WO2022036662A1 - Method for enzymatic synthesis of 3-hydroxybutyrate - Google Patents

Method for enzymatic synthesis of 3-hydroxybutyrate Download PDF

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
seq
mutant
amino acid
acid sequence
alcohol dehydrogenase
Prior art date
Application number
PCT/CN2020/110402
Other languages
French (fr)
Chinese (zh)
Inventor
范文超
高书良
王金刚
梁岩
杨海锋
任亮
袁圣伦
Original Assignee
浙江华睿生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 浙江华睿生物技术有限公司 filed Critical 浙江华睿生物技术有限公司
Priority to PCT/CN2020/110402 priority Critical patent/WO2022036662A1/en
Priority to CN202080103912.5A priority patent/CN116157509A/en
Publication of WO2022036662A1 publication Critical patent/WO2022036662A1/en

Links

Classifications

    • 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
    • C12P7/62—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.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Provided is a method for the enzymatic synthesis of 3-hydroxybutyrate. According to the method, acetoacetate is taken as a substrate, and a reduction reaction is catalyzed with an alcohol dehydrogenase of SEQ ID No:1 or a mutant thereof of SEQ ID Nos:3-27, to obtain 3-hydroxybutyrate.

Description

酶法合成3-羟基丁酸酯的方法Method for enzymatic synthesis of 3-hydroxybutyrate 技术领域technical field
本发明属于基因工程和酶催化技术领域，具体地说，涉及一种酶法合成3-羟基丁酸酯的方法。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.
背景技术Background technique
近年来，生酮饮食概念逐渐成为大家认可的一种健康的生活方式，通过摄取含酮体食物，可实现为机体补充酮体，进而用于机体的酮类代谢。乙酰乙酸、3-羟基丁酸和丙酮是人类机体所需的三种酮体形式，其中3-羟基丁酸(3-Hydroxybutyrate，3-HB)作为主要原料的酮体补充产品已成功商业化，并且市场需求逐年增加。In recent years, the concept of ketogenic diet has gradually become a healthy lifestyle recognized by everyone. By ingesting ketone-containing foods, 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.
3-羟基丁酸的制备主要包括化学合成法、酶促转化法和微生物发酵法。目前的酶法生产3-羟基丁酸酯工艺基本是以化工原料乙酰乙酸甲酯或乙酰乙酸乙酯作为底物，经醇脱氢酶(EC 1.1.1.1)，或羰基还原酶(EC 1.1.1.148)催化，以NADPH或NADH为辅酶，可发生酮基的还原成羟基，生成产物3-羟基丁酸甲酯或3-羟基丁酸乙酯。3-羟基丁酸甲酯或3-羟基丁酸乙酯进一步经过酯水解反应后，可实现3-羟基丁酸的制备。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.
但是，目前工业上使用的醇脱氢酶和羰基还原酶的酶活性普遍较低，导致产物生产成本较高；而且由于这些醇脱氢酶和羰基还原酶的底物特异性即底物专一性过高，导致其适用底物范围太过狭窄。However, the enzyme activities of alcohol dehydrogenases and carbonyl reductases currently used in industry are generally low, resulting in high product production costs; and because of the substrate specificity of these alcohol dehydrogenases and carbonyl reductases The property is too high, resulting in a too narrow range of suitable substrates.
发明内容SUMMARY OF THE INVENTION
为了改进3-羟基丁酸酯的酶法生产工艺，本发明对于醇脱氢酶和羰基还原酶进行了大量的筛选，研究它们对乙酰乙酸甲酯和乙酰乙酸乙酯的催化性能，并且通过随机突变、组合突变等技术对底物适用范围较大的Lactobacillus kefiri DSM 20587来源的醇脱氢酶(SEQ ID NO:1)进行改造，获得了酶活力显著提高的突变体，以便高效地催化乙酰乙酸酯生成3-羟基丁酸酯。具体而言，本发明包括如下技术方案。In order to improve the enzymatic production process of 3-hydroxybutyrate, 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. Specifically, the present invention includes the following technical solutions.
一种酶法合成3-羟基丁酸酯的方法，其特征在于，以乙酰乙酸酯作为底物，使用醇脱氢酶SEQ ID NO:1或者其突变体催化还原反应，得到3-羟基丁酸酯：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:
Figure PCTCN2020110402-appb-000001
Figure PCTCN2020110402-appb-000001
其中R是C1-C4烷基，选自甲基、乙基、丙基、异丙基、正丁基、仲丁基、异丁基、叔丁基。即，所述3-羟基丁酸酯选自3-羟基丁酸甲酯、3-羟基丁酸乙酯、3-羟基丁酸丙酯、3-羟基丁酸异丙酯、3-羟基丁酸丁酯、3-羟基丁酸仲丁酯、3-羟基丁酸异丁酯、3-羟基丁酸叔丁酯；wherein 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;
上述醇脱氢酶突变体是SEQ ID NO:1氨基酸序列经过一个以上位点氨基酸残基的突变(包括但不限于取代、缺失或添加)而成、且具有醇脱氢酶SEQ ID NO:1功能的多肽；或者是与SEQ ID NO:1氨基酸序列有85％以上同源性、优选地90％以上同源性、更优地95％以上同源性、且具有醇脱氢酶SEQ ID NO:1功能的多肽。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.
上述的醇脱氢酶SEQ ID NO:1功能是指能够催化乙酰乙酸甲酯还原为3-羟基丁酸甲酯、且催化乙酰乙酸乙酯还原为3-羟基丁酸乙酯的功能。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.
优选上述醇脱氢酶突变体的酶活性高于SEQ ID NO:1。Preferably, the enzymatic activity of the above-mentioned alcohol dehydrogenase mutant is higher than that of SEQ ID NO:1.
优选地，上述底物乙酰乙酸酯是乙酰乙酸甲酯或乙酰乙酸乙酯，相应地，上述产物3-羟基丁酸酯是3-羟基丁酸甲酯或3-羟基丁酸乙酯。Preferably, the above-mentioned substrate acetoacetate is methyl acetoacetate or ethyl acetoacetate, and correspondingly, the above-mentioned product 3-hydroxybutyrate is methyl 3-hydroxybutyrate or ethyl 3-hydroxybutyrate.
上述3-羟基丁酸酯包括3-羟基丁酸甲酯或3-羟基丁酸乙酯，尤其是指R-构型的3-羟基丁酸酯，包括(R)-3-羟基丁酸甲酯或(R)-3-羟基丁酸乙酯。Above-mentioned 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.
在一种优选的实施方式中，酶催化反应体系中还添加有异丙醇和辅酶NADP+(烟酰胺腺嘌呤二核苷酸磷酸，辅酶II)。NADP+的作用是，作为氧化剂掠夺电子，醇脱氢酶利用异丙醇将NADP+还原为NADPH，产生充足的NADPH作为生物合成的还原剂，从而促进还原反应。In a preferred embodiment, isopropanol and coenzyme NADP+ (nicotinamide adenine dinucleotide phosphate, coenzyme II) are also added to the enzyme-catalyzed reaction system. The role of NADP+ is to snatch electrons as an oxidant, and alcohol dehydrogenase uses isopropanol to reduce NADP+ to NADPH, producing sufficient NADPH as a reducing agent for biosynthesis, thereby promoting the reduction reaction.
本发明的酶催化反应体系的pH可以为7.0-8.0，优选pH 7.2-7.8，更优选pH 7.4-7.5。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.
酶催化反应温度为25-45℃，优选28-40℃，更优选30-35℃。The temperature of the enzyme-catalyzed reaction is 25-45°C, preferably 28-40°C, more preferably 30-35°C.
上述方法中，所述醇脱氢酶突变体中的突变位点可以是SEQ ID NO:1氨基酸序列中选自下组的位点：第6位、第19位、第25位、第57位、第77位、第89位、第97位、第123位、第147位、第149位、第151位、第155位、第190位、第197位、第202位、第220位、第221位、第235位、或者它们两种以上的组合。In the above method, 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.
本发明的第二个方面提供了一种醇脱氢酶突变体，其为上述的醇脱氢酶突变体。例如是SEQ ID NO:1氨基酸序列中下述位点发生突变后形成的突变体：第6位、 第19位、第25位、第57位、第77位、第89位、第97位、第123位、第147位、第149位、第151位、第155位、第190位、第197位、第202位、第220位、第221位、第235位、或者它们两种以上的组合。The second aspect of the present invention provides an alcohol dehydrogenase mutant, which is the above-mentioned alcohol dehydrogenase mutant. For example, 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.
在一种优选的实施方式中，上述醇脱氢酶突变体中的突变选自下组：K6N、I19L、D25G、I57N或I57T、T77N或T77S、N89T或N89K、K97R或K97N、R123S或R123H、F147I或F147C、G149D或G149R、P151L、A155D、Y190F或Y190G、D197E、A202V或A202T、P220Q、N221T或N221I或N221V、S235Y、或者它们两种以上的组合。In a preferred embodiment, 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.
例如，上述醇脱氢酶突变体选自下组：For example, the above-mentioned alcohol dehydrogenase mutant is selected from the group consisting of:
SEQ ID NO:3，其为SEQ ID NO:1氨基酸序列A202T、K97R的突变体；SEQ ID NO:3, which is a mutant of SEQ ID NO:1 amino acid sequence A202T, K97R;
SEQ ID NO:4，其为SEQ ID NO:1氨基酸序列A202V、K97R、Y190G的突变体；SEQ ID NO:4, which is a mutant of SEQ ID NO:1 amino acid sequence A202V, K97R, Y190G;
SEQ ID NO:5，其为SEQ ID NO:1氨基酸序列A202T、K97R、F147I、K6N的突变体；SEQ ID NO:5, which is a mutant of SEQ ID NO:1 amino acid sequence A202T, K97R, F147I, K6N;
SEQ ID NO:6，其为SEQ ID NO:1氨基酸序列A202T、K97R、N89T、R123H、N221T的突变体；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，其为SEQ ID NO:1氨基酸序列A202T、D25G的突变体；SEQ ID NO:7, which is a mutant of SEQ ID NO:1 amino acid sequence A202T, D25G;
SEQ ID NO:8，其为SEQ ID NO:1氨基酸序列A202T、K97R、S235Y、I57N、R123H的突变体；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，其为SEQ ID NO:1氨基酸序列A202V、K97R、N221I、Y190F的突变体；SEQ ID NO:9, which is a mutant of SEQ ID NO:1 amino acid sequence A202V, K97R, N221I, Y190F;
SEQ ID NO:10，其为SEQ ID NO:1氨基酸序列A202V、K97R、N221I、Y190F、D25G、K6N、R123S的突变体；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，其为SEQ ID NO:1氨基酸序列A202V、N221I、Y190F、G149D、D25G的突变体；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，其为SEQ ID NO:1氨基酸序列A202V、Y190F、D25G的突变体；SEQ ID NO:12, which is a mutant of SEQ ID NO:1 amino acid sequence A202V, Y190F, D25G;
SEQ ID NO:13，其为SEQ ID NO:1氨基酸序列A202V、Y190F、D25G、I57T的突变体；SEQ ID NO: 13, which is a mutant of SEQ ID NO: 1 amino acid sequence A202V, Y190F, D25G, I57T;
SEQ ID NO:14，其为SEQ ID NO:1氨基酸序列A202V、N221I、Y190F、F147I的突变体；SEQ ID NO: 14, which is a mutant of SEQ ID NO: 1 amino acid sequence A202V, N221I, Y190F, F147I;
SEQ ID NO:15，其为SEQ ID NO:1氨基酸序列K97R、N221I、Y190F、F147I的突变体；SEQ ID NO: 15, which is a mutant of SEQ ID NO: 1 amino acid sequence K97R, N221I, Y190F, F147I;
SEQ ID NO:16，其为SEQ ID NO:1氨基酸序列A202V、N221I、Y190F、F147I、D197E、P151L的突变体；SEQ ID NO: 16, which is a mutant of the amino acid sequence A202V, N221I, Y190F, F147I, D197E, P151L of SEQ ID NO:1;
SEQ ID NO:17，其为SEQ ID NO:1氨基酸序列A202V、N221V、Y190F、F147I、I19L、G149R的突变体；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，其为SEQ ID NO:1氨基酸序列A202V、N221I、Y190F、F147I、K97N、N89K、R123S的突变体；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，其为SEQ ID NO:1氨基酸序列A202T、N221I、Y190F、K6N的突变体；SEQ ID NO: 19, which is a mutant of SEQ ID NO: 1 amino acid sequence A202T, N221I, Y190F, K6N;
SEQ ID NO:20，其为SEQ ID NO:1氨基酸序列A202V、N221I、Y190F、F147I、K97N、N89K、R123S、A155D、T77N的突变体；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，其为SEQ ID NO:1氨基酸序列A202V、N221I、Y190F、F147I、K97N、N89K、R123S、T77S、G149R、P151L的突变体；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，其为SEQ ID NO:1氨基酸序列Y190F的突变体；SEQ ID NO:22, which is a mutant of SEQ ID NO:1 amino acid sequence Y190F;
SEQ ID NO:23，其为SEQ ID NO:1氨基酸序列K97R的突变体；SEQ ID NO:23, which is a mutant of SEQ ID NO:1 amino acid sequence K97R;
SEQ ID NO:24，其为SEQ ID NO:1氨基酸序列P220Q、F147C的突变体；SEQ ID NO:24, which is a mutant of SEQ ID NO:1 amino acid sequence P220Q, F147C;
SEQ ID NO:25，其为SEQ ID NO:1氨基酸序列I57N的突变体；SEQ ID NO:25, which is a mutant of SEQ ID NO:1 amino acid sequence I57N;
SEQ ID NO:26，其为SEQ ID NO:1氨基酸序列G149D的突变体；SEQ ID NO:26, which is a mutant of SEQ ID NO:1 amino acid sequence G149D;
SEQ ID NO:27，其为SEQ ID NO:1氨基酸序列R123S的突变体。SEQ ID NO: 27, which is a mutant of the amino acid sequence R123S of SEQ ID NO: 1.
尤其优选上述醇脱氢酶突变体是SEQ ID NO:18、SEQ ID NO:20或者SEQ ID NO:21。It is especially preferred that the above-mentioned alcohol dehydrogenase mutant is SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:21.
本发明的第三个方面提供一种表达醇脱氢酶SEQ ID NO:1或上述醇脱氢酶突变体SEQ ID NOs:3-27之一的微生物。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.
上述微生物选自大肠杆菌、毕赤酵母、枯草芽孢杆菌，优选是大肠杆菌，更优选是大肠杆菌BL21(DE3)。当微生物是大肠杆菌时，野生型醇脱氢酶SEQ ID NO:1的编码基因可以是核苷酸序列SEQ ID NO:2。The microorganism is selected from Escherichia coli, Pichia pastoris, and Bacillus subtilis, preferably Escherichia coli, more preferably Escherichia coli BL21(DE3). When the microorganism is Escherichia coli, the gene encoding wild-type alcohol dehydrogenase SEQ ID NO:1 may be the nucleotide sequence SEQ ID NO:2.
上述微生物可作为醇脱氢酶的天然固定化形式而直接用于生产3-羟基丁酸酯。The microorganisms described above can be used directly for the production of 3-hydroxybutyrate as a natural immobilized form of alcohol dehydrogenase.
本发明从众多醇脱氢酶和羰基还原酶1#-23#中筛选出的野生型醇脱氢酶SEQ ID NO:1和以其为基础构建的突变体SEQ ID NOs:3-27应用于3-羟基丁酸酯的酶 法合成中时，能够催化乙酰乙酸甲酯还原为3-羟基丁酸甲酯、并且催化乙酰乙酸乙酯还原为3-羟基丁酸乙酯，拓宽了乙酰乙酸酯底物范围，具有工业化应用前景。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.
具体实施方式detailed description
本发明筛选的野生型醇脱氢酶SEQ ID NO:1来源于开菲尔乳杆菌Lactobacillus kefiri DSM 20587，在实施例中编号为19#，其在催化乙酰乙酸酯还原为3-羟基丁酸酯时，需要辅酶NADPH的参与。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氨基酸序列为：SEQ ID NO:1 amino acid sequence is:
Figure PCTCN2020110402-appb-000002
Figure PCTCN2020110402-appb-000002
通过对SEQ ID NO:1进行多轮突变，发现一系列突变点，构建得到多个酶活力提到的突变体，包括SEQ ID NOs:3-27，它们都能够以乙酰乙酸甲酯和乙酰乙酸乙酯为底物，催化得到相应的3-羟基丁酸甲酯和3-羟基丁酸乙酯。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.
有些位点的突变并非是单一的突变，比如第202位突变可以是A202T，也可以是A202V。其中A202T突变是指SEQ ID NO:1氨基酸序列第202位的丙氨酸(A或Ala)残基被苏氨酸(T或Thr)替换的突变，A202V突变是指第202位的丙氨酸(A或Ala)残基被缬氨酸(V或Val)替换的突变。The mutation of some sites is not a single mutation, for example, the mutation at position 202 can be A202T or A202V. Wherein 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), and 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).
在实施例中，术语“野生(型)”、“野生酶”、“野生型酶”表示相同的意义，都是指醇脱氢酶的野生序列SEQ ID NO:1。为了与突变体(突变酶)相区别和表述方便起见，在本发明中可以将野生型醇脱氢酶称为“野生(型)醇脱氢酶”或者“野生(型)酶”。In the embodiment, the terms "wild (type)", "wild enzyme" and "wild-type enzyme" have the same meaning, and all refer to the wild-type sequence SEQ ID NO: 1 of alcohol dehydrogenase. For the convenience of distinguishing from mutants (mutant enzymes) and expressing convenience, the wild-type alcohol dehydrogenase may be referred to as "wild (type) alcohol dehydrogenase" or "wild (type) enzyme" in the present invention.
由于醇脱氢酶突变体SEQ ID NOs:3-27的功能没有改变，为了描述方便起见，有时也将“醇脱氢酶突变体”简称为“醇脱氢酶”，这是本领域技术人员容易理解的。Since the functions of the alcohol dehydrogenase mutants SEQ ID NOs: 3-27 have not changed, for the convenience of description, the "alcohol dehydrogenase mutant" is sometimes abbreviated as "alcohol dehydrogenase". easy to understand.
为了在基因工程中最常用的大肠杆菌中表达醇脱氢酶SEQ ID NO:1，本发明对其表达基因进行了密码子优化，以此作为构建醇脱氢酶突变体的基础模板，野生型醇脱氢酶SEQ ID NO:1的编码基因可以是核苷酸序列SEQ ID NO:2：In order to express 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:
Figure PCTCN2020110402-appb-000003
Figure PCTCN2020110402-appb-000003
Figure PCTCN2020110402-appb-000004
Figure PCTCN2020110402-appb-000004
通过多轮易错PCR随机突变技术获得了多个突变体序列，即本发明中具有氨基酸序列SEQ ID NOs:3-27的突变体。Through multiple rounds of error-prone PCR random mutation technology, multiple mutant sequences were obtained, that is, mutants with amino acid sequences SEQ ID NOs: 3-27 in the present invention.
本发明的醇脱氢酶突变体的氨基酸数量只有252个，且序列明确，因此本领域技术人员很容易获得其编码基因、包含这些基因的表达盒和质粒、以及包含该质粒的转化体。The number of amino acids of the alcohol dehydrogenase mutant of the present invention is only 252, and the sequence is clear, so those skilled in the art can easily obtain its encoding genes, expression cassettes and plasmids containing these genes, and transformants containing the plasmids.
为了在不同微生物中进行蛋白质SEQ ID NOs:3-27的最佳表达，可以针对特定的微生物比如大肠杆菌进行密码子优化。密码子优化是可用于通过增加感兴趣基因的翻译效率使生物体中蛋白质表达最大化的一种技术。不同的生物体由于突变倾向和天然选择而通常示出对于编码相同氨基酸的一些密码子之一的特殊偏好性。例如，在生长快速的微生物如大肠杆菌中，优化密码子反映出其各自的基因组tRNA库的组成。因此，在生长快速的微生物中，氨基酸的低频率密码子可以用用于相同氨基酸的但高频率的密码子置换。因此，优化的DNA序列的表达在快速生长的微生物中得以改良。For optimal expression of the proteins SEQ ID NOs: 3-27 in different microorganisms, 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. For example, in fast growing microorganisms such as E. coli, 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. Thus, the expression of optimized DNA sequences is improved in fast growing microorganisms.
这些基因、表达盒、质粒、转化体可以通过本领域技术人员所熟知的基因工程构建方式获得。These genes, expression cassettes, plasmids, and transformants can be obtained by genetic engineering construction methods well known to those skilled in the art.
上述转化体宿主可以使任何适合表达多聚磷酸激酶的微生物，包括细菌和真菌。优选微生物是大肠杆菌、毕赤酵母、酿酒酵母、或者枯草芽孢杆菌，优选大肠杆菌，更优选大肠杆菌BL21(DE3)。The above transformant host can be any microorganism suitable for the expression of polyphosphokinase, including bacteria and fungi. Preferred microorganisms are Escherichia coli, Pichia, Saccharomyces cerevisiae, or Bacillus subtilis, preferably Escherichia coli, more preferably Escherichia coli BL21(DE3).
在该反应体系中，醇脱氢酶也可以呈现酶的形式或者菌体的形式。所述酶的形式包括游离酶、固定化酶，包括纯化酶、粗酶、发酵液、载体固定的酶等；所述菌体的形式包括存活菌体和死亡菌体。In this reaction system, 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.
上述菌体形式本身就是一种天然的固定化酶，而且不需要进行破碎处理、甚至提取纯化处理，就可以作为一种酶制剂用于催化反应。由于反应底物和反应产物都是小分子化合物，可以很方便地穿过菌体的生物屏障--细胞膜，因此不需要对菌体进行破碎处理，这在经济方面是有利的。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.
以下结合具体实施例对本发明做进一步详细说明。应理解，以下实施例仅用于说明本发明而非用于限定本发明的范围。The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following examples are only used to illustrate the present invention and not to limit the scope of the present invention.
本文中涉及到多种物质的添加量、含量及浓度，其中所述的百分含量，除特别说明外，皆指质量百分含量。The addition amount, content and concentration of various substances are involved in this article, and the percentage content, unless otherwise specified, refers to the mass percentage content.
实施例Example
材料和方法Materials and Method
LB培养基：10g/L胰蛋白胨，5g/L酵母提取物，10g/L氯化钠，pH7.2。(LB固体培养基另加20g/L琼脂粉。)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培养基：24g/L酵母提取物、12g/L胰蛋白胨、16.43g/L K 2HPO 4.3H 2O、2.31g/L KH 2PO 4、5g/L甘油，pH7.0-7.5。(TB固体培养基另加20g/L琼脂粉。) 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培养基：按配方分别配置下述母液：ZY培养基，50×M培养基，50×5052培养基，1M MgSO 4，1000×微量元素，1000×抗生素。 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培养基：10g蛋白胨，5g酵母粉，加水定容到950ml，121℃，20min灭菌。ZY medium: 10g peptone, 5g yeast powder, add water to make up to 950ml, 121℃, sterilize for 20min.
50×M培养基：223g Na 2HPO 4·12H 2O，85g KH 2PO 4，66.88g NH 4Cl，17.7g Na 2SO 4，加水定容到500ml，121℃，20min灭菌。 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培养基：125g甘油，12.5g葡萄糖，50gα-乳糖，加水定容到500ml，121℃，20min灭菌。50×5052 medium: 125g glycerol, 12.5g glucose, 50g α-lactose, add water to make up to 500ml, sterilize at 121°C for 20min.
1M MgSO 4：24.65g MgSO 4·7H 2O，定容到100ml，121℃，20min灭菌。 1M MgSO 4 : 24.65g MgSO 4 ·7H 2 O, dilute to 100ml, sterilize at 121°C for 20min.
1000×微量元素：使用50ml 0.12M的HCl溶解1.35g FeCl 3·6H 2O，再分别加入0.32g CaCl 2·2H 2O，0.2g MnCl 2·4H 2O，0.3g ZnSO 4·7H 2O，0.05g CoCl 2·6H 2O，0.04g CuCl 2·2H 2O，0.05g NiCl 2·6H 2O，0.05g Na 2MoO 4·2H 2O，0.04g Na 2SeO 3，0.02g H 3BO 3，加水定容到100ml，过滤除菌。 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×抗生素：500mg卡那霉素，加水定容到10ml，过滤除菌。1000× antibiotics: 500mg kanamycin, add water to make up to 10ml, filter and sterilize.
将灭过菌的各母液按950ml ZY培养基、20ml 50×M、20ml 50×5052、2ml 1M MgSO 4、2ml 1000×微量元素、1ml 1000×抗生素混合均匀，即得ZYM自诱导培养基。 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.
实施例中的全基因合成由苏州金唯智生物科技有限公司完成，并装载到载体pET24a。引物合成及测序皆由苏州金唯智生物科技有限公司公司完成。The whole gene synthesis in the examples was completed by Suzhou Jinweizhi Biotechnology Co., Ltd. and loaded into the vector pET24a. Primer synthesis and sequencing were completed by Suzhou Jinweizhi Biotechnology Co., Ltd.
实施例中的分子生物学实验包括质粒构建、酶切、连接、感受态细胞制备、转化、培养基配制等等，主要参照《分子克隆实验指南》(Molecular Cloning:A Laboratory Manual)(第三版)，J.萨姆布鲁克，D.W.拉塞尔(美)编著，黄培堂等译，科学出版社，北京，2002)进行。必要时可以通过简单试验确定具体实验条件。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扩增实验根据质粒或DNA模板供应商提供的反应条件或试剂盒说明书进行。必要时可以通过简单试验予以调整。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.
需说明的是，为描述方便起见，在实施例中，可将菌株编号、质粒编号、酶编号、酶编码基因编号共用一个编号，这是本领域技术人员容易理解的，即同一个编号在不同环境中可以指代不同的生物形式。比如19#既可以代表菌株Lactobacillus kefiri DSM 20587，也可以代表质粒pET24a-19#编号、酶SEQ ID NO:1编号、酶编码基因SEQ ID NO:2编号。It should be noted that, for the convenience of description, in the examples, the 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. For example, 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.
实施例1筛选用于合成3-羟基丁酸酯的酶Example 1 Screening of enzymes for synthesizing 3-hydroxybutyrate
考察用于将乙酰乙酸甲酯/乙酰乙酸乙酯还原为3-羟基丁酸甲酯/3-羟基丁酸乙酯的微生物来源酶。自NCBI数据库检索，选取共计23个酶基因，如表1所示。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.
表1、微生物来源的野生型酶库构建列表Table 1. List of wild-type enzyme library construction from microorganisms
Figure PCTCN2020110402-appb-000005
Figure PCTCN2020110402-appb-000005
Figure PCTCN2020110402-appb-000006
Figure PCTCN2020110402-appb-000006
酶表达工程菌的构建：使用密码子优化工具Codon Adaptation Tool( http://www.jcat.de/)对23个酶进行适应大肠杆菌密码子优化，序列规避排除NdeI/XhoI位点特征，进而获得对应编码基因碱基序列。例如第19#号醇脱氢酶SEQ ID NO:1的编码基因可以是核苷酸序列SEQ ID NO:2。将上述23个酶的基因委托苏州金唯智生物科技有限公司进行基因合成，并按需求将合成的基因片段装载到大肠杆菌表达质粒系统pET24a载体的NdeI/XhoI位点中，获得23个表达质粒，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#，用于后续的蛋白表达。 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. For example, 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.
实施例2工程菌酶活力检测Embodiment 2 Enzyme activity detection of engineering bacteria
用电转化法将上述23个表达质粒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#分别转化入大肠杆菌BL21(DE3)感受态细胞中，涂含卡那霉素的LB培养基平板，37℃培养过夜。各自挑选2个单菌落，接种到含有LB培养基的试管中，培养过夜，离心收集菌体，抽提质粒，基因测序确定正确，得到重组菌株。在基因工程菌种的平板上挑选单克隆，接种到5mL LB培养基中，37℃培养过；按1％v/v接种到含有20mL TB培养基的250mL摇瓶中培养4-6小时，OD600达到1.2-1.5后， 加入0.2mM的IPTG诱导，降温到25℃培养10-16小时，离心获得菌体，-80℃冻存24小时备用。The above 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 transformed into E. coli BL21 (DE3) competent cells, and coated with kanamycin-containing LB medium plates, Incubate overnight at 37°C. Two single colonies were selected respectively, inoculated into test tubes containing LB medium, cultured overnight, collected by centrifugation, plasmids were extracted, and the gene sequencing was confirmed to be correct to obtain recombinant strains. 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.
使用下述方法测定酶活：5ml反应体系溶液(100g/L乙酰乙酸甲酯或乙酰乙酸乙酯，100mL/L异丙醇，3mM NADP或NAD，pH＝7.5)，加入准确称取的0.05g湿菌体，30℃水浴保温30min，最后使用1M的盐酸进行终止反应。反应液取样，进行HPLC检测产物3-羟基丁酸甲酯或3-羟基丁酸乙酯产物浓度。Use the following method to measure enzyme activity: 5ml reaction system solution (100g/L methyl acetoacetate or ethyl acetoacetate, 100mL/L isopropanol, 3mM NADP or NAD, pH=7.5), add accurately weighed 0.05g 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.
HPLC检测条件，进样量10ul；色谱柱，SB-AQ；流动相A，0.3％磷酸，流动相B，乙腈，A:B＝80:20；流速，1ml/min；柱温40℃；检测波长210nm；检测时长15min。根据产物浓度，计算单位菌体的反应酶活。HPLC detection conditions, injection volume 10ul; chromatographic column, SB-AQ; mobile phase A, 0.3% phosphoric acid, mobile phase B, acetonitrile, A:B=80:20; flow rate, 1ml/min; column temperature 40°C; detection Wavelength 210nm; detection time 15min. According to the product concentration, the reaction enzyme activity of the unit cell was calculated.
酶活定义：单位时间内(min)生成1μM产物所需要的菌体量为一个酶活单位(U)。Definition of enzyme activity: the amount of bacteria required to generate 1 μM product per unit time (min) is one unit of enzyme activity (U).
对23个野生酶工程菌的酶活力进行对比，结果如表2所示。The enzyme activities of 23 wild enzyme engineered bacteria were compared, and the results are shown in Table 2.
表2、野生型酶活评估结果Table 2. Results of wild-type enzyme activity evaluation
Figure PCTCN2020110402-appb-000007
Figure PCTCN2020110402-appb-000007
Figure PCTCN2020110402-appb-000008
Figure PCTCN2020110402-appb-000008
*备注：测活数据均以1#酶催化乙酰乙酸乙酯底物的酶活作为00％进行计算。*Remarks: The activity data are calculated with the enzyme activity of 1# enzyme catalyzing ethyl acetoacetate substrate as 00%.
其中19#酶对两种底物的活力较为均衡，根据该结果，后续使用pET24a-19#质粒，进行易错PCR突变体库(简称易错突变库)的构建和筛选评估。Among them, 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).
实施例3第一轮突变库的构建和筛选Example 3 Construction and Screening of the First Round Mutation Library
以pET24a-19#质粒为模板，设计如下引对物进行扩增：Using the pET24a-19# plasmid as a template, the following primers were designed for amplification:
正向19#-F：5’-CATATGACCGACCGTCTGAAAGG-3’，Forward 19#-F: 5'-CATATGACCGACCGTCTGAAAGG-3',
反向19#-R：5’-CTCGAG TTACTGAGCGGTGTAACCACCG-3’。Reverse 19#-R: 5'-CTCGAG TTACTGAGCGGTGTAACCACCG-3'.
使用易错PCR技术构建随机突变体库。50μL易错PCR反应体系包括：500ng质粒模板，500pmol的19#-F引物，500pmol的19#-R引物，1x PCR buffer，0.2mM dGTP，0.2mM dATP，1mM dCTP，1mM dTTP，7mM MgCl 2，0.1mM MnCl 2，2.5个单位的Taq酶(Invitrogen TM)。 Random mutant libraries were constructed using error-prone PCR techniques. 50μL 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 ™ ).
易错PCR反应条件为：95℃ 5min；94℃ 30s，55℃ 30s，72℃ 2min/kbp，30个循环；72℃ 10min。胶回收上述随机突变片段作为下一轮PCR的megaprimer，用KOD FX DNA聚合酶(TOYOBO)做MegaPrimer PCR，50μl反应体系，1X PCR buffer，2mM dNTPs，megaprimer 250ng，pET24a-19#质粒50ng，1个单位KOD FX，PCR反应程序：94℃ 5min；98℃ 10s，60℃ 30s，68℃ 1min，25个循环；68℃ 10min。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 μl 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.
PCR产物经DpnI消化10h后，进行电转化大肠杆菌BL21(DE3)感受态细胞，涂含卡那霉素的LB培养基平板，37℃培养过夜得到随机突变库克隆。After the PCR product was digested with DpnI for 10 h, the competent cells of Escherichia coli BL21 (DE3) were electro-transformed, plated on LB medium plates containing kanamycin, and cultured at 37°C overnight to obtain random mutation library clones.
获得突变库克隆后，进行克隆挑取、培养、反应筛选。取无菌96孔板，每个孔加入400μl的LB培养基(含卡那霉素50μg/ml)，使用无菌牙签挑取突变库单克隆，以分别转化了pET24a或pET24a-19#质粒的BL21(DE3)工程菌作为空白和阴性对照。上述孔板于37℃孔板摇床280rpm培养20h，用作种子液。新取一块96孔板，各孔分别加入400μl的ZYM培养基(含卡那霉素50μg/ml)，从种子孔板接种50μl菌液，30℃，280rpm培养24h。剩余种子液添加终浓度为15％的甘油后，-80℃保藏，备用。ZYM孔板培养结束，测定菌浓OD600。同时各孔取50μL，转移至另一新的96孔板，4000rpm离心10min，沉淀菌体孔板冷冻保存16h。冻存后的96孔板菌体，室温解冻20min，加入200μl配置好的反应体系溶液(100g/L乙酰乙酸乙酯，100mL/L异丙醇，3mM NADP，pH＝7.5)，充分震荡重悬，放置于摇床，30℃，280rpm反应40min，反应结束，放置冰上，用排枪向96孔板中加入200μl的稀盐酸，终止反应，4000rpm离心10min 后得到上清。取离心上清，用纯水稀释5-50倍，使用酶标仪测定OD240值。通过对各孔的吸光值进行读数对比，酶活越高，OD240读数越低。After the mutant library clones are obtained, clone picking, culture and reaction screening are carried out. Take a sterile 96-well plate, add 400 μl of LB medium (containing kanamycin 50 μg/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. Take a new 96-well plate, add 400 μl of ZYM medium (containing 50 μg/ml of kanamycin) to each well, inoculate 50 μl of bacterial liquid from the seed well plate, and cultivate at 30° C., 280 rpm for 24 h. After adding glycerol with a final concentration of 15%, the remaining seed solution was stored at -80°C for later use. After the ZYM plate culture was completed, the bacterial concentration OD600 was determined. At the same time, 50 μL was taken from each well, transferred to another new 96-well plate, centrifuged at 4000 rpm for 10 min, and the cell plate was precipitated and stored frozen for 16 h. The frozen 96-well plate cells were thawed at room temperature for 20 min, and 200 μl of the prepared reaction system solution (100 g/L ethyl acetoacetate, 100 mL/L isopropanol, 3 mM NADP, pH=7.5) was added, and resuspended with sufficient shaking. , placed on a shaker, 30°C, 280rpm for 40min reaction, the reaction was over, placed on ice, 200μl of dilute hydrochloric acid was added to the 96-well plate with a row gun to stop the reaction, and the supernatant was obtained after centrifugation at 4000rpm for 10min. Take the centrifugation supernatant, dilute it 5-50 times with pure water, and use a microplate reader to measure the OD240 value. By comparing the readings of the absorbance values of each well, the higher the enzyme activity, the lower the OD240 reading.
经过第一轮的筛选，优势克隆从种子孔板转接TB摇瓶，接种200μl到含有20mL TB培养基的250mL摇瓶中37℃培养4-6小时，OD600达到1.2-1.5后，加入0.2mM的IPTG诱导，降温到25℃培养10-16小时，离心获得菌体，一部分于-80℃冻存24小时备用，另一部分进行质粒抽提，送测序，进行突变位点判定。对相应突变体分别进行两种底物的酶活测定，结果见表3。After the first round of screening, the dominant clones were transferred from the seed well plate to a TB shake flask, inoculated with 200 μl 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.
表3、不同克隆株的酶蛋白测序及酶活Table 3. Enzyme protein sequencing and enzyme activity of different clones
Figure PCTCN2020110402-appb-000009
Figure PCTCN2020110402-appb-000009
*备注：测活数据均以19#酶催化乙酰乙酸乙酯底物的酶活作为100％进行计算。*Remarks: The activity data are calculated with the enzyme activity of 19# enzyme catalyzing ethyl acetoacetate substrate as 100%.
由表3可以看出，1024号酶(SEQ ID NO:3)对两种底物的催化活力都是最高的，且较为均衡。It can be seen from Table 3 that the catalytic activity of No. 1024 enzyme (SEQ ID NO: 3) to both substrates is the highest and more balanced.
根据上述对比结果，参照实施例1中的方法，设计1024号酶的编码基因并构建pET24a-1024质粒，后续使用pET24a-1024质粒，进行易错PCR突变体库的构建和筛选评估。According to the above comparison results, referring to the method in 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.
实施例4第二轮突变库的构建和筛选Example 4 Construction and Screening of the Second Round Mutation Library
以质粒pET24a-1024为模板，按照实施例3中的方法，进行第二轮的易错PCR突变体库构建和筛选，其中以分别转化了pET24a或pET24a-1024质粒的BL21(DE3)工程菌作为空白和阴性对照，结果见表4。Using plasmid pET24a-1024 as a template, according to the method in Example 3, a second round of error-prone PCR mutant library construction and screening was performed, wherein the BL21 (DE3) engineered bacteria transformed with pET24a or pET24a-1024 plasmids were used as Blank and negative controls, the results are shown in Table 4.
表4、第二轮突变库中不同菌株的酶蛋白测序及酶活Table 4. Enzyme protein sequencing and enzyme activity of different strains in the second round of mutation library
Figure PCTCN2020110402-appb-000010
Figure PCTCN2020110402-appb-000010
由表4可以看出，30231号酶(SEQ ID NO:9)对两种底物的催化活力相对较高，且较为均衡。As can be seen from Table 4, the catalytic activity of No. 30231 enzyme (SEQ ID NO: 9) to the two substrates is relatively high and balanced.
根据上述对比结果，参照实施例1中的方法，设计30231号酶的编码基因并构建pET24a-30231质粒，后续使用pET24a-30231质粒，进行易错PCR突变体库的构建和筛选评估。According to the above comparison results, referring to the method in 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.
实施例5第三轮突变库的构建和筛选Example 5 Construction and Screening of the Third Round Mutation Library
以质粒pET24a-30231为模板，按照实施例3中的方法，进行第三轮的易错PCR突变体库构建和筛选，其中以分别转化了pET24a或pET24a-30231质粒的BL21(DE3)工程菌作为空白和阴性对照，结果见表5。Using plasmid pET24a-30231 as a template, according to the method in Example 3, the third round of error-prone PCR mutant library construction and screening was carried out, wherein the BL21 (DE3) engineering bacteria transformed with pET24a or pET24a-30231 plasmids were used as Blank and negative controls, the results are shown in Table 5.
表5、第三轮突变库中不同菌株的酶蛋白测序及酶活Table 5. Enzyme protein sequencing and enzyme activity of different strains in the third round of mutation library
Figure PCTCN2020110402-appb-000011
Figure PCTCN2020110402-appb-000011
由表5可以看出，55786号酶(SEQ ID NO:14)对两种底物的催化活力相对较高，且较为均衡。As can be seen from Table 5, the catalytic activity of enzyme No. 55786 (SEQ ID NO: 14) to the two substrates is relatively high and balanced.
根据上述对比结果，参照实施例1中的方法，设计55786号酶的编码基因并构建pET24a-55786质粒，后续使用pET24a-55786质粒，进行易错PCR突变体库的构建和筛选评估。According to the above comparison results, referring to the method in 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.
实施例6第四轮突变库的构建和筛选Example 6 Construction and Screening of the Fourth Round Mutation Library
以质粒pET24a-55786为模板，按照实施例3中的方法，进行第四轮的易错PCR突变体库构建和筛选，其中以分别转化了pET24a或pET24a-55786质粒的BL21(DE3)工程菌作为空白和阴性对照，结果见表6。Using plasmid pET24a-55786 as a template, according to the method in Example 3, the fourth round of error-prone PCR mutant library construction and screening was carried out, wherein the BL21 (DE3) engineering bacteria transformed with pET24a or pET24a-55786 plasmid were used as Blank and negative controls, the results are shown in Table 6.
表6、第四轮突变库中不同菌株的酶蛋白测序及酶活Table 6. Enzyme protein sequencing and enzyme activity of different strains in the fourth round of mutation library
Figure PCTCN2020110402-appb-000012
Figure PCTCN2020110402-appb-000012
由表6可以看出，65781号酶(SEQ ID NO:18)对两种底物的催化活力相对较高，且较为均衡。As can be seen from Table 6, the catalytic activity of No. 65781 enzyme (SEQ ID NO: 18) to the two substrates is relatively high and balanced.
根据上述对比结果，参照实施例1中的方法，设计65781号酶的编码基因并构建pET24a-65781质粒，后续使用pET24a-65781质粒，进行易错PCR突变体库的构建和筛选评估。According to the above comparison results, referring to the method in Example 1, 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.
实施例7第五轮突变库的构建和筛选Example 7 Construction and Screening of the Fifth Round Mutation Library
以质粒pET24a-65781为模板，按照实施例3中的方法，进行第四轮的易错PCR突变体库构建和筛选，其中以分别转化了pET24a或pET24a-65781质粒的BL21(DE3)工程菌作为空白和阴性对照，结果见表7。Using plasmid pET24a-65781 as a template, according to the method in Example 3, the fourth round of error-prone PCR mutant library construction and screening was performed, wherein the BL21 (DE3) engineered bacteria transformed with pET24a or pET24a-65781 plasmid were used as Blank and negative controls, the results are shown in Table 7.
表7、第五轮突变库中不同菌株的酶蛋白测序及酶活Table 7. Enzyme protein sequencing and enzyme activity of different strains in the fifth round of mutation library
Figure PCTCN2020110402-appb-000013
Figure PCTCN2020110402-appb-000013
Figure PCTCN2020110402-appb-000014
Figure PCTCN2020110402-appb-000014
由表7可以看出，76789号酶(SEQ ID NO:20)和78932号酶(SEQ ID NO:21)对两种底物的催化活力都有一定提高，且较为均衡。As can be seen from Table 7, the catalytic activity of the enzyme No. 76789 (SEQ ID NO: 20) and the enzyme No. 78932 (SEQ ID NO: 21) to the two substrates has been improved to a certain extent and is relatively balanced.
参照实施例1中的方法，分别设计76789号酶和78932号酶的编码基因并构建pET24a-76789质粒和pET24a-78932质粒。按照实施例2中的方法，电转化法将这两种质粒分别转化入大肠杆菌BL21(DE3)感受态细胞中，得到基因工程菌pET24a-76789/BL21(DE3)和pET24a-78932/BL21(DE3)，用于催化乙酰乙酸甲酯和乙酰乙酸乙酯反应制备3-羟基丁酸酯。Referring to the method in 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.
实施例8突变酶催化制备3-羟基丁酸酯Example 8 Catalytic preparation of 3-hydroxybutyrate by mutant enzyme
分别将转化了突变体质粒pET24a-76789、pET24a-78932的BL21(DE3)的工程菌pET24a-76789/BL21(DE3)和pET24a-78932/BL21(DE3)接种到含有LB培养基的试管中，37℃培养过夜，然后按1％v/v比例接种到含有100mL TB培养基的500mL摇瓶中，37℃培养4-6小时，OD600达到1.2-1.5后，加入0.2mM的IPTG诱导，降温到25℃培养10-16小时，离心获得菌体，-80℃冻存24小时，备用。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 ℃, then inoculated into a 500mL shake flask containing 100mL TB medium at a ratio of 1% v/v, cultured at 37℃ for 4-6 hours, after the OD600 reached 1.2-1.5, added 0.2mM IPTG for induction, and cooled to 25 Cultivated at ℃ for 10-16 hours, centrifuged to obtain bacterial cells, and frozen at -80 ℃ for 24 hours for use.
催化反应采用1L反应体系：底物乙酰乙酸甲酯或乙酰乙酸乙酯50g/L，异丙醇80ml/L，NADP辅因子10mM，pH7.5，湿菌体1.5％。摇床30℃，230rpm震荡反应，15h，加盐酸终止反应，定量检测产物浓度以及底物浓度，计算底物转化率。同步取样，进行3-羟基丁酸酯手性检测，转化液经12000rpm离心3min，取上清。上清液中加入乙酸乙酯，在涡旋振荡器上震荡5min。12000rpm离心3min，取乙酸乙酯相，加入0.2克无水硫酸钠，摇床震荡过夜。干燥后乙酸乙酯相高速离心10min，吸取上清液进行GC检测。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. Centrifuge at 12,000 rpm for 3 min, take the ethyl acetate phase, add 0.2 g of anhydrous sodium sulfate, and shake on a shaker overnight. After drying, the ethyl acetate phase was centrifuged at high speed for 10 min, and the supernatant was drawn for GC detection.
GC检测条件为：色谱柱Gamma DEXTM 225 Capillary Column 30m*0.25nm*0.25μm film thickness；进样量：0.1μL；进样器温度：250℃；分流比：190:1；载气压力：10.795psi；流速：1mL/min；升温程序：初始温度40℃，保持5min，以10℃/min的升温速率升温至170℃，保持2min；运行时间：20min；检测器：FID，300℃；空气流速：400mL/min；氢气流速：30mL/min；尾吹气(N2)：25mL/min。GC detection conditions are: chromatographic column Gamma DEXTM 225 Capillary Column 30m*0.25nm*0.25μm film thickness; injection volume: 0.1μL; injector temperature: 250℃; 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.
两个菌株pET24a-76789和pET24a-78932催化乙酰乙酸甲酯/乙酰乙酸乙酯制备 3-羟基丁酸酯的实验结果列于表8中。The experimental results of the two strains pET24a-76789 and pET24a-78932 catalyzing the production of 3-hydroxybutyrate from methyl acetoacetate/ethyl acetoacetate are listed in Table 8.
表8、酶催化制备3-羟基丁酸酯的反应结果Table 8. The reaction results of enzyme-catalyzed preparation of 3-hydroxybutyrate
Figure PCTCN2020110402-appb-000015
Figure PCTCN2020110402-appb-000015
表8结果显示，两种突变酶76789号酶(SEQ ID NO:20)和78932号酶(SEQ ID NO:21)都能够催化两种底物反应得到R-构型的3-羟基丁酸酯，两种底物的转化率都能够达到90％以上，产物手性纯度皆高于99％，具有工业化应用前景。The results in Table 8 show that both mutant enzymes No. 76789 (SEQ ID NO: 20) and No. 78932 (SEQ ID NO: 21) can catalyze the reaction of two substrates to obtain 3-hydroxybutyrate in the R-configuration , the conversion rate of the two substrates can reach more than 90%, and the chiral purity of the product is higher than 99%, which has the prospect of industrial application.

Claims (10)

  1. 一种酶法合成3-羟基丁酸酯的方法，其特征在于，以乙酰乙酸酯作为底物，使用醇脱氢酶SEQ ID NO:1或者其突变体催化还原反应，得到3-羟基丁酸酯，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 acid ester,
    其中所述醇脱氢酶突变体是SEQ ID NO:1氨基酸序列经过一个以上位点氨基酸残基的突变、且具有醇脱氢酶SEQ ID NO:1功能的多肽；或者是与SEQ ID NO:1氨基酸序列有85％以上同源性、且具有醇脱氢酶SEQ ID NO:1功能的多肽。Wherein the alcohol dehydrogenase mutant is a polypeptide whose amino acid sequence of SEQ ID NO: 1 undergoes mutation of amino acid residues at one or more sites and has the function of alcohol dehydrogenase SEQ ID NO: 1; or is the same as SEQ ID NO: 1. The amino acid sequence has more than 85% homology and has the function of alcohol dehydrogenase SEQ ID NO: 1.
  2. 如权利要求1所述的方法，其特征在于，所述乙酰乙酸酯是乙酰乙酸甲酯或乙酰乙酸乙酯，相应地，所述3-羟基丁酸酯是3-羟基丁酸甲酯或3-羟基丁酸乙酯。The method of claim 1, wherein the acetoacetate is methyl acetoacetate or ethyl acetoacetate, and correspondingly, the 3-hydroxybutyrate is methyl 3-hydroxybutyrate or Ethyl 3-hydroxybutyrate.
  3. 如权利要求1所述的方法，其特征在于，反应体系中添加有异丙醇和辅酶NADP+。The method of claim 1, wherein isopropanol and coenzyme NADP+ are added to the reaction system.
  4. 如权利要求1所述的方法，其特征在于，所述醇脱氢酶突变体是SEQ ID NO:1氨基酸序列中下述位点发生突变后所形成的突变体：第6位、第19位、第25位、第57位、第77位、第89位、第97位、第123位、第147位、第149位、第151位、第155位、第190位、第197位、第202位、第220位、第221位、第235位、或者它们两种以上的组合。The method of claim 1, wherein the alcohol dehydrogenase mutant 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 , No. 25, No. 57, No. 77, No. 89, No. 97, No. 123, No. 147, No. 149, No. 151, No. 155, No. 190, No. 197, No. 202nd, 220th, 221st, 235th, or a combination of two or more of them.
  5. 一种醇脱氢酶突变体，其为如权利要求4中所述的醇脱氢酶突变体。An alcohol dehydrogenase mutant, which is the alcohol dehydrogenase mutant as claimed in claim 4.
  6. 如权利要求5所述的醇脱氢酶突变体，其特征在于，所述醇脱氢酶突变体中的突变选自下组：K6N、I19L、D25G、I57N或I57T、T77N或T77S、N89T或N89K、K97R或K97N、R123S或R123H、F147I或F147C、G149D或G149R、P151L、A155D、Y190F或Y190G、D197E、A202V或A202T、P220Q、N221T或N221I或N221V、S235Y、或者它们两种以上的组合。The alcohol dehydrogenase mutant of claim 5, wherein the mutation in the 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 of them.
  7. 如权利要求6所述的醇脱氢酶突变体，其特征在于，所述醇脱氢酶突变体选自下组：The alcohol dehydrogenase mutant of claim 6, wherein the alcohol dehydrogenase mutant is selected from the group consisting of:
    SEQ ID NO:3，其为SEQ ID NO:1氨基酸序列A202T、K97R的突变体；SEQ ID NO:3, which is a mutant of SEQ ID NO:1 amino acid sequence A202T, K97R;
    SEQ ID NO:4，其为SEQ ID NO:1氨基酸序列A202V、K97R、Y190G的突变体；SEQ ID NO:4, which is a mutant of SEQ ID NO:1 amino acid sequence A202V, K97R, Y190G;
    SEQ ID NO:5，其为SEQ ID NO:1氨基酸序列A202T、K97R、F147I、K6N的突变体；SEQ ID NO:5, which is a mutant of SEQ ID NO:1 amino acid sequence A202T, K97R, F147I, K6N;
    SEQ ID NO:6，其为SEQ ID NO:1氨基酸序列A202T、K97R、N89T、R123H、N221T的突变体；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，其为SEQ ID NO:1氨基酸序列A202T、D25G的突变体；SEQ ID NO:7, which is a mutant of SEQ ID NO:1 amino acid sequence A202T, D25G;
    SEQ ID NO:8，其为SEQ ID NO:1氨基酸序列A202T、K97R、S235Y、I57N、R123H的突变体；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，其为SEQ ID NO:1氨基酸序列A202V、K97R、N221I、Y190F的突变体；SEQ ID NO:9, which is a mutant of SEQ ID NO:1 amino acid sequence A202V, K97R, N221I, Y190F;
    SEQ ID NO:10，其为SEQ ID NO:1氨基酸序列A202V、K97R、N221I、Y190F、D25G、K6N、R123S的突变体；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，其为SEQ ID NO:1氨基酸序列A202V、N221I、Y190F、G149D、D25G的突变体；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，其为SEQ ID NO:1氨基酸序列A202V、Y190F、D25G的突变体；SEQ ID NO:12, which is a mutant of SEQ ID NO:1 amino acid sequence A202V, Y190F, D25G;
    SEQ ID NO:13，其为SEQ ID NO:1氨基酸序列A202V、Y190F、D25G、I57T的突变体；SEQ ID NO: 13, which is a mutant of SEQ ID NO: 1 amino acid sequence A202V, Y190F, D25G, I57T;
    SEQ ID NO:14，其为SEQ ID NO:1氨基酸序列A202V、N221I、Y190F、F147I的突变体；SEQ ID NO: 14, which is a mutant of SEQ ID NO: 1 amino acid sequence A202V, N221I, Y190F, F147I;
    SEQ ID NO:15，其为SEQ ID NO:1氨基酸序列K97R、N221I、Y190F、F147I的突变体；SEQ ID NO: 15, which is a mutant of SEQ ID NO: 1 amino acid sequence K97R, N221I, Y190F, F147I;
    SEQ ID NO:16，其为SEQ ID NO:1氨基酸序列A202V、N221I、Y190F、F147I、D197E、P151L的突变体；SEQ ID NO: 16, which is a mutant of the amino acid sequence A202V, N221I, Y190F, F147I, D197E, P151L of SEQ ID NO:1;
    SEQ ID NO:17，其为SEQ ID NO:1氨基酸序列A202V、N221V、Y190F、F147I、I19L、G149R的突变体；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，其为SEQ ID NO:1氨基酸序列A202V、N221I、Y190F、F147I、K97N、N89K、R123S的突变体；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，其为SEQ ID NO:1氨基酸序列A202T、N221I、Y190F、K6N的突变体；SEQ ID NO: 19, which is a mutant of SEQ ID NO: 1 amino acid sequence A202T, N221I, Y190F, K6N;
    SEQ ID NO:20，其为SEQ ID NO:1氨基酸序列A202V、N221I、Y190F、F147I、K97N、N89K、R123S、A155D、T77N的突变体；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，其为SEQ ID NO:1氨基酸序列A202V、N221I、Y190F、F147I、K97N、N89K、R123S、T77S、G149R、P151L的突变体；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，其为SEQ ID NO:1氨基酸序列Y190F的突变体；SEQ ID NO:22, which is a mutant of SEQ ID NO:1 amino acid sequence Y190F;
    SEQ ID NO:23，其为SEQ ID NO:1氨基酸序列K97R的突变体；SEQ ID NO:23, which is a mutant of SEQ ID NO:1 amino acid sequence K97R;
    SEQ ID NO:24，其为SEQ ID NO:1氨基酸序列P220Q、F147C的突变体；SEQ ID NO:24, which is a mutant of SEQ ID NO:1 amino acid sequence P220Q, F147C;
    SEQ ID NO:25，其为SEQ ID NO:1氨基酸序列I57N的突变体；SEQ ID NO:25, which is a mutant of SEQ ID NO:1 amino acid sequence I57N;
    SEQ ID NO:26，其为SEQ ID NO:1氨基酸序列G149D的突变体；SEQ ID NO:26, which is a mutant of SEQ ID NO:1 amino acid sequence G149D;
    SEQ ID NO:27，其为SEQ ID NO:1氨基酸序列R123S的突变体。SEQ ID NO: 27, which is a mutant of the amino acid sequence R123S of SEQ ID NO: 1.
  8. 一种微生物，其表达如权利要求7所述的醇脱氢酶突变体SEQ ID NOs:3-27之一。A microorganism expressing one of the alcohol dehydrogenase mutant SEQ ID NOs:3-27 of claim 7.
  9. 如权利要求8所述的微生物，其特征在于，所述微生物选自大肠杆菌、毕赤酵母、枯草芽孢杆菌。The microorganism of claim 8, wherein the microorganism is selected from Escherichia coli, Pichia pastoris, and Bacillus subtilis.
  10. 如权利要求5所述醇脱氢酶突变体或者如权利要求8所述微生物在生产3-羟基丁酸酯中的用途。Use of the alcohol dehydrogenase mutant of claim 5 or the microorganism of claim 8 in the production of 3-hydroxybutyrate.
PCT/CN2020/110402 2020-08-21 2020-08-21 Method for enzymatic synthesis of 3-hydroxybutyrate WO2022036662A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN2020/110402 WO2022036662A1 (en) 2020-08-21 2020-08-21 Method for enzymatic synthesis of 3-hydroxybutyrate
CN202080103912.5A CN116157509A (en) 2020-08-21 2020-08-21 Method for synthesizing 3-hydroxybutyrate by enzyme method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2020/110402 WO2022036662A1 (en) 2020-08-21 2020-08-21 Method for enzymatic synthesis of 3-hydroxybutyrate

Publications (1)

Publication Number Publication Date
WO2022036662A1 true WO2022036662A1 (en) 2022-02-24

Family

ID=80323364

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/110402 WO2022036662A1 (en) 2020-08-21 2020-08-21 Method for enzymatic synthesis of 3-hydroxybutyrate

Country Status (2)

Country Link
CN (1) CN116157509A (en)
WO (1) WO2022036662A1 (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109295019A (en) * 2018-10-29 2019-02-01 浙江大学 A kind of Alcohol dehydrogenase mutant and its application
CN110093302A (en) * 2019-06-13 2019-08-06 浙江华睿生物技术有限公司 A kind of lactobacillus mutant strain and its application
CN111172124A (en) * 2020-02-26 2020-05-19 复旦大学 Carbonyl reductase mutant and application thereof in preparation of (R) -4-chloro-3-hydroxy-butyrate
CN111454921A (en) * 2019-12-30 2020-07-28 南京朗恩生物科技有限公司 Ketoreductase mutant with improved enzyme activity and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109295019A (en) * 2018-10-29 2019-02-01 浙江大学 A kind of Alcohol dehydrogenase mutant and its application
CN110093302A (en) * 2019-06-13 2019-08-06 浙江华睿生物技术有限公司 A kind of lactobacillus mutant strain and its application
CN111454921A (en) * 2019-12-30 2020-07-28 南京朗恩生物科技有限公司 Ketoreductase mutant with improved enzyme activity and application thereof
CN111172124A (en) * 2020-02-26 2020-05-19 复旦大学 Carbonyl reductase mutant and application thereof in preparation of (R) -4-chloro-3-hydroxy-butyrate

Also Published As

Publication number Publication date
CN116157509A (en) 2023-05-23

Similar Documents

Publication Publication Date Title
EP2222841B1 (en) Ketol-acid reductoisomerase using nadh
CN111254129B (en) Polyphosphate kinase mutant and application thereof
CN110791493B (en) Aspartate ammonia lyase mutant and application thereof
WO2016138641A1 (en) Generation and use of candida and carbonyl reductase thereof
CN113528606B (en) Method for preparing 17 beta-hydroxysteroid by enzyme catalysis
CN109055324B (en) Improved ketoreductase and application thereof
EP2821485A1 (en) Hydrocarbon synthase gene, and use thereor
Chang et al. A combined strategy of metabolic pathway regulation and two-step bioprocess for improved 4-androstene-3, 17-dione production with an engineered Mycobacterium neoaurum
KR101262999B1 (en) Recombinant yeast by overexpression of pyruvate decarboxylase and alcohol dehydrogenase for improving productivity of bioethanol and Method for producing ethanol using the same
CN112746067A (en) Lysine decarboxylase mutants for producing D-ornithine
CN113061593B (en) L-malate dehydrogenase mutant and application thereof
CN111349644A (en) Bacterial strain and method for biosynthesis of isoprene glycol
Radoš et al. Stereospecificity of Corynebacterium glutamicum 2, 3-butanediol dehydrogenase and implications for the stereochemical purity of bioproduced 2, 3-butanediol
US10889840B2 (en) Conversion of methylglyoxal into hydroxyacetone using novel enzymes and applications thereof
CN114908129B (en) Dehydrogenase for the preparation of (R) -4-chloro-3-hydroxybutyric acid ethyl ester
WO2022036662A1 (en) Method for enzymatic synthesis of 3-hydroxybutyrate
CN115806923A (en) Engineering bacterium containing fatty acyl-coenzyme A oxidase gene and application of engineering bacterium in preparation of 10-hydroxy-2-decenoic acid
WO2017202193A1 (en) Applications of recombinant ketoreductase in preparation of (r)-3-quinuclidinol
Cheng et al. Strain isolation and study on process parameters for xylose-to-xylitol bioconversion
JP5671339B2 (en) Yeast producing ethanol from xylose
Li et al. Characterization and Application of a Novel Glucose Dehydrogenase with Excellent Organic Solvent Tolerance for Cofactor Regeneration in Carbonyl Reduction
CN113122563A (en) Method for constructing R-3-aminobutyric acid production strain
CN112592904A (en) 17 beta-hydroxysteroid dehydrogenase mutant of mycobacterium and heterologous expression thereof
US20210017526A1 (en) Xylose metabolizing yeast
CN112410353A (en) fkbS gene, genetic engineering bacterium containing fkbS gene, and preparation method and application of fkbS gene

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20949872

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20949872

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC

122 Ep: pct application non-entry in european phase

Ref document number: 20949872

Country of ref document: EP

Kind code of ref document: A1