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Recombinant industrial brewing yeast strains with ADH2 interruption using self-cloning GSH1+CUP1 cassette
Article first published online: 1 APR 2009
DOI: 10.1111/j.1567-1364.2009.00502.x
© 2009 Chinese Academy of Sciences. Journal compilation © 2009 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd
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How to Cite
Wang, Z.-Y., Wang, J.-J., Liu, X.-F., He, X.-P. and Zhang, B.-R. (2009), Recombinant industrial brewing yeast strains with ADH2 interruption using self-cloning GSH1+CUP1 cassette. FEMS Yeast Research, 9: 574–581. doi: 10.1111/j.1567-1364.2009.00502.x
Publication History
- Issue published online: 8 MAY 2009
- Article first published online: 1 APR 2009
- Received 1 September 2008; revised 23 December 2008; accepted 23 February 2009.First published online 1 April 2009.
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Keywords:
- industrial brewing yeast;
- self-cloning;
- acetaldehyde;
- glutathione;
- ADH2;
- GSH1
Abstract
A self-cloning module for gene knock-out and knock-in in industrial brewing yeast strain was constructed that contains copper resistance and γ-glutamylcysteine synthetase gene cassette, flanked by alcohol dehydrogenase II gene (ADH2) of Saccharomyces cerevisiae. The module was used to obtain recombined strains RY1 and RY2 by targeting the ADH2 locus of host Y1. RY1 and RY2 were genetically stable. PCR and enzyme activity analysis of RY1 and RY2 cells showed that one copy of ADH2 was deleted by GSH1+CUP1 insertion, and an additional copy of wild type was still present. The fermentation ability of the recombinants was not changed after genetic modification, and a high level of glutathione (GSH) was secreted, resulting from GSH1 overexpression, which codes for γ-glutamylcysteine synthetase. A pilot-scale brewing test for RY1 and RY2 indicated that acetaldehyde content in fermenting liquor decreased by 21–22%, GSH content increased by 20–22% compared with the host, the antioxidizability of the recombinants was improved, and the sensorial evaluation was also better than that of the host. No heterologous DNA was harbored in the recombinants; therefore, they could be applied in the beer industry in terms of their biosafety.