Alcohol dehydrogenase mutant compositions and uses thereof

Abstract

The present application relates to the technical fields of genetic engineering and enzyme engineering, and discloses an alcohol dehydrogenase mutant composition and application thereof, wherein 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. The mutants are used for one-pot whole-cell biocatalysis of 6-chloro-3,5-dicarbonyl hexyl acid tert-butyl ester to synthesize (3R,5S)-6-chloro-3,5-dihydroxy hexyl acid tert-butyl ester, and have high catalytic efficiency, high catalytic activity in a slightly acidic environment and long half-life.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and enzyme engineering, specifically to alcohol dehydrogenase mutant compositions and their applications. Background Technology

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

[0003] Currently, (3R,5S)-CDHH primarily uses either the prochiral ethyl 4-chloro-3-carbonylbutyrate (COBE) (Route 1) or tert-butyl 6-chloro-3,5-dicarbonylhexanoate (CDOH) (Route 2) as the starting substrate. Compared to COBE, Route 2 not only reduces one chemical step, but both reactions can be synthesized via biocatalysis. Its reaction conditions are mild and facilitate direct cascade catalysis or even "one-pot" synthesis without product separation, making it more convenient and efficient. The main challenge of Route 2 lies in the fact that the starting substrate CDOH is a reactive γ-chloro-β,δ-diketone ester compound, sensitive to heat, acids, bases, and heavy metal ions. In aqueous solution, it readily undergoes irreversible cyclization, producing a furanone byproduct. Furthermore, CDOH remains relatively stable in a buffer environment at pH 5.5.

[0004] Route 1:

[0005]

[0006] Route 2:

[0007]

[0008] The commonly used whole-cell method involves using a wild-type strain (Lactobacillus kefir) to synthesize the target product by continuously catalyzing CDOH using two naturally occurring alcohol dehydrogenases. The main drawbacks of this type of wild-type strain whole-cell production include low fermentation cell density, high production costs, high reaction pH leading to substrate damage, low catalytic efficiency of the alcohol dehydrogenases, and low yield of the target product. Summary of the Invention

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

[0010] To address the above problems, the present invention provides the following technical solution:

[0011] In a first aspect, this application provides an alcohol dehydrogenase mutant composition, including 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, and 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, and position 230.

[0014] In one embodiment of this application, this application provides an alcohol dehydrogenase mutant composition, including an ADHA mutant and an ADHB mutant;

[0015] ADHA mutants are based on the amino acid sequence shown in SEQ ID NO.1 and contain 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 one embodiment of this application, the alcohol dehydrogenase mutant composition includes 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 from G19T, E48T, N90T, P150Y, K200E, and 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 from S31L, W70F, N114T and V162A.

[0020] In one embodiment of this 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, with combined mutations in K7A, G19T, V35L, E48T, H62R, N90T, Q127S, P150Y, C173A, K200E, G219A and D237R, named ADHA_M. 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, with combined mutations in T12S, S31L, K51E, W70F, G92S, N114T, S136I, V162A, P187G, A204I, and Y230F, and is named ADHB_M. The amino acid sequence of ADHB_M is shown in SEQ ID NO.4.

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

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

[0025] In one embodiment of this application, the 7th lysine in ADHA_K7A, i.e. SEQ ID NO.1, is mutated to alanine.

[0026] In one embodiment of this application, the 19th glycine in ADHA_G19T, i.e., SEQ ID NO.1, is mutated to threonine.

[0027] In one embodiment of this application, the valine at position 35 of ADHA_V35L, i.e. SEQ ID NO.1, is mutated to leucine.

[0028] In one embodiment of this application, the glutamic acid at position 48 of ADHA_E48T, i.e., SEQ ID NO.1, is mutated to threonine.

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

[0030] In one embodiment of this application, the 90th aspartic acid in ADHA_N90T, i.e., SEQ ID NO.1, is mutated to threonine.

[0031] In one embodiment of this application, ADHA_Q127S, i.e., glutamine at position 127 of SEQ ID NO.1, is mutated to serine.

[0032] In one embodiment of this application, the 150th proline in ADHA_P150Y, i.e., SEQ ID NO.1, is mutated to tyrosine.

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

[0034] In one embodiment of this application, the 200th lysine residue of ADHA_K200E, i.e., SEQ ID NO.1, is mutated to glutamic acid.

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

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

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

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

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

[0040] In one embodiment of this application, the 70th tryptophan in ADHB_W70F, i.e., SEQ ID NO.2, is mutated to phenylalanine.

[0041] In one embodiment of this application, the 92nd glycine in ADHB_G92S, i.e., SEQ ID NO.2, is mutated to serine.

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

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

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

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

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

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

[0048] In one embodiment of this application, ADHA_M, i.e., SEQ ID NO.1, is mutated as follows: lysine at position 7 is mutated to alanine, glycine at position 19 is mutated to threonine, valine at position 35 is mutated to leucine, glutamic acid at position 48 is mutated to threonine, histidine at position 62 is mutated to arginine, aspartic acid 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 glutamic acid, glycine at position 219 is mutated to alanine, and aspartic acid at position 237 is mutated to arginine.

[0049] In one embodiment of this application, ADHB_M, i.e., SEQ ID NO.2, is mutated as follows: threonine at position 12 is mutated to serine, serine at position 31 is mutated to leucine, lysine at position 51 is mutated to glutamic acid, 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 this application, fusion proteins obtained by attaching tags to the protein ends defined by ADHA mutants and ADHB mutants are also within the scope of protection of this application.

[0051] Secondly, this application provides a set of genes encoding compositions of alcohol dehydrogenase mutants.

[0052] In one embodiment of this application, a set of genes encoding ADHA_M and ADHB_M is provided.

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

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

[0055] Thirdly, this application provides a recombinant plasmid containing the said gene.

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

[0057] Fourthly, this application provides a recombinant engineered bacterium comprising an alcohol dehydrogenase mutant composition.

[0058] In one embodiment of this application, a recombinant engineered bacterium comprising ADHA mutant and ADHB mutant is provided.

[0059] In one embodiment of this application, a recombinant engineered bacterium comprising ADHA_M and ADHB_M is provided.

[0060] In one embodiment of this application, the recombinant engineered bacteria may be selected from Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, or Corynebacterium glutamicum as the host bacteria.

[0061] Fifthly, this application provides the use of the alcohol dehydrogenase mutant composition in the one-pot biocatalytic synthesis of tert-butyl 6-chloro-3,5-dicarbonyl hexanoate from (3R,5S)-6-chloro-3,5-dihydroxyhexanoate.

[0062] In one embodiment of this application, in the presence of a hydrogen donor, ADHA_M and ADHB_M are used as catalysts to catalyze the reaction of the substrate tert-butyl 6-chloro-3,5-dicarbonylhexanoate to synthesize (3R,5S)-6-chloro-3,5-dihydroxyhexanoate tert-butyl ester.

[0063] In one embodiment of this application, the synthetic route is as follows:

[0064]

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

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

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

[0068] The recombinant engineered bacteria were activated, transferred to an induction medium, and induced with an inducer. The bacteria were then centrifuged and collected, and the bacterial cells were resuspended in a buffer solution to obtain the final product.

[0069] In one embodiment of this 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 this application, the concentration of tert-butyl 6-chloro-3,5-dicarbonylhexanoate is 1-10000 g / L.

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

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

[0073] In one embodiment of this application, the amount of isopropanol added may be selected from 2.5 eq.

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

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

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

[0077] In one embodiment of this 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) This application constructs a dual-enzyme system that can be heterologously expressed in high-density fermentation host bacteria and synthesize (3R,5S)-6-chloro-3,5-dihydroxyhexanoate tert-butyl ester by one-pot whole-cell biocatalysis of 6-chloro-3,5-dicarbonylhexanoate tert-butyl ester, achieving 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 of biotechnology industry.

[0080] (2) The alcohol dehydrogenase mutant composition provided in this application can catalyze the synthesis of (3R,5S)-6-chloro-3,5-dihydroxyhexanoate tert-butyl ester in a short time and with high efficiency. Moreover, the bio-enzyme catalysis 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 in this application has high catalytic efficiency, retains higher catalytic activity in a slightly acidic environment of pH 5.5, and has a longer half-life. Detailed Implementation

[0082] The technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments.

[0083] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of the particular range. The range defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range.

[0084] Unless otherwise stated, when this invention relates to percentages between liquids, the percentage is volume / volume percentage; when this invention relates to percentages between liquids and solids, the percentage is volume / weight percentage; when this invention relates to percentages between solids and liquids, the percentage is weight / volume percentage; and the remainder is weight / weight percentage.

[0085] The present invention will be further described below with reference to specific embodiments. Molecular biology experimental methods not specifically described in the following embodiments can be performed according to the methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition) or conventional methods in the art, or according to the kit and product instructions.

[0086] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0087] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0088] Due to the unique nature of amino acid sequences, any protein that shares more than 90% identity with the amino acid sequence shown in this invention and has the same function falls within the scope of protection of this invention.

[0089] Due to the special nature of nucleotide sequences, any nucleotide that has more than 90% identity with the nucleotide sequence shown in this invention and has the same function is within the scope of protection of this invention.

[0090] Example 1: Preparation of recombinant bacteria expressing alcohol dehydrogenase ADHA and alcohol dehydrogenase ADHB and their mutants

[0091] 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) was selected. A nucleotide sequence of the alcohol dehydrogenase ADHA encoding gene from Gemmatimonadota bacterium or a mutant encoding gene was inserted into the NdeI / XhoI site of the vector. A nucleotide sequence of the alcohol dehydrogenase ADHB encoding gene from Scopulibacillus cellulosilyticus or a mutant encoding gene was inserted into the NcoI / EcoRI site. When the inserted sequences were both the nucleotide sequences of the alcohol dehydrogenase ADHA and ADHB encoding genes, the recombinant expression plasmid pRSFDuet-1-ADHA-ADHB was obtained. The recombinant plasmid was introduced into Escherichia coli BL21(DE3) for induction and expression (when the fermentation broth OD... 600 When the concentration reached 0.6, an inducer was added to induce expression (the final concentration of the inducer was 0.02-1 g / L, and the induction time was 4-50 h), yielding BL21-pRSFDuet-1-ADHA-ADHB. 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, and ADHA_D237R.

[0093] Using plasmids or E. coli BL21(DE3) genome as templates, PCR amplification was performed using the 2×PhantaR Max Master Mix kit from Nanjing Novizan Biotechnology Co., Ltd. The reaction process was as follows: 95℃, 1 min; 68℃, 2 min; 72℃, 2 min, for a total of 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 endonucleases from TaKaRa. The linearized plasmid and the target gene fragment were ligated using the CloneEZR recombinant cloning kit from Nanjing Genscript Biotech Co., Ltd. The ligation system is shown in Table 2. The ligation conditions were 22°C for 30 min and 4°C for 5 min.

[0097] Table 2. Connection System

[0098]

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

[0100] PCR amplification of the site-directed mutant encoding gene of ADHA: Rapid mutation was performed using unmutated strain pRSFDuet-1-ADHA-ADHB as template DNA via PCR amplification.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[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] 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, and ADHB_Y230F.

[0139] PCR amplification of the site-directed mutant encoding gene of ADHB: Rapid mutation was performed using unmutated strain pRSFDuet-1-ADHA-ADHB as template DNA via PCR amplification.

[0140] The primers for site-directed mutagenesis of T12S are:

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

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

[0143] The primers for site-directed mutagenesis of S31L are:

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

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

[0146] The primers for site-directed mutagenesis of K51E are:

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

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

[0149] The primers for site-directed mutagenesis of W70F are:

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

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

[0152] The primers for site-directed mutagenesis of G92S are:

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

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

[0155] The primers for site-directed mutagenesis of N114T are:

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

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

[0158] The primers for site-directed mutagenesis of S136I are:

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

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

[0161] The primers for site-directed mutagenesis of V162A are:

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

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

[0164] The primers for site-directed mutagenesis of P187G are:

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

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

[0167] The primers for site-directed mutagenesis of A204I are:

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

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

[0170] The primers for site-directed mutagenesis of Y230F are:

[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 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 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 plated onto LB agar plates containing 50 μg / L kanamycin (NaCl 10 g / L, yeast extract 5 g / L, peptone 10 g / L, agar 20 g / L) and incubated at 37°C for 12 h. The next day, single colonies were selected from the plates and transferred to shake tubes containing 5 mL of LB liquid medium (containing 50 μg / L kanamycin) and cultured at 37°C and 200 rpm for 12 h as seed culture. The seed culture was then transferred at a 1% (v:v) inoculation rate to 100 mL of TB medium (yeast extract 25 g / L, peptone 15 g / L, NaCl 10 g / L, glucose 2 g / L, lactose 0.5 g / L, containing 50 μg / L kanamycin). The medium was then incubated at 37°C and 200 rpm with shaking. After 2 hours, adjust the temperature to 25℃ and continue culturing for 20-22 hours.

[0176] Collect the fermentation broth and freeze-centrifuge (4℃, 7000rpm, 6min). Resuspend the broth in a buffer (1g of wet bacterial sludge re-dissolved in 5mL buffer) as the whole cell product and store it at 4℃ 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 was as follows: 0.1 g of CDOH, 2 eq of isopropanol, and 0.5 mg of crude enzyme were added to a 1.5 mL enzyme-catalyzed reaction system, followed by replenishment with 100 mM sodium carbonate buffer. The reaction conditions were 30℃ and 200 rpm; sampling times were 0 min, 20 min, and 30 min. Sample preparation: extraction with ethyl acetate, followed by 100-fold dilution of the extract for GC detection. Enzyme activity (U): The amount of enzyme required to convert 1 μmol of (s)-CHOH within 1 minute is defined as one enzyme activity unit. The relative enzyme activities of other mutant enzymes were calculated using the wild-type enzyme activity as 100%.

[0179] The method for determining the enzyme activity of alcohol dehydrogenase ADHB and its mutants was as follows: 0.1 g of (s)-CHOH, 2 eq of isopropanol, and 0.5 mg of crude enzyme were added to a 1.5 mL enzyme-catalyzed reaction system, followed by replenishment with 100 mM sodium carbonate buffer. The reaction conditions were 30℃ and 200 rpm; sampling times were 0 min, 20 min, and 30 min. Sample preparation: extraction with ethyl acetate, followed by 100-fold dilution of the extract for GC detection. Enzyme activity (U): The amount of enzyme required to convert 1 μmol of (3R,5S)-CDHH within 1 minute is defined as one enzyme activity unit. The relative enzyme activities of other mutant enzymes were calculated using the wild-type enzyme activity as 100%.

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

[0181] Comparison of enzyme activity changes of alcohol dehydrogenase ADHA, alcohol dehydrogenase ADHB and their mutants showed that the catalytic activity of the alcohol dehydrogenase ADHA mutant (ADHA_M) was 58.9 times that of ADHA, and the catalytic activity of the alcohol dehydrogenase ADHB mutant (ADHB_M) was 43.89 times that of ADHB. The enzyme modification effect was 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 activity and half-life of alcohol dehydrogenases ADHA, ADHB, and their mutants under slightly acidic conditions.

[0187] The catalytic activity and half-life were determined under slightly acidic conditions as follows: Wild-type and mutant enzymes were incubated for different times at a pH 5.5 buffer, and enzyme activity was measured. Half-life curves were plotted, and the half-life time was calculated. The enzyme activity assay was performed using the same method as above. Enzyme activity assays showed that the multi-point mutant ADHA_M not only exhibited 8201% of the wild-type enzyme activity at a pH 5.5 buffer, but also had a half-life extended by 129.4 h. Its ability to catalyze substrates under pH 5.5 buffer conditions was significantly superior to that of the wild-type enzyme. Specific data are shown in Table 5. Similarly, the mutant ADHB_M not only exhibited 7361% of the wild-type enzyme activity at a pH 5.5 buffer, but also had a half-life extended by 225.7 h. Its ability to catalyze substrates under pH 5.5 buffer conditions was significantly superior to that of the wild-type enzyme. 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: Optimization of the reaction temperature of the whole-cell reaction system

[0194] For ADHA and ADHB, while increasing the reaction temperature can yield higher enzyme activity, it often comes at the cost of enzyme protein inactivation. Therefore, selecting a suitable reaction temperature to balance the effects of enzyme activity and protein inactivation is a crucial step in optimizing the catalytic reaction system. The reaction system was incubated at 25℃, 30℃, 35℃, and 40℃, with other conditions kept constant, including 1000 g / L CDOH, 2.5 eq isopropanol, 100 mM sodium carbonate buffer, and 300 mU / L whole-cell bacterial sludge. The reaction solution was analyzed by GC after 12 h of reaction. The results showed that the yields at 25℃, 30℃, 35℃, and 40℃ were 10.2%, 65.6%, 99.5%, and 77.3%, respectively. Therefore, the optimal catalytic reaction temperature is 35℃.

[0195] Example 7: Optimization of the reaction pH in the whole-cell reaction system

[0196] While increasing the pH can yield higher enzyme activities for ADHA and ADHB, it also leads to irreversible substrate cyclization, resulting in substrate damage. Therefore, selecting a suitable pH to balance enzyme activity and substrate loss is crucial for optimizing the catalytic reaction system. The reaction system was incubated at pH 4.5, 5, 5.5, 6, 6.5, and 7, with other conditions remaining constant, including 1000 g / L CDOH, 2.5 eq isopropanol, 100 mM sodium carbonate buffer, and 300 mU / L whole-cell bacterial sludge. The reaction solution was analyzed by GC after 12 h at 35°C. 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, pH 5.5 is the optimal pH for the catalytic reaction.

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

[0198] Increasing the amount of isopropanol can improve the reaction rate, but it also increases the cost. Therefore, a suitable amount of isopropanol needs to be selected to balance the efficiency between reaction rate and production cost. The reaction system was incubated with isopropanol at amounts of 0.5, 1, 1.5, 2, 2.5, and 3 eq, respectively, while keeping other conditions the same, including 1000 g / L CDOH, a pH 5.5 buffer environment, and 300 mU / L whole-cell bacterial sludge. The reaction solution was analyzed by GC after reacting at 35°C for 12 h. The results showed that the yields at isopropanol additions 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 optimal isopropanol addition amount is 2.5 eq.

[0199] Example 9: Biocatalytic synthesis of tert-butyl 6-chloro-3,5-dicarbonyl hexanoate (3R,5S)-6-chloro-3,5-dihydroxyhexanoate.

[0200] A one-pot whole-cell biocatalytic synthesis of (3R,5S)-6-chloro-3,5-dihydroxyhexanoate tert-butyl ester was achieved. The reaction system used 3000 g / L CDOH, a pH 5.5 buffer environment, 2.5 eq isopropanol, and 300 mU / L whole-cell bacterial sludge. After reaction at 35 °C for 4, 8, 12, and 16 h, the yields were 64.7%, 99.4%, 99.8%, and 99.9%, respectively. This means that 3000 g / L CDOH, catalyzed by 300 mU / L whole-cell bacterial sludge, can synthesize 99.4% of the product after 8 h.

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

Claims

1. An alcohol dehydrogenase mutant composition, 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, with combined mutations in K7A, G19T, V35L, E48T, H62R, N90T, Q127S, P150Y, C173A, K200E, G219A, and D237R. 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, with combined mutations in T12S, S31L, K51E, W70F, G92S, N114T, S136I, V162A, P187G, A204I, and Y230F. The amino acid sequence of the ADHB mutant is shown in SEQ ID NO.

4.

2. The gene encoding the alcohol dehydrogenase mutant composition as described in claim 1.

3. A recombinant plasmid comprising the gene as described in claim 2.

4. A recombinant engineered bacterium comprising the alcohol dehydrogenase mutant composition as described in claim 1.

5. The alcohol dehydrogenase mutant composition of claim 1 is used for the one-pot biocatalytic synthesis of tert-butyl 6-chloro-3,5-dicarbonylhexanoate (3 R 5 S Its application in tert-butyl 6-chloro-3,5-dihydroxyhexanoate is characterized by, In the presence of a hydrogen donor, using ADHA and ADHB mutants as catalysts, the substrate tert-butyl 6-chloro-3,5-dicarbonyl hexanoate was catalyzed to synthesize (3) R 5 S 6-chloro-3,5-dihydroxyhexanoate tert-butyl ester.

6. The one-pot biocatalytic synthesis of tert-butyl 6-chloro-3,5-dicarbonylhexanoate from the alcohol dehydrogenase mutant composition according to claim 5 (3 R 5 S Its application in tert-butyl 6-chloro-3,5-dihydroxyhexanoate is characterized by, The hydrogen donor is isopropanol; the ADHA mutant and ADHB mutant are derived from the inducible expression enzyme products of recombinant engineered bacteria.

7. The one-pot biocatalytic synthesis of tert-butyl 6-chloro-3,5-dicarbonylhexanoate from the alcohol dehydrogenase mutant composition according to claim 6 (3 R 5 S Its application in tert-butyl 6-chloro-3,5-dihydroxyhexanoate is characterized by, The final concentration of the inducer added during the induced expression was 0.02-1 g / L, and the induction time was 4-50 h.

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