Enzyme reductase mutant and its application in the synthesis of dimethyl (R)-2-methylsuccinate

Abstract

The present invention provides an alkene reductase mutant and its application in the synthesis of dimethyl ( R )-dimethyl 2-methylsuccinate. The present invention conducts directed evolution on the alkene reductase to obtain an alkene reductase mutant with improved enzyme activity and substrate tolerance. The obtained mutant can efficiently reduce dimethyl itaconate to prepare highly optically pure ( R )-dimethyl 2-methylsuccinate, and the reaction substrate concentration, reaction yield, and optical purity of the product are high. The reaction process is simple to operate, has low energy consumption, and meets the requirements of green chemistry. Therefore, the present invention can be applied to the biotransformation of industrial production of ( R )-dimethyl 2-methylsuccinate.

Description

Technical Field

[0001] The present invention belongs to the fields of biotechnology and biocatalysis, and particularly relates to an enoate reductase mutant and its use in the synthesis of dimethyl (R)-2-methylsuccinate. Background Art

[0002] Dimethyl 2-methylsuccinate with a chiral carbon atom is an important structural unit, commonly found in natural products, drug molecules, and synthetic intermediates. R Dimethyl (S)-2-methylsuccinate is a precursor for the synthesis of sacubitril, which is a neprilysin inhibitor that can block the degradation of two polypeptides responsible for vasodilation, achieving the effect of lowering blood pressure. Sacubitril / valsartan sodium, a combination of sacubitril and valsartan in a 1:1 ratio, is the first dual inhibitor of angiotensin receptor and neprilysin developed by Novartis and used clinically. This drug is a compound preparation with functions such as vasodilation, prevention and reversal of cardiovascular remodeling, and natriuresis. It is a cardiovascular drug with a brand-new mechanism of action. Sacubitril / valsartan sodium was successively approved for marketing by the US FDA and the EU EMA in 2015 and by the Chinese NMPA in 2017, with the trade name Novartis (CN108602785 B).

[0003] Dimethyl itaconate is cheaper and more readily available than dimethyl citraconate and dimethyl mesaconate, and is an important raw material for the synthesis of R ()-dimethyl 2-methylsuccinate. The synthetic methods of R ()-dimethyl 2-methylsuccinate mainly include chemical methods and biocatalytic methods. The chemical synthesis of chiral dimethyl 2-methylsuccinate uses 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, which has 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 a more green and efficient biocatalytic method.

[0004] The enzymes used in the 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, and the highest theoretical yield is 50%. Ene-reductase is a class of oxidoreductases that can reduce activated alkenes, with high stereo- and regioselectivity. It is ubiquitous in nature and widely exists in fungi, bacteria, and plants. It can catalyze the asymmetric reduction of conjugated alkene compounds with electron-withdrawing groups (EWG groups: esters, acids, nitriles, aldehydes, ketones, nitro, etc.). Ene-reductase is a nicotinamide adenine dinucleotide (NAD(P)H)-dependent reductase. Many ene-reductases from different sources, such as Thermus scotoductus SA-01 , Saccharomyces carlsbergensis , Rattus norvegicus , Pseudomonas putida etc. have been reported to be used in the synthesis of chiral dimethyl 2-methylsuccinate, but most of the reactions are analytical-grade reactions, and the substrate concentration does not exceed 10 mM. There are two reported preparative-scale reactions: In 2012, a preparative-scale reaction for the asymmetric reduction synthesis of ([[]] R )-dimethyl 2-methylsuccinate by ene-reductase was published. Through screening of the enzyme library and optimization of the reaction conditions, the concentration of the substrate dimethyl citraconate finally reached 200 mM, and a large amount of toluene was used as a co-solvent (28%). The conversion rate was 53% in 20 hours and 73% in 44 hours [D. Mangan, A Three-Enzyme SystemInvolving 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 freeze-dried powder of ene-reductase ENE-102 as a catalyst to catalyze the asymmetric reduction of dimethyl itaconate to ( R )-dimethyl 2-methylsuccinate at a high substrate concentration (730 mM) [B. B. 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 Af 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:

[0009] In another preferred embodiment, the tryptophan (W) at position 117 is mutated to isoleucine (I), tyrosine (Y), or histidine (H), preferably tyrosine (Y).

[0010] In another preferred embodiment, the leucine (A) at position 293 is mutated to threonine (T) or proline (P), preferably proline (P).

[0011] 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).

[0012] 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 ee Value>99%.

[0013] In a third aspect, the enoyl reductase mutant described in the first aspect catalyzes the following reaction: Specifically, the catalytic reaction uses the culture, bacterial cells, or cell lysate after fermentation of a genetically engineered bacterium containing the wild-type enoyl reductase or its mutant encoding gene as a catalyst, dimethyl itaconate as a substrate, with a substrate concentration of 0.5 - 2 M, NADP(H) as a coenzyme, a buffer with a pH of 6.0 - 9.0 as the reaction medium, at 25°C - 40°C, and 200 rpm for 12 hours. After the reaction is complete, it is centrifuged, extracted, and the solvent is removed under reduced pressure to obtain R )-dimethyl 2-methylbutanedioate with a yield of more than 85%.

[0014] The vector series used in the genetically engineered bacterium producing enoyl reductase in the present invention includes: pET series plasmids, pTXB1 series, pGEX series, pETduet series, pTYB series.

[0015] The genetically engineered bacterium producing enoyl reductase in the present invention is characterized in that the host bacterium capable of highly expressing foreign genes is one of the following: BL21 series, Rosetta series, Origami series, Tuner series.

[0016] In the present invention, the transformant obtained by transforming the host with the plasmid can grow based on known information and produce the enoyl reductase described in the present invention. Any artificial or natural medium containing appropriate carbon sources, nitrogen sources, inorganic and other nutrients can be used as long as it can support the growth of the host bacteria and express the target protein. There are no specific limitations on the culture method and culture conditions, and appropriate selection can be made according to the differences in the culture method and type, etc., as long as it can support the growth of the host and produce the corresponding active enoyl reductase.

[0017] The enoyl reductase and mutant used for preparing R )-dimethyl 2-methylbutanedioate in the present invention can be the culture of the above-mentioned genetically engineered recombinant enoyl reductase bacteria, or the bacterial cells obtained by centrifuging the culture medium or their processed products. The processed products refer to the extracts obtained from the bacteria, cell lysates, or the separated products obtained by separating and / or purifying the enoyl reductase from the extracts, or the immobilized products obtained by immobilizing the extracts or processed products.

[0018] The applicable medium in the reaction can be water, fermentation broth, or an aqueous medium containing different buffers. The buffers used can be one or several appropriate phosphates, Tris hydrochloride, bicarbonates, carbonates, etc. added to water.

[0019] The substrate concentration in the present invention is not limited. Usually, the substrate is 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 or continuously removed by in-situ separation methods.

[0020] The mutant obtained by the present invention can efficiently reduce dimethyl itaconate to prepare highly optically pure dimethyl ( R )-2-methylsuccinate, and the reaction substrate concentration, reaction yield, and optical purity of the product are high. The reaction process is simple to operate, has low energy consumption, and meets the requirements of green chemistry. Therefore, the present invention can be applied to the biotransformation of dimethyl ( R )-2-methylsuccinate in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the GC chromatogram of the racemate of diethyl 2-methylsuccinate and the GC chromatogram of the product ( R )-diethyl 2-methylsuccinate. Among them, (a) is the GC chromatogram of the racemate of dimethyl 2-methylsuccinate, (b) is the GC chromatogram of (R)-dimethyl 2-methylsuccinate, and (c) is the chromatogram of the product catalyzed by AfER. DETAILED DESCRIPTION OF THE INVENTION

[0022] The following is further illustrated by specific examples, aiming to better understand the content of the invention, but these examples do not constitute a limitation to the present invention.

[0023] In the preferred embodiment of the present invention, the preparation method of the ene reductase mutant is as follows:

[0024] (1) Ene reductase Af 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.

[0025] (2) The recombinant plasmid was transferred into the host cell Escherichia coli BL21(DE3) to obtain the corresponding engineering strain.

[0026] (3) The engineering strain was inoculated into 4 mL of LB liquid medium containing kanamycin (50 mg / L) (peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L), and cultured overnight at 37 °C and 200 rpm to obtain a culture solution. The culture solution was inoculated into the fermentation medium (LB liquid medium) at an inoculation amount of 1% (v / v), and cultured in a shaker at 37 °C and 200 rpm until the OD 600 reached 0.6 - 0.8, and IPTG was added to a final concentration of 0.1 mM, and induced in a shaker at 25 °C and 200 rpm for 8 - 12 h.

[0027] (4) The cells were collected by centrifugation and subjected to high-pressure crushing.

[0028] Example 1, ene reductase Af Construction of ER mutant library

[0029] Obtained by homology modeling Af The simulated protein structure of ER was obtained. Non-conserved residues in its substrate-binding pocket were selected for saturation mutagenesis respectively. Degenerate codon NNK was used to design mutant primers, with pET21a(+)- Af ER as the template. The obtained monoclonal colonies were picked into 96-well deep-well plates for cultivation, and the expressed proteins were screened for high-throughput activity. The screening method was to detect the decrease of NADPH at 340 nm. The specific method of the reaction was as follows: substrate concentration 50 mM, NADPH 0.5 mg / mL, crude enzyme solution 30 μL, glucose oxidase 20 U / mL, glucose 50 mM, and made up to 200 μL with sodium phosphate buffer. The decrease of NADPH at 340 nm was detected by a microplate reader. If the enzyme activity was relatively high, the consumption of NADPH was faster and the slope of the decrease curve was larger.

[0030] The sites for library construction mutation were 35, 38, 39, 40, 71, 72, 73, 86, 115, 117, 241, 293, 349, 372 and 373, and the beneficial mutation sites with improved enzyme activity were 117 and 293.

[0031] Example 2, ene reductase Af Construction of ER combinatorial mutant library

[0032] According to the mutation results, sites 117 and 293 with significantly improved activity were selected to construct a two-site combinatorial mutant library, and mutant 1-11 with further improved activity was obtained. The conversion rate and stereoselectivity of the mutant were investigated by establishing a conversion reaction for dimethyl itaconate as the substrate. The substrate concentration was 500 mM, the bacterial liquid was not concentrated, and the reaction was detected after 2 h. The enzyme activity determination and conversion reaction results are shown in Table 1.

[0033] Table 1 Af Enzyme activities of ER and its mutants, conversion rate and stereoselectivity for substrates

[0034]

[0035] Example 3, Induced expression of ene reductase mutants

[0036] Prepare 50 mL of seed liquid. The medium is LB liquid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl). Use an inoculation loop to pick a single colony of genetically engineered bacteria and inoculate it into the medium. Incubate overnight at 37°C and 200 rpm. Transfer the overnight cultured seed liquid to the fermentation medium at an inoculation amount of 1%, and culture at 37°C and 200 rpm until 600 the OD is about 0.6 - 0.8. Add 0.1 mM IPTG and induce for more than 12 h. Centrifuge the culture solution at 6000 rpm to collect the bacterial cells, and perform high-pressure crushing to obtain the crude enzyme solution of enol reductase.

[0037] Example 4: Catalytic synthesis of dimethyl ( Af )-2-methylsuccinate by enol reductase R using wild-type enol reductase whole cells

[0038] 10 ml of phosphate buffer (pH 7.0), enol reductase Af 50 mg / ml of wild-type enol reductase cells, 1.3 eq of glucose, NADP + 0.5 mg / ml, GDH 3 U / mL, substrate concentration 1 M (158 g / L), react at 37°C, and judge the reaction progress by TLC spotting. After 12 h, add ethyl acetate for extraction, centrifuge, dry the organic phase, rotary evaporate to collect the product, and detect by GC. The yield is 95%, ee >99%.

[0039] Example 5: Catalytic synthesis of dimethyl ( Af )-2-methylsuccinate by enol reductase R using wild-type enol reductase whole cells

[0040] 10 ml of phosphate buffer (pH 7.0), enol reductase Af 50 mg / ml of wild-type enol reductase cells, 1.3 eq of glucose, NADP + 0.5 mg / ml, GDH 3 U / mL, substrate concentration 1.4 M, react at 37°C, and judge the reaction progress by TLC spotting. After 12 h, add ethyl acetate for extraction, centrifuge, dry the organic phase, rotary evaporate to collect the product, and detect by GC. The yield is 70%, ee >99%.

[0041] Example 6: Catalytic synthesis of dimethyl ( R )-2-methylsuccinate by whole cells of enol reductase mutant 6

[0042] 10 ml of phosphate buffer (pH 7.0), 50 mg / ml of enol reductase mutant 6 cells, 1.3 eq of glucose, NADP +0.5 mg / ml, GDH 3 U / mL, substrate concentration 1.4 M, react at 37 °C, judge the reaction progress by TLC plate spotting. After 12 hours, add ethyl acetate for extraction, centrifuge, dry the organic phase, rotary evaporate to collect the product, detect by GC, the yield is 85%, ee >99%.

[0043] Example 7: Use the whole cells of enoyl reductase mutant 9 to catalyze the synthesis of R dimethyl (S)-2-methylbutanedioate

[0044] 10 ml of phosphate buffer, 50 mg / ml of the cells of enoyl reductase mutant 9, 1.3 eq of glucose, NADP + 0.5 mg / ml, GDH 3 U / mL, substrate concentration 1.4 M, react at 37 °C, judge the reaction progress by TLC plate spotting. After 12 hours, add ethyl acetate for extraction, centrifuge, dry the organic phase, rotary evaporate to collect the product, detect by GC, the yield is 95%, ee >99%.

[0045] Example 8: Use the crude enzyme solution of enoyl reductase mutant 9 to catalyze the synthesis of R dimethyl (S)-2-methylbutanedioate

[0046] 10 ml of phosphate buffer, 50 mg / ml of the crude enzyme solution of enoyl reductase mutant 9, 1 - 1.3 eq of glucose, NADP + 0.1 - 0.5 mg / ml, GDH 3 U / mL, substrate concentration 1 - 2 M (see Table 2), react at 37 °C, judge the reaction progress by TLC plate spotting. After 12 hours, add ethyl acetate for extraction, centrifuge, dry the organic phase, rotary evaporate to collect the product, detect by GC, the yield and the optical purity of the product are shown in Table 2.

[0047] Table 2: Optimization of reaction conditions

[0048]

[0049] Example 9: Use the whole cells of enoyl reductase mutant 9 to catalyze the synthesis of R dimethyl (S)-2-methylbutanedioate

[0050] 1 L of phosphate buffer (pH 7.0), 50 g / L of the whole cells of enoyl reductase mutant 9, 1.2 eq of glucose, NADP + 0.5 mg / ml, GDH 3 U / mL, substrate concentration 1.8 M, react at 37 °C, judge the reaction progress by TLC plate spotting. After 12 hours, add ethyl acetate for extraction, centrifuge, dry the organic phase, rotary evaporate, collect the product, detect by GC, the conversion rate is 95%, the yield is 90%, e.e.Value 99%.

[0051] Example 10: Using the crude enzyme solution of enoyl reductase mutant 9 to catalyze the synthesis of R dimethyl (

[0052] )-2-methylbutanedioate + 1 L of phosphate buffer (pH 7.0), 50 g / L of crude enzyme solution obtained by disrupting enoyl reductase cells 9, 1.2 eq of glucose, NADP e.e. Value 99%.

Claims

1. An alkene reductase mutant protein, characterized in that, It is obtained by having only any one of the following mutations based on the amino acid sequence shown in SEQ ID NO.1: Leucine A at position 293 is mutated to proline P; Position 117 is mutated to isoleucine I, tyrosine Y or histidine H; Position 117 is mutated to isoleucine I and position 293 is mutated to threonine T; Position 117 is mutated to tyrosine Y and position 293 is mutated to threonine T; Position 117 is mutated to histidine H and position 293 is mutated to threonine T; Position 117 is mutated to isoleucine I and position 293 is mutated to proline P; Position 117 is mutated to tyrosine Y and position 293 is mutated to proline P; Position 117 is mutated to histidine H and position 293 is mutated to proline P.

2. The coding gene of the enoyl reductase mutant protein as claimed in claim 1.

3. An expression vector containing the coding gene as claimed in claim 2.

4. The expression vector according to claim 3, characterized in that, It is a pET series plasmid, pTXB1 series, pGEX series, pETduet series or pTYB series.

5. A genetically engineered bacterium containing the coding gene as claimed in claim 2 or the expression vector as claimed in claim 3.

6. The genetically engineered bacterium according to claim 5, wherein Its starting bacterium is one of the following: BL21 series, Rosetta series, Origami series, Tuner series.

7. A method for synthesizing dimethyl (R)-2-methylsuccinate, characterized in that, Using the enoyl reductase mutant protein as claimed in claim 1, dimethyl itaconate is used as a substrate, and (R)-dimethyl 2-methylbutanedioate is generated through a catalytic reaction.

8. The method according to claim 7, wherein The catalytic reaction uses the culture, bacterial cells or lysate after fermentation of the genetically engineered bacterium as claimed in claim 5 or 6 as a catalyst, the substrate concentration of dimethyl itaconate is 0.5 - 2 M, NADP(H) is used as a coenzyme, 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 with shaking, and after the reaction is complete, (R)-dimethyl 2-methylbutanedioate is obtained after centrifugation, extraction and solvent removal under reduced pressure.

9. The method according to claim 8, wherein The buffer solution used is water added with phosphate, Tris hydrochloride, bicarbonate or carbonate.

10. The application of the enoyl reductase mutant protein as claimed in claim 1 in the synthesis of (R)-dimethyl 2-methylbutanedioate.

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