Modified glutamate dehydrogenase and use thereof

Abstract

To provide a modified glutamate dehydrogenase (GluDH).SOLUTION: A modified GluDH of the present invention shows an increased activity for catalyzing the reaction of 4-(hydroxymethylphosphinyl)-2-oxobutanoic acid (PPO) and an amino donor to generate L-glufosinate and / or an improved dynamic property. The present invention also refers to a polynucleotide encoding the modified GluDH of the present invention, a vector and a host cell for expressing the modified GluDH of the present invention, and a method of producing L-glufosinate using the modified GluDH and the host cell of the present invention.SELECTED DRAWING: None

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 Art

[0002] Glufosinate (also called 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 herbicide that disrupts the nitrogen metabolism of plants by inhibiting the activity of L-glutamine synthetase, ultimately killing the plants. Glufosinate has greater advantages than glyphosate, such as a wide application range, rapid efficacy, long-term persistence, low toxicity, and safety. Therefore, the sales of glufosinate are increasing rapidly, and a large demand is expected in the next period, making it very promising.

[0003] However, the process for producing glufosinate is complex and the production difficulty is high. Due to the high price, it is hindered 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 chiral pure L-glufosinate by deracemization of D,L-glufosinate is practically important and has been becoming common in the synthesis of L-glufosinate in recent years.

[0004] In recent years, numerous methods for preparing L-glufosinate from D,L-glufosinate have been reported. Traditional separation methods by chemical modification are not competitive due to the high cost and the fact that D-glufosinate cannot be used. Currently, 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 the selective hydrolysis of L-N-acetylglufosinate catalyzed by carboxypeptidase. However, D-N-acetylglufosinate cannot be hydrolyzed and can be reused in the hydrolysis step after chemical or enzymatic racemization (see, for example, Chinese Patent Application Publication No. 108690854). The disadvantages of this method include that there are multiple reaction steps and it is necessary to separate L-glufosinate obtained from hydrolysis from the N-acetyl substrate.

[0006] D-glufosinate is oxidized to 2-oxo-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-supported carbon to produce D,L-glufosinate, and D,L-glufosinate is gradually converted to L-glufosinate due to the stereoselectivity of DAAO (see, for example, Chinese Patent Application Publication No. 105567780). The disadvantages of this solution include that a large amount of palladium-carbon catalyst is required and the reaction raw materials (such as oxygen and ammonium formate) are wasted.

[0007] In addition, PPO can be converted to L-glufosinate by a stereoselective aminotransfer reaction catalyzed by L-amino acid transaminase (L-TA) (see, for example, U.S. Patent Application Publication No. 20180030487). This solution has the drawback that the aminotransfer step is an equilibrium reaction, and thus an excess amount of an amino donor (amino acid or organic amine) needs to be supplied to achieve a high conversion rate (for example, when supplying 3 equivalents of the amino donor, the conversion rate is 90%), and the excess amount of the amino donor and the corresponding by-products will have a profound impact on 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 an inorganic ammonium salt as an amino donor under the assistance of an NAD(P)H cycle that consumes formic acid, glucose, or a simple alcohol. The reaction catalyzed by L-AADH does not require a very excessive hydrogen donor and can achieve a high conversion rate.

[0009] It has been reported that L-glufosinate is prepared using a native or modified glutamate dehydrogenase (GluDH) that catalyzes the asymmetric reductive amination of PPO (see, for example, Chinese Patent No. 107630052, Chinese Patent No. 106978453, Chinese Patent No. 108588045, and Chinese Patent Application Publication No. 109609474).

[0010] However, in the art, there is still a need to provide a GluDH that shows an increase in activity and / or improvement in kinetic properties (for example, an increase in the Vmax value, a decrease in the Km value, or an increase in Vmax / Km) for catalyzing the reaction between PPO and an amino donor to produce L-glufosinate. SUMMARY OF THE INVENTION

[0011] In a first aspect, the present invention provides a modified glutamate dehydrogenase comprising amino acid substitutions at two or more positions compared to an initial GluDH, which shows an increase in activity and / or improvement in kinetic properties (e.g., an increase in Vmax, a decrease in Km, or an increase in Vmax / Km) for catalyzing the reaction of PPO with an amino donor to produce L-glufosinate.

[0012] In some embodiments, the modified GluDH, compared to the initial GluDH, the amino acid at position 104 is substituted with C and the amino acid at position 175 is substituted with G; the amino acid at position 132 is substituted with L and the amino acid at position 175 is substituted with G; the amino acid at position 133 is substituted with V and the amino acid at position 175 is substituted with G; 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 175 is substituted with G and the amino acid at position 181 is substituted with K or R; 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 175 is substituted with G and the amino acid at position 203 is substituted with I; 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; the amino acid at position 132 is substituted with L, 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 175 is substituted with G, and the amino acid at position 182 is substituted 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 is substituted with G, and the amino acid at position 182 is substituted with R; 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; 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 is substituted with S, 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 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 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 comprising a combination of amino acid substitutions selected from The positions are numbered with reference to SEQ ID NO: 1.

[0013] In some embodiments, the modified GluDH includes substitutions at positions 173, 175, and 182, as numbered with reference to SEQ ID NO: 1, compared to its native GluDH. 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 includes amino acid substitutions 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.

[0014] In some embodiments, the modified GluDH includes 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 includes 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.

[0015] In some embodiments, the initial GluDH is wild-type GluDH. In some embodiments, the initial GluDH is derived from a microorganism of the family Bacillaceae, preferably a microorganism of the genus Lysinibacillus or Bacillus, more preferably Lysinibacillus sphaericus or Bacillus velezensis. In a preferred embodiment, the initial GluDH includes the amino acid sequence of SEQ ID NO: 1 or 2.

[0016] In some embodiments, the modified GluDH includes 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.

[0017] In some embodiments, the activity of the modified GluDH for catalyzing the reaction of PPO and an amino donor to produce L-glufosinate is at least 100%, 105%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or higher than the activity of SEQ ID NO: 3 that catalyzes this reaction.

[0018] In a second aspect, the present invention provides a polynucleotide encoding the modified GluDH of the present invention and a vector comprising the polynucleotide of the present invention.

[0019] In a third aspect, the present invention provides a host cell comprising the modified GluDH of the present invention, its coding polynucleotide, or a vector comprising the above polynucleotide.

[0020] 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.

Mode for Carrying Out the Invention

[0021] The present invention mainly relates to a modified GluDH for catalyzing the reaction of PPO and an amino donor to produce L-glufosinate. Unless otherwise specified, the terms used herein have meanings commonly understood by those skilled in the art.

[0022] 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-ketoglutaric acid. Further, GluDH has the activity of catalyzing the reaction of PPO with an amino donor to produce L-glufosinate. The present invention provides a modified GluDH polypeptide that exhibits an increase in activity for catalyzing the reaction of PPO with an amino donor to produce L-glufosinate, and / or an improvement in kinetic properties including, but not limited to, an increase in the Vmax value, a decrease in the Km value, or an increase in Vmax / Km.

[0023] As used herein, the term "peptide" means a chain containing at least two amino acids linked by peptide bonds. The term "polypeptide" can be interchanged with "protein" and means a chain containing 10 or more amino acid residues. In this specification, the chemical formulas or sequences of all peptides and polypeptides are described in the left-to-right order indicating the direction from the amino terminus to the carboxyl terminus.

[0024] The term "amino acid" includes amino acids that are naturally present in proteins and non-natural amino acids. The conventional nomenclature (one-letter and three-letter) of amino acids that are naturally present in proteins is used, and this nomenclature can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989). Amino Acid One-Letter Three-Letter Alanine A Ala Arginine R Arg Asparagine N Asn Aspartic Acid D Asp Cysteine C Cys Glutamine Q Gln Glutamic Acid E Glu Glycine G Gly Histidine H His Isoleucine I Ile Leucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe Proline P Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y Tyr Valine V Val

[0025] As used herein, the term "modification" refers to any modification to a polypeptide, such as substitution, deletion, insertion, and / or addition of amino acid(s).

[0026] In some embodiments, the modified GluDH of the present invention contains amino acid substitutions at two or more positions compared to its native GluDH, and shows an increase in activity for catalyzing the reaction of PPO with an amino donor to produce L-glutamate, and / or an improvement in kinetic properties including, but not limited to, an increase in Vmax value, a decrease in Km value, or an increase in Vmax / Km.

[0027] In some embodiments, the modified GluDH has the following combinations of amino acid substitutions compared to its native GluDH: The amino acid at position 104 is substituted with C and the amino acid at position 175 is substituted with G; The amino acid at position 132 is substituted with L and the amino acid at position 175 is substituted with G; The amino acid at position 133 is substituted with V and the amino acid at position 175 is substituted with G; 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 175 is substituted with G and the amino acid at position 181 is substituted with K or R; 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 175 is substituted with G, and the amino acid at position 203 is substituted with I; 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; The amino acid at position 132 is substituted with L, 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 175 is substituted with G, and the amino acid at position 182 is substituted 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 is substituted with G, and the amino acid at position 182 is substituted with R; 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; 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 is substituted with S, 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 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 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 has The positions are numbered with reference to SEQ ID NO: 1

[0028] In some embodiments, the modified GluDH of the present invention includes substitutions at positions 132 and 175 compared to its initial GluDH, and the positions are 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 includes substitutions at one or more positions selected from positions 104, 133, 173, 181, and 182 compared to its 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

[0029] In some embodiments, the modified GluDH of the present invention includes substitutions at positions 133 and 175 compared to its initial GluDH, and the positions are 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 includes substitutions at one or more positions selected from positions 104, 132, 173, 181, and 182 compared to its 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

[0030] In some embodiments, the modified GluDH of the present invention contains substitutions at positions 173 and 175, with the positions numbered with reference to SEQ ID NO: 1, as compared to its initial GluDH. 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 contains substitutions at one or more positions selected from positions 104, 132, 133, 181, and 182, as compared to its 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.

[0031] In some embodiments, the modified GluDH of the present invention contains substitutions at positions 175 and 182, with the positions numbered with reference to SEQ ID NO: 1, as compared to its initial GluDH. 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 contains substitutions at one or more positions selected from positions 104, 132, 133, 173, and 181, as compared to its 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.

[0032] In some embodiments, the modified GluDH of the present invention includes substitutions at positions 132, 133, and 175, as numbered with reference to SEQ ID NO: 1, compared to its initial GluDH. 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 includes substitutions at one or more positions selected from positions 104, 173, 181, and 182 compared to its 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.

[0033] In some embodiments, the modified GluDH of the present invention includes substitutions at positions 132, 173, and 175, as numbered with reference to SEQ ID NO: 1, compared to its initial GluDH. 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 includes substitutions at one or more positions selected from positions 104, 133, 181, and 182 compared to its 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.

[0034] In some embodiments, the modified GluDH of the present invention contains substitutions at positions 133, 173, and 175 compared to its initial GluDH, and the 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 contains substitutions at one or more positions selected from positions 104, 132, 181, and 182 compared to its 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.

[0035] In some embodiments, the modified GluDH of the present invention contains substitutions at positions 132, 175, and 182 compared to its initial GluDH, and the 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 contains substitutions at one or more positions selected from positions 104, 133, 173, and 181 compared to its 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.

[0036] In some embodiments, the modified GluDH of the present invention includes substitutions at positions 133, 175, and 182 compared to its initial GluDH, and the 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 includes substitutions at one or more positions selected from positions 104, 132, 173, and 181 compared to its 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.

[0037] In some embodiments, the modified GluDH of the present invention includes substitutions at positions 173, 175, and 182 compared to its initial GluDH, and the 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 includes substitutions at one or more positions selected from positions 104, 132, 133, and 181 compared to its 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.

[0038] The GluDH polypeptide on which the amino acid modification is carried out is referred to herein as the starting GluDH. The starting GluDH may be a wild-type GluDH or a variant of the wild-type GluDH. For example, when the modification is initiated 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 when the modification is initiated based on a variant polypeptide of SEQ ID NO: 1 (e.g., SEQ ID NOs: 3 to 30), the variant polypeptide is the "starting GluDH" for the modified GluDH.

[0039] As used herein, the term "wild-type GluDH" refers to a naturally occurring GluDH. In some embodiments, the initial GluDH is a GluDH derived from a microorganism of the family Bacillaceae. In some embodiments, the wild-type GluDH is a GluDH derived from a microorganism of the genus Lysinibacillus or Bacillus. Preferably, the wild-type GluDH is a GluDH derived from Lysinibacillus sphaericus (SEQ ID NO: 2) or a GluDH derived from Bacillus velezensis (SEQ ID NO: 1).

[0040] For the purposes of the present invention, to determine the percentage identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced into the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid sequence or nucleic acid sequence). Subsequently, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. If the position of the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position of the second sequence, such molecules are identical at this position. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage identity = number of identical positions / total number of positions (i.e., overlapping positions) × 100). Preferably, the two sequences are of the same length.

[0041] One skilled in the art is aware that various computer programs can be used to determine the identity between two arrays.

[0042] "Percentage of amino acid identity" or "percentage of amino acid sequence identity" refers to the comparison between the amino acids of two polypeptides. When optimally aligned, the two polypeptides have approximately the specified percentage of identical amino acids. For example, "95% amino acid identity" refers to the comparison between the amino acids of two polypeptides, and when optimally aligned, 95% of the amino acids of the two polypeptides are identical.

[0043] 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%, particularly preferably at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1 or 2. In some embodiments, the modified GluDH has the following combinations of amino acid substitutions compared to its initial GluDH: The amino acid at position 104 is substituted with C and the amino acid at position 175 is substituted with G; The amino acid at position 132 is substituted with L and the amino acid at position 175 is substituted with G; The amino acid at position 133 is substituted with V and the amino acid at position 175 is substituted with G; 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 175 is substituted with G and the amino acid at position 181 is substituted with K or R; 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 175 is substituted with G and the amino acid at position 203 is substituted with I; 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; The amino acid at position 132 is substituted with L, 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 175 is substituted with G, and the amino acid at position 182 is substituted 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 is substituted with G, and the amino acid at position 182 is substituted with R; 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; 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 is substituted with S, 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 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 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 have, The position is 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.

[0044] In some embodiments, the modified GluDH of the present invention comprises substitutions at positions 132 and 175 as compared to its initial GluDH, and the positions are 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 as compared to its 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.

[0045] In some embodiments, the modified GluDH of the present invention includes substitutions at positions 133 and 175, numbered with reference to SEQ ID NO: 1, compared to its initial GluDH. 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 includes substitutions at one or more positions selected from positions 104, 132, 173, 181, and 182, compared to its 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.

[0046] In some embodiments, the modified GluDH of the present invention contains substitutions at positions 173 and 175, numbered with reference to SEQ ID NO: 1, compared to its initial GluDH. 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 contains substitutions at one or more positions selected from positions 104, 132, 133, 181, and 182 compared to its 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.

[0047] In some embodiments, the modified GluDH of the present invention contains substitutions at positions 175 and 182, numbered with reference to SEQ ID NO: 1, compared to its initial GluDH. 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 contains substitutions at one or more positions selected from positions 104, 132, 133, 173, and 181 compared to its 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.

[0048] In some embodiments, the modified GluDH of the present invention contains substitutions at positions 132, 133, and 175, numbered with reference to SEQ ID NO: 1, compared to its initial GluDH. 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 contains substitutions at one or more positions selected from positions 104, 173, 181, and 182 compared to its 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.

[0049] In some embodiments, the modified GluDH of the present invention includes substitutions at positions 132, 173, and 175, numbered with reference to SEQ ID NO: 1, compared to its initial GluDH. 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 includes substitutions at one or more positions selected from positions 104, 133, 181, and 182, compared to its 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.

[0050] In some embodiments, the modified GluDH of the present invention contains substitutions at positions 133, 173, and 175, with the positions numbered with reference to SEQ ID NO: 1, as compared to its initial GluDH. 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 contains substitutions at one or more positions selected from positions 104, 132, 181, and 182, as compared to its 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.

[0051] In some embodiments, the modified GluDH of the present invention includes substitutions at positions 132, 175, and 182, numbered with reference to SEQ ID NO: 1, compared to its initial GluDH. 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 includes substitutions at one or more positions selected from positions 104, 133, 173, and 181 compared to its 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.

[0052] In some embodiments, the modified GluDH of the present invention contains substitutions at positions 133, 175, and 182, numbered with reference to SEQ ID NO: 1, compared to its initial GluDH. 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 contains substitutions at one or more positions selected from positions 104, 132, 173, and 181 compared to its 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.

[0053] In some embodiments, the modified GluDH of the present invention contains substitutions at positions 173, 175, and 182, numbered with reference to SEQ ID NO: 1, compared to its initial GluDH. 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 contains substitutions at one or more positions selected from positions 104, 132, 133, and 181, compared to its 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.

[0054] In some embodiments, the modified GluDH contains substitutions at positions 173, 175, and 182, numbered with reference to SEQ ID NO: 1, compared to its native GluDH. 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 contains amino acid substitutions 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 native 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 native GluDH comprises or consists of SEQ ID NO: 1.

[0055] In some embodiments, the modified GluDH contains 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 contains 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. For example, the initial GluDH comprises or consists of SEQ ID NO: 1.

[0056] 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%, 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 initial GluDH differs from SEQ ID NO: 1 or 2 in that it contains one or more amino acid substitutions, deletions, insertions, and / or additions. In some embodiments, the initial GluDH contains one or more conservative amino acid substitutions compared to SEQ ID NO: 1 or 2. In some embodiments, the initial GluDH contains one or more amino acid insertions or deletions compared to SEQ ID NO: 1 or 2.

[0058] The term "conservative substitution" is also referred to as substitution by "homologous" amino acid residues, where an amino acid is replaced by an amino acid residue having a similar side chain, such as an amino acid having a basic side chain (e.g., lysine, arginine, and histidine), an amino acid having an acidic side chain (e.g., aspartic acid, glutamic acid), an amino acid having an uncharged polar side chain (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), an amino acid having a nonpolar side chain (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), an amino acid having a beta-branched side chain (e.g., threonine, valine, isoleucine), and an amino acid having an aromatic side chain (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0059] Generally, conservative substitution of an amino acid has a minimal impact on the activity of the resulting protein. Such substitutions are described below. A conservative substitution is replacing an amino acid with an amino acid that has similar 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.

[0060] As used herein, "homologous" amino acid residues refer to amino acid residues having 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 positively charged lysine, arginine, and histidine; negatively charged glutamic acid and aspartic acid; hydrophobic glycine, alanine, valine, leucine, isoleucine, proline, and phenylalanine; polar serine, threonine, cysteine, methionine, tryptophan, tyrosine, asparagine, glutamine; aromatic phenylalanine, tyrosine, and tryptophan; serine and threonine or glutamine and asparagine or leucine and isoleucine having chemically similar side chain groups.

[0061] Examples of conservative amino acid substitutions in proteins include substitution of Ala with Ser, Arg with Lys, Asn with Gln or His, Asp with Glu, Cys with Ser, Gln with Asn, Glu with Asp, Gly with Pro, His with Asn or Gln, Ile with Leu or Val, Leu with Ile or Val, Lys with Arg or Gln, Met with Leu or Ile, Phe with Met, Leu, or Tyr, Ser with Thr, Thr with Ser, Trp with Tyr, Tyr with Trp or Phe, and Val with Ile or Leu.

[0062] 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 other than positions 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, and the modified GluDH exhibits an increase in activity for catalyzing the reaction of PPO with an amino donor to produce L-glufosinate and / or an improvement in kinetic properties, such as an increase in Vmax, a decrease in Km, or an increase in Vmax / Km, compared to its native 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, the substitutions being at positions other than positions 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%, particularly preferably at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1 or 2.

[0063] As used herein, the activity of an enzyme refers to the decrease in substrate or increase in product per unit time in a chemical reaction catalyzed by a unit mass of the enzyme under certain specific conditions. For example, the activity of the modified GluDH of the present invention can be represented by the amount of decrease in PPO or increase in L-glufosinate per unit time under the catalysis of a unit mass of the modified GluDH under certain specific conditions.

[0064] Also, the activity of an enzyme in this specification may refer to the relative activity of the enzyme, which is expressed as a ratio of the activity of the enzyme of interest to the activity of a given enzyme catalyzing the same reaction, such as a relative activity percentage.

[0065] In some embodiments, the activity of the modified GluDH of the present invention is expressed as a relative activity percentage compared to SEQ ID NO: 3. In some embodiments, the activity of the modified GluDH that catalyzes 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 higher than the activity of SEQ ID NO: 3 that catalyzes this reaction.

[0066] In some embodiments, the activity of the modified GluDH of the present invention is expressed as a relative activity percentage compared to SEQ ID NO: 31. In some embodiments, the activity of the modified GluDH that catalyzes 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 higher than the activity of SEQ ID NO: 31 that catalyzes this reaction.

[0067] As used herein, the term "amino donor" refers to a compound that provides an amino group, including inorganic and organic compounds. Examples of "amino donors" include, but are not limited to, ammonium salts (e.g., NH 4 Cl, NH 4 NO 3 , (NH 4 )2 SO 4 、 ammonium acetate, etc.), amino acids, or organic amines. In some embodiments, the amino donor is an ammonium salt, such as NH 4 Cl.

[0068] To produce, in addition to the enzyme activity, it is necessary to consider the kinetic properties. The kinetic properties of the enzyme in this specification include, but are not limited to, the Vmax, Km, and Vmax / Km of the enzyme. The improvement of the kinetic properties in this specification includes, but is not limited to, an increase in Vmax, a decrease in Km, and an increase in Vmax / Km.

[0069] As used herein, the term "Vmax" refers to the maximum rate of the catalytic reaction that can be achieved with a specific concentration of the enzyme. In particular, under the condition that the enzyme concentration is constant, when the substrate concentration is within a specific range, the reaction rate generally increases with the increase of the substrate concentration. When the substrate concentration reaches a specific value, the reaction rate reaches the maximum value, and the reaction rate will not increase even if the substrate concentration increases.

[0070] As used herein, the term "Km" refers to the substrate concentration at which the reaction rate reaches half of the maximum rate of catalysis, i.e., Vmax, at a specific enzyme concentration.

[0071] II. Polynucleotides Encoding Modified GluDH As used herein, the terms "polynucleotide" or "nucleic acid molecule" include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), and analogs of DNA or RNA produced using nucleotide analogs. The nucleic acid molecule may be single-stranded or double-stranded, and preferably may be double-stranded DNA. Nucleotide analogs or derivatives (e.g., inosine nucleotides or phosphorothioate nucleotides) can be used in the synthesis of nucleic acids. Such nucleotides can be used to prepare, for example, nucleic acids with altered base pairing ability or increased nuclease resistance.

[0072] In addition, the present invention provides a polynucleotide encoding the modified GluDH of the present invention. Thus, in the present invention, the term "modification" also includes genetic manipulation of the polynucleotide encoding the GluDH polypeptide of the present invention. The modification may be a substitution, deletion, insertion, and / or addition of one or more nucleotides.

[0073] 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 that begins with an ATG start codon or other start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA. The coding sequence may be DNA, cDNA, or a recombinant nucleotide sequence.

[0074] In addition, nucleic acid molecules encompassing all or part of the 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.

[0075] The polynucleotides of the present invention can be amplified according to standard PCR amplification techniques using cDNA, mRNA, or genomic DNA as a template, and suitable oligonucleotide primers. The nucleic acids amplified as described above can be cloned into a suitable vector and characterized by DNA sequence analysis.

[0076] The polynucleotides of the present invention can be prepared by standard synthetic techniques, for example, by using an automated DNA synthesizer.

[0077] The present invention also relates to the complementary strands of the nucleic acid molecules described herein. Nucleic acid molecules complementary to other nucleotide sequences are molecules that are sufficiently complementary to the nucleotide sequence so as to be able to hybridize with the other nucleotide sequence to form a stable double-strand.

[0078] As used herein, generally, the term "hybridization" means that 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 maintain hybridization to each other under a given stringent hybridization condition and washing condition.

[0079] Those skilled in the art are aware of various conditions for hybridization, such as stringent hybridization conditions and highly stringent hybridization conditions. See, for example, 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.

[0080] Of course, the polynucleotides of the present invention do not include polynucleotides that merely hybridize to a polyA sequence (such as the 3'-terminal poly(A) of mRNA) or a complementary stretch of polyT (or U) residues. III. Expression and production of modified GluDH To express the modified GluDH of the present invention, nucleic acid constructs and vectors containing the polynucleotides of the present invention, such as expression vectors, are also provided.

[0081] 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.

[0082] The term "nucleic acid construct" refers to a single-stranded or double-stranded nucleic acid molecule that has been isolated from a naturally occurring gene or modified to include a nucleic acid segment that does not occur naturally. When a nucleic acid construct contains the control sequences necessary for the expression of the coding sequence of the present invention, the term nucleic acid construct is synonymous with the term "expression cassette".

[0083] The term "expression vector" refers herein to a linear or circular DNA molecule that contains additional nucleotides provided for the expression of a polynucleotide, such as a polynucleotide encoding a polypeptide of the present invention operably linked to a control sequence. Expression vectors include viral vectors or plasmid vectors.

[0084] As used herein, the term "control sequences" includes all elements necessary or beneficial for the expression of a polynucleotide encoding a polypeptide of the 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, the control sequences include a promoter and signals to terminate transcription and translation.

[0085] For example, the control sequence may be a suitable promoter sequence that is recognized by a host cell and causes the nucleotide sequence encoding the polypeptide of the invention to be expressed. The promoter sequence contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, such as the lac operon of E. coli. Also included are mutant, truncated, and hybrid promoters, which can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0086] As used herein, the term "operably linked" refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide sequence such that the control sequence directs the expression of the polypeptide coding sequence.

[0087] The polynucleotide encoding a polypeptide of the invention can be subjected to various manipulations to enable the expression of the polypeptide. It may be desirable or necessary to manipulate the polynucleotide according to the expression vector before inserting it into the vector. Techniques for modifying polynucleotide sequences by recombinant DNA methods are well known in the art.

[0088] To identify and select host cells containing the expression vector of the present invention, the vector of the present invention preferably contains one or more selection markers that enable easy selection such as transformed cells, transfected cells, transduced cells, etc. The selection marker is a gene whose product provides a biocide or confers virus resistance, heavy metal resistance, auxotrophy, etc. For example, bacterial selection markers are the dal gene derived from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin resistance, kanamycin resistance, chloramphenicol resistance, or tetracycline resistance.

[0089] The vector of the present invention may be integrated into the genome of the host cell or may replicate autonomously in the cell independently of the genome. Elements necessary for integration into the host cell genome or autonomous replication are known in the art (see, for example, Sambrook et al., 1989, supra).

[0090] Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are well known in the art and refer to various techniques for introducing exogenous nucleic acids (such as DNA) into host cells, which can be found, for example, in Sambrook et al., 1989, supra; Davis et al., Basic Methods in Molecular Biology (1986); and other laboratory manuals.

[0091] The present invention also relates to recombinant host cells containing the polynucleotide of the present invention, which are advantageously used in the recombinant production of GluDH polypeptide. A vector containing the polynucleotide of the present invention is introduced into the host cell, whereby the vector is retained as an integrated chromosomal entity or as an autonomously replicating extrachromosomal vector. Those skilled in the art are familiar with conventional vectors and host cells for expressing proteins.

[0092] In some embodiments, the host cell of the present invention is an E. coli cell such as E. coli BL21(DE3). In some embodiments, the expression vector is pET-30a(+).

[0093] 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 containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian cells and yeast host cells), the use of a heterologous signal sequence can increase the expression and / or secretion of GluDH.

[0094] IV. Production of L-glufosinate Furthermore, the present invention provides a method for preparing L-glufosinate, the method comprising contacting the modified GluDH or host cell of the present invention with PPO. In some embodiments, the method for preparing L-glufosinate-ammonium of the present invention is (a) supplying 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 contains an NADPH / NADP cycling system, (b) incubating the reaction medium to produce L-glufosinate and includes.

[0095] In some embodiments, a cell-free catalysis method is used to produce L-glufosinate, and the modified GluDH of the present invention is supplied in step (a). In some embodiments, the free or immobilized modified GluDH of the present invention can be used.

[0096] In some embodiments, the amino donor is an ammonium salt, such as NH 4 Cl.

[0097] In some embodiments, the reaction medium contains 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 contains alcohol dehydrogenase. Also, in some embodiments, the alcohol dehydrogenase may be immobilized.

[0098] 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.

[0099] In some embodiments, the medium is a buffer such as PBS buffer and Tris-HCl buffer. In one embodiment, the medium is a PBS buffer such as 100 mM PBS buffer. In some embodiments, the pH of the reaction medium is 7.5 - 8.

[0100] In some embodiments, the reaction medium is a medium that is partially or entirely composed of a cell culture medium, and the activity of the modified GluDH of the present invention is provided by the host cells of the present invention cultured in the reaction medium.

[0101] In some embodiments, the reaction medium is a medium that is partially or entirely composed of a cell culture medium, and the NADPH / NADP cycle system such as alcohol dehydrogenase activity is provided by the host cells of the present invention or the second host cells cultured in the reaction medium.

[0102] In some embodiments, the host cell and / or the second host cell of the present invention 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. Before, during, or after the addition of the grown host cells, PPO is provided to the buffer or water.

[0103] In some embodiments, bacterial cells such as E. coli cells can be used.

Example

[0104] Those skilled in the art will understand the present invention more clearly through the following examples. It should be understood that the examples are only for illustration and do not limit the scope of the present invention.

[0105] Example 1. Substances and Methods Unless otherwise specified, all experimental methods used in the present invention are conventional methods. Specific gene cloning operations can be found in Sambrook et al., 1989, mentioned above.

[0106] i) Reagents: DNA polymerase (PrimeSTAR Max DNA polymerase) and DpnI endonuclease were purchased from TaKaRa. The 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). NH 4 Cl 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), and this dehydrogenase is recombinantly expressed in E. coli.

[0107] ii) Vectors and Strains: The expression vector used was pET-30a(+). The plasmid was purchased from Novagen. The host cell used was Escherichia coli BL21(DE3) purchased from Tiangen BioTech(Beijing) Co., Ltd.

[0108] iii) Sequencing and primer synthesis were performed by Synbio Technologies Co., Ltd.

[0109] iv) Site-directed mutagenesis: A specific primer pair was designed to introduce the desired substitution into the base corresponding to the amino acid position where mutation was required. The isolated pre-mutated plasmid (containing the coding sequence of wild-type GluDH and the pET-30a(+) backbone) was used as a template, and mutation was introduced by PCR using the Quickchange technique (Nucleic Acids Research, 2004, Vol. 32(14): e115). After PCR amplification, the amplification product was digested with DpnI for 4 hours to remove the template plasmid. The digested product was transformed into Escherichia coli BL21(DE3) competent cells, followed by plating on LB agar (containing 50 mg / L kanamycin), inoculating a single colony into LB broth (containing 50 mg / L kanamycin) for culture, and sequencing to confirm that it was the correct mutant. The confirmed clones were stored at -80 °C for future use.

[0110] v) Protein expression and preparation of crude enzyme solution: The stored clones were activated on LB agar. Then, a single colony was inoculated into LB broth (containing 50 mg / L kanamycin) and incubated at 37 °C with shaking for 12 hours. 1 mL of the culture solution was transferred to 50 mL of fresh LB broth (containing 50 mg / L kanamycin), incubated at 37 °C with shaking until the OD600 reached approximately 0.6, and after adding IPTG (final concentration 0.4 mM), incubated at 25 °C for 16 hours to induce protein expression.

[0111] After incubation, the culture was centrifuged at 4,000 g for 10 minutes at 4°C, the supernatant was discarded, and Escherichia coli cells were collected. The collected Escherichia coli cells were resuspended in 15 mL of pre-cooled 50 mM PBS, pH 7.0, and disrupted by sonication at 4°C. The cell disruption solution was centrifuged at 6,000 g for 15 minutes at 4°C to remove the precipitate. The resulting supernatant was a crude enzyme solution containing the recombinant enzyme.

[0112] vi) Determination of enzyme activity NH 4 Cl and NADP+ were added to the PPO solution (100 mM) in PBS, and the pH of the solution was adjusted to 8 with aqueous ammonia solution. This solution contained PPO at a final concentration of 100 mM, NH 4 Cl at a final concentration of 50 mM, and NADP+ at a final concentration of 0.2 g / L. The crude enzyme solution obtained as described in v) and alcohol dehydrogenase were added to the above solution. The final concentration of GluDH was 0.02 g / L, and the final concentration of alcohol dehydrogenase was 0.2 g / L. This solution was continuously shaken at 30°C for 2 hours (400 rpm) on a shaker, then samples were taken, and the amount of L-glutamine produced was detected by OPA pre-column derivatization HPLC, thereby determining the initial rate of the catalytic reaction.

[0113] vii) Determination of the kinetic properties of the enzyme A plurality of reaction systems (200 μL) containing 100 mM PBS (pH adjusted with aqueous ammonia solution to pH 7.5), 0.15 mM NADPH (reduced), 50 mM NH 4 Cl, 10% by volume diluted (500-fold) crude enzyme solution, and various concentrations (5 - 100 mM) of the substrate PPO were prepared in a 96-well plate. The intensity of UV absorption at 340 nm was detected at 30°C, and the rate of change in absorption 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 absorption.

[0114] Example 2. Preparation and detection of mutants of GluDH (BvGluDH) derived from Bacillus belezensis Mutants were prepared using the coding nucleic acid of BvGluDH (SEQ ID NO: 1) as a template, and the enzyme activity and the 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 with respect to the enzyme activity of the mutant of SEQ ID NO: 3.

[0115]

Table 1

[0116] 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 the 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 with respect to the enzyme activity of the mutant of SEQ ID NO: 31.

[0117]

Table 2

[0118] 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 with respect to the enzyme activity of the mutant of SEQ ID NO: 21 when no heat treatment is performed. The relative enzyme activity after heat treatment means the percentage of the enzyme activity of the mutant with respect to the enzyme activity of the mutant of SEQ ID NO: 21 after incubation at 45 °C for 30 minutes.

[0119]

Table 3

[0120] Example 5. Preparation and detection of BvGluDH variants Variants were prepared by introducing additional mutations based on the variant of SEQ ID NO: 65, and the enzyme activity was measured according to the method of Example 1. The obtained variants and their enzyme activities are shown in Table 4. The relative enzyme activity means the percentage of the enzyme activity of the variant with respect to the enzyme activity of the variant of SEQ ID NO: 65.

[0121] [Table 4]

Claims

1. A modified glutamate dehydrogenase (GluDH), which, compared to the original GluDH, has a substitution of the amino acid at position 104 with C and a substitution of the amino acid at position 175 with G; an amino acid substitution at position 132 to L and an amino acid substitution at position 175 to 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 to G or S and an amino acid substitution at position 175 to 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; an amino acid substitution at position 175 with G and an amino acid substitution at position 203 with I; 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; the amino acid at position 132 is substituted with L, 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 175 is substituted with G, and the amino acid at position 182 is substituted 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 is substituted with G, and the amino acid at position 182 is substituted 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 is substituted with S, 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 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 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 a modified GluDH, wherein the positions are numbered with reference to SEQ ID NO:1, and wherein the modified GluDH exhibits increased activity for catalyzing the reaction of 4-(hydroxymethylphosphinyl)-2-oxobutanoic acid (PPO) with an amino donor to produce L-glufosinate, and / or 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. A modified GluDH comprising 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.

6. 1. A modified GluDH comprising substitutions at positions 173, 175, and 182, relative to the initial GluDH, wherein 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, said positions being numbered with reference to SEQ ID NO:1, wherein the modified GluDH exhibits increased activity for catalyzing the reaction of PPO with an amino donor to produce L-glufosinate, relative to the initial GluDH.

7. The modified GluDH according to claim 6, wherein the initial GluDH is a wild-type GluDH.

8. 8. The modified GluDH according to claim 6 or 7, 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.

9. and further comprising 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, wherein the amino acid at position 9 is substituted with S, L, or Y, the amino acid at position 22 is substituted with W or E, 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 31 is substituted with H, and the amino acid at position 56 is substituted with Q.

9. The modified GluDH according to claim 6, wherein the amino acid at position 124 is substituted with L, the amino acid at position 143 is substituted with E, the amino acid at position 199 is substituted with W or Y, the amino acid at position 216 is substituted with G, 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 420 is substituted with R, the amino acid at position 431 is substituted with S, and the amino acid at position 437 is substituted with K.

10. The modified GluDH according to any one of claims 6 to 8, further comprising substitutions of amino acids at positions 22, 56, 199, and 420, wherein 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 199 is substituted with Y, and the amino acid at position 420 is substituted with R.

11. 11. The modified GluDH of claim 10, 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.

12. The modified GluDH according to any one of claims 6 to 11, wherein the initial GluDH has the amino acid sequence of SEQ ID NO:

1.

13. 13. The modified GluDH of claim 12, wherein the activity of the modified GluDH to catalyze the reaction of PPO with an amino donor to produce L-glufosinate is at least 130% of the activity of SEQ ID NO:3 to catalyze the reaction.

14. A polynucleotide encoding the modified GluDH according to any one of claims 1 to 13.

15. An expression vector comprising the polynucleotide of claim 14.

16. A host cell comprising the modified GluDH according to any one of claims 1 to 13, the polynucleotide according to claim 14, or the vector according to claim 15.

17. A method for producing L-glufosinate, comprising contacting a modified GluDH according to any one of claims 1 to 13 or a host cell according to claim 16 with PPO.

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