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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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Official URL: http://dx.doi.org/10.1016/j.nbt.2015.08.003

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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
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:
DateEvent
25 January 2016Published
31 September 2015Available
17 August 2015Accepted
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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