Fungal Diversity DOI 10.1007/s13225-012-0192-7 Phylogenetically diverse endozoic fungi in the South China Sea sponges and their potential in synthesizing bioactive natural products suggested by PKS gene and cytotoxic activity analysis Zhisheng Yu & Baohua Zhang & Wei Sun & Fengli Zhang & Zhiyong Li Received: 20 June 2012 /Accepted: 20 July 2012 # Mushroom Research Foundation 2012 Abstract Sponges are well documented to harbor large Aspergillus as the predominant component in the culturable amounts of microbes. Though it is known that sponge- fungal community. Particularly, genera Schizophyllum, derived fungi are important sources for marine natural prod- Sporidiobolus, and Bjerkandera in phylum Basidiomycota ucts, the phylogenetic diversity and biological function of and genus Yarrowia in phylum Ascomycota were isolated sponge-associated fungi remain largely unknown. In this from marine sponges for the first time. PKS genes were study, the diversity of culturable endozoic fungi in sponges detected in 12 isolates suggesting their potential for synthe- from the South China Sea was revealed based on the ITS sizing PKS compounds. Among the 12 isolates with PKS phylogenetic analysis. Meanwhile the fungal potential for genes, 9 isolates displayed strong in vitro cytotoxic activity producing bioactive natural products was estimated accord- (e.g. IC50<50 μg/ml) against human cancer cell lines A- ing to the detection of Beta-ketosynthase in the polyketide 549, Bel-7402, A-375 and MRC-5. This study demonstrates synthase (PKS) gene cluster and cytotoxic activity bioassay. the phylogenetically diverse endozoic fungi in South China As a result, diverse fungi including 14 genera (Aspergillus, Sea sponges, and highlights the potential of sponge- Penicillium, Scolecobasidium, Eurotium, Alternaria, Fusa- associated fungi in producing biologically active natural rium, Hypocreales, Yarrowia, Candida, Hypoxylon, Spori- products. diobolus, Schizophyllum, Bjerkandera, and Trichosporon) in ten orders (Xylariales, Moniliales, Pleosporales, Saccha- Keywords Cytotoxic activity . Fungal diversity . Polyketide romycetales, Hypocreales, Eurotiales, Sporidiobolales, synthase (PKS) gene . Sponge Agaricales, Aphyllophorales and Tremellales) of phyla Ascomycota and Basidiomycota were isolated with Introduction Zhisheng Yu and Baohua Zhang contributed equally to this paper. Z. Yu : W. Sun : F. Zhang : Z. Li Since the pioneering work of Barghoorn and Linder (1944), Marine Biotechnology Laboratory, State Key Laboratory significant progress has been made on marine-derived fungi of Microbial Metabolism and School of Life Sciences (Bugni and Ireland 2004; Raghukumar 2008; Debbab et al. and Biotechnology, Shanghai Jiao Tong University, 2011; Jones 2011). However, compared with their terrestrial Shanghai 200240, People’s Republic China counterparts, marine fungi remain one of the most under B. Zhang explored groups in the marine environment, especially ma- Eastern Hepatobiliary Surgery Hospital, rine fungi associated with marine macro-organisms (Wang Second Military Medical University, et al. 2008; Li and Wang 2009). Shanghai 200438, People’s Republic China As the oldest animal e.g. over 600 million years old, Z. Li (*) marine sponges (Porifera) form close association with a School of Life Sciences and Biotechnology, wide variety of microorganisms including bacteria, archaea Shanghai Jiao Tong University, and fungi (Hentschel et al. 2006; Taylor et al. 2007a, b; Gao 800 Dongchuan Road, Shanghai 200240, People’s Republic China et al. 2008; Liu et al. 2010; Ding et al. 2011; Lee et al. 2011; e-mail: [email protected] Paz et al. 2010; Zhou et al. 2011; Schmitt et al. 2012; Fungal Diversity Webster and Taylor 2012). In contrast to the understanding Cinachyrella australiensis (3–10), Diplastrella megastellat of the sponge bacterial diversity (Lee et al. 2011; Webster (1–9) and Geodia neptuni (1–11) were collected nearby and Taylor 2012; Schmitt et al. 2012), little is known about Yongxin Island and LingShui in the South China Sea at a the fungal diversity due to the lack of direct evidence of depth of ca.10–20 m. Sponges were transferred directly to fungal mycelia, for example, microscopic, immunological, zip-lock bags containing sea water to prevent the contact of or fluorescence in situ hybridization detection. At present, sponge tissue with the air. The samples were transported to studies on the fungal diversity in sponges are relatively few the laboratory on ice and processed immediately for fungal than those addressing the metabolic versatility of sponge- isolation. derived fungi (Lang et al. 2007; Proksch et al. 2008; Xie et Before the isolation of endozoic fungi, sponge sample al. 2008; Abdel-Lateffa et al. 2009; Zhang et al. 2009a; Lee was cut into small pieces and rinsed three times with sterile et al. 2010). Meanwhile, the biological and ecological func- artificial seawater (ASW) (Li and Liu 2006) to get rid of tions of sponge-associated fungi remain largely unanswered fungi on the sponge surface and in the sponge inner cavity. (Maldonado et al. 2005; Rot et al. 2006; Baker et al. 2009; Sponge inner issue was cut into pieces (1–3cm3) with a Paz et al. 2010). sterile scalpel and immersed in sterile calcium-and- Fungi associated with sponges display diverse biological magnesium-free ASW for 10 min. Then, the small sponge activities and represent the single most prolific source for pieces were homogenized by a high-speed pulse-type ho- marine fungi-derived bioactive compounds (Proksch et al. mogenizer machine. The homogenate was diluted with ster- 2003, 2008, 2010; Bugni and Ireland 2004; Amagata et al. ile ASW at three dilutions (1:10, 1:100, 1:1000). One 2006a; Bhadury et al. 2006; Lang et al. 2007; Saleem et al. hundred micro liters of each dilution was plated onto Martin 2007; Xie et al. 2008; Abdel-Lateffa et al. 2009; Lee et al. and MYPG media (Ding et al. 2011) in triplicate and incu- 2010; Wiese et al. 2011), suggesting their importance in the bated at 28 °C for 7–15 days. All the media were prepared development of marine drugs. Polyketides have been im- with ASW and adjusted to pH 7.4–7.6. Media were auto- mensely concerned over the past decades. Various novel claved at 121 °C for 20 min except the medium containing polyketide compounds with biological activities and ecolog- glucose which was autoclaved at 115 °C for 30 min. Thirty ical functions have been found from marine-derived micrograms per liter of streptomycin and ampicillin were microbes (Blunt et al. 2009). The presence of polyketide added to the media to inhibit bacterial growth. synthase (PKS) in sponge-associated fungi suggests the potential in producing related polyketide compounds and Genomic DNA extraction, PCR amplification of rDNA-ITS guides the production of related natural products. The filtra- fragment and phylogenetic analysis tion of PKS genes from sponge-associated bacteria has started (Schirmer et al. 2005; Kim and Fuerst 2006; Jiang After cultivation in Martin medium at 180 rpm, 28 °C for 5– et al. 2007; Hochmuth and Piel 2009; Zhang et al. 2009b, c; 7 days, the cultures were centrifuged at 5,000×g for 5 min. Siegl and Hentschel 2010), but until now, the screening of The precipitate was grinded in a mortar containing 600 μl PKS genes in sponge-associated fungi is still very scarce CTAB lysis buffer (2 % CTAB, 1.4 M NaCl, 100 mM Tris, except our recent report (Zhou et al. 2011). 20 mM EDTA, 1 % PVP). The mycelial mixture was trans- In this study, the culturable endozoic fungal associated ferred into a 1.5-ml Eppendorf tube and heated to 65 °C for with marine sponges in the South China Sea were isolated 30 min, extracted twice with an equal volume of phenol/ and their phylogenetical diversity was investigated using chloroform/isoamyl alcohol (25:24:1) and washed with ITS phylogenetic analysis. Meanwhile, their potential for chloroform/isoamyl alcohol (24:1). After centrifugation at producing bioactive natural products was evaluated accord- 10,000×g for 5 min, the supernatant was transferred to a ing to the detection of the conserved Beta-ketosynthase of new microtube and precipitated by adding equal volume of polyketide synthase (PKS) gene and the bioassay of cyto- isopropanol at −20 °C for 1 h. Finally, the DNA pellets were toxic activity. collected by centrifugation (12,000×g, 15 min), washed with 75 % ethanol twice and re-suspended in 40 μlTE Buffer (10 mM Tris, 1 mM EDTA, pH 8.0). RNA was Materials and methods removed by adding 2 μl of RNase A (10 mg/ml; Invitrogen) at 60 °C for 10 min. Sponge sampling and isolation of sponge-associated fungi The resulting genomic DNA solution was used as a template to amplify the fungal rDNA-ITS fragment (500– Ten species known marine sponges Amphimedon queens- 800 bp) using the primers ITS1 and ITS4 (White et al. landica (2–1), Holoxea sp. (3–1), Phyllospongia foliascens 1990). The PCR reaction mixture (40 μl) was compose of (3–2), Iotrochota sp. (3–3), Ircinia felix (3–5), Aplysina Premix Taq 20 μl (TaKaRa Taq 1.25U/25 μl, dNTP Mixture aerophoba (3–6), Xestospongia testudinari (3–7), 2×conc.; 0.4 mM, PCR Buffer 2×conc.; 3 mM Mg2+), 1 μl Fungal Diversity of each primer (10 μM), 1 μl of fungal DNA, 2 μl DMSO proteins in the nr protein database using the BLASTP algo- and 15 μl of ddH2O. PCR was carried out as follows: initial rithm. Unrooted phylogenetic tree based on amino acid denaturation (94 °C for 5 min), 30 cycles of denaturation sequences of KS domain was constructed using neighbor- (94 °C for 30 s), primer annealing (56 °C for 30 s), and joining method in MEGA 4.0 combined with bootstrap elongation (72 °C for 1 min), with a final longation at 72 °C analysis with 1,000 replications. for 10 min. PCR products were purified using Cycle-pure Kit (Omega) according to the manufacturer’s instructions.