Many bacteria use protein-based organelles known as bacterial microcompartments (BMCs) to organize and sequester sequential enzymatic reactions. Regardless of their specialized metabolic function, all BMCs are delimited by a shell made of multiple structurally redundant, yet functionally diverse, hexameric (BMC-H), pseudohexameric/trimeric (BMC-T), or pentameric (BMC-P) shell protein paralogs. When expressed without their native cargo, shell proteins have been shown to self-assemble into 2D sheets, open-ended nanotubes, and closed shells of ≈40 nm diameter that are being developed as scaffolds and nanocontainers for applications in biotechnology. Here, by leveraging a strategy for affinity-based purification, it is demonstrated that a wide range of empty synthetic shells, many differing in end-cap structures, can be derived from a glycyl radical enzyme-associated microcompartment. The range of pleomorphic shells observed, which span ≈2 orders of magnitude in size from ≈25 nm to ≈1.8 µm, reveal the remarkable plasticity of BMC-based biomaterials. In addition, new capped nanotube and nanocone morphologies are observed that are consistent with a multicomponent geometric model in which architectural principles are shared among asymmetric carbon, viral protein, and BMC-based structures.

Heme is a versatile redox cofactor that has considerable potential for synthetic biology and bioelectronic applications. The capacity to functionalize non-heme-binding proteins with covalently bound heme moieties in vivo could expand the variety of bioelectronic materials, particularly if hemes could be attached at defined locations so as to facilitate position-sensitive processes like electron transfer. In this study, we utilized the cytochrome maturation system I to develop a simple approach that enables incorporation of hemes into the backbone of target proteins in vivo. We tested our methodology by targeting the self-assembling bacterial microcompartment shell proteins, and inserting functional hemes at multiple locations in the protein backbone. We found substitution of three amino acids on the target proteins promoted heme attachment with high occupancy. Spectroscopic measurements suggested these modified proteins covalently bind low-spin hemes, with relative low redox midpoint potentials (about -210 mV vs. SHE). Heme-modified shell proteins partially retained their self-assembly properties, including the capacity to hexamerize, and form inter-hexamer attachments. Heme-bound shell proteins demonstrated the capacity to integrate into higher-order shell assemblies, however, the structural features of these macromolecular complexes was sometimes altered. Altogether, we report a versatile strategy for generating electron-conductive cytochromes from structurally-defined proteins, and provide design considerations on how heme incorporation may interface with native assembly properties in engineered proteins.

Formate has great potential to function as a feedstock for biorefineries because it can be sustainably produced by a variety of processes that don't compete with agricultural production. However, naturally formatotrophic organisms are unsuitable for large-scale cultivation, difficult to engineer, or have inefficient native formate assimilation pathways. Thus, metabolic engineering needs to be developed for model industrial organisms to enable efficient formatotrophic growth. Here, we build a prototype synthetic formate utilizing bacterial microcompartment (sFUT) encapsulating the oxygen-sensitive glycyl radical enzyme pyruvate formate lyase and a phosphate acyltransferase to convert formate and acetyl-phosphate into the central biosynthetic intermediate pyruvate. This metabolic module offers a defined environment with a private cofactor coenzyme A that can cycle efficiently between the encapsulated enzymes. To facilitate initial design-build-test-refine cycles to construct an active metabolic core, we used a "wiffleball" architecture, defined as an icosahedral bacterial microcompartment (BMC) shell with unoccupied pentameric vertices to freely permit substrate and product exchange. The resulting sFUT prototype wiffleball is an active multi enzyme synthetic BMC functioning as platform technology.