Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 4: DISCUSSIONA) B)Figure 38. The chemical structure of A) actinopyrone, and B) trichostatin A(Adapted from Chapman and Hall DNP, 1982-2001).4.9 ConclusionsPrescreening environmental cultures using PCR prior to the commencement offermentative studies was shown to be a reliable indicator that an isolate contained thecapability to produce biologically active secondary metabolite(s) and that all of theisolates contain multiple biosynthetic genes. Screening by conventional PCR withnon-degenerate primers was found to be applicable to isolating similar sequencesfrom environmental isolates, however this approach may not be suitable for isolatingnovel SMBG genes. Degenerate-PCR would be better suited in isolating divergentbiosynthetic genes from environmental microbial sources due to the degeneracy inannealing of the primers to similar sequences and can lead to the discovery of novelSMBG (Seow et al., 1997).The degree of SM productivity is reliant on the type of biological screen beingimplemented (Franco & Coutinho, 1991). Antimicrobial screens were successfullyused to detect the highly productive isolates from the less productive isolates.Antifungal metabolites derived from A0350, A1113 and A3675 were shown tocontain spectral properties similar to polyene type compounds. Extract A1488showing no antimicrobial activities, was shown by TLC to express metabolites andcontain SMBG by PCR indicating the biosynthetic capability to produce a aromaticpolyketide. This observation highlights the importance of using a multi-directionalapproach to screening and that the type of compound to be discovered is dependent ondetection method used for screening.Realisation of an isolates full potential for producing secondary metabolites,fermentative media design for SM screening needs to well-defined for each individualisolate. It was shown that certain constituents in the liquid media such as dextrin and_____________________________________________________________________132