A portable pathway specific transcription factor-promoter circuit enables heterologous production of fungal nonribosomal peptides.

Most fungal nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters remain transcriptionally silent, severely restricting the discovery of bioactive peptide metabolites. Standard constitutive promoters fail to support robust expression of large multidomain NRPS enzymes. Here, we identify a conserved subgroup of Zn(ii)2Cys6 transcription factors (TFs) encoded within fungal NRPS clusters and functionally characterize TdaN, which triggers transcription from its cognate promoter P tdaA by recognizing paired CGG-type cis-regulatory motifs. The TdaN-P tdaA pair is repurposed as a portable transcriptional circuit capable of activating five out of seven heterologous NRPS/NRPS-like genes in Trichoderma hypoxylon, notably the recalcitrant silent synthetase Afpes1 that previously demanded screening over 90 distinct promoters for trace product yields. Circuit-driven overexpression of ftmA yields abundant cyclo-l-Trp-l-Pro (cWP) and seven structurally diversified cWP derivatives. This study reveals a conserved regulatory paradigm for fungal nonribosomal peptide biosynthesis and delivers a versatile transcriptional module to expand cryptic peptide chemical diversity.