PPT1-mediated plastidic phosphoenolpyruvate import enhances fatty acid biosynthesis in sugar-rich tissues.

Metabolic engineering of vegetative tissues for lipid production holds transformative potential for sustainable biofuels, yet achieving sufficient yields remains challenging. Here, we present a strategy to enhance fatty acid synthesis by redirecting cytosolic phosphoenolpyruvate (PEP) into plastids through overexpression of the plastidial phosphoenolpyruvate/phosphate translocator (PPT1) in vegetative tissues of Arabidopsis thaliana. Integrated metabolomic and transcriptomic analyses revealed that AtPPT1 overexpression alleviated metabolite overaccumulation in high-sugar tissues, consistent with enhanced carbon flux coordination between the cytosol and chloroplast. Notably, phosphofructokinase activity, a key step in glycolysis, was elevated, linking plastidic PEP import to increased glycolytic throughput. In Arabidopsis, overexpression of AtPPT1 increased fatty acid content and lipid droplet accumulation in the sugar-accumulating sweet11;12;13 mutant, but not in wild-type Col-0. Together, these findings establish PEP redirection as an effective strategy to boost fatty acid and lipid production in sugar-rich vegetative tissues and provide a complementary metabolic module for future lipid-engineering efforts.