Engineering early-branching anaerobic gut fungi for lignocellulose breakdown and bioproduction

To develop new technologies to break down plant material into sugar, much can be learned by studying how microbes digest lignocellulose in biomass-rich environments, such as the digestive tract of large herbivores. Anaerobic gut fungi (Neocallimastigales) are native to the gut and rumen of these animals, where they have evolved powerful enzymes to degrade plant biomass. Our goal is to develop new experimental tools to engineer anaerobic fungi and anaerobic microbial consortia for lignocellulose breakdown and chemical production. To accomplish this goal, we isolated a panel of anaerobic fungi and associated microbes from different herbivores and screened for their ability to degrade several types of lignin-rich grasses and agricultural waste. By focusing on model anaerobic fungi from the Piromyces, Neocallimastix, and Anaeromyces genera, we have employed next-generation sequencing to discover thousands of new genes, revealing hundreds of novel biomass-degrading enzymes. Additionally, we have characterized key regulatory patterns for these enzymes, which depend on the environment of the fungus. Using this information, we have piloted new genetic engineering strategies to manipulate gut fungi at the molecular level, along with ‘bottom-up’ strategies to synthesize microbial consortia for compartmentalized breakdown and bioproduction.