Abstract

Realizing the biotechnological potential of fungal cellulosomes Enzymatic conversion of readily available plant biomass to chemicals offers a greener, more resilient alternative to traditional petrochemical synthesis. However, innovations enabling use of cheaper, lower quality inputs and enhancing conversion yield and throughput are needed to catalyze their broader adoption. Fungal cellulosomes are modular protein machines that drive biomass hydrolysis by incorporating enzymes into complexes via protein-protein interactions involving enzyme-fused dockerin domains. Engineered fungal cellulosomes and synthetic protein complexes constructed using modular fungal dockerin and cohesin domains represent two promising technologies for bioprocess innovation. However, the mechanism by which cellulosomes self-assemble as well as the composition-activity relationships underlying cellulosome function remain unknown, precluding the development of fungal cellulosomes into biocatalytic technologies with real word applications. This thesis describes a two-pronged approach to characterizing the composition, activity, and structure of native fungal cellulosomes and designing synthetic protein complexes assembled with cellulosome parts. Enabled by a robust cellulosome purification method we developed, we catalog the major enzyme components of a native fungal cellulosome and measure the biomass hydrolysis activity of cellulosome complexes with different enzyme content. By developing a synthetic, dockerin-binding protein, we engineer a suite of modular interacting parts for constructing designer protein complexes with fungal cellulosome proteins. 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.