Engineering Saccharomyces cerevisiae for high-level terpenoid production

Malaria is a global health problem that threatens 300-500 million people and kills more than one million people annually. The threat is intensified by the fact that the malaria parasite Plasmodium falciparum is showing increasing resistance to drugs that have been successfully employed in the past. Artemisinin-based combination therapies (ACTs) have become the WHO-recommended treatment for malaria because they are effective against Plasmodium spp. resistant to other anti-malarial drugs. The critical component to these therapies, artemisinin, is a sesquiterpene lactone endoperoxide, extracted from Sweet Wormwood (Artemisia annua ), but is unfortunately in short supply and unaffordable to most malaria sufferers. With the ongoing state of the malaria epidemic compounded by these difficulties, the world needs an alternative source of artemisinin. While total synthesis of artemisinin is difficult and costly, the semi-synthesis of artemisinin from microbially-sourced artemisinic acid, its immediate precursor, could be a cost-effective, environmentally-friendly, high quality, and reliable source for artemisinin.; Saccharomyces cerevisiae (Baker's yeast) was selected as a microbial host for artemisinic acid production because of its long history of industrial use and its tractable genetic qualities. Initial engineering efforts focused on the production of amorphadiene, the sesquiterpene backbone of artemisinic acid, in yeast. Amorphadiene is just one of over 40,000 identified terpenes, all of which are derived from the five carbon building block isopentenyl pyrophosphate (IPP). Farnesyl pyrophosphate (FPP), derived from IPP, is enzymatically converted to amorphadiene by amorphadiene synthase (ADS). Introduction of ADS from A. annua into yeast facilitated the production of amorphadiene in this heterologous host; however, because most of the limited endogenous FPP pool within the cell is diverted to sterol synthesis, the quantity produced was low.; To increase FPP production in S. cerevisiae, the expression of several genes responsible for FPP synthesis were upregulated, and one gene responsible for FPP conversion to sterols was downregulated. In construction of a final production host, all of these modifications to the host strain were made by chromosomal integration to ensure the genetic stability of the host strain. Overexpression of a truncated, soluble form of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (tHMGR) improved amorphadiene production approximately five-fold. Downregulation of ERG9, which encodes squalene synthase (the first step after FPP in the sterol biosynthetic pathway), using a methionine-repressible promoter (PMET3) increased amorphadiene production an additional two-fold. Expression of upc2-1 , a semi-dominant mutant allele that enhances the activity of UPC2 (a global transcription factor regulating the biosynthesis of sterols in S. cerevisiae), had increased amorphadiene production still further by two-fold. Integration of an additional copy of tHMGR combined with the overexpression of the gene encoding FPP synthase raised the total amorphadiene production on a per cell basis to 50-fold that of the original yeast strain expressing only ADS.; Additional sterol metabolites derived from FPP were analyzed to determine the flux around the FPP branch-point in an engineered strain tuned to a range of squalene synthase activities. These data added insight to understanding the optimal culturing conditions for amorphadiene production as well as suggesting future engineering efforts. Subsequent work to increase amorphadiene production focused on increasing ADS activity by increasing plasmid stability and increasing transcription of the gene.; The high-level amorphadiene production achieved in the engineered yeast strain was then leveraged for high-level production of artemisinic acid through the introduction of a cytochrome P450 monooxygenase from A. annua . This work illustrates the heterologous expression of two dis...