Development of high-copy number plasmids in Pseudoalteromonas haloplanktis TAC125
ABSTRACT: The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) is considered an interesting alternative host for the recombinant protein production, that can be explored when the conventional bacterial expression systems fail. Indeed, the manufacture of all the difficult-to-expre...
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Online Access: | http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10033558/ http://www.ncbi.nlm.nih.gov/pubmed/36912903 https://doi.org/10.1007/s00253-023-12448-w |
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ftpubmed:oai:pubmedcentral.nih.gov:10033558 2023-05-15T13:47:45+02:00 Development of high-copy number plasmids in Pseudoalteromonas haloplanktis TAC125 Calvanese, Marzia Balestra, Cecilia Colarusso, Andrea Lauro, Concetta Riccardi, Christopher Fondi, Marco Parrilli, Ermenegilda Tutino, Maria Luisa 2023-03-13 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10033558/ http://www.ncbi.nlm.nih.gov/pubmed/36912903 https://doi.org/10.1007/s00253-023-12448-w en eng Springer Berlin Heidelberg http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10033558/ http://www.ncbi.nlm.nih.gov/pubmed/36912903 http://dx.doi.org/10.1007/s00253-023-12448-w © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . Appl Microbiol Biotechnol Applied Genetics and Molecular Biotechnology Text 2023 ftpubmed https://doi.org/10.1007/s00253-023-12448-w 2023-03-26T02:16:51Z ABSTRACT: The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) is considered an interesting alternative host for the recombinant protein production, that can be explored when the conventional bacterial expression systems fail. Indeed, the manufacture of all the difficult-to-express proteins produced so far in this bacterial platform gave back soluble and active products. Despite these promising results, the low yield of recombinant protein production achieved is hampering the wider and industrial exploitation of this psychrophilic cell factory. All the expression plasmids developed so far in PhTAC125 are based on the origin of replication of the endogenous pMtBL plasmid and are maintained at a very low copy number. In this work, we set up an experimental strategy to select mutated OriR sequences endowed with the ability to establish recombinant plasmids at higher multiplicity per cell. The solution to this major production bottleneck was achieved by the construction of a library of psychrophilic vectors, each containing a randomly mutated version of pMtBL OriR, and its screening by fluorescence-activated cell sorting (FACS). The selected clones allowed the identification of mutated OriR sequences effective in enhancing the plasmid copy number of approximately two orders of magnitude, and the production of the recombinant green fluorescent protein was increased up to twenty times approximately. Moreover, the molecular characterization of the different mutant OriR sequences allowed us to suggest some preliminary clues on the pMtBL replication mechanism that deserve to be further investigated in the future. KEY POINTS: • Setup of an electroporation procedure for Pseudoalteromonas haloplanktis TAC125. • Two order of magnitude improvement of OriR-derived psychrophilic expression systems. • Almost twenty times enhancement in Green fluorescent protein production. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00253-023-12448-w. Text Antarc* Antarctic PubMed Central (PMC) Antarctic The Antarctic Applied Microbiology and Biotechnology 107 7-8 2469 2481 |
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PubMed Central (PMC) |
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ftpubmed |
language |
English |
topic |
Applied Genetics and Molecular Biotechnology |
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Applied Genetics and Molecular Biotechnology Calvanese, Marzia Balestra, Cecilia Colarusso, Andrea Lauro, Concetta Riccardi, Christopher Fondi, Marco Parrilli, Ermenegilda Tutino, Maria Luisa Development of high-copy number plasmids in Pseudoalteromonas haloplanktis TAC125 |
topic_facet |
Applied Genetics and Molecular Biotechnology |
description |
ABSTRACT: The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) is considered an interesting alternative host for the recombinant protein production, that can be explored when the conventional bacterial expression systems fail. Indeed, the manufacture of all the difficult-to-express proteins produced so far in this bacterial platform gave back soluble and active products. Despite these promising results, the low yield of recombinant protein production achieved is hampering the wider and industrial exploitation of this psychrophilic cell factory. All the expression plasmids developed so far in PhTAC125 are based on the origin of replication of the endogenous pMtBL plasmid and are maintained at a very low copy number. In this work, we set up an experimental strategy to select mutated OriR sequences endowed with the ability to establish recombinant plasmids at higher multiplicity per cell. The solution to this major production bottleneck was achieved by the construction of a library of psychrophilic vectors, each containing a randomly mutated version of pMtBL OriR, and its screening by fluorescence-activated cell sorting (FACS). The selected clones allowed the identification of mutated OriR sequences effective in enhancing the plasmid copy number of approximately two orders of magnitude, and the production of the recombinant green fluorescent protein was increased up to twenty times approximately. Moreover, the molecular characterization of the different mutant OriR sequences allowed us to suggest some preliminary clues on the pMtBL replication mechanism that deserve to be further investigated in the future. KEY POINTS: • Setup of an electroporation procedure for Pseudoalteromonas haloplanktis TAC125. • Two order of magnitude improvement of OriR-derived psychrophilic expression systems. • Almost twenty times enhancement in Green fluorescent protein production. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00253-023-12448-w. |
format |
Text |
author |
Calvanese, Marzia Balestra, Cecilia Colarusso, Andrea Lauro, Concetta Riccardi, Christopher Fondi, Marco Parrilli, Ermenegilda Tutino, Maria Luisa |
author_facet |
Calvanese, Marzia Balestra, Cecilia Colarusso, Andrea Lauro, Concetta Riccardi, Christopher Fondi, Marco Parrilli, Ermenegilda Tutino, Maria Luisa |
author_sort |
Calvanese, Marzia |
title |
Development of high-copy number plasmids in Pseudoalteromonas haloplanktis TAC125 |
title_short |
Development of high-copy number plasmids in Pseudoalteromonas haloplanktis TAC125 |
title_full |
Development of high-copy number plasmids in Pseudoalteromonas haloplanktis TAC125 |
title_fullStr |
Development of high-copy number plasmids in Pseudoalteromonas haloplanktis TAC125 |
title_full_unstemmed |
Development of high-copy number plasmids in Pseudoalteromonas haloplanktis TAC125 |
title_sort |
development of high-copy number plasmids in pseudoalteromonas haloplanktis tac125 |
publisher |
Springer Berlin Heidelberg |
publishDate |
2023 |
url |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10033558/ http://www.ncbi.nlm.nih.gov/pubmed/36912903 https://doi.org/10.1007/s00253-023-12448-w |
geographic |
Antarctic The Antarctic |
geographic_facet |
Antarctic The Antarctic |
genre |
Antarc* Antarctic |
genre_facet |
Antarc* Antarctic |
op_source |
Appl Microbiol Biotechnol |
op_relation |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10033558/ http://www.ncbi.nlm.nih.gov/pubmed/36912903 http://dx.doi.org/10.1007/s00253-023-12448-w |
op_rights |
© The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
op_doi |
https://doi.org/10.1007/s00253-023-12448-w |
container_title |
Applied Microbiology and Biotechnology |
container_volume |
107 |
container_issue |
7-8 |
container_start_page |
2469 |
op_container_end_page |
2481 |
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1766247810619932672 |