標題: 紅麴菌膽固醇抑制劑 (Monacolin K)、橘黴素及轉位子相關基因之研究
Characterization of Monacolin K, Citrinin, and Non-LTR Retrotransposon Related Genes in Monascus pilosus
作者: 陳煜沛
Yi-Pei Chen
曾慶平
袁國芳
Ching-Ping Tseng
Gwo-Fang Yuan
生物科技學系
關鍵字: 紅麴;膽固醇抑制劑;橘黴素;轉位子;Monascus;Monacolin K;Citrinin;non-LTR retrotransposon
公開日期: 2008
摘要: 紅麴菌可生產各種不同的二次代謝物,包括膽固醇抑制劑(monacolin K),橘黴素(citrinin)及色素等。紅麴菌所生產的膽固醇抑制劑monacolin K與Aspergillus terreus產生的lovastatin為結構相同的物質。橘黴素則是一種腎肝毒素,亦是針對格蘭氏陽性菌的抗菌劑,橘黴素除了紅麴菌會產生之外,於Aspergillus及Penicillium中也有發現。根據前人的研究指出,lovastatin與citrinin基因群已分別在A. terreus及M. purpureus真菌中發現。因此為了解紅麴菌monacolin K基因群,我們從細菌人工染色體基因庫(Bacterial Artificial Chromosome library, BAC)篩選出含有monacolin K基因群之BAC clones,並完成定序及註解分析,monacolin K生合成基因群全長為42 kb包含9個基因,命名為mokA-mokI,其與已知lovastatin基因群具有54%的相似度。然而為確認所選殖基因確實參與monacolin K的生合成,本研究首先利用核酸□抑制劑(aurintricarboxylic acid)建立具方便及效率性之紅麴基因轉殖技術,藉由基因轉殖篩選出mokA基因的突變株,並證實此突變株不會產生monacolin K,進一步於紅麴菌中表現mokH基因,確認轉型株中含有兩套mokH基因,分析轉型株的monacolin K產量及RNA表現,發現其表現量皆高於野生株,因此結果明確說明所篩選的基因參與monacolin K的生合成。此外,選殖出mokA基因,並與4’-phosphopantetheine transferase (PPTase) 於Escherichia coli中共同表現,發現E. coli中大量表現出色胺酸(tryptophan)的前驅物anthranilic acid,此結果顯示mokA基因於E. coli中表現出的產物視同誘發物(elicitor),可調控誘導anthranilic acid在E. coli中表現。 再者,本研究於紅麴菌的細菌人工染色體基因庫定序中發現新的non-LTR逆轉位子,並命名為MRT,其序列長度約為5.5 kb,包含有兩個開放讀架(ORFs)。此兩個開放讀架與gag及pol基因相似,並且於pol相似基因的3’端具有很多的腺嘌呤。第一個開放讀架為517個氨基酸的蛋白質,包含有多量半胱氨酸的鋅指區域。第二個開放讀架為1181個氨基酸的蛋白質,包含有脫嘌呤/脫嘧啶核酸內切□、反轉錄□、核糖核酸□H及CCHC區域。根據MRT non-LTR逆轉位子的氨基酸序列所建立的親緣關係,可將其歸屬於Tad1群叢。南方雜交法則進一步發現只有四種紅麴菌M. pilosus,M. ruber,M. sanguineus與M. barkeri具有MRT non-LTR逆轉位子。除此之外,利用β-tubulin基因所建立的親緣關係可歸群出MRT non-LTR逆轉位子之存在與否。 紅麴菌廣泛應用於食品發酵及保健藥物中,因此如何分辨紅麴菌中是否含有橘黴素(citrinin)則相對性的重要,本研究分析了18株不同的紅麴菌種,發現只有M. purpureus與M. kaoliang具橘黴素的基因群,並且也只有此兩個菌種能偵測到橘黴素的表現量,相反的其他物種包括M. pilosus,M. ruber,M. barkeri,M. floridanus,M. lunisporas及M. pallens皆缺乏橘黴素基因群,此研究結果顯示橘黴素只有紅麴菌M. purpureus與M. kaoliang會產生,另外,利用β-tubulin基因所建立的親緣關係則亦可歸群出橘黴素基因群之存在與否。
Monascus species can produce various secondary metabolites with polyketide structures as monacolin K, citrinin, and pigments. Monacolin K which inhibits cholesterol synthesis is also known as lovastatin isolated from Aspergillus terreus. Citrinin, the hepato-nephrotoxic agent and antibiotic against gram-positive bacteria, has been identified in Aspergillus and Penicillium spp. In previous studies, lovastatin and citrinin biosynthetic gene clusters have been characterized in Aspergillus terreus and Monascus purpureus respectively. To explore the monacolin K biosynthetic gene cluster in M. pilosus BCRC38072 producing monacolin K, construction of a BAC library was carried out. The putative monacolin K biosynthetic gene cluster was found within a 42-kb region in the mps01 clone. 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 A. terreus, were assumed to be involved in monacolin K biosynthesis. In order to characterize the putative monacolin K biosynthetic gene cluste, the valid and convenient gene transformation in Monascus pilosus BCRC38072 was established using nuclease inhibitor, aurintricarboxylic acid (ATA). A gene disruption constructed to replace the central part of mokA, a polyketide synthase gene, in wild type M. pilosus BCRC38072 with a hygromycin B resistance gene through homologous recombination resulted in a mokA-disrupted strain. The disruptant did not produce monacolin K, indicating that mokA encoded the PKS responsible for monacolin K biosynthesis in M. pilosus BCRC38072. Moreover, the transformant containing two copies 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 mokA and mokH involved in the monacolin K biosynthesis. In addition, the mokA gene and sfp gene were coexpressed in Escherichia coli. The sfp gene obtained from Bacillus subtilis is a phosphopantetheinyl transferase required to convert the expressed apo-PKS to its holo form. Interestingly, anthranilic acid which is the precursor of tryptophan was found. This result revealed that that expression product of polyketide synthase (mokA) in E. coli was responsible for the elicitor to regulate anthranilate flux through the anthranilate synthase gene specific to the tryptophan biosynthetic pathway. During the BAC (mps01) sequencing of M. pilosus BCRC38072, a new non-LTR retrotransposon, named MRT, was discovered. The entire nucleotide sequence of the MRT element was 5.5-kb long, including two open reading frames. These two ORFs showed homologies to gag-like and pol-like gene products, and an A-rich sequence at the 3’ end of pol-like gene. ORF1 encoded a protein of 517 amino acids and contained a cysteine-rich zinc finger motif. ORF2 encoded a protein of 1181 amino acids and contained apurinic/apyrimidinic endonuclease (APE), reverse transcriptase (RT), RNaseH domains, and a CCHC motif. The phylogenetic analyses demonstrated that the MRT element was classified into the Tad1 clade. The results of Southern hybridizations showed that MRT elements were distributed within M. pilosus, M. ruber, M. sanguineus, and M. barkeri. Also, the species of Monascus can be grouped by the presence or absence of MRT elements in the hybridization pattern according to phylogenetic subgroups established with the partial β-tubulin gene. Monascus species has been widely used in food fermentation, and has shown a highly promising application in medicine development. Therefore, it is important to identify the non-citrinin producing Monascus strains. Eighteen Monascus strains were investigated for the distribution of mycotoxin citrinin biosynthesis genes. These results showed that the genotype of citrinin genes was observed only in M. purpureus and M. kaoliang, while the phenotype of citrinin productivity was detected in M. purpureus and M. kaoliang. In contrast, the PCR and Southern blot results suggested that citrinin biosythesis genes were absent or significantly different in M. pilosus, M. ruber, M. barkeri, M. floridanus, M. lunisporas, and M. pallens. These results clearly indicated that the highly conserved citrinin gene cluster in M. purpureus and M. kaoliang carried out the citrinin biosynthesis. In addition, according to the phylogenetic subgroups established with the β-tubulin gene, the citrinin gene cluster can respectively group the species of Monascus.
URI: http://140.113.39.130/cdrfb3/record/nctu/#GT009028805
http://hdl.handle.net/11536/38358
Appears in Collections:Thesis


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