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Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme of the oxidative part of the pentose phosphate pathway and serves as the major source of NADPH for metabolic reactions and oxidative stress response in pro- and eukaryotic cells. We here report on a strain of the model yeast <i>Saccharomyces cerevisiae</i> which lacks the G6PD-encoding <i>ZWF1</i> gene and displays distinct growth retardation on rich and synthetic media, as well as a strongly reduced chronological lifespan. This strain was used as a recipient to introduce plasmid-encoded heterologous <i>G6PD</i> genes, synthesized in the yeast codon usage and expressed under the control of the native <i>PFK2</i> promotor. Complementation of the hypersensitivity of the <i>zwf1</i> mutant towards hydrogen peroxide to different degrees was observed for the genes from humans (<i>HsG6PD1</i>), the milk yeast <i>Kluyveromyces lactis</i> (<i>KlZWF1</i>), the bacteria <i>Escherichia coli</i> (<i>EcZWF1</i>) and <i>Leuconostoc mesenteroides</i> (<i>LmZWF1</i>), as well as the genes encoding three different plant G6PD isoforms from <i>Arabidopsis thaliana</i> (<i>AtG6PD1, AtG6PD5, AtG6PD6</i>). The plastidic AtG6PD1 isoform retained its redox-sensitive activity when produced in the yeast as a cytosolic enzyme, demonstrating the suitability of this host for determination of its physiological properties. Mutations precluding the formation of a disulfide bridge in AtG6PD1 abolished its redox-sensitivity but improved its capacity to complement the yeast <i>zwf1</i> deletion. Given the importance of G6PD in human diseases and plant growth, this heterologous expression system offers a broad range of applications.