The present invention relates in a first aspect to a method for producing L-glufosinate. The method comprises a step (b) in which bialaphos is reacted to give L-glufosinate, wherein the reaction according to step (b) is catalyzed by a C-terminal processing peptidase Ei, which may be categorized in the EC class 3.4.21.102. The L-glufosinate obtained in the method may be used as herbicide.

In a second aspect, the present invention relates to a method for controlling a weed plant W. In the method according to the second aspect of the invention, bialaphos is cleaved to give an herbicidally active amount of L-glufosinate, wherein the reaction is catalyzed by the C-terminal processing peptidase Ei. The herbicidally active amount of L-glufosinate which is thus produced contacts the weed plant, thereby impairing its growth or leading to its dieback.

Background of the Invention

Organic phosphorous compounds, i.e. chemical agents comprising a carbon-phosphor bond, are widely applied as herbicides in the area of plant protection. Agents such as the herbicides glyphosate (Roundup®, Touchdown®) and glufosinate (Basta®, Liberty®) as well as the growth regulator glyphosine (Polaris®) are used for this purpose (as described for example by G. Horlein, Rev. Environ. Contam. Toxicol. 1994, 138, 73 - 145).

The esters of P-methyl phosphinic acid (for example, P-methyl phosphinic acid butyl ester; “MPBE”; CAS-No: 6172-80-1) have a key role as synthetic building blocks in the synthesis of the non-selective herbicide glufosinate. These esters are accessible via two fundamental synthetic pathways (summarized in Figures 3 a and 3 b, page 130, of the article of K. Haack, Chem. Unserer Zeit 2003, 37, 128 - 138): a. Reacting diethyl chlorophosphite [CIP(OC2H5)2] with CHsMgCI provides methyl diethoxy phosphine [H3CP(OC2Hs)2; “DEMP”; CAS-No. 15715-41-0], which is partially hydrolyzed to give the corresponding P-methyl phosphinic acid ethyl ester (M PEE; CAS-Nr: 16391-07-4). b. Alternatively, methane can be reacted with phosphor trichloride at 500 °C to give methyl dichloro phosphane H3CPCI2. The latter can then be solvolyzed in alcohols to give the corresponding P-methyl phosphinic acid esters.

The esters of P-methyl phosphinic acid add to carbon-carbon double bonds regioselectively. This property is used in the synthesis of glufosinate for the formation of the second phosphor-carbon bond. For example, H3CPH(O)OR (R = Alkyl) reacts with 1 -cyano allyl acetate in an addition reaction to provide an intermediate. Subsequent exchange of the acetate substituent with ammonia and hydrolysis of the cyano group and the ester group of the phosphinic acid moiety give glufosinate. Acrylic acid ester is a cheaper alternative starting material. It can react with the ester of P-methyl phosphinic acid to 3-[alkoxy(methyl)phosphinyl]propionic acid alkyl ester. Claisen reaction of this diester with diethyl oxalate, hydrolysis and decarboxylation provide the corresponding a-keto acid, which can be reductively aminated to give glufosinate.

These and further synthetic routes towards glufosinate are also described in the art, e.g. in WO 1999/009039 A1 , EP 0 508 296 A1 .

A general disadvantage of all synthetic routes to glufosinate is that the obtained glufosinate is a racemic mixture. However, as there is no herbicidal activity of the D-enantiomer, L-glufosinate [hereinafter “LGA”; CAS-Nr. 35597-44-5; other names “(S)-glufosinate”, “(-)-glufosinate”] is the enantiomer of economic interest.

LGA

For example, CN 111662325 A discloses a synthetic pathway in which L-homoserine is reacted to the respective hydantoin, followed by addition of methane phosphor dichloride, which results in methane phosphine which is disubstituted with L-homoserine hydantoin. After a final Arbuzov reaction and hydrolysis, LGA is obtained. Although the disclosure describes a high enantiomeric excess (= “ee”), this ee could not be reproduced by the inventors of the present invention. A reason could be that during this synthesis pathway, strong acidic (HCI for saponification of the hydantoin phosphor bond) and alkaline conditions (NaOH, 100 °C for ring opening) are applied. Such conditions usually lead to racemization, as described by M. Bovarnick & H.T. Clarke, Journal of the American Chemical Society 1938, 60, 2426 - 2430, by R. A. Lazarus, J. Org. Chem. 1990, 55, 4755 - 4757, and by A. S. Bommarius, M. Kottenhahn, H. Klenk, K. Drauz: “A direct route from hydantoins to D-amino acids employing a resting cell biocatalyst with D-hydantoinase and D- carbamoylase acitivity” on pages 164 and 167 in “Microbial Reagents in Organic Synthesis” Series C: Mathematical and Physical Sciences - Vol. 381 , S. Servi (Ed.), 1992, Springer Science+Business Media, B.V., Dordrecht.

For enantioselective syntheses of LGA, chemical and enzymatic pathways are described in the art.

WO 2020/145513 A1 and WO 2020/145514 A1 describe a chemical route to LGA. In this route, an L-homoserine derivative such as O-acetyl L-homoserine or O-succinyl L-homoserine is used as starting material and L-glufosinate is obtained by a sequence of reactions including lactonization and halogenation.

WO 2020/145627 A1 describes a similar route, wherein during halogenation, a bromo derivative is obtained. The route disclosed by CN 106083922 A resembles these synthetic pathways but starts off from L-methionine.

CN 108516991 A describes another synthetic pathway to LGA, starting from the azeotropic dehydration of L-homoserine to give L-3,6-bis(2-haloethyl)-2,5-diketopiperazine, followed by introduction of a methyl phosphonate diester group, and hydrolysis.

WO 2017/151573 A1 discloses a two-step enzymatic synthesis of LGA from D-glufosinate. In the first step, D-glufosinate is oxidatively deaminated to give 2-oxo-4-[hydroxy(methyl)phosphinoyl]- butyric acid (“PPG”), followed by the specific amination of PPO to LGA as the second step. The first step is carried out by catalysis of a D-amino acid oxidase, the second step is catalyzed by a transaminase.

WO 2020/051188 A1 discloses a similar method of converting racemic glufosinate to the L-enantiomer. In addition, it discloses a step in which the a-ketoacid or ketone byproduct formed during amination of PPO with an amine donor is converted by ketoglutarate decarboxylase to further shift the equilibrium to LGA.

WO 2019/018406 A1 discloses a method of purifying LGA from a mixture comprising LGA and glutamate. Glutamate is converted to pyroglutamate enzymatically by glutaminyl-peptidyl cyclotransferase, and LGA is then purified from the resulting mixture with an ion-exchange resin.

WO 2013/072486 A1 disclose hydantoinase mutants which have a greater activity towards D-amino acids.

WO 00/58449 A1 discloses hydantoinase mutants which have a greater activity towards L-amino acids.

The object of the present invention is to provide a further enzymatic process for producing L-glufosinate in high enantiomeric excess. In particular, such process should allow to use new substrates which heretofore were not used in the enzymatic synthesis of L-glufosinate.

A further object of the present invention is to provide a method for controlling weeds, wherein the herbicidal activity may be controlled site- und time-specifically.

Summary of the Invention

The present invention solves the problems mentioned above by providing a method for producing L-glufosinate from a substrate that has not been used in such an enzymatic production of L-glufosinate before. In particular, the present invention provides a method for producing L-glufosinate from bialaphos by using a specific, enzymatically catalyzed pathway. Bialaphos thus serves as alternative substrate in the production of L-glufosinate, allowing for flexibility of production where there is no reliance on the known substrates that are currently being used for L-glufosinate production.

In particular, this object is achieved by the present invention which relates in a first aspect to a method for producing L-glufosinate, comprising a step (b) in which bialaphos is reacted to give LGA, wherein the reaction according to step (b) is catalyzed by a C-terminal processing peptidase Ei.

In a second aspect, the present invention relates to a method for controlling a weed plant W, comprising:

(i) contacting the weed plant W with bialaphos and a C-terminal processing peptidase Ei;

(ii) reacting bialaphos to give L-glufosinate in an amount that is herbicidally effective for weed W, wherein the reaction is catalyzed by the C-terminal processing peptidase Ei;

(iii) contacting the weed plant W with LGA in an amount that is herbicidally effective for weed plant W, causing an herbicidal effect on the weed plant W.

Detailed Description of the Invention

It was surprisingly found that certain enzymes, namely C-terminal processing peptidases Ei, can catalyze the reaction in which bialaphos is cleaved to give L-glufosinate (= “LGA”), thus opening a new synthetic pathway to L-glufosinate.

1 . First Aspect: Method for Producing L-glufosinate

In a first aspect, the present invention relates to a method for producing L-glufosinate, comprising a step (b) in which BIAP is reacted to give LGA, wherein the reaction according to step (b) is catalyzed by a C-terminal processing peptidase Ei. “Method according to the first aspect of the invention” is hereinafter abbreviated as “Method I”

1.1 LGA

L-glufosinate [herein “LGA”; CAS-Nr. 35597-44-5; other names “(S)-glufosinate”, “(-)-glufosinate”] has the structure according to the following formula (I):

1.2 BIAP

Bialaphos [„L-alanyl-L-alanyl-phosphinothricine“; herein “BIAP”; CAS-Nr. 35597-43-4] has the structure according to the following formula (II):

BIAP is an antibiotic which is for example described in the book “Comprehensive Natural Products Chemistry”, Editor(s): Sir Derek Barton, Koji Nakanishi, Otto Meth-Cohn, Pergamon, 1999, pages 865 - 880, ISBN 9780080912837. It is produced e.g. by Streptomyces hygroscopicus as described by A. Raibaud, M. Zalacain, T.G. Holt, R. Tizard, C.J. Thompson, J. Bacteriol. 1991 , 173, 4454 - 4463.

The BIAP used in step (b) of Method I may be exogenous.

In a preferred embodiment of Method I, the BIAP used in step (b) is produced by chemical or biotechnological synthesis in a step (a1). Logically, step (a1) precedes step (b). More preferably, in step (a1), BIAP is produced by biotechnological synthesis.

The chemical synthesis of BIAP is described for example by M.D. Armstrong, J. Am. Chem. Soc. 1948, 70, 1756- 1759.

The biotechnological synthesis of BIAP is described for example by

T. Murakami, H. Anzai, S. Imai, A. Satoh, K. Nagaoka, C.J. Thompson, MGG Molecular & General Genetics 1986, 205, 42 - 53;

E. Bayer, K.H. Gugel, K. Hagele, H. Hagenmaier, S. Jessipow, W.A. Konig, H. Zahner, Helv Chim Acta 1 72, 55, 224 - 239 (hereinafter “Bayer et al.“)

J.A.V. Blodgett, P.M. Thomas, G. Li, J.E. Velasquez, W.A. van der Donk, N.L. Kelleher, W.W. Metcalf, Nat Chem Biol. 2007; 3, 480 - 485; H. Seto, T. Sasaki, S. Imai, T. Tsuruoka, H. Ogawa, A. Satoh, S. Inouye, T. Niida, N. Otake, J Antibiot (Tokyo) 1983, 36, 96 - 98;

K. Bartsch, R. Schneider, A. Schulz, Appl. Environ. Microbiol.1996, 62, 3794 - 3799.

In an even more preferred embodiment of Method I, the BIAP employed in step (b) is produced by biotechnological synthesis in a step (a1).

In this even more preferred embodiment, bacteria producing BIAP, which are, in particular, Streptomyces, preferably one of Streptomyces meliloti, Streptomyces viridochromogenes, Streptomyces hygroscopicus, wherein Streptomyces meliloti is most preferred, are cultivated to produce BIAP.

The cultivation of bacteria and in particular Streptomyces may be carried out by the skilled person. Typically, the bacteria are cultivated in an appropriate culture medium, which is preferably aqueous. “Culture medium” is in particular an aqueous solution that preferably contains all nutrients essential so that the respective bacteria grow and produce the desired product, in this case BIAP. The choice of the culture medium is known to the skilled person. They are e.g. described in M. Bonnet, J.C. Lagier, D. Raoult, S. Khelaifia, New Microbe and New Infect 2020, 34 100622.

Even more preferably, in step (a1), BIAP is produced by biotechnological synthesis from Streptomyces, in particular from one of Streptomyces meliloti, Streptomyces viridochromogenes, Streptomyces hygroscopicus, wherein Streptomyces meliloti is most preferred.

Hence, in a preferred embodiment of step (a1), BIAP is produced by the following method:

Cells CBIAP, which are capable of producing BIAP, which are in particular bacterial cells, preferably Streptomyces cells, even more preferably cells from at least one of Streptomyces meliloti, Streptomyces viridochromogenes, Streptomyces hygroscopicus, wherein Streptomyces meliloti is most preferred, are cultivated in an aqueous medium Aq1 to produce an aqueous medium Aq1 comprising BIAP. This BIAP is then employed in step (b) of Method I.

Optionally, the cells CBIAP are at least partially lysed in the aqueous medium Aq1 comprising BIAP after step (a1) so that intracellular BIAP is liberated into Aq1.

In a further preferred embodiment, the BIAP is separated from the aqueous medium Aq1 before it is employed in step (b) of Method I. 1.3 Enzyme Ei

Method I, step (b), is enzymatically catalyzed.

1.3.1 General

The term “enzyme” means any substance composed wholly or largely of protein or polypeptides that catalyzes or promotes, more or less specifically, one or more chemical or biochemical reactions.

Any of the enzymes used according to any aspect of the present invention may be an isolated enzyme. In particular, the enzymes 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 activating polypeptides or factors essential for its activity.

In particular, this also means that the term “C-terminal processing peptidase” comprises the respective enzymes in combination with all the cofactors necessary for their function.

A “polypeptide” is a chain of chemical building blocks called amino acids that are 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, respectively. These terms are sometimes used interchangeably. A polypeptide may or may not be glycosylated.

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 is encoded by such a molecule, particularly a polypeptide or nucleic acid that, as such, does not occur naturally but is the result of genetic engineering or refers to a cell that comprises a recombinant molecule. For example, a nucleic acid molecule is recombinant if it comprises a promoter functionally linked to a sequence encoding a catalytically active polypeptide and the promoter has been engineered such that the catalytically active polypeptide is overexpressed relative to the level of the polypeptide in the corresponding wild type cell that comprises the original unaltered nucleic acid molecule. As a further example, a polypeptide is recombinant if it is identical to a polypeptide sequence occurring in nature but has been engineered to contain one or more point mutations that distinguish it from any polypeptide sequence occurring in nature.

The term “overexpressed”, as used herein, means that the respective polypeptide encoded or expressed is expressed at a level higher or at higher activity than would normally be found in the cell under identical conditions in the absence of genetic modifications carried out to increase the expression, for example in the respective wild type cell. The polypeptide of the C-terminal processing peptidase Ei used in the method of the present invention may be isolated. The term “isolated”, as used herein, means that the enzyme of interest is enriched compared to the cell in which it occurs naturally. The enzyme may be enriched, in particular, by at least one method selected from centrifugation, column chromatography, filtration, electrophoresis, which is preferably SDS polyacrylamide electrophoresis, activity assays, preferably by at least one method selected from centrifugation, column chromatography. For example, the enzyme of interest may constitute more than 5, 10, 20, 50, 75, 80, 85, 90, 95 or 99 percent of all the polypeptides present in the preparation as judged by visual inspection of a polyacrylamide gel following staining with Coomassie blue dye.

The term “an aqueous solution” or “an aqueous medium” comprises any solution comprising water, mainly water as solvent that may be used to keep the cell according to any aspect of the present invention, at least temporarily, in a metabolically active and/or viable state and comprises, if such is necessary, any additional substrates. The person skilled in the art is familiar with the preparation of numerous aqueous solutions, usually referred to as “media”, that may be used to keep cells used in the methods according to the invention Typical examples are LB medium in the case of E. coli or Bacillus, HA medium in the case of Streptomyces. It is advantageous to use as an aqueous solution a minimal medium, i.e. a medium of reasonably simple composition that comprises only the minimal set of salts and nutrients indispensable for keeping the cell in a metabolically active and/or viable state, by contrast to complex mediums, to avoid dispensable contamination of the products with unwanted side products. For example, M9 medium may be used as a minimal medium. The cells are incubated sufficiently long enough to produce the desired product, i.e. BIAP (in case of Streptomyces) or enzyme Ei, for example for at least 1 , 2, 4, 5, 10 or 20 hours. The temperature chosen must be such that the cells according to any aspect of the present invention remains catalytically competent and/or metabolically active, for example 10 to 42 °C, preferably 30 to 40 °C, in particular, 32 to 38 °C.

1.3.2 C-terminal processing peptidases

Step (b) of Method I is catalysed by a C-terminal processing peptidase Ei.

Herein, C-terminal processing peptidases are also abbreviated as “CtpA”.

C-terminal processing peptidases are known to the skilled person as enzymes that show specific recognition of a C-terminal tripeptide, Xaa-Yaa-Zaa, in which Xaa is preferably Ala or Leu, Yaa is preferably Ala or Tyr, and Zaa is preferably Ala. These enzymes preferably cleave at a variable distance from the C-terminus. A typical cleavage is -Ala-Ala-/-Arg-Ala-Ala-Lys-Glu-Asn-Tyr-Ala- Leu-Ala-Ala. The C-terminal processing peptidase Ei that may be used in step (b) of Method I may be derived from Bacillus sp., in particular Bacillus subtilis, which is preferably Bacillus subtilis subsp. spizizenii, Bacillus velezensis, Bacillus pumilus.

The C-terminal processing peptidase Ei that may be used in step (b) of Method I may be a C-terminal processing peptidases categorized in the EC class 3.4.21.102.

These enzymes are part of the DNA damage repair mechanism, as described in P.E. Burby, Z.W. Simmons, J.W. Schroeder, L.A. Simmons PLoS Genet 2018, 14, e1007512.

These enzymes are further described e.g. by the review by C.R. Harwood & Y. Kikuchi, FEMS Microbiology Reviews 2022, fuab046, 46, 1-20 (in particular on page 9 thereof).

The C-terminal processing peptidase Ei suitable for the method according to the present invention may originate from Bacillus sp., in particular Bacillus subtilis, which is preferably Bacillus subtilis subsp. spizizenii, Bacillus velezensis, Bacillus pumilus.

The finding that a C-terminal processing peptidase Ei may be used for the catalysis of cleaving BIAP was surprising and provides for advantageous applications.

Namely, the prior art only discloses catalysis of the intracellular cleavage of bialaphos to give LGA in Bacillus, such as described by J. Solomon, L. Su, S. Shyn, A.D. Grossman, J. Bacteriol. 2003, 185, 6425 - 6433 and A. Koide, J.A. Hoch, Mol. Microbiol. 1994, 13, 417 - 426, and in particular on page 235, 1 ^st paragraph of Bayer et al. In contrast, the C-terminal processing peptidases Ei employed in the method according to the present invention are secreted into the environment of the Bacillus cells and thus are easier to isolate.

The respective sequences can be derived from databases such as the Braunschweig Enzyme Database (BRENDA, Germany, available underwww.brenda-enzymes.org/index.php), the National Center for Biotechnological 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/kegg/).

The following table 1 gives preferred examples for C-terminal processing peptidases that may be used in step (b) of Method I. able 1

In a preferred embodiment of the method of the present invention, the reaction in step (b) of Method I is catalyzed by a C-terminal processing peptidase Ei, wherein the polypeptide sequence of the C-terminal processing peptidase Ei is selected from the group consisting of SEQ ID NO: 2 and variants of SEQ ID NO: 2, SEQ ID NO: 4 and variants of SEQ ID NO: 4, SEQ ID NO: 6 and variants of SEQ ID NO: 6, SEQ ID NO: 8 and variants of SEQ ID NO: 8, SEQ ID NO: 10 and variants of SEQ ID NO: 10, SEQ ID NO: 12 and variants of SEQ ID NO: 12, SEQ ID NO: 14 and variants of SEQ ID NO: 14, SEQ ID NO: 16 and variants of SEQ ID NO: 16, SEQ ID NO: 18 and variants of SEQ ID NO: 18, SEQ ID NO: 20 and variants of SEQ ID NO: 20, SEQ ID NO: 22 and variants of SEQ ID NO: 22, SEQ ID NO: 24 and variants of SEQ ID NO: 24, SEQ ID NO: 26 and variants of SEQ ID NO: 26, SEQ ID NO: 28 and variants of SEQ ID NO: 28, more preferably is selected from the group consisting of SEQ ID NO: 2 and variants of SEQ ID NO: 2.

In an even more preferred embodiment of the method of the present invention, the reaction in step (b) of Method I is catalyzed by a C-terminal processing peptidase Ei, wherein the polypeptide sequence of the C-terminal processing peptidase Ei is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, more preferably SEQ ID NO: 2.

The term “variant” is further explained below (item 1.3.4.1). In the context of the present application, it is understood to mean a polypeptide sequences with at least 60 % sequence identity to the respective polypeptide sequence.

1.3.3 Methods for obtaining enzymes

The enzymes that can be used in the method according to the present invention may be synthesized by methods that are known to the skilled person.

One approach is to express the enzyme(s) in microorganism(s) such as Bacillus, Escherichia coll (= “E. coir), Saccharomyces cerevisiae, Pichia pastoris, Corynebacterium glutamicum and others, and to add the whole cells to the reactions as whole cell biocatalysts. Another approach is to express the enzyme(s), lyse the microorganisms, and add the cell lysate. Yet another approach is to purify, or partially purify, the enzyme(s) from a lysate and add pure or partially pure enzyme(s) to the reaction. If multiple enzymes are required for a reaction, the enzymes can be expressed in one or several microorganisms, including expressing all enzymes within a single microorganism.

For example, the skilled person can obtain the enzymes according to the invention by expression, in particular, overexpression, (hereinafter, “expression, in particular overexpression” is abbreviated as (over)expression”, and “express, in particular overexpress” is abbreviated as “(over)express”) of these enzymes in a cell and subsequent isolation thereof, e.g. as described in DE 100 31 999 A1. Episomal plasmids, for example, are employed for increasing the expression of the respective genes. In such plasmids, the nucleic acid molecule to be (over)expressed or encoding the polypeptide or enzyme to be (over)expressed 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 consisting of about 40 to 50 base pairs which constitutes the binding site for an RNA polymerase holoenzyme and the transcriptional start point (M. Patek, J. Holatko, T. Busche, J. Kalinowski, J. Nesvera, Microb. Biotechnol. 2013, 6, 103-117; hereinafter “Patek et al., 2013”), whereby the strength of expression of the controlled polynucleotide or gene can be influenced. A “functional linkage” is obtained by the sequential arrangement of a promoter with a gene, which leads to a transcription of the gene.

Suitable strong promoters or methods of producing such promoters for increasing expression are known from the literature (e.g. S. Lisser & H. Margalit, Nucleic Acid Res. 1993, 21, 1507-1516; M. Patek & J. Nesvera in H. Yukawa and M Inui (eds.), Corynebacterium glutamicum, Microbiology Monographs 23, Springer Verlag Berlin Heidelberg 2013, 51-88; B. J. Eikmanns, E. Kleinertz, W. Liebl, H. Sahm, Gene 1991 , 102, 93-98). For instance, native promoters may be optimized by altering the promoter sequence in the direction of known consensus sequences with respect to increasing the expression of the genes functionally linked to these promoters (M. Patek, B. J. Eikmanns, J. Patek, H. Sahm, Microbiology 1996, 142, 1297-1309; Patek et al., 2013).

Constitutive promoters are also suitable for the (over)expression, in which the gene encoding the enzyme activity is expressed continuously under the control of the promoter such as, for example, the glucose dependent deo promoter. Chemically induced promoters are also suitable, such as tac, lac or trp. The most widespread system for the induction of promoters is the lac operon of E. coll. In this case, either lactose or isopropyl B-D-thiogalactopyranoside (IPTG) is used as inducer. Also, systems using arabinose (e.g. the pBAD system) or rhamnose (e.g. E. coll KRX) are common as inducers. A system for physical induction is, for example, the temperature-induced cold shock promoter system based on the E. coll cspA promoter from Takara or Lambda PL and also osmotically inducible promoters, for example, osmB (e.g. WO 95/25785 A1).

Suitable plasmids or vectors are in principle all embodiments available for this purpose to the person skilled in the art. The state of the art describes standard plasmids that may be used for this purpose, for example the pET system of vectors exemplified by pET-3a or pET-26b(+) (commercially available from Novagen). Further plasmids and vectors can be taken, for example, from the brochures of the companies Novagen, Promega, New England Biolabs, Clontech or Gibco BRL. Further preferred plasmids and vectors can be found 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.

The plasmid vector, which contains the gene to be amplified, is then converted to the desired strain, e.g. by conjugation or transformation. The method of conjugation is described, for example, by A. Schafer, J. Kalinowski, A. Piihler, Applied and Environmental Microbiology 1994, 60, 756- 759. Methods for transformation are described, for example, in G. Thierbach, A. Schwarzer, A. Piihler, Applied Microbiology and Biotechnology 1988, 29, 356-362, L. K. Dunican & E. Shivnan, Bio/Technology 1989, 7, 1067-1070 and A. Tauch, O. Kirchner, L. Wehmeier, J. Kalinowski, A. Piihler, FEMS Microbiology Letters 1994, 123, 343-347. After homologous recombination by means of a “cross-over” event, the resulting strain contains at least two copies of the gene concerned.

The desired enzyme can be isolated by disrupting cells which contain the desired activity in a manner known to the person skilled in the art, for example with the aid of a ball mill, a French press or of an ultrasonic disintegrator and subsequently separating off cells, cell debris and disruption aids, such as, for example, glass beads, by centrifugation for 10 minutes at 13000 rpm and 4 °C. Using the resulting cell-free crude extract, enzyme assays with subsequent LC-ESI-MS detection of the products can then be carried out. Alternatively, the enzyme can be enriched in the manner known to the person skilled in the art by chromatographic methods (such as nickel-nitrilotriacetic acid affinity chromatography, streptavidin affinity chromatography, gel filtration chromatography or ion-exchange chromatography) or else purified to homogeneity.

Whether or not a nucleic acid or polypeptide is (over)expressed, may be determined by way of quantitative PCR reaction in the case of a nucleic acid molecule, SDS polyacrylamide electrophoreses, Western blotting or comparative activity assays in the case of a polypeptide. Genetic modifications may be directed to transcriptional, translational, and/or post-translational modifications that result in a change of enzyme activity and/or selectivity under selected and/or identified culture conditions.

Therefore, in a preferred embodiment of Method I, the C-terminal processing peptidase Ei employed in step (b) is obtained in a step (a2) comprising steps (21) and optionally comprising step (a22). Logically, step (a2) precedes step (b).

In step (a21), cells C _Ei capable of producing the C-terminal processing peptidase Ei, which are in particular bacterial cells, and more preferably selected from the group consisting of Escherichia, Bacillus, Corynebacterium, more preferably from Bacillus, Escherichia coll, even more preferably from Bacillus, which is most preferably selected from Bacillus subtilis, which is preferably Bacillus subtilis subsp. spizizenii, Bacillus velezensis, Bacillus pumilus, are cultivated in an aqueous medium Aq2 to produce an aqueous medium Aq2 comprising a C-terminal processing peptidase Ei.

The aqueous medium Aq2 comprising a C-terminal processing peptidase Ei obtained in step (a21) may then directly be employed in Method I, step (b). In an alternative embodiment, the C-terminal processing peptidase Ei comprised by Aq2 is at least partially separated from Aq2 and the cells C _Ei . Such separation might be performed by the skilled person, in particular by methods described herein.

Optionally, the cells C _Ei are at least partially lysed in the aqueous medium Aq2 comprising a C-terminal processing peptidase Ei after step (a21).

In an alternative preferred embodiment of step (a2), in a step (a22) following step (a21), the C-terminal processing peptidase Ei in the aqueous medium Aq2 is at least partially separated from the aqueous medium Aq2, so that a purified C-terminal processing peptidase Ei and an aqueous medium Aq2* are obtained, wherein Aq2* comprises at least 1 %, preferably at least 5 %, more preferably at least 10 %, more preferably at least 25 %, more preferably at least 35 %, more preferably at least 50 %, more preferably at least 60 %, more preferably at least 70 %, even more preferably at least 80 %, even more preferably at least 85 %, even more preferably at least 90 %, even more preferably at least 95 %, even more preferably at least 99% of the cells C _Ei employed in step (a21) that retain their capacity to produce a C-terminal processing peptidase Ei, and further C-terminal processing peptidase Ei may then be produced by the cells C _Ei retaining their capacity to produce a C-terminal processing peptidase Ei.

Optionally, before or during step (a22), further aqueous medium Aq21 may be added to Aq21*.

The C-terminal processing peptidase Ei obtained in step (a2), and in particular after step (a21) or (a22), respectively, may then be used in step (b) of Method I.

1.3.4 Definitions

1.3.4.1 “Variants"

In the context of the present invention and in particular to the Methods I and II, the term “variant” with respect to polypeptide sequences refers to a polypeptide sequence with a degree of identity to the reference sequence of at least 60%, more preferably at least 61 %, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71 %, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably 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 further particular 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 goes without saying that a “variant” of a certain polypeptide sequence is not identical to the polypeptide sequence.

Such variants may be prepared by introducing deletions, insertions, substitutions, or combinations thereof, in particular in amino acid sequences, as well as fusions comprising such macromolecules or variants thereof.

Modifications of amino acid residues of a given polypeptide sequence which lead to no significant modifications of the properties and function of the given polypeptide are known to those skilled in the art. Thus for example many amino acids can often be exchanged for one another without problems; examples of such suitable amino acid substitutions are: Ala by Ser; Arg by Lys; Asn by Gin or His; Asp by Glu; Cys by Ser; Gin by Asn; Glu by Asp; Gly by Pro; His by Asn or Gin; He by Leu or Vai; Leu by Met or Vai; Lys by Arg or Gin or Glu; Met by Leu or He; Phe by Met or Leu or Tyr; Ser by Thr; Thr by Ser; Trp by Tyr; Tyr by Trp or Phe; Vai by lie or Leu. It is also known that modifications, particularly at the N- or C-terminus of a polypeptide in the form of for example amino acid insertions or deletions, often exert no significant influence on the function of the polypeptide.

In line with this, preferable variants according to the invention of any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, respectively, have a polypeptide sequence that comprises the amino acids of the respective sequence that are essential for the function, for example the catalytic activity of a protein, or the fold or structure of the protein. The other amino acids may be deleted, substituted or replaced by insertions or essential amino acids are replaced in a conservative manner to the effect that the activity of the enzyme, in particular the C-terminal processing peptidase, is preserved.

The person skilled in the art is aware that various computer programs are available for the calculation of similarity or identity between two nucleotide or amino acid sequences.

Preferred methods for determining the identity initially generate the greatest alignment between the sequences to be compared. Computer programs for determining the identity include, but are not limited to, the GCG program package including

GAP [J. Devereux, P. Haeberli, O. Smithies, Nucleic Acid Res. 1984, 12, 387 - 395, Genetics Computer Group University of Wisconsin, Medicine (Wl)], and

BLASTP, BLASTN and FASTA (S.F. Altschul, W. Gish, W. Miller, E.W. Myers, D.J. Lipman, J. Mol. Biol. 1990, 215, 403 - 410; doi: 10.1016/S0022-2836(05)80360-2. PMID: 2231712; hereinafter “Altschul etal.’"). The BLAST program can be obtained from the National Center for Biotechnology Information (NCBI) and from other sources (BLAST Handbook, Altschul et al., NCBI NLM NIH Bethesda ND 22894).

For instance, the percentage identity between two amino acid sequences can be determined by the algorithm developed by S. B. Needleman & C. D. Wunsch, J. Mol. Biol. 1970, 48, 443-453 (hereinafter “Needle & Wunsch”), which has been integrated into the GAP program in the GCG software package, using either a BLOSUM62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1 , 2, 3, 4, 5 or 6. The person skilled in the art will recognize that the use of different parameters will lead to slightly different results, but that the percentage identity between two amino acid sequences overall will not be significantly different. The BLOSUM62 matrix is typically used applying the default settings (gap weight: 12, length weight: 1). In the context of the present invention, a sequence identity of 80% according to the above algorithm means 80% homology. The same applies to higher identities.

Most preferably, the degree of identity between sequences is determined in the context of the present invention by the programme “Needle” using the substitution matrix BLOSUM62, the gap opening penalty of 10, and the gap extension penalty of 0.5. The Needle program implements the global alignment algorithm described by Needle & Wunsch. The substitution matrix used according to the present invention is BLOSUM62, gap opening penalty is 10, and gap extension penalty is 0.5. The preferred version used in the context of this invention is the one presented by F. Madeira, Y.M. Park, J. Lee, N. Buso, T. Gur, N. Madhusoodanan, P. Basutkar, A.R.N. Tivey, S.C. Potter, R.D. Finn, Nucleic Acids Res. 2019, 47, W636-W641 , Web Server issue (preferred version accessible online on May 17, 2022 via https://www.ebi.ac.uk/Tools/psa/emboss_needle/). In a particular embodiment, the percentage of identity of an amino acid sequence of a polypeptide with, or 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 percentage.

1.3.4.3 “Preferred assay to identify particularly active variants”

1.3.4.3.1 Assay A

Especially preferable polypeptide variants of any SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, respectively, in the context of the present invention may be identified by the skilled person as those displaying activity in the following assay (“Assay A”).

Assay A is carried out by 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) 150 pl of an aqueous solution containing phosphate buffer (0.1 M, pH 7.0) and 1.0 nmol of the polypeptide to be tested are prepared and heated to 30 °C.

A1 .2) The reaction is started by adding 150 pl of an aqueous solution of bialaphos (30 °C) so that the initial concentration of bialaphos in the reaction solution is 20 mg/l.

A1 .3) After addition of the bialaphos, the reaction is conducted for 120 minutes at 30 ^oo C. Then, the reaction is stopped by adding 50 pl 1 % formate solution and cooling on ice.

A2) Then, a blank test is carried out by the following steps A2.1), A2.2), and A2.3) as follows:

A2.1) 150 pl of an aqueous solution containing phosphate buffer (0.1 M, pH 7.0) prepared and heated to 30 °C.

A2.2) The reaction is started by adding 150 pl of an aqueous solution of bialaphos (30 °C) so that the initial concentration of bialaphos in the reaction solution is 20 mg/l.

A2.3) After addition of the bialaphos, the reaction is conducted for 120 minutes at 30 ^oo C. Then, the reaction is stopped by adding 50 pl 1 % formate solution and cooling on ice. A3) Finally, the amount (in mole) of LGA obtained in the reaction solution in A1 .3) and A2.3), respectively, is determined and compared, preferably by LC-MS analysis.

A4) If the amount of LGA determined for A1.3) is more than the amount determined for A2.3), then the variant to be tested displays activity in Assay A.

If the amount of LGA determined for A1 .3) is the same or less than the amount determined for A2.3), then the variant to be tested does not display activity in Assay A.

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

1.3.4.3.2 Assay B

In a further assay (“Assay B”), the activity of a polypeptide variants of any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, respectively, with respect to the polypeptide may be determined.

Assay B is carried out by the following steps:

B1) First, the activity of the “standard” polypeptide standard (i.e., one polypeptide sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28), is determined by the following steps B1 .1), B1 .2), and B1 .3) as follows:

B1.1) 150 pl of an aqueous solution containing phosphate buffer (0.1 M, pH 7.0) and 1.0 nmol of the polypeptide to be tested are prepared and heated to 30 °C.

B1 .2) The reaction is started by adding 150 pl of an aqueous solution of bialaphos (30 °C) so that the initial concentration of bialaphos in the reaction solution is 20 mg/l.

B1 .3) After addition of the bialaphos, the reaction is conducted for 120 minutes at 30 °C. Then, the reaction is stopped by adding 50 pl 1 % formate solution and cooling on ice.

B2) Then, steps B1 .1), B1 .2), and B1 .3) are repeated with the variant: B2.1) 150 pl of an aqueous solution containing phosphate buffer (0.1 M, pH 7.0) and 1.0 nmol of the “variant” polypeptide to be tested are prepared and heated to 30 °C.

B2.2) The reaction is started by adding 150 pl of an aqueous solution of bialaphos (30 °C) so that the initial concentration of bialaphos in the reaction solution is 20 mg/l.

B2.3) After addition of the bialaphos, the reaction is conducted for 120 minutes at 30 °C. Then, the reaction is stopped by adding 50 pl 1 % formate solution and cooling on ice.

B3) Finally, the amount of LGA obtained in the reaction solution in B1 .3) and B2.3) is determined and compared, preferably by LC-MS analysis described under item 1 .6.

B4) Then, the ratio of the amount (in mole) of LGA obtained in B2.3) is divided by the amount (in mole) of LGA obtained in B1 .3). This ratio is then multiplied with a factor of 100, giving the relative activity of the variant polypeptide with respect to the “standard” polypeptide in percent.

Preferable 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.

1.3.4.4 “Preferred variants”

Preferred variants of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28 that may be used in Method I as well as Method II according to the invention are as follows:

1.3.4.4.1 “Variants of SEQ ID NO: 2”

In particular, a variant of SEQ ID NO: 2 is a polypeptide with sequence identity of > 60 %, more preferably > 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 polypeptide sequence SEQ ID NO: 2.

Preferred variants of SEQ ID NO: 2 display activity in Assay A under item 1 .3.4.3.1 .

Even more preferably, the activity of the respective variant of SEQ ID NO: 2 is at least 1 %, preferably at least 10 %, more preferably at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 % relative to the activity of SEQ ID NO: 2 as determined in Assay B under item 1 .3.4.3.2.

Even more preferably, the activity of the respective variant of SEQ ID NO: 2 is in the range of 1 to 1000 %, preferably in the range of 5 to 500 %, more preferably in the range of 10 to 400 %, more preferably in the range of 40 to 200 %, more preferably in the range of 50 to 150 %, more preferably in the range of 60 to 140 %, more preferably in the range of 70 to 130 %, more preferably in the range of 80 to 120 %, more preferably in the range of 90 to 110 %, more preferably 100 % relative to the activity of SEQ ID NO: 2 as determined in Assay B under item

1.3.4.3.2.

1.3.4.4.2 “Variants of SEQ ID NO: 4”

In particular, a variant of SEQ ID NO: 4 is a polypeptide with sequence identity of > 60 %, more preferably > 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 polypeptide sequence SEQ ID NO: 4.

Preferred variants of SEQ ID NO: 4 display activity in Assay A under item 1 .3.4.3.1 .

Even more preferably, the activity of the respective variant of SEQ ID NO: 4 is at least 1 %, preferably at least 10 %, more preferably at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 % relative to the activity of SEQ ID NO: 4 as determined in Assay B under item 1 .3.4.3.2.

Even more preferably, the activity of the respective variant of SEQ ID NO: 4 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: 4 as determined in Assay B under item

1.3.4.3.2. 1.3.4.4.3 “Variants ofSEQ ID NO: 6”

In particular, a variant of SEQ ID NO: 6 is a polypeptide with sequence identity of > 60 %, more preferably > 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 polypeptide sequence SEQ ID NO: 6.

Preferred variants of SEQ ID NO: 6 display activity in Assay A under item 1 .3.4.3.1 .

Even more preferably, the activity of the respective variant of SEQ ID NO: 6 is at least 1 %, preferably at least 10 %, more preferably at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 % relative to the activity of SEQ ID NO: 6 as determined in Assay B under item 1 .3.4.3.2.

Even more preferably, the activity of the respective 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 as determined in Assay B under item

1.3.4.3.2.

1.3.4.4.4 “Variants ofSEQ ID NO: 8’’

In particular, a variant of SEQ ID NO: 8 is a polypeptide with sequence identity of > 60 %, more preferably > 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 polypeptide sequence SEQ ID NO: 8.

Preferred variants of SEQ ID NO: 8 display activity in Assay A under item 1 .3.4.3.1 .

Even more preferably, the activity of the respective variant of SEQ ID NO: 8 is at least 1 %, preferably at least 10 %, more preferably at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 % relative to the activity of SEQ ID NO: 8 as determined in Assay B under item 1 .3.4.3.2.

Even more preferably, the activity of the respective 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 as determined in Assay B under item

1.3.4.3.2.

1.3.4.4.5 “Variants of SEQ ID NO: 10”

In particular, a variant of SEQ ID NO: 10 is a polypeptide with sequence identity of > 60 %, more preferably > 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 polypeptide sequence SEQ ID NO: 10.

Preferred variants of SEQ ID NO: 10 display activity in Assay A under item 1 .3.4.3.1 .

Even more preferably, the activity of the respective variant of SEQ ID NO: 10 is at least 1 %, preferably at least 10 %, more preferably at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 % relative to the activity of SEQ ID NO: 10 as determined in Assay B under item 1 .3.4.3.2.

Even more preferably, the activity of the respective variant of SEQ ID NO: 10 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: 10 as determined in Assay B under item

1.3.4.3.2. 1.3.4.4.6 “Variants ofSEQ ID NO: 12’’

In particular, a variant of SEQ ID NO: 12 is a polypeptide with sequence identity of > 60 %, more preferably > 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 polypeptide sequence SEQ ID NO: 12.

Preferred variants of SEQ ID NO: 12 display activity in Assay A under item 1 .3.4.3.1 .

Even more preferably, the activity of the respective variant of SEQ ID NO: 12 is at least 1 %, preferably at least 10 %, more preferably at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 % relative to the activity of SEQ ID NO: 12 as determined in Assay B under item 1 .3.4.3.2.

Even more preferably, the activity of the respective variant of SEQ ID NO: 12 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: 12 as determined in Assay B under item

1.3.4.3.2.

1.3.4.4.7 “Variants ofSEQ ID NO: 14’’

In particular, a variant of SEQ ID NO: 14 is a polypeptide with sequence identity of > 60 %, more preferably > 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 polypeptide sequence SEQ ID NO: 14.

Preferred variants of SEQ ID NO: 14 display activity in Assay A under item 1 .3.4.3.1 .

Even more preferably, the activity of the respective variant of SEQ ID NO: 14 is at least 1 %, preferably at least 10 %, more preferably at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 % relative to the activity of SEQ ID NO: 14 as determined in Assay B under item 1 .3.4.3.2.

Even more preferably, the activity of the respective variant of SEQ ID NO: 14 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: 14 as determined in Assay B under item

1.3.4.3.2.

1.3.4.4.8 “Variants of SEQ ID NO: 16”

In particular, a variant of SEQ ID NO: 16 is a polypeptide with sequence identity of > 60 %, more preferably > 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 polypeptide sequence SEQ ID NO: 16.

Preferred variants of SEQ ID NO: 16 display activity in Assay A under item 1 .3.4.3.1 .

Even more preferably, the activity of the respective variant of SEQ ID NO: 16 is at least 1 %, preferably at least 10 %, more preferably at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 % relative to the activity of SEQ ID NO: 16 as determined in Assay B under item 1 .3.4.3.2.

Even more preferably, the activity of the respective variant of SEQ ID NO: 16 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: 16 as determined in Assay B under item

1.3.4.3.2.

1.3.4.4.9 “Variants of SEQ ID NO: 18’’

In particular, a variant of SEQ ID NO: 18 is a polypeptide with sequence identity of > 60 %, more preferably > 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 polypeptide sequence SEQ ID NO: 18.

Preferred variants of SEQ ID NO: 18 display activity in Assay A under item 1 .3.4.3.1 .

Even more preferably, the activity of the respective variant of SEQ ID NO: 18 is at least 1 %, preferably at least 10 %, more preferably at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 % relative to the activity of SEQ ID NO: 18 as determined in Assay B under item 1 .3.4.3.2.

Even more preferably, the activity of the respective variant of SEQ ID NO: 18 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: 18 as determined in Assay B under item

1.3.4.3.2.

1.3.4.4.10 “Variants of SEQ ID NO: 20’’

In particular, a variant of SEQ ID NO: 20 is a polypeptide with sequence identity of > 60 %, more preferably > 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 polypeptide sequence SEQ ID NO: 20.

Preferred variants of SEQ ID NO: 20 display activity in Assay A under item 1 .3.4.3.1 .

Even more preferably, the activity of the respective variant of SEQ ID NO: 20 is at least 1 %, preferably at least 10 %, more preferably at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 % relative to the activity of SEQ ID NO: 20 as determined in Assay B under item 1 .3.4.3.2. Even more preferably, the activity of the respective variant of SEQ ID NO: 20 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: 20 as determined in Assay B under item

1.3.4.3.2.

1.3.4.4.11 “Variants of SEQ ID NO: 22”

In particular, a variant of SEQ ID NO: 22 is a polypeptide with sequence identity of > 60 %, more preferably > 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 polypeptide sequence SEQ ID NO: 22.

Preferred variants of SEQ ID NO: 22 display activity in Assay A under item 1 .3.4.3.1 .

Even more preferably, the activity of the respective variant of SEQ ID NO: 22 is at least 1 %, preferably at least 10 %, more preferably at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 % relative to the activity of SEQ ID NO: 22 as determined in Assay B under item 1 .3.4.3.2.

Even more preferably, the activity of the respective variant of SEQ ID NO: 22 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: 22 as determined in Assay B under item

1.3.4.3.2.

1.3.4.4.12 “Variants of SEQ ID NO: 24”

In particular, a variant of SEQ ID NO: 24 is a polypeptide with sequence identity of > 60 %, more preferably > 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 polypeptide sequence SEQ ID NO: 24.

Preferred variants of SEQ ID NO: 24 display activity in Assay A under item 1 .3.4.3.1 .

Even more preferably, the activity of the respective variant of SEQ ID NO: 24 is at least 1 %, preferably at least 10 %, more preferably at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 % relative to the activity of SEQ ID NO: 24 as determined in Assay B under item 1 .3.4.3.2.

Even more preferably, the activity of the respective variant of SEQ ID NO: 24 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: 24 as determined in Assay B under item

1.3.4.3.2.

1.3.4.4.13 “Variants of SEQ ID NO: 26”

In particular, a variant of SEQ ID NO: 26 is a polypeptide with sequence identity of > 60 %, more preferably > 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 polypeptide sequence SEQ ID NO: 26.

Preferred variants of SEQ ID NO: 26 display activity in Assay A under item 1 .3.4.3.1 .

Even more preferably, the activity of the respective variant of SEQ ID NO: 26 is at least 1 %, preferably at least 10 %, more preferably at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 % relative to the activity of SEQ ID NO: 26 as determined in Assay B under item 1 .3.4.3.2.

Even more preferably, the activity of the respective variant of SEQ ID NO: 26 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: 26 as determined in Assay B under item

1.3.4.3.2.

1.3.4.4.14 “Variants of SEQ ID NO: 28’’

In particular, a variant of SEQ ID NO: 28 is a polypeptide with sequence identity of > 60 %, more preferably > 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 polypeptide sequence SEQ ID NO: 28.

Preferred variants of SEQ ID NO: 28 display activity in Assay A under item 1 .3.4.3.1 .

Even more preferably, the activity of the respective variant of SEQ ID NO: 28 is at least 1 %, preferably at least 10 %, more preferably at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 % relative to the activity of SEQ ID NO: 28 as determined in Assay B under item 1 .3.4.3.2.

Even more preferably, the activity of the respective variant of SEQ ID NO: 28 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: 28 as determined in Assay B under item

1.3.4.3.2.

1.3.4.5 Method conditions

The reaction in step (b) according to Method I may be carried out under conditions known to the skilled person.

The reaction medium in which bialaphos is reacted to give L-glufosinate is preferably aqueous, more preferably an aqueous buffer. Exemplary buffers commonly used in biotransformation reactions and advantageously used herein include Tris, phosphate, or any of Good's buffers, such as 2-(/V-morpholino)ethanesulfonic acid (“MES”), /V-(2-acetamido)iminodiacetic acid (“ADA”), piperazine-/V,/\/'-bis(2-ethanesulfonic acid) (“PIPES”), /V-(2-acetamido)-2- aminoethanesulfonic acid (“ACES”), P-hydroxy-4- morpholinepropanesulfonic acid (“MOPSO”), cholamine chloride, 3-(/V-morpholino)propanesulfonic acid (“MOPS”), /V,/V-Bis(2-hydroxyethyl)- 2-aminoethanesulfonic acid (“BES”), 2-[[1 ,3-dihydroxy-

2-(hydroxymethyl)propan-2- yl]amino]ethanesulfonic acid (“TES”), 4-(2-hydroxyethyl)-

1 -piperazineethanesulfonic acid (“HEPES”), 3-(Bis(2-hydroxyethyl)amino)-2-hydroxypropane- 1 -sulfonic acid (“DIPSO”), acetamidoglycine,

3-(/V-Tris(hydroxymethyl)methylamino(-2-hydroxypropane)su lfonic acid (“TAPSO”), piperazine- /V,/\/'-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”).

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

Preferably, step (b) according to Method I is carried out in a phosphate buffer.

The pH of the reaction medium in step (b) of the method is preferably in the range of from 2 to 10, more preferably in the range of from 5 to 8, most preferably 6.5 to 7.5, more preferably 7.0.

The method according to the invention is preferably carried out at a temperature in the range of from 20 °C to 70 °C, more preferably in the range of from 30 °C to 55 °C, more preferably 30 °C to 50 °C, more preferably 30 °C.

2. Second Aspect: Method for controlling a weed plant W

The present invention may advantageously be applied in the context of agriculture. It allows for a precise application of LGA to crops and weed. In this context, BIAP may be applied to weed as the “masked” herbicide which is “armed” (or “demasked”) by reacting it to give LGA under the catalysis of a C-terminal processing peptidase Ei. This specific activation of the herbicidal effect improves the efficiency of the treatment, because less herbicide needs to be applied.

Therefore, in a second aspect, the present invention relates to a method for controlling a weed plant W, comprising

(i) contacting the weed plant W with bialaphos and a C-terminal processing peptidase Ei; (ii) reacting bialaphos to give L-glufosinate in an amount that is herbicidally effective for weed W, wherein the reaction is catalyzed by the C-terminal processing peptidase Ei;

(iii) contacting the weed plant W is contacted with LGA in an amount that is herbicidally effective for weed plant W, causing an herbicidal effect on the weed plant W.

Hereinafter, the “method for controlling a weed plant W” according to the second aspect of the invention is also abbreviated as “Method II”.

In a preferred embodiment of Method II, also a glufosinate-tolerant crop plant C is contacted with bialaphos and a C-terminal processing peptidase Ei in step (i). In a particular embodiment, the glufosinate-tolerant crop plant C is resistant to the effect of LGA, i.e. the L-glufosinate, does not cause an herbicidal effect on the crop plant C. In particular, in step (ii) of Method II, bialaphos is reacted to give L-glufosinate in an amount that is herbicidally effective for weed W but not herbicidally effective for crop plant C wherein the reaction is catalyzed by the C-terminal processing peptidase E and in step (iii) the weed plant W is contacted with LGA in an amount that is herbicidally effective for weed plant W, causing an herbicidal effect on the weed plant W, while the crop plant C is either not contacted by L-glufosinate or contacted with LGA in an amount that is not herbicidally effective for weed plant W. More preferably, the crop plant C is then contacted with LGA in an amount that is not herbicidally effective for crop plant C.

2.1 Definitions

The term herbicide, as used herein, means an active ingredient that kills, controls or otherwise adversely modifies the growth of plants.

According to the invention, a “herbicidally effective amount” is an amount of active ingredient that causes a “herbicidal effect”, i.e. an adversely modifying effect and includes deviations from natural development, killing, regulation, desiccation, retardation. In particular, the herbicidal effect is exerted on weed W. The weed W is thus not glufosinate-tolerant. “Weed W” or “Weed plant W” may be used interchangeably herein.

According to the invention, “plants” and “vegetation” includes germinant seeds, emerging seedlings, plants emerging from vegetative propagules, and established vegetation.

According to the invention, “immature vegetation” refers to small vegetative plants prior to reproductive stage, and “mature vegetation” refers to vegetative plants during and after reproductive stage. According to the invention, a glufosinate-tolerant plant or crop refers to plants or crops, in particular crop C, that is genetically modified to be tolerant to glufosinate, i.e. L-glufosinate and D-glufosinate, in particular L-glufosinate. “Crop C” or “Crop plant C” may be used interchangeably herein.

Glufosinate tolerance can be provided, for example, by the pat gene (US 5,587,903 A) or by other genes providing transgenic crop tolerance to glufosinate, e.g., bar (US 5,561 ,236 A) and dsm2 (WO 2008/070845 A2).

Likewise, tolerance to other herbicides such 2,4-dichlorophenoxyacetic acid 2-hydroxy- /V,/V,/V-trimethylethanaminium 2-(2,4-dichlorophenoxy)acetate (abbreviated as “2,4-D”) may be conferred to plants or crops, as described e.g. in WO 2015/089014 A1 and WO 2015/089015 A1.

For example, 2,4-D-tolerant soybeans refer to soybeans that are genetically modified to be tolerant to 2,4-D. Examples of 2,4-D tolerant soybeans include soybeans containing the aad-12 gene which confers tolerance to 2,4-D (US 8,283,522 B2). As used herein, 2,4-D-tolerant corn refers to corn that is genetically modified to be tolerant to 2,4-D. Examples of 2,4-D tolerant corn include corn containing the aad-1 gene which confers tolerance to 2,4-D (US 7,838,733 B2). As used herein, 2,4-D-tolerant cotton refers to cotton that is genetically modified to be tolerant to 2,4-D. Examples of 2,4-D tolerant cotton include cotton containing the aad-12 gene which confers tolerance to 2,4-D. However, tolerance in each of these crops by the aad-1 or aad-12 genes or with alternative genes providing additional or alternative tolerance to transgenic crops [e.g., aad-13 (US 8,278,505 B2), tfdA (US 6,153,401 A), or 24dt02 (CN 103060279 A)] is considered to be included within the scope of the 2,4-D-tolerant plants or crops such as soybeans, corn, or cotton.

In the context of the invention, “a weed plant W” (or simply “weed W”) refers to any undesired plant in an outdoor or indoor cultivation. In particular, a weed plant W grows in concurrence to crop plants C. In particular, the weed W is selected from the group consisting of Abutilon theophrasti, Alopecurus myosuroides, Amaranthus species, in particular Amaranthus palmeri, Ambrosia artemisiifolia, Ambrosia psilostachya, Ambrosia trifida, Anoda cristata, Asclepias syriaca, Avena fatua, Bidens Pilosa, Borreria species, in particular Borreria alata (or Spermacoce alata or Spermacoce latifolia), Brachiaria decumbens (or Urochloa decumbens), Brachiaria brizantha (or Urochloa brizantha), Brachiaria platyphylla (or Urochloa platyphylla), Brachiaria plantaginea (or Urochloa plantaginea), Cassia obtusifolia, Cenchrus echinatus, Centaurea maculosa, Chenopodium album, Cirsium arvense, Commelina benghalensis, Convolvulus arvensis, Conyza canadensis, Conyza sumatrensis, Cyperus esculentus, Cyperus rotundus, Datura stramonium, Daucus carota, Digitaria horizontalis, Digitaria insularis, Digitaria sanguinalis, Echinochloa crus- galli, Echinochloa coIonum, Eleusine indica, Erigeron bonariensis (or Conyza bonariensis), Erigeron canadensis (Conyza canadensis), Euphorbia dentata, Euphorbia esula, Euphorbia heterophylla, Euphorbia hirta (or Chamaesyce hirta), Ipomoea hederacea, Ipomoea lacunosa, Jacquemontia tamnifolia, Lactuca serriola, Lolium multiflorum, Panicum dichotomiflorum, Panicum miliaceum, Plantago lanceolata, Portulaca oleracea, Richardia species, Rumex obtusifolius, Salsola tragus, Sesbania exaltata, Setaria faberi, Setaria viridis, Sida species, in particular Sida spinosa, Sinapis arvensis, Solanum ptychanthum, Solidago species, Sonchus arvensis, Sorghum halepense, Sorghum bicolor, Taraxacum officinale, Tridax procumbens, Trifolium repens, Urtica dioica, Xanthium strumarium. Weed plants W are, in particular, not tolerant to LGA, which means that LGA, when applied to them in a “herbicidally effective amount” causes a “herbicidal effect” on them.

In the context of the invention, “a crop plant C” (or simply “crop C”) refers to any desired plant in an outdoor or indoor cultivation. In particular, the crop C is selected from the group consisting of tree and vine orchards, fruiting crops, cereal crops, plantation crops.

In particular, tree and vine orchards are selected from the group consisting of citrus, grapes, almond, apple, apricot, avocado, beechnut, Brazil nut, butternut, cashew, cherry, chestnut, chinquapin, crab apple, date, feijoa, fig, filbert, hickory nut, juniper, kiwi, lemon, lime, loquat, macadamia nut, mandarins, mayhaws, nectarine, olives, oranges, peach, pear, pecan, persimmon, pistachio, plum, pomegranates, pome fruit, prune, pumpkin, rose hip, sea buckthorn, service tree, sorb tree, stone fruit, tree nuts, quince, walnut.

In particular, fruiting crops are selected from the group consisting of blueberries, guava, papaya, strawberries, taro, blackberries, raspberries.

In particular, plantation crops are selected from the group consisting of coffee, cotton, cacao, palm oil, rubber, soybean, tea.

In particular, cereal crops are selected from the group consisting of barley, corn, emmer, lentils, oats, rice, rye, sorghum, spelt, sunflower, wheat.

Crop plants C are, in particular, tolerant to LGA, which means in particular that the herbicidally effect amount of LGA needed to cause a “herbicidal effect” on them is larger than, preferably at least 2 times, even more preferably at least 5 times, even more preferably at least 10 times, even more preferably at least 50 times, than the herbicidally effective amount of LGA needed to cause a “herbicidal effect” on the weed plant W which is not LGA-tolerant. 2.2 Step (i) of the method for controlling weed

In step (i) of Method II, the weed W is contacted with bialaphos and a C-terminal processing peptidase Ei.

BIAP employed in step (i) of Method II may be obtained as described under item 1 .2 above for Method I.

The C-terminal processing peptidase Ei employed in step (i) of Method II is as described under item 1 .3.2 above for Method I.

In particular, the C-terminal processing peptidase Ei that may be used in step (i) of Method II may be a C-terminal processing peptidases categorized in the EC class 3.4.21.102.

In particular, the polypeptide sequence of the C-terminal processing peptidase Eithat may be used in step (i) of Method II may be selected from the group consisting of SEQ ID NO: 2 and variants of SEQ ID NO: 2, SEQ ID NO: 4 and variants of SEQ ID NO: 4, SEQ ID NO: 6 and variants of SEQ ID NO: 6, SEQ ID NO: 8 and variants of SEQ ID NO: 8, SEQ ID NO: 10 and variants of SEQ ID NO: 10, SEQ ID NO: 12 and variants of SEQ ID NO: 12, SEQ ID NO: 14 and variants of SEQ ID NO:

14, SEQ ID NO: 16 and variants of SEQ ID NO: 16, SEQ ID NO: 18 and variants of SEQ ID NO:

18, SEQ ID NO: 20 and variants of SEQ ID NO: 20, SEQ ID NO: 22 and variants of SEQ ID NO:

22, SEQ ID NO: 24 and variants of SEQ ID NO: 24, SEQ ID NO: 26 and variants of SEQ ID NO:

26, SEQ ID NO: 28 and variants of SEQ ID NO: 28, more preferably is selected from the group consisting of SEQ ID NO: 2 and variants of SEQ ID NO: 2.

In an even more preferred embodiment of the method of the present invention, the polypeptide sequence of the C-terminal processing peptidase Eithat may be used in step (i) of Method II may be selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, more preferably SEQ ID NO: 2.

The C-terminal processing peptidase Ei that may be used in step (i) of Method II may be obtained as described under item 1 .3.3 above for Method I.

The bialaphos and the C-terminal processing peptidase Ei used in step (i) of Method II may be applied to the weed W by any means known to the skilled person.

In one preferred embodiment of step (i) of Method II, the weed W may be contacted with two separate solutions S _Bi and S _B 2, wherein S _Bi contains bialaphos and is in particular an aqueous solution, and S _B 2 contains a C-terminal processing peptidase Ei, and is in particular an aqueous solution, so that solutions S _Bi and S _B2 are mixed on the weed to give solution S _B . In this preferred embodiment, the solutions S _Bi and S _B2 may be applied to the weed sequentially or simultaneously.

In an alternative embodiment of step (i) of Method II, solution S _B containing bialaphos and a C-terminal processing peptidase Ei is mixed before it contacts the weed. In this alternative embodiment, two separate solutions S _Bi and S _B2 , wherein S _Bi contains bialaphos and is in particular an aqueous solution, and S _B2 contains an C-terminal processing peptidase Ei, and is in particular an aqueous solution, are prepared separately and then mixed to give solution S _B which is then applied to the weed W in step (i) of Method II.

The way of contacting the weed with solution S _B or solutions S _Bi and S _B2 may be carried out by means known to the skilled person.

For example, in a greenhouse, solution S _B (or solutions S _Bi and S _B2 ) may be sprayed on the weed, in particular by humans or machines (robots). In the outdoor area, solution S _B may be applied to the weed by agricultural machinery, such as tractors, or by planes such as crop dusters.

It is advantageous that solution S _B is a buffer in which the reaction according to step (ii) of Method II may be carried out.

2.3 Step (ii) of the method for controlling weed

In step (ii) of Method II, bialaphos is reacted to give L-glufosinate in an amount that is herbicidally effective for weed W, wherein the reaction is catalyzed by the C-terminal processing peptidase Ei. The reaction in step (ii) according to Method II may be carried out under conditions known to the skilled person.

In particular, the reaction medium in which bialaphos is reacted to give L-glufosinate is preferably aqueous, more preferably an aqueous buffer.

Exemplary buffers commonly used in biotransformation reactions and advantageously used herein include Tris, phosphate, or any of Good's buffers, such as 2-(/V-morpholino)ethanesulfonic acid (“MES”), /V-(2-acetamido)iminodiacetic acid (“ADA”), piperazine-/V,/\/'-bis(2-ethanesulfonic acid) (“PIPES”), /V-(2-acetamido)-2- aminoethanesulfonic acid (“ACES”), P-hydroxy- 4-morpholinepropanesulfonic acid (“MOPSO”), cholamine chloride, 3-(/V- morpholino)propanesulfonic acid (“MOPS”), /V,/V-Bis(2-hydroxyethyl)- 2-aminoethanesulfonic acid (“BES”), 2-[[1 ,3-dihydroxy-2-(hydroxymethyl)propan-2- yl]amino]ethanesulfonic acid (“TES”), 4-(2- hydroxyethyl)-1 -piperazineethanesulfonic acid (“HEPES”), 3-(Bis(2-hydroxyethyl)amino)-2- hydroxypropane-1 -sulfonic acid (“DIPSO”), acetamidoglycine, 3-(/V-Tris(hydroxymethyl)methylamino(-2-hydroxypropane)sulfo nic acid (“TAPSO”), piperazine- /V,/\^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”).

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

Preferably, step (ii) according to Method II is carried out in a phosphate buffer.

The pH of the reaction medium in step (b) of the method is preferably from 2 to 10, more preferably from 5 to 8, more preferably from 6.5 to 7.5, more preferably from 6.8 to 7.2, most preferably 7.0.

The method according to the invention is preferably carried out at a temperature from 20 °C to 70 °C, more preferably from 30 °C to 55 °C, more preferably from 30 °C to 50 °C, more preferably of 30 °C.

As these are the usual environmental conditions, the reaction according to step (ii), Method II, may take place on the weed W outdoors as well indoors as soon as BIAP and Ei are applied to the weed W so that BIAP reacts to LGA under the catalytic effect of the C-terminal processing peptidase Ei.

2.4 Step (Hi) of the method for controlling weed

In step (iii) of Method II, the weed plant W is contacted with LGA in an amount that is herbicidally effective for weed plant W, causing an herbicidal effect on the weed plant W.

The absolute amount of LGA necessary to be herbicidally effective depends on the weed W to be treated but may be determined in each case by the skilled person.

In a preferred embodiment of Method II, and in particular of step (i), also a glufosinate-tolerant crop plant C is contacted with bialaphos and a C-terminal processing peptidase Ei. In a particular embodiment, the glufosinate-tolerant crop plant C is resistant to the effect of LGA. This means that in particular, the amount of LGA produced in step (ii) is not sufficient to cause an herbicidal effect on the crop plant C, but is sufficient to cause an herbicidal effect on the weed plant W.

By the Method II, site-specific application of the herbicide LGA is possible, allowing for a more ecological and economic application of this herbicide. Examples

1. Materials and Methods

1.1 Bacillus strains used for the invention

1.2 Streptomyces strain for Bialaphos production

Streptomyces viridochromogenes DSM40110

1.3 Incubation of Bacillus strains

Selected single colonies of each Bacillus strain were used to start cultivation in LB broth (MILLER) from Merck (Darmstadt, Germany; Cat. no. 110285). The liquid cultures (10 ml liquid medium per 100 ml Erlenmeyer flask with 3 baffles) were incubated overnight up to an OD600 of 5.0 in the Infers HT Multitron standard incubator shaker from Infers GmbH (Einsbach, Germany) at 30 °C and 200 rpm. The concentration of the polypeptide in the supernatant may be determined by SDS page and analysis of the respective bands via the software GelQuant® (BiochemLabSolutions).

1.4 Incubation of Streptomyces viridochromogenes DSM40110

Selected single colonies of Streptomyces viridochromogenes DSM40110 strain was used to start cultivation in HA liquid medium. The liquid cultures (10 ml liquid medium per 100 ml Erlenmeyer flask with 3 baffles) were incubated overnight in the Infers HT Multitron standard incubator shaker from Infers GmbH (Einsbach, Germany) at 28 °C and 120 rpm. The HA liquid medium had the following composition: HA medium (pH = 7.3)

1.5 Production of L-glufosinate

After overnight incubation the cultures were centrifuged for 6 min at 4 °C and 4000 rpm and 150 pl of each Bacillus supernatant were used to react with 21 mg/l Bialaphos produced by Streptomyces viridochromogenes DSM40110. Therefore, 150 pl of the different Bacillus supernatants were each mixed with 150 pl of Streptomyces viridochromogenes DSM40110 supernatant. After 16 h incubation at 30 °C the suspension was analyzed by LC-MS QQQ.

1.6 Analytical Method

All measurements were carried out with an LC-MS QQQ. The HPLC used belongs to the 1260 Infinity series, which is coupled with a 6420 Triple-Quadropol mass spectrometer with electrospray ionization. A Luna HILIC (100 x 2 mm; 3 pm) from Phenomenex at 35 °C was used as the separation column. By means of a gradient (0.50 min, 5 % A - 95 % B, 1 .2 min 45 % A - 55 % B, 4.0 min 45 % A - 55 % B, 4.1 min 100 % A, 7.0 min 100 % A, 7.1 min 5 % A-95 % B, 11 min 5 % A - 95 % B) of buffer A (water + 10OmM ammonium acetate + 0.1 % formic acid) and buffer B (acetonitrile + 0.1% formic acid) and a flow of 0.6 mL/min, the elution of glufosinate occurs at 3.6 minutes. The injection volume was 2 pL.

Glufosinate was measured in positive MRM mode (Precursor Ion 182.06, Product Ion 136) with a fragmentor voltage of 88 V and a collisions energy of 5 V. The gas temperature of the source was 350°C with a flow of 12 L/min, nebulizer 15psi and the capillary voltage 4000 V. To calculate the data, the peak area was used in a quadratic function without zero pass.

2. Results

The following table shows whether conversion of BIAP to LGA could be observed for the respective Bacillus strain in the supernatant.

Tab. 2: Conversion of bialaphos to L-glufosinate using supernatants of different Bacillus strains described in the invention. (*> In column “conversion bialaphos L-glufosinat?”, “yes” or “no” is defined by analyzing each sample by the above described analytical method; n The respective sequences were identified via EDGAR as described under chapter 4.3; (***> For Bacillus subtilis subsp. spizizenii, the concentration of LGA was also quantified and found to be 16.27 mg/l (mean value of two independent cultivations). 3. Identification of the extracellular protease by genomic data based analysis using software toll EDGAR and SignalP

For each of the Bacillus strains used for the described experiments whole genome seguencing was done. For analyzing the genome seguence the software tool EDGAR (Efficient Database framework for comparative Genome Analyses using BLAST score Ratios) was used.

The software tool EDGAR facilitates comparative genome analysis covering the computation of genomic subsets like the core genome, singletons and pan-genomes (Blom et al.; Dieckmann et al.). EDGAR was applied for the calculation of a gene set for 16 Bacillus strains showing a specific wet lab phenotype. Therefore, a private EDGAR project was established containing these strains. The genome seguences were established by “Single Molecule, Real-Time” (SMRT) seguencing using polymerase version six and chemistry version four (P6-C4) (Pacific Biosciences) and Illumina paired-end seguencing (2 x 300 bp or 2 x 150 bp). The genome assembly was performed with Unicycler (vO.4.7; Wick et al.) or HGAP (v3; Chin et al.). Genome seguences (unpublished data) of all strains were annotated using Prokka version 1.14.5 (Seemann).

The EDGAR “gene set” calculation generates complex genomic subsets based on the calculation of orthologous genes. To define an orthologous gene, EDGAR is designed to use bidirectional hits of the alignment tool BLAST (Altschul et al.) and selects a specific threshold over a given alignment length. In practice, for the “gene set” calculation genomes are selected as “INCLUDE”, “EXCLUDE”, or “IGNORE” (which is the default). Gene sets are calculated such that there has to be a set of orthologous genes in all “included” genomes while there must not be any ortholog to one of the “excluded” genomes. More specifically, genes which are included in the 14 “active wetlab” strains were selected as “INCLUDE” and 2 “inactive wet-lab” strains were selected as “EXCLUDE”. Finally, the computed gene set covered 319 orthologous gene groups. Automatic annotations of the underlying genes were filtered to „proteases“ reducing the set to five orthologous gene groups. Associated genes were translated into proteins using CLC Genomics Workbench 21.0.4 (Qiagen) and were checked for signal peptide signatures using SignalP version 5. SignalP is a bioinformatic tool that can predict signal peptide sequences in the amino terminus of many newly synthesized proteins that target proteins into, or across, membranes (Almagro Armenteros et al.). With signal peptide prediction, one orthologous gene group was identified encoding C-terminal processing peptidases.

Finally, these C-terminal processing peptidases (“CtpA”; polypeptide sequences of SEQ ID No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and SEQ ID No. 28) were identified to catalyze the conversion of Bialaphos to L-glufosinate. This opens a new pathway for the enantioselective synthesis of LGA, as it makes use of a new starting material (BIAP), from which LGA may be synthesized.

Also, the LGA synthesis according to the invention may advantageously be used in the area of weed control, as it allows for site-specific application of LGA as herbicide. Namely, BIAP is selectively “activated” by cleaving it to give LGA only at those locations where the enzyme Ei is applied. By this, application of LGA in areas where its effects are not desired is avoided.

4. Further References

Almagro Armenteros JJ, Tsirigos KD, S0nderby CK, Petersen TN, Winther O, Brunak S, von Heijne G, Nielsen H. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat Biotechnol. 2019 Apr;37(4):420-423. doi: 10.1038/s41587-019-0036-z. Epub 2019 Feb 18. PMID: 30778233. Abbreviated as “Almagro Armenteros et al."

Blom J, Albaum SP, Doppmeier D, Piihler A, Vorholter FJ, Zakrzewski M, Goesmann A. EDGAR: a software framework for the comparative analysis of prokaryotic genomes. BMC Bioinformatics.

2009 May 20; 10: 154. doi: 10.1186/1471-2105-10-154. PMID: 19457249; PMCID: PMC2696450. Abbreviated as “Blom et al."

Chin CS, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, Clum A, Copeland A, Huddleston J, Eichler EE, Turner SW, Korlach J. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat Methods. 2013 Jun;10(6):563-9. doi:

10.1038/nmeth.2474. Epub 2013 May 5. PMID: 23644548. Abbreviated as “Chin et al."

Dieckmann MA, Beyvers S, Nkouamedjo-Fankep RC, Hanel PHG, Jelonek L, Blom J, Goesmann A. EDGAR3.0: comparative genomics and phylogenomics on a scalable infrastructure. Nucleic Acids Res. 2021 Jul 2;49(W1):W185-W192. doi: 10.1093/nar/gkab341. PMID: 33988716; PMCID: PMC8262741. Abbreviated as “Dieckmann et al."

Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014 Jul 15;30(14):2068-9. doi: 10.1093/bioinformatics/btu153. Epub 2014 Mar 18. PMID: 24642063. Abbreviated as “Seemann”.

Wick RR, Judd LM, Gorrie CL, Holt KE. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol. 2017 Jun 8;13(6):e1005595. doi:

10.1371/journal.pcbi.1005595. PMID: 28594827; PMCID: PMC5481147. Abbreviated as “Wick et al."