Efficient and Cost-Effective\nSynthesis of <i>N</i>‑Acetyllactosamine by Sequential\nModular Enzymatic\nCascade Reactions Involving NTP Regeneration

Human milk oligosaccharides (HMOs)\nplay important roles\nin the\ndevelopment of infants, which are the third most abundant component\nin human milk. <i>N</i>-Acetyllactosamine (LacNAc) is an\nimportant intermediate for the biosynthesis of other HMOs and antigens.\nSince currently appropriate synthetic methods for large-scale production\nof LacNAc are not available, it is urgently needed to develop an efficient\nand cost-effective synthetic pathway for LacNAc preparation. In this\nstudy, a cost-effective pathway of LacNAc synthesis involving regeneration\nof adenosine triphosphate (ATP) and uridine 5′-triphosphate\n(UTP) was established. After optimizing the reaction conditions, LacNAc\nwas synthesized at a yield of >90% via a sequential one-pot multienzyme\n(OPME) method with crude enzymes at 100 mM substrates. Finally, LacNAc\nwas produced efficiently and cost-effectively at a 5 L scale. This\nstrategy would possibly meet the requirements of potential industrial\nproduction of LacNAc and would provide guidance for the production\nof other structurally complex HMOs or functional oligosaccharides\nin the future.