An Aspergillus nidulans Platform for the Complete Cluster Refactoring and Total Biosynthesis of Fungal Natural Products.

Fungal natural products (NPs) comprise a vast number of bioactive molecules with diverse activities, and among them are many important drugs. However, the yields of fungal NPs from native producers are usually low, and total synthesis of structurally complex NPs is challenging. As such, downstream derivatization and optimization of lead fungal NPs can be impeded by the high cost of obtaining sufficient starting material. In recent years, reconstitution of NP biosynthetic pathways in heterologous hosts has become an attractive alternative approach to produce complex NPs. Here, we present an efficient, cloning-free strategy for the cluster refactoring and total biosynthesis of fungal NPs in Aspergillus nidulans. Our platform places our genes of interest (GOIs) under the regulation of the robust asperfuranone afo biosynthesis gene machinery, allowing for their concerted activation upon induction. We demonstrated the utility of our system by creating strains that can synthesize high-value NPs, citreoviridin (1), mutilin (2), and pleuromutilin (3), with good to high yield and purity. This platform can be used not only for producing NPs of interests (i.e., total biosynthesis) but also for elucidating cryptic biosynthesis pathways.