Identification and characterization of acetyl-CoA carboxylase gene cluster in Streptomyces toxytricini.
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 AccAl 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.
Demirev
Atanas V
AV
Faculty of Biotechnology, Yeungnam University, Gyongsan 712-749, Republic of Korea.
Lee
Ji Seon
JS
Sedai
Bhishma R
BR
Ivanov
Ivan G
IG
Nam
Doo Hyun
DH
D000103:Acetyl-CoA Carboxylase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D019921:Biotinylation; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000103:Acetyl-CoA Carboxylase