High-Level Production of Lacto- N -neotetraose in Escherichia coli by Stepwise Optimization of the Biosynthetic Pathway.

Lacto- N -neotetraose (LNnT), an abundant human milk oligosaccharide (HMO), has been approved as a novel functional additive for infant formulas. Therefore, LNnT biosynthesis has attracted extensive attention. Here, a high LNnT-producing, low lacto- N -triose II (LNT II)-residue Escherichia coli strain was constructed. First, an initial LNnT-producing chassis strain was constructed by blocking lactose, UDP- N -acetylglucosamine, and UDP-galactose competitive consumption pathways and introducing β -1,3- N -acetylglucosaminyltransferase LgtA and β -1,4-galactosyltransferase LgtB. Subsequently, the supply of LNnT precursors was increased by enhancing UDP- N -acetylglucosamine and UDP-galactose synthesis, inactivating LNT II extracellular transporter SetA, and improving UTP synthesis. Then, modular engineering strategy was used to optimize LNnT biosynthetic pathway fluxes. Moreover, pathway fluxes were fine-tuned by modulating translation initiation strength of essential genes lgtB , prs, and lacY . Finally, LNnT production reached 6.70 g/L in a shake flask and 19.40 g/L in a 3 L bioreactor with 0.47 g/(L h) productivity, with 1.79 g/L LNT II residue, highest productivity level, and lowest LNT II residue thus far.