Pathway Optimization and Uridine 5'-Triphosphate Regeneration for Enhancing Lacto- N -Tetraose Biosynthesis in Engineered Escherichia coli .

Recently, human milk oligosaccharides (HMOs) have attracted increasing attention and display great commercial importance, especially for the infant formula industry. Lacto- N -tetraose (LNT) is an important neutral HMO commercially added in infant formula and a core structure for synthesizing complex HMOs. Previously, a novel LNT-generating β-1,3-galactosyltransferase from Pseudogulbenkiania ferrooxidans was identified and used for construction of an LNT-producing engineered Escherichia coli . In this work, LNT biosynthesis was further enhanced by pathway optimization and uridine 5'-triphosphate (UTP) regeneration. The main strategies included genomic integration of UDP-glucose 4-epimerase-encoding gene, fine-tuning of the LNT pathway-related genes, blocking of competitive pathways related to UDP-galactose, and overexpression of UTP supply related genes. The maximal LNT titer reached 6.16 and 57.5 g/L by shake-flask and fed-batch fermentation, respectively.