CLONING, SEQUENCE AND EXPRESSION OF THE TYROSINASE GENE FROM STREPTOMYCES-LAVENDULAE MA406 A-1

A black diffusible pigment produced by Streptomyces lavendulae MA406 A-1 was shown to be melanin-like and formed by the action of extracellular tyrosinase. Southern analysis revealed the presence of sequences (designated as TYR1 and TYR2) homologous to the tyrosinase structural gene of Streptomyces antibioticus in two different regions of the S. lavendulae DNA. The TYR1 sequence was absent in all the melanin-negative (Mel-) mutants of this organism, and they showed no tyrosinase activity either extracellularly or intracellularly; in contrast, the TYR2 sequence was detected in some of the Mel- mutants. The TYR1 and the TYR2 region was cloned as 8.0- and 4.5-kilobase (kb) BamHI-generated DNA fragments, respectively, from the wildtype strain. The restriction maps of the cloned fragments were, in fact, different from each other. Only the TYR1 fragment conferred the Mel+ phenotype upon the naturally Mel- Streptomyces lividans when introduced with the Streptomyces-E. coli shuttle vector pRES18. When plasmid pTYDELTA33 with the 2.3 kb insert of TYR1 essential for conferring the Mel+ phenotype was transformed into S. lividans and a Mel- mutant of S. lavendulae possessing only the TYR2 sequence, the transformants of both organisms showed inducible tyrosinase activity. Most of the tyrosinase activity was secreted rapidly during the growth of the S. lavendulae transformant; in contrast, the majority remained intracellular in the S. lividans transformant. DNA sequence analysis revealed that the 2.3 kb fragment contained two open reading frames (ORFs) on the same strand with coding capacities of 156 amino acids (the first ORF) and 273 amino acids (the second ORF). The calculated molecular weights of the deduced proteins were 16,993 and 30,935, respectively. The larger ORF was found to specify tyrosinase. The protein sequence showed a striking homology (83%) to that reported for S. antibioticus tyrosinase. The smaller ORF seems to be a homologue of ORF438, preceding the tyrosinase gene in the mel operon of S. antibioticus. The mel operon of S. lavendulae had some interesting structural features distinct from that of S. antibioticus: the initiation codon of the tyrosinase gene and the termination codon of the preceding gene overlapped and the smaller ORF contained a TTA codon which is rare in Streptomyces, suggesting the involvement of a bldA-like gene in the regulation of the expression of this gene at the translational level.