Towards using bacterial microcompartments as a platform for spatial metabolic engineering in the industrially important and metabolically versatile Zymomonas mobilis

Abstract

Advances in synthetic biology have enabled the incorporation of novel biochemical pathways for the production of high-value products into industrially important bacterial hosts. However, attempts to redirect metabolic fluxes towards desired products often lead to the buildup of toxic or undesirable intermediates or, more generally, unwanted metabolic cross-talk. The use of shells derived from self-assembling protein-based prokaryotic organelles, referred to as bacterial microcompartments (BMCs), as a scaffold for metabolic enzymes represents a sophisticated approach that can both insulate and integrate the incorporation of challenging metabolic pathways into industrially important bacterial hosts. Here we took a synthetic biology approach and introduced the model shell system derived from the myxobacterium Haliangium ochraceum (HO shell) into the industrially relevant organism Zymomonas mobilis with the aim of constructing a BMC-based spatial scaffolding platform. SDS-PAGE, transmission electron microscopy, and dynamic light scattering analyses collectively demonstrated the ability to express and purify empty capped and uncapped HO shells from Z. mobilis. As a proof of concept to internally load or externally decorate the shell surface with enzyme cargo, we have successfully targeted fluorophores to the surfaces of the BMC shells. Overall, our results provide the foundation for incorporating enzymes and constructing BMCs with synthetic biochemical pathways for the future production of high-value products in Z. mobilis.

In Vitro Encapsulation of Functionally Active Abiotic Photosensitizers Inside a Bacterial Microcompartment Shell

“…Of recent interest are photosynthetic biohybrid structures that use solar energy to transform abundant chemicals into fuels and other useful compounds. − BMC shells are opportune systems for biohybrid development due to their modular protein architecture and self-assembling control of interior microenvironments. However, there are no reported examples of synthetic molecular complexes incorporated within BMC shells to date, as existing cargo loading methods have largely focused on in vivo encapsulation of protein cargo, often through fusion of cargo enzymes to shell proteins by using encapsulation peptides or orthogonal adaptor domains like the SpyTag/SpyCatcher system. ,,− As a first step toward employing BMC shell architectures for photons-to-fuels catalysis, we investigate adding light-driven electron transfer capabilities by encapsulation of ruthenium photosensitizers (RuPS). BMCs provide a unique microenvironment to investigate the molecular photochemistry of RuPS, namely the excited state dynamics and photoinduced electron transfer properties upon encapsulation.…”