Presence of copalyl diphosphate synthase gene in an actinomycete possessing the mevalonate pathway.
We have previously shown that gene clusters for biosyntheses of terpentecin and BE-40644, a diterpene antibiotic and a sesquiterpene antibiotic, respectively, were located in the adjacent mevalonate pathway gene clusters. In this study, a mevalonate pathway gene cluster was cloned from Streptomyces sp. strain KO-3988, which was known to produce furaquinocin A, employing a hybridization experiment using a 3-hydroxy-3-methyl glutaryl CoA (HMG-CoA) reductase gene, which had been previously cloned from the strain KO-3988, as a probe. By sequencing flanking regions, we found four open reading frames that could encode a putative cytochrome P450 (ORF1), an isoprenoid cyclase (ORF2), an unknown protein (ORF3), and a polyprenyl diphosphate synthase gene (ORF4) in the upstream region of the mevalonate pathway gene cluster, though we did not find any genes related to furaquinocin A biosynthesis. The two ORFs (ORF2 and 4) were expressed as recombinant enzymes in E. coli and used for studies to investigate functions of these products. The ORF4 product was confirmed to be a geranylgeranyl diphosphate (GGDP, C20) synthase. The ORF2 product proved to catalyze a conversion of GGDP into copalyl diphosphate, the first example of an enzyme with this function of prokaryotic origin. These results again showed that actinomycetes possessing the mevalonate pathway usually produce an isoprenoid and that its biosynthetic gene cluster exists in adjacent the mevalonate pathway gene cluster.
Kawasaki
Takashi
T
Biotechnology Research Center, Toyama Prefectural University, Toyama 939-0398, Japan.
Kuzuyama
Tomohisa
T
Kuwamori
Yuko
Y
Matsuura
Nobuyasu
N
Itoh
Nobuya
N
Furihata
Kazuo
K
Seto
Haruo
H
Dairi
Tohru
T
D019883:Alkyl and Aryl Transferases / Q000235:genetics*; D000900:Anti-Bacterial Agents / Q000096:biosynthesis; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D015245:Deoxyribonuclease BamHI / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D008798:Mevalonic Acid / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D010940:Plant Proteins; D019883:Alkyl and Aryl Transferases; C090963:ent-kaurene synthetase A; D015245:Deoxyribonuclease BamHI; D008798:Mevalonic Acid