The Genetic and Metabolic Regulation of Rhamnolipids Biosynthesis in Pseudomonas Aeruginosa Reveals New Engineering Strategies for Heterologous Expression

Rhamnolipids (RLs) are bacterially-produced surfactants of growing biotechnological importance. The first-known producer is the opportunistic 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 insufficient for the development of a successful RLs metabolic engineering strategy in non-pathogenic host. In this study, we characterize the metabolic and genetic changes naturally taking place in P. aeruginosa PA14 in mineral salts medium (MSM) in which RLs is 100 times more produced compared to a rich, undefined medium, tryptic soy broth (TSB). In MSM, production of the lipid precursor of RLs 3-hydroxydecanoate is at least 30 times higher and exclusively in the R- chiral form, compared to growth in TSB. We hypothesize this is attributed to an increase (2-3 times) in R-specific enoyl-CoA hydratase (ECH) activity. Interestingly, the expression of rhlYZ, encoding the R-ECH proposed to be implicated in R-3-hydroxydecanoate precursor supply, is under control of the quorum sensing system together with the other precursor supplying operon, rmlBDAC, coding for rhamnose, and with the genes coding for precursor linking enzymes, rhlAB. Furthermore, comparative expression experiments using qRT-PCR in MSM compared to TSB demonstrated that RhlAB is the key element in RLs overproduction in MSM as it is 85 times more expressed than in TSB. This transcriptional data, together with the results of homologous expression of the two operons coding for RLs precursors, rhlYZ and rmlBDAC, 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 and P. fluorescens. 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.