Engineered ketoreductase mutant for synthesizing tergorazan intermediate and application of engineered ketoreductase mutant

Abstract

The invention provides an engineered ketoreductase mutant, which comprises a polypeptide sequence, a gene sequence, a recombinant expression vector containing the gene, an engineering strain, a protein expression method of the engineering strain, and a reaction process for selectively synthesizing (R)-5, 7-difluorochroman-4-ol by using the engineered ketoreductase mutant.

Description

Technical Field

[0001] This application belongs to the field of industrial applications of biotechnology, specifically relating to an engineered ketone reductase mutant that catalyzes the selective synthesis of (R)-5,7-difluorobenzodihydropyran-4-one (Tegorazan intermediate) and its application. Background Technology

[0002] Tegoprazan, also known as ticoraxan, is a small molecule drug developed by Pfizer Inc. It belongs to the class of potassium-competitive acid blockers (P-CABs) and, as a novel acid-suppressing drug, possesses a novel mechanism of action. Tegoprazan acts on resting or activated H+ / K+-ATPase through hydrogen and ionic bonds, thereby inhibiting the conformational change of this enzyme, preventing it from completing hydrogen-potassium exchange, and thus exerting its acid-suppressing effect. Furthermore, the drug takes effect directly after absorption into the bloodstream, without requiring activation in a strongly acidic environment, rapidly reaching peak plasma concentrations, and is unaffected by food intake. Tegoprazan is primarily used to treat gastroesophageal reflux, upper gastrointestinal bleeding, and to prevent stress-induced gastric mucosal damage. Compared to current mainstream proton pump inhibitors such as esomeprazole, rabeprazole, pantoprazole, omeprazole, and lansoprazole, tegoprazan has advantages such as a longer half-life and is unaffected by the CYP2C19 enzyme. The approval of this drug provides a new treatment option for this type of disease and, to some extent, compensates for the shortcomings of other drugs, enabling better treatment outcomes. Teglasazan was first approved in South Korea in 2018, and subsequently received approval from China's National Medical Products Administration (NMPA) in 2022, under the brand name Tasinza. This drug is classified as a Class I chemical drug in China.

[0003] The preparation of tegorazan mainly involves two structural segments, one of which is a chiral fluorool segment, namely (R)-5,7-difluorobenzodihydropyran-4-ol. (R)-5,7-difluorobenzodihydropyran-4-ol is an important intermediate in the synthesis of tegorazan, and its chirality is the determining factor in the molecular configuration of tegorazan. Patent CN101341149B discloses a chemical preparation method for (R)-5,7-difluorobenzodihydropyran-4-ol. This method uses 5,7-difluorobenzodihydropyran-4-one as a substrate and asymmetricly reduces the substrate in tetrahydrofuran solution using chiral reagents (S)-1-methyl-3,3-diphenyl-1H,3H-pyrrolo[1,2-c][1,3,2]oxazoloborane and borane-dimethyl sulfide complexes to obtain (R)-5,7-difluorobenzodihydropyran-4-ol. However, the chiral reagents used in this method are expensive and difficult to recover, and the borane reagents used are highly toxic, posing safety hazards.

[0004] Patent CN114907303A discloses a method for preparing (R)-5,7-difluorobenzodihydropyran-4-ol, which involves using 5,7-difluorobenzodihydropyran-4-one as a raw material, reducing it to obtain racemic 5,7-difluorobenzodihydropyran-4-ol, and then subjecting it to stereoselective acylation with vinyl acetate via an enzyme catalyst to obtain (R)-5,7-difluorobenzodihydropyran-4-ol. This method involves numerous steps, and after the enzyme-catalyzed reaction, column chromatography is used to purify and separate (R)-5,7-difluorobenzodihydropyran-4-ol acetate and (S)-5,7-difluorobenzodihydropyran-4-ol, thus lacking economic viability for large-scale production.

[0005] Patent CN115029397A discloses a method for preparing (R)-5,7-difluorobenzodihydropyran-4-ol using ketone reductase, but it does not disclose the sequence of any ketone reductase with R-selectivity. Generally, wild-type ketone reductases in nature exhibit low activity and stereoselectivity towards non-natural ketone substrates (such as 5,7-difluorobenzodihydropyran-4-one). Engineering modifications, such as directed protein evolution, are necessary to obtain highly active and stereoselective catalytic enzymes, thus enabling their application in industrial production. Furthermore, the coenzyme recycling system described in patent CN115029397A uses glucose as a substrate to generate gluconic acid. The gluconic acid produced by this method is difficult to separate from the product (R)-5,7-difluorobenzodihydropyran-4-ol, and gluconic acid causes a decrease in pH during the reaction process. Therefore, pH control is required during the reaction, increasing the number of steps in the process.

[0006] This application relates to engineered ketoreductase mutants and their supporting isopropanol-based coenzyme cycling system, in order to achieve the industrial application of ketoreductase selectively catalyzing the synthesis of (R)-5,7-difluorobenzodihydropyran-4-ol. Summary of the Invention

[0007] To achieve the above objectives, this application provides an engineered ketone reductase mutant, comprising a polypeptide sequence, a gene sequence, a recombinant expression vector containing the gene, an engineered strain and its protein expression method, and a reaction process for selectively synthesizing (R)-5,7-difluorobenzodihydropyran-4-ol using the engineered ketone reductase mutant, as detailed below. Figure 1 As shown.

[0008] This application provides an improved engineered ketone reductase mutant, which is modified through a process of directed evolution, resulting in mutations such as substitution, insertion, or deletion of a certain number of amino acid residues. The engineered ketone reductase mutant of this application is derived from a ketone reductase mutant of *Empedobacter stercoris* (containing 5 mutations compared to the wild type: G94A; S153I; Y188A; S199D; H202M), with the gene sequence shown in SEQ ID NO: 1 and the amino acid sequence shown in SEQ ID NO: 2. It was obtained by further mutations into its amino acid sequence, resulting in any one of the following amino acid mutations compared to SEQ ID NO: 2: (a) A188G; (b) L198N; (c) A188G; L198N; (d) A188G; L198Q; (e) A97F; A188G; L198Q; (f) A97F; P151T; A188G; L198Q.

[0009] More specifically, in some embodiments, the engineered ketone reductase polypeptide improved based on SEQ ID NO: 2 comprises a polypeptide consisting of the amino acid sequences corresponding to SEQ ID NO: 42, 44, 46, 48, 50, and 52.

[0010] On the other hand, this application provides a polynucleotide sequence encoding the aforementioned engineered ketone reductase mutant or an expression vector capable of expressing the aforementioned engineered ketone reductase mutant and a host cell. In some embodiments, the host cell may be a bacterial host cell, such as *Escherichia coli*. The host cell can be used to express and isolate the engineered ketone reductase mutant described herein, or optionally directly used to react and transform substrates into products.

[0011] This application also provides a method for asymmetric catalytic synthesis of (R)-5,7-difluorobenzodihydropyran-4-ol using the engineered ketone reductase mutant disclosed herein. Modified engineered ketone reductase mutant polypeptides that can be used in the above method may include amino acid sequences selected from the corresponding SEQ ID NOs: 42, 44, 46, 48, 50, and 52.

[0012] Any of the methods disclosed herein for preparing (R)-5,7-difluorobenzodihydropyran-4-ol using engineered reductase mutants can be performed under a range of suitable reaction conditions, including but not limited to substrate loading, peptide loading, cofactor loading, pH, and temperature. For example, in some embodiments, the preparation of (R)-5,7-difluorobenzodihydropyran-4-ol can be performed under suitable reaction conditions including: (a) about 5 g / L to 100 g / L of substrate 5,7-difluorobenzodihydropyran-4-one; (b) about 1 g / L to 10 g / L of engineered ketone reductase mutant peptide; (c) 10% (v / v) isopropanol; (d) about 0.01 g / L to 0.5 g / L of NAD+ coenzyme concentration; (e) a reaction pH of about 6 to 8; and (f) a temperature of about 30°C to 50°C.

[0013] In currently available literature, patent databases, and protein or enzyme databases, the inventors have not found any specific wild-type ketone reductase sequences capable of catalyzing the asymmetric synthesis of (R)-5,7-difluorobenzodihydropyran-4-ol from 5,7-difluorobenzodihydropyran-4-one. Patent application CN115029397A discloses a method for preparing (R)-5,7-difluorobenzodihydropyran-4-ol using ketone reductases (including one or more combinations of short-chain dehydrogenases SDR, medium-chain dehydrogenases MDR, or aldehyde-ketone reductases AKR), but does not disclose the specific sequences of any of these enzymes. During the research and development process, the inventors extensively selected reported and typical wild-type short-chain dehydrogenases SDR, MDR, and AKR and designed and constructed several mutants of these three enzymes. These mutants were expressed and extensively screened in the laboratory, but the results differed significantly from expectations. Under a substrate concentration of 5 g / L for 5,7-difluorobenzodihydropyran-4-one, no ketoreductase was found that could completely convert 5,7-difluorobenzodihydropyran-4-one to the chiral product (R)-5,7-difluorobenzodihydropyran-4-ol with an ee value >99% (see Example 4 for some experimental data). The results showed that an active enzyme was found in the SDR category, but with poor stereoselectivity. Among them, the sequence of a ketoreductase with relatively high catalytic activity and relatively good stereoselectivity is shown in SEQ ID NO:2. SEQ ID NO:2 was obtained by adding 5 mutations to the wild-type ketoreductase derived from *Empedobacter stercoris*, but the ee value of (R)-5,7-difluorobenzodihydropyran-4-ol catalyzed by SEQ ID NO:2 was <30%. Most of the SDR enzymes detected by the inventors exhibited poor stereoselectivity, with (S)-5,7-difluorobenzodihydropyran-4-one being the predominant product. Furthermore, among the MDR and AKR enzymes detected by the inventors, very few wild-type ketone reductases showed high activity towards the target reaction, and their confirmed stereoselectivity was also S-type product selectivity. Therefore, they cannot be used for the industrial production of (R)-5,7-difluorobenzodihydropyran-4-ol.

[0014] Although the preparation of (R)-5,7-difluorobenzodihydropyran-4-ol using the currently selected optimal ketoreductase SEQ ID NO:2 still cannot meet the requirements for stereoenzyme activity and selectivity in industrial production, there is still room for improvement and enhancement. The inventors used protein structure modeling and molecular docking to connect the R- and S-selective intermediate complex, which binds to the NAD+ coenzyme, into the enzyme activity pocket of ketoreductase SEQ ID NO:2. By studying the conformational differences between the R-selective and S-selective intermediate complexes in the activity pocket, key amino acid sites related to selectivity were identified. Mutations of these key amino acid sites yielded mutants of ketoreductase SEQ ID NO:2, and these mutants showed improved stereoselectivity and enzyme activity. For example, the A188G mutation (A to G mutation) and the L198Q mutation (L to Q mutation) based on SEQ ID NO:2 both reduced the steric hindrance of the conformational binding of the R-selective intermediate complex. Based on this principle, by designing combined mutations at multiple key sites and screening the mutant enzymes for enzyme activity and stereoselectivity, some improved engineered ketone reductase mutants were finally obtained, which significantly improved the enzyme's catalytic activity and selectivity for R-configuration products.

[0015] In some embodiments of this application, the above-mentioned ketone reductase mutant is selected from the sequence obtained by amino acid mutation of SEQ ID NO:2, and the amino acid mutation is any one of the following: (a) A188G; (b) L198N; (c) A188G; L198N; (d) A188G; L198Q; (e) A97F; A188G; L198Q; (f) A97F; P151T; A188G; L198Q.

[0016] Compared with SEQ ID NO:2, the catalytic activity and stereoselectivity of the above-mentioned ketone reductase mutants for ketone substrates are significantly improved, which is beneficial to the industrial application of ketone reductase-catalyzed synthesis of (R)-5,7-difluorobenzodihydropyran-4-ol with high chiral purity.

[0017] In some embodiments of this application, a DNA molecule is also provided that encodes any of the aforementioned ketoreductase mutants. The ketoreductase mutant encoded by this DNA molecule exhibits enhanced catalytic activity or stereoselectivity towards ketone compounds, facilitating the synthesis of (R)-5,7-difluorobenzodihydropyran-4-ol via enzymatic catalysis. In some embodiments, the polynucleotide encoding the engineered ketoreductase mutant comprises a sequence selected from odd-numbered sequence identifiers of SEQ ID NO:41-51.

[0018] In some embodiments of this application, a recombinant plasmid is also provided, wherein the aforementioned DNA molecule is linked. The DNA molecule in the recombinant plasmid is positioned at an appropriate location within the plasmid, enabling the DNA molecule to be correctly and smoothly replicated, transcribed, or expressed. The recombinant expression vector can be any vector (e.g., plasmid or virus) that can be readily used in the recombinant DNA step and can result in the expression of a polynucleotide sequence. The choice of vector will generally depend on the compatibility of the vector with the host cell to which it is to be introduced. Many expression vectors useful for embodiments of this disclosure are commercially available. Exemplary expression vectors can be prepared by operatively linking a polynucleotide encoding a modified ketoreductase polypeptide to the plasmid pET28a(+).

[0019] In some embodiments of this application, a host cell is also provided, wherein the recombinant plasmid containing DNA is operatively transformed into the host cell for the expression of ketone reductase. Host cells used to express polypeptides encoded by the expression vectors of this disclosure are well known in the art and include, but are not limited to, *Escherichia coli*, yeast cells (e.g., *Saccharomyces cerevisiae* or *Pichia pastoris*). An exemplary host cell is *Escherichia coli* BL21(DE3). The aforementioned host cells can be wild-type or genome-edited engineered cells, such as those by knocking out wild-type transaminase genes carried in the host cell genome. Suitable culture media and growth conditions for the aforementioned host cells are well known in the art.

[0020] In some embodiments of this application, a method for synthesizing the chiral compound (R)-5,7-difluorobenzodihydropyran-4-ol is also provided. This method involves using any of the ketone reductase mutants described above to perform an asymmetric ketone reduction reaction on 5,7-difluorobenzodihydropyran-4-one under the action of the cyclic coenzyme NAD+ with isopropanol as a substrate, to obtain (R)-5,7-difluorobenzodihydropyran-4-ol compound with high chiral purity.

[0021] Since the ketone reductase mutant of this application has higher enzyme catalytic activity and stereoselectivity than the reported ketone reductase, the preparation of chiral (R)-5,7-difluorobenzodihydropyran-4-ol compounds using the ketone reductase mutant of this application can not only reduce production costs, but also obtain products with higher purity and ee value.

[0022] Considering the improved enzyme activity and stereoselectivity of engineered ketone reductase mutants, the process conditions such as substrate concentration, reaction temperature, coenzyme concentration, and reaction pH were investigated.

[0023] In some embodiments of this application, a suitable substrate concentration range for engineered ketone reductases is also provided, the suitable substrate concentration range being about 5 g / L to 100 g / L of substrate 5,7-difluorobenzodihydropyran-4-one.

[0024] In some embodiments of this application, a suitable temperature range for engineered ketone reductase is also provided, with a suitable reaction temperature range of about 30°C to 50°C.

[0025] In some embodiments of this application, a suitable coenzyme concentration range for engineered ketone reductase is also provided, with a suitable NAD+ coenzyme concentration range of about 0.01 g / L to 0.5 g / L.

[0026] In some embodiments of this application, suitable pH ranges for engineered ketone reductases are also provided, including reaction pH of about 6 to 8.

[0027] In some embodiments of this application, a method for the catalytic preparation of (R)-5,7-difluorobenzodihydropyran-4-ol compounds by engineered ketone reductase under optimal reaction conditions is also provided. The method comprises 100 g / L of substrate 5,7-difluorobenzodihydropyran-4-one, 10 g / L of wet bacterial cells expressing the engineered ketone reductase mutant of SEQ ID NO:52, 0.05 g / L NAD+, 10% (v / v) isopropanol, reacting at 50°C and a stirring speed of 200 rpm for 20 h. Following a post-processing step, a 90% yield of the product (R)-5,7-difluorobenzodihydropyran-4-ol is obtained, with an ee value >99%.

[0028] As described herein and illustrated in the examples, this disclosure envisions a range of suitable reaction conditions that can be used in the methods described herein, including, but not limited to, ranges of substrate loading, temperature, coenzyme loading, pH, etc. Further suitable reaction conditions for performing the method of biocatalytically converting a substrate compound into a product compound using the engineered ketoreductase mutant peptide described herein can be readily optimized through routine experiments, including but not limited to contacting the engineered reductase mutant with the substrate compound under experimental reaction conditions of substrate loading, temperature, coenzyme loading, and pH, and detecting the product compound, for example, using the methods described in the examples provided herein.

[0029] The above-described embodiments of this application achieve the following technical effects:

[0030] 1) The use of engineered ketone reductase mutants to catalyze the preparation of (R)-5,7-difluorobenzodihydropyran-4-ol significantly improved the reaction activity and selectivity, and the product ee value was greater than 99%.

[0031] 2) Using engineered ketone reductase mutants for catalytic reactions, the substrate concentration can reach 100 g / L and the conversion rate can reach 98%, which greatly improves production efficiency.

[0032] 3) Using engineered ketone reductase mutants, it is possible to withstand 50℃ and reduce the amount of coenzyme to 0.01-0.05 g / L.

[0033] 4) Using engineered ketone reductase mutants, combined with isopropanol to decycle coenzyme NAD+, avoids pH regulation during the reaction process compared to the glucose cycling system, reduces reaction operation steps, and the generated acetone can be recycled and reused, which facilitates post-processing and reduces production costs, thus achieving green chemical production. Attached Figure Description

[0034] Figure 1 Ketoreductase-catalyzed asymmetric synthesis of (R)-5,7-difluorobenzodihydropyran-4-ol from 5,7-difluorobenzodihydropyran-4-one.

[0035] Figure 2 HPLC analysis chromatograms - product yield detection: standard samples 5,7-difluorobenzodihydropyran-4-ol, 5,7-difluorobenzodihydropyran-4-one (sorted in order of peak elution time)

[0036] Figure 3 HPLC Analysis Chromatograms - Chirality Detection: Standard Samples 5,7-difluorobenzodihydropyran-4-one, (S)-5,7-difluorobenzodihydropyran-4-ol, (R)-5,7-difluorobenzodihydropyran-4-ol (sorted in order of elution time)

[0037] Figure 4 HPLC Analysis - Product Detection: (R)-5,7-difluorobenzodihydropyran-4-ol prepared by engineered ketone reductase method

[0038] Figure 5 HPLC Analysis - Chirality Detection (ee Value): Preparation of (R)-5,7-difluorobenzodihydropyran-4-ol by Engineered Ketone Reductase Method Detailed Implementation

[0039] The following examples further illustrate this application, but the application is not limited thereto. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0040] Example 1: Construction of gene cloning and expression vectors

[0041] The DNA corresponding to the amino acid sequence of ketone reductase was synthesized using common techniques in the field and cloned into the expression vector pET28a(+). The recombinant expression plasmid was transformed into competent E. coil BL21(DE3) cells under the following conditions: heat shock at 42°C for 90 s, ice bath for 5 min, followed by recovery in an LB culture medium at 37°C in a shaker for 1 h. Finally, the plating was spread onto LB plates containing kanamycin and incubated inverted at 37°C overnight to obtain the recombinant transformant.

[0042] Example 2: Expression of ketone reductase

[0043] A single colony of *E. coli* BL21(DE3) containing a ketone reductase expression plasmid was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of LB medium (containing 30 μg / mL kanamycin) and incubated overnight at 30 °C with shaking. When the OD of the culture medium... 600 When it reaches 2, at 5% (v / v) The inoculum was transferred to a 1000 mL Erlenmeyer flask containing 250 mL of TB medium, and IPTG was added to a final concentration of 0.1 mM to induce ketone reductase expression. The flask was then placed in a shaker at 28 °C. After 20 h of shaking culture, the culture was centrifuged at 8000 rpm for 10 min, and the supernatant was discarded to collect the cells as wet cells. The wet cells successfully expressed soluble ketone reductase. The wet cells can be used directly for enzyme-catalyzed reactions or stored frozen at -20 °C until use.

[0044] Example 3: Site-directed mutagenesis to obtain engineered ketone reductase mutants

[0045] All reagents used here are commercial, with Quikchange kit (supplier: Agilent) being the preferred choice. The sequence design of the mutation primers was performed according to the kit instructions. The construction of site-directed mutagenesis is used as an example. The PCR system is as follows: 10 μL of 5x Buffer, 1 μL of 10 mM dNTP, 1 μL of plasmid DNA template (50 ng / μL), 0.75 μL each of upstream and downstream primers and mutation primers (10 μM), 0.5 μL of high-fidelity enzyme, and 36 μL of ddH2O. The PCR amplification steps are as follows: (1) 98 °C, pre-denaturation for 3 min; (2) 98 °C, denaturation for 10 s; (3) 72 °C, annealing and extension for 3 min; steps (2) to (3) are repeated 25 times; (5) 72 °C, extension for 10 min, and cooling to 4 °C. 2 μL of LDpnI is added to the PCR product and the plasmid template is eliminated by enzyme digestion at 37 °C overnight. The PCR product after enzyme digestion was transformed into E. coli BL21(DE3) competent cells and plated onto LB plates containing kanamycin to obtain the mutant at the target residue position.

[0046] Example 4: Screening and stereoselectivity detection of ketone reductases (short-chain dehydrogenase SDR, medium-chain dehydrogenase MDR, or aldehyde-ketone reductase AKR) catalyzing the synthesis of (R)-5,7-difluorobenzodihydropyran-4-ol. Reaction conditions: 200 μl reaction system included 5 g / L substrate, 0.5 g / L NAD+ and NADP+, 10% (v / v) DMSO, 10% (v / v) isopropanol, 60 μl ketone reductase wet bacterial suspension, 0.1 M phosphate buffer, pH 6, 30℃, reaction time 20 h. Detection results are shown in Table 1:

[0047] Table 1

[0048]

[0049]

[0050] The polynucleotide sequences encoding the above amino acid sequences SEQ ID NO: 2-40 are the sequences shown in SEQ ID NO: 1-39. Note: A negative ee result indicates that the enzyme's stereoselectivity is S-selective (i.e., it is more inclined to produce (S)-5,7-difluorobenzodihydropyran-4-ol), and NA indicates that chirality was not tested (because the conversion rate is low, so chirality determination is unnecessary).

[0051] Conversion rate testing methods:

[0052] The HPLC instrument used to determine the yield of the above reaction products was a commercially available Agilent 1260 liquid chromatograph. The chromatographic column was an Ultimate XB-C18, 250*4.6mm, 5µm. The mobile phase was a mixture of water and acetonitrile in different proportions, the flow rate was 1.0mL / min, and the column temperature was 35℃. The running time was 0-5min: 10% acetonitrile; 5-7min: 90% acetonitrile; 7-10min: 10% acetonitrile, with a total run time of 10min. The injection volume was 10µL, the solvent was 50% acetonitrile, and the detection wavelength was 222nm. The retention time of (R)-5,7-difluorobenzodihydropyran-4-ol was 7.6min, and the retention time of 5,7-difluorobenzodihydropyran-4-one was 7.3min. Figure 2 (As shown).

[0053] Chirality detection method:

[0054] The HPLC instrument used to determine the ee value of the above reaction was a commercially available Agilent 1260 liquid chromatograph. The chromatographic column was an AD-H250*4.6mm 5µm column. The mobile phase was a mixture of n-hexane and isopropanol in different proportions. The flow rate was 2.0 mL / min, and the column temperature was 35℃. The run times were as follows: 0-15 min, 100% n-hexane; 15-20 min, 98% n-hexane; 20-25 min, 95% n-hexane; 25-35 min, 50% n-hexane; 35-50 min, 100% n-hexane. The total run time was 50 min. The injection volume was 10 µL, the solvent was isopropanol:n-hexane 1:9, and the detection wavelength was 222 nm. The retention times for 5,7-difluorobenzodihydropyran-4-one were 15.0 min, for (S)-5,7-difluorobenzodihydropyran-4-ol was 16.2 min, and for (R)-5,7-difluorobenzodihydropyran-4-ol was 20.1 min. Figure 3 (As shown).

[0055] Example 5: Screening reaction and chiral detection of (R)-5,7-difluorobenzodihydropyran-4-ol synthesized by engineered ketone reductase mutant. Reaction conditions: 5 mL system containing 20 g / L substrate, 0.5 g / L NAD+ and NADP+, 10% (V / V) isopropanol, 10 g / L ketone reductase wet cells, 0.1 M phosphate buffer, pH 6, 30℃, reaction time 20 h. Detection results are shown in Table 2:

[0056] Table 2

[0057]

[0058] Amino acid mutations are shown in Table 3:

[0059] Table 3

[0060] amino acid sequence number amino acid residue differences relative to SEQ ID NO:2 SEQ ID NO:42 A188G SEQ ID NO:44 L198N; SEQ ID NO:46 A188G; L198N; SEQ ID NO:48 A188G; L198Q; SEQ ID NO:50 A97F; A188G; L198Q; SEQ ID NO:52 A97F; P151T; A188G; L198Q;

[0061] Example 6: Substrate concentration range of engineered ketone reductase mutants

[0062] In a 5 mL reaction system, four concentrations of 20 / 50 / 80 / 100 g / L of 5,7-difluorobenzodihydropyran-4-one substrate were added, while maintaining other conditions unchanged: 10 g / L of SEQ ID NO:52 engineered ketone reductase mutant wet cells, 0.5 g / L NAD, and 10% (v / v) isopropanol. Finally, 0.1 M pH 6.0 phosphate buffer was added to bring the reaction system to a volume of 5 mL. The reaction was carried out at 30 °C for 20 h, quenched with 5 mL of acetonitrile, and the yield of (R)-5,7-difluorobenzodihydropyran-4-ol was determined by HPLC.

[0063] The test results are shown in Table 4:

[0064] Table 4

[0065] substrate g / L Product yield % 20 >99％ 50 >99％ 80 >99％ 100 80％

[0066] The test results show that the engineered ketone reductase mutant of SEQ ID NO:52 can completely transform approximately 20 to 80 g / L of substrate under the conditions of 0.5 g / L NAD+, 10% isopropanol, 30°C, and 10 g / L wet cell feed, and achieves a product yield of 80% at a substrate concentration of 100 g / L.

[0067] Example 7: Reaction temperature range of engineered ketone reductase mutants

[0068] In a 5 mL reaction system, the reaction was carried out at 30 / 40 / 50 °C for 20 h in 1 g / L of SEQ ID NO:52 engineered ketone reductase mutant wet cells, 20 g / L of 5,7-difluorobenzodihydropyran-4-one substrate, 0.5 g / L NAD+, 10% (v / v) isopropanol, and 0.1 M pH 6.0 phosphate buffer. The reaction was quenched with 5 mL of acetonitrile, and the yield of (R)-5,7-difluorobenzodihydropyran-4-ol was determined by HPLC.

[0069] The test results are shown in Table 5:

[0070] Table 5

[0071] temperature Product yield % 30℃ 49.2％ 40℃ 90.6％ 50℃ 99.8％

[0072] The test results indicate that the engineered ketone reductase mutant of SEQ ID NO:52 can tolerate temperatures of approximately 30°C to 50°C, with an optimal reaction temperature of 50°C.

[0073] Example 8: Coenzyme Concentration Range of Engineered Ketoreductase Mutants

[0074] In a 5 mL reaction system, with 2 g / L of SEQ ID NO:52 engineered ketone reductase mutant wet cells, 100 g / L of 5,7-difluorobenzodihydropyran-4-one substrate, and 10% (v / v) isopropanol, 0.5 / 0.1 / 0.05 / 0.025 / 0.01 g / L of coenzyme NAD+ were added, and the reaction was carried out at 40 °C for 20 h in 0.1 M pH 6.0 phosphate buffer. The reaction was quenched with 5 mL of acetonitrile, and the yield of (R)-5,7-difluorobenzodihydropyran-4-ol was determined by HPLC.

[0075] The test results are shown in Table 6:

[0076] Table 6

[0077] NAD+ (g / L) Product yield % 0.5 24％ 0.1 31％ 0.05 31％ 0.025 30％ 0.01 29％

[0078] The test results show that the coenzyme concentration range of the engineered ketone reductase mutant of SEQ ID NO:52 is about 0.01 to 0.5 g / L, and the optimal coenzyme concentration is 0.05 g / L NAD+. This also indicates that the engineered ketone reductase mutant can reduce the amount of coenzyme required, thereby further reducing costs and increasing profits.

[0079] Example 9: Reaction pH range of engineered ketone reductase mutants

[0080] In a 5 mL reaction system, with 1 g / L of SEQ ID NO:52 engineered ketone reductase mutant wet cells, 20 g / L of 5,7-difluorobenzodihydropyran-4-one substrate, 0.5 g / L NAD+, and 10% (v / v) isopropanol, the reaction was carried out at 30 °C for 20 h in 0.1 M phosphate buffer at pH 6.0, pH 7.0, and pH 8.0. The reaction was quenched with 5 mL of acetonitrile, and the yield of (R)-5,7-difluorobenzodihydropyran-4-ol was determined by HPLC.

[0081] The test results are shown in Table 7:

[0082] Table 7

[0083] pH Product yield % 6.0 49.2％ 7.0 57.5％ 8.0 52.3％

[0084] The test results show that the engineered ketone reductase mutant of SEQ ID NO:52 can tolerate reaction conditions of pH 6.0-8.0, and the optimal pH is 7.

[0085] Example 10: Method for the catalytic preparation of (R)-5,7-difluorobenzodihydropyran-4-ol by engineered ketone reductase mutant under optimized reaction conditions

[0086] Weigh 25 g of the substrate 5,7-difluorobenzodihydropyran-4-one into a 500 mL glass bottle, add 0.0125 g of NAD, then add 225 mL of 0.1 M phosphate buffer (pH 7.0) and 25 mL of isopropanol; finally add 2.5 g of wet bacterial cells of SEQ ID NO:52. The final reaction concentration is: 100 g / L substrate 5,7-difluorobenzodihydropyran-4-one, 10 g / L SEQ ID NO:52 engineered ketone reductase mutant wet bacterial cells, 0.05 g / L NAD, and 10% (v / v) isopropanol. Add a stir bar to the glass bottle, set the reaction temperature to 50 °C, the stirring speed to 200 rpm, and react for 20 h. The product yield was detected to be approximately 98%. Figure 4As shown), inactivate at 70℃ for 1 hour, turn off heating, allow to cool to room temperature, and sterilize with diatomaceous earth. Wash the diatomaceous earth with anhydrous ethanol and combine the filtrates. Rotary evaporate the filtrate at 40℃ until solid precipitates, stop rotary evaporation, cool, and obtain 22.5 g of product. Calculations show that the separation yield is >90% and the ee value is >99.0%. Figure 5 (As shown).

[0087] It should be understood that after reading the foregoing content of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An engineered ketone reductase polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, 42, 44, 46, 48, 50, 52, and capable of catalyzing the production of (R)-5,7-difluorobenzodihydropyran-4-one from 5,7-difluorobenzodihydropyran-4-ol under suitable reaction conditions.

2. The ketone reductase polypeptide of claim 1, wherein the suitable reaction conditions include 5 g / L-100 g / L of substrate 5,7-difluorobenzodihydropyran-4-one; 1 g / L-10 g / L of engineered ketone reductase polypeptide; and 10% (v / v) isopropanol. 0.01 g / L - 0.5 g / L NAD+ coenzyme; pH 6 - 8; temperature 30℃ - 50℃.

3. A polynucleotide encoding a polypeptide according to any one of claims 1-2, wherein the polynucleotide sequence comprises sequences corresponding to SEQ ID Nos: 1, 41, 43, 45, 47, 49, 51.

4. An expression vector comprising the polynucleotide of claim 3, wherein the expression vector comprises a plasmid, a granule, a bacteriophage, or a viral vector.

5. A host cell comprising the expression vector of claim 4, wherein the host cell is preferably *Escherichia coli* (E. coli). coli).

6. A method for preparing (R)-5,7-difluorobenzodihydropyran-4-ol, the method comprising, in a suitable solvent and in the presence of coenzyme NAD+, performing an asymmetric ketone reduction reaction on 5,7-difluorobenzodihydropyran-4-one using an engineered ketone reductase polypeptide of any one of claims 1-2, to obtain (R)-5,7-difluorobenzodihydropyran-4-ol compound with high chiral purity.

7. The method of claim 6, wherein the reaction solvent comprises water, isopropanol, and dimethyl sulfoxide (DMSO).

8. The method of claim 6, wherein the reaction conditions include a temperature of 30°C to 50°C and a pH of 6.0 to 8.

0.

9. The method of claim 6, wherein the reaction conditions include 1 g / L-10 g / L of engineered ketone reductase polypeptide and 0.01 g / L-0.5 g / L of NAD+ coenzyme.

10. The method of claim 6, wherein the substrate loading of 5,7-difluorobenzodihydropyran-4-one is from 5 g / L to 100 g / L.

Images