紅麴菌轉錄因子MokH對DNA鍵結之分析及探討不同紅麴菌種之膽固醇抑制劑基因群

Abstract

Monacolin K是紅麴菌中由polyketide synthases (PKS)生成的二級代謝產物，並與土黴菌所產生的Lovastatin具相同結構。Monacolin K可藉由競爭型抑制來抑制膽固醇生合成中hydroxymethyl glutaryl-coenzyme A (HMG-CoA)與速率決定酵素HMGCA reductase的作用，進而抑制膽固醇合成。MokH是Zn(II)2Cys6型蛋白轉錄因子，此轉錄因子的DNA結合序列尚未被研究。為了要辨別出在Monacolin K基因群中MokH的結合序列，本研究將mokH基因構築於pET101/D-TOPO質體中，於Escherichia coli BL21 StarTM (DE3)中大量表現蛋白。將純化後的MokH利用電泳遷移率變動分析(electrophoretic mobility shift assay, EMSA)來鑑別其可能結合區域，並進一步藉由足跡法(footprinting)來分辨MokH的結合序列。EMSA的結果顯示在膽固醇抑制劑基因mokA、mokB、mokC、mokD及mokE的上游皆有MokH的結合區域。足跡法的分析結果則顯示出mokA上游146到176及209到235鹼基具有MokH的結合位。這些結果顯示出MokH會結合到特定的區域並且可能具有多個結合位。為了進一步探討不同紅麴菌種之膽固醇抑制劑基因群的分布，以南方墨點法分析後發現只有M. pilosus、M. ruber及M. barkeri具有完整的膽固醇抑制劑基因群。而在定序及分析過MokH、兩個polyketide synthase MokA及MokB的ketoreductase (KR) 結構域之胺基酸序列後顯示出只有M. ruber BCRC33314及BCRC33323在MokH的序列中有相異的胺基酸。在進一步利用MokH之胺基酸序列進行親緣關係分析顯示出，M. pilosus及M. ruber BCRC31523、BCRC31533、BCRC31534和BCRC33314被分在同一演化支上，而M. barkeri則與M. ruber BCRC31535及BCRC33323被分在另一演化支，這樣的結果初步推測可是因為生長環境所造成演化上的分歧。

Monacolin K, a secondary metabolite synthesized by polyketide synthases (PKS) in Monascus pilosus, has the same structure of Lovastatin produced by Aspergillus terreus. It can inhibit the synthesis of cholesterol by competitive inhibition of monacolin K and HMG-CoA with HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. mokA and mokB genes have been identified as polyketide synthases; also, the mokH gene has been known as the transcription factor. These genes are critical for the monacolin K synthesis. The transcription factor mokH of Monascus pilosus was also examined. The electrophoretic mobility shift assay (EMSA) and footprinting were carried out to analyze the MokH binding regions. The results of EMSA indicated that the MokH binding regions were present at the upstream of mokA, mokB, mokC, mokD, and mokE. The MokH binding sites at the 146 to 176 bp and 209 to 235 bp upstream of mokA were identified by footprinting analysis. These results indicated that the transcription factor encoded by the mokH gene can bind to the specific regions and contained more than one binding site. In order to further identify the monacolin K biosynthesis related gene among 18 different Monascus strains, the Southern blot was brought up to analyze the existence of related genes. Results of the Southern blot indicated that 9 out of 18 Monascus strains lacked the genes related to monacolin K biosynthesis. Also, real-time polymerase chain reaction was carried out to analyze the expression of those genes. To further investigate the diversity of mokA, mokB and mokH, polymerase chain reaction was used to amplify specific regions. The result of the sequenced fragment indicated that only M. ruber BCRC33314 and BCRC33323 showed difference in MokH.