Biochemical and genetic basis of oligocyclic aromatic polyketide formation in basidiomycetes

就结构多样性及其有益或有毒的生物活性而言，聚酮类化合物是一类杰出的天然产物。它们由聚酮合成酶（pks）产生，即复杂的多功能酶。细菌和子囊菌聚酮生物合成的遗传和生化基础已经很清楚。相反，关于担子菌（蘑菇型真菌）的知识非常匮乏。它们中寡环聚酮的形成（例如具有这种结构特征的色素和毒素）仍然完全未被研究。这种知识差距更令人惊讶，因为担子菌代表了大约40,000种的门，是生物活性天然产物的丰富来源。网帽菇属是担子菌中最大的属之一。本属的物种产生大量的寡环聚酮。申请人以香网帽（Cortinarius odorifer）为模型，研究了其基因组。这种蘑菇产生粘液素A1和B1，它们是二聚体，甲基化的低环多酮。鉴定出6个高度相似的基因（pks1-pks6），它们编码一个迄今未知的、进化上分离的群体的PKSs。据推测，这些基因/酶控制寡环多酮的形成。这一假设得到了两个甲基转移酶和一个芳香过氧化物酶的邻近基因的支持，这两个基因可能在痰素生物合成过程中催化甲基化和二聚化。本项目旨在通过对PKS1-PKS6酶的功能表征来验证上述假设。为了达到这一目标，酶将在真菌宿主（黑曲霉）中异种产生，然后在体内和体外形成产品。产品将通过液相色谱、质谱和核磁共振谱分析。此外，还将对这两种甲基转移酶进行异源合成和表征。成功完成的项目具有模型性质，为进一步了解毒性或药用相关担子菌聚酮的结构多样性是如何产生的奠定了基础。

Regarding structural diversity and their useful or toxic bioactivities, the polyketides are an outstanding group of natural products. They are produced by polyketide synthases (PKSs), i.e. complex, multifunctional Enzymes. The genetic and biochemical basis of polyketide biosynthesis in bacteria and ascomycetes is well understood. Conversely, the knowledge for basidiomycetes (mushroom-type fungi) is very scarce. The formation of oligocyclic polyketides (e.g. pigments and toxins with this structural feature) in them has remained completely uninvestigated. This knowledge gap is even more surprising, as the basidiomycetes represent a phylum of some 40,000 species and an abundant source of bioactive natural products.The genus Cortinarius (Webcap Mushroom) is one of the largest genera of the basidiomycetes. Species within this genus produce numerous oligocyclic polyketides. Using the Fragrant Webcap (Cortinarius odorifer) as a model, the applicant investigated its genome. This mushroom produces phlegmacins A1 and B1, which are dimeric, methylated oligocyclic polyketides. Six highly similar genes (pks1-pks6) were identified which encode PKSs of a hitherto unknown, evolutionarily separate group. It is hypothesized that these genes/enzymes control the formation of oligocyclic polyketides. This hypothesis is supported by adjacent genes for two methyl transferases and an aromatic peroxidase that may catalyze methylation and dimerization during phlegmacin biosynthesis.This project aims at functional characterization of the enzymes PKS1-PKS6 to test the above hypothesis. To reach this goal, the enzymes will be produced heterologously in a fungal host (Aspergillus niger), followed by product formation in vivo and in vitro. The products will be analyzed by liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy. In addition, the two methyl transferases will be produced heterologously and characterized. The successfully completed project has model character and lays the foundation for future work to generally understand how structural diversity of toxic or pharmaceutically relevant basidiomycete polyketides is generated.