Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region

ChemBioChem, vol. 23, pp. e202100702

Abstract

Type 1 secretion systems (T1SS) have a relatively simple architecture compared to other classes of secretion systems and therefore, are attractive to be optimized by protein engineering. Here, we report a KnowVolution campaign for the hemolysin (Hly) enhancer fragment, an untranslated region upstream of the hlyA gene, of the hemolysin T1SS of Escherichia coli to enhance its secretion efficiency. The best performing variant of the Hly enhancer fragment contained five nucleotide mutations at five positions (A30U, A36U, A54G, A81U, and A116U) resulted in a 2-fold increase in the secretion level of a model lipase fused to the secretion carrier HlyA1. Computational analysis suggested that altered affinity to the generated enhancer fragment towards the S1 ribosomal protein contributes to the enhanced secretion levels. Furthermore, we demonstrate that involving a native terminator region along with the generated Hly enhancer fragment increased the secretion levels of the Hly system up to 5-fold.

Authors 8

1. Zohreh Pourhassan N.

   Heinrich Heine University Düsseldorf

   Affiliation as printed

   Institute of Biochemistry Heinrich Heine University Universitätsstrasse 1 40225 Düsseldorf Germany

2. Haiyang Cui Aachen Institute of Biotechnology

   University of Illinois Urbana-Champaign · DWI – Leibniz Institute for Interactive Materials · Carl R. Woese Institute for Genomic Biology · RWTH Aachen University

   Affiliation as printed

   DWI-Leibniz Institute for Interactive Materials Forckenbeckstraße 50 52056 Aachen Germany

   Institute of Biotechnology RWTH Aachen University Worringerweg 3 52074 Aachen Germany

   Present address: Carl R. Woese Institute for Genomic Biology University of Illinois at Urbana-Champaign 1206 West Gregory Drive Urbana Il 61801 USA

   Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany

   Present address: Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 West Gregory Drive, Urbana, Il 61801 USA

3. Sakshi Khosa

   Heinrich Heine University Düsseldorf

   Affiliation as printed

   Institute of Biochemistry Heinrich Heine University Universitätsstrasse 1 40225 Düsseldorf Germany

4. Mehdi D. Davari

   Leibniz Institute of Plant Biochemistry

   Affiliation as printed

   Department of Bioorganic Chemistry Leibniz Institute of Plant Biochemistry Weinberg 3 06120 Halle Germany

   Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle, Germany

5. Karl‐Erich Jaeger

   Forschungszentrum Jülich

   Affiliation as printed

   Institute of Molecular Enzyme Technology Heinrich Heine University and Institute of Bio- and Geosciences IBG-1 Biotechnology, Forschungszentrum Jülich GmbH 52426 Jülich Germany

6. Sander H. J. Smits

   Heinrich Heine University Düsseldorf

   Affiliation as printed

   Institute of Biochemistry Heinrich Heine University Universitätsstrasse 1 40225 Düsseldorf Germany

7. Ulrich Schwaneberg Aachen Institute of Biotechnology

   DWI – Leibniz Institute for Interactive Materials · RWTH Aachen University

   Affiliation as printed

   DWI-Leibniz Institute for Interactive Materials Forckenbeckstraße 50 52056 Aachen Germany

   Institute of Biotechnology RWTH Aachen University Worringerweg 3 52074 Aachen Germany

   Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany

8. Lutz Schmitt corresponding

   Heinrich Heine University Düsseldorf

   Affiliation as printed

   Institute of Biochemistry Heinrich Heine University Universitätsstrasse 1 40225 Düsseldorf Germany