Cloning And Characterization Of The EryA Promoter Region Of Erythromycin Biosynthetic Genes From Saccharopolyspora Erythraea

The eryA genes play a very important role in the biosynthesis of erythromycin macrolactone ring (6-deoxy-erythronolide B, 6-dEB). In this paper, an Escherichia coli-Saccharopolyspora erythraea shuttle vector containing the eryA promoter region was constructed using the enhanced green fluorescent protein (EGFP) gene as a reporter. Fluorescence microscopy showed that the eryA promoter was not only functional in Sac. erythraea but also in heterologous host Streptomyces lividans. Subsequently, characteristics of the eryA promoter region as a promoter of Streptomyces expression vector were deep studied, revealing that the predicted-35 region was not necessary for the promoter and the 41-bp promoter DNA fragment only carrying predicted-10 region was still active in Streptomyces. Site-directed mutant demonstrated that the predicted-10 region was indispensable for the promoter. Thus, the 41-bp promoter segment can function as an effective promoter of Streptomyces expression vector, which is one of the shortest promoters hitherto found in Streptomyces and is very useful for constructing new Streptomyces expression vectors.Moreover, neighbouring regions of the eryA promoter were preliminarily analyzed and the results confirmed that exogenous genes couldn't be expressed in Sac. erythraea and S. lividans under the promoter including 300bp DNA sequences of downstream of eryA translation start point. Illumination of the phenomenon will take a foundation in further exploring the regulation of erythromycin biosynthetic gene.