Resolving cofactor imbalance in triacylglycerol biosynthesis in oilseeds through glycolytic shunts: a modeling study

During seed development, carbohydrates are rapidly converted into triacylglycerols (TAGs), with glycolysis and the oxidative pentose phosphate pathway (OPPP) traditionally considered key sources of acetyl-CoA, ATP, NADH, and NADPH for fatty acid synthesis. However, how these classical pathways integrate into an overall stoichiometrically balanced conversion of sugars to TAGs remains unclear. Previous biochemical and isotope-tracing studies in oilseeds across species have revealed that glycolysis is partially bypassed by alternative routes involving ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) and NADPH-dependent malic enzyme (NADPH-ME). The role of these glycolytic shunts in the overall conversion of carbohydrates to TAG is not fully resolved. Here, we evaluate a minimal stoichiometric model for the carbohydrate-to-TAG conversion that satisfies complete cofactor balancing. Conversion solely through glycolysis and OPPP leads to NADH overproduction and cofactor imbalance. Balanced scenarios require inclusion of the malate shunt, the oxidative RubisCO shunt, or an NADPH-producing glyceraldehyde 3-phosphate dehydrogenase variant. All balanced routes also necessitate active mitochondrial oxidative phosphorylation to convert excess NADH to ATP. Applying a large-scale model of central metabolism in developing Brassica napus seeds, I further predict that glycolytic bypass usage increases in parallel with seed oil content, supporting their role in enabling high lipid biosynthetic fluxes under physiological constraints. HighlightConventional carbohydrate-to-triacylglycerol conversion is cofactor-imbalanced; flux balance analysis shows glycolytic bypasses help correct this, enabling higher biosynthetic rates.