Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 1: INTRODUCTIONTable 8: DNA Probes used in the detection of SMBG in Actinobacteria spp.SMBGGroupActinobacteria Compound Class Probe Reference6 - DeoxysugarStreptomyces spp.AminoglycosidesMacrolidesstrD, strE, strLM,strHStockmann &Piepersberg, 1992S . argillaceus Anthracycline strD, strE, strM Lombó et al., 1996,actI & actIIIS. rishiriensis Aminocoumarin strE Wang et al., 2000S. spheroids Aminocoumarin strE Steffensky et al., 2000S. viridochromogenes Tü57 Orthosomycin strE Gaisser et al., 1997S. violaceoruber Tü22 Benzoisochromane strE Bechthold et al., 1995Actinoplanes sp. 50/10 Acarbose strE, AS2,AS5 Stratmann et al., 1999S. cyanogenus S136 Angucycline strE Westrich et al., 1999S. antibioticus Macrolide strDEM Aguirrezabalaga et al.,2000S. globisporus Enediyne strE Liu & Shen, 2000S. noursei Polyene macrolide gdhA Zotchev et al., 2000*KS - authors designed probes based on active site of ketosynthase and acyltransferase enzymes fromtype II polyketide synthases.AHBA = 3-amino-5-hydroxybenzoic acid involved in shikimate biosynthesislat = lysine 6-aminotransferaseIPNS = Isopenicillin N synthasePKR = bifunctional cyclase/dehydrataseACMS = actinomycin synthetasesNGDH/gdhA = dNTP-glucose 4,6 dehydratase involved in deoxysugar biosynthesisNGS = dNTP-glucose synthase involved in deoxysugar biosynthesisSimilarly the strE and strD probes derived from the streptomycin deoxysugar geneshave also been efficient in cloning corresponding genes in various actinobacteriaproducing different compounds. The erythromycin PKS genes have proved to beefficient in the cloning of corresponding PKS genes in macrolide producingactinobacteria. However, in certain cases we see that PKS genes from similar classesof secondary metabolites are the preferred option for cloning, such is the case foransamycin producing actinobacteria using rifamycin (ansamycin compound) PKS asthe probe (Yu et al., 2002).1.6.3 Detection of secondary metabolite biosynthetic genes using thepolymerase chain reaction (PCR)The major focus of this study is concerned with the molecular screening of naturalactinobacteria isolated from soil and using PCR in predicting bioactive populations.Degenerate PCR has provided an attractive option in the recovery of secondarymetabolite genes in actinobacteria species and other microorganisms (Seow et al.,1997; Nicholson et al., 2001). Degenerate primer design involves performing a_____________________________________________________________________32