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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.

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Alkyl sophorosides, a special type of sophorolipids, can be produced using Candida bombicola cultures with glucose as main carbon source and 2-dodecanol as co-substrate. The dominating component, 2-dodecylsophoroside SL-E 2-12 , was purified via medium pressure liquid chromatography (MPLC) and used as substrate for further enzymatic modifications. Using b-glucuronidase in aqueous media the first modification gave a glycosidic cleavage of the acetyl glucose unit leading to 2-dodecyl-glucoside GL-A 2-12 . In a subsequent lipase-catalyzed acylation with sebacic acid in toluene this compound was functionalized regioselectively at the primary C-6 0 position of glucose. Similar lipase-catalyzed reactions in toluene, but now using SL-E 2-12 as acyl acceptor and unusual hydroxyl fatty acids -3-hydroxy decanoic acid and 17-hydroxy stearic acid -as acyl donors, resulted in mono-and diacylations of primary hydroxyl positions of the sophorose unit (C-6 0 and C-6 00 ) as shown by NMR and MS studies. In physicochemical characterization experiments the new glycoconjugates lowered the surface tension of water from 72 to 27-32 mN/m. Moreover it was observed that the new products inhibit the growth of particular Gram-positive bacteria. Additionally they indicate potential for anti-tumor promoting activity.Practical applications: According to literature data 2-alkyl-sophorosides can be produced using Candida bombicola cultures with yields of more than 20 g/L. Based on this starting material, 2-dodecyl-sophoroside, several regioselective enzymatic modifications were performed. Initially, b-glucuronidase catalysis in aqueous buffer solution gave 2-dodecyl-glucoside which was then successfully acylated at the C-6 position of glucose by lipase catalysis using a common dicarboxylic acid, sebacic acid. The resulting free carboxylic function of the acidic derivative opens up the possibility of achieving additional chemo-enzymatic reactions at this position for conversion, for instance, into polyesters. In addition lipase catalysis in organic solvents of 2-dodecyl-sophoroside allows acylation with unusual hydroxyl fatty acids of microbial origin. Physicochemical studies showed that these new glycoconjugates may be of interest for the cosmetic industry due to moisture conservation of the skin. For practical applications the conditions of the above biocatalytic reactions have been improved and scaled up.

To produce novel types of sophorose lipids containing an odd number of carbon atoms in the lipophilic moiety, Candida bombicola ATCC 22214 was grown in 500-ml flask cultures with glucose as main carbon source, and additionally, 2-tridecanone as co-substrate. After solvent extraction, the crude product mixture was separated into pure fractions, and each fraction was analysed via NMR and mass spectroscopy. This effective strategy generated five new glycolipids, 2-tridecyl sophorosides, which differed in the number of glucose units, and acetyl and hydroxy groups, respectively. Based on these compounds, a proposal for the possible biosynthetic pathway was deduced. Two compounds of the mixture, mono- and diacetylated 2-tridecyl sophorosides, respectively, were able to lower the surface tension of water from 72 mN m(-1) to 32 mN m(-1) and the interfacial tension between water and n-hexadecane from 43 mN m(-1) down to 4 and 3 mN m(-1). Thus, both compounds possess a very good surfactant behaviour. Moreover, it was observed that the new products inhibit the growth of particular Gram-positive bacteria, and they indicate potential for antitumour-promoting activity.