Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 4: DISCUSSION4.1.5 Isopenicillin N SynthaseOne of the most promising approaches in the search for novel β-lactams have focusedon the presence of isopenicillin N synthase (IPNS) which is common to allβ-lactam antibiotics (Kralis and Kirby, 1998). The IPNS catalyzes the main reactionto form isopenicillin N and is encoded by the pcbC gene. The conserved nature of theDNA and amino acid sequence corresponding to β-lactam biosynthetic genes (Smithet al., 1990) has made it possible to utilize PCR-mediated approaches for in detectingpcbC genes from natural actinobacterial populations (Kralis and Kirby, 1998; Niebla-Perez and Wellington, 1997; Sim et al., 1997). Studies using DNA probes fordetection of IPNS gene in β-lactam producing Streptomyces have shown that theyshare more than 70 % amino acid sequence similarity (Shiffman et al., 1988). In thisstudy, the pcbC gene was selected to design primers. This gene exhibited severalconserved regions as was to be expected as this gene contains greater than 80 %sequence similarity at the nucleotide level (Shiffman et al., 1988). PCR experimentsusing primers pcbC03f and pcbC03r amplified the predicted 0.35 kb product, howeveronly two of the four actinobacterial species tested produced the expected amplifiedproduct. The 0.35 kb product was amplified in S.cattleya and S.griseus, but noproduct was detected in the Nocardia species and S.clavuligerus. The 0.35 kbamplified product was only obtained from streptomyces species which is reflected inthe sequence bias of both primers and indicates that the primers are highly specific(see table 45).Amino acid sequences translated from the DNA sequence amplified from bothβ-lactam producing Streptomycete spp. type strains showed approximately 70 %sequence similarity (Table 47). The pcbC gene was the least abundant SMBG,detected in 1 of 22 of the environmental isolates tested. As no DNA sequencing wascarried out on the amplified product in one the positive environmental isolate(A2360), sequence correlation could not be made on the degree of similaritiesbetween the amplified DNA and pcbC genes. Single PCR products were amplified inthe 0.4 – 0.55 kb range using the pcbC01f/pcbC01r primer set in four of the Cerylidcultures; the amplification of these higher molecular weight products may indicatethat they possess similar genes._____________________________________________________________________118