Enzymatic and cell factory approaches to the production of human milk oligosaccharides

β-N-Acetylhexosaminidases for Carbohydrate Synthesis via Trans-Glycosylation

“…Most noteworthy in this context is the recently reported synthesis of LNT II where an isolated yield of 86% (281 g/L) was achieved in a reaction using Lac and Glc-oxa in a ratio of 1:1 at a concentration of 600 mM each (Table 9) [100]. [51] NoHex (Nocardia orientalis 1 donor based; 2 isolated yield(s); 3 isolated as deprotected conjugates; 4 purified from β-galactosidase preparation (grade XI, Sigma-Aldrich); 5 2% diglycoside observed; 6 enzyme source not specified; 7 carried out in a plasticized glass phase: 10% H 2 O, 5% EtOH, 5% n-PrOH; 8 donor added stepwise; 9 isolated from Taka-diastase (Sankyo); 10 t-butanol gave no product; 11 not isolated; 12 only detected by TLC; 13 or Amycolatopsis orientalis IFO12806T; 14 only aliphatic OH of coniferyl alcohol glycosylated; 15 1,3-butanediol and 1,2,4-butanetriol gave no product, but enhanced chitotriose formation; 16 no product formation with dulcitol; 17 Phenyl-and 2-chlorophenylglycosides were cleaved overnight, the other two were stable; 18 thioglycoligase reaction; 19 30% (v/v) MeCN added; 20 5% (v/v) DMSO added; 21 10 mM DTT added.…”

“…GlcNAc is probably the hexosamine of the biggest industrial interest since it can be isolated from large industrial waste streams, e.g., from shrimp and crab waste (because chitin is a main constituent of the shrimp and crab shells) and either serve as a renewable carbon and nitrogen source for bio-ethanol production [5] or be used as a constituent for higher value functional applications, e.g., in medicine, high-value food ingredients, or cosmetics [6]. Prominent examples of such high value GlcNAc-containing food-ingredient products are the human milk oligosaccharides (HMOs, Figure 1) [7,8]. Apart from the simplest HMOs such as 2 -and 3-fucosyllactose as well as 3 -and 6 -sialyllactose, all HMOs contain at least one GlcNAc moiety [9].…”

“…donor based; 2 not isolated; 3 isolated yield; 4 no product hydrolysis; 5 thioglycoligase reaction; 6 5% (v/v) DMSO and 10 mM DTT added;7 probably Gal-β-1,4-(GlcNAc-β-1,2-)Glc or Gal-β-1,4-(GlcNAc-β-1,3-)Glc; 8 only analyzed by TLC; 9 specified loop sequence inserted in between W354 and R355 of Hex1.…”

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

“…In contrast, LNnT is less abundant both on its own and as an HMO core, and is also present in human milk in lower levels than, e.g., lacto- N -tetraose (LNT) [11,12], but it appears that LNnT is easier to synthesize in large scale and was therefore marketed first [9,13,14]. Indeed, for more complex and larger structures, fermentation yields are often low [13,15].…”

“…Currently, many HMOs are not available in sufficient quantities and there is no efficient route to their production. The status of enzymatic HMO production—both in vitro and in cell factories—was recently thoroughly reviewed [15], and while great progress has been made over the past two decades, the discrepancy between human milk composition and currently obtainable HMOs is evident, providing a continued impetus to produce a wider span of true HMO structures by controlled enzymatic synthesis. One way to accomplish production of larger and more complex HMOs is to employ transglycosylation catalyzed by glycoside hydrolases (or glycosidases, GHs), possibly in combination with use of fermentation-derived backbone structures such as LNnT or LNT as acceptor substrates for the enzymatic glycosylation.…”