Engineered l-Lactate Responding Promoter System Operating in Glucose-Rich and Anoxic Environments

Area of Science:

• Synthetic biology
• Microbial engineering
• Biosensor development

Background:

• Genetically encoded lactate biosensors are valuable for biopharmaceutical production, diagnostics, and cellular therapies.
• Existing biosensors exhibit poor performance in glucose-rich, anoxic environments, limiting their applications.
• Optimizing biosensor function in these challenging conditions is crucial for expanding their utility.

Purpose of the Study:

• To engineer an optimized, synthetic lactate biosensor system capable of operating in glucose-rich and anoxic conditions.
• To enhance the signal-to-noise ratio and dynamic range of whole-cell lactate biosensors.
• To develop a versatile biosensor for applications in bioproduction and advanced cellular therapies.

Main Methods:

• Repurposing the natural LldPRD promoter regulated by the LldR transcriptional regulator.
• Designing a hybrid promoter to remove glucose catabolite and anoxic repression.
• Tuning regulator and reporter gene expressions to optimize biosensor performance.
• Testing the biosensor in *Escherichia coli* Nissle 1917 and 3D tumor spheroids.

Main Results:

• Developed ALPaGA (A Lactate Promoter Operating in Glucose and Anoxia), a novel synthetic lactate biosensor.
• ALPaGA functions effectively in glucose-rich, aerobic, and anoxic environments.
• Demonstrated reliable performance in *Escherichia coli* Nissle 1917.
• Successfully detected endogenous l-lactate produced by 3D tumor spheroids with an improved dynamic range.

Conclusions:

• The ALPaGA system offers a robust solution for lactate detection in challenging environments.
• This engineered biosensor has potential applications in monitoring bioproduction processes.
• ALPaGA can enhance the specificity of engineered bacterial cancer therapies by targeting lactate-rich tumor microenvironments.