Synthesis of Human Milk Oligosaccharides: Protein Engineering Strategies for Improved Enzymatic Transglycosylation

Abstract

Human milk oligosaccharides (HMOs) signify a unique group of oligosaccharides in breast milk, which is of major importance for infant health and development. The functional benefits of HMOs create an enormous impetus for biosynthetic production of HMOs for use as additives in infant formula and other products. HMO molecules can be synthesized chemically, via fermentation, and by enzymatic synthesis. This treatise discusses these different techniques, with particular focus on harnessing enzymes for controlled enzymatic synthesis of HMO molecules. In order to foster precise and high-yield enzymatic synthesis, several novel protein engineering approaches have been reported, mainly concerning changing glycoside hydrolases to catalyze relevant transglycosylations. The protein engineering strategies for these enzymes range from rationally modifying specific catalytic residues, over targeted subsite -1 mutations, to unique and novel transplantations of designed peptide sequences near the active site, so-called loop engineering. These strategies have proven useful to foster enhanced transglycosylation to promote different types of HMO synthesis reactions. The rationale of subsite -1 modification, acceptor binding site matching, and loop engineering, including changes that may alter the spatial arrangement of water in the enzyme active site region, may prove useful for novel enzyme-catalyzed carbohydrate design in general.

Keywords: casein glycomacropeptide; fucosidase; human milk oligosaccharides; protein engineering; sialidase; transfucosylation; transglycosylation; transsialylation; β-N-acetylhexosaminidase.

Figure 1

Human milk oligosaccharide (HMO) blueprint structure [1]. Gal: galactose, Glc: glucose, GlcNAc: N-acetylglucosamine, Fuc: fucose, Sia: sialic acid (N-acetylneuraminic acid). Lactose is at the reducing end of all HMO structures, which may be elongated with β-N-acetyllactosamine (LacNAc) or lacto-N-biose units. Both lactose and elongated structures may be decorated with Fuc and/or Sia. The colored shapes indicate the Symbol Nomenclature for Glycans (SNFG [8], https://www.ncbi.nlm.nih.gov/glycans/snfg.html), which is commonly used for presenting the numerous HMO structures.

Figure 2

Reaction scheme sketches for glycosidase-catalyzed transglycosylation, which takes place in competition with substrate hydrolysis [49,68]. (A) Classical Koshland double-displacement mechanism exemplified by the α-l-fucosidase reaction: The intermediate, which is in the opposite anomeric configuration compared to the substrate and product as per the double displacement mechanism of retaining glycoside hydrolases (GHs), is attacked by a nucleophile. If this nucleophile is water, (primary) hydrolysis occurs. If a glycosyl acceptor performs the nucleophilic attack, transglycosylation occurs. (B) Substrate-assisted reaction mechanism of the GH20 β-N-acetylhexosaminidases [65]. For both reaction mechanisms, the resulting glycosylated product may also be subject to (secondary) hydrolysis catalyzed by the same glycosidase. The balance between the transglycosylation rate (r_T) and the hydrolytic rate (r_H) is governed by the reaction conditions as well as by enzyme properties, which can be altered through protein engineering. Regioselectivity in the product formation may vary. In HMO synthesis, R₁ and R₂ are glycosides, but for transglycosylation, in general, they can be other compounds, e.g., primary alcohols.