Active Compounds From Metagenomic Library Of The Marine Sponge Discodermia Calyx

Marine sponges are rich and important sources for secondary bioactive metabolites,many of which are proved to be produced by symbiotic bacteria. However, the vastmajority of the sponge microbial community remains uncultured on laboratoryconditions. Functional metagenomics explores uncultured environmentalmicroorganisms by extracting genomic DNA directly from samples without any cultureor isolation steps. Considering the chemical and biological diversity present in thesponge microbial community, the metagenomic library of marine sponges should be aninteresting resource to search for unique bioactive small molecules and thecorresponding unknown functional genes. Therefore, we constructed the metagenomiclibrary of a marine sponge, Discodermia calyx (D. calyx). Functional screening of thislibrary resulted in the detection of the three different positive clones and furtherresearch led to identification of four porphyrins (1-4) and the corresponding functionalgenes, three fatty acids (5-7) and the corresponding functional genes, eight indolederivatives (8-15) from the liquid culture, and one amide (16), eleven cyclodipeptides(17-27) from plate culture of positive clones. The main results were shown as follows:①The metagenomic library of marine sponge was successfully constructed.Functional screening of the library afforded3different positive clones: pDC113byanti-Bacillus cereus (B. cereus) activity, pDC112and pDC114by their brownish-redcolour.②Four porphyrins: Zn-coproporphyrin III (1), coproporphyrin III (2),Zn-protoporphyrin IX (3) and protoporphyrin IX (4) were produced by brownish-redclone pDC112and pDC114.1was the major red pigment and was elucidated by itscharacteristic1H NMR,13C NMR,1H-1H COSY, HMQC, HMBC, MS and UV data.The sequence analysis of the fosmid DNA in pDC112identified31putative ORFsencoded in40.639kb including cytochrome c-type biogenesis protein CcsB (261aa),glutamyl-tRNA reductase (hemA,420amino acids, aa), porphobilinogen deaminase(hemC,322aa), and porphobilinogen synthase (hemB,322aa), which involved in thebiosynthesis of porphyrins. Sequence analysis of pDC114revealed32putative ORFsencoded in37.753kb containing the homologs involved in the formation of linear andcyclic tetrapyrroles, the enzymes required for the porphyrin biosynthetic C5pathway, such as precorrin-4C(11)-methyltransferase (175aa), cobalamin biosynthesis protein(391aa), precorrin-3B methylase (358aa), hemA (463aa), andglutamate-1-semialdehyde-2,1-aminomutase (hemL,428aa). The hemA commonlypresent in both pDC112and pDC114would be an essential ORF for porphyrinproduction. Considering the importance of porphyrin derivatives for medicinal anddietary uses, the continuous exploitation of the genes with the intent of futureengineering studies on the production of porphyrins in E. coli would be quite valuable.③Three β-hydroxyl fatty acids:3-hydroxypalmitic acid (5),3-hydroxymyristicacid (6) and3-hydroxylauric acid (7) were heterologously expressed in an anti-B.cereus clone pDC113from liquid culture.5showed moderate antibacterial activity(MIC, μg/mL) against B. cereus (6.25) and Candida albicans (C. albicans,12.5). Asequence analysis of the insert DNA of pDC113revealed42putative ORFs encoded in43.32kb. Among them23putative ORFs covering24.813kb involved in thebiosynthesis of fatty acids and lipid A and shared homology to bacterial fatty acidsynthaseⅡ: ACP (acyl carrier protein), plsX (fatty acid/phospholipid synthesis protein),fabD (ACP S-malonyltransferase), fabF (-ketoacyl-acyl carrier protein synthase II),fabG (3-oxoacyl-ACP reductase), and fabZ (-hydroxyacyl-ACP dehydratase), andenzymes involving in lipid A biosynthesis: LpxA, LpxD, LpxB, LpxK, KdtA, LpxL andLpxM. Other ORFs were likely transmembrane proteins involved in the biosynthesis oflipid A. The biosynthetic pathways of fatty acids and lipid A are potentially attractivetargets for the development of antimicrobial agents, especially agents againstdrug-resistant bacteria. The microbes and their interesting genes harbored in marinesponges are a unique source for these drug targets.④Seven indole derivatives (8-14) were also identified from other active fractionsof liquid culture of antibacterial pDC113including12owing strong antibacterialactivity. Their corresponding gene information could not be easily discovered throughdirect sequence blast analysis of42ORFs encoded in43.32kb of the insert DNA ofclone pDC113. Clone pDC113was re-cultured in the presence of tryptamine to checkwhether the production of compound8(Harman) was increased. Unexpectedly,compound15was detected and identified as a novel tris-indole derivative exhibitingstrong antibacterial activity (MIC, μg/mL) against B. cereus (<0.78), MSSA (<0.78) andmoderate activity against C. albicans (6.25), and E. coli (25). It also showed strongcytotoxicity against P388Cells (IC50,1.52μg/mL). ⑤Plate culture was also tried to pDC112and pDC113and different compoundswere detected and separated. One clone specific amide (16) was detected from activefraction of plate culture of pDC112although only its part structure was determined dueto its scarcity and instable. Eleven cyclodipeptides (17-27) were afforded bybioassay-guided fractionation of plate culture of pDC113. To our best knowledge, theseare first cyclodepeptides isolated from metagenomic library. Sequence analysissuggested that isolated cyclodipeptides were synthesized by neither nonribosomalpeptide synthetases (NRPS) nor enzymes sharing homology with reportedcyclodipeptides synthases (CDPSs). It was high possibility that they werebiosynthesized by novel enzymes encoded by interesting genes. This result corroboratesthe most attractive theoretical potential of metagenomics-the improved chance to findnew genes.Twenty seven compounds and functional genes of compound1-7were identifiedfrom metagenomic library of marine sponge D. calyx by functional screening. Resultsdemonstrated that functional screening of metagenomic library of marine sponge D.calyx was practical and quite valuable to discover active compounds and interestingfunctional genes from unique marine sources.