Rerouting phosphoenolpyruvate in sugar-rich tissues enables increased fatty acid biosynthesis

Metabolic engineering in the vegetative tissues of highly productive C4 crop, such as sugarcane, to produce oil is a promising strategy to convert solar energy into biofuel. However, biofuel production needs to be increased to meet the commercial requirements. To address this challenge, we deployed a novel approach to redirect phosphoenolpyruvate flux into plastids via overexpressing a plastidial phosphoenolpyruvate/phosphate translocator (PPT1). The concept was first tested in Arabidopsis. Excitingly, the results showed overexpressing AtPPT1 boosted fatty acid levels in leaves of sugar-rich sweet11;12;13 mutant rather than in the wild type. Increased fatty acid and oil accumulation were observed in older leaves of the triple mutant rather than in young leaves. Metabolomic analysis revealed that overexpressing AtPPT1 enabled the transition of the glycolytic intermediate metabolites from accumulating in sweet11;12;13 mutant back to a similar level as wild type. Under sugar-rich physiological conditions, overexpressing AtPPT1 triggers systematic transcriptomic changes involving class II trehalose-6P synthase (TPS) mediated signaling pathway, which regulates fatty acid synthesis. The strategy was further confirmed in sugarcane, where overexpressing sorghum SbPPT1 driven by a cane stem-specific promoter boosted fatty acid levels (>20% increase) in mature internodes of transgenic sugarcane plants without leading to obvious growth penalty. Positive correlations were observed between fatty acid level and SbPPT1 expression level. In summary, our work reported that directing phosphoenolpyruvate into plastids of sugar-rich tissues can stimulate fatty acid biosynthesis in vegetative tissues. Our strategy promises to achieve commercial-scale lipid production in engineered sugarcane. Overall design: Wild Type and Sugar Rich Triple mutant arabidopsis were grown under normal conditions, one set as control and the other overexpressing AtPPT1