Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 4: DISCUSSIONdegenerate PCR technique (Okuta et al., 1998). As screening DNA is becoming moreefficient, as is the case with colony PCR whereby cultures are used directly in PCRreactions with no need for lengthy DNA extractions (Sheu et al., 2000; Ishikawa etal., 2000), detection of SMBG from environmentally isolated microorganisms shouldbecome economically feasible for high throughput screening.4.2 Secondary metabolite production of actinobacteria4.2.1 Solid AgarTwenty two environmental actinobacterial isolates from the Cerylid culture collectionwere initially screened by agar-type antimicrobial assays. Mycelial extracts fromisolates grown on yeast-malt extract (YME) solid agar, showed that fungi and grampositive bacteria were sensitive to the secondary metabolites (SM) produced by theCerylid cultures (Table 49).4.2.2 Submerged FermentationsEnhancement of antimicrobial activity was achieved by applying a variety offermentation techniques and using complex media with known constituents whichenhance SM production (see discussion below). Complex media was selected due tothe beneficial properties imposed on antibiotic submerged fermentation studies theseinclude, promotion of homogeneous dispersal of mycelial growth, variable chemicalexpression, faster growth and higher quantities of antibiotics (Dekleva et al., 1985;Doull & Vining, 1989; Whitaker, 1992). However the disadvantage of using complexmedia include difficulty in distinguishing constituents inducing production of adesired SM. Utilisation of general SM screening conditions is a common dilemmaposed in many screening programs, the conditions are often empirically determinedwhich requires high technical resources and is costly (Huang et al., 1999). However,it is becoming apparent that culture conditions favoring production of SM are beingidentified and implemented in screening for novel SM (Iwai & Ōmura, 1982; Ōmura,1986).4.2.2.1 Carbon SourcesOf the major constituents which are known to influence the efficacy of SMproduction, include carbon and nitrogen sources (Iwai & Ōmura, 1982). The carbon_____________________________________________________________________121