Bacterial microcompartments (MCPs) are large, inducible, proteinaceous organelles that encapsulate the enzymes, substrates, and cofactors required for specific metabolic pathways in certain species of bacteria. MCPs allow for the spatial organization of pathways containing volatile or toxic intermediates that would be detrimental to the cell if produced at the same levels in the cytosol. One example of an MCP is the 1,2-propanediol utilization (Pdu) MCP found in Salmonella enterica, which sequesters the 1,2-propanediol degradation pathway, including the toxic intermediate propionaldehyde. This sequestration is desirable to repurpose for other heterologous pathways that may suffer from similar inefficiencies, but important questions about how exactly the compartments function to enhance pathway performance must be answered before compartmentalization can be widely used in metabolic engineering strategies. One function of MCPs that is hypothesized to improve pathway performance is the creation of a private encapsulated cofactor pool that is internally recycled. By purifying and then studying the MCPs in vitro using bacterial extracts, we evaluated how model pathways are impacted by controlling the compartment environment directly, including external presence or absence of required cofactors, and addition or removal of pathway and recycling enzymes internally. This method allowed us to study a suite of conditions that would normally not be accessible using in vivo methods. From these experiments we found that, surprisingly, redox recycling is not always required, and we gained unexpected insights into the permeability of the proteinaceous shell. We then used these insights to investigate the benefits of encapsulation on isoprenoid production by encapsulating three steps of the mevalonate pathway.

Investigating the impact of cofactor availability and enzyme composition on metabolic pathway performance in bacterial microcompartments

ACS Fall 2021

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