Modular Systems Engineering Enables Streamlined Development of Escherichia coli MG1655 Strains for Neutral Core Human...

Area of Science:

• Microbial Engineering
• Synthetic Biology
• Glycobiology

Background:

• Human milk oligosaccharides (HMOs) are crucial bioactive components of human milk with significant health implications.
• Current methods for HMO production are often complex and costly, limiting their accessibility.
• Developing efficient microbial production systems for HMOs is a key goal in biotechnology.

Purpose of the Study:

• To establish a modular systems engineering framework in *Escherichia coli* for the efficient synthesis of neutral core HMOs.
• To demonstrate the adaptability of this framework for producing different HMO structures.
• To achieve high-titer production of specific HMOs using engineered microbial cell factories.

Main Methods:

• Developed a modular framework in *Escherichia coli* MG1655 involving optimization of glycosyltransferase expression, nucleotide sugar donor supply, and transport.
• Utilized sequential module optimization and chromosomal integration for plasmid-free and inducer-free strain construction.
• Adapted the modular logic by swapping terminal glycosyltransferases to produce different target HMOs.

Main Results:

• Engineered *E. coli* strains achieved high production titers: 81.96 g/L for lacto-N-triose II (LNTri II), 78.52 g/L for lacto-N-tetraose (LNT), and 43.17 g/L for lacto-N-neotetraose (LNnT).
• The modular strategy enabled efficient and adaptable synthesis of multiple neutral core HMOs.
• Stable, plasmid-free strains were developed for robust industrial application.

Conclusions:

• The modular systems engineering approach provides a streamlined and adaptable paradigm for developing microbial cell factories for complex oligosaccharide synthesis.
• This work facilitates the scalable production of essential HMOs, potentially impacting infant nutrition and therapeutic applications.
• The developed framework can be extended for the production of other complex glycans.