Improved production of two anti-Candida lipopeptide homologues co-produced by the wild-type Bacillus subtilis RLID 12.1 under optimized conditions

Antifungal cyclic lipopeptides, bioactive metabolites produced by many species of the genus Bacillus are promising alternatives to synthetic fungicide and antibiotics for the biocontrol of human pathogenic fungi. In our previous study, we reported the co- production of five antifungal lipopeptides homologues (designated as AF1, AF2, AF3, AF4 and AF5) by the producer strain Bacillus subtilis RLID 12.1 using unoptimized medium; though two homologues AF3 and A5 differed by 14 Da and in fatty acid chain length were found effective in antifungal action, the production/yield rate of these two lipopeptides determined by High-Performance Liquid Chromatography was less in the unoptimized media. In this study, we specifically targeted the production/yield enhancement of these two compounds. Following the statistical optimization (Plackett-Burman and Box- Behnken designs) of media formulation, temperature and growth conditions, the production of AF3 and AF5 was improved by about 25.8- and 7.4-folds, respectively under static condition. To boost the production of these two homologous lipopeptides in the optimized media heat-inactivated Candida albicans cells were used as supplement resulting in 34- and 14-fold increase of AF3 and AF5 respectively. Four clinical Candida auris isolates had AF3 and AF5 MICs (100 % inhibition) ranging between 4 and 16 microg/ml indicating the lipopeptide's clinical potential. To determine the in vitro pharmacodynamic potential of AF3 and AF5, time kill assays were conducted which showed that AF3 (at 4X and 8X concentrations) at 48h exhibited mean log reductions of 2.31 and 3.14 CFU/ml of C. albicans SC 5314 respectively whereas AF5 at 8X concentration showed a mean log reduction of 2.14 CFU/ml. With the increasing threat of multidrug-resistant yeasts and fungi, these antifungal lipopeptides produced by optimized method promise to aid in the development of novel antifungal that target disease-causing fungi with improved efficacy.