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High-Level Difucosyllactose Biosynthesis via Metabolic and Transporter Engineering.

Lizhi Zhu1,2, Caiwen Lao3, Lixia Yuan1,2

  • 1University of Science and Technology of China, Hefei 230026, China.

Journal of Agricultural and Food Chemistry
|April 15, 2026
PubMed
Summary
This summary is machine-generated.

This study engineered *Escherichia coli* to produce difucosyllactose (DFL), a prebiotic oligosaccharide. A record titer of 110.3 g/L was achieved through metabolic and transporter engineering, demonstrating a robust platform for industrial production.

Keywords:
Escherichia colidifucosyllactosehuman milk oligosaccharidemetabolic engineering

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Area of Science:

  • Metabolic Engineering
  • Biotechnology
  • Microbial Fermentation

Background:

  • Difucosyllactose (DFL) is a complex human milk oligosaccharide with significant prebiotic potential.
  • Efficient biosynthesis of DFL is crucial for its industrial application.
  • Existing methods for DFL production are limited in titer and efficiency.

Purpose of the Study:

  • To engineer *Escherichia coli* for high-titer biosynthesis of DFL.
  • To optimize the metabolic pathways and transporter systems for DFL production.
  • To establish a robust platform for industrial-scale DFL manufacturing.

Main Methods:

  • Engineered *Escherichia coli* using glycosyltransferases (WbgL and FutM1) for DFL synthesis.
  • Optimized enzyme catalytic efficiency and gene copy numbers.
  • Disrupted competing pathways (*wcaI*, *wcaH*, *nudK*) and deleted *pykA* to enhance precursor availability.
  • Engineered transporters by deleting *emrE* and overexpressing *ompF*.
  • Implemented continuous low-concentration lactose feeding in a 5-L bioreactor.

Main Results:

  • Achieved a record-breaking DFL titer of 110.3 g/L.
  • Identified optimal enzyme pair (WbgL and FutM1) and maximized their catalytic efficiency.
  • Successfully redirected carbon flux and enhanced precursor availability.
  • Improved synthesis efficiency through targeted transporter engineering.
  • Demonstrated high-level DFL production under optimized fermentation conditions.

Conclusions:

  • Systematic metabolic engineering and refined process control provide a robust platform for industrial production of complex oligosaccharides like DFL.
  • The engineered *E. coli* strain and fermentation strategy represent a significant advancement in DFL manufacturing.
  • This study paves the way for large-scale, cost-effective production of DFL for prebiotic applications.