Amplification of the entire kanamycin biosynthetic gene cluster during empirical strain improvement of Streptomyces kanamyceticus

doi:10.1073/pnas.0603251103

Amplification of the entire kanamycin biosynthetic gene cluster during empirical strain improvement of Streptomyces kanamyceticus

*Department of Molecular Microbiology, John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom; and
^†Microbiological Resources and Technology Laboratories, Meiji Seika Kaisha Ltd., 788 Kayama, Odawara-shi, Kanagawa 2500852, Japan

Communicated by H. Boyd Woodruff, Soil Microbiology Associates, Watchung, NJ, April 21, 2006 (received for review March 6, 2006)

Abstract

Streptomyces kanamyceticus 12-6 is a derivative of the wild-type strain developed for industrial kanamycin (Km) production. Southern analysis and DNA sequencing revealed amplification of a large genomic segment including the entire Km biosynthetic gene cluster in the chromosome of strain 12-6. At 145 kb, the amplifiable unit of DNA (AUD) is the largest AUD reported in Streptomyces. Striking repetitive DNA sequences belonging to the clustered regularly interspaced short palindromic repeats family were found in the AUD and may play a role in its amplification. Strain 12-6 contains a mixture of different chromosomes with varying numbers of AUDs, sometimes exceeding 36 copies and producing an amplified region >5.7 Mb. The level of Km production depended on the copy number of the Km biosynthetic gene cluster, suggesting that DNA amplification occurred during strain improvement as a consequence of selection for increased Km resistance. Amplification of DNA segments including entire antibiotic biosynthetic gene clusters might be a common mechanism leading to increased antibiotic production in industrial strains.

Footnotes

^‡To whom correspondence should be addressed. E-mail: mervyn.bibb{at}bbsrc.ac.uk
Author contributions: K.Y., T.M., and M.B. designed research; K.Y. performed research; and K.Y. and M.B. analyzed data; and K.Y. and M.B. wrote the paper.
Conflict of interest statement: No conflicts declared.
Data deposition: The sequences reported in this paper have been deposited in the Genbank database (accession nos. AB254080, AB254081, and AB254082).
Abbreviations:

Abbreviations:

AUD,

amplifiable unit of DNA;

Rs,

recombination site;

PFGE,

pulsed-field gel electrophoresis;

CRISPR,

clustered regularly interspaced short palindromic repeats;

Km,

kanamycin.
Freely available online through the PNAS open access option.