Part C | 17.2
462 Part C Marine Genomics
Therefore, it is emphasized that the direct method
should be avoided when constructing the metagenomic
library for natural product synthesis.
Metagenomic library construction methods have
been extensively optimized for cloning in BAC and
FOSMID (F1 origin-based cosmid vector); both have
been successfully used in sponge metagenomics. Compared with fosmid libraries only capable of screening up
to 40 kb inserts, environmental BAC libraries enable the
screening of larger DNA inserts up to 200 kb [17.29],
which can facilitate the reconstruction and functional
analyses of environmental genomes [17.30].
17.2.3 Function-Based Screening
of the Metagenomic Library
One of the simplest strategies used to detect antibiotic producing environmental DNA (eDNA) clones is
top agar overlay assays. Metagenomic enzymes are
usually screened on agar plates supplemented with substrates. Positive clones from the metagenomic library
can be identified by visual screening for the appearance of a clear zone or color. Functional screening of
a metagenomic library requires no special devices and
high throughput can be performed, but the signals are
often faint due to low hit rates.
Alternative approaches of cell lysates are applied
to improve the sensitivity of agar plate-based screening. By growing the clones of a metagenomic library
in 96-well plates, followed by preparing the lysates by
either a chemical or a physical procedure, and mixing them with substrates, the sensitivity is improved.
This method is frequently applied for screening gene
resistance to toxic compounds such as antibiotics and
heavy metals. This method can also be applied to screen
essential genes using hosts that lack the target genes.
A third approach is based on the reporter assay. By
linking the target gene with the reporter gene, such as
green fluorescent protein, ˇ-galactosidase, and tetracycline resistance genes, screening can be conducted
based on the expression of reporter genes [17.31].
17.2.4 Sequence-Based Screening
of the Metagenomic Library
Type II PKS Biosynthetic Genes
ThePKSs are responsible for the production of bacterial polyketides, a diverse group of pharmacologically
important natural products which include antibiotics,
antitumor, immunosuppressive, and cholesterol lowering agents. Bacterial type II PKS biosynthetic gene
clusters encode three enzymes, including two ketoacyl
synthases, KS a (catalyzes the Claisen type condensation between the growing polyketide and incoming
acyl-CoA subunits) and KSb (anchors the growing
polyketide chain during chain elongation). The minimal PKS gene cassette is adequate to produce the
polyketide in type II PKS systems. Additional enzymes
such as ketoreductases (KR), cyclases, and aromatases
are required to fold and cyclize the poly b-ketoacyl
intermediate into the final structure. The structural diversity of the natural products originates from type
II PKS, from divergence within the PKS domains,
and their arrangement with assorted modification enzymes. With this knowledge of the functions of all
three constituents of the minimal PKS (ketosynthase
alpha subunit-ketosyntahase beta subunit-acyl carrier
protein, KSa-KSb-ACP), a scheme was designed to
determine overall polyketide chain length and structure. Degenerate PCR primers were designed based
on conserved regions within KSa and ACP genes in
type II PKS were used to amplify KSb sequences directly from metagenomic DNA. The discovery of novel
small molecules from metagenomic DNA reveals the
enormous genetic diversity encoded within the uncultured bacterial genomes for the discovery of natural
products [17.32].
Type I PKS
Type I PKS generally consist of a set of multifunctional
modules. Each module is responsible for one cycle of
polyketide chain elongation. The module contains ketosynthase (KS), acyltransferase (AT), and acyl carrier
protein (ACP) domains that together catalyze a two-carbon extension of the chain. After such an extension, the
resulting ketone can either be left as is or converted
to a ˇ-hydroxyl, a double bond, or an alkane group
by stepwise processing of ketoreductase (KR), dehydratase (DH), and enoyl reductase (ER) domains. The
substrate specificities of the ATs, the order and number of the modules, and the composition of catalytic
domains within each module provide a code for the
structure of the synthesized polyketide. Furthermore,
the PKS genes for a particular polyketide are usually
clustered and colinear with the order of its biosynthesis.
Consequently, the structure of the polyketide product
of an unknown PKS can be approximated from the sequence of its genes [17.33].
Nonribosomal Peptide Synthetases (NRPS)
NRPS gene clusters encode for a wide range of
nonribosomal peptides, ranging from antibiotics, tox-
462 Part C Marine Genomics
Therefore, it is emphasized that the direct method
should be avoided when constructing the metagenomic
library for natural product synthesis.
Metagenomic library construction methods have
been extensively optimized for cloning in BAC and
FOSMID (F1 origin-based cosmid vector); both have
been successfully used in sponge metagenomics. Compared with fosmid libraries only capable of screening up
to 40 kb inserts, environmental BAC libraries enable the
screening of larger DNA inserts up to 200 kb [17.29],
which can facilitate the reconstruction and functional
analyses of environmental genomes [17.30].
17.2.3 Function-Based Screening
of the Metagenomic Library
One of the simplest strategies used to detect antibiotic producing environmental DNA (eDNA) clones is
top agar overlay assays. Metagenomic enzymes are
usually screened on agar plates supplemented with substrates. Positive clones from the metagenomic library
can be identified by visual screening for the appearance of a clear zone or color. Functional screening of
a metagenomic library requires no special devices and
high throughput can be performed, but the signals are
often faint due to low hit rates.
Alternative approaches of cell lysates are applied
to improve the sensitivity of agar plate-based screening. By growing the clones of a metagenomic library
in 96-well plates, followed by preparing the lysates by
either a chemical or a physical procedure, and mixing them with substrates, the sensitivity is improved.
This method is frequently applied for screening gene
resistance to toxic compounds such as antibiotics and
heavy metals. This method can also be applied to screen
essential genes using hosts that lack the target genes.
A third approach is based on the reporter assay. By
linking the target gene with the reporter gene, such as
green fluorescent protein, ˇ-galactosidase, and tetracycline resistance genes, screening can be conducted
based on the expression of reporter genes [17.31].
17.2.4 Sequence-Based Screening
of the Metagenomic Library
Type II PKS Biosynthetic Genes
ThePKSs are responsible for the production of bacterial polyketides, a diverse group of pharmacologically
important natural products which include antibiotics,
antitumor, immunosuppressive, and cholesterol lowering agents. Bacterial type II PKS biosynthetic gene
clusters encode three enzymes, including two ketoacyl
synthases, KS a (catalyzes the Claisen type condensation between the growing polyketide and incoming
acyl-CoA subunits) and KSb (anchors the growing
polyketide chain during chain elongation). The minimal PKS gene cassette is adequate to produce the
polyketide in type II PKS systems. Additional enzymes
such as ketoreductases (KR), cyclases, and aromatases
are required to fold and cyclize the poly b-ketoacyl
intermediate into the final structure. The structural diversity of the natural products originates from type
II PKS, from divergence within the PKS domains,
and their arrangement with assorted modification enzymes. With this knowledge of the functions of all
three constituents of the minimal PKS (ketosynthase
alpha subunit-ketosyntahase beta subunit-acyl carrier
protein, KSa-KSb-ACP), a scheme was designed to
determine overall polyketide chain length and structure. Degenerate PCR primers were designed based
on conserved regions within KSa and ACP genes in
type II PKS were used to amplify KSb sequences directly from metagenomic DNA. The discovery of novel
small molecules from metagenomic DNA reveals the
enormous genetic diversity encoded within the uncultured bacterial genomes for the discovery of natural
products [17.32].
Type I PKS
Type I PKS generally consist of a set of multifunctional
modules. Each module is responsible for one cycle of
polyketide chain elongation. The module contains ketosynthase (KS), acyltransferase (AT), and acyl carrier
protein (ACP) domains that together catalyze a two-carbon extension of the chain. After such an extension, the
resulting ketone can either be left as is or converted
to a ˇ-hydroxyl, a double bond, or an alkane group
by stepwise processing of ketoreductase (KR), dehydratase (DH), and enoyl reductase (ER) domains. The
substrate specificities of the ATs, the order and number of the modules, and the composition of catalytic
domains within each module provide a code for the
structure of the synthesized polyketide. Furthermore,
the PKS genes for a particular polyketide are usually
clustered and colinear with the order of its biosynthesis.
Consequently, the structure of the polyketide product
of an unknown PKS can be approximated from the sequence of its genes [17.33].
Nonribosomal Peptide Synthetases (NRPS)
NRPS gene clusters encode for a wide range of
nonribosomal peptides, ranging from antibiotics, tox-