Encapsulation of select violacein pathway enzymes in the 1,2-propanediol utilization bacterial microcompartment to divert pathway flux

A continual goal in metabolic engineering is directing pathway flux to desired products and avoiding loss of pathway intermediates to competing pathways. Encapsulation of the pathway is a possible solution, as it creates a diffusion barrier between pathway intermediates and competing enzymes. It is hypothesized that bacteria use organelles known as bacterial microcompartments - proteinaceous shells encapsulating a metabolic pathway - for this purpose. We aim to determine to what degree this hypothesized benefit is conferred to encapsulated pathways. To this end, we used bacterial microcompartments to encapsulate select enzymes from the violacein pathway, which is composed of five enzymes that produce violacein as the main product and deoxyviolacein as a side product. Importantly, we studied the pathway in a cell-free context, allowing us to hold constant the concentration of unencapsulated and encapsulated enzymes and increase our control over reaction conditions. The VioE enzyme is a branch point in that it makes the precursor for both violacein and deoxyviolacein, the VioC enzyme is required for production of deoxyviolacein, and the VioD enzyme is required for violacein production. When we encapsulated VioE and VioC and left VioD unencapsulated, the product profile shifted toward deoxyviolacein and away from violacein compared to when VioC and VioD were both unencapsulated. This work provides the first fully quantitative evidence that microcompartment-based encapsulation can be used to divert pathway flux to the encapsulated pathway. It provides insight into why certain pathways are encapsulated natively and could be leveraged for metabolic engineering applications.