Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 1: INTRODUCTION1.4.1.2 Codon Usage (CU)Codon usage (CU) of genes in actinobacterial coding sequences exhibit strong biasfor G or C in the third position and in the first position, but no obvious preference inposition two. This codon bias is significant as it is possible that codon preference mayreflect the relative abundance of particular charged tRNA species and that this mightbe a means of regulating the expression of certain biosynthetic genes (Wright &Bibb., 1992). This finding is supported by Leskiw et al. (1991) who found thatmutational loss of tRNA that translates UUA leucine codons prevents the productionof secondary metabolites. The bias towards G and C in the degenerate position ofamino acid codons has greatly facilitated the construction of effective DNA probesbased upon amino acid sequences of protein products (Fishman et al., 1987).Section 5: Genetic Factors Affecting theProduction of Secondary Metabolites1.5.1 Plasmid InstabilityPlasmids harbouring SM biosynthetic genes have been known to undergo aphenomenon known as genetic instability where frequent deletions arise, which canbe spontaneous/induced causing a decrease/loss in SM production (Thomas et al.,1991; Dary et al. 1992) or a favourable deletion leading to an increase in SMproduction (Cullum et al., 1988). Often the mutants generated by genetic instabilitydecrease the rate of SM production by altering gene expression patterns (Matsushima& Baltz, 1996).1.5.2 Induced MutagenesisMutagenesis involves the alteration of genes causing an altered state of function. InSM producing actinobacterial strains, mutagenesis can either be used to abolishunwanted intermediates or to increase yields of a desired product (Queener et al.,1978., Ivanova et al., 1995). By inducing mutations by UV light exposure, coldstorage or chemical treatments such as N-methyl-N’-nitro-N-nitrosoguanidine(MNNG) or ethidium bromide it has been possible to identify biosynthetic genes onchromosomal DNA (Baltz, 1986; Crameri et al., 1986; Adamidis et al., 1990; Volff etal., 1993; Ikeno et al., 1996), the genes that block biosynthesis are cloned andsequenced and placed in plasmids to determine the extent to which they control_____________________________________________________________________27