Multienzyme Platform for the Synthesis of UDP Sugars and Human Milk Oligosaccharides

Naturally occurring in breast milk, human milk oligosaccharides (HMOs) are of great interest as an ingredient for infant nutrition due to numerous associated health benefits. Current commercial production relies mainly on microbial fermentation, while enzymatic synthesis is used to produce milligram scales for scientific studies. Enzymatic synthesis using glycosyltransferases and nucleotide sugars is especially promising due to high reaction yields, but is limited by low activity of glycosyltransferases and the high cost of nucleotide sugars. This study presents a novel approach that uses a single engineered E. coli BL21(DE3) strain to simultaneously express six recombinant enzymes (UMPK, PPK3, GALK, NAHK, GALU, and PPA). This enables dual-nucleotide sugar synthesis through two integrated multienzyme cascades. The system uses cost-effective substrates, including uridine 5'-monophosphate (UMP), N-acetylglucosamine (GlcNAc), galactose (Gal), and ATP. In situ ATP regeneration is achieved through polyphosphate (PolyP_n) breakdown. Comparative studies of three different expression strain configurations demonstrated that crude cell lysate could serve as an effective biocatalyst. This eliminates the need for expensive enzyme purification while maintaining high catalytic activity. Using crude cell lysate, conversion yields approaching 100% were obtained. Both UDP-GlcNAc and UDP-Gal were successfully purified using anion-exchange chromatography. Based on the UV spectrum, purities of 85-99% and recovery yields exceeding 90%, respectively, were achieved. The practical application of this system was demonstrated by the successful synthesis of two HMOs: Lacto-N-triose II (LNTII) and lacto-N-neotetraose (LNnT), which demonstrates an effective nucleotide sugar recycling in coupled enzymatic reactions and paves the way toward larger scale production.