Defining Paxilline Biosynthesis in Penicillium paxilli

Abstract

Indole diterpenes are a large, structurally and functionally diverse group of secondary metabolites produced by filamentous fungi. Biosynthetic schemes have been proposed for these metabolites but until recently none of the proposed steps had been validated by biochemical or genetic studies. Using Penicillium paxilli as a model experimental system to study indole diterpene biosynthesis we previously showed by deletion analysis that a cluster of seven genes is required for paxilline biosynthesis. Two of these pax genes, paxP and paxQ (encoding cytochrome P450 monooxygenases), are required in the later steps in this pathway. Here, we describe the function of paxP and paxQ gene products by feeding proposed paxilline intermediates to strains lacking the pax cluster but containing ectopically integrated copies of paxP or paxQ. Transformants containing paxP converted paspaline into 13-desoxypaxilline as the major product and ␤-PC-M6 as the minor product. ␤-PC-M6, but not ␣-PC-M6, was also a substrate for PaxP and was converted to 13-desoxypaxilline. paxQ-containing transformants converted 13-desoxypaxilline into paxilline. These results confirm that paspaline, ␤-PC-M6, and 13-desoxypaxilline are paxilline intermediates and that paspaline and ␤-PC-M6 are substrates for PaxP, and 13-desoxypaxilline is a substrate for PaxQ. PaxP and PaxQ also utilized ␤-paxitriol and ␣-PC-M6 as substrates converting them to paxilline and ␣-paxitriol, respectively. These findings have allowed us to delineate clearly the biosynthetic pathway for paxilline for the first time.

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

“…paxQ -containing transformants converted 13-desoxypaxilline into paxilline. These results confirmed that paspaline, β-PC-M6 and 13-desoxypaxilline are paxilline intermediates and that paspaline and β-PC-M6 are substrates for PaxP, and 13-desoxypaxilline is a substrate for PaxQ [10]. Stepwise introduction of the pax genes into A. oryzae showed that addition of paxG-C-M-B-P-Q was sufficient to reconstitute the machinery for paxilline biosynthesis [12].…”

“…While deletion mutants of paxP and paxQ were blocked for paxilline biosynthesis, they accumulated paspaline and 13-desoxypaxilline, respectively, confirming that both genes were required for paxilline biosynthesis and that paspaline and 13-desoxypaxilline were the most likely substrates for the corresponding enzymes [9]. This was confirmed by feeding these compounds to strains lacking the pax cluster but containing ectopically integrated copies of paxP and paxQ [10]. Transformants containing paxP converted paspaline into 13-desoxypaxilline as the major product and β-PC-M6 as the minor product.…”

“…Using a combination of plasmid insertional mutagenesis and chromosome walking, a cluster of genes was isolated and shown to be required for paxilline biosynthesis [8]. Gene disruption and chemical complementation experiments have shown that paxG , paxP and paxQ are required for paxilline biosynthesis [8,9,10]. …”

“…Stepwise introduction of paxG , paxC , paxM and paxB into A. oryzae , combined with in vitro protein expression studies, demonstrated that PaxC is a prenyl transferase required for formation of 3-GGI and that PaxM and PaxB catalyze the stepwise epoxidation and cyclization of 3-GGI to paspaline [12]. Two cytochrome P450 monooxygenases, PaxP and PaxQ are involved in the later steps of the pathway in which paspaline is converted to paxilline [9,10]. While deletion mutants of paxP and paxQ were blocked for paxilline biosynthesis, they accumulated paspaline and 13-desoxypaxilline, respectively, confirming that both genes were required for paxilline biosynthesis and that paspaline and 13-desoxypaxilline were the most likely substrates for the corresponding enzymes [9].…”

Natural products from filamentous fungi and production by heterologous expression

“…In other works, Tagami et al (2013) successfully synthesised the indole-diterpene paxilline (21) in A. oryzae , a bioactive natural product that shows MRSA growth inhibition. Six genes had first been identified as being involved in biosynthesis of paxilline in the native producer Penicillium paxilli (Saikia et al 2006, 2007) and were expressed heterologously by Tagami et al (2013). Also in this case, the authors adopted a stepwise transformation approach and were therefore able to isolate and identify the intermediate compounds of the paxilline biosynthetic pathway.…”