title: The vitamin-sensitive promoter PTHI11enables pre-defined autonomous induction of recombinant protein production in Pichia pastoris.
abstract: The methylotrophic yeast Pichia pastoris is widely used for production of recombinant proteins. Here we characterize a vitamin-sensitive regulatory sequence, which can be controlled independently of the main culture medium compounds such as carbon, nitrogen, or phosphor source. The THI11 promoter (PTHI11) sequence derives from a gene involved in biosynthesis of thiamine. For characterization, a P. pastoris strain expressing recombinant human serum albumin under control of PTHI11was grown in the controlled environment of a bioreactor. The thiamine sensitivity of PTHI11was proven and specified in batch cultures containing different amounts of extracellular thiamine. Under non-repressing conditions PTHI11offers a constitutive expression pattern with growth rate dependent product formation. Furthermore, promoter activity and thus product formation can be repressed for a desired period of time by supplementing the culture with a pre-defined amount of exogenous thiamine. Once a threshold of biomass is reached, PTHI11driven expression starts autonomously without external intervention. Based on these findings a tailor-made process strategy was developed and experimentally verified. Additionally, we compared the THI11 promoter with the commonly used GAP promoter. In conclusion, the THI11 promoter is a versatile and easy to control regulatory sequence which enables the realization of novel protein production strategies. Biotechnol. Bioeng. 2016;113: 2633-2643. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Biotechnology, BOKU-University of Natural Resources and Life Sciences Vienna, Muthgasse 18, Vienna 1190, Austria.|Austrian Centre of Industrial Biotechnology (ACIB GmbH), Vienna, Austria.
authors.forename: Nils
authors.initials: N
authors.lastname: Landes
authors.affiliation: Department of Biotechnology, BOKU-University of Natural Resources and Life Sciences Vienna, Muthgasse 18, Vienna 1190, Austria.|Austrian Centre of Industrial Biotechnology (ACIB GmbH), Vienna, Austria.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Gasser
authors.affiliation: Department of Biotechnology, BOKU-University of Natural Resources and Life Sciences Vienna, Muthgasse 18, Vienna 1190, Austria.
authors.forename: Karola
authors.initials: K
authors.lastname: Vorauer-Uhl
authors.affiliation: Department of Biotechnology, BOKU-University of Natural Resources and Life Sciences Vienna, Muthgasse 18, Vienna 1190, Austria.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Lhota
authors.affiliation: Department of Biotechnology, BOKU-University of Natural Resources and Life Sciences Vienna, Muthgasse 18, Vienna 1190, Austria. diethard.mattanovich@boku.ac.at.|Austrian Centre of Industrial Biotechnology (ACIB GmbH), Vienna, Austria. diethard.mattanovich@boku.ac.at.
authors.forename: Diethard
authors.initials: D
authors.lastname: Mattanovich
authors.affiliation: Austrian Centre of Industrial Biotechnology (ACIB GmbH), Vienna, Austria.|School of Bioengineering, University of Applied Sciences, FH Campus Vienna, Austria.
authors.forename: Michael
authors.initials: M
authors.lastname: Maurer
mesh_terms: D024861:Genetic Enhancement / Q000379:methods; D010843:Pichia / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D038621:Regulatory Sequences, Ribonucleic Acid / Q000235:genetics*; D013831:Thiamine / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D038621:Regulatory Sequences, Ribonucleic Acid; D013831:Thiamine
keywords: Komagataella; Pichia pastoris; THI11 promoter; recombinant protein; thiamine; yeast