MTT solution was added into the wells and incubated for 2 h. After the medium was removed, DMSO was added to each well. GSK1120212 The plates were gently agitated until the color reaction was uniform, and the OD570 was determined using a microplate reader (Wellscan MK3; Labsystems Dragon). Media-only treated cells with DMSO served as the indicator of 100% cell viability. Antifungal activity tests showed that inhibition zone diameters of the crude extract against Phomopsis asparagi, Polystigma deformans, Cladosporium cucumerinum, Monilinia fructicola, and Colletotrichum lagenarium were 15, 25, 20,
15, and 20 mm, respectively; however, the inhibition zone diameters of blank groups were only 6 mm, which implied great learn more potential of Streptomyces sp. W007 in agricultural fungal disease control. Genome sequence of Streptomyces sp. W007 revealed the presence of 149 open reading frames (ORFs) in the contig 151. A homology search showed that some ORFs were homologous to angucyclinone derivatives biosynthesis genes reported previously. Based on their positions and deduced functions, we identified 20 ORFs (from ORF 4216 to 4235, named ang 1 to ang 20) probably involved in the biosynthesis of angucyclinone antibiotics (Fig. 1). The putative functions of ORFs and the closest homologues are shown
in Table 1. According to blastp results with NCBI nr database, ang 2 shows high percent identity (93%) to SAM-dependent methyltransferase from Streptomyces griseus IFO 13350 (Ohnishi et al., 2008), which can regulate spore development and antibiotic synthesis (Bao et al., 2010). Ang 4 is identified as hydrolase (Ohnishi et al., 2008). Ang 7 and ang 5 are in accordance with short-chain dehydrogenase/reductase (SDR) and 3-oxoacyl-(acyl carrier protein) reductase, which catalyze the reduction in ketone group. Ang 10 shares 56% amino acid identity with O-methyltransferase of
Streptomyces sp. 2238-SVT4 (Kawasaki et al., 2010). Ang 11 is a FAD-binding hydroxylase similar to the Phenylethanolamine N-methyltransferase type II polyketide gene cluster from Streptomyces fradiae (Decker & Haag, 1995). Ang 12 has high similarity of 89% to cyclase in Streptomyces sp. SCC2136 (Basnet et al., 2006). In the gene cluster of angucyclinone antibiotics, the mini PKS is found to be composed of ang 13, 14, and 15 that present the functions of ketoacyl synthase (KSα), chain length factor (KSβ), and ACP, respectively. Ang 16 shows similarity to ketone group reductase of urdamycin A biosynthesis and can be assigned to reduce C-9 (Decker & Haag, 1995). Ang 17 and 20 show high percent identities to aromatase from Streptomyces sp. SCC 2136 (Basnet et al., 2006) and acetyl-coenzyme A carboxyl transferase alpha chain in Streptomyces venezuelae ATCC 10712 (Pullan et al., 2011), respectively. Ang 18 and 19 show sequence similarities to oxygenase reductase and ketoacyl reductase from Streptomyces sp. 2238-SVT4 (Kawasaki et al., 2010).