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Expression of the Aspergillus niger glucose oxidase gene in Saccharomyces cerevisiae and its potential applications in wine production

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Abstract

There is a growing consumer demand for wines containing lower levels of alcohol and chemical preservatives. The objectives of this study were to express the Aspergillus niger gene encoding a glucose oxidase (GOX; β-d-glucose:oxygen oxidoreductase, EC 1.1.3.4) in Saccharomyces cerevisiae and to evaluate the transformants for lower alcohol production and inhibition of wine spoilage organisms, such as acetic acid bacteria and lactic acid bacteria, during fermentation. The A. niger structural glucose oxidase (gox) gene was cloned into an integration vector (YIp5) containing the yeast mating pheromone α-factor secretion signal (MFα1 S ) and the phosphoglycerate-kinase-1 gene promoter (PGK1 P ) and terminator (PGK1 T ). The PGK1 P -MFα1 S -gox-PGK1 T cassette (designated GOX1) was introduced into a laboratory strain (Σ1278) of S. cerevisiae. Yeast transformants were analysed for the production of biologically active glucose oxidase on selective agar plates and in liquid assays. The results indicated that the recombinant glucose oxidase was active and was produced beginning early in the exponential growth phase, leading to a stable level in the stationary phase. The yeast transformants also displayed antimicrobial activity in a plate assay against lactic acid bacteria and acetic acid bacteria. This might be explained by the fact that a final product of the GOX enzymatic reaction is hydrogen peroxide, a known antimicrobial agent. Microvinification with the laboratory yeast transformants resulted in wines containing 1.8–2.0% less alcohol. This was probably due to the production of d-glucono-δ-lactone and gluconic acid from glucose by GOX. These results pave the way for the development of wine yeast starter culture strains for the production of wine with reduced levels of chemical preservatives and alcohol.

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Abbreviations

GOX:

Glucose oxidase enzyme

gox :

Aspergillus niger glucose oxidase gene

GOX1 :

Aspergillus niger gox gene expressed in Saccharomyces cerevisiae as the PGK1 P -MFα1 S -gox-PGK1 T gene cassette

MFα1 S :

Yeast mating pheromone α-factor secretion signal

PGK1 P :

Yeast phosphoglycerate-kinase-1 gene promoter

PGK1 T :

Yeast phosphoglycerate-kinase-1 gene terminator

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Acknowledgements

The authors wish to thank the South African wine industry (Winetech) and the National Research Foundation (NRF) for funding this study. We would also like to thank Dr. R. Geisen of the Federal Research Centre for Nutrition in Germany for sending us the Aspergillus niger glucose oxidase gene, and Dr. Miranda Waldron of the Electron Microscope Unit at the University of Cape Town for her time and patience with the scanning electron microscopy. In addition, we want to thank Dr. Elisabeth Rohwer of the Institute for Wine Biotechnology at Stellenbosch University, for her time and help with the high-performance capillary electrophoresis system.

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  1. Institute for Wine Biotechnology, Department of Viticulture and Oenology, Stellenbosch University, 7600 , Stellenbosch, South Africa

    D. F. Malherbe, M. du Toit, R. R. Cordero Otero, P. van Rensburg & I. S. Pretorius

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  1. D. F. Malherbe
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  2. M. du Toit
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  3. R. R. Cordero Otero
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  4. P. van Rensburg
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  5. I. S. Pretorius
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Correspondence to I. S. Pretorius.

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Malherbe, D.F., du Toit, M., Cordero Otero, R.R. et al. Expression of the Aspergillus niger glucose oxidase gene in Saccharomyces cerevisiae and its potential applications in wine production. Appl Microbiol Biotechnol 61, 502–511 (2003). https://doi.org/10.1007/s00253-002-1208-0

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  • DOI: https://doi.org/10.1007/s00253-002-1208-0

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