Novel insights into biosynthesis and uptake of rhamnolipids and their precursors

The human pathogenic bacterium Pseudomonasaeruginosa produces rhamnolipids, glycolipids with functionsfor bacterial motility, biofilm formation, and uptake of hydrophobicsubstrates. Rhamnolipids represent a chemically heterogeneousgroup of secondary metabolites composed of one ortwo rhamnose molecules linked to one or mostly two 3-hydroxyfatty acids of various chain lengths. The biosyntheticpathway involves rhamnosyltransferase I encoded by the rhlABoperon, which synthesizes 3-(3-hydroxyalkanoyloxy)alkanoicacids (HAAs) followed by their coupling to one rhamnose moiety.The resulting mono-rhamnolipids are converted to dirhamnolipidsin a third reaction catalyzed by therhamnosyltransferase II RhlC. However, the mechanism behindthe biosynthesis of rhamnolipids containing only a singlefatty acid is still unknown. To understand the role of proteinsinvolved in rhamnolipid biosynthesis the heterologous expressionof rhl-genes in non-pathogenic Pseudomonas putidaKT2440 strains was used in this study to circumvent the complexquorum sensing regulation in P. aeruginosa. Our resultsreveal that RhlA and RhlB are independently involved inrhamnolipid biosynthesis and not in the form of a RhlAB heterodimercomplex as it has been previously postulated.Furthermore, we demonstrate that mono-rhamnolipids providedextracellularly as well as HAAs as their precursors are generallytaken up into the cell and are subsequently converted todi-rhamnolipids by P. putida and the native host P. aeruginosa.Finally, our results throw light on the biosynthesis ofrhamnolipids containing one fatty acid,which occurs by hydrolyzationof typical rhamnolipids containing two fatty acids,valuable for the production of designer rhamnolipids with desiredphysicochemical properties