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Improved activity of the Cel5A endoglucanase in Saccharomyces cerevisiae deletion mutants defective in oxidative stress defense mechanisms

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Abstract

Objectives

Developing a Saccharomyces cerevisiae system for optimizing the expression of recombinant eukaryotic proteins.

Results

Two deletion mutants, which were hypersensitive to H2O2, were obtained by knocking out CTT1 and SOD2, respectively. The mutation rate of the mutants was up to over 4000 times of the spontaneous mutation rate when treated with H2O2. Endoglucanase Cel5A was used as a model enzyme to evaluate the system for improving the expression of the recombinant protein. Sixteen mutants of the RDKY3615 (ctt1∆) transformant and seven mutants of the RDKY3615 (sod2∆) transformant had significantly high Cel5A activity, while none mutants of the RDKY3615 transformant had significantly high enzyme activity.

Conclusion

The combination of deletion mutagenesis and H2O2 treatment greatly accelerate the generation of genetic variants and will be a useful tool in improving the heterologous expression in S. cerevisiae.

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Acknowledgement

This work was supported by the National Basic Research Program (2003CB716000 and 2010CB630902), the National Science Found (Nos. 30370036 and 30670039), the National Key Technology R&D Program (2011BAC02B04), the National High Technology Research and Development Program (2007AA05Z455) of China and the Fund of Glycosylation Research Group in Shandong University. We thank Prof. Tianhong Wang for kindly providing the pUCmT-Cel5A vector.

Supporting information

Supplementary Table 1—Strains and plasmids used in this study.

Supplementary Table 2—PCR primers used in this study.

Supplementary Figure 1—Screen of the mutants for high Cel5A activity on activity indicator plate. Each colony was formed from a single cell. CMC around the colonies was hydrolyzed by Cel5A secreted by the colonies. The size of the hydro-halo represented the secreted endoglucanase activity of the colony.

Author information

Authors and Affiliations

  1. Research Center for Eco-Environmental Science, Chinese Academy of Science, Beijing, People’s Republic of China

    Guochao Wu & Guoqiang Zhuang

  2. Institute of Basic Medical Sciences, Qilu Hospital, Shandong University, Jinan, People’s Republic of China

    Jintang Sun

  3. State Key Laboratory of Microbial Technology, Shandong University, Jinan, People’s Republic of China

    Guochao Wu, Jintang Sun, Shanshan Yu, Guoqiang Zhuang, Weifeng Liu, Jianqiang Lin & Yinbo Qu

  4. China Science and Technology Exchange Center, Beijing, People’s Republic of China

    Quanchao Dong

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  1. Guochao Wu
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  2. Jintang Sun
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  5. Guoqiang Zhuang
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Corresponding author

Correspondence to Guoqiang Zhuang.

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Wu, G., Sun, J., Yu, S. et al. Improved activity of the Cel5A endoglucanase in Saccharomyces cerevisiae deletion mutants defective in oxidative stress defense mechanisms. Biotechnol Lett 37, 1081–1089 (2015). https://doi.org/10.1007/s10529-015-1771-y

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  • DOI: https://doi.org/10.1007/s10529-015-1771-y

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