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Extracellular recombinant protein production under continuous culture conditions with Escherichia coli using an alternative plasmid selection mechanism

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Abstract

The secretion of recombinant proteins into the extracellular space by Escherichia coli presents advantages like easier purification and protection from proteolytic degradation. The controlled co-expression of a bacteriocin release protein aids in moving periplasmic proteins through the outer membrane. Since such systems have rarely been applied in continuous culture it seemed to be attractive to study the interplay between growth-phase regulated promoters controlling release protein genes and the productivity of a chemostat process. To avoid the use of antibiotics and render this process more sustainable, alternative plasmid selection mechanisms were required. In the current study, the strain E. coli JM109 harboring plasmid p582 was shown to stably express and secrete recombinant β-glucanase in continuous culture using a minimal medium. The segregational instability of the plasmid in the absence of antibiotic selection pressure was demonstrated. The leuB gene, crucial in the leucine biosynthetic pathway, was cloned onto plasmid p582 and the new construct transformed into an E. coli Keio (ΔleuB) knockout strain. The ability of the construct to complement the leucine auxotrophy was initially tested in shake-flasks and batch cultivation. Later, this strain was successfully grown for more than 200 h in a chemostat and was found to be able to express the recombinant protein. Significantly, it showed a stable maintenance of the recombinant plasmid in the absence of any antibiotics. The plasmid stability in a continuously cultivated E. coli fermentation, in the absence of antibiotics, with extracellular secretion of recombinant protein provides an interesting model for further improvements.

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Abbreviations

t :

Batch/fed-batch/chemostat operating time, h

X :

Dry biomass concentration, g L−1

OD 600 :

Optical density

S Gly :

Residual glycerol concentration in medium, g L−1

E :

Recombinant β-glucanase activity in extracellular medium, kU mL−1

c etPr :

Total protein concentration in extracellular medium, g L−1

S AS :

Residual ammonium sulphate concentration in medium, g L−1

c nPl :

Eluted plasmid concentration normalized to optical density of sample, ng μL−1

L V,E :

Volumetric recombinant β-glucanase productivity, kU L−1 h−1

D :

Space velocity during chemostat operation, h−1

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Acknowledgments

The excellent guidance from Dr. Gerhard Miksch on discussions of the cloning strategy and for the gift of plasmid p582 is deeply acknowledged. Technical assistance from Eberhard Wünsch and Thomas Schäffer is gratefully acknowledged. R.S.V.S thanks the Deutscher Akademischer Austauschdienst (DAAD) for financial support.

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Authors and Affiliations

  1. Fermentation Engineering, Faculty of Technology, Bielefeld University, Universitätsstrasse 25, 33615, Bielefeld, Germany

    Ram Shankar Velur Selvamani, Karl Friehs & Erwin Flaschel

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  1. Ram Shankar Velur Selvamani
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  2. Karl Friehs
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  3. Erwin Flaschel
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Correspondence to Ram Shankar Velur Selvamani.

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Velur Selvamani, R.S., Friehs, K. & Flaschel, E. Extracellular recombinant protein production under continuous culture conditions with Escherichia coli using an alternative plasmid selection mechanism. Bioprocess Biosyst Eng 37, 401–413 (2014). https://doi.org/10.1007/s00449-013-1005-4

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  • DOI: https://doi.org/10.1007/s00449-013-1005-4

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