Cloning of the tryptophan operon of Brevibacterium divaricatum and its expression in E. coli.

Abstract

A genomic bank from Brevibacterium divaricatum has been prepared using lambda EMBL3 as a vector. The genomic bank's titers are 2.2 x 10(6) pfu/micrograms. Through screening by plaque hybridization, a 9.6 kb NcoI fragment which contains the entire trp operon has been isolated. Polymerase chain reaction amplification and restriction endonuclease analysis of PCR fragments indicated that there is homology between the coryneform bacteria; however, some genetic diversity among the species still exists. By complementation tests using subcloning of the 9.6 kb NcoI fragments and various E. coli tryptophan auxotrophs, this fragment was found to contain a gene cluster composed of trpE, trpD, trpC, trpB and trpA in this order. This revealed that the tryptophan biosynthesis genes in B. divaricatum may be an operon.