(568c) Engineering E.Coli to Synthesize Simvastatin

Simvastatin (Zocor) is a pharmaceutically important semisynthetic compound because of its potent inhibitory activity towards hydroxymethylglutaryl coenzyme A reductase (HMGR), the rate-limiting step of cholesterol biosynthesis. We had previously developed an Escherichia coli-based, whole-cell biocatalytic process that can convert a precursor molecule Monacolin J (MJ) to simvastatin in one-step, utilizing a readily available dimethylbutyryl thioester substrate. The enzyme that catalyzes the conversion is the acyltransferase LovD from Aspergillus terreus. In this presentation, we will present recent metabolic engineering and protein engineering work that have resulted in significant enhancement in the efficiency and throughput of the whole cell system, including 1) identification of BioH as the carboxylesterase that hydrolyzes the essential thioester substrate; 2) deletion of the bioH gene that led to doubling of the whole cell catalytic conversion rate; 3) protein engineering efforts to improve the thermal stability of LovD; and 4) manipulating the E. coli multidrug transporter systems towards achieving higher product throughput. Together, these efforts have led to a biocatalytic approach towards simvastatin synthesis that can be economically competitive with the currently adopted synthetic processes.