Abstract
This study aimed to optimize culture media to enhance vancomycin production by Amycolatopsis orientalis. Using a combination of one-variable-at-a-time (OVAT) analysis, nutrient screening, and knockout experiments, we identified critical factors influencing biomass formation and antibiotic yield. Among tested carbon sources, maltodextrin significantly increased vancomycin production (bioassay zone: 13.33 mm), while glycerol favored biomass growth but had minimal impact on antibiotic synthesis. For nitrogen sources, soybean meal and soya flour optimally supported both biomass and vancomycin yield. Salt supplementation revealed that CaCO₃ and MgSO₄ improved mycelial growth, whereas knockout studies demonstrated that dextrose and soya peptone were essential for vancomycin production. Notably, omitting phosphate (K₂HPO₄) enhanced both biomass and antibiotic yield, suggesting phosphate repression of secondary metabolism. Seed media trials identified SS-6 and SS-10 + A as optimal for mycelial growth, highlighting the importance of early-stage culture conditions for fermentation outcomes. These findings provide a cost-effective, scalable strategy for maximizing vancomycin production, with potential for further optimization using statistical or metabolic flux approaches.