Olefin reductase, coding gene, vector, engineering bacterium and application thereof

Abstract

The invention discloses an olefin reductase, a coding gene, a carrier, an engineering bacterium and an application of the olefin reductase. An olefin reductase SeER mutant is obtained by carrying out single-point or multi-site site-directed mutagenesis or site-directed saturation mutagenesis on the 38th site, the 117th site, the 248th site, the 293th site and the 373th site of an amino acid sequence shown in SEQ ID NO.2. The invention provides a series of novel olefin reductase SeER mutants capable of efficiently, stereocomplementarily reducing an olefin compound to prepare a chiral compound, chiral GABA derivative drugs such as phenibuterol, baclofen, tolybuterol, pregabalin and the like are successfully synthesized through a chemical-enzyme coupling method, and compared with an existing method, the novel olefin reductase SeER mutants have the advantages that the novel olefin reductase SeER mutants can be used for efficiently and stereocomplementarily reducing the olefin compound to prepare the chiral compound; the strain disclosed by the invention can flexibly regulate and control the configuration of a product, and is high in stereoselectivity and conversion rate.

Description

(I) Technical field

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

[0002] Olefin reductase (ERs) is a oxidoreductase belonging to the NAD(P)H-dependent oxidoreductase superfamily. It can catalyze the asymmetric reduction of olefins and produce chiral molecules with two possible stereocenters. Due to its mild reaction conditions and high selectivity, olefin reductase has gradually replaced the metal-catalyzed high-pressure hydrogenation method and become an important tool for the construction of stereocenters of olefin substrates. However, natural olefin reductase is limited by its narrow substrate spectrum, and most ERs usually show a single stereoselectivity for substrates, while enantiocomplementary ERs are rare, which limits its application in the synthesis of stereocomplementary chiral molecules. In addition, wild-type olefin reductase often shows low activity and stereoselectivity for non-natural (synthetic) substrates, so it is necessary to implement enzyme molecular modification to enhance its activity and stereocomplementary selectivity for non-natural substrates.

[0003] Chiral γ-amino acid butyric acid (GABA) and its derivatives are a class of molecules with multiple 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)-, (S)-β-aryl-γ-lactams, providing a simple and atom-economical method for synthesizing stereocomplementary chiral GABA derivatives (Adv. Synth. Catal. 2015, 357, 1849–1860). However, the catalytic efficiency of the olefin reductase is low and the stereocomplementary selectivity is poor, which makes it difficult to meet the synthesis requirements of highly optically pure chiral molecules. (III) Summary of the invention

[0004] The present invention aims to provide an olefin reductase SeER mutant, an encoding gene, a vector, an engineering bacterium and an application thereof in stereocomplementary reduction of olefin compounds to synthesize chiral compounds. The present invention screens and obtains mutants capable of catalytic reduction of olefin compounds with high activity (>99% conversion rate) and high stereocomplementary selectivity (ee value 99% R-selectivity and 99% S-selectivity) by molecularly modifying the olefin reductase SeER from S.eubayanus, such as (Z)-3-cyano-3-phenylacrylate and its series of non-natural substrates, and further converts the optically pure product into chiral GABA derivatives such as fenibute, baclofen, tolibut, pregabalin and the like by coupling chemical cyanide reduction and ester hydrolysis, thereby solving the problems of low activity and poor stereoselectivity of existing olefin reductases for asymmetric stereocomplementary reduction of (Z)-3-cyano-3-phenylacrylate and its derivatives.

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

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

[0007] Furthermore, the olefin reductase SeER mutant is preferably one 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, the amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence is 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, the amino acid sequence is shown in SEQ ID NO.6, and the nucleotide sequence is shown in SEQ ID NO. NO.5); (6) the tryptophan at position 117 is mutated to histidine, and the leucine at position 293 is mutated to histidine (W117H / L293H, the amino acid sequence is shown in SEQ ID NO.8, and the nucleotide sequence is shown in SEQ ID NO.7); (7) the tryptophan at position 117 is mutated to glycine, and the threonine at position 38 is mutated to tryptophan (W117G / T38W, the amino acid sequence is shown in SEQ ID NO.10, and the nucleotide sequence is shown in SEQ ID NO.9); (8) the tryptophan at position 117 is mutated to glycine, and the tyrosine at position 373 is mutated to alanine (W117G / Y373A, the amino acid sequence is shown in SEQ ID NO.12, and the nucleotide sequence is shown in SEQ ID NO.11); (9) the tryptophan at position 117 is mutated to alanine, and the threonine at position 38 is mutated to tryptophan (W117A / T38W, the amino acid sequence is shown in SEQ ID NO.14, and the nucleotide sequence is shown in SEQ ID NO.15). 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 application of the olefin reductase mutant in the synthesis of chiral compounds by stereocomplementary reduction of olefin compounds. The application method is: using wet bacteria or purified enzymes obtained by induction culture of recombinant genetic engineering bacteria of olefin reductase mutants as catalysts, wet bacteria or purified enzyme freeze-dried powder obtained by induction culture of glucose dehydrogenase genetic engineering bacteria as coenzymes, prochiral olefin compounds as substrates, glucose as cosubstrate, dimethyl sulfoxide as cosolvent, pH 5-9 buffer (preferably pH 6.0, 100mM potassium phosphate buffer) as reaction medium to form a reaction system, reacting at 25-40°C (preferably 30°C) and 1000rpm, and the reaction is completed, and the reaction solution is separated and purified to obtain a chiral product.

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

[0011] Further, 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), 1f), methyl (Z)-3-cyano-3-(m-tolyl)acrylate (1g), methyl (Z)-3-cyano-3-(4-methoxyphenyl)acrylate (1h), methyl (Z)-3-cyano-3-(4-methylphenyl)acrylate (1i), methyl (Z)-3-cyano-3-(4-fluorophenyl)acrylate (1j), methyl (Z)-3-cyano-3-(4-chlorophenyl)acrylate (1k), methyl (Z)-3-cyano-3-(4-bromophenyl)acrylate (1h), methyl (Z)-3-cyano-3-(4-methylphenyl)acrylate (1i), methyl (Z)-3-cyano-3-(4-fluorophenyl)acrylate (1j), methyl (Z)-3-cyano-3-(4-chlorophenyl)acrylate (1k), methyl (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-dichloro 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-(thiophen-2-yl)acrylate (1u), (Z)-3-cyano-5-methylhex-2-enoic acid ethyl ester (1v).

[0015] Further, in the reaction system, the substrate is added to a final concentration of 1-20 mM (preferably 5-10 mM); the 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 in 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 the volume of dimethyl sulfoxide is added at a concentration of 5-10%. The glucose dehydrogenase gene (GenBank No. KM817194.1) is from Exiguobacterium sibirium DSM17290, the nucleotide sequence is shown in SEQ ID NO.15, and the amino acid sequence is shown in SEQ ID NO.16.

[0016] Further, the wet cell was prepared as follows: the engineered bacteria containing the olefin reductase SeER mutant gene was inoculated into an 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 isopropyl thiogalactoside (IPTG) with a final concentration of 0.15mM into the culture medium, culture at 18°C ​​for 18h, and centrifuge at 4°C and 8000rpm for 10min to obtain wet cells of olefin reductase SeER mutant; the preparation method of wet cells obtained by induction culture of the engineered bacteria containing glucose dehydrogenase gene is the same as that of wet cells containing olefin reductase SeER mutant gene.

[0017] Further, the SeER mutant purified enzyme was prepared as follows: the wet bacteria of the engineered bacteria containing the olefin reductase SeER mutant gene were resuspended in a pH 6.0, 100mM potassium phosphate buffer at an amount of 100g / L, and ultrasonically disrupted on an ice-water mixture for 25min, the ultrasonic disruption conditions were: power of 400W, disruption for 2s, pause for 3s, and the disruption mixture was taken to obtain a crude enzyme solution; 0.1mg / mL of riboflavin sodium phosphate (FMN) was added to the obtained crude enzyme solution, and the solution was allowed to stand on ice for 30min, followed by centrifugation at 8000rpm and 4°C for 20min, the supernatant was collected, and after filtering through a 0.45μm microfiltration membrane, the mutant protein was purified by a nickel affinity column (1.6×10cm, Bio-Rad, USA), and the purification operation was as follows: ① The sample was loaded at a flow rate of 1.0mL / min, and a pH 7.0 column containing 0.5M NaCl and 20mM imidazole was used. ② Then use pH 6.0, 20mM potassium phosphate buffer to gravity elute to remove unbound impurities at a flow rate of 1.0mL / min until the conductivity is stable; ② Then use pH 6.0, 20mM potassium phosphate containing 0.5M NaCl and 500mM imidazole to gravity elute 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 a replacement buffer to remove 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 needs to be 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 also provides an application of an olefin reductase SeER mutant in catalyzing stereocomplementary reduction of (Z)-3-cyano-3-phenylacrylate to synthesize phenibut, wherein the application is: using wet bacteria or purified enzyme obtained by fermentation culture of an engineered bacterium containing an olefin reductase SeER mutant encoding gene as a catalyst, using purified enzyme freeze-dried powder obtained by fermentation culture of an engineered bacterium containing a glucose dehydrogenase encoding gene as a coenzyme, and using (Z)-3-cyano-3-phenylacrylate as a substrate. , using dimethyl sulfoxide (DMSO) as a cosolvent and a pH 5-9 buffer as a reaction medium, reacting 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] Further, the synthesis method of phenibut hydrochloride is 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, the solution turns black, and after the reaction of the raw materials is completed by monitoring by TLC, the methanol is removed by reduced pressure distillation, and the concentrated solid is refluxed in 6M HCl solution for 12 hours. After the reaction is completed, water is removed by reduced pressure distillation, and the concentrated solid is recrystallized with isopropanol to obtain the drug phenibut 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 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 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 co-solvent is added to a final concentration of 10% by volume, the substrate is first dissolved in the co-solvent 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 wet bacteria; 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 enzymes 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 pH 5-9 buffer 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 mutant is preferably SeER-W117H / F248A, SeER-W117H / T38S, SeER-W117H / L293H, and the amino acid sequence is shown in SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.8.

[0022] Further, the synthesis method of baclofen hydrochloride is as follows: (R)-3-(4-chlorophenyl)-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 see whether the raw materials are completely reacted. After the reaction is completed, 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 with 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 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 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 co-solvent is added to a final concentration of 10% by volume, the substrate is first dissolved in the co-solvent 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 wet bacteria; 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, purified enzyme lyophilized 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] Further, the synthesis method of tolibut hydrochloride is 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 see whether the raw materials are completely reacted. After the reaction is completed, 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 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 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 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 co-solvent is added to a final concentration of 10% by volume, the substrate is first dissolved in the co-solvent 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 wet bacteria; 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 A cosolvent is used, a pH 5-9 buffer is used as a reaction medium, and a 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, SeER-W117A / T38W, and the amino acid sequence is shown in SEQ ID NO.10, SEQ ID NO.12, and SEQ ID NO.14.

[0028] Further, the synthesis method of pregabalin hydrochloride is as follows: (S)-3-cyano-5-methylhexanoic acid ethyl 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 continues to be stirred at 0°C for 15 minutes. The solution turns black and is monitored by TLC to see whether the raw materials are completely reacted. After the reaction is completed, 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 with isopropanol to obtain the drug pregabalin hydrochloride. The mass ratio of (S)-3-cyano-5-methylhexanoic acid ethyl ester to NiCl2·6H2O is 1:2.5, and the volumetric amount of methanol is 0.05 mL / mg based on the mass of (R)-3-cyano-3-phenylpropionic acid methyl ester; the mass ratio of (S)-3-cyano-5-methylhexanoic acid ethyl ester to NaBH4 is 1:1.8-2; and the volumetric amount of HCl solution is 0.1 mL / mg based on the mass of (S)-3-cyano-5-methylhexanoic acid ethyl ester.

[0029] Furthermore, the substrate is added to a final concentration of 10 mM, the co-solvent is added to a final concentration of 10% by volume, the substrate is first dissolved in the co-solvent 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 wet bacteria; 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, the stop codon is TAA, and 397 amino acids are encoded.

[0031] The olefin reductase SeER mutant of the present invention is obtained by protein engineering, and finally 8 beneficial mutants T38W, T38S, W117H, W117G, W117A, F248A, L293H, Y373A are obtained; through further iterative combination mutation, a pair of stereo complementary olefin reductase SeER mutants (W117H / F248A, W117G / T38W) are obtained. The obtained mutant recombinant plasmid is transferred into E.coli BL21 (DE3) competent cells by heat shock, and the obtained strain is inoculated, transferred, induced culture, and bacterial cell recovery. The inoculation, transfer, induction culture, and bacterial cell recovery of the olefin reductase mutant of the present invention can be any culture medium in the art that can grow the bacterial cell of the present invention, preferably LB culture medium: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, water dissolution. There is no particular limitation on the culture method and culture conditions, which can be appropriately selected according to common knowledge in the art based on factors such as the host type and culture method.

[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 activity of the mutant SeER-W117H / F248A is increased by 160 times compared with 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 the wild-type WT-SeER; the mutant strains (SeER-W117H / F248A, SeER-W117G / T38W) also show high activity and excellent stereocomplementary selectivity for 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 fenibute, baclofen, tolibut, pregabalin, etc. through a chemical-enzyme coupling method. Compared with the existing method, the strain of the present invention can flexibly regulate the configuration of the product, and has high selectivity and conversion rate. (IV) Description of the drawings

[0037] Figure 1 It 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-phenylpropanoic acid methyl ester and (S)-3-cyano-3-phenylpropanoic acid methyl ester by coupling the olefin reductase SeER mutant with glucose dehydrogenase EsGDH.

[0038] Figure 2 HPLC chromatograms for the preparation of (R)-3-cyano-3-phenylpropionic acid methyl ester and (S)-3-cyano-3-phenylpropionic acid methyl ester products 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] Figure 3 The effect of temperature on the specific activity of SeER-W117H / F248A.

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

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

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

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

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

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

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

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

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

[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), i.e., 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 solution; the seed solution was inoculated into a 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 OD 600 =0.6-0.8, then add IPTG with a final concentration of 0.15 mM to the culture solution, culture at 18°C ​​for 18 h, centrifuge at 4°C and 8000 rpm for 10 min, and collect the wet cells of the engineered bacteria.

[0054] (3) Enzyme purification

[0055] The wet bacterial cells in 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 as follows: power of 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, followed by centrifugation 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 containing 0.5 M NaCl, 20 mM imidazole, pH 6.0, 20 mM potassium phosphate buffer at a flow rate of 1.0 mL / min to remove unbound impurities until the conductivity is stable; ② Then use a gravity elution containing 0.5 M NaCl, 500 mM imidazole, pH 6.0, 20 mM potassium phosphate buffer for 1 column volume; Pour all the collected eluate (one column volume, 10 mL) into a 30 kDa ultrafiltration tube, use 20 mM pH 6.0 potassium phosphate as the replacement buffer to remove imidazole, and collect the retained protein, which is the SeER purified enzyme, recorded as WT-SeER.

[0058] (4) Enzyme activity assay

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

[0060] Enzyme activity detection conditions: 1mM NADPH, appropriate amount of purified enzyme (enzyme protein content is 200mg), 1mM substrate (Z)-3-cyano-3-phenylacrylate, volume concentration of 5% DMSO, the substrate is first dissolved in DMSO and then added, pH 6.0, 100mM potassium phosphate buffer is added to the final volume of 200μL, react at 30℃, 1000rpm for 2min, and use Biotek Epoch microplate reader to detect NADPH consumption.

[0061] The substrate conversion rate, product ee value and yield were detected by Agilent 1260 HPLC: 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 30 °C.

[0062] 2. EsGDH enzyme purification

[0063] The method of step 1 was used to artificially synthesize the glucose dehydrogenase gene (GenBank No. KM817194.1) from Exiguobacterium sibirium DSM 17290 in GenBank, the nucleotide sequence of which is shown in SEQ ID NO. 15, and the same conditions (except that no riboflavin sodium phosphate was added to the EsGDH crude enzyme solution and the molecular weight cutoff of the ultrafiltration tube was 10 kDa) were used to prepare EsGDH wet bacteria and EsGDH purified enzyme. The EsGDH purified enzyme was freeze-dried at -80°C to prepare a purified enzyme freeze-dried 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] Definition of enzyme activity: 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 unit of enzyme activity.

[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 docking software YASARA was used to dock the substrate (Z)-3-cyano-3-phenylacrylate into the active pocket of WT-SeER, and the distance from the substrate was selected to be Among the amino acid residues in the range, these amino acids are most likely to affect the selectivity and activity of the enzymatic reaction. After excluding the conservative catalytic triad amino acids (H189, N192, Y194), T38, W117, F248, L293, and Y373 were 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 of site 117: The plasmid pET21a(+)-seer of the wild-type olefin reductase genetic engineering bacterium E. coli BL21(DE3) / pET21a(+)-seer of Example 1 was extracted, and the tryptophan at site 117 was mutated to other 19 amino acids using the primers in Table 1. The PCR product was digested with enzymes to remove the original template and cleaned up, and then transformed into E. coli BL21(DE3) competent cells, and the clones were inoculated into LB solid culture medium containing 100 μg / mL ampicillin and cultured at 37°C for 12 hours. Then, the clones were picked out and transferred to 10 mL of LB liquid culture medium containing 100 μg / mL ampicillin, and cultured at 37°C and 200 rpm for 10 hours. The culture solution was centrifuged at 8000 rpm for 10 minutes to collect the wet cells.

[0071] Table 1. Primer design for site-directed saturation mutagenesis of olefin reductase 117

[0072]

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

[0074] PCR reaction system (25 μL): 0.5 μL forward primer (100 μM), 0.5 μL reverse primer (100 μM), 12.5 μL 2×Phanta buffer, 0.5 μL dNTP mixture (10 mM each), 0.5 μL plasmid template, 0.5 μL DNA polymerase and 10 μL ultrapure water. According to the Phanta Super-Fidelity DNA polymerase operation manual, the PCR program was set as follows: 95°C pre-denaturation for 5 min, then 30 cycles (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, 5 g / L EsGDH wet cells prepared by the method of Example 1, the reaction medium is pH 6.0, 100 mM potassium phosphate buffer, constituting a 1 mL reaction system, reacting at 30°C, 1000 rpm for 24 h, extracting the reaction solution with ethyl acetate, concentrating and drying the organic phase, and dissolving the sample with chromatographic grade isopropanol, using liquid chromatography in Example 1 to detect the product, substrate concentration, calculate substrate conversion rate, enzyme specific activity and stereoselectivity, and 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. The 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 of F248

[0080] The plasmid of the engineering bacteria E. coli BL21 (DE3) / pET21a (+) -seer of Extraction Example 1 was used as a template, and PCR amplification was performed using the primers in Table 3 and the method and conditions in step 2. The conversion rate and stereoselectivity of each mutant wet bacteria to the substrate were tested. The results showed that the substrate conversion rate of SeER-F248A was 99%, and the R-isomer ee value was 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] Extraction Example 1 The plasmid of the engineering bacteria E. coli BL21 (DE3) / pET21a (+) -seer was used as a template, and the primers in Table 4 were used to perform PCR amplification using the method and conditions in step 2, and the conversion rate and stereoselectivity of each mutant wet cell to the substrate were tested. The results showed that the substrate conversion rate of SeER-T38S was 99%, and the ee value of the R-isomer was 99%. The substrate conversion rate of SeER-T38W was 99%, and the ee value of the S-isomer was 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 engineering bacteria E. coli BL21 (DE3) / pET21a (+) -seer of Extraction Example 1 was used as a template, and PCR amplification was performed using the primers in Table 5 and the method and conditions in step 2, and the conversion rate and stereoselectivity of each mutant wet bacteria to the substrate were tested. The results showed that the substrate conversion rate of SeER-L293H was 99%, and the R-isomer ee value was 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 engineering bacteria E. coli BL21 (DE3) / pET21a (+) -seer of Extraction Example 1 was used as a template, and PCR amplification was performed using the primers in Table 6 and the method and conditions in step 2, and the conversion rate and stereoselectivity of each mutant wet bacteria to the substrate were tested. The results showed that the substrate conversion rate of SeER-Y373A was 99%, and the S-isomer ee value was 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 used as a template, and PCR amplification was performed using the primers in Table 7 and the method and conditions of Example 2 to construct 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 in a -80°C refrigerator. Prepare wet cells and purified enzymes of each mutant using the method of Example 1. The amount of purified enzyme added is calculated based on protein content.

[0102] The substrate conversion rate and stereoselectivity were tested using the method and conditions of Example 2, wherein 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 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: In 1 mL of 100 mM buffer at different pH (pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 9.0), add 0.02 mg / mL SeER-W117H / F248A or SeER-W117G / T38W purified enzyme, 10 mM substrate (Z)-3-cyano-3-phenylacrylate (the substrate was first dissolved in DMSO and then added), 5% DMSO in final volume concentration, 30 mM glucose, 1 mg / mL EsGDH purified enzyme lyophilized powder. React at 30°C and 1000 rpm for 24 h, detect the product and substrate concentrations according to the method in Example 1, and calculate the conversion rate and product ee value. As shown in Table 8, the optimal reaction pH of SeER-W117H / F248A and SeER-W117G / T38W is 6.0.

[0110] Table 8 Optimal reaction pH

[0111]

[0112] 2. Optimum reaction temperature

[0113] In 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), 5% DMSO by volume, 1 mM NADPH, the mixture was reacted 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 Figure 3 , Figure 4 The optimal reaction temperature of SeER-W117H / F248A and SeER-W117G / T38W was 30℃.

[0114] 3. Reaction cosolvent

[0115] In 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 respectively. The substrate was first dissolved in an 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 under the cosolvent was detected according to the method in Example 1. The results are as follows: Figure 5 As shown, the best reaction co-solvent for SeER-W117H / F248A and SeER-W117G / T38W is DMSO.

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

[0117] The kinetic parameters were determined using the 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, the content of WT-SeER is 0.5 mg, the content of SeER-W117H and SeER-W117G is 0.05 mg, the content of SeER-W117H / F248A and SeER-W117G / T3W is 0.02 mg), the concentration of substrate (Z)-3-cyano-3-phenylacrylate ranges from 0 to 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 of the mutant was 3 min and the reaction time of 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 by 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 the 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. Among them, the catalytic efficiency of the mutants SeER-W117H / F248A and SeER-W117G / T3W was increased by 359 times compared with the wild type WT-SeER.

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

[0121]

[0122]

[0123] Example 6: Characterization of substrate spectra of wild-type SeER and its mutants (SeER-W117H / F248A, SeER-W117G / T3W)

[0124] In 200 μL of pH 6.0, 100 mM potassium phosphate buffer, WT-SeER, SeER-W117H / F248A or SeER-W117G / T38W purified enzyme (based on protein content, WT-SeER feed amount is 0.5 mg, mutant SeER-W117H / F248A and SeER-W117G / T38W feed amount is 0.02 mg), 1 mM NADPH, 1 mM substrate, final volume concentration 5% DMSO, the substrate was first dissolved and then added to the reaction system, reacted at 30°C, 1000 rpm for 3 min, and the enzyme activity was detected according to the method in Example 1. Each substrate was set up in parallel 3 times.

[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 Substrate spectrum of wild-type SeER and its mutants (SeER-W117H / F248A, SeER-W117G / T3W)

[0127]

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

[0129] Example 7: Catalytic performance of SeER-W117H / F248A and SeER-W117G / T3W for a series of olefin compounds

[0130] SeER-W117H / F248A or SeER-W117G / T38W purified enzyme (based on protein content, the feed amount of mutant SeER-W117H / F248A and SeER-W117G / T38W was 0.02 mg), 1 mM NADPH, 1 mM substrate ( Figure 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 detected according to the method of Example 1. The results are shown in Figure 6 . The mutant SeER-W117H / F248A showed good catalytic activity and excellent R-selectivity for most substrates. SeER-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, SeER-W117G / T38W showed excellent S-selectivity for other tested substrates.

[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 6 M KOH was used to control pH. The reaction was carried out at 30 ° C and 1000 rpm for 24 h. After the reaction was completed, it 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 ( Figure 7 ).

[0134] 90 mg of (R)-3-cyano-3-phenylpropionic acid methyl ester and NiCl2·6H2O (2 equiv, 226.1 mg) were dissolved in methanol (MeOH) (5 mL), cooled to 0°C, and stirred for 15 min. NaBH4 (10 equiv, 180 mg) was then added in portions. The final solution was stirred at 0°C for 15 min. The solution turned black and the reaction of the raw materials was monitored by TLC. After the reaction was completed, methanol was removed by vacuum distillation. The concentrated solid was refluxed in 10 mL of 6M HCl solution for 12 h. After the reaction was completed, water was removed by vacuum distillation. The concentrated solid was recrystallized from isopropanol to obtain 85 mg of the drug phenibut hydrochloride.

[0135] 2. Reduction of (Z)-3-(4-chlorophenyl)-3-cyanoacrylate to synthesize baclofen

[0136] The substrate in step 1 was changed to (Z)-3-(4-chlorophenyl)-3-cyanoacrylate, and the other conditions and operations were the same to obtain 95 mg of optically pure product (99% ee) (R)-3-(4-chlorophenyl)-3-cyanopropionic acid methyl ester ( Figure 8 ).

[0137] Using the conditions and method of step 1, (R)-3-cyano-3-phenylpropionic acid methyl ester was replaced with (R)-3-(4-chlorophenyl)-3-cyanopropionic acid methyl ester to obtain 90 mg of the drug baclofen hydrochloride.

[0138] 3. Reduction of (Z)-3-(4-methylphenyl)-3-cyanoacrylate to synthesize Tolibut

[0139] The substrate in step 1 was changed to (Z)-3-(4-methylphenyl)-3-cyanoacrylate, and the other conditions and operations were the same to obtain 87 mg of optically pure product (99% ee) (R)-3-(4-methylphenyl)-3-cyanopropionic acid methyl ester ( Fig. 9 ).

[0140] Using the conditions and method of step 1, (R)-3-cyano-3-phenylpropionic acid methyl ester was replaced with (R)-3-(4-methylphenyl)-3-cyanopropionic acid methyl ester to obtain 80 mg of the drug tolibut hydrochloride.

[0141] Example 9: Synthesis of baclofen by stereoreduction of (Z)-3-(4-chlorophenyl)-3-cyanoacrylate methyl ester catalyzed by different mutants

[0142] 1. SeER-W117H / T38S

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

[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 catalytic stereoreduction of (Z)-3-cyano-5-methylhex-2-enoic acid ethyl ester 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 SeER mutant, characterized in that: The olefin reductase SeER mutant is obtained by performing single-point or multi-point site-directed mutagenesis or site-directed saturation mutagenesis at positions 38, 117, 248, 293 and 373 of the amino acid sequence of the olefin reductase SeER from S. eubayanus shown in SEQ ID NO.

2.

2. The olefin reductase SeER mutant according to claim 1, characterized in 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) the tryptophan at position 117 is mutated into histidine; (2) the tryptophan at position 117 is mutated into glycine; (3) the tryptophan at position 117 is mutated into alanine; (4) the tryptophan at position 117 is mutated into histidine, and the phenylalanine at position 248 is mutated into alanine; (5) the tryptophan at position 117 is mutated into histidine, and the threonine at position 38 is mutated into serine; (6) the tryptophan at position 117 is mutated into histidine, and the leucine at position 293 is mutated into histidine; (7) the tryptophan at position 117 is mutated into glycine, and the threonine at position 38 is mutated into tryptophan; (8) the tryptophan at position 117 is mutated into glycine, and the tyrosine at position 373 is mutated into alanine; (9) the tryptophan at position 117 is mutated into alanine, and the threonine at position 38 is mutated into tryptophan.

3. Use of the olefin reductase mutant according to claim 1 in synthesizing chiral compounds by stereocomplementary reduction of olefin compounds.

4. The use according to claim 3, characterized in that The application method comprises: using wet bacteria or purified enzyme obtained by induction culture of recombinant genetic engineering bacteria of olefin reductase mutant as a catalyst, using wet bacteria or purified enzyme freeze-dried powder obtained by induction culture of glucose dehydrogenase genetic engineering bacteria as a coenzyme, using a prochiral olefin compound as a substrate, using glucose as a cosubstrate, using dimethyl sulfoxide as a cosolvent, 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 after the reaction is completed, separating and purifying the reaction solution to obtain a chiral product; The structure of the olefin compound is shown in 1: 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.

5. The use according to claim 4, characterized in that When the olefin reductase SeER mutant is a mutant in which the amino acid sequence shown in SEQ ID NO.2 is mutated to one of the following, the chiral product obtained by catalyzing the substrate is an R-configuration: the 117th tryptophan is mutated to histidine, the 117th tryptophan is mutated to histidine and the 248th phenylalanine is mutated to alanine, the 117th tryptophan is mutated to histidine and the 38th threonine is mutated to serine, the 117th tryptophan is mutated to histidine and the 293rd leucine is mutated to histidine; when the olefin reductase SeER mutant is a mutant in which the amino acid sequence shown in SEQ ID NO.2 is mutated to one of the following, the chiral product obtained by catalyzing the substrate is an R-configuration When the amino acid sequence shown in NO.2 mutates into one of the following, the chiral product obtained by catalyzing the substrate is S-configuration: the 117th tryptophan mutates to glycine, the 117th tryptophan mutates to glycine and the 38th threonine mutates to tryptophan, the 117th tryptophan mutates to glycine and the 373rd tyrosine mutates to alanine, the 117th tryptophan mutates to alanine, the 117th tryptophan mutates to alanine and the 38th tryptophan mutates to tryptophan.

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

15.

7. Use of the olefin reductase SeER mutant according to claim 1 in catalyzing stereocomplementary reduction of (Z)-3-cyano-3-phenylacrylate to synthesize phenibut, characterized in that: The application is as follows: using wet bacteria or purified enzyme obtained by fermentation culture of engineered bacteria containing olefin reductase SeER mutant encoding genes as catalyst, using purified enzyme freeze-dried powder obtained by fermentation culture of engineered bacteria containing glucose dehydrogenase encoding genes as coenzyme, using (Z)-3-cyano-3-phenylacrylate as substrate, using dimethyl sulfoxide as cosolvent, using pH 5-9 buffer as reaction medium, reacting at 25-40°C and 1000rpm, and after the reaction is finished, separating and purifying the reaction liquid to obtain (R)-3-cyano-3-phenylpropionic acid methyl ester; reducing (R)-3-cyano-3-phenylpropionic acid methyl ester with sodium borohydride and hydrolyzing with hydrochloric acid to synthesize phenibut hydrochloride; the amino acid sequence of the olefin reductase SeER mutant is shown in SEQ ID NO.

4.

8. Use of the olefin reductase SeER mutant according to claim 1 in catalyzing stereocomplementary reduction of (Z)-3-(4-chlorophenyl)-3-cyanoacrylate to synthesize baclofen, characterized in that: The application uses wet bacteria or purified enzyme obtained by fermentation culture of engineered bacteria containing olefin reductase SeER mutant encoding genes as a catalyst, purified enzyme lyophilized powder obtained by fermentation culture of engineered bacteria containing glucose dehydrogenase encoding genes as a coenzyme, (Z)-3-(4-chlorophenyl)-3-cyanoacrylate methyl ester as a substrate, dimethyl sulfoxide as a cosolvent, pH 5-9 buffer as a reaction medium, and reacts at 25-40° 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 amino acid sequences of the olefin reductase SeER mutant are shown in SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.

8.

9. Use of the olefin reductase SeER mutant according to claim 1 in catalyzing the stereoreduction of (Z)-3-(4-methylphenyl)-3-cyanoacrylate to synthesize tolibut, characterized in that: The application uses wet bacteria or purified enzymes obtained by fermentation culture of engineered bacteria containing olefin reductase SeER mutant encoding genes as catalysts, purified enzyme freeze-dried powder obtained by fermentation culture of engineered bacteria containing glucose dehydrogenase encoding genes as coenzymes, (Z)-3-(4-methylphenyl)-3-cyanoacrylate methyl ester as substrate, dimethyl sulfoxide as cosolvent, pH 5-9 buffer as reaction medium, and reacts at 25-40°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 amino acid sequence of the olefin reductase SeER mutant is shown in SEQ ID NO.

4.

10. Use of the olefin reductase SeER mutant according to claim 1 in catalyzing the stereoreduction of (Z)-3-cyano-5-methylhex-2-enoic acid ethyl ester to synthesize pregabalin, characterized in that: The application uses wet bacteria or purified enzyme obtained by fermentation culture of engineered bacteria containing olefin reductase SeER mutant encoding genes as a catalyst, purified enzyme lyophilized powder obtained by fermentation culture of engineered bacteria containing glucose dehydrogenase encoding genes as a coenzyme, (Z)-3-cyano-5-methylhex-2-enoic acid ethyl ester as a substrate, dimethyl sulfoxide as a cosolvent, pH 5-9 buffer as a reaction medium, and reacts at 25-40° C. and 1000 rpm. After the reaction is completed, the reaction liquid 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 amino acid sequence of the olefin reductase SeER mutant is shown in SEQ ID NO.10, SEQ ID NO.12, and SEQ ID NO.14.

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