Rapid One-Step Inactivation of Single or Multiple Genes in Escherichia coli

We developed an integrated helper plasmid-based gene manipulation system for more efficient and rapid engineering of Escherichia coli. The integrated helper plasmid, pCW611, contains two recombinases which are expressed in reverse direction by two independent inducible systems. One is Red recombinase under the control of arabinose inducible system to induce recombination event by using linear gene knockout DNA fragment while the other is Crerecombinase which is controlled by IPTG inducible system to obtain marker-less mutant strains.

The main advantage of this system is that the time and effort required can be significantly reduced because the iterative transformation of the helper plasmid and curing steps are not required. We could delete one target gene in 3 days by using pCW611. To verify the usefulness of this gene manipulation system, the deletion experiments were performed for knocking out four target genes individually (adhE, sfcA, frdABCD, and ackA) and two genes simultaneously for two cases (adhE-aspA and sfcA-aspA). Also, sequential deletion of four target genes (fumB, iclR, fumA, and fumC) was successfully performed for the construction of fumaric acid producing strain. The efficiencies of target gene replacement and removal of the resistance marker were nearly 50% and 100%, respectively. This rapid and efficient gene manipulation system successfully developed and validated should be useful for the metabolic engineering of E. coli.(Development of systems metabolic engineering platform technologies for biorefineries; 

NRF-2012-C1AAA001-2012M1A2A2026556) funded by the Ministry of Education, Science and Technology)