紅麴菌中 Monacolin K 生合成相關基因 cDNA 之選殖與分析

Abstract

Polyketides 為微生物或真菌所產生結構多樣的天然化合物，它們往往具有許多生理活性，例如抗生素，毒素，香味及色素分子，在許多的植物中亦可發現。Polyketides 是由 polyketide synthases (PKSs) 所合成，它在演化上和 fatty acid synthase (FAS) 類似，但是催化情況更加複雜，才能合成結構複雜的二級代謝物。monacolin K (lovastatin) 為一種真菌合成的 polyketide 類化合物，它是膽固醇合成的關鍵酵素 HMG-CoA reductase 的抑制劑。最初在 Aspergillus terreus 中發現，後來在中國傳統醱酵菌種 Monascus spp. 中亦可發現。A. terreus 中的 lovastatin 合成酵素的基因序列已被發現 (lovB, lovF)，分別可轉譯出 lovastatin nonaketide synthase (LNKS) 及 lovastatin diketide synthase (LDKS)，此二蛋白質合成 lovastatin 之主體結構，另外需要其他酵素之協助才能合成完整的 lovastatin。先前的研究建立了紅麴染色體基因組庫 (黃, 2004)，並且篩選出可能含有 LNKS 或 LDKS 基因之正反應株 pDASHB3。它的全長約有 17kb，以限制酶作用為五個片段後個別分析，並利用不同的引子對可將其完整序列拼湊出。其中內含一可能之 PKS 基因 (putPKS)，一個可能的 ORF，一個蛋白質功能可能為 cytochrome P450 monooxygenase 之 mLovA 基因及功能可能為 hydoxylase 活性之 mlovX。根據 pDASHB3 已知基因序列設計專一性引子針對紅麴菌進行 RT-PCR 可放大出部分 putPKS 序列及輔助基因 mlovA。本論文另外測定了紅麴菌液態培養之 monacolin K 生合成曲線，並參考建構了可利用酵母菌雙雜合實驗研究蛋白質交互作用的紅麴菌 cDNA 庫。我們亦建構了輔助蛋白質 MLovA 的蛋白質表現系統，並且成功將原態 MLovA 及突變 MLovA 蛋白質進行初步表現。

Polyketides are structure-diversified natural products, which are produced by microorganisms, fungi, and also by some plants. They display a wealth of physiologically activites, including antibiotic, toxin, aroma and pigments. Polyketides are produced by polyketide synthases (PKSs) which are evolutionarily related to fatty acid synthase (FAS), but much more sophicated to synthesize complicated secondary metabolites. monacolin K (lovastatin), a polyketide from fungus, is an inhibitor of HMG-CoA reductase, which is a key enzyme in the biosynthesis pathway of cholesterol. They are found in Aspergillus terreus, and also found in fungi Monascus spp., which is applied to Chinese traditional fermentation. At least two lovastatin biosynthesis genes from A. terreus have been cloned (lovB, lovF), and they encode lovastatin nonaketide synthase (LNKS) and lovastatin diketide synthase (LDKS). LNKS and LDKS together are responsible for the production of the core structure of lovastatin, while other enzymes are required to produce the full-functional lovastatin. In previous study we constructed the Monascus genomic library (Huang, 2004), and isolated a positive clone that contained putative LNKS or LDKS. The 17kb insert in the positive clone, pDASHB3, was digested to 5 fragments and subcloned. The gene arrangements were determined by using different primer sets. The genes contained in this clone were predicted to have a putative PKS gene, an unclassified ORF, a putative cytochrome P450 monooxygenase MLovA, and a putative hydroxylase gene mlovX. According to the genomic sequences ontained from pDASHB3, we designed several specific primers to amplify the partial putative Moncolin K biosynthesis gene putPKS and an auxiliary protein mlovA from Monascus by RT-PCR. In addition, we measured the production curve of monacolin K in Monascus broth culture, and construced a Monascus cDNA library which can be used to fish interacting proteins of PKSs using yeast-two hybrid techniques. We also construced the protein expression system of a putative auxiliary protein MLovA, and heterologous protein expression of MLovA was performed.