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Engineering High Value Oil Production into Biofuel Crops
Department of Plant and Soil Sciences
Assuming biofuels generated via the fermentation of sugars derived from cellulosic and non-cellulosic constituents of biofuels crops will provide a substantial contribution to our future energy needs, augmenting and amending the productivity of these biofuel crops is now a major research thrust worldwide.
One way of enhancing these biofuels crops will be to engineer them for value-added components such as oils that can be used for efficient fuel production and the manufacturing of other high-value products currently derived from petroleum oils.
Towards this end, we are proposing to engineer optimized production of long, branched-chain hydrocarbon biosynthesis into plants suitable as biofuels crops. Branched chain hydrocarbons, like methylated triterpenes, are readily cracked into paraffins and naphthenes that can either be distilled to combustible fuels (gasoline, jet fuel and diesel), or can be used directly for the synthesis of plastics, nylons, paints and other oil-derived products manufactured by diverse chemical industries.
2011 Project Description
During the initial funding period for this application, we have developed a series of expression vectors to the production of squalene in transgenic tobacco plants. These vectors have been designed to evaluate two key parameters for optimized expression - cell specific expression and targeting of the designed biochemistry to the cytosolic compartment versus the chloroplast compartment.
To examine the first parameter, promoters regulating constitutive gene expression have been compared to those directing trichome specific expression. To target the biochemistry to different cellular compartments, amino terminal targeting signal sequences have been appended to the respective genes. The constructs have thus comprised a prenyltranferase to augment the biosynthesis of FPP and squalene synthase to convert the FPP to squalene, targeted to either the cytoplasm of the cell (to divert carbon from the mevalonate isoprenoid pathway) or the chloroplast (to divert carbon from the methyl erythritol phosphate pathway) compartments, and for these genes to be expressed in all cell types in comparison to expression in secretory trichomes. Control constructs include those without the prenyltransferase gene, and thus testing for the availability of FPP for unique oil biosynthesis in the cytoplasm and chloroplasts Twenty to forty independent transgenic lines have been generated per construct and these initial lines screen for their squalene content. Control non-trangenic lines accumulate very little squalene in the range of 0.1 to 0.4 microgm/gm fresh weight.
Plants engineered for squalene production in the cytoplasm of all cell types exhibited a broad range of squalene accumulation from that found in the control non-transformed plants to upwards of 150 microgm/gm fr. wt. When constitutive expression was targeted to the chloroplasts the levels of squalene accumulation increased approximately 4-fold to 600 microgm/gm fr wt. and exhibited a developmental accumulation pattern. That is squalene accumulation levels in young levels was lower than in mature levels. When expression of the prenyltransferase and squalene synthase were targeted to the chloroplasts of trichomes, the highest level of squalene accumulation was observed, greater than 1,700 microgm/gm fr. wt. and approximately 1.7% of the dry weight of the plant.
A few of the transgenic lines were grown in a field experiment to assess general growth, biomass accumulation and squalene accumulation. High squalene accumulating lines for both constitutive and trichome specific expression were evaluated with genetically segregating populations. The plants with the transgenes constitutively expressed in all cell types exhibited overall agronomic performance comparable to the control checks and accumulated significant levels of squalene.
However, the trichome specific engineered sibling plants accumulated substantial quantities of squalene, but the growth performance of these plants were severely impacted. Plant height and photosynthesis rates, for example, were approximately 20 to 25% the level of the control plants.
The results obtained suggest that engineering triterpene accumulation specific to trichome cells of plants does result in high squalene production, but may adversely impact plant performance in field settings. In contrast, engineering this pathway for expression throughout the plant does result in significant squalene accumulation without impacting agronomic performance.