Gene Mining and Optimization of the Biosynthesis of Cannabidiol in <i>Fusarium solani</i> XG4-2

Cannabidiol (CBD) exhibits various pharmacological properties, including antidepressant, antioxidant, antipsychotic, analgesic, and neuroprotective effects. However, the low natural yields of CBD and the restricted biosynthetic pathways currently limit its commercial viability and demand in the marketplace. This study evaluated the potential of 101 endophytic fungi from <i>Cannabis sativa</i> for biotransformation and CBD synthesis. Notably, strain XG4-2 (CGMCC 40476) stably produced 50.07 μg/L CBD in a basic medium and shared 99.42% ITS sequence homology with <i>Fusarium solani</i>, representing the first endophytic fungal reported to synthesize CBD. To enhance CBD production, a triple-strategy approach was implemented, comprising cocultivation with engineered <i>Saccharomyces cerevisiae</i>, optimization of fermentation conditions, and the addition of host-derived plant inducers. This integrated strategy led to a significant increase in CBD yield, reaching 526.9 μg/L, a 9.52-fold enhancement compared to the initial output. Whole-genome sequencing of XG4-2 revealed 44 putative secondary metabolite biosynthetic gene clusters and 23 genes potentially involved in CBD biosynthesis, indicating a unique metabolic pathway distinct from that of plants. This study demonstrates a novel approach for microbial CBD production by leveraging endophytic fungal resources, cocultivation strategies, fermentation optimization, and gene discovery. The findings provide a foundation for sustainable and efficient biotechnological production of CBD.