Abstract
Linear, branch-chained triterpenes, including squalene (C30), botryococcene (C30), and their methylated derivatives (C31-C37), generated by the green alga Botryococcus braunii race B have received significant attention because of their utility as chemical and biofuel feedstocks. However, the slow growth habit of B. braunii makes it impractical as a production system. In this study, we evaluated the potential of generating high levels of botryococcene in tobacco (Nicotiana tabacum) plants by diverting carbon flux from the cytosolic mevalonate pathway or the plastidic methylerythritol phosphate pathway by the targeted overexpression of an avian farnesyl diphosphate synthase along with two versions of botryococcene synthases. Up to 544 µg g(-1) fresh weight of botryococcene was achieved when this metabolism was directed to the chloroplasts, which is approximately 90 times greater than that accumulating in plants engineered for cytosolic production. To test if methylated triterpenes could be produced in tobacco, we also engineered triterpene methyltransferases (TMTs) from B. braunii into wild-type plants and transgenic lines selected for high-level triterpene accumulation. Up to 91% of the total triterpene contents could be converted to methylated forms (C31 and C32) by cotargeting the TMTs and triterpene biosynthesis to the chloroplasts, whereas only 4% to 14% of total triterpenes were methylated when this metabolism was directed to the cytoplasm. When the TMTs were overexpressed in the cytoplasm of wild-type plants, up to 72% of the total squalene was methylated, and total triterpene (C30+C31+C32) content was elevated 7-fold. Altogether, these results point to innate mechanisms controlling metabolite fluxes, including a homeostatic role for squalene.
Highlights
Linear, branch-chained triterpenes, including squalene (C30), botryococcene (C30), and their methylated derivatives (C31–C37), generated by the green alga Botryococcus braunii race B have received significant attention because of their utility as chemical and biofuel feedstocks
An earlier study demonstrated that plastid-targeted engineering of a foreign squalene synthase (SQS) and an farnesyl diphosphate synthase (FPS) can successfully divert carbon flux from the methylerythritol phosphate (MEP) pathway to accumulate a high level of squalene in transgenic tobacco (Wu et al, 2012)
That study revealed that the availability of isopentenyl diphosphate (IPP)/DMAPP precursors was adequate but that strong regulatory mechanisms were absent in the chloroplast for novel squalene (C30) production to occur
Summary
Branch-chained triterpenes, including squalene (C30), botryococcene (C30), and their methylated derivatives (C31–C37), generated by the green alga Botryococcus braunii race B have received significant attention because of their utility as chemical and biofuel feedstocks. When the TMTs were overexpressed in the cytoplasm of wild-type plants, up to 72% of the total squalene was methylated, and total triterpene (C30+C31+C32) content was elevated 7-fold. These results point to innate mechanisms controlling metabolite fluxes, including a homeostatic role for squalene. Methylated derivatives are the dominant triterpene species generated by B. braunii race B (Metzger, 1985; Metzger et al, 1988), and these derivatives are known to yield higher quality fuels due to their high energy content and the hydrocracking products derived by virtue of having more hydrocarbon branches. These TMTs are believed to be insoluble enzymes; they exhibit large hydrophobic areas, and their activities were only observed in vitro using yeast microsomal preparations (no activity was observed when expressed in bacteria; Niehaus et al, 2012)
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