Establishing a straight-forward I-SceI mediated recombination one plasmid system for efficient genome editing in P. putida KT2440

Pseudomonas putida has become an increasingly important chassis for the production of valuable bioproducts. This development is not at least due to the ever-improving genetic toolbox, including gene and genome editing techniques. Here, we present a novel, one plasmid design of a key genetic tool, the pEMG/pSW system, guaranteeing one engineering cycle to be finalized in three days. The pEMG/pSW system proved in the last decade to be valuable for targeted genome engineering in Pseudomonas, as it enables the deletion of large regions of the genome, the integration of heterologous gene clusters or targeted generation of point mutations. Here, to expedite genetic engineering, two alternative plasmids were constructed: 1) the sacB gene from Bacillus subtilis was integrated into the I-SceI expressing plasmid pSW-2 as counterselection marker to accelerated plasmid curing; 2) double strand break introducing gene I-SceI and SacB counterselection marker were integrated into the backbone of the original pEMG vector, named pEMG-RIS. The single plasmid of pEMG-RIS allows rapid genome editing despite the low transcriptional activity of a single copy of the I-SceI encoding gene. Here, the usability of the pEMG-RIS is shown in P. putida KT2440 by integrating an expression cassette including a msfGFP gene in three days. In addition, a large fragment of almost 16 kb was also integrated. In summary, an updated pEMG/pSW genome editing system is presented that allows efficient and rapid genome editing in P. putida. The pEMG-RIS will be available via the Addgene platform. ### Competing Interest Statement The authors have declared no competing interest.