Untersuchungen zur Biosynthese von Indolalkaloiden aus den Basidiomyceten der Gattung Psilocybe

The goal of this doctoral thesis was the close examination of the biosynthesis of different indole alkaloides produced by different species of the fungal genus Psilocybe. This included the first characterization of the biosynthesis of psilocybin as the main psychotropic agent and the first discovery of the simultaneous production of β-carbolines. In the first step, the genome of P. cyanescens was sequenced and analyzed for characteristic genes and clusters or modifying enzymes. After the detection of the psilocybin biosynthesis cluster in Psilocybe, the cluster was expressed heterologous in E. coli and A. niger and an in vitro characterization of the psilocybin biosynthesis was done. Because of the raised pharmaceutical interest as well as some collaboration with the company Promega and the non-profit organization Usona, an expanded biocatalytic route for the in vitro production of psilocybin was established. The solution was the development of a continuous in vitro synthesis through a connection of the tryptophan synthase (TS) out of the basidiomycete P. cubensis with the biosynthesis route of psilocybin. Additionally, the substrate specifity of the TS were tested against different other halogenated indoles and for the first time different analogues of psilocybin could be produced biocatalytically with the same set of enzymes (Blei, Baldeweg et al. 2018). The generation of another derivate the 6-methylpsilocybin succeeded through an exchange with the TS of S. enterica (Fricke, Sherwood et al. 2019). Furthermore, the biosynthesis of the indole alkaloid l-Hypaphorin was characterized using the methyltransferase TrpM. Surprisingly no interference occured between the substrates of TrpM and the methyltransferase from the psilocybin biosynthesis PsiM because of strict substrate specifics. Supplemental to the already addressed indole alkaloides my work could identify further natural products out of the β-carboline class in the fruiting bodies and mycelia of different Psilocybe species. This was a big discovery in regards to the possible synergistic effects this could have on the psychoactive effect of Psilocybin through an inhibition of the monoaminoxidase enzyme (Blei, Dörner et al. 2019)

«Золота» пора кооперації в умовах непу

Рецензія на книгу: Голець В.В. Кооперація і неп (20-і рр. XX ст.). - Чернігів: Просвіта, 2006. - 244 с

Simultaneous Production of Psilocybin and a Cocktail of β‐Carboline Monoamine Oxidase Inhibitors in “Magic” Mushrooms

The psychotropic effects of Psilocybe “magic” mushrooms are caused by the l ‐tryptophan‐derived alkaloid psilocybin. Despite their significance, the secondary metabolome of these fungi is poorly understood in general. Our analysis of four Psilocybe species identified harmane, harmine, and a range of other l ‐tryptophan‐derived β‐carbolines as their natural products, which was confirmed by 1D and 2D NMR spectroscopy. Stable‐isotope labeling with 13 C 11 ‐ l ‐tryptophan verified the β‐carbolines as biosynthetic products of these fungi. In addition, MALDI‐MS imaging showed that β‐carbolines accumulate toward the hyphal apices. As potent inhibitors of monoamine oxidases, β‐carbolines are neuroactive compounds and interfere with psilocybin degradation. Therefore, our findings represent an unprecedented scenario of natural product pathways that diverge from the same building block and produce dissimilar compounds, yet contribute directly or indirectly to the same pharmacological effects

Bacillus subtilis attachment to Aspergillus niger hyphae results in mutually altered metabolism

Interaction between microbes affects the growth, metabolism and differentiation of members of the microbial community. While direct and indirect competition, like antagonism and nutrient consumption have a negative effect on the interacting members of the population, microbes have also evolved in nature not only to fight, but in some cases to adapt to or support each other, while increasing the fitness of the community. The presence of bacteria and fungi in soil results in various interactions including mutualism. Bacilli attach to the plant root and form complex communities in the rhizosphere. Bacillus subtilis, when grown in the presence of Aspergillus niger, interacts similarly with the fungus, by attaching and growing on the hyphae. Based on data obtained in a dual transcriptome experiment, we suggest that both fungi and bacteria alter their metabolism during this interaction. Interestingly, the transcription of genes related to the antifungal and putative antibacterial defence mechanism of B. subtilis and A. niger, respectively, are decreased upon attachment of bacteria to the mycelia. Analysis of the culture supernatant suggests that surfactin production by B. subtilis was reduced when the bacterium was co-cultivated with the fungus. Our experiments provide new insights into the interaction between a bacterium and a fungus

Enhanced surface colonisation and competition during bacterial adaptation to a fungus

Bacterial-fungal interactions influence microbial community performance of most ecosystems and elicit specific microbial behaviours, including stimulating specialised metabolite production. Here, we use a co-culture experimental evolution approach to investigate bacterial adaptation to the presence of a fungus, using a simple model of bacterial-fungal interactions encompassing the bacterium Bacillus subtilis and the fungus Aspergillus niger. We find in one evolving population that B. subtilis was selected for enhanced production of the lipopeptide surfactin and accelerated surface spreading ability, leading to inhibition of fungal expansion and acidification of the environment. These phenotypes were explained by specific mutations in the DegS-DegU two-component system. In the presence of surfactin, fungal hyphae exhibited bulging cells with delocalised secretory vesicles possibly provoking an RlmA-dependent cell wall stress. Thus, our results indicate that the presence of the fungus selects for increased surfactin production, which inhibits fungal growth and facilitates the competitive success of the bacterium