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Explore or exploit? A model-based screening strategy for PETase secretion by Corynebacterium glutamicum
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  • Laura M. Helleckes,
  • Carolin Müller,
  • Tim Griesbach,
  • Vera Waffenschmidt,
  • Matthias Moch,
  • Michael Osthege,
  • Wolfgang Wiechert,
  • Marco Oldiges
Laura M. Helleckes
Forschungszentrum Julich Institut fur Bio und Geowissenschaften

Corresponding Author:[email protected]

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Carolin Müller
Forschungszentrum Julich Institut fur Bio und Geowissenschaften
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Tim Griesbach
Forschungszentrum Julich Institut fur Bio und Geowissenschaften
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Vera Waffenschmidt
Forschungszentrum Julich Institut fur Bio und Geowissenschaften
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Matthias Moch
Forschungszentrum Julich Institut fur Bio und Geowissenschaften
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Michael Osthege
Forschungszentrum Julich Institut fur Bio und Geowissenschaften
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Wolfgang Wiechert
Forschungszentrum Julich Institut fur Bio und Geowissenschaften
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Marco Oldiges
Forschungszentrum Julich Institut fur Bio und Geowissenschaften
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Abstract

Extracellular production of target proteins simplifies downstream processing due to obsolete cell disruption. However, optimal combinations of a heterologous protein, suitable signal peptide and secretion host can currently not be predicted, resulting in large strain libraries that need to be tested. On the experimental side, this challenge can be tackled by miniaturization, parallelization and automation, which provide high-throughput screening data. These data need to be condensed into a candidate ranking for decision making to focus bioprocess development on the most promising candidates. We screened for Bacillus subtilis signal peptides mediating Sec secretion of two polyethylene terephthalate degrading enzymes (PETases), leaf-branch compost cutinase (LCC) and polyester hydrolase (PE-H) mutants, by Corynebacterium glutamicum. We developed a fully automated screening process and constructed an accompanying Bayesian statistical modeling framework, which we applied in screenings for highest activity in 4-nitrophenyl palmitate degradation. In contrast to classical evaluation methods, batch effects and biological errors are taken into account and their uncertainty is quantified. Within only two rounds of screening, the most suitable signal peptide was identified for each PETase. Results from LCC secretion in microliter-scale cultivation were shown to be scalable to laboratory-scale bioreactors. This work demonstrates an experiment-modeling loop that can accelerate early-stage screening in a way that experimental capacities are focused to the most promising strain candidates. Combined with high-throughput cloning, this paves the way for using large strain libraries of several hundreds of strains in a Design-Build-Test-Learn approach.
28 Jul 2022Submitted to Biotechnology and Bioengineering
28 Jul 2022Submission Checks Completed
28 Jul 2022Assigned to Editor
31 Jul 2022Reviewer(s) Assigned
21 Aug 2022Editorial Decision: Revise Major
21 Aug 2022Review(s) Completed, Editorial Evaluation Pending
30 Sep 20221st Revision Received
30 Sep 2022Submission Checks Completed
30 Sep 2022Assigned to Editor
09 Oct 2022Review(s) Completed, Editorial Evaluation Pending
09 Oct 2022Editorial Decision: Accept
Jan 2023Published in Biotechnology and Bioengineering volume 120 issue 1 on pages 139-153. 10.1002/bit.28261