| Isoprenoids are a highly diverse class of natural products from which numerous commercial chemicals and medicines are derived. Although these compounds are highly valuable in medicine and industry, their production often either suffers from low yields for those molecules derived from natural sources or requires complex and inefficient synthetic routes. Engineering metabolic pathways to produce large quantities of complex isoprenoids in a tractable microbial host presents an attractive alternative to extraction from environmental sources or chemical syntheses.; A major obstacle to efficient microbial biosynthesis of isoprenoids is the production of the universal isoprenoid precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The biosynthesis of these precursors is highly regulated in all organisms, and previous research on increasing the in vivo supply has focused on engineering a host's native isoprenoid biosynthesis pathway to avoid known regulation.; To better address this limitation, we have taken the novel approach of engineering Escherichia coli to over-produce IPP and DMAPP by cloning and expressing the heterologous mevalonate isoprenoid pathway from Saccharomyces cerevisiae. When co-expressed with a condon-optimized amorphadiene synthase, this system successfully demonstrated high-level production of terpenes such as amorpha-4,11-diene, the sesquiterpene olefin precursor to the antimalarial drug artemisinin.; Expression of the heterologous mevalonate pathway circumvented native regulation of isoprenoid biosynthesis in E. coli, by providing a second, unregulated route to the isoprenoid precursors. However, initial optimization efforts demonstrated that unregulated carbon flux through the pathway is detrimental to both production and cell growth. Gene titration studies and metabolite profiling linked growth inhibition phenotypes with the accumulation of pathway intermediates, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA), and the isoprenyl pyrophosphates, IPP, DMAPP, and farnesyl pyrophosphate. Further study of growth inhibited cells through metabolite and mRNA analysis suggested that the accumulation of HMG-CoA caused an inhibition in the early steps of E. coli's fatty acid biosynthetic pathway. By modulating expression of HMG-CoA reductase and amorphadiene synthase, the pathway bottlenecks were eliminated, growth inhibition was alleviated and production increased. The synthetic mevalonate pathway operons were further optimized by laboratory evolution.; By engineering E. coli for high level production of isoprenoids, we demonstrate that balancing carbon flux through the engineered biosynthetic pathway is a key determinant in optimizing isoprenoid biosynthesis in microbial hosts. |
