Anaerobic fungi (<i>Neocallimastigomycota</i>) are native to the digestive tracts of ruminant and hindgut fermenting animals where they are central to the digestion of crude lignocellulosic materials. Anaerobic fungi secrete the largest known diversity of lignocellulolytic carbohydrate active enzymes (CAZymes) in the fungal kingdom (&gt;300 CAZymes), which unaided can degrade up to 60% of the ingested plant material. Thus these microorganisms have rich biotechnological potential ranging from developing renewable energy technologies to improving animal nutrition. To this end, we are working to build a suite of genetic engineering tools for these organisms. This tool box includes a variety of promoters, antibiotic resistance markers, fluorescent reporters, as well as methods to transform anaerobic fungi. We have also identified subcellular localization signal sequences for targeting proteins to specific organelles (e.g nuclear localization sequences). Currently we are working to isolate an Autonomously Replicating Sequence to enhance plasmid stability. Preliminary efforts show addition of these putative ARS sequences to unstable fungal plasmids rescues growth to nearly positive control levels. Simultaneously we are working to identify a minimal centromeric sequence to develop a truly stable plasmid. By leveraging chromatin conformation capture (HiC) we have determined the degree of both structural and sequence conservation in gut fungal centromeres across 2 genera. Future efforts will involve testing these putative minimal centromeres in anaerobic fungi for assessing plasmid maintenance. Once validated, these parts will form the foundational tools to generate a deeper systems-level understanding of anaerobic fungal physiology while establishing fundamental knowledge about regulation of gut fungal CAZymes.

Building genetic engineering tools for anaerobic gut fungi

ACS Fall 2021

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