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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ftdoajarticles:oai:doaj.org/article:20aaad40a2524ea083b28581fdc89256 2023-05-15T13:51:51+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. Natsuki Omae Yuka Sameshima-Yamashita Kazunori Ushimaru Hideaki Koike Hiroko Kitamoto Tomotake Morita 2021-01-01T00:00:00Z https://doi.org/10.1371/journal.pone.0247462 https://doaj.org/article/20aaad40a2524ea083b28581fdc89256 EN eng Public Library of Science (PLoS) https://doi.org/10.1371/journal.pone.0247462 https://doaj.org/toc/1932-6203 1932-6203 doi:10.1371/journal.pone.0247462 https://doaj.org/article/20aaad40a2524ea083b28581fdc89256 PLoS ONE, Vol 16, Iss 3, p e0247462 (2021) Medicine R Science Q article 2021 ftdoajarticles https://doi.org/10.1371/journal.pone.0247462 2022-12-31T13:08:25Z 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. Article in Journal/Newspaper Antarc* Antarctica antarcticus Directory of Open Access Journals: DOAJ Articles PLOS ONE 16 3 e0247462 |
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Medicine R Science Q Natsuki Omae Yuka Sameshima-Yamashita Kazunori Ushimaru Hideaki Koike Hiroko Kitamoto Tomotake Morita 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. |
topic_facet |
Medicine R Science Q |
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 |
Article in Journal/Newspaper |
author |
Natsuki Omae Yuka Sameshima-Yamashita Kazunori Ushimaru Hideaki Koike Hiroko Kitamoto Tomotake Morita |
author_facet |
Natsuki Omae Yuka Sameshima-Yamashita Kazunori Ushimaru Hideaki Koike Hiroko Kitamoto Tomotake Morita |
author_sort |
Natsuki Omae |
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 (PLoS) |
publishDate |
2021 |
url |
https://doi.org/10.1371/journal.pone.0247462 https://doaj.org/article/20aaad40a2524ea083b28581fdc89256 |
genre |
Antarc* Antarctica antarcticus |
genre_facet |
Antarc* Antarctica antarcticus |
op_source |
PLoS ONE, Vol 16, Iss 3, p e0247462 (2021) |
op_relation |
https://doi.org/10.1371/journal.pone.0247462 https://doaj.org/toc/1932-6203 1932-6203 doi:10.1371/journal.pone.0247462 https://doaj.org/article/20aaad40a2524ea083b28581fdc89256 |
op_doi |
https://doi.org/10.1371/journal.pone.0247462 |
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PLOS ONE |
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16 |
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3 |
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e0247462 |
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1766255881077391360 |