Engineering Synthetic Systems Inspired By Anaerobic Fungi

Anaerobic fungi in the hindgut of large herbivores are among the most robust organisms at degrading crude lignocellulose. Their remarkable cellulolytic capabilities have great potential for use in biomass breakdown and biofuel processing. Anaerobic fungi achieve cellulolytic efficiency through the production of large, multi-enzyme complexes called fungal cellulosomes. In isolation, anaerobic fungi metabolize some of the released sugars and convert them into fermentation products. In nature, however, they exist in a community with archaea, bacteria, and protozoa, which drastically alter the behavior of the fungi. By elucidating the parts responsible for efficient biomass degradation at both the protein and cellular level, we seek to replicate this efficiency in synthetic systems.

Previously, anaerobic fungi have been shown to interact closely with methane producing archaea (methanogens). Methanogens reduce CO₂ with H₂ for growth and methanogenesis,which maintains a low partial pressure of H₂ in their environments, allowing the fungi to more efficiently metabolize sugars. To further investigate this mechanism, native fungal/methanogen consortia were isolated from herbivore fecal materials. ITS profiling and genomic sequencing revealed the presence of one fungus, two methanogens, and one bacterium in one consortium, which was stable under continuous passage for over 20 months. The consortium demonstrated faster and more complete degradation of cellulosic substrates, as well as a wider range of utilized substrates compared to the monocultured fungus alone. By introducing the methanogens into cultures of other well-characterized anaerobic fungi, stable synthetic co-cultures were established. These stable synthetic consortia demonstrated similar efficiency, and suggest a promising option for conversion of crude biomass into sustainable chemicals. 

Anaerobic fungi create large enzyme complexes called fungal cellulosomes, which they utilize for biomass degradation. Fungal cellulosomes are similar to bacterial cellulosomes in that the protein-protein interactions are mediated through parts termed the dockerin and cohesin, however the exact sequence for the cohesin module has yet to be established. Through a combination of –OMICs approaches and traditional biochemical assays, a large putative scaffoldin molecule was identified. The scaffoldin was heterologously expressed and screened for interaction with recombinant dockerin through an ELISA. The K_D,app was determined using Equilibrium Surface Plasmon Resonance. A transcriptomic survey of dockerin domain-containing proteins revealed some degree of conservation in dockerin location on classes of CAZymes. Using this observation, the dockerin domains were adapted to thermostable cellulases, demonstrating its applicability as a novel protein scaffolding systems and suggesting the possibility of synthetic cellulosomes for biomass degradation.