Co‑cultivation of the anaerobic fungusCaecomyces churroviswithMethanobacterium bryantiienhances transcription of carbohydrate binding modules

AbstractAnaerobic fungi and methanogenic archaea are two classes of microorganisms found in the rumen microbiome that metabolically interact during lignocellulose breakdown. Here, stable synthetic co-cultures of the anaerobic fungusCaecomyces churrovisand the methanogenMethanobacterium bryantii(not native to the rumen) were formed, demonstrating that microbes from different environments can be paired based on metabolic ties. Transcriptional and metabolic changes induced by methanogen co-culture were evaluated inC. churrovisacross a variety of substrates to identify mechanisms that impact biomass breakdown and sugar uptake. A high-quality genome ofC. churroviswas obtained and annotated, which is the first sequenced genome of a non-rhizoid forming anaerobic fungus.C. churrovispossess an abundance of CAZymes and carbohydrate binding modules and, in agreement with previous studies of early-diverging fungal lineages, N6-methyldeoxyadenine (6mA) was associated with transcriptionally active genes. Co-culture with the methanogen increased overall transcription of CAZymes, carbohydrate binding modules, and dockerin domains in co-cultures grown on both lignocellulose and cellulose and caused upregulation of genes coding associated enzymatic machinery including carbohydrate binding modules in family 18 and dockerin domains across multiple growth substrates relative toC. churrovismonoculture. Two other fungal strains grown on a reed canary grass substrate in co-culture with the same methanogen also exhibited high log2fold change values for upregulation of genes encoding carbohydrate binding modules in families 1 and 18. Transcriptional upregulation indicated that co-culture of theC. churrovisstrain with a methanogen may enhance pyruvate formate lyase (PFL) function for growth on xylan and fructose and production of bottleneck enzymes in sugar utilization pathways, further supporting the hypothesis that co-culture with a methanogen may enhance certain fungal metabolic functions. Upregulation of CBM18 may play a role in fungal-methanogen physical associations and fungal cell wall development and remodeling.