No abstract

Biosurfactants are amphiphilic secondary metabolites produced by microorganisms. Marine bacteria have recently emerged as a rich source for these natural products which exhibit surface-active properties, making them useful for diverse applications such as detergents, wetting and foaming agents, solubilisers, emulsifiers and dispersants. Although precise structural data are often lacking, the already available information deduced from biochemical analyses and genome sequences of marine microbes indicates a high structural diversity including a broad spectrum of fatty acid derivatives, lipoamino acids, lipopeptides and glycolipids. This review aims to summarise biosyntheses and structures with an emphasis on low molecular weight biosurfactants produced by marine microorganisms and describes various biotechnological applications with special emphasis on their role in the bioremediation of oil-contaminated environments. Furthermore, novel exploitation strategies are suggested in an attempt to extend the existing biosurfactant portfolio.

SummaryThe present study provides a deeper view of protein functionality as a function of temperature, salt and pressure in deep-sea habitats. A set of eight different enzymes from five distinct deep-sea (3040-4908 m depth), moderately warm (14.0-16.5°C) biotopes, characterized by a wide range of salinities (39-348 practical salinity units), were investigated for this purpose. An enzyme from a 'superficial' marine hydrothermal habitat (65°C) was isolated and characterized for comparative purposes. We report here the first experimental evidence suggesting that in salt-saturated deepsea habitats, the adaptation to high pressure is linked to high thermal resistance (P value = 0.0036). Salinity might therefore increase the temperature window for enzyme activity, and possibly microbial growth, in deep-sea habitats. As an example, Lake Medee, the largest hypersaline deep-sea anoxic lake of the Eastern Mediterranean Sea, where the water temperature is never higher than 16°C, was shown to contain halopiezophilic-like enzymes that are most active at 70°C and with denaturing temperatures of 71.4°C. The determination of the crystal structures of five proteins revealed unknown molecular mechanisms involved in protein adaptation to polyextremes as well as distinct active site architectures and substrate preferences relative to other structurally characterized enzymes.

Marine habitats represent a prolific source for molecules of biotechnological interest. In particular, marine bacteria have attracted attention and were successfully exploited for industrial applications. Recently, a group of Pseudomonas species isolated from extreme habitats or living in association with algae or sponges were clustered in the newly established Pseudomonas pertucinogena lineage. Remarkably for the predominantly terrestrial genus Pseudomonas, more than half (9) of currently 16 species within this lineage were isolated from marine or saline habitats. Unlike other Pseudomonas species, they seem to have in common a highly specialized metabolism. Furthermore, the marine members apparently possess the capacity to produce biomolecules of biotechnological interest (e.g. dehalogenases, polyester hydrolases, transaminases). Here, we summarize the knowledge regarding the enzymatic endowment of the marine Pseudomonas pertucinogena bacteria and report on a genomic analysis focusing on the presence of genes encoding esterases, dehalogenases, transaminases and secondary metabolites including carbon storage compounds.

Amine transaminases of the class III ω-TAs are key enzymes for modification of chemical building blocks, but finding those capable of converting bulky ketones and (R) amines is still challenging. Here, by an extensive analysis of the substrate spectra of 10 class III ω-TAs, we identified a number of residues playing a role in determining the access and positioning of bulky ketones, bulky amines, and (R)- and (S) amines, as well as of environmentally relevant polyamines, particularly putrescine. The results presented can significantly expand future opportunities for designing (R)-specific class III ω-TAs to convert valuable bulky ketones and amines, as well as for deepening the knowledge into the polyamine catabolic pathways.

The functional expression of heterologous genes in standard hosts such as Escherichia coli is often hampered by various limitations including improper folding, incomplete targeting, and missassembly of the corresponding enzymes. This observation led to the development of numerous expression systems that are based on alternative, metabolic versatile hosts. One such organism is the Gram-negative phototrophic nonsulfur purple bacterium Rhodobacter capsulatus. During photosynthetic growth, R. capsulatus exhibits several unique properties including the formation of an intracytoplasmic membrane system as well as the synthesis of various metal-containing cofactors. These properties make R. capsulatus a promising expression host particularly suited for difficult-to-express proteins such as membrane proteins. In this chapter, we describe a novel R. capsulatus expression system and its application.

Functional expression of genes from metagenomic libraries is limited by various factors including inefficient transcription and/or translation of target genes as well as improper folding and assembly of the corresponding proteins caused by the lack of appropriate chaperones and cofactors. It is now well accepted that the use of different expression hosts of distinct phylogeny and physiology can dramatically increase the rate of success. In the following chapter, we therefore describe tools and protocols allowing for the comparative heterologous expression of genes in five bacterial expression hosts, namely Escherichia coli, Pseudomonas putida, Bacillus subtilis, Burkholderia glumae, and Rhodobacter capsulatus. Different broad-host-range shuttle vectors are described that allow activity-based screening of metagenomic DNA in these bacteria. Furthermore, we describe the newly developed transfer-and-expression system TREX which comprises genetic elements essential to allow for expression of large clusters of functionally coupled genes in different microbial species.

The Gram-negative proteobacterium Pseudomonas oleovorans DSM 1045 is considered a promising source for enzymes of biotechnological interest, e.g., hydrolases and transaminases. Here, we present a draft sequence of its 4.86-Mb genome, enabling the identification of novel biocatalysts.