Chapter 12 : DNA stable Isotope Probing and Gene Mining

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This chapter reviews the state of the art in isolation of new and valuable industrial enzymes from natural biodiversity, and discusses the strengths and weaknesses of each approach and shows how results with DNA stable isotope probing (DNA-SIP) indicate that this technique has substantial promise for improving the effectiveness of strategies for discovering new and valuable enzymes. The many successes of microbial enzymes from extreme environments as biocatalysts have generated an increasing demand for new, robust, and highly specific enzymes to perform all manner of transformations. Metagenomics has emerged as a powerful approach to access genes from uncultivated microbes by direct cloning of microbial DNA extracted from the environment. The study by Jeon and colleagues combined DNA-SIP and traditional microbiological methods to discover novel microorganisms involved in the degradation of the polyaromatic pollutant naphthalene. This study is an excellent example of how such techniques could be combined to discover new enzymes. The DNA-SIP gene mining approach allows the enrichment and recovery of functional genes from active and potentially uncultivated microorganisms and therefore is of great interest to the biotechnology industry. DNA-SIP is a powerful approach that can be used to enrich target genes for gene mining and thus improve and facilitate their screening in clone libraries. DNA-SIP can be combined with recent mutagenesis techniques such as gene shuffling in order to create large numbers of potentially novel biocatalysts.

Citation: Moussard H, Smith T, Murrell J. 2011. DNA stable Isotope Probing and Gene Mining, p 259-276. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch12

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Polycyclic Aromatic Hydrocarbons
16s rRNA Sequencing
DNA Synthesis
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Image of FIGURE 1

Flow chart illustrating the DNA-SIP gene mining approach. Starting from the SIP incubation of environmental samples followed by purification of the “heavy” DNA (C-DNA), three routes are then available to identify novel genes: (1) through PCR detection using functional gene primers; (2) through sequence-based or function-based screening of large- or small-insert metagenomic libraries; and (3) through mining of metabolic homologous genes in the genome of active microorganisms (if genome is available in databases). As a downstream application, various re-engineering strategies can also be carried out for creating protein diversity (e.g., in order to obtain highly selective and efficient biocatalysts for catalytic processes under various conditions).

Citation: Moussard H, Smith T, Murrell J. 2011. DNA stable Isotope Probing and Gene Mining, p 259-276. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch12
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Image of FIGURE 2

Schematic representation of cost-effective production of complex C-labeled compounds from plant or microbial biomass. In the first step, plants or microorganisms are incubated with simple and readily available C-labeled compounds (e.g., C-labeled carbon dioxide, bicarbonate, or glucose). Following incubation, complex C-labeled components of the plant (long carbohydrate biopolymers such as lignin and cellulose) or the microorganisms (e.g., membrane lipids and proteins or chitin from the cell wall of fungi) are extracted and are now available to be used for DNA-SIP experiments and gene mining of enzymes responsible for the degradation of these labeled substrates (e.g., cellulases, xylanases, ligninases, proteases, lipases, chitinases).

Citation: Moussard H, Smith T, Murrell J. 2011. DNA stable Isotope Probing and Gene Mining, p 259-276. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch12
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Examples of studies that have used DNA-SIP for gene mining

Citation: Moussard H, Smith T, Murrell J. 2011. DNA stable Isotope Probing and Gene Mining, p 259-276. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch12

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