Co‑cultivation of the anaerobic fungus Caecomyces churrovis with Methanobacterium bryantii enhances transcription of carbohydrate binding modules, dockerins, and pyruvate formate lyases on specific substrates

Abstract

Anaerobic 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 fungus Caecomyces churrovis and the methanogen Methanobacterium 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 in C. churrovis across a variety of substrates to identify mechanisms that impact biomass breakdown and sugar uptake. A high-quality genome of C. churrovis was obtained and annotated, which is the first sequenced genome of a non-rhizoid-forming anaerobic fungus. C. churrovis possess 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 to C. churrovis monoculture. Two other fungal strains grown on a reed canary grass substrate in co-culture with the same methanogen also exhibited high log2-fold change values for upregulation of genes encoding carbohydrate binding modules in families 1 and 18. Transcriptional upregulation indicated that co-culture of the C. churrovis strain 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.

Co‑cultivation of anaerobic fungi with Clostridium acetobutylicum bolsters butyrate and butanol production from cellulose and lignocellulose

“…The anaerobic gut fungal strains, Neocallimastix californiae (Haitjema et al., 2017 ; Solomon et al., 2016 ), Anaeromyces robustus (Haitjema et al., 2017 ; Solomon et al., 2016 ), and Caecomyces churrovis (Brown et al., 2021 ; Henske et al., 2017 ), were isolated as described previously. The fungal cultures were anaerobically cultivated at 39°C in Hungate tubes with 100% CO 2 headspace, 10 ml of Medium C (MC) or Minimal Medium 2 (M2), and 0.5 g reed canary grass as the carbon source as described previously (Teunissin et al, 1991 ; Theodorou et al., 2005 ).…”

“…The anaerobic fungi also release excess sugars during lignocellulose breakdown (Henske et al., 2018 ) as well as other fermentation products such as acetate, lactate, H 2 , and ethanol. In the herbivore rumen, these fungal metabolites are understood to support the growth of other gut microbes such as archaeal methanogens to release methane (Bauchop & Mountfort, 1981 ; Hess et al., 2020 ; Theodorou et al., 1996 ), but they can also be used to drive growth and bioproduction in microbes that are not native to the rumen microbiome (Brown et al., 2021 ; Henske et al., 2018 ; Li et al., 2022 ; Swift et al., 2019 ). While the anaerobic fungi lack reliable genetic tools (Hooker et al., 2019 ), they can still be harnessed for bioproduction as members of synthetic consortia.…”

Metabolic influence of core ciliates within the rumen microbiome

“…Nine available fungal genomes downloaded from Joint Genome Institute (JGI) Mycocosm 51 were added to RUDB-C ( Anaeromyces sp. S4 21 , Caecomyces churrovis 24 , Neocallimastix californiae 21 , Neocallimastix lanati 25 , Piromyces finnis 21 , Piromyces sp. E2 21 , Piromyces UH3-1 20,21,23 , Piromyces eukMAG 52 , Orpinomyces sp.…”

Carbohydrate active enzyme system in rumen fungi: a review

“…has the enzyme activity of β-glucosidase (EC 3.2.1.21), while β-galactosidase ( EC 3.2.1.23 related to the GH family) has no activity. Brown et al (2021) preferred Caecomyces sp. in co-culture studies with methanogenic microorganisms because CAZy enzyme activity and the effect of carbohydrate-binding module (CBM) are significantly increased.…”