Biology Reference
In-Depth Information
FIGURE 10.2
Techniques used to activate cryptic pathways.
studies. Another method is the genomisotopic approach. 148 This method employs a
combination of genomic sequence analysis and isotope-guided fractionation to identify
unknown compounds synthesized from orphan gene clusters. Identification of an orphan
gene cluster which led to the discovery of orfamide A, the model for a group of bioactive
cyclic lipopeptides, was done through analysis of the Pseudomonas fluorescens Pf-5 genome.
A third approach is the in vitro reconstitution approach. 149 This strategy was applied to a
terpene synthase encoded by the SCO5222 (SC7E4.19) gene of S. coelicolor . Incubation of the
recombinant protein, SCO5222p, with farnesyl diphosphate in the presence of Mg(II) gave
a new sesquiterpene, (
194
)-epi-isozizaene. The fourth approach is the biosynthetic gene
inactivation/comparative metabolic profiling approach. 150 Gcs, a type III PKS encoded by
the SCO7221 ORF of the bacterium S. coelicolor , required for biosynthesis of the germicidin
family of 3,6-dialkyl-4-hydroxypyran-2-one natural products, was discovered through this
method. The fifth approach is the genome mining approach. 151 In this approach, genes that
encode tailoring enzymes from natural product biosynthesis pathways serve as indicator
genes for the identification of strains that have the genetic potential to produce natural
products of interest. One example is from halogenases, which are known to be involved in
the synthesis of halometabolites. From PCR screening of 550 randomly selected Actinomycetes
strains, 103 novel putative halogenase genes were identified. The sixth approach is to activate
silent clusters by manipulation of regulatory genes. Homologous overexpression of a putative
activator gene apdR led to activation of a silent PKS-NRPS gene cluster from Aspergillus
nidulans . 145 Another recent example to activate silent fungal secondary metabolite gene
clusters was done by deletion of hdaA , the gene encoding an Aspergillus nidulans histone
deacetylase (HDAC). 152 Using this approach, two telomere-proximal gene clusters were
transcriptionally activated, leading to the production of multiple secondary metabolites not
previously known to be produced by A. nidulans . 153
1
Production of Valuable Compounds
ERYTHROMYCIN A AND ITS DERIVATIVES
Polyketides are a diverse group of natural products with important applications in the
pharmaceutical industry. They can be found in a variety of organisms including plants,
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