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Rhamnolipids (RLs) are bacterially-produced biosurfactants of growing biotechnological importance. The first-known producer is the human pathogen Pseudomonas aeruginosa. Although much is known about RLs biosynthesis, the exact metabolic and genetic regulation of RLs biosynthesis in this prototypic producer is still required for the development of a successful RLs metabolic engineering strategy in non-pathogenic strains. In this study, we characterize the metabolic and genetic changes naturally taking place by P. aeruginosa PA14 in minimal salts medium (MSM) in which RLs is 100 times more produced compared to the rich medium, tryptic soy broth (TSB). In MSM, the lipid precursors of RLs, 3-hydroxydecanoate, are at least 30 times more produced and are exclusively in the R- chiral form compared to TSB. The higher R-3-hydroxydecanoate precursor concentration in MSM is suggested to be attributed to the increase (2-3 times) of the R-specific ECH activity compared to that in TSB. Interestingly, the expression of rhlYZ, the proposed R-ECH implicated in R-3-hydroxydecanoate precursor supply, is under control of quorum sensing together with the other precursor supplying gene, rmlBDAC, coding for rhamnose, and the gene coding for precursor linking enzyme, rhlAB. Furthermore, comparative expression experiments using qRT-PCR in MSM compared to TSB demonstrates that RhlAB is the key element in the resulting RLs overproduction in MSM as it is 85 times more expressed in MSM than in TSB. This together with the results of the native-promterless homologous expression of the three genes, alone or combined with rhlAB, further corroborate that the RhlAB-mediated step is rate limiting in the biosynthesis of RLs rather than the precursor supply step. Although low RLs yields were obtained, heterologous expression of rhlAB was successful in hosts furnishing the two RLs precursors, namely P. putida KT2440 and P. fluorescens LMG1794. Yet, E. coli DH5α host required rmlBDAC co-expression. This study enhances the understanding of regulation of RLs biosynthesis and proposes a diagnostic tool that helps in the selection of the appropriate non-pathogenic hosts for heterologous RLs production.