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Exchange of endogenous and heterogeneous yeast terminators in Pichia pastoris to tune mRNA stability and gene expression

In the yeast Saccharomyces cerevisiae, terminator sequences not only terminate transcription but also affect expression levels of the protein-encoded upstream of the terminator. The non-conventional yeast Pichia pastoris (syn. Komagataella phaffii) has frequently been used as a platform for metaboli...

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Autores principales: Ito, Yoichiro, Terai, Goro, Ishigami, Misa, Hashiba, Noriko, Nakamura, Yasuyuki, Bamba, Takahiro, Kumokita, Ryota, Hasunuma, Tomohisa, Asai, Kiyoshi, Ishii, Jun, Kondo, Akihiko
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736810/
https://www.ncbi.nlm.nih.gov/pubmed/33257988
http://dx.doi.org/10.1093/nar/gkaa1066
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author Ito, Yoichiro
Terai, Goro
Ishigami, Misa
Hashiba, Noriko
Nakamura, Yasuyuki
Bamba, Takahiro
Kumokita, Ryota
Hasunuma, Tomohisa
Asai, Kiyoshi
Ishii, Jun
Kondo, Akihiko
author_facet Ito, Yoichiro
Terai, Goro
Ishigami, Misa
Hashiba, Noriko
Nakamura, Yasuyuki
Bamba, Takahiro
Kumokita, Ryota
Hasunuma, Tomohisa
Asai, Kiyoshi
Ishii, Jun
Kondo, Akihiko
author_sort Ito, Yoichiro
collection PubMed
description In the yeast Saccharomyces cerevisiae, terminator sequences not only terminate transcription but also affect expression levels of the protein-encoded upstream of the terminator. The non-conventional yeast Pichia pastoris (syn. Komagataella phaffii) has frequently been used as a platform for metabolic engineering but knowledge regarding P. pastoris terminators is limited. To explore terminator sequences available to tune protein expression levels in P. pastoris, we created a ‘terminator catalog’ by testing 72 sequences, including terminators from S. cerevisiae or P. pastoris and synthetic terminators. Altogether, we found that the terminators have a tunable range of 17-fold. We also found that S. cerevisiae terminator sequences maintain function when transferred to P. pastoris. Successful tuning of protein expression levels was shown not only for the reporter gene used to define the catalog but also using betaxanthin production as an example application in pathway flux regulation. Moreover, we found experimental evidence that protein expression levels result from mRNA abundance and in silico evidence that levels reflect the stability of mRNA 3′-UTR secondary structure. In combination with promoter selection, the novel terminator catalog constitutes a basic toolbox for tuning protein expression levels in metabolic engineering and synthetic biology in P. pastoris.
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spelling pubmed-77368102020-12-17 Exchange of endogenous and heterogeneous yeast terminators in Pichia pastoris to tune mRNA stability and gene expression Ito, Yoichiro Terai, Goro Ishigami, Misa Hashiba, Noriko Nakamura, Yasuyuki Bamba, Takahiro Kumokita, Ryota Hasunuma, Tomohisa Asai, Kiyoshi Ishii, Jun Kondo, Akihiko Nucleic Acids Res Synthetic Biology and Bioengineering In the yeast Saccharomyces cerevisiae, terminator sequences not only terminate transcription but also affect expression levels of the protein-encoded upstream of the terminator. The non-conventional yeast Pichia pastoris (syn. Komagataella phaffii) has frequently been used as a platform for metabolic engineering but knowledge regarding P. pastoris terminators is limited. To explore terminator sequences available to tune protein expression levels in P. pastoris, we created a ‘terminator catalog’ by testing 72 sequences, including terminators from S. cerevisiae or P. pastoris and synthetic terminators. Altogether, we found that the terminators have a tunable range of 17-fold. We also found that S. cerevisiae terminator sequences maintain function when transferred to P. pastoris. Successful tuning of protein expression levels was shown not only for the reporter gene used to define the catalog but also using betaxanthin production as an example application in pathway flux regulation. Moreover, we found experimental evidence that protein expression levels result from mRNA abundance and in silico evidence that levels reflect the stability of mRNA 3′-UTR secondary structure. In combination with promoter selection, the novel terminator catalog constitutes a basic toolbox for tuning protein expression levels in metabolic engineering and synthetic biology in P. pastoris. Oxford University Press 2020-12-01 /pmc/articles/PMC7736810/ /pubmed/33257988 http://dx.doi.org/10.1093/nar/gkaa1066 Text en © The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Synthetic Biology and Bioengineering
Ito, Yoichiro
Terai, Goro
Ishigami, Misa
Hashiba, Noriko
Nakamura, Yasuyuki
Bamba, Takahiro
Kumokita, Ryota
Hasunuma, Tomohisa
Asai, Kiyoshi
Ishii, Jun
Kondo, Akihiko
Exchange of endogenous and heterogeneous yeast terminators in Pichia pastoris to tune mRNA stability and gene expression
title Exchange of endogenous and heterogeneous yeast terminators in Pichia pastoris to tune mRNA stability and gene expression
title_full Exchange of endogenous and heterogeneous yeast terminators in Pichia pastoris to tune mRNA stability and gene expression
title_fullStr Exchange of endogenous and heterogeneous yeast terminators in Pichia pastoris to tune mRNA stability and gene expression
title_full_unstemmed Exchange of endogenous and heterogeneous yeast terminators in Pichia pastoris to tune mRNA stability and gene expression
title_short Exchange of endogenous and heterogeneous yeast terminators in Pichia pastoris to tune mRNA stability and gene expression
title_sort exchange of endogenous and heterogeneous yeast terminators in pichia pastoris to tune mrna stability and gene expression
topic Synthetic Biology and Bioengineering
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736810/
https://www.ncbi.nlm.nih.gov/pubmed/33257988
http://dx.doi.org/10.1093/nar/gkaa1066
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