Apicidin F: Characterization and Genetic Manipulation of a New Secondary Metabolite Gene Cluster in the Rice Pathogen Fusarium fujikuroi
Apicidin F: Characterization and Genetic Manipulation of a New Secondary Metabolite Gene Cluster in the Rice Pathogen Fusarium fujikuroi
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DOI：
10.1371/journal.pone.0103336
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发表时间：
2014-07-24
期刊：
影响因子：
3.7
通讯作者：
Tudzynski, Bettina
中科院分区：
文献类型：
--
作者：
Niehaus, Eva-Maria;Janevska, Slavica;Tudzynski, Bettina
真菌F.藤仓以生产引起水稻“恶苗病”的赤霉素而闻名。除了这些植物激素，它还能产生其他次级代谢产物（SM），如色素和真菌毒素。基因组测序共发现了45个潜在的SM基因簇，其中大部分是隐蔽的和沉默的。在这项研究中，我们的特点是一个新的非核糖体肽合成酶（NRPS）基因簇，负责生产环四肽apicidin F（APF）。这种新的SM与已知的组蛋白去乙酰化酶抑制剂apicidin具有结构相似性。为了深入了解生物合成途径，删除了11个簇基因中的大多数，并通过HPLC-DAD和HPLC-HRMS分析突变体产生APF或新衍生物的能力。通过HPLC-HRMS和NMR分析进行结构鉴定。我们鉴定了两个新的APF衍生物，命名为apicidin J和K。此外，我们研究了APF生物合成的调节，并表明簇基因在高氮和酸性pH条件下以依赖于氮调节剂AreB和pH调节剂PacC的方式表达。此外，过度表达的非典型途径特异性转录因子（TF）编码基因APF 2导致升高的表达下诱导，甚至抑制条件下的簇基因和显着增加的产品产量。生物信息学分析允许在APF基因的启动子中鉴定推定的Apf 2 DNA结合（“Api-box”）基序。该序列基序中的点突变导致APF产生急剧减少，表明该基序对于激活簇基因是必需的。最后，我们提供了一个模型的APF生物合成途径的基础上，在文化的缺失突变体的衍生物的化学鉴定。
The fungus F. fujikuroi is well known for its production of gibberellins causing the 'bakanae' disease of rice. Besides these plant hormones, it is able to produce other secondary metabolites (SMs), such as pigments and mycotoxins. Genome sequencing revealed altogether 45 potential SM gene clusters, most of which are cryptic and silent. In this study we characterize a new non-ribosomal peptide synthetase (NRPS) gene cluster that is responsible for the production of the cyclic tetrapeptide apicidin F (APF). This new SM has structural similarities to the known histone deacetylase inhibitor apicidin. To gain insight into the biosynthetic pathway, most of the 11 cluster genes were deleted, and the mutants were analyzed by HPLC-DAD and HPLC-HRMS for their ability to produce APF or new derivatives. Structure elucidation was carried out be HPLC-HRMS and NMR analysis. We identified two new derivatives of APF named apicidin J and K. Furthermore, we studied the regulation of APF biosynthesis and showed that the cluster genes are expressed under conditions of high nitrogen and acidic pH in a manner dependent on the nitrogen regulator AreB, and the pH regulator PacC. In addition, over-expression of the atypical pathway-specific transcription factor (TF)-encoding gene APF2 led to elevated expression of the cluster genes under inducing and even repressing conditions and to significantly increased product yields. Bioinformatic analyses allowed the identification of a putative Apf2 DNA-binding ("Api-box'') motif in the promoters of the APF genes. Point mutations in this sequence motif caused a drastic decrease of APF production indicating that this motif is essential for activating the cluster genes. Finally, we provide a model of the APF biosynthetic pathway based on chemical identification of derivatives in the cultures of deletion mutants.