Waste management and sustainable energy production are two major concerns of modern society. The use of microbial consortia for waste treatment has the potential to address both of these concerns simultaneously, since consortia possess the ability to convert crude biomass into biofuels and bio-based chemicals. The organic fraction of municipal solid waste (OFMSW) or food waste is an abundant and inexpensive carbon substrate that can be utilized by microbial systems to generate useful products. Anaerobic consortia containing fungi, bacteria, protozoa, and methanogenic archaea capable of converting wet waste materials into valuable substances already exist in nature and have been isolated from the guts of herbivores. Although bioreactors utilizing undefined natural consortia to digest wet waste and generate biogas have been constructed, the failure rate is high due to instability and death of the microbial community. The development of biotechnology capable of handling variable input, recovering from environmental disturbances, and producing consistent products is dependent upon engineering stability and robustness among consortia members. To achieve this, it is necessary to understand the genomic basis for stable interaction between members of these microbial communities.In this work, transcriptional and metabolic changes induced by methanogen co-culture were evaluated in the anaerobic fungal strain C. churrovis across a variety of substrates to identify mechanisms that impact biomass breakdown and sugar uptake. Co-culture with the methanogen increased overall transcription of carbohydrate active enzymes (CAZymes), carbohydrate binding modules, and dockerin domains in co-cultures grown on both lignocellulose and cellulose. Next, a system for simultaneous and sequential co-cultivation of the anaerobic fungus Anaeromyces robustus and the anaerobic bacterium C. acetobutylicum was established based on lactate cross-feeding to produce butyrate and butanol from lignocellulose. Higher levels of butyrate and butanol in fungal and C. acetobutylicum cultures reveal that creating consortia that include these two microbes could be a promising future avenue of industrial bio-butyrate and biobutanol production. Finally, a method to extract high-quality RNA from anaerobic fungi at multiple timepoints in the fungal growth phase was developed to fully characterize differential expression in both fungal monocultures and fungal-methanogen co-cultures. The fungal strain Anaeromyces robustus co-cultivated with the methanogen Methanobacterium bryantii upregulates genes encoding fungal carbohydrate active enzymes and other cellulosome components relative to fungal monocultures when grown on a cellulose substrate, but expression patterns changed at 24-hour intervals throughout the fungal growth phase. Overall, this work indicates that anaerobic fungi can be successfully combined with non-native microbes in consortia capable of converting low-cost biomass substrates into value-added products.

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.

The development of a renewable, bio-based economy requires efficient methods to extract fermentable sugars from complex plant material. Currently, bioprocessing from crude biomass requires multiple steps including pretreatment to separate lignin from sugar-rich cellulose and hemicellulose, enzymatic hydrolysis to release simple sugars, and microbial fermentation to produce value-added chemicals. Consolidated bioprocessing seeks to improve bioprocessing efficiency by reducing the number of steps required to get from plant biomass to chemical product. To address this challenge, we derived inspiration from natural microbial communities known for degrading biomass. Within the rumen microbiome of large herbivores, anaerobic gut fungi are the primary colonizers of plant material and present an untapped opportunity for consolidated bioprocessing. These unique microorganisms efficiently hydrolyze lignocellulosic biomass into simple sugars, but remain relatively uncharacterized in comparison to industrial production organisms. We implemented Next Generation Sequencing (NGS) technologies alongside biochemical studies to develop a deeper understanding of gut fungi, their metabolism, and the mechanisms by which they break down complex biomass to identify a path forward for their industrial application. We also developed simple, rapid methodologies for cryopreservation and DNA extraction that are critical for the development of industrial microbes.

Sequencing and functional annotation of transcriptomes and genomes of novel isolated species of gut fungi has elucidated their large repertoire of biomass degrading enzymes including cellulases, hemicellulases, and accessory enzymes. These enzymes allow them to efficiently degrade crude biomass, yielding similar growth rates on complex plant material and simple sugars. Remarkably, in isolated batch culture, the biomass degrading power of gut fungi is sufficient to generate surplus fermentable sugars for the growth of additional microorganisms. This ability has been exploited to develop a novel two-stage consolidated bioprocessing scheme that uses anaerobic gut fungi to consolidate the pretreatment and hydrolysis steps in traditional bioprocessing to hydrolyze sugars directly from crude biomass. These sugars can then be fed to the easily metabolically engineered model yeast, Saccharomyces cerevisiae, to support growth and bioproduction in a two-stage fermentation scheme.

Further, RNA sequencing studies have provided critical insight into the regulation of biomass degrading activity. Gene expression during growth on varying substrates and in response to a carbon catabolite repressor has revealed conditions required to optimize expression of biomass degrading enzymes. Unannotated sequences that co-regulate with predicted biomass degrading enzymes have also been identified as candidate genes that may host novel biomass degrading function. Together these results reveal important process considerations for the use of gut fungi in industrial bioprocessing to maximize the production of enzymes and the degradation of biomass. While challenges remain for the implementation of gut fungi in industrial bioprocessing, we have demonstrated their potential to consolidate pretreatment and hydrolysis either through engineered culturing schemes or development of improved enzyme cocktails for biomass hydrolysis.

Microbiota in the digestive tracts of herbivores are complex, highly-structured communities that degrade lignocellulosic biomass into simple sugars and then metabolize it into fatty acids, gases, and other small molecules, approximately three times as efficiently as the most advanced human-engineered processes in use (Godon et al., 2013). The degradative power of these communities arises from partnerships between organisms carrying out complementary metabolic tasks, which together constitute the processes of anaerobic digestion. Cooperative strategies arising from these partnerships that microbiota deploy during anaerobic digestion of complex substrates are not well understood. A fuller understanding of these strategies will shed light on underlying factors driving the division of labor at each stage of lignocellulolysis and aid in identifying genomic and transcriptional mechanisms that predict patterns of labor division in a given consortium. Applying this knowledge will enable the development of approaches to modulate the activity of existing microbiota and construct synthetic consortia to optimally transform complex substrates into desired products.In this body of work, we examine microbial relationships and consequent digestive strategies in the degradation of complex biomass as they manifest through both composition and activity, through employing enrichment to study the gut microbiota of captive individuals of three herbivore species – the San Clemente Island goat (Capra hircus), a foregut-fermenting hoofed mammal, the Eastern black and white colobus monkey (Colobus guereza), a foregut-fermenting primate, and the Western lowland gorilla (Gorilla gorilla gorilla), a hindgut-fermenting primate. In the first of two studies, we enriched a goat fecal microbiome in parallel cultures on three lignocellulosic substrates and hemicellulose, and then sequenced the actively transcribed genes from the microbes enriched on each substrate. We demonstrated that parallel enrichment on complex biomass subsrates and purified hemicellulose enriches consortia whose transcriptional activity is shaped by the substrate. However, this activity was likely limited by genetically-encoded factors inherent to the consortium. We also observed a previously-uncharacterized strategy for division of carbohydrate-active enzyme expression labor exhibited across all lignocellulosic substrates. We then investigated the composition and structure of lignocellulolytic communities in C. guereza and G. gorilla gorilla fecal microbiota, and attempted to enrich anaerobic fungi of the phylum Neocallimastigomycota from fecal samples of captive C. guereza and G. gorilla gorilla. This attempt was unsuccessful at enriching anaerobic fungi, but instead enriched a diverse community of other fungal taxa with diverse roles in lignocellulolysis, metabolism, and regulation of the microbial community and its host. We found that the C. guereza and G. gorilla gorilla gut microbiome contain rare bacterial and fungal taxa that may have important roles in lignocellulolysis in the gut, and that enrichment on complex substrates with the aid of antibiotics is an underutilized but crucial method for fully characterizing the diversity of herbivore gut microbiota by enabling the growth and sequencing of rare taxa in the microbiome. Taken together, these studies investigate relationships between microbes in lignocellulolytic gut consortia, through the lenses of community composition and activity, to yield new insights about how these consortia work together.

Natural products, or secondary metabolites, are small molecules produced primarily by bacteria, fungi, and plants. Their chemical diversity has conferred many useful bioactivities, such as antibacterial, antifungal, cholesterol-lowering, and immunosuppressant. In addition to their value as medicinal drugs, these molecules also serve important ecological roles in their native environments, by mediating interactions between microorganisms. Natural products are crucial to future microbiome engineering efforts for their natural ability to modulate microbial communities. The digestive tracts of large herbivores, including the rumen, harbor complex microbial communities, consisting of fungi, bacteria, protozoa, and methanogenic archaea. This vast reservoir of chemical diversity is almost completely untapped with regards to the discovery of natural products. These microbiomes are of interest both for their potential to yield new drug candidates and as a microbial platform for chemical production from renewable feedstocks. In this work, we explore the biosynthetic potential of anaerobic fungi native to the digestive tracts of herbivores to synthesize natural products. The genomes, transcriptomes, proteomes, and metabolomes of anaerobic fungi reveal that these underexplored fungi synthesize novel natural products, some of which are used to compete with rumen bacteria. Groundbreaking dual transcriptomics of rumen fungi co-cultured with rumen bacteria provides evidence that the relationship between these two organisms is antagonistic and that the presence of bacteria stimulates the expression of biosynthetic genes encoding enzymes that synthesize potential antibiotics. By sequencing the active genes of microbiomes inoculated from a source microbiome (goat fecal pellet), we demonstrated that the bacterial and fungal biosynthetic genes of natural products were active in sequential cultures passaged in vitro. We found that fungal biosynthetic genes were upregulated at later generations of batch passaging. This finding revealed that the expression of fungal biosynthetic genes is dynamic and suggested that natural products may function in the stability of the microbial community. Overall this dissertation points to the potential of natural products sourced from the rumen as drug candidates, as well as their importance in future microbiome engineering efforts.

The global climate is in crisis, and change through disruptive technological innovation is the only option for humanity to avoid catastrophic collapses of ecosystems, economies, and, ultimately, the ability of our planet to support human life on its current magnitude. Replacing unsustainable industrial practices with novel and green processes is one area of technology that could help mitigate petroleum dependence and, by extension, damage to the biosphere. Biorefineries use synthetic biology to achieve chemical production from renewable feedstocks. Of these, lignocellulosic biorefineries that convert plant cell walls into valuable chemicals are one especially attractive concept, as lignocellulose is the most abundant renewable carbon pool in the biosphere. A central technical challenge of the lignocellulosic biorefinery concept is depolymerizing recalcitrant polymers so they can be upgraded using complementary biological and abiotic processes. Lignin is the most recalcitrant faction of plant cell walls, and this polymer is chemically distinct from the polysaccharides it protects. The aromaticity of lignin presents both a challenge and opportunity since this chemistry imparts essential properties to plant cell walls, such as hydrophobicity and resistance to degradation, that are also desirable in important consumer products like plastics. Innovation in lignin deconstruction is a current focus of lignocellulosic biorefinery research, and novel ways to degrade this polymer that are inspired by biology could help address this challenge. Large herbivores and their microbial gut communities convert lignocellulose into other chemicals at a rate and yield that eclipses our current best lignocellulosic biorefineries. My work seeks to discover the unknown aspects of biological lignin degradation as it occurs in herbivore gut microbiomes by focusing on a group of highly specialized lignocellulose degraders, The anaerobic gut fungi or Neocallimastigomycetes are some of the most proficient lignocellulose degraders documented to date, and in my work, I interrogate interactions between these organisms and lignin. The work is designed to increase understanding of how Neocallimastigomycetes depolymerize and eventually use carbon in lignin. During this pursuit, I have participated in every aspect of investigating these unique organisms, from enrichment and isolation to the heterologous expression of Neocallimastigomycete proteins in model organisms. My approach has been to match measurements of biochemistry and polymer chemistry, such as nuclear magnetic resonance, to measurements of biological activity. Along the way, I leveraged my extensive experience in biochemistry and metabolism to increase knowledge pertaining to the basic biology of the Neocallimastigomycetes. The most notable finding I present in this thesis is the discovery that anaerobic fungi from class Neocallimastigomycetes can deconstruct lignin, and this is the first instance in which anyone has measured this bond scission by an anaerobic microbe. Surrounding this finding, I also present advances in understanding syntrophic relationships between bacteria, fungi, and archaea and novel aspects of the responsive metabolism of Neocallimastigomycete fungi. The presented findings increase understanding of herbivore digestion, anaerobic microbiology, and refinement of lignocellulose into value-added chemicals. Many of my publications raise more questions than they answer, but this is the nature of creating new knowledge. Despite the new questions raised and extensive progress still required for biotechnology to help mitigate the climate crisis, the work in this dissertation is a small step forward in creating sustainable biotechnology inspired by nature.

In the Tauopathy subfamily of neurodegenerative diseases, the intrinsically disordered protein (IDP), Tau, undergoes a transition from its native disordered state into insoluble, pathological amyloid fibrils, such as those found in the neurofibrillary tangles of Alzheimer’s disease (AD). The mechanisms of Tau aggregation are still unclear, despite decades of research. This dissertation describes how Tau interacts with it surrounding environment and the cofactors that lead to its pathological aggregation. Chapter 1 provides an overview of disordered proteins, aggregation, Tau, neurodegeneration, and the biophysical techniques used throughout this manuscript. Chapter 2 describes the fibril structures formed by recombinant Tau when incubated with heparin, a polysulphated glycosaminoglycan, and how the structures are heterogeneous and dissimilar to the fibrils observed in AD. Chapter 3 investigates the nature of the interaction between Tau and the anionic cofactors routinely used to generate fibrils in vitro. The cofactors were found to be integral to the fibrils and stoichiometric quantities of cofactor were required to form the fibril. In Chapter 4, fragments of Tau were produced that were found to exist in pathological brain tissue. These proteolytic products of Tau that are observed in AD patients form potent, aggregation-prone fragments of Tau that may be involved in initiating aggregation of larger species of full-length Tau. In Chapter 5, shorter peptides of Tau are used to demonstrate the effect of a hereditary mutation (P301L) observed in chromosome 17 linked frontotemporal dementia and Parkinsonism (FTDP-17). The primary effect of the mutation, in a short peptide of Tau (HP301L), is to create a slower-moving hydration shell with a lower entropy than in the WT form. The lower entropy around the hydrophobic leucine makes the dehydration required for amyloid formation more energetically favorable, leading to higher aggregation propensity. Chapter 6 describes ongoing work to determine structure of the fibrils formed by the P301L peptide in Chapter 5. A structure is presented showing a fibril core at 4.6 Å resolution with a C2 symmetry composed of 2 hairpin-shaped protofilaments. Chapter 7 investigates the proline rich domain of Tau and its role in liquid liquid phase separation and identifies the SH3 domain as a binding partner that may be involved in the removal of Tau from microtubules. Chapter 8 suggests future directions for the field and outstanding questions from this body of work.

Rising risks of climate change and supply chain insecurity highlight the need to develop alternative, greener synthesis routes to common materials currently sourced from petroleum. Biological systems excel at interconverting chemicals with exquisite specificity and speed, using networks of enzymes that perform catalysis at mild conditions. Protein complexes in nature colocalize complementary subunits to perform sophisticated biochemistry, and artificial, spatial organization of enzyme systems into synthetic complexes is an attractive strategy for improving biocatalytic process throughputs in industrial settings. While some sets of modular parts that enable designer protein complex construction exist, there is still a need to develop new components that are widely compatible with different enzymes and that are highly engineerable to impart desired self-assembly properties. Fungal cellulosomes, modular protein machines produced by anaerobic fungi in the guts of herbivores to rapidly free sugars from plant matter, represent an unexplored framework for synthetic protein complex construction. Cellulosomes synergistically incorporate enzymes involved in biomass degradation into discrete complexes via modular protein-protein interactions between enzyme fused dockerin domains and cohesin domains repeated on a central scaffoldin protein. Over 80% of the degradative power anaerobic fungi possess is attributed to cellulosomes, but the mechanistic nature of their activity and their assembly mechanism remain unknown. These knowledge gaps have precluded the development of fungal cellulosomes or their parts as biocatalytic technologies with real world applications. We apply a range of experimental techniques towards addressing how cellulosomes are produced in native anaerobic fungal cultures and characterizing the composition, nanostructure, and biochemical activity of purified, native cellulosomes. Immunofluorescence microscopy with cellulosome-labeling antibodies shows cellulosomes localize to the surfaces of cells, but that only cells at certain stages of the multi-staged life cycle produce cellulosomes under specific growth conditions. A robust cellulosome purification method we developed, in conjunction with mass spectrometry-based proteomics and biomass hydrolysis kinetic assays, provides high resolution details into the composition and lignocellulolytic activities of isolated cellulosomes produced by an anaerobic fungus, advancing our understanding of how cellulosomes can be engineered to enhance biomass hydrolysis rates. Towards leveraging the modular cellulosome assembly framework for synthetic biology applications, we develop a suite of modular interacting parts for constructing protein complexes with fungal cellulosome proteins. Through a combination of molecular modeling and high-throughput screening, we engineer interacting domains with a range of pH dependent binding behaviors for building protein complexes whose composition and therefore function are modulated with and environmental trigger, pH. Together, these tools and insights shed light on how cellulosomes make anaerobic fungi prolific biomass degraders and provide a framework for engineering protein complexes inspired by fungal cellulosomes designed for a wide range of applications.