Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Organic Chemistry Regular Papers
Cloning of a Gene Cluster Responsible for the Biosynthesis of Diterpene Aphidicolin, a Specific Inhibitor of DNA Polymerase α
Tomonobu TOYOMASUKentaro NAKAMINAMIHiroaki TOSHIMATakashi MIEKenji WATANABEHiroyuki ITOHirokazu MATSUIWataru MITSUHASHITakeshi SASSAHideaki OIKAWA
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2004 Volume 68 Issue 1 Pages 146-152

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Abstract

  The fungal diterpene, aphidicolin, is a well-known specific inhibitor of DNA polymerase α. Terpenoids are an important class of natural products. However, identification of the biosynthetic gene cluster in terpenoids is relatively rare compared with another important class of natural products, polyketides. To explore a reliable identification method for the biosynthetic gene cluster in fungal diterpenoids, cloning of the biosynthetic gene cluster of aphidicolin was employed. The application of a simple PCR method for genome walking based on the sequence of cDNA encoding aphidicolan-16β-ol synthase (ACS) allowed us to analyze a 15.6-kb region of the Phoma betae genomic DNA. Six ORFs, PbGGS, ACS, PbP450-1, PbP450-2, PbTP, and PbTF were found in this region, and respectively expected to encode geranylgeranyl diphosphate synthase, diterpene synthase, two cytochrome P-450s, the transporter and transcription factor. Their amino acid sequences and introns were deduced by a corresponding cDNA analysis. This study shows that simple PCR-based genome walking without constructing a genomic DNA library is useful for identification of a small gene cluster. We propose a general strategy for the cloning the biosynthetic genes of fungal diterpenoids by using fungal GGS.

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© 2004 by Japan Society for Bioscience, Biotechnology, and Agrochemistry
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