Alcohol dehydrogenase mutant composition and application thereof

Abstract

The invention relates to the technical field of gene engineering and enzyme engineering, and discloses an alcohol dehydrogenase mutant composition and application thereof, an ADHA mutant is based on an amino acid sequence as shown in SEQ ID NO.1 and comprises one or more amino acid mutations selected from the following sites: K7A, V35L, H62R, Q127S, C173A and G219A; the ADHB mutant is based on an amino acid sequence as shown in SEQ ID NO.2, and comprises one or more amino acid mutations selected from the following sites: T12S, K51E, G92S, S136I, P187G, A204I and Y230F. The catalyst is used for synthesizing (3R, 5S)-6-chloro-3, 5-dihydroxy hexanoic acid tert-butyl ester through one-pot whole-cell biological catalysis of 6-chloro-3, 5-dicarbonyl hexanoic acid tert-butyl ester, the catalytic efficiency is high, the catalytic activity in a slightly acidic environment is high, and the half-life period time is long.

Description

Technical Field

[0001] The present invention relates to the technical fields of genetic engineering and enzyme engineering, and particularly relates to an alcohol dehydrogenase mutant composition and its application. Background Art

[0002] Statins are one of the most successful lipid-lowering drugs developed in recent years. As a hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, it can competitively inhibit the activity of the endogenous cholesterol synthesis rate-limiting enzyme (HMG-CoA reductase), thereby blocking the mevalonic acid metabolic pathway in cells, reducing cholesterol synthesis in the liver, and promoting an increase in the number and activity of low-density lipoprotein receptors on the cell surface, accelerating the plasma cholesterol clearance rate. While lowering blood lipids, it can also prevent and treat cardiovascular diseases such as atherosclerosis and coronary heart disease. Based on the structure-activity relationship of statins, the indispensable pharmacophore in this class of drugs is the chiral β,δ-dihydroxyvaleric acid moiety. (3R,5S)-6-chloro-3,5-dihydroxyhexanoic acid tert-butyl ester, abbreviated as (3R,5S)-CDHH, is a β,δ-dihydroxyvalerate.

[0003] Currently, (3R,5S)-CDHH mainly uses prochiral ethyl 4-chloro-3-oxobutyrate (COBE) (Route 1) or tert-butyl 6-chloro-3,5-dioxohexanoate (CDOH) (Route 2) as the starting substrate. In comparison, Route 2 not only reduces one chemical step, but both steps can be catalytically synthesized by biological methods. Its reaction conditions are mild and conducive to direct cascade catalysis or even "one-pot" without product separation steps, which is more convenient and efficient. The main difficulty of Route 2 is that the starting substrate CDOH is a reactive γ-chloro-β,δ-diketone ester compound, which is sensitive to heat, acids and bases, heavy metal ions, and is prone to irreversible cyclization reaction in aqueous solution and produces a furanone by-product. In addition, CDOH remains relatively stable in a buffer environment with a pH of 5.5.

[0004] Route 1:

[0005]

[0006] Route 2:

[0007]

[0008] The currently commonly used whole-cell method is to use wild-type strains (Lactobacillus kefir) to continuously catalyze CDOH to synthesize the target product through two alcohol dehydrogenases carried by themselves. The main disadvantages of such wild-type strain whole-cell production mainly include low fermentation cell density, high production cost, high reaction pH leading to easy damage of the substrate, low catalytic efficiency of alcohol dehydrogenase, and low empty yield of the target product. Summary of the Invention

[0009] In view of the above problems existing in the prior art, the present application provides an alcohol dehydrogenase mutant composition and its application, which can be heterologously expressed in a high-density fermentation host bacterium, has high enzyme activity, and has a long half-life in a pH 5.5 environment.

[0010] To solve the above problems, the present invention provides the following technical solutions:

[0011] In a first aspect, the present application provides an alcohol dehydrogenase mutant composition, comprising an ADHA mutant and an ADHB mutant;

[0012] The ADHA mutant is based on the amino acid sequence shown in SEQ ID NO.1 and contains one or more amino acid mutations selected from the following sites: position 7, position 35, position 62, position 127, position 219;

[0013] The ADHB mutant is based on the amino acid sequence shown in SEQ ID NO.2 and contains one or more amino acid mutations selected from the following sites: position 12, position 51, position 92, position 136, position 187, position 204, position 230.

[0014] In an embodiment of the present application, the present application provides an alcohol dehydrogenase mutant composition, comprising an ADHA mutant and an ADHB mutant;

[0015] The ADHA mutant is based on the amino acid sequence shown in SEQ ID NO.1 and contains one or more amino acid mutations selected from the following sites: K7A, V35L, H62R, Q127S, C173A, and G219A;

[0016] The ADHB mutant is based on the amino acid sequence shown in SEQ ID NO.2 and contains one or more amino acid mutations selected from the following sites: T12S, K51E, G92S, S136I, P187G, A204I, and Y230F.

[0017] In an embodiment of the present application, the alcohol dehydrogenase mutant composition comprises an ADHA mutant and an ADHB mutant;

[0018] The ADHA mutant is based on the amino acid sequence shown in SEQ ID NO.1 and contains one or more amino acid mutations selected from the following sites: K7A, V35L, H62R, Q127S, C173A, and G219A; and contains one or more amino acid mutations selected from G19T, E48T, N90T, P150Y, K200E, D237R;

[0019] The ADHB mutant is based on the amino acid sequence shown in SEQ ID NO.2 and contains one or more amino acid mutations selected from the following sites: T12S, K51E, G92S, S136I, P187G, A204I, and Y230F; and contains one or more amino acid mutations of S31L, W70F, N114T, and V162A.

[0020] In one embodiment of the present application, the alcohol dehydrogenase mutant composition includes an ADHA mutant and an ADHB mutant;

[0021] The ADHA mutant is based on the amino acid sequence shown in SEQ ID NO.1, and joint mutations of K7A, G19T, V35L, E48T, H62R, N90T, Q127S, P150Y, C173A, K200E, G219A, and D237R are performed, named ADHA_M, and the amino acid sequence of ADHA_M is shown in SEQ ID NO.3;

[0022] The ADHB mutant is based on the amino acid sequence shown in SEQ ID NO.2, and joint mutations of T12S, S31L, K51E, W70F, G92S, N114T, S136I, V162A, P187G, A204I, and Y230F are performed, named ADHB_M, and the amino acid sequence of ADHB_M is shown in SEQ ID NO.4.

[0023] In one embodiment of the present application, the alcohol dehydrogenase ADHA is derived from Gemmatimonadota bacterium.

[0024] In one embodiment of the present application, the alcohol dehydrogenase ADHB is derived from Scopulibacillus cellulosilyticus.

[0025] In one embodiment of the present application, ADHA_K7A means that the 7th lysine of SEQ ID NO.1 is mutated to alanine.

[0026] In one embodiment of the present application, ADHA_G19T means that the 19th glycine of SEQ ID NO.1 is mutated to threonine.

[0027] In one embodiment of the present application, ADHA_V35L means that the 35th valine of SEQ ID NO.1 is mutated to leucine.

[0028] In one embodiment of the present application, ADHA_E48T means that the 48th glutamate of SEQ ID NO.1 is mutated to threonine.

[0029] In one embodiment of the present application, in ADHA_H62R, the histidine at position 62 of SEQ ID NO.1 is mutated to arginine.

[0030] In one embodiment of the present application, in ADHA_N90T, the aspartic acid at position 90 of SEQ ID NO.1 is mutated to threonine.

[0031] In one embodiment of the present application, in ADHA_Q127S, the glutamine at position 127 of SEQ ID NO.1 is mutated to serine.

[0032] In one embodiment of the present application, in ADHA_P150Y, the proline at position 150 of SEQ ID NO.1 is mutated to tyrosine.

[0033] In one embodiment of the present application, in ADHA_C173A, the cysteine at position 173 of SEQ ID NO.1 is mutated to alanine.

[0034] In one embodiment of the present application, in ADHA_K200E, the lysine at position 200 of SEQ ID NO.1 is mutated to glutamic acid.

[0035] In one embodiment of the present application, in ADHA_G219A, the glycine at position 219 of SEQ ID NO.1 is mutated to alanine.

[0036] In one embodiment of the present application, in ADHA_D237R, the aspartic acid at position 237 of SEQ ID NO.1 is mutated to arginine.

[0037] In one embodiment of the present application, in ADHB_T12S, the threonine at position 12 of SEQ ID NO.2 is mutated to serine.

[0038] In one embodiment of the present application, in ADHB_S31L, the serine at position 31 of SEQ ID NO.2 is mutated to leucine.

[0039] In one embodiment of the present application, in ADHB_K51E, the lysine at position 51 of SEQ ID NO.2 is mutated to glutamic acid.

[0040] In one embodiment of the present application, in ADHB_W70F, the tryptophan at position 70 of SEQ ID NO.2 is mutated to phenylalanine.

[0041] In one embodiment of the present application, in ADHB_G92S, the glycine at position 92 of SEQ ID NO.2 is mutated to serine.

[0042] In one embodiment of the present application, asparagine at position 114 of ADHB_N114T, i.e., SEQ ID NO.2, is mutated to threonine.

[0043] In one embodiment of the present application, serine at position 136 of ADHB_S136I, i.e., SEQ ID NO.2, is mutated to isoleucine.

[0044] In one embodiment of the present application, valine at position 162 of ADHB_V162A, i.e., SEQ ID NO.2, is mutated to alanine.

[0045] In one embodiment of the present application, proline at position 187 of ADHB_P187G, i.e., SEQ ID NO.2, is mutated to glycine.

[0046] In one embodiment of the present application, alanine at position 204 of ADHB_A204I, i.e., SEQ ID NO.2, is mutated to isoleucine.

[0047] In one embodiment of the present application, tyrosine at position 230 of ADHB_Y230F, i.e., SEQ ID NO.2, is mutated to phenylalanine.

[0048] In one embodiment of the present application, lysine at position 7 of ADHA_M, i.e., SEQ ID NO.1, is mutated to alanine, glycine at position 19 is mutated to threonine, valine at position 35 is mutated to leucine, glutamate at position 48 is mutated to threonine, histidine at position 62 is mutated to arginine, aspartate at position 90 is mutated to threonine, glutamine at position 127 is mutated to serine, proline at position 150 is mutated to tyrosine, cysteine at position 173 is mutated to alanine, lysine at position 200 is mutated to glutamate, glycine at position 219 is mutated to alanine, and aspartate at position 237 is mutated to arginine.

[0049] In one embodiment of the present application, threonine at position 12 of ADHB_M, i.e., SEQ ID NO.2, is mutated to serine, serine at position 31 is mutated to leucine, lysine at position 51 is mutated to glutamate, tryptophan at position 70 is mutated to phenylalanine, glycine at position 92 is mutated to serine, asparagine at position 114 is mutated to threonine, serine at position 136 is mutated to isoleucine, valine at position 162 is mutated to alanine, proline at position 187 is mutated to glycine, alanine at position 204 is mutated to isoleucine, and tyrosine at position 230 is mutated to phenylalanine.

[0050] In one embodiment of the present application, fusion proteins obtained by connecting tags to the protein termini defined by the ADHA mutant and the ADHB mutant are also within the protection scope of the present application.

[0051] In a second aspect, the present application provides a group of genes encoding an alcohol dehydrogenase mutant composition.

[0052] In one embodiment of the present application, the present application provides a group of genes encoding ADHA_M and ADHB_M.

[0053] In one embodiment of the present application, the nucleotide sequence of the gene encoding ADHA_M is as shown in SEQ ID NO.7.

[0054] In one embodiment of the present application, the nucleotide sequence of the gene encoding ADHB_M is as shown in SEQ ID NO.8.

[0055] In a third aspect, the present application provides a recombinant plasmid containing the said gene.

[0056] In one embodiment of the present application, the plasmid vector can be selected from pRSFDuet-1.

[0057] In a fourth aspect, the present application provides a recombinant engineered bacterium containing an alcohol dehydrogenase mutant composition.

[0058] In one embodiment of the present application, the present application provides a recombinant engineered bacterium containing an ADHA mutant and an ADHB mutant.

[0059] In one embodiment of the present application, the present application provides a recombinant engineered bacterium containing ADHA_M and ADHB_M.

[0060] In one embodiment of the present application, the recombinant engineered bacterium can select Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris or Corynebacterium glutamicum as the host bacterium.

[0061] In a fifth aspect, the present application provides the use of the alcohol dehydrogenase mutant composition in the one-pot biocatalytic synthesis of (3R,5S)-tert-butyl 6-chloro-3,5-dihydroxyhexanoate from tert-butyl 6-chloro-3,5-dioxohexanoate.

[0062] In one embodiment of the present application, in the presence of a hydrogen donor, using ADHA_M and ADHB_M as catalysts, the substrate tert-butyl 6-chloro-3,5-dioxohexanoate is catalytically reacted to synthesize (3R,5S)-tert-butyl 6-chloro-3,5-dihydroxyhexanoate.

[0063] In one embodiment of the present application, the synthesis route is as follows:

[0064]

[0065] In one embodiment of the present application, the hydrogen donor is isopropanol.

[0066] In one embodiment of the present application, the catalyst is derived from the induced expression enzyme product of a recombinant engineered bacterium.

[0067] In one embodiment of the present application, the obtaining of the induced expression product of the recombinant engineered bacterium includes the following steps:

[0068] The recombinant engineered bacterium is activated, transferred to an induction medium, an inducer is added for induction culture, centrifuged and the cells are collected, and the cell mass is resuspended in a buffer solution.

[0069] In one embodiment of the present application, the final concentration of the added inducer is 0.02 - 1 g / L, and the induction time is 4 - 50 h.

[0070] In one embodiment of the present application, the concentration of tert-butyl 6-chloro-3,5-dioxohexanoate is 1 - 10000 g / L.

[0071] In one embodiment of the present application, the addition amount of the induced expression enzyme product is 1 - 10000 mU / L.

[0072] In one embodiment of the present application, the addition amount of isopropanol is 0 - 10 eq.

[0073] In one embodiment of the present application, the addition amount of isopropanol can be selected from 2.5 eq.

[0074] In one embodiment of the present application, the pH value of the reaction is 5.0 - 7.5.

[0075] In one embodiment of the present application, the pH value of the reaction can be selected from 5.5.

[0076] In one embodiment of the present application, the reaction temperature is 30 - 40 °C.

[0077] In one embodiment of the present application, the reaction temperature can be selected from 35 °C.

[0078] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0079] (1) By constructing a dual-enzyme system in the present application, it can be heterologously expressed in a high-density fermentation host bacterium, and (3R,5S)-tert-butyl 6-chloro-3,5-dihydroxyhexanoate can be synthesized by one-pot whole-cell biocatalysis of tert-butyl 6-chloro-3,5-dioxohexanoate, realizing continuous catalysis, effectively solving the problem of unstable substrate raw materials, improving production efficiency, reducing substrate loss, simplifying the production process, and promoting the application in the biotechnology industry.

[0080] (2) The alcohol dehydrogenase mutant composition provided by the present application can efficiently catalyze the synthesis of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate in a short time, and the biocatalytic method is green, environmentally friendly and pollution-free, and is more suitable for green industrial processing and production.

[0081] (3) The alcohol dehydrogenase mutant composition provided by the present application has high catalytic efficiency, retains higher catalytic activity in a slightly acidic environment with a pH of 5.5, and has a longer half-life. Detailed implementation manners

[0082] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments.

[0083] The "range" disclosed in the present application is defined in the form of a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of a specific range. The ranges defined in this way can include the end values or not include the end values, and can be combined arbitrarily, that is, any lower limit can be combined with any upper limit to form a range.

[0084] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between a liquid and a solid, the percentage is volume / weight percentage; when the present invention relates to the percentage between a solid and a liquid, the percentage is weight / volume percentage; the rest are weight / weight percentages.

[0085] The present invention will be further described below in conjunction with specific embodiments. For the molecular biology experimental methods not specifically described in the following embodiments, reference can be made to the methods listed in "Molecular Cloning: A Laboratory Manual" (Third Edition) by J. Sambrook or the conventional methods in the art, or carried out according to the kits and product instructions.

[0086] The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0087] The test materials used in the following embodiments are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0088] Due to the particularity of the amino acid sequence, any protein having more than 90% identity with the amino acid sequence shown in the present invention and having the same function belongs to the protection scope of the present invention.

[0089] Due to the particularity of the nucleotide sequence, any nucleotide having more than 90% identity with the nucleotide sequence shown in the present invention and having the same function belongs to the protection scope of the present invention.

[0090] Example 1 Preparation of Recombinant Bacteria Expressing Alcohol Dehydrogenase ADHA and Alcohol Dehydrogenase ADHB and Their Mutants

[0091] Select the dual-gene expression vector pRSFDuet-1 (disclosed in Han Guangwei. Co-expression and Immunogenicity Study of Clostridium perfringens α, β_1, β_2, ε Toxin Proteins [D]. Chinese Academy of Agricultural Sciences, 2014). Insert the nucleotide sequence of the alcohol dehydrogenase ADHA-encoding gene or the nucleotide sequence of the mutant-encoding gene derived from Gemmatimonadota bacterium at the NdeI / XhoI site of the vector, and insert the nucleotide sequence of the alcohol dehydrogenase ADHB-encoding gene or the nucleotide sequence of the mutant-encoding gene derived from Scopulibacillus cellulosilyticus at the NcoI / EcoRI site. When the inserted sequences are the nucleotide sequence of the alcohol dehydrogenase ADHA-encoding gene and the nucleotide sequence of the alcohol dehydrogenase ADHB-encoding gene, the recombinant expression plasmid pRSFDuet-1-ADHA-ADHB is obtained. The recombinant plasmid is introduced into Escherichia coli BL21(DE3) for induction and expression (when the OD 600 of the fermentation broth reaches 0.6, an inducer is added for induction expression, and the final concentration of the added inducer is 0.02 - 1 g / L, and the induction time is 4 - 50 h), and BL21-pRSFDuet-1-ADHA-ADHB is obtained. The amino acid sequence of alcohol dehydrogenase ADHA is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.5. The amino acid sequence of alcohol dehydrogenase ADHB is shown in SEQ ID NO.2, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.6.

[0092] ADHA mutants include the following single-point mutations: ADHA_K7A, ADHA_G19T, ADHA_V35L, ADHA_E48T, ADHA_H62R, ADHA_N90T, ADHA_Q127S, ADHA_P150Y, ADHA_C173A, ADHA_K200E, ADHA_G219A, ADHA_D237R.

[0093] Using the plasmid or the E. coli BL21(DE3) genome as a template, PCR amplification is carried out using the 2×PhantaR Max Master Mix kit from Nanjing Novoprotein Biotechnology Co., Ltd. The reaction process is: 95°C, 1 min; 68°C, 2 min; 72°C, 2 min, with 30 cycles. The PCR reaction system is shown in Table 1.

[0094] Table 1. PCR System

[0095]

[0096] The plasmid vector was digested overnight at 30 °C using restriction enzymes from TaKaRa. The CloneEZR recombinant cloning kit from Nanjing GenScript Biotech Co., Ltd. was used to ligate the linearized plasmid and the target gene fragment. The ligation system is shown in Table 2, and the ligation conditions were 22 °C for 30 min and 4 °C for 5 min.

[0097] Table 2. Ligation system

[0098]

[0099] The ligation solution was transferred into E. coli BL21(DE3) competent cells. Single colonies were picked from a plate containing kanamycin (50 mg / L) and inoculated into LB medium containing the same concentration of kanamycin, and cultured overnight at 37 °C and 200 rpm.

[0100] PCR amplification of the site-directed mutant encoding gene of ADHA: Using PCR amplification technology, rapid mutagenesis was performed with the unmutated strain pRSFDuet-1-ADHA-ADHB as the template DNA:

[0101] The primers for site-directed mutagenesis of K7A were:

[0102] Forward primer: 5'-CgctAGCGCGGTGATTACCGGCGGCGCGCTGG-3';

[0103] Reverse primer: 5'-TAATCACCGCGCTagcGCCGCGCATGCGGTTCAT-3'.

[0104] The primers for site-directed mutagenesis of G19T were:

[0105] Forward primer: 5'-GGGCATTactCGCGCGTGCGCGCTGAAATTTG-3';

[0106] Reverse primer: 5'-ACGCGCGagtAATGCCCAGCGCGCCGCCGGTA-3'.

[0107] The primers for site-directed mutagenesis of V35L were:

[0108] Forward primer: 5'-CGcttACCGATGTGGATGTGGATAAAGGCAGC-3';

[0109] Reverse primer: 5'-ATCCACATCGGTaagCGCCACCGCCGCGCCTTC-3'.

[0110] The primers for site-directed mutagenesis of E48T are as follows:

[0111] Forward primer: 5'-TGTGGCGaccGAAATTCGCGATCGCGGCGGCG-3';

[0112] Reverse primer: 5'-GAATTTCggtCGCCACAATGCTGCCTTTATCC-3'.

[0113] The primers for site-directed mutagenesis of H62R are as follows:

[0114] Forward primer: 5'-TGTGGAAagaGATGTGGCGGATGAAGCGGGCT-3';

[0115] Reverse primer: 5'-CCACATCtctTTCCACAAACACCGCATCGCCG-3'.

[0116] The primers for site-directed mutagenesis of N90T are as follows:

[0117] Forward primer: 5'-TGTGCTGGTGAACacaGCGGGCGTGGCGCTGAGC-3';

[0118] Reverse primer: 5'-CtgtGTTCACCAGCACATCCAGTTTTTTATAG-3'.

[0119] The primers for site-directed mutagenesis of Q127S are as follows:

[0120] Forward primer: 5'-TGCGATTtctACCATGAAAACCAACGGCAGCG-3';

[0121] Reverse primer: 5'-TCATGGTagaAATCGCATGTTTGGTGCCCAGA-3'.

[0122] The primers for site-directed mutagenesis of P150Y are as follows:

[0123] Forward primer: 5'-GGGCGATtatAACCTGGCGGCGTATAACGCGA-3';

[0124] Reverse primer: 5'-CCAGGTTataATCGCCCACCAGGCCTTCAATG-3'.

[0125] The primers for site-directed mutagenesis of C173A are as follows:

[0126] Forward primer: 5'-GCTGTATgctGCGAAAGCGGGCTATAACATTC-3';

[0127] Reverse primer: 5'-CTTTCGCagcATACAGCGCCGCGCTTTTGGTC-3'.

[0128] The primers for site-directed mutagenesis of K200E are:

[0129] Forward primer: 5'-CTTTCTGgaaAGCCAGGGCGATGTGGCGCAGG-3';

[0130] Reverse primer: 5'-CCTGGCTttcCAGAAAGTTTTCCACCATCGGG-3'.

[0131] The primers for site-directed mutagenesis of G219A are:

[0132] Forward primer: 5'-ATCCGATTgccCATGTGGGCGAACCGGATGAT-3';

[0133] Reverse primer: 5'-CACATGggcAATCGGATGCAGGCTATCCAGCA-3'.

[0134] The primers for site-directed mutagenesis of D237R are:

[0135] Forward primer: 5'-CGAGCaggGAAAGCAAATTTGTGACCGGCACC-3';

[0136] Reverse primer: 5'-TTTGCTTTCcctGCTCGCCAGATACAGCACGC-3'.

[0137] The ADHA_K7A / G19T / V35L / E48T / H62R / N90T / Q127S / P150Y / C173A / K200E / G219A / D237R protein mutant includes all the above mutations and is named ADHA_M. Its amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence of the nucleic acid molecule encoding the mutant protein is shown in SEQ ID NO.7.

[0138] The ADHB mutants include the following single-point mutations: ADHB_T12S, ADHB_S31L, ADHB_K51E, ADHB_W70F, ADHB_G92S, ADHB_N114T, ADHB_S136I, ADHB_V162A, ADHB_P187G, ADHB_A204I, ADHB_Y230F.

[0139] PCR Amplification of the Gene Encoding Site-Directed Mutants of ADHB: Using the PCR amplification technique, the unmutated strain pRSFDuet-1-ADHA-ADHB was used as the template DNA for rapid mutagenesis:

[0140] The primers for the T12S site-directed mutation were:

[0141] Forward primer: 5'-TGAGCCTGATTagtGGCGGCGCGAGCGGCATT-3';

[0142] Reverse primer: 5'-GCCactAATCAGGCTCACTTTGCTATCCAGGC-3'.

[0143] The primers for the S31L site-directed mutation were:

[0144] Forward primer: 5'-GAAGGCcttAAAATTGCGATTACCGATATTAACA-3';

[0145] Reverse primer: 5'-GCAATTTTaagGCCTTCTTTGCTAAACAGCATCG-3'.

[0146] The primers for the K51E site-directed mutation were:

[0147] Forward primer: 5'-AATTgaaAACAGCGGCGGCGAAGCGATTTTTA-3';

[0148] Reverse primer: 5'-CGCCGCTGTTttcAATTTCATCCACCACGCTCTG-3'.

[0149] The primers for the W70F site-directed mutation were:

[0150] Forward primer: 5'-CCAAAGAAGATGAAtttAAAAAAGCGATTGATATTGTGCAG-3';

[0151] Reverse primer: 5'-aaaTTCATCTTCTTTGGTCACATCATGCTGAA-3'.

[0152] The primers for the G92S site-directed mutation were:

[0153] Forward primer: 5'-ACAACGCGagtATTGGCCTGGCGGCGAACGTG-3';

[0154] Reverse primer: 5'-GCCAATactCGCGTTGTTCACCAGCACATCCA-3'.

[0155] The primers for the N114T site-directed mutation are as follows:

[0156] Forward primer: 5'-GAGCATTactCTGGATGGGCGTGTTTCTGGGC-3';

[0157] Reverse primer: 5'-CATCCAGagtAATGCTCAGCACTTTGCGCCAC-3'.

[0158] The primers for the S136I site-directed mutation are as follows:

[0159] Forward primer: 5'-CCAGAGCGGattCATTATTAACATGAGCAGCATTGAAG-3';

[0160] Reverse primer: 5'-TAATGaatCCGCTCTGGTTGTTTTTCATCGCC-3'.

[0161] The primers for the V162A site-directed mutation are as follows:

[0162] Forward primer: 5'-GccGCGCATTCTGACCAAAAGCGCGGCGCTGC-3';

[0163] Reverse primer: 5'-TGGTCAGAATGCGCggcCCGCCTTTGCTCGCGTTA-3'.

[0164] The primers for the P187G site-directed mutation are as follows:

[0165] Forward primer: 5'-GTGCAggtGGGCTATATTAAAACCCCGATGGT-3';

[0166] Reverse primer: 5'-ATATAGCCCaccTGCACGCTGTTCACGCGCAC-3'.

[0167] The primers for the A204I site-directed mutation are as follows:

[0168] Forward primer: 5'-AAAGCAAatcGGTGGTGAAATATCTGGAAAGCC-3';

[0169] Reverse primer: 5'-CACCACCgatTTGCTTTTTTCATCATCTTTTTCC-3'.

[0170] The primers for the Y230F site-directed mutation are as follows:

[0171] Forward primer: 5'-TATGGCGCGCTttcTCTGGCGAGCGATGAAAGC-3';

[0172] Reverse primer: 5'-AGAgaaAGCGCGCCATACGCAATATCAATCGG-3'.

[0173] The ADHB_T12S / S31L / K51E / W70F / G92S / N114T / S136I / V162A / P187G / A204I / Y230F protein mutant includes all of the above mutations and is named ADHB_M. Its amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence of the nucleic acid molecule encoding the mutant protein is shown in SEQ ID NO.8.

[0174] Example 2 Obtaining of Whole-Cell Fermentation Products of Alcohol Dehydrogenase ADHA, Alcohol Dehydrogenase ADHB and Their Mutants

[0175] The recombinant expression strains containing alcohol dehydrogenase ADHA, alcohol dehydrogenase ADHB and their mutants prepared in Example 1 were respectively spread on LB solid plates (10 g / L NaCl, 5 g / L yeast extract, 10 g / L peptone, 20 g / L agar) containing 50 μg / L kanamycin and incubated in a 37°C incubator for 12 h. The next day, single colonies were picked from the plates into shake tubes containing 5 mL of LB liquid medium (containing 50 μg / L kanamycin) and cultured in a shaker at 37°C and 200 rpm for 12 h as seed solutions. The seed solutions were transferred to 100 mL of TB medium (25 g / L yeast extract, 15 g / L peptone, 10 g / L NaCl, 2 g / L glucose, 0.5 g / L lactose, containing 50 μg / L kanamycin) at an inoculation amount of 1% (v:v). They were placed in a shaker at 37°C and 200 rpm for oscillatory culture. After 2 h, the temperature was adjusted to 25°C and the culture continued for 20 - 22 h.

[0176] The fermentation broth was collected and centrifuged by freezing (4°C, 7000 rpm, 6 min), and resuspended with buffer (1 g of wet cell pellet was dissolved in 5 mL of buffer) as the whole-cell product, which was stored at 4°C in a refrigerator for later use.

[0177] Example 3 Enzyme Activity Assay of Alcohol Dehydrogenase ADHA, Alcohol Dehydrogenase ADHB and Their Mutants

[0178] The method for determining the enzyme activity of alcohol dehydrogenase ADHA and its mutants is as follows: Add 0.1 g of CDOH, 2 eq of isopropanol, and 0.5 mg of crude enzyme to a 1.5 mL enzyme-catalyzed reaction system, and then supplement it with 100 mM sodium carbonate buffer. The reaction conditions are 30 °C and 200 rpm; the sampling times are 0 min, 20 min, and 30 min. Sample treatment: Extract with ethyl acetate, and then dilute the extract 100 times for GC detection. Enzyme activity definition (U): The amount of enzyme required to convert 1 μmol of (s)-CHOH in 1 minute is defined as 1 enzyme activity unit. Taking the enzyme activity of the wild enzyme as 100%, the relative enzyme activities of other mutant enzymes are obtained.

[0179] The method for determining the enzyme activity of alcohol dehydrogenase ADHB and its mutants is as follows: Add 0.1 g of (s)-CHOH, 2 eq of isopropanol, and 0.5 mg of crude enzyme to a 1.5 mL enzyme-catalyzed reaction system, and then supplement it with 100 mM sodium carbonate buffer. The reaction conditions are 30 °C and 200 rpm; the sampling times are 0 min, 20 min, and 30 min. Sample treatment: Extract with ethyl acetate, and then dilute the extract 100 times for GC detection. Enzyme activity definition (U): The amount of enzyme required to convert 1 μmol of (3R,5S)-CDHH in 1 minute is defined as 1 enzyme activity unit. Taking the enzyme activity of the wild enzyme as 100%, the relative enzyme activities of other mutant enzymes are obtained.

[0180] Example 4 Comparison of the catalytic activities of alcohol dehydrogenase ADHA, alcohol dehydrogenase ADHB and their mutants

[0181] By comparing the changes in the enzyme activities of alcohol dehydrogenase ADHA, alcohol dehydrogenase ADHB and their mutants, the results show that the catalytic activity of the alcohol dehydrogenase ADHA mutant (ADHA_M) is 58.9 times that of ADHA, and the catalytic activity of the alcohol dehydrogenase ADHB mutant (ADHB_M) is 43.89 times that of ADHB. The effect of enzyme modification is significant, greatly improving the catalytic efficiency of alcohol dehydrogenase ADHA and alcohol dehydrogenase ADHB.

[0182] Table 3. Relative enzyme activities of alcohol dehydrogenase ADHA and its mutants

[0183]

[0184] Table 4. Relative enzyme activities of alcohol dehydrogenase ADHB and its mutants

[0185]

[0186] Example 5 Catalytic activities and half-life times of alcohol dehydrogenase ADHA, alcohol dehydrogenase ADHB and their mutants under slightly acidic conditions

[0187] The detection methods for catalytic activity and half-life under slightly acidic conditions are as follows: The wild-type enzyme and mutant enzymes were incubated for different times under pH 5.5 buffer conditions, and the enzyme activity was measured. The half-life curve was plotted and the half-life time was obtained. The enzyme activity detection protocol was the same as above. Enzyme activity detection showed that the multi-site mutant ADHA_M not only had an enzyme activity 8201% that of the wild-type enzyme under pH 5.5 buffer conditions, but also had an extended half-life of 129.4 h. Its ability to catalyze the substrate under pH 5.5 buffer conditions was far superior to that of the wild-type enzyme. The specific data are shown in Table 5. Similarly, the mutant ADHB_M not only had an enzyme activity 7361% that of the wild-type enzyme under pH 5.5 buffer conditions, but also had an extended half-life of 225.7 h. Its ability to catalyze the substrate under pH 5.5 buffer conditions was far superior to that of the wild-type enzyme. The specific data are shown in Table 6.

[0188] Table 5. Relative enzyme activity and half-life of alcohol dehydrogenase ADHA and its mutants under pH 5.5 buffer conditions

[0189]

[0190]

[0191] Table 6. Relative enzyme activity and half-life of alcohol dehydrogenase ADHB and its mutants under pH 5.5 buffer conditions

[0192]

[0193] Example 6 Optimizing the reaction temperature of the whole-cell reaction system

[0194] For ADHA and ADHB, although higher enzyme activity can be obtained by increasing the reaction temperature, it is often accompanied by the inactivation of the enzyme protein. Therefore, selecting a suitable reaction temperature to balance the effects between enzyme activity and protein inactivation is also an important part of optimizing the catalytic reaction system. The reaction system was incubated at 25 °C, 30 °C, 35 °C and 40 °C respectively, and other conditions were kept the same, including 1000 g / L CDOH, 2.5 eq isopropanol, 100 mM sodium carbonate buffer and 300 mU / L whole-cell bacterial sludge. After reacting for 12 h, the reaction solution was analyzed by GC. The results showed that the yields were 10.2%, 65.6%, 99.5% and 77.3% under the conditions of 25 °C, 30 °C, 35 °C and 40 °C respectively. Therefore, the catalytic reaction temperature was preferably 35 °C.

[0195] Example 7 Optimizing the reaction pH of the whole-cell reaction system

[0196] For the reaction pH, although increasing the pH can obtain higher enzyme activities of ADHA and ADHB, the substrate undergoes irreversible cyclization, resulting in substrate damage. Therefore, selecting an appropriate reaction pH to balance the utility between enzyme activity and substrate loss is also an important part of optimizing the catalytic reaction system. The reaction system was incubated at pH 4.5, 5, 5.5, 6, 6.5, and 7, respectively, with other conditions remaining the same, including 1000 g / L CDOH, 2.5 eq isopropanol, 100 mM sodium carbonate buffer, and 300 mU / L whole-cell bacterial sludge. After reacting for 12 h at 35 °C, the reaction solution was analyzed by GC. The results showed that the yields at pH 4.5, 5, 5.5, 6, 6.5, and 7 were 0.2%, 43.5%, 99.7%, 65.4%, 53.4%, and 26.7%, respectively. Therefore, the preferred catalytic reaction pH is 5.5.

[0197] Example 8 Optimization of the Isopropanol Addition Amount in the Whole-Cell Reaction System

[0198] For the isopropanol addition amount, increasing the addition amount can increase the reaction rate, but the corresponding cost also increases. Therefore, selecting an appropriate isopropanol addition amount to balance the utility between the reaction rate and production cost. The reaction system was incubated with isopropanol addition amounts of 0.5, 1, 1.5, 2, 2.5, and 3 eq, respectively, with other conditions remaining the same, including 1000 g / L CDOH, a pH 5.5 buffer environment, and 300 mU / L whole-cell bacterial sludge. After reacting for 12 h at 35 °C, the reaction solution was analyzed by GC. The results showed that the yields at isopropanol addition amounts of 0.5, 1, 1.5, 2, 2.5, and 3 eq were 58.3%, 66.4%, 79.2%, 86.3%, 99.5%, and 99.6%, respectively. Therefore, the preferred isopropanol addition amount is 2.5 eq.

[0199] Example 9 Biocatalytic Synthesis of tert-Butyl (3R,5S)-6-Chloro-3,5-dihydroxyhexanoate from tert-Butyl 6-Chloro-3,5-dioxohexanoate

[0200] The one-pot whole-cell biocatalytic synthesis of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate from tert-butyl 6-chloro-3,5-dioxohexanoate was carried out with a reaction system of 3000 g / L CDOH, a pH 5.5 buffer environment, an isopropanol addition amount of 2.5 eq, and 300 mU / L whole-cell bacterial sludge. After reacting for 4, 8, 12, and 16 h at 35 °C, the yields were 64.7%, 99.4%, 99.8%, and 99.9%, respectively. That is, 3000 g / L CDOH can be catalytically converted to synthesize 99.4% of the product after 8 h by 300 mU / L whole-cell bacterial sludge.

[0201] The present application has been described in detail above in connection with specific embodiments and exemplary examples. However, these descriptions should not be construed as limiting the present application. Those skilled in the art understand that various equivalent substitutions, modifications or improvements can be made to the technical solutions and their implementation manners of the present application without departing from the spirit and scope of the present application, and all of these fall within the scope of the present application.

Claims

1. An alcohol dehydrogenase mutant composition, characterized in that: Includes ADHA mutants and ADHB mutants; The ADHA mutant is based on the amino acid sequence shown in SEQ ID NO.1, and comprises one or more amino acid mutations selected from the following sites: K7A, V35L, H62R, Q127S, C173A and G219A; The ADHB mutant is based on the amino acid sequence shown in SEQ ID NO.2, and comprises one or more amino acid mutations selected from the following sites: T12S, K51E, G92S, S136I, P187G, A204I and Y230F.

2. The alcohol dehydrogenase mutant composition according to claim 1, characterized in that: The mutants include ADHA mutants and ADHB mutants; The ADHA mutant is based on the amino acid sequence shown in SEQ ID NO.1, and comprises one or more amino acid mutations selected from the following sites: K7A, V35L, H62R, Q127S, C173A and G219A; and comprises one or more amino acid mutations selected from G19T, E48T, N90T, P150Y, K200E, D237R; The ADHB mutant is based on the amino acid sequence shown in SEQ ID NO.2, and comprises one or more amino acid mutations selected from the following sites: T12S, K51E, G92S, S136I, P187G, A204I and Y230F; and comprises one or more amino acid mutations of S31L, W70F, N114T, V162A.

3. The alcohol dehydrogenase mutant composition according to claim 2, characterized in that: The mutants include ADHA mutants and ADHB mutants; The ADHA mutant is based on the amino acid sequence shown in SEQ ID NO.1, and K7A, G19T, V35L, E48T, H62R, N90T, Q127S, P150Y, C173A, K200E, G219A, and D237R are jointly mutated. The amino acid sequence of the ADHA mutant is shown in SEQ ID NO.3; The ADHB mutant is based on the amino acid sequence shown in SEQ ID NO.2, and T12S, S31L, K51E, W70F, G92S, N114T, S136I, V162A, P187G, A204I, and Y230F are jointly mutated. The amino acid sequence of the ADHB mutant is shown in SEQ ID NO.

4.

4. A gene encoding the alcohol dehydrogenase mutant composition according to any one of claims 1 to 3.

5. A recombinant plasmid comprising the gene according to claim 4.

6. A recombinant engineered bacterium comprising the alcohol dehydrogenase mutant composition according to any one of claims 1 to 3.

7. Use of the alcohol dehydrogenase mutant composition according to claim 1 in one-pot biocatalytic synthesis of (3R,5S)-6-chloro-3,5-dihydroxyhexanoic acid tert-butyl ester from 6-chloro-3,5-dicarbonylhexanoate, characterized in that: In the presence of a hydrogen donor, ADHA mutants and ADHB mutants were used as catalysts to catalyze the reaction of substrate tert-butyl 6-chloro-3,5-dicarbonylhexanoate to synthesize tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate.

8. Use of the alcohol dehydrogenase mutant composition according to claim 7 in one-pot biocatalytic synthesis of (3R,5S)-6-chloro-3,5-dihydroxyhexanoic acid tert-butyl ester from 6-chloro-3,5-dicarbonylhexanoate, characterized in that: The hydrogen donor is isopropanol; the ADHA mutant and the ADHB mutant are derived from the induced expression enzyme products of recombinant engineering bacteria.

9. Use of the alcohol dehydrogenase mutant composition according to claim 8 in one-pot biocatalysis for synthesizing (3R,5S)-6-chloro-3,5-dihydroxyhexanoic acid tert-butyl ester from 6-chloro-3,5-dicarbonylhexanoate, characterized in that: The final concentration of the inducer added in the induced expression is 0.02-1 g / L, and the induced expression time is 4-50 h.

10. Use of the alcohol dehydrogenase mutant composition according to claim 8 in one-pot biocatalysis for synthesizing (3R,5S)-6-chloro-3,5-dihydroxyhexanoic acid tert-butyl ester from 6-chloro-3,5-dicarbonylhexanoate, characterized in that: The concentration of tert-butyl 6-chloro-3,5-dicarbonylhexanoate is 1-10000 g / L, the amount of isopropanol added is 0-10 eq, the amount of the induced expression enzyme product added is 1-10000 mU / L, the pH value of the reaction is 5.0-7.5, and the reaction temperature is 30-40°C.

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