Hiroyasu Onaka

The biosynthetic gene cluster for rebeccamycin, an indolocarbazole antibiotic, from Lechevalieria aerocolonigenes ATCC 39243 has 11 ORFs. To clarify their functions, mutants with rebG, rebD, rebC, rebP, rebM, rebR, rebH, rebT, or orfD2_disrupted were constructed, and the gene products were examined. rebP disruptants produced 11,11'-dichlorochromopyrrolic acid, found to be a biosynthetic intermediate by a bioconversion experiment. Other genes encoded N-glycosyltransferase (rebG), monooxygenase (rebC), methyltransferase (rebM), a transcriptional activator (rebR), and halogenase (rebH). Staurosporine is a representative member of indolocarbazole antibiotics. The entire staurosporine biosynthetic and regulatory gene cluster spanning 20-kb was cloned from Streptomyces sp. TP-A0274 and sequenced. The gene cluster consists of 14 ORFs and the amino acid sequence homology search revealed that it contains three genes, staO, staD, and staP, coding for the enzymes involved in the indolocarbazole aglycone biosynthesis, two genes, staG and staN, for the bond formation between the aglycone and deoxysugar, eight genes, staA, staB, staE, staJ, staI, staK, staMA, and staMB, for the deoxysugar biosynthesis and one gene, staR is a transcriptional regulator. staO, staD, and staP were similar to rehO, rebD, and rebP, respectively, all of which are responsible for indolocarbazole biosynthesis, But a rebC homolog, encoding a putative enzyme oxidizing the C-7 site of pyrrole rings, was not found in the staurosporine biosynthetic gene cluster. These results suggest that indolocarbazole is constructed by oxidative decarboxylation of chromopyrrolic acid (11,11'-dichlorochromopyrrolic acid in rebeccamycin) generated from two molecules of tryptophan by coupling and that the oxidation state at the C-7 position depends on the additional enzyme(s) encoded by the biosynthetic genes. Moreover, heterologous gene expression of a complete set of the biosynthetic genes for staurosporine and rebeccamycin cloned into pTOYAMAcos confirmed their role in staurosporine and rebeccamycin biosynthesis.

To survey endophytic actinomycetes as potential biocontrol agents against fungal diseases of rhododendron, young plants of rhododendron were surface-sterilized for use as an isolation source. Nine, six and two isolates, with distinguishing characteristics based on the macroscopic appearance of colonies, were obtained from roots, stems and leaves, respectively, suggesting that various species of actinomycetes grow in the respective organs of this plant as symbionts or parasites. On an agar medium, only isolate R-5 commonly formed a clear growth-inhibition zone against two major fungal pathogens of rhododendron, Phytophthora cinnamomi and Pestalotiopsis sydowiana, indicating that this isolate can produce antifungal material (s) . Acetone extracts of a liquid culture of R-5 had a broad antimicrobial spectrum against Gram-positive bacteria, yeast and filamentous fungi. Isolate R-5 was identified as a Streptomyces sp. based on morphological, physiological and chemotaxonomical characteristics. The present results indicate that isolate R-5 is a suitable candidate for the biocontrol of diseases of rhododendron.