Discovery of unprecedented prenylated indole piperazines and pyrazines through cryptic biosynthetic gene cluster heterologous expression.

Prenylation modifications of natural products typically introduce greater structural complexity and enhance their biological activities. Yet, the modification of piperazine alkaloids by dimethylallyl tryptophan synthases (DMATS) remains unreported. In this study, we identified and activated a silent DMATS-containing piperazine biosynthetic gene cluster (BGC), flz, in Aspergillus flavipes via heterologous expression and in vitro enzymatic assay, leading to the isolation and identification of sixteen metabolites. Among these, twelve are new, including five tryptophan-valine-derived alkaloids (2-6) and seven previously unreported prenylated analogs (8-13, 17). Notably, 8 and 9 represent novel prenylated piperazines featuring a unique 6-5-5-6 ring system. Most significantly, we uncovered a versatile DMATS, FlzE, capable of catalyzing mono-prenylation on flexible substrates, such as piperazine, pyrazine, and diketopiperazine, at multiple sites in either regular or reverse manners. This study not only expands the chemical space of indole alkaloid derivatives but also provides a versatile and engineerable biocatalyst for the prenylation of both natural and synthetic products.