2′-fucosyllactose (2′-FL), one of the simplest but most abundant oligosaccharides in human milk, has been demonstrated to have many positive benefits for the healthy development of newborns. However, the high-cost production and limited availability restrict its widespread use in infant nutrition and further research on its potential functions. In this study, on the basis of previous achievements, we developed a powerful cell factory by using a lacZ-mutant Escherichia coli C41 (DE3)ΔZ to ulteriorly increase 2′-FL production by feeding inexpensive glycerol. Initially, we co-expressed the genes for GDP-L-fucose biosynthesis and heterologous α-1,2-fucosyltransferase in C41(DE3)ΔZ through different plasmid-based expression combinations, functionally constructing a preferred route for 2′-FL biosynthesis. To further boost the carbon flux from GDP-L-fucose toward 2′-FL synthesis, deletion of chromosomal genes (wcaJ, nudD, and nudK) involved in the degradation of the precursors GDP-L-fucose and GDP-mannose were performed. Notably, the co-introduction of two heterologous positive regulators, RcsA and RcsB, was confirmed to be more conducive to GDP-L-fucose formation and thus 2′-FL production. Further a genomic integration of an individual copy of α-1,2-fucosyltransferase gene, as well as the preliminary optimization of fermentation conditions enabled the resulting engineered strain to achieve a high titer and yield. By collectively taking into account the intracellular lactose utilization, GDP-L-fucose availability, and fucosylation activity for 2′-FL production, ultimately a highest titer of 2′-FL in our optimized conditions reached 6.86 g/L with a yield of 0.92 mol/mol from lactose in the batch fermentation. Moreover, the feasibility of mass production was demonstrated in a 50-L fed-batch fermentation system in which a maximum titer of 66.80 g/L 2′-FL was achieved with a yield of 0.89 mol 2′-FL/mol lactose and a productivity of approximately 0.95 g/L/h 2′-FL. As a proof of concept, our preliminary 2′-FL production demonstrated a superior production performance, which will provide a promising candidate process for further industrial production.

3-fucosyllactose (3-FL) and 2'-fucosyllactose (2'-FL), are two important fucosylated oligosaccharides in human milk. Extensive studies on 2'-FL enabled its official approval for use in infant formula. However, development of 3-FL has been somewhat sluggish due to its low content in human milk and poor yield in enlarged production. Here, an α-1,3-fucosyltransferase mutant was introduced into an engineered Escherichia coli (E. coli) capable of producing GDP-L-fucose, leading to a promising 3-FL titer in a 5.0-L bioreactor. To increase the availability of cofactors (NADPH and GTP) for optimized 3-FL production, zwf, pntAB, and gsk genes were successively overexpressed, finally resulting in a higher 3-FL level with a titer of 35.72 g/L and a yield of 0.82 mol 3-FL/mol lactose. Unexpectedly, the deletion of pfkA gene led to a much lower performance of 3-FL production than the control strain. Still, our strategy achieved the highest 3-FL level in E. coli to date.

The objective of this study was to investigate the stability and degradation kinetics of N-acetylneuraminic acid (Neu5Ac). The pH of the solution strongly influenced the stability of Neu5Ac, which was more stable at neutral pH and low temperatures. Here, we provide detailed information on the degradation kinetics of Neu5Ac at different pH values (1.0, 2.0, 11.0 and 12.0) and temperatures (60, 70, 80 and 90 °C). The study of the degradation of Neu5Ac under strongly acidic conditions (pH 1.0–2.0) is highly pertinent for the hydrolysis of polysialic acid. The degradation kinetics of alkaline deacetylation were also studied. Neu5Ac was highly stable at pH 3.0–10.0, even at high temperature, but the addition of H₂O₂ greatly reduced its stability at pH 5.0, 7.0 and 9.0. Although Neu5Ac has a number of applications in products of everyday life, there are no reports of rigorous shelf-life studies. This research provides kinetic data that can be used to predict product shelf lives at different temperatures and pH values.

The probiotic Weizmannia coagulans (W. coagulans) BC2000 can increase the abundance of intestinal transforming ellagic acid (EA) bacteria and inhibit metabolic disorders caused by hyperlipidemia by activating liver autophagy. This study aimed to investigate the inhibitory effects of W. coagulans BC2000 and EA on hyperlipidemia-induced cholesterol metabolism disorders. C57BL/6J mice (n = 10 in each group) were fed a low-fat diet, high-fat diet (HFD), HFD supplemented with EA, HFD supplemented with EA and W. coagulans BC77, HFD supplemented with EA, and W. coagulans BC2000. EA and W. coagulans BC2000 supplementation prevented HFD-induced hypercholesterolemia and promoted fecal cholesterol excretion. Transcriptome analysis showed that primary bile acid biosynthesis in the liver was significantly activated by EA and W. coagulans BC2000 treatments. EA and W. coagulans BC2000 treatment also significantly increased the intestinal Eggerthellaceae abundance and the liver EA metabolites, iso-urolithin A, Urolithin A, and Urolithin B. Therefore, W. coagulans BC2000 supplementation promoted the intestinal transformation of EA, which led to the upregulation of liver bile synthesis, thus preventing hypercholesterolemia.

2'-Fucosyllactose (2'-FL) is one of the most important oligosaccharides in human milk. In this study, a glucose–glycerol mixed carbon source and the deletion of the genes arcA and iclR were attempted for the first time in engineered Escherichia coli to improve 2'-FL production. The highest 2'-FL titre of 5.04 g/L, almost 1.4 times higher than that in the control strain, was obtained in the arcA mutant strain using shake-flask culture. In addition, the arcA mutant showed a significantly reduced accumulation of acetate. However, the deletion of iclR and the double deletion of arcA/iclR provided no benefit in terms of 2'-FL biosynthesis. Overall, these results suggest that deletion of arcA could efficiently increase the 2'-FL yield in E. coli and could lay a foundation for more efficient and larger-scale production. Moreover, in shake-flask culture under optimal conditions, the 2'-FL titer reached a maximum value of 7.14 g/L.

Among the human milk oligosaccharides (HMOs), one of the most abundant oligosaccharides and has great benefits for human health is 3′-sialyllactose (3′-SL). Given its important physiological functions and the lack of cost-effective production processes, we constructed an in vitro multi-enzymatic cofactor recycling system for the biosynthesis of 3′-SL from a low-cost substrate. First, we constructed the biosynthetic pathway and increased the solubility of cytidine monophosphate kinase (CMK) with chaperones. We subsequently identified that β-galactosidase (lacZ) affects the yield of 3′-SL, and hence with the lacZ gene knocked out, a 3.3-fold increase in the production of 3′-SL was observed. Further, temperature, pH, polyphosphate concentration, and concentration of divalent metal ions for 3′-SL production were optimized. Finally, an efficient biotransformation system was established under the optimized conditions. The maximum production of 3′-SL reached 38.7 mM, and a molar yield of 97.1% from N-acetylneuraminic acid (NeuAc, sialic acid, SA) was obtained. The results demonstrate that the multi-enzymatic cascade biosynthetic pathway with cofactor regeneration holds promise as an industrial strategy for producing 3′-SL.