组蛋白乙酰化修饰对红色红曲菌M-7基因组中聚酮合酶基因簇全局调控

Monascus spp., a kind of China-typical microorganism resources,is the producer of Hongqu,which has been used for thousands of years in China. In addition to polyketide synthases(PKSA) coding for monakolin K(a kind of blood-lipid lowering agent) and citrinin (a kind of mycotoxin), there are more than ten other kinds of PKSAs which encode for unknown polyketides based on the predicted results for the genome of M.ruber M-7 by bioinformatics analysis. But we learn very little information about them,which restricts the further development of Monascus spp..It is an effective measure to modulate the expression of secondary gene clusters in fungi by changing the activities of histone deacetylases. In this study, two kinds of measures will be taken to inhibit the activities of histone deacetylases in order to improve the transcriptional level of gene clusters of unkonw polyketide synthases(PKSAs) in Monascus ruber M-7.One is adding chemical inhibitors of histone deacetylase to the different kinds of cultures, and the other is knocking out the genes and overexpressing the genes congding for the histone deactylases.Then, the conditions or the genetically modified mutants will be selected with the actived unknown PKSAs by comparing the transcriptional level of PKSAs.Then, the activeted unknown PKSA will be deleted in the seleced mutants above, and the level of transcription of PKSAs, metabolites and proteins of the mutants with PKSA-deletion will be analyzed. At last, the unkonwn polyketide will be isolated and identified based on theose results above. In short,all the results in this study will provide theoretical guidance to modulate the expression of unknown PKSAs,to discover new polyketide and to utilize of the Monascus spp.

红曲菌(Monascus spp.)是我国药食两用的特色微生物资源，其发酵产品红曲已有上千年应用历史。课题组对红色红曲菌（M.ruber）M-7基因组解析表明，该菌株除含有降血脂成分莫纳呵啉K等聚酮合酶（PKSA）基因簇外，还含有10多种其它PKSAs基因簇，而且发现有些PKSAs基因簇是沉默的。目前关于这些基因簇的了解还是空白，这极大限制了红曲菌的深度开发利用。改变组蛋白去乙酰化酶（HDAC）活性是调控真菌次级代谢基因簇表达活性的有效手段。本研究将采用化学修饰与基因敲除（过表达）相结合方法调节HDAC活性，分析调节前后红曲菌株PKSA转录活性与代谢谱变化；在此基础上以PKSA被激活的菌株为受体，构建对应PKSA缺失突变株，分析突变株PKSA转录活性、蛋白谱及代谢谱的变化，分离鉴定新的聚酮化合物；探讨HDAC对红曲菌PKSAs的调控机理。研究结果将为合理高效开发红曲菌资源提供理论指导。

组蛋白翻译后修饰对染色质的结构和功能具有重要影响，并调控基因的表达。组蛋白乙酰化是目前研究的最为深入的修饰类型，其通过组蛋白乙酰基转移酶和去乙酰化酶共同维持动态平衡。近年来，大量研究表明抑制组蛋白去乙酰化酶活性或敲除之是激活真菌基因组中未知基因簇表达或发现新的次级代谢产物的有效途径。本项目以红色红曲菌基因组中预测的聚酮合酶(polyketide synthase)基因簇为研究对象，采用化学抑制剂和分子生物学相结合的方法，抑制或敲除红色红曲菌M-7中组蛋白去乙酰化酶活性，分析红曲菌组蛋白乙酰化修饰变化对红色红曲菌PKSs基因簇表达及次级代谢的影响。结果显示在选用的5种常用的组蛋白去乙酰化酶抑制剂中，DHC能显著影响实验菌株代谢产物种类，并分离鉴定出1种未曾报道的新的化合物（乙酰红曲荧光醇a）；real-time RT-PCR分析显示在设定的时间点内，DHC的添加下调参与桔霉素合成的PKS(基因号GME2757)的表达，并测得桔霉素分泌量降低，上调未知基因号为GME7426的PKS的表达，对其它7个PKSs调控作用没有确定的趋势；代谢组学分析表明DHC显著扰动红曲菌代谢稳态和能量的变化。采用基因敲除技术成功敲除了Mrhda1，Mrhos2和Mrsir2三个预测的组蛋白去乙酰化酶编码基因，real-time RT- PCR分析表明不同组蛋白去乙酰化酶缺失菌株对不同PKS的转录活性影响不同，相应次级代谢产物产率也发生了不同变化。转录组数据表明Mrhda1，Mrhos2和Mrsir2不仅调节红曲菌次级代谢转录，也参与了包括初级代谢、应激、细胞发育等多个生物学过程。与原始菌株相比，HPLC分析结果显示ΔMrhda1、ΔMrhos2和ΔMrsir2菌株均能不同程度增加红曲菌次级代谢产物的种类。研究结果为深入探讨组蛋白去乙酰化酶介导的组蛋白乙酰化修饰对红曲菌不同次级代谢产物调控差异的机制、次级代谢与初级代谢之间的关系等生物学过程提供了重要基础。