Annals of Microbiology

, Volume 64, Issue 3, pp 1457–1462 | Cite as

Use of the glyceraldehyde-3-phosphate dehydrogenase promoter from a thermotolerant yeast, Pichia thermomethanolica, for heterologous gene expression, especially at elevated temperature

  • Piyanun Harnpicharnchai
  • Peerada Promdonkoy
  • Kittapong Sae-Tang
  • Niran Roongsawang
  • Sutipa Tanapongpipat
Short Communication
First Online:
Received:
Accepted:

Abstract

The glyceraldehyde-3-phosphate dehydrogenase (GAP) gene from the thermotolerant yeast strain Pichia thermomethanolica BCC16875 was characterized. To investigate the efficiency of the GAP promoter for heterologous expression, especially at high temperature in various carbon sources, the promoter was employed for constitutive expression of a phytase reporter gene. The results showed that this promoter was able to drive efficient expression of phytase at 30 °C; the native promoter was highly robust compared with the heterologous GAP promoter from Pichia pastoris. More importantly, the GAP promoter was shown to be able to function at higher temperatures up to 42 °C, which could be useful for large-scale protein production to help reduce cooling costs in the fermenter. Expression in different carbon sources revealed that the GAP promoter was functional in glucose-, glycerol-, and methanol-containing media, with the highest level of expression in YPD medium. This strong promoter will help promote high expression of heterologous protein expression in P. thermomethanolica, especially in large-scale fermentation. In addition, a new tool for heterologous expression in yeast has been gained.

Keywords

Thermotoloerant yeast Glyceraldehyde-3-phosphate dehydrogenase Heterologous gene expression Carbon source 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

We thank Mr. Wuttichai Mhuantong for technical assistance and Dr. Philip J. Shaw for critically editing the manuscript. Financial support (P-09-00108) from National Center for Genetic Engineering and Biotechnology, Thailand is greatly appreciated.

Supplementary material

13213_2013_765_MOESM1_ESM.pdf (1.3 mb)
Supplementary Figure 1 (PDF 1313 kb)
13213_2013_765_MOESM2_ESM.pdf (253 kb)
Supplementary Figure 2 (PDF 252 kb)

References

  1. Bitter GA, Egan KM (1984) Expression of heterologous genes in Saccharomyces cerevisiae from vectors utilizing the glyceraldehyde-3-phosphate dehydrogenase gene promoter. Gene 32:263–274PubMedCrossRefGoogle Scholar
  2. Bitter GA, Chang KK, Egan KM (1991) A multi-component upstream activation sequence of the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene promoter. Mol Gen Genet 231:22–32PubMedCrossRefGoogle Scholar
  3. Böer E, Steinborn G, Kunze G, Gellissen G (2007) Yeast expression platforms. Appl Microbiol Biotechnol 77:513–523PubMedCrossRefGoogle Scholar
  4. Cai F, Li T, Xie Y, He X (2013) Expression of functional single-chain variable domain fragment (scFv) antibody against Metolcarb in Pichia pastoris. Ann Microbiol. doi: 10.10007/s13213-013-0692-z Google Scholar
  5. Cartharius K, Frech K, Grote K, Klocke B, Haltmeier M, Klingenhoff A, Frisch M, Bayerlein M, Werner T (2005) MatInspector and beyond: promoter analysis based on transcription factor binding sites. Bioinformatics 21:2933–2942PubMedCrossRefGoogle Scholar
  6. Chen JM, Zhu GY, Xia WT, Zhao ZQ (2012) Proteomic analysis of rat retina after methanol intoxication. Toxicology 293:89–96PubMedCrossRefGoogle Scholar
  7. Delroisse J-M, Dannau M, Gilsoul J-J, El Mejdoub T, Destain J, Portetelle D, Thonart P, Haubruge E, Vandenbol M (2005) Expression of a synthetic gene encoding a Tribolium castaneum carboxylesterase in Pichia pastoris. Protein Expr Purif 42:286–294PubMedCrossRefGoogle Scholar
  8. Hensing MC, Rouwenhorst RJ, Heijnen JJ, van Dijken JP, Pronk JT (1995) Physiological and technological aspects of large-scale heterologous-protein production with yeasts. Antonie Van Leeuwenhoek 67:261–279PubMedCrossRefGoogle Scholar
  9. Heo J-H, Hong WK, Cho EY, Kim MW, Kim J-Y, Kim CH, Rhee SK, Kang HA (2003) Properties of the Hansenula polymorpha-derived constitutive GAP promoter, assessed using an HAS reporter gene. FEMS Yeast Res 4:175–184PubMedCrossRefGoogle Scholar
  10. Kumar NV, Rangarajan PN (2012) The zinc finger proteins Mxr1p and ROP have the same DNA binding specificity but regulate methanol metabolism antagonistically in Pichia pastoris. J Biol Chem 287:34465–34473PubMedCentralPubMedCrossRefGoogle Scholar
  11. Kuroda S, Otaka S, Fujisawa Y (1994) Fermentable and nonfermentable carbon sources sustain constitutive levels of expression of yeast triosephosphate dehydrogenase gene 3 gene from distinct promoter elements. J Biol Chem 269:6153–6162PubMedGoogle Scholar
  12. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) ClustalW and ClustalX version 2. Bioinformatics 23:2947–2948PubMedCrossRefGoogle Scholar
  13. Limtong S, Srisuk N, Yongmanitchai W, Yurimoto H, Nakase T, Kato N (2005) Pichia thermomethanolica sp. nov., a novel thermotolerant, methylotrophic yeast isolated in Thailand. Int J Syst Evol Microbiol 55:2225–2229PubMedCrossRefGoogle Scholar
  14. Limtong S, Srisuk N, Yongmanitchai W, Yurimoto H, Takashi Nakase T (2008) Ogataea chonburiensis sp. nov. and Ogataea nakhonphanomensis sp. nov., thermotolerant, methylotrophic yeast species isolated in Thailand, and transfer of Pichia siamensis and Pichia thermomethanolica to the genus Ogataea. Int J Syst Evol Microbiol 58:302–307PubMedCrossRefGoogle Scholar
  15. Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22:249–270PubMedCrossRefGoogle Scholar
  16. Promdonkoy P, Tang K, Sornlake W, Harnpicharnchai P, Kobayashi Sriprang R, Ruanglek V, Upathanpreecha T, Vesaratchavest M, Eurwilaichitr L, Tanapongpipat S (2009) Expression and characterization of Aspergillus thermostable phytases in Pichia pastoris. FEMS Microbiol Lett 290:18–24PubMedCrossRefGoogle Scholar
  17. Reese MG (2001) Application of a time-delay neural network to promoter annotation in the Drosophila melanogaster genome. Comput Chem 26:51–56PubMedCrossRefGoogle Scholar
  18. Sandhu SK, Oberoi HS, Dhaliwal SS, Babbar N, Kaur U, Nanda D, Kumar D (2012) Ethanol production from Kinnow mandarin (Citrus reticulata) peels via simultaneous saccharification and fermentation using crude enzyme produced by Aspergillus oryzae and the thermotolerant Pichia kudriavzevii strain. Ann Microbiol 62:655–666CrossRefGoogle Scholar
  19. Tanapongpipat S, PromdonkoyP WT, Tirasophon W, Roongsawang N, Chiba Y, Eurwilaichitr L (2012) Heterologous protein expression in Pichia thermomethanolica BCC16875, a thermotolerant methylotrophic yeast and characterization of N-linked glycosylation in secreted protein. FEMS Microbiol Lett 334:127–134PubMedCrossRefGoogle Scholar
  20. Van Bogaert INA, De Maeseneire SL, Develter D, Soetaert W, Vandamme EJ (2008) Cloning and characterisation of the glyceraldehyde 3-phosphate dehydrogenase gene of Candida bombicola and use of its promoter. J Ind Microbiol Biotechnol 35:1085–1092PubMedCrossRefGoogle Scholar
  21. van Zutphen T, Baerends RJ, Susanna KA, de Jong A, Kuipers OP, Veenhuis M, van der Klei IJ (2010) Adaptation of Hansenula polymorpha to methanol: a transcriptome analysis. BMC Genomics 11:1PubMedCentralPubMedCrossRefGoogle Scholar
  22. Wang X, Sun Y, Ke F, Zhao H, Liu T, Xu L, Liu Y, Yan Y (2012) Constitutive expression of Yarrowia lipolytica lipase LIP2 in Pichia pastoris using GAP as promoter. Appl Biochem Biotechnol 166:1355–1367PubMedCrossRefGoogle Scholar
  23. Waterham HR, Digan ME, Koutz PJ, Lair SV, Cregg JM (1997) Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene 186:37–44PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg and the University of Milan 2013

Authors and Affiliations

  • Piyanun Harnpicharnchai
    • 1
  • Peerada Promdonkoy
    • 1
  • Kittapong Sae-Tang
    • 1
  • Niran Roongsawang
    • 1
  • Sutipa Tanapongpipat
    • 1
  1. 1.Bioresources Technology Unit, National Center for Genetic Engineering and BiotechnologyNational Science and Technology Development AgencyKhlong LuangThailand

Personalised recommendations