Modified DAAO Enzyme and Its Applications

Abstract

The present invention relates to a modified D-amino acid oxidase (DAAO). In particular, the modified DAAO of the present invention has activity for catalyzing the oxidation of D-glufosinate to PPO. Additionally, the modified DAAO of the present invention has increased activity and / or increased stability for catalyzing the oxidation of D-glufosinate to PPO compared to SEQ ID NO. 4. The present invention also relates to a polynucleotide encoding the modified DAAO of the present invention, a vector and a host cell expressing the modified DAAO of the present invention, and a method for producing L-glufosinate using the modified DAAO and the host cell of the present invention.

Description

Technology Field

[0001] The present invention relates to the field of enzyme engineering. In particular, the present invention relates to a modified D-amino acid oxidase (DAAO) 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 transgenic crops have developed resistance. Glufosinate is a broad-spectrum contact-killing herbicide that disrupts plant nitrogen metabolism by inhibiting the activity of L-glutamine synthase, ultimately killing the plants. Glufosinate offers significant advantages over glyphosate, such as broad coverage, rapid action, long duration, low toxicity, and safety. Consequently, sales of glufosinate are increasing rapidly, and demand for it is very high and the outlook is good for the following period.

[0003] However, the manufacturing process of glufosinates is complex, making production difficult. The high price prevents rapid replacement of glyphosate. Currently available glufosinates are racemic mixtures containing equal amounts of two optical isomers (D,L-glufosinates), of which only L-glufosinates are biologically active. Therefore, the production of chiral pure L-glufosinates by deracemization of D,L-glufosinates is of substantial importance and has become popular in the synthesis of L-glufosinates in recent years.

[0004] Recently, many methods for producing L-glufosinate from D,L-glufosinate have been reported. Conventional separation methods based on chemical modification are not competitive due to high costs and the inability to utilize D-glufosinate. Currently, the major technical pathways reported for converting D-glufosinate-ammonium into L-glufosinate are as follows.

[0005] 1. After D,L-glufosinate is converted to N-acetylglufosinate, L-glufosinate is obtained by selective hydrolysis of LN-acetylglufosinate, which is catalyzed by carboxypeptidase, whereas DN-acetylglufosinate cannot be hydrolyzed and can be recycled to the hydrolysis step after chemical or enzymatic racemization (see, for example, CN108690854A). The disadvantage of this method is that it involves multiple reaction steps and requires separating the L-glufosinate obtained by hydrolysis from the N-acetylated substrate.

[0006] 2. D-glufosinate is oxidized to 2-carbonyl-4-(hydroxymethylphosphono)butyric acid (PPO), which is then reduced or transamined to produce L-glufosinate-ammonium. In most references, D-amino acid oxidase (DAAO) is used to catalyze the oxidation of D-glufosinate to PPO, and catalase (CAT) is typically added to remove the hydrogen peroxide produced. Since PPO can be reduced by formic acid under the catalytic action of palladium on carbon to produce D,L-glufosinate, D,L-glufosinate can be gradually converted to L-glufosinate due to the stereoselectivity of DAAO (see, e.g., CN105567780A). The disadvantage of this solution is that it requires a large amount of palladium-carbon catalyst, and raw materials for the reaction (e.g., oxygen and ammonium formate) are wasted.

[0007] PPO can also be converted to L-glufosinate by a stereoselective transamination reaction catalyzed by L-amino acid transaminase (L-TA) (see, e.g., US20180030487A1).

[0008] This solution has the disadvantage that the amino transfer step is a balanced reaction, so in order to achieve a high conversion rate (e.g., 90% conversion rate when 3 equivalents of amino donor are provided), an excess amount of amino donor (amino acid or organic amine) must be provided, and the excess amount of amino donor and the corresponding byproducts will have a serious impact on subsequent separation and purification steps.

[0009] Additionally, PPO can be converted to L-glufosinate through a stereoselective reduction reaction catalyzed by L-amino acid dehydrogenase (L-AADH) (see, e.g., CN107502647A, CN109576236A, and CN109609582A). In this solution, the concentration of the converted substrate is low or the loss is large.

[0010] Solutions using D-amino acid oxidase and L-amino acid dehydrogenase have a potential cost advantage over the aforementioned solutions. However, currently reported methods generally result in high production costs due to low concentrations of convertible substrates or excessive losses. Achieving high concentrations of D,L-glufosinate thalassamining is the choke point of the current process.

[0011] Major factors that hinder the conversion of high-concentration substrates may include the following: the recombinant DAAO enzyme has poor stability, is unstable under reactor conditions, and becomes inactivated during the reaction.

[0012] Another limiting factor is the selective catalytic activity of the enzyme for D-glufosinate. Some modifications of DAAO were performed in the prior art to confer activity for D-glufosinate.

[0013] ㆍFrom US 7,939,709 Rhodotorula toruloides ( Rhodotorula gracilis A DAAO mutation of (also known as, see https: / / www.atcc.org / products / all / 10788.aspx) was used to synthesize PPO from D-glufosinate, as mentioned in the patent. Rhodotorula toruloidesThe DAAO mutations include the F58K mutation, a mutation with substitutions of H, S, T, C, Q, G, N, and A at position M213, and mutations at positions 223 and 238. Tim Hawks et al. 2011 (D-glufosinate as a male sterility agent for hybrid seed production, Plant Biotechnology Journal, (2011) 9, pp. 301-314) reported content similar to the aforementioned patent, where DAAO is Rhodotorula toruloides It contains mutations at positions 58 and 213.

[0014] In US 9,834,802 Rhodotorula toruloides L-glufosinate was synthesized from D,L-glufosinate using DAAO mutations and TA (transaminase). Additionally, DAAO is described as containing one or more mutations at positions 54, 56, 58, 213, and 238, and several specific combinations are exemplified.

[0015] In CN109576236A Rhodotorula toruloides A mutant capable of oxidizing D-glufosinate based on DAAO was also constructed. The mutant has one or more mutations at amino acid positions 52, 54, 58, 213, and 335.

[0016] In other patent applications such as CN105567780A and CN109609582A, the oxidation of D-glufosinate and DAAO was also involved, but the order of enzymes used was not indicated.

[0017] However, DAAO with higher stability and / or higher activity for D-glufosinate is still needed. The problem to be solved

[0018] In the first embodiment, the present invention comprises amino acid substitutions at 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more positions compared to wild-type DAAO, wherein the modified DAAO has activity for catalyzing the oxidation of D-glufosinate to PPO.

[0019] In some embodiments, the modified DAAO comprises amino acid substitutions at positions 54, 56, 58, and 213, said positions numbered with reference to SEQ ID NO. 2. Preferably, at position 54, the amino acid is substituted with I, V, T, or L, and more preferably with I or V. Preferably, at position 56, the amino acid is substituted with N. Preferably, at position 58, the amino acid is substituted with H or Q, and more preferably with H. Preferably, at position 213, the amino acid is substituted with S or T, and more preferably with S. In some embodiments, the modified DAAO further comprises amino acid substitutions at positions 210 and / or 221. Preferably, at position 210, the amino acid is substituted with A, G, or P, and more preferably with A. Preferably, at position 221, the amino acid is substituted with R.

[0020] In some embodiments, the modified DAAO comprises amino acid substitutions at positions 54, 58, 213, and 221, said positions numbered with reference to SEQ ID NO. 2. Preferably, the amino acid at position 54 is substituted with V, the amino acid at position 58 is substituted with Q, the amino acid at position 213 is substituted with S, and the amino acid at position 221 is substituted with R. In some embodiments, the modified DAAO further comprises amino acid substitutions at positions 210 and / or 56. Preferably, the amino acid at position 56 is substituted with N, and the amino acid at position 210 is substituted with A.

[0021] In some embodiments, the modified DAAO of the present invention further comprises amino acid substitutions at one or more positions selected from the group consisting of 2, 81, 97, 193, 194, 237, 265, 273, 274, 300, 317, 319, 337, and 342. Preferably, at position 2, the amino acid is substituted with C or S, at position 81, the amino acid is substituted with Y, at position 97, the amino acid is substituted with V, at position 193, the amino acid is substituted with T, at position 194, the amino acid is substituted with V or C, at position 237, the amino acid is substituted with V, at position 265, the amino acid is substituted with C, at position 273, the amino acid is substituted with D, at position 274, the amino acid is substituted with E, at position 300, the amino acid is substituted with S or T, at position 317, the amino acid is substituted with Y or W, at position 319, the amino acid is substituted with K, at position 337, the amino acid is substituted with S, and at position 342, the amino acid is substituted with S or H.

[0022] Alternatively, in some embodiments, a modified DAAO of the present invention further comprises amino acid substitutions at one or more positions selected from the group consisting of 194, 237, 265, 273, 274, 300, 317, and 319. Preferably, at position 194, the amino acid is substituted with V or C; at position 237, the amino acid is substituted with V; at position 265, the amino acid is substituted with C; at position 273, the amino acid is substituted with D; at position 274, the amino acid is substituted with E; at position 300, the amino acid is substituted with S; at position 317, the amino acid is substituted with Y or W; and at position 319, the amino acid is substituted with K.

[0023] Alternatively, in some embodiments, a modified DAAO of the present invention further comprises amino acid substitutions at one or more positions selected from the group consisting of 2, 81, 97, 193, 300, 337, and 342, wherein at position 2, the amino acid is substituted with C or S, at position 81, the amino acid is substituted with Y, at position 97, the amino acid is substituted with V, at position 193, the amino acid is substituted with T, at position 300, the amino acid is substituted with T, at position 337, the amino acid is substituted with S, and at position 342, the amino acid is substituted with S.

[0024] In some embodiments, the modified DAAO comprises amino acid substitutions at positions 54, 58, 194, and 213 compared with SEQ ID NO. 1, wherein the modified DAAO has activity for catalyzing the oxidation of D-glufosinate to PPO. Preferably, at position 54, the amino acid is substituted with I, V, T, or L; at position 58, the amino acid is substituted with H or Q; at position 194, the amino acid is substituted with V or C; and at position 213, the amino acid is substituted with S or T. In some embodiments, the modified DAAO further comprises amino acid substitutions at one or more positions selected from the group consisting of 56, 210, 221, 237, 265, 273, 274, 300, 317, and 319. Preferably, at position 56, the amino acid is substituted with N, at position 210, the amino acid is substituted with A, G, or P, at position 221, the amino acid is substituted with R, at position 237, the amino acid is substituted with V, at position 265, the amino acid is substituted with C, at position 273, the amino acid is substituted with D, at position 274, the amino acid is substituted with E, at position 300, the amino acid is substituted with S, at position 317, the amino acid is substituted with Y or W, and at position 319, the amino acid is substituted with K.

[0025] In some embodiments, the modified DAAO comprises or is composed of one amino acid sequence of SEQ ID NOs 5-86, or the modified DAAO comprises 1-10 amino acid substitutions at positions other than 54, 56, 58, 194, 210, 213, 221, 237, 265, 273, 274, 300, 317, and 319 compared to one of SEQ ID NOs 5-30 and 66-76, comprises 1-10 amino acid substitutions at positions other than 2, 54, 56, 58, 81, 97, 193, 210, 213, 221, 300, 337, and 342 compared to one of SEQ ID NOs 31-57 and 77-86, or 54, 56, 58, compared to one of SEQ ID NOs 58-65 It includes 1-10 amino acid substitutions at positions other than 210, 213 and 221, wherein the modified DAAO has activity that catalyzes the oxidation of D-glufosinate to PPO.

[0026] In a second aspect, the present invention provides a polynucleotide encoding a modified DAAO of the present invention, and a vector comprising the polynucleotide of the present invention.

[0027] In a third aspect, the present invention provides a host cell comprising a modified DAAO of the present invention, its coding polynucleotide, or a vector comprising a polynucleotide.

[0028] In a fourth aspect, the present invention provides a method for producing L-glufosinate comprising the step of contacting a modified DAAO of the present invention or a host cell of the present invention with D-glufosinate. means of solving the problem

[0029] The present invention relates primarily to a modified DAAO for catalyzing the oxidation of D-glufosinate to produce L-glufosinate. Unless otherwise specified, terms used herein have the meanings generally understood by those skilled in the art.

[0030] I. Modified D-Amino Acid Oxidase

[0031] As used herein, the terms "D-amino acid oxidase" and "DAAO" refer to enzymes that catalyze the oxidation of D-amino acids to produce keto acids (EC 1.4.3.3). In general, naturally occurring DAAO cannot catalyze the oxidation of D-glufosinate. Accordingly, the present invention provides a modified DAAO capable of catalyzing the oxidation of D-glufosinate to PPO. Preferably, the modified DAAO polypeptide has increased stability and / or increased activity for catalyzing the oxidation of D-glufosinate to PPO.

[0032] As used herein, the term "peptide" means a chain comprising at least two amino acids connected by peptide bonds. The term "polypeptide" may be replaced with "protein" and means a chain comprising at least 10 amino acid residues. The chemical formulas or sequences of all peptides and polypeptides in this invention are described from left to right, indicating the direction from the amino terminus to the carboxyl terminus.

[0033] The term "amino acid" above includes naturally occurring amino acids and non-natural amino acids in proteins. The conventional naming conventions (one-letter and three-letter) for naturally occurring amino acids in proteins are used, as seen 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).

[0034] Amino acid, one letter, three letters

[0035] Alanine A Ala

[0036] Arginine R Arg

[0037] Asparagine N Asn

[0038] Aspartic acid D Asp

[0039] Cysteine ​​C Cys

[0040] Glutamine Q Gln

[0041] Glutamic acid (Glutamic acid) E Glu

[0042] Glycine (G) Gly

[0043] Histidine H His

[0044] Isoleucine I Ile

[0045] Leucine (Leucine) L Leu

[0046] Lysine K Lys

[0047] Methionine M Met

[0048] Phenylalanine (Phenylalanine) F Phe

[0049] Proline P Pro

[0050] Serine S Ser

[0051] Threonine T Thr

[0052] Tryptophan W Trp

[0053] Tyrosine Y Tyr

[0054] Valine V Val

[0055] As used herein, the term “modification” means any chemical modification to a polypeptide, e.g., substitution, deletion, insertion, and / or addition of amino acid(s).

[0056] In some embodiments, the modified DAAO of the present invention comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions compared to the wild-type DAAO, and said modified DAAO has activity for catalyzing the oxidation of D-glufosinate to PPO. In some embodiments, said modified DAAO comprises amino acid substitutions at positions 54, 56, 58, and 213 compared to its wild-type DAAO, said positions numbered with reference to SEQ ID NO. 2. Preferably, at position 54, the amino acid is substituted with I, V, T, or L, and more preferably with I or V. Preferably, at position 56, the amino acid is substituted with N. Preferably, at position 58, the amino acid is substituted with H or Q, and more preferably with H. Preferably, at the 213 position, the amino acid is substituted with S or T, more preferably with S. In some embodiments, the modified DAAO comprises a combination of substituents 54V, 56N, 58H, and 213S, or 54I, 56N, 58H, and 213S, compared to its wild-type DAAO. In some embodiments, the modified DAAO further comprises amino acid substitution(s) at the 210 and / or 221 positions. Preferably, at the 210 position, the amino acid is substituted with A, G, or P, more preferably with A. Preferably, at the 221 position, the amino acid is substituted with R.

[0057] In some embodiments, the modified DAAO comprises amino acid substitutions at positions 54, 58, 213, and 221, said positions numbered with reference to SEQ ID NO. 2. Preferably, at position 54, the amino acid is substituted with V, at position 58, the amino acid is substituted with Q, at position 213, the amino acid is substituted with S, and at position 221, the amino acid is substituted with R. In some embodiments, the modified DAAO further comprises amino acid substitutions at positions 210 and / or 56. Preferably, at position 56, the amino acid is substituted with N, and at position 210, the amino acid is substituted with A.

[0058] In some embodiments, the modified DAAO of the present invention further comprises amino acid substitutions at one or more positions selected from the group consisting of 2, 81, 97, 193, 194, 237, 265, 273, 274, 300, 317, 319, 337 and 342, said positions numbered with reference to SEQ ID NO. 2, and said modified DAAO has activity for catalyzing the oxidation of D-glufosinate to PPO. Preferably, at position 2, the amino acid is substituted with C or S, at position 81, the amino acid is substituted with Y, at position 97, the amino acid is substituted with V, at position 193, the amino acid is substituted with T, at position 194, the amino acid is substituted with V or C, at position 237, the amino acid is substituted with V, at position 265, the amino acid is substituted with C, at position 273, the amino acid is substituted with D, at position 274, the amino acid is substituted with E, at position 300, the amino acid is substituted with S or T, at position 317, the amino acid is substituted with Y or W, at position 319, the amino acid is substituted with K, at position 337, the amino acid is substituted with S, and at position 342, the amino acid is substituted with S or H.

[0059] Alternatively, the modified DAAO of the present invention further comprises an amino acid at one or more positions selected from the group consisting of 194, 237, 265, 273, 274, 300, 317, and 319, said positions numbered with reference to SEQ ID NO. 2, and said modified DAAO has activity for catalyzing the oxidation of D-glufosinate to PPO. Preferably, at position 194, the amino acid is substituted with V or C; at position 237, the amino acid is substituted with V; at position 265, the amino acid is substituted with C; at position 273, the amino acid is substituted with D; at position 274, the amino acid is substituted with E; at position 300, the amino acid is substituted with S; at position 317, the amino acid is substituted with Y or W; and at position 319, the amino acid is substituted with K.

[0060] Alternatively, the modified DAAO further comprises amino acid substitutions at one or more positions selected from the group consisting of 2, 81, 97, 193, 300, 337, and 342, said positions numbered with reference to SEQ ID NO. 2, and said modified DAAO has activity for catalyzing the oxidation of D-glufosinate to PPO. Preferably, at position 2, the amino acid is substituted with C or S; at position 81, the amino acid is substituted with Y; at position 97, the amino acid is substituted with V; at position 193, the amino acid is substituted with T; at position 300, the amino acid is substituted with T; at position 337, the amino acid is substituted with S; and at position 3234, the amino acid is substituted with S.

[0061] In some embodiments, the modified DAAO of the present invention comprises amino acid substitutions at positions 54, 58, 194, and 213 compared with SEQ ID NO. 1, wherein the modified DAAO has activity for catalyzing the oxidation of D-glufosinate to PPO. Preferably, at position 54, the amino acid is substituted with I, V, T, or L; at position 58, the amino acid is substituted with H or Q; at position 194, the amino acid is substituted with V or C; and at position 213, the amino acid is substituted with S or T. In some embodiments, the modified DAAO further comprises amino acid substitutions at one or more positions selected from the group consisting of positions 56, 210, 221, 237, 265, 273, 274, 300, 317, and 319. Preferably, at position 56, the amino acid is substituted with N; at position 210, the amino acid is substituted with A, G, or P; at position 221, the amino acid is substituted with R; at position 237, the amino acid is substituted with V; at position 265, the amino acid is substituted with C; at position 273, the amino acid is substituted with D; at position 274, the amino acid is substituted with E; at position 300, the amino acid is substituted with S; at position 317, the amino acid is substituted with Y or W; and at position 319, the amino acid is substituted with K. In some embodiments, the modified DAAO of the present invention further comprises, compared with SEQ ID NO. 1, the insertion of one or more conserved substitutions of amino acids or the deletion of one or more amino acids.

[0062] In some embodiments, a modified DAAO of the present invention comprises a combination of amino acid substitutions selected from the group consisting of the following compared to its wild type (said positions are numbered with reference to SEQ ID NO. 2):

[0063] - 54V, 58Q, 194V, 213S;

[0064] - 54V, 58Q, 194C, 213S;

[0065] - 54V, 58Q, 213S, 273D;

[0066] - 54V, 58Q, 213S, 317Y;

[0067] - 54V, 58Q, 213S, 317W;

[0068] - 54V, 58Q, 213S, 274E;

[0069] - 54V, 58Q, 213S, 319K;

[0070] - 54V, 58Q, 194C, 213S, 317Y;

[0071] - 54V, 58Q, 194C, 213S, 265C, 317Y;

[0072] - 54V, 58Q, 194C, 213S, 265C, 300S, 317Y;

[0073] - 54V, 58Q, 194C, 213T, 265C, 300S, 317Y;

[0074] - 54V, 58Q, 194C, 213S, 210G, 265C, 300S, 317Y;

[0075] - 54V, 58Q, 194C, 213S, 210P, 265C, 300S, 317Y;

[0076] - 54V, 58Q, 194C, 213S, 210A, 265C, 300S, 317Y;

[0077] - 54V, 58Q, 194C, 213S, 221R, 265C, 300S, 317Y;

[0078] - 54V, 58Q, 194C, 213S, 237A, 265C, 300S, 317Y;

[0079] - 54V, 58Q, 194C, 213S, 237V, 265C, 300S, 317Y;

[0080] - 54V, 56N, 58Q, 194C, 213S, 265C, 300S, 317Y;

[0081] - 54T, 56N, 58Q, 194C, 213S, 265C, 300S, 317Y;

[0082] - 54I, 56N, 58Q, 194C, 213S, 265C, 300S, 317Y;

[0083] - 54V, 56N, 58H, 194C, 213S, 265C, 300S, 317Y;

[0084] - 54L, 56N, 58Q, 194C, 213S, 265C, 300S, 317Y;

[0085] - 54I, 56N, 58H, 194C, 213S, 265C, 300S, 317Y;

[0086] - 54V, 56N, 58H, 194C, 213S, 237V, 265C, 300S, 317Y;

[0087] - 54V, 56N, 58H, 194C, 213S, 210A, 237V, 265C, 300S, 317Y;

[0088] - 54I, 56N, 58H, 194C, 213S, 210A, 221R, 265C, 300S, 317Y;

[0089] - 54L, 56N, 58Q;

[0090] - 54T, 56N, 58Q;

[0091] - 54I, 56N, 58H;

[0092] - 54V, 56N, 58H;

[0093] - 54L, 56N, 58Q, 213S;

[0094] - 54T, 56N, 58Q, 213S;

[0095] - 54I, 56N, 58H, 213S;

[0096] - 54V, 56N, 58H, 213S;

[0097] - 2C, 54V, 56N, 58H, 213S;

[0098] - 2S, 54V, 56N, 58H, 213S;

[0099] - 54V, 56N, 58H, 81Y, 213S;

[0100] - 54V, 56N, 58H, 97V, 213S;

[0101] - 54V, 56N, 58H, 193T, 213S;

[0102] - 54V, 56N, 58H, 213S, 300T;

[0103] - 54V, 56N, 58H, 213S, 337S;

[0104] - 54V, 56N, 58H, 213S, 342S;

[0105] - 2S, 54V, 56N, 58H, 81Y, 97V, 193T, 213S, 337S;

[0106] - 54V, 56N, 58H, 97V, 193A, 213S, 337S, 342H;

[0107] - 2C, 54V, 56N, 58H, 81Y, 97V, 213S, 337S;

[0108] - 2C, 54V, 56N, 58H, 81Y, 97V, 193A, 213S, 342S;

[0109] - 54V, 56N, 58H, 97V, 193T, 213S, 337S, 342H;

[0110] - 54V, 56N, 58H, 81Y, 97V, 193T, 213S, 337S, 342H;

[0111] - 54V, 56N, 58H, 97V, 193T, 213S, 300T, 337S, 342H;

[0112] - 54V, 56N, 58H, 81Y, 97V, 193T, 213S, 300T, 337S, 342H;

[0113] - 54V, 56N, 58H, 97V, 193T, 210A, 213S, 300T, 337S, 342H;

[0114] - 54V, 56N, 58H, 97V, 193T, 213S, 221R, 300T, 337S, 342H;

[0115] - 54V, 56N, 58H, 97V, 193T, 210A, 213S, 221R, 300T, 337S, 342H;

[0116] - 58K, 213T;

[0117] - 54V, 56N, 58H, 210A, 213S;

[0118] - 54V, 56N, 58H, 213S, 221R;

[0119] - 54V, 56N, 58H, 210A, 213S, 221R;

[0120] - 54I, 56N, 58H, 210A, 213S;

[0121] - 54I, 56N, 58H, 213S, 221R;

[0122] - 54I, 56N, 58H, 210A, 213S, 221R;

[0123] - 54V, 58Q, 213S;

[0124] - 54V, 58Q, 210A, 213S;

[0125] - 54V, 58Q, 213S, 221R; 및

[0126] - 54V, 58Q, 210A, 213S, 221R.

[0127] A DAAO polypeptide based on the performance of amino acid modification is referred to herein as an initiating DAAO. The initiating DAAO may be a wild-type DAAO or a variant of the wild-type DAAO. For example, if the modification is initiated based on the polypeptide of SEQ ID NO. 1, the polypeptide of SEQ ID NO. 1 is the “initiating DAAO” for the modified DAAO; and if the modification is initiated based on the variant polypeptide of SEQ ID NO. 1 (SEQ ID NO. 4-30), the variant polypeptide is the “initiating DAAO” for the modified DAAO.

[0128] As used herein, the term "wild-type DAAO" refers to naturally occurring DAAO. In some embodiments, the wild-type DAAO is Rhodotorula It is a DAAO of the genus. In some embodiments, the wild-type DAAO is one of SEQ ID NOs 1-3. SEQ ID NO 1 is Rhodotorula toruloides It is the DAAO amino acid sequence from (GenBank Assembly No. CAJ87425.1), and Sequence No. 2 is Rhodotorula It is the DAAO amino acid sequence from sp. JG-1b (GenBank Assembly No. KWU45700.1), and Sequence No. 3 is Rhodotorula taiwanensis This is the estimated DAAO amino acid sequence from (GenBank Assembly No. POY70719.1).

[0129] In the present invention, to determine the percentage identity between two amino acid sequences or two nucleic acid sequences, said sequences are aligned for the purpose of optimal comparison (e.g., a gap may be introduced into the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). Then, amino acid residues or nucleotides are compared at the corresponding amino acid position or nucleotide position. When a position of the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position of the second sequence, these molecules are identical at this position. The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage identity = number at identical positions / total number at total positions (i.e., overlapping positions × 100)). Preferably, said two sequences are of the same length.

[0130] Those skilled in the art know that various computer programs can be used to determine identity between two sequences.

[0131] "Amino acid identity percentage" or "amino acid sequence identity percentage" represents a comparison between the amino acids of two polypeptides, and when optimally aligned, the two polypeptides have an approximate specified percentage of identical amino acids. For example, "95% amino acid identity" represents a comparison between the amino acids of two polypeptides, and when optimally aligned, 95% of the amino acids of the two polypeptides are identical.

[0132] In some embodiments, the wild-type DAAO is at least 65% identical to one of SEQ ID NOs 1-3, preferably at least 70%, 75%, or 80%, more preferably at least 85%, 90%, or 95%, particularly 96%, 97%, 98%, or 99% identical.

[0133] In some embodiments, the modified DAAO of the present invention comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions compared to the wild type, and said modified DAAO has activity that catalyzes the oxidation of D-glufosinate to PPO. In some embodiments, the modified DAAO of the present invention comprises amino acid substitutions at positions 54, 56, 58, and 213, said positions numbered with reference to SEQ ID NO. 2. Preferably, at position 54, the amino acid is substituted with I, V, T, or L, more preferably with I or V. Preferably, at position 56, the amino acid is substituted with N. Preferably, at position 58, the amino acid is substituted with H or Q, more preferably with H. Preferably, at position 213, the amino acid is substituted with S or T, more preferably with S. In some preferred embodiments, the modified DAAO comprises substituents 54V, 56N, 58H, and 213S, or substituents 54I, 56N, 58H, and 213S, compared to its wild-type DAAO. In some embodiments, the modified DAAO further comprises amino acid substitution(s) at the 210 and / or 221 positions. Preferably, at the 210 position, the amino acid is substituted with A, G, or P, more preferably with A. Preferably, at the 221 position, the amino acid is substituted with R. Preferably, the modified DAAO of the present invention further comprises amino acid substitutions at one or more positions selected from the group consisting of 194, 237, 265, 273, 274, 300, 317, and 319.Preferably, at position 194, the amino acid is substituted with V or C, at position 237, the amino acid is substituted with V, at position 265, the amino acid is substituted with C, at position 273, the amino acid is substituted with D, at position 274, the amino acid is substituted with E, at position 300, the amino acid is substituted with S, at position 317, the amino acid is substituted with Y or W, and at position 319, the amino acid is substituted with K. Preferably, the wild-type DAAO is at least 65% identical to SEQ ID NO. 1, preferably at least 70%, 75%, or 80%, more preferably 85%, 90%, or 95%, particularly 96%, 97%, 98%, or 99% identical.

[0134] In some embodiments, the modified DAAO comprises amino acid substitutions at positions 54, 58, 213, and 221, said positions numbered with reference to SEQ ID NO. 2. Preferably, at position 54, the amino acid is substituted with V, at position 58, the amino acid is substituted with Q, at position 213, the amino acid is substituted with S, and at position 221, the amino acid is substituted with R. In some embodiments, the modified DAAO further comprises amino acid substitutions at positions 210 and / or 56. Preferably, at position 56, the amino acid is substituted with N, and at position 210, the amino acid is substituted with A. Preferably, the modified DAAO of the present invention further comprises amino acid substitutions at one or more positions selected from the group consisting of 194, 237, 265, 273, 274, 300, 317, and 319, wherein the amino acid at position 194 is substituted with V or C, the amino acid at position 237 is substituted with V, the amino acid at position 265 is substituted with C, the amino acid at position 273 is substituted with D, the amino acid at position 274 is substituted with E, the amino acid at position 300 is substituted with S, the amino acid at position 317 is substituted with Y or W, and the amino acid at position 319 is substituted with K. Preferably, the wild-type DAAO is identical to SEQ ID NO. 1 by at least 65%, preferably at least 70%, 75%, or 80%, more preferably at least 85%, 90%, or 95%, particularly 96%, 97%, 98%, or 99%.

[0135] In some embodiments, the modified DAAO of the present invention comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions compared to the wild type, and said modified DAAO has activity for catalyzing the oxidation of D-glufosinate to PPO. In some embodiments, the modified DAAO of the present invention comprises amino acid substitutions at positions 54, 56, 58, and 213, said positions numbered with reference to SEQ ID NO. 2. Preferably, at position 54, the amino acid is substituted with I, V, T, or L, more preferably with I or V. Preferably, at position 56, the amino acid is substituted with N. Preferably, at position 58, the amino acid is substituted with H or Q, more preferably with H. Preferably, at position 213, the amino acid is substituted with S or T, more preferably with S. In some preferred embodiments, the modified DAAO comprises substituents 54V, 56N, 58H, and 213S, or substituents 54I, 56N, 58H, and 213S, compared to its wild-type DAAO. In some embodiments, the modified DAAO further comprises amino acid substitution(s) at the 210 and / or 221 positions. Preferably, at the 210 position, the amino acid is substituted with A, G, or P, more preferably A. Preferably, at the 221 position, the amino acid is substituted with R. Preferably, the modified DAAO of the present invention further comprises amino acid substitutions at one or more positions selected from the group consisting of 2, 81, 97, 193, 300, 337, and 342, wherein the amino acid at position 2 is substituted with C or S, the amino acid at position 81 is substituted with Y, the amino acid at position 97 is substituted with V, the amino acid at position 193 is substituted with T, the amino acid at position 300 is substituted with T, the amino acid at position 337 is substituted with S, and the amino acid at position 342 is substituted with S or H.Preferably, the wild-type DAAO is at least 65% identical to SEQ ID NO. 2, preferably at least 70%, 75%, or 80%, more preferably at least 85%, 90%, or 95%, particularly 96%, 97%, 98%, or 99% identical.

[0136] In some embodiments, the modified DAAO comprises amino acid substitutions at positions 54, 58, 213, and 221, said positions numbered with reference to SEQ ID NO. 2.

[0137] Preferably, at position 54, the amino acid is substituted with V, at position 58, the amino acid is substituted with Q, at position 213, the amino acid is substituted with S, and at position 221, the amino acid is substituted with R. In some embodiments, the modified DAAO further comprises amino acid substitution(s) at positions 210 and / or 56. Preferably, at position 56, the amino acid is substituted with N, and at position 210, the amino acid is substituted with A. Preferably, the modified DAAO of the present invention further comprises amino acid substitutions at one or more positions selected from the group consisting of 2, 81, 97, 193, 300, 337, and 342, wherein the amino acid at position 2 is substituted with C or S, the amino acid at position 81 is substituted with Y, the amino acid at position 97 is substituted with V, the amino acid at position 193 is substituted with T, the amino acid at position 300 is substituted with T, the amino acid at position 337 is substituted with S, and the amino acid at position 342 is substituted with S or H. Preferably, the wild-type DAAO is identical to SEQ ID NO. 2 by at least 65%, preferably at least 70%, 75%, or 80%, more preferably at least 85%, 90%, or 95%, particularly 96%, 97%, 98%, or 99%.

[0138] In some embodiments, the modified DAAO comprises 4-20, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, or 4-5 amino acid substitutions compared to the wild type. In some embodiments, the modified DAAO comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions compared to the wild type.

[0139] In some embodiments, the wild-type DAAO differs from one of SEQ ID NOs 1-3 in that it comprises a substitution, deletion, insertion, and / or addition of one or more amino acids. In some embodiments, the wild-type DAAO comprises a conserved substitution of one or more amino acids compared to one of SEQ ID NOs 1-3. In some embodiments, the wild-type DAAO comprises an insertion or deletion of one or more amino acids compared to one of SEQ ID NOs 1-3.

[0140] In some embodiments, the modified DAAO comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions compared to its wild-type DAAO, wherein the modified DAAO has activity for catalyzing the oxidation of D-glufosinate to PPO. In some embodiments, the modified DAAO comprises amino acid substitutions at positions 54, 56, 58, and 213, wherein the positions are numbered with reference to SEQ ID NO. 2, wherein the modified DAAO is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, or 98% identical to one of SEQ ID NOs 1-3. Preferably, the amino acid at position 54 is substituted with I, V, T, or L, more preferably with I or V. Preferably, the amino acid at position 56 is substituted with N. Preferably, the amino acid at position 58 is substituted with H or Q, more preferably with H. Preferably, the amino acid at position 213 is substituted with S or T, more preferably with S. In some preferred embodiments, the modified DAAO polypeptide comprises a combination of substituents 54V, 56N, 58H, and 213S, or 54I, 56N, 58H, and 213S, compared to its wild-type DAAO. In some embodiments, the modified DAAO further comprises amino acid substitution(s) at positions 210 and / or 221. Preferably, the amino acid at position 210 is substituted with A, G, or P, more preferably with A. Preferably, the amino acid at position 221 is substituted with R.

[0141] In some embodiments, the modified DAAO comprises amino acid substitutions at positions 54, 58, 213, and 221, said positions numbered with reference to SEQ ID NO. 2, wherein the modified DAAO is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, or 98% or more identical to one of SEQ ID NOs 1-3. Preferably, at position 54, the amino acid is substituted with V; at position 58, the amino acid is substituted with Q; at position 213, the amino acid is substituted with S; and at position 221, the amino acid is substituted with R. In some embodiments, the modified DAAO further comprises amino acid substitutions at positions 210 and / or 56. Preferably, at position 56, the amino acid is substituted with N; and at position 210, the amino acid is substituted with A.

[0142] In some embodiments, the modified DAAO of the present invention further comprises amino acid substitutions at one or more positions selected from the group consisting of 2, 81, 97, 193, 194, 237, 265, 273, 274, 300, 317, 319, 337 and 342, said positions numbered with reference to SEQ ID NO. 2, wherein the modified DAAO has activity for catalyzing the oxidation of D-glufosinate to PPO. Preferably, at position 2, the amino acid is substituted with C or S, at position 81, the amino acid is substituted with Y, at position 97, the amino acid is substituted with V, at position 193, the amino acid is substituted with T, at position 194, the amino acid is substituted with V or C, at position 237, the amino acid is substituted with V, at position 265, the amino acid is substituted with C, at position 273, the amino acid is substituted with D, at position 274, the amino acid is substituted with E, at position 300, the amino acid is substituted with S or T, at position 317, the amino acid is substituted with Y or W, at position 319, the amino acid is substituted with K, at position 337, the amino acid is substituted with S, and at position 342, the amino acid is substituted with S or H.

[0143] Alternatively, in some embodiments, the modified DAAO of the present invention further comprises substitutions at one or more positions selected from the group consisting of 194, 237, 265, 273, 274, 300, 317, and 319, the positions numbered with reference to SEQ ID NO. 2, wherein the modified DAAO has activity for catalyzing the oxidation of D-glufosinate to PPO. Preferably, at position 194, the amino acid is substituted with V or C; at position 237, the amino acid is substituted with V; at position 265, the amino acid is substituted with C; at position 273, the amino acid is substituted with D; at position 274, the amino acid is substituted with E; at position 300, the amino acid is substituted with S; at position 317, the amino acid is substituted with Y or W; and at position 319, the amino acid is substituted with K.

[0144] Alternatively, in some embodiments, the modified DAAO of the present invention further comprises substitutions at one or more positions selected from the group consisting of 2, 81, 97, 193, 300, 337, and 342, said positions numbered with reference to SEQ ID NO. 2, wherein the modified DAAO has activity for catalyzing the oxidation of D-glufosinate to PPO. Preferably, at position 2, the amino acid is substituted with C or S; at position 81, the amino acid is substituted with Y; at position 97, the amino acid is substituted with V; at position 193, the amino acid is substituted with T; at position 300, the amino acid is substituted with T; at position 337, the amino acid is substituted with S; and at position 342, the amino acid is substituted with S or H.

[0145] In some embodiments, the modified DAAO comprises amino acid substitutions at positions 54, 58, 194, and 213 compared with SEQ ID NO. 1, wherein the modified DAAO has activity for catalyzing the oxidation of D-glufosinate to PPO, and wherein the modified DAAO is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, or 98% or more identical to SEQ ID NO. 1. Preferably, the amino acid at position 54 is substituted by I, V, T, or L, the amino acid at position 58 is substituted by H or Q, the amino acid at position 194 is substituted by V or C, and the amino acid at position 213 is substituted by S or T. In some embodiments, the modified DAAO further comprises amino acid substitutions at one or more positions selected from the group consisting of positions 56, 210, 221, 237, 265, 273, 274, 300, 317, and 319. Preferably, at position 56, the amino acid is substituted with N; at position 210, the amino acid is substituted with A, G, or P; at position 221, the amino acid is substituted with R; at position 237, the amino acid is substituted with V; at position 265, the amino acid is substituted with C; at position 273, the amino acid is substituted with D; at position 274, the amino acid is substituted with E; at position 300, the amino acid is substituted with S; at position 317, the amino acid is substituted with Y or W; and at position 319, the amino acid is substituted with K. In some embodiments, the modified DAAO of the present invention further comprises one or more conserved substitutions of amino acids, or comprises one or more insertions or deletions of amino acids compared to SEQ ID NO. 1.

[0146] The above term "conserved substitution" is also referred to as substitution by "homologous" amino acid residues, which are amino acid residues having similar side chains, e.g., amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). It means a substitution that is replaced.

[0147] In general, conserved substitutions of amino acids have minimal effect on the activity of the resulting protein. These substitutions are described below. Conserved substitution involves replacing an amino acid with one that is similar in size, hydrophobicity, charge, polarity, spatial characteristics, and orientation. Substitutions are generally conserved when precise control of protein properties is desired.

[0148] As used herein, "homologous" amino acid residues refer to amino acid residues having similar chemical properties related to hydrophobicity, charge, polarity, stereochemical properties, aromatic properties, etc.

[0149] Examples of amino acids homologous to one another 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, and glutamine; aromatic phenylalanine, tyrosine, and tryptophan; serine and threonine, or glutamine and asparagine, or leucine and isoleucine, which have chemically similar side chain groups.

[0150] Examples of conservative amino acid substitutions in proteins are as follows: Ala is substituted with Ser, Arg is substituted with Lys, Asn is substituted with Gln or His, Asp is replaced with Glu, Cys is substituted with Ser, Gln is substituted with Asn, Glu is substituted with Asp, Gly is substituted with Pro, His is substituted with Asn or Gln, Ile is substituted with Leu or Val, Leu is substituted with Ile or Val, Lys is substituted with Arg or Gln, Met is substituted with Leu or Ile, Phe is substituted with Met, Leu, or Tyr, Ser is substituted with Thr, Thr is substituted with Ser, Trp is substituted with Tyr, Tyr is substituted with Trp or Phe, and Val is substituted with Ile or Leu.

[0151] In some embodiments, the modified DAAO comprises or consists of one amino acid sequence of SEQ ID NOs 5-86, or the modified DAAO comprises 1-10 amino acid substitutions at positions other than 54, 56, 58, 194, 210, 213, 221, 237, 265, 273, 274, 300, 317, and 319 compared to one of SEQ ID NOs 5-30 and 66-76, and comprises 1-10 amino acid substitutions at positions other than 2, 54, 56, 58, 81, 97, 193, 210, 213, 221, 300, 337, and 342 compared to one of SEQ ID NOs 31-57 and 77-86, or 54, 56, compared to one of SEQ ID NOs 58-65 It includes 1-10 amino acid substitutions at positions other than 58, 210, 213 and 221, wherein the modified DAAO has activity that catalyzes the oxidation of D-glufosinate to PPO. In some embodiments, the modified DAAO comprises or consists of one amino acid sequence of SEQ ID NOs 5-86, or the modified DAAO comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid substitutions at positions other than 54, 56, 58, 194, 210, 213, 221, 237, 265, 273, 274, 300, 317, and 319 compared to one of SEQ ID NOs 5-30 and 66-76, and 1, 2, 3, at positions other than 2, 54, 56, 58, 81, 97, 193, 210, 213, 221, 300, 337, and 342 compared to one of SEQ ID NOs 31-57 and 77-86 It comprises 4, 5, 6, 7, 8, 9, or 10 or more amino acid substitutions, or comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid substitutions at positions other than 54, 56, 58, 210, 213, and 221 compared to one of SEQ ID NOs 58-65, and said modified DAAO has activity for catalyzing the oxidation of D-glufosinate to PPO.In some embodiments, the modified DAAO is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to one of SEQ ID NOs 1-3.

[0152] As used herein, enzyme activity refers to a decrease in substrate or an increase in product per unit time in a chemical reaction catalyzed by a unit mass of enzyme under specific conditions. For example, the activity of the modified DAAO of the present invention may be expressed as a decrease in D-glufosinate or an increase in PPO per unit time under the catalytic action of a unit mass of the modified DAAO under specific conditions.

[0153] In the present invention, the activity of an enzyme may also represent the relative activity of the enzyme, expressed as the ratio of the activity of the enzyme of interest to the activity of a given enzyme catalyzing the same reaction, such as the relative activity percentage.

[0154] In some embodiments, the activity of the modified DAAO of the present invention is expressed as a percentage relative activity compared with SEQ ID NO. 4. In some embodiments, the activity of the modified DAAO for catalyzing the oxidation of D-glufosinate to PPO is at least 100%, 105%, 110%, 120%, 130%, 150%, 170%, 200%, 250%, 300% or more of the activity of SEQ ID NO. 4 for catalyzing the oxidation of D-glufosinate to PPO.

[0155] In addition, improving the stability of the modified DAAO is advantageous for industrial production. In some embodiments, the stability is thermal stability, which refers to the ability of an enzyme to maintain activity after incubation at a specific temperature (e.g., 40-60°C or higher) for a specific time (e.g., 10 minutes to 1 hour). In some embodiments, the modified DAAO has better thermal stability than the polypeptide of SEQ ID NO. 4. For example, after incubation at 43-45°C for 20 minutes, the activity of the modified DAAO of the present invention is at least 100%, 105%, 110%, 120%, 130%, 150%, 170%, 200%, 250%, 300%, or more of the activity of the polypeptide of SEQ ID NO. 4. Alternatively, the modified DAAO of the present invention has a higher T50, where T50 refers to the temperature at which enzyme activity decreases by 50% after 1 hour of incubation. In some embodiments, the T50 of the modified DAAO of the present invention is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10°C higher than that of the polypeptide of SEQ ID NO. 4.

[0156] In some embodiments, the modified DAAO has better thermal stability than the polypeptide of SEQ ID NO. 4, and its activity for catalyzing the oxidation of D-glufosinate to PPO is at least 100%, 105%, 110%, 120%, 130%, 150%, 170%, 200%, 250%, 300% or more than the activity of SEQ ID NO. 4 for catalyzing the oxidation of D-glufosinate to PPO.

[0157] II. Polynucleotide encoding modified DAAO

[0158] As used herein, the terms “polynucleotide” or “nucleic acid molecule” include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and DNA or RNA analogs produced using nucleotide analogs. The nucleic acid molecules may be single-stranded or double-stranded, preferably double-stranded DNA. The synthesis of the nucleic acid may use nucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). These nucleotides may be used to produce nucleic acids having, for example, modified base pairing ability or increased nuclease resistance.

[0159] The present invention also provides a polynucleotide encoding a modified DAAO of the present invention. Accordingly, in the present invention, the term modification also includes genetic manipulation of a polynucleotide encoding the DAAO polypeptide of the present invention. The modification may be a substitution, deletion, insertion, and / or addition of nucleotides.

[0160] As used herein, the term “encoding” means that a polynucleotide directly specifies the amino acid sequence of its protein product. The boundaries of said coding sequence are generally determined by an open read frame that starts 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. said coding sequence may be DNA, cDNA, or a recombinant nucleotide sequence.

[0161] In addition, nucleic acid molecules covering all or part of the nucleic acid sequence of the present invention can be separated by a polymerase chain reaction (PCR) using oligonucleotide primers designed and synthesized based on sequence information included in the sequence.

[0162] The polynucleotide 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 acid amplified as described above can be cloned into a suitable vector and characterized by DNA sequencing analysis.

[0163] The polynucleotide of the present invention can be manufactured using standard synthesis techniques, for example, an automated DNA synthesizer.

[0164] The present invention also relates to a complementary strand of a nucleic acid molecule as described herein. A nucleic acid molecule complementary to another nucleotide sequence is a molecule sufficiently complementary to the nucleotide sequence to be able to hybridize with the other nucleotide sequence to form a stable duplex.

[0165] As used herein, the term “hybridization” refers to nucleotide sequences that are homologous to each other by at least about 90%, preferably at least about 95%, more preferably at least about 96%, more preferably at least 98%, and generally maintain hybridization to each other under given strict hybridization and washing conditions.

[0166] Those skilled in the art are aware of various conditions for hybridization, such as strict hybridization conditions and highly strict hybridization conditions. For example, see Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, NY; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, John Wiley & Sons, NY.

[0167] Of course, the polynucleotides of the present invention do not include polynucleotides that hybridize only to poly A sequences (such as the 3' end poly(A) of mRNA) or poly T (or U) residues.

[0168] III. Expression and Production of Modified DAAO

[0169] To express the modified DAAO of the present invention, a vector comprising a nucleic acid construct and a polynucleotide of the present invention, for example, an expression vector, is also provided.

[0170] As used herein, the term “expression” includes any step relating to the production of a polypeptide and includes, but is not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0171] The term "nucleic acid construct" means a single-stranded or double-stranded nucleic acid molecule, which is isolated from a naturally occurring gene or modified to include a nucleic acid segment that does not occur naturally. Where the nucleic acid construct contains a regulatory sequence necessary to express the coding sequence of the present invention, the term nucleic acid construct is synonymous with the term "expression cassette."

[0172] The above term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide of the present invention, operably linked to an additional nucleotide provided for the expression of a regulatory sequence, for example. The expression vector comprises a viral vector or a plasmid vector.

[0173] In this document, the term "control sequence" comprises all elements necessary or beneficial for the expression of a polynucleotide encoding the polypeptide of the present invention. Each control sequence may be natural or foreign with respect to the nucleotide sequence encoding the polypeptide, or may be natural or foreign to one another. 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 promoters and signals for the termination of transcription and translation.

[0174] For example, the regulatory sequence may be a suitable promoter sequence that is a nucleotide sequence recognized by a host cell to express a polynucleotide encoding the polypeptide of the present invention. The promoter sequence comprises a transcriptional regulatory sequence that mediates the expression of the polypeptide. The promoter is any nucleotide sequence that exhibits transcriptional activity in a selected host cell, for example E. coli It may be a lac operon. The promoter may also include mutant, truncated, and hybrid promoters and may be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0175] In the present invention, the term "operably linked" refers to a configuration in which a regulatory sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide sequence, and said regulatory sequence directs the expression of the polypeptide coding sequence.

[0176] The polynucleotide encoding the polypeptide of the present invention may undergo various manipulations to allow for the expression of the polypeptide. Manipulation of the polynucleotide according to the expression vector is desirable or necessary before insertion into the vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art.

[0177] To identify and select host cells containing the expression vector of the present invention, the vector of the present invention preferably includes one or more selection markers capable of simply selecting cells such as those undergoing transformation, transfection, or transduction. The selectable marker is a gene that provides the product with biocide or virus resistance, heavy metal resistance, supplemental nutrition requirements, etc. For example, the bacterial selection marker is Bacillus subtilis or Bacillus licheniformis It is a marker that confers antibiotic resistance, such as the dal gene, or resistance to ampicillin, kanamycin, chloramphenicol, or tetracycline.

[0178] The vector of the present invention can be incorporated into the genome of a host cell or replicate autonomously within a cell independent of the genome. The elements required for integration into the host cell genome or autonomous replication are known in the art (see, for example, the aforementioned Sambrook et al., 1989).

[0179] Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques.

[0180] As used herein, the terms “transformation” and “transfection” refer to various techniques for introducing foreign nucleic acids (e.g., DNA) into a host cell, which are well known in the art and can be found, for example, in the aforementioned Sambrook et al., 1989; Davis et al., Basic Methods in Molecular Biology (1986) and other laboratory manuals.

[0181] The present invention also relates to a recombinant host cell comprising the polynucleotide of the present invention, which is advantageously used for the recombinant production of DAAO polypeptides. A vector comprising the polynucleotide of the present invention is introduced into a host cell, thereby maintaining the vector as a chromosomal conjugate or as a self-replicating extrachromosomal vector. Those skilled in the art are aware of conventional vectors and host cells for expressing proteins.

[0182] In some embodiments, the host cell of the present invention E. coli BL21(DE3) and like E. coli It is a cell. In some embodiments, the expression vector is pET-30a(+).

[0183] The modified DAAO of the present invention can be operably linked to a non-DAAO polypeptide (e.g., a heterogeneous amino acid sequence) to form a fusion protein. For example, in one embodiment, the fusion protein is a GST-DAAO fusion protein, wherein the DAAO sequence is fused to the C-terminus of the GST sequence. This fusion protein can facilitate the purification of recombinant DAAO. In another embodiment, the fusion protein is a DAAO protein containing a heterogeneous signal sequence at its N-terminus. In certain host cells (e.g., mammalian and yeast host cells), the expression and / or secretion of DAAO can be increased by the use of the heterogeneous signal sequence.

[0184] IV. Production of L-glufosinate

[0185] In addition, the present invention provides a method for producing L-glufosinate comprising the step of contacting the modified DAAO or host cell of the present invention with D-glufosinate.

[0186] In some embodiments, the method for preparing L-glufosinate-ammonium of the present invention comprises the following steps:

[0187] (a) providing the activity of the modified DAAO and D-glufosinate of the present invention to a reaction medium, and optionally, providing catalase activity to the reaction medium,

[0188] (b) a step of incubating the reaction medium to oxidize D-glufosinate to PPO, and

[0189] (c) A step of generating L-glufosinate by reduction of PPO or amino group transfer.

[0190] In some embodiments, L-glufosinate is produced using a cell-free catalytic method, and the modified DAAO of the present invention is provided in step (a). In some embodiments, the modified DAAO of the present invention, either free or immobilized, may be used. Catalase may also be immobilized.

[0191] In some embodiments, the incubation is performed at 20-50°C, preferably 25-40°C, more preferably 28-35°C, for example 30°C.

[0192] In some embodiments, the medium is a buffer such as PBS and Tris-HCl buffer. In one embodiment, the medium is a Tris-HCl buffer such as 50 mM, pH 8.0 Tris-HCl buffer.

[0193] In some embodiments, the reaction medium is a medium composed of a cell culture medium, either partially or wholly, and the activity of the modified DAAO of the present invention is provided by the host cells of the present invention cultured in the reaction medium.

[0194] In some embodiments, the reaction medium is a medium composed of a cell culture medium, either partially or wholly, and the catalase activity is provided by a second host cell, which is a host cell of the present invention or cultured in the reaction medium.

[0195] In some embodiments, the host cells and / or second host cells of the present invention are cultured and expanded in a cell culture medium, and then the expanded host cells are separated from the cell culture medium and the biomass is resuspended using a buffer or water. D-glufosinate is added to the buffer or water before, during, or after the addition of the expanded host cells.

[0196] In some embodiments, E. coli Bacterial cells, such as cells, can be used. Specific details for implementing the invention

[0197] Those skilled in the art will be able to understand the present invention more clearly through the following examples. It should be understood that the examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0198] Example 1: Materials and Method

[0199] Unless otherwise specified, all experimental methods used in the present invention are conventional methods. Specific gene cloning operations can be seen in the aforementioned Sambrook et al., 1989.

[0200] i) Reagent:

[0201] DNA polymerase (PrimeSTAR Max DNA polymerase) and DpnI endonuclease were purchased from TaKaRa; the plasmid isolation kit was purchased from Axygen; catalase was purchased from Zaozhuang Quanding Biological Technology Co., Ltd., Item No. QD-001; and D,L-Glufosinate was purchased from Lier Chemical Co., Ltd.

[0202] ii) Vector and strain:

[0203] The expression vector used was pET-30a(+) and the plasmid was purchased from Novagen; the host cells used were purchased from Tiangen BioTech (Beijing) Co., Ltd. E. coli It was BL21(DE3).

[0204] iii) Sequencing and primer synthesis were performed by Synbiotechnology Co., Ltd.

[0205] iv) Site-directed mutation:

[0206] Specific primer pairs were designed to introduce desired substituents at bases corresponding to the amino acid positions required for the mutation. The isolated pre-mutation plasmid (containing coding sequences for wild-type DAAO and pET-30a(+) backbones) was used as a template, and mutations were introduced by PCR using Quickchange technology (Nucleic Acids Research, 2004, 32 (14):e115). After PCR amplification, the amplified product was digested with DpnI for 4 hours to remove the template plasmid. The digested product E. coliAfter transforming into BL21(DE3) soluble cells, the cells were plated on LB agar (containing 50 mg / L kanamycin), and a single colony was collected in LB medium (containing 50 mg / L kanamycin), cultured, and then sequenced to confirm the correct mutation. The verified clone was stored at -80℃ for future use.

[0207] v) Protein expression and preparation of coenzyme solution:

[0208] The stored clones were activated on LB agar. Then, a single colony was inoculated into LB medium (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 medium (containing 50 mg / L kanamycin), incubated with shaking at 37°C until the OD600 reached approximately 0.6, and then incubated at 25°C for 16 hours to induce protein expression after the addition of IPTG (final concentration 0.4 mM).

[0209] After incubation, the above culture is centrifuged at 4,000g for 10 minutes at 4℃, the supernatant is discarded, and E. coli Cells were collected. The collected E. coli Cells were resuspended in 15 mL of pre-cooled phosphate-buffered saline (PBS) at pH 7.0 and sonicated at 4°C. The cell lysis solution was centrifuged at 6,000 g, 4°C, for 15 minutes to remove the precipitate, and the obtained supernatant was a crude enzyme solution (13 g / L) containing recombinant enzyme.

[0210] vi) Measurement of enzyme activity

[0211] D,L-glufosinate was dissolved in 50 mM Tris-HCl buffer, pH=8, and the final concentration of D,L-glufosinate in the solution was 100 mM. After adding 2 g / L coenzyme and 2 g / L catalase to the solution, the solution was continuously shaken (400 rpm) in a shaker at 30°C for 2 hours. The decrease in D-glufosinate and ee values ​​was detected by OPA pre-column derivatization high-performance liquid chromatography to determine the sampling and initial rates of the catalytic reaction.

[0212] Example 2. Rhodotorula toruloides Preparation and detection of DAAO (RtDAAO) mutations

[0213] Mutants were prepared using a nucleic acid encoding RtDAAO (SEQ No. 1) as a template according to the method of Example 1. The generated mutants are shown in Table 1, wherein the mutant of SEQ No. 4 is the mutant reported in US 9,834,802 (RtDAAO N54V, F58Q, M213S). The generated mutants were incubated at 45°C for 20 minutes, and enzyme activity was measured according to the method described in Example 1. The results are shown in Table 1, where relative enzyme activity represents the percentage of the activity of the mutant (after incubation) versus the activity of SEQ No. 4 (indicated as "+" for 100–150%, "++" for 150–200%, and "+++" for 200% or more).

[0214] Mutations in the mutants Sequence number Relative enzyme activity N54V, F58Q, A194V, M213S 5 +++ N54V, F58Q, A194C, M213S 6 +++ N54V, F58Q, M213S, E273D 7 ++ N54V, F58Q, M213S, A317Y 8 ++ N54V, F58Q, M213S, A317W 9 +++ N54V, F58Q, M213S, G274E 10 ++ N54V, F58Q, M213S, A319K 11 + N54V, F58Q, A194C, M213S, A317Y 12 +++

[0215] Next, additional mutations were introduced based on SEQ ID NO. 12, and the resulting mutations are shown in Table 2. The relative enzyme activity of the mutants was measured (percentage of the activity of SEQ ID NO. 4 versus the activity of the mutants without heat treatment). Then, the generated mutants were incubated at a series of temperatures (40-60°C) for 1 hour, and T50 was measured (enzyme activity decreased by 50% after 1 hour of incubation). The results are shown in Table 2.

[0216] Mutations in the mutants Sequence number T50 * Relative enzyme activity N54V, F58Q, M213S 4 41-43℃ 100% N54V, F58Q, A194C, M213S, A317Y 12 47-48℃ 103% N54V, F58Q, A194C, M213S, T265C, A317Y 13 49-50℃ 124% N54V, F58Q, A194C, M213S, T265C, V300S, A317Y 14 52-53℃ 128%

[0217] Amino acid substitutions were additionally introduced based on SEQ ID NO. 14, and the enzymatic activity of the mutants was tested (without heat treatment). The results are shown in Table 3. Relative enzymatic activity refers to the percentage of the activity of the mutant versus the activity of SEQ ID NO. 4 without heat treatment (indicated as "+" for 110–120%, "++" for 120–150%, "+++" for 150–200%, and "++++" for 200% or more).

[0218] Mutations in the mutants Sequence number Relative enzyme activity N54V, F58Q, A194C, M213T, T265C, V300S, A317Y 15 ++ N54V, F58Q, A194C, M213S, R210G, T265C, V300S, A317Y 16 ++ N54V, F58Q, A194C, M213S, R210P, T265C, V300S, A317Y 17 ++ N54V, F58Q, A194C, M213S, R210A, T265C, V300S, A317Y 18 ++ N54V, F58Q, A194C, M213S, P221R, T265C, V300S, A317Y 19 +++ N54V, F58Q, A194C, M213S, T237A, T265C, V300S, A317Y 20 ++ N54V, F58Q, A194C, M213S, T237V, T265C, V300S, A317Y 21 ++ N54V, T56N, F58Q, A194C, M213S, T265C, V300S, A317Y 22 +++ N54T, T56N, F58Q, A194C, M213S, T265C, V300S, A317Y 23 +++ N54I, T56N, F58Q, A194C, M213S, T265C, V300S, A317Y 24 ++++ N54V, T56N, F58H, A194C, M213S, T265C, V300S, A317Y 25 ++++ N54L, T56N, F58Q, A194C, M213S, T265C, V300S, A317Y 26 ++++ N54I, T56N, F58H, A194C, M213S, T265C, V300S, A317Y 27 ++++ N54V, T56N, F58H, A194C, M213S, T237V, T265C, V300S, A317Y 28 ++++ N54V, T56N, F58H, A194C, M213S, R210A, T237V, T265C, V300S, A317Y 29 ++++ N54I, T56N, F58H, A194C, M213S, R210A, P221R, T265C, V300S, A317Y 30 ++++

[0219] Example 3. Rhodotorula sp. Preparation and detection of DAAO mutations from JG-1b (RsDAAO)

[0220] According to the method of Example 1, a mutant was prepared using the nucleic acid encoding RsDAAO (Sequence No. 2) as a template, and its enzyme activity was measured. The generated mutant and its enzyme activity are shown in Table 4, where relative enzyme activity refers to the percentage of the activity of the mutant versus the activity of Sequence No. 4 without heat treatment (indicated as "--" for less than 70%, "-" for 70–100%, "+" for 110–120%, "++" for 120–150%, "+++" for 150–200%, and "++++" for 200% or more), and the activity of the wild-type RsDAAO (Sequence No. 2) is 0.

[0221] 돌연변이의 변이체(Mutations in the mutants) 서열번호 상대 효소 활성(Relative enzyme activity) N54L, T56N, F58Q 31 -- N54T, T56N, F58Q 32 -- N54I, T56N, F58H 33 +++ N54V, T56N, F58H 34 ++ N54L, T56N, F58Q, M213S 35 +++ N54T, T56N, F58Q, M213S 36 +++ N54I, T56N, F58H, M213S 37 ++++ N54V, T56N, F58H, M213S 38 ++++

[0222] Amino acid substitutions were further introduced based on SEQ ID NO. 38, and the enzymatic activity of the resulting mutants was measured. The resulting mutants are shown in Table 5. The activity of the mutants (without heat treatment) is identical to that of SEQ ID NO. 38. The mutant of SEQ ID NO. 38 and the mutant with additionally introduced amino acid substitutions were incubated at 43°C for 20 minutes, and the activity of the incubated mutants was measured. The results are shown in Table 5, where the relative enzymatic activity (after incubation at 43°C for 20 minutes) represents the percentage of the mutant's activity versus SEQ ID NO. 38's activity (150–200% is indicated as "++", and 200% or more as "++++").

[0223] Mutations in the mutants Sequence number Relative enzyme activity T2C, N54V, T56N, F58H, M213S 39 +++ T2S, N54V, T56N, F58H, M213S 40 ++ N54V, T56N, F58H, F81Y, M213S 41 ++ N54V, T56N, F58H, A97V, M213S 42 +++ N54V, T56N, F58H, E193T, M213S 43 ++ N54V, T56N, F58H, M213S, S300T 44 +++ N54V, T56N, F58H, M213S, A337S 45 +++ N54V, T56N, F58H, M213S, G342S 46 +++ T2S, N54V, T56N, F58H, F81Y, A97V, E193T, M213S, A337S 47 +++ N54V, T56N, F58H, A97V, E193A, M213S, A337S, G342H 48 +++ T2C, N54V, T56N, F58H, F81Y, A97V, M213S, A337S 49 +++ T2C, N54V, T56N, F58H, F81Y, A97V, E193A, M213S, G342S 50 +++ N54V, T56N, F58H, A97V, E193T, M213S, A337S, G342H 51 +++ N54V, T56N, F58H, F81Y, A97V, E193T, M213S, A337S, G342H 52 +++ N54V, T56N, F58H, A97V, E193T, M213S, S300T, A337S, G342H 53 +++ N54V, T56N, F58H, F81Y, A97V, E193T, M213S, S300T, A337S, G342H 54 +++

[0224] Additional amino acid substitutions were introduced based on SEQ ID NO. 54, and the enzymatic activity of the resulting mutants was measured. The resulting mutants and their enzymatic activities are shown in Table 6, where relative enzymatic activity (without heat treatment) represents the percentage of mutant activity versus the activity of SEQ ID NO. 4 (indicated as "++" for 120–150%, "+++" for 150–200%, and "++++" for 200% or more).

[0225] Mutations in the mutants Sequence number Relative enzyme activity N54V, T56N, F58H, A97V, E193T, R210A, M213S, S300T, A337S, G342H 55 ++++ N54V, T56N, F58H, A97V, E193T, M213S, P221R, S300T, A337S, G342H 56 ++++ N54V, T56N, F58H, A97V, E193T, R210A, M213S, P221R, S300T, A337S, G342H 57 ++++

[0226] Example 4. Rhodotorula taiwanensis Preparation and detection of DAAO (RtnDAAO) mutations

[0227] Mutants were prepared using the nucleic acid encoding RtnDAAO (SEQ No. 3) as a template according to the method of Example 1, and their enzymatic activity was measured. The generated mutants and their enzymatic activities are shown in Table 7, where relative enzymatic activity represents the percentage of the mutant's activity versus the activity of SEQ No. 4 (indicated as "--" for 70% or less, "-" for 70–100%, "+" for 110–120%, and "++" for 120–150%), and the activity of the wild-type RtnDAAO (SEQ No. 3) is 0.

[0228] Mutations in the mutants Sequence number Relative enzyme activity F58K, M213T 58 -- N54L, T56N, F58Q, M213S 59 -- N54T, T56N, F58Q, M213S 60 -- N54I, T56N, F58H, M213S 61 - N54V, T56N, F58H, M213S 62 + N54V, T56N, F58H, R210A, M213S 63 ++ N54V, T56N, F58H, M213S, P221R 64 ++ N54V, T56N, F58H, R210A, M213S, P221R 65 ++

[0229] Example 5. Preparation of mutants with various substitution combinations based on different wild types

[0230] Mutants were prepared by introducing various combinations of mutations into wild-type RtDAAO and RsDAAO according to the method described in the above examples, and enzyme activity was measured. The RtDAAO-based mutants and their enzyme activities are shown in Table 8, where relative enzyme activity represents the percentage of the activity of the mutant versus the activity of SEQ ID NO. 4 (indicated as "+" for 110–120%, "++" for 120–150%, "+++" for 150–200%, and "++++" for 200% or more).

[0231] Mutations in the mutants Sequence number Relative enzyme activity N54V, F58Q, M213S 4 N54I, T56N, F58H, M213S 66 ++++ N54V, T56N, F58H, M213S 67 ++++ N54V, F58Q, R210A, M213S 68 + N54V, F58Q, M213S, P221R 69 ++++ N54V, F58Q, R210A, M213S, P221R 70 ++++ N54V, T56N, F58H, R210A, M213S 71 ++++ N54V, T56N, F58H, M213S, P221R 72 ++++ N54V, T56N, F58H, R210A, M213S, P221R 73 +++ N54I, T56N, F58H, R210A, M213S 74 ++++ N54I, T56N, F58H, M213S, P221R 75 ++ N54I, T56N, F58H, R210A, M213S, P221R 76 ++++

[0232] RsDAAO-based mutants and their enzymatic activities are shown in Table 9, where relative enzymatic activity represents the percentage of the activity of the mutant versus the activity of SEQ ID NO. 77 (indicated as "+" for 110–120%, "++" for 120–150%, "+++" for 150–200%, and "++++" for 200% or more).

[0233] Mutations in the mutants Sequence number Relative enzyme activity N54V, F58Q, M213S 77 N54I, T56N, F58H, M213S 37 ++++ N54V, T56N, F58H, M213S 38 ++++ N54V, F58Q, R210A, M213S 78 + N54V, F58Q, M213S, P221R 79 +++ N54V, F58Q, R210A, M213S, P221R 80 ++++ N54V, T56N, F58H, R210A, M213S 81 ++++ N54V, T56N, F58H, M213S, P221R 82 ++++ N54V, T56N, F58H, R210A, M213S, P221R 83 +++ N54I, T56N, F58H, R210A, M213S 84 ++++ N54I, T56N, F58H, M213S, P221R 85 +++ N54I, T56N, F58H, R210A, M213S, P221R 86 ++

Claims

Claim 1 A modified D-amino acid oxidase (DAAO) comprising amino acid substitutions at positions 54, 56, 58 and 213 compared to wild-type DAAO, wherein the positions are numbered with reference to SEQ ID NO. 2, wherein the amino acid at position 54 is substituted with I, V, T or L, the amino acid at position 56 is substituted with N, the amino acid at position 58 is substituted with H or Q, and the amino acid at position 213 is substituted with S or T, and the modified DAAO is a modified D-amino acid oxidase (DAAO) having activity for catalyzing the oxidation of D-glufosinate to PPO. Claim 2 A modified DAAO according to claim 1, characterized in that the amino acid at position 54 is substituted with I or V, the amino acid at position 58 is substituted with H, and the amino acid at position 213 is substituted with S. Claim 3 The modified DAAO of claim 1, wherein the modified DAAO further comprises amino acid substitution(s) at positions 210, 221, or both, wherein the amino acid at position 210 is substituted with A, G, or P, and the amino acid at position 221 is substituted with R. Claim 4 In paragraph 3, a modified DAAO characterized in that the amino acid at the 210 position is substituted with A. Claim 5 A modified DAAO comprising amino acid substitutions at positions 54, 58, 213 and 221 compared to a wild-type DAAO, wherein the positions are numbered with reference to SEQ ID NO. 2, wherein the amino acid at position 54 is substituted with V, the amino acid at position 58 is substituted with Q, the amino acid at position 213 is substituted with S, and the amino acid at position 221 is substituted with R, and the modified DAAO has activity for catalyzing the oxidation of D-glufosinate to PPO. Claim 6 In claim 5, the modified DAAO further comprises amino acid substitution(s) at positions 210, 56, or both, wherein the amino acid at position 56 is substituted with N and the amino acid at position 210 is substituted with A. Claim 7 In any one of claims 1 to 6, the modified DAAO further comprises amino acid substitutions at one or more positions selected from the group consisting of 2, 81, 97, 193, 194, 237, 265, 273, 274, 300, 317, 319, 337, and 342, wherein at position 2, the amino acid is substituted with C or S; at position 81, the amino acid is substituted with Y; at position 97, the amino acid is substituted with V; at position 193, the amino acid is substituted with T; at position 194, the amino acid is substituted with V or C; at position 237, the amino acid is substituted with V; at position 265, the amino acid is substituted with C; at position 273, the amino acid is substituted with D; at position 274, the amino acid is substituted with E; at position 300, the amino acid is substituted with S or T; at position 317, the amino acid is substituted with Y or W; and at position 319, the amino acid is K. A modified DAAO characterized by being substituted, wherein the amino acid at position 337 is substituted with S and the amino acid at position 342 is substituted with S or H. Claim 8 A modified DAAO according to any one of claims 1 to 6, wherein the modified DAAO further comprises amino acid substitutions at one or more positions selected from the group consisting of 194, 237, 265, 273, 274, 300, 317, and 319, wherein the amino acid at position 194 is substituted with V or C, the amino acid at position 237 is substituted with V, the amino acid at position 265 is substituted with C, the amino acid at position 273 is substituted with D, the amino acid at position 274 is substituted with E, the amino acid at position 300 is substituted with S, the amino acid at position 317 is substituted with Y or W, and the amino acid at position 319 is substituted with K. Claim 9 A modified DAAO according to any one of claims 1 to 6, wherein the modified DAAO further comprises amino acid substitutions at one or more positions selected from the group consisting of 2, 81, 97, 193, 300, 337, and 342, wherein the amino acid at position 2 is substituted with C or S, the amino acid at position 81 is substituted with Y, the amino acid at position 97 is substituted with V, the amino acid at position 193 is substituted with T, the amino acid at position 300 is substituted with T, the amino acid at position 337 is substituted with S, and the amino acid at position 342 is substituted with S or H. Claim 10 A modified DAAO comprising amino acid substitutions at positions 54, 58, 194, and 213 compared with SEQ NO. 1, wherein the amino acid at position 54 is substituted with I, V, T, or L, the amino acid at position 58 is substituted with H or Q, the amino acid at position 194 is substituted with V or C, and the amino acid at position 213 is substituted with S or T, and the modified DAAO has activity for catalyzing the oxidation of D-glufosinate to PPO. Claim 11 The modified DAAO according to claim 10 further comprises amino acid substitutions at one or more positions selected from the group consisting of 56, 210, 221, 237, 265, 273, 274, 300, 317, and 319, wherein the amino acid at position 56 is substituted with N, the amino acid at position 210 is substituted with A, G, or P, the amino acid at position 221 is substituted with R, the amino acid at position 237 is substituted with V, the amino acid at position 265 is substituted with C, the amino acid at position 273 is substituted with D, the amino acid at position 274 is substituted with E, the amino acid at position 300 is substituted with S, the amino acid at position 317 is substituted with Y or W, and the amino acid at position 319 is substituted with K. Claim 12 A modified DAAO comprising or composed of the following: an amino acid sequence of one of SEQ ID NOs 5-86, or 1-10 amino acid substitutions at positions other than 54, 56, 58, 194, 210, 213, 221, 237, 265, 273, 274, 300, 317, and 319 compared to one of SEQ ID NOs 5-30 and 66-76, 1-10 amino acid substitutions at positions other than 2, 54, 56, 58, 81, 97, 193, 210, 213, 221, 300, 337, and 342 compared to one of SEQ ID NOs 31-57 and 77-86, or other than 54, 56, 58, 210, 213, and 221 compared to one of SEQ ID NOs 58-65 A modified DAAO comprising or composed of an amino acid sequence including 1 to 10 amino acid substitutions at a position, wherein the modified DAAO has activity for catalyzing the oxidation of D-glufosinate to PPO. Claim 13 A polynucleotide encoding a modified DAAO of any one of claims 1 to 6 and claims 10 to 12. Claim 14 An expression vector comprising the polynucleotide of claim 13. Claim 15 A host cell comprising a modified DAAO according to any one of claims 1 to 6 and claims 10 to 12. Claim 16 A method for producing L-glufosinate comprising the step of contacting a modified DAAO of any one of claims 1 to 6 and claims 10 to 12 with D-glufosinate. Claim 17 A method for producing L-glufosinate comprising the step of contacting the host cell of claim 15 with D-glufosinate.