Abstract
Lacto-N-neotetraose (LNnT) is a critical component of human milk oligosaccharides. This study introduces a systems metabolic engineering method to produce LNnT in Escherichia coli. First, 12 target genes contributing to LNnT production were identified using a double-plasmid system. Subsequently, combinatorial optimization of the copy number was performed to tune the target gene expression strength. Next, the CRISPR/Cas9 system was used to block the UDP-Gal and UDP-GlcNAc competitive pathways, and the titer of LNnT reached 1.16 g/L (E27). Moreover, the lactoylglutathione lyase (GloA) was deleted to block the competing metabolite pathway from glycerol to lactate, and the titer of LNnT (1.46 g/L) was 26% higher than that of strain E27. Finally, the LNnT productivity was increased to 0.34 g/L/h in a 3 L bioreactor, which was 36% higher than the recently reported LNnT productivity. This research work opens an innovative framework for the effective production of LNnT.
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