The selective marker in the plasmid-based expression system is usually a gene that encodes an antibiotic-resistant protein; therefore, the antibiotic has to add to maintain the plasmid when growing the bacteria. This antibiotic addition would lead to increase of production cost and the environment contamination. In this study, a novel Escherichia coli expression system, the lpxA deletion mutant harboring an lpxA-carrying vector, was developed. To develop this system, three plasmids pCas9Cre, pTF-A-UD, and pRSFCmlpxA were constructed. The plasmid pCas9Cre produces enzymes Cas9, lambda-Red, and Cre and can be cured by growing at 42 degrees C; pTF-A-UD contains several DNA fragments required for deleting the chromosomal lpxA and can be cured by adding isopropyl-D-thiogalactopyranoside; pRSFCmlpxA contains the lpxA mutant lpxA123 and CamR. When E. coli were transformed with these three plasmids, the chromosomal lpxA and the CamR in pRSFCmlpxA can be efficiently removed, resulting in an E. coli lpxA mutant harboring pRSFlpxA. The lpxA is essential for the growth of E. coli; its relocation from chromosome to a constitutive expression vector is an ideal strategy to maintain the vector without antibiotic addition. The lpxA123 in pRSFlpxA can complement the deletion of the chromosomal lpxA and provide a strong selective pressure to maintain the plasmid pRSFlpxA. This study provides an experimental evidence that this novel expression system is convenient and efficient to use and can be used to improve l-threonine biosynthesis in the wild type E. coli MG1655 and an l-threonine producing E. coli TWF006.