Building a toolbox of protein scaffolds for future immobilization of biocatalysts.

Biological materials that are genetically encoded and can self-assemble offer great potential as immobilization platforms in industrial biocatalysis. Protein-based scaffolds can be used for the spatial organization of enzymes, to stabilize the catalysts and provide optimal microenvironments for reaction sequences. In our previous work, we created a protein scaffold for enzyme localization by engineering the bacterial microcompartment shell protein EutM from Salmonella enterica. Here, we sought to expand this work by developing a toolbox of EutM proteins with different properties, with the potential to be used for future immobilization of enzymes. We describe the bioinformatic identification of hundreds of homologs of EutM from diverse microorganisms. We specifically select 13 EutM homologs from extremophiles for characterization, based on phylogenetic analyses. We synthesize genes encoding the novel proteins, clone and express them in E. coli, and purify the proteins. In vitro characterization shows that the proteins self-assemble into robust nano- and micron-scale architectures including protein nanotubes, filaments, and scaffolds. We explore the self-assembly characteristics from a sequence-based approach and create a synthetic biology platform for the coexpression of different EutM homologs as hybrid scaffolds with integrated enzyme attachment points. This work represents a step towards our goal of generating a modular toolbox for the rapid production of self-assembling protein-based materials for enzyme immobilization.