A simple proficient method for targeted genome editing in Escherichia coli

Abstract

Metabolic engineering and synthetic biology aim to create a potent bio-systems including bacteria for many useful purposes to produce value added products etc. Here, we report that a more promising method for targeted genome editing in E. coli was developed based on λ Red recombinase system by combining Multiplex automated genomic engineering (MAGE) and TetA dual selection system. This method could be successfully and rapidly used for targeted genome engineering including deletions, insertions, replacement and point mutation without mutS inactivation and plasmid cloning. It was observed that even one MAGE cycle was sufficient to get the positive genome edited mutants with the efficiencies of over 90% (insertion) or 70% (replacement/deletion) by the optimized selection process on NiCl2. To demonstrate the efficiency of our system we optimized metabolic flux through the deoxyxylulose-5- phosphate (DXP) biosynthesis pathway by changing the native promoters of two rate limiting genes with the constitutive synthetic promoter. The representative clones of genetic variants E.coli strains produced higher amount of lycopene than that of the control strain. This method could achieve a scarless proficient targeted genome editing with a seamless screening process.