Part B | 11.3
318 Part B Tools and Methods in Marine Biotechnology
a metagenomic library [11.123]. The main advantage
of this approach is that the target can be identified even
if it is not being expressed by the host [11.128, 140].
Subcloning using the appropriate host and vector can
result in the functional expression of the gene or gene
cluster of interest, allowing the functional characterization and biotechnological application of the product.
A critical drawback of molecular screenings is that
they depend on sequence database information, which
is currently biased, and as a consequence usually results in the retrieval of variations of previously known
genes. In order to maximize the discovery process in
molecular screenings, sequences of genes identified in
a metagenomic library by a functional approach or gene
fragments retrieved by PCR from the same environment can also be used as a source of de novo, unbiased
genetic information for primer design [11.141]. An interesting approach to mine for gene clusters involved in
the biosynthesis of bioactive molecules is to perform
a retrobiosynthetic analysis on the structure of these
compounds in order to predict the enzymes involved in
the biosynthetic pathway [11.128]. This information is
then used to design degenerate primer sets for the retrieval of gene fragments by PCR [11.128].
Besides the construction of metagenomic libraries,
DNA isolated from environmental samples can be
sequenced directly using next-generation sequencing
technologies, resulting in the random generation of sequence information from the genomes contained in the
microbial community [11.142]. This approach has been
extensively used for the analysis of microbial communities from marine environments, providing unprecedented insights into their genetic potential [11.120].
Continuous improvements in sequencing technologies
is resulting in longer read lengths, larger sequence
outputs, as well as lower costs, allowing a deeper
analysis of the microbial communities. As individual
reads are usually assembled, genome fragments containing whole operons, and even draft genomes from
uncultured bacteria can be obtained [11.143, 144]. This
information is critical in bioprospecting efforts, as it can
be used as a basis for the recovery of the genome fragment from the same community, or alternatively, for its
synthesis. The term synthetic metagenomics has been
proposed to define the discovery approach that involves
in silico identification of hypothetical target sequences
followed by automated chemical DNA synthesis and
heterologous expression [11.145]. This approach has
recently been used to obtain de novo functional methyl
halide transferases using information from the GenBank database, enzymes that are useful for biofuel production [11.145]. This approach allows the exploitation
of existing sequence databases, currently underexplored
and underexploited [11.146]. Synthetic metagenomics
has the additional advantage of allowing codon optimization, which may significantly improve gene expression [11.145]. Sharma and collaborators [11.147]
have developed a resource called MetaBioME with the
goal of facilitating the discovery of novel commercially useful enzymes from metagenome information
(Table 11.2).
Other Meta-Omics
Next-generation sequencing technologies can also be
used to analyze the subset of genes in a microbial assemblage that is being transcribed under a particular environmental condition [11.148, 149]. For this approach,
total RNA is extracted from the environmental sample,
rRNA is removed in order to enrich for the mRNA fraction, and copy DNA (cDNA) synthesis is performed
before sequencing [11.148]. When cDNA yield is insufficient for analysis, however, an amplification step
can be included. Metatranscriptome sequencing represents a powerful tool to analyze microbial communities,
albeit with considerable challenges. Not only environmental cells present low mRNA contents, but also their
half-lives are very short, in the range of a few minutes [11.150]. In addition, mRNA constitutes a very
small fraction of the total RNA in bacterial cells and
the enrichment of the mRNA fraction in prokaryotes is
challenging [11.151]. Limitation in environmental sample quality and quantity, as well as low mRNA integrity
and purity can also affect metatranscriptomic analyses [11.149, 151]. Despite these methodological challenges, this approach has been increasingly applied in
fundamental research on microbial communities from
various marine habitats [11.152–157].
Like shotgun sequencing metagenomics, sequencebased metatranscriptomics presents no significant bias
towards known sequences, and is considered highly
informative concerning ongoing ecologically relevant
processes [11.149, 150]. Other advantages of metatranscriptomics over metagenomics are that only ecologically relevant information is retrieved and fewer
resources are required for this analysis [11.148]. However, sequence reads are still too short for bioprospecting efforts. Metatranscriptomics represents a powerful
approach for the discovery of metabolically relevant
enzymes that are actively involved in particular biochemical pathways [11.148, 158]. Another application
is the analysis of community-specific variants of functional genes; this information is highly relevant to the
318 Part B Tools and Methods in Marine Biotechnology
a metagenomic library [11.123]. The main advantage
of this approach is that the target can be identified even
if it is not being expressed by the host [11.128, 140].
Subcloning using the appropriate host and vector can
result in the functional expression of the gene or gene
cluster of interest, allowing the functional characterization and biotechnological application of the product.
A critical drawback of molecular screenings is that
they depend on sequence database information, which
is currently biased, and as a consequence usually results in the retrieval of variations of previously known
genes. In order to maximize the discovery process in
molecular screenings, sequences of genes identified in
a metagenomic library by a functional approach or gene
fragments retrieved by PCR from the same environment can also be used as a source of de novo, unbiased
genetic information for primer design [11.141]. An interesting approach to mine for gene clusters involved in
the biosynthesis of bioactive molecules is to perform
a retrobiosynthetic analysis on the structure of these
compounds in order to predict the enzymes involved in
the biosynthetic pathway [11.128]. This information is
then used to design degenerate primer sets for the retrieval of gene fragments by PCR [11.128].
Besides the construction of metagenomic libraries,
DNA isolated from environmental samples can be
sequenced directly using next-generation sequencing
technologies, resulting in the random generation of sequence information from the genomes contained in the
microbial community [11.142]. This approach has been
extensively used for the analysis of microbial communities from marine environments, providing unprecedented insights into their genetic potential [11.120].
Continuous improvements in sequencing technologies
is resulting in longer read lengths, larger sequence
outputs, as well as lower costs, allowing a deeper
analysis of the microbial communities. As individual
reads are usually assembled, genome fragments containing whole operons, and even draft genomes from
uncultured bacteria can be obtained [11.143, 144]. This
information is critical in bioprospecting efforts, as it can
be used as a basis for the recovery of the genome fragment from the same community, or alternatively, for its
synthesis. The term synthetic metagenomics has been
proposed to define the discovery approach that involves
in silico identification of hypothetical target sequences
followed by automated chemical DNA synthesis and
heterologous expression [11.145]. This approach has
recently been used to obtain de novo functional methyl
halide transferases using information from the GenBank database, enzymes that are useful for biofuel production [11.145]. This approach allows the exploitation
of existing sequence databases, currently underexplored
and underexploited [11.146]. Synthetic metagenomics
has the additional advantage of allowing codon optimization, which may significantly improve gene expression [11.145]. Sharma and collaborators [11.147]
have developed a resource called MetaBioME with the
goal of facilitating the discovery of novel commercially useful enzymes from metagenome information
(Table 11.2).
Other Meta-Omics
Next-generation sequencing technologies can also be
used to analyze the subset of genes in a microbial assemblage that is being transcribed under a particular environmental condition [11.148, 149]. For this approach,
total RNA is extracted from the environmental sample,
rRNA is removed in order to enrich for the mRNA fraction, and copy DNA (cDNA) synthesis is performed
before sequencing [11.148]. When cDNA yield is insufficient for analysis, however, an amplification step
can be included. Metatranscriptome sequencing represents a powerful tool to analyze microbial communities,
albeit with considerable challenges. Not only environmental cells present low mRNA contents, but also their
half-lives are very short, in the range of a few minutes [11.150]. In addition, mRNA constitutes a very
small fraction of the total RNA in bacterial cells and
the enrichment of the mRNA fraction in prokaryotes is
challenging [11.151]. Limitation in environmental sample quality and quantity, as well as low mRNA integrity
and purity can also affect metatranscriptomic analyses [11.149, 151]. Despite these methodological challenges, this approach has been increasingly applied in
fundamental research on microbial communities from
various marine habitats [11.152–157].
Like shotgun sequencing metagenomics, sequencebased metatranscriptomics presents no significant bias
towards known sequences, and is considered highly
informative concerning ongoing ecologically relevant
processes [11.149, 150]. Other advantages of metatranscriptomics over metagenomics are that only ecologically relevant information is retrieved and fewer
resources are required for this analysis [11.148]. However, sequence reads are still too short for bioprospecting efforts. Metatranscriptomics represents a powerful
approach for the discovery of metabolically relevant
enzymes that are actively involved in particular biochemical pathways [11.148, 158]. Another application
is the analysis of community-specific variants of functional genes; this information is highly relevant to the