Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 1: INTRODUCTIONsoil DNA, cloning it into a culturable organism and screening the resultant clones forthe production of new secondary metabolites (Handelsman et al., 1998). The majorchallenges facing the application of this technology to soil DNA is maintaining thelarge size (70-120kb) of the DNA fragments while removing nonDNA soil materialthat inhibits cloning and expressing the soil DNA in suitable heterologous hosts(MacNeil et al., 2001).Figure 15. Cloning soil DNA for isolating new biosynthetic pathways for thesynthesis of bioactive molecules from noncultured soil microorganisms. Step 1.Separation of intact bacteria from soil, and then DNA extracted directly from thebacteria; Step 2. DNA is cut using a restriction enzyme and cloned into a bacterialartificial chromosome (BAC); Step 3. transformation of E.coli cells with BAC vector;Step 4. BAC clones screened for biological activity and for the production of novelsecondary metabolites (adapted from Handelsman et al., 1998).Section 7: Production of Secondary MetabolitesThe production of secondary metabolites in actinobacteria can be initiated orenhanced by the manipulation of fermentation conditions (Ōmura & Tanaka, 1984).The need to manipulate fermentation conditions has arisen due to the minimalquantities of SM produced by natural actinobacterial cultures, which may not besufficient for further down-stream chemical and biological activity characterisation_____________________________________________________________________36