While copper is an essential micronutrient and a technologically indispensable heavy metal, it is toxic at high concentrations, harming the environment and human health. Currently, copper is monitored with costly and low-throughput analytical techniques that do not evaluate bioavailability, a crucial parameter which can be measured only with living cells. We overcame these limitations by building upon yeast S. cerevisiae's native copper response and constructed a promising next-generation eukaryotic whole-cell copper biosensor. We combined a dual-reporter fluorescent system with an engineered CUP1 promoter and overexpressed Cup2 transactivator, constructing through four iterations a total of 16 variants of the biosensor, with the best one exhibiting a linear range of 10^-8 to 10^-3 M of bioavailable copper. The engineered variant distinguishes itself through superior specificity, detection limit, and linear range, compared to other currently reported eukaryotic and prokaryotic whole-cell copper biosensors. Moreover, the variant serves as a dual-sensing reporter for Cu²⁺ detection and cell viability, disregards non-bioavailable copper and other heavy metals, is relatively independent of the cell's physiological status, and was validated on real-world samples which contained interfering substances. Finally, by re-engineering the transactivator, we altered the system's sensitivity and growth rate while assessing the performance of Cup2 with heterologous activation domains. Thus, in addition to presenting the next-generation whole-cell copper biosensor, this work urges for an iterative design of eukaryotic biosensors and paves the way toward higher sensitivity through transactivator engineering.

• PMID: 35785751
• DOI full text
• PubMed

Reference Type

Journal Article

Authors

Žunar B, Mosrin C, Bénédetti H, Vallée B

Primary Lit For

Additional Lit For

Review For