A curcumin direct protein (DiPro) biosensor for cell-free prototyping

In synthetic biology, biosensors are routinely coupled to a gene expression cascade for detecting small molecules and physical signals. We posit that an alternative direct protein (DiPro) biosensor mechanism, could provide a new opportunity for rapid detection of specific chemicals. Herein, we reveal a fluorescent curcumin DiPro biosensor, based on the Escherichia coli double bond reductase ( Ec CurA) as a detection system. We characterise the Ec CurA DiPro biosensor and propose enhanced curcumin fluorescence is generated through π-π stacking between protein and ligand. Using a cell-free synthetic biology approach, we use the Ec CurA DiPro biosensor to fine tune 10 reaction parameters (cofactor, substrate, and enzyme levels) for cell-free biosynthesis, assisted through acoustic liquid handling robotics. Overall, we increase Ec CurA-curcumin fluorescence by 80-fold. We speculate that a generic DiPro biosensor fluorescence mechanism can be further exploited for a wider range of chemicals that share intrinsic fluorescence and have a suitable binding protein.