A ketoreductase mutant and its use in the synthesis of (r)-1-(2,6-dichlorophenyl)ethanol

Abstract

The application provides a ketoreductase mutant and application thereof in synthesis of (R)-1-(2,6-dichlorophenyl)ethanol. Specifically, the application provides a ketoreductase mutant, the amino acid sequence of the ketoreductase mutant is an amino acid sequence obtained by mutating the amino acid sequence shown in SEQ ID NO. 2, and the mutation at least includes one or more of the following mutation sites: phenylalanine at the 147th position is mutated into leucine, tyrosine at the 190th position is mutated into proline, and valine at the 196th position is mutated into leucine. Compared with the wild-type enzyme, the activity and selectivity of the mutant in the process of catalyzing 2',6'-dichlorophenylacetophenone into (R)-1-(2,6-dichlorophenyl)ethanol are both improved, the whole process is high in efficiency and low in cost, and can be applied to industrial production.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a ketone reductase mutant and its application in the synthesis of (R)-1-(2,6-dichlorophenyl)ethanol. Background Technology

[0002] Chiral secondary alcohols, especially those with their ortho-carbon atoms replaced by aromatic compounds or heteroatom-containing aromatic compounds, are synthetic intermediates for many physiologically active chiral compounds and have many important industrial applications. 1-(2,6-dichlorophenyl)ethanol is one such secondary alcohol, and its various optically active isomers are widely used in various antibacterial drugs and pesticides. However, the synthesis of these chiral secondary alcohols, especially (R)-1-(2,6-dichlorophenyl)ethanol, is rarely reported.

[0003] Current reports indicate that (R)-1-(2,6-dichlorophenyl)ethylpyrrolidine-2-carboxylate is used as a starting material, and alkaline hydrolysis yields (R)-1-(2,6-dichlorophenyl)ethanol. However, the starting material itself is chiral, making its synthesis difficult. Additionally, some reports describe the use of the prochiral ketone 2',6'-dichloroacetophenone as a starting material, followed by reduction with a metal catalyst to obtain the corresponding product. However, these methods all suffer from the same drawback: poor selectivity, resulting in products with insufficient optical purity.

[0004] Therefore, there is still a need in the field to find a ketone reductase catalyst that can synthesize (R)-1-(2,6-dichlorophenyl)ethanol efficiently and selectively. Summary of the Invention

[0005] The purpose of this invention is to provide a ketone reductase catalyst capable of efficiently and selectively synthesizing (R)-1-(2,6-dichlorophenyl)ethanol. Biocatalysts can achieve a variety of chemical reactions under mild reaction conditions, exhibiting unparalleled selectivity, particularly in the synthesis of chiral alcohols and chiral amines.

[0006] In a first aspect, the present invention provides a ketone reductase mutant, wherein the amino acid sequence of the ketone reductase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.2, and the mutation includes at least one or more of the following mutation sites: phenylalanine at position 147 is mutated to leucine, tyrosine at position 190 is mutated to proline, and valine at position 196 is mutated to leucine.

[0007] In a preferred embodiment, the amino acid sequence of the ketone reductase mutant is an amino acid series with more than 95% homology to the amino acid sequence shown in SEQ ID NO: 4, preferably 96% homology, and more preferably 97%, 98%, 99% or 100% homology.

[0008] In a second aspect of the invention, a ketone reductase mutant gene is provided, wherein the nucleotide sequence of the gene is a nucleotide series having more than 95% homology with the nucleotide sequence shown in SEQ ID NO: 3, preferably 96% homology, more preferably 97%, 98%, 99% or 100% homology.

[0009] In a third aspect of the present invention, a recombinant expression vector containing the ketone reductase mutant gene described in the second aspect of the present invention is provided, the recombinant expression vector including the pET series, pQE series, pRSET series, pGEX series, pBV series, pTrc series, pTwin series, pEZZ series, pKK series, pUC series, pPIC series, pHIL series and pYAM series.

[0010] In a fourth aspect of the present invention, a genetically engineered bacterium containing a ketone reductase mutant gene as described in the first aspect of the present invention is provided, wherein the genetically engineered bacterium contains the ketone reductase mutant gene as described in the second aspect of the present invention.

[0011] In a fifth aspect of the invention, a genetically engineered bacterium according to the fourth aspect of the invention is provided, wherein the host cell of the genetically engineered bacterium is selected from one of Escherichia coli, Saccharomyces cerevisiae, or Pichia pastoris.

[0012] As a preferred embodiment, the vector system of the genetically engineered bacteria is an ET series vector or a pPIC series vector.

[0013] In a sixth aspect of the present invention, a method for preparing a ketone reductase mutant polypeptide is provided, comprising the following steps: culturing the genetically engineered bacteria described in the fourth aspect of the present invention to obtain the ketone reductase mutant polypeptide.

[0014] As a preferred embodiment, the ketone reductase mutant polypeptide is prepared by industrial fermentation of the genetically engineered bacteria described in the fourth aspect of the present invention under certain fermentation conditions in a production tank.

[0015] As a preferred embodiment, the fermentation conditions in the production tank are: DO ≥ 30%, air flow rate 1:1.5vvm, and glucose residue ≤ 1%.

[0016] In a seventh aspect of the invention, a method for producing (R)-1-(2,6-dichlorophenyl)ethanol is provided, comprising the step of catalytic reduction of a ketone compound by a ketone reductase, wherein the ketone reductase is a mutant of the ketone reductase described in the first aspect of the invention.

[0017] In a preferred embodiment, the ketone compound is 2',6'-dichloroacetophenone, and the concentration of the 2',6'-dichloroacetophenone is 10-250 g / L; the product of the reduction reaction is (R)-1-(2,6-dichlorophenyl)ethanol.

[0018] In a preferred embodiment, the conversion rate of the 2',6'-dichloroacetophenone is greater than 99%; the ee value is greater than 99%, preferably 99.9%.

[0019] In a preferred embodiment, the ketone reductase is an enzyme powder containing the ketone reductase mutant, or a whole cell or cell lysate containing the ketone reductase mutant.

[0020] In a preferred embodiment, the concentration of the ketone reductase mutant enzyme powder is 1–10 g / L; and the concentration of the ketone reductase cells is 5–50 g / L.

[0021] In a preferred embodiment, the catalytic reduction reaction further includes a cofactor, which is selected from any one or a combination of NADP salt, NADPH, NADH or NAD salt, and the concentration of the cofactor is 0.05 to 1 g / L.

[0022] In a preferred embodiment, the catalytic reduction reaction is carried out in a buffer solution selected from phosphate buffer or tris-hydrochloric acid buffer, and the concentration of the buffer solution is 50 to 500 mmol / L.

[0023] In a preferred embodiment, the catalytic reduction reaction is carried out at a pH of 5.5–8.5 and a temperature of 25–45°C.

[0024] In an eighth aspect of the invention, the use of the ketone reductase mutant as described in the first aspect of the invention as a catalyst in the synthesis of (R)-1-(2,6-dichlorophenyl)ethanol is provided.

[0025] As a preferred embodiment, the application is carried out by the following method: in a buffer solution, in the presence of a coenzyme cycling system, a coenzyme, 2',6'-dichloroacetophenone and a solubilizer, under the action of the ketone reductase mutant described in the first method of the present invention, (R)-1-(2,6-dichlorophenyl)ethanol is synthesized to obtain an optically chiral alcohol.

[0026] In a preferred embodiment, the buffer solution is a phosphate buffer solution, preferably a phosphate buffer solution with a pH of 7.0.

[0027] In a preferred embodiment, the coenzyme cycle system is selected from isopropanol or a combination of glucose and GDH enzyme.

[0028] In a preferred embodiment, the coenzyme is selected from the group consisting of any one or a combination of NADP salt, NADPH, NADH, or NAD salt.

[0029] In a preferred embodiment, the co-solvent includes DMSO.

[0030] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0031] Compared with the prior art, the present invention has the following technical effects:

[0032] (1) The ketone reductase mutant provided by the present invention has improved enzyme activity and selectivity in the process of catalyzing the synthesis of (R)-1-(2,6-dichlorophenyl)ethanol compared with wild-type ketone reductase.

[0033] (2) The ketone reductase mutant provided by the present invention can efficiently and selectively transform the target reaction, and the product has high optical activity and high reaction yield.

[0034] (3) The ketone reductase mutant provided by the present invention makes the whole process highly efficient and low in cost, and can be applied to industrial production. Attached Figure Description

[0035] none Detailed Implementation

[0036] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0037] According to one embodiment of the present invention, the ketone reductase mutant gene is derived from Lentilactobacillus kefiri Dehydrogenase (LkADH), and the NCBI accession number of the wild-type template is AY267012.1.

[0038] Example 1: Construction and Screening of Mutants

[0039] To improve the activity and selectivity of wild-type LkADH ketone reductase (its nucleotide sequence is shown in SEQ ID NO:1, and its amino acid sequence is shown in SEQ ID NO:2) for its substrate, site-directed mutagenesis was performed on residues 147, 190, and 196 of the wild-type ketone reductase sequence shown in SEQ ID NO:2. The mutation sites were: phenylalanine at position 147 was mutated to leucine (F147L), tyrosine at position 190 was mutated to proline (Y190P), and valine at position 196 was mutated to leucine (V196L). The designed primers are shown in Table 1 below.

[0040] Table 1

[0041]

[0042]

[0043] In this embodiment, PrimeSTAR max high-fidelity DNA polymerase was used to amplify the LkADH expression plasmid. The PCR system consisted of: 1 μL forward primer (20 μM), 1 μL reverse primer (20 μM), 1 μL template DNA (50 μg / ml), 22 μL ddH2O, and 25 μL PrimeSTAR Max premix (2×). The PCR experimental procedure is shown in Table 2 below.

[0044] Table 2

[0045]

[0046] The obtained full-plasmid mutant fragment was digested with Dpn I and transformed into E. coli BL21(DE3) competent cells. After transformation, single colonies were picked for sequencing verification. Correct mutants were selected for culture and viability and selectivity tests were performed. The performance-enhancing mutants were stacked, and the mutation method was the same as above.

[0047] Example 2: Expression of ketoreductase and preparation of crude enzyme solution

[0048] Based on Example 1, in this example, wild-type ketoreductase LkADH and its mutant strains were streaked onto LB agar plates containing kanamycin. Single colonies were picked and inoculated into LB tubes containing kanamycin and cultured at 37°C and 200 rpm for 16 h to obtain primary seed culture. Then, the primary seed culture was inoculated at a rate of 1% into TB shake flasks containing kanamycin and cultured at 37°C and 200 rpm until the OD600 was between 0.6 and 0.8. IPTG solution (final concentration 0.1 mM) was added to each shake flask for induction, and the cells were cultured at 25°C and 200 rpm for 16 h. The cells were collected by centrifugation and stored at -20°C for later use. To prepare the crude enzyme solution, a certain amount of frozen cells was weighed and suspended in 100 mM, pH 7.0 phosphate buffer, followed by sonication. After disruption, the cells were centrifuged at 4°C and 8000 × g for 25 min, and the resulting supernatant was the crude enzyme solution.

[0049] Example 3 Enzyme activity detection and analytical grade bioreduction reaction

[0050] First, using the prochiral ketone 2',6'-dichloroacetophenone corresponding to the target product (R)-1-(2,6-dichlorophenyl)ethanol as a substrate, the catalytic activity of ketone reductase LkADH was determined. Then, a coenzyme cycle system was constructed for bioreduction, and the ee value of the product was detected.

[0051] The enzyme activity assay procedure is as follows: Add 50 μL of crude ketoreductase LkADH or its mutant, 2700 μL of phosphate buffer (100 mM, pH 7.0), and 150 μL of NADPH aqueous solution (2 mM) to a quartz cuvette. Incubate at 25°C for 2 min. Then, use the preset program of the enzyme activity assay instrument to scan at a wavelength of 340 nm for 3 min with a scan interval of 10 s. After the system stabilizes (and the obtained curve flattens), add 100 μL of 2',6'-dichloroacetophenone isopropanol solution (300 mM) to the reaction system, and then perform the enzyme activity assay according to the above procedure. Calculate the enzyme activity during the time period when the absorbance value changes uniformly. Repeat 3 times and take the average value.

[0052] The analytical-grade bioreduction reaction procedure is as follows: Add 750 μL of phosphate buffer (100 mM, pH 7.0), 50 μL of NADPH aqueous solution (2 mM), 100 μL of 2',6'-dichloroacetophenone isopropanol solution (300 mM), 7.5 mg of glucose, and 50 μL each of crude enzyme solutions of ketone reductase and glucose dehydrogenase to a 2 mL centrifuge tube. Then, place the centrifuge tube in a constant-temperature shaker at 30°C and 200 rpm for 24 h. After the reaction, extract with an equal volume of ethyl acetate and perform liquid chromatography to determine the ee value of the product.

[0053] The enzyme activity and selectivity of wild-type (WT) ketone reductase and its mutants are shown in Table 3 below:

[0054] Table 3

[0055] mutant Enzyme activity / U / g Product EE value / % WT 156 92.5％ F147L 260 91.8％ Y190P 235 88.7％ V196L 186 99.8％ F147L / Y190P 500 88.2％ F147L / V196L 380 >99.9％ Y190P / V196L 320 >99.9％ F147L / Y190P / V196L 402 >99.9％

[0056] As shown in Table 3, compared with wild-type (WT) ketone reductase, the enzyme activity of single-point mutant ketone reductase mutants (such as F147L, Y190P, or V196L) is increased by more than 20%; the enzyme activity of double-point mutant ketone reductase mutants (such as F147L / Y190P, F147L / V196L, or Y190P / V196L) is increased by more than 100%, especially the enzyme activity of the F147L / Y190P double-point mutant ketone reductase mutant is increased by about 2200%; the enzyme activity of triple-point mutant ketone reductase (F147L / Y190P / V196L, encoding its nucleotide sequence as shown in SEQ ID NO: 3, and its amino acid sequence as shown in SEQ ID NO: 4) is increased by more than 1600%, and the ee value of the purified product is >99.9%.

[0057] Example 4: Enzymatic preparation of (R)-1-(2,6-dichlorophenyl)ethanol at gram rate

[0058] Add 180 mL of 0.1 M pH 7.0 potassium phosphate buffer to a 200 mL reaction system. The final concentration of the substrate 2',6'-dichloroacetophenone is 10 g / L, the volume percentage of isopropanol is 10%, the volume percentage of DMSO is 10%, the final concentration of disodium NADP is 0.2 g / L, and the final concentration of ketone reductase mutant F147L / Y190P / V196L (encoding its nucleotide sequence as shown in SEQ ID NO: 3, and its amino acid sequence as shown in SEQ ID NO: 4) is 1 g / L. React at 35 °C with stirring in a water bath for 18 h. The substrate was completely converted, and the ee value of the purified product was >99.9%.

[0059] Example 5: Enzymatic kilogram-scale preparation of (R)-1-(2,6-dichlorophenyl)ethanol

[0060] To a 10 L reaction system, add 0.1 M pH 7.0 potassium phosphate buffer, 2',6'-dichloroacetophenone substrate to a final concentration of 100 g / L, DMSO (10% by volume), NADP disodium salt to a final concentration of 0.2 g / L, glucose to a final concentration of 20 g / L, GDH enzyme powder to a final concentration of 0.5 g / L, and ketone reductase mutant F147L / Y190P / V196L (encoding its nucleotide sequence as shown in SEQ ID NO: 3, amino acid sequence as shown in SEQ ID NO: 4) enzyme powder to a final concentration of 1 g / L. React at 35 °C with stirring in a water bath for 20 h, and add 10% Na2CO3 solution dropwise to maintain the pH of the system between 6.5 and 7.5. Testing showed complete substrate conversion, and the purified product had an ee value >99.9%.

[0061] The relevant sequence information involved in this invention is as follows:

[0062] SEQ ID NO: 1 (nucleotide sequence of wild-type LkADH ketone reductase)

[0063] ATGACTGATCGTTTAAAAGGCAAAGTAGCAATTGTAACTGGCGGTACCTTGGGAATTGGCTTGGCAATCGCTGATAAGTTTGTTGAAGAAGGCGCAAAGGTTGTTATTACCGGCCGTCACGCTGATGTAGGTGAAAAAGCTGCCAAATCAATCGGCGGCACAGACGTTATCCGTTTTGTCCAACACGATGCTTCTGATGAAGCCGGCTGGACTAAGTTGTTTGATACGACTGAAGAAGCATTTGGCCCAGTTACCACGGTTGTCAACAATGCCGGAATTGCGGTCAGCAAGAGTGTTGAAGATACCACAACTGAAGAATGGCGCAAGCTGCTCTCAGTTAACTTGGATGGTGTCTTCTTCGGTACCCGTCTTGGAATCCAACGTATGAAGAATAAAGGACTCGGAGCATCAATCATCAATATGTCATCTATCGAAGGTTTTGTTGGTGATCCAACTCTGGGTGCATACAACGCTTCAAAAGGTGCTGTCAGAATTATGTCTAAATCAGCTGCCTTGGATTGCGCTTTGAAGGACTACGATGTTCGGGTTAACACTGTTCATCCAGGTTATATCAAGACACCATTGGTTGACGATCTTGAAGGGGCAGAAGAAATGATGTCACAGCGGACCAAGACACCAATGGGTCATATCGGTGAACCTAACGATATCGCTTGGATCTGTGTTTACCTGGCATCTGACGAATCTAAATTTGCCACTGGTGCAGAATTCGTTGTCGATGGTGGATACACTGCTCAATAA

[0064] SEQ ID NO: 2 (Amino acid sequence of wild-type LkADH ketoreductase)

[0065] MTDRLKGKVAIVTGGTLGLAIADKFVEEGAKVVITGRHADVGEKAAKSIGGTDVIRFVQHDASDEAGWTKLFDTTEEAFGPVTTVVNNAGIAVSKSVEDTTTEEWRKLLSVNLDGVFFGTRLGI QRMKNKGLGASIINMSSIEGFVGDPTLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLEGAEEMMSQRTKTPMGHIGEPNDIAWICVYLASDESKFATGAEFVVDGGYTAQ

[0066] SEQ ID NO: 3 (nucleotide sequence of F147L / Y190P / V196L ketone reductase mutant)

[0067] ATGACTGATCGTTTAAAAGGCAAAGTAGCAATTGTAACTGGCGGTACCTTGGGAATTGGCTTGGCAATCGCTGATAAGTTTGTTGAAGAAGGCGCAAAGGTTGTTATTACCGGCCGTCACGCTGATGTAGGTGAAAAAGCTGCCAAATCAATCGGCGGCACAGACGTTATCCGTTTTGTCCAACACGATGCTTCTGATGAAGCCGGCTGGACTAAGTTGTTTGATACGACTGAAGAAGCATTTGGCCCAGTTACCACGGTTGTCAACAATGCCGGAATTGCGGTCAGCAAGAGTGTTGAAGATACCACAACTGAAGAATGGCGCAAGCTGCTCTCAGTTAACTTGGATGGTGTCTTCTTCGGTACCCGTCTTGGAATCCAACGTATGAAGAATAAAGGACTCGGAGCATCAATCATCAATATGTCATCTATCGAAGGTctgGTTGGTGATCCAACTCTGGGTGCATACAACGCTTCAAAAGGTGCTGTCAGAATTATGTCTAAATCAGCTGCCTTGGATTGCGCTTTGAAGGACTACGATGTTCGGGTTAACACTGTTCATCCAGGTCCTATCAAGACACCATTGCTGGACGATCTTGAAGGGGCAGAAGAAATGATGTCACAGCGGACCAAGACACCAATGGGTCATATCGGTGAACCTAACGATATCGCTTGGATCTGTGTTTACCTGGCATCTGACGAATCTAAATTTGCCACTGGTGCAGAATTCGTTGTCGATGGTGGATACACTGCTCAATAA

[0068] SEQ ID NO: 4 (Amino acid sequence of the ketoreductase mutant F147L / Y190P / V196L)

[0069] MTDRLKGKVAIVTGGTLGIGLAIADKFVEEGAKVVITGRHADVGEKAAKSIGGTDVIRFVQHDASDEAGWTKLFDTTEEAFGPVTTVVNNAGIAVSKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKNKGLGASIINMSSIEGLVGDPTLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGPIKTPLLDDLEGAEEMMSQRTKTPMGHIGEPNDIAWICVYLASDESKFATGAEFVVDGGYTAQ

[0070] SEQ ID NO:5(F147L-F)

[0071] ATATGTCATCTATCGAAGGTCTGGTTGGTGATCCAACTCTGGG

[0072] SEQ ID NO:6(F147L-R)

[0073] CCCAGAGTTGGATCACCAACcagACCTTCGATAGATGACATAT

[0074] SEQ ID NO:7(Y190P-F)

[0075] GGTTAACACTGTTCATCCAGGTCCTATCAAGACACCATTGGTTGAC

[0076] SEQ ID NO:8(Y190P-R)

[0077] GTCAACCAATGGTGTCTTGATAGGACCTGGATGAACAGTGTTAACC

[0078] SEQ ID NO:9(V196L-F)

[0079] ATCAAGACACCATTGCTGGACGATCTTGA

[0080] SEQ ID NO:10(V196L-R)

[0081] TCAAGATCGTCCAGCAATGGTGTCTTGAT

[0082] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Use of a ketoreductase mutant in catalyzing the reduction of 2',6'- dichloroacetophenone to (R)-1-(2,6-dichlorophenyl)ethanol, characterized in that, The ketoreductase is derived from Lactobacillus kefiri, and its wild-type amino acid sequence is shown as SEQ ID NO: 2, and only the following combined mutations occur in the amino acid sites: F147L, Y190P and V196L.

2. Use according to claim 1, characterized in that, The application is carried out by synthesizing (R) -1-(2, 6-dichlorophenyl) ethanol under the action of the ketoreductase mutant of claim 1 in the presence of a coenzyme cycle system, coenzyme, 2', 6'-dichloroacetophenone and a cosolvent in a buffer solution to prepare an optically chiral alcohol.

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