Modular Design of a Copper Ion Biosensor Based on the MAPK Signaling Pathway in .

Copper ion poses serious threats to both the environment and human health. To develop a yeast biosensor with reduced background noise and enhanced detection sensitivity, we constructed a quorum-sensing module with amplified positive feedback. This biosensor employs a copper ion-pheromone communication system, which allows haploid -type yeast () to express the α-pheromone gene () under the control of the copper ion-inducible promoter . The α-pheromone activates the mitogen-activated protein kinase (MAPK) signaling pathway, which in turn induces the expression of the green fluorescent protein (GFP) gene via the pheromone-inducible promoter pprm1. To improve the performance of the biosensor, we optimized the promoter and constructed the Ste5ΔN-CTM chassis. Specifically, the promoter intensity was improved by converting the three nonconsensus Pheromone Response Elements (PRE) in into consensus PRE sequences, resulting in the Pro promoter. The Ste5ΔN-CTM strain continuously activates the MAPK signaling pathway. Next, to offset the loss of sensitivity and dynamic response range caused by endogenous pheromone degradation, we knocked out the pheromone degradation gene using CRISPR-Cas9 gene editing technology. Additionally, we established a functional model relating the copper ion concentration to the GFP signal output. In conclusion, this study designed a modular copper ion biosensor system by integrating sensing, amplification, and signal-reporting components, laying a foundation for the development of biosensors for other heavy metals.