Pathway Optimization and Uridine 5′-Triphosphate Regeneration for Enhancing Lacto-<i>N</i>-Tetraose Biosynthesis in Engineered <i>Escherichia coli</i>
Zeyu Li, Yingying Zhu, Pan Zhang, Wenli Zhang, Wanmeng Mu
Abstract
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.