Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 1: INTRODUCTION(v) utilisation of combinatorial biochemistry approaches in cloning SM biosyntheticgenes from a producing strain and introducing it into another producing strainproducing a similar compound; (vi) directed evolution in accelerating enhancedenzymatic activity and broader substrate specificity. (Ōmura, 1986; Lazzarini et al.,2000; Zähner & Fiedler, 1995; Bull et al., 2000; Strohl, 1997). In order for theadvancement of SM screening programs, new lead substances are required which canbe chemically transformed in which the bioactivity and pharmacological propertiesare modified to suit particular therapeutic needs (Verpoorte, 1998; Bull et al., 2000).1.2.2.1 Rapid identification of microbial metabolitesMicrobial bioactive compounds have proven elusive in industrial screening programs,as culture conditions are not always well defined (Table 2) and often incapable ofinducing appropriate biosynthetic pathways. Furthermore, there is a limited capacityof detectable systems being unable to selectively identify the effector compounds.Traditionally thin layer chromatography (TLC) has been used to determine themetabolic fingerprint in response to effects of culture conditions on SM production.However, the current approaches used by Zahn et al (2001) show that electrospraymass spectrometry (EMS) is a effective and more accurate approach used indetermining the metabolic fingerprint of strains and identifying culture conditionsinducing the expression of secondary metabolites. The culture conditions used in thescreening for SM production are often the conditions that permit optimum growth, asboth are mutually exclusive.1.2.2.2 Chemical screening using chromatography and spectroscopyChemical screens such as high performance liquid chromatography (HPLC) and TLCare effective at identifying known compounds and their congeners. However, they arelimited in their usefulness only to known classes of compounds which allows foridentification or group allocation of an unknown compound at an early stage inscreening (Fiedler, 1993). However, coupling HPLC with diode array detection, massspectrometry (MS) or nuclear magnetic resonance spectrometry (NMR) have beenefficient methods used for screening and identifying microbial metabolites fromfermentation broths (Abel et al., 1999; Higgs et al., 2001). When LC-NMR/MS isused in conjunction with biological activity assays new leads can be detected in a very_____________________________________________________________________9