Transporter Engineering Enables the Efficient Production of Lacto-N-triose II and Lacto-N-tetraose in Escherichia coli

Lacto-N-triose (LNT II) and lacto-N-tetraose (LNT) are human milk oligosaccharides (HMOs) with various potential functions for infants. HMO production by Escherichia coli fermentation has attracted attention in recent years. However, little is known about the cellular export of HMOs. In this study, we identified four endogenous E. coli transporter genes (setA, setB, ydeA, and mdfA), overexpression of which significantly increased the efficiency of LNT II production. The setA-enhanced strain accumulated 34.2 g/L LNT II in a 3 L bioreactor. In the production of LNT, which uses LNT II as an intermediate, disruption of setA remarkably decreased the LNT II accumulation and enhanced the titer of LNT. Furthermore, by heterologous expression of extracellular β-1,3-N-acetylglucosaminidase from Bifidobacterium bifidum, which degrades LNT II, we eliminated LNT II completely. This study shows that regulation of sugar efflux transporters in E. coli can increase the production of HMOs and decrease the amounts of undesired byproducts.