Modular pathway engineering of key precursor supply pathways for lacto-N-neotetraose production in Bacillus subtilis

BACKGROUND: Lacto-N-neotetraose (LNnT) is one of the important ingredients of human milk oligosaccharides, which can enhance immunity, regulate intestinal bacteria and promote cell maturation. RESULTS: In this study, the synthetic pathway of LNnT was constructed by co-expressing the lactose permease (LacY) β-1,3-N-acetylglucosaminyltransferase (LgtA) and β-1,4-galactostltransferase (LgtB) in Bacillus subtilis, resulting in an LNnT titer of 0.61 g/L. Then, by fine-tuning the expression level of LgtB, the growth inhibition was reduced and the LNnT titer was increased to 1.31 g/L. In addition, by modular pathway engineering, the positive-acting enzymes of the UDP-GlcNAc and UDP-Gal pathways were strengthened to balance the two key precursors supply, and the LNnT titer was improved to 1.95 g/L. Finally, the LNnT titer reached 4.52 g/L in a 3-L bioreactor with an optimal glucose and lactose feeding strategy. CONCLUSIONS: In general, this study showed that the LNnT biosynthesis could be significantly increased by optimizing enzymes expression levels and modular pathway engineering for balancing the precursors supply in B. subtilis.