Resistance Gene-Guided Genome Mining: Serial Promoter Exchanges in Aspergillus nidulans Reveal the Biosynthetic Pathway for Fellutamide B, a Proteasome Inhibitor

Fungal genome projects are revealing thousands of cryptic secondary metabolism (SM) biosynthetic gene clusters that encode pathways that potentially produce valuable compounds. Heterologous expression systems should allow these clusters to be expressed and their products obtained, but approaches are needed to identify the most valuable target clusters. The inp cluster of Aspergillus nidulans contains a gene, inpE, that encodes a proteasome subunit, leading us to hypothesize that the inp cluster produces a proteasome inhibitor and inpE confers resistance to this compound. Previous efforts to express this cluster have failed, but by sequentially replacing the promoters of the genes of the cluster with a regulatable promotor, we have expressed them successfully. Expression reveals that the product of the inp cluster is the proteasome inhibitor fellutamide B, and our data allow us to propose a biosynthetic pathway for the compound. By deleting inpE and activating expression of the inp cluster, we demonstrate that inpE is required for resistance to internally produced fellutamide B. These data provide experimental validation for the hypothesis that some fungal SM clusters contain genes that encode resistant forms of the enzymes targeted by the compound produced by the cluster.