Enabling tools for metabolic engineering in anaerobic fungi

Early-diverging anaerobic fungi (phylum Neocallimastigomycota) are attractive emerging platforms for metabolic engineering due to their robust degradation of untreated plant feedstocks and novel biosynthetic capabilities. More importantly, anaerobic fungi are naturally competent during portions of their lifecycle and are genetically tractable. To effectively leverage these abilities, however, an engineering toolbox is required. My lab is pursuing a number of plasmid-, genomic-, and epigenomic- strategies for tool development to domesticate anaerobic fungi for biotech. We have identified several gene regulatory elements from transcriptomic and genomic resources to drive gene expression for the first time in either the nucleus or cytoplasm of anaerobic fungi. Validation studies with flavin-based fluorescent reporters and selection markers confirm sequence dependent increases in gene expressing in a controllable manner. These tools enable library-based screening approaches to construct the first stable anaerobic fungal plasmid and the introduction of CRISPR-based endonucleases for genomic integration. We have also pioneered epigenetic regulation of anaerobic fungi and identified several chemical inhibitors that can perturb the epigenome of anaerobic fungi with concurrent phenotypic changes in biomass-degrading enzyme secretion. Efforts are currently underway to capitalize on systems biology methods to rapidly elucidate and expand parts available for engineering applications. In parallel, we have also examined the feasibility of anaerobic fungal process development with partner microbes such as K. marxianus. Anaerobic fungi readily release a suite of fermentable sugars and organic acids that support the growth of engineered K. marxianus to make value-added esters and fragrance compounds from crude biomass. These examples showcase a few of the metabolic engineering opportunities currently available with anaerobic fungi. More importantly, our growing toolbox of genetic and epigenetic strategies can be readily generalized to other non-model systems, and will soon enable direct metabolic engineering in anaerobic fungi.