Combinatorial Strategy of Modular Metabolic Engineering and Fermentation Optimization Jointly Improved Gibberellic Acid Production in Fusarium fujikuroi

ABSTRACT Gibberellic acid 3 (GA 3 ), a diterpenoid phytohormone industrially biosynthesized by Fusarium fujikuroi , serves as a pivotal plant growth regulator with extensive agricultural applications. Currently, industrial GA 3 production predominantly relies on prolonged submerged microbial fermentation with F. fujikuroi as the main production strain, valued for its native biosynthetic capacity. Nevertheless, large‐scale industrialization of GA 3 remains constrained by low production yields. In this study, a systematic multimodular metabolic engineering framework was implemented to enhance GA₃ biosynthesis in F. fujikuroi . The engineering strategy encompassed four synergistic modules: reinforcement of fatty acid biosynthesis, augmentation of acetyl‐CoA metabolic flux, optimization of redox cofactor homeostasis, and overexpression of the positive transcriptional regulator. This integrated approach yielded the engineered strain OE: Lae1‐AGP3 demonstrating a 2.58 g/L GA₃ titer in shake‐flask fermentation. Subsequent bioprocess optimization through exogenous fatty acid supplementation further elevated GA 3 production to 2.86 g/L, representing a 10.9% increase. This study demonstrates the feasibility of coordinated metabolic modifications for improving GA 3 biosynthesis in F. fujikuroi , offering practical insights for overcoming productivity limitations in fungal secondary metabolite fermentation processes.