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Novel secondary secondary metabolites metabolites from from the the deep deep sea sea
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Figure 5: Chemical structures of novel secondary metabolites from the deep sea (≥ 1000m).
Further information about the compounds are listed in Table 2
The dermacozines exemplarily demonstrate the enormous unexplored potential of deep-sea bacteria for recent drug discovery. Most of the novel chemical structures from deep-sea microorganisms originate from Gram-positive bacteria and in particular from members of the Actinobacteria (see Table 2). Like their terrestrial counterparts, representatives of marine Actinobacteria, even from deep-sea environments, appear to have a promising capacity for the production of secondary metabolites (Bull et al., 2005; Lam, 2006; Fenical and Jensen, 2006). Especially
members of the Streptomycetes appear to be well represented producers of novel bioactive structures from the deep ocean (Table 1). Streptokordin e.g. was produced by a Streptomyces sp. obtained from Ayu Trough sediment and displayed significant cytotoxicity against human tumor cell lines (IC50 < 10 µg/ml) (Jeong et al., 2006).
Another strain of Streptomyces sp. that has been isolated from 4680 m depth at Marshall Islands produces Gamma-indomycinone, a new compound of the class of pluramycine antibiotics (Schumacher et al., 1995). Anti-angionesis activity is attributed to streptopyrrolidine which was produced by another deep-sea isolate of Streptomyces sp.. Further deep-sea natural products are ammosamides A and B (Hughes et al., 2009). These compounds originated form a Streptomyces strain from 1618 m depth and exhibit remarkable cytotoxicity against colon carcinoma cells HCT-116. As it becomes obvious, natural products of Streptomyces sp. are clearly dominating the list of novel deep-sea substances. This might be due to the fact, that terrestrial Streptomycetes produce the main part of bioactive microbial compounds, and thus marine members of Streptomycetes are in special focus of natural product research. There are of course also biologically active compounds from other Gram-positive and Gram-negative strains as well (Table 2), but obviously representatives of the Actinobacteria appear to be especially promising for novel natural product discovery from deep-sea habitats.
The importance of marine actinomycetes for the discovery of natural products is also reflected by the substantial fraction of the genome (5-10%) that is allocated to mostly cryptic secondary metabolites (Baltz, 2008). Screening for secondary metabolite biosynthetic gene clusters like the non-ribosomal peptide synthetase (nrps) and the polyketide synthases (pks) biosynthesis genes became a common tool for the selection of promising producer strains from various habitats (Metsä Ketelä et al., 1999; Martens et al., 2007; Jiang et al., 2008; Zhao et al., 2008; Schneemann et al., 2010). Non-ribosomal polypeptides, such as the antibiotic vancomycin as well as various polyketides, such as aromatic polyketides (like tetracycline), macrolides (like erythromycine), polyether (like salinomycine) and polyenes (like nystatin), are complex natural products with an immense structural diversity. They are produced by multifunctional enzymes, termed non-ribosomal peptide synthetase (NRPS) and polyketide synthases (PKS) (Cane and Walsh, 1999; Schwarzer and Marahiel, 2001).
Bacterial strains from deep-sea sediments have also been effectively screened for nrps or pks gene fragments by PCR-amplification (Pathom-aree et al., 2006). Such
approaches reinforced the presence of a large amount of secondary metabolites producing bacteria in the deep-sea realm. In contrast to the relatively few secondary metabolites isolated from deep-sea environments yet, novel enzymes, lipids and exopolysaccharides with specific properties for biotechnological application have already been isolated from deep-sea bacteria, and in particular from those derived from deep-sea hydrothermal vent fields (for reviews see (Wilson and Brimble, 2009;
Pettit, 2010)). Due to the extreme working conditions of biotechnological production steps (like e.g. extreme temperature and pressure), bacteria from extreme environments revealed to be a suitable source for novel compounds with biotechnological use. Thus it might also be auspicious to explore the diversity of secondary metabolites from deep-sea bacteria of different and particularly of contrasting extreme habitats.
Aim of the thesis
The Aim of this thesis was the characterization of deep-sea bacteria and the elucidation of their potential to produce natural products. Accordingly, the major part of this study was focused on cultured bacteria. As bacterial strains from deep-sea habitats are barely investigated for the production of bioactive natural products to date, the research on deep-sea bacteria was conducted in two contrasting deep-sea ecosystems: an extremely oligotrophic environment and a deep-sea hydrothermal vent field (characteristic features are summarized in Table 1).
In both habitats the focus was laid on (a) the deep-sea bacterial communities (b) the physiology and metabolic capabilities of the indigenous bacteria and (c) the discovery of novel natural products from deep-sea bacteria:
(a) Investigation of deep-sea bacterial communities:
The bacterial communities of the LHF and the Eastern Mediterranean deep sea were analyzed by culture-dependent approaches. Especially representatives of novel taxa were of interest, because they might be specially adapted to the specific living conditions and might provide novel natural products. Since the search for and the subsequent production of bioactive natural products requires up-scaling of fermentation processes and a variety of test assays running under standard
laboratory conditions, special interest was laid on the mesophilic fraction of the bacterial communities. The LHF as well as the Eastern Mediterranean deep-sea bacterial community are supposed to comprise a considerable fraction of mesophiles which were the basis of this study. In addition, the barely investigated bacterial community of diffusive fluids from the LHF was analyzed by the onstruction of a 16S rRNA gene library.
(b) Physiological and metabolic capabilities:
Deep-sea bacteria need to be physiologically adapted to the abiotic prerequisites of their habitat, e.g., elevated hydrostatic pressure. Possibly, the production of secondary metabolites may also be affected by these living conditions. One objective of this thesis was therefore the investigation of bacterial taxa cultured after incubation at in situ pressure, since bacteria recovered after this treatment can be expected to be specifically adapted to the deep-sea. Further objectives were up-scaled cultivation, extraction and analysis of the respective metabolite spectra as well as the presence of gene clusters responsible for the production of secondary metabolite pathways (e.g. nrps and pks) to evaluate the potential of selected strains for the production of secondary metabolites. Besides these culture-dependent approaches of the metabolic capabilities, the barely investigated bacterial community of the LHF was analyzed for functional genes responsible for autotrophic CO2-fixation and sulfur oxidation / reduction.
(c) Discovery of novel active compounds from the deep sea:
Since the deep sea appeared to be barely investigated for active compounds to date, this thesis focused on two quite different deep-sea environments. Almost no prior knowledge was available concerning the potential of bioactive metabolites from deep-sea habitats in general and especially from the chosen habitats. Therefore, a major objective was to evaluate the potential of bacteria isolated from the deep sea for the synthesis of novel secondary metabolites.
Thesis outline
The results of this thesis are presented in the following chapters. All chapters are composed in form of manuscripts for publication, some are published (Chapter III, IV and V) others are submitted (Chapter I) or in preparation for submission (Chapter II and IV). Bacteria of the Eastern Mediterranean deep sea are characterized in Chapters I – III, while those of the Logatchev hydrothermal vent field are part of Chapters IV – VI.
Finally, the methods, results and future perspectives of the two investigated environments will be discussed (Discussion).
(a) Investigation of deep-sea bacterial communities
Chapter I and IV outline the isolation of heterotrophic aerobic bacteria from the two