Gaining access to enzymes that are able to optimally degrade glycosides of interest under specific conditions has been achieved using  two high-throughput methodologies. One involves the synthesis of libraries of genes from within glycoside hydrolase families that contain the general activity of interest. Representation of specificity diversity within that family is achieved bioinformatically by creating phylogenetically distinct sub-families of genes. Representative genes from each sub-family are then synthesised and expressed in E coli, and the library is screened.<br/><br/>In the other approach we screen the diverse gene repertoire present within the “silent majority” of microorganisms that have not been cultured through metagenomic approaches. Total DNA from an environment of interest is extracted, fragmented into chunks containing either ~ 1 gene (3-5 kB) or ~30-40 genes (40-50 kB) and transformed into E. coli. After picking colonies into 384 well plates we screen them for the activities of interest using custom-synthesized substrates. After hits are validated we sequence interesting fosmids and clone and express any obvious CAZymes: if not obvious we identify the responsible gene through small insert libraries.<br/><br/>Using this approach we have identified useful glycosidases of novel specificity from environments ranging from forest soils to human and beaver feces. In particular we have screened the human gut metagenomic libraries for enzymes that remove the Gal or GalNAc residues that function as the antigenic determinants from A and B type red blood cells, thereby generating “universal” O type blood.  High-throughput screening of libraries for glycoside phosphorylases will also be discussed if time permits

Discovery of CAZYmes for cell surface glycan removal through metagenomics: Towards universal blood

256th National Meeting (2018)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.