Carbonyl reductase mutant, coding gene and application of carbonyl reductase mutant in synthesis of 4-tert-butylcyclohexanol

Abstract

The invention discloses a carbonyl reductase mutant, a coding gene and application of the carbonyl reductase mutant in synthesis of 4-tert-butylcyclohexanol, and belongs to the technical field of bioengineering. The carbonyl reductase mutant is obtained by carrying out amino acid mutation on short-chain dehydrogenase from escherichia coli K12 with an amino acid sequence shown as SEQ ID NO.1 or carrying out amino acid mutation on medium-chain dehydrogenase from candida parapsilosis with an amino acid sequence shown as SEQ ID NO.2. Compared with wild type carbonyl reductase, the carbonyl reductase mutant provided by the invention has higher enzyme activity, cis-4-tert-butyl cyclohexanol or trans-4-tert-butyl cyclohexanol can be prepared by taking 4-tert-butyl cyclohexanone as a substrate, the product yield is high, no by-product is generated, and the carbonyl reductase mutant has a very good industrial application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering technology, and particularly relates to a carbonyl reductase mutant, a coding gene and application in synthesis of 4-tert-butylcyclohexanol. BACKGROUND

[0002] 4-tert-butylcyclohexanol (CAS No. 98-52-2) includes two configuration isomers, cis-4-tert-butylcyclohexanol (CAS No. 937-05-3) and trans-4-tert-butylcyclohexanol (CAS No. 21862-63-5). 4-tert-butylcyclohexanol can be used for acetylation to synthesize a commonly used fragrance 4-tert-butylcyclohexyl acetate (commonly known as Iris ester), which has a rich woody and iris flower fragrance and stable product properties, and is widely used in perfume essence, cosmetic essence, soap essence and other daily chemical essence formulations. The fragrance synthesized by 4-tert-butylcyclohexanol is usually a cis / trans isomer mixture, and the presence of the trans isomer can significantly reduce the fragrance quality, and high cis-4-tert-butylcyclohexyl acetate (cis content greater than 90%) can present a richer floral and softer woody characteristics, and the commercial value is significantly improved. Therefore, high cis-4-tert-butylcyclohexanol should be preferentially selected as a synthetic raw material.

[0003] Trans-4-tert-butylcyclohexanol is a highly specific TRPV1 inhibitor in vitro and in vivo, and can relieve the over-activation caused by the increase of TRPV1 expression level or signal intensity in sensitive skin, and is widely used in skin care products, pesticides, oral sprays and scalp care essential oils to relieve skin irritation. As the main core ingredient of SymSitiveTM, it has been commercialized in the medical skin care product OptimEyes soothing cream. With the increasing demand of consumers for sensitive skin care, the market potential of trans-4-tert-butylcyclohexanol will be further expanded.

[0004] Currently, the preparation of 4-tert-butylcyclohexanol mainly has two methods, chemical method and biocatalysis method. The chemical method mainly includes the reduction of 4-tert-butylcyclohexanone, such as using LiAlH4, AlCl3 catalyst to prepare trans-4-tert-butylcyclohexanol (Organic Syntheses, 1973, 5, 175), using proton exchange membrane (PEM) reactor, rhodium as catalyst to prepare cis-4-tert-butylcyclohexanol by electrocatalytic hydrogenation (ACS Energy Letters, 2023, 8: 1010-1017); or the hydrogenation of 4-tert-butylphenol, such as patent document CN115888814A discloses the use of a composite catalyst containing metal elements to prepare trans-4-tert-butylcyclohexanol, and patent document CN117534542A discloses the use of a supported catalyst comprising a carrier, a nickel-based alloy and an alkaline earth metal oxide to prepare cis-4-tert-butylcyclohexanol; or separation from cis / trans isomer mixture, such as using phthalic acid to prepare trans-4-tert-butylcyclohexanol by repeated crystallization, then saponification with sodium hydroxide in water, followed by extraction with pentane (Journal of the American Chemical Society, 1955, 77: 5562-5578). However, the chemical synthesis method has problems such as complicated catalyst synthesis route, harsh conditions (high temperature and high pressure), toxic reagents, etc.

[0005] The biocatalysis method can obtain the corresponding high stereoselectivity product in one step, reduce the use of noble metal or metal-containing composite catalyst, simplify the operation process and reduce the production cost. Carbonyl reductase is divided into three superfamilies according to its characteristics: short-chain dehydrogenase, medium-chain dehydrogenase and aldehyde-ketone reductase, which has high stereoselectivity and wide substrate spectrum. According to the report of Brown et al., using 4-tert-butylcyclohexanone as substrate, through screening Almac'CESK 5000 carbonyl reductase kit, cis-4-tert-butylcyclohexanol synthesis enzyme A161 with complete conversion of 500 g / L 4-tert-butylcyclohexanone in 22 hours, yield ≥98%, and carbonyl reductase N151 with high selectivity to synthesize trans-4-tert-butylcyclohexanol in 24 hours under 65 mM substrate concentration, conversion rate is 95% (Organic Process Research & Development, 2011, 15, 1036-1039). However, the sequence information of this commercial enzyme has not been disclosed, and it is not clear whether the high-trans selectivity carbonyl reductase can maintain excellent performance at higher substrate concentration.

[0006] Therefore, it is a problem to be solved by those skilled in the art to develop a carbonyl reductase with high catalytic activity and high stereoselectivity for the synthesis of cis-4-tert-butylcyclohexanol and trans-4-tert-butylcyclohexanol at high substrate load. SUMMARY

[0007] The present application aims to provide a carbonyl reductase with high catalytic activity and high stereoselectivity for the preparation of cis-4-tert-butylcyclohexanol or trans-4-tert-butylcyclohexanol with high optical purity, meeting the requirements of industrial production.

[0008] To achieve the above-mentioned object, the present application adopts the following technical solutions: The present application obtains several carbonyl reductase mutants with significantly improved catalytic activity and high stereoselectivity by site-directed mutagenesis of the coding gene (SEQ ID NO. 22) of short-chain dehydrogenase UCPA from Escherichia coli K12 and the coding gene (SEQ ID NO. 25) of medium-chain dehydrogenase CpSADH from Candida parapsilosis.

[0009] Therefore, the present application provides a carbonyl reductase mutant obtained by amino acid mutation of short-chain dehydrogenase from Escherichia coli K12 with an amino acid sequence as shown in SEQ ID NO. 1 or obtained by amino acid mutation of medium-chain dehydrogenase from Candida parapsilosis with an amino acid sequence as shown in SEQ ID NO. 2; In the short-chain dehydrogenase, at least one of positions 93, 143, 149, 187, 193, and 212 is mutated, wherein the cysteine at position 93 is mutated to isoleucine; the threonine at position 143 is mutated to alanine or valine; the aspartic acid at position 149 is mutated to valine; the tyrosine at position 187 is mutated to alanine, serine, glycine, or methionine; the alanine at position 193 is mutated to isoleucine; and the methionine at position 212 is mutated to tryptophan. In the medium-chain dehydrogenase, at least one of positions 59 and 77 is mutated, wherein the aspartic acid at position 59 is mutated to histidine or leucine; and the aspartic acid at position 77 is mutated to histidine.

[0010] Specifically, the mutant UCPA-C93I of short-chain dehydrogenase with cysteine at position 93 mutated to isoleucine has an amino acid sequence as shown in SEQ ID NO. 3; The mutant UCPA-T143A of short-chain dehydrogenase with threonine at position 143 mutated to alanine has an amino acid sequence as shown in SEQ ID NO. 4; a mutant of short-chain dehydrogenase with threonine at position 143 mutated to valine, UCPA-T143V, having the amino acid sequence shown in SEQ ID NO. 5; a mutant of short-chain dehydrogenase with aspartic acid at position 149 mutated to valine, UCPA-D149V, having the amino acid sequence shown in SEQ ID NO. 6; a mutant of short-chain dehydrogenase with tyrosine at position 187 mutated to alanine, UCPA-Y187A, having the amino acid sequence shown in SEQ ID NO. 7; a mutant of short-chain dehydrogenase with tyrosine at position 187 mutated to serine, UCPA-Y187S, having the amino acid sequence shown in SEQ ID NO. 8; a mutant of short-chain dehydrogenase with tyrosine at position 187 mutated to glycine, UCPA-Y187G, having the amino acid sequence shown in SEQ ID NO. 9; a mutant of short-chain dehydrogenase with tyrosine at position 187 mutated to methionine, UCPA-Y187M, having the amino acid sequence shown in SEQ ID NO. 10; a mutant of short-chain dehydrogenase with alanine at position 193 mutated to isoleucine, UCPA-A193I, having the amino acid sequence shown in SEQ ID NO. 11; a mutant of short-chain dehydrogenase with methionine at position 212 mutated to tryptophan, UCPA-M212W, having the amino acid sequence shown in SEQ ID NO. 12; a mutant of short-chain dehydrogenase with aspartic acid at position 149 mutated to valine and alanine at position 193 mutated to isoleucine, UCPA-D149V / A193I, having the amino acid sequence shown in SEQ ID NO. 13; a mutant of short-chain dehydrogenase with threonine at position 143 mutated to valine and tyrosine at position 187 mutated to alanine, UCPA-T143V / Y187A, having the amino acid sequence shown in SEQ ID NO. 14; a mutant of short-chain dehydrogenase with threonine at position 143 mutated to valine and methionine at position 212 mutated to tryptophan, UCPA-T143V / M212W, having the amino acid sequence shown in SEQ ID NO. 15; a mutant of short-chain dehydrogenase with threonine at position 143 mutated to valine, cysteine at position 93 mutated to isoleucine, and tyrosine at position 187 mutated to alanine, UCPA-T143V / C93I / Y187A, having the amino acid sequence shown in SEQ ID NO. 16; a mutant of medium-chain dehydrogenase with aspartic acid at position 59 mutated to histidine, CpSADH-D59H, having the amino acid sequence shown in SEQ ID NO. 17; CpSADH-D59L, the amino acid sequence of which is shown as SEQ ID NO. 18, in which the aspartic acid at position 59 of the medium-chain dehydrogenase is mutated into leucine; CpSADH-D77H, the amino acid sequence of which is shown as SEQ ID NO. 19, in which the aspartic acid at position 77 of the medium-chain dehydrogenase is mutated into histidine; CpSADH-D59H / D77H, the amino acid sequence of which is shown as SEQ ID NO. 20, in which the aspartic acid at position 59 of the medium-chain dehydrogenase is mutated into histidine and the aspartic acid at position 77 of the medium-chain dehydrogenase is mutated into histidine; CpSADH-D59L / D77H, the amino acid sequence of which is shown as SEQ ID NO. 21, in which the aspartic acid at position 59 of the medium-chain dehydrogenase is mutated into leucine and the aspartic acid at position 77 of the medium-chain dehydrogenase is mutated into histidine; Studies have shown that the catalytic activity of the above-mentioned carbonyl reductase mutants is significantly improved compared with the wild-type carbonyl reductase. Through amino acid sequence alignment, the above-mentioned carbonyl reductase mutants all have a typical Rossmann fold domain of the dehydrogenase superfamily, which contains a conserved coenzyme binding motif, and constitutes the core structural basis for the carbonyl reductase mutants to bind coenzyme and perform catalytic function.

[0011] Conservative substitution forms, forms with one or more amino acids added or deleted, amino-terminal truncated forms, carboxy-terminal truncated forms of other amino acid positions of the above-mentioned carbonyl reductase mutants are also included in the scope of the present application.

[0012] The present application also provides a coding gene encoding the carbonyl reductase mutant. When the carbonyl reductase mutant is a mutant of short-chain dehydrogenase after amino acid mutation, the coding gene is obtained by mutating the codon encoding the corresponding amino acid based on the nucleotide sequence shown in SEQ ID NO. 22. Specifically, C93I is the codon TGT encoding the 93rd cysteine mutated into the codon ATT encoding isoleucine, T143A is the codon ACT encoding the 143rd threonine mutated into the codon GCG encoding alanine, T143V is the codon ACT encoding the 143rd threonine mutated into the codon GTA encoding valine, D149V is the codon GAT encoding the 149th aspartic acid mutated into the codon GTT encoding valine, Y187A is the codon TAC encoding the 187th tyrosine mutated into the codon GCG encoding alanine, Y187S is the codon TAC encoding the 187th tyrosine mutated into the codon TCC encoding serine, Y187G is the codon TAC encoding the 187th tyrosine mutated into the codon GGC encoding glycine, Y187M is the codon TAC encoding the 187th tyrosine mutated into the codon ATG encoding methionine, A193I is the codon GCG encoding the 193rd alanine mutated into the codon ATC encoding isoleucine, and M212W is the codon ATG encoding the 212th methionine mutated into the codon TGG encoding tryptophan.

[0013] When the carbonyl reductase mutant is a mutant of medium-chain dehydrogenase after amino acid mutation, the coding gene is obtained by mutating the codon encoding the corresponding amino acid based on the nucleotide sequence shown in SEQ ID NO. 25. Specifically, D59H is the codon GAC encoding the 59th aspartic acid mutated into the codon CAC encoding histidine, D59L is the codon GAC encoding the 59th aspartic acid mutated into the codon CTG encoding leucine, and D77H is the codon GAC encoding the 77th aspartic acid mutated into the codon CAT encoding histidine.

[0014] The present application also provides a recombinant expression plasmid containing the coding gene. The recombinant expression plasmid comprises a primary vector and the coding gene encoding the carbonyl reductase mutant inserted at the multiple cloning site of the primary vector.

[0015] When the carbonyl reductase mutant is a mutant of short-chain dehydrogenase after amino acid mutation, the recombinant expression plasmid contains the coding gene of the mutant of short-chain dehydrogenase after amino acid mutation and the glucose-1-dehydrogenase coding gene. Preferably, the primary vector adopts pCDFDuet plasmid.

[0016] When the carbonyl reductase mutant is a mutant of short-chain dehydrogenase after amino acid mutation, the original vector uses pET30a plasmid.

[0017] The application also provides genetically engineered bacteria containing the recombinant expression plasmid. The genetically engineered bacteria are used to produce the carbonyl reductase mutant. The recombinant expression plasmid is obtained by transforming a host cell to obtain genetically engineered bacteria, and the host cell can be various conventional host cells in the art, and as a preferred, the host bacteria of the genetically engineered bacteria use Escherichia coli BL21. E. coli BL21.

[0018] Another object of the application is to provide the use of the carbonyl reductase mutant in the preparation of cis-4-tert-butylcyclohexanol or trans-4-tert-butylcyclohexanol.

[0019] Specifically, when the carbonyl reductase mutant is a mutant of short-chain dehydrogenase after amino acid mutation, the use includes: under the condition of providing coenzyme and / or auxiliary coenzyme regeneration, the mutant catalyzing the reduction of 4-tert-butylcyclohexanone to generate cis-4-tert-butylcyclohexanol or trans-4-tert-butylcyclohexanol; When the carbonyl reductase mutant is a mutant of medium-chain dehydrogenase after amino acid mutation, the use includes: under the condition of providing coenzyme and / or auxiliary coenzyme regeneration and adding Zn 2+ , catalyzing the reduction of 4-tert-butylcyclohexanone to generate cis-4-tert-butylcyclohexanol; The coenzyme can be, but is not limited to, NAD + , NADH, NADP + , NADPH; the auxiliary coenzyme regeneration is adding a co-substrate such as glucose.

[0020] The short-chain dehydrogenase mutant provided by the application catalyzes the reduction of 4-tert-butylcyclohexanone to generate high-optical-purity cis-4-tert-butylcyclohexanol ( de ≥99%) or trans-4-tert-butylcyclohexanol ( de ≥99%) in the presence of coenzyme NAD + and glucose; the medium-chain dehydrogenase mutant catalyzes the reduction of 4-tert-butylcyclohexanone to generate high-optical-purity cis-4-tert-butylcyclohexanol ( de ≥98.2%) in the presence of coenzyme NAD + and Zn 2+ solution, which has a good prospect of industrial application.

[0021] As preferred, the application comprises: using wet bacteria obtained by centrifugation after fermentation culture of genetically engineered bacteria containing the coding gene of the carbonyl reductase mutant, wet bacteria immobilized cells, enzymes extracted after ultrasonic disruption of wet bacteria, or immobilized enzymes as catalyst, using 4-tert-butyl cyclohexanone as substrate, adding coenzyme NAD + , adding glucose or Zn 2+ , using buffer solution containing organic solvent with pH value ranging from 6.0 to 8.0 as reaction medium, oscillating the reaction under the condition of 25 to 45℃ and 150 to 300 rpm, and separating cis-4-tert-butyl cyclohexanol or trans-4-tert-butyl cyclohexanol from the reaction solution after the reaction is completed.

[0022] The carbonyl reductase mutant catalyst of the present application can be used in the form of whole cells of engineered bacteria, in the form of crude enzymes without purification, or in the form of partially purified or completely purified enzymes. The carbonyl reductase mutant of the present application can also be made into a biocatalyst in the form of immobilized enzymes or immobilized cells by using immobilization techniques known in the art.

[0023] As preferred, when the carbonyl reductase mutant is a mutant of short-chain dehydrogenase after amino acid mutation, the initial concentration of substrate in the catalytic reaction system is 50 to 1000 mM; the concentration of coenzyme NAD + is 1 to 3 mM; the concentration of glucose is 1.5 times the concentration of substrate; the organic solvent is isopropyl alcohol or acetonitrile, with a volume fraction of 5 to 15%; the amount of catalyst is 50 to 100 g / L based on the weight of wet bacteria, wherein the water content of wet bacteria is 70 to 90%; and the catalytic reaction time is 4 to 24 h.

[0024] As a specific embodiment of the present application, the wet bacteria are E. coli BL21 / pCDFDuet-GDH-UCPA-D149V / A193I. The catalytic reaction conditions are: the initial concentration of substrate is 50 mM; the concentration of coenzyme NAD + is 2 mM; the concentration of glucose is 75 mM; the buffer solution is phosphate buffer, i.e. NaH2PO4-Na2HPO4 buffer solution, with pH of 7.0; the volume fraction of isopropyl alcohol in the buffer solution is 10%; the catalytic reaction temperature is 30℃, and the reaction time is 6 h. The yield of cis-4-tert-butyl cyclohexanol of the mutant can reach 92.0%, and the optical purity de is ≥99%.

[0025] As another specific embodiment of the present application, the wet bacteria are E. coliBL21 / pCDFDuet-GDH-UCPA-Y187A. The catalytic reaction conditions are as follows: the initial concentration of the substrate is 1 M; the concentration of coenzyme NAD + is 2 mM; the concentration of glucose is 1.5 M; the buffer is phosphate buffer, i.e. NaH2PO4-Na2HPO4 buffer, the pH is 7.0; the volume fraction of acetonitrile in the buffer solution is 10%; the temperature of the catalytic reaction is 40℃, and the reaction time is 10 h. The yield of trans-4-tert-butylcyclohexanol produced by the mutant can reach 99.7%, which is 15.6 times that of the wild-type short-chain dehydrogenase under the same catalytic conditions, and the optical purity de is ≥99%.

[0026] As a preference, when the carbonyl reductase mutant is a mutant of medium-chain dehydrogenase after amino acid mutation, in the catalytic reaction system, the initial concentration of the substrate is 200 ~ 1000 mM; the concentration of coenzyme NAD + is 1 ~ 3 mM; Zn 2+ is added in the form of ZnCl2 solution, the concentration is 0.05 ~ 0.15 mM; the organic solvent is isopropanol, the volume fraction is 2.5 ~ 20%; and the amount of the catalyst is 50 ~ 100 g / L based on the weight of the wet bacteria, wherein the water content of the wet bacteria is 70 ~ 90%.

[0027] As a specific embodiment of the present application, the wet bacteria are E.coli BL21 / pET30a-CpSADH-D59H / D77H. The catalytic reaction conditions are as follows: the initial concentration of the substrate is 1 M; the concentration of ZnCl2 solution is 0.1 mM; the buffer is phosphate buffer, i.e. NaH2PO4-Na2HPO4 buffer, the pH is 7.0; the volume fraction of isopropanol in the buffer solution is 20%; the temperature of the catalytic reaction is 30℃, and the reaction time is 24 h. The yield of cis-4-tert-butylcyclohexanol produced by the mutant can reach 99.1%, which is 1.5 times that of the wild-type medium-chain dehydrogenase under the same catalytic conditions, and the optical purity de is 98.2%.

[0028] The preparation method of the wet bacteria is as follows: the genetically engineered bacteria are inoculated into LB liquid medium containing corresponding antibiotics (short-chain dehydrogenase, 50 μg / mL streptomycin; medium-chain dehydrogenase, 50 μg / mL kanamycin), and the bacteria are cultured at 37℃ for 15-16 h under oscillation; the seed liquid is inoculated into fresh LB liquid medium containing corresponding antibiotics (short-chain dehydrogenase, 50 μg / mL streptomycin; medium-chain dehydrogenase, 50 μg / mL kanamycin) at a volume ratio of 1%, and the bacteria are cultured at 37℃ under oscillation at 220 rpm until the OD 600The wet bacterial cells were collected by centrifugation at 4000 rpm for 10 min.

[0029] The present application has the following beneficial effects: (1) The carbonyl reductase mutant provided by the present application has higher enzyme activity than the wild-type carbonyl reductase, and can be used to prepare cis-4-tert-butylcyclohexanol or trans-4-tert-butylcyclohexanol with 4-tert-butylcyclohexanone as the substrate, with high yield of the product and no by-product.

[0030] (2) The present application uses the carbonyl reductase mutant as a biological catalyst to make the obtaining of high-optical-purity 4-tert-butylcyclohexanol more economical and simple, and the production method has the advantages of simple operation and low cost, greatly reduces the production cost, and has good industrial application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is the amino acid sequence alignment of short-chain dehydrogenase UCPA and all mutants thereof and medium-chain dehydrogenase CpSADH and all mutants thereof, and the triangle mark in the figure represents the coenzyme binding motif of the two enzymes (short-chain dehydrogenase: Gly 13 -X-X-X-Gly 17 -X-Gly 19 ; medium-chain dehydrogenase: Gly 114 -X-X-X-Gly 118 -X-Gly 120 ).

[0032] Figure 2 is the standard product cis / trans-4-tert-butylcyclohexanol, E. coli is the gas chromatogram of the reaction products of BL21 blank control, short-chain dehydrogenase UCPA and substrate 4-tert-butylcyclohexanone, and medium-chain dehydrogenase CpSADH and substrate 4-tert-butylcyclohexanone. DETAILED DESCRIPTION

[0033] The present application will be further described below in conjunction with specific examples. The following examples are only used to illustrate the present application, and are not used to limit the application scope of the present application. Modifications or replacements of the methods, steps or conditions of the present application without departing from the spirit and essence of the present application all belong to the scope of the present application.

[0034] The test methods used in the following examples are conventional methods unless otherwise specified; and the materials, reagents, etc. used are reagents and materials available from commercial channels unless otherwise specified.

[0035] Nucleotide sequences are written from left to right in the 5' to 3' direction, and amino acid sequences are written from left to right in the amino-terminal to carboxyl-terminal direction.

[0036] Example 1: Construction of genetically engineered bacteria expressing short-chain or medium-chain dehydrogenase wild type and each mutant 1. Construction of genetically engineered bacteria expressing short-chain dehydrogenase and medium-chain dehydrogenase wild type The amino acid sequence of the wild type of short-chain dehydrogenase (UCPA) from E. coli K12 is shown in SEQ ID NO. 1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 22; the amino acid sequence of glucose dehydrogenase (GDH) from Bacillus is shown in SEQ ID NO. 23, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 24; the amino acid sequence of the wild type of medium-chain dehydrogenase (CpSADH) from C. parapsilosis is shown in SEQ ID NO. 2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 25.

[0037] According to the nucleotide sequence corresponding to UCPA (www.uniprot.org / uniprotkb / P37440, GenBank: WP_000517431.1) in the Uniprot protein library, a pair of primers was designed, and the genomic DNA of E. coli K12 (strain K12) was used as a template to amplify a small fragment of UCPA with homologous arms at both ends by PCR. In addition, a plasmid pCDFDuet-GDH containing the GDH gene was used as a template, and a pair of primers was designed to amplify a large fragment of pCDFDuet-GDH with homologous arms at both ends by PCR. Escherichia coli The genomic DNA of E. coli K12 (strain K12) was used as a template to amplify a small fragment of UCPA with homologous arms at both ends by PCR. In addition, a plasmid pCDFDuet-GDH containing the GDH gene was used as a template, and a pair of primers was designed to amplify a large fragment of pCDFDuet-GDH with homologous arms at both ends by PCR. pn After digestion with I, the small fragment and the large fragment were connected by Gibson and transformed into E. coli BL21 to obtain the genetically engineered recombinant strain carrying the wild type short-chain dehydrogenase encoding gene E. coli BL21 / pCDFDuet-GDH-UCPA. E.coli BL21 / pCDFDuet-GDH-UCPA.

[0038] According to the nucleotide sequence corresponding to SADH (i.e. CpSADH) (www.uniprot.org / uniprotkb / O42703, GenBank: BAA24528.1) in the Uniprot protein library, a pair of primers was designed, and the genomic DNA was used as a template to amplify the target gene by PCR. Candida parapsilosis The genomic DNA was used as a template to amplify the target gene by PCR, and the PCR product was cleaned by a PCR product cleaning kit. The restriction endonucleases B amH I and X hoI The PCR product and pET30a plasmid were digested with enzymes, and the digested PCR product and plasmid pET30a were connected using T4 DNA fast ligase and transformed into E. coli E. coli BL21, to obtain an engineered recombinant strain carrying a wild-type medium-chain dehydrogenase-encoding gene E.coli BL21 / pET30a-CpSADH.

[0039] The recombinant genetically engineered strain was activated on an LB plate containing the corresponding antibiotic and cultured at 37°C for 15-16 h, and a single colony was picked and inoculated in 5 mL of LB liquid test tube containing the corresponding antibiotic (50 μg / mL kanamycin) and cultured at 37°C and 220 rpm until the OD E.coli BL21 / pCDFDuet-GDH-UCPA recombinant strain: 50 μg / mL streptomycin; E.coli BL21 / pET30a-CpSADH recombinant strain: 50 μg / mL kanamycin) and cultured at 37°C and 220 rpm until the OD 600 was about 0.6, and the plasmid was extracted according to the instructions of the plasmid extraction kit.

[0040] 2. Construction of short-chain dehydrogenase and medium-chain dehydrogenase single-point mutant The extracted plasmid pCDFDuet-GDH-UCPA or pET30a-CpSADH of step 1 was used as a template, and the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent, United States) was used to complete the construction of the single-point mutant plasmid.

[0041] Specifically, the cysteine (C) at position 93, the threonine (T) at position 143, the aspartic acid (D) at position 149, the tyrosine (Y) at position 187, the alanine (A) at position 193, and the methionine (M) at position 212 in the wild-type amino acid sequence of short-chain dehydrogenase were subjected to single-point mutation; the aspartic acid (D) at position 59 and the aspartic acid (D) at position 77 in the wild-type amino acid sequence of medium-chain dehydrogenase were subjected to single-point mutation. The corresponding primers were designed, as shown in Table 1.

[0042] Table 1. Primers used for mutant construction

[0043] The plasmid of the single-point mutant constructed above was transformed into E. coli E. coli BL21 competent cells, mixed and placed on ice for 15 min, and then the E. coli E. coliBL21 competent was heat shocked at 42℃ for 90 s, then placed on ice for 5 min, 1 mL LB medium without antibiotics was added and incubated at 37℃ for 45 min. After centrifugation at 12000 rpm for 1 min, the supernatant was discarded, and the remaining 100 μL supernatant was resuspended with bacteria, then spread on LB plate containing corresponding antibiotics (short-chain dehydrogenase: 50 μg / mL streptomycin; medium-chain dehydrogenase: 50 μg / mL kanamycin), and incubated at 37℃ for 18 h.

[0044] A single colony on the plate was picked into a test tube containing 5 mL LB medium with corresponding resistance (short-chain dehydrogenase, 50 μg / mL streptomycin; medium-chain dehydrogenase, 50 μg / mL kanamycin), and incubated for 6 h. Then 1 mL was taken for sequencing. The remaining bacteria were added with an equal volume of 40% glycerol solution, and stored in a 80℃ refrigerator for later use. In this way, short-chain dehydrogenase and medium-chain dehydrogenase single-point mutant engineering strains were obtained. The names of the single-point mutant engineering strains and the corresponding amino acid sequences are shown in Table 2.

[0045] Table 2. Names of single-point mutant engineering strains and corresponding amino acid sequences

[0046] 3. Construction of double-point mutant of short-chain dehydrogenase and medium-chain dehydrogenase The plasmid of the single-point mutant engineering strain was extracted by using the plasmid extraction kit. The alanine (A) at position 193 in the wild-type amino acid sequence of short-chain dehydrogenase was single-point mutated using the pCDFDuet-GDH-UCPA-D149V plasmid constructed in step 2 as a template; the tyrosine (Y) at position 187 in the wild-type amino acid sequence of short-chain dehydrogenase was single-point mutated using the pCDFDuet-GDH-UCPA-T143V plasmid constructed in step 2 as a template; the methionine (M) at position 212 in the wild-type amino acid sequence of short-chain dehydrogenase was single-point mutated using the pCDFDuet-GDH-UCPA-T143V plasmid constructed in step 2 as a template; the aspartic acid (D) at position 59 in the wild-type amino acid sequence of medium-chain dehydrogenase was single-point mutated using the pET30a-CpSADH-D77H plasmid constructed in step 2 as a template. The corresponding primers are shown in Table 1. The double-point mutant plasmid was constructed using the point mutation kit. The method for constructing the double-point mutant engineering strain is the same as above. The corresponding name and amino acid sequence number are shown in Table 3.

[0047] Table 3. Names of double-point mutant engineering strains and corresponding amino acid sequences

[0048] 4. Construction of triple-point mutant of short-chain dehydrogenase The plasmid of the double-point mutant strain was extracted by using the plasmid mini-preparation kit, and the cysteine (C) at the 93rd position in the wild-type amino acid sequence of the short-chain dehydrogenase was single-point mutated by using the pCDFDuet-GDH-UCPA-T143V / Y187A plasmid constructed in step 3 as a template. The corresponding primers are shown in Table 1, and the construction of the three-point mutant plasmid is completed by using the point mutation kit. The three-point mutant genetic engineering strain is constructed E. coli The method of BL21 / pCDFDuet-GDH-UCPA-T143V / C93I / Y187A is the same as above, and the amino acid sequence number is SEQ ID NO. 16.

[0049] Through amino acid sequence alignment, the short-chain dehydrogenase and the medium-chain dehydrogenase mutant both have the typical Rossmann fold domain of the dehydrogenase superfamily, and the domain contains a conserved coenzyme binding motif, such as Figure 1 The short-chain dehydrogenase is indicated by the middle triangular mark: Gly 13 -X-X-X-Gly 17 -X-Gly 19 The medium-chain dehydrogenase is: Gly 114 -X-X-X-Gly 118 -X-Gly 120 , which constitutes the core structural basis for the binding of the two dehydrogenases and their mutants to coenzymes to perform catalytic functions.

[0050] Example 2: Induced expression of wild-type and each mutant of short-chain or medium-chain dehydrogenase The engineering bacteria expressing wild-type enzymes and the engineering bacteria expressing each mutant constructed in Example 1 were inoculated into 5 mL LB liquid medium test tubes containing corresponding antibiotics (short-chain dehydrogenase: containing 50 μg / mL streptomycin; medium-chain dehydrogenase: containing 50 μg / mL kanamycin), and cultured at 37°C for 15 ~ 16 h. Then, 1% inoculation amount (v / v) was inoculated into 50 mL LB medium containing corresponding antibiotics (short-chain dehydrogenase: containing 50 μg / mL streptomycin; medium-chain dehydrogenase: containing 50 μg / mL kanamycin), and cultured at 37°C, 220 rpm until the bacterial concentration OD 600 was about 0.6, and then 0.1 mM IPTG was added, and the culture was induced at 25°C, 220 rpm for 6 h. The wet bacteria were collected by centrifugation at 4000 rpm for 10 min, and the wet bacteria of the engineering bacteria expressing wild-type enzymes and the engineering bacteria expressing each mutant were obtained, respectively.

[0051] Example 3: Preparation of cis- or trans-4-tert-butylcyclohexanol by using wild-type and mutant of short-chain dehydrogenase Reaction solution one: containing 2 mM coenzyme NAD+ 75 mM glucose solution, 50 mM 4-tert-butylcyclohexanone, 10% isopropanol (v / v) in 0.1 M NaH2PO4-Na2HPO4 buffer, pH 7.0.

[0052] Reaction solution two: contained 2 mM coenzyme NAD + 450 mM glucose solution, 300 mM 4-tert-butylcyclohexanone, 10% isopropanol (v / v) in 0.1 M NaH2PO4-Na2HPO4 buffer, pH 7.0.

[0053] Reaction solution three: contained 2 mM coenzyme NAD + 1.5 M glucose solution, 1 M 4-tert-butylcyclohexanone, 10% acetonitrile (v / v) in 0.5 M NaH2PO4-Na2HPO4 buffer, pH 7.0.

[0054] The short-chain dehydrogenase wild type and mutants obtained in Example 2 were resuspended in the above reaction solutions (see Table 4 for details) to give a wet cell concentration of 50 g / L (100 g / L for reaction solution three) to give a reaction system solution. 1 mL of the reaction system solution (10 mL for reaction solution three) was placed in a 30°C (40°C for reaction solution three) constant temperature shaker at 220 rpm for 6 h (10 h for reaction solution three). After the reaction, an equal volume of ethyl acetate was added and centrifuged, and the upper organic phase was taken and an internal standard solution was added.

[0055] The yield and de values were determined by gas chromatography (GC) analysis, and the gas chromatograms of the cis-4-tert-butylcyclohexanol and trans-4-tert-butylcyclohexanol standards are shown in Figure 2 The final measured product de values and yields are shown in Table 4.

[0056] Table 4. Yields and de values of cis / trans-4-tert-butylcyclohexanol produced by short-chain dehydrogenase wild type and mutants

[0057] Example 4: Medium-chain dehydrogenase mutants for the production of cis-4-tert-butylcyclohexanol Reaction solution four: contained 2 mM coenzyme NAD + 0.1 mM ZnCl2, 200 mM 4-tert-butylcyclohexanone, 20% isopropanol (v / v) in 0.1 M NaH2PO4-Na2HPO4 buffer, pH 7.0.

[0058] Reaction solution five: 2 mM coenzyme NAD + , 0.1 mM ZnCl2, 1 M 4-tert-butylcyclohexanone, 20% isopropanol (v / v) in 0.1 M NaH2PO4-Na2HPO4 buffer with pH 7.0.

[0059] The wild type and mutant meso-dehydrogenase wet bacteria obtained in Example 2 were resuspended with the above reaction solutions (see Table 5 for details) to make the wet bacteria content 50 g / L, and the reaction system solution was prepared. 1 mL of the reaction system solution was taken and reacted at 30°C in a constant temperature shaker at 220 rpm for 1 h (24 h when using reaction solution five). After the reaction was completed, an equal volume of ethyl acetate was extracted and centrifuged, and the upper organic phase was taken and an internal standard solution was added.

[0060] The yield and de value of cis-4-tert-butylcyclohexanol standard were determined by gas chromatography (GC) analysis, and the gas chromatogram of cis-4-tert-butylcyclohexanol standard is shown in Figure 2 The final measured product de value and yield are shown in Table 5.

[0061] Table 5. The de value and yield of cis-4-tert-butylcyclohexanol prepared by meso-dehydrogenase wild type and each mutant

[0062] Analysis of experimental results: Compared with the wild type, the mutants of Escherichia coli K12-derived short-chain dehydrogenase UCPA and the mutants of Candida parapsilosis-derived meso-dehydrogenase CpSADH provided by the present application all have better catalytic activity. The yield of trans-4-tert-butylcyclohexanol synthesized by the UCPA mutant is up to 99.7%, de the value is up to 99%, and the optical purity is high; the yield of cis-4-tert-butylcyclohexanol synthesized is up to 92.0%, de the value is up to 99%. The yield of cis-4-tert-butylcyclohexanol synthesized by the CpSADH mutant is up to 99.1%, de the value is up to 98.2%.

[0063] Moreover, the above-mentioned biocatalyst is easy to prepare, has mild reaction conditions, wide substrate adaptability, is environmentally friendly, can efficiently catalyze the reduction reaction of para-alkyl substituted cyclohexanone, and has good industrial application and development prospects.

[0064] The above shows and describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the above embodiments do not limit the present application in any form, and any technical solutions obtained by equivalent replacement or equivalent transformation fall within the protection scope of the present application.

Claims

1. A carbonyl reductase mutant, characterized in that, The carbonyl reductase mutant is obtained by amino acid mutation of a short-chain dehydrogenase from Escherichia coli K12 with an amino acid sequence as shown in SEQ ID NO. 1 or a medium-chain dehydrogenase from Candida parapsilosis with an amino acid sequence as shown in SEQ ID NO. 2; wherein the amino acid mutation site of the short-chain dehydrogenase is at least one of positions 93, 143, 149, 187, 193 and 212, the cysteine at position 93 is mutated into isoleucine; the threonine at position 143 is mutated into alanine or valine; the aspartic acid at position 149 is mutated into valine; the tyrosine at position 187 is mutated into alanine, serine, glycine or methionine; the alanine at position 193 is mutated into isoleucine; and the methionine at position 212 is mutated into tryptophan; the amino acid mutation site of the medium-chain dehydrogenase is at least one of positions 59 and 77, the aspartic acid at position 59 is mutated into histidine or leucine; and the aspartic acid at position 77 is mutated into histidine.

2. The carbonyl reductase mutant of claim 1, wherein The amino acid sequence of the mutant of the short-chain dehydrogenase after amino acid mutation is shown in any one of SEQ ID NO. 3 ~ SEQ ID NO. 16; and the amino acid sequence of the mutant of the medium-chain dehydrogenase after amino acid mutation is shown in any one of SEQ ID NO. 17 ~ SEQ ID NO.

21.

3. A coding gene encoding the carbonyl reductase mutant of claim 1 or 2.

4. A recombinant expression plasmid, characterized in that, The recombinant expression plasmid comprises the coding gene of claim 3.

5. The recombinant expression plasmid of claim 4, wherein, The recombinant expression plasmid comprises the coding gene of the mutant of the short-chain dehydrogenase after amino acid mutation and a coding gene of glucose-1-dehydrogenase.

6. A genetically engineered bacterium comprising the recombinant expression plasmid of claim 4 or 5.

7. The genetically engineered bacteria as described in claim 6, characterized in that, The host cell of the genetically engineered bacterium is Escherichia coli.

8. Use of the carbonyl reductase mutant of claim 1 or 2 in the preparation of cis-4-tert-butylcyclohexanol or trans-4-tert-butylcyclohexanol.

9. The use according to claim 7, wherein the compound is ###00003### 8 or ###00004### 9. The application comprises: using wet bacteria, wet bacteria immobilized cells, enzymes or immobilized enzymes obtained by centrifugation of wet bacteria, wet bacteria immobilized cells, enzymes or immobilized enzymes extracted after ultrasonic crushing of the wet bacteria after fermentation culture of genetically engineered bacteria containing the coding gene of the carbonyl reductase mutant as a catalyst, using 4-tert-butyl cyclohexanone as a substrate, adding coenzyme NAD + , adding glucose or Zn 2+ , using a buffer solution containing an organic solvent with a pH value ranging from 6.0 to 8.0 as a reaction medium, oscillating the reaction under the conditions of 25 to 45℃ and 150 to 300 rpm, and separating cis-4-tert-butyl cyclohexanol or trans-4-tert-butyl cyclohexanol from the reaction solution after the reaction is completed.

10. Use according to claim 8 or 9, characterized in that, When the carbonyl reductase mutant is a mutant of short-chain dehydrogenase after amino acid mutation, the initial concentration of the substrate in the catalytic reaction system is 50-1000 mM; the concentration of coenzyme NAD + is 1-3 mM; the concentration of glucose is 1.5 times that of the substrate; the organic solvent is isopropyl alcohol or acetonitrile, and the volume fraction is 5-15%; the amount of catalyst is 50-100 g / L based on the weight of wet bacteria, wherein the water content in the wet bacteria is 70-90%. When the carbonyl reductase mutant is a mutant of medium-chain dehydrogenase after amino acid mutation, the initial concentration of the substrate in the catalytic reaction system is 200 ~ 1000 mM; the concentration of coenzyme NAD + Is 1 ~ 3 mM; Zn 2+ Is added in the form of ZnCl2 solution, the concentration is 0.05 ~ 0.15 mM; the organic solvent is isopropanol, the volume fraction is 2.5 ~ 20%; the amount of catalyst is 50 ~ 100 g / L based on the weight of wet bacteria, and the water content in the wet bacteria is 70 ~ 90%.

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