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Development of a mixed feed strategy for a recombinant Pichia pastoris strain producing with a de-repression promoter
BACKGROUND: Recombinant protein production in the yeast Pichia pastoris is usually based on the alcohol oxidase promoters pAOX1 and pAOX2, which are regulated by methanol and strongly repressed by other C-sources, like glycerol and glucose. However, the use of methanol brings several disadvantages,...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4561368/ https://www.ncbi.nlm.nih.gov/pubmed/26156850 http://dx.doi.org/10.1186/s12934-015-0292-7 |
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author | Capone, Simona Horvat, Jernej Herwig, Christoph Spadiut, Oliver |
author_facet | Capone, Simona Horvat, Jernej Herwig, Christoph Spadiut, Oliver |
author_sort | Capone, Simona |
collection | PubMed |
description | BACKGROUND: Recombinant protein production in the yeast Pichia pastoris is usually based on the alcohol oxidase promoters pAOX1 and pAOX2, which are regulated by methanol and strongly repressed by other C-sources, like glycerol and glucose. However, the use of methanol brings several disadvantages, which is why current trends in bioprocess development with P. pastoris are focussing on minimizing the required amount of methanol or even avoid its employment. In this respect novel promoter systems which do not rely on methanol have been investigated and promoter variants were designed to fine-tune gene expression. Amongst these novel promoter systems, mutated AOX promoters, which are regulated by available carbon source concentration (so-called de-repressed promoters), are currently raising attention. However, the main disadvantage of such a production system is that expression and growth usually cannot happen concomitantly resulting in low space–time-yields. RESULTS: Here we show the development of a mixed-feed strategy for an industrial recombinant P. pastoris de-repression strain aiming at increased productivity and maximum space–time-yield. By doing dynamic experiments we determined a ratio between the specific substrate uptake rates of glycerol and sorbitol allowing a more than 2-fold increased productivity compared to the conventional single substrate de-repression strategy. CONCLUSION: Based on our results we recommend adjusting q(s glycerol) = 0.04 g g(−1) h(−1) and q(s sorbitol) = 0.055 g g(−1) h(−1) to obtain highest productivity with a P. pastoris de-repression strain. Our methodological approach of designing mixed-feed strategies based on physiological strain characterization using dynamic experiments proved to be beneficial. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-015-0292-7) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-4561368 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-45613682015-09-08 Development of a mixed feed strategy for a recombinant Pichia pastoris strain producing with a de-repression promoter Capone, Simona Horvat, Jernej Herwig, Christoph Spadiut, Oliver Microb Cell Fact Research BACKGROUND: Recombinant protein production in the yeast Pichia pastoris is usually based on the alcohol oxidase promoters pAOX1 and pAOX2, which are regulated by methanol and strongly repressed by other C-sources, like glycerol and glucose. However, the use of methanol brings several disadvantages, which is why current trends in bioprocess development with P. pastoris are focussing on minimizing the required amount of methanol or even avoid its employment. In this respect novel promoter systems which do not rely on methanol have been investigated and promoter variants were designed to fine-tune gene expression. Amongst these novel promoter systems, mutated AOX promoters, which are regulated by available carbon source concentration (so-called de-repressed promoters), are currently raising attention. However, the main disadvantage of such a production system is that expression and growth usually cannot happen concomitantly resulting in low space–time-yields. RESULTS: Here we show the development of a mixed-feed strategy for an industrial recombinant P. pastoris de-repression strain aiming at increased productivity and maximum space–time-yield. By doing dynamic experiments we determined a ratio between the specific substrate uptake rates of glycerol and sorbitol allowing a more than 2-fold increased productivity compared to the conventional single substrate de-repression strategy. CONCLUSION: Based on our results we recommend adjusting q(s glycerol) = 0.04 g g(−1) h(−1) and q(s sorbitol) = 0.055 g g(−1) h(−1) to obtain highest productivity with a P. pastoris de-repression strain. Our methodological approach of designing mixed-feed strategies based on physiological strain characterization using dynamic experiments proved to be beneficial. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-015-0292-7) contains supplementary material, which is available to authorized users. BioMed Central 2015-07-10 /pmc/articles/PMC4561368/ /pubmed/26156850 http://dx.doi.org/10.1186/s12934-015-0292-7 Text en © Capone et al. 2015 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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 Capone, Simona Horvat, Jernej Herwig, Christoph Spadiut, Oliver Development of a mixed feed strategy for a recombinant Pichia pastoris strain producing with a de-repression promoter |
title | Development of a mixed feed strategy for a recombinant Pichia pastoris strain producing with a de-repression promoter |
title_full | Development of a mixed feed strategy for a recombinant Pichia pastoris strain producing with a de-repression promoter |
title_fullStr | Development of a mixed feed strategy for a recombinant Pichia pastoris strain producing with a de-repression promoter |
title_full_unstemmed | Development of a mixed feed strategy for a recombinant Pichia pastoris strain producing with a de-repression promoter |
title_short | Development of a mixed feed strategy for a recombinant Pichia pastoris strain producing with a de-repression promoter |
title_sort | development of a mixed feed strategy for a recombinant pichia pastoris strain producing with a de-repression promoter |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4561368/ https://www.ncbi.nlm.nih.gov/pubmed/26156850 http://dx.doi.org/10.1186/s12934-015-0292-7 |
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