Metabolic Engineering of Photorespiratory Bypass Pathways to Enhance Novel Biofuel Production in Transgenic Plants

Industrialization and the global population growth rate have placed a tremendous burden on food, energy, and other natural resources, including land and water. The current world population of ~7.1 billion is expected to increase to 9.6 billion by 2050, while the global energy demand is anticipated to increase more than 3-fold over the same time period. In contrast, global oil production will peak before 2030, leading to diminishing supply and increasing cost. Hence, there is a clear and urgent need to harness the full potential of sustainable energy sources and convert these efficiently to resources required for human and industrial applications.

The current project is addressing this worldwide challenge by focusing on the development of plant systems to capture sunlight energy into more direct biofuels rather than sugars or fatty acids requiring extensive downstream processing. Our work is also focused on remediating a longstanding inefficiency in photosynthesis, the process known as photorespiration. About 20% of the time, photorespiration rather than photosynthesis occurs wherein instead of CO2 being fixed and converted to precursors for carbohydrate biosynthesis, O2 is condensed with RuBP to yield glycolate. The glycolate is subsequently decarboxylated in an energy intensive process resulting in the net loss of one CO2 returned to the atmosphere.
While attempts to engineer the oxygenation reaction out of the RuBP Carboxylase/Oxygenase have been unsuccessful, attempts to recycle carbon in the photorespiratory glycolate have shown some promise. We too have pursued the latter strategy as illustrated in Fig. 1 with the intent of recycling the carbon into the production of high-value, triterpene biofuels. Our focus on production of linear, branched-chain hydrocarbon triterpenes is driven by the ease of their catalytic cracking into all class of fuels: gasoline, disease, and jet fuel.
To evaluate various photorespiratory constructs, we first generated transgenic lines engineered for novel triterpene production in the chloroplast (G1 line). These transgenic lines were then re-engineered with 3 different constructs as depicted in Fig. 1, and the regenerated lines evaluated for triterpene accumulation by standard GC-MS analyses and for the operation of the putative bypass pathways by measuring the incorporation of radioactivity from glycolate into triterpenes.
pT1 lines containing a partial bypass pathway showed a slight increase (33%) in the glycolate incorporation into triterpenes as well as triterpene accumulation (44%) compared to the G1 control line. These levels were further enhanced in the pT3 lines incorporating a complete bypass pathway where higher levels of glycolate incorporation into triterpenes (500%) correlated with higher triterpene accumulation (97%) compared to the G1 controls. The highest triterpene levels (~2500 µg/g FW) were obtained in the pT5 lines, where glycolate incorporation and triterpene accumulation were enhanced by 1200% and 220%, respectively, compared to G1 controls.
Our data provides evidence for engineering known and novel photorespiratory bypass pathways in plants for efficient biofuel production. These metabolic engineering strategies demonstrate the use of sustainable energy to meet the growing need for fuels and renewable resources.