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Quantifying the accessibility of the metagenome by random expression cloning techniques

Authors

  • Esther M. Gabor,

    1. Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, the Netherlands.
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    • Both authors contributed equally to this work.

  • Wynand B. L. Alkema,

    1. Center for Genomics and Bioinformatics, Karolinska Institute, Berzelius väg 35, 17177 Stockholm, Sweden.
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    • Both authors contributed equally to this work.

  • Dick B. Janssen

    Corresponding author
    1. Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, the Netherlands.
      *E-mail D.B.Janssen@chem.rug.nl; Tel. (+31) 50 3634209; Fax: (+31) 50 3634165.
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*E-mail D.B.Janssen@chem.rug.nl; Tel. (+31) 50 3634209; Fax: (+31) 50 3634165.

Summary

The exploitation of the metagenome for novel biocatalysts by functional screening is determined by the ability to express the respective genes in a surrogate host. The probability of recovering a certain gene thereby depends on its abundance in the environmental DNA used for library construction, the chosen insert size, the length of the target gene, and the presence of expression signals that are functional in the host organism. In this paper, we present a set of formulas that describe the chance of isolating a gene by random expression cloning, taking into account the three different modes of heterologous gene expression: independent expression, expression as a transcriptional fusion and expression as a translational fusion. Genes of the last category are shown to be virtually inaccessible by shotgun cloning because of the low frequency of functional constructs. To evaluate which part of the metagenome might in this way evade exploitation, 32 complete genome sequences of prokaryotic organisms were analysed for the presence of expression signals functional in E. coli hosts, using bioinformatics tools. Our study reveals significant differences in the predicted expression modes between distinct taxonomic groups of organisms and suggests that about 40% of the enzymatic activities may be readily recovered by random cloning in E. coli.

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