Transcriptome-assisted sugar transporter screening for enhanced lacto-N-tetraose biosynthesis in Escherichia coli MG1655.

The biocatalytic upgrading of renewable substrates to human milk oligosaccharides (HMOs) via cell factory offers a green and safety synthesis pathway toward value-added chemicals. The manipulation of cell factory and transporter is the key to realize the green synthesis of Lacto-N-tetraose (LNT), a neutral non-fucosylated oligosaccharide in breast milk. In current study, we present a plasmid-free approach for the de novo synthesis of LNT with increased sugar transport efficiency. A total 3 transporter genes including tauB, nikD, and livM were screened based on the transcriptome analysis, and approved to be capacity of enhancing the LNT production. Through multidimensional metabolic engineering approaches, including overexpressing the rate-limiting glycosyltransferases, manipulating sugar transport efficiency, and increasing the supply of UDP-galactose-N-acetyl-glucosamine and UDP-galactose, LNT production was enhanced more than 1.0-fold in contrast to the control strain. The optimized strain MG3WB produced LNT with titers reaching 6.13 g/L in shake flask and 40.35 g/L in a 5 L bioreactor without the use of any antibiotics or plasmids. More importantly, the development of a sugar transport-based cell factory will advance biosynthesis of LNT and other HMO products.