Modified glutamate dehydrogenase and uses thereof

Abstract

The present invention relates to a modified glutamate dehydrogenase (GluDH). In particular, the modified GluDH of the present invention exhibits increased activity and / or improved kinetic properties for catalyzing the reaction of 4-(hydroxymethylphosphinyl)-2-oxobutanoic acid (PPO) with an amino donor to produce L-glufosinate. The present invention also relates to polynucleotides encoding the modified GluDH of the present invention, vectors and host cells for expressing the modified GluDH of the present invention, and methods for producing L-glufosinate using the modified GluDH and host cells of the present invention.

Description

[Technical field]

[0001] The present invention relates to the field of enzyme engineering. In particular, the present invention relates to a modified glutamate dehydrogenase (GluDH) and its use in the production of glufosinate. [Background technology]

[0002] Glufosinate (also known as 4-[hydroxy(methyl)phosphono]-D,L-homoalanine) is the world's second-best-selling herbicide to which genetically modified crops are resistant. Glufosinate is a broad-spectrum contact kill herbicide that disrupts plant nitrogen metabolism by inhibiting the activity of L-glutamine synthetase, ultimately killing the plant. Glufosinate has great advantages over glyphosate, including a wide range of application, rapid effect, long-lasting effect, low toxicity, and safety. Therefore, the sales of glufosinate are increasing rapidly and are very promising, with a large demand expected in the next fiscal year.

[0003] However, the process for producing glufosinate is complicated, making it difficult to produce. Its high price prevents it from rapidly replacing glyphosate. Currently, commercially available glufosinate is a racemic mixture containing equal amounts of two optical isomers (D,L-glufosinate), and only L-glufosinate is biologically active. Therefore, the preparation of chirally pure L-glufosinate by deracemization of D,L-glufosinate is of practical importance and has become common in the synthesis of L-glufosinate in recent years.

[0004] In recent years, many methods have been reported for preparing L-glufosinate from D,L-glufosinate. Traditional separation methods by chemical modification are not competitive due to the high cost and the fact that D-glufosinate cannot be used. At present, the main and representative technical routes reported for converting D-glufosinate-ammonium to L-glufosinate are as follows:

[0005] D,L-glufosinate is converted to N-acetylglufosinate, and then L-glufosinate is obtained by selective hydrolysis of LN-acetylglufosinate catalyzed by carboxypeptidase, while DN-acetylglufosinate cannot be hydrolyzed and can be recycled to the hydrolysis step after chemical or enzymatic racemization (see, for example, China Patent Application Publication No. 108690854). The disadvantages of this method include the multiple reaction steps and the need to separate L-glufosinate obtained from hydrolysis from the N-acetylated substrate.

[0006] D-glufosinate is oxidized to 2-carbonyl-4-(hydroxymethylphosphono)butyric acid (PPO), and then PPO is reduced or transaminated to produce L-glufosinate-ammonium. In most literature, D-amino acid oxidase (DAAO) is used to catalyze the oxidation of D-glufosinate to PPO. PPO is reduced by formic acid under the catalysis of palladium-on-carbon to produce D,L-glufosinate, which is gradually converted to L-glufosinate due to the stereoselectivity of DAAO (see, for example, China Patent Application Publication No. 105567780). The disadvantages of this solution include the need for a large amount of palladium-on-carbon catalyst and the waste of raw materials for the reaction (such as oxygen and ammonium formate).

[0007] Alternatively, PPO can be converted to L-glufosinate by stereoselective transamination catalyzed by L-amino acid transaminase (L-TA) (see, for example, US Patent Publication No. 20180030487). This solution has the disadvantage that the transamination step is an equilibrium reaction, and therefore an excess amount of amino donor (amino acid or organic amine) needs to be provided to achieve a high conversion rate (e.g., 90% conversion rate when 3 equivalents of amino donor are provided), and the excess amount of amino donor and corresponding by-products will seriously affect the subsequent separation and purification steps.

[0008] In addition, PPO can be converted to L-glufosinate by a stereoselective reduction reaction catalyzed by L-amino acid dehydrogenase (L-AADH) using inorganic ammonium salts as amino donors with the assistance of the NAD(P)H cycle consuming formic acid, glucose, or simple alcohols. The reaction catalyzed by L-AADH does not require a large excess of hydrogen donor and can achieve a high conversion rate.

[0009] The use of native or engineered glutamate dehydrogenase (GluDH) to catalyze the asymmetric reductive amination of PPO to prepare L-glufosinate has been reported (see, for example, CN Patent No. 107630052, CN Patent No. 106978453, CN Patent No. 108588045, and CN Patent Publication No. 109609474). Summary of the Invention [Problem to be solved by the invention]

[0010] However, there remains a need in the art to provide GluDHs that exhibit increased activity and / or improved kinetic properties (e.g., increased Vmax value, decreased Km value, or increased Vmax / Km) for catalyzing the reaction of PPO with an amino donor to produce L-glufosinate. [Means for solving the problem]

[0011] In a first aspect, the present invention provides a modified glutamate dehydrogenase comprising amino acid substitutions at two or more positions compared to a starting GluDH, the modified glutamate dehydrogenase exhibiting increased activity and / or improved kinetic properties (e.g., increased Vmax, decreased Km, or increased Vmax / Km) for catalyzing the reaction of PPO with an amino donor to produce L-glufosinate. In some embodiments, the modified GluDH has, compared to the initial GluDH: a substitution of the amino acid at position 104 with C and a substitution of the amino acid at position 175 with G; a substitution of the amino acid at position 132 with L and a substitution of the amino acid at position 175 with G; a substitution of the amino acid at position 133 with V and a substitution of the amino acid at position 175 with G; an amino acid substitution at position 173 with G or S and an amino acid substitution at position 175 with G; the amino acid at position 175 is substituted with G and the amino acid at position 181 is substituted with K or R; a substitution of the amino acid at position 175 with G and the amino acid at position 182 with R; a substitution of the amino acid at position 175 with G and the amino acid at position 203 with I; the amino acid at position 132 is substituted with L, the amino acid at position 133 with V, and the amino acid at position 175 with G; the amino acid at position 132 is substituted with L, the amino acid at position 173 with S, and the amino acid at position 175 with G; the amino acid at position 132 is substituted with L, the amino acid at position 175 with G, and the amino acid at position 182 with R; the amino acid at position 133 is substituted with V, the amino acid at position 173 is substituted with G or S, and the amino acid at position 175 is substituted with G; the amino acid at position 133 is substituted with V, the amino acid at position 175 with G, and the amino acid at position 182 with R; an amino acid substitution at position 173 to G, an amino acid substitution at position 175 to G, and an amino acid substitution at position 182 to R; the amino acid at position 132 is substituted with L, the amino acid at position 133 is substituted with V, the amino acid at position 173 is substituted with S, and the amino acid at position 175 is substituted with G; the amino acid at position 132 is substituted with L, the amino acid at position 133 is substituted with V, the amino acid at position 175 is substituted with G, and the amino acid at position 182 is substituted with R; the amino acid at position 132 is substituted with L, the amino acid at position 173 with S, the amino acid at position 175 with G, and the amino acid at position 182 with R; an amino acid substitution at position 133 to V, an amino acid substitution at position 173 to G or S, an amino acid substitution at position 175 to G, and an amino acid substitution at position 182 to R; and The amino acid at position 132 is substituted with L, the amino acid at position 133 is substituted with V, the amino acid at position 173 is substituted with G or S, the amino acid at position 175 is substituted with G, and the amino acid at position 182 is substituted with R. and a combination of amino acid substitutions selected from These amino acid positions are numbered with reference to SEQ ID NO:1.

[0012] In some embodiments, the modified GluDH comprises substitutions at positions 173, 175, and 182 compared to the initial GluDH, these amino acid positions being numbered with reference to SEQ ID NO: 1. In some embodiments, the amino acid at position 173 is substituted with G. Preferably, the amino acid at position 175 is substituted with G. Preferably, the amino acid at position 182 is substituted with R. In some embodiments, the modified GluDH further comprises an amino acid substitution at one or more positions selected from positions 9, 22, 23, 25, 31, 56, 124, 143, 199, 216, 242, 263, 339, 420, 431, and 437. Preferably, the amino acid at position 9 is substituted with S, L, or Y. Preferably, the amino acid at position 22 is substituted with W or E. Preferably, the amino acid at position 23 is substituted with M. Preferably, the amino acid at position 25 is substituted with D. Preferably, the amino acid at position 31 is substituted with H. Preferably, the amino acid at position 56 is substituted with Q. Preferably, the amino acid at position 124 is substituted with L. Preferably, the amino acid at position 143 is substituted with E. Preferably, the amino acid at position 199 is substituted with W or Y. Preferably, the amino acid at position 216 is substituted with G. Preferably, the amino acid at position 263 is substituted with S. Preferably, the amino acid at position 339 is substituted with Q. Preferably, the amino acid at position 420 is substituted with R. Preferably, the amino acid at position 431 is substituted with S. Preferably, the amino acid at position 437 is substituted with K.

[0013] In some embodiments, the modified GluDH comprises amino acid substitutions at positions 22, 56, 173, 175, 182, 199, and 420. Preferably, the amino acid at position 22 is substituted with E. Preferably, the amino acid at position 56 is substituted with Q. Preferably, the amino acid at position 173 is substituted with G. Preferably, the amino acid at position 175 is substituted with G. Preferably, the amino acid at position 182 is substituted with R. Preferably, the amino acid at position 199 is substituted with Y. Preferably, the amino acid at position 420 is substituted with R. In some embodiments, the modified GluDH further comprises amino acid substitutions at one or more positions selected from positions 31, 124, and 216. Preferably, the amino acid at position 31 is substituted with H. Preferably, the amino acid at position 124 is substituted with L. Preferably, the amino acid at position 216 is substituted with G.

[0014] In some embodiments, the initial GluDH is a wild-type GluDH. In some embodiments, the initial GluDH is derived from a microorganism of the Bacillaceae family, preferably a microorganism of the genus Lysinibacillus or Bacillus, more preferably Lysinibacillus sphaericus or Bacillus velezensis. In a preferred embodiment, the initial GluDH comprises the amino acid sequence of SEQ ID NO: 1 or 2.

[0015] In some embodiments, the modified GluDH comprises one of the amino acid sequences of SEQ ID NOs: 4-14, 16-19, 21, 22, 24, 25, 27-30, 32-48, 50, 51, and 53-72.

[0016] The present invention relates to a modified GluDH comprising amino acid substitutions at positions 22, 56, 173, 175, 182, 199, 381, and 420 compared to the initial GluDH, in which the amino acid at position 22 is substituted with E, the amino acid at position 56 is substituted with Q, the amino acid at position 173 is substituted with G, the amino acid at position 175 is substituted with G, the amino acid at position 182 is substituted with R, the amino acid at position 199 is substituted with Y, and the amino acid at position 381 is substituted with C. The present invention further provides a modified GluDH, wherein the amino acid at position 420 is substituted with R, and the amino acid positions are numbered with reference to SEQ ID NO:1, wherein the modified GluDH exhibits increased activity for catalyzing the reaction of 2-carbonyl-4-(hydroxymethylphosphono)butyric acid (PPO) with an amino donor to produce L-glufosinate, and / or exhibits improved stability, and / or exhibits increased Vmax, decreased Km, or increased Vmax / Km, compared to initial GluDH.

[0017] In some embodiments, the initial GluDH is a wild-type GluDH. In some embodiments, the initial GluDH is derived from a microorganism of the Bacillaceae family, preferably a microorganism of the genus Lysinibacillus or Bacillus, more preferably Lysinibacillus sphaericus or Bacillus velezensis. In some embodiments, the initial GluDH comprises the amino acid sequence of SEQ ID NO: 1 or 2.

[0018] In some embodiments, the modified GluDH further comprises an amino acid substitution at one or more positions selected from 31, 124, and 216, wherein the amino acid at position 31 is substituted with H, the amino acid at position 124 is substituted with L, and the amino acid at position 216 is substituted with G.

[0019] In some embodiments, the modified GluDH further comprises an amino acid substitution at one or more positions selected from 9, 23, 25, 143, 263, 339, 431, and 437, wherein the amino acid at position 9 is substituted with S, L, or Y, the amino acid at position 23 is substituted with M, the amino acid at position 25 is substituted with D, the amino acid at position 143 is substituted with E, the amino acid at position 263 is substituted with S, the amino acid at position 339 is substituted with Q, the amino acid at position 431 is substituted with S, and the amino acid at position 437 is substituted with K.

[0020] In some embodiments, the modified GluDH comprises the amino acid sequence of one of SEQ ID NOs: 73-80.

[0021] In some embodiments, the activity of the modified GluDH to catalyze the reaction of PPO with an amino donor to produce L-glufosinate is at least 100%, 105%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more than the activity of SEQ ID NO:3 to catalyze this reaction.

[0022] In a second aspect, the present invention provides a polynucleotide encoding a modified GluDH of the present invention, and a vector comprising a polynucleotide of the present invention.

[0023] In a third aspect, the present invention provides a host cell comprising a modified GluDH of the present invention, a coding polynucleotide thereof, or a vector comprising said polynucleotide.

[0024] In a fourth aspect, the present invention provides a method for producing L-glufosinate, the method comprising the step of contacting the modified GluDH of the present invention or the host cell of the present invention with PPO. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The present invention primarily relates to a modified GluDH for catalyzing the reaction of PPO with an amino donor to produce L-glufosinate. Unless otherwise specified, the terms used herein have the meanings commonly understood by those skilled in the art.

[0026] I. Modified glutamate dehydrogenase As used herein, the terms "glutamate dehydrogenase" and "GluDH" refer to an enzyme that catalyzes the dehydrogenation of glutamate to produce α-ketoglutarate. Additionally, GluDH has an activity to catalyze the reaction of PPO with an amino donor to produce L-glufosinate. The present invention provides modified GluDH polypeptides that exhibit increased activity for catalyzing the reaction of PPO with an amino donor to produce L-glufosinate and / or improved kinetic properties, including, but not limited to, increased Vmax value, decreased Km value, or increased Vmax / Km.

[0027] As used herein, the term "peptide" refers to a chain containing at least two amino acids linked by a peptide bond. The term "polypeptide" is interchangeable with "protein" and refers to a chain containing 10 or more amino acid residues. All peptide and polypeptide formulas or sequences herein are written in left-to-right order, indicating the direction from the amino terminus to the carboxyl terminus.

[0028] The term "amino acid" includes amino acids that occur naturally in proteins and non-naturally occurring amino acids. Conventional nomenclature for amino acids that occur naturally in proteins (one-letter and three-letter) is used and can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Amino acid 1 letter 3 letters Alanine A Ala Arginine R Arg Asparagine N Asn Aspartic Acid D Asp Cysteine ​​C Cys Glutamine Q Gln Glutamic Acid E Glu Glycine Gly Histidine H His Isoleucine I Ile Leucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe Proline P Pro Serine S Threonine T Thr Tryptophan W Trp Tyrosine Y Tyr Valin V Val

[0029] As used herein, the term "modification" refers to any alteration to a polypeptide, for example, substitution, deletion, insertion, and / or addition of an amino acid(s).

[0030] In some embodiments, the modified GluDH of the present invention contains amino acid substitutions at two or more positions compared to its initial GluDH and exhibits increased activity for catalyzing the reaction of PPO with an amino donor to produce L-glufosinate and / or improved kinetic properties, including, but not limited to, an increased Vmax value, a decreased Km value, or an increased Vmax / Km.

[0031] In some embodiments, the modified GluDH has the following combination of amino acid substitutions compared to its initial GluDH: a substitution of the amino acid at position 104 with C and a substitution of the amino acid at position 175 with G; a substitution of the amino acid at position 132 with L and a substitution of the amino acid at position 175 with G; a substitution of the amino acid at position 133 with V and a substitution of the amino acid at position 175 with G; an amino acid substitution at position 173 with G or S and an amino acid substitution at position 175 with G; the amino acid at position 175 is substituted with G and the amino acid at position 181 is substituted with K or R; a substitution of the amino acid at position 175 with G and the amino acid at position 182 with R; a substitution of the amino acid at position 175 with G and the amino acid at position 203 with I; the amino acid at position 132 is substituted with L, the amino acid at position 133 with V, and the amino acid at position 175 with G; the amino acid at position 132 is substituted with L, the amino acid at position 173 with S, and the amino acid at position 175 with G; the amino acid at position 132 is substituted with L, the amino acid at position 175 with G, and the amino acid at position 182 with R; the amino acid at position 133 is substituted with V, the amino acid at position 173 is substituted with G or S, and the amino acid at position 175 is substituted with G; the amino acid at position 133 is substituted with V, the amino acid at position 175 with G, and the amino acid at position 182 with R; an amino acid substitution at position 173 to G, an amino acid substitution at position 175 to G, and an amino acid substitution at position 182 to R; the amino acid at position 132 is substituted with L, the amino acid at position 133 is substituted with V, the amino acid at position 173 is substituted with S, and the amino acid at position 175 is substituted with G; the amino acid at position 132 is substituted with L, the amino acid at position 133 is substituted with V, the amino acid at position 175 is substituted with G, and the amino acid at position 182 is substituted with R; the amino acid at position 132 is substituted with L, the amino acid at position 173 with S, the amino acid at position 175 with G, and the amino acid at position 182 with R; an amino acid substitution at position 133 to V, an amino acid substitution at position 173 to G or S, an amino acid substitution at position 175 to G, and an amino acid substitution at position 182 to R; and The amino acid at position 132 is substituted with L, the amino acid at position 133 is substituted with V, the amino acid at position 173 is substituted with G or S, the amino acid at position 175 is substituted with G, and the amino acid at position 182 is substituted with R. having These amino acid positions are numbered with reference to SEQ ID NO:1.

[0032] In some embodiments, the modified GluDH of the present invention comprises substitutions at positions 132 and 175 compared to the initial GluDH, these amino acid positions being numbered with reference to SEQ ID NO: 1. Preferably, the amino acid at position 132 is substituted with L, and the amino acid at position 175 is substituted with G. In some embodiments, the modified GluDH of the present invention further comprises substitutions at one or more positions selected from positions 104, 133, 173, 181, and 182 compared to the initial GluDH. In some embodiments, the amino acid at position 104 is substituted with C. In some embodiments, the amino acid at position 133 is substituted with V. In some embodiments, the amino acid at position 173 is substituted with G or S. In some embodiments, the amino acid at position 181 is substituted with K or R. In some embodiments, the amino acid at position 182 is substituted with R.

[0033] In some embodiments, the modified GluDH of the present invention comprises substitutions at positions 133 and 175 compared to the initial GluDH, these amino acid positions being numbered with reference to SEQ ID NO: 1. Preferably, the amino acid at position 133 is substituted with V, and the amino acid at position 175 is substituted with G. In some embodiments, the modified GluDH of the present invention further comprises substitutions at one or more positions selected from positions 104, 132, 173, 181, and 182 compared to the initial GluDH. In some embodiments, the amino acid at position 104 is substituted with C. In some embodiments, the amino acid at position 132 is substituted with L. In some embodiments, the amino acid at position 173 is substituted with G or S. In some embodiments, the amino acid at position 181 is substituted with K or R. In some embodiments, the amino acid at position 182 is substituted with R.

[0034] In some embodiments, the modified GluDH of the present invention comprises substitutions at positions 173 and 175 compared to the initial GluDH, these amino acid positions being numbered with reference to SEQ ID NO: 1. Preferably, the amino acid at position 173 is substituted with G or S, and the amino acid at position 175 is substituted with G. In some embodiments, the modified GluDH of the present invention further comprises substitutions at one or more positions selected from positions 104, 132, 133, 181, and 182 compared to the initial GluDH. In some embodiments, the amino acid at position 104 is substituted with C. In some embodiments, the amino acid at position 132 is substituted with L. In some embodiments, the amino acid at position 133 is substituted with V. In some embodiments, the amino acid at position 181 is substituted with K or R. In some embodiments, the amino acid at position 182 is substituted with R.

[0035] In some embodiments, the modified GluDH of the present invention comprises substitutions at positions 175 and 182 compared to the initial GluDH, these amino acid positions being numbered with reference to SEQ ID NO: 1. Preferably, the amino acid at position 175 is substituted with G, and the amino acid at position 182 is substituted with R. In some embodiments, the modified GluDH of the present invention further comprises substitutions at one or more positions selected from positions 104, 132, 133, 173, and 181 compared to the initial GluDH. In some embodiments, the amino acid at position 104 is substituted with C. In some embodiments, the amino acid at position 132 is substituted with L. In some embodiments, the amino acid at position 133 is substituted with V. In some embodiments, the amino acid at position 173 is substituted with G or S. In some embodiments, the amino acid at position 181 is substituted with K or R.

[0036] In some embodiments, the modified GluDH of the present invention comprises substitutions at positions 132, 133, and 175, compared to the initial GluDH, where these amino acid positions are numbered with reference to SEQ ID NO: 1. Preferably, the amino acid at position 132 is substituted with L, the amino acid at position 133 is substituted with V, and the amino acid at position 175 is substituted with G. In some embodiments, the modified GluDH of the present invention further comprises substitutions at one or more positions selected from positions 104, 173, 181, and 182, compared to the initial GluDH. In some embodiments, the amino acid at position 104 is substituted with C. In some embodiments, the amino acid at position 173 is substituted with G or S. In some embodiments, the amino acid at position 181 is substituted with K or R. In some embodiments, the amino acid at position 182 is substituted with R.

[0037] In some embodiments, the modified GluDH of the present invention comprises substitutions at positions 132, 173, and 175, compared to the initial GluDH, where these amino acid positions are numbered with reference to SEQ ID NO: 1. Preferably, the amino acid at position 132 is substituted with L, the amino acid at position 173 is substituted with G or S, and the amino acid at position 175 is substituted with G. In some embodiments, the modified GluDH of the present invention further comprises substitutions at one or more positions selected from positions 104, 133, 181, and 182, compared to the initial GluDH. In some embodiments, the amino acid at position 104 is substituted with C. In some embodiments, the amino acid at position 133 is substituted with V. In some embodiments, the amino acid at position 181 is substituted with K or R. In some embodiments, the amino acid at position 182 is substituted with R.

[0038] In some embodiments, the modified GluDH of the present invention comprises substitutions at positions 133, 173, and 175, compared to the initial GluDH, where these amino acid positions are numbered with reference to SEQ ID NO: 1. Preferably, the amino acid at position 133 is substituted with V, the amino acid at position 173 is substituted with G or S, and the amino acid at position 175 is substituted with G. In some embodiments, the modified GluDH of the present invention further comprises substitutions at one or more positions selected from positions 104, 132, 181, and 182, compared to the initial GluDH. In some embodiments, the amino acid at position 104 is substituted with C. In some embodiments, the amino acid at position 132 is substituted with L. In some embodiments, the amino acid at position 181 is substituted with K or R. In some embodiments, the amino acid at position 182 is substituted with R.

[0039] In some embodiments, the modified GluDH of the present invention comprises substitutions at positions 132, 175, and 182, compared to the initial GluDH, where these amino acid positions are numbered with reference to SEQ ID NO: 1. Preferably, the amino acid at position 132 is substituted with L, the amino acid at position 175 is substituted with G, and the amino acid at position 182 is substituted with R. In some embodiments, the modified GluDH of the present invention further comprises substitutions at one or more positions selected from positions 104, 133, 173, and 181, compared to the initial GluDH. In some embodiments, the amino acid at position 104 is substituted with C. In some embodiments, the amino acid at position 133 is substituted with V. In some embodiments, the amino acid at position 173 is substituted with G or S. In some embodiments, the amino acid at position 181 is substituted with K or R.

[0040] In some embodiments, the modified GluDH of the present invention comprises substitutions at positions 133, 175, and 182, compared to the initial GluDH, where these amino acid positions are numbered with reference to SEQ ID NO: 1. Preferably, the amino acid at position 133 is substituted with V, the amino acid at position 175 is substituted with G, and the amino acid at position 182 is substituted with R. In some embodiments, the modified GluDH of the present invention further comprises substitutions at one or more positions selected from positions 104, 132, 173, and 181, compared to the initial GluDH. In some embodiments, the amino acid at position 104 is substituted with C. In some embodiments, the amino acid at position 132 is substituted with L. In some embodiments, the amino acid at position 173 is substituted with G or S. In some embodiments, the amino acid at position 181 is substituted with K or R.

[0041] In some embodiments, the modified GluDH of the present invention comprises substitutions at positions 173, 175, and 182, compared to the initial GluDH, where these amino acid positions are numbered with reference to SEQ ID NO: 1. Preferably, the amino acid at position 173 is substituted with G or S, the amino acid at position 175 is substituted with G, and the amino acid at position 182 is substituted with R. In some embodiments, the modified GluDH of the present invention further comprises substitutions at one or more positions selected from positions 104, 132, 133, and 181, compared to the initial GluDH. In some embodiments, the amino acid at position 104 is substituted with C. In some embodiments, the amino acid at position 132 is substituted with L. In some embodiments, the amino acid at position 133 is substituted with V. In some embodiments, the amino acid at position 181 is substituted with K or R.

[0042] In some embodiments, the modified GluDH of the present invention comprises amino acid substitutions at positions 22, 56, 173, 175, 182, 199, 381, and 420, compared to the initial GluDH, where these amino acid positions are numbered with reference to SEQ ID NO: 1, and the modified GluDH exhibits increased activity for catalyzing the reaction of 2-carbonyl-4-(hydroxymethylphosphono)butyric acid (PPO) with an amino donor to produce L-glufosinate, and / or exhibits improved stability, and / or exhibits increased Vmax, decreased Km, or increased Vmax / Km, compared to the initial GluDH. Preferably, the amino acid at position 22 is substituted with E. Preferably, the amino acid at position 56 is substituted with Q. Preferably, the amino acid at position 173 is substituted with G. Preferably, the amino acid at position 175 is substituted with G. Preferably, the amino acid at position 182 is substituted with R. Preferably, the amino acid at position 199 is substituted with Y. Preferably, the amino acid at position 381 is substituted with C. Preferably, the amino acid at position 420 is substituted with R. In some embodiments, the modified GluDH further comprises an amino acid substitution at one or more positions selected from positions 31, 124, and 216. Preferably, the amino acid at position 31 is substituted with H. Preferably, the amino acid at position 124 is substituted with L. Preferably, the amino acid at position 216 is substituted with G.

[0043] In some embodiments, the modified GluDH of the present invention comprises, as compared to the initial GluDH, the following amino acid substitutions: 22E-56Q-173G-175G-182R-199Y-381C-420R; 22E-31H-56Q-173G-175G-182R-199Y-381C-420R; 22E-56Q-124L-173G-175G-182R-199Y-381C-420R; 22E-56Q-173G-175G-182R-199Y-216G-381C-420R; 22E-31H-56Q-124L-173G-175G-182R-199Y-381C-420R; 22E-31H-56Q-173G-175G-182R-199Y-216G-381C-420R; 22E-56Q-124L-173G-175G-182R-199Y-216G-381C-420R; or 22E-31H-56Q-124L-173G-175G-182R-199Y-216G-381C-420R Including, These amino acid positions are numbered with reference to SEQ ID NO:1.

[0044] In some embodiments, the modified GluDH further comprises an amino acid substitution at one or more positions selected from 9, 23, 25, 143, 263, 339, 431, and 437. Preferably, the amino acid at position 9 is substituted with S, L, or Y. Preferably, the amino acid at position 23 is substituted with M. Preferably, the amino acid at position 25 is substituted with D. Preferably, the amino acid at position 143 is substituted with E. Preferably, the amino acid at position 263 is substituted with S. Preferably, the amino acid at position 339 is substituted with Q. Preferably, the amino acid at position 431 is substituted with S. Preferably, the amino acid at position 437 is substituted with K.

[0045] The GluDH polypeptide on which the amino acid modifications are based is referred to herein as starting GluDH. The starting GluDH may be a wild-type GluDH or a variant of the wild-type GluDH. For example, if the modifications are based on the polypeptide of SEQ ID NO: 1, the polypeptide of SEQ ID NO: 1 is the "starting GluDH" for the modified GluDH, and if the modifications are based on a variant polypeptide of SEQ ID NO: 1 (e.g., SEQ ID NOs: 3-30), the variant polypeptide is the "starting GluDH" for the modified GluDH.

[0046] As used herein, the term "wild-type GluDH" refers to naturally occurring GluDH. In some embodiments, the initial GluDH is GluDH derived from a microorganism of the Bacillaceae family. In some embodiments, the wild-type GluDH is GluDH derived from a microorganism of the Lysinibacillus or Bacillus genus. Preferably, the wild-type GluDH is GluDH derived from Lysinibacillus sphaericus (SEQ ID NO: 2) or GluDH derived from Bacillus beresensis (SEQ ID NO: 1).

[0047] In the present invention, to determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide in the corresponding position of the second sequence, then such molecules are identical at this position. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage of identity=number of identical positions / total number of positions (i.e., overlapping positions)×100). Preferably, the two sequences are identical in length.

[0048] Those skilled in the art know that various computer programs can be used to determine identity between two sequences.

[0049] "Percentage of amino acid identity" or "percentage of amino acid sequence identity" refers to a comparison between the amino acids of two polypeptides, which, when optimally aligned, have approximately the specified percentage of identical amino acids. For example, "95% amino acid identity" refers to a comparison between the amino acids of two polypeptides, which, when optimally aligned, have approximately 95% of the amino acids of the two polypeptides that are identical.

[0050] In some embodiments, wild-type GluDH has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, and particularly preferably at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1 or 2.

[0051] In some embodiments, the modified GluDH has the following combination of amino acid substitutions compared to its initial GluDH: a substitution of the amino acid at position 104 with C and a substitution of the amino acid at position 175 with G; a substitution of the amino acid at position 132 with L and a substitution of the amino acid at position 175 with G; a substitution of the amino acid at position 133 with V and a substitution of the amino acid at position 175 with G; an amino acid substitution at position 173 with G or S and an amino acid substitution at position 175 with G; the amino acid at position 175 is substituted with G and the amino acid at position 181 is substituted with K or R; a substitution of the amino acid at position 175 with G and the amino acid at position 182 with R; a substitution of the amino acid at position 175 with G and the amino acid at position 203 with I; the amino acid at position 132 is substituted with L, the amino acid at position 133 with V, and the amino acid at position 175 with G; the amino acid at position 132 is substituted with L, the amino acid at position 173 with S, and the amino acid at position 175 with G; the amino acid at position 132 is substituted with L, the amino acid at position 175 with G, and the amino acid at position 182 with R; the amino acid at position 133 is substituted with V, the amino acid at position 173 is substituted with G or S, and the amino acid at position 175 is substituted with G; the amino acid at position 133 is substituted with V, the amino acid at position 175 with G, and the amino acid at position 182 with R; an amino acid substitution at position 173 to G, an amino acid substitution at position 175 to G, and an amino acid substitution at position 182 to R; the amino acid at position 132 is substituted with L, the amino acid at position 133 is substituted with V, the amino acid at position 173 is substituted with S, and the amino acid at position 175 is substituted with G; the amino acid at position 132 is substituted with L, the amino acid at position 133 is substituted with V, the amino acid at position 175 is substituted with G, and the amino acid at position 182 is substituted with R; the amino acid at position 132 is substituted with L, the amino acid at position 173 with S, the amino acid at position 175 with G, and the amino acid at position 182 with R; an amino acid substitution at position 133 to V, an amino acid substitution at position 173 to G or S, an amino acid substitution at position 175 to G, and an amino acid substitution at position 182 to R; and The amino acid at position 132 is substituted with L, the amino acid at position 133 is substituted with V, the amino acid at position 173 is substituted with G or S, the amino acid at position 175 is substituted with G, and the amino acid at position 182 is substituted with R. having These amino acid positions are numbered with reference to SEQ ID NO: 1. Preferably, the initial GluDH has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1 or 2.

[0052] In some embodiments, the modified GluDH of the present invention comprises substitutions at positions 132 and 175 compared to the initial GluDH, these amino acid positions being numbered with reference to SEQ ID NO: 1. Preferably, the amino acid at position 132 is substituted with L, and the amino acid at position 175 is substituted with G. In some embodiments, the modified GluDH of the present invention further comprises substitutions at one or more positions selected from positions 104, 133, 173, 181, and 182 compared to the initial GluDH. In some embodiments, the amino acid at position 104 is substituted with C. In some embodiments, the amino acid at position 133 is substituted with V. In some embodiments, the amino acid at position 173 is substituted with G or S. In some embodiments, the amino acid at position 181 is substituted with K or R. In some embodiments, the amino acid at position 182 is substituted with R. Preferably, the initial GluDH has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1 or 2.

[0053] In some embodiments, the modified GluDH of the present invention comprises substitutions at positions 133 and 175 compared to the initial GluDH, these amino acid positions being numbered with reference to SEQ ID NO: 1. Preferably, the amino acid at position 133 is substituted with V, and the amino acid at position 175 is substituted with G. In some embodiments, the modified GluDH of the present invention further comprises substitutions at one or more positions selected from positions 104, 132, 173, 181, and 182 compared to the initial GluDH. In some embodiments, the amino acid at position 104 is substituted with C. In some embodiments, the amino acid at position 132 is substituted with L. In some embodiments, the amino acid at position 173 is substituted with G or S. In some embodiments, the amino acid at position 181 is substituted with K or R. In some embodiments, the amino acid at position 182 is substituted with R. Preferably, the initial GluDH has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1 or 2.

[0054] In some embodiments, the modified GluDH of the present invention comprises substitutions at positions 173 and 175 compared to the initial GluDH, these amino acid positions being numbered with reference to SEQ ID NO: 1. Preferably, the amino acid at position 173 is substituted with G or S, and the amino acid at position 175 is substituted with G. In some embodiments, the modified GluDH of the present invention further comprises substitutions at one or more positions selected from positions 104, 132, 133, 181, and 182 compared to the initial GluDH. In some embodiments, the amino acid at position 104 is substituted with C. In some embodiments, the amino acid at position 132 is substituted with L. In some embodiments, the amino acid at position 133 is substituted with V. In some embodiments, the amino acid at position 181 is substituted with K or R. In some embodiments, the amino acid at position 182 is substituted with R. Preferably, the initial GluDH has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1 or 2.

[0055] In some embodiments, the modified GluDH of the present invention comprises substitutions at positions 175 and 182 compared to the initial GluDH, these amino acid positions being numbered with reference to SEQ ID NO: 1. Preferably, the amino acid at position 175 is substituted with G, and the amino acid at position 182 is substituted with R. In some embodiments, the modified GluDH of the present invention further comprises substitutions at one or more positions selected from positions 104, 132, 133, 173, and 181 compared to the initial GluDH. In some embodiments, the amino acid at position 104 is substituted with C. In some embodiments, the amino acid at position 132 is substituted with L. In some embodiments, the amino acid at position 133 is substituted with V. In some embodiments, the amino acid at position 173 is substituted with G or S. In some embodiments, the amino acid at position 181 is substituted with K or R. Preferably, the initial GluDH has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1 or 2.

[0056] In some embodiments, the modified GluDH of the present invention comprises substitutions at positions 132, 133, and 175, compared to the initial GluDH, where these amino acid positions are numbered with reference to SEQ ID NO: 1. Preferably, the amino acid at position 132 is substituted with L, the amino acid at position 133 is substituted with V, and the amino acid at position 175 is substituted with G. In some embodiments, the modified GluDH of the present invention further comprises substitutions at one or more positions selected from positions 104, 173, 181, and 182, compared to the initial GluDH. In some embodiments, the amino acid at position 104 is substituted with C. In some embodiments, the amino acid at position 173 is substituted with G or S. In some embodiments, the amino acid at position 181 is substituted with K or R. In some embodiments, the amino acid at position 182 is substituted with R. Preferably, the initial GluDH has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1 or 2.

[0057] In some embodiments, the modified GluDH of the present invention comprises substitutions at positions 132, 173, and 175, compared to the initial GluDH, where these amino acid positions are numbered with reference to SEQ ID NO: 1. Preferably, the amino acid at position 132 is substituted with L, the amino acid at position 173 is substituted with G or S, and the amino acid at position 175 is substituted with G. In some embodiments, the modified GluDH of the present invention further comprises substitutions at one or more positions selected from positions 104, 133, 181, and 182, compared to the initial GluDH. In some embodiments, the amino acid at position 104 is substituted with C. In some embodiments, the amino acid at position 133 is substituted with V. In some embodiments, the amino acid at position 181 is substituted with K or R. In some embodiments, the amino acid at position 182 is substituted with R. Preferably, the initial GluDH has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1 or 2.

[0058] In some embodiments, the modified GluDH of the present invention comprises substitutions at positions 133, 173, and 175, compared to the initial GluDH, where these amino acid positions are numbered with reference to SEQ ID NO: 1. Preferably, the amino acid at position 133 is substituted with V, the amino acid at position 173 is substituted with G or S, and the amino acid at position 175 is substituted with G. In some embodiments, the modified GluDH of the present invention further comprises substitutions at one or more positions selected from positions 104, 132, 181, and 182, compared to the initial GluDH. In some embodiments, the amino acid at position 104 is substituted with C. In some embodiments, the amino acid at position 132 is substituted with L. In some embodiments, the amino acid at position 181 is substituted with K or R. In some embodiments, the amino acid at position 182 is substituted with R. Preferably, the initial GluDH has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1 or 2.

[0059] In some embodiments, the modified GluDH of the present invention comprises substitutions at positions 132, 175, and 182, compared to the initial GluDH, where these amino acid positions are numbered with reference to SEQ ID NO: 1. Preferably, the amino acid at position 132 is substituted with L, the amino acid at position 175 is substituted with G, and the amino acid at position 182 is substituted with R. In some embodiments, the modified GluDH of the present invention further comprises substitutions at one or more positions selected from positions 104, 133, 173, and 181, compared to the initial GluDH. In some embodiments, the amino acid at position 104 is substituted with C. In some embodiments, the amino acid at position 133 is substituted with V. In some embodiments, the amino acid at position 173 is substituted with G or S. In some embodiments, the amino acid at position 181 is substituted with K or R. Preferably, the initial GluDH has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1 or 2.

[0060] In some embodiments, the modified GluDH of the present invention comprises substitutions at positions 133, 175, and 182, compared to the initial GluDH, where these amino acid positions are numbered with reference to SEQ ID NO: 1. Preferably, the amino acid at position 133 is substituted with V, the amino acid at position 175 is substituted with G, and the amino acid at position 182 is substituted with R. In some embodiments, the modified GluDH of the present invention further comprises substitutions at one or more positions selected from positions 104, 132, 173, and 181, compared to the initial GluDH. In some embodiments, the amino acid at position 104 is substituted with C. In some embodiments, the amino acid at position 132 is substituted with L. In some embodiments, the amino acid at position 173 is substituted with G or S. In some embodiments, the amino acid at position 181 is substituted with K or R. Preferably, the initial GluDH has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1 or 2.

[0061] In some embodiments, the modified GluDH of the present invention comprises substitutions at positions 173, 175, and 182, compared to the initial GluDH, where these amino acid positions are numbered with reference to SEQ ID NO: 1. Preferably, the amino acid at position 173 is substituted with G or S, the amino acid at position 175 is substituted with G, and the amino acid at position 182 is substituted with R. In some embodiments, the modified GluDH of the present invention further comprises substitutions at one or more positions selected from positions 104, 132, 133, and 181, compared to the initial GluDH. In some embodiments, the amino acid at position 104 is substituted with C. In some embodiments, the amino acid at position 132 is substituted with L. In some embodiments, the amino acid at position 133 is substituted with V. In some embodiments, the amino acid at position 181 is substituted with K or R. Preferably, the initial GluDH has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1 or 2.

[0062] In some embodiments, the modified GluDH comprises substitutions at positions 173, 175, and 182 compared to the initial GluDH, these amino acid positions being numbered with reference to SEQ ID NO: 1. Preferably, the amino acid at position 173 is substituted with G, the amino acid at position 175 is substituted with G, and the amino acid at position 182 is substituted with R. In some embodiments, the modified GluDH further comprises an amino acid substitution at one or more positions selected from positions 9, 22, 23, 25, 31, 56, 124, 143, 199, 216, 242, 263, 339, 420, 431, and 437. Preferably, the amino acid at position 9 is substituted with S, L, or Y. Preferably, the amino acid at position 22 is substituted with W or E. Preferably, the amino acid at position 23 is substituted with M. Preferably, the amino acid at position 25 is substituted with D. Preferably, the amino acid at position 31 is substituted with H. Preferably, the amino acid at position 56 is substituted with Q. Preferably, the amino acid at position 124 is substituted with L. Preferably, the amino acid at position 143 is substituted with E. Preferably, the amino acid at position 199 is substituted with W or Y. Preferably, the amino acid at position 216 is substituted with G. Preferably, the amino acid at position 263 is substituted with S and the amino acid at position 339 is substituted with Q. Preferably, the amino acid at position 420 is substituted with R. Preferably, the amino acid at position 431 is substituted with S. Preferably, the amino acid at position 437 is substituted with K. Preferably, the initial GluDH has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1 or 2.

[0063] In some embodiments, the modified GluDH comprises amino acid substitutions at positions 22, 56, 173, 175, 182, 199, and 420. Preferably, the amino acid at position 22 is substituted with E. Preferably, the amino acid at position 56 is substituted with Q. Preferably, the amino acid at position 173 is substituted with G. Preferably, the amino acid at position 175 is substituted with G. Preferably, the amino acid at position 182 is substituted with R. Preferably, the amino acid at position 199 is substituted with Y. Preferably, the amino acid at position 420 is substituted with R. In some embodiments, the modified GluDH further comprises amino acid substitutions at one or more positions selected from positions 31, 124, and 216. Preferably, the amino acid at position 31 is substituted with H. Preferably, the amino acid at position 124 is substituted with L. Preferably, the amino acid at position 216 is substituted with G. Preferably, the initial GluDH has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1 or 2.

[0064] In some embodiments, the modified GluDH of the present invention comprises amino acid substitutions at positions 22, 56, 173, 175, 182, 199, 381, and 420, compared to the initial GluDH, where these amino acid positions are numbered with reference to SEQ ID NO: 1, and the modified GluDH exhibits increased activity for catalyzing the reaction of 2-carbonyl-4-(hydroxymethylphosphono)butyric acid (PPO) with an amino donor to produce L-glufosinate, and / or exhibits improved stability, and / or exhibits increased Vmax, decreased Km, or increased Vmax / Km, compared to the initial GluDH. Preferably, the amino acid at position 22 is substituted with E. Preferably, the amino acid at position 56 is substituted with Q. Preferably, the amino acid at position 173 is substituted with G. Preferably, the amino acid at position 175 is substituted with G. Preferably, the amino acid at position 182 is substituted with R. Preferably, the amino acid at position 199 is substituted with Y. Preferably, the amino acid at position 381 is substituted with C. Preferably, the amino acid at position 420 is substituted with R. In some embodiments, the modified GluDH further comprises an amino acid substitution at one or more positions selected from positions 31, 124, and 216. Preferably, the amino acid at position 31 is substituted with H. Preferably, the amino acid at position 124 is substituted with L. Preferably, the amino acid at position 216 is substituted with G. Preferably, the initial GluDH has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1 or 2. For example, the initial GluDH comprises or consists of SEQ ID NO: 1.

[0065] In some embodiments, the modified GluDH of the present invention comprises, as compared to the initial GluDH, the following amino acid substitutions: 22E-56Q-173G-175G-182R-199Y-381C-420R; 22E-31H-56Q-173G-175G-182R-199Y-381C-420R; 22E-56Q-124L-173G-175G-182R-199Y-381C-420R; 22E-56Q-173G-175G-182R-199Y-216G-381C-420R; 22E-31H-56Q-124L-173G-175G-182R-199Y-381C-420R; 22E-31H-56Q-173G-175G-182R-199Y-216G-381C-420R; 22E-56Q-124L-173G-175G-182R-199Y-216G-381C-420R; or 22E-31H-56Q-124L-173G-175G-182R-199Y-216G-381C-420R Including, These amino acid positions are numbered with reference to SEQ ID NO: 1. Preferably, the initial GluDH has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1 or 2. For example, the initial GluDH comprises or consists of SEQ ID NO: 1.

[0066] In some embodiments, the modified GluDH further comprises an amino acid substitution at one or more positions selected from 9, 23, 25, 143, 263, 339, 431, and 437. Preferably, the amino acid at position 9 is substituted with S, L, or Y. Preferably, the amino acid at position 23 is substituted with M. Preferably, the amino acid at position 25 is substituted with D. Preferably, the amino acid at position 143 is substituted with E. Preferably, the amino acid at position 263 is substituted with S. Preferably, the amino acid at position 339 is substituted with Q. Preferably, the amino acid at position 431 is substituted with S. Preferably, the amino acid at position 437 is substituted with K. Preferably, the initial GluDH has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1 or 2. For example, the initial GluDH comprises or consists of SEQ ID NO: 1.

[0067] In some embodiments, the modified GluDH of the present invention has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, and particularly preferably at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1 or 2.

[0068] In some embodiments, the initial GluDH differs from SEQ ID NO: 1 or 2 by including one or more amino acid substitutions, deletions, insertions, and / or additions. In some embodiments, the initial GluDH includes one or more amino acid conservative substitutions compared to SEQ ID NO: 1 or 2. In some embodiments, the initial GluDH includes one or more amino acid insertions or deletions compared to SEQ ID NO: 1 or 2.

[0069] The term "conservative substitution", also referred to as substitution with a "homologous" amino acid residue, means that an amino acid is replaced with an amino acid residue having a similar side chain, such as amino acids having basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0070] In general, conservative substitution of amino acids has minimal effect on the activity of the resulting protein. Such substitutions are described below. Conservative substitution is replacing an amino acid with an amino acid that is similar in size, hydrophobicity, charge, polarity, spatial characteristics, and aromaticity. When it is desired to precisely adjust the properties of a protein, the substitution is generally conservative.

[0071] As used herein, "homologous" amino acid residues refer to amino acid residues that have similar chemical properties with respect to hydrophobicity, charge, polarity, steric characteristics, aromatic characteristics, etc. Examples of amino acids that are homologous to each other include lysine, arginine, and histidine, which are positively charged; glutamic acid and aspartic acid, which are negatively charged; glycine, alanine, valine, leucine, isoleucine, proline, and phenylalanine, which are hydrophobic; serine, threonine, cysteine, methionine, tryptophan, tyrosine, asparagine, glutamine, which are polar; phenylalanine, tyrosine, and tryptophan, which are aromatic; serine and threonine or glutamine and asparagine or leucine and isoleucine, which have chemically similar side chain groups.

[0072] Examples of conservative amino acid substitutions in proteins include Ala to Ser, Arg to Lys, Asn to Gln or His, Asp to Glu, Cys to Ser, Gln to Asn, Glu to Asp, Gly to Pro, His to Asn or Gln, Ile to Leu or Val, Leu to Ile or Val, Lys to Arg or Gln, Met to Leu or Ile, Phe to Met, Leu, or Tyr, Ser to Thr, Thr to Ser, Trp to Tyr, Tyr to Trp or Phe, and Val to Ile or Leu.

[0073] In some embodiments, the modified GluDH comprises or consists of one of SEQ ID NOs: 4-14, 16-19, 21, 22, 24, 25, 27-30, 32-48, 50, 51, and 53-72, or the modified GluDH comprises 1-10 amino acid substitutions (e.g., conservative substitutions) compared to one of SEQ ID NOs: 4-14, 16-19, 21, 22, 24, 25, 27-30, 32-48, 50, 51, and 53-72, the substitutions being at positions 9, 22, 23, 25, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 51, and 53-72. The modified GluDH is present at a position other than positions 6, 104, 124, 132, 133, 143, 173, 175, 181, 182, 199, 203, 216, 242, 263, 339, 420, 431, and 437, and exhibits increased activity for catalyzing the reaction of PPO with an amino donor to produce L-glufosinate and / or improved kinetic properties, e.g., increased Vmax, decreased Km, or increased Vmax / Km, compared to the initial GluDH. In some embodiments, the modified GluDH comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions (e.g., conservative substitutions) compared to one of SEQ ID NOs: 4-14, 16-19, 21, 22, 24, 25, 27-30, 32-48, 50, 51, and 53-72, where the substitutions are at positions other than 9, 22, 23, 25, 31, 56, 104, 124, 132, 133, 143, 173, 175, 181, 182, 199, 203, 216, 242, 263, 339, 420, 431, and 437. In some embodiments, the modified GluDH of the present invention has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, and particularly preferably at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1 or 2.

[0074] In some embodiments, the modified GluDH comprises or consists of one of SEQ ID NOs: 73-80, or the modified GluDH comprises 1-10 amino acid substitutions (e.g., conservative substitutions) compared to one of SEQ ID NOs: 73-80, where the substitutions are at positions other than 22, 31, 56, 124, 173, 175, 182, 199, 216, 381, and 420, and the modified GluDH exhibits increased activity for catalyzing the reaction of PPO with an amino donor to produce L-glufosinate, and / or exhibits improved stability, and / or exhibits improved kinetic properties, e.g., increased Vmax, decreased Km, or increased Vmax / Km, compared to SEQ ID NOs: 1, 3, 21, or 65. In some embodiments, the modified GluDH of the present invention has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, and particularly preferably at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1 or 2.

[0075] As used herein, the activity of an enzyme refers to the reduction in substrate or increase in product per unit time in a chemical reaction catalyzed by a unit mass of an enzyme under certain conditions. For example, the activity of the modified GluDH of the present invention can be represented by the reduction in PPO or increase in L-glufosinate per unit time catalyzed by a unit mass of modified GluDH under certain conditions.

[0076] The activity of an enzyme herein may also refer to the relative activity of the enzyme, such as the relative activity percentage, expressed as the ratio of the activity of an enzyme of interest to the activity of a given enzyme that catalyzes the same reaction.

[0077] In some embodiments, the activity of the modified GluDH of the invention is expressed as a relative activity percentage compared to SEQ ID NO: 3. In some embodiments, the activity of the modified GluDH to catalyze the reaction of PPO with an amino donor to produce L-glufosinate is at least 100%, 105%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, or more than the activity of SEQ ID NO: 3 to catalyze this reaction.

[0078] In some embodiments, the activity of the modified GluDH of the invention is expressed as a relative activity percentage compared to SEQ ID NO: 31. In some embodiments, the activity of the modified GluDH to catalyze the reaction of PPO with an amino donor to produce L-glufosinate is at least 100%, 105%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more than the activity of SEQ ID NO: 31 to catalyze this reaction.

[0079] As used herein, the term "amino donor" refers to a compound that provides an amino group, including inorganic and organic compounds. "Amino donors" include, but are not limited to, ammonium salts (e.g., NH4Cl, NH4NO3, (NH4)2SO4, ammonium acetate, etc.), amino acids, or organic amines. In some embodiments, the amino donor is an ammonium salt, such as NH4Cl.

[0080] For production, the activity of the enzyme as well as its stability and kinetic properties must be taken into consideration.

[0081] Improved stability, as used herein, includes increased stability. In some embodiments of the present invention, the stability of an enzyme is thermostability, which means that the activity does not decrease or decreases only slightly after incubation at a certain temperature (e.g., 40-60°C or higher) for a certain period of time (e.g., 10 minutes to 1 hour). Taking modified glutamate dehydrogenase (mGluDH) as an example, the initial activity of mGluDH A is A1, its activity after incubation at 43°C for 20 minutes is A2, the initial activity of mGluDH B is B1, and its activity after incubation at 43°C for 20 minutes is B2. If A1 / A2 is greater than B1 / B2, the thermostability of mGluDH B is higher than that of mGluDH A.

[0082] Kinetic properties of an enzyme herein include, but are not limited to, Vmax, Km, and Vmax / Km of the enzyme. Improvements in kinetic properties herein include, but are not limited to, an increase in Vmax, a decrease in Km, and an increase in Vmax / Km.

[0083] As used herein, the term "Vmax" refers to the maximum rate of catalytic reaction that can be achieved at a certain concentration of enzyme. In particular, under the condition of constant enzyme concentration, when the substrate concentration is within a certain range, the rate of reaction generally increases with increasing substrate concentration, and when the substrate concentration reaches a certain value, the rate of reaction will reach a maximum value, and the rate of reaction will not increase with increasing substrate concentration.

[0084] As used herein, the term "Km" refers to the concentration of substrate at which the rate of a reaction reaches half the maximum rate of catalysis, or Vmax, at a particular enzyme concentration.

[0085] II. Polynucleotides encoding modified GluDH As used herein, the term "polynucleotide" or "nucleic acid molecule" includes DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA), as well as analogs of DNA or RNA produced using nucleotide analogs. Nucleic acid molecules can be single-stranded or double-stranded, preferably double-stranded DNA. Nucleic acid synthesis can use nucleotide analogs or derivatives (e.g., inosine nucleotides or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acids with altered base pairing capabilities or increased nuclease resistance.

[0086] The present invention also provides a polynucleotide encoding a modified GluDH of the present invention. Thus, in the present invention, the term modification also includes genetic manipulation of a polynucleotide encoding a GluDH polypeptide of the present invention. The modification may be a substitution, deletion, insertion and / or addition of one or more nucleotides.

[0087] As used herein, the term "encoding" means that a polynucleotide directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which generally begins with the ATG start codon or other start codon such as GTG and TTG, and ends with a stop codon such as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA, or recombinant nucleotide sequence.

[0088] In addition, a nucleic acid molecule encompassing all or a portion of a nucleic acid sequence of the present invention can be isolated by polymerase chain reaction (PCR) using oligonucleotide primers designed and synthesized based on the sequence information contained in the sequence.

[0089] Polynucleotides of the invention can be amplified using cDNA, mRNA, or genomic DNA as a template and suitable oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid thus amplified can be cloned into a suitable vector and characterized by DNA sequence analysis.

[0090] The polynucleotides of the invention can be prepared by standard synthetic techniques, eg, by using an automated DNA synthesizer.

[0091] The present invention also relates to the complementary strands of the nucleic acid molecules described herein. A nucleic acid molecule that is complementary to another nucleotide sequence is a molecule that is sufficiently complementary to the nucleotide sequence such that it can hybridize with the other nucleotide sequence to form a stable duplex.

[0092] As used herein, generally, the term "hybridization" refers to nucleotide sequences that are at least about 90%, preferably at least about 95%, more preferably at least about 96%, more preferably at least 98% homologous to each other, maintaining hybridization to each other under given stringent hybridization and wash conditions.

[0093] Those skilled in the art are aware of various conditions for hybridization, such as stringent hybridization conditions and highly stringent hybridization conditions. See, e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, New York; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, John Wiley & Sons, New York.

[0094] Of course, the polynucleotides of the present invention do not include polynucleotides that merely hybridize to polyA sequences (such as the 3' terminal poly(A) of mRNA) or to complementary stretches of polyT (or U) residues. III. Expression and Production of Modified GluDH Also provided are nucleic acid constructs and vectors comprising the polynucleotides of the invention, such as expression vectors, for expressing a modified GluDH of the invention.

[0095] As used herein, the term "expression" includes any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0096] The term "nucleic acid construct" refers to a single- or double-stranded nucleic acid molecule that has been isolated from a naturally occurring gene or modified to contain a non-naturally occurring nucleic acid segment. When the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention, the term nucleic acid construct is synonymous with the term "expression cassette."

[0097] The term "expression vector" as used herein refers to a linear or circular DNA molecule that contains a polynucleotide encoding a polypeptide of the invention operably linked to additional nucleotides, such as control sequences, that provide for expression of the polynucleotide. Expression vectors include viral vectors or plasmid vectors.

[0098] The term "control sequences" as used herein includes all elements necessary or beneficial for expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide, or native or foreign to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters and signals for terminating transcription and translation.

[0099] For example, the control sequence may be a suitable promoter sequence, which is a nucleotide sequence that is recognized by a host cell and causes expression of a polynucleotide encoding a polypeptide of the present invention. The promoter sequence includes a transcription control sequence that mediates the expression of the polypeptide. The promoter may be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, for example, the lac operon of E. coli. Promoters also include mutant, truncated, and hybrid promoters and may be derived from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0100] The term "operably linked" as used herein refers to a configuration in which a control sequence is positioned in appropriate relation to a coding sequence of a polynucleotide sequence such that the control sequence directs expression of the polypeptide coding sequence.

[0101] The polynucleotide encoding the polypeptide of the present invention can be subjected to various manipulations to allow expression of the polypeptide. It may be desirable or necessary to manipulate the polynucleotide depending on the expression vector before inserting it into the vector. Techniques for modifying polynucleotide sequences by recombinant DNA methods are well known in the art.

[0102] To identify and select host cells containing an expression vector of the invention, the vector of the invention preferably contains one or more selectable markers that allow for easy selection of transformed, transfected, transduced cells, etc. A selectable marker is a gene whose product results in a biocide or provides viral resistance, heavy metal resistance, auxiliary auxotrophy, etc. For example, a bacterial selectable marker is the dal gene from Bacillus subtilis or Bacillus licheniformis, or a marker that confers antibiotic resistance, such as ampicillin resistance, kanamycin resistance, chloramphenicol resistance, or tetracycline resistance.

[0103] The vectors of the invention may integrate into the genome of a host cell or may autonomously replicate within the cell independently of the genome. Elements necessary for integration into the genome or for autonomous replication of a host cell are known in the art (see, for example, Sambrook et al., 1989, supra).

[0104] Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" refer to various techniques for introducing foreign nucleic acid (such as DNA) into a host cell, which are well known in the art and can be found, for example, in Sambrook et al., 1989, supra; Davis et al., Basic Methods in Molecular Biology (1986); and other laboratory manuals.

[0105] The present invention also relates to recombinant host cells comprising the polynucleotides of the present invention, which are advantageously used in the recombinant production of GluDH polypeptides. A vector comprising the polynucleotide of the present invention is introduced into a host cell, whereby the vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector. Those skilled in the art are aware of conventional vectors and host cells for expressing proteins.

[0106] In some embodiments, a host cell of the invention is an E. coli cell, such as E. coli BL21(DE3). In some embodiments, the expression vector is pET-30a(+).

[0107] The modified GluDH of the present invention can be operably linked to a non-GluDH polypeptide (e.g., a heterologous amino acid sequence) to form a fusion protein. For example, in one embodiment, the fusion protein is a GST-GluDH fusion protein in which the GluDH sequence is fused to the C-terminus of the GST sequence. This fusion protein can facilitate the purification of recombinant GluDH. In another embodiment, the fusion protein is a GluDH protein that includes a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian and yeast host cells), the use of a heterologous signal sequence can increase expression and / or secretion of GluDH.

[0108] IV. Production of L-glufosinate Furthermore, the present invention provides a method for preparing L-glufosinate, the method comprising the step of contacting the modified GluDH or a host cell of the present invention with PPO. In some embodiments, the method for preparing L-glufosinate-ammonium of the present invention comprises: (a) providing the activity of the modified GluDH of the present invention to a reaction medium containing PPO and an amino donor, and optionally the reaction medium containing an NADPH / NADP cycle system; (b) incubating the reaction medium to produce L-glufosinate. Includes.

[0109] In some embodiments, a cell-free catalytic method is used to produce L-glufosinate, and the modified GluDH of the present invention is provided in step (a). In some embodiments, free or immobilized modified GluDH of the present invention can be used.

[0110] In some embodiments, the amino donor is an ammonium salt, such as NH4Cl.

[0111] In some embodiments, the reaction medium comprises NADPH / NADP. NADPH / NADP cycle systems suitable for the present invention are known in the art and include, but are not limited to, alcohol dehydrogenase, glucose dehydrogenase (GDH), or glucose-6-phosphate dehydrogenase (G6PD). In some embodiments, the NADPH / NADP cycle system comprises alcohol dehydrogenase. Also, in some embodiments, the alcohol dehydrogenase may be immobilized.

[0112] In some embodiments, the incubation is carried out at 20-50°C, preferably 25-40°C, more preferably 28-35°C, for example 30°C.

[0113] In some embodiments, the medium is a buffer solution, such as PBS buffer and Tris-HCl buffer. In one embodiment, the medium is a PBS buffer solution, such as 100 mM PBS buffer. In some embodiments, the pH of the reaction medium is 7.5-8.

[0114] In some embodiments, the reaction medium is a medium that is partially or entirely composed of cell culture medium, and the activity of the modified GluDH of the present invention is provided by a host cell of the present invention cultured in the reaction medium.

[0115] In some embodiments, the reaction medium is a medium that is partially or entirely composed of cell culture medium, and the NADPH / NADP cycle system, such as alcohol dehydrogenase activity, is provided by a host cell of the invention or by a second host cell cultured in the reaction medium.

[0116] In some embodiments, the host cells of the invention and / or the second host cells are cultured and grown in a cell culture medium, then the grown host cells are separated from the cell culture medium and the biomass is resuspended using a buffer or water, and a PPO is provided to the buffer or water before, during, or after the addition of the grown host cells.

[0117] In some embodiments, bacterial cells, such as E. coli cells, can be used. EXAMPLES

[0118] Those skilled in the art will understand the present invention more clearly through the following examples. It should be understood that the examples do not limit the scope of the present invention, but are for illustrative purposes only.

[0119] Example 1. Materials and Methods Unless otherwise specified, all laboratory methods used in the present invention are conventional. Specific gene cloning procedures can be found in Sambrook et al., 1989, supra.

[0120] i) Reagents: DNA polymerase (PrimeSTAR Max DNA polymerase) and DpnI endonuclease were purchased from TaKaRa. Plasmid isolation kit was purchased from Axygen. PPO was synthesized by the applicant according to the prior art (see J.Org.Chem. 1991, vol. 56, pp. 1783-1788). NH4Cl was purchased from Sinopharm Chemical Reagent Co.Ltd., Beijing. NADP+ / NADPH was purchased from Aladdin. Alcohol dehydrogenase is a dehydrogenase derived from Lactobacillus kefiri (NCBI accession number WP_054768785.1), which is recombinantly expressed in E. coli.

[0121] ii) Vectors and strains: The expression vector used was pET-30a(+). The plasmids were purchased from Novagen. The host cells used were E. coli BL21(DE3) purchased from Tiangen BioTech (Beijing) Co., Ltd.

[0122] iii) Sequencing and primer synthesis were performed by Synbio Technologies Co., Ltd.

[0123] iv) Site-directed mutagenesis: Specific primer pairs were designed to introduce the desired substitutions at the bases corresponding to the amino acid positions where mutations were required. The isolated pre-mutagenesis plasmid (containing the coding sequence of wild-type GluDH and the pET-30a(+) backbone) was used as a template to introduce the mutations by PCR using the Quickchange technology (Nucleic Acids Research, 2004, vol. 32(14):e115). After PCR amplification, the amplified products were digested with DpnI for 4 hours to remove the template plasmid. The digestion products were transformed into E. coli BL21(DE3) competent cells, followed by plating on LB agar (containing 50 mg / L kanamycin), and a single colony was inoculated and grown in LB broth (containing 50 mg / L kanamycin), and sequenced to confirm that it was the correct mutant. The confirmed clones were stored at -80°C for future use.

[0124] v) Protein expression and preparation of crude enzyme solutions: The stored clones were activated on LB agar. A single colony was then inoculated into LB broth (containing 50 mg / L kanamycin) and incubated at 37°C with shaking for 12 hours. 1 mL of the culture was transferred to 50 mL of fresh LB broth (containing 50 mg / L kanamycin) and incubated at 37°C with shaking until the OD600 reached approximately 0.6, and then incubated at 25°C for 16 hours after adding IPTG (final concentration 0.4 mM) to induce protein expression.

[0125] After incubation, the culture was centrifuged at 4,000g for 10 min at 4°C, the supernatant was discarded, and the E. coli cells were harvested. The harvested E. coli cells were resuspended in 15 mL of pre-chilled 50 mM PBS, pH 7.0, and disrupted by sonication at 4°C. The cell disruption solution was centrifuged at 6,000g for 15 min at 4°C to remove the precipitate. The resulting supernatant was a crude enzyme solution containing the recombinant enzyme.

[0126] vi) Determination of enzyme activity NH4Cl and NADP+ were added to a PPO solution (100 mM) in PBS, and the pH of the solution was adjusted to 8 with aqueous ammonia. The solution contained a final concentration of 100 mM PPO, a final concentration of 50 mM NH4Cl, and a final concentration of 0.2 g / L NADP+. The crude enzyme solution and alcohol dehydrogenase obtained as described in v) were added to the above solution. The final concentration of GluDH is 0.02 g / L, and the final concentration of alcohol dehydrogenase is 0.2 g / L. The solution was kept shaking (400 rpm) at 30°C for 2 hours on a shaker, and then sampled to detect the amount of L-glutamine produced by OPA pre-column derivatization HPLC, thereby determining the initial rate of the catalytic reaction.

[0127] vii) Determination of the kinetic properties of enzymes Several reaction systems (200 μL) containing 100 mM PBS at pH 7.5 (pH adjusted with aqueous ammonia), 0.15 mM NADPH (reduced), 50 mM NH4Cl, 10% by volume diluted (500-fold) crude enzyme solution, and various concentrations (5-100 mM) of substrate PPO were prepared in a 96-well plate. The intensity of UV absorbance at 340 nm was detected at 30°C, and the rate of change in absorbance was recorded over time (mA / min). The obtained parameters were substituted into the Michaelis-Menten equation, and the reaction rate was calculated from the rate of change in absorbance.

[0128] Example 2. Preparation and detection of mutants of GluDH (BvGluDH) derived from Bacillus verense Mutants were prepared using the coding nucleic acid of BvGluDH (SEQ ID NO: 1) as a template, and the enzyme activity and kinetic parameters Vmax and Km were measured according to the method of Example 1. The obtained mutants and their enzyme activities and kinetic parameters are shown in Table 1. The relative enzyme activity means the percentage of the enzyme activity of the mutant to the enzyme activity of the mutant of SEQ ID NO: 3.

[0129] [Table 1]

[0130] Example 3. Preparation and detection of mutants of GluDH (LsGluDH) derived from Lysinibacillus sphaericus Mutants were prepared using the coding nucleic acid of LsGluDH (SEQ ID NO: 2) as a template, and the enzyme activity and kinetic parameters Vmax and Km were measured according to the method of Example 1. The obtained mutants and their enzyme activities and kinetic parameters are shown in Table 2. The relative enzyme activity means the percentage of the enzyme activity of the mutant to the enzyme activity of the mutant of SEQ ID NO: 31.

[0131] [Table 2]

[0132] Example 4. Preparation and detection of mutants of BvGluDH Mutants were prepared by introducing additional mutations based on the mutant of SEQ ID NO:21, and the enzyme activity was measured according to the method of Example 1. The obtained mutants and their enzyme activities are shown in Table 3. The initial relative enzyme activity means the percentage of the enzyme activity of the mutant relative to the enzyme activity of the mutant of SEQ ID NO:21 without heat treatment. The relative enzyme activity after heat treatment means the percentage of the enzyme activity of the mutant relative to the enzyme activity of the mutant of SEQ ID NO:21 after incubation at 45°C for 30 minutes.

[0133] [Table 3]

[0134] Example 5. Preparation and detection of mutants of BvGluDH Mutants were prepared by introducing additional mutations based on the mutant of SEQ ID NO:65, and the enzyme activity was measured according to the method of Example 1. The obtained mutants and their enzyme activities are shown in Table 4. The relative enzyme activity means the percentage of the enzyme activity of the mutant to the enzyme activity of the mutant of SEQ ID NO:65.

[0135] [Table 4]

Claims

1. A modified glutamate dehydrogenase (GluDH) comprising amino acid substitutions at positions 22, 56, 173, 175, 182, 199, 381, and 420 compared to an initial glutamate dehydrogenase (GluDH), in which the amino acid at position 22 is substituted with E, the amino acid at position 56 is substituted with Q, the amino acid at position 173 is substituted with G, the amino acid at position 175 is substituted with G, the amino acid at position 182 is substituted with R, the amino acid at position 199 is substituted with Y, the amino acid at position 381 is substituted with C, and the amino acid at position 420 is substituted with R; The positions of these amino acids are numbered with reference to SEQ ID NO:1, and the modified GluDH exhibits increased activity for catalyzing the reaction of 2-carbonyl-4-(hydroxymethylphosphono)butyric acid (PPO) with an amino donor to produce L-glufosinate, and / or exhibits improved stability, and / or exhibits increased Vmax, decreased Km, or increased Vmax / Km, compared to the initial GluDH.

2. The modified GluDH according to claim 1, wherein the initial GluDH is a wild-type GluDH.

3. 3. The modified GluDH according to claim 1 or 2, wherein the initial GluDH is derived from a microorganism of the Bacillaceae family, preferably from a microorganism of the genus Lysinibacillus or Bacillus, more preferably from Lysinibacillus sphaericus or Bacillus velezensis.

4. The modified GluDH according to any one of claims 1 to 3, wherein the initial GluDH comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

5. 5. The modified GluDH of claim 1, further comprising an amino acid substitution at one or more positions selected from 31, 124, and 216, wherein the amino acid at position 31 is substituted with H, the amino acid at position 124 is substituted with L, and the amino acid at position 216 is substituted with G.

6. 4. The modified GluDH of claim 1, further comprising an amino acid substitution at one or more positions selected from positions 9, 23, 25, 143, 263, 339, 431, and 437, wherein the amino acid at position 9 is substituted with S, L, or Y, the amino acid at position 23 is substituted with M, the amino acid at position 25 is substituted with D, the amino acid at position 143 is substituted with E, the amino acid at position 263 is substituted with S, the amino acid at position 339 is substituted with Q, the amino acid at position 431 is substituted with S, and the amino acid at position 437 is substituted with K.

7. A modified GluDH comprising the amino acid sequence of any one of SEQ ID NOs:73-80.

8. A polynucleotide encoding the modified GluDH according to any one of claims 1 to 7.

9. An expression vector comprising the polynucleotide of claim 8.

10. A host cell comprising the modified GluDH according to any one of claims 1 to 7, the polynucleotide according to claim 8, or the vector according to claim 9.

11. A method for producing L-glufosinate, comprising contacting a modified GluDH according to any one of claims 1 to 7 or a host cell according to claim 10 with PPO.

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