Unveiling relationships among triterpene synthases in higher plants through enhanced heterologous expression in yeast

Natural product biosynthesis is heavily influenced by external stimuli; therefore, at any given time the host may contain only a subset of the products that these organisms can biosynthesize. Consequently, compounds isolated from a native source may not be a full collection of the organism's biosynthetic capacity. Genome mining offers the first comprehensive approach to investigating natural product biosynthesis. Genomic sequence provides the ability to obtain, for our purposes, all of the triterpene synthases (oxidosqualene cyclases, OSCs) that exist in a given plant. The origins of triterpenoid skeletal diversity were explored by characterizing OSCs through heterologous expression in the yeast Saccharomyces cerevisiae . The work of this thesis utilized two approaches to increase OSC substrate accumulation and production in Saccharomyces through genetic engineering to increase substrate concentration for heterologous expression of OSCs. Metabolic engineering efforts focused on modifying Saccharomyces to accumulate oxidosqualene (OS) and dioxidosqualene (DOS), the precursors for triterpene biosynthesis. A particularly useful aspect of this work is that several cyclases that showed little to no activity in vitro are quite active in these hosts, which has allowed us to characterize several genes that were previously thought not to encode OSCs. The generated strains facilitated the second aspect of this thesis, which characterized several OSCs from Arabidopsis thaliana, Sorghum bicolor, Lactuca sativa and Lactuca serriola . Genome-mining approaches have uncovered new triterpene skeletons, broadened the metabolic profile of several plants, and illuminated evolutionary relationships between these genes