Development of surface-engineered yeast cells displaying phytochelatin synthase and their application to cadmium biosensors by the combined use of pyrene-excimer fluorescence.
The development of simple, portable, inexpensive, and rapid analytical methods for detecting and monitoring toxic heavy metals are important for the safety and security of humans and their environment. Herein, we describe the application of phytochelatin (PC) synthase, which plays a critical role in heavy metal responses in higher plants and green algae, in a novel fluorescent sensing platform for cadmium (Cd). We first created surface-engineered yeast cells on which the PC synthase from Arabidopsis (AtPCS1) was displayed with retention of enzymatic activity. The general concept for the sensor is based on the Cd level-dependent synthesis of PC2 from glutathiones by AtPCS1-displaying yeast cells, followed by simple discriminative detection of PC2 via sensing of excimer fluorescence of thiol-labeling pyrene probes. The intensity of excimer fluorescence increased in the presence of Cd up to 1.0 μM in an approximately dose-dependent manner. This novel biosensor achieved a detection limit of as low as 0.2 μM (22.5 μg/L) for Cd. Although its use may be limited by the fact that Cu and Pb can induce cross-reaction, the proposed simple biosensor holds promise as a method useful for cost-effective screening of Cd contamination in environmental and food samples. The AtPCS1-displaying yeast cells also might be attractive tools for dissection of the catalytic mechanisms of PCS.
Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, 565-0871, Japan.
Hideyuki
H
Matsuura
Yosuke
Y
Yamamoto
Misa
M
Muraoka
Kenji
K
Akaishi
Yasuhisa
Y
Hori
Kanoko
K
Uemura
Naoki
N
Tsuji
Kazuo
K
Harada
Kazumasa
K
Hirata
Takeshi
T
Bamba
Hitoshi
H
Miyasaka
Kouichi
K
Kuroda
Mitsuyoshi
M
Ueda
D019881:Aminoacyltransferases / Q000737:chemistry* / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000378:metabolism; D015374:Biosensing Techniques; D002104:Cadmium / Q000737:chemistry; D003001:Cloning, Molecular; D005453:Fluorescence; D005818:Genetic Engineering / Q000379:methods; D005978:Glutathione; D007218:Industrial Microbiology; D011721:Pyrenes / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
D029681:Arabidopsis Proteins; D011721:Pyrenes; D002104:Cadmium; C030984:pyrene; D019881:Aminoacyltransferases; C502365:AT5G44070 protein, Arabidopsis; C093784:glutathione gamma-glutamylcysteinyltransferase; D005978:Glutathione
Saccharomyces cerevisiae; cadmium biosensor; cell surface display; phytochelatin synthase; pyrene excimer