Metabolic Engineering of Arabidopsis Plant to Produce Triterpenoid Hydrocarbons for Biofuel

The microalga Botryococcus braunii (B. braunii) can produce extracellular oil in the form of triterpenoid hydrocarbons up to 40 precent of their dry mass. These hydrocarbons with carbon chain lengths ranging from C30 to C40 including botryococcene, squalene and methylated squalenes, are ideal for the production of high quality liquid transport fuels and petrochemical alternatives. However, commercial-scale production of B. braunii is hampered by its slow growth rate. Other fast growing microalgae and terrestrial plants, unlike B. braunii, do not accumulate triterpene hydrocarbons, because squalene, a key metabolite of the plant triterpene pathway, is rapidly converted to downstream products. With the aim of modifying plants to accumulate substantial amounts of squalene-like triterpenes for biofuel, we used a metabolic engineering approach, to divert the plant triterpene pathway for hydrocarbon production. A B. braunii triterpene methyltransferase 3 (BbTMT-3) gene was cloned and transferred into Arabidopsis thaliana. The function of BbTMT-3 was found to add methyl groups to squalene, which prevented squalene from being further metabolized. Further chemical analysis indicated that the transgenic plants contained 30μg/g fresh weight of monomethylsqualene and 50μg/g of dimethylsqualene, and these new hydrocarbon molecules were not present in untransformed plants. The accumulation of the triterpenoid hydrocarbons indicated that the intended transformation was successful, and demonstrated the feasibility of new biofuel production via metabolic engineering. Further research is now aimed to engineer the upstream genes to direct greater carbon flux into the triterpene pathway in order to increase the hydrocarbon content.