Part A | 8.6
236 Part A Marine Flora and Fauna
dazine (50 M) to standard bacterial growth allows the
detection of laccase activity due to the appearance of
a purple halo resulting from the oxidation of the substrate.
Syringaldazine has been successfully employed in
the functional screening of a bovine rumen metagenomic library to clone a novel laccase gene [8.261],
while guaicol has similarly been used in a functional
screen to clone a novel laccase from a mangrove soil
metagenomic library [8.262]; detailed protocols have
been developed for the functional screening of metagenomic libraries from numerous environmental sources
by the Golyshin group [8.263]. Thus, with the availability of these functional screens coupled with the fact
that sequence-based screens have already resulted in the
cloning of novel sponge-derived laccases, which will be
discussed later in this chapter, it is likely that metagenomic libraries constructed from marine sponges will
contain large numbers of novel laccases, many with
potentially biotechnologically important biochemical
characteristics.
Function-based screens continue to be developed
as evidenced by the recent functional screen involving the use of chrome azurol S (CAS) as an indicator.
This screen involves CAS changing color from orange
to blue in the presence of iron and has recently been
used in the cloning of gene clusters for two known
siderophores, namely vibrioferrin and bisucaberin from
marine metagenomic libraries [8.264].
8.6.2 Problems Associated
with Functional Screening
of Metagenomic Libraries
A number of problems continue to hamper the discovery of novel genes and gene products from metagenomic clone libraries. These include the choice of
an appropriate heterologous host system and appropriate screens for the detection of activities.
E. coli is the heterologous host of choice in most
cases [8.265]. Uchiyama and colleagues reported that
40% of foreign genes can successfully be expressed
in E. coli [8.224]. However, the expression of foreign
genes can be impeded by host codon usage preferences,
problems with gene promoter recognition, transcription initiation factors, improper protein folding, and
the inability to export gene products from the host
cell [8.265]. In addition, the expression of foreign gene
products can sometimes be toxic to the heterologous
host [8.266]. The abundance of genes of interest in the
source environment and the cloned insert size and library size can also have an effect on the probability of
cloning particular genes [8.266].
Efforts to increase the rate of gene and product
discovery can be improved by the use of alternative heterologous host expression systems other than E. coli.
In this respect, an increasing number of host systems
are now becoming available, including Agrobacterium
tumefaciens, Bacillus subtilis, Burkholderia graminis,
Caulobacter vibrioides, Pseudoalteromonas haloplanktis, Pseudomonas putida, Ralstonia metallidurans, Rhizobium leguminosarum, Streptomyces spp., Sulfolobus
solfataricus, Thermus thermophilus, and Thiocapsa
roseopersicina [8.267, 268]. In addition, increases in
the rate of novel gene discovery are likely to be improved through the use of multiple heterologous host
expression systems, such as through the use of shuttle vectors that can be transformed from E. coli into
different hosts, thereby increasing the likelihood of expression [8.265]. In this respect, the authors’ group has
developed a novel cloning vector pCCERI (Fig. 8.7),
which incorporates elements from Streptomyces phage
'C31 for chromosomal integration into alternative
hosts and an inducible replicon for ease of handling.
pCCERI incorporates the 'C31 integrase gene and attachment site for site specific integration into hosts
containing the cognate attB site. The vector also incorporates the oriV from the inducible copy number
system developed by Wild et al. [8.269], allowing induction from single to multicopy in a modified E. coli
host. In addition, the vector has apramycin and chloramphenicol resistance genes for selection in different
hosts, and oriT for conjugal transfer. A helper plasmid pERI3-50-1 was also constructed to allow efficient
high-throughput conjugation of entire metagenomic libraries to the alternative host strains Pseudomonas
putida and Streptomyces lividans, thereby increasing
the likelihood of obtaining the expression of metagenomic inserts.
While, as previously mentioned, functional screening of marine sponge metagenomic libraries has been
successful in the isolation of numerous industrially
relevant biocatalysts, primarily involving the use of
screens that employ colony color or colony appearance
changes. However, many enzymes are not amenable to
identification using these types of approaches. In an
attempt to overcome this problem approaches involving the use of gene-expression reporter-based assays
have been developed. This involves the use of a transcriptional regulator that responds to the product of
a reaction, which is catalyzed by the enzyme being
targeted, resulting in the transcription factor-dependent
236 Part A Marine Flora and Fauna
dazine (50 M) to standard bacterial growth allows the
detection of laccase activity due to the appearance of
a purple halo resulting from the oxidation of the substrate.
Syringaldazine has been successfully employed in
the functional screening of a bovine rumen metagenomic library to clone a novel laccase gene [8.261],
while guaicol has similarly been used in a functional
screen to clone a novel laccase from a mangrove soil
metagenomic library [8.262]; detailed protocols have
been developed for the functional screening of metagenomic libraries from numerous environmental sources
by the Golyshin group [8.263]. Thus, with the availability of these functional screens coupled with the fact
that sequence-based screens have already resulted in the
cloning of novel sponge-derived laccases, which will be
discussed later in this chapter, it is likely that metagenomic libraries constructed from marine sponges will
contain large numbers of novel laccases, many with
potentially biotechnologically important biochemical
characteristics.
Function-based screens continue to be developed
as evidenced by the recent functional screen involving the use of chrome azurol S (CAS) as an indicator.
This screen involves CAS changing color from orange
to blue in the presence of iron and has recently been
used in the cloning of gene clusters for two known
siderophores, namely vibrioferrin and bisucaberin from
marine metagenomic libraries [8.264].
8.6.2 Problems Associated
with Functional Screening
of Metagenomic Libraries
A number of problems continue to hamper the discovery of novel genes and gene products from metagenomic clone libraries. These include the choice of
an appropriate heterologous host system and appropriate screens for the detection of activities.
E. coli is the heterologous host of choice in most
cases [8.265]. Uchiyama and colleagues reported that
40% of foreign genes can successfully be expressed
in E. coli [8.224]. However, the expression of foreign
genes can be impeded by host codon usage preferences,
problems with gene promoter recognition, transcription initiation factors, improper protein folding, and
the inability to export gene products from the host
cell [8.265]. In addition, the expression of foreign gene
products can sometimes be toxic to the heterologous
host [8.266]. The abundance of genes of interest in the
source environment and the cloned insert size and library size can also have an effect on the probability of
cloning particular genes [8.266].
Efforts to increase the rate of gene and product
discovery can be improved by the use of alternative heterologous host expression systems other than E. coli.
In this respect, an increasing number of host systems
are now becoming available, including Agrobacterium
tumefaciens, Bacillus subtilis, Burkholderia graminis,
Caulobacter vibrioides, Pseudoalteromonas haloplanktis, Pseudomonas putida, Ralstonia metallidurans, Rhizobium leguminosarum, Streptomyces spp., Sulfolobus
solfataricus, Thermus thermophilus, and Thiocapsa
roseopersicina [8.267, 268]. In addition, increases in
the rate of novel gene discovery are likely to be improved through the use of multiple heterologous host
expression systems, such as through the use of shuttle vectors that can be transformed from E. coli into
different hosts, thereby increasing the likelihood of expression [8.265]. In this respect, the authors’ group has
developed a novel cloning vector pCCERI (Fig. 8.7),
which incorporates elements from Streptomyces phage
'C31 for chromosomal integration into alternative
hosts and an inducible replicon for ease of handling.
pCCERI incorporates the 'C31 integrase gene and attachment site for site specific integration into hosts
containing the cognate attB site. The vector also incorporates the oriV from the inducible copy number
system developed by Wild et al. [8.269], allowing induction from single to multicopy in a modified E. coli
host. In addition, the vector has apramycin and chloramphenicol resistance genes for selection in different
hosts, and oriT for conjugal transfer. A helper plasmid pERI3-50-1 was also constructed to allow efficient
high-throughput conjugation of entire metagenomic libraries to the alternative host strains Pseudomonas
putida and Streptomyces lividans, thereby increasing
the likelihood of obtaining the expression of metagenomic inserts.
While, as previously mentioned, functional screening of marine sponge metagenomic libraries has been
successful in the isolation of numerous industrially
relevant biocatalysts, primarily involving the use of
screens that employ colony color or colony appearance
changes. However, many enzymes are not amenable to
identification using these types of approaches. In an
attempt to overcome this problem approaches involving the use of gene-expression reporter-based assays
have been developed. This involves the use of a transcriptional regulator that responds to the product of
a reaction, which is catalyzed by the enzyme being
targeted, resulting in the transcription factor-dependent