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

Gibberellic acid 3 (GA), a diterpenoid phytohormone industrially biosynthesized by Fusarium fujikuroi, serves as a pivotal plant growth regulator with extensive agricultural applications. Currently, industrial GA 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 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 production to 2.86 g/L, representing a 10.9% increase. This study demonstrates the feasibility of coordinated metabolic modifications for improving GA biosynthesis in F. fujikuroi, offering practical insights for overcoming productivity limitations in fungal secondary metabolite fermentation processes.