Investigation of Heterologously Expressed Glucose-6-Phosphate Dehydrogenase Genes in a Yeast zwf1 Deletion.

Jürgen J Heinisch,Johannes Knuesting,Renate Scheibe

doi:10.3390/microorganisms8040546

Jürgen J Heinisch, Johannes Knuesting + Show 1 more

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https://doi.org/10.3390/microorganisms8040546

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Journal: Microorganisms	Publication Date: Apr 9, 2020
Citations: 19	License type: CC BY 4.0

Affiliation: Osnabrück University

Abstract

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 Saccharomyces cerevisiae which lacks the G6PD-encoding ZWF1 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 G6PD genes, synthesized in the yeast codon usage and expressed under the control of the native PFK2 promotor. Complementation of the hypersensitivity of the zwf1 mutant towards hydrogen peroxide to different degrees was observed for the genes from humans (HsG6PD1), the milk yeast Kluyveromyces lactis (KlZWF1), the bacteria Escherichia coli (EcZWF1) and Leuconostoc mesenteroides (LmZWF1), as well as the genes encoding three different plant G6PD isoforms from Arabidopsis thaliana (AtG6PD1, AtG6PD5, AtG6PD6). 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 zwf1 deletion. Given the importance of G6PD in human diseases and plant growth, this heterologous expression system offers a broad range of applications.

Highlights

All cells from bacteria to humans have developed mechanisms to cope with oxidative stress caused by reactive oxygen species (ROS), which are a by-product of respiratory metabolism and cause damage to membranes and DNA [1]
In order to establish a versatile expression system for Glucose-6-phosphate dehydrogenase (G6PD) genes from organisms spanning the kingdoms of bacteria, fungi, plants, and animals, we obtained a deletion mutant in the sole S. cerevisiae ZWF1 gene, substituting its open reading frame (ORF) for a kanMX deletion cassette in one allele of the wild-type diploid strain DHD5 (Figure 1A)
The zwf1 deletion was lethal in another common background of strain Σ1278b [57], underlining the substantial degree of nucleotide polymorphisms observed between commonly employed yeast strains [45]

Summary

Introduction

All cells from bacteria to humans have developed mechanisms to cope with oxidative stress caused by reactive oxygen species (ROS), which are a by-product of respiratory metabolism and cause damage to membranes and DNA [1]. The importance of corresponding signaling pathways that elicit a proper cellular response to ROS accumulation, especially in tumor cells, has been underlined by studies earning last year’s award of the Nobel prize for physiology and medicine (see [2], and references therein). Glucose-6-phosphate dehydrogenase (G6PD), a key enzyme of the pentose phosphate pathway (PPP) generates NADPH required to detoxify ROS, and for methionine biosynthesis [3,4]). The human G6PD gene resides on the X-chromosome and heterozygosity for some mutations may protect against malaria infections [11]. This correlates with the observation that the malaria parasite, Plasmodium falciparum, is very sensitive to oxidative stress, and drugs targeted towards its native G6PD are used to combat infections [12]

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