Identification and characterization of a novel diterpene gene cluster in Aspergillus nidulans.
Identification and characterization of a novel diterpene gene cluster in Aspergillus nidulans.
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DOI：
10.1371/journal.pone.0035450
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Nakari-Setälä T
中科院分区：
文献类型：
--
作者：
Bromann K;Toivari M;Viljanen K;Vuoristo A;Ruohonen L;Nakari-Setälä T
由于真菌次生代谢产物具有药用和毒性特性，它是医疗有用化合物的丰富来源。真菌基因组测序揭示了众多次生代谢物基因簇，但由于这些基因簇中的基因在正常实验室条件下通常不表达，许多生物合成途径的产物尚不明确。因此，为了发现新的代谢产物，找到诱导这些原本不表达的生物合成基因簇中基因表达的方法至关重要。我们通过基因组挖掘预测一个生物合成基因簇，在构巢曲霉中发现了一种新的次生代谢产物。一种锌（II）2半胱氨酸6型转录因子PbcR被鉴定出来，并且其作为预测基因簇的途径特异性激活因子的作用得到了证实。pbcR的过表达上调了所鉴定基因簇中七个基因的转录，并导致一种二萜化合物的产生，通过气相色谱/质谱法将其鉴定为对映-海松-8(14),15 -二烯。在过表达pbcR的菌株中还观察到形态变化。通过过表达其假定的锌（II）2半胱氨酸6型转录因子激活一个隐性基因簇，从而在构巢曲霉中发现了一种新的次生代谢产物。实时定量聚合酶链反应和DNA芯片分析使我们能够预测生物合成基因簇的边界。此外，我们鉴定出一个新的真菌海松二烯环化酶基因以及编码3 -羟基- 3 -甲基-戊二酰辅酶A（HMG - CoA）还原酶和牻牛儿基牻牛儿基焦磷酸（GGPP）合酶的基因。这些基因以前都没有被认为与构巢曲霉中萜类的生物合成有关。这些结果确定了第一个构巢曲霉二萜基因簇，并提出了对映-海松-8(14),15 -二烯的生物合成途径。
Fungal secondary metabolites are a rich source of medically useful compounds due to their pharmaceutical and toxic properties. Sequencing of fungal genomes has revealed numerous secondary metabolite gene clusters, yet products of many of these biosynthetic pathways are unknown since the expression of the clustered genes usually remains silent in normal laboratory conditions. Therefore, to discover new metabolites, it is important to find ways to induce the expression of genes in these otherwise silent biosynthetic clusters. We discovered a novel secondary metabolite in Aspergillus nidulans by predicting a biosynthetic gene cluster with genomic mining. A Zn(II)2Cys6–type transcription factor, PbcR, was identified, and its role as a pathway-specific activator for the predicted gene cluster was demonstrated. Overexpression of pbcR upregulated the transcription of seven genes in the identified cluster and led to the production of a diterpene compound, which was characterized with GC/MS as ent-pimara-8(14),15-diene. A change in morphology was also observed in the strains overexpressing pbcR. The activation of a cryptic gene cluster by overexpression of its putative Zn(II)2Cys6–type transcription factor led to discovery of a novel secondary metabolite in Aspergillus nidulans. Quantitative real-time PCR and DNA array analysis allowed us to predict the borders of the biosynthetic gene cluster. Furthermore, we identified a novel fungal pimaradiene cyclase gene as well as genes encoding 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase and a geranylgeranyl pyrophosphate (GGPP) synthase. None of these genes have been previously implicated in the biosynthesis of terpenes in Aspergillus nidulans. These results identify the first Aspergillus nidulans diterpene gene cluster and suggest a biosynthetic pathway for ent-pimara-8(14),15-diene.
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