Combinatorial Metabolic Engineering for Enhanced Gibberellic Acid Biosynthesis in <i>Fusarium fujikuroi</i>

Gibberellic acid (GA₃), a quintessential diterpenoid phytohormone, is indispensable in agronomic practices, horticulture, and the wine industry. This study implemented a combinatorial metabolic engineering strategy within Fusarium fujikuroi (F. fujikuroi) by integrating the potentiation of global regulatory factors (GRFs), and amplification of biosynthetic precursors, alongside dynamic modulation of cofactors with dissolved oxygen supply, to precisely enhance GA₃ biosynthesis. Transcriptomic analyses revealed that positive GRFs (AreB, Hat1, and Ada3) enhanced carbon and nitrogen metabolism, increased biomass accumulation, and upregulated transcription levels of the GA₃ biosynthetic gene cluster. The use of endogenous nitrogen-responsive promoters ensured a balanced supply of cofactors and oxygen, thereby preventing the accumulation of terpenoid by-products. These combinatorial metabolic engineering strategies presented in this study make a significant step toward the enhancement of GA₃ yield (3.22 g/L) via submerged fermentation of F. fujikuroi, offering novel insights to enable high-level biosynthesis of secondary metabolites in fungal chassis.