Lacto-N-neotetraose (LNnT) is a crucial neutral core human milk oligosaccharide (HMO). In this study, we established a LNnT-producing Saccharomyces cerevisiae cell factory through comprehensive metabolic engineering. Specifically, the de novo biosynthetic pathway of LNnT was assembled by heterologously expressing the lactose permease (lac12) from Kluyveromyces lactis and the glycosyltransferase from Neisseria meningitidis in S. cerevisiae. Subsequently, carbon source regulation based on the glucose-sensitive GAL regulatory system was employed to optimize the expression time of heterologous genes, achieving a production of 15.61 mg/L of LNnT in shake-flask fermentation. In addition, the key rate-limiting steps involved in LNnT synthesis pathway were identified and the corresponding genes were overexpressed to enhance LNnT production, resulting in an 8-fold increase in LNnT titer compared to that of parental strain. To our knowledge, this is the first report on LNnT biosynthesis in S. cerevisiae, opening up the possibility of green production of LNnT using food-safe microorganisms.

This study aimed to explore the improvement effect of xylooligosaccharides (XOS) on chronic diarrhea. According to the principle of randomized parallel controlled experiments, patients with clinical chronic diarrhea were recruited and randomly divided into three groups, a placebo group (CK, taking 3 g of maltodextrin per day), a low-dose XOS group (3X, taking 3 g of XOS per day) and a high-dose XOS group (6X, taking 6 g of XOS per day). The diarrhea symptom scores, serum biochemical indexes, fecal short-chain fatty acids and gut microbiota were analyzed after four weeks of intervention. Then, the fecal bacterial supernatant of the patients in each intervention group was administered by gavage to mice for 24 consecutive days, and the intestinal permeability and the levels of inflammatory factors were measured after 12 h fasting on the 25^th day. The results showed that diarrhea symptoms and lipid metabolism tended to be improved in the intervention groups compared to the placebo group, and fecal butyric acid levels increased. The relative abundance of Blautia, Bifidobacterium, and Lachnospiraceae_unclassified were increased, while the relative abundance of Prevotella was decreased. Compared to the placebo group, the intestinal barrier tended to be stable in the mice gavaged with the fecal bacterial supernatant of the XOS intervention groups, colonic inflammation was significantly alleviated, the level of serum D-lactic acid (D-LA) was significantly decreased (P＜0.05 or 0.01), and the expression of tight junction protein (zonula occludens-1 (ZO-1) and occludin) was up-regulated. These results indicate that XOS are effective in improving chronic diarrhea, and are expected to alleviate chronic diarrhea.

The purpose of this study was to explore the ability of different bifidobacterial species to utilize xylooligosaccharide (XOS). It focused on the mechanism for the efficient utilization of XOS by Bifidobacterium pseudolongum JNFEN6 through cell physiological analysis and transcriptomic analysis. The results showed that XOS had different effects on the growth of different Bifidobacterium species, and it had the most significant effect on the growth and acid-producing capacity of B. pseudolongum JNFEN6. When B. pseudolongum JNFEN6 was cultured for 36 h on XOS as the sole carbon source, the activity of β-xylosidase produced by this strain was 0.97 U/mL, and the concentration of propionic acid in the fermentation system was 85.26 μg/mL. The transcriptomic results showed that a total of 297 differentially expressed genes were identified, including 136 up-regulated genes and 161 down-regulated genes. Among them, the ATP-binding cassette (ABC) transport permease, substrate-binding protein and MFS transporter genes promoted the transport of XOS, while the XOS metabolic hydrolase, acetate kinase and lactate dehydrogenase genes promoted the metabolism of XOS. In summary, B. pseudolongum JNFEN6 can efficiently utilize XOS, which can take in XOS, mainly through the ABC transport system and the MFS transport system, and metabolize XOS through the ‘bifidus shunt’ pathway, finally producing short-chain fatty acids or other organic compounds. This study provides a theoretical basis for the application of XOS as a bifidus factor.