Engineering Escherichia coli for highly efficient production of lacto-N-triose II from N-acetylglucosamine, the monomer of chitin

Background: Lacto-N-triose II (LNT II), an important backbone for the synthesis of different human milk oligosaccharides, such as lacto-N-neotetraose and lacto-N-tetraose, has recently received significant attention. The production of LNT II from renewable carbon sources has attracted worldwide attention from the perspective of sustainable development and green environmental protection. Results: In this study, we first constructed an engineered E. coli cell factory for producing LNT II from N-acetylglucosamine (GlcNAc) feedstock, a monomer of chitin, by introducing heterologous beta-1,3-acetylglucosaminyltransferase, resulting in a LNT II titer of 0.12 g L-1. Then, lacZ (lactose hydrolysis) and nanE (GlcNAc-6-P epimerization to ManNAc-6-P) were inactivated to further strengthen the synthesis of LNT II, and the titer of LNT II was increased to 0.41 g L-1. To increase the supply of UDP-GlcNAc, a precursor of LNT II, related pathway enzymes including GlcNAc-6-P deacetylase, glucosamine synthase, and UDP-N-acetylglucosamine pyrophosphorylase, were overexpressed in combination, optimized, and modulated. Finally, a maximum titer of 15.8 g L-1 of LNT II was obtained in a 3-L bioreactor with optimal enzyme expression levels and beta-lactose and GlcNAc feeding strategy. Conclusions: Metabolic engineering of E. coli is an effective strategy for LNT II production from GlcNAc feedstock. The titer of LNT II could be significantly increased by modulating the gene expression strength and blocking the bypass pathway, providing a new utilization for GlcNAc to produce high value-added products.

Automated summary

This study successfully engineered an E. coli cell factory to produce LNT II from GlcNAc and significantly increased the titer of LNT II through various strategies, providing a new direction for producing high value-added products from GlcNAc feedstock.