Abstract
SummarySqualene 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 μg/g seed for botryococcene and 227.30 μg/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 μg/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.
Highlights
Isoprenoids or terpenoids comprise one of the largest classes of natural products with tens of thousands of unique molecules currently identified and estimates that these represent 1%–10% of the total number of possible species
The forty carbon tetraterpenes are derived from the condensation of two geranylgeranyl diphosphate (GGPP) to yield phytoene by the squalene synthase-like reaction catalysed by phytoene synthase
An interesting subcellular division of isoprenoid biosynthesis exists: the mevalonic acid (MVA) pathway resides in the cytosol, whereas the methylerythritol phosphate (MEP) pathway is localized to the plastids, both of which give rise to the initial prenyl units, isopentenyl diphosphate (IPP) and DMAPP
Summary
Isoprenoids or terpenoids comprise one of the largest classes of natural products with tens of thousands of unique molecules currently identified and estimates that these represent 1%–10% of the total number of possible species This large catalog of compounds arises, in part, from regio- and stereo-chemistry diversification imposed during their biosynthesis, as well secondary modifications like hydroxylation, acylation, aroylation and glycosylation. Sesqui- and tri-terpenes are derived from FPP biosynthesized in the cytosol nearly exclusively from the MVA pathway, whereas mono-, di- and tetra-terpenes are biosynthesized in the plastids from precursors provided by the MEP pathway These two pathways are understood to normally operate independently of one another, but there have been documented cases of cross-talk and possible sharing of intermediates or basic prenyl units (Arigoni et al, 1997; Flu€gge and Gao, 2005; Hemmerlin et al, 2003; Opitz et al, 2014)
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