A Stereospecific Pathway Diverts β-Oxidation Intermediates to the Biosynthesis of Rhamnolipid Biosurfactants

• β-oxidation is the main direct supplier of lipid precursors for rhamnolipids (RLs)
• R-enoyl-CoA hydratases, RhlY/Z, shunt β-oxidation intermediates to RLs
• RhlYZ provides 70% of the total 3-hydroxyalkanoates intracellular pool in PA14
• Polyhydroxyalkanoates and RLs share the same 3-hydroxyalkanoate precursor pool

SummaryRhamnolipids are multipurpose surface-active molecules produced by the bacterium Pseudomonas aeruginosa from L-rhamnose and R-3-hydroxyalkanoate (C10±2) precursors. R-3-hydroxyalkanoate precursor is believed to be synthesized de novo. We demonstrate, however, that β-oxidation is the predominant source of this precursor. Inhibition of β-oxidation sharply decreases rhamnolipids production, even when using a nonfatty acid carbon source (glycerol). Isotope tracing shows that β-oxidation intermediates are direct precursors of rhamnolipids. A mutant-based survey revealed an operon coding for enoyl-CoA hydratases/isomerases (ECH/I), named RhlYZ, implicated in rhamnolipids production via an axial role in 3-hydroxyalkanoate synthesis. In vitro, RhlZ is an R-ECH/I transforming 2-decenoyl-CoA, a β-oxidation intermediate, into R-3-hydroxydecanoyl-CoA, the potential rhamnolipids precursor. Interestingly, polyhydroxyalkanoates share with rhamnolipids the RhlYZ-generated R-3-hydroxyalkanoates pool, as demonstrated by the decrease of polyhydroxyalkanoates upon mutation of rhlYZ and the increase of rhamnolipids in a polyhydroxyalkanoates-defective mutant.