Marine Microbial Bioprospecting 11.3 Methods for Microbial Bioprospecting in Marine Environments 317
Part B | 11.3
ple, which is called metagenomic DNA [11.120]. This
DNA can be used for the construction of a metagenomic library [11.121] or alternatively, it can be randomly sequenced using next-generation sequencing
technologies [11.122]. Due to the uneven distribution
and high species richness of most microbial communities, metagenomic analyses are very challenging.
Despite the difficulties still affecting metagenomics,
in recent years this discipline has been fundamental
in increasing our understanding of microbial communities and has become an important tool for mining
novel biomolecules or activities with biotechnological
potential [11.123, 124]. For instance, the construction
and screening of metagenomic libraries have resulted
in the identification of many novel biocatalysts, including lipases/esterases, cellulases, chitinases, DNA
polymerases, proteases, and antibiotics [11.123, 125].
Marine sediments, microbial communities from marine
invertebrates, and cold marine environments are among
the most commonly studied habitats, due to their high
biotechnological potential [11.126–128].
The cloning of fragments of metagenomic DNA using the appropriate vectors and suitable hosts allows
us to store and mine the genetic potential contained
in a microbial community [11.121]. The selection of
the vector for library construction (plasmids, cosmids,
fosmids, or BACs – bacterial artificial chromosomes)
depends mainly on the desired insert length. For example, large-insert libraries are required for recovering
large gene clusters [11.123]. Other factors to consider are the desired cell copy number, the quality
of the metagenomic DNA, the genes that are being
targeted, the chosen host, as well as the selected screening strategy [11.121, 123]. In order to reach sufficient
coverage of metagenomes of highly diverse microbial
communities, such as those from soils or sediments,
metagenomic libraries need to contain a large number
of clones [11.129]. To increase hit rates, enrichment
cultures were used prior to metagenomic DNA extraction, although this approach can result in an overall loss
of diversity [11.130]. Another possible strategy is the
use of stable-isotope-labeled substrates to enrich the
functionally-relevant fraction of the microbial community. In this case, density centrifugation of metagenomic
DNA is performed after labeling, before the construction of the metagenomic library [11.121, 131].
Two different strategies can be used for the screening of a metagenomic library: a function-based approach (detection or selection for metabolic activity)
or a sequence-based approach (detection of a specific target gene). The first strategy, called functional metagenomics, does not require previous knowledge of sequence information and can, therefore, result in the identification of entirely novel classes of
genes [11.123]. It presents the additional advantage
that the identified gene or gene cluster is already being
functionally expressed in the host. However, functionalbased screenings can be problematic due to low-level
gene expression, lack of post-translational modifications, the formation of insoluble aggregated folding
intermediates, as well as detrimental effects that the
products can have on the host cell [11.132, 133]. Currently, the most commonly used vector and host for
constructing metagenomic libraries are fosmids and
Escherichia coli [11.121]. The use of other hosts and
the development of vectors able to replicate in various species are particularly useful for expression-based
analyses of metagenomic libraries [11.129, 134, 135].
In addition, hosts can be engineered to improve gene
expression [11.136].
Different function-driven approaches can be used
for the screening of metagenomic libraries. One of
them is the detection of the desired phenotype in agarplate assays, for instance, an enzymatic activity or
colony pigmentation [11.124]. Agar-plate based screenings have the advantage of not requiring expensive
devices. However, they are usually labor intensive and
they tend to have low hit rates due to the generation
of weak signals [11.129]. In addition, this strategy depends on the availability of assays able to detect the
desired metabolic function, of which there are, unfortunately, very few [11.131]. In order to increase the
sensitivity of the assays, the enzymatic activity can be
measured in cell lysates [11.124, 129]. This is usually
performed using colony picking robots and microplate
readers to shorten the processing time. Another strategy, called heterologous complementation, significantly
simplifies functional screening by taking advantage of
gene targets for which the desired phenotype is required
for the survival of the host, such as genes that confer
resistance to metals or antibiotics [11.129, 137]. On the
other hand, the ability of some substrates to induce gene
expression through closely located regulatory elements
has been used to engineer vectors containing reporter
genes [11.124]. Interestingly, Uchiyama and collaborators [11.138, 139] have created specific reporter assays
based on fluorescent proteins in order to screen for
enzyme-encoding genes in metagenomic libraries.
In contrast to functional metagenomics, molecular screenings involve the use of primers or probes
that have been designed based on conserved regions
of already-known genes or protein families to mine
Part B | 11.3
ple, which is called metagenomic DNA [11.120]. This
DNA can be used for the construction of a metagenomic library [11.121] or alternatively, it can be randomly sequenced using next-generation sequencing
technologies [11.122]. Due to the uneven distribution
and high species richness of most microbial communities, metagenomic analyses are very challenging.
Despite the difficulties still affecting metagenomics,
in recent years this discipline has been fundamental
in increasing our understanding of microbial communities and has become an important tool for mining
novel biomolecules or activities with biotechnological
potential [11.123, 124]. For instance, the construction
and screening of metagenomic libraries have resulted
in the identification of many novel biocatalysts, including lipases/esterases, cellulases, chitinases, DNA
polymerases, proteases, and antibiotics [11.123, 125].
Marine sediments, microbial communities from marine
invertebrates, and cold marine environments are among
the most commonly studied habitats, due to their high
biotechnological potential [11.126–128].
The cloning of fragments of metagenomic DNA using the appropriate vectors and suitable hosts allows
us to store and mine the genetic potential contained
in a microbial community [11.121]. The selection of
the vector for library construction (plasmids, cosmids,
fosmids, or BACs – bacterial artificial chromosomes)
depends mainly on the desired insert length. For example, large-insert libraries are required for recovering
large gene clusters [11.123]. Other factors to consider are the desired cell copy number, the quality
of the metagenomic DNA, the genes that are being
targeted, the chosen host, as well as the selected screening strategy [11.121, 123]. In order to reach sufficient
coverage of metagenomes of highly diverse microbial
communities, such as those from soils or sediments,
metagenomic libraries need to contain a large number
of clones [11.129]. To increase hit rates, enrichment
cultures were used prior to metagenomic DNA extraction, although this approach can result in an overall loss
of diversity [11.130]. Another possible strategy is the
use of stable-isotope-labeled substrates to enrich the
functionally-relevant fraction of the microbial community. In this case, density centrifugation of metagenomic
DNA is performed after labeling, before the construction of the metagenomic library [11.121, 131].
Two different strategies can be used for the screening of a metagenomic library: a function-based approach (detection or selection for metabolic activity)
or a sequence-based approach (detection of a specific target gene). The first strategy, called functional metagenomics, does not require previous knowledge of sequence information and can, therefore, result in the identification of entirely novel classes of
genes [11.123]. It presents the additional advantage
that the identified gene or gene cluster is already being
functionally expressed in the host. However, functionalbased screenings can be problematic due to low-level
gene expression, lack of post-translational modifications, the formation of insoluble aggregated folding
intermediates, as well as detrimental effects that the
products can have on the host cell [11.132, 133]. Currently, the most commonly used vector and host for
constructing metagenomic libraries are fosmids and
Escherichia coli [11.121]. The use of other hosts and
the development of vectors able to replicate in various species are particularly useful for expression-based
analyses of metagenomic libraries [11.129, 134, 135].
In addition, hosts can be engineered to improve gene
expression [11.136].
Different function-driven approaches can be used
for the screening of metagenomic libraries. One of
them is the detection of the desired phenotype in agarplate assays, for instance, an enzymatic activity or
colony pigmentation [11.124]. Agar-plate based screenings have the advantage of not requiring expensive
devices. However, they are usually labor intensive and
they tend to have low hit rates due to the generation
of weak signals [11.129]. In addition, this strategy depends on the availability of assays able to detect the
desired metabolic function, of which there are, unfortunately, very few [11.131]. In order to increase the
sensitivity of the assays, the enzymatic activity can be
measured in cell lysates [11.124, 129]. This is usually
performed using colony picking robots and microplate
readers to shorten the processing time. Another strategy, called heterologous complementation, significantly
simplifies functional screening by taking advantage of
gene targets for which the desired phenotype is required
for the survival of the host, such as genes that confer
resistance to metals or antibiotics [11.129, 137]. On the
other hand, the ability of some substrates to induce gene
expression through closely located regulatory elements
has been used to engineer vectors containing reporter
genes [11.124]. Interestingly, Uchiyama and collaborators [11.138, 139] have created specific reporter assays
based on fluorescent proteins in order to screen for
enzyme-encoding genes in metagenomic libraries.
In contrast to functional metagenomics, molecular screenings involve the use of primers or probes
that have been designed based on conserved regions
of already-known genes or protein families to mine