抄録
In the ocean, many structurally unique compounds that have significant biological activities have been isolated from various marine invertebrates. Especially, sponges, belonging to the porifera, are also isolated many natural products. Recent research suggests that many marine sponges harbor various microbial symbionts, by which many bioactive compounds are produced, and that the number of cultivable bacteria represents 1% or fewer of the total environmental bacteria. So, to take advantage of sponge symbionts efficiently, the metagenomic analysis is optimal as culture independent analysis technique. In this study, we isolate and analyze metagenomic DNA from bacterial symbionts of Japanese marine sponge, H. okadai, and obtained genomic information for bacterial symbionts. Marine sponge, H. okadai, was crushed with buffer and separated by centrifuge and the genomic DNA was extracted from bacteria fraction. Firstly, isolated genomic DNA was sequenced on a Roche FLX pyrosequencer (Roche, Mannheim, Germany). Pyrosequencing of this DNA yielded 230,000 reads (50 Mbp). Then, metagenomic DNA was ligated into fosmid vector pCC1FOS vector (Epicentre) and the ligated vectors were transformed into E. coli EPI300 (Epicentre). The transformants were spread onto LB medium. A total of 150000 fosmid clones metagenomic library were constructed. Constructed metagenomic library derived from H. okadai, was screened for pigment production to obtain several active clones. Then, tinted clones were screened and the compound was purified from culture broth of clone by several chromatography steps to afford single isolated compound. We clarified that yellow pigment shows cytotoxicity to B16 cell(IC50 11.8 μM).
