Combinatorial Strategy of Modular Metabolic Engineering and Fermentation Optimization Jointly Improved Gibberellic...

Gibberellic acid 3 (GA₃), a diterpenoid phytohormone industrially biosynthesized by Fusarium fujikuroi, serves as a pivotal plant growth regulator with extensive agricultural applications. Currently, industrial GA₃ 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 GA₃ 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 GA₃ production to 2.86 g/L, representing a 10.9% increase. This study demonstrates the feasibility of coordinated metabolic modifications for improving GA₃ biosynthesis in F. fujikuroi, offering practical insights for overcoming productivity limitations in fungal secondary metabolite fermentation processes.