A Minispidroin Guides the Molecular Design for Cellular Condensation Mechanisms in S. cerevisiae

[Image: see text] Structural engineering of molecules for condensation is an emerging technique within synthetic biology. Liquid–liquid phase separation of biomolecules leading to condensation is a central step in the assembly of biological materials into their functional forms. Intracellular condensates can also function within cells in a regulatory manner to facilitate reaction pathways and to compartmentalize interactions. We need to develop a strong understanding of how to design molecules for condensates and how their in vivo–in vitro properties are related. The spider silk protein NT2RepCT undergoes condensation during its fiber-forming process. Using parallel in vivo and in vitro characterization, in this study, we mapped the effects of intracellular conditions for NT2RepCT and its several structural variants. We found that intracellular conditions may suppress to some extent condensation whereas molecular crowding affects both condensate properties and their formation. Intracellular characterization of protein condensation allowed experiments on pH effects and solubilization to be performed within yeast cells. The growth of intracellular NT2RepCT condensates was restricted, and Ostwald ripening was not observed in yeast cells, in contrast to earlier observations in E. coli. Our results lead the way to using intracellular condensation to screen for properties of molecular assembly. For characterizing different structural variants, intracellular functional characterization can eliminate the need for time-consuming batch purification and in vitro condensation. Therefore, we suggest that the in vivo–in vitro understanding will become useful in, e.g., high-throughput screening for molecular functions and in strategies for designing tunable intracellular condensates.