Enabling growth-decoupled Komagataella phaffii recombinant protein production based on the methanol-free P(DH) promoter
The current transition towards the circular bioeconomy requires a rational development of biorefineries to sustainably fulfill the present demands. The use of Komagataella phaffii (Pichia pastoris) can meet this challenge, since it has the capability to use crude glycerol as a carbon-source, a by-pr...
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ftpubmed:oai:pubmedcentral.nih.gov:10076887 2023-05-15T14:00:03+02:00 Enabling growth-decoupled Komagataella phaffii recombinant protein production based on the methanol-free P(DH) promoter Bernat-Camps, Núria Ebner, Katharina Schusterbauer, Veronika Fischer, Jasmin Elgin Nieto-Taype, Miguel Angel Valero, Francisco Glieder, Anton Garcia-Ortega, Xavier 2023-03-23 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10076887/ https://doi.org/10.3389/fbioe.2023.1130583 en eng Frontiers Media S.A. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10076887/ http://dx.doi.org/10.3389/fbioe.2023.1130583 Copyright © 2023 Bernat-Camps, Ebner, Schusterbauer, Fischer, Nieto-Taype, Valero, Glieder and Garcia-Ortega. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Front Bioeng Biotechnol Bioengineering and Biotechnology Text 2023 ftpubmed https://doi.org/10.3389/fbioe.2023.1130583 2023-04-09T01:06:11Z The current transition towards the circular bioeconomy requires a rational development of biorefineries to sustainably fulfill the present demands. The use of Komagataella phaffii (Pichia pastoris) can meet this challenge, since it has the capability to use crude glycerol as a carbon-source, a by-product from the biodiesel industry, while producing high- and low-added value products. Recombinant protein production (RPP) using K. phaffii has often been driven either by the methanol induced AOX1 promoter (P( AOX1 )) and/or the constitutive GAP promoter (P( GAP )). In the last years, strong efforts have been focused on developing novel expression systems that expand the toolbox variety of K. phaffii to efficiently produce diverse proteins that requires different strategies. In this work, a study was conducted towards the development of methanol-free expression system based on a heat-shock gene promoter (P(DH)) using glycerol as sole carbon source. Using this promoter, the recombinant expression is strongly induced in carbon-starving conditions. The classical P( GAP ) was used as a benchmark, taking for both strains the lipase B from Candida antarctica (CalB) as model protein. Titer of CalB expressed under P(DH) outperformed P( GAP ) controlled expression in shake-flask cultivations when using a slow-release continuous feeding technology, confirming that P(DH) is induced under pseudo-starving conditions. This increase was also confirmed in fed-batch cultivations. Several optimization rounds were carried out for P(DH) under different feeding and osmolarity conditions. In all of them the P(DH) controlled process outperformed the P( GAP ) one in regard to CalB titer. The best P(DH) approach reached 3.6-fold more specific productivity than P( GAP ) fed-batch at low μ. Compared to the optimum approach for P( GAP )-based process, the best P(DH) fed-batch strategy resulted in 2.3-fold higher titer, while the specific productivity was very similar. To summarize, P(DH) is an inducible promoter that exhibited a non-coupled ... Text Antarc* Antarctica PubMed Central (PMC) Frontiers in Bioengineering and Biotechnology 11 |
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Bioengineering and Biotechnology |
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Bioengineering and Biotechnology Bernat-Camps, Núria Ebner, Katharina Schusterbauer, Veronika Fischer, Jasmin Elgin Nieto-Taype, Miguel Angel Valero, Francisco Glieder, Anton Garcia-Ortega, Xavier Enabling growth-decoupled Komagataella phaffii recombinant protein production based on the methanol-free P(DH) promoter |
topic_facet |
Bioengineering and Biotechnology |
description |
The current transition towards the circular bioeconomy requires a rational development of biorefineries to sustainably fulfill the present demands. The use of Komagataella phaffii (Pichia pastoris) can meet this challenge, since it has the capability to use crude glycerol as a carbon-source, a by-product from the biodiesel industry, while producing high- and low-added value products. Recombinant protein production (RPP) using K. phaffii has often been driven either by the methanol induced AOX1 promoter (P( AOX1 )) and/or the constitutive GAP promoter (P( GAP )). In the last years, strong efforts have been focused on developing novel expression systems that expand the toolbox variety of K. phaffii to efficiently produce diverse proteins that requires different strategies. In this work, a study was conducted towards the development of methanol-free expression system based on a heat-shock gene promoter (P(DH)) using glycerol as sole carbon source. Using this promoter, the recombinant expression is strongly induced in carbon-starving conditions. The classical P( GAP ) was used as a benchmark, taking for both strains the lipase B from Candida antarctica (CalB) as model protein. Titer of CalB expressed under P(DH) outperformed P( GAP ) controlled expression in shake-flask cultivations when using a slow-release continuous feeding technology, confirming that P(DH) is induced under pseudo-starving conditions. This increase was also confirmed in fed-batch cultivations. Several optimization rounds were carried out for P(DH) under different feeding and osmolarity conditions. In all of them the P(DH) controlled process outperformed the P( GAP ) one in regard to CalB titer. The best P(DH) approach reached 3.6-fold more specific productivity than P( GAP ) fed-batch at low μ. Compared to the optimum approach for P( GAP )-based process, the best P(DH) fed-batch strategy resulted in 2.3-fold higher titer, while the specific productivity was very similar. To summarize, P(DH) is an inducible promoter that exhibited a non-coupled ... |
format |
Text |
author |
Bernat-Camps, Núria Ebner, Katharina Schusterbauer, Veronika Fischer, Jasmin Elgin Nieto-Taype, Miguel Angel Valero, Francisco Glieder, Anton Garcia-Ortega, Xavier |
author_facet |
Bernat-Camps, Núria Ebner, Katharina Schusterbauer, Veronika Fischer, Jasmin Elgin Nieto-Taype, Miguel Angel Valero, Francisco Glieder, Anton Garcia-Ortega, Xavier |
author_sort |
Bernat-Camps, Núria |
title |
Enabling growth-decoupled Komagataella phaffii recombinant protein production based on the methanol-free P(DH) promoter |
title_short |
Enabling growth-decoupled Komagataella phaffii recombinant protein production based on the methanol-free P(DH) promoter |
title_full |
Enabling growth-decoupled Komagataella phaffii recombinant protein production based on the methanol-free P(DH) promoter |
title_fullStr |
Enabling growth-decoupled Komagataella phaffii recombinant protein production based on the methanol-free P(DH) promoter |
title_full_unstemmed |
Enabling growth-decoupled Komagataella phaffii recombinant protein production based on the methanol-free P(DH) promoter |
title_sort |
enabling growth-decoupled komagataella phaffii recombinant protein production based on the methanol-free p(dh) promoter |
publisher |
Frontiers Media S.A. |
publishDate |
2023 |
url |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10076887/ https://doi.org/10.3389/fbioe.2023.1130583 |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_source |
Front Bioeng Biotechnol |
op_relation |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10076887/ http://dx.doi.org/10.3389/fbioe.2023.1130583 |
op_rights |
Copyright © 2023 Bernat-Camps, Ebner, Schusterbauer, Fischer, Nieto-Taype, Valero, Glieder and Garcia-Ortega. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
op_doi |
https://doi.org/10.3389/fbioe.2023.1130583 |
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Frontiers in Bioengineering and Biotechnology |
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