Alkene reductase mutant and application thereof in synthesis of (R)-2-dimethyl methylsuccinate

Abstract

The invention provides an alkene reductase mutant and an application of the alkene reductase mutant in synthesis of (R)-2-dimethyl methylsuccinate. According to the method, the olefin reductase is subjected to directed evolution to obtain the olefin reductase mutant with improved enzyme activity and substrate tolerance, the obtained mutant can be used for efficiently reducing dimethyl itaconate to prepare the high-optical-purity (R)-2-dimethyl methylsuccinate, and the reaction substrate concentration, the reaction yield and the optical purity of the product are high; the reaction process is simple to operate and low in energy consumption, and meets green chemical requirements. Therefore, the method disclosed by the invention can be applied to the biotransformation of the industrial production of the (R)-2-methyl dimethyl succinate.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and biocatalysis, and in particular relates to an ene reductase mutant and its use in synthesizing (R)-2-methylsuccinic acid dimethyl ester. Background Art

[0002] Dimethyl 2-methylsuccinate with a chiral carbon atom is an important structural unit and is commonly found in natural products, drug molecules, and synthetic intermediates. R Dimethyl methyl succinate is the precursor for the synthesis of sacubitril, which is an enkephalinase inhibitor that can block the degradation of two peptides responsible for vasodilation, thereby lowering blood pressure. Sacubitril-valsartan sodium, which is a combination of sacubitril and valsartan in a 1:1 ratio, is the first dual-acting inhibitor of angiotensin receptors and enkephalinase developed by Novartis and used clinically. The drug is a compound preparation that has the effects of vasodilation, prevention and reversal of cardiovascular remodeling and urinary sodium excretion. It is a cardiovascular drug with a completely new mechanism of action. Sacubitril-valsartan sodium was approved for marketing by the US FDA and the EU EMA in 2015, and was approved for marketing by China's NMPA in 2017. The trade name is Entresto (CN108602785 B).

[0003] Dimethyl itaconate is cheaper and easier to obtain than dimethyl citraconic acid and dimethyl mesaconic acid. R )-An important raw material for dimethyl 2-methylsuccinate. R )-2-methylsuccinate synthesis methods mainly include chemical method and biocatalytic method. Chemical synthesis of chiral 2-methylsuccinate requires expensive chiral complex catalysts composed of metals such as Rh, Ru, Pd, and Ir. The reaction conditions are harsh, the pollution is serious, and the yield is low. There are many limitations in actual large-scale production [F. Parmeggiani, "A Study in Yellow": Investigations in the Stereoselectivity of Ene-Reductases, Chembiochem 2022, 23 , e202100445]. Therefore, it is particularly important to explore greener and more efficient bio-enzyme catalysis methods.

[0004] The enzymes for biosynthesis of chiral dimethyl 2-methylsuccinate mainly include lipase and ene reductase. Lipase can be used for the resolution of the racemate of methyl 2-methylsuccinate, with the highest theoretical yield of 50%. Enene reductase is a class of oxidoreductases that can reduce activated alkenes. It has high stereo and regioselectivity and is ubiquitous in nature. It is widely present in fungi, bacteria, and plants. It can catalyze the asymmetric reduction of conjugated olefin compounds with electron-withdrawing groups (EWG groups: esters, acids, nitriles, aldehydes, ketones, nitro groups, etc.). Enene reductase is a nicotinamide adenine dinucleotide (NAD(P)H)-dependent reductase. There are many ene reductases from different sources, such as Thermus scotoductus SA-01 , Saccharomyces carlsbergensis , Rattus norvegicus , Pseudomonas putida It has been reported that it can be used to synthesize chiral dimethyl 2-methylsuccinate, but most of the reactions are analytical-scale reactions with substrate concentrations not exceeding 10 mM. There are two reported preparative-scale reactions: In 2012, a paper on the asymmetric reduction synthesis of ene reductase was published ( R )-2-methylsuccinate preparative reaction, by screening the enzyme library and optimizing the reaction conditions, the final substrate dimethyl citraconic acid ester concentration reached 200 mM, and a large amount of toluene was used as a cosolvent (28%). The conversion rate was 53% in 20 hours and 73% in 44 hours [D. Mangan, A Three-Enzyme System Involving an Ene-Reductase for Generating Valuable Chiral Building Blocks, Advanced Synthesis & Catalysis 2012, 354 , 2185-2190]; In 2016, Domínguez et al. reported the use of ene reductase ENE-102 freeze-dried powder as a catalyst to catalyze the asymmetric reduction of dimethyl itaconate to ( R )-2-Methylsuccinic acid dimethyl ester [BB Domnguez, Reduction of Activated Carbon-Carbon Double Bonds using Highly Active and Enantioselective Double BondReductases, Johnson Matthey Technology Review 2016, 60 , 243-249]. Summary of the invention

[0005] In the present invention, the activity of a series of ene reductases was systematically explored, and ene reductase mutant proteins with high substrate tolerance were successfully obtained through screening and directed evolution, which can efficiently convert dimethyl itaconate into ( R )-2-methylsuccinic acid dimethyl ester, with high separation yield and high optical purity.

[0006] Specifically, in order to efficiently synthesize ( R )-2-methylsuccinic acid dimethyl ester, the present invention is derived from Aspergillus flavus Oleoreductase from NRRL3357 Of ER (NCBI XP_041146091, SEQ ID NO.1), and directed evolution was performed to obtain olefin reductase mutant proteins with improved enzyme activity and substrate tolerance, and olefin substrates were reduced to prepare high optical purity ( R )-2-methylsuccinic acid dimethyl ester. The reaction process is shown below.

[0007]

[0008] The first aspect of the present invention provides a wild-type olefin reductase or a mutant protein thereof, wherein the amino acid sequence of the wild-type is shown in SEQ ID NO.1, the mutant protein is a non-natural protein, and the mutant protein is an olefin reductase mutant with mutations at one or more sites including sites 117 and 293 in the amino acid sequence shown in SEQ ID NO.1: 1-439: In another preferred embodiment, the tryptophan (W) at position 117 is mutated to isoleucine (I), tyrosine (Y), or histidine (H), preferably tyrosine (Y).

[0009] In another preferred embodiment, the leucine (A) at position 293 is mutated to threonine (T) or proline (P), preferably proline (P).

[0010] More specifically, the following combined mutations are present: position 117 is mutated to isoleucine (I), tyrosine (Y) or histidine (H) and position 293 is mutated to threonine (T) or proline (P).

[0011] In the second aspect, a synthesis of high optical purity ( R )-2-methylsuccinate dimethyl ester, using an engineered wild-type olefin reductase or a mutant thereof as a catalyst, wherein the engineered olefin reductase has at least 90% identity with SEQ ID NO.1, and the mutant transforms the product ( R )-2-methylsuccinic acid dimethyl ester oh Value>99%.

[0012] In a third aspect, the olefin reductase mutant described in the first aspect catalyzes the following reaction: Specifically, the catalytic reaction uses a culture, bacterial cells or a broken liquid of a genetically engineered bacterium encoding a wild-type olefin reductase or a mutant thereof after fermentation as a catalyst, dimethyl itaconate as a substrate, a substrate concentration of 0.5-2M, NADP (H) as a coenzyme, a buffer solution with a pH of 6.0-9.0 as a reaction medium, and is converted at 25°C-40°C and 200 rpm for 12 hours. After the reaction is complete, the reaction is centrifuged, extracted, and the solvent is removed under reduced pressure to obtain ( R )- dimethyl 2-methylsuccinate, with a yield greater than 85%.

[0013] The vector series used by the genetically engineered bacteria producing ene reductase in the present invention include: pET series plasmids, pTXB1 series, pGEX series, pETduet series, and pTYB series.

[0014] The genetically engineered bacteria producing olefin reductase described in the present invention is characterized in that the host bacteria capable of efficiently expressing foreign genes is one of the following: BL21 series, Rosetta series, Origami series, and Tuner series.

[0015] In the present invention, the transformant obtained by transforming the host with the plasmid can grow and produce the alkene reductase of the present invention based on known information. Any artificial or natural medium containing suitable carbon source, nitrogen source, inorganic and other nutrients can be used as long as it can meet the growth of the host bacteria and can express the target protein. There is no clear limitation on the culture method and culture conditions, and they can be appropriately selected according to the different culture methods and types, as long as they can meet the growth of the host and can produce the corresponding active alkene reductase.

[0016] For preparing the present invention ( R The olefin reductase and mutants of dimethyl 2-methylsuccinate may be the culture of the above-mentioned olefin reductase genetically engineered recombinant bacteria, or may be bacterial cells obtained by centrifuging the culture medium or their processed products. The processed products refer to extracts, broken liquids obtained from bacterial cells, or isolated products obtained by separation and / or purification of olefin reductase from extracts, or immobilized products of immobilized extracts or processed products.

[0017] The medium suitable for the reaction can be water, fermentation broth or an aqueous medium containing different buffers. The buffer used can be water with one or more appropriate phosphates, Tris hydrochloride, bicarbonate, carbonate, etc. added thereto.

[0018] The substrate concentration of the present invention is not limited, and is usually 0.5-2 M. Considering the reaction effect, the substrate concentration is preferably greater than or equal to 1.5 M. The reaction product can also be separated after the reaction is completed or the product can be continuously removed by an in-situ separation method.

[0019] The mutant obtained by the present invention can be used to efficiently reduce dimethyl itaconate to prepare highly optically pure ( R )-2-methylsuccinic acid dimethyl ester, and the reaction substrate concentration, reaction yield and product optical purity are high, the reaction process is simple to operate, the energy consumption is low, and it meets the requirements of green chemistry. Therefore, the present invention can be applied to industrial production ( R )-Biological transformation of dimethyl 2-methylsuccinate. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the GC spectrum of the racemic form of diethyl 2-methylsuccinate and the product ( R GC spectrum of (a)-2-methylsuccinic acid diethyl ester. Among them, (b) GC spectrum of (R)-2-methylsuccinic acid dimethyl ester, (c) spectrum of AfER catalytic synthesis product. DETAILED DESCRIPTION

[0021] The invention is further described below by means of specific examples, the purpose of which is to provide a better understanding of the invention, but these examples do not constitute a limitation to the invention.

[0022] In a preferred embodiment of the present invention, the preparation method of the alkene reductase mutant is as follows: (1) Oleoreductase Of The gene of the corresponding mutation site of ER was constructed into the pET 21a (+) expression vector to obtain a recombinant plasmid carrying the target enzyme gene.

[0023] (2) The recombinant plasmid was transferred into the host bacterial cell Escherichia coli BL21 (DE3) to obtain the corresponding engineered strain.

[0024] (3) The engineered strain was inoculated into 4 mL of LB liquid medium (peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L) containing kanamycin (50 mg / L), and cultured overnight at 37°C, 200 rpm to obtain a culture solution. The culture solution was inoculated into the fermentation medium (LB liquid medium) at an inoculum rate of 1% (v / v), and cultured in a shaker at 37°C, 200 rpm until the OD 600 The concentration of IPTG was 0.6-0.8, and IPTG was added to a final concentration of 0.1 mM. The mixture was induced at 25°C and 200 rpm in a shaking incubator for 8-12 h.

[0025] (4) Collect the bacteria by centrifugation and crush them under high pressure.

[0026] Example 1, olefin reductase Of Construction of ER mutant library Obtained through homology modeling Of The protein structure of ER was simulated, and non-conserved residues in its substrate binding pocket were selected for saturation mutation. The degenerate codon NNK was used to design mutation primers. Of ER was used as a template. The obtained monoclonal colonies were picked up and cultured in a 96-well deep-well plate, and the expressed proteins were subjected to high-throughput activity screening by detecting the decrease of NADPH at 340 nm. The specific reaction method was: substrate concentration 50 mM, NADPH 0.5 mg / mL, crude enzyme solution 30 μL, glucose oxidase 20 U / mL, glucose 50 mM, sodium phosphate buffer was made up to 200 μL, and the microplate reader was used to detect the decrease of NADPH at 340 nm. If the enzyme activity is relatively high, the consumption of NADPH is fast, and the slope of the decline curve is large.

[0027] The sites for mutation in library construction were 35, 38, 39, 40, 71, 72, 73, 86, 115, 117, 241, 293, 349, 372 and 373, and the beneficial mutation sites for improved enzyme activity were 117 and 293.

[0028] Example 2, olefin reductase Of Construction of ER combination mutant library According to the mutation results, sites 117 and 293 with significantly improved activity were selected to construct a combined mutant library of the two sites, and mutants 1-11 with further improved activity were obtained. The conversion rate and stereoselectivity of the mutants were investigated by establishing a conversion reaction for the substrate dimethyl itaconate. The substrate concentration was 500 mM, the bacterial solution was not concentrated, and the reaction was tested for 2 h. The enzyme activity determination and conversion reaction results are shown in Table 1.

[0029] Table 1 Of Enzyme activity, substrate conversion rate and stereoselectivity of ER and its mutants

[0030] Example 3: Inducible expression of olefin reductase mutants Prepare 50 mL of seed solution, the culture medium is LB liquid medium (peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L), pick a single colony of genetically engineered bacteria with an inoculation loop and inoculate it into the culture medium, and culture it at 37°C, 200 rpm overnight. Transfer the overnight cultured seed solution to the fermentation medium at a 1% inoculation rate, and culture it at 37°C, 200 rpm until OD 600When the p-value is about 0.6-0.8, 0.1 mM IPTG is added and induced for more than 12 h. The culture solution is centrifuged at 6000 rpm to collect the bacteria, which are crushed under high pressure to obtain the crude enzyme solution of ene reductase.

[0031] Example 4: Using olefin reductase Of ER wild-type whole-cell catalytic synthesis ( R )-2-Methylsuccinate dimethyl ester Phosphate buffer (pH 7.0) 10 ml, ene reductase Of ER wild-type bacteria 50 mg / ml, 1.3eq glucose, NADP + 0.5mg / ml, GDH 3 U / mL, substrate concentration 1 M (158g / L), reaction at 37℃, TLC spot plate to determine the reaction progress. After 12 hours, ethyl acetate was added for extraction, centrifuged, the organic phase was dried, and the product was collected by spin drying. GC detection showed a yield of 95%. oh >99%.

[0032] Example 5: Using olefin reductase Of ER wild-type whole-cell catalytic synthesis ( R )-2-Methylsuccinate dimethyl ester Phosphate buffer (pH 7.0) 10 ml, ene reductase Of ER wild-type bacteria 50 mg / ml, 1.3eq glucose, NADP + 0.5mg / ml, GDH 3 U / mL, substrate concentration 1.4 M, reaction at 37℃, TLC spot plate to determine the reaction progress. After 12 hours, ethyl acetate was added for extraction, centrifuged, the organic phase was dried, and the product was collected by spin drying. GC detection showed a yield of 70%. oh >99%.

[0033] Example 6: Whole-cell catalytic synthesis of olefin reductase mutant 6 ( R )-2-Methylsuccinate dimethyl ester Phosphate buffer (pH 7.0) 10 ml, ene reductase mutant 6 bacteria 50 mg / ml, 1.3 eq glucose, NADP + 0.5mg / ml, GDH 3 U / mL, substrate concentration 1.4 M, reaction at 37℃, TLC spot plate to determine the reaction progress. After 12 hours, ethyl acetate was added for extraction, centrifuged, the organic phase was dried, and the product was collected by spin drying. GC detection showed a yield of 85%. oh >99%.

[0034] Example 7: Synthesis of ( R )-2-Methylsuccinate dimethyl ester 10 ml phosphate buffer, 50 mg / ml ene reductase mutant 9 bacteria, 1.3 eq glucose, NADP + 0.5mg / ml, GDH 3 U / mL, substrate concentration 1.4 M, reaction at 37℃, TLC spot plate to determine the reaction progress. After 12 hours, ethyl acetate was added for extraction, centrifuged, the organic phase was dried, and the product was collected by spin drying. GC detection showed a yield of 95%. oh >99%.

[0035] Example 8: Synthesis of ( R )-2-Methylsuccinate dimethyl ester 10 ml phosphate buffer, 50 mg / ml crude enzyme solution of ene reductase mutant 9, 1-1.3eq glucose, NADP + 0.1-0.5mg / ml, GDH 3 U / mL, substrate concentration 1-2 M (see Table 2), reaction at 37℃, TLC spot plate to judge the reaction progress. After 12 hours, ethyl acetate was added for extraction, centrifuged, the organic phase was dried, and the product was collected by spin drying. GC detection, the yield and product optical purity are shown in Table 2.

[0036] Table 2. Optimization of reaction conditions

[0037]

[0038] Example 9: Whole-cell catalytic synthesis of olefin reductase mutant 9 ( R )-2-Methylsuccinate dimethyl ester 1 L phosphate buffer (pH 7.0), 50 g / L ene reductase mutant 9 whole cell, 1.2 eq glucose, NADP + 0.5mg / ml, GDH 3 U / mL, substrate concentration 1.8 M, reaction at 37℃, TLC spot plate to judge the reaction progress. After 12 hours, ethyl acetate was added for extraction, centrifuged, the organic phase was dried, and the product was collected by spin drying. GC detection showed a conversion rate of 95% and a yield of 90%. oh Worth 99%.

[0039] Example 10: Synthesis of ( R )-2-Methylsuccinate dimethyl ester 1 L phosphate buffer (pH 7.0), 50 g / L crude enzyme solution of olefin reductase cell 9, 1.2 eq glucose, NADP +0.5mg / ml, GDH 3 U / mL, substrate concentration 1.8 M, reaction at 37℃, TLC spot plate to judge the reaction progress. After 12 hours, ethyl acetate was added for extraction, centrifuged, the organic phase was dried, and the product was collected by spin drying. GC detection showed a conversion rate of 99% and a yield of 93%. oh Worth 99%.

Claims

1. An alkene reductase mutant protein, characterized in that The 117th tryptophan W in the amino acid sequence shown in SEQ ID NO.1 is mutated to isoleucine I, tyrosine Y or histidine H, and / or the 293rd leucine A is mutated to threonine T or proline P.

2. The olefin reductase mutant protein according to claim 1, characterized in that The tryptophan W at position 117 is mutated to tyrosine Y, or the leucine A at position 293 is mutated to proline P, or the 117 is mutated to isoleucine I, tyrosine Y or histidine H and the 293 is mutated to threonine T or proline P.

3. A gene encoding the olefin reductase mutant protein as claimed in claim 1 or 2.

4. An expression vector containing the coding gene as claimed in claim 3.

5. The expression vector according to claim 4, characterized in that It is a pET series plasmid, a pTXB1 series, a pGEX series, a pETduet series or a pTYB series.

6. A genetically engineered bacterium containing the coding gene as claimed in claim 3 or the expression vector as claimed in claim 4.

7. The genetically engineered bacterium according to claim 6, characterized in that The starting bacteria is one of the following: BL21 series, Rosetta series, Origami series, Tuner series.

8. A synthesis ( R )-dimethyl 2-methylsuccinate, characterized in that Using the olefin reductase mutant protein as described in claim 1 or 2, or the olefin reductase with the amino acid sequence shown in SEQ ID NO.1 as a catalyst, dimethyl itaconate as a substrate, and generating ( R )-dimethyl 2-methylsuccinate.

9. The method according to claim 8, characterized in that The catalytic reaction uses the culture, bacterial cells or broken liquid of the genetically engineered bacteria as claimed in claim 6 or 7 after fermentation as a catalyst, the dimethyl itaconate substrate concentration is 0.5-2M, NADP (H) is used as a coenzyme, and a buffer solution with a pH of 6.0-9.0 is used as a reaction medium. The reaction is carried out at 25°C-40°C under shaking. After the reaction is complete, the reaction is centrifuged, extracted, and the solvent is removed under reduced pressure to obtain ( R )- dimethyl 2-methylsuccinate.

10. The method according to claim 9, characterized in that The buffer used was water with phosphate, Tris hydrochloride, bicarbonate or carbonate added.

11. The olefin reductase mutant protein as claimed in claim 1 or 2 or the olefin reductase having the amino acid sequence as shown in SEQ ID NO.1 is synthesized ( R )-Application of dimethyl 2-methylsuccinate.

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