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.

Alkylimidazolium-based ionic liquids (AILs), once hailed as ″green solvents,″ have seen widespread use, but recent concerns have emerged regarding their environmental and health risks. This study integrates in vitro and in vivo dose-response metabolomics to investigate liver metabolic disturbances and pathway sensitivity to 1-octyl-3-methylimidazolium (M8OI) exposure. Important liver function indicators, including catalase, alanine aminotransferase, aspartate aminotransferase, and glycosylated serum protein, showed significant alterations (P < 0.05), indicating liver dysfunction. Metabolomics analysis revealed dose-dependent changes in energy metabolism and oxidative stress pathways in both cell and rat models, characterized by increased levels of thiamine and lipopolysaccharides, and decreased levels of nicotinamide and adenine. Key intermediates of the tricarboxylic acid cycle, such as citrate and isocitrate, exhibited significant alterations (P < 0.05). Pathway analysis identified disruptions in arginine, proline, and purine metabolism. Quantitative risk characterization based on effective concentration (EC) values identified key metabolites─adenine (EC_-10 = 0.004 mg/kg), (±)12(13)-DiHOME (EC_-10 = 0.024 mg/kg), and nicotinamide (EC_-10 = 0.05 mg/kg) in vivo, and isocitrate (EC_-10 = 0.22 μM), d-threo-isocitric acid (EC_-10 = 0.23 μM), and citric acid (EC_-10 = 0.40 μM) in vitro─as potential biomarkers of M8OI-induced metabolic disruption. These findings highlight hepatic metabolic disturbances induced by M8OI, with dose-response metabolomics identifying benchmark dose values based on regression models, thereby providing a basis for health risk assessment.