Characterization and Optimization of Lovd towards Enzymatic Biosynthesis of Simvastatin

Simvastatin (Zocor) is a pharmaceutically important semisynthetic compound due to its potent inhibitory activity towards hydroxymethylglutaryl coenzyme A reductase (HMGR), the rate-limiting step of cholesterol biosynthesis. We recently discovered an acyl-transferase, LovD (46 kDa), which is involved in the lovastatin biosynthetic pathway. LovD catalyzes the acyltransfer of the side chain α-methylbutyrate synthesized by LovF to the C-8 hydroxyl of the direct biosynthetic precursor monacolin J sodium salt (MJSS) to yield lovastatin sodium salt. We demonstrated that LovD has broad substrate specificity towards: 1) the acyl group, 2) the thioester acyl carrier, and 3) the decalin core. We showed an Escherichia coli based whole-cell platform can readily convert MJSS to simvastatin sodium salt using the cost-effective -dimethylbutyryl-S-methyl-3-mercaptopropionate (DMB-S-MMP) as the thioester acyl donor. Further optimization of the E. coli expression host through deletion of a competing esterase bioH led to significant improvement in the kinetics of the biocatalyst. In addition, we rationally engineered LovD by using a combination of homology structural prediction and site-directed mutagenesis. Through systematic cysteine replacement with other amino acids, we created a double mutant C40A/C60N which displayed > 50% improvement in protein solubility and whole cell activity. An optimized fed-batch fermentation process that combines the above improvements was developed to quantitatively convert > 18 g/L MJSS to simvastatin in 18 hours. Taken together, the whole process can be economically competitive with the optimized synthetic methods currently used to manufacture simvastatin.