紅麴菌膽固醇抑制劑(Monacolin K)之轉錄調控基因(MokH)研究

Abstract

過去我們的研究發現紅麴菌的膽固醇抑制劑MonacolinK生合成基因群全長約為42kb，包含有9個基因，命名為mokA—mokI，其與已知的土黴菌(Aspergillusterreus)具有54%以上的相似度，其中mokH基因為一轉錄因子，含有典型的胱氨酸鋅指區域，如同其他真菌調控因子AflR，Ctb8，Gal4與Lac9，當mokH基因於紅麴菌中過量表現，其轉型株的MonacolinK產量及相關合成基因的RNA表現皆高於野生株，此結果說明mokH基因參與MonacolinK之生合成，推測其會與啟動子區域的DNA鍵結進而調控下游MonacolinK基因表現(Chen et al.,2010)。目前我們已在大腸桿菌中成功表現出MokH蛋白（見圖五)，初步利用EMSA分析亦證實MokH蛋白會與MonacolinK基因(mokA)啟動子區域進行鍵結(見圖六）。

本研究計畫為兩年期計畫，第一年將鑑別出MokH轉錄因子對Monacolin K共九個基因的鍵結保留區域，之後我們會將此鍵結序列針對紅麴菌全基因體啟動子區域進行比對，將更了解紅麴基因的調控機制。第二年利用次世代定序方式(Next generation sequencing - targetresequencing)分析不同紅麴菌的膽固醇抑制劑生合成基因，鑑別在不同紅麴菌種是否也包含有相同鍵結序列，進一步將MokH異源表現於土黴菌(A. terreus)及不同紅麴菌種，預期結果將對紅麴菌在生產膽固醇抑制劑的調控機制有更精確掌握，同時可獲得高產量膽固醇抑制劑轉型株，對後續紅麴菌膽固醇抑制劑之基因調控機制的研究，可提供更多資訊與了解。

Previously, we have demonstrated that Monascus pilosus contains the monacolinK biosynthetic gene cluster within a 42-kb region. The deduced amino acid sequences encoded by the nine genes designated as mokA-mokI, which share over 54% similarity with the lovastatin biosynthetic gene cluster in Aspergillus terreus, were involved in monacolin K biosynthesis. The mokH gene encoding Zn(II)2Cys6 binuclear DNA binding protein, similar to AflR, Ctb8, Gal4 and Lac9 in fungi, is assumed to be an activator for monacolin K production. The transformant containing overexpression of the mokH gene-encoded transcription factor showed higher production of monacolin K than wild type strain. Real-time RT-PCR analysis also demonstrated that the transcripts of monacolin K biosynthetic genes in the transformant were higher than those in wild type strain. These results suggested that mokH involved in the monacolin K biosynthesis (J. Agric. Food Chem. 2010.). Furthermore, we have successfully overexpressed MokH protein in E. coli (Fig. 5). The preliminary data of MokH-DNA binding were also confirmed by Electrophoretic Mobility Shift Assay (EMSA) (Fig. 6).

This project serves as two years. During the first year, Electrophoretic Mobility Shift Assay (EMSA), Isothermal Titration Calorimetry(iTC) and Footprint will be carried out to explore the binding sequence of monacolin K genes. The binding sequencing will be further analyzed within the whole genomic sequence of M. pilosus. During the second year, the monacolin K biosynthetic genes will be analyzed in different Monascus species by target resequencing of Next generation sequencing (NGS). The consensus binding sites will be explored in M. spp. In addition, MokH will be over-expressed in Aspergillus terreus and M. spp. to examine monacolin K production. The transformants with high monacolin K production will be obtained. These results for the monacolin K biosynthesis can provide the information of transcription factor regulation in Monascus and increase the yields of cholesterol synthesis inhibitor.