Directed transfer of large DNA fragments between Streptomyces species.
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 approximately 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.
Hu
Z
Z
Departments of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
Hopwood
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DA
Khosla
C
C
D001426:Bacterial Proteins / Q000235:genetics; D003227:Conjugation, Genetic; D029968:Escherichia coli Proteins; D018014:Gene Transfer Techniques; D005798:Genes, Bacterial; D005818:Genetic Engineering / Q000379:methods; D005810:Multigene Family; D013302:Streptomyces / Q000235:genetics*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C067119:kilA protein, E coli