Engineering triterpene metabolism in the oilseed of Arabidopsis thaliana

Squalene and botryococcene are linear, hydrocarbon triterpenes that have industrial and medicinal values. While natural sources for these compounds exist, there is a pressing need for robust, renewable production platforms. Oilseeds are an excellent target for heterologous production because of their roles as natural storage repositories and their capacity to produce precursors from photosynthetically-derived carbon. We generated transgenic Arabidopsis thaliana plants using a variety of engineering strategies (subcellular targeting and gene stacking) to assess the potential for oilseeds to produce these two compounds. Constructs used seed-specific promoters and evaluated expression of a triterpene synthase alone and in conjunction with a farnesyl diphosphate synthase (FPS) plus 1-deoxyxylulose 5-phosphate synthase (DXS). Constructs directing biosynthesis to the cytosol to harness isoprenoid precursors from the mevalonic acid (MVA) pathway were compared to those directing biosynthesis to the plastid compartment diverting precursors from the methylerythritol phosphate (MEP) pathway. On average, the highest accumulation for both compounds was achieved by targeting the triterpene synthase, FPS and DXS to the plastid (526.84 mu g/g seed for botryococcene and 227.30 mu g/g seed for squalene). Interestingly, a higher level accumulation of botryococcene (a non-native compound) was observed when the biosynthetic enzymes were targeted to the cytosol (>1000 mu g/g seed in one line), but not squalene (natively produced in the cytosol). Not only do these results indicate the potential of engineering triterpene accumulation in oilseeds, but they also uncover some the unique regulatory mechanisms controlling triterpene metabolism in different cellular compartments of seeds.