| Abstract |
Secondary metabolism in actinomycetes has been known to be controlled by a small molecule, γ-butyrolactone
autoregulator, the binding of which to each corresponding receptor leads to the regulation of the transcriptional expression of the
secondary metabolites. We expected that expression of an autoregulator receptor or a pleiotropic regulator in a non-host was to be
gained insight of effective production of new metabolic materials. In order to study the function of the receptor protein (seaR), which is
isolated from Saccharopolyspora erythraea, we introduced the seaR gene to Streptomyces coelicolor A3(2) as host strains. An effective
transformation procedure for S. coelicolor A3(2) was established based on transconjugation by Escherichia coli ET12567/pUZ8002 with a
φC31-derived integration vector, pSET152, which contained int, oriT, attP and ermEp* (erythromycin promotor). Therefore, the pEV615
was introduced into S. coelicolor A3(2) by conjugation and integrated at the attB locus in the chromosome of the recipients by the φC31
integrase (int) function. Exconjugant of S. coelicolor A3(2) containing the seaR gene was confirmed by PCR and transcriptional expression
of the seaR gene in the transformant was analyzed by RT-PCR. In case of S. coelicolor A3(2), a phenotype microarray was used to analyze
the phenotype of transformant compared with wild type by seaR expression. After that, in order to confirm the accuracy of the results
obtained from the phenotype microarray, an antimicrobial susceptibility test was carried out. This test indicated that sensitivity of the
transformant was higher than wild type in tetracycline case. These results indicated that some biosynthesis genes or resistance genes
for tetracycline biosynthesis in transformant might be repressed by seaR expression. Therefore, subsequent experiments, analysis of
transcriptional pattern of genes for tetracycline production or resistance, are needed to confirm whether biosynthesis genes or
resistance genes for tetracycline are repressed or not. |
