Abstract
Controlling the recombinant protein production rate in Escherichia coli is of utmost importance to ensure product quality and quantity. Up to now, only the genetic construct, introduced into E. coli, and the specific growth rate of the culture were used to influence and stir the productivity. However, bioprocess technological means to control or even tune the productivity of E. coli are scarce. Here, we present a novel method for the process-technological control over the recombinant protein expression rate in E. coli. A mixed-feed fed-batch bioprocess based on the araBAD promoter expression system using both d-glucose and l-arabinose as assimilable C-sources was designed. Using the model product green fluorescent protein, we show that the specific product formation rate can be efficiently tuned even on the cellular level only via the uptake rate of l-arabinose. This novel approach introduces an additional degree of freedom for the design of recombinant bioprocesses with E. coli. We anticipate that the presented method will result in significant quality and robustness improvement as well as cost and process time reduction for recombinant bacterial bioprocesses in the future.
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Acknowledgments
The authors acknowledge Prof. Ingrid Steiner and Timo Langemann for their assistance with the fluorescence analytics (flourimeter and flow cytometer). Furthermore, the authors acknowledge BIRD-C GmbH & CoKEG (Kritzendorf, Austria) for the provision of the E. coli C41 strain.
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The authors declare that they have no conflict of interest.
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Patrick Sagmeister and Clemens Schimek contributed equally to this work.
Total soluble intracellular protein separated by 12% SDS PAGE and stained with Coomassie Brilliant Blue. Lane 1 to 6 shows the time related expression of EGFP in one center point experiment from the time point of induction (lane 1) to the end of the process (lane 6). The lower box indicates the accumulation of EGFP at the anticipated molecular weight of 33 kDa. The upper box indicates the accumulation of a second protein which is supposed to be Ribulokinase, a 61 kDa protein involved in the L-arabinose catabolism. Lane 7: Molecular weight standard (SeeBlue® Plus2 Pre-stained Protein Standard).
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Sagmeister, P., Schimek, C., Meitz, A. et al. Tunable recombinant protein expression with E. coli in a mixed-feed environment. Appl Microbiol Biotechnol 98, 2937–2945 (2014). https://doi.org/10.1007/s00253-013-5445-1
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DOI: https://doi.org/10.1007/s00253-013-5445-1