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Applied and Environmental Microbiology, May 2000, p. 2274-2277, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Directed Transfer of Large DNA Fragments between Streptomyces Species

Zhihao Hu,1 David A. Hopwood,2 and Chaitan Khosla1,3,4,*

Departments of Chemical Engineering,1 Chemistry,3 and Biochemistry,4 Stanford University, Stanford, California 94305-5025, and Department of Genetics, John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom2

Received 6 January 2000/Accepted 20 February 2000

The biosynthesis of complex natural products in bacteria is invariably encoded within large gene clusters. Although this facilitates the cloning of such gene clusters, their heterologous expression in genetically amenable hosts remains a challenging problem, principally due to the difficulties associated with manipulating large DNA fragments. Here we describe a new method for the directed transfer of a gene cluster from one Streptomyces species to another. The method takes advantage of tra gene-mediated conjugal transfer of chromosomal DNA between actinomycetes. As proof of principle, we demonstrate transfer of the entire ~22-kb actinorhodin gene cluster, and also the high-frequency cotransfer of two loci that are 150 to 200 kb apart, from Streptomyces coelicolor to an engineered derivative of Streptomyces lividans.

* Corresponding author. Mailing address: Departments of Chemical Engineering, Chemistry, and Biochemistry, Stanford University, Stanford, CA 94305-5025. Phone and fax: (650) 723-6538. E-mail: ck{at}chemeng.stanford.edu.

Applied and Environmental Microbiology, May 2000, p. 2274-2277, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.




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