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 β-PC-M6 as the minor product. β-PC-M6, but not α-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, 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. PaxP and PaxQ also utilized β-paxitriol and α-PC-M6 as substrates converting them to paxilline and α-paxitriol, respectively. These findings have allowed us to delineate clearly the biosynthetic pathway for paxilline for the first time. 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 β-PC-M6 as the minor product. β-PC-M6, but not α-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, 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. PaxP and PaxQ also utilized β-paxitriol and α-PC-M6 as substrates converting them to paxilline and α-paxitriol, respectively. These findings have allowed us to delineate clearly the biosynthetic pathway for paxilline for the first time. Indole diterpenes are a large, structurally and functionally diverse group of secondary metabolites produced mainly by the Penicillium, Aspergillus, Claviceps, and Neotyphodium species of filamentous fungi (1Parker E.J. Scott B. An Z. Handbook of Industrial Mycology. Marcel Dekker, New York2004: 405-426Google Scholar, 2Scott B. Jameson G.B. Parker E.J. Tkacz J.S. Lange L. Advances in Fungal Biotechnology for Industry, Agriculture, and Medicine. Kluwer Academic/Plenum Publishers, New York2004: 163-198Crossref Google Scholar). Most of these metabolites are potent tremorgenic mammalian toxins (3Steyn P.S. Vleggaar R. Fortschr. Chem. Org. Naturst. 1985; 48: 1-80PubMed Google Scholar) and some have anti-insect properties (4Gloer J.B. Acc. Chem. Res. 1995; 28: 343-350Crossref Scopus (82) Google Scholar). Although much work has been done to demonstrate the origins of indole and diterpene components of these metabolites from tryptophan or a tryptophan precursor and mevalonate, respectively (5Acklin W. Weibel F. Arigoni D. Chimia. 1977; 31: 63Google Scholar, 6Byrne K.M. Smith S.K. Ondeyka J.G. J. Am. Chem. Soc. 2002; 124: 7055-7060Crossref PubMed Scopus (60) Google Scholar, 7de Jesus A.E. Gorst-Allman C.P. Steyn P.S. van Heerden F.R. Vleggaar R. Wessels P.L. Hull W.E. J. Chem. Soc. Perkin Trans. 1983; 1: 1863-1868Crossref Scopus (75) Google Scholar, 8Laws I. Mantle P.G. J. Gen. Microbiol. 1989; 135: 2679-2692Google Scholar), very little is known about their biosynthetic pathways. Biosynthetic schemes have been proposed for these metabolites based on chemical identification of likely intermediates and precursor-feeding studies from the organism of interest and related filamentous fungi (9Gatenby W.A. Munday-Finch S.C. Wilkins A.L. Miles C.O. J. Agric. Food Chem. 1999; 47: 1092-1097Crossref PubMed Scopus (47) Google Scholar, 10Mantle P.G. Weedon C.M. Phytochemistry. 1994; 36: 1209-1217Crossref Scopus (45) Google Scholar, 11Munday-Finch S.C. Wilkins A.L. Miles C.O. Phytochemistry. 1996; 41: 327-332Crossref Scopus (63) Google Scholar). However, until recently none of the proposed steps had been validated by biochemical or genetic studies. The cloning and characterization of a cluster of genes from Penicillium paxilli necessary for biosynthesis of the indole diterpene, paxilline, has helped to understand the genetics and biochemistry of this important class of secondary metabolites (12Young C. McMillan L. Telfer E. Scott B. Mol. Microbiol. 2001; 39: 754-764Crossref PubMed Scopus (125) Google Scholar). Analysis of the pax gene cluster has also facilitated the isolation of pax gene orthologues from Aspergillus flavus and Neotyphodium lolii, fungi that produce the related, but more complex indole diterpenes aflatrem and lolitrems, respectively (13Young C.A. Bryant M.K. Christensen M.J. Tapper B.A. Bryan G.T. Scott B. Mol. Genet. Genomics. 2005; 274: 13-29Crossref PubMed Scopus (153) Google Scholar, 14Zhang S. Monahan B.J. Tkacz J.S. Scott B. Appl. Environ. Microbiol. 2004; 70: 6875-6883Crossref PubMed Scopus (82) Google Scholar). Based on gene disruption and chemical complementation studies, a cluster of seven genes including paxG (encoding a geranylgeranyl diphosphate synthase), paxM (encoding a FAD-dependent monooxygenase), paxC (encoding a prenyltransferase), paxP and paxQ (encoding two cytochrome P450 monooxygenases), and paxA and paxB (encoding two putative membrane proteins) 3B. Monahan, S. Saikia, and B. Scott, unpublished results. , has been shown to be essential for paxilline biosynthesis (12Young C. McMillan L. Telfer E. Scott B. Mol. Microbiol. 2001; 39: 754-764Crossref PubMed Scopus (125) Google Scholar, 15McMillan L.K. Carr R.L. Young C.A. Astin J.W. Lowe R.G. Parker E.J. Jameson G.B. Finch S.C. Miles C.O. McManus O.B. Schmalhofer W.A. Garcia M.L. Kaczorowski G.J. Goetz M. Tkacz J.S. Scott B. Mol. Genet. Genomics. 2003; 270: 9-23Crossref PubMed Scopus (47) Google Scholar). Recently, we have shown that four of these genes, paxG, paxM, paxB, and paxC, are required for the biosynthesis of the proposed first stable indole diterpene intermediate, paspaline (16Saikia S. Parker E.J. Koulman A. Scott B. FEBS Lett. 2006; 580: 1625-1630Crossref PubMed Scopus (52) Google Scholar). Later steps in the paxilline biosynthesis pathway involve two cytochrome P450 monooxygenases, PaxP and PaxQ, which are proposed to utilize paspaline and 13-desoxypaxilline as their respective substrates (15McMillan L.K. Carr R.L. Young C.A. Astin J.W. Lowe R.G. Parker E.J. Jameson G.B. Finch S.C. Miles C.O. McManus O.B. Schmalhofer W.A. Garcia M.L. Kaczorowski G.J. Goetz M. Tkacz J.S. Scott B. Mol. Genet. Genomics. 2003; 270: 9-23Crossref PubMed Scopus (47) Google Scholar). These compounds together with paspaline B, PC-M6, and β-paxitriol have been proposed to form a metabolic grid for paxilline biosynthesis (11Munday-Finch S.C. Wilkins A.L. Miles C.O. Phytochemistry. 1996; 41: 327-332Crossref Scopus (63) Google Scholar). The formation of paxilline from PC-M6 was proposed to occur either via 13-desoxypaxilline or viaβ-paxitriol suggesting a bifurcation at the penultimate step to paxilline biosynthesis (11Munday-Finch S.C. Wilkins A.L. Miles C.O. Phytochemistry. 1996; 41: 327-332Crossref Scopus (63) Google Scholar). The objectives of this study were to identify the substrates and products for PaxP and PaxQ and to assess the involvement of proposed paxilline intermediates. Given the versatility of cytochrome P450 enzymes to catalyze multiple steps (17Ro D.K. Arimura G. Lau S.Y. Piers E. Bohlmann J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8060-8065Crossref PubMed Scopus (149) Google Scholar, 18Tudzynski B. Appl. Microbiol. Biotechnol. 2005; 66: 597-611Crossref PubMed Scopus (171) Google Scholar), we sought to define the roles that PaxP and PaxQ play in the conversion of paspaline to paxilline. Bacterial Strains and Plasmids—The bacterial strains and plasmids used are listed in Table 1. Escherichia coli strain XL 1-Blue (19Bullock W.O. Fernandez J.M. Short J.M. BioTechniques. 1987; 5: 376-379Google Scholar) served as the host for routine cloning. The transformants of this host were grown on LB agar plates supplemented with ampicillin (100 μg/ml) for selection. pGEM®-T Easy (Promega) and pII99 (20Namiki F. Matsunaga M. Okuda M. Inoue I. Nishi K. Fujita Y. Tsuge T. Mol. Plant Microbe Interact. 2001; 14: 580-584Crossref PubMed Scopus (207) Google Scholar) plasmids were used for cloning.TABLE 1Strains and plasmidsStrains and plasmidsPlasmid/IdentifierRelevant characteristicsaAmpR, ampicillin-resistant; GenR, Geneticin-resistant; HygR, hygromycin-resistant.Source/Ref.Plasmids pGEM®-T EasyAmpRPromegapII99AmpR/GenR (PtrpC-nptII-TtrpC)20Namiki F. Matsunaga M. Okuda M. Inoue I. Nishi K. Fujita Y. Tsuge T. Mol. Plant Microbe Interact. 2001; 14: 580-584Crossref PubMed Scopus (207) Google ScholarpSS1pII99 containing paxP on a 2.68-kb EcoRI fragment; AmpR/GenRThis studypSS2pII99 containing paxQ on a 3.02-kb HindIII fragment; AmpR/GenRThis studyFungal strains (P. paxilli) PN2013Wild-type; paxilline-positive21Itoh Y. Johnson R. Scott B. Curr. Genet. 1994; 25: 508-513Crossref PubMed Scopus (70) Google Scholar PN2253LM662PN2013/ΔpaxT-paxD; HygR; paxilline-negative12Young C. McMillan L. Telfer E. Scott B. Mol. Microbiol. 2001; 39: 754-764Crossref PubMed Scopus (125) Google Scholar PN2250CY2PN2013/Δ(pax cluster and flanking sequences); HygR; paxilline-negative22Young C. Itoh Y. Johnson R. Garthwaite I. Miles C.O. Munday-Finch S.C. Scott B. Curr. Genet. 1998; 33: PubMed Scopus (47) Google Scholar HygR; L.K. Carr R.L. Young C.A. Astin J.W. Lowe R.G. Parker E.J. Jameson G.B. Finch S.C. Miles C.O. McManus O.B. Schmalhofer W.A. Garcia M.L. Kaczorowski G.J. Goetz M. Tkacz J.S. Scott B. Mol. Genet. Genomics. 2003; 270: 9-23Crossref PubMed Scopus (47) Google Scholar study HygR; L.K. Carr R.L. Young C.A. Astin J.W. Lowe R.G. Parker E.J. Jameson G.B. Finch S.C. Miles C.O. McManus O.B. Schmalhofer W.A. Garcia M.L. Kaczorowski G.J. Goetz M. Tkacz J.S. Scott B. Mol. Genet. Genomics. 2003; 270: 9-23Crossref PubMed Scopus (47) Google Scholar study study study study study study study study study study study study strains XL W.O. Fernandez J.M. Short J.M. BioTechniques. 1987; 5: 376-379Google Scholar study ampicillin-resistant; GenR, Geneticin-resistant; HygR, in a Fungal Strains and strains used are listed in Table 1. of paxilli Y. Johnson R. Scott B. Curr. Genet. 1994; 25: 508-513Crossref PubMed Scopus (70) Google Scholar), paxP and paxQ deletion strains and respectively (15McMillan L.K. Carr R.L. Young C.A. Astin J.W. Lowe R.G. Parker E.J. Jameson G.B. Finch S.C. Miles C.O. McManus O.B. Schmalhofer W.A. Garcia M.L. Kaczorowski G.J. Goetz M. Tkacz J.S. Scott B. Mol. Genet. Genomics. 2003; 270: 9-23Crossref PubMed Scopus (47) Google Scholar), pax cluster deletion strains C. Itoh Y. Johnson R. Garthwaite I. Miles C.O. Munday-Finch S.C. Scott B. Curr. Genet. 1998; 33: PubMed Scopus (47) Google Scholar) and (12Young C. McMillan L. Telfer E. Scott B. Mol. Microbiol. 2001; 39: 754-764Crossref PubMed Scopus (125) Google Scholar), and their were grown in Aspergillus at for as previously (16Saikia S. Parker E.J. Koulman A. Scott B. FEBS Lett. 2006; 580: 1625-1630Crossref PubMed Scopus (52) Google Scholar). Fungal used for the isolation of of and indole diterpene analysis were also grown as previously (16Saikia S. Parker E.J. Koulman A. Scott B. FEBS Lett. 2006; 580: 1625-1630Crossref PubMed Scopus (52) Google Scholar). The for used for the isolation of were to for indole diterpene was and by a was a of the of Plant Scopus Google Scholar) as previously C. Itoh Y. Johnson R. Garthwaite I. Miles C.O. Munday-Finch S.C. Scott B. Curr. Genet. 1998; 33: PubMed Scopus (47) Google Scholar). was from used was with in containing of and of the and products were and a were by the F. S. Proc. Natl. Acad. Sci. U. S. A. 1977; PubMed Scopus Google Scholar) with were on and of paxP and paxQ for cloning was in containing a of a of of and of for for paxP and paxQ was in containing a of a of of and of for paxP and paxQ was in containing of a of of and of The used with and were at for at for for and for and at for and with at for and for at for for and for and at for were in a or The used for paxP and paxQ for cloning were as paxP and and paxQ and The in to the to either EcoRI or HindIII as at These were also used for the transformants for of paxP and paxQ. The used for analysis of paxP and paxQ were as paxP and and paxQ and The of the that from or be of PaxP and PaxQ PaxP, was used as a in a the and the The 2.68-kb was with EcoRI and into pII99 the PaxQ, was used as a in a the and The 3.02-kb was with HindIII and into pII99 to the paxilli of paxilli paxP and paxQ deletion strains and and pax cluster deletion strains and were and with of plasmids or as previously (16Saikia S. Parker E.J. Koulman A. Scott B. FEBS Lett. 2006; 580: 1625-1630Crossref PubMed Scopus (52) Google Scholar). Transformants were on supplemented with either (100 μg/ml) or μg/ml) of PC-M6 and diterpenes PC-M6 and were by of 13-desoxypaxilline and paxilline, the by Miles C.O. Wilkins A.L. S.C. J. Agric. Food Chem. Scopus Google Scholar). of of 13-desoxypaxilline and paxilline New was for with and of in of respectively. The products of the were by to of was to the by the of of The products were four with of and the was by the of and The was in and the in of this was on as previously (16Saikia S. Parker E.J. Koulman A. Scott B. FEBS Lett. 2006; 580: 1625-1630Crossref PubMed Scopus (52) Google Scholar), which two with a in the The in the paxilline had of and and in the 13-desoxypaxilline had of and The to the two were and by The products of paxilline were as and β-paxitriol based on to New The products of 13-desoxypaxilline were as α-PC-M6 and β-PC-M6 based on the analysis of β-PC-M6 with and the conversion of α-PC-M6 to were by as previously (16Saikia S. Parker E.J. Koulman A. Scott B. FEBS Lett. 2006; 580: 1625-1630Crossref PubMed Scopus (52) Google Scholar). of to Fungal from of paxilli was grown for as previously (16Saikia S. Parker E.J. Koulman A. Scott B. FEBS Lett. 2006; 580: 1625-1630Crossref PubMed Scopus (52) Google Scholar). was with of and of to two containing of containing precursor was in two of of at at with at the of was as was for indole diterpene analysis as previously (16Saikia S. Parker E.J. Koulman A. Scott B. FEBS Lett. 2006; 580: 1625-1630Crossref PubMed Scopus (52) Google Scholar). Indole was for indole diterpenes by and as previously (16Saikia S. Parker E.J. Koulman A. Scott B. FEBS Lett. 2006; 580: 1625-1630Crossref PubMed Scopus (52) Google Scholar). the of indole at and at was to confirm the of indole diterpene in a However, are shown for of paxilli paxP and paxQ with was shown that the deletion of paxP and paxQ were for paxilline biosynthesis and paspaline and respectively (15McMillan L.K. Carr R.L. Young C.A. Astin J.W. Lowe R.G. Parker E.J. Jameson G.B. Finch S.C. Miles C.O. McManus O.B. Schmalhofer W.A. Garcia M.L. Kaczorowski G.J. Goetz M. Tkacz J.S. Scott B. Mol. Genet. Genomics. 2003; 270: 9-23Crossref PubMed Scopus (47) Google Scholar). confirm that disruption of paxP and paxQ genes was for the in paxilline biosynthesis complementation were with and The complementation and were by cloning copies of paxP and paxQ genes into the respectively of deletion and were with and respectively. from were by not and for their to paxilline. These showed that the of and into and was to paxilline biosynthesis in the strains However, the deletion containing the were not for paxilline biosynthesis not These results that the of paxP and paxQ genes the for paxilline biosynthesis in and and that the paxP and paxQ complementation are These complementation were used for analysis of paxP and complementation of paxilline biosynthesis in paxP and paxQ deletion of paxilli paxP and paxQ deletion and their containing ectopically integrated copies of paxP and paxQ genes, were by for indole paxP deletion deletion containing paxP paxilline and paspaline B, paxQ deletion deletion containing paxQ paxilline and 13-desoxypaxilline Transformants for Analysis of paxP and confirm the function of paxP and complementation and were into the of the deletion (12Young C. McMillan L. Telfer E. Scott B. Mol. Microbiol. 2001; 39: 754-764Crossref PubMed Scopus (125) Google Scholar) that the pax gene cluster and also into C. Itoh Y. Johnson R. Garthwaite I. Miles C.O. Munday-Finch S.C. Scott B. Curr. Genet. 1998; 33: PubMed Scopus (47) Google Scholar) that also the pax gene cluster flanking An of transformants was by not for of paxP or paxQ. Transformants containing a product of the were for paxP or paxQ by analysis that of the that from or be for paxP or paxQ was in the transformants in for the transformants that were used for for paxP or paxQ was in and analysis that paxP and paxQ were but at in the in the Transformants that showed of paxP or paxQ were for paxP and paxQ analysis Analysis of that paspaline is a substrate for PaxP, and strains containing paxP were with paspaline for and of these transformants for indole diterpenes by not and of and transformants had two indole 13-desoxypaxilline and β-PC-M6, as major and minor that were in and and transformants containing the pII99 not was converted to 13-desoxypaxilline by the the transformants showed conversion of the paspaline to indole diterpene Analysis of transformants showed that the of paspaline conversion with the of paxP These results that paspaline is a substrate for PaxP and is converted to 13-desoxypaxilline as the major product and β-PC-M6 as the minor product and that PaxP is of more steps proposed (15McMillan L.K. Carr R.L. Young C.A. Astin J.W. Lowe R.G. Parker E.J. Jameson G.B. Finch S.C. Miles C.O. McManus O.B. Schmalhofer W.A. Garcia M.L. Kaczorowski G.J. Goetz M. Tkacz J.S. Scott B. Mol. Genet. Genomics. 2003; 270: 9-23Crossref PubMed Scopus (47) Google Scholar). Analysis of 13-desoxypaxilline is a substrate for PaxQ, and strains that paxQ were with together with not and analysis of of and strains containing paxQ paxilline as the indole diterpene product indole diterpenes the were in of and deletion and and strains containing the pII99 not to the paspaline feeding the transformants were more at converting the 13-desoxypaxilline to paxilline the with the of conversion with the of paxQ These that 13-desoxypaxilline is a substrate for PaxQ. PC-M6 PC-M6 has been proposed as a paxilline (11Munday-Finch S.C. Wilkins A.L. Miles C.O. Phytochemistry. 1996; 41: 327-332Crossref Scopus (63) Google Scholar), is biochemical to this PC-M6 is for paxilline biosynthesis and a substrate for PaxP, the and of PC-M6 were and with the transformants of strains containing a PaxP, and were to β-PC-M6 to 13-desoxypaxilline the strains containing PaxQ or the were to the substrate The showed of 13-desoxypaxilline which is likely the of by PaxP Although the paxP deletion was to utilize β-PC-M6 as a showed of paspaline in to β-PC-M6 these results that β-PC-M6 is a substrate for PaxP and for paxilline biosynthesis. analysis of of strains containing a PaxQ, and showed of a indole diterpene, However, the strains containing PaxP, and and the were to utilize the α-PC-M6 as a substrate a of to is in the of these strains this not have the of indole at and of paxP deletion showed of paspaline to that in of Although these results that α-PC-M6 is a substrate for PaxQ, is to be for paxilline biosynthesis paxilline have a and β-paxitriol are intermediates in the paxilline biosynthesis pathway and substrates for either PaxP or PaxQ, the two were and to the strains the of strains containing a PaxP, and showed a indole diterpene, paxilline These results that β-paxitriol is a substrate for The conversion of β-paxitriol to paxilline by the paxQ deletion that the of β-paxitriol the steps by PaxQ. these results that β-paxitriol is not a in paxilline biosynthesis. These results confirm that the at is a function of PaxP, which the conversion of β-PC-M6 to 13-desoxypaxilline and β-paxitriol to paxilline, studies with showed conversion of the substrate by the strains that was not for paxilline biosynthesis. this study we have the two cytochrome P450 monooxygenases, PaxP and PaxQ, and that these enzymes are for the conversion of paspaline to paxilline. of paspaline and β-PC-M6, and 13-desoxypaxilline with two pax cluster deletion and containing either PaxP or PaxQ in the conversion of the compounds into indole diterpene products that these compounds are substrates for PaxP and PaxQ, and intermediates for paxilline biosynthesis. P450 enzymes were more in their to their respective substrates in the in the be to a of of the genes in the in the which in be to the of S. B. Appl. Environ. Microbiol. 2005; PubMed Scopus Google Scholar, M. F. Appl. Environ. Microbiol. 2002; PubMed Scopus Google Scholar). Given that is the of a with a deletion and that of this deletion is not C. Itoh Y. Johnson R. Garthwaite I. Miles C.O. Munday-Finch S.C. Scott B. Curr. Genet. 1998; 33: PubMed Scopus (47) Google Scholar), the of a gene a the be filamentous genes have been to be with paxilline biosynthesis M. B. Mol. Microbiol. 2003; 47: PubMed Scopus Google Scholar, B. B. Mol. Gen. Genet. 1999; PubMed Scopus Google Scholar, J.W. 2004; PubMed Scopus Google Scholar, J.W. D. 2005; PubMed Scopus Google Scholar, J.W. D. R. Chem. 2006; PubMed Scopus Google Scholar). Although was shown that two putative genes, and that putative are with the pax cluster (12Young C. McMillan L. Telfer E. Scott B. Mol. Microbiol. 2001; 39: 754-764Crossref PubMed Scopus (125) Google Scholar), for these genes are to paxilline suggesting that these two genes not have a in paxilline biosynthesis. S. and B. Scott, unpublished results. precursor-feeding paxilline was the conversion product of 13-desoxypaxilline by PaxQ, PaxP converted paspaline to 13-desoxypaxilline and These results demonstrate that PaxP is to catalyze multiple steps at a and at of the group by via of a group by a P450 has been in biosynthesis Johnson R.L. J.M. J. Res. 1989; PubMed Google Scholar, FEBS Lett. 1999; PubMed Scopus Google Scholar). the cytochrome P450 was shown to catalyze four at four in a of from to B. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). at multiple by a P450 has also been shown for the in which the cytochrome P450 and of to form K. K. Y. E. PubMed Scopus Google Scholar). F. cytochrome P450 at a in the conversion of to B. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar, B. E. Appl. Environ. Microbiol. 2001; PubMed Scopus (82) Google Scholar). multiple have been for the cytochrome P450 enzymes by genes the cluster in Biotechnol. 2006; 70: PubMed Scopus Google Scholar). indole diterpene paspaline has previously been from paxilli and as the first of paspaline (11Munday-Finch S.C. Wilkins A.L. Miles C.O. Phytochemistry. 1996; 41: 327-332Crossref Scopus (63) Google Scholar). However, in precursor-feeding indole diterpenes β-PC-M6 and 13-desoxypaxilline were as the conversion products of paspaline by of β-PC-M6 with a pax cluster strain containing PaxP in conversion to 13-desoxypaxilline that β-PC-M6 is also a substrate for Although PaxP was shown to catalyze the of β-paxitriol to produce paxilline, β-paxitriol was not as a pathway in the feeding These results clearly that the of the group not a as proposed previously (15McMillan L.K. Carr R.L. Young C.A. Astin J.W. Lowe R.G. Parker E.J. Jameson G.B. Finch S.C. Miles C.O. McManus O.B. Schmalhofer W.A. Garcia M.L. Kaczorowski G.J. Goetz M. Tkacz J.S. Scott B. Mol. Genet. Genomics. 2003; 270: 9-23Crossref PubMed Scopus (47) Google Scholar), but is by the of PaxP a study on the biosynthesis of paxilline from paspaline was proposed that a bifurcation at PC-M6 and were two from PC-M6 to paxilline this was proposed that paxilline be either from β-paxitriol or from 13-desoxypaxilline (11Munday-Finch S.C. Wilkins A.L. Miles C.O. Phytochemistry. 1996; 41: 327-332Crossref Scopus (63) Google Scholar). of PC-M6 as the of However, precursor-feeding have shown that this to the PaxQ conversion of 13-desoxypaxilline to paxilline, is not by PaxQ. of β-paxitriol with a paxQ deletion paxilline biosynthesis. these precursor-feeding studies that β-paxitriol is not a of the paxilline biosynthesis pathway but be in steps is that the pax cluster is of converting paxilline to of with strains containing pax cluster and also with a strain containing PaxP that was not a substrate for However, α-PC-M6 was shown to be a substrate for PaxQ converted to Given that the paxilline intermediates have a at of a substrate that has a by a is is more likely a precursor for and that have the at (9Gatenby W.A. Munday-Finch S.C. Wilkins A.L. Miles C.O. J. Agric. Food Chem. 1999; 47: 1092-1097Crossref PubMed Scopus (47) Google Scholar, C.O. Wilkins A.L. S.C. J. Agric. Food Chem. Scopus Google Scholar, J. Mantle P.G. Phytochemistry. 1994; Scopus Google Scholar). The formation of by PaxQ be a or a of PaxQ. The biosynthetic of this is not Penicillium and Penicillium of β-paxitriol and not α-paxitriol, into and and and that β-paxitriol is precursor for the complex indole diterpenes with J. Mantle P.G. Phytochemistry. 1994; Scopus Google Scholar). These the substrate of enzymes in the biosynthesis of indole indole diterpenes that have been from fungi to the and have and to the and have (1Parker E.J. Scott B. An Z. Handbook of Industrial Mycology. Marcel Dekker, New York2004: 405-426Google Scholar). identification of PaxP and PaxQ in the cluster C.A. S. K. G. Johnson Bryan G.T. S. Scott B. Fungal Genet. 2006; PubMed Scopus Google Scholar) in the substrate of these P450 enzymes in indole diterpene biosynthesis. study for the first paspaline is to paxilline and the pathway and the gene products for paxilline biosynthesis. also a for PaxP by in the of the indole diterpene intermediates. is to this by either or and in paxilli and this paxilli to form indole diterpenes with S. Munday-Finch and C. Miles for of paxilline, paspaline, α-paxitriol, and with