WO2023222226A1 - Procédé enzymatique pour la production de l-glufosinate - Google Patents

Procédé enzymatique pour la production de l-glufosinate Download PDF

Info

Publication number
WO2023222226A1
WO2023222226A1 PCT/EP2022/063546 EP2022063546W WO2023222226A1 WO 2023222226 A1 WO2023222226 A1 WO 2023222226A1 EP 2022063546 W EP2022063546 W EP 2022063546W WO 2023222226 A1 WO2023222226 A1 WO 2023222226A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
variants
epr
range
glufosinate
Prior art date
Application number
PCT/EP2022/063546
Other languages
English (en)
Inventor
Stefan Pelzer
Alexandra TILKER
Frank JANKOWITSCH
Ludger Lautenschütz
Markus PÖTTER
Original Assignee
Evonik Operations Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Evonik Operations Gmbh filed Critical Evonik Operations Gmbh
Priority to PCT/EP2022/063546 priority Critical patent/WO2023222226A1/fr
Priority to ARP230101208A priority patent/AR129337A1/es
Publication of WO2023222226A1 publication Critical patent/WO2023222226A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P13/00—Herbicides; Algicides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C12N15/8277—Phosphinotricin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)

Definitions

  • 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 an Epr serine protease Ei, which may be categorized in the EC class 3.4.21.-.
  • the L-glufosinate obtained in the method may be used as herbicide.
  • the present invention relates to a method for controlling a weed plant W.
  • bialaphos is cleaved to give an herbicidally active amount of L-glufosinate, wherein the reaction is catalyzed by the Epr serine protease 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.
  • Organic phosphorous compounds i.e. chemical agents comprising a carbon-phosphor bond
  • herbicides i.e. chemical agents comprising a carbon-phosphor bond
  • 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).
  • esters of P-methyl phosphinic acid 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. Republicer Zeit 2003, 37, 128 - 138): a.
  • 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.
  • Acrylic acid ester is a cheaper alternative starting material.
  • L-glufosinate 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.
  • 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.
  • WO 2020/145513 A1 and WO 2020/145514 A1 describe a chemical route to LGA.
  • 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.
  • 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.
  • PPG 2-oxo-4-[hydroxy(methyl)phosphinoyl]- butyric acid
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 an Epr serine protease Ei.
  • the present invention relates to a method for controlling a weed plant W, comprising:
  • 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 an Epr serine protease Ei.
  • Method I “Method according to the first aspect of the invention” is hereinafter abbreviated as “Method I”
  • L-glufosinate [herein “LGA”; CAS-Nr. 35597-44-5; other names “(S)-glufosinate”, “(-)-glufosinate”] has the structure according to the following formula (I):
  • Bialaphos [ althoughL-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 Arthur 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.
  • the BIAP used in step (b) is produced by chemical or biotechnological synthesis in a step (a1).
  • step (a1) precedes step (b). More preferably, in step (a1), BIAP is produced by biotechnological synthesis.
  • the BIAP employed in step (b) is produced by biotechnological synthesis in a step (a1).
  • 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.
  • 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.
  • 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.
  • BIAP is produced by the following method:
  • 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.
  • the BIAP is separated from the aqueous medium Aq1 before it is employed in step (b) of Method I. 1.3 Enzyme Ei
  • Step (b) is enzymatically catalyzed.
  • 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.
  • 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.
  • Epr serine protease 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.
  • 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.
  • 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 Epr serine protease Ei used in the method of the present invention may be isolated.
  • isolated 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.
  • 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.
  • 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.
  • media 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.
  • 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.
  • Step (b) of Method I is catalysed by an Epr serine protease Ei.
  • Epr serine protease 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.
  • Epr serine protease Ei that may be used in step (b) of Method I may be an Epr serine protease categorized in the EC class 3.4.21 .-.
  • the Epr serine protease 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.
  • Epr serine protease Ei may be used for the catalysis of cleaving BIAP was surprising and provides for advantageous applications.
  • 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/).
  • BRENDA Braunschweig Enzyme Database
  • NCBI National Center for Biotechnological Information
  • KEGG Kyoto Encyclopedia of Genes and Genomes
  • the reaction in step (b) of Method I is catalyzed by an Epr serine protease Ei, wherein the polypeptide sequence of the Epr serine protease 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: 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 variant
  • SEQ ID NO: 2 and variants of SEQ ID NO: 2.
  • the reaction in step (b) of Method I is catalyzed by an Epr serine protease Ei, wherein the polypeptide sequence of the Epr serine protease 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 polypeptide sequence of the Epr serine protease 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
  • 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.
  • 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.
  • 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.
  • 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.
  • expression, in particular overexpression is abbreviated as (over)expression”
  • expression, in particular overexpress is abbreviated as “(over)express”
  • (over)express) 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.
  • 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
  • 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).
  • 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.
  • tac lac operon of E. coll.
  • lactose or isopropyl B-D-thiogalactopyranoside (IPTG) is used as inducer.
  • systems using arabinose e.g. the pBAD system
  • rhamnose e.g. E. coll KRX
  • 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.
  • 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.
  • enzyme assays with subsequent LC-ESI-MS detection of the products can then be carried out.
  • 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.
  • chromatographic methods such as nickel-nitrilotriacetic acid affinity chromatography, streptavidin affinity chromatography, gel filtration chromatography or ion-exchange chromatography
  • nucleic acid or polypeptide 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.
  • step (b) the Epr serine protease 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).
  • cells C Ei capable of producing the Epr serine protease 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 an Epr serine protease Ei.
  • the aqueous medium Aq2 comprising an Epr serine protease Ei obtained in step (a21) may then directly be employed in Method I, step (b).
  • the Epr serine protease Ei comprised by Aq2 is at least partially separated from Aq2 and the cells C E i. Such separation might be performed by the skilled person, in particular by methods described herein.
  • the cells C Ei are at least partially lysed in the aqueous medium Aq2 comprising an Epr serine protease Ei after step (a21).
  • step (a2) in a step (a22) following step (a21), the Epr serine protease Ei in the aqueous medium Aq2 is at least partially separated from the aqueous medium Aq2, so that a purified Epr serine protease 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 Epr serine protease Ei , and further Epr serine protease Ei may
  • step (a22) further aqueous medium Aq21 may be added to Aq21*.
  • Epr serine protease Ei obtained in step (a2), and in particular after step (a21) or (a22), respectively, may then be used in step (b) of Method I.
  • 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 65%, more preferably at least 66%, more preferably
  • 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.
  • 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 Epr serine protease, is preserved.
  • 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 et al.”).
  • 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).
  • 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 BLOSUM62 matrix is typically used applying the default settings (gap weight: 12, length weight: 1).
  • a sequence identity of 80% according to the above algorithm means 80% homology. The same applies to higher identities.
  • 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/).
  • 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.
  • 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:
  • the activity of the variant to be tested is determined by the following steps A1 .1), A1 .2), and A1 .3) as follows:
  • 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.
  • 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.
  • A2.1 150 pl of an aqueous solution containing phosphate buffer (0.1 M, pH 7.0) prepared and heated to 30 °C.
  • 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.
  • 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.
  • 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.
  • the reaction in steps A 1 .3) and A2.3) can also be stopped by adding methanol, preferably 1 ml of methanol.
  • 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. is carried out by the following steps:
  • 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
  • B1 .1 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
  • 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.
  • steps B1 .1), B1 .2), and B1 .3) are repeated with the variant:
  • 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.
  • Preferable formate solutions in steps B1.3) and B2.3) are ammonium formate or sodium formate solutions.
  • the reaction in steps B1.3) and B2.3) can also be stopped by adding methanol, preferably 1 ml of methanol.
  • 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:
  • 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 .
  • 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.
  • 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
  • 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 .
  • 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.
  • 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
  • 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 .
  • 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.
  • 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
  • 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 .
  • 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.
  • 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
  • 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 .
  • 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.
  • 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
  • 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 .
  • 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.
  • 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
  • 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 .
  • 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.
  • 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
  • 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 .
  • 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.
  • 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
  • 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 .
  • 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.
  • 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
  • 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 .
  • 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.
  • 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
  • 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 .
  • 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.
  • 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
  • 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 .
  • 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.
  • 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
  • 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 .
  • 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.
  • 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
  • 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 .
  • 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.
  • 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
  • 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]ethanes
  • ammonium can act as a buffer.
  • One or more organic solvents can also be added to the reaction.
  • 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.
  • 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.
  • 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 an Epr serine protease Ei. This specific activation of the herbicidal effect improves the efficiency of the treatment, because less herbicide needs to be applied.
  • the present invention relates to a method for controlling a weed plant W, comprising
  • Method II the “method for controlling a weed plant W” according to the second aspect of the invention is also abbreviated as “Method II”.
  • a glufosinate-tolerant crop plant C is contacted with bialaphos and an Epr serine protease Ei in step (i).
  • 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.
  • 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 Epr serine protease 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.
  • herbicide means an active ingredient that kills, controls or otherwise adversely modifies the growth of plants.
  • 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.
  • 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.
  • plants and “vegetation” includes germinant seeds, emerging seedlings, plants emerging from vegetative propagules, and established vegetation.
  • “immature vegetation” refers to small vegetative plants prior to reproductive stage
  • “mature vegetation” refers to vegetative plants during and after reproductive stage
  • 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).
  • pat gene US 5,587,903 A
  • other genes providing transgenic crop tolerance to glufosinate, e.g., bar (US 5,561 ,236 A) and dsm2 (WO 2008/070845 A2).
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • a weed plant W refers to any undesired plant in an outdoor or indoor cultivation.
  • a weed plant W grows in concurrence to crop plants C.
  • 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
  • Crop C refers to any desired plant in an outdoor or indoor cultivation.
  • the crop C is selected from the group consisting of tree and vine orchards, fruiting crops, cereal crops, plantation crops.
  • 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.
  • fruiting crops are selected from the group consisting of blueberries, guava, papaya, strawberries, taro, blackberries, raspberries.
  • plantation crops are selected from the group consisting of coffee, cotton, cacao, palm oil, rubber, soybean, tea.
  • 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.
  • step (i) of Method II the weed W is contacted with bialaphos and an Epr serine protease Ei.
  • BIAP employed in step (i) of Method II may be obtained as described under item 1 .2 above for Method I.
  • Epr serine protease Ei employed in step (i) of Method II is as described under item 1 .3.2 above for Method I.
  • the Epr serine protease Ei that may be used in step (i) of Method II may be an Epr serine protease categorized in the EC class 3.4.21 .-.
  • the polypeptide sequence of the Epr serine protease 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: 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
  • 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.
  • the polypeptide sequence of the Epr serine protease 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.
  • Epr serine protease 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 Epr serine protease Ei used in step (i) of Method II may be applied to the weed W by any means known to the skilled person.
  • 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 B2 contains an Epr serine protease 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 .
  • the solutions S Bi and S B2 may be applied to the weed sequentially or simultaneously.
  • solution S B containing bialaphos and an Epr serine protease Ei is mixed before it contacts the weed.
  • 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 Epr serine protease 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.
  • solution S B or solutions S Bi and S B2
  • solution S B may be sprayed on the weed, in particular by humans or machines (robots).
  • solution S B may be applied to the weed by agricultural machinery, such as tractors, or by planes such as crop dusters.
  • solution S B is a buffer in which the reaction according to step (ii) of Method II may be carried out.
  • 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 Epr serine protease Ei.
  • the reaction in step (ii) according to Method II may be carried out under conditions known to the skilled person.
  • 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]ethanes
  • ammonium can act as a buffer.
  • One or more organic solvents can also be added to the reaction.
  • 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.
  • 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 Epr serine protease Ei.
  • 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.
  • a glufosinate-tolerant crop plant C is contacted with bialaphos and an Epr serine protease Ei.
  • 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.
  • HA liquid medium 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.
  • 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.
  • the software tool EDGAR facilitates comparative genome analysis covering the computation of genomic subsets like the core genome, singletons and pan-genomes (Blom etal. Dieckmann etal.). 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 EDGAR “gene set” calculation generates complex genomic subsets based on the calculation of orthologous genes.
  • 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.
  • BLAST Altschul et al.
  • 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.
  • genes which are included in the 14 “active wet-lab” strains were selected as “INCLUDE” and 2 “inactive wet-lab” strains were selected as “EXCLUDE”.
  • the computed gene set covered 319 orthologous gene groups. Automatic annotations of the underlying genes were filtered to choirproteases“ 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 extracellular minor serine proteases Epr.
  • Epr serine proteases polypeptide sequences of SEQ ID No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and SEQ ID No. 28
  • BIAP new starting material
  • 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.
  • EDGAR a software framework for the comparative analysis of prokaryotic genomes. BMC Bioinformatics.
  • 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.”

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • Pest Control & Pesticides (AREA)
  • Environmental Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne, selon un premier aspect, un procédé de production de L-glufosinate. Le procédé comprend une étape (b) dans laquelle du bialaphos est soumis à une réaction pour donner du L-glufosinate, la réaction selon l'étape (b) étant catalysée par une sérine protéase Epr E1 qui peut être classée dans la catégorie EC 3.4.21.-. Le L-glufosinate obtenu dans le procédé peut être utilisé en tant qu'herbicide. Selon un second aspect, la présente invention concerne un procédé de lutte contre une plante adventice (W). Dans le procédé selon le second aspect de l'invention, le bialaphos est clivé pour produire une quantité à action herbicide de L-glufosinate, la réaction étant catalysée par la sérine protéase Epr E1. La quantité à action herbicide de L-glufosinate qui est ainsi produite entre en contact avec la plante adventice, ce qui permet d'altérer sa croissance ou de conduire à son dépérissement terminal.
PCT/EP2022/063546 2022-05-19 2022-05-19 Procédé enzymatique pour la production de l-glufosinate WO2023222226A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/EP2022/063546 WO2023222226A1 (fr) 2022-05-19 2022-05-19 Procédé enzymatique pour la production de l-glufosinate
ARP230101208A AR129337A1 (es) 2022-05-19 2023-05-16 Método enzimático para producir l-glufosinato

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/EP2022/063546 WO2023222226A1 (fr) 2022-05-19 2022-05-19 Procédé enzymatique pour la production de l-glufosinate

Publications (1)

Publication Number Publication Date
WO2023222226A1 true WO2023222226A1 (fr) 2023-11-23

Family

ID=82067743

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2022/063546 WO2023222226A1 (fr) 2022-05-19 2022-05-19 Procédé enzymatique pour la production de l-glufosinate

Country Status (2)

Country Link
AR (1) AR129337A1 (fr)
WO (1) WO2023222226A1 (fr)

Citations (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995025785A1 (fr) 1994-03-22 1995-09-28 Bio-Technology General Corp. PLASMIDES D'EXPRESSION REGULES PAR UN PROMOTEUR osmB
US5561236A (en) 1986-03-11 1996-10-01 Plant Genetic Systems Genetically engineered plant cells and plants exhibiting resistance to glutamine synthetase inhibitors, DNA fragments and recombinants for use in the production of said cells and plants
US5587903A (en) 1994-06-22 1996-12-24 Yale; Thomas W. Artificial intelligence language program
WO2000058449A1 (fr) 1999-03-29 2000-10-05 California Institute Of Technology Variants d'hydantoinase a proprietes ameliorees et leur utilisation pour la production d'acides amines
US6153401A (en) 1986-08-29 2000-11-28 Hoechst Schering Agrevo Gnbh Microorganisms and plasmids for 2,4-dichlorophenoxyacetic acid (2,4-D) monooxygenase formation and process for the production of these plasmids and strains
DE10031999A1 (de) 1999-09-09 2001-04-19 Degussa Verfahren zur fermentativen Herstellung von D-Pantothensäure unter Verwendung coryneformer Bakterien
WO2008070845A2 (fr) 2006-12-07 2008-06-12 Dow Agrosciences Llc Nouveaux gènes marqueurs selectionnables
US7838733B2 (en) 2004-04-30 2010-11-23 Dow Agrosciences Llc Herbicide resistance genes
US8278505B2 (en) 2007-05-09 2012-10-02 Dow Agrosciences, Llc. Herbicide resistance genes for resistance to aryloxyalkanoate herbicides
US8283522B2 (en) 2005-10-28 2012-10-09 Dow Agrosciences, Llc. Herbicide resistance genes
CN103060279A (zh) 2012-12-25 2013-04-24 北京大北农科技集团股份有限公司 除草剂抗性蛋白质、其编码基因及用途
WO2013072486A1 (fr) 2011-11-16 2013-05-23 Evonik Industries Ag Mutants de la hydantoinase
WO2015089014A1 (fr) 2013-12-10 2015-06-18 Dow Agrosciences Llc Désherbage herbicide synergique et tolérance améliorée de combinaisons de 2,4-d-choline et de glufosinate dans des sojas, du maïs et du coton tolérants au 2,4-d et au glufosinate
CN106083922A (zh) 2016-08-23 2016-11-09 山东省农药科学研究院 一种精草铵膦的制备方法
WO2017151573A1 (fr) 2016-03-02 2017-09-08 Agrimetis, Llc Procédés de production de l-glufosinate
CN108516991A (zh) 2018-05-28 2018-09-11 山东省农药科学研究院 一种精草铵膦的制备方法
WO2019018406A1 (fr) 2017-07-18 2019-01-24 Agrimetis, Llc Procédés de purification de l-glufosinate
WO2020051188A1 (fr) 2018-09-05 2020-03-12 Agrimetis, Llc Procédés d'amélioration des rendements de l-glufosinate
WO2020145513A1 (fr) 2019-01-11 2020-07-16 씨제이제일제당(주) Intermédiaire de l-glufosinate et procédé de préparation de l-glufosinate
WO2020145627A1 (fr) 2019-01-11 2020-07-16 씨제이제일제당(주) Procédé de préparation de glufosinate
CN111662325A (zh) 2019-03-05 2020-09-15 利尔化学股份有限公司 一种制备l-草铵膦的方法

Patent Citations (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5561236A (en) 1986-03-11 1996-10-01 Plant Genetic Systems Genetically engineered plant cells and plants exhibiting resistance to glutamine synthetase inhibitors, DNA fragments and recombinants for use in the production of said cells and plants
US6153401A (en) 1986-08-29 2000-11-28 Hoechst Schering Agrevo Gnbh Microorganisms and plasmids for 2,4-dichlorophenoxyacetic acid (2,4-D) monooxygenase formation and process for the production of these plasmids and strains
WO1995025785A1 (fr) 1994-03-22 1995-09-28 Bio-Technology General Corp. PLASMIDES D'EXPRESSION REGULES PAR UN PROMOTEUR osmB
US5587903A (en) 1994-06-22 1996-12-24 Yale; Thomas W. Artificial intelligence language program
WO2000058449A1 (fr) 1999-03-29 2000-10-05 California Institute Of Technology Variants d'hydantoinase a proprietes ameliorees et leur utilisation pour la production d'acides amines
DE10031999A1 (de) 1999-09-09 2001-04-19 Degussa Verfahren zur fermentativen Herstellung von D-Pantothensäure unter Verwendung coryneformer Bakterien
US7838733B2 (en) 2004-04-30 2010-11-23 Dow Agrosciences Llc Herbicide resistance genes
US8283522B2 (en) 2005-10-28 2012-10-09 Dow Agrosciences, Llc. Herbicide resistance genes
WO2008070845A2 (fr) 2006-12-07 2008-06-12 Dow Agrosciences Llc Nouveaux gènes marqueurs selectionnables
US8278505B2 (en) 2007-05-09 2012-10-02 Dow Agrosciences, Llc. Herbicide resistance genes for resistance to aryloxyalkanoate herbicides
WO2013072486A1 (fr) 2011-11-16 2013-05-23 Evonik Industries Ag Mutants de la hydantoinase
CN103060279A (zh) 2012-12-25 2013-04-24 北京大北农科技集团股份有限公司 除草剂抗性蛋白质、其编码基因及用途
WO2015089014A1 (fr) 2013-12-10 2015-06-18 Dow Agrosciences Llc Désherbage herbicide synergique et tolérance améliorée de combinaisons de 2,4-d-choline et de glufosinate dans des sojas, du maïs et du coton tolérants au 2,4-d et au glufosinate
WO2015089015A1 (fr) 2013-12-10 2015-06-18 Dow Agrosciences Llc Désherbage herbicide synergique à partir de combinaisons de 2,4-d-choline et glufosinate
WO2017151573A1 (fr) 2016-03-02 2017-09-08 Agrimetis, Llc Procédés de production de l-glufosinate
CN106083922A (zh) 2016-08-23 2016-11-09 山东省农药科学研究院 一种精草铵膦的制备方法
WO2019018406A1 (fr) 2017-07-18 2019-01-24 Agrimetis, Llc Procédés de purification de l-glufosinate
CN108516991A (zh) 2018-05-28 2018-09-11 山东省农药科学研究院 一种精草铵膦的制备方法
WO2020051188A1 (fr) 2018-09-05 2020-03-12 Agrimetis, Llc Procédés d'amélioration des rendements de l-glufosinate
WO2020145513A1 (fr) 2019-01-11 2020-07-16 씨제이제일제당(주) Intermédiaire de l-glufosinate et procédé de préparation de l-glufosinate
WO2020145514A1 (fr) 2019-01-11 2020-07-16 씨제이제일제당(주) Procédé de production de l-glufosinate
WO2020145627A1 (fr) 2019-01-11 2020-07-16 씨제이제일제당(주) Procédé de préparation de glufosinate
CN111662325A (zh) 2019-03-05 2020-09-15 利尔化学股份有限公司 一种制备l-草铵膦的方法

Non-Patent Citations (36)

* Cited by examiner, † Cited by third party
Title
"Microbiology Monographs", vol. 23, 2013, SPRINGER VERLAG, article "Corynebacterium glutamicum", pages: 51 - 88
A. KOIDEJ.A. HOCH, MOL. MICROBIOL., vol. 13, 1994, pages 417 - 426
A. S. BOMMARIUSM. KOTTENHAHNH. KLENKK. DRAUZ: "Microbial Reagents in Organic Synthesis'' Series C: Mathematical and Physical Sciences", vol. 381, 1992, SPRINGER SCIENCE+BUSINESS MEDIA, article "A direct route from hydantoins to D-amino acids employing a resting cell biocatalyst with D-hydantoinase and D-carbamoylase acitivity"
A. SCHAFERJ. KALINOWSKIA. PIIHLER, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 60, 1994, pages 756 - 759
A. TAUCHO. KIRCHNERL. WEHMEIERJ. KALINOWSKIA. PIIHLER, FEMS MICROBIOLOGY LETTERS, vol. 123, 1994, pages 343 - 347
ALMAGRO ARMENTEROS JJTSIRIGOS KDSONDERBY CKPETERSEN TNWINTHER OBRUNAK SVON HEIJNE GNIELSEN H.: "SignalP 5.0 improves signal peptide predictions using deep neural networks", NAT BIOTECHNOL., vol. 37, no. 4, 18 February 2019 (2019-02-18), pages 420 - 423, XP036900634, DOI: 10.1038/s41587-019-0036-z
B. J. EIKMANNSE. KLEINERTZW. LIEBLH. SAHM, GENE, vol. 102, 1991, pages 93 - 98
BLOM JALBAUM SPDOPPMEIER DPÜH ER AVORHOLTER FJZAKRZEWSKI MGOESMANN A: "EDGAR: a software framework for the comparative analysis of prokaryotic genomes", BMC BIOINFORMATICS, vol. 10, 20 May 2009 (2009-05-20), pages 154, XP021055600, DOI: 10.1186/1471-2105-10-154
C.R. HARWOODY. KIKUCHI: "FEMS Microbiology Reviews", 2022, pages: 1 - 20
CAO CHENG-HAO ET AL: "Enzyme cascade for biocatalytic deracemization of D,L-phosphinothricin", JOURNAL OF BIOTECHNOLOGY, ELSEVIER, AMSTERDAM NL, vol. 325, 29 September 2020 (2020-09-29), pages 372 - 379, XP086425115, ISSN: 0168-1656, [retrieved on 20200929], DOI: 10.1016/J.JBIOTEC.2020.09.024 *
CHENG FENG ET AL: "Tuning amino acid dehydrogenases with featured sequences for L-phosphinothricin synthesis by reductive amination", JOURNAL OF BIOTECHNOLOGY, ELSEVIER, AMSTERDAM NL, vol. 312, 3 March 2020 (2020-03-03), pages 35 - 43, XP086092028, ISSN: 0168-1656, [retrieved on 20200303], DOI: 10.1016/J.JBIOTEC.2020.03.001 *
CHIN CSALEXANDER DHMARKS PKLAMMER AADRAKE JHEINER CCLUM ACOPELAND AHUDDLESTON JEICHLER EE: "Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data", NAT METHODS., vol. 10, no. 6, 5 May 2013 (2013-05-05), pages 563 - 9, XP055185340, DOI: 10.1038/nmeth.2474
DIECKMANN MABEYVERS SNKOUAMEDJO-FANKEP RCHANEL PHGJELONEK LBLOM J: "Goesmann A. EDGAR3.0: comparative genomics and phylogenomics on a scalable infrastructure", NUCLEIC ACIDS RES., vol. 49, no. W1, 2 July 2021 (2021-07-02), pages W185 - W192
E. BAYERK.H. GUGELK. HAGELEH. HAGENMAIERS. JESSIPOWW.A. KONIGH. ZAHNER, HELV CHIM ACTA, vol. 55, 1972, pages 224 - 239
F. MADEIRAY.M. PARKJ. LEEN. BUSOT. GURN. MADHUSOODANANP. BASUTKARA.R.N. TIVEYS.C. POTTERR.D. FINN, NUCLEIC ACIDS RES., vol. 47, 2019, pages W636 - W641
G. HORLEIN, REV. ENVIRON. CONTAM. TOXICOL., vol. 138, 1994, pages 73 - 145
G. THIERBACHA. SCHWARZERA. PIIHLER, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 29, 1988, pages 356 - 362
H. SETOT. SASAKIS. IMAIT. TSURUOKAH. OGAWAA. SATOHS. INOUYET. NIIDAN. OTAKE, J ANTIBIOT (TOKYO, vol. 36, 1983, pages 96 - 98
J. DEVEREUXP. HAEBERLIO. SMITHIES: "Nucleic Acid Res.", vol. 12, 1984, GENETICS COMPUTER GROUP UNIVERSITY OF WISCONSIN, MEDICINE (WL, pages: 387 - 395
J. SOLOMONL. SUS. SHYNA.D. GROSSMAN, J. BACTERIOL., vol. 185, 2003, pages 6425 - 6433
J.A.V. BLODGETTP.M. THOMASG. LIJ.E. VELASQUEZW.A. VAN DER DONKN.L. KELLEHERW.W. METCALF, NAT CHEM BIOL., vol. 3, 2007, pages 480 - 485
K. BARTSCHR. SCHNEIDERA. SCHULZ, APPL. ENVIRON. MICROBIOL., vol. 62, 1996, pages 3794 - 3799
K. HAACK, CHEM. UNSERER ZEIT, vol. 37, 2003, pages 128 - 138
L. K. DUNICANE. SHIVNAN, BIO/TECHNOLOGY, vol. 7, 1989, pages 1067 - 1070
M. BONNETJ.C. LAGIERD. RAOULTS. KHELAIFIA, NEW MICROBE AND NEW INFECT, vol. 34, 2020, pages 100622
M. BOVARNICKH.T. CLARKE, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 60, 1938, pages 2426 - 2430
M. PATEKB. J. EIKMANNSJ. PATEKH. SAHM, MICROBIOLOGY, vol. 142, 1996, pages 1297 - 1309
M. PATEKJ. HOLATKOT. BUSCHEJ. KALINOWSKIJ. NESVERA, MICROB. BIOTECHNOL., vol. 6, 2013, pages 103 - 117
M.D. ARMSTRONG, J. AM. CHEM. SOC., vol. 70, 1948, pages 1756 - 1759
R. A. LAZARUS, J. ORG. CHEM., vol. 55, 1990, pages 4755 - 4757
S. B. NEEDLEMANC. D. WUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 443 - 453
S. LISSERH. MARGALIT, NUCLEIC ACID RES., vol. 21, 1993, pages 1507 - 1516
S.F. ALTSCHULW. GISHW. MILLERE.W. MYERSD.J. LIPMAN, J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
SEEMANN T. PROKKA: "rapid prokaryotic genome annotation", BIOINFORMATICS, vol. 30, no. 14, 18 March 2014 (2014-03-18), pages 2068 - 9
T. MURAKAMIH. ANZAIS. IMAIA. SATOHK. NAGAOKAC.J. THOMPSON, MGG MOLECULAR & GENERAL GENETICS, vol. 205, 1986, pages 42 - 53
WICK RRJUDD LMGORRIE CLHOLT KE: "Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads", PLOS COMPUT BIOL., vol. 13, no. 6, 8 June 2017 (2017-06-08), pages e1005595

Also Published As

Publication number Publication date
AR129337A1 (es) 2024-08-14

Similar Documents

Publication Publication Date Title
US10023886B2 (en) (R)-selective amination
Kim et al. Cloning and characterization of a novel β-transaminase from Mesorhizobium sp. strain LUK: a new biocatalyst for the synthesis of enantiomerically pure β-amino acids
Martinez et al. Biochemical and spectroscopic characterization of the non-heme Fe (II)-and 2-oxoglutarate-dependent ethylene-forming enzyme from Pseudomonas syringae pv. phaseolicola PK2
US11667897B2 (en) D-amino acid oxidative enzyme mutant and application thereof
Dilworth et al. Acid tolerance in legume root nodule bacteria and selecting for it
EP4105335A1 (fr) Procédé enzymatique pour la production d'esters p-alkyl du l-glufosinate
AU2022250692B2 (en) Enzymatic method for producing l-glufosinate and its phosphoesters
JP5516664B2 (ja) Ｎ−アセチル−（Ｒ，Ｓ）−β−アミノ酸アシラーゼ遺伝子
CN107119084B (zh) 一种利用转氨酶和乙烯合成酶生产l-草铵膦的方法
Torrens-Spence et al. Diverse functional evolution of serine decarboxylases: identification of two novel acetaldehyde synthases that uses hydrophobic amino acids as substrates
Westendorf et al. The two enantiospecific dichlorprop/α-ketoglutarate-dioxygenases from Delftia acidovorans MC1–protein and sequence data of RdpA and SdpA
WO2023222226A1 (fr) Procédé enzymatique pour la production de l-glufosinate
WO2023222227A1 (fr) Procédé enzymatique de production de l-glufosinate
JP5119783B2 (ja) Ｎ−アセチル−（Ｒ，Ｓ）−β−アミノ酸アシラーゼ遺伝子
AU2022446842A1 (en) Enzymatic method for the production of l-glufosinate p-esters.
WO2023174511A1 (fr) Procede enzymatique de production de p-esters de l-glufosinate
WO2023232225A1 (fr) Procédé enzymatique de production diastéréosélective de p-esters de l-glufosinate
WO2024061455A1 (fr) Procédé enzymatique de production de l-glufosinate et de ses phosphoesters
Brodegger et al. Novel thioredoxin targets in Dictyostelium discoideum identified by two-hybrid analysis: Interactions of thioredoxin with elongation factor 1α and yeast alcohol dehydrogenase
WO2024061456A1 (fr) Procédé enzymatique de production de l-glufosinate et de ses phosphoesters
SK500632021A3 (sk) Spôsob navýšenia permeability vonkajších bunkových štruktúr pri Vibrionatriegens z pohľadu extracelulárneho transportu rekombinantných proteínov
Jiwaji Regulation of hyu gene expression in Agrobacterium tumefaciens strains RU-AE01 and RU-OR
WO2001018233A9 (fr) Procede de recherche systematique d'inhibiteurs de la biosynthese de la riboflavine

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22730719

Country of ref document: EP

Kind code of ref document: A1