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Heterologous Expression of the Carrot Hsp17.7 gene Increased Growth, Cell Viability, and Protein Solubility in Transformed Yeast (Saccharomyces cerevisiae) under Heat, Cold, Acid, and Osmotic Stress Conditions

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Abstract

In industrial fermentation of yeast (Saccharomyces cerevisiae), culture conditions are often modified from the optimal growth conditions of the cells to maintain large-scale cultures and/or to increase recombinant protein production. However, altered growth conditions can be stressful to yeast cells resulting in reduced cell growth and viability. In this study, a small heat shock protein gene from carrot (Daucus carota L.), Hsp17.7, was inserted into the yeast genome via homologous recombination to increase tolerance to stress conditions that can occur during industrial culture. A DNA construct, Translational elongation factor gene promoter—carrot Hsp17.7 gene—Phosphoribosyl-anthranilate isomerase gene (an auxotrophic marker), was generated by a series of PCRs and introduced into the chromosome IV of the yeast genome. Immunoblot analysis showed that carrot Hsp17.7 accumulated in the transformed yeast cell lines. Growth rates and cell viability of these cell lines were higher than control cell lines under heat, cold, acid, and hyperosmotic stress conditions. Soluble protein levels were higher in the transgenic cell lines than control cell lines under heat and cold conditions, suggesting the molecular chaperone function of the recombinant Hsp17.7. This study showed that a recombinant DNA construct containing a HSP gene from carrot was successfully expressed in yeast by homologous recombination and increased tolerances to abiotic stress conditions.

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References

  1. Ahn Y, Song N (2012) A cytosolic heat shock protein expressed in carrot (Daucus carota L.) enhances cell viability under oxidative and osmotic stress conditions. HortScience 47:143–148

    CAS  Google Scholar 

  2. Ahn Y, Zimmerman JL (2006) Introduction of the carrot HSP17.7 into potato (Solanum tuberosum L.) enhances cellular membrane stability and tuberization in vitro. Plant Cell Environ 29:95–104

    Article  CAS  PubMed  Google Scholar 

  3. Basílio ACM, Araújo PRL, Morais JOF, Silva-Filho EA, de Morais MA Jr, Simões DA (2008) Detection and identification of wild yeast contaminants of the industrial fuel ethanol fermentation process. Curr Microbiol 56:322–326

    Article  PubMed  Google Scholar 

  4. Bentley NJ, Fitch IT, Tuite MF (1992) The small heat-shock protein Hsp26 of Saccharomyces cerevisiae assembles into a high molecular weight aggregate. Yeast 8:95–106

    Article  CAS  PubMed  Google Scholar 

  5. Bonander N, Bill MR (2012) Optimising yeast as a host for recombinant protein production. Methods MolBiol 866:1–9

    CAS  Google Scholar 

  6. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  7. Brosnan MP, Donnelly D, James TC, Bond U (2000) The stress response is repressed during fermentation in brewery strains of yeast. J Appl Microbiol 88:746–755

    Article  CAS  PubMed  Google Scholar 

  8. Conzelmann A, Riezman H, Desponds C, Bron C (1988) A major 125-kd membrane glycoprotein of Saccharomyces cerevisiae is attached to the lipid bilayer through an inositol-containing phospholipid. EMBO J 7:2233–2240

    CAS  PubMed  PubMed Central  Google Scholar 

  9. de Melo HF, Bonini BM, Thevelein J, Simões DA, Morais MA Jr (2009) Physiological and molecular analysis of the stress response of Saccharomyces cerevisiae imposed by strong inorganic acid with implication to industrial fermentations. J Appl Microbiol 109:116–127

    PubMed  Google Scholar 

  10. Gasser B, Saloheimo M, Rinas U, Dragosits M, Rodríguez-Carmona E, Baumann K, Giuliani M, Parrilli E, Branduardi P, Lang C, Porro D, Ferrer P, Tutino ML, Mattanovich D, Villaverde A (2008) Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. Microb Cell Fact 7:11

    Article  PubMed  PubMed Central  Google Scholar 

  11. Gatignol A, Dassain M, Tiraby G (1990) Cloning of Saccharomyces cerevisiae promoters using a probe vector based on phleomycin resistance. Gene 91:35–41

    Article  CAS  PubMed  Google Scholar 

  12. Gietz RD, Schiestl RH (2007) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2:31–34

    Article  CAS  PubMed  Google Scholar 

  13. Hohmann S (2002) Osmotic stress signaling and osmoadaptation in yeast. Microbiol Mol Biol Rev 66:300–372

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Jahic M, Veide A, Charoenrat T, Teeri T, Enfors SO (2006) Process technology for production and recovery of heterologous proteins with Pichia pastoris. Biotechnol Prog 22:1465–1473

    Article  CAS  PubMed  Google Scholar 

  15. Jiang C, Xu J, Zhang H, Zhang X, Shi J, Li M, Ming F (2009) A cytosolic class I small heat shock protein, RcHSP17.8, of Rosa chinensis confers resistance to a variety of stresses to Escherichia coli, yeast and Arabidopsis thaliana. Plant Cell Environ 32:1046–1059

    Article  CAS  PubMed  Google Scholar 

  16. Kim H, Ahn Y (2009) Expression of a gene encoding the carrot HSP17.7 in Escherichia coli enhances cell viability and protein solubility under heat stress. HortScience 44:866–869

    Google Scholar 

  17. Kitagawa M, Miyakawa M, Matsumura Y, Tsuchido T (2002) Escherichia coli small heat shock proteins, IbpA and IbpB, protect enzymes from inactivation by heat and oxidants. Eur J Biochem 269:2907–2917

    Article  CAS  PubMed  Google Scholar 

  18. Kregel KC (2002) Invited review: heat shock proteins: modifying factors in physiological stress responses and acquired thermotolerance. J Appl Physiol 92:2177–2186

    Article  CAS  PubMed  Google Scholar 

  19. Malik MK, Slovin JP, Hwang CH, Zimmerman JL (1999) Modified expression ofa carrot small heat shock protein gene, Hsp17.7, results in increased or decreased thermotolerance. Plant J 20:89–99

    Article  CAS  PubMed  Google Scholar 

  20. Mattanovich D, Gasser B, Hohenblum H, Sauer M (2004) Stress in recombinant protein producing yeasts. J Biotechnol 113:121–135

    Article  CAS  PubMed  Google Scholar 

  21. Pacheco A, Pereira C, Almeida MJ, Sousa MJ (2009) Small heat-shock protein Hsp12 contributes to yeast tolerance to freezing stress. Microbiology 155:2021–2028

    Article  CAS  PubMed  Google Scholar 

  22. Park H, Ahn Y (2016) Development of transgenic Escherichia coli with improved viability by heterologous expression of a heat shock protein gene from carrot (Daucus carota L.). HortScience 51:305–310

    Article  CAS  Google Scholar 

  23. Piper PW, Ortiz-Calderon C, Holyoak C, Coote P, Cole M (1997) Hsp30, the integral plasma membrane heat shock protein of Saccharomyces cerevisiae, is a stress-inducible regulator of plasma membrane H(+)-ATPase. Cell Stress Chaperon 2:12–24

    Article  CAS  Google Scholar 

  24. Rao MB, Tanksale AM, Ghatge MS, Deshpande VV (1998) Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev 62:597–635

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:172

    PubMed  PubMed Central  Google Scholar 

  26. Saito H, Posas F (2012) Response to hyperosmotic stress. Genetics 192:289–318

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Tiwari S, Thakur R, Shankar J (2015) Role of heat-shock proteins in cellular function and in the biology of fungi. Biotechnol Res Int 2015: Article ID 132635

  28. van Leeuwen J, Andrews B, Boone C, Tan G (2015) Rapid and efficient plasmid construction by homologous recombination in yeast. Cold Spring Harb Protoc 9:853–861

    Google Scholar 

  29. Varela JC, Praekelt UM, Meacock PA, Planta RJ, Mager WH (1995) The Saccharomyces cerevisiae HSP12 gene is activated by the high-osmolarity glycerol pathway and negatively regulated by protein kinase A. Mol Cell Biol 15:6232–6245

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Verghese J, Abrams J, Wang Y, Moranoa KA (2012) Biology of the heat shock response and protein chaperones: budding yeast (Saccharomyces cerevisiae) as a model system. Microbiol Mol Biol Rev 76:115–158

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Vierling E (1991) The Roles of Heat Shock Proteins in Plants. Annu Rev Plant Biol 42:579–620

    Article  CAS  Google Scholar 

  32. Wang W, Vinocur B, Shoseyov O, Altman A (2004) Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci 9:244–252

    Article  CAS  PubMed  Google Scholar 

  33. Xue Y, Peng R, Xiong A, Li X, Zha D, Yao Q (2009) Yeast heat-shock protein gene HSP26 enhances freezing tolerance in Arabidopsis. J Plant Physiol 166:844–850

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2015R1C1A2A01053040).

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Authors and Affiliations

  1. Department of Biotechnology, College of Engineering for Future Convergence, Sangmyung University, 20 Hongjimun 2-gil, Jongno-gu, Seoul, 03016, South Korea

    Eunhye Ko, Minhye Kim, Yunho Park & Yeh-Jin Ahn

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  1. Eunhye Ko
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Correspondence to Yeh-Jin Ahn.

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Ko, E., Kim, M., Park, Y. et al. Heterologous Expression of the Carrot Hsp17.7 gene Increased Growth, Cell Viability, and Protein Solubility in Transformed Yeast (Saccharomyces cerevisiae) under Heat, Cold, Acid, and Osmotic Stress Conditions. Curr Microbiol 74, 952–960 (2017). https://doi.org/10.1007/s00284-017-1269-z

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  • DOI: https://doi.org/10.1007/s00284-017-1269-z

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