Climate change is an urgent and collective challenge, and a portfolio of solutions is needed to reduce CO₂ emissions or to increase carbon capture and utilization from the atmosphere. Nature has been evolving CO₂ utilization pathways for billions of years and offers a promising repository of novel metabolisms and enzymes capable of CO₂ fixation. However, non-model bacteria are recalcitrant to genetic engineering, and the application of modern genetic tools is cumbersome. One of the main barriers is the low transformation efficiency, as most tools and technologies require the delivery of DNA molecules to tune and modify the host metabolism. This transformation barrier is a common feature of all wild-type bacteria, which employ a variety of defense systems to avoid phages, plasmids, and other mobile genetic elements in their native ecological niche. To transform them with recombinant DNA, this arsenal has to be predicted, characterized and circumvented.

In our study, we performed an in-depth analysis of Cupriavidus necator H16 using bioinformatic tools to study its restriction enzymes and defense systems. By using tailored plasmids, we confirmed the functional role of three systems encoded in the genome, and through a combination of plasmid mutation and demethylation, we transformed large plasmids with higher efficiency. We also succeeded in transforming suicide plasmids via electroporation, deleting the native defense systems and creating a domesticated strain.

These findings will benefit both the C. necator H16 community and researchers working with other non-model bacteria by providing a roadmap that can be followed to increase transformation efficiency.