Heterologous rhamnolipid biosynthesis by <i>P. putida</i> KT2440—ACP dependency and the role of fatty acid metabolism

Rhamnolipids are glycolipids and, as such, biosurfactants with high surface activity, good biodegradability, low toxicity, and thus manifold applications. Although rhamnolipids have been extensively researched and are already produced industrially, the biosynthesis pathway has not yet been resolved beyond doubt. While the origin of the hydrophilic moiety is clear and all responsible genes and enzymes have been identified, the origin of the hydrophobic moiety, 3-(3-hydroxyalkanoyloxy) alkanoic acid, remains to be identified. Here, the involvement of the two central metabolic pathways, that is, fatty acid <i>de novo</i> synthesis (FAS) and β-oxidation in the biosynthesis of rhamnolipids from different carbon sources was investigated in the heterologous production host <i>Pseudomonas putida</i> KT2440. Besides using mutants deficient in β-oxidation for this purpose, the absolute configuration, that is, the spatial arrangement of the substituents at the stereocenter of the produced rhamnolipid molecules, was investigated for <i>P. putida</i> KT2440, as well as for the native rhamnolipid producer <i>Pseudomonas aeruginosa</i> PAO1. Furthermore, ¹³C labeling experiments were performed to gain further insights into the metabolic network. The combined results suggested that the acyltransferase RhlA shows a strong preference for ACP-activated substrates, and that fatty acids are broken down to acetyl-CoA before the C₂-fragments can be incorporated into rhamnolipids via FAS.IMPORTANCEThis study contributes to resolving the incomplete understanding of rhamnolipid biosynthesis, particularly the origin of its hydrophobic moiety. Although rhamnolipids have been extensively studied and are already produced industrially, gaps in knowledge regarding their metabolic pathway limit further optimization for large-scale, sustainable production. By investigating the role of fatty acid <i>de novo</i> synthesis and β-oxidation in rhamnolipid biosynthesis, this study provides crucial insights that could enable metabolic engineering strategies to enhance production efficiency, especially in the heterologous host <i>Pseudomonas putida</i> KT2440, as this strain is considered a safer and more suitable alternative to <i>Pseudomonas aeruginosa</i>. These findings collectively pave the way for improved biotechnological production of rhamnolipids, facilitating their broader industrial application as environmentally friendly biosurfactants.