Tunable Display of Proteins on Bacterial Spore Surface

Bacteria endospores are the most robust and durable type of cells found in nature, attributed to the multiple protective layers of proteins that encase the spore. Some spore coat proteins on the outmost layer have been used as anchors to display proteins for various applications ranging from vaccine delivery to enzyme display. Since spores are highly robust systems and can survive various extreme environments, such as extreme heat or cold, toxic chemicals, and radiations, these proteins displayed on the spore surface often demonstrate enhanced robustness and longer shelf life. Being part of the spore, these proteins can be easily produced through sporulation. No further purification is needed and this significantly reduces the production costs. Moreover, for enzymes used in industrial processes, spore based enzymes can be reused as they are part of the spore and easy to separate from the reaction mixture. Altogether spores are an attractive platform for protein display. However, the assembly of spores is a delicate and complex system. Currently protein display on spore surface relies on a few anchor proteins and the expressions of them are all under native regulatory elements. No expression system is available to display multiple enzymes and modulate their expression level in response to external signals. In this work, we designed tunable expression systems that integrate the natural regulatory elements for tight sporulation control and binding sites for non-sporulation transcription factors or CRISPR-dCas9. Using GFP and mCherry as reporters, it was shown that the designed systems allow for functional expression and display of heterologous proteins in the range of 0.2 to 15 times of those subject to the native regulatory control. The development of the tunable expression and display systems enable the fine tuning of the display of multiple protein complexes on the spore surface, which is critical to investigate the synergy between proteins in a pathway and optimize the overall efficiency.