Olefin reductase, encoding gene, vector, engineered bacteria and application thereof

Abstract

The application discloses an olefin reductase, a coding gene, a carrier, an engineering bacterium and application thereof, wherein the olefin reductase SeER mutant is obtained by single-point or multi-point site-directed mutation or site-directed saturation mutation of the 38th, 117th, 248th, 293rd and 373rd sites of the amino acid sequence shown in SEQ ID NO. 2. The application provides a series of new olefin reductase SeER mutants capable of efficiently stereocomplementary reduction of olefin compounds to prepare chiral compounds, and successfully synthesizes chiral GABA derivative drugs such as phenibut, baclofen, tolperisone and pregabalin through a chemical-enzyme coupling method. Compared with existing methods, the strain can flexibly regulate the configuration of the product, and has high stereoselectivity and conversion rate.

Description

(1) Technical field

[0001] The invention belongs to the field of drug synthesis, and particularly relates to an alkene reductase SeER mutant and its application in reducing alkene compounds to synthesize chiral compounds. (2) Background technology

[0002] Olefin reductases (ERs) are oxidoreductases belonging to the NAD(P)H-dependent oxidoreductase superfamily. They catalyze the asymmetric reduction of alkenes and produce chiral molecules with two possible stereocenters. Due to their mild reaction conditions and high selectivity, olefin reductases have gradually replaced metal-catalyzed high-pressure hydrogenation methods and become an important tool for constructing stereocenters in olefin substrates. However, natural olefin reductases are limited by their narrow substrate spectrum, and most ERs typically exhibit a single stereoselectivity for substrates, while enantiocomplementary ERs are rare, which limits their application in the synthesis of stereocomplementary chiral molecules. In addition, wild-type olefin reductases often exhibit low activity and stereoselectivity towards non-natural (synthetic) substrates. Therefore, enzyme molecular modification is needed to enhance their activity and stereocomplementary selectivity towards non-natural substrates.

[0003] The chiral γ-amino acid butyric acid (GABA) and its derivatives are a class of molecules with diverse biological activities. In 2015, Jon D. Stewart et al. first reported the use of olefin reductase OYE1 and its mutants to catalyze the stereocomplementary reduction of (Z)-3-cyano-3-phenylacrylate to synthesize (R)- and (S)-β-aryl-γ-lactams, providing a facile and atom-economical method for synthesizing stereocomplementary chiral GABA derivatives (Adv. Synth. Catal. 2015, 357, 1849–1860). However, the catalytic efficiency of this olefin reductase was low, and its stereocomplementary selectivity was poor, making it difficult to synthesize highly optically pure chiral molecules. (3) Summary of the invention

[0004] The present invention aims to provide an olefin reductase SeER mutant, encoding gene, vector, engineered bacteria, and use thereof in the stereocomplementary reduction of olefin compounds to synthesize chiral compounds. The present invention molecularly modifies the olefin reductase SeER from S. eubayanus to screen and obtain mutants capable of catalytically reducing olefin compounds with high activity (>99% conversion rate) and high stereocomplementary selectivity (ee values ​​of 99% R-selectivity and 99% S-selectivity), such as (Z)-3-cyano-3-phenylacrylate and a series of non-natural substrates thereof. Furthermore, by coupling chemical cyanide reduction and ester hydrolysis, the optically pure products can be converted into chiral GABA derivatives, such as fenibute, baclofen, tolibut, and pregabalin. This solves the problems of low activity and poor stereoselectivity of existing olefin reductases for the asymmetric stereocomplementary reduction of (Z)-3-cyano-3-phenylacrylate and its derivatives.

[0005] The technical solution adopted in the present invention is:

[0006] In a first aspect, the present invention provides a SeER olefin reductase mutant, obtained by subjecting the SeER olefin reductase mutant to single-site or multi-site mutagenesis or site-directed saturation mutagenesis at positions 38, 117, 248, 293, and 373 of the amino acid sequence of SeER olefin reductase from S. eubayanus as shown in SEQ ID NO. 2. The nucleotide sequence of the gene encoding the SeER olefin reductase of the present invention is shown in SEQ ID NO. 1.

[0007] Furthermore, it is preferred that the olefin reductase SeER mutant is a mutant in which the amino acid sequence shown in SEQ ID NO.2 is mutated into one of the following: (1) tryptophan at position 117 is mutated into histidine (W117H); (2) tryptophan at position 117 is mutated into glycine (W117G); (3) tryptophan at position 117 is mutated into alanine (W117A); (4) tryptophan at position 117 is mutated into histidine, and phenylalanine at position 248 is mutated into alanine (W117H / F248A, amino acid sequence as shown in SEQ ID NO.4, nucleotide sequence as shown in SEQ ID NO.3); (5) tryptophan at position 117 is mutated into histidine, and threonine at position 38 is mutated into serine (W117H / T38S, amino acid sequence as shown in SEQ ID NO.6, nucleotide sequence as shown in SEQ ID NO.5); (6) tryptophan at position 117 is mutated to histidine, and leucine at position 293 is mutated to histidine (W117H / L293H, amino acid sequence as shown in SEQ ID NO.8, nucleotide sequence as shown in SEQ ID NO.7); (7) tryptophan at position 117 is mutated to glycine, and threonine at position 38 is mutated to tryptophan (W117G / T38W, amino acid sequence as shown in SEQ ID NO.10, nucleotide sequence as shown in SEQ ID NO.9); (8) tryptophan at position 117 is mutated to glycine, and tyrosine at position 373 is mutated to alanine (W117G / Y373A, amino acid sequence as shown in SEQ ID NO.12, nucleotide sequence as shown in SEQ ID NO.11); (9) tryptophan at position 117 is mutated to alanine, and threonine at position 38 is mutated to tryptophan (W117A / T38W, amino acid sequence as shown in SEQ ID NO.14, nucleotide sequence as shown in SEQ ID Shown in NO.13).

[0008] The present invention also relates to the olefin reductase SeER mutant encoding gene, recombinant expression vector, enzyme mutant and recombinant genetic engineering bacteria. The recombinant expression vector is preferably vector pET21a(+), and the recombinant genetic engineering bacteria is preferably host bacteria E. coli BL21(DE3).

[0009] In a second aspect, the present invention also relates to the use of the olefin reductase mutant in the synthesis of chiral compounds by stereocomplementary reduction of olefin compounds. The method of the application is as follows: wet cells or purified enzymes obtained by induction culture of recombinant genetically engineered bacteria of the olefin reductase mutant are used as catalysts, wet cells or lyophilized powder of purified enzymes obtained by induction culture of genetically engineered bacteria of glucose dehydrogenase are used as coenzymes, a prochiral olefin compound is used as substrate, glucose is used as cosubstrate, dimethyl sulfoxide is used as cosolvent, a pH 5-9 buffer solution (preferably pH 6.0, 100mM potassium phosphate buffer solution) is used as reaction medium to form a reaction system, the reaction is carried out at 25-40°C (preferably 30°C) and 1000rpm, and after the reaction is completed, the reaction solution is separated and purified to obtain a chiral product.

[0010] Furthermore, when the olefin reductase SeER mutant is a mutant in which the amino acid sequence shown in SEQ ID NO. 2 is mutated into one of the following, the chiral product obtained by catalyzing the substrate is an R-configuration: the 117th tryptophan is mutated into histidine, the 117th tryptophan is mutated into histidine and the 248th phenylalanine is mutated into alanine, the 117th tryptophan is mutated into histidine and the 38th threonine is mutated into serine, the 117th tryptophan is mutated into histidine and the 293rd leucine is mutated into histidine; when the olefin reductase SeER mutant is a mutant in which the amino acid sequence shown in SEQ ID NO. When the amino acid sequence shown in NO.2 mutates into one of the following, the chiral product obtained by catalysis of the substrate is S-configuration: tryptophan at position 117 mutates to glycine, tryptophan at position 117 mutates to glycine and threonine at position 38 mutates to tryptophan, tryptophan at position 117 mutates to glycine and tyrosine at position 373 mutates to alanine, tryptophan at position 117 mutates to alanine, tryptophan at position 117 mutates to alanine and tryptophan at position 38 mutates to tryptophan.

[0011] Furthermore, the structure of the olefin compound is shown in 1:

[0012]

[0013] In formula 1, R2 is methyl, ethyl, isopropyl, or tert-butyl; R1 is one of the following: phenyl, 2-fluorophenyl, 3-chlorophenyl, m-tolyl, 4-methoxyphenyl, 4-methylphenyl, 4-methoxyphenyl, 4-methylphenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 4-trifluoromethylphenyl, [1,1'-biphenyl]-4-yl, naphthalene-2-yl, 3-bromo-4-methoxyphenyl, 3,4-dichlorophenyl, 3,4-difluorophenyl, 3,5-dichlorophenyl, 3,5-difluorophenyl, thiophen-2-yl, or methylhexyl.

[0014] Furthermore, the olefin compound is one of the following: (Z)-3-cyano-3-phenylacrylate methyl ester (1a), (Z)-3-cyano-3-phenylacrylate ethyl ester (1b), (Z)-3-cyano-3-phenylacrylate isopropyl ester (1c), (Z)-3-cyano-3-phenylacrylate tert-butyl ester (1d), (Z)-3-cyano-3-(2-fluorophenyl)acrylate methyl ester (1e), (Z)-3-cyano-3-(3-chlorophenyl)acrylate methyl ester ( 1f), (Z)-3-cyano-3-(m-tolyl) acrylate (1g), (Z)-3-cyano-3-(4-methoxyphenyl) acrylate (1h), (Z)-3-cyano-3-(4-methylphenyl) acrylate (1i), (Z)-3-cyano-3-(4-fluorophenyl) acrylate (1j), (Z)-3-cyano-3-(4-chlorophenyl) acrylate (1k), (Z)-3-cyano-3-(4-bromophenyl) acrylate Methyl (Z)-3-cyano-3-(4-trifluoromethylphenyl)acrylate (1l), Methyl (Z)-3-cyano-3-(4-trifluoromethylphenyl)acrylate (1m), Methyl (Z)-3-([1,1'-biphenyl]-4-yl)-3-cyanoacrylate (1n), Methyl (Z)-3-cyano-3-(naphthalen-2-yl)acrylate (1o), Methyl (Z)-3-(3-bromo-4-methoxyphenyl)-3-cyanoacrylate (1p), Methyl (Z)-3-cyano-3-(3,4-dichlorophenyl)acrylate phenyl)acrylate (1q), (Z)-3-cyano-3-(3,4-difluorophenyl)acrylate (1r), (Z)-3-cyano-3-(3,5-dichlorophenyl)acrylate (1s), (Z)-3-cyano-3-(3,5-difluorophenyl)acrylate (1t), (E)-3-cyano-3-(thien-2-yl)acrylate (1u), (Z)-3-cyano-5-methylhex-2-enoic acid ethyl ester (1v).

[0015] Furthermore, in the reaction system, the substrate is added to a final concentration of 1-20 mM (preferably 5-10 mM); glucose is added to a final concentration of 1-50 mM (preferably 30 mM); the catalyst is used in an amount of 10-30 g / L (preferably 20 g / L) based on the weight of the wet bacteria or 0.01-2.5 g / L (preferably 0.1 g / L) based on the protein content of the purified enzyme; the coenzyme EsGDH is used in an amount of 0.5-2 g / L (preferably 1 g / L) based on the weight of the purified enzyme lyophilized powder; and dimethyl sulfoxide is added at a concentration of 5-10% by volume. The glucose dehydrogenase gene (GenBank No. KM817194.1) is derived from Exiguobacterium sibirium DSM17290, with the nucleotide sequence shown in SEQ ID NO. 15 and the amino acid sequence shown in SEQ ID NO. 16.

[0016] Furthermore, the wet cell was prepared as follows: the engineered bacteria containing the olefin reductase SeER mutant gene were inoculated into LB liquid culture medium containing a final concentration of 100 μg / mL ampicillin, and cultured at 37°C and 200 rpm for 10 h to obtain a seed solution; the seed solution was inoculated into a sterilized LB liquid culture medium containing a final concentration of 100 μg / mL ampicillin at an inoculum volume concentration of 1.0% (v / v), and cultured at 37°C and 200 rpm until the OD 600 =0.6~0.8, add isopropylthiogalactoside (IPTG) with a final concentration of 0.15mM to the culture medium, culture at 18°C ​​for 18h, and centrifuge at 4°C and 8000rpm for 10min to obtain wet cells of the olefin reductase SeER mutant; the preparation method of the wet cells obtained by induction culture of the engineered bacteria containing the glucose dehydrogenase gene is the same as the wet cells containing the olefin reductase SeER mutant gene.

[0017] Furthermore, the SeER mutant purified enzyme was prepared as follows: the wet cells of the engineered bacteria containing the olefin reductase SeER mutant gene were resuspended in a pH 6.0, 100 mM potassium phosphate buffer at 100 g / L, and ultrasonically disrupted on an ice-water mixture for 25 min. The ultrasonic disruption conditions were: power 400 W, disruption for 2 s, pause for 3 s, and the disrupted mixture was taken to obtain a crude enzyme solution; 0.1 mg / mL of riboflavin sodium phosphate (FMN) was added to the obtained crude enzyme solution, and the solution was allowed to stand on ice for 30 min, followed by centrifugation at 8000 rpm and 4°C for 20 min. The supernatant was collected and filtered through a 0.45 μm microfiltration membrane. The mutant protein was purified by a nickel affinity column (1.6×10 cm, Bio-Rad, USA). The purification operation was as follows: ① The sample was loaded at a flow rate of 1.0 mL / min and filtered using a pH 7.0 column containing 0.5 M NaCl and 20 mM imidazole. ② Gravity elution with 6.0, 20mM potassium phosphate buffer at a flow rate of 1.0mL / min to remove unbound impurities until the conductivity stabilizes; ② Then gravity elute with pH 6.0, 20mM potassium phosphate containing 0.5M NaCl and 500mM imidazole for 1 column volume; collect all the eluate and transfer it to an ultrafiltration tube with a molecular weight cutoff of 30kDa, use 20mM, pH 6.0 potassium phosphate as the replacement buffer to remove the imidazole, collect the retained protein, and obtain the SeER mutant purified enzyme. The preparation method of the EsGDH purified enzyme is the same as that of the SeER mutant purified enzyme, but no riboflavin sodium phosphate is added to the crude enzyme solution and the ultrafiltration tube used has a molecular weight cutoff of 10kDa.

[0018] In a third aspect, the present invention further provides an application of an olefin reductase SeER mutant in catalyzing the stereocomplementary reduction of (Z)-3-cyano-3-phenylacrylate to synthesize phenibut, wherein the application is as follows: wet bacteria or purified enzyme obtained by fermentation culture of an engineered bacterium containing an olefin reductase SeER mutant encoding gene is used as a catalyst, a purified enzyme freeze-dried powder obtained by fermentation culture of an engineered bacterium containing a glucose dehydrogenase encoding gene is used as a coenzyme, and (Z)-3-cyano-3-phenylacrylate is used as a substrate. , using dimethyl sulfoxide (DMSO) as a cosolvent and a pH 5-9 buffer as a reaction medium, the reaction is carried out at 25-40° C. (preferably 30° C.) and 1000 rpm. After the reaction is completed, the reaction solution is separated and purified to obtain (R)-3-cyano-3-phenylpropionic acid methyl ester; (R)-3-cyano-3-phenylpropionic acid methyl ester is reduced with sodium borohydride and hydrolyzed with hydrochloric acid to synthesize phenibut hydrochloride; the olefin reductase SeER mutant is preferably SeER-W117H / F248A, and the amino acid sequence is shown in SEQ ID NO.4.

[0019] Furthermore, a method for synthesizing fenibute hydrochloride is provided as follows: (R)-3-cyano-3-phenylpropionic acid methyl ester and NiCl2·6H2O are dissolved in methanol, cooled to 0°C, stirred for 15 minutes, and then NaBH4 is added in portions. The final solution is further stirred at 0°C for 15 minutes, and the solution turns black. After the reaction of the raw materials is completed as monitored by TLC, the methanol is removed by vacuum distillation, and the concentrated solid is refluxed in 6M HCl solution for 12 hours. After the reaction is completed, water is removed by vacuum distillation, and the concentrated solid is recrystallized from isopropanol to obtain the drug fenibute hydrochloride. The mass ratio of the (R)-3-cyano-3-phenylpropionic acid methyl ester to NiCl2·6H2O is 1:2.5, and the volumetric amount of methanol used is 0.05 mL / mg based on the mass of the (R)-3-cyano-3-phenylpropionic acid methyl ester; the mass ratio of the (R)-3-cyano-3-phenylpropionic acid methyl ester to NaBH4 is 1:1.8-2; and the volumetric amount of the HCl solution used is 0.1 mL / mg based on the mass of the (R)-3-cyano-3-phenylpropionic acid methyl ester.

[0020] Furthermore, the substrate is added to a final concentration of 10 mM, the cosolvent is added to a final concentration of 10% by volume, and the substrate is first dissolved in the cosolvent and then added; the glucose is added to a final concentration of 30 mM; the coenzyme is added to a final concentration of 1 mg / mL; the catalyst is added to a final concentration of 20 g / L based on the weight of the wet cells; and the buffer is 100 mM, pH 6.0 potassium phosphate buffer.

[0021] In a fourth aspect, the present invention provides an application of an olefin reductase SeER mutant in catalyzing the stereocomplementary reduction of (Z)-3-(4-chlorophenyl)-3-cyanoacrylate to synthesize baclofen, wherein the application uses wet bacteria or purified enzyme obtained by fermentation culture of an engineered bacterium containing an olefin reductase SeER mutant encoding gene as a catalyst, a purified enzyme freeze-dried powder obtained by fermentation culture of an engineered bacterium containing a glucose dehydrogenase encoding gene as a coenzyme, (Z)-3-(4-chlorophenyl)-3-cyanoacrylate as a substrate, dimethyl sulfoxide (DMSO) as a cosolvent, and A pH 5-9 buffer solution is used as the reaction medium, and the reaction is carried out at 25-40° C. (preferably 30° C.) and 1000 rpm. After the reaction is completed, the reaction solution is separated and purified to obtain (R)-3-(4-chlorophenyl)-3-cyanopropionic acid methyl ester; (R)-3-(4-chlorophenyl)-3-cyanopropionic acid methyl ester is reduced with sodium borohydride and hydrolyzed with hydrochloric acid to synthesize baclofen hydrochloride; the olefin reductase SeER mutants are preferably SeER-W117H / F248A, SeER-W117H / T38S, and SeER-W117H / L293H, and the amino acid sequences are shown in SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.8.

[0022] Furthermore, a method for synthesizing baclofen hydrochloride is provided as follows: (R)-3-(4-chlorophenyl)-3-cyanopropionic acid methyl ester and NiCl2·6H2O are dissolved in methanol (MeOH), cooled to 0°C, and stirred for 15 minutes. NaBH4 is then added in portions, and the final solution is further stirred at 0°C for 15 minutes. The solution turns black, and TLC is used to monitor whether the raw materials have reacted completely. After the reaction is completed, methanol is removed by vacuum distillation, and the concentrated solid is refluxed in 6M HCl solution for 12 hours. After the reaction is completed, water is removed by vacuum distillation, and the concentrated solid is recrystallized from isopropanol to obtain the drug baclofen hydrochloride. The mass ratio of the (R)-3-(4-chlorophenyl)-3-cyanopropionic acid methyl ester to NiCl2·6H2O is 1:2.5, and the volumetric amount of methanol used is 0.05 mL / mg based on the mass of the (R)-3-cyano-3-phenylpropionic acid methyl ester; the mass ratio of the (R)-3-(4-chlorophenyl)-3-cyanopropionic acid methyl ester to NaBH4 is 1:1.8-2; and the volumetric amount of the HCl solution used is 0.1 mL / mg based on the mass of the (R)-3-(4-chlorophenyl)-3-cyanopropionic acid methyl ester.

[0023] Furthermore, the substrate is added to a final concentration of 10 mM, the cosolvent is added to a final concentration of 10% by volume, and the substrate is first dissolved in the cosolvent and then added; the glucose is added to a final concentration of 30 mM; the coenzyme is added to a final concentration of 1 mg / mL; the catalyst is added to a final concentration of 20 g / L based on the weight of the wet cells; and the buffer is 100 mM, pH 6.0 potassium phosphate buffer.

[0024] In a fifth aspect, the present invention further provides an application of the olefin reductase SeER mutant in catalyzing the stereoreduction of (Z)-3-(4-methylphenyl)-3-cyanoacrylate to synthesize Tolibut, wherein the application uses wet bacteria or purified enzyme obtained by fermentation culture of an engineered bacterium containing an olefin reductase SeER mutant encoding gene as a catalyst, a purified enzyme freeze-dried powder obtained by fermentation culture of an engineered bacterium containing a glucose dehydrogenase encoding gene as a coenzyme, (Z)-3-(4-methylphenyl)-3-cyanoacrylate as a substrate, and Dimethyl sulfoxide (DMSO) is used as a cosolvent, a pH 5-9 buffer solution is used as a reaction medium, and the reaction is carried out at 25-40°C (preferably 30°C) and 1000rpm. After the reaction is completed, the reaction solution is separated and purified to obtain (R)-3-(4-methylphenyl)-3-cyanopropionic acid methyl ester; (R)-3-(4-methylphenyl)-3-cyanopropionic acid methyl ester is reduced with sodium borohydride and hydrolyzed with hydrochloric acid to synthesize tolibut hydrochloride; the olefin reductase SeER mutant is preferably SeER-W117H / F248A, and the amino acid sequence is shown in SEQ ID NO.4.

[0025] Furthermore, a method for synthesizing tolibut hydrochloride is provided as follows: (R)-3-(4-methylphenyl)-3-cyanopropionic acid methyl ester and NiCl2·6H2O are dissolved in methanol (MeOH), cooled to 0°C, stirred for 15 minutes, and then NaBH4 is added in portions. The final solution is further stirred at 0°C for 15 minutes. The solution turns black and is monitored by TLC to determine whether the raw materials have reacted completely. After the reaction is completed, methanol is removed by vacuum distillation and the concentrated solid is refluxed in 6M HCl solution for 12 hours. After the reaction is completed, water is removed by vacuum distillation and the concentrated solid is recrystallized from isopropanol to obtain the drug tolibut hydrochloride. The mass ratio of the (R)-3-(4-methylphenyl)-3-cyanopropionic acid methyl ester to NiCl2·6H2O is 1:2.5, and the volumetric amount of methanol used is 0.05 mL / mg based on the mass of the (R)-3-cyano-3-phenylpropionic acid methyl ester; the mass ratio of the (R)-3-(4-methylphenyl)-3-cyanopropionic acid methyl ester to NaBH4 is 1:1.8-2; and the volumetric amount of the HCl solution used is 0.1 mL / mg based on the mass of the (R)-3-(4-methylphenyl)-3-cyanopropionic acid methyl ester.

[0026] Furthermore, the substrate is added to a final concentration of 10 mM, the cosolvent is added to a final concentration of 10% by volume, and the substrate is first dissolved in the cosolvent and then added; the glucose is 30 mM; the coenzyme is added to a final concentration of 1 mg / mL; the catalyst is added to a final concentration of 20 g / L based on the weight of the wet bacteria; and the buffer is 100 mM, pH 6.0 potassium phosphate buffer.

[0027] In a sixth aspect, the present invention provides an application of the olefin reductase SeER mutant in catalyzing the stereoreduction of (Z)-3-cyano-5-methylhex-2-enoic acid ethyl ester to synthesize pregabalin, wherein the application uses wet bacteria or purified enzyme obtained by fermentation culture of an engineered bacterium containing an olefin reductase SeER mutant encoding gene as a catalyst, purified enzyme lyophilized powder obtained by fermentation culture of an engineered bacterium containing a glucose dehydrogenase encoding gene as a coenzyme, (Z)-3-cyano-5-methylhex-2-enoic acid ethyl ester as a substrate, and dimethyl sulfoxide (DMSO) as a catalyst. A cosolvent is used, and a pH 5-9 buffer solution is used as a reaction medium. The reaction is carried out at 25-40° C. (preferably 30° C.) and 1000 rpm. After the reaction is completed, the reaction solution is separated and purified to obtain (S)-3-cyano-5-methylhexanoic acid ethyl ester; (S)-3-cyano-5-methylhexanoic acid ethyl ester is reduced with sodium borohydride and hydrolyzed with hydrochloric acid to synthesize pregabalin hydrochloride; the olefin reductase SeER mutant is preferably SeER-W117G / T38W, SeER-W117G / Y373A, or SeER-W117A / T38W, and the amino acid sequences are shown in SEQ ID NO.10, SEQ ID NO.12, and SEQ ID NO.14.

[0028] Furthermore, a method for synthesizing pregabalin hydrochloride is provided as follows: (S)-3-cyano-5-methylhexanoic acid ethyl ester and NiCl2·6H2O are dissolved in methanol, cooled to 0°C, and stirred for 15 minutes. NaBH4 is then added in portions, and the final solution is further stirred at 0°C for 15 minutes. The solution turns black, and TLC is used to monitor whether the raw materials have reacted completely. After the reaction is completed, methanol is removed by vacuum distillation, and the concentrated solid is refluxed in 6M HCl solution for 12 hours. After the reaction is completed, water is removed by vacuum distillation, and the concentrated solid is recrystallized from isopropanol to obtain the drug pregabalin hydrochloride. The mass ratio of the (S)-3-cyano-5-methylhexanoate to NiCl2·6H2O is 1:2.5, and the volumetric amount of methanol used is 0.05 mL / mg based on the mass of the (R)-3-cyano-3-phenylpropionic acid methyl ester; the mass ratio of the (S)-3-cyano-5-methylhexanoate to NaBH4 is 1:1.8-2; and the volumetric amount of the HCl solution used is 0.1 mL / mg based on the mass of the (S)-3-cyano-5-methylhexanoate.

[0029] Furthermore, the substrate is added to a final concentration of 10 mM, the cosolvent is added to a final concentration of 10% by volume, and the substrate is first dissolved in the cosolvent and then added; the glucose is added to a final concentration of 30 mM; the coenzyme is added to a final concentration of 1 mg / mL; the catalyst is added to a final concentration of 20 g / L based on the weight of the wet cells; and the buffer is 100 mM, pH 6.0 potassium phosphate buffer.

[0030] The base sequences of the olefin reductase SeER and the olefin reductase mutant of the present invention are both 1194 bp in full length. From the first base to the 1194th base, the start codon is ATG and the stop codon is TAA, encoding 397 amino acids.

[0031] The olefin reductase SeER mutants described in the present invention were obtained through protein engineering, ultimately yielding eight beneficial mutants: T38W, T38S, W117H, W117G, W117A, F248A, L293H, and Y373A. Further iterative combination mutations yielded a pair of stereocomplementary olefin reductase SeER mutants (W117H / F248A, W117G / T38W). The resulting mutant recombinant plasmids were heat-shocked into E. coli BL21 (DE3) competent cells, and the resulting strains were inoculated, transferred, induced, and recovered. The culture medium for inoculation, transfer, induction, and cell recovery of the olefin reductase mutants of the present invention can be any culture medium known in the art that can grow the cells of the present invention, preferably LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, dissolved in water. There is no particular limitation on the culture method and culture conditions, which can be appropriately selected based on factors such as the host type and culture method, and according to common knowledge in the art.

[0032] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0033] (1) The activity and stereoselectivity of the olefin reductase SeER mutant were significantly improved.

[0034] The present invention provides a series of novel olefin reductase SeER mutants capable of efficiently stereocomplementarily reducing olefin compounds to prepare chiral compounds; when the substrate is (Z)-3-cyano-3-phenylacrylate, the mutant SeER-W117H / F248A has a 160-fold increase in activity compared to the wild-type WT-SeER, and the R-stereoselectivity is improved from good (ee P value 95%) to strict (ee P value 99%); the mutant strain SeER-W117G / T38W has strict S-selectivity, and the enzyme activity is 75 times higher than that of wild-type WT-SeER; the mutant strains (SeER-W117H / F248A, SeER-W117G / T38W) also show high activity and excellent stereocomplementary selectivity towards other non-natural test olefin substrates.

[0035] (2) The conversion rate of olefin reductase SeER mutants in synthesizing chiral GABA derivatives was improved.

[0036] The olefin reductase SeER mutant screened by the present invention successfully synthesized chiral GABA derivative drugs such as fenibut, baclofen, tolibut, pregabalin, etc. through a chemical-enzyme coupling method. Compared with existing methods, the strain described in the present invention can flexibly regulate the configuration of the product and has high selectivity and conversion rate. (IV) Description of the accompanying drawings

[0037] Picture 1 This is a schematic diagram of the reaction formula for the asymmetric stereocomplementary reduction of (Z)-3-cyano-3-phenylacrylate to prepare (R)-3-cyano-3-phenylpropionic acid methyl ester and (S)-3-cyano-3-phenylpropionic acid methyl ester by coupling the olefin reductase SeER mutant with glucose dehydrogenase EsGDH.

[0038] Picture 2 HPLC chromatograms showing the product detection of (R)-3-cyano-3-phenylpropionic acid methyl ester and (S)-3-cyano-3-phenylpropionic acid methyl ester prepared by asymmetric stereocomplementary reduction of (Z)-3-cyano-3-phenylacrylate by SeER mutants; a represents the liquid phase diagram of the racemic form of 3-cyano-3-phenylpropionic acid methyl ester, b represents the liquid phase diagram of R-chiral 3-cyano-3-phenylpropionic acid methyl ester, and c represents the liquid phase diagram of S-chiral 3-cyano-3-phenylpropionic acid methyl ester.

[0039] Picture 3 The effect of temperature on the specific activity of SeER-W117H / F248A.

[0040] Picture 4 The effect of temperature on the specific activity of SeER-W117G / T38W.

[0041] Picture 5 The effect of cosolvent on the specific activity of SeER-W117H / F248A and SeER-W117G / T38W.

[0042] Picture 6 The catalytic efficiency of SeER-W117H / F248A and SeER-W117G / T38W for different olefin substrates.

[0043] Picture 7 This is the hydrogen spectrum of the reaction solution for synthesizing phenibut hydrochloride.

[0044] Picture 8 This is the hydrogen spectrum of the reaction solution for the synthesis of baclofen hydrochloride.

[0045] Picture 9 This is the hydrogen spectrum of the reaction solution for synthesizing Tolibut hydrochloride.

[0046] Picture 10 This is the hydrogen spectrum of the reaction solution for synthesizing pregabalin. (V) Specific implementation methods

[0047] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0048] The LB liquid medium used in the present invention is: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, dissolved in water. The LB solid medium is LB liquid medium with 18 g / L agar added.

[0049] Example 1: Preparation of SeER purified enzyme and enzyme activity determination

[0050] 1. SeER enzyme purification

[0051] (1) Wild-type olefin reductase genetically engineered bacteria: The olefin reductase gene sequence from Saccharomyces eubayanus in GenBank (GenBank No. XP_018218866.1), namely the olefin reductase SeER gene (nucleotide sequence as shown in SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.2), was artificially synthesized and ligated between the NdeI and XhoI restriction sites of the vector pET21a(+) to construct a recombinant expression vector, which was then transferred into the host bacteria E. coli BL21(DE3) to obtain the wild-type olefin reductase genetically engineered bacteria E. coli BL21(DE3) / pET21a(+)-seer.

[0052] (2) Wet bacteria

[0053] The wild-type olefin reductase genetically engineered bacteria E. coli BL21 (DE3) / pET21a (+) -seer constructed in step 1 was inoculated into LB liquid medium containing a final concentration of 100 μg / mL ampicillin, and cultured at 37 ° C, 200 rpm for 10 h to obtain seed liquid; the seed liquid was inoculated into fresh LB liquid medium containing a final concentration of 100 μg / mL ampicillin at a volume concentration of 1% (v / v), and cultured at 37 ° C, 200 rpm until the OD 600 =0.6-0.8, and then add IPTG to the culture solution with a final concentration of 0.15mM. After culturing at 18°C ​​for 18h, centrifuge at 4°C and 8000rpm for 10min to collect the wet cells of the engineered bacteria.

[0054] (3) Enzyme purification

[0055] The wet bacterial cells from step 2 were dispersed in a pH 6.0, 100 mM potassium phosphate buffer at a rate of 100 g wet bacterial cells / L buffer and resuspended. The cells were ultrasonically disrupted on an ice-water mixture for 25 min. The ultrasonic disruption conditions were: power 400 W, disruption for 2 s, and pause for 3 s. The ultrasonic disruption mixture was the crude enzyme solution.

[0056] To the obtained crude enzyme solution, 1 mg / g of wet bacterial cells of riboflavin sodium phosphate (FMN) was added, and the solution was allowed to stand on ice for 30 min. The solution was then centrifuged at 8000 rpm and 4°C for 20 min. The supernatant was collected and filtered through a 0.45 μm filter membrane. The permeate was then purified using a nickel affinity column to purify the mutant enzyme.

[0057] The enzyme purification operation is as follows: ① Load the sample at a flow rate of 1.0 mL / min, and use a gravity system to remove unbound impurities with a pH 6.0, 20 mM potassium phosphate buffer containing 0.5 M NaCl and 20 mM imidazole at a flow rate of 1.0 mL / min until the conductivity is stable; ② Then use a pH 6.0, 20 mM potassium phosphate buffer containing 0.5 M NaCl and 500 mM imidazole to gravity elute for one column volume; all the collected eluate (one column volume, 10 mL) is poured into a 30 kDa ultrafiltration tube, 20 mM pH 6.0 potassium phosphate is used as the replacement buffer to remove imidazole, and the retained protein is collected, which is the SeER purified enzyme, recorded as WT-SeER.

[0058] (4) Enzyme activity determination

[0059] One unit of enzyme activity (U) is defined as the amount of enzyme required to consume 1 micromole of NADPH or produce 1 micromole of product per minute under optimal conditions. Specific activity is defined as the number of international units of enzyme activity per milligram of enzyme protein, U / mg.

[0060] Enzyme activity assay conditions: 1 mM NADPH, appropriate amount of purified enzyme (enzyme protein content 200 mg), 1 mM substrate (Z)-3-cyano-3-phenylacrylate), 5% DMSO (volume concentration). The substrate was first dissolved in DMSO before addition. The final volume was made up to 200 μL with 100 mM potassium phosphate buffer, pH 6.0. The reaction was incubated at 30°C, 1000 rpm for 2 min. NADPH consumption was measured using a Biotek Epoch microplate reader.

[0061] The substrate conversion rate, product ee value and yield were detected by Agilent 1260 high performance liquid chromatograph: chiral column (OD-H, 5 μm, 4.6 mm × 250 mm), flow rate 1 mL / min, mobile phase of n-hexane / isopropanol = 95 / 5 (v / v), column temperature at 30 °C.

[0062] 2. EsGDH enzyme purification

[0063] Using the method of step 1, the glucose dehydrogenase gene (GenBank No. KM817194.1) from Exiguobacterium sibirium DSM 17290, listed in GenBank, was synthesized. The nucleotide sequence is shown in SEQ ID NO. 15. Wet EsGDH cells and purified EsGDH enzyme were prepared using the same conditions (except that riboflavin sodium phosphate was not added to the crude EsGDH enzyme solution and the molecular weight cutoff of the ultrafiltration tube was 10 kDa). The purified EsGDH enzyme was lyophilized at -80°C to produce a lyophilized powder for subsequent experiments.

[0064] Enzyme activity assay conditions: 1 mM NADP + , 0.01 mg protein content of EsGDH purified enzyme, 1 mM glucose, pH 6.0, 100 mM potassium phosphate buffer to a final volume of 200 μL, react at 30°C for 2 min, and use Biotek Epoch microplate reader to detect the amount of NADPH generated.

[0065] Enzyme activity definition: Under optimal conditions, the amount of enzyme required to generate 1 micromole of NADPH or consume 1 micromole of glucose per minute is defined as 1 enzyme activity unit.

[0066] Example 2: Selection of WT-SeER mutant sites and construction of single-site mutants

[0067] 1. Selection of WT-SeER mutant sites

[0068] The substrate (Z)-3-cyano-3-phenylacrylate was docked into the active pocket of WT-SeER using the docking software YASARA. Amino acid residues within the range are most likely to affect the selectivity and activity of the enzymatic reaction. After excluding the conserved catalytic triad amino acids (H189, N192, Y194), T38, W117, F248, L293, and Y373 are selected as mutation sites. At the same time, since W117 has a larger side chain group and is most likely to affect the reaction between the enzyme and the substrate, it is preferentially subjected to saturation mutation.

[0069] 2. Site-directed saturation mutagenesis of W117

[0070] (1) Mutation at site 117: The plasmid pET21a(+)-seer of the wild-type olefin reductase genetically engineered bacteria E. coli BL21(DE3) / pET21a(+)-seer of Example 1 was extracted, and PCR amplification was performed using the primers in Table 1 to mutate the tryptophan at site 117 into 19 other amino acids. After enzyme digestion to remove the original template and Clean up, the PCR product was transformed into E. coli BL21(DE3) competent cells, and the clones were inoculated into LB solid medium containing 100 μg / mL ampicillin and incubated at 37°C for 12 h. Then the clones were picked and transferred into 10 mL of LB liquid medium containing 100 μg / mL ampicillin, and incubated at 37°C, 200 rpm for 10 h. The culture was centrifuged at 8000 rpm for 10 min, and the wet bacteria were collected.

[0071] Table 1. Design of primers for site-directed saturation mutation at site 117 of olefin reductase

[0072]

[0073] Note: a W117TY represents the upstream universal primer for mutation at site 117.

[0074] The PCR reaction system (25 μL) was composed of 0.5 μL of forward primer (100 μM), 0.5 μL of reverse primer (100 μM), 12.5 μL of 2x Phanta buffer, 0.5 μL of dNTP mixture (10 mM each), 0.5 μL of plasmid template, 0.5 μL of DNA polymerase and 10 μL of ultrapure water. According to the operation manual of Phanta Super-Fidelity DNA polymerase, the PCR program was set as follows: 95°C pre-denaturation for 5 min, then 30 cycles of 95°C denaturation for 15 s, 55-65°C annealing for 15 s, 72°C extension for 10 s, 72°C final extension for 10 min, and 16°C incubation.

[0075] (2) Screening of mutants

[0076] Mutant screening reaction system: 50 g / L of each wet cell prepared in step 1, 5 g / L (Z)-3-cyano-3-phenylacrylate, 15 g / L glucose, and 5 g / L of EsGDH wet cells prepared according to the method of Example 1. The reaction medium was 100 mM potassium phosphate buffer at pH 6.0, forming a 1 mL reaction system. After reacting at 30°C and 1000 rpm for 24 h, the reaction solution was extracted with ethyl acetate. The organic phase was concentrated and dried, and the sample was dissolved in chromatography-grade isopropanol. The product and substrate concentrations were determined using liquid chromatography according to Example 1. The substrate conversion rate, enzyme specific activity, and stereoselectivity were calculated. The results are shown in Table 2. Three replicates were set for each mutant characterization. The dominant mutant strains screened were E. coli BL21(DE3) / pET21a(+)-seer-W117H, E. coli BL21(DE3) / pET21a(+)-seer-W117G, and E. coli BL21(DE3) / pET21a(+)-seer-W117A. Wet cells and purified enzyme solutions of each mutant were prepared using the method of Example 1.

[0077] Table 2 Catalytic performance of beneficial mutants

[0078]

[0079] 3. Mutation at F248

[0080] The plasmid of the engineered bacteria E. coli BL21(DE3) / pET21a(+)-seer of Example 1 was used as a template, and PCR amplification was performed using the primers in Table 3, the method and conditions in step 2, and the conversion rate and stereoselectivity of each mutant wet bacteria for the substrate were tested. The results showed that SeER-F248A had a substrate conversion rate of 99% and an R-isomer ee value of 99%.

[0081] Table 3. Primer design for SeER-F248 site mutation

[0082]

[0083] Note: In Table 3, NNK represents a degenerate codon, and MNN represents a degenerate codon paired with NNK.

[0084] 4. Mutation at T38

[0085] The plasmid of the engineered bacterium E. coli BL21(DE3) / pET21a(+)-seer from Example 1 was used as a template. PCR amplification was performed using the primers in Table 4, the method and conditions in step 2, and the conversion efficiency and stereoselectivity of each mutant wet cell were tested. The screening results showed that SeER-T38S had a substrate conversion efficiency of 99% and an R-isomer ee value of 99%. SeER-T38W had a substrate conversion efficiency of 99% and an S-isomer ee value of 99%.

[0086] Table 4. Primer design for SeER-T38 site mutation

[0087]

[0088] Note: In Table 4, NNK represents a degenerate codon, and MNN represents a degenerate codon paired with NNK.

[0089] 5. Mutation of L293

[0090] The plasmid of the engineered bacteria E. coli BL21(DE3) / pET21a(+)-seer of Example 1 was used as a template, and PCR amplification was performed using the primers in Table 5, the method and conditions in step 2, and the conversion rate and stereoselectivity of each mutant wet bacteria for the substrate were tested. The results showed that SeER-L293H had a substrate conversion rate of 99% and an R-isomer ee value of 99%.

[0091] Table 5. Primer design for SeER-L293 site mutation

[0092]

[0093]

[0094] Note: In Table 5, NNK represents a degenerate codon, and MNN represents a degenerate codon paired with NNK.

[0095] 6. Mutation at Y373

[0096] The plasmid of the engineered bacteria E. coli BL21 (DE3) / pET21a (+) -seer of Example 1 was used as a template, and PCR amplification was performed using the primers in Table 6, the method and conditions in step 2, and the conversion rate and stereoselectivity of each mutant wet bacteria for the substrate were tested. The results showed that SeER-Y373A had a substrate conversion rate of 99% and an S-isomer ee value of 99%.

[0097] Table 6 SeER-Y373 site mutation primer design

[0098]

[0099] Note: In Table 6, NNK represents a degenerate codon, and MNN represents a degenerate codon paired with NNK.

[0100] Example 3: Construction of combined mutants

[0101] The plasmid of the engineering bacteria E. coli BL21 (DE3) / pET21a (+) -seer-W117H of Example 2 was extracted as a template, and PCR amplification was performed using the primers in Table 7 and the method and conditions of Example 2 to construct the engineering bacteria E. coli BL21 (DE3) / pET21a (+) -seer-W117H / F248A, E. coli BL21 (DE3) / pET21a (+) -seer-W117H / T38S, E. coli BL21 (DE3) / pET21a (+) -seer-W117H / L293H, E. coli BL21 (DE3) / pET21a (+) -seer-W117G / T38W, E. coli BL21(DE3) / pET21a(+)-seer-W117G / Y373A, E. coli BL21(DE3) / pET21a(+)-seer-W117A / T38W. Store at -80°C. Wet cells and purified enzymes of each mutant were prepared using the method described in Example 1. The amount of purified enzyme added was calculated based on protein content.

[0102] The substrate conversion rate and stereoselectivity were tested using the method and conditions of Example 2. The substrate conversion rates of SeER-W117H / F248A mutant, SeER-W117H / F248A mutant, and SeER-W117H / L293H mutant were all 99%, and the ee values ​​of the R-isomer were all 99%.

[0103] The substrate conversion rates of SeER-W117G / T38W mutant, SeER-W117G / Y373A mutant, and SeER-W117A / T38W mutant were all 99%, and the S-stereoselectivity was all 99%.

[0104] Table 7 Combination mutation primers

[0105]

[0106] Example 4: Optimal reaction pH, temperature, and catalytic reaction aids for SeER-W117H / F248A and SeER-W117G / T38W

[0107] 1. Optimal reaction pH

[0108] The optimal reaction pH of the mutants was screened using acetate buffer at pH 5.0-5.5, phosphate buffer at pH 5.5-7.5, and Tris-hydrochloric acid buffer at pH 7.5-9.0.

[0109] Reaction system: 1 mL of 100 mM buffer at different pH values ​​(pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 9.0) was added with 0.02 mg / mL SeER-W117H / F248A or SeER-W117G / T38W purified enzyme, 10 mM substrate ((Z)-3-cyano-3-phenylacrylate) (dissolved in DMSO before addition), 5% DMSO, 30 mM glucose, and 1 mg / mL lyophilized EsGDH purified enzyme. The reaction was carried out at 30°C and 1000 rpm for 24 h. The product and substrate concentrations were determined according to the method in Example 1, and the conversion and product ee values ​​were calculated. As shown in Table 8, the optimal reaction pH for both SeER-W117H / F248A and SeER-W117G / T38W was 6.0.

[0110] Table 8 Optimal reaction pH

[0111]

[0112] 2. Optimum reaction temperature

[0113] To 200 μL of pH 6.0, 100 mM potassium phosphate buffer, 0.02 mg / mL protein concentration of SeER-W117H / F248A or SeER-W117G / T38W purified enzyme, 1 mM substrate (Z)-3-cyano-3-phenylacrylate (the substrate was first dissolved in DMSO and then added), a final volume concentration of 5% DMSO, 1 mM NADPH, and the reaction was carried out at 20-45°C (20°C, 25°C, 30°C, 35°C, 40°C, 45°C) and 1000 rpm for 3 min. The enzyme activity at different temperatures was detected according to the method of Example 1. The detection results are shown in FIG. Picture 3 、 Picture 4 The optimal reaction temperature for SeER-W117H / F248A and SeER-W117G / T38W was 30°C.

[0114] 3. Reaction cosolvent

[0115] To 200 μL of pH 6.0, 100 mM potassium phosphate buffer, 0.02 mg / mL protein concentration of SeER-W117H / F248A or SeER-W117G / T38W purified enzyme, 1 mM NADPH, 1 mM substrate (Z)-3-cyano-3-phenylacrylate, and 5% volume final concentration of different organic solvents (DMSO, DMF, CH3CN, THF, i-PrOH, MeOH) were added. The substrates were first dissolved in the organic solvent and then added to the reaction system. The reaction was carried out at 30°C and 1000 rpm for 3 min. The enzyme activity in the presence of the cosolvent was detected according to the method in Example 1. The results are as follows: Picture 5 As shown in the figure, the best reaction co-solvent for SeER-W117H / F248A and SeER-W117G / T38W was DMSO.

[0116] Example 5: Determination of enzyme catalytic reaction kinetic parameters of SeER and its mutants

[0117] Kinetic parameters were determined using purified enzymes of WT-SeER and its mutants SeER-W117G, SeER-W117G / T38W, SeER-W117H, and SeER-W117H / F248A as catalysts. Specifically:

[0118] Add 1 mM NADP to 500 μL of potassium phosphate (100 mM, pH 6.0) buffer. + , 1 mg / mL EsGDH purified enzyme lyophilized powder, 30 mM glucose, an appropriate amount of purified enzyme (in terms of protein content, WT-SeER content is 0.5 mg, SeER-W117H and SeER-W117G content is 0.05 mg, SeER-W117H / F248A and SeER-W117G / T3W content is 0.02 mg), the concentration of substrate (Z)-3-cyano-3-phenylacrylate is in the range of 0-6 mM (0 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.8 mM, 1 mM, 1.5 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM), the substrate is first dissolved in DMSO and then added, and the final volume concentration of DMSO is 5%. The reaction was carried out at 30°C and 1000 rpm for 3-60 min (the reaction time for the mutant was 3 min and the reaction time for WT-SeER was 60 min). The conversion rate of the substrate (Z)-3-cyano-3-phenylacrylate or the yield of the product 3-cyano-3-phenylpropionic acid methyl ester was determined using the method of Example 1. The Michaelis-Menten equation in Origin 2024b software was used for fitting to calculate the Michaelis-Menten constant K of SeER and its mutants for the substrate. M , turnover number k cat and catalytic efficiency k cat / K M.

[0119] The results are shown in Table 9. Compared with the wild type, the turnover number and catalytic efficiency of the four mutants were significantly increased. Se ER-W117H / F248A and Se The catalytic efficiency of ER-W117G / T38W was significantly higher than that of wild-type WT- Se ER increased by 359 times.

[0120] Table 9 Kinetic parameters of wild-type SeER and mutants

[0121]

[0122]

[0123] Example 6: Wild type Se ER and its mutants ( Se ER-W117H / F248A, Se ER-W117G / T38W) substrate spectrum characterization

[0124] In 200 μL of pH 6.0, 100 mM potassium phosphate buffer, WT- Se ER, Se ER-W117H / F248A or Se ER-W117G / T38W purified enzyme (based on protein content, WT- Se The amount of ER feed was 0.5 mg, and the mutant Se ER-W117H / F248A and Se ER-W117G / T38W was added (0.02 mg of feed), 1 mM NADPH, 1 mM substrate, and a final volume concentration of 5% DMSO. The substrate was dissolved in water and then added to the reaction system. The reaction was incubated at 30°C and 1000 rpm for 3 min. Enzyme activity was determined according to the method in Example 1. Three replicates were performed for each substrate.

[0125] The results are shown in Table 10. The catalytic activity of the mutant enzyme for substrates in the substrate spectrum is significantly higher than that of WT-SeER.

[0126] Table 10 Wild type Se ER and its mutants ( Se ER-W117H / F248A, Se ER-W117G / T38W) substrate spectrum

[0127]

[0128] Note: ND stands for no viability detected.

[0129] Example 7: Se ER-W117H / F248A and Se Catalytic Performance of ER-W117G / T38W for a Series of Olefin Compounds

[0130] In 200 μL of pH 6.0, 100 mM potassium phosphate buffer, add Se ER-W117H / F248A or Se ER-W117G / T38W purified enzyme (based on protein content, mutant Se ER-W117H / F248A and Se ER-W117G / T38W feed amount is 0.02 mg), 1 mM NADPH, 1 mM substrate ( Picture 6 ), the final volume concentration was 5% DMSO, the substrate was first dissolved in DMSO and then added to the reaction system, and the reaction was carried out at 30°C and 1000 rpm for 3 min. The substrate conversion rate and stereoselectivity were tested according to the method of Example 1. The results are shown in Picture 6 Mutants Se ER-W117H / F248A exhibited good catalytic activity and excellent R -Selective. Se ER-W117G / T38W also showed good catalytic activity for the tested substrates; except for (Z)-3-cyano-3-(3,5-dichlorophenyl)acrylate and (Z)-3-cyano-3-(3,5-difluorophenyl)acrylate, Se ER-W117G / T38W showed excellent S -Selective.

[0131] Example 8: SeER-W117H / F248A catalyzes stereoreduction of olefin substrates

[0132] 1. Reduction of (Z)-3-cyano-3-phenylacrylate to synthesize phenibut

[0133] 10 mM (Z)-3-cyano-3-phenylpropionic acid methyl ester (dissolved in DMSO before adding) was added to 50 mL potassium phosphate buffer (100 mM, pH 6.0), 10% DMSO, 30 mM glucose, 1 mg / mL EsGDH purified enzyme lyophilized powder, 20 g / L SeER-W117H / F248A wet cells, and pH was controlled using 6 M KOH. The reaction was carried out at 30 ° C and 1000 rpm for 24 h. After the reaction was completed, the mixture was extracted with ethyl acetate and concentrated to dryness to obtain 90 mg of optically pure (99% ee) product (R)-3-cyano-3-phenylpropionic acid methyl ester ( Picture 7 ).

[0134] (R)-3-cyano-3-phenylpropionic acid methyl ester 90 mg and NiCl2-6H2O (2 equiv, 226.1 mg) were dissolved in methanol (MeOH) (5 mL) and cooled to 0 °C, stirred for 15 min, then NaBH4(10 equiv, 180 mg) was added portionwise, the final solution was continued to stir at 0 °C for 15 min, the solution turned black, the reaction was monitored by TLC until the starting material was consumed, after the reaction was completed, the methanol was removed by distillation under reduced pressure, the concentrated solid was refluxed in 10 mL 6M HCl solution for 12 h, after the reaction was completed, the water was removed by distillation under reduced pressure, the concentrated solid was recrystallized with isopropanol to give 85 mg of the drug phenibut hydrochloride.

[0135] 2. Reduction of (Z)-3-(4-chlorophenyl)-3-cyanocrotonic acid methyl ester to synthesize baclofen

[0136] Using the conditions and procedures of Step 1, the substrate was changed to (Z)-3-(4-chlorophenyl)-3-cyanocrotonic acid methyl ester, and the other conditions and operations were the same, to give 95 mg of optically pure product (99% ee) (R)-3-(4-chlorophenyl)-3-cyanopropionic acid methyl ester ( Picture 8 ).

[0137] Using the conditions and procedures of Step 1, (R)-3-cyano-3-phenylpropionic acid methyl ester was changed to (R)-3-(4-chlorophenyl)-3-cyanopropionic acid methyl ester, and the drug baclofen hydrochloride 90 mg was obtained.

[0138] 3. Reduction of (Z)-3-(4-methylphenyl)-3-cyanocrotonic acid methyl ester to synthesize tolperisone

[0139] Using the conditions and procedures of Step 1, the substrate was changed to (Z)-3-(4-methylphenyl)-3-cyanocrotonic acid methyl ester, and the other conditions and operations were the same, to give 87 mg of optically pure product (99% ee) (R)-3-(4-methylphenyl)-3-cyanopropionic acid methyl ester ( Picture 9 ).

[0140] Using the conditions and procedures of Step 1, (R)-3-cyano-3-phenylpropionic acid methyl ester was changed to (R)-3-(4-methylphenyl)-3-cyanopropionic acid methyl ester, and the drug tolperisone hydrochloride 80 mg was obtained.

[0141] Example 9: Different mutants catalyze the stereoselective reduction of (Z)-3-(4-chlorophenyl)-3-cyanocrotonic acid methyl ester to synthesize baclofen

[0142] 1. SeER-W117H / T38S

[0143] The mutant in step 2 of Example 8 was changed to SeER-W117H / T38S. Other conditions and operations were the same to obtain 81 mg of optical product (98% ee). After sodium borohydride reduction and hydrochloric acid hydrolysis, 76 mg of baclofen hydrochloride was synthesized.

[0144] 2. SeER-W117H / L293H

[0145] The mutant in step 2 of Example 8 was changed to SeER-W117H / L293H. Other conditions and operations were the same to obtain 77 mg of optical product (97% ee). After sodium borohydride reduction and hydrochloric acid hydrolysis, 70 mg of baclofen hydrochloride was synthesized.

[0146] Example 10: Synthesis of Pregabalin by Stereochemical Reduction of (Z)-3-Cyano-5-methylhex-2-enoic Acid Ethyl Ester Catalyzed by Different Mutants

[0147] 1. SeER-W117G / T38W

[0148] The mutant in step 1 of Example 8 was changed to SeER-W117G / T38W, and the substrate was changed to (Z)-3-cyano-5-methylhex-2-enoic acid ethyl ester. Other conditions and operations were the same to obtain 80 mg of optical product (99% ee, S). After sodium borohydride reduction and hydrochloric acid hydrolysis, 75 mg of pregabalin hydrochloride was synthesized.

[0149] 2. SeER-W117G / Y373A

[0150] The mutant in step 1 of Example 8 was changed to SeER-W117G / Y373A, and the substrate was changed to (Z)-3-cyano-5-methylhex-2-enoic acid ethyl ester. Other conditions and operations were the same to obtain 78 mg of optical product (99% ee, S). After sodium borohydride reduction and hydrochloric acid hydrolysis, 70 mg of pregabalin hydrochloride was synthesized.

[0151] 3. SeER-W117A / T38W

[0152] The mutant in step 1 of Example 8 was changed to SeER-W117G / T38W, and the substrate was changed to (Z)-3-cyano-5-methylhex-2-enoic acid ethyl ester. Other conditions and operations were the same to obtain 80 mg of optical product (99% ee, S). After sodium borohydride reduction and hydrochloric acid hydrolysis, 75 mg of pregabalin hydrochloride was synthesized.

Claims

1. An olefin reductase Se ER mutants, characterized in that The olefin reductase Se The amino acid sequence of the ER mutant is shown as one of SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, and SEQ ID NO.

14.

2. A mutant of the olefin reductase according to claim 1 for synthesizing olefin compounds by stereocomplementary reduction R -Application in configurational chiral compounds, characterized in that, The application method comprises the following steps: using wet bacteria or purified enzyme obtained by inducing and culturing recombinant genetically engineered bacteria of an olefin reductase mutant as a catalyst, using wet bacteria or purified enzyme freeze-dried powder obtained by inducing and culturing glucose dehydrogenase genetically engineered bacteria as a coenzyme, using a prochiral olefin compound as a substrate, using glucose as a cosubstrate, using dimethyl sulfoxide as a cosolvent, and using a buffer solution of pH 5 to 9 as a reaction medium to form a reaction system, carrying out the reaction at 25 to 40° C. and 1000 rpm, and separating and purifying the reaction solution after the reaction is completed to obtain R -Configurational chiral product; The olefin compound is one of the following: (Z)-3-cyano-3-phenyl methyl acrylate, (Z)-3-cyano-3-phenyl ethyl acrylate, (Z)-3-cyano-3-phenyl isopropyl acrylate, (Z)-3-cyano-3-phenyl tert-butyl acrylate, (Z)-3-cyano-3-(2-fluorophenyl) methyl acrylate, (Z)-3-cyano-3-(3-chlorophenyl) methyl acrylate, (Z)-3-cyano-3-(m-tolyl) methyl acrylate, (Z)-3-cyano-3-(4-methoxyphenyl) methyl acrylate, (Z)-3-cyano-3-(4-methylphenyl) methyl acrylate, (Z)-3-cyano-3-(4-fluorophenyl) methyl acrylate, (Z)-3-cyano-3-(4-chlorophenyl) methyl acrylate, (Z)-3-cyano-3-(4-bromophenyl) methyl acrylate Methyl (Z)-3-cyano-3-(4-trifluoromethylphenyl)acrylate, Methyl (Z)-3-([1,1'-biphenyl]-4-yl)-3-cyanoacrylate, Methyl (Z)-3-cyano-3-(naphthalen-2-yl)acrylate, Methyl (Z)-3-(3-bromo-4-methoxyphenyl)-3-cyanoacrylate, Methyl (Z)-3-cyano-3-(3,4- Methyl (Z)-3-cyano-3-(3,4-difluorophenyl)acrylate, methyl (Z)-3-cyano-3-(3,5-dichlorophenyl)acrylate, methyl (Z)-3-cyano-3-(3,5-difluorophenyl)acrylate, methyl (E)-3-cyano-3-(thien-2-yl)acrylate, ethyl (Z)-3-cyano-5-methylhex-2-enoate; The olefin reductase Se The amino acid sequence of the ER mutant is shown in SEQ ID NO.

4.

3. A mutant of the olefin reductase according to claim 1 for synthesizing olefin compounds by stereocomplementary reduction S -Application in configurational chiral compounds, characterized in that, The application method comprises the following steps: using wet bacteria or purified enzyme obtained by inducing and culturing recombinant genetically engineered bacteria of an olefin reductase mutant as a catalyst, using wet bacteria or purified enzyme freeze-dried powder obtained by inducing and culturing glucose dehydrogenase genetically engineered bacteria as a coenzyme, using a prochiral olefin compound as a substrate, using glucose as a cosubstrate, using dimethyl sulfoxide as a cosolvent, and using a buffer solution of pH 5 to 9 as a reaction medium to form a reaction system, carrying out the reaction at 25 to 40° C. and 1000 rpm, and separating and purifying the reaction solution after the reaction is completed to obtain S -Configurational chiral product; The olefin compound is one of the following: (Z)-3-cyano-3-phenyl methyl acrylate, (Z)-3-cyano-3-phenyl ethyl acrylate, (Z)-3-cyano-3-phenyl isopropyl acrylate, (Z)-3-cyano-3-phenyl tert-butyl acrylate, (Z)-3-cyano-3-(2-fluorophenyl) methyl acrylate, (Z)-3-cyano-3-(3-chlorophenyl) methyl acrylate, (Z)-3-cyano-3-(m-tolyl) methyl acrylate, (Z)-3-cyano-3-(4-methoxyphenyl) methyl acrylate, (Z)-3-cyano-3-(4-methylphenyl) methyl acrylate, (Z)-3-cyano-3-(4-fluorophenyl) methyl acrylate Acrylic acid methyl ester, (Z)-3-cyano-3-(4-chlorophenyl) acrylate, (Z)-3-cyano-3-(4-bromophenyl) acrylate, (Z)-3-cyano-3-(4-trifluoromethylphenyl) acrylate, (Z)-3-cyano-3-(naphthalen-2-yl) acrylate, (Z)-3-(3-bromo-4-methoxyphenyl)-3-cyanoacrylate, (Z)-3-cyano-3-(3,4-dichlorophenyl) acrylate, (Z)-3-cyano-3-(3,4-difluorophenyl) acrylate, (E)-3-cyano-3-(thiophen-2-yl) acrylate, (Z)-3-cyano-5-methylhex-2-enoic acid ethyl ester; The olefin reductase Se The amino acid sequence of the ER mutant is shown in SEQ ID NO.

10.

4. The use according to claim 2 or 3, characterized in that In the reaction system, the substrate is added to a final concentration of 1-20 mM; the glucose is added to a final concentration of 1-50 mM; the catalyst dosage is 10-30 g / L based on the weight of the wet cells or 0.01-2.5 g / L based on the protein content of the purified enzyme; the coenzyme Es The weight of GDH is 0.5-2 g / L based on the weight of the purified enzyme lyophilized powder; the volume concentration of dimethyl sulfoxide is 5-10%; the nucleotide sequence of the glucose dehydrogenase gene is shown in SEQ ID NO.

15.

5. An olefin reductase according to claim 1 Se The application of ER mutant in catalyzing stereocomplementary reduction of (Z)-3-cyano-3-phenylacrylate to synthesize phenibut is characterized in that: The application is: containing olefin reductase Se The wet bacteria or purified enzyme obtained by fermentation culture of an engineered bacterium encoding an ER mutant gene was used as a catalyst, the purified enzyme freeze-dried powder obtained by fermentation culture of an engineered bacterium containing a glucose dehydrogenase encoding gene was used as a coenzyme, (Z)-3-cyano-3-phenylacrylate was used as a substrate, dimethyl sulfoxide was used as a cosolvent, and a pH 5-9 buffer solution was used as a reaction medium. The reaction was carried out at 25-40°C and 1000 rpm. After the reaction was completed, the reaction solution was separated and purified to obtain ( R )-3-cyano-3-phenylpropionic acid methyl ester; ( R )-3-cyano-3-phenylpropionic acid methyl ester is reduced with sodium borohydride and hydrolyzed with hydrochloric acid to synthesize phenibut hydrochloride; the olefin reductase Se The amino acid sequence of the ER mutant is shown in SEQ ID NO.

4.

6. An olefin reductase according to claim 1 Se The application of an ER mutant in catalyzing the stereocomplementary reduction of (Z)-3-(4-chlorophenyl)-3-cyanoacrylate to synthesize baclofen is characterized in that: The application is based on olefin reductase Se The wet bacteria or purified enzyme obtained by fermentation culture of an engineered bacterium encoding an ER mutant gene was used as a catalyst, the purified enzyme freeze-dried powder obtained by fermentation culture of an engineered bacterium encoding a glucose dehydrogenase gene was used as a coenzyme, (Z)-3-(4-chlorophenyl)-3-cyanoacrylate was used as a substrate, dimethyl sulfoxide was used as a cosolvent, and a pH 5-9 buffer solution was used as a reaction medium. The reaction was carried out at 25-40°C and 1000 rpm. After the reaction was completed, the reaction solution was separated and purified to obtain ( R )-3-(4-chlorophenyl)-3-cyanopropionic acid methyl ester; ( R )-3-(4-chlorophenyl)-3-cyanopropionic acid methyl ester is reduced with sodium borohydride and hydrolyzed with hydrochloric acid to synthesize baclofen hydrochloride; the olefin reductase Se The amino acid sequence of the ER mutant is shown in SEQ ID NO.

4.

7. An olefin reductase according to claim 1 Se The application of ER mutant in catalyzing stereoreduction of (Z)-3-(4-methylphenyl)-3-cyanoacrylate to synthesize tolibut is characterized in that: The application is based on olefin reductase Se The wet bacteria or purified enzyme obtained by fermentation culture of the engineered bacteria containing the gene encoding the ER mutant was used as a catalyst, the purified enzyme freeze-dried powder obtained by fermentation culture of the engineered bacteria containing the gene encoding the glucose dehydrogenase was used as a coenzyme, (Z)-3-(4-methylphenyl)-3-cyanoacrylate was used as a substrate, dimethyl sulfoxide was used as a cosolvent, and a pH 5-9 buffer solution was used as a reaction medium. The reaction was carried out at 25-40°C and 1000 rpm. After the reaction was completed, the reaction solution was separated and purified to obtain ( R )-3-(4-methylphenyl)-3-cyanopropionic acid methyl ester; ( R )-3-(4-methylphenyl)-3-cyanopropionic acid methyl ester is reduced with sodium borohydride and hydrolyzed with hydrochloric acid to synthesize tolibut hydrochloride; the olefin reductase Se The amino acid sequence of the ER mutant is shown in SEQ ID NO.

4.

8. An olefin reductase according to claim 1 Se The application of an ER mutant in catalyzing the stereoreduction of (Z)-3-cyano-5-methylhex-2-enoic acid ethyl ester to synthesize pregabalin is characterized in that: The application is based on olefin reductase Se The wet bacteria or purified enzyme obtained by fermentation culture of an engineered bacterium encoding an ER mutant gene is used as a catalyst, the purified enzyme freeze-dried powder obtained by fermentation culture of an engineered bacterium containing a glucose dehydrogenase encoding gene is used as a coenzyme, (Z)-3-cyano-5-methylhex-2-enoic acid ethyl ester is used as a substrate, dimethyl sulfoxide is used as a cosolvent, and a pH 5-9 buffer solution is used as a reaction medium. The reaction is carried out at 25-40°C and 1000 rpm. After the reaction is completed, the reaction solution is separated and purified to obtain ( S )-ethyl 3-cyano-5-methylhexanoate; ( S )-3-cyano-5-methylhexanoic acid ethyl ester is reduced with sodium borohydride and hydrolyzed with hydrochloric acid to synthesize pregabalin hydrochloride; the olefin reductase Se The amino acid sequence of the ER mutant is shown in SEQ ID NO. 10.

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