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아타나스데미레프,Ji Seon Lee,Bhishma R. Sedai,Ivan G. Ivanov,남두현 한국미생물학회 2009 The journal of microbiology Vol.47 No.4
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.
Demirev, Atanas V.,Khanal, Anamika,Sedai, Bhishma R.,Lim, Si Kyu,Na, Min Kyun,Nam, Doo Hyun Springer-Verlag 2010 Applied microbiology and biotechnology Vol.87 No.3
<P><I>Streptomyces toxytricini</I> produces lipstatin, a specific inhibitor of pancreatic lipase, which is derived from two fatty acid moieties with eight and 14 carbon atoms. The <I>pccB</I> gene locus in 10.6 kb fragment of <I>S. toxytricini</I> chromosomal DNA contains three genes for acyl-coenzyme A carboxylase (ACCase) complex <I>accA3</I>, <I>pccB</I>, and <I>pccE</I> that are presumed to be involved in secondary metabolism. The <I>pccB</I> gene encoding a β subunit of ACCase [carboxyltransferase (CT)] was identified upstream of <I>pccE</I> gene for a small protein of ε subunit. The <I>accA3</I> encoding the α subunit of ACCase [biotin carboxylase (BC)] was also identified downstream of <I>pccB</I> gene. When the <I>pccB</I> and <I>pccE</I> genes were inactivated by homologous recombination, the lipstatin production was reduced as much as 80%. In contrast, the accumulation of another compound, tetradeca-5.8-dienoic acid (the major lipstatin precursor), was 4.5-fold increased in disruptant compared with wild-type. It implies that PccB of <I>S. toxytricini</I> is involved in the activation of octanoic acid to hexylmalonic acid for lipstatin biosynthesis.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (doi:10.1007/s00253-010-2587-2) contains supplementary material, which is available to authorized users.</P>