The gene cluster for chloramphenicol biosynthesis in Streptomyces venezuelae ISP5230 includes novel shikimate pathway homologues and a monomodular non-ribosomal peptide synthetase gene

Regions of the Streptomyces venezuelae ISP5230 chromosome flanking pabAB, a n amino-deoxychorismate synthase gene needed for chloramphenicol (Cm) produ ction, were examined for involvement in biosynthesis of the antibiotic. Thr ee of four ORFs in the sequence downstream of pabAB resembled genes involve d in the shikimate pathway. BLASTX searches of GenBank showed that the dedu ced amino acid sequences of ORF3 and ORF4 were similar to proteins encoded by monofunctional genes for chorismate mutase and prephenate dehydrogenase, respectively, while the sequence of the ORFS product resembled deoxy-arabi no-heptulosonate-7-phosphate (DAHP) synthase, the enzyme that initiates the shikimate pathway. A relationship to Cm biosynthesis was indicated by sequ ence similarities between the ORF6 product and membrane proteins associated with Cm export. BLASTX searches of GenBank for matches with the translated sequence of ORF1 in chromosomal DNA immediately upstream of pabAB did not detect products relevant to Cm biosynthesis. However, the presence of Cm bi osynthesis genes in a 7.5 kb segment of the chromosome beyond ORF1 was infe rred when conjugal transfer of the DNA into a blocked S. venezuelae mutant restored Cm production. Deletions in the 7.5 kb segment of the wild-type ch romosome eliminated Cm production, confirming the presence of Cm biosynthes is genes in this region. Sequencing and analysis located five ORFs, one of which (ORFS) was deduced from BLAST searches of GenBank, and from character istic motifs detected in alignments of its deduced amino acid sequence, to be a monomodular nonribosomal peptide synthetase. GenBank searches did not identify ORF7, but matched the translated sequences of ORFs 9, 10 and 11 wi th short-chain ketoreductases, the ATP-binding cassettes of ABC transporter s, and coenzyme A ligases, respectively. As has been shown for ORF2, disrup ting ORF3, ORF7, ORFS or ORF9 blocked Cm production.