Biosynthetic gas vesicles (GVs), as novel nanoscale ultrasound contrast agents, exhibit unique potential in biomedical ultrasound imaging. For example, they are expected to have better tissue penetration through the tumor vasculature for detecting tumor cells by the design of GV-based acoustic probes. Of all these GVs, GVs from Halobacterium sp. NRC-1 possess the largest size (over 200 nm) and are nearly spherical in shape, endowing them with stronger acoustic signals and better tumor penetration. However, their genetic manipulation is relatively difficult due to the requirement of a high-salt cytoplasmic environment for their expression and assembly, limiting the application of biosynthetic technology for modulating their structural features in heterologous host cells. In this study, we cloned the gene cluster encoding GVs from Halobacterium sp. NRC-1 and transformed it into Haloferax volcanii, an archaeal species naturally incapable of producing GVs. The genetically engineered Haloferax volcanii successfully synthesized functional GVs (GV_vol) with a similar size and shape to naturally synthesized GVs from Halobacterium sp. NRC-1 (GV_halo). The ultrasound imaging properties of GV_vol heterologously synthesized in Haloferax volcanii were compared with naturally synthesized GV_halo in vitro and in vivo, showing that GV_vol could achieve a mean signal intensity of 113.6 ± 0.9 a.u. in vitro and a peak intensity of 121.5 ± 0.8 a.u. in vivo in the kidney, compared with 115.7 ± 0.5 a.u. and 119.0 ± 0.5 a.u. for GV_halo, respectively. These findings confirm the functional integrity of heterologously synthesized GV_vol and its potential for biomedical applications. Our study provides a solid experimental foundation for genetically tailoring Halobacterium GV properties to optimize biomedical imaging performance.

Gas vesicles (GVs) are hollow protein nanostructures derived from microorganisms and show significant potential for ultrasound imaging applications. However, the direct production of gas vesicles (GVs) from their native hosts faces several limitations: poor contrast imaging performance, insufficient yield, and high costs associated with extraction and purification. These challenges heavily hinder their clinical translation and application. The heterologous expression of GV genes varies significantly among different chassis strains due to their distinct intracellular environments, which ultimately affects GV performance and yield. Therefore, it is crucial to select an appropriate chassis cell that can produce GVs with excellent imaging performance. In this study, the GV gene cluster from Serratia sp. ATCC 39006 was heterologously expressed in five different bacterial chassis strains: Escherichia coli BL21 (AI), Escherichia coli K-12 MG1655, Escherichia coli Nissle 1917, Salmonella YB1, and Vibrio natriegens. By systematically comparing the yield, particle morphology, and ultrasound imaging performance of GVs produced by these strains, we elucidated the impact of chassis cells on GV synthesis and function. This work provides experimental evidence and theoretical support for screening robust GV-producing strains and facilitates future biomedical applications of GVs.