LDOB: multi-dimensional programmable lactose-derived oligosaccharide biosensors.

Area of Science:

• Biotechnology
• Microbial Engineering
• Synthetic Biology

Background:

• Human milk oligosaccharides (HMOs) possess crucial health benefits but their scalable production via microbial synthesis is hindered by a lack of efficient analytical tools.
• Strain improvement for HMOs production requires rapid and universal methods to assess biosynthesis efficiency.

Purpose of the Study:

• To develop a novel whole-cell biosensor system for real-time monitoring and high-throughput screening of HMO-producing microbial strains.
• To engineer Escherichia coli with a lactose-derived oligosaccharide biosensor (LDOB) for efficient strain selection.

Main Methods:

• Construction of genetically engineered Escherichia coli strains expressing lactose-derived oligosaccharide biosensors (LDOB).
• Integration of multidimensional negative-feedback modules (multi-repression, targeted protein degradation, oRBS) into the LDOB sensing circuit.
• Validation of LDOB signal correlation with HMO yields (2'-FL, LNnT) and coupling with fluorescence-activated droplet sorting (FADS) for high-throughput screening.

Main Results:

• LDOB systems translate lactose consumption into inversely correlated biomass and fluorescence read-outs, enabling real-time HMO synthesis monitoring.
• LDOB signals showed strong consistency with yields of key HMOs like 2'-fucosyllactose (2'-FL) and lacto-N-neotetraose (LNnT).
• High-throughput screening using LDOB and FADS resulted in significant titer enhancements: 42.8% for 2'-FL and 86.4% for LNnT.

Conclusions:

• LDOB provides a superior tool for monitoring HMO synthesis and screening high-yield producing strains, overcoming previous bottlenecks in microbial biomanufacturing.
• This engineered biosensor system significantly advances the field of HMO biomanufacturing by enabling efficient strain improvement and accelerating production processes.