Disentangling Metabolite Detection from Pathway Stress Using Biosensors.

Area of Science:

• Synthetic Biology
• Metabolic Engineering
• Biotechnology

Background:

• Genetically encoded biosensors link metabolite production to fluorescent outputs.
• Metabolite production pathways can impose stress, confounding biosensor signals.
• Protein tyrosine phosphatase 1B (PTP1B) is a therapeutic target for cancer and diabetes.

Purpose of the Study:

• Develop and compare fluorescence-based biosensors for PTP1B inhibitors.
• Assess biosensor performance in the presence of heterologous terpenoid pathways.
• Identify a biosensor that functions reliably despite pathway-induced stress.

Main Methods:

• Engineered four fluorescence-based biosensors for PTP1B inhibitors in *Escherichia coli*.
• Tested biosensor functionality with and without co-expressed terpenoid pathways.
• Evaluated biosensor performance based on dynamic range, toxicity, and stress tolerance.

Main Results:

• All four biosensors detected exogenous PTP1B inhibitors.
• Only one biosensor remained functional alongside terpenoid pathways, with reduced fluorescence due to stress.
• This sensor, utilizing split T7 RNA polymerase, showed high dynamic range and low toxicity.
• Sensor variants enabled differentiation between terpenoid pathways producing PTP1B inhibitors and those that did not.

Conclusions:

• Pathway-specific stresses significantly alter biosensor signals, comparable to target analyte effects.
• A robust biosensor framework was developed to disentangle analyte signals from pathway-induced stress.
• This work provides a method for identifying PTP1B inhibitors using biosensors in high-throughput screens.