Disruption of protease A and B orthologous genes in the basidiomycetous yeast Pseudozyma antarctica GB-4(0) yields a stable extracellular biodegradable plastic-degrading enzyme
The yeast Pseudozyma antarctica (currently designated Moesziomyces antarcticus) secretes a xylose-induced biodegradable plastic-degrading enzyme (PaE). To suppress degradation of PaE during production and storage, we targeted the inhibition of proteolytic enzyme activity in P. antarctica. Proteases...
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ftpubmed:oai:pubmedcentral.nih.gov:7968665 2023-05-15T13:31:40+02:00 Disruption of protease A and B orthologous genes in the basidiomycetous yeast Pseudozyma antarctica GB-4(0) yields a stable extracellular biodegradable plastic-degrading enzyme Omae, Natsuki Sameshima-Yamashita, Yuka Ushimaru, Kazunori Koike, Hideaki Kitamoto, Hiroko Morita, Tomotake 2021-03-17 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968665/ http://www.ncbi.nlm.nih.gov/pubmed/33730094 https://doi.org/10.1371/journal.pone.0247462 en eng Public Library of Science http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968665/ http://www.ncbi.nlm.nih.gov/pubmed/33730094 http://dx.doi.org/10.1371/journal.pone.0247462 © 2021 Omae et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. CC-BY PLoS One Research Article Text 2021 ftpubmed https://doi.org/10.1371/journal.pone.0247462 2021-04-04T00:42:11Z The yeast Pseudozyma antarctica (currently designated Moesziomyces antarcticus) secretes a xylose-induced biodegradable plastic-degrading enzyme (PaE). To suppress degradation of PaE during production and storage, we targeted the inhibition of proteolytic enzyme activity in P. antarctica. Proteases A and B act as upper regulators in the proteolytic network of the model yeast, Saccharomyces cerevisiae. We searched for orthologous genes encoding proteases A and B in the genome of P. antarctica GB-4(0) based on the predicted amino acid sequences. We found two gene candidates, PaPRO1 and PaPRO2, with conserved catalytically important domains and signal peptides indicative of vacuolar protease function. We then prepared gene-deletion mutants of strain GB-4(0), ΔPaPRO1 and ΔPaPRO2, and evaluated PaE stability in culture by immunoblotting analysis. Both mutants exhibited sufficient production of PaE without degradation fragments, while the parent strain exhibited the degradation fragments. Therefore, we concluded that the protease A and B orthologous genes are related to the degradation of PaE. To produce a large quantity of PaE, we made a PaPRO2 deletion mutant of a PaE-overexpression strain named XG8 by introducing a PaE high-production cassette into the strain GB-4(0). The ΔPaPRO2 mutant of XG8 was able to produce PaE without the degradation fragments during large-scale cultivation in a 3-L jar fermenter for 3 days at 30°C. After terminating the agitation, the PaE activity in the XG8 ΔPaPRO2 mutant culture was maintained for the subsequent 48 h incubation at 25°C regardless of remaining cells, while activity in the XG8 control was reduced to 55.1%. The gene-deleted mutants will be useful for the development of industrial processes of PaE production and storage. Text Antarc* Antarctica antarcticus PubMed Central (PMC) PLOS ONE 16 3 e0247462 |
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Research Article Omae, Natsuki Sameshima-Yamashita, Yuka Ushimaru, Kazunori Koike, Hideaki Kitamoto, Hiroko Morita, Tomotake Disruption of protease A and B orthologous genes in the basidiomycetous yeast Pseudozyma antarctica GB-4(0) yields a stable extracellular biodegradable plastic-degrading enzyme |
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Research Article |
description |
The yeast Pseudozyma antarctica (currently designated Moesziomyces antarcticus) secretes a xylose-induced biodegradable plastic-degrading enzyme (PaE). To suppress degradation of PaE during production and storage, we targeted the inhibition of proteolytic enzyme activity in P. antarctica. Proteases A and B act as upper regulators in the proteolytic network of the model yeast, Saccharomyces cerevisiae. We searched for orthologous genes encoding proteases A and B in the genome of P. antarctica GB-4(0) based on the predicted amino acid sequences. We found two gene candidates, PaPRO1 and PaPRO2, with conserved catalytically important domains and signal peptides indicative of vacuolar protease function. We then prepared gene-deletion mutants of strain GB-4(0), ΔPaPRO1 and ΔPaPRO2, and evaluated PaE stability in culture by immunoblotting analysis. Both mutants exhibited sufficient production of PaE without degradation fragments, while the parent strain exhibited the degradation fragments. Therefore, we concluded that the protease A and B orthologous genes are related to the degradation of PaE. To produce a large quantity of PaE, we made a PaPRO2 deletion mutant of a PaE-overexpression strain named XG8 by introducing a PaE high-production cassette into the strain GB-4(0). The ΔPaPRO2 mutant of XG8 was able to produce PaE without the degradation fragments during large-scale cultivation in a 3-L jar fermenter for 3 days at 30°C. After terminating the agitation, the PaE activity in the XG8 ΔPaPRO2 mutant culture was maintained for the subsequent 48 h incubation at 25°C regardless of remaining cells, while activity in the XG8 control was reduced to 55.1%. The gene-deleted mutants will be useful for the development of industrial processes of PaE production and storage. |
format |
Text |
author |
Omae, Natsuki Sameshima-Yamashita, Yuka Ushimaru, Kazunori Koike, Hideaki Kitamoto, Hiroko Morita, Tomotake |
author_facet |
Omae, Natsuki Sameshima-Yamashita, Yuka Ushimaru, Kazunori Koike, Hideaki Kitamoto, Hiroko Morita, Tomotake |
author_sort |
Omae, Natsuki |
title |
Disruption of protease A and B orthologous genes in the basidiomycetous yeast Pseudozyma antarctica GB-4(0) yields a stable extracellular biodegradable plastic-degrading enzyme |
title_short |
Disruption of protease A and B orthologous genes in the basidiomycetous yeast Pseudozyma antarctica GB-4(0) yields a stable extracellular biodegradable plastic-degrading enzyme |
title_full |
Disruption of protease A and B orthologous genes in the basidiomycetous yeast Pseudozyma antarctica GB-4(0) yields a stable extracellular biodegradable plastic-degrading enzyme |
title_fullStr |
Disruption of protease A and B orthologous genes in the basidiomycetous yeast Pseudozyma antarctica GB-4(0) yields a stable extracellular biodegradable plastic-degrading enzyme |
title_full_unstemmed |
Disruption of protease A and B orthologous genes in the basidiomycetous yeast Pseudozyma antarctica GB-4(0) yields a stable extracellular biodegradable plastic-degrading enzyme |
title_sort |
disruption of protease a and b orthologous genes in the basidiomycetous yeast pseudozyma antarctica gb-4(0) yields a stable extracellular biodegradable plastic-degrading enzyme |
publisher |
Public Library of Science |
publishDate |
2021 |
url |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968665/ http://www.ncbi.nlm.nih.gov/pubmed/33730094 https://doi.org/10.1371/journal.pone.0247462 |
genre |
Antarc* Antarctica antarcticus |
genre_facet |
Antarc* Antarctica antarcticus |
op_source |
PLoS One |
op_relation |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968665/ http://www.ncbi.nlm.nih.gov/pubmed/33730094 http://dx.doi.org/10.1371/journal.pone.0247462 |
op_rights |
© 2021 Omae et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
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CC-BY |
op_doi |
https://doi.org/10.1371/journal.pone.0247462 |
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