Bacterial inclusion bodies (IBs) have long been considered as inactive, unfolded waste material produced by heterologous overexpression of recombinant genes. In industrial applications, they are occasionally used as an alternative in cases where a protein cannot be expressed in soluble form and in high enough amounts. Then, however, refolding approaches are needed to transform inactive IBs into active soluble protein. While anecdotal reports about IBs themselves showing catalytic functionality/ activity (CatIB) are found throughout literature, only recently, the use of protein engineering methods has facilitated the ondemand production of CatIBs. CatIB formation is induced usually by fusing short peptide tags or aggregation-inducing protein domains to a target protein. The resulting proteinaceous particles formed by heterologous expression of the respective genes can be regarded as a biologically produced bionanomaterial or, if enzymes are used as target protein, carrier-free enzyme immobilizates. In the present contribution, we review general concepts important for CatIB production, processing, and application. Key points • Catalytically active inclusion bodies (CatIBs) are promising bionanomaterials. • Potential applications in biocatalysis, synthetic chemistry, and biotechnology. • CatIB formation represents a generic approach for enzyme immobilization. • CatIB formation efficiency depends on construct design and expression conditions.

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.

Summary The formation of new replication origins (cSDR) and repair of DNA double‐strand breaks (DSBs) in E. coli share a commonality. We find that the two processes require the RNAP‐associated factor, DksA. However, whereas cSDR also relies on (p)ppGpp, the alarmone molecule is dispensable for the repair of topoisomerase type II (Top II) DNA adducts and associated DSBs. The requirement for DksA in repair of nalidixic acid (Nal)‐induced DSBs or for the formation of new origins is not suppressed by a greA deletion mutation, indicating an active role of DksA rather than competition with GreA for insertion into the RNAP secondary channel. Like dksA mutations, transcription termination factor Rho mutations also confer sensitivity to Nal. The rho and dksA mutations are not epistatic, suggesting they involve different repair pathways. The roles of DksA in DSB repair and cSDR differ; certain DksA and RNAP mutants are able to support the first process, but not the latter. We suggest that new origin formation and DNA repair of protein adducts with DSBs may both involve the removal of RNAP without destruction of the RNA:DNA hybrid.