| Popis: |
Uncultured microbes represent a huge untapped biological resource of novel genes and gene products. Although recent genomic and metagenomic sequencing efforts have led to the identification of numerous genes that are homologous to existing annotated genes, there remains, yet, an enormous pool of unannotated genes that do not find significant sequence homology to existing annotated genes. Functional metagenomics offers a way to identify and annotate novel gene products. Here, we have constructed a metagenomic phage display library from healthy human fecal samples, and performed functional carbohydrate binding screens against dietary, microbial and host polysaccharides/glycoconjugates in order to mine novel carbohydrate binding domains that might aid gut commensals in the metabolism of complex carbohydrates and in adherence and colonization of the gut. We obtained several protein sequences that do not find a hit to any known Pfam domain but show carbohydrate binding module-like folds as per Alphafold2 prediction. We heterologously expressed, purified and biochemically characterized some of these protein domains and demonstrated their carbohydrate-binding function. Our study reveals several previously unannotated carbohydrate-binding domains, including a levan binding domain and four complex N-glycan binding domains that might be useful for the labeling, visualization, and isolation of these glycans. |
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