Biosynthesis of Sialyllacto-N-tetraose c in Engineered Escherichia coli

Area of Science:

• Microbiology
• Metabolic Engineering
• Biochemistry

Background:

• Human milk oligosaccharides (HMOs) are crucial for infant health.
• Sialyllacto-N-tetraose c (LST c) is a complex HMO with significant developmental and application importance.
• Efficient biosynthesis of LST c is challenging.

Purpose of the Study:

• To develop a microbial cell factory for efficient LST c production.
• To optimize metabolic pathways for LST c biosynthesis using multivariate modular metabolic engineering (MMME).
• To enhance precursor supply and cofactor regeneration for increased LST c yield.

Main Methods:

• Utilized multivariate modular metabolic engineering (MMME) to screen sialyltransferases and balance metabolic fluxes.
• Engineered an Escherichia coli strain (W15) by blocking competing pathways and enhancing precursor (UDP-GlcNAc, UDP-Gal) supply.
• Established a CTP cofactor regeneration system.

Main Results:

• Constructed engineered strains capable of LST c biosynthesis.
• Achieved an LST c titer of 220.9 mg/L in shake flask cultures with strain W15.
• Reached 922.2 mg/L LST c in a 3-L fed-batch fermentation, with 10.25 mg/L/h productivity and 38.70 mg/g DCW specific yield.

Conclusions:

• MMME is an effective strategy for developing microbial cell factories for complex HMOs like LST c.
• The engineered E. coli strain W15 demonstrates high-level LST c production.
• This research provides a viable method for the industrial production of LST c.