Function Of TetR-family Regulator SACE₃986in Saccharopolyspora Erythraea

Saccharopolyspora erythraea, a soil-dwelling life camp Gram-positive actinomycetes, produces a variety of complex secondary metabolites. Erythromycin is one of a class of secondary metabolites with broad-spectrum macrolide antibacterial activity. Since clinical applications, erythromycin antimicrobial drugs is the main one of the widely used anti-inflammatory drugs. In recent years, many TetR family regulatory genes （SCOI712, SAV151etc.） involved in the synthesis of antibiotics was discovered in other Streptomyces, suggesting that there may be the same regulatory genes involved in the regulation of erythromycin biosynthesis in S. erythraea.In this study,△SACE₃986mutant was constructed by a fragment homologous recombination. Firstly1500bp homologous arms of SACE₃986were obtained by PCR, and then insert into the appropriate location in the plasmid pUCTSR carrying resistance gene tsr to construct the plasmid pUCTSRA3986; SACE₃986gene was replaced with resistance gene tsr to construct ASACE₃986by a fragment homologous recombination. By the fermentation of ASACE₃986and the original strain A226at30℃, bacteriostatic test of bacillus subtilis analysis revealed that compared to A226, erythromycin yield of△SACE₃986mutant increased significantly. The preliminary result showed that SACE₃986may be negatively regulatory gene involved in the biosynthesis of erythromycin.In order to determine the impact on erythromycin biosynthesis, the pIB139vector carrying SACE₃986gene was introduced into△SACE₃986mutant and A226severally, and we obtained the△SACE₃986/pIB1393986、△SACE₃986/plB139、 A226/pIB1393986and A226/pIB139mutants. HPLC analysis revealed erythromycin A yield in△SACE₃986/pW1393986was restored to86.2%the yeild of A226, while the yield of A226/pIB1393986reduced46.7%over A226/pIB139. These results indicated that SACE₃986was a negatively regulatory gene participate in erythromycin biosynthesis.To investigate whether the deletion of SACE₃986influenced the morphological differentiation and the biomass, the results showed that spore formation in△SACE3986mutant was not significantly different from A226, indicating that SACE₃986gene did not regulate spore formation; cell concentration of△SACE₃986was the mainly same to A226, displayed the improvement of erythromycin production was not own to variation of cell density in△SACE₃986strain.We further inactivated SACE3986in an industrial strain WB. The result indicated erythromycin production increased by37.6%-40.3%in WB/△SACE₃986, and showed that SACE₃986was also able to regulate the production of erythromycin in the industrial strain WB.In the study, eryAI, ermE and SACE₃985were selected for transcriptional analysis among A226and△SACE₃986by qRT-PCR. The results showed that the transcription of eryAI, ermE and SACE₃985in△SACE₃986were increased by3.7-,1.7-and5.7-folds relative to that of its parent strain, respectively. The finding revealed the improvement of erythromycin yield may be own to increasing the amount of transcription of the three genes.In order to study the mechanism of erythromycin biosynthesis, which was regulated by SACE₃986gene, we expressed and purified His6-SACE₃986protein, and compounded SACE₃986protein with eryAI promoter, ermE resistance gene promoter and SACE₃985-SACE₃986intergenic region. EMSAs showed that in vitro SACE₃986bound SACE₃985-SACE₃986intergenic region, while it uncombined with eryAI and ermE promoters, indicating SACE₃986did not control them directly, SACE₃985, as the target gene of SACE₃986, was regulated to influence erythromycin production secondhand.SACE₃985gene was introduced into the original strain A226by the integrating vector pIB139in order to verify the function of the target gene, HPLC analysis showed that the erythromycin yield of the mutant improved by37.8%compared with that of its parent strain, indicating that the improvement of erythromycin production was own to the overexpression of SACE₃985.As TetR family of transcriptional regulator, SACE₃986was identified to negatively regulate the yield of erythromycin, and to a certain extent it clarified the molecular mechanism, which SACE₃986regulated erythromycin biosynthesis. The research provides a new idea and theoretical basis for exploring regulatory mechanisms of erythromycin biosynthesis and using genetic engineering to improve the production of erythromycin.