Metabolic Engineering of Escherichia coil for Efficient Biosynthesis of Lacto-N-tetraose Using a Novel β-1,3-Galactosyltransferase from Pseudogulbenkiania ferrooxidans

Human milk oligosaccharides (HMOs) attract considerable interest in recent years because of their particular role in infant health. Lacto-N-tetraose (LNT), one of the most abundant HMOs, has been commercially added in the infant formula as a functional fortifier. In this study, a novel LNT-producing beta-1,3-galactosyltransferase (beta-1,3-GalT) from Pseudogulbenkiania ferrooxidans was screened from 14 putative candidates, and a highly LNT-producing metabolically engineered Escherichia coli strain was constructed based on a previously constructed lacto-N-triose II (LNT II)-producing strain, by strengthening UDP-galactose synthesis and introduction of P. ferrooxidans beta-1,3-GalT. The engineered strain produced 3.11 and 25.49 g/L LNT in shake-flask and fed-batch cultivation, with the molar conversion ratio of LNT II to LNT of 88.15 and 85.09%, respectively. The productivity and specific yield of LNT in fed-batch cultivation were measured to be 0.61 g/L.h and 0.76 g/g dry cell weight, respectively. To the best of our knowledge, it is the highest LNT yield ever reported.

Automated summary

This study identified a novel LNT-producing beta-1,3-galactosyltransferase from a bacterium and engineered a high LNT-producing E. coli strain. The engineered strain achieved the highest reported LNT yield through shake-flask and fed-batch cultivation, with enhanced productivity and specific yield of LNT.