Detection and Expression of Biosynthetic Genes in Actinobacteria ...

_____________________________________________________________________LIST OF FIGURESFigure 1: Primary metabolic pathways leading to the formation of secondarymetabolites _______________________________________________5Figure 2: Schematic representation of a bacterial cell eliciting resistancemechanisms_______________________________________________7Figure 3: Clustering of biosynthetic and resistance genes, a segment of theStreptomyces fradiae genome containing tylosin biosynthetic genes(EDHFJ) and tylosin resistance genes [tlrB,tlrC] __________________7Figure 4: Screening process for a new bioactive microbial metabolite _________8Figure 5: Illustration depicting the key enzyme reactions in polyketidebiosynthesis_______________________________________________13Figure 6: General modular organization of the type I PKS __________________14Figure 7: Modular organization of two polyketide synthases ________________16Figure 8: Aromatic polyketide biosynthesis of the gene products from theactinorhodin PKS cluster ____________________________________17Figure 9: Structural organization of aromatic polyketide synthase genes ofvarious secondary metabolites produced by actinobacteria __________18Figure 10A: Streptomycin (str) gene clusters of S. griseus N2-3-11 andS. glaucescens GLA.0_____________________________________20Figure 10B: Streptomycin biosynthetic pathway in Streptomyces griseus _______20Figure 11: The general structure of β-lactams ____________________________21Figure 12: β-lactam biosynthetic gene cluster _____________________________23Figure 13: Illustration depicting the key enzymatic reactions in the β-lactambiosynthetic pathway _______________________________________23Figure 14: Diagram depicting the regulatory network in response to a stimulusand the changes accompanying a cells activities and functions ______25Figure 15: Cloning soil DNA for isolating new biosynthetic pathways for thesynthesis of bioactive molecules from noncultured soilmicroorganisms ___________________________________________36Figure 16: The influence of temperatures permitting vegetative growth onsecondary metabolism ______________________________________43Figure 17: Flow chart depicting the strategy employed for selecting andvalidating appropriate primer sequences ________________________54Figure 18: Schematic flow diagram of the isolation of microbial bioactivesecondary metabolites ______________________________________62Figure 19: Illustration depicting the conserved minimal PKS gene organizationin Type II PKS ____________________________________________71Figure 20: Comparison of the amino acid sequences of KSα genes ____________73Figure 21: Comparison of the nucleic acid sequences of KSα genes ___________75Figure 22: PCR amplification of 0.47 kb KSα internal fragment by PCR fromenvironmental actinobacterial isolates__________________________78Figure 23: Multiple sequence alignment of β-Ketoacyl synthase (KSα) genesfrom type II PKS of spore antibiotic producing actinobacteria withamplified environmental KSα genes from actinobacterial isolatesA1488 and A3023 _________________________________________79Figure 24: Unrooted neighbour-joining phylogenetic tree constructed from aminoacid sequences of KSα gene fragments from type II polyketidesynthases ________________________________________________81_____________________________________________________________________v