Cloning of a gene cluster responsible for the biosynthesis of diterpene aphidicolin, a specific inhibitor of DNA polymerase alpha.
The fungal diterpene, aphidicolin, is a well-known specific inhibitor of DNA polymerase alpha. 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-16beta-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.
Toyomasu
Tomonobu
T
Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Yamagata, Japan.
Nakaminami
Kentaro
K
Toshima
Hiroaki
H
Mie
Takashi
T
Watanabe
Kenji
K
Ito
Hiroyuki
H
Matsui
Hirokazu
H
Mitsuhashi
Wataru
W
Sassa
Takeshi
T
Oikawa
Hideaki
H
D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D016590:Aphidicolin / Q000096:biosynthesis* / Q000494:pharmacology; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D004256:DNA Polymerase I / Q000037:antagonists & inhibitors; D004798:Enzymes / Q000235:genetics* / Q000378:metabolism; D051231:Farnesyltranstransferase; D006418:Heme / Q000378:metabolism; D003904:Mitosporic Fungi / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction / Q000379:methods; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D004798:Enzymes; D014157:Transcription Factors; D016590:Aphidicolin; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D019883:Alkyl and Aryl Transferases; C433216:aphidicolan-16beta-ol synthase; D051231:Farnesyltranstransferase; D004256:DNA Polymerase I