Enzymatic process for the production of l-glufosinate and its phosphonate

Abstract

The present invention relates to a method for the enzymatic production of L-glufosinate or a phosphate ester thereof. The method comprises a step wherein activated L-homoserine H A is reacted with a substrate S selected from the group consisting of methyl phosphinic acid and methyl phosphinate. The present invention enables new substrates in the enzymatic process preparation of L-glufosinate and phosphate esters thereof.

Description

[0001] This invention relates to an enzymatically catalyzed method for the production of L-glufosinate (“L-GA” or “LGA”) or its phosphate esters. The method includes the activation of L-homoserine H therein. A The step involves reacting with a substrate S selected from methylphosphonic acid and methylphosphonate esters. This invention enables the production of novel substrates in the enzymatic production of L-glufosinate and its phosphate esters. 1. Background Technology

[0002] Organophosphorus compounds, or chemical reagents containing carbon-phosphorus bonds, are widely used as herbicides in plant protection. Glyphosate is one such herbicide. and glufosinate and the growth regulator glyphosate For this purpose (e.g., G). As described in Rev. Environ. Contam. Toxicol. 1994, 138, 73-145.

[0003] p-Methylphosphonates (e.g., butyl p-methylphosphonate; "MPBE"; CAS No.: 6172-80-1) play a crucial role as building blocks in the synthesis of the non-selective herbicide glufosinate. These esters can be obtained via two basic synthetic routes (summarized on page 130 of the article by K. Haack, Chem. Unserer Zeit 2003, 37, 128-138). Figure 3 a and 3b):

[0004] a. The reaction of diethylphosphononyl chloride [ClP(OC2H5)2] with CH3MgCl yields methyldiethoxyphosphine [H3CP(OC2H5)2; "DEMP"; CAS No. 15715-41-0], which is partially hydrolyzed to give the corresponding ethyl methylphosphonate (MPEE; CAS No. 16391-07-4).

[0005] b. Alternatively, methane can react with phosphorus trichloride at 500°C to produce methyldichlorophosphine (H3CPCl2). The latter can be solvated in an alcohol to give the corresponding methylphosphonate.

[0006] methylphosphonates selectively add carbon-carbon double bonds. This property is used in the synthesis of glufosinate to form a second phosphorus-carbon bond. For example, H3CPH(O)OR (R = alkyl) reacts with 1-cyanoallylic acetate in an addition reaction to provide an intermediate. Subsequently, the exchange of the acetate subcomponent with ammonia and the hydrolysis of the cyano and ester groups of the phosphonate moiety yield glufosinate.

[0007] Acrylates are a cheaper alternative. They react with p-methylphosphonate to form alkyl 3-[alkoxy(methyl)phosphono]propionate. This diester undergoes a Claisen reaction, hydrolysis, and decarboxylation with diethyl oxalate to produce the corresponding α-keto acid, which can be reductively amination to generate glufosinate.

[0008] These and other routes for the synthesis of L-glufosinate are also described in the art, for example in WO 1999 / 009039 A1 and EP 0 508 296 A1.

[0009] WO 2020 / 145513 A1 and WO 2020 / 145514 A1 describe a chemical route for the production of L-glufosinate. In this route, homoserine derivatives such as O-acetylhomoserine or O-succinylhomoserine are used as starting materials, and L-glufosinate is obtained through a series of reactions including lactoneation and halogenation.

[0010] WO 2020 / 145627 A1 describes a similar route in which bromine derivatives are obtained during halogenation.

[0011] The route disclosed in CN 106083922 A is similar, but it starts with L-methionine.

[0012] EP 2402453 A2 describes an enzymatic method for producing methionine by reacting a mixture of methanethiol and dimethyl sulfide with O-acetylhomoserine or O-succinylhomoserine.

[0013] CN 108516991 A describes another route for the synthesis of L-glufosinate, starting with the azeotropic dehydration of L-homoserine to obtain L-3,6-bis(2-haloethyl)-2,5-diketopiramate, followed by the introduction of a methylphosphonic acid diester group and hydrolysis.

[0014] A general drawback of all synthetic routes for glufosinate is that the resulting glufosinate is a racemic mixture. However, since the D-enantiomer lacks herbicidal activity, the L-glufosinate is an economically viable enantiomer.

[0015] For the enantioselective synthesis of L-glufosinate, an enzymatic pathway has been described in this art.

[0016] WO 2017 / 151573 A1 discloses a two-step enzymatic synthesis of L-glufosinate from D-glufosinate. In the first step, D-glufosinate is oxidatively deaminated to give 2-oxo-4-[hydroxy(methyl)phosphono]butyric acid (“PPO”), which is then specifically amination to L-glufosinate as the second step. The first step is catalyzed by a D-amino acid oxidase, and the second step is catalyzed by a transaminase.

[0017] WO 2020 / 051188 A1 discloses a similar method for converting racemic glufosinate into the L-glufosinate enantiomer. Furthermore, it discloses a step in which the α-keto acid or ketone byproduct formed during the amination of PPO with an amine donor is converted by a ketoglutarate decarboxylase to further shift the equilibrium to L-glufosinate.

[0018] WO 2019 / 018406 A1 discloses a method for purifying L-glufosinate from a mixture containing L-glufosinate and glutamate. The glutamate is enzymatically converted to pyroglutamate by gamma-glutamyl cyclotransferase, and then L-glufosinate is purified from the resulting mixture by ion exchange.

[0019] The object of this invention is to provide another enzymatic process for the high enantiomeric overproduction of L-glufosinate. In particular, this method should allow the use of novel substrates not previously used for the enzymatic synthesis of L-glufosinate. 2. Brief Description of the Invention

[0020] This invention addresses the aforementioned problems by providing a method for producing L-glufosinate from substrates not previously used for enzymatic production of L-glufosinate. Specifically, this invention provides a method for producing L-glufosinate or L-glufosinate phosphate esters from methylphosphonic acid and its esters using an enzymatic catalytic pathway. Therefore, these phosphorus compounds are used as alternative substrates in the production of L-glufosinate, allowing for production flexibility that does not depend on known substrates currently used for L-glufosinate production.

[0021] Specifically, this objective is achieved by the present invention, which relates to an enzyme-catalyzed method for producing L-glufosinate or its phosphate ester, comprising step (a), wherein activated L-homoserine H A These compounds are produced by reacting with substrate S. 3. Description of the attached drawings

[0022] Figure 1 The pET-26b(+)_metY-Cg plasmid map is shown.

[0023] Figure 2 The pET-26b(+)_metY_P2T plasmid map is shown.

[0024] Figure 3 The pET-26b(+)_metZ-Cv plasmid map is shown.

[0025] Figure 4 The pET-26b(+)_metZ-Hn plasmid map is shown. 4. Detailed Description of the Invention

[0026] Surprisingly, certain phosphorus-containing compounds, namely methylphosphonic acid and methylphosphonate, can react with activated L-homoserine under enzymatic catalysis, thus opening up new pathways for the synthesis of L-glufosinate and L-glufosinate phosphates. This is particularly surprising because similar compounds, such as DEMP, do not undergo similar reactions with activated L-homoserine.

[0027] Therefore, the present invention relates to an enzyme-catalyzed method for producing L-glufosinate or its phosphate ester, comprising step (a), wherein activated L-homoserine H A Reacts with substrate S of structure (I) to produce a compound of structure (III).

[0028]

[0029] Where R 1 Selected from hydrogen, alkyl, alkenyl, alkynyl, hydroxyalkyl, and aryl.

[0030] According to the present invention, compounds represented as "L-glufosinate or its phosphate ester" are represented by structure (III). When R in structure (III) 1 When hydrogen is present, the compound is L-GA.

[0031] When R in structure (III) 1 When the compound is selected from alkyl, alkenyl, alkynyl, hydroxyalkyl, or aryl, it is a phosphate ester of L-GA.

[0032] Activated L-homoserine H A It has the following structure (II):

[0033]

[0034] Where R 2 It is a hydrocarbon group having 1 to 15 carbon atoms, which optionally includes at least one functional group selected from OH, COOH, and NH.

[0035] The reaction in step (a) is catalyzed by at least one enzyme selected from hydrogen sulfide hydrolase E1 and cystathionine γ-synthase E2.

[0036] 4.1 Substrate S

[0037] The substrate S according to the present invention is selected from methylphosphonic acid and methylphosphonate.

[0038] The substrate S has structure (I). In structure (I), R 1 Selected from hydrogen, alkyl, alkenyl, alkynyl, hydroxyalkyl, aryl,

[0039] Preferably selected from hydrogen and alkyl groups,

[0040] Preferably selected from hydrogen, alkyl groups having 1 to 6, preferably 1 to 4 carbon atoms,

[0041] More preferably, it is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, isobutyl, tert-butyl, even more preferably from hydrogen, methyl, ethyl, n-butyl, even more preferably from hydrogen, methyl, n-butyl, and most preferably from methyl, n-butyl. Most preferably, R 1 It is n-butyl.

[0042] When R in structure (I) 1 When hydrogen is present, the compound is methylphosphonic acid.

[0043] When R in structure (I) 1 When the compounds are selected from alkyl, alkenyl, alkynyl, hydroxyalkyl, or aryl groups, the compound is an ester of methylphosphonic acid.

[0044] R in structure (I) and structure (III) 1 They are the same.

[0045] 4.2 Activated L-homoserine H A

[0046] Another reactant in the reaction according to the invention is activated L-homoserine H. A .

[0047] This technician is aware of activated L-homoserine H A (Sometimes also referred to as "L-methionine precursor", for example in WO2008 / 013432A1), it specifically refers to O-acyl L-homoserine.

[0048] 4.2.1 Activated L-homoserine H A

[0049] Activated L-homoserine has the following chemical structure (II):

[0050]

[0051] Where R 2 It is a hydrocarbon group having 1 to 15 carbon atoms, which optionally includes at least one functional group selected from OH, COOH, and NH.

[0052] More preferably, the activated L-homoserine is selected from O-acetyl-L-homoserine [structure (II-A)], O-succinyl-L-homoserine [structure (II-B)], O-propionyl-L-homoserine [structure (II-C)], O-acetoacetyl-L-homoserine [structure (II-D)], O-coumaryl-L-homoserine [structure (II-E)], O-malonyl-L-homoserine [structure (II-F)], O-hydroxymethylglutaryl-L-homoserine [structure (II-G)], and O-glutaryl-L-homoserine [structure (II-H)].

[0053] Even more preferably, the activated L-homoserine is selected from O-acetyl-L-homoserine [structure (II-A)] and O-succinyl-L-homoserine [structure (II-B)].

[0054] Most preferably, the activated L-homoserine is O-acetyl-L-homoserine [structure (II-A)].

[0055]

[0056] 4.2.2 Activated L-homoserine H A Chemical synthesis

[0057] The activated L-homoserine H used in the method of this invention A It can be obtained through organic chemical synthetic routes known to those skilled in the art. For example, the synthesis of O-succinylhomoserine is described in M. Flavin, C. Slaughter, Biochemistry 1965, 4, 1370-1375. The synthesis of O-acetylhomoserine is described in S. Nagai, M. Flavin, Methods in Enzymology, Metabolism of Amino Acids and Amines Part B 1971, 17(Part B), 423-424.

[0058] The chemical synthesis of potential precursors is described, for example, in M.D. Armstrong, J. Am. Chem. Soc. 1948, 70, 1756-1759.

[0059] 4.2.3 Activated L-homoserine H A Biotechnology Synthesis

[0060] Optionally, preferably, the activated L-homoserine H used in this invention AObtained through biotechnological means. For example, as described in WO 2008 / 013432 A1 or by H. Kase, K. Nakayama, Agr. BioI. Chem. 1974, 38, 2021-2030.

[0061] Generate activated L-homoserine H A The strains are preferably selected from Escherichia sp., Erwinia sp., Serratia sp., Providencia sp., Corynebacterium sp., Pseudomonas sp., Leptospira sp., Salmonella sp., Brevibacterium sp., Hypomononas sp., Chromobacterium sp., Nocardia sp., fungi, especially yeasts.

[0062] Biotechnical methods for obtaining L-homoserine are also available in the art, for example, as described in US 3,598,701, US 6,303,348B1, EP 0 994 190 A2, EP 1 149 911 A2, and WO 2004 / 067757 A1.

[0063] 4.3 Enzymes

[0064] The method according to the present invention is enzyme-catalyzed.

[0065] The term "enzyme" refers to any substance that is composed entirely or mostly of proteins or polypeptides, which more or less catalyze or promote one or more chemical or biochemical reactions.

[0066] Any enzyme used according to any aspect of the invention may be an isolated enzyme. In particular, the enzyme used according to any aspect of the invention may be used in its active state and may contain all coenzyme factors, substrates, auxiliary and / or activating peptides or factors necessary for their activity.

[0067] In particular, this also means that the terms “cystathionine γ-synthase” and “hydrogen sulfide hydrolase”, especially “O-acetylhomoserine hydrogen sulfide hydrolase” or “O-succinylhomoserine hydrogen sulfide hydrolase”, contain all the coenzyme factors required for the respective enzymes and their functions. In particular, this coenzyme factor is pyridoxal phosphate monohydrate (“PMP”).

[0068] A polypeptide is a series of chemical building blocks called amino acids linked together by chemical bonds called peptide bonds. Proteins or polypeptides, including enzymes, can be "natural" or "wild-type," meaning they exist in nature or have the amino acid sequence of natural proteins, respectively. These terms are sometimes used interchangeably. Polypeptides can be glycosylated or not.

[0069] The enzymes used in any aspect of the invention can be recombinant. As used herein, the term "recombinant" refers to a molecule or the molecule encoded by such a molecule, particularly a polypeptide or nucleic acid, which is not naturally occurring but is the result of genetic engineering, or to a cell containing the recombinant molecule. For example, a nucleic acid molecule is recombinant if it contains a promoter functionally linked to a sequence encoding a catalytically active polypeptide, and that promoter has been engineered such that the catalytically active polypeptide is overexpressed at polypeptide levels relative to the polypeptide in a corresponding wild-type cell containing the original, unaltered nucleic acid molecule. As another example, a polypeptide is recombinant if it has the same sequence as a naturally occurring polypeptide but has been modified to include one or more point mutations to distinguish it from any naturally occurring polypeptide sequence.

[0070] The term “overexpression” as used in this article refers to an expression level or activity of the corresponding polypeptide that is higher than that normally found in the corresponding wild-type cells under the same conditions without genetic modification to increase expression.

[0071] As used herein, the term "isolated" refers to an enzyme of interest that is enriched compared to its naturally occurring form in cells. Enrichment can be achieved by SDS-PAGE and / or activity assays. For example, by visually examining the polyacrylamide gel after staining with Coomassie blue, it can be determined that the enzyme of interest constitutes 5, 10, 20, 50, 75, 80, 85, 90, 95, or more than 99% of all peptides present in the formulation.

[0072] 4.3.1 Enzymes E1 and E2

[0073] Step (a) of the method according to the invention is catalyzed by at least one enzyme selected from hydrogen sulfide hydrolase E1 and cystathionine γ-synthase E2.

[0074] This person skilled in the art knows that hydrogen sulfide hydrolase is an enzyme that catalyzes at least one of the following reactions <1A> and <1B>:

[0075] <1A>: O-acetyl-L-homoserine + methanethiol → L-methionine + acetic acid.

[0076] <1B>: O-succinyl-L-homoserine + methanethiol → L-methionine + succinic acid.

[0077] The hydrogen sulfide hydrolase that exhibits higher catalytic activity in reaction <1A> than in reaction <1B> can be termed "O-acetyl-L-homoserine hydrogen sulfide hydrolase".

[0078] The hydrogen sulfide hydrolase that exhibits higher catalytic activity for reaction <1B> than for reaction <1A> can be called "O-succinyl-L-homoserine hydrogen sulfide hydrolase".

[0079] Those skilled in the art know that cystathionine γ-synergase catalyzes at least the following reactions <2> Enzymes:

[0080] <2> O-Succinyl-L-homoserine + L-cysteine ​​→ L-cystathionine + succinic acid

[0081] In a preferred embodiment, step (a) of the method according to the invention is catalyzed by hydrogen sulfide hydrolase E1, which is even more preferably O-acetylhomoserine hydrogen sulfide hydrolase or O-succinylhomoserine hydrogen sulfide hydrolase, and most preferably O-acetylhomoserine hydrogen sulfide hydrolase.

[0082] The hydrogen sulfide hydrolase or cystathionine γ-synthase that can be used in step (a) of the method of the present invention may be derived from Bacillus, particularly Bacillus subtilis; Bradyrhizobium sp., particularly Bradyrhizobium japonicum; Brachybacterium; Colwellia sp., particularly Colwellia psychrerythraea; Corynebacterium, particularly Corynebacterium glutamicum and Corynebacterium humireducens; Chromobacterium, particularly Chromobacterium violaceum; Erwinia; Escherichia, particularly Escherichia coli; Hyphomonas sp., particularly Hyphomonas moniliforme. * *Neptunium*; *Klebsiella* sp., especially *Klebsiella pneumoniae*; *Leptospira* sp., especially *Leptospira interrogans*; *Methylobacillus* sp., especially *Methylobacillus flavorlates*; *Methylococcus* sp., especially *Methylococcus capsulatus*; *Nitrosomonas* sp., especially *Nitrosomonas europaea*; *Nocardia* sp., especially *Nocardia farcinica*; *Providencenia* sp.; *Pseudomonas* sp., especially *Pseudomonas aeruginosa*. aeruginosa; Rhodobacter sp., especially Rhodobacter sphaeroides; Salmonella sp., especially Salmonella enterica; Serratia sp.; Shigella sp.Especially *Shigella flexneri*; fungi, preferably yeasts, more preferably *Saccharomyces* p., and even more preferably *Saccharomyces cerevisiae*.

[0083] The hydrogen sulfide hydrolase that can be used in the method according to the present invention can be O-acetyl-L-homoserine hydrogen sulfide hydrolase classified in EC class EC 2.5.1.49 or O-succinyl-L-homoserine hydrogen sulfide hydrolase classified in EC class EC 2.5.1-.

[0084] These enzymes are part of the direct thioacylation pathway in methionine biosynthesis and are PMP-dependent. They are described, for example, by MP Ferla and WMPatrick, Microbiology 2014, 160, 1571-1584.

[0085] WO 02 / 18613A1, WO 2007 / 024933 A2, EP 2 657 345 A1, EP 2 657 250 A2, WO2015 / 165746A1 and WO 2008 / 013432 A1 disclose examples of enzymes having O-acetyl-L-homoserine hydrogen sulfide hydrolase and O-succinyl-L-homoserine hydrogen sulfide hydrolase activities according to the present invention.

[0086] The O-acetyl-L-homoserine hydrogen sulfide hydrolase suitable for the method according to the invention can be derived from *Corynebacterium*, particularly *Corynebacterium glutamicum* or *Corynebacterium terpenoides*; *Leptospira*, particularly *Leptospira interrogans*; *Pseudomonas*, particularly *Pseudomonas aeruginosa*; yeast, particularly *Saccharomyces*, preferably *Saccharomyces cerevisiae*. Preferred O-acetyl-L-homoserine hydrogen sulfide hydrolases suitable for the method according to the invention can be derived from *Corynebacterium*. More preferably *Corynebacterium glutamicum* or *Corynebacterium terpenoides*, even more preferably *Corynebacterium glutamicum*, even more preferably *Corynebacterium glutamicum* ATCC 13032.

[0087] The O-succinyl-L-homoserine hydrogen sulfide hydrolase suitable for the method according to the invention can be derived from *Rhizobium* spp., particularly *Rhizobium spp.*; *Chromobacterium* spp., particularly *Chromobacterium violaceum*; *Hydromycota* spp., particularly *Hydromycota spp.*; *Methylobacillus* spp., particularly *Methylobacillus flagellates*; *Methylcoccus* spp., particularly *Methylcoccus capsulatum*; *Nitrosomonas* spp., particularly *Nitrosomonas cerevisiae*; *Nocardia* spp., *Nocardia glandulae*; *Pseudomonas* spp., particularly *Pseudomonas aeruginosa* and *Pseudomonas putida*; and *Rhodophyton* spp., particularly *Rhodophyton floccosum*.

[0088] The preferred O-succinyl-L-homoserine hydrogen sulfide hydrolase suitable for the method according to the invention can be derived from the genus *Chromobacterium*, particularly *Chromobacterium violaceum*; or the genus *Micromonas*, particularly *Micromonas chrysogenus*. Even more preferably, it can be derived from *Chromobacterium violaceum* ATCC 12472 or *Micromonas chrysogenus* ATCC 15444.

[0089] The cystathionine γ-synthase that can be used in the method according to the present invention can be a cystathionine γ-synthase classified in EC class EC 2.5.1.48.

[0090] These enzymes are PMP-dependent, as described, for example, by T. Clausen, R. Huber, L. Prade, MCWahl and A. Messerschmidt, The EMBO Journal 1998, 17, 6827-6838.

[0091] The cystathionine γ-synthase suitable for the method according to the invention can be derived from Escherichia coli, particularly Escherichia coli; Corynebacterium, particularly Corynebacterium glutamicum; Klebsiella, particularly Klebsiella pneumoniae; Bacillus, particularly Bacillus subtilis; Shigella, particularly Shigella flexneri; Colwellella, particularly Colwellella chilli; Salmonella, particularly Salmonella enterica.

[0092] The sequences can be obtained from databases such as the Braunschweig Enzyme Database (BRENDA, Germany, available for download at www.brenda-enzymes.org / index.php), the National Center for Biotechnological Information (NCBI, available for download at https: / / www.ncbi.nlm.nih.gov / ), or the Kyoto Encyclopedia of Genes and Genomes (KEGG, Japan, available for download at www.https: / / www.genome.jp / kegg / ).

[0093] Table 1 below provides preferred examples of hydrogen sulfide hydrolases and cystathionine γ-synthases that can be used in step (a) of the method according to the invention. Genes encoding hydrogen sulfide hydrolases are designated as “metY”, “met17”, and “Met17” for O-acetyl-L-homoserine hydrogen sulfide hydrolases (“AHS”), and as “metZ” for O-succinyl-L-homoserine hydrogen sulfide hydrolases (“SHS”). Genes encoding cystathionine γ-synthase (“CGS”) are labeled as “metB” and “metI”.

[0094] Table 1

[0095]

[0096]

[0097] In a preferred embodiment of the method of the present invention, the reaction in step (a) is catalyzed by at least one enzyme selected from hydrogen sulfide hydrolase E1 and cystathionine γ-synthase E2, wherein the polypeptide sequence of hydrogen sulfide hydrolase E1 is selected from:

[0098] -O-acetylhomoserine hydrogen sulfide hydrolase selected from variants of SEQ ID NO: 2 and SEQ ID NO: 2, variants of SEQ ID NO: 6 and SEQ ID NO: 6, variants of SEQ ID NO: 19 and SEQ ID NO: 19, variants of SEQ ID NO: 22 and SEQ ID NO: 22, variants of SEQ ID NO: 23 and SEQ ID NO: 23, and

[0099] -O-succinylhomoserine hydrogen sulfide hydrolase selected from variants of SEQ ID NO: 10 and SEQ ID NO: 10, variants of SEQ ID NO: 14 and SEQ ID NO: 14, variants of SEQ ID NO: 18 and SEQ ID NO: 18, variants of SEQ ID NO: 20 and SEQ ID NO: 20, variants of SEQ ID NO: 24 and SEQ ID NO: 24, variants of SEQ ID NO: 25 and SEQ ID NO: 25, variants of SEQ ID NO: 26 and SEQ ID NO: 26, variants of SEQ ID NO: 27 and SEQ ID NO: 27, variants of SEQ ID NO: 28 and SEQ ID NO: 28, and variants of SEQ ID NO: 34 and SEQ ID NO: 34.

[0100] -And the polypeptide sequence of cystathionine γ-synthetase E2 is selected from SEQ ID NO: 17 and its variants, SEQ ID NO: 21 and its variants, SEQ ID NO: 29 and its variants, SEQ ID NO: 30 and its variants, SEQ ID NO: 31 and its variants, SEQ ID NO: 32 and its variants, SEQ ID NO: 33 and its variants.

[0101] In a more preferred embodiment of the method of the present invention, the reaction in step (a) is selected from those catalyzed by hydrogen sulfide hydrolase E1:

[0102] O-acetylhomoserine hydrogen sulfide hydrolase selected from variants of SEQ ID NO: 2 and SEQ ID NO: 2, variants of SEQ ID NO: 6 and SEQ ID NO: 6, variants of SEQ ID NO: 19 and SEQ ID NO: 19, variants of SEQ ID NO: 22 and SEQ ID NO: 22, and variants of SEQ ID NO: 23 and SEQ ID NO: 23, and

[0103] O-succinylhomoserine hydrogen sulfide hydrolase selected from variants of SEQ ID NO: 10 and SEQ ID NO: 10, variants of SEQ ID NO: 14 and SEQ ID NO: 14, variants of SEQ ID NO: 18 and SEQ ID NO: 18, variants of SEQ ID NO: 20 and SEQ ID NO: 20, variants of SEQ ID NO: 24 and SEQ ID NO: 24, variants of SEQ ID NO: 25 and SEQ ID NO: 25, variants of SEQ ID NO: 26 and SEQ ID NO: 26, variants of SEQ ID NO: 27 and SEQ ID NO: 27, variants of SEQ ID NO: 28 and SEQ ID NO: 28, and variants of SEQ ID NO: 34 and SEQ ID NO: 34.

[0104] In a more preferred embodiment of the method of the present invention, the reaction in step (a) is catalyzed by O-acetylhomoserine hydrogen sulfide hydrolase E1, wherein the O-acetylhomoserine hydrogen sulfide hydrolase is selected from variants of SEQ ID NO: 2 and SEQ ID NO: 2, variants of SEQ ID NO: 6 and SEQ ID NO: 6, and the O-succinylhomoserine hydrogen sulfide hydrolase is selected from variants of SEQ ID NO: 10 and SEQ ID NO: 10, variants of SEQ ID NO: 14 and SEQ ID NO: 14.

[0105] In a more preferred embodiment of the method of the present invention, the reaction in step (a) is catalyzed by hydrogen sulfide hydrolase E1 selected from O-acetylhomoserine hydrogen sulfide hydrolase selected from SEQ ID NO: 2 and SEQ ID NO: 6 and O-succinylhomoserine hydrogen sulfide hydrolase selected from SEQ ID NO: 10 and SEQ ID NO: 14.

[0106] The term "variant" is further explained below (section 4.3.3.1). In the context of this application, it should be understood to refer to a polypeptide sequence that has at least 80% sequence identity with the corresponding polypeptide sequence.

[0107] 4.3.2 Methods for obtaining enzymes

[0108] The enzymes that can be used in the methods of the present invention can be synthesized by methods known to those skilled in the art.

[0109] One approach is to express the enzyme in microorganisms such as *E. coli*, *Saccharomyces cerevisiae*, and *Pichia pastoris*, and then add the whole cell as a whole-cell biocatalyst to the reaction. Another approach is to express the enzyme, lyse the microorganism, and add the cell lysate. Yet another approach is to purify or partially purify the enzyme from the lysate and add the pure or partially purified enzyme to the reaction. If a reaction requires multiple enzymes, these enzymes can be expressed in one or more microorganisms, including expressing all enzymes in a single microorganism.

[0110] For example, a technician can obtain the enzyme according to the invention, such as that described in DE 100 31 999A1, by expressing, particularly overexpressing (hereinafter, "expression, particularly overexpression" is abbreviated as (over)expression) and subsequently isolating it in cells. For example, free plasmids are used to increase the expression of their respective genes. In such plasmids, the nucleic acid molecule to be (over)expressed or encoding the polypeptide or enzyme to be (over)expressed can be placed under the control of a strongly inducible promoter, such as the lac promoter, located upstream of the gene. The promoter is a DNA sequence of about 40 to 50 base pairs that constitutes the binding site of the RNA polymerase holoenzyme and the transcription start point (M. Pátek, J. Holátko, T. Busche, J. Kalinowski, J. (Microbial Biotechnology 2013, 6, 103-117), thereby influencing the expression intensity of controlled polynucleotides or genes. "Functional linkage" is obtained through the sequential arrangement of promoters and genes, which leads to gene transcription.

[0111] Suitable strong promoters for increasing expression or methods for generating such promoters are known from the literature (e.g., S. Lisser & H. Margalit, Nucleic Acid Research 1993, 21, 1507-1516; M. Patèk and J. Nesvera in H. Yukawa and M. Inui (eds.), Corynebacterium glutamicum, Microbiology Monographs 23, Springer Verlag Berlin Heidelberg 2013, 51-88; BJ Eikmanns, E. Kleinertz, W. Liebl, H. Sahm, Gene 1991, 102, 93-98). For example, natural promoters can be optimized by altering promoter sequences in directions with known common sequences to increase the expression of genes functionally linked to these promoters (M. Pátek, BJ Eikmanns, J. Pátek, H. Sahm, Microbiology 1996, 142, 1297-1309; M. Pátek, J. Holátko, T. Busche, J. Kalinowski, J. Microbial Biotechnology 2013,6,103-117).

[0112] Constitutive promoters are also suitable for (over)expression, where the gene encoding enzyme activity is expressed sequentially under the control of promoters such as glucose-dependent deo promoters. Chemically induced promoters are also suitable, such as tac, lac, or trp. The most widely used system for inducing promoters is the lac operon of E. coli. In this case, lactose or isopropyl β-D-thiogalactopyranoside (IPTG) is used as an inducer. In addition, systems using arabinose (e.g., the pBAD system) or rhamnose (e.g., E. coli KRX) as inducers are common. Systems used for physical induction include, for example, the E. coli cspA promoter based on Takara or Lambda PL and the temperature-induced cold shock promoter system based on osmB (e.g., WO 95 / 25785A1), an osmotically inducible promoter.

[0113] Suitable plasmids or vectors are, in principle, all embodiments available to those skilled in the art for this purpose. Prior art describes standard plasmids that can be used for this purpose, such as the pET system vectors exemplified by pET-3a or pET-26b(+) (commercially available from Novagen). Other plasmids and vectors are available from brochures of companies such as Novagen, Promega, New England Biolabs, Clontech, or Gibco BRL. Further optimized plasmids and vectors can be found in the following literature: Glover, DM (1985) DNA cloning: a practical approach, Vol. I-III, IRL Press Ltd., Oxford; Rodriguez, R.L. and Denhardt, DT (eds) (1988) Vectors: a survey of molecular cloning vectors and their uses, 179-204, Butterworth, Stoneham; Goeddel, DV (1990) Systems for heterologous gene expression, Methods Enzymol. 185, 3-7; Sambrook, J.; Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York.

[0114] The plasmid vector containing the gene to be amplified is then transformed into the desired strain, for example, by conjugation or transformation. Conjugation methods include, for example, A. The transformation method is described, for example, in J. Kalinowski, A. Pühler, *Applied and Environmental Microbiology*, 1994, 60, 756-759. It is also described, for instance, in G. Thierbach, A. Schwarzer, A. Pühler, *Applied Microbiology and Biotechnology*, 1988, 29, 356-362; LK Dunican & E. Shivnan, *Bio / Technology*, 1989, 7, 1067-1070; and A. Tauch, O. Kirchner, L. Wehmeier, J. Kalinowski, A. Pühler, *FEMS Microbiology Letters*, 1994, 123, 343-347. Following homologous recombination via a "crossover" event, the resulting strain contains copies of at least two related genes.

[0115] The desired enzyme can be isolated by lysing cells containing the desired activity in a manner known to those skilled in the art, for example by means of a ball mill, Freund's crusher, or an ultrasonic grinder, followed by separation of cells, cell debris, and lysis aids, such as glass beads, by centrifugation at 13,000 rpm and 4 °C for 10 minutes. The resulting cell-free crude extract can then be used for enzyme analysis and subsequent LC-ESI-MS detection of the product. Alternatively, the enzyme can be enriched or purified to homogeneity by chromatographic methods (e.g., nickel-nitrilotriacetic acid affinity chromatography, streptavidin affinity chromatography, gel filtration chromatography, or ion exchange chromatography) in a manner known to those skilled in the art.

[0116] Whether a nucleic acid or peptide is (over)expressed can be determined by quantitative PCR (for nucleic acid molecules), SDS-PAGE, Western blotting, or comparative activity assays (for peptides). Genetic modifications can target transcription, translation, and / or post-translational modifications, resulting in changes in enzyme activity and / or selectivity under selected and / or identified culture conditions.

[0117] 4.3.3 Definition

[0118] 4.3.3.1 “Variants”

[0119] In the context of this invention, the term "variant" in relation to a polypeptide sequence refers to a polypeptide sequence that has at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identity with the reference sequence. In a further specific embodiment, the degree of similarity is at least 98.0%, more preferably at least 98.2%, more preferably at least 98.4%, more preferably at least 98.6%, more preferably at least 98.8%, more preferably at least 99.0%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, or at least more preferably at least 99.9%. It goes without saying that a "variant" of a certain polypeptide sequence is not identical to that polypeptide sequence.

[0120] This variant can be prepared by introducing deletions, insertions, substitutions, or combinations thereof, particularly in the amino acid sequence, as well as by incorporating fusions of such macromolecules or variants thereof.

[0121] Those skilled in the art will know that modifications to amino acid residues in a given polypeptide sequence do not result in significant modifications to the properties and functions of the given polypeptide. Therefore, for example, many amino acids can often be interchanged with each other without any problems; examples of such suitable amino acid substitutions are: Ala replaced by Ser; Arg replaced by Lys; Asn replaced by Gln or His; Asp replaced by Glu; Cys replaced by Ser; Gln replaced by Asn; Glu replaced by Asp; Gly replaced by Pro; His replaced by Asn or Gln; Ile replaced by Leu or Val; Leu replaced by Met or Val; Lys replaced by Arg, Gln, or Glu; Met replaced by Leu or Ile; Phe replaced by Met, Leu, or Tyr; Ser replaced by Thr; Thr replaced by Ser; Trp replaced by Tyr; Tyr replaced by Trp or Phe; Val replaced by Ile or Leu. It is also known that modifications, particularly at the N- or C-terminus of a polypeptide, in the form of, for example, amino acid insertions or deletions, generally have no significant effect on the function of the polypeptide.

[0122] Based on this, preferred variants of the invention according to any one of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33 have polypeptide sequences containing functions essential to the said sequences, such as catalytic activity of the protein, or the folding or structure of the protein. Other amino acids may be deleted, substituted, or inserted, or essential amino acids may be substituted in a conserved manner to maintain enzyme activity, particularly the activity of hydrogen sulfide hydrolase or cystathionine γ-synthase.

[0123] 4.3.3.2 "Sequence Identities"

[0124] Those skilled in the art will know that various computer programs can be used to calculate the similarity or identity between two nucleotide or amino acid sequences.

[0125] The preferred method for determining identity initially produces the largest possible alignment between the sequences to be compared. Computer programs for determining identity include, but are not limited to, the GCG package, which includes:

[0126] -GAP [J. Deveroy et al., Nucleic Acid Research 1984, 12, page 387, Genetics Computer Group University of Wisconsin, Medicine (WI)], and

[0127] -BLASTP, BLASTN and FASTA (S. Altschul et al., Journal of Molecular Biology 1990, 215, 403-410). This BLAST procedure can be obtained from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Handbook, S. Altschul et al., NCBI NLM NIH Bethesda ND 22894; S. Altschul et al., see above).

[0128] For example, the percentage identity between two amino acid sequences can be determined using the GAP program, which is already integrated into the GCG software package, SBNeedleman and CDWunsch, J.Mol.Biol.1970,48,443-453, using either the BLOSUM62 or PAM250 matrix with interstitial weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. Those skilled in the art will recognize that using different parameters will lead to slightly different results, but the percentage identity between the two amino acid sequences will not differ significantly overall. The BLOSUM62 matrix is ​​typically applied with the default settings (interstitial weight: 12, length weight: 1).

[0129] In the context of this invention, according to the above algorithm, 80% sequence similarity means 80% homology. This also applies to higher similarities.

[0130] Most preferably, in the context of this invention, the degree of similarity between sequences is determined by the procedure "Needle" using a substitution matrix BLOSUM62, a gap opening penalty of 10, and a gap expansion penalty of 0.5. The Needle procedure performs the global alignment described in SB Needleman and CD Wunsch, J. Mol. Biol. 1970, 48, 443-453. The substitution matrix used according to this invention is BLOSUM62, the gap opening penalty is 10, and the gap expansion penalty is 0.5. The preferred version used in the context of this invention is the version proposed by F. Madeira, YMPark, J. Lee, N. Buso, T. Gur, N. Madhusoodanan, P. Basutkar, ARN Tivey, SCPotter, RDBinn, Nucleic Acids Research 2019, 47, W636–W641, Web Server issue (accessible online as of March 31, 2021 via https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ).

[0131] In one specific implementation, the percentage of similarity between the amino acid sequence of the polypeptide and the reference polypeptide sequence is determined by: i) aligning the two amino acid sequences using the Needle program with a BLOSUM62 replacement matrix, with a vacancy opening penalty of 10 and a vacancy expansion penalty of 0.5; ii) calculating the number of exact matches in the alignment; iii) dividing the number of exact matches by the length of the longest of the two amino acid sequences; and iv) converting the result of the division in iii) into a percentage.

[0132] 4.3.3.3 "Preferred assay method for identifying particularly active variants"

[0133] 4.3.3.3.1 Determination of A

[0134] In the context of this invention, any particularly preferred polypeptide variant of any one of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33 can be identified by a person skilled in the art as a polypeptide variant that exhibits activity in the following analysis (“Assay A”).

[0135] Determination of A is performed through the following steps:

[0136] A1) First, the activity of the variant to be tested is determined through the following steps: A1.1), A1.2), and A1.3):

[0137] A1.1) Prepare a 990 μl reaction solution containing phosphate buffer (0.1 M, pH 7.5), 3 mM O-acetyl-L-homoserine-HCl, 10 μM pyridoxal 5' phosphate monohydrate and 1.0 nmol of the test peptide, and heat to 50 °C.

[0138] A1.2) The reaction was initiated by adding 10 μl of a 200 mM butyl methylphosphonate solution (MPBE, CAS#6172-80-1). The concentration of MPBE in the reaction solution was 2 mM.

[0139] A1.3) After adding MPBE, the reaction was carried out at 50°C for 120 minutes. Then, the reaction was stopped by adding 10 μl of 1% formate solution and cooling on ice.

[0140] A2) Then, perform a blank test using the following steps: A2.1), A2.2), and A2.3):

[0141] A2.1) Prepare a 990 μl reaction solution containing phosphate buffer (0.1 M, pH 7.5), 3 mM O-acetyl-L-homoserine-HCl, and 10 μM pyridoxal 5' phosphate monohydrate, and heat to 50 °C.

[0142] A2.2) The reaction was initiated by adding 10 μl of a 200 mM butyl methylphosphonate solution (MPBE, CAS#6172-80-1).

[0143] A2.3) After adding MPBE, the reaction was carried out at 50°C for 120 minutes. Then, the reaction was stopped by adding 10 μl of 1% formate solution and cooling on ice.

[0144] A3) Finally, preferably by LC-MS analysis as described under section 5.7, the butyl phosphate of L-GA obtained in the reaction solutions of A1.3) and A2.3) respectively (i.e., the compound according to formula (III), wherein R) is determined and compared. 1 = The amount of n-butyl (in moles).

[0145] A4) If the amount of L-GA butyl phosphate determined in A1.3) is greater than the amount determined in A2.3), then the variant to be tested shows activity in assay A.

[0146] If the amount of L-GA butyl phosphate determined in A1.3) is equal to or less than the amount determined in A2.3), the variant to be tested does not show activity in assay A.

[0147] In steps A1.3) and A2.3), the preferred formate solution is an ammonium formate or sodium formate solution. Alternatively, the reaction in steps A1.3) and A2.3) can also be stopped by adding methanol, preferably 1 ml of methanol.

[0148] 4.3.3.3.2 Determination of B

[0149] In a further assay (“Assay B”), the activity of any one of the peptide variants of the peptide, namely SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33, can be determined.

[0150] The determination of B is carried out through the following steps:

[0151] B1) First, the activity of the “standard” peptide standard (i.e., a peptide sequence selected from SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33) is determined by the following steps B1.1, B1.2), and B1.3):

[0152] B1.1) Prepare a 990 μl reaction solution containing phosphate buffer (0.1 M, pH 7.5), 3 mM O-acetyl-L-homoserine-HCl, 10 μM pyridoxal 5' phosphate monohydrate and 1.0 nmol of the test "standard" peptide, and heat to 50 °C.

[0153] B1.2) The reaction was initiated by adding 10 μl of a 200 mM butyl methylphosphonate solution (MPBE, CAS#6172-80-1). The concentration of MPBE in the reaction solution was 2 mM.

[0154] B1.3) After adding MPBE, the reaction was carried out at 50°C for 120 minutes. Then, the reaction was stopped by adding 10 μl of 1% formate solution and cooling on ice.

[0155] B2) Then, repeat steps B1.1), B1.2), and B1.3) using the variants:

[0156] B2.1) Prepare a 990 L reaction solution containing phosphate buffer (0.1 M, pH 7.5), 3 mM O-acetyl-L-homoserine-HCl, 10 μM pyridoxal 5' phosphate monohydrate, and 1.0 nmol of the test "variant" peptide, and heat to 50 °C.

[0157] B2.2) The reaction was initiated by adding 10 μl of a 200 mM butyl methylphosphonate solution (MPBE, CAS#6172-80-1). The concentration of MPBE in the reaction solution was 2 mM.

[0158] B2.3) After adding MPBE, the reaction was carried out at 50°C for 120 minutes. Then, the reaction was stopped by adding 10 μl of 1% formate solution and cooling on ice.

[0159] B3) Finally, preferably by LC-MS analysis as described under section 5.7, the butyl phosphate of L-GA obtained in the reaction solutions of B1.3) and B2.3) (i.e., compound of formula (III), wherein R) is determined and compared. 1 = the amount of n-butyl).

[0160] B4) Then, divide the amount (in moles) of butyl phosphate of L-GA obtained in B2.3) by the amount (in moles) of butyl phosphate of L-GA obtained in B1.3). Then multiply this ratio by a factor of 100 to give the percentage of relative activity of the variant peptide relative to the "standard" peptide.

[0161] In steps B1.3) and B2.3), the preferred formate solution is an ammonium formate or sodium formate solution. Alternatively, the reaction in steps B1.3) and B2.3) can also be stopped by adding methanol, preferably 1 ml of methanol.

[0162] 4.3.3.4 "Preferred Variants"

[0163] 4.3.3.4.1 "Variants of SEQ ID NO:2"

[0164] In particular, the variant of SEQ ID NO:2 is a polypeptide having ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity with the polypeptide sequence SEQ ID NO:2.

[0165] The preferred variant of SEQ ID NO: 2 showed activity in assay A under section 4.3.3.3.1.

[0166] Even more preferably, the activity of the corresponding variant of SEQ ID NO:2 relative to the activity of SEQ ID NO:2 determined in determination B under 4.3.3.3.2 is at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%.

[0167] Even more preferably, the activity of the corresponding variant of SEQ ID NO:2 relative to the activity of SEQ ID NO:2 determined in determination B under 4.3.3.3.2 is in the range of 1 to 1000%, preferably in the range of 5 to 500%, more preferably in the range of 10 to 400%, more preferably in the range of 40 to 200%, more preferably in the range of 50 to 150%, more preferably in the range of 60 to 140%, more preferably in the range of 70 to 130%, more preferably in the range of 80 to 120%, more preferably in the range of 90 to 110%, more preferably 100%.

[0168] 4.3.3.4.2 "Variants of SEQ ID NO:6"

[0169] In particular, the variant of SEQ ID NO: 6 is a polypeptide having ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity with the polypeptide sequence SEQ ID NO: 6.

[0170] The preferred variant of SEQ ID NO: 6 showed activity in assay A under section 4.3.3.3.1.

[0171] Even more preferably, the activity of the corresponding variant of SEQ ID NO: 6 relative to the activity of SEQ ID NO: 6 determined in determination B under 4.3.3.3.2 is at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%.

[0172] Even more preferably, the activity of the corresponding variant of SEQ ID NO: 6 relative to the activity of SEQ ID NO: 6 determined in determination B under 4.3.3.3.2 is in the range of 1 to 1000%, preferably in the range of 5 to 500%, more preferably in the range of 10 to 400%, more preferably in the range of 40 to 200%, more preferably in the range of 50 to 150%, more preferably in the range of 60 to 140%, more preferably in the range of 70 to 130%, more preferably in the range of 80 to 120%, more preferably in the range of 90 to 110%, more preferably 100%.

[0173] 4.3.3.4.3 "Variants of SEQ ID NO:10"

[0174] In particular, the variant of SEQ ID NO: 10 is a polypeptide having ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity with the polypeptide sequence SEQ ID NO: 10.

[0175] The preferred variant of SEQ ID NO: 10 shows activity in assay A under section 4.3.3.3.1.

[0176] Even more preferably, the activity of the corresponding variant of SEQ ID NO: 10 relative to the activity of SEQ ID NO: 10 determined in determination B under 4.3.3.3.2 is at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%.

[0177] Even more preferably, the activity of the corresponding variant of SEQ ID NO: 10 relative to the activity of SEQ ID NO: 10 determined in determination B under 4.3.3.3.2 is in the range of 1 to 1000%, preferably in the range of 5 to 500%, more preferably in the range of 10 to 400%, more preferably in the range of 40 to 200%, more preferably in the range of 50 to 150%, more preferably in the range of 60 to 140%, more preferably in the range of 70 to 130%, more preferably in the range of 80 to 120%, more preferably in the range of 90 to 110%, more preferably 100%.

[0178] 4.3.3.4.4 "Variants of SEQ ID NO:14"

[0179] In particular, the variant of SEQ ID NO: 14 is a polypeptide having ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity with the polypeptide sequence SEQ ID NO: 14.

[0180] The preferred variant of SEQ ID NO: 14 showed activity in assay A under section 4.3.3.3.1.

[0181] Even more preferably, the activity of the corresponding variant of SEQ ID NO: 14 relative to the activity of SEQ ID NO: 14 as determined in assay B under 4.3.3.3.2 is at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%.

[0182] Even more preferably, the activity of the corresponding variant of SEQ ID NO: 14 relative to the activity of SEQ ID NO: 14 determined in determination B under 4.3.3.3.2 is in the range of 1 to 1000%, preferably in the range of 5 to 500%, more preferably in the range of 10 to 400%, more preferably in the range of 40 to 200%, more preferably in the range of 50 to 150%, more preferably in the range of 60 to 140%, more preferably in the range of 70 to 130%, more preferably in the range of 80 to 120%, more preferably in the range of 90 to 110%, and more preferably in the range of 100%.

[0183] 4.3.3.4.5 Other variants

[0184] Preferred variants of any one of SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33 may be determined by making corresponding adjustments to SEQ ID NO:2 as described in section 4.3.3.4.1.

[0185] Preferred variants of SEQ ID NO:19 can be determined by the procedure that occurs when the word “SEQ ID NO:2” is replaced by the word “SEQ ID NO:19” in the description of item 4.3.3.4.1.

[0186] Preferred variants of SEQ ID NO:22 can be determined by the procedure that occurs when the word “SEQ ID NO:2” is replaced by the word “SEQ ID NO:22” in the description of item 4.3.3.4.1.

[0187] Preferred variants of SEQ ID NO:23 can be determined by the procedure that occurs when the word “SEQ ID NO:2” is replaced by the word “SEQ ID NO:23” in the description of item 4.3.3.4.1.

[0188] Preferred variants of SEQ ID NO:18 can be determined by the procedure that occurs when the word “SEQ ID NO:2” is replaced by the word “SEQ ID NO:18” in the description of item 4.3.3.4.1.

[0189] Preferred variants of SEQ ID NO:20 can be determined by the procedure that occurs when the word “SEQ ID NO:2” is replaced by the word “SEQ ID NO:20” in the description of item 4.3.3.4.1.

[0190] Preferred variants of SEQ ID NO:24 can be determined by the procedure that occurs when the word “SEQ ID NO:2” is replaced by the word “SEQ ID NO:24” in the description of item 4.3.3.4.1.

[0191] Preferred variants of SEQ ID NO:25 can be determined by the procedure that occurs when the word “SEQ ID NO:2” is replaced by the word “SEQ ID NO:25” in the description of item 4.3.3.4.1.

[0192] Preferred variants of SEQ ID NO:26 can be determined by the procedure that occurs when the word “SEQ ID NO:2” is replaced by the word “SEQ ID NO:26” in the description of item 4.3.3.4.1.

[0193] Preferred variants of SEQ ID NO:27 can be determined by the procedure that occurs when the word “SEQ ID NO:2” is replaced by the word “SEQ ID NO:27” in the description of item 4.3.3.4.1.

[0194] Preferred variants of SEQ ID NO:28 can be determined by the procedure that occurs when the word “SEQ ID NO:2” is replaced by the word “SEQ ID NO:28” in the description of item 4.3.3.4.1.

[0195] Preferred variants of SEQ ID NO:34 can be determined by the procedure that occurs when the word “SEQ ID NO:2” is replaced by the word “SEQ ID NO:34” in the description of item 4.3.3.4.1.

[0196] Preferred variants of SEQ ID NO:17 can be determined by the procedure that occurs when the word “SEQ ID NO:2” is replaced by the word “SEQ ID NO:17” in the description of item 4.3.3.4.1.

[0197] Preferred variants of SEQ ID NO:21 can be determined by the procedure that occurs when the word “SEQ ID NO:2” is replaced by the word “SEQ ID NO:21” in the description of item 4.3.3.4.1.

[0198] Preferred variants of SEQ ID NO:29 can be determined by the procedure that occurs when the word “SEQ ID NO:2” is replaced by the word “SEQ ID NO:29” in the description of item 4.3.3.4.1.

[0199] Preferred variants of SEQ ID NO:30 can be determined by the procedure that occurs when the word “SEQ ID NO:2” is replaced by the word “SEQ ID NO:30” in the description of item 4.3.3.4.1.

[0200] Preferred variants of SEQ ID NO:31 can be determined by the procedure that occurs when the word “SEQ ID NO:2” is replaced by the word “SEQ ID NO:31” in the description of item 4.3.3.4.1.

[0201] Preferred variants of SEQ ID NO:32 can be determined by the procedure that occurs when the word “SEQ ID NO:2” is replaced by the word “SEQ ID NO:32” in the description of item 4.3.3.4.1.

[0202] Preferred variants of SEQ ID NO:33 can be determined by the procedure that occurs when the word “SEQ ID NO:2” is replaced by the word “SEQ ID NO:33” in the description of item 4.3.3.4.1.

[0203] 4.4 Method Conditions

[0204] The reaction in step a) of the method according to the invention can be carried out under conditions known to a person skilled in the art.

[0205] Activated L-homoserine H A The reaction medium that reacts with the substrate S is preferably aqueous, and more preferably an aqueous buffer.

[0206] Exemplary buffers commonly used in biotransformation reactions and advantageously used herein include Tris, phosphates, or any Good's buffers such as 2-(N-morpholino)ethanesulfonic acid (“MES”), N-(2-acetamido)iminodiacetic acid (“ADA”), piperazine-N,N'-bis(2-ethanesulfonic acid) (“PIPES”), N-(2-acetamido)-2-aminosulfonic acid (“ACES”), p-hydroxy-4-morpholinopropanesulfonic acid (“MOPSO”), and cholochoamine. chloride), 3-(N-morpholino)propanesulfonic acid (“MOPS”), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (“BES”), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl]amino]ethanesulfonic acid (“TES”), 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (“HEPES”), 3-(bis(2-hydroxyethyl)amino)-2-hydroxypropane-1-sulfonic acid (“DIPSO”), acetamide glycine, 3-(N-tris(hydroxymethyl) Methylamino(-2-hydroxypropane)sulfonic acid (“TAPSO”), piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) (“POPSO”), 4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid) (“HEPPSO”), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (“HEPPS”), trimethylglycine, glycine, dihydroxyethylglycine, or 3-[[1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl]amino]prop-1-sulfonic acid (“TAPS”).

[0207] In some implementations, ammonium can be used as a buffer. One or more organic solvents may also be added to the reaction.

[0208] Preferably, step a) of the method according to the invention is carried out in a phosphate buffer solution.

[0209] The pH of the reaction medium in step a) of this method is preferably in the range of 2 to 10, more preferably in the range of 5 to 8, and most preferably 7.5.

[0210] The method according to the invention is preferably carried out in the range of 20°C to 70°C, more preferably in the range of 30°C to 55°C, and most preferably in the range of 50°C.

[0211] 4.5L-Glufosinate-ammonium phosphate

[0212] The product of the method according to the present invention is a compound having the following structure (III):

[0213]

[0214] The compound based on structure (III) is L-glufosinate (for R... 1=H) or L-glufosinate phosphate (for R 1 =alkyl, alkenyl, alkynyl, hydroxyalkyl, or aryl).

[0215] According to compounds with structure (III), where R 1 = n-Butyl, abbreviated as "L-GA-Bu" or "LGA-Bu".

[0216] This person skilled in the art understands that, according to the compound with structure (III), residue R 1 The properties depend on residues R in the substrate structure (I). 1 Its characteristics.

[0217] If R 1 If it is hydrogen, then L-glufosinate is obtained directly in the method according to the present invention.

[0218] In a preferred embodiment, R 1 The compound (III) is L-glufosinate phosphate, selected from alkyl, alkenyl, alkynyl, hydroxyalkyl, aryl, preferably alkyl, more preferably methyl, ethyl, n-butyl.

[0219] In these embodiments, the method according to the invention further preferably includes a further step b), wherein the compound of structure (III) obtained in step (a) is saponified, wherein R 1 L-Glufosinate is obtained by selecting from alkyl, alkenyl, alkynyl, hydroxyalkyl, and aryl groups, preferably alkyl, and more preferably methyl, ethyl, and n-butyl groups.

[0220] This can be done using methods known to technicians.

[0221] Preferably, this saponification is carried out under acidic conditions, more preferably by mixing 1 volume of reaction medium containing the structure (III) compound obtained in step (a) with 4 volumes of 6N HCl for 2 hours and incubating the resulting mixture at a temperature of 50°C to 150°C, preferably at 100°C.

[0222] Alternatively, enzymatic saponification can be performed. 5. Examples

[0223] The ability of genes encoding hydrogen sulfide hydrolase from different sources (EC 2.5.1.- and EC 2.5.1.49) to react with active homoserine derivatives and different substrates according to structure (I) to form glufosinate derivatives was tested.

[0224] 5.1 Example 1: Identification of suitable enzymes and construction of plasmids

[0225] 5.1.1 Detected hydrogen sulfide hydrolase

[0226] Table 2 summarizes the reference details for the O-acetylhomoserine hydrogen sulfide hydrolase and O-succinylhomoserine hydrogen sulfide hydrolase genes used in the examples.

[0227] Table 2

[0228]

[0229] 5.1.2 Preparation of gene expression plasmids

[0230] 5.1.2.1 O-acetylhomoserine hydrogen sulfide hydrolase metY derived from Corynebacterium glutamicum

[0231] The metY gene, derived from Corynebacterium glutamicum ATCC 13032, encodes O-acetylhomoserine hydrogen sulfide hydrolase and can be found in the genomic sequence accessible under NCBI NC_003450 under the locus_tag NCgl0625.

[0232] To achieve the expression of this enzyme, the expression vector pET-26b(+)(Novagen EMD Millipore) was used. Therefore, the metY_Cg polynucleotide of SEQ ID NO: 3 was synthesized by GeneArt (ThermoFisher Scientific (Waltham, USA)).

[0233] To assemble the expression vector pET-26b(+)_metY-Cg, both the pET-26(+) and metY_Cg polynucleotides were treated with NdeI and NotI, ligated, and the ligation mixture was used to transform E. coli. The resulting vector pET-26b(+)_metY-Cg is shown below. Figure 1 As shown.

[0234] DNA of the expression vector pET-26b(+)_metY-Cg was isolated from the transformants.

[0235] 5.1.2.2 O-acetylhomoserine hydrogen sulfide hydrolase metY_P2T derived from Corynebacterium glutamicum

[0236] The O-acetylhomoserine hydrogen sulfide hydrolase variant metY_P2T is encoded by the polynucleotide of SEQ ID NO: 5, which is identical to the polynucleotide of SEQ ID NO: 1, except that the nucleic acid bases from position 4 to 6 are aca.

[0237] The polynucleotide of SEQ ID NO: 5 encodes a polypeptide containing the amino acid sequence of SEQ ID NO: 6, which is identical to the polynucleotide of SEQ ID NO: 1, except that the amino acid proline at position 2 is replaced by threonine.

[0238] To achieve expression of the enzyme variant metY_P2T, the expression vector pET-26b(+)(Novagen EMDMillipore) was used. Therefore, the metY_P2T polynucleotide of SEQ ID NO: 7 was synthesized by GeneArt (ThermoFisherScientific (Waltham, USA)).

[0239] To assemble the expression vector pET-26b(+)_metY_P2T, both the pET-26(+) and metY_P2T polynucleotides were treated with NdeI and NotI, ligated, and the ligation mixture was used to transform E. coli. The resulting vector pET-26b(+)_metY_P2T is shown below. Figure 2 As shown.

[0240] DNA of the expression vector pET-26b(+)_metY_P2T was isolated from the transformants.

[0241] 5.1.2.3 O-succinylhomoserine hydrogen sulfide hydrolase metZ derived from Chromobacterium violaceum

[0242] The metZ gene, derived from Chromobacterium violaceum ATCC 12472, encodes O-succinylhomoserine hydrogen sulfide hydrolase and can be found in the genomic sequence accessible under locus_tag CV_2725 at NCBI AE016825.

[0243] To achieve the expression of this enzyme, the expression vector pET-26b(+)(Novagen EMD Millipore) was used. Therefore, the metZ_Cv polynucleotide of SEQ ID NO: 11 was synthesized by GeneArt (ThermoFisher Scientific (Waltham, USA)).

[0244] To assemble the expression vector pET-26b(+)_metZ-Cv, both the pET-26(+) and metZ-Cv polynucleotides were treated with NdeI and XhoI, ligated, and then transformed into E. coli using a ligation mixture. The resulting vector pET-26b(+)_metZ-Cv is shown below. Figure 3 As shown.

[0245] DNA of the expression vector pET-26b(+)_metZ-Cv was isolated from the transformants.

[0246] 5.1.2.4 O-succinylhomoserine hydrogen sulfide hydrolase metZ derived from *Hylocereus undatus*

[0247] The metZ gene, derived from *Hylocereus uncinata* ATCC 15444, encodes O-succinylhomoserine hydrogen sulfide hydrolase and can be found in the genomic sequence accessible under locus_tag HNE_2672 at NCBI CP000158.

[0248] To achieve the expression of this enzyme, the expression vector pET-26b(+)(Novagen EMD Millipore) was used. Therefore, the metZ_Hn polynucleotide of SEQ ID NO: 15 was synthesized by GeneArt (ThermoFisher Scientific (Waltham, USA)).

[0249] To assemble the expression vector pET-26b(+)_metZ-Hn, both the pET-26(+) and metZ_Hn polynucleotides were treated with XbaI and XhoI, ligated, and then transformed into E. coli using a ligation mixture. The resulting vector pET-26b(+)_metZ-Hn is shown below. Figure 4 As shown.

[0250] DNA of the expression vector pET-26b(+)_metZ-Hn was isolated from the transformants.

[0251] 5.2 Transformation and Expression of Hydrogen Sulfide Hydrolase

[0252] These vectors carrying the hydrogen sulfide hydrolase gene were transformed into *Escherichia coli* BL21(DE3) (New England Biolabs), and subsequently cultured on LB agar plates containing 50 mg / L kanamycin at 37°C for 16 h. Strains of BL21 carrying the vector pET26b(+) without any inserted fragment were used as negative controls.

[0253] The strains were named Ec BL21 pET-26b(+)_metY-Cg, Ec BL21 pET-26b(+)_metY-Cg_P2T, Ec BL21 pET-26b(+)_metZ-Cv, Ec BL21 pET-26b(+)_metZ-Hn and Ec BL21 pET-26b(+).

[0254] In each case, one colony was selected and inoculated into 10 ml of LB medium containing 50 mg / L kanamycin, and cultured at 37°C and 250 rpm for 6 hours. Subsequently, 50 μl of LB medium was treated with 50 mg / L kanamycin, and 50 μl of the growing cell culture was inoculated and incubated at 28°C and 250 rpm for 16 hours. This cell culture was then diluted in a 2 L flask with 200 ml of fresh LB medium containing 50 μg / L kanamycin to an OD of 0.15, and cultured further under the same conditions until an OD of 0.5 (approximately 4 hours). The initiation of gene expression induction was then influenced by adding 200 μl of 300 mM IPTG stock solution (final concentration 300 μM isopropyl-β-D-thiogalactopyranoside (IPTG), Sigma-Aldrich, Germany). Induction was performed at 28°C and 250 rpm for 4 h, followed by harvesting of the culture (8 ml, normalized to OD=1). The supernatant was removed by centrifugation (20 min, 4000 rpm, 4°C). The cell pellet was washed twice with 800 μl of 0.1 M potassium phosphate buffer (pH 7.5) and maintained in 1 ml buffer. Mechanical cell digestion was performed using a FastPrep FP120 instrument (QBiogene, Heidelberg), where cells were agitated four times at 6.5 m / s for 30 s in a digestion vessel containing 300 mg of glass beads (0.2–0.3 mm). The crude extract was then centrifuged at 12000 rpm, 4°C, for 20 min to remove undigested cells and cell debris. The supernatant was used for enzyme analysis. The concentration of peptides in the supernatant was determined using SDS-PAGE and analyzed by software. (BiochemLabSolutions) analyzes the corresponding bands.

[0255] 5.3 Embodiments I1 to I4 of the present invention

[0256] The following assays were performed to determine whether the corresponding peptide catalyzes the reaction between the corresponding phosphorus-containing substrate and activated L-homoserine. O-acetyl-L-homoserine was used as the activated L-homoserine substrate.

[0257] To 880 μL of phosphate buffer (0.1 M, pH 7.5) containing 1 nmol of the analyte polpypetide, add 100 μL of 30 mM O-acetylhomoserine-HCl aqueous solution, 10 μL of 1 mM pyridoxal phosphate monohydrate aqueous solution (1 mM), and 10 μL of 200 mM butyl p-methylphosphonate aqueous solution (CAS No. 6172-80-1; "MPBE"). The reaction is carried out at 50 °C for 120 min. Then, 100 min fractions of the solution are diluted in 100 min methanol and applied to LC-MS QQQ (section 5.7) for LGA-Bu analysis.

[0258] 5.4 Embodiment I5 of the present invention

[0259] The catalytic activity of the peptide isolated from pET-26b(+)_metY-Cg_P2T was further tested in a reaction with methyl p-methylphosphonate (“MPME”; CAS No. 16391-06-3).

[0260] To 880 μL of phosphate buffer (0.1 M, pH 7.5) containing 1 nmol of this peptide, 100 μL of 30 mM acetylhomoserine-HCl aqueous solution, 10 μL of 1 mM pyridoxal 5'-phosphate monohydrate aqueous solution (1 mM), and 10 μL of 200 mM MPME aqueous solution were added. The reaction was carried out at 50 °C for 120 min. Then, 100 μL of the fractional solution was diluted in 100 μL of methanol and applied to LC-MS QQQ (section 5.7) for analysis of LGA-Bu.

[0261] 5.4 Comparative Examples C1 to C4

[0262] Further investigation was conducted to determine whether other phosphorus compounds could serve as substrates for the above reactions. For this purpose, diethyl sodium methyl phosphite (DEMP; CAS No. 15715-41-0) was tested.

[0263] To 880 μL of phosphate buffer (0.1 M, pH 7.5) containing 1 nmol of the analyte, add 100 μL of 30 mM acetylhomoserine-HCl aqueous solution, 10 μL of 1 mM pyridoxal 5'-phosphate monohydrate aqueous solution (1 mM), and 10 μL of 200 mM DEMP aqueous solution. The reaction is carried out at 50 °C for 120 min. Then, 100 μL of the fractional solution is diluted in 100 μL of methanol and applied to LC-MS QQQ (section 5.7) for LGA-Bu analysis.

[0264] 5.5 Comparative Examples C5 to C8

[0265] Further investigation was conducted to determine whether the peptides corresponding to the above reaction <1A> would catalyze the reaction of O-acetylhomoserine with methanethiol (“MM”; CAS No. 74-93-1).

[0266] To 880 μL of phosphate buffer (0.1 M, pH 7.5) containing 1 nmol of the analyte, add 100 μL of 30 mM acetylhomoserine-HCl aqueous solution, 10 μL of 1 mM pyridoxal 5'-phosphate monohydrate aqueous solution (1 mM), and 10 μL of 200 mM aqueous solution. The reaction is carried out at 50 °C for 120 min. Then, 100 μL of the fractional solution is diluted in 100 μL of methanol and analyzed by LC-MS QQQ (section 5.7) to determine the corresponding products.

[0267] In the cases of Examples I1 to I4 of the present invention, the product is a compound according to structure (III), wherein R 1 = n-Butyl (“LGA-Bu”).

[0268] In the case of Example I5 of the present invention, the product is a compound according to structure (III), wherein R 1 =Methyl.

[0269] In comparative examples C1 to C4, the product is a compound according to structure (III), wherein R 1 = Ethyl (“LGA-Et”).

[0270] In the case of comparative examples C5 to C6, the product is methionine.

[0271] Table 3 below summarizes these results. The abbreviations used are...

[0272] “DEMP” = methyldiethoxyphosphine (CAS No. 15715-41-0);

[0273] “MPBE” = p-methylphosphonic acid butyl ester (CAS No. 6172-80-1);

[0274] “MPME” = methyl para-methylphosphonate (CAS No. 16391-06-3);

[0275] “MM” = Methanethiol (CAS No. 74-93-1).

[0276] Table 3

[0277] Example enzymes polypeptide Substrate product I1 Cg_MetY SEQ ID NO:2 MPBE yes I2 Cg_MetY_P2T SEQ ID NO:6 MPBE yes I3 Cv_MetZ SEQ ID NO:10 MPBE yes I4 Hn_MetZ SEQ ID NO:14 MPBE yes I5 Cg_MetY_P2T SEQ ID NO:6 MPME yes C1 Cg_MetY SEQ ID NO:2 DEMP no C2 Cg_MetY_P2T SEQ ID NO:6 DEMP no C3 Cv_MetZ SEQ ID NO:10 DEMP no C4 Hn_MetZ SEQ ID NO:14 DEMP no C5 Cg_MetY SEQ ID NO:2 MM yes C6 Cg_MetY_P2T SEQ ID NO:6 MM yes C7 Cv_MetZ SEQ ID NO:10 MM yes C8 Hn_MetZ SEQ ID NO:14 MM yes

[0278] 5.6 Results

[0279] The results summarized in Table 3 surprisingly show that the tested peptides accept other substrate methylphosphonic acid compounds, such as MPBE and MPME, and catalyze their reaction with activated L-homoserine to the corresponding LGA phosphate n-butyl or methyl esters.

[0280] This discovery is even more surprising because technicians would not have expected these enzymes to catalyze these reactions, since other phosphate compounds such as DEMP do not function as substrates.

[0281] This discovery opens up new enzymatic pathways for LGA and its derivatives.

[0282] 5.7 Analytical Methods

[0283] All analytical measurements in the experiment were performed by scanning on an LC-MS QQQ system. The samples were diluted in methanol (v:v = 1:2).

[0284] The HPLC system used was an Agilent 1260 Infinity series, connected to a 6420 triple quadrupole mass spectrometer with electrospray ionization. Peak identification was performed in positive detection mode by retention time and molecular weight.

[0285] Data evaluation was conducted using peak area and zero-zero secondary calibration.

[0286] Detailed information on data collection methods

[0287] Ion source ESI (electrospray ionization)

[0288] Time parameters

[0289] Mass range m / z = 50-300

[0290] 400 scans per second

[0291] Dissociation 40V

[0292] Polar positive ions (scanning mode)

[0293] Source parameters

[0294] Gas temperature 350℃

[0295] airflow 12 l / min

[0296] 50psi sprayer

[0297] 4000V capillary tube

[0298] Binary pump

[0299] Injection volume 2.00 μL

[0300] Flow rate 0.60 mL / min

[0301] Solvent components

[0302] Solvent A: 100mM ammonium acetate plus 0.1% (v / v) formic acid in H2O

[0303] Solvent B: Acetonitrile containing 0.1% formic acid

[0304] The gradients are shown in Table 4.

[0305] Table 4

[0306] Time (min) Solvent A (%) Solvent B (%) 0.5 5 95 1.2 45 55 4.0 45 55 4.1 95 5 7.0 95 5 7.1 5 95 10.0 5 95

[0307] column

[0308] Type: Luna HILIC; 100 x 2 mm; 3 μm; Phenomenex 00D-4449-BO

[0309] Temperature 30.0℃

[0310] 6. Sequence Overview

[0311] The following table provides an overview of the DNA and protein sequences mentioned in this application:

[0312] The abbreviations used are: "AHS" = O-acetylhomoserine hydrogen sulfide hydrolase; "SHS" = O-succinylhomoserine hydrogen sulfide hydrolase; "CGS" = cystathionine γ-synthase.

[0313]

[0314]

[0315]

Claims

1. An enzyme-catalyzed method for producing L-glufosinate or its phosphate ester, comprising step (a), wherein activated L-homoserine H A Reacts with substrate S of structure (I) to produce a compound of structure (III). Where R 1 Selected from hydrogen, methyl, ethyl, n-butyl, And the activated L-homoserine H A Compounds with the following structure (II): Where R 2 It is a hydrocarbon group having 1 to 15 carbon atoms, which optionally includes at least one functional group selected from OH, COOH, and NH. Furthermore, the reaction in step (a) is catalyzed by hydrogen sulfide hydrolase. The polypeptide sequence of the hydrogen sulfide hydrolase is selected from SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10 and SEQ ID NO:

14.

2. The method according to claim 1, wherein the activated L-homoserine H A Selected from O-succinyl-L-homoserine and O-acetyl-L-homoserine.

3. The method according to claim 2, wherein the activated L-homoserine H A It is O-acetyl-L-homoserine.

4. The method according to any one of claims 1 to 3, further comprising step (b), wherein the compound of structure (III) obtained in step (a) is saponified to obtain L-glufosinate.

5. The method according to claim 1, wherein the hydrogen sulfide hydrolase is O-acetylhomoserine hydrogen sulfide hydrolase or O-succinylhomoserine hydrogen sulfide hydrolase.

6. The method according to any one of claims 1 to 3, wherein the activated L-homoserine H A By generating activated L-homoserine H A Preparation by fermentation of strains.

7. The method according to claim 6, wherein the strain producing activated L-homoserine HA is selected from Escherichia sp., Erwinia sp., Serratia sp., Providencia sp., Corynebacterium sp., Pseudomonas sp., Leptospira sp., Salmonella sp., Brevibacterium sp., Hypomononas sp., Chromobacterium sp., and Norcardia sp., and fungi.

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