Identification of Functional Promoters in Anaerobic Gut Fungi

Lignocellulose is the most abundant renewable carbon source on Earth, yet its recalcitrance remains a major bottleneck in biofuel and bioproduct production. Anaerobic gut fungi (AGF; phylum Neocallimastigomycota) initiate lignocellulose deconstruction in herbivore gut microbiomes under anaerobic conditions. Although they represent a minority of the community, AGF encode expansive repertoires of carbohydrate-active enzymes with broad substrate specificity, making them promising platforms for lignocellulose bioprocessing. However, functional characterization and engineering of AGF remain limited by genetic intractability. Highly AT-rich and repetitive genomes, horizontal gene transfer, complex life cycles, and transformation barriers have hindered molecular tool development, and robust promoters for heterologous expression are lacking. To address this gap, we are pursuing two complementary strategies leveraging JGI construct generation and sequencing capabilities. First, we are developing a highthroughput promoter screening pipeline targeting ~200 candidate promoters identified from upstream regions of highly expressed AGF genes across transcriptomic datasets. JGI-generated promoter–reporter constructs will be evaluated in Saccharomyces cerevisiae and AGF using fluorescent reporters to quantify promoter strength across heterologous and native contexts. Second, we are applying DAP-seq to characterize AGF transcription factors and identify high-confidence binding motifs within candidate promoter regions. Of 176 TFs analyzed (92 from Caecomyces churrovis and 84 from Neocallimastix californiae), ~16% passed quality thresholds (FRiP > 0.5), with six datasets exhibiting strong enrichment (>100 peaks per genome). While not sufficient for regulatory network reconstruction, these data enable motif discovery and prioritization of candidate promoters for functional validation. Together, these efforts establish a promoter toolkit to enable AGF genetic engineering for bioenergy and biomanufacturing applications.