Development of a DNA endonuclease I-SceI-based scarless genome editing system for Cupriavidus necator

Cupriavidus necator is a promising microbial chassis capable of fixing CO₂ and producing high polyhydroxyalkanoate yields. Consequently, various genetic engineering methods have been explored. While sacB-based homologous recombination (HR) and CRISPR-Cas9 have shown both advantages and disadvantages in C. necator, alternative tools, including the DNA endonuclease I-SceI-mediated HR system could enable precise, scarless genome editing without requiring a large database. We developed a two-plasmid-based I-SceI HR system for efficient gene deletion and insertion in C. necator by altering origin replication and induction systems. The pOUO-1 plasmid was designed for conjugation-based genome integration via first HR, whereas the pOH-4 plasmid was constructed to express I-SceI, inducing second HR. Unlike conventional I-SceI expression strategies, which fail to trigger second HR in C. necator, transformation with pOH-4 alone was sufficient for recombination. A plasmid-curing strategy was optimized to eliminate the highly stable pOH-4 by increasing the incubation temperature to 37°C. Using this optimized system, the phaC₁ gene was successfully knocked out; the phaC_BP-M-CPF4 was inserted at the same site, resulting in a novel poly(3-hydroxybutyrate-co-5-hydroxyvalerate)-producing strain. This newly established I-SceI HR technique significantly simplifies genome engineering in C. necator, reducing the timeframe to a few weeks and facilitating its further applications in synthetic biology.