High-Level Production of Lacto‑<i>N</i>‑neotetraose in <i>Escherichia coli</i> 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

High-Yield Synthesis of 2′-Fucosyllactose from Glycerol and Glucose in Engineered <i>Escherichia coli</i>

2′-Fucosyllactose (2′-FL) is vital for the growth and development of newborns. In this study, we developed a synthesis pathway for 2′-FL in Escherichia coli BL21 (DE3). Then, we optimized the solubility of α-1,2-fucosyltransferase, thereby enhancing the production yield of 2′-FL. Based on this finding, we further enhanced the expression of guanosine inosine kinase Gsk and knocked out the isocitrate lyase regulator gene iclR. This strategy reduced the formation of byproduct acetate during the metabolic process and alleviated carbon source overflow effects in the strain, resulting in further improvement of the yield of 2′-FL. In a 3 L bioreactor, employing fed-batch fermentation with glycerol and glucose as substrates, the engineered strain BWLAI-RSZL exhibited impressive 2′-FL titers of 121.9 and 111.56 g/L, along with productivity levels of 1.57 and 1.31 g/L/h, respectively. The reported 2′-FL titers reached a groundbreaking level, irrespective of the carbon source employed (glycerol or glucose), highlighting the significant potential for large-scale industrial synthesis of 2′-FL