微生物活性天然产物可促进产生菌株的生态效益，并在临床和农业中被广泛应用于具有明确定义目标和潜在机制的情况。然而，它们的生物合成对产生菌株的生理效应仍然大部分未知。Actinosynnema pretiosum ATCC 31280产生的抗肿瘤青蒿菌素P-3（AP-3）被发现在高浓度下可以抑制产生菌株的生长，并靶向与细胞分裂有关的FtsZ蛋白。之前的研究表明ATCC 31280中AP-3可能存在其他隐藏的靶标。在本研究中，我们使用源自AP-3的光亲和探针进行药物蛋白组学方法，以分析AP-3与蛋白质组间的相互作用。AP-3与看似无关的脱氧胸腺苏糖二磷酸酶化酶、醛脱氢酶和依赖黄素的胸腺苷酸合酶等蛋白质发生特定的结合，分别参与细胞壁组装、中心碳代谢和核苷酸生物合成。AP-3作为这三个靶标蛋白的非竞争性抑制剂，在干扰多样化的代谢途径中对产生菌株产生生理应激。过表达这些靶标蛋白增加了菌株生物量并显著提高了AP-3的效价。这一发现表明，能够识别并改造活性天然产物的隐藏靶标可以深入了解微生物生理学，并提高产品效价。© 2023. Springer Nature Limited.

Microbial bioactive natural products mediate ecologically beneficial functions to the producing strains, and have been widely used in clinic and agriculture with clearly defined targets and underlying mechanisms. However, the physiological effects of their biosynthesis on the producing strains remain largely unknown. The antitumor ansamitocin P-3 (AP-3), produced by Actinosynnema pretiosum ATCC 31280, was found to repress the growth of the producing strain at high concentration and target the FtsZ protein involved in cell division. Previous work suggested the presence of additional cryptic targets of AP-3 in ATCC 31280. Herein we use chemoproteomic approach with an AP-3-derived photoaffinity probe to profile the proteome-wide interactions of AP-3. AP-3 exhibits specific bindings to the seemingly unrelated deoxythymidine diphosphate glucose-4,6-dehydratase, aldehyde dehydrogenase, and flavin-dependent thymidylate synthase, which are involved in cell wall assembly, central carbon metabolism and nucleotide biosynthesis, respectively. AP-3 functions as a non-competitive inhibitor of all three above target proteins, generating physiological stress on the producing strain through interfering diverse metabolic pathways. Overexpression of these target proteins increases strain biomass and markedly boosts AP-3 titers. This finding demonstrates that identification and engineering of cryptic targets of bioactive natural products can lead to in-depth understanding of microbial physiology and improved product titers.© 2023. Springer Nature Limited.