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.

Growth of engineered Pseudomonas putida KT2440 on glucose, xylose, and arabinose: Hemicellulose hydrolysates and their major sugars as sustainable carbon sources

“…In this present study, we chose P. putida KT2440 as a host for generating optimized expression strains by heterologous expression of the xylAB and araBAD operons to enlarge the available substrate spectrum for this remarkable platform organism. P. putida KT2440 has developed into an excellent and robust workhorse for the expression of heterologous genes (Loeschcke & Thies, 2015;Martins Dos Santos, Heim, Moore, Strätz, & Timmis, 2004), possesses an outstanding tolerance toward numerous organic compounds and has been extensively studied for the biosynthesis of biotechnological relevant products, for example, rhamnolipids (Cha, Lee, Kim, Kim, & Lee, 2008;Tiso et al, , 2018Wittgens et al, 2017Wittgens et al, , 2018Wittgens et al, , 2011. Its genome has been completely sequenced, which provides complete insights into its metabolic potential (Nelson et al, 2002;Poblete-Castro, Becker, Dohnt, Santos, & Wittmann, 2012), and especially in Germany, the strain KT2440 is of great importance, since it is the only P. putida, which remained in the biosafety level 1 (S1) being a key prerequisite for its use in many industrial applications (BVL, 2012).…”

Host-Pathogen Interaction in the Lung of Patients Infected withPseudomonas aeruginosa

“…Upon infection, the host decreases iron levels in the blood [99]; this iron deficiency regulates a great number of bacterial virulent genes like alginate, the most relevant virulence factor, for P. aeruginosa survival [100]. In the lung, iron deficiency turns on AlgQ, the bacterial biofilm production gene, also known as AlgR2 [101,102], under the Pfr A regulation that assists to the formation of two kinds of cytoplasmic aggregates: large vacuole-like bodies and smaller granules containing iron in association with oxygen or phosphate, very likely polyPi [103]. This leads to the RSCV type of P. aeruginosa.…”

“…Studies on the biosynthetic pathway of biofilms show that chelation of iron by lactoferrin destabilizes the bacterial membrane [113], which combined with xylitol hinders the ability of the bacteria to respond to iron deficiency [101], showing some promise for CF treatment.…”

Potential of biotechnological conversion of lignocellulose hydrolyzates by Pseudomonas putida KT2440 as a model organism for a bio‐based economy

“…This gram‐negative, ubiquitous, saprophytic soil bacterium has become a remarkable workhorse for biotechnical processes (Loeschcke & Thies, ; Martins Dos Santos et al, ). As an example, it is a suitable host for the production of the biosurfactant rhamnolipid (Arnold, Henkel, et al, ; Beuker, Barth, et al, , Beuker, Steier, et al, ; Cha, Lee, Kim, Kim, & Lee, ; Tiso et al, ; Wittgens et al, , , ). Unlike many other Pseudomonads , P. putida KT2440 is classified as biosafety level 1 according to American Type Culture Collection.…”