Metabolic engineering of Escherichia coli for the production of Lacto- N -neotetraose (LNnT)

Lacto-N-neotetraose (LNnT), one of the most important human milk oligosaccharides, can be used as infants’ food additives. Nowadays, extraction, chemical and biological synthesis were utilized to obtain LNnT, while these methods still face some problems such as low yield and high cost. The aim of current work is to construct a de novo biosynthesis pathway of LNnT in E. coli K12 MG1655. The lgtA and lgtB were first expressed by a plasmid, resulting in a LNnT titer of 0.04 g/L. To improve the yield of LNnT on substrate lactose, lacZ and lacI were knocked out, and lacY was over-expressed. As a result, the yield of LNnT on lactose increased from 0.01 to 0.09 mol/mol, and the titer of LNnT elevated to 0.41 g/L. In addition, the pathway was regulated using the titer of Lacto-N-triose II (LNTII) as a measure, and obtained a high titer strain of LNnT for 1.04 g/L. Finally, the gene expressions were fine-tuned, the titer of LNnT reached 1.2 g/L, which was 93% higher than the control strain, and the yield on lactose reached 0.28 mol/mol. The engineering strategy of pathway construction and modulation used in this study is applicable to facilitate the microbial production of other metabolites in E. coli.