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Superior protein titers in half the fermentation time: Promoter and process engineering for the glucose‐regulated GTH1 promoter of Pichia pastoris

Protein production in Pichia pastoris is often based on the methanol‐inducible P (AOX1) promoter which drives the expression of the target gene. The use of methanol has major drawbacks, so there is a demand for alternative promoters with good induction properties such as the glucose‐regulated P (GTH...

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Autores principales: Prielhofer, Roland, Reichinger, Michaela, Wagner, Nina, Claes, Katrien, Kiziak, Christoph, Gasser, Brigitte, Mattanovich, Diethard
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6221138/
https://www.ncbi.nlm.nih.gov/pubmed/30016537
http://dx.doi.org/10.1002/bit.26800
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author Prielhofer, Roland
Reichinger, Michaela
Wagner, Nina
Claes, Katrien
Kiziak, Christoph
Gasser, Brigitte
Mattanovich, Diethard
author_facet Prielhofer, Roland
Reichinger, Michaela
Wagner, Nina
Claes, Katrien
Kiziak, Christoph
Gasser, Brigitte
Mattanovich, Diethard
author_sort Prielhofer, Roland
collection PubMed
description Protein production in Pichia pastoris is often based on the methanol‐inducible P (AOX1) promoter which drives the expression of the target gene. The use of methanol has major drawbacks, so there is a demand for alternative promoters with good induction properties such as the glucose‐regulated P (GTH1) promoter which we reported recently. To further increase its potential, we investigated its regulation in more details by the screening of promoter variants harboring deletions and mutations. Thereby we could identify the main regulatory region and important putative transcription factor binding sites of P (GTH1). Concluding from that, yeast metabolic regulators, monomeric Gal4‐class motifs, carbon source‐responsive elements, and yeast GC‐box proteins likely contribute to the regulation of the promoter. We engineered a P (GTH1) variant with greatly enhanced induction properties compared with that of the wild‐type promoter. Based on that, a model‐based bioprocess design for high volumetric productivity in a limited time was developed for the P (GTH1) variant, to employ a glucose fed‐batch strategy that clearly outperformed a classical methanol fed‐batch of a P (AOX1) strain in terms of titer and process performance.
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spelling pubmed-62211382018-11-15 Superior protein titers in half the fermentation time: Promoter and process engineering for the glucose‐regulated GTH1 promoter of Pichia pastoris Prielhofer, Roland Reichinger, Michaela Wagner, Nina Claes, Katrien Kiziak, Christoph Gasser, Brigitte Mattanovich, Diethard Biotechnol Bioeng Articles Protein production in Pichia pastoris is often based on the methanol‐inducible P (AOX1) promoter which drives the expression of the target gene. The use of methanol has major drawbacks, so there is a demand for alternative promoters with good induction properties such as the glucose‐regulated P (GTH1) promoter which we reported recently. To further increase its potential, we investigated its regulation in more details by the screening of promoter variants harboring deletions and mutations. Thereby we could identify the main regulatory region and important putative transcription factor binding sites of P (GTH1). Concluding from that, yeast metabolic regulators, monomeric Gal4‐class motifs, carbon source‐responsive elements, and yeast GC‐box proteins likely contribute to the regulation of the promoter. We engineered a P (GTH1) variant with greatly enhanced induction properties compared with that of the wild‐type promoter. Based on that, a model‐based bioprocess design for high volumetric productivity in a limited time was developed for the P (GTH1) variant, to employ a glucose fed‐batch strategy that clearly outperformed a classical methanol fed‐batch of a P (AOX1) strain in terms of titer and process performance. John Wiley and Sons Inc. 2018-08-08 2018-10 /pmc/articles/PMC6221138/ /pubmed/30016537 http://dx.doi.org/10.1002/bit.26800 Text en © 2018 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
spellingShingle Articles
Prielhofer, Roland
Reichinger, Michaela
Wagner, Nina
Claes, Katrien
Kiziak, Christoph
Gasser, Brigitte
Mattanovich, Diethard
Superior protein titers in half the fermentation time: Promoter and process engineering for the glucose‐regulated GTH1 promoter of Pichia pastoris
title Superior protein titers in half the fermentation time: Promoter and process engineering for the glucose‐regulated GTH1 promoter of Pichia pastoris
title_full Superior protein titers in half the fermentation time: Promoter and process engineering for the glucose‐regulated GTH1 promoter of Pichia pastoris
title_fullStr Superior protein titers in half the fermentation time: Promoter and process engineering for the glucose‐regulated GTH1 promoter of Pichia pastoris
title_full_unstemmed Superior protein titers in half the fermentation time: Promoter and process engineering for the glucose‐regulated GTH1 promoter of Pichia pastoris
title_short Superior protein titers in half the fermentation time: Promoter and process engineering for the glucose‐regulated GTH1 promoter of Pichia pastoris
title_sort superior protein titers in half the fermentation time: promoter and process engineering for the glucose‐regulated gth1 promoter of pichia pastoris
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6221138/
https://www.ncbi.nlm.nih.gov/pubmed/30016537
http://dx.doi.org/10.1002/bit.26800
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