CN116157509A

CN116157509A - Method for synthesizing 3-hydroxybutyrate by enzyme method

Info

Publication number: CN116157509A
Application number: CN202080103912.5A
Authority: CN
Inventors: 范文超; 高书良; 王金刚; 梁岩; 杨海锋; 任亮; 袁圣伦
Original assignee: Zhejiang Huarui Biotechnology Co ltd
Current assignee: Zhejiang Huarui Biotechnology Co ltd
Priority date: 2020-08-21
Filing date: 2020-08-21
Publication date: 2023-05-23
Also published as: WO2022036662A1

Abstract

A method for synthesizing 3-hydroxybutyrate by an enzymatic method is provided, wherein acetoacetate is used as a substrate, and alcohol dehydrogenase SEQ ID NO. 1 or mutants thereof SEQ ID NOs 3-27 are used for catalyzing and reducing reaction to obtain the 3-hydroxybutyrate.

Description

Method for synthesizing 3-hydroxybutyrate by enzyme method

Technical Field

The invention belongs to the technical fields of genetic engineering and enzyme catalysis, and particularly relates to a method for synthesizing 3-hydroxybutyrate by an enzyme method.

Background

In recent years, the concept of ketogenic diet has become a well-established life style, and the intake of foods containing ketone bodies can be used for supplementing ketone bodies to organisms and further for ketone metabolism of the organisms. Acetoacetate, 3-Hydroxybutyrate and acetone are three ketone body forms required for the human body, of which 3-Hydroxybutyrate (3-HB) has been successfully commercialized as a ketone body supplementary product of a main raw material, and market demands have increased year by year.

The preparation of 3-hydroxybutyric acid mainly comprises a chemical synthesis method, an enzymatic conversion method and a microbial fermentation method. The existing technology for producing 3-hydroxybutyrate by an enzymatic method basically takes methyl acetoacetate or ethyl acetoacetate as a chemical raw material, takes NADPH or NADH as a coenzyme and can generate ketone group to reduce into hydroxyl by being catalyzed by alcohol dehydrogenase (EC 1.1.1.1) or carbonyl reductase (EC 1.1.1.148) to generate products of 3-hydroxybutyrate methyl or 3-hydroxybutyrate ethyl. The preparation of 3-hydroxybutyric acid can be realized after the 3-hydroxybutyric acid methyl ester or 3-hydroxybutyric acid ethyl ester is further subjected to ester hydrolysis reaction.

However, the enzymatic activities of alcohol dehydrogenases and carbonyl reductases currently used industrially are generally low, resulting in high production costs of the products; moreover, the substrate specificity, i.e., the substrate specificity, of these alcohol dehydrogenases and carbonyl reductases is too high, resulting in too narrow a range of applicable substrates.

Disclosure of Invention

In order to improve the enzymatic production process of 3-hydroxybutyrate, a large number of screening is carried out on alcohol dehydrogenase and carbonyl reductase, the catalytic performances of the alcohol dehydrogenase and carbonyl reductase on methyl acetoacetate and ethyl acetoacetate are researched, and the alcohol dehydrogenase (SEQ ID NO: 1) with a relatively large substrate application range and a Lactobacillus kefiri DSM 20587 source is modified by random mutation, combined mutation and other technologies, so that mutants with remarkably improved enzyme activity are obtained, and the acetoacetate is efficiently catalyzed to generate the 3-hydroxybutyrate. Specifically, the invention comprises the following technical scheme.

A method for synthesizing 3-hydroxybutyrate by an enzymatic method is characterized in that acetoacetate is used as a substrate, and alcohol dehydrogenase SEQ ID NO. 1 or a mutant thereof is used for catalytic reduction reaction to obtain 3-hydroxybutyrate:

wherein R is a C1-C4 alkyl group selected from methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl. Namely, the 3-hydroxybutyrate is selected from the group consisting of methyl 3-hydroxybutyrate, ethyl 3-hydroxybutyrate, propyl 3-hydroxybutyrate, isopropyl 3-hydroxybutyrate, butyl 3-hydroxybutyrate, sec-butyl 3-hydroxybutyrate, isobutyl 3-hydroxybutyrate, tert-butyl 3-hydroxybutyrate;

the alcohol dehydrogenase mutant is a polypeptide which is formed by mutating (including but not limited to substituting, deleting or adding) more than one site amino acid residues of the amino acid sequence of SEQ ID NO. 1 and has the function of the alcohol dehydrogenase SEQ ID NO. 1; or a polypeptide having 85% or more homology, preferably 90% or more homology, more preferably 95% or more homology to the amino acid sequence of SEQ ID No. 1 and having the function of alcohol dehydrogenase SEQ ID No. 1.

The alcohol dehydrogenase SEQ ID NO. 1 functions as described above refer to a function capable of catalyzing the reduction of methyl acetoacetate to methyl 3-hydroxybutyrate and ethyl acetoacetate to ethyl 3-hydroxybutyrate.

Preferably, the above alcohol dehydrogenase mutant has an enzyme activity higher than that of SEQ ID NO. 1.

Preferably, the substrate acetoacetate is methyl acetoacetate or ethyl acetoacetate, and the product 3-hydroxybutyrate is methyl 3-hydroxybutyrate or ethyl 3-hydroxybutyrate, respectively.

The above 3-hydroxybutyrate includes methyl 3-hydroxybutyrate or ethyl 3-hydroxybutyrate, and particularly, R-configured 3-hydroxybutyrate includes methyl (R) -3-hydroxybutyrate or ethyl (R) -3-hydroxybutyrate.

In a preferred embodiment, isopropanol and the coenzyme NADP+ (nicotinamide adenine dinucleotide phosphate, coenzyme II) are also added to the enzyme-catalyzed reaction system. The role of NADP+ is to scavenge electrons as an oxidant, and alcohol dehydrogenase reduces NADP+ to NADPH using isopropanol, producing sufficient NADPH as a reducing agent for biosynthesis, thereby promoting the reduction reaction.

The pH of the enzyme-catalyzed reaction system of the invention may be 7.0 to 8.0, preferably pH7.2 to 7.8, more preferably pH 7.4 to 7.5.

The enzyme-catalyzed reaction temperature is 25-45 ℃, preferably 28-40 ℃, more preferably 30-35 ℃.

In the above method, the mutation site in the alcohol dehydrogenase mutant may be a site selected from the group consisting of SEQ ID NO:1 amino acid sequence: bit 6, 19, 25, 57, 77, 89, 97, 123, 147, 149, 151, 155, 190, 197, 202, 220, 221, 235, or a combination of two or more thereof.

In a second aspect of the present invention there is provided an alcohol dehydrogenase mutant as described above. For example, the mutant is formed by mutating the following sites in the amino acid sequence of SEQ ID NO: 1: bit 6, 19, 25, 57, 77, 89, 97, 123, 147, 149, 151, 155, 190, 197, 202, 220, 221, 235, or a combination of two or more thereof.

In a preferred embodiment, the mutation in the alcohol dehydrogenase mutant is selected from the group consisting of: K6N, I19L, D G, I N or I57T, T77N or T77S, N89T or N89K, K97R or K97N, R S or R123H, F147I or F147C, G149D or G149R, P151L, A155D, Y190F or Y190G, D197E, A V or A202T, P220Q, N221T or N221I or N221V, S235Y or a combination of two or more thereof.

For example, the above alcohol dehydrogenase mutants are selected from the group consisting of:

SEQ ID NO. 3, which is a mutant of SEQ ID NO. 1 amino acid sequence A202T, K R;

SEQ ID NO. 4, which is a mutant of amino acid sequence A202V, K, 97, R, Y, 190G of SEQ ID NO. 1;

SEQ ID NO. 5, which is a mutant of SEQ ID NO. 1 amino acid sequence A202T, K97R, F147I, K N;

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, a mutant of SEQ ID NO. 1 amino acid sequence A202T, D G;

SEQ ID NO. 8, which is a mutant of SEQ ID NO. 1 amino acid sequence A202T, K97R, S235Y, I57N, R H;

SEQ ID NO. 9, a mutant of SEQ ID NO. 1 amino acid sequence A202V, K97R, N221I, Y190F;

10, which is a mutant of amino acid sequence A202V, K97R, N221I, Y190F, D G, K6N, R123S of SEQ ID NO 1;

11, which is a mutant of amino acid sequence A202V, N221I, Y190F, G149D, D G of SEQ ID NO. 1;

SEQ ID NO. 12, a mutant of SEQ ID NO. 1 amino acid sequence A202V, Y190F, D G;

13, which is a mutant of amino acid sequence A202V, Y190F, D G, I57T of SEQ ID NO. 1;

SEQ ID NO. 14, a mutant of SEQ ID NO. 1 amino acid sequence A202V, N221I, Y190F, F147I;

15, which is a mutant of amino acid sequence K97R, N221I, Y190F, F147I of SEQ ID NO. 1;

16, which is a mutant of amino acid sequence A202V, N221I, Y190F, F147I, D197E, P151L of SEQ ID NO. 1;

17, which is a mutant of amino acid sequence A202V, N221V, Y190F, F147I, I5219L, G149R of SEQ ID NO. 1;

18, which is a mutant of amino acid sequence A202V, N221I, Y190F, F147I, K3897N, N89K, R123S of SEQ ID NO 1;

SEQ ID NO. 19, a mutant of SEQ ID NO. 1 amino acid sequence A202T, N221I, Y190F, K N;

20, which is a mutant of amino acid sequence A202V, N221I, Y190F, F147I, K97N, N89K, R123S, A155D, T N of SEQ ID NO 1;

21, which is a mutant of amino acid sequence A202V, N221I, Y190F, F147I, K N, N89K, R123S, T77S, G149R, P L of SEQ ID NO 1;

SEQ ID NO. 22, a mutant of the amino acid sequence Y190F of SEQ ID NO. 1;

SEQ ID NO. 23, a mutant of SEQ ID NO. 1 amino acid sequence K97R;

24, which is a mutant of amino acid sequence P220Q, F147C of SEQ ID NO 1;

25, which is a mutant of amino acid sequence I57N of SEQ ID NO. 1;

26, which is a mutant of amino acid sequence G149D of SEQ ID NO. 1;

SEQ ID NO. 27 is a mutant of the amino acid sequence R123S of SEQ ID NO. 1.

Particularly preferred mutants of the above alcohol dehydrogenase are SEQ ID NO. 18, SEQ ID NO. 20 or SEQ ID NO. 21.

In a third aspect, the invention provides a microorganism expressing an 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 (DE 3). When the microorganism is E.coli, the gene encoding the wild-type alcohol dehydrogenase SEQ ID NO. 1 may be the nucleotide sequence SEQ ID NO. 2.

The microorganisms described above can be used directly as naturally immobilized forms of alcohol dehydrogenase for the production of 3-hydroxybutyrate.

The wild alcohol dehydrogenase SEQ ID NO 1 and the mutant SEQ ID NOs 3-27 constructed based on the wild alcohol dehydrogenase SEQ ID NO 1 can catalyze the reduction of methyl acetoacetate into methyl 3-hydroxybutyrate and the reduction of ethyl acetoacetate into ethyl 3-hydroxybutyrate when being applied to the enzymatic synthesis of 3-hydroxybutyrate, thus widening the substrate range of the acetoacetate and having industrial application prospect.

Detailed Description

The wild-type alcohol dehydrogenase selected according to the invention, SEQ ID NO. 1, is derived from Lactobacillus kefir Lactobacillus kefiri DSM, 20587, numbered 19 in the examples, which requires the involvement of the coenzyme NADPH in catalyzing the reduction of acetoacetates to 3-hydroxybutyrate.

The amino acid sequence of SEQ ID NO. 1 is:

through carrying out multiple rounds of mutation on SEQ ID NO. 1, a series of mutation points are found, a plurality of mutants with the enzyme activities mentioned are constructed, wherein the mutants comprise SEQ ID NOs 3-27, and the mutants can be catalyzed by taking methyl acetoacetate and ethyl acetoacetate as substrates to obtain corresponding methyl 3-hydroxybutyrate and ethyl 3-hydroxybutyrate.

Some of the mutations are not single mutations, for example, the mutation at position 202 may be A202T or A202V. Wherein the A202T mutation refers to a mutation in which the alanine (A or Ala) residue at position 202 of the amino acid sequence of SEQ ID NO. 1 is replaced with threonine (T or Thr), and the A202V mutation refers to a mutation in which the alanine (A or Ala) residue at position 202 is replaced with valine (V or Val).

In the examples, the terms "wild type", "wild-type enzyme" and "wild-type enzyme" are intended to mean the same meaning and all refer to the wild-type sequence of the alcohol dehydrogenase SEQ ID NO. 1. For the sake of distinction and convenience of expression from mutants (mutant enzymes), the wild-type alcohol dehydrogenase may be referred to as "wild-type (type) alcohol dehydrogenase" or "wild-type (type) enzyme" in the present invention.

Since the function of the alcohol dehydrogenase mutants SEQ ID NOs:3-27 is not changed, the "alcohol dehydrogenase mutant" is sometimes also referred to simply as "alcohol dehydrogenase" for convenience of description, as will be readily understood by those skilled in the art.

In order to express the alcohol dehydrogenase SEQ ID NO. 1 in escherichia coli which is most commonly used in genetic engineering, the invention optimizes codons of the expressed genes, and takes the genes as a basic template for constructing an alcohol dehydrogenase mutant, wherein the encoding genes of the wild alcohol dehydrogenase SEQ ID NO. 1 can be nucleotide sequences SEQ ID NO. 2:

a plurality of mutant sequences, namely mutants with amino acid sequences SEQ ID NOs of 3-27 in the invention, are obtained through a plurality of rounds of error-prone PCR random mutation technology.

The alcohol dehydrogenase mutants of the present invention have only 252 amino acids in number and are well-defined in sequence, and thus the genes encoding them, expression cassettes and plasmids containing these genes, and transformants containing the plasmids can be readily obtained by those skilled in the art.

For optimal expression of the proteins SEQ ID NOs 3-27 in different microorganisms, codon optimization can be carried out 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 translational efficiency of a gene of interest. Different organisms often show a special preference for one of some codons encoding the same amino acid due to mutation propensity and natural selection. For example, in a fast-growing microorganism such as E.coli, the optimized codons reflect the composition of their respective genomic tRNA pool. Thus, in fast-growing microorganisms, the low frequency codons of an amino acid can be replaced with codons for the same amino acid but at a high frequency. Thus, the expression of the optimized DNA sequence is improved in fast growing microorganisms.

These genes, expression cassettes, plasmids, transformants can be obtained by genetic engineering construction methods well known to those skilled in the art.

The transformant host can be any microorganism suitable for expressing the polyphosphate kinase, including bacteria and fungi. Preferably the microorganism is E.coli, pichia pastoris, saccharomyces cerevisiae, or Bacillus subtilis, preferably E.coli, more preferably E.coli BL21 (DE 3).

In this reaction system, the alcohol dehydrogenase may be in the form of an enzyme or in the form of a bacterial cell. The enzyme forms include free enzyme, immobilized enzyme, including purified enzyme, crude enzyme, fermentation broth, carrier immobilized enzyme, etc.; the forms of the bacterial cells include viable bacterial cells and dead bacterial cells.

The bacterial form is a natural immobilized enzyme, and can be used as an enzyme preparation for catalytic reaction without crushing treatment or even extraction and purification treatment. Since the reaction substrate and the reaction product are both small molecular compounds, the biological barrier-cell membrane of the cells can be conveniently crossed, so that the cells do not need to be crushed, which is economically advantageous.

The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

The amounts, amounts and concentrations of various substances are referred to herein, wherein the percentages refer to percentages by mass unless otherwise specified.

Examples

Materials and methods

LB medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, pH7.2. (LB solid Medium additionally 20g/L agar powder.)

TB medium: 24g/L yeast extract, 12g/L tryptone, 16.43g/L K ₂ HPO ₄ .3H ₂ O、2.31g/L KH ₂ PO ₄ 5g/L glycerol, pH7.0-7.5. (TB solid Medium additionally 20g/L agar powder.)

ZYM medium: the following mother solutions are respectively prepared according to the formula: ZY medium, 50 XM medium, 50 X5052 medium, 1M MgSO ₄ 1000 x trace elements, 1000 x antibiotics.

ZY medium: 10g peptone, 5g yeast powder, water was added to a volume of 950ml, and the mixture was sterilized at 121℃for 20 min.

50 XM 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 Adding water to 500ml, sterilizing at 121deg.C for 20 min.

50×5052 medium: 125g glycerol, 12.5g glucose, 50g alpha-lactose, and water to 500ml,121 ℃ for 20min sterilization.

1M MgSO ₄ ：24.65g MgSO ₄ ·7H ₂ O, constant volume to 100ml,121 ℃, sterilizing for 20 min.

1000 x trace elements: 1.35g FeCl was dissolved using 50ml of 0.12M HCl ₃ ·6H ₂ O, 0.32g CaCl was added separately ₂ ·2H ₂ O，0.2g MnCl ₂ ·4H ₂ O，0.3g ZnSO ₄ ·7H ₂ O，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 Adding water to a volume of 100ml, filtering and sterilizing.

1000 x antibiotics: 500mg kanamycin, water is added to a volume of 10ml, and the solution is filtered for sterilization.

The sterilized mother solutions were treated with 950ml of ZY medium, 20ml of 50 XM, 20ml of 50X 5052 and 2ml of 1M MgSO ₄ 2ml of 1000 Xtrace elements and 1ml of 1000 Xantibiotics are uniformly mixed to obtain the ZYM self-induction culture medium.

The total gene synthesis in the examples was performed by su Jin Weizhi biotechnology limited and loaded into vector pET24a. Primer synthesis and sequencing were all performed by the company Jin Weizhi biotechnology, su.

The molecular biology experiments in the examples include plasmid construction, digestion, ligation, competent cell preparation, transformation, medium preparation, etc., and are mainly performed with reference to the "molecular cloning Experimental guidelines" (Molecular Cloning: A Laboratory Manual) (third edition), J.Sam Brooks, J.W. Lassel (America) code, huang Peitang et al, science Press, beijing, 2002). The specific experimental conditions can be determined by simple experiments, if necessary.

The PCR amplification experiments were performed according to the reaction conditions or kit instructions provided by the plasmid or DNA template suppliers. Can be adjusted if necessary by simple tests.

It should be noted that, for convenience of description, in the embodiment, the strain number, the plasmid number, the enzyme number, and the enzyme coding gene number may be used together by one number, which is easily understood by those skilled in the art, that is, the same number may refer to different biological forms in different environments. For example, 19# may represent strain Lactobacillus kefiri DSM 20587, or plasmid pET24a-19# numbering, enzyme SEQ ID NO:1 numbering, enzyme encoding gene SEQ ID NO:2 numbering.

Example 1 screening of enzymes for the Synthesis of 3-hydroxybutyrate

The enzyme of microbial origin for reducing methyl acetoacetate/ethyl acetoacetate to methyl 3-hydroxybutyrate/ethyl 3-hydroxybutyrate was examined. A total of 23 enzyme genes were selected from NCBI database search, as shown in Table 1.

TABLE 1 construction list of microorganism-derived wild type enzyme library

Construction of enzyme expression engineering bacteria: codon Adaptation Tool using codon optimization toolhttp://www.jcat.de/) E.coli codon optimization for 23 enzymesThe NdeI/XhoI locus characteristics are eliminated by the column evasion, and then the corresponding coding gene base sequence is obtained. For example, the coding gene for alcohol dehydrogenase # 19 SEQ ID NO. 1 may be the nucleotide sequence SEQ ID NO. 2. The genes of the 23 enzymes are entrusted to the Sizhou intelligent biotechnology limited company for gene synthesis, and the synthesized gene fragments are loaded into NdeI/XhoI sites of an escherichia coli expression plasmid system pET24a vector 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# for subsequent expression of proteins.

Example 2 detection of enzyme Activity of engineering bacteria

The 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 (DE 3) competent cells, respectively, and cultured overnight at 37 ℃.2 single colonies are respectively selected, inoculated into a test tube containing LB culture medium, cultured overnight, thalli are centrifugally collected, plasmids are extracted, and gene sequencing is determined to be correct, so that the recombinant strain is obtained. Selecting a monoclonal on a flat plate of a genetic engineering strain, inoculating the monoclonal into 5mL of LB culture medium, and culturing at 37 ℃; inoculating to 250mL shake flask containing 20mL TB culture medium at 1%v/v, culturing for 4-6 hr, adding 0.2mM IPTG to induce OD600 to 1.2-1.5, cooling to 25deg.C, culturing for 10-16 hr, centrifuging to obtain thallus, and freezing at-80deg.C for 24 hr.

The enzyme activity was determined using the following method: 5mL of the reaction system solution (100 g/L of methyl acetoacetate or ethyl acetoacetate, 100mL/L of isopropyl alcohol, 3mM NADP or NAD, pH=7.5), 0.05g of wet bacterial cells were added to the reaction system solution, the reaction system was incubated in a water bath at 30℃for 30 minutes, and finally the reaction was terminated by using 1M hydrochloric acid. The reaction solution was sampled and the concentration of the product methyl 3-hydroxybutyrate or ethyl 3-hydroxybutyrate was detected by HPLC.

HPLC detection conditions, sample injection amount of 10ul; a 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 ℃; the detection wavelength is 210nm; the detection time is 15min. And calculating the reactive enzyme activity of the unit thalli according to the concentration of the product.

Definition of enzyme activity: the amount of cells required to produce 1. Mu.M of the product per unit time (min) was one enzyme activity unit (U).

The enzyme activities of the 23 wild-type enzyme engineering bacteria were compared, and the results are shown in Table 2.

TABLE 2 evaluation results of wild type enzyme activity

* Remarks: the activity data were calculated as 00% for the enzyme activity of the 1# enzyme for the acetoacetate substrate.

Wherein the activity of the 19# enzyme on the two substrates is balanced, and according to the result, the construction, screening and evaluation of an error-prone PCR mutant library (error-prone mutant library for short) are carried out by using pET24a-19# plasmid.

EXAMPLE 3 first round mutant library construction and screening

Using pET24a-19# plasmid as a template, designing the following primers for amplification:

forward 19# -F:5'-CATATGACCGACCGTCTGAAAGG-3' the number of the individual pieces of the plastic,

reversing 19# -R:5'-CTCGAG TTACTGAGCGGTGTAACCACCG-3'.

Random mutant libraries were constructed using error-prone PCR techniques. The 50. Mu.L error-prone PCR reaction system comprises: 500ng of plasmid template, 500pmol of 19# -F primer, 500pmol of 19# -R primer，1x PCR buffer，0.2mM dGTP，0.2mM dATP，1mM dCTP，1mM dTTP，7mM MgCl ₂ ，0.1mM MnCl ₂ Taq enzyme (Invitrogen) of 2.5 units ^TM )。

The error-prone PCR reaction conditions were: 95 ℃ for 5min;94℃for 30s,55℃for 30s,72℃for 2min/kbp,30 cycles; and at 72℃for 10min. The random mutant fragment was recovered as MegaPrimer for the next round of PCR, megaPrimer PCR was performed using KOD FX DNA polymerase (TOYOBO), 50. Mu.l reaction system, 1X PCR buffer,2mM dNTPs,megaprimer 250ng,pET24a-19# plasmid 50ng,1 unit KOD FX, PCR reaction program: 94 ℃ for 5min;98℃10s,60℃30s,68℃1min,25 cycles; and at 68℃for 10min.

After the PCR product was digested with DpnI for 10 hours, competent cells of E.coli BL21 (DE 3) were electrotransformed, plated on LB medium containing kanamycin, and cultured overnight at 37℃to obtain random mutant library clones.

After obtaining mutant library clone, carrying out clone picking, culturing and reaction screening. Sterile 96-well plates were taken, 400. Mu.l of LB medium (containing 50. Mu.g/ml kanamycin) was added to each well, and mutant library monoclonal was picked using sterile toothpicks to transform BL21 (DE 3) engineering bacteria of pET24a or pET24a-19# plasmids, respectively, as blank and negative controls. The above-mentioned well plate was cultured at 37℃in a well plate shaker at 280rpm for 20 hours and used as a seed solution. A96-well plate was newly prepared, 400. Mu.l of ZYM medium (containing 50. Mu.g/ml kanamycin) was added to each well, and 50. Mu.l of the bacterial liquid was inoculated from the seed well plate and cultured at 30℃and 280rpm for 24 hours. And (3) after glycerol with the final concentration of 15% is added into the residual seed liquid, preserving at-80 ℃ for later use. After the culture in ZYM well plate, the OD600 of the concentration was measured. Simultaneously, 50 mu L of each well is transferred to another new 96-well plate, centrifuged at 4000rpm for 10min, and the precipitated cell plates are frozen and stored for 16h. Thawing frozen 96-well plate thalli at room temperature for 20min, adding 200 μl of prepared reaction system solution (100 g/L ethyl acetoacetate, 100mL/L isopropanol, 3mM NADP, pH=7.5), fully shaking for resuspension, placing on a shaking table, reacting at 30 ℃ at 280rpm for 40min, placing on ice after the reaction is finished, adding 200 μl of diluted hydrochloric acid into the 96-well plate by a discharge gun, stopping the reaction, centrifuging at 4000rpm for 10min, and obtaining the supernatant. The centrifugal supernatant was diluted 5-50 times with pure water, and OD240 value was measured using an ELISA reader. By comparing the absorbance values of each well, the higher the enzyme activity, the lower the OD240 reading.

Through the first round of screening, the dominant clone is transferred from a seed pore plate to a TB shake flask, inoculated with 200 mu l to 250mL shake flask containing 20mL TB culture medium, cultured for 4-6 hours at 37 ℃, after OD600 reaches 1.2-1.5, added with 0.2mM IPTG for induction, cooled to 25 ℃ for culturing for 10-16 hours, centrifuged to obtain thalli, one part is frozen at-80 ℃ for 24 hours for standby, the other part is subjected to plasmid extraction, sequencing and mutation site judgment. The enzyme activities of the two substrates were measured for the respective mutants, and the results are shown in Table 3.

TABLE 3 enzyme protein sequencing and enzyme Activity of different clones

* Remarks: the activity data were calculated as 100% for the enzyme activity of the 19# enzyme catalyzed acetoacetate substrate.

As can be seen from Table 3, the catalytic activity of enzyme No. 1024 (SEQ ID NO: 3) was the highest and balanced for both substrates.

According to the above comparison result, referring to the method in example 1, the coding gene of 1024 # enzyme was designed and pET24a-1024 plasmid was constructed, and then construction and screening evaluation of error-prone PCR mutant library were performed using pET24a-1024 plasmid.

EXAMPLE 4 construction and screening of the second round mutant library

A second round of error-prone PCR mutant library construction and selection was performed in accordance with the method of example 3 using plasmid pET24a-1024 as a template, with BL21 (DE 3) engineering bacteria transformed with pET24a or pET24a-1024 plasmid, respectively, as blank and negative control, results are shown in Table 4.

TABLE 4 sequencing and enzymatic Activity of different strains in the second round of mutant library

As can be seen from Table 4, the catalytic activities of enzyme 30231 (SEQ ID NO: 9) on both substrates were relatively high and balanced.

According to the above comparison result, referring to the method in example 1, the coding gene of 30231 type enzyme was designed and pET24a-30231 plasmid was constructed, and then construction and screening evaluation of error-prone PCR mutant library were performed using pET24a-30231 plasmid.

EXAMPLE 5 construction and screening of third round mutant library

A third round of error-prone PCR mutant library construction and selection was performed in accordance with the method of example 3 using plasmid pET24a-30231 as a template, with BL21 (DE 3) engineering bacteria transformed with pET24a or pET24a-30231 plasmids, respectively, as blank and negative controls, and the results are shown in Table 5.

TABLE 5 sequencing of enzyme proteins and enzyme Activity of different strains in third round mutant library

As can be seen from Table 5, the catalytic activity of the 55786 enzyme (SEQ ID NO: 14) was relatively high and balanced for both substrates.

According to the comparison result, the coding gene of 55786 enzyme is designed and pET24a-55786 plasmid is constructed by referring to the method in the example 1, and then the construction and screening evaluation of error-prone PCR mutant library are carried out by using pET24a-55786 plasmid.

EXAMPLE 6 construction and screening of fourth round mutant library

A fourth round of error-prone PCR mutant library construction and selection was performed in the same manner as in example 3 using plasmid pET24a-55786 as a template, with BL21 (DE 3) engineering bacteria transformed with pET24a or pET24a-55786 plasmids, respectively, as blank and negative controls, and the results are shown in Table 6.

Table 6 enzyme protein sequencing and enzyme Activity of different strains in fourth round mutant library

As can be seen from Table 6, the catalytic activity of the 65781 enzyme (SEQ ID NO: 18) was relatively high and balanced for both substrates.

According to the comparison result, the coding gene of 65781 enzyme is designed and pET24a-65781 plasmid is constructed by referring to the method in example 1, and then the construction and screening evaluation of error-prone PCR mutant library are carried out by using pET24a-65781 plasmid.

EXAMPLE 7 construction and screening of fifth round mutant library

A fourth round of error-prone PCR mutant library construction and selection was performed in the same manner as in example 3 using plasmid pET24a-65781 as a template, with BL21 (DE 3) engineering bacteria transformed with pET24a or pET24a-65781 plasmid, respectively, as blank and negative controls, and the results are shown in Table 7.

TABLE 7 sequencing and enzymatic Activity of different strains in the fifth round of mutant library

As can be seen from Table 7, the catalytic activities of the 76789 enzyme (SEQ ID NO: 20) and the 78932 enzyme (SEQ ID NO: 21) on the two substrates are improved to some extent and balanced.

With reference to the method in example 1, the coding genes for the 76789 and 78932 enzymes were designed and pET24a-76789 plasmids and pET24a-78932 plasmids were constructed, respectively. According to the method in example 2, the two plasmids are respectively transformed into competent cells of escherichia coli BL21 (DE 3) by an electrotransformation method to obtain genetically engineered bacteria pET24a-76789/BL21 (DE 3) and pET24a-78932/BL21 (DE 3) which are used for catalyzing the reaction of methyl acetoacetate and ethyl acetoacetate to prepare 3-hydroxybutyrate.

EXAMPLE 8 mutant enzyme catalyzed preparation of 3-hydroxybutyrate

Engineering bacteria pET24a-76789/BL21 (DE 3) and pET24a-78932/BL21 (DE 3) transformed with mutant plasmids pET24a-76789 and pET24a-78932 are respectively inoculated into a test tube containing LB culture medium, cultured overnight at 37 ℃, then inoculated into a 500mL shake flask containing 100mL TB culture medium according to the proportion of 1%v/v, cultured for 4-6 hours at 37 ℃, after OD600 reaches 1.2-1.5, added with 0.2mM IPTG for induction, cooled to 25 ℃ for 10-16 hours, and centrifuged to obtain thalli, and frozen for 24 hours at-80 ℃ for later use.

The catalytic reaction adopts a 1L reaction system: the substrate methyl acetoacetate or ethyl acetoacetate 50g/L, isopropyl alcohol 80ml/L, NADP cofactor 10mM, pH7.5, wet cell 1.5%. Shaking reaction is carried out at 30 ℃ by a shaking table at 230rpm for 15 hours, hydrochloric acid is added to stop the reaction, the concentration of the product and the concentration of the substrate are quantitatively detected, and the substrate conversion rate is calculated. Synchronously sampling, carrying out chiral detection on 3-hydroxybutyrate, centrifuging the conversion solution at 12000rpm for 3min, and taking a supernatant. Ethyl acetate was added to the supernatant and the mixture was shaken on a vortex shaker for 5min. Centrifuge at 12000rpm for 3min, take ethyl acetate phase, add 0.2 g anhydrous sodium sulfate, shake overnight. After drying, the ethyl acetate phase was centrifuged at high speed for 10min, and the supernatant was aspirated for GC detection.

The GC detection conditions were: column Gamma DEXTM 225 Capillary Column 30m*0.25nm*0.25 μm film thickness; sample injection amount: 0.1 μl; injector temperature: 250 ℃; split ratio: 190:1; carrier gas pressure: 10.795psi; flow rate: 1mL/min; heating program: the initial temperature is 40 ℃, the temperature is kept for 5min, the temperature is increased to 170 ℃ at the heating rate of 10 ℃/min, and the temperature is kept for 2min; run time: 20min; a detector: FID,300 ℃; air flow rate: 400mL/min; hydrogen flow rate: 30mL/min; tail blow (N2): 25mL/min.

The experimental results of the two strains pET24a-76789 and pET24a-78932 catalyzing methyl acetoacetate/ethyl acetoacetate to produce 3-hydroxybutyrate are set forth in Table 8.

TABLE 8 reaction results of enzyme-catalyzed preparation of 3-hydroxybutyrate

The results in Table 8 show that both the mutant enzyme 76789 (SEQ ID NO: 20) and the mutant enzyme 78932 (SEQ ID NO: 21) can catalyze the reaction of two substrates to obtain the 3-hydroxybutyrate with R-configuration, the conversion rate of the two substrates can reach more than 90%, the chiral purity of the product is higher than 99%, and the industrial application prospect is achieved.

Claims

A method for synthesizing 3-hydroxybutyrate by an enzymatic method is characterized in that acetoacetate is used as a substrate, alcohol dehydrogenase SEQ ID NO. 1 or a mutant thereof is used for catalyzing and reducing reaction to obtain 3-hydroxybutyrate,

wherein the alcohol dehydrogenase mutant is a polypeptide with the function of the alcohol dehydrogenase SEQ ID NO. 1, wherein the amino acid sequence of the polypeptide is mutated by more than one site amino acid residues; or a polypeptide which has more than 85% homology with the amino acid sequence of SEQ ID NO. 1 and has the function of alcohol dehydrogenase SEQ ID NO. 1.
The method of claim 1, wherein the acetoacetate is methyl acetoacetate or ethyl acetoacetate, and the 3-hydroxybutyrate is methyl 3-hydroxybutyrate or ethyl 3-hydroxybutyrate, respectively.
The method according to claim 1, wherein isopropyl alcohol and coenzyme NADP+ are added to the reaction system.
The method of claim 1, wherein the alcohol dehydrogenase mutant is a mutant formed by mutating the amino acid sequence of SEQ ID NO. 1 at: bit 6, 19, 25, 57, 77, 89, 97, 123, 147, 149, 151, 155, 190, 197, 202, 220, 221, 235, or a combination of two or more thereof.
An alcohol dehydrogenase mutant as set forth in claim 4.
The alcohol dehydrogenase mutant of claim 5, wherein the mutation in the alcohol dehydrogenase mutant is selected from the group consisting of: K6N, I19L, D G, I N or I57T, T77N or T77S, N89T or N89K, K97R or K97N, R S or R123H, F147I or F147C, G149D or G149R, P151L, A155D, Y190F or Y190G, D197E, A V or A202T, P220Q, N221T or N221I or N221V, S235Y or a combination of two or more thereof.
The alcohol dehydrogenase mutant of claim 6, wherein the 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, K R;

SEQ ID NO. 4, which is a mutant of amino acid sequence A202V, K, 97, R, Y, 190G of SEQ ID NO. 1;

SEQ ID NO. 5, which is a mutant of SEQ ID NO. 1 amino acid sequence A202T, K97R, F147I, K N;

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, a mutant of SEQ ID NO. 1 amino acid sequence A202T, D G;

SEQ ID NO. 8, which is a mutant of SEQ ID NO. 1 amino acid sequence A202T, K97R, S235Y, I57N, R H;

SEQ ID NO. 9, a mutant of SEQ ID NO. 1 amino acid sequence A202V, K97R, N221I, Y190F;

10, which is a mutant of amino acid sequence A202V, K97R, N221I, Y190F, D G, K6N, R123S of SEQ ID NO 1;

11, which is a mutant of amino acid sequence A202V, N221I, Y190F, G149D, D G of SEQ ID NO. 1;

SEQ ID NO. 12, a mutant of SEQ ID NO. 1 amino acid sequence A202V, Y190F, D G;

13, which is a mutant of amino acid sequence A202V, Y190F, D G, I57T of SEQ ID NO. 1;

SEQ ID NO. 14, a mutant of SEQ ID NO. 1 amino acid sequence A202V, N221I, Y190F, F147I;

15, which is a mutant of amino acid sequence K97R, N221I, Y190F, F147I of SEQ ID NO. 1;

16, which is a mutant of amino acid sequence A202V, N221I, Y190F, F147I, D197E, P151L of SEQ ID NO. 1;

17, which is a mutant of amino acid sequence A202V, N221V, Y190F, F147I, I5219L, G149R of SEQ ID NO. 1;

18, which is a mutant of amino acid sequence A202V, N221I, Y190F, F147I, K3897N, N89K, R123S of SEQ ID NO 1;

SEQ ID NO. 19, a mutant of SEQ ID NO. 1 amino acid sequence A202T, N221I, Y190F, K N;

20, which is a mutant of amino acid sequence A202V, N221I, Y190F, F147I, K97N, N89K, R123S, A155D, T N of SEQ ID NO 1;

21, which is a mutant of amino acid sequence A202V, N221I, Y190F, F147I, K N, N89K, R123S, T77S, G149R, P L of SEQ ID NO 1;

SEQ ID NO. 22, a mutant of the amino acid sequence Y190F of SEQ ID NO. 1;

SEQ ID NO. 23, a mutant of SEQ ID NO. 1 amino acid sequence K97R;

24, which is a mutant of amino acid sequence P220Q, F147C of SEQ ID NO 1;

25, which is a mutant of amino acid sequence I57N of SEQ ID NO. 1;

26, which is a mutant of amino acid sequence G149D of SEQ ID NO. 1;

SEQ ID NO. 27 is a mutant of the amino acid sequence R123S of SEQ ID NO. 1.
A microorganism expressing an alcohol dehydrogenase mutant of claim 7, SEQ ID NOs, one of SEQ ID NOs 3-27.
The microorganism of claim 8, wherein the microorganism is selected from the group consisting of escherichia coli, pichia pastoris, and bacillus subtilis.
Use of an alcohol dehydrogenase mutant according to claim 5 or a microorganism according to claim 8 for the production of 3-hydroxybutyrate.