Nucleotide sequence of the macromomycin apoprotein gene and its expression in Streptomyces macromomyceticus.

A 1.6 kb Sph I-Sac I DNA fragment from Streptomyces macromomyceticus, expected to include the macromomycin (MCM) apoprotein gene, was sequenced. The fragment (1,556 bp) was found to include a putative promoter, an ORF directing pre-apoprotein which should be split into the leader peptide (Met1 to Gly32) and the MCM apoprotein (Ala33 to Ala144), and a putative terminator. The amino acid sequence deduced from the base sequence of the DNA is consistent with the amino acid sequence previously determined by the Edman degradation and other procedures applied to the protein, except base sequence AAC coding for Asn was found rather than Asp111 previously reported. The GC content of the 3rd letters throughout the ORF was 92% in contrast to the sum of the first and the second letters, 62%. There was a low GC content stretch of 20 bp (30% GC) at about 120 bp upstream of the ORF. The Pst I-Sph I 620 bp fragment including the low GC content stretch showed promoter activity when subcloned in a promoter probe vector. About 700 nucleotides long mRNA, which is long enough to span the ORF and the bordering regions, was identified using the Northern blot analysis. A primer extension experiment showed that the transcriptional starting point was A at 89 bp upstream of the ORF. Dot blot analysis of expression of MCM apoprotein gene indicated that the gene was expressed nearly constitutively, while production of holo MCM (the complex consisting of MCM apoprotein and a specific chromophore) depended greatly on culture conditions.