(523d) Multi-Enzyme Display on Bacterial Spore Surface for Plastic Degradation

Bacterial endospores released from cells as a survival mechanism are resistant to various harsh environments. Spores from Bacillus subtilis have been used to display diverse proteins including enzymes and vaccines. Typically the displayed protein is translationally fused to a surface anchor protein and the fusion protein is assembled onto the spore surface during the sporulation process. Thus, no further purification is needed and this significantly reduces the production costs. In addition, due to the robustness of spores, the displayed proteins often gain resistance to a wider range of temperatures and pHs as well as other unfavorable conditions. The spore surface display platform proves to be an ideal protein expression and display system either for easier long term storage or for enhanced robustness. However, so far only a single protein has been displayed on one spore surface. For multiple proteins involved in the same pathway, localizing them on the same spore surface with close proximity has obvious advantages to promote the overall efficiency. In this project, we take a two-enzyme pathway to break down plastic PET to its monomers as an example to show how to modulate their expression level on the spore surface. Regulation using synthetic inducible promoters at the transcription level and designer scaffold based regulation at the post-translational level are both used to vary the ratio of the two enzymes: PETase and MHETase. These are further compared with a mixture of spores carrying the two enzymes separately. Depending on the choice of anchor proteins, certain pairs do offer spatial advantages to improve the overall efficiency. This platform for display multiple proteins can be adapted to more complex pathways so that bacterial spores can be used as robust biocatalysts for in vitro biosynthesis or biodegradation.