(505b) Elucidating the Mode of Cytotoxicity in an Escherichia Coli Strain Engineered for Isoprenoid Production Using Transcriptomics and Metabolite Profiling

Engineering synthetic metabolic pathways in microbes for the production of pharmaceuticals and complex chemicals is an attractive alternative to chemical synthesis. However, in transferring large biosynthetic pathways to alternate hosts and manipulating expression levels, the native regulation of carbon flux through the pathway may be lost, leading to the accumulation of toxic intermediates. With the maturation of ?omics'-style analysis it is now technically feasible to identify modes of toxicity associated with the accumulation of foreign molecules in engineered bacterium. Previously, Escherichia coli was engineered to produce large quantities of isoprenoids by creating a mevalonate-based isopentenyl pyrophosphate biosynthetic pathway (Martin et al. 2003. Nat. Biotechnol. 21:796). The engineered E. coli produced high levels of isoprenoids, but further optimization lead 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. Using both DNA microarray analysis and metabolite profiling we have studied E. coli strains inhibited by the intracellular accumulation of HMG-CoA. Our results indicate HMG-CoA inhibits a specific pathway in the microbial host leading to a generalized membrane stress. This work exhibits the utility of using transcriptomic and metabolomic methods to resolve biological roadblocks when engineering microbial systems.