Omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), such as eicosapentaenoic acid (EPA) (20:5n-3) and docosahexaenoic acid (DHA) (22:6n-3), are considered essential for human health. Microorganisms are the primary producers of omega-3 fatty acids in marine ecosystems, representing a sustainable source of these lipids, as an alternative to the fish industry. Some marine bacteria can produce LC-PUFAs de novo via the Polyunsaturated Fatty Acid (Pfa) synthase/ Polyketide Synthase (PKS) pathway, which does not require desaturation and elongation of saturated fatty acids. Cultivation-independent surveys have revealed that the diversity of microorganisms harboring a molecular marker of the pfa gene cluster (i.e., pfaA-KS domain) is high and their potential distribution in marine systems is widespread, from surface seawater to sediments. However, the isolation of PUFA producers from marine waters has been typically restricted to deep or cold environments. Here, we report a phenotypic and genotypic screening for the identification of omega-3 fatty acid producers in free-living bacterial strains isolated from 5, 500, and 1000 m deep coastal seawater from the Bay of Biscay (Spain). We further measured EPA production in pelagic Vibrio sp. strains collected at the three different depths. Vibrio sp. EPA-producers and non-producers were simultaneously isolated from the same water samples and shared a high percentage of identity in their 16S rRNA genes, supporting the view that the pfa gene cluster can be horizontally transferred. Within a cluster of EPA-producers, we found intraspecific variation in the levels of EPA synthesis for isolates harboring different genetic variants of the pfaA-KS domain. The maximum production of EPA was found in a Vibrio sp. strain isolated from a 1000 m depth (average 4.29% ± 1.07 of total fatty acids at 10 °C, without any optimization of culturing conditions).

As previously reported, P. aeruginosa genes PA2077 and PA2078 code for 10S-DOX (10S-Dioxygenase) and 7,10-DS (7,10-Diol Synthase) enzymes involved in long-chain fatty acid oxygenation through the recently described oleate-diol synthase pathway. Analysis of the amino acid sequence of both enzymes revealed the presence of two heme-binding motifs (CXXCH) on each protein. Phylogenetic analysis showed the relation of both proteins to bacterial di-heme cytochrome c peroxidases (Ccps), similar to Xanthomonas sp. 35Y rubber oxidase RoxA. Structural homology modelling of PA2077 and PA2078 was achieved using RoxA (pdb 4b2n) as a template. From the 3D model obtained, presence of significant amino acid variations in the predicted heme-environment was found. Moreover, the presence of palindromic repeats located in enzyme-coding regions, acting as protein evolution elements, is reported here for the first time in P. aeruginosa genome. These observations and the constructed phylogenetic tree of the two proteins, allow the proposal of an evolutionary pathway for P. aeruginosa oleate-diol synthase operon. Taking together the in silico and in vivo results obtained we conclude that enzymes PA2077 and PA2078 are the first described members of a new subfamily of bacterial peroxidases, designated as Fatty acid-di-heme Cytochrome c peroxidases (FadCcp).

Two Alteromonas sp. strains isolated from deep seawater were grown to promote the production of exopolysaccharides (EPS, E611 and E805), which were incorporated into chitosan solutions to develop films. The combination of the major marine polysaccharides (chitosan and the isolated bacterial EPS) resulted in the formation of homogenous, transparent, colorless films, suggesting good compatibility between the two components of the film-forming formulation. With regards to optical properties, the films showed low values of gloss, in the range of 5–10 GU, indicating the formation of non-glossy and rough surfaces. In addition to the film surface, both showed hydrophobic character, with water contact angles higher than 100 º, regardless of EPS addition. Among the two EPS under analysis, chitosan films with E805 showed better mechanical performance, leading to resistant, flexible, easy to handle films.

A search for extremophile enzymes from ancient volcanic soils in El Hierro Island (Canary Islands, Spain) allowed isolation of a microbial sporulated strain collection from which several enzymatic activities were tested. Isolates were obtained after sample cultivation under several conditions of nutrient contents and temperature. Among the bacterial isolates, supernatants from the strain designated JR3 displayed high esterase activity at temperatures ranging from 30 to 100°C, suggesting the presence of at least a hyper-thermophilic extracellular lipase. Sequence alignment of known thermophilic lipases allowed design of degenerated consensus primers for amplification and cloning of the corresponding lipase, named LipJ. However, the cloned enzyme displayed maximum activity at 30°C and pH 7, showing a different profile from that observed in supernatants of the parental strain. Sequence analysis of the cloned protein showed a pentapeptide motif -GHSMG- distinct from that of thermophilic lipases, and much closer to that of esterases. Nevertheless, the 3D structural model of LipJ displayed the same folding as that of thermophilic lipases, suggesting a common evolutionary origin. A phylogenetic study confirmed this possibility, positioning LipJ as a new member of the thermophilic family of bacterial lipases I.5. However, LipJ clusters in a clade close but separated from that of Geobacillus sp. thermophilic lipases. Comprehensive analysis of the cloned enzyme suggests a common origin of LipJ and other bacterial thermophilic lipases, and highlights the most probable divergent evolutionary pathway followed by LipJ, which during the harsh past times would have probably been a thermophilic enzyme, having lost these properties when the environment changed to more benign conditions.