The TetR-like regulator Sco4385 and Crp-like regulator Sco3571 modulate heterologous production of antibiotics in <i>Streptomyces coelicolor</i> M512

Heterologous expression in well-studied model strains is a routinely applied method to investigate biosynthetic pathways. Here, we pursue a comparative approach of large-scale DNA-affinity-capturing assays (DACAs) coupled with semi-quantitative mass spectrometry (MS) to identify putative regulatory proteins from <i>Streptomyces coelicolor</i> M512, which bind to the heterologously expressed biosynthetic gene clusters (BGCs) of the liponucleoside antibiotics caprazamycin and liposidomycin. Both gene clusters share an almost identical genetic arrangement, including the location of promoter regions, as detected by RNA sequencing. A total of 2,214 proteins were trapped at the predicted promoter regions, with only three binding to corresponding promoters in both gene clusters. Among these, the overexpression of a yet uncharacterized TetR-family regulator (TFR), Sco4385, increased caprazamycin but not liposidomycin production. Protein-DNA interaction experiments using biolayer interferometry confirmed the binding of Sco4385 to P<i>cpz10</i> and P<i>lpmH</i> at different locations within both promoter regions, which might explain its functional variance. Sequence alignment allowed the determination of a consensus sequence present in both promoter regions, to which Sco4385 was experimentally shown to bind. Furthermore, we found that the overexpression of the Crp regulator, Sco3571, leads to a threefold increase in caprazamycin and liposidomycin production yields, possibly due to an increased expression of a precursor pathway.IMPORTANCEStreptomycetes are well-studied model organisms for the biosynthesis of pharmaceutically, industrially, and biotechnologically valuable metabolites. Their naturally broad repertoire of natural products can be further exploited by heterologous expression of biosynthetic gene clusters (BGCs) in non-native host strains. This approach forces the host to adapt to a new regulatory and metabolic environment. In our study, we demonstrate that a host regulator not only interacts with newly incorporated gene clusters but also regulates precursor supply for the produced compounds. We present a comprehensive study of regulatory proteins that interact with two genetically similar gene clusters for the biosynthesis of liponucleoside antibiotics. Thereby, we identified regulators of the heterologous host that influence the production of the corresponding antibiotic. Surprisingly, the regulatory interaction is highly specific for each biosynthetic gene cluster, even though they encode largely structurally similar metabolites.