The Estremadura Spur pockmarks are a unique and unexplored ecosystem located in the North Atlantic, off the coast of Portugal. A total of 85 marine-derived actinomycetes were isolated and cultured from sediments collected from this ecosystem at a depth of 200 to 350 m. Nine genera, Streptomyces, Micromonospora, Saccharopolyspora, Actinomadura, Actinopolymorpha, Nocardiopsis, Saccharomonospora, Stackebrandtia, and Verrucosispora were identified by 16S rRNA gene sequencing analyses, from which the first two were the most predominant. Non-targeted LC-MS/MS, in combination with molecular networking, revealed high metabolite diversity, including several known metabolites, such as surugamide, antimycin, etamycin, physostigmine, desferrioxamine, ikarugamycin, piericidine, and rakicidin derivatives, as well as numerous unidentified metabolites. Taxonomy was the strongest parameter influencing the metabolite production, highlighting the different biosynthetic potentials of phylogenetically related actinomycetes; the majority of the chemical classes can be used as chemotaxonomic markers, as the metabolite distribution was mostly genera-specific. The EtOAc extracts of the actinomycete isolates demonstrated antimicrobial and antioxidant activity. Altogether, this study demonstrates that the Estremadura Spur is a source of actinomycetes with potential applications for biotechnology. It highlights the importance of investigating actinomycetes from unique ecosystems, such as pockmarks, as the metabolite production reflects their adaptation to this habitat.

This study investigated the fatty acids (FA) profile of 54 actinomycete strains isolated from marine sediments collected off the Portugal continental coast, specifically from the Estremadura Spur pockmarks field, by GC/MS. Fatty acid methyl esters (FAMEs) were prepared from the ethyl acetate lipidic extracts of these strains and analyzed by gas chromatography–mass spectrometry (GC/MS), with FA identification performed using the NIST library. The identified FAs varied from C12:0 to C20:0, where 32 distinct FAs were identified, including 7 branched-chain fatty acids (BCFAs), 9 odd-chain fatty acids (OCFAs), 8 monounsaturated fatty acids (MUFAs), 6 saturated fatty acids (SFAs), 1 polyunsaturated fatty acid (PUFA), and 1 cyclic chain fatty acid (CCFA). The average expressed content was BCFA (47.54%), MUFA (28.49%), OCFA (26.93%), and SFA (22.16%), of which i-C16:0, C18:1ω9, and C16:0 were predominant, while PUFA (3.58%) and CCFA (0.41%) were identified as minor components. The identified BCFA were i-C16:0, a-C15:0, i-C15:0, i-C15:1ω6, a-C16:0, a-C14:0, and i-C17:0, which include combined branching and unsaturation and branching and odd. SFAs were present in all species, with C16:0 and C18:0 being the most representative. Rare OCFAs C19:1ω9, C17:1ω7, C15:0, and C17:0 were expressed. PUFA C18:1ω9 was detected; within this class, omega families ω9, ω7, ω6, and ω5 were identified, and no ω3 was detected. The only CCFA was benzene-butanoic acid (benzene-C4:0). These findings highlight the metabolic versatility of actinomycetes, providing valuable insights into microbial chemotaxonomy and offering promising biochemical leads for the development of biofuel, nutraceutical, and antifungal agents. Furthermore, these results underline the diversity and biotechnological potential of FAs in actinomycetes, uncovering their potential to be used as microbial cell factories, and paving the way for innovations in biofuels, pharmaceuticals, and eco-friendly industrial products.

Introduction: Actinomycetes Streptomyces genus is recognized for their ability to produce two of the most valuable families of natural products: non-ribosomal peptides (NRPs) and poliketides (PKs) [ 1 Bumpus SB , Evans BS , Thomas PM , et al. A proteomics approach to discovering natural products and their biosynthetic pathways. Nat Biotechnol. 2009;27:951–956. [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] ]. In particular, Streptomyces aculeolatus (Figure 1), belonging to the MAR4 group, is known to produce a specific group of bioactive secondary metabolites named hybrid isoprenoids (HI) [ 2–4 Gallagher KA , Rauscher K , Ioca LP , et al. Phylogenetic and chemical diversity of a hybrid-isoprenoid-producing streptomycete lineage. Appl and Environm Microbiol. 2013;79:6894–6902. Gallagher KA , Jensen PR . Genomic insights into the evolution of hybrid isoprenoid biosynthetic gene clusters in the MAR4 marine streptomycete clade. BMC Genomics. 2015;16:1–13. Prieto-Davó A , Dias T , Gomes S, et al. The Madeira archipelago as a significant source of marine-derived actinomycete diversity with anticancer and antimicrobial potential. Frontiers Microbiol. 2016;7:1–12. ]. These compounds have great bioactive potential as antibacterial, antifungal, and anticancer agents making them potentially attractive to the pharmaceutical industry [ 3–5 Gallagher KA , Jensen PR . Genomic insights into the evolution of hybrid isoprenoid biosynthetic gene clusters in the MAR4 marine streptomycete clade. BMC Genomics. 2015;16:1–13. Prieto-Davó A , Dias T , Gomes S, et al. The Madeira archipelago as a significant source of marine-derived actinomycete diversity with anticancer and antimicrobial potential. Frontiers Microbiol. 2016;7:1–12. Bin Cheng Y , Jensen P , Fenical W , et al. Cytotoxic and antimicrobial napyradiomycins from two marine-derived Streptomyces strains. Eur J Org Chem. 2013;3751–3757. doi:10.1002/ejoc.201300349 ]. Four S. aculeolatus stains isolated from marine sediments collected off the Madeira Archipelago (coded PTM-29, PTM-129, PTM-346 and PTM-398) revealed the production of secondary metabolites with distinct bioactivities. To date, small organic compounds are the most widely explored but now, our main goal is the screening of extracellular water soluble protein species with biological activities secreted by S. aculeolatus. Materials and methods: The antimicrobial activity of S. aculeolatus extracellular extracts (aqueous phase) was evaluated by screening the biological activity against two Methicillin Resistant strains of the human pathogen Staphyloccocus aureus, namely COL and MW2. Minimal inhibitory concentrations (MIC) were determined by growth inhibition halos and microdilution analysis. Results: Preliminary results showed antimicrobial activity against MRSA from extracellular extracts of strains PTM-29, PTM-129, PTM-346 and PTM-398, with MIC values ranging from 0.010-0.020 µg/µl. Discussion and conclusions: All the aqueous extracellular extracts exhibited inhibitory growth effect against COL and MW2 MRSA strains. In addition, the extract from PTM-346 showed the highest inhibitory activity whereas PTM-29 showed the lowest level of activity. Interestingly, PTM-129, whose organic crude extract did not show biological activity, revealed antimicrobial activity in the aqueous extracellular extract obtained in this study. In the future, the bioactive peptide species will be isolated from the extracellular extracts, purified and characterized by biochemical techniques. Ultimately, this work will deliver novel bioactive peptides that might be conducted to clinical trials, in the pursuing of lead-like agents.

The marine-obligate actinomycetes of the genus Salinispora are well-known for their ability to produce natural products with important biological activities (e.g. antineoplastic, antifungal, antimicrobial, antitumor, immunosuppressive, etc.).[1–3] Three species of this genus have been characterized: S. pacifica, S. tropica and S. arenicola.[4] Previous genomics studies of the latter two revealed that the genes responsible for the synthesis of natural products occupy 8.8 and 10.9% of their genomes, respectively, and suggest that the production of secondary metabolites may be associated with environmental adaptation.[5] In this context, the present work aims to characterize the proteins involved in the synthesis of the bioactive compounds produced by S. arenicola and S. pacifica strains (coded PTM-99 and PTM-93, respectively), isolated from marine sediments collected from the Madeira Archipelago, and to understand the relationship between the global protein expression and the production of secondary metabolites in these actinomycetes. To achieve this purpose, we performed a comparative proteomic analysis based on two-dimensional electrophoresis (2DE) associated to mass spectrometry techniques. According to the image analysis with Melanie 7.06 software, the 2D gels (16 x 16 cm; 12,5% acrylamide) of S. arenicola and S. pacifica cells showed 650 and 510 spots, respectively in a pH range 4–7. The protein profiles of the two actinomycetes display significant differences, a very interesting observation, since these species possess a similarity of 99% according to the 16S rRNA gene sequence analysis. The gels were compared to identify the common protein spots in each sample, which could be involved in the synthesis of secondary metabolites in these two bacterial species. After gel alignment and matching, 37 protein spots were identified with the same molecular weight and isoelectric point. The identification of these proteins is being performed by mass spectrometry (MS) techniques.