A Journey through the Arabidopsis thaliana Genome: Discovering the Origins of Novel Triterpene Metabolites
Castillo-Rivera, Dorianne A
Matsuda, Seiichi P. T.
Doctor of Philosophy
Plants produce a large variety of natural products, including over 20,000 different triterpenoids. Triterpenoid functions range from roles as membrane sterols and hormones in primary metabolism, to defense compounds in secondary metabolism. Beyond these roles in the plant, triterpenoids have substantial human value as flavors, fragrances, and medicines. This thesis explores the enzymatic formation of triterpenoids through cationic cyclization of a linear precursor oxidosqualene and further metabolism by radical oxidation. Thirteen different oxidosqualene cyclases (OSCs) are encoded by the model plant Arabidopsis thaliana and collectively produce a plethora of triterpene skeletons. This structural diversity of triterpenes was investigated through a comprehensive analysis of the A. thaliana PEN6 product profile. This product profile contained 33 compounds that were found by combining genome mining, heterologous expression in yeast and HSQC analysis. Some of these compounds were novel to Arabidopsis: isoursenol, (13R,14Z,17E)-malabarica-14,17,21-trien-3β-ol, nematocyphol, (20R,S) dammarenediols, Δ8(26)-seco-β-amyrin, and 9αH-Δ8(26)-polypodatetraenol. After the cyclization, triterpenes can be modified by a number of enzymes, including cytochrome P450 monooxygenases (CYP450s). Candidate CYP450s for a given triterpene were identified by gene cluster analysis combined with microarray databases. Heterologous expression of the OSC, ATR2 and CYP450 together with GC-MS and NMR techniques allowed the elucidation of metabolic pathways and structures of a variety of oxygenated triterpenes. These experimental techniques led to the identification of new oxidized metabolites produced by the co-expression of Arabidopsis clusters: THAS1 with CYP708A2 and CYP705A5, PEN1 with CYP705A2 and MRN1 with CYP71A16. The thalianol cluster gave rise to several unexpected side chain and ring oxidized metabolites. This thesis also describes the side chain cleavage of arabidiol by CYP705A1 to give a C19 methyl ketone, and the hydroxylation of an allylic methyl of marneral/marnerol to 23-hydroxymarneral/23-hydroxymarnerol by CYP71A16. This work sheds light on the metabolic fate of some Arabidopsis triterpenes. When applied more generally, this strategy may begin to fill a large knowledge gap in metabolomics and functional genomics.