A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria

Abstract

When fed to a β-galactosidase-negative (lacZ-) Escherichia coli strain that was grown on an alternative carbon source (such as glycerol), lactose accumulated intracellularly on induction of the lactose permease. We showed that intracellular lactose was efficiently glycosylated when genes of glycosyltransferase that use lactose as acceptor were expressed. High-cell-density cultivation of lacZ- strains that overexpressed the β1,3 N acetyl glucosaminyl-transferase lgtA gene of Neisseria meningitidis resulted in the synthesis of 6 g · L-1 of the expected trisaccharide (GlcNAcβ1-3Galβ1-4Glc). When the β1,4 galactosyl-transferase lgtB gene of N. meningitidis was coexpressed with lgtA, the trisaccharide was further converted to lacto-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc) and lacto-N-neoheaxose with a yield higher than 5 g · L-1. In a similar way, the nanA- E. coli strain that was devoid of NeuAc aldolase activity accumulated NeuAc on induction of the NanT permease and the lacZ-nanA- strain that overexpressed the N. meningitidis genes of the α2,3 sialyltransferase and of the CMP-NeuAc synthase efficiently produced sialyllactose (NeuAcα2-3Galβ1-4Glc) from exogenous NeuAc and lactose.