WO2024017405A1 - Mutant de carbonyle réductase thermostable et tolérant à l'isopropanol et son utilisation - Google Patents

Mutant de carbonyle réductase thermostable et tolérant à l'isopropanol et son utilisation Download PDF

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WO2024017405A1
WO2024017405A1 PCT/CN2023/114764 CN2023114764W WO2024017405A1 WO 2024017405 A1 WO2024017405 A1 WO 2024017405A1 CN 2023114764 W CN2023114764 W CN 2023114764W WO 2024017405 A1 WO2024017405 A1 WO 2024017405A1
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Prior art keywords
carbonyl reductase
mutant
reductase mutant
formula
seq
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PCT/CN2023/114764
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English (en)
Chinese (zh)
Inventor
陈少欣
汤佳伟
张露文
吴远杰
张正玉
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上海医药工业研究院有限公司
中国医药工业研究总院有限公司
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Publication of WO2024017405A1 publication Critical patent/WO2024017405A1/fr

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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
    • 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
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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.)
    • 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
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
    • 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)
    • C12Y101/01184—Carbonyl reductase (NADPH) (1.1.1.184)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
    • 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 present invention relates to the field of pharmaceutical compound preparation, and in particular to a thermostable and isopropyl alcohol-tolerant carbonyl reductase mutant and its application.
  • Carbonyl reductase (Ketoredutase, KRED) can directionally reduce prochiral carbonyl compounds into chiral hydroxy compounds and is an important tool for the synthesis of chiral drugs. Carbonyl reductase, which has the advantages of high selectivity and high conversion rate under mild conditions, has long been valued by the pharmaceutical industry.
  • the antiplatelet therapeutic drug Ticagrelor is an effective drug for the treatment of acute coronary syndrome and was launched in my country in November 2012.
  • (S)-2-Chloro-1-(3,4-difluorophenyl)ethanol (Formula II) is an important chiral intermediate in the synthesis of ticagrelor.
  • LSADH carbonyl reductase
  • Leifsonia sp.strain S749 can efficiently reduce 2-chloro-1-(3,4-difluorophenyl)ethanone (formula I) to synthesize alternative Graylo chiral alcohol intermediate (S)-2-chloro-1-(3,4-difluorophenyl)ethanol (ee>99.9%).
  • S Graylo chiral alcohol intermediate
  • LSADH is deactivated prematurely in high concentrations of substrates, products and isopropanol, and cannot perform as it should. Therefore, it is of great significance to improve the stability of enzymes so that they can withstand and adapt to harsh industrial environments.
  • Enzyme stability modification has always been a hot and difficult point in modification.
  • Existing modification strategies include the introduction of disulfide bonds, modification of salt bridges, surface charge engineering, and enzyme cyclization engineering. But no matter which strategy is used to modify the enzyme, the biggest problem in enzyme stability modification is that while improving the rigidity of the enzyme structure, it will also reduce its original catalytic activity. Therefore, in the process of enzyme modification, it is extremely challenging to effectively balance stability and activity.
  • the present invention provides a thermally stable and isopropyl alcohol tolerant carbonyl reductase mutant and its application.
  • the present invention carries out molecular evolution of the enzyme through rational design to obtain a mutant with greatly improved thermal stability and isopropyl alcohol tolerance, which is more conducive to large-scale industrial application.
  • a first aspect of the present invention provides a carbonyl reductase mutant, which has S148L and/or Q169K mutations in the amino acid sequence shown in SEQ ID NO:1.
  • the carbonyl reductase mutant further includes one or more mutations of I145V, A163G and L207V.
  • the carbonyl reductase mutant further includes one or more mutations of T100P, T111R and V183N.
  • the mutant is selected from the group consisting of:
  • amino acid sequence of the carbonyl reductase mutant is as shown in SEQ ID NO: 4.
  • a second aspect of the invention provides another carbonyl reductase mutant, which has a mutation selected from the following group on the amino acid sequence shown in SEQ ID NO: 1:
  • a third aspect of the invention provides an isolated nucleic acid encoding a carbonyl reductase mutant as described in the first aspect of the invention.
  • nucleotide sequence of the nucleic acid is as shown in SEQ ID NO: 3 or 5.
  • the fourth aspect of the present invention provides a recombinant expression vector comprising the isolated nucleic acid as described in the third aspect of the present invention.
  • the fifth aspect of the present invention provides a genetically engineered bacterium, which contains the isolated nucleic acid as described in the third aspect of the present invention, or the recombinant expression vector as described in the fourth aspect of the present invention.
  • a sixth aspect of the present invention provides a carbonyl reductase combination, which includes at least one carbonyl reductase mutant according to the first aspect of the present invention and a wild-type carbonyl reductase, or includes at least two of the first aspect of the present invention.
  • the carbonyl reductase mutant includes at least one carbonyl reductase mutant according to the first aspect of the present invention and a wild-type carbonyl reductase, or includes at least two of the first aspect of the present invention.
  • the seventh aspect of the present invention provides a method for preparing a compound represented by formula II, which includes using the carbonyl reductase mutant described in the first aspect of the present invention or the genetically engineered bacterium described in the fifth aspect of the present invention or the present invention.
  • the carbonyl reductase combination according to the sixth aspect of the invention catalyzes the compound represented by Formula I.
  • the carbonyl reductase mutant has a T100P/T111R/I145V/S148L/A163G/Q169K/V183N/L207V mutation on the amino acid sequence shown in SEQ ID NO:1.
  • the method includes the following steps:
  • the buffer is a phosphate buffer with a concentration of 0.1M, a pH of 7.0, and a reaction temperature of 40-50°C, such as 45°C.
  • the method also includes extracting the compound represented by Formula II, and the extraction includes the following steps:
  • reaction conditions of the reaction are as follows:
  • the protein inactivation temperature is 55-65°C; preferably 60°C; the time is 0.5-1.5h, for example 1h; diatomite is used for filtration, and the amount of diatomite is preferably 8%-12%. For example 10%;
  • step (2) dry with anhydrous sodium sulfate, and wash the organic layer with water and 5% brine.
  • the eighth aspect of the present invention provides the carbonyl reductase mutant described in the first aspect of the present invention or the genetically engineered bacterium described in the fifth aspect of the present invention or the carbonyl reductase combination described in the sixth aspect of the present invention in the preparation of formula II Applications of the compounds shown.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the carbonyl reductase mutant of the present invention has improved catalytic efficiency compared with the wild type, and has good thermal stability and isopropyl alcohol tolerance.
  • the catalytic efficiency of mutant 35 is increased by 41 times compared with wild-type LSADH; Tm is increased by 23.3°C, and the half-life at 55°C is increased from 3 minutes to 60 hours; the tolerance to isopropyl alcohol is increased to 80%; Relative to WT, mutant 35 has a thermal stability of 2077.7 times and an isopropyl alcohol tolerance of 209.1 times.
  • Figure 1 shows the half-life of wild-type carbonyl reductase LSADH and its mutants at 55°C.
  • the carbonyl reductase LSADH (SEQ ID NO:1) gene derived from Leifsonia sp.strain S749 was codon optimized (SEQ ID NO:3) and synthesized by Nanjing GenScript into the pET28a(+) vector. Subsequently, the plasmid containing the LSADH gene was introduced into the host E. coli BL21 (DE3) competent cells to obtain a recombinant genetically engineered strain of carbonyl reductase (LSADH).
  • coli BL21 (DE3), and then spread it onto LB solid medium (containing 50 ⁇ g/mL kanamycin), and incubate at 37 Incubate overnight at °C. The next day, single colonies were picked into a 96-well plate containing 400 ⁇ L LB medium (containing 50 ⁇ g/mL kanamycin) and cultured at 37°C overnight. Then transfer 10 ⁇ L of seed solution from the 96-well plate cultured overnight to a new 96-well plate (containing 400 ⁇ L of fermentation medium containing 50 ⁇ g/mL kanamycin), and culture with shaking at 37°C until the OD 600 value is >0.8.
  • IPTG with a final concentration of 1mM was added and cultured at 28°C for 20 hours to induce expression of the LSADH mutant. Finally, the 96-well plate was moved to a centrifuge, centrifuged at 4000g for 30 minutes to collect the bacteria, and stored at -20°C for later use.
  • the fermentation medium formula is as follows: yeast extract (2.4%), soy peptone (1.2%), sodium chloride (0.3%), glycerol (0.5%), dipotassium hydrogen phosphate (0.2%), magnesium sulfate heptahydrate (0.05 %).
  • lysis buffer 0.1 M phosphate buffer containing 1000 U lysozyme, pH 7.0
  • lysis buffer 0.1 M phosphate buffer containing 1000 U lysozyme, pH 7.0
  • lyse it at 30°C for 1 hour, put it into a centrifuge at 4°C, 4000g, centrifuge for 30 minutes, and remove the clarification
  • the supernatants were used to determine mutant activity.
  • LSADH and mutant crude After the enzyme solution was soaked at 55°C and isopropyl alcohol with concentrations of 40%, 60%, and 80%, the stability test was performed.
  • the test system (total volume 200 ⁇ L) is as follows: 4mM 2-chloro-1-(3,4-difluorophenyl)ethanone substrate, 1mM NADH, 20% DMSO (v/v), K 2 HPO 4 -KH 2 PO 4 phosphate buffer (100mM, pH 7.0) and 10 ⁇ L LSADH or mutant enzyme solution.
  • the protein concentration of crude enzyme solution of LSADH and mutants was diluted and adjusted according to the actual activity measurement.
  • the activity of LSADH and its mutants was calculated and characterized based on the change value of NADH absorbance at 340 nm.
  • Mutants with significantly improved stability were retested in a 2mL activity measurement system, specifically: 20g/L 2-chloro-1-(3,4-difluorophenyl)ethanone substrate, 20% isopropyl alcohol (v /v), 0.1g/L NAD + , K 2 HPO 4 -KH 2 PO 4 phosphate buffer (100mM, pH 7.0) and 100 ⁇ l crude enzyme solution before or after treatment. After reacting at 28°C for 15 minutes, the conversion rate was detected by HPLC.
  • mutant 35 the amino acid sequence is shown in SEQ ID NO: 4, the nucleotide sequence is shown in SEQ ID NO: 5
  • the inventor further investigated the comparison of mutant 35 Kinetic parameters, half-life at 55°C, Tm and changes (Table 2). From the K m and k cat /K m values, it can be seen that the affinity of mutant 35 for 2-chloro-1-(3,4-difluorophenyl)ethanone is increased by 8 times compared with wild-type LSADH. Catalytic efficiency increased by 40.5 times.
  • mutant 35 In terms of thermal stability, the half-life (t1/2) of mutant 35 at 55°C is 60 hours, while the half-life (t1/2) of wild-type LSADH is only 3 minutes. As shown in Figure 1, mutant 35 can still maintain 80% of the catalytic activity after incubation at 55°C for 24 hours, and 20% of the catalytic activity remains after 144 hours of incubation. while wild type LSADH is no longer active after incubation at 55°C for 15 minutes. At the same time, we also measured the melting temperature of mutant 35 and found that the Tm value of mutant 35 increased from 39.5°C of wild-type LSADH to 62.8°C. This shows that mutant 35 unfolds and gradually destroys the secondary structure at a higher temperature than wild-type LSADH.

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Abstract

L'invention concerne un mutant de carbonyle réductase thermostable et tolérant à l'isopropanol et son utilisation. Le mutant de carbonyl réductase est soumis à des mutations S148L et/ou Q169K sur une séquence d'acides aminés telle que représentée dans SEQ ID NO : 1. L'invention concerne en outre une combinaison de carbonyle réductase et un procédé de préparation d'un composé tel que représenté dans la formule II, et une utilisation de la combinaison de carbonyle réductase dans la préparation du composé tel que représenté dans la formule II. La stabilité thermique du mutant 35 de la présente invention est de 2077,7 fois celle d'une enzyme WT, et la tolérance à l'isopropanol du mutant 35 est de 209,1 fois celle de l'enzyme WT.
PCT/CN2023/114764 2022-07-19 2023-08-24 Mutant de carbonyle réductase thermostable et tolérant à l'isopropanol et son utilisation WO2024017405A1 (fr)

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CN202210851795.1 2022-07-19
CN202210851795.1A CN117417909A (zh) 2022-07-19 2022-07-19 一种热稳定和异丙醇耐受的羰基还原酶突变体及其应用

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110177579A1 (en) * 2008-09-26 2011-07-21 Ma Kesen Thermostable Alcohol Dehydrogenase Derived From Thermococcus Guaymasensis
CN108949707A (zh) * 2017-05-24 2018-12-07 武汉大学 一种热稳定性提高的醇脱氢酶突变体
CN111321129A (zh) * 2018-12-15 2020-06-23 宁波酶赛生物工程有限公司 工程化酮还原酶多肽及其应用
CN113981013A (zh) * 2021-12-02 2022-01-28 寰酶生物技术(上海)有限公司 一种手性四氢萘-2-醇化合物的生物催化制备方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110177579A1 (en) * 2008-09-26 2011-07-21 Ma Kesen Thermostable Alcohol Dehydrogenase Derived From Thermococcus Guaymasensis
CN108949707A (zh) * 2017-05-24 2018-12-07 武汉大学 一种热稳定性提高的醇脱氢酶突变体
CN111321129A (zh) * 2018-12-15 2020-06-23 宁波酶赛生物工程有限公司 工程化酮还原酶多肽及其应用
CN113981013A (zh) * 2021-12-02 2022-01-28 寰酶生物技术(上海)有限公司 一种手性四氢萘-2-醇化合物的生物催化制备方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein 28 May 2021 (2021-05-28), ANONYMOUS : "LS-ADH, partial [synthetic construct] ", XP093129962, retrieved from NCBI Database accession no. QVQ68835.1 *

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