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Controlling Specific Growth Rate for Recombinant Protein Production by Pichia pastoris Under Oxidation Stress in Fed-batch Fermentation

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Abstract

Methanol can be used by Pichia pastoris as the carbon source and inducer to produce recombinant proteins in high-cell-density fermentations. However, methanol oxidation at high specific growth rates can lead to the reactive oxygen species (ROS) accumulation, resulting in cell damage. Here, we study the relationship between methanol feeding and ROS accumulation by controlling specific growth rate during the induction phase. A higher specific growth rate increased the level of ROS accumulation caused by methanol oxidation. While the cell growth rate was proportional to specific growth rate, maximum total protein production and highest enzyme activity were achieved at a specific growth rate of 0.05 1/h as compared to that of 0.065 1/h. Moreover, oxidative damage induced by over-accumulation of ROS in P. pastoris during the methanol induction phase caused cell death and reduced protein expression ability. ROS scavenging system analysis revealed that the higher specific growth rate, especially 0.065 1/h, resulted in increased intracellular catalase activity and decreased glutathione content significantly. Finally, Spearman’s correlation analysis further revealed that the reduced glutathione might be beneficial for maintaining cell viability and increasing protein production under oxidative stress caused by ROS toxic accumulation. Our findings suggest an integrated strategy to control the feeding of the essential substrate based on analyzing its response to oxidative stress caused by ROS toxic accumulation, as well as develop a strategy to optimize fed-batch fermentation.

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Data Availability

The data produced and/or analyzed in the current study are available from the corresponding author on reasonable request.

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References

  1. Shang, T., Si, D., Zhang, D., Liu, X., Zhao, L., Hu, C., Fu, Y., & Zhang, R. (2014). Enhancement of thermoalkaliphilic xylanase production by Pichia pastoris through novel fed-batch strategy in high cell-density fermentation. BMC Biotechnology, 17(1), 311–316.

    Google Scholar 

  2. Calik, P., Bayraktar, E., Inankur, B., Soyaslan, E. S., Sahin, M., Taspinar, H., & Ozdamar, T. H. (2010). Influence of pH on recombinant human growth hormone production by Pichia pastoris. Journal of Chemical Technology and Biotechnology, 85(12), 1628–1635.

    Article  CAS  Google Scholar 

  3. Wang, J., Wu, Z., Zhang, T., Wang, Y., & Yang, B. (2019). High-level expression of Thermomyces dupontii thermophilic lipase in Pichia pastoris via combined strategies. 3 Biotech, 9(2), 1–9.

    Article  Google Scholar 

  4. Su, X., Geng, X., Fu, M., Wu, Y., Yin, L., Zhao, F., & Chen, W. (2017). High-level expression and purification of a Mollusca endoglucanase from Ampullaria crossean in Pichia pastoris. Protein Expression & Purification, 139, 8–13.

    Article  CAS  Google Scholar 

  5. Yu, Y., Zhou, X., Wu, S., Wei, T., & Yu, L. (2014). High-yield production of the human lysozyme by Pichia pastoris SMD1168 using response surface methodology and high-cell-density fermentation. Electronic Journal of Biotechnology, 17(6), 311–316.

    Article  Google Scholar 

  6. Landolfo, S., Politi, H., Angelozzi, D., & Mannazzu, I. (2008). ROS accumulation and oxidative damage to cell structures in Saccharomyces cerevisiae wine strains during fermentation of high-sugar-containing medium. Biochimica et Biophysica Acta-General Subjects, 1780(6), 892–898.

    Article  CAS  Google Scholar 

  7. Ribeiro, T. P., Fernandes, C., Melo, K. V., Ferreira, S. S., & Horn, A. (2015). Iron, copper, and manganese complexes with in vitro superoxide dismutase and/or catalase activities that keep Saccharomyces cerevisiae cells alive under severe oxidative stress. Free Radical Biology and Medicine, 80, 67–76.

    Article  PubMed  CAS  Google Scholar 

  8. Zhang, W., Bevins, M. A., Plantz, B. A., Smith, L. A., & Meagher, M. M. (2000). Modeling Pichia pastoris growth on methanol and optimizing the production of a recombinant protein, the heavy-chain fragment C of botulinum neurotoxin, serotype A. Biotechnology and Bioengineering, 70(1), 1–8.

    Article  PubMed  CAS  Google Scholar 

  9. Kupcsulik, B., Sevella, B., Ballagi, A., & Kozma, J. (2001). Evaluation of three methanol feed strategies for recombinant Pichia pastoris muts fermentation. Acta Alimentaria, 30(1), 99–111.

    Article  CAS  Google Scholar 

  10. Gao, M., Dong, S., Yu, R., Wu, J., Zheng, Z., Shi, Z., & Zhan, X. (2011). Improvement of ATP regeneration efficiency and operation stability in porcine interferon-α production by Pichia pastoris under lower induction temperature. Korean Journal of Chemical Engineering, 28(6), 1412–1419.

    Article  CAS  Google Scholar 

  11. Gao, M., Li, Z., Yu, R., Wu, J., Zheng, Z., Shi, Z., Zhan, X., & Lin, C. (2012). Methanol/sorbitol co-feeding induction enhanced porcine interferon-α production by P. pastoris associated with energy metabolism shift. Bioprocess and Biosystems Engineering, 35(7), 1125–1136.

    Article  PubMed  CAS  Google Scholar 

  12. Gellissen, G. (2000). Heterologous protein production in methylotrophic yeasts. Applied Microbiology and Biotechnology, 54(6), 741–750.

    Article  PubMed  CAS  Google Scholar 

  13. Barrigón, J. M., Montesinos, J. L., & Valero, F. (2013). Searching the best operational strategies for Rhizopus oryzae lipase production in Pichia pastoris Mut+ phenotype: Methanol limited or methanol non-limited fed-batch cultures? Biochemical Engineering Journal, 75(1), 47–54.

    Article  Google Scholar 

  14. Dan, W., Chu, J., Hao, Y., Wang, Y., Zhuang, Y., & Zhang, S. (2011). High efficient production of recombinant human consensus interferon mutant in high cell density culture of Pichia pastoris using two phases methanol control. Process Biochemistry, 46(8), 1663–1669.

    Article  Google Scholar 

  15. Ren, H., & Yuan, J. (2005). Model-based specific growth rate control for Pichia pastoris to improve recombinant protein production. Journal of Chemical Technology & Biotechnology, 80(11), 1268–1272.

    Article  CAS  Google 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. Electronic Journal of Biotechnology, 22(4), 62–67.

    Article  Google Scholar 

  17. Yuan, D., Lan, D., Xin, R., Yang, B., & Wang, Y. (2016). Screening and characterization of a thermostable lipase from marine Streptomyces sp. strain W007. Biotechnology & Applied Biochemistry, 63(1), 41–50.

    Article  CAS  Google Scholar 

  18. Li, H., & Xia, Y. (2019). High-yield production of spider short-chain insecticidal neurotoxin Tx4(6–1) in Pichia pastoris and bioactivity assays in vivo. Protein Expression and Purification, 154, 66–73.

    Article  PubMed  CAS  Google Scholar 

  19. Zhang, X., Ai, Y., Xu, Y., & Yu, X. (2019). High-level expression of Aspergillus niger lipase in Pichia pastoris: Characterization and gastric digestion in vitro. Food chemistry, 247, 305–313.

    Article  Google Scholar 

  20. Sinha, J., Plantz, B. A., Inan, M., & Meagher, M. M. (2010). Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: Case study with recombinant ovine interferon-tau. Biotechnology and bioengineering, 89(1), 102–112.

    Article  Google Scholar 

  21. Liu, T., Zhu, L., Wang, J., Wang, J., Zhang, J., Sun, X., & Zhang, C. (2015). Biochemical toxicity and DNA damage of imidazolium-based ionic liquid with different anions in soil on Vicia faba seedlings. Scientific reports, 5(1), 18444–18453.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Zepeda, A. B., Figueroa, C. A., Pessoa, A., & Farías, J. G. (2018). Free fatty acids reduce metabolic stress and favor a stable production of heterologous proteins in Pichia pastoris. Brazilian journal of microbiology, 49(4), 856–864.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Livak, K., & Schmittgen, T. (2000). Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△Ct method. Methods, 25(4), 402–408.

    Article  Google Scholar 

  24. Zhang, W., Potter, K. J. H., Plantz, B. A., Schlegel, V. L., Smith, L. A., & Meagher, M. M. (2003). Pichia pastoris fermentation with mixed-feeds of glycerol and methanol: Growth kinetics and production improvement. Journal of industrial microbiology & biotechnology, 30(4), 210–215.

    Article  CAS  Google Scholar 

  25. Rebnegger, C., Vos, T., Graf, A. B., Valli, M., Pronk, J. T., Daran-Lapujade, P. A. S., & Mattanovicha, D. (2016). Pichia Pastoris exhibits high viability and a low maintenance energy requirement at near-zero specific growth rates. Applied & Environmental Microbiology, 82(15), 4570–4583.

    Article  CAS  Google Scholar 

  26. Sinha, J., Plantz, B. A., Zhang, W., Gouthro, M., Schlegel, V., Liu, C. P., & Meagher, M. M. (2003). Improved production of recombinant ovine interferon-τ by Mut+ strain of Pichia pastoris using an optimized methanol feed profile. Biotechnology Progress, 19(3), 794–802.

    Article  PubMed  CAS  Google Scholar 

  27. Rahimi, A., Hosseini, S. N., Jauidanbardan, A., & Khatami, M. (2019). Continuous fermentation of recombinant Pichia pastoris Mut(+) producing HBsAg: Optimizing dilution rate and determining strain-specific parameters. Food & Bioproducts Processing, 118(part C), 248–257.

    Article  CAS  Google Scholar 

  28. Gao, M., & Shi, Z. (2013). Process control and optimization for heterologous protein production by methylotrophic Pichia pastoris. Chinese Journal of Chemical Engineering, 21(2), 216–226.

    Article  CAS  Google Scholar 

  29. Liu, W., Inwood, S., Gong, T., Sharma, A., Yu, L., & Zhu, P. (2019). Fed-batch high-cell-density fermentation strategies for Pichia pastoris growth and production. Critical Reviews in Biotechnology, 39(2), 258–271.

    Article  PubMed  CAS  Google Scholar 

  30. Gasser, B., Prielhofer, R., & Marx, H. (2013). Pichia pastoris: Protein production host and model organism for biomedical research. Future Microbiology, 8(2), 191–208.

    Article  PubMed  CAS  Google Scholar 

  31. Liu, L., Yang, H., Shin, H., Chen, R., Li, J., Du, G., & Chen, J. (2013). How to achieve high-level expression of microbial enzymes Strategies and perspectives. Bioengineered, 4(4), 212–223.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Wu, D., Zhu, H., Chu, J., & Wu, J. (2019). N-acetyltransferase co-expression increases α-glucosidase expression level in Pichia pastoris. Journal of biotechnology, 289, 26–30.

    Article  PubMed  CAS  Google Scholar 

  33. Lee, J., Won, Y., Park, K., Lee, M., Tachibana, H., Yamada, K., & Seo, K. (2012). Celastrol inhibits growth and induces apoptotic cell death in melanoma cells via the activation ROS-dependent mitochondrial pathway and the suppression of PI3K/AKT signaling. Apoptosis, 17(12), 1275–1286.

    Article  PubMed  CAS  Google Scholar 

  34. Costa, V. M. V., Amorim, M. A., Quintanilha, A., & Moradas-Ferreira, P. (2002). Hydrogen peroxide-induced carbonylation of key metabolic enzymes in Saccharomyces cerevisiae: The involvement of the oxidative stress response regulators Yap1 and Skn7. Free Radical Biology and Medicine, 33(11), 1507–1515.

    Article  PubMed  CAS  Google Scholar 

  35. Halliwell, B., & Gutteridge, J. M. C. (1985). Free radicals in biology and medicine (5th ed.). Oxford University Press.

    Google Scholar 

  36. Fahey, C. R. (2001). Novel Thiols of Prokaryotes. Annual Review of Microbiology, 55, 333–356.

    Article  PubMed  CAS  Google Scholar 

  37. Delic, M., Rebnegger, C., Wanka, F., Puxbaum, V., Haberhauer-Troyer, C., Hann, S., Kollensperger, G., Mattanovich, D., & Gasser, B. (2012). Oxidative protein folding and unfolded protein response elicit differing redox regulation in endoplasmic reticulum and cytosol of yeast. Free Radical Biology and Medicine, 52(9), 2000–2012.

    Article  PubMed  CAS  Google Scholar 

  38. Tessoulin, B., Descamps, G., Moreau, P., Maiga, S., Lode, L., Godon, C., Marionneau-Lambot, S., Oullier, T., Le, G. S., Amiot, M., & Pellat-Deceunynck, C. (2014). PRIMA-1Met induces myeloma cell death independent of p53 by impairing the GSH/ROS balance. Blood, 124(10), 1626–1636.

    Article  PubMed  CAS  Google Scholar 

  39. Liu, Z., Zhang, M., Han, X., Xu, H., Zhang, B., Yu, Q., & Li, M. (2016). TiO2 nanoparticles cause cell damage independent of apoptosis and autophagy by impairing the ROS-scavenging system in Pichia pastoris. Chemico-Biological Interactions, 252, 9–18.

    Article  PubMed  CAS  Google Scholar 

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Funding

This work was supported by the National Key R & D Program of China (2018YFC0311104), National Science Fund for Distinguished Young Scholars (31725022), Key Program of Natural Science Foundation of China (31930084), Guangdong marine economy promotion projects (MEPP) Fund (no. GDOE[2019]A20).

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Authors and Affiliations

  1. School of Food Science and Engineering, South China University of Technology, Guangzhou, Guangdong, 510640, People’s Republic of China

    Rongkang Hu, Ruiguo Cui, Qingqing Xu, Dongming Lan & Yonghua Wang

  2. College of Life Sciences, Anhui Normal University, Wuhu, Anhui, 241000, People’s Republic of China

    Rongkang Hu

  3. Guangdong Youmei Institute of Intelligent Bio-Manufacturing Co., Ltd, Foshan, Guangdong, 528200, People’s Republic of China

    Rongkang Hu, Ruiguo Cui, Qingqing Xu, Dongming Lan & Yonghua Wang

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  1. Rongkang Hu
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  4. Dongming Lan
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  5. Yonghua Wang
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Contributions

Rongkang Hu and Yonghua Wang designed the study. Rongkang Hu did experimental work. Rongkang Hu, Ruiguo Cui, and Yonghua Wang collected and analyzed the data. Rongkang Hu wrote the manuscript with support from Qingqing Xu, Dongming Lan, Ruiguo Cui, and Yonghua Wang. All the authors read and approved the manuscript for publication.

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Correspondence to Yonghua Wang.

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Hu, R., Cui, R., Xu, Q. et al. Controlling Specific Growth Rate for Recombinant Protein Production by Pichia pastoris Under Oxidation Stress in Fed-batch Fermentation. Appl Biochem Biotechnol 194, 6179–6193 (2022). https://doi.org/10.1007/s12010-022-04022-3

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  • DOI: https://doi.org/10.1007/s12010-022-04022-3

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