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Small-scale slow glucose feed cultivation of Pichia pastoris without repression of AOX1 promoter: towards high throughput cultivations

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Abstract

Recombinant protein synthesis in Pichia pastoris is generally controlled by the strong methanol inducible AOX1 promoter which is repressed by glucose and glycerol. In shake flasks, commonly one or two methanol pulses are added per day for induction. Such pulse feeding procedure leads to carbon starvation phases, which may enhance proteolytic activities and, therefore, cause product losses. Starvation between the methanol pulses could be avoided with a continuous enzymatic feed of glucose from a glucose-based polymer. The amount of glucose was low enough to prevent AOX1 repression by glucose. Energy and carbon were continuously supplied for cell maintenance resulting in significantly increased cell densities and product activities, as shown here at the example of a fungal lipase expressed in P. pastoris. A threefold improvement in measured product activity was obtained by applying enzymatic glucose feed and a further improvement was achieved by applying a defined mixture of ammonium compounds. The strategy described here simplifies the general procedure in shaken cultures by allowing the direct continuation of the cultivation from glucose to the methanol-based production phase without a medium change. It is easily applicable to multiwell plates and thus beneficial for high throughput applications.

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Acknowledgments

The authors wish to thank Mrs. Lilja Tuohimaa, Mrs. Tuula Karppinen, B.Sc Henna Karppinen, and M.Sc Ville-Hermanni Sotaniemi for their skillful technical assistance. Prof. Pau Ferrer is gratefully acknowledged for providing the Pichia pastoris strains and the anti-ROL antibody. Dr. David Resina, Dr. Monika Bollok and the other members of the ERA-IB EngBioCat consortium are thanked for many helpful discussions. This study was funded by the Finnish Funding Agency for Technology and Innovation (Tekes), decision number 40345. The project was part of ERA-IB-project implementing an enzyme engineering technology platform for the provision of tailor-made enzymes (EngBioCat). Also Finnish Foundation for Technology Promotion and Tauno Tönning Research Foundation are thanked for the financial support.

Conflict of interest

JPP, AV and PN are co-founders and minor shareholders of BioSilta Oy. AV is R&D director, and PN is a scientific advisor of the company. AM, HO and JK declare no competing interests.

Author information

Author notes

  1. Heikki Ojamo

    Present address: Department of Biotechnology and Chemical Technology, School of Chemical Technology, Aalto University, P.O. BOX 16100, 00076, Aalto, Finland

  2. Anu Mursula

    Present address: Department of Biochemistry and Biocenter Oulu, University of Oulu, P.O. Box 3000, 90014, Oulu, Finland

Authors and Affiliations

  1. Bioprocess Engineering Laboratory, Department of Process and Environmental Engineering, University of Oulu, P.O. Box 4300, 90014, Oulu, Finland

    Johanna Panula-Perälä, Janne Karhunen, Heikki Ojamo & Anu Mursula

  2. BioSilta Oy, Biologintie 1H4, 90570, Oulu, Finland

    Antti Vasala

  3. Chair of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Ackerstr. 71-76, ACK24, 13355, Berlin, Germany

    Peter Neubauer

Authors
  1. Johanna Panula-Perälä
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Corresponding author

Correspondence to Johanna Panula-Perälä.

Electronic supplementary material

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449_2013_1098_MOESM1_ESM.tif

Supplementary Fig. 1 Western blot analysis of expressed ROL after 64 h of cultivation. Samples were taken from induced cultivations (BMEB or BSEB) made with different concentrations of the glucose-releasing enzyme (0.5, 1 or 2 U l−1), from negative control cultivation (WT) and reference cultivation with BMM medium. Equal volumes of culture supernatant were loaded to the gel. BMEB buffered minimal medium including the glucose polymer but no glycerol. BSEB BioSilta’s prototype EnBase medium version. BMM buffered minimal methanol medium. ROL was detected by using suitable antibodies and a horseradish peroxidase-coupled color reagent (a) and by ECL (b). In b, the higher concentrations of ROL were detected as white bands because of over exposure of the detection film. (TIFF 117 kb)

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Panula-Perälä, J., Vasala, A., Karhunen, J. et al. Small-scale slow glucose feed cultivation of Pichia pastoris without repression of AOX1 promoter: towards high throughput cultivations. Bioprocess Biosyst Eng 37, 1261–1269 (2014). https://doi.org/10.1007/s00449-013-1098-9

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  • DOI: https://doi.org/10.1007/s00449-013-1098-9

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