal bath at 100°C for 2 hours, and the pH was adjusted to 7.0 after hydrolysis. LC-MS was used to detect the hydrolyzed samples, and 1 mM L-glufosinate was added as an internal standard. The peak with a molecular weight of 182 was extracted in the positive mode to determine the production of L-glufosinate.

[0078] Example 2. Preparation and detection of MetY (GsMetY) from Bacillus stearothermophilus According to the procedures of Example 1, a clone expressing GsMetY (SEQ ID NO: 5) was activated, a purified enzyme was prepared, and L-glufosinate produced by the catalytic action of the purified enzyme was detected.

[0079] According to LC-MS detection, a peak with a molecular weight of 182 was extracted from the reaction product sample catalyzed by GsMetY (see Figure 1A). A single peak with a molecular weight of 182 was extracted from the sample to which 1 mM L-glufosinate was added as an internal standard, and the peak height was increased (see Figure 1B). This indicates that the reaction product sample contained L-glufosinate, i.e., L-glufosinate was produced by the catalytic action of the purified GsMetY enzyme.

[0080] Example 3. Preparation and detection of whole cells expressing GsMetY Following the procedures of Example 1, clones expressing GsMetY (SEQ ID NO: 5) were activated, whole cells were prepared, and L-glufosinate produced by whole cell catalysis was determined.

[0081] LC-MS detection revealed that a peak with a molecular weight of 182 was extracted from the reaction product sample catalyzed by whole cells expressing GsMetY (see Figure 2A). In the sample spiked with 1 mM L-glufosinate as an internal standard, no new peak was detected, and the height and area of ​​the peak near the retention time of 1.8 minutes increased (see Figure 2B), indicating that the reaction product sample contained L-glufosinate, i.e., L-glufosinate was produced by the catalytic action of whole cells expressing GsMetY.

[0082] Example 4. Preparation and detection of whole cells expressing CGL (SpCGL) from Streptomyces phaeochromogenes A clone expressing SpCGL (SEQ ID NO: 20) was activated, whole cells were prepared according to the procedure of Example 1, and L-glufosinate produced by whole cell catalysis was determined.

[0083] According to LC-MS detection, a peak with a molecular weight of 182 was extracted from the reaction product sample catalyzed by whole cells expressing SpCGL (see FIG. 3A). In the sample to which 1 mM L-glufosinate was added as an internal standard, no new peak was detected, and the height and area of ​​the peak near the retention time of 1.8 minutes increased (see FIG. 3B), indicating that the reaction product sample contained L-glufosinate, i.e., L-glufosinate was produced by the catalytic action of whole cells expressing SpCGL.

[0084] Example 5. Production of L-glufosinate in E. coli cells expressing exogenous enzymes This example was performed to determine whether E. coli cells expressing exogenous enzymes could catalyze the conversion of L-homoserine to L-glufosinate.

[0085] Recombinant E. coli cells expressing each of the enzymes in Table 1 were prepared as described in Example 1 and tested for whole-cell catalyzed production of L-glufosinate.

[0086] As shown in Table 2, recombinant E. coli cells expressing any of the enzymes of SEQ ID NOs: 1 to 31 can catalyze the conversion of L-homoserine to L-glufosinate, where catalytic ability is represented by a black star, with more black stars indicating stronger catalytic ability.

[0087] [Table 2-1] [Table 2-2]

[0088] array SEQ ID NO: 1 MRVEGAIGKTPVVRLAKVVEPDMAEVWVKLEGLNPGGSIKDRPAWYMIKDAEERGILRPGSGQVIVEPTSGNTGIGLAMIAASRGYRLILTMPAQMSEERKRVLKAFGAELVLTDPERRMLAAREEALRLKEELGAFMPDQFKNPANVRAHY ETTGPELYEALEGRIDAFVYGSGTGGTITGVGRYLKERIPHVKVIAVEPARSNVLSGGKMGQHGFQGMGPGFIPENLDLSLLDGVIQVWEEDAFPLARRLAREEGLFLGMSSGGIVWAALQVARELGPGKRVACISPDGGWKYLSTPLYAEP SEQ ID NO: 2 MTLNRSIETKLVQLGNLSDPTTGAVSPPIHLSTAYKHAGIGESTGFDYTRTKNPTRALLEAGFADLEGGDMGFACSSGMAAIQLVLSIFKPGDELVVPDDLYGGTYRLFNFFQETYNIKPVYSKFESVEQVEALINEHTRALFIETPTNPLMQEFDLQVYAELAHKHGALLIVDNTFYTPYFQRP IDLGADIVLHSATKYIGGHNDVLAGIVVAKGTELAERIGFIHNGSGMVLGAMDSWLLIRGLKTMHLRLKQHDANAKAIAAYLEEEALVTDVLYTGKGGMLSFRLQKPEWIDPFLRNLKLITFAESLGGVESFITYPATQTHADMPYEERVERGVCDRLLRFSVGIEEAEDLIADLRQVFDILRKEA SEQ ID NO: 3 MDNKETICVQGNYKPGNGEPRVLPLYQSTTFKYSSIDQLAELFDLKVDGHIYSRISNPTIQAFEEKISLLEGGVSSVAVSSGQSANMLAVLNICKSGDSILCSSKVYGGTFNLLGPSLKKFGIDLISFDLDSSEDEIVELAKENTKVVFAETLANPTLEVIDFEKIANVAKRINVPFIVDNSLASPVLCNPLKYGANIVTHSTTKYLDGH ASSVGGIIVDGGNFNWDNGKFPELVEPDPTYHGISYTQKFGNAAYATKARVQLLRDYGNCLSPFNAYLTNLNVETLHLRMERHSENALKIARFLEKHENVDWINY PGLEDNKYYENAKKYLSRGCSGVLSFGVRGGLENAKKFVEKLQIASLVTHVSDVRTCVIHPASTTHRQLTEEQLIASGVLPSLIRLSVGIENVEDLIADLNQALNF SEQ ID NO:4 MNNWKKGTICIQGGYTPKSGEPRVLPIYQSTTYKYNDPDEVAALFDLKAEGHMYSRISNPTVAAFEEKIAALEGGVGALAVASGQSASTLSILNVCKSGEHVVAASTLYGGTYSLFSTTLKKFGIDVTFVDPEATEEEILKNCRENTKAVFGETIGNPGLNVLDFEKFSNVAKKIKAPFIVDNTIATPYLCNPLKLGANVVVHSATKYIDGH ATTVGGVIIDGGNFDWNNGRFPDFTEPDPSYHGIKYTETFGDSAYIVKARVQLLRDLGVCTSPFNAFLFSLGLETLHLRMERHSDNAIKLGRFLEKHSKVSWVSYP LLESHSTYETAKKYLPKGASGILTFGVKGGVEAGKQFIRNLKLAALVVHLGDARTSVLHPASTTHRQLTEEEQLSAGVTPDLIRVSVGIEDIEDIIEDFDQALNNIK SEQ ID NO:5 MSNEQTFRPETLAIHAGQKPDAETGARAVPIYQTSSYVFRDSEHAANLFGLKEEGFIYTRIMNPTNDVFEKRIAALEGGIGALALSSGQAAVFYSIIINAISAGDEIVSSSSIYGGTYNLFAHTLRKFGITVKFVDPSPENFERAITDKTKALFAETIGNPKNDVLDIEAVADIAHRHAIPLIVDNTVASPYLLRPIEFGADIVVHSATKFIGGHGN SIGGVIVDSGKFDWKGSGKFPEFTEDPPSYHGLVYVDAVGEAAYITKARIQLLRDLGAALSPFNAFLLLQGLETLHLRMQRHSENALAVAKFLEEEEAVESVNYPGLPSHPSHELAKKYLPNGQGAIVTFEIKGVEAGKKLIDSVKLFSHLANIGDSKSLIIHPASTTHEQLSPDEQLSAGVTPGLVRLSVGTEAIDDILDDLRQAIRQSQTVGVK sequence number 6 MDWKKYGYNTRALHAGYEPPEQATGSRAVPIYQTTSYVFRDSDHAARLFALEEPGFIYTRIGNPTVSVLEERIAALEEGVGALAVASGQAAITYAILNIAGPGDEIVSGSALYGGTYNLFRHTLYKKSGIIVKFVDETDPKNIEEAITEKTKAVYLETIGNPGLTVPDFEAIAEIAHRHGVPLIVDNTVAPYIFRPFEHGADIVVYSATKFIGGH GTSIGGLIVDSGKFDWTNGKFPELVEPDPSYHGVSYVETFKEAAYIAKCRTQLLRDLGSCMSPFNAFLFILGLETSLSLRMKKHCENALKIVEFLKSHPAVSWVNYPIAEGNKTRENALKYLKEGYGAIVTFGVKGGKEAGKKFIDSLTLISHLANIGDARTLAIHPASTTHQQLTEEEQLKTGVTPDMIRLSVGIEDVEDIIADLDQALRKSQEG sequence no.7 MKLETLAVHAGYSPDPTTRAVAVPIYQTTSYAFDDTQHGADLFDLKVPGNIYTRIMNPTNDVLEQRVAALEGGVGALAVASGMAAITYAIQTVAEAGDNIVSVAKLYGGTYNLLAHTLPRIGIQARFAAHDDVAALEALIDERTKAVFCETIGNPAGNIIDLQALADAAHRHGVPLIVDNTVATPVLCRPFEHGADIVVHSLTKYMGGHGTS IGGIVVDSGKFDWAANKSRFPLLNTPDPSYHGVTYTEAFGPAAFIGRCRVVPLRNMGAALSPFNAFLILQGLETLALRMERHCDNALAVARYLQQHPQVAWVKYAGLADNPEHALARRYLGGRPAAILSFGIQGGSAAGARFIDALKLVVRLVNIGDAKSLACHPASTTHRQLNAEELARAGVSDDMVRLSIGIEHIDDILADQALAAAAR sequence no.8 MSNNIKAYNNVLELIGETPLIKLSQTVASFPGNYYAKAEAFNPGHSSKDRIALHIIEQAEKKGILKPGDTIIETTSGNTGFSIAMVSRIKGYDCILAVSSKSSADKIDMLKSMGAKVYVCPANVSADDPRSYYQVAKRLHDEIKNSIYINQYFNDLNTEAHYLTTGPEIWNQT AGEITHLVACSGTGGTISGTARFLKEQNPNIKILGVDAYGSVLKKYHETQEFDKDEIYPYRIEGLGKNLIPTADTDFDVIDSFTKVKDEDAAHMARKISQTEGLFVGYTSGAAMQAVKQLVVEGFDKNSNVVVIFPDHGSRYMSKIYNDQWMQEQGFVDSKSTANEQHIEYIK sequence number 9 MAVEWTGIANDVTELVGKTPLVYLNKVVDGCVGRIVAKLEFMEPLSSVKDRIGYSMIADAEEKGLITPGKSVLIEPTSGNTGIGLAFTAAAKGYRLIIVMPASMTLERRMVLKALGAELVLTDPEKGIRGAFQKVEEIYAKTPNSYVLQQFENPSNPKMHYD TTGPEIWKATGGKIDALVAGIGTGGTITGSGKFLKEQNPNIKVCAVEPAESPVLVGGQPGSHGIYGIGAGFIPKVVDVSLLDEVVHVTTSEAIETAKLLAVKEGLFVGVSSGAAAAAAIKVAKRPEMAGKLIVTIFPSCGERYLSSALFESIREESENMTFEP SEQ ID NO: 10 MVFPSPLDFFERGKPTPLVRSRLQLPNGVRVWLKLEWYNPFSLSVKDRPAVEIISRLSRRVEKGSLVADATSSNFGVALSAVARLYGYRARVYLPGAAEEFGKLLPRLLGAQVIVDPEAPSTVHLLPRVMKDSKNEGFVHVNQFYNDANFEAHM RGTAREIFVQSRRGGLALRGVAGSLGTSGHMSAAAFYLQSVDPSIRAVLVQPAQGDSIPGIRRVETGMLWINMLDISYTLAEVTLEEAMEAVVEVARSDGLVIGPSGGAAVKALAKKAAEGDLEPGDYVVVVPDTGFKYLSLVQNALEGAGDSV SEQ ID NO: 11 MGRFILKCLKCGREYSQEYRLTCENDDSFLRAEYLEKKLELRKQPGIGRFHSWLPVQEELTTEAGPITYKSEALARELGLSNLYIGFSGYWPEKGAFIKTCSFKELEAHPTMQLLKESGGKAIVLASAGNTGRAFAHVSALTGTDVYIVVPDSGIPKLWLPEEPTDSIHLISMTPGNDYTDAINLAGRIAKLPGMVPEGGARNVARRE GMGTVMLDAAVTIGKMPDHYFQAVGSGTGGISAWEASLRLREDGRFGSKLPKLQLTQNLPFVPMYNAWQEGRRDIIPEIDMKDAKKRIEETYATVLTNRAPPYSVTGGLYDALVDTTDGIMYAVSKEEALDAKALFESLEGIDILPPSAVAAASLLKAVEAGNVGKDDTILLNIAGGGFKRLKEDFTLFQIEPEITVSNPDVPLEELKL SEQ ID NO: 12 MTRYDSLLQALGNTPLVGLQRLSPRWDDGRDGPHVRLWAKLEDRNPTGSIKDRPAVRMIEQAEADGLLRPGATILEPTSGNTGISLAMAARLKGYRLICVMPENTSVERRQLLELYGAQIIFSAAEGGSNTAVATAKELAATNPSWVMLYQYGNPANTDSH YCGTGPELLADLPEITHFVAGLGTTGTLMGTGRFLREHVANVKIVAAEPRYGEGVYALRNMDEGVPELYDPEILTARYSVGAVDAVRRTRELVHTEGIFAGISTGAVLHAALGVGAGALAAGERADIALVVADAGWKYLSTGAYAGSLDDAETALEGQLWA SEQ ID NO: 13 MTVITDITELIGNTPLLRLKNFDVPEGVAVYAKLEMMNPGGSIKDRLGDMLIRDALDSGKVKPGGVIIEATAGNTGIGLALSARKYGLKAIFCVPEHFSREKQQIMQALGASIIHTPRQDGMQGAIQKAIQLETEIENSYCVLQFKNRVNPST YYKTLGPEMWEALDGNIHTFVAGAGSGGTFAGTASFLKEKNPAVKTVIVEPVGSILNGGEPHAHKTEGIGMEFIPDYMDKSHDEIYTVTDENAFRLVKEAAEKEGLLIGSSSGAALYAALEEAKKASAGTNIVTVFPDSSDRYISKQIYEGGI SEQ ID NO: 14 MVQLHQLFPKHEVFAKLEYMNPGGSMKDRPAKYIIEHGIKHGLITENTHLIESTSGNLGIALAMIAKIKGLKLTCVVDPKISPTNLKIIKSYGANVEMVEEPDAHGGYLMTRIAKVQELLATIDDAYWINQYANELNWQSHYHGAGTEIVETI KQPIDYFVAPVSTTGSIMGMSRKIKEVHPNAQIVAVDAKGSVIFGDKPINRELPGIGASRVPEILNRSEINQVIHVDDYQSALGCRKLIDYEGIFAGGSTGSIIAAIEQLITSIEEGATIVTILPDRGDRYLDLVYSDTWLEKMKSRQGVKSE SEQ ID NO: 15 MTLQYPTIADCVGNTPLVRLQRMAGNTSNTLLLKLEGNNPAGSVKDRPALSMITRAELRGQIKPGDTLIEATSGNTGIALAMAAAIKGYKMILIMPDNGSAERKAAMTAYGAELLLVTPEEGMEGARDLAERMAAEGRGQVLDQFANGDN PEAHYTSTGPEIWRQTQGTITHFVSSMGTTGTIMGNSRYLKEQNPAIQIVGLQPMEGAAIPGIRRWPQEYLPKIYNAARVDRIIDMAQREAEDTTRRLAREEGIFCGVSSGGAVAGMLRLSAELENAVIVAIICDRGDRYLSTGIFDAPN SEQ ID NO: 16 MSVFSLKIDIADNKFFNGETSPLFSQSQAKLARQFHQKIAGYRPTPLCALDDLANLFGVKKILVKDESKRFGLNAFKMLGGAYAIAQLLCEKYHLDIET LSFEHLKNAIGEKMTFATTTDGNHGRGVAWAAQQLGQNAVIYMPKGSAQERVDAILNLGAECIVTDMNYDDTVRLTMQHAQQHGWEVVQDTAWEGYTKIP TWIMQGYATLADEAVEQMREMGVTPTHVLLQAGVGAMAGGVLGYLVDVYSPQNLHSIIVEPDKADCIYRSGVKGDIVNVGGDMATIMAGLACGEPNPLGWEILRNCATQFISCQDSVAALGMRVLGNPYGNDPRIISGESGAVGLGVLAAVHYHPQRQSLMEKLALNKDAVVLVISTEGDTDVKHYREVVWEGKHAVAP SEQ ID NO: 17 MISAFDIFKIGIGPSSSHTVGPMNAGKSFIDRLESSGLLTATSHIVVDLYGSLSLTGKGHATDVAIIMGLAGNSPQDVVIDEIPAFIELVTRSGRLPVASGAHIVDFPVAKNI IFHPEMLPRHENGMRITAWKGQEELLSKTYYSVGGGFIVEEEHFGLSHDVETSVPYDFHSAGELLKMCDYNGLSISGLMMHNELALRSKAEIDAGFARIWQVMHDGIERGMNTE GVLPGPLNVPRRAVALRRQLVSSDNISNDPMNVIDWINMYALAVSEENAAGGRVVTAPTNGACGIIPAVLAYYDKFRRPVNERSIARYFLAAGAIGALYKMNASISGAEVGCQ GEIGVACSMAAAGLTELLGGSPAQVCNAAEIAMEHNLGLTCDPVAGQVQIPCIERNAINAVKAVNAARMAMRRTSAPRVSLDKVIETMYETGKDMNDKYRETSRGGLAIKVVCG SEQ ID NO: 18 MENAKMNSLIAQYPLVKDLVALKETTTWFNPGTTSLAEGLPYVGLTEQDVQDAHARLSRFAPYLAKAFPETAATGGIIESELVAIPAMQKRLEKEYQQPISGQLLLKKDSH LPISGSIKARGGIYEVLAHAEKLALEAGLLTLDDDYSKLLSPEFKQFFSQYSIAVGSTGNLGLSIGIMSARIGFKVTVHMSADARAWKKAKLRSHGVTVVEYEQDYGVAVE EGRKAAQSDPNCFFIDDENSRTLFLGYSVAGQRLKAQFAQQGRIVDADNPLFVYLPCGVGGGPGGVAFGLKLAFGDHVHCFFAEPTHSPCMLLGVHTGLHDQISVQDIGI DNLTAADGLAVGRASGFVGRAMERLLDGFYTLSDQTMYDMLGWLAQEEGIRLEPSALAGMAGPQRVCASVSYQQMHGFSAEQLRNTTHLVWATGGGMVPEEEMNQYLAKGR SEQ ID NO: 19 MSIVRLYTARQLPRYRPLRFVRFNSTQVLSPKQRWTELKDTDFIKDSNGEEQPDYVKLILTSRVYDVVDDAGSPLTYAINLSHRCGTNVYLKREDLLPVFSFKLRGAYNMIAHLHSNSPTPVPGV IACSAGNHAQGVAFSANKLNIPATIVMPTPTPSIKYTNVSRLGSQVVLYGDDFDSAKQECARLSAEQNLIDIPPFNHPYVIAGQGTVALEITRQLRLDKLDAIFVPVGGGGLIAGIAVYLKQIAPH VKIIGVETYDADAMYQSLKNHKSTVLDKVGLFADGTAVKVLGDETWRLAKDLVDEVVLVTTDELCAAIKDIFEDTRSITEPSGALSVAGLKRYIEDHSEIDHKGKTYVPVLSGANMNFDRLRFVS ERAVLGEGREVSLAVTIPEQPGEFAKLQKLINPRAITEFSYRTSGEPTANIFVSFNVIDKKKEKALVINAMTEAGYKVVDISENELAKSHGRYLVGGKSSIMVILVKSCVDSISQKDSKKNSNNS SEQ ID NO: 20 MTDSVTGAGDGTRAVRAGLPEPVKHEPTLPGPVFAAHFHLPGDPTGPYTYGRDENPTWTHLERAIGELEAPGRDDVETLVFASGMAAISAVLFSQLSAGDTVVLPDDGYQVLPLVRAQLEAYGVEVRTAPTGGDAQLDVLDGAKLLWIETPSNPGLDVCDVRRLVEAAHARGCLVAVDNTLATPLGQRPLEL GADFSVASGTKQLTGHGDILLGYVTGGDAAAMASVRRWRKIVGAIPGPMEAWLAHRSIATLQLRVDRQNANALVIAEALRDRHEDLGVRYPGLPGDPSHKIASQQMRRFGCVVSFTLPTRARADRFLDALRLVDDATSFGGVRSTAERRGRWGGDAVPEGFIRFSAGAEDPQDLVADVLRALNESSQPTPSE SEQ ID NO: 21 MTASSDHPITTPPRAPSPVHGFGTLAVHAGSPHDPATGAVIEAISLSTTFAQTAVGKPVGEFEYSRSSNPNRANFEKMVAALEHAKYALAYSSGSATTANILQSLAAGSHVISVSDVYGGTHRYFTQVAKAHGVKVTFTPEIEVDIRDHITDATKLVWIETPSNPTLRLVDIRAVATAAHERGILVVVDNTFLSPYVQNPLDHGADIV VHSVTKYINGHSDVVMGVAAFNSDELYARLSFLQNAIGAVPSAFDSWLAHRGAKTLHLRAREATTNATAIAHALEASPLVISVNYPGLESHPHRAIALKQHRNGMGGGMLSFRIHGGHAAAEKFCQYTKIFTLAESLGGVESLCEIPSSMTHAGIPKAQREAVGIFDDLVRISCGVEDAEDLKADVLQALERAVADAANGVSNGVNGTH SEQ ID NO: 22 MKKKTLMIHGGITGDEKTGAVSVPIYQVSTYKQPKAGQHTGYEYSRTANPTRTALEALVTELESGEAGYAFSSGMAAITAVMMLFNSGDHVVLTDDVYGGTYRVMTKVLNRLGIESTFVDTSSREEVKAIRPNTKAIYIETPTNPLLKITDLTLMADIAKKAGVLLIVDNTFNTPYFQQPLTLGADIV LHSATKYLGGHSDVVGGLVVTASKELGEELHFVQNSTGGVLGPQDSWLLMRGIKTLGLRMEAIDQNARKIASFLENHPAVQTLYYPGSSNHPGHELAKTQGAGFGGMISFDIGSEERVDAFLGNLKLFTIAESLGAVESLISVPARMTHASIPRERRLELGITDGLIRISVGIEDAEDLLEDIGQALENI SEQ ID NO: 23 MATVSSCLLRRSRTASRIFKTSLRCFSTTSSSAQTVSGSSPFPFTGTNIKTNVSQLIGRTPLVYLSKISEGSGAYIAVKQEMMQPTASVKDRPALAMIEDAEKKGLISPGKTVLIEPTSGNMGISMAFMAAMKGYKMVLTMPSYTSMERRVVMRAFGADLILTDPDKGMGGTVKKANQLLDSTP DGFMLQQFNNPANTQVHFETTGPEIWEDTQGKVDIFVMGIGSGGTVSGVGRYLKSQNPNVKIYGVEPAESNILNGGKPGPHLITGNGVGFKPEILDMDVMDAVLEVKSDDAVKMARQLALQEGLLVGISSGANTIAALDLAKRPENKGKLIVTIHPSFGERYLSSALFKELREEAENMQPVPVE SEQ ID NO: 24 MSGIYPAPVRLESHRQQFPALANKAYFNYGGQGTLPQASLEAIQQAYEYIQRHGPFSAGVNAWIIEEANKTREAIASELGAPPEAIAITEDVTVGCNIAMWGINWQAGDHLLLTDCEHPSVIATAQEIARRFDVEVSTCPILSTLNQGDPTAVIAQHLRTQTRLVVLSHILWNTGQVLPVKEIVEVCRNYSSSSQTIRVL VDAAQSVGCLPLNLTELGADFYAFTGHKWWCGPEGIGGLYVRPDAWESLNPTFIGWRGIVSNQAGKPTGWKPNAQRFEVATSAYPLYAGVRSAIATHQQW GTPQERYQQICQISEYLWQRLSQLDFVKCLRTSPPQAGLVSFVLTNEHSHQQLVKSLEQQNLMLRTILDPNCVRACVHYFTQHAEIDKLIEAMKKIKSKG SEQ ID NO: 25 MRDSHHNTGFSTRAIHHGYDPLSHGGALVPPVYQTATYAFPTVEYGAACFAGEEPGHFYSRISNPTLALLEQRMASLEGGEAGLALASGMGAITSTLWTLLRPGDELIVGRTLYGCTFAFLHHGIGEFGVKVRHVDLNDAKALKAAISSKTRMIYFETPANNPNMQLVDIAAVVEAVRGHDVHVVVDNTYCTPYLQRPLE LGADLVVHSATKYLSGHGDITAGLVVGRKVLVDRIRLEGLKDMTGAVLSPHDAALLMRGIKTLALRMDRHCANAQQVAEFLVRQPQVELIHYPGLPSFAQYALAQRQMRLPGGMIAFELKGGIEAGRRFMNALQLFARAVSLGDAESLAQHPASMTHSSYTPQERAHHGISEGLVRLSVGLEDVEDLLADVALQACC SEQ ID NO: 26 MTSLKTKVIHGGISTDRTTGAVSVPIYQTSTYKQNGLGKPKEYEYSRSGNPTRHALEELIADLEGGVQGFAFSSGLAGIHAVLSLFSAGDHIILADDVYGGTFRLMDKVLTKTGIIYDLVDLSNLEDLKAAFKAETKAVYFETPSNPLLKVLDIKEISSIAKAHNALTLVDNTFATPYLQQPIALGADIV LHSATKYLGGHSDVVAGLVTTNSNELASEIGFLQNSIGAVLGPQDSWLVQRGIKTLALRMEAHSANAQKIAEFLEASQAVSKVYYPGLVNHEGHEIAKKQMTAFGGMISFELTDENAVKNFVENLRYFTLAESLGGVESLIEVPAVMTHASIPKELREEIGIKDGLIRLSVGVEALEDLLTDLKEALEKE SEQ ID NO: 27 MPLHNLTRFPRLEFIGAPTPLEYLPRFSDYLGREIFIKRDDVTPMAMGGNKLRKLEFLAADALREGADTLITAGAIQSNHVRQTAAVAAKLGLHCVALLENPIGTTAENYLTNGNRLLLDLFNTQIEMCDALTDPNAQLEELATRVEAQGFRPYVIPVGGSNAL GALGYVESALEIAQQCEGAVNISSVVVASGSAGTHAGLAVGLEHLMPESELIGVTVSRSVADQLPKVVNLQQAIAKELELTASAEILLWDDYFAPGYGVPNDEGMEAVKLLARLEGILLDPVYTGKAMAGLIDGISQKRFKDEGPILFIHTGGAPALFAYHPHV SEQ ID NO: 28 MIFSVDKVRADFPVLSREVNGLPLAYLDSAASAQKPSQVIDAEAEFYRHGYAAVHRGIHTLSAQATEKMENVRKRASLFINARSAEELVFVRGTTEGINLVANSWGNSNVRAGDNIIISQMEHHANIVPWQMLCARVGAELRVIPLNPDGTLQLETLPTLFDEKTRLLAITHVSNVLGTENPLAEMITLAHQHGAKVLVDGAQ AVMHHPVDVQALDCDFYVFSGHKLYGPTGIGILYVKEALLQEMPPWEGGGSMIATVSLSEGTTWTKAPWRFEAGTPNTGGIIGLGAALEYVSALGLNNIAEYEQNLMHYALSQLESVPDLTLYGPQNRLGVIAFNLGKHHAYDVGSFLDNYGIAVRTGHHCAMPLMAYYNVPAMCRASLAMYNTHEEVDRLVTGLQRIHRLLG SEQ ID NO: 29 MHLARYPRRFIAHLPTPLERLDRLTAELGGPEIWIKRDDCTGLSTGGNKTRKLEFLMAEAELQGADMVMTQGATQSNHARQTAAFAAKLGMDCHILLEDRTGSNNANYNNNGNVLLDHLHGATTEKRPGSGLDMNAEMEKVAEKFRADGRKVYTIPGGGSNPTGALGYV NCAFEMLNQFNERGLKVDHIVHATGSAGTQAGLITGLQAMNAQIPLLIGVRAPKPKQEENVYNLACATAEKLGCPGVVAREDVVANTDYVGEGYGIPTESGLEAIRMFAELEAILLDPVYSAKGAAGFIDLIRKGHFKKGERVVFLHTGGAVALFGYDNAFDYSGRWVA SEQ ID NO: 30 MDDYKRILITKILKNEVTEALGCTEVGLIGYAVSLCNISDPFSIEKIELTLNNGSFKNAYAVGVPNTKKYGILPAVVGGLLGDHKNKLLVFNGIKYSQKLEDFIKERLKIRVINSPLYCGVKIKDNSGNTFESLIKDNHLNVVIPKINNKLISEINGSEKEEYKNLELLDFLEYIDEIPEEIIQLVEKTIYTNNNLIK GDFLNFGNDCLSNMVNKTTSACNTRMIGENMPAMSVAKSGNMGIMATLPIIAYDYSNEQNQEKLIKSILLSVLVTIYATYKSSYLSSMCGCVSKGGMGAVIGLCYYKNGKNIKKLDSAARTFTANLPGIICDGGKVGCALKLASGCFAAYSSLFVDISYENGIVGKNFKECVENISEISKIMGDLSDIVKIMSKKEI SEQ ID NO: 31 MANDRSVDWKFSGSEAAKEASALGTYSSKLFALCDPLGKPILPPRSKSAETSHTAEKAVVEAVLCGTGNAYAPSLGLPVARSAVAEYLNRDLPKKLTADDVFMTVGGKQAIELTVDILAKPKANVLLPKPGFPWDVVRTIYKNLEVRKYDFIPEQNYEIDFDSVRALVDENTFAIFIINPHNPNGNTYSEVHLKKLAELARELRIMVVS DEVFRWTVFGSNPFVPMGKFSSIVPVVTLGSISKGWIVPGWRTGWVALHDLDGVFKCTKVLTAAKQFLVINSKPPTVIQAALPTILENTPEEFFHKRQKFLKEKADFAYSKLKEIPTLTCYLKPEACTFLWTELNLSRFADIKDDEEFCEKLATEENLVLLPGIAFGLKNWARHSIDMEISTLEDALGRLKSFCERHSIIIEVPLKDVNGVK

Claims

1. 1. A process for preparing L-glufosinate, comprising: a) providing a reaction medium comprising L-homoserine, or a salt, ester, amide, or anhydride thereof, methylphosphinic acid, or a salt or ester thereof, and an enzyme selected from the group consisting of a transferase that transfers an alkyl or aryl other than methyl, 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-glufosinate or a phosphonate thereof; and Optionally, c) recovering L-glufosinate or its phosphonate from the reaction medium The process includes:

2. 2. The process of claim 1, wherein the enzyme is selected from the group consisting of enzymes having the following EC numbers: EC 2.5.1.-, EC 4.3.1.-, and EC 4.4.1.-.

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

4. The enzyme is selected from the group consisting of EC2.5.1.47, EC2.5.1.48, EC2.5.1.49, EC2.5.1.51, EC2.5.1.52, EC2.5.1.65, EC2.5.1.76, EC2.5.1.113, EC2.5.1.134, EC2.5.1.140, EC2.5.1.144, EC4.3.1.15, EC4.3.1.17, EC4.3.1.18, EC4.3.1 4. The process of any one of claims 1 to 3, wherein the enzyme is selected from the group consisting of enzymes having the EC numbers EC 4.4.1.19, 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 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 enzyme is capable of inhibiting the activity of bacteria 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, and the like. tuberculosis, Bacillus subtilis, Staphylococcus aureus, Pseudomonas fluorescens, Escherichia coli, Candida maltosa, Streptomyces phaeochromogenes, Neurospora crassa, Spinacia oleracea, Cyanobacteria bacterium, Pseudomonas putida, Lactococcus lactis, Ruegeria pomeroyi, Methanococcus maripaludis, and Camelina sativa. The process according to any one of claims 1 to 4, wherein the organism is selected from the group consisting of:

6. 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 Clostridioides difficile, O-acetylhomoserine aminocarboxypropyltransferase derived from Clostridium novyi, O-acetylhomoserine aminocarboxypropyltransferase derived from Geobacillus stearothermophilus, O-acetylhomoserine aminocarboxypropyltransferase derived from Thermotoga maritima, and O-acetylhomoserine aminocarboxypropyltransferase derived from Pseudomonas aeruginosa. O-acetylhomoserine aminocarboxypropyltransferase from A. aeruginosa, β-pyrazolylalanine synthase from Nonlabens dokdonensis, L-mimosine synthase from Leucaena leucocephala, O-phosphoserine sulfhydrylase from Aeropyrum pernix, cysteine ​​synthase from Methanosarcina acetivorans, [CysO sulfur carrier protein]-thiocarboxylate-dependent cysteine ​​synthase from Mycobacterium tuberculosis, cystathionine β-synthase from Bacillus subtilis, Staphylococcus aureus N-(2-amino-2-carboxyethyl)-L-glutamate synthase from Pseudomonas aureus, S-sulfo-L-cysteine ​​synthase from Pseudomonas fluorescens, and Escherichia coli.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 gamma-lyase from Streptomyces phaeochromogenes, cystathionine gamma-lyase from Neurospora Crassa, homocysteine ​​desulfhydrase from Bacillus subtilis, L-3-cyanoalanine synthase from spinach (Spinacia oleracea), cysteine ​​lyase from Cyanobacteria bacterium, Pseudomonas putida 6. The process of any one of claims 1 to 5, wherein the selected enzyme is selected from the group consisting of methionine gamma-lyase from Lactococcus putida, cysteine-S-conjugate beta-lyase from Lactococcus lactis, D-cysteine ​​desulfhydrase from Escherichia coli, selenocysteine ​​lyase from Escherichia coli, L-cysteate sulfolyase from Ruegeria pomeroyi, L-cysteine ​​desulfidase from Methanococcus maripaludis, and L-cystine beta-lyase from Camelina sativa.

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

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

9. 8. The process of claim 1, wherein the reaction medium in step a) comprises a host cell expressing the enzyme.

10. 10. The process of claim 9, further comprising culturing the host cells.

11. 11. The process 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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