Establishment of a nonhomologous end joining-mediated genomic library in Fusarium fujikuroi for high-level production of gibberellic acid

Gibberellic acid (GA3) is primarily produced by the liquid fermentation of Fusarium fujikuroi , yet its complex metabolic network limits rational strain engineering. Here, we strengthened the nonhomologous end joining (NHEJ) pathway and mapped the distribution of integration to establish a genome-scale insertional mutagenesis library in F. fujikuroi . The library enabled rapid identification of ten novel genomic loci that support stable heterologous gene expression, thereby expanding the available genetic toolkit. We further applied the library to uncover functional determinants of GA3 biosynthesis and acetic acid tolerance, identifying previously uncharacterized gene targets. Reverse engineering of these targets yielded a GA3-overproducing strain capable of tolerating and utilizing acetic acid wastewater. In a 5-l bioreactor, the engineered strain NH4 reached a GA3 titer of 3.32 g/l, 3.05-fold higher than the control strain. This study establishes an efficient NHEJ-mediated genome-scale mutagenesis platform for F. fujikuroi and provides more genetic targets for constructing high-yield GA3 cell factories.