Efficient and Cost-Effective Synthesis of N-Acetyllactosamine by Sequential Modular Enzymatic Cascade Reactions Involving NTP Regeneration

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