Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris
Abstract 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...
Published in: | Microbial Cell Factories |
---|---|
Main Authors: | , , , , , , , , , , , , , |
Other Authors: | |
Format: | Article in Journal/Newspaper |
Language: | English |
Published: |
Springer Science and Business Media LLC
2021
|
Subjects: | |
Online Access: | http://dx.doi.org/10.1186/s12934-021-01564-9 http://link.springer.com/content/pdf/10.1186/s12934-021-01564-9.pdf http://link.springer.com/article/10.1186/s12934-021-01564-9/fulltext.html |
id |
crspringernat:10.1186/s12934-021-01564-9 |
---|---|
record_format |
openpolar |
spelling |
crspringernat:10.1186/s12934-021-01564-9 2023-05-15T14:12:50+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 H2020 Research Infrastructures 2021 http://dx.doi.org/10.1186/s12934-021-01564-9 http://link.springer.com/content/pdf/10.1186/s12934-021-01564-9.pdf http://link.springer.com/article/10.1186/s12934-021-01564-9/fulltext.html en eng Springer Science and Business Media LLC http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ CC-BY Microbial Cell Factories volume 20, issue 1 ISSN 1475-2859 Applied Microbiology and Biotechnology Bioengineering Biotechnology journal-article 2021 crspringernat https://doi.org/10.1186/s12934-021-01564-9 2022-01-04T16:48:19Z Abstract 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 level of gene expression can be adjusted, or tuned, which is interesting when using Pichia pastoris as a cell factory for different products of interest. Article in Journal/Newspaper Antarc* Antarctica Springer Nature (via Crossref) Microbial Cell Factories 20 1 |
institution |
Open Polar |
collection |
Springer Nature (via Crossref) |
op_collection_id |
crspringernat |
language |
English |
topic |
Applied Microbiology and Biotechnology Bioengineering Biotechnology |
spellingShingle |
Applied Microbiology and Biotechnology Bioengineering Biotechnology 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 |
Applied Microbiology and Biotechnology Bioengineering Biotechnology |
description |
Abstract 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 level of gene expression can be adjusted, or tuned, which is interesting when using Pichia pastoris as a cell factory for different products of interest. |
author2 |
H2020 Research Infrastructures |
format |
Article in Journal/Newspaper |
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 |
Springer Science and Business Media LLC |
publishDate |
2021 |
url |
http://dx.doi.org/10.1186/s12934-021-01564-9 http://link.springer.com/content/pdf/10.1186/s12934-021-01564-9.pdf http://link.springer.com/article/10.1186/s12934-021-01564-9/fulltext.html |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_source |
Microbial Cell Factories volume 20, issue 1 ISSN 1475-2859 |
op_rights |
http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ |
op_rightsnorm |
CC-BY |
op_doi |
https://doi.org/10.1186/s12934-021-01564-9 |
container_title |
Microbial Cell Factories |
container_volume |
20 |
container_issue |
1 |
_version_ |
1766285191325679616 |