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A novel E. coli strain shows controllable leakiness for extracellular production of recombinant proteins
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  • Jens Kastenhofer,
  • Lukas Rettenbacher,
  • Lukas Feuchtenhofer,
  • Jürgen Mairhofer,
  • Oliver Spadiut
Jens Kastenhofer
Vienna University of Technology
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Lukas Rettenbacher
Vienna University of Technology
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Lukas Feuchtenhofer
enGenes Biotech GmbH
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Jürgen Mairhofer
enGenes Biotech GmbH
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Oliver Spadiut
Vienna University of Technology
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Abstract

Recombinant proteins in Escherichia coli are usually expressed inside the cell. With the growing interest in continuous cultivation, secretion of product to the medium is not only a benefit, but a necessity in future bioprocessing. In this study, we present the X-press strain, a novel E. coli production host for growth decoupled, extracellular recombinant protein production. We investigated the effect of the process parameters temperature and specific glucose uptake rate (qS) on the strain’s growth, productivity, lysis and leakiness, to find the parameter space allowing extracellular protein production. Two model proteins were used, Protein A and a VHH single-domain antibody, and performance was compared to the industrial standard strain BL21(DE3). We show that inducible growth repression in the X-press strain greatly mitigates the effect of metabolic burden under different process conditions. Furthermore, temperature and qS were used to control productivity and leakiness. In the X-press strain, extracellular Protein A and VHH titer reached up to 349 mg/g and 19.6 mg/g, respectively, comprising up to 90% of total soluble product, while keeping cell lysis at a minimum. Our findings demonstrate that the X-press strain constitutes a valuable host for extracellular production of recombinant protein with E. coli.