Metagenome-Based Functionalgene (Cluster)Study Of Sponge And Its Associatied Bacterias

Since 1950s, many active materials have been found from marine sponges. It was suggested that some of active materials are from bacteria associated with sponge, but direct proof need to be found. Many nicrobes with antimicrobial function have been isolated from sponge, it is of great value to study functional gene (cluster) with the aim to reveal the molecular mechanism of the antimicrobial compounds. Meanwhile, because that the majority of sponge-associated microbes are still not culturable at present, therefore, the construction of metagenome library is valuable for the utilization of uncultured microbial functional gene.To date, researches on PKS and NRPS gene clusters are rarely found, although some PKS and NRPS compounds with anti-cancer, antimicrobe had been found from sponge. This paper aim to get PKS and NRPS gene (cluster) using metagenome-based technique in order to find molecular basis for antimicrobial function and proof to the sponge-associated microbial role in the synthesis of active compound. At the same time, the construction of sponge metagenome library containing large segment DNA will be helpful for the screening of functional gene cluster of uncultured microbes.Genomic DNA of Staphylococcus lentus A75, with broad anti-microbial function according to our previous investigation, was extracted for the construction of Fosmid library. The size of insert DNA is from 25kb to 43kb in this library. Through PCR and anti-microbial activity screening,a positive clone against Bacillus subtilis was isolated from metagenome library and a gene cluster about 26kb in length was sequenced. Finally, the entire PKS gene about 21.8kb in length that probably coded a polyketide synthase cotaining 22 DH, AT, ACP, KS, KR which catalyzed the synthesis of a polyketide with 12C in its main chain was obtained.Genomic DNA of Bacillus sp. B114, with broad anti-microbial function according to our previous study, was extracted for the construction of Fosmid library. Through PCR a NRPS gene fragment about 974bp in length, which belonged to Adenylation domain, was obtained. Metagenomic DNA of four sponges Stelletta tenui,Halichondria regosa,Dysidea avara and Craniella australiensis from the South China Sea were extracted for the screening functional genes by means of PCR. As a result, a KS gene about 671bp in length was obtained from Halichondria regosa. BLAST analysis showed that the homology of the amino acid sequence with that of the KS domain of PKS gene in Rhodobacterales bacterium was up to 96%. It provided powerful proof to the microbial origin hypothesis of sponge active compounds. Phylogenetic analysis indicated that the obtained PKS gene belongs to trans-AT KS domains. Consequently, metagenomic DNA of Halichondria regosa was extracted to construct Fosmid library. The size of insert DNA is from 26kb to 39kb in this library, that can meet the need of gene cluster screening.In this paper, (1) a Fosmid metagenome library of Stelletta tenui associated Staphylococcus lentus A75 was constructed and a 21.8kb PKS gene cluster cotaining AT,ACP,KS,KR was obtained, which lay a basis for PKS active compound study and its biological combined synthesis. (2) 974bp NRPS gene fragment belonging to Adenylation domain was found in the metagenome library of Bacillus sp. B114 associated with Halichondria regosa, which lay a basis for NRPS compound study. (3) The finding of KS domain of PKS gene in sponge Halichondria regosa provided powerful proof to the microbial origin hypothesis of sponge active compounds. (4) The construction of metagenomic Fosmid library of Halichondria regosa containing large segement DNA from 26kb to 39kb provide a resource for functional gene cluster screening.This investigation is with great innovation because that the study on the functional gene (cluster) of sponge and its associated microbes has just begun in the world.