Regulator-Guided Strain Prioritization and Genome Engineering Uncovers Concanamycin Analogs.

Bafilomycin and concanamycin analogs (1-3) are microbial natural products with potent V-ATPase inhibitory activity. Despite their promising therapeutic potential, limited access to these plecomacrolide scaffolds has constrained their broader bioactivity evaluation. Here, we report an integrative approach combining genome mining, strain engineering, and molecular networking to expand the chemical diversity of natural concanamycin analogs. Genome analysis identified a concanamycin-like biosynthetic gene cluster in Streptomyces humidus with a unique genetic architecture, suggesting that it may encode previously uncharacterized analogs. Coupling strain engineering, untargeted metabolomics, and feature-based molecular networking of the engineered S. humidus strain expedited dereplication and visualization of an underexplored subnetwork of concanamycin-like metabolites. This prioritization strategy enabled the isolation of five known analogs (4-7, 9) and three new analogs that we termed tanamycin E, X, and F (10-12). Optimization of cultivation conditions for the engineered S. humidus strain facilitated the isolation of 93.0 mg of TAN-1323 D (9) and 24.7 mg of the newly identified compound 10, per liter of culture. Collectively, this work provides scalable strategies for accessing and prioritizing new derivatives, overcoming traditional limitations in the production and discovery of bioactive natural products.