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Discovery and Characterization of a Novel Carbohydrate-Active Enzyme from the Anaerobic Fungus Piromyces finnis

ABSTRACT: Dwelling in the gut of herbivores, anaerobic gut fungi (AGF) have evolved several strategies to efficiently degrade unpretreated biomass. Their genomes encode a diverse array of carbohydrate-active enzymes (CAZymes), yet exceedingly few of these enzymes have been validated. Here, we developed a predictive bioinformatic pipeline to both annotate novel putative CAZymes and validate their activity through heterologous expression. 173 fungal proteins from Piromyces finnis associated with biomass degradation were synthesized and expressed in E. coli, and 9.8% were soluble with expression levels exceeding 5% of total proteome. Among the 17 heterologously expressed proteins, a combination of sequence and structure-based homology annotation identified many multifunctional proteins having catalytic domains, such as glycoside hydrolase (GH) and carbohydrate esterase (CE), fused with repetitive dockerins. Screening across three cellulose and hemicellulose substates experimentally verified six predicted functions. One promising enzyme, celsome_012, exhibited robust and optimized activity against beechwood xylan at 37°C and pH 6.4, producing five-times more products than other recombinant proteins screened here. Overall, this study represents the first large-scale screening campaign of putative AGF CAZymes, highlighting proteins amenable to E. coli overexpression, integrating advanced sequence and structural annotation, and identifying a robust, novel fungal xylanase for detailed biochemical characterization.

INSTRUMENT(S):

ORGANISM(S): Escherichia Coli

SUBMITTER: Christopher Petzold

LAB HEAD: Christopher J. Petzold

PROVIDER: PXD061558 | Pride | 2025-07-31

REPOSITORIES: Pride

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			Action	DRS
	A10_Celsome_023-R1.raw	Raw
	A10_celsome_72-R1.raw	Raw
	A11_Celsome_019-R1.raw	Raw
	A11_celsome_8-R1.raw	Raw
	A12_Celsome_049-R1.raw	Raw

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