A programmable Aeromonas chassis, AMAX2, for advanced biomanufacturing.

Area of Science:

• Synthetic biology
• Microbial engineering
• Biotechnology

Background:

• Engineered bacterial chassis are crucial for synthetic biology, requiring robust biosynthesis and tailored functionalities.
• The previously developed AMAX chassis offered fast growth and high protein yield.
• Enhancing biosafety while maintaining performance is essential for advanced biomanufacturing.

Purpose of the Study:

• To develop AMAX2, a next-generation bacterial chassis with improved biosafety and expanded functionalities.
• To engineer a chassis for diverse applications including protein production, minicell generation, and targeted delivery.
• To comprehensively assess the biosafety and performance of the AMAX2 chassis.

Main Methods:

• Genetic engineering of the AMAX chassis by deleting virulence and nonessential genes.
• Genome-wide CRISPRi-seq to identify essential genes.
• Engineering for DNA-free minicells, surface display (SpyCatcher-SpyTag), and targeted secretion (T6SS).
• In vitro and in vivo assays to evaluate biosafety (hemolysis, cytotoxicity, pathogenicity).

Main Results:

• AMAX2 exhibits enhanced biosafety with no detectable hemolysis, cytotoxicity, or pathogenicity.
• The chassis supports diverse inducible systems, multiple plasmid types, and rare codon expression.
• AMAX2 enables DNA-free minicell production, surface display, and targeted protein translocation.
• Genome-wide essential gene mapping identified core and conditionally essential genes.

Conclusions:

• AMAX2 is a versatile, programmable chassis for advanced biomanufacturing and biotherapeutic development.
• Its combination of high performance, biosafety, and multifunctionality makes it a competitive alternative platform.
• AMAX2 represents a next-generation microbial platform for synthetic biology applications.