Construction and comprehensive characterization of an EcLDCc-CatIB set—varying linkers and aggregation inducing tags

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.

Construction and characterization of BsGDH-CatIB variants and application as robust and highly active redox cofactor regeneration module for biocatalysis

“…Whilst PT-18AWT did not form any IBs, the corresponding SG-18AWT variant displayed only 1% IB carrying cells. Interestingly, the same phenomenon was shown for the 18AWT-Tag with Ec LDCc and strengthened the hypothesis that this tag has a higher tendency to bind to cell membrane components rather than forming isolated IBs [ 4 , 21 ]. Moreover, comparing microscopic analysis data of Bs GDH with previous results from Ec LDCc revealed similar CatIB and cell morphology effects, such as abnormal cell structures in combination with L6KD-Tag or a looser CatIB structure with 3HAMP-Tag, but it is less dominant with Bs GDH than with Ec LDCc.…”

“…The microscopic analysis of the Bs GDH-CatIB variants confirmed the hypothesis that combinations of different linkers and aggregation inducing tags lead to different cell and IB morphologies [ 4 ]. The variants with PT-L6KD and SG-ELK16 formed dense inclusion bodies with 63% and 75% of the cells carrying CatIBs, respectively (Fig.…”

“…Strikingly, the influence of the linker/aggregation inducing tag combination on CatIB formation was different between the two enzymes. With Ec LDCc-SG-TDoT 78% of the cells carried CatIBs, whereas with Bs GDH-SG-TDoT it was only 17% [ 4 ]. This strongly supports the hypothesis that there is no optimal linker/aggregation inducing tag combination, but rather a more complex interaction between the three parts, i.e., including the nature of the target protein in focus.…”

“…However, for some enzymes the formation of natural CatIBs, like lysine decarboxylase of Escherichia coli ( Ec LDC c ), adenosine 5′-monophosphate phosphorylase of Thermococcus kodakarensis and lipase A of Bacillus subtilis were reported. Instead of inactive inclusion bodies, the protein aggregates retained some catalytical activity [ 4 – 6 ]. Enzymatic activity of such natural CatIBs or of enzymes which do not form any IBs can be enhanced or enforced by generating synthetic tailor-made CatIBs.…”

“…Recent studies revealed that different combinations of linkers and aggregation inducing tags lead to cooperative effects and activity level variations for each specific enzyme. For example, Ec LDCc showed higher activity levels in combination with the rigid PT-Linker instead of the flexible SG-Linker [ 4 ]. For alcohol dehydrogenase of Ralstonia sp .…”