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.ResultsHere, 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.ConclusionsOur 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.
Highlights
In recent years, the production of inclusion bodies that retained substantial catalytic activity was demonstrated
The ten different Catalytically active inclusion body (CatIB) variants were produced in E. coli BL21(DE3) using M9 autoinduction medium (See Additional file 1: Table S2)
CatIBs appear as white refractive particles or granule-like structures at the cell poles (Fig. 1), which is typical for Inclusion body (IB) [38]
Summary
The production of inclusion bodies that retained substantial catalytic activity was demonstrated. Enzymes produced by microbial systems becoming increasingly important, e.g., for the sustainable production of platform chemicals and bio-based polymers [1,2,3,4] Due to their advantages, like heat resistance, tensile strength and electrical insulation, polyamides are interesting for diverse applications in the electrical, automotive and textile industry as well as for consumer articles and in the medical sector [5]. DAP can be biotechnologically produced from l-lysine by enzymatic decarboxylation through the constitutive lysine decarboxylase (LDCc) [7] or the acid-induced variant CadA [8] from Escherichia coli. Both enzymes use pyridoxal 5’-phosphate (PLP) as a cofactor. The l-lysine was enzymatically decarboxylated to yield DAP by the native EcLDCc, which was overproduced in E. coli, [9]
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