NADH-dependent biosensor in Saccharomyces cerevisiae: principle and validation at the single cell level.
A reporter system was constructed to measure perturbations in the NADH/NAD(+) co-factor balance in yeast, by using the green fluorescent protein gene under the control of the GPD2 promoter that is induced under conditions of excess of NADH. High fluorescence levels were obtained in a glycerol 3-phosphate dehydrogenase double deletion strain (gpd1Δgpd2Δ), which is deficient in the ability to regenerate NAD(+) via glycerol formation. The responsiveness of the reporter system to externally induced perturbations in NADH oxidation was also evaluated in the gpd1Δgpd2Δ strain background by addition of acetoin, as well as by introduction of a set of heterologous xylose reductases (XRs) having different selectivities for NADH. Addition of acetoin during cell proliferation under oxygen-limited conditions resulted in a more than 2-fold decrease in mean fluorescence intensity as compared to the control experiment. Strains carrying XRs with different selectivities for NADH could be distinguished at the single cell level, so that the XR with the highest selectivity for NADH displayed the lowest fluorescence. In conclusion, the designed system successfully allowed for monitoring perturbations in the cellular redox metabolism caused by environmental changes, or by heterologous gene expression. The reporter system displayed high resolution in distinguishing cytosolic NADH oxidation capacity and hence has potential to be used for high-throughput screening based on the fluorescence of single cells.
Division of Applied Microbiology, Department of Chemistry, Lund University, Lund, 221 00, SE, Sweden.
Jan Dines
JD
Knudsen
Division of Applied Microbiology, Department of Chemistry, Lund University, Lund, 221 00, SE, Sweden.
Magnus
M
Carlquist
Division of Applied Microbiology, Department of Chemistry, Lund University, Lund, 221 00, SE, Sweden.
Marie
M
Gorwa-Grauslund
NADH biosensor; Redox balance; Saccharomyces cerevisiae; Single cell analysis