Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 1: INTRODUCTIONbiosynthesis (Table 7). The limitation of suitable plasmids has hampered efforts indetermining the effects of multiple mutations in polyketide synthases (McDaniel etal., 1999). In some cases generation of unexpected structures are produced, due toexpression of previously latent downstream genes or upstream genes due to insertedfragment.Table 7. Mutagenesis of secondary metabolite producing actinobacteria.Mutant Type ofGene Effects on Referencesstrain mutagenesis effected secondarymetabolismNSA205 Chromosomal ADS205* Blocking Schauner et al.,. 1999S. coelicolorC542deletionsInducedmutations ^* ADS205 Amplified DNA Sequence^ UV light & chemical exposureabsA Blocking Adamidis et al., 1990Recent efforts by McDaniel et al. (1999) have shown that by selectively inducingsingle or multiple mutations in the catalytic site of polyketide synthases, the synthesisof unnatural compounds are produced (which could by chemical or natural).Section 6: Molecular Techniques used to DetectSecondary Metabolite Biosynthetic GenesCurrent advances in molecular detection techniques are providing valuable screeningtools in determining the taxonomic diversity of antibiotic producing microorganismsand determining the biosynthetic capabilities of microorganisms (Dalbǿge & Lange,1998; August et al., 1999). The application of the polymerase chain reaction (PCR)technique has been successfully used to selectively amplify biosynthetic genes usingdegenerate primers, independent of culture conditions and shown to provide a reliableindication of actinobacteria able to produce secondary metabolites of an expectedgroup, or novel compounds (Seow et al., 1997; Morris et al., 1999). Alternativemolecular techniques which have also been successful include the use of DNAprobes, to screen genomic or cDNA libraries for biosynthetic genes to identifycandidate secondary metabolite producing clones (Stockmann & Piepersberg, 1992;August et al., 1999)._____________________________________________________________________28