3'-Sialyllactose (3'-SL), a key component of human milk oligosaccharides, provides significant health benefits and immune modulation, and is increasingly used in infant formula and dietary supplements. This study presents a novel approach for the efficient biosynthesis of 3'-SL using Escherichia coli BL21star(DE3)ΔlacZ through genomic integration. We first addressed the issue of metabolic competition by deleting crucial genes, nanA, nanK, nanE, and nanT, that are involved in the degradation of N-acetylneuraminic acid. This strategic gene knockout minimized the flux through competing pathways. The engineered Escherichia coli strain was subsequently transformed with the exogenous genes neuBCA and nST, enabling the de novo synthesis of 3'-SL. A modular metabolic engineering strategy was utilized to optimize the expression of key enzymes within the MSU module, enhancing and balancing the carbon flux distribution. Additionally, a cofactor regeneration strategy was implemented to increase CTP availability, which improved cofactor recycling and fine-tuned the metabolic pathway for maximal 3'-SL production. Transport protein screening was incorporated to further increase the extracellular concentration of 3'-SL, resulting in an unprecedented yield of 56.8 g/L in a 5L bioreactor fermentation, setting a new benchmark in the field.

Levilactobacillus brevis strains can be isolated from traditional Chinese pickles and used as the starter cultures to improve the nutritional profiles of fermented juices. Three L. brevis strains (LBG-29, LBG-24, LBD–14) that produce high levels of gamma-aminobutyric acid (GABA; >300 mg/L) were isolated from traditional Chinese pickles. The strains showed tolerance to low pH and high bile salts and exhibited safety in vitro. Litchi juice was fermented using each strain at 37 °C for 48 h. The litchi juice was determined to be a good substrate for fermentation as the process enhanced its functional profile. Overall, cell vitality increased (above 8.7 log₁₀ CFU/mL), the antioxidant activities of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric ion-reducing antioxidant power (FRAP) were significantly increased, and the antioxidant capacity of the 2,2′-amino-di(3-ethyl-benzothiazoline sulphonic acid-6)ammonium salt (ABTS) was decreased. There was also a significant increase in the GABA and acetic acid content after LBG-29 and LBG-24 fermentation. It was thus determined that the LBG-29 and LBG-24 strains could be used to improve beverage functionality and aid in the development of new products. This is the first report of litchi fermentation using L. brevis as a starter culture. Further research is required to elucidate the functional benefits for the human body and the nutritional and functional properties during its shelf life.

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.

(1) Background: Ellagic acid (EA) acts as a product of gut microbiota transformation to prevent insulin resistance, which is limited by high-fat diet (HFD)-induced dysbiosis. The aim of this study was to investigate the synergistic effects and mechanisms of supplementation with the probiotic Weizmannia coagulans (W. coagulans) on the prevention of insulin resistance by EA; (2) Methods: C57BL/6J mice were divided into five groups (n = 10/group): low-fat-diet group, high-fat-diet group, EA intervention group, EA + W. coagulans BC77 group, and EA + W. coagulans BC2000 group; (3) Result: W. coagulans BC2000 showed a synergistic effect on EA’s lowering insulin resistance index and inhibiting high-fat diet-induced endotoxemia. The combined effect of BC2000 and EA activated the autophagy pathway in the mouse liver, a urolithin-like effect. This was associated with altered β-diversity of gut microbiota and increased Eggerthellaceae, a potential EA-converting family. Ellagic acid treatment alone and the combined use of ellagic acid and W. coagulans BC77 failed to activate the hepatic autophagy pathway; (4) Conclusions: W. coagulans BC2000 can assist EA in its role of preventing insulin resistance. This study provides a basis for the development of EA-rich functional food supplemented with W. coagulans BC2000.

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.