LAUSR

Significance Streptomyces genomes harbor a trove of biosynthetic gene clusters (BGCs) that encode for drug-like molecules. However, only a fraction of these readily yield expected products. To investigate why this is, we used polycyclic tetramate macrolactam (PTM) antibiotic production as a model system. By comparing the genomes and PTM production profiles of several closely related Streptomyces griseus clade members, we uncovered two mechanisms that differentiate more robust producers from weaker ones. The first involves small insertion–deletion lesions in PTM BGC promoters that significantly modulate production. The second mechanism involves biosynthetic pathway interactions, in which robust PTM producers unexpectedly benefit from griseorhodin coproduction, and weaker producers lack the pathway. We highlight comparative metabologenomics as a powerful approach to understand antibiotic crypticity. Streptomyces genomes harbor numerous, biosynthetic gene clusters (BGCs) encoding for drug-like compounds. While some of these BGCs readily yield expected products, many do not. Biosynthetic crypticity represents a significant hurdle to drug discovery, and the biological mechanisms that underpin it remain poorly understood. Polycyclic tetramate macrolactam (PTM) antibiotic production is widespread within the Streptomyces genus, and examples of active and cryptic PTM BGCs are known. To reveal further insights into the causes of biosynthetic crypticity, we employed a PTM-targeted comparative metabologenomics approach to analyze a panel of S. griseus clade strains that included both poor and robust PTM producers. By comparing the genomes and PTM production profiles of these strains, we systematically mapped the PTM promoter architecture within the group, revealed that these promoters are directly activated via the global regulator AdpA, and discovered that small promoter insertion–deletion lesions (indels) differentiate weaker PTM producers from stronger ones. We also revealed an unexpected link between robust PTM expression and griseorhodin pigment coproduction, with weaker S. griseus–clade PTM producers being unable to produce the latter compound. This study highlights promoter indels and biosynthetic interactions as important, genetically encoded factors that impact BGC outputs, providing mechanistic insights that will undoubtedly extend to other Streptomyces BGCs. We highlight comparative metabologenomics as a powerful approach to expose genomic features that differentiate strong, antibiotic producers from weaker ones. This should prove useful for rational discovery efforts and is orthogonal to current engineering and molecular signaling approaches now standard in the field.