A Cloningless and Scarless Genome Editing Method for Metabolic Pathway Engineering in Escherichia coli

Genome modification is a prerequisite in the field of synthetic biology and metabolic engineering that demand the construction of potent strains for basic and applied research including therapeutic and industrial applications in addition to understanding the effects of evolved mutations and, genetic and functional genome analyses (for functional genomic experiments). Genetic engineering methods, such as zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeat (CRISPR) – Cas (CRISPR-associated systems), λ Red recombinase system mediated genetic engineering, multiplex automated genomic engineering (MAGE), have their own merits and demerits. An efficient chromosome engineering method for Escherichia coli is required to make mutants useful in basic research and applied science. In this context, we have developed a more promising method based on λ Red recombinase system by coupling the antibiotic marker amplification flanked with target homologous sequence, MAGE and TetA dual selection system. This method could be successfully used for targeted genome engineering including deletion, insertion, replacement and point mutations. The basic strategy is the PCR amplification of antibiotic marker (Tet) flanked with target homologous sequence and transformation into the λ Red recombinase expressing system enabling to select the antibiotic resistant transformants and subsequently transformation of MAGE primers exclusively designed for insertion, replacement, and deletion in the target site. After MAGE, the modified transformants are enriched by tet^S/metal ion (Ni⁺) resistant on liquid culture and final selection by tet^S/tet^R on LB and LB-tet plates. To demonstrate the efficiency of our system we optimized metabolic flux through the deoxyxylulose-5-phosphate (DXP) biosynthesis and beta-oxidation pathways. We hope that the above described method could achieve a scarless proficient targeted genome editing with a seamless screening process.