Metagenomics-Based Cloning of Amilase-Encoding Genes from the Uncultured Symbiotic Bacteria of a Marine Sponge Theonella swinhoei from Kapoposang Island, South Sulawesi

Franciscus Edi Priyono; Dewi Seswita Zilda; Yudi Kusnadi; Tri A Hadi; Irvina Nurrachmi; Agustinus Robert Uria

doi:10.15578/squalen.272

Metagenomics-Based Cloning of Amilase-Encoding Genes from the Uncultured Symbiotic Bacteria of a Marine Sponge Theonella swinhoei from Kapoposang Island, South Sulawesi

Franciscus Edi Priyono, Dewi Seswita Zilda, Yudi Kusnadi, Tri A Hadi, Irvina Nurrachmi, Agustinus Robert Uria

Abstract

Marine sponges have recently been recognized as the source ofenzymes, including members of hydrolases. Hydrolytic enzymes are extracellularly produced by sponge-associated bacteria to mediate the metabolism of complex organic matters, thereby assisting the sponge hosts in nutrition and metabolic processes. Among hydrolytic enzymes, amilaseshas attracted increasing attention due to their potential industrial applications. This research work was aimed atutilizing functional metagenomicsapproach for the discovery of amilases derived from the uncultured symbiotic bacteriaof the Indonesian marine sponge Theonella swinhoei. Weinitially constructed a small-insert metagenomiclibrary in Escherichia coliby cloning of metagenome in the size range of5-20 kb prepared from the sponge’s microbiome. Further functional screening of the resulting metagenomic library led to the isolation of two recombinant E. coli clones potentially harboring amilase genes, as indicated by the presence of clearing zones surroinding the selective medium containing 1% amilum.

Keywords

Theonella swinhoei; Uncultured symbiotic bacteria; DNA cloning; amylase gene

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References

Amann R.I., Ludwig W., and Schleifer K.-H. (1995). Phylogenetic identification and in situ detection of individual microbial cells without cultivation.Microbiol. Rev. 59(1):143-169.

Bradford M.M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding.Analytical Biochemistry 72, 248-254.

Chasanah E., Zilda D.S., and Uria A.R. (2009).Screening and characterization of bacterial chitosanase from marine environment.Journal of Coastal Development 12 (2):64-72.

FebyA., and Nair S. (2010). Sponge-associated bacteria of Lakshadweep coral reefs, India: resource for extracellular hydrolytic enzymes. Advances in Bioscience and Biotechnology 1:330-337

Friedrich A.B., I. Fischer, P. Proksch, J. Hacker, and U. Hentschel.2001. Temporal variation of the microbial community associated with the Mediterranean sponge Aplysinaaerophoba. FEMS Microbiol. Ecol. 38(2-3):105–113.

Gray, J.S. (1868). Note on Theonella, a new genus of coralloid sponges from Formosa. Proceedings of the Zoological Society of London (3): 565-566.

Gupta R., Gigras P., Mohapatra H., Goswami V.K., and Chauhan B. (2003). Microbial α-amylases: a biotechnological perspective. Process Biochem. 38:1599–1616.

Hooper, J.N.A. (2003).Sponguide: guide to sponge collection and identification. Queensland Museum, Brisbane. 26pp.

Hugenholtz P., Goebel B.M., and Pace N.R. (1998).Impact of culture - independent studies on the emerging phylogenetic view of bacterial diversity.J. Bacteriol. 180(18):4765-4774.

Marx, J.C., Collins, T., D'Amico, S., Feller, G. and Gerday, C. (2007).Cold-adapted enzymes from marine antarctic microorganisms. Marine Biotechnology, 9(3):293-304.

Pandey A., Nigam P., Soccol C.R., Soccol V.T., Singh D., and Mohan R. (2003). Advances in microbial amylases. Biotechnol Appl Biochem. 31(Pt 2):135–152

Pabel, C. T., Joachim, V., W ilde, C., Franke, P.,Hofemeister, J., Adler, B., Bringmann, G., Hacker, J.,and Hentschel, V. 2003. Antimicrobialactivities andmatrix assisted laser desorption/ ionization massspectrometry of Baccilus isolated from the marine spons Aplysinaaerophoba. Marine Biotechnology.p. 424-434.

Sanger F., Nicklen S., and Coulson A.R. (1977).DNA sequencing with chain-terminating inhibitors.ProcNatlAcadSci USA 74, 5463-5467.

SjofjanO., and Ardyati T. (2011). Extracellular amylase activity of amylolyticbacteria isolated from Quail’s (Coturnix japonica) intestinal tract in Corn Flour Medium. International Journal of Poultry Science 10 (5): 411-415, 2011

Taylor M.W., Radax R., Steger D., Wagner M. (2007)..Sponge-associatedmicroorganisms: evolution, ecology, and biotechnological potential.MicrobiolMolBiol Rev. 71:295-347.

UriaA.R. and Piel J. (2009). Cultivation-independent approaches to investigate thechemistry of marine symbiotic bacteria. Phytochemistry Rev. 8:401–414.

UriaA.R., and Chasanah E. (2005). Chitinase and chitosanase from microorganisms associated with marine sponges. Proceeding of the 9th ASEAN Food Conference on 8-9th August 2015 in Jakarta.

UriaA.R., and Zilda D.S. (2016). Metagenomics-guided mining of commercially useful enzymes from marine microorganisms.Chapter - Advances in Food and Nutrition Research in the book “marine enzymes biotechnology: production and industrial applications, part I“– published by Elsevier. doi:10.1016/bs.afnr.2016.05.001.

Vaseekaran, S., Balakumar, S., and Arasaratnam, V. 2010.Isolation and Identificationof a Bacterial Strain Producing Thermostable α-Amylase.Tropical Agricultural Research22: 1, 1-11.

Webster N.S, and Hill R.T. (2001). The culturable microbial community of the Great Barrier Reef sponge Rhopaloeidesodorabile is dominated by an α-Proteobacterium. Mar. Biol. (Berl) 138:843–851.

Zhang C., and Kim S-K. (2010). Research and application of marine microbial enzymes: status and prospects. Mar. Drugs 8(6): 1920-1934.

DOI: https://doi.org/10.15578/squalen.272