Carbonyl reductase mutants and their application in stereoselective synthesis of aromatic polysubstituted chiral γ-butyrolactones

Abstract

The present invention relates to a carbonyl reductase mutant and its application in the stereoselective synthesis of aromatic multi-substituted chiral γ-butyrolactone. The carbonyl reductase mutant is a protein derived from carbonyl reductase SsCR, whose amino acid sequence is substituted, deleted or added with several amino acids as shown in SEQ ID No.2 and can catalyze the asymmetric reduction of aromatic α- / β-unsaturated ketoesters, and whose catalytic activity is higher than that of carbonyl reductase SsCR, wherein the amino acid sequence of carbonyl reductase SsCR is shown in SEQ ID No.2. The present invention also provides a gene encoding the carbonyl reductase mutant, a recombinant expression vector and a recombinant expression transformant containing the gene, and the application of the carbonyl reductase mutant in the synthesis of chiral aromatic multi-substituted γ-butyrolactone. Compared with the prior art, the present invention has the significant advantages of simple process, high atom economy, high product optical purity and environmental friendliness, and has good industrial application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and in particular relates to a modified mutant of the carbonyl reductase SsCR of the yeast Pichia stipitis, a gene encoding the carbonyl reductase mutant, a recombinant expression vector and a recombinant expression transformant containing the gene, and the use of the carbonyl reductase mutant or the recombinant expression transformant as a catalyst in the stereoselective synthesis of aromatic polysubstituted chiral γ-butyrolactones. A method for the stereoselective synthesis of aromatic polysubstituted chiral γ-butyrolactones is also provided. Background Art

[0002] Flavors and fragrances are a class of substances with pleasant odors that are widely used in the food, daily chemical, chemical, and pharmaceutical industries. Initially, natural flavors were isolated and extracted from plants. However, due to factors such as low concentration, seasonal variations, and plant diseases, natural flavors were expensive and marketed at very high prices (Food & Bioprocess Technology, 2018, 11:2217-2228). In the 1850s, scientists began using chemical methods to synthesize these compounds, leading to a rapid decline in the market price of artificial flavors and fragrances. US and EU regulations (CFR 1990 and EEC 1334 / 2008) define natural flavors as follows: natural flavor molecules can only be obtained through physical processes (extraction from natural resources) or produced using enzymes or microorganisms, and the precursors involved must be obtained from nature.

[0003] Chiral γ-lactones are an important class of structural units and synthetic building blocks, widely present in more than 15,000 natural products, and have important industrial application value. For example, chiral γ-lactones are an important class of flavor molecules, widely used in food flavors, flavor enhancers and daily chemical products. Data show that the sales of the global flavor and fragrance market increased from US$18 billion in 2006 to US$26.3 billion in 2017. The scale of the global flavor and fragrance market continued to grow from 2017 to 2022, reaching approximately 39 billion euros in 2022, the highest value in the past six years, an increase of 4% over 2021 (J.Agric.FoodChem.2024,72(33),18305-18320). In recent years, China's flavor and fragrance industry has also developed rapidly. In 2018, the industry sales exceeded 40 billion yuan, and the market size reached 43.9 billion yuan in 2023. It is expected to exceed 50 billion yuan by 2026.

[0004] Chiral lactones are well-known natural flavor and fragrance compounds found in a variety of natural products, such as mango (Food Chemistry. 2013, 136(2):585-594.2), peach (Food Chemistry. 2009, 116(1):356-364.), and dairy products (Journal of Dairy Science. 2012, 95(3):1128-1139.). The function of chiral lactones is closely related to their chemical structure. Studies have shown that γ-decalactone has the aroma of peach and strawberry, while δ-decalactone has the aroma of coconut milk (Trends in Biotechnology. 2005, 23(4):193-198.). The type of side chain, the position of the functional group, and the length of the carbon chain of the lactone all affect its flavor properties. For example, γ-octanolactone has a milky aroma, while γ-undecalactone has a strong peach and almond aroma (J. Agric. Food Chem. 1989, 37: 413-418.). The stereo configuration of the lactone compound also affects the purity and intensity of the aroma. Natural lactones are mainly (R)-enantiomers, which have a stronger odor than the (S)-enantiomer and are the main source of fruity aroma (J. Agric. Food Chem. 1989, 37 (2): 413-418.). Lactones produced by chemical methods usually have poor enantioselectivity and low optical purity, so the aroma quality and intensity are weaker and consumer acceptance is also low. For example, chemically synthesized γ-decalactone is significantly inferior to naturally extracted γ-decalactone in aroma intensity. Therefore, as food additives, lactone compounds with a single configuration are in greater demand, have higher added value, and are very popular in the market.

[0005] Chiral lactone compounds have very important biological functions and pharmaceutical activities. These compounds are widely present in nature and play a key role in many biological processes. About 10% of natural products contain lactone ring structures. [], including alkaloids, pheromones, antibiotics and antitumor agents, etc. Chiral lactone compounds are the core components of a variety of bioactive molecules (Tetrahedron.2021,84,132001–13039.). In the field of medicinal chemistry, chiral lactones are regarded as a special structure because they have a variety of pharmacological properties, such as antifungal, antibiotic, antitumor, anti-inflammatory and cytotoxic activities (Angewandte Chemie.2014,53(16):4164-4167.). These properties make chiral lactones have a wide range of application potential in the development of new drugs and the treatment of various diseases. Many natural chiral lactone compounds, such as erythromycin, exhibit antibacterial and antifungal activities and can inhibit the growth of pathogenic microorganisms. In addition, some chiral lactones, such as paclitaxel, have anticancer properties and prevent cancer cell proliferation by interfering with cell division. At the same time, these compounds also have anti-inflammatory effects, such as ginkgolide, which can reduce inflammation and oxidative damage. Furthermore, chiral lactones can act as pheromones in plants and microorganisms, participating in intercellular signaling, affecting plant growth and development or bacterial quorum sensing. Therefore, the structural and chiral characteristics of chiral lactone compounds enable them to play an important role in various biological systems.

[0006] Chiral polysubstituted γ-butyrolactones are core building blocks of many natural products and high-value-added chemicals used in the pharmaceutical and food industries. Currently, their synthesis is primarily based on chemical methods. The precise construction of multiple chiral centers within polysubstituted five-membered heterocycles presents significant challenges, particularly for bulky aromatically substituted chiral γ-butyrolactones, which face challenges such as low stereoselectivity, expensive metal catalysts, harsh reaction conditions, and low atom economy. Summary of the Invention

[0007] In response to the problems faced by the chemical synthesis of chiral polysubstituted γ-butyrolactones in the prior art, such as low stereoselectivity, expensive metal catalysts, harsh reaction conditions, and low atom economy, the present invention provides a carbonyl reductase mutant and its application in the stereoselective synthesis of aromatic polysubstituted chiral γ-butyrolactones. That is, the carbonyl reductase mutant is used to perform asymmetric reduction of the potential chiral carbon of a chiral γ-ketoester, and finally the polysubstituted chiral γ-butyrolactone is obtained through intramolecular cyclization.

[0008] More specifically, the present invention molecularly modifies the carbonyl reductase SsCR required for the synthesis of polysubstituted chiral lactones, thereby providing a carbonyl reductase mutant, a gene encoding the carbonyl reductase mutant, a recombinant expression vector and a recombinant expression transformant containing the gene, and the use of the carbonyl reductase mutant in the synthesis of chiral aromatic polysubstituted γ-butyrolactones.

[0009] The carbonyl reductase mutant provided by the present invention has high stereoselectivity and high activity and can expand the substrate spectrum.

[0010] The purpose of the present invention can be achieved by the following technical solutions:

[0011] One of the technical solutions of the present invention:

[0012] The present invention provides a carbonyl reductase SsCR mutant, wherein the carbonyl reductase mutant is a protein derived from the carbonyl reductase SsCR, wherein the amino acid sequence shown in SEQ ID No. 2 is substituted, deleted or added with several amino acids, and the protein is capable of catalyzing the asymmetric reduction of aromatic α- / β-unsaturated ketoesters and has a higher catalytic activity than the carbonyl reductase SsCR. The amino acid sequence of the carbonyl reductase SsCR is shown in SEQ ID No. 2.

[0013] In one embodiment of the present invention, the carbonyl reductase SsCR mutant is one of the proteins with the following amino acid sequence:

[0014] (1) replacing the valine at position 126 of the amino acid sequence shown in SEQ ID No. 2 with alanine;

[0015] (2) the valine at position 126 of the amino acid sequence shown in SEQ ID No. 2 was replaced with alanine, and the methionine at position 215 was replaced with glycine;

[0016] (3) the valine at position 126 of the amino acid sequence shown in SEQ ID No. 2 was replaced with alanine, and the asparagine at position 210 was replaced with histidine;

[0017] (4) The valine at position 126 of the amino acid sequence shown in SEQ ID No. 2 is replaced by alanine, the methionine at position 215 is replaced by glycine, and the asparagine at position 210 is replaced by histidine.

[0018] The amino acid sequence of carbonyl reductase SsCR (PDB: 5GMO) is shown in SEQ ID No. 2. Carbonyl reductase SsCR is known and is derived from the yeast Scheffersomyces stipitis. This application used it as a parent to perform molecular engineering and screened for mutants with improved catalytic performance and stability, as well as those that relieve substrate inhibition.

[0019] The present invention also provides a method for obtaining the carbonyl reductase mutant: through gene mining and large-scale screening of laboratory-preserved microbial strains, the gene for the carbonyl reductase SsCR (PDB: 5GMO) from Pichia stipitis was discovered. The gene was artificially synthesized, directly linked to the PET-28a expression vector, and directly transformed into Escherichia coli Ecoli.BL 21 (DE3). Through induced expression and purification, a carbonyl reductase capable of catalyzing the asymmetric reduction of α- / β-saturated ketoesters was obtained, which is NADPH-dependent. The amino acid sequence of the carbonyl reductase SsCR is shown in SEQ ID No. 2.

[0020] In the present invention, the gene is artificially synthesized, directly connected to the PET-28a expression vector, and directly transformed into Escherichia coli Ecoli.BL 21 (DE3) for induced expression and purification.

[0021] The carbonyl reductase mutant provided by the present invention has the following properties: it can catalyze the asymmetric reduction of α- / β-saturated ketoesters and stereoselectively synthesize multi-substituted chiral γ-butyrolactone.

[0022] The present invention also provides a method for semi-rational design and construction of a carbonyl reductase mutant library. The crystal structure of SsCR can be downloaded by inputting the protein ID (5GMO) in ProteinData Bank, and then the protein model is used for molecular docking with (R)-3-methyl-4-oxooctanoic acid ethyl ester (R)-2t. According to the catalytic mechanism and binding energy of short-chain dehydrogenase, a suitable docking model is selected. The amino acids at these sites were subjected to site-directed saturation mutagenesis to further improve the activity of the enzyme. In the three-dimensional structure of the SsCR amino acid sequence shown in SEQ ID No. 2, the amino acid residues surrounding the substrate (R)-2t binding site include: Y13, L14, P83, V84, F86, T123, S124, V126, V127, F164, N191, D192, V193, Y194, I195, F196, N210, L210, S212, M215, I236, and V241. A semi-rational design was used to perform site-directed saturation mutagenesis on the amino acids at these sites. The primers used are shown in Table 1:

[0023] Table 1 Primers for SsCR mutation amino acid sites

[0024]

[0025]

[0026] The second technical solution of the present invention:

[0027] The present invention provides an isolated nucleic acid, which is a nucleic acid molecule encoding the carbonyl reductase mutant as described in technical solution one.

[0028] The nucleotide sequence of the nucleic acid encoding carbonyl reductase (SsCR) is shown in SEQ ID No. 1, with a total length of 1005 nucleotides. The coding sequence (CDS) from base 1 to base 1005 has an ATG start codon, no stop codon, and is expressed as a fusion of upstream and downstream histidine tags. The protein encoded by this gene is carbonyl reductase (SsCR), and its amino acid sequence is shown in SEQ ID No. 2.

[0029] The present invention also provides the source of the nucleic acid: the nucleic acid is obtained by an artificial full sequence synthesis method.

[0030] The third technical solution of the present invention:

[0031] The present invention provides a recombinant expression vector comprising the nucleic acid encoding the carbonyl reductase mutant.

[0032] The recombinant expression vector of the present invention can be constructed by ligating the nucleic acid of the carbonyl reductase mutant to various expression vectors using conventional methods in the art. Such expression vectors include various plasmid vectors conventional in the art, preferably plasmid pET-28a. The gene of the carbonyl reductase mutant can be ligated to the pET-28a vector during artificial gene synthesis to obtain the corresponding recombinant expression vector.

[0033] The fourth technical solution of the present invention:

[0034] The present invention provides a recombinant expression transformant comprising the nucleic acid encoding the carbonyl reductase mutant or the carbonyl reductase recombinant expression vector.

[0035] The recombinant expression transformant of the present invention can be obtained by transforming the recombinant expression vector described in Technical Solution 3 into a host cell. The host cell is a conventional host cell in the art, as long as the recombinant expression vector can stably replicate itself and the gene of the enzyme it carries can be effectively expressed. The host cell is preferably Escherichia coli, more preferably Escherichia coli E.coli BL21 (DE3) or Escherichia coli E.coli DH5α. This solution preferably transforms the recombinant expression vector into Escherichia coli E.coli BL21 (DE3) to obtain the preferred genetically engineered strain of the present invention (E.coliBL21 (DE3) / pET28a-SsCR).

[0036] The fifth technical solution of the present invention is:

[0037] The present invention provides a carbonyl reductase mutant catalyst, wherein the enzyme catalyst is in any of the following forms:

[0038] (1) culturing the recombinant expression transformant as described in Technical Solution 4, and isolating transformant cells containing the carbonyl reductase mutant;

[0039] (2) culturing the recombinant expression transformant as described in Technical Solution 4, isolating the transformant cells containing the carbonyl reductase mutant, and disrupting the transformant cells containing the carbonyl reductase mutant to obtain a cell disrupted liquid;

[0040] (3) freeze-drying the cell disrupted liquid of the carbonyl reductase mutant to obtain a freeze-dried enzyme powder;

[0041] (4) Pure enzyme of the carbonyl reductase mutant.

[0042] The present invention also provides a method for preparing the enzyme catalyst, which is preferably: culturing the recombinant expression transformant as described in Technical Solution 4, and isolating and obtaining a recombinantly expressed carbonyl reductase mutant. The culture medium used for culturing the recombinant expression transformant is any culture medium in the art that can enable the transformant to grow and produce the recombinant carbonyl reductase of the present invention. The culture medium is preferably LB culture medium, and its formula is: peptone 10g / L, yeast extract 5g / L, NaCl 10g / L, pH 7.0. There are no special restrictions on the culture method and culture conditions. They can be appropriately selected according to conventional knowledge in the art based on factors such as the host cell type and culture method, as long as the transformant can grow and produce the carbonyl reductase mutant. The specific operations for culturing the recombinant expression transformant can be carried out according to conventional operations in the art. Preferably, the several recombinant Escherichia coli described in the present invention are inoculated into LB culture medium containing kanamycin respectively, and cultured at 37°C. When the optical density OD 600 Reach 0.5~1.0 (preferably OD 600 When the concentration of isopropyl-β-D-thiogalactopyranoside (IPTG) is 0.6), a final concentration of 0.1 to 1.0 mmol / L (preferably 0.5 mmol / L) is added to induce enzyme production, and the culture is continued at 16°C for 24 hours to efficiently express the carbonyl reductase mutant of the present invention. After the culture is completed, the precipitated bacterial cells are collected by centrifugation to obtain the resting cells of the recombinant expression transformant; the harvested cells are suspended in PBS buffer (100 mM, pH 7.0), ultrasonically disrupted, the disrupted liquid is centrifuged, and the supernatant is collected to obtain the crude enzyme solution of the recombinant carbonyl reductase SsCR and its mutants; the cell pellet harvested by centrifugation is freeze-dried to obtain freeze-dried cells, which are convenient for long-term storage and future use.

[0043] Enzyme activity determination: Preheat 1 ml of reaction system (100 mmol / L sodium phosphate buffer, pH 6.5) containing 2 mmol / L α- / β-unsaturated ketoester and 0.1 mmol / L NADPH to 30°C, then add an appropriate amount of the corresponding pure enzyme, mix well, and incubate at 30°C for reaction. Measure the absorbance change of NADPH at 340 nm on a spectrophotometer and record the absorbance change over a certain period of time.

[0044] The enzyme activity was calculated according to the following formula:

[0045] Enzyme activity (U) = EW × V × 10 3 / (6220×l)

[0046] Where EW is the change in absorbance at 340 nm over 1 minute; V is the volume of the reaction solution in mL; 6220 is the molar extinction coefficient of NADPH in L / (mol·cm); and l is the optical path length in cm. One unit (U) of enzyme activity corresponds to the amount of enzyme required to oxidize 1 μmol of NADPH per minute under the above conditions.

[0047] Technical solution six of the present invention:

[0048] The present invention provides use of a carbonyl reductase mutant and a carbonyl reductase mutant catalyst in synthesizing chiral gamma-butyrolactone by asymmetric reduction of alpha- / beta-saturated gamma-ketoester.

[0049] The chemical structure of the α- / β-saturated γ-ketoester 2 is shown below:

[0050]

[0051] The asymmetric reduction of α- / β-saturated γ-ketoester to synthesize chiral γ-butyrolactone can be carried out according to the following preferred method: in a phosphate buffer solution at pH 6.5, in the presence of glucose dehydrogenase (2 g / L), glucose (1.5 equiv) and NADP + In the presence of 0-1.0 mmol / L, the asymmetric reduction reaction of the α- / β-saturated γ-ketoester intermediate is catalyzed by the carbonyl reductase mutant and the carbonyl reductase mutant catalyst (10 g / L freeze-dried enzyme powder).

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] (1) The present invention innovatively constructs a process route for stereoselective synthesis of chiral aromatic polysubstituted γ-butyrolactones.

[0054] (2) The carbonyl reductase SsCR-V126A / N210H / M215G provided by the present invention is a mutant based on the existing enzyme and can be efficiently used in the catalytic system described in the present invention, thereby efficiently synthesizing a variety of multi-substituted chiral γ-butyrolactones.

[0055] (3) The lactone compound of the present invention can be used as an intermediate for fragrance products or natural products, and has wide application value in the fragrance market and in the synthesis of natural products.

[0056] (4) The present invention has mild reaction conditions, high conversion rate, high catalytic activity, good product optical purity, high atom economy, and ee and dr values ​​that can be higher than 99%, which has good industrial application prospects. DETAILED DESCRIPTION

[0057] The present invention is described in detail below through specific examples, but the invention is not limited to the scope of these examples. Experimental methods in the following examples, where specific conditions are not specified, were performed according to conventional methods and conditions, or according to the product specifications. The reaction or detection conditions described in the Summary of the Invention can be combined or modified according to common knowledge in the art and can be verified through experimentation.

[0058] The sources of materials in the following examples are:

[0059] The recombinant plasmid pET28a-SsCR contains the nucleic acid sequence shown in SEQ ID No. 1 in the sequence listing and is constructed by Shanghai Qingke Biotechnology Co., Ltd.

[0060] The expression plasmid pET28a was purchased from Novagen.

[0061] E. coli BL21 (DE3) competent cells were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.

[0062] Example 1: Preparation of recombinant expression plasmid and recombinant expression transformant of carbonyl reductase SsCR

[0063] The recombinant expression plasmid of carbonyl reductase SsCR was directly synthesized by Shanghai Qingke Biotechnology Co., Ltd. and the corresponding gene sequence was connected to the PET-28a vector.

[0064] The resulting recombinant plasmid was transformed into E. coli BL21(DE3) competent cells and plated onto LB medium containing 50 μg / mL kanamycin. The cells were cultured at 37°C for at least 8 hours. The resulting colonies were verified by colony PCR, and positive clones corresponding to the successfully amplified target band of the correct length were selected. After sequencing verification, the positive clones were selected to obtain the recombinant expression transformants (E. coli BL21(DE3) / pET28a-SsCR).

[0065] Example 2: Inducible expression of carbonyl reductase SsCR

[0066] The recombinant expression transformant obtained in Example 1 was inoculated into LB medium containing 50 μg / mL kanamycin and cultured on a shaker at 37°C for 12 h. Then, the inoculum was inoculated into a 500 ml conical flask containing 100 ml LB medium (containing 50 μg / mL kanamycin) at a 1% (v / v) inoculum volume and cultured on a shaker at 37°C and 220 rpm. When the OD of the culture solution reached 600 When the p-value reached 0.6, IPTG was added to a final concentration of 0.2 mmol / L for induction. After induction at 16°C for 24 h, the culture medium was centrifuged at 7500 rpm, the cell pellet was collected, and washed with physiological saline to obtain resting cells.

[0067] The resting cells of 0.5g as above method gained are suspended in the sodium phosphate buffer (100mM, pH 6.5) of 100mL, and ultrasonication is carried out in an ice-water bath. The centrifugal collection supernatant is the crude enzyme liquid of corresponding recombinant carbonyl reductase. The gained crude enzyme liquid is through polyacrylamide gel electrophoresis analysis, and recombinant carbonyl reductase exists with the form of soluble expression. In addition the crude enzyme liquid of the recombinant carbonyl reductase that can be obtained can be carried out freeze drying to obtain the crude enzyme powder of recombinant carbonyl reductase.

[0068] Example 3 Site-directed saturation mutagenesis of carbonyl reductase SsCR

[0069] PCR amplification was performed using PrimeStar HSDNA Polymerase using pET28a-SsCR as a template. The PCR system was as follows: pET28a-SsCR plasmid (100 nM, 1 uL), upstream and downstream primers (10 nM, 1 uL), 5× PrimeStar Buffer (MgCl2 2+Plus, 4uL), dNTP Mixture (2.5mM, 1.6uL), PrimeStar HS DNA Polymerase (0.2uL), and add sterile distilled water to 20uL. PCR reaction program: (1) 95℃ pre-denaturation for 3min; (2) 98℃ denaturation for 10s; (3) set gradient temperature annealing for 10s; (4) 72℃ extension for 6min40s. Steps (2) to (4) were extended for 30 cycles in total; and finally extended at 72℃ for 10min. 0.5μL of Dpn I enzyme was added to 8.5μL of PCR product and 1uL of rSmart cut buffer and incubated at 37℃ for 3h to digest the template. The digestion product was transformed into E. coli BL21 (DE3) competent cells and evenly spread on LB agar plates containing 50μg / ml kanamycin. The plates were placed in a 37℃ incubator and incubated for about 12h. The resulting monoclonal colonies were picked and cultured in 96-well deep-well plates. The cells were disrupted with lysozyme, and the expressed proteins were screened for high-throughput activity in the 96-well plates using NADPH as a coenzyme. Mutants with higher activity were purified and characterized, and the corresponding genes were sequenced. High-throughput screening using a microplate reader revealed that replacing valine at position 126 with alanine (V126A); replacing methionine at position 215 with glycine, and combining this with replacing valine at position 126 with alanine (V126A / M215G); and the final mutant (V126A / N210H / M215G), resulting from a combination of replacing valine at position 126 with alanine, methionine at position 215 with glycine, and asparagine at position 210 with histidine, all improved the activity of (R)-2t. Among them, SsCR-V126A has the highest activity, but both the first-generation mutant (V126A) and the second-generation mutant (V126A / M215G) have substrate inhibition effects. At low concentrations, SsCR-V126A may have a better catalytic effect, while at high concentrations, the final mutant SsCR-V126A / N210H / M215G, which eliminates the substrate inhibition effect, has the best effect. Therefore, the desired mutant can be flexibly selected according to the substrate concentration. It was purified and characterized, and the enzyme activity and kinetic parameters of the mutants were determined (shown in Tables 2-3).

[0070] High-throughput activity screening assay for SsCR mutants: Sodium phosphate buffer (100 mmol / L, pH 6.5) containing 2 mmol / L substrate (R)-3-methyl-4-oxooctenoic acid ethyl ester (R)-2t and 0.1 mmol / L NADPH was dispensed into a 96-well plate and preheated to 30°C. An appropriate amount of SsCR mutant enzyme solution was then added using a dispenser. The reaction was shaken at 30°C. The absorbance change of NADPH at 340 nm was detected on a microplate reader. The absorbance change within 10 minutes was recorded and the corresponding enzyme activity was calculated.

[0071] Method for determining the enzyme activity of SsCR mutants: preheat 1 mL of reaction system (100 mmol / L sodium phosphate buffer, pH 6.5) containing 2 mmol / L different substrates and 0.1 mmol / L NADPH to 30°C, then add an appropriate amount of SsER mutant enzyme solution, incubate at 30°C, detect the absorbance change at 340 nm on a spectrophotometer, record the absorbance change within 1 min, and calculate the enzyme activity.

[0072] Table 2 Kinetic parameters of mutants against (R)-2t

[0073]

[0074] a Fold change in k cat / K m over the WT. b Fold change in K i over the V1. c NI:Not Inhibited.

[0075] Table 3 Specific activity of mutants against (R)-2t

[0076]

[0077] Next, we screened for carbonyl reductases that could catalyze the reduction of aromatic α- / β-saturated γ-ketoester intermediates. M5 (E23K / K61V / K127E / N145R / A149T / E104K, Chem. Commun., 2021, 57, 10584-10587) and SmCR V4(R123C / L209P / F183Y / V61K, ChemCatChem., 2019, 11, 2600–2606) have been shown to have high specific activity and stereoselectivity for aliphatic γ-keto acids and γ-ketoesters. Among them, SsCR-V3 is a mutant obtained by molecular modification. Therefore, these four CRs were selected for comparative screening, and their activities and enantioselectivities for 2a-2s were determined (Table 4). SsCR-V3 showed high specific activity for substrates 2a / 2b / 2d (1.17–9.05 U mg -1 ) and excellent diastereoselectivity (94:6–>99:1dr). Especially for the bulky substrate 2c, the specific activity of SsCR-V3 reached 54.3 U mg -1 However, its diastereoselectivity was poor (56:44dr), possibly due to the large steric hindrance of 2c. Compared with the parent SsCR, the mutant SsCR-V3 significantly improved the activity of 2a-2d, increasing it to 1-2 orders of magnitude.

[0078]

[0079] Table 4 Screening of carbonyl reductase

[0080] Table 4.Screening of several carbonyl reductases.

[0081]

[0082]

[0083] Table 4

[0084]

[0085]

[0086] Table 4

[0087]

[0088]

[0089] Specific activity (U mg -1):35℃,substrates(2mM),PBS(pH 6.5),NADPH(0.1mM),purifiedenzymes.The enantioselectivity of different ene reductases was analyzed by GC.

[0090] “-”:No reaction.“nd”:not determined.

[0091] Furthermore, the data in these tables indicate that SsCR and its mutant SsCR-V3 catalyze the reaction of most aromatic α- / β-saturated γ-ketoester intermediates. In particular, mutant SsCR-V3 exhibits significantly improved activity toward some substrates. SsCR-V3 exhibits high specific activity (1.2–7.2 U mg⁻¹) and good diastereoselectivity (89:11–99:1 dr) toward substrates 2h-2k bearing halogen atoms on the phenyl ring. However, SsCR-V3 exhibits lower activity (0.26–0.78 U mg⁻¹) toward substrates 2l-2o bearing alkyl substituents on the phenyl ring, but this activity is sufficient for small-scale preparations. This demonstrates the high tolerance of SsCR-V3 toward substrates bearing electron-withdrawing substituents on the phenyl ring.

[0092] In addition, SsCR-V3 was also able to catalyze the bulky substrate 2p-2s, although the activity was weak (0.05–0.33 U mg -1 A carbonyl reductase-catalyzed process has successfully synthesized bulky trisubstituted aromatic chiral γ-butyrolactones (4p-4s) for the first time via a bioenzymatic method, achieving 98-99% ee and >99:1 dr. Compared to traditional methods, this asymmetric reduction reaction represents a simple, atom-economical, and enantioselective approach to afford large quantities of aromatic multisubstituted chiral γ-butyrolactones in an inexpensive and efficient manner.

[0093] Large-scale preparation of γ-butyrolactone catalyzed by the carbonyl reductase mutant described in Example 4

[0094] The reaction is an asymmetric reduction of an α- / β-saturated γ-ketoester intermediate by a carbonyl reductase (CRs) mutant, and can be carried out according to the following preferred method: in a phosphate buffer at pH 6.5, in the presence of glucose dehydrogenase, glucose and NADP + In the presence of, under the action of the carbonyl reductase catalyst, catalyzes the asymmetric reduction reaction of the α- / β-saturated γ-ketoester intermediate.

[0095] In this example, the amount of the carbonyl reductase (SsCR-V126A / M215G / N210H mutant) added to the system was lyophilized enzyme powder (10 g / L). During the enzymatic asymmetric reduction of the α- / β-saturated γ-ketoester intermediate, the coenzyme NADPH is oxidized to generate NADP. + In order to recycle the coenzyme NADPH, glucose and glucose dehydrogenase from Bacillus megaterium were added to the reaction system (J Ind Microb Biotechnol, 2011, 38: 633–641). The molar ratio of glucose to substrate was 1.5, and the additional NADP + The dosage is 0.2mmol / L. The buffer is sodium phosphate buffer, preferably at pH 6.5. The concentration of phosphate buffer is 0.2mol / L. The temperature of the enzymatic asymmetric reduction reaction is preferably 30°C. During the reaction, intermittent sampling is performed to determine the reaction conversion rate. The reaction time is based on the time when the substrate is completely converted or the reaction conversion rate stops increasing, which is generally 1 to 24h. In this reaction, the reaction is carried out in a 100mL beaker, and 1.5equiv of glucose and 0.2mM NADP are directly added to the beaker after the first step reaction. + The carbonyl reductase (SsCR-V126A / M215G / N210H mutant) lyophilized enzyme powder (10 g / L) and glucose dehydrogenase lyophilized enzyme powder (2 g / L) were used. The reaction was carried out in a water bath at 30°C. The reaction was allowed to proceed for 1 to 24 hours, terminated with a 2M sulfuric acid solution, extracted with an equal volume of ethyl acetate, and dried over anhydrous sodium sulfate overnight. The substrate conversion and the ee value of the reduction product were determined. The product yield is the isolated yield, as shown below.

[0096]

[0097] The reaction conversion and product enantiomeric excess (ee) can be analyzed by gas chromatography, preferably using Conversion analysis was performed using a MS capillary column (30 m × 0.25 mm × 0.25 μm) with nitrogen as the carrier gas and a flame ionization detector (FID) as the detector. The inlet and detector temperatures were 280°C. EE analysis of the reduction products was performed using a GC / CP-Chirasil-Dex CB capillary column (25 m × 0.25 mm × 0.25 μm) with nitrogen as the carrier gas and a flame ionization detector (FID) as the detector. The inlet and detector temperatures were 280°C.

[0098] After the enzymatic asymmetric reduction reaction is completed, extraction is performed with an equal amount of a water-insoluble organic solvent commonly used in the art, such as ethyl acetate, butyl acetate, toluene, dichloromethane, chloroform, isopropyl ether, or methyl tert-butyl ether. The extraction is repeated twice, and the extracts are combined and dried over anhydrous sodium sulfate. In this example, ethyl acetate is used for extraction. The resulting dried crude organic layer is subjected to rotary evaporation to remove the solvent, thereby obtaining the corresponding optically active chiral lactone crude product. Purified γ-butyrolactone is then obtained by column chromatography (eluent: petroleum ether / ethyl acetate = 20:1).

[0099] The various reaction or detection conditions described in the present invention can be combined or modified according to common knowledge in the art and can be verified through experiments.

[0100] The sequences involved in the present invention are as follows:

[0101] SEQ ID NO.1

[0102] atgactacctcagttttcgtttcaggtgcaaccggttaccttgcccaacaaattattgcacttgttctctccaagggctacaa

[0103] ggtcgttggttcggtcagatctgaagaaaagggtgcaaacttaaaaaaattgtatggtgacgatttctcctatgaagttgtcaagg

[0104] tcttggaacagaagggtgctttcgatgaagccttgaagaagcaccgaagttacaattttcttacacactgcctctccagttacc

[0105] ttcgaagttgaagataccgaaaaggaaatcttgattcctgccattaatggaacaaagtacgtcttgcaatctatcaaggacgttgc

[0106] tcctcaaatcaccagagttgtttacaccagttctgtcgttgctatgagcgtcccagaggaattaggtagcccagatgtggtcctct

[0107] ctgaagcttcttggagtagtctctcttacgagcaatccaagactcatggagttttggcttacttcggttcgaagcaatttgctgaaa

[0108] gggctgcatgggagtttgttgaacaggaaaagccaaactttgctctctcgaccgtaaaccctgtctacatttttggtcctcaagct

[0109] aaggacgaggaagttaagggtaccttgaacctttctgccgaaatggttaattccgtattgaagttgaataaggacgacgatgttc

[0110] cagcaactactggtactttcattgatgttagagatgtggctaaagctcaccttgcagccttcgaaaaggacgaagcaaagggtg

[0111] aaagacttctcctctctaacaccagattcaatggtcaaactcttttggacgttgttagaaagaacttcccacaacttgctgacaagc

[0112] ttccagttggaaagccacattctgacgatttctctgcttttaaggaatggaacgacaagaagaccaagaagattcttggatttgaat

[0113] acttcgactttgaaacttctgttgttgactcaatcaagcaagttttgaaggtacaaggttaa

[0114] SEQ ID NO. 2

[0115] MTTSVFVSGATGYLAQQIIALVLSKGYKVVGSVRSEEKGANLKKLYGDDFS

[0116] YEVVKVLEQKGAFDEALKKHPEVTIFLHTASPVTFEVEDTEKEILIPAINGTKYV

[0117] LQSIKDVAPQITRVVYTSSVVAMSVPEELGSPDVVLSEASWSSLSYEQSKTHGVL

[0118] AYFGSKQFAERAAWEFVEQEKPNFALSTVNPVYIFGPQAKDEEVKGTLNLSAEM

[0119] VNSVLKLNKDDDVPATTGTFIDVRDVAKAHLAAFEKDEAKGERLLLSNTRFNG

[0120] QTLLDVVRKNFPQLADKLPVGKPHSDDFSAFKEWNDKKTKKILGFEYFDFETS

[0121] VVDSIKQVLKVQG*

[0122] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A carbonyl reductase mutant, characterized in that The carbonyl reductase mutant is one of the proteins with the following amino acid sequence: (1) replacing valine at position 126 of the amino acid sequence shown in SEQ ID No. 2 with alanine; (2) The valine at position 126 of the amino acid sequence shown in SEQ ID No. 2 is replaced by alanine, and the methionine at position 215 is replaced by glycine; (3) The valine at position 126 of the amino acid sequence shown in SEQ ID No. 2 is replaced by alanine, and the asparagine at position 210 is replaced by histidine; (4) The valine at position 126 of the amino acid sequence shown in SEQ ID No. 2 is replaced by alanine, the methionine at position 215 is replaced by glycine, and the asparagine at position 210 is replaced by histidine.

2. An isolated nucleic acid, characterized in that The nucleic acid encodes the carbonyl reductase mutant according to claim 1.

3. A recombinant expression vector, characterized in that: Comprising the nucleic acid according to claim 2.

4. A recombinant expression transformant, characterized in that: Comprising the recombinant expression vector according to claim 3.

5. A carbonyl reductase mutant catalyst, characterized in that: Select from any of the following forms: (1) Cultivating the recombinant expression transformant according to claim 4, and isolating transformant cells containing the carbonyl reductase mutant according to claim 1; (2) culturing the recombinant expression transformant according to claim 4, isolating transformant cells containing the carbonyl reductase mutant according to claim 1, and disrupting the transformant cells containing the carbonyl reductase mutant to obtain a cell disrupted liquid; (3) Cultivating the recombinant expression transformant according to claim 4, isolating transformant cells containing the carbonyl reductase mutant according to claim 1, disrupting the transformant cells containing the carbonyl reductase mutant, obtaining a cell disrupted liquid, and freeze-drying the cell disrupted liquid of the carbonyl reductase mutant to obtain a freeze-dried enzyme powder.

6. Use of the carbonyl reductase mutant according to claim 1 or the carbonyl reductase mutant catalyst according to claim 5, characterized in that: A method for using the carbonyl reductase mutant according to claim 1 or the carbonyl reductase mutant catalyst according to claim 5 for asymmetric reduction of α- / β-saturated γ-ketoesters to synthesize chiral γ-butyrolactone.

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