途径特异性转录因子MPPR2调控红曲色素组分转化的分子机制研究

Monascus pigments (MPs), an important secondary metabolite of Monascus, are widely used in food industry as natural colorants. MPs are mainly composed of three kinds of pigments, red, orange and yellow pigments, and transformations between them are concerned by many researchers. Nowadays more various color MPs are needed as development of food industry. Gene mppR2 which encoded Zn(II)2Cys6 zinc cluster transcription factor (TF) in MPs gene cluster, showed significantly differentially expressions when conversions of MPs components were induced by blue light in previous studies. Additionally, several specific DNA binding sites of Zn(II)2Cys6 TF were found in upstream sequences of the oxidoreductase encoded gene mppE involved in MPs components transformations. Therefore, we speculated that MPPR2 might be a pathway specific TF that regulated MPs components transformations induced by blue light. The function of gene mppR2 is intended to analyze by gene knockout and overexpression technology. DNA binding sites of MPPR2 in MPs gene cluster will be identified by EMSA and yeast one-hybrid assay. Co-regulator protein interacted with MPPR2 will be screened by protein interaction assay. Finally, the regulatory mechanisms of MPs transformation by a pathway specific regulator MPPR2 would be clarified, which could lay a foundation for various color MPs production.

红曲色素（MPs）为天然食品着色剂，是红曲霉的重要次级代谢产物，主要由红、橙、黄三类色素组成，三者间的生物转化是目前研究的热点，生产不同色调的MPs已成为适应市场需求的发展趋势。申请人前期研究发现，在红曲霉响应蓝光调控MPs组分转化时，位于MPs基因簇内的Zn(II)2Cys6锌簇蛋白转录因子（TF）基因mppR2发生显著性差异表达；同时，参与MPs组分转化的氧化还原酶基因mppE上游有多个Zn(II)2Cys6型TF专一性结合位点。所以，初步推测MPPR2是蓝光诱导下调控MPs组分转化的途径特异性TF。本项目拟借助基因敲除和过表达技术，分析mppR2基因功能；其次，利用EMSA实验结合酵母单杂交系统，确定MPPR2在MPs基因簇中的DNA结合位点。然后，通过蛋白质互作实验，查找与MPPR2密切结合的共调节因子，解析其调控MPs组分转化的分子机制，为调控生产不同色调的MPs奠定理论基础。

红曲色素（MPs）是红曲霉（Monascus spp.）主要的次级代谢产物，作为天然食品着色剂已有上千年的食用历史。MPs的生物合成途径和调控机制一直是研究的热点。前期研究发现，MPPR2可能是MPs合成途径的特异性转录调控因子（TF），但其具体功能和调控机制尚不明确。本项目采用基因敲除和过表达技术构建了mppR2基因突变株，比较了突变株与野生型在菌落形态、生长发育及次级代谢产物合成方面的差异，明确了mppR2基因的功能；借助转录组学技术分析了敲除菌株和野生型的差异表达基因，初步说明了MPPR2调控色素合成的机制。结果表明，mppR2基因编码了813个氨基酸，属于Zn(II)2Cys6型转录因子。敲除mppR2基因使紫色红曲霉M8的菌落颜色变红，分生孢子和子囊孢子数减少，生物量降低，但MPs的产量显著提高，尤其是红色素组分，固态培养条件下红色素产量是野生型的3.3倍，同时影响了桔霉素的合成。过表达菌株的对应指标与敲除菌株正好相反。转录组测序结果表明，与M8相比mppR2基因敲除菌株上调了糖酵解途径和MPs合成途径的基因表达水平，下调了莫那可林K和桔霉素合成途径基因的表达水平，促使更多的MPs合成前体物（乙酰辅酶A和丙二酰辅酶A）通过代谢流进入了MPs合成途径，从而产生了更多的MPs。由此表明，MPPR2是红曲色素合成途径中的负调控转录因子。另外，敲除mppR2使部分全局性TF和调控孢子发育相关基因的表达水平下调，说明敲除mppR2一定程度上延缓了细胞的初级代谢，进一步促使了次级代谢发生。研究结果为调控MPs的生物合成提供了理论参考，为提高MPs的产量提供了新策略。