Method for directly preparing L-glufosinate-ammonium from L-homoserine

Abstract

The invention provides a method for preparing L-glufosinate-ammonium by catalyzing homoserine and methylphosphinic acid to react through an enzyme. The method does not include a step of producing O-acyl-L-homoserine, and the method does not use O-acyl-L-homoserine.

Description

A method for directly preparing L-phosphinothricin 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-phosphinothricin using L-homoserine as a substrate. Background Art

[0002] Glufosinate (also known as 4-[hydroxy(methyl)phosphonyl]-D,L-homoalanine) is the world's second-best-selling genetically modified crop-tolerant herbicide. Glufosinate is a broad-spectrum contact herbicide that inhibits the activity of L-glutamine synthetase in plants, disrupting nitrogen metabolism and ultimately killing the plant. Compared to glyphosate, glufosinate offers significant advantages, including a wider range of applications, rapid onset of action, long-lasting effect, lower toxicity, and greater safety. Consequently, glufosinate sales are growing rapidly, and the market demand is expected to be huge in the foreseeable future, with promising prospects.

[0003] However, the complex production process for glufosinate ammonium makes its production technically challenging. Its high price hinders its rapid replacement for glyphosate. Furthermore, chemically synthesized glufosinate ammonium is a racemic mixture containing equal amounts of two optical isomers (D,L-glufosinate ammonium), but only the L-configuration is physiologically active.

[0004] In recent years, numerous methods for preparing L-glufosinate from D,L-glufosinate have been reported. For example, D-glufosinate is oxidized to 2-carbonyl-4-(hydroxymethylphosphono)butyric acid (PPO), which is then reduced or transaminated to produce L-glufosinate. However, chemically synthesizing D / L-glufosinate and then enzymatically converting D-glufosinate to L-glufosinate is complex and expensive. Therefore, a more efficient method for synthesizing L-glufosinate remains unmet.

[0005] WO 2022 / 207543 A1 reports a method for synthesizing L-phosphinothricin or its phosphonate using activated homoserine (O-acetylhomoserine or O-succinylhomoserine) and methylphosphinic acid or its ester as substrates under the catalysis of an enzyme (sulfhydrylase or cystathionine γ-synthase).

[0006] However, this method requires the prior production (e.g., by fermentation) of O-acyl homoserine, which is limited by i) the amount of O-acyl homoserine accumulated; ii) the instability of O-acyl homoserine (towards acid, alkali, and heat); iii) the inhibition of enzyme activity by by-product organic acids; and iv) if the pH is adjusted during the reaction, the production cost, process complexity, and product separation difficulty are increased.

[0007] Therefore, it is necessary to develop a new method for preparing L-phosphinothion, 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 methylphosphinic acid or its ester under enzyme catalysis to generate L-phosphinothricin or its phosphonate 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-glufosinate-ammonium, comprising the following steps:

[0011] 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 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-phosphinothricin or a phosphonate thereof; and optionally

[0013] c) recovering L-phosphinothricin or its phosphonate 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 (EC 4.3.1.19-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 4.4.1.3-hydroxybenzoic acid). 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 shows the LC-MS results of the reaction product catalyzed by purified GsMetY. A: Detection results of the reaction product sample, B: Detection results after adding internal standard.

[0024] Figure 2 shows the LC-MS results of the reaction products catalyzed by E. coli expressing GsMetY. A: Sample test results, B: Test results after adding internal standard.

[0025] Figure 3 shows the LC-MS results of the reaction products catalyzed by E. coli expressing SpCGL. A: Sample test results, B: Test results after adding internal standard.

[0026] Detailed Description of the Invention

[0027] The present invention provides a novel method for preparing L-glufosinate-ammonium. Unless otherwise specified, the terms used herein have the meanings generally understood by those skilled in the art.

[0028] Prior art reports indicate that 4-hydroxy-activated L-homoserine (such as O-acylhomoserine) can react with methylphosphinic acid or its ester under enzyme catalysis to produce L-phosphinothricin or its phosphonate.

[0029] Surprisingly, the inventors discovered that L-homoserine can react with methylphosphinic acid or its ester under enzyme catalysis to generate L-phosphinothricin or its phosphonate and water (as shown in Formula I).

[0030] 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.

[0031] Therefore, the present invention provides a method for preparing L-glufosinate-ammonium, comprising the following steps:

[0032] 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 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;

[0033] b) incubating the reaction medium to produce a reaction product comprising L-phosphinothricin or a phosphonate thereof; and optionally

[0034] c) recovering L-phosphinothricin or its phosphonate from the reaction product.

[0035] 1. Substrate

[0036] 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.

[0037] 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.

[0038] 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.

[0039] Methylphosphinic acid or its ester is shown in Formula II.

[0040] wherein R is selected from hydrogen, alkyl, alkenyl, alkynyl, hydroxyalkyl, and aryl.

[0041] In some embodiments, the reaction medium comprises methylphosphinate.In some embodiments, the method further comprises the step of hydrolyzing L-glufosinate-ammonium phosphonate.

[0042] 2. Enzymes

[0043] As described above, the reaction of the present invention is carried out under the catalysis of an enzyme, that is, an enzymatic reaction.

[0044] 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.

[0045] 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.

[0046] 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).

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.-.

[0051] 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.

[0052] 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). .

[0053] 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.

[0054] 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 (EC 4.3.1.19-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 4.4.1.3-hydroxybenzoic acid). 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] Those skilled in the art are aware that various computer programs can be used to determine the identity between two sequences.

[0059] "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.

[0060] 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.

[0061] 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.

[0062] 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).

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 3. Preparation conditions

[0067] The enzyme of the present invention may be provided in the form of an isolated polypeptide.

[0068] 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).

[0069] 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.

[0070] 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 (+).

[0071] 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.

[0072] 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

[0073] 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.

[0074] Example 1. Materials and methods

[0075] 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.

[0076] i) Reagents: Isopropyl β-D-thiogalactopyranoside (IPTG) was purchased from Beyotime Biotechnology Co., Ltd., L-homoserine was purchased from Jiangsu Aikon Biopharmaceutical R&D Co., Ltd., butyl methylphosphinate was synthesized in-house according to the method reported in patent CN106674275B, L-glufosinate, pyridoxal phosphate (PLP), and acetonitrile were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and Triton-X100 was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0077] ii) Vectors and strains:

[0078] The expression vector used in the examples was pET-29b(+), purchased from Sangon Biotech (Shanghai) Co., Ltd.;

[0079] The host cells used in the examples were Escherichia coli BL21 (DE3), purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0080] iii) Gene cloning:

[0081] 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.

[0082] Table 1

[0083] iv) Recombinant host cell preparation

[0084] 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.

[0085] v) Protein expression and preparation of whole-cell catalysts:

[0086] 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.

[0087] 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.

[0088] vi) Enzyme preparation

[0089] 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.

[0090] vii) Purified enzyme-catalyzed reactions

[0091] To a 20 mM solution of L-homoserine in Tris, butyl methylphosphinate and PLP were added, and the pH of the solution was adjusted to 7.4 with hydrochloric acid. The final concentrations of L-homoserine, butyl methylphosphinate, and PLP were 84 mM, 168 mM, and 0.3 mM, respectively. The purified enzyme prepared as described in step vi) was added to the solution to a final concentration of 0.5 mg / mL. The mixture was shaken continuously (220 rpm) at 37°C for 12 hours. A sample was then hydrolyzed using 6N HCl in a 100°C metal bath for 2 hours. After hydrolysis, the pH was adjusted to 7.0. The hydrolyzed sample and a sample supplemented with 1 mM L-phosphinothricin as an internal standard were analyzed by LC-MS. The peak with a molecular weight of 182 was extracted in positive mode to determine the formation of L-phosphinothricin.

[0092] viii) Whole-cell catalytic reactions

[0093] To a 20 mM solution of L-homoserine in Tris, butyl methylphosphinate, PLP, and Triton X-100 were added, and the pH of the solution was adjusted to 7.4 with hydrochloric acid. The final concentrations of L-homoserine, butyl methylphosphinate, PLP, and Triton X-100 were 84 mM, 168 mM, 0.3 mM, and 0.2%, respectively. Whole cells prepared as described in v) were added to the above solution, with a final concentration of 30 OD 600 The mixture was shaken continuously (220 rpm) at 37°C for 12 hours. Samples were then taken and hydrolyzed in a 100°C metal bath using 6N HCl for 2 hours. After hydrolysis, the pH was adjusted to 7.0. The hydrolyzed samples and samples supplemented with 1 mM L-glufosinate as an internal standard were analyzed by LC-MS. The peak with a molecular weight of 182 was extracted in the positive mode to determine the formation of L-glufosinate.

[0094] Example 2: Preparation and Detection of MetY from Bacillus stearothermophilus (GsMetY)

[0095] A clone expressing GsMetY (SEQ ID NO: 5) was activated, and a purified enzyme was prepared according to the method of Example 1. L-glufosinate produced by the purified enzyme was detected. LC-MS analysis revealed a peak with a molecular weight of 182 extracted from the GsMetY-catalyzed reaction product sample (see Figure 1A ). Furthermore, only a peak with a molecular weight of 182 was extracted from the sample containing 1 mM L-glufosinate as an internal standard, and the peak height was increased (see Figure 1B ), indicating that the reaction product sample contained L-glufosinate and that pure GsMetY enzyme catalyzed the production of L-glufosinate.

[0096] Example 3: Preparation and detection of whole cells expressing GsMetY

[0097] A clone expressing GsMetY (SEQ ID NO: 5) was activated, and whole cells were prepared according to the method of Example 1, and L-glufosinate production catalyzed by the whole cells was detected. LC-MS analysis revealed that a peak with a molecular weight of 182 was extracted from the reaction product sample catalyzed by the GsMetY-expressing whole cells (see FIG2A ). No new peak was detected in the sample supplemented with 1 mM L-glufosinate as an internal standard, and the peak height and area of ​​the peak with a retention time of approximately 1.8 min increased (see FIG2B ), indicating that the reaction product sample contained L-glufosinate, i.e., L-glufosinate was produced catalyzed by the GsMetY-expressing whole cells.

[0098] Example 4. Preparation and detection of whole cells expressing CGL (SpCGL) from Streptomyces fuscachromogenes

[0099] A clone expressing SpCGL (SEQ ID NO: 20) was activated, whole cells were prepared according to the method of Example 1, and L-glufosinate production catalyzed by the whole cells was detected. LC-MS analysis revealed a peak with a molecular weight of 182 extracted from the reaction product sample catalyzed by the SpCGL-expressing whole cells (see Figure 3A). No new peak was detected in the sample supplemented with 1 mM L-glufosinate as an internal standard. Furthermore, the peak height and area of ​​the peak with a retention time of approximately 1.8 min increased (see Figure 3B), indicating that the reaction product sample contained L-glufosinate, indicating that the SpCGL-expressing whole cells catalyzed the production of L-glufosinate.

[0100] Example 5: Production of L-phosphinothricin using E. coli cells expressing exogenous enzymes

[0101] The purpose of this example is to determine whether E. coli cells expressing exogenous enzymes can catalyze the conversion of L-homoserine to L-glufosinate.

[0102] As described in Example 1, recombinant E. coli cells expressing the enzymes in Table 1 were prepared, and the L-phosphinothricin produced by the whole cells was detected.

[0103] As shown in Table 2, recombinant E. coli cells expressing any one of the enzymes of SEQ ID NOs: 1-31 can catalyze the conversion of L-homoserine into L-phosphinothricin, wherein the catalytic ability of the enzyme is represented by ★, and the more ★, the stronger the catalytic ability.

[0104] Table 2

[0105] sequence

Claims

1. A method for preparing L-glufosinate-ammonium, comprising the following steps: a) providing a reaction medium, which comprises L-homoserine or a salt, ester or amide thereof, methylphosphinic acid or a salt or ester thereof 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-phosphinothricin or a phosphonate thereof; and optionally c) recovering L-phosphinothricin or its phosphonate 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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