Protein Translocation Control in via Temperature-Dependent Aggregation: Application to a Conditionally Lethal Enzyme, Levansucrase.

Precise control of protein translocation is essential for synthetic biology and protein engineering. Here, we present a temperature-responsive system using elastin-like polypeptides (ELPs) to regulate the translocation of a conditionally lethal enzyme in . The enzyme, levansucrase, whose activity becomes lethal in the presence of sucrose, was engineered with an N-terminal signal peptide and a C-terminal ELP tag. At 37 °C, the ELP tag induced intracellular aggregation of the fusion protein, preventing its secretion and allowing cell survival, as indicated by translucent colony formation. In contrast, at 16 °C, the ELP remained soluble, permitting levansucrase secretion into the medium. The resulting conversion of sucrose into levan by the secreted enzyme led to host cell death. These findings highlight ELP-mediated aggregation as a reversible and tunable strategy for regulating protein localization and secretion in , with potential applications in synthetic biology, metabolic engineering, and biocontainment systems.