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Quantifying the accessibility of the metagenome by random expression cloning techniques
Esther M Gabor1, Wynand B L Alkema, Dick B Janssen
1Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, the Netherlands.
Environmental Microbiology
|August 13, 2004
Summary
Discovering novel biocatalysts from environmental DNA relies on gene expression in surrogate hosts. This study presents formulas to predict gene recovery probability, finding ~40% of enzymatic activities are accessible via random cloning in E. coli.
Area of Science:
- Metagenomics
- Molecular Biology
- Bioinformatics
Background:
- Functional screening of metagenomes for novel biocatalysts requires efficient gene expression in surrogate hosts.
- Gene recovery probability depends on DNA abundance, insert size, gene length, and functional expression signals in the host.
Purpose of the Study:
- To develop a predictive model for the probability of isolating genes via random expression cloning.
- To analyze the accessibility of metagenomic genes based on different heterologous gene expression modes.
- To assess the potential for exploiting metagenomic data for biocatalyst discovery.
Main Methods:
- Formulas were derived to calculate the probability of gene isolation considering independent, transcriptional fusion, and translational fusion expression modes.
- Bioinformatic analysis of 32 complete prokaryotic genome sequences was performed to identify functional expression signals in E. coli.
- Metagenomic data was analyzed to predict the proportion of genes recoverable through random cloning.
Main Results:
- Genes expressed as translational fusions are largely inaccessible through shotgun cloning due to low functional construct frequency.
- Significant variations in predicted expression modes were observed across different prokaryotic taxonomic groups.
- Approximately 40% of enzymatic activities are predicted to be readily recoverable by random cloning in E. coli.
Conclusions:
- Random expression cloning efficiency is significantly influenced by the mode of heterologous gene expression.
- A substantial portion of metagenomic potential for biocatalyst discovery may be overlooked due to limitations in current cloning strategies.
- Targeted approaches may be necessary to access genes not readily recoverable by random cloning in E. coli.