Primary metabolites are compounds essential to an organism’s survival at all times, whereas secondary metabolites perform more specialized functions, such as plant defense. Specialized metabolism therefore tends to be less conserved across species and this more diverse array of natural products includes molecules that have evolved biological activities and which are consequently developed as medicines. These molecules also reflect evolutionary change and may be used to study relationships between species. Terpenes and their derivatives comprise a significant portion of this metabolism and oxidosqualene, a linear triterpene, is metabolized to over 20,000 triterpenoids. Oxidosqualene cyclases (OSCs) are a class of enzymes that cyclize oxidosqualene into a variety of mono- and polycyclic triterpenes. All 13 OSCs encoded by the model plant A. thaliana have been previously characterized; we aim to study putative OSCs in several relatives to establish differences in their triterpenoid compositions and to illuminate change in that family. Comparison of our findings with previously characterized OSCs will give better understanding of how enzymes evolve into closely related homologs. The determination of a complete product profile for these OSCs will also aid in developing a potential mechanism for product formation through analysis of pathways leading to numerous minor products. OSCs involved in secondary metabolism prove difficult to study in their plant of origin because the enzymes are often differentially expressed and the background of other plant metabolites prevents accurate assignment of products to specific enzymes. Therefore we have used a combination of genome mining, heterologous expression in yeast, and analysis of complex mixtures by GC-MS and HSQC to identify and characterize several new OSCs. Determination of the triterpene biosynthetic capacities of close relatives of A. thaliana and subsequent comparison of those to that of A. thaliana can give insight into evolution and the potential triterpene biosynthetic capacity of their common ancestors. The difference in enzymatic capabilities in species as recently diverged as these also underscores the limitations of relying on a model organism such as A. thaliana in questions of secondary metabolism.

Characterization of enzyme product profiles in specialized metabolism through heterologous expression

251st National Meeting (2016)

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