Maize remains a dietary cornerstone worldwide, yet its nutritional profile is constrained by intrinsically low lysine content an essential amino acid for human and monogastric animal nutrition due to the predominance of lysine-deficient zein storage proteins. The opaque2 (o2) mutation confers increased lysine accumulation via transcriptional suppression of zein-encoding genes, yet the metabolic perturbations underlying this phenotype, particularly trade-offs with starch biosynthesis, remain incompletely elucidated. Here, we employed a genome-scale metabolic model of developing maize endosperm (iZM-Endosperm), quantitatively integrated with temporal transcriptomic profiles spanning 6 to 30 days after pollination (DAP), to characterize genotype-specific metabolic bottlenecks in wild-type and o2 tissues. At 15 DAP coinciding with the grain-filling transition o2 endosperm exhibited intensified flux constraints across aspartate/glutamate pathways, the tricarboxylic acid (TCA) cycle, and cofactor-coupled reactions involving folate and NADPH. These were driven by nitrogen and carbon flux reallocation following zein repression. Notably, rate-limiting control points at aspartate kinase and pyruvate dehydrogenase were saturated by precursor demand rather than catalytic insufficiency, culminating in lysine hyperaccumulation. In parallel, starch biosynthetic fluxes were attenuated via limitations in pyruvate-to-acetyl-CoA conversion, malonyl-CoA turnover, and redox cofactor availability, revealing reprogrammed carbon allocation architecture. By contrast, the wild-type prioritized zein and secondary metabolite biosynthesis, with constraints in aromatic amino acid and lipid metabolism that limited lysine and starch fluxes. By 18 DAP, persistent cofactor and TCA cycle constraints in o2 revealed sustained competition with carotenoid and fatty acid elongation pathways. These findings designate 15 DAP as a critical developmental inflection point where o2 reallocates metabolic capacity to favor lysine over starch. The co-localization of dual-function bottlenecks namely, aspartate kinase and pyruvate dehydrogenase establishes rational targets for endosperm-specific metabolic engineering. Future work will pursue experimental validation and implement cofactor regeneration, precursor channeling, and synthetic bypasses to simultaneously enhance lysine content and restore starch biosynthetic efficiency.
