3 Biotech

, 9:62 | Cite as

High-level expression of Thermomyces dupontii thermophilic lipase in Pichia pastoris via combined strategies

  • Jianrong Wang
  • Zongze Wu
  • Tianyu Zhang
  • Yonghua Wang
  • Bo YangEmail author
Original Article


In this study, a combined strategy was used to improve the production of Thermomyces dupontii lipase (TDL) in Pichia pastoris. First, the native gene of TDL was optimized based on the codon usage of P. pastoris, ligated to pPICZαA and transformed in P. pastoris X33. A recombinant strain designated X33-T23 with the highest activity (1020 U/mL in shake flasks) amongst 216 recombinant colonies was selected for further investigations. To further increase the production of TDL, nine different secretion helper factor genes were transformed in the recombinant strain, X33-T23. The recombinant strain co-expression with the gene encoding protein disulfide isomerase, designated X33-T23-PDI, exhibited the highest activity in shake flasks (1760 U/mL) and in 5 L bioreactor (57521 U/mL) which were 1.67- and 1.46-fold higher, respectively, than for strain X33-T23. Additionally, the optimization of the inducers (temperature and pH) for the recombinant strain X33-T23-PDI in 5 L bioreactor produced, as expected, much higher lipase activity (81203 U/mL). The results of this study will provide an effective method to produce TDL and give some clues on how to improve production of heterologous proteins in P. pastoris.


Codon optimization Pichia pastoris Secretion helper factor Thermophilic lipase Thermomyces dupontii 



This work was supported by the National High Technology Project of P.R. China (No. 2014AA093514).

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest.

Supplementary material

13205_2019_1597_MOESM1_ESM.docx (248 kb)
Supplementary material 1 (DOCX 248 KB)


  1. Ahmad M, Hirz M, Pichler H, Schwab H (2014) Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol 98:5301–5317CrossRefGoogle Scholar
  2. Borrelli GM, Trono D (2015) Recombinant lipases and phospholipases and their use as biocatalysts for industrial applications. Int J Mol Sci 16:20774–20840CrossRefGoogle Scholar
  3. Chuang SM, Chen L, Lambertson D, Anand M, Kinzy TG, Madura K (2005) Proteasome-mediated degradation of cotranslationally damaged proteins involves translation elongation factor 1A. Mol Cell Biol 25:403–413CrossRefGoogle Scholar
  4. Coghlan A, Wolfe KH (2000) Relationship of codon bias to mRNA concentration and protein length in Saccharomyces cerevisiae. Yeast 16:1131–1145CrossRefGoogle Scholar
  5. Damasceno LM, Anderson KA, Ritter G, Cregg JM, Old LJ, Batt CA (2007) Cooverexpression of chaperones for enhanced secretion of a single-chain antibody fragment in Pichia pastoris. Appl Microbiol Biotechnol 74:381–389CrossRefGoogle Scholar
  6. Delic M, Valli M, Graf AB, Pfeffer M, Mattanovich D, Gasser B (2013) The secretory pathway: exploring yeast diversity. FEMS Microbiol Rev 37:872–914CrossRefGoogle Scholar
  7. Delic M, Göngrich R, Mattanovich D, Gasser B (2014) Engineering of protein folding and secretion-strategies to overcome bottlenecks for efficient production of recombinant proteins. Antioxid Redox Signal 21:414–437CrossRefGoogle Scholar
  8. Ding X, Zheng RC, Tang XL, Zheng YG (2018) Engineering of Talaromyces thermophilus lipase by altering its crevice-like binding site for highly efficient biocatalytic synthesis of chiral intermediate of Pregablin. Bioorg Chem 77:330–338CrossRefGoogle Scholar
  9. Dragosits M, Stadlmann J, Albiol J, Baumann K, Maurer M, Gasser B, Sauer M, Altmann F, Ferrer P, Mattanovich D (2009) The effect of temperature on the proteome of recombinant Pichia pastoris. J Proteome Res 8:1380–1392CrossRefGoogle Scholar
  10. Gasser B, Sauer M, Maurer M, Stadlmayr G, Mattanovich D (2007) Transcriptomics-based identification of novel factors enhancing heterologous protein secretion in yeasts. Appl Environ Microbiol 73:6499–6507CrossRefGoogle Scholar
  11. Gu L, Zhang J, Du G, Chen J (2015) Multivariate modular engineering of the protein secretory pathway for production of heterologous glucose oxidase in Pichia pastoris. Enzyme Microb Tech 68:33–42CrossRefGoogle Scholar
  12. Guerfal M, Ryckaert S, Jacobs PP, Ameloot P, Van Craenenbroeck K, Derycke R, Callewaert N (2010) The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed and membrane proteins. Microb Cell Fact 9:49CrossRefGoogle Scholar
  13. Idiris A, Tohda H, Kumagai H, Takegawa K (2010) Engineering of protein secretion in yeast: strategies and impact on protein production. Appl Microbiol Biotechnol 86:403–417CrossRefGoogle Scholar
  14. Jaeger KE, Eggert T (2002) Lipases for biotechnology. Curr Opin Biotechnol 13:390–397CrossRefGoogle Scholar
  15. Klabunde J, Kleebank S, Piontek M, Hollenberg CP, Hellwig S, Degelmann A (2007) Increase of calnexin gene dosage boosts the secretion of heterologous proteins by Hansenula polymorpha. FEMS Yeast Res 7:1168–1180CrossRefGoogle Scholar
  16. Lan D, Qu M, Yang B, Wang Y (2016) Enhancing production of lipase MAS1 from marine Streptomyces sp. strain in Pichia pastoris by chaperones co-expression. Electron J Biotechn 22:62–67CrossRefGoogle Scholar
  17. Li Z, Xiong F, Lin Q, d’Anjou M, Daugulis AJ, Yang DS, Hew CL (2001) Low-temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris. Protein Expr Purif 21:438–445CrossRefGoogle Scholar
  18. Li X, Liu Z, Wang G, Pan D, Jiao L, Yan Y (2016) Overexpression of Candida rugosa lipase Lip1 via combined strategies in Pichia pastoris. Enzyme Microb Tech 82:115–124CrossRefGoogle Scholar
  19. Li J, Sun C, Chen L, Sun L, Duan L, Zheng Q, Hu X (2017) Optimization of the secretory expression of recombinant human C-reactive protein in Pichia pastoris. 3 Biotech 7:291CrossRefGoogle Scholar
  20. Marín A, Gallardo M, Kato Y, Shirahige K, Gutiérrez G, Ohta K, Aguilera A (2003) Relationship between G + C content, ORF-length and mRNA concentration in Saccharomyces cerevisiae. Yeast 20:703–711CrossRefGoogle Scholar
  21. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45CrossRefGoogle Scholar
  22. Romdhane IB, Fendri A, Gargouri Y, Gargouri A, Belghith H (2010) A novel thermoactive and alkaline lipase from Talaromyces thermophilus fungus for use in laundry detergents. Biochem Eng J 53:112–120CrossRefGoogle Scholar
  23. Romdhane IB, Romdhane ZB, Gargouri A, Belghith H (2011) Esterification activity and stability of Talaromyces thermophilus lipase immobilized onto chitosan. J Mol Catal B-Enzym 68:230–239CrossRefGoogle Scholar
  24. Romdhane IB, Romdhane ZB, Bouzid M, Gargouri A, Belghith H (2013) Application of a chitosan-immobilized Talaromyces thermophilus lipase to a batch biodiesel production from waste frying oils. Appl Biochem Biotechnol 171:1986–2002CrossRefGoogle Scholar
  25. Samuel P, Prasanna Vadhana AK, Kamatchi R, Antony A, Meenakshisundaram S (2013) Effect of molecular chaperones on the expression of Candida antarctica lipase B in Pichia pastoris. Microbiol Res 168:615–620CrossRefGoogle Scholar
  26. Sha C, Yu XW, Lin NX, Zhang M, Xu Y (2013a) Enhancement of lipase r27RCL production in Pichia pastoris by regulating gene dosage and co-expression with chaperone protein disulfide isomerase. Enzyme Microb Tech 53:438–443CrossRefGoogle Scholar
  27. Sha C, Yu XW, Li F, Xu Y (2013b) Impact of gene dosage on the production of lipase from Rhizopus chinensis CCTCC M201021 in Pichia pastoris. Appl Biochem Biotechnol 169:1160–1172CrossRefGoogle Scholar
  28. Singh R, Kumar M, Mittal A, Mehta PK (2016) Microbial enzymes: industrial progress in 21st century. 3 Biotech 6:174CrossRefGoogle Scholar
  29. Wang Y, Wang Z, Xu Q, Du G, Hua Z (2009) Lowering induction temperature for enhanced production of polygalacturonate lyase in recombinant Pichia pastoris. Process Biochem 44:949–954CrossRefGoogle Scholar
  30. Wang JR, Li YY, Xu SD, Li P, Liu JS, Liu DN (2013) High-level expression of pro-form lipase from Rhizopus oryzae in Pichia pastoris and its purification and characterization. Int J Mol Sci 15:203–217CrossRefGoogle Scholar
  31. Wang JR, Li YY, Liu D (2016) Improved production of Aspergillus usamii endo-β-1,4-xylanase in Pichia pastoris via combined strategies. Biomed Res Int 2:3265895Google Scholar
  32. Wu M, Liu W, Yang G, Yu D, Lin D, Sun H, Chen S (2014) Engineering of a Pichia pastoris expression system for high-level secretion of HSA/GH fusion protein. Appl Biochem Biotechnol 172:2400–2411CrossRefGoogle Scholar
  33. Yang Z, Zhang Z (2018) Engineering strategies for enhanced production of protein and bio-products in Pichia pastoris: A review. Biotechnol Adv 36:182–195CrossRefGoogle Scholar
  34. Yu P, Zhu Q, Chen K, Lv X (2015) Improving the secretory production of the heterologous protein in Pichia pastoris by focusing on protein folding. Appl Biochem Biotechnol 175:535–548CrossRefGoogle Scholar
  35. Zhang X, Li X, Xia L (2015) Expression of a thermo-alkaline lipase gene from Talaromyces thermophilus in recombinant Pichia pastoris. Biochem Eng J 103:263–269CrossRefGoogle Scholar
  36. Zhao X, Huo KK, Li YY (2000) Synonymous codon usage in Pichia pastoris. Chin J Biotechnol 16:308–311. (in Chinese)Google Scholar
  37. Zhong Y, Yang L, Guo Y, Fang F, Wang D, Li R, Jiang M, Kang W, Ma J, Sun J, Xiao W (2014) High-temperature cultivation of recombinant Pichia pastoris increases endoplasmic reticulum stress and decreases production of human interleukin-10. Microb Cell Fact 13:163CrossRefGoogle Scholar

Copyright information

© King Abdulaziz City for Science and Technology 2019

Authors and Affiliations

  • Jianrong Wang
    • 1
  • Zongze Wu
    • 1
  • Tianyu Zhang
    • 1
  • Yonghua Wang
    • 2
  • Bo Yang
    • 1
    Email author
  1. 1.School of Bioscience and BioengineeringSouth China University of TechnologyGuangzhouPeople’s Republic of China
  2. 2.School of Food Science and EngineeringSouth China University of TechnologyGuangzhouPeople’s Republic of China

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