Cargando…
Improvement of Pseudoalteromonas haloplanktis TAC125 as a Cell Factory: IPTG-Inducible Plasmid Construction and Strain Engineering
Our group has used the marine bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) as a platform for the successful recombinant production of “difficult” proteins, including eukaryotic proteins, at low temperatures. However, there is still room for improvement both in the refinement of PhTAC12...
Autores principales: | , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598627/ https://www.ncbi.nlm.nih.gov/pubmed/32987756 http://dx.doi.org/10.3390/microorganisms8101466 |
_version_ | 1783602665793519616 |
---|---|
author | Colarusso, Andrea Lauro, Concetta Calvanese, Marzia Parrilli, Ermenegilda Tutino, Maria Luisa |
author_facet | Colarusso, Andrea Lauro, Concetta Calvanese, Marzia Parrilli, Ermenegilda Tutino, Maria Luisa |
author_sort | Colarusso, Andrea |
collection | PubMed |
description | Our group has used the marine bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) as a platform for the successful recombinant production of “difficult” proteins, including eukaryotic proteins, at low temperatures. However, there is still room for improvement both in the refinement of PhTAC125 expression plasmids and in the bacterium’s intrinsic ability to accumulate and handle heterologous products. Here, we present an integrated approach of plasmid design and strain engineering finalized to increment the recombinant expression and optimize the inducer uptake in PhTAC125. To this aim, we developed the IPTG-inducible plasmid pP79 and an engineered PhTAC125 strain called KrPL LacY(+). This mutant was designed to express the E. coli lactose permease and to produce only a truncated version of the endogenous Lon protease through an integration-deletion strategy. In the wild-type strain, pP79 assured a significantly better production of two reporters in comparison to the most recent expression vector employed in PhTAC125. Nevertheless, the use of KrPL LacY(+) was crucial to achieving satisfying production levels using reasonable IPTG concentrations, even at 0 °C. Both the wild-type and the mutant recombinant strains are characterized by an average graded response upon IPTG induction and they will find different future applications depending on the desired levels of expression. |
format | Online Article Text |
id | pubmed-7598627 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-75986272020-10-31 Improvement of Pseudoalteromonas haloplanktis TAC125 as a Cell Factory: IPTG-Inducible Plasmid Construction and Strain Engineering Colarusso, Andrea Lauro, Concetta Calvanese, Marzia Parrilli, Ermenegilda Tutino, Maria Luisa Microorganisms Article Our group has used the marine bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) as a platform for the successful recombinant production of “difficult” proteins, including eukaryotic proteins, at low temperatures. However, there is still room for improvement both in the refinement of PhTAC125 expression plasmids and in the bacterium’s intrinsic ability to accumulate and handle heterologous products. Here, we present an integrated approach of plasmid design and strain engineering finalized to increment the recombinant expression and optimize the inducer uptake in PhTAC125. To this aim, we developed the IPTG-inducible plasmid pP79 and an engineered PhTAC125 strain called KrPL LacY(+). This mutant was designed to express the E. coli lactose permease and to produce only a truncated version of the endogenous Lon protease through an integration-deletion strategy. In the wild-type strain, pP79 assured a significantly better production of two reporters in comparison to the most recent expression vector employed in PhTAC125. Nevertheless, the use of KrPL LacY(+) was crucial to achieving satisfying production levels using reasonable IPTG concentrations, even at 0 °C. Both the wild-type and the mutant recombinant strains are characterized by an average graded response upon IPTG induction and they will find different future applications depending on the desired levels of expression. MDPI 2020-09-24 /pmc/articles/PMC7598627/ /pubmed/32987756 http://dx.doi.org/10.3390/microorganisms8101466 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Colarusso, Andrea Lauro, Concetta Calvanese, Marzia Parrilli, Ermenegilda Tutino, Maria Luisa Improvement of Pseudoalteromonas haloplanktis TAC125 as a Cell Factory: IPTG-Inducible Plasmid Construction and Strain Engineering |
title | Improvement of Pseudoalteromonas haloplanktis TAC125 as a Cell Factory: IPTG-Inducible Plasmid Construction and Strain Engineering |
title_full | Improvement of Pseudoalteromonas haloplanktis TAC125 as a Cell Factory: IPTG-Inducible Plasmid Construction and Strain Engineering |
title_fullStr | Improvement of Pseudoalteromonas haloplanktis TAC125 as a Cell Factory: IPTG-Inducible Plasmid Construction and Strain Engineering |
title_full_unstemmed | Improvement of Pseudoalteromonas haloplanktis TAC125 as a Cell Factory: IPTG-Inducible Plasmid Construction and Strain Engineering |
title_short | Improvement of Pseudoalteromonas haloplanktis TAC125 as a Cell Factory: IPTG-Inducible Plasmid Construction and Strain Engineering |
title_sort | improvement of pseudoalteromonas haloplanktis tac125 as a cell factory: iptg-inducible plasmid construction and strain engineering |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598627/ https://www.ncbi.nlm.nih.gov/pubmed/32987756 http://dx.doi.org/10.3390/microorganisms8101466 |
work_keys_str_mv | AT colarussoandrea improvementofpseudoalteromonashaloplanktistac125asacellfactoryiptginducibleplasmidconstructionandstrainengineering AT lauroconcetta improvementofpseudoalteromonashaloplanktistac125asacellfactoryiptginducibleplasmidconstructionandstrainengineering AT calvanesemarzia improvementofpseudoalteromonashaloplanktistac125asacellfactoryiptginducibleplasmidconstructionandstrainengineering AT parrilliermenegilda improvementofpseudoalteromonashaloplanktistac125asacellfactoryiptginducibleplasmidconstructionandstrainengineering AT tutinomarialuisa improvementofpseudoalteromonashaloplanktistac125asacellfactoryiptginducibleplasmidconstructionandstrainengineering |