REFERENCE GENOMES AND GENETIC TOOLS FOR ANAEROBIC FUNGI
Creators: name: Hooker, Casey A.
Publisher: name: Purdue University Graduate School
Year: 2022
Subjects: Industrial microbiology (incl. biofeedstocks)
Fermentation
Synthetic biology
Genomics

description: Non-model microorganisms offer a wealth of biotechnological potential that may be leveraged to address a variety of global grand challenges. These include challenges in carrying out complex or altogether new chemistries, discovery and production of bioactive molecules, sustainable production of biochemicals and bioproducts from renewable feedstocks, and improving agricultural practices for responsible management of carbon. Specifically, using renewable plant biomass as a substrate for production of fuels and or chemicals offers a near ubiquitous supply that does not compete with food or petrochemicals. Alternatively, identifying new natural products will be essential to addressing the ever-increasing occurrence of antibiotic resistance. Non-model organisms may provide elegant solutions to many of these challenges, whether by possessing new or more efficient strategies to depolymerize lignocellulose, by encoding enzymes with increased stabilities and or specific activities, or perhaps by containing rich biosynthetic capabilities for production of previously unidentified natural products, among others. Yet efforts to leverage non-model microorganisms for their diverse biotechnological potential remain limited to a variety of often difficult, yet not insurmountable challenges. In this work, I propose anaerobic gut fungi (Neocallimastigomycota) as a robust microbial system that may be leveraged to efficiently depolymerize crude lignocellulose, increase animal nutrition, or identify novel natural products. To this end, I detail the first chromosomally resolved genome assembly of anaerobic fungi (<em>Piromyces communis </em>var. <em>indianae</em> UH3-1). I investigate the genome organization of this isolate and describe how acquisition of Carbohydrate Active EnZymes (CAZymes) contribute to the robust lignocellulolytic activity of gut fungi. I then detail efforts to build a nascent genetic engineering toolbox for these anaerobic organisms. With the acquisition of the first chromosomally resolved genome assemblies, I identify a basic set of genetic parts needed for a genetic engineering toolkit. I show these parts are functional and detail methods to enable higher throughput testing in vivo. I subsequently detail efforts to construct the first preliminary CRISPR tools for anaerobic fungi as these will be essential to establish precise DNA targeting in future strain engineering efforts. I then describe the role of epigenetics in anaerobic fungi, detailing the extent to which it may be leveraged to control gene expression. Finally, I provide a discussion of this work and describe how it may guide future efforts to domesticate these organisms. Collectively, this work provides the first chromosomally resolved genome assembly as a resource for the community, along with genetic tools and techniques to begin domesticating these non-model organisms. Importantly, this work reveals that despite the challenges associated with anaerobic microbes of relatively high complexity, they are not insurmountable, and thus efforts to domesticate them are feasible.