A Programmable Finite-Replicated Organism Framework for Balanced Safety and Functionality

Live-attenuated vaccines face a critical challenge in balancing immunogenicity with safety. To address this, we engineered programmable finite-replicated organisms (FROs) by depositing a limited number of indispensable components (such as noncanonical amino acids, ncAAs) within the cell, consuming the coenabling precise control of bacterial replication capability while preserving antigenic breadth. Two strategies were adopted to achieve the following purposes: (1) encoding ncAA in essential genes; (2) encoding ncAA in antitoxin of toxin-antitoxin (TA) systems. As noncanonical amino acids, 3,5-dichlorotyrosine (Cl2Y) was encoded by the amber codon (TAG) and inserted into the essential genes (e.g., <i>serS</i>, <i>murG</i>, and <i>dnaA</i>) or antitoxin genes. After optimizing expression and the number of amber codons in the storage genes, the FRO cells can grow up to six generations, achieving amplification approaching 100 times after depletion of the ncAA in the growth medium. The escape frequencies are 10^-5 to 10^-7, which need to be optimized by combining multiple storage genes in the same genome in the future. This work holds the potential to amplify the amounts of antigens for vaccines, potentially accelerating the development of next-generation vaccines against antibiotic-resistant threats.