UC Santa Barbara

It is urgent that we develop and implement bio-based alternatives to our existing petroleum chemical and energy infrastructure. Lignocellulose is the most abundant renewable carbon resource on the planet, making it a promising biofeedstock. However, its recalcitrance to degradation via chemicals and model microbes like E. coli and yeast precludes its utilization for bioenergy or production of commodity and specialty chemicals. So-called “non-model” anaerobic microbial consortia found in the rumen of large herbivores have evolved as specialized biomass degraders and have potential for lignocellulose-based bioproduction if they can be onboarded, characterized, and deployed at scale.Anaerobic gut fungi (AGF) in rumen consortia produce nature’s greatest known variety and abundance of lignocellulose-degrading carbohydrate-active enzymes (CAZymes). This, combined with their mixed-acid fermentation profile, makes them interesting candidates for industrial CAZyme production and/or biomass deconstruction and conversion. However, AGF are not genetically tractable, and their physiology and primary metabolism are poorly understood, which limits our ability to predict and manipulate phenotypes for user-specified culture outcomes. AGF could therefore be deployed in communities alongside genetically tractable workhorse strains, wherein AGF specialize in degradation of lignocellulose to sugars and conversion to bioproduct precursors. Microbial communities are, in principle, capable of virtually limitless chemical transformations. In practice, designing consortia with predictable, prescribed functions is challenging, especially with a largely uncharacterized constituent species such as an AGF. Before we can deploy anaerobic consortia industrially, we must understand AGF physiology and metabolism. Specifically, we must know the entire space of achievable AGF phenotypes and how to accentuate the functions that we desire (fast growth, production of CAZymes, high flux of certain metabolites, etc.). Without genetic tools, we require creative and multifaceted approaches to characterize and tune AGF growth and metabolism. Toward this goal, we synthesized multi-omic and biochemical data into the first AGF genome-scale metabolic model, offering the most complete description of AGF growth and metabolism available. The model established the theoretical AGF phenotype space; from there, we exposed AGF to myriad culture conditions (some resembling their natural habitat and some more artificial) to explore which phenotypes are both biotechnologically useful and achievable in practice. Using a non-rhizoidal AGF, Caecomyces churrovis, we developed simple, yet vital methods for quantification of AGF growth and metabolic flux that are routine in model systems but have been unavailable to AGF. By stirring C. churrovis cultures, we elicited a suspended culture morphology that grows faster and expresses significantly more CAZymes per cell than typical biofilm cultures. We leveraged these well-mixed suspended cultures to develop methods for non-destructive quantification of AGF growth and flux in co-culture with prokaryotes, and showed that methanogens significantly increased AGF growth rate and altered AGF metabolic flux to yield different fermentation product profiles. In a significant step towards industrial deployment of AGF, we demonstrated the first steady state continuous culture of AGF using a DIY Arduino-based continuous flow bioreactor. Turbidostat bioreactor operation uncovered relationships between setpoint titer and AGF growth rate and flux, enabling users to specify continuous production rates of target metabolites and enzymes and vary them depending on the application at hand. Our understanding of AGF physiology remains far from comprehensive. However, the research presented in this dissertation has elucidated design rules for AGF cultures with measurable, predictable, and tunable growth and metabolite production rates, moving us closer to deployment of AGF and anaerobic consortia for industrial lignocellulose valorization.