Combinatorial Metabolic and Process Engineering for Enhanced GA₄₊₇ Production and Tunable GA₄/GA₇ Ratio in Fusarium...

Gibberellins (GAs) are ubiquitous phytohormones that regulate plant growth and are widely used in agriculture. Among these, GA₃ and GA₄₊₇ are the only commercially available products, yet GA₄₊₇ commands a much higher price than GA₃, primarily due to its low titer in the industrial fungus Fusarium fujikuroi. To engineer a high-yielding GA₄₊₇ producer, we first deleted p450-3 to block the conversion of GA₄ and GA₇ to GA₁ and GA₃. This led to the accumulation of GA₄₊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 Δp450-3 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 GA₄₊₇ production. Under optimized conditions, the engineered strain achieved a final titer of 6.08 g/L (a 6.51-fold increase over the Δp450-3 parent), comprising 2.13 g/L GA₄ and 3.96 g/L GA₇, representing 5.30- and 7.44-fold increases, respectively. Preliminary optimization in a 10 L fermenter yielded 3.51 g/L of GA₄₊₇. Finally, a solid-state fermentation system was developed on wheat bran, yielding 17.34 g GA₄₊₇ per kg and enabling green, in-house, in-situ gibberellin production. In addition to substantially increasing the GA₄₊₇ titer and total GA accumulation, this study demonstrated that the GA₄/GA₇ ratio can be modulated through both molecular and fermentation strategies.