Highly regio- and enantioselective synthesis of chiral intermediate for pregabalin using one-pot bienzymatic cascade of nitrilase and amidase.

Nitrilase-mediated hydrolysis of isobutylsuccinonitrile (IBSN) is a highly attractive approach for (S)-3-cyano-5-methylhexanoic acid ((S)-CMHA), the critical chiral intermediate of pregabalin. In this study, a robust nitrilase from Arabis alpina (AaNIT) was screened and engineered. The N258D mutant was obtained with high catalytic activity and excellent enantioselectivity (E > 300) towards IBSN at a high substrate concentration of 100 g L. Byproduct (S)-3-cyano-5-methyl hexanoic amide ((S)-CMHM) was detected and identified for the first time during the catalytic process. By employing a feasible one-pot bienzymatic cascade of mutant N258D and amidase from Pantoea sp. (Pa-Ami) expressed separately in recombinant Escherichia coli cells, the byproduct (S)-CMHM was eliminated and (S)-CMHA was obtained with a conversion of 45.0% and ee of 99.3%. These results provided the novel plant-derived nitrilase as a promising biocatalyst for (S)-CMHA biosynthesis and demonstrated the feasibility of one-pot bienzymatic cascade reaction for large-scale production of the pregabalin precursor.