A host-vector toolbox for improved secretory protein overproduction in Bacillus subtilis

Applied Microbiology and Biotechnology, vol. 106, pp. 5137–5151

Abstract

Target proteins in biotechnological applications are highly diverse. Therefore, versatile flexible expression systems for their functional overproduction are required. In order to find the right heterologous gene expression strategy, suitable host-vector systems, which combine different genetic circuits, are useful. In this study, we designed a novel Bacillus subtilis expression toolbox, which allows the overproduction and secretion of potentially toxic enzymes. This toolbox comprises a set of 60 expression vectors, which combine two promoter variants, four strong secretion signals, a translation-enhancing downstream box, and three plasmid backbones. This B. subtilis toolbox is based on a tailor-made, clean deletion mutant strain, which is protease and sporulation deficient and exhibits reduced autolysis and secondary metabolism. The appropriateness of this alternative expression platform was tested for the overproduction of two difficult-to-produce eukaryotic model proteins. These included the sulfhydryl oxidase Sox from Saccharomyces cerevisiae, which forms reactive hydrogen peroxide and undesired cross-linking of functional proteins, and the human interleukin-1β, a pro-inflammatory cytokine. For the best performing Sox and interleukin, overproducing and secreting variants of these new B. subtilis toolbox fermentation strategies were developed and tested. This study demonstrates the suitability of the prokaryotic B. subtilis host-vector system for the extracellular production of two eukaryotic proteins with biotechnological relevance. KEY POINTS: • Construction of a versatile Bacillus subtilis gene expression toolbox. • Verification of the toolbox by the secretory overproduction of two difficult-to-express proteins. • Fermentation strategy for an acetoin-controlled overproduction of heterologous proteins.

Authors 13

1. Anna Krüger

   Hamburg University of Technology

   Affiliation as printed

   Institute of Technical Microbiology, Hamburg University of Technology, Kasernenstr. 12, 21073, Hamburg, Germany

2. Norma Welsch

   Universität Greifswald · Institute of Marine Biotechnology

   Affiliation as printed

   Institute of Marine Biotechnology, Walther-Rathenau-Str. 49, 17489, Greifswald, Germany

   Pharmaceutical Biotechnology, Institute of Pharmacy, University of Greifswald, Felix-Hausdorff-Str. 3, 17487, Greifswald, Germany

3. Alexandra Dürwald

   Universität Greifswald

   Affiliation as printed

   Pharmaceutical Biotechnology, Institute of Pharmacy, University of Greifswald, Felix-Hausdorff-Str. 3, 17487, Greifswald, Germany

4. Henrike B. Brundiek
   Affiliation as printed

   Enzymicals AG, Walther-Rathenau-Straße 49a, 17489, Greifswald, Germany

5. Rainer Wardenga
   Affiliation as printed

   Enzymicals AG, Walther-Rathenau-Straße 49a, 17489, Greifswald, Germany

6. Henning Piascheck

   Hamburg University of Technology

   Affiliation as printed

   Institute of Technical Microbiology, Hamburg University of Technology, Kasernenstr. 12, 21073, Hamburg, Germany

7. Hendrik G. Mengers Aachen Institute of Applied Microbiology - iAMB

   RWTH Aachen University

   Affiliation as printed

   Institute of Applied Microbiology - iAMB, Aachen Biology and Biotechnology - ABBt, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany

8. Jana Krabbe

   Universität Greifswald · Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie e. V. - Hans-Knöll-Institut (HKI)

   Affiliation as printed

   Department of Biomolecular Chemistry, Leibniz Institute for Natural Product Research and Infection Biology, HKI, Beutenbergstr. 11a, 07745, Jena, Germany

   Pharmaceutical Biotechnology, Institute of Pharmacy, University of Greifswald, Felix-Hausdorff-Str. 3, 17487, Greifswald, Germany

9. Johannes Kabisch

   Norwegian University of Science and Technology · Universität Greifswald

   Affiliation as printed

   Department of Biotechnology and Food Science, NTNU, Sem Sælands vei 6, 7034, Trondheim, Norway

   Pharmaceutical Biotechnology, Institute of Pharmacy, University of Greifswald, Felix-Hausdorff-Str. 3, 17487, Greifswald, Germany

10. Lutz Popper
    Affiliation as printed

    Stern Enzym GmbH & Co. KG, Kurt-Fischer-Str. 55, 22926, Ahrensburg, Germany

11. Tanno Hübel

    Miltenyi Biotec (Germany)

    Affiliation as printed

    Miltenyi Biotec GmbH, Robert-Koch-Str. 1, 17166, Teterow, Germany

12. Garabed Antranikian

    Hamburg University of Technology

    Affiliation as printed

    Institute of Technical Microbiology, Hamburg University of Technology, Kasernenstr. 12, 21073, Hamburg, Germany

13. Thomas Schweder corresponding

    Universität Greifswald · Institute of Marine Biotechnology

    Affiliation as printed

    Institute of Marine Biotechnology, Walther-Rathenau-Str. 49, 17489, Greifswald, Germany. schweder@uni-greifswald.de

    Pharmaceutical Biotechnology, Institute of Pharmacy, University of Greifswald, Felix-Hausdorff-Str. 3, 17487, Greifswald, Germany. schweder@uni-greifswald.de

    Institute of Marine Biotechnology, Walther-Rathenau-Str. 49, 17489, Greifswald, Germany