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Human milk oligosaccharides (HMOs) are a structurally complex group of unbound polysaccharides, representing the third-largest solid component in breast milk. They play a crucial role in the intestinal health and immune system development of infants. Sialylated HMOs, including 3'-sialactose (3'-SL) and 6'-sialactose (6'-SL), are major components of HMOs, playing significant roles in immune regulation, anti-inflammatory processes, and promotion of probiotic growth. Currently, the cost-effective production of high-value sialactose by microbial fermentation with readily available raw materials has become a research hotspot due to the high nutritional value and potential applications of sialylated HMOs in infant food. This paper summarizes the functions and biosynthesis of 3'-SL and 6'-SL. Furthermore, it reviews the research progress in the synthesis of sialactose by Escherichia coli , offering valuable insights for future industrial production.

Lacto- N -neotetraose (LNnT) is a neutral human milk oligosaccharide with important biological functions. However, the low LNnT productivity and the incomplete conversion of the intermediate lacto- N -tetraose II (LNT II) currently limited the sustainable biosynthesis of LNnT. First, the LNnT biosynthetic module was integrated in Escherichia coli . Next, the LNnT export system was optimized to alleviate the inhibition of intracellular LNnT synthesis. Furthermore, by utilizing rate-limiting enzyme diagnosis, the expressions of LNnT synthesis pathway genes were finely regulated to further enhance the production yield of LNnT. Subsequently, a strategy of cofermentation using a glucose/glycerol (4:6, g/g) mixed feed was employed to regulate carbon flux distribution. Finally, by overexpressing key transferases, LNnT and LNT II titers reached 112.47 and 7.42 g/L, respectively, in a 5 L fermenter, and 107.4 and 2.08 g/L, respectively, in a 1000 L fermenter. These are the highest reported titers of LNnT to date, indicating its significant potential for industrial production.

Lacto- N -neotetraose (LNnT) and lacto- N -tetraose (LNT) are important oligosaccharides found in breast milk and are commonly used as nutritional supplements in infant formula. We used metabolic engineering techniques to optimize the modified Escherichia coli BL21 star (DE3) strain for efficient synthesis of LNnT and LNT using β-1,4-galactosyltransferase (HpgalT) from Helicobacter pylori and β-1,3-galactosyltransferase (SewbdO) from Salmonella enterica subsp. salamae serovar , respectively. Further, we optimized the expression of three key genes, lgtA , galE , and HpgalT ( SewbdO ), to synthesize LNnT or LNT and deleted several genes ( ugd , ushA , agp , wcaJ , otsA , and wcaC ) to block competition in the UDP-galactose synthesis pathway. The optimized strain produced LNnT or LNT with a titer of 22.07 or 48.41 g/L, respectively, in a supplemented batch culture, producing 0.41 or 0.73 g/L/h, respectively. The strategies used in this study contribute to the development of cell factories for high-level LNnT and LNT and their derivatives.

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 Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

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.

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. [ABSTRACT FROM AUTHOR]