(451f) Reverse Engineering of an Erythromycin Overproducing Strain

Actinomycetes are an industrially important species of bacteria because they produce 40% of the antibiotics in the pharmaceutical market. However, in nature these bacteria produce limited amounts of antibiotics. To increase the amount of products made by actinomycetes, pharmaceutical companies employ many rounds of random mutagenesis and screening to develop an antibiotic overproducer. Our goal is to understand the overproduction mechanisms of these mutated strains at the molecular level and to use the knowledge to rationally engineer more efficient and productive overproducing strains. Previously we have shown using DNA microarrays that the industrial overproducer strain of Saccharopolyspora erythraea, the natural producer of erythromycin, expresses erythromycin gene cluster genes significantly longer than the wild type strain, accounting, at least partially, for the increase in antibiotic titers observed for the overproducer. Recently, we discovered a regulator for the erythromycin gene cluster (eryR) using an electrophoretic mobility shift assay (EMSA). EryR has high homology to bldD, a global regulator involved in morphological development and antibiotic production in Streptomyces coelicolor. To study the function of eryR, we complemented a ΔBldD S. coelicolor strain with eryR, and were able to restore the sporulation and antibiotic production lost in the knockout strain. This suggests that eryR is able to function similar to bldD. Initial binding studies on eryR showed that it binds to all the erythromycin gene cluster promoters, as well as its own promoter. When we compared the gel shift profile using the eryR promoter region probe caused by wild type and overproducer total protein, we found two shifts, and the top shift is lengthened in the overproducer. We have determined the bottom shift is eryR binding to its own promoter, but we have not yet determined what is causing the top shift. Current efforts to elucidate the two shifts include introducing mutations and deletions into the probe, protein identification by mass spectrometry, and DNase I footprinting