Enzymatic methods for producing L-glufosinate and its phosphoesters

Abstract

The present invention relates to an enzyme-catalyzed method for producing L-glufosinate or its phosphate esters, which method comprises the step of reacting activated L-homoserine H A The method comprises reacting L-glufosinate (e.g., acetyl or succinyl L-homoserine) with a substrate S selected from methylphosphinic acid and esters of methylphosphinic acid. The sulfhydrylase enzyme E1 (aminocarboxypropyltransferase, EC 2.5.1) is used as the enzymatic catalyst. The present invention makes available new substrates for the enzymatic production of L-glufosinate and its phosphoesters.

Description

[Technical Field]

[0001] The present invention relates to an enzyme-catalyzed method for producing L-glufosinate ("L-GA" or "LGA") or a phosphoester thereof. The method comprises the step of converting activated L-homoserine H A with a substrate S selected from methylphosphinic acid and esters of methylphosphinic acid. The sulfhydrylase enzyme E1 is used as the enzymatic catalyst. The present invention makes available new substrates for the enzymatic production of L-glufosinate and its phosphoesters.

[0002] 1. Background technology Organophosphorus compounds, i.e. chemicals containing a carbon-phosphorus bond, are widely used as herbicides in the field of plant protection. Agents such as the herbicides glyphosate (Roundup®, Touchdown®) and glufosinate (Basta®, Liberty®) and the growth regulator glyphosine (Polaris®) are used for this purpose (for example, as described by G. Hoerlein, Rev. Environ. Contam. Toxicol. 1994, 138, 73-145).

[0003] Esters of p-methylphosphinic acid (e.g., p-methylphosphinic acid butyl ester; "MPBE", CAS number: 6172-80-1) play an important role as synthetic building blocks in the synthesis of the non-selective herbicide glufosinate. These esters are obtained via two basic synthetic routes (summarized in Figures 3a and 3b on page 130 of K. Haack's paper (Chem. Unserer Zeit 2003, 37, 128-138)): a. Diethylchlorophosphite [ClP(OC2H5)2] reacts with CH3MgCl to provide methyldiethoxyphosphine [H3CP(OC2H5)2; "DEMP"; CAS No. 15715-41-0], which can be partially hydrolyzed to give the corresponding methylphosphinic acid ethyl ester (MPEE; CAS No.: 16391-07-4). b. Alternatively, methane can be reacted with phosphorus trichloride at 500 °C to give methyldichlorophosphane H3CPCl2, which can be solvolyzed in alcohol to give the corresponding methylphosphinate.

[0004] Esters of p-methylphosphinic acid regioselectively add to carbon-carbon double bonds. This property is used to form a second phosphorus-carbon bond in the synthesis of glufosinate. For example, H3CPH(O)OR (R = alkyl) reacts with 1-cyanoallyl acetate in an addition reaction to give an intermediate. Subsequently, the acetate substituent is exchanged with ammonia, and the cyano group and the ester group of the phosphinic acid moiety are hydrolyzed to give glufosinate.

[0005] Acrylic acid esters are a cheaper alternative starting material. They can be reacted with esters of p-methylphosphinic acid to give alkyl 3-[alkoxy(methyl)phosphinyl]propionic acid esters. Claisen reaction of this diester with diethyl oxalate, hydrolysis, and decarboxylation affords the corresponding α-keto acid, which undergoes reductive amination to give glufosinate.

[0006] These and further synthetic routes for L-glufosinate are also described in the literature, for example in WO 1999 / 009039 and EP 0 508 296 A1.

[0007] WO 2020 / 145513 and WO 2020 / 145514 describe chemical pathways for L-glufosinate, in which a homoserine derivative, such as O-acetylhomoserine or O-succinylhomoserine, is used as a starting material to obtain L-glufosinate through a series of reactions including lactonization and halogenation.

[0008] WO 2020 / 145627 describes a similar route in which bromine derivatives are obtained during halogenation.

[0009] The route disclosed by CN106083922 is similar but starts from L-methionine.

[0010] EP 2402453 A1 describes an enzymatic method for producing methionine by enzymatically reacting a mixture of methyl mercaptan and dimethyl sulfide with O-acetylhomoserine or O-succinylhomoserine.

[0011] CN108516991 describes another route to synthesize L-glufosinate by starting from the azeotropic dehydration of L-homoserine to give L-3,6-bis(2-haloethyl)-2,5-diketopiperazine, followed by the introduction of a methylphosphinate diester group and hydrolysis.

[0012] One drawback of all synthetic routes to glufosinate is that the resulting glufosinate is a racemic mixture. However, L-glufosinate is the economically interesting enantiomer because the D-enantiomer has no herbicidal activity.

[0013] Enzymatic routes have been described in the art for the enantioselective synthesis of L-glufosinate.

[0014] WO 2017 / 151573 discloses a two-step enzymatic synthesis of L-glufosinate from D-glufosinate. In the first step, D-glufosinate is oxidatively deaminated to produce 2-oxo-4-[hydroxy(methyl)phosphinoyl]butyric acid ("PPO"), followed by the specific amination of PPO to L-glufosinate in the second step. The first step is catalyzed by a D-amino acid oxidase, and the second step is catalyzed by a transaminase.

[0015] WO 2020 / 051188 discloses a similar method for converting racemic glufosinate to the L-glufosinate enantiomer, and further discloses the conversion of α-keto acid or ketone by-products formed during the amination of PPO with an amine donor by ketoglutarate decarboxylase to further shift the equilibrium toward L-glufosinate.

[0016] WO 2019 / 018406 discloses a method for purifying L-glufosinate from a mixture containing L-glufosinate and glutamate: Glutamate is enzymatically converted to pyroglutamate by glutaminyl peptidyl cyclotransferase, and L-glufosinate is purified from the resulting mixture by ion exchange.

[0017] The object of the present invention is to provide a further enzymatic method for producing L-glufosinate with high enantiomeric excess. In particular, such a method should enable the use of new substrates that have not been used so far in the enzymatic synthesis of L-glufosinate.

[0018] 2. Summary of the Invention The present invention solves the aforementioned problems by providing a method for producing L-glufosinate from substrates not previously used in the enzymatic production of L-glufosinate. In particular, the present invention provides a method for producing L-glufosinate or a phosphoester of L-glufosinate from methylphosphinic acid and its esters using an enzyme-catalyzed pathway. Thus, these phosphorus compounds serve as alternative substrates in the production of L-glufosinate, allowing for production flexibility without relying on known substrates currently used for the production of L-glufosinate.

[0019] In particular, this aim is to activate L-homoserine H A This is achieved by the present invention, which relates to an enzyme-catalyzed method for producing L-glufosinate or a phosphoester thereof, comprising the step (a) of reacting

[0020] 3. Brief description of the invention The present invention therefore provides an enzyme-catalyzed method for producing L-glufosinate or a phosphoester thereof, comprising the step of: A with a substrate S of structure (I) below to produce a compound of structure (III) below: [ka] [In the formula, R 1 is selected from hydrogen, alkyl, alkenyl, alkynyl, hydroxyalkyl, and aryl; Activated L-homoserine H A has the following structure (II): [ka] [In the formula, R 2 is a hydrocarbon group having 1 to 15 carbon atoms optionally containing at least one functional group selected from OH, COOH, and NH; the reaction of step (a) is enzymatically catalyzed by sulfhydrylase E1; The present invention relates to an enzymatically catalyzed method for producing L-glufosinate or a phosphoester thereof, wherein the polypeptide sequence of the sulfhydrylase enzyme E1 is selected from the group consisting of SEQ ID NO:5 and variants of SEQ ID NO:5, SEQ ID NO:6 and variants of SEQ ID NO:6, SEQ ID NO:7 and variants of SEQ ID NO:7, and SEQ ID NO:8 and variants of SEQ ID NO:8.

[0021] 4. Detailed Description of the Invention Surprisingly, it has been found that certain phosphorus-containing compounds, namely, methylphosphinic acid and esters of methylphosphinic acid, can react with activated L-homoserine under enzyme catalysis, opening a new synthetic route to L-glufosinate and L-glufosinate phosphoesters. This was particularly surprising because similar compounds, such as DEMP, did not react in a similar reaction with activated L-homoserine.

[0022] The present invention therefore provides activated L-homoserine H A with a substrate S of the following structure (I) to produce a compound of the following structure (III): [ka] [In the formula, R 1 is selected from hydrogen, alkyl, alkenyl, alkynyl, hydroxyalkyl, aryl].

[0023] The compound designated as "L-glufosinate or a phosphoester thereof" according to the present invention is represented by structural formula (III). 1 When is hydrogen, the compound is L-GA.

[0024] In structure (III), R 1 When is selected from alkyl, alkenyl, alkynyl, hydroxyalkyl, aryl, the compound is a phosphoester of L-GA.

[0025] Activated L-homoserine H A has the following structure (II): [ka] [In the formula, R 2 is a hydrocarbon group having 1 to 15 carbon atoms optionally containing at least one functional group selected from OH, COOH, and NH.

[0026] The reaction in step (a) is catalyzed by the enzyme sulfhydrylase E1.

[0027] 4.1 Substrate S The substrate S according to the invention is selected from the group consisting of methylphosphinic acid and esters of methylphosphinic acid.

[0028] The substrate S has the structure (I). In structure (I), R 1 is selected from hydrogen, alkyl, alkenyl, alkynyl, hydroxyalkyl, aryl, preferably selected from hydrogen, alkyl, preferably selected from hydrogen, alkyl having 1 to 6, preferably 1 to 4 carbon atoms, more preferably selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, isobutyl, tert-butyl, even more preferably selected from hydrogen, methyl, ethyl, n-butyl, even more preferably selected from hydrogen, methyl, n-butyl, preferably selected from methyl, n-butyl. Most preferably, R 1 is n-butyl.

[0029] In structure (I), R 1 When is hydrogen, the compound is methylphosphinic acid (also called "P-methylphosphinic acid," "PMEA").

[0030] In structure (I), R 1 When is selected from alkyl, alkenyl, alkynyl, hydroxyalkyl, aryl, the compound is an ester of methylphosphinic acid.

[0031] R in Structure (I) and Structure (III) 1 are identical.

[0032] 4.2 Activated L-homoserine H A The other reaction partner in the reaction of the present invention is activated L-homoserine H A is.

[0033] Those skilled in the art will recognize that activated L-homoserine H A (sometimes referred to as "L-methionine precursor" e.g. in WO 2008 / 013432), which specifically refers to O-acyl L-homoserine.

[0034] 4.2.1 Activated L-homoserine H A Activated L-homoserine has the following chemical structure (II): [ka] [In the formula, R 2 is a hydrocarbon group having 1 to 15 carbon atoms optionally containing at least one functional group selected from OH, COOH, and NH.

[0035] More preferably, the activated L-homoserine is selected from the group consisting of 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-coumaroyl-L-homoserine [structure (II-E)], O-malonyl-L-homoserine [structure (II-F)], O-hydroxymethylglutaryl-L-homoserine [structure (II-G)], and O-pimelyl-L-homoserine [structure (II-H)].

[0036] Even more preferably, the activated L-homoserine is selected from the group consisting of O-acetyl-L-homoserine [structure (II-A)], O-succinyl-L-homoserine [structure (II-B)].

[0037] Most preferably, the activated L-homoserine is O-acetyl-L-homoserine [structure (II-A)].

[0038] [ka]

[0039] 4.2.2 Activated L-homoserine H A Chemical synthesis of Activated L-homoserine H used in the method of the present invention A can be obtained by organic chemical synthesis routes known to those skilled in the art. For example, the synthesis of O-succinylhomoserine is described in M. Flavin and C. Slaughter, Biochemistry 1965, 4, 1370-1375. The synthesis of O-acetylhomoserine is described in S. Nagai and M. Flavin, Methods in Enzymology, Metabolism of Amino Acids and Amines Part B 1971, 17(Part B), 423-424.

[0040] The chemical synthesis of potential precursors is described, for example, in MD Armstrong, J. Am. Chem. Soc. 1948, 70, 1756-1759.

[0041] 4.2.3 Activated L-homoserine H A Biotechnological synthesis of Alternatively and preferably, activated L-homoserine H is used in the present invention. Acan be obtained by biotechnological means, as described, for example, in WO 2008 / 013432 or H. Kase, K. Nakayama, Agr. Biol. Chem. 1974, 38, 2021-2030.

[0042] Activated L-homoserine H A The bacterial species producing the above are preferably selected from the group consisting of Escherichia spp., Erwinia spp., Serratia spp., Providencia spp., Corynebacterium spp., Pseudomonas spp., Leptospira spp., Salmonella spp., Brevibacterium spp., Hypomononas spp., Chromobacterium spp., Norcardia spp., fungi, in particular yeasts.

[0043] Biotechnological processes for obtaining L-homoserine have also been described in the art, e.g., U.S. Pat. No. 3,598,701, U.S. Pat. No. 6,303,348, EP 0994190, EP 1149911, WO 2004 / 067757.

[0044] 4.3 Enzymes The process according to the invention is enzyme catalyzed.

[0045] The term "enzyme" refers to a substance, composed mostly of proteins or polypeptides, that catalyzes or facilitates, in any way, one or more chemical or biochemical reactions.

[0046] Any of the enzymes used in accordance with any aspect of the invention may be isolated enzymes. In particular, the enzymes used in accordance with any aspect of the invention may be used in an active state and in the presence of all cofactors, substrates, auxiliary polypeptides and / or activating polypeptides or factors essential for their activity.

[0047] In particular, this also means that the term "sulfhydrylase", in particular "O-acetylhomoserine sulfhydrylase" or "O-succinylhomoserine sulfhydrylase", includes the respective enzyme in combination with all cofactors necessary for its function, in particular pyridoxal 5'-phosphate ("PLP").

[0048] A "polypeptide" is a chain of chemical building blocks called amino acids that are joined together by chemical bonds called peptide bonds. Proteins or polypeptides, including enzymes, may be "native" or "wild-type," meaning that they occur in nature or have the amino acid sequence of a native protein, respectively. These terms are sometimes used interchangeably. Polypeptides may be glycosylated or non-glycosylated.

[0049] The enzymes used in accordance with any aspect of the present invention may be recombinant. As used herein, the term "recombinant" refers to a molecule or a molecule encoded by such a molecule, particularly a polypeptide or nucleic acid that is not naturally occurring but is the result of genetic engineering, or to a cell containing a recombinant molecule. For example, if a nucleic acid molecule is recombinant, it contains a promoter operably linked to a sequence encoding a catalytically active polypeptide, and the promoter has been engineered to overexpress the catalytically active polypeptide compared to the level of the polypeptide in a corresponding wild-type cell containing the original, unmodified nucleic acid molecule. As a further example, if a polypeptide is recombinant, it is identical to a naturally occurring polypeptide sequence but has been engineered to contain one or more differences that distinguish it from any naturally occurring polypeptide sequence.

[0050] As used herein, the term "overexpression" means that the respective encoded or expressed polypeptide is expressed at a higher level or activity than would normally be found in a cell under the same conditions, e.g., in the respective wild-type cell, in the absence of genetic modifications made to increase expression.

[0051] As used herein, the term "isolated" means that the target enzyme is enriched compared to the cells in which it naturally occurs. The enzyme may be enriched by SDS-polyacrylamide gel electrophoresis and / or activity assay. For example, the target enzyme may constitute more than 5, 10, 20, 50, 75, 80, 85, 90, 95, or 99% of all polypeptides present in the preparation, as determined by visual inspection of a polyacrylamide gel after staining with Coomassie Blue dye.

[0052] 4.3.1 Enzyme E1 Step (a) of the method according to the invention is enzymatically catalyzed by sulfhydrylase E1.

[0053] Sulfhydrylase is known to those skilled in the art as an enzyme that catalyzes at least one of the following reactions <1A> and <1B>: <1A>: O-acetyl-L-homoserine + methanethiol → L-methionine + acetate. <1B>: O-succinyl-L-homoserine + methanethiol → L-methionine + succinate.

[0054] A sulfhydrylase having a higher catalytic activity for reaction <1A> than for reaction <1B> may be designated as "O-acetyl-L-homoserine sulfhydrylase."

[0055] A sulfhydrylase having a higher catalytic activity for reaction <1B> than for reaction <1A> may be referred to as "O-succinyl-L-homoserine sulfhydrylase."

[0056] Step (a) of the method according to the invention is catalyzed by sulfhydrylase E1, which is even more preferably O-acetylhomoserine sulfhydrylase or O-succinylhomoserine sulfhydrylase, most preferably O-acetylhomoserine sulfhydrylase.

[0057] Sulfhydrylases that may be used in step (a) of the method according to the invention are derived from Elusimicrobia species, in particular Elusimicrobia bacterium, Hyphomonas species, Myobacterium species, Pseudonocardia species, in particular Pseudonocardia thermophila.

[0058] The sulfhydrylase enzyme that may be used in the method according to the invention may be O-acetyl-L-homoserine sulfhydrylase, which is classified in the EC class EC2.5.1.49, or O-succinyl-L-homoserine sulfhydrylase, which is classified in the EC class EC2.5.1.-.

[0059] These enzymes are part of the direct sulfurylation pathway for methionine biosynthesis and depend on PMP, as described, for example, by M.P. Ferla and W.M. Atrick (Microbiology 2014, 160, 1571-1584).

[0060] WO 02 / 18613, WO 2007 / 024933, EP 2 657 345, EP 2 657 250, WO 2015 / 165746, and WO 2008 / 013432 disclose examples of enzymes having O-acetyl-L-homoserine sulfhydrylase activity and O-succinyl-L-homoserine sulfhydrylase activity according to the present invention.

[0061] O-acetyl-L-homoserine sulfhydrylases suitable for the method according to the invention may be derived from Elusimicrobia species, in particular Elusimicrobia bacterium, Myobacterium species, Pseudonocardia species, in particular Pseudonocardia thermophila.

[0062] An O-succinyl-L-homoserine sulfhydrylase suitable for the method according to the invention may be derived from a Hyphomonas species.

[0063] Respective sequences are obtained from databases such as the Braunschweig Enzyme Database (BRENDA, Germany, available at www.brenda-enzymes.org / index.php), the National Center for Biotechnological Information (NCBI, available at https: / / www.ncbi.nlm.nih.gov / ) or the Kyoto Encyclopedia of Genes and Genomes (KEGG, Japan, available at www.https: / / www.genome.jp / kegg / ).

[0064] Table 1 below shows preferred examples of sulfhydrylases that may be used in step (a) of the method according to the invention. The genes encoding the sulfhydrylases are designated as "MET43," "MET46," and "MET52" for O-acetyl-L-homoserine sulfhydrylase ("AHS"), and "MET17" for O-succinyl-L-homoserine sulfhydrylase ("SHS").

[0065] [Table 1]

[0066] Step (a) is catalyzed by at least one sulfhydrylase E1, wherein the polypeptide sequence of the sulfhydrylase enzyme E1 is selected from the group consisting of SEQ ID NO:5 and variants of SEQ ID NO:5, SEQ ID NO:6 and variants of SEQ ID NO:6, SEQ ID NO:7 and variants of SEQ ID NO:7, and SEQ ID NO:8 and variants of SEQ ID NO:8.

[0067] In an even more preferred embodiment of the method of the present invention, the reaction of step (a) is catalyzed by a sulfhydrylase E1 selected from the group consisting of an O-acetylhomoserine sulfhydrylase selected from the group consisting of SEQ ID NO:6 and a variant of SEQ ID NO:6, SEQ ID NO:7 and a variant of SEQ ID NO:7, SEQ ID NO:8 and a variant of SEQ ID NO:8, and an O-succinylhomoserine sulfhydrylase selected from the group consisting of SEQ ID NO:5 and a variant of SEQ ID NO:5.

[0068] The term "variant" is further explained below (section 4.3.3.1). In the context of the present application, "variant" is understood to mean a polypeptide sequence that has at least 80% sequence identity to the respective polypeptide sequence.

[0069] 4.3.2 Methods for obtaining enzymes The enzymes that can be used in the method according to the invention can be synthesized by methods known to those skilled in the art.

[0070] One approach is to express the enzyme in a microorganism, such as Escherichia coli (E. coli), Saccharomyces cerevisiae, or Pichia pastoris, and add the whole cells to the reaction as a whole-cell biocatalyst. 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 pure enzyme to the reaction. If multiple enzymes are required for the reaction, the enzymes can be expressed in one or more microorganisms, including expressing all enzymes in a single microorganism.

[0071] For example, those skilled in the art can obtain enzymes according to the present invention by expressing, in particular overexpressing, these enzymes in cells (hereinafter, "expression, in particular overexpression" will be abbreviated as "(over)expression" and "expression, in particular overexpress" will be abbreviated as "(over)expressing") and subsequently isolating them, as described, for example, in DE 10031999 A1. Episomal plasmids are used, for example, to increase the expression of the respective genes. In such plasmids, the nucleic acid molecule to be (over)expressed or the nucleic acid molecule encoding the (over)expressed polypeptide or enzyme may be placed under the control of a strong inducible promoter, such as the lac promoter, located upstream of the gene. A promoter is a DNA sequence of approximately 40 to 50 base pairs that constitutes the binding site for RNA polymerase holoenzyme and the transcription start site (M. Patek, J. Holatko, T. Busche, J. Kalinowski, J. Nesvera, Microbial Biotechnology 2013, 6, 103-117), which can influence the intensity of expression of the controlled polynucleotide or gene. "Operable linkage" results from the contiguous placement of a promoter and a gene, directing transcription of the gene.

[0072] Suitable strong promoters for increasing expression or methods for producing such promoters are known from the literature (e.g., S. Lisser & H. Margalit, Nucleic Acid Research 1993, 21, 1507-1516; M. Patek 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, native promoters may be optimized by altering the promoter sequence toward known consensus sequences for increasing expression of genes operably linked to these promoters (M. Patek, BJ Eikmanns, J. Patek, H. Sahm, Microbiology 1996, 142, 1297-1309; M. Patek, J. Holatko, T. Busche, J. Kalinowski, J. Nesvera, Microbial Biotechnology 2013, 6, 103-117).

[0073] Constitutive promoters are also suitable for (over)expression; in this case, the gene encoding the enzyme activity is continuously expressed under the control of a promoter, such as the glucose-dependent deo promoter. Chemically inducible promoters, such as tac, lac, and trp, are also suitable. The most common system for promoter induction is the lac operon of E. coli. In this case, either lactose or isopropyl β-D-thiogalactopyranoside (IPTG) is used as the inducer. Systems using arabinose (e.g., the pBAD system) or rhamnose (e.g., E. coli KRX) as the inducer are also common. Systems for physical induction are, for example, the temperature-inducible cold-shock promoter system based on the E. coli cspA promoter from Takara or Lambda PL, and osmotically inducible promoters, such as osmB (e.g., WO 95 / 25785).

[0074] Suitable plasmids or vectors are in principle all embodiments available to a person skilled in the art for this purpose. The prior art describes standard plasmids that may be used for this purpose, such as the pET system of vectors, exemplified by pET-3a or pET-28a(+) (available from Novagen). Other plasmids and vectors can be obtained, for example, from the brochures of Novagen, Promega, New England Biolabs, Clontech or Gibco BRL. Other preferred plasmids and vectors are described in: Glover, D. M. (1985) DNA cloning: a practical approach, Vol. I-III, IRL Press Ltd., Oxford; Rodriguez, R. L. and Denhardt, D. T. (eds) (1988) Vectors: a survey of molecular cloning vectors and their uses, 179-204, Butterworth, Stoneham; Goeddel, D. V. (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.

[0075] The plasmid vector containing the gene to be amplified is then transformed into the strain of interest, for example, by conjugation or transformation. Conjugation methods are described, for example, in A. Schaefer, J. Kalinowski, and A. Puehler, Applied and Environmental Microbiology 1994, 60, 756-759. Transformation methods are described, for example, in G. Thierbach, A. Schwarzer, and A. Puehler, Applied Microbiology and Biotechnology 1988, 29, 356-362; L. K. Dunican and E. Shivnan, Bio / Technology 1989, 7, 1067-1070; and A. Tauch, O. Kirchner, L. Wehmeier, J. Kalinowski, and A. Puehler, FEMS Microbiology Letters 1994, 123, 343-347. After homologous recombination by a "crossover" event, the resulting strain contains at least two copies of the relevant gene.

[0076] The desired enzyme can be isolated by disrupting cells containing the desired activity using methods known to those skilled in the art, for example, using a ball mill, French press, or ultrasonicator, followed by centrifugation at 13,000 rpm and 4°C for 10 minutes to separate the cells, cell debris, and disruption aids, such as glass beads. The resulting crude cell-free extract can be used to perform an enzyme assay, followed by product detection by LC-ESI-MS. Alternatively, the enzyme can be concentrated or purified to homogeneity by methods known to those skilled in the art, for example, by chromatographic methods (e.g., nickel-nitrilotriacetic acid affinity chromatography, streptavidin affinity chromatography, gel filtration chromatography, or ion exchange chromatography).

[0077] Whether a nucleic acid or polypeptide is (over)expressed may be determined by quantitative PCR reactions in the case of nucleic acid molecules, and by SDS-polyacrylamide electrophoresis, Western blotting or comparative activity assays in the case of polypeptides. Genetic modifications may be directed to transcriptional, translational and / or post-translational modifications that result in altered enzymatic activity and / or selectivity under selected and / or identified culture conditions.

[0078] 4.3.3 Definition 4.3.3.1 "Variant" In the context of the present invention, the term "variant" in relation to a polypeptide sequence refers to a polypeptide sequence having a degree of identity to a reference sequence of 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%. In still other specific embodiments, the degree of identity 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 preferably at least 99.9%. Of course, a "variant" of a polypeptide sequence is not identical to that polypeptide sequence.

[0079] Such variants may be prepared by introducing deletions, insertions, substitutions, or combinations thereof, particularly in the amino acid sequence, as well as by introducing fusions comprising such macromolecules or variants thereof.

[0080] Modifications of amino acid residues in a given polypeptide sequence that do not significantly alter the properties and functions of the given polypeptide are known to those skilled in the art. Thus, for example, many amino acids can often be substituted for each other without problems, and examples of such suitable amino acid substitutions are Ala to Ser, Arg to Lys, Asn to Gln or His, Asp to Glu, Cys to Ser, Gln to Asn, Glu to Asp, Gly to Pro, His to Asn or Gln, Ile to Leu or Val, Leu to Met or Val, Lys to Arg, Gln, or Glu, Met to Leu or Ile, Phe to Met, Leu, or Tyr, Ser to Thr, Thr to Ser, Trp to Tyr, Tyr to Trp or Phe, and Val to Ile or Leu. It is also known that modifications, for example in the form of insertions or deletions of amino acids, particularly at the N- or C-terminus of a polypeptide, often do not significantly affect the function of the polypeptide.

[0081] Consistent with this, inventive variants of any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 each have a polypeptide sequence that includes amino acids of the respective sequence that are essential for the function of the protein, e.g., the catalytic activity of the protein, or the folding or structure of the protein. Other amino acids may be replaced by deletion, substitution, or insertion, or essential amino acids are replaced in a conservative manner that preserves the activity of the enzyme, particularly the sulfhydrylase.

[0082] 4.3.3.2 "Sequence identity" Those skilled in the art will recognize that various computer programs are available to calculate the similarity or identity between two nucleotide or amino acid sequences.

[0083] A preferred method for determining identity is to first produce a maximal alignment between the sequences being compared. Computer programs for determining identity include, but are not limited to, the GCG program package, which includes: - GAP [J. Deveroy et al., Nucleic Acid Research 1984, 12, page 387, Genetics Computer Group, University of Wisconsin, Medicine (WI)], and - BLASTP, BLASTN and FASTA (S. Altschul et al., Journal of Molecular Biology 1990, 215, 403-410). BLAST programs are available 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., supra).

[0084] For example, the percentage identity between two amino acid sequences can be determined by the algorithm developed by SB Needleman and CD Wunsch (J. Mol. Biol. 1970, 48, 443-453), which is integrated into the GAP program in the GCG software package, and uses either the BLOSUM62 matrix or the PAM250 matrix, gap 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 the use of different parameters will lead to slightly different results, but the overall percentage identity between two amino acid sequences will not differ significantly. The BLOSUM62 matrix is ​​typically used with the default settings (gap weight: 12, length weight: 1).

[0085] In the context of the present invention, 80% sequence identity according to said algorithm means 80% homology. The same applies to higher identities.

[0086] Most preferably, the degree of identity between sequences is determined in the present context by the program "Needle" using the substitution matrix BLOSUM62, a gap opening penalty of 10, and a gap extension penalty of 0.5. The Needle program implements the global alignment algorithm described by SB Needleman and CD Wunsch (J. Mol. Biol. 1970, 48, 443-453). The substitution matrix used in accordance with the present invention is BLOSUM62, the gap opening penalty is 10, and the gap extension penalty is 0.5. The preferred version used in the present description is F. Madeira, YM Park, J. Lee, N. Buso, T. Gur, N. Madhusoodanan, P. Basutkar, ARN Tivey, SC Potter, RD Finn, Nucleic Acids Research 2019, 47, W636-W641, Web Server issue (recommended version accessible online at https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / on March 31, 2021).

[0087] In certain embodiments, the percentage of identity between or to a reference polypeptide sequence is determined by: i) aligning the two amino acid sequences using the Needle program with a BLOSUM62 substitution matrix, a gap opening penalty of 10, and a gap extension penalty of 0.5; ii) counting 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) to a percentage.

[0088] 4.3.3.3 Preferred Assays for Identifying Particularly Active Variants 4.3.3.3.1 Assay A In the context of the present invention, particularly preferred polypeptide variants of any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 may be identified by one of skill in the art as exhibiting activity in the following assay ("Assay A").

[0089] Assay A is carried out in the following steps: A1) First, the activity of the variant to be tested is determined by the following steps A1.1), A1.2) and A1.3) as follows: 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 polypeptide to be tested and heat to 50°C. A1.2) The reaction is initiated by adding 10 μl of a 200 mM solution of butyl-p-methylphosphinate (MPBE, CAS#6172-80-1). The concentration of MPBE in the reaction solution is 2 mM. A1.3) After adding MPBE, the reaction is carried out at 50°C for 120 minutes, and then stopped by adding 10 μl of 1% formic acid solution and cooling on ice.

[0090] A2) Next, a blank test is performed in the following steps A2.1), A2.2), and A2.3): A2.1) Prepare 990 μl of 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. A2.2) The reaction is initiated by adding 10 μl of a 200 mM solution of butyl-P-methylphosphinate (MPBE, CAS#6172-80-1). A2.3) After adding MPBE, the reaction is carried out at 50°C for 120 minutes, and then stopped by adding 10 μl of 1% formic acid solution and cooling on ice.

[0091] A3) Finally, the butyl phosphoester of L-GA (i.e., R 1 = n-butyl) are measured and compared, preferably by LC-MS analysis as described in item 5.7.

[0092] A4) If the amount of butyl phosphoester of L-GA measured in A1.3) is greater than the amount measured in A2.3), the tested variant is active in Assay A.

[0093] If the amount of butyl phosphoester of L-GA measured in A1.3) is equal to or less than the amount measured in A2.3), the tested variant does not show activity in Assay A.

[0094] Preferred formic acid solutions in steps A1.3) and A2.3) are ammonium formate or sodium formate solutions. Alternatively, the reactions in steps A1.3) and A2.3) can also be stopped by adding methanol, preferably 1 ml of methanol.

[0095] 4.3.3.3.2 Assay B In a further assay ("Assay B"), the activity of polypeptide variants of any of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively, may be determined relative to the polypeptide.

[0096] Assay B is carried out in the following steps: B1) First, the activity of a "standard" polypeptide standard (i.e., one polypeptide sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8) is determined by the following steps B1.1), B1.2), and B1.3) as follows: 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 "standard" polypeptide to be tested and heat to 50°C. B1.2) The reaction is initiated by adding 10 μl of a 200 mM solution of butyl-p-methylphosphinate (MPBE, CAS#6172-80-1). The concentration of MPBE in the reaction solution is 2 mM. B1.3) After adding MPBE, the reaction is carried out at 50°C for 120 minutes, and then stopped by adding 10 μl of 1% formic acid solution and cooling on ice.

[0097] B2) Then repeat steps B1.1), B1.2), and B1.3) with the variant: B2.1) 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 "variant" polypeptide to be tested and heat to 50°C. B1.2) The reaction is initiated by adding 10 μl of a 200 mM solution of butyl-p-methylphosphinate (MPBE, CAS#6172-80-1). The concentration of MPBE in the reaction solution is 2 mM. B1.3) After adding MPBE, the reaction is carried out at 50°C for 120 minutes, and then stopped by adding 10 μl of 1% formic acid solution and cooling on ice.

[0098] B3) Finally, the butyl phosphoester of L-GA (i.e., R 1 = n-butyl) are preferably measured and compared by LC-MS analysis as described in item 5.7.

[0099] B4) The ratio of the amount (moles) of butyl phosphoester of L-GA obtained in B2.3) is then divided by the amount (moles) of butyl phosphoester of L-GA obtained in B1.3), and this ratio is multiplied by a factor of 100 to obtain the relative activity of the variant polypeptide compared to the "standard" polypeptide in percent.

[0100] Preferred formic acid solutions in steps B1.3) and B2.3) are ammonium formate or sodium formate solutions. Alternatively, the reactions in steps B1.3) and B2.3) can also be stopped by adding methanol, preferably 1 ml of methanol.

[0101] 4.3.3.4 "Preferred Variant" 4.3.3.4.1 "Variant of SEQ ID NO: 5" In particular, a variant of SEQ ID NO: 5 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 to the polypeptide sequence SEQ ID NO: 5.

[0102] Preferred variants of SEQ ID NO:5 exhibit activity in Assay A of Section 4.3.3.3.1.

[0103] Even more preferably, the activity of each variant of SEQ ID NO:5 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% of the activity of SEQ ID NO:5 as measured in Assay B of Section 4.3.3.3.2.

[0104] Even more preferably, the activity of each variant of SEQ ID NO: 5 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 100%, relative to the activity of SEQ ID NO: 5 measured in Assay B of Section 4.3.3.3.2.

[0105] 4.3.3.4.2 "Variant of SEQ ID NO: 6" In particular, a 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 to the polypeptide sequence SEQ ID NO: 6.

[0106] Preferred variants of SEQ ID NO: 6 exhibit activity in Assay A of Section 4.3.3.3.1.

[0107] Even more preferably, the activity of each variant of SEQ ID NO: 6 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% of the activity of SEQ ID NO: 6 as measured in Assay B of Section 4.3.3.3.2.

[0108] Even more preferably, the activity of each variant of SEQ ID NO: 6 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 100%, relative to the activity of SEQ ID NO: 6 measured in Assay B of Section 4.3.3.3.2.

[0109] 4.3.3.4.3 "Variant of SEQ ID NO: 7" In particular, a variant of SEQ ID NO: 7 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 to the polypeptide sequence SEQ ID NO: 7.

[0110] Preferred variants of SEQ ID NO: 7 exhibit activity in Assay A of Section 4.3.3.3.1.

[0111] Even more preferably, the activity of each variant of SEQ ID NO: 7 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% of the activity of SEQ ID NO: 7 as measured in Assay B of Section 4.3.3.3.2.

[0112] Even more preferably, the activity of each variant of SEQ ID NO: 7 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 100%, relative to the activity of SEQ ID NO: 7 measured in Assay B of Section 4.3.3.3.2.

[0113] 4.3.3.4.4 "Variant of SEQ ID NO: 8" In particular, a variant of SEQ ID NO: 8 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 to the polypeptide sequence SEQ ID NO: 8.

[0114] Preferred variants of SEQ ID NO: 8 exhibit activity in Assay A of Section 4.3.3.3.1.

[0115] Even more preferably, the activity of each variant of SEQ ID NO:8 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% of the activity of SEQ ID NO:8 as measured in Assay B of Section 4.3.3.3.2.

[0116] Even more preferably, the activity of each variant of SEQ ID NO: 8 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 100%, relative to the activity of SEQ ID NO: 8 measured in Assay B of Section 4.3.3.3.2.

[0117] 4.4 Method conditions The reaction in step a) of the process according to the invention may be carried out under conditions known to those skilled in the art.

[0118] Activated L-homoserine H A The reaction medium in which is reacted with the substrate S is preferably aqueous, more preferably an aqueous buffer.

[0119] Exemplary buffers routinely used in biotransformations and advantageously used herein include any of Tris, phosphate, or 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-aminoethanesulfonic acid ("ACES"), P-hydroxy-4-morpholinepropanesulfonic acid ("MOPSO"), chloramine chloride, 3-(N-morpholino)propanesulfonic acid ("MOPS"), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid ("BES"), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid ("TES"), These include 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ("HEPES"), 3-(bis(2-hydroxyethyl)amino)-2-hydroxypropane-1-sulfonic acid ("DIPSO"), acetamidoglycine, 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"), tricine, glycinamide, bicine, or 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonic acid ("TAPS").

[0120] In some embodiments, ammonium may act as a buffer. One or more organic solvents may be added to the reaction.

[0121] Preferably, step a) of the method according to the invention is carried out in a phosphate buffer.

[0122] The pH of the reaction medium in step a) of the process of the invention is preferably in the range of 2 to 10, more preferably in the range of 5 to 8, most preferably 7.5.

[0123] The process according to the invention is preferably carried out at a temperature in the range of 20°C to 70°C, more preferably in the range of 30°C to 55°C, most preferably at 50°C.

[0124] 4.5 L-Glufosinate phosphoester The product of the process according to the present invention has the following structure (III): [ka] is a compound of

[0125] The compound according to structure (III) is L-glufosinate (R 1 is H) or L-glufosinate phosphoester (R 1 is alkyl, alkenyl, alkynyl, hydroxyalkyl or aryl).

[0126] R 1 The compound of structure (III) where is n-butyl is abbreviated as "L-GA-Bu" or "LGA-Bu."

[0127] Those skilled in the art will recognize the residue R in the compound according to structure (III) 1 The identity of residue R in the substrate structure (I) 1 I understand that this depends on the identity of

[0128] R 1 is hydrogen, L-glufosinate is obtained directly in the method according to the invention.

[0129] R 1 In a preferred embodiment, wherein is selected from alkyl, alkenyl, alkynyl, hydroxyalkyl, aryl, preferably alkyl, more preferably methyl, ethyl, n-butyl, compound (III) is L-glufosinate phosphoester.

[0130] In these embodiments, the method according to the invention comprises the step (a) of obtaining a compound having a hydroxyl group and R 1 is selected from alkyl, alkenyl, alkynyl, hydroxyalkyl, aryl, preferably alkyl, more preferably methyl, ethyl, n-butyl, to obtain L-glufosinate.

[0131] This can be done by methods known to those skilled in the art.

[0132] Preferably, such saponification is carried out under acidic conditions, more preferably by mixing 1 volume of the reaction medium containing the compound of structure (III) obtained in step (a) with 4 volumes of 6N HCl for 2 hours and incubating the resulting mixture at 50°C to 150°C, preferably 100°C.

[0133] Alternatively, enzymatic saponification may be carried out.

[0134] 5. Working Example Genes from different sources encoding sulfhydrylases (EC 2.5.1.- and EC 2.5.1.49) were tested for their ability to react with activated homoserine derivatives and different substrates according to structure (I) to form glufosinate derivatives.

[0135] 5.1 Example 1 Identification of suitable enzymes and construction of plasmids 5.1.1 Enzymes tested Bibliographic details of the genes encoding O-succinylhomoserine sulfhydrylase ("SHS") or O-acetylhomoserine sulfhydrylase ("AHS") used in the examples are summarized in Table 2 ("AA" = polypeptide).

[0136] [Table 2]

[0137] 5.1.2 Preparation of gene expression plasmids 5.1.2.1 O-Succinylhomoserine Sulfhydrase MET27 from Hypomonas sp. The MET17 gene from Hypomonas encodes O-succinylhomoserine sulfhydrylase, and the encoded polypeptide sequence can be found at NCBI Reference Sequence: WP_011647651.1.

[0138] To achieve the expression of the enzyme, the expression vector pET-28a(+) (Novagen EMD Millipore) was used. Therefore, the MET17_Hy polynucleotide according to SEQ ID NO: 9 was synthesized by GeneArt (Thermo Fisher Scientific, Waltham, USA).

[0139] To construct the expression vector pET-28a(+)_MET17_Hy, the vector pET-28a(+) and the MET17_Hy polynucleotide were both treated with NdeI and XhoI, ligated, and the ligation mixture was used to transform E. coli.

[0140] DNA of the expression vector pET-28a(+)_MET17_Hy was isolated from the transformants.

[0141] 5.1.2.2 O-Acetylhomoserine sulfhydrase MET43 from Mycobacterium species The MET43 gene from Mycobacterium species encodes O-acetylhomoserine sulfhydrylase. The encoded polypeptide sequence can be found at NCBI Reference Sequence: MCB0926676.1.

[0142] To achieve the expression of the enzyme, the expression vector pET-28a(+) (Novagen EMD Millipore) was used. Therefore, the MET43_Ms polynucleotide according to SEQ ID NO: 10 was synthesized by GeneArt (Thermo Fisher Scientific, Waltham, USA).

[0143] To construct the expression vector pET-28a(+)_MET43_Ms, the vector pET-28a(+) and the MET43_Ms polynucleotide were both treated with NdeI and XhoI, ligated, and the ligation mixture was used to transform E. coli.

[0144] DNA of the expression vector pET-28a(+)_MET43_Ms was isolated from the transformants.

[0145] 5.1.2.3 O-Acetylhomoserine sulfhydrase MET46 from Pseudonocardia thermophila The MET46 gene from Pseudonocardia thermophila encodes O-acetylhomoserine sulfhydrylase. The encoded polypeptide sequence can be found in NCBI Reference Sequence: WP_073459782.1.

[0146] To achieve the expression of the enzyme, the expression vector pET-28a(+) (Novagen EMD Millipore) was used. Therefore, the MET46_Pt polynucleotide according to SEQ ID NO: 11 was synthesized by GeneArt (Thermo Fisher Scientific, Waltham, USA).

[0147] To construct the expression vector pET-28a(+)_MET46_Pt, the vector pET-28a(+) and the MET46_Pt polynucleotide were both treated with NdeI and XhoI, ligated, and the ligation mixture was used to transform E. coli.

[0148] DNA of the expression vector pET-28a(+)_MET46_Pt was isolated from the transformants.

[0149] 5.1.2.4 O-Acetylhomoserine sulfhydrase ME52 from Elusimicrobia bacterium The MET52 gene from Elusimicrobia bacterium encodes O-acetylhomoserine sulfhydrylase. The encoded polypeptide sequence can be found in NCBI Reference Sequence: MBI4397379.1.

[0150] To achieve the expression of the enzyme, the expression vector pET-28a(+) (Novagen EMD Millipore) was used. Therefore, the MET52_Eb polynucleotide according to SEQ ID NO: 12 was synthesized by GeneArt (Thermo Fisher Scientific, Waltham, USA).

[0151] To construct the expression vector pET-28a(+)_MET52_Eb, the vector pET-28a(+) and the MET52_Eb polynucleotide were both treated with NdeI and XhoI, ligated, and the ligation mixture was used to transform E. coli.

[0152] DNA of the expression vector pET-28a(+)_MET52_Eb was isolated from the transformants.

[0153] 5.2 Transformation and Expression of O-Succinylhomoserine Sulfhydrase or O-Acetylhomoserine Sulfhydrase These vectors carrying the O-succinylhomoserine sulfhydrylase or O-acetylhomoserine sulfhydrylase gene were transformed into Escherichia coli BL21(DE3) (New England Biolabs), followed by cultivation on LB medium agar plates containing 50 mg / L kanamycin at 37°C for 16 hours. A BL21 strain carrying the vector pET-28a(+) without an insert was used as a negative control.

[0154] The resulting strains were designated Ec BL21 pET-28a(+)_MET17-Hy, Ec BL21 pET-28a(+)_MET43-Ms, Ec BL21 pET-28a(+)_MET46-Pt, and Ec BL21 pET-28a(+)_MET52-Eb, respectively.

[0155] In each case, a colony was selected and inoculated into 10 ml of LB medium containing 50 mg / L kanamycin and grown at 37°C for 6 hours at 250 rpm. Subsequently, 50 μl of LB medium was treated with 50 mg / L kanamycin, inoculated with 50 μl of the grown cell culture, and grown at 28°C for 16 hours at 250 rpm. This cell culture was diluted to an OD of 0.15 in 200 ml of fresh LB medium containing 50 μg / L kanamycin in a 2-L flask and further grown under the same conditions until an OD of 0.5 was reached (approximately 4 hours). The onset of gene expression induction was then affected by the addition of 200 μl of a 300 mM IPTG stock solution (final concentration 300 μM isopropyl-β-D-thiogalactopyranoside (IPTG), Sigma-Aldrich, Germany). Induction was carried out at 28°C for 4 hours at 250 rpm. The culture was then harvested (8 ml, normalized to OD = 1), the supernatant removed by centrifugation (20 min, 4000 rpm, 4°C), and the pelleted cells were washed twice with 800 μl of 0.1 M potassium phosphate buffer (pH 7.5) and taken up in 1 ml of buffer. Mechanical cell digestion was performed using a FastPrep FP120 device (QBiogene, Heidelberg). The cells were shaken four times for 30 seconds at 6.5 m / s in a digestion vessel containing 300 mg of glass beads (0.2–0.3 mm diameter). The crude extract was then centrifuged at 12000 rpm for 20 minutes at 4°C to remove undigested cells and cell debris.

[0156] The indicated proteins containing HisTag sequences were purified using HisPur cobalt resin (Thermo Fischer Scientific, Germany). The purification process was carried out using standard procedures available from Thermo Fischer Scientific. Freshly purified protein lysates were used for enzyme assays. The concentrations of polypeptides in the lysates were determined by SDS-PAGE and analysis of the respective bands using the software GelQuant® (BiochemLabSolutions).

[0157] 5.3 Examples I1 to I4 of the present invention To determine whether each polypeptide catalyzes the reaction of each phosphorus-containing substrate with activated L-homoserine, the following assay was performed: O-acetyl L-homoserine was used as the activated L-homoserine substrate.

[0158] To 880 μl of phosphate buffer (0.1 M, pH 7.5) containing 1 nmol of the test polypeptide, 100 μl of 30 mM O-acetylhomoserine-HCl solution, 10 μl of 1 mM pyridoxal 5'-phosphate monohydrate (1 mM) solution, and 10 μl of 200 mM butyl p-methylphosphinate (CAS No. 6172-80-1, "MPBE") solution were added. The reaction was carried out at 50°C for 120 minutes. Then, 100 μl of the batch solution was diluted with 100 μl of methanol and applied to LC-MS QQQ (item 5.7) to analyze LGA-Bu.

[0159] 5.4 Inventive Examples I5 and I6 The polypeptides of SEQ ID NO:5 and SEQ ID NO:8 are P-methylphosphinic acid ("PMEA"; R 1 To determine whether β-L-homoserine catalyzes the reaction of activated L-homoserine with structure (I) where is H, the following assay was performed. O-acetyl L-homoserine was used as the activated L-homoserine substrate.

[0160] To 880 μl of phosphate buffer (0.1 M, pH 7.5) containing 1 nmol of the test polypeptide, 100 μl of a 30 mM aqueous solution of O-acetylhomoserine-HCl, 10 μl of a 1 mM aqueous solution of pyridoxal 5'-phosphate monohydrate (1 mM), and 10 μl of a 200 mM aqueous solution of PMEA were added. The reaction was carried out at 50°C for 120 minutes. Then, 100 μl of the batch solution was diluted with 100 μl of methanol and applied to LC-MS QQQ (Section 5.7) to analyze LGA-Bu.

[0161] The results are summarized in Table 3 below. The abbreviations used are: "MPBE" = Butyl p-methylphosphinate (CAS number 6172-80-1) "PMEA" = p-methylphosphinic acid (CAS number 4206-94-4) is.

[0162] [Table 3]

[0163] 5.6 Results The results summarized in Table 3 surprisingly show that the tested polypeptides accept methylphosphinic acid compounds, such as MPBE and PMEA, as substrates and catalyze their reaction with activated L-homoserine to give the n-butyl P-ester of LGA or free LGA, respectively.

[0164] This finding is even more surprising because one of skill in the art would not have expected these enzymes to catalyze these reactions since other phosphate compounds, such as DEMP, do not function as substrates.

[0165] This discovery opens up a new enzymatic pathway for LGA and its derivatives.

[0166] 5.7 Analysis method All analytical measurements in the experiments were performed by scanning an LC-MS QQQ system. Samples were diluted with methanol (v:v=1:2).

[0167] The HPLC used belonged to the Agilent 1260 Infinity series and was connected to a mass spectrometer 6420 triple quadrupole equipped with electrospray ionization. Peak identification was performed by retention time and molecular weight in positive detection mode.

[0168] Data evaluation was performed by peak area and quadratic calibration without zero.

[0169] Details of how to obtain Ion source: ESI (electrospray ionization) Time parameters Mass range m / z=50~300 Scans / sec 400 Fragmentation 40V Polarity: Positive (scan mode) Source Parameters Gas temperature 350℃ Gas flow rate 12 l / min Nebulizer 50psi Capillary 4000V Binary Pump Injection volume 2.00μL Flow rate 0.60mL / min Solvent Composition Solvent A: 100 mM ammonium acetate + 0.1% (v / v) formic acid in HO Solvent B: 0.1% formic acid in acetonitrile Slope See Table 4 [Table 4] column Type: Luna HILIC; 100 x 2 mm; 3 μm; Phenomenex 00D-4449-BO Temperature 30.0℃

[0170] 6. Sequence Overview The following table provides a summary of the DNA and protein sequences referred to in the present application: [Table 5]

Claims

1. An enzyme-catalyzed method for producing L-glufosinate or its phosphoester, comprising the step of: A with a substrate S of structure (I) below to produce a compound of structure (III) below: 【Chemical 1】 [In the formula, R 1 is selected from hydrogen, alkyl, alkenyl, alkynyl, hydroxyalkyl, and aryl; Activated L-homoserine H A has the following structure (II): 【Chemistry 2】 [In the formula, R 2 is a hydrocarbon group having 1 to 15 carbon atoms optionally containing at least one functional group selected from OH, COOH, and NH; The reaction of step (a) is carried out by sulfhydrylase E 1 is enzymatically catalyzed by Sulfhydrylase enzyme E 1 is selected from the group consisting of SEQ ID NO:5 and variants of SEQ ID NO:5, SEQ ID NO:6 and variants of SEQ ID NO:6, SEQ ID NO:7 and variants of SEQ ID NO:7, SEQ ID NO:8 and variants of SEQ ID NO:8, An enzyme-catalyzed process for producing L-glufosinate or its phosphoester.

2. Activated L-homoserine H A The method of claim 1, wherein is selected from O-succinyl-L-homoserine and O-acetyl-L-homoserine.

3. Activated L-homoserine H A The method of claim 2, wherein is O-acetyl-L-homoserine.

4. R 1 4. The method of claim 1, wherein is selected from hydrogen, alkyl.

5. The method of claim 4, wherein the alkyl group is selected from methyl, ethyl, and n-butyl.

6. R 1 is selected from alkyl, alkenyl, alkynyl, hydroxyalkyl, aryl, and further comprising step (b) of saponifying the compound of structure (III) obtained in step (a) to obtain L-glufosinate.

7. Activated L-homoserine H A However, activated L-homoserine H A 7. The method according to claim 1, wherein the yeast is prepared by fermentation of a bacterial strain producing

8. Activated L-homoserine H A 8. The method of claim 7, wherein the bacterial species producing the formula (I) is selected from the group consisting of Escherichia species, Erwinia species, Serratia species, Providencia species, Corynebacterium species, Pseudomonas species, Leptospira species, Salmonella species, Brevibacterium species, Hypomonas species, Chromobacterium species, and Norcardia species, and fungi.

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