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Pichia pastoris Aft1 - a novel transcription factor, enhancing recombinant protein secretion
BACKGROUND: The methylotrophic yeast Pichia pastoris is frequently used for the production of recombinant proteins. However, expression levels can vary depending on the target protein. Allowing for simultaneous regulation of many genes, which may elicit a desired phenotype like increased protein pro...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161868/ https://www.ncbi.nlm.nih.gov/pubmed/25205197 http://dx.doi.org/10.1186/s12934-014-0120-5 |
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author | Ruth, Claudia Buchetics, Markus Vidimce, Viktorija Kotz, Daniela Naschberger, Stefan Mattanovich, Diethard Pichler, Harald Gasser, Brigitte |
author_facet | Ruth, Claudia Buchetics, Markus Vidimce, Viktorija Kotz, Daniela Naschberger, Stefan Mattanovich, Diethard Pichler, Harald Gasser, Brigitte |
author_sort | Ruth, Claudia |
collection | PubMed |
description | BACKGROUND: The methylotrophic yeast Pichia pastoris is frequently used for the production of recombinant proteins. However, expression levels can vary depending on the target protein. Allowing for simultaneous regulation of many genes, which may elicit a desired phenotype like increased protein production, overexpression of transcription factors can be used to overcome expression bottlenecks. Here, we present a novel P. pastoris transcription factor currently annotated as Aft1, activator of ferrous transport. RESULTS: The promoter regions of key secretory P. pastoris genes were screened for fungal transcription factor binding sites, revealing Aft1 as an interesting candidate for improving secretion. Genome wide analysis of transcription factor binding sites suggested Aft1 to be involved in the regulation of many secretory genes, but also indicated possible novel functions in carbohydrate metabolism. No Aft binding sites were found in promoters of characteristic iron homeostasis genes in P. pastoris. Microarrays were used to study the Aft1 regulon in detail, confirming Aft1 involvement in the regulation of carbon-responsive genes, and showing that iron regulation is dependent on FEP1, but not AFT1 expression levels. The positive effect of AFT1 overexpression on recombinant protein secretion was demonstrated for a carboxylesterase from Sphingopyxis sp. MTA144, for which secretion was improved 2.5-fold in fed batch bioreactor cultivations. CONCLUSION: This study demonstrates that the transcription factor Aft1 can be used to improve recombinant protein secretion in P. pastoris. Furthermore, we discovered possible novel functions of Aft1 in carbohydrate metabolism and provide evidence arguing against a direct role of Aft1 in P. pastoris iron regulation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-014-0120-5) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-4161868 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-41618682014-09-13 Pichia pastoris Aft1 - a novel transcription factor, enhancing recombinant protein secretion Ruth, Claudia Buchetics, Markus Vidimce, Viktorija Kotz, Daniela Naschberger, Stefan Mattanovich, Diethard Pichler, Harald Gasser, Brigitte Microb Cell Fact Research BACKGROUND: The methylotrophic yeast Pichia pastoris is frequently used for the production of recombinant proteins. However, expression levels can vary depending on the target protein. Allowing for simultaneous regulation of many genes, which may elicit a desired phenotype like increased protein production, overexpression of transcription factors can be used to overcome expression bottlenecks. Here, we present a novel P. pastoris transcription factor currently annotated as Aft1, activator of ferrous transport. RESULTS: The promoter regions of key secretory P. pastoris genes were screened for fungal transcription factor binding sites, revealing Aft1 as an interesting candidate for improving secretion. Genome wide analysis of transcription factor binding sites suggested Aft1 to be involved in the regulation of many secretory genes, but also indicated possible novel functions in carbohydrate metabolism. No Aft binding sites were found in promoters of characteristic iron homeostasis genes in P. pastoris. Microarrays were used to study the Aft1 regulon in detail, confirming Aft1 involvement in the regulation of carbon-responsive genes, and showing that iron regulation is dependent on FEP1, but not AFT1 expression levels. The positive effect of AFT1 overexpression on recombinant protein secretion was demonstrated for a carboxylesterase from Sphingopyxis sp. MTA144, for which secretion was improved 2.5-fold in fed batch bioreactor cultivations. CONCLUSION: This study demonstrates that the transcription factor Aft1 can be used to improve recombinant protein secretion in P. pastoris. Furthermore, we discovered possible novel functions of Aft1 in carbohydrate metabolism and provide evidence arguing against a direct role of Aft1 in P. pastoris iron regulation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-014-0120-5) contains supplementary material, which is available to authorized users. BioMed Central 2014-09-03 /pmc/articles/PMC4161868/ /pubmed/25205197 http://dx.doi.org/10.1186/s12934-014-0120-5 Text en © Ruth et al.; licensee BioMed Central Ltd. 2014 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. 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. |
spellingShingle | Research Ruth, Claudia Buchetics, Markus Vidimce, Viktorija Kotz, Daniela Naschberger, Stefan Mattanovich, Diethard Pichler, Harald Gasser, Brigitte Pichia pastoris Aft1 - a novel transcription factor, enhancing recombinant protein secretion |
title | Pichia pastoris Aft1 - a novel transcription factor, enhancing recombinant protein secretion |
title_full | Pichia pastoris Aft1 - a novel transcription factor, enhancing recombinant protein secretion |
title_fullStr | Pichia pastoris Aft1 - a novel transcription factor, enhancing recombinant protein secretion |
title_full_unstemmed | Pichia pastoris Aft1 - a novel transcription factor, enhancing recombinant protein secretion |
title_short | Pichia pastoris Aft1 - a novel transcription factor, enhancing recombinant protein secretion |
title_sort | pichia pastoris aft1 - a novel transcription factor, enhancing recombinant protein secretion |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161868/ https://www.ncbi.nlm.nih.gov/pubmed/25205197 http://dx.doi.org/10.1186/s12934-014-0120-5 |
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