Collaborative Research: EDGE FGT: Establishing functional genomics in anaerobic fungi for applications in agriculture, sustainability, and carbon cycling

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Anaerobic fungi are powerful microorganisms native to the digestive tracts of ruminants and hindgut fermenters, including important livestock such as cattle, sheep, and goats. Within their hosts, these fungi provide essential nutrients and compounds that drive animal growth and behavior. Specifically, they promote the digestion of fiber-rich feedstocks, secrete metabolites that sustain the growth of other microbes critical for digestion, produce compounds that enhance host health, and control the production of methane. This project develops and optimizes methods to engineer anaerobic fungi allowing scientists to study and control these molecular processes for diverse applications. Undergraduate and graduate students, postdoctoral fellows, K-12 students, and the public will be integrated into the studies through coursework, laboratory research, YouTube videos and local outreach to communities in Southern California and the Mid Atlantic. Training materials will also be developed to broadly disseminate project findings to the scientific community, accelerating research into these unusual microbes. This project has far reaching impacts on plant biomass breakdown, economical biofuels, renewable chemical production, climate change, animal nutrition and health, and drug discovery.This project develops foundational genetic tools to study anaerobic gut fungi. To date, only a handful of reports exist for the transient transformation of these species. However, the advent of CRISPR-Cas technology and the recent acquisition of complete genomes for these species enable new strategies for permanent genetic modification. Focusing on representative isolates from several genera within the Neocallimastigomycota phylum, this project optimizes methods to deliver genetic materials to fungal zoospores, the juvenile life stage of this phylum, which will be quantified through selection and fluorescence-activated cell sorting. Next, the project develops two strategies l to introduce or knock down/out genes. In the first strategy, CRISPR-based tools will be deployed and optimized to engineer novel cellular phenotypes. In the complementary second approach, the project leverages LTR retrotransposons distributed throughout anaerobic fungal genomes as platforms to enhance DNA integration frequency and identify fungal promoters. Research findings will be distilled into training materials that will be disseminated through international and interdisciplinary research communities such as the Rumen Microbial Genomics (RMG) Network and the Anaerobic Fungi Network (AFN) to catalyze scientific advancement in a number of allied fields. Similarly, the public will be engaged through partnerships with organizations such as the Santa Barbara Zoo. This research will result in the first experimental tools to test gene function in the anaerobic fungi, enabling insight into their lifestyle and providing a path to microbial engineering and hypothesis testing.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.