Beyond direct pathway engineering: reprogramming Fusarium fujikuroi from a GA₃ producer into a GA₄₊₇ factory

Synthetic biology is emerging as a key approach in chemical synthesis, whose efficiency hinges crucially on the direct engineering of metabolic pathways. In this study, we propose a dual-intervention paradigm to reprogram the industrial fungus Fusarium fujikuroi from a default gibberellic acid (GA₃) producer into an exclusive factory for the higher-value gibberellin GA₄₊₇, providing a complementary and orthogonal approach to traditional intra-pathway manipulations. First, by introducing Arabidopsis-derived transporters (Npfs and Sweets), we successfully created a thermodynamic sink that actively depletes intracellular GA₄/GA₇ pools. With the best candidate protein, Sweet1, the parent strain was converted into an exclusive producer of GA₄₊₇ (with GA₃ levels undetectable), a conversion driven by the significantly accelerated dissociation rate (K_dis) for GA₇. Concurrently, we uncovered a non-canonical, highly specific regulatory mechanism: overexpression of the Sfp-type 4'-phosphopantetheinyl transferase Ppt1 triggered targeted post-transcriptional silencing of up to 99.9 % of P450-3 mRNA, thereby completely silencing GA₃ biosynthesis and again yielding an exclusive producer of GA₄₊₇. Synergistic integration of transporter-driven spatial pulling and Ppt1-mediated gene silencing, coupled with fermentation optimization, propelled the final GA₄₊₇ titer to an unprecedented 3.29 g/L (reaching 0.4 g/L for GA₄ and 2.89 g/L for GA₇, representing 17.39-, 962.33-, and 125.54-fold increases over the parent strain, respectively). This study achieves indirect metabolic reprogramming not via conventional pathway engineering, but by targeting a membrane transport bottleneck and employing a putative post-transcriptional silencing mechanism.