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Siderophores are small molecular iron chelators and participate in the multiple cellular processes in fungi. In this study, biosynthesis gene clusters of coprogens and dimerumic acids were identified by transcriptional level differences of genes related to iron deficiency conditions in Metarhizium robertsii. This leads to the characterization of new coprogen metachelin C (1) and five known siderophores metachelin A (2), metachelin A-CE (3), metachelin B (4), dimerumic acid 11-mannoside (5), and dimerumic acid (6). The structure of metachelin C (1) was elucidated by NMR spectroscopy and HR-ESI-MS analysis. Genetic deletions of mrsidA, and mrsidD abolished the production of compounds 1–6 that implied their involvement in the biosynthesis of coprogen and dimerumic acid. Interestingly, NRPS gene mrsidD is responsible for biosynthesis of both coprogen and dimerumic acid, thus we proposed plausible biosynthetic pathways for the synthesis of coprogen and dimerumic acid siderophores. Therefore, our study provides the genetic basis for understanding the biosynthetic pathway of coprogen and dimerumic acid in Metarhizium robertsii.

As an indispensable essential amino acid in the human body, lysine is extremely rich in edible mushrooms. The α-aminoadipic acid (AAA) pathway is regarded as the biosynthetic pathway of lysine in higher fungal species in Agaricomycetes. However, there is no deep understanding about the molecular evolutionary relationship between lysine biosynthesis and species in Agaricomycetes. Herein, we analyzed the molecular evolution of lysine biosynthesis in Agaricomycetes. The phylogenetic relationships of 93 species in 34 families and nine orders in Agaricomycetes were constructed with six sequences of LSU, SSU, ITS (5.8 S), RPB1, RPB2, and EF1-α datasets, and then the phylogeny of enzymes involved in the AAA pathway were analyzed, especially homocitrate synthase (HCS), α-aminoadipate reductase (AAR), and saccharopine dehydrogenase (SDH). We found that the evolution of the AAA pathway of lysine biosynthesis is consistent with the evolution of species at the order level in Agaricomycetes. The conservation of primary, secondary, predicted tertiary structures, and substrate-binding sites of the enzymes of HCS, AAR, and SDH further exhibited the evolutionary conservation of lysine biosynthesis in Agaricomycetes. Our results provide a better understanding of the evolutionary conservation of the AAA pathway of lysine biosynthesis in Agaricomycetes.

Fungi from unique environments exhibit special physiological characters and plenty of bioactive natural products. However, the recalcitrant genetics or poor transformation efficiencies prevent scientists from systematically studying molecular biological mechanisms and exploiting their metabolites. In this study, we targeted a guanophilic fungus Amphichorda guana LC5815 and developed a genetic transformation system. We firstly established an efficient protoplast preparing method by conditional optimization of sporulation and protoplast regeneration. The regeneration rate of the protoplast is up to about 34.6% with 0.8 M sucrose as the osmotic pressure stabilizer. To develop the genetic transformation, we used the polyethylene glycol-mediated protoplast transformation, and the testing gene AG04914 encoding a major facilitator superfamily transporter was deleted in strain LC5815, which proves the feasibility of this genetic manipulation system. Furthermore, a uridine/uracil auxotrophic strain was created by using a positive screening protocol with 5-fluoroorotic acid as a selective reagent. Finally, the genetic transformation system was successfully established in the guanophilic fungus strain LC5815, which lays the foundation for the molecular genetics research and will facilitate the exploitation of bioactive secondary metabolites in fungi.