Bacteria equipped with genetically encoded lactate biosensors are promising tools for biopharmaceutical production, diagnostics, and cellular therapies. However, many applications involve glucose-rich and anoxic environments, in which current whole-cell lactate biosensors show low performance. Here we engineer an optimized, synthetic lactate biosensor system by repurposing the natural LldPRD promoter regulated by the LldR transcriptional regulator. We removed glucose catabolite and anoxic repression by designing a hybrid promoter, containing LldR operators and tuned both regulator and reporter gene expressions to optimize biosensor signal-to-noise ratio. The resulting lactate biosensor, termed ALPaGA (A Lactate Promoter Operating in Glucose and Anoxia), can operate in glucose-rich, aerobic and anoxic conditions. We show that ALPaGA works reliably in the probiotic chassis Escherichia coli Nissle 1917 and can detect endogenous l -lactate produced by 3D tumor spheroids with an improved dynamic range. In the future, the ALPaGA system could be used to monitor bioproduction processes and improve the specificity of engineered bacterial cancer therapies by restricting their activity to the lactate-rich microenvironment of solid tumors.
Bacteria equipped with genetically-encoded lactate biosensors would support several applications in biopharmaceutical production, diagnosis, or therapeutics. However, many applications involve glucose-rich and anaerobic environments, in which current whole-cell lactate biosensors have low performance. Here we engineered a synthetic lactate biosensor system by repurposing the natural LldPRD promoter regulated by the LldR transcriptional regulator. We removed glucose catabolite repression by designing a hybrid promoter, containing LldR operators and tuned both regulator and reporter gene expression to optimize biosensor signal-to-noise ratio. The resulting lactate biosensor, termed ALPaGA (ALactate Promoter Operating in Glucose and Anaerobia) can operate in glucose rich, aerobic and anaerobic conditions. Our work provides a versatile lactate biosensing platform suitable for many environmental conditions.
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