Metabolic Engineering of Saccharomyces cerevisiae for De Novo Biosynthesis of Gibberellin A 4

Gibberellin A 4 (GA 4 ), a bioactive diterpenoid phytohormone with important agricultural uses, was efficiently biosynthesized in engineered Saccharomyces cerevisiae to overcome traditional fungal fermentation limitations. We established an ent -kaurenoic acid (KA) foundation by introducing plant/fungal enzymes (e.g., Xd GGPPS, Gf CPS/KS, and Sr KO) and enhancing the mevalonate pathway precursor supply. To optimize the metabolic flux distribution, dynamic ERG9 regulation was implemented alongside ergosterol-responsive promoter P ERG1 to balance growth and production. For GA 4 synthesis, fungal-derived cytochrome P450 systems demonstrated superior heterologous expression compared to plant counterparts. Multi-copy chromosomal integration and endoplasmic reticulum engineering ( INO2 / ICE2 overexpression and PAH1 knockout) alleviated P450 membrane saturation bottlenecks. Under optimized fermentation with nitrogen supplementation in a 5 L bioreactor, the engineered strain achieved 48.6 mg/L of GA 4 production. We have established a robust platform integrating modular design, dynamic regulation, and organellar engineering for complex terpenoid biosynthesis, providing valuable insights for microbial gibberellin production.