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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

Extracellular xylitol (A, B) and glycerol (C, D) concentrations (in g/L) over time for the control background (A, C) or thegpd1Δgpd2Δbackground (B, D) carrying no XR, wtXR or mutXR. Legend: TMB4140 (control, no XR “red dot”), TMB4141 (control, wtXR “blue x”), TMB4143 (control, mutXR(green square)), TMB4144 (gpd1Δgpd2Δ, no XR “red plus”), TMB4145 (gpd1Δgpd2Δ, wtXR “blue o”), TMB4147 (gpd1Δgpd2Δ, mutXR “green asterisk”).
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Figure 4: Extracellular xylitol (A, B) and glycerol (C, D) concentrations (in g/L) over time for the control background (A, C) or thegpd1Δgpd2Δbackground (B, D) carrying no XR, wtXR or mutXR. Legend: TMB4140 (control, no XR “red dot”), TMB4141 (control, wtXR “blue x”), TMB4143 (control, mutXR(green square)), TMB4144 (gpd1Δgpd2Δ, no XR “red plus”), TMB4145 (gpd1Δgpd2Δ, wtXR “blue o”), TMB4147 (gpd1Δgpd2Δ, mutXR “green asterisk”).

Mentions: Strains were cultivated under oxygen-limited conditions in sealed vials containing minimal medium with galactose as the carbon source and xylose as the substrate for the XR-catalysed reduction. Galactose was used because S. cerevisiae has a specific galactose uptake system, Gal2p (Cirillo [1968]) that also transports xylose, and whose expression benefits xylose utilization (Johnston [1987]; Hamacher et al. [2002]). So, even though the yeast does not metabolize galactose as fast as glucose, the use of galactose allows the cell to have co-consumption of hexose and xylose (Bro et al. [2005]; Garcia Sanchez et al. [2010]). The growth profiles of the different strains are shown in Figures 3A and B. During the first 24 hours, gpd1Δ gpd2Δ strains reached an OD of around 3, which was only slightly lower than the corresponding control strains. During the next 48 hours, however, TMB4147 strain (gpd1Δ gpd2Δ, mutXR) continued growing and reached an OD of 5.2 whereas strain TMB4145 (gpd1Δ gpd2Δ, wtXR) only reached OD 3.7 while the OD of strain TMB4144 (gpd1Δ gpd2Δ, no XR) decreased to 2 (Figure 3B). Also TMB4147 strain carrying the XR with the highest selectivity for NADH (mutXR) almost reached the same OD as the corresponding control strain TMB4143 (control, mutXR) (Figures 3A, B), which was not the case for the other strains carrying either no XR or wtXR. Improved NADH reoxidisation was expected to be coupled to the NADH-dependent xylose reduction to xylitol. Indeed high xylitol production, between 3 and 5 g/L, was measured during the first 48 hours of cultivations in all strains harboring a heterologous XR (Figure 4). Also the deletion strains produced xylitol faster, and in larger quantities than the control strains. Within the first 48 hours, there was no significant difference in xylitol production between strains carrying wtXR and mutXR, neither in the control strain background (Figure 4A) nor in the gpd1Δ gpd2Δ background (Figure 4B). However, the strains carrying mutXR reached slightly higher xylitol concentrations at the end of the cultivations. Xylitol was also produced in strains lacking heterologous XR, but to a significantly lower degree (<0.8 g/L after 70h). Xylitol may actually arise from the NADPH-dependent conversion of xylose by endogenous NADPH-dependent XR, such as the one encoded by GRE3 gene (Träff et al. [2002]). In the case of XR-carrying strains, xylitol may also originate from the NADPH-catalysed reaction of the introduced XR. Glycerol production was observed for the double deletion strains carrying the control or mutated XR (Figure 4C, D), which may be explained by the fact that P. stipitis XR can use DHAP as substrate, which leads to glycerol formation (Jeppsson et al. [2003]).


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

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

Extracellular xylitol (A, B) and glycerol (C, D) concentrations (in g/L) over time for the control background (A, C) or thegpd1Δgpd2Δbackground (B, D) carrying no XR, wtXR or mutXR. Legend: TMB4140 (control, no XR “red dot”), TMB4141 (control, wtXR “blue x”), TMB4143 (control, mutXR(green square)), TMB4144 (gpd1Δgpd2Δ, no XR “red plus”), TMB4145 (gpd1Δgpd2Δ, wtXR “blue o”), TMB4147 (gpd1Δgpd2Δ, mutXR “green asterisk”).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Extracellular xylitol (A, B) and glycerol (C, D) concentrations (in g/L) over time for the control background (A, C) or thegpd1Δgpd2Δbackground (B, D) carrying no XR, wtXR or mutXR. Legend: TMB4140 (control, no XR “red dot”), TMB4141 (control, wtXR “blue x”), TMB4143 (control, mutXR(green square)), TMB4144 (gpd1Δgpd2Δ, no XR “red plus”), TMB4145 (gpd1Δgpd2Δ, wtXR “blue o”), TMB4147 (gpd1Δgpd2Δ, mutXR “green asterisk”).
Mentions: Strains were cultivated under oxygen-limited conditions in sealed vials containing minimal medium with galactose as the carbon source and xylose as the substrate for the XR-catalysed reduction. Galactose was used because S. cerevisiae has a specific galactose uptake system, Gal2p (Cirillo [1968]) that also transports xylose, and whose expression benefits xylose utilization (Johnston [1987]; Hamacher et al. [2002]). So, even though the yeast does not metabolize galactose as fast as glucose, the use of galactose allows the cell to have co-consumption of hexose and xylose (Bro et al. [2005]; Garcia Sanchez et al. [2010]). The growth profiles of the different strains are shown in Figures 3A and B. During the first 24 hours, gpd1Δ gpd2Δ strains reached an OD of around 3, which was only slightly lower than the corresponding control strains. During the next 48 hours, however, TMB4147 strain (gpd1Δ gpd2Δ, mutXR) continued growing and reached an OD of 5.2 whereas strain TMB4145 (gpd1Δ gpd2Δ, wtXR) only reached OD 3.7 while the OD of strain TMB4144 (gpd1Δ gpd2Δ, no XR) decreased to 2 (Figure 3B). Also TMB4147 strain carrying the XR with the highest selectivity for NADH (mutXR) almost reached the same OD as the corresponding control strain TMB4143 (control, mutXR) (Figures 3A, B), which was not the case for the other strains carrying either no XR or wtXR. Improved NADH reoxidisation was expected to be coupled to the NADH-dependent xylose reduction to xylitol. Indeed high xylitol production, between 3 and 5 g/L, was measured during the first 48 hours of cultivations in all strains harboring a heterologous XR (Figure 4). Also the deletion strains produced xylitol faster, and in larger quantities than the control strains. Within the first 48 hours, there was no significant difference in xylitol production between strains carrying wtXR and mutXR, neither in the control strain background (Figure 4A) nor in the gpd1Δ gpd2Δ background (Figure 4B). However, the strains carrying mutXR reached slightly higher xylitol concentrations at the end of the cultivations. Xylitol was also produced in strains lacking heterologous XR, but to a significantly lower degree (<0.8 g/L after 70h). Xylitol may actually arise from the NADPH-dependent conversion of xylose by endogenous NADPH-dependent XR, such as the one encoded by GRE3 gene (Träff et al. [2002]). In the case of XR-carrying strains, xylitol may also originate from the NADPH-catalysed reaction of the introduced XR. Glycerol production was observed for the double deletion strains carrying the control or mutated XR (Figure 4C, D), which may be explained by the fact that P. stipitis XR can use DHAP as substrate, which leads to glycerol formation (Jeppsson et al. [2003]).

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