title: Novel insights into biosynthesis and uptake of rhamnolipids and their precursors.
abstract: The human pathogenic bacterium Pseudomonas aeruginosa produces rhamnolipids, glycolipids with functions for bacterial motility, biofilm formation, and uptake of hydrophobic substrates. Rhamnolipids represent a chemically heterogeneous group of secondary metabolites composed of one or two rhamnose molecules linked to one or mostly two 3-hydroxyfatty acids of various chain lengths. The biosynthetic pathway involves rhamnosyltransferase I encoded by the rhlAB operon, which synthesizes 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) followed by their coupling to one rhamnose moiety. The resulting mono-rhamnolipids are converted to di-rhamnolipids in a third reaction catalyzed by the rhamnosyltransferase II RhlC. However, the mechanism behind the biosynthesis of rhamnolipids containing only a single fatty acid is still unknown. To understand the role of proteins involved in rhamnolipid biosynthesis the heterologous expression of rhl-genes in non-pathogenic Pseudomonas putida KT2440 strains was used in this study to circumvent the complex quorum sensing regulation in P. aeruginosa. Our results reveal that RhlA and RhlB are independently involved in rhamnolipid biosynthesis and not in the form of a RhlAB heterodimer complex as it has been previously postulated. Furthermore, we demonstrate that mono-rhamnolipids provided extracellularly as well as HAAs as their precursors are generally taken up into the cell and are subsequently converted to di-rhamnolipids by P. putida and the native host P. aeruginosa. Finally, our results throw light on the biosynthesis of rhamnolipids containing one fatty acid, which occurs by hydrolyzation of typical rhamnolipids containing two fatty acids, valuable for the production of designer rhamnolipids with desired physicochemical properties.
authors.affiliation: Ulm Center for Peptide Pharmaceuticals (U-PEP), Ulm University, Albert-Einstein-Allee 11, 89081, Ulm, Germany. andreas.wittgens@uni-ulm.de.|Institute for Molecular Enzyme Technology (IMET), Heinrich-Heine-University Düsseldorf, Forschungszentrum Jülich, Wilhelm-Johnen-Straße, 52428, Jülich, Germany. andreas.wittgens@uni-ulm.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Wittgens
authors.affiliation: Institute for Molecular Enzyme Technology (IMET), Heinrich-Heine-University Düsseldorf, Forschungszentrum Jülich, Wilhelm-Johnen-Straße, 52428, Jülich, Germany.
authors.forename: Filip
authors.initials: F
authors.lastname: Kovacic
authors.affiliation: Boehringer Ingelheim Pharma GmbH & Co. KG, Biopharmaceutical and Analytical Development, Birkendorfer Straße 65, 88400, Biberach an der Riß, Germany.
authors.forename: Markus Michael
authors.initials: MM
authors.lastname: Müller
authors.affiliation: Institute of Process Engineering in Life Sciences, Section II: Technical Biology, Karlsruhe Institute of Technology (KIT), Engler-Bunte-Ring 1, 76131, Karlsruhe, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Gerlitzki
authors.affiliation: Central Institute for Engineering, Electronics and Analytics, Section Analytics (ZEA-3), Forschungszentrum Jülich, Wilhelm-Johnen-Straße, 52428, Jülich, Germany.
authors.forename: Beatrix
authors.initials: B
authors.lastname: Santiago-Schübel
authors.affiliation: Institute for Bio- and Geosciences, IBG-3: Agrosphere, Forschungszentrum Jülich, Wilhelm-Johnen-Straße, 52428, Jülich, Germany.
authors.forename: Diana
authors.initials: D
authors.lastname: Hofmann
authors.affiliation: Institute of Applied Microbiology (iAMB), Aachen Biology and Biotechnology (ABBt), RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany.
authors.forename: Till
authors.initials: T
authors.lastname: Tiso
authors.affiliation: Institute of Applied Microbiology (iAMB), Aachen Biology and Biotechnology (ABBt), RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany.
authors.forename: Lars Mathias
authors.initials: LM
authors.lastname: Blank
authors.affiliation: Institute of Food Science and Biotechnology, Department of Bioprocess Engineering (150k), University of Hohenheim, Fruwirthstraße 12, 70599, Stuttgart, Germany.
authors.forename: Marius
authors.initials: M
authors.lastname: Henkel
authors.affiliation: Institute of Food Science and Biotechnology, Department of Bioprocess Engineering (150k), University of Hohenheim, Fruwirthstraße 12, 70599, Stuttgart, Germany.
authors.forename: Rudolf
authors.initials: R
authors.lastname: Hausmann
authors.affiliation: Institute of Process Engineering in Life Sciences, Section II: Technical Biology, Karlsruhe Institute of Technology (KIT), Engler-Bunte-Ring 1, 76131, Karlsruhe, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Syldatk
authors.affiliation: Institute for Molecular Enzyme Technology (IMET), Heinrich-Heine-University Düsseldorf, Forschungszentrum Jülich, Wilhelm-Johnen-Straße, 52428, Jülich, Germany.|iQu Collegiate-Didactics, Heinrich-Heine-University Düsseldorf, Universitätsstraße 1, 40225, Düsseldorf, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Wilhelm
authors.affiliation: Ulm Center for Peptide Pharmaceuticals (U-PEP), Ulm University, Albert-Einstein-Allee 11, 89081, Ulm, Germany.|Institute for Molecular Enzyme Technology (IMET), Heinrich-Heine-University Düsseldorf, Forschungszentrum Jülich, Wilhelm-Johnen-Straße, 52428, Jülich, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Rosenau
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D053898:Biosynthetic Pathways / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D003651:Decanoates / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism*; D006017:Glycolipids / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009154:Mutation; D009876:Operon; D011550:Pseudomonas aeruginosa / Q000235:genetics; D016958:Pseudomonas putida / Q000235:genetics* / Q000378:metabolism*; D053038:Quorum Sensing; D012210:Rhamnose / Q000031:analogs & derivatives / Q000378:metabolism; D013501:Surface-Active Agents
chemical_list: D001426:Bacterial Proteins; D003651:Decanoates; D005227:Fatty Acids; D006017:Glycolipids; D013501:Surface-Active Agents; C418382:rhamnolipid; C085734:rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoate; D012210:Rhamnose
keywords: Biosurfactant; Biosynthesis pathway; Pseudomonas aeruginosa; Pseudomonas putida; Rhamnolipids