Abstract
Natural tools for recombinant protein production show technological limitations. Available natural promoters for gene expression in Pichia pastoris are either constitutive, weak or require the use of undesirable substances or procedures for induction. Here we show the application of deletion variants based on the well known methanol inducible AOX1 promoter and small synthetic promoters, where cis-acting elements were fused to core promoter fragments. They enable differently regulated target protein expression and at the same time to replace methanol induction by a glucose or glycerol feeding strategy. Trypsinogen, the precursor of the serine protease trypsin, was expressed using these different promoters. Depending on the applied promoter the production window (i.e. the time of increasing product concentration) changed significantly. In fedbatch processes trypsinogen yields before induction with methanol were up to 10 times higher if variants of the AOX1 promoter were applied. In addition, the starting point of autoproteolytic product degradation can be predetermined by the promoter choice.
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Acknowledgments
We want to express our gratitude to VTU Technology for scientific and financial support. Furthermore the research was partially supported by the Swiss Academy of Engineering Sciences (SATW) in scope of the “Industrial Biotechnology” program (TK-07-19).
Ethical standards
We herewith declare that the experiments comply with the current laws of the country Austria.
Conflict of interest statement
This work was financed by VTU Technology. R. Weis is employed by VTU Technology. VTU Technology holds the patent WO2006089329 (Hartner and Glieder (2005) Mutant AOX1 promoters. WO2006089329). The majority of the promoters described in this study are part of this patent. Except those facts the authors for this journal article herewith declare that there is no conflict of interest and no financial relationship between the authors and the funding organization.
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Ruth, C., Zuellig, T., Mellitzer, A. et al. Variable production windows for porcine trypsinogen employing synthetic inducible promoter variants in Pichia pastoris . Syst Synth Biol 4, 181–191 (2010). https://doi.org/10.1007/s11693-010-9057-0
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DOI: https://doi.org/10.1007/s11693-010-9057-0