Levan production and comparative genomic analysis of lichen-associated Bacillus licheniformis strains

Levan, a fructose homopolymer produced by many bacterial genera, particularly Bacillus, offers exceptional physicochemical properties and broad industrial potential. Lichens represent remarkable yet underexplored microhabitats which harbor many bacterial genera. Therefore, lichens may offer a unique opportunity to discover efficient biopolymer producing strains and bioactive compounds. Lichen-associated bacteria TLs4 and TLs25, isolated from Platismatia sp. and Pseudevernia sp., respectively, were cultivated in sucrose-rich medium and their exopolysaccharides were analyzed. Both strains produced exopolysaccharide yields (46.48 ± 3.46 and 43.44 ± 1.72 g/L) under in vitro conditions. NMR spectroscopy confirmed the exopolysaccharide is homogeneous β-(2/6)-linked levan, and GPC-MALLS analysis revealed a bimodal molecular weight distribution. Whole-genome sequence analysis revealed single circular chromosomes of 4.34 Mb (TLs4) and 4.27 Mb (TLs25), with >99% average nucleotide identity to Bacillus licheniformis. CAZyme profiling revealed rich carbohydrate-active enzyme repertoires in both genomes. GH68 levansucrases of both isolates are strictly conserved and identical to DSM13, indicating they are not unique to lichen-associated strains. Comparative genomic analysis revealed strain-specific features in prophage-associated regions, sugar nucleotide metabolism, ABC transporters, and biosynthetic gene clusters. AntiSMASH analysis revealed a beta-lactone-associated gene cluster potentially associated with a distinct fatty-acid/polyketide metabolite that requires experimental validation. While levan production within the lichen thallus and its ecological role remain unclear, this study establishes a valuable genomic foundation for future research on the biosynthetic potential of these bacteria, offering promising prospects for industrial applications and biotechnological development.