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Adaptive evolution in producing microtiter cultivations generates genetically stable Escherichia coli production hosts for continuous bioprocessing
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  • Artur Schuller,
  • Monika Cserjan-Puschmann,
  • Christoph Köppl,
  • Reingard Grabherr,
  • Martin Wagenknecht,
  • Matteo Schiavinato,
  • Juliane Dohm,
  • Heinz Himmelbauer,
  • Gerald Striedner
Artur Schuller
Christian Doppler Laboratory for production of next-level biopharmaceuticals in E. coli, University of Natural Resources and Life Sciences
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Monika Cserjan-Puschmann
Christian Doppler Laboratory for production of next-level biopharmaceuticals in E. coli, University of Natural Resources and Life Sciences
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Christoph Köppl
Christian Doppler Laboratory for production of next-level biopharmaceuticals in E. coli, University of Natural Resources and Life Sciences
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Reingard Grabherr
Christian Doppler Laboratory for production of next-level biopharmaceuticals in E. coli, University of Natural Resources and Life Sciences
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Martin Wagenknecht
Boehringer Ingelheim RCV GmbH und Co KG
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Matteo Schiavinato
Institute of Computational Biology, University of Natural Resources and Life Sciences
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Juliane Dohm
Institute of Computational Biology, University of Natural Resources and Life Sciences
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Heinz Himmelbauer
Institute of Computational Biology, University of Natural Resources and Life Sciences
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Gerald Striedner
Christian Doppler Laboratory for production of next-level biopharmaceuticals in E. coli, University of Natural Resources and Life Sciences
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Abstract

The production of recombinant proteins usually reduces cell fitness and the growth rate of producing cells. The growth disadvantage favors faster-growing non-producer mutants. Therefore, continuous bioprocessing is hardly feasible in Escherichia coli due to the high escape rate. We investigated the stability of E. coli expression systems under long-term production conditions and how metabolic load triggered by recombinant gene expression influences the characteristics of mutations. We conducted iterated fed-batch-like microbioreactor cultivations under production conditions. We used the easy-to-produce green fluorescent protein (GFP) and a challenging antigen-binding fragment (Fab) as model proteins, and BL21(DE3) and BL21Q strains as expression hosts. In comparative whole genome sequencing analyses, we identified mutations that allowed cells to grow unhindered despite recombinant protein production. A T7 RNA polymerase expression system is only conditionally suitable for long-term cultivation under production conditions. Mutations leading to non-producers occur in either the T7 RNA polymerase gene or the T7 promoter. The host RNA polymerase-based BL21Q expression system remained stable in the production of GFP in long-term cultivations. For the production of Fab, mutations in lacI of the BL21Q derivatives had positive effects on long-term stability. Our results indicate that adaptive evolution carried out with genome-integrated E. coli expression systems in microtiter cultivations under industrial relevant production conditions is an efficient strain development tool for production hosts.

Peer review status:UNDER REVIEW

10 Aug 2020Submitted to Biotechnology Journal
15 Aug 2020Submission Checks Completed
15 Aug 2020Assigned to Editor
23 Aug 2020Reviewer(s) Assigned
21 Sep 2020Editorial Decision: Revise Minor
23 Sep 20201st Revision Received
23 Sep 2020Assigned to Editor
23 Sep 2020Submission Checks Completed
23 Sep 2020Reviewer(s) Assigned