Molecular basis of the negative regulator MihR2 in Miharamycin biosynthesis

Miharamycins (MIHs) are a structurally unique class of antifungal peptidyl nucleoside antibiotics from Streptomyces miharaensis, exhibiting potent antifungal activity against rice blast disease caused by Magnaporthe oryzae. Among the MIH family members, Miharamycin A (MIH A) is the major bioactive component. Elucidating the regulatory mechanism underlying its biosynthesis is critical for enhancing its production via metabolic engineering. Genetic deletion of mihR2 boosts MIH A production, identifying MihR2 as a novel transcription factor whose regulatory mechanism warrant further investigation. Here we demonstrate that MihR2 directly represses the MIH A biosynthesis by binding to PmihC2/K and PmihT/U promoters in Miharamycin A biosynthesis gene cluster. EMSA and fluorescence polarization assays defined the MihR2-binding DNA motif as a specific arrangement pattern 5'-GTCA-X_n-TGAC-3', while structural and mutagenesis analyses pinpointed the key DNA-binding residues in MihR2. Further functional validation showed that alanine substitution of R15, Q22, or R43 abolished repression, underscoring their essential role in MIH A biosynthesis control. The elucidation of the MihR2-mediated repression mechanism reveals a potentially novel transcriptional regulatory paradigm of natural product biosynthesis in Actinobacteria. This work reveals a previously uncharacterized regulatory mechanism in Streptomyces, expands the known diversity of transcriptional regulators in Actinobacteria, and provides a molecular basis for engineering secondary metabolite production by targeting repressive pathway control.