Abstract
In vitro glycorandomization is a powerful strategy to alter the glycosylation patterns of natural products and small molecule therapeutics. Yet, such in vitro methods are often difficult to scale and can be costly given the requirement to provide various nucleotides and cofactors. Here, we report the construction of several recombinant E. coli prototype strains that allow the facile production of a range of small molecule glycosides. This strategy relies on the engineered promiscuity of three key enzymes, an anomeric kinase, a sugar-1-phosphate nucleotidyltransferase, and a glycosyltransferase, as well as the ability of diverse small molecules to freely enter E. coli. Subsequently, this work is the first demonstration of "in vivo glycorandomization" and offers vast combinatorial potential by simple fermentation.