Enhancement of chemical diversity in fungal endophytes from arid plants of Andalusia

Abstract

It has been widely described that fungi represent one of the most prolific sources of novel NPs, but low production yields and the lack of expression of cryptic gene clusters in laboratory conditions are frequently key limiting factors for exploiting the full secondary metabolite potential of fungi in vitro. Native plant communities from arid areas present distinctive characteristics to survive in extreme conditions. The large number of poorly studied endemic plants represents a unique potential source for the discovery of novel fungal symbionts as well as host-specific endophytes not yet described. This work represents the first study culturing and characterizing the fungal symbiont community of an extensive number of halophytic and xerophytic plant specimens from arid areas of Andalusia. Moreover, once the populations of isolates were characterized, the aim of the study has been to stimulate and characterize their chemical diversity on secondary metabolites production by applying diverse culture-based methodologies: adding adsorptive polymeric resins, using small-molecule epigenetic elicitors and/or co-culturing microorganisms isolated from the same ecological niche. A total of 349 fungal strains isolated from 63 selected plant species from arid ecosystems located in the southeast of Andalusia, were characterized morphologically as well as based on their ITS/28S ribosomal gene sequences. The fungal communities isolated were distributed among 19 orders including Basidiomycetes and Ascomycetes, with Pleosporales as the most abundant order. In total, 107 different genera were identified being Neocamarosporium the most frequently isolated genus, followed by Preussia and Alternaria. In the case of the Preussia isolates, a specific study was carried out to review their taxonomy and to characterize occurring chemotypes of Preussia species from arid and other environmental samples of the Iberian Peninsula. Sixteen natural compounds were identified and based on combined analyses, 11 chemotypes were inferred and could be used to resolve groups of closely related Preussia species. Initially, strains were grown in four different media in presence and absence of selected resins to promote chemical diversity in the generation of new secondary metabolites. Fermentation extracts were chemically characterized and evaluated looking for new antifungal activities against plant and human fungal pathogens, as well as, cytotoxic activities against the human liver cancer cell line HepG2. From the 349 isolates tested, 126 (36%) exhibited significant bioactivities including 58 strains with exclusive antifungal properties and 33 strains with exclusive activity against the HepG2 hepatocellular carcinoma cell line. After LCMS analysis of the active extracts, 68 known bioactive secondary metabolites could be identified as produced by 96 strains, and 12 likely unknown compounds were found in a subset of 14 fungal endophytes. Small molecule histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors are commonly used to perturb the production of fungal metabolites leading to the induction of the expression of silent biosynthetic pathways. Thus, a systematic approach to evaluate and identify the possible effects of HDAC and DNMT inhibitors on the metabolic profiles of wild type fungal endophytes was developed. Metabolomics by volcano plot representations based on LC/MS was used to determine significant differences between two sets of samples from two different conditions of interest. Chemical identification and characterization of the most significant SMs induced by these epigenetic modifiers was performed with part of the isolates obtained. Co-culture approach was also tested as a third way of inducing antagonist relationships between the isolates from plants of arid zones of Andalusia. For this purpose, a screening looking for antagonistic interactions among the population of isolates was performed by co-culturing the strains on agar plates in pairs. An automated extraction method was designed and developed to determine if these antagonistic effects observed between the isolates were induced by co-culturing and if the antifungal compounds were accumulating spatially in the interaction zone. Ultraviolet (UV) and Mass Spectrometry (MS) imaging techniques were developed to visualize the spatial distribution of metabolites and facilitated the identification of induced secondary metabolites upon the microbial interactions. The chemical profiles of the differential expression after applications of these three culture-based approaches were compared. As proof of concept of induced new chemistry, fourteen induced secondary metabolites only produced under these approaches were purified and identified. The structures of six of these compounds, some of them bioactives, were new and herein elucidated. Results confirmed that the exploitation of the microbial richness associated to arid areas of Andalusia, combined with the application of integrated culturing methods, can favour the activation of fungal cryptic metabolic pathways towards the discovery of new bioactive natural products with the potential to become new leads for the development of future therapeutic agents.