Carbonyl reductase mutant and application thereof
Through the directed transformation of Kluyveromyces lactis carbonyl reductase, carbonyl reductase mutants with high enzyme activity and high stereoselectivity were obtained, which solved the problems of low chemical separation efficiency and low biological stereoselectivity in the synthesis of fluoxetine, and achieved efficient and environmentally friendly industrial production.
Patent Information
- Application Number
- CN202510504588.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-22
- Publication Date
- 2025-07-18
AI Technical Summary
In the process of fluoxetine synthesis, the chemical separation efficiency is low, the cost is high, and the biological method is low in three-dimensional selectivity, making it difficult to achieve efficient and environmentally friendly industrial production.
By directed modification of the carbonyl reductase from Kluyveromyces lactis-derived carbonyl reductase mutant K1SDRV198A/N213D/T215K with high enzyme activity and high stereoselectivity, used for catalytic synthesis of (S)-3-hydroxy-3-phenyl propionate.
High enzyme activity and high stereoselectivity were achieved, and the enantiomer excess value of the product (S)-3-hydroxy-3-phenylpropionate was >99%, which was 975.6% higher than that of wild type, and had good industrial application potential.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and particularly relates to a carbonyl reductase mutant and its application in the enzymatic synthesis of (S)-ethyl 3-hydroxy-3-phenylpropionate. Background Art
[0002] Fluoxetine (FXT), also known as Prozac, is a selective serotonin reuptake inhibitor (SSRIs) with the chemical formula C 17 H 19 ClF3NO, with a molecular weight of 345.787, is in the form of white or off-white powder and is soluble in water. Fluoxetine can significantly inhibit the reuptake of serotonin by nerve synapses, thereby increasing the extracellular level of serotonin, and regulating physiological functions such as human mood, motor control, and sleep, so as to achieve an antidepressant effect. Because of its small side effects and the characteristic of not inhibiting monoamine oxidase, it is called "the best psychotropic drug in the 20th century". Fluoxetine can also be used to treat mental diseases such as obsessive-compulsive disorder and anxiety disorder. At the same time, there are also studies showing that it has a certain therapeutic effect on malignant tumors, binge eating disorder, etc.
[0003] With the trend of high incidence, strong recurrence, and high fatality rate of depression, the market demand for fluoxetine is also increasing continuously. However, as a chiral compound, fluoxetine has two enantiomeric racemic molecular forms of R(-) and S(+). The construction of chiral centers is also a difficult point in its synthesis process. Although both structures can inhibit the reuptake of serotonin, the inhibitory ability of the S(+) enantiomer is stronger than that of the R(-) enantiomer. Similar to fluoxetine, the synthesis precursor of fluoxetine, ethyl 3-hydroxy-3-phenylpropionate (EHPP), also has two enantiomeric structures of R(-) and S(+), and both can be obtained by asymmetric reduction of ethyl benzoyl acetate (EBA). Currently, the commonly used methods are chemical methods and biological methods. The chemical method obtains the EHPP racemate through the reduction reaction of EBA, and then resolves it to obtain two single-configuration EHPPs. However, this method has disadvantages such as low resolution efficiency, complex reaction conditions, high actual cost, and environmental unfriendliness, which limit its industrial application. The biological method uses carbonyl reductase or enzyme-containing cells as catalysts to asymmetrically reduce to obtain a single-configuration EHPP. Compared with the chemical method, this method not only omits the cumbersome resolution steps, but also has mild reaction conditions, high conversion efficiency, and most importantly, can obtain products with high optical purity. Therefore, it is considered the most suitable production method to replace the chemical method in the future.
[0004] Carbonyl reductases (CRs), also known as alcohol dehydrogenases (EC 1.1.1.1, ADHs), are the largest subclass of oxidoreductases. According to their amino acid sequences and structural characteristics, they can be divided into three categories: short-chain dehydrogenases / reductases (SDRs), medium-chain dehydrogenases / reductases (MDRs), and aldo-keto reductases (AKRs). Carbonyl reductases are widely present in various organisms, and hundreds of crystal structures have been resolved in the PDB database. However, most current studies are still limited to small molecule substrates. Due to the relatively complex side chain groups (benzene ring and ester side chain) in EBA, most known carbonyl reductases show problems such as inactivity and low stereoselectivity towards it. With the development of bioinformatics, directed modification of wild carbonyl reductases for substrates has become a hot topic in enzymology research. Therefore, obtaining a carbonyl reductase that can efficiently catalyze the reduction of EBA to enantiopure EHPP through enzyme modification technology is the key to realizing the industrial production of fluoxetine by enzymatic method. Summary of the Invention
[0005] The purpose of the present invention is to provide a carbonyl reductase mutant and its application.
[0006] In order to improve the production efficiency and product optical purity of ethyl 3-hydroxy-3-phenylpropionate, a precursor for the synthesis of fluoxetine, the present invention has conducted in-depth research on the carbonyl reductase (KlSDR) derived from Kluyveromyces lactis. Through directed modification, a novel carbonyl reductase mutant with high enzyme activity and high stereoselectivity, its coding gene, a recombinant vector containing the mutant coding gene, a genetically engineered bacterium, and the application of the carbonyl reductase mutant in the biocatalytic synthesis of (S)-ethyl 3-hydroxy-3-phenylpropionate are obtained.
[0007] To achieve the above purpose, the present invention adopts the following technical solutions:
[0008] The present invention first provides a carbonyl reductase mutant, which is obtained by mutation at least one amino acid at position 198, position 213, and position 215 of the amino acid sequence shown in SEQ ID NO.1; the mutation is one or more combinations of the following:
[0009] (1) Valine at position 198 is mutated to any one of alanine and glycine;
[0010] (2) The asparagine at position 213 is mutated to any one of aspartic acid, glutamic acid, and arginine;
[0011] (3) The threonine at position 215 is mutated to any one of aspartic acid, glutamic acid, and lysine.
[0012] Preferably, the carbonyl reductase mutant is a mutant KlSDR with a superimposed effect obtained by mutating the 198th position of the amino acid sequence shown in SEQ ID NO.1 to alanine, mutating the 213th position to aspartic acid, and mutating the 215th position to lysine. V198A / N213D / T215K .
[0013] The present invention also provides a gene encoding the carbonyl reductase mutant, and the gene is obtained by base mutation of the nucleotide sequence shown in SEQ ID NO.2.
[0014] The present invention also provides an expression vector containing the nucleic acid sequence of the carbonyl reductase mutant. Preferably, the expression vector is a pET series expression vector.
[0015] The present invention also provides a genetically engineered recombinant bacterium containing the expression vector. Preferably, the genetically engineered recombinant bacterium is Escherichia coli E.coli BL21(DE3).
[0016] The present invention also provides the application of the carbonyl reductase mutant in the enzymatic preparation of (S)-ethyl 3-hydroxy-3-phenylpropionate. Specifically, the application is as follows: using the above-mentioned carbonyl reductase mutant or a catalyst containing the above-mentioned carbonyl reductase mutant and glucose dehydrogenase, using ethyl benzoylacetate as a substrate, glucose as a co-substrate, and NADH as a reducing coenzyme, to synthesize the fluoxetine chiral precursor (S)-ethyl 3-hydroxy-3-phenylpropionate.
[0017] Preferably, the above catalytic reaction is carried out in 100 mM phosphate buffer at 35 °C and pH = 6.5, with an initial concentration of ethyl benzoylacetate of 0.5 mol / L.
[0018] Advantages of the present invention: The present invention provides a carbonyl reductase mutant with high enzyme activity and high stereoselectivity and its application in the catalytic synthesis of ethyl 3-hydroxy-3-phenylpropionate. Compared with the wild-type carbonyl reductase, the novel carbonyl reductase mutant KlSDR provided by the present invention V198A / N213D / T215K not only has better stereoselectivity and thermal stability, but also has a specific enzyme activity of 91.67 U / mg, which is 975.6% higher than that of the wild type. The enantiomeric excess value (e.e.%) of the product (S)-ethyl 3-hydroxy-3-phenylpropionate > 99%, which is higher than 66.7% of the wild type, and has good potential for industrial application. Description of the Drawings
[0019] Figure 1 It is a schematic diagram of the asymmetric reduction synthesis of fluoxetine from ethyl benzoylacetate; the light gray part is the chiral structure.
[0020] Figure 2 It is a molecular docking result diagram of carbonyl reductase and the small molecule substrate ethyl benzoylacetate (EBA).
[0021] Figure 3 It is an electrophoresis diagram of carbonyl reductase and mutant proteins; Lane M: 14 - 200KDa Protein Marker; Lane 1: wild-type carbonyl reductase; Lane 2: carbonyl reductase mutant N213D / T215K; Lane 3: carbonyl reductase mutant V198A / N213D / T215K.
[0022] Figure 4 It is the specific enzyme activity of carbonyl reductase and mutants N213D / T215K, V198A / N213D / T215K.
[0023] Figure 5 It is the optimal reaction temperature of carbonyl reductase and mutant V198A / N213D / T215K.
[0024] Figure 6 It is the optimal reaction pH of carbonyl reductase and mutant V198A / N213D / T215K.
[0025] Figure 7 It is a time - conversion rate diagram of carbonyl reductase and mutant V198A / N213D / T215K catalyzing EBA. Detailed Description of the Invention
[0026] Example 1 Construction of the Strain E.coli BL21(DE3) / pET - 28a - sdr Producing Carbonyl Reductase
[0027] The carbonyl reductase gene (sdr) derived from Kluyveromyces lactis was synthesized by Beijing Tsingke Biotechnology Co., Ltd. after codon optimization and constructed onto the pET-28a plasmid, named pET-28a-sdr, and the vector was transformed into competent E. coli DH5α. The recombinant bacterium E. coli DH5α was inoculated into a 5 ml LB test tube medium and cultured with shaking at 37 °C and 220 rpm for 12 h. After the culture was completed, the cells were collected by centrifugation at 12,000 rpm for 1 min, and the plasmid was extracted using a plasmid miniprep kit. The obtained plasmid was transferred into the expression host of E. coli BL21(DE3) to obtain the strain E. coli BL21(DE3) / pET-28a-sdr producing carbonyl reductase. A glycerol strain was prepared by adding sterile glycerol at a final concentration of 20% and stored at -80 °C.
[0028] Example 2 Construction and Screening of Carbonyl Reductase Mutants
[0029] The three-dimensional protein structure of KlSDR (PDB ID: 7C1E) was downloaded from the RCSB PDB database. The small molecule EBA was searched and downloaded in sdf format 3D structure file through the PubChem website https: / / pubchem.ncbi.nlm.nih.gov / and converted to mol2 format by OpenBanel software. After dehydrating and removing ligands from the KlSDR structure using pymol software, semi-flexible molecular docking of KlSDR and EBA was performed by AutoDock 4 software. The docking results were visually analyzed by pymol as shown in Figure X. After result analysis, it was determined that the key factor affecting the catalytic properties of carbonyl reductase was the steric hindrance of its substrate channel, and the key sites affecting the binding of KlSDR and EBA were Val-198, Asn-213, and Thr-215 respectively. Taking the above three sites as the mutation sites to improve the catalytic activity of carbonyl reductase, V198 was replaced with small molecule residue amino acids (Ala and Gly), and N213 and T215 were replaced with more hydrophilic amino acids (Asp, Glu, Lys, and Arg). Using the plasmid pET-28a-sdr obtained in Example 1 as a template, primers were designed to introduce the above mutations by inverse PCR. The PCR primers are shown in the following table:
[0030] Table 1 Primers for Site-Directed Mutagenesis by Inverse PCR
[0031]
[0032] Note: The underlined part is the mutation site.
[0033] The PCR reaction procedure is as follows: 95°C for 2 min, followed by 35 cycles, which are 95°C for 10 s, 55°C for 5 s, and 72°C for 10 s, respectively. The PCR products are digested with Dpn I enzyme for 3 h to remove the initial plasmid template, and then transformed into E. coli BL21(DE3) competent cells. The cells are spread on the surface of LB solid medium containing 50 μg / ml kanamycin and cultured overnight at 37°C. The next day, the successfully constructed positive transformants are identified by colony PCR, and the plasmids are extracted and sent to Beijing Tsingke Biotechnology Co., Ltd. for sequencing to verify the correctness of the mutation sites. After verification, glycerol stocks are prepared by adding sterile glycerol at a final concentration of 20% and stored at -80°C.
[0034] Add an appropriate amount of LB medium containing 50 μg / ml kanamycin to a 96-well deep-well plate. Inoculate the initial carbonyl reductase strain and the transformants from the stored glycerol tubes into the 96-well deep-well plate. After culturing at 37°C and 220 rpm for 2.5 h, add 0.1 mM IPTG and continue to induce culture at 28°C for 16 h. After the culture is completed, centrifuge at low temperature and high speed to discard the supernatant. The wet cells are resuspended in 100 mM phosphate buffer (pH = 6.5) and an appropriate amount of lysozyme is added. Incubate on a shaker at room temperature for 40 min, and centrifuge at 4°C and 10,000 rpm for 10 min to collect the crude enzyme solution in the supernatant. Set up a 200 μl reaction system on a 96-well microplate reader, including: 0.05 mmol / L NADH, 1 mmol / L EBA, 0.1 mol / L phosphate buffer (pH = 6.5), and finally add an appropriate amount of crude enzyme to activate the reaction. Set the temperature of the microplate reader to 35°C and detect the change in absorbance at a wavelength of 340 nm. The amount of enzyme required to catalyze the oxidation of 1 μmol NADH per minute is defined as one enzyme activity unit (U). Calculate the crude enzyme activity of the mutant enzyme. Taking the enzyme activity of the wild-type carbonyl reductase as 100% relative enzyme activity, screen for high enzyme activity mutants. The results are shown in the following table.
[0035] Table 2 Relative enzyme activities of carbonyl reductase and single-site mutants
[0036]
[0037] As can be seen from the results in the table, the four mutants V198A, N213D, N213R, and T215K achieved good positive feedback mutations at the corresponding mutation sites. Using the single-site mutant plasmid as a template, the four forward mutations were combined and arranged according to the above operations to construct plasmids V198A / N213D, V198A / N213R, V198A / T215K, N213D / T215K, N213R / T215K, V198A / N213D / T215K, and V198A / N213R / T215K respectively. The enzyme activities of the multi-combination mutants were measured by the same method. Taking the enzyme activity of the wild-type carbonyl reductase as 100% relative enzyme activity, high-enzyme-activity mutants were screened. The results are shown in the following table.
[0038] Table 3 Relative Enzyme Activities of Carbonyl Reductase and Multi-Site Combinations Mutations
[0039]
[0040] As can be seen from the results in the table, through stacking mutations, the enzyme activities of N213D / T215K and V198A / N213D / T215K were further improved. These mutants were selected as the objects for the second screening of enzyme activity for subsequent experiments.
[0041] Example 3 Preparation of Pure Enzyme of Carbonyl Reductase Mutant and Second Screening of Enzyme Activity
[0042] Take out the recombinant Escherichia coli glycerol bacteria obtained in Example 1 and Example 2, and inoculate them into 5 ml of LB medium containing 50 μg / ml kanamycin respectively. Incubate at 37 °C with shaking at 220 rpm for 14 h, and then inoculate at an inoculation amount of 1% (v / v) into 50 ml of LB medium containing 50 μg / ml kanamycin. Incubate at 37 °C with shaking at 220 rpm until the OD 600 reaches 0.6 - 0.8. Add IPTG with a final concentration of 0.1 mM, and continue to induce and culture at 28 °C with shaking at 200 rpm for 16 h. After the induction is completed, collect the bacterial liquid, centrifuge at 8000 rpm for 10 min at 4 °C, discard the supernatant, collect the bacterial cells, wash twice with PBS buffer (pH = 7.0) and then resuspend, and place on an ice-water mixture for ultrasonic disruption. The working conditions of the ultrasonic cell disruptor are: power 800 W, working time 8 s, interval time 10 s, for a total of 99 times. After the disruption is completed, centrifuge at 8000 rpm for 10 min at 4 °C, and collect the supernatant as the crude enzyme solution. Filter it with a 0.45 μm microporous filter membrane for standby.
[0043] Use nickel ion affinity chromatography column for protein purification, rinse the column with deionized water (about 10 column volumes) at 4°C, and then balance the column with low salt concentration buffer (500mmol / L NaCl, 50mM Tris, pH=7.0). When the effluent at the lower end of the column is consistent with the pH value of the low salt concentration buffer pumped into the column (about 5 column volumes of buffer), add the reserved membrane-passed crude enzyme solution to the column. First, rinse with a buffer containing a low concentration of imidazole (500mmol / L NaCl, 50mmol / L imidazole, 50mM Tris, pH=7.0) to remove impurities until baseline equilibrium, and then elute the target protein with an eluent containing a high concentration of imidazole (500mmol / L NaCl, 500mmol / L imidazole, 50mM Tris, pH=7.0). The eluate at the absorption peak was collected, which was the pure enzyme solution of the target protein, and the bands were verified by SDS-PAGE protein gel electrophoresis. After verification, the concentration was determined using a Bradford protein concentration determination kit (with BSA protein as the standard curve) and stored at -80°C for subsequent enzyme activity determination and catalytic reaction.
[0044] Enzyme activity rescreening: Set up a 1ml reaction system in a 1.5ml EP centrifuge tube, including: 0.5mmol / L NADH, 50mmol / L EBA, 100mmol / L phosphate buffer (pH=6.5), and finally add 20μL of pure enzyme solution diluted to 20mg / L with phosphate buffer to activate the reaction, and detect the change in absorbance at a wavelength of 340nm. The amount of enzyme required to catalyze the oxidation of 1μmol NADH per minute is one activity unit (U). The specific enzyme activity (U / mg) is defined as the number of enzyme activity units per unit weight (mg) of protein.
[0045] The re-screening results are as follows Figure 4 As shown, the specific enzyme activity of the wild-type carbonyl reductase is 8.52 U / mg, the specific enzyme activity of the mutant N213D / T215K is 56.72 U / mg, and the specific enzyme activity of the mutant V198A / N213D / T215K is 91.67 U / mg. After multi-site combined mutation superposition, the specific enzyme activity is increased by 975.9% compared with the wild-type carbonyl reductase.
[0046] Example 4 Effects of temperature and pH on the activity of carbonyl reductase and its mutant V198A / N213D / T215K
[0047] A phosphate buffer solution with a pH of 6.5 was used to prepare the reaction system. According to the enzyme activity detection method in Example 3, the reaction temperature was controlled at 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, and 55°C, respectively. The enzyme activity of carbonyl reductase was measured under different temperature conditions, and the highest enzyme activity was set as 100%, and the relative enzyme activity was compared. The results are shown in FIG.Figure 5 As shown in the figure, the optimal reaction temperature of wild-type carbonyl reductase is 30 °C, and the enzyme activity decreases significantly after 45 °C. The optimal reaction temperature of carbonyl reductase mutant V198A / N213D / T215K is 35 °C, and it has certain heat resistance, and the enzyme activity is still 78.3% at 50 °C.
[0048] The reaction systems were prepared using citrate-citrate buffer system (pH = 4.0 - 6.0), phosphate buffer system (pH = 6.0 - 8.0), and Tris-HCl buffer system (pH 8.0 - 9.0) respectively. The activity of carbonyl reductase was measured at 30 °C according to the enzyme activity detection method in Example 3. The highest enzyme activity was set as 100%, and the relative enzyme activities were compared. The results are as Figure 6 shown, the optimal reaction pH of both wild-type carbonyl reductase and carbonyl reductase mutant V198A / N213D / T215K is 6.5.
[0049] Example 5 Enzymatic Synthesis of Ethyl (S)-3-Hydroxy-3-phenylpropionate by Carbonyl Reductase and Mutant V198A / N213D / T215K
[0050] A dual-enzyme coupled reaction system was prepared in a 500 ml beaker: 3 mol / L EBA, 0.2 mmol / L NADH, 0.2 mol / L glucose, carbonyl reductase (final concentration of 200 mg / L), glucose dehydrogenase (final concentration of 100 mg / L), and supplemented to 300 ml with phosphate buffer (100 mM, pH = 6.5). It was placed on a magnetic stirrer and heated in a water bath at 35 °C with a stirring speed of 400 rpm. After the reaction started, the pH of the reaction system was adjusted using a 4 M sodium carbonate solution to maintain it at pH 6.5. Samples were taken at intervals of 2 h and the conversion was monitored. After 4 h of reaction, 0.03 mol of glucose was added to the reaction system at intervals of 2 h. When the conversion of EBA no longer increased, ethyl acetate with twice the volume of the reaction mixture was added to the reaction system to terminate the reaction. After thorough shaking, it was sonicated for 2 min, centrifuged at 15 °C and 7000 rpm for 5 min, and the supernatant was collected. The precipitate was resuspended with one volume of ethyl acetate, shaken and sonicated again for 2 min, centrifuged at 15 °C and 7000 rpm for 5 min, and the two supernatants were mixed to obtain the crude product.
[0051] The crude product was purified by rotary evaporation to remove impurities and solvents at a rotary evaporation temperature of 65 °C. Gas chromatography was used to detect the product concentration and chirality. The gas detection method is as follows: chromatographic column, supelcoβ-120 (250×2.5 mm); injection volume, 1 μL; column temperature, 125 °C; injection port temperature, 250 °C; detection port temperature, 255 °C; carrier gas is nitrogen; carrier gas flow rate, 2 mL / min; split ratio, 1:15; detector, flame ionization detector (FID). The formula for the molar conversion rate of the substrate is:
[0052]
[0053] wherein, p is the mass of the product at the end of the reaction; M p is the molecular weight of the product; M s is the molecular weight of the substrate; q is the mass of the substrate input in total for the reaction.
[0054] The calculation formula for the enantiomeric excess value (e.e.%) of the product is:
[0055]
[0056] wherein, C S is the concentration of the S-configuration product; C R is the concentration of the R-configuration product.
[0057] The results are as Figure 7 shown. After 18 h of catalytic reaction, the conversion rate of the wild-type carbonyl reductase for catalyzing the substrate EBA is 44.6%, and the enantiomeric excess value of the product is 66.7%. While for the mutant V198A / N213D / T215K, the conversion rate is 99.6%, and the enantiomeric excess value > 99.9%. It is proved that this mutant not only has better catalytic conversion ability, but also has good stereoselectivity, and has the application potential for realizing the industrial production of (S)-ethyl 3-hydroxy-3-phenylpropionate.
[0058] Although the present invention has been disclosed above with preferred examples, it is not intended to limit the present invention. For those skilled in the art, any equivalent modifications and substitutions made to the above-described embodiments are also within the scope of this application. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of this application should be covered within the scope of this application. For those conditions not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For all reagents or instruments without indicating the manufacturer, they are all conventional products that can be purchased commercially. In order to better illustrate the invention, numerous specific details are given in the following detailed description. Those skilled in the art should understand that the present invention can also be implemented without some specific details. In other embodiments, the methods, means, equipment and steps well-known to those skilled in the art are not described in detail so as to highlight the gist of this application.
Claims
1. A carbonyl reductase mutant, characterized in that, The carbonyl reductase mutant is obtained by mutation at at least one amino acid at positions 198, 213, and 215 in the amino acid sequence shown in SEQ ID NO.1, and the mutation is one or more combinations of the following: (1) Valine at position 198 is mutated to any one of alanine and glycine; (2) Asparagine at position 213 is mutated to any one of aspartic acid, glutamic acid, and arginine; (3) Threonine at position 215 is mutated to any one of aspartic acid, glutamic acid, and lysine.
2. The carbonyl reductase mutant according to claim 1, wherein The carbonyl reductase mutant described above is a mutant KlSDR with a superimposed effect obtained by mutating the 198th amino acid in the amino acid sequence shown in SEQ ID NO.1 to alanine, mutating the 213th amino acid to aspartic acid, and mutating the 215th amino acid to lysine V198A / N213D / T215K .
3. The coding gene of the carbonyl reductase mutant according to claim 1.
4. The coding gene according to claim 2, wherein The coding gene is obtained by base substitution of the nucleotide sequence shown in SEQ ID NO.
2.
5. An expression vector, characterized in that, The expression vector contains the nucleotide sequence according to claim 4.
6. An expression vector according to claim 5, wherein The expression vector is a pET series expression vector.
7. A genetically engineered recombinant bacterium, characterized in that, The genetically engineered recombinant bacterium contains the expression vector according to claim 4.
8. The application of the carbonyl reductase mutant according to claim 1 in the enzymatic synthesis of ethyl (S)-3-hydroxy-3-phenylpropionate.
9. The application according to claim 8, wherein Using the carbonyl reductase mutant or a catalyst containing the carbonyl reductase mutant and glucose dehydrogenase, ethyl benzoylacetate as a substrate, glucose as a co-substrate, and NADH as a reducing coenzyme, to catalytically synthesize ethyl (S)-3-hydroxy-3-phenylpropionate.
10. The application according to claim 9, characterized in that, The catalytic reaction is carried out in 100 mM phosphate buffer at 35 °C and pH = 6.5 with the concentration of ethyl benzoylacetate being 3 mol / L.