crustosum abolished the synthesis of penitrems A, B, D, E, and F, and led to accumulation of paspaline, a key intermediate for paxilline biosynthesis in P. Similarly, deletion of janP and janD in P. janthinellum abolished the synthesis of prenyl-elaborated indole-diterpenes, and led to accumulation in the latter of 13-desoxypaxilline, a key intermediate for the synthesis of the structurally related aflatremanes synthesized by Aspergillus flavus. This study helps resolve the genetic basis for the complexity of indole-diterpene natural products found within the Penicillium and Aspergillus species.
All indole-diterpene gene clusters identified to date have a core set of genes for the synthesis of paspaline and a suite of genes encoding multi-functional cytochrome P450 monooxygenases, FAD dependent monooxygenases, and prenyl transferases that catalyse various regio- and stereo- specific oxidations that give rise to the diversity of indole-diterpene products synthesized by this group of fungi.
Using Penicillium paxilli as a model experimental system we have identified and functionally characterized the genes required for the synthesis of paxilline, a potent inhibitor of calcium activated BK channels. paxilli has established that a cluster of seven genes is required for paxilline biosynthesis. paxilli mutant deleted for the entire pax gene cluster we showed by gene reconstitution experiments that just four of these genes, paxG, paxM, paxB, and paxC, are required for the synthesis of paspaline, the first cyclic indole-diterpene intermediate in this pathway ( Figure 1). Based on this study we proposed a biosynthetic scheme for paspaline biosynthesis. This scheme has recently been experimentally validated by reconstitution of the pathway in the heterologous host A. Increased chemical complexity is achieved through enzyme-specific decorations of this core structure through the action of two cytochrome P450 monooxygenases, PaxP, and PaxQ. These additional steps have also been experimentally validated by reconstitution of paxilline biosynthesis in the heterologous host A. Successful amplification was achieved for both species using degenerate primers conC1 and conC2 resulting in putative gene fragments that were similar to the corresponding region of the P. These amplification products were 529-bp for P. crustosum, the paxC-like fragment was extended using primers 2402F1 (specific for the conC1-conC2 product) and degenerate primer PBR2 to produce a contiguous sequence of 2020-bp, which included 813-bp that was similar to the 3' end of paxC, an intergenic region of 865-bp, and 342-bp that was similar to the 3' end of paxP from P. Janthinellum a second product of 601-bp was amplified using degenerate primers PPF1 and PPR2 that was similar to a fragment of paxP from P.
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