Alkenel reductase mutants and their use in the synthesis of chiral, poly-substituted aromatic gamma-butyrolactones

Abstract

The present application relates to an ene reductase mutant and its application in the synthesis of chiral aromatic polysubstituted gamma-butyrolactone. The ene reductase mutant is a protein formed by replacing the tryptophan at position 100 of the amino acid sequence shown in SEQ ID No. 2 with alanine. The present application provides a new ene reductase mutant SsER-A314Y / W100A, and a double enzyme cascade catalytic system is constructed with the mutant SsCR-V3 (V126A / N210H / M215G) of the carbonyl reductase SsCR for the efficient stereoselective synthesis of various aromatic chiral gamma-butyrolactone. Compared with the prior art, the aromatic chiral gamma-butyrolactone double enzyme cascade system provided by the present application has the significant advantages of simple process, high atom economy, high optical purity of product and environmental friendliness, and has good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and in particular relates to olefin reductase mutants, genes encoding the olefin reductase mutants, recombinant expression vectors containing the genes and recombinant expression transformants, and the application of olefin reductase mutants in the synthesis of chiral aromatic polysubstituted γ-butyrolactones. Background Technology

[0002] Chiral γ-lactones are an important structural unit and building block in synthesis, widely found in over 13,000 natural products and having significant industrial applications. For example, chiral γ-lactones are important flavor molecules, widely used in food flavorings, flavor enhancers, and daily chemical products. Data shows that the global flavor and fragrance market grew from US$18 billion in 2006 to US$26.3 billion in 2017. The global flavor and fragrance market continued to grow from 2017 to 2022, reaching approximately €39 billion in 2022, the highest value in nearly six years, representing a 4% increase over 2021 (J. Agric. Food Chem. 2024, 72(33), 18305-18320). In recent years, China's flavor and fragrance industry has also developed rapidly, with industry sales exceeding RMB 40 billion in 2018, and the market size reaching RMB 43.9 billion in 2023, and is expected to exceed RMB 50 billion by 2026.

[0003] Chiral lactones are well-known natural flavor and aroma 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-decyl lactone has the aroma of peach and strawberry, while δ-decyl lactone 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 all affect their flavor characteristics. For example, γ-octyl lactone has a milky aroma, while γ-undecaprolactone has a strong peach and almond aroma (J. Agric. Food Chem. 1989, 37: 413-418.). The stereoconfiguration of lactone compounds also affects the purity and intensity of aroma. Natural lactones are mainly (R)-enantiomers, which have a stronger aroma than (S)-enantiomers and are the main source of fruit aroma (J. Agric. Food Chem. 1989, 37(2): 413-418.). Chemically produced lactones usually have poor enantioselectivity and low optical purity, resulting in weaker aroma quality and intensity, and lower consumer acceptance. For example, chemically synthesized γ-decyl lactone is significantly less intense in aroma than naturally extracted γ-decyl lactone. Therefore, as food additives, single-configuration lactone compounds are in greater demand, have higher added value, and are popular in the market.

[0004] Chiral lactones possess crucial biological functions and pharmacological activities. These compounds are widely distributed in nature and play key roles in numerous biological processes. Approximately 10% of natural products contain lactone ring structures[], including alkaloids, pheromones, antibiotics, and antitumor agents. Chiral lactones are core components of many bioactive molecules (Tetrahedron. 2021, 84, 132001–13039.). In medicinal chemistry, chiral lactones are considered a special structure due to their diverse pharmacological properties, such as antifungal, antibiotic, antitumor, anti-inflammatory, and cytotoxic activities (Angewandte Chemie. 2014, 53(16): 4164-4167.). These properties give chiral lactones broad application potential in developing new drugs and treating various diseases. Many natural chiral lactones, such as erythromycin, exhibit antibacterial and antifungal activities, inhibiting the growth of pathogenic microorganisms. Furthermore, some chiral lactones, such as paclitaxel, possess anticancer properties, inhibiting cancer cell proliferation by interfering with cell division. These compounds also possess anti-inflammatory properties; for example, ginkgolides can reduce inflammation and oxidative damage. Furthermore, chiral lactones can act as pheromones in plants and microorganisms, participating in intercellular signaling and influencing plant growth and development or bacterial quorum sensing. Therefore, the structure and chiral characteristics of chiral lactone compounds enable them to play important roles in a variety of biological systems.

[0005] Chiral polysubstituted γ-butyrolactones are core structural units in many natural products and high-value-added chemicals used in the pharmaceutical and food industries. Currently, the synthesis of chiral polysubstituted γ-butyrolactones is still mainly based on chemical methods. Precisely constructing multiple chiral centers on a five-membered heterocycle with multiple substituents presents significant challenges, especially for bulky aromatic-substituted chiral γ-butyrolactones, which suffer from low stereoselectivity, expensive metal catalysts, harsh reaction conditions, and low atom economy.

[0006] Polysubstituted chiral lactones contain substituents at the α-, β-, and γ- positions, and contain more than two chiral carbon atoms, resulting in richer structures and functions and high application value. However, the efficient and stereoselective construction of multiple chiral centers remains a significant challenge in their synthesis. A currently feasible synthetic route involves asymmetric hydrogenation of the C=C double bond using olefin reductases (ERs) or enone reductases (EnoRs) to construct the α,β-chiral carbon, followed by asymmetric reduction using carbonyl reductases (CRs) or alcohol dehydrogenases (ADHs) to obtain the α,β-substituted γ-hydroxy acid (ester), and finally intramolecular cyclization to obtain the polysubstituted chiral γ-lactone. However, existing carbonyl reductases (CRs) or alcohol dehydrogenases (ADHs) suffer from poor activity and stereoselectivity in the synthesis of chiral γ-lactones. Summary of the Invention

[0007] To address the problems of low stereoselectivity, expensive metal catalysts, harsh reaction conditions, and low atom economy in the chemical synthesis of chiral γ-lactones in existing technologies, as well as the problems of poor activity and poor stereoselectivity in the existing biological synthesis of chiral γ-lactones, this invention provides an olefin reductase mutant and its application in the synthesis of chiral aromatic polysubstituted γ-butyrolactones.

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

[0009] This invention starts with α- / β-unsaturated γ-keto esters and obtains highly stereoselective α- / β-substituted chiral γ-keto ester intermediates through asymmetric reduction using an olefin reductase mutant. Then, it further performs asymmetric reduction of the latent chiral carbon of the chiral γ-keto ester using a carbonyl reductase mutant. Finally, intramolecular cyclization yields multisubstituted chiral γ-butyrolactones. The olefin reductase mutants provided by this invention exhibit high stereoselectivity and high activity, expanding the substrate spectrum.

[0010] The objective of this invention can be achieved through the following technical solutions:

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

[0012] This invention provides an olefin reductase mutant, which is:

[0013] A protein whose amino acid sequence as shown in SEQ ID No. 2 has been substituted, deleted, or added with several amino acids and is capable of catalyzing the asymmetric reduction of aromatic α- / β-unsaturated ketone esters, and whose catalytic activity is higher than that of olefin reductase SsER-A314Y or exhibits new catalytic activity, wherein the amino acid sequence of olefin reductase SsER-A314Y is shown in SEQ ID No. 2.

[0014] In one embodiment of the present invention, the olefin reductase mutant is a protein composed of a new amino acid sequence formed by replacing tryptophan at position 100 of the amino acid sequence shown in SEQ ID No. 2 with a new amino acid, and the olefin reductase mutant can catalyze the asymmetric reduction of aromatic α- / β-unsaturated ketone esters, and its catalytic activity is higher than that of olefin reductase SsER-A314Y.

[0015] In one embodiment of the present invention, the alkene reductase mutant is a protein composed of a new amino acid sequence formed by replacing tryptophan at position 100 of the amino acid sequence shown in SEQ ID No. 2 with alanine.

[0016] The olefin reductase mutant described in this invention has the following properties and applications: it can stereoselectively synthesize aromatic polysubstituted γ-butyrolactones through a dual-enzyme cascade catalysis.

[0017] This invention uses the SsER-A314Y olefin reductase mutant as the parent organism and performs molecular modification to obtain a mutant with novel catalytic activity, exhibiting activity against a variety of aromatic substrates from non-existent to fully developed. Specifically, the SsER-A314Y olefin reductase mutant is a mutant with improved catalytic performance and stability obtained through semi-rational modification of the SsER olefin reductase from *Swingsia samuiensi*. This SsER-A314Y olefin reductase mutant has been disclosed in patent CN115927219A.

[0018] The present invention also provides a method for obtaining the olefin reductase mutant. In one embodiment of the present invention, the olefin reductase mutant can be obtained by the following method: through gene mining and large-scale screening of microbial strains preserved in the laboratory, the gene of olefin reductase (SsER-A314Y) from Swingsia samuiensi is discovered, and through artificial synthesis, it is directly linked to the PET-28a expression vector and directly transformed into Escherichia coli. BL 21(DE3). Through induced expression and purification, an olefin reductase mutant that can catalyze the asymmetric reduction of medium- and long-chain α- / β-unsaturated ketone esters is obtained. This olefin reductase mutant is NADPH dependent.

[0019] In this invention, the olefin reductase mutant can be directly linked to the PET-28a expression vector by artificially synthesizing the gene, and then directly transformed into Escherichia coli BL 21(DE3) for induced expression and purification.

[0020] The present invention also provides a method for obtaining the olefin reductase mutant, which constructs a mutant library of olefin reductase SsER-A314Y based on alanine scanning, and then screens to obtain the olefin reductase mutant.

[0021] Specifically, the method for constructing the mutant library of olefin reductase SsER-A314Y is as follows:

[0022] Input the amino acid sequence of olefin reductase SsER-A314Y (as shown in SEQ ID No. 2) into SWISS-MODEL for homology modeling or directly predict the protein structure using AlphaFold2. Then, perform molecular docking between the modeled protein and ethyl 2-p-phenylfluoro-4-oxoprenoate (E)-1b. Select a suitable docking model based on the catalytic mechanism and binding energy of the short-chain dehydrogenase. By analyzing the substrate pocket area... Amino acid scanning was performed to detect alanine residues, further enhancing enzyme activity and revealing new catalytic activities. In the stereochemical structure of the amino acid sequence SsER-A314Y shown in SEQ ID No. 2, the amino acid residues surrounding the substrate (E)-1b binding site include: W100, M102, R104, V106, H107, T122, P124, D125, E126, V127, T129, K133, K134, N175, S231, Q232, G233, V244, F269, L270, V340A, and Y344A. Using alanine scanning technology, all amino acids at these sites were mutated to alanine. The primers used are shown in Table 1.

[0023] Table 1 Primers for SsER mutant amino acid sites

[0024]

[0025] The second technical solution of the present invention:

[0026] The present invention provides an isolated nucleic acid, wherein the nucleic acid is a nucleic acid molecule encoding an alkene reductase mutant as described in technical solution one.

[0027] The nucleotide sequence of the nucleic acid encoding olefin reductase SsER-A314Y is shown in SEQ ID No. 1, and is 1068 nucleotides in length. Its coding sequence (CDS) extends from the first base to the 1068th base, with the start codon being ATG and no stop codon. It is expressed with upstream and downstream histidine tags fused together.

[0028] The third technical solution of the present invention:

[0029] The present invention provides a recombinant expression vector containing the olefin reductase mutant nucleic acid.

[0030] The recombinant expression vector of the present invention can be constructed by linking the nucleic acid of the olefin reductase mutant to various expression vectors using conventional methods in the art. The expression vectors include various plasmid vectors conventional in the art, with plasmid pET-28a being preferred. The gene of the olefin reductase mutant can be linked to the pET-28a vector during artificial gene synthesis to obtain the corresponding recombinant expression vector.

[0031] The fourth technical solution of the present invention:

[0032] The present invention also provides a recombinant expression transformant comprising the alkene reductase mutant nucleic acid gene or its recombinant expression vector.

[0033] 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 it satisfies the requirement that the recombinant expression vector can stably replicate itself and that the gene carrying the enzyme can be effectively expressed. The host cell is preferably *Escherichia coli*, more preferably *E. coli* BL21(DE3) or *E. coli* DH5α. Preferably, the recombinant expression vector is transformed into *E. coli* BL21(DE3) to obtain the preferred genetically engineered strains of the present invention (E. coli BL21(DE3) / pET28a-SsER-A314Y or E. coli BL21(DE3) / pET28a-SsER-A314Y / W100A and E. coli BL21(DE3) / pET28a-SsCR-V3).

[0034] The fifth technical solution of this invention:

[0035] This invention provides an olefin reductase mutant catalyst, wherein the olefin reductase mutant catalyst is in any of the following forms:

[0036] (1) Cultivate the recombinant expression transformant as described in technical solution 4, and isolate the transformant cells containing the olefin reductase mutant;

[0037] (2) Cultivate the recombinant expression transformant as described in technical solution 4, isolate the transformant cells containing the olefin reductase mutant, and break the transformant cells containing the olefin reductase mutant to obtain the cell lysate.

[0038] (3) The freeze-dried enzyme powder obtained by freeze-drying the cell lysate of the olefin reductase mutant;

[0039] (4) The purified enzyme of the olefin reductase mutant.

[0040] This invention also provides a method for preparing the olefin reductase mutant catalyst, preferably by: culturing the recombinant expression transformant as described in technical solution four, and isolating and obtaining a recombinant olefin 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 olefin reductase of this invention. The culture medium is preferably LB medium, with the following formula: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0. There are no special limitations on the culture method and conditions; appropriate selection can be made according to the host cell type and culture method, based on conventional knowledge in the art, as long as the transformant can grow and produce the olefin reductase mutant. The specific operation of culturing the recombinant expression transformant can be performed according to conventional operation in the art. Preferably, the several recombinant Escherichia coli strains described in this invention are inoculated into LB medium containing kanamycin and cultured at 37°C. When the optical density OD of the culture medium reaches a certain value... 600 Achieving 0.5–1.0 (preferred OD) 600 When the concentration is 0.6%, isopropyl-β-D-thiogalactopyranoside (IPTG) is added to a final concentration of 0.1–1.0 mmol / L (preferably 0.5 mmol / L) to induce enzyme production. After culturing at 16°C for 24 h, the alkene reductase mutant described in this invention can be efficiently expressed. After culturing, the precipitated bacterial cells are collected by centrifugation; these are the resting cells of the recombinant expression transformant. The harvested cells are suspended in PBS buffer (100 mM, pH 7.0), sonicated, and the lysate is centrifuged. The supernatant is collected to obtain the crude enzyme solution of the recombinant alkene reductase SsER. The centrifuged cell pellet is freeze-dried to obtain frozen stem cells, which are beneficial for long-term storage and convenient for future use.

[0041] Enzyme activity assay: A 1 ml reaction system containing 2 mmol / L α- / β-unsaturated keto esters and 0.1 mmol / L NADPH (100 mmol / L sodium phosphate buffer, pH 6.5) was preheated to 35°C. Then, an appropriate amount of the corresponding purified enzyme was added, mixed thoroughly, and the reaction was incubated at 35°C. The absorbance change of NADPH at 340 nm was detected on a spectrophotometer, and the absorbance change value was recorded over a certain period of time.

[0042] Enzyme activity can be calculated using the following formula:

[0043] Enzyme activity (U) = EW × V × 10 3 / (6220×l)

[0044] In the formula, 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); and l is the optical path distance in cm. One unit of enzyme activity (U) corresponds to the amount of enzyme required to oxidize 1 μmol of NADPH per minute under the above conditions.

[0045] The sixth technical solution of the present invention:

[0046] This invention provides the application of an olefin reductase mutant and an olefin reductase mutant catalyst in the stereoselective synthesis of chiral aromatic polysubstituted γ-butyrolactones via a two-enzyme cascade catalysis.

[0047] Preferably, the method for the stereoselective synthesis of chiral aromatic polysubstituted γ-butyrolactones via a dual-enzyme cascade catalysis is as follows:

[0048] S1. An olefin asymmetric reduction reaction of α- / β-unsaturated γ-keto esters is carried out using an olefin reductase mutant or an olefin reductase mutant catalyst to obtain α- / β-saturated γ-keto ester intermediates.

[0049] S2. The proximal chiral carbon of the α- / β-saturated γ-keto ester intermediate is asymmetrically reduced using carbonyl reductase, a carbonyl reductase mutant, or a carbonyl reductase mutant catalyst, and finally polysubstituted chiral γ-butyrolactone is obtained through intramolecular cyclization.

[0050] The chemical structure of the α- / β-unsaturated γ-keto ester is shown below:

[0051]

[0052] The process for the stereoselective synthesis of chiral aromatic polysubstituted γ-butyrolactones via a dual-enzyme cascade catalysis is shown below:

[0053] In one embodiment of the present invention, the carbonyl reductase is selected as SsCR, which is derived from the carbonyl reductase SsCR (PDB: 5GMO) of Scheffersomyces stipitis, and the amino acid sequence of carbonyl reductase SsCR is as shown in SEQ ID No. 4.

[0054] In one embodiment of the present invention, the carbonyl reductase mutant is carbonyl reductase SsCR-V3 (SsCR-V126A / M215G / N210H mutant, i.e., replacing valine at position 126 with alanine, methionine at position 215 with glycine, and asparagine at position 210 with histidine in the amino acid sequence shown in SEQ ID No. 4). The nucleotide sequence of the nucleic acid encoding carbonyl reductase SsCR is shown in SEQ ID No. 3, with a total length of 1005 nucleotide bases. Its coding sequence (CDS) extends from the first base to the 1005th base, with a start codon of ATG and no stop codon. It is expressed with upstream and downstream histidine tags fused together. The amino acid sequence of the protein encoded by this gene is shown in SEQ ID No. 4.

[0055] In one embodiment of the present invention, in the stereoselective synthesis of chiral aromatic polysubstituted γ-butyrolactones via a dual-enzyme cascade catalysis...

[0056] The first step, the asymmetric reduction of α- / β-unsaturated keto esters, can be carried out as follows (preferred method): in phosphate buffer at pH 6.5, with glucose dehydrogenase (2 g / L), glucose (1.5 equiv), and NADP... + In the presence of (0–1.0 mmol / L), and with the action of an olefin reductase mutant or an olefin reductase mutant catalyst (5.2–48 mg of pure enzyme), the asymmetric reduction reaction of the α- / β-unsaturated ketone ester is catalyzed.

[0057] The second step is the asymmetric reduction of the α- / β-saturated γ-keto ester intermediate by a carbonyl reductase (CR) mutant, which can be carried out as follows (preferred method): in phosphate buffer at pH 6.5, with glucose dehydrogenase (2 g / L), glucose (1.5 equiv), and NADP... + In the presence of (0–1.0 mmol / L), the carbonyl reductase catalyst (10 g / L lyophilized enzyme powder) catalyzes the asymmetric reduction reaction of the α- / β-saturated γ-keto ester intermediate.

[0058] Unless otherwise specified, all raw materials and reagents used in this invention are commercially available. Furthermore, this invention also provides a chemical synthesis method for the starting substrate α- / β-unsaturated ketone ester, as shown below:

[0059]

[0060] In one embodiment of the present invention, method (1) can be implemented as follows: Add the corresponding α-oxoester (10-15 mmol) and 1-triphenylphosphine methylene acetone (typically 1.5 equiv) to 25 mL of benzene, and stir at room temperature for 20-24 hours. If the conversion of the α-oxoester is not ideal, stir the reaction mixture at 50°C for another 5-10 hours. Then cool the mixture, evaporate the benzene to 1 / 3 of its volume under vacuum, add approximately (20 mL × 3) hexane, and filter the precipitate with diatomaceous earth. The filtrate is concentrated and purified by column chromatography on silica gel (eluent: petroleum ether / ethyl acetate = 20:1). (That is, the structure shown in α- / β-unsaturated γ-keto esters 1a-1h is obtained.)

[0061] Method (2): A solution of 1-phenyl-1,2-propanedione (10-15 mmol) and an appropriate amount of triphenylphosphine ylide (usually 1.5 equiv) in CH2Cl2 (100 mL) was stirred at room temperature for 24 hours, then concentrated under reduced pressure. Hexane (50 mL × 3) was added to the cooled crude mixture. Triphenylphosphine oxide was precipitated through a diatomaceous earth filter. The filtrate was concentrated and purified by column chromatography on silica gel (elution: petroleum ether / ethyl acetate = 50:1). (That is, the structure shown in α- / β-unsaturated γ-keto ester 1i was obtained.)

[0062] Method (3): Add the corresponding α-oxoester (10-15 mmol) and 1-triphenylphosphine ethyl acetone (usually 1.5 equiv) to 25 mL of benzene and stir at room temperature for 20-24 hours. Subsequent procedures are as described in Method (1). (That is, the structure shown in 1 g-1 l of α- / β-unsaturated γ-keto ester is obtained).

[0063] This invention provides an olefin reductase mutant, a gene encoding the olefin reductase mutant, a recombinant expression vector containing the gene and a recombinant expression transformant, and the application of the olefin reductase mutant in the synthesis of chiral aromatic polysubstituted γ-butyrolactone.

[0064] Compared with the prior art, the present invention has the following beneficial effects:

[0065] (1) This invention innovatively constructs a process route for the stereoselective synthesis of chiral aromatic polysubstituted γ-butyrolactones by a dual-enzyme cascade catalysis.

[0066] (1) The olefin reductase SsER-A314Y / W100A provided by the present invention is a mutant based on existing enzymes. It can combine with the carbonyl reductase mutant SsCR-V3 and be used efficiently in the dual enzyme cascade system described in the present invention, thereby efficiently synthesizing a variety of multisubstituted chiral γ-butyrolactones.

[0067] (2) The final product of the present invention is a multi-substituted chiral lactone compound, which can be used as an intermediate for fragrance products or natural products and has wide application value in the fragrance market and natural product synthesis.

[0068] (3) The reaction conditions of this invention are mild, the conversion rate is high, the catalytic activity is high, the product has good optical purity, the atom economy is high, and the ee and dr values ​​can be higher than 99%, which has a good prospect for industrial application. Attached Figure Description

[0069] Figure 1 Alanine scan results for SsER-A314Y;

[0070] Figure 2 Substrate spectrum of mutant SsER-A314Y / W100A. Detailed Implementation

[0071] The present invention will now be described in detail with reference to specific embodiments, but this does not limit the invention to the scope of the embodiments described. Experimental methods in the following embodiments that do not specify specific conditions are performed according to conventional methods and conditions, or as selected according to the product instructions. The reaction or detection conditions described in the invention can be combined or modified based on common knowledge in the art, and can be verified experimentally.

[0072] The materials used in the following embodiments are sourced from:

[0073] The recombinant plasmids pET28a-SsER-A314Y and pET28a-SsCR-V3 contain nucleic acid sequences as shown in SEQ ID No. 1 and SEQ ID No. 3 in the sequence listing. They were constructed and deposited in our laboratory, and the sequences can also be constructed by Shanghai Qingke Biotechnology Co., Ltd.

[0074] The expression plasmid pET28a was purchased from Novagen.

[0075] E. coli BL21(DE3) competent cells were purchased from Beijing Tiangen Biotech Co., Ltd.

[0076] Example 1: Preparation of the recombinant expression plasmid and recombinant expression transformant of the enzyme

[0077] The recombinant expression plasmid of the enzyme (recombinant plasmid pET28a-SsER-A314Y) was directly synthesized by Shanghai Qingke Biotechnology Co., Ltd. and the corresponding gene sequence was ligated into the PET-28a vector.

[0078] The obtained recombinant plasmid was transformed into E. coli BL21(DE3) competent cells and plated on LB agar plates containing 50 μg / mL kanamycin. The cells were incubated at 37°C for at least 8 hours. Colony PCR was performed to verify the growth of the colonies, and positive clones corresponding to the successfully amplified target band of the correct length were selected. After sequencing verification, positive clones were selected to obtain the recombinant expression transformants (E. coli BL21(DE3) / pET28a-SsER-A314Y and E. coli BL21(DE3) / pET28a-SsCR-V3).

[0079] Example 2: Induced expression of the enzyme

[0080] 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, at an inoculation rate of 1% (v / v), it was inoculated into 500 mL Erlenmeyer flasks containing 100 mL of LB medium (containing 50 μg / mL kanamycin) and cultured on a shaker at 37°C and 220 rpm. When the OD of the culture medium... 600 When the concentration 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 the cells were washed with physiological saline to obtain resting cells.

[0081] 0.5 g of resting cells obtained as described above were suspended in 100 mL of sodium phosphate buffer (100 mM, pH 6.5), and sonicated in an ice-water bath. The supernatant was collected by centrifugation, which yielded the crude enzyme solution of recombinant olefin reductase. Analysis of the crude enzyme solution by polyacrylamide gel electrophoresis showed that the recombinant olefin reductase existed in a soluble expression form. Subsequently, purification by nickel column chromatography followed by ultrafiltration concentration yielded a pure enzyme solution of recombinant olefin reductase. Alternatively, the crude enzyme solution of recombinant olefin reductase could be freeze-dried to obtain crude enzyme powder of recombinant olefin reductase.

[0082] Example 3: Alanine scan of olefin reductase SsER-A314Y (single-point mutation)

[0083] Using pET28a-SsER-A314Y as a template, PCR amplification was performed using PrimeStar HSDNA Polymerase. The PCR system consisted of: pET28a-SsER-A314Y plasmid (100 nM, 1 μL), forward and reverse primers (10 nM, 1 μL), and 5× PrimeStar Buffer (MgCl₂). 2+Add 4 μL of dNTP Mixture (2.5 mM, 1.6 μL), PrimeStar HSDNA Polymerase (0.2 μL), and sterile distilled water to make up to 20 μL. PCR reaction procedure: (1) 95℃ pre-denaturation for 3 min; (2) 98℃ denaturation for 10 s; (3) set gradient temperature annealing for 10 s; (4) 72℃ extension for 6 min 40 s. Steps (2) to (4) were performed for a total of 30 cycles; the final extension was performed at 72℃ for 10 min. Add 0.5 μL of Dpn I enzyme to 8.5 μL of PCR product and 1 μL of rSmart cut buffer and incubate at 37℃ for 3 h to digest the template. Transform the digested product into E. coli BL21(DE3) competent cells and spread it evenly on LB agar plates containing 50 μg / ml kanamycin. Incubate at 37℃ for about 12 h. The obtained single-clone colonies were picked and cultured in LB test tubes, and then induced for expression and purified for activity testing as shown in Example 2.

[0084] Activity assays of different mutants revealed that replacing tryptophan at position 100 with serine (W100A) not only increased activity against the model substrate (E)-1b but also increased activity against various structurally similar substrates, resulting in novel catalytic activity. Figure 1 and Figure 2 The enzyme was purified and characterized, and its enzyme activity and kinetic parameters were determined (Tables 2-3). As shown in Table 3, the enzyme activity assay for SsER and its mutants was performed as follows: A reaction system containing 2 mmol / L of different substrates and 0.1 mmol / L NADPH (100 mmol / L sodium phosphate buffer, pH 7.0) was preheated to 35°C. Then, an appropriate amount of purified SsER-A314Y or its mutant enzyme was added, bringing the total system to 1 mL. The reaction was incubated at 35°C, and the absorbance change at 340 nm was detected using a spectrophotometer. The absorbance change within 1 minute was recorded, and the enzyme activity was calculated. In Table 3, data without (%) represent specific activity (U mg). -1 ), where (%) represents enantioselectivity, and nd represents not measured.

[0085]

[0086] Table 2 Kinetic parameters of SsER and its mutants to substrate (E)-1b

[0087] Table 3. Specific activity and enantioselectivity of mutant SsER-A314Y / W100A for various substrates.

[0088]

[0089] Asymmetric reduction-scale preparation of the olefin reductase mutant described in Example 4

[0090] The first step, the asymmetric reduction of α- / β-unsaturated keto esters, can be carried out using the following preferred method: in phosphate buffer at pH 6.5, with glucose dehydrogenase, glucose, and NADP... + In the presence of the olefin reductases (ERs) catalyst, the asymmetric reduction reaction of the α- / β-unsaturated ketone ester is catalyzed.

[0091] In this embodiment, the concentration of the substrate in the reaction solution can be 20 mmol / L. Depending on the reaction system used, the amount of purified olefin reductase (SsER-A314Y / W100A mutant) used is 10.5–80 mg, as shown below. During the enzymatic asymmetric reduction of medium- and long-chain α- / β-unsaturated keto esters, the coenzyme NADPH is oxidized to generate NADP. + To facilitate the cyclic regeneration of the coenzyme NADPH, glucose and glucose dehydrogenase from Bacillus megaterium were added to the reaction system (JInd Microb Biotechnol, 2011, 38:633–641). Depending on the reaction system, the activity units of glucose dehydrogenase could be equal to those of the olefin reductase. The molar ratio of glucose to substrate was 1.5, and the added NADP... + The amount used is 0.2 mmol / L. The buffer solution is sodium phosphate buffer, preferably with a pH of 6.5. The concentration of the phosphate buffer is 0.2 mol / L. The temperature of the enzymatic asymmetric reduction reaction is 35℃. During the reaction, the reaction conversion rate is measured intermittently, and the reaction time is based on the time when the substrate is completely converted or the reaction conversion rate stops increasing, generally 6–48 h. In this reaction, the reaction is carried out in a 100 mL beaker. Different substrates with a final concentration of 20 mM, 30 mM glucose, and 0.2 mM NADP are added to 50 mL of sodium phosphate buffer (100 mM, pH 6.5). + The purified olefin reductase (SsER-A314Y / W100A mutant) enzyme (10.5-80 mg) and 2 g / L glucose dehydrogenase lyophilized enzyme powder were used. The reaction was carried out in a water bath at 35°C. The reaction was terminated with 2M sulfuric acid solution after 6-48 h, extracted with an equal volume of ethyl acetate, and dried overnight with anhydrous sodium sulfate. The substrate conversion and ee value of the reduction product were determined, and the product yield was the separation yield, as shown below.

[0092]

[0093] Large-scale preparation of γ-butyrolactone catalyzed by the alkylene reductase and carbonyl reductase dual-enzyme cascade described in Example 5.

[0094] The second step is the asymmetric reduction of the α- / β-saturated γ-keto ester intermediate by a carbonyl reductase (CR) mutant, which can be carried out as follows (preferred method): in phosphate buffer at pH 6.5, with glucose dehydrogenase, glucose, and NADP... + In the presence of the olefin reductase catalyst, the asymmetric reduction reaction of the α- / β-saturated γ-keto ester intermediate is catalyzed. The two-enzyme cascade reaction involves directly adding the reaction mixture required for the second step to the reaction system after the first step reaction is completed.

[0095] In this embodiment, after the first step reaction, the amount of carbonyl reductase (SsCR-V126A / M215G / N210H mutant) added to the system is lyophilized enzyme powder (10 g / L). During the enzymatic asymmetric reduction of the α- / β-saturated γ-keto ester intermediate, the coenzyme NADPH is oxidized to generate NADP. + To facilitate the cyclic regeneration of 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 added NADP... + The dosage is 0.2 mmol / L. The buffer solution is sodium phosphate buffer, preferably pH 6.5. The concentration of the phosphate buffer is 0.2 mol / L. The temperature of the enzymatic asymmetric reduction reaction is preferably 30℃. During the reaction, the conversion rate is measured intermittently, and the reaction time is based on the time when the substrate is completely converted or the conversion rate stops increasing, generally 1–24 h. In this reaction, the reaction is carried out in a 100 mL beaker, and 1.5 equiv of glucose and 0.2 mM NADP are added directly 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 terminated with 2 M sulfuric acid solution after 1–24 h, extracted with an equal volume of ethyl acetate, and dried overnight with anhydrous sodium sulfate. The substrate conversion and 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 enantiomeric excess value (ee) of the product can be analyzed by gas chromatography, preferably using... Conversion analysis was performed using an MS capillary column (30 m × 0.25 mm × 0.25 μm) with nitrogen as the carrier gas and a flame ionization detector (FID). The injection port temperature and detector temperature were both 280 °C. ee values ​​of the reduction products were analyzed 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). The injection port temperature and detector temperature were both 280 °C.

[0098] After the enzymatic asymmetric reduction reaction, the product was extracted with an equal volume of a conventional water-insoluble organic solvent, such as ethyl acetate, butyl acetate, toluene, dichloromethane, chloroform, isopropyl ether, or methyl tert-butyl ether. The extraction was repeated twice, and the extracts were combined and dried with anhydrous sodium sulfate. In this example, ethyl acetate was used for extraction. The dried crude organic layer was then evaporated using a rotary evaporator to remove the solvent, yielding the corresponding optically active chiral lactone crude product. This crude product was then purified by column chromatography to obtain pure γ-butyrolactone (eluent: petroleum ether / ethyl acetate = 20:1).

[0099] The reaction or detection conditions described in this invention can be combined or modified based on common knowledge in the field, and can be verified through experiments.

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

[0101] SEQ ID NO.1

[0102]

[0103] SEQ ID NO.2

[0104] MADLFSSIKIGDIITKNRIFMAPLTRARVERDAVPVPMMAEYYAQRAQAGLIISEATGISREGLGWPYAPGIWTDEQVEAWKSVTQAVHDKGGKIVCQLWHMGRAVHSSVTGLQPVSASETTAPDEVHTYDGKKPYEKARALTKSDITRILNDYEQAARNAMRAGFDGVQIHAANGYLIDQFLRDGTNHRSDEYGGSLENRVRLLVEVTQRVIATVGAEKTGVRLSPNGDSQGVIDSAPEKIFVLAAQELERLGVAWLELRENSTTGTFLAPTDQPKLSPEIRKVFHRSLVLNQDYSFEEAQAAIRDGHADAIYFGRKFISNPDLPDRFAKNIPLQESNVSTWYSRGEEGYIDYPF

[0105] SEQ ID NO.3

[0106]

[0107] SEQ ID NO.4

[0108] MTTSVFVSGATGYLAQQIIALVLSKGYKVVGSVRSEEKGANLKKLYGDDFSYEVVKVLEQKGAFDEALKKHPEVTIFLHTASPVTFEVEDTEKEILIPAINGTKYVLQSIKDVAPQITRVVYTSSAVAMSVPEELGSPDVVLSEASWSSLSYEQSKTHGVLAYFGSK QFAERAAWEFVEQEKPNFALSTVNPVYIFGPQAKDEEVKGTLNHSAEVVNSVLKLNKDDDVPATTGTFIDVRDVAKAHLAAFEKDEAKGERLLLSNTRFNGQTLLDVVRKNFPQLADKLPVGKPHSDDFSAFKEWNDKKTKKILGFEYFDFETSVVDSIKQVLKVQG

[0109] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any 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 invention should be within the protection scope of the present invention.

Claims

1. An ene reductase mutant, characterized in that, The ene reductase mutant is a protein consisting of a new amino acid sequence formed after the tryptophan at position 100 of the amino acid sequence shown in SEQ ID No. 2 is replaced by alanine, and can catalyze the asymmetric reduction of aromatic alpha- / beta-unsaturated ketone ester.

2. An isolated nucleic acid, comprising, The nucleic acid is a nucleic acid molecule encoding the ene reductase mutant according to claim 1.

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

4. A recombinant expression transformant, characterized by, The recombinant expression vector according to claim 3.

5. An ene reductase mutant catalyst characterized in that, Selected from any one of the following forms: (1) culturing the recombinant expression transformant according to claim 4, isolating the transformant cells containing the ene reductase mutant according to claim 1; (2) culturing the recombinant expression transformant according to claim 4, isolating the transformant cells containing the ene reductase mutant according to claim 1, crushing the transformant cells containing the ene reductase mutant to obtain a cell crushing solution; (3) freeze-drying the cell crushing solution of the ene reductase mutant to obtain a freeze-dried enzyme powder; (4) the pure enzyme of the ene reductase mutant.

6. Use of the ene reductase mutant according to claim 1 or the ene reductase mutant catalyst according to claim 5 in the stereoselective synthesis of chiral aromatic multi-substituted gamma-butyrolactone in a two-enzyme cascade catalysis with a carbonyl reductase.

7. Use according to claim 6, characterized in that, The method for the stereoselective synthesis of chiral aromatic multi-substituted gamma-butyrolactone in a two-enzyme cascade catalysis is: S1, using the ene reductase mutant or the ene reductase mutant catalyst to perform an asymmetric olefin reduction reaction on an aromatic alpha- / beta-unsaturated gamma-ketone ester to obtain an aromatic alpha- / beta-saturated gamma-ketone ester intermediate; S2, using a carbonyl reductase mutant SsCR-V126A / M215G / N210H to perform an asymmetric reduction reaction on the latent chiral carbon of the aromatic alpha- / beta-saturated gamma-ketone ester intermediate, and finally obtaining a stereoselective synthesis of chiral aromatic multi-substituted chiral gamma-butyrolactone through intramolecular cyclization.

8. Use according to claim 7, characterized in that, The chemical structure of the aromatic alpha- / beta-unsaturated gamma-ketone ester is as follows: 。

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