Nagahama Institute of Bio-Science and Technology and Nagoya University convert genetically intractable bacteria into tractable form — Potential use in biomanufacturing

The research group of Associate Professor Masahito Ishikawa of the Department of Bioscience at Nagahama Institute of Bio-Science and Technology, and Professor Katsutoshi Hori of the Graduate School of Engineering at Nagoya University, have announced that they successfully modified Acinetobacter sp. strain Tol5, a bacterium useful for biomanufacturing but unresponsive to genetic recombination, to a genetically tractable bacterium. The obtained Tol5 mutant strain is expected to be useful as a microbial platform in biomanufacturing. The application of the knowledge obtained in this study to other bacteria is expected to increase the variety of bacteria used in biomanufacturing, allow for the utilization of unused raw materials, diversify products, and improve production efficiency. The results were published in the May 9 issue of Applied and Environmental Microbiology, a scientific journal of the American Society for Microbiology.

Biomanufacturing uses the cells of microorganisms, animals, plants, and other organisms to produce chemicals, fuels, foods, and other products from biomass resources and carbon dioxide in the atmosphere. It is expected to be a technology that will contribute to the realization of a sustainable society. Currently, Escherichia coli is mainly used as a microbial platform, but there is a need for microbial platforms that can grow in diverse environments where E. coli cannot survive and produce more complex compounds. Acinetobacter sp. strain Tol5 is a toluene-degrading bacterium isolated from the natural environment and is characterized by high tolerance to organic solvents, ability to convert diverse substances, and high adhesiveness and agglutination, but is resistant to genetic recombination.

In this study, the research group revealed that a factor responsible for the genetic intractability of the Tol5 strain is its defense mechanism against foreign DNA. They created a Tol5 mutant that lacks two restriction enzyme genes constituting the defense mechanism and performed gene transfer to the mutant by electroporation. The results showed that the transformation efficiency of the Tol5 mutant was approximately 57,000 times as high as that of E. coli. Acinetobacter sp. strain Tol5 was successfully modified into a genetically tractable bacterium. Moreover, they confirmed its applicability to DNA assembly (combination of DNA fragments) in vitro and in cells. The construction of recombinant DNA, which has been done in E. coli, is expected to be possible in the Tol5 mutant strain.

Ishikawa said, "Genetic modification of bacteria is like breed improvement to enhance the biological functions of bacteria. For bacteria, however, it is hacking biological functions by foreign genes introduced from outside the cell. It is similar to a viral infection. While the coronavirus disease was raging during the 2019 pandemic, I conceived of this study from the standpoint of bacteria. Moving forward, from the perspective of viruses infecting bacteria (phages) this time, I would like to develop a technology mimicking a strategy used by phages to evade the immune mechanism of bacteria."

Journal Information
Publication: Applied and Environmental Microbiology
Title: 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
DOI: 10.1128/aem.00400-24