Title Identification and Characterization of Acetyl-CoA Carboxylase Gene Cluster in Streptomyces toxytricini
Author Atanas V. Demirev1, Ji Seon Lee2, Bhishma R. Sedai2, Ivan G. Ivanov3, and Doo Hyun Nam2*
Address 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
Bibliography Journal of Microbiology, 47(4),473-478, 2009,
Key Words acetyl-CoA carboxylase, biotin carboxylase, carboxyltransferase, biotin apo-protein ligase, Streptomyces toxytricini
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.