Developing stable plasmid transformation techniques for anaerobic gut fungi

Abstract

Lignocellulosic feedstock degradation is a critical process in acquiring sustainable fuel and high-value chemicals. Anaerobic gut fungi (AGF), microorganisms residing in herbivore guts, have promising potential in the lignocellulose biodegradation process, because of their distinctive plant carbohydrate binding and biomass degradation ability through multiple carbohydrate-activating enzymes and their assembly into fungal cellulosomes that synergistically enhance biodegradation rates. Although there has been a successful attempt to develop a genetic engineering toolkit for transforming gene cassettes into an AGF system, CAZyme expression through this system was never investigated, and it is also transient, relying on periodic DNA dosing. ☐ Hence, we attempted to improve this pre-developed genetic engineering technique for AGF. First, we tried expressing CAZymes through this system. At the same time, we investigated candidate sequences that would support autonomous replication of gene cassette on AGF, ranging from yeast 2-micron plasmid, Neocallimastix giraffe GF-Ma genomic DNA library, and random AT sequence library. Although we were not fully able to confirm the expression of CAZymes through the current AGF transformation platform, we confirmed that out of 3 candidate sequences, yeast 2-micron plasmid exhibits longer and higher expression of proteins compared to the current transformation platform for AGF, opening the possibility for further research.