Engineering β-1,4-galactosyltransferase to Enhance Lacto-N-neotetraose Production in Corynebacterium glutamicum

Lacto- N -neotetraose (LNnT), a pivotal oligosaccharide in human milk, play a significant role in modulating the intestinal microbiota of infants, and exhibit potential applications in antiviral therapy. Corynebacterium glutamicum is a widely used model microorganism in industrial fermentation; however, it is incapable of synthesizing LNnT under natural conditions. Previously, we constructed a C. glutamicum strain CL014 for LNnT production, but the yield was limited. In this study, the key enzyme β-1,4-galactosyltransferase LgtB in CL014 was engineered to improve LNnT biosynthesis. 14 mutant strains were constructed by replacing the critical residual Arg ²⁴ of LgtB to different amino acids and screened for the optimal LNnT production, and the highest LNnT production was obtained in the mutant CL014-T. CL014-T could produce 1.40 g/L LNnT in shake flask cultivation, which is 33.3% increase comparing to the control strain CL014. Molecular docking simulations demonstrated that the mutation promotes the formation of additional hydrogen bonds in the flexible loop region, enhancing conformational flexibility and optimizing substrate interactions. Further mechanistic insights were obtained through molecular dynamics simulations, which uncovered that increased dynamic flexibility in two functional regions and the formation of more core hydrogen bonds collectively contribute to enhanced binding stability and improved product yield. After 96 h feed-batch fermentation, CL014-T produced 2.29 g/L LNnT. This work demonstrates that C. glutamicum has the potential to be engineered as an efficient cell factory for human milk oligosaccharide production.