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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.

Additional file 1: Table S1. NREL compositional analysis of the renewable plant biomass used in this study. Table S2. NREL Compositional analysis of poplar constructs used in this study. Table S3. Syringyl lignin content of the poplar constructs used in this study. Table S4. Sugar conversion percentages for untreated plant biomass used in this study. Figure S1. UH3-1 DNA controls: A) In lane 2, the bacterial V4/V5 primers do not amplify genomic DNA of this organism. However, in lane 3, the JB206/JB205 primers amplify the ITS1 region of this Piromyces isolate. B) The NL1/NL4 primers amplify UH3-1 28s rDNA. Figure S3. Expanded LSU phylogenetic tree with accession numbers. Figure S4. UH3-1 growth curves on various carbon sources. Figure S5. UH3-1 shows visible fungal biomass accumulation on media containing xylose. The top tube was inoculated and shows a high amount of fungal biomass, while the bottom tube was used as a negative control, and was not inoculated. Figure S6. UH3-1 shows strong xylanolytic activity on xylan from beechwood at 50 °C, pH 7 for six hours of hydrolysis. Values normalized to Viscozyme. Figure S7. UH3-1 growth curves on lignocellulosic substrates and the genetically modified lines of poplar used for the S lignin analysis. Figure S8. Autoclaving corn stover at 120 °C for 30 min does not significantly enhance fungal growth rate or total accumulated pressure. This autoclaved corn stover was not washed to remove any potential fermentation inhibitors that would be expected to reduce fungal growth. N = 4.