PCR and Omics Based Techniques to Study the Diversity, Ecology and Biology of Anaerobic Fungi: Insights, Challenges and Opportunities

Abstract

Anaerobic fungi (phylum Neocallimastigomycota) are common inhabitants of the digestive tract of mammalian herbivores, and in the rumen, can account for up to 20% of the microbial biomass. Anaerobic fungi play a primary role in the degradation of lignocellulosic plant material. They also have a syntrophic interaction with methanogenic archaea, which increases their fiber degradation activity. To date, nine anaerobic fungal genera have been described, with further novel taxonomic groupings known to exist based on culture-independent molecular surveys. However, the true extent of their diversity may be even more extensively underestimated as anaerobic fungi continue being discovered in yet unexplored gut and non-gut environments. Additionally many studies are now known to have used primers that provide incomplete coverage of the Neocallimastigomycota. For ecological studies the internal transcribed spacer 1 region (ITS1) has been the taxonomic marker of choice, but due to various limitations the large subunit rRNA (LSU) is now being increasingly used. How the continued expansion of our knowledge regarding anaerobic fungal diversity will impact on our understanding of their biology and ecological role remains unclear; particularly as it is becoming apparent that anaerobic fungi display niche differentiation. As a consequence, there is a need to move beyond the broad generalization of anaerobic fungi as fiber-degraders, and explore the fundamental differences that underpin their ability to exist in distinct ecological niches. Application of genomics, transcriptomics, proteomics and metabolomics to their study in pure/mixed cultures and environmental samples will be invaluable in this process. To date the genomes and transcriptomes of several characterized anaerobic fungal isolates have been successfully generated. In contrast, the application of proteomics and metabolomics to anaerobic fungal analysis is still in its infancy. A central problem for all analyses, however, is the limited functional annotation of anaerobic fungal sequence data. There is therefore an urgent need to expand information held within publicly available reference databases. Once this challenge is overcome, along with improved sample collection and extraction, the application of these techniques will be key in furthering our understanding of the ecological role and impact of anaerobic fungi in the wide range of environments they inhabit.

Beef cattle that respond differently to fescue toxicosis have distinct gastrointestinal tract microbiota

“…Anaerobic fungi of the phylum Neocallimastigomycota are effective fiber degrading organisms in the herbivore gut and have been reported to improve feed intake, feed digestibility, feed efficiency, and daily weight gain and milk production [39, 40]. Of the 50 most abundant OTUs, OTUs 14, 22, 25, 26, 31, 34, 35, and 45 were classified as members of the Neocallimastigomycota.…”

Biomass‐degrading enzymes are catabolite repressed in anaerobic gut fungi

“…Within this consortium of anaerobic microbes, anaerobic fungi are the least well‐characterized, but they are some of the most active microbes against lignin‐infused substrates, containing the largest repertoire of biomass‐degrading genes in sequenced fungi . The low abundance of gut fungi in a rumen supplied with high‐starch diets, and conversely high abundance under high‐fiber conditions suggests that these fungi are highly adapted to fiber degradation, which is of particular interest for the hydrolysis of recalcitrant biomass encountered in bioprocessing . However, the first genomes of the anaerobic fungi were sequenced only in the past 5 years due to the problems associated with fungal culture, and difficulties that stem from AT‐richness and extensive genomic repeats .…”

Rumen anaerobic fungi

“…21 While they can also produce lactate and ethanol as waste products, hydrogenosome-driven metabolism is likely more favorable. 11 identified cluster in a separate clade, which have been assigned to their own family (Neocallimastigaceae), order (Neocallimastigales), class (Neocallimastigomycetes), and phylum (Neocallimastigomycota). 23 They include three monocentric genera (Neocallimastix, Caecomyces and Piromyces) and three polycentric genera (Anaeromyces, Orpinomyces and Cyllamyces), with two additional proposed genera (Oontomyces and Buwchfawromyces) based on molecular data from other gut environments.…”

“…24,25 Comparative genomic studies have revealed that horizontal gene transfer appears to have played an important role in the adaptation of fungi to gut environments, which have included carbohydrate degrading enzymes as well as certain hydrogenosome enzymes. 11 Slowly, but surely, interest in anaerobic fungi has continued to gain in momentum, in large part driven by interest in their biotechnological potential. 11 With continued improvements in culturing, phylogenetic profiling, metagenomics, and gene annotation, more insights are likely yet to come for these fascinating fungi.…”

“…11 Slowly, but surely, interest in anaerobic fungi has continued to gain in momentum, in large part driven by interest in their biotechnological potential. 11 With continued improvements in culturing, phylogenetic profiling, metagenomics, and gene annotation, more insights are likely yet to come for these fascinating fungi.…”