Establishing a straightforward I-SceI-mediated recombination one-plasmid system for efficient genome editing in P....

Pseudomonas putida has become an increasingly important chassis for producing valuable bioproducts. This development is not least due to the ever-improving genetic toolbox, including gene and genome editing techniques. Here, we present a novel, one-plasmid design of a critical genetic tool, the pEMG/pSW system, guaranteeing one engineering cycle to be finalized in 3 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 the 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 a 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 an msfGFP gene in 3 days. In addition, a large fragment of 12.1 kb was also integrated. In summary, we present an updated pEMG/pSW genome editing system that allows efficient and rapid genome editing in P. putida. All plasmids designed in this study will be available via the Addgene platform.