Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 4: DISCUSSIONan indication of metabolite production in isolate A1488 and no chemical expressiontests were conducted it cannot be assumed conclusively that this isolate does notproduce any metabolites. Detection of KS α related DNA sequences in environmentalactinobacteria has provided evidence for the presence of a aromatic polyketidepathway (Seow et al., 1997; Metsä-Ketelä et al., 1999), this may be the case forisolate A1488 however fermentation studies may have not been adequate to inducethe appropriate pathway.4.1.3 Type I Polyketide SynthaseThe KS gene is involved in the biosynthesis of aliphatic compounds such aserythromycin and tylosin. The KS gene encodes a condensing enzyme (ketosynthase),which is part of a large polypeptide unit known as a modular PKS. These modularPKS contain all the necessary enzyme activities, present as discrete catalytic domains(Khosla, 1997). The highly conserved KS domain containing 60-84% identity over thewhole domain between different actinobacterial species (MacNeil et al., 1993), waschosen as the target region for the PCR screening assay in this study. Degenerate PCRhas provided a useful approach in cloning and detecting modular PKS genes in pureisolates in various microorganisms (Brautaset et al., 2000; Nicholson et al., 2001),though there have been limited reports concerning the detection of modular PKSgenes from natural actinobacterial populations, using non-degenerate or degeneratePCR primers (Thamchaipenet et al., 1997).A non-degenerate primer set ole01f/ole01r was designed which passed all the primerdesign criterion (chapter 2 section 2.2.1.7) and was shown by computer simulationexperiments to amplify a 0.84 kb product. Application of these primers in PCRexperiments showed that they were effective in detecting the putative ketosynthase(KS) gene fragment in only one of four type cultures tested. A 0.84 kb product wasamplified in S. hygroscopicus but not in S. erythreae, S. avermitilis and S. fradiae. Aexplanation of this result may be due to the partially variable 3’ end of the ole1f andole01r primers (Table 38). The limited detection capabilities of primer setole01f/ole01r indicated that they were not suitable and were omitted fromimplementation for SMBG screening._____________________________________________________________________115