A New Expression System Based on Psychrotolerant Debaryomyces macquariensis Yeast and Its Application to the Production of Cold-Active β-d-Galactosidase from Paracoccus sp. 32d
Yeasts provide attractive host/vector systems for heterologous gene expression. The currently used yeast-based expression platforms include mesophilic and thermotolerant species. A eukaryotic expression system working at low temperatures could be particularly useful for the production of thermolabil...
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ftmdpi:oai:mdpi.com:/1422-0067/23/19/11691/ 2023-08-20T04:02:24+02:00 A New Expression System Based on Psychrotolerant Debaryomyces macquariensis Yeast and Its Application to the Production of Cold-Active β-d-Galactosidase from Paracoccus sp. 32d Marta Wanarska Ewelina Krajewska-Przybyszewska Monika Wicka-Grochocka Hubert Cieśliński Anna Pawlak-Szukalska Aneta M. Białkowska Marianna Turkiewicz Tomasz Florczak Ewa Gromek Joanna Krysiak Natalia Filipowicz agris 2022-10-02 application/pdf https://doi.org/10.3390/ijms231911691 EN eng Multidisciplinary Digital Publishing Institute Molecular Microbiology https://dx.doi.org/10.3390/ijms231911691 https://creativecommons.org/licenses/by/4.0/ International Journal of Molecular Sciences; Volume 23; Issue 19; Pages: 11691 Debaryomyces macquariensis strain D50 psychrotolerant yeast host/vector expression system constitutive GAP promoter constitutive TEF1 promoter CYC1 transcriptional terminator Text 2022 ftmdpi https://doi.org/10.3390/ijms231911691 2023-08-01T06:44:04Z Yeasts provide attractive host/vector systems for heterologous gene expression. The currently used yeast-based expression platforms include mesophilic and thermotolerant species. A eukaryotic expression system working at low temperatures could be particularly useful for the production of thermolabile proteins and proteins that tend to form insoluble aggregates. For this purpose, an expression system based on an Antarctic psychrotolerant yeast Debaryomycesmacquariensis strain D50 that is capable of growing at temperatures ranging from 0 to 30 °C has been developed. The optimal physical culture conditions for D. macquariensis D50 in a fermenter are as follows: temperature 20 °C, pH 5.5, aeration rate of 1.5 vvm, and a stirring speed of 300 rpm. Four integrative plasmid vectors equipped with an expression cassette containing the constitutive GAP promoter and CYC1 transcriptional terminator from D. macquariensis D50 were constructed and used to clone and express a gene-encoding cold-active β-d-galactosidase of Paracoccus sp. 32d. The yield was 1150 U/L of recombinant yeast culture. Recombinant D. macquariensis D50 strains were mitotically stable under both selective and non-selective conditions. The D. macquariensis D50 host/vector system has been successfully utilized for the synthesis of heterologous thermolabile protein, and it can be an alternative to other microbial expression systems. Text Antarc* Antarctic MDPI Open Access Publishing Antarctic International Journal of Molecular Sciences 23 19 11691 |
institution |
Open Polar |
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MDPI Open Access Publishing |
op_collection_id |
ftmdpi |
language |
English |
topic |
Debaryomyces macquariensis strain D50 psychrotolerant yeast host/vector expression system constitutive GAP promoter constitutive TEF1 promoter CYC1 transcriptional terminator |
spellingShingle |
Debaryomyces macquariensis strain D50 psychrotolerant yeast host/vector expression system constitutive GAP promoter constitutive TEF1 promoter CYC1 transcriptional terminator Marta Wanarska Ewelina Krajewska-Przybyszewska Monika Wicka-Grochocka Hubert Cieśliński Anna Pawlak-Szukalska Aneta M. Białkowska Marianna Turkiewicz Tomasz Florczak Ewa Gromek Joanna Krysiak Natalia Filipowicz A New Expression System Based on Psychrotolerant Debaryomyces macquariensis Yeast and Its Application to the Production of Cold-Active β-d-Galactosidase from Paracoccus sp. 32d |
topic_facet |
Debaryomyces macquariensis strain D50 psychrotolerant yeast host/vector expression system constitutive GAP promoter constitutive TEF1 promoter CYC1 transcriptional terminator |
description |
Yeasts provide attractive host/vector systems for heterologous gene expression. The currently used yeast-based expression platforms include mesophilic and thermotolerant species. A eukaryotic expression system working at low temperatures could be particularly useful for the production of thermolabile proteins and proteins that tend to form insoluble aggregates. For this purpose, an expression system based on an Antarctic psychrotolerant yeast Debaryomycesmacquariensis strain D50 that is capable of growing at temperatures ranging from 0 to 30 °C has been developed. The optimal physical culture conditions for D. macquariensis D50 in a fermenter are as follows: temperature 20 °C, pH 5.5, aeration rate of 1.5 vvm, and a stirring speed of 300 rpm. Four integrative plasmid vectors equipped with an expression cassette containing the constitutive GAP promoter and CYC1 transcriptional terminator from D. macquariensis D50 were constructed and used to clone and express a gene-encoding cold-active β-d-galactosidase of Paracoccus sp. 32d. The yield was 1150 U/L of recombinant yeast culture. Recombinant D. macquariensis D50 strains were mitotically stable under both selective and non-selective conditions. The D. macquariensis D50 host/vector system has been successfully utilized for the synthesis of heterologous thermolabile protein, and it can be an alternative to other microbial expression systems. |
format |
Text |
author |
Marta Wanarska Ewelina Krajewska-Przybyszewska Monika Wicka-Grochocka Hubert Cieśliński Anna Pawlak-Szukalska Aneta M. Białkowska Marianna Turkiewicz Tomasz Florczak Ewa Gromek Joanna Krysiak Natalia Filipowicz |
author_facet |
Marta Wanarska Ewelina Krajewska-Przybyszewska Monika Wicka-Grochocka Hubert Cieśliński Anna Pawlak-Szukalska Aneta M. Białkowska Marianna Turkiewicz Tomasz Florczak Ewa Gromek Joanna Krysiak Natalia Filipowicz |
author_sort |
Marta Wanarska |
title |
A New Expression System Based on Psychrotolerant Debaryomyces macquariensis Yeast and Its Application to the Production of Cold-Active β-d-Galactosidase from Paracoccus sp. 32d |
title_short |
A New Expression System Based on Psychrotolerant Debaryomyces macquariensis Yeast and Its Application to the Production of Cold-Active β-d-Galactosidase from Paracoccus sp. 32d |
title_full |
A New Expression System Based on Psychrotolerant Debaryomyces macquariensis Yeast and Its Application to the Production of Cold-Active β-d-Galactosidase from Paracoccus sp. 32d |
title_fullStr |
A New Expression System Based on Psychrotolerant Debaryomyces macquariensis Yeast and Its Application to the Production of Cold-Active β-d-Galactosidase from Paracoccus sp. 32d |
title_full_unstemmed |
A New Expression System Based on Psychrotolerant Debaryomyces macquariensis Yeast and Its Application to the Production of Cold-Active β-d-Galactosidase from Paracoccus sp. 32d |
title_sort |
new expression system based on psychrotolerant debaryomyces macquariensis yeast and its application to the production of cold-active β-d-galactosidase from paracoccus sp. 32d |
publisher |
Multidisciplinary Digital Publishing Institute |
publishDate |
2022 |
url |
https://doi.org/10.3390/ijms231911691 |
op_coverage |
agris |
geographic |
Antarctic |
geographic_facet |
Antarctic |
genre |
Antarc* Antarctic |
genre_facet |
Antarc* Antarctic |
op_source |
International Journal of Molecular Sciences; Volume 23; Issue 19; Pages: 11691 |
op_relation |
Molecular Microbiology https://dx.doi.org/10.3390/ijms231911691 |
op_rights |
https://creativecommons.org/licenses/by/4.0/ |
op_doi |
https://doi.org/10.3390/ijms231911691 |
container_title |
International Journal of Molecular Sciences |
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23 |
container_issue |
19 |
container_start_page |
11691 |
_version_ |
1774712843350835200 |