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

Abstract

The invention relates to an alkene reductase mutant and application of the alkene reductase mutant in synthesis of chiral aromatic polysubstituted gamma-butyrolactone. The alkene reductase mutant is a protein composed of a new amino acid sequence formed by replacing tryptophan at the 100th site of the amino acid sequence as shown in SEQ ID No.2 with alanine. The invention provides a novel alkene reductase mutant SsER-A314Y / W100A, the alkene reductase mutant SsER-A314Y / W100A and a mutant SsCR-V3 (V126A / N210H / M215G) of carbonyl reductase SsCR construct a double-enzyme cascade catalytic system, and the double-enzyme cascade catalytic system is used for 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 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 an ene reductase mutant, a gene encoding the ene reductase mutant, a recombinant expression vector and a recombinant expression transformant containing the gene, and application of the ene reductase mutant in synthesizing chiral aromatic multi-substituted gamma-butyrolactone. Background Art

[0002] Chiral γ-lactones are an important class of structural units and synthetic building blocks, widely present in more than 13,000 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 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. 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 scale reached 43.9 billion yuan in 2023. It is expected to exceed 50 billion yuan by 2026.

[0003] 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.

[0004] 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, and 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 ginkgolides, 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 structural and chiral characteristics of chiral lactone compounds make them play an important role in a variety of biological systems.

[0005] 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.

[0006] Polysubstituted chiral lactones have substituents at the α-, β-, and γ-positions, contain more than two chiral carbon atoms, have richer structures and functions, and have high application value. How to accurately construct multiple chiral centers with high efficiency and high stereoselectivity is a very challenging problem in its synthesis. Starting from α-, β-unsaturated γ-keto acids (esters), the α, β-chiral carbon is constructed once by asymmetric hydrogenation of the C=C double bond by ene reductases (ERs) or enoketone reductases (EnoRs), and then asymmetric reduction is performed by carbonyl reductases (CR) or alcohol dehydrogenases (ADH) to obtain α, β-substituted γ-hydroxy acids (esters), and finally, multi-substituted chiral γ-lactones are obtained by intramolecular cyclization. This is a currently feasible synthetic route. However, the existing carbonyl reductases (CR) or alcohol dehydrogenases (ADH) have problems such as poor activity and poor stereoselectivity in the synthesis of chiral γ-lactones. Summary of the invention

[0007] In view of the problems faced by the chemical synthesis of chiral γ-lactones in the prior art, such as low stereoselectivity, expensive metal catalysts, harsh reaction conditions and low atom economy, as well as the problems faced by the existing biological synthesis of chiral γ-lactones, such as poor activity and poor stereoselectivity, the present invention provides ene reductase mutants and their use in the synthesis of chiral aromatic multi-substituted γ-butyrolactones.

[0008] More specifically, the present invention conducts resource mining and molecular modification of the ene reductase required for the synthesis of polysubstituted chiral lactones, and further provides an ene reductase mutant, a gene encoding the ene reductase mutant, a recombinant expression vector and a recombinant expression transformant containing the gene, and the use of the ene reductase mutant in the synthesis of chiral aromatic polysubstituted γ-butyrolactones.

[0009] The present invention starts from α- / β-unsaturated γ-ketoester, obtains a high stereoselective α- / β-substituted chiral γ-ketoester intermediate by asymmetric reduction of ene reductase mutant, then further asymmetric reduction of the potential chiral carbon of the chiral γ-ketoester by carbonyl reductase mutant, and finally obtains multi-substituted chiral γ-butyrolactone by intramolecular cyclization. The ene reductase mutant provided by the present invention has high stereoselectivity and high activity, and can expand the substrate spectrum range.

[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 an alkene reductase mutant, which is:

[0013] A protein in which the amino acid sequence shown in SEQ ID No. 2 is substituted, deleted or added with several amino acids and can catalyze the asymmetric reduction of aromatic α- / β-unsaturated ketoesters, and the catalytic activity is higher than that of olefin reductase SsER-A314Y or a new catalytic activity appears, 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 ene reductase mutant is a protein composed of a new amino acid sequence formed by replacing the tryptophan at position 100 of the amino acid sequence shown in SEQ ID No. 2 with a new amino acid, and the ene reductase mutant can catalyze the asymmetric reduction of aromatic α- / β-unsaturated ketoesters, and the catalytic activity is higher than that of the ene reductase SsER-A314Y.

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

[0016] The olefin reductase mutant of the present invention has the following properties and applications: it can stereoselectively synthesize aromatic multi-substituted gamma-butyrolactone through double enzyme cascade catalysis.

[0017] The present invention uses the olefin reductase SsER-A314Y mutant as the parent, performs molecular modification, and obtains a mutant with new catalytic activity, and the activity on a variety of aromatic substrates is improved from scratch. Among them, the olefin reductase SsER-A314Y mutant is a mutant with improved catalytic performance and stability screened out through semi-rational modification using the olefin reductase SsER from Swingsia samuiensi as the parent. The olefin reductase SsER-A314Y mutant has been disclosed in patent CN115927219A.

[0018] The present invention also provides a method for obtaining the ene reductase mutant. In one embodiment of the present invention, the ene reductase mutant can be obtained by the following method: that is, through gene mining and large-scale screening of laboratory-preserved microbial strains, the gene of the ene reductase (SsER-A314Y) from Swingsia samuiensi 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 through induced expression and purification, an ene reductase mutant capable of catalyzing the asymmetric reduction of medium- and long-chain α- / β-unsaturated ketoesters is obtained, and the ene reductase mutant is NADPH-dependent.

[0019] In the present invention, the olefin reductase mutant can be directly connected to the PET-28a expression vector by means of artificial gene synthesis, and directly transformed into Escherichia coli Ecoli.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 the olefin reductase SsER-A314Y based on alanine scanning, and then screens and obtains the olefin reductase mutant.

[0021] Specifically, the method for constructing a 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, perform homology modeling or use AlphaFold2 to directly predict the protein structure, and then use the modeled protein model to perform molecular docking with 2-p-phenylfluoro-4-oxopentenoic acid ethyl ester (E)-1b. According to the catalytic mechanism and binding energy of short-chain dehydrogenase, select the appropriate docking model. Alanine scanning is performed on the amino acids of the substrate (E)-1b to further improve the activity of the enzyme and generate new catalytic activity. In the three-dimensional structure of SsER-A314Y with the amino acid sequence shown in SEQ ID No. 2, the amino acid residues around 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. The amino acids at these sites are all mutated to alanine using alanine scanning technology. The primers used are shown in Table 1:

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

[0024]

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

[0026] The present invention provides an isolated nucleic acid, which is a nucleic acid molecule encoding the olefin 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, with a total length of 1068 nucleotide bases. Its coding sequence (CDS) starts from the first base to the 1068th base, the start codon is ATG, there is no stop codon, and the upstream and downstream histidine tags are fused and expressed.

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

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

[0030] The recombinant expression vector of the present invention can be constructed by connecting the nucleic acid of the olefin 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 olefin reductase mutant can be connected 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 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. The scheme of the present invention preferably transforms the recombinant expression vector into Escherichia coli 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 the present invention is:

[0035] The present invention provides an alkene reductase mutant catalyst, wherein the alkene reductase mutant catalyst is in any of the following forms:

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

[0037] (2) culturing the recombinant expression transformant as described in technical solution 4, isolating the transformant cells containing the olefin reductase mutant, and disrupting the transformant cells containing the olefin reductase mutant to obtain a cell disrupted liquid;

[0038] (3) freeze-drying the cell disrupted liquid of the olefin reductase mutant to obtain a lyophilized enzyme powder;

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

[0040] The present invention also provides a method for preparing the olefin reductase mutant catalyst, which is preferably: culturing the recombinant expression transformant as described in technical solution four, and isolating and obtaining a recombinantly expressed 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 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 olefin 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 the ene 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 ene reductase SsER; the cell precipitate harvested by centrifugation is freeze-dried to obtain freeze-dried cells, which are conducive to long-term storage and convenient for future use.

[0041] 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 35°C, then add an appropriate amount of the corresponding pure enzyme, mix well, and keep the reaction at 35°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.

[0042] The enzyme activity was calculated according to the following formula:

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

[0044] 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.

[0045] Technical solution six of the present invention:

[0046] The invention provides an application of an ene reductase mutant and an ene reductase mutant catalyst in stereoselective synthesis of chiral aromatic multi-substituted gamma-butyrolactone by double enzyme cascade catalysis.

[0047] Preferably, the method for stereoselective synthesis of chiral aromatic polysubstituted γ-butyrolactone by dual enzyme cascade catalysis is:

[0048] S1. using an olefin reductase mutant or an olefin reductase mutant catalyst to perform an olefin asymmetric reduction reaction on an α- / β-unsaturated γ-ketoester to obtain an α- / β-saturated γ-ketoester intermediate;

[0049] S2. Asymmetric reduction reaction of the potential chiral carbon of the α- / β-saturated γ-ketoester intermediate is carried out using carbonyl reductase or a carbonyl reductase mutant or a carbonyl reductase mutant catalyst, and finally a multi-substituted chiral γ-butyrolactone is obtained through intramolecular cyclization.

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

[0051]

[0052] The process of stereoselective synthesis of chiral aromatic polysubstituted γ-butyrolactone by dual enzyme cascade catalysis is as follows:

[0053] In one embodiment of the present invention, the carbonyl reductase is selected from SsCR, which is derived from the carbonyl reductase SsCR of Scheffersomyces stipitis (PDB: 5GMO), and the amino acid sequence of the carbonyl reductase SsCR is as 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., the 126th valine of the amino acid sequence shown in SEQ ID No.4 is replaced by alanine, the 215th methionine is replaced by glycine, and the 210th asparagine is replaced by histidine), and 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) starts from the 1st 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 amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.4.

[0055] In one embodiment of the present invention, in the reaction of stereoselective synthesis of chiral aromatic polysubstituted γ-butyrolactone catalyzed by dual enzyme cascade,

[0056] The first step of the asymmetric reduction of α- / β-unsaturated ketoesters 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 an ene reductase mutant or an ene reductase mutant catalyst (pure enzyme 5.2-48 mg), the asymmetric reduction reaction of the α- / β-unsaturated ketoester is catalyzed.

[0057] The second step is the asymmetric reduction of the α- / β-saturated γ-ketoester intermediate by the carbonyl reductase (CRs) mutant, which 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 catalyst (10 g / L freeze-dried enzyme powder), the asymmetric reduction reaction of the α- / β-saturated γ-ketoester intermediate is catalyzed.

[0058] Unless otherwise specified, the raw materials or reagents used in the present invention are all commercially available. The present invention also provides a chemical synthesis method of the starting substrate α- / β-unsaturated ketoester, as shown below:

[0059]

[0060] In one embodiment of the present invention, the above method (1) can be implemented as follows: in 25 mL of benzene, add the corresponding α-oxoester (10-15 mmol) and 1-triphenylphosphine methylideneacetone (usually 1.5 equiv), and stir at room temperature for 20-24 hours. If the conversion rate of the α-oxoester is not ideal, the reaction mixture is stirred at 50°C for another 5-10 hours. Then the mixture is cooled, benzene is evaporated to 1 / 3 of its volume in a vacuum, about (20 mL×3) hexane is added, and the precipitate is filtered with diatomaceous earth. After the filtrate is concentrated, column chromatography is performed on silica gel for purification (effluent: petroleum ether / ethyl acetate = 20:1). (That is, the structure shown in α- / β-unsaturated γ-ketoester 1a-1h is obtained)

[0061] Method (2): A solution of 1-phenyl-1,2-propanedione (10-15 mmol) and an appropriate triphenylphosphine ylide (usually 1.5 equiv) in CH2Cl2 (100 mL) was stirred at room temperature for 24 hours and then concentrated under reduced pressure. n-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 (eluent: petroleum ether / ethyl acetate = 50:1). (The structure obtained is the α- / β-unsaturated γ-ketoester 1i)

[0062] Method (3): Add the corresponding α-oxoester (10-15 mmol) and 1-triphenylphosphine ethylidene acetone (usually 1.5 equiv) to 25 mL of benzene and stir at room temperature for 20-24 hours. The subsequent operation is as in method (1). (The structure obtained is α- / β-unsaturated γ-ketoester 1g-1l).

[0063] The invention provides an ene reductase mutant, a gene encoding the ene reductase mutant, a recombinant expression vector and a recombinant expression transformant containing the gene, and application of the ene reductase mutant in synthesizing chiral aromatic multi-substituted gamma-butyrolactone.

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

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

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

[0067] (2) The final product, the multi-substituted chiral 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.

[0068] (3) 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. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0070] Figure 2 Substrate spectrum of mutant SsER-A314Y / W100A. DETAILED DESCRIPTION

[0071] 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.

[0072] The sources of materials in the following examples are:

[0073] The recombinant plasmids pET28a-SsER-A314Y and pET28a-SsCR-V3 contain the nucleic acid sequences shown in SEQ ID No. 1 and SEQ ID No. 3 in the sequence listing, which are constructed and preserved in this laboratory and 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 Biochemical Technology 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 connected to the PET-28a vector.

[0078] 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-SsER-A314Y and E.coli BL21 (DE3) / pET28a-SsCR-V3).

[0079] Example 2: Inducible 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 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 pellet was collected, and washed with physiological saline to obtain resting cells.

[0081] 0.5g of resting cells obtained by the above method are suspended in 100mL of sodium phosphate buffer (100mM, pH 6.5), ultrasonically broken in an ice-water bath, and the supernatant is collected by centrifugation to obtain the crude enzyme liquid of the corresponding recombinant ene reductase. The obtained crude enzyme liquid is analyzed by polyacrylamide gel electrophoresis, and the recombinant ene reductase exists in the form of soluble expression. Subsequently, the pure enzyme liquid of the recombinant ene reductase can be obtained by nickel column purification and ultrafiltration concentration. In addition, the crude enzyme liquid of the recombinant ene reductase obtained can be freeze-dried to obtain the crude enzyme powder of the recombinant ene reductase.

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

[0083] Using pET28a-SsER-A314Y as template, PrimeStar HSDNA Polymerase was used for PCR amplification. The PCR system was: pET28a-SsER-A314Y plasmid (100nM, 1uL), upstream and downstream primers (10nM, 1uL), 5× PrimeStar Buffer (Mg 2+plus, 4uL), dNTP Mixture (2.5mM, 1.6uL), PrimeStar HSDNA 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 6min 40s. Steps (2) to (4) were extended for 30 cycles in total; 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 LB test tubes, and then induced for expression and purified for activity testing as shown in Example 2.

[0084] By measuring the activity of different mutants, it was found that replacing the tryptophan at position 100 with serine (W100A) not only increased the activity for the model substrate (E)-1b from zero to one, but also increased the activity for a variety of structurally similar substrates from zero to one, generating new catalytic activity ( Figure 1 and Figure 2 ). It was purified and characterized, and the mutants were subjected to enzyme activity determination and kinetic parameter determination (as shown in Tables 2-3). As shown in Table 3, the method for determining the enzyme activity of SsER and its mutants: the reaction system containing 2mmol / L different substrates and 0.1mmol / L NADPH (100mmol / L sodium phosphate buffer, pH 7.0) was preheated to 35°C, and then an appropriate amount of SsER-A314Y or its mutant pure enzyme was added, the total system was 1mL, and the reaction was kept at 35°C. The absorbance change at 340nm was detected on a spectrophotometer, and the absorbance change value within 1min was recorded to calculate the enzyme activity. In Table 3, the data without (%) represents the specific activity (U mg -1 ), the data with (%) represent enantioselectivity, and nd means not determined.

[0085]

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

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

[0088]

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

[0090] The first step of the asymmetric reduction of α- / β-unsaturated ketoester 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 ene reductase (ERs) catalyst, the asymmetric reduction reaction of the α- / β-unsaturated ketoester is catalyzed.

[0091] In this embodiment, the concentration of the substrate in the reaction solution can be 20 mmol / L. According to the reaction system used, the pure enzyme dosage of the olefin reductase (SsER-A314Y / W100A mutant) is 10.5-80 mg, and the specific dosage is as follows. During the enzymatic asymmetric reduction of medium- and long-chain α- / β-unsaturated ketoesters, 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 (JInd Microb Biotechnol, 2011, 38: 633–641). Depending on the reaction system, the activity unit loading of glucose dehydrogenase can be equal to that of the ene reductase. 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 with a pH of 6.5. The concentration of the phosphate buffer is 0.2mol / L. The temperature of the enzymatic asymmetric reduction reaction is 35°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 6 to 48h. In this reaction, the reaction is carried out in a 100mL beaker, and 50mL of sodium phosphate buffer (100mM, pH 6.5) is added with different substrates with a final concentration of 20mM, 30mM glucose, and 0.2mM NADP. + , the olefin reductase (SsER-A314Y / W100A mutant) pure enzyme (10.5-80 mg), 2 g / L glucose dehydrogenase freeze-dried enzyme powder. The reaction was carried out in a water bath at 35°C. The reaction was continued for 6 to 48 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.

[0092]

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

[0094] The second step reaction is the asymmetric reduction of the α- / β-saturated γ-ketoester intermediate by the carbonyl reductase (CRs) mutant, which 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 olefin reductase catalyst, catalyze the asymmetric reduction reaction of the α- / β-saturated γ-ketoester intermediate. The double enzyme cascade reaction is to directly add the reaction system required for the second step reaction to the reaction system after the first step reaction is completed.

[0095] In this embodiment, after the first step of the reaction, 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 removed by a rotary evaporator to obtain the corresponding optically active chiral lactone crude product, and then pure γ-butyrolactone is obtained by column chromatography (eluent: petroleum ether / ethyl acetate = 20:1).

[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]

[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 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. An olefin reductase mutant, characterized in that for: A protein in which the amino acid sequence shown in SEQ ID No. 2 is substituted, deleted or added with several amino acids and can catalyze the asymmetric reduction of aromatic α- / β-unsaturated ketoesters, and the catalytic activity is higher than that of olefin reductase SsER-A314Y or a new catalytic activity appears, wherein the amino acid sequence of olefin reductase SsER-A314Y is shown in SEQ ID No.

2.

2. An olefin reductase mutant according to claim 1, characterized in that: The ene reductase mutant is a protein composed of a new amino acid sequence formed by replacing the tryptophan at position 100 of the amino acid sequence shown in SEQ ID No. 2 with a new amino acid, and the ene reductase mutant can catalyze the asymmetric reduction of aromatic α- / β-unsaturated ketoesters, and the catalytic activity is higher than that of the ene reductase SsER-A314Y.

3. An olefin reductase mutant according to claim 1, characterized in that: The olefin reductase mutant is a protein composed of a new amino acid sequence formed by replacing the tryptophan at position 100 of the amino acid sequence shown in SEQ ID No. 2 with alanine.

4. An isolated nucleic acid, characterized in that The nucleic acid is a nucleic acid molecule encoding the olefin reductase mutant according to claim 1, 2 or 3.

5. A recombinant expression vector, characterized in that: Comprising the nucleic acid as described in claim 4.

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

7. An olefin 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 olefin reductase mutant according to any one of claims 1 to 3; (2) culturing the recombinant expression transformant according to claim 6, isolating transformant cells containing the olefin reductase mutant according to any one of claims 1 to 3, and disrupting the transformant cells containing the olefin reductase mutant to obtain a cell disrupted liquid; (3) freeze-drying the cell disrupted liquid of the olefin reductase mutant to obtain a lyophilized enzyme powder; (4) The pure enzyme of the olefin reductase mutant.

8. Use of the olefin reductase mutant according to any one of claims 1 to 3 or the olefin reductase mutant catalyst according to claim 7 in the stereoselective synthesis of chiral aromatic multi-substituted γ-butyrolactones by dual enzyme cascade catalysis.

9. The use according to claim 8, characterized in that: The method for stereoselective synthesis of chiral aromatic polysubstituted γ-butyrolactone by dual enzyme cascade catalysis is: S1. using an olefin reductase mutant or an olefin reductase mutant catalyst to perform an olefin asymmetric reduction reaction on an α- / β-unsaturated γ-ketoester to obtain an α- / β-saturated γ-ketoester intermediate; S2. Asymmetric reduction reaction of the potential chiral carbon of the α- / β-saturated γ-ketoester intermediate is carried out using carbonyl reductase or a carbonyl reductase mutant or a carbonyl reductase mutant catalyst, and finally a multi-substituted chiral γ-butyrolactone is obtained through intramolecular cyclization.

10. The use according to claim 9, characterized in that: The chemical structure of the α- / β-unsaturated γ-ketoester is shown below:

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