Identification of putative producers of rhamnolipids/glycolipids and their transporters using genome mining

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• A total of 71 new putative rhamnolipid (RL)-producing species were discovered with genome mining. • Industrially interesting new RL producers included non-pathogenic and anaerobic species. • The identified RhlABC orthologs categorized into 11 distinct phylogenetic clades. • Most RL producers harbored a conserved putative RL transport mechanism, RhlDEF. • Transport genes (rhlDEF) co-localized primarily with biosynthetic gene clusters of RL. Rhamnolipids (RLs) are microbial glycolipids (GLs) with interesting structure-dependent bioactivities and physicochemical properties making them suitable for diverse medical and industrial applications. The discovery of RLs with more interesting bioactivities and properties has relied on laborious screening of new RL producers isolated from the environment, and has resulted in the redundant identification of already known RL producers and structures. Here, we present a genome mining approach that enabled the identification of 80 RL-producing species (including the two reference species), 71 of which were previously unreported. Distance trees of two of their RL biosynthetic enzymes, RhlAB, allowed for the identification of 11 distinct clades. Preliminary experimental validation with thin layer chromatography on one non-pathogenic RL/GL producer, Nevskia soli , confirmed its putative production of RLs. Additionally, this study led to the discovery of the putative RL transport mechanism involving three transmembrane proteins whose coding genes are highly conserved and clustered with one of the RL biosynthetic gene clusters in most RL/GL producers identified in this study.