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Journal of Bacteriology, October 2001, p. 6085-6094, Vol. 183, No. 20
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.20.6085-6094.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Eubacterial Diterpene Cyclase Genes Essential for Production of the Isoprenoid Antibiotic Terpentecin

Tohru Dairi,1,* Yoshimitsu Hamano,1 Tomohisa Kuzuyama,2 Nobuya Itoh,1 Kazuo Furihata,3 and Haruo Seto2

Biotechnology Research Center, Toyama Prefectural University, Toyama 939-0398,1 and Institute of Molecular and Cellular Biosciences2 and Division of Agriculture and Agricultural Life Science,3 The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan

Received 26 March 2001/Accepted 25 July 2001

A gene cluster containing the mevalonate pathway genes (open reading frame 2 [ORF2] to ORF7) for the formation of isopentenyl diphosphate and a geranylgeranyl diphosphate (GGDP) synthase gene (ORF1) had previously been cloned from Streptomyces griseolosporeus strain MF730-N6, a diterpenoid antibiotic, terpentecin (TP) producer (Y. Hamano, T. Dairi, M. Yamamoto, T. Kawasaki, K Kaneda, T. Kuzuyama, N. Itoh, and H. Seto, Biosci. Biotech. Biochem. 65:1627-1635, 2001). Sequence analysis in the upstream region of the cluster revealed seven new ORFs, ORF8 to ORF14, which were suggested to encode TP biosynthetic genes. We constructed two mutants, in which ORF11 and ORF12, which encode a protein showing similarities to eukaryotic diterpene cyclases (DCs) and a eubacterial pentalenene synthase, respectively, were inactivated by gene disruptions. The mutants produced no TP, confirming that these cyclase genes are essential for the production of TP. The two cyclase genes were also expressed in Streptomyces lividans together with the GGDP synthase gene under the control of the ermE* constitutive promoter. The transformant produced a novel cyclic diterpenoid, ent-clerod-3,13(16),14-triene (terpentetriene), which has the same basic skeleton as TP. The two enzymes, each of which was overproduced in Escherichia coli and purified to homogeneity, converted GGDP into terpentetriene. To the best of our knowledge, this is the first report of a eubacterial DC.

* Corresponding author. Mailing address: Biotechnology Research Center, Toyama Prefectural University, Toyama 939-0398, Japan. Phone: 81-766-56-7500. Fax: 81-766-56-2498. E-mail: dairi{at}pu-toyama.ac.jp.

Journal of Bacteriology, October 2001, p. 6085-6094, Vol. 183, No. 20
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.20.6085-6094.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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