Rapid Gene Knockout Method Using Single Helper Plasmid Expressing Red and Cre Recombinases

Abstract

Development of rapid gene manipulation tools is essential for efficientmetabolic engineering approaches. In this study, integration helper plasmidwas developed for rapid gene manipulation. The integration helper plasmidpCW611 expresses two recombinases (Red and Cre) by using twoindependent (IPTG and Arabinose) inducible systems. Required time andeffort can be significantly reduced compared to conventional method byusing integration helper plasmid because the iterative transformation ofthe helper plasmid and curing steps are not required. We could deleteone target gene in 3 days by using pCW611. To verify the usefulnessof this gene manipulation system, the deletion experiments were performedfor knocking out four target genes individually (adhE, sfcA, frdABCD,and ackA) and two genes simultaneously for two cases (adhE-aspA andsfcA-aspA). Also, fumaric acid producing E. coil strain was developedby deleting four target genes (fumB, iclR, fumA, and fumC) in 10 daysas a proof-of-concept study.(Technology Development Program to SolveClimate Changes on Systems Metabolic Engineering for Biorefineries fromthe Ministry of Science, ICT and Future Planning (MSIP) through theNational Research Foundation (NRF) of Korea)