Cyclosporine Biosynthesis in Tolypocladium inflatum Benefits Fungal Adaptation to the Environment

The cycloundecapeptide cyclosporin A (CsA) was first isolated from the insect-pathogenic fungus Tolypocladium inflatum for its antifungal activity and later developed as an immunosuppressant drug. However, the full biosynthetic mechanism of CsA remains unknown and has puzzled researchers for decades. In this study, the biosynthetic gene cluster is suggested to include 12 genes encoding enzymes, including the nonribosomal peptide synthetase (NRPS) (SimA) responsible for assembling the 11 amino acid substrates of cyclosporine and a polyketide synthase (PKS) (SimG) to mediate the production of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-l-threonine (Bmt). Individual deletion of 10 genes, isolation of intermediates, and substrate feeding experiments show that Bmt is biosynthesized by three enzymes, including SimG, SimI, and SimJ. The substrate d-alanine is catalyzed from l-alanine by alanine racemase SimB. Gene cluster transcription is regulated by a putative basic leucine zipper (bZIP)-type protein encoded by the cluster gene SimL. We also found that the cluster cyclophilin (SimC) and transporter (SimD) genes contribute to the tolerance of CsA in the CsA-producing fungus. We also found that cyclosporine production could enable the fungus to outcompete other fungi during cocultivation tests. Deletion of the CsA biosynthetic genes also impaired fungal virulence against insect hosts. Taking all the data together, in addition to proposing a biosynthetic pathway of cyclosporines, the results of this study suggest that CsA produced by this fungus might play important ecological roles in fungal environment interactions.