Native Promoter Strategy for High-Yielding Synthesis and Engineering of Fungal Secondary Metabolites

[Image: see text] Strategies are needed for the robust production of cryptic, silenced, or engineered secondary metabolites in fungi. The filamentous fungus Fusarium heterosporum natively synthesizes the polyketide equisetin at >2 g L(–1) in a controllable manner. We hypothesized that this production level was achieved by regulatory elements in the equisetin pathway, leading to the prediction that the same regulatory elements would be useful in producing other secondary metabolites. This was tested by using the native eqxS promoter and eqxR regulator in F. heterosporum, synthesizing heterologous natural products in yields of ∼1 g L(–1). As proof of concept for the practical application, we resurrected an extinct pathway from an endophytic fungus with an initial yield of >800 mg L(–1), leading to the practical synthesis of a selective antituberculosis agent. Finally, the method enabled new insights into the function of polyketide synthases in filamentous fungi. These results demonstrate a strategy for optimally employing native regulators for the robust synthesis of secondary metabolites.