Marine Sponges – Molecular Biology and Biotechnology 8.6 Metagenomic Strategies for Natural Product Discovery 239
Part A | 8.6
proaches are often successful, they at best lead to the
identification of new variants of already well-known
classes of proteins, with very few really novel genes being detected.
Notwithstanding this, such homology-based strategies have led to the identification of genes encoding
novel enzymes from marine metagenomic libraries. Examples include the use of degenerate PCR primers
designed based on the conserved regions of alkane hydroxylase gene sequences, to clone two alkane hydroxylase (alkB) genes from a Pacific deep-sea sediment
metagenomic library [8.270]. Moreover, the recent
cloning of four unique FADH 2 -dependent halogenases
from the metagenome of the Mediterranean sponge
Aplysina aerophoba using PCR primers designed based
on conserved sequences from previously cloned halogenases indicates that the microbial consortia of sponges
are likely to be a valuable source for novel halogenases [8.271].
Other examples include the cloning of a novel
peptidase encoding gene using PCR primers designed
to amplify cellulase genes, from the metagenome of
the western Arctic Ocean [8.272]. In addition, a new
laccase gene was cloned from a marine microbial
metagenomic library from the South China Sea, using
PCR primers based on the relatively highly conserved
sequence coding for copper binding sites in bacterial laccases (Cu1F ACMWCKGTTCAYTGGCACGG;
Cu4R TGNTCNAGNAWGTGRCARTG) [8.260]. This
novel laccase shared less than 40% sequence identity
with previously characterized multicopper oxidases and
when heterologously expressed in E. coli resulted in
the production of a recombinant protein that exhibited alkalescence-dependent, chloride tolerant laccase
activity. Similar primers were employed by the authors’ group to clone two candidate laccase encoding
genes (SNB11 and SNB12) from the metagenomic
library of the sponge Stelletta normani, which was
collected from a depth of 1348 m from the Atlantic
Ocean in Irish waters. Basic Local Alingment Search
Tool (BLAST) analyses and sequence alignments of
the cloned sequences show that although the copper
binding domains in these novel genes are conserved,
the overall gene sequences are quite divergent from
known gene sequences (Fig. 8.8). The quite marked
difference between these newly cloned SNB11 and
SNB12 genes from S. normani and already known
genes suggests that marine sponges and those from
deep sea ecosystems in particular may prove a valuable
source for laccases with potentially new and improved
functionality.
8.6.4 Sequence-Based Screening
of Metagenomic Libraries to Identify
Genes Involved in the Biosynthesis
of Natural Products
As previously mentioned, sponges are a rich source
of natural products, many of which are now widely
believed to be metabolites, which are, in fact, synthesized by symbiotic bacteria. There are a number of
examples of marine sponge-associated bacterial isolates
which produce compounds that are similar and in many
cases identical to sponge-derived compounds. These
include jasplakinolide (jaspamide) from the sponge
Jaspis sp. and cyclodepsipeptide chondramide D isolated from the myxobacterium Chrondromyces crocatus
and salicylihalamide A produced by Haliclona sp.,
which is almost identical to the myxobacterial metabolite apicularen A. In addition, there are a number of
sponge-associated bacteria that synthesize important
natural products, including the Salinispora spp., which
produces rifamycins and a Micromonospora sp., from
Acanthostrongylophora sp., which produces the antimalarial compound manzamine A.
Many of the structurally diverse classes of natural products most commonly associated with microorganisms include polyketides, non-ribosomal peptides,
or hybrid peptide-polyketides, which are synthesized
by polyketide synthases (PKS), non-ribosomal peptide
synthetases (NRPS), or hybrid PKS-NRPS synthases
which are encoded in gene clusters in the producing
bacteria. These multifunctional biosynthetic enzymes
typically consist of a series of modules which are
usually distributed among several polypeptides. These
modules consist of a series of enzymatic domains
each of which is involved in the catalysis of individual biosynthetic steps. Biosynthesis occurs in an
assembly line-like process with the nascent polyketide or peptide chain passing from module to module,
with each module adding extender units until synthesis of the compound is complete. Polyketide-derived
natural products which are believed to be produced
by symbiotic sponges microbes include halichondrin
B, discodermolide, peloruside, and laulimalide; while
polyketide/non-ribosomal peptides include hemiasterlins, the aforementioned jaspamide, and the salicylihalimides. Thus, perhaps it is not surprising that numerous sequence-based screens, many of which have
been PCR-based, have been undertaken on sponge
metagenomic libraries; specifically targeting PKS and
NRPS gene clusters as a means of assessing overall diversity and the secondary metabolite/chemical
Part A | 8.6
proaches are often successful, they at best lead to the
identification of new variants of already well-known
classes of proteins, with very few really novel genes being detected.
Notwithstanding this, such homology-based strategies have led to the identification of genes encoding
novel enzymes from marine metagenomic libraries. Examples include the use of degenerate PCR primers
designed based on the conserved regions of alkane hydroxylase gene sequences, to clone two alkane hydroxylase (alkB) genes from a Pacific deep-sea sediment
metagenomic library [8.270]. Moreover, the recent
cloning of four unique FADH 2 -dependent halogenases
from the metagenome of the Mediterranean sponge
Aplysina aerophoba using PCR primers designed based
on conserved sequences from previously cloned halogenases indicates that the microbial consortia of sponges
are likely to be a valuable source for novel halogenases [8.271].
Other examples include the cloning of a novel
peptidase encoding gene using PCR primers designed
to amplify cellulase genes, from the metagenome of
the western Arctic Ocean [8.272]. In addition, a new
laccase gene was cloned from a marine microbial
metagenomic library from the South China Sea, using
PCR primers based on the relatively highly conserved
sequence coding for copper binding sites in bacterial laccases (Cu1F ACMWCKGTTCAYTGGCACGG;
Cu4R TGNTCNAGNAWGTGRCARTG) [8.260]. This
novel laccase shared less than 40% sequence identity
with previously characterized multicopper oxidases and
when heterologously expressed in E. coli resulted in
the production of a recombinant protein that exhibited alkalescence-dependent, chloride tolerant laccase
activity. Similar primers were employed by the authors’ group to clone two candidate laccase encoding
genes (SNB11 and SNB12) from the metagenomic
library of the sponge Stelletta normani, which was
collected from a depth of 1348 m from the Atlantic
Ocean in Irish waters. Basic Local Alingment Search
Tool (BLAST) analyses and sequence alignments of
the cloned sequences show that although the copper
binding domains in these novel genes are conserved,
the overall gene sequences are quite divergent from
known gene sequences (Fig. 8.8). The quite marked
difference between these newly cloned SNB11 and
SNB12 genes from S. normani and already known
genes suggests that marine sponges and those from
deep sea ecosystems in particular may prove a valuable
source for laccases with potentially new and improved
functionality.
8.6.4 Sequence-Based Screening
of Metagenomic Libraries to Identify
Genes Involved in the Biosynthesis
of Natural Products
As previously mentioned, sponges are a rich source
of natural products, many of which are now widely
believed to be metabolites, which are, in fact, synthesized by symbiotic bacteria. There are a number of
examples of marine sponge-associated bacterial isolates
which produce compounds that are similar and in many
cases identical to sponge-derived compounds. These
include jasplakinolide (jaspamide) from the sponge
Jaspis sp. and cyclodepsipeptide chondramide D isolated from the myxobacterium Chrondromyces crocatus
and salicylihalamide A produced by Haliclona sp.,
which is almost identical to the myxobacterial metabolite apicularen A. In addition, there are a number of
sponge-associated bacteria that synthesize important
natural products, including the Salinispora spp., which
produces rifamycins and a Micromonospora sp., from
Acanthostrongylophora sp., which produces the antimalarial compound manzamine A.
Many of the structurally diverse classes of natural products most commonly associated with microorganisms include polyketides, non-ribosomal peptides,
or hybrid peptide-polyketides, which are synthesized
by polyketide synthases (PKS), non-ribosomal peptide
synthetases (NRPS), or hybrid PKS-NRPS synthases
which are encoded in gene clusters in the producing
bacteria. These multifunctional biosynthetic enzymes
typically consist of a series of modules which are
usually distributed among several polypeptides. These
modules consist of a series of enzymatic domains
each of which is involved in the catalysis of individual biosynthetic steps. Biosynthesis occurs in an
assembly line-like process with the nascent polyketide or peptide chain passing from module to module,
with each module adding extender units until synthesis of the compound is complete. Polyketide-derived
natural products which are believed to be produced
by symbiotic sponges microbes include halichondrin
B, discodermolide, peloruside, and laulimalide; while
polyketide/non-ribosomal peptides include hemiasterlins, the aforementioned jaspamide, and the salicylihalimides. Thus, perhaps it is not surprising that numerous sequence-based screens, many of which have
been PCR-based, have been undertaken on sponge
metagenomic libraries; specifically targeting PKS and
NRPS gene clusters as a means of assessing overall diversity and the secondary metabolite/chemical