Directed Evolution and Structural Characterization of A Simvastatin Synthase

Simvastatin is the active pharmaceutical ingredient of the blockbuster cholesterol lowering drug Zocor. We have previously developed an Escherichia coli based whole-cell biocatalytic platform towards the synthesis of simvastatin acid starting from the precursor monacolin J (MJ) acid and the side chain donor α-dimethylbutyryl-S-methyl-mercaptopropionate (DMB-SMMP). The key part of the process is simvastatin synthase LovD, which is marginally stable when expressed in E. coli and moderately efficient towards DMB-SMMP. In this study, we employed directed protein evolution to improve LovD activity and stability as a whole cell biocatalyst. After multiple rounds of random mutagenesis and two rounds of saturated mutagenesis, mutants with higher thermostablity and and significantly improved activities were isolated. The best mutant exhibited eleven-fold improvement in whole cell activity as a simvastatin synthase. To understand the basis of improved catalytic efficiency, X-ray crystal structures of the wild type and an improved mutant were obtained. The crystal structures provided atomic resolution of the mechanism of catalysis, substrate and product binding, and a likely basis of beneficial mutations. The significantly improved efficiency of the mutant LovD enzyme enables futher commercial development of the biocatalytic synthesis of simvastatin.