Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris
BACKGROUND: Pichia pastoris is a powerful and broadly used host for recombinant protein production (RPP), where past bioprocess performance has often been directed with the methanol regulated AOX1 promoter (P(AOX1)), and the constitutive GAP promoter (P(GAP)). Since promoters play a crucial role in...
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ftpubmed:oai:pubmedcentral.nih.gov:7986505 2023-05-15T14:00:14+02:00 Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris Garrigós-Martínez, Javier Vuoristo, Kiira Nieto-Taype, Miguel Angel Tähtiharju, Juha Uusitalo, Jaana Tukiainen, Pauliina Schmid, Christian Tolstorukov, Ilya Madden, Knut Penttilä, Merja Montesinos-Seguí, José Luis Valero, Francisco Glieder, Anton Garcia-Ortega, Xavier 2021-03-23 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986505/ https://doi.org/10.1186/s12934-021-01564-9 en eng BioMed Central http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986505/ http://dx.doi.org/10.1186/s12934-021-01564-9 © The Author(s) 2021 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. CC0 PDM CC-BY Microb Cell Fact Research Text 2021 ftpubmed https://doi.org/10.1186/s12934-021-01564-9 2021-03-28T01:48:33Z BACKGROUND: Pichia pastoris is a powerful and broadly used host for recombinant protein production (RPP), where past bioprocess performance has often been directed with the methanol regulated AOX1 promoter (P(AOX1)), and the constitutive GAP promoter (P(GAP)). Since promoters play a crucial role in an expression system and the bioprocess efficiency, innovative alternatives are constantly developed and implemented. Here, a thorough comparative kinetic characterization of two expression systems based on the commercial PDF and UPP promoters (P(PDF), P(UPP)) was first conducted in chemostat cultures. Most promising conditions were subsequently tested in fed-batch cultivations. These new alternatives were compared with the classical strong promoter P(GAP), using the Candida antarctica lipase B (CalB) as model protein for expression system performance. RESULTS: Both the P(PDF) and P(UPP)-based expression systems outperformed similar P(GAP)-based expression in chemostat cultivations, reaching ninefold higher specific production rates (q(p)). CALB transcription levels were drastically higher when employing the novel expression systems. This higher expression was also correlated with a marked upregulation of unfolded protein response (UPR) related genes, likely from an increased protein burden in the endoplasmic reticulum (ER). Based on the chemostat results obtained, best culture strategies for both P(PDF) and P(UPP) expression systems were also successfully implemented in 15 L fed-batch cultivations where q(p) and product to biomass yield (Y(P/X)*) values were similar than those obtained in chemostat cultivations. CONCLUSIONS: As an outcome of the macrokinetic characterization presented, the novel P(PDF) and P(UPP) were observed to offer much higher efficiency for CalB production than the widely used P(GAP)-based methanol-free alternative. Thus, both systems arise as highly productive alternatives for P. pastoris-based RPP bioprocesses. Furthermore, the different expression regulation patterns observed indicate the ... Text Antarc* Antarctica PubMed Central (PMC) Microbial Cell Factories 20 1 |
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Research Garrigós-Martínez, Javier Vuoristo, Kiira Nieto-Taype, Miguel Angel Tähtiharju, Juha Uusitalo, Jaana Tukiainen, Pauliina Schmid, Christian Tolstorukov, Ilya Madden, Knut Penttilä, Merja Montesinos-Seguí, José Luis Valero, Francisco Glieder, Anton Garcia-Ortega, Xavier Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris |
topic_facet |
Research |
description |
BACKGROUND: Pichia pastoris is a powerful and broadly used host for recombinant protein production (RPP), where past bioprocess performance has often been directed with the methanol regulated AOX1 promoter (P(AOX1)), and the constitutive GAP promoter (P(GAP)). Since promoters play a crucial role in an expression system and the bioprocess efficiency, innovative alternatives are constantly developed and implemented. Here, a thorough comparative kinetic characterization of two expression systems based on the commercial PDF and UPP promoters (P(PDF), P(UPP)) was first conducted in chemostat cultures. Most promising conditions were subsequently tested in fed-batch cultivations. These new alternatives were compared with the classical strong promoter P(GAP), using the Candida antarctica lipase B (CalB) as model protein for expression system performance. RESULTS: Both the P(PDF) and P(UPP)-based expression systems outperformed similar P(GAP)-based expression in chemostat cultivations, reaching ninefold higher specific production rates (q(p)). CALB transcription levels were drastically higher when employing the novel expression systems. This higher expression was also correlated with a marked upregulation of unfolded protein response (UPR) related genes, likely from an increased protein burden in the endoplasmic reticulum (ER). Based on the chemostat results obtained, best culture strategies for both P(PDF) and P(UPP) expression systems were also successfully implemented in 15 L fed-batch cultivations where q(p) and product to biomass yield (Y(P/X)*) values were similar than those obtained in chemostat cultivations. CONCLUSIONS: As an outcome of the macrokinetic characterization presented, the novel P(PDF) and P(UPP) were observed to offer much higher efficiency for CalB production than the widely used P(GAP)-based methanol-free alternative. Thus, both systems arise as highly productive alternatives for P. pastoris-based RPP bioprocesses. Furthermore, the different expression regulation patterns observed indicate the ... |
format |
Text |
author |
Garrigós-Martínez, Javier Vuoristo, Kiira Nieto-Taype, Miguel Angel Tähtiharju, Juha Uusitalo, Jaana Tukiainen, Pauliina Schmid, Christian Tolstorukov, Ilya Madden, Knut Penttilä, Merja Montesinos-Seguí, José Luis Valero, Francisco Glieder, Anton Garcia-Ortega, Xavier |
author_facet |
Garrigós-Martínez, Javier Vuoristo, Kiira Nieto-Taype, Miguel Angel Tähtiharju, Juha Uusitalo, Jaana Tukiainen, Pauliina Schmid, Christian Tolstorukov, Ilya Madden, Knut Penttilä, Merja Montesinos-Seguí, José Luis Valero, Francisco Glieder, Anton Garcia-Ortega, Xavier |
author_sort |
Garrigós-Martínez, Javier |
title |
Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris |
title_short |
Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris |
title_full |
Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris |
title_fullStr |
Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris |
title_full_unstemmed |
Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris |
title_sort |
bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with pichia pastoris |
publisher |
BioMed Central |
publishDate |
2021 |
url |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986505/ https://doi.org/10.1186/s12934-021-01564-9 |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_source |
Microb Cell Fact |
op_relation |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986505/ http://dx.doi.org/10.1186/s12934-021-01564-9 |
op_rights |
© The Author(s) 2021 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
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CC0 PDM CC-BY |
op_doi |
https://doi.org/10.1186/s12934-021-01564-9 |
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