To bolster renewable technologies, more efficient methods to extract sugars from crude plant biomass are required. Plants contain cellulose that can be depolymerized into fermentable sugars for microbial biofuel production. However, crude biomass contains hemicellulose, lignin, and other biopolymers that complicate enzymatic hydrolysis and sugar release. To address this problem, one can turn to nature for inspiration, particularly to microbes that thrive on crude biomass. Large herbivores, such as cows and horses, harbor a consortium of microbes in their digestive tract that convert recalcitrant biomass to sugars. Of these microbes, anaerobic gut fungi represent a rich, understudied source of biomass degrading enzymes, including those required for cellulose and hemicellulose degradation, as well as lignin modification. We have isolated two novel strains of gut fungi from animals at the Santa Barbara Zoo to characterize their ability to release sugars from crude biomass. Neocallimastix sp. G1 and Anaeromyces sp. S4, have demonstrated their ability to grow on a variety of carbon sources ranging from simple sugars to woody biomass. Transcriptomic analyses have identified specific enzymatic domains required for the breakdown of plant material that are enriched in these isolates, including cellulases (GH5, GH9, GH48), hemicellulases (GH10, GH11, GH43), and polysaccharide deacetylases. During growth on cellulose, an excess of simple sugars (to concentrations of 5-8 g/L) are produced by the fungi, suggesting that other facultative microorganisms may be able to subsist on the excess sugar in co-culture. Strains of S. cerevisiae and E. coli were engineered to produce Flavin based fluorescent protein (FbFP) as a reporter of co-culture growth on excess sugars left over from fungal cellulose breakdown. We will discuss stability of these synthetic co-culture platforms, and strategies we have used to optimize production of n-butanol by an engineered E. coli strain in co-culture with anaerobic fungi. As anaerobic fungi are not yet genetically tractable, our co-culture strategy offers a path forward to make value-added products directly from crude lignocellulose. By coupling the degradative capabilities of gut fungi with the versatility of model microbes, a limitless array of products can likely be generated.

Novel co-culture approach to compartmentalize biomass deconstruction and biofuel production

249th National Meeting (2015)

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