Accessing new and improved enzymes for unnatural glycoside synthesis and cell surface antigen removal through metagenomics, gene library synthesis and directed evolution
September 24-28, 2017
Gaining access to enzymes that are able to degrade or synthesise specific glycosides, especially those containing modified sugars, can be very challenging. In theory this could be achieved by re-design of the active site of existing enzymes, but very few such approaches have been successful in producing enzymes that are of practical value. Likewise, modification of selectivity via directed evolution approaches has not been as fruitful as had been hoped. Both such approaches could benefit from access to more and better “start points” for modification.
Mother Nature has already supplied an enormous diversity of biocatalysts, each of which could serve as a starting point for directed evolution studies. The problem can be in accessing this diversity in a reasonably efficient manner. Here I shall describe two ways in which we have approached this problem. One of these is through synthesis of libraries of genes from within promising glycoside hydrolase families, selected to represent phylogenetically distinct sub-families of genes. In the other approach we have used activity-based, or functional metagenomics to generate a library of over 600 expressed glycosidases. These libraries have been subjected to “first-pass” high-throughput characterisation for substrate specificity, thermal stability, pH profile and mechanism. This sub-library has then been screened to identify preferred catalysts for cleavage of specific unnaturally modified sugars (e.g. azido sugars). Once candidate enzymes are identified they are then mutated to "glycosynthase" versions that can be used to "tag" glycans. In parallel we have screened the human gut metagenomic libraries for enzymes that can be used to 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. Such libraries can also be used to select the optimal candidate for further improvements through directed evolution.
Steve Withers, "Accessing new and improved enzymes for unnatural glycoside synthesis and cell surface antigen removal through metagenomics, gene library synthesis and directed evolution" in "Enzyme Engineering XXIV", Pierre Monsan, Toulouse White Biotechnology, France Magali Remaud-Simeon, LISBP-INSA, University of Toulouse, France Eds, ECI Symposium Series, (2017). http://dc.engconfintl.org/enzyme_xxiv/131