DEVELOPING ENGINEERING TOOLS FOR ANAEROBIC FUNGI
Renewable plant biomass represents a rich source of fixed carbon that is poised to accelerate the growth of the bioeconomy because it is widely available, underused, and inexpensive. Similarly, because it is a ubiquitous, carbohydrate-rich feedstock that can be used in a broad range of bioprocesses, current and emerging technologies are being designed to transform these feedstocks into a variety of products including surfactants, food additives, pigments, plastics, and biofuels. However, current strategies to deconstruct recalcitrant plant materials rely on expensive enzymes with inefficient and harsh pretreatment steps. However, anaerobic fungi degrade a variety of crude, untreated biomass materials into fermentable sugars that can be converted into various products making them an appealing, low-cost solution to this problem. Although there are potential applications in industry for anaerobic fungi, it remains untapped because of the difficulties in cultivating them, sequencing their genomes, and genetically engineering them.In this work, three novel anaerobic fungi were isolated, and their genomes were sequenced to identify their genomic potential that was then leveraged to develop bioprocesses and engineering tools. Specifically, I developed methods to acquire the first gapless genomes for anaerobic fungi to provide more comprehensive insight into their capabilities. The biomass hydrolyzing abilities of one strain were characterized and leveraged as a pretreatment system for plant biomass; by partnering these anaerobic fungi with K. marxianus yeast, higher carbon conversion to fine and commodity chemicals was achieved as part of a two-stage bioproduction system. Similarly, the genomes were leveraged to identify novel genes for mevalonate production. My analysis of codon utilization due to the unusual GC composition of these genomes overcome one of the challenges with heterologous gene expression, leading to a hybrid pathway in E. coli with titers up to 2.5 g/L of mevalonate. Finally, a basic set of genetic tools was created including promoters, reporters, selection markers, and a gene-editing system that are still in development but form the fundamental toolbox for genetic engineering in anaerobic fungi. Together, this work provides a foundation for future genetic and metabolic engineering approaches that can enhance the efficiency and production of chemicals and fuels from renewable plant biomass.