Polyketide Synthase (PKS) And Non-Ribosomal Peptide Synthase (NRPS) Genes From Phylogenetically Diverse Cultivable Fungi Associated With The South China Sea Sponges

In this study, six different fungal media (Martins, 2216, M1, Gause, MYPG and Czapek Sabouraud media) and three temperature gradient (20℃,25℃,30℃) were used to investigate the phylogenetic diversity of culturable fungi associated with ten sponges collected from the South China Sea based on RFLP analysis and 18S rRNA gene sequences. Sterile oxford cup-based method was used to examine antimicrobial activities against E. coli, P. fluorescens, S. aureus, B. subtilis and A. nigers, C. albicans. Meanwhile, PKS and NRPS genes of the 177 culturable fungi were screened by PCR method.The results revealed that 177 fungi were isolated from 10 sponges collected from the South China Sea using 6 media at 3 different temperature, where 8 from X4, 23 from X5, 32 from X6, 1 from X7, 4 from X8, 35 from X9, 22 from X10, 17 from X11, 25 from X12 totally. There was no obvious difference among three cultivation temperature 20℃, 25℃and 30℃. Among the tested 6 media, Martins medium and MYPG medium cultivated the most fungal isolates (n=45 and n=44 respectively). In Czapek's medium and M1 medium, 32 and 24 isolates from marine sponge, respectively. Based on fungi 18S rDNA specific primers, we amplify 760bp fragments with PCR. Used Taq I, Hinf I and Hae III to accomplish RFLP enzyme-digestion differing in patterens combined with colony morphology, 24 independent fungi strains were selected. Partial sequencing of the 18S rRNA gene of these fungal isolates and analyzed by BLAST revealed that: 24 representative isolates were classified into 4 orders(Microascales,Phyllachorales,Hypocreales and Eurotiales) and 10 genera of Ascomycota. The prodominant isolates were Eurotiales (37.5%) and Hypocreales (41.6%).All the 177 fungal isolates were examined for activity against E. coli, P. fluorescens, S. aureus, B. subtilis and A. nigers, C. albicans.The fungal isolates showed antibacterial activity with 3(x4-167,x11-75,x12-91) against B. subtilis, 8(x6-13,x10-49,x10-63,x12-94,x12-95,x12-109,x13-105,x13-118) against P. fluorescens, especially X13-105, X12-94 and X13-118 displayed antibacterial activity against both P. fluorescens and S. aureus.15 polyketide synthases gene sequences 790bp in length were detected in these fungal isolates. The PKS genes showed high sequence similarity to parts of Penicillium chrysogenum Wisconsin 54-1255 complete genome and Aspergillus fumigatus Af293 polyketide synthase gene by BLAST analyzed. Meanwhile, 4 nonribosomal peptides synthesis gene sequences 1200bp in length were detected in these fungal isolates. The NRPS genes showed high sequence similarity to parts of Penicillium chrysogenum Wisconsin 54-1255 complete genome by BLAST analyzed.In this study, a great rich and diverse culturable fungi associated with sponges collected from the South China Sea was revealed by molecular identification and phylogenetic analysis.As the largest source of activity products produced by marine fungi, some strains of Penicillium and Aspergillus isolated in this study showed a broad-spectrum antimicrobial activity against bacteria.The PKS and NRPS genes were found existed in some strians,which suggested that the fungi with PKS and NRPS may play a role in sponge chemical joint defense. In the research of fungal PKS and NRPS genes, a variety of PKS or NRPS genes were found in the same strains, based on the diversity and novelty of PKS and NRPS genes which can guide the selection of bacteria with medicinal activity. Based on antibiotic activity assay, some strains showed antimicrobial activities towards P.fluorescens which had a great guidance of the screening of sponge-associated fungi with new compounds synthesis ability.Our study suggests the role of sponge-symbiosis bacteria played in the host's chemical defense and provides further proof for the hypothesis that sponge-associated bacteria are the true producers of many novel bioactive compounds.