Using promoter libraries to reduce metabolic burden due to plasmid-encoded proteins in recombinant Escherichia coli

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.


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 (mgg-1DCW) and 4.5-fold in terms of FucA activity (AUg-1DCW) compared to previous expression. Moreover, acetic acid production was reduced to 50%, expressed as gAcgDCW-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. © 2015 Elsevier B.V.

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Divisions: Engineering & Applied Sciences
Engineering & Applied Sciences > European Bioenergy Research Institute (EBRI)
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Published Date: 2016-01-25
Authors: Pasini, Martina
Fernández-castané, Alfred ( 0000-0002-2572-7797)
Jaramillo, Alfonso
De Mas, Carles
Caminal, Gloria
Ferrer, Pau

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