Evaluation of Bacterial Microcompartment Cofactor Recycling and Permeability with a Model Guided In Vitro Assay

Biomanufacturing is a promising strategy for sustainable chemical production. However, challenges such as cofactor competition and low pathway flux prevent competitive titers. Some bacteria address these challenges by encapsulating metabolic pathways in bacterial microcompartments (MCPs), many of which contain dedicated cofactor recycling enzymes. We sought to determine how pathway cofactor recycling and intermediate sequestration in MCPs benefit pathway performance using an in vitro assay and kinetic model of the 1,2-propanediol utilization (Pdu) system. Guided by model simulations, we performed experimental design to characterize permeability, a key and difficult-to-measure property of MCPs. Using our model and measurements of metabolite concentrations over time, we estimate MCP permeability values in the range of 10 ⁻⁵ cm/s. We also demonstrated that NAD+/NADH recycling in the Pdu MCP benefits increased pathway flux. This study integrates experiments and systems modeling to advance our understanding of why pathways are encapsulated and to inform bioengineering applications.