Annotation of glycoside hydrolases in unassembled metagenomes using CAZyO_GH

Functional characterization of microbiomes often relies on the sequencing of metagenomic DNA extracted from environmental samples, with current approaches using metagenome-assembled genomes (MAGs). Although glycoside hydrolases (GHs) are central to carbon cycling, accurate annotation of GHs in metagenomic datasets remains challenging due to the multidomain architecture of carbohydrate-active enzymes and the prevalence of unassembled short reads due to limitations in the MAG-generation process.

Here, we present CAZyO_GH (CAZymes Open-source GH annotation), a curated reference database for the domain-specific identification of 135 protein domains spanning 99 GH families with well-defined catalytic domain signatures. CAZyO_GH focuses on individual GH domains, enabling robust annotation of both assembled and unassembled metagenomic data. We validated CAZyO_GH by reanalyzing genomes listed in CAZy db, where predicted GH profiles closely matched reported values. Next, we used CAZyO_GH to analyze 12 human gut metagenomes and 12 newly sequenced soil microbiomes to reveal environment-specific GH repertoires. By accurately detecting catalytic domains independent of the genomic context, CAZyO_GH improves sensitivity and specificity in short-read metagenomic annotation. This framework provides a scalable and reproducible approach to investigate carbohydrate-active enzymes across ecosystems, advancing our capacity to characterize microbial functional potential in global carbon cycling.

CAZyO_GH data is available on figshare (https://figshare.com/projects/CAZyO_GH/267770).