WO2022228505A1 - D-氨基酸氧化酶突变体及其在制备l-草铵膦中的应用 - Google Patents

D-氨基酸氧化酶突变体及其在制备l-草铵膦中的应用 Download PDF

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WO2022228505A1
WO2022228505A1 PCT/CN2022/089830 CN2022089830W WO2022228505A1 WO 2022228505 A1 WO2022228505 A1 WO 2022228505A1 CN 2022089830 W CN2022089830 W CN 2022089830W WO 2022228505 A1 WO2022228505 A1 WO 2022228505A1
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amino acid
glufosinate
acid oxidase
oxidase mutant
seq
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PCT/CN2022/089830
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French (fr)
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魏东芝
王华磊
吴承骏
刘清海
张舰
罗中华
张长雷
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永农生物科学有限公司
华东理工大学
宁夏永农生物科学有限公司
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Publication of WO2022228505A1 publication Critical patent/WO2022228505A1/zh
Priority to US18/469,527 priority Critical patent/US20240101974A1/en

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
    • C12N9/0024—D-Amino acid oxidase (1.4.3.3)
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    • 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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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
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    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/03—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
    • C12Y104/03003—D-Amino-acid oxidase (1.4.3.3)
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    • 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)
    • C12Y104/01005—L-Amino-acid dehydrogenase (1.4.1.5)
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    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/01—Peroxidases (1.11.1)
    • C12Y111/01006—Catalase (1.11.1.6)

Definitions

  • the application relates to the field of biotechnology; in particular, the application relates to a D-amino acid oxidase mutant, the use of the D-amino acid oxidase mutant in the preparation of L-glufosinate, and a method utilizing the D-amino acid oxidase Method for preparing L-glufosinate from oxidase mutants.
  • Glufosinate-ammonium also known as bialaphos, glufosinate, the trade names include Baoshida, Besutun, etc., the English name is phosphinothricin (abbreviated as PPT), the chemical name is 2-amino-4-[hydroxy (methyl) ) phosphono]butyric acid
  • PPT phosphinothricin
  • Glufosinate-ammonium can inhibit glutamine synthase after acting on plants, thereby interrupting the reversible reaction of glutamate in plants, causing metabolic disorders, causing plants to accumulate excess ammonia and poisoning, and at the same time, plants cannot synthesize chlorophyll and photosynthesis is inhibited , resulting in plant death.
  • Glufosinate-ammonium is mainly used in orchards, potato fields, non-arable land, etc. to control annual and perennial grass and dicotyledonous weeds, such as crabgrass, foxtail, wild wheat; perennial grass weeds and sedges, such as Fescue, duck sprouts, etc.
  • glufosinate-ammonium There are two optical isomers of glufosinate-ammonium, namely L-glufosinate-ammonium and D-glufosinate-ammonium, but only the L-type has herbicidal activity, and it is easily decomposed in the soil and is less toxic to humans and animals. Weeding has a wide spectrum and little damage to the environment.
  • glufosinate-ammonium sold on the market is generally a racemic mixture. If the glufosinate-ammonium product can be used in the form of pure optical isomer of L-configuration, the usage amount of glufosinate-ammonium can be significantly reduced, which is of great significance for improving atom economy, reducing use cost and reducing environmental pressure.
  • the chiral resolution method requires the use of an expensive chiral resolving agent (such as quinine), and the resolution steps are very cumbersome (for example, it needs to undergo steps such as salt formation, induced crystallization, desalination, etc.), and the theoretical yield of separation is Only 50%, which leads to a relatively low industrial value of this route.
  • an expensive chiral resolving agent such as quinine
  • the resolution steps are very cumbersome (for example, it needs to undergo steps such as salt formation, induced crystallization, desalination, etc.), and the theoretical yield of separation is Only 50%, which leads to a relatively low industrial value of this route.
  • Chemical synthesis methods include asymmetric synthesis methods and natural amino acid chiral source methods.
  • the disadvantages include the need to use expensive precious metals and ligands or starting materials or reaction routes that require highly toxic substances or long reaction synthesis routes.
  • Biocatalytic production of glufosinate has the advantages of strict stereoselectivity, mild reaction conditions and high yield, and is an advantageous method for the production of L-glufosinate. It mainly includes the following two categories: (1) The derivative of L-glufosinate-ammonium is used as the substrate, and it is obtained by direct enzymatic hydrolysis. The main advantages are high conversion rate and high ee value of the product, but it requires expensive and difficult to obtain hand. (2) The precursor of racemic glufosinate-ammonium is used as the substrate, which is obtained by selective separation of enzymes. The main advantages are that the raw materials are relatively easy to obtain and the catalyst activity is high, but the theoretical yield can only reach 50%, resulting in waste of raw materials.
  • the deracemization synthesis method using D,L-glufosinate-ammonium as raw material highlights a huge cost advantage. Since the commercially available glufosinate is D,L-glufosinate-ammonium, its industrial production technology is very mature, and the deracemization synthesis method directly uses D,L-glufosinate-ammonium as the raw material, which is simple and easy to obtain, low in cost, and can It is better to connect with the existing glufosinate-ammonium industrial production system.
  • This application is based on the identification of D-amino acid oxidase mutants that can be used to improve the bioenzymatic production of L-glufosinate-ammonium.
  • the present application relates to a D-amino acid oxidase mutant having D-amino acid oxidase (DAAO) activity, wherein, when compared with the amino acid sequence of the D-amino acid oxidase comprising the sequence shown in SEQ ID NO.1
  • DAAO D-amino acid oxidase
  • the amino acid sequence of the D-amino acid oxidase mutant comprises substitutions of amino acid residues corresponding to the 62nd and/or 226th positions
  • the 62nd and 226th positions are with reference to SEQ ID NO.1 defined
  • the amino acid sequence of the D-amino acid oxidase mutant is at least 90% identical to the sequence shown in SEQ ID NO. 1.
  • the substitution of the amino acid residue at position 62 is F62K, that is, the amino acid residue at position 62 is replaced by F with K.
  • the substitution of the amino acid residue at position 226 is M226T, that is, the amino acid residue at position 226 is replaced by M with T.
  • the D-amino acid oxidase mutant comprises the F62K amino acid substitution when aligned with the amino acid sequence of the D-amino acid oxidase comprising the sequence set forth in SEQ ID NO. 1, wherein the amino acid position is with reference to SEQ ID NO. 1 Limited by ID NO.1.
  • the D-amino acid oxidase mutant comprises the M226T amino acid substitution when aligned with the amino acid sequence of the D-amino acid oxidase comprising the sequence set forth in SEQ ID NO. 1, wherein the amino acid position is with reference to SEQ ID NO. 1 Limited by ID NO.1.
  • the D-amino acid oxidase mutant comprises F62K and M226T amino acid substitutions when aligned with the amino acid sequence of a D-amino acid oxidase comprising the sequence set forth in SEQ ID NO. 1, wherein the amino acid positions are Defined with reference to SEQ ID NO.1.
  • the amino acid sequence of the D-amino acid oxidase comprising the sequence shown in SEQ ID NO. 1 may be referred to as a wild-type enzyme having D-amino acid oxidase activity in the present application.
  • the nucleotide sequence of the wild-type enzyme can be the nucleotide sequence shown in SEQ ID NO.6.
  • the "D-amino acid oxidase mutants" described in this application have D-amino acid oxidase activity, that is, the activity of converting D-amino acids into ammonia, ketoacids (the type of ketoacid is determined by the D-amino acid substrate) and hydrogen peroxide
  • D-amino acid oxidase mutant described in the present application has the activity of converting D-glufosinate into 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid (PPO for short).
  • amino acid position is defined with reference to SEQ ID NO. 1
  • amino acid in the D-amino acid oxidase mutant is aligned with the amino acid sequence of SEQ ID NO. 1 at a specific amino acid position (e.g., position 62, 226th) alignment.
  • D-amino acid oxidase mutants of the present application may have improved activity compared to wild-type enzymes having D-amino acid oxidase activity, such as having D-glufosinate in D,L-glufosinate conversion Higher catalytic efficiency in the catalytic reaction of 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid, high utilization efficiency of oxygen, and optionally a beneficial effect in the production of L-glufosinate-ammonium Biocatalysis, especially the better stability used in the biocatalysis described in this application, etc.
  • catalytic efficiency refers to the property of a D-amino acid oxidase that allows it to convert D-glufosinate to 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid.
  • the D-amino acid oxidase mutants of the present application have enhanced catalytic efficiency compared to wild-type or reference D-amino acid oxidase.
  • the catalytic efficiency of the D-amino acid oxidase mutant of the present application is at least 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 of the catalytic efficiency of the wild-type or reference D-amino acid oxidase , 2.5, 2.6, 2.7, 2.8, 2.9, 3.0 or 3.1 times.
  • the D-amino acid oxidase mutant is derived from Microbotryum intermedium.
  • the amino acid sequence of the D-amino acid oxidase mutant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence shown in SEQ ID NO. 1 %, 97%, 98% or 99% identity.
  • the nucleotide sequence of the D-amino acid oxidase mutant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • the D-amino acid oxidase mutant comprises one or several (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) compared to the wild-type enzyme
  • the amino acid sequence of amino acid substitutions, deletions and/or insertions and/or has one or more truncated amino acid sequences compared to the wild-type enzyme.
  • the D-amino acid oxidase mutant has an amino acid substitution at position 62 and/or 226 when aligned with the amino acid sequence of the D-amino acid oxidase comprising the sequence shown in SEQ ID NO. 1 substitution of amino acid residues.
  • the application provides a nucleic acid or polynucleotide sequence comprising a sequence encoding a D-amino acid oxidase mutant as described above.
  • the nucleic acid or polynucleotide sequence may be isolated.
  • nucleic acid or “polynucleotide” are intended to include DNA molecules (eg, cDNA or genomic DNA) and RNA molecules (eg, mRNA) and analogs of DNA or RNA produced using nucleotide analogs. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA.
  • the application provides an expression vector comprising the nucleic acid or polynucleotide sequence described above.
  • the expression vector may comprise operably linked to one or more control sequences capable of directing expression of the D-amino acid oxidase mutants described above in a suitable expression host.
  • operably linked refers to the joining of polynucleotide elements (or coding sequences or nucleic acid sequences) in a functional relationship.
  • a nucleic acid sequence is "operably linked” when it is placed in a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • the control sequences may include promoters, enhancers, terminators, and the like.
  • the expression vector can be any vector (eg, a plasmid or virus) that can conveniently carry out recombinant DNA procedures and can result in the expression of a polynucleotide of the D-amino acid oxidase mutant.
  • the choice of expression vector generally depends on the compatibility of the vector with the cells into which it is to be introduced.
  • the expression vector may exist as an extrachromosomal entity, a vector whose replication is independent of chromosomal replication, eg, a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
  • the expression vector may be one that, when introduced into a host cell, integrates into the genome and replicates with the chromosome into which it has been integrated.
  • More than one copy (eg, 2, 3, or 4) of the expression vector of the present application can be inserted into a host cell to increase the production of D-amino acid oxidase mutants encoded by the nucleic acid sequences contained within the expression vector ( Overexpression).
  • the expression vector of the present application may also comprise nucleic acid sequences encoding formate dehydrogenase, glucose dehydrogenase or alcohol dehydrogenase, to achieve the expression of these dehydrogenases and mutants, preferably with L-amino acids Dehydrogenase mutants were co-expressed.
  • the present application provides a recombinant host cell comprising the nucleic acid described herein or the expression vector described herein.
  • the recombinant host cell can be a prokaryotic or eukaryotic cell.
  • the host cell belongs to the genus Saccharomyces, Aspergillus, Pichia, Kluyveromyces, Candida, Hansenula Hansenula, Humicola, Issatchenkia, Trichosporon, Brettanomyces, Pachysolen, Yarrowia Yarrowia, Actinomycetes, Streptomyces, Bacillus or Escherichia; preferably Saccharomyces cerevisiae, Yarrowia lipolytica (Yarrowia lipolitica), one of Candida krusei (Candida krusei), Issatchenkia orientalis, Bacillus subtilis (Bacillus subtilis) or Escherichia coli (Escherichia coli).
  • the expression vectors of the present application can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to various art-recognized techniques for introducing foreign nucleic acid (eg, DNA) into host cells known to those of skill in the art. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Davis et al., Basic Methods in Molecular Biology (1986) and other laboratory manuals.
  • the recombinant host cell is co-expressing (a) an L-amino acid dehydrogenation having activity to convert 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid to L-glufosinate The enzyme and (b) a host cell of a dehydrogenase selected from formate dehydrogenase, glucose dehydrogenase or alcohol dehydrogenase.
  • the present application provides the use of the D-amino acid oxidase mutant, nucleic acid, expression vector or recombinant host cell described in the present application in preparing L-glufosinate-ammonium.
  • the present application provides a method for preparing L-glufosinate, comprising converting D-glufosinate to L-glufosinate in the presence of an enzyme catalyzing system, wherein the enzyme catalyzes
  • the system includes a D-amino acid oxidase mutant for the conversion of D-glufosinate to 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid.
  • the D-glufosinate is initially present in a racemic mixture of D- and L-glufosinate or a salt thereof.
  • the racemic glufosinate starting material can be provided in a variety of forms.
  • Various salts of racemic glufosinate, such as the ammonium and hydrochloride salts, or zwitterions can be used.
  • the enzymatic catalytic system further comprises an L-amino acid dehydrogenase for converting 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid to L-glufosinate.
  • the L-amino acid dehydrogenase is a Glu/Leu/Phe/Val dehydrogenase.
  • the L-amino acid dehydrogenase or Glu/Leu/Phe/Val dehydrogenase can be any enzyme known in the art having L-amino acid dehydrogenase or Glu/Leu/Phe/Val dehydrogenase activity or a variant thereof .
  • catalase is further included in the enzymatic catalytic system.
  • the catalase is used to remove the by-product hydrogen peroxide because the accumulation of hydrogen peroxide can have a toxic effect on the enzyme catalyst.
  • the catalase can be any enzyme with catalase activity known in the art, such as catalase purchased from Ningxia Xiasheng Industrial Group Co., Ltd. with the commodity code of CAT-400.
  • the enzymatic catalytic system further comprises a coenzyme recycling system, and the coenzyme recycling system is selected from at least one of the following:
  • Formate dehydrogenase coenzyme cycle system including formate dehydrogenase, formate and coenzyme;
  • Glucose dehydrogenase coenzyme cycle system including glucose dehydrogenase, glucose and coenzyme;
  • Alcohol dehydrogenase coenzyme cycle system including alcohol dehydrogenase, isopropanol and coenzyme.
  • the coenzyme is NADH.
  • the formate dehydrogenase (FDH) described herein can be any enzyme or enzyme variant known in the art having formate dehydrogenase activity.
  • the formate dehydrogenase is derived from Lactobacillus buchneri.
  • the amino acid sequence of the formate dehydrogenase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence shown in SEQ ID No. 2 %, 98% or 99% identity.
  • the nucleotide sequence of the formate dehydrogenase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • the glucose dehydrogenase (GDH) described herein can be any enzyme or enzyme variant known in the art having glucose dehydrogenase activity.
  • the glucose dehydrogenase is derived from Exiguobacterium sibiricum.
  • the amino acid sequence of the glucose dehydrogenase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% with the amino acid sequence shown in SEQ ID NO. 3 %, 98% or 99% identity.
  • the nucleotide sequence of the glucose dehydrogenase and the nucleotide sequence shown in SEQ ID NO. 8 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • the alcohol dehydrogenase (ADH) described herein can be any enzyme or enzyme variant known in the art having alcohol dehydrogenase activity.
  • the alcohol dehydrogenase is derived from Lactobacillus brevis.
  • the amino acid sequence of the alcohol dehydrogenase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% with the amino acid sequence shown in SEQ ID NO. 4 %, 98% or 99% identity.
  • the nucleotide sequence of the alcohol dehydrogenase has at least 90%, 91%, 92%, 93%, 94%, 95%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • the enzymes described herein may be in the form of In: purified enzyme; partially purified enzyme; cell-free extract or crude cell extract; liquid, powder or fixed form; permeabilized cells, whole cells or whole fermentation broth or any other suitable form containing enzyme.
  • the form of each enzyme in the enzymatic catalytic system is independently selected from the group consisting of: a free enzyme and a recombinant host cell expressing the enzyme.
  • the recombinant host cells expressing the enzyme are each independently selected from the group consisting of: Saccharomyces, Aspergillus, Pichia, Kluyveromyces, Candida, Hansenula, Humicola, Issatchenkia, Trichosporon, Brettanomyces, Cysts Pachysolen, Yarrowia, Actinomycetes, Streptomyces, Bacillus or Escherichia; for example selected from Saccharomyces cerevisiae (Saccharomyces cerevisiae), Yarrowia lipolitica, Candida krusei, Issatchenkia orientalis, Bacillus subtilis or Escherichia coli.
  • the conversion reaction is carried out in a reaction solution.
  • the pH of the reaction solution is 7-10, and the pH of the reaction solution is preferably 8-9.
  • the reaction solution with pH 7-10 preferably in the reaction solution with pH 8-9, better reaction efficiency can be obtained.
  • the methods described herein may comprise step a) of taking place an oxidation reaction catalyzed by a mutant D-amino acid oxidase and step b) taking place of a transamination reaction catalyzed by a Glu/Leu/Phe/Val dehydrogenase.
  • the temperature of the oxidation reaction in step a) is 25-45°C, such as 30-45°C, 35-45°C, etc.; the time is 6-24 hours, such as 6-12 hours, 12-24 hours, For example, 6 hours, 12 hours, etc.
  • step b) the PPO produced in step a) is catalyzed and reduced to L-glufosinate-ammonium by L-amino acid dehydrogenase, so as to realize the in-situ de-racemization of D,L-glufosinate-ammonium, and the obtained ee value is greater than 99% L-Glufosinate-ammonium.
  • the reaction system of step b) further includes coenzyme NADH.
  • the molar ratio of NADH to substrate is from 1:10 to 1:5000.
  • NADH is added in an amount of 0.1-2 mM on a molar basis; more preferably 0.5 mM.
  • the temperature of the reductive amination reaction in step b) is 25-45°C, such as 30-45°C, 35-45°C, etc.; the time is 6-24 hours, such as 6-12 hours, 12-24 hours hours, such as 6 hours, 12 hours, etc.
  • step b) the molar ratio of inorganic ammonium donor to substrate at the start of the reaction is 1:1 to 10:1.
  • the inorganic ammonium donor in step b), can be ammonium phosphate, ammonium chloride, ammonium sulfate, ammonium formate, ammonium acetate, ammonia water; preferably, the inorganic ammonium donor can be ammonium phosphate, ammonium formate , ammonia water; more preferably, the inorganic ammonium donor can be ammonia water.
  • no additional hydrogen peroxide is added to the methods of the present application.
  • D-amino acid oxidase or its mutant has high utilization efficiency of oxygen when catalyzing the reaction, and can meet the requirement of realizing high-efficiency catalytic oxidation reaction only under the oxygen concentration provided by the aeration and stirring process.
  • the methods described herein can be carried out in one or more reaction vessels. Preferably, the methods described herein are carried out in one reaction vessel (ie "one pot two step process").
  • step a) may include: in the presence of the first recombinant microorganism and oxygen, oxidizing D,L-glufosinate-ammonium to obtain 2-carbonyl-4-[hydroxy(methyl)phosphono]butanoic acid. Utilizing the first recombinant microorganism can impart higher catalytic efficiency to the method of the present application.
  • the first recombinant microorganism can be constructed using any method known in the art.
  • the first recombinant microorganism can be constructed as follows: constructing a recombinant expression vector comprising the D-amino acid oxidase mutant gene, transforming the recombinant expression vector into the microorganism, inducing and culturing the obtained recombinant microorganism, and isolating and culturing it. liquid to obtain the first recombinant microorganism containing the D-amino acid oxidase mutant gene.
  • the added amount of the first recombinant microorganism is 1g/L-200g/L reaction solution; more preferably, 10g/L-100g/L reaction solution; Preferably, it is 30g/L reaction solution.
  • step b) may comprise: in a second recombinant microorganism co-expressing Glu/Leu/Phe/Val dehydrogenase and an enzyme for coenzyme recycling (eg formate dehydrogenase, glucose dehydrogenase or alcohol dehydrogenase)
  • an enzyme for coenzyme recycling eg formate dehydrogenase, glucose dehydrogenase or alcohol dehydrogenase
  • the 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid obtained in step a) is subjected to reductive amination reaction to obtain L-glufosinate-ammonium.
  • the second recombinant microorganism can confer a higher catalytic efficiency to the method of the present application.
  • the second recombinant microorganism can be constructed using any method known in the art.
  • the second recombinant microorganism can be constructed by constructing a recombinant expression vector containing the genes for the Glu/Leu/Phe/Val dehydrogenase and enzymes for coenzyme cycling, transforming the recombinant expression vector into the microorganism, The obtained recombinant microorganism is induced and cultured, and the culture medium is separated to obtain a second recombinant microorganism containing the genes of Glu/Leu/Phe/Val dehydrogenase and enzymes for coenzyme recycling.
  • the addition amount of the second recombinant microorganism is 1g/L-200g/L reaction solution; more preferably, 3g/L-100g/L reaction solution; most Preferably, it is 30g/L reaction solution.
  • the first and second recombinant microorganisms can be any engineered bacteria suitable for enzyme expression.
  • the first and second recombinant microorganisms each independently belong to one of the following genera: Saccharomyces, Aspergillus, Pichia, Kluyveromyces Genus (Kluyveromyces), Candida (Candida), Hansenula (Hansenula), Humicola (Humicola), Issatchenkia (Issatchenkia), Trichosporon (Trichosporon), Brettanomyces ( Brettanomyces), Pachysolen, Yarrowia, Actinomycetes, Streptomyces, Bacillus or Escherichia.
  • the first and second recombinant microorganisms are each independently selected from the group consisting of Saccharomyces cerevisiae, Yarrowia lipolitica, Candida krusei, Issatchenkia orientalis, Bacillus subtilis or Escherichia coli. In some more preferred embodiments, both the first and second recombinant microorganisms are E. coli.
  • the yield of the methods of the present application can be measured by any method known in the art.
  • the content of both configurations in the glufosinate product obtained can be measured by chiral HPLC.
  • the L-glufosinate product is obtained in an enantiomeric excess (e.e.) of at least 99.0%, 99.5%, or 99.9%.
  • the L-glufosinate product is obtained in at least 95%, 96%, or 97% yield.
  • the experimental methods in the present invention are conventional methods unless otherwise specified, and the specific gene cloning operation can be found in the "Molecular Cloning Experiment Guide" edited by J. Sambrook et al.
  • SEQ ID NO. 1 is the amino acid sequence annotated as D-amino acid oxidase (DAAO) derived from Microbotryum intermedium.
  • DAAO D-amino acid oxidase
  • SEQ ID NO. 2 is the amino acid sequence annotated as formate dehydrogenase (FDH) derived from Lactobacillus buchneri.
  • FDH formate dehydrogenase
  • SEQ ID NO. 3 is the amino acid sequence annotated as glucose dehydrogenase (GDH) derived from Exiguobacterium sibiricum.
  • SEQ ID NO. 4 is the amino acid sequence annotated as alcohol dehydrogenase (ADH) derived from Lactobacillus brevis.
  • ADH alcohol dehydrogenase
  • SEQ ID NO. 5 is the amino acid sequence derived from Delftia acidovorans annotated as Glu/Leu/Phe/Val dehydrogenase.
  • SEQ ID NO. 6 is the nucleotide sequence annotated as D-amino acid oxidase (DAAO) derived from Microbotryum intermedium.
  • DAAO D-amino acid oxidase
  • SEQ ID NO. 7 is the nucleotide sequence annotated as formate dehydrogenase (FDH) derived from Lactobacillus buchneri.
  • FDH formate dehydrogenase
  • SEQ ID NO. 8 is the nucleotide sequence annotated as glucose dehydrogenase (GDH) derived from Exiguobacterium sibiricum.
  • SEQ ID NO. 9 is the nucleotide sequence annotated as alcohol dehydrogenase (ADH) derived from Lactobacillus brevis.
  • ADH alcohol dehydrogenase
  • SEQ ID NO. 10 is the nucleotide sequence derived from Delftia acidovorans annotated as Glu/Leu/Phe/Val dehydrogenase.
  • the D-amino acid oxidase mutant of the present application enables the reaction system to have better catalytic efficiency.
  • the conversion rate is much higher than that of the wild type, and the PPO yield is also higher than that of the wild type. Greatly improved.
  • Fig. 1 exemplarily shows the reaction formula for producing L-glufosinate by the multi-enzyme system splitting method adopted in the method of the present application.
  • FIG. 2 exemplarily shows the reaction formula of deracemization to synthesize L-glufosinate (glucose dehydrogenase coenzyme cycle system).
  • Fig. 3 exemplarily shows the reaction formula of deracemization to synthesize L-glufosinate (formate dehydrogenase coenzyme cycle system).
  • FIG. 4 exemplarily shows the reaction process of preparing L-glufosinate-ammonium by double-bacteria multi-enzyme one-pot two-step deracemization.
  • Reagents used in upstream genetic engineering were purchased from Corning Life Sciences (Wujiang) Co., Ltd.; One-step cloning kits were purchased from Novozymes Co., Ltd.; E .coli BL21(DE3), plasmid pET-28a(+), etc.
  • the reagent 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid (PPO) used in the downstream catalytic process was from Yongnong Bioscience Co., Ltd.; D,L-glufosinate-ammonium was from Yongnong Bioscience Co., Ltd.; Other commonly used reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.
  • the progress of the reaction was detected by high performance liquid chromatography (HPLC), and the PPO was analyzed.
  • HPLC analysis method is: chromatographic column PBR; column temperature/30° C.; flow rate/1 mL/min; detection wavelength/210 nm; mobile phase: 5mM (NH 4 ) 2 HPO 4 .
  • the content of the two configurations of glufosinate-ammonium was detected by chiral HPLC analysis method.
  • the chiral HPLC analysis method was: chromatographic column/OA-5000L; mobile phase/0.5g/L cupric ammonium sulfate pentahydrate solution, and added 0.3% v/v acetonitrile; detection wavelength/254 nm; flow rate/1 mL/min; column temperature/35°C.
  • D-amino acid oxidase derived from Microbotryum intermedium (DAAO, GenBank No.: FMSP01000004.1, the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.6) was subjected to complete analysis. After gene synthesis, it was inserted into the expression plasmid pET-28a(+) to obtain pET-28a-daao. After sequencing and verification, pET-28a-daao was transferred into the expression host E. coli BL21 (DE3) for subsequent expression of the recombinase.
  • DAAO D-amino acid oxidase derived from Microbotryum intermedium
  • FDH formic acid dehydrogenation
  • GDH glucose dehydrogenase
  • Exiguobacterium sibiricum The sequence of the glucose dehydrogenase (GDH) derived from Exiguobacterium sibiricum (the amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.8) is subjected to full gene synthesis and inserted into the expression plasmid pET -28a(+), yields pET-28a-gdh. After sequencing and verification, pET-28a-gdh was transferred into the expression host E. coli BL21 (DE3) for subsequent expression of the recombinase.
  • GDH glucose dehydrogenase
  • ADH alcohol dehydrogenase
  • Lactobacillus brevis The amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.9 is subjected to full gene synthesis and inserted into the expression plasmid pET -28a(+), yields pET-28a-adh. After sequencing and verification, the pET-28a-adh was transferred into the expression host E. coli BL21 (DE3) for subsequent expression of the recombinase.
  • L-amino acid dehydrogenase from Delftia acidovorans (LAADH, GenBank No.: WP_012202150.1, NCBI annotated as Glu/Leu/Phe/Val dehydrogenase, the amino acid sequence is shown in SEQ ID NO.5, the nucleotide The gene sequence shown in SEQ ID NO.10) was subjected to full gene synthesis and inserted into the expression plasmid pET-28a(+) to obtain plasmid pET-28a-laadh. After sequencing and verification, it was transferred to the expression host E. coli BL21 (DE3) for subsequent expression of the recombinase.
  • Example 3 Construction of D-amino acid oxidase (DAAO) mutants (positions 62, 226)
  • positions 62 and/or 226 were mutated (specifically, F62K, M226T).
  • the primer sequences for PCR were designed for mutants mutated at positions 62 and 226 of the mutated D-amino acid oxidase sequence, as shown in Table 1:
  • PCR (25 ⁇ L) amplification system is as follows:
  • the cells were transformed into BL21 competent cells, spread on LB solid medium containing 50 ⁇ g/mL kanamycin, and cultured at 37°C overnight. Single colonies were picked to obtain mutant transformants. Bacteria were obtained as described in Example 2.
  • the catalytic efficiency of D-amino acid oxidase and its mutants were compared by measuring the amount of PPO produced.
  • the reaction system is: 300mM racemic glufosinate-ammonium salt, 100mM pH8.0 phosphate buffer, 8000U/L catalase, 20g/L D-amino acid oxidation Enzyme or its mutant freeze-dried cells.
  • a sample of the reaction solution was taken for processing, the concentration of PPO was measured by HPLC, and the conversion rate (product PPO concentration/substrate D-PPT concentration ⁇ 100%) was calculated, as shown in Table 2.
  • positions 91 and 168 were mutated (specifically V91I, N168G).
  • the primer sequences for the mutant PCR design of the mutated mutants at the 91st and 168th positions of the mutated LAADH sequence are specifically shown in Table 3:
  • the cells were transformed into BL21 competent cells, spread on LB solid medium containing 50 ⁇ g/mL kanamycin, and cultured at 37°C overnight. Single colonies were picked to obtain mutant transformants. Cells were obtained as described in Example 2.
  • the glucose dehydrogenase gene fragment was connected to the multi-cloning site by a seamless cloning kit, and the restriction enzyme site was Hind III, and the plasmid pET-28a-LAADH V91I was constructed.
  • -N168G-GDH the expression strain E.coli BL21(DE3)/pET-28a-LAADH V91I-N168G-GDH was constructed.
  • the formate dehydrogenase gene fragment was connected to the multiple cloning site by a seamless cloning kit, and the restriction enzyme site was Hind III, and the plasmid pET-28a-LAADH V91I was constructed -N168G-FDH, the expression strain E.coli BL21(DE3)/pET-28a-LAADH V91I-N168G-FDH was constructed.
  • the alcohol dehydrogenase gene fragment was connected to the multi-cloning site by a seamless cloning kit, and the restriction site was Hind III to construct the plasmid pET-28a-LAADH V91I -N168G-ADH, the expression strain E.coli BL21(DE3)/pET-28a-LAADH V91I-N168G-ADH was constructed.
  • Example 8 Preparation of L-glufosinate-ammonium (containing formate dehydrogenase FDH coenzyme cycle system) by double bacteria multi-enzyme de-racemization
  • Example 9 Preparation of L-glufosinate-ammonium (alcohol dehydrogenase ADH coenzyme cycle system) by de-racemization of double bacteria multi-enzyme
  • reaction solution (adjusted to pH8.0 with ammonia water), containing 400mM D,L-PPT, 8000U/L catalase, 0.5% (v/v) defoamer, 20g/L E .coli BL21(DE3)/pET-28a-DAAO F62K-M226 lyophilized cells, 20g/L E.coli BL21(DE3)/pET-28a-LAADH V91I-N168G-ADH lyophilized cells, 0.5mM NADH and 250mM isopropanol, ventilated with air, ventilation rate 2L/min, ammonia water to control pH to 8, temperature 30°C, and react for 24 hours.
  • the consumption of D-glufosinate and the generation of L-glufosinate and the like in the reaction process were detected by the liquid phase detection method shown in the examples, and the reaction progress curve was shown in FIG. 4 .
  • the graph shows that the concentration of D-glufosinate gradually decreased and the concentration of L-glufosinate gradually increased over time.
  • the liquid phase detection L-PPT was 380 mM, the e.e. value of the product L-glufosinate-ammonium was greater than 99%, and the L-PPT conversion yield was 95%.

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Abstract

公开了D-氨基酸氧化酶突变体及其在制备L-草铵膦中的应用。该D-氨基酸氧化酶突变体当与包含SEQ ID NO.1所示序列的D-氨基酸氧化酶的氨基酸序列比对时，所述D-氨基酸氧化酶突变体的氨基酸序列包含对应于第62位和/或第226位的氨基酸残基的替换，该第62位和第226位是参照SEQ ID NO.1限定的，并且该D-氨基酸氧化酶突变体的氨基酸序列与SEQ ID NO.1所示序列具有至少90％的同一性。

Description

D-氨基酸氧化酶突变体及其在制备L-草铵膦中的应用
相关申请的交叉引用
本申请要求于2021年04月29日提交中国专利局的申请号为202110475247.9、名称为“D-氨基酸氧化酶突变体及其在制备L-草铵膦中的应用”的中国专利申请的优先权，并将其全部内容通过引用结合在本申请中。
技术领域
本申请涉及生物技术领域；特别地，本申请涉及D-氨基酸氧化酶突变体，所述D-氨基酸氧化酶突变体在制备L-草铵膦中的用途，以及一种利用所述D-氨基酸氧化酶突变体制备L-草铵膦的方法。
背景技术
草铵膦(又名双丙氨膦、草丁膦，商品名包括保试达、百速顿等，英文名为phosphinothricin(简称PPT)，化学名为2-氨基-4-[羟基(甲基)膦酰基]丁酸)是德国赫斯特公司(现属于拜耳公司)在20世纪80年代开发的一种低毒、高效、非选择性触杀型有机磷除草剂。草铵膦作用于植株后可以抑制谷氨酰胺合成酶，从而中断植物体内谷氨酸的可逆反应，造成代谢紊乱，使植物堆积过量的氨而中毒，同时导致植物无法合成叶绿素从而光合作用受到抑制，导致植物死亡。草铵膦主要用于果园、马铃薯田、非耕地等，用于防治一年生和多年生的禾本科及双子叶杂草，如马唐、狗尾草、野小麦；多年生的禾本科杂草和莎草，如羊茅、鸭芽等。
灭生性除草剂市场巨大。目前，世界三大除草剂分别为百草枯，草甘膦，草铵膦。在市场使用方面，草甘膦独占鳌头，但是由于其长期使用，使得大量杂草产生抗性，而草甘膦也趋于失效；百草枯由于其剧毒性，已被列入《鹿特丹公约》，全球越来越多国家禁用或限用，中国农业部已发布公告说明，百草枯在2014年7月1日停止生产，2016年7月1日禁止使用；而目前草铵膦产 量虽小，却具有优异的除草性能和较小的药害副作用，因此，在未来一段时间内拥有巨大的市场潜力。
草铵膦有两种光学异构体，分别为L-草铵膦和D-草铵膦，但只有L-型具有除草活性，且在土壤中易分解，对人类和动物的毒性较小，除草谱广，对环境的破坏力小。
目前，市场上销售的草铵膦一般都是外消旋混合物。若草铵膦产品能以L-构型的纯光学异构体形式使用，可显著降低草铵膦的使用量，这对于提高原子经济性、降低使用成本、减轻环境压力具有重要意义。
手性纯L-草铵膦的主要制备方法主要由三种：手性拆分法，化学合成法和生物催化法。
手性拆分法需要使用价格昂贵的手性拆分剂(例如奎宁)，拆分的步骤非常繁琐(例如需要经历成盐、诱导结晶、解盐等步骤)，并且拆分的理论收率只有50％，这导致此路线的工业价值比较低。
化学合成法包括不对称合成法和天然氨基酸手性源法等，其缺点包括需要用到昂贵的贵金属和配体或起始原料或反应路线需要剧毒物质或反应合成路线较长等。
生物催化法生产草铵膦则具有立体选择性严格、反应条件温和、收率高等优点，是生产L-草铵膦的优势方法。主要包括以下两类：(1)以L-草铵膦的衍生物为底物，通过酶法直接水解获得，主要优点是转化率高，产物ee值较高，但需要昂贵且不易获得的手性原料为前体；(2)以外消旋草铵膦的前体为底物，通过酶的选择性拆分获得，主要优点为原料相对易得，催化剂活力高，但是理论收率只能达到50％，造成原料浪费。
除了这两种传统的生物催化法，以D,L-草铵膦为原料的去消旋合成方法凸显了巨大的成本优势。由于市售的草铵膦为D,L-草铵膦，其工业化生产技术已十分成熟，去消旋合成方法直接以D,L-草铵膦为原料，简单易得，成本较低，能够较好对接现有草铵膦工业生产体系。
发明内容
本申请基于D-氨基酸氧化酶突变体的鉴定，这些突变体可用于改进L-草铵膦的生物酶法生产。
在第一方面，本申请涉及具有D-氨基酸氧化酶(DAAO)活性的D-氨基酸氧化酶突变体，其中，当与包含SEQ ID NO.1所示序列的D-氨基酸氧化酶的氨基酸序列比对时，所述D-氨基酸氧化酶突变体的氨基酸序列包含对应于第62位和/或第226位的氨基酸残基的替换，所述第62位和第226位是参照SEQ ID NO.1限定的，并且所述D-氨基酸氧化酶突变体的氨基酸序列与SEQ ID NO.1所示序列具有至少90％的同一性。
在一些实施方式中，所述第62位的氨基酸残基的替换为F62K，即第62位的氨基酸残基由F替换为K。在一些实施方式中，所述第226位的氨基酸残基的替换为M226T，即第226位的氨基酸残基由M替换为T。
在一些实施方式中，所述D-氨基酸氧化酶突变体当与包含SEQ ID NO.1所示序列的D-氨基酸氧化酶的氨基酸序列比对时包含F62K氨基酸替换，其中氨基酸的位置是参照SEQ ID NO.1限定的。在一些实施方式中，所述D-氨基酸氧化酶突变体当与包含SEQ ID NO.1所示序列的D-氨基酸氧化酶的氨基酸序列比对时包含M226T氨基酸替换，其中氨基酸的位置是参照SEQ ID NO.1限定的。在一些实施方式中，所述D-氨基酸氧化酶突变体当与包含SEQ ID NO.1所示序列的D-氨基酸氧化酶的氨基酸序列比对时包含F62K和M226T氨基酸替换，其中氨基酸的位置是参照SEQ ID NO.1限定的。
包含SEQ ID NO.1所示序列的D-氨基酸氧化酶的氨基酸序列在本申请中可以被称为具有D-氨基酸氧化酶活性的野生型酶。所述野生型酶的核苷酸序列可以为SEQ ID NO.6所示的核苷酸序列。
本申请所述的“D-氨基酸氧化酶突变体”具有D-氨基酸氧化酶活性，即将D-氨基酸转化成氨、酮酸(酮酸种类由D-氨基酸底物决定)和过氧化氢的活性，特别地，本申请所述的D-氨基酸氧化酶突变体具有将D-草铵膦转化为2-羰基-4-[羟基(甲基)膦酰基]丁酸(简称PPO)的活性。
“氨基酸的位置是参照SEQ ID NO.1限定的”是指D-氨基酸氧化酶突变体中的氨基酸在与SEQ ID NO.1的氨基酸序列进行比对时与特定氨基酸位置(例如第62位、第226位)对齐。
与具有D-氨基酸氧化酶活性的野生型酶相比，本申请的D-氨基酸氧化酶突变体可以具有改进的活性，例如具有在将D,L-草铵膦中的D-草铵膦转化为2-羰基-4-[羟基(甲基)膦酰基]丁酸的催化反应中的更高的催化效率，对氧气的利用效率高，以及任选地具有利于在生产L-草铵膦的生物催化法，特别是本申请所描述的生物催化法中使用的更佳的稳定性等。
本申请中所用的术语“催化效率”是指D-氨基酸氧化酶允许其将D-草铵膦转化为2-羰基-4-[羟基(甲基)膦酰基]丁酸的性质。在一个实施方案中，与野生型或参照D-氨基酸氧化酶相比，本申请的D-氨基酸氧化酶突变体的催化效率得以增强。优选地，本申请的D-氨基酸氧化酶突变体的催化效率是野生型或参照D-氨基酸氧化酶的催化效率的至少1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0或3.1倍。
在一些实施方式中，所述D-氨基酸氧化酶突变体来源于Microbotryum intermedium。
在一些实施方式中，所述D-氨基酸氧化酶突变体的氨基酸序列与SEQ ID NO.1所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。在一些实施方式中，所述D-氨基酸氧化酶突变体的核苷酸序列与SEQ ID NO.6所示的核苷酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。在一些实施方式中，所述D-氨基酸氧化酶突变体包含与野生型酶相比具有一个或几个(例如1、2、3、4、5、6、7、8、9或10个)氨基酸替换、缺失和/或插入的氨基酸序列和/或与野生型酶相比具有一个或多个截短的氨基酸序列。
在一些实施方式中，所述D-氨基酸氧化酶突变体当与包含SEQ ID NO.1所示序列的D-氨基酸氧化酶的氨基酸序列比对时的氨基酸替换为第62位和/或第226位的氨基酸残基的替换。
在第二方面，本申请提供了包含编码上述D-氨基酸氧化酶突变体的序列的核酸或多核苷酸序列。所述核酸或多核苷酸序列可以是经分离的。
术语“核酸”或“多核苷酸”旨在指包括DNA分子(例如cDNA或基因组DNA)和RNA分子(例如mRNA)以及使用核苷酸类似物产生的DNA或RNA的类似物。核酸分子可以是单链或双链的，但优选是双链DNA。
在第一方面中描述的特征、定义和优选项同样适用于第二方面。
在第三方面，本申请提供了包含上述核酸或多核苷酸序列的表达载体。所述表达载体可以包含与能够指导上述D-氨基酸氧化酶突变体在合适表达宿主中表达的一种或更多种控制序列可操作的连接。
术语“可操作的连接”是指处于功能关系的多核苷酸元件(或编码序列或核酸序列)的连接。当核酸序列被置于与另一核酸序列的功能关系中时，其是“可操作的连接”的。例如，如果启动子或增强子影响编码序列的转录，则其与编码序列可操作地连接。在一些实施方式中，所述控制序列可以包括启动子、增强子、终止子等。
表达载体可以是可方便地进行重组DNA程序并且可引起D-氨基酸氧化酶突变体的多核苷酸表达的任何载体(例如质粒或病毒)。表达载体的选择通常取决于载体与待引入所述载体的细胞的相容性。所述表达载体可以作为染色体外实体而存在、其复制独立于染色体复制的载体，例如，质粒、染色体外元件、微型染色体或人工染色体。或者，表达载体可以是当被引入宿主细胞时整合至基因组中并与其已整合至其中的染色体一起复制的载体。
可将多于一个拷贝(例如2个、3个或4个)的本申请的表达载体插入宿主细胞中以增加由包含在表达载体内的核酸序列编码的D-氨基酸氧化酶突变体的产生(过表达)。
在一些实施方式中，本申请的表达载体还可以包含编码甲酸脱氢酶、葡萄糖脱氢酶或醇脱氢酶的核酸序列，以实现这些脱氢酶与突变体的表达，优选与L-氨基酸脱氢酶突变体共表达。
在第一和第二方面中描述的特征、定义和优选项同样适用于第三方面。
在第四方面，本申请提供了一种重组宿主细胞，其包含本申请所述的核酸或者本申请所述的表达载体。
在一些实施方式中，所述重组宿主细胞可以是原核或真核细胞。在一些实施方式中，所述宿主细胞属于酵母属(Saccharomyces)、曲霉属(Aspergillus)、毕赤酵母属(Pichia)、克鲁维酵母属(Kluyveromyces)、假丝酵母属(Candida)、汉逊酵母属(Hansenula)、腐质霉属(Humicola)、伊萨酵母属(Issatchenkia)、毛孢子菌属(Trichosporon)、酒香酵母属(Brettanomyces)、管囊酵母属(Pachysolen)、耶氏酵母属(Yarrowia)、放线菌属(Actinomycetes)、链霉菌属(Streptomyces)、芽孢杆菌属(Bacillus) 或埃希氏杆菌属(Escherichia)；优选地属于酿酒酵母(Saccharomyces cerevisiae)、解脂耶氏酵母(Yarrowia lipolitica)、克鲁斯假丝酵母(Candida krusei)、东方伊萨酵母(Issatchenkia orientalis)、枯草芽孢杆菌(Bacillus subtilis)或大肠杆菌(Escherichia coli)中的一种。
可以通过常规转化或转染技术将本申请的表达载体引入原核或真核细胞中。在本文中使用时，术语“转化”和“转染”旨在指用于将外来核酸(例如DNA)引入本领域技术人员公知的宿主细胞中的多种本领域公认的技术。用于转化或转染宿主细胞的合适方法可见于Sambrook等(Molecular Cloning:A Laboratory Manual,第2版.Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY,1989)，Davis等,Basic Methods in Molecular Biology(1986)和其他实验室手册。
在一些实施方式中，所述重组宿主细胞是共表达(a)具有将2-羰基-4-[羟基(甲基)膦酰基]丁酸转化为L-草铵膦活性的L-氨基酸脱氢酶和(b)选自甲酸脱氢酶、葡萄糖脱氢酶或醇脱氢酶的脱氢酶的宿主细胞。
在第一、第二、第三方面中描述的特征、定义和优选项同样适用于第四方面。
在第五方面，本申请提供了本申请所述的D-氨基酸氧化酶突变体、核酸、表达载体或重组宿主细胞在制备L-草铵膦中的用途。
在第一、第二、第三、第四方面中描述的特征、定义和优选项同样适用于第五方面。
在第六方面，本申请提供了一种制备L-草铵膦的方法，其包括在酶催化体系的存在下使D-草铵膦发生转化反应生成L-草铵膦，其中所述酶催化体系包括用于将D-草铵膦转化为2-羰基-4-[羟基(甲基)膦酰基]丁酸的D-氨基酸氧化酶突变体。
在一些实施方式中，所述D-草胺膦最初存在于D-和L-草胺膦或其盐的外消旋混合物中。外消旋的草胺膦起始原料可以以多种形式提供。可使用外消旋草胺膦的各种盐，诸如铵盐和盐酸盐，或两性离子。
在一些实施方式中，所述酶催化体系还包含用于将2-羰基-4-[羟基(甲基)膦酰基]丁酸转化为L-草铵膦的L-氨基酸脱氢酶。在一些实施方式中，所述L-氨基酸脱氢酶是Glu/Leu/Phe/Val脱氢酶。L-氨基酸脱氢酶或Glu/Leu/Phe/Val脱 氢酶可以为本领域已知的任何具有L-氨基酸脱氢酶或Glu/Leu/Phe/Val脱氢酶活性的酶或其变体。例如，CN109750009B、CN109609475B、CN110791484B中描述的L-氨基酸脱氢酶。
在一些实施方式中，所述酶催化体系中还包括过氧化氢酶。所述过氧化氢酶用于去除副产物过氧化氢，因为过氧化氢积累会对酶催化剂有毒害作用。过氧化氢酶可以为本领域已知的任何具有过氧化氢酶活性的酶，例如购自宁夏夏盛实业集团有限公司，商品编号为CAT-400的过氧化氢酶。
在一些实施方式中，所述酶催化体系还包括辅酶循环系统，所述辅酶循环系统选自以下中的至少一种：
(1)甲酸脱氢酶辅酶循环系统：包括甲酸脱氢酶，甲酸盐和辅酶；
(2)葡萄糖脱氢酶辅酶循环系统：包括葡萄糖脱氢酶，葡萄糖和辅酶；
(3)醇脱氢酶辅酶循环系统：包括醇脱氢酶，异丙醇和辅酶。
在一些优选的实施方式中，所述辅酶是NADH。本申请所述的甲酸脱氢酶(FDH)可以为本领域已知的任何具有甲酸脱氢酶活性的酶或酶变体。在一些实施方式中，所述甲酸脱氢酶来源于Lactobacillus buchneri。在一些实施方式中，所述甲酸脱氢酶的氨基酸序列与SEQ ID No.2所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。在一些实施方式中，所述甲酸脱氢酶的核苷酸序列与SEQ ID No.7所示的核苷酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。
本申请所述的葡萄糖脱氢酶(GDH)可以为本领域已知的任何具有葡萄糖脱氢酶活性的酶或酶变体。在一些实施方式中，所述葡萄糖脱氢酶来源于Exiguobacterium sibiricum。在一些实施方式中，所述葡萄糖脱氢酶的氨基酸序列与SEQ ID NO.3所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。在一些实施方式中，所述葡萄糖脱氢酶的核苷酸序列与SEQ ID NO.8所示的核苷酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。
本申请所述的醇脱氢酶(ADH)可以为本领域已知的任何具有醇脱氢酶活性的酶或酶变体。在一些实施方式中，所述醇脱氢酶来源于Lactobacillus brevis。在一些实施方式中，所述醇脱氢酶的氨基酸序列与SEQ ID NO.4所示的氨基酸 序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。在一些实施方式中，所述醇脱氢酶的核苷酸序列与SEQ ID NO.9所示的核苷酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。
本申请所述的酶(例如D-氨基酸氧化酶突变体、Glu/Leu/Phe/Val脱氢酶、过氧化氢酶、甲酸脱氢酶、葡萄糖脱氢酶或醇脱氢酶)的形式可以为：纯化的酶；部分纯化的酶；无细胞提取物或粗细胞提取物；液体、粉末或固定形式；含有酶的可透化处理的细胞、完整细胞或完整发酵液或其他任何合适形式。在一些实施方式中，所述酶催化体系中的每种酶的形式各自独立地选自：游离酶和表达酶的重组宿主细胞。
在一些实施方式中，其中所述表达酶的重组宿主细胞各自独立地选自：酵母属(Saccharomyces)、曲霉属(Aspergillus)、毕赤酵母属(Pichia)、克鲁维酵母属(Kluyveromyces)、假丝酵母属(Candida)、汉逊酵母属(Hansenula)、腐质霉属(Humicola)、伊萨酵母属(Issatchenkia)、毛孢子菌属(Trichosporon)、酒香酵母属(Brettanomyces)、管囊酵母属(Pachysolen)、耶氏酵母属(Yarrowia)、放线菌属(Actinomycetes)、链霉菌属(Streptomyces)、芽孢杆菌属(Bacillus)或埃希氏杆菌属(Escherichia)；例如选自酿酒酵母(Saccharomyces cerevisiae)、解脂耶氏酵母(Yarrowia lipolitica)、克鲁斯假丝酵母(Candida krusei)、东方伊萨酵母(Issatchenkia orientalis)、枯草芽孢杆菌(Bacillus subtilis)或大肠杆菌(Escherichia coli)。
在一些实施方式中，所述转化反应在反应液中进行。优选地，所述反应液的pH为7-10，优选pH为8-9的反应液。在pH为7-10的反应液中，优选在pH为8-9的反应液中进行反应时可以获得更优的反应效率。
本申请所述的方法可以包括：发生由D-氨基酸氧化酶突变体催化的氧化反应的步骤a)和发生由Glu/Leu/Phe/Val脱氢酶催化的转氨反应的步骤b)。
在一些实施方式中，步骤a)的氧化反应的温度为25-45℃，例如30-45℃，35-45℃等；时间为6-24小时，例如6-12小时，12-24小时，例如6小时、12小时等。
在步骤b)中，步骤a)中产生的PPO被L-氨基酸脱氢酶催化还原为L-草铵膦，从而实现D,L-草铵膦的原位去消旋化，得到ee值大于99％的L-草铵膦。
在一些实施方式中，步骤b)的反应体系中还包括辅酶NADH。在一些实施方式中，NADH与底物的摩尔比为1:10-1:5000。在一些实施方式中，以摩尔浓度计，NADH的添加量为0.1-2mM；更优选为0.5mM。在一些实施方式中，步骤b)的还原胺化反应的温度为25-45℃，例如30-45℃，35-45℃等；时间为6-24小时，例如6-12小时，12-24小时，例如6小时、12小时等。
在一些实施方式中，在步骤b)中，反应开始时无机铵供体与底物的摩尔比为1:1-10:1。
在一些实施方式中，在步骤b)中，无机铵供体可以为磷酸铵、氯化铵、硫酸铵、甲酸铵、乙酸铵、氨水；优选地，无机铵供体可以为磷酸铵、甲酸铵、氨水；更优选，无机铵供体可以为氨水。
在一些实施方式中，本申请的方法中不额外添加过氧化氢。本方法中D-氨基酸氧化酶或其突变体在催化反应时，对氧气的利用效率高，仅在通过通气搅拌过程所提供的氧气浓度下，即可满足实现高效催化氧化反应的需求。
本申请所述的方法可以在一个或更多个反应容器中进行。优选地，本申请所述的方法在一个反应容器中进行(即“一锅两步法”)。
在一些优选的实施方式中，步骤a)中使用的D-氨基酸氧化酶突变体由第一重组微生物表达。因此，步骤a)可以包括：在第一重组微生物和氧气的存在下，使D,L-草铵膦发生氧化反应得到2-羰基-4-[羟基(甲基)膦酰基]丁酸。利用所述第一重组微生物能够赋予本申请方法更高的催化效率。可以利用本领域已知的任何方法构建所述第一重组微生物。例如，所述第一重组微生物可以如下构建：构建包含所述D-氨基酸氧化酶突变体基因的重组表达载体，将所述重组表达载体转化至微生物，对获得的重组微生物进行诱导培养，分离培养液得到含有D-氨基酸氧化酶突变体基因的第一重组微生物。优选地，按照10000rpm离心10min后的菌体湿重计，所述第一重组微生物的添加量为1g/L-200g/L反应液；更优选地，10g/L-100g/L反应液；最优选地，为30g/L反应液。
在一些优选的实施方式中，步骤b)中使用的Glu/Leu/Phe/Val脱氢酶和用于辅酶循环的酶由第二重组微生物共表达。因此，步骤b)可以包括：在共表达Glu/Leu/Phe/Val脱氢酶和用于辅酶循环的酶(例如甲酸脱氢酶、葡萄糖脱氢酶或醇脱氢酶)的第二重组微生物和无机铵盐的存在下，使步骤a)获得的2-羰基-4-[羟基(甲基)膦酰基]丁酸发生还原胺化反应得到L-草铵膦。利用所述第 二重组微生物能够赋予本申请方法更高的催化效率。可以利用本领域已知的任何方法构建所述第二重组微生物。例如，所述第二重组微生物可以如下构建：构建含所述Glu/Leu/Phe/Val脱氢酶和用于辅酶循环的酶的基因的重组表达载体，将所述重组表达载体转化至微生物，对获得的重组微生物进行诱导培养，分离培养液得到含有Glu/Leu/Phe/Val脱氢酶和用于辅酶循环的酶的基因的第二重组微生物。优选地，按照10000rpm离心10min后的菌体湿重计，所述第二重组微生物的添加量为1g/L-200g/L反应液；更优选地，3g/L-100g/L反应液；最优选地，为30g/L反应液。
所述第一和第二重组微生物可以是任何适用于酶表达的工程菌。在一些实施方式中，所述第一和第二重组微生物各自独立地属于以下属中的一种：酵母属(Saccharomyces)、曲霉属(Aspergillus)、毕赤酵母属(Pichia)、克鲁维酵母属(Kluyveromyces)、假丝酵母属(Candida)、汉逊酵母属(Hansenula)、腐质霉属(Humicola)、伊萨酵母属(Issatchenkia)、毛孢子菌属(Trichosporon)、酒香酵母属(Brettanomyces)、管囊酵母属(Pachysolen)、耶氏酵母属(Yarrowia)、放线菌属(Actinomycetes)、链霉菌属(Streptomyces)、芽孢杆菌属(Bacillus)或埃希氏杆菌属(Escherichia)。在一些优选的实施方式中，所述第一和第二重组微生物各自独立地选自酿酒酵母(Saccharomyces cerevisiae)、解脂耶氏酵母(Yarrowia lipolitica)、克鲁斯假丝酵母(Candida krusei)、东方伊萨酵母(Issatchenkia orientalis)、枯草芽孢杆菌(Bacillus subtilis)或大肠杆菌(Escherichia coli)。在一些更优选的实施方式中，所述第一和第二重组微生物均是大肠杆菌。
本申请方法的产率可以通过本领域已知的任何方法测量。例如，可以通过手性HPLC来测量所获得的草铵膦产物中两个构型含量。在一些实施方式中，获得的L-草铵膦产物的对映体过量(e.e.)至少为99.0％、99.5％或99.9％。在一些实施方式中，获得的L-草铵膦产物的收率至少为95％、96％或97％。
本发明大写英文字母代表如本领域技术人员熟知的氨基酸，根据本申请，在此代表的是相应的氨基酸残基。
本发明中的实验方法如无特别说明均为常规方法，基因克隆操作具体可参见J.萨姆布鲁克等编的《分子克隆实验指南》。
对序列表的描述：
SEQ ID NO.1是来源于Microbotryum intermedium的注释为D-氨基酸氧化酶(DAAO)的氨基酸序列。
SEQ ID NO.2是来源于Lactobacillus buchneri的注释为甲酸脱氢酶(FDH)的氨基酸序列。
SEQ ID NO.3是来源于Exiguobacterium sibiricum的注释为葡萄糖脱氢酶(GDH)的氨基酸序列。
SEQ ID NO.4是来源于Lactobacillus brevis的注释为醇脱氢酶(ADH)的氨基酸序列。
SEQ ID NO.5是来源于Delftia acidovorans注释为Glu/Leu/Phe/Val脱氢酶的氨基酸序列。
SEQ ID NO.6是来源于Microbotryum intermedium的注释为D-氨基酸氧化酶(DAAO)的核苷酸序列。
SEQ ID NO.7是来源于Lactobacillus buchneri的注释为甲酸脱氢酶(FDH)的核苷酸序列。
SEQ ID NO.8是来源于Exiguobacterium sibiricum的注释为葡萄糖脱氢酶(GDH)的核苷酸序列。
SEQ ID NO.9是来源于Lactobacillus brevis的注释为醇脱氢酶(ADH)的核苷酸序列。
SEQ ID NO.10是来源于Delftia acidovorans注释为Glu/Leu/Phe/Val脱氢酶的核苷酸序列。
本申请的D-氨基酸氧化酶突变体使反应体系具有更好的催化效率，以外消旋D,L-草铵膦为底物进行催化反应时，转化率远高于野生型，PPO产率也大幅提升。
附图说明
图1示例性示出本申请方法采用的多酶体系拆分法生产L-草铵膦的反应式。
图2示例性示出去消旋合成L-草铵膦的反应式(葡萄糖脱氢酶辅酶循环系统)。
图3示例性示出去消旋合成L-草铵膦的反应式(甲酸脱氢酶辅酶循环系统)。
图4示例性示出双菌多酶一锅两步法去消旋化制备L-草铵膦的反应进程。
具体实施方式
实施例
材料和方法
上游基因工程所用试剂：实施例中使用的基因组提取试剂盒、质粒提取试剂盒、DNA纯化回收试剂盒购自康宁生命科学(吴江)有限公司；一步克隆试剂盒购自诺唯赞有限公司；E.coli BL21(DE3)、质粒pET-28a(+)等购自上海旭冠生物科技发展有限公司；DNA标记、低分子量标准蛋白、蛋白预制胶购自北京GenStar有限公司；ClonExpress II One Step Cloning Kit无缝克隆试剂盒购自南京诺唯赞生物科技股份有限公司；pfu DNA聚合酶和Dpn I内切酶购自赛默飞世尔科技(中国)有限公司；引物合成，序列测序工作由杭州擎科梓熙生物技术有限公司完成，全基因合成由生工生物工程(上海)股份有限公司完成。以上试剂使用方法参考商品说明书。
下游催化工艺所用试剂2-羰基-4-[羟基(甲基)膦酰基]丁酸(PPO)来自于永农生物科学有限公司；D,L-草铵膦来自于永农生物科学有限公司；其他常用试剂购自国药集团化学试剂有限公司。
实施例中通过高效液相色谱(HPLC)检测反应的进行，并对PPO进行分析。HPLC分析方法为：色谱柱PBR；柱温/30℃；流速/1mL/min；检测波长/210nm；流动相：5mM(NH 4) 2HPO 4。
通过手性HPLC分析方法检测草铵膦的两个构型的含量，手性HPLC分析方法为：色谱柱/OA-5000L；流动相/0.5g/L五水硫酸铜铵溶液，另加入0.3％v/v乙腈；检测波长/254nm；流速/1mL/min；柱温/35℃。
实施例1：基因工程菌的构建
将来源于Microbotryum intermedium的D-氨基酸氧化酶(DAAO，GenBank号：FMSP01000004.1，氨基酸序列为SEQ ID NO.1所示，核苷酸序列为SEQ ID NO.6所示)的基因序列进行全基因合成后，插入表达质粒pET-28a(+)，得到pET-28a-daao。测序验证无误后将pET-28a-daao转入表达宿主大肠杆菌E.coli BL21(DE3)中用于后续重组酶的表达。
将来源于Lactobacillus buchneri的甲酸脱氢(FDH)的序列(氨基酸序列为SEQ ID NO.2所示，核苷酸序列为SEQ ID NO.7所示)进行全基因合成后，插入表达质粒pET-28a(+)得到pET-28a-fdh。测序验证无误后将pET-28a-fdh转入表达宿主大肠杆菌E.coli BL21(DE3)中用于后续重组酶的表达。
将来源于Exiguobacterium sibiricum的葡萄糖脱氢酶(GDH)的序列(氨基酸序列为SEQ ID NO.3所示，核苷酸序列为SEQ ID NO.8所示)进行全基因合成后，插入表达质粒pET-28a(+)，得到pET-28a-gdh。测序验证无误后将pET-28a-gdh转入表达宿主大肠杆菌E.coli BL21(DE3)中用于后续重组酶的表达。
将来源于Lactobacillus brevis的醇脱氢酶(ADH)的序列(氨基酸序列为SEQ ID NO.4所示，核苷酸序列为SEQ ID NO.9所示)进行全基因合成后，插入表达质粒pET-28a(+)，得到pET-28a-adh。测序验证无误后将pET-28a-adh转入表达宿主大肠杆菌E.coli BL21(DE3)中用于后续重组酶的表达。
将来源于Delftia acidovorans的L-氨基酸脱氢酶(LAADH，GenBank号：WP_012202150.1，NCBI注释为Glu/Leu/Phe/Val脱氢酶，氨基酸序列为SEQ ID NO.5所示，核苷酸序列为SEQ ID NO.10所示)的基因序列进行全基因合成，插入表达质粒pET-28a(+)上，得到质粒pET-28a-laadh。测序验证无误后转入表达宿主大肠杆菌E.coli BL21(DE3)中用于后续重组酶的表达。
实施例2：工程菌菌体的培养
分别将工程菌重组大肠杆菌E.coli BL21(DE3)/pET-28a-DAAO、E.coli BL21(DE3)/pET-28a-LAADH、E.coli BL21(DE3)/pET-28a-FDH、E.coli BL21(DE3)/pET-28a-GDH和E.coli BL21(DE3)/pET-28a-ADH经平皿划线活化后，挑单菌落接种至含有50μg/mL卡那霉素的10mL LB液体培养基中，37℃震荡培养10h。按2％的接种量转接至50mL同样含有50μg/mL卡那霉素的LB液体培养基中，37℃震荡培养至OD600达到0.8左右时，加入终浓度为0.1mM的IPTG，25℃下震荡培养12h。培养结束后，将培养液8000rpm离心10min，弃上清，收集菌体，放到-80℃超低温冰箱中保存，待用。
实施例3：D-氨基酸氧化酶(DAAO)突变体(62位、226位)的构建
在实施例1中所述野生型DAAO序列的基础上突变了第62位和/或226位(具体为F62K、M226T)。针对突变的D-氨基酸氧化酶序列的第62位、第226位进行突变的突变体设计PCR的引物序列，具体如表1所示：
表1
序号 引物名 引物序列
1 F62KF gattcttgcgggtccaccttggggcaccagttcgctc
2 F62KR gagcgaactggtgccccaaggtggacccgcaagaatc
3 M226TF ggggtctgacgcatcggtagtgcacagcttgac
4 M226TR gtcaagctgtgcactaccgatgcgtcagacccc
PCR(25μL)扩增体系如下：
Pfu缓冲液12.5μL，引物2μL，模板质粒1μL，dNTP 0.5μL，Pfu 1μL，加入ddH 2O补足至25μL。
PCR扩增条件：
(1)95℃预变性3min，(2)95℃变性30秒，(3)65℃退火30秒，(4)72℃延伸5min，20个循环，(5)72℃延伸10min，(6)4℃保存。
PCR后取5μL对扩增产物进行核酸凝胶电泳分析，得到的目标条带清晰，将剩余产物加入0.5μL Dpn I内切酶，放于37℃下消化模板DNA 3h。
反应完成转化至BL21感受态细胞，涂布在含有50μg/mL卡那霉素的LB固体培养基，37℃培养过夜。挑单菌落，获得突变体转化子。按照实施例2所述获得菌体。
实施例4：D-氨基酸氧化酶突变体酶活的比较
通过测定PPO的生成量来比较D-氨基酸氧化酶与其突变体的催化效率。当只有D-氨基酸氧化酶及突变体时，反应体系为：300mM外消旋草铵膦铵盐，100mM pH8.0磷酸盐缓冲液，8000U/L过氧化氢酶，20g/L D-氨基酸氧化酶或其突变体冻干细胞。反应16h后，取反应液样品进行处理，使用HPLC测定PPO的浓度并计算转化率(产物PPO浓度/底物D-PPT浓度×100％)，具体如表2所示。
表2
Figure PCTCN2022089830-appb-000001
由表2可以看出，所得突变体的转化率均高于野生型DAAO的转化率。其中，转化率最高的为DAAO突变体4，其突变位点第62位的F突变为K，第226位的M突变为T。
实施例5：Glu/Leu/Phe/Val脱氢酶突变体(91位、168位)的构建
在实施例1中所述野生型LAADH序列的基础上突变了第91位和168位(具体为V91I、N168G)。针对突变的LAADH序列的第91位、第168位进行突变的突变体PCR设计引物序列，具体如表3所示：
表3
序号 引物名 引物序列
1 V91IF cctggtggaaacggatgccgcccttgccg
2 V91IR cggcaagggcggcatccgtttccaccagg
3 N168GF gggtccgaagaatcggtcgtgcagcgcttgc
4 N168GR gcaagcgctgcacgaccgattcttcggaccc
PCR扩增体系及条件同实施例3所述步骤。
反应完成转化至BL21感受态细胞，涂布在含有50μg/mL卡那霉素的LB固体培养基，37℃培养过夜。挑单菌落，获得突变体转化子。按照实施例2所述获得菌体。
实施例6：LAADH表达菌株的构建
一、含葡萄糖脱氢酶辅酶循环系统的表达菌株的构建
在载体pET-28a-LAADH V91I-N168G上，通过无缝克隆试剂盒将葡萄糖脱氢酶基因片段连接到多克隆位点上，酶切位点为Hind III，构建得到质粒pET-28a-LAADH V91I-N168G-GDH，构建得到表达菌株E.coli BL21(DE3)/pET-28a-LAADH V91I-N168G-GDH。
二、含甲酸脱氢酶辅酶循环系统的表达菌株的构建
在载体pET-28a-LAADH V91I-N168G上，通过无缝克隆试剂盒将甲酸脱氢酶基因片段连接到多克隆位点上，酶切位点为Hind III，构建得到质粒pET-28a-LAADH V91I-N168G-FDH，构建得到表达菌株E.coli BL21(DE3)/pET-28a-LAADH V91I-N168G-FDH。
三、醇脱氢酶辅酶循环系统的表达菌株的构建
在载体pET-28a-LAADH V91I-N168G上，通过无缝克隆试剂盒将醇脱氢酶基因片段连接到多克隆位点上，酶切位点为Hind III，构建得到质粒pET-28a-LAADH V91I-N168G-ADH，构建得到表达菌株E.coli BL21(DE3)/pET-28a-LAADH V91I-N168G-ADH。
实施例7：双菌多酶去消旋制备L-草铵膦(含葡萄糖脱氢酶GDH辅酶循环体系)
按照实施例2的方法培养能够表达D-氨基酸氧化酶菌株E.coli BL21(DE3)/pET-28a-DAAO F62K-M226和能够表达L-氨基酸脱氢酶和葡萄糖脱氢酶的表达菌株E.coli BL21(DE3)/pET-28a-LAADH V91I-N168G-GDH，离心收集菌体细胞并冻干。
在1L反应器中，加入600mL的磷酸铵缓冲液(pH8.0，100mM)，含有400mM D,L-PPT，8000U/L过氧化氢酶，5％(v/v)消泡剂、20g/L E.coli BL21(DE3)/pET-28a-DAAO F62K-M226菌体，20g/L E.coli BL21(DE3)/pET-28a-LAADH V91I-N168G-GDH菌体，0.5mM NADH和250mM葡萄糖，通入空气，通气量2L/min，再加入氨水控制pH为8，温度30℃，反应24小时。反应结束后，液相检测L-PPT为388mM，产品L-草铵膦的e.e.值大于99％，L-PPT转化收率为97％。
实施例8：双菌多酶去消旋制备L-草铵膦(含甲酸脱氢酶FDH辅酶循环体系)
按照实施例2的方法培养能够表达D-氨基酸氧化酶菌株E.coli BL21(DE3)/pET-28a-DAAO F62K-M226和能够表达L-氨基酸脱氢酶和甲酸脱氢酶的表达菌株E.coli BL21(DE3)/pET-28a-LAADH V91I-N168G-FDH，离心收集菌体细胞并冻干。
在1L反应器中，加入600mL的磷酸铵缓冲液(pH8.0，100mM)，含有400mM D,L-PPT，8000U/L过氧化氢酶，5％(v/v)消泡剂，20g/L E.coli BL21(DE3)/pET-28a-DAAO F62K-M226冻干菌体，20g/L E.coli BL21(DE3)/pET-28a-LAADH V91I-N168G-FDH冻干菌体，0.5mM NADH和250mM甲酸铵，通入空气，通气量2L/min，氨水控制pH为8，温度30℃，反应24小时。反应结束后，液相检测L-PPT为382mM，产品L-草铵膦的e.e.值大于99％，L-PPT转化收率为95.5％。
实施例9：双菌多酶去消旋制备L-草铵膦(醇脱氢酶ADH辅酶循环体系)
按照实施例2的方法培养能够表达D-氨基酸氧化酶菌株E.coli BL21(DE3)/pET-28a-DAAO F62K-M226和能够表达L-氨基酸脱氢酶和醇脱氢酶的表达菌株E.coli BL21(DE3)/pET-28a-LAADH V91I-N168G-ADH，离心收集菌体细胞并冻干。
在1L反应器中，加入600mL反应液(氨水调节至pH8.0)，含有400mM D,L-PPT，8000U/L过氧化氢酶，0.5％(v/v)消泡剂，20g/L E.coli BL21(DE3)/pET-28a-DAAO F62K-M226冻干菌体，20g/L E.coli BL21(DE3)/pET-28a-LAADH V91I-N168G-ADH冻干菌体，0.5mM NADH和250mM异丙醇，通入空气，通气量2L/min，氨水控制pH为8，温度30℃，反应24小时。以实施例所示液相检测方法检测反应过程中D-草铵膦的消耗和L-草铵膦等的生成，反应进程曲线如图4所示。该图显示，随着时间的推移D-草铵膦的浓度逐渐降低，L-草铵膦的浓度逐渐升高。反应结束后，液相检测L-PPT为380mM，产品L-草铵膦的e.e.值大于99％，L-PPT转化收率为95％。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其 它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (18)

  1. 一种D-氨基酸氧化酶突变体，其中，当与包含SEQ ID NO.1所示序列的D-氨基酸氧化酶的氨基酸序列比对时，所述D-氨基酸氧化酶突变体的氨基酸序列包含对应于第62位和/或第226位的氨基酸残基的替换，所述第62位和第226位是参照SEQ ID NO.1限定的，并且所述D-氨基酸氧化酶突变体的氨基酸序列与SEQ ID NO.1所示序列具有至少90％的同一性。
  2. 根据权利要求1所述的D-氨基酸氧化酶突变体，其中第226位的氨基酸残基的替换为M226T；优选的，第226位的氨基酸残基的替换为M226T，第62位的氨基酸残基的替换为F62K。
  3. 根据权利要求1或2所述的D-氨基酸氧化酶突变体，其来源于Microbotryum intermedium。
  4. 根据权利要求1至3中任一项所述的D-氨基酸氧化酶突变体，其氨基酸序列与SEQ ID NO.1所示序列具有至少99％的同一性。
  5. 核酸，其包含编码权利要求1至4中任一项所述的D-氨基酸氧化酶突变体的序列。
  6. 表达载体，其包含权利要求5所述的核酸。
  7. 重组宿主细胞，其包含权利要求5所述的核酸或者权利要求6所述的表达载体。
  8. 根据权利要求7所述的重组宿主细胞，其中所述宿主细胞属于酿酒酵母(Saccharomyces cerevisiae)、解脂耶氏酵母(Yarrowia lipolitica)、克鲁斯假丝酵母(Candida krusei)、东方伊萨酵母(Issatchenkia orientalis)、放线菌(Actinomycetes)、链霉菌(Streptomyces)、枯草芽孢杆菌(Bacillus subtilis)或大肠杆菌(Escherichia coli)中的一种。
  9. 权利要求1至4中任一项所述的D-氨基酸氧化酶突变体、权利要求5所述的核酸、权利要求6所述的表达载体、或者权利要求7-8任一项所述的重组宿主细胞在制备L-草铵膦中的用途。
  10. 一种制备L-草铵膦的方法，其包括在酶催化体系的存在下使D-草铵膦发生转化反应生成L-草铵膦，其中所述酶催化体系包括用于将D-草铵膦转化 为2-羰基-4-[羟基(甲基)膦酰基]丁酸的权利要求1至4中任一项所述的D-氨基酸氧化酶突变体；特别地，所述D-草胺膦最初存在于D-和L-草胺膦或其盐的外消旋混合物中。
  11. 根据权利要求10所述的方法，其中所述酶催化体系还包含用于将2-羰基-4-[羟基(甲基)膦酰基]丁酸转化为L-草铵膦的L-氨基酸脱氢酶。
  12. 根据权利要求10或11所述的方法，其中所述酶催化体系还包括过氧化氢酶。
  13. 根据权利要求10至12中任一项所述的方法，其中所述酶催化体系还包括辅酶循环系统，所述辅酶循环系统选自以下中的至少一种：
    (1)甲酸脱氢酶辅酶循环系统：包括甲酸脱氢酶，甲酸盐和辅酶；
    (2)葡萄糖脱氢酶辅酶循环系统：包括葡萄糖脱氢酶，葡萄糖和辅酶；
    (3)醇脱氢酶辅酶循环系统：包括醇脱氢酶，异丙醇和辅酶。
  14. 根据权利要求10至13中任一项所述的方法，其中所述酶催化体系中的每种酶的形式各自独立地选自：游离酶和表达酶的重组宿主细胞。
  15. 根据权利要求14所述的方法，其中所述表达酶的重组宿主细胞各自独立地选自：酿酒酵母(Saccharomyces cerevisiae)、解脂耶氏酵母(Yarrowia lipolitica)、克鲁斯假丝酵母(Candida krusei)、东方伊萨酵母(Issatchenkia orientalis)、放线菌(Actinomycetes)、链霉菌(Streptomyces)、枯草芽孢杆菌(Bacillus subtilis)或大肠杆菌(Escherichia coli)。
  16. 根据权利要求14或15所述的方法，其中，以湿菌体重量计，所述重组宿主细胞的总添加量为1-200g/L反应液。
  17. 根据权利要求10至16中任一项所述的方法，其中所述转化反应在pH为7-10的反应液中进行，优选地，所述反应液是pH为8-9的反应液。
  18. 根据权利要求10至17中任一项所述的方法，所述D-氨基酸氧化酶突变体催化的氧化反应的反应温度为25-45℃，时间为6-24h。
PCT/CN2022/089830 2021-04-29 2022-04-28 D-氨基酸氧化酶突变体及其在制备l-草铵膦中的应用 WO2022228505A1 (zh)

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