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.

Keywords: Biosurfactant; Biosynthesis pathway; Pseudomonas aeruginosa; Pseudomonas putida; Rhamnolipids.

Fig. 1

Chemical structures of rhamnolipids. Rhamnolipids are separated into mono- and di-rhamnolipids based on the number of L-rhamnose residues. Beside typical rhamnolipid species containing two 3-hydroxyfatty acids (mono-rhamno-di-lipid and di-rhamno-di-lipid), there exist species containing only one fatty acid chain (mono-rhamno-mono-lipid and di-rhamno-mono-lipid). Rhamnolipids from P. aeruginosa typically contain fatty acids with chain lengths between C₈ and C₁₄ (n = 1–7) while organisms from the genus Burkholderia produce rhamnolipids with longer alkyl chains and typical lengths between C₁₂ and C₁₆ (n = 5–9)

Fig. 2

Rhamnolipids and HAAs produced by recombinant P. putida. a Thin layer chromatography (TLC) of extracts from single rhlA or rhlB expression shows no detectable amounts of rhamnolipids after 24 h (HPLC results not shown). b HPLC analysis of HAAs reveals their production in a rhlA expressing P. putida strain. c HPLC analysis of HAAs (squares) and mono-rhamnolipids (triangles) and TLC of P. putida cultures carrying rhlAB operon. Rhamnolipids are visible as brown bands on TLC plates as in the rhamnolipid-standard. Samples extracted from P. putida cultures show an additional violet spot descending from IPTG as in extracts of IPTG containing LB media (IPTG control). Samples were taken every 6 h for a period of 24 h from three independent cultures

Fig. 3

Identification of the catalytic triade of RhlA. a The three-dimensional structure of RhlA from P. aeruginosa was modeled using the chloroperoxidase L (CpoL; PDB code: 1A88) from Streptomyces lividans as template. Despite low sequence identity (14%), the catalytic triad Ser, Asp, and His (indicated by an asterisk underneath the sequences) are strongly conserved among these two proteins. Sequences identical and similar were shaded in black and yellow, respectively. b Structural superimposition of CpoL (brown) and RhlA (blue) shows a high conservation of secondary structure elements. The catalytic triad of CpoL (Ser96, His255 and Asp226) and the putative catalytic triad of RhlA (Ser102, His251, and Asp223) are structurally strongly conserved. Dashed lines indicate catalytically important interactions of the active site residues

Fig. 4

Production of mono-rhamnolipids by recombinant P. putida in HAA containing conditioned medium. P. putida strains expressing single rhlB (a) or the rhlA*B operon (b), containing inactive RhlA, were cultivated in HAA containing conditioned medium, obtained from a rhlA expressing P. putida strain. Extracts were analyzed via HPLC revealing HAAs (squares) and mono-rhamnolipids (triangles) and thin layer chromatography. Rhamnolipids are visible as brown bands on TLC plates as in the rhamnolipid-standard. Samples extracted from P. putida cultures show an additional violet spot descending from IPTG as in extracts of IPTG containing LB media (IPTG control). Samples were taken every 6 h for a period of 24 h from three independent cultures

Fig. 5

Production of di-rhamnolipids by recombinant P. putida in mono-rhamnolipid containing conditioned medium. P. putida strains expressing single rhlC (a) or the PA1131-rhlC operon (b) were cultivated in mono-rhamnolipid containing conditioned medium, obtained from a rhlAB expressing P. putida strain. c For comparison, P. putida expressing the biosynthetic rhlABC operon cultivated in fresh LB media. Extracts were analyzed via HPLC revealing HAAs (squares), mono-rhamnolipids (triangles), and di-rhamnolipids (circles) and thin layer chromatography. Rhamnolipids are visible as brown bands on TLC plates as in the rhamnolipid-standard. Samples extracted from P. putida cultures show an additional violet spot descending from IPTG as in extracts of IPTG containing LB media (IPTG control). Samples were taken every 6 h for a period of 24 h from three independent cultures

Fig. 6

Production of rhamnolipids by P. aeruginosa rhl-mutant strains. Thin layer chromatography was performed to analyze rhamnolipid biosynthesis by P. aeruginosa ΔrhlA and P. aeruginosa ΔrhlC cultivated in PPGAS medium. In addition, P. aeruginosa ΔrhlA was cultivated in mono-rhamnolipid containing conditioned medium, obtained from a P. aeruginosa ΔrhlC culture. Samples were taken after 24 h. Rhamnolipids are visible as brown bands on TLC plates as in the rhamnolipid-standard