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 (PAOX1), and the constitutive GAP promoter (PGAP). Since promoters play a crucial role...
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ftdatacite:10.6084/m9.figshare.c.5350849 2023-05-15T14:01:41+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 https://dx.doi.org/10.6084/m9.figshare.c.5350849 https://springernature.figshare.com/collections/Bioprocess_performance_analysis_of_novel_methanol-independent_promoters_for_recombinant_protein_production_with_Pichia_pastoris/5350849 unknown figshare https://dx.doi.org/10.1186/s12934-021-01564-9 Creative Commons Attribution 4.0 International https://creativecommons.org/licenses/by/4.0/legalcode cc-by-4.0 CC-BY Biochemistry Genetics FOS Biological sciences Molecular Biology Biotechnology 69999 Biological Sciences not elsewhere classified Developmental Biology 110309 Infectious Diseases FOS Health sciences Collection article 2021 ftdatacite https://doi.org/10.6084/m9.figshare.c.5350849 https://doi.org/10.1186/s12934-021-01564-9 2021-11-05T12:55:41Z 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 (PAOX1), and the constitutive GAP promoter (PGAP). 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 (PPDF, PUPP) 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 PGAP, using the Candida antarctica lipase B (CalB) as model protein for expression system performance. Results Both the PPDF and PUPP-based expression systems outperformed similar PGAP-based expression in chemostat cultivations, reaching ninefold higher specific production rates (qp). 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 PPDF and PUPP expression systems were also successfully implemented in 15 L fed-batch cultivations where qp and product to biomass yield (YP/X*) values were similar than those obtained in chemostat cultivations. Conclusions As an outcome of the macrokinetic characterization presented, the novel PPDF and PUPP were observed to offer much higher efficiency for CalB production than the widely used PGAP-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 DataCite Metadata Store (German National Library of Science and Technology) |
institution |
Open Polar |
collection |
DataCite Metadata Store (German National Library of Science and Technology) |
op_collection_id |
ftdatacite |
language |
unknown |
topic |
Biochemistry Genetics FOS Biological sciences Molecular Biology Biotechnology 69999 Biological Sciences not elsewhere classified Developmental Biology 110309 Infectious Diseases FOS Health sciences |
spellingShingle |
Biochemistry Genetics FOS Biological sciences Molecular Biology Biotechnology 69999 Biological Sciences not elsewhere classified Developmental Biology 110309 Infectious Diseases FOS Health sciences 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 |
Biochemistry Genetics FOS Biological sciences Molecular Biology Biotechnology 69999 Biological Sciences not elsewhere classified Developmental Biology 110309 Infectious Diseases FOS Health sciences |
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 (PAOX1), and the constitutive GAP promoter (PGAP). 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 (PPDF, PUPP) 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 PGAP, using the Candida antarctica lipase B (CalB) as model protein for expression system performance. Results Both the PPDF and PUPP-based expression systems outperformed similar PGAP-based expression in chemostat cultivations, reaching ninefold higher specific production rates (qp). 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 PPDF and PUPP expression systems were also successfully implemented in 15 L fed-batch cultivations where qp and product to biomass yield (YP/X*) values were similar than those obtained in chemostat cultivations. Conclusions As an outcome of the macrokinetic characterization presented, the novel PPDF and PUPP were observed to offer much higher efficiency for CalB production than the widely used PGAP-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. |
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 |
figshare |
publishDate |
2021 |
url |
https://dx.doi.org/10.6084/m9.figshare.c.5350849 https://springernature.figshare.com/collections/Bioprocess_performance_analysis_of_novel_methanol-independent_promoters_for_recombinant_protein_production_with_Pichia_pastoris/5350849 |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_relation |
https://dx.doi.org/10.1186/s12934-021-01564-9 |
op_rights |
Creative Commons Attribution 4.0 International https://creativecommons.org/licenses/by/4.0/legalcode cc-by-4.0 |
op_rightsnorm |
CC-BY |
op_doi |
https://doi.org/10.6084/m9.figshare.c.5350849 https://doi.org/10.1186/s12934-021-01564-9 |
_version_ |
1766271727380201472 |