A step forward to the optimized HlyA type 1 secretion system through directed evolution

Applied Microbiology and Biotechnology, vol. 107, pp. 5131–5143

Abstract

Secretion of proteins into the extracellular space has great advantages for the production of recombinant proteins. Type 1 secretion systems (T1SS) are attractive candidates to be optimized for biotechnological applications, as they have a relatively simple architecture compared to other classes of secretion systems. A paradigm of T1SS is the hemolysin A type 1 secretion system (HlyA T1SS) from Escherichia coli harboring only three membrane proteins, which makes the plasmid-based expression of the system easy. Although for decades the HlyA T1SS has been successfully applied for secretion of a long list of heterologous proteins from different origins as well as peptides, but its utility at commercial scales is still limited mainly due to low secretion titers of the system. To address this drawback, we engineered the inner membrane complex of the system, consisting of HlyB and HlyD proteins, following KnowVolution strategy. The applied KnowVolution campaign in this study provided a novel HlyB variant containing four substitutions (T36L/F216W/S290C/V421I) with up to 2.5-fold improved secretion for two hydrolases, a lipase and a cutinase. KEY POINTS: • An improvement in protein secretion via the use of T1SS • Reaching almost 400 mg/L of soluble lipase into the supernatant • A step forward to making E. coli cells more competitive for applying as a secretion host.

Authors 7

1. Zohreh Pourhassan N.

   Heinrich Heine University Düsseldorf

   Affiliation as printed

   Institute of Biochemistry, Heinrich Heine University, Universitätsstr. 1, 40225, Düsseldorf, Germany

   Institute of Biochemistry, Heinrich Heine University, Düsseldorf, Germany

2. Haiyang Cui Aachen DWI - Leibniz Institute for Interactive Materials 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

   DWI-Leibniz Institute for Interactive Materials, Aachen, Germany

   Institute of Biotechnology, RWTH Aachen University, Aachen, Germany

   Present Address: Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, USA

3. Neele Muckhoff

   Heinrich Heine University Düsseldorf

   Affiliation as printed

   Institute of Biochemistry, Heinrich Heine University, Universitätsstr. 1, 40225, Düsseldorf, Germany

   Institute of Biochemistry, Heinrich Heine University, 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, Halle, Germany

5. Sander H. J. Smits

   Heinrich Heine University Düsseldorf

   Affiliation as printed

   Institute of Biochemistry, Heinrich Heine University, Universitätsstr. 1, 40225, Düsseldorf, Germany

   Institute of Biochemistry, Heinrich Heine University, Düsseldorf, Germany

6. Ulrich Schwaneberg Aachen DWI - Leibniz Institute for Interactive Materials 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

   DWI-Leibniz Institute for Interactive Materials, Aachen, Germany

   Institute of Biotechnology, RWTH Aachen University, Aachen, Germany

7. Lutz Schmitt corresponding

   Heinrich Heine University Düsseldorf

   Affiliation as printed

   Institute of Biochemistry, Heinrich Heine University, Universitätsstr. 1, 40225, Düsseldorf, Germany. lutz.schmitt@hhu.de

   Institute of Biochemistry, Heinrich Heine University, Düsseldorf, Germany