Rhamnolipids: diversity of structures, microbial origins and roles

Ahmad Mohammad Abdel-Mawgoud Franois Lpine Eric Dziel 0 ) INRS-Institut Armand-Frappier , 531 Boulevard des Prairies, Laval, Qc H7V 1B7, Canada Rhamnolipids are glycolipidic biosurfactants produced by various bacterial species. They were initially found as exoproducts of the opportunistic pathogen Pseudomonas aeruginosa and described as a mixture of four congeners: -L-rhamnopyranosyl--L-rhamnopyranosyl-hydroxydecanoyl--hydroxydecanoate (Rha-Rha-C10-C10), -L-rhamnopyranosyl--L-rhamnopyranosyl--hydroxydecanoate (Rha-Rha-C10), as well as their mono-rhamnolipid congeners Rha-C10-C10 and Rha-C10. The development of more sensitive analytical techniques has lead to the further discovery of a wide diversity of rhamnolipid congeners and homologues (about 60) that are produced at different concentrations by various Pseudomonas species and by bacteria belonging to other families, classes, or even phyla. For example, various Burkholderia species have been shown to produce rhamnolipids that have longer alkyl chains than those produced by P. aeruginosa. In P. aeruginosa, three genes, carried on two distinct operons, code for the enzymes responsible for the final steps of rhamnolipid synthesis: one operon carries the rhlAB genes and the other rhlC. Genes highly similar to rhlA, rhlB, and rhlC have also been found in various Burkholderia species but grouped within one putative operon, and they have been shown to be required for rhamnolipid production as well. The exact physiological function of these secondary metabolites is still unclear. Most identified activities are derived from the surface activity, wetting ability, detergency, and other amphipathic-related properties of these molecules. Indeed, rhamnolipids promote the uptake and biodegradation of poorly soluble substrates, act as immune modulators and virulence factors, have antimicrobial activities, and are involved in surface motility and in bacterial biofilm development. - Surfactants are chemical compounds that (as entailed by their name) display surface activity. They have predilection for interfaces of dissimilar polarities (liquidair or liquid liquid) and are soluble in both organic (non-polar) and aqueous (polar) solvents. This property originates from their amphiphilic (or amphipathic) structures, which comprise both hydrophilic (head) and hydrophobic groups (tail) (Desai and Banat 1997). Biosurfactants are surfactants of biological origin. Microorganisms like bacteria, yeasts, and fungi are known to produce various types of biosurfactants. Their hydrophilic part is usually composed of sugars, amino acids, or polar functional groups like carboxylic acid groups. The hydrophobic part is typically an aliphatic hydrocarbon chain of -hydroxy fatty acids (Lang and Wullbrandt 1999). Surfactants are generally classified according to the charge carried by their polar groups (head) into cationic, anionic, amphoteric, and nonionic. Biosurfactants are attracting much attention because they represent ecological alternatives to their synthetic counterparts: they exhibit lower toxicity, potentially high activities, and stability at extremes of temperature, pH, and salinity. They have a wide variety of structures, and they can be produced from renewable feedstocks by a wide variety of microorganisms. Most importantly, they are biodegradable, making them environmentally friendly, green chemicals. Biosurfactants are classified according to their molecular structure into mainly glycolipids (e.g., rhamnolipids (RLs) and sophorolipids), lipopeptides (e.g., surfactin), polymeric biosurfactants (e.g., emulsan and alasan), fatty acids ((e.g., 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs)), and phospholipids (e.g., phosphatidylethanolamine) (Desai and Banat 1997; Lang and Wullbrandt 1999). Rhamnolipids are surface-active glycolipids. They have been intensively investigated and extensively reviewed (Maier and Sobern-Chvez 2000; Nitschke et al. 2005; Ochsner et al. 1996; Sobern-Chvez 2004; SobernChvez et al. 2005). However, two questions about this class of biosurfactants are emerging as important topics that have yet been poorly reviewed. First, what is the intrinsic, natural role of RLs for the producing organisms? Second, what actually are these producers? Indeed, while the opportunistic pathogen Pseudomonas aeruginosa has traditionally been considered the primary RL-producing microorganism, many other bacterial species, especially in recent years, have been reported to produce RLs as well. Still, apart from some clear cases, it appears that the ability to produce RLs is in fact restricted to a limited number of species, and that many reports are anecdotal. Although initially only four RL species had been described, the development of more sensitive analytical techniques has revealed the co-production of a far wider variety of RL congeners. Thus, this review focuses on the diversity of reported RL bacterial producers, the chemical structure of identified RLs, and the various physiological functions and roles attributed to RLs for the producing bacteria. Diversity of chemical structures of rhamnolipids The initial discovery of RLs goes back to 1946 when Bergstrm et al. (1946a, b) reported an oily glycolipid produced by Pseudomonas pyocyanea (now P. aeruginosa) after growth on glucose that was named pyolipic acid and whose structural units were identified as L-rhamnose and -hydroxydecanoic acid (Hauser and Karnovsky 1954; Jarvis and Johnson 1949). Jarvis and Johnson (1949) further elucidated the structure of a RL isolated from P. aeruginosa and showed that it was composed of two -hydroxydecanoic acids linked through a glycosidic bond to two rhamnose moieties, with the two -hydroxy fatty acid portions linked through an ester bond while the disaccharide portion contained a putative 1,3-glycosidic linkage. Edwards and Hayashi (1965) rather found that the linkage between the two rhamnose moieties is an -1,2glycosidic linkage, as determined by periodate oxidation and methylation. Based on that, they chemically described this RL as 2-O--1,2-L-rhamnopyranosyl--L-rhamnopyranosyl--hydroxydecanoyl--hydroxydecanoate (di-RL, structure 39 in Table 1). This was the first discovered glycolipid containing a link between a sugar and a hydroxylated fatty acid residue (Shaw 1970). Later on, Itoh et al. (1971) isolated and identified a new RL congener produced concurrently with the aforementioned di-RL by several strains of P. aeruginosa after growth on nparaffin, and it was identified as -L-rhamnopyranosyl-hydroxydecanoyl--hydroxydecanoate (mono-RL, structure 13 in Table 1). Moreover, they postulated that this mono-RL is the precursor of the di-RL (Itoh et al. 1971), although this had been suggested previously (Burger et al. 1963). A few years later, two additional RL congeners were identified in cultures of Pseudomonas sp. DSM 2874 grown as resting cells on n-alkanes or glycerol (Syldatk et al. 1985). These were similar to the previously identified mon (...truncated)