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NADH-dependent biosensor in Saccharomyces cerevisiae: principle and validation at the single cell level.

Knudsen JD, Carlquist M, Gorwa-Grauslund M - AMB Express (2014)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Applied Microbiology, Department of Chemistry, Lund University, Lund, 221 00, SE, Sweden.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Mean fluorescence intensities (FI) for the different strains (wild type,gpd1Δ, gpd2Δ and gpd1Δgpd2Δ) carrying either the multicopy (YEp)- or the integrative (YIp)- based GFP reporter. Fluorescence intensities are given in linear values. All values are normalized to the control strain TMB4120 carrying YEp-based GFP reporter. Acetoin was added at a concentration of 0.5 g/L.
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Figure 1: Mean fluorescence intensities (FI) for the different strains (wild type,gpd1Δ, gpd2Δ and gpd1Δgpd2Δ) carrying either the multicopy (YEp)- or the integrative (YIp)- based GFP reporter. Fluorescence intensities are given in linear values. All values are normalized to the control strain TMB4120 carrying YEp-based GFP reporter. Acetoin was added at a concentration of 0.5 g/L.

Mentions: Strains were cultivated in batch mode under oxygen-limited conditions for 5 hours in defined mineral medium with glucose as sole carbon source, after which the FI was measured (Figure 1). At this time point, cells were in exponential growth phase and could be considered as being in pseudo-steady state with regards to regulation of the glucose catabolic pathways. All redox engineered strain had higher FI than the control strain TMB4120. However the double deletion strain TMB4123 (gpd1Δ gpd2Δ) had the highest FI (21.5 fold higher than the control), whereas each single deletion had moderate increase in FI (1.5 and 1.2 fold for gpd1Δ and gpd2Δ, respectively) (Figure 1). To further evaluate the biosensor responsiveness to perturbations in NADH/NAD+ ratio, the FI of TMB4123 (gpd1Δ gpd2Δ) was measured after addition of acetoin (3-hydroxy-2-butanone) during respiro-fermentative growth under oxygen-limited conditions. Acetoin is an alternative redox sink to the glycerol shunt/pathway through its NADH-dependent conversion to 2,3-butanediol (Bruinenberg et al. [1983]; Björkqvist et al. [1997]). The reaction is catalyzed by a NADH-dependent 2,3-butanediol dehydrogenase encoded by BDH1 (Gonzalez et al. [2000]). Indeed the addition of acetoin led to reduced FI as the relative fluorescence decreased from 21.5 to 8.0 (Figure 1). Altogether our results demonstrated that the constructed biosensor reported on the cells capacity to oxidize NADH, and more generally on the NAD(H) cofactor balance.


NADH-dependent biosensor in Saccharomyces cerevisiae: principle and validation at the single cell level.

Knudsen JD, Carlquist M, Gorwa-Grauslund M - AMB Express (2014)

Mean fluorescence intensities (FI) for the different strains (wild type,gpd1Δ, gpd2Δ and gpd1Δgpd2Δ) carrying either the multicopy (YEp)- or the integrative (YIp)- based GFP reporter. Fluorescence intensities are given in linear values. All values are normalized to the control strain TMB4120 carrying YEp-based GFP reporter. Acetoin was added at a concentration of 0.5 g/L.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230897&req=5

Figure 1: Mean fluorescence intensities (FI) for the different strains (wild type,gpd1Δ, gpd2Δ and gpd1Δgpd2Δ) carrying either the multicopy (YEp)- or the integrative (YIp)- based GFP reporter. Fluorescence intensities are given in linear values. All values are normalized to the control strain TMB4120 carrying YEp-based GFP reporter. Acetoin was added at a concentration of 0.5 g/L.
Mentions: Strains were cultivated in batch mode under oxygen-limited conditions for 5 hours in defined mineral medium with glucose as sole carbon source, after which the FI was measured (Figure 1). At this time point, cells were in exponential growth phase and could be considered as being in pseudo-steady state with regards to regulation of the glucose catabolic pathways. All redox engineered strain had higher FI than the control strain TMB4120. However the double deletion strain TMB4123 (gpd1Δ gpd2Δ) had the highest FI (21.5 fold higher than the control), whereas each single deletion had moderate increase in FI (1.5 and 1.2 fold for gpd1Δ and gpd2Δ, respectively) (Figure 1). To further evaluate the biosensor responsiveness to perturbations in NADH/NAD+ ratio, the FI of TMB4123 (gpd1Δ gpd2Δ) was measured after addition of acetoin (3-hydroxy-2-butanone) during respiro-fermentative growth under oxygen-limited conditions. Acetoin is an alternative redox sink to the glycerol shunt/pathway through its NADH-dependent conversion to 2,3-butanediol (Bruinenberg et al. [1983]; Björkqvist et al. [1997]). The reaction is catalyzed by a NADH-dependent 2,3-butanediol dehydrogenase encoded by BDH1 (Gonzalez et al. [2000]). Indeed the addition of acetoin led to reduced FI as the relative fluorescence decreased from 21.5 to 8.0 (Figure 1). Altogether our results demonstrated that the constructed biosensor reported on the cells capacity to oxidize NADH, and more generally on the NAD(H) cofactor balance.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Applied Microbiology, Department of Chemistry, Lund University, Lund, 221 00, SE, Sweden.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus