Abstract
Caves are a unique ecosystem that harbor diverse microorganisms, and provide a challenging environment to the dwelling microbial communities, which may boost gene expression and can lead to the production of inimitable bioactive natural products. In this study, we obtained 59 actinobacteria from four different caves located in Bahadurkhel, District Karak, Pakistan. On the basis of taxonomic characteristics, 30 isolates were selected and screened for secondary metabolites production and bioactivity profiling. The extracts of all the isolates exhibited promising antibacterial activity against several pathogenic bacteria, with the best outcome seen in the extract of isolate SNK 21. The metabolomic analysis of the extracts by LC-MS/MS-based molecular networking and whole genome sequencing (WGS) followed by antiSMASH analysis revealed the presence of diverse secondary metabolites and biosynthetic gene clusters (BGCs) in SNK 21. Purification of compounds by manual chromatography, HPLC, and characterization by NMR, HR-MS, led to the identification of the active compounds, actinomycin D and its isomer. In addition, metabolomic analysis and genome mining of morphologically distinct isolates, SNK 202 and SNK 329, also showed diverse secondary metabolites and BGCs, underscoring the potential of actinobacteria from undisturbed caves in Pakistan as a new source of bioactive compounds.