Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 4: DISCUSSION4.1 PCR screening assays for detecting biosyntheticcapability in environmental actinobacteriaThe distribution of microorganisms in nature is influenced to a great extent to thesurrounding physico-chemical environment, which in turn effects the metabolism ofmicroorganisms to adapt to change (Finlay et al., 1997). Actinobacteria which aremajor components of the soil microbiota have been isolated from diverse naturalhabitats (Xu et al., 1996; Groth et al., 1999) and many exhibit the metaboliccapability to produce a diverse array of bioactive secondary metabolites (Osada, 1995;Sanglier et al., 1993). The chemical diversity generated by actinobacterial species isunparalled to that observed in any other microorganism. Molecular based screeninghas become an efficient means of accessing this chemical diversity through thedetection of secondary metabolite biosynthetic genes (SMBG) in bacterial genomes(Seow et al., 1997). The application of gene homology based screening inactinobacteria was first demonstrated by Hopwood et al. (1985) who showed that ahomologous probe corresponding to the antibiotic actinorhodin biosynthetic gene,could be used to detect similar biosynthetic gene clusters in actinobacterial species.The cloning of SMBG has been facilitated by using conserved domains of SMBG asDNA probes or as templates for the design of PCR primers (Julien et al., 2000;Brautaset et al., 2000). In this study PCR-mediated screening was used to determinethe prevalence of secondary metabolite biosynthetic genes in natural actinobacterialpopulations isolated from the Australian environment. Appropriate design ofconserved non-degenerate primers, specific for SMBG permitted the detection ofamplifiable DNA corresponding to presumptive SMBG fragments. Screening withmultiple sets of PCR primers targeting individual SMBG of the four major classes ofsecondary metabolites in actinobacteria allowed for the detection of biosyntheticcapabilities of the environmental isolates to be determined.The diverse chemical structures generated by actinobacteria are derived from commonbiosynthetic pathways. These consist of the aromatic and aliphatic polyketides, 6-deoxysugars and beta-lactam pathways. In this study, attention was focused on thesefour biosynthetic pathways and their associated SMBG encoding synthases which areinvolved in catalytic reactions in these biosynthetic pathways. These synthasescommonly occur in the early phases of SM biosynthetic pathways and form common_____________________________________________________________________112