PPT1 ‐mediated plastidic phospho enol pyruvate 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.