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.

链霉菌基因组包含许多编码类药物化合物的生物合成基因簇 (BGC)。虽然这些 BGC 中的一些很容易产生预期的产品，但许多没有。生物合成的神秘性是药物发现的一个重大障碍，支撑它的生物学机制仍然知之甚少。多环四环内酰胺 (PTM) 抗生素生产在链霉菌属中广泛存在，并且已知活性和隐秘 PTM BGC 的例子。为了进一步深入了解生物合成隐秘性的原因，我们采用了 PTM 靶向比较代谢基因组学方法来分析一组灰色 S.进化枝菌株，包括较差和强大的 PTM 生产者。通过比较这些菌株的基因组和 PTM 产生谱，我们系统地绘制了该组内的 PTM 启动子结构，揭示这些启动子是通过全局调节因子 AdpA 直接激活的，并发现小的启动子插入-缺失病变（indels）分化较弱来自更强大的 PTM 生产商。我们还揭示了强大的 PTM 表达与灰鞘色素联合生产之间的意外联系，较弱的S. griseus – clade PTM 生产者无法生产后一种化合物。这项研究强调启动子插入缺失和生物合成相互作用是影响 BGC 输出的重要的遗传编码因素，提供的机制见解无疑将扩展到其他链霉菌BGC。我们强调比较代谢基因组学是一种强大的方法，可以揭示区分强抗生素生产者与弱抗生素生产者的基因组特征。这应该证明对合理的发现工作很有用，并且与目前该领域标准的当前工程和分子信号传导方法是正交的。