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Heterologous Expression of Novobiocin and Clorobiocin Biosynthetic Gene Clusters

Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany,¹ Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom²

Received 30 September 2004/ Accepted 24 November 2004

A method was developed for the heterologous expression of biosynthetic gene clusters in different Streptomyces strains and for the modification of these clusters by single or multiple gene replacements or gene deletions with unprecedented speed and versatility. -Red-mediated homologous recombination was used for genetic modification of the gene clusters, and the attachment site and integrase of phage C31 were employed for the integration of these clusters into the heterologous hosts. This method was used to express the gene clusters of the aminocoumarin antibiotics novobiocin and clorobiocin in the well-studied strains Streptomyces coelicolor and Streptomyces lividans, which, in contrast to the natural producers, can be easily genetically manipulated. S. coelicolor M512 derivatives produced the respective antibiotic in yields comparable to those of natural producer strains, whereas S. lividans TK24 derivatives were at least five times less productive. This method could also be used to carry out functional investigations. Shortening of the cosmids' inserts showed which genes are essential for antibiotic production.

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