A base-editing and auxotrophic selection platform for linking epigenetic regulators to silent biosynthetic gene...

Filamentous fungi harbor numerous silent biosynthetic gene clusters (BGCs), representing an underexplored reservoir of natural products. Chemical inhibition of epigenetic regulators is an effective strategy for activating silent BGCs, but it often lacks specificity and cannot readily link activated clusters to their cognate regulators. Here, we developed a coupled cytosine base editing and auxotrophic selection platform in Aspergillus nidulans to systematically identify epigenetic regulators of silent gene clusters. An sgRNA library targeting 24 core epigenetic regulators was introduced, together with a cytosine base editor, into 21 pyrG-based PKS reporter strains, enabling growth-based selection and direct sgRNA recovery by Sanger sequencing. This screen identified five regulator-cluster activation pairs, including known interactions and two previously uncharacterized mechanisms. Specifically, KdmB represses the AN7084 gene cluster by erasing promoter H3K4me3, whereas Kmt5 silences the cichorine core pks gene through H4K20me3 deposition. This scalable platform enables rapid matching of silent BGCs with their epigenetic regulators and advances chromatin-guided fungal genome mining.