Applications of transcriptional profiling in the engineering of Escherichia coli for the production of anti-malarial therapeutics

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 an accumulation of toxic intermediates and consumption of cellular cofactors, energy or vital biosynthetic precursors by the heterologous pathway. With the maturation of functional genomics analysis it is now feasible to identify modes of toxicity associated with the accumulation of foreign molecules in the engineered bacterium and elucidate the exact nature of metabolic burden associated with the expression of a novel biochemical pathway.; Previously, Escherichia coli was engineered to produce large quantities of the sesquiterpene precursor to artemisinic acid, amorpha-4-11-diene by creating a mevalonate-based isopentenyl pyrophosphate biosynthetic pathway 1 (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 to E. coli. 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 fatty acid biosynthesis in the microbial host leading to a generalized membrane stress.; In a second series of experiments we combined transcriptomic analysis with proteomic and metabolite profiling to explore the metabolic burden on the E. coli host induced by producing amorpha-4-11-diene via the exogenous isoprenoid pathway. This work discovered the alterations in the intracellular prenyl phosphate pool was leading to under-modification of tRNA's and inducing a high serine flux. This serine flux was detrimental to overall productivity and media designed to address the serine requirement increased amorpha-4-11-diene production 2-fold.; This body of work suggests that DNA microarrays, when combined with other complementary analysis methods, can be a powerful tool for the design of novel biochemical pathways in microbial hosts.