Naslov
Re-engineering of CUP1 promoter and Cup2/Ace1 transactivator to convert Saccharomyces cerevisiae into a whole-cell eukaryotic biosensor capable of detecting 10 nM of bioavailable copper

Autori
Žunar, Bojan ; Mosrin, Christine ; Bénédetti, Héléne ; Vallée, Béatrice

Izvornik
Biosensors and Bioelectronics (0956-5663) 214 (2022); 114502, 10

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
whole-cell biosensor, copper biosensor, bioavailability, Saccharomyces cerevisiae, transactivator engineering

Sažetak
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.

Izvorni jezik
Engleski

Znanstvena područja
Biotehnologija

POVEZANOST RADA