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Biotransformation of Flavone by CYP105P2 from Streptomyces peucetius
( Niraula ),( Narayan Prasad ),( Saurabh Bhattarai ),( Na Rae Lee ),( Jae Kyung Sohng ),( Tae Jin Oh ) 한국미생물 · 생명공학회 2012 Journal of microbiology and biotechnology Vol.22 No.8
Biocatalytic transfer of oxygen in isolated cytochrome P450 or whole microbial cells is an elegant and efficient way to achieve selective hydroxylation. Cytochrome P450 CYP105P2 was isolated from Streptomyces peucetius that showed a high degree of amino acid identity with hydroxylases. Previously performed homology modeling, and subsequent docking of the model with flavone, displayed a reasonable docked structure. Therefore, in this study, in a pursuit to hydroxylate the flavone ring, CYP105P2 was co-expressed in a two-vector system with putidaredoxin reductase (camA) and putidaredoxin (camB) from Pseudomonas putida for efficient electron transport. HPLC analysis of the isolated product, together with LCMS analysis, showed a monohydroxylated flavone, which was further established by subsequent ESI/MS-MS, A successful 10.35% yield was achieved with the whole-cell bioconversion reaction in Escherichia coli. We verified that CYP105P2 is a potential bacterial hydroxylase.
In-silico and In-vitro based studies of Streptomyces peucetius CYP107N3 for oleic acid epoxidation
( Saurabh Bhattarai ),( Narayan Prasad Niraula ),( Jae Kyung Sohng ),( Tae Jin Oh ) 생화학분자생물학회(구 한국생화학분자생물학회) 2012 BMB Reports Vol.45 No.12
Certain members of the cytochromes P450 superfamily metabolize polyunsaturated long-chain fatty acids to several classes of oxygenated metabolites. An approach based on in silico analysis predicted that Streptomyces peucetius CYP107N3 might be a fatty acid-metabolizing enzyme, showing high homology with epoxidase enzymes. Homology modeling and docking studies of CYP107N3 showed that oleic acid can fit directly into the active site pocket of the double bond of oleic acid within optimum distance of 4.6 □ from the Fe. In order to confirm the epoxidation activity proposed by in silico analysis, a gene coding CYP107N3 was expressed in Escherichia coli. The purified CYP107N3 was shown to catalyze C9-C10 epoxidation of oleic acid in vitro to 9,10-epoxy stearic acid confirmed by ESI-MS, HPLC-MS and GC-MS spectral analysis. [BMB Reports 2012; 45(12): 736-741]
( Ariungerel Mandakh ),( Narayan Prasad Niraula ),( Eung Pil Kim ),( Jae Kyung Sohng ) 한국미생물 · 생명공학회 2010 Journal of microbiology and biotechnology Vol.20 No.12
Pantothenate kinase (PanK) catalyzes the first step in the biosynthesis of the essential and ubiquitous cofactor coenzyme A (CoA) in all organisms. Here, we report the identification, cloning, and characterization of panK-sp from Streptomyces peucetius ATCC 27952. The gene encoded a protein of 332 amino acids with a calculated molecular mass of 36.8 kDa and high homology with PanK from S. avermitilis and S. coelicolor A3(2). To elucidate the putative function of PanK-sp, it was cloned into pET32a(+) to construct pPKSP32, and the PanK-sp was then expressed in E. coli BL21(DE3) as a His-tag fusion protein and purified by immobilized metal affinity chromatography. The enzyme assay of PanK-sp was carried out as a coupling assay. The gradual decrease in NADH concentration with time clearly indicated the phosphorylating activity of PanK-sp. Furthermore, the ca. 1.4-fold increase of DXR and the ca. 1.5-fold increase of actinorhodin by in vivo overexpression of panK-sp, constructed in pIBR25 under the control of a strong ermE* promoter, established its positive role in secondary metabolite production from S. peucetius and S. coelicolor, respectively.
Malla, Sailesh,Niraula, Narayan Prasad,Liou, Kwangkyoung,Sohng, Jae Kyung Elsevier 2009 Journal of bioscience and bioengineering Vol.108 No.2
<P><B>Abstract</B></P><P>To enhance doxorubicin (DXR) production, the structural sugar biosynthesis genes <I>desIII</I> and <I>desIV</I> from <I>Streptomyces venezuelae</I> ATCC 15439 and the glycosyltransferase pair <I>dnrS/dnrQ</I> from <I>Streptomyces peucetius</I> ATCC 27952 were cloned into the expression vector pIBR25, which contains a strong <I>ermE⁎</I> promoter. The recombinant plasmids pDnrS25 and pDnrQS25 were constructed for overexpression of <I>dnrS</I> and the <I>dnrS/dnrQ</I> pair, whereas pDesSD25 and pDesQS25 were constructed to express <I>desIII/desIV</I> and <I>dnrS/dnrQ–desIII/desIV</I>, respectively. All of these recombinant plasmids were introduced into <I>S. peucetius</I> ATCC 27952. The recombinant strains produced more DXR than the <I>S. peucetius</I> parental strain: a 1.2-fold increase with pDnrS25, a 2.8-fold increase with pDnrQS25, a 2.6-fold increase with pDesSD25, and a 5.6-fold increase with pDesQS25. This study showed that DXR production was significantly enhanced by overexpression of potential biosynthetic sugar genes and glycosyltransferase.</P>