Biosynthesis of ansamitocin P-3 incurs stress on the producing strain Actinosynnema pretiosum at multiple targets

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.