Lipid A is a major component in the outer membrane of most Gram-negative bacteria. Monophosphoryl lipid A contains no phosphate group at 1-position and can be used as an adjuvant. We constructed an Escherichia coli mutant CW001 by integrating a gene lpxE into the chromosome of E. coli W3110. The gene lpxE encodes an enzyme LpxE which removes the 1-phosphate group of lipid A. CW001 predominantly produces 1-dephosphorylated lipid A in vivo, as adjudged by thin layer chromatography and electro-spray ionization mass spectrometry. This study not only is important for the development of lipid A adjuvants but also provides a novel method for integration of heterologous genes into the chromosome of E. coli.