Hydantoinase variants for chiral biocatalytic synthesis of l-glufosinate

Abstract

Engineered hydantoinase variants and uses of the engineered hydantoinase variants in biocatalytic production of L-glufosinate are provided. L-glufosinate is produced by hydrolyzing a 5-substituted hydantoin with a hydantoinase engineered to exhibit enhanced L-enantioselectivity to form a carbamoyl-containing compound and then cleaving the carbamoyl group with an L-stereospecific carbamoylase. The L-glufosinate thus produced may be utilized as an herbicide for agriculture.

Description

[0001] Docket No. 30275 / 2024351 / PC

[0002] HYDANTOINASE VARIANTS FOR CHIRAL BIOCATALYTIC SYNTHESIS OF L- GLUFOSINATE

[0003] INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0004] The Sequence Listing, which is a part of the present disclosure, is submitted concurrently with the specification as a text file. The name of the XML file containing the Sequence Listing is “2024351_SeqListing.xml", which was created on February 3, 2026, and is 96,638 bytes in size. The subject matter of the Sequence Listing is incorporated herein in its entirety by reference.

[0005] FIELD

[0006] Described herein are hydantoinase variants and methods for producing a single stereoisomer of glufosinate, particularly for the production of L-glufosinate.

[0007] BACKGROUND

[0008] Phosphinothricin (4-(hydroxymethylphosphinyl)butyric acid), also called glufosinate, is a phosphorus-containing herbicide which targets glutamine synthetase. It is a non-selective, foliarly-applied herbicide.

[0009] Of the two enantiomers of glufosinate, only the L-enantiomer has herbicidal activity.

[0010] Therefore, there is a need for development of techniques to efficiently produce the active L-glufosinate form.

[0011] SUMMARY

[0012] Hydantoinase variants and uses of the hydantoinase variants in biocatalytic production of L-glufosinate are provided. L-glufosinate is produced by hydrolyzing a 5-substituted hydantoin with a hydantoinase engineered to exhibit enhanced L-enantioselectivity to form a carbamoyl-containing compound and then cleaving the carbamoyl group with an L-stereospecific carbamoylase to form an herbicide for agricultural use. The hydantoinase variants are more active and / or enantioselective than wild type hydantoinases, making the biocatalytic route more efficient by having a shorter reaction time and / or by reduction of unwanted byproducts and / or contaminants.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the amino acid sequence of hydantoinase hyb 2-17 (SEQ ID NO: 1).

[0014] FIG. 2 shows the amino acid sequences (SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, and 52) and the corresponding nucleicDocket No. 30275 / 2024351 / PC

[0015] acid sequences (3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, and 53), respectively, for variant hydantoinases.

[0016] FIG 3 shows the amino acid sequence of ncs 1-23 carbamoylase (Uniprot A0A535Y1H2) (SEQ ID NO: 54)

[0017] DETAILED DESCRIPTION

[0018] Definitions

[0019] The term “hydantoinase” as used herein refers to an enzyme which hydrolyzes a hydantoin, such as a 5-substituted hydantoin.

[0020] A “variant” of a polypeptide comprises an amino acid sequence in which one or more amino acid residues are inserted into, deleted from, and / or substituted into the amino acid sequence relative to another polypeptide sequence.

[0021] As used herein, the term “engineered” can generally refer to a non-naturally occurring organism, nucleic acid, nucleic acid construct, or polypeptide. Such non-naturally occurring nucleic acids may include natural nucleic acids that have been modified, for example that have deletions, substitutions, inversions, insertions, etc., and / or combinations of nucleic acid sequences of different origin that are joined using molecular biology technologies (e.g., a nucleic acid sequences encoding a fusion protein) (e.g., a protein or polypeptide formed from the combination of two different proteins or protein fragments), the combination of a nucleic acid encoding a polypeptide to a promoter sequence, where the coding sequence and promoter sequence are from different sources or otherwise do not typically occur together naturally (e.g., a nucleic acid and a constitutive promoter), etc. Recombinant or engineered can also refer to the polypeptide encoded by the recombinant nucleic acid. Non-naturally occurring nucleic acids or polypeptides include nucleic acids and polypeptides modified by man.

[0022] “Percentage of sequence identity,” “% sequence identity,” “percent identity,” or “% identity” is calculated by comparing two optimally aligned sequences over a particular region, determining the number of positions at which the identical base or amino acid occurs in both sequences in order to yield the number of matched positions, dividing the number of such positions by the total number of positions in the region being compared and multiplying the result by 100. The result of this calculation can also be described as “percent identical” or “% identical.”

[0023] A sequence identity can be calculated from a pairwise alignment showing only a local region of the first sequence or the second sequence (“Local Identity”). For example, program BlastDocket No. 30275 / 2024351 / PC

[0024] (NCBI) produces such alignments; % sequence identity=(# of Identical residues / length of alignment)x100)].

[0025] A sequence identity can also be calculated from a pairwise alignment showing both sequences over the full length, thus showing the first sequence and the second sequence in their full length (“Global sequence identity”). For example, program Needle (EMBOSS) produces such alignments (i.e. , % sequence identity=(# of identical residues / length of alignment)x100)). EMBOSS Needle reads two input sequences and writes their optimal global sequence alignment to file, then it uses the Needleman-Wunsch alignment algorithm (Needleman and Wunsch (J. Mol. Biol. (1979) 48, p. 443-453)) to find the optimum alignment (including gaps) of two sequences along their entire length.

[0026] Preferably, “% sequence identity” is calculated using the program NEEDLE (The European Molecular Biology Open Software Suite (EMBOSS)) with the program’s default parameters (i.e., gap open=10.0, gap extend=0.5, matrix=EBLOSUM62, end gap=false, end gap open=10, and end gap extend=0.5), such that sequence identity is calculated from the alignment showing both sequences over the full length.

[0027] The term "alkyl" as used herein denotes in each case a straight-chain or branched alkyl group having usually from 1 to 20 carbon atoms, preferably from 1 to 8 carbon atoms, frequently from 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms, e.g. 2 or 4 carbon atoms. Examples of alkyl groups are methyl, ethyl, n-propyl, iso-propyl, n-butyl, 2-butyl, isobutyl, tert-butyl, n- pentyl, 1 -methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1 -ethyl propyl, and n- hexyl.

[0028] A “conservative amino acid substitution” or “conservative substitution” means replacement of one amino acid residue in an amino acid sequence with a different amino acid residue having a similar property. Conservative substitutions are well known in the art. Some examples of a conservative amino acid substitution include but are not limited to replacing a positively charged amino acid residue with a different positively charged amino acid residue; replacing a polar amino acid residue with a different polar amino acid residue; replacing a non-polar amino acid residue with a different non-polar amino acid residue, replacing a basic amino acid residue with a different basic amino acid residue, or replacing an aromatic amino acid residue with a different aromatic amino acid residue. Some examples of conserved amino acid substitutions are shown below:Docket No. 30275 / 2024351 / PC

[0029]

[0030] Variant Hydantoinase Enzymes

[0031] The present invention provides new hydantoinase enzymes with increased catalytic activity and / or enantioselectivity.

[0032] In one embodiment, an engineered enzyme having hydantoinase activity (i.e. , a variant hydantoinase) is provided comprising at least one mutation (relative to the wild-type or parent enzyme sequence) at one or more positions corresponding to the following positions of SEQ ID NO: 1: 66, 67, 73, 74, 128, 129, 152, 156, 159, 161, 239, 245, and 313.Docket No. 30275 / 2024351 / PC

[0033] The at least one mutation at the recited positions may be amino acid substitutions, deletions, and / or insertions. In one embodiment, all of the mutations in the wild-type or parent enzyme sequence at the indicated positions are amino acid substitutions.

[0034] Thus, in another embodiment, a variant hydantoinase is provided comprising an amino acid sequence with an amino acid substitution (relative the wild-type or parent enzyme sequence) at one or more positions corresponding to the following positions of SEQ ID NO: 1: 66, 67, 73, 74, 128, 129, 152, 156, 159, 161, 239, 245, and 313.

[0035] The engineered enzymes may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid substitutions at positions corresponding to the following positions of SEQ ID NO: 1: 66, 67, 73, 74, 128, 129, 152, 156, 159, 161, 239, 245, and 313.

[0036] In some embodiments, the amino acid substitution at the position corresponding to position 66 of SEQ ID NO: 1 is other than leucine, the amino acid substitution at the position corresponding to position 67 of SEQ ID NO: 1 is other than valine, the amino acid substitution at the position corresponding to position 73 of SEQ ID NO: 1 is other than arginine, the amino acid substitution at the position corresponding to position 74 of SEQ ID NO: 1 is other than serine, the amino acid substitution at the position corresponding to position 128 of SEQ ID NO: 1 is other than leucine, the amino acid substitution at the position corresponding to position 129 of SEQ ID NO: 1 is other than isoleucine, the amino acid substitution at the position corresponding to position 152 of SEQ ID NO: 1 is other than leucine, the amino acid substitution at the position corresponding to position 156 of SEQ ID NO: 1 is other than threonine, the amino acid substitution at the position corresponding to position 159 of SEQ ID NO: 1 is other than glutamic acid, the amino acid substitution at the position corresponding to position 161 of SEQ ID NO: 1 is other than aspartic acid, the amino acid substitution at the position corresponding to position 239 of SEQ ID NO: 1 is other than valine, the amino acid substitution at the position corresponding to position 245 of SEQ ID NO: 1 is other than valine, and / or the amino acid substitution at the position corresponding to position 313 of SEQ ID NO: 1 is other than leucine.

[0037] For example, the variant hydantoinase may have one or more of the following amino acids at positions corresponding to the following positions of SEQ ID NO: 1: 66A / R / D / G, 67T, 73K / P, 74V, 1281, 129C, 1521, 156M / L, 159K, 161E / M, 239L, 245G, 313A / V.

[0038] The amino acid substitutions may be made to any hydantoinase at one or more positions that correspond to the positions of SEQ ID NO: 1 discussed above.

[0039] In some embodiments, the hydantoinase having the amino acid sequence of SEQ ID NO: 1 may act as the parent, and the engineered hydantoinase may have one or more of the following amino acid substitutions: L66A / R / D / G, V67T, R73K / P, S74V, L128I, L129C, L152I,Docket No. 30275 / 2024351 / PC

[0040] T156M / L, E159K, D161E / M, V239L, V245G, L313A / V (where L66A / R / D / G indicates position 66 may be substituted with any of A, R, D, or G).

[0041] In another embodiment, a variant hydantoinase enzyme is provided comprising an amino acid sequence with at least one of the following amino acids at position(s) corresponding to the following positions of SEQ ID NO: 1: 66A / R / D / G, 67T, 73K / P, 74V, 1281, 129C, 1521, 156M / L, 159K, 161 E / M, 239L, 245G, 313A / V (where 66A / R / D / G indicates position 66 is any of A, R, D, or G). The variant hydantoinase enzymes may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 (e.g., 1-3, 1-5, 2-8, 3-5, etc., or no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10) of the indicated amino acids at the indicated positions corresponding to the positions of SEQ ID NO: 1.

[0042] The variant hydantoinases discussed herein may have an amino acid sequence with at least 70%, at least 75%, at least 70, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, identical to SEQ ID NO: 1, preferably over the full length of the amino acid sequence of SEQ ID NO: 1. For the amino acid residues in the variant hydantoinases outside of positions 66, 67, 73, 74, 128, 129, 152, 156, 159, 161, 239, 245, and 313 of SEQ I D NO: 1 , some or all of the non-identical amino acids (relative to SEQ ID NO: 1) may be conservative amino acid substitutions. For example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 of the non-identical amino acid residues, or 1-10, 10-20, 20-30, 40-50, 50-60, 60-70, 70-80, 80-90 or 90-100 or even all of the non-identical amino acid residues may be conservative amino acid substitutions.

[0043] In a further embodiment, a variant hydantoinase enzyme is provided comprising an amino acid sequence at least 80% identical but less than 100% identical (e.g., at least 85% identical, at least 90% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, etc.) to SEQ ID NO: 1 (preferably over the full length of the amino acid sequence of SEQ ID NO: 1), wherein the amino acid sequence comprises an amino acid substitution, as compared to SEQ ID NO: 1, at one or more positions corresponding to the following positions of SEQ ID NO: 1: 66, 67, 73, 74, 128, 129, 152, 156, 159, 161, 239, 245, and 313. When present, the amino acid substitution at the position corresponding to position 66 of SEQ ID NO: 1 is other than leucine, the amino acid substitution at the position corresponding to position 67 of SEQ ID NO: 1 is other than valine, the amino acid substitution at the position corresponding to position 73 of SEQ ID NO: 1 isDocket No. 30275 / 2024351 / PC

[0044] other than arginine, the amino acid substitution at the position corresponding to position 74 of SEQ ID NO: 1 is other than serine, the amino acid substitution at the position corresponding to position 128 of SEQ ID NO: 1 is other than leucine, the amino acid substitution at the position corresponding to position 129 of SEQ ID NO: 1 is other than isoleucine, the amino acid substitution at the position corresponding to position 152 of SEQ ID NO: 1 is other than leucine, the amino acid substitution at the position corresponding to position 156 of SEQ ID NO: 1 is other than threonine, the amino acid substitution at the position corresponding to position 159 of SEQ ID NO: 1 is other than glutamic acid, the amino acid substitution at the position corresponding to position 161 of SEQ ID NO: 1 is other than aspartic acid, the amino acid substitution at the position corresponding to position 239 of SEQ ID NO: 1 is other than valine, the amino acid substitution at the position corresponding to position 245 of SEQ ID NO: 1 is other than valine, and / or the amino acid substitution at the position corresponding to position 313 of SEQ ID NO: 1 is other than leucine. For example, the variant hydantoinase may have one or more of the following amino acids at positions corresponding to the following positions of SEQ ID NO: 1:

[0045] 66A / R / D / G, 67T, 73K / P, 74V, 1281, 129C, 1521, 156M / L, 159K, 161E / M, 239L, 245G, 313A / V. For the amino acid residues in the variant hydantoinases outside of positions 66, 67, 73, 74, 128, 129, 152, 156, 159, 161, 239, 245, and 313 of SEQ ID NO: 1, some or all of the non-identical amino acids (relative to SEQ ID NO: 1) may be conservative amino acid substitutions. For example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 of the non-identical amino acid residues, or wherein 1-10, 10-20, 20-30, 40-50, 50-60, 60-70, 70-80, 80-90 or 90-100 or even all of the non-identical amino acid residues may be conservative amino acid substitutions.

[0046] In yet another embodiment, a variant hydantoinase is provided comprising an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52.

[0047] In one embodiment, the catalytic activity of the variant hydantoinase is higher by a factor of at least 1.5, 2, 3, 4, 5, 10, 15, 20, 25 or 30 than the catalytic activity of

[0048] the hydantoinase having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the catalytic activity of the variant hydantoinase is higher by a factor of from 1.5 - 30, from 1.5 - 10, from 1.5 -5, from 2 - 3, from 1.5 - 3.5, or from 1.5 -2.5, than the catalytic activity of the hydantoinase having the amino acid sequence of SEQ ID NO: 1.Docket No. 30275 / 2024351 / PC

[0049] In another embodiment, the enantioselectivity of the variant hydantoinase for L-hydantoin is higher by a factor of at least 1.2, 1.5, 2, 3, 4, 5, 10, 15, 20, 25, 30, 50, 100 or 150 than the enantioselectivity of the hydantoinase having amino acid sequence SEQ ID NO: 1. In a further embodiment, the enantioselectivity of the variant hydantoinase for L-hydantoin butyl glufosinate has been increased by a factor of at least 1.2, preferably at least a factor of 1.5, 2, 2.5, 3, 3.5, 4, 5, 10, 15, 20, 25, 30, 50, 100 or 150, compared to the hydantoinase having the amino acid sequence shown in SEQ ID NO: 1. In yet a further embodiment, the enantioselectivity of the variant hydantoinase for L-hydantoin or L-hydantoin butyl glufosinate has been increased by a factor of from 1.2 - 50, from 1.2 - 25, from 1.2 - 15, from 1.2 - 10, from 1.2 -8, from 1.2 -5, from 1.5 - 4.5, from 2 - 4, or from 2.5 - 3. 5 as compared to the hydantoinase having the amino acid sequence shown in SEQ ID NO: 1.

[0050] Preparation of L-Glufosinate using Engineered Hydantoinase Enzymes

[0051] L-glufosinate, an alkyl ester thereof, or a salt of L-glufosinate or an alkyl ester thereof may be prepared using a variant hydantoinase enzyme as described herein.

[0052] Thus, a method is provided for manufacturing L-glufosinate, an alkyl ester thereof, or a salt thereof having the formula (III):

[0053]

[0054] wherein R is H, a salt (e.g., ammonium or sodium), or Ci-Cs alkyl.

[0055] When R is hydrogen in formula (III), the compound is L-glufosinate of the formula (Illa):

[0056]

[0057] L-glufosinate (Illa).

[0058] The method comprises the steps of: a) hydrolyzing a racemic mixture of a hydantoin having the formula (I)

[0059]

[0060] Docket No. 30275 / 2024351 / PC

[0061] or an L-hydantoin of formula (la)

[0062]

[0063] using a variant hydantoinase enzyme described herein to form an N-carbamoyl amino acid having the formula (Ila)

[0064]

[0065] b) cleaving off the carbamoyl moiety of the N-carbamoyl amino acid having the formula (Ila) to produce the compound of formula (III).

[0066] In some embodiments, R is H or Ci-Cs alkyl. In other embodiments, R is H or Ci-Ce alkyl. In some embodiments, R is H or C2-C4 alkyl. In further embodiments, R is H, ethyl, or butyl. In some embodiments, R is Ci-Cs alkyl. In other embodiments, R is Ci-Ce alkyl. In some embodiments, R is C2-C4 alkyl. In further embodiments, R is ethyl or butyl. In such embodiments, the method may further comprise a step (c) of deprotecting the compound. Such deprotection may occur under acidic conditions using any suitable acid. For example, hydrochloric acid or sulfuric acid may be used.

[0067] In some embodiments, step a) may also result in the compound of formula lib:

[0068]

[0069] (lib).

[0070] In a preferred embodiment, the cleaving step b) provides the glufosinate, its alkyl ester or the salts thereof in form of an enantiomeric excess of L-glufosinate, its alkyl ester or the salts thereof having the formula (III) (e.g., an enantiomeric excess of greater than 70%, 80%, 90%, 95%, 96%, 97%, 98%, or99%):

[0071] In some embodiments, at least 40%, at least 50%, at least 60%, at least 70% at least 80%, at least 90%, or at least 95% of the hydantoin of formula 1) is converted to L-glufosinate, its alkyl ester or the salts thereof having the formula (III).Docket No. 30275 / 2024351 / PC

[0072] In some embodiments, the hydrolyzing step a) is performed at a pH of 6 to 11 (e.g., 6.5 to 10, 7 to 9.5, or 7.5 to 9). The pH may be adjusted using alkali hydroxide (e.g. sodium hydroxide or potassium hydroxide).

[0073] In some embodiments, the hydrolyzing step a) is performed at a temperature of 20 to 50 °C (e.g., 25 to 45 °C, 30 to 42 °C, or 32 to 40 °C).

[0074] The hydrolyzing step a) may be performed under aqueous conditions (e.g., degassed aqueous phosphate buffer or degassed aqueous potassium phosphate buffer).

[0075] The hydrolyzing step a) may also be performed during stirring (e.g., at 50 to 1000 rpm, at 100 to 800 rpm, at 150 to 600 rpm, at 180 to 400 rpm, or at 200 to 300 rpm).

[0076] The cleaving step b) may be performed under enzymatic conditions using an N-carbamoyl amino acid hydrolase enzyme (e.g., an L-N-Carbamoyl amino acid hydrolase enzyme) or may be performed under chemical conditions (e.g., using sodium nitrite and / or hydrogen chloride).

[0077] Suitable N-Carbamoyl amino acid hydrolase enzymes are selected from the group consisting of EC 3.5.1 Hydrolases acting on linear amides, EC 3.5.1.87 N-carbamoyl-L-amino-acid hydrolase, 3.5.1.77 N-carbamoyl-D- amino-acid hydrolase, and mixtures thereof. Suitable N-Carbamoyl amino acid hydrolase enzymes that can be used in the method include those selected from the group consisting of A0A535Y1H2 (SEQ ID NO: 54) and variants thereof, A0A7Y0T4N7 and variants thereof, Q88FQ3 and variants thereof, Q88Q81 and variants thereof, A0A126S6J4 and variants thereof, Q8VLIL6 and variants thereof, H9B8T5 and variants thereof, Q9FB05 and variants thereof, C0ZCM8 and variants thereof, C0Z7R5 and variants thereof, A0A0K9YX84 and variants thereof, E3HLIL6 and variants thereof, A0A1V9BSS3 and variants thereof, A0A1V9BSS3 and variants thereof, AOA4D7Q548 and variants thereof, Q9F464 and variants thereof, A0A2S9D976 and variants thereof, AOA1 I6VZZ4 and variants thereof, A0A1 L6RE91 and variants thereof, A0A3E0C996 and variants thereof, AOA3M7BGJ4 and variants thereof, A0A2D7YQN7 and variants thereof, A0A535Y1 H2 and variants thereof, AOA223E4I5 and variants thereof, M2VSE9 and variants thereof, A0A3T0K6C0 and variants thereof, AOA416FGE1 and variants thereof, D1 P143 and variants thereof, A0A6P2ISL4 and variants thereof, A0A3S6Z2M9 and variants thereof, A0A0C1 LIS49 and variants thereof, A0A1Y4GC62 and variants thereof, A0A3D3VMN7 and variants thereof, AOA2K8L549 and variants thereof, A0A1G0MC89 and variants thereof, A0A1 M6WYS1 and variants thereof, AOA2K2BYI3 and variants thereof, A0A510DYR5 and variants thereof, A0A5Y3XFN7 and variants thereof, AOA381 IB54 and variants thereof, AOA2V3IQW6 and variants thereof, and mixtures thereof, wherein variants are defined as polypeptide sequences with at least 80 %, preferably 90%, and most preferably 95%,Docket No. 30275 / 2024351 / PC

[0078] sequence identity to the respective polypeptide sequence. Most preferably the N-Carbamoyl amino acid hydrolase enzyme is selected from the group consisting of A0A3E0C996 and variants thereof, A0A535Y1 H2 and variants thereof, A0A6P2ISL4 and variants thereof, A0A1Y4GC62, and variants thereof, wherein variants are defined as polypeptide sequences with at least 80 %, preferably 90%, and most preferably 95%, sequence identity to the respective polypeptide sequence. It is to be understood that the above outlined N-Carbamoyl amino acid hydrolase enzymes are indicated in the nomenclature of the database identifier according to the Uniprot database (www.UniProt.orq), accessed 2ndof December 2022. In this connection, the cleaving step b) is preferably performed at a temperature of 20 to 50 °C, preferably of 25 to 45 °C, more preferably of 30 to 42 °C, and in particular of 32 to 40 °C. Further, the reaction pressure is preferably ambient pressure. Preferably, the reaction pressure is in the range of 0.995 to 1 .030 mbar, more preferably of 1 .005 to 1.020 mbar, and in particular of about 1.013 mbar. In a preferred embodiment of the present invention, the cleaving step b) is performed at a pH of 5 to 10, preferably of 6 to 9, and in particular of about 7.

[0079] In a preferred embodiment of the present invention, the cleaving step b) is performed during stirring, preferably at 50 to 1000 rpm, more preferably at 100 to 800 rpm, even more preferably at 150 to 600 rpm, still more preferably at 180 to 400 rpm, and in particular at 200 to 300 rpm.

[0080] In another preferred embodiment of the present invention, the cleaving step b) is performed under chemical conditions. It is to be understood that the term “chemical condition” or “chemically cleaving” refers to a cleaving step that is not performed under enzymatic conditions. Any suitable chemical approach is possible. The cleavage may exemplarily be performed using sodium nitrite and / or hydrogen chloride. The N-carbamoyl amino acid having the formula (II) may exemplarily be treated with concentrated hydrogen chloride at elevated temperature. Alternatively, the N-carbamoyl amino acid having the formula (2) as defined above may be treated with sodium nitrite and hydrogen chloride under aqueous conditions. In this connection, the cleaving step b) is preferably performed at a temperature of 25 to 120 °C, more preferably of 50 to 110 °C, and in particular of 60 to 105 °C. Further, the reaction pressure is preferably ambient pressure. Preferably, the reaction pressure is in the range of 0.995 to 1.030 mbar, more preferably of 1.005 to 1.020 mbar, and in particular of about 1 .013 mbar. In a preferred embodiment of the present invention, the cleaving step b) is performed at a pH of 0 to 5, preferably of 0 to 3. The reaction mixture can be worked-up under standard procedure (i.e. washing and purifying).Docket No. 30275 / 2024351 / PC

[0081] In another embodiment, the product of the cleaving step is isolated as the sodium or ammonium salt of L-glufosinate (i.e. , formula III with R = sodium or ammonium). For this, the product of the cleavage reaction is treated with a base such as sodium hydroxide or ammonia. The reaction can be performed in aqueous or non-aqueous media including mixtures of water with solvents that are miscible with water, such as alcohols, preferably methanol, ethanol or isopropanol. In a preferred embodiment of the invention, the product is L-glufosinate ammonium, and it is purified by crystallization from mixtures of water and methanol or ethanol.

[0082] In some embodiments, the method further comprises the addition of a Hydantoin Racemase enzyme and an N-Carbamoyl amino acid racemase enzyme.

[0083] In a preferred embodiment of the present invention, step a) and step b) are performed in a single container, wherein step b) is performed under enzymatic conditions. In this connection, all reagents are preferably substantially added at the start of the reaction.

[0084] Alternatively, the reagents for step a) and the reagents for step b) are preferably added to the single container at different times.

[0085] In a preferred embodiment of the present invention, the method comprises the addition of a Hydantoinase enzyme, a Hydantoin Racemase enzyme, and an N-Carbamoyl amino acid hydrolase enzyme, wherein all reaction steps are performed in a single container (also known as “One-Pot” conditions), preferably wherein all reagents are substantially added at the start of the reaction or wherein the reagents are added to the single container at different times.

[0086] In a preferred embodiment of the present invention, the method comprises the addition of a Hydantoinase enzyme, a Hydantoin Racemase enzyme, and an N-Carbamoyl amino acid hydrolase enzyme, wherein all reaction steps are performed in a single container (also known as “One-Pot” conditions), preferably wherein all reagents are substantially added at the start of the reaction or wherein the reagents are added to the single container at different times.

[0087] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0088] While the present teachings have been described in conjunction with various embodiments and examples, it is not intended that the present teachings be limited to such embodiments or examples. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.Docket No. 30275 / 2024351 / PC

[0089] While various inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0090] The claims should not be read as limited to the described order or elements unless stated to that effect. It should be understood that various changes in form and detail may be made by one of ordinary skill in the art without departing from the spirit and scope of the appended claims. All embodiments that come within the spirit and scope of the following claims and equivalents thereto are claimed.

[0091] EXAMPLES

[0092] Example 1 : Variant Screening

[0093] Site-saturation mutagenesis was used to find beneficial mutations among the closest 96 residues to the active site of the hydantoinase hyb 2-17 (SEQ ID NO: 1), barring those with over 99% conservation among similar enzymes.

[0094] Variants were screened in a two-enzyme cascade assay with 2.5 mM racemic hydantoin butyl glufosinate as a substrate. Variant hydantoinase and ncs 1-23 carbamoylase (Uniprot A0A535Y1H2) (SEQ ID NO: 54) were produced in whole E. coli, with each enzyme in a different strain under a rhamnose induction system in a pDHE plasmid. Reactions were carried out in sealed plates in 100 mM HEPES buffer with 100 mM NaCI and 1 mM MnCh. As ncs 1-23 cannot hydrolyze butyl D-glufosinate carbamoylic acid, the ratio of glufosinate to total product as measured by mass spectrometry was used as a proxy for enantioselectivity.Docket No. 30275 / 2024351 / PC

[0095] To adjust for rate differences the screening conditions were optimized to ODeoo=10 for carbamoylase, ODeoo=0.1 for hydantoinase, and a 24 hour reaction time.

[0096] Three rounds of directed evolution were conducted. The first round consisted exclusively of site-saturation mutagenesis targeting the 96 residues chosen as described above. The second and third rounds featured site-saturation mutagenesis in tandem with combinatorial site-directed mutagenesis.

[0097] Samples were screened by LC-MS on an Agilent 6230 LC / TOF system with a Kinetex 100x2.1 mm C18 column with a 1.7 |im particle size and a 100 A pore size, using a 3 minute separation method at 0.2 mL / min and 40°C with an isocratic eluent of 80% solution A / 20% solution B, where solution A was 95% water / 5% acetonitrile with 0.1% formic acid and solution B was 5% water / 95% acetonitrile with 0.1% formic acid. Detection was in positive mode via the M+H+ion using a 140 V fragmentor voltage. . The top 18 variants of each round as determined by LCMS were then streaked on new plates from glycerol stocks and used in a validation assay along template controls. These reactions were analyzed both by achiral LCMS as above and by chiral LCMS with an Astec Chirobiotic-T2250x4.6 mm teicoplanin column with 5 |iM particle size to differentiate the L- and D-enantiomers of butyl glufosinate and carbamoylic acid. Chiral LC-MS used the same instrument and the same detection parameters as above, but elution was at 0.7 mL / min at 25°C and used a 16 minute gradient of 10% A / 90% B to 35% A / 65% B, with solutions A and B being the same as above, followed by a 1 minute gradient back to 10% A / 90% B and a 5 min reequlibration at 10% A / 90% B.

[0098] The variants resulting from the experiments are listed in Table 1. The Table shows the increase in activity and increase in enantioselectivity (for L-hydantoin butyl glufosinate) as compared to the parent hydantoinase hyb 2-17 (SEQ ID NO: 1) using racemic hydantoin butyl glufosinate as the substrate. The amino acid and nucleic acid sequences of the variant hydantoinases are shown in Figure 2.

[0099] TABLE 1: ENGINEERED VARIANT HYDANTOINASES

[0100]

[0101] Docket No. 30275 / 2024351 / PC

[0102]

Claims

Docket No. 30275 / 2024351 / PCCLAIMSWhat is claimed is:

1. A variant hydantoinase comprising at least one mutation at one or more positions corresponding to the following positions of SEQ ID NO: 1: L66, V67, R73, S74, L128, L129, L152, T156, E159, D161, V239, V245, L313.

2. A variant hydantoinase comprising an amino acid sequence with an amino acid substitution at one or more positions corresponding to the following positions of SEQ ID NO: 1: 66, 67, 73, 74, 128, 129, 152, 156, 159, 161, 239, 245, and 313.

3. The variant hydantoinase of claim 2, wherein the amino acid sequence comprises at least two amino acid substitutions at positions corresponding to the following positions of SEQ ID NO: 1: 66, 67, 73, 74, 128, 129, 152, 156, 159, 161, 239, 245, and 313.

4. The variant hydantoinase of claim 2, wherein the amino acid sequence comprises at least three amino acid substitutions at positions corresponding to the following positions of SEQ ID NO: 1: 66, 67, 73, 74, 128, 129, 152, 156, 159, 161, 239, 245, and 313.

5. A variant hydantoinase comprising an amino acid sequence with at least one of the following amino acids at a position or positions corresponding to the following positions of SEQ ID NO: 1: 66A / R / D / G, 67T, 73K / P, 74V, 1281, 129C, 1521, 156M / L, 159K, 161 E / M, 239L, 245G, 313A / V.

6. A variant hydantoinase comprising an amino acid sequence with at least 80% sequence identity but less than 100% sequence identity to SEQ ID NO: 1 over the full length of SEQ I D NO: 1 , wherein the amino acid sequence comprises an amino acid substitution, as compared to SEQ ID NO: 1, at one or more positions corresponding to the following positions of SEQ ID NO: 1: 66, 67, 73, 74, 128, 129, 152, 156, 159, 161, 239, 245, and 313.

7. The variant hydantoinase of any of claims 1-6, wherein the variant has hydantoinase activity.

8. The variant hydantoinase of any of claims 1-7, wherein the variant has an amino acid sequence at least 85% identical to SEQ ID NO: 1 over the full length of SEQ ID NO: 1.

9. The variant hydantoinase of any of claims 1-8, wherein the variant has an amino acid sequence at least 90% identical to SEQ ID NO: 1 over the full length of SEQ ID NO: 1.Docket No. 30275 / 2024351 / PC10. The variant hydantoinase of any of claims 1-9, wherein the variant has an amino acid sequence at least 95% identical to SEQ ID NO: 1 over the full length of SEQ ID NO: 1.

11. The variant hydantoinase of any of claims 1-10, wherein the variant has an amino acid sequence at least 96% identical to SEQ ID NO: 1 over the full length of SEQ ID NO: 1.

12. The variant hydantoinase of any of claims 1-11, wherein the variant has an amino acid sequence at least 97% identical to SEQ ID NO: 1 over the full length of SEQ ID NO: 1.

13. The variant hydantoinase of any of claims 1-12, wherein the variant has an amino acid sequence at least 98% identical to SEQ ID NO: 1 over the full length of SEQ ID NO: 1.

14. The variant hydantoinase of any of claims 1-13, wherein the variant has an amino acid sequence at least 99% identical to SEQ ID NO: 1 over the full length of SEQ ID NO: 1.

15. The variant hydantoinase of any of claims 1-14, wherein any amino acids in the variant hydantoinase outside of positions corresponding to positions 66, 67, 73, 74, 128, 129, 152, 156, 159, 161, 239, 245, and 313 of SEQ ID NO: 1, that are not identical with corresponding amino acids in SEQ ID NO: 1 are conservative substitutions of the corresponding amino acid in SEQ ID NO: 1.

16. A variant hydantoinase comprising an amino acid sequence of SEQ ID NO: 1 with one or more substitutions selected from the following: L66A / R / D / G, V67T, R73K / P, S74V, L128I, L129C, L152I, T156M / L, E159K, D161E / M, V239L, V245G, and / or L313A / V.

17. The variant hydantoinase of claim 16, comprising no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, or no more than 10 substitutions selected from the following:L66A / R / D / G, V67T, R73K / P, S74V, L128I, L129C, L152I, T156M / L, E159K, D161E / M, V239L, V245G, and / or L313A / V.

18. The variant hydantoinase of any of claims 1-17, wherein the variant has an amino acid sequence selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, and 52.

19. A method of preparing L-glufosinate, an alkyl ester thereof, or a salt of L-glufosinate or alkyl ester thereof comprising:admixing racemic hydantoin glufosinate, an alkyl ester thereof, or a salt of hydantoin glufosinate or alkyl ester thereof with the variant hydantoinase of any one of claims 1-18 to form a carbamoyl intermediate andDocket No. 30275 / 2024351 / PCreacting the carbamoyl intermediate with a carbamoylase to form L- glufosinate, an alkyl ester thereof, or a salt of L-glufosinate or alkyl ester thereof.

20. The method of claim 19, wherein the racemic hydantoin is racemic hydantoin butyl glufosinate.

21. The method of claim 19 or 20, wherein L-glufosinate, the alkyl ester thereof, or the salt of L-glufosinate or the alkyl ester thereof has an enantiomeric excess of 95% or more.

22. The method of claim 19 or 20, wherein L-glufosinate, the alkyl ester thereof, or the salt of L-glufosinate or the alkyl ester thereof has an enantiomeric excess of 98% or more.

23. The method of claim 19 or 20, wherein L-glufosinate, the alkyl ester thereof, or the salt of L-glufosinate or the alkyl ester thereof has an enantiomeric excess of 99% or more.

24. The method of claim 19 or 20, wherein L-glufosinate, the alkyl ester thereof, or the salt of L-glufosinate or the alkyl ester thereof has an enantiomeric excess of 99.5% or more.

25. The method of any one of claims 19 to 24, wherein the method produces a salt of L-glufosinate.

26. The method of claim 25, wherein the salt is L-glufosinate ammonium.

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