Antje Labes

Bio-mining the microbial treasures of the ocean: New natural products

The biological resources of the oceans have been exploited since ancient human history, mainly by catching fish and harvesting algae. Research on natural products with special emphasis on marine animals and also algae during the last decades of the 20th century has revealed the importance of marine organisms as producers of substances useful for the treatment of human diseases. Though a large number of bioactive substances have been identified, some many years ago, only recently the first drugs from the oceans were approved. Quite astonishingly, the immense diversity of microbes in the marine environments and their almost untouched capacity to produce natural products and therefore the importance of microbes for marine biotechnology was realized on a broad basis by the scientific communities only recently. This has strengthened worldwide research activities dealing with the exploration of marine microorganisms for biotechnological applications, which comprise the production of bioactive compounds for pharmaceutical use, as well as the development of other valuable products, such as enzymes, nutraceuticals and cosmetics. While the focus in these fields was mainly on marine bacteria, also marine fungi now receive growing attention. Although culture-dependent studies continue to provide interesting new chemical structures with biological activities at a high rate and represent highly promising approaches for the search of new drugs, exploration and use of genomic and metagenomic resources are considered to further increase this potential. Many efforts are made for the sustainable exploration of marine microbial resources. Large culture collections specifically of marine bacteria and marine fungi are available. Compound libraries of marine natural products, even of highly purified substances, were established. The expectations into the commercial exploitation of marine microbial resources has given rise to numerous institutions worldwide, basic research facilities as well as companies. In Europe, recent activities have initiated a dynamic development in marine biotechnology, though concentrated efforts on marine natural product research are rare. One of these activities is represented by the Kieler Wirkstoff-Zentrum KiWiZ, which was founded in 2005 in Kiel (Germany).

Comprehensive Investigation of Marine Actinobacteria Associated with the Sponge Halichondria panicea

ABSTRACT Representatives of Actinobacteria were isolated from the marine sponge Halichondria panicea collected from the Baltic Sea (Germany). For the first time, a comprehensive investigation was performed with regard to phylogenetic strain identification, secondary metabolite profiling, bioactivity determination, and genetic exploration of biosynthetic genes, especially concerning the relationships of the abundance of biosynthesis gene fragments to the number and diversity of produced secondary metabolites. All strains were phylogenetically identified by 16S rRNA gene sequence analyses and were found to belong to the genera Actinoalloteichus, Micrococcus, Micromonospora, Nocardiopsis, and Streptomyces. Secondary metabolite profiles of 46 actinobacterial strains were evaluated, 122 different substances were identified, and 88 so far unidentified compounds were detected. The extracts from most of the cultures showed biological activities. In addition, the presence of biosynthesis genes encoding polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) in 30 strains was established. It was shown that strains in which either PKS or NRPS genes were identified produced a significantly higher number of metabolites and exhibited a larger number of unidentified, possibly new metabolites than other strains. Therefore, the presence of PKS and NRPS genes is a good indicator for the selection of strains to isolate new natural products.

Algae as an important environment for bacteria – phylogenetic relationships among new bacterial species isolated from algae

Goecke F., Thiel V., Wiese J., Labes A. and Imhoff J.F. 2013. Algae as an important environment for bacteria – phylogenetic relationships among new bacterial species isolated from algae. Phycologia 52: 14–24. DOI: 10.2216/12-24.1.s1. Bacteria are an inherent part of the biotic environment of algae. Recent investigations revealed that bacterial communities associated with algae were generally highly host specific. Several new bacterial species and genera were isolated from algae, which suggested that algae were an interesting environment for discovery of new bacterial taxa; however, the distribution of the different phylogenetic groups among those isolates remained unclear, and this information could help to explain specific associations. We conducted a phylogenetic study based on 16S rRNA gene sequences available in GenBank, including 101 validly described bacterial species that were isolated from eukaryotic macro- and micro-algae from marine and freshwater environments. These species were distributed among six bacterial phyla, including: Bacteroidetes (42 species), Proteobacteria (36 species), and Firmicutes, Actinobacteria, Verrucomicrobia and Planctomycetes (23 species). Bacterial species and strains that carried out similar metabolic functions were found to colonize similar algal taxa or algal groups. This assumption was supported by information available from bacterial species descriptions: (1) Most of the bacteria described from microalgae grouped into the Roseobacter clade (Alphaproteobacteria), which indicated that members of this group were well adapted for life in close association with phytoplankton; and (2) 32% of the bacterial species, mainly isolates from macroalgae, were able to decompose macroalgal polysaccharides. Because algal-bacterial association are still under-studied in various algal groups, we expect a great number of new bacterial taxa to be discovered in the future.

Calcarides A-E, antibacterial macrocyclic and linear polyesters from a Calcarisporium strain.

Bioactive compounds were detected in crude extracts of the fungus, Calcarisporium sp. KF525, which was isolated from German Wadden Sea water samples. Purification of the metabolites from the extracts yielded the five known polyesters, 15G256α, α-2, β, β-2 and π (1–5), and five new derivatives thereof, named calcarides A–E (6–10). The chemical structures of the isolated compounds were elucidated on the basis of one- and two-dimensional NMR spectroscopy supported by UV and HRESIMS data. The compounds exhibited inhibitory activities against Staphylococcus epidermidis, Xanthomonas campestris and Propionibacterium acnes. As the antibacterial activities were highly specific with regard to compound and test strain, a tight structure-activity relationship is assumed.

Unusual Starch Degradation Pathway via Cyclodextrins in the Hyperthermophilic Sulfate-Reducing Archaeon Archaeoglobus fulgidus Strain 7324

The hyperthermophilic archaeon Archaeoglobus fulgidus strain 7324 has been shown to grow on starch and sulfate and thus represents the first sulfate reducer able to degrade polymeric sugars. The enzymes involved in starch degradation to glucose 6-phosphate were studied. In extracts of starch-grown cells the activities of the classical starch degradation enzymes, alpha-amylase and amylopullulanase, could not be detected. Instead, evidence is presented here that A. fulgidus utilizes an unusual pathway of starch degradation involving cyclodextrins as intermediates. The pathway comprises the combined action of an extracellular cyclodextrin glucanotransferase (CGTase) converting starch to cyclodextrins and the intracellular conversion of cyclodextrins to glucose 6-phosphate via cyclodextrinase (CDase), maltodextrin phosphorylase (Mal-P), and phosphoglucomutase (PGM). These enzymes, which are all induced after growth on starch, were characterized. CGTase catalyzed the conversion of starch to mainly beta-cyclodextrin. The gene encoding CGTase was cloned and sequenced and showed highest similarity to a glucanotransferase from Thermococcus litoralis. After transport of the cyclodextrins into the cell by a transport system to be defined, these molecules are linearized via a CDase, catalyzing exclusively the ring opening of the cyclodextrins to the respective maltooligodextrins. These are degraded by a Mal-P to glucose 1-phosphate. Finally, PGM catalyzes the conversion of glucose 1-phosphate to glucose 6-phosphate, which is further degraded to pyruvate via the modified Embden-Meyerhof pathway.

First crenarchaeal chitinase found in Sulfolobus tokodaii

This is the first description of a functional chitinase gene within the crenarchaeotes. Here we report of the heterologues expression of the ORF BAB65950 from Sulfolobus tokodaii in E. coli. The resulting protein degraded chitin and was hence classified as chitinase (EC 3.2.4.14). The protein characterization revealed a specific activity of 75 mU/mg using colloidal chitin as substrate. The optimal activity of the enzyme was measured at pH 2.5 and 70°C, respectively. A dimeric enzyme configuration is proposed. According to amino acid sequence similarities chitinases are attributed to the two glycoside hydrolase families 18 and 19. The derived amino acid sequence of the S. tokodaii gene differed from sequences of these two glycoside hydrolase families. However, within a phylogenetic tree of protein sequences, the crenarchaeal sequence of S. tokodaii clustered in close proximity to members of the glycoside hydrolase family 18.

Novel Members of Glycoside Hydrolase Family 13 Derived from Environmental DNA

ABSTRACT Starch and pullulan-modifying enzymes of the α-amylase family (glycoside hydrolase family 13) have several industrial applications. To date, most of these enzymes have been derived from isolated organisms. To increase the number of members of this enzyme family, in particular of the thermophilic representatives, we have applied a consensus primer-based approach using DNA from enrichments from geothermal habitats. With this approach, we succeeded in isolating three new enzymes: a neopullulanase and two cyclodextrinases. Both cyclodextrinases displayed significant maltogenic amylase side activity, while one showed significant neopullulanase side activity. Specific motifs and domains that correlated with enzymatic activities were identified; e.g., the presence of the N domain was correlated with cyclodextrinase activity. The enzymes exhibited stability under thermophilic conditions and showed features appropriate for biotechnological applications.

Phylogenetic analysis and antibiotic activity of bacteria isolated from the surface of two co-occurring macroalgae from the Baltic Sea

Bacteria associated with Fucus vesiculosus and Delesseria sanguinea, two macroalgae from the Kiel Fjord were investigated seasonally over two years by cultivation-based methods. A total of 166 bacterial strains were isolated from the macroalgae, affiliated to seven classes of bacteria (Actinobacteria, Bacilli, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Cytophagia and Flavobacteria). According to 16S rRNA gene sequence similarities they were arranged in 82 phylotypes of > 99.0% sequence identity. Assuming that chemical factors rule the bacteria–macroalga and bacteria–bacteria interactions on algal surfaces, we tested the antibiotic activity of the bacterial isolates not only against a panel of four standard test organisms (Bacillus subtilis, Candida glabrata, Escherichia coli and Staphylococcus lentus) but also four macroalga-associated microorganisms: Algicola bacteriolytica and Pseudoalteromonas elyakovii (macroalgal pathogens), and Bacillus algicola and Formosa algae (strains associated with algal surfaces). Organic extracts of more than 51% of the isolates from the two macroalgae inhibited the growth of at least one of the tested microorganisms. As much as 46% and 45% of the isolates derived from F. vesiculosus and D. sanguinea, respectively, showed antimicrobial activity against the set of macroalga-associated bacteria, compared with 13 and 19% against a standard set of microorganisms. High antibacterial activity against macroalgal pathogens and bacterial competitors support the assumption that complex chemical interactions shape the relationships of bacteria associated with macroalgae and suggest that these bacteria are a rich source of antimicrobial metabolites.

From Discovery to Production: Biotechnology of Marine Fungi for the Production of New Antibiotics

Filamentous fungi are well known for their capability of producing antibiotic natural products. Recent studies have demonstrated the potential of antimicrobials with vast chemodiversity from marine fungi. Development of such natural products into lead compounds requires sustainable supply. Marine biotechnology can significantly contribute to the production of new antibiotics at various levels of the process chain including discovery, production, downstream processing, and lead development. However, the number of biotechnological processes described for large-scale production from marine fungi is far from the sum of the newly-discovered natural antibiotics. Methods and technologies applied in marine fungal biotechnology largely derive from analogous terrestrial processes and rarely reflect the specific demands of the marine fungi. The current developments in metabolic engineering and marine microbiology are not yet transferred into processes, but offer numerous options for improvement of production processes and establishment of new process chains. This review summarises the current state in biotechnological production of marine fungal antibiotics and points out the enormous potential of biotechnology in all stages of the discovery-to-development pipeline. At the same time, the literature survey reveals that more biotechnology transfer and method developments are needed for a sustainable and innovative production of marine fungal antibiotics.

Lindgomycin, an Unusual Antibiotic Polyketide from a Marine Fungus of the Lindgomycetaceae.

An unusual polyketide with a new carbon skeleton, lindgomycin (1), and the recently described ascosetin (2) were extracted from mycelia and culture broth of different Lindgomycetaceae strains, which were isolated from a sponge of the Kiel Fjord in the Baltic Sea (Germany) and from the Antarctic. Their structures were established by spectroscopic means. In the new polyketide, two distinct domains, a bicyclic hydrocarbon and a tetramic acid, are connected by a bridging carbonyl. The tetramic acid substructure of compound 1 was proved to possess a unique 5-benzylpyrrolidine-2,4-dione unit. The combination of 5-benzylpyrrolidine-2,4-dione of compound 1 in its tetramic acid half and 3-methylbut-3-enoic acid pendant in its decalin half allow the assignment of a new carbon skeleton. The new compound 1 and ascosetin showed antibiotic activities with IC50 value of 5.1 (±0.2) µM and 3.2 (±0.4) μM, respectively, against methicillin-resistant Staphylococcus aureus.