Excavation of acoustic nanostructures biosynthesis gene clusters by combinatorial strategy

Abstract

Gas vesicles (GVs) produced by microorganisms are genetically engineered, air-filled protein nanostructures that have widespread applications in ultrasound imaging and ultrasound-mediated drug delivery. However, constrained by the shape and size, most of them are difficult to be imaged by clinical ultrasound machines, which limits their biomedical applications. Here, we constructed a hybrid gene cluster of the structural gene cluster from Serratia sp. ATCC 39006 and the accessory gene cluster from Bacillus megaterium in Escherichia coli to synthesize a novel gene-encoded gas vesicle with a width of approximately 70 nm and a length of about 100 nm, using a synthetic biology strategy, termed as ARG_S1B. This new type of GVs can be stably produced in bacteria and is able to be imaged by clinical ultrasound machines in vivo and in vitro. Furthermore, the novel nanostructure can be easily engineered for different particle sizes through point saturation mutation, expanding the sources of GVs and providing new insights into the biosynthesis mechanism of GVs.

Keywords: Escherichia coli; Acoustic nanostructures; Combinatorial biosynthesis strategy; Gas vesicles; Site-saturation mutagenesis; Ultrasound imaging.

Fig. 1

Workflow for mining GVs biosynthetic gene clusters (BGCs) by combinatorial biosynthesis technique

Fig. 2

a The final BGCs after modular analysis in this study. b The combination of different gene clusters. (St, structural gene; Ac, accessory genes; Af, Anabaena flos-aquae, Bm, Bacillus megaterium, Sc, Streptomyces coelicolor A3(2), Hs, Halobacterium sp. NRC-1 pNRC100, HC, Halobacterium salinarium C-GVP, Se, Serratia sp. ATCC 39006). c Flotation assay results after stewing 48 h. d Average OD₆₀₀ in Flotation Assay, three biological replicates

Fig. 3

Characterization results of ARG_S1B in vitro. a TEM images of E. coli cells expressing different gene combinations (Scale bars, 500 nm), see also Figure S1. b Organization of GVs biosynthetic gene clusters; the region highlighted in grey was varied. c TEM images of ARG_S1B and ARG1 (Scale bars, 200 nm). d Sequence alignment of GvpA from A. flos-aquae and Serratia sp. ATCC 39006. e, f Violin plots showing the particle width and length. Red dashed horizontal lines indicate the interquartile range and a red solid horizontal line indicates medians; significance levels, ****p < 0.0001. g Hydrodynamic diameters of ARG_S1B and ARG1. h Zeta potential analysis was conducted with N = 3 biological replicates

Fig. 4

Characterization of ARG_S1B in vitro. a The ultrasound image of a phantom containing serial dilutions of GVs with different OD₅₀₀. b The ‘BURST’ images of a phantom containing GVs of ARG_S1B (OD₅₀₀ = 3.2) and ARG1 (OD₅₀₀ = 2.7), before and after acoustic collapse. c, d Quantification of the signal intensity target ROIs from images in (a) and other replicates. e Mean ultrasound contrast from ARG1 and ARG_S1B at various GV densities. Error bars representing mean ± SD for N = 3 replicates

Fig. 5

Characterization of ARG_S1B in vivo. a The ultrasound contrast images of the liver received with ARG_S1B or ARG1 at different time points, OD₅₀₀ = 3.0. b, c Contrast signal intensity in the liver (white-dotted rectangular regions). d Mean ultrasound contrast from ARG1 and ARG_S1B at different injection time. Error bars representing mean ± SD for N = 3 replicates

Fig. 6

Controllable modification of GVs particle size. a Sequence alignment of GvpA derived from different species using ESPript 3.0. b Engineered GV mutants. Subscripted names stand for mutations, for example, T8 A. c Protein structure prediction of GvpA derived from Serratia sp. ATCC 39006 and A. flos-aquae using Alpha Fold. d TEM images of ARG_S1B and GvpA mutations (Scale bars, bacteria, 500 nm; GVs, 200 nm). e, f Violin plots showing the width and length of particle respectively. Black dashed horizontal lines indicate the interquartile range and a red solid horizontal line indicates medians (significance levels, ****p < 0.0001; ns, not significant)