Enhanced Lichenysin Production in Bacillus licheniformis by Physicochemical Mutagenesis Combined with High-Throughput...

Lichenysin is a cyclic lipopeptide secondary metabolite produced by Bacillus licheniformis, with excellent biocompatibility and diverse biological activities. It has broad application potential in biomedicine, agricultural pest control, energy production, and environmental remediation. Nevertheless, the improvement of wild strain with complex genetic backgrounds through genetic and metabolic pathway modifications poses significant challenges in terms of time and cost, which has become a bottleneck limiting the diversified development of lichenysin-producing strains. In this research, ultraviolet (UV) irradiation, N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethyl methanesulfonate (EMS) and composite mutagenesis strategies were systematically utilized to conduct multiple rounds of mutagenesis using Bacillus licheniformis Ali5 as the parental strain. Subsequently, the mutants were screened through high-throughput screening in a 96-well plate based on the hexadecylpyridinium chloride-fluorescein (CPC-FL) fluorescence detection principle. After two rounds of mutagenesis and genetic stability assessment, five high-yielding mutant strains with stable genetic traits (UV40-4, NT382-5, EMS191-1, NV341-3 and EV52-1) were successfully screened. The results of flask culture illustrated that the mutants were superior to the parental strain in biomass, the titer/OD₆₀₀ and lichenysin yield (up to 20.6%). This study established a quick and scalable mutagenesis and high-throughput screening system, which provided high-quality strains for its industrial-scale production.