(67b) Synthetic Regulation of Sporulation Genes for Tunable Display of Proteins on Bacterial Spore Surface

Bacteria endospores, with multiple protein layers encasing and protecting the genetic material in the core, are the most robust type of cells found in nature. A few proteins on the outmost layer of the spore have been used as anchors to display proteins such as enzymes and vaccines. These displayed proteins benefit from the protective protein layer and gain enhanced robustness. In addition, they are assembled to the spore surface during sporulation and no further purification or immobilization steps are needed, which significantly reduces the production costs. In all, bacterial spores are an attractive platform for protein display. However, the assembly of spores is delicately regulated and even the outermost layer consists of over 70 different proteins. How these proteins are interacted and organized is elusive. Currently display of a protein on the spore surface relied on a few abundant surface proteins, the expression level of which is completely regulated by native sporulation regulators. In order to display multiple enzymes on the same spore surface and regulate their relative abundance, a tunable expression system in response to non-sporulation signals are needed. In this work, we designed tunable expression systems that integrate the sporulation regulatory elements and binding sites for non-sporulation regulators, including transcription factors LacI and TetR, as well as a series of dCas9:sgRNA complexes targeting at various sites of the spore surface gene. Using GFP and mCherry as reporters, the expression level of the spore surface anchor was evaluated under various induction conditions using the wildtype as a control. Most of the designed synthetic systems show a wide range of expression levels without impeding the integrity of the spores. These synthetic regulation systems allow the fine tuning of displayed proteins on the spore surface, which is critical to optimize a multi-protein pathway.