Cooperative Chemical Biosynthesis of Tetrodotoxin: Evidence from Marine Microbial Contributors.

Tetrodotoxin (TTX) is a potent neurotoxin widely distributed in pufferfish and other marine organisms, yet its microbial biosynthetic basis remains unresolved because no definitive bacterial TTX gene cluster has been identified. This study develops a hypothesis-generating comparative-genomic framework to prioritize bacterial genomes for experimental investigation of TTX-related metabolism; it does not demonstrate bacterial TTX biosynthesis. Thirteen genomes representing Pseudoalteromonas, Cytobacillus, Shewanella, and Vibrio were analyzed using whole-genome comparison, average amino acid identity (AAI), and a weighted functional-prioritization score. Candidate homologous gene families were assigned to four mechanistic groups: scaffold formation and nitrogen incorporation (Group A), redox tailoring and polyoxygenation (Group B), structural tailoring and rearrangement (Group C), and transport, regulation, and ecological support (Group D). The AAI structure revealed both closely related Vibrio lineages and deeply divergent genera, enabling interpretation of functional enrichment against contrasting genomic backgrounds. Cytobacillus gottheilii 1839 and Pseudoalteromonas tetraodonis DSM 16,099 had the highest cumulative scores, whereas Vibrio representatives showed moderate or partial enrichment profiles. These rankings reflect the distribution of hypothesized functional markers, rather than validated TTX-production capacity. Groups A and B were more discriminating than the broadly distributed Groups C and D. Accordingly, C. gottheilii 1839 and P. tetraodonis DSM 16,099 are proposed as high-priority targets for integrated metabolomics, transcriptomics, targeted gene disruption, and pathway-mining studies. The results support a dispersed, multi-module working hypothesis for microbial contribution to TTX-associated ecology, while emphasizing that the pathway, its products, and the causal role of individual strains remain to be experimentally resolved.