[Cloning and characterization of melanin biosynthesis genes from Streptomyces lincolnensis 78-11]

Using mel C1-C2 gene from pIJ702 as probe to hybridize wity chromosome DNA of S. lincolnensis 78-11, a series of positive bands were found, including a 3.2 kb BanHI fragment and a 2.6 kb SphI fragment. So a gene library of S. lincolnensis, containg 3.0-3.5 kb BamHI fragment, was constructed. Melanin biosynthesis genes, mel C1 and mel C2, were then cloned and the fragment, inserted in recombinant plasmid pRSB336 were sequenced. Among five ORFs of the 3152 bp BamHI fragment, mel C1 and mel C2 were highly homologous to the respective genes of the other three strains of Streptomyces, morever, the product of mel C2 gene from S. lincolnensis 78-11 was slightly homologous to tyrosinase from human-being by 17.3% and from rat by 24.5%. All of the above results indicated that mel C1, mel C2 and orf 3 constitute the melanin biosynthesis operon of S. lincolnensis. In the further study of the cloned melanin biosynthesis genes of S. lincolnensis 78-11, recombinant plasmids, pPZ518 and pPZ519, which contain promoter of neo gene and mel gene in different orientation respectively, were constructed, and then were used to transform S. lividans TK23. 12 transformants of pPZ518, selected at random, secreted light brown pigment in R2YE culture medium, but all transformants of pPZ519 and pES1 were white.