(5a) Engineering Anaerobic Gut Fungi for Lignocellulose Hydrolysis and Biofuel Production

Biofuels derived from lignocellulosic materials are an attractive, renewable alternative to petroleum-based fuels, yet the recalcitrance of lignocellulose to enzymatic degradation remains a significant barrier to biofuel development.  Anaerobic gut fungi that reside within the rumen and hindgut of large herbivores are among the most efficient digesters of lignocellulose in nature due to their invasive, filamentous growth and broad range of fibrolytic enzymes.  Despite the powerful cellulose degrading capacity of gut fungi, remarkably little is known about the cellulolytic enzymes from these organisms due to difficulties associated with their isolation and culture.  In order to adapt anaerobic fungi for biotechnology applications, and to explore the attributes of attractive cellulolytic enzymes that they produce, we have implemented methods to isolate and sustain anaerobic gut fungi in batch culture.  Following isolation of fungi from horse and cow digestive tracts, we have supported fungal growth in a rumen fluid-rich medium, and coupled cellular proliferation to the generation of fermentation gases that accumulate during growth.  Through cultivation of anaerobic fungi and the exploitation of catabolic regulation patterns, we have been able to apply powerful metagenomic strategies to discover new cellulase genes and novel cellulolytic protein complexes in these organisms.  En route to adapting anaerobic gut fungi for direct cellulosic bioprocessing, we are also exploring several methods to achieve stable genetic transformation in these microbes.