Biology Reference
In-Depth Information
Combinatorial biosynthesis has been particularly successful with PKS genes. Novel
combinations of type I and type II PKS genes produced numerous derivatives of medically
important macrolide antibiotics and unusual polycyclic aromatic compounds.
103,105
108
The initial demonstration was comprised of novel polycyclic aromatic metabolites produced
by hybrid forms of the actinorhodin producing genes and tetracenomycin type II PKS
genes,
109
113
resulting in rational design of new analogues.
114
A large number of novel
compounds were produced through such a strategy,
115
including tetracenomycin M resulting
from the combination of mithramycin and tetracenomycin genes,
116
and a novel 18-carbon
polyketide made by hybrid forms of tetracenomycin and griseusin cyclases.
117
The modular
PKS fosters the real excitement of using combinatorial biosynthesis for drug discovery and
development. One comprehensively studied example is the 6-deoxyerythronolide B synthase
(DEBS), which will be discussed in detail in the following section. Deletion, inactivation, or
shuffling of domains or modules within or outside the system generated many novel
compounds.
118,119
Another strategy to generate novel compounds is to change the starter or
extension units.
120
133
This approach has been extended to other drugs.
134
136
The potential of combinatorial biosynthesis was further expanded by the addition of the
deoxysugar (DOS) biosynthesis genes. Usually the formation of DOSs as glycosides, which
are made by the glucose-1-phosphate key metabolic intermediate, thymidine diphospho
4-keto-6-deoxyglucose or one of its derivatives,
137
generates biological activities. Since the
genes involved in these DOS-producing pathways have been identified, attempts have been
made to use combinatorial biosynthesis to make analogues of known antibiotic glycosides
or novel metabolites.
138
141
ACTIVATION OF SILENT GENE CLUSTERS
The increasing availability of whole-genome sequences has revised our view of the metabolic
capabilities of microorganisms. Analyses of more than 500 microbial genome sequences
currently in the publicly accessible databases have revealed numerous examples of gene
clusters encoding enzymes similar to those known to be involved in the biosynthesis of many
important natural products.
142
Examples of such enzymes include NRPSs, PKSs, and terpene
synthases, as well as enzymes belonging to less thoroughly investigated families. Many of
these gene clusters are hypothesized to produce novel natural products. However, most
of them are cryptic biosynthetic pathways, for which the encoded natural product is
unknown, or the pathway is silent and the product cannot be detected under general growth
conditions. Since genomic sequencing projects are mostly focused on bacteria and fungi,
cryptic pathways related to valuable secondary metabolites are often revealed from these
genome databases. For example, while
Streptomyces coelicolor
was known to produce only
four secondary metabolites,
143
genome analysis revealed 18 additional cryptic biosynthetic
pathways. It is intriguing to note that this is not a special case, because analyses of other
microbial genomes originating from myxobacteria, cyanobacteria, and filamentous fungi
shows the presence of comparable or even larger numbers of cryptic pathways.
144,145
These pathways represent an untapped treasure trove, which is likely to grow
exponentially in the future, uncovering many novel and possibly bioactive
compounds.
146
193
Various strategies have been developed to discover the products of cryptic biosynthetic gene
clusters (
Fig. 10.2
). Bioinformatics tools are usually used to predict the putative function of
a target gene cluster from sequenced genomes and metagenomes before any experiments.
Several techniques have been used to activate the cryptic pathways. One is to identify
the product through prediction of physicochemical properties.
147
By using this approach,
17 novel biosynthetic loci were identified from the genus
Salinispora
, and bioinformatic
analysis elucidated the structure of the polyene macrolactam salinilactam A. This provides
a powerful bridge between genomic analysis and traditional natural product isolation
