Engineering the enzyme toolbox to tailor glycosylation in small molecule natural products and protein biologics

Abstract

Many glycosylated small molecule natural products and glycoprotein biologics are important in a broad range of therapeutic and industrial applications. The sugar moieties that decorate these compounds often show a profound impact on their biological functions, thus biocatalytic methods for controlling their glycosylation are valuable. Enzymes from nature are useful tools to tailor bioproduct glycosylation but these sometimes have limitations in their catalytic efficiency, substrate specificity, regiospecificity, stereospecificity, or stability. Enzyme engineering strategies such as directed evolution or semi-rational and rational design have addressed some of the challenges presented by these limitations. In this review, we highlight some of the recent research on engineering enzymes to tailor the glycosylation of small molecule natural products (including alkaloids, terpenoids, polyketides, and peptides), as well as the glycosylation of protein biologics (including hormones, enzyme replacement therapies, enzyme inhibitors, vaccines, and antibodies).

Biosynthetic production of anticoagulant heparin polysaccharides through metabolic and sulfotransferases engineering strategies

“…The expression of eukaryotic sulfotransferases in prokaryotic systems presents significant challenges. First, the absence of glycosylation modification in proteins recombinantly expressed in prokaryotic systems necessitates compensatory mutations to maintain protein stability 43 . Second, the membrane localization of the Golgi apparatus results in the proteins being situated on a large hydrophobic surface in eukaryotes, posing difficulties for sulfotransferase expression in E. coli and leading to issues of activity instability 44 .…”

Non-Native Site-Selective Enzyme Catalysis

“…Glycosylation of small molecules can also dramatically alter their biological function . Many natural glycosyl acceptors, including polyphenols, catechols, and sugars, often bear multiple similarly reactive hydroxyl groups, so site-selective glycosylation is required to ensure that only the biologically relevant isomer is formed. , Glycosyl transferases (GTs) must also control the stereoselectivity at the anomeric carbon of the glycosyl donor. While this selectivity is typically accompanied by relatively high substrate specificity, researchers have found that some GTs and engineered variants can accept a range of glycosyl donors and acceptors to enable site-selective, non-native glycosylation. − For example, while UGT71A15 was found to catalyze nonselective glycosylation of some polyphenolic acceptors, it catalyzes site-selective addition of glucose to 84 (Scheme A) .…”

Non-Native Site-Selective Enzyme Catalysis

“…97 Many natural glycosyl acceptors, including polyphenols, catechols, and sugars, often bear multiple similarly reactive hydroxyl groups, so site-selective glycosylation is required to ensure that only the biologically relevant isomer is formed. 98,99 Glycosyl transferases (GTs) must also control the stereoselectivity at the anomeric carbon of the glycosyl donor. While this selectivity is typically accompanied by relatively high substrate specificity, researchers have found that some GTs and engineered variants can accept a range of glycosyl donors and acceptors to enable site-selective, non-native glycosylation.…”