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Improving freeze-tolerance of baker’s yeast through seamless gene deletion of NTH1 and PUT1

  • Genetics and Molecular Biology of Industrial Organisms
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Baker’s yeast strains with freeze-tolerance are highly desirable to maintain high leavening ability after freezing. Enhanced intracellular concentration of trehalose and proline in yeast is linked with freeze-tolerance. In this study, we constructed baker’s yeast with enhanced freeze-tolerance by simultaneous deletion of the neutral trehalase-encoded gene NTH1 and the proline oxidase-encoded gene PUT1. We first used the two-step integration-based seamless gene deletion method to separately delete NTH1 and PUT1 in haploid yeast. Subsequently, through two rounds of hybridization and sporulation-based allelic exchange and colony PCR-mediated tetrad analysis, we obtained strains with restored URA3 and deletion of NTH1 and/or PUT1. The resulting strain showed higher cell survival and dough-leavening ability after freezing compared to the wild-type strain due to enhanced accumulation of trehalose and/or proline. Moreover, mutant with simultaneous deletion of NTH1 and PUT1 exhibits the highest relative dough-leavening ability after freezing compared to mutants with single-gene deletion perhaps due to elevated levels of both trehalose and proline. These results verified that it is applicable to construct frozen dough baker’s yeast using the method proposed in this paper.

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Acknowledgments

This work was supported by program for National Natural Science Foundation of China (Grant Number 31301536), Changjiang Scholars and Innovative Research Team in University (IRT1166), the National High Technology Research, the Development Program of China (863 Program) (Grant Number 2012AA022108), the Youth Foundation of Application Base and Frontier Technology Project of Tianjin, China (12JCQNJC06500), and Science and Research Foundation of Tianjin University of Science and Technology (20120114).

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Authors and Affiliations

  1. Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Industrial Microbiology Key Laboratory, College of Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, China

    Jian Dong, Didi Chen, Guanglu Wang, Cuiying Zhang, Liping Du, Shanshan Liu, Yu Zhao & Dongguang Xiao

  2. Tianjin Food Safety and Low Carbon Manufacturing Collaborative Innovation Center, Tianjin, 300457, People′s Republic of China

    Jian Dong, Didi Chen, Cuiying Zhang, Liping Du, Shanshan Liu, Yu Zhao & Dongguang Xiao

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  1. Jian Dong
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  2. Didi Chen
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  3. Guanglu Wang
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  8. Dongguang Xiao
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Correspondence to Dongguang Xiao.

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Dong, J., Chen, D., Wang, G. et al. Improving freeze-tolerance of baker’s yeast through seamless gene deletion of NTH1 and PUT1 . J Ind Microbiol Biotechnol 43, 817–828 (2016). https://doi.org/10.1007/s10295-016-1753-7

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  • DOI: https://doi.org/10.1007/s10295-016-1753-7

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