Studies on the gene cluster for oxytetracycline biosynthesis from Streptomyces rimosus

Prior to the work described in this thesis, a great deal of the oxytetracycline cluster (otc) had been sequenced and the putative functions of deduced gene products assigned, based on similarity to known gene products in the databases. The 3.0 kb KpnI23-MluI26b fragment located between the otcD and otcX loci was sequenced. Three open reading frames with the same direction of transcription were found, otcD-ORF3, -ORF4 and -ORF5. OtcD-ORF3 has good similarity to oxidoreductases reported previously in other polyketide gene clusters. The biosynthesis of OTC requires three oxidoreductase steps, one at position C9 which removes the keto function, one at position C6 in the conversion of 4-keto-ATC to ATC and another at the last step [OTC dehydrogenase]. A ketoreductase has been reported previously within the otcY locus that shows stronger similarity to actIII of S.coelicolor. OtcD-ORF3 has been tentatively assigned as catalysing the last step of the pathway. OtcD-ORF4 shows good end-to-end similarity to a coding region of unknown function within the gene cluster for daunorubicin biosynthesis in S.griseus. OtcD-ORF5 is clearly defined as a potential protein coding region that shows good similarity to a small region of an open reading frame within the B.subtilis genome which again has unknown function. The B.subtilis coding region is very hydrophobic and carries a series of repeat sequences, the significance of which is not yet understood. Preliminary transcriptional analysis of this 3.0 kb KpnI23-MluI26b fragment using low-resolution S1 mapping has revealed a single transcript which encompasses OtcD-ORF3 and OtcD-ORF4. A stable stem-loop immediately after the translational stop codon of OtcD-ORF4 was located with high-resolution mapping. A potential promoter sequence was located immediately preceding OtcD-ORF5, however no transcript was detected with low-resolution mapping at the time point when the RNA was isolated