Isolation and structure elucidation of bioactive secondary metabolites from marine-derived bacteria

Due to the uniqueness of the marine environment, natural products isolated from marine organisms often present differentiated chemical structures exhibiting a wide range of biological activities through specialized mechanisms. Several pharmaceutical drugs with the active substance being of marine origin are already on the market, while a significant number is currently in preclinical and clinical trials. In addition, marine natural products find applications in the fields of cosmetology, nutrition, biotechnology and materials science. In recent years, marine microorganisms have attracted constantly increasing attention as they have been proven to be a rich source of novel bioactive molecules with novel carbon skeletons or uncommon functional groups. In the framework of the present PhD thesis, the chemical composition of the organic extracts obtained after large scale cultivation of four selected bacterial strains isolated from marine sediments collected in the Aegean (Milos) and Ionian (Kefalonia and Kerkyra) Seas was investigated. The extracts were subjected to a series of chromatographic separations to result in the isolation of their secondary metabolites, the chemical structures of which were elucidated on the basis of the analysis of their spectroscopic data (mainly NMR and MS). In total, 78 different secondary metabolites were isolated, 75 of which have so far been identified. Among these, 12 are new natural products, while 6 were isolated for the first time from a natural source.Twenty-five secondary metabolites were isolated and identified from Bacillus endophyticus BI0327 strain, including: (i) nine 2,5-diketopiperazines (2,5-DKPs), (ii) three indole derivatives, (iii) one naphthoquinone, (iv) two triterpenes, (v) eight simple aromatic derivatives and (vi) two methacrylic acid derivatives, two of which are new natural products, whereas one was isolated for the first time from a natural source. Two secondary metabolites were isolated and identified from Streptomyces althioticus BI0850 strain, including one 2,5-DKP and one simple nitrogenous compound. Forty-four secondary metabolites were isolated and identified from Streptomyces smyrnaeus BI0918 strain, including: (i) twenty-four 2,5-DKPs, one of which is a new natural product, (ii) seven benzodiazepine derivatives, four of which are new natural products, whereas two were isolated from the first time from a natural source, (iii) an indole derivative, (iv) two nucleoside derivatives, (v) two other nitrogen compounds, one of which was isolated for the first time from a natural source, (vi) a simple sulfur-containing compound, (vii) two derivatives of benzoic acid, (viii) one simple glycerol derivative, (ix) a methacrylic acid derivative which is a new natural product and (x) three high molecular weight polyether metabolites. Thirty-four secondary metabolites were isolated and identified from Bacillus subtilis strain BI0980, including: (i) nineteen 2,5-DKPs, two of which are new natural products and one was isolated for the first time from a natural source, (ii) three indole derivatives, (iii) one nucleoside derivative, (iv) one flavonoid, (v) five benzoic acid derivatives, (vi) four metabolites of different chemical classes, two of which are new natural products and one was isolated for the first time from a natural source and (vii) a methacrylic acid derivative. The evaluation of the biological activity of the metabolites isolated in sufficient quantities is currently in progress.Λόγω της ιδιαιτερότητας του θαλάσσιου περιβάλλοντος, τα φυσικά προϊόντα που απομονώνονται από θαλάσσιους οργανισμούς παρουσιάζουν συχνά διαφοροποιημένες χημικές δομές με ευρύ φάσμα βιολογικών δράσεων μέσω εξειδικευμένων μηχανισμών. Στο εμπόριο κυκλοφορούν ήδη αρκετά φαρμακευτικά σκευάσματα με τη δραστική ουσία να είναι θαλάσσιας προέλευσης, ενώ πολλά βρίσκονται σε στάδιο προκλινικών και κλινικών δοκιμών. Ακόμη, τα θαλάσσια φυσικά προϊόντα βρίσκουν εφαρμογή στην κοσμητολογία, στη διατροφή, στη βιοτεχνολογία και στην επιστήμη υλικών. Τα τελευταία χρόνια, οι θαλάσσιοι μικροοργανισμοί παρουσιάζουν ιδιαίτερο ενδιαφέρον, καθώς έχουν αποδειχθεί πλούσιες πηγές νέων βιοδραστικών μορίων με πρωτότυπους ανθρακικούς σκελετούς ή σπάνιες δραστικές ομάδες. Στα πλαίσια της παρούσας διδακτορικής διατριβής μελετήθηκε η χημική σύσταση των οργανικών εκχυλισμάτων που παραλήφθηκαν έπειτα από υγρή καλλιέργεια σε μεγάλη κλίμακα τεσσάρων επιλεγμένων βακτηριακών στελεχών, τα οποία έχουν απομονωθεί από θαλάσσια ιζήματα που συλλέχθηκαν στο Αιγαίο (Μήλος) και το Ιόνιο (Κεφαλονιά και Κέρκυρα) Πέλαγος. Τα εκχυλίσματα υποβλήθηκαν σε σειρά χρωματογραφικών διαχωρισμών με σκοπό την απομόνωση των δευτερογενών μεταβολιτών τους, οι οποίοι ταυτοποιήθηκαν με βάση την ανάλυση των φασματοσκοπικών τους δεδομένων (κυρίως NMR και MS). Συνολικά, απομονώθηκαν 78 διαφορετικοί δευτερογενείς μεταβολίτες, εκ των οποίων μέχρι στιγμής έχουν ταυτοποιηθεί οι 75. Εξ αυτών, οι 12 αποτελούν νέα φυσικά προϊόντα, ενώ οι 6 απομονώθηκαν για πρώτη φορά από φυσική πηγή. Από το στέλεχος Bacillus endophyticus ΒΙ0327 απομονώθηκαν και ταυτοποιήθηκαν 25 δευτερογενείς μεταβολίτες: (α) εννέα 2,5-δικετοπιπεραζίνες (2,5-DKPs), (β) τρία παράγωγα ινδολίου, (γ) μία ναφθοκινόνη, (δ) δύο τριτερπένια, (ε) οκτώ απλά αρωματικά παράγωγα και (στ) δύο παράγωγα μεθακρυλικού οξέος, εκ των οποίων δύο αποτελούν νέα φυσικά προϊόντα, ενώ ένας απομονώθηκε πρώτη φορά από φυσική πηγή. Από το στέλεχος Streptomyces althioticus ΒΙ0850 απομονώθηκαν και ταυτοποιήθηκαν 2 δευτερογενείς μεταβολίτες, εκ των οποίων μία 2,5-DKP και μία απλή αζωτούχος ένωση.Από το στέλεχος Streptomyces smyrnaeus ΒΙ0918 απομονώθηκαν και ταυτοποιήθηκαν 44 δευτερογενείς μεταβολίτες: (α) εικοσιτέσσερις 2,5-DKPs, εκ των οποίων μία αποτελεί νέο φυσικό προϊόν, (β) επτά παράγωγα βενζοδιαζεπινών, εκ των οποίων τέσσερα αποτελούν νέα φυσικά προϊόντα και δύο απομονώθηκαν πρώτη φορά από φυσική πηγή, (γ) ένα παράγωγο ινδολίου, (δ) δύο νουκλεοσιδικά παράγωγα, (ε) δύο άλλες αζωτούχες ενώσεις, εκ των οποίων μία απομονώθηκε πρώτη φορά από φυσική πηγή, (στ) μία απλή θειούχος ένωση, (ζ) δύο παράγωγα βενζοϊκού οξέος, (η) ένα απλό παράγωγο γλυκερόλης, (θ) ένα παράγωγο μεθακρυλικού οξέος, το οποίο αποτελεί νέο φυσικό προϊόν και (ι) τρεις πολυαιθερικοί μεταβολίτες μεγάλου μοριακού βάρους. Από το στέλεχος Bacillus subtilis ΒΙ0980 απομονώθηκαν και ταυτοποιήθηκαν 34 δευτερογενείς μεταβολίτες: (α) δεκαεννέα 2,5-DKPs, εκ των οποίων δύο αποτελούν νέα φυσικά προϊόντα και μία απομονώθηκε πρώτη φορά από φυσική πηγή, (β) τρία παράγωγα ινδολίου, (γ) ένα νουκλεοσιδικό παράγωγο, (δ) ένα φλαβονοειδές, (ε) πέντε παράγωγα βενζοϊκού οξέος, (στ) τέσσερεις μεταβολίτες διαφορετικών κατηγοριών, εκ των οποίων δύο αποτελούν νέα φυσικά προϊόντα και ένα απομονώθηκε πρώτη φορά από φυσική πηγή και (ζ) ένα παράγωγο μεθακρυλικού οξέος. Η αξιολόγηση της βιολογικής δράσης των μεταβολιτών που έχουν απομονωθεί σε ικανές ποσότητες βρίσκεται επί του παρόντος σε εξέλιξη

New Chlorinated 2,5-Diketopiperazines from Marine-Derived Bacteria Isolated from Sediments of the Eastern Mediterranean Sea

From the organic extracts of five bacterial strains isolated from marine sediments collected in the East Mediterranean Sea, three new (15, 16, 31) and twenty-nine previously reported (1–14, 17–30, 32) metabolites bearing the 2,5-diketopiperazine skeleton were isolated. The structures of the chlorinated compounds 15, 16, and 31 were elucidated by extensive analysis of their spectroscopic data (NMR, MS, UV, IR). Compounds 15 and 16 were evaluated for their antifungal activity against Candida albicans and Aspergillus niger but were proven inactive. The relevant literature is supplemented with complete NMR assignments and revisions for the 29 previously reported compounds

New C15 Acetogenins from Two Species of Laurencia from the Aegean Sea

The chemical diversity of the approximately 1,200 natural products isolated from red algae of the genus Laurencia, in combination with the wide range of their biological activities, have placed species of Laurencia in the spotlight of marine chemists’ attention for over 60 years. The chemical investigation of the organic (CH2Cl2/MeOH) extracts of Laurencia microcladia and Laurencia obtusa, both collected off the coasts of Tinos island in the Aegean Sea, resulted in the isolation of 32 secondary metabolites, including 23 C15 acetogenins (1–23), 7 sesquiterpenes (24–30) and 2 diterpenes (31 and 32). Among them, six new C15 acetogenins, namely 10-acetyl-sagonenyne (2), cis-sagonenyne (3), trans-thuwalenyne C (4), tinosallene A (11), tinosallene B (12) and obtusallene XI (17), were identified and their structures were elucidated by extensive analysis of their spectroscopic data. Compounds 1–3, 5–11, 13 and 15–32 were evaluated for their antibacterial activity against Staphylococcus aureus and Escherichia coli

Neorogioltriol and Related Diterpenes from the Red Alga <i>Laurencia</i> Inhibit Inflammatory Bowel Disease in Mice by Suppressing M1 and Promoting M2-Like Macrophage Responses

Macrophages are central mediators of inflammation, orchestrating the inflammatory response through the production of cytokines and nitric oxide. Macrophages obtain pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes, which can be modulated by soluble factors, including natural products. Despite the crucial protective role of inflammation, chronic or deregulated inflammation can lead to pathological states, such as autoimmune diseases, metabolic disorders, cardiovascular diseases, and cancer. In this case, we studied the anti-inflammatory activity of neorogioltriol (1) in depth and identified two structurally related diterpenes, neorogioldiol (2), and O11,15-cyclo-14-bromo-14,15-dihydrorogiol-3,11-diol (3), with equally potent activity. We investigated the mechanism of action of metabolites 1&#8315;3 and found that all three suppressed macrophage activation and promoted an M2-like anti-inflammatory phenotype by inducing expression of Arginase1, MRC1, IRAK-M, the transcription factor C/EBP&#946;, and the miRNA miR-146a. In addition, they suppressed iNOS induction and nitric oxide production. Importantly, treatment of mice with 2 or 3 suppressed DSS-induced colitis by reducing tissue damage and pro-inflammatory cytokine production. Thus, all these three diterpenes are promising lead molecules for the development of anti-inflammatory agents targeting macrophage polarization mechanisms

In Vivo Evaluation of the Wound Healing Activity of Extracts and Bioactive Constituents of the Marine Isopod Ceratothoa oestroides

Wound healing is a fundamental response to tissue injury and a number of natural products has been found to accelerate the healing process. Herein, we report the preparation of a series of different polarity (organic and aqueous) extracts of the marine isopod Ceratothoa oestroides and the in vivo evaluation of their wound healing activity after topical administration of ointments incorporating the various extracts on wounds inflicted on SKH-hr1 hairless mice. The most active extract was fractionated for enrichment in the bioactive constituents and the fractions were further evaluated for their wound healing activity, while their chemical profiles were analyzed. Wound healing was evaluated by clinical assessment, photo-documentation, histopathological analysis and measurement of biophysical skin parameters, such as transepidermal water loss (TEWL), hydration, elasticity, and skin thickness. The highest levels of activity were exerted by treatment of the wounds with a fraction rich in eicosapentaenoic acid (EPA), as well as myristic and palmitoleic acids. Topical application of the bioactive fraction on the wounds of mice resulted in complete wound closure with a skin of almost normal architecture without any inflammatory elements

Expanding the terpene biosynthetic code with non-canonical 16 carbon atom building blocks

Humankind relies on specialized metabolites for medicines, flavors, fragrances, and numerous other valuable biomaterials. However, the chemical space occupied by specialized metabolites, and, thus, their application potential, is limited because their biosynthesis is based on only a handful of building blocks. Engineering organisms to synthesize alternative building blocks will bypass this limitation and enable the sustainable production of molecules with non-canonical chemical structures, expanding the possible applications. Herein, we focus on isoprenoids and combine synthetic biology with protein engineering to construct yeast cells that synthesize 10 non-canonical isoprenoid building blocks with 16 carbon atoms. We identify suitable terpene synthases to convert these building blocks into C(16) scaffolds and a cytochrome P450 to decorate the terpene scaffolds and produce different oxygenated compounds. Thus, we reconstruct the modular structure of terpene biosynthesis on 16-carbon backbones, synthesizing 28 different non-canonical terpenes, some of which have interesting odorant properties