The metabolic diversity of natural products in higher plants appears to be driven out of necessity to adapt and survive in different ecological environments. Among the vast amount of natural products found in nature, triterpenes are widely distributed among higher plants. Triterpenes serve as precursors of essential membrane sterols in primary metabolism, and as compounds involved in defense in secondary metabolism. These complex chemical structures can be leads in drug discovery. Oxidosqualene cyclases (OSCs) are enzymes that generate the ring systems of triterpenoids through cyclization of (3S)-oxidosqualene. With the arrival of the genomic era, genome mining has become a reliable strategy to give a comprehensive accounting of triterpenoids in organisms whose genome is available. Arabidopsis thaliana, a model plant organism has provided, through genome mining, the first complete picture of triterpene skeletons characteristic of plants. Now, the availability of different genomes opens up the possibility of exploring the metabolic capabilities of other plant clades. This thesis uses genome mining and heterologous expression in the yeast Saccharomyces cerevisiae to explore the biosynthetic diversity of oxidosqualene cyclases in two small clades of Brasicaceae and Asteraceae, Arabidopsis and Lactuca. OSCs have been widely studied in Arabidopsis thaliana but barely been identified in the Arabidopsis lyrata genome. Phylogenetic analysis comparing both organisms was used to discover the presence of nine putative OSCs. Three A. lyrata OSCs with interesting differences from their A. thaliana orthologs were expressed in yeast. The product profile of each enzyme was characterized by analytical techniques such as NMR and GC-MS. AlyPEN4 shows a similar product profile to its ortholog in A.thaliana, making as its major product thalianol. In the process of analysis a novel triterpene, 14(S)-25-nor-9β-methylpodioda-5(10),17,21-trienol, was discovered. Expression of AlyPEN6 illustrates the first example in secondary metabolism of orthologous genes with different function; the ratio between the two major products seco-β-amyrin and seco-α-amyrin was significantly different. Finally AlyPEN9, an OSC characteristic of A. lyrata turn out to be a β-amyrin synthase. Analysis of the genome shows that AlyPEN9 seems to be the only product specific β-amyrin of this organism.
Lactuca sativa, the first organism of the Asteraceae family to have its genome available, is a promising model system for the study of triterpenes synthesis. Here, I present the progress toward the characterization of the product profile of the Lactuca genus in order to understand the evolutionary relationships among different plant clades. Manual splicing of its genome uncovered 17 putative OSC. An improved product profile of six previously characterized oxidosqualene cyclases from Lactuca is reported. Also, a novel cyclase was discovered; it makes nematocyphol as its major product and 39 other compounds, including lupane type rearranged products. One of these compounds, lup-19(21)-en-3β-ol was found for the first time as part of the product profile of an OSC. This work shows that, although Arabidopsis and Lactuca have a unique set of oxidosqualene cyclases, most of the triterpenes that have been characterized within the Brasicaceae clade are present in the Asteraceae clade. The ability to synthesize unique sets of secondary metabolites may be related to the mechanism of adaptation and defense of different species.