Enhanced microbiota-derived mucinases in colorectal cancer patients revealed by gut metagenome probing coupled with functional validation.

Mucinases produced by the gut microbiota play a dual role in regulating the integrity and renewal of the mucus layer, which is essential for maintaining gut homeostasis and human health. In this study, we constructed protein hidden Markov models based on 11 known mucinases and used them to systematically identify mucinase sequences from gut metagenome-assembled genomes derived from 80 colorectal cancer (CRC) patients and 86 healthy (Healthy) subjects. A total of 1,869 mucinases were detected, widely distributed across the studied cohorts, with the majority originating from , , and species. Further analysis identified 42 mucinases that differed significantly in abundance between the two groups, all of which were enriched in CRC patients. Taxonomic attribution revealed that, in CRC patients, these mucinases were primarily derived from (36.0%), (30.6%), (8.8%), (8.6%), and (6.4%), whereas in Healthy subjects, they mainly originated from (26.1%), (22.7%), and (20.3%), with a notably higher proportion from . Among the 42 mucinases, WL42 and LLN1 exhibited significantly higher abundance levels compared to the others. Phylogenetic and predicted structural analyses suggested that these two mucinases belonged to the M60 and M98 families, respectively. Functional validation through co-incubation experiments demonstrated that both mucinases could cleave the glycosylated MUC1 and MUC2 substrates, but not the corresponding non-glycosylated proteins. These findings confirm the feasibility of discovering novel mucinases directly from gut metagenomic data and provide insights into their potential roles in health and disease.IMPORTANCEOur study established a feasible bioinformatics pipeline for the systematic identification of microbial mucinases within the gut microbiome, providing a methodological foundation for large-scale mining of functionally active mucin-degrading enzymes. We identified 42 mucinases significantly enriched in CRC patients, suggesting their potential involvement in CRC pathogenesis. Among them, two mucinases were experimentally validated for their ability to degrade mucin, offering direct functional evidence of their capacity to disrupt the mucosal barrier. Genus-level metagenomic profiling further identified , , and as major mucinase-producing genera. Maintaining the secretory balance of these mucinase-producing bacteria might be crucial for ameliorating intestinal barrier dysfunction in CRC patients. The findings of this study offer critical insights into the microbial origins and potential mechanistic contributions of mucinases in colorectal cancer, underscoring their relevance in mucus barrier breakdown and disease progression.