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Effect of secretory pathway gene overexpression on secretion of a fluorescent reporter protein in Aspergillus nidulans
BACKGROUND: The considerable capacity of filamentous fungi for the secretion of proteins is the basis for multi-billion dollar industries producing enzymes and proteins with therapeutic value. The stepwise pathway from translation to secretion is therefore well studied, and genes playing major roles...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5611598/ https://www.ncbi.nlm.nih.gov/pubmed/28955462 http://dx.doi.org/10.1186/s40694-016-0021-y |
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author | Schalén, Martin Anyaogu, Diana Chinyere Hoof, Jakob Blæsbjerg Workman, Mhairi |
author_facet | Schalén, Martin Anyaogu, Diana Chinyere Hoof, Jakob Blæsbjerg Workman, Mhairi |
author_sort | Schalén, Martin |
collection | PubMed |
description | BACKGROUND: The considerable capacity of filamentous fungi for the secretion of proteins is the basis for multi-billion dollar industries producing enzymes and proteins with therapeutic value. The stepwise pathway from translation to secretion is therefore well studied, and genes playing major roles in the process have been identified through transcriptomics. The assignment of function to these genes has been enabled in combination with gene deletion studies. In this work, 14 genes known to play a role in protein secretion in filamentous fungi were overexpressed in Aspergillus nidulans. The background strain was a fluorescent reporter secreting mRFP. The overall effect of the overexpressions could thus be easily monitored through fluorescence measurements, while the effects on physiology were determined in batch cultivations and surface growth studies. RESULTS: Fourteen protein secretion pathway related genes were overexpressed with a tet-ON promoter in the RFP-secreting reporter strain and macromorphology, physiology and protein secretion were monitored when the secretory genes were induced. Overexpression of several of the chosen genes was shown to cause anomalies on growth, micro- and macro-morphology and protein secretion levels. While several constructs exhibited decreased secretion of the model protein, the overexpression of the Rab GTPase RabD resulted in a 40 % increase in secretion in controlled bioreactor cultivations. Fluorescence microscopy revealed alterations of protein localization in some of the constructed strains, giving further insight into potential roles of the investigated genes. CONCLUSIONS: This study demonstrates the possibility of significantly increasing cellular recombinant protein secretion by targeted overexpression of secretion pathway genes. Some gene targets investigated here, including genes from different compartments of the secretory pathway resulted in no significant change in protein secretion, or in significantly lowered protein titres. As the 14 genes selected in this study were previously shown to be upregulated during protein secretion, our results indicate that increased expression may be a way for the cell to slow down secretion in order to cope with the increased protein load. By constructing a secretion reporter strain, the study demonstrates a robust way to study the secretion pathway in filamentous fungi. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s40694-016-0021-y) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-5611598 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-56115982017-09-27 Effect of secretory pathway gene overexpression on secretion of a fluorescent reporter protein in Aspergillus nidulans Schalén, Martin Anyaogu, Diana Chinyere Hoof, Jakob Blæsbjerg Workman, Mhairi Fungal Biol Biotechnol Research BACKGROUND: The considerable capacity of filamentous fungi for the secretion of proteins is the basis for multi-billion dollar industries producing enzymes and proteins with therapeutic value. The stepwise pathway from translation to secretion is therefore well studied, and genes playing major roles in the process have been identified through transcriptomics. The assignment of function to these genes has been enabled in combination with gene deletion studies. In this work, 14 genes known to play a role in protein secretion in filamentous fungi were overexpressed in Aspergillus nidulans. The background strain was a fluorescent reporter secreting mRFP. The overall effect of the overexpressions could thus be easily monitored through fluorescence measurements, while the effects on physiology were determined in batch cultivations and surface growth studies. RESULTS: Fourteen protein secretion pathway related genes were overexpressed with a tet-ON promoter in the RFP-secreting reporter strain and macromorphology, physiology and protein secretion were monitored when the secretory genes were induced. Overexpression of several of the chosen genes was shown to cause anomalies on growth, micro- and macro-morphology and protein secretion levels. While several constructs exhibited decreased secretion of the model protein, the overexpression of the Rab GTPase RabD resulted in a 40 % increase in secretion in controlled bioreactor cultivations. Fluorescence microscopy revealed alterations of protein localization in some of the constructed strains, giving further insight into potential roles of the investigated genes. CONCLUSIONS: This study demonstrates the possibility of significantly increasing cellular recombinant protein secretion by targeted overexpression of secretion pathway genes. Some gene targets investigated here, including genes from different compartments of the secretory pathway resulted in no significant change in protein secretion, or in significantly lowered protein titres. As the 14 genes selected in this study were previously shown to be upregulated during protein secretion, our results indicate that increased expression may be a way for the cell to slow down secretion in order to cope with the increased protein load. By constructing a secretion reporter strain, the study demonstrates a robust way to study the secretion pathway in filamentous fungi. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s40694-016-0021-y) contains supplementary material, which is available to authorized users. BioMed Central 2016-04-12 /pmc/articles/PMC5611598/ /pubmed/28955462 http://dx.doi.org/10.1186/s40694-016-0021-y Text en © Schalén et al. 2016 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 Schalén, Martin Anyaogu, Diana Chinyere Hoof, Jakob Blæsbjerg Workman, Mhairi Effect of secretory pathway gene overexpression on secretion of a fluorescent reporter protein in Aspergillus nidulans |
title | Effect of secretory pathway gene overexpression on secretion of a fluorescent reporter protein in Aspergillus nidulans |
title_full | Effect of secretory pathway gene overexpression on secretion of a fluorescent reporter protein in Aspergillus nidulans |
title_fullStr | Effect of secretory pathway gene overexpression on secretion of a fluorescent reporter protein in Aspergillus nidulans |
title_full_unstemmed | Effect of secretory pathway gene overexpression on secretion of a fluorescent reporter protein in Aspergillus nidulans |
title_short | Effect of secretory pathway gene overexpression on secretion of a fluorescent reporter protein in Aspergillus nidulans |
title_sort | effect of secretory pathway gene overexpression on secretion of a fluorescent reporter protein in aspergillus nidulans |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5611598/ https://www.ncbi.nlm.nih.gov/pubmed/28955462 http://dx.doi.org/10.1186/s40694-016-0021-y |
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