Enzymatic catalytic method for producing L-glufosinate-ammonium and phospholipids thereof

Abstract

The present invention relates to an enzymatically catalyzed process for the production of L-glufosinate-ammonium or its phosphate esters. The process comprises a step in which activated L-homoserine HA, such as acetyl or succinyl L-homoserine, is reacted with a substrate S selected from the group consisting of methylphosphinic acid and esters of methylphosphinic acid. The hydrogen sulfide hydrolytic enzyme E1 (aminocarboxypropyl transferase, EC 2.5. 1) is used for enzymatic catalysis. A new substrate can be obtained in the enzymatic production of the L-glufosinate-ammonium phosphonate and the phosphoesterase of the L-glufosinate-ammonium phosphonate. The invention makes it possible to use a new substrate in the enzymatic production of L-glufosinate-ammonium and its phosphate esters.

Description

Field of the Invention

[0001] The present invention relates to an enzymatic process for producing L-phosphinothricin ("L-GA" or "LGA") or its phosphate. The process comprises: A The step of reacting with a substrate S selected from methylphosphinic acid and esters of methylphosphinic acid. Sulfhydrylase E1 is used for enzymatic catalysis. The present invention can make new substrates available in the enzymatic production of L-phosphinothricin and its phosphate ester. Background of the Invention

[0003] Organic phosphor compounds, i.e. chemical agents containing a carbon-phosphor bond, are widely used as herbicides in plant protection. Agents such as the herbicide glyphosate and glufosinate and the growth regulator glyphosate For this purpose (e.g. by G. Rev. Environ. Containm. Toxicol. 1994, 138, 73-145).

[0004] Esters of P-methylphosphinic acid (e.g. butyl P-methylphosphinic acid ester; "MPBE"; CAS-No: 6172-80-1) play a key role as building blocks in the synthesis of the non-selective herbicide glufosinate. These esters can be obtained by two basic synthetic routes (summarized in the article of K. Haack, Chem. Unserer Zeit 2003, 37, 128-138, p. 130, Figures 3a and 3b):

[0005] a. Reaction of diethyl chlorophosphite [ClP(OC2H5)2] with CH3MgCl provides methyldiethoxyphosphine [H3CP(OC2H5)2; "DEMP"; CAS-No.15715-41-0], which is partially hydrolyzed to give the corresponding methyl phosphinate (MPEE; CAS-Nr: 16391-07-4).

[0006] b. Alternatively, methane can be reacted with phosphor trichloride at 500°C to give methyldichlorophosphine H3CPCl2. The latter can then be solvolyzed in alcohol to give the corresponding methylphosphinate.

[0007] Esters of p-methylphosphinic acid regioselectively add to carbon-carbon double bonds. This property is used to synthesize glufosinate for forming a second phosphor-carbon bond. For example, H3CPH(O)OR (R = alkyl) reacts with 1-cyanoallyl acetate in an addition reaction to provide an intermediate. Subsequent exchange of the acetic acid substituent with ammonia and hydrolysis of the ester and cyano groups of the phosphinic acid moiety yield glufosinate.

[0008] Acrylate is a cheaper alternative starting material. It can be reacted with an ester of p-methylphosphinic acid to produce alkyl 3-[alkoxy(methyl)phosphinyl]propionate. Claisen reaction of the diester with diethyl oxalate, hydrolysis and decarboxylation provides the corresponding α-keto acid, which can be reductively aminated to give glufosinate.

[0009] These and further synthesis routes to L-glufosinate are also described in the art (eg in WO 1999 / 009039 A1, EP 0508296 A1).

[0010] WO 2020 / 145513 A1 and WO 2020 / 145514 A1 describe a chemical route for synthesizing L-phosphinothricin. In this route, homoserine derivatives such as O-acetylhomoserine or O-succinylhomoserine are used as starting materials, and L-phosphinothricin is obtained by a series of reactions including lactonization and halogenation.

[0011] WO 2020 / 145627 A1 describes a similar route, in which bromo derivatives are obtained during the halogenation.

[0012] CN 106083922 A discloses a similar pathway, but starting from L-methionine.

[0013] EP 2402453 A2 describes an enzymatic process for producing methionine by enzymatically reacting a mixture of methyl mercaptan and dimethyl sulfide with O-acetyl homoserine or O-succinyl homoserine.

[0014] CN108516991 A describes another synthetic route for L-phosphinothricin, starting from the azeotropic dehydration of L-homoserine to obtain L-3,6-bis(2-haloethyl)-2,5-diketopiperazine, followed by the introduction of a methylphosphite diester group and hydrolysis.

[0015] A general disadvantage of all glufosinate-ammonium synthetic routes is that the glufosinate-ammonium obtained is a racemic mixture. However, since the D-enantiomer has no herbicidal activity, L-glufosinate-ammonium is the economically interesting enantiomer.

[0016] For the enantioselective synthesis of L-glufosinate, enzymatic pathways are described in the art.

[0017] WO 2017 / 151573 A1 discloses a two-step enzymatic synthesis of L-phosphinothricin by D-phosphinothricin. In the first step, D-phosphinothricin is oxidatively deaminated to obtain 2-oxo-4-[hydroxy(methyl)phosphono]butyric acid ("PPO"), followed by specific amination of PPO to L-phosphinothricin as the second step. The first step is carried out by catalysis of D-amino acid oxidase, and the second step is catalyzed by transaminase.

[0018] WO 2020 / 051188 A1 discloses a similar method for converting racemic glufosinate to L-glufosinate enantiomers. In addition, a step is disclosed in which the α-keto acid or ketone byproduct formed during the amination of PPO with an amine donor is converted by ketoglutarate decarboxylase to further transfer the equilibrium to L-glufosinate.

[0019] WO 2019 / 018406 A1 discloses a method for purifying L-phosphinothricin from a mixture comprising L-phosphinothricin and glutamate. Glutamate is enzymatically converted to pyroglutamate by glutaminyl-peptidyl cell cyclotransferase, and then L-phosphinothricin is purified from the resulting mixture by ion exchange.

[0020] The object of the present invention is to provide a further enzymatic process for producing L-phosphinothricin in high enantiomeric excess. In particular, such a process should allow the use of new substrates that have not been used for the enzymatic synthesis of L-phosphinothricin to date. SUMMARY OF THE INVENTION

[0022] The present invention solves the above problems by providing a method for producing L-glufosinate from a substrate that has not been used in the enzymatic production of L-glufosinate before. In particular, the present invention provides a method for producing L-glufosinate or a phosphate ester of L-glufosinate using an enzymatic catalytic pathway from methylphosphinic acid and its ester. Therefore, these phosphor compounds are used as alternative substrates in the production of L-glufosinate, thereby allowing production flexibility that does not rely on known substrates currently used for L-glufosinate production.

[0023] In particular, this object is achieved by the present invention, which relates to an enzymatic method for producing L-phosphinothricin or its phosphate, comprising step (a), wherein activated L-homoserine H A Reacts with substrate S to produce these compounds.

[0024] Therefore, the present invention relates to an enzymatic method for producing L-phosphinothricin or its phosphate, comprising step (a), wherein activated L-homoserine H A Reacting with a substrate S of the following structure (I) to produce a compound of the following structure (III),

[0025]

[0026] Where R 1 is selected from hydrogen, alkyl, alkenyl, alkynyl, hydroxyalkyl, aryl,

[0027] And the activated L-homoserine H A Has the following structure (II):

[0028]

[0029] Where R 2 is a hydrocarbon group having 1 to 15 carbon atoms, which optionally contains at least one functional group selected from OH, COOH, NH.

[0030] and wherein the reaction in step (a) is enzymatically catalyzed by sulfhydrylase E1,

[0031] The polypeptide sequence of the sulfhydrylase E1 is selected from 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. DETAILED DESCRIPTION OF THE INVENTION

[0033] Surprisingly, it has been found that certain phosphor-containing compounds, namely methylphosphinic acid and esters of methylphosphinic acid, can react with activated L-homoserine under enzymatic catalysis, thereby opening up new synthetic routes for L-phosphinothricin and L-phosphinothricin phosphates. This is particularly surprising because similar compounds such as DEMP do not react in similar reactions with activated L-homoserine.

[0034] Therefore, the present invention relates to an enzymatic method for producing L-phosphinothricin or its phosphate, which comprises step (a), wherein activated L-homoserine H A Reacting with a substrate S of the following structure (I) to produce a compound of the following structure (III),

[0035]

[0036] Where R 1 Selected from hydrogen, alkyl, alkenyl, alkynyl, hydroxyalkyl, aryl.

[0037] The compound represented by "L-phosphinothricin or its phosphate" according to the present invention is represented by structure (III). 1 = hydrogen, the compound is L-GA.

[0038] When R in structure (III) 1When the group is selected from alkyl, alkenyl, alkynyl, hydroxyalkyl and aryl, the compound is a phosphate ester of L-GA.

[0039] Activated L-homoserine H A Has the following structure (II):

[0040]

[0041] Where R 2 is a hydrocarbon group having 1 to 15 carbon atoms, which optionally contains at least one functional group selected from OH, COOH, NH.

[0042] The reaction in step (a) is catalyzed by an enzyme, which is sulfhydrylase E1.

[0043] 4.1 Substrate S

[0044] The substrate S according to the invention is selected from methylphosphinic acid and esters of methylphosphinic acid.

[0045] The substrate S has structure (I). In structure (I), R 1 is selected from hydrogen, alkyl, alkenyl, alkynyl, hydroxyalkyl, aryl,

[0046] Preferably selected from hydrogen, alkyl,

[0047] is preferably selected from hydrogen, alkyl having 1 to 6, preferably 1 to 4, carbon atoms,

[0048] More preferably, R1 is 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, R1 is n-butyl.

[0049] When R in structure (I) 1 = hydrogen, the compound is methylphosphinic acid (also known as "P-methylphosphinic acid", "PMEA").

[0050] When R in structure (I) 1 When the group is selected from alkyl, alkenyl, alkynyl, hydroxyalkyl and aryl, the compound is an ester of methylphosphinic acid.

[0051] R in structures (I) and (III) 1 same.

[0052] 4.2 Activated L-homoserine H A

[0053] Another reaction partner in the reaction according to the invention is activated L-homoserine H A .

[0054] Those skilled in the art know that activated L-homoserine H A (sometimes also indicated as "L-methionine precursor", for example WO 2008 / 013432 A1), which refers in particular to O-acyl L-homoserine.

[0055] 4.2.1 Activated L-homoserine H A

[0056] Activated L-homoserine has the following chemical structure (II):

[0057]

[0058] Where R 2 is a hydrocarbon group having 1 to 15 carbon atoms, which optionally contains at least one functional group selected from OH, COOH, NH.

[0059] 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-coumaroyl-L-homoserine [Structure (II-E)], O-malonyl-L-homoserine [Structure (II-F)], O-hydroxymethylglutaryl-L-homoserine [Structure (II-G)] and O-pimeloyl-L-homoserine [Structure (II-H)].

[0060] Even more preferably, the activated L-homoserine is selected from O-acetyl-L-homoserine [Structure (II-A)], O-succinyl-L-homoserine [Structure (II-B)].

[0061] Most preferably, the activated L-homoserine is O-acetyl-L-homoserine [Structure (II-A)].

[0062]

[0063] 4.2.2 Activated L-homoserine H A Chemical synthesis

[0064] Activated L-homoserine H used in the method of the present invention AIt can be obtained by organic chemical synthesis routes known to those skilled in the art. For example, the synthesis of O-succinyl homoserine is described in M. Flavin, C. Slaughter, Biochemistry 1965, 4, 1370-1375. The synthesis of O-acetyl-homoserine is described in Nagai, M. Flavin, Methods in Enzymology, Metabolism of Amino Acids and Amines Part B 1971, 17 (Part B), 423-424.

[0065] The chemical synthesis of potential precursors is described, for example, by MD Armstrong, J. Am. Chem. Soc. 1948, 70, 1756-1759.

[0066] 4.2.3 Biotechnological synthesis of activated L-homoserine HA

[0067] Alternatively and preferably, the activated L-homoserine HA used in the present invention is obtained by biotechnological means. For example, this is described in WO 2008 / 013432 A1 or by H. Kase, K. Nakayama, Agr. BioI. Chem. 1974, 38, 2021-2030.

[0068] Produces activated L-homoserine H A The strain is preferably selected from the group consisting of Escherichia sp., Erwinia sp., Serratia sp., Provideia sp., Corynebacterium sp., Pseudomonas sp., Leptospira sp., Salmonella sp., Brevibacterium sp., Hypomononas sp., Chromobacterium sp., Norcardia sp., fungi (especially yeasts).

[0069] Biotechnological processes for obtaining L-homoserine are also described in the art (eg in US 3,598,701, US 6,303,348 B1, EP 0994190 A2, EP 1149911 A2, WO 2004 / 067757 A1).

[0070] 4.3 Enzymes

[0071] The process according to the invention is enzymatically catalyzed.

[0072] The term "enzyme" is intended to mean a substance consisting entirely or largely of proteins or polypeptides which catalyzes or promotes one or more chemical or biochemical reactions more or less specifically.

[0073] Any enzyme used according to any aspect of the present invention may be an isolated enzyme. In particular, the enzyme used according to any aspect of the present invention may be used in an active state and in the presence of all cofactors, substrates, auxiliary and / or activation polypeptides or factors necessary for their activity.

[0074] 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 their function. In particular, this cofactor is pyridoxal 5'-phosphate ("PLP").

[0075] A "polypeptide" is a chain of chemical building blocks called amino acids, linked together by chemical bonds called peptide bonds. A protein or polypeptide, including an enzyme, may be "native" or "wild type," meaning that it occurs in nature or has the amino acid sequence of a native protein. These terms are sometimes used interchangeably. A polypeptide may or may not be glycosylated.

[0076] The enzyme used according to any aspect of the present invention may be recombinant. The term "recombinant" as used herein refers to a molecule or particularly a polypeptide or nucleic acid encoded by such a molecule that does not exist naturally but is the result of genetic engineering, or refers to a cell containing a recombinant molecule. For example, if a nucleic acid molecule comprises a promoter functionally linked to a sequence encoding a catalytically active polypeptide and the promoter has been engineered so that the catalytically active polypeptide is overexpressed relative to the polypeptide level in a corresponding wild-type cell containing the original unaltered nucleic acid molecule, the nucleic acid molecule is recombinant. As a further example, if a polypeptide is identical to a naturally occurring polypeptide sequence but is engineered to contain one or more point mutations that distinguish it from any naturally occurring polypeptide sequence, the polypeptide is recombinant.

[0077] As used herein, the term "overexpression" means that the encoded or expressed corresponding polypeptide is expressed at a higher level or higher activity than normally found in a cell (e.g., in a corresponding wild-type cell) under the same conditions in the absence of the genetic modification to increase expression.

[0078] As used herein, the term "isolated" means that the enzyme of interest is enriched compared to the cells in which it naturally occurs. The enzyme can be enriched by SDS polyacrylamide electrophoresis and / or activity assays. For example, the enzyme of interest may constitute 5%, 10%, 20%, 50%, 75%, 80%, 85%, 90%, 95% or 99% of all polypeptides present in the preparation as judged by visual inspection of a polyacrylamide gel after staining with Coomassie blue dye.

[0079] 4.3.1 Enzyme E1

[0080] Step (a) of the process according to the invention is enzymatically catalyzed by sulfhydrylase E1.

[0081] Sulfhydrylase is known to those skilled in the art as an enzyme that catalyzes at least one of the following reactions <1A>, <1B>:

[0082] <1A> O-acetyl-L-homoserine + methyl mercaptan → L-methionine + acetate.

[0083] <1B> O-succinyl-L-homoserine + methyl mercaptan → L-methionine + succinate.

[0084] A sulfhydrylase having a higher catalytic activity for reaction <1A> than for reaction <1B> can be expressed as "O-acetyl-L-homoserine sulfhydrylase".

[0085] A sulfhydrylase having a higher catalytic activity for reaction <1B> than for reaction <1A> can be expressed as "O-succinyl-L-homoserine sulfhydrylase".

[0086] Step (a) of the process according to the invention is catalyzed by a sulfhydrylase E1, which is even more preferably O-acetyl homoserine sulfhydrylase or O-succinyl homoserine sulfhydrylase, most preferably O-acetyl homoserine sulfhydrylase.

[0087] The sulfhydrylase that can be used in step (a) of the method according to the invention originates from Elusimicrobia sp., in particular Elusimicrobia bacterium; Mycomonas sp.; Myobacterium sp.; Pseudonocardia sp., in particular Pseudonocardia thermophila.

[0088] The sulfhydrylase that can be used in the method according to the invention may be an O-acetyl-L-homoserine sulfhydrylase classified in EC class EC 2.5.149 or an O-succinyl-L-homoserine sulfhydrylase classified in EC class EC 2.5.1.

[0089] These enzymes are part of the direct sulfurylation pathway of methionine biosynthesis and are PMP-dependent. They are described, for example, by MP Ferla and WM Patrick, Microbiology 2014, 160, 1571-1584.

[0090] WO 02 / 18613 A1, WO 2007 / 024933 A2, EP 2657345 A1, EP 2657250 A2, WO 2015 / 165746 A1 and WO 2008 / 013432 A1 disclose examples of the enzyme having O-acetyl-L-homoserine sulfhydrylase and O-succinyl-L-homoserine sulfhydrylase activity according to the present invention.

[0091] The O-acetyl-L-homoserine sulfhydrylase suitable for use in the method according to the invention may be derived from the genus Aglaobras, in particular Aglaobras; the genus Mycobacterium; the genus Pseudonocardia, in particular Pseudonocardia thermophila.

[0092] O-succinyl-L-homoserine sulfhydrylase suitable for use in the method according to the invention may be derived from Mycena spp.

[0093] The corresponding sequences can be derived from databases such as the Braunschweig Enzyme Database (BRENDA, Germany, available under www.benda-enzyme.org / index.php), the National Center for Biotechnology Information (NCBI, available under https: / / www.ncbi.nlm.nih.gov / ) or the Kyoto Encyclopedia of Genes and Genomes (KEGG, Japan, available under www.https: / / www.genome.jp / keg / ).

[0094] Table 1 below gives preferred examples of sulfhydrylases that can be used in step (a) of the method according to the present invention. The genes encoding sulfhydrylases are represented as "MET43", "MET46" and "MET52" for O-acetyl-L-homoserine sulfhydrylase ("AHS"), and as "MET17" for O-succinyl-L-homoserine sulfhydrylase ("SHS").

[0095] Table 1

[0096] strain Gene name NCBI accession number SEQ ID NO of polypeptide: Mycelium MET17 WP_011647651.1 SEQ ID NO:5 Mycobacterium MET43 MCB0926676.1 SEQ ID NO:6 Pseudonocardia thermophila MET46 WP_073459782.1 SEQ ID NO:7 Mysterious bacteria MET52 MBI4397379.1 SEQ ID NO:8

[0097] Step (a) is catalyzed by at least sulfhydrylase E1, wherein the polypeptide sequence of sulfhydrylase hydrolase E1 is selected from 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.

[0098] In an even more preferred embodiment of the method of the present invention, the reaction in step (a) is catalyzed by a sulfhydrylase E1 selected from the group consisting of O-acetyl homoserine sulfhydrylases selected from 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, and O-succinyl homoserine sulfhydrylases selected from the group consisting of SEQ ID NO: 5 and variants of SEQ ID NO: 5.

[0099] The term "variant" is further explained below (item 4.3.3.1). In the context of the present application, it is understood to mean a polypeptide sequence having at least 80% sequence identity with the corresponding polypeptide sequence.

[0100] 4.3.2 Methods for obtaining enzymes

[0101] The enzymes which can be used in the method according to the invention can be synthesized by methods known to those skilled in the art.

[0102] One approach is to express the enzyme in a microorganism such as Escherichia coli (="E. coli"), Saccharomyces cerevisiae, Pichia pastoris, etc., and add the entire 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 the reaction requires multiple enzymes, the enzymes can be expressed in one or several microorganisms, including expressing all enzymes in a single microorganism.

[0103] For example, a person skilled in the art can obtain these enzymes by expressing, in particular overexpressing (hereinafter, "expression, in particular overexpression" is abbreviated as "(over) expression") the enzymes of the present invention in cells and their subsequent isolation, as described, for example, in DE10031999A1. For example, liposome plasmids are used to increase the expression of the corresponding gene. 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 strong inducible promoter such as the lac promoter located upstream of the gene. The promoter is a DNA sequence consisting of about 40-50 base pairs, which constitutes the binding site of the RNA polymerase holoenzyme and the transcription start point (M. Pátek, J. Hollátko, T. Busche, J. Kalinowski, J. Needera, Microbial Biotechnology 2013, 6, 103-117), whereby the expression intensity of the controlled polynucleotide or gene may be affected. The "functional connection" is obtained by the sequential arrangement of the promoter and the gene, which leads to the transcription of the gene.

[0104] Suitable strong promoters or methods for increasing expression of such promoters are known from the literature (e.g. S. Liser & H. Margalit, Nucleic Acid Research 1993, 21, 1507-1516; M. Patho and J. Nesvera in H. Yukawa and M Inui (eds.), Corynebacterium gluticum, Microbiology Monograph 23, Springer Verlag Berlin Heidelberg 2013, 51-88; BJ Eikmanns, E. Kleinertz, W. Liebl, H. Sahm, Gene 1991, 102, 93-98). For example, these promoters can be optimized by changing the promoter sequence in the direction of the known consensus sequence with respect to increasing the expression of a gene functionally linked to the native promoter (M. Pátek, BJ Eigkmann, J. Pátek, H. Saham, Microbiology 1996, 142, 1297-1309; M. Pátek, J. Hollátko, T. Busche, J. Kalinowski, J. Needera, Microbial Biotechnology 2013, 6, 103-117).

[0105] Constitutive promoters are also suitable for (over) expression, wherein the gene encoding the enzyme activity is continuously expressed under the control of a promoter such as the glucose-dependent deo promoter. Chemically induced promoters are also suitable, such as tac, lac or trp. The most extensive system for inducing promoters is the lac operon of Escherichia coli. In this case, lactose or isopropyl β-D-thiogalactoside (IPTG) is used as an inducer. In addition, systems using arabinose (e.g., pBAD system) or rhamnose (e.g., Escherichia coli KRX) as inducers are common. Systems for physical induction are, for example, temperature-induced cold shock promoter systems based on the Escherichia coli cspA promoter from Takara or Lambda PL and osmotic inducible promoters such as osmB (e.g., WO95 / 25785A1).

[0106] Suitable plasmids or vectors are in principle all embodiments available to the skilled person for this purpose. The state of the art describes standard plasmids that can be used for this purpose, for example with pET-3a or pET-28a (+) (commercially available from Novagen) as typical vector pET systems. Further plasmids and vectors can be obtained, for example, from the manuals of the companies Novagen, Promega, NewEngland Biolabs, Clontech or Gibco BRL. Further preferred plasmids and vectors can be found in Glover, DM (1985) DNA cloning: a practical approach, Vol. I-III, IRL Press Ltd., Oxford; Rodriguez, RL 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, EF and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York.

[0107] The plasmid vector containing the gene to be amplified is then transformed into the desired strain, for example by conjugation or transformation. Conjugation methods are used, for example, by A. J. Kalinowski, A. Pühler, Applied and Environmental Microbiology 1994, 60, 756-759. Methods for transformation are described, for example, 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. After homologous recombination by a "cross-over" event, the resulting strain contains at least two copies of the gene of interest.

[0108] The desired enzyme can be isolated by disrupting cells containing the desired activity in a manner known to those skilled in the art, for example by means of a ball mill, a French press or an ultrasonic pulverizer, followed by separation of cells, cell debris and disruption aids such as glass beads by centrifugation at 13000 rpm and 4° C. for 10 minutes. The resulting cell-free crude extract can then be used for enzyme assays of the product and subsequent LC-ESI-MS detection. Alternatively, the enzyme can be enriched in a manner known to those skilled in the art, or purified to homogeneity, by chromatographic methods (such as nickel-nitrilotriacetic acid affinity chromatography, streptavidin affinity chromatography, gel filtration chromatography or ion exchange chromatography).

[0109] Whether a nucleic acid or polypeptide is (over) expressed can be determined by quantitative PCR reaction (in the case of nucleic acid molecules), SDS polyacrylamide electrophoresis, Western blotting or comparative activity assay (in the case of polypeptides). The genetic modification may involve transcription, translation and / or post-translational modification that results in changes in enzyme activity and / or selectivity under selected and / or specified culture conditions.

[0110] 4.3.3 Definition

[0111] 4.3.3.1 "Variants"

[0112] In the context of the present invention, the term "variant" with respect 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 yet further 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 more preferably at least 99.9%. It will be understood that certain "variants" of a polypeptide sequence are not identical to the polypeptide sequence.

[0113] Such variants can be prepared by introducing deletions, insertions, substitutions or a combination thereof, particularly in amino acid sequence, as well as fusions comprising such macromolecules or variants thereof.

[0114] It is known to the skilled person that modifications of the amino acid residues of a given polypeptide sequence do not significantly alter the properties and functions of a given polypeptide. Thus, for example, many amino acids can often be exchanged for one another without problems; 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 or Gln or Glu; Met to Leu or Ile; Phe to Met or Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; Val to Ile or Leu. It is also known that modifications, in particular modifications made at the N- or C-terminus of a polypeptide, for example in the form of amino acid insertions or deletions, often do not exert a significant effect on the function of the polypeptide.

[0115] In line with this, the variants according to any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 of the present invention respectively have a polypeptide sequence comprising amino acids of the corresponding sequence that are essential for function (e.g., catalytic activity of the protein or folding or structure of the protein). Other amino acids may be deleted, inserted, substituted or replaced, or essential amino acids may be replaced in a conservative manner to the effect that the activity of the enzyme (particularly sulfhydrylase) is retained.

[0116] 4.3.3.2 “Sequence identity”

[0117] Those skilled in the art are aware of a variety of computer programs that can be used to calculate the similarity or identity between two nucleotide or amino acid sequences.

[0118] Preferred methods for determining identity initially generate a maximum alignment between the sequences being compared. Computer programs for determining identity include, but are not limited to, the GCG program package, including

[0119] -GAP [j.] Devey et al., Nucleic Acid Research 1984, 12, p. 387, Genetics Computer Group University of Wisconsin, Medicine (WI)], and

[0120] - BLASTP, BLASTN and FASTA (S. Altschul et al., Journal of Molecular Biology 1990, 215, 403-410). The BLAST programs are available from the National Center for Biotechnology Information (NCBI) and from other sources (BLAST Handbook, S. Altschul et al., NCBI NLM NIH Beethesda ND22894; S. Altschul et al., above).

[0121] For example, the percent identity between two amino acid sequences can be determined by an algorithm developed by SB Needleman and CD Wunsch, J. Mol. Biol. 1970, 48, 443-453, which has been integrated into the GAP program in the GCG software package using a BLOSUM62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length of 1, 2, 3, 4, 5 or 6. Those skilled in the art will recognize that using different parameters will result in slightly different results, but the percent identity between two amino acid sequences will not be significantly different overall. The BLOSUM62 matrix is ​​typically used to apply default settings (gap weight: 12, length weight: 1).

[0122] In the context of the present invention, a sequence identity of 80% according to the above algorithm means 80% homology. The same conclusion applies to higher identities.

[0123] Most preferably, in the context of the present invention, the degree of identity between sequences is determined 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 in SB Needleman and CD Wunsch, J. Mol. Biol. 1970, 48, 443-453. The substitution matrix used according to 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 context of the present invention is presented by F. Madeira, YM Park, J. Lee, N. Buso, T. Gur, N. Madhusoodanan, P. Basutkar, ARN Tivey, S Cotter, R D Finn, Nucleic Acids Research 2019, 47, W636–W641, Web Server issue (the preferred version was accessible online at https: / / www.ebio.ac.uk / Tools / psa / emboss_needle / on March 31, 2021).

[0124] In a specific embodiment, the percent identity of the amino acid sequence of a polypeptide to a reference polypeptide sequence is determined by: i) aligning the two amino acid sequences using the Needle program with the 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 of iii) into a percentage.

[0125] 4.3.3.3 "Preferred assays for identifying specific active variants"

[0126] 4.3.3.3.1 Determination of A

[0127] 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, respectively, may be recognized by a person skilled in the art as those showing activity in the following assay ("Assay A").

[0128] Assay A was performed by the following steps:

[0129] 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:

[0130] A1.1) Prepare 990 μl of a 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.

[0131] A1.2) The reaction was started by adding 10 μl of 200 mM butyl-P-methylphosphinate (MPBE, CAS# 6172-80-1) solution. The concentration of MPBE in the reaction solution was 2 mM.

[0132] A1.3) After addition of MPBE, the reaction was carried out for 120 minutes at 50° C. Then, the reaction was stopped by adding 10 μl of 1% formate solution and cooling on ice.

[0133] A2) Then perform a blank test by following the steps A2.1), A2.2) and A2.3) as follows:

[0134] A2.1) 990 μl of a 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 was prepared and heated to 50°C.

[0135] A2.2) The reaction was started by adding 10 μl of a 200 mM butyl-P-methylphosphinate (MPBE, CAS# 6172-80-1) solution.

[0136] A2.3) After adding MPBE, the reaction was carried out for 120 minutes at 50° C. Then, the reaction was stopped by adding 10 μl of 1% formate solution and cooling on ice.

[0137] A3) Finally, the butyl-phosphate of L-GA (i.e. the compound according to formula (III), R 1 =n-butyl) amount (in moles).

[0138] A4) The variant to be tested shows activity in assay A if the amount of butyl-phosphate of L-GA determined for A1.3) is greater than the amount of butyl-phosphate determined for A2.3).

[0139] If the amount of butyl-phosphate of L-GA determined for A1.3) is the same as or less than the amount of butyl-phosphate determined for A2.3), the variant to be tested shows no activity in assay A.

[0140] Preferred formate solutions in steps A1.3) and A2.3) are ammonium formate or sodium formate solutions. Alternatively, the reaction in steps A1.3) and A2.3) can also be stopped by adding methanol (preferably 1 ml of methanol).

[0141] 4.3.3.3.2 Determination of B

[0142] In a further assay ("Assay B"), the activity of a polypeptide variant of any one of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 relative to the respective polypeptide may be determined.

[0143] Assay B was performed by the following steps:

[0144] B1) First, the activity of the "standard" polypeptide standard (i.e., a polypeptide sequence selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8) is determined by following the steps B1.1), B1.2) and B1.3) as follows:

[0145] B1.1) Prepare 990 μl of a 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.

[0146] B1.2) The reaction was started by adding 10 μl of 200 mM butyl-P-methylphosphinate (MPBE, CAS# 6172-80-1) solution. The concentration of MPBE in the reaction solution was 2 mM.

[0147] B1.3) After adding MPBE, the reaction was carried out for 120 minutes at 50° C. Then, the reaction was stopped by adding 10 μl of 1% formate solution and cooling on ice.

[0148] B2) Then, repeat steps B1.1), B1.2) and B1.3) with the variant:

[0149] B2.1) Prepare 990 μl of a 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.

[0150] B2.2) The reaction was started by adding 10 μl of 200 mM butyl-P-methylphosphinate (MPBE, CAS# 6172-80-1) solution. The concentration of MPBE in the reaction solution was 2 mM.

[0151] B2.3) After adding MPBE, the reaction was carried out for 120 minutes at 50° C. Then, the reaction was stopped by adding 10 μl of 1% formate solution and cooling on ice.

[0152] B3) Finally, the butyl-phosphate of L-GA obtained in the reaction solutions of B1.3) and B2.3) (i.e. the compound according to formula (III), R 1 =n-butyl).

[0153] B4) Then, the amount of butyl-phosphate of L-GA obtained in B2.3) (in moles) is divided by the amount of butyl-phosphate of L-GA obtained in B1.3) (in moles) to obtain a ratio. This ratio is then multiplied by a factor of 100 to give a percentage of the relative activity of the variant polypeptide relative to the "standard" polypeptide.

[0154] Preferred formate solutions in steps B1.3) and B2.3) are ammonium formate or sodium formate solutions. Alternatively, the reaction in steps B1.3) and B2.3) can also be stopped by adding methanol (preferably 1 ml of methanol).

[0155] 4.3.3.4 "Preferred variant"

[0156] 4.3.3.4.1 “Variants of SEQ ID NO: 5”

[0157] 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 with the polypeptide sequence SEQ ID NO:5.

[0158] Preferred variants of SEQ ID NO: 5 show activity in Assay A under item 4.3.3.3.1.

[0159] Even more preferably, the activity of the corresponding 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 determined in assay B under item 4.3.3.3.2.

[0160] Even more preferably, the activity of the corresponding 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%, more preferably 100% relative to the activity of SEQ ID NO:5 determined in assay B under item 4.3.3.3.2.

[0161] 4.3.3.4.2 "Variants of SEQ ID NO: 6"

[0162] 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 with the polypeptide sequence SEQ ID NO:6.

[0163] Preferred variants of SEQ ID NO: 6 show activity in Assay A under item 4.3.3.3.1.

[0164] Even more preferably, the activity of the corresponding 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 determined in assay B under item 4.3.3.3.2.

[0165] Even more preferably, the activity of the corresponding 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%, more preferably 100% relative to the activity of SEQ ID NO:6 determined in assay B under item 4.3.3.3.2.

[0166] 4.3.3.4.3 “Variants of SEQ ID NO: 7”

[0167] 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 with the polypeptide sequence SEQ ID NO:7.

[0168] Preferred variants of SEQ ID NO: 7 show activity in Assay A under item 4.3.3.3.1.

[0169] Even more preferably, the activity of the corresponding 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 determined in assay B under item 4.3.3.3.2.

[0170] Even more preferably, the activity of the corresponding 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%, more preferably 100% relative to the activity of SEQ ID NO:7 determined in assay B under item 4.3.3.3.2.

[0171] 4.3.3.4.4 “Variants of SEQ ID NO: 8”

[0172] 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 with the polypeptide sequence SEQ ID NO:8.

[0173] Preferred variants of SEQ ID NO: 8 show activity in Assay A under item 4.3.3.3.1.

[0174] Even more preferably, the activity of the corresponding 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 determined in assay B under item 4.3.3.3.2.

[0175] Even more preferably, the activity of the corresponding 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%, more preferably 100% relative to the activity of SEQ ID NO:8 determined in assay B under item 4.3.3.3.2.

[0176] 4.4 Method conditions

[0177] The reaction in step a) of the process according to the invention can be carried out under conditions known to those skilled in the art.

[0178] Activated L-homoserine H A The reaction medium for the reaction with the substrate S is preferably aqueous, more preferably an aqueous buffer.

[0179] Exemplary buffers commonly used in biotransformation reactions 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-morpholinopropanesulfonic acid ("MOPSO"), ammonium 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"), S”), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (“HEPES”), 3-(bis(2-hydroxyethyl)amino)-2-hydroxypropyl-1-sulfonic acid (“DIPSO”), acetylaminoglycine, 3-(N-tris(hydroxymethyl)methylamino(2-hydroxypropyl)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, diglycine, or 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propyl-1-sulfonic acid (“TAPS”).

[0180] In some embodiments, ammonium can act as a buffer.One or more organic solvents can also be added to the reaction.

[0181] Preferably, step a) of the method according to the invention is carried out in phosphate buffer.

[0182] The pH of the reaction medium in step a) of the process is preferably 2-10, more preferably 5-8, most preferably 7.5.

[0183] The process according to the present invention is preferably carried out at a temperature of 20°C to 70°C, more preferably 30°C to 55°C, most preferably 50°C.

[0184] 4.5L-Glufosinate Phosphate

[0185] The product of the process according to the invention is a compound of the following structure (III):

[0186]

[0187] The compound according to structure (III) is L-phosphinothricin (R 1 =H) or L-phosphinothricin phosphate (R 1 =alkyl, alkenyl, alkynyl, hydroxyalkyl or aryl).

[0188] According to the compound of structure (III) (wherein R 1 =n-butyl) is abbreviated as "L-GA-Bu" or "LGA-Bu".

[0189] A person skilled in the art will appreciate that according to the compound of structure (III) the residue R 1 The identity of is determined by the residue R in the substrate structure (I). 1 identity.

[0190] If R 1 is hydrogen, and L-phosphinothionyl ammonium is directly obtained according to the method of the present invention.

[0191] In a preferred embodiment (where R 1 is selected from alkyl, alkenyl, alkynyl, hydroxyalkyl, aryl, preferably alkyl, more preferably methyl, ethyl, n-butyl), and compound (III) is L-phosphinothricin phosphate.

[0192] In these embodiments, it is further preferred that the method according to the invention comprises a further step b), wherein the compound of structure (III) obtained in step (a) (and wherein R 1 A group selected from alkyl, alkenyl, alkynyl, hydroxyalkyl, aryl, preferably alkyl, more preferably methyl, ethyl, n-butyl) is saponified to obtain L-phosphinothricin.

[0193] This can be done by methods known to those skilled in the art.

[0194] Preferably, such saponification is carried out under acidic conditions, more preferably by mixing 1 volume of a reaction medium containing a compound of structure (III) (which is obtained in step (a)) and 4 volumes of 6N HCl for 2 h, and incubating the resulting mixture at a temperature of 50° C. to 150° C. (preferably at 100° C.).

[0195] Alternatively, enzymatic saponification may be performed. Example

[0196] Genes encoding sulfhydrylases (EC 2.5.1.- and EC 2.5.1.49) from different sources were tested for their ability to react with activated homoserine derivatives and different substrates according to structure (I) to form glufosinate derivatives.

[0197] 5.1 Identification of suitable enzymes and construction of plasmids

[0198] 5.1.1 Enzymes examined

[0199] 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).

[0200] Table 2

[0201]

[0202] 5.1.2 Preparation of gene expression plasmid

[0203] 5.1.2.1 O-succinylhomoserine sulfhydrylase MET17 from Mycena

[0204] The MET17 gene from Mycena encodes O-succinylhomoserine sulfhydrylase. The encoded polypeptide sequence can be found at NCBI as NCBI reference sequence WP_0116476511:

[0205] To achieve enzyme expression, the expression vector pET-28a(+) (Novagen EMD Millipore) was used. Thus, the MET17_Hy polynucleotide according to SEQ ID NO: 9 was synthesized by Geneart (Thermo Fisher Scientific (Waltham, USA)).

[0206] To assemble the expression vector pET-28a(+)_MET17_Hy, both the vector pET-28a(+) and the MET17_Hy polynucleotide were treated with NdeI and XhoI, ligated, and the ligation mixture was used to transform E. coli.

[0207] The DNA of the expression vector pET-28a(+)_MET17_Hy was isolated from the transformant.

[0208] 5.1.2.2 O-Acetylhomoserine Sulfhydrylase MET43 from Mycobacterium

[0209] The MET43 gene from Mycobacterium encodes O-acetylhomoserine sulfhydrylase. The encoded polypeptide sequence can be found at NCBI as NCBI reference sequence MCB0926676.1.

[0210] To achieve enzyme expression, the expression vector pET-28a(+) (Novagen EMD Millipore) was used. Thus, the MET43_Ms polynucleotide according to SEQ ID NO: 10 was synthesized by Geneart (Thermo Fisher Scientific (Waltham, USA)).

[0211] To assemble the expression vector pET-28a(+)_MET43_Ms, both the vector pET-28a(+) and the MET43_Ms polynucleotide were treated with NdeI and XhoI, ligated, and the ligation mixture was used to transform E. coli.

[0212] The DNA of the expression vector pET-28a(+)_MET43_Ms was isolated from the transformant.

[0213] 5.1.2.3 O-Acetylhomoserine sulfhydrylase MET46 from Pseudonocardia thermophila

[0214] The MET46 gene from Pseudonocardia thermophila encodes O-acetylhomoserine sulfhydrylase. The encoded polypeptide sequence can be found at NCBI as NCBI reference sequence WP_073459782.1.

[0215] To achieve enzyme expression, the expression vector pET-28a(+) (Novagen EMD Millipore) was used. Thus, the MET46_Pt polynucleotide according to SEQ ID NO: 11 was synthesized by Geneart (Thermo Fisher Scientific (Waltham, USA)).

[0216] To assemble the expression vector pET-28a(+)_MET46_Pt, both the vector pET-28a(+) and the MET46_Pt polynucleotide were treated with NdeI and XhoI, ligated, and the ligation mixture was used to transform E. coli.

[0217] The DNA of the expression vector pET-28a(+)_MET46_Pt was isolated from the transformants.

[0218] 5.1.2.4 O-Acetylhomoserine sulfhydrylase MET52 from Bacteria

[0219] The MET52 gene from the bacterium Mycobacterium styrabilis encodes O-acetylhomoserine sulfhydrylase. The encoded polypeptide sequence can be found at NCBI as NCBI reference sequence MBI4397379.1.

[0220] To achieve enzyme expression, the expression vector pET-28a(+) (Novagen EMD Millipore) was used. Thus, the MET52_Eb polynucleotide according to SEQ ID NO: 12 was synthesized by Geneart (Thermo Fisher Scientific (Waltham, USA)).

[0221] To assemble the expression vector pET-28a(+)_MET52_Eb, both the vector pET-28a(+) and the MET52_Eb polynucleotide were treated with NdeI and XhoI, ligated, and the ligation mixture was used to transform E. coli.

[0222] The DNA of the expression vector pET-28a(+)_MET52_Eb was isolated from the transformants.

[0223] 5.2 Transformation and expression of O-succinylhomoserine sulfhydrylase or O-acetylhomoserine sulfhydrylase

[0224] These vectors carrying the O-succinylhomoserine sulfhydrylase or O-acetylhomoserine sulfhydrylase gene were transformed in E. coli BL21 (DE3) (New England Biolabs), respectively, which were then cultured on LB medium agar plates with 50 mg / l kanamycin at 37° C. for 16 h. Strain BL21 carrying the vector pET-28a (+) without any insert was used as a negative control.

[0225] The obtained strains were named Ec BL21pET-28a(+)_MET17-Hy, Ec BL21pET-28a(+)_MET43-Ms, Ec BL21pET-28a(+)_MET46-Pt and Ec BL21pET-28a(+)_MET52-Eb.

[0226] In each case, colonies were selected, which were inoculated into 10 ml LB medium with 50 mg / l kanamycin and cultivated for 6 hours at 37 ° C, 250 rpm. 50 μ l LB medium was subsequently treated with 50 mg / l kanamycin and inoculated with 50 μ l growing cell cultures and incubated for 16 h at 28 ° C, 250 rpm. It was 0.15 to dilute this cell culture to OD in a 2L flask with 200 ml fresh LB medium containing 50 μ g / l kanamycin, and further cultivated under the same conditions until OD reached was 0.5 (circa 4 h). The starting point of gene expression induction was then affected by adding 200 μ l 300 mM IPTG stock solution (final concentration 300 μ M isopropyl-β-D-thiogalactoside (IPTG), Sigma-Aldrich, Germany). Induce 4 h at 28 ° C, 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 precipitated cells were washed twice with 800 μl of 0.1 M potassium phosphate buffer (pH 7.5) and removed in 1 ml of buffer. Mechanical cell digestion was performed in a FastPrep FP120 instrument (QBiotene, Heidelberg) with 300 mg of glass beads. The cells were shaken four times at 6.5 m / s for 30 s in the digestion vessel. The crude extract was then centrifuged at 12000 rpm, 4°C for 20 min to remove undigested cells and cell debris.

[0227] The protein containing the HisTag sequence was purified using HisPur Cobalt Resin (Thermo Fischer Scientific, Germany). The purification process was performed using standard procedures available from Thermo Fischer Scientific. Freshly purified protein lysates were used for enzyme assays. The concentration of polypeptides in the lysate was determined by SDS PAGE and analyzed by software (Biochem Lab Solutions) to analyze the corresponding bands.

[0228] 5.3 Inventive Examples I1 to I4

[0229] The following assay was performed to determine whether the corresponding polypeptide catalyzes the reaction of the corresponding phosphor-containing substrate and activated L-homoserine. O-Acetyl L-homoserine was used as the activated L-homoserine substrate.

[0230] To 880 μl of phosphate buffer (0.1 M, pH 7.5) containing 1 nmol of the polypeptide to be tested, 100 μl of 30 mM O-acetylhomoserine-HCl aqueous solution, 10 μl of 1 mM pyridoxal 5'-phosphate monohydrate aqueous solution (1 mM), and 10 μl of 200 mM butyl P-methylphosphinate (CAS-No. 6172-80-1; "MPBE") aqueous solution were added. The reaction was carried out at 50° C. for 120 minutes. Then, 100 μl of the batch solution was diluted in 100 μl of methanol and applied to LC-MS QQQ (item 5.7) to analyze LGA-Bu.

[0231] 5.4 Inventive Examples I5 and I6

[0232] The following assay was performed to determine whether the polypeptides according to SEQ ID NO:5 and SEQ ID NO:8 catalyze the reaction of P-methyl-phosphinic acid ["PMEA"; structure (I) with R1 = H] and activated L-homoserine. O-Acetyl L-homoserine was used as the activated L-homoserine substrate.

[0233] To 880 μl of phosphate buffer (0.1M, pH 7.5) containing 1 nmol of the polypeptide, 100 μl of 30 mM O-acetylhomoserine-HCl aqueous solution, 10 μl of 1 mM pyridoxal 5'-phosphate monohydrate aqueous solution (1 mM), and 10 μl of 200 mM PMEA aqueous solution were added. The reaction was carried out at 50° C. for 120 minutes. Then, 100 μl of the batch solution was diluted in 100 μl of methanol and applied to LC-MS QQQ (item 5.7) to analyze LGA-Bu.

[0234] The results are summarized in Table 3 below. The abbreviations used are

[0235] "MPBE" = butyl p-methylphosphinate (CAS-No. 6172-80-1);

[0236] "PMEA" = P-methylphosphinic acid (CAS-No. 4206-94-4).

[0237] Table 3

[0238] Examples Enzymes Peptides Substrate product I1 MET17 SEQ ID NO:5 MTBE yes I2 MET43 SEQ ID NO:6 MTBE yes I3 MET46 SEQ ID NO:7 MTBE yes I4 MET52 SEQ ID NO:8 MTBE yes I5 MET17 SEQ ID NO:5 PMEA yes I6 MET52 SEQ ID NO:8 PMEA yes

[0239] 5.6 Results

[0240] The results summarized in Table 3 indicate that the tested polypeptides accept methylphosphinic acid compounds, such as MPBE and PMEA, as substrates and catalyze their reaction with activated L-homoserine to the corresponding n-butyl P-ester of LGA or free LGA.

[0241] This finding is even more surprising since one skilled in the art would not expect these enzymes to catalyze these reactions since other phosphate compounds such as DEMP do not function as substrates.

[0242] This discovery opens new enzymatic pathways for LGA and its derivatives.

[0243] 5.7 Analysis methods

[0244] All analytical measurements of the experiments have been performed by scanning on a LC-MS QQQ system. The samples were diluted in methanol (v:v=1:2).

[0245] The HPLC applied was of the 1260 Infinity series from Agilent connected to a mass spectrometer 6420 triple quadrupole with electrospray ionization. Peak detection was performed by retention time and molecular mass in positive detection mode.

[0246] Data evaluation was performed by secondary calibration without zero and peak areas.

[0247] Get method details

[0248] Ion source ESI (electrospray ionization)

[0249] Time parameters

[0250] Mass range m / z = 50-300

[0251] 400 scans per second

[0252] Segment 40V

[0253] Polarity positive (scan mode)

[0254] Source Parameters

[0255] Gas temperature 350℃

[0256] Airflow 12 L / min

[0257] Sprayer 50psi

[0258] Capillary 4000V

[0259] Binary Pump

[0260] Injection volume 2.00 μL

[0261] Flow 0.60mL / min

[0262] Solvent composition

[0263] Solvent A 100 mM ammonium acetate + 0.1% (v / v) formic acid in H2O

[0264] Solvent B 0.1% formic acid in acetonitrile

[0265] gradient See Table 4.

[0266] Table 4

[0267] 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

[0268] column

[0269] Type Luna HILIC; 100×2mm; 3μm; Phenomenex 00D-4449-BO Temperature 30.0℃

[0270] 6. Sequence Overview

[0271] The following table provides an overview of the DNA and protein sequences mentioned in the context of this application:

[0272]

[0273]

Claims

1. An enzymatic method for producing L-phosphinothricin or its phosphate, comprising step (a), wherein activated L-homoserine H A Reacting with a substrate S of the following structure (I) to produce a compound of the following structure (III), Where R 1 is selected from hydrogen, alkyl, alkenyl, alkynyl, hydroxyalkyl, aryl, And wherein the activated L-homoserine H A Has the following structure (II): Where R 2 is a hydrocarbon group having 1 to 15 carbon atoms, which optionally contains at least one functional group selected from OH, COOH, NH. and wherein the reaction in step (a) is enzymatically catalyzed by sulfhydrylase E1, The polypeptide sequence of the sulfhydrylase E1 is selected from 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.

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

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

4. The method according to any one of claims 1 to 3, wherein R 1 Selected from hydrogen and alkyl.

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

6. The method according to any one of claims 1 to 3, wherein R 1 is selected from the group consisting of alkyl, alkenyl, alkynyl, hydroxyalkyl, and aryl, and further comprises a step (b), wherein the compound of structure (III) obtained in step (a) is saponified to obtain L-phosphinothricin.

7. according to the method according to any one of claims 1 to 6, wherein the activated L-homoserine H A By generating activated L-homoserine H A It is prepared by fermentation of strains.

8. A method according to claim 7. wherein the activated L-homoserine H is produced A The strain is selected from Escherichia sp., Erwinia sp., Serratia sp., Providenia sp., Corynebacterium sp., Pseudomonas sp., Leptospira sp., Salmonella sp., Brevibacterium sp., Hypomononas sp., Chromobacterium sp. and Norcardia sp., fungi.

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