UC Santa Barbara

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.