N-acetyl-D-mannosamine

3′ -Sialyllactose (3′ -SL, Neu5Acα2,3 Galβ1,4Glc), a prominent sialylated human milk oligosaccharide (HMO), has attracted significant attention due to its diverse physiol. properties.The efficient α2,3-sialyltransferase (α2,3-SiaT) is crucial for the biosynthesis of 3 ′ -SL.In this study, the 3 ′ -SL biosynthetic pathway was constructed in EZAK (E. coli BL21(DE3) ΔlacZΔnanAΔnanK). 406NST, which exhibits a high 3 ′ -SL yield and low byproduct formation, was chosen for mol. modification.Five amino acid differences between 406NST and NST were targeted for site-directed mutagenesis.Subsequently, saturation mutagenesis was carried out at the D40 position, followed by superimposed multipoint mutagenesis to generate the optimal strain Z28 (406NST-D40R-N113D-P120H), resulting in an extracellular 3 ′-SL yield of 4.67 g/L in shake flasks.In a 5 L bioreactor, the extracellular 3 ′-SL yield reached 29.54 g/L, achieving an overall 3 ′-SL yield of 0.52 g/L/h and a lactose conversion yield of 0.62 mol 3 ′-SL/mol.In conclusion, a highly active and specific α2,3-SiaT was successfully constructed, significantly improving the yield of 3 ′ -SL.

This study defines biochemical mechanisms that contribute to novel neural-regenerative activities we recently demonstrated for thiol-modified ManNAc analogs in human neural stem cells (hNSCs) by comparing our lead drug candidate for brain repair, "TProp," to a "size-matched" N-alkyl control analog, "But." These analogs biosynthetically install non-natural sialic acids into cell surface glycans, altering cell surface receptor activity and adhesive properties of cells. In this study, TProp modulated sialic acid-related biology in hNSCs to promote neuronal differentiation through modulation of cell adhesion molecules (integrins α6, β1, E-cadherin, and PSGL-1) and stem cell markers. By comparison, But elicited minimal change to these endpoints, indicating dependence on the chemical properties of the thiol group of non-natural sialic acids and not the size of this sugar's N-acyl group. Conversely, But elicited distinct intracellular responses including increased nestin expression (~6-fold) and the modulation of several metabolites identified through cell-wide screening. Metabolites up-regulated by But included dopamine and norfenenfrine, suggesting that this analog may be a drug candidate for treating neural damage associated with conditions such as Parkinson's disease. The metabolomics data also provided new insights into the neuroprotective effects of TProp when used to treat brain injury by upregulation of anti-inflammatory metabolites (e.g., α- & γ-linolenic acids) valuable for dampening injury- and treatment-related inflammation. Finally, these analogs modulate compounds that control proline (e.g., 1-pyrroline-2-carboxylate), a master regulator of many cellular activities. Overall, this study presents new mechanisms and pathways to exploit metabolic glycoengineering for neural repair and treatment of neurodegenerative diseases.