ゲノムワイド超変異原性工学的手法によるシアネコシスティス属PCC 6803の膜媒介型トリクロサン耐性の解明

Cyanobacteria represent an ancient group of photosynthetic microorganisms that offer unparalleled insights into evolutionarily conserved stress adaptation mechanisms essential for plant resilience. To investigate how photosynthetic organisms mitigate chemical stressors, we employed Synechocystis sp. PCC 6803-a keystone model for photosynthetic research due to its plant-like electron transport chain and stress-responsive plasticity. By implementing a genomic hypermutation strategy, we synergistically knocked out DNA replication fidelity genes and overexpressed error-prone replication elements, generating hypermutable strains HM24 and HM33 with relative mutation rates of 97 and 116-fold, respectively. Following triclosan (TCS) stress screening, the CRISPR-Cpf1 strategy was used to complement mutations and yielded transformants R-HM24 and R-HM33 that exhibited 96h EC50 values of 4.963 mg/L and 5.238 mg/L-322- and 340-fold increases over wild-type levels. The strains demonstrated enhanced TCS and multidrug antibiotic tolerance. Whole-genome resequencing identified consistent missense mutation in fabI across resistant strains. Mechanistic analyses revealed that the hypermutated Synechocystis strains acquired resistance primarily by mutating the essential fabI protein to decrease its affinity for TCS. This study establishes the application of hypermutation-driven evolution for rapid dissection of pollutant resistance in photosynthetic microbes, thereby advocating for stricter regulation of antimicrobial pollutants in aquatic environments.