Functional analysis of Penicillium paxilli genes required for biosynthesis of paxilline : this thesis is presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Biochemistry at Massey University, Palmerston North, New Zealand

dc.contributor.authorSaikia, Sanjay
dc.date.accessioned2010-07-26T01:50:15Z
dc.date.availableNO_RESTRICTIONen_US
dc.date.available2010-07-26T01:50:15Z
dc.date.issued2006
dc.description.abstractPaxilline belongs to a large, structurally and functionally diverse group of indole-diterpenes and is synthesised by the filamentous fungus Penicillium paxilli. A gene cluster for paxilline biosynthesis in P. paxilli has been identified and characterised. However, none of the steps proposed in the biosynthesis of paxilline or paxilline-like indole-diterpenes have been validated. In some diterpene-producing filamentous fungi, including P. paxilli, two distinct copies of geranylgeranyl diphosphate (GGPP) synthase, that catalyses the committed step in diterpene biosynthesis, have been identified. However, the biological significance of the presence of two distinct GGPP synthases is not known. In this study, biochemical analysis of the paxilline gene products in P. paxilli and subcellular localisation of the two P. paxilli GGPP synthases, Ggs1 and PaxG, were carried out. Transfer of constructs containing different combinations of pax genes into a pax cluster negative deletion derivative of P. paxilli identified four Pax proteins that are required for the biosynthesis of a paxilline intermediate, paspaline. These proteins are PaxG, a GGPP synthase, PaxM, a FAD-dependent monooxygenase, PaxB, a putative membrane protein, and PaxC, a prenyltransferase. Using precursor feeding experiments, it was confirmed that the indole-diterpenes paspaline and β-PC-M6 are substrates for the cytochrome P450 monooxygenase, PaxP, and are converted to 13-desoxypaxilline. Further, it was confirmed that the indole-diterpene 13-desoxypaxilline is a substrate for PaxQ, a cytochrome P450 monooxygenase, and is converted to paxilline. Unlike PaxQ, PaxP is specific for indole-diterpene substrates that have a β-stereochemistry. The detection of the indole-diterpene products was related to the expression of the transgene in the pax cluster negative background. Reporter fusion studies of the two P. paxilli GGPP synthases, Ggs1 and PaxG, showed that the Ggs1-EGFP fusion protein was localised to punctuate structures whose identity could not be established, and the EGFP-GRV fusion protein, containing the C-terminal tripeptide GRV of PaxG, was localised to peroxisomes.en_US
dc.identifier.urihttp://hdl.handle.net/10179/1489
dc.language.isoenen_US
dc.publisherMassey Universityen_US
dc.rightsThe Authoren_US
dc.subjectPenicillium analysisen_US
dc.subjectPenicillium genesen_US
dc.subject.otherFields of Research::270000 Biological Sciences::270100 Biochemistry and Cell Biologyen_US
dc.titleFunctional analysis of Penicillium paxilli genes required for biosynthesis of paxilline : this thesis is presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Biochemistry at Massey University, Palmerston North, New Zealanden_US
dc.typeThesisen_US
massey.contributor.authorSaikia, Sanjay
thesis.degree.disciplineBiochemistryen_US
thesis.degree.grantorMassey Universityen_US
thesis.degree.levelDoctoralen_US
thesis.degree.levelDoctoralen
thesis.degree.nameDoctor of Philosophy (Ph.D.)en_US
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