Combinatorial Metabolic and Process Engineering for Enhanced GA 4+7 Production and Tunable GA 4 /GA 7 Ratio in Fusarium fujikuroi

ABSTRACT Gibberellins (GAs) are ubiquitous phytohormones that regulate plant growth and are widely used in agriculture. Among these, GA 3 and GA 4+7 are the only commercially available products, yet GA 4+7 commands a much higher price than GA 3 , primarily due to its low titer in the industrial fungus Fusarium fujikuroi . To engineer a high‐yielding GA 4+7 producer, we first deleted p450‐3 to block the conversion of GA 4 and GA 7 to GA 1 and GA 3 . This led to the accumulation of GA 4+7 at 0.934 g/L, a 37‐fold increase over the wild‐type strain, albeit with over half reduction in total GA accumulation. Using this Δ p450‐3 mutant as a platform, we combined metabolic engineering (overexpressing rate‐limiting enzymes) with process optimization (pH, medium composition and fermentation duration). These combinatorial interventions synergistically boosted GA 4+7 production. Under optimized conditions, the engineered strain achieved a final titer of 6.08 g/L (a 6.51‐fold increase over the Δ p450‐3 parent), comprising 2.13 g/L GA 4 and 3.96 g/L GA 7 , representing 5.30‐ and 7.44‐fold increases, respectively. Preliminary optimization in a 10 L fermenter yielded 3.51 g/L of GA 4+7 . Finally, a solid‐state fermentation system was developed on wheat bran, yielding 17.34 g GA 4+7 per kg and enabling green, in‐house, in‐situ gibberellin production. In addition to substantially increasing the GA 4+7 titer and total GA accumulation, this study demonstrated that the GA 4 /GA 7 ratio can be modulated through both molecular and fermentation strategies.