Glycosyltransferase engineering for carbohydrate synthesis

Abstract

Glycosyltransferases are powerful tools for the synthesis of complex and biologically important carbohydrates. Wild-type glycosyltransferases may not have all the properties and functions that are desired for large-scale production of carbohydrates that exist in nature and those with non-natural modifications. With the increasing availability of crystal structures of glycosyltransferases, especially those in the presence of donor and acceptor analogs, crystal structure-guided rational design has been quite successful in obtaining mutants with desired functionalities. With current limited understanding of the structure-activity relationship of glycosyltransferases, directed evolution continues to be a useful approach for generating additional mutants with functionality that can be screened for in a high-throughput format. Mutating the amino acid residues constituting or close to the substrate binding sites of glycosyltransferases by structure-guided directed evolution further explores the biotechnological potential of glycosyltransferases that can only be realized through enzyme engineering. This mini-review discusses the progress made towards glycosyltransferase engineering and the lessons learned for future engineering efforts and assay development.

Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs

“…34 Previous studies have demonstrated that glycosyltransferases are amenable to enzyme-substrate engineering, wherein the active site is modified to accept a non-native substrate. 35 Of particular relevance, the enzymes -1,4-galactosyltransferase 1 (4Gal-T1) and O-linked -Nacetylglucosamine transferase (O-GlcNAc transferase) have been rationally designed to accommodate nucleotide sugars bearing chemical handles. [36][37][38] Both of these engineered glycosyltransferases demonstrate expanded substrate specificity, as they still use their native substrates, UDP-galactose and UDP-GlcNAc, respectively.…”

Production of Plant Secondary Metabolites: Examples, Tips and Suggestions for Biotechnologists

“…The technique of directed evolution (DE) may be more useful in such cases. To restrict the need to screen a large mutational library, variation of this semi-rational method can be employed [ 148 ]. For example, iterative site-saturation mutagenesis (ISM), where a critical amino acid is replaced by all other 19 amino acids at that position, can be used to select for the most efficient GT which can be further improved in the second round, by targeting another residue in the active-site.…”

In vitro biosynthesis of poly-β-1,4-glucan derivatives using a pro-miscuous glycosyltransferase

“…However, glycosyltransferases can be promiscuous in acceptor and donor substrates, thereby providing a rich source of biocatalysts with potentially exploitable side reactions. For example, human blood group galactosyltransferase and bovine β4GalT1 have been reported to display activity with multiple UDP-donor substrates including UDP-Glc [42][43][44] , while Neisseria meningitidis LgtB displays activity towards multiple acceptor substrates 45 . The rates of activity on alternative acceptors and donors have in many cases been demonstrated to be sufficient for exploitation of the promiscuous activity in synthesis of glycosides 42,[44][45][46][47][48] .…”