Triggering bioactive metabolite production in fungi

Worldwide, spreading drug resistance of pathogens in medicine or agriculture has become a huge problem in the last decades. It is all the more serious, since at the same time the number of novel, effective drugs decreased considerably. Fungi are amongst those organisms, which have been successfully exploited to produce bioactive compounds like the antibiotic penicillin. However, recent research indicated that the potential of these organisms to produce such powerful bioactive compounds was vastly underestimated. Many biosynthetic pathways remained dormant under laboratory conditions so far and thus the real potential of even well- investigated organisms has yet to be determined. To unlock this potential and to produce novel compounds for medicine and agriculture it is vital to increase our currently limited understanding of fungal physiology. One major challenge is to activate these dormant biosynthetic pathways in order to stimulate the production of novel compounds. This project aims to exactly do this for the two biotechnologically interesting fungi Metarhizium brunneum and Penicillium ochrochloron. The former, a fungus which has been successfully used as biocontrol agent, is known to produce the so-called destruxins a broad range of highly interesting compounds with anti-insectal, phytotoxic and anti-cancer activity. The second fungus explored in this project is a fungus which has been used for bioleaching of heavy metal ores and which produces the novel and only little investigated antibiotic xanthoepocin. For this study, both organisms will be grown under highly standardized cultivation conditions and exposed to a range of different stimuli known to activate dormant biosynthetic pathways such as the simultaneous cultivation of two organisms or the presence of an epigenetic modifier. By correlating metabolic shifts with the bioactivity (e.g. test against multi-drug resistant bacteria and fungi) of samples, it will be possible to prioritize potential new bioactive metabolites for later isolation and identification and gain already first insights into the underlying physiology. Another aim of this project is to explore how these stimuli influence the production of 22 different destruxins and xanthoepocin. Also purified xanthoepocin and selected destruxins will be tested for their cytotoxicity and their bioactivity against a broad range of multi-drug resistant pathogens. The results of this study will be of interest for several fields of research including medicinal and agricultural applications and will allow unique new insights into the complex network of fungal metabolism.

Fungi are a rich source of bioactive natural compounds, which is of particular importance in times of increasing multi-resistance to antibiotics. One of many strategies for obtaining new secondary metabolites, but also for exploring fundamental physiological aspects, is the targeted manipulation of the environmental conditions under which fungi grow. This project, based on a physiological approach using standardised bioreactor batch cultures, exploited this strategy and sought to obtain novel bioactive natural products in combination with a deeper understanding of fungal biology. This study had two main objectives: (1) First, to pre-select sample fractions with potentially novel bioactive compounds produced by bioreactor-grown cultures of Penicillium ochrochloron and Metarhizium brunneum. The strains were exposed to different culture conditions, including nutrient limitation, different light conditions or co-cultivation. (2) Secondly, to investigate how the aforementioned stimuli affect the biotechnologically and medically interesting metabolites xanthoepocin (P. ochrochloron) and destruxins (M. brunneum). In this study, a number of trigger factors of the medically interesting secondary metabolite xanthoepocin were identified. For example, the highest yield was obtained under nitrogen limitation and in the dark. Furthermore, we were able to prove that xanthoepocin is effective against more multi-resistant bacterial species than previously assumed. If the light-sensitive molecule is protected from visible light, its effectiveness was even increased. We were furthermore able to observe an unexpected light-dependent production of the agriculturally and medically relevant destruxins in M. brunneum. By simultaneous cultivation (co-cultivation) of P. ochrochloron and M. brunneum, we achieved an additional significant increase in the destruxin yield. Several crude extracts of tested fungi showed promising bioactivity and will be the subject of future research. Due to the physiological approach, we were able to obtain a wealth of insights into basic physiological processes such as growth, production of secondary metabolites or excretion of organic acids. In general, the stimulus light proved to be an extremely effective trigger factor for manipulating fungal metabolism, whereby we could find evidence of profound changes at the physiological level. In addition to xanthoepocin and the destruxins, we were able to demonstrate for the first time that the production of a number of other already known secondary metabolites is light-dependent. Surprisingly, primary metabolism (e.g. organic acid excretion) also proved to be very light-sensitive in some cases, and we were able to demonstrate nutrient-dependent light sensitivity in several occasions. The results of this project provide unique insights into the (light) metabolism of fungi and form the basis for future research that will address both the primary and secondary metabolism of single and co-cultures of biotechnologically relevant fungi. In addition to numerous fundamental findings regarding selected medically relevant secondary metabolites, the light-dependent production of organic acids in fungi is likely to be of particular biotechnological relevance.