Modulating growth and secondary metabolite patterns in lichen under stress

Many organisms containing high-value compounds are difficult to culture or are becoming endangered or even extinct by over-harvesting. Lichens, in general, are slow growing organisms and the extraction of the naturally grown, composite thalli, in many cases is economically not feasible and profitable and can be also very limited. Mycobiont cultures are an attractive alternative to the extraction of naturally grown thalli. The applicant`s laboratory at the University of Salzburg has established a world -wide recognized and also for Europe unique culture collection of c. 150 different mycobionts. The modulation ("regulative manipulation") of growth conditions of microorganism and fungi is a common strategy used in biotechnology and applied microbiology to improve yields and diversity of secondary metabolites of therapeutic value. Interest in polyketide â type metabolites is considerable, as many of these natural products are of medical, industrial and/or agricultural importance. In case studies, we have started to "modulate" growth and culture conditions. We have optimized culture conditions to obtain increased biomass production for several selected mycobionts (e.g. Roccella decipiens, species of the genus Xanthoparmelia) by adopting particular environmental conditions in one of our culture chambers. In recent investigations, by exploring further possibilities to optimize culture conditions and biomass production it turned out that axenically cultured mycobionts can be triggered to produce single or a whole pattern of secondary metabolites. Polyketides and shikimate derivatives, have been demonstrated, to be only biosynthesized under "permissive" ecological conditions. By using the knowledge from preliminary investigations and doing further extensive test series in the planned project we would be able to achieve the production of one particular polyketide and even the production of a predictable pattern of polyketides, depending upon the investigated lichen chemotypes. Such studies could also help to elucidate the often observed variation in secondary products (chemosyndromic variation) within a lichen population growing under heterogeneous conditions. Variations in chemistry actually mirror physiological, ecological and even evolutionary responses to changes in the environment and climate. The repeatable and in-vitro production of higher quantities of lichen metabolites in fungal cell cultures has already and could further become a milestone elucidating the architecture and function of PKS-genes that are involved in polyketide and "still unknown" genes that control shikimate production. In a novel and holistic approach, functional genomics will be used to understand the molecular mechanisms that are involved in the desiccation tolerance of some lichens. By constructing a cDNA library from the lichen Xanthoparmelia conspersa and its cultured mycobiont , we plan to perform trancriptome sampling which could be used to detect and identify further "new genes" responsible for the control of desiccation resistance in lichens.

As most lichens are slow-growing organisms, the extraction of valuable biologically active secondary metabolites from lichen thalli growing in the natural environment (since 2016, recognized as a 2 fungi & algae symbiosis) is economically not profitable and is/was, if done, only performed for perfume industry. The major objective of the project was to find an alternative to the collection of thalli from nature and use axenically cultured mycobionts, which would lead to higher quantities and diversity of secondary metabolites by effective modulation of growth and culture conditions. As most of these metabolites produced by lichens are polyketide-type compounds and in some cases, they are shikimate/pulvinic acid derivatives (the latter are often thought to act as sunscreens filtering harmful UV light on exposed rock surfaces), which could have a potential medical and industrial application, our test series, especially with selected species of Xanthoparmelia exhibited very interesting and exciting results. With new combinations of nutrients in the culture media and well defined microclimatic conditions in the culture chambers (simulations of desiccation stress and shifts of temperatures), we succeeded growing mycobionts in higher quantitites, which then predictably produced single or more complex patterns of secondary metabolites, sometimes even complete patterns of related and unrelated lichen substances. Particularly, the Xanthoparmelia species, which grew under natural conditions in semiarid localities of Australia, revealed an exceptional degree of chemosyndromic variation. Under specific simulation of stress (drought and low temperature treatments) some of the major substances became satellite substances and vice versa. By changing the environmental conditions in the culture chambers, in several cases, related and similar secondary metabolite patterns as found within thalli from nature, were found. It became obvious by comparing numerous test series that the mycobionts produced only typical chemosyndromes under considerable desiccation stress and low concentrations of carbohydrates in the nutrient media. It was the first time to get direct support for our hypotheses. From the point of view of the project leader this was an essential mile stone to understand and reinterpret the outcome of the test series. In further studies we were able to decipher the enzyme architecture, localization and function of polyketide-synthase genes of selected species of Xanthoparmelia. The search for desiccation resistance/tolerance genes was initiated, but was not terminated, for this reason could be the major objective in a follow-up future proposal.