Tuning the transcription rate of recombinant protein in strong Escherichia coli expression systems through repressor titration.
The main goal of this work was to develop a strategy that enables tuning of recombinant gene expression relative to the metabolic capacity of the host cell synthesis machinery. In the past, strong expression systems have been developed in order to maximize recombinant gene expression. However, these systems exert an extremely high metabolic burden onto the host cell, which may even lead to cell death. Hence, the period of recombinant gene expression is significantly reduced, and therefore, maximal yield cannot be attained. To extend the production phase and to achieve optimal yields, adjustment of recombinant gene expression by modulation of the transcription rate is required. To control transcription, we designed a feed regime, which continuously supplies limiting amounts of inducer in a constant ratio to biomass. For the accurate determination of appropriate amounts of inducer, a time shifted exponential substrate and inducer feed strategy has been developed. The potential of this metabolic and engineering integrated approach was proven in fed-batch cultivation experiments using E. coli HMS174(DE3)(pET11ahSOD) as model system. Furthermore, our strategy enables the use of lactose as inducer, since its consumption can be compensated by appropriate feed profiles. The attained results fully comply with all requirements of industrial large scale cultivation and improve the applicability of strong expression systems.
Striedner
Gerald
G
Institute of Applied Microbiology, University of Agricultural Sciences Vienna, Muthgasse 18, A-1190 Vienna, Austria.
Cserjan-Puschmann
Monika
M
Pötschacher
Florentina
F
Bayer
Karl
K
D000222:Adaptation, Physiological / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D005246:Feedback / Q000502:physiology; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D006801:Humans; D007785:Lactose / Q000378:metabolism*; D010865:Pilot Projects; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000502:physiology*; D013482:Superoxide Dismutase / Q000096:biosynthesis / Q000235:genetics; D014158:Transcription, Genetic / Q000502:physiology*; D015533:Transcriptional Activation / Q000502:physiology
D011994:Recombinant Proteins; D012097:Repressor Proteins; D013482:Superoxide Dismutase; D007785:Lactose