基因探勘並解析麴黴菌屬真菌PSU-RSPG185之炔類asperpentyn的上游生合成途徑

Abstract

乙炔天然物含有炔烴官能基，根據報導其具有多種生物活性。炔烴可用於標記天然物，在藥物開發過程中是一種重要的工具。儘管炔烴官能基很重要，但目前對於其生合成途徑卻了解甚少，尤其有關炔環己內酯的中間炔生合成機制仍為未知。 Aspergillus sp. PSU-RSPG185為麴黴菌屬之土壤真菌，可生產一系列炔環己內酯代謝物，其中包含asperpentyn。為了研究中間炔代謝物asperpentyn的生合成，最初我們根據結構推測異戊烯轉移酶可能參與，所以利用此酶序列做基因組探勘，找到了六個可能的生合成基因群，並且發現這些基因群中都具有與調控生合成基因相關的轉錄因子。因此，為了活化生合成基因表現並誘導相關二次代謝物的產生，我們在宿主過度表達轉錄因子，將過度表達轉錄因子的DNA載體轉殖入真菌的原生質體，其中三組顯示出正確的菌落篩選。另外，我們將六個基因群中最有可能負責asperpentyn生合成的scaffold322分別在小巢狀麴菌和米麴菌進行表達，成功地在小巢狀麴菌轉殖入可能參與上游生合成的兩個基因，以及將整個scaffold322生合成基因群轉殖在小巢狀麴菌；在米麴菌則成功轉殖五個生合成基因。然而，利用液相層析進行代謝物分析卻沒有偵測到可能的中間產物。根據此結果，我們推測asperpentyn的生合成可能由其他的生合成基因群負責。 我們再做了一次更深入的基因探勘，比較另外兩個可生產asperpentyn的真菌Eutypa lata及Pestalotiopsis fici，發現aty生合成基因群編碼在這三個真菌的基因組中。根據餵養實驗以及酶體外試驗結果，我們確定了aty基因群中的苯丙胺酸氨裂合酶（AtyH）將苯丙胺酸催化成反式肉桂酸，作為對羥基苯甲酸的前驅物，接下來對羥基苯甲酸的異戊二烯化則是意外地由聚異戊烯轉移酶（AtyB）催化，再由细胞色素P450單加氧酶（AtyI）進行催化產生可能含有炔官能基的產物（m/z 201），此外我們發現水楊酸羥化酶（AtyG）會消耗m/z 201產物，未來我們將會分離m/z 201產物以鑑定其化學結構。總結來說，我們確定了asperpentyn上游生合成途徑及其參與的酶，而未來也將會繼續探索關鍵的炔合成作用機制及解析下游生合成途徑。此研究也為往後研究真菌中間炔生合成機制奠定了基礎。

Acetylenic natural products contain alkyne functionality and are reported to show a variety of bioactivities. Alkyne-tagging of natural products is an important strategy for pharmaceutical development. Despite the importance of the alkyne moiety, the biosynthetic routes to alkynes are poorly understood. To date, the biosynthetic machinery of internal alkyne formation in acetylenic cyclohexanoid remains unknown. Aspergillus sp. PSU-RSPG185 is a soil fungus that produces a series of acetylenic cyclohexanoids including asperpentyn. To investigate the biosynthesis of asperpentyn, we initially performed genome mining by searching prenyltransferase to scan for the biosynthetic gene cluster candidates, based on the structure of asperpentyn. Six gene cluster candidates were obtained. Interestingly, all of them encode transcriptional factors (TFs) which may regulate the expression of biosynthetic genes. Therefore, we performed TF overexpression to activate gene expression and to induce the production of corresponding secondary metabolites. Protoplasts preparation of this strain was developed and six TF overexpression cassettes were transformed to Aspergillus sp. PSU-RSPG185, respectively. Three of them showed correct selectivity based on medium selection. For candidate scaffold322, we succeeded in transforming two genes in Aspergillus nidulans, five genes into Aspergillu oryzae or even whole scaffold322 in A.nidulans based on PCR screening. However, no intermediates of asperpentyn were detected. Considering the possibilities of other gene clusters as candidates involved in asperpentyn biosynthesis, we did more thorough genome mining by comparing the genome with another two asperpentyn-producing strains, Eutypa lata and Pestalotiopsis fici. One gene cluster candidate aty was found and encoded in the genome of each strain. Here we elucidated the upstream pathway of asperpentyn. Based on the result of in vivo feeding experiments and in vitro test, we characterized a phenylalanine ammonia lyase (AtyH) that converts L-phenylalanine to trans-cinnamic acid, as the precursor of 4-hydroxybenzoic acid (4-HBA). The following prenylation on 4-HBA was catalyzed by unexpected polyprenyl transferase (AtyB). One cytochrome P450 monooxygenase (AtyI) was identified to convert prenyl 4-HBA into a product (m/z 201) which may correspond to eutypinic acid and possess an alkyne moiety. Besides, we found that salicylate hydroxylase (AtyG) might consume the product m/z201 in yeast feeding experiment of co-expressing AtyI together with AtyG. The product m/z 201 will be isolated and its structure will be characterized. In summary, we have characterized the genes involved in the upstream biosynthetic pathway of asperpentyn. The property and mechanism of the key enzymes that install the alkyne functionality and the other enzymes involved in the downstream pathway still need to be characterized. Our study paved the foundation for the study of the biosynthetic machinery of internal alkyne in fungi.