Temporal gene regulation enables controlled expression of gas vesicles and preserves bacterial viability.

Gas vesicles (GVs) are genetically encodable, air-filled protein nanostructures that have rapidly emerged as a versatile platform for biomedical imaging, cell tracking, and therapeutic delivery. However, in non-native hosts such as Escherichia coli, heterologous expression is hampered by a complex assembly involving ~10 proteins, provoking proteotoxic stress and impaired growth. Here we report a reproducible drop in cell density and viability 8-16 h after GV induction. To address these, we develop a dual-inducer transcriptional system that orthogonally controls assembly factors and the shell protein GvpA2 over a range of stoichiometries. Sequential expression by initiating assembly factors before GvpA2 restores growth without compromising GV production. We further show that the interval between inductions tunes both GV yield and cellular stress. Orthogonal decoupling plus a ~2-3 h head-start for assembly factors prevents proteotoxicity, preserves yields, and restores viability, establishing a generalizable temporal-stoichiometric design for heterologous expression of multimeric protein nanostructures.