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 (mg g(-1)DCW) and 4.5-fold in terms of FucA activity (AU g(-1)DCW) 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.
Department of Chemical Engineering, Escola d'Enginyeria, Universitat Autònoma de Barcelona, Bellaterra, Cerdanyola del Vallès, Spain. Electronic address: Martina.pasini@uab.cat.
Martina
M
Pasini
School of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham, UK.
Alfred
A
Fernández-Castané
School of Life Sciences, University of Warwick, Coventry CV4 7AL, UK; Institute of Systems and Synthetic Biology, Université d'Évry Val d'Essonne, CNRS, F-91000 Évry, France.
Alfonso
A
Jaramillo
Department of Chemical Engineering, Escola d'Enginyeria, Universitat Autònoma de Barcelona, Bellaterra, Cerdanyola del Vallès, Spain.
Carles
C
de Mas
Institute of Advanced Chemical of Catalonia, IQAC-CSIC, Spain.
Gloria
G
Caminal
Department of Chemical Engineering, Escola d'Enginyeria, Universitat Autònoma de Barcelona, Bellaterra, Cerdanyola del Vallès, Spain.
Pau
P
Ferrer
D019342:Acetic Acid / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D018533:Biomass; D018929:Cell Culture Techniques; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005822:Genetic Vectors / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D008780:Methyltransferases / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics*
D000900:Anti-Bacterial Agents; D029968:Escherichia coli Proteins; D008780:Methyltransferases; D005947:Glucose; D019342:Acetic Acid