Compared to aerobic microbes that thrive in the presence of oxygen, live cell imaging of anaerobic microorganisms is exceptionally difficult as we lack tools for high-resolution imaging that are compatible with oxygen-free workflows. This project will develop novel anaerobic imaging tools and workflows that will be applied to understand the biomass degrading abilities of anaerobic gut fungi, which were recently discovered to deconstruct lignin in the absence of oxygen through unknown mechanisms. Anaerobic fungi degrade plant biomass through invasive, filamentous growth, and the secretion of multi-protein biomass-degrading complexes called fungal cellulosomes. Found within the rumen of large herbivores, anaerobic fungi are difficult to culture, but their genomes contain the largest collection of biomass-degrading enzymes in any sequenced organism, and a vast array of proteins of yet unknown function. We will develop new multi-modal bioimaging approaches to learn how anaerobic fungi orchestrate plant cell wall (lignocellulose) degradation through their unique multi-protein cellulosomes – and how these fungi are able to access carbohydrate biopolymers encased in lignin. We will employ a multifaceted and multimodal approach to interrogate anaerobic fungal and cellulosome interactions on the nanoscale both inside and outside live anaerobic fungi. This approach will be enabled by (1) new genetically encoded anaerobic fluorescence reporters, (2) custom nanobody and quantum dot (QD) probes to improve our ability to localize cellulosomes and track their dynamics through the fungal secretory pathway without suffering photobleaching artifacts, and (3) automated anaerobic liquid handling and multi- modal imaging to unmask proteins associated with anaerobic lignin breakdown. We will use new probes and innovative bioimaging platforms to visualize and track lignin breakdown by anaerobic fungi, unmask fungal proteins of unknown function, and to open the way for new mechanistic insights that can be leveraged for lignin conversion and bioproduction.
