Presentation Information
[3ASBA-16]Genetic domestication of the non-model bacterium Acinetobacter sp. Tol 5 for advanced synthetic biology applications
○Masahito Ishikawa1, Katsutoshi Hori2 (1. Nagahama Inst. Bio-Sci. Tech. (Japan), 2. Nagoya Univ. (Japan))
Keywords:
Non-model bacteria,Transformation,Restriction-modification,CRISPR-Cas9
[Purpose]
Environmental bacterial isolates are increasingly recognized as promising chassis for synthetic biology due to their inherent robustness and unique metabolic capabilities compared with conventional laboratory strains. Acinetobacter sp. Tol 5 is a particularly attractive candidate for industrial and environmental applications, such as bioremediation and immobilized biocatalysis, owing to its high adhesiveness mediated by the nanofiber protein AtaA and its ability to degrade various aromatic compounds. However, its broader application has been limited by its low transformation efficiency via electroporation, which reflects its intrinsic resistance to foreign DNA. In this study, we aimed to domesticate Tol 5 by overcoming its defense systems against exogenous DNA.
[Method]
To improve genetic tractability, we investigated the impact of restriction–modification (R–M) systems on electrotransformation efficiency. Genome analysis identified four putative R–M systems in Tol 5. The genes encoding type I and type III restriction enzymes were deleted using a conjugation-based suicide plasmid method. The resulting mutant was evaluated for electrotransformation efficiency and compatibility with in vitro and in vivo DNA assembly techniques. In addition, the CRISPR–Cas9-based genome editing tools tailored for Acinetobacter species were applied.
[Results]
Deletion of the type I and III R–M systems markedly improved electrotransformation efficiency compared with the wild-type strain. This improvement enabled direct application of in vitro and in vivo DNA assembly within Tol 5 cells, eliminating the need for intermediate construction in Escherichia coli. Although the CRISPR–Cas9 genome editing system was ineffective, a CRISPR-based base editing system successfully introduced targeted mutations that suppressed ataA expression. These results demonstrate that genetically refined Tol 5 can serve as a versatile and engineerable chassis for synthetic biology.
[Consideration]
The improved transformation efficiency observed after deleting type I and III R–M systems indicates that restriction barriers are a major factor limiting genetic manipulation in Tol 5. Our results suggest that overcoming these barriers, rather than optimizing transformation conditions alone, is key to improving genetic accessibility in environmental bacteria. In addition, the successful use of DNA assembly directly in Tol 5 demonstrates that practical genetic engineering can be achieved without relying on Escherichia coli.
[Conclusion]
This study overcomes the genetic intractability of an environmental bacterium by removing key restriction barriers. Our approach expands the applicability of advanced genetic tools to non-model bacteria and provides a foundation for developing robust microbial chassis for industrial, environmental, and biomedical applications.
Environmental bacterial isolates are increasingly recognized as promising chassis for synthetic biology due to their inherent robustness and unique metabolic capabilities compared with conventional laboratory strains. Acinetobacter sp. Tol 5 is a particularly attractive candidate for industrial and environmental applications, such as bioremediation and immobilized biocatalysis, owing to its high adhesiveness mediated by the nanofiber protein AtaA and its ability to degrade various aromatic compounds. However, its broader application has been limited by its low transformation efficiency via electroporation, which reflects its intrinsic resistance to foreign DNA. In this study, we aimed to domesticate Tol 5 by overcoming its defense systems against exogenous DNA.
[Method]
To improve genetic tractability, we investigated the impact of restriction–modification (R–M) systems on electrotransformation efficiency. Genome analysis identified four putative R–M systems in Tol 5. The genes encoding type I and type III restriction enzymes were deleted using a conjugation-based suicide plasmid method. The resulting mutant was evaluated for electrotransformation efficiency and compatibility with in vitro and in vivo DNA assembly techniques. In addition, the CRISPR–Cas9-based genome editing tools tailored for Acinetobacter species were applied.
[Results]
Deletion of the type I and III R–M systems markedly improved electrotransformation efficiency compared with the wild-type strain. This improvement enabled direct application of in vitro and in vivo DNA assembly within Tol 5 cells, eliminating the need for intermediate construction in Escherichia coli. Although the CRISPR–Cas9 genome editing system was ineffective, a CRISPR-based base editing system successfully introduced targeted mutations that suppressed ataA expression. These results demonstrate that genetically refined Tol 5 can serve as a versatile and engineerable chassis for synthetic biology.
[Consideration]
The improved transformation efficiency observed after deleting type I and III R–M systems indicates that restriction barriers are a major factor limiting genetic manipulation in Tol 5. Our results suggest that overcoming these barriers, rather than optimizing transformation conditions alone, is key to improving genetic accessibility in environmental bacteria. In addition, the successful use of DNA assembly directly in Tol 5 demonstrates that practical genetic engineering can be achieved without relying on Escherichia coli.
[Conclusion]
This study overcomes the genetic intractability of an environmental bacterium by removing key restriction barriers. Our approach expands the applicability of advanced genetic tools to non-model bacteria and provides a foundation for developing robust microbial chassis for industrial, environmental, and biomedical applications.
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