WO2022007881A1 - 经修饰的谷氨酸脱氢酶及其应用 - Google Patents

经修饰的谷氨酸脱氢酶及其应用 Download PDF

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WO2022007881A1
WO2022007881A1 PCT/CN2021/105179 CN2021105179W WO2022007881A1 WO 2022007881 A1 WO2022007881 A1 WO 2022007881A1 CN 2021105179 W CN2021105179 W CN 2021105179W WO 2022007881 A1 WO2022007881 A1 WO 2022007881A1
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amino acid
acid substitution
gludh
modified
positions
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PCT/CN2021/105179
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English (en)
French (fr)
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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 MX2023000450A priority Critical patent/MX2023000450A/es
Priority to AU2021306531A priority patent/AU2021306531A1/en
Priority to US18/004,768 priority patent/US20230332113A1/en
Priority to BR112023000234A priority patent/BR112023000234A2/pt
Priority to JP2022580791A priority patent/JP2023532699A/ja
Priority to EP21837954.3A priority patent/EP4180524A1/en
Priority to IL299379A priority patent/IL299379A/en
Priority to CA3184703A priority patent/CA3184703A1/en
Priority to KR1020237003987A priority patent/KR20230031956A/ko
Publication of WO2022007881A1 publication Critical patent/WO2022007881A1/zh

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
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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)
    • C12N9/0016—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 NAD or NADP as acceptor (1.4.1)
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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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
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    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
    • C12Y104/01002—Glutamate dehydrogenase (1.4.1.2)

Definitions

  • the present invention relates to the field of enzyme engineering.
  • the present invention relates to modified glutamate dehydrogenase (GluDH) and its use in the production of glufosinate-ammonium.
  • GluDH modified glutamate dehydrogenase
  • Glufosinate also known as 4-[hydroxy(methyl)phosphono]-D,L-homoalanine
  • Glufosinate-ammonium is a broad-spectrum contact herbicide.
  • L-glutamine synthase By inhibiting the activity of L-glutamine synthase in plants, it leads to nitrogen metabolism disorders in plants and eventually kills plants.
  • glyphosate glufosinate has significant advantages, such as wide application range, quick effect, long lasting effect, lower toxicity and safety. Therefore, the sales volume of glufosinate-ammonium is growing rapidly, and the market demand will be huge in the next period of time, and the prospect is very broad.
  • glufosinate-ammonium is a racemic mixture (D,L-glufosinate-ammonium) containing equal amounts of two optical isomers, but only the L-configuration is physiologically active. Therefore, the preparation of chiral pure L-glufosinate by deracemization of D,L-glufosinate has important practical significance, and has become a popular direction for the synthesis of L-glufosinate in recent years.
  • L-glufosinate Convert D, L-glufosinate into N-acetyl glufosinate, and then catalyzed by carboxypeptidase, L-type N-acetyl glufosinate is selectively hydrolyzed to obtain L-glufosinate, while D-type N -Acetyl glufosinate is not hydrolyzed and can be recycled into the hydrolysis step after chemical or enzymatic racemization (see eg Chinese patent application CN108690854A).
  • the disadvantage of this method is the need for multi-step reactions and the need to separate the L-glufosinate-ammonium obtained by hydrolysis from the N-acetylated substrate.
  • D-glufosinate is oxidized to 2-carbonyl-4-(hydroxymethylphosphono) butyric acid (PPO), and then PPO is reduced or transaminated to form L-glufosinate.
  • PPO D-amino acid oxidase
  • DAAO D-amino acid oxidase
  • PPO can be reduced to D,L-glufosinate by formic acid under palladium-carbon catalysis.
  • DAAO D-amino acid oxidase
  • D,L-glufosinate is gradually converted to L-glufosinate (see, eg, 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.
  • PPO can also be converted to L-glufosinate using a stereoselective transamination reaction catalyzed by L-amino acid transaminase (L-TA) (see, eg, US20180030487A1).
  • L-TA L-amino acid transaminase
  • the disadvantage of this scheme is that the transamination step is an equilibrium reaction, and an excess of amino donor (amino acid or organic amine) needs to be provided to achieve high conversion (eg, 3-fold equivalent of amino donor, 90% conversion), while excess The amino donor and the corresponding by-products will seriously affect the subsequent separation and purification steps.
  • L-amino acid dehydrogenase L-AADH
  • NAD(P)H recycling system can consume formic acid, glucose or simple alcohols , which converts PPO to L-glufosinate-ammonium.
  • the reaction catalyzed by L-AADH does not require a large excess of hydrogen donor and can achieve high conversion.
  • GluDH glutamate dehydrogenase
  • the present invention provides a modified glutamate dehydrogenase (GluDH) comprising amino acid substitutions at two or more positions compared to its starting GluDH, wherein the modified GluDH has increased and/or have improved kinetic properties (eg, increased Vmax value, decreased Km value, or increased Vmax/Km).
  • GluDH glutamate dehydrogenase
  • the modified GluDH has an amino acid substitution selected from the group consisting of:
  • the amino acid substitution at position 104 is C and the amino acid substitution at position 175 is G;
  • the amino acid substitution at position 132 is L and the amino acid substitution at position 175 is G;
  • the amino acid substitution at position 133 is V and the amino acid substitution at position 175 is G;
  • the amino acid substitution at position 173 is G or S, and the amino acid substitution at position 175 is G;
  • amino acid substitution at position 175 is G and the amino acid substitution at position 181 is K or R;
  • amino acid substitution at position 175 is G and the amino acid substitution at position 182 is R;
  • the amino acid substitution at position 175 is G and the amino acid substitution at position 203 is I;
  • the amino acid substitution at position 132 is L, the amino acid substitution at position 133 is V, and the amino acid substitution at position 175 is G;
  • the amino acid substitution at position 132 is L, the amino acid substitution at position 173 is S, and the amino acid substitution at position 175 is G;
  • the amino acid substitution at position 132 is L, the amino acid substitution at position 175 is G, and the amino acid substitution at position 182 is R;
  • the amino acid substitution at position 133 is V
  • the amino acid substitution at position 173 is G or S
  • the amino acid substitution at position 175 is G;
  • the amino acid substitution at position 133 is V, the amino acid substitution at position 175 is G, and the amino acid substitution at position 182 is R;
  • the amino acid substitution at position 173 is G, the amino acid substitution at position 175 is G, and the amino acid substitution at position 182 is R;
  • the amino acid substitution at position 132 is L, the amino acid substitution at position 133 is V, the amino acid substitution at position 173 is S, and the amino acid substitution at position 175 is G;
  • the amino acid substitution at position 132 is L, the amino acid substitution at position 133 is V, the amino acid substitution at position 175 is G, and the amino acid substitution at position 182 is R;
  • the amino acid substitution at position 132 is L, the amino acid substitution at position 173 is S, the amino acid substitution at position 175 is G, and the amino acid substitution at position 182 is R;
  • the amino acid substitution at position 133 is V, the amino acid substitution at position 173 is G or S, the amino acid substitution at position 175 is G, and the amino acid substitution at position 182 is R; and
  • the amino acid substitution at position 132 is L
  • the amino acid substitution at position 133 is V
  • the amino acid substitution at position 173 is G or S
  • the amino acid substitution at position 175 is G
  • the amino acid substitution at position 182 is R
  • the modified GluDH comprises substitutions at positions 173, 175, and 182, wherein the positions are numbered with reference to SEQ ID NO: 1, compared to its starting GluDH.
  • the amino acid substitution at position 173 is G.
  • the amino acid substitution at position 175 is G.
  • the amino acid substitution at position 182 is R.
  • the modified GluDH further comprises positions selected from the group consisting of positions 9, 22, 23, 25, 31, 56, 124, 143, 199, 216, 242, 263, 339, 420, 431 and 437 amino acid substitutions at one or more positions.
  • the amino acid substitution at position 9 is S, L or Y.
  • the amino acid substitution at position 22 is W or E.
  • the amino acid substitution at position 23 is M.
  • the amino acid substitution at position 25 is D.
  • the amino acid substitution at position 31 is H.
  • the amino acid substitution at position 56 is Q.
  • the amino acid substitution at position 124 is L.
  • the amino acid substitution at position 143 is E.
  • the amino acid substitution at position 199 is W or Y.
  • the amino acid substitution at position 216 is G.
  • the amino acid substitution at position 263 is S.
  • the amino acid substitution at position 339 is Q.
  • the amino acid substitution at position 420 is R.
  • the amino acid substitution at position 431 is S.
  • the amino acid substitution at position 437 is K.
  • the modified GluDH comprises amino acid substitutions at positions 22, 56, 173, 175, 182, 199, and 420 compared to its starting GluDH.
  • the amino acid substitution at position 22 is E.
  • the amino acid substitution at position 56 is Q.
  • the amino acid substitution at position 173 is G.
  • the amino acid substitution at position 175 is G.
  • the amino acid substitution at position 182 is R.
  • the amino acid substitution at position 199 is Y.
  • the amino acid substitution at position 420 is R.
  • the modified GluDH further comprises amino acid substitutions at one or more positions selected from positions 31, 124, and 216.
  • the amino acid substitution at position 31 is H.
  • the amino acid substitution at position 124 is L.
  • the amino acid substitution at position 216 is G.
  • the starting GluDH is wild-type GluDH.
  • the starting GluDH is derived from a microorganism of the family Bacillaceae, preferably a microorganism of the genus Lysinibacillus or Bacillus, more preferably Bacillus sphaericus (Lysinibacillus sphaericus) or Bacillus velezensis.
  • the starting GluDH comprises the amino acid sequence of SEQ ID NO: 1 or 2.
  • the modified GluDH comprises one of SEQ ID NOs: 4-14, 16-19, 21, 22, 24, 25, 27-30, 32-48, 50, 51, and 53-72 amino acid sequence.
  • the activity of the modified GluDH to catalyze the reaction of PPO with an amino donor to L-glufosinate is at least 100%, 105%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or higher.
  • the present invention provides polynucleotides encoding the modified GluDHs of the present invention, and vectors comprising the polynucleotides of the present invention.
  • the present invention provides a host cell comprising a modified GluDH of the present invention, a polynucleotide encoding the same, or a vector comprising the polynucleotide.
  • the present invention also provides a method for producing L-glufosinate-ammonium, comprising contacting the modified GluDH of the present invention or the host cell of the present invention with PPO.
  • the present invention mainly relates to modified GluDH for catalyzing the reaction of PPO with an amino donor to produce L-glufosinate.
  • terms used herein have the meanings commonly understood by those skilled in the art.
  • the terms "glutamate dehydrogenase” and “GluDH” refer to enzymes that catalyze the dehydrogenation of glutamate to produce alpha-ketoglutarate.
  • GluDH also has the activity of catalyzing the reaction between PPO and amino donor to generate L-glufosinate.
  • the present invention provides modified GluDH polypeptides having increased activity for catalyzing the reaction of PPO with an amino donor to L-glufosinate-ammonium and/or having improved kinetic properties including, but not limited to, increased Vmax, decreased Km and Improved Vmax/Km.
  • peptide refers to a chain of at least two amino acids linked by peptide bonds.
  • polypeptide is used interchangeably herein with the term “protein” and refers to a chain containing ten or more amino acid residues. All peptide and polypeptide formulas or sequences herein are written left to right, indicating the orientation from the amino terminus to the carboxy terminus.
  • amino acid includes both naturally occurring amino acids and unnatural amino acids in proteins.
  • the one-letter and three-letter names of amino acids naturally occurring in proteins use names commonly used in the art, as 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 modification to a polypeptide consisting of a polypeptide of the invention or a homologous sequence thereof, including, but not limited to, substitution, deletion, insertion and/or addition of one or more amino acids.
  • the modified GluDHs of the invention comprise amino acid substitutions at two or more positions compared to their starting GluDHs, wherein the modified GluDHs have an increased catalytic reaction of PPO with an amino donor to form L - Activity and/or improved kinetic properties of glufosinate-ammonium including, but not limited to, increased Vmax, decreased Km, and increased Vmax/Km.
  • the modified GluDH has an amino acid substitution selected from the group consisting of:
  • the amino acid substitution at position 104 is C and the amino acid substitution at position 175 is G;
  • the amino acid substitution at position 132 is L and the amino acid substitution at position 175 is G;
  • the amino acid substitution at position 133 is V and the amino acid substitution at position 175 is G;
  • the amino acid substitution at position 173 is G or S, and the amino acid substitution at position 175 is G;
  • amino acid substitution at position 175 is G and the amino acid substitution at position 181 is K or R;
  • amino acid substitution at position 175 is G and the amino acid substitution at position 182 is R;
  • the amino acid substitution at position 175 is G and the amino acid substitution at position 203 is I;
  • the amino acid substitution at position 132 is L, the amino acid substitution at position 133 is V, and the amino acid substitution at position 175 is G;
  • the amino acid substitution at position 132 is L, the amino acid substitution at position 173 is S, and the amino acid substitution at position 175 is G;
  • the amino acid substitution at position 132 is L, the amino acid substitution at position 175 is G, and the amino acid substitution at position 182 is R;
  • the amino acid substitution at position 133 is V
  • the amino acid substitution at position 173 is G or S
  • the amino acid substitution at position 175 is G;
  • the amino acid substitution at position 133 is V, the amino acid substitution at position 175 is G, and the amino acid substitution at position 182 is R;
  • the amino acid substitution at position 173 is G, the amino acid substitution at position 175 is G, and the amino acid substitution at position 182 is R;
  • the amino acid substitution at position 132 is L, the amino acid substitution at position 133 is V, the amino acid substitution at position 173 is S, and the amino acid substitution at position 175 is G;
  • the amino acid substitution at position 132 is L, the amino acid substitution at position 133 is V, the amino acid substitution at position 175 is G, and the amino acid substitution at position 182 is R;
  • the amino acid substitution at position 132 is L, the amino acid substitution at position 173 is S, the amino acid substitution at position 175 is G, and the amino acid substitution at position 182 is R;
  • the amino acid substitution at position 133 is V, the amino acid substitution at position 173 is G or S, the amino acid substitution at position 175 is G, and the amino acid substitution at position 182 is R; and
  • the amino acid substitution at position 132 is L
  • the amino acid substitution at position 133 is V
  • the amino acid substitution at position 173 is G or S
  • the amino acid substitution at position 175 is G
  • the amino acid substitution at position 182 is R
  • the modified GluDHs of the invention comprise substitutions at positions 132 and 175, where the positions are numbered with reference to SEQ ID NO: 1, compared to their starting GluDH.
  • the amino acid substitution at position 132 is L and the amino acid substitution at position 175 is G.
  • the modified GluDHs of the invention further comprise substitutions at one or more positions selected from positions 104, 133, 173, 181 and 182 compared to their starting GluDH.
  • the amino acid substitution at position 104 is C.
  • the amino acid substitution at position 133 is V.
  • the amino acid substitution at position 173 is G or S.
  • the amino acid substitution at position 181 is K or R.
  • the amino acid substitution at position 182 is R.
  • the modified GluDHs of the invention comprise substitutions at positions 133 and 175, where the positions are numbered with reference to SEQ ID NO: 1, compared to their starting GluDH.
  • the amino acid substitution at position 133 is V and the amino acid substitution at position 175 is G.
  • the modified GluDHs of the invention further comprise substitutions at one or more positions selected from positions 104, 132, 173, 181 and 182 compared to their starting GluDH.
  • the amino acid substitution at position 104 is C.
  • the amino acid substitution at position 132 is L.
  • the amino acid substitution at position 173 is G or S.
  • the amino acid substitution at position 181 is K or R.
  • the amino acid substitution at position 182 is R.
  • the modified GluDHs of the invention comprise substitutions at positions 173 and 175, where the positions are numbered with reference to SEQ ID NO: 1, compared to their starting GluDH.
  • the amino acid substitution at position 173 is G or S
  • the amino acid substitution at position 175 is G.
  • the modified GluDHs of the invention further comprise substitutions at one or more positions selected from positions 104, 132, 133, 181 and 182 compared to their starting GluDH.
  • the amino acid substitution at position 104 is C.
  • the amino acid substitution at position 132 is L.
  • the amino acid substitution at position 133 is V.
  • the amino acid substitution at position 181 is K or R.
  • the amino acid substitution at position 182 is R.
  • the modified GluDHs of the invention comprise substitutions at positions 175 and 182, where the positions are numbered with reference to SEQ ID NO: 1, compared to their starting GluDH.
  • the amino acid substitution at position 175 is G and the amino acid substitution at position 182 is R.
  • the modified GluDHs of the invention further comprise substitutions at one or more positions selected from positions 104, 132, 133, 173 and 181 compared to their starting GluDH.
  • the amino acid substitution at position 104 is C.
  • the amino acid substitution at position 132 is L.
  • the amino acid substitution at position 133 is V.
  • the amino acid substitution at position 173 is G or S.
  • the amino acid substitution at position 181 is K or R.
  • the modified GluDHs of the invention comprise substitutions at positions 132, 133, and 175, where the positions are numbered with reference to SEQ ID NO: 1, compared to their starting GluDH.
  • the amino acid substitution at position 132 is L
  • the amino acid substitution at position 133 is V
  • the amino acid substitution at position 175 is G.
  • the modified GluDHs of the invention further comprise substitutions at one or more positions selected from positions 104, 173, 181 and 182 compared to their starting GluDH.
  • the amino acid substitution at position 104 is C.
  • the amino acid substitution at position 173 is G or S.
  • the amino acid substitution at position 181 is K or R.
  • the amino acid substitution at position 182 is R.
  • the modified GluDHs of the invention comprise substitutions at positions 132, 173, and 175, where the positions are numbered with reference to SEQ ID NO: 1, compared to their starting GluDH.
  • the amino acid substitution at position 132 is L
  • the amino acid substitution at position 173 is G or S
  • the amino acid substitution at position 175 is G.
  • the modified GluDHs of the invention further comprise substitutions at one or more positions selected from positions 104, 133, 181 and 182 compared to their starting GluDH.
  • the amino acid substitution at position 104 is C.
  • the amino acid substitution at position 133 is V.
  • the amino acid substitution at position 181 is K or R.
  • the amino acid substitution at position 182 is R.
  • the modified GluDHs of the invention comprise substitutions at positions 133, 173, and 175, where the positions are numbered with reference to SEQ ID NO: 1, compared to their starting GluDH.
  • the amino acid substitution at position 133 is V
  • the amino acid substitution at position 173 is G or S
  • the amino acid substitution at position 175 is G.
  • the modified GluDHs of the invention further comprise substitutions at one or more positions selected from positions 104, 132, 181 and 182 compared to their starting GluDH.
  • the amino acid substitution at position 104 is C.
  • the amino acid substitution at position 132 is L.
  • the amino acid substitution at position 181 is K or R.
  • the amino acid substitution at position 182 is R.
  • the modified GluDHs of the invention comprise substitutions at positions 132, 175, and 182, where the positions are numbered with reference to SEQ ID NO: 1, compared to their starting GluDH.
  • the amino acid substitution at position 132 is L
  • the amino acid substitution at position 175 is G
  • the amino acid substitution at position 182 is R.
  • the modified GluDHs of the invention further comprise substitutions at one or more positions selected from positions 104, 133, 173, and 181 compared to their starting GluDH.
  • the amino acid substitution at position 104 is C.
  • the amino acid substitution at position 133 is V.
  • the amino acid substitution at position 173 is G or S.
  • the amino acid substitution at position 181 is K or R.
  • the modified GluDHs of the invention comprise substitutions at positions 133, 175, and 182, where the positions are numbered with reference to SEQ ID NO: 1, compared to their starting GluDH.
  • the amino acid substitution at position 133 is V
  • the amino acid substitution at position 175 is G
  • the amino acid substitution at position 182 is R.
  • the modified GluDHs of the invention further comprise substitutions at one or more positions selected from positions 104, 132, 173 and 181 compared to their starting GluDH.
  • the amino acid substitution at position 104 is C.
  • the amino acid substitution at position 132 is L.
  • the amino acid substitution at position 173 is G or S.
  • the amino acid substitution at position 181 is K or R.
  • the modified GluDHs of the invention comprise substitutions at positions 173, 175, and 182, where the positions are numbered with reference to SEQ ID NO: 1, compared to their starting GluDH.
  • the amino acid substitution at position 173 is G or S
  • the amino acid substitution at position 175 is G
  • the amino acid substitution at position 182 is R.
  • the modified GluDHs of the invention further comprise substitutions at one or more positions selected from positions 104, 132, 133, and 181 compared to their starting GluDH.
  • the amino acid substitution at position 104 is C.
  • the amino acid substitution at position 132 is L.
  • the amino acid substitution at position 133 is V.
  • the amino acid substitution at position 181 is K or R.
  • the GluDH polypeptide on which the amino acid modification is made is referred to as the starting GluDH.
  • the starting GluDH can be wild-type GluDH or a variant of wild-type GluDH.
  • the polypeptide of SEQ ID NO: 1 is the "starting GluDH” relative to the modified GluDH; SEQ ID NOs: 3-30)
  • the variant polypeptide is the "starting GluDH” relative to the modified GluDH.
  • wild-type GluDH refers to naturally occurring GluDH.
  • the starting GluDH is derived from a microorganism of the family Bacillus.
  • the wild-type GluDH is a GluDH from a microorganism of the genus Bacillus lysine or Bacillus.
  • the wild-type GluDH is GluDH from Bacillus sphaericus (SEQ ID NO:2) or GluDH from Bacillus velesi (SEQ ID NO:1).
  • the sequences are aligned for optimal comparison (eg gaps can be introduced in the first amino acid or nucleic acid sequence to match the second amino acid sequence) or nucleic acid sequences 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 at the corresponding position in the second sequence, then the molecules are identical at that position.
  • percent identity number of identical positions/total number of positions (ie, overlapping positions) x 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 that, when optimally aligned, have approximately the specified percentage of amino acids identical. For example, “95% amino acid identity” refers to comparing the amino acids of two polypeptides which, when optimally aligned, are 95% identical.
  • wild-type GluDH as used herein has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94% of SEQ ID NO: 1 or 2 , 95%, 96%, 97%, 98% or 99% sequence identity.
  • the modified GluDH has an amino acid substitution selected from the group consisting of:
  • the amino acid substitution at position 104 is C and the amino acid substitution at position 175 is G;
  • the amino acid substitution at position 132 is L and the amino acid substitution at position 175 is G;
  • the amino acid substitution at position 133 is V and the amino acid substitution at position 175 is G;
  • the amino acid substitution at position 173 is G or S, and the amino acid substitution at position 175 is G;
  • amino acid substitution at position 175 is G and the amino acid substitution at position 181 is K or R;
  • amino acid substitution at position 175 is G and the amino acid substitution at position 182 is R;
  • the amino acid substitution at position 175 is G and the amino acid substitution at position 203 is I;
  • the amino acid substitution at position 132 is L, the amino acid substitution at position 133 is V, and the amino acid substitution at position 175 is G;
  • the amino acid substitution at position 132 is L, the amino acid substitution at position 173 is S, and the amino acid substitution at position 175 is G;
  • the amino acid substitution at position 132 is L, the amino acid substitution at position 175 is G, and the amino acid substitution at position 182 is R;
  • the amino acid substitution at position 133 is V
  • the amino acid substitution at position 173 is G or S
  • the amino acid substitution at position 175 is G;
  • the amino acid substitution at position 133 is V, the amino acid substitution at position 175 is G, and the amino acid substitution at position 182 is R;
  • the amino acid substitution at position 173 is G, the amino acid substitution at position 175 is G, and the amino acid substitution at position 182 is R;
  • the amino acid substitution at position 132 is L, the amino acid substitution at position 133 is V, the amino acid substitution at position 173 is S, and the amino acid substitution at position 175 is G;
  • the amino acid substitution at position 132 is L, the amino acid substitution at position 133 is V, the amino acid substitution at position 175 is G, and the amino acid substitution at position 182 is R;
  • the amino acid substitution at position 132 is L, the amino acid substitution at position 173 is S, the amino acid substitution at position 175 is G, and the amino acid substitution at position 182 is R;
  • the amino acid substitution at position 133 is V, the amino acid substitution at position 173 is G or S, the amino acid substitution at position 175 is G, and the amino acid substitution at position 182 is R; and
  • the amino acid substitution at position 132 is L
  • the amino acid substitution at position 133 is V
  • the amino acid substitution at position 173 is G or S
  • the amino acid substitution at position 175 is G
  • the amino acid substitution at position 182 is R
  • the starting GluDH has at least 65% or 70% of SEQ ID NO: 1 or 2, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the modified GluDHs of the invention comprise substitutions at positions 132 and 175, where the positions are numbered with reference to SEQ ID NO: 1, compared to their starting GluDH.
  • the amino acid substitution at position 132 is L and the amino acid substitution at position 175 is G.
  • the modified GluDHs of the invention further comprise substitutions at one or more positions selected from positions 104, 133, 173, 181 and 182 compared to their starting GluDH.
  • the amino acid substitution at position 104 is C.
  • the amino acid substitution at position 133 is V.
  • the amino acid substitution at position 173 is G or S.
  • the amino acid substitution at position 181 is K or R.
  • the amino acid substitution at position 182 is R.
  • the starting GluDH has at least 65% or 70% of SEQ ID NO: 1 or 2, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the modified GluDHs of the invention comprise substitutions at positions 133 and 175, where the positions are numbered with reference to SEQ ID NO: 1, compared to their starting GluDH.
  • the amino acid substitution at position 133 is V and the amino acid substitution at position 175 is G.
  • the modified GluDHs of the invention further comprise substitutions at one or more positions selected from positions 104, 132, 173, 181 and 182 compared to their starting GluDH.
  • the amino acid substitution at position 104 is C.
  • the amino acid substitution at position 132 is L.
  • the amino acid substitution at position 173 is G or S.
  • the amino acid substitution at position 181 is K or R.
  • the amino acid substitution at position 182 is R.
  • the starting GluDH has at least 65% or 70% of SEQ ID NO: 1 or 2, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the modified GluDHs of the invention comprise substitutions at positions 173 and 175, where the positions are numbered with reference to SEQ ID NO: 1, compared to their starting GluDH.
  • the amino acid substitution at position 173 is G or S
  • the amino acid substitution at position 175 is G.
  • the modified GluDHs of the invention further comprise substitutions at one or more positions selected from positions 104, 132, 133, 181 and 182 compared to their starting GluDH.
  • the amino acid substitution at position 104 is C.
  • the amino acid substitution at position 132 is L.
  • the amino acid substitution at position 133 is V.
  • the amino acid substitution at position 181 is K or R.
  • the amino acid substitution at position 182 is R.
  • the starting GluDH has at least 65% or 70% of SEQ ID NO: 1 or 2, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the modified GluDHs of the invention comprise substitutions at positions 175 and 182, where the positions are numbered with reference to SEQ ID NO: 1, compared to their starting GluDH.
  • the amino acid substitution at position 175 is G and the amino acid substitution at position 182 is R.
  • the modified GluDHs of the invention further comprise substitutions at one or more positions selected from positions 104, 132, 133, 173 and 181 compared to their starting GluDH.
  • the amino acid substitution at position 104 is C.
  • the amino acid substitution at position 132 is L.
  • the amino acid substitution at position 133 is V.
  • the amino acid substitution at position 173 is G or S.
  • the amino acid substitution at position 181 is K or R.
  • the starting GluDH has at least 65% or 70% of SEQ ID NO: 1 or 2, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the modified GluDHs of the invention comprise substitutions at positions 132, 133, and 175, where the positions are numbered with reference to SEQ ID NO: 1, compared to their starting GluDH.
  • the amino acid substitution at position 132 is L
  • the amino acid substitution at position 133 is V
  • the amino acid substitution at position 175 is G.
  • the modified GluDHs of the invention further comprise substitutions at one or more positions selected from positions 104, 173, 181 and 182 compared to their starting GluDH.
  • the amino acid substitution at position 104 is C.
  • the amino acid substitution at position 173 is G or S.
  • the amino acid substitution at position 181 is K or R.
  • the amino acid substitution at position 182 is R.
  • the starting GluDH has at least 65% or 70% of SEQ ID NO: 1 or 2, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the modified GluDHs of the invention comprise substitutions at positions 132, 173, and 175, where the positions are numbered with reference to SEQ ID NO: 1, compared to their starting GluDH.
  • the amino acid substitution at position 132 is L
  • the amino acid substitution at position 173 is G or S
  • the amino acid substitution at position 175 is G.
  • the modified GluDHs of the invention further comprise substitutions at one or more positions selected from positions 104, 133, 181 and 182 compared to their starting GluDH.
  • the amino acid substitution at position 104 is C.
  • the amino acid substitution at position 133 is V.
  • the amino acid substitution at position 181 is K or R.
  • the amino acid substitution at position 182 is R.
  • the starting GluDH has at least 65% or 70% of SEQ ID NO: 1 or 2, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the modified GluDHs of the invention comprise substitutions at positions 133, 173, and 175, where the positions are numbered with reference to SEQ ID NO: 1, compared to their starting GluDH.
  • the amino acid substitution at position 133 is V
  • the amino acid substitution at position 173 is G or S
  • the amino acid substitution at position 175 is G.
  • the modified GluDHs of the invention further comprise substitutions at one or more positions selected from positions 104, 132, 181 and 182 compared to their starting GluDH.
  • the amino acid substitution at position 104 is C.
  • the amino acid substitution at position 132 is L.
  • the amino acid substitution at position 181 is K or R.
  • the amino acid substitution at position 182 is R.
  • the starting GluDH has at least 65% or 70% of SEQ ID NO: 1 or 2, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the modified GluDHs of the invention comprise substitutions at positions 132, 175, and 182, where the positions are numbered with reference to SEQ ID NO: 1, compared to their starting GluDH.
  • the amino acid substitution at position 132 is L
  • the amino acid substitution at position 175 is G
  • the amino acid substitution at position 182 is R.
  • the modified GluDHs of the invention further comprise substitutions at one or more positions selected from positions 104, 133, 173, and 181 compared to their starting GluDH.
  • the amino acid substitution at position 104 is C.
  • the amino acid substitution at position 133 is V.
  • the amino acid substitution at position 173 is G or S.
  • the amino acid substitution at position 181 is K or R.
  • the starting GluDH has at least 65% or 70% of SEQ ID NO: 1 or 2, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the modified GluDHs of the invention comprise substitutions at positions 133, 175, and 182, where the positions are numbered with reference to SEQ ID NO: 1, compared to their starting GluDH.
  • the amino acid substitution at position 133 is V
  • the amino acid substitution at position 175 is G
  • the amino acid substitution at position 182 is R.
  • the modified GluDHs of the invention further comprise substitutions at one or more positions selected from positions 104, 132, 173 and 181 compared to their starting GluDH.
  • the amino acid substitution at position 104 is C.
  • the amino acid substitution at position 132 is L.
  • the amino acid substitution at position 173 is G or S.
  • the amino acid substitution at position 181 is K or R.
  • the starting GluDH has at least 65% or 70% of SEQ ID NO: 1 or 2, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the modified GluDHs of the invention comprise substitutions at positions 173, 175, and 182, where the positions are numbered with reference to SEQ ID NO: 1, compared to their starting GluDH.
  • the amino acid substitution at position 173 is G or S
  • the amino acid substitution at position 175 is G
  • the amino acid substitution at position 182 is R.
  • the modified GluDHs of the invention further comprise substitutions at one or more positions selected from positions 104, 132, 133, and 181 compared to their starting GluDH.
  • the amino acid substitution at position 104 is C.
  • the amino acid substitution at position 132 is L.
  • the amino acid substitution at position 133 is V.
  • the amino acid substitution at position 181 is K or R.
  • the starting GluDH has at least 65% or 70% of SEQ ID NO: 1 or 2, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the modified GluDHs of the invention comprise substitutions at positions 173, 175 and 182, wherein the positions are numbered with reference to SEQ ID NO:1, compared to their starting GluDH.
  • the amino acid substitution at position 173 is G
  • the amino acid substitution at position 175 is G
  • the amino acid substitution at position 182 is R.
  • the modified GluDH further comprises a group selected from positions 9, 22, 23, 25, 31, 56, 124, 143, 199, 216, 263, 339, 420, 431 and 437 or amino acid substitutions at multiple positions.
  • the amino acid substitution at position 9 is S, L or Y.
  • the amino acid substitution at position 22 is W or E.
  • the amino acid substitution at position 23 is M.
  • the amino acid substitution at position 25 is D.
  • the amino acid substitution at position 31 is H.
  • the amino acid substitution at position 56 is Q.
  • the amino acid substitution at position 124 is L.
  • the amino acid substitution at position 143 is E.
  • the amino acid substitution at position 199 is W or Y.
  • the amino acid substitution at position 216 is G.
  • the amino acid substitution at position 263 is S.
  • the amino acid substitution at position 339 is Q.
  • the amino acid substitution at position 420 is R.
  • the amino acid substitution at position 431 is S.
  • the amino acid substitution at position 437 is K.
  • the starting GluDH has at least 65% or 70% of SEQ ID NO: 1 or 2, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the starting GluDH comprises or consists of SEQ ID NO:1.
  • the modified GluDH comprises amino acid substitutions at positions 22, 56, 173, 175, 182, 199, and 420 compared to its starting GluDH.
  • the amino acid substitution at position 22 is E.
  • the amino acid substitution at position 56 is Q.
  • the amino acid substitution at position 173 is G.
  • the amino acid substitution at position 175 is G.
  • the amino acid substitution at position 182 is R.
  • the amino acid substitution at position 199 is Y.
  • the amino acid substitution at position 420 is R.
  • the modified GluDH further comprises amino acid substitutions at one or more positions selected from the group consisting of positions 31, 124, and 216.
  • the amino acid substitution at position 31 is H.
  • the amino acid substitution at position 124 is L.
  • the amino acid substitution at position 216 is G.
  • the starting GluDH has at least 65% or 70% of SEQ ID NO: 1 or 2, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the starting GluDH comprises or consists of SEQ ID NO:1.
  • the modified GluDH of the present invention has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94% of SEQ ID NO: 1 or 2 %, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the starting GluDH differs from SEQ ID NO: 1 or 2 by having one or more amino acid substitutions, deletions, insertions and/or additions. In some embodiments, the starting GluDH has a conservative substitution of one or more amino acids compared to SEQ ID NO: 1 or 2. In some embodiments, the starting GluDH has one or more amino acid insertions or deletions compared to SEQ ID NO: 1 or 2.
  • substitution also referred to as a substitution by a "homologous" amino acid residue, refers to a substitution in which the amino acid residue is replaced by an amino acid residue with a similar side chain, e.g., an amino acid with a basic side chain (e.g., lysine). , arginine and histidine), amino acids with acidic side chains (eg aspartic acid, glutamic acid), non-charged polar side chain amino acids (eg glycine, asparagine, glutamine, serine, threonine) acid, tyrosine, cysteine), non-polar side chain amino acids (e.g.
  • beta-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 minimal effect on the activity of the resulting protein. This substitution is described below.
  • a conservative substitution is the replacement of an amino acid with an amino acid that is similar in size, hydrophobicity, charge, polarity, steric characteristics, aromaticity, etc. Such substitutions are usually conservative when it is desired to fine-tune the properties of the protein.
  • homologous amino acid residues refer to amino acid residues that have 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, 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 for Ala, Lys for Arg, Gln or His for Asn, Glu for Asp, Ser for Cys, Asn for Gln, Asp for Glu, Pro for Gly, Asn or Gln for 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 GluDH comprises a The amino acid sequence or consists of the amino acid sequence of one of SEQ ID NOs: 4-14, 16-19, 21, 22, 24, 25, 27-30, 32-48, 50, 51 and 53-72, or the The modified GluDH contains 1-10 amino acids compared to one of SEQ ID NOs: 4-14, 16-19, 21, 22, 24, 25, 27-30 and, 32-48, 50, 51 and 53-72 Substitution (eg, conservative substitution), wherein the substitution is at positions 9, 22, 23, 25, 31, 56, 104, 124, 132, 133, 143, 173, 175, 181, 182, 199, 203, 216, positions other than 242, 263, 339, 420, 431 and 437, wherein the modified GluDH has increased activity for catalyzing the reaction of PPO with an amino donor to L-glufosinate compared to its starting GluDH, and /or the modified GluDH has improved kinetic properties such as increased Vmax value, decreased Km value or increased Vmax/Km.
  • the modified GluDH is one of the A comparison comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions (eg, conservative substitutions), wherein the substitutions are located at positions 9, 22, 23, 25 , 31, 56, 104, 124, 132, 133, 143, 173, 175, 181, 182, 199, 203, 216, 242, 263, 339, 420, 431, and 437.
  • the modified GluDH of the present invention has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94% of SEQ ID NO: 1 or 2 %, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the activity of an enzyme refers to the decrease in the amount of substrate or the increase in the amount of product per unit time in a chemical reaction catalyzed by a unit mass of enzyme under certain conditions.
  • the activity of the modified GluDH of the present invention is represented by the amount of PPO decrease or the amount of L-glufosinate increase per unit time under the catalysis of unit mass of modified GluDH under certain conditions.
  • the activity of an enzyme may also refer to the relative activity of the enzyme, expressed as a ratio of the activity of the enzyme of interest to the activity of a given enzyme that catalyzes the same reaction, such as a percent relative activity.
  • the activity of the modified GluDHs of the invention is expressed as a percent relative activity compared to SEQ ID NO:3.
  • the activity of the modified GluDH to catalyze the reaction of PPO with an amino donor to L-glufosinate is at least 100%, 105%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or higher.
  • the activity of the modified GluDHs of the invention is expressed as a percent relative activity compared to SEQ ID NO:31.
  • the activity of the modified GluDH to catalyze the reaction of PPO with an amino donor to L-glufosinate-ammonium is at least 100%, 105%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or higher.
  • amino donor refers to a compound that provides an amino group, including inorganic and organic compounds.
  • Ammonium salts e.g. NH 4 Cl, NH 4 NO 3 , (NH 4) 2 SO 4, ammonium acetate, etc.
  • amino acid e.g. NH 4 Cl, NH 4 NO 3 , (NH 4) 2 SO 4, ammonium acetate, etc.
  • amino donor is an ammonium salt, for example, NH 4 Cl.
  • kinetic properties of an enzyme include, but are not limited to, the Vmax, Km, and Vmax/Km of the enzyme.
  • improved kinetic properties include, for example, but not limited to, increased Vmax, decreased Km, and increased Vmax/Km.
  • Vmax refers to the maximum velocity of a catalytic reaction that can be achieved at a given enzyme concentration. Specifically, under the condition of constant enzyme concentration, when the substrate concentration is within a certain range, the reaction speed usually increases with the increase of the substrate concentration; and when the substrate concentration reaches a certain value, the reaction speed reaches The maximum value (ie Vmax) no longer increases with the increase of substrate concentration.
  • Km refers to the substrate concentration at which the catalytic rate reaches half of the maximum catalytic rate (ie, Vmax) at a given enzyme concentration.
  • nucleic acid molecule includes DNA molecules (eg, cDNA or genomic DNA) and RNA molecules (eg, 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 nucleic acid can be synthesized using nucleotide analogs or derivatives (eg, 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 GluDHs of the present invention. Therefore, in the present invention, the term modification also includes the genetic manipulation of the polynucleotide encoding the GluDH polypeptide of the present invention. The modifications may be substitutions, deletions, insertions and/or additions of one or more nucleotides.
  • the term "encode” refers to a polynucleotide that directly specifies the amino acid sequence of its protein product.
  • the boundaries of the coding sequences are generally defined by open reading frames, which typically begin with the ATG start codon or additional start codons such as GTG and TTG and end with stop codons such as TAA, TAG and TGA.
  • the coding sequence may be a DNA, cDNA or recombinant nucleotide sequence.
  • nucleic acid molecules encompassing all or part of the nucleic acid sequences of the invention can be isolated by polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based on the sequence information contained in the sequences.
  • PCR polymerase chain reaction
  • the polynucleotides of the present invention can be amplified according to standard PCR amplification techniques using cDNA, mRNA or genomic DNA as template and suitable oligonucleotide primers.
  • the nucleic acid thus amplified can be cloned into a suitable vector and characterized by DNA sequence analysis.
  • polynucleotides of the present invention can be prepared by standard synthetic techniques, eg, using an automated DNA synthesizer.
  • the present invention also relates to complementary strands of the nucleic acid molecules described herein.
  • a nucleic acid molecule that is complementary to another nucleotide sequence is one that is sufficiently complementary to that nucleotide sequence so that it can hybridize to the other nucleotide sequence to form a stable duplex.
  • hybridize is a nucleotide that is at least about 90%, preferably at least about 95%, more preferably at least about 96%, more preferably at least 98% homologous to each other under given stringent hybridization and washing conditions Sequences generally remain hybridized to each other.
  • polynucleotides of the present invention do not include polynucleotides that hybridize only to a poly A sequence (such as the 3' end poly(A) of mRNA) or to a complementary stretch of poly T (or U) residues.
  • nucleic acid constructs and vectors such as expression vectors, comprising the polynucleotides 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- or double-stranded nucleic acid molecule isolated from a naturally occurring gene or modified to contain a nucleic acid segment that does not occur in nature.
  • nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains the control sequences required for expression of the coding sequences of the invention.
  • expression vector refers herein to a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide of the invention together with additional nucleotides provided for expression of the polynucleotide, eg, Control sequences, operably linked.
  • the expression vector includes viral vector or plasmid vector.
  • control sequences is meant herein to include all elements required or advantageous for the expression of a polynucleotide encoding a polypeptide of the invention.
  • Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide, or native or foreign to each other.
  • control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include a promoter and transcriptional and translational stop signals.
  • control sequence may be a suitable promoter sequence, a nucleotide sequence recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the invention.
  • the promoter sequence contains transcriptional control sequences that mediate the expression of the polypeptide.
  • the promoter can be any nucleotide sequence that exhibits transcriptional activity in the host cell of choice, eg, the Escherichia coli lac operon.
  • the promoters also include mutated, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
  • operably linked refers herein to a configuration in which control sequences are placed in place relative to the coding sequence of a polynucleotide sequence, whereby the control sequences direct expression of the polypeptide coding sequence.
  • polynucleotides encoding the polypeptides of the present invention can undergo various manipulations to allow expression of the polypeptides. Manipulation of the polynucleotide may be desirable or necessary depending on the expression vector prior to its insertion into the vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art.
  • the vectors of the present invention preferably contain one or more selectable markers that allow for simple selection of transformed, transfected, transduced, etc. cells.
  • a selectable marker is a gene whose product provides biocide or virus resistance, heavy metal resistance, complement auxotrophy, and the like.
  • a bacterial selectable marker is the dal gene from Bacillus subtilis or Bacillus licheniformis, or a marker conferring resistance to antibiotics such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance.
  • the vectors of the present invention can integrate into the host cell genome or replicate autonomously in the cell independent of the genome.
  • the elements required for integration into the host cell genome or for autonomous replication are known in the art (see, eg, Sambrook et al., 1989, supra).
  • 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 exogenous nucleic acid (eg, DNA) into a host cell, and can be found, for example, in Sambrook et al., 1989, supra; Davis et al. ., Basic Methods in Molecular Biology (1986) and other laboratory manuals.
  • the present invention also relates to recombinant host cells comprising the polynucleotides of the present invention which are advantageously used in the recombinant production of GluDH polypeptides.
  • a vector comprising a polynucleotide of the 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 are aware of 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 GluDHs of the invention can be operably linked to non-GluDH polypeptides (eg, heterologous amino acid sequences) to form fusion proteins.
  • the fusion protein is a GST-GluDH fusion protein, wherein the GluDH sequence is fused to the C-terminus of the GST sequence. This fusion protein aids in the purification of recombinant GluDH.
  • the fusion protein is a GluDH protein containing a heterologous signal sequence at its N-terminus. In certain host cells (eg, mammalian and yeast host cells), expression and/or secretion of GluDH can be increased through the use of heterologous signal sequences.
  • the present invention provides a method for preparing L-glufosinate-ammonium, comprising contacting the modified GluDH or host cell of the present invention with PPO.
  • the method for preparing L-glufosinate-ammonium of the present invention comprises the steps:
  • cell-free catalytic methods are used to produce L-glufosinate-ammonium, and in step (a), the modified GluDHs of the present invention are provided. In some embodiments, free or immobilized modified GluDHs of the invention may be used.
  • the amino donor is an ammonium salt, for example, NH 4 Cl.
  • the reaction medium comprises NADPH/NADP.
  • NADPH/NADP cycle systems suitable for use in the present invention are known in the art and include, but are not limited to, alcohol dehydrogenase, glucose dehydrogenase (GDH) or glucose-6-phosphate dehydrogenase (G6PD) .
  • the NADPH/NADP cycle system comprises an alcohol dehydrogenase.
  • the alcohol dehydrogenase can also be immobilized.
  • the incubation is performed at 20-50°C, preferably 25-40°C, more preferably 28-35°C, eg, 30°C.
  • the medium comprises a buffer, eg, PBS, Tris-HCl buffer.
  • the medium comprises PBS, eg, 100 mM PBS.
  • the pH of the reaction medium is 7.5-8.
  • the reaction medium is a medium consisting in part or in whole of the cell culture medium in which the activity of the modified GluDHs of the invention is provided by the host cells of the invention in which the host cells are cultured.
  • the reaction medium is a medium consisting partly or entirely of cell culture medium, and the NADPH/NADP cycle system, such as alcohol dehydrogenase activity, is provided by the host cell of the invention or by a second host cell , the host cells are cultured in the reaction medium.
  • the NADPH/NADP cycle system such as alcohol dehydrogenase activity
  • the host cells of the invention and/or the second host cells are cultured and expanded in cell culture medium, and the expanded host cells are isolated from the cell culture medium, using buffers or water to make Biomass resuspension.
  • PPO is provided to the buffer or water before, during, or after addition of the expanded host cells.
  • bacterial cells such as E. coli cells, can be used.
  • DNA polymerase (PrimeSTAR Max DNA Polymerase) and DpnI endonuclease were purchased from TaKaRa company, plasmid extraction kit was purchased from Axygen company, PPO was the applicant according to the prior art (see J.Org.Chem.1991, 56, 1783-1788), NH 4 Cl was purchased from Sinopharm Chemical Reagent Beijing Co., Ltd., NADP+/NADPH was purchased from Aladdin, and alcohol dehydrogenase was recombinantly expressed by E. NCBI Accession No. WP_054768785.1).
  • the used expression vector was pET-30a(+), the plasmid was purchased from Novagen Company, and the used host cell was Escherichia coli BL21 (DE3), purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.
  • 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. with shaking for 12 h. Transfer 1 mL of the culture to 50 mL of fresh LB liquid medium (containing 50 mg/L kanamycin), incubate at 37 °C with shaking until the OD600 reaches about 0.6, add IPTG (final concentration 0.4mM) at 25 °C Incubate for 16 h to induce protein expression.
  • LB liquid medium containing 50 mg/L kanamycin
  • the culture was centrifuged at 4,000 g for 10 min at 4°C, the supernatant was discarded, and the E. coli cells were collected.
  • the collected E. coli cells were resuspended in 15 mL of pre-chilled 50 mM PBS, pH 7.0, and the E. coli cells were sonicated at 4°C.
  • the cell disrupted liquid was centrifuged at 6,000 g at 4°C for 15 min to remove the precipitate, and the obtained supernatant was the crude enzyme liquid containing recombinant enzyme.
  • reaction systems were prepared on a 96-well microtiter plate, which contained 100 mM pH 7.5 PBS (adjusted pH with ammonia), 0.15 mM NADPH (reduced form), 50 mM NH 4 Cl, 10% volume diluted crude enzyme solution (diluted 500 times), different concentrations of substrate PPO (5-100mM).
  • mutants were prepared according to the method of Example 1, and the enzymatic activity and kinetic parameters Vmax and Km were measured.
  • the obtained mutants and their activity and kinetic parameters are shown in Table 1, and the relative enzyme activity refers to the percentage of the activity of the mutant vs. the activity of the mutant of SEQ ID NO: 3.
  • A175G-G181R 10 100% 77 84 A175G-A182R 11 111% 61 46 A175G-V203I 12 72% 53 62 A175G-P132L-I133V 13 131% 34 41 A175G-P132L-V173S 14 132% 53 26 A175G-P132L-V173G 15 61% twenty two 36 A175G-P132L-A182R 16 115% 36 29 A175G-I133V-V173S 17 178% 78 51 A175G-I133V-V173G 18 164% 54 71 A175G-I133V-A182R 19 112% 49 26 A175G-V173S-A182R 20 35% 10 56 A175G-V173G-A182R twenty one 160% 71 twenty three A175G-P132L-I133V-V173S twenty two 95% 46 32 A175G-P132L-I133V-V173G twenty
  • mutants were prepared according to the method of Example 1, and the enzymatic activity and kinetic parameters Vmax and Km were measured.
  • the obtained mutant and its activity and kinetic parameters are shown in Table 2, wherein the mutant of SEQ ID NO: 31 is the mutant (LsGluDH A175G) reported in CN108588045B, and the relative enzyme activity refers to the activity of the mutant vs.
  • SEQ ID Percentage of activity of mutants of NO:31 are shown in Table 2, wherein the mutant of SEQ ID NO: 31 is the mutant (LsGluDH A175G) reported in CN108588045B, and the relative enzyme activity refers to the activity of the mutant vs.
  • Mutants were prepared according to the method of Example 1, additional mutations were introduced on the basis of the mutant of SEQ ID NO: 21, and the enzymatic activity was measured. The obtained mutants and their activities are shown in Table 3.
  • the initial relative enzyme activity refers to the percentage of the activity of the mutant vs. the activity of the mutant of SEQ ID NO: 21 without heat treatment; the relative enzyme activity after heat treatment refers to the Percentage of mutant activity vs. mutant of SEQ ID NO: 21 after 30 min incubation at 45°C.
  • Mutants were prepared according to the method of Example 1, additional mutations were introduced on the basis of the mutant of SEQ ID NO: 65, and the enzymatic activity was measured.
  • the obtained mutants and their activities are shown in Table 4, and the initial relative enzyme activity refers to the percentage of the activity of the mutant vs. the activity of the mutant of SEQ ID NO: 65 without heat treatment.
  • mutants SEQ ID NO: Relative enzyme activity A175G-V173G-A182R-Q22E-K56Q-N199Y-A420R 65 100% A175G-V173G-A182R-Q22E-K56Q-N199Y-A420R-A31H 66 165% A175G-V173G-A182R-Q22E-K56Q-N199Y-A420R-F124L 67 106% A175G-V173G-A182R-Q22E-K56Q-N199Y-A420R-A216G 68 117% A175G-V173G-A182R-Q22E-K56Q-N199Y-A420R-A31H-F124L 69 143% A175G-V173G-A182R-Q22E-K56Q-N199Y-A420R-A31H-A216G 70 132% A175G-V173G-A182

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Abstract

提供经修饰的谷氨酸脱氢酶（GluDH）。具体而言，提供经修饰的GluDH具有提高的催化PPO与氨基供体反应生成L-草铵膦的活性和／或改进的动力学性质。还提供编码所述经修饰的GluDH的多核苷酸、表达本发明的经修饰的GluDH的载体和宿主细胞以及使用所述经修饰的GluDH和宿主细胞生产L-草铵膦的方法。

Description

经修饰的谷氨酸脱氢酶及其应用 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谷氨酸脱氢酶(GluDH)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：
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。
将D-草铵膦氧化成2-羰基-4-(羟基甲基膦酰基)丁酸(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，而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，以无机铵盐为氨基供体，辅以NAD(P)H循环系统，消耗甲酸、葡萄糖或简单醇类，将PPO转化 为L-草铵膦。L-AADH催化的反应并不需要大大过量的氢给体，可以实现高转化率。
有报道利用天然的或经修饰的谷氨酸脱氢酶(GluDH)催化PPO不对称还原胺化制备L-草铵膦(参见，例如，CN107630052B、CN106978453B、CN108588045B和CN109609474A)。
但是，本领域仍需要提供具有提高的催化PPO与氨基供体反应生成L-草铵膦的活性和/或具有改进的动力学性质(例如，提高的Vmax值、降低的Km值或增加的Vmax/Km)的GluDH。
发明内容
在第一方面，本发明提供一种经修饰的谷氨酸脱氢酶(GluDH)，与其起始GluDH相比，包含两个或多个位置的氨基酸取代，其中所述经修饰的GluDH具有提高的催化PPO与氨基供体反应生成L-草铵膦的活性和/或具有改进的动力学性质(例如，提高的Vmax值、降低的Km值或增加的Vmax/Km)。
在一些实施方案中，所述经修饰的GluDH与其起始GluDH相比，具有选自以下组合的氨基酸取代：
第104位的氨基酸取代为C，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，且第175位的氨基酸取代为G；
第133位的氨基酸取代为V，且第175位的氨基酸取代为G；
第173位的氨基酸取代为G或S，且第175位的氨基酸取代为G；
第175位的氨基酸取代为G，且第181位的氨基酸取代为K或R；
第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第175位的氨基酸取代为G，且第203位的氨基酸取代为I；
第132位的氨基酸取代为L，第133位的氨基酸取代为V，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，第173位的氨基酸取代为S，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，且第175位的氨基酸取代为G；
第133位的氨基酸取代为V，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第173位的氨基酸取代为G，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第132位的氨基酸取代为L，第133位的氨基酸取代为V，第173位的氨基酸取代为S，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，第133位的氨基酸取代为V，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第132位的氨基酸取代为L，第173位的氨基酸取代为S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；和
第132位的氨基酸取代为L，第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R，
其中所述位置参照SEQ ID NO:1进行编号。
在一些实施方案中，所述经修饰的GluDH与其起始GluDH相比，包含第173、175和182位的取代，其中所述位置参照SEQ ID NO:1进行编号。在一些实施方案中，第173位的氨基酸取代为G。优选地，第175位的氨基酸取代为G。优选地，第182位的氨基酸取代为R。在一些实施方案中，所述经修饰的GluDH，还包含选自第9、22、23、25、31、56、124、143、199、216、242、263、339、420、431和437位的一或多个位置的氨基酸取代。优选地，第9位的氨基酸取代为S、L或Y。优选地，第22位的氨基酸取代为W或E。优选地，第23位的氨基酸取代为M。优选地，第25位的氨基酸取代为D。优选地，第31位的氨基酸取代为H。优选地，第56位的氨基酸取代为Q。优选地，第124位的氨基酸取代为L。优选地，第143位的氨基酸取代为E。优选地，第199位的氨基酸取代为W或Y。优选地，第216位的氨基酸取代为G。优选地，第263位的氨基酸取代为S。优选地，第339位的氨基酸取代为Q。优选地，第420位的氨基酸取代为R。优选地，第431位的氨基酸取代为S。优选地，第437位的氨基酸取代为K。
在一些实施方案中，所述经修饰的GluDH与其起始GluDH相比，包含第22、56、173、175、182、199和420位的氨基酸取代。优选地，第22位的氨基酸取代为E。优选地，第56位的氨基酸取代为Q。优选地，第173位的氨基酸取代为G。优选地，第175位的氨基酸取代为G。优选地，第182位的氨基酸取代为R。优选地，第199位的氨基酸取代为Y。优选地，第420位的氨基酸取代为R。在一些实施方案中，所述经修饰的GluDH还包含选自第31、124和216位的一或多个位置的氨基酸取代。优选地，第31位的氨基酸取代为H。优选地，第124位的氨基酸取代为L。优选地，第216位的氨基酸取代为G。在一些实施方案中，所述起始GluDH是野生型GluDH。在一些实施方案中，所述起始GluDH来源于芽胞杆菌科(Bacillaceae)的微生物，优选赖氨酸芽孢杆菌属(Lysinibacillus)或芽胞杆菌属(Bacillus)的微生物，更优选球形赖氨酸芽胞杆菌(Lysinibacillus sphaericus)或贝莱斯芽胞杆菌(Bacillus velezensis)。在优选的实施方案中，所述起始GluDH包含SEQ ID NO:1或2的氨基酸序列。
在一些实施方案中，所述经修饰的GluDH包含SEQ ID NO:4-14、16-19、21、22、24、25、27-30、32-48、50、51和53-72之一的氨基酸序列。
在一些实施方案中，所述经修饰的GluDH催化PPO与氨基供体反应生成L-草铵膦的活性是SEQ ID NO:3的催化此反应的活性的至少100％、105％、110％、120％、130％、140％、150％、160％、170％、180％、190％、200％或更高。
在第二方面，本发明提供编码本发明的经修饰的GluDH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GluDH、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GluDH或本发明的宿主细胞与PPO接触。
发明详述
本发明主要涉及经修饰的GluDH，用于催化PPO与氨基供体反应，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、经修饰的谷氨酸脱氢酶
如本文所用，术语“谷氨酸脱氢酶”和“GluDH”是指催化谷氨酸脱氢产生α-酮戊二酸的酶。此外，GluDH还具有催化PPO与氨基供体反应，生成L-草铵膦的活性。本发明提供经修饰的GluDH多肽，其具有提高的催化PPO与氨基供体反应生成L-草铵膦的活性和/或具有改进的动力学性质，包括但不限于提高的Vmax、降低的Km和提高的Vmax/Km。
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”在本文中可以与术语“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 PCTCN2021105179-appb-000001
Figure PCTCN2021105179-appb-000002
如本文所用，术语“修饰”是指对由本发明的多肽或其同源序列组成的多肽的任何修饰，包括但不限于，取代、删除、插入和/或添加一或多个氨基酸。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含两个或多个位置的氨基酸取代，其中所述经修饰的GluDH具有提高的催化PPO与氨基供体反应生成L-草铵膦的活性和/或具有改进的动力学性质，包括但不限于提高的Vmax、降低的Km和提高的Vmax/Km。
在一些实施方案中，所述经修饰的GluDH与其起始GluDH相比，具有选自以下组合的氨基酸取代：
第104位的氨基酸取代为C，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，且第175位的氨基酸取代为G；
第133位的氨基酸取代为V，且第175位的氨基酸取代为G；
第173位的氨基酸取代为G或S，且第175位的氨基酸取代为G；
第175位的氨基酸取代为G，且第181位的氨基酸取代为K或R；
第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第175位的氨基酸取代为G，且第203位的氨基酸取代为I；
第132位的氨基酸取代为L，第133位的氨基酸取代为V，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，第173位的氨基酸取代为S，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，且第175位的氨基酸取代为G；
第133位的氨基酸取代为V，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第173位的氨基酸取代为G，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第132位的氨基酸取代为L，第133位的氨基酸取代为V，第173位的氨基酸取代为S，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，第133位的氨基酸取代为V，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第132位的氨基酸取代为L，第173位的氨基酸取代为S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；和
第132位的氨基酸取代为L，第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R，
其中所述位置参照SEQ ID NO:1进行编号。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置132和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置132的氨基酸取代为L，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、133、173、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置133的氨基酸取代为V。在一些实施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置133和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置133的氨基酸取代为V，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、173、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置173和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置173的氨基酸取代为G或S，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、133、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置133的氨基酸取代为V。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置175 和182的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置175的氨基酸取代为G，且位置182的氨基酸取代为R。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、133、173和181的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置133的氨基酸取代为V。在一些实施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置132、133和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置132的氨基酸取代为L，位置133的氨基酸取代为V，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、173、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置132、173和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置132的氨基酸取代为L，位置173的氨基酸取代为G或S，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、133、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置133的氨基酸取代为V。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置133、173和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置133的氨基酸取代为V，位置173的氨基酸取代为G或S，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置132、175和182的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置132的氨基酸取代为L，位置175的氨基酸取代为G，且位置182的氨基酸取代为R。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、133、173和181的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置133的氨基酸取代为V。在一些实施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置133、 175和182的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置133的氨基酸取代为V，位置175的氨基酸取代为G，且位置182的氨基酸取代为R。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、173和181的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。
在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，包含位置173、175和182的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置173的氨基酸取代为G或S，位置175的氨基酸取代为G，且位置182的氨基酸取代为R。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、133、和181的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置133的氨基酸取代为V。在一些实施方案中，位置181的氨基酸取代为K或R。
在本文中，在其基础上进行氨基酸修饰的GluDH多肽称为起始GluDH。所述起始GluDH可以是野生型GluDH，也可以是野生型GluDH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GluDH，SEQ ID NO:1的多肽是“起始GluDH”；而如果从SEQ ID NO:1的变体多肽(例如SEQ ID NO:3-30)开始进行修饰，则相对于经修饰的GluDH，所述变体多肽是“起始GluDH”。
如本文所用，术语“野生型GluDH”是指天然存在的GluDH。在一些实施方案中，所述起始GluDH来源于芽胞杆菌科的微生物。在一些实施方案中，所述野生型GluDH是来自赖氨酸芽孢杆菌属或芽胞杆菌属的微生物的GluDH。优选地，所述野生型GluDH是来自球形赖氨酸芽胞杆菌的GluDH(SEQ ID NO:2)或来自贝莱斯芽胞杆菌的GluDH(SEQ ID NO: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，本文所用的野生型GluDH与SEQ ID NO:1或2具有至少65％ 或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、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GluDH与其起始GluDH相比，具有选自以下组合的氨基酸取代：
第104位的氨基酸取代为C，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，且第175位的氨基酸取代为G；
第133位的氨基酸取代为V，且第175位的氨基酸取代为G；
第173位的氨基酸取代为G或S，且第175位的氨基酸取代为G；
第175位的氨基酸取代为G，且第181位的氨基酸取代为K或R；
第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第175位的氨基酸取代为G，且第203位的氨基酸取代为I；
第132位的氨基酸取代为L，第133位的氨基酸取代为V，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，第173位的氨基酸取代为S，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，且第175位的氨基酸取代为G；
第133位的氨基酸取代为V，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第173位的氨基酸取代为G，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第132位的氨基酸取代为L，第133位的氨基酸取代为V，第173位的氨基酸取代为S，且第175位的氨基酸取代为G；
第132位的氨基酸取代为L，第133位的氨基酸取代为V，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第132位的氨基酸取代为L，第173位的氨基酸取代为S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；和
第132位的氨基酸取代为L，第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R，
其中所述位置参照SEQ ID NO:1进行编号。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、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GluDH与其起始GluDH相比，包含位置132和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置132的氨基酸取代为L，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、133、173、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置133的氨基酸取代为V。在一些实施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、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GluDH与其起始GluDH相比，包含位置133和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置133的氨基酸取代为V，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、173、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、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GluDH与其起始GluDH相比，包含位置173和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置173的氨基酸取代为G或S，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、133、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置133的氨基酸取代为V。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、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GluDH与其起始GluDH相比，包含位置175和182的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置175的氨基酸取代为G，且位置182的氨基酸取代为R。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、133、173和181的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置133的氨基酸取代为V。在一些实 施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、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GluDH与其起始GluDH相比，包含位置132、133和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置132的氨基酸取代为L，位置133的氨基酸取代为V，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、173、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、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GluDH与其起始GluDH相比，包含位置132、173和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置132的氨基酸取代为L，位置173的氨基酸取代为G或S，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、133、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置133的氨基酸取代为V。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、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GluDH与其起始GluDH相比，包含位置133、173和175的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置133的氨基酸取代为V，位置173的氨基酸取代为G或S，且位置175的氨基酸取代为G。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、181和182的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置181的氨基酸取代为K或R。在一些实施方案中，位置182的氨基酸取代为R。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、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GluDH与其起始GluDH相比，包含位置132、175和182的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置132的氨基酸取代为L，位置175的氨基酸取代为G，且位置182的氨基酸取代为R。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、133、173和181的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。 在一些实施方案中，位置133的氨基酸取代为V。在一些实施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、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GluDH与其起始GluDH相比，包含位置133、175和182的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置133的氨基酸取代为V，位置175的氨基酸取代为G，且位置182的氨基酸取代为R。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、173和181的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置173的氨基酸取代为G或S。在一些实施方案中，位置181的氨基酸取代为K或R。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、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GluDH与其起始GluDH相比，包含位置173、175和182的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，位置173的氨基酸取代为G或S，位置175的氨基酸取代为G，且位置182的氨基酸取代为R。在一些实施方案中，本发明的经修饰的GluDH与其起始GluDH相比，还包含选自位置104、132、133、和181的一或多个位置的取代。在一些实施方案中，位置104的氨基酸取代为C。在一些实施方案中，位置132的氨基酸取代为L。在一些实施方案中，位置133的氨基酸取代为V。在一些实施方案中，位置181的氨基酸取代为K或R。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、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GluDH与其起始GluDH相比，包含第173、175和182位的取代，其中所述位置参照SEQ ID NO:1进行编号。优选地，第173位的氨基酸取代为G，第175位的氨基酸取代为G，且第182位的氨基酸取代为R。在一些实施方案中，所述经修饰的GluDH，还包含选自第9、22、23、25、31、56、124、143、199、216、263、339、420、431和437位的一或多个位置的氨基酸取代。优选地，第9位的氨基酸取代为S、L或Y。优选地，第22位的氨基酸取代为W或E。优选地，第23位的氨基酸取代为M。优选地，第25位的氨基酸取代为D。优选地，第31位的氨基酸取代为H。优选地，第56位的氨基酸取代为Q。优选地，第124位的氨基酸取代为L。优选地，第143位的氨基酸取代为E。优选地，第199位的氨基酸取代为W或Y。优选地，第216位的氨基酸取代为G。优选地，第263位的氨基酸取代为S。优选地，第339位的氨基酸取代为Q。优选地，第420位的氨基酸取代为R。优选地，第431位的氨基酸取代为S。优选地，第437位的氨基酸取代为K。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、96％、97％、98％或99％的序列相同性。例如，所述起始GluDH 包含SEQ ID NO:1或由SEQ ID NO:1组成。
在一些实施方案中，所述经修饰的GluDH与其起始GluDH相比，包含第22、56、173、175、182、199和420位的氨基酸取代。优选地，第22位的氨基酸取代为E。优选地，第56位的氨基酸取代为Q。优选地，第173位的氨基酸取代为G。优选地，第175位的氨基酸取代为G。优选地，第182位的氨基酸取代为R。优选地，第199位的氨基酸取代为Y。优选地，第420位的氨基酸取代为R。在一些实施方案中，所述经修饰的GluDH还包含选自第31、124和216位的一或多个位置的氨基酸取代。优选地，第31位的氨基酸取代为H。优选地，第124位的氨基酸取代为L。优选地，第216位的氨基酸取代为G。优选地，所述起始GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、96％、97％、98％或99％的序列相同性。例如，所述起始GluDH包含SEQ ID NO:1或由SEQ ID NO:1组成。
在一些实施方案中，本发明的经修饰的GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、95％、96％、97％、98％或99％的序列相同性。
在一些实施方案，所述起始GluDH与SEQ ID NO:1或2的区别在于，具有一或多个氨基酸的取代、删除、插入和/或添加。在一些实施方案中，所述起始GluDH与SEQ ID NO:1或2相比，具有一或多个氨基酸的保守取代。在一些实施方案中，所述起始GluDH与SEQ ID NO:1或2相比，具有一或多个氨基酸的插入或删除。
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，经修饰的GluDH，包含SEQ ID NO:4-14、16-19、21、22、24、25、27-30、32-48、50、51和53-72之一的氨基酸序列或由SEQ ID NO:4-14、16-19、21、22、24、25、27-30、32-48、50、51和53-72之一的氨基酸序列组成，或者所述经修饰的GluDH与SEQ ID NO:4-14、16-19、21、22、24、25、27-30和、32-48、50、51和53-72之一相比包含1-10个氨基酸取代(例如，保守取代)，其中所述取代位于位置9、22、23、25、31、56、104、124、132、133、143、173、175、181、182、199、203、216、242、263、339、420、431和437之外的位置，其中与其起始GluDH相比，所述经修饰的GluDH具有提高的催化PPO与氨基供体反应生成L-草铵膦的活性，和/或所述经修饰的GluDH具有改进的动力学性质，例如提高的Vmax值、降低的Km值或增加的Vmax/Km。在一些实施方案中，所述经修饰的GluDH与与SEQ ID NO:4-14、16-19、21、22、24、25、27-30、32-48、50、51和53-72之一相比，包含1、2、3、4、5、6、7、8、9、10或更多个氨基酸取代(例如，保守取代)，其中所述取代位于位置9、22、23、25、31、56、104、124、132、133、143、173、175、181、182、199、203、216、242、263、339、420、431和437之外的位置。在一些实施方案中，本发明的经修饰的GluDH与SEQ ID NO:1或2具有至少65％或70％，优选至少75％或80％，更优选至少85％或90％，特别优选至少94％、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GluDH的活性，用一定条件下，在单位质量的经修饰的GluDH催化下，单位时间内PPO减少的量或L-草铵膦增加的量来表示。
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GluDH的活性以与SEQ ID NO:3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GluDH催化PPO与氨基供体反应生成L-草铵膦的活性是SEQ ID NO:3的催化此反应的活性的至少100％、105％、110％、120％、130％、140％、150％、160％、170％、180％、190％、200％或更高。
在一些实施方案中，本发明的经修饰的GluDH的活性以与SEQ ID NO:31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GluDH催化PPO与氨基供体反应生成L-草铵膦的活性是SEQ ID NO:31的催化此反应的活性的至少100％、105％、110％、120％、130％、140％、150％、160％、170％、180％、190％、200％或更高。
如本文所用，术语“氨基供体”是指提供氨基的化合物，包括无机化合物和有机化合 物。“氨基供体”包括但不限于铵盐(例如NH 4Cl、NH 4NO 3、(NH 4) 2SO 4、醋酸铵等)、氨基酸或有机胺。在一些实施方案中，所述氨基供体是铵盐，例如NH 4Cl。
在生产中，除了酶活性之外，还需要考虑酶的动力学性质。在本文中，酶的动力学性质包括但不限于酶的Vmax、Km和Vmax/Km。在本文中，改进的动力学性质包括，例如但不限于，提高的Vmax、降低的Km和提高的Vmax/Km。
如本文所用，术语“Vmax”是指在一定酶浓度下，所能达到的催化反应的最大速度。具体而言，在酶浓度不变的条件下，当底物浓度在一定范围内时，反应速度通常会随着底物浓度的提升而提升；而当底物浓度达到一定值时，反应速度达到最大值(即Vmax)，不再随着底物浓度的提升而提升。
如本文所用，术语“Km”是指在一定酶浓度下，当催化速度达到最大催化速度(即Vmax)的一半时的底物浓度。
二、编码经修饰的GluDH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GluDH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GluDH多肽的多核苷酸的遗传操作。所述修饰可以是取代、删除、插入和/或添加一或多个核苷酸。
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GluDH
为表达本发明的经修饰的GluDH，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GluDH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GluDH可以与非-GluDH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-GluDH融合蛋白，其中GluDH序列与GST序列的C-末端融合。这种融合蛋白可帮助重组GluDH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GluDH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GluDH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GluDH或宿主细胞与PPO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)向包含PPO和氨基供体的反应介质提供本发明的经修饰的GluDH的活性，任选地，所述反应介质包含NADPH/NADP循环系统，及
(b)温育所述反应介质以生成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GluDH。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GluDH。
在一些实施方案中，所述氨基供体是铵盐，例如NH 4Cl。
在一些实施方案中，所述反应介质包含NADPH/NADP。适用于本发明的NADPH/NADP循环系统是本领域已知的，其包含，包括但不限于，醇脱氢酶、葡萄糖脱氢酶(GDH)或葡糖-6-磷酸脱氢酶(G6PD)。在一些实施方案中，所述NADPH/NADP循环系统包含醇脱氢酶。在一些实施方案中，所述醇脱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，所述介质包含缓冲液，例如PBS、Tris-HCl缓冲液。在一个实施方案中，所述介质包含PBS，例如100mM的PBS。在一些实施方案中，所述反应介质的pH值为7.5-8。
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GluDH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NADPH/NADP循环系统，如醇脱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、期间或之后向所述缓冲液或水提供PPO。
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，PPO是申请人根据现有技术(参见J.Org.Chem.1991,56,1783-1788)合成的，NH 4Cl购自国药集团化学试剂北京有限公司，NADP+/NADPH购自阿拉丁，醇脱氢酶是大肠杆菌重组表达的来自高加索酸奶乳杆菌的脱氢酶(NCBI登录号WP_054768785.1)。
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的碱基引入所需的取代。用提取的突变前质粒(包含野生型GluDH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)蛋白质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50mM 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。
vi)酶活性测定
向PPO的PBS(100mM)溶液，中加入NH 4Cl、NADP+，并用氨水调节溶液pH值至8，溶液中PPO的终浓度为100mM，NH 4Cl的终浓度为50mM，而NADP+的终浓度为0.2g/L。向上述溶液加入如v)中描述的方法制备的GluDH粗酶液和醇脱氢酶，GluDH终浓度为0.02g/L，而醇脱氢酶的终浓度为0.2g/L。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L-草铵膦的生成量，从而测定催化反应初始速度。
vii)酶动力学测定
在96孔酶标板上配置多个200μL反应体系，其中含100mM pH 7.5PBS(用氨水调pH)、0.15mM的NADPH(还原型)、50mM的NH 4Cl，10％体积稀释后的粗酶液(稀释500倍)、不同浓度底物PPO(5-100mM)。在30℃检测340nm紫外吸光强度的变化，记录并计算吸收随时间的变化率mA/min。所得参数带入Michaelis-Menten方程，其中的反应速度用吸光度变化率计。
实施例2、制备和检测来自贝莱斯芽胞杆菌的GluDH(BvGluDH)的突变体
以BvGluDH(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，并测量酶活性和动力学参数Vmax和Km。所得突变体及其活性和动力学参数如表1所示，相对酶活是指突变体的活性vs.SEQ ID NO:3的突变体的活性的百分比。
表1
突变体中引入的取代 SEQ ID NO: 相对酶活 Vmax(mA/min) Km(mM)
A175G 3 100％ 60 73
A175G-L104C 4 90％ 60 34
A175G-P132L 5 131％ 56 32
A175G-I133V 6 113％ 59 43
A175G-V173S 7 111％ 79 52
A175G-V173G 8 123％ 80 53
A175G-G181K 9 93％ 70 80
A175G-G181R 10 100％ 77 84
A175G-A182R 11 111％ 61 46
A175G-V203I 12 72％ 53 62
A175G-P132L-I133V 13 131％ 34 41
A175G-P132L-V173S 14 132％ 53 26
A175G-P132L-V173G 15 61％ 22 36
A175G-P132L-A182R 16 115％ 36 29
A175G-I133V-V173S 17 178％ 78 51
A175G-I133V-V173G 18 164％ 54 71
A175G-I133V-A182R 19 112％ 49 26
A175G-V173S-A182R 20 35％ 10 56
A175G-V173G-A182R 21 160％ 71 23
A175G-P132L-I133V-V173S 22 95％ 46 32
A175G-P132L-I133V-V173G 23 53％ 14 47
A175G-P132L-I133V-A182R 24 97％ 39 35
A175G-P132L-V173S-A182R 25 79％ 40 14
A175G-P132L-V173G-A182R 26 34％ 19 22
A175G-I133V-V173S-A182R 27 139％ 65 28
A175G-I133V-V173G-A182R 28 139％ 73 26
A175G-P132L-I133V-V173S-A182R 29 70％ 37 24
A175G-P132L-I133V-V173G-A182R 30 138％ 71 53
实施例3、制备和检测来自球形赖氨酸芽胞杆菌的GluDH(LsGluDH)的突变体
以LsGluDH(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，并测量酶活性和动力学参数Vmax和Km。所得突变体及其活性和动力学参数如表2所示，其中SEQ ID NO:31的突变体是CN108588045B中报道的突变体(LsGluDH A175G)，相对酶活是指突变体的活性vs.SEQ ID NO:31的突变体的活性的百分比。
表2
突变体中引入的取代 SEQ ID NO: 相对酶活 Vmax(mA/min) Km(mM)
A175G 31 100％ 39 49
A175G-P132L 32 127％ 45 35
A175G-I133V 33 106％ 39 35
A175G-V173S 34 185％ 106 62
A175G-V173G 35 132％ 64 49
A175G-A182R 36 102％ 46 38
A175G-V173G-A182R 37 169％ 58 31
A175G-I133V-V173S-A182R 38 131％ 38 26
实施例4、制备和检测BvGluDH的突变体
根据实施例1的方法制备突变体，在SEQ ID NO:21的突变体的基础上引入额外的突变，并测量酶活性。所得突变体及其活性如表3所示，初始相对酶活是指未经热处理时，突变体的活性vs.SEQ ID NO:21的突变体的活性的百分比；热处理后相对酶活是指在45℃温育30min之后，突变体的活性vs.SEQ ID NO:21的突变体的活性的百分比。
表3
Figure PCTCN2021105179-appb-000003
Figure PCTCN2021105179-appb-000004
实施例5、制备和检测BvGluDH的突变体
根据实施例1的方法制备突变体，在SEQ ID NO:65的突变体的基础上引入额外的突变，并测量酶活性。所得突变体及其活性如表4所示，初始相对酶活是指未经热处理时，突变体的活性vs.SEQ ID NO:65的突变体的活性的百分比。
表4
突变体中的突变 SEQ ID NO: 相对酶活
A175G-V173G-A182R-Q22E-K56Q-N199Y-A420R 65 100％
A175G-V173G-A182R-Q22E-K56Q-N199Y-A420R-A31H 66 165％
A175G-V173G-A182R-Q22E-K56Q-N199Y-A420R-F124L 67 106％
A175G-V173G-A182R-Q22E-K56Q-N199Y-A420R-A216G 68 117％
A175G-V173G-A182R-Q22E-K56Q-N199Y-A420R-A31H-F124L 69 143％
A175G-V173G-A182R-Q22E-K56Q-N199Y-A420R-A31H-A216G 70 132％
A175G-V173G-A182R-Q22E-K56Q-N199Y-A420R-F124L-A216G 71 97％
A175G-V173G-A182R-Q22E-K56Q-N199Y-A420R-A31H-F124L-A216G 72 103％

Claims (17)

  1. 一种经修饰的谷氨酸脱氢酶(GluDH)，与其起始GluDH相比，具有选自以下组合的氨基酸取代：
    第104位的氨基酸取代为C，且第175位的氨基酸取代为G；
    第132位的氨基酸取代为L，且第175位的氨基酸取代为G；
    第133位的氨基酸取代为V，且第175位的氨基酸取代为G；
    第173位的氨基酸取代为G或S，且第175位的氨基酸取代为G；
    第175位的氨基酸取代为G，且第181位的氨基酸取代为K或R；
    第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
    第175位的氨基酸取代为G，且第203位的氨基酸取代为I；
    第132位的氨基酸取代为L，第133位的氨基酸取代为V，且第175位的氨基酸取代为G；
    第132位的氨基酸取代为L，第173位的氨基酸取代为S，且第175位的氨基酸取代为G；
    第132位的氨基酸取代为L，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
    第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，且第175位的氨基酸取代为G；
    第133位的氨基酸取代为V，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
    第173位的氨基酸取代为G，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
    第132位的氨基酸取代为L，第133位的氨基酸取代为V，第173位的氨基酸取代为S，且第175位的氨基酸取代为G；
    第132位的氨基酸取代为L，第133位的氨基酸取代为V，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
    第132位的氨基酸取代为L，第173位的氨基酸取代为S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；
    第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R；和
    第132位的氨基酸取代为L，第133位的氨基酸取代为V，第173位的氨基酸取代为G或S，第175位的氨基酸取代为G，且第182位的氨基酸取代为R，
    其中所述位置参照SEQ ID NO:1进行编号，且其中与其起始GluDH相比，所述经修饰的GluDH具有提高的催化2-羰基-4-(羟基甲基膦酰基)丁酸(PPO)与氨基供体反应生成L-草铵膦的活性，和/或所述经修饰的GluDH具有提高的Vmax、降低的Km或增加 的Vmax/Km。
  2. 权利要求1的经修饰的GluDH，其中所述起始GluDH是野生型GluDH。
  3. 权利要求1或2的经修饰的GluDH，其中所述起始GluDH来源于芽胞杆菌科的微生物，优选赖氨酸芽孢杆菌属或芽胞杆菌属的微生物，更优选球形赖氨酸芽胞杆菌(Lysinibacillus sphaericus)或贝莱斯芽胞杆菌(Bacillus velezensis)。
  4. 权利要求1-3任一项的经修饰的GluDH，其中所述起始GluDH包含SEQ ID NO:1或2的氨基酸序列。
  5. 一种经修饰的GluDH，包含SEQ ID NO:4-14、16-19、21、22、24、25、27-30、32-48、50、51和53-72之一的氨基酸序列。
  6. 一种经修饰的GluDH，与其起始GluDH相比，包含第173、175和182位的取代，其中第173位的氨基酸取代为G，第175位的氨基酸取代为G，且第182位的氨基酸取代为R，其中所述位置参照SEQ ID NO:1进行编号，且其中与其起始GluDH相比，所述经修饰的GluDH具有提高的催化PPO与氨基供体反应生成L-草铵膦的活性。
  7. 权利要求6的经修饰的GluDH，其中所述起始GluDH是野生型GluDH。
  8. 权利要求6或7的经修饰的GluDH，其中所述起始GluDH来源于芽胞杆菌科的微生物，优选赖氨酸芽孢杆菌属或芽胞杆菌属的微生物，更优选球形赖氨酸芽胞杆菌或贝莱斯芽胞杆菌。
  9. 权利要求6-8任一项的经修饰的GluDH，还包含选自第9、22、23、25、31、56、124、143、199、216、242、263、339、420、431和437位的一或多个位置的氨基酸取代，其中第9位的氨基酸取代为S、L或Y，第22位的氨基酸取代为W或E，第23位的氨基酸取代为M，第25位的氨基酸取代为D，第31位的氨基酸取代为H，第56位的氨基酸取代为Q，第124位的氨基酸取代为L，第143位的氨基酸取代为E，第199位的氨基酸取代为W或Y，第216位的氨基酸取代为G，第263位的氨基酸取代为S，第339位的氨基酸取代为Q，第420位的氨基酸取代为R，第431位的氨基酸取代为S，第437位的氨基酸取代为K。
  10. 权利要求6-8任一项的经修饰的GluDH，还包含第22、56、199和420位的氨基酸取代，其中第22位的氨基酸取代为E，第56位的氨基酸取代为Q，第199位的氨基酸取代为Y，第420位的氨基酸取代为R。
  11. 权利要求10的经修饰的GluDH，还包含选自第31、124和216位的一或多个位置的氨基酸取代，其中第31位的氨基酸取代为H，第124位的氨基酸取代为L，第216位的氨基酸取代为G。
  12. 权利要求6-11任一项的经修饰的GluDH，其中所述起始GluDH具有SEQ ID NO:1的氨基酸序列。
  13. 权利要求12的经修饰的GluDH，其中所述经修饰的GluDH催化PPO与氨基供体反应生成L-草铵膦的活性是SEQ ID NO:3的催化此反应的活性的至少130％。
  14. 一种多核苷酸，编码权利要求1-13任一项的经修饰的GluDH。
  15. 一种表达载体，包含权利要求14的多核苷酸。
  16. 一种宿主细胞，包含权利要求1-13任一项的经修饰的GluDH、权利要求14多核苷酸或权利要求15的载体。
  17. 一种生产L-草铵膦的方法，包括使1-13任一项的经修饰的GluDH或权利要求16的宿主细胞与PPO接触。
PCT/CN2021/105179 2020-07-09 2021-07-08 经修饰的谷氨酸脱氢酶及其应用 WO2022007881A1 (zh)

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