WO2021115256A1 - 经修饰的daao酶及其应用 - Google Patents

经修饰的daao酶及其应用 Download PDF

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Publication number
WO2021115256A1
WO2021115256A1 PCT/CN2020/134540 CN2020134540W WO2021115256A1 WO 2021115256 A1 WO2021115256 A1 WO 2021115256A1 CN 2020134540 W CN2020134540 W CN 2020134540W WO 2021115256 A1 WO2021115256 A1 WO 2021115256A1
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substituted
daao
modified
positions
amino acid
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PCT/CN2020/134540
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English (en)
French (fr)
Inventor
谢新开
徐伟
范俊英
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四川利尔生物科技有限公司
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Priority to MX2022006534A priority Critical patent/MX2022006534A/es
Application filed by 四川利尔生物科技有限公司 filed Critical 四川利尔生物科技有限公司
Priority to CN202080032605.2A priority patent/CN113825841B/zh
Priority to AU2020401155A priority patent/AU2020401155A1/en
Priority to JP2022534287A priority patent/JP2023504869A/ja
Priority to BR112022010266A priority patent/BR112022010266A2/pt
Priority to EP20898200.9A priority patent/EP4074837A4/en
Priority to IL293679A priority patent/IL293679A/en
Priority to CA3160673A priority patent/CA3160673A1/en
Priority to KR1020227023206A priority patent/KR20220136350A/ko
Priority to CN202210880427.XA priority patent/CN116286699A/zh
Publication of WO2021115256A1 publication Critical patent/WO2021115256A1/zh
Priority to US17/836,773 priority patent/US11667896B2/en
Priority to US18/137,430 priority patent/US20230416696A1/en

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
    • C12N9/0024—D-Amino acid oxidase (1.4.3.3)
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/002—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by oxidation/reduction reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity
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    • C12N2330/00—Production
    • C12N2330/50—Biochemical production, i.e. in a transformed host cell
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    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/03—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
    • C12Y104/03003—D-Amino-acid oxidase (1.4.3.3)

Definitions

  • the invention relates to the field of enzyme engineering. Specifically, the present invention relates to a modified D-amino acid oxidase (DAAO) and its application in the production of glufosinate-ammonium.
  • DAAO D-amino acid oxidase
  • Glufosinate (glufosinate, also known as 4-[hydroxy(methyl)phosphono]-D,L-homoalanine) is the world's second-selling transgenic crop tolerant herbicide.
  • Glufosinate-ammonium is a broad-spectrum contact-killing herbicide that inhibits the activity of L-glutamine synthetase in plants, causing disturbances in nitrogen metabolism in plants and ultimately killing plants.
  • glyphosate glufosinate has significant advantages, such as wide application range, quick effect, long lasting period, lower toxicity, and safety. Therefore, the sales of glufosinate-ammonium are growing rapidly, and the market demand will be huge in the future, and the prospects are very broad.
  • glufosinate-ammonium is a racemic mixture (D,L- glufosinate-ammonium) containing equal amounts of two optical isomers, but only the L-configuration has physiological activity. Therefore, the preparation of chiral pure L- glufosinate-ammonium by deracemization of D,L- glufosinate-ammonium has important practical significance and has become a popular direction for the synthesis of L- glufosinate-ammonium in recent years.
  • D-glufosinate-ammonium 2-carbonyl-4-(hydroxymethylphosphono) butyric acid (PPO for short), and then reduce or transaminate the PPO to produce L- glufosinate-ammonium.
  • D-amino acid oxidase DAAO
  • CAT catalase
  • PPO can be reduced by formic acid under the catalysis of palladium-carbon to produce D,L-glufosinate-ammonium.
  • D,L-glufosinate-ammonium is gradually converted to L-glufosinate-ammonium (see, for example, CN105567780A).
  • the disadvantage of this scheme is that the amount of palladium-carbon catalyst is large and the reaction raw materials (such as oxygen and ammonium formate) are wasted.
  • L-TA L-amino acid transaminase
  • L-TA L-amino acid transaminase
  • the disadvantage of this scheme is that the transamination step is a balanced reaction, and excess amino donors (amino acids or organic amines) need to be provided to achieve a high conversion rate (for example, to provide 3 times the equivalent of amino donors, the conversion rate is 90%), and the excess The amino donor and the corresponding by-products will seriously affect the subsequent separation and purification steps.
  • PPO can be converted into L-glufosinate-ammonium by a stereoselective reduction reaction catalyzed by L-amino acid dehydrogenase (L-AADH) (see, for example, CN107502647A, CN109576236A and CN109609582A).
  • L-AADH L-amino acid dehydrogenase
  • the scheme of using D-amino acid oxidase and L-amino acid dehydrogenase has potential cost advantages.
  • the concentration of the substrate that can be transformed is generally not high, or the loss is too large, resulting in excessive production costs.
  • the realization of high concentration of D,L-glufosinate-ammonium deracemization has become the bottleneck of the current process.
  • the main factors hindering the conversion of higher concentrations of substrates may be: the recombinant DAAO enzyme has poor stability and is unstable under the conditions of the reactor, and the enzyme is inactivated during the reaction.
  • Another limiting factor is the selective catalytic activity of the enzyme on D-glufosinate-ammonium.
  • some modifications have been made to DAAO to endow DAAO with activity on D-glufosinate-ammonium:
  • Rhodotorula toruloides also known as Rhodotorula gracilis, see https://www.atcc.org/products/all/10788.aspx
  • Rhodotorula gracilis also known as Rhodotorula gracilis, see https://www.atcc.org/products/all/10788.aspx
  • the DAAO mutant of Rhodosporidium parvum mentioned in the patent contains the F58K mutation, which is substituted with H, S, T, C, Q, G, N, and A mutations at position M213, and mutations at positions 223 and 238 .
  • CN109576236A based on DAAO from Rhodosporidium vulgaris, a mutant capable of oxidizing D-glufosinate was also constructed.
  • the mutant has single or multiple mutations at amino acid positions 52, 54, 58, 58, 213, and 335.
  • DAAO enzymes are also involved to oxidize D-glufosinate-ammonium, but the sequence of the enzymes used is not indicated.
  • the present invention provides a modified D-amino acid oxidase (DAAO), which contains 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, compared with its wild-type DAAO Amino acid substitutions at 14, 15, 16, 17, 18, 19, 20 or more positions, wherein the modified DAAO has the activity of catalyzing the oxidation of D- glufosinate to PPO.
  • DAAO D-amino acid oxidase
  • the modified DAAO includes amino acid substitutions at positions 54, 56, 58, and 213, and the positions are numbered with reference to SEQ ID NO: 2.
  • position 54 is substituted with I, V, T or L, more preferably I or V.
  • position 56 is substituted with N.
  • position 58 is substituted with H or Q, more preferably H.
  • position 213 is substituted with S or T, more preferably S.
  • the modified DAAO further comprises amino acid substitutions at positions 210 and/or 221.
  • position 210 is substituted with A, G or P, more preferably A.
  • position P221 is substituted with R.
  • the modified DAAO includes amino acid substitutions at positions 54, 58, 213, and 221, and the positions are numbered with reference to SEQ ID NO: 2.
  • position 54 is substituted with V
  • position 58 is substituted with Q
  • position 213 is substituted with S
  • position 221 is substituted with R.
  • the modified DAAO further comprises amino acid substitutions at positions 210 and/or 56.
  • position 56 is substituted with N and position 210 is substituted with A.
  • the modified DAAO of the present invention further comprises one or more positions selected from 2, 81, 97, 193, 194, 237, 265, 273, 274, 300, 317, 319, 337, and 342 Replacement.
  • position 2 is substituted with C or S
  • position 81 is substituted with Y
  • position 97 is substituted with V
  • position 193 is substituted with T
  • position 194 is substituted with V or C
  • position 237 is substituted with V
  • position 265 is substituted with C
  • position Replace 273 with D, position 274 with E position 300 with S or T
  • position 317 with Y or W position 319 with K
  • position 337 with S and position 342 with S or H.
  • the modified DAAO of the present invention further comprises substitutions at one or more positions selected from 194, 237, 265, 273, 274, 300, 317, and 319.
  • position 194 is substituted with V or C
  • position 237 is substituted with V
  • position 265 is substituted with C
  • position 273 is substituted with D
  • position 274 is substituted with E
  • position 300 is substituted with S
  • position 317 is substituted with Y or W
  • position 319 was replaced by K.
  • the modified DAAO of the present invention further comprises one or more substitutions selected from positions 2, 81, 97, 193, 300, 337, and 342, wherein the substitution at position 2 is C or S , Position 81 is substituted with Y, position 97 is substituted with V, position 193 is substituted with T, position 300 is substituted with T, position 337 is substituted with S, and position 342 is substituted with S.
  • the modified DAAO comprises amino acid substitutions at positions 54, 58, 194, and 213 compared to SEQ ID NO:1, wherein the modified DAAO has a catalytic D-glufosinate Oxidation is the activity of PPO.
  • position 54 is substituted with I, V, T or L
  • position 58 is substituted with H or Q
  • position 194 is substituted with V or C
  • position 213 is substituted with S or T.
  • the modified DAAO further comprises one or more amino acid substitutions selected from positions 56, 210, 221, 237, 265, 273, 274, 300, 317, and 319.
  • position 56 is substituted with N
  • position 210 is substituted with A
  • G or P position P221 is substituted with R
  • position 237 is substituted with V
  • position 265 is substituted with C
  • position 273 is substituted with D
  • position 274 is substituted with E
  • position 300 is replaced by S
  • position 317 is replaced by Y or W
  • position 319 is replaced by K.
  • the modified DAAO includes the amino acid sequence of one of SEQ ID NO: 5-86 or consists of the amino acid sequence of one of SEQ ID NO: 5-86, or the modified DAAO is combined with SEQ ID NO: Compared with one of 5-30 and 66-76, at positions other than positions 54, 56, 58, 194, 210, 213, 221, 237, 265, 273, 274, 300, 317, and 319 Contains 1-10 amino acid substitutions, compared with one of SEQ ID NO: 31-57 and 77-86, except for positions 2, 54, 56, 58, 81, 97, 193, 210, 213, 221, 300, Positions other than 337 and 342 contain 1-10 amino acid substitutions, or, compared with one of SEQ ID NO: 58-65, contain 1- 10 amino acid substitutions, wherein the modified DAAO has the activity of catalyzing the oxidation of D- glufosinate to PPO.
  • the present invention provides a polynucleotide encoding the modified DAAO of the present invention, and a vector containing the polynucleotide of the present invention.
  • the present invention provides a host cell containing the modified DAAO of the present invention, its encoding polynucleotide or a vector containing the polynucleotide.
  • the present invention also provides a method for producing L-glufosinate-ammonium, which comprises contacting the modified DAAO of the present invention or the host cell of the present invention with D-glufosinate-ammonium.
  • the present invention mainly relates to modified DAAO, which is used to catalyze the oxidation of D-glufosinate-ammonium to produce L-glufosinate-ammonium.
  • modified DAAO which is used to catalyze the oxidation of D-glufosinate-ammonium to produce L-glufosinate-ammonium.
  • 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).
  • DAAO oxidation of D-amino acids to produce keto acids (EC 1.4.3.3).
  • the naturally occurring DAAO cannot catalyze the oxidation of D- glufosinate. Therefore, the present invention provides modified DAAO polypeptides that are capable of catalyzing the oxidation of D- glufosinate to PPO.
  • the modified DAAO polypeptide has increased stability and/or increased activity of catalyzing the oxidation of D- glufosinate to PPO.
  • peptide means a chain of at least two amino acids connected by peptide bonds.
  • polypeptide is used interchangeably with the term “protein” herein, and refers to a chain containing ten or more amino acid residues. All peptide and polypeptide chemical formulas or sequences herein are written from left to right, indicating the direction from the amino terminal to the carboxy terminal.
  • amino acid includes naturally occurring amino acids and unnatural amino acids in proteins.
  • the one-letter and three-letter names of amino acids naturally occurring in proteins are commonly used in the field, and can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • modification refers to any chemical modification of a polypeptide, such as amino acid substitutions, deletions, insertions, and/or additions.
  • the modified DAAO polypeptide of the present invention comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, compared to its wild-type DAAO. 18, 19, 20 or more amino acid substitutions, wherein the modified DAAO polypeptide has the activity of catalyzing the oxidation of D- glufosinate to PPO.
  • the modified DAAO polypeptide of the present invention includes amino acid substitutions at positions 54, 56, 58, and 213 compared to its wild-type DAAO, and the positions are numbered with reference to SEQ ID NO: 2.
  • position 54 is substituted with I, V, T or L, more preferably I or V.
  • position 56 is substituted with N.
  • position 58 is substituted with H or Q, more preferably H.
  • position 213 is substituted with S or T, more preferably S.
  • the modified DAAO polypeptide comprises a combination of the following substitutions compared to its wild-type DAAO: 54V, 56N, 58H, and 213S, or 54I, 56N, 58H, and 213S.
  • the modified DAAO further comprises amino acid substitutions at positions 210 and/or 221.
  • position 210 is substituted with A, G or P, more preferably A.
  • position P221 is substituted with R.
  • the modified DAAO includes amino acid substitutions at positions 54, 58, 213, and 221, and the positions are numbered with reference to SEQ ID NO: 2.
  • position 54 is substituted with V
  • position 58 is substituted with Q
  • position 213 is substituted with S
  • position 221 is substituted with R.
  • the modified DAAO further comprises amino acid substitutions at positions 210 and/or 56.
  • position 56 is substituted with N and position 210 is substituted with A.
  • the modified DAAO of the present invention further comprises one or more positions selected from 2, 81, 97, 193, 194, 237, 265, 273, 274, 300, 317, 319, 337, and 342 The position is numbered with reference to SEQ ID NO: 2, wherein the modified DAAO polypeptide has the activity of catalyzing the oxidation of D- glufosinate-ammonium to PPO.
  • position 2 is substituted with C or S
  • position 81 is substituted with Y
  • position 97 is substituted with V
  • position 193 is substituted with T
  • position 194 is substituted with V or C
  • position 237 is substituted with V
  • position 265 is substituted with C
  • position Replace 273 with D position 274 with E
  • position 300 with S or T
  • position 317 with Y or W
  • position 319 with K
  • position 337 with S and position 342 with S or H.
  • the modified DAAO of the present invention further includes substitutions at one or more positions selected from 194, 237, 265, 273, 274, 300, 317, and 319, and the positions are numbered with reference to SEQ ID NO: 2, wherein
  • the modified DAAO polypeptide has the activity of catalyzing the oxidation of D- glufosinate to PPO.
  • position 194 is substituted with V or C
  • position 237 is substituted with V
  • position 265 is substituted with C
  • position 273 is substituted with D
  • position 274 is substituted with E
  • position 300 is substituted with S
  • position 317 is substituted with Y or W
  • Position 319 was substituted with K.
  • the modified DAAO of the present invention further comprises one or more substitutions selected from positions 2, 81, 97, 193, 300, 337, and 342, and the positions are numbered with reference to SEQ ID NO: 2, wherein
  • the modified DAAO polypeptide has the activity of catalyzing the oxidation of D-glufosinate to PPO.
  • position 2 is substituted with C or S
  • position 81 is substituted with Y
  • position 97 is substituted with V
  • position 193 is substituted with T
  • position 300 is substituted with T
  • position 337 is substituted with S
  • position 342 is substituted with S.
  • the modified DAAO of the present invention includes amino acid substitutions at positions 54, 58, 194, and 213, wherein the modified DAAO has a catalytic D-glufosinate oxidation Is the activity of PPO.
  • position 54 is substituted with I, V, T or L
  • position 58 is substituted with H or Q
  • position 194 is substituted with V or C
  • position 213 is substituted with S or T.
  • the modified DAAO further comprises one or more amino acid substitutions selected from positions 56, 210, 221, 237, 265, 273, 274, 300, 317, and 319.
  • position 56 is substituted with N
  • position 210 is substituted with A
  • G or P position P221 is substituted with R
  • position 237 is substituted with V
  • position 265 is substituted with C
  • position 273 is substituted with D
  • position 274 is substituted with E
  • position 300 is replaced by S
  • position 317 is replaced by Y or W
  • position 319 is replaced by K.
  • the modified DAAO of the present invention also has conservative substitutions of one or more amino acids, or insertions or deletions of one or more amino acids.
  • the modified DAAO of the present invention has amino acid substitutions selected from the following combinations (positions are numbered with reference to SEQ ID NO: 2):
  • the DAAO polypeptide on which amino acid modifications are made is referred to as the starting DAAO.
  • the starting DAAO can be wild-type DAAO or a variant of wild-type DAAO.
  • the polypeptide of SEQ ID NO:1 is the "starting DAAO"; and if the variant polypeptide of SEQ ID NO:1 (for example SEQ ID NO: 4-30) starts to be modified, and compared to the modified DAAO, the variant polypeptide is the "starting DAAO".
  • wild-type DAAO refers to naturally occurring DAAO.
  • the wild-type DAAO is DAAO from Rhodotorula.
  • the wild-type DAAO is one of SEQ ID NO: 1-3.
  • SEQ ID NO: 1 is the amino acid sequence of DAAO from Rhodotorula sp.
  • JG-1b (GenBank accession number: CAJ87425.1), and SEQ ID NO: 2 is the amino acid sequence of DAAO from Rhodotorula sp.JG-1b (GenBank accession number: KWU45700.1), and SEQ ID NO: 3 is the putative DAAO amino acid sequence from Rhodotorula taiwanensis (GenBank accession number: POY70719.1).
  • the sequences are aligned for the purpose of optimal comparison (for example, gaps can be introduced in the first amino acid or nucleic acid sequence to match the second amino acid sequence). Or nucleic acid sequence for optimal alignment).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide in the corresponding position in the second sequence, then these molecules are the same at this position.
  • percent identity number of identical positions/total number of positions (i.e. overlapping positions) ⁇ 100.
  • the two sequences are the same length.
  • Percent amino acid identity or “percent amino acid sequence identity” refers to comparing the amino acids of two polypeptides, and when optimally aligned, the two polypeptides have approximately the specified percentage of identical amino acids. For example, “95% amino acid identity” refers to comparing the amino acids of two polypeptides. When the two polypeptides are optimally aligned, 95% of the amino acids of the two polypeptides are identical.
  • the wild-type DAAO and one of SEQ ID NO: 1-3 have at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, 90% or 95%, particularly Preferably at least 96%, 97%, 98% or 99% sequence identity.
  • the modified DAAO polypeptide of the present invention comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, compared to its wild-type DAAO. 18, 19, 20, or more amino acid substitutions, wherein the modified DAAO polypeptide has the activity of catalyzing the oxidation of D- glufosinate to PPO.
  • the modified DAAO polypeptide of the present invention includes amino acid substitutions at positions 54, 56, 58, and 213 compared to its wild-type DAAO, and the positions are numbered with reference to SEQ ID NO: 2.
  • position 54 is substituted with I, V, T or L, more preferably I or V.
  • position 56 is substituted with N.
  • position 58 is substituted with H or Q, more preferably H.
  • position 213 is substituted with S or T, more preferably S.
  • the modified DAAO polypeptide comprises the following substitutions compared to its wild-type DAAO: 54V, 56N, 58H, and 213S, or 54I, 56N, 58H, and 213S.
  • the modified DAAO further comprises amino acid substitutions at positions 210 and/or 221.
  • position 210 is substituted with A, G or P, more preferably A.
  • position P221 is substituted with R.
  • the modified DAAO of the present invention further comprises one or more substitutions selected from 194, 237, 265, 273, 274, 300, 317 and 319, wherein the substitution at position 194 is V or C, and the substitution at position 237 Is V, position 265 is replaced with C, position 273 is replaced with D, position 274 is replaced with E, position 300 is replaced with S, position 317 is replaced with Y or W, and position 319 is replaced with K.
  • the wild-type DAAO and SEQ ID NO:1 have at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, 90% or 95%, particularly preferably at least 96%, 97% , 98% or 99% sequence identity.
  • the modified DAAO includes amino acid substitutions at positions 54, 58, 213, and 221, and the positions are numbered with reference to SEQ ID NO: 2.
  • position 54 is substituted with V
  • position 58 is substituted with Q
  • position 213 is substituted with S
  • position 221 is substituted with R.
  • the modified DAAO further comprises amino acid substitutions at positions 210 and/or 56.
  • position 56 is substituted with N and position 210 is substituted with A.
  • the modified DAAO of the present invention further comprises one or more substitutions selected from 194, 237, 265, 273, 274, 300, 317 and 319, wherein the substitution at position 194 is V or C, and the substitution at position 237 Is V, position 265 is replaced with C, position 273 is replaced with D, position 274 is replaced with E, position 300 is replaced with S, position 317 is replaced with Y or W, and position 319 is replaced with K.
  • the wild-type DAAO and SEQ ID NO:1 have at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, 90% or 95%, particularly preferably at least 96%, 97% , 98% or 99% sequence identity.
  • the modified DAAO polypeptide of the present invention comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, compared to its wild-type DAAO. 18, 19, 20, or more amino acid substitutions, wherein the modified DAAO polypeptide has the activity of catalyzing the oxidation of D- glufosinate to PPO.
  • the modified DAAO polypeptide of the present invention includes amino acid substitutions at positions 54, 56, 58, and 213 compared to its wild-type DAAO, and the positions are numbered with reference to SEQ ID NO: 2.
  • position 54 is substituted with I, V, T or L, more preferably I or V.
  • position 56 is substituted with N.
  • position 58 is substituted with H or Q, more preferably H.
  • position 213 is substituted with S or T, more preferably S.
  • the modified DAAO polypeptide comprises the following substitutions compared to its wild-type DAAO: 54V, 56N, 58H, and 213S, or 54I, 56N, 58H, and 213S.
  • the modified DAAO further comprises amino acid substitutions at positions 210 and/or 221.
  • position 210 is substituted with A, G or P, more preferably A.
  • position P221 is substituted with R.
  • the modified DAAO of the present invention further comprises one or more substitutions selected from positions 2, 81, 97, 193, 300, 337 and 342, wherein the substitution at position 2 is C or S, and the substitution at position 81 is Y, position 97 is substituted with V, position 193 is substituted with T, position 300 is substituted with T, position 337 is substituted with S, and position 342 is substituted with S.
  • the wild-type DAAO and SEQ ID NO: 2 have at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, 90% or 95%, particularly preferably at least 96%, 97% , 98% or 99% sequence identity.
  • the modified DAAO includes amino acid substitutions at positions 54, 58, 213, and 221, and the positions are numbered with reference to SEQ ID NO: 2.
  • position 54 is substituted with V
  • position 58 is substituted with Q
  • position 213 is substituted with S
  • position 221 is substituted with R.
  • the modified DAAO further comprises amino acid substitutions at positions 210 and/or 56.
  • position 56 is substituted with N and position 210 is substituted with A.
  • the modified DAAO of the present invention further comprises one or more substitutions selected from positions 2, 81, 97, 193, 300, 337 and 342, wherein the substitution at position 2 is C or S, and the substitution at position 81 is Y, position 97 is substituted with V, position 193 is substituted with T, position 300 is substituted with T, position 337 is substituted with S, and position 342 is substituted with S.
  • the wild-type DAAO and SEQ ID NO: 2 have at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, 90% or 95%, particularly preferably at least 96%, 97% , 98% or 99% sequence identity.
  • the modified DAAO comprises 4-20, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9 compared to wild-type , 4-8, 4-7, 4-6 or 4-5 amino acid substitutions. In some embodiments, the modified DAAO comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 compared to the wild type. , 20 or more amino acid substitutions.
  • the wild-type DAAO differs from one of SEQ ID NO: 1-3 in that it has one or more amino acid substitutions, deletions, insertions and/or additions. In some embodiments, the wild-type DAAO has conservative substitutions of one or more amino acids compared with one of SEQ ID NO: 1-3. In some embodiments, the wild-type DAAO has one or more amino acid insertions or deletions compared with one of SEQ ID NO: 1-3.
  • the modified DAAO polypeptide of the present invention comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, compared to its wild-type DAAO. 18, 19, 20, or more amino acid substitutions, wherein the modified DAAO polypeptide has the activity of catalyzing the oxidation of D- glufosinate to PPO.
  • the modified DAAO polypeptide of the present invention includes amino acid substitutions at positions 54, 56, 58, and 213 compared to its wild-type DAAO, and the positions are numbered with reference to SEQ ID NO: 2, wherein the The modified DAAO has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity with one of SEQ ID NO: 1-3.
  • position 54 is substituted with I, V, T or L, more preferably I or V.
  • position 56 is substituted with N.
  • position 58 is substituted with H or Q, more preferably H.
  • position 213 is substituted with S or T, more preferably S.
  • the modified DAAO polypeptide comprises a combination of the following substitutions compared to its wild-type DAAO: 54V, 56N, 58H, and 213S, or 54I, 56N, 58H, and 213S.
  • the modified DAAO further comprises amino acid substitutions at positions 210 and/or 221.
  • position 210 is substituted with A, G or P, more preferably A.
  • position P221 is substituted with R.
  • the modified DAAO comprises amino acid substitutions at positions 54, 58, 213, and 221, and the positions are numbered with reference to SEQ ID NO: 2, wherein the modified DAAO is identical to SEQ ID NO: 1
  • One of -3 has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity.
  • position 54 is substituted with V
  • position 58 is substituted with Q
  • position 213 is substituted with S
  • position 221 is substituted with R.
  • the modified DAAO further comprises amino acid substitutions at positions 210 and/or 56.
  • position 56 is substituted with N and position 210 is substituted with A.
  • the modified DAAO of the present invention further comprises one or more positions selected from 2, 81, 97, 193, 194, 237, 265, 273, 274, 300, 317, 319, 337, and 342 The position is numbered with reference to SEQ ID NO: 2, wherein the modified DAAO polypeptide has the activity of catalyzing the oxidation of D- glufosinate-ammonium to PPO.
  • position 2 is substituted with C or S
  • position 81 is substituted with Y
  • position 97 is substituted with V
  • position 193 is substituted with T
  • position 194 is substituted with V or C
  • position 237 is substituted with V
  • position 265 is substituted with C
  • position Replace 273 with D position 274 with E
  • position 300 with S or T
  • position 317 with Y or W
  • position 319 with K
  • position 337 with S and position 342 with S or H.
  • the modified DAAO of the present invention further includes substitutions at one or more positions selected from 194, 237, 265, 273, 274, 300, 317, and 319, and the positions are numbered with reference to SEQ ID NO: 2, wherein
  • the modified DAAO polypeptide has the activity of catalyzing the oxidation of D- glufosinate to PPO.
  • position 194 is substituted with V or C
  • position 237 is substituted with V
  • position 265 is substituted with C
  • position 273 is substituted with D
  • position 274 is substituted with E
  • position 300 is substituted with S
  • position 317 is substituted with Y or W
  • Position 319 was substituted with K.
  • the modified DAAO of the present invention further comprises one or more substitutions selected from positions 2, 81, 97, 193, 300, 337, and 342, and the positions are numbered with reference to SEQ ID NO: 2, wherein
  • the modified DAAO polypeptide has the activity of catalyzing the oxidation of D-glufosinate to PPO.
  • position 2 is substituted with C or S
  • position 81 is substituted with Y
  • position 97 is substituted with V
  • position 193 is substituted with T
  • position 300 is substituted with T
  • position 337 is substituted with S
  • position 342 is substituted with S.
  • the modified DAAO of the present invention includes amino acid substitutions at positions 54, 58, 194, and 213, wherein the modified DAAO has a catalytic D-glufosinate oxidation Is the activity of PPO, wherein the modified DAAO has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% or 98% sequence identity with SEQ ID NO:1 .
  • position 54 is substituted with I, V, T or L
  • position 58 is substituted with H or Q
  • position 194 is substituted with V or C
  • position 213 is substituted with S or T.
  • the modified DAAO further comprises one or more amino acid substitutions selected from positions 56, 210, 221, 237, 265, 273, 274, 300, 317, and 319.
  • position 56 is substituted with N
  • position 210 is substituted with A
  • G or P position P221 is substituted with R
  • position 237 is substituted with V
  • position 265 is substituted with C
  • position 273 is substituted with D
  • position 274 is substituted with E
  • position 300 is replaced by S
  • position 317 is replaced by Y or W
  • position 319 is replaced by K.
  • the modified DAAO of the present invention further has conservative substitutions of one or more amino acids, or insertions or deletions of one or more amino acids.
  • amino acid residue substitution refers to a substitution in which the amino acid residue is replaced by an amino acid residue having a similar side chain, for example, an amino acid with a basic side chain (e.g., lysine) , Arginine and histidine), acidic side chain amino acids (e.g. aspartic acid, glutamic acid), non-charged electroactive side chain amino acids (e.g. glycine, asparagine, glutamine, serine, threonine) Acid, tyrosine, cysteine), non-polar side chain amino acids (e.g.
  • a basic side chain e.g., lysine
  • acidic side chain amino acids e.g. aspartic acid, glutamic acid
  • non-charged electroactive side chain amino acids e.g. glycine, asparagine, glutamine, serine, threonine
  • Acid tyrosine, cysteine
  • non-polar side chain amino acids
  • ⁇ -branched side chain amino acids e.g. threonine, valine, isoleucine
  • aromatic side chain amino acids e.g. tyrosine, phenylalanine, tryptophan, histidine
  • Conservative amino acid substitutions generally have the least impact on the activity of the resulting protein. This substitution is described below. Conservative substitution is to replace an amino acid with an amino acid that is similar in size, hydrophobicity, charge, polarity, spatial characteristics, and aromaticity. When it is desired to fine-tune the properties of the protein, such substitutions are usually conservative.
  • homologous amino acid residues refer to amino acid residues with similar chemical properties related to hydrophobicity, charge, polarity, steric characteristics, aromatic characteristics, and the like.
  • amino acids that are homologous to each other include positively charged lysine, arginine, histidine, negatively charged glutamic acid, aspartic acid, hydrophobic glycine, alanine, valine, and leucine Acid, isoleucine, proline, phenylalanine, polar serine, threonine, cysteine, methionine, tryptophan, tyrosine, asparagine, glutamine , Aromatic phenylalanine, tyrosine, tryptophan, serine and threonine with chemically similar side chain groups, or glutamine and asparagine, or leucine and isoleucine.
  • Examples of conservative amino acid substitutions in proteins include: Ser replaces Ala, Lys replaces Arg, Gln or His replaces Asn, Glu replaces Asp, Ser replaces Cys, Asn replaces Gln, Asp replaces Glu, Pro replaces Gly, Asn or Gln replaces His, Leu Or Val replaces Ile, Ile or Val replaces Leu, Arg or Gln replaces Lys, Leu or Ile replaces Met, Met, Leu or Tyr replaces Phe, Thr replaces Ser, Ser replaces Thr, Tyr replaces Trp, Trp or Phe replaces Tyr, and Ile or Leu replaces Val.
  • the modified DAAO includes the amino acid sequence of one of SEQ ID NO: 5-86 or consists of the amino acid sequence of one of SEQ ID NO: 5-86, or the modified DAAO is combined with SEQ ID NO: Compared with one of 5-30 and 66-76, at positions other than positions 54, 56, 58, 194, 210, 213, 221, 237, 265, 273, 274, 300, 317, and 319 Contains 1-10 amino acid substitutions, compared with one of SEQ ID NO: 31-57 and 77-86, except for positions 2, 54, 56, 58, 81, 97, 193, 210, 213, 221, 300, Positions other than 337 and 342 contain 1-10 amino acid substitutions, or, compared with one of SEQ ID NO: 58-65, contain 1- 10 amino acid substitutions, wherein the modified DAAO has the activity of catalyzing the oxidation of D- glufosinate to PPO.
  • the modified DAAO is compared with one of SEQ ID NOs: 5-30 and 66-76, except for positions 54, 56, 58, 194, 210, 213, 221, 237, 265, Positions other than 273, 274, 300, 317, and 319 contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions; and SEQ ID NO: 31-57 and 77 Compared with one of -86, the positions other than positions 2, 54, 56, 58, 81, 97, 193, 210, 213, 221, 300, 337, and 342 include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions; or, compared with one of SEQ ID NO: 58-65, contains 1 at positions other than positions 54, 56, 58, 210, 213, and 221 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions. In some embodiments, the modified DAAO and one of SEQ ID NO: 1-3 have at least 70%, 75%, 80%, 85%, 90%
  • the activity of an enzyme refers to a decrease in substrate or an increase in product per unit time in a chemical reaction catalyzed by an enzyme per unit mass under certain conditions.
  • the activity of the modified DAAO of the present invention can be expressed by the amount of decrease in D-glufosinate-ammonium or the increase in PPO per unit time under certain conditions under the catalysis of a unit mass of modified DAAO.
  • the activity of an enzyme can also refer to 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 that catalyzes the same reaction, such as percentage relative activity.
  • the activity of the modified DAAO of the present invention is expressed as a percentage relative activity compared to SEQ ID NO:4.
  • the activity of the modified DAAO to catalyze the oxidation of D- glufosinate to PPO is at least 100%, 105%, 110% of the activity of SEQ ID NO: 4 to catalyze the oxidation of D- glufosinate to PPO. %, 120%, 130%, 150%, 170%, 200%, 250%, 300% or higher.
  • the stability is thermostability, which refers to the ability of an enzyme to maintain activity after incubating at a certain temperature (such as 40-60° C. or higher) for a certain period of time (such as 10 minutes to 1 hour).
  • the modified DAAO has better thermal stability than the polypeptide of SEQ ID NO: 4.
  • the activity of the modified DAAO of the present invention is at least 100%, 105%, 110%, 120%, 130%, 150% of the activity of the polypeptide of SEQ ID NO: 4 %, 170%, 200%, 250%, 300% or higher.
  • the modified DAAO of the present invention has a higher T50, where T50 refers to the temperature at which the enzyme activity decreases by 50% after incubation at this temperature for one hour.
  • T50 refers to the temperature at which the enzyme activity decreases by 50% after incubation at this temperature for one hour.
  • the T50 of the modified DAAO of the present invention is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10°C or higher than the polypeptide of SEQ ID NO: 4.
  • the modified DAAO has better thermal stability than the polypeptide of SEQ ID NO: 4, and its activity of catalyzing the oxidation of D- glufosinate-ammonium to PPO is the catalytic D- of SEQ ID NO: 4
  • the oxidation of glufosinate to at least 100%, 105%, 110%, 120%, 130%, 150%, 170%, 200%, 250%, 300% or more of the activity of PPO.
  • nucleic acid molecule includes DNA molecules (such as cDNA or genomic DNA) and RNA molecules (such as mRNA) and analogs of DNA or RNA produced using nucleotide analogs.
  • the nucleic acid molecule may be single-stranded or double-stranded, preferably double-stranded DNA.
  • the synthesis of the nucleic acid may use nucleotide analogs or derivatives (for example, inosine or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acids with altered base pairing ability or increased nuclease resistance.
  • the present invention also provides polynucleotides encoding the modified DAAO of the present invention. Therefore, in the present invention, the term modification also includes genetic manipulation of the polynucleotide encoding the DAAO polypeptide of the present invention. The modification may be a substitution, deletion, insertion and/or addition of nucleotides.
  • the term "encoding" refers to the amino acid sequence of a polynucleotide directly specifying its protein product.
  • the boundaries of the coding sequence are generally determined by an open reading frame, which usually starts with the ATG start codon or other start codons such as GTG and TTG, and ends with stop codons such as TAA, TAG, and TGA.
  • the coding sequence can be a DNA, cDNA or recombinant nucleotide sequence.
  • nucleic acid molecules covering all or part of the nucleic acid sequence of the present invention can be separated by polymerase chain reaction (PCR), which uses the design of synthetic oligonucleotide primers based on the sequence information contained in the sequence.
  • PCR polymerase chain reaction
  • the polynucleotide of the present invention can be amplified using cDNA, mRNA or genomic DNA as a template and suitable oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into a suitable vector and characterized by DNA sequence analysis.
  • the polynucleotide of the present invention can be prepared by standard synthesis techniques, for example, by using an automated DNA synthesizer.
  • the invention also relates to the complementary strands of the nucleic acid molecules described herein.
  • a nucleic acid molecule that is complementary to other nucleotide sequences is a molecule that is sufficiently complementary to the nucleotide sequence so that it can hybridize with other nucleotide sequences to form a stable duplex.
  • hybridization refers to nucleotides that are at least about 90%, preferably at least about 95%, more preferably at least about 96%, and more preferably at least 98% homologous to each other under given stringent hybridization and washing conditions. The sequences generally remain hybridized to each other.
  • the polynucleotide of the present invention does not include a polynucleotide that only hybridizes to a poly A sequence (such as the 3'end poly (A) of mRNA) or a complementary stretch of poly T (or U) residues.
  • nucleic acid constructs and vectors containing the polynucleotide of the present invention are also provided.
  • 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.
  • nucleic acid construct refers to a single-stranded or double-stranded nucleic acid molecule, which is isolated from a naturally occurring gene or modified to contain a nucleic acid segment that does not occur in nature.
  • nucleic acid construct contains the control sequences required to express the coding sequence of the present invention, the term nucleic acid construct is synonymous with the term "expression cassette”.
  • expression vector refers herein to a linear or circular DNA molecule, which comprises a polynucleotide encoding a polypeptide of the present invention, the polynucleotide and additional nucleotides provided for the expression of the polynucleotide, for example, Control sequence, operably connected.
  • the expression vector includes a viral vector or a plasmid vector.
  • control sequence refers herein to include all elements necessary or advantageous for the expression of the polynucleotide encoding the polypeptide of the present invention.
  • Each control sequence may be natural or foreign to the nucleotide sequence encoding the polypeptide, or natural or foreign to each other.
  • control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminator. At a minimum, control sequences include promoters and transcription and translation termination signals.
  • control sequence may be a suitable promoter sequence, a nucleotide sequence recognized by the host cell to express the polynucleotide encoding the polypeptide of the present invention.
  • the promoter sequence contains transcription 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, for example, the Escherichia coli lac operon.
  • the promoters also include mutant, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell.
  • operably linked refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence, whereby the control sequence directs the expression of the polypeptide coding sequence.
  • the polynucleotide encoding the polypeptide of the present invention can be subjected to various operations to allow expression of the polypeptide. Before inserting it into the vector, it is desirable or necessary to manipulate the polynucleotide according to the expression vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art.
  • the vector of the present invention preferably contains one or more selectable markers, which allow simple selection of transformed, transfected, transduced, etc. cells.
  • a selectable marker is a gene whose product provides biocide or virus resistance, heavy metal resistance, supplementation of auxotrophs, etc.
  • the bacterial selectable marker is the dal gene from Bacillus subtilis or Bacillus licheniformis, or a marker that confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance.
  • the vector of the present invention can be integrated into the genome of the host cell or can replicate autonomously in the cell without relying on the genome. 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).
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques.
  • transformation and “transfection” refer to various art-recognized techniques for introducing foreign nucleic acids (such as DNA) into host cells, which can be found in, for example, the aforementioned Sambrook et al., 1989; Davis et al. .,Basic Methods in Molecular Biology (1986) and other laboratory manuals.
  • the present invention also relates to a recombinant host cell, which contains the polynucleotide of the present invention, which is advantageously used in the recombinant production of DAAO polypeptides.
  • the vector containing the polynucleotide of the present invention is introduced into a host cell, whereby the vector is retained as a chromosomal integrant or as a self-replicating extrachromosomal vector.
  • Those skilled in the art know conventional vectors and host cells for expressing proteins.
  • the host cell of the present invention is an E. coli cell, such as E. coli BL21 (DE3).
  • the expression vector is pET-30a(+).
  • the modified DAAO of the present invention can be operably linked to a non-DAAO polypeptide (for example, a heterologous amino acid sequence) to form a fusion protein.
  • a non-DAAO polypeptide for example, a heterologous amino acid sequence
  • 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 help the purification of recombinant DAAO.
  • the fusion protein is a DAAO protein containing a heterologous signal sequence at its N-terminus.
  • the expression and/or secretion of DAAO can be increased through the use of heterologous signal sequences.
  • the present invention provides a method for preparing L- glufosinate-ammonium, which comprises contacting the modified DAAO or host cell of the present invention with D- glufosinate-ammonium.
  • the method for preparing L-glufosinate-ammonium of the present invention includes the following steps:
  • a cell-free catalytic method is used to produce L-glufosinate-ammonium, and in step (a), the modified DAAO of the present invention is provided.
  • free or immobilized modified DAAO of the present invention can be used. Catalase can also be immobilized.
  • the incubation is performed at 20-50°C, preferably 25-40°C, more preferably 28-35°C, such as 30°C.
  • the medium is a buffer, such as PBS, Tris-HCl buffer.
  • the medium is a Tris-HCl buffer, such as a 50 mM, pH 8.0 Tris-HCl buffer.
  • the reaction medium is a medium partially or entirely composed of a cell culture medium, and the activity of the modified DAAO of the present invention is provided by the host cell of the present invention, and the host cell is cultured in the reaction medium.
  • the reaction medium is a medium partially or entirely composed of a cell culture medium, and the catalase activity is provided by the host cell of the present invention or by a second host cell, and the host cell is provided in the host cell of the present invention. Culture in the reaction medium.
  • the host cell of the present invention and/or the second host cell are cultured and expanded in a cell culture medium, and then the expanded host cell is separated from the cell culture medium, and a buffer or water is used to make The biomass is resuspended. D-Glufosinate-ammonium is added to the buffer or water before, during, or after adding the amplified host cell.
  • bacterial cells may be used, such as E. coli cells.
  • DNA polymerase (PrimeSTAR Max DNA Polymerase) and DpnI endonuclease were purchased from TaKaRa company, plasmid extraction kit was purchased from Axygen company, catalase was purchased from Zaozhuang Troceting Biotechnology Co., Ltd., item number QD-001 , D,L-Glufosinate-ammonium was purchased from Lier Chemical Co., Ltd.
  • the expression vector used was pET-30a(+), the plasmid was purchased from Novagen, and the host cell used was E. coli BL21 (DE3), purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.
  • coli BL21 (DE3) competent cells spread on LB agar medium (containing 50 mg/L kanamycin), and picked a single colony to LB liquid medium (containing 50 mg/L card Namycin) was cultured and sequenced to verify the correctness of the mutation.
  • the verified clones are stored at -80°C for later use.
  • the deposited clones were activated on LB agar medium. Then, a single colony was inoculated into LB liquid medium (containing 50 mg/L kanamycin), and incubated at 37° C. for 12 h with shaking. Transfer 1mL culture to 50mL fresh LB liquid medium (containing 50mg/L kanamycin), incubate with shaking at 37°C until OD600 reaches about 0.6, add IPTG (final concentration 0.4mM) at 25°C Incubate for 16h to induce protein expression.
  • LB liquid medium containing 50 mg/L kanamycin
  • E. coli cells were collected.
  • the collected E. coli cells were resuspended in 15 mL of pre-cooled phosphate buffered saline (PBS) with a pH of 7.0, and the E. coli cells were sonicated at 4°C.
  • PBS phosphate buffered saline
  • the cell disruption solution was centrifuged at 6,000 g at 4°C for 15 min to remove the precipitate, and the resulting supernatant was a crude enzyme solution (13 g/L) containing recombinant enzyme.
  • a mutant was prepared according to the method of Example 1.
  • the resulting mutants are shown in Table 1, wherein the mutant of SEQ ID NO: 4 is the mutant reported in US 9,834,802 (RtDAAO N54V, F58Q, M213S).
  • the enzyme activity was measured according to the method described in Example 1. The results are shown in Table 1.
  • the relative enzyme activity refers to the activity of the mutant after incubation vs.
  • SEQ ID NO: 38 On the basis of SEQ ID NO: 38, amino acid substitutions were further introduced, and the enzyme activity of the obtained mutant was measured. The resulting mutants are shown in Table 5. The activity of the mutants (without heat treatment) is equivalent to SEQ ID NO:38. The mutant of SEQ ID NO: 38 and the mutant obtained by further introducing 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 enzyme activity refers to 43°C After incubation for 20 minutes, the activity of the mutant vs. SEQ ID NO: 38 is the percentage of activity (150-200% is expressed as "++", and more than 200% is expressed as "+++").
  • a mutant was prepared according to the method of Example 1, and the enzyme activity was measured.
  • the obtained mutants and their enzyme activities are shown in Table 7, where the relative enzyme activity refers to the percentage of the activity of the mutant vs.
  • the activity of SEQ ID NO: 4 (less than 70% is expressed as "--", 70-100% is expressed) Is "-", 110-120% is expressed as "+”, 120-150% is expressed as "++"), the activity of wild-type RtnDAAO (SEQ ID NO: 3) is 0.
  • RtDAAO-based mutants and their enzyme activities are shown in Table 8, where the relative enzyme activity refers to the percentage of the activity of the mutant vs.
  • SEQ ID NO: 4 110-120% is expressed as “+”, 120-150 % Is expressed as “++”, 150-200% is expressed as “+++”, and more than 200% is expressed as "++++").

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Abstract

涉及经修饰的D-氨基酸氧化酶（DAAO）。具体而言，经修饰的DAAO具有催化D草铵膦氧化为PPO的活性。此外，经修饰的DAAO与SEQ ID NO:4相比具有提高的催化D草铵膦氧化为PPO的活性和/或提高的稳定性。还涉及编码经修饰的DAAO的多核苷酸、表达经修饰的DAAO的载体和宿主细胞以及使用经修饰的DAAO和宿主细胞生产L-草铵膦的方法。

Description

经修饰的DAAO酶及其应用 技术领域
本发明涉及酶工程领域。具体而言，本发明涉及经修饰的D-氨基酸氧化酶(DAAO)及其在生产草铵膦中的应用。
背景技术
草铵膦(glufosinate，也称为4-[羟基(甲基)膦酰基]-D,L-高丙氨酸)是世界销量第二的转基因作物耐受的除草剂。草铵膦是一种广谱触杀型除草剂，通过抑制植物体内的L-谷氨酰胺合成酶的活性，导致植物体内氮代谢紊乱，最终杀死植物。与草甘膦相比，草铵膦具有显著优势，如应用范围广、见效快、持效期长、更低毒、安全等。因此，草铵膦的销量增长迅速，在未来一段时间内市场需求巨大，前景非常广阔。
但是，草铵膦的工艺路线复杂，导致产品生产技术难度高。高昂的价格阻碍其迅速取代草甘膦。目前市售的草铵膦是包含等量的两种光学异构体的外消旋混合物(D,L-草铵膦)，但其中只有L-构型具有生理活性。因此，通过D,L-草铵膦去消旋化制备手性纯的L-草铵膦具有重要现实意义，成为近年来合成L-草铵膦的热门方向。
近年来报道从D,L-草铵膦制备L-草铵膦的方法众多。传统的化学修饰拆分法因成本高且不能利用D-型草铵膦而不具备竞争性。目前报道了下列将D-草铵膦转化成为L-草铵膦的主要代表性技术路线：
1.将D,L-草铵膦转化成N-乙酰基草铵膦，再经羧肽酶催化，L型N-乙酰基草铵膦经选择性水解，获得L-草铵膦，而D型N-乙酰基草铵膦不水解，可经化学或酶法消旋化后再次循环进入水解步骤(参见例如中国专利申请CN108690854A)。该方法的缺点是需多步反应，还有需要将经水解获得的L-草铵膦与N-乙酰化的底物分离。
2.将D-草铵膦氧化成2-羰基-4-(羟基甲基膦酰基)丁酸(简称PPO)，再对PPO进行还原或转氨生成L-草铵膦。在大部分文献中，使用D-氨基酸氧化酶(DAAO)催化将D-草铵膦氧化成PPO，其中通常还需要添加过氧化氢酶(CAT)以移除生成的过氧化氢。PPO可以在钯碳催化下被甲酸还原生成D,L-草铵膦，利用DAAO的立体选择性，将D,L-草铵膦逐渐转化为L-草铵膦(参见，例如，CN105567780A)。这个方案的缺点是钯碳催化剂用量大，并且浪费反应原料(如氧气和甲酸铵)。
还可以采用L-氨基酸转氨酶(L-TA)催化的立体选择性转氨反应将PPO转化成L-草铵膦(参见，例如，US20180030487A1)。这个方案的缺点在于，转氨步骤是平衡反应，需要提供过量的氨基供体(氨基酸或有机胺)来实现高转化率(例如提供3倍当量的氨基供体，转化率90％)，而过量的氨基供体和对应的副产物将严重影响后续的分离纯化步骤。
此外，可以通过L-氨基酸脱氢酶(L-AADH)催化的立体选择性还原反应，将PPO转 化为L-草铵膦(参见，例如，CN107502647A、CN109576236A和CN109609582A)。这种方案中，转化的底物浓度低或损失大。
比较上述方案，采用D-氨基酸氧化酶配合L-氨基酸脱氢酶的方案，具有潜在的成本优势。但目前报道过的方法中，可以转化的底物浓度普遍不高，或是损失过大，造成生产成本过高。实现高浓度的D,L-草铵膦去消旋化，成为目前的工艺瓶颈。
阻碍更高浓度的底物转化的主要因素可能是：重组的DAAO酶稳定性较差而在反应釜的条件下不稳定，反应过程中酶失活。
另一个限制性因素是酶对D-草铵膦的选择性催化活性。现有技术中对DAAO进行了一些修饰来赋予DAAO对D-草铵膦的活性：
·在US7,939,709中，使用来源于圆红冬孢酵母(Rhodotorula toruloides，又名Rhodotorula gracilis，参见https://www.atcc.org/products/all/10788.aspx)的DAAO的突变体实现了从D-草铵膦合成PPO的目的。该专利提及的圆红冬孢酵母DAAO突变体中包含F58K突变，在位置M213取代为H、S、T、C、Q、G、N、和A的突变，以及在位置223和238的突变。Tim Hawks等在2011年发表的文章(D-glufosinate as a male sterility agent for hybrid seed production,Plant Biotechnology Journal,(2011)9,pp.301-314)，报道了上述专利的类似内容，采用圆红冬孢酵母的DAAO，其包含58、213位的突变。
·在US9,834,802中，使用圆红冬孢酵母来源的DAAO的突变体配合转氨酶(TA)实现了从D,L-草铵膦合成L-草铵膦。还描述了该DAAO的位置54、56、58、213和238包含一个或多个突变，并例举了几种具体组合方式。
·在CN109576236A中，基于来自圆红冬孢酵母的DAAO，也构建了可以氧化D-草铵膦的突变体。所述突变体在氨基酸第52位、第54位、第58位、第213位和第335位进行单突变或多突变。
·在其他专利申请，如CN105567780A、CN109609582A中也涉及DAAO酶来氧化D-草铵膦，但未注明所用酶的序列。
但是，本领域仍需要提供具有更高的稳定性和/或对D-草铵膦具有更高活性的DAAO。
发明内容
在第一方面，本发明提供一种经修饰的D-氨基酸氧化酶(DAAO)，与其野生型DAAO相比，包含4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或更多个位置的氨基酸取代，其中所述经修饰的DAAO具有催化D-草铵膦氧化为PPO的活性。
在一些实施方案中，所述经修饰的DAAO包含位置54、56、58和213的氨基酸取代，所述位置参照SEQ ID NO:2进行编号。优选地，位置54取代为I、V、T或L，更优选I或V。优选地，位置56取代为N。优选地，位置58取代为H或Q，更优选H。优选地，位置213取代为S或T，更优选S。在一些实施方案中，所述经修饰的DAAO 还包含位置210和/或221的氨基酸取代。优选地，位置210取代为A、G或P，更优选A。优选地，位置P221取代为R。
在一些实施方案中，所述经修饰的DAAO包含位置54、58、213和221的氨基酸取代，所述位置参照SEQ ID NO:2进行编号。优选地，位置54取代为V，位置58取代为Q，位置213取代为S，位置221取代为R。在一些实施方案中，所述经修饰的DAAO还包含位置210和/或56的氨基酸取代。优选地，位置56取代为N，位置210取代为A。
在一些实施方案中，本发明的经修饰的DAAO还包含选自2、81、97、193、194、237、265、273、274、300、317、319、337和342的一或多个位置的取代。优选地，位置2取代为C或S，位置81取代为Y，位置97取代为V，位置193取代为T，位置194取代为V或C，位置237取代为V，位置265取代为C，位置273取代为D，位置274取代为E，位置300取代为S或T，位置317取代为Y或W，位置319取代为K，位置337取代为S，位置342取代为S或H。
或者，在一些实施方案中，本发明的经修饰的DAAO还包含选自194、237、265、273、274、300、317和319的一或多个位置的取代。优选地，位置194取代为V或C，位置237取代为V，位置265取代为C，位置273取代为D，位置274取代为E，位置300取代为S，位置317取代为Y或W，位置319取代为K。
或者，在一些实施方案中，本发明的经修饰的DAAO还包含选自位置2、81、97、193、300、337和342的一或多个位置的取代，其中位置2取代为C或S，位置81取代为Y，位置97取代为V，位置193取代为T，位置300取代为T，位置337取代为S，和位置342取代为S。
在一些实施方案中，所述经修饰的DAAO，其与SEQ ID NO:1相比，包含位置54、58、194和213的氨基酸取代，其中所述经修饰的DAAO具有催化D-草铵膦氧化为PPO的活性。优选地，位置54取代为I、V、T或L，位置58取代为H或Q，位置194取代为V或C，位置213取代为S或T。在一些实施方案中，所述经修饰的DAAO还包含选自位置56、210、221、237、265、273、274、300、317和319的一或多个位置的氨基酸取代。优选地，位置56取代为N，位置210取代为A、G或P，位置P221取代为R，位置237取代为V，位置265取代为C，位置273取代为D，位置274取代为E，位置300取代为S，位置317取代为Y或W，位置319取代为K。
在一些实施方案中，所述经修饰的DAAO，包含SEQ ID NO:5-86之一的氨基酸序列或由SEQ ID NO:5-86之一的氨基酸序列组成，或者所述经修饰的DAAO与SEQ ID NO:5-30和66-76之一相比，在除位置54、56、58、194、210、213、221、237、265、273、274、300、317和319之外的位置包含1-10个氨基酸取代，与SEQ ID NO:31-57和77-86之一相比，在除位置2、54、56、58、81、97、193、210、213、221、300、337和342之外的位置包含1-10个氨基酸取代，或与SEQ ID NO:58-65之一相比，在除位置54、56、58、210、213和221之外的位置包含1-10个氨基酸取代，其中所述经修饰的DAAO具有催化D-草铵膦氧化为PPO的活性。
在第二方面，本发明提供编码本发明的经修饰的DAAO的多核苷酸，以及包含本发明的多核苷酸的载体。
在第三方面，本发明提供包含本发明的的经修饰的DAAO、其编码多核苷酸或包含所述多核苷酸的载体的宿主细胞。
在第四方面，本发明还提供一种生产L-草铵膦的方法，包括使本发明的经修饰的DAAO或本发明的宿主细胞与D-草铵膦接触。
发明详述
本发明主要涉及经修饰的DAAO，用于催化D-草铵膦氧化，以生产L-草铵膦。除非另有说明，本文中使用的术语具有本领域技术人员一般理解的含义。
一、经修饰的D-氨基酸氧化酶
如本文所用，术语“D-氨基酸氧化酶”和“DAAO”是指催化D-氨基酸氧化生产酮酸的酶(EC 1.4.3.3)。通常，天然存在的DAAO不能催化D-草铵膦的氧化。因此，本发明提供经修饰的DAAO多肽，其能够催化D-草铵膦氧化成PPO。优选地所述经修饰的DAAO多肽具有提高的稳定性和/或提高的催化D-草铵膦氧化成PPO的活性。
如本文所用，术语“肽”表示通过肽键连接的至少两个氨基酸的链。术语“多肽”在本文中可以与属于“蛋白质”互换使用，是指含有十个或更多个氨基酸残基的链。本文中的所有肽和多肽化学式或序列均是从左至右书写的，表示从氨基末端至羧基末端的方向。
术语“氨基酸”包括蛋白质中天然存在的氨基酸和非天然氨基酸。蛋白质中天然存在的氨基酸的单字母和三字母命名采用本领域惯用名，可见于Sambrook,et al.(Molecular Cloning:A Laboratory Manual,2nd,ed.Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1989)。
Figure PCTCN2020134540-appb-000001
Figure PCTCN2020134540-appb-000002
如本文所用，术语“修饰”是指对多肽的任何化学修饰，例如氨基酸的取代、缺失、插入和/或添加。
在一些实施方案中，本发明的经修饰的DAAO多肽与其野生型DAAO相比，包含4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或更多个氨基酸取代，其中所述经修饰的DAAO多肽具有催化D-草铵膦氧化成PPO的活性。在一些实施方案中，本发明的经修饰的DAAO多肽与其野生型DAAO相比，包含位置54、56、58和213的氨基酸取代，所述位置参照SEQ ID NO:2进行编号。优选地，位置54取代为I、V、T或L，更优选I或V。优选地，位置56取代为N。优选地，位置58取代为H或Q，更优选H。优选地，位置213取代为S或T，更优选S。在一些优选的实施方案中，所述经修饰的DAAO多肽与其野生型DAAO相比，包含以下取代的组合：54V、56N、58H和213S，或54I、56N、58H和213S。在一些实施方案中，所述经修饰的DAAO还包含位置210和/或221的氨基酸取代。优选地，位置210取代为A、G或P，更优选A。优选地，位置P221取代为R。
在一些实施方案中，所述经修饰的DAAO包含位置54、58、213和221的氨基酸取代，所述位置参照SEQ ID NO:2进行编号。优选地，位置54取代为V，位置58取代为Q，位置213取代为S，位置221取代为R。在一些实施方案中，所述经修饰的DAAO还包含位置210和/或56的氨基酸取代。优选地，位置56取代为N，位置210取代为A。
在一些实施方案中，本发明的经修饰的DAAO还包含选自2、81、97、193、194、237、265、273、274、300、317、319、337和342的一或多个位置的取代，所述位置参照SEQ ID NO:2进行编号，其中所述经修饰的DAAO多肽催化D-草铵膦氧化成PPO的活性。优选地，位置2取代为C或S，位置81取代为Y，位置97取代为V，位置193取代为T，位置194取代为V或C，位置237取代为V，位置265取代为C，位置273取代为D，位置274取代为E，位置300取代为S或T，位置317取代为Y或W，和位置319取代为K，位置337取代为S，和位置342取代为S或H。
或者，本发明的经修饰的DAAO还包含选自194、237、265、273、274、300、317和319的一或多个位置的取代，所述位置参照SEQ ID NO:2进行编号，其中所述经修饰的DAAO多肽催化D-草铵膦氧化成PPO的活性。优选地，位置194取代为V或C，位置237取代为V，位置265取代为C，位置273取代为D，位置274取代为E，位置300取代为S，位置317取代为Y或W，和位置319取代为K。
或者，本发明的经修饰的DAAO还包含选自位置2、81、97、193、300、337和342的一或多个位置的取代，所述位置参照SEQ ID NO:2进行编号，其中所述经修饰的 DAAO多肽催化D-草铵膦氧化成PPO的活性。优选地，位置2取代为C或S，位置81取代为Y，位置97取代为V，位置193取代为T，位置300取代为T，位置337取代为S，和位置342取代为S。
在一些实施方案中，与SEQ ID NO:1相比，本发明的经修饰的DAAO包含位置54、58、194和213的氨基酸取代，其中所述经修饰的DAAO具有催化D-草铵膦氧化为PPO的活性。优选地，位置54取代为I、V、T或L，位置58取代为H或Q，位置194取代为V或C，位置213取代为S或T。在一些实施方案中，所述经修饰的DAAO还包含选自位置56、210、221、237、265、273、274、300、317和319的一或多个位置的氨基酸取代。优选地，位置56取代为N，位置210取代为A、G或P，位置P221取代为R，位置237取代为V，位置265取代为C，位置273取代为D，位置274取代为E，位置300取代为S，位置317取代为Y或W，位置319取代为K。在一些实施方案中，与SEQ ID NO:1相比，本发明的经修饰的DAAO还具有一或多个氨基酸的保守取代，或具有一或多个氨基酸的插入或缺失。
在一些实施方案中，本发明的经修饰的DAAO与其野生型相比，具有选自如下组合的氨基酸取代(位置参照SEQ ID NO:2进行编号)：
-54V、58Q、194V、213S；
-54V、58Q、194C、213S；
-54V、58Q、213S、273D；
-54V、58Q、213S、317Y；
-54V、58Q、213S、317W；
-54V、58Q、213S、274E；
-54V、58Q、213S、319K；
-54V、58Q、194C、213S、317Y；
-54V、58Q、194C、213S、265C、317Y；
-54V、58Q、194C、213S、265C、300S、317Y；
-54V、58Q、194C、213T、265C、300S、317Y；
-54V、58Q、194C、213S、210G、265C、300S、317Y；
-54V、58Q、194C、213S、210P、265C、300S、317Y；
-54V、58Q、194C、213S、210A、265C、300S、317Y；
-54V、58Q、194C、213S、221R、265C、300S、317Y；
-54V、58Q、194C、213S、237A、265C、300S、317Y；
-54V、58Q、194C、213S、237V、265C、300S、317Y；
-54V、56N、58Q、194C、213S、265C、300S、317Y；
-54T、56N、58Q、194C、213S、265C、300S、317Y；
-54I、56N、58Q、194C、213S、265C、300S、317Y；
-54V、56N、58H、194C、213S、265C、300S、317Y；
-54L、56N、58Q、194C、213S、265C、300S、317Y；
-54I、56N、58H、194C、213S、265C、300S、317Y；
-54V、56N、58H、194C、213S、237V、265C、300S、317Y；
-54V、56N、58H、194C、213S、210A、237V、265C、300S、317Y；
-54I、56N、58H、194C、213S、210A、221R、265C、300S、317Y；
-54L、56N、58Q；
-54T、56N、58Q；
-54I、56N、58H；
-54V、56N、58H；
-54L、56N、58Q、213S；
-54T、56N、58Q、213S；
-54I、56N、58H、213S；
-54V、56N、58H、213S；
-2C、54V、56N、58H、213S；
-2S、54V、56N、58H、213S；
-54V、56N、58H、81Y、213S；
-54V、56N、58H、97V、213S；
-54V、56N、58H、193T、213S；
-54V、56N、58H、213S、300T；
-54V、56N、58H、213S、337S；
-54V、56N、58H、213S、342S；
-2S、54V、56N、58H、81Y、97V、193T、213S、337S；
-54V、56N、58H、97V、193A、213S、337S、342H；
-2C、54V、56N、58H、81Y、97V、213S、337S；
-2C、54V、56N、58H、81Y、97V、193A、213S、342S；
-54V、56N、58H、97V、193T、213S、337S、342H；
-54V、56N、58H、81Y、97V、193T、213S、337S、342H；
-54V、56N、58H、97V、193T、213S、300T、337S、342H；
-54V、56N、58H、81Y、97V、193T、213S、300T、337S、342H；
-54V、56N、58H、97V、193T、210A、213S、300T、337S、342H；
-54V、56N、58H、97V、193T、213S、221R、300T、337S、342H；
-54V、56N、58H、97V、193T、210A、213S、221R、300T、337S、342H；
-58K、213T；
-54V、56N、58H、210A、213S；
-54V、56N、58H、213S、221R；
-54V、56N、58H、210A、213S、221R；
-54I、56N、58H、210A、213S；
-54I、56N、58H、213S、221R；
-54I、56N、58H、210A、213S、221R；
-54V、58Q、213S；
-54V、58Q、210A、213S；
-54V、58Q、213S、221R；和
-54V、58Q、210A、213S、221R。
在本文中，在其基础上进行氨基酸修饰的DAAO多肽称为起始DAAO。所述起始DAAO可以是野生型DAAO，也可以是野生型DAAO的变体。例如，从SEQ ID NO:1的多肽开始进行修饰，则相对于经修饰的DAAO，SEQ ID NO:1的多肽是“起始DAAO”；而如果从SEQ ID NO:1的变体多肽(例如SEQ ID NO:4-30)开始进行修饰，则相对于经修饰的DAAO，所述变体多肽是“起始DAAO”。
如本文所用，术语“野生型DAAO”是指天然存在的DAAO。在一些实施方案中，所述野生型DAAO是来自红酵母属的DAAO。在一些实施方案中，所述野生型DAAO是SEQ ID NO:1-3之一。SEQ ID NO:1是来自圆红冬孢酵母的DAAO的氨基酸序列(GenBank登录号：CAJ87425.1)，SEQ ID NO:2是来自Rhodotorula sp.JG-1b的DAAO的氨基酸序列(GenBank登录号：KWU45700.1)，且SEQ ID NO:3是来自Rhodotorula taiwanensis的推定的DAAO的氨基酸序列(GenBank登录号：POY70719.1)。
对于本发明，为确定两个氨基酸序列或两个核酸序列的相同性百分比，以最佳比较为目的比对序列(例如在第一个氨基酸或核酸序列中可导入缺口，以与第二个氨基酸或核酸序列进行最佳比对)。然后比较在相应氨基酸位置或核苷酸位置的氨基酸残基或核苷酸。当第一个序列中的位置在第二个序列中相应位置由相同氨基酸残基或核苷酸占据时，则这些分子在这个位置是相同的。两个序列之间的相同性百分比是所述序列共有的相同位置的数量的函数(即相同性百分比＝相同位置的数量/位置(即重叠位置)的总数量×100)。优选地，这两个序列是相同长度的。
本领域技术人员知晓，可以使用不同的计算机程序确定两个序列之间的相同性。
“氨基酸相同性百分比”或者“氨基酸序列相同性百分比”是指比较两个多肽的氨基酸，当最佳比对时，所述两个多肽具有大约指定的相同氨基酸百分比。例如，“95％的氨基酸相同性”是指比较两个多肽的氨基酸，当最佳比对时，所述两个多肽有95％的氨基酸相同。
在一些实施方案中，所述野生型DAAO与SEQ ID NO:1-3之一具有至少65％，优选至少70％、75％或80％，更优选至少85％、90％或95％，特别优选至少96％、97％、98％或99％的序列相同性。
在一些实施方案中，本发明的经修饰的DAAO多肽与其野生型DAAO相比，包含4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或更多个氨基酸取代，其中所述经修饰的DAAO多肽催化D-草铵膦氧化成PPO的活性。在一些实施方案 中，本发明的经修饰的DAAO多肽与其野生型DAAO相比，包含位置54、56、58和213的氨基酸取代，所述位置参照SEQ ID NO:2进行编号。优选地，位置54取代为I、V、T或L，更优选I或V。优选地，位置56取代为N。优选地，位置58取代为H或Q，更优选H。优选地，位置213取代为S或T，更优选S。在一些优选的实施方案中，所述经修饰的DAAO多肽与其野生型DAAO相比，包含以下取代：54V、56N、58H和213S，或54I、56N、58H和213S。在一些实施方案中，所述经修饰的DAAO还包含位置210和/或221的氨基酸取代。优选地，位置210取代为A、G或P，更优选A。优选地，位置P221取代为R。优选地，本发明的经修饰的DAAO还包含选自194、237、265、273、274、300、317和319的一或多个位置的取代，其中位置194取代为V或C，位置237取代为V，位置265取代为C，位置273取代为D，位置274取代为E，位置300取代为S，位置317取代为Y或W，和位置319取代为K。优选地，所述野生型DAAO与SEQ ID NO:1具有至少65％，优选至少70％、75％或80％，更优选至少85％、90％或95％，特别优选至少96％、97％、98％或99％的序列相同性。
在一些实施方案中，所述经修饰的DAAO包含位置54、58、213和221的氨基酸取代，所述位置参照SEQ ID NO:2进行编号。优选地，位置54取代为V，位置58取代为Q，位置213取代为S，位置221取代为R。在一些实施方案中，所述经修饰的DAAO还包含位置210和/或56的氨基酸取代。优选地，位置56取代为N，位置210取代为A。优选地，本发明的经修饰的DAAO还包含选自194、237、265、273、274、300、317和319的一或多个位置的取代，其中位置194取代为V或C，位置237取代为V，位置265取代为C，位置273取代为D，位置274取代为E，位置300取代为S，位置317取代为Y或W，和位置319取代为K。优选地，所述野生型DAAO与SEQ ID NO:1具有至少65％，优选至少70％、75％或80％，更优选至少85％、90％或95％，特别优选至少96％、97％、98％或99％的序列相同性。
在一些实施方案中，本发明的经修饰的DAAO多肽与其野生型DAAO相比，包含4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或更多个氨基酸取代，其中所述经修饰的DAAO多肽催化D-草铵膦氧化成PPO的活性。在一些实施方案中，本发明的经修饰的DAAO多肽与其野生型DAAO相比，包含位置54、56、58和213的氨基酸取代，所述位置参照SEQ ID NO:2进行编号。优选地，位置54取代为I、V、T或L，更优选I或V。优选地，位置56取代为N。优选地，位置58取代为H或Q，更优选H。优选地，位置213取代为S或T，更优选S。在一些优选的实施方案中，所述经修饰的DAAO多肽与其野生型DAAO相比，包含以下取代：54V、56N、58H和213S，或54I、56N、58H和213S。在一些实施方案中，所述经修饰的DAAO还包含位置210和/或221的氨基酸取代。优选地，位置210取代为A、G或P，更优选A。优选地，位置P221取代为R。优选地，本发明的经修饰的DAAO还包含选自位置2、81、97、193、300、337和342的一或多个位置的取代，其中位置2取代为C或S，位置81取代为Y，位置97取代为V，位置193取代为T，位置300取代为T，位置337取代为 S，和位置342取代为S。优选地，所述野生型DAAO与SEQ ID NO:2具有至少65％，优选至少70％、75％或80％，更优选至少85％、90％或95％，特别优选至少96％、97％、98％或99％的序列相同性。
在一些实施方案中，所述经修饰的DAAO包含位置54、58、213和221的氨基酸取代，所述位置参照SEQ ID NO:2进行编号。优选地，位置54取代为V，位置58取代为Q，位置213取代为S，位置221取代为R。在一些实施方案中，所述经修饰的DAAO还包含位置210和/或56的氨基酸取代。优选地，位置56取代为N，位置210取代为A。优选地，本发明的经修饰的DAAO还包含选自位置2、81、97、193、300、337和342的一或多个位置的取代，其中位置2取代为C或S，位置81取代为Y，位置97取代为V，位置193取代为T，位置300取代为T，位置337取代为S，和位置342取代为S。优选地，所述野生型DAAO与SEQ ID NO:2具有至少65％，优选至少70％、75％或80％，更优选至少85％、90％或95％，特别优选至少96％、97％、98％或99％的序列相同性。
在一些实施方案中，所述经修饰的DAAO与野生型相比，包含4-20、4-15、4-14、4-13、4-12、4-11、4-10、4-9、4-8、4-7、4-6或4-5个氨基酸取代。在一些实施方案中，所述经修饰的DAAO与野生型相比，包含4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或更多个氨基酸取代。
在一些实施方案，所述野生型DAAO与SEQ ID NO:1-3之一的区别在于，具有一或多个氨基酸的取代、缺失、插入和/或添加。在一些实施方案中，所述野生型DAAO与SEQ ID NO:1-3之一相比，具有一或多个氨基酸的保守取代。在一些实施方案中，所述野生型DAAO与SEQ ID NO:1-3之一相比，具有一或多个氨基酸的插入或缺失。
在一些实施方案中，本发明的经修饰的DAAO多肽与其野生型DAAO相比，包含4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或更多个氨基酸取代，其中所述经修饰的DAAO多肽催化D-草铵膦氧化成PPO的活性。在一些实施方案中，本发明的经修饰的DAAO多肽与其野生型DAAO相比，包含位置54、56、58和213的氨基酸取代，所述位置参照SEQ ID NO:2进行编号，其中所述经修饰的DAAO与SEQ ID NO:1-3之一具有至少70％、75％、80％、85％、90％、95％、96％、97％或98％的序列相同性。优选地，位置54取代为I、V、T或L，更优选I或V。优选地，位置56取代为N。优选地，位置58取代为H或Q，更优选H。优选地，位置213取代为S或T，更优选S。在一些优选的实施方案中，所述经修饰的DAAO多肽与其野生型DAAO相比，包含以下取代的组合：54V、56N、58H和213S，或54I、56N、58H和213S。在一些实施方案中，所述经修饰的DAAO还包含位置210和/或221的氨基酸取代。优选地，位置210取代为A、G或P，更优选A。优选地，位置P221取代为R。
在一些实施方案中，所述经修饰的DAAO包含位置54、58、213和221的氨基酸取代，所述位置参照SEQ ID NO:2进行编号，其中所述经修饰的DAAO与SEQ ID NO:1-3之一具有至少70％、75％、80％、85％、90％、95％、96％、97％或98％的序列相同性。优选地，位置54取代为V，位置58取代为Q，位置213取代为S，位置221取代 为R。在一些实施方案中，所述经修饰的DAAO还包含位置210和/或56的氨基酸取代。优选地，位置56取代为N，位置210取代为A。
在一些实施方案中，本发明的经修饰的DAAO还包含选自2、81、97、193、194、237、265、273、274、300、317、319、337和342的一或多个位置的取代，所述位置参照SEQ ID NO:2进行编号，其中所述经修饰的DAAO多肽催化D-草铵膦氧化成PPO的活性。优选地，位置2取代为C或S，位置81取代为Y，位置97取代为V，位置193取代为T，位置194取代为V或C，位置237取代为V，位置265取代为C，位置273取代为D，位置274取代为E，位置300取代为S或T，位置317取代为Y或W，和位置319取代为K，位置337取代为S，和位置342取代为S或H。
或者，本发明的经修饰的DAAO还包含选自194、237、265、273、274、300、317和319的一或多个位置的取代，所述位置参照SEQ ID NO:2进行编号，其中所述经修饰的DAAO多肽催化D-草铵膦氧化成PPO的活性。优选地，位置194取代为V或C，位置237取代为V，位置265取代为C，位置273取代为D，位置274取代为E，位置300取代为S，位置317取代为Y或W，和位置319取代为K。
或者，本发明的经修饰的DAAO还包含选自位置2、81、97、193、300、337和342的一或多个位置的取代，所述位置参照SEQ ID NO:2进行编号，其中所述经修饰的DAAO多肽催化D-草铵膦氧化成PPO的活性。优选地，位置2取代为C或S，位置81取代为Y，位置97取代为V，位置193取代为T，位置300取代为T，位置337取代为S，和位置342取代为S。
在一些实施方案中，与SEQ ID NO:1相比，本发明的经修饰的DAAO包含位置54、58、194和213的氨基酸取代，其中所述经修饰的DAAO具有催化D-草铵膦氧化为PPO的活性，其中所述经修饰的DAAO与SEQ ID NO:1具有至少70％、75％、80％、85％、90％、95％、96％、97％或98％的序列相同性。优选地，位置54取代为I、V、T或L，位置58取代为H或Q，位置194取代为V或C，位置213取代为S或T。在一些实施方案中，所述经修饰的DAAO还包含选自位置56、210、221、237、265、273、274、300、317和319的一或多个位置的氨基酸取代。优选地，位置56取代为N，位置210取代为A、G或P，位置P221取代为R，位置237取代为V，位置265取代为C，位置273取代为D，位置274取代为E，位置300取代为S，位置317取代为Y或W，位置319取代为K。在一些实施方案中，与SEQ ID NO:1相比，本发明的经修饰的DAAO还具有一或多个氨基酸的保守取代，或具有一或多个氨基酸的插入或缺失。
术语“保守取代”也称为由“同源”氨基酸残基取代，是指其中氨基酸残基由具有相似侧链的氨基酸残基置换的取代，例如，碱性侧链的氨基酸(例如赖氨酸、精氨酸和组氨酸)、酸性侧链的氨基酸(例如天冬氨酸、谷氨酸)、非荷电极性侧链氨基酸(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、β-分支的侧链氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)及芳香侧链氨基酸(例如酪氨酸、苯丙氨 酸、色氨酸、组氨酸)。
保守氨基酸取代通常对所得蛋白质的活性的影响最小。这种取代在下文描述。保守取代是用大小、疏水性、电荷、极性、空间特征、芳香性等相似的氨基酸置换一个氨基酸。当希望精细调节蛋白质的特性时，这种取代通常是保守的。
如本文所用，“同源”氨基酸残基是指具有相似化学性质的氨基酸残基，所述化学性质涉及疏水性、电荷、极性、空间特征、芳香性特征等。彼此同源的氨基酸的例子包括正电荷的赖氨酸、精氨酸、组氨酸，负电荷的谷氨酸、天冬氨酸，疏水性的甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸，极性的丝氨酸、苏氨酸、半胱氨酸、甲硫氨酸、色氨酸、酪氨酸、天冬酰胺、谷氨酰胺，芳香性的苯丙氨酸、酪氨酸、色氨酸，化学相似侧链基团的丝氨酸与苏氨酸，或者谷氨酰胺和天冬酰胺，或者亮氨酸和异亮氨酸。
蛋白质中氨基酸保守取代的例子包括：Ser取代Ala，Lys取代Arg，Gln或His取代Asn，Glu取代Asp，Ser取代Cys，Asn取代Gln，Asp取代Glu，Pro取代Gly，Asn或Gln取代His，Leu或Val取代Ile，Ile或Val取代Leu，Arg或Gln取代Lys，Leu或Ile取代Met，Met、Leu或Tyr取代Phe，Thr取代Ser，Ser取代Thr，Tyr取代Trp，Trp或Phe取代Tyr，及Ile或Leu取代Val。
在一些实施方案中，所述经修饰的DAAO，包含SEQ ID NO:5-86之一的氨基酸序列或由SEQ ID NO:5-86之一的氨基酸序列组成，或者所述经修饰的DAAO与SEQ ID NO:5-30和66-76之一相比，在除位置54、56、58、194、210、213、221、237、265、273、274、300、317和319之外的位置包含1-10个氨基酸取代，与SEQ ID NO:31-57和77-86之一相比，在除位置2、54、56、58、81、97、193、210、213、221、300、337和342之外的位置包含1-10个氨基酸取代，或与SEQ ID NO:58-65之一相比，在除位置54、56、58、210、213和221之外的位置包含1-10个氨基酸取代，其中所述经修饰的DAAO具有催化D-草铵膦氧化为PPO的活性。在一些实施方案中，所述经修饰的DAAO与SEQ ID NO:5-30和66-76之一相比，在除位置54、56、58、194、210、213、221、237、265、273、274、300、317和319之外的位置包含1、2、3、4、5、6、7、8、9、10或更多个氨基酸取代；与SEQ ID NO:31-57和77-86之一相比，在除位置2、54、56、58、81、97、193、210、213、221、300、337和342之外的位置包含1、2、3、4、5、6、7、8、9、10或更多个氨基酸取代；或与SEQ ID NO:58-65之一相比，在除位置54、56、58、210、213和221之外的位置包含1、2、3、4、5、6、7、8、9、10或更多个氨基酸取代。在一些实施方案中，所述经修饰的DAAO与SEQ ID NO:1-3之一具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％或99％的序列相同性。
如本文所用，酶的活性指在一定条件下，在单位质量的酶催化的化学反应中，单位时间内底物的减少量或产物的增加量。例如，本发明的经修饰的DAAO的活性，用一定条件下，在单位质量的经修饰的DAAO催化下，单位时间内D-草铵膦减少的量或PPO增加的量来表示。
在本文中，酶的活性也可以指酶的相对活性，以感兴趣的酶的活性与催化相同反应的给定的酶的活性的比值表示，如百分比相对活性。
在一些实施方案中，本发明的经修饰的DAAO的活性以与SEQ ID NO:4相比的百分比相对活性表示。在一些实施方案中，所述经修饰的DAAO催化D-草铵膦氧化为PPO的活性是SEQ ID NO:4的催化D-草铵膦氧化为PPO的活性的至少100％、105％、110％、120％、130％、150％、170％、200％、250％、300％或更高。
提高经修饰的DAAO的稳定性对于应用于工业生产也是有利的。在一些实施方案中，所述稳定性是热稳定性，指酶在一定温度(如40-60℃或更高)温育一定时间(如10分钟至1小时)后的保持活性的能力。在一些实施方案中，所述经修饰的DAAO具有优于SEQ ID NO:4的多肽的热稳定性。例如，在43-45℃温育20分钟后，本发明的经修饰的DAAO的活性是SEQ ID NO:4的多肽的活性的至少100％、105％、110％、120％、130％、150％、170％、200％、250％、300％或更高。或者，本发明的经修饰的DAAO具有更高的T50，其中T50是指在该温度温育一小时后，酶的活性下降50％的温度。在一些实施方案中，本发明的经修饰的DAAO的T50比SEQ ID NO:4的多肽高约1、2、3、4、5、6、7、8、9、10℃或更高。
在一些实施方案中，所述经修饰的DAAO具有优于SEQ ID NO:4的多肽的热稳定性，且其催化D-草铵膦氧化为PPO的活性是SEQ ID NO:4的催化D-草铵膦氧化为PPO的活性的至少100％、105％、110％、120％、130％、150％、170％、200％、250％、300％或更高。
二、编码经修饰的DAAO的多核苷酸。
如本文所用，术语“多核苷酸”或者“核酸分子”包括DNA分子(例如cDNA或基因组DNA)和RNA分子(例如mRNA)及使用核苷酸类似物产生的DNA或RNA的类似物。所述核酸分子可以是单链或双链的，优选双链DNA。所述核酸的合成可以使用核苷酸类似物或衍生物(例如肌苷或硫代磷酸核苷酸)。这种核苷酸可以用于，例如，制备具有改变的碱基配对能力或者增加的核酸酶抗性的核酸。
本发明还提供编码本发明的经修饰的DAAO的多核苷酸。因此，在本发明中，术语修饰还包括对编码本发明的DAAO多肽的多核苷酸的遗传操作。所述修饰可以是核苷酸的取代、缺失、插入和/或添加。
如本文所用，术语“编码”是指多核苷酸直接指定其蛋白质产物的氨基酸序列。编码序列的边界一般由开放读框确定，所述开放读框通常以ATG起始密码子或另外的起始密码子如GTG和TTG开始，以终止密码子如TAA、TAG和TGA结束。所述编码序列可以是DNA、cDNA或重组核苷酸序列。
此外，涵盖本发明的全部或部分核酸序列的核酸分子可以通过聚合酶链反应(PCR)分离，所述PCR使用基于所述序列中包含的序列信息设计合成的寡核苷酸引物。
本发明的多核苷酸可以使用cDNA、mRNA或者基因组DNA作为模板及合适的寡核苷酸引物根据标准PCR扩增技术进行扩增。如此扩增的核酸可以克隆进合适的载体 中，并通过DNA序列分析进行表征。
本发明的多核苷酸可以通过标准的合成技术制备，例如使用自动化DNA合成仪制备。
本发明还涉及本文描述的核酸分子的互补链。与其它核苷酸序列互补的核酸分子是与该核苷酸序列充分互补的分子，使得其可以与其他核苷酸序列杂交，从而形成稳定双链体。
如本文所用，术语“杂交”是在给定的严格杂交和洗涤条件下，彼此至少大约90％、优选至少大约95％、更优选至少大约96％、更优选至少98％同源的核苷酸序列通常保持彼此杂交。
本领域技术人员知道各种用于杂交的条件，如严格杂交条件和高度严格杂交条件。参见，例如，Sambrook et al.,1989,Molecular Cloning,A Laboratory Manual,Cold Spring Harbor Press,N.Y.；和Ausubel et al.(eds.),1995,Current Protocols in Molecular Biology,John Wiley&Sons,N.Y.。
当然，本发明的多核苷酸不包括仅与poly A序列(如mRNA的3’末端poly(A))或者与互补的一段poly T(或U)残基杂交的多核苷酸。
三、表达和生产经修饰的DAAO
为表达本发明的经修饰的DAAO，还提供包含本发明的多核苷酸的核酸构建体和载体，如表达载体。
如本文所用，术语“表达”包括多肽生产中包含的任何步骤，包括但不限于转录、转录后修饰、翻译、翻译后修饰和分泌。
术语“核酸构建体”是指单链或双链的核酸分子，其分离自天然存在的基因或者被修饰为含有天然不存在的核酸区段。当所述核酸构建体含有表达本发明编码序列所需的控制序列时，术语核酸构建体与术语“表达盒”同义。
术语“表达载体”在本文是指线性或环形DNA分子，其包含编码本发明多肽的多核苷酸，所述多核苷酸与为所述多核苷酸表达而提供的另外的核苷酸，例如，控制序列，可操纵地连接。所述表达载体包括病毒载体或质粒载体。
术语“控制序列”在本文是指包括表达编码本发明多肽的多核苷酸所需或有利的所有元件。各控制序列对于编码多肽的核苷酸序列可以是天然的或者是外来的，或者彼此是天然或者外来的。这种控制序列包括但不限于前导序列、聚腺苷酸化序列、前肽序列、启动子、信号肽序列及转录终止子。最低限度，控制序列包括启动子和转录及翻译终止信号。
例如，所述控制序列可以是合适的启动子序列，一种由宿主细胞识别以表达编码本发明多肽的多核苷酸的核苷酸序列。所述启动子序列含有介导所述多肽的表达的转录控制序列。所述启动子可以是在所选择的宿主细胞中表现出转录活性的任何核苷酸序列，例如，大肠杆菌(Escherichia coli)lac操纵子。所述启动子还包括突变的、截短的和杂合的启动子，并且可以从与宿主细胞同源或异源的编码胞外或胞内多肽的基因获得。
术语“可操纵地连接”在本文是指这样的构型，其中控制序列置于相对于多核苷酸序列的编码序列的适当位置，由此所述控制序列指导多肽编码序列的表达。
编码本发明多肽的多核苷酸可以进行各种操作，以使得多肽表达。在将其插入载体之前，根据表达载体对多核苷酸的操作是可取的或必需的。利用重组DNA方法修饰多核苷酸序列的技术为本领域熟知。
为了鉴定和选择包含本发明的表达载体的宿主细胞，本发明的载体优选含有一或多个可选择标记，其使得可以对转化、转染、转导等的细胞进行简单的选择。可选择标记是一种基因，其产物提供生物杀灭剂或病毒抗性、重金属抗性、补充营养缺陷型等。例如，细菌的可选择标记是来自枯草芽孢杆菌或地衣芽孢杆菌的dal基因，或者赋予抗生素抗性如氨苄青霉素、卡那霉素、氯霉素或四环素抗性的标记。
本发明的载体可整合进宿主细胞基因组中或者在细胞中不依赖于基因组而自主复制。为了整合进宿主细胞基因组中或者自主复制所需的元件是本领域已知的(参见例如前述Sambrook et al.,1989)。
载体DNA可以通过常规转化或转染技术导入原核或真核细胞中。如本文所用，术语“转化”和“转染”是指将外源核酸(例如DNA)导入宿主细胞中的各种本领域公认的技术，可见于例如前述Sambrook et al.,1989；Davis et al.,Basic Methods in Molecular Biology(1986)及其它实验室手册。
本发明还涉及重组宿主细胞，其包含本发明的多核苷酸，所述多核苷酸有利地用于DAAO多肽的重组产生中。包含本发明多核苷酸的载体被导入宿主细胞中，由此所述载体作为染色体整合体或作为自身复制染色体外载体被保留。本领域技术人员知晓表达蛋白质的常规载体和宿主细胞。
在一些实施方案中，本发明的宿主细胞是大肠杆菌细胞，如大肠杆菌BL21(DE3)。在一些实施方案中，所述表达载体是pET-30a(+)。
本发明的经修饰的DAAO可以与非-DAAO多肽(例如异源氨基酸序列)可操纵地连接，形成融合蛋白。例如，在一个实施方案中，所述融合蛋白是GST-DAAO融合蛋白，其中DAAO序列与GST序列的C-末端融合。这种融合蛋白可帮助重组DAAO的纯化。在另一实施方案中，所述融合蛋白是在其N末端含有异源信号序列的DAAO蛋白。在某些宿主细胞中(例如哺乳动物和酵母宿主细胞)，可以通过使用异源信号序列增加DAAO的表达和/或分泌。
四、生产L-草铵膦
此外，本发明提供一种制备L-草铵膦的方法，包括使本发明的经修饰的DAAO或宿主细胞与D-草铵膦接触。
在一些实施方案中，本发明的制备L-草铵膦的方法包括如下步骤：
(a)向反应介质提供本发明的经修饰的DAAO的活性和D-草铵膦，任选地，向所述反应介质提供过氧化氢酶活性，
(b)温育所述反应介质以使得D-草铵膦氧化为PPO，及
(c)对PPO进行还原或转氨生成L-草铵膦。
在一些实施方案中，使用无细胞催化方法生产L-草铵膦，在步骤(a)中，提供本发明的经修饰的DAAO。在一些实施方案中，可以使用游离或固定化的本发明的修饰的DAAO。过氧化氢酶也可以被固定化。
在一些实施方案中，所述温育在20-50℃，优选25-40℃，更优选28-35℃，例如30℃进行。
在一些实施方案中，所述介质是缓冲液，例如PBS、Tris-HCl缓冲液。在一个实施方案中，所述介质是Tris-HCl缓冲液，例如50mM、pH8.0的Tris-HCl缓冲液。
在一些实施方案中，反应介质是部分或全部由细胞培养基组成的介质，本发明的经修饰的DAAO的活性由本发明的宿主细胞提供，所述宿主细胞在所述反应介质中培养。
在一些实施方案中，所述反应介质是部分或全部由细胞培养基组成的介质，且所述过氧化氢酶活性由本发明的宿主细胞或者由第二种宿主细胞提供，所述宿主细胞在所述反应介质中培养。
在一些实施方案中，将本发明的宿主细胞和/或所述第二宿主细胞在细胞培养基中培养并扩增，然后从细胞培养基分离经扩增的宿主细胞，使用缓冲液或水使生物量重悬浮。在加入所述经扩增的宿主细胞之前、期间或之后在所述缓冲液或水中加入D-草铵膦。
在一些实施方案中，可以使用细菌细胞，例如大肠杆菌细胞。
实施例
通过以下实施例，本领域技术人员会更清楚地理解本发明。应理解，实施例只是用于说明，而非限制本发明的范围
实施例1、材料和方法
如无特别说明，本发明中使用的实验方法均为常规方法，基因克隆操作具体可参见前述Sambrook et al.,1989。
i)试剂：DNA聚合酶(PrimeSTAR Max DNA Polymerase)和DpnI内切酶购自TaKaRa公司，质粒提取试剂盒购自Axygen公司，过氧化氢酶购自枣庄全鼎生物科技有限公司，货号QD-001，D,L-草铵膦购自利尔化学股份有限公司。
ii)载体和菌株：所使用的表达载体为pET-30a(+)，质粒购自Novagen公司，所使用的宿主细胞为大肠杆菌BL21(DE3)，购自天根生化科技(北京)有限公司。
iii)测序与引物合成由苏州泓迅生物科技股份有限公司完成。
iv)定点突变：
设计特异性引物对，在所需突变的氨基酸位置对应碱基引入所需的取代。用提取的突变前质粒(包含野生型DAAO编码序列，pET-30a(+)骨架)为模版，利用Quickchange技术(Nucleic Acids Research,2004,32(14):e115)通过PCR引入突变。PCR扩增结束后，扩增产物用Dpn Ⅰ消化4h去除模版质粒。将消化产物转化至大肠杆菌BL21(DE3)感受 态细胞中，涂布于LB琼脂培养基(含有50mg/L的卡那霉素)、挑单菌落至LB液体培养基(含有50mg/L的卡那霉素)中培养，测序验证突变正确性。经验证的克隆置于-80℃保藏备用。
v)蛋白表达及粗酶液的制备：
在LB琼脂培养基上将保藏的克隆活化。然后，将单菌落接种至LB液体培养基(含有50mg/L的卡那霉素)中，37℃震荡温育12h。将1mL培养物转接至50mL新鲜的LB液体培养基(含有50mg/L的卡那霉素)中，37℃震荡温育至OD600达到0.6左右，加入IPTG(终浓度为0.4mM)在25℃温育16h以诱导蛋白质表达。
温育后，将培养物以4,000g在4℃离心10min，弃上清，收集大肠杆菌细胞。将收集的大肠杆菌细胞重悬于预冷的15mL pH 7.0的磷酸盐缓冲液(PBS)中，在4℃超声破碎大肠杆菌细胞。细胞破碎液以6,000g在4℃离心15min去除沉淀，得到的上清为含重组酶的粗酶液(13g/L)。
vi)酶活性测定
将D,L-草铵膦溶解于50mM pH＝8的Tris-HCl缓冲液中，溶液中D,L-草铵膦的终浓度为100mM。向上述溶液加入2g/L的粗酶和2g/L的过氧化氢酶。在30℃，于振荡器上持续震荡(400rpm)2小时，取样并用OPA柱前衍生化高效液相色谱检测D-草铵膦的减少量与ee值，从而测定催化反应初始速度。
实施例2、制备和检测来自圆红冬孢酵母的DAAO(RtDAAO)的突变体
以RtDAAO(SEQ ID NO:1)的编码核酸为模板，根据实施例1的方法制备突变体。所得突变体如表1所示，其中SEQ ID NO:4的突变体是US 9,834,802中报道的突变体(RtDAAO N54V、F58Q、M213S)。将得到的突变体在45℃温育20min后根据实施例1描述的方法测量酶活，结果示于表1，其中相对酶活是指温育后，突变体的活性vs.SEQ ID NO:4的活性的百分比(100-150％表示为“+”，150-200％表述为“++”，大于200％表述为“+++”)。
表1
突变体中的取代 SEQ ID NO: 相对酶活
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 +++
然后在SEQ ID NO:12的基础上进一步进行突变，所得突变体如表2所示。测量所 的突变体的相对酶活(不经热处理，突变体的活性vs.SEQ ID NO:4的活性的百分比)。然后，并将所得突变体在一系列温度(40-60℃)温育1h，并测量T50(在该温度温育1h，酶活性降低50％)，结果示于表2。
表2
Figure PCTCN2020134540-appb-000003
在SEQ ID NO:14的基础上进一步引入氨基酸取代，并检测突变体的酶活性(不经热处理)，所得结果示于表3，其中相对酶活是指，不经热处理，突变体的活性vs.SEQ ID NO:4的活性的百分比(110-120％表示为“+”，120-150％表示为“++”，150-200％表示为“+++”，大于200％表示为“++++”)。
表3
Figure PCTCN2020134540-appb-000004
实施例3、制备和检测来自Rhodotorula sp.JG-1b的DAAO(RsDAAO)的突变体
以RsDAAO(SEQ ID NO:2)的编码核酸为模板，根据实施例1的方法制备突变体，并测量酶活性。所得突变体及其酶活性如表4所示，其中相对酶活是指，不经热处理，突变体的活性vs.SEQ ID NO:4的活性的百分比(小于70％表述为“--”，70-100％表示为“-”，110-120％表示为“+”，120-150％表示为“++”，150-200％表示为“+++”，大于200％表示为“++++”)，野生型RsDAAO(SEQ ID NO:2)的活性为0。
表4
突变体中的取代 SEQ ID NO: 相对酶活
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 ++++
在SEQ ID NO:38的基础上进一步引入氨基酸取代，并测量所得突变体的酶活。所得突变体如表5所示，(不经热处理)突变体的活性与SEQ ID NO:38相当。将SEQ ID NO:38的突变体和进一步引入氨基酸取代所得的突变体在43℃温育20min，测量经温育的突变体的活性，结果示于表5，其中相对酶活是指在43℃温育20min后，突变体的活性vs.SEQ ID NO:38的活性的百分比(150-200％表述为“++”，大于200％表述为“+++”)。
表5
突变体中的取代 SEQ ID NO: 相对酶活
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 +++
在SEQ ID NO:54的基础上进一步引入氨基酸取代，并测量所得突变体的酶活。所得突变体及其酶活性如表6所示，其中相对酶活是指，不经热处理，突变体的活性vs.SEQ ID NO:4的活性的百分比(120-150％表示为“++”，150-200％表示为“+++”，大于200％表示为“++++”)。
表6
Figure PCTCN2020134540-appb-000005
实施例4、制备和检测来自Rhodotorula taiwanensis的DAAO(RtnDAAO)的突变体
以RtnDAAO(SEQ ID NO:3)的编码核酸为模板，根据实施例1的方法制备突变体，并测量酶活性。所得突变体及其酶活性如表7所示，其中相对酶活是指突变体的活性vs.SEQ ID NO:4的活性的百分比(小于70％表述为“--”，70-100％表示为“-”，110-120％表示为“+”，120-150％表示为“++”)，野生型RtnDAAO(SEQ ID NO:3)的活性为0。
表7
突变体中的取代 SEQ ID NO: 相对酶活
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 ++
实施例5、基于不同的野生型采用各种取代的组合制备突变体
根据以上实施例描述的方法，在野生型RtDAAO和RsDAAO中分别引入多种突变组合制备突变体，并测量酶活性。
基于RtDAAO的突变体及其酶活如表8所示，其中相对酶活是指突变体的活性vs.SEQ ID NO:4的活性的百分比(110-120％表示为“+”，120-150％表示为“++”，150-200％表示为“+++”，大于200％表示为“++++”)。
表8
突变体中的取代 SEQ ID NO: 相对酶活
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 ++++
基于RsDAAO的突变体及其酶活如表9所示，其中相对酶活是指突变体的活性vs.SEQ ID NO:77的活性的百分比(110-120％表示为“+”，120-150％表示为“++”，150-200％表示为“+++”，大于200％表示为“++++”)。
表9
突变体中的取代 SEQ ID NO: 相对酶活
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 (16)

  1. 一种经修饰的D-氨基酸氧化酶(DAAO)，
    与其野生型DAAO相比，包含位置54、56、58和213的氨基酸取代，其中所述位置参照SEQ ID NO:2进行编号，其中位置54取代为I、V、T或L，位置56取代为N，位置58取代为H或Q，位置213取代为S或T，且
    其中所述经修饰的DAAO具有催化D-草铵膦氧化为PPO的活性。
  2. 权利要求1的经修饰的DAAO，其中位置54取代为I或V，位置58取代为H，位置213取代为S。
  3. 权利要求1或2的经修饰的DAAO，其中所述经修饰的DAAO还包含位置210和/或221的氨基酸取代，其中位置210取代为A、G或P，位置P221取代为R。
  4. 权利要求3的经修饰的DAAO，其中位置210取代为A。
  5. 一种经修饰的DAAO，与其野生型DAAO相比，包含位置54、58、213和221的氨基酸取代，所述位置参照SEQ ID NO:2进行编号，其中位置54取代为V，位置58取代为Q，位置213取代为S，位置221取代为R，且其中所述经修饰的DAAO具有催化D-草铵膦氧化为PPO的活性。
  6. 权利要求5的经修饰的DAAO，其中所述经修饰的DAAO还包含位置210和/或56的氨基酸取代，位置56取代为N，位置210取代为A。
  7. 权利要求1-6任一项的经修饰的DAAO，其中所述经修饰的DAAO还包含选自2、81、97、193、194、237、265、273、274、300、317、319、337和342的一或多个位置的取代，其中位置2取代为C或S，位置81取代为Y，位置97取代为V，位置193取代为T，位置194取代为V或C，位置237取代为V，位置265取代为C，位置273取代为D，位置274取代为E，位置300取代为S或T，位置317取代为Y或W，位置319取代为K，位置337取代为S，位置342取代为S或H。
  8. 权利要求1-6任一项的经修饰的DAAO，其中所述经修饰的DAAO还包含选自194、237、265、273、274、300、317和319的一或多个位置的取代，其中位置194取代为V或C，位置237取代为V，位置265取代为C，位置273取代为D，位置274取代为E，位置300取代为S，位置317取代为Y或W，位置319取代为K。
  9. 权利要求1-6任一项的经修饰的DAAO，其中所述经修饰的DAAO还包含选自位置2、81、97、193、300、337和342的一或多个位置的取代，其中位置2取代为C或S，位置81取代为Y，位置97取代为V，位置193取代为T，位置300取代为T，位置337取代为S，位置342取代为S或H。
  10. 一种经修饰的DAAO，其与SEQ ID NO:1相比，包含位置54、58、194和213的氨基酸取代，其中位置54取代为I、V、T或L，位置58取代为H或Q，位置194取代为V或C，位置213取代为S或T，且其中所述经修饰的DAAO具有催化D-草铵膦氧化为PPO的活性。
  11. 权利要求10的经修饰的DAAO，其中所述经修饰的DAAO还包含选自位置56、210、221、237、265、273、274、300、317和319的一或多个位置的氨基酸取代，其中位置56取代为N，位置210取代为A、G或P，位置221取代为R，位置237取代为V，位置265取代为C，位置273取代为D，位置274取代为E，位置300取代为S，位置317取代为Y或W，位置319取代为K。
  12. 一种经修饰的DAAO，包含SEQ ID NO:5-86之一的氨基酸序列或由SEQ ID NO:5-86之一的氨基酸序列组成，或者所述经修饰的DAAO与SEQ ID NO:5-30和66-76之一相比，在除位置54、56、58、194、210、213、221、237、265、273、274、300、317和319之外的位置包含1-10个氨基酸取代，与SEQ ID NO:31-57和77-86之一相比，在除位置2、54、56、58、81、97、193、210、213、221、300、337和342之外的位置包含1-10个氨基酸取代，或与SEQ ID NO:58-65之一相比，在除位置54、56、58、210、213和221之外的位置包含1-10个氨基酸取代，其中所述经修饰的DAAO具有催化D-草铵膦氧化为PPO的活性。
  13. 一种多核苷酸，编码权利要求1-12任一项的经修饰的DAAO。
  14. 一种表达载体，包含权利要求13的多核苷酸。
  15. 一种宿主细胞，包含权利要求1-12任一项的经修饰的DAAO、权利要求13多核苷酸或权利要求14的载体。
  16. 一种生产L-草铵膦的方法，包括使1-12任一项的经修饰的DAAO或权利要求15的宿主细胞与D-草铵膦接触。
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Free format text: 1) EXPLIQUE EM ATE 60 (SESSENTA) DIAS, COM DOCUMENTOS COMPROBATORIOS SE NECESSARIO, A DIVERGENCIA NO NOME DO INVENTOR CONSTANTE NO PEDIDO INTERNACIONAL COMO ?WEI XU? CONSTANTE NO FORMULARIO DA PETICAO INICIAL NO 870220045886 COMO ?WE XU?. 2) FAVOR EFETUAR, EM ATE 60 (SESSENTA) DIAS, O PAGAMENTO DE GRU CODIGO DE SERVICO 260 PARA A REGULARIZACAO DO PEDIDO, CONFORME ART 2O 1O DA RESOLUCAO 189/2017 E NOTA DE ESCLARECIMENTO PUBLICADA NA RPI 2421 DE 30/05/2017, UMA VEZ QUE A PETICAO NO 870220049644 DE 06/06/2022 APRESENTA DOCUMENTOS REFERENTES A 2 SERVICOS DIVERSOS (COMPLEMENTACAO DA TRADUCAO E MODIFICACAO DE PAGINAS JA ENVIADAS NO PEDIDO) TENDO SIDO PAGA SOMENTE 1 RETRIBUICAO. DEVERA SER PAGA MA

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