CLONING AND SEQUENCE-ANALYSIS OF THE HIGHLY EXPRESSED MELANIN-SYNTHESIZING GENE OPERON FROM STREPTOMYCES-CASTANEOGLOBISPORUS

Streptomyces castaneoglobisporus HUT6202 overproduces a diffusible mel anin-like pigment. An operon, designated mel, containing a gene that e ncodes tyrosinase, which is involved in the synthesis of melanin pigme nt, was cloned from the chromosomal DNA of the microorganism into the high-copy plasmid pAK114 and expressed in S. lividans. The tyrosinase activity of the transformed cells was at approximately a 110-fold high er level than that of the same host carrying the plasmid pIJ702, which has the same replication origin as pAK114 and carries the mel operon from S. antibioticus. The sequence analysis of the S. castaneoglobispo rus mel operon revealed that an open-reading frame consisting of 378 b ase pairs(bp), designated ORF378, was found upstream of the tyrosinase gene (TYRC) consisting of 819 bp. In the present study, we constructe d a chimeric mel operon consisting of ORF378 from S. castaneoglobispor us and the tyrosinase gene (TYRA) from S. antibioticus. The chimeric m el operon or the S. antibioticus mel operon, which consists of ORF438 and TYRA, expressed the tyrosinase activity in Escherichia coli intrac ellularly when located under the control of lacZ promoter, and the tyr osinase activity from the former was at a 30-fold higher level than th at from the latter. This suggests that the gene contributing to the hi gh expression of the tyrosinase activity in S. castaneoglobisporus is ORF378, rather than TYRC.