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Effects of Rho1, a small GTPase on the production of recombinant glycoproteins in Saccharomyces cerevisiae
BACKGROUND: To humanize yeast N-glycosylation pathways, genes involved in yeast specific hyper-mannosylation must be disrupted followed by the introduction of genes catalyzing the synthesis, transport, and addition of human sugars. However, deletion of these genes, for instance, OCH1, which initiate...
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/PMC5073930/ https://www.ncbi.nlm.nih.gov/pubmed/27769287 http://dx.doi.org/10.1186/s12934-016-0575-7 |
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author | Xu, Sha Zhang, Ge-Yuan Zhang, Huijie Kitajima, Toshihiko Nakanishi, Hideki Gao, Xiao-Dong |
author_facet | Xu, Sha Zhang, Ge-Yuan Zhang, Huijie Kitajima, Toshihiko Nakanishi, Hideki Gao, Xiao-Dong |
author_sort | Xu, Sha |
collection | PubMed |
description | BACKGROUND: To humanize yeast N-glycosylation pathways, genes involved in yeast specific hyper-mannosylation must be disrupted followed by the introduction of genes catalyzing the synthesis, transport, and addition of human sugars. However, deletion of these genes, for instance, OCH1, which initiates hyper-mannosylation, could cause severe defects in cell growth, morphogenesis and response to environmental challenges. RESULTS: In this study, overexpression of RHO1, which encodes the Rho1p small GTPase, is confirmed to partially recover the growth defect of Saccharomyces cerevisiae Δalg3Δoch1 double mutant strain. In addition, transmission electron micrographs indicated that the cell wall structure of RHO1-expressed cells have an enhanced glucan layer and also a recovered mannoprotein layer, revealing the effect of Rho1p GTPase on cell wall biosynthesis. Similar complementation phenotypes have been confirmed by overexpression of the gene that encodes Fks2 protein, a catalytic subunit of a 1,3-β-glucan synthase. Besides the recovery of cell wall structure, the RHO1-overexpressed Δalg3Δoch1 strain also showed improved abilities in temperature tolerance, osmotic potential and drug sensitivity, which were not observed in the Δalg3Δoch1-FKS2 cells. Moreover, RHO1 overexpression could also increase N-glycan site occupancy and the amount of secreted glycoproteins. CONCLUSIONS: Overexpression of RHO1 in ‘humanized’ glycoprotein producing yeasts could significantly facilitate its future industrial applications for the production of therapeutic glycoproteins. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-016-0575-7) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-5073930 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-50739302016-10-26 Effects of Rho1, a small GTPase on the production of recombinant glycoproteins in Saccharomyces cerevisiae Xu, Sha Zhang, Ge-Yuan Zhang, Huijie Kitajima, Toshihiko Nakanishi, Hideki Gao, Xiao-Dong Microb Cell Fact Research BACKGROUND: To humanize yeast N-glycosylation pathways, genes involved in yeast specific hyper-mannosylation must be disrupted followed by the introduction of genes catalyzing the synthesis, transport, and addition of human sugars. However, deletion of these genes, for instance, OCH1, which initiates hyper-mannosylation, could cause severe defects in cell growth, morphogenesis and response to environmental challenges. RESULTS: In this study, overexpression of RHO1, which encodes the Rho1p small GTPase, is confirmed to partially recover the growth defect of Saccharomyces cerevisiae Δalg3Δoch1 double mutant strain. In addition, transmission electron micrographs indicated that the cell wall structure of RHO1-expressed cells have an enhanced glucan layer and also a recovered mannoprotein layer, revealing the effect of Rho1p GTPase on cell wall biosynthesis. Similar complementation phenotypes have been confirmed by overexpression of the gene that encodes Fks2 protein, a catalytic subunit of a 1,3-β-glucan synthase. Besides the recovery of cell wall structure, the RHO1-overexpressed Δalg3Δoch1 strain also showed improved abilities in temperature tolerance, osmotic potential and drug sensitivity, which were not observed in the Δalg3Δoch1-FKS2 cells. Moreover, RHO1 overexpression could also increase N-glycan site occupancy and the amount of secreted glycoproteins. CONCLUSIONS: Overexpression of RHO1 in ‘humanized’ glycoprotein producing yeasts could significantly facilitate its future industrial applications for the production of therapeutic glycoproteins. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-016-0575-7) contains supplementary material, which is available to authorized users. BioMed Central 2016-10-21 /pmc/articles/PMC5073930/ /pubmed/27769287 http://dx.doi.org/10.1186/s12934-016-0575-7 Text en © The Author(s) 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 Xu, Sha Zhang, Ge-Yuan Zhang, Huijie Kitajima, Toshihiko Nakanishi, Hideki Gao, Xiao-Dong Effects of Rho1, a small GTPase on the production of recombinant glycoproteins in Saccharomyces cerevisiae |
title | Effects of Rho1, a small GTPase on the production of recombinant glycoproteins in Saccharomyces cerevisiae |
title_full | Effects of Rho1, a small GTPase on the production of recombinant glycoproteins in Saccharomyces cerevisiae |
title_fullStr | Effects of Rho1, a small GTPase on the production of recombinant glycoproteins in Saccharomyces cerevisiae |
title_full_unstemmed | Effects of Rho1, a small GTPase on the production of recombinant glycoproteins in Saccharomyces cerevisiae |
title_short | Effects of Rho1, a small GTPase on the production of recombinant glycoproteins in Saccharomyces cerevisiae |
title_sort | effects of rho1, a small gtpase on the production of recombinant glycoproteins in saccharomyces cerevisiae |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5073930/ https://www.ncbi.nlm.nih.gov/pubmed/27769287 http://dx.doi.org/10.1186/s12934-016-0575-7 |
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