Identification of a novel β-1,4-galactosyltransferase from Haemophilus parainfluenzae for efficient biosynthesis of lacto-N-neotetraose in Escherichia coli

As an important member of human milk oligosaccharides (HMOs), lacto-<i>N</i>-neotetraose (LNnT) has been recognized as a promising HMO with many physiological activities for infants. β-1,4-Galactosyltransferases play a vital role in the final step of LNnT biosynthesis in microbial cell factory. Herein, we identified a novel β-1,4-galactosyltransferase (Hpa-GalT) from <i>Haemophilus parainfluenzae</i> for the efficient biosynthesis of LNnT in <i>E. coli</i> BL21 star(DE3). After overexpression of the key genes in the synthetic pathway, the titer of LNnT reached 1.86 g/L in shake flask fermentation. Moreover, a variant A186P/E146Q of Hpa-GalT was obtained by molecular modification, resulting in an increase of the LNnT titer to 2.15 g/L. Subsequently, a series of genes related to competitive pathways were deleted, including <i>lacZ</i>, <i>ugd</i>, <i>wcaJ</i>, and <i>wecB</i>, the engineered strain produced a LNnT titer of 2.23 g/L. After optimization of LNnT transport along with LNT II utilization, the engineered strain AH31 produced 2.93 g/L of LNnT in shake flask fermentation. Furthermore, it produced a maximum titer of 32.6 g/L of total LNnT and 27.1 g/L of extracellular LNnT in a 5-L fermentor with the productivity for 0.52 g/L/h. Overall, this study provides a novel β-1,4-galactosyltransferase and lays a foundation for the efficient biosynthesis of LNnT.