The elimination of two restriction enzyme genes allows for electroporation-based transformation and CRISPR-Cas9-based base editing in the non-competent Gram-negative bacterium Acinetobacter sp. Tol 5.

Environmental isolates are promising candidates for new chassis of synthetic biology because of their inherent capabilities, which include efficiently converting a wide range of substrates into valuable products and resilience to environmental stresses; however, many remain genetically intractable and unamenable to established genetic tools tailored for model bacteria. Acinetobacter sp. Tol 5, an environmentally isolated Gram-negative bacterium, possesses intriguing properties for use in synthetic biology applications. Despite the previous development of genetic tools for the engineering of strain Tol 5, its genetic manipulation has been hindered by low transformation efficiency via electroporation, rendering the process laborious and time-consuming. This study demonstrated the genetic refinement of the Tol 5 strain, achieving efficient transformation via electroporation. We deleted two genes encoding type I and type III 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 without reliance on intermediate Escherichia coli constructs. In addition, we successfully adapted a CRISPR-Cas9-based base-editing platform developed for other Acinetobacter species. Our findings provide genetic modification strategies that allow for the domestication of environmentally isolated bacteria, streamlining their utilization in synthetic biology applications.IMPORTANCERecent synthetic biology has sought diverse bacterial chassis from environmental sources to circumvent the limitations of laboratory Escherichia coli strains for industrial and environmental applications. One of the critical barriers in cell engineering of bacterial chassis is their inherent resistance to recombinant DNA, propagated either in vitro or within E. coli cells. Environmental bacteria have evolved defense mechanisms against foreign DNA as a response to the constant threat of phage infection. The ubiquity of phages in natural settings accounts for the genetic intractability of environmental isolates. The significance of our research is in demonstrating genetic modification strategies for the cell engineering of such genetically intractable bacteria. This research marks a pivotal step in the domestication of environmentally isolated bacteria, promising candidates for emerging synthetic biology chassis. Our work thus significantly contributes to advancing their applications across industrial, environmental, and biomedical fields.