LuxR家族调控因子DptR1调节达托霉素生物合成的转录调控机制研究

项目名称： LuxR家族调控因子DptR1调节达托霉素生物合成的转录调控机制研究

项目编号： No.21506048

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 有机化学

项目作者： 宇光海

作者单位： 河南工业大学

项目金额： 21万元

中文摘要： 达托霉素（Dap）是以非核糖体蛋白合成酶机制合成的抗多种耐药病菌的新型大环内酯类抗生素，应用前景广阔。研究表明，LuxR家族蛋白广泛存在于链霉菌属抗生素合成基因簇中，对抗生素合成起重要的调控作用。且研究表明，Dap基因簇中的DeoR家族蛋白DptR2是达托霉素合成的必须正调控因子。而与DptR2相邻的 DptR1蛋白是C末端具有LuxR-HTH保守结构域，N末端具有Winged-HTH结构域的新型LuxR家族调控蛋白，但其对Dap合成的转录调控机制尚不明确。本课题拟利用免疫共沉淀技术捕获与DptR1互作的调控蛋白；利用RT-PCR、凝胶迁移及DNaseI足迹等技术解析DptR1达托霉素合成途径专一性转录调控机制；利用染色质免疫共沉淀技术，在基因组尺度上锁定DptR1调控靶标，多尺度解析DptR1调节达托霉素生物合成的转录调控机制，以期为构建具有我国自主知识产权的高产工程菌株奠定理论基础。

中文关键词： 达托霉素；玫瑰孢链霉菌；转录调控；LuxR家族调控蛋白；DptR1

英文摘要： Daptomycin is a member of the A21978C family of the cyclic anionic 13-amino acid lipopeptide, produced by a non-ribosomal peptide synthetase (NRPS) mechanism in Streptomyces roseosporus, which has a novel, calcium-dependent mechanism of antibiosis and has antibacterial activity against multi-antibiotic resistant pathogens. The market prospect of daptomycin has been bright and promising, since approved by FDA in 2003.. It was reported that LuxR family proteins exist widely in Streptomyces antibiotic biosynthesis gene clusters and play important regulatory roles in the transcription regulation of the biosynthesis of antibiotics..DptR1, a novel-type LuxR family regulator, has a LuxR family helix-turn-helix (LuxR-HTH) DNA binding domain at its C-terminus and the N-terminus belonged to the Winged-helix-turn-helix DNA binding domain. However, the transcription regulation mechanism of DptR1 on the biosynthesis of daptomycin is unknown. . It was reported that DeoR-type regulator DptR2 which encoding gene dptR2 is adjacent to the dptR1, had important positive transcription regulation function on daptomycin production, but it did not regulate the expression of dpt gene cluster. The dptR2 disruption mutant did not produce daptomycin at all, and complementation with dptR2 under its native promoter could restore the production of daptomycin.. In summary, DptR1 may play important roles in the transcription regulation of daptomycin biosynthesis in consideration of its novel structure of domains and the important transcription regulation function of DptR2 on the biosynthesis of daptomycin.. In this study, the transcription regulation mechanism of the novel-type LuxR family regulator DptR1 on the biosynthesis of daptomycin will be systematically researched. In summary, the real-time-PCR, electrophoretic mobility shift assay (EMSA) and DNaseI footprint technologies will be used to clarify the dpt gene cluster-specific regulation mechanism of DptR1; while the co-immunoprecipitation (Co-IP) be utilized to capture the interacting regulatory proteins of DptR1 in vivo and the chromatin immunoprecipitation with the second generation gene sequencing technology (Chip-seq) will be utilized to study the potential regulatory target genes of DptR1 in the whole genome.

英文关键词： Daptomycin;Streptomyces roseosporus;Transcription regulation;LuxR Family Regulatory protein;DptR1