Deletion and Gene Expression Analyses Define the Paxilline Biosynthetic Gene Cluster in Penicillium paxilli

Abstract

The indole-diterpene paxilline is an abundant secondary metabolite synthesized by Penicillium paxilli. In total, 21 genes have been identified at the PAX locus of which six have been previously confirmed to have a functional role in paxilline biosynthesis. A combination of bioinformatics, gene expression and targeted gene replacement analyses were used to define the boundaries of the PAX gene cluster. Targeted gene replacement identified seven genes, paxG, paxA, paxM, paxB, paxC, paxP and paxQ that were all required for paxilline production, with one additional gene, paxD, required for regular prenylation of the indole ring post paxilline synthesis. The two putative transcription factors, PP104 and PP105, were not co-regulated with the pax genes and based on targeted gene replacement, including the double knockout, did not have a role in paxilline production. The relationship of indole dimethylallyl transferases involved in prenylation of indole-diterpenes such as paxilline or lolitrem B, can be found as two disparate clades, not supported by prenylation type (e.g., regular or reverse). This paper provides insight into the P. paxilli indole-diterpene locus and reviews the recent advances identified in paxilline biosynthesis.

Biosynthesis of fungal meroterpenoids

“…The function of the biosynthetic gene cluster for paxilline (179) has been investigated by gene deletion, complementation, and heterologous reconstitution experiments, which established the complete biosynthetic route of 179. 121,[128][129][130][131][132] In the paxilline pathway, indole-2-glycerol phosphate (192) serves as the indole donor, which is geranylgeranylated by the PTase PaxC to yield 3-geranylgeranylindole (193). The prenylation reaction differs from the above-described meroterpenoid pathways in two aspects.…”

“…134 Furthermore, the involvement of paxA in paxilline biosynthesis is indicated, since the paxA-deletion mutant is unable to produce paxilline (179) and the deciency is complemented by the reintroduction of paxA. 132 PaxA is predicted to be a sixtransmembrane protein and is also present in the other indolediterpenoid biosynthetic gene clusters. However, reconstitution studies have revealed that paxA was not required for the production of 179, 131 and therefore the role of paxA remains enigmatic.…”

Molecular Cloning and Functional Analysis of Gene Clusters for the Biosynthesis of Indole-Diterpenes in Penicillium crustosum and P. janthinellum

“…Nine of these were putative orthologs of genes in the PAX cluster. All seven of the genes that are necessary for paxilline biosynthesis in P. paxilli [ paxG (geranygeranyl diphosphate synthase), paxA (integral membrane protein), paxM (FAD-dependent monooxygenase), paxB (integral membrane protein) paxC (prenyl transferase) paxP (cytochrome P450 monooxygenase) and paxQ (cytochrome P450 monooxygenase)], had homologs in the PEN cluster plus homologs of two genes [ paxD (aromatic prenyl transferase) and paxO (oxidoreductase)] that have demonstrated or possible functions in paxilline modification [ 11 ]. These genes were tandemly arranged in the cluster from PC-07 to PC-13 and PC-18 to PC-19, interrupted by genes PC-14 to PC-17.…”

“…Using Penicillium paxilli as a model experimental system we have identified and functionally characterized the genes required for the synthesis of paxilline [ 10 , 11 ], a potent inhibitor of calcium activated BK channels [ 7 ]. Genetic analysis of P. paxilli has established that a cluster of seven genes is required for paxilline biosynthesis [ 10 , 11 , 12 ].…”

“…Using Penicillium paxilli as a model experimental system we have identified and functionally characterized the genes required for the synthesis of paxilline [ 10 , 11 ], a potent inhibitor of calcium activated BK channels [ 7 ]. Genetic analysis of P. paxilli has established that a cluster of seven genes is required for paxilline biosynthesis [ 10 , 11 , 12 ]. Using a P. 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 [ 13 ], the first cyclic indole-diterpene intermediate in this pathway ( Figure 1 ).…”