Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 4: DISCUSSIONpresence of two SMBG (Table 48). This may indicate that the isolate(s) have thepotential to produce more than one type of secondary metabolite or that it contains acomponent of other biosynthetic pathways (Stockmann & Piepersberg, 1992). Ofparticular interest are isolates A2010, A1113, A2707, A0350 and A2056 whichcontain both polyketide and deoxysugar genes. Such a profile is indicative of thegenetic composition of a number of bioactive microbial metabolites, particularlymacrolides and anthracyclines (Staunton & Wilkinson, 1999; Richardson & Khosla,1999).PCR screening of the Cerylid isolates showed that ten of the eleven isolates classifiedas Streptomycete strains were positive for containing secondary metabolitebiosynthetic genes. Whereas only 6 of the 11 non-Streptomyces isolates testedpositive for secondary metabolite biosynthetic genes.The PCR screens applied to the actinobacterial culture collection provided by CerylidBiosciences (Melbourne, Australia), were benefical in identifying environmentalisolates harboring putative SMBG. Thus providing a useful selection criterion inconcentrating fermentative efforts on those isolates shown to possess the geneticmachinery necessary for the synthesis of certain classes of secondary metabolites.This is a major advantage in screening microbial sources for secondary metabolites,as identifying the biosynthetic capabilities of the isolate prior to the commencementof fermentation studies, offers clues to elucidating production media which can betailored for the identified class of compounds. Empirical approaches have beencontinually used for the determination of suitable fermentative media and selectingthe most appropriate media where chemical metabolites are expressed (Bu’Lock etal., 1982; Zahn et al., 2001). Due to the declining rate at which novel secondarymetabolites are being discovered using empirical screening (Strohl, 1997), moredirected approaches have evolved such as PCR based screens which discriminateproductive cultures from redundant ones. Degenerate PCR has been a powerfulapproach in detecting divergent SMBG in cultured and uncultured microorganisms(Seow et al., 1997), however based on the SMBG sequences retrieved from theenvironmental isolates using the KSM/ATM degenerate primer set highly similargenes could only be detected. Thus, in order to detect novel SMBG this primer setmay not be appropriate, or may need to be modified or used in variations of the_____________________________________________________________________120