Background Escherichia coli does not produce n-butanol naturally, but can be butanologenic when related enzymes were expressed using inducible elements on plasmids. In this study we attempted to confer E. coli strain capability of automatic excretion of the chemical by employing a native anaerobic promoter. Also, a novel DNA kit was designed for PCR preparation of linear DNA fragments to perform strain modification. The kit is primarily composed of two mother vectors, co-transformation of linear DNAs into E. coli can simultaneously introduce two butanol synthetic operons into the chromosome and create two in-frame gene deletions at targeted native loci. Results E. coli strain Bw2V carries plasmid pCNA-PHC and pENA-TA, both utilizes native anaerobic promoter Phya for the expression of butanol synthetic enzymes. When Bw2V was subjected in anaerobic fermentation using medium containing extra glucose, the accumulated n-butanol in the broth was up to 2.8 g/L in bioreactor; as the genetic element expressing the same pathway was introduced into the genome, the titer of butanol was 1.4 g/L. Conclusions The expression system using Phya is effective in applications that involve expression plasmids as also applicable in ectopic expression as single copy on the chromosome. Results imply that Phya can be subjected for broader application in bioproduction of more feedstock chemicals.