Identification and Characterization of Acetyl-CoA Carboxylase Gene Cluster in Streptomyces toxytricini

Atanas V. Demirev; Ji Seon Lee; Bhishma R. Sedai; Ivan G. Ivanov; Doo Hyun Nam

doi:10.1007/s12275-009-0135-5

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Identification and Characterization of Acetyl-CoA Carboxylase Gene Cluster in Streptomyces toxytricini: Atanas V. Demirev ¹, Ji Seon Lee ², Bhishma R. Sedai ², Ivan G. Ivanov ³, Doo Hyun Nam ²; Journal of Microbiology 2009;47(4):473-478.
DOI: https://doi.org/10.1007/s12275-009-0135-5
Published online: September 9, 2009

¹Faculty of Biotechnology, Yeungnam University, Gyongsan 712-749, Republic of Korea, ²Faculty of Pharmacy, Yeungnam University, Gyongsan 712-749, Republic of Korea, ³Department of Gene Regulation, Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria

Corresponding author: Doo Hyun Nam , Tel: 82-53-810-2825,

Received: 27 April 2009 • Accepted: 18 June 2009

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Abstract

The gene locus for acetyl-CoA carboxylase (ACC) involved in the primary metabolism was identified from the genomic library of Streptomyces toxytricini which produces a lipase inhibitor lipstatin. The 7.4 kb cloned gene was comprised of 5 ORFs including accD1, accA1, hmgL, fadST1, and stsF. In order to confirm the biochemical characteristics of AccA1, the gene was overexpressed in Escherichia coli cells, and the recombinant protein was purified through Ni2+ affinity chromatography. Because most of the expressed AccA1 was biotinylated by host E. coli BirA in the presence of D-biotin, the non-biotinylated apo-AccA1 was purified after gene induction without D-biotin, followed by exclusion of holo-AccA1 using streptavidin beads. The separated apo-AccA1 was post-translationally biotinylated by S. toxytricini biotin apo-protein ligase (BPL) in a time- and enzyme-dependent manner. This result supports that this gene cluster of S. toxytricini encodes the functional ACC enzyme subunits to be biotinylated.

Keywords: acetyl-CoA carboxylase;biotin carboxylase;carboxyltransferase;biotin apo-protein ligase;Streptomyces toxytricini

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Identification and Characterization of Acetyl-CoA Carboxylase Gene Cluster in Streptomyces toxytricini

J. Microbiol. 2009;47(4):473-478. Published online September 9, 2009

DOI: https://doi.org/10.1007/s12275-009-0135-5

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