Carbonyl reductase mutant and application thereof in synthesis of chiral 2, 2-disubstituted-1, 3-cyclic diol

Abstract

The invention discloses a carbonyl reductase mutant and application thereof in synthesis of chiral 2, 2-disubstituted-1, 3-cyclic diol, and belongs to the field of enzyme engineering and organic synthesis. The carbonyl reductase mutant is obtained by carrying out single-point mutation, double-point mutation, three-point mutation, four-point mutation, five-point mutation or six-point mutation on the 1st site to the 251st site of an amino acid sequence as shown in SEQ ID NO.1 by wild type carbonyl reductase from lactobacillus brevis, and the carbonyl reductase mutant is obtained by carrying out single-point mutation, double-point mutation, three-point mutation, four-point mutation, five-point mutation or six-point mutation on the 1st site to the 251st site of the amino acid sequence as shown in SEQ ID NO.1. The carbonyl reductase mutant can directly, efficiently and highly stereoselectively catalyze the reaction of a 2, 2-disubstituted-1, 3-cyclic diketone compound to synthesize the chiral 2, 2-disubstituted-1, 3-cyclic diol, and has a wide application prospect in the aspect of synthesizing the chiral 2, 2-disubstituted-1, 3-cyclic diol.

Description

Technical Field

[0001] The invention belongs to the field of enzyme engineering and organic synthesis, and particularly relates to a carbonyl reductase mutant and an application thereof in synthesizing chiral 2,2-disubstituted-1,3-cyclodiol. Background Art

[0002] Chiral diols have unique stereo configuration and physicochemical properties, which make them have broad application prospects in asymmetric synthesis, catalytic reaction and drug synthesis. There are many methods for preparing chiral diols, including chemical synthesis, biological transformation and natural extraction, among which the chemical synthesis method may have the problems of cumbersome steps and low conversion rate, and the yield of natural extraction is low. The biological transformation method uses enzymes in biological systems as catalysts to convert precursor compounds into chiral diols with specific stereo configurations, which have high stereoselectivity and are green and environmentally friendly. For example, the Chinese patent document with publication number CN118652864A discloses a carbonyl reductase mutant and its application. The invention transforms the wild-type carbonyl reductase of Novosphingobium aromaticivorans by directed evolution, and the transformed product can be used to catalyze the high yield and high purity generation of the target diol product.

[0003] 2,2-Disubstituted-1,3-cyclodiols are very important organic synthetic intermediates that can be further transformed into many natural products or steroidal compounds containing multiple chiral centers, such as ouabagenin, trewianin, 2,3-dihydrohygrophorone H, etc. However, the precise construction of single stereoisomers of 2,2-disubstituted-1,3-cyclodiols with two or three chiral centers remains a major challenge in synthetic chemistry. Direct reduction (dicarbonyl reduction) of 2,2-disubstituted-1,3-cyclodiones is the most direct and effective method for synthesizing 2,2-disubstituted-1,3-cyclodiols. However, there are as many as 8 carbonyl reduction products of 2,2-disubstituted-1,3-cyclodione compounds, including 4 products with one carbonyl group reduced and 4 products with both carbonyl groups reduced, as shown in the following formula, where n is an integer ≥1; R is an aromatic group; this makes the stereoselective synthesis of 2,2-disubstituted-1,3-cyclodiols more difficult.

[0004]

[0005] In addition, the current synthesis methods have the disadvantages of harsh reaction conditions, poor reaction stereoselectivity, and complex reaction steps (Angew.Chem.Int.Ed.2013,52,5300–5304,J.Org.Chem.2018,83,13888-13910,J.Org.Chem.2020,85,9599-9606). Therefore, it is necessary to develop a simple and efficient method for synthesizing chirally pure 2,2-disubstituted-1,3-cyclodiols. Summary of the invention

[0006] In order to solve the deficiencies in the above-mentioned prior art, the present invention provides a carbonyl reductase mutant, which can directly, efficiently and stereoselectively catalyze the reaction of 2,2-disubstituted-1,3-cyclodione compounds to synthesize chiral 2,2-disubstituted-1,3-cyclodiols.

[0007] The specific technical solutions adopted are as follows:

[0008] The carbonyl reductase mutant is obtained by subjecting the wild-type carbonyl reductase from Lactobacillus brevis to single-point mutation, double-point mutation, triple-point mutation, quadruple-point mutation, quintuple-point mutation or hexa-point mutation at positions 1 to 251 of the amino acid sequence shown in SEQ ID NO.1;

[0009] The specific single point mutation site is any of the following:

[0010] mutation at position 95;

[0011] mutation at position 144;

[0012] mutation at position 189;

[0013] mutation at position 198;

[0014] mutation at position 205;

[0015] mutation at position 248;

[0016] The specific double point mutation site is any of the following:

[0017] Combined mutations at positions 95 and 198;

[0018] Combined mutations at positions 143 and 144;

[0019] Combined mutations at positions 144 and 189;

[0020] Combined mutations at positions 189 and 248;

[0021] The specific three-point mutation site is any of the following:

[0022] combined mutations at positions 143, 144, and 189;

[0023] combined mutations at positions 144, 189, and 248;

[0024] The specific four mutation sites are:

[0025] combined mutations at positions 143 and 144 and 189 and 248;

[0026] The specific five mutation sites are:

[0027] combined mutations at positions 93 and 143 and 144 and 189 and 248;

[0028] combined mutations at positions 143 and 144 and 189 and 205 and 248;

[0029] The specific six mutation sites are:

[0030] Combined mutations at positions 143 and 144 and 189 and 198 and 205 and 248.

[0031] The carbonyl reductase mutant has the catalytic activity of catalyzing 2,2-disubstituted-1,3-cyclodione compounds to generate chiral 2,2-disubstituted-1,3-cyclodiols, and is obtained by transforming the wild-type carbonyl reductase from Lactobacillus brevis. The nucleotide sequence encoding the wild-type carbonyl reductase is shown in SEQ ID NO.2.

[0032] Further, the specific single point mutation is any one of the following:

[0033] N95I; N95Q; E144G; E144A; Y189G; Y189A; Y189C; L198F; L198N; M205G; M205A; Y248G; Y248A.

[0034] Further, the specific double point mutation is any one of the following:

[0035] N95Q and L198F; N95Q and L198S; N95Q and L198A; I143G and E144G; I143G and E144A; E144G and Y189A; E144A and Y189A; Y189C and M205A; Y189A and Y248A; Y189A and Y248G; E144G and Y189L.

[0036] Further, the specific three-point mutation is any one of the following:

[0037] I143G and E144G and Y189G; I143G and E144G and Y189A; I143G and E144L and Y189C; E144G and Y189A and Y248G; E144G and Y189A and Y248A.

[0038] Further, the specific four-point mutation is any one of the following:

[0039] I143G and E144G and Y189A and Y248A; I143G and E144A and Y189A and Y248A; I143G and E144L and Y189A and Y248A; I143G and E144G and Y189G and Y248A; I143G and E144G and Y189A and Y248L.

[0040] Further, the specific five-point mutation is any one of the following:

[0041] A93L and I143G and E144G and Y189A and Y248A; I143G and E144G and Y189A and M205A and Y248A; I143G and E144G and Y189A and M205A and Y248F; I143G and E144G and Y189A and M205F and Y248A.

[0042] Further, the specific six-point mutation is any one of the following:

[0043] I143G and E144G and Y189C and L198N and M205A and Y248A.

[0044] Preferably, the single point mutation is: M205A, and the corresponding amino acid sequence is shown in SEQ ID NO.3.

[0045] Preferably, the double point mutations are: N95Q and L198F, and the corresponding amino acid sequence is shown in SEQ ID NO.4.

[0046] Preferably, the three point mutations are: I143G, E144G and Y189A, and the corresponding amino acid sequence is shown in SEQ ID NO.5.

[0047] Preferably, the four point mutations are: I143G, E144G, Y189A and Y248L; the corresponding amino acid sequences are shown in SEQ ID NO.6.

[0048] The present invention also provides a gene encoding the carbonyl reductase mutant.

[0049] The present invention also provides a recombinant expression vector, which contains a gene encoding the carbonyl reductase mutant.

[0050] The present invention also provides a genetically engineered bacterium, wherein the genetically engineered bacterium expresses the carbonyl reductase mutant, which can be obtained by transforming the recombinant expression vector into a host microorganism.

[0051] The present invention also provides the use of the carbonyl reductase mutant or the genetically engineered bacteria in synthesizing chiral 2,2-disubstituted-1,3-cyclodiols.

[0052] Specifically, the carbonyl reductase mutant or the genetically engineered bacteria is used to directly reduce the 2,2-disubstituted-1,3-cyclodione shown in Formula I to the chiral 2,2-disubstituted-1,3-cyclodiol shown in Formula II;

[0053]

[0054] wherein n is 1 or 2; and R is phenyl, naphthyl, p-methoxyphenyl, m-methoxyphenyl, o-methoxyphenyl, p-methylphenyl, m-methylphenyl, o-methylphenyl, p-fluorophenyl, m-fluorophenyl, o-fluorophenyl, p-chlorophenyl, m-chlorophenyl, o-chlorophenyl, p-bromophenyl, m-bromophenyl or o-bromophenyl.

[0055] Furthermore, the chiral 2,2-disubstituted-1,3-cyclodiol is (1S,3S)-2,2-disubstituted-1,3-cyclodiol or (1R,3R)-2,2-disubstituted-1,3-cyclodiol.

[0056] The present invention also provides a method for synthesizing chiral 2,2-disubstituted-1,3-cyclodiol, the method comprising: taking 2,2-disubstituted-1,3-cyclodione as substrate, adding crude enzyme solution obtained after fermentation and culture of the genetically engineered bacteria, and NADP + , glucose dehydrogenase and glucose, react at 20-50° C. for 8-12 hours. After the reaction is complete, the reaction solution is separated and purified to obtain chiral 2,2-disubstituted-1,3-cyclodiol.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] The present invention transforms the wild-type carbonyl reductase from Lactobacillus brevis, and the obtained carbonyl reductase mutant has a wide application prospect in synthesizing chiral 2,2-disubstituted-1,3-cyclodiols, especially being able to achieve high-selective synthesis of (1S,3S)-2,2-disubstituted-1,3-cyclodiols or (1R,3R)-2,2-disubstituted-1,3-cyclodiols. In a specific embodiment, the carbonyl reductase mutant uses 2,2-disubstituted-1,3-cyclopentanedione as a substrate to catalyze the conversion rate of (1S,3S)-2,2-disubstituted-1,3-cyclodiols to reach 99%, the ee value reaches 99%, and the dr value is greater than 97 / 3, and the conversion rate of (1R,3R)-2,2-disubstituted-1,3-cyclodiols to reach 99%, the ee value reaches 99%, and the dr value is greater than 99 / 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 A is the reference HPLC spectrum of eight reduction products of substrate 1a; B is the HPLC spectrum of the reaction products of substrate 1a catalyzed by wild-type carbonyl reductase.

[0060] Figure 2 A is the reference HPLC spectrum of eight reduction products of substrate 1a; B is the HPLC spectrum of the reaction of substrate 1a generating (1R, 3R)-3a catalyzed by the carbonyl reductase mutant "M205A"; C is the HPLC spectrum of the reaction of substrate 1a generating (1S, 3S)-3a catalyzed by the carbonyl reductase mutant "I143G and E144G and Y189A and Y248L". DETAILED DESCRIPTION

[0061] In order to make the purpose, features and advantages of the present invention more obvious and easy to understand, the following is a detailed description through specific embodiments. In the following description, many specific details are set forth in order to fully understand the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below. The technical features in each embodiment of the present invention can be combined accordingly without conflicting with each other.

[0062] The operating methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. The contents not described in detail in this specification belong to the prior art known to professionals in the field. The experimental materials used in the following examples, unless otherwise specified, can be purchased from conventional biochemical reagent companies.

[0063] Wherein, as used herein, the term "AxxB" means that the amino acid A at position xx is changed to B, for example, "M205A" means that the methionine (M) at position 205 is mutated to alanine (A); "I143G and E144G and Y189A and Y248L" means a combination of mutations at positions 143, 144, 189 and 248, wherein the isoleucine (I) at position 143 is mutated to glycine (G), the glutamic acid (E) at position 144 is mutated to glycine (G), the tyrosine (Y) at position 189 is mutated to alanine (A), the tyrosine (Y) at position 248 is mutated to leucine (L), and so on.

[0064] The carbonyl reductase mutants constructed in the present invention are all mutants of the wild-type carbonyl reductase (LbADH, PDB ID: 1zk4) SEQ ID NO.1 derived from Lactobacillus brevis.

[0065] Example 1: Construction of a carbonyl reductase LbADH mutant library and screening of mutants

[0066] 1. Construction of pET28a(+)-LbADH plasmid and genetically engineered bacteria E. coli BL21(DE3) / pET28a(+)-LbADH

[0067] The amino acid sequence of the wild-type carbonyl reductase from Lactobacillus brevis is shown in SEQ ID NO. 1, and the corresponding nucleotide sequence is shown in SEQ ID NO. 2. The corresponding nucleotide sequence was fully synthesized and cloned between the restriction endonuclease sites NdeI and XhoI of the pET-28a(+) vector to obtain the recombinant plasmid pET28a(+)-LbADH, which was further transformed into the expression host E. coli BL21(DE3), and the positive clones were picked to obtain the E. coli BL21(DE3) / pET28a(+)-LbADH genetic engineering bacteria.

[0068] 2. Construction of carbonyl reductase mutant library

[0069] Using pET28a(+)-LbADH as a template, the amino acids at positions 95, 143, 144, 189, 198, 205, and 248 in the catalytic active pocket were analyzed and selected for site-directed mutagenesis. Primers were designed (primer sequences are shown in Table 1), and mutants were constructed using the plasmid rolling circle amplification method, and PCR was performed using the high-fidelity polymerase KOD-plus.

[0070] The PCR reaction conditions were as follows: 5 μL 10× KOD buffer, 5 μL dNTP (2 mM), and 2 μL MgSO were added to a 50 μL PCR reaction system.4 (25mM), template 20-100ng, forward primer 1μL (10mM), reverse universal primer T7-ter 1μL (10mM), 1μL KOD polymerase, add sterile distilled water to 50μL. PCR reaction procedure: (1) 94℃ pre-denaturation for 2min, (2) 98℃ denaturation for 15sec, (3) 55℃ annealing for 30sec, (4) 68℃ extension for 3min 10sec, steps (2)-(4) for a total of 40 cycles. PCR products were stored at 4℃.

[0071] After the PCR product was verified by agarose gel electrophoresis analysis, restriction endonuclease DpnⅠ was added and digested at 37℃ for 2h. The digestion product was transferred into E.coli BL21(DE3) competent cells and spread on a plate containing kanamycin sulfate, and placed in a 37℃ incubator for 12h. Single clones were selected for sequencing, and the corresponding mutant strains were obtained if the sequencing was correct. The mutation site and the amino acid after mutation are shown in Table 2.

[0072] For the construction of a single point mutant at a certain site, the forward primer sequence design is shown in Table 1. It is only necessary to replace NNK with the nucleotide codon of the corresponding mutation in Table 2. For the construction of a combination mutant, it is necessary to introduce other mutations step by step on the basis of a single point mutant to obtain a combination mutant of multiple sites. For example, in the construction of "N95Q and L198F", the primers of N95Q are used. Using the wild-type nucleotide sequence shown in SEQ ID NO.2 as a template, the AAT in the nucleotide sequence shown in SEQ ID NO.2 was mutated to CAG (N95Q) by PCR, and the N95Q mutant was obtained after sequencing was correct; further, the N95Q plasmid was used as a template and the primer L198F was used to generate the N95Q mutant. By mutating CTG in the nucleotide sequence shown in SEQ ID NO. 2 to TTT (L198F) by PCR, a double mutant of N95Q and L198F can be obtained. Other mutants can be constructed by a similar step-by-step mutation introduction method.

[0073] Table 1 Primer sequences of mutation sites (SEQ ID NO.7 to SEQ ID NO.15)

[0074] Location Primer sequence (5'-3' direction) Reverse universal primer T7-ter GCTAGTTATTGCTCAGCGG 93 AATGCAGGTATTNNKGTTAATAAAAGC 95 GGTATTGCAGTTNNKAAAAGCGTTGAA 143 AATATGAGCAGCNNKGAAGGTTTTGTTG 144 ATGAGCAGCATTNNKGGTTTTGTTGGT 189 GTTCATCCGGGTNNKATTAAAACACCG 198 CTGGTTGATGATNNKCCGGGTGCAGAA 205 GCAGAAGAAGCANNKAGTCAGCGTACC 248 GTGGATGGTGGTNNKACCGCGCAG

[0075] Table 2 Mutation sites and amino acids after mutation

[0076]

[0077] Example 2: Inducible expression of carbonyl reductase mutants

[0078] The single colonies of the genetically engineered bacteria expressing the carbonyl reductase mutant prepared in Example 1 were inoculated into 2 mL of LB liquid medium (10 g / L peptone, 5 g / L yeast powder, 10 g / L sodium chloride) containing kanamycin sulfate antibiotics, and cultured overnight at 37°C, 200 rpm in a shaking incubator to obtain seed liquid. The seed liquid cultured overnight was transferred to 200 mL of LB medium containing kanamycin sulfate at an inoculum of 1%, and cultured at 37°C, 200 rpm until OD 600 When the concentration of IPTG was about 0.6 to 0.8, IPTG (isopropyl-β-D-thiogalactose, final concentration was 0.5 mM) was added and induced at 18°C, 200 rpm for 18 h. The cells were collected by centrifugation at 4°C, 6000 rpm. The cells were washed with Tris-HCl buffer (Tris 50 mM, NaCl 100 mM, MgCl 2 2mM, pH 7.5) (1g wet cells use 5mL buffer), and disrupt with an ultrasonic cell disruptor, centrifuge at 4°C, 12000rpm to retain the supernatant, i.e., the crude enzyme solution, for subsequent reaction detection.

[0079] Example 3: Synthesis of chiral 2,2-disubstituted-1,3-cyclodiols

[0080] The wild-type carbonyl reductase and the carbonyl reductase mutant were cultured and expressed according to the induction expression method in Example 2, and then the crude enzyme solution was collected. The crude enzyme solution was used as a biocatalyst to catalyze the synthesis of chiral 2,2-disubstituted-1,3-cyclodiols from 2,2-disubstituted-1,3-cyclodiones.

[0081] The structural formula of substrate 1a is

[0082] (1) Wild-type carbonyl reductase

[0083] Add 100uL of dimethyl sulfoxide solution containing 1mg / 100uL substrate 1a and 2mg NADP to a 5mL PE tube. + , 20U glucose dehydrogenase, 120mg glucose and 900uL wild-type carbonyl reductase crude enzyme solution were placed on an oscillator and reacted at 30°C and 1500rpm for 10h. After the reaction, it was extracted with ethyl acetate three times and then the solvent was dried. The substrate conversion rate was detected to be 99%, but the content of 2,2-disubstituted-1,3-cyclodiol in the product was <5%, reference Figure 1 As can be seen from A in the figure, the reaction products are almost all hydroxyketones. The HPLC spectrum of the reaction is shown in Figure 1 As shown in B.

[0084] (2) Carbonyl reductase mutant single point mutation L198N

[0085] Add 100uL of dimethyl sulfoxide solution containing 1mg / 100uL substrate 1a and 2mg NADP to a 5mL PE tube. + , 20U glucose dehydrogenase, 120mg glucose and 900uL mutant "L198N" carbonyl reductase crude enzyme solution were placed on an oscillator and reacted at 30°C and 1500rpm for 10h. After the reaction, it was extracted with ethyl acetate three times and then the solvent was dried. The substrate conversion rate was detected to be 99%, and the product content of (1R,3R)-2,2-disubstituted-1,3-cyclodiol was 36%.

[0086] (3) Carbonyl reductase mutant single point mutation N95I

[0087] Add 100uL of dimethyl sulfoxide solution containing 1mg / 100uL substrate 1a and 2mg NADP to a 5mL PE tube. + , 20U glucose dehydrogenase, 120mg glucose and 900uL mutant "N95I" carbonyl reductase crude enzyme solution were placed on an oscillator and reacted at 30°C and 1500rpm for 10h. After the reaction, it was extracted with ethyl acetate three times and then the solvent was dried. The substrate conversion rate was detected to be 99%, and the product content of (1R,3R)-2,2-disubstituted-1,3-cyclodiol was 57%.

[0088] (4) Carbonyl reductase mutant with double point mutations N95Q and L198F

[0089] Add 100uL of dimethyl sulfoxide solution containing 1mg / 100uL substrate 1a and 2mg NADP to a 5mL PE tube. + , 20U glucose dehydrogenase, 120mg glucose and 900uL mutant "N95Q and L198F" carbonyl reductase crude enzyme solution, placed on an oscillator, 30℃, 1500rpm for 10h. After the reaction, extract with ethyl acetate 3 times, then spin dry the solvent. The substrate conversion rate was detected to be 99%, (1R,3R)-2,2-disubstituted-1,3-cyclodiol accounted for 95% of the product content, ee value>99%, dr value>98 / 2.

[0090] (5) Carbonyl reductase mutant single point mutation M205A

[0091] Add 100uL of dimethyl sulfoxide solution containing 1mg / 100uL substrate 1a and 2mg NADP to a 5mL PE tube. + , 20U glucose dehydrogenase, 120mg glucose and 900uL mutant "M205A" carbonyl reductase crude enzyme solution were placed on an oscillator and reacted at 30°C and 1500rpm for 10h. After the reaction, it was extracted with ethyl acetate three times and then the solvent was dried. The substrate conversion rate was detected to be 99%, reference Figure 2 From A in the figure, it can be seen that the reaction product is mainly (1R,3R)-2,2-disubstituted-1,3-cyclodiol, and the (1R,3R)-2,2-disubstituted-1,3-cyclodiol accounts for 95% of the product content, the ee value is 99%, and the dr value is>99 / 1. The reaction HPLC spectrum is shown in Figure 2 As shown in B.

[0092] (6) Carbonyl reductase mutant with three point mutations E144G, Y189A and Y248A

[0093] Add 100uL of dimethyl sulfoxide solution containing 1mg / 100uL substrate 1a and 2mg NADP to a 5mL PE tube. + , 20U glucose dehydrogenase, 120mg glucose and 900uL mutant "E144G and Y189A and Y248A" carbonyl reductase crude enzyme solution were placed on an oscillator and reacted at 30°C and 1500rpm for 10h. After the reaction, it was extracted with ethyl acetate three times and then the solvent was dried. The substrate conversion rate was detected to be 99%, and the product content of (1S,3S)-2,2-disubstituted-1,3-cyclodiol was 63%.

[0094] (7) Carbonyl reductase mutant with three point mutations I143G, E144G and Y189A

[0095] Add 100uL of dimethyl sulfoxide solution containing 1mg / 100uL substrate 1a and 2mg NADP to a 5mL PE tube. + , 20U glucose dehydrogenase, 120mg glucose and 900uL mutant "I143G and E144G and Y189A" carbonyl reductase crude enzyme solution were placed on an oscillator and reacted at 30°C and 1500rpm for 10h. After the reaction, it was extracted with ethyl acetate three times and then the solvent was dried. The substrate conversion rate was detected to be 99%, and the product content of (1S,3S)-2,2-disubstituted-1,3-cyclodiol was 77%.

[0096] (8) Carbonyl reductase mutant with four mutations I143G, E144G, Y189A and Y248L

[0097] Add 100uL of dimethyl sulfoxide solution containing 1mg / 100uL substrate 1a and 2mg NADP to a 5mL PE tube. +, 20U glucose dehydrogenase, 120mg glucose and 900uL mutant "I143G and E144G and Y189A and Y248L" carbonyl reductase crude enzyme solution, placed on an oscillator, 30°C, 1500rpm for 10h. After the reaction, extract with ethyl acetate three times, then spin dry the solvent. The substrate conversion rate was detected to be 99%, (1S,3S)-2,2-disubstituted-1,3-cyclodiol accounted for 96% of the product content, the ee value was 99%, the dr value>97 / 3, and the reaction HPLC spectrum was as follows Figure 2 As shown in C.

[0098] (9) Carbonyl reductase mutant with four mutations I143G, E144G, Y189A and Y248A

[0099] Add 100uL of dimethyl sulfoxide solution containing 1mg / 100uL substrate 1a and 2mg NADP to a 5mL PE tube. + , 20U glucose dehydrogenase, 120mg glucose and 900uL mutant "I143G and E144G and Y189A and Y248A" carbonyl reductase crude enzyme solution were placed on an oscillator and reacted at 30°C and 1500rpm for 10h. After the reaction, it was extracted with ethyl acetate three times and then the solvent was dried. The substrate conversion rate was detected to be 99%, and the product content of (1S,3S)-2,2-disubstituted-1,3-cyclodiol was 70.7%.

[0100] (10) Carbonyl reductase mutant five-point mutation I143G and E144G and Y189A and M205A and Y248A

[0101] Add 100uL of dimethyl sulfoxide solution containing 1mg / 100uL substrate 1a and 2mg NADP to a 5mL PE tube. + , 20U glucose dehydrogenase, 120mg glucose and 900uL mutant "I143G and E144G and Y189A and M205A and Y248A" carbonyl reductase crude enzyme solution were placed on an oscillator and reacted at 30°C and 1500rpm for 10h. After the reaction, it was extracted with ethyl acetate three times and then the solvent was dried. The substrate conversion rate was detected to be 99%, and the product content of (1S,3S)-2,2-disubstituted-1,3-cyclodiol was 86.8%.

[0102] (11) Carbonyl reductase mutant five-point mutation I143G and E144G and Y189A and M205A and Y248F

[0103] Add 100uL of dimethyl sulfoxide solution containing 1mg / 100uL substrate 1a and 2mg NADP to a 5mL PE tube. +, 20U glucose dehydrogenase, 120mg glucose and 900uL mutant "I143G and E144G and Y189A and M205A and Y248F" carbonyl reductase crude enzyme solution were placed on an oscillator and reacted at 30°C and 1500rpm for 10h. After the reaction, it was extracted with ethyl acetate three times and then the solvent was dried. The substrate conversion rate was detected to be 99%, and the product content of (1S,3S)-2,2-disubstituted-1,3-cyclodiol was 57.6%.

[0104] (12) Carbonyl reductase mutant five-point mutation I143G and E144G and Y189A and M205F and Y248A

[0105] Add 100uL of dimethyl sulfoxide solution containing 1mg / 100uL substrate 1a and 2mg NADP to a 5mL PE tube. + , 20U glucose dehydrogenase, 120mg glucose and 900uL mutant "I143G and E144G and Y189A and M205F and Y248A" carbonyl reductase crude enzyme solution were placed on an oscillator and reacted at 30°C and 1500rpm for 10h. After the reaction, it was extracted with ethyl acetate three times and then the solvent was dried. The substrate conversion rate was detected to be 99%, and the product content of (1S,3S)-2,2-disubstituted-1,3-cyclodiol was 64.1%.

[0106] (13) Carbonyl reductase mutant five-point mutation A93L and I143G and E144G and Y189A and Y248A

[0107] Add 100uL of dimethyl sulfoxide solution containing 1mg / 100uL substrate 1a and 2mg NADP to a 5mL PE tube. + , 20U glucose dehydrogenase, 120mg glucose and 900uL mutant "A93L and I143G and E144G and Y189A and Y248A" carbonyl reductase crude enzyme solution were placed on an oscillator and reacted at 30°C and 1500rpm for 10h. After the reaction, it was extracted with ethyl acetate three times and then the solvent was dried. The substrate conversion rate was detected to be 99%, and the product content of (1S,3S)-2,2-disubstituted-1,3-cyclodiol was 73%.

[0108] (14) Carbonyl reductase mutant with two point mutations E144G and Y189L

[0109] Add 100uL of dimethyl sulfoxide solution containing 1mg / 100uL substrate 1a and 2mg NADP to a 5mL PE tube. +, 20U glucose dehydrogenase, 120mg glucose and 900uL mutant "E144G and Y189L" carbonyl reductase crude enzyme solution were placed on an oscillator and reacted at 30°C and 1500rpm for 10h. After the reaction, it was extracted with ethyl acetate three times and then the solvent was dried. The substrate conversion rate was detected to be 99%, and the product content of (1S,3S)-2,2-disubstituted-1,3-cyclodiol was 61.4%.

[0110] (15) Carbonyl reductase mutant single point mutation Y189C

[0111] Add 100uL of dimethyl sulfoxide solution containing 1mg / 100uL substrate 1a and 2mg NADP to a 5mL PE tube. + , 20U glucose dehydrogenase, 120mg glucose and 900uL mutant "Y189C" carbonyl reductase crude enzyme solution were placed on an oscillator and reacted at 30°C and 1500rpm for 10h. After the reaction, it was extracted with ethyl acetate three times and then the solvent was dried. The substrate conversion rate was detected to be 99%, and the product content of (1R,3R)-2,2-disubstituted-1,3-cyclodiol was 78.5%.

[0112] (16) Carbonyl reductase mutant with double point mutations Y189C and M205A

[0113] Add 100uL of dimethyl sulfoxide solution containing 1mg / 100uL substrate 1a and 2mg NADP to a 5mL PE tube. + , 20U glucose dehydrogenase, 120mg glucose and 900uL mutant "Y189C and M205A" carbonyl reductase crude enzyme solution were placed on an oscillator and reacted at 30°C and 1500rpm for 10h. After the reaction, it was extracted with ethyl acetate three times and then the solvent was dried. The substrate conversion rate was detected to be 99%, and the product content of (1R,3R)-2,2-disubstituted-1,3-cyclodiol was 35.5%.

[0114] (17) Carbonyl reductase mutant six-point mutation I143G and E144G and Y189C and L198N and M205A and Y248A

[0115] Add 100uL of dimethyl sulfoxide solution containing 1mg / 100uL substrate 1a and 2mg NADP to a 5mL PE tube. +, 20U glucose dehydrogenase, 120mg glucose and 900uL mutant "I143G and E144G and Y189C and L198N and M205A and Y248A" carbonyl reductase crude enzyme solution were placed on an oscillator and reacted at 30°C and 1500rpm for 10h. After the reaction, it was extracted with ethyl acetate three times and then the solvent was dried. The substrate conversion rate was detected to be 99%, and the product content of (1R,3R)-2,2-disubstituted-1,3-cyclodiol was 40.5%.

[0116] (18) Carbonyl reductase mutant with three point mutations I143G, E144L and Y189C

[0117] Add 100uL of dimethyl sulfoxide solution containing 1mg / 100uL substrate 1a and 2mg NADP to a 5mL PE tube. + , 20U glucose dehydrogenase, 120mg glucose and 900uL mutant "I143G and E144L and Y189C" carbonyl reductase crude enzyme solution were placed on an oscillator and reacted at 30°C and 1500rpm for 10h. After the reaction, it was extracted with ethyl acetate three times and then the solvent was dried. The substrate conversion rate was detected to be 99%, and the product content of (1S,3S)-2,2-disubstituted-1,3-cyclodiol was 70.1%.

[0118] The above-described embodiments provide a detailed description of the technical solutions of the present invention. It should be understood that the above descriptions are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A carbonyl reductase mutant, characterized in that: The wild-type carbonyl reductase from Lactobacillus brevis is obtained by subjecting the wild-type carbonyl reductase from Lactobacillus brevis to single-point mutation, double-point mutation, triple-point mutation, quadruple-point mutation, quintuple-point mutation or hexa-point mutation at positions 1 to 251 of the amino acid sequence shown in SEQ ID NO.1; The specific single point mutation site is any of the following: mutation at position 95; mutation at position 144; mutation at position 189; mutation at position 198; mutation at position 205; mutation at position 248; The specific double point mutation site is any of the following: Combined mutations at positions 95 and 198; Combined mutations at positions 143 and 144; Combined mutations at positions 144 and 189; Combined mutations at positions 189 and 248; The specific three-point mutation site is any of the following: combined mutations at positions 143, 144, and 189; combined mutations at positions 144, 189, and 248; The specific four mutation sites are: combined mutations at positions 143 and 144 and 189 and 248; The specific five mutation sites are: combined mutations at positions 93 and 143 and 144 and 189 and 248; combined mutations at positions 143 and 144 and 189 and 205 and 248; The specific six mutation sites are: Combined mutations at positions 143 and 144 and 189 and 198 and 205 and 248.

2. The carbonyl reductase mutant according to claim 1, characterized in that The specific single point mutation is any of the following: N95I; N95Q; E144G; E144A; Y189G; Y189A; Y189C; L198F; L198N; M205G; M205A; Y248G; Y248A; And / or, the specific double point mutation is any one of the following: N95Q and L198F; N95Q and L198S; N95Q and L198A; I143G and E144G; I143G and E144A; E144G and Y189A; E144A and Y189A; Y189C and M205A; Y189A and Y248A; Y189A and Y248G; E144G and Y189L; And / or, the specific three-point mutation is any one of the following: I143G and E144G and Y189G; I143G and E144G and Y189A; I143G and E144L and Y189C; E144G and Y189A and Y248G; E144G and Y189A and Y248A; And / or, the specific four-point mutation is any one of the following: I143G and E144G and Y189A and Y248A; I143G and E144A and Y189A and Y248A; I143G and E144L and Y189A and Y248A; I143G and E144G and Y189G and Y248A; I143G and E144G and Y189A and Y248L; And / or, the specific five-point mutation is any one of the following: A93L and I143G and E144G and Y189A and Y248A; I143G and E144G and Y189A and M205A and Y248A; I143G and E144G and Y189A and M205A and Y248F; I143G and E144G and Y189A and M205F and Y248A; And / or, the specific six-point mutation is any one of the following: I143G and E144G and Y189C and L198N and M205A and Y248A.

3. A gene encoding the carbonyl reductase mutant according to claim 1 or 2.

4. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the gene according to claim 3.

5. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria express the carbonyl reductase mutant according to claim 1 or 2.

6. Use of the carbonyl reductase mutant according to claim 1 or 2 or the genetically engineered bacteria according to claim 5 in synthesizing chiral 2,2-disubstituted-1,3-cyclodiols.

7. The use according to claim 6, characterized in that: Using the carbonyl reductase mutant or the genetically engineered bacteria, the 2,2-disubstituted-1,3-cyclodione represented by formula I is reduced to a chiral 2,2-disubstituted-1,3-cyclodiol represented by formula II; wherein n is 1 or 2; and R is phenyl, naphthyl, p-methoxyphenyl, m-methoxyphenyl, o-methoxyphenyl, p-methylphenyl, m-methylphenyl, o-methylphenyl, p-fluorophenyl, m-fluorophenyl, o-fluorophenyl, p-chlorophenyl, m-chlorophenyl, o-chlorophenyl, p-bromophenyl, m-bromophenyl or o-bromophenyl.

8. The use according to claim 6, characterized in that: The chiral 2,2-disubstituted-1,3-cyclodiol is (1S,3S)-2,2-disubstituted-1,3-cyclodiol or (1R,3R)-2,2-disubstituted-1,3-cyclodiol.

9. A method for synthesizing chiral 2,2-disubstituted-1,3-cyclodiols, characterized in that: The method comprises: taking 2,2-disubstituted-1,3-cyclodiketone as substrate, adding crude enzyme solution obtained by fermentation culture of genetically engineered bacteria as claimed in claim 5, and NADP + , glucose dehydrogenase and glucose, react at 20-50° C. for 8-12 hours. After the reaction is complete, the reaction solution is separated and purified to obtain chiral 2,2-disubstituted-1,3-cyclodiol.

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