Construction and Optimization of Mevalonate Pathway for Production of Isoprenoids in Escherichia coli

The isoprenoid family, containing over 50,000 members, constitutes one of the most structurally diverse groups of natural products. They range from essential and relatively universal primary metabolites, such as sterols, carotenoids, and hormones, to more unique secondary metabolites that serve roles in plant defense and communication and cellular and organismal development. Although these molecules have vast potential in medicine and industry their production is limited by two factors: 1- The yields from harvest and extraction of these compounds from their native sources are low2- Due to their complex structure, synthetic routes to most isoprenoids are difficult and inefficientTherefore engineering metabolic pathways for production of large quantities of isoprenoids in a microbial host is an attractive approach. 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 codon-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 it was shown that unregulated flux through the bottom part of mevalonate pathway is is detrimental to both production and cell growth. Accumulation of some the mevalonate pathway intermediates isoprenyl pyrophosphates, IPP, DMAPP, and farnesyl pyrophosphate causes severe growth inhibition and affects production of amorphadiene. Further studies of growth inhibited cells through metabolite and protein analysis suggested that accumulation of these intermediates results is down-regulation of parts of the pathway that are responsible for production of these toxic intermediates. After the cells successfully decrease protein expression and metabolite production the growth is restored.By engineering E. coli for high level production of isoprenoids, we demonstrate that balancing carbon flux through the engineered biosynthetic pathway is the key factor in optimization efforts towards high level production of isoprenoids.