Enzyme

Bacteria

Biology

Microbiology

Antibody

Chemistry

Immunology

Lactose

Bacillus subtilis

Food science

Titer

Genetics

Biochemistry

Biotechnology for Biofuels

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 .

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