Carbonyl reductase mutants and their use in chromanone reduction

Abstract

The application belongs to the field of molecular biology and enzyme engineering, and discloses a carbonyl reductase mutant and application thereof in chromanone reduction. The mutant protein has significantly improved activity of catalyzing reduction of a carbonyl in 5,7-difluorochroman-4-one to generate an R-configuration alcohol relative to wild-type protein. The mutant strain of the carbonyl reductase with improved activity is constructed, and can be applied to the production process of tegrazan intermediates, and can realize complete conversion of 260 g / L substrate to generate (R)-5,7-difluorochroman-4-ol.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and enzyme engineering, and relates to carbonyl reductase mutants and their application in reduced chromones. Background Technology

[0002] Carbonyl reductases, also known as alcohol dehydrogenases, are a class of biocatalysts that reversibly reduce carbonyl groups to alcohols. To achieve catalytic activity, carbonyl reductases require a coenzyme to transfer hydride ions to the carbonyl substrate. In the catalytic process of carbonyl reductases, the amount of coenzyme used is equal to the molar amount of the substrate. From an economic perspective, appropriate coenzyme regeneration systems are matched to the carbonyl reductase catalytic reaction. With the development of directed evolution technology, the application of carbonyl reductases in the preparation of chiral alcohols is becoming increasingly widespread.

[0003] Cromolol, the reduced form of cromoglycan, is a key intermediate in many drugs, such as tegoprazan, a novel selective reversible H2O drug. + / K + - ATPase proton pump inhibitors competitively block potassium ion binding sites on the proton pump, thus inhibiting ion exchange and preventing the pumping of hydrogen ions, thereby suppressing gastric acid secretion. In 2018, Teglazan was approved in South Korea for the treatment of gastroesophageal reflux disease (GERD), erosive esophagitis (EE), and non-erosive reflux disease (NERD). (R)-5,7-difluorobenzodihydropyran-4-ol is a key intermediate in the synthesis of its chiral module. Figure 1 .

[0004] Currently, there are methods for synthesizing (R)-5,7-difluorobenzodihydropyran-4-ol. For example, using 5,7-difluorobenzodihydropyran-4-one as a substrate, under argon protection, a boron dimethyl sulfide complex is injected into a toluene solution of S-Me-CBS, and an anhydrous tetrahydrofuran solution of the substrate is slowly added dropwise. After standing overnight, the reaction solution is slowly poured into methanol cooled in an ice-water bath. Extraction and recrystallization yield the product (R)-5,7-difluorobenzodihydropyran-4-ol (CN112851646A). However, chemical catalysis inevitably involves the use of hazardous reagents, making the development of green and efficient catalytic technologies imperative. This invention, through directed evolution, obtained a mutant with significantly enhanced activity in converting 5,7-difluorobenzodihydropyran-4-one, laying the foundation for the green biomanufacturing of (R)-5,7-difluorobenzodihydropyran-4-ol. Summary of the Invention

[0005] The purpose of this invention is to provide a carbonyl reductase mutant, its encoding gene, and its applications, thereby improving enzyme activity and substrate structural diversity to meet industrial application requirements. This invention functionally determined and directed the evolution of the enzyme MdADH (GenBank: KAF2156407.1) derived from Myriangium duriaei, obtaining a mutant with significantly enhanced enzyme activity. This mutant is then reduced to (R)-chromanol using chromone and its structural analogues, such as 5,7-difluorobenzodihydropyran-4-one, as substrates.

[0006] The present invention provides a mutant of carbonyl reductase whose protein sequence has at least 90% identity with SEQ ID NO: 1, and is a mutant obtained by substitution mutation at one or more of the sites S12, R17, N20, K80, N199, P201, Q202, Q204, Y238, S244, T251, and W255 in the amino acid sequence 1 to 301 shown in SEQ ID NO: 1.

[0007] Specifically, its amino acid sequence corresponds to the amino acid sequence shown in SEQ ID NO: 1 and contains any of the following substitution mutations: The 199th position of asparagine N is mutated to alanine A and glycine G; The proline P at position 201 is mutated to serine S, threonine T, and tyrosine Y. The glutamine Q at position 202 is mutated to alanine A, glycine G, and valine V; The glutamine Q at position 204 is mutated to methionine M, alanine A, and leucine L; The tyrosine Y at position 238 is mutated to histidine H, serine S, and threonine T. The threonine S at position 244 is mutated to proline P; The threonine T mutation at position 251 is replaced by asparagine N, histidine H, and lysine K. The tryptophan W at position 255 is mutated into alanine A, valine V, and isoleucine I; The glutamine Q at position 204 is mutated to methionine M, and the tyrosine Y at position 238 is mutated to histidine H; The asparagine N at position 199 is mutated to alanine A, the glutamine Q at position 204 is mutated to methionine M, and the tyrosine Y at position 238 is mutated to histidine H. The asparagine N at position 199 is mutated to alanine A, the glutamine Q at position 204 is mutated to methionine M, and the tyrosine Y at position 238 is mutated to histidine H. The glutamine Q at position 204 is mutated to methionine M, the tyrosine Y at position 238 is mutated to histidine H, and the threonine T at position 251 is mutated to alanine A. Arginine R at position 17 is mutated to histidine H, glutamine Q at position 204 is mutated to methionine M, and tyrosine Y at position 238 is mutated to histidine H. The asparagine N at position 199 is mutated to alanine A, the glutamine Q at position 204 is mutated to methionine M, the tyrosine Y at position 238 is mutated to histidine H, and the threonine T at position 251 is mutated to alanine A. The 12th threonine S was mutated to proline P, the 17th arginine R was mutated to histidine H, the 204th glutamine Q was mutated to methionine M, and the 238th tyrosine Y was mutated to histidine H. The 12th threonine S was mutated to proline P, the 17th arginine R was mutated to histidine H, the 199th asparagine N was mutated to alanine A, the 204th glutamine Q was mutated to methionine M, and the 238th tyrosine Y was mutated to histidine H. The 12th threonine S was mutated to proline P, the 17th arginine R was mutated to histidine H, the 80th lysine K was mutated to serine S, the 199th asparagine N was mutated to alanine A, the 204th glutamine Q was mutated to methionine M, and the 238th tyrosine Y was mutated to histidine H. The following mutations occurred: threonine at position 12 (S) was replaced by proline (P); arginine at position 17 (R) was replaced by histidine (H); lysine at position 80 (K) was replaced by serine (S); asparagine at position 199 (N) was replaced by alanine (A); glutamine at position 204 (Q) was replaced by methionine (M); tyrosine at position 238 (Y) was replaced by histidine (H); and threonine at position 251 (T) was replaced by alanine (A).

[0008] The present invention also provides a nucleic acid encoding a mutant of the carbonyl reductase described above.

[0009] The present invention further provides a recombinant expression vector encoding the nucleic acid of the aforementioned carbonyl reductase mutant.

[0010] The present invention also provides recombinant bacteria encoding nucleic acids of the aforementioned carbonyl reductase mutant.

[0011] The present invention also provides a method for reducing chromones, particularly reducing 5,7-difluorobenzodihydropyran-4-one, comprising the following steps:

[0012] The carbonyl reductase mutant protein is contacted with a reaction substrate, and a coenzyme cycle system is added to catalyze the reaction, thereby obtaining the corresponding (R)-chromanol, especially (R)-5,7-difluorobenzodihydropyran-4-ol.

[0013] Specifically, the pH of the reaction system is 6.0-8.0, the reaction time is 12-48 hours, and the substrate concentration is 80-270 g / L.

[0014] The optimal reaction conditions are: pH 6.5; reaction time 15-36 hours.

[0015] Optionally, the method further includes the step of separating and purifying the (R)-5,7-difluorobenzodihydropyran-4-ol.

[0016] Applying the carbonyl reductase mutant strain with enhanced activity from this invention to the production process of Tegrazan intermediates can achieve complete conversion of 80-270 g / L of substrate to generate (R)-5,7-difluorobenzodihydropyran-4-ol, thus making it more practical. Attached Figure Description

[0017] Figure 1 The structures of Teglazan and (R)-5,7-difluorobenzodihydropyran-4-one are given.

[0018] Figure 2 The liquid phase spectrum is that of sodium borohydride reduced from 5,7-difluorobenzodihydropyran-4-one.

[0019] Figure 3 The image shows the liquid phase spectrum of the substrate 5,7-difluorobenzodihydropyran-4-one.

[0020] Figure 4 This is a liquid phase spectrum of an enzyme-catalyzed conversion reaction. Detailed Implementation

[0021] Unless otherwise specified, the experimental techniques and methods used in this embodiment are conventional techniques and methods. For example, experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the embodiments can be obtained through legitimate commercial channels.

[0022] Example 1: Construction of a carbonyl reductase mutant library and screening of mutants

[0023] I. Screening of carbonyl reductase library

[0024] Transformation reactions were performed to construct a laboratory-preserved enzyme library. The substrate was prepared using phosphate buffered solution (pH 7.5): 10 mM (1.8 g / L), 5% DMSO, 50 mM (10 g / L) glucose, 5 U / mL glucose dehydrogenase (GDH), 10 g / L of wet E. coli cells expressing the carbonyl reductase gene, and coenzyme NAD(P). +0.3 g / L, reacted at 30℃ for 24 hours. Extracted with an equal volume of ethyl acetate, primary screening by TLC, and secondary screening by HPLC. The product was reduced with sodium borohydride ( Figure 2 The comparison shows that the carbonyl reductase MdADH can convert the substrate 5,7-difluorobenzodihydropyran-4-one ( Figure 3 Highly efficient and selective reduction. The amino acid sequence of the carbonyl reductase MdADH, SEQ ID NO: 1, is as follows: MPSTDSSDSPLSGGVFRHNNTVAPPASIGVLKLFDLTGKTAIVTGAGAGIGLAVARALAEAGANVAIWYHGNDAALKRAKEISEKYGVKCEAYKSDTTDEAAVKKLVNQNVKDFNGRLDIFVANAGIPWTQGPALDGEASHYRKVVEVDLD GTFYCARAAGDVFRRQKELGTDLAGNELRNWRGGSFIATASMSGHIVNVPQLQAAYNAAKAGVIHLCKSLAVEWVRFARVNTISPGYIATEISNFVPPETKDQWRDRTPMGREGEAEELGGAFLYLASDASSYTTGTDIIVDGGYCLPLE.

[0025] II. First Round of Revitalization Transformation

[0026] MdADH and coenzyme NADP were obtained by RoseTTAFold All-Atom simulation in Science 384, 291 (2024). + Docking results with the substrate 5,7-difluorobenzodihydropyran-4-one.

[0027] The amino acid sites within an 8 Å range were analyzed, with the substrate as the central focus, revealing S192 / Y207 / K211 as a catalytic triplet. Structural analysis was performed, and the following sites were selected for constructing a mutant library: N199, Q202, L203, Q204, A205, Y238, S244, F246, P248, T251, and W255 were subjected to saturation mutations. Degenerate codon NNK was used to design mutation primers, with pET21a-MdADH as the template. A two-step PCR method was employed, using high-fidelity polymerase Pfx.

[0028] 1. Construction of pET21a-MdADH plasmid

[0029] The wild-type amino acid sequence of carbonyl reductase is shown in SEQ ID NO:1. The corresponding nucleotide sequence was fully synthesized and cloned into the restriction endonuclease sites NdeI and XhoI of the pET-21a vector to obtain the recombinant plasmid pET21a-MdADH. This plasmid was further transformed into the expression host E. coli BL21(DE3), and positive clones were selected to obtain the recombinant expression transformant E. coli BL21(DE3) / pET21a-MdADH.

[0030] 2. Construction of a carbonyl reductase mutant library

[0031] Using pET21a-MdADH as a template, amplification was performed using the primers in Table 1. PCR was performed by combining forward primer 42F with reverse primer 199-205-R, and PCR was performed by combining forward primer 174-F with reverse primer 238-255-R. The PCR reaction system and conditions are as follows:

[0032] 1. Step-by-step PCR reaction system and reaction conditions

[0033] Round 1: In a PCR reaction system with a total volume of 25 μL, add 15 ng of template, 12.5 μL of 2×Pfx Mix, 0.5 μL (10 μM) of each of the two mutant primers, and add sterile distilled water to a final volume of 25 μL.

[0034] The first step of the PCR reaction procedure is as follows: ① Pre-denaturation at 98℃ for 2 min, ② Denaturation at 98℃ for 30 sec, ③ Annealing at Tm-5℃ for 30 sec, ④ Extension at 72℃ for 30 sec, ⑤ Final extension at 72℃ for 5 min. Steps ② to ④ are performed for a total of 30 cycles.

[0035] 2. Second step PCR reaction system and reaction conditions

[0036] Round 2: In a PCR reaction system with a total volume of 50 μL, add 30 ng of template, 25 μL of 2×Pfx Mix, 1 μL of primer fragment (the product of the first round of PCR), and distilled water to a final volume of 50 μL.

[0037] The second step of the PCR reaction procedure is as follows: ① Pre-denaturation at 98℃ for 2 min, ② Denaturation at 98℃ for 30 sec, ③ Annealing at 55℃ for 30 sec, ④ Extension at 72℃ for 3 min, ⑤ Final extension at 72℃ for 10 min. Steps ② to ④ are performed for a total of 25 cycles.

[0038] After verifying the PCR products obtained in the above steps by agarose gel electrophoresis, the restriction endonuclease DpnI was added and digested at 37°C for 2 h. The digested products were then transformed into E. coli BL21(DE3) competent cells and plated on plates containing 10 μg / mL ampicillin antibiotic. The plates were then incubated statically at 37°C for about 12 h, resulting in single colonies and the carbonyl reductase mutant library.

[0039] Meanwhile, the pET21a-MdADH mutant library plasmid was transformed into E. coli BL21(DE3) competent cells and plated on a plate containing 100 mg / mL ampicillin antibiotic. The plate was then incubated at 37°C for about 12 h, and a single colony was grown, thus obtaining a strain expressing the carbonyl reductase mutant.

[0040] Table 1. Primer sequences from the first round of modification

[0041]

[0042] 3. Induced expression of carbonyl reductase mutants

[0043] The monoclonal colonies obtained after culturing in step 2 above were picked and transferred to LB liquid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl) containing 4 mL of ampicillin (100 mg / L) and cultured overnight at 37°C and 200 rpm to obtain the culture medium. The culture medium was then inoculated into fermentation medium (LB liquid medium) at a 1% (v / v) inoculation rate and cultured on a shaker at 37°C and 200 rpm until OD reached... 600 The concentration was 0.6-0.8, and IPTG was added to a final concentration of 0.1 mM. The mixture was induced at 25°C and 200 rpm for 8-12 hours. The bacterial cells were collected by centrifugation at 6000 g, and then subjected to high-pressure disruption to obtain a crude carbonyl reductase solution for subsequent enzyme activity assays.

[0044] 4. Screening of carbonyl reductase mutants

[0045] The screening method involved detecting the decrease in NADPH at 340 nm. The specific reaction method was as follows: 10 mM substrate, 0.25 mg / mL NADPH, 10 μL crude enzyme solution, and potassium phosphate buffer to a final volume of 200 μL. NADPH exhibits characteristic absorbance at 340 nm. The decrease in absorbance at 340 nm was detected using a microplate reader; higher enzyme activity resulted in faster NADPH consumption and a steeper slope in the decrease curve. Beneficial mutation sites that enhanced enzyme activity were identified through screening: S12, R17, N20, K80, N199, P201, Q202, Q204, Y238, S244, T251, and W255.

[0046] III. The Second Round of Revitalization Transformation

[0047] Based on the results of saturation mutation, single-mutation site combination mutants were constructed by selecting sites with significantly improved activity. The resulting mutants were cultured in test tubes containing 4 mL of LB medium. The activity of the expressed protein was detected. The results of significantly improved activity are shown in Table 2. Single mutants 9 and 11 had the highest activity, which was 35.3 and 30.8 times higher than that of wild type, respectively.

[0048] Table 2. Relative activity of KaCR and its mutants against substrates

[0049]

[0050] Using the single mutant 9Q204M as a template, double mutants of N199, P201, Q202, Y238, S244, T251, and W255 were constructed, yielding the optimal double mutant 22Q204M / Y238H, which increased enzyme activity to 48.2 times that of the wild type. Using double mutants 22 and Q204M / Y238H as templates, saturation mutations were further performed on N199, P201, Q202, S244, T251, and W255, resulting in triple mutants, etc. The enzyme activities of N199A / Q204M / Y238H and Q204M / Y238H / T251A were increased by 68.2 and 79.8 times, respectively. Using the triple mutant 24Q204M / Y238H / T251A as a template, further mutations were performed on N199, P201, Q202, S244, and W255, resulting only in the quadruple mutant 25, N199A / Q204M / Y238H / T251A, which showed an increase in activity of 92.6 times.

[0051] Using the machine learning model EvolveProScience, 2025, 387(6482), the optimal three-mutant 26, R17H / Q204M / Y238H, four-mutant 27, S12P / R17H / Q204M / Y238H, five-mutant 28, S12P / R17H / N199A / Q204M / Y238H, and six-mutant were obtained through data feeding. 29. S12P / R17H / K80S / N199A / Q204M / Y238H, and seven mutants 30 and S12P / R17H / K80S / N199A / Q204M / Y238H / T251A, showed increased activity of 126.5, 145.6, 159.8, 153.6, and 185.3%, respectively. All mutants exhibited stereoselectivity in the R configuration, with ee > 98%.

[0052] Example 2: Substrate spectrum expansion of mutants

[0053] Transformation reactions were constructed using the obtained mutants 26-30 to transform the substrates. Specific enzyme activity was measured in 100 mM potassium phosphate buffer at pH 6.5. Substrate concentrations were as follows: 5 mM for 1a, 2a, 3a, 8a, 12a, and 14a; 4 mM for 5a, 10a, and 13a; 3 mM for 20a; 2 mM for 7a and 11a; 1 mM for 6a, 9a, 17a, 18a, and 19a; 0.5 mM for 16a and 21a; 0.4 mM for 4a; and 0.2 mM for 15a.

[0054] The structural formula of the substrate is as follows:

[0055] .

[0056] The conversion results are shown in Table 3.

[0057] Table 3. Enzyme activity detection results of wild-type enzymes and mutants.

[0058]

[0059] Example 3: Whole-cell catalytic synthesis of (R)-5,7-difluorobenzodihydropyran-4-ol using mutant 26

[0060] Mutant 26 was obtained by inducing expression according to the method in Example 1. The bacterial cells were collected by centrifugation (6000 rpm) and used as a biocatalyst. The bacterial cells were resuspended in 100 mL of potassium phosphate buffer (pH 7.0, 100 mM), 80 g / L 5,7-difluorobenzodihydropyran-4-one, 10% / v DMSO, 100 g / L glucose, and 0.3 g / L NADP. + The mixture was prepared with 30 U / mL LGDH and 10 g / L wet bacterial cells, and reacted at 30°C for 12 hours, with the pH controlled between 6.5 and 7.2 during the reaction. After the reaction, HPLC analysis showed a conversion rate of 98% and an ee value of 99.28%.

[0061] Example 4: Synthesis of (R)-5,7-difluorobenzodihydropyran-4-ol catalyzed by the lysis broth of mutant 26.

[0062] Mutant 26 was obtained by inducing expression according to the method in Example 1. The bacterial cells were collected by centrifugation (6000 rpm), and the crude enzyme solution after lysis was used as a biocatalyst. Other conditions were the same as in Example 2, with a reaction time of 10 hours, a conversion rate of 99%, and a product de value of 99.59%.

[0063] Example 5: Whole-cell catalytic synthesis of (R)-5,7-difluorobenzodihydropyran-4-ol using mutant 27

[0064] Mutant 10 was obtained by inducing expression according to the method in Example 1. The bacterial cells were collected by centrifugation (6000 rpm) and used as a biocatalyst. The bacterial cells were resuspended in 100 mL of potassium phosphate buffer (pH 7.0, 100 mM), 160 g / L 5,7-difluorobenzodihydropyran-4-one, 200 g / L glucose, and 0.2 g / L NADP. + The reaction was carried out at 30 U / mL LGDH and 10 mg / mL whole cells at 30°C for 15 hours, with the pH controlled between 6.5 and 7.2 during the reaction. After the reaction, HPLC analysis showed a conversion rate of 99% and an ee value of 99.67% for the product.

[0065] Example 6: Whole-cell catalytic synthesis of (R)-5,7-difluorobenzodihydropyran-4-ol using mutant 28

[0066] Following the method described in Example 1, mutant 10 was obtained by inducing expression. The bacterial cells were collected by centrifugation (6000 rpm) and used as a biocatalyst. The bacterial cells were resuspended in 300 mL of potassium phosphate buffer (pH 7.0, 100 mM), 1405,7-difluorobenzodihydropyran-4-one, 180 g / L glucose, and 0.2 g / L NADP. + The reaction was carried out at 25°C for 15 hours with 30 U / mL LGDH and 5 mg / mL wet bacterial cells, while maintaining the pH between 6.5 and 7.2 during the reaction. After the reaction, HPLC analysis showed a conversion rate of 97% and an ee value of 99.78% for the product.

[0067] Example 7: Whole-cell catalytic synthesis of (R)-5,7-difluorobenzodihydropyran-4-ol using mutant 29

[0068] Mutant 29 was obtained by inducing expression according to the method in Example 1. The bacterial cells were collected by centrifugation (6000 rpm) and used as a biocatalyst. The bacterial cells were resuspended in 300 mL of potassium phosphate buffer (pH 7.0, 100 mM), 240 g / L 5,7-difluorobenzodihydropyran-4-one, 280 g / L glucose, and 0.2 g / L NADP. + The mixture was prepared with 30 U / mL LGDH and 10 mg / mL wet bacterial cells, and reacted at 35°C for 10 hours, with the pH controlled between 6.5 and 7.2 during the reaction. After the reaction, HPLC analysis showed a conversion rate of 98% and an ee value of 99.77% for the product.

[0069] Example 8: Whole-cell catalytic synthesis of (R)-5,7-difluorobenzodihydropyran-4-ol using mutant 30

[0070] Mutant 8 was obtained by inducing expression according to the method in Example 1. The bacterial cells were collected by centrifugation (6000 rpm) and used as a biocatalyst. The bacterial cells were resuspended in 300 mL of potassium phosphate buffer (pH 7.0, 100 mM), 270 g / L 5,7-difluorobenzodihydropyran-4-one, 300 g / L glucose, and 0.2 g / L NADP. + 30 U / mL LGDH and 20 mg / mL wet bacterial cells were used to react at 30℃ for 10 hours, with the pH controlled between 6.5 and 7.2 during the reaction. After the reaction, HPLC analysis showed a conversion rate of 98% and a product ee value of 99.79%. Figure 4 ).

Claims

1. A mutant of a carbonyl reductase, characterized in that, Its amino acid sequence corresponds to the amino acid sequence shown in SEQ ID NO: 1 and contains any of the following substitution mutations: The 199th position of asparagine N is mutated to alanine A and glycine G; The proline P at position 201 is mutated to serine S, threonine T, and tyrosine Y. The glutamine Q at position 202 is mutated to alanine A, glycine G, and valine V; The glutamine Q at position 204 is mutated to methionine M, alanine A, and leucine L; The tyrosine Y at position 238 is mutated to histidine H, serine S, and threonine T. The threonine S at position 244 is mutated to proline P; The threonine T mutation at position 251 is replaced by asparagine N, histidine H, and lysine K. The tryptophan W at position 255 is mutated into alanine A, valine V, and isoleucine I; The glutamine Q at position 204 is mutated to methionine M, and the tyrosine Y at position 238 is mutated to histidine H; The asparagine N at position 199 is mutated to alanine A, the glutamine Q at position 204 is mutated to methionine M, and the tyrosine Y at position 238 is mutated to histidine H. The asparagine N at position 199 is mutated to alanine A, the glutamine Q at position 204 is mutated to methionine M, and the tyrosine Y at position 238 is mutated to histidine H. The glutamine Q at position 204 is mutated to methionine M, the tyrosine Y at position 238 is mutated to histidine H, and the threonine T at position 251 is mutated to alanine A. Arginine R at position 17 is mutated to histidine H, glutamine Q at position 204 is mutated to methionine M, and tyrosine Y at position 238 is mutated to histidine H. The asparagine N at position 199 is mutated to alanine A, the glutamine Q at position 204 is mutated to methionine M, the tyrosine Y at position 238 is mutated to histidine H, and the threonine T at position 251 is mutated to alanine A. The 12th threonine S was mutated to proline P, the 17th arginine R was mutated to histidine H, the 204th glutamine Q was mutated to methionine M, and the 238th tyrosine Y was mutated to histidine H. The 12th threonine S was mutated to proline P, the 17th arginine R was mutated to histidine H, the 199th asparagine N was mutated to alanine A, the 204th glutamine Q was mutated to methionine M, and the 238th tyrosine Y was mutated to histidine H. The 12th threonine S was mutated to proline P, the 17th arginine R was mutated to histidine H, the 80th lysine K was mutated to serine S, the 199th asparagine N was mutated to alanine A, the 204th glutamine Q was mutated to methionine M, and the 238th tyrosine Y was mutated to histidine H. The following mutations occurred: threonine at position 12 (S) was replaced by proline (P); arginine at position 17 (R) was replaced by histidine (H); lysine at position 80 (K) was replaced by serine (S); asparagine at position 199 (N) was replaced by alanine (A); glutamine at position 204 (Q) was replaced by methionine (M); tyrosine at position 238 (Y) was replaced by histidine (H); and threonine at position 251 (T) was replaced by alanine (A).

2. A nucleic acid encoding a mutant of the carbonyl reductase as described in claim 1.

3. A recombinant expression vector containing the encoded nucleic acid as described in claim 2.

4. A recombinant bacterium containing the encoded nucleic acid as described in claim 2.

5. The application of the mutant carbonyl reductase as described in claim 1 in the reduction of chromone.

6. The application as described in claim 5, characterized in that, It is used to prepare (R)-5,7-difluorobenzodihydropyran-4-ol.

7. A method for preparing chromone from reduced chromone, comprising the following steps: The mutant carbonyl reductase as described in claim 1 is contacted with the reaction substrate chromone, and a coenzyme cycle system is added to catalyze the reaction, thereby obtaining the corresponding (R)-chromone.

8. The method as described in claim 7, characterized in that, The chromone is 5,7-difluorobenzodihydropyran-4-one, and the (R)-chromol is (R)-5,7-difluorobenzodihydropyran-4-ol.

9. The method as described in claim 7, characterized in that, The pH of the reaction system is 6.0-8.0; the substrate concentration is 80-270 g / L; and the reaction time is 12-48 hours.

10. The method as described in claim 9, characterized in that, The reaction system has a pH of 6.5; a reaction time of 15-36 hours; and also includes the step of separating and purifying the (R)-chromanol.

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