A programmable Aeromonas chassis, AMAX2, for advanced biomanufacturing

Synthetic biology increasingly demands engineered bacterial chassis that combine robust biosynthesis with tailored functionalities. We previously developed AMAX, an Aeromonas-derived chassis with fast growth, high GC content, and exceptional recombinant protein yield. Here, we report AMAX2, a next-generation chassis generated by deleting additional virulence-associated and nonessential genes in AMAX to enhance biosafety while preserving robust growth and protein expression. AMAX2 supports diverse inducible systems, multiple plasmid types, and tRNA supplementation to enable rare codon-enriched gene expression. Genome-wide CRISPRi-seq identified 153 core and 289 conditionally essential genes. Beyond expression, AMAX2 can be engineered for the production of DNA-free minicells, surface display for targeted protein decoration via SpyCatcher-SpyTag, and protein translocation into specific target cells via nanobody display and a plasmid-borne type VI secretion system. These features establish AMAX2 as a programmable chassis for advanced biomanufacturing. Comprehensive in vitro and in vivo assays showed that AMAX2 exhibits superior biosafety, with no detectable hemolysis, cytotoxicity, or pathogenicity in cellular and animal models. These multifunctional capabilities position AMAX2 as a versatile platform for industrial biomanufacturing and biotherapeutic development.IMPORTANCEAMAX2 is a genetically programmable chassis that bridges industrial protein production and advanced functional applications, including targeted secretion, surface display, and minicellgeneration. Its unique combination of high performance, broad genetic compatibility, and multifunctionality provides a competitive alternative to existing platforms. By integrating genome-wide essential gene mapping and customizable delivery pathways, AMAX2 holds the potential for serving as a next-generation microbial platform in synthetic biology.