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...
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ftdoajarticles:oai:doaj.org/article:65fec0020c894a709e03afcf4f3128d7 2023-05-15T13:48:41+02:00 Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris Javier Garrigós-Martínez Kiira Vuoristo Miguel Angel Nieto-Taype Juha Tähtiharju Jaana Uusitalo Pauliina Tukiainen Christian Schmid Ilya Tolstorukov Knut Madden Merja Penttilä José Luis Montesinos-Seguí Francisco Valero Anton Glieder Xavier Garcia-Ortega 2021-03-01T00:00:00Z https://doi.org/10.1186/s12934-021-01564-9 https://doaj.org/article/65fec0020c894a709e03afcf4f3128d7 EN eng BMC https://doi.org/10.1186/s12934-021-01564-9 https://doaj.org/toc/1475-2859 doi:10.1186/s12934-021-01564-9 1475-2859 https://doaj.org/article/65fec0020c894a709e03afcf4f3128d7 Microbial Cell Factories, Vol 20, Iss 1, Pp 1-12 (2021) Komagataella phaffii (Pichia pastoris) Recombinant protein production Methanol-free bioprocesses Expression system characterisation Promoter regulation Bioprocess development Microbiology QR1-502 article 2021 ftdoajarticles https://doi.org/10.1186/s12934-021-01564-9 2022-12-31T13:31:57Z 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 ... Article in Journal/Newspaper Antarc* Antarctica Directory of Open Access Journals: DOAJ Articles Microbial Cell Factories 20 1 |
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Directory of Open Access Journals: DOAJ Articles |
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ftdoajarticles |
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English |
topic |
Komagataella phaffii (Pichia pastoris) Recombinant protein production Methanol-free bioprocesses Expression system characterisation Promoter regulation Bioprocess development Microbiology QR1-502 |
spellingShingle |
Komagataella phaffii (Pichia pastoris) Recombinant protein production Methanol-free bioprocesses Expression system characterisation Promoter regulation Bioprocess development Microbiology QR1-502 Javier Garrigós-Martínez Kiira Vuoristo Miguel Angel Nieto-Taype Juha Tähtiharju Jaana Uusitalo Pauliina Tukiainen Christian Schmid Ilya Tolstorukov Knut Madden Merja Penttilä José Luis Montesinos-Seguí Francisco Valero Anton Glieder Xavier Garcia-Ortega Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris |
topic_facet |
Komagataella phaffii (Pichia pastoris) Recombinant protein production Methanol-free bioprocesses Expression system characterisation Promoter regulation Bioprocess development Microbiology QR1-502 |
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 ... |
format |
Article in Journal/Newspaper |
author |
Javier Garrigós-Martínez Kiira Vuoristo Miguel Angel Nieto-Taype Juha Tähtiharju Jaana Uusitalo Pauliina Tukiainen Christian Schmid Ilya Tolstorukov Knut Madden Merja Penttilä José Luis Montesinos-Seguí Francisco Valero Anton Glieder Xavier Garcia-Ortega |
author_facet |
Javier Garrigós-Martínez Kiira Vuoristo Miguel Angel Nieto-Taype Juha Tähtiharju Jaana Uusitalo Pauliina Tukiainen Christian Schmid Ilya Tolstorukov Knut Madden Merja Penttilä José Luis Montesinos-Seguí Francisco Valero Anton Glieder Xavier Garcia-Ortega |
author_sort |
Javier Garrigós-Martínez |
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 |
BMC |
publishDate |
2021 |
url |
https://doi.org/10.1186/s12934-021-01564-9 https://doaj.org/article/65fec0020c894a709e03afcf4f3128d7 |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_source |
Microbial Cell Factories, Vol 20, Iss 1, Pp 1-12 (2021) |
op_relation |
https://doi.org/10.1186/s12934-021-01564-9 https://doaj.org/toc/1475-2859 doi:10.1186/s12934-021-01564-9 1475-2859 https://doaj.org/article/65fec0020c894a709e03afcf4f3128d7 |
op_doi |
https://doi.org/10.1186/s12934-021-01564-9 |
container_title |
Microbial Cell Factories |
container_volume |
20 |
container_issue |
1 |
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1766249585222615040 |