The enantioselective biocatalytic hydrolysis of rac-2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester to obtain a key (S)-intermediate is crucial for the synthesis of Pregabalin (a lipophilic 4-aminobutyric acid (GABA) analogue and has been developed to a new blockbuster drug for the treatment of several central nervous system disorders including neuropathic pain, epilepsy, anxiety and social phobia). It is also substrate of EstZF172 A0A0N7CSD6 from Psudomonas which is not an Alpha/beta hydrolase but a Beta-lactamase, family VIII Xu et al.
The scissile fatty acid binding site of lipases is divided into different sub-groups and plays an important role in the catalytic properties of the enzymes. In this study, the Talaromyces thermophilus lipase was engineered by altering its crevice-like binding site for efficient synthesis of chiral intermediate of Pregablin through kinetic resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE). The substitution of residues located at the crevice-like binding site with phenylalanine (Phe) resulted in significantly increased hydrolysis activity. The variant L206F/P207F/L259F exhibited a 37.23-fold and 47.02-fold improvement in the specific activity and turnover number (kcat) toward CNDE, respectively. Simultaneously, the optimum temperature and substrate preference were both altered in the variants. The study herein successfully engineered the TTL with improved catalytic properties for efficient biosynthesis of Pregablin intermediate. The investigation of structure-functional relationship provided important guidance for further modification of lipases with crevice-like binding site domain.
Title: Engineering of Thermomyces lanuginosus lipase Lip: creation of novel biocatalyst for efficient biosynthesis of chiral intermediate of Pregabalin Li XJ, Zheng RC, Ma HY, Zheng YG Ref: Applied Microbiology & Biotechnology, 98:2473, 2014 : PubMed
Efficient and highly enantioselective hydrolysis of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE) is the most crucial step in chemoenzymatic synthesis of Pregabalin. By using site-saturation mutagenesis and high-throughput screening techniques, lipase Lip from Thermomyces lanuginosus DSM 10635 was engineered to improve its activity towards CNDE. The triple mutant, S88T/A99N/V116D exhibited a 60-fold improvement in specific activity for CNDE (2.35 U/mg) over the wild-type Lip (0.039 U/mg). Modeling and docking studies demonstrated that the mutant could more effectively stabilize oxygen anions in transition states and the lid of Lip in the open conformation. Additionally, the kinetic resolution of CNDE catalyzed by Escherichia coli cell overexpressing S88T/A99N/V116D mutant afforded (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid in 42.4 % conversion and 98 % ee within 20 h with a substrate loading of 1 M (255 g/l). These results demonstrated that a novel and promising biocatalyst was created for efficient chemoenzymatic manufacturing of Pregabalin.
Title: Biocatalytic synthesis of chiral intermediate of pregabalin with high substrate loading by a newly isolated Morgarella morganii ZJB-09203 Zheng RC, Wang TZ, Fu DJ, Li AP, Li XJ, Zheng YG Ref: Applied Microbiology & Biotechnology, 97:4839, 2013 : PubMed
The chemoenzymatic process involving biocatalytic resolution of rac-2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE, 1) has been the most competitive and attractive route for pregabalin. A new esterase-producing strain ZJB-09203, which exhibited high hydrolytic activity, excellent enantioselectivity, and diastereoselectivity towards CNDE, has been successfully isolated from soil samples with a pH indicator agar plate method. The isolate was identified as Morgarella morganii by the ATB system (ID 32 GN) and the 16S rDNA sequence. In order to suppress product inhibition during enzymatic hydrolysis of CNDE, an adsorptive biocatalytic process was developed by utilizing anion-exchange resin D201 as adsorbent for selective removal of (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid (2) from the reaction medium. This approach allowed the substrate loading to be increased up to 1.5 M and the chiral intermediate 2 was produced in 682 mM, 45.3 % conversion, and 95 % ee. These results imply that M. morganii ZJB-09203 esterase is a promising biocatalyst in the development of chemenzymatic manufacturing process for pregabalin.
1 lessTitle: Engineering of Talaromyces thermophilus lipase by altering its crevice-like binding site for highly efficient biocatalytic synthesis of chiral intermediate of Pregablin Ding X, Zheng RC, Tang XL, Zheng YG Ref: Bioorg Chem, 77:330, 2018 : PubMed
The scissile fatty acid binding site of lipases is divided into different sub-groups and plays an important role in the catalytic properties of the enzymes. In this study, the Talaromyces thermophilus lipase was engineered by altering its crevice-like binding site for efficient synthesis of chiral intermediate of Pregablin through kinetic resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE). The substitution of residues located at the crevice-like binding site with phenylalanine (Phe) resulted in significantly increased hydrolysis activity. The variant L206F/P207F/L259F exhibited a 37.23-fold and 47.02-fold improvement in the specific activity and turnover number (kcat) toward CNDE, respectively. Simultaneously, the optimum temperature and substrate preference were both altered in the variants. The study herein successfully engineered the TTL with improved catalytic properties for efficient biosynthesis of Pregablin intermediate. The investigation of structure-functional relationship provided important guidance for further modification of lipases with crevice-like binding site domain.
Title: Discovery and expression of a Pseudomonas sp. esterase as a novel biocatalyst for the efficient biosynthesis of a chiral intermediate of pregabalin Xu F, Chen S, Xu G, Wu J, Yang L Ref: Biotechnol Bioprocess Eng, 20:473, 2015 : PubMed
Efficient and highly enantioselective hydrolysis of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE) is the most crucial step in chemoenzymatic synthesis of Pregabalin. By using site-saturation mutagenesis and high-throughput screening techniques, lipase Lip from Thermomyces lanuginosus DSM 10635 was engineered to improve its activity towards CNDE. The triple mutant, S88T/A99N/V116D exhibited a 60-fold improvement in specific activity for CNDE (2.35 U/mg) over the wild-type Lip (0.039 U/mg). Modeling and docking studies demonstrated that the mutant could more effectively stabilize oxygen anions in transition states and the lid of Lip in the open conformation. Additionally, the kinetic resolution of CNDE catalyzed by Escherichia coli cell overexpressing S88T/A99N/V116D mutant afforded (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid in 42.4 % conversion and 98 % ee within 20 h with a substrate loading of 1 M (255 g/l). These results demonstrated that a novel and promising biocatalyst was created for efficient chemoenzymatic manufacturing of Pregabalin.
Title: Biocatalytic synthesis of chiral intermediate of pregabalin with high substrate loading by a newly isolated Morgarella morganii ZJB-09203 Zheng RC, Wang TZ, Fu DJ, Li AP, Li XJ, Zheng YG Ref: Applied Microbiology & Biotechnology, 97:4839, 2013 : PubMed
The chemoenzymatic process involving biocatalytic resolution of rac-2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE, 1) has been the most competitive and attractive route for pregabalin. A new esterase-producing strain ZJB-09203, which exhibited high hydrolytic activity, excellent enantioselectivity, and diastereoselectivity towards CNDE, has been successfully isolated from soil samples with a pH indicator agar plate method. The isolate was identified as Morgarella morganii by the ATB system (ID 32 GN) and the 16S rDNA sequence. In order to suppress product inhibition during enzymatic hydrolysis of CNDE, an adsorptive biocatalytic process was developed by utilizing anion-exchange resin D201 as adsorbent for selective removal of (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid (2) from the reaction medium. This approach allowed the substrate loading to be increased up to 1.5 M and the chiral intermediate 2 was produced in 682 mM, 45.3 % conversion, and 95 % ee. These results imply that M. morganii ZJB-09203 esterase is a promising biocatalyst in the development of chemenzymatic manufacturing process for pregabalin.
