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WO2019011237A1 - Utilisation de lactate déshydrogénase dans la synthèse asymétrique d'un composé hydroxyle chiral - Google Patents

Utilisation de lactate déshydrogénase dans la synthèse asymétrique d'un composé hydroxyle chiral Download PDF

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Publication number
WO2019011237A1
WO2019011237A1 PCT/CN2018/095149 CN2018095149W WO2019011237A1 WO 2019011237 A1 WO2019011237 A1 WO 2019011237A1 CN 2018095149 W CN2018095149 W CN 2018095149W WO 2019011237 A1 WO2019011237 A1 WO 2019011237A1
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polypeptide
seq
amino acid
compound
formula
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Chinese (zh)
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田振华
程占冰
丁少南
张涛
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Abiochem Biotechnology Co Ltd
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Abiochem Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01028—D-Lactate dehydrogenase (1.1.1.28)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention belongs to the technical field of bioengineering, and particularly relates to a lactate dehydrogenase, a preparation method of the lactate dehydrogenase, and the use of the lactate dehydrogenase as a catalyst in asymmetric synthesis of a chiral hydroxy compound.
  • Ply drugs are mainly used to treat heart failure and high blood pressure.
  • antihypertensive drugs such as ACE inhibitors, calcium antagonists, angiotensin II receptor antagonists and ⁇ -blockers in the Chinese antihypertensive drug market.
  • Puli drugs have been affected by sartan drugs, and sales have declined.
  • the prices are relatively stable.
  • Methods of synthesizing R-HPBE include chemical methods and biological methods. Among them, the chemical method is based on cheap and readily available raw materials, and finally synthesizes R-HPBE through a multi-step reaction. For example, benzoic acid and pyruvic acid are subjected to a multi-step reaction to form 2-hydroxy-4-phenylbutyric acid, followed by racemization and esterification to synthesize (R)-HPBE; and as by diethyl oxalate and Ethyl phenylpropionate was used as a starting material to synthesize ethyl 2-carbonyl-4-phenylbutanoate and then subjected to asymmetric hydrogenation to obtain (R)-HPBE.
  • benzoic acid and pyruvic acid are subjected to a multi-step reaction to form 2-hydroxy-4-phenylbutyric acid, followed by racemization and esterification to synthesize (R)-HPBE; and as by diethyl oxalate
  • the chemical method has obvious advantages in product scale and can reach a production scale of 500 kg.
  • the chemical method uses a catalyst contaminated with heavy metals such as Pt, which cannot meet the requirements of green chemistry and has high cost.
  • chemical methods have disadvantages such as low yield, relatively harsh reaction conditions, high purity of the substrate, and low optical activity of the obtained product, and thus are not suitable for scale production.
  • the Applicant has applied for a patent for the asymmetric reduction of ketone reductase (R)-HPBE (CN2014105418998), wherein the ketone ester can achieve an optical purity of 98% or more by the action of the ketoreductase.
  • R ketone reductase
  • CN2014105418998 ketone reductase
  • the stability of the raw material OPBE is poor, the separation and purification process of the product is complicated, and the production cost is high.
  • One of the objects of the present invention is to provide a use of a polypeptide in the production of a compound of formula A or a downstream product of a compound of formula A as a precursor.
  • Another object of the invention is to provide a process for the production of a compound of formula A.
  • a further object of the invention is to provide a compound producing strain of formula A.
  • a further object of the present invention is to provide a method of constructing a strain of a compound of formula A.
  • amino acid sequence shown by SEQ ID NO: 1 is passed through one or several, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1- a polypeptide derived from a polypeptide having the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1) formed by substitution, deletion or addition of three, most preferably one amino acid residues;
  • R 1 represents hydrogen, or 1, 2, 3 or 4 substituents selected from the group consisting of halogen, -OH, substituted or unsubstituted C 1 -C 8 alkyl, substituted or unsubstituted C 3 -C 8 a cycloalkyl, substituted or unsubstituted C 1 -C 8 alkoxy group, or a substituted or unsubstituted C 3 -C 8 cycloalkoxy group, wherein said substitution has one or more selected from the group consisting of Substituents: halogen, -OH, -NH 2 , -CN, C 1 -C 3 alkyl, C 1 -C 3 haloalkyl, -NH(C 1 -C 3 alkyl), -N(C 1 -C 3 alkyl) 2 ;
  • the compound of formula A is formed by asymmetric reduction of a prochiral carbonyl acid compound.
  • the prochiral carbonyl acid compound is a compound of formula B:
  • R 1 and R 2 are as defined above.
  • the prochiral carbonyl acid compound comprises the following compound or a pharmaceutically acceptable salt thereof:
  • R 1 is H or Cl.
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • R 1 is H and n is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • R 1 is Cl and n is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • R 1 is H and p is 1.
  • R 1 is Cl and p is 1.
  • the polypeptide is one or more, preferably 1-20, more preferably 1-15, more preferably 1-10, at either end of the amino acid sequence shown in SEQ ID NO: 1. More preferably, the addition of 1-8, more preferably 1-3, most preferably 1 amino acid residue, derived from the polypeptide of the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1) Peptide.
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO: 1.
  • the polypeptide has an amino acid sequence of at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96 of the amino acid sequence set forth in SEQ ID NO:1. %, 97%, 98%, 99% or more of any polypeptide sequence of sequence identity.
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO: 3.
  • the compound of formula A comprises the following compound or a pharmaceutically acceptable salt thereof:
  • downstream products of the compound of formula A include: enalapril, benazepril, ramipril, cilazapril, cepril, and spironolide.
  • amino acid sequence shown by SEQ ID NO: 1 is passed through one or several, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-
  • the polypeptide is one or more, preferably 1-20, more preferably 1-15, more preferably 1-10, at either end of the amino acid sequence shown in SEQ ID NO: 1. More preferably, the addition of 1-8, more preferably 1-3, most preferably 1 amino acid residue, derived from the polypeptide of the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1) Peptide.
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO: 1.
  • the polypeptide has an amino acid sequence of at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96 of the amino acid sequence set forth in SEQ ID NO:1. %, 97%, 98%, 99% or more of any polypeptide sequence of sequence identity.
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO: 3.
  • the production strain is a bacterium.
  • the production strain is Escherichia coli.
  • the E. coli is selected from the group consisting of E. coli BL21 (DE3), E. coli C2566.
  • R 1 and R 2 are as defined above;
  • the concentration of the compound of the formula B in the reaction system is from 1 g/L to 1000 g/L, preferably from 5 g/L to 500 g/L, more preferably from 10 g/L to 100 g. /L, optimally 20g/L-80g/L.
  • the concentration of the stereoselective lactate dehydrogenase in the reaction system is 1 ⁇ 10 2 U / L - 1 ⁇ 10 5 U / L, preferably 1 ⁇ 10 3 U /L-1 ⁇ 10 5 U / L, more preferably 5 ⁇ 10 3 U / L - 1 ⁇ 10 5 U / L, and even more preferably 1 ⁇ 10 4 U / L - 1 ⁇ 10 5 U / L, preferably 5 ⁇ 10 4 U - 1 ⁇ 10 5 U / L.
  • stereoselective lactate dehydrogenase is:
  • amino acid sequence shown by SEQ ID NO: 1 is passed through one or several, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-
  • stereoselective lactate dehydrogenase is:
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO: 1.
  • the polypeptide has an amino acid sequence of at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96 of the amino acid sequence set forth in SEQ ID NO:1. %, 97%, 98%, 99% or more of any polypeptide sequence of sequence identity.
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO: 3.
  • the compound of formula A is R-HPBA.
  • the compound of formula A is (R)-(E)-2-hydroxy-4-phenyl-3-butenoic acid.
  • a coenzyme is also present in the reaction system.
  • the coenzyme is selected from the group consisting of NADH, NADPH, NAD, NADP, or a combination thereof.
  • the concentration of the coenzyme in the reaction system is from 5 mg/L to 1000 mg/L, preferably from 10 mg/L to 800 mg/L, more preferably from 20 mg/L to 600 mg/L. More preferably, it is 50 mg/L to 500 mg/L, and most preferably 100 mg/L to 300 mg/L.
  • an enzyme for coenzyme regeneration is also present in the reaction system.
  • the concentration of the enzyme for coenzyme regeneration is 1 ⁇ 10 2 U / L - 1 ⁇ 10 6 U / L, preferably 1 ⁇ 10 3 U / L - 1 ⁇ 10 5 U/L, more preferably 5 ⁇ 10 3 U / L - 2 ⁇ 10 5 U / L, most preferably 1.5 ⁇ 10 4 U / L - 1.5 ⁇ 10 5 U / L.
  • the enzyme for coenzyme regeneration is selected from the group consisting of formate dehydrogenase, glucose dehydrogenase, or a combination thereof.
  • the concentration of the glucose dehydrogenase is 1 ⁇ 10 2 U / L - 1 ⁇ 10 6 U / L, preferably 1 ⁇ 10 3 U / L - 5 ⁇ 10 5 U / L, more preferably 1 ⁇ 10 4 U / L - 1 ⁇ 10 5 U / L, most preferably 4 ⁇ 10 4 U / L - 5 ⁇ 10 4 U / L.
  • the formate dehydrogenase concentration is 1 ⁇ 10 2 U / L - 1 ⁇ 10 6 U / L, preferably 1 ⁇ 10 3 U / L - 5 ⁇ 10 5 U / L, more preferably 1 ⁇ 10 4 U / L - 1 ⁇ 10 5 U / L, most preferably 4 ⁇ 10 4 U / L - 5 ⁇ 10 4 U / L.
  • the temperature is from 10 ° C to 50 ° C, preferably from 15 ° C to 40 ° C, more preferably from 20 ° C to 30 ° C.
  • the pH is from 6 to 10, preferably from 6.5 to 9.0, more preferably from 7.5 to 8.0.
  • the compound of formula A is isolated from the culture system of 1).
  • a method of constructing a strain of a compound of formula A comprising:
  • the strain is made to comprise an expression vector expressing a polypeptide or such that a gene expressing the following polypeptide is integrated into the genome of the strain, the polypeptide being:
  • amino acid sequence shown by SEQ ID NO: 1 is passed through one or several, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-
  • the polypeptide is one or more, preferably 1-20, more preferably 1-15, more preferably 1-10, at either end of the amino acid sequence shown in SEQ ID NO: 1. More preferably, the addition of 1-8, more preferably 1-3, most preferably 1 amino acid residue, derived from the polypeptide of the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1) Peptide.
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO: 1.
  • the polypeptide has an amino acid sequence of at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96 of the amino acid sequence set forth in SEQ ID NO:1. %, 97%, 98%, 99% or more of any polypeptide sequence of sequence identity.
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO: 3.
  • sequence of the gene is selected from the group consisting of:
  • sequence of the gene is set forth in SEQ ID NO: 7.
  • the gene is constructed on an expression vector.
  • D-lactate dehydrogenase which is capable of stereoselectively catalyzing the reaction of formula I, such as OPBA, E. -2--Oxo-4-phenyl-3-butenoic acid
  • a compound of formula A eg R-HPBA, (R)-(E)-2-hydroxy-4-phenyl-3-butyl Acetate
  • conversion rate ⁇ 98%, chiral ee value ⁇ 99% thereby greatly improving production efficiency and reducing production costs.
  • the raw material for the reduction reaction by using the lactate dehydrogenase of the present invention is easy to obtain, has high yield, low cost, and is easy to be enlarged, and is suitable for large-scale production.
  • the present invention has been completed on this basis.
  • the term “about” means that the value can vary by no more than 1% from the recited value.
  • the expression “about 100” includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the terms "containing” or “including” may be open, semi-closed, and closed. In other words, the terms also include “consisting essentially of,” or “consisting of.”
  • reaction can be carried out and purified using the manufacturer's instructions for use of the kit, or in a manner well known in the art or as described in the present invention.
  • the above techniques and methods can generally be carried out according to conventional methods well known in the art, as described in the various summaries and more specific references cited and discussed in this specification.
  • group and its substituents can be selected by those skilled in the art to provide stable structural moieties and compounds.
  • substituent When a substituent is described by a conventional chemical formula written from left to right, the substituent also includes the chemically equivalent substituent obtained when the structural formula is written from right to left.
  • substituent -CH 2 O- is equivalent to -OCH 2 -.
  • C1-C6 alkyl refers to an alkyl group as defined below having a total of from 1 to 6 carbon atoms.
  • the total number of carbon atoms in the simplified symbol does not include carbon that may be present in the substituents of the group.
  • halogen means fluoro, chloro, bromo or iodo.
  • Haldroxy means an -OH group.
  • alkyl means a fully saturated straight or branched hydrocarbon chain group, It consists only of carbon atoms and hydrogen atoms, has, for example, 1 to 7 carbon atoms, and is linked to the rest of the molecule by a single bond, including, for example, but not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl Base, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, heptyl and the like.
  • Enantiomeric excess (ee) is generally used to characterize the excess value of one enantiomer relative to the other enantiomer in a chiral molecule.
  • polypeptide or “polypeptide of the invention” or “polypeptide of the invention” or “D-lactate dehydrogenase” or “stereoselective lactate dehydrogenase” as used herein have the same meaning and are used interchangeably herein. , both refer to a protein having a catalytically active compound which produces a compound of formula A. This polypeptide is naturally absent from E. coli and is an exogenous protein.
  • the polypeptide of the present invention may be: (a1) a polypeptide having the amino acid sequence shown in SEQ ID NO: 1; or (b1) passing one or more of the amino acid sequence shown in SEQ ID NO: 1. Substituents, deletions or additions of preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-3, most preferably 1 amino acid residues A polypeptide derived from a polypeptide having the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1).
  • the polypeptide is one or more, preferably 1-20, more preferably 1-15, more preferably 1-10, at either end of the amino acid sequence set forth in SEQ ID NO: 1. More preferably, the addition of 1-8, more preferably 1-3, most preferably 1 amino acid residue, derived from the polypeptide of the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1) Peptide.
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO: 1.
  • the polypeptide has an amino acid sequence of at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96 of the amino acid sequence set forth in SEQ ID NO:1. %, 97%, 98%, 99% or more of any polypeptide sequence of sequence identity.
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO:3.
  • polypeptide of the invention represents a protein having the amino acid sequence set forth in SEQ ID NO: 1, the coding sequence of which is set forth in SEQ ID NO: 5.
  • polypeptide of the invention represents a protein having the amino acid sequence set forth in SEQ ID NO: 3, the coding sequence of which is set forth in SEQ ID NO: 7.
  • the polypeptide of the present invention comprises up to 20, preferably up to 10, preferably up to 8, and preferably up to 3, compared to the polypeptide of the amino acid sequence of SEQ ID NO: 1. More preferably up to two, preferably up to one amino acid is replaced by a similar or similar amino acid. Mutants of these conservative variations can be produced according to, for example, amino acid substitutions as shown in the table below.
  • polynucleotide encoding a polypeptide can be a polynucleotide comprising the polypeptide, or a polynucleotide further comprising additional coding and/or non-coding sequences.
  • the homology or sequence identity may be 80% or more, preferably 90% or more, more preferably 95% to 98%, and most preferably 99% or more.
  • Methods for determining sequence homology or identity include, but are not limited to, Computational Molecular Biology, Lesk, AM, Oxford University Press, New York, 1988; Biocomputing: Information Biocomputing: Informatics and Genome Projects, Smith, DW, Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM and Griffin, HG , Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux , J. M. Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J.
  • the preferred method of determining identity is to obtain the largest match between the sequences tested.
  • the method of determining identity is compiled in a publicly available computer program.
  • Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al, 1990).
  • the BLASTX program is available to the public from NCBI and other sources (BLAST Handbook, Altschul, S. et al, NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al, 1990).
  • the well-known Smith Waterman algorithm can also be used to determine identity.
  • polypeptide of the present invention has D-lactate dehydrogenase activity and can be used to produce a compound of formula A or a downstream product of a compound of formula A as a precursor.
  • polypeptide is:
  • amino acid sequence shown by SEQ ID NO: 1 is passed through one or several, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1- a polypeptide derived from a polypeptide having the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1) formed by substitution, deletion or addition of three, most preferably one amino acid residues;
  • R 1 and R 2 are as defined above.
  • the compound of formula A is formed by asymmetric reduction of a prochiral carbonyl acid compound.
  • the prochiral carbonyl acid compound is a compound of formula B:
  • R 1 and R 2 are as defined above.
  • the prochiral carbonyl acid compound comprises the following compound or a pharmaceutically acceptable salt thereof:
  • the polypeptide is one or more, preferably 1-20, more preferably 1-15, more preferably 1-10, at either end of the amino acid sequence set forth in SEQ ID NO: 1. More preferably, the addition of 1-8, more preferably 1-3, most preferably 1 amino acid residue, derived from the polypeptide of the amino acid sequence of SEQ ID NO: 1 having the function of the polypeptide of (a1) Peptide.
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO: 1 or SEQ ID NO: 3.
  • polypeptide of the invention represents a protein having the amino acid sequence set forth in SEQ ID NO: 1, the coding sequence of which is set forth in SEQ ID NO: 5.
  • polypeptide of the invention represents a protein having the amino acid sequence set forth in SEQ ID NO: 3, the coding sequence of which is set forth in SEQ ID NO: 7.
  • the compound of formula A comprises R-HPBA or a pharmaceutically acceptable salt thereof.
  • the compound of formula A comprises (R)-(E)-2-hydroxy-4-phenyl-3-butenoic acid or a pharmaceutically acceptable salt thereof.
  • downstream products of the compound of formula A include: enalapril, benazepril, ramipril, cilazapril, cepril, sulpiride.
  • a strain expressing a polypeptide of the present invention is capable of stereoselectively catalyzing a reaction of the formula I, such as OPBA, E-2-oxo-4-phenyl-3- Butenoic acid) is efficiently converted to a compound of formula A (eg R-HPBA, (R)-(E)-2-hydroxy-4-phenyl-3-butenoic acid), conversion ⁇ 98%, chiral ee The value is ⁇ 99%.
  • the strain expresses the following polypeptide:
  • amino acid sequence shown by SEQ ID NO: 1 is passed through one or several, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-
  • the polypeptide is one or more, preferably 1-20, more preferably 1-15, more preferably 1-10 at either end of the amino acid sequence set forth in SEQ ID NO:1.
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO: 1.
  • the polypeptide has an amino acid sequence of at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95% of the amino acid sequence set forth in SEQ ID NO:1. 96%, 97%, 98%, 99% or more of any polypeptide sequence of sequence identity.
  • amino acid sequence of the polypeptide is set forth in SEQ ID NO:3.
  • the production strain is a bacterium, preferably E. coli (e.g. E. coli BL21 (DE3), E. coli C2566).
  • E. coli e.g. E. coli BL21 (DE3), E. coli C2566).
  • the stereoselective lactate dehydrogenase can be used in various forms.
  • resting cells or wet cells expressing the stereoselective lactate dehydrogenase of the present invention may be used, or various forms such as a crude enzyme solution, a pure enzyme or a crude enzyme powder may be used, or an immobilized enzyme may be used.
  • the method of producing a compound of formula A comprises:
  • R 1 and R 2 are as defined above;
  • the method of producing a compound of formula A comprises:
  • the compound of formula A is isolated from the culture system of 1).
  • a compound producing strain of the formula A having high conversion can be constructed by including the strain comprising an expression vector expressing the polypeptide of the present invention or by integrating a gene expressing the polypeptide of the present invention into the genome of the strain.
  • the method further comprises determining the conversion of the resulting strain and/or the yield of the compound of formula A to verify the resulting strain.
  • the reduction reaction using the lactate dehydrogenase of the present invention is low in cost and easy to enlarge, and is suitable for large-scale production.
  • the reagents and starting materials used in the present invention are commercially available.
  • PCR was carried out using genomic DNA as a template.
  • the experimental conditions were as follows:
  • the cloning enzyme was ligated into the NdeI&HindIII site of the expression vector pET28a to obtain two expression plasmids.
  • the gene sequences SEQ ID NO: 7 and SEQ ID NO: 8 were obtained by gene synthesis.
  • Example 1 The recombinant expression plasmid of Example 1 was transformed into E. coli BL21 (DE3) competent cells in E. coli BL21 (DE3) competent cells under the conditions of 42 ° C, heat shock for 90 seconds, and the positive recombinants were plated on a plate containing kanamycin antibiotics. Screening, picking monoclonal, colony PCR to verify positive clones.
  • the recombinant recombinant strain was cultured to obtain the positive recombinant transformant E.coli BL21(DE3)/pET28a-LDH1(NP_765629.1), E.coli BL21(DE3)/pET28a-LDH2(NP_765676.1), E.coli BL21(DE3 ) / pET28a-LDH3 (WP_002484424.1), and E. coli BL21 (DE3) / pET28a - LDH4 (WP_002456698.1).
  • High-density fermentation of LDH The above recombinant Escherichia coli was inoculated into 200 mL of LB medium containing 50 ug/mL kanamycin, and cultured at 37 ° C, 180-220 rpm for 10-16 h.
  • the cultured seeds were inoculated in a 3 L upper tank fermentation medium (M9) at a ratio of 10% (v/v) (glucose 4 g/L, disodium hydrogen phosphate 12.8 g/L, potassium dihydrogen phosphate 3 g/L).
  • ammonium chloride 1g / L ammonium chloride 1g / L, sodium sulfate 0.5g / L, calcium chloride 0.0152g / L, magnesium chloride hexahydrate 0.41g / L), at 25-35 ° C, 300-800rpm, air flow 2-6L / min Culture under conditions.
  • a feed medium containing 60% glycerol was fed at a rate of 5-20 mL/h until the end of the fermentation.
  • induction was started by adding 0.1-1 mM IPTG. After induction for 5-15 hours, the cells were placed in a can, and the cells were collected by centrifugation at 5000 rpm.
  • the enzyme activity (U) is defined as the amount of enzyme required to consume 1 ⁇ mol of NADH per minute.
  • the enzyme activity (U) was measured by measuring the NADH consumption rate at a wavelength of 340 nm using a spectrophotometer at 50 ° C in a 50 mM phosphate buffer, an OPBA concentration of 1 mM, and a NADH concentration of 1 mM.
  • the concentration of OPBA is 0.1M
  • the concentration of NAD+ is 0.02 mM
  • D-lactate dehydrogenase is 1 to 10 g/L
  • the glucose dehydrogenase GDH is 0.1 to 3 g/L.
  • Reaction temperature 20 to 35 ° C
  • reaction time 1 h
  • pH between 6.5 and 7.5 was controlled with 1 M NaOH or saturated Na 2 CO 3 .
  • the pH was adjusted to 2 to 3 with 1 M hydrochloric acid, extracted with 2 times volume of ethyl acetate three times, and dried by rotary evaporation to obtain HPBA.
  • the reaction conversion rate was determined by HPLC, and the ee value was detected by chiral HPLC.
  • reaction temperature 20-30 ° C
  • reaction time 8 h
  • pH value 6.5-7.5 was controlled with 1 M NaOH.
  • the pH was adjusted to 2 to 3 with 1 M hydrochloric acid, extracted with 2 times volume of ethyl acetate three times, and dried by rotary evaporation to obtain crude R-HPBA.
  • the reaction conversion rate was 99% and the ee value was 99.9% by HPLC.
  • reaction temperature 20 ⁇ 30 ° C, with 1M NaOH control pH between 7.5 ⁇ 8.0.
  • the progress of the reaction was monitored according to the amount of NaOH.
  • 30 g of OPBA was added and the reaction was continued for 10 hours.
  • the pH was adjusted to 2 to 3 with 1 M hydrochloric acid, extracted with 2 times volume of ethyl acetate three times, and dried by rotary evaporation to obtain crude R-HPBA.
  • the reaction conversion rate was 100% and the ee value was 99.9% by HPLC.

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Abstract

L'invention concerne l'utilisation d'une lactate déshydrogénase dans la synthèse asymétrique d'un composé hydroxyle chiral. En particulier, l'invention concerne l'utilisation d'un polypeptide dans la production d'un composé de formule A ou d'un produit en aval ayant le composé de formule A comme précurseur. L'invention concerne en outre un procédé de production d'un composé de formule A, le procédé consistant à cultiver une souche bactérienne qui exprime le polypeptide pour obtenir le composé de formule A. L'invention concerne en outre une souche bactérienne qui produit le composé de formule A, et un procédé de production de la souche bactérienne qui produit le composé de formule A. Au moyen du procédé, le composé de formule A peut être produit de manière efficace et peu coûteuse. Le procédé permet de préparer le composé de formule A obtenu à une concentration élevée, et présente les avantages d'une pureté optique de produit élevée, de conditions de réaction douces, d'être respectueux de l'environnement, d'avoir un fonctionnement simple et un développement industriel facile.
PCT/CN2018/095149 2017-07-11 2018-07-10 Utilisation de lactate déshydrogénase dans la synthèse asymétrique d'un composé hydroxyle chiral Ceased WO2019011237A1 (fr)

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CN105567652A (zh) * 2014-10-14 2016-05-11 南京博优康远生物医药科技有限公司 一种酮还原酶及其在不对称合成手性羟基化合物中的应用

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CN102260664A (zh) * 2011-06-10 2011-11-30 尚科生物医药（上海）有限公司 固定化全细胞催化剂及其制备方法和应用
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