Optimization and Characterization of Bioactive Metabolites from Cave-Derived Rhodococcus jialingiae C1

Extremophilic microorganisms offer an untapped potential for producing unique bioactive metabolites with therapeutic applications. In the current study, bacterial isolates were obtained from samples collected from Chamalang cave located in Kohlu District, Balochistan, Pakistan. The cave-derived isolate C1 (<i>Rhodococcus jialingiae</i>) exhibits prominent antibacterial activity against multidrug-resistant pathogens (MDR), including <i>Escherichia coli</i>, <i>Staphylococcus aureus</i>, and <i>Micrococcus luteus</i>. It also demonstrates substantial antioxidant activity, with 71% and 58.39% DPPH radical scavenging. Optimization of physicochemical conditions, such as media, pH, temperature, and nitrogen and carbon sources and concentrations substantially enhanced both biomass and metabolite yields. Optimal conditions comprise specialized media, a pH of 7, a temperature of 30 °C, peptone (1.0 g/L) as the nitrogen source, and glucose (0.5 g/L) as the carbon source. HPLC and QTOF-MS analyses uncovered numerous metabolites, including a phenolic compound, 2-[(E)-3-hydroxy-3-(4-methoxyphenyl) prop-2-enoyl]-4-methoxyphenolate, Streptolactam C, Puromycin, and a putative aromatic polyketide highlighting the C1 isolate chemical. Remarkably, one compound (C₁₄H₃₆N₇) demonstrated a special molecular profile, signifying structural novelty and warranting further characterization by techniques such as ¹H and ¹³C NMR. These findings highlight the biotechnological capacity of the C1 isolate as a source of novel antimicrobials and antioxidants, linking environmental adaptation to metabolic potential and supporting natural product discovery pipelines against antibiotic resistance.