目的 分离分析内脐蠕孢真菌(Drchslera catenaria)的次级聚酮类代谢产物,初步分析其非还原型Ⅰ型聚酮合成酶基因组成,为该菌株中大黄酚生物合成机制的阐明奠定理论基础.方法 应用Czapek Dox培养基发酵培养,酸化乙酸乙酯提取,硅胶柱层析及薄层制备等方法,对该菌株产生的聚酮类代谢产物进行分离纯化;利用紫外(UV),液相-质谱联用(LC-MS)和核磁共振谱(1H-NMR)鉴定化学结构;基于真菌聚酮合成酶保守序列设计引物,克隆与代谢产物合成相关的聚酮合成酶基因.结果与结论 从试验菌株菌丝体及发酵液中分离获得4个芳香聚酮类化合物,其结构分别为大黄酚(chrysophanol),长蠕孢素(helminthosporin),冰岛青霉素(islandicin),链蠕孢素(catenarin),其中后3个为首次从该真菌中得到.从基因组DNA中得到一段非还原性聚酮合成酶(PKS)基因序列(约5.3kb),与Pyrenophora tritici-repentis Pt-1C-BFP中黄色色素合成相关的基因片段有较高类似度,很有可能与内脐蠕孢真菌中大黄酚的生物合成有关;在距离该基因2.5kb处克隆到一段基因序列(0.79kb),与P.triticirepentis Pt-1C-BFP中β-酮酯酰基-ACP-还原酶(KR)基因完全相似,其很可能负责大黄素的6位羰基经还原形成大黄酚.%Objective To isolate and identify the secondary metabolites of Drechslera catenaria, preliminarily analyze non-reducing type I polyketide synthases (PKSs), which will provide theoretic basis for the biosynthetic mechanism. Methods The tested fungus was fermented in Czapek Dox broth; fermentation cultures including mycelia were homogenized and the pH of the suspension was adjusted to around 3.0, then extracted with ethyl acetate, isolated and purified with silica column chromatography and thin layer chromatography; compounds were identified using UV, LC-MS and 'H-NMR. Primer pairs were designed based on the conserved domain sequence of type I PKSs, and the PKS genes related to the biosynthesis of metabolites were cloned. Results and Conclusion Four metabolites and their chemical structures were the same as chrysophanol, helminthosporin, islandicin, catenarin, the latter three compounds had not been previously reported in this organism. A 5.3kb DNA fragment was obtained from genomic DNA of D. Catenaria, which is identical to a conidial yellow pigment biosynthesis PKS in Pyrenophora tritici-repentis Pt-lC-BFP, this is likely the PKS gene responsible for the biosynthesis of chrysophanol. Additionally, a 0.79kb DNA fragment, likely a keto-reductase encoding gene, was cloned at the position of 2.5kb downstream from the above PKS gene, as required for chrysophanol formation with the reduction at 6-carbonyl of emodin.
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