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22 PROCEEDINGS OF INTERNATIONAL SCIENTIFIC CONFERENCE ON APPLIED BIOTECHNOLOGYThe mechanisms of action of anti-TB compounds derived from actinomycetes are as diverse as their chemical structures. These mechanisms often target essential biological processes in M. tuberculosis, such as cell wall synthesis, protein translation, and energy metabolism. For example, rifamycin inhibits RNA polymerase, while streptomycin disrupts protein synthesis. Recent studies have identified additional modes of action, including interference with quorum sensing, disruption of lipid metabolism, and inhibition of virulence factors. The exploration of such mechanisms not only enhances our understanding of TB pathogenesis but also aids in overcoming resistance by targeting novel pathways [5]. This study focuses on leveraging the vast actinomycetes library to identify and characterize novel anti-tuberculosis compounds. By integrating advanced screening techniques and mechanistic studies, we aim to uncover new chemical entities that can contribute to the global fight against TB. 2. MATERIALS AND METHODS 2.1. Construction of ECUM libraryTo establish the extract library, soil samples were collected from diverse sites around southeastern Asia, focusing on environments with unusual weather conditions and ecologies, e.g., alpine, tropical, polar, and desert regions. Pure microbe was isolated from soil by using humic acid agar plate, a medium specific designed for isolation of actinomycetes. For long term storage, glycerol stock were made and stored at -80%u00b0C. Every microbe was fermented by using three different liquid media, Bennett%u2019s (BN) medium, glucose-soybean starch (GSS) medium, and dextrin-yeast-corn steep liquor (DYC) medium medium and extracted with organic solvent. After incubation at