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WO2022036662A1 - Procédé de synthèse enzymatique de 3-hydroxybutyrate - Google Patents

Procédé de synthèse enzymatique de 3-hydroxybutyrate Download PDF

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Publication number
WO2022036662A1
WO2022036662A1 PCT/CN2020/110402 CN2020110402W WO2022036662A1 WO 2022036662 A1 WO2022036662 A1 WO 2022036662A1 CN 2020110402 W CN2020110402 W CN 2020110402W WO 2022036662 A1 WO2022036662 A1 WO 2022036662A1
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seq
mutant
amino acid
acid sequence
alcohol dehydrogenase
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Inventor
范文超
高书良
王金刚
梁岩
杨海锋
任亮
袁圣伦
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Zhejiang Huarui Biotechnology Co Ltd
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Zhejiang Huarui Biotechnology Co Ltd
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Priority to PCT/CN2020/110402 priority Critical patent/WO2022036662A1/fr
Priority to CN202080103912.5A priority patent/CN116157509A/zh
Publication of WO2022036662A1 publication Critical patent/WO2022036662A1/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
    • 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
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12P7/62—Carboxylic acid esters
    • 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
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
    • 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
    • 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
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae

Definitions

  • the invention belongs to the technical field of genetic engineering and enzyme catalysis, in particular to a method for synthesizing 3-hydroxybutyrate by an enzymatic method.
  • ketogenic diet has gradually become a healthy lifestyle recognized by everyone.
  • ketone bodies can be supplemented for the body, and then used for ketone metabolism in the body.
  • Acetoacetate, 3-hydroxybutyrate and acetone are the three forms of ketone bodies required by the human body, of which 3-hydroxybutyrate (3-Hydroxybutyrate, 3-HB) as the main raw material for ketone body supplementation products has been successfully commercialized, And the market demand is increasing year by year.
  • the preparation of 3-hydroxybutyric acid mainly includes chemical synthesis, enzymatic conversion and microbial fermentation.
  • the current enzymatic production of 3-hydroxybutyrate basically uses chemical raw material methyl acetoacetate or ethyl acetoacetate as a substrate, and is processed by alcohol dehydrogenase (EC 1.1.1.1), or carbonyl reductase (EC 1.1.1. 1.148) Catalysis, with NADPH or NADH as coenzyme, the reduction of ketone group into hydroxyl group can occur, and the product methyl 3-hydroxybutyrate or ethyl 3-hydroxybutyrate can be generated. After methyl 3-hydroxybutyrate or ethyl 3-hydroxybutyrate is further subjected to ester hydrolysis reaction, 3-hydroxybutyric acid can be prepared.
  • the present invention conducts a large number of screenings for alcohol dehydrogenase and carbonyl reductase to study their catalytic performance on methyl acetoacetate and ethyl acetoacetate, and randomly Mutation, combined mutation and other techniques were used to transform the alcohol dehydrogenase (SEQ ID NO: 1) derived from Lactobacillus kefiri DSM 20587, which has a wide range of substrates, and obtained mutants with significantly improved enzyme activity, so as to efficiently catalyze acetoacetyl The ester yields 3-hydroxybutyrate.
  • the present invention includes the following technical solutions.
  • a method for enzymatic synthesis of 3-hydroxybutyrate characterized in that, using acetoacetate as a substrate, using alcohol dehydrogenase SEQ ID NO: 1 or a mutant thereof to catalyze a reduction reaction to obtain 3-hydroxybutyrate Ester:
  • R is a C1-C4 alkyl group selected from methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl. That is, the 3-hydroxybutyrate is selected from methyl 3-hydroxybutyrate, ethyl 3-hydroxybutyrate, propyl 3-hydroxybutyrate, isopropyl 3-hydroxybutyrate, and 3-hydroxybutyric acid Butyl, sec-butyl 3-hydroxybutyrate, isobutyl 3-hydroxybutyrate, tert-butyl 3-hydroxybutyrate;
  • the above-mentioned alcohol dehydrogenase mutant is formed by the amino acid sequence of SEQ ID NO:1 through the mutation (including but not limited to substitution, deletion or addition) of amino acid residues at more than one site, and has the alcohol dehydrogenase SEQ ID NO:1 A functional polypeptide; or it has more than 85% homology, preferably more than 90% homology, more preferably more than 95% homology with the amino acid sequence of SEQ ID NO: 1, and has alcohol dehydrogenase SEQ ID NO :1 functional peptide.
  • the above-mentioned alcohol dehydrogenase SEQ ID NO:1 function refers to the function capable of catalyzing the reduction of methyl acetoacetate to methyl 3-hydroxybutyrate and the reduction of ethyl acetoacetate to ethyl 3-hydroxybutyrate.
  • the enzymatic activity of the above-mentioned alcohol dehydrogenase mutant is higher than that of SEQ ID NO:1.
  • the above-mentioned substrate acetoacetate is methyl acetoacetate or ethyl acetoacetate
  • the above-mentioned product 3-hydroxybutyrate is methyl 3-hydroxybutyrate or ethyl 3-hydroxybutyrate.
  • 3-hydroxybutyrate includes methyl 3-hydroxybutyrate or ethyl 3-hydroxybutyrate, especially 3-hydroxybutyrate in R-configuration, including (R)-methyl 3-hydroxybutyrate ester or (R)-ethyl 3-hydroxybutyrate.
  • isopropanol and coenzyme NADP+ are also added to the enzyme-catalyzed reaction system.
  • NADP+ is to snatch electrons as an oxidant
  • alcohol dehydrogenase uses isopropanol to reduce NADP+ to NADPH, producing sufficient NADPH as a reducing agent for biosynthesis, thereby promoting the reduction reaction.
  • the pH of the enzyme-catalyzed reaction system of the present invention can be 7.0-8.0, preferably pH 7.2-7.8, more preferably pH 7.4-7.5.
  • the temperature of the enzyme-catalyzed reaction is 25-45°C, preferably 28-40°C, more preferably 30-35°C.
  • the mutation site in the alcohol dehydrogenase mutant can be a site selected from the following group in the amino acid sequence of SEQ ID NO: 1: the 6th position, the 19th position, the 25th position, the 57th position , No. 77, No. 89, No. 97, No. 123, No. 147, No. 149, No. 151, No. 155, No. 190, No. 197, No. 202, No. 220, No. 221st bit, 235th bit, or a combination of two or more of them.
  • the second aspect of the present invention provides an alcohol dehydrogenase mutant, which is the above-mentioned alcohol dehydrogenase mutant.
  • it is a mutant formed by mutating the following positions in the amino acid sequence of SEQ ID NO: 1: the 6th position, the 19th position, the 25th position, the 57th position, the 77th position, the 89th position, the 97th position, No. 123, No. 147, No. 149, No. 151, No. 155, No. 190, No. 197, No. 202, No. 220, No. 221, No. 235, or two or more of them combination.
  • the mutation in the above-mentioned alcohol dehydrogenase mutant is selected from the group consisting of K6N, I19L, D25G, I57N or I57T, T77N or T77S, N89T or N89K, K97R or K97N, R123S or R123H, F147I or F147C, G149D or G149R, P151L, A155D, Y190F or Y190G, D197E, A202V or A202T, P220Q, N221T or N221I or N221V, S235Y, or a combination of two or more thereof.
  • the above-mentioned alcohol dehydrogenase mutant is selected from the group consisting of:
  • SEQ ID NO:3 which is a mutant of SEQ ID NO:1 amino acid sequence A202T, K97R;
  • SEQ ID NO:4 which is a mutant of SEQ ID NO:1 amino acid sequence A202V, K97R, Y190G;
  • SEQ ID NO:5 which is a mutant of SEQ ID NO:1 amino acid sequence A202T, K97R, F147I, K6N;
  • SEQ ID NO:6 which is a mutant of SEQ ID NO:1 amino acid sequence A202T, K97R, N89T, R123H, N221T;
  • SEQ ID NO:7 which is a mutant of SEQ ID NO:1 amino acid sequence A202T, D25G;
  • SEQ ID NO:8 which is a mutant of SEQ ID NO:1 amino acid sequence A202T, K97R, S235Y, I57N, R123H;
  • SEQ ID NO:9 which is a mutant of SEQ ID NO:1 amino acid sequence A202V, K97R, N221I, Y190F;
  • SEQ ID NO:10 which is a mutant of SEQ ID NO:1 amino acid sequence A202V, K97R, N221I, Y190F, D25G, K6N, R123S;
  • SEQ ID NO:11 which is a mutant of the amino acid sequence A202V, N221I, Y190F, G149D, D25G of SEQ ID NO:1;
  • SEQ ID NO:12 which is a mutant of SEQ ID NO:1 amino acid sequence A202V, Y190F, D25G;
  • SEQ ID NO: 13 which is a mutant of SEQ ID NO: 1 amino acid sequence A202V, Y190F, D25G, I57T;
  • SEQ ID NO: 14 which is a mutant of SEQ ID NO: 1 amino acid sequence A202V, N221I, Y190F, F147I;
  • SEQ ID NO: 15 which is a mutant of SEQ ID NO: 1 amino acid sequence K97R, N221I, Y190F, F147I;
  • SEQ ID NO: 17 which is a mutant of SEQ ID NO: 1 amino acid sequence A202V, N221V, Y190F, F147I, I19L, G149R;
  • SEQ ID NO: 18 which is a mutant of SEQ ID NO: 1 amino acid sequence A202V, N221I, Y190F, F147I, K97N, N89K, R123S;
  • SEQ ID NO: 19 which is a mutant of SEQ ID NO: 1 amino acid sequence A202T, N221I, Y190F, K6N;
  • SEQ ID NO:20 which is a mutant of SEQ ID NO:1 amino acid sequence A202V, N221I, Y190F, F147I, K97N, N89K, R123S, A155D, T77N;
  • SEQ ID NO:21 which is a mutant of SEQ ID NO:1 amino acid sequence A202V, N221I, Y190F, F147I, K97N, N89K, R123S, T77S, G149R, P151L;
  • SEQ ID NO:22 which is a mutant of SEQ ID NO:1 amino acid sequence Y190F;
  • SEQ ID NO:23 which is a mutant of SEQ ID NO:1 amino acid sequence K97R;
  • SEQ ID NO:24 which is a mutant of SEQ ID NO:1 amino acid sequence P220Q, F147C;
  • SEQ ID NO:25 which is a mutant of SEQ ID NO:1 amino acid sequence I57N;
  • SEQ ID NO:26 which is a mutant of SEQ ID NO:1 amino acid sequence G149D;
  • SEQ ID NO: 27 which is a mutant of the amino acid sequence R123S of SEQ ID NO: 1.
  • the above-mentioned alcohol dehydrogenase mutant is SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:21.
  • a third aspect of the present invention provides a microorganism expressing alcohol dehydrogenase SEQ ID NO: 1 or one of the above-mentioned alcohol dehydrogenase mutants SEQ ID NOs: 3-27.
  • the microorganism is selected from Escherichia coli, Pichia pastoris, and Bacillus subtilis, preferably Escherichia coli, more preferably Escherichia coli BL21(DE3).
  • the gene encoding wild-type alcohol dehydrogenase SEQ ID NO:1 may be the nucleotide sequence SEQ ID NO:2.
  • microorganisms described above can be used directly for the production of 3-hydroxybutyrate as a natural immobilized form of alcohol dehydrogenase.
  • the wild-type alcohol dehydrogenase SEQ ID NO: 1 and the mutants SEQ ID NOs: 3-27 constructed on the basis of the wild-type alcohol dehydrogenases 1#-23# screened out in the present invention are applied to In the enzymatic synthesis of 3-hydroxybutyrate, it can catalyze the reduction of methyl acetoacetate to methyl 3-hydroxybutyrate, and catalyze the reduction of ethyl acetoacetate to ethyl 3-hydroxybutyrate, which broadens the scope of acetoacetate.
  • the range of ester substrates has industrial application prospects.
  • the wild-type alcohol dehydrogenase SEQ ID NO: 1 screened in the present invention is derived from Lactobacillus kefiri DSM 20587, and is numbered 19# in the examples, which catalyzes the reduction of acetoacetate to 3-hydroxybutyric acid In the case of esters, the participation of the coenzyme NADPH is required.
  • SEQ ID NO:1 amino acid sequence is:
  • SEQ ID NO: 1 Through multiple rounds of mutation of SEQ ID NO: 1, a series of mutation points were found, and a number of mutants mentioned in enzyme activity were constructed, including SEQ ID NOs: 3-27, which were all capable of methyl acetoacetate and acetoacetate Ethyl ester is used as the substrate to catalyze the corresponding methyl 3-hydroxybutyrate and ethyl 3-hydroxybutyrate.
  • the mutation at position 202 can be A202T or A202V.
  • the A202T mutation refers to the mutation in which the alanine (A or Ala) residue at position 202 of the amino acid sequence of SEQ ID NO: 1 is replaced by threonine (T or Thr)
  • the A202V mutation refers to the alanine at position 202 Mutations in which the (A or Ala) residue is replaced by a valine (V or Val).
  • wild (type) wild enzyme
  • wild-type enzyme wild-type enzyme
  • alcohol dehydrogenase SEQ ID NO: 1 in Escherichia coli, which is the most commonly used in genetic engineering, the present invention has carried out codon optimization on its expression gene, and used it as a basic template for constructing alcohol dehydrogenase mutants.
  • Wild-type The gene encoding alcohol dehydrogenase SEQ ID NO:1 can be the nucleotide sequence SEQ ID NO:2:
  • mutant sequences were obtained, that is, mutants with amino acid sequences SEQ ID NOs: 3-27 in the present invention.
  • codon optimization can be performed for specific microorganisms such as E. coli.
  • Codon optimization is a technique that can be used to maximize protein expression in an organism by increasing the translation efficiency of the gene of interest. Different organisms often show a particular preference for one of several codons encoding the same amino acid due to mutational propensity and natural selection.
  • optimized codons reflect the composition of their respective genomic tRNA pools. Thus, in fast growing microorganisms, codons of low frequency for amino acids can be replaced with codons of high frequency for the same amino acid.
  • the expression of optimized DNA sequences is improved in fast growing microorganisms.
  • genes, expression cassettes, plasmids, and transformants can be obtained by genetic engineering construction methods well known to those skilled in the art.
  • the alcohol dehydrogenase may also be in the form of an enzyme or a bacterial cell.
  • the form of the enzyme includes free enzyme, immobilized enzyme, including purified enzyme, crude enzyme, fermentation broth, enzyme immobilized on a carrier, etc.
  • the form of the bacterial cell includes surviving bacterial cell and dead bacterial cell.
  • the above-mentioned bacterial form itself is a natural immobilized enzyme, and can be used as an enzyme preparation for catalyzing reactions without the need for crushing treatment or even extraction and purification treatment. Since both the reaction substrate and the reaction product are small molecular compounds, they can easily pass through the cell membrane, the biological barrier of the bacteria, so the bacteria do not need to be disrupted, which is advantageous in terms of economy.
  • LB medium 10 g/L tryptone, 5 g/L yeast extract, 10 g/L sodium chloride, pH 7.2. (Add 20g/L agar powder to LB solid medium.)
  • TB medium 24 g/L yeast extract, 12 g/L tryptone, 16.43 g/L K 2 HPO 4 .3H 2 O, 2.31 g/L KH 2 PO 4 , 5 g/L glycerol, pH 7.0-7.5. (Add 20g/L agar powder to TB solid medium.)
  • ZYM medium The following mother liquors were prepared according to the formula: ZY medium, 50 ⁇ M medium, 50 ⁇ 5052 medium, 1M MgSO 4 , 1000 ⁇ trace elements, 1000 ⁇ antibiotics.
  • ZY medium 10g peptone, 5g yeast powder, add water to make up to 950ml, 121°C, sterilize for 20min.
  • 50 ⁇ M medium 223g Na 2 HPO 4 ⁇ 12H 2 O, 85g KH 2 PO 4 , 66.88g NH 4 Cl, 17.7g Na 2 SO 4 , add water to dilute to 500ml, sterilize at 121°C for 20min.
  • 50 ⁇ 5052 medium 125g glycerol, 12.5g glucose, 50g ⁇ -lactose, add water to make up to 500ml, sterilize at 121°C for 20min.
  • 1000 ⁇ trace elements dissolve 1.35g FeCl 3 ⁇ 6H 2 O with 50ml 0.12M HCl, then add 0.32g CaCl 2 ⁇ 2H 2 O, 0.2g MnCl 2 ⁇ 4H 2 O, 0.3g ZnSO 4 ⁇ 7H 2 O respectively , 0.05g CoCl 2 6H 2 O, 0.04g CuCl 2 2H 2 O, 0.05g NiCl 2 6H 2 O, 0.05g Na 2 MoO 4 2H 2 O, 0.04g Na 2 SeO 3 , 0.02g H 3 BO 3 , add water to make up to 100ml, filter and sterilize.
  • 1000 ⁇ antibiotics 500mg kanamycin, add water to make up to 10ml, filter and sterilize.
  • the sterilized mother liquors were mixed evenly with 950ml ZY medium, 20ml 50 ⁇ M, 20ml 50 ⁇ 5052, 2ml 1M MgSO 4 , 2ml 1000 ⁇ trace elements, and 1ml 1000 ⁇ antibiotics to obtain ZYM autoinduction medium.
  • the molecular biology experiments in the examples include plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, medium preparation, etc., mainly with reference to "Molecular Cloning: A Laboratory Manual” (Third Edition) ), edited by J. Sambrook, DW Russell (US), translated by Huang Peitang et al., Science Press, Beijing, 2002). If necessary, specific experimental conditions can be determined by simple experiments.
  • PCR amplification experiments were carried out according to the reaction conditions or kit instructions provided by the plasmid or DNA template supplier. If necessary, it can be adjusted by simple experimentation.
  • strain number, plasmid number, enzyme number, and enzyme-encoding gene number can be shared with one number, which is easily understood by those skilled in the art, that is, the same number is in different Different biological forms can be referred to in the environment.
  • 19# can represent both the strain Lactobacillus kefiri DSM 20587, the plasmid pET24a-19# number, the enzyme SEQ ID NO: 1 number, and the enzyme encoding gene SEQ ID NO: 2 number.
  • Microbial derived enzymes for the reduction of methyl acetoacetate/ethyl acetoacetate to methyl 3-hydroxybutyrate/ethyl 3-hydroxybutyrate were investigated.
  • a total of 23 enzyme genes were selected from the NCBI database search, as shown in Table 1.
  • the construction of enzyme expression engineering bacteria using the codon optimization tool Codon Adaptation Tool ( http://www.jcat.de/ ) to adapt 23 enzymes to E. coli codon optimization, the sequence avoidance excludes NdeI/XhoI site features, and then Obtain the base sequence of the corresponding coding gene.
  • the gene encoding alcohol dehydrogenase SEQ ID NO:1 of No. 19 can be the nucleotide sequence SEQ ID NO:2.
  • the genes of the above 23 enzymes were entrusted to Suzhou Jinweizhi Biotechnology Co., Ltd. for gene synthesis, and the synthesized gene fragments were loaded into the NdeI/XhoI site of the E.
  • coli expression plasmid system pET24a vector as required to obtain 23 expression plasmids, pET24a-1#, pET24a-2#, pET24a-3#, pET24a-4#, pET24a-5#, pET24a-6#, pET24a-7#, pET24a-8#, pET24a-9#, pET24a-10#, pET24a-11#, pET24a-12#, pET24a-13#, pET24a-14#, pET24a-15#, pET24a-16#, pET24a-17#, pET24a-18#, pET24a-19#, pET24a-20#, pET24a-21#, pET24a-22#, pET24a-23# were used for subsequent protein expression.
  • Embodiment 2 Enzyme activity detection of engineering bacteria
  • Single clones were selected on the plates of genetically engineered strains, inoculated into 5mL LB medium, and cultured at 37°C; inoculated into 250mL shake flasks containing 20mL TB medium at 1% v/v and cultured for 4-6 hours, OD600 After reaching 1.2-1.5, add 0.2 mM IPTG for induction, cool down to 25°C for 10-16 hours, centrifuge to obtain bacterial cells, and freeze at -80°C for 24 hours for use.
  • the wet cells were incubated in a water bath at 30°C for 30 min, and finally 1M hydrochloric acid was used to terminate the reaction.
  • the reaction solution was sampled, and HPLC was performed to detect the product concentration of methyl 3-hydroxybutyrate or ethyl 3-hydroxybutyrate.
  • enzyme activity the amount of bacteria required to generate 1 ⁇ M product per unit time (min) is one unit of enzyme activity (U).
  • the activity data are calculated with the enzyme activity of 1# enzyme catalyzing ethyl acetoacetate substrate as 00%.
  • the activity of the 19# enzyme on the two substrates was relatively balanced. According to the results, the pET24a-19# plasmid was used to construct and screen and evaluate the error-prone PCR mutant library (referred to as the error-prone mutation library).
  • Random mutant libraries were constructed using error-prone PCR techniques.
  • 50 ⁇ L error-prone PCR reaction system includes: 500ng plasmid template, 500pmol 19#-F primer, 500pmol 19#-R primer, 1x PCR buffer, 0.2mM dGTP, 0.2mM dATP, 1mM dCTP, 1mM dTTP, 7mM MgCl 2 , 0.1 mM MnCl2, 2.5 units of Taq enzyme (Invitrogen TM ).
  • the error-prone PCR reaction conditions were: 95°C for 5 min; 94°C for 30s, 55°C for 30s, 72°C for 2min/kbp, 30 cycles; 72°C for 10min.
  • the above random mutation fragment was recovered by gel as the megaprimer of the next round of PCR, and KOD FX DNA polymerase (TOYOBO) was used for MegaPrimer PCR, 50 ⁇ l reaction system, 1X PCR buffer, 2mM dNTPs, megaprimer 250ng, pET24a-19# plasmid 50ng, 1 piece Unit KOD FX, PCR reaction program: 94°C 5min; 98°C 10s, 60°C 30s, 68°C 1min, 25 cycles; 68°C 10min.
  • the competent cells of Escherichia coli BL21 were electro-transformed, plated on LB medium plates containing kanamycin, and cultured at 37°C overnight to obtain random mutation library clones.
  • mutant library clones are obtained, clone picking, culture and reaction screening are carried out. Take a sterile 96-well plate, add 400 ⁇ l of LB medium (containing kanamycin 50 ⁇ g/ml) to each well, and use a sterile toothpick to pick the single clone of the mutant library to transform the plasmids of pET24a or pET24a-19# respectively.
  • BL21(DE3) engineered bacteria were used as blank and negative controls.
  • the above-mentioned orifice plates were cultured at 37° C. orifice plate shaker at 280 rpm for 20 h and used as seed solution.
  • the dominant clones were transferred from the seed well plate to a TB shake flask, inoculated with 200 ⁇ l into a 250 mL shake flask containing 20 mL of TB medium, and cultured at 37°C for 4-6 hours. After the OD600 reached 1.2-1.5, 0.2 mM was added. Induced by IPTG, cooled to 25°C for 10-16 hours, centrifuged to obtain bacterial cells, part of which was frozen at -80°C for 24 hours for use, and the other part was subjected to plasmid extraction and sequencing to determine mutation sites. The corresponding mutants were tested for the enzymatic activities of the two substrates, and the results are shown in Table 3.
  • the activity data are calculated with the enzyme activity of 19# enzyme catalyzing ethyl acetoacetate substrate as 100%.
  • Example 1 the encoding gene of enzyme No. 1024 was designed and the pET24a-1024 plasmid was constructed, and the pET24a-1024 plasmid was subsequently used to construct and evaluate the error-prone PCR mutant library.
  • Example 1 the coding gene of No. 30231 enzyme was designed and the pET24a-30231 plasmid was constructed, and the pET24a-30231 plasmid was subsequently used to construct and evaluate the error-prone PCR mutant library.
  • Example 1 the encoding gene of enzyme No. 55786 was designed and the pET24a-55786 plasmid was constructed, and the pET24a-55786 plasmid was subsequently used to construct and evaluate the error-prone PCR mutant library.
  • the coding gene of enzyme No. 65781 was designed and the pET24a-65781 plasmid was constructed, and the pET24a-65781 plasmid was subsequently used to construct and screen and evaluate the error-prone PCR mutant library.
  • Example 1 the encoding genes of the enzyme No. 76789 and the enzyme No. 78932 were designed and the plasmids pET24a-76789 and pET24a-78932 were constructed. According to the method in Example 2, these two plasmids were transformed into Escherichia coli BL21(DE3) competent cells by electroporation method to obtain genetically engineered bacteria pET24a-76789/BL21(DE3) and pET24a-78932/BL21(DE3 ), used to catalyze the reaction of methyl acetoacetate and ethyl acetoacetate to prepare 3-hydroxybutyrate.
  • the engineered strains pET24a-76789/BL21(DE3) and pET24a-78932/BL21(DE3) transformed with mutant plasmids pET24a-76789 and BL21(DE3) of pET24a-78932 were inoculated into test tubes containing LB medium, 37 Cultivated overnight at °C, then inoculated into a 500mL shake flask containing 100mL TB medium at a ratio of 1% v/v, cultured at 37°C for 4-6 hours, after the OD600 reached 1.2-1.5, added 0.2mM IPTG for induction, and cooled to 25 Cultivated at °C for 10-16 hours, centrifuged to obtain bacterial cells, and frozen at -80 °C for 24 hours for use.
  • the catalytic reaction adopts a 1L reaction system: substrate methyl acetoacetate or ethyl acetoacetate 50g/L, isopropanol 80ml/L, NADP cofactor 10mM, pH 7.5, wet cell 1.5%. Shake the reaction at 30°C, 230rpm for 15h, add hydrochloric acid to stop the reaction, quantitatively detect the product concentration and substrate concentration, and calculate the substrate conversion rate. Simultaneous sampling was performed to detect the chirality of 3-hydroxybutyrate. The transformation solution was centrifuged at 12,000 rpm for 3 min, and the supernatant was taken. Ethyl acetate was added to the supernatant and shaken on a vortex shaker for 5 min.
  • GC detection conditions are: chromatographic column Gamma DEXTM 225 Capillary Column 30m*0.25nm*0.25 ⁇ m film thickness; injection volume: 0.1 ⁇ L; injector temperature: 250°C; split ratio: 190:1; carrier gas pressure: 10.795psi ; Flow rate: 1 mL/min; Heating program: initial temperature of 40°C, hold for 5min, heating up to 170°C at a heating rate of 10°C/min, hold for 2min; Running time: 20min; Detector: FID, 300°C; Air flow rate: 400mL/min; hydrogen flow rate: 30mL/min; makeup gas (N2): 25mL/min.

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Abstract

L'invention concerne un procédé de synthèse enzymatique de 3-hydroxybutyrate. Selon le procédé, l'acétoacétate est pris comme substrat, et une réaction de réduction est catalysée avec une alcool déshydrogénase de SEQ ID No : 1 ou un mutant de celle-ci de SEQ ID No : 3-27, pour obtenir du 3-hydroxybutyrate.
PCT/CN2020/110402 2020-08-21 2020-08-21 Procédé de synthèse enzymatique de 3-hydroxybutyrate Ceased WO2022036662A1 (fr)

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CN119776302A (zh) * 2024-04-03 2025-04-08 杭州微远生物科技有限公司 醇脱氢酶突变体及其在合成高光学纯度（s）-6-羟基-8-氯辛酸乙酯中的应用

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CN111172124A (zh) * 2020-02-26 2020-05-19 复旦大学 一种羰基还原酶突变体及其在制备(r)-4-氯-3-羟基-丁酸酯中的应用
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CN110093302A (zh) * 2019-06-13 2019-08-06 浙江华睿生物技术有限公司 一种乳杆菌突变菌株及其应用
CN111454921A (zh) * 2019-12-30 2020-07-28 南京朗恩生物科技有限公司 一种酶活提高的酮还原酶突变体及其应用
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