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Using promoter libraries to reduce metabolic burden due to plasmid-encoded proteins in recombinant Escherichia coli
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Pasini, Martina, Fernández-Castané, Alfred , Jaramillo, Alfonso, de Mas, Carles , Caminal, Gloria and Ferrer, Pau (2016) Using promoter libraries to reduce metabolic burden due to plasmid-encoded proteins in recombinant Escherichia coli. New Biotechnology, 33 (1). pp. 78-90. doi:10.1016/j.nbt.2015.08.003
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WRAP_0380721-lf-270815-5f36aa53-843d-4dec-9d2a-9652b3c4ebaa.pdf - Accepted Version - Requires a PDF viewer. Download (2810Kb) | Preview |
Official URL: http://dx.doi.org/10.1016/j.nbt.2015.08.003
Abstract
The over-expression of proteins in recombinant host cells often requires a significant amount of resources causing an increase in the metabolic load for the host. This results in a variety of physiological responses leading to altered growth parameters, including growth inhibition or activation of secondary metabolism pathways. Moreover, the expression of other plasmid-encoded genes such as antibiotic resistance genes or repressor proteins may also alter growth kinetics. In this work, we have developed a second-generation system suitable for Escherichia coli expression with an antibiotic-free plasmid maintenance mechanism based on a glycine auxotrophic marker (glyA). Metabolic burden related to plasmid maintenance and heterologous protein expression was minimized by tuning the expression levels of the repressor protein (LacI) and glyA using a library of promoters and applying synthetic biology tools that allow the rapid construction of vectors. The engineered antibiotic-free expression system was applied to the l-fuculose phosphate aldolase (FucA) over-production, showing an increase in production up to 3.8-fold in terms of FucA yield (mg g−1DCW) and 4.5-fold in terms of FucA activity (AU g−1DCW) compared to previous expression. Moreover, acetic acid production was reduced to 50%, expressed as gAc gDCW−1. Our results showed that the aforementioned approaches are of paramount importance in order to increment the protein production in terms of mass and activity.
| Item Type: | Journal Article | ||||||||
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| Subjects: | T Technology > TP Chemical technology | ||||||||
| Divisions: | Faculty of Science > Life Sciences (2010- ) | ||||||||
| Library of Congress Subject Headings (LCSH): | Proteins -- Research, Synthetic biology , Recombinant proteins, Escherichia coli, Cells -- Growth, Antibiotics | ||||||||
| Journal or Publication Title: | New Biotechnology | ||||||||
| Publisher: | Elsevier | ||||||||
| ISSN: | 1871-6784 | ||||||||
| Official Date: | 25 January 2016 | ||||||||
| Dates: |
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| Volume: | 33 | ||||||||
| Number: | 1 | ||||||||
| Page Range: | pp. 78-90 | ||||||||
| DOI: | 10.1016/j.nbt.2015.08.003 | ||||||||
| Status: | Peer Reviewed | ||||||||
| Publication Status: | Published | ||||||||
| Access rights to Published version: | Restricted or Subscription Access | ||||||||
| Funder: | Universidad Autónoma de Barcelona (UAdB) | ||||||||
| Grant number: | Novel Alternatives for Microbial Production of Enzymes and Multi Enzymatic stereoselective Synthesis (EzProSyn) |
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