麴菌中二次代謝物生合成產物之鑑定與解析研究

Abstract

麴菌屬(Aspergillus)是產生具藥用二次代謝產物的重要來源。隨著越來越多的麴菌屬物種完成基因組定序，顯示負責二次代謝產物的生物合成基因數量，遠多於已被鑑定出的二次代謝產物的數目。這意指著許多生合成基因的化學產物仍有待進一步鑑定。因此，我們有興趣研究與解析這些生物合成基因所負責的化學產物，尤其是針對具有特殊蛋白結構區域的生物合成基因。 至今在小巢狀麴菌(Aspergillus nidulans)中，所有類非核醣體胜肽合成酶 (nonribosomal peptide synthetase-like; NRPS-like)的基因裡，目前唯一已被證實的是tdiA基因參與terrequinone A的生物合成反應，而此TdiA具有一特殊的硫酯酶(thioesterase; TE)結構區域，已知這樣的結構區域有助於產物的釋出。利用蛋白質保留區域資料庫分析得知，AN3396.4是個含有938個胺基酸且具有此特殊硫酯酶結構區域的類非核醣體胜肽合成酶。為鑑定此基因的化學產物，首先，我們利用可誘導性的乙醇脱氫酶(alcohol dehydrogenase; alcA)啟動子取代內生性的啟動子，實驗結果顯示，誘導AN3396.4基因的表現，可明顯促進microperfuranone的產量，因此我們將此基因命名為micA。進一步將micA基因放入黑麴菌(A. niger)中進行異源表現(heterologous expression)，更證實在單一啟動micA的表現下，即可產生microperfuranone。 此外，已知非還原性聚酮合成酶(nonreducing polyketide synthases; NR-PKSs)可藉由硫酯酶/Claisen型環化酶(thioesterase/Claisen-cyclase; TE/CLC)、硫酯酶(TE)以及還原酶(reductase; R)等不同結構區域的調控進而幫助產物的釋放。利用生物演化分析，我們發現第六族的非還原性聚酮合成酶，都具有一典型的硫酯酶結構區域。此族群中唯一例外的，是一個缺少硫酯酶結構區域的黑麴菌基因(e_gw1_19.204),藉由反轉錄聚合酶鏈鎖反應與核酸定序並結合異源表現等方法，我們發現此基因與緊鄰且具有還原酶結構區域的下游基因est_GWPlus_C_190476，其實合為一個完整基因，我們重新註解此新基因為dtbA。實驗結果顯示，當啟動dtbA可產生兩聚酮化合物2,4-dihydroxy-3,5,6-trimethylbenzaldehyde 與6-ethyl-2,4-dihydroxy-3,5- dimethylbenzaldehyde，若進一步將DtbA的還原酶結構區域與AusA (austinol生物合成; AN8383.4) 或PkbA (cichorine生物合成; AN6448.4)的硫酯酶結構區域進行置換，則可順利使得產物的官能基從醛基轉換成羧酸。此研究結果推論，合理的結構區域置換除了能有效地達到官能基的改造，且能藉此產生更具價值的化學物質或藥物。

The Aspergillus species are well-known producers of medicinally important secondary metabolites. As more and more Aspergillus species genomes were sequenced, it is clear that there are far more secondary metabolite biosynthetic genes than there are known secondary metabolites. This implies that these organisms can produce additional secondary metabolites, which have not yet been identified. Thus, we are interested in deciphering the chemical product of the putative biosynthesis genes, especially ones with non-canonical domain architectures. The only one nonribosomal peptide synthetase-like (NRPS-like) gene in A. nidulans with a known product is tdiA, containing a thioesterase (TE) domain, which is involved in terrequinone A biosynthesis. The AN3396.4, with 938 amino acid residues, is another NRPS-like potential candidate that possesses a TE domain which facilitates product release by conserved domain database searches. To identify the products of this gene, we replaced the native promoter with the inducible alcohol dehydrogenase (alcA) promoter. Our results demonstrated that induction of AN3396.4 led to the enhanced production of microperfuranone. We named the gene AN3396.4 micA for microperfuranone synthase. Furthermore, the heterologous expression of micA in A. niger confirmed that only one NRPS-like gene, micA, is necessary for the production of microperfuranone. In addition, release of the polyketide chain from a non-reducing polyketide synthase (NR-PKS) could be mediated by several different types of domains including□thioesterase/Claisen-cyclase (TE/CLC), TE, and reductase (R) domains. Through bioinformatic analyses of A. niger, we identified a putative TE-less NR-PKS encoded by e_gw1_19.204, belonging to a group VI NR-PKSs typically has a TE domain for their product releasing. Using reverse transcription polymerase chain reaction (RT-PCR), DNA sequencing, and heterologous expression approaches, we re-annotated the NR-PKS gene, e_gw1_19.204 and its downstream R domain gene, est_GWPlus_C_190476 as a single gene which we name dtbA. Our results demonstrated that DtbA protein produces two polyketides, 2,4-dihydroxy-3,5,6-trimethylbenzaldehyde and 2-ethyl-4,6-dihydroxy-3,5-dimethylbenzaldehyde. Generation of DtbA□R+TE chimeric PKSs by swapping the DtbA R domain with the AusA (austinol biosynthesis; AN8383.4) or PkbA (cichorine biosynthesis; AN6448.4) TE domain enabled the production of two metabolites with carboxylic acids replacing the corresponding aldehydes. These studies allow us to propose the biosynthetic pathways for secondary metabolites. Furthermore, our data indicated that rational domain swaps may provide a route for engineering the functionalization of valuable chemicals.