真菌的非核糖體胜肽合成酶(nonribosomal peptide synthetases, NRPSs)屬於多功能蛋白質複合體，其二次代謝產物廣泛被應用在醫療製藥界，這些藥用價值歸因於二次代謝產物特殊功能，例如著名的紅麴菌素K(monacolin K)是由紅麴菌(Monascus pilosus)之聚酮合成酶(polyketide synthases, PKSs)負責生合成，紅麴菌素K是一種膽固醇合成抑制劑，能調節人體內的膽固醇。另外有研究顯示，麴菌所產生的黃酮酚(flavonoid)可增加天然抗氧化能力。已知紅麴菌(Monascus)屬於子囊菌門(Ascomycota)，其中的Monascus ruber及Monascus purpureus已完成基因組定序，目前依紅麴菌進行基因探勘的結果顯示，所鑑定出的二次代謝產物並不多，意味著許多生合成基因的產物有待進一步鑑定。小巢狀麴菌(Aspergillus nidulans)屬於子囊菌門之絲狀真菌，因其生長快速且易於培養、架構及遺傳背景簡單且易於分析等優勢，使其成為真核細胞生物學上重要的研究工具。本計畫利用小巢狀麴菌為異源表現(heterologous expression)系統，表現M. ruber的非核糖體胜肽合成酶基因(包含PID 398062, PID3 83194, PID 391237, PID 500739, PID 441704 and PID 499644等)，並分別偵測其蛋白所負責的生合成產物，本研究結果顯示，在透過可調節的啟動子alcA(P)進行轉錄活化後，PID 500739非核糖體胜肽合成酶的異源表達株產生化合物1 (m/z 314 MH) 與化合物2 (m/z 466 MH)，此結果顯示透過異源表達系統確實可針對紅麴菌中二次代謝產物之基因體功能進行研究與解析，除有利於進一步鑑定其結構並深入探討其生合成叢集之產物外，對真菌在研製保健與醫療相關應用上也提供重要資訊。 Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes that produce a diverse group of natural products. The efficacy as medicines is due to the biological activities of secondary metabolites (SMs). Monacolin K, an antihypercholestrolemia agent, is a secondary metabolite synthesized by PKSs from Monascus. Recent reports showed that the enhanced contents of flavonoids in the Monascus-fermented rice bran (MRB) may contribute to the antioxidant activity in MRB. Monascus is species of mold and belongs to the phylum Ascomycota. The genomes of two species of Monascus, Monascus ruber and Monascus purpureus, which are commonly used for Monascus-fermented rice (MFR), have been sequenced. The number of known secondary metabolites (SM) produced by M. ruber and M. purpureus is much less than the SM genes that have been identified. Our preliminary results in heterologous overexpression system revealed that Monascus polyketide synthase (PKS) genes can be expressed in A. nidulans and produced analyzable products. To further confirm if this is the case in NRPS, we further use A. nidulans as a heterologous overexpress host to express the NRPS genes of M. ruber. Among them, we found that the overexpression of PID 500739 results in the production of compound 1 (m/z 314 MH) and 2 (m/z 466 MH) using liquid chromatography/mass spectroscopy (LC/MS) analysis. Our data suggested that the identification of the important secondary metabolites from M. ruber will provide a basis for the clues of NRPs biosynthetic pathways. This grant will help to unlock the metabolites from the M. ruber and provide useful development resources for healthcare and medical centers.
