Kira Küsters1,2, Martina Pohl1, Ulrich Krauss1,3, Gizem Ölçücü1,3, Sandor Albert1,4, Karl-Erich Jaeger1,3, Wolfgang Wiechert1,5, Marco Oldiges6,7. 1. Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany. 2. Institute of Biotechnology, RWTH Aachen University, 52074, Aachen, Germany. 3. Institute of Molecular Enzyme Technology, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany. 4. Faculty of Biotechnology, University of Applied Sciences Mannheim, 68163, Mannheim, Germany. 5. Computational Systems Biotechnology (AVT.CSB), RWTH Aachen University, 52074, Aachen, Germany. 6. Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany. m.oldiges@fz-juelich.de. 7. Institute of Biotechnology, RWTH Aachen University, 52074, Aachen, Germany. m.oldiges@fz-juelich.de.
Abstract
BACKGROUND: In recent years, the production of inclusion bodies that retained substantial catalytic activity was demonstrated. These catalytically active inclusion bodies (CatIBs) were formed by genetic fusion of an aggregation inducing tag to a gene of interest via short linker polypeptides and overproduction of the resulting gene fusion in Escherichia coli. The resulting CatIBs are known for their high stability, easy and cost efficient production, and recyclability and thus provide an interesting alternative to conventionally immobilized enzymes. RESULTS: Here, we present the construction and characterization of a CatIB set of the lysine decarboxylase from Escherichia coli (EcLDCc), constructed via Golden Gate Assembly. A total of ten EcLDCc variants consisting of combinations of two linker and five aggregation inducing tag sequences were generated. A flexible Serine/Glycine (SG)- as well as a rigid Proline/Threonine (PT)-Linker were tested in combination with the artificial peptides (18AWT, L6KD and GFIL8) or the coiled-coil domains (TDoT and 3HAMP) as aggregation inducing tags. The linkers were fused to the C-terminus of the EcLDCc to form a linkage between the enzyme and the aggregation inducing tags. Comprehensive morphology and enzymatic activity analyses were performed for the ten EcLDCc-CatIB variants and a wild type EcLDCc control to identify the CatIB variant with the highest activity for the decarboxylation of L-lysine to 1,5-diaminopentane. Interestingly, all of the CatIB variants possessed at least some activity, whilst most of the combinations with the rigid PT-Linker showed the highest conversion rates. EcLDCc-PT-L6KD was identified as the best of all variants allowing a volumetric productivity of 457 g L- 1 d- 1 and a specific volumetric productivity of 256 g L- 1 d- 1 gCatIB-1. Noteworthy, wild type EcLDCc, without specific aggregation inducing tags, also partially formed CatIBs, which, however showed lower activity compared to most of the newly constructed CatIB variants (volumetric productivity: 219 g L- 1 d- 1, specific volumetric activity: 106 g L- 1 d- 1 gCatIB- 1). Furthermore, we demonstrate that microscopic analysis can serve as a tool to find CatIB producing strains and thus allow for prescreening at an early stage to save time and resources. CONCLUSIONS: Our results clearly show that the choice of linker and aggregation inducing tag has a strong influence on the morphology and the enzymatic activity of the CatIBs. Strikingly, the linker had the most pronounced influence on these characteristics.
BACKGROUND: In recent years, the production of inclusion bodies that retained substantial catalytic activity was demonstrated. These catalytically active inclusion bodies (CatIBs) were formed by genetic fusion of an aggregation inducing tag to a gene of interest via short linker polypeptides and overproduction of the resulting gene fusion in Escherichia coli. The resulting CatIBs are known for their high stability, easy and cost efficient production, and recyclability and thus provide an interesting alternative to conventionally immobilized enzymes. RESULTS: Here, we present the construction and characterization of a CatIB set of the lysine decarboxylase from Escherichia coli (EcLDCc), constructed via Golden Gate Assembly. A total of ten EcLDCc variants consisting of combinations of two linker and five aggregation inducing tag sequences were generated. A flexible Serine/Glycine (SG)- as well as a rigid Proline/Threonine (PT)-Linker were tested in combination with the artificial peptides (18AWT, L6KD and GFIL8) or the coiled-coil domains (TDoT and 3HAMP) as aggregation inducing tags. The linkers were fused to the C-terminus of the EcLDCc to form a linkage between the enzyme and the aggregation inducing tags. Comprehensive morphology and enzymatic activity analyses were performed for the ten EcLDCc-CatIB variants and a wild type EcLDCc control to identify the CatIB variant with the highest activity for the decarboxylation of L-lysine to 1,5-diaminopentane. Interestingly, all of the CatIB variants possessed at least some activity, whilst most of the combinations with the rigid PT-Linker showed the highest conversion rates. EcLDCc-PT-L6KD was identified as the best of all variants allowing a volumetric productivity of 457 g L- 1 d- 1 and a specific volumetric productivity of 256 g L- 1 d- 1 gCatIB-1. Noteworthy, wild type EcLDCc, without specific aggregation inducing tags, also partially formed CatIBs, which, however showed lower activity compared to most of the newly constructed CatIB variants (volumetric productivity: 219 g L- 1 d- 1, specific volumetric activity: 106 g L- 1 d- 1 gCatIB- 1). Furthermore, we demonstrate that microscopic analysis can serve as a tool to find CatIB producing strains and thus allow for prescreening at an early stage to save time and resources. CONCLUSIONS: Our results clearly show that the choice of linker and aggregation inducing tag has a strong influence on the morphology and the enzymatic activity of the CatIBs. Strikingly, the linker had the most pronounced influence on these characteristics.
Entities:
Keywords:
Catalytically active inclusion bodies; Downstream processing; Enzymes; Immobilization; Microscopic analysis; Protein aggregates; Protein engineering
Authors: Hannah Chung; Jung Eun Yang; Ji Yeon Ha; Tong Un Chae; Jae Ho Shin; Martin Gustavsson; Sang Yup Lee Journal: Curr Opin Biotechnol Date: 2015-08-28 Impact factor: 9.740
Authors: Ramona Kloss; Michael H Limberg; Ursula Mackfeld; Doris Hahn; Alexander Grünberger; Vera D Jäger; Ulrich Krauss; Marco Oldiges; Martina Pohl Journal: Sci Rep Date: 2018-04-11 Impact factor: 4.379