Anchoring Proteins for Spore-Display of Enzymes Are Best Selected Using Surface Availability of the Anchoring Terminus As Opposed to Loading Density of the Anchor Protein

Cell-free metabolic engineering has many advantages over traditional cellular approaches: cell-free systems suffer less from toxicity constraints and competing enzymatic reactions, can achieve near theoretical yields, and can be easily modeled due to their simplicity. However, the free enzymes of cell-free systems are unstable and vulnerable to inactivation, require arduous purification steps, and have relatively unproven scalability. Bacillus subtilis spore-display has been used to circumvent some of these issues. Enzymes fused to the spore coat often have increased stability due to their immobilization. Moreover, the self-assembling functionalized spores are easy to purify using centrifugation. Because of this, we have been working towards using spore-display as a platform for the rapid prototyping of in vitro enzymatic systems. A key part of this investigation is selecting the correct spore coat protein to act as a fusion anchor for the displayed enzymes. Many groups have selected anchor proteins based on loading density, evaluated by measuring the fluorescence of spores with a GFP-anchor fusion. However, many spore coat proteins have high loading densities yet make poor enzyme anchors. We posit this is because of the layered nature of the spore coat. Many copies of an enzyme may exist in the spore, but their active sites may be inaccessible to their substrate. For this reason, we have begun evaluating spore-display anchor proteins by creating N and C terminal his-tag fusions of each spore coat protein and evaluating the availability of those his-tags on the spore surface. We then compare the surface availability of each protein’s terminus to the measured activity of enzymes fused to the same terminus. Using this method, we have demonstrated that terminus surface-availability is a better indicator of a spore coat protein’s ability to act as a fusion anchor than loading density and have identified optimal fusion partners for enzyme spore-display.