Metabolomic studies of plant-associated fungi: method development for the increased production and identification of microbial bioactive secondary metabolites

Author(s)

Denise Medeiros Selegato

• Document type: Doctoral Thesis
• Press: Araraquara. 2019-05-06.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Química. Araraquara
• Defense date: 2019-03-29
• Advisor: Ian Castro Gamboa

Abstract

The discovery of new drug leads from natural sources has decreased over the last decades, emerging the use of new and uncommon matrices for the screening of bioactive secondary metabolites. Among those, the plant microbiota and marine microorganisms have demonstrated a great potential to provide new pharmaceutical leads, producing a high diversity of chemical structures encountered in little-explored and extensive microbial population. Conventionally, microbial metabolite screening is performed in monocultures, in the absence of biotic and abiotic interactions. However, the lack of these communications commonly found in nature seriously limit the chemical diversity that can be obtained by one single strain, remaining silenced many other genes encoded for new secondary metabolites. Over the last decade, several methods have been developed aiming to understand the conditions under which biosynthetic cryptic genes are activated. Among those, the post genomic strategies have revealed widespread potential for the enhancement of chemical diversity, modifying different levels of the cellular machinery for a comprehensive regulation of the microbial metabolome. These strategies include co-cultivation, biotransformation and One Strain Many Compounds (OSMAC) and have been successfully used as a fast and inexpensive alternative for the induction of new bioactive compounds. Notwithstanding these strategies for the expansion of the microbial metabolome, in the screening of these complex matrices, the large dynamic range and diversity of metabolites still hamper their identification and biological correlation. These analytical obstacles require increasingly robust techniques and the use of algorithms for a more accurate and multivariate analysis of the chemical data. In this work, we present an integrative strategy for the enhancement of the identification and detection of secondary metabolites in plant-associated microbes. For this, we have applied different post-genomic strategies and computational tools to accurately elucidate known and novel bioactive metabolites in crude extracts. Results showed that post genomic strategies provided a significant increase in the chemical diversity of all selected fungi, in which co-culture resulted in the highest and more diverse metabolic induction. Moreover, the application of Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS)-dereplication methodologies, combined with deconvolution-based 1HNMR quantification and MS/MS Molecular Networking, enabled the extraction of a broader chemical data, accurate biological correlation and the identification of several secondary metabolites never reported for the targeted species. Overall, microbes have demonstrated a multitude of biosynthetic pathways. Hence, the activation of cryptic genes led not only to the identification of novel biologically promising leads, but also a better understanding of how the biotic and abiotic interactions can interfere on these fungal metabolomes. (AU)

• FAPESP's process: 14/05935-0 - Co-culture of microorganisms isolated from the rhizosphere of Senna spectabilis aiming to produce new bioactive metabolites
• Grantee: Denise Medeiros Selegato
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)