Reconstructing Anaerobic Microbiomes from the ‘Bottom-up': New Techniques to Decipher Interwoven Metabolism

Anaerobic microbiomes are among the most diverse microbial communities on earth, yet the mechanisms that control community structure and stability are poorly understood. For example, the rumen microbiome within large herbivores consists of four interdependent microbial populations (bacteria, protozoans, fungi, and methanogens) that work together to drive crude biomass into sugars and fermentation waste products. However, the interdependency of these microbes and their impact on microbial metabolism has been difficult to characterize since the vast majority of these microbes cannot be cultured. To characterize interwoven metabolism of anaerobes, we have focused on an interdependent sub-population within the rumen microbiome – anaerobic fungi and their associated hydrogenotrophic archaea (methanogens). In this community, gut fungi hydrolyze cellulose-rich biomass in the herbivore rumen, and a syntrophic partnership with methanogens allows fungi to thrive by siphoning hydrogen to methane. Our goal is to understand this natural syntrophy, and mimic it to engineer synthetic, stable anaerobic consortia that funnel crude biomass to sustainable chemicals.

To construct anaerobic consortia from the “bottom-up”, we have overcome culture challenges by isolating anaerobic fungi with dependent methanogens from herbivore fecal materials. These environmental co-cultures establish a simplified system to model the cooperative action of the anaerobes, and our prokaryotic/eukaryotic systems are stable in culture > 60 weeks. Using RNA-Seq, we have modeled the regulatory patterns for fungal genes from the Piromyces, Neocallimastix, and Anaeromyces genera during hydrolysis of reed canary grass and discovered several conserved regulons of novel genes that govern function, which are catabolite repressed. Addition of methanogens (e.g. Methanocorpusculum) during biomass breakdown shows that fungal-methanogen syntrophy drastically accelerates cellulose and lignocellulose breakdown by the fungi. Furthermore, the excess sugar hydrolysates and metabolites from an Anaeromyces/Methanocorpusculum co-culture enable metabolic linkage of non-native facultative anaerobes to the consortium. Strains of S. cerevisiae and E. coli were engineered to produce Flavin based fluorescent protein (FbFPs) as a reporter of co-culture growth on excess sugars (5-8 g/L) 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 strategy offers a path forward to make value-added products directly from crude lignocellulose by compartmentalizing lignocellulose breakdown and production in co-cultured microbes.