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Applied and Environmental Microbiology, June 1999, p. 2703-2709, Vol. 65, No. 6
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Development of a Self-Cloning System for Actinomadura verrucosospora and Identification of Polyketide Synthase Genes Essential for Production of the Angucyclic Antibiotic Pradimicin

Tohru Dairi,* Yoshimitsu Hamano, Tamotsu Furumai, and Toshikazu Okidagger

Biotechnology Research Center, Toyama Prefectural University, Kurokawa 5180, Kosugi, Toyama 939-0398, Japan

Received 23 December 1998/Accepted 11 March 1999

A self-cloning system for Actinomadura verrucosospora, a producer of the angucyclic antibiotic pradimicin A (PRM A), has been developed. The system is based on reproducible and reliable protoplasting and regeneration conditions for A. verrucosospora and a novel plasmid vector that consists of a replicon from a newly found Actinomadura plasmid and a selectable marker cloned from the Actinomadura strain. The system has an efficiency of more than 105 CFU/microgram of DNA. Using this system, we have cloned and identified the polyketide synthase (PKS) genes essential for PRM A biosynthesis from A. verrucosospora. Nucleotide sequence analysis of the 3.5-kb SalI-SphI fragment showed that ketosynthase subunits (open reading frame 1 [ORF1] and ORF2) of the essential PKS genes have strong similarities (59 to 89%) to those for angucyclic antibiotic biosynthesis.

* Corresponding author. Mailing address: Biotechnology Research Center, Toyama Prefectural University, Kurokawa 5180, Kosugi, Toyama 939-0398, Japan. Phone: 81-766-56-7500, ext. 561. Fax: 81-766-56-2498. E-mail: dairi{at}pu-toyama.ac.jp.

dagger Present address: Applied Life Science Research, Tamagawa University, 6-1-1, Tamagawa-Gakuen, Machida, Tokyo 194-8610, Japan.

Applied and Environmental Microbiology, June 1999, p. 2703-2709, Vol. 65, No. 6
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.




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