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 . Proteas...
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crplos:10.1371/journal.pone.0247462 2024-05-19T07:31:28+00:00 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 Hagiwara, Daisuke NARO Bio-oriented Technology Research Advancement Institution 2021 http://dx.doi.org/10.1371/journal.pone.0247462 https://dx.plos.org/10.1371/journal.pone.0247462 en eng Public Library of Science (PLoS) http://creativecommons.org/licenses/by/4.0/ PLOS ONE volume 16, issue 3, page e0247462 ISSN 1932-6203 journal-article 2021 crplos https://doi.org/10.1371/journal.pone.0247462 2024-05-01T06:59:57Z 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, Pa PRO1 and Pa PRO2 , with conserved catalytically important domains and signal peptides indicative of vacuolar protease function. We then prepared gene-deletion mutants of strain GB-4(0), ΔPa PRO1 and ΔPa PRO2 , 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 Pa PRO2 deletion mutant of a PaE-overexpression strain named XG8 by introducing a PaE high-production cassette into the strain GB-4(0). The ΔPa PRO2 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 ΔPa PRO2 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 PLOS PLOS ONE 16 3 e0247462 |
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Open Polar |
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PLOS |
op_collection_id |
crplos |
language |
English |
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, Pa PRO1 and Pa PRO2 , with conserved catalytically important domains and signal peptides indicative of vacuolar protease function. We then prepared gene-deletion mutants of strain GB-4(0), ΔPa PRO1 and ΔPa PRO2 , 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 Pa PRO2 deletion mutant of a PaE-overexpression strain named XG8 by introducing a PaE high-production cassette into the strain GB-4(0). The ΔPa PRO2 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 ΔPa PRO2 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. |
author2 |
Hagiwara, Daisuke NARO Bio-oriented Technology Research Advancement Institution |
format |
Article in Journal/Newspaper |
author |
Omae, Natsuki Sameshima-Yamashita, Yuka Ushimaru, Kazunori Koike, Hideaki Kitamoto, Hiroko Morita, Tomotake |
spellingShingle |
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 |
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 (PLoS) |
publishDate |
2021 |
url |
http://dx.doi.org/10.1371/journal.pone.0247462 https://dx.plos.org/10.1371/journal.pone.0247462 |
genre |
Antarc* Antarctica antarcticus |
genre_facet |
Antarc* Antarctica antarcticus |
op_source |
PLOS ONE volume 16, issue 3, page e0247462 ISSN 1932-6203 |
op_rights |
http://creativecommons.org/licenses/by/4.0/ |
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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1799469323146756096 |