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.
Highlights
The relative concentrations of NADH/NAD+ redox couple determine the thermodynamic feasibility of more than 100 reactions in cellular metabolism which places it at the core of the metabolic network of every organism including Saccharomyces cerevisiae (Förster et al 2003)
In S. cerevisiae, it is well known that a skewed ratio of cytosolic NADH/NAD+ can be compensated by glycerol production regulated by a fine-tuned expression of the GPD2 gene (Ansell et al 1997; Björkqvist et al 1997)
Flourescence intensity (FI) were in the same order of magnitude in single glycerol-3phosphate dehydrogenase (GPD) deletion strain and in the control strain, confirming the previously observed compensatory pattern between Gpdp isoenzymes in order to maintain NAD+ regeneration via glycerol production (Ansell et al 1997)
Summary
The relative concentrations of NADH/NAD+ redox couple determine the thermodynamic feasibility of more than 100 reactions in cellular metabolism which places it at the core of the metabolic network of every organism including Saccharomyces cerevisiae (Förster et al 2003). Quantification of redox couples in cells is complicated by several factors: the NADH and NAD+ are compartmentalized in S. cerevisiae and the cofactors are to a large extent in a protein bound form (Veech et al 1969; Bücher et al 1972). In S. cerevisiae, the cytosolic enzyme glycerol-3phosphate dehydrogenase (GPD) 2 encoded by the GPD2 gene plays a central role in redox metabolism. GPD2 expression increases as the need for cytosolic reoxidation of NADH increases because Gpd2p acts as redox sink via the NADH-coupled reduction of dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate (G3P). The genes can complement each other’s functions, a double deletion renders the yeast cell osmosensitive as well as incapable of anaerobic growth, due to the inability to reoxidize cytosolic NADH arising from biomass formation (van Dijken and Scheffers 1986; Nissen et al 1997)
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