Simple glycolipids of microbes: Chemistry, biological activity and metabolic engineering.

Glycosylated lipids (GLs) are added-value lipid derivatives of great potential. Besides their interesting surface activities that qualify many of them to act as excellent ecological detergents, they have diverse biological activities with promising biomedical and cosmeceutical applications. Glycolipids, especially those of microbial origin, have interesting antimicrobial, anticancer, antiparasitic as well as immunomodulatory activities. Nonetheless, GLs are hardly accessing the market because of their high cost of production. We believe that experience of metabolic engineering (ME) of microbial lipids for biofuel production can now be harnessed towards a successful synthesis of microbial GLs for biomedical and other applications. This review presents chemical groups of bacterial and fungal GLs, their biological activities, their general biosynthetic pathways and an insight on ME strategies for their production.

Liquid chromatography/mass spectrometry for the identification and quantification of rhamnolipids.

Rhamnolipids (RL) are surface-active glycolipids produced by Pseudomonas aeruginosa. They are always produced by this bacterium as a complex mixture of congeners, each composed of one or two rhamnose molecules linked to a dimer of 3-hydroxyfatty acids with a chain length of 8-12 carbons. Increasing interest for RL drives the need for efficient analytical methods to characterize these mixtures of molecules. High-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) is a very precise and relatively high-throughput method for the identification of each congener and their quantification in bacterial cultures. Using (13)C-labeled RL as internal standards can further enhance the precision of the quantification. Collision-induced dissociation (CID) experiments by MS/MS is a powerful tool for the detection and identification of structural variations in RL produced by various Pseudomonas strains or by a specific strain under different culture conditions. CID even allows the discrimination between isomers with subtle structural variations, like Rha-C8-C10 and Rha-C10-C8, which are almost inseparable chromatographically. We are presenting here the detailed protocols for HPLC/MS and HPLC/MS/MS analysis of RL and their lipid precursors, the 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAA), directly in bacterial culture supernatants.

A stereospecific pathway diverts ß-oxidation intermediates to the biosynthesis of rhamnolipid biosurfactants.

Rhamnolipids are multipurpose surface-active molecules produced by the bacterium Pseudomonas aeruginosa from L-rhamnose and R-3-hydroxyalkanoate (C10±2) precursors. R-3-hydroxyalkanoate precursor is believed to be synthesized de novo. We demonstrate, however, that ß-oxidation is the predominant source of this precursor. Inhibition of ß-oxidation sharply decreases rhamnolipids production, even when using a nonfatty acid carbon source (glycerol). Isotope tracing shows that ß-oxidation intermediates are direct precursors of rhamnolipids. A mutant-based survey revealed an operon coding for enoyl-CoA hydratases/isomerases (ECH/I), named RhlYZ, implicated in rhamnolipids production via an axial role in 3-hydroxyalkanoate synthesis. In vitro, RhlZ is an R-ECH/I transforming 2-decenoyl-CoA, a ß-oxidation intermediate, into R-3-hydroxydecanoyl-CoA, the potential rhamnolipids precursor. Interestingly, polyhydroxyalkanoates share with rhamnolipids the RhlYZ-generated R-3-hydroxyalkanoates pool, as demonstrated by the decrease of polyhydroxyalkanoates upon mutation of rhlYZ and the increase of rhamnolipids in a polyhydroxyalkanoates-defective mutant.

Rhamnolipids: diversity of structures, microbial origins and roles.

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: alpha-L-rhamnopyranosyl-alpha-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydecanoate (Rha-Rha-C(10)-C(10)), alpha-L-rhamnopyranosyl-alpha-L-rhamnopyranosyl-beta-hydroxydecanoate (Rha-Rha-C(10)), as well as their mono-rhamnolipid congeners Rha-C(10)-C(10) and Rha-C(10). 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.

Characterization of rhamnolipid produced by Pseudomonas aeruginosa isolate Bs20.

Rhamnolipid produced by Pseudomonas aeruginosa isolate Bs20 is viscous sticky oily yellowish brown liquid with a fruity odor. It showed solubility at aqueous pH > 4 with optimum solubility at pH 7-7.5 and freely soluble in ethyl acetate. This biosurfactant has a very high surface activity as it could lower the surface tension of water to 30 mN/m at about 13.4 mg/L, and it exhibited excellent stabilities at high temperatures (heating at 100 degrees C for 1 h and autoclaving at 121 degrees C for 10 min), salinities (up to 6% NaCl), and pH values (up to pH 13). The produced biosurfactant can be used in the crude form either as cell-free or cell-containing culture broth of the grown bacteria, since both preparations showed high emulsification indices ranged between 59% and 66% against kerosene, diesel, and motor oil. These characters make the test rhamnolipid a potential candidate for use in bioremediation of hydrocarbon-contaminated sites or in the petroleum industry. High-performance thin-layer chromatography densitometry revealed that the extracted rhamnolipid contained the two most active rhamnolipid homologues dirhamno dilipidic rhamnolipid and monorhamno dilipidic rhamnolipid at 44% and 56%, respectively, as compared to 51% and 29.5%, respectively, in a standard rhamnolipid preparation. The nature and ratio of these two rhamnolipid homologues showed to be strain dependent rather than medium-component dependent.

Characterization of surfactin produced by Bacillus subtilis isolate BS5.

Physical and chromatographic characterization of the surfactin biosurfactant produced by Bacillus subtilis isolate BS5 has been conducted to study its potentiality for industrial application. The crude extract of test surfactin appeared as off-white to buff flake-like amorphous residue with bad odor similar to sour pomegranate. Test surfactin showed solubility in aqueous solution at pH>5 with optimum solubility at pH 8-8.5. It was also soluble in organic solvents like ethanol, acetone, methanol, butanol, chloroform, and dichloromethane. Surfactin crystals appeared rectangular with blunt corners and were arranged perpendicular to each other making a plus sign. Extracted surfactin showed high surface activity, as it could lower the surface tension of water from about 70 to 36 mN/m at approximately 15.6 mg/l. Moreover, test surfactin exhibited excellent stabilities at high temperatures (100 degrees C for up to 1 h at and autoclaving at 121 degrees C for 10 min), salinities (up to 6% NaCl), and over a wide range of pH (5-13). Test surfactin in the cell-free supernatant or crude culture broth forms showed high emulsification indices against kerosene (62.5% and 59%, respectively), diesel (62.5% and 66%, respectively), and motor oil (62% and 66%, respectively). These characters can effectively make test surfactin, in its crude forms, a potential candidate for the use in bioremediation of hydrocarbon-contaminated sites or in the petroleum industry. Chromatographic characterization of test surfactin, using high-performance liquid chromatography technique, revealed that the extracted surfactin contained numerous isoforms, of which six were found in the standard surfactin preparation (Fluka). Additional peaks appeared in the test surfactin and not in the standard one. These peaks may correspond to new surfactin isoforms that may be present in the test surfactin produced by B. subtilis isolate BS5.