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Novel helper factors influencing recombinant protein production in Pichia pastoris based on proteomic analysis under simulated microgravity

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Abstract

Microgravity and simulated microgravity (SMG) have quite significant effects on numerous microbial cellular processes. The effects of SMG on the production of recombinant proteins and transcription profiling in prokaryotic and eukaryotic expression host have been investigated. The present study showed that SMG significantly enhanced the specific productivities and activities of the reporter enzymes PGUS and AtXYN that were expressed in recombinant Pichia pastoris. Proteomic profiling revealed that 21 proteins were significantly up-regulated and 35 proteins were drastically down-regulated at the stationary phase, when the recombinant P. pastoris responded to SMG. Six strongly up-regulated genes, TPX, FBA, PGAM, ENO, SBA1, and AKR-E, involved in the oxidative stress response, methanol metabolism, glycolytic pathway, and protein folding, were selected to analyze their impacts on recombinant protein production by co-overexpression in the shaker flask fermentation. The co-overexpressed strains, particularly TPX, FBA, and PGAM, demonstrated promising results with approximately 2.46-fold, 1.58-fold, and 1.33-fold increases in the specific yields of PGUS compared to the control after 48 h of methanol induction, respectively. In the meantime, the corresponding PGUS specific activities were increased by 2.33-fold, 2.09-fold, and 1.32-fold, respectively. Thiol peroxidase (TPX), which is involved in the oxidative stress response, significantly influenced the transcriptional levels of the reporter gene PGUS. The present study provides valuable information for further exploration of the molecular mechanism of P. pastoris response to SMG and facilitates simulated microgravity for finding novel helper factors to rationally engineer the strains in normal fermentation by using proteomic studies.

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Acknowledgments

This work was financially supported by the National High Technology Research and Development Program of China (863 Program) (No. 2012AA02A704), the Major State Basic Research Development Program of China (973 Program) (No. 2013CB733900), and the Natural Science Foundation of Beijing (No. 2112035).

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The authors declare that no competing financial interests exist.

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Authors and Affiliations

  1. School of Life Science, Beijing Institute of Technology, 100081, Beijing, China

    Jie Huangfu, Feng Qi, Muhammad Saad Ahmed & Chun Li

  2. National Chromatography R&A Centre, Dalian Institute of Chemical Physics, CAS, Dalian, 116023, China

    Hongwei Liu & Hanfa Zou

  3. College of Life Sciences/Engineering Research Center of Industrial Microbiology, Fujian Normal University, Fuzhou, 350108, China

    Feng Qi

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  1. Jie Huangfu
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  2. Feng Qi
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  3. Hongwei Liu
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  4. Hanfa Zou
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  5. Muhammad Saad Ahmed
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  6. Chun Li
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Correspondence to Chun Li.

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Jie Huangfu and Feng Qi contributed equally to this study

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Huangfu, J., Qi, F., Liu, H. et al. Novel helper factors influencing recombinant protein production in Pichia pastoris based on proteomic analysis under simulated microgravity. Appl Microbiol Biotechnol 99, 653–665 (2015). https://doi.org/10.1007/s00253-014-6175-8

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  • DOI: https://doi.org/10.1007/s00253-014-6175-8

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