Overcoming the limited availability of human milk oligosaccharides: challenges and opportunities for research and application

Abstract

Human milk oligosaccharides (HMOs) are complex sugars highly abundant in human milk but currently not present in infant formula. Rapidly accumulating evidence from in vitro and in vivo studies, combined with epidemiological associations and correlations, suggests that HMOs benefit infants through multiple mechanisms and in a variety of clinical contexts. Until recently, however, research on HMOs has been limited by an insufficient availability of HMOs. Most HMOs are found uniquely in human milk, and thus far it has been prohibitively tedious and expensive to isolate and synthesize them. This article reviews new strategies to overcome this lack of availability by generating HMOs through chemoenzymatic synthesis, microbial metabolic engineering, and isolation from human donor milk or dairy streams. Each approach has its advantages and comes with its own challenges, but combining the different methods and acknowledging their limitations creates new opportunities for research and application with the goal of improving maternal and infant health.

Keywords: bioengineering; carbohydrates; human milk oligosaccharides; milk; pediatrics; synthesis.

Figure 1

Symbol representation of (A) human milk oligosaccharides (HMOs) and (B) nonhuman milk oligosaccharides currently added to infant formula. (A) HMOs contain lactose at the reducing end and can be elongated by up to 15 disaccharide units of either lacto- N -biose (Gal β1–3-linked to GlcNAc) or N -acetyllactosamine (Gal β1–4-linked to GlcNAc). In addition, HMO can be fucosylated in α1–2-, α1–3-, or α1–4-linkage or/and sialylated in α2–3- or α2–6-linkage. (B) Galactooligosaccharides (GOS), which are not present in human milk, are oligomers of galactose that often carry glucose at the reducing end. Fructooligosaccharides (FOS), which are also not present in human milk, are oligomers of fructose that also carry glucose at the reducing end. Fructose itself is not a building block of the oligosaccharides naturally found in human milk. GOS and FOS are currently added to some infant formulas but are structurally very different from human milk oligosaccharides.

Figure 2

Chemoenzymatic synthesis of sialyllacto- N -tetraose (LST-a). (A) Chemical glycosylation. (B) Removal of all ester- and acetamido-protecting groups. (C) α2–3-sialyltransferase.

Figure 3

LNnT diversification. (A) Helicobacter pylori α1–3-fucosyltransferase; (B) ST3Gal4; (C) ST3Gal, H. pylori α1–4-fucosyltransferase. Abbreviations : HO, hydroxyl; SEt, thioethyl; TFA, trifluoroacetimide.

Figure 4

Gene targets of metabolic engineering to produce 2′-fucosyllactose ( 2′-FL) in E. coli. Three elements for producing 2′-FL in E. coli are illustrated. Either extracellularly fed or intracellularly synthesized lactose can be used as a backbone. De novo and/or salvage pathways for producing GDP- _L -fucose can be used. Fucosyltransferases from various sources can be used for fucosylation of lactose. Abbreviations : ATP, adenosine triphosphate; DHA-P, dihydroxyacetone phosphate; FKP, _L -fucokinase/guanosine 5 ′ -diphosphate- l -fucose pyrophosphorylase; Fru-6-P, fructose-6-phosphate; fucT2 , α1–2-fucosyltransferase; Gal, galactose; galE , UDP-glucose 4 epimerase; galT1 , galactose-1-phosphate uridylyltransferase; galU , UTP-glucose-1-phosphate uridylyltransferase; GDP-Fuc, guanosine diphosphate fucose; GDP-Man, guanosine diphosphate mannose; Glc, glucose; gmd , GDP-mannose 4,6-dehydratase; GDP-4-keto-6-dMan, GDP-4-keto-6-deoxy- d -mannose epimerase/reductase; Glc-6-P, glucose-6-phosphate; GTP, guanosine triphosphate; lacZ , β-galactosidase; manA , mannose-6-phosphate isomerase; manB , phosphomannomutase, manC , mannose-1-phosphate guanylyltransferase; Man-6-P, mannose-6-phosphate; pgi , glucose-6-phosphate isomerase; pgm , phosphoglucomutase; wcaG , GDP-4-keto-6-deoxy-d-mannose-3,5- epimerase/4-reductase.