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Inhibition of extracellular proteases improves the production of a xylanase in Parageobacillus thermoglucosidasius

BACKGROUND: Parageobacillus thermoglucosidasius is a thermophilic and ethanol-producing bacterium capable of utilising both hexose and pentose sugars for fermentation. The organism has been proposed to be a suitable organism for the production of bioethanol from lignocellulosic feedstocks. These fee...

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Autores principales: Holland, Alexandria T. N., Danson, Michael J., Bolhuis, Albert
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6425571/
https://www.ncbi.nlm.nih.gov/pubmed/30894163
http://dx.doi.org/10.1186/s12896-019-0511-0
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author Holland, Alexandria T. N.
Danson, Michael J.
Bolhuis, Albert
author_facet Holland, Alexandria T. N.
Danson, Michael J.
Bolhuis, Albert
author_sort Holland, Alexandria T. N.
collection PubMed
description BACKGROUND: Parageobacillus thermoglucosidasius is a thermophilic and ethanol-producing bacterium capable of utilising both hexose and pentose sugars for fermentation. The organism has been proposed to be a suitable organism for the production of bioethanol from lignocellulosic feedstocks. These feedstocks may be difficult to degrade, and a potential strategy to optimise this process is to engineer strains that secrete hydrolases that liberate increased amounts of sugars from those feedstocks. However, very little is known about protein transport in P. thermoglucosidasius and the limitations of that process, and as a first step we investigated whether there were bottlenecks in the secretion of a model protein. RESULTS: A secretory enzyme, xylanase (XynA1), was produced with and without its signal peptide. Cell cultures were fractionated into cytoplasm, membrane, cell wall, and extracellular milieu protein extracts, which were analysed using immunoblotting and enzyme activity assays. The main bottleneck identified was proteolytic degradation of XynA1 during or after its translocation. A combination of mass spectrometry and bioinformatics indicated the presence of several proteases that might be involved in this process. CONCLUSION: The creation of protease-deficient strains may be beneficial towards the development of P. thermoglucosidasius as a platform organism for industrial processes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12896-019-0511-0) contains supplementary material, which is available to authorized users.
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spelling pubmed-64255712019-03-29 Inhibition of extracellular proteases improves the production of a xylanase in Parageobacillus thermoglucosidasius Holland, Alexandria T. N. Danson, Michael J. Bolhuis, Albert BMC Biotechnol Research Article BACKGROUND: Parageobacillus thermoglucosidasius is a thermophilic and ethanol-producing bacterium capable of utilising both hexose and pentose sugars for fermentation. The organism has been proposed to be a suitable organism for the production of bioethanol from lignocellulosic feedstocks. These feedstocks may be difficult to degrade, and a potential strategy to optimise this process is to engineer strains that secrete hydrolases that liberate increased amounts of sugars from those feedstocks. However, very little is known about protein transport in P. thermoglucosidasius and the limitations of that process, and as a first step we investigated whether there were bottlenecks in the secretion of a model protein. RESULTS: A secretory enzyme, xylanase (XynA1), was produced with and without its signal peptide. Cell cultures were fractionated into cytoplasm, membrane, cell wall, and extracellular milieu protein extracts, which were analysed using immunoblotting and enzyme activity assays. The main bottleneck identified was proteolytic degradation of XynA1 during or after its translocation. A combination of mass spectrometry and bioinformatics indicated the presence of several proteases that might be involved in this process. CONCLUSION: The creation of protease-deficient strains may be beneficial towards the development of P. thermoglucosidasius as a platform organism for industrial processes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12896-019-0511-0) contains supplementary material, which is available to authorized users. BioMed Central 2019-03-20 /pmc/articles/PMC6425571/ /pubmed/30894163 http://dx.doi.org/10.1186/s12896-019-0511-0 Text en © The Author(s). 2019 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 Article
Holland, Alexandria T. N.
Danson, Michael J.
Bolhuis, Albert
Inhibition of extracellular proteases improves the production of a xylanase in Parageobacillus thermoglucosidasius
title Inhibition of extracellular proteases improves the production of a xylanase in Parageobacillus thermoglucosidasius
title_full Inhibition of extracellular proteases improves the production of a xylanase in Parageobacillus thermoglucosidasius
title_fullStr Inhibition of extracellular proteases improves the production of a xylanase in Parageobacillus thermoglucosidasius
title_full_unstemmed Inhibition of extracellular proteases improves the production of a xylanase in Parageobacillus thermoglucosidasius
title_short Inhibition of extracellular proteases improves the production of a xylanase in Parageobacillus thermoglucosidasius
title_sort inhibition of extracellular proteases improves the production of a xylanase in parageobacillus thermoglucosidasius
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6425571/
https://www.ncbi.nlm.nih.gov/pubmed/30894163
http://dx.doi.org/10.1186/s12896-019-0511-0
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AT bolhuisalbert inhibitionofextracellularproteasesimprovestheproductionofaxylanaseinparageobacillusthermoglucosidasius