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

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.
This work was supported by the Spanish MICINN, project number CTQ2011-28398-CO2-01 and the research group 2009SGR281 and by the Bioprocess Engineering and Applied Biocatalisys Group, department of Chemical Engineering of the Universitat Autonoma de Barcelona, Cerdanyola del Valles (Spain). M.P. acknowledges the Universitat Autònoma de Barcelona for the pre-doctoral fellowship. Appendix A