Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 4: DISCUSSIONintermediatery chemical structures from which a number of diverse secondarymetabolites are derived. The DNA and protein sequences of these synthases arehighly conserved across different actinobacteria genera, and DNA homology-baseddetection methodologies have been successfully adapted in cloning SMBG anddetermining the biosynthetic origins of secondary metabolites in differentactinobacteria (Metsä-Ketelä et al., 1999; August et al., 1999; Miao et al., 2001).4.1.1 Design of PCR primersThe validity of the PCR assays involved applying various controls. A negative control(blank) containing all the reaction components except the actinobacterial DNA wasimplemented in all PCR assays to ensure that the PCR reactions were contaminantfreeand that no spurious amplifiable products appeared. The specificity of the primersets were tested against DNA extracted from two known non-producing SM bacteria(Escherichia coli and Bacillus pumilus), no amplifiable product was obtained in bothcases. All DNA extracted from the actinobacterial pure strains and Cerylid isolateswere tested by 16S rDNA PCR using actinobacterial-biased primers to ensure that theDNA was amplifiable and to avoid false-negative results. The designed primer setswere tested against actinobacterial pure cultures known to contain the respectiveSMBG, and all primers sets except ole01f/ole02r were effective in amplifying thecorrect sized product in two or more pure strains. Direct DNA sequencing ofamplified fragments of SMBG was performed to ensure that the correct sequence wasobtained.4.1.2 Type II Polyketide SynthaseAromatic polyketides are synthesized by monofunctional proteins, which containcatalytic activities, of which the β-ketoacyl synthase, which is encoded by the KS αgene, is involved in condensation reactions. The highly conserved KS α gene sequencewas used to design non-degenerate primers, which were used in the screens conductedin detecting similar DNA sequences in actinobacterial pure cultures and the Cerylidactinobacterial isolates. Multiple sequence alignments of amino acid andcorresponding nucleic acid sequences of the KS α gene revealed several highlyconserved regions. Two regions were chosen as suitable target sites for primerdevelopment as shown in figures 20 and 21, these two regions passed the required_____________________________________________________________________113