Ketoreductase mutants and uses thereof

Abstract

This application relates to the field of genetic engineering technology, specifically to a ketone reductase mutant and its uses. The ketone reductase mutant is formed by one or more amino acid mutations in the amino acid sequence of the wild-type ketone reductase shown in SEQ ID NO. 2, which is derived from *Chryseobacterium* sp. When the ketone reductase and its mutant of this invention are used to resolve milobalin intermediates, the chiral purity of the target product is greater than 99.9%. Compared with currently reported methods for synthesizing target products, this invention avoids the use of metal catalysts when using ketone reductase and its mutant for target product synthesis, making the process greener, more environmentally friendly, and reducing costs. Furthermore, this ketone reductase mutant simplifies the resolution process while improving the chiral purity of the target product, resulting in a simple synthesis method.

Description

Technical Field

[0001] This application relates to the field of genetic engineering technology, specifically to a ketone reductase mutant and its uses. Background Technology

[0002] Currently, the synthesis of milobalin intermediate (compound II, (1R,5S)-3-ethylbicyclo[3.2.0]hepten-3-en-6-one) is mostly carried out using chemical methods (CN103987687B). In these chemical processes, the use of metal catalysts leads to high production costs, and in order to obtain a product with high chiral purity, some of the target product is lost, ultimately resulting in a low yield of the final product. Enzyme catalysis is a mild, efficient, and green chemical technology that has attracted much attention from industry and academia in recent years due to its excellent performance in asymmetric synthesis and chiral resolution. Utilizing the high specificity, exactness, and stereoselectivity of enzymes, the efficient resolution of key milobalin intermediates can be achieved. CN104245951B provides a commercial ketone reductase for resolving compound II, an intermediate of milobalin. However, the chiral purity of the final product (compound II) obtained by this enzyme is low at 98.85% (ee: 97.7%), which requires additional purification steps in subsequent synthesis steps and does not meet the current requirement of chiral purity greater than 99.5% (ee: 99%) for this type of pharmaceutical intermediate.

[0003] Therefore, it is of great significance to provide a highly active and selective ketone reductase for the separation of key intermediates of milobalin. Summary of the Invention

[0004] Technical issues

[0005] Metal catalysts used for the resolution of milobalin intermediates result in high production costs and low final product yields, while existing ketone reductases for the resolution of milobalin intermediates lead to complex subsequent synthetic steps and fail to meet the current requirement of chiral purity greater than 99.5% for this type of pharmaceutical intermediate. This invention aims to provide a ketone reductase for the resolution of key milobalin intermediates, which not only possesses high activity and selectivity but also simplifies subsequent resolution process steps.

[0006] Technical solution

[0007] The first aspect of this application provides a ketone reductase mutant, which is formed by any one of the following mutations in the amino acid sequence of the wild-type ketone reductase shown in SEQ ID NO. 2:

[0008] H145X1+H42X+Q97X+S153X+A150X+L194X, wherein X1 is one of H (histidine), W (tryptophan), Y (tyrosine) or F (phenylalanine); X is one of V (valine), G (glycine), A (alanine), L (leucine), I (isoleucine), F (phenylalanine), Y (tyrosine), P (proline), C (cysteine), M (methionine), S (serine), D (aspartic acid), N (asparagine), T (threonine), E (glutamic acid), Q (glutamine), H (histidine), K (lysine), R (arginine) or W (tryptophan).

[0009] In some embodiments, the ketone reductase mutant is any one of the following mutations occurring on the amino acid sequence of the wild-type ketone reductase shown in SEQ ID NO.2: H145W, H145Y, H145F, H145, H145W+H42S, H145W+H42T, H145W+H42I, H145W+H42V, H145Y+H42S, H145Y+H42T, H145Y+H42I, H145Y+H42V, H145F+H42S, H145F+H42T, H145F+H42I, H145F+H42V, H145+H42S, H145+H42T, H145+H42I, H145+H42V, H145W+H 42S+Q97A, H145W+H42T+Q97L, H145W+H42I+Q97I, H145W+H42V+Q97V, H1 45F+H42S+Q97A, H145F+H42T+Q97L, H145F+H42I+Q97I, H145F+H42V+Q9 7V, H145+H42S+Q97A, H145+H42T+Q97L, H145+H42I+Q97I, H145+H42V+Q 97V, H145W+H42S+Q97A+S153I, H145W+H42T+Q97L+S153L, H145W+H42I+Q 97I+S153T, H145W+H42V+Q97V+S153V, H145Y+H42S+Q97A+S153I, H145Y +H42T+Q97L+S153L、H145Y+H42I+Q97I+S153T、H145Y+H42V+Q97V+S153 V、H145Y+A91V+S153L+Q97L、H145Y+A91T+S153L+Q97L、H145+H42S+Q97 A+S153I, H145+H42T+Q97L+S153L, H145+H42I+Q97I+S153T, H145+H42V+ Q97V+S153V+A91T, H145Y+H42S+Q97A+S153I+A91V, H145Y+H42T+Q97L+ S153L+A91F, H145Y+H42S+Q97A+S153V+A150L+L194T, H145Y+H42S+Q97 A+S153V+A150L+A91T, H145Y+H42T+Q97V+S153V+A150L+A91A, H145F+H 42S+Q97A+S153V+A150L+L194T, H145F+H42S+Q97A+S153V+A150L+A91T,The following amino acids are listed: H145F+H42T+Q97V+S153V+A150L+A91A, H145+H42S+Q97A+S153V+A150L+L194T, H145+H42S+Q97A+S153V+A150L+A91T, H145+H42T+Q97V+S153V+A150L+A91A, H145+H42S+Q97A+S153V+A150L+L194T, H145W+H42S+Q97A+S153V+A150L+A91T, and H145W+H42T+Q97V+S153V+A150L+A91A; where H145 indicates that the amino acid at position 145 remains unchanged. ,

[0010] In some embodiments, the wild-type ketone reductase is derived from Chryseobacterium sp., Flavobacterium sp., and Lentilactobacillus sp. or other ketone reductases with a sequence similarity greater than 90%.

[0011] A second aspect of this application provides a DNA molecule, said DNA molecule being a nucleotide sequence encoding the above-mentioned ketone reductase mutant or its complementary sequence.

[0012] A third aspect of this application provides a host cell containing the aforementioned DNA molecules.

[0013] In some embodiments, the host cell is a competent cell.

[0014] In some embodiments, the host cell is Escherichia coli BL21(DE3).

[0015] The fourth aspect of this application provides the use of the aforementioned ketone reductase mutant in the preparation of milobalin intermediates.

[0016] The fifth aspect of this application provides the use of wild-type ketone reductase in the preparation of milobalin intermediates, the amino acid sequence of which is shown in SEQ ID NO.2.

[0017] The sixth aspect of this application provides a method for preparing the milobalin intermediate (1R,5S)-3-ethylbicyclo[3.2.0]hepta-3-en-6-one, comprising the following steps: using a wild-type ketone reductase with the amino acid sequence shown in SEQ ID NO.2 or using any of the above-mentioned ketone reductase mutants, in the presence of a coenzyme, a hydrogen donor, and a dehydrogenase, catalyzing the reduction reaction of compound III in a racemic mixture to compound IV in a buffer solution, and then separating the milobalin intermediate (1R,5S)-3-ethylbicyclo[3.2.0]hepta-3-en-6-one of formula II:

[0018]

[0019] In some embodiments, the coenzyme is NAD. + / NADH or NADP + / NADPH; the hydrogen donor is one or more of formic acid, formate, glucose or isopropanol.

[0020] In some embodiments, the dehydrogenase is glucose dehydrogenase or formate dehydrogenase; wherein the glucose dehydrogenase is used simultaneously with glucose, and the formate dehydrogenase is used simultaneously with formic acid or formate salt; when the hydrogen donor is isopropanol, no dehydrogenase needs to be added.

[0021] Technical effect

[0022] This invention provides a ketone reductase derived from natural enzyme mutations with better activity and selectivity. Its catalytic target product achieves 100% chiral purity resolution and a yield of 48%, representing a significant improvement.

[0023] This invention uses a bioenzymatic method to replace the chemical method, avoiding the use of metal catalysts and organic reagents such as pyridine, thus achieving the green chemical synthesis of advanced pharmaceutical intermediates while reducing production costs, which has significant advantages.

[0024] The ketone reductase mutant of the present invention simplifies the process steps for resolving the milobalin intermediate, and the synthesis method is simple. This route is a proposed commercial production route and is practical. Attached Figure Description

[0025] Figure 1 The image shows the controlled HPLC chromatogram of the target product in Example 6;

[0026] Figure 2 The image shows the controlled HPLC chromatogram of the target product in Example 7;

[0027] Figure 3 The image shows the chiral HPLC chromatogram of the target product in Example 7. Detailed Implementation

[0028] To facilitate the explanation of the technical solution of this application, the following is a general explanation and definition of the terms and expressions used in this application.

[0029] The terms “comprising,” “including,” or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase “comprising one…” does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0030] In the comparative experiments provided in this application, unless otherwise specified, all other experimental conditions and materials are kept consistent to ensure comparability.

[0031] Unless otherwise specified, all reagents and instruments used in the embodiments of this invention can be purchased from the market.

[0032] The following provides a further description of a ketone reductase mutant provided in this application and its uses.

[0033] Example 1: Screening of target ketone reductase

[0034]

[0035] Screening principle: Compound I is a racemic mixture comprising compounds of formula II and formula III. Ideally, the target ketone reductase can selectively reduce compound III in compound I to compound IV, from which the target compound, formula II, can be obtained. When over-reduction occurs, compound II in compound I will be reduced to compound V. This can lead to synthesis failure of the target compound or a decrease in the chiral purity of the product. Therefore, by analyzing the composition and content of the product, target ketone reductases can be screened.

[0036] Measure 20 mL of phosphate buffer (pH 7.0), then weigh out the coenzyme NADP. +20 mg of nicotinamide adenine dinucleotide phosphate (NAPDP), 40 mg of glucose dehydrogenase, and 600 mg of glucose were added to a buffer solution and mixed well. 200 μL of this buffer solution was added to each well of a 96-well plate containing the enzyme. 10 μL of compound I was added to each well of the same 96-well plate. After reacting at 30°C for 18 h, the mixture was quenched and diluted with 800 μL of methanol before being sent for analysis. The screening results are shown in Table 1. Enzyme #7, which showed the best overall catalytic performance, was a ketone reductase derived from *Chryseobacterium* sp. (its sequence was retrieved from the NCBI database and synthesized by General Biotech).

[0037] Table 1: Ketone reductase screening

[0038]

[0039] Example 2: Construction of a ketone reductase mutant library derived from Chryseobacterium sp.

[0040] Using the ketone reductase gene of *Chryseobacterium* sp. as a template, the nucleotide sequence of the parental template LG-23 is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. Structural analysis was performed using the molecular structure simulation tool (Chimera). Based on the protein structure analysis results, target amino acid mutation sites were selected: H145, H42, Q97, S153, A150, A91, and L194. Multiple primers targeting these mutation sites were designed and synthesized (see Table 2). After obtaining the primers, the PCR reaction conditions were optimized (pre-denaturation: 98℃, 3 min; denaturation: 98℃, 10 s; annealing: 57℃, 15 s; extension: 72℃, 3 min; final extension: 72℃, 5 min; number of cycles: 30) to amplify the gene and obtain the PCR product. The PCR products were digested, concentrated and desalted using a DNA purification kit (Beyotime, product number: D0033), and then transformed into *E. coli* competent cells BL21(DE3). The cells were plated on kanamycin-resistant plates and incubated overnight at 37°C inverted mode to obtain single colonies. The next day, the single colonies were picked and transferred to 96-well plates (H145, H42, Q97, S153, and L194), respectively. 300 μL of LB (Luria-Bertani) medium was added, and the cells were incubated overnight. After preserving 100 μL of the culture, 400 μL of LB medium was added, and the cells were incubated at 37°C for approximately 5 hours. Then, 100 μL of LB medium containing isopropyl-β-D-thiogalactoside (IPTG) was added, and the cells were induced to culture overnight at 25°C. The supernatant was removed by centrifugation to obtain the ketone reductase mutant library.

[0041] Table 2 Sequences of upstream and downstream mutant primers

[0042] Primer Name Primer Sequence (5’-3’) H145-F GGCATCTATCNNKGGTATCGTTGCGGCGCCGCT H145-R AGCGGCGCCGCAACGATACCMNNGATAGATGCC H42-F TCAACGAAGATNNKGGTAACAAAGCGGTT H42-R AACCGCTTTGTTACCMNNATCTTCGTTGA Q97-F TATCGGTGGTGAANNKGCGCTGGCGGGTGA Q97-R TCACCCGCCAGCGCMNNTTCACCACCGATA S153-F GCGCCGCTGNNKAGCGCTT S153-R AAGCGCTMNNCAGCGGCGC A150-F CGTTGCGNNKCCGCTGTCTAGCGCTT A150-R AAGCGCTAGACAGCGGMNNCGCAACG A91-F GCATGCAACAACNNKGGTATCGGTGGTGAACAGG A91-R CCTGTTCACCACCGATACCMNNGTTGTTGCATGC L194-F CATCGAAACCCCGCTGNNKGAATCTCT L194-R AGAGATTCMNNCAGCGGGGTTTCGATG

[0043] Note: H145-F: SEQ ID NO.3; H145-R: SEQ ID NO.4; H42-F: SEQ ID NO.5; H42-R: SEQ ID NO.6; Q97-F: SEQ ID NO.7; Q97-R: SEQ ID NO.8; S153-F: SEQ ID NO.9; S153-R: SEQ ID NO.10; A150-F: SEQ ID NO.11; A150-R: SEQ ID NO.12; A91-F: SEQ ID NO.13; A91-R: SEQ ID NO.14; L194-F: SEQ ID NO.15; L194-F: SEQ ID NO.16.

[0044] Example 3: Screening and Sequencing Identification of Mutant Libraries

[0045] Measure 20 mL of phosphate buffer (pH 7.0), then weigh out NADP. + 20 mg of glucose dehydrogenase, 40 mg of glucose, and 600 mg of glucose were added to phosphate buffer (pH 5.0-9.0) and mixed well. 200 μL of the above mixture was added to each well of the prepared Chryseobacterium sp. ketoreductase mutant library. The bacterial cells were resuspended, and then 5 μL of compound I was added. The mixture was shaken and incubated at 15-30°C and 300 rpm for approximately 8-18 hours. The reaction was quenched with 800 μL of methanol, centrifuged, and analyzed by liquid chromatography to detect the compound results. Bacteria from the wells with the best reaction were then recultured and sent to General Biotechnology for gene sequencing. Analysis showed that the amino acid at position 145 was the key amino acid controlling the enzyme's selectivity (affecting the resolution). Amino acid steric hindrance analysis, as shown in Table 3, showed that when histidine at position 145 was replaced with sterically more sterically hindered tyrosine (H145Y), tryptophan (H145W), or phenylalanine (H145F), the enzyme selectivity was better, and fewer over-reduction products (compound V) were detected in the product. When the amino acid is mutated to a less sterically hindered amino acid (H145A, H145G), the selectivity becomes significantly worse, the over-reduction product (compound V) increases, and compound V may even be the main reduction product.

[0046] Table 3: Partial experimental results and sequencing results of one round of saturation mutation

[0047]

[0048] Note: ++ indicates that under the experimental and detection conditions, the main compounds in the system are II (target compound) and IV; - indicates that under the experimental and detection conditions, in addition to compounds II and IV, the proportion of compound V increases; -- indicates that under the experimental and detection conditions, the selectivity of the enzyme undergoes a fundamental change, that is, the main products are compounds III and V.

[0049] Example 4: Preparation and Screening of Combinatorial Mutants

[0050] Multiple target primers were designed based on a single-point mutation approach. PCR reaction conditions were optimized for gene amplification. The PCR product was digested, concentrated and desalted using the kit, and then transferred to *E. coli* competent cells BL21(DE3). The cells were plated on kanamycin-containing plates and incubated overnight at 37°C inverted to obtain single colonies. The next day, the single colonies were transferred to 96-well plates, 300 μL of LB medium was added, and the cells were incubated overnight. After preserving 100 μL of the culture, 400 μL of LB medium was added, and the cells were incubated at 37°C for approximately 5 hours. Then, 100 μL of LB medium containing IPTG was added, and the cells were induced to grow overnight at 25°C. The supernatant was removed by centrifugation to obtain the mutated enzyme library.

[0051] Measure 20 mL of phosphate buffer (pH 7.0), then weigh 20 mg NADP, 40 mg glucose dehydrogenase, and 600 mg glucose, and add them to the buffer solution and mix well. Take the prepared Chryseobacterium sp. ketoreductase mutant library, add 200 μL of the above mixture to each well, resuspend the bacterial cells, add 5 μL of compound I, shake well, and react at 30°C and 300 rpm for about 16 h. Quench the reaction with 800 μL of methanol, centrifuge, and send for sample analysis. The test results of the combined mutants are shown in Table 4.

[0052] Table 4. Results of activity tests on the combined mutants

[0053]

[0054]

[0055]

[0056] Note: H145 indicates that the amino acid at position 145 remains unchanged; + indicates an activity increase of 20%-50% compared to the first round, ++ indicates an activity increase of 50%-100%, and +++ indicates an activity increase of more than 100%. / indicates that there is no significant difference in enzyme selectivity after mutation; * indicates that under the experimental and detection conditions, the proportion of V increases, except for compounds II and IV; ** indicates that under the experimental and detection conditions, the enzyme selectivity undergoes a fundamental change, i.e., the main products are compounds III and V.

[0057] Example 5: Comparison of dominant mutants and wild-type enzymes

[0058] Weigh out 50 mL of phosphate buffer (200 mM, pH 7.0), and add 7.3 g of glucose, 0.5 g of glucose dehydrogenase, and 50 mg of NAD+ sequentially. + 3.3 g of ketoreductase mutant H145Y+H42S+Q97A+S153V+A150L+L194T cells and wild-type enzyme cells were used. Finally, 5.0 g of compound I was weighed and added to each reaction system, and the reaction was carried out at 30°C. During the reaction, the pH was adjusted using 10% sodium hydroxide solution to maintain the pH of the reaction system at 7.0-7.5. When the conversion rate of the mutant ketoreductase was 30.89%, the conversion rate of the wild-type enzyme was 17.85%, indicating that the mutant ketoreductase has higher activity. At this point, no obvious over-reduction product (compound V) was produced by the mutant ketoreductase, indicating that the mutant enzyme has higher activity and substrate specificity.

[0059] Example 6: Reaction test using isopropanol as a hydrogen source in the system

[0060] Weigh out 10g of isopropanol and 50mg of NAD in 50mL of phosphate buffer (200Mm, pH 7.0). + The enzyme solution was broken up with 10g of the ketone reductase mutant H145Y+H42S+Q97A+S153V+A150L+L194T. Finally, 5.0g of compound I was weighed and added to the reaction system, and the reaction was carried out at 30℃. The intermediate control results showed that (…). Figure 1 The ketone reductase provided by this invention can catalyze reactions using isopropanol as a hydrogen source, and exhibits good selectivity (few over-reduction products). When isopropanol is used as a hydrogen donor, there is no need to add dehydrogenase, and the step of adjusting pH is also eliminated. Experiments showed that the conversion rate is slower when isopropanol is used as a hydrogen source compared to the glucose system.

[0061] Example 7: Scale-up Validation of Dominant Mutants

[0062] Add 3L of water, 14.97L of sodium dihydrogen phosphate dihydrate, 180.54g of disodium hydrogen phosphate dodecahydrate, and 438g of glucose monohydrate to a 10L reactor, followed by 3.0g of NAD. +30g of glucose dehydrogenase, 600g of the lysed mutant enzyme H145Y+H42S+Q97A+S153V+A150L+L194T, and finally 300g of compound I were added to the reaction system. The reaction was started at 30℃. During the reaction, the pH was adjusted with 10% sodium hydroxide solution to maintain the pH of the reaction system at 7.0-7.5. Samples were taken during the reaction for monitoring. After the reaction was completed, the pH was adjusted by 1-2 with concentrated hydrochloric acid, stirred for 30 min, and then 100g of diatomaceous earth was added and filtered. The aqueous phase was extracted with 300mL of methyl tert-butyl ether (MTBE), and 500mL of MTBE was used to slurry the diatomaceous earth. The organic phases were separated and combined. The organic solvent was removed by rotary evaporation of the organic phase to obtain a mixture of milobalin intermediates. When the chiral purity of the target product compound II was 99.89%, the content of the over-reduction product (compound V) was 0.76%. See the monitoring chart below. Figure 2 .

[0063] DMAP (26 g) and succinic anhydride (210 g) were added to the mixture in this order, and the mixture was stirred at 60 °C for 8 hours. 400 mL of water was added, and the mixture was extracted twice with TBME (400 mL each time). The mixture was then washed with 5% potassium carbonate aqueous solution (2.0 L each time) and concentrated. Compound II was given in 145.1 g, chiral purity 99.96%, yield 48.3% (liquid chromatography, theoretical yield 50%). Figure 3 ).

[0064] Scale-up verification experiments show that the ketone reductase mutant of the present invention is adapted to commercial production routes and has practical applications.

[0065] The above specific embodiments further illustrate the purpose, technical solution and beneficial effects of this application. It should be understood that the above are only specific embodiments of this application and are not intended to limit the scope of protection of this application. Any modifications, equivalent substitutions, improvements, etc., made on the basis of the technical solution of this application should be included within the scope of protection of this application.

Claims

1. A ketone reductase mutant, characterized in that, The ketone reductase mutant is formed by any one of the following mutations in the amino acid sequence of the wild-type ketone reductase shown in SEQ ID NO.2: H145X1+H42X+Q97X+S153X+A150X+L194X, where X1 is one of H, W, Y or F; X is one of V, G, A, L, I, F, Y, P, C, M, S, D, N, T, E, Q, H, K, R or W.

2. The ketone reductase mutant according to claim 1, characterized in that, The ketoreductase mutant is any one of the following mutations occurring in the amino acid sequence of the wild-type ketoreductase shown in SEQ ID NO.2: H145W, H145Y, H145F, H145, H145W+H42S, H145W+H42T, H145W+H42I, H145W+H42V, H145Y+H42S, H145Y+H42T, H145Y+H42I, H145Y+H42V, H145F+H42S, H145F+H42T, H145F+H42I, H145F+H42V. H145+H42S, H145+H42T, H145+H42I, H145+H42V, H145W+H42S+Q97A, H145W+H42T+Q97L, H145W+H42I+Q97I, H145W+ H42V+Q97V, H145F+H42S+Q97A, H145F+H42T+Q97L, H145F+H42I+Q97I, H145F+H42V+Q97V, H145+H42S+Q97A, H145+H 42T+Q97L, H145+H42I+Q97I, H145+H42V+Q97V, H145W+H42S+Q97A+S153I, H145W+H42T+Q97L+S153L, H145W+H42I+ Q97I+S153T, H145W+H42V+Q97V+S153V, H145Y+H42S+Q97A+S153I, H145Y+H42T+Q97L+S153L, H145Y+H42I+Q97I+S1 53T, H145Y+H42V+Q97V+S153V, H145+H42S+Q97A+S153I, H145+H42T+Q97L+S153L, H145+H42I+Q97I+S153T, H145Y+H42S+Q97A+S153V+A150L+L194T, H145F+H42S+Q97A+S153V+A150L+L194T or H145+H42S+Q97A+S153V+A150L+L194T.

3. The ketone reductase mutant according to claim 1, characterized in that, The wild-type ketone reductase is derived from Chryseobacterium sp.

4. A DNA molecule, characterized in that, The DNA molecule is a nucleotide sequence encoding the ketone reductase mutant as described in claim 1 or 2, or its complementary sequence.

5. A host cell, characterized in that, The host cell contains the DNA molecule as described in claim 4; wherein the host cell is Escherichia coli BL21(DE3).

6. Use of the ketone reductase mutant of claim 1 or 2 in the preparation of the milobalin intermediate (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one.

7. The use of wild-type ketone reductase in the preparation of the milobalin intermediate (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one, characterized in that, The amino acid sequence of the wild-type ketone reductase is shown in SEQ ID NO.

2.

8. A method for preparing the milobalin intermediate (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one, characterized in that, Includes the following steps: Using a wild-type ketone reductase with the amino acid sequence shown in SEQ ID NO.2 or a ketone reductase mutant according to any one of claims 1-2, in the presence of a coenzyme, a hydrogen donor, and a dehydrogenase, the reduction of compound III in a racemic mixture to compound IV is catalyzed in a buffer solution, yielding the milobalin intermediate (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one of formula II:

9. The method according to claim 8, characterized in that, The coenzyme is NAD. + / NADH or NADP + / NADPH; the hydrogen donor is one or more of formic acid, formate, glucose or isopropanol.

10. The method according to claim 9, characterized in that, The dehydrogenase is glucose dehydrogenase or formate dehydrogenase; wherein the glucose dehydrogenase is used simultaneously with glucose, and the formate dehydrogenase is used simultaneously with formic acid or formate salt; when the hydrogen donor is isopropanol, no dehydrogenase needs to be added.

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