The gut microbiomes of large ruminant herbivores are extremely complex, and harbor a population of anaerobic fungi that persist despite being overwhelmed by bacteria. Recently, omics analyses have revealed a wealth of potential, novel natural products in four culturable anaerobic gut fungal species of the phylum Neocallimastigomycota: Anaeromyces robustus, Caecomyces churrovis, Neocallimastix californiae, and Piromyces finnis. The putative natural products likely enable these fungi to survive in a competitive habitat. Across the four gut fungi, in silico genome mining tools predict 146 genes that encode biosynthetic enzymes for a variety of natural products, including nonribosomal peptides and polyketides that can be repurposed for applications in pharmaceutical discovery and as biomaterial building blocks. During standard laboratory growth, 26% of predicted core biosynthetic genes were transcribed, and 30% of predicted gene products were translated across the four gut fungi. Metabolomics with liquid chromatography-tandem mass spectrometry (LC-MS/MS) detected hundreds of potential natural products from fungal supernatants, with a putatively identified polyketide-like styrylpyrone (baumin) detected across all four strains. With the goal of isolating and characterizing gut fungal natural products, a variety of culturing techniques are used to amplify secondary metabolism in the cultured gut fungi. One such technique is synthetic co-culturing of gut fungal strains with the rumen gut bacterium Fibrobacter sp. UWB7 to act as competition in the growth environment. Heterologous expression, or the expression of non-native genes in model hosts, is an additional route to access gut fungal natural products. Heterologous expression constructs were designed for several polyketide and several nonribosomal peptide biosynthetic genes for expression in the fungal microbial hosts of Saccharomyces cerevisiae and Aspergillus nidulans. Samples from cultured gut fungi and heterologous expression hosts undergo a metabolomics screen with LC-MS/MS to prioritize natural product isolation and characterization. Overall, this study describes a wealth of novel natural products sourced from the anaerobic fungi, and prioritizes candidates for biotech application.

Discovery and characterization of novel natural products from the rumen microbiome

ACS Spring 2022

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