Design of Modular Parts for Synthetic Systems Inspired By Anaerobic Fungi

Anaerobic fungi in the hindgut of large herbivores are among the most robust organisms at degrading crude lignocellulose. They achieve this efficiency through the production of large, multi-enzyme complexes called fungal cellulosomes. The fungi also act synergistically with other microorganisms in the microbiome, such as archaea, bacteria, and protozoa. 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. 

At the protein level, fungal cellulosomes are similar to bacterial cellulosomes in that the protein-protein interactions are mediated through parts termed the dockerin and cohesin. However, many differences exist. The dockerin domains exist in tandem repeats and bear no species specificity like those in the bacterial systems. Furthermore, the exact sequence for the cohesin module has yet to be established. Through analysis of transcriptomic data for three fungal isolates, patterns governing the native placement of dockerin domains on fungal cellulases were characterized. By recombinantly grafting these dockerin domains onto similar enzymes from other organisms, the original activity of the enzymes were retained while allowing for incorporation of these exogenous enzymes into fungal cellulosomes. This was demonstrated for the TmCel5A, TmManB, TmXynA, and TmXynB from Thermotoga maritima. These incorporated enzymes demonstrated a greater level of synergy with the native cellulosomes when compared to the catalytic domain without the grafted dockerins. Similarly, the newly grafted dockerin modules were replaced with carbohydrate binding modules, demonstrating that this approach can be extended to other protein binding domains. The eventual goal is to create entirely synthetic cellulosomes, which could be applied to any biocatalytic process.

At the cellular level, the anaerobic fungi have also been shown to interact closely with methane producing archaea (methanogens). The methanogens siphon hydrogen and other metabolites from the fungi, allowing the fungi to more efficiently produce energy by increasing the flux through their hydrogenosomes. This increased energy is hypothesized to increase production of cellulases, accelerating the degradation of lignocellulose in co-culture. To further investigate this mechanism, native fungal/methanogen co-cultures were isolated from herbivore fecal materials. These co-cultures were maintained together and also separated into monocultures, effectively creating parts for synthetic co-cultures. By introducing the methanogens into cultures of other well-characterized anaerobic fungi, stable synthetic co-cultures were established. With this proof of concept, other parts to the consortia can be introduced, such as non-native methanogens capable of funneling other accumulating metabolites like acetate. These stable synthetic consortia should increase the efficiency of conversion of crude biomass into sustainable chemicals.