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A Mitochondrial Autonomously Replicating Sequence from Pichia pastoris for Uniform High Level Recombinant Protein Production
Pichia pastoris is a non-conventional methylotrophic yeast that is widely used for recombinant protein production, typically by stably integrating the target gene into the genome as part of an expression cassette. However, the comparatively high clonal variability associated with this approach usual...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5411459/ https://www.ncbi.nlm.nih.gov/pubmed/28512458 http://dx.doi.org/10.3389/fmicb.2017.00780 |
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author | Schwarzhans, Jan-Philipp Luttermann, Tobias Wibberg, Daniel Winkler, Anika Hübner, Wolfgang Huser, Thomas Kalinowski, Jörn Friehs, Karl |
author_facet | Schwarzhans, Jan-Philipp Luttermann, Tobias Wibberg, Daniel Winkler, Anika Hübner, Wolfgang Huser, Thomas Kalinowski, Jörn Friehs, Karl |
author_sort | Schwarzhans, Jan-Philipp |
collection | PubMed |
description | Pichia pastoris is a non-conventional methylotrophic yeast that is widely used for recombinant protein production, typically by stably integrating the target gene into the genome as part of an expression cassette. However, the comparatively high clonal variability associated with this approach usually necessitates a time intense screening step in order to find strains with the desired productivity. Some of the factors causing this clonal variability can be overcome using episomal vectors containing an autonomously replicating sequence (ARS). Here, we report on the discovery, characterization, and application of a fragment of mitochondrial DNA from P. pastoris for use as an ARS. First encountered as an off-target event in an experiment aiming for genomic integration, the newly created circular plasmid named “pMito” consists of the expression cassette and a fragment of mitochondrial DNA. Multiple matches to known ARS consensus sequence motifs, but no exact match to known chromosomal ARS from P. pastoris were detected on the fragment, indicating the presence of a novel ARS element. Different variants of pMito were successfully used for transformation and their productivity characteristics were assayed. All analyzed clones displayed a highly uniform expression level, exceeding by up to fourfold that of a reference with a single copy integrated in its genome. Expressed GFP could be localized exclusively to the cytoplasm via super-resolution fluorescence microscopy, indicating that pMito is present in the nucleus. While expression levels were homogenous among pMito clones, an apparent upper limit of expression was visible that could not be explained based on the gene dosage. Further investigation is necessary to fully understand the bottle-neck hindering this and other ARS vectors in P. pastoris from reaching their full capability. Lastly, we could demonstrate that the mitochondrial ARS from P. pastoris is also suitable for episomal vector transformation in Saccharomyces cerevisiae, widening the potential for biotechnological application. pMito displayed strong potential to reduce clonal variability in experiments targeting recombinant protein production. These findings also showcase the as of yet largely untapped potential of mitochondrial ARS from different yeasts for biotechnological applications. |
format | Online Article Text |
id | pubmed-5411459 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-54114592017-05-16 A Mitochondrial Autonomously Replicating Sequence from Pichia pastoris for Uniform High Level Recombinant Protein Production Schwarzhans, Jan-Philipp Luttermann, Tobias Wibberg, Daniel Winkler, Anika Hübner, Wolfgang Huser, Thomas Kalinowski, Jörn Friehs, Karl Front Microbiol Microbiology Pichia pastoris is a non-conventional methylotrophic yeast that is widely used for recombinant protein production, typically by stably integrating the target gene into the genome as part of an expression cassette. However, the comparatively high clonal variability associated with this approach usually necessitates a time intense screening step in order to find strains with the desired productivity. Some of the factors causing this clonal variability can be overcome using episomal vectors containing an autonomously replicating sequence (ARS). Here, we report on the discovery, characterization, and application of a fragment of mitochondrial DNA from P. pastoris for use as an ARS. First encountered as an off-target event in an experiment aiming for genomic integration, the newly created circular plasmid named “pMito” consists of the expression cassette and a fragment of mitochondrial DNA. Multiple matches to known ARS consensus sequence motifs, but no exact match to known chromosomal ARS from P. pastoris were detected on the fragment, indicating the presence of a novel ARS element. Different variants of pMito were successfully used for transformation and their productivity characteristics were assayed. All analyzed clones displayed a highly uniform expression level, exceeding by up to fourfold that of a reference with a single copy integrated in its genome. Expressed GFP could be localized exclusively to the cytoplasm via super-resolution fluorescence microscopy, indicating that pMito is present in the nucleus. While expression levels were homogenous among pMito clones, an apparent upper limit of expression was visible that could not be explained based on the gene dosage. Further investigation is necessary to fully understand the bottle-neck hindering this and other ARS vectors in P. pastoris from reaching their full capability. Lastly, we could demonstrate that the mitochondrial ARS from P. pastoris is also suitable for episomal vector transformation in Saccharomyces cerevisiae, widening the potential for biotechnological application. pMito displayed strong potential to reduce clonal variability in experiments targeting recombinant protein production. These findings also showcase the as of yet largely untapped potential of mitochondrial ARS from different yeasts for biotechnological applications. Frontiers Media S.A. 2017-05-02 /pmc/articles/PMC5411459/ /pubmed/28512458 http://dx.doi.org/10.3389/fmicb.2017.00780 Text en Copyright © 2017 Schwarzhans, Luttermann, Wibberg, Winkler, Hübner, Huser, Kalinowski and Friehs. http://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) or licensor 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. |
spellingShingle | Microbiology Schwarzhans, Jan-Philipp Luttermann, Tobias Wibberg, Daniel Winkler, Anika Hübner, Wolfgang Huser, Thomas Kalinowski, Jörn Friehs, Karl A Mitochondrial Autonomously Replicating Sequence from Pichia pastoris for Uniform High Level Recombinant Protein Production |
title | A Mitochondrial Autonomously Replicating Sequence from Pichia pastoris for Uniform High Level Recombinant Protein Production |
title_full | A Mitochondrial Autonomously Replicating Sequence from Pichia pastoris for Uniform High Level Recombinant Protein Production |
title_fullStr | A Mitochondrial Autonomously Replicating Sequence from Pichia pastoris for Uniform High Level Recombinant Protein Production |
title_full_unstemmed | A Mitochondrial Autonomously Replicating Sequence from Pichia pastoris for Uniform High Level Recombinant Protein Production |
title_short | A Mitochondrial Autonomously Replicating Sequence from Pichia pastoris for Uniform High Level Recombinant Protein Production |
title_sort | mitochondrial autonomously replicating sequence from pichia pastoris for uniform high level recombinant protein production |
topic | Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5411459/ https://www.ncbi.nlm.nih.gov/pubmed/28512458 http://dx.doi.org/10.3389/fmicb.2017.00780 |
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