Noncanonical Amino Acid Incorporation Modulates Condensate States of Intrinsically Disordered Proteins in <i>Escherichia coli</i> Cells

Biomolecular condensates are distinct subcellular structures with on-demand material states and dynamics in living cells. However, strategies for modulating their material states and physicochemical properties are lacking. Here, we report a chemical strategy for modulating the condensate states of intrinsically disordered proteins in bacterial <i>Escherichia coli</i> cells. This is achieved by noncanonical amino acid (DOPA) incorporation into model resilin-like proteins (RLPs) to endow autonomous oxidative and coordinative cross-linking mechanisms. Biogenesis of spherical gel-like condensates is achieved upon heterologous expression of the DOPA-incorporated RLP in the cells at 30 °C. We reveal that liquid-liquid phase separation underlies the formation of liquid condensates, and this liquid-like state is metastable and its dynamics is compromised by the oxidative and coordinative cross-linkings that ultimately drive the liquid-to-gel transition. Therefore, this study has deepened our understanding of biomolecular condensation and offers a new chemical strategy to expand the landscape of condensation phenotypes of living cells.