Development of a Gene Knockout System in E. coli Using Integration-Helper Plasmid

In this study, more rapid and efficient engineering method using integration helper plasmid in E. coli was developed. The integration helper plasmid, pCW611, has two recombinases which are expressed in reverse direction by two independent inducible systems. By using this system, required time and effort 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, fumaric acid producing E. coli strain was developed by deleting four target genes (fumB, iclR, fumA, and fumC) in 10 days as a proof-of-concept study.

(This work was supported by the Advanced Biomass Research and Development Center of Korea (NRF-2010-0029799) through the Global Frontier Research Program of the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF). Development of systems metabolic engineering platform technologies for biorefineries; NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557) funded by the Ministry of Education, Science and Technology).