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

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.

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。

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。LuxR家族调控蛋白DptR1对Dap合成的转录调控机制尚不明确。本项目比较研究了WT与dptR1基因敲除、回补及过表达突变株在达托霉素合成、菌体生长、形态学等表型差异：敲除或过表达dptR1基因，都会引起达托霉素产量不同程度下降；WT与dptR1突变株间不存在明显的形态学差异，DptR1不参与玫瑰孢链霉菌的形态分化。比较研究了WT与dptR1突变株dpt基因簇关键基因的转录水平差异：敲除或过表达dptR1基因，都会使达托霉素合成关键基因在第3天转录水平提高，而在第4天转录水平大幅下降，这说明dptR1参与的达托霉素合成调控机制较复杂且具有时序性。在第3天，DptR1对dptR2转录具有抑制作用；dptR3的转录水平在敲除菌株和过表达菌株都不同幅度下降。在第4天dptR2，dptR3的转录水平均显著下降。邻苯二酚氧化酶报告系统实验结果表明，DptR1激活自身的启动子活性，正调控自身编码基因dptR1的转录水平；而抑制dptR3启动子活性，负调控dptR3的转录水平。对dptR1敲除突变株与WT进行了比较转录组测序分析：转录差异基因共511个，其中上调基因169个，下调基因342个。GO富集分析和KEGG富集分析表明，差异基因主要富集于：精氨酸及脯氨酸代谢，谷氨酰胺合成，色氨酸代谢，甘氨酸、丝氨酸及苏氨酸代谢，ABC膜转运蛋白。氨基酸是达托霉素合成的前体，而ABC膜转运蛋白则可能参与前体氨基酸和达托霉素的跨膜转运。此外编码 MbtH 的同源蛋白dptG和编码甲基转移酶参与特殊前体3m-Glu合成的dptI的转录水平也显著提高。而MbtH 蛋白与A 结构域紧密结合激活腺苷酰化反应，起到激活和招募NRPS作用。综上，DptR1可能通过参与调控重要氨基酸前体及特殊前体3m-Glu合成，ABC膜转运蛋白，以及MbtH 的同源蛋白dptG ，来调控达托霉素的生物合成。综上，本项目在表型、在dpt基因簇转录水平及全转录组等不同水平初步揭示了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，为进一步了解达托霉素合成的转录调控机制，提供了理论基础。