基于氧化还原酶作用的柔红霉素生物合成负调控机制研究

Daunorubicin (DNR) is one of the widely used anticancer drugs in clinic, which isolated from the secondary metabolites of Streptomyces coeruleorubidus. In our previous work, dauW was considered as the negative regulator, the transcriptional level of the specific pathway regulator dnrI was increased, and the productivity of DNR was also improved significantly by completely knock out dauW , however the mechanism of negative regulation mediated by dauW was not clear. DauW did not have the basic characteristic of transcriptional factor from Streptomyces by bioinformatics analysis, however dauW encodes putative flavin-dependent oxidoreductase. In this study, the oxidoreductase catalysis mechanism of DauW was investigated, DauW was expressed and purified to establish the enzymatic assay system in vitro and the catalysate of DauW was prepared. DnrO was the key transcriptional factor in DNR regulation , therefore the effect of catalysate of DauW on DnrO was investigated by the electrophoretic mobility shift assay EMSA、Lux reporter system and RT-PCR in vitro and vivo. Furthermore, The catalysate of DauW was investigated as the specific chemical signals to bind with the DnrO for modulating the regulatory network of DNR. In conclusion, the mechanism of specific negative regulation of DNR mediated by the action of oxidoreductase can be elucidated in this work , which may be helpful for better understanding of DNR biosynthesis, and provide the theoretical guidance for improvement of DNR productivity.

柔红霉素（Daunorubicin，DNR）是临床广泛使用的抗肿瘤药物，是来源于天蓝淡红链霉菌的重要次级代谢产物。前期研究认为dauW是一类负调控基因，将其完全敲除可以提高途径特异性调控基因dnrI的转录水平，DNR产量大幅度提高，但其负调控机制一直未能阐明。通过生物信息学分析DauW不具有链霉菌转录调控因子的基本特征，却具有与黄素腺嘌呤二核苷酸（FAD）共价结合的氧化还原酶特征性结构域。本研究通过体外表达DauW，构建酶反应体系探索DauW的氧化还原酶反应机制并制备DauW催化产物。通过凝胶迁移、荧光素酶报告系统、实时定量PCR等体内外实验探索DauW催化产物是否与DNR核心调控蛋白DnrO特异性结合，并进一步探索DauW催化产物对DNR生物合成的特异性调控途径。本研究将阐明基于氧化还原酶作用的DNR负调控机制，进一步解析DNR的整个生物合成过程，为提高DNR的发酵水平提供理论指导。

柔红霉素（Daunorubicin，DNR）是临床广泛使用的抗肿瘤药物，是来源于天蓝淡红链霉菌的重要次级代谢产物。前期研究认为dauW是一类负调控基因，但通过生物信息学分析DauW具有与黄素腺嘌呤二核苷酸 （FAD）共价结合的氧化还原酶特征性结构域。本研究通过基因敲除、蛋白表达、底物分离、体外酶反应的构建，探索DauW的氧化还原酶功能。实验结果初步表明DauW以鲍霉素产物B4为底物，对其糖基C4位上的羟基氧化脱氢，从而获得其氧化产物。并且通过凝胶迁移，实时定量PCR等体内外实验探索DauW催化产物是否与DNR核心调控蛋白DnrO特异性结合。实验结果表明，DauW催化产物并不能与柔红霉素关键调控蛋白DnrO结合，因此该产物未能作为信号分子参与柔红霉素的途径特异性调控，也反应了链霉菌次级代谢产物调控机制的复杂性。但本研究在对天蓝淡红链霉菌基因组测序的基础上，首次发现了柔红霉素产生菌基因组上γ-丁内酯类信号分子的生物合成基因簇。通过基因敲除和发酵产物分析，发现该基因簇中的关键转录调控因子对柔红霉素的生物合成有着直接影响，从而为发现和探索柔红霉素的全局调控机制奠定基础。通过本研究的初步探索，从而更深入理解柔红霉素的整个生物合成过程，为进一步提高柔红霉素的发酵水平提供理论指导。