Industrial-scale production of Lacto-N-tetraose in engineered Bacillus subtilis via modular pathway optimization

Lacto-N-tetraose (LNT), an important human milk oligosaccharide with prebiotic benefits, was successfully produced de novo in Bacillus subtilis, establishing this Generally Recognized as Safe (GRAS) organism as a suitable platform for infant nutritional ingredients. A detailed enzyme screening identified three key enzymes: β-1,3-galactosyltransferase from Pseudogulbenkiania ferrooxidans, β-1,3-N-acetylglucosaminyltransferase from Neisseria polysaccharea, and β-galactoside permease (LacY) from Escherichia coli. The co-expression of these enzymes in strain BPPY enabled the first complete biosynthesis of LNT in B. subtilis, achieving a yield of 1.42 g/L in shake-flask cultures. Advanced metabolic engineering strategies, such as disrupting competing pathways, enhancing UDP-GlcNAc/Gal precursor flow, and optimizing heterologous pathways, led to the development of strain BPPY31, which produced 7.83 g/L of LNT, a 5.5-fold increase. To efficiently regulate carbon flux, a cost-effective CRISPR interference (CRISPRi) system was created to downregulate essential competing genes (zwf, pfkA, murAB). The engineered strain BD7 yielded 12.51 g/L in flask cultures and an impressive 80.48 g/L in 5-L fed-batch bioreactors, with only 4.43 g/L of the byproduct lacto-N-triose II, achieving lactose and glucose conversion rates of 92.25 % and 24 %, respectively. This study reports the highest documented LNT titer to date using a GRAS-compliant biomanufacturing platform characterized by precise metabolic regulation, scalability, and significant potential for industrial production of human milk oligosaccharides.