Genetic modification strategies for electroporation and CRISPR-Cas-based technologies in the non-competent Gram-negative bacteriumAcinetobactersp. Tol 5

Environmental isolates are promising candidates for new chassis of synthetic biology because of their inherent conversion capabilities and resilience to environmental stresses; however, many remain genetically intractable and unamenable to established genetic tools tailored for model bacteria. Acinetobacter sp. Tol 5 possesses intriguing properties for use in synthetic biology applications. However, genetic manipulation via electroporation is hindered by its low transformation efficiency. This study demonstrated the genetic refinement of the Tol 5 strain, achieving efficient transformation via electroporation. We deleted two genes encoding restriction enzymes. The resulting mutant strain not only exhibited marked efficiency of electrotransformation but also proved receptive to both in vitro and in vivo DNA assembly technologies, thereby facilitating the construction of recombinant DNA. In addition, we successfully adapted a CRISPR-Cas9-based base-editing platform developed for other Acinetobacter species. Our genetic modification strategy allows for the domestication of non-model bacteria, streamlining their utilization in synthetic biology applications.