The Genome of Tolypocladium inflatum: Evolution, Organization, and Expression of the Cyclosporin Biosynthetic Gene Cluster

The ascomycete fungus Tolypocladium inflatum, a pathogen of beetle larvae, is best known as the producer of the immunosuppressant drug cyclosporin. The draft genome of T. inflatum strain NRRL 8044 (ATCC 34921), the isolate from which cyclosporin was first isolated, is presented along with comparative analyses of the biosynthesis of cyclosporin and other secondary metabolites in T. inflatum and related taxa. Phylogenomic analyses reveal previously undetected and complex patterns of homology between the nonribosomal peptide synthetase (NRPS) that encodes for cyclosporin synthetase (simA) and those of other secondary metabolites with activities against insects (e.g., beauvericin, destruxins, etc.), and demonstrate the roles of module duplication and gene fusion in diversification of NRPSs. The secondary metabolite gene cluster responsible for cyclosporin biosynthesis is described. In addition to genes necessary for cyclosporin biosynthesis, it harbors a gene for a cyclophilin, which is a member of a family of immunophilins known to bind cyclosporin. Comparative analyses support a lineage specific origin of the cyclosporin gene cluster rather than horizontal gene transfer from bacteria or other fungi. RNA-Seq transcriptome analyses in a cyclosporin-inducing medium delineate the boundaries of the cyclosporin cluster and reveal high levels of expression of the gene cluster cyclophilin. In medium containing insect hemolymph, weaker but significant upregulation of several genes within the cyclosporin cluster, including the highly expressed cyclophilin gene, was observed. T. inflatum also represents the first reference draft genome of Ophiocordycipitaceae, a third family of insect pathogenic fungi within the fungal order Hypocreales, and supports parallel and qualitatively distinct radiations of insect pathogens. The T. inflatum genome provides additional insight into the evolution and biosynthesis of cyclosporin and lays a foundation for further investigations of the role of secondary metabolite gene clusters and their metabolites in fungal biology.作为甲虫幼虫的病原菌，膨大弯颈霉（Tolypocladium inflatum）因生产免疫抑制剂环孢素（cyclosporin）而广为人知。本研究报道了环孢素首次分离得到的宿主菌株——膨大弯颈霉NRRL 8044（ATCC 34921）的基因组草图，并附带针对该菌及其近缘类群中环孢素与其他次级代谢产物生物合成过程的比较分析。系统基因组学分析揭示了编码环孢素合成酶（simA）的非核糖体肽合成酶（nonribosomal peptide synthetase，NRPS）与其他具有昆虫拮抗活性的次级代谢产物相关NRPS（如白僵菌素、绿僵菌素等）之间此前未被发现的复杂同源模式，并证实了模块重复与基因融合在NRPS家族多样化过程中的关键作用。本研究阐明了负责环孢素生物合成的次级代谢基因簇：该基因簇除包含环孢素生物合成所需的核心基因外，还携带一个亲环蛋白（cyclophilin）编码基因——亲环蛋白属于一类可结合环孢素的免疫亲和蛋白家族成员。比较分析表明，环孢素基因簇起源于该菌的谱系特异性演化，而非通过细菌或其他真菌的水平基因转移获得。在环孢素诱导培养基中开展的RNA测序（RNA-Seq）转录组分析明确了环孢素基因簇的边界，并揭示了该基因簇内亲环蛋白基因的高表达水平；在含有昆虫血淋巴的培养基中，研究人员观察到环孢素基因簇内多个基因（包括高表达的亲环蛋白基因）出现了较弱但显著的上调表达。膨大弯颈霉同时也是线虫草科（Ophiocordycipitaceae）首个可供参考的基因组草图，该科是真菌肉座菌目（Hypocreales）下第三类昆虫病原真菌类群，这一资源支持了昆虫病原菌的平行演化以及具有本质差异的辐射演化事件。膨大弯颈霉的基因组草图为环孢素的演化与生物合成研究提供了新的视角，并为后续探究次级代谢基因簇及其产物在真菌生物学中的功能奠定了基础。