Research & Teaching Faculty

Systematic Screening of Synthetic Gene-Encoded Enzymes for Synthesis of Modified Glycosides

TitleSystematic Screening of Synthetic Gene-Encoded Enzymes for Synthesis of Modified Glycosides
Publication TypeJournal Article
Year of Publication2019
AuthorsArmstrong, Z, Liu, F, Chen, H-M, Hallam, SJ, Withers, SG
Date PublishedAPR

The construction of large, phylogenetically diverse libraries of synthetic genes from a sequence-based family allows rapid evaluation of the substrate specificity encoded within the gene products and identification of rare catalytic capabilities. For carbohydrate-active enzymes such as glycosidases, this opens up approaches to search for enzymes that are capable of catalyzing the hydrolysis and transfer of modified sugars. Herein we report the screening of a library of E. coli clones each harboring 1 of 175 synthetic genes that represent the subfamilies within the glycoside hydrolase family GH1 to identify a subset of enzymes that cleave amino- and azido-glucosides modified at the 3-, 4-, and 6-positions. Eight of the most active such enzymes were further characterized through detailed kinetic analysis and plate-based aglycone specificity screening. Three glycosynthase mutants of each of these enzymes were generated, in which their catalytic nucleophile glutamate was substituted by alanine, glycine, or serine, and their synthetic capabilities were explored with alpha-glucosyl fluoride and 3-, 4-, and 6-amino and -azido derivatives. The utility of seven of the generated mutants was then demonstrated through the assembly of taggable disaccharides, trisaccharides, glycolipid analogues, and activity-based probes. Not only does this work present the creation of several useful biocatalysts, it also provides a general framework that can be exploited to develop catalysts from other glycosidehydrolase-containing clone libraries.