Natural Product Discovery from Anaerobic Gut Fungi

Abstract

Secondary metabolites – also termed natural products – are small, organic compounds that often possess bioactivities and diverse chemical structures. These metabolites hold significant biotechnological and therapeutic potential, with applications in pesticides, preservatives, biopolymers, biofuels, and pharmaceuticals. Secondary metabolism is largely unexplored in obligate anaerobes – organisms that do not survive in the presence of molecular oxygen. In fact, nearly all known secondary metabolites are produced by aerobes. Anaerobic gut fungi (phylum Neocallimastigomycota) are obligate anaerobes that reside in the complex gut microbiomes of some large herbivores. In silico genome mining and previous omics analyses (transcriptomic, proteomic, metabolomic) indicate that anaerobic gut fungi possess untapped potential to produce secondary metabolites.

In this talk, I will outline two complementary approaches to explore secondary metabolites from anaerobic gut fungi: (1) direct, omics-based study of cultured anaerobic gut fungi and (2) heterologous expression of predicted biosynthetic genes in better-studied microbial hosts, Saccharomyces cerevisiae (yeast) and Aspergillus nidulans (a mold). Altogether, these studies provide foundational, omics-based datasets that advance our understanding of secondary metabolism in anaerobic gut fungi.

For the first approach, I profiled metabolites of two anaerobic gut fungal species (Anaeromyces robustus and Caecomyces churrovis) using untargeted gas chromatography with mass spectrometry (GC-MS). In parallel, I profiled metabolites of C. churrovis cultured under various growth conditions, using untargeted liquid chromatography with tandem mass spectrometry (LC-MS/MS). These metabolomic analyses show that anaerobic gut fungi produce likely terpene and terpenoid secondary metabolites. Additionally, I generated the first transcriptomic profile of the young, zoosporic life stage of anaerobic gut fungi (Neocallimastix californiae), which provides evidence for potential life stage-specific secondary metabolites.

For the heterologous expression-based approach, I expressed an array of predicted biosynthetic genes for polyketide synthases and non-ribosomal peptide synthetases in S. cerevisiae and A. nidulans. In several expression groups, heterologously expressed RNA or protein was successfully detected, and a metabolomics screen with LC-MS/MS determined metabolites that were differentially detected between expression groups and their empty vector negative control. This effort establishes the first expression platform to investigate biosynthetic pathways for anaerobic gut fungal secondary metabolites.