Metabolic Engineering of Escherichia coli for High-Titer Biosynthesis of 3′-Sialyllactose

3′-Sialyllactose (3′-SL) is among the foremost and simplest sialylated breast milk oligosaccharides. In this study, an engineered Escherichia coli for high-titer 3′-SL biosynthesis was developed by introducing a multilevel metabolic engineering strategy, including (1) the introduction of precursor CMP-Neu5Ac synthesis pathway and high-performance α2,3-sialyltransferase (α2,3-SiaT) genes into strain BZ to achieve de novo synthesis of 3′-SL; (2) optimizing the expression of glmS-glmM-glmU involved in the UDP-GlcNAc and CMP-Neu5Ac synthesis pathways, and constructing a glutamine cycle system, balancing the precursor pools; (3) analysis of critical intermediates and inactivation of competitive pathway genes to redirect carbon flux to 3′-SL biosynthesis; and (4) enhanced catalytic performance of rate-limiting enzyme α2,3-SiaT by RBS screening, protein tag cloning. The final strain BZAPKA14 yielded 9.04 g/L 3′-SL in a shake flask. In a 3 L bioreactor, fed-batch fermentation generated 44.2 g/L 3′-SL, with an overall yield and lactose conversion of 0.53 g/(L h) and 0.55 mol 3′-SL/mol, respectively.