Combinatorial metabolic engineering and whole-cell biocatalysis enable high-level lacto-N-neotetraose production in Corynebacterium glutamicum

Abstract

Lacto-N-neotetraose (LNnT), a functional oligosaccharide abundant in human milk, holds significant nutritional and biomedical value. This study engineered the industrial workhorse Corynebacterium glutamicum ATCC13032, which lacks native LNnT biosynthesis capability, through systematic metabolic engineering. Initial strain CL001 was constructed by heterologously expressing LgtA and LgtB from Neisseria meningitidis and LacY from Escherichia coli, achieving 0.084 g/L LNnT. Subsequent introduction of E. coli galactokinase GalK (strain CL002) enhanced production to 0.286 g/L. Metabolic flux optimization through zwf gene knockout to suppress the pentose phosphate pathway (strain CL013) further increased titers to 0.477 g/L. Implementation of an ABC transporter system (strain CL014) elevated LNnT production to 1.05 g/L. Whole-cell catalysis of CL014 for LNnT production was optimized after 54 h cultivation at 37 °C. Substrate optimization established ideal concentrations at 60 mM lactose, 60 mM galactose, and 240 mM glucose. Under these conditions, CL014 achieved 9.12 g/L LNnT production, representing a 108-fold improvement over the initial engineered strain. This work demonstrates the potential of engineered C. glutamicum as an efficient platform for human milk oligosaccharide biosynthesis.

Keywords

Corynebacterium glutamicum / Human milk oligosaccharide / LNnT / zwf / ABC transporter

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Funding

Government of Jiangsu Province(No. BK20233003)

RIGHTS & PERMISSIONS

Jiangnan University