Carbonyl reductase mutant and application thereof in stereoselective synthesis of aromatic polysubstituted chiral gamma-butyrolactone

Abstract

The invention relates to a carbonyl reductase mutant and application of the carbonyl reductase mutant in stereoselective synthesis of aromatic polysubstituted chiral gamma-butyrolactone. The carbonyl reductase mutant is a protein which is obtained by substitution, deletion or addition of several amino acids on an amino acid sequence as shown in SEQ ID No.2, can catalyze asymmetric reduction of aromatic alpha- / beta-unsaturated ketone ester, is derived from carbonyl reductase SsCR and has catalytic activity higher than that of the carbonyl reductase SsCR, and the amino acid sequence of the carbonyl reductase SsCR is as shown in SEQ ID No.2. The invention also provides a gene for coding the carbonyl reductase mutant, a recombinant expression vector and a recombinant expression transformant containing the gene, and application of the carbonyl reductase mutant in synthesis of chiral aromatic polysubstituted gamma-butyrolactone. Compared with the prior art, the method disclosed by the invention has the remarkable advantages of simple process, high atom economy, high optical purity of the product and environmental friendliness, and has a very good industrial application prospect.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering technology, and in particular relates to a mutant of a carbonyl reductase SsCR of 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 stereoselective synthesis of aromatic multi-substituted chiral γ-butyrolactone. A method for stereoselective synthesis of aromatic multi-substituted chiral γ-butyrolactone is also provided. Background Art

[0002] Flavors and fragrances are a class of substances with pleasant odors, which are widely used in the fields of food, daily chemicals, chemistry and pharmaceuticals. Initially, people separated and extracted natural flavors from plants. Due to factors such as low concentration, seasonal changes and plant diseases, natural flavors are costly and the market price is very expensive (Food & Bioprocess Technology, 2018, 11: 2217-2228). In the 1850s, scientists began to use chemical methods to synthesize such compounds, and the market price of artificial flavors and fragrances fell rapidly. 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 (extracted from natural resources) or prepared using enzymes and microorganisms, and the precursors involved should 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 application value in industry. 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 global flavor and fragrance market size 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. Food Chem. 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, present 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-decanoic acid has the aroma of peach and strawberry, while δ-decanoic acid has the aroma of coconut milk (Trends in Biotechnology. 2005, 23 (4): 193-198.). The type of side chain of lactone, the position of functional group and the length of carbon chain will affect its flavor characteristics. For example, γ-octanolactone has a milky flavor, while γ-undecanolide has a strong peach and almond aroma (J. Agric. Food Chem. 1989, 37: 413-418.). The stereo configuration of lactone compounds will also affect the purity and intensity of the aroma. Natural lactones are mainly (R)-enantiomers, which have a stronger smell than (S)-enantiomers 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 weak, and consumer acceptance is also low. For example, chemically synthesized γ-decanolide is obviously inferior to naturally extracted γ-decanolide in aroma intensity. Therefore, as a food additive, lactone compounds with a single configuration are in greater demand, have higher added value, and are well received by 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 biologically active 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. In addition, chiral lactones can act as pheromones in plants and microorganisms, participate in intercellular signal transduction, affect plant growth and development or bacterial quorum sensing. Therefore, the structure and chirality of chiral lactone compounds make them play an important role in a variety of biological systems.

[0006] Chiral polysubstituted γ-butyrolactones are core building blocks of many natural products and high value-added chemicals in the pharmaceutical and food industries. At present, the synthesis of chiral polysubstituted γ-butyrolactones is still dominated by chemical methods. The precise construction of multiple chiral centers on five-membered heterocycles substituted with multiple groups faces great challenges, especially for bulky aromatic substituted chiral γ-butyrolactones, which face problems such as low stereoselectivity, expensive metal catalysts, harsh reaction conditions and low atom economy. Summary of the invention

[0007] In view of the problems faced by the chemical synthesis of chiral polysubstituted γ-butyrolactone 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-butyrolactone, that is, the carbonyl reductase mutant is used to perform asymmetric reduction on the potential chiral carbon of 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, and further provides 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 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 is substituted, deleted or added with several amino acids as shown in SEQ ID No. 2 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, wherein the amino acid sequence of the carbonyl reductase SsCR is as shown in SEQ ID No. 2.

[0013] In one embodiment of the present invention, the carbonyl reductase SsCR mutant is one of the proteins having 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 is replaced by alanine, and the methionine at position 215 is replaced by glycine;

[0016] (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;

[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 Scheffersomyces stipitis. The present application uses it as the parent to carry out molecular modification and screens mutants with improved catalytic performance and stability and relieved substrate inhibition effect.

[0019] The present invention also provides a method for obtaining the carbonyl reductase mutant: that is, through gene mining and large-scale screening of laboratory preserved microbial strains, the gene of carbonyl reductase SsCR (PDB: 5GMO) from Pichia stipitis is found, and it is directly connected to the PET-28a expression vector by artificial synthesis, and directly transformed into Escherichia coli Ecoli.BL 21 (DE3), and a carbonyl reductase capable of catalyzing asymmetric reduction of α- / β-saturated ketoesters is obtained by inducing expression and purification, 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 directly connected to the PET-28a expression vector by means of artificial synthesis, and is directly transformed into Escherichia coli Ecoli.BL 21 (DE3) for induced expression and purification.

[0021] The carbonyl reductase mutant provided by the invention has the following properties: it can catalyze the asymmetric reduction of α- / β-saturated ketoester 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 the short-chain dehydrogenase, a suitable docking model is selected. The amino acids of the substrate (R)-2t binding site are subjected to site-directed saturation mutation 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 around 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, etc. Semi-rational design was used to perform site-directed saturation mutation on the amino acids of 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, wherein the nucleic acid is a nucleic acid molecule encoding the carbonyl reductase mutant as described in technical solution one.

[0028] Among them, the nucleotide sequence of the nucleic acid encoding carbonyl reductase SsCR is shown in SEQ ID No.1, with a total length of 1005 nucleotide bases. Its coding sequence (CDS) starts from the first base to the 1005th base, the start codon is ATG, there is no stop codon, and the upstream and downstream histidine tags are fused and expressed. The protein encoded by the gene is carbonyl reductase SsCR, and the 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 connecting the nucleic acid of the carbonyl reductase mutant to various expression vectors by conventional methods in the art, and the expression vector includes various plasmid vectors conventional in the art, preferably plasmid pET-28a. The gene of the carbonyl reductase mutant can be connected 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 three 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 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) A 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 four, 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 grow the transformant 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, and they can be appropriately selected according to the common knowledge in the art according to factors such as the host cell type and the culture method, as long as the transformant can grow and produce the carbonyl reductase mutant. The specific operation of culturing the recombinant expression transformant can be carried out according to the conventional operation in the art. Preferably, the several recombinant Escherichia coli described in the present invention are respectively inoculated into LB culture medium containing kanamycin 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 0.1-1.0 mmol / L (preferably 0.5 mmol / L) at a final concentration is added to induce enzyme production, and the culture is continued at 16°C for 24 hours to efficiently express a carbonyl reductase mutant described in the present invention. After the culture is completed, the precipitated bacterial cells are collected by centrifugation, which are 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 precipitate harvested by centrifugation is freeze-dried to obtain freeze-dried cells, which is conducive to long-term storage and convenient for future use.

[0043] Enzyme activity determination: Preheat 1 ml 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 keep the reaction at 30°C. Detect the absorbance change of NADPH at 340 nm on a spectrophotometer and record the change in absorbance within 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 within 1 min; V is the volume of the reaction solution, in mL; 6220 is the molar extinction coefficient of NADPH, in L / (mol·cm); l is the optical path length, in cm. One enzyme activity unit (U) corresponds to the amount of enzyme required to oxidize 1 μmol NADPH per minute under the above conditions.

[0047] Technical solution six of the present invention:

[0048] The invention provides use of a carbonyl reductase mutant and a carbonyl reductase mutant catalyst in asymmetric reduction of α- / β-saturated γ-ketoester to synthesize chiral γ-butyrolactone.

[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 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), under the action of the carbonyl reductase mutant and the carbonyl reductase mutant catalyst (10 g / L freeze-dried enzyme powder), the asymmetric reduction reaction of the α- / β-saturated γ-ketoester intermediate is catalyzed.

[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-butyrolactone.

[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 of a fragrance product or a natural product, and has a wide range of application value in the fragrance market and in the synthesis of natural products.

[0056] (4) The reaction conditions of the present invention are mild, the conversion rate is high, the catalytic activity is high, the product has good optical purity, high atom economy, and the ee value and dr value can be higher than 99%, which has a good industrial application prospect. DETAILED DESCRIPTION

[0057] The present invention is described in detail below through specific examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product specifications. The various reactions or detection conditions described in the summary of the invention can be combined or changed according to common knowledge in the art, and can be verified by experiments.

[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 table, and is a sequence 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 connected to the PET-28a vector.

[0064] The obtained recombinant plasmid was transformed into E.coli BL21 (DE3) competent cells, spread on LB medium plates containing 50 μg / mL kanamycin, and cultured at 37°C for more than 8 hours. The grown colonies were verified by colony PCR, and the positive clones corresponding to the successfully amplified target bands with the correct length were picked. After sequencing verification, the positive clones were picked 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 in a shaking incubator 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 rate of 1% (v / v), and the flask was placed in a shaking incubator at 37°C and 220 rpm for shaking culture. When the OD of the culture solution reached 600 When it 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 solution was centrifuged at 7500 rpm, the cell precipitate was collected, and washed with physiological saline to obtain resting cells.

[0067] 0.5 g of resting cells obtained by the above method were suspended in 100 mL of sodium phosphate buffer (100 mM, pH 6.5), ultrasonically broken in an ice-water bath, and the supernatant was collected by centrifugation to obtain the crude enzyme solution of the corresponding recombinant carbonyl reductase. The obtained crude enzyme solution was analyzed by polyacrylamide gel electrophoresis, and the recombinant carbonyl reductase existed in the form of soluble expression. In addition, the crude enzyme solution of the recombinant carbonyl reductase obtained can be freeze-dried to obtain the crude enzyme powder of the recombinant carbonyl reductase.

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

[0069] Using pET28a-SsCR as template, PrimeStar HSDNA Polymerase was used for PCR amplification. The PCR system was: pET28a-SsCR plasmid (100nM, 1uL), upstream and downstream primers (10nM, 1uL), 5× PrimeStar Buffer (Mg 2+plus, 4uL), dNTP Mixture (2.5mM, 1.6uL), PrimeStar HS DNA Polymerase (0.2uL), add sterile distilled water to make up 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 performed for a total of 30 cycles; and finally extended at 72℃ for 10min. Add 0.5μL of Dpn I enzyme to 8.5μL of PCR product and 1uL of rSmart cut buffer and incubate 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 and placed in a 37℃ incubator for about 12h. The obtained monoclonal colonies were picked up and cultured in 96-well deep-well plates, and the cultured cells were broken by lysozyme. The expressed proteins were screened for high-throughput activity in 96-well plates with NADPH as a coenzyme. The mutants with higher activity were purified and characterized, and the corresponding genes were sequenced. Through high-throughput screening by microplate reader, it was found that replacing valine at position 126 with alanine (V126A); replacing methionine at position 215 with glycine, and combining valine at position 126 with alanine (V126A / M215G); replacing valine at position 126 with alanine, methionine at position 215 with glycine, and asparagine at position 210 with histidine to obtain the final mutant (V126A / N210H / M215G) 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 mutant were determined (as 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, preheated to 30°C, and then an appropriate amount of SsCR mutant enzyme solution was added using a dispenser. The reaction was shaken at 30°C, and the absorbance change of NADPH at 340 nm was detected on a microplate reader. The absorbance change within 10 min 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, keep the reaction 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 carbonyl reductases that can 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, due to the large steric hindrance of 2c, its diastereoselectivity is poor (56:44dr). Compared with the parent SsCR, the mutant SsCR-V3 significantly improves the activity of 2a-2d, which is 1-2 orders of magnitude higher than that of the parent.

[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] In addition, from the data in these tables, it can be seen that SsCR and its mutant SsCR-V3 can catalyze most aromatic α- / β-saturated γ-ketoester intermediates. In particular, the mutant SsCR-V3 has significantly improved activity for some substrates. SsCR-V3 showed high specific activity (1.2–7.2 U mg-1) and good diastereoselectivity (89:11–99:1 dr) for substrates 2h-2k with halogen atoms on the benzene ring. However, SsCR-V3 has lower activity (0.26–0.78 U mg-1) for substrates 2l-2o with alkyl substitutions on the benzene ring, but it can also meet the needs of small-scale preparation. This shows that SsCR-V3 has a high tolerance for substrates with electron-withdrawing substituents on the benzene 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 ). For the first time, bulky trisubstituted aromatic chiral γ-butyrolactones (4p-4s) were successfully synthesized by a bioenzymatic method via a carbonyl reductase-catalyzed process with ee values ​​of 98-99% and dr>99:1. Compared with traditional methods, this asymmetric reduction reaction represents a simple, atom-economical and enantioselective approach to provide large amounts of aromatic multi-substituted chiral γ-butyrolactones in a cheap and efficient manner.

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

[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 is lyophilized enzyme powder (10 g / L). When the enzymatic α- / β-saturated γ-ketoester intermediate is asymmetric reduced, 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 a sodium phosphate buffer, preferably pH 6.5. The concentration of the 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) freeze-dried enzyme powder (10g / L), 2g / L glucose dehydrogenase freeze-dried enzyme powder. The reaction was carried out in a water bath at 30°C. The reaction was continued for 1 to 24 hours, and the reaction was terminated with a 2M sulfuric acid solution, extracted with an equal volume of ethyl acetate, and dried overnight with anhydrous sodium sulfate. The substrate conversion rate and the ee value of the reduction product were determined, and the product yield was the separation yield, as shown below.

[0096]

[0097] The reaction conversion rate and the product enantiomeric excess (ee) can be analyzed by gas chromatography, preferably using The conversion rate was analyzed by MS capillary column (30m×0.25mm×0.25μm), the carrier gas was nitrogen, and the detector was a hydrogen flame ionization detector (FID). The injection port temperature was 280℃, and the detector temperature was 280℃. The ee value of the reduction product was analyzed by GC / CP-Chirasil-Dex CB capillary column (25m×0.25mm×0.25μm), the carrier gas was nitrogen, and the detector was a hydrogen flame ionization detector (FID). The injection port temperature was 280℃, and the detector temperature was 280℃.

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

[0099] The various reactions 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] aaaaggacgaggaagttaagggtaccttgaacctttctgccgaaatggttaattccgtattgaagttgaataaggacgacgatgttc

[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 to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and 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 a protein derived from carbonyl reductase SsCR, in which the amino acid sequence shown in SEQ ID No. 2 is replaced, deleted or several amino acids are added, and the protein is capable of catalyzing the asymmetric reduction of aromatic α- / β-unsaturated ketoesters, and the 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.

2. A carbonyl reductase mutant according to claim 1, characterized in that: The carbonyl reductase SsCR mutant is one of the proteins with the following amino acid sequence: (1) replacing the 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.

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

4. An isolated nucleic acid according to claim 3, characterized in that The nucleotide sequence of carbonyl reductase SsCR is shown in SEQ ID No.

1.

5. A recombinant expression vector, characterized in that: Comprising the nucleic acid as described in claim 3.

6. A recombinant expression transformant, characterized in that: Comprising the recombinant expression vector as described in claim 5.

7. A carbonyl reductase mutant catalyst, characterized in that: Choose from any of the following: (1) culturing the recombinant expression transformant according to claim 6, and isolating transformant cells containing the carbonyl reductase mutant according to claim 1 or 2; (2) culturing the recombinant expression transformant according to claim 6, isolating transformant cells containing the carbonyl reductase mutant according to claim 1 or 2, 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 6, isolating transformant cells containing the carbonyl reductase mutant according to claim 1 or 2, 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 lyophilized enzyme powder.

8. Use of the carbonyl reductase mutant according to claim 1 or 2 or the carbonyl reductase mutant catalyst according to claim 7, characterized in that: A method for synthesizing chiral γ-butyrolactone by asymmetric reduction of α- / β-saturated γ-ketoester using the carbonyl reductase mutant according to claim 1 or 2 or the carbonyl reductase mutant catalyst according to claim 7.

9. The use according to claim 8, characterized in that: The process route for stereoselective synthesis of chiral aromatic polysubstituted γ-butyrolactone is as follows:

10. The use according to claim 8, characterized in that: The α- / β-saturated γ-ketoester is selected from one of the following:

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