Endophytic fungi are promising producers of bioactive small molecules. Bioinformatic analysis of the genome of an endophytic fungus Penicillium dangeardii revealed 43 biosynthetic gene clusters, exhibited its strong ability to produce numbers of secondary metabolites. However, this strain mainly produce rubratoxins alone with high yield in varied culture conditions, suggested most gene clusters are silent. Efforts for mining the cryptic gene clusters in P. dangeardii, including epigenetic regulation and one-strain-many-compounds (OSMAC) approach were failed probably due to the high yield of rubratoxins. A metabolic shunting strategy by deleting the key gene for rubratoxins biosynthesis combining with optimization of culture condition successfully activated multiple silent genes encoding for other polyketide synthases (PKSs), and led to the trace compounds detectable. As a result, a total of 23 new compounds including azaphilone monomers, dimers, trimers with unprecedented polycyclic bridged heterocycle and spiral structures, as well as siderophores were identified. Some compounds showed significant cytotoxicities, anti-inflammatory or antioxidant activities. The attractive dual PKSs gene clusters for azaphilones biosynthesis were mined by bioinformatic analysis and overexpression of a pathway specific transcription factor. Our work therefor provides an efficient approach to mine the chemical diversity of endophytic fungi.

内生真菌是生物活性小分子的有前途的生产者。对内生真菌青霉菌（Penicillium dangeardii）的基因组进行生物信息学分析，揭示了其含有43个生物合成基因簇，显示出其产生大量次级代谢产物的强大能力。然而，该菌株在不同的培养条件下主要产生红曲霉素（rubratoxins），且产量较高，这表明大多数基因簇处于沉默状态。通过表观遗传调控和“一菌多用”（one-strain-many-compounds，OSMAC）方法挖掘P. dangeardii中的隐秘基因簇的努力未能成功，可能是因为红曲霉素的高产量。通过代谢分流策略——删除红曲霉素生物合成的关键基因，并结合优化培养条件，成功激活了多个沉默基因，这些基因编码其他聚酮合酶（polyketide synthases，PKSs），并使得微量化合物得以检测。结果，共鉴定出23种新化合物，包括具有前所未有的多环桥联杂环和螺旋结构的单体、二聚体和三聚体的氮杂菲酮（azaphilone）以及铁载体（siderophores）。其中一些化合物显示出显著的细胞毒性、抗炎或抗氧化活性。通过生物信息学分析和过表达途径特异性转录因子，挖掘到了用于氮杂菲酮生物合成的双聚酮合酶基因簇。因此，我们的工作为挖掘内生真菌的化学多样性提供了一种有效的方法。