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.

Most of the Earth’s biosphere is cold and is populated by cold-adapted microorganisms. To explore the natural enzyme diversity of these environments and identify new carboxylesterases, we have screened three marine metagenome gene libraries for esterase activity. The screens identified 23 unique active clones, from which five highly active esterases were selected for biochemical characterization. The purified metagenomic esterases exhibited high activity against α-naphthyl and p-nitrophenyl esters with different chain lengths. All five esterases retained high activity at 5 °C indicating that they are cold-adapted enzymes. The activity of MGS0010 increased more than two times in the presence of up to 3.5 M NaCl or KCl, whereas the other four metagenomic esterases were inhibited to various degrees by these salts. The purified enzymes showed different sensitivities to inhibition by solvents and detergents, and the activities of MGS0010, MGS0105 and MGS0109 were stimulated three to five times by the addition of glycerol. Screening of purified esterases against 89 monoester substrates revealed broad substrate profiles with a preference for different esters. The metagenomic esterases also hydrolyzed several polyester substrates including polylactic acid suggesting that they can be used for polyester depolymerization. Thus, esterases from marine metagenomes are cold-adapted enzymes exhibiting broad biochemical diversity reflecting the environmental conditions where they evolved.

DNA derived from environmental samples is a rich source of novel bioactive molecules. The choice of the habitat to be sampled predefines the properties of the biomolecules to be discovered due to the physiological adaptation of the microbial community to the prevailing environmental conditions. We have constructed a metagenomic library in Escherichia coli DH10b with environmental DNA (eDNA) isolated from the microbial community of a slaughterhouse drain biofilm consisting mainly of species from the family Flavobacteriaceae. By functional screening of this library we have identified several lipases, proteases and two clones (SA343 and SA354) with biosurfactant and hemolytic activities. Sequence analysis of the respective eDNA fragments and subsequent structure homology modelling identified genes encoding putative N-acyl amino acid synthases with a unique two-domain organisation. The produced biosurfactants were identified by NMR spectroscopy as N-acyltyrosines with N-myristoyltyrosine as the predominant species. Critical micelle concentration and reduction of surface tension were similar to those of chemically synthesised N-myristoyltyrosine. Furthermore, we showed that the newly isolated N-acyltyrosines exhibit antibiotic activity against various bacteria. This is the first report describing the successful application of functional high-throughput screening assays for the identification of biosurfactant producing clones within a metagenomic library.

TesA from Pseudomonas aeruginosa belongs to the GDSL hydrolase family of serine esterases and lipases that possess a broad substrate- and regiospecificity. It shows high sequence homology to TAP, a multifunctional enzyme from Escherichia coli exhibiting thioesterase, lysophospholipase A, protease and arylesterase activities. Recently, we demonstrated high arylesterase activity for TesA, but only minor thioesterase and no protease activity. Here, we present a comparative analysis of TesA and TAP at the structural, biochemical and physiological levels. The crystal structure of TesA was determined at 1.9 Å and structural differences were identified, providing a possible explanation for the differences in substrate specificities. The comparison of TesA with other GDSL-hydrolase structures revealed that the flexibility of active-site loops significantly affects their substrate specificity. This assumption was tested using a rational approach: we have engineered the putative coenzyme A thioester binding site of E. coli TAP into TesA of P. aeruginosa by introducing mutations D17S and L162R. This TesA variant showed increased thioesterase activity comparable to that of TAP. TesA is the first lysophospholipase A described for the opportunistic human pathogen P. aeruginosa. The enzyme is localized in the periplasm and may exert important functions in the homeostasis of phospholipids or detoxification of lysophospholipids.

The adaptation of microorganisms to extreme living temperatures requires the evolution of enzymes with a high catalytic efficiency under these conditions. Such extremophilic enzymes represent valuable tools to study the relationship between protein stability, dynamics and function. Nevertheless, the multiple effects of temperature on the structure and function of enzymes are still poorly understood at the molecular level. Our analysis of four homologous esterases isolated from bacteria living at temperatures ranging from 10°C to 70°C suggested an adaptation route for the modulation of protein thermal properties through the optimization of local flexibility at the protein surface. While the biochemical properties of the recombinant esterases are conserved, their thermal properties have evolved to resemble those of the respective bacterial habitats. Molecular dynamics simulations at temperatures around the optimal temperatures for enzyme catalysis revealed temperature-dependent flexibility of four surface-exposed loops. While the flexibility of some loops increased with raising the temperature and decreased with lowering the temperature, as expected for those loops contributing to the protein stability, other loops showed an increment of flexibility upon lowering and raising the temperature. Preserved flexibility in these regions seems to be important for proper enzyme function. The structural differences of these four loops, distant from the active site, are substantially larger than for the overall protein structure, indicating that amino acid exchanges within these loops occurred more frequently thereby allowing the bacteria to tune atomic interactions for different temperature requirements without interfering with the overall enzyme function.