Limits...
Interplay of metagenomics and in vitro compartmentalization.

Ferrer M, Beloqui A, Vieites JM, Guazzaroni ME, Berger I, Aharoni A - Microb Biotechnol (2008)

Bottom Line: In recent years, the application of approaches for harvesting DNA from the environment, the so-called, 'metagenomic approaches' has proven to be highly successful for the identification, isolation and generation of novel enzymes.Functional screening for the desired catalytic activity is one of the key steps in mining metagenomic libraries, as it does not rely on sequence homology.In particular, we focus on the use of in vitro compartmentalization (IVC) approaches to address potential advantages and problems the merger of culture-independent and IVC techniques might bring on the mining of enzyme activities in microbial communities.

View Article: PubMed Central - PubMed

Affiliation: CSIC, Institute of Catalysis, Department of Applied Biocatalysis, Madrid, Spain. mferrer@icp.csic.es

Show MeSH
Mining genomes and metagenomes for novel enzymes. A gene library is created from environmental samples (Step 1–3) and used to screen for novel genes (Step 4) cloned into bacteria which can be sequenced (Step 5a). The encoded proteins expressed in appropriate host are then subjected to structure‐function analyses (central panel). Alternatively, large‐scale sequencing of bulk DNA is used for archiving and sequence homology screening purposes to capture the largest amount of the available genetic resources present in environmental samples (Step 5b).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3815420&req=5

f2: Mining genomes and metagenomes for novel enzymes. A gene library is created from environmental samples (Step 1–3) and used to screen for novel genes (Step 4) cloned into bacteria which can be sequenced (Step 5a). The encoded proteins expressed in appropriate host are then subjected to structure‐function analyses (central panel). Alternatively, large‐scale sequencing of bulk DNA is used for archiving and sequence homology screening purposes to capture the largest amount of the available genetic resources present in environmental samples (Step 5b).

Mentions: There are two distinct mining strategies taken in metagenomics to capture the largest amount of the available enzymatic resources in environmental DNA (Fig. 2). First, the application of the sequence‐based approach that involves the design of PCR primers or hybridization probes for the target genes that are derived from conserved regions of already known protein families (Gabor et al., 2007). The use of microarrays to profile libraries offers to this strategy an effective approach for characterizing many clones rapidly in a HT fashion (Sebat et al., 2003). This format is referred to as a metagenome microarray (MGA). In the MGA format, the ‘probe’ and ‘target’ concept is a reversal of those of general cDNA and oligonucleotide microarrays: targets (fosmid clones) are spotted on a slide and a specific gene probe is labelled and used for hybridization. This format of microarray may offer an effective HTS approach for identifying clones from metagenome libraries rapidly without the need of laborious procedures for screening various target genes. As an example, Sebat and colleagues (2003) and Park and colleagues (2008) used microarray platforms to screen microbial genomes and whole community genomes. However, the difficulty and limitation of this approach is related to achieving high hybridization efficiency and that the target genes derived from conserved regions of already known protein families reduce our chances for obtaining fundamentally new proteins (Gabor et al., 2007). On the other hand, based on the few metagenome surveys of microbial and viral communities completed to date, a consensus is emerging that environmental communities are extraordinarily diverse and contain a high proportion (over 60%) of novel sequences with unknown functions that are relatively distant from better known representatives in sequenced genomes (see examples shown in Tables 1 and 2).


Interplay of metagenomics and in vitro compartmentalization.

Ferrer M, Beloqui A, Vieites JM, Guazzaroni ME, Berger I, Aharoni A - Microb Biotechnol (2008)

Mining genomes and metagenomes for novel enzymes. A gene library is created from environmental samples (Step 1–3) and used to screen for novel genes (Step 4) cloned into bacteria which can be sequenced (Step 5a). The encoded proteins expressed in appropriate host are then subjected to structure‐function analyses (central panel). Alternatively, large‐scale sequencing of bulk DNA is used for archiving and sequence homology screening purposes to capture the largest amount of the available genetic resources present in environmental samples (Step 5b).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815420&req=5

f2: Mining genomes and metagenomes for novel enzymes. A gene library is created from environmental samples (Step 1–3) and used to screen for novel genes (Step 4) cloned into bacteria which can be sequenced (Step 5a). The encoded proteins expressed in appropriate host are then subjected to structure‐function analyses (central panel). Alternatively, large‐scale sequencing of bulk DNA is used for archiving and sequence homology screening purposes to capture the largest amount of the available genetic resources present in environmental samples (Step 5b).
Mentions: There are two distinct mining strategies taken in metagenomics to capture the largest amount of the available enzymatic resources in environmental DNA (Fig. 2). First, the application of the sequence‐based approach that involves the design of PCR primers or hybridization probes for the target genes that are derived from conserved regions of already known protein families (Gabor et al., 2007). The use of microarrays to profile libraries offers to this strategy an effective approach for characterizing many clones rapidly in a HT fashion (Sebat et al., 2003). This format is referred to as a metagenome microarray (MGA). In the MGA format, the ‘probe’ and ‘target’ concept is a reversal of those of general cDNA and oligonucleotide microarrays: targets (fosmid clones) are spotted on a slide and a specific gene probe is labelled and used for hybridization. This format of microarray may offer an effective HTS approach for identifying clones from metagenome libraries rapidly without the need of laborious procedures for screening various target genes. As an example, Sebat and colleagues (2003) and Park and colleagues (2008) used microarray platforms to screen microbial genomes and whole community genomes. However, the difficulty and limitation of this approach is related to achieving high hybridization efficiency and that the target genes derived from conserved regions of already known protein families reduce our chances for obtaining fundamentally new proteins (Gabor et al., 2007). On the other hand, based on the few metagenome surveys of microbial and viral communities completed to date, a consensus is emerging that environmental communities are extraordinarily diverse and contain a high proportion (over 60%) of novel sequences with unknown functions that are relatively distant from better known representatives in sequenced genomes (see examples shown in Tables 1 and 2).

Bottom Line: In recent years, the application of approaches for harvesting DNA from the environment, the so-called, 'metagenomic approaches' has proven to be highly successful for the identification, isolation and generation of novel enzymes.Functional screening for the desired catalytic activity is one of the key steps in mining metagenomic libraries, as it does not rely on sequence homology.In particular, we focus on the use of in vitro compartmentalization (IVC) approaches to address potential advantages and problems the merger of culture-independent and IVC techniques might bring on the mining of enzyme activities in microbial communities.

View Article: PubMed Central - PubMed

Affiliation: CSIC, Institute of Catalysis, Department of Applied Biocatalysis, Madrid, Spain. mferrer@icp.csic.es

Show MeSH