Producing complex chemicals using synthetic metabolic pathways in microbial hosts can have many advantages over chemical synthesis but is often complicated by deleterious interactions between pathway intermediates and the host cell metabolism. With the maturation of functional genomic analysis, it is now technically feasible to identify modes of toxicity associated with the accumulation of foreign molecules in the engineered bacterium. Previously, Escherichia coli was engineered to produce large quantities of isoprenoids by creating a mevalonate-based isopentenyl pyrophosphate biosynthetic pathway (V. J. J. Martin et al., Nat. Biotechnol. 21:796-802, 2003). The engineered E. coli strain produced high levels of isoprenoids, but further optimization led to an imbalance in carbon flux and the accumulation of the pathway intermediate 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA), which proved to be cytotoxic to E. coli. Using both DNA microarray analysis and targeted metabolite profiling, we have studied E. coli strains inhibited by the intracellular accumulation of HMG-CoA. Our results indicate that HMG-CoA inhibits fatty acid biosynthesis in the microbial host, leading to generalized membrane stress. The cytotoxic effects of HMG-CoA accumulation can be counteracted by the addition of palmitic acid (16:0) and, to a lesser extent, oleic acid (cis-Delta(9)-18:1) in the growth medium. This work demonstrates the utility of using transcriptomic and metabolomic methods to optimize synthetic biological systems.