Mit der Kraft des Meeres gegen Krebs

Für die Behandlung schwerer Erkrankungen wie Krebs und entzündlicher Leiden gibt es dringenden Bedarf an neuen Substanzen für effizientere und schonenderer Therapien mit weniger Nebenwirkunge

Ein ungewöhnlicher Stärkeabbauweg im hyperthermophilen archaeellen Sulfatreduzierer Archaeoglobus fulgidus Stamm 7324 und Charakterisierung Stärke-abbauender Enzyme aus geothermalen Habitaten

Comparative analyses of sugar catabolism in archaea revealed that degradation of glucose and hexose polymers proceeds via modified glycolytic pathways, mainly via modified Embden-Meyerhof pathways. The utilization of sugars by Archaeoglobus fulgidus species, which represent the first isolated archaeal sulfat reducer, has not unequivocally been demonstrated so far. Thus, in this thesis it was found that Archaeoglobus fulgidus strain 7324, rather than the type strain VC16, was able to grow on starch and sulfate as energy and carbon source. Starch was degraded to acetate via an unusual starch degradation pathway. Some key enzymes were purified and characterized. Furthermore, starch degrading and modifying enzymes from geothermal habitats were characterized to find properties useful for biotechnological applications. 2.1 Starch degradation in A. fulgidus strain 7324 A. fulgidus strain 7324 was found to grow on starch (1 g/l) and sulfate (30 mM) in presence of yeast extract (0.5 g/l) as energy and carbon source. During exponential growth on starch, 1 mol of glucose-equivalent was incompletely oxidized with sulfate to approximately 2 mol acetate, 2 mol CO2 and 1 mol H2S. Beside starch, amylose and β-cyclodextrin were used as substrates. Other sugars, e.g. glucose, fructose and maltose were not utilized. This is the first report of growth of a sulfate reducer on starch, i.e. on a polymeric sugar. Extracts of starch-grown cells of A. fulgidus Stamm 7324 contained all activites of an unusual starch degradation pathway to acetate, which includes the conversion of starch to glucose 6-phosphate as well as the conversion of glucose to pyruvate via a modified Embden-Meyerhof pathway. Specific activities of the key enzymes of the starch conversion to acetate were significantly higher in starch-grown cells than in lactate-grown cells, indicating induction of these enzymes during starch catabolism. In A. fulgidus strain VC16 neither enzyme activities of starch degradation nor the corresponding homologous genes were detected. Starch degradation to glucose 6-phosphate. Extracts of starch-grown A. fulgidus strain 7324 contained all enzymes of a starch degradation pathway so far only described for Klebsiella oxytoca and Thermococcus sp. B1001. In this pathway starch is converted to cyclodextrins by means of a cyclodextrin glucanotransferase. After transport into the cell cyclodextrins are linearised via a cyclodextrinase. By means of a maltodextrin phosphorylase the resulting maltooligodextrins are cleaved by phosporylation. The resulting glucose 1-phosphat is converted via phosphoglucomutase to glucose 6-phosphat, an intermediate of glycolysis. These four enzymes were purified and characterized. The N-terminal amino acid sequences and the molecular and catalytic properties of the enzymes were very similar to their homologs of the Thermococcales. The classical enzymes of starch degradation, α-amylase and pullulanase, could not be detected in A. fulgidus strain 7324 Glucose degradation to pyruvate via a modified Embden-Meyerhof pathway. In extracts of starch-grown cells the following enzymes of a modified Embden-Meyerhof pathway were detected: ADP-dependent glucokinase, phosphoglucose isomerase, ADP-dependent 6-phosphofructokinase, fructose-1,6-phosphate aldolase, glyceraldehyde-3-phosphate: ferredoxin oxidoreductase, phosphoglycerate mutase, enolase, and pyruvate kinase. So far, this pathway was only described for Thermococcales. The first enzyme of this modified Embden-Meyerhof pathway, ADP-dependent glucokinase, was purified and characterized. The properties of the glucokinase were similar to the glucokinases from Thermococcales. So far, formation of glucose as substrate of the ADP-dependent glucokinase can not be explained by the described starch degradation pathway. Pyruvate conversion to acetate. Pyruvate conversion to acetate involved pyruvate: ferredoxin oxidoreductase and ADP-forming acetyl-CoA synthetase. Activities of phosphotransacetylase and acetate kinase could not be detected. The findings indicate that the archaeal sulfate reducer A. fulgidus strain 7324 converts starch to acetate via an unsual starch degradation pathway, a modified Embden-Meyerhof pathway and acetyl-CoA synthetase (ADP-forming). This is the first report of a starch degradation pathway in a sulfate reducer. 2.2 Characterization of starch degrading enzymes from geothermal habitats Furthermore, starch degrading and modifying enzymes from geothermal habitats were characterized within the EC project �Thermogenes�. The genes of these enzymes were isolated from environmental DNA and organisms, respectively, originating from geothermal habitats in Iceland. The intention of these experiments was the characterization of enzymes from the glycosylhydrolase family 13 in respect to their substrate specificity, cation dependence and stability in order to find properties of biotechnological interest. Starting from seven different isolated, sequenced and cloned. Seven different genes of starch degrading enzymes were expressed in E. coli, purified and characterized. One protein showed pullulanase activity and one neopullulanase activity, respectively; five proteins exhibited cyclodextrinase activity. The characterized proteins were highly stable at moderate thermophilic conditions and showed a broad substrate spectrum, useful properties for biotechnological applications. Differences in substrate specificity can be explained by differences in amino acid sequences, e.g. the existence of a N-terminal domain correlated with cyclodextrinase activity

Higher yields of cyclodepsipetides from Scopulariopsis brevicaulis by random mutagenesis

The ascomycete Scopulariopsis brevicaulis, which was isolated from the marine sponge Tethya aurantium, produces two cyclodepsipeptides, scopularides A and B [1]. Both peptides exhibit activity against several tumor cell lines. Within the EU-project MARINE FUNGI (EU FP7, 265926) one of our aims is to enhance the production of these secondary metabolites. We are in the process to establish two ways of random mutagenesis by both UV radiation and transposon-mediated. To this end we created UV-mutants and a miniaturised screening method was developed. UV-radiation was performed at 312 nm and the survival rate was set to 1 %. With this method a mutant library was established. To screen these mutants for higher secondary metabolites production, we developed a miniaturised screening method which includes decreased cultivation volume, fast extraction and an optimised LC-MS analysis format. Using the UV mutagenesis, we were able to identify several mutants with a higher scopularide production in comparison to the wild type. One of these mutants, which produces three times more biomass and more than double the amount of scopularide A, has been used for another round of mutation. Next generation sequencing is being employed to identify the molecular genetic basis of the observed mutations. In parallel we employ transposable elements to introduce mutants [2]. The impact of transposons on gene expression as well as their ability to cause major mutations within the genome or single genes makes them an interesting tool for random mutagenesis [3, 4, 5]. We employ the Vader transposon in its homologous host and found that Vader mostly integrates within or very close to genes. Thus it appears to be a useful tool for transposon-mediated mutagenesis in A. niger (6). At current we try to enhance its usability by modifying the Vader element

Production of scopularide A in submerged culture with Scopulariopsis brevicaulis

Background: Marine organisms produce many novel compounds with useful biological activity, but are currently underexploited. Considerable research has been invested in the study of compounds from marine bacteria, and several groups have now recognised that marine fungi also produce an interesting range of compounds. During product discovery, these compounds are often produced only in non-agitated culture conditions, which are unfortunately not well suited for scaling up. A marine isolate of Scopulariopsis brevicaulis, strain LF580, produces the cyclodepsipeptide scopularide A, which has previously only been produced in non-agitated cultivation. Results: Scopulariopsis brevicaulis LF580 produced scopularide A when grown in batch and fed-batch submerged cultures. Scopularide A was extracted primarily from the biomass, with approximately 7% being extractable from the culture supernatant. By increasing the biomass density of the cultivations, we were able to increase the volumetric production of the cultures, but it was important to avoid nitrogen limitation. Specific production also increased with increasing biomass density, leading to improvements in volumetric production up to 29-fold, compared with previous, non-agitated cultivations. Cell densities up to 36 g L-1 were achieved in 1 to 10 L bioreactors. Production of scopularide A was optimised in complex medium, but was also possible in a completely defined medium. Conclusions: Scopularide A production has been transferred from a non-agitated to a stirred tank bioreactor environment with an approximately 6-fold increase in specific and 29-fold increase in volumetric production. Production of scopularide A in stirred tank bioreactors demonstrates that marine fungal compounds can be suitable for scalable production, even with the native production organism

Molecular Networking-Based Metabolome and Bioactivity Analyses of Marine-Adapted Fungi Co-cultivated With Phytopathogens

Fungi represent a rich source of bioactive metabolites and some are marketed as alternatives to synthetic agrochemicals against plant pathogens. However, the culturability of fungal strains in artificial laboratory conditions is still limited and the standard mono-cultures do not reflect their full spectrum chemical diversity. Phytopathogenic fungi and bacteria have successfully been used in the activation of cryptic biosynthetic pathways to promote the production of new secondary metabolites in co-culture experiments. The aim of this study was to map the fungal diversity of Windebyer Noor, a brackish lake connected to Baltic Sea (Germany), to induce the chemical space of the isolated marine-adapted fungi by co-culturing with phytopathogens, and to assess their inhibitory potential against six commercially important phytopathogens. Out of 123 marine-adapted fungal isolates obtained, 21 were selected based on their phylogenetic and metabolite diversity. They were challenged with two phytopathogenic bacteria (Pseudomonas syringae and Ralstonia solanacearum) and two phytopathogenic fungi (Magnaporthe oryzae and Botrytis cinerea) on solid agar. An in-depth untargeted metabolomics approach incorporating UPLC–QToF–HRMS/MS-based molecular networking (MN), in silico MS/MS databases, and manual dereplication was employed for comparative analysis of the extracts belonging to nine most bioactive co-cultures and their respective mono-cultures. The phytopathogens triggered interspecies chemical communications with marine-adapted fungi, leading to the production of new compounds and enhanced expression of known metabolites in co-cultures. MN successfully generated a detailed map of the chemical inventory of both mono- and co-cultures. We annotated overall 18 molecular clusters (belonging to terpenes, alkaloids, peptides, and polyketides), 9 of which were exclusively produced in co-cultures. Several clusters contained compounds, which could not be annotated to any known compounds, suggesting that they are putatively new metabolites. Direct antagonistic effects of the marine-adapted fungi on the phytopathogens were observed and anti-phytopathogenic activity was demonstrated.The untargeted metabolomics approach combined with bioactivity testing allowed prioritization of two co-cultures for purification and characterization of marine fungal metabolites with crop-protective activity. To our knowledge, this is the first study employing plant pathogens to challenge marine-adapted fungi

Observación de bacterias sobre la superficie de oogonios liberados de Fucus vesiculosus L. (Phaeophyceae)

Pese al enorme número de bacterias que se encuentran normalmente en la columna de agua, se registra por primera vez su presencia sobre oogonios del alga Fucus vesiculosus. En cortes transversales, sin embargo, los oogonios se encontraban libres de epibiosis. Las algas fucoides son fundamentales en ciertos ambientes costeros, mas el impacto microbiano sobre su ciclo de vida y colonización es completamente desconocido.Pese al enorme número de bacterias que se encuentran normalmente en la columna de agua, se registra por primera vez su presencia sobre oogonios del alga Fucus vesiculosus. En cortes transversales, sin embargo, los oogonios se encontraban libres de epibiosis. Las algas fucoides son fundamentales en ciertos ambientes costeros, mas el impacto microbiano sobre su ciclo de vida y colonización es completamente desconocido

Development and Validation of a Fast and Optimized Screening Method for Enhanced Production of Secondary Metabolites Using the Marine Scopulariopsis brevicaulis Strain LF580 Producing Anti-Cancer Active Scopularide A and B

Natural compounds from marine fungi are an excellent source for the discovery and development of new drug leads. The distinct activity profiles of the two cyclodepsipeptides scopularide A and B against cancer cell lines set their marine producer strain Scopulariopsis brevicaulis LF580 into the focus of the EU project MARINE FUNGI. One of the main goals was the development of a sustainable biotechnological production process for these compounds. The secondary metabolite production of strain LF580 was optimized by random mutagenesis employing UV radiation. For a fast and reliable detection of the intracellular secondary metabolite production level, a miniaturized bioactivity-independent screening method was developed, as the random mutagenesis yielded a large number of mutants to be analysed quantitatively and none of the existing hyphenated bioassay-dependent screening systems could be applied. The method includes decreased cultivation volume, a fast extraction procedure as well as an optimized LC-MS analysis. We show that deviation could be specifically reduced at each step of the process: The measuring deviation during the analysis could be minimized to 5% and technical deviation occurring in the downstream part to 10–15%. Biological variation during the cultivation process still has the major influence on the overall variation. However, the approach led to a 10-fold reduction of time and similar effects on costs and effort compared to standard reference screening methods. The method was applied to screen the UV-mutants library of Scopulariopsis brevicaulis LF580. For validation purposes, the occurring variations in the miniaturized scale were compared to those in the classical Erlenmeyer flask scale. This proof of concept was performed using the wild type strain and 23 randomly selected mutant strains. One specific mutant strain with an enhanced production behavior could be obtained

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